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Orchid seed diversity Orchid seed diversity A scanning electron microscopy survey A scanning electron microscopy survey

Wilhelm Barthlott Bernadette Große-Veldmann Nadja Korotkova

The orchid family (), with some 22 000 , is one of the two largest families of . In spite of the vast availa- ble literature on orchids, rather little is known about their seeds, which are generally considered as wind dispersed, small and re- duced “dust seeds”. Wilhelm Barthlott For this fi rst monograph of orchid seeds, the authors have ana- Bernadette Große-Veldmann lysed some 1400 collections of orchid seeds over the last four dec- Nadja Korotkova ades by scanning electron microscopy (SEM) and other methods. The material studied represents about 1100 species from 350 gen- era (out of about 880 recognized orchid genera).

This monograph provides a unique and indispensable resource – 135 for all those studying orchid systematics, those seeking to identify A B orchid seeds, as well as those with more general interests in the Orchidaceae.

What is included? C D • 620 scanning electron micrographs of orchid seeds • detailed seed descriptions for 350 genera, as well as for 14 tribes and the fi ve orchid subfamilies E F • an introduction summarizing earlier studies on orchid seeds and current knowledge of orchid phylogeny

G H • a consistent terminology for characters of the orchid seed coat • the defi nition of 17 orchid seed types

Fig. 55. Epidendroideae – Cymbidieae – , Cyrtopodiinae – A, B: paludosum, length: 800 µm – C, D: lurida, length: 300 µm – E, F: Porphyroglottis maxwelliae, length: 500 µm – G, H: • a discussion on the systematic and diagnostic value and evo- parviflorum, length: 700 µm. lution of orchid seed characters • seed characters and seed types visualized on 26 phylogenet- ic trees

Shape of testa cells elongated, rectangular elongated, rounded at the end • references Pachyphyllum Cymbidieae Cyrtopodium • an appendix listing the sources of the orchid seed material Graphorkis studied Dipodium Calypso Calypsoeae • an index to genera and higher taxa Barthlott W., Große-Veldmann B. & Korotkova N. 2014: Orchid Agrostophyllinae Cadetia Dendrobiinae Epigeneium seed diversity: A scanning electron microscopy survey. – Berlin: Epidendroideae Cymbidieae Botanic Garden and Botanical Museum Berlin-Dahlem. – Englera 32. – ISBN 978-3-921800-92-8. – Softcover, 17.6 × 25 cm (B5), Agrostophyllinae 245 pages, 620 micrographs, 26 phylogenetic trees and 7 other Collabieae fi gures. – Price: EUR 25. Tropidieae Sobralia For information on ordering, please visit: www.bgbm.org/englera Monophyllorchis Triphoreae Calypsoeae Xerorchis Xerorchideae Orchid seed diversity A scanning electron microscopy survey

Wilhelm Barthlott1 Bernadette Große-Veldmann1 Nadja Korotkova1,2

1 Nees-Institut für Biodiversität der Pflanzen, Rheinische Friedrich-Wilhelms-Universität Bonn

2 Institut für Biologie/Botanik, Systematische Botanik und Pflanzengeographie, Freie Universität Berlin und Botanischer Garten und Botanisches Museum Berlin-Dahlem

Published by the

Botanic Garden and Botanical Museum Berlin-Dahlem as Englera 32

Serial publication of the Botanic Garden and Botanical Museum Berlin-Dahlem

December 2014 Englera is a monographic series, published at irregular intervals by the Botanic Garden and Botanical Museum Berlin-Dahlem (BGBM), Freie Universität Berlin. In scope it is an international series, pub- lishing peer-reviewed original material from the entire fields of , algal and fungal and systematics, also covering related fields such as floristics, plant geography and history of , provided that it is monographic in approach and of considerable volume.

Editor: Nicholas J. Turland Production Editor: Michael Rodewald Printing and bookbinding: LASERLINE Digitales Druckzentrum Bucec & Co. Berlin KG

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Submission of manuscripts: Authors should contact by e-mail Nicholas J. Turland, Editor of Englera, Botanischer Garten und Botanisches Museum Berlin-Dahlem, Freie Universität Berlin, Königin-Luise-Str. 6 – 8, 14195 Berlin, Germany; e-mail: [email protected]

Subscription and exchange: BGBM Press, Att. of Susanne Schmutzler, Botanischer Garten und Botanisches Museum Berlin-Dahlem, Freie Universität Berlin, Köni­­gin-Luise-Str. 6 – 8, 14195 Berlin, Germany; e-mail: [email protected]

© Botanischer Garten und Botanisches Museum Berlin-Dahlem, 2014 All rights (including translations into other languages) reserved. No part of this issue may be repro- duced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mecha- nical, photocopying, recording or otherwise, without the prior written permission of the publishers.

