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IZVESTIYA TSKhA, special issue, 2013
FRUIT STRUCTURE OF THE GENUS TORICELLIA DC. (TORICELLIACEAE) AND ITS TAXONOMIC POSITION IN THE ORDER APIALES
E.Yu. Yembaturova1, A.I. Konstantinova2
(1RSAU-MTAA, 2MSU named in honour of M.V. Lomonosov)
Abstract: The genus Toricellia DC. with 2 species - T. tiliifolia DC. and T. angulata Oliv. - is represented by dioecious trees or shrubs with alternate exstipulate leaves and morphological differences between male and female flowers. Its fruit (pyrenarium) is ovoid or obliquely ovoid, crowned by a persistent calyx. Toricellia has 2-4-loculed yet 1-seeded pyrenaria, developed from the inferior ovary (pseudomonocarpous fruits). Outer walls of sterile locules have rounded openings. A very peculiar stone ("pyrene") consists of two zones: the outer is very thick, consisting of multilayered lignified sclereids; the inner is one-layered, fiber-formed, inconspicuous. Thus, in species of Toricellia, the endocarp proper composes only the inner part of the pyrene, while the outer part is made of mesocarp elements, so the "pyrene" is of combined mesoendocarpic origin. The only seed is of the same shape as the locule - oblong and curved. Seed coat of a few cell layers. Some features shared by Toricellia and other related genera (Aralidium Miq., Griselinia Forst., Melanophylla Baker) are discussed along with Toricellia's possible taxonomic relations.
Key words: Toricellia, Toricelliaceae, fruit anatomy, pyrenaria, Apiales, Cornales, taxonomic position
Introduction
The genus Toricellia DC. with 2 species - Toricellia tiliifolia DC. and T. angulata Oliv. - is represented by dioecious trees or shrubs with alternate exstipulate leaves and morphological differences between male and female flowers. The pentamerous flowers are in lax axillary paniculate inflorescences. These plants' distribution range is confined to southeastern Asia, including Bhutan, China, northern India, Nepal and Sikkim; one of the two species is endemic to China.
Toricellia, described and validated by A.P. de Candolle in late 1830, was named after an Italian naturalist, physicist and mathematician Evangelista Torricelli. This peculiar Chinese-Himalayan genus was attributed to the family Cornaceae by H. Harms in 1898 [4] and W. Wangerin in 1910 [14] - they placed it in the tribe Toricellieae. However, W. Wangerin pointed out marked morphological differences existing between the only genus of this tribe and other 15 representatives of Cornaceae sensu Harms [4] (Cornus L., Nyssa L., Helwingia Willd., Aucuba Thunb., Garrya Douglas ex Lindl., Mastixia Blume, etc.). Based on these differences (multi-celled glandular trichomes, specific hanging axillary paniculate inflorescences, free stylodia, etc.), X.S. Hu was able to describe a new family Toricelliaceae in 1934. Later, the advisability of this placement was confirmed by wood and bark anatomy research by Adams [1] who noted relative primitiveness of Toricellia's characters treating the genus as a climax point of a long and independent evolutionary line. Out of genera of Cornaceae sensu Harms (1898), Griselinia Forst. and Helwingia were most often related to Toricellia, and some authors would remove Toricellia from Cornaceae circle of affinity and relate it to Apiales. A.L. Takhtajan [9, 10] was convinced
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that Toricellia is even more distant from Cornales and related orders, therefore placing the genus in the order of its own, Toricelliales, between Aralidiales and Araliales (Apiales).
The results of recent molecular research provided new insights into Toricellia's taxonomic position [2, 6]. Based on these data, currently the family Toricelliaceae is described to include three genera - Toricellia, Melanophylla Baker (formerly a member of Cornaceae sensu Harms and Melanophyllaceae within no longer existing order Hydrangeales) and Aralidium Miq. (previously representing a family and an order of its own, Aralidiaceae and Aralidiales, respectively).
Even though Harms's concept of Cornaceae of 1898 [4] is now considered outdated and more molecular-based classifications have been and are being proposed, the clear-cut circumscription of the this circle of related or unrelated genera is still to be completed. Therefore, for the purposes of convenience and methodology, hereinafter we are still going to use "Cornaceae sensu Harms", which is known to include 15 genera (Cornus, Aucuba, Alangium Lam., Camptotheca Decne, Corokia A. Cunn., Curtisia Aiton, Davidia Baill., Helwingia, Garrya, Griselinia, Kaliphora Hook. f., Mastixia, Melanophylla, Nyssa and Toricellia). We do acknowledge thought that this concept cannot be valid in the phylogenetic sense.
