Pericarp development and fruit structure in borassoid palms (Arecaceae Coryphoideae Borasseae)

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1 Annals of Botany 108: , 2011 doi: /aob/mcr148, available online at PART OF A SPECIAL ISSUE ON PALM BIOLOGY Pericarp development and fruit structure in borassoid palms (Arecaceae Coryphoideae Borasseae) Mikhail S. Romanov 1, *, Alexey V. F. Ch. Bobrov 2, D. Siril A. Wijesundara 3 and Ekaterina S. Romanova 4 1 N. V. Tcitcin Main Botanical Garden RAS, 4 Botanical Street, Moscow, , Russia, 2 Department of Biogeography, Geographical Faculty, M. V. Lomonosov Moscow State University, 1 Vorobjevi Gori, Moscow, , Russia, 3 Royal Botanic Gardens, Peradeniya, Sri Lanka and 4 Botanical Garden, Biological Faculty, M. V. Lomonosov Moscow State University, 1 Vorobjevi Gori, Moscow, , Russia * For correspondence. romanovmikhail@hotmail.com Received: 23 December 2010 Returned for revision: 24 January 2011 Accepted: 28 March 2011 Published electronically: 10 August 2011 Background and Aims The Borasseae form a highly supported monophyletic clade in the Arecaceae Coryphoideae. The fruits of Coryphoideae are small, drupaceous with specialized anatomical structure of the pericarp and berries. The large fruits of borassoid palms contain massive pyrenes, which develop from the middle zone of the mesocarp. The pericarp structure and mode of its development in Borasseae are similar to those of Eugeissona and Nypa. A developmental carpological study of borassoid palms will allow us to describe the process of pericarp development and reveal the diagnostic fruit features of borassoid palms, determine the morphogenetic fruit type in Borasseae genera, and describe similarities in fruit structure and pericarp development with other groups of palms. Methods The pericarp anatomy was studied during development with light microscopy based on the anatomical sections of fruits of all eight Borasseae genera. Key Results The following general features of pericarp structure in Borasseae were revealed: (1) differentiation of the pericarp starts at early developmental stages; (2) the exocarp is represented by a specialized epidermis; (3) the mesocarp is extremely multilayered and is differentiated into several topographical zones a peripheral parenchymatous zone(s) with scattered sclerenchymatous elements and vascular bundles, a middle zone (the stony pyrene comprising networks of elongated sclereids and vascular bundles) and an inner parenchymatous zone(s); (4) differentiation and growth of the pyrene tissue starts at early developmental stages and ends long before maturation of the seed; (5) the inner parenchymatous zone(s) of the mesocarp is dramatically compressed by the mature seed; (6) the endocarp (unspecialized epidermis) is not involved in pyrene formation; and (7) the spermoderm is multilayered in Hyphaeninae and obliterated in Lataniinae. Conclusions The fruits of Borasseae are pyrenaria of Latania-type. This type of pericarp differentiation is also found only in Eugeissona and Nypa. The fruits of other Coryphoideae dramatically differ from Borasseae by the pericarp anatomical structure and the mode of its development. Key words: Fruit development, pericarp anatomy, mesocarpic pyrene, unspecialized endocarp, Borasseae, pyrenarium of Latania-type. INTRODUCTION The Borasseae form a tribe of eight palm genera distributed on the lands bordering the Indian Ocean and on its islands (Dransfield et al., 2008, p. 308). According to recent molecular data (Bayton, 2005; Asmussen et al., 2006; Dransfield et al., 2008; Baker et al., 2009) the tribe Borasseae is a wellsupported monophyletic clade of the Arecaceae Coryphoideae. Ontogenetic studies of pericarp anatomy in the Coryphoideae (Murray, 1973; Bobrov and Romanov, 2007; Bobrov et al., 2007, 2008a, b, 2010) have shown that representatives of the tribes Trachycarpeae, Corypheae and Caryoteae share both a complex pericarp structure and a specialized mode of pyrene development in their drupaceous fruits, whereas the genera of Cryosophileae, Phoeniceae, Chuniophoeniceae and Sabaleae produce berries (Biradar and Mahabale, 1969; Landsberg, 1981). The fruits of the Borasseae, with one to three massive pyrenes, are much larger than in other genera of the Coryphoideae. Our original carpological data have shown that the pyrene in borassoid palms develops in the middle zone of the mesocarp. Thus, a broad-scale carpological study of all eight genera of borassoid palms, including a developmental study of pericarp histogenesis, would allow us to describe the process of pericarp development (including the diagnostic fruit features of borassoid palms), to determine the morphogenetic fruit type in the Borasseae, and to identify similarities in fruit structure and pericarp development with other groups of palms. Such a study is described here. From the data obtained, three histogenetic zones of the pericarp the exocarp, the mesocarp and the endocarp derived from the outer epidermis, mesophyll and inner epidermis of the gynoecium were recognized at all developmental stages. The recognition of these zones as histogenetic, but not as topographic, is an important concept in developmental studies (Cave, 1869; Garcin, 1890; Bobrov et al., 2009). We # The Author Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. 