ISSN 0170-4818 ISBN 978-3-921800-92-8

Addresses of the authors: Prof. Dr. Wilhelm Barthlott, Nees-Institut für Biodiversität der Pflanzen, Rheinische Friedrich-Wil- helms-Universität Bonn, Venusbergweg 22, 53115 Bonn, Germany; e-mail: [email protected] Bernadette Große-Veldmann, Nees-Institut für Biodiversität der Pflanzen, Rheinische Friedrich- Wilhelms-Universität Bonn, Meckenheimer Allee 170, 53115 Bonn, Germany; e-mail: b.grosse- [email protected] Dr. Nadja Korotkova, Institut für Biologie/Botanik, Systematische Botanik und Pflanzengeographie, Freie Universität Berlin & Botanischer Garten und Botanisches Museum Berlin-Dahlem, Köni­­gin- Luise-Str. 6 – 8, 14195 Berlin, Germany; e-mail: [email protected]

Cover design: From Beer J. G. 1863: Beiträge zur Morphologie und Biologie der Familie der Orchideen (Tab. III & IV). – Wien: Druck und Verlag von Carl Gerold’s Sohn. Contents 5

Contents

Summary and key words ...... 8

Preface and acknowledgements 9

Introduction Current knowledge of orchid phylogeny ...... 12 Seeds of orchids 13 Earlier seed morphology studies in Orchidaceae 14 Aims of this study 18

Descriptors and terminology Seed characters Seed shape ...... 19 Seed size (length of seed) 20 Seed colour ...... 20 Number of testa cells (along longitudinal axis of seed) ...... 20 Shape of individual testa cells ...... 20 Testa cell pattern ...... 20 Anticlinal wall curvature ...... 21 Transverse anticlinal walls ...... 21 Intercellular gaps ...... 22 Surface structure of periclinal walls ...... 23 Cuticular layer ...... 23 Modifications of testa cell corners ...... 23 Seed types 24

Material and methods Seed material ...... 27 Taxonomic coverage ...... 27 Scanning electron microscopy (SEM) ...... 27 Orchidaceae systematics ...... 27 Character coding ...... 27 Tracing of characters ...... 28

Seed descriptions Apostasioideae ...... 29 29 ...... 29 30 Cypripedioideae ...... 32 ...... 32 Chloraeeae 33 Codonorchideae ...... 33 33 Cranichidinae ...... 34 Galeottiellinae ...... 34 6 Contents

Goodyerinae ...... 34 Manniellinae ...... 36 Pterostylidinae ...... 36 Spiranthinae ...... 36 Diseae ...... 38 Brownleeinae 38 Coryciinae ...... 39 Disinae ...... 39 Huttonaeinae 40 Satyriinae 40 Diurideae ...... 40 Acianthinae 40 Caladeniinae ...... 41 Cryptostylidinae ...... 41 Diuridinae ...... 41 Drakaeinae 42 Megastylidinae ...... 42 Prasophyllinae ...... 43 Rhizanthellinae 43 Thelymitrinae 43 Orchideae ...... 43 Epidendroideae ...... 48 Arethuseae 49 Arethusinae 49 49 Calypsoeae 51 Collabieae ...... 52 Cymbidieae ...... 53 53 Coeliopsidinae ...... 54 Cymbidiinae ...... 54 Cyrtopodiinae ...... 55 Eriopsidinae ...... 55 Eulophiinae 55 56 ...... 57 62 Vargasiellinae ...... 63 ...... 63 Epidendreae ...... 65 ...... 66 Chysinae ...... 66 Coeliinae ...... 66 ...... 66 70 71 Gastrodieae ...... 72 Malaxideae ...... 72 Neottieae 73 Nervilieae ...... 74 Contents 7