As we can see from the above cited references, Toricellia has been studied well in terms of its vegetative structure and molecular aspects, but very regretfully, fruit structure has been omitted in these studies. However, it is broadly accepted that fruit structure (especially fruit anatomy) is an important taxonomic marker because carpological traits are known to be very constant and not much variable under the influence of environmental factors. Our present work in fruit and seed structure of Toricellia and related genera is aimed at bridging this gap and searching for traits of high taxonomic value that would be able to contribute to the clarification of Toricellia's status and position within flowering plants.
Materials and methods
For morphological and anatomical research, mature fruits of Toricellia tiliifolia and T. angulata were used. The material was obtained from botanic gardens and carpological collections of herbaria (carpological collection of Komarov Botanical Institute, St. Petersburg, Russia (LE), Herbarium of Royal Botanic Gardens Edinburgh, UK (E).
Plant material was fixed in 70% ethyl alcohol and freehand transverse (cross) sections were made. Dry fruits were previously rehydrated in Strassbourger mixture (water, glycerol and 70% ethyl alcohol in equal proportions) at 50°C or boiled and cooled down repeatedly. Further treatment of sections was performed according to traditional anatomical techniques [5, 8] and included the following:
1. Phloroglucinol + HCl (concentrated) - test-reaction to reveal lignification;
2. I2 + KI - test-reaction for proteins and starch;
3. Sudan III (IV) - test- reaction for lipids (including cutin and suberin).
After test-reactions and staining, if needed, the sections were embedded in glycerolgelatin and studied with the help of light microscope (Primo Star by Carl Zeiss). Suitable sections were photographed and drawn.
Transverse fruit sections and surface ultrasculpture of Toricellia fruit was studied at the Inter-department laboratory of electron microscopy of the Faculty of Biology, MSU named in honour of M.V. Lomonosov, by means of scanning electron microscopes (SEM) Hitachi S-405A and JSM-6380 LA. The plant material was prepared for SEM investigations according to the techniques used in the Inter-department lab of MSU. SEM research was documented by microphotographs.
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Results
Fruit morphology
Toricellia fruits are often referred to as drupes (or 'drupaceous') but this term is imprecise as true drupes are only known to occur in the subfamily Prunoideae of the family Rosaceae where the fruit develops from a single-carpelled apocarpous gynoeceum [7]. For plants with syncarpous gynocea, just like the majority of Cornaceae sensu Harms, a more suitable term "pyrenarium" is used (for a detailed substantiation of the terminology applied to Cornaceae and related families as well as explanation of the crucial developmental aspects of such fruits see works by E. Yu. Yembaturova and co-authors [15, 17]).
The fruits of Toricellia are small inferior pyrenaria, 5-8 mm in length and 4-5 mm in diameter in dry condition; their shape is somewhat asymmetrical (flattened on one side and bulging on the other. Two to three protruding projections (unrelated to vascular bundles) are found on the bulging side. Dry fruits are black in colour; in natural conditions, a mature fruit is dark purple [12]. The fruit's single stone possesses 2 to 4 locules (triangle in cross section) with only one of them being fertile and bearing a single seed capable of reaching full maturity [12; original data].
Pericarp histology
At the first glance, the pericarp of Toricellia is very simple in structure (Figure 1B, 2B), but since the fruit develops from the inferior ovary, five histological zones can be distinguished in it:
1. Exocarp is represented by the epiderm (originating from the outer hypanthial epiderm) and subepidermal underlying tissue. The pigment determining the fruit colour is localized in exocarp cells.
2. Parenchymous mesocarp of tissues of hypanthial origin.
3. Parenchymous mesocarp of outer carpellary tissues with vascular bundles and their derivatives.
4. Stone, or pyrene, made of supporting tissue (sclereids).
5. Endocarp proper - the inner layer of fibers (different from the sclereids) derived from the inner carpellary epiderm. The endocarp is an integral part of the stone.