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2 1490 Romanov et al. Pericarp development and fruit structure in Borasseae purposely do not use the term endocarp in any morphological descriptions to designate the hard zone of the pericarp [as Roth (1977) did]. The term pyrene is recommended for use in this context to indicate the stony zone of the pericarp which may be of either endocarpic or mesocarpic origin. MATERIALS AND METHODS A list of species and specimens that were investigated is given in Table 1. Material of borassoid palm species was fixed in formalin acetic acid alcohol or 70 % ethanol and dry herbarium specimens were also used in the present study (see below). Standard anatomical protocols were used for anatomical investigations (Prozina, 1960; O Brien and McCully, 1981). No methods were used for softening hard tissues of the pericarp before sectioning. Transverse (TS) and longitudinal sections (LS) (15 30 mm) of Borassoid fruit developmental stages were studied from sections obtained with a slide microtome from samples embedded in paraffin. These sections were stained with phloroglucinol and hydrochloric acid to study details of lignification of cell walls in different topographical zones of the pericarp and were preserved in glycerine. Some sections were stained with ruthenium red and toluidine blue. All sections were visualized by light microscopy. RESULTS Fruit morphology and anatomy at different developmental stages were studied in all genera of the Borasseae (Figs 1 6, Table 2). Detailed morphological descriptions are available from different sources, particularly from the two editions of Genera Palmarum (Uhl and Dransfield, 1987; Dransfield et al., 2008); to avoid repetition only short descriptions of fruit morphology are given here. In all genera of the Borasseae the fruits develop from a trimerous syncarpous gynoecium with a single ovule in each locule. The fruits of subtribe Hyphaeninae (Hyphaene, Satranala, Bismarckia and Medemia) usually develop from one carpel, the other two remaining undeveloped. In this case the fruits are monomerous, as described below. Rarely, two or even all three carpels develop into a fruit, which becomes bi- or trilobed in these cases. These dimerous and trimerous fruits are hemisyncarpous. Each lobe of the fruit contains a single pyrene. In all cases the primarily terminal stigma of the gynoecium remains very close to its original position and the sub-basal stigmatic remains are found in a proximal position on the fruit surface the lateral surface in the case of monomerous fruits or between two (three) fruitlets at their base in the case of the development of di- and trimerous fruits (cf. Fig. 1E and 1M). In the subtribe Lataniinae (Latania, Lodoicea, Borassus and Borassodendron) all three carpels always contribute to fruit development and the fruits are entire (not lobate) at all developmental stages. These fruits are typical tri-, di- or pseudomonomerous pyrenaria with three to one pyrenes (and correspondingly seeds) inside (Figs 1U and 2B). The stigma remains in an apical position until maturity (Figs 1T and 2G). There are three apical pores around the stigma (Fig. 2D, F, G); from these pores narrow lenticelate ducts descend down to the ovoid-shaped bodies of septal nectaries at the base of the fruit (Fig. 2F). In all genera of the Borasseae except Satranala, seed germination occurs through the apical pore of the pyrene (cf. Fig. 1F and 1I). Hyphaene The fruits of the Hyphaene species studied (H. coriacea, H. compressa, H. dichotoma and H. guineensis, Fig. 1A F) usually develop from only one carpel to produce a monospermous, somewhat pear-like (truncate at the distal end) fruit(let) (Fig. 1F), the two remaining carpels developing into small appendages at the base. Six stages of fruit development were identified for H. coriacea and through them the differentiation of the fruit wall was studied (Table 2). All the descriptions of Hyphaene below are based on this species. At the first TABLE 1. List of investigated species and specimens Species Bismarckia nobilis Bismarckia nobilis Borassodendron machadonis Borassus flabellifer Borassus flabellifer Borassus aethiopum Hyphaene compressa Hyphaene coriacea Hyphaene dichotoma Hyphaene guineensis Latania loddigesii Latania lontaroides Latania lontaroides Lodoicea maldivica Lodoicea maldivica Medemia argun Medemia argun Medemia argun Satranala decussilvae Specimen details MR and AB, # 844, , Montgomery Botanical Center, USA, MWG MR and AB, # 1355, , Montgomery Botanical Center, USA s.n. BH MR and AB, # 953, , Jardín Botánico Nacional de Cuba, Cuba, MWG MR and AB, # 1378, , Fairchild Tropical Botanical Garden, USA, MWG MR and AB, # 847, , Montgomery Botanical Center, USA, MWG MR and AB, # 831, , Montgomery Botanical Center, USA, MWG MR and AB, # 864, , Montgomery Botanical Center, USA, MWG MR and AB, # 900, , Jardín Botánico Nacional de Cuba, Cuba, MWG MR and AB, # 567, , Jardín Botánico Nacional de Cuba, Cuba, MWG MR and AB, # 1371, , Fairchild Tropical Botanical Garden, USA, MWG MR and AB, # 1601, , Singapore Botanical Garden, Singapore, MWG MR and AB, # 1651, , Royal Botanic Gardens Peradeniya, Sri Lanka, MWG Moore and Cuého, s. n., 1972, Vallée de Mai, Seichelles, BH Ibrahin, , Western Desert, near Aswan Oasis, Egypt, RBG Kew Spirit collections, UK Darian, s.n., BH carpological collection, P OUEM 15, July August 1994, Mananara Biosphere Reserve, Madagascar, RBG Kew Palm group collection, UK

3 Romanov et al. Pericarp development and fruit structure in Borasseae 1491 A B C D E F G H I J K L M N O P Q R S T U FIG. 1. Fruit morphology and macro sections. (A G) Hyphaene coriacea, (H K) Satranala decussivalvae, (L P) Bismarckia nobilis, (Q S) Medemia argun, (T U) Latania loddigesii. (A E) Monomerous fruit developmental stages (first to fifth, respectively); arrows point to the stigma. (F) Longitudinal section (LS) of fruit at the fifth developmental stage (arrow points to the apical pore). (G) Transverse section (TS) of the pericarp. (H) Bilobed mature fruit. (I) Dehiscent pyrene with germinated seed; arrow points to the slit between the pyrene valves. (J) TS of fruit at the fifth developmental stage. (K) Close up of TS of fruit from (J). (L) Fourth fruit developmental stage. (M) Bilobed fruit; the fourth stage of fruit development. (N) LS of the mature fruit. (O) TS of the mature fruit. (P) Close up of TS of fruit from (O). (Q) Monomerous fruit. (R) TS of fruit. (S) Close up of TS of fruit from (R). (T) Mature fruit with apical position of stigma (arrow). (U) TS of fruit at the fourth developmental stage. Abbreviations: m1, first zone of mesocarp; m2, second zone of mesocarp; m3, third zone of mesocarp. Scale bars (A C,F,I,J,L,N,O,Q,R,U)¼ 1 cm; (D, E, H, M, T) ¼ 2 cm; (G) ¼ 0.2 cm; (K, P, S) ¼ 0.5 cm.