Epipogiinae 74 Nerviliinae ...... 74 Podochileae ...... 75 ...... 75 Thelasinae ...... 76 Sobralieae ...... 76 Triphoreae ...... 77 Diceratostelinae ...... 77 77 Tropidieae ...... 77 Xerorchideae 77 Vandeae ...... 77 78 ...... 80 ...... 81 ...... 82 Epidendroideae unplaced genera ...... 82

Discussion The systematic and diagnostic value and evolution of orchid seed characters ...... 87 Are orchid seeds systematically relevant? – Earlier evidence from comparative studies . . . 91 Are orchid seeds systematically relevant? – Earlier evidence from cladistic studies 92 Summary of the character reconstruction 93 Concluding remarks 94

SEM micrographs ...... 95

Selected seed characters visualized on phylogenetic trees Seed shape ...... 186 Seed size ...... 188 Seed colour ...... 190 Number of testa cells ...... 192 Shape of testa cells ...... 194 Testa cell pattern ...... 196 Anticlinal wall curvature ...... 198 Transverse anticlinal walls ...... 199 Intercellular gaps ...... 200 Ridges on periclinal walls 202 Verrucosities on periclinal walls ...... 204 Perforations on periclinal walls 206 Cuticular layer ...... 207 Wax caps ...... 209 Testa cell extensions ...... 210 Seed types 211

References ...... 213

Appendix: Sources of material ...... 217

Index ...... 239 8 Summary

Summary The Orchidaceae with some 22 000 species is ported molecular phylogenetic trees and were one of the two largest plant families. Despite found to be largely consistent with the major of the vast literature on orchids, rather little is clades. Combinations of characters classified known about their seeds, which are generally into 17 seed types often delimit tribes (e.g. Cym­ considered as wind dispersed, small and reduced bidieae, Epidendreae, Vandeae). Highly special- “dust seeds”. ized features like polyembryony (up to 12 em- Based on some 1400 collections of orchid bryos per seed in ) or highly adaptive seeds analysed by SEM and other methods over (with respect to dispersal biology) seed-coat the last four decades, about 7000 micrographs features (e.g. in , or the sophisticated of some 1100 species from 352 (out of c. 880) seed-attachment mechanism of ) are genera were evaluated for this first monograph restricted to only a few genera. on orchid seeds. This monograph provides a first atlas (624 mi- Orchid seeds exhibit an astonishing diversity. crographs) and data to identify the major groups This is not only reflected in their sizes (between of orchid seeds and a terminology for taxonomic 0.1 mm in and 6 mm in Epidendrum) purposes. The first character reconstruction of and shapes, but especially in the complexity of seed characters based on modern molecular phy- their lightweight seed-coat architecture and hi- logenetic hypotheses allows an application for erarchical surface sculpturing. A consistent ter- further systematic studies of the family. minology for characters of the orchid seed coat is proposed. Key words – Orchidaceae, orchids, seeds, testa, Taxa at subtribal to tribal levels are often well surface, micromorphology, SEM, taxonomy, characterized by seed-coat characters. Fifteen phylogeny, dispersal biology selected characters were mapped on well-sup- Preface and acknowledgements 9