The border between the hypanthial and the carpellary zone of the fruit wall is quite pronounced: the former consists of larger cells whereas the latter is in general made of minute, often compress cells that partially obliterate in the mature fruit (T. tiliifolia). Vascular bundles derivatives form one or two circles. They tend to be located towards the inner part of the fleshy mesocarp of carpellary origin. The above mentioned projections appear to be formed out of all tissues present in the fleshy part of the pericarp. In total, the fleshy part of the pericarp (of exomesocarpic origin) is constituted by 6 to 10 cell layers.
The stone in most studied specimens is composed of10 to 12 layers of well-lignified sclerenchymous elements (of rounded or irregular shape in cross section) with moderately thickened walls (Figure 1D, 2D) while the endocarp proper is not very conspicuous but nevertheless present; fibers in its structure are readily distinguishable from other sclereids of the pyrene (Figure 1C, 2C); their walls are canaliculated. No secretory structures were found in mature fruits of T. tiliifolia The pericarp histology of both studied species is found to be very similar.
As far as the fruit surface ultrasculpture (Figure 1A, 2A) is concerned, the primary sculpture is flattened, showing rhombic or trapeziform outlines of epidermal cells. The
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Fig. 1. Fruit structure of T. tiliifolia (SEM photographs): A - fruit surface, x150; B - fruit cross section, x25; C - pyrene structure: endocarp fibers and mesocarp sclereids, x70; D - sclereid-like lignified elements of mesocarpic origin
cuticle is slightly plicate. A few stomata or sparse thin simple trichomes can be observed. Very few globoid or differently shaped granules of epicuticular wax are seen on the fruit surface (Figure 1A, 2A).
Seed structure
The seed structure in Toricellia has been described previously [12]. The seed develops from a single ovule hanging down from the top of the fertile locule. Its short funiculus possesses an outgrowth. At maturity, the seed can easily be removed from the locule. According to our observations, in certain parts of the fertile locule wall, the stone is not fully continuous and apparently, the seed simply falls out of the stone when full maturity is reached and the outer fleshy tissues are destroyed. This peculiar trait has not been observed in any representatives of Cornales or Apiales studied to date.
The seed coat is rather thin and filmy however, it is much more pronounced than in representatives of Cornaceae and related families with thick bony endocarps (Cornus, Alangium, Nyssa, etc.) due to the correlation between the endocarp and seed coat thickness characteristic to fleshy drupaceous fruits [13].
Discussion
As shown by our investigation, fruit structure of Toricellia is very peculiar and differs greatly from that of most other Apiales or Cornales genera studied previously. The most striking features are: quite thin fleshy part of the pericarp (exocarp and part of mesocarp)
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Fig. 2. Fruit structure of T. angulata (SEM photographs): A - fruit surface, x150; B - fruit cross section through the middle part, x25; C - pyrene structure: endocarp fibers and mesocarp sclereids, x70; D - lignified elements of mesocarpic origin
and a four-loculed pyrene of relatively thin-walled and moderately lignified (in comparison to most other Cornaceae sensu Harms) sclereids. In the pyrene, only one locule is fertile and seed-bearing, but the other two or three sterile locules retain their shape and do not compress. Moreover, it was interesting to observe almost discontinuous structure of the stone wall which could allow the seed to literarily fall out of it.
Pyrene structure. The majority of Cornaceae sensu Harms (except Curtisia, Kaliphora and Helwingia [17]) are known to possess pseudomonomerous pyrenaria (with only one of two or more locules and seeds reaching full development). In many genera, (Cornus and its segregates, Nyssa, Corokia, Davidia) all locules besides the fertile one are markedly compressed. However, there are some taxa in this circle of affinity where sterile locules retain their shape and size. One of them isMelanophylla, in which the seed-bearing locule is the smallest of three, two other ones being large and somewhat inflated [11]. Just like in Toricellia, the seed of Melanophylla copies the shape of the locule. It is known to have copious endosperm and a tiny embryo, differently from Toricellia, where the embryo is much bigger in size. Another feature shared by the two discussed genera is the relatively small thickness of the pyrene wall.