4 1492 Romanov et al. Pericarp development and fruit structure in Borasseae A B C D E F G H I J K L M N O FIG. 2. Fruit morphology and macro sections. (A D) Lodoicea maldivica, (E H, L) Borassus flabellifer, (I K) Borassus flabellifer Borassus aethiopum, (M O) Borassodendron machadonis. (A) Gynoecium and fruit morphology. (B) TS of mature fruit with ventral (top arrow) and dorsal (bottom arrow) internal flanges. (C) Close up of TS of fruit from B. (D) TS of gynoecium with three TS of ducts of septal nectaries (arrow). (E) TS of fruit at the first developmental stage; dorsal vascular bundles are forming their flanges (arrow). (F) LS of fruit at the second developmental stage and LS of septal nectary with secretory body (bottom arrow), lenticelate duct (middle arrow) and apical pore (top arrow). (G) Fruit morphology at the second developmental stage; note the apical position of stigma (top arrow) and apical pores of septal nectarines (bottom arrow). (H) TS of the fruit fragment at the second developmental stage at the locule level; note ventral (top arrow) and dorsal (bottom arrow) flanges. (I) LS of fruit at the fourth developmental stage. (J) Morphology of fruit at the fifth developmental stage. (K) Fragment of TS of fruit at the fifth developmental stage. (L) TS of pyrene of mature fruit; note ventral (top arrow) and dorsal (bottom arrow) flanges. (M) Morphology of the pyrene ( peripheral softer tissues of pericarp removed). (N) TS section of the pyrene; note the rudiment of the ventral suture (top arrow), ventral (middle arrow) and dorsal (bottom arrow) flanges. (OL) Close up of TS of pyrene from (N); note the rudimentary ventral slit (arrow). Abbreviations: m1, first zone of mesocarp; m2, second zone of mesocarp; m3, third zone of mesocarp; m4, fourth zone of mesocarp. Scale bars (A, B) ¼ 10 cm; (C E, H) ¼ 1 cm; (F, G, I, L N) ¼ 2 cm; (J) ¼ 5 cm; (K, O) ¼ 0.5 cm.

5 Romanov et al. Pericarp development and fruit structure in Borasseae 1493 A B E F C D FIG. 3. Anatomical TS of pericarp. (A E) Hyphaene coriacea, (F) Satranala decussivalvae. (A E) Fruit developmental stages (first to fifth, respectively). (F) Fifth stage of fruit development. Abbreviations: ex, exocarp; m1, first zone of mesocarp; m2, second zone of mesocarp; m3, third zone of mesocarp; en, endocarp. Scale bars ¼ 500 mm.

6 1494 Romanov et al. Pericarp development and fruit structure in Borasseae A B L O C D E F G M H N I J K FIG. 4. Anatomical TS of pericarp. (A, F H) Bismarckia nobilis, (B, C) Hyphaene coriacea, (D, E) Satranala decussivalvae, (I M) Medemia argun, (N) Latania lontaroides, (O) Lodoicea maldivica. (A) Mature fruit, peripheral zones of pericarp. (B, C) Innermost layers of pericarp at the first (B) and the third (C) developmental stages. (D) Hypodermis structure. (E) Close up of the pyrene fragment from Fig. 3F. (F) Hypodermis structure. (G) Fragment of pyrene structure. (H) Inner zones of pericarp. (I) Hypodermis structure. (J) Structure of the pyrene. (K) Close up of the pyrene structure from J. (L) Exocarp, outer and middle zones of mesocarp. (M) Inner zone of mesocarp and endocarp. (N) Fourth developmental stage. (O) Stage of pollinated gynoecium. Abbreviations: ex, exocarp; hy, hypodermis; m1, first zone of mesocarp; m2, second zone of mesocarp; m3, third zone of mesocarp; m4, fourth zone of mesocarp; en, endocarp. Scale bars (A, F, G, I, L, M, O) ¼ 500 mm; (B, D) ¼ 100 mm; (C, E, H, K) ¼ 200 mm; (J, N) ¼ 1000 mm.

7 Romanov et al. Pericarp development and fruit structure in Borasseae 1495 A C D E F G B H I J K FIG. 5. Anatomical TS of pericarp. (A C, H K) Lodoicea maldivica, (D) Borassus flabellifer, (E G) Latania lontaroides. (A) Fourth developmental stage; inner zones of pericarp. (B, C) Mature fruit. (D) First developmental stage. (E) Fourth developmental stage. (F) Fifth developmental stage. (G) Close up of the inner zones of pericarp from Fig. 4N. (H) Structure of the peripheral layers of pericarp in pollinated gynoecium. (I) Structure of the peripheral layers of pericarp in mature fruit. (J) Close up of aerenchyma structure from (C) (third zone of mesocarp, mature fruit). (K) Endocarp structure in the mature fruit. Abbreviations: ex, exocarp; hy, hypodermis; m1, first zone of mesocarp; m2, second zone of mesocarp; m3, third zone of mesocarp; m4, fourth zone of mesocarp; m5, fifth zone of mesocarp; m6, sixth zone of mesocarp; en, endocarp. Scale bars (A D, I) ¼ 1000 mm; (E H, J, K) ¼ 200 mm.

8 1496 Romanov et al. Pericarp development and fruit structure in Borasseae A G H I B J C D E K F FIG. 6. Anatomical TS of pericarp. (A F) Borassus flabellifer, (G K) Borassus flabellifer Borassus aethiopum. (A) Exocarp and peripheral zone of mesocarp structure at the locule level at the second stage of fruit development. (B) Exocarp and peripheral zone of mesocarp structure in the distal part of the fruit (above the locule level) at the second stage of fruit development. (C) Structure of the second zone of the pericarp (pyrene), at the second stage of fruit development. (D) Endocarp structure at the second stage of fruit development. (E) Structure of inner zones of pericarp in mature fruits. (F) Close up of endocarp structure in mature fruit. (G, H) Fourth stage of fruit development. (I) Peripheral zones of pericarp at the fifth stage of fruit development. (J) Outer zone of mesocarp at the fifth stage of fruit development. (K) Inner zones of pericarp at the fifth stage of fruit development. Abbreviations: ex, exocarp; hy, hypodermis; m1, first zone of mesocarp; m2, second zone of mesocarp; m3, third zone of mesocarp; m4, fourth zone of mesocarp; en, endocarp. Scale bars (A, D, E, F, I) ¼ 100 mm; (B, C, G, H, J) ¼ 500 mm.