Preface and acknowledgements

Orchids, one of the two largest families of flow- editor of the new edition of ’s ering plants, have fascinated botanists and plant “Die Orchideen”, the most comprehensive or- enthusiasts over centuries. The literature fills chid monograph at that time. shelves in libraries and is almost unmanageable. I collected the first orchid seeds in the green- There are countless pictures of the fascinating houses in 1972 and was fascinated by the un- splendour of orchid . Only few people expected diversity that opened up under the have studied the morphology of the seeds of the electron microscope. With the curator Karlheinz about 22 000 orchid species: the usual notion in Senghas and my two postdoctoral friends we textbooks says orchid seeds are tiny, reduced and published the very first SEM study of orchid uniform – not very inspiring for a botanist. seeds in 1974. Then I discovered the small mon- The monograph presented here has a very long ograph on orchid seeds by Joseph Georg Beer history – more than 40 years after our research be- (1863) in our library. Though hardly taken notice gan a comprehensive description of the surprising of, it is a unique pioneering work in this field. diversity of orchid seed coats is finally presented. Josef Georg Beer (1803 – 1873) was a famous Therefore, a brief review of this history as well as Viennese fashion designer and worked until the persons involved is worthy of mention. 1843 in his father’s company, and then devoted In 1971 one of the first scanning electron mi- himself to private extensive bromeliad and or- croscopes (a Cambridge Stereoscan 500) was chid collection. He was Secretary General of the established from DFG (Deutsche Forschungsge- Austrian Imperial and Royal Horticultural Soci- meinschaft) funds at the Institute of Systematic ety and among other things organized the horti- Botany and Plant Geography at the University cultural part of the world exhibition in Vienna in of Heidelberg. I myself was able to work with 1867 on behalf of the Austrian government. His the SEM for my dissertation on the taxonomy work on orchid seeds published in 1863 includ- of epiphytic Cactaceae that also relied on char- ed remarkable astonishing coloured lithographic acters I could observe in the SEM. For us Hei- plates, self-designed by Beer, giving an impres- delberg postdocs (Nesta Ehler, Rainer Schill sion of the variety of shapes, colours, as well as and myself), these were the pioneering days of transparency and lightness of orchid seeds that scanning electron microscopy. Already in 1977, a SEM image cannot convey. One of his unsur- we could present a first monograph on the SEM passed plates is reproduced here (Fig. 7); it and analysis of the surfaces of 2200 plant species, one other from the same work have been used also discussing functional aspects, the most im- for the cover design. portant of which later became widely known as The investigations were intensified after 1974. the lotus effect. Karlheinz Senghas pollinated the orchids in the Heidelberg was also an international centre of living collection systematically for seed pro- orchid research in the seventies. The Institute of duction and identified the material. The Swiss Systematic Botany and Plant Geography and the pharmacist Othmar J. Wildhaber in Zurich had Botanical Garden were linked through Werner collected seeds of almost all European orchids Rauh who was the director of both these institu- over decades and introduced us to his material. tions. He built up an extraordinary collection of Gunnar Seidenfaden provided material from plants on his many expeditions to tropical coun- . Friedhelm Butzin regularly sent sam- tries. Besides the succulent and bromeliad col- ples from the rich stock of the Botanic Garden lection, the orchids alone occupied three large and Botanical Museum Berlin-Dahlem, and Pe- greenhouses. This was perhaps the world’s big- ter Taylor sent material from the of gest living collection of orchids at that time. This the Royal Botanic Gardens, Kew. Berlin and collection was curated by Karlheinz Senghas, an Kew have contributed a significant portion of the internationally renowned orchid taxonomist and material examined. 10 Preface and acknowledgements

In Robert L. Dressler (Florida and Panama), ceived new material from the growing Heidel- we found an enthusiastic scientist to support the berg collection, and additionally from collectors work in various ways, and he visited us several and herbaria around the world. In addition, the times in Heidelberg. One had the impression that general work on scanning electron microscopy everyone was happy that someone finally dealt of cuticular plant surfaces was continued – to- with the so long-neglected seeds of orchids. I day around 150 000 micrographs of surfaces of was invited to give a talk at the 8th World Orchid about 20 000 species have accumulated in our Conference in Frankfurt in 1976. archives. The old Cambridge Stereoscan micrographs To illustrate this alone for the Orchidaceae: from that time are still unsurpassed in quality: this work is based on the analysis of about 1100 the images of or Satyrium odo­ species from 350 genera. For this purpose, a total rum and many others presented here date from of about 1400 collections was examined in the the year 1973. Structural analysis followed in SEM; on average five micrographs were taken which we could clarify the nature of the stabiliz- per collection. In particularly complex cases – ers of the balloon-shaped seed coats as helical such as in Chiloschista lunifera – up to 100 pic- wall thickening by using an oxygen ion etching tures were taken per taxon. A total of about 7000 method. Shortly after, a publication on the func- micrographs were evaluated for this monograph. tional morphology of dust-seeds followed. It was clear that a publication of such data Our collection of orchid seeds had now grown nowadays would require a phylogenetic con- to over 500 taxa, and the SEM micrographs – in text – that was also available because the orchids pre-digital times on photo paper – filled many are very well studied with molecular system- file folders. I was still a postdoc, with the aim to atic approaches and their relationships are well qualify as professor. And I realized that I could understood. The processing of the data started not handle the seed morphology of the huge from about 2009 by Nadja Korotkova, who was orchid family alone. And a lucky circumstance familiar with the methods for character recon- helped: a study friend, Bernhard Ziegler, today struction in a phylogenetic context from her dis- a retired