Discontinuous pyrene. The opening (or discontinuity) in the wall of the fertile locule, noted in Toricellia, is seemingly a peculiar trait not found in any studied representatives, related to Toricellia now or before. One must note though that openings, more or less rounded in shape, have been reported by R. Eyde [3] in the walls of sterile locules. According to this botanist, the germination in Toricellia occurs by means of a triangle valve. Almost discontinuous pyrene (however, much bigger in thickness) was observed in
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Kaliphora [16], which was closely related to Melanophylla within Hydrangeales [11] in the past. Similar traits (locules discontinuous from outside) have been revealed in a saniculoid genus Petagnaea Caruel [A.I. Konstantinova, unpublished data].
Fleshy part of the pericarp. Even though in Toricellia's supposed relatives by Cornales (Cornus, Alangium, Nyssa, Curtisia, Mastixia, Corokia, etc.) the fleshy zone of the fruit wall is always very well-developed, there are some representatives in which the reduction of this part has been noted. More distinctively this trend of exomesocarp reduction, together with more pronounced histological zonality and distinct specialization of the zones, is observed in more specialized fruits of Apiales.
The absence of secretory structures in the mature pericarp points out Toricellia's difference from Apiales, where they are present in abundance. However, they have not been found in mature fruits of Helwingia, Aralidium, Kaliphora and Griselinia.
All carpological traits taken together suggest comparing the studied genus with yet another representative of Cornaceae sensu Harms - Griselinia. These plants have many features in common, e.g., they both tend towards pyrene reduction in thickness and the degree of cell wall lignification of sclereid-like elements (Figure 1D). Both of them have rather uniform structure of the fleshy exomesocarp, where no secretory canals are found at maturity and derivatives of vascular bundles can be seen and help to identify the borderline between the carpellary and hypanthial zones of the fruit wall. However, differences are present, too - most importantly, in Toricellia all three or four locules are well-developed in the stone whereas in Griselinia only one of them is pronounced and obviously dominating. G. scandens Taub. is known to possess sclereids in the fleshy part of the pericarp - this feature is common for many Cornales but not found in Toricellia. The seed of Griselinia is bigger in size and the embryo develops to a much more advanced stage. The seed coat structure is similar in both of the discussed genera, but it seems thicker and less compressed in Griselinia, suggesting a parenchotestal type of the seed in the genus.
Apart from carpological traits, there is a biochemical feature shared by Griselinia and Toricellia. Both of them have been reported to accumulate an iridoid compound
Comparative analysis of fruit structure traits in Toricellia and possibly related genera
Traits Toricellia Griselinia Aralidium Melanophylla* Kaliphora Schefflera
Number of locules 3-4 1 1 2-3 2 From 2 to many
Number of fully developed seeds per fruit 1 1 1 1 2 From 2 to many, seeds may mature only in some of the locules
Borderline between hypanthial and carpellary zone Present; more pronounced in T.angulata Poorly defined, the most pronounced in G. jodinifolia Present, well seen Present, well seen
Secretory system Absent Absent Absent Absent Usually well-developed
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Table - ending
Traits Toricellia Griselinia Aralidium Melanophylla* Kaliphora Schefflera
Discontinuity of the stone Present, in the distal part of the fruit (outside) None None Present, in the proximal part of the fruit (inside) Present, in the proximal part of the fruit (inside) In some species (rarely) in the proximal part (inside), in the distal part or both
Presence of endocarp (as carpel's inner epiderm) Present as a single layer of fibers lining the pyrene made of sclereids Absent, obliterated or indistinct in the mass of pyrene-composing sclereids (G. lucida) Present as a layer of obliterated parenchyma cells Absent, obliterated or indistinct in the mass of pyrene-composing fibers Absent, obliterated or indistinct in the mass of pyrene-com-posing fibers (from several layers to one)
Seed coat Thin, non-differentiated Thin, non-differentiated More or less differentiated into exo-, meso- and endotesta Thin, non-differentiated Thin, non-differentiated
Presence of griselinoside Present Present Present
* Data on fruit structure of Melanophylla are taken from V.I. Trifonova's paper [11].
griselinoside [3, 9]. This substance has also been found in Aralidium [9], which is united with Toricellia within one family - Toricelliaceae according to one of the most recent treatments of Apiales based on molecular research [2, 6].
Data on fruit structure in some of the above discussed genera, currently placed in or related to Apiales are given in the Table. Schefflera J.R. Forst. & G. Forst. is shown here as a typical and the most numerous representative of the order Apiales.