9 Romanov et al. Pericarp development and fruit structure in Borasseae 1497 TABLE 2. Fruit developmental stages in borassoid palms: number of cell layers of pericarp at every studied stage Genus Stage Hyphaene Satranala 210 Bismarckia 150 Medemia 120 Latania Lodoicea Borassus Borassodendron 500 developmental stage (soon after pollination, Fig. 1A) one of three carpels begins to dominate over the others; it is twice as large at this point. The pericarp is thick (Fig. 3A, Table 2) and differentiated into an exocarp (outer epidermis), mesocarp (mutlilayered parenchyma) and endocarp (locular epidermis, Fig. 4B). Below the outer epidermis, several irregular layers of tannin-containing cells comprise the hypodermis. The mesocarp starts its differentiation into three topographic zones: the outer one (isodiametric parenchymatous cells with numerous radial nests of tannin-containing cells and nests of sclereids scattered among them), the middle one (larger parenchymatous cells with scattered solitary cells or small nests of tannin-containing cells; rather numerous large central longitudinal provascular bundles and numerous initials of additional small protovascular bundles on the inner border) and the inner zone ( parenchyma with scattered tannin-containing cells and oil-containing cells). At the second developmental stage fruits are larger (Fig. 1B) and the pericarp is thicker (Fig. 3B), due to cell multiplication in the mesocarp. Tannin-containing cells are grouped into larger nests in the outer and inner zones of the mesocarp, and the nests of sclereids in the peripheral zone are also larger. The large vascular bundles in the middle part of the mesocarp central zone are seen to be differentiating into vascular and bundle sheath elements. The inner provascular bundles have become larger and more pronounced, resulting in a regular circle seen in the transverse section of the fruitlet. At the third stage the fruits are similar in shape to the mature fruits: the two reduced carpels are easily recognized (Fig. 1C). The exo- and endocarp (Fig. 4C) are still single-layered; the cells of the endocarp are elongated in a tangential direction and covered with a cuticle. New nests of radially elongated cells with thickened walls differentiate in the outer zone of the mesocarp (Fig. 3C). The cells of the middle zone multiply and become elongated in a tangential direction; formerly spherical tannin-containing cells also become elongated in a tangential direction. The inner vascular bundles are now well differentiated and have massive sheaths composed of longitudinally elongated fibre-like cells with thickened walls. At the fourth stage the fruits are orange brown (Fig. 1D, differing from previous stages, when the fruits were green). The walls of most of the fibre-like cells nested in the mesocarp peripheral zone are now seen to be lignified (Fig. 3D). This lignification is a centripetal process. Further development and differentiation of tangentially elongated cells takes place in the middle zone of the mesocarp. It becomes increasingly multilayered, larger and harder due to thickening of cell walls. The vascular bundle sheath of the inner circle becomes more massive. The parenchyma cells of the inner mesocarp zone become larger compared with the previous stage. The fifth stage of fruit development identified represents nearly mature fruits (Fig. 1E, F) showing the final stage of differentiation of the pericarp (Figs 1G and 3E). The cells of the exocarp in mature fruits have thickened and lignified walls. Cells of the mesocarpic tanniniferous hypoderm also have thickened lignified walls by the time maturity is reached. Scattered brachysclereids originate in the outer zone of the mesocarp in mature fruits, some of them have differentiated from tannin-containing cells and still contain tannins. The numerous nests of fibre-like sclereids are connected with the cells of the second zone, which shows dramatic changes by now the walls of all cells (except the tannin-containing cells) are thickened and lignified. Spherical stegmata (silica bodies) are observed on the periphery of some fibre-like sclereids. This zone is in fact differentiated into two sub-zones. The outer one is composed of stout vascular bundles with massive sclerenchymatous sheaths and numerous twisted networks of fibre-like, mostly tangentially elongated sclereids with thickened lignified walls. The nests of tannin-containing cells are observed between some of these sclerenchymatous networks. The inner sub-zone comprises large longitudinal vascular bundles with stout U-shaped sheaths composed of longitudinally elongated fibre-like sclereids and adheres to the networks of sclereids from the outer sclerenchymatous sub-zone. Thus, the pyrene of the fruitlet is formed from interlaced networks of sclereids, which are interspersed with nests of parenchymatous cells. In its overall form, the structure of the pyrene in H. coriacea could be likened to two spherical baskets tightly packed inside each other. The pyrene of Hyphaene is not absolutely rigid. The main ventral and dorsal vascular bundles participate in pyrene formation in fruits of Hyphaene (and all other genera of the Borasseae). The inner zone of the mesocarp is still composed of multilayered parenchyma with scattered tannin- and oil-containing cells. This zone plays a very important role at the sixth and final stage of fruit and seed maturation: the seed dramatically enlarges and compresses this inner soft (spongy) zone of the pericarp. Thus, the final stages of seed development take place under the protection of the pyrene, which becomes lignified long before seed maturation and fruit abscission. In all other borassoid palm genera the innermost parenchymatous zone of the mesocarp functions in the same way and provides extra space for seed growth inside the pyrene. Satranala In most cases only one carpel (sometimes two: Fig. 1H) develops into a monospermous spherical fruit in Satranala decussilvae. The surface of the pyrene is covered with longitudinally orientated flanges the main one, which is dorsiventral and circular, plus additional regularly distributed lateral ones rising from the base of the pyrene and partly anastomosing in the distal part (Fig. 1G K). When the

10 1498 Romanov et al. Pericarp development and fruit structure in Borasseae process of seed germination starts, the pyrene dehisces into two valves (Fig. 1I). The pericarp of the studied fruit of Satranala displays complete differentiation with all sclerenchymatous zones displaying lignification prior to the seed reaching maturity (Figs 1J, K and 3F, Table 2). The pericarp of S. decussilvae is multilayered (Table 2) and differentiated into an exocarp (epidermis composed of cells with thickened and partly lignified walls and tannins in the lumens, Fig. 4D), a mesocarp (multilayered and differentiated into three topographic zones) and an endocarp (locular epidermis composed of thin-walled, tangentially elongated fusiform cells) (Fig. 3F). Below the exocarp, several layers of tannincontaining brachysclereids mixed with scattered small tannin-containing parenchymatous cells comprise the hypodermis (Fig. 4E). The outer zone of the mesocarp is formed of parenchymatous cells with insufficiently thickened walls, and scattered among them small brachysclereids (solitary or in nests), solitary huge oval-shaped sclereids and radially elongated nests of radially elongated fibre-like sclereids. Many cells in this zone contain tannins or secretory substances. There are scattered schizogenous secretory cavities, which are spherical or oval-shaped, in the peripheral zone of the mesocarp. The second zone of the mesocarp, the pyrene, is composed of fibre-like sclereids with heavily thickened walls and numerous spherical stegmata on the periphery of the cells (Fig. 4E). The fibre-like sclereids comprise networks, interlaced in different directions, and separated by parenchymatous layers. Stout vascular bundles participate in the formation of the pyrene. The pyrene contains many cell layers organized to form flanges. The dehiscence zone of the pyrene has a peculiar structure: fibre-like cells are orientated parallel to the plane of dehiscence and they are partly separated by a parenchymatous zone. The third zone of the mesocarp is composed of parenchymatous cells and numerous scattered large, spherical, schizogenous secretory cavities, which may be aggregated into groups (separated by thin layers of parenchymatous cells) in the innermost part of this mesocarp zone (Fig. 1J). Vascular bundles are regularly distributed in this zone just inside the pyrene. Bismarckia The mature fruit(let)s of Bismarckia nobilis are ellipsoidal (Fig. 1L, M). The rumination of the single seed is achieved by irregular longitudinal invaginations of the pyrene (approx. ten irregular inner flanges) and a deep radial central protrusion at the base of the seed (Fig. 1N P). The pericarp of a mature fruit is multilayered (Fig. 4A, F H, Table 2) and differentiated into an exocarp (single-layered epidermis of small tannincontaining cells with thickened and lignified walls), a mesocarp (differentiated into four topographic zones) and an endocarp (single-layered epidermis compressed at maturity by the growing seed). Below the exocarp the hypodermis, which is composed of several layers of brachysclereids ( partly interspaced with parenchymatous cells), is differentiated (Fig. 4F). The outer zone of the mesocarp is formed from parenchymatous cells and scattered among them solitary tannincontaining and oil-containing cells, numerous brachysclereids (single or in small nests) and large radially elongated nests of fibre-like sclereids. Next, a very soft and fleshy mesocarp zone is formed of 4 7 layers of strongly radially elongated parenchymatous cells; sometimes oil bodies can be seen within the lumen. The third topographic zone is the pyrene with a wavy outer surface (Fig. 1P). The pyrene is composed of fibre-like sclereids, which comprise the elongated networks, interlaced in different directions (Fig. 4G, H). These networks also make the invaginations inside the seeds which constitute the rumination. The stout longitudinal vascular bundles (including dorsal and ventral ones) with massive sclerenchymatous sheaths also participate in pyrene formation and are mostly regularly scattered between the networks of sclereids at the periphery of this zone. The fourth zone of the mesocarp is composed of numerous layers of parenchymatous cells, the cells of the innermost layers containing tannins (Fig. 4H). Medemia The fruits of Medemia argun are ellipsoidal (Fig. 1Q) The pericarp is thick (Fig. 1R, S) and multilayered (Fig. 4L, M, Table 2) and differentiated into an exocarp (single-layered epidermis formed of small tannin-containing cells with thickened and lignified walls), a mesocarp (differentiated into three topographic zones) and an endocarp (single-layered epidermis compressed by the growing seed by the time maturity is reached). The outer zone of the mesocarp is composed of parenchymatous cells and scattered among them nests of tannincontaining cells and elongated nests of fibre-like sclereids. Just below the exocarp the well-developed hypodermis composed of several layers of tightly packed brachysclereids can be distinguished (Fig. 4I). The next zone (the pyrene) is formed of fibre-like sclereids. The peripheral cells are mostly tangentially elongated and are in tight contact with the inner longitudinally elongated networks of fibre-like sclereids, which are more or less regularly arranged (Fig. 4J L). The dorsal and ventral vascular bundles with massive sclerenchymatous sheaths also participate in pyrene formation. The pyrene in Medemia is thinner than in Bismarckia, Satranala and Hyphaene. The third zone of the mesocarp is composed of large parenchymatous cells interspersed with scattered, solitary tannin-containing cells (Fig. 4M). Latania The fruits of Latania species are obovate with three (two to one) pyrenes inside (Fig. 1T U). The pyrenes are covered with several dorsal longitudinal flanges (Fig. 1U) irregularly anastomosing at the distal end. The mature pericarp of Latania lontaroides is multilayered (Table 2) and differentiated into an exocarp (single-layered epidermis composed of large palisade tannin-containing cells with thickened walls), a mesocarp (differentiated into four topographic zones) and an endocarp (single-layered epidermis compressed by the growing seed at maturity) (Figs 4N and 5E G). Several layers of small brachysclereids comprise the hypodermis (Fig. 5E, F). The outer zone of the mesocarp comprises parenchymatous cells with numerous elongated nests of radially elongated brachysclereids scattered among them. These nests are radially interconnected