Conclusions
The genus Toricellia has demonstrated a unique combination of features that does not give an immediate suggestion of its close relationship to any of the genera ever considered as Toricellia's possible relatives. The genera of its family, Aralidium and Melanophylla, do share some traits with Toricellia however, still remaining rather different.
Our carpological investigation has revealed some interesting features, one of the being the locules discontinuous from outside. A similar trait has been found in Grise-linia, which has also been transferred from Cornales to Apiales, and a saniculoid genus Petagnaea.
The absence of secretory structures in mature fruits of Toricellia links it to Aralidium and Griselinia. However, this carpological evidence is not in favour of Toricellia's affin-
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ity with Apiales as representatives of this order are known to have a variety of secretory structures in their fruits.
An interesting peculiarity of Toricellia is the accumulation of griselinoside. This iridoid compound is also found in Griselinia and Toricellia's suggested family-mate Ara-lidium. This peculiar trait, as well as the other above listed characters, definitely requires further investigations.
Acknowledgements
The present research was supported by the grant № 12-04-01298 of Russian Foundation for Basic Research (RFBR).
The authors are much indebted to Dr. Alexander P. Suchorukov (Moscow State University) for his assistance in obtaining plant material for research.
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1. Adams J.E. Studies in the comparative anatomy of the Cornaceae // J. Elisha Mitchell Sci. Soc. 1949. Vol. 65. P. 218-244.
2. Chandler G.T., Plunkett, G.M. Evolution in Apiales: nuclear and chloroplast markers together in (almost) perfect harmony // Bot. Journ. Linn. Soc. 2004. Vol. 144. P. 123-147.
3. Eyde R.H. Comprehending Cornus: puzzle and progress in the systematics of Dogwoods // Bot. Rev. 1988. Vol. 54. № 3. P. 251-300.
4. Harms H. Cornaceae // A. Engler, K. Prantl Die Naturlichen Pflanzenfamilien. 1898. T1. 3. Abt. 8. P. 250-270.
5. O'Brien T.P., McCullyM.E. The study of plant structure: principles and selected methods. Melbourne: Termarcarphi and Pty. Ltd., 1981. 352 p.
6. Plunkett G.M., Chandler G.T., Lowry II P.P., Pinney S.M., Sprenkle T.S. Recent advances in understanding Apiales and a revised classification. // S. Afr. Journ. Bot. 2004. Vol. 70. P. 371-381.
7. Shibakina G.V. Kostyanka kak ekologicheskyi tip ploda i nekotorye voprosy terminologii pri opisanii plodov v semeistve Araliaceae (A drupe as an ecological fruit type and some terminological matters in the description of fruits in the family Araliaceae) // Botanycheskii Zhurnal (Bot. Journ.). 1984. Vol. 69. P. 1076-1083.
8. Sozonova L.I., Trusov N.A. Plant cells and tissues, light microscopy. Moscow: Izdatelstvo RUDN (Publishing House of the University of Peoples' Friendship), 2007. 64 p.
9. Takhtajan A.L. Sistema magnoliofitov [The system ofMagnoliophyta]. Leningrad: Nauka, 1987. 439 p.
10. Takhtajan A.L. Diversity and classification of flowering plants. New York: Columbia Univ. Press, 1997. 643 p.
11. Trifonova VI. The family Melanophyllaceae // Sravnitelnaya anatomiya semyan (Comparative seed anatomy). Vol. 6. Saint Petersburg: Nauka (Science), 2000. P. 262-267.
12. Trifonova V.I. The family Toricelliaceae // Sravnitelnaya anatomiya semyan (Comparative seed anatomy). Vol. 6. Saint Petersburg: Nauka (Science), 2000. P. 317-318.
13. Vasilevskaya V.K., Melikian A.P. On the origin and main trends in the evolution of angiosperm fruits and seeds // Vestnik LSU (Messenger of Leningrad State Univ.). 1982. № 9. P. 23-30.
14. Wangerin W. Cornaceae // A. Engler Das Pflanzenreich. Leipzig, 1910. Hf. 41. Ser. 4. P. 43-92.
15. Yembaturova E.Yu. Sravnitelnaya carpologiya predstavitelei Cornales Dumort. i sblizhae-mykh s nim poryadkov v svyazi s voprosami ikh sistematiki (The comparative carpology of the representatives of Cornales Dumort. and related orders in connection with their systematics). Ph.D. thesis. Moscow, 2001. 445 p.