11 Romanov et al. Pericarp development and fruit structure in Borasseae 1499 by nests of tangentially elongated brachysclereids. The second zone of the mesocarp is composed solely of parenchymatous cells with poorly thickened walls. The next zone, the pyrene (Fig. 5G), is composed of fibre-like sclereids which are mostly longitudinally elongated with numerous vascular bundles scattered among them. The flanges on the pyrene s outer surface correspond to some of these vascular bundles with massive sclerenchymatous sheaths. The peripheral layers of sclereids are mostly tangentially elongated. The fourth zone of the mesocarp is composed of large parenchymatous cells with numerous vascular bundles with mechanical sheaths scattered among them. Lodoicea The fruits of Lodoicea maldivica (J.F. Gmel.) Pers. are the biggest monospermous fruits of the plant kingdom (Fig. 2A). The ultimately heavy and volumetric fruit of the double coconut develops for 5 7 years and dramatic structural changes occur in the pericarp during this time with respect to the carpel wall (Fig. 2B D, Table 2). At the stage of the pollinated gynoecium the fruit wall is multilayered (Table 2) and differentiated into an exocarp (single-layered epidermis formed of palisade cells with thickened walls), a mesocarp (differentiated into three topographic zones) and an endocarp (single-layered epidermis) (Figs. 4O). Below the exocarp the hypodermis, composed of several layers of brachysclereids, can be distinguished (Figs 4O and 5H); brachysclereids (solitary or in small nests) are also scattered in the peripheral part of the outer mesocarp zone. The outer zone of the mesocarp is formed of parenchymatous cells and scattered among them are numerous mostly longitudinally arranged vascular bundles. The next zone of the mesocarp is composed of small parenchymatous cells, most of which are elongated (in one of many different directions); numerous small vascular bundles are differentiated in the peripheral part of this zone. The innermost zone of the mesocarp is composed of large isodiametric parenchymatous cells and scattered among them are numerous small vascular bundles orientated in different directions. At the next developmental stage the pericarp is much thicker (Table 2) and the mesocarp is differentiated into six topographic zones (exocarp and endocarp still represented by a single-layered epidermis composed of palisade cells). Generally the differentiation of the mesocarp is similar in mature fruit, a detailed description of which is given below. The main differences in fruit structure at the fourth and sixth developmental stages are apparent in the structure of zones 4 6 of the mesocarp (Fig. 5A). The fourth zone of the mesocarp comprises the pyrene and is composed of approx. 100 layers of fibre-like sclereids with thickened walls. These sclereids form thick elongated networks which are interlaced in different directions, and are partly separated by layers of parenchymatous cells containing tannins. The fifth zone of the mesocarp is very thick (more than 3 cm) and consists of more than 100 layers of large parenchymatous cells with small nests of tannincontaining cells and numerous vascular bundles scattered among them. The sixth, innermost mesocarp zone is represented by layers of parenchymatous cells with solitary tannin-containing cells and numerous vascular bundles scattered among them. The inner hypodermis is differentiated from the innermost layers of this zone; it is formed of several layers of tannin-containing cells. The mature pericarp is differentiated into a single-layered exocarp ( palisade cells with thickened walls covered with thick cuticle, Fig. 5H), an extremely multilayered mesocarp (differentiated into six topographic zones) and a singlelayered endocarp (epidermal cells) (Fig. 5B, J, K). The first, peripheral zone is composed of three types of sclereids with thickened lignified walls (Fig. 5H). The outermost layers of small irregularly distributed brachysclereids form the hypodermis. Below them are the huge tangentially elongated sclereids forming vertical rows of cell layers. They are regularly interspersed with nests of radially elongated sclereids. The peripheral zone of the mesocarp gradually transforms into a second zone, which is composed of mostly tangentially elongated parenchymatous cells with thickened walls and numerous brachysclereids (either solitary or in nests), as well as regularly developed vascular bundles with stout sheaths. The brachysclereids become mostly solitary and fewer in number and the bundles become smaller and less numerous towards the inner side of this zone. Gradually, the second topographic zone transforms into the third one, which consists of aerenchyma formed from thin-walled cells developing in tangential rows interspersed with tangentially elongated intercellular spaces of varying shape (Fig. 5J). Numerous vascular bundles are present in this zone. The fourth zone of the mesocarp (the pyrene) consists of up to 100 layers of fibrelike sclereids, which form large elongated networks. The latter are interlaced in different directions and separated by layers of parenchymatous cells. The fifth zone of the mesocarp is composed of parenchymatous cells containing tannins in the walls and numerous vascular bundles that predominate in the central region. This zone is dramatically flattened in mature fruits in comparison with previous developmental stages. The innermost layer of cells in this zone is an interrupted layer of brachysclereids. Parenchymatous cells and numerous vascular bundles represent the sixth mesocarp zone. Numerous large secretory cells are seen to have developed in this zone and form tangential nests (rows). Borassus Borassus species have the second largest fruits among borassoid palms (Fig. 2E L). The description given here is based on two studied species Borassus flabellifer and a hybrid Borassus (B. flabellifer B. aethiopum), which displays a very similar pericarp structure. When cleaned of the peripheral pericarp, the pyrenes remain covered with numerous fragments of vascular bundles, which participate in the formation of the outer mesocarp zones. At the first stage the pericarp is highly multilayered (Table 2) and is composed of parenchymatous cells (Figs 2E and 5D) and numerous ( pro-)vascular bundles. In transverse sections of immature fruits, the dorsal vascular bundle is notable. The exocarp and endocarp are represented by a poorly differentiated epidermis and the mesocarp is poorly

12 1500 Romanov et al. Pericarp development and fruit structure in Borasseae differentiated into three parenchymatous zones the outer one ( parenchymatous cells with scattered vascular bundles), the middle one (smaller cells containing different granulate deposits in the lumens and scattered protovascular bundles) and the innermost zone (structure similar to the second zone, but the parenchymatous cells are larger). The mesocarp structure is very different with regard to the locules and in the central region of the fruit (the distal region is much more developed). At the second stage of fruit development (Fig. 2F H and 6A D), the exocarp is composed of undifferentiated epidermal tannin-containing cells, the outer mesocarp zone also shows poor locule differentiation (Fig. 6A) and the exocarp is represented by palisade epidermal cells with thickened walls and numerous vascular bundles with massive sheath differentiated in the peripheral mesocarp zone in the central region of the fruit (Fig. 6B). The endocarp is represented by undifferentiated epidermal cells (Fig. 6D). At the fourth stage (Fig. 2I), several layers of brachysclereids comprise the hypodermis below the exocarp. The mesocarp is differentiated into four topographic zones. The outer zone is composed of parenchymatous cells with numerous large longitudinal, stout, sheathed vascular bundles scattered among them and numerous smaller vascular bundles. The middle part of this parenchymatous zone is similar in structure to aerenchyma. The second zone of the mesocarp (the pyrene) consists of fibre-like sclereids with heavily thickened and partly lignified walls. These sclereids form large elongated networks which are interlaced in different directions. Scattered layers of parenchymatous cells are observed between the nests of sclereids. Dorsal and ventral vascular bundles are seen to produce longitudinal ridges on the inner surface of the pyrene. The third zone of the mesocarp is composed of layers of large parenchymatous cells with numerous vascular bundles scattered among them. The fourth zone is formed of small parenchymatous cells and numerous scattered vascular bundles. The endocarp is represented by a single-layered epidermis, which makes contact from the inside with the reduced and partly obliterated one- to two-layered seed coat. At the fifth and sixth stages of fruit development (Fig. 2J L and 6E, F, I K) the exocarp is finally differentiated and represented by typical tannin-containing palisade cells with thickened lignified walls (Fig. 6I). Several layers of tannincontaining brachysclereids make up the hypodermis (Fig. 6I). The pyrene (the second mesocarp zone) is composed of fibrelike sclereids with fully lignified walls. The third zone of the mesocarp is composed of tannin-containing parenchymatous cells (Fig. 6K) and scattered vascular bundles dramatically compressed by the growing seed. The inner mesocarp zone is formed from parenchymatous cells with thickened walls, numerous vascular bundles and a stout sheath (Fig. 6E, K). The endocarp is represented by the inner epidermis, which is composed of ellipsoidal tannin-containing parenchymatous cells (Fig. 6E, F). Borassodendron The fruits of Borassodendron machadonis (Fig. 2M O) and their pericarp structure are very similar to those of Borassus. The most important and notable difference lies in the structure of the pyrene, which consists of about ten additional inner flanges (ridges) (besides the two main ones formed from two ventral and one dorsal bundle) developed on its inner surface (Fig. 2N, O). Large longitudinal vascular bundles (including dorsal and ventral ones) with their massive sheaths participate in the formation of these ridges. The ridges make invaginations into the seed and thus the observed rumination. There is a short rudiment of the ventral suture on the adaxial face of every pyrene. The ventral slit could be traced throughout the second zone of the mesocarp inside the rudimental zone on the surface (Fig. 2O). Two types of seed coat differentiation are found in borassoid palms. In the first case, common to all genera of the Hyphaeninae, the seed coats are very well developed, multilayered (25 50 layers of cells) and composed of parenchymatous cells (sometimes containing tannins or secretory substances) and numerous vascular bundles scattered among them. In the Lataniinae at early developmental stages, the seed coats are represented by 1 2 layers of parenchymatous cells and they are fully obliterated at fruit maturity. Thus, the single-layered parenchymatous endocarp is in contact with cells of the endosperm in mature fruit. DISCUSSION Our anatomical study of fruits of the Coryphoideae Borasseae at different developmental stages revealed the following general features in their pericarp structure: (1) the anatomical differentiation of the pericarp starts at early developmental stages ( just after pollination), and the pericarp development is progressive and regular; (2) the exocarp is formed of specialized epidermal tannin-containing cells with thickened walls, the latter being isodiametric in the Hyphaeninae and palisade in the Lataniinae; (3) the mesocarp (constituting most of the pericarp) is heavily multilayered and is differentiated into several topographical histological zones one or more peripheral parenchymatous zone(s) with scattered sclerenchymatous elements and vascular bundles, a middle zone comprising the woody (stony) pyrene and one or more inner parenchymatous zone(s); (4) the differentiation and growth of the pyrene tissue starts in the early developmental stages (shortly after pollination) and finishes long before maturation of the seed the pyrene is composed of networks of fibre-like sclereids orientated in different directions and sheaths of vascular bundles which together form a large, tightly packed and interlaced network; (5) main, dorsal and ventral vascular bundles participate in pyrene formation and may form internal flanges on its inner surface; (6) the inner parenchymatous zone of the mesocarp plays an important role in seed growth and maturation following the growth of the pyrene this soft zone progressively becomes thicker and fills the free space within the locule, thereby securing a fixed position for the seed; when the seed starts its dramatic enlargement prior to maturation, it compresses the inner parenchymatous zone(s) of the mesocarp and finally fills the entire space inside the pyrene; (7) the endocarp is represented by a single-layered unspecialized epidermis and does not participate in pyrene formation. A number of outstanding and specific fruit features for palms are found in the different genera of the borassoid palms. The dehiscence of the pyrene in Satranala is unique