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16. Yembaturova E. Yu., Konstantinova A.I. Ispol'zovanie dannykh sravnitel'noi karpologii dlya utochneniya polozheniya madagaskarskogo roda Kaliphora (Kaliphoraceae) v sisteme pokry-tosemennykh (Using carpological evidence to clarify the position of a Madagascan genus Kaliphora (Kaliphoraceae) in the system of angiosperms // Carpology and reproductive biology of higher plants. Proceedings of Russian national scientific conference with international participants dedicated to the memory of Prof. A.P. Melikian (October, 18th - 19th, 2011, Moscow). Moscow: OJSC "Astra-Polyraphia", 2011. P. 105-108.
17. Yembaturova E.Yu., Van Wyk B-E., Tilney P.M. A review of the genus Curtisia (Curti-siaceae) // Bothalia. 2009. Vol. 39. № 1. P. 87-96.
ОСОБЕННОСТИ СТРОЕНИЯ ПЛОДОВ ПРЕДСТАВИТЕЛЕЙ РОДА TORICELLIA DC. (TORICELLIACEAE) И ВЗГЛЯДЫ НА ПОЛОЖЕНИЕ ТАКСОНА В СИСТЕМЕ ПОРЯДКА APIALES
Е.Ю. Ембатурова1, А.И. Константинова2
(1-РГАУ-МСХА имени К.А. Тимирязева, 2-МГУ имени М.В. Ломоносова)
Аннотация: представители рода Toricellia DC.- T. tiliifolia DC. и T. angulata Oliv. -двудомные деревья и кустарники с очередными листьями без прилистников и отличающимися по морфологии мужскими и женскими цветками. Плоды этих растений (пиренарии) неравнобокие, яйцевидные или почти шаровидные, на верхушке с неопадающей чашечкой. Отличительная особенность Toricellia - развитие из нижней завязи плода с 2-4 гнездами, только одно из которых несет семя (псевдомономерный плод). Характерно наличие округлых отверстий в наружных стенках стерильных гнезд. В косточке («пирене») различаются две гистологические зоны: наружная, часто очень мощная, состоящая из склереид, и слабо выраженная внутренняя зона из одного слоя волокон. Таким образом, у видов Toricellia собственно эндокарпий составляет лишь внутреннюю часть пирены, тогда как наружная образована элементами мезокарпия, так что происхождение пирены мезоэндокарпическое. Единственное семя повторяет форму фертильного гнезда - продолговатое и в разной степени согнутое. Спермодерма малослойная. В статье обсуждаются признаки, отмеченные у рода Toricellia и сближаемых с ним родов (Aralidium Miq., Griselinia Forst., Melanophylla Baker), а также взгляды на систематическое положение изучаемого таксона.
Ключевые слова: Toricellia, Toricelliaceae, анатомия плода, пиренарии, Apiales, Cornales, систематическое положение.
Ембатурова Елена Юрьевна — к. б. н., доцент кафедры ботаники РГАУ-МСХА имени К.А. Тимирязева (127550, г. Москва, ул. Тимирязевская, д. 49; тел.: (499)976-16-18; e-mail: drleena@inbox.ru).
Константинова Александра Игоревна — к. б. н., ассистент кафедры высших растений биологического факультета МГУ имени М.В. Ломоносова (119991, г. Москва, ГСП-1, Ленинские горы, МГУ, д. 1 стр. 12, биологический факультет; тел.: (495)939-16-03; e-mail: alex.i.konstantinova@gmail.com).
Dr. Elena Yurievna Yembaturova — Candidate of biological Sciences, Associate Professor of the Dept. of Botany, RSAU-MTAA (ul. Timiryazevskaya, 49, Moscow 127550, Russian Federation; phone: +7(499)976-16-18; e-mail: drleena@inbox.ru).
Dr. Alexandra Igorevna Konstantinova — Candidate of biological Sciences, Assistant Professor of the Dept. of Higher Plants, MSU named in honour of M.V. Lomonosov (Faculty of Biology, MSU, Leninskiye Gory 1 str. 12, Moscow 119991, Russian Federation; phone: + 7(495)939-1603; e-mail: alex.i.konstantinova@gmail.com).
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