13 Romanov et al. Pericarp development and fruit structure in Borasseae 1501 amongst borassoid palms (and the Arecaceae as the whole). The development of a multilayered aerenchyma in the middle topographic zone of the mesocarp in Lodoicea provides an adaptation of the fruits for hydrochory by increasing buoyancy. Similar (but atypical and irregular) aerenchyma develops in the middle of the outer mesocarp zone in Borassus fruits. The pyrenes in some genera of the Borasseae have external (Satranala, Latania) or internal (Bismarckia, Lodoicea, Borassus, Borassodendron) flanges. These internal flanges are constructed from either dorsal and ventral bundles alone (i.e. the main ones) with massive sheaths (Borassus, Lodoicea), or from both the main and additional vascular bundles (Borassodendron), or in other cases the flanges are formed from the interspaced networks of sclereids only (Bismarckia). Coenocarpous fruits with pericarp differentiation as seen in Borassoid palms are classified as a distinct morphogenetic fruit type the pyrenarium of Latania-type (Bobrov et al., 2009). The structure of the pericarp in borassoid palms and its mode of development are considered to constitute a symplesiomorphy with respect to the Borasseae, for which monophyly within the Coryphoideae has been consistently supported (Asmussen et al., 2006; Dransfield et al., 2008; Baker et al., 2009). Similar carpological features and the same mode of pericarp development have also been revealed in Eugeissona (Calamoideae Eugeissoneae: A. V. F. Ch. Bobrov, M. V. Lomonosov, Moscow State University, Moscow, Russia, and J. Dransfield, RBG Kew, Richmond, UK, unpubl. res.), which was recently inferred to be the most basally branching palm genus (Asmussen et al., 2006; Dransfield et al., 2008; Baker et al., 2009; Takhtajan, 2009), and Nypa fruticans (Nypoideae) fruits (M. S. Romanov, unpubl. res.), which is sister to all other palms except the subfamily Calamoideae (Asmussen et al., 2006; Dransfield et al., 2008; Baker et al., 2009). The gynoecium and fruit of Eugeissona are covered with scales, which are a synapomorphous feature of the Calamoideae (Baker et al., 2000; Asmussen et al., 2006; Dransfield et al., 2008; A. V. F. Ch. Bobrov, J. Dransfield, RBG Kew, Richmond, UK, and M. S. Romanov, unpubl. res.). There are internal flanges on the pyrene surface formed from the vascular bundles in Eugeissona fruits, which develop in the same mode as in some borassoid palms (Lodoicea, Borassus, Borassodendron). Based on the anatomical differentiation of their pericarp, the coenocarpous fruits of Eugeissona are classified as the same morphogenetic fruit type as borassoid palms the pyrenarium of Latania-type (Bobrov et al., 2009). The fruits of Nypa are drupes of Rhapis-type, the apocarpous variation of the pyrenarium of Latania-type. The pyrenarium of Latania-type is the plesiomorphic fruit type for the Calamoideae, all other taxa of which have berries with a specialized pericarp structure (A. V. F. Ch. Bobrov, J. Dransfield, RBG Kew, Richmond, UK, and M. S. Romanov, unpubl. res.). Thus, the pyrenarium of Latania-type is revealed in three groups of palms from different subfamilies, including the basal-most palm Eugeissona plus Nypa and the Borasseae. The similarities in structure and identity seen in the pericarp development of borassoid palms, Eugeissona and Nypa are even more striking if we take into consideration the different structural gynoecium types in these taxa: apocarpous made from free carpels in Nypa; paracarpous (unilocular, symplicate) in Eugeissona; and eusyncarpous (trilocular, synascidiate) in the Borasseae (Uhl and Moore, 1971; Uhl and Dransfield, 1987; Endress, 1994; Dransfield et al., 2008). Fruit structure and the mode of pericarp development in other representatives of the Coryphoideae studied differ from those of borassoid palms. The pericarp anatomy of the mature fruits of Chamaerops (Bobrov and Romanov, 2007), Rhapis, Guihaia, Trachycarpus (Bobrov et al., 2007, 2008a), Licuala (A. V. F. Ch. Bobrov and M. S. Romanov, unpubl. res.), Rhapidophyllum, Livistona, Pritchardia and Washingtonia (Murray, 1973) is generally similar: the exocarp is represented by a singlelayered epidermis, the mesocarp is differentiated into three main topographic zones [ peripheral; parenchymatous with vascular system; middle (¼ the pyrene) made of (one) two to four layers of woody sclereids; and the inner parenchymatous zone] and a single-layered parenchymatous endocarp. The pyrene in these fruits is formed from the middle zone of the mesocarp, but it is composed of sclereids of different shape (and origin see below): most cells are isodiametric in outline with heavily thickened walls, but some sclereids are partly radially elongated and have thinner walls. Pericarp development in these genera is similar to the processes described for coryphoid fruit type established by Murray (1973). Differentiation of the carpel walls starts from development of the pyrene in the middle zone of the mesocarp, then the peripheral zone of the mesocarp develops. Further fruitlet/fruit growth gives rise to lacunae in the sclerenchymatous pyrene. These lacunae are progressively filled via multiplication of cells of the peripheral and inner mesocarp zones. Soon the walls of these new cells become thickened (although less so than the walls of the original cells of this zone) and lignified. The inner zone of the mesocarp and the unspecialized endocarp suffer partial compression at maturity due to the growing seed. Fruits displaying this type of pericarp differentiation are classified as drupes of the Rhapis-type (Bobrov et al., 2009). The mode of development of the middle (sclerenchymatous) zone of the mesocarp in Chamaerops, Rhapis, Guihaia, Trachycarpus, Licuala, Rhapidophyllum, Livistona, Pritchardia and Washingtonia on the one hand and in borassoid palms on the other differs considerably. The structure of the mature pericarp and the mode of its development in Phoenix (Lloyd, 1910; Landsberg, 1981), Schippia concolor, Chuniophoenix and Sabal differ from the above-mentioned taxa of the Coryphoideae (Bobrov et al., 2008b, 2010). In this case the exocarp is represented by a typical epidermis, the mesocarp is differentiated into a hypodermis (comprising several layers of thin-walled cells and layers of sclereids with very thick lignified walls) and a main parenchymatous zone, containing two concentric circles of vascular bundles, whilst the endocarp is singlelayered and relatively unspecialized. Differentiation of the carpel wall in Phoenix (Landsberg, 1981), Schippia, Chuniophoenix and Sabal starts with the multiplication of the mesophyll and the formation of the multilayered mesocarp, which is originally composed of regular parenchymatous cells. Next, the hypodermis differentiates into tannin-containing cells which form the peripheral layers of the hypodermis and the sclerenchymatous cells of the inner hypodermal layers. The endocarp undergoes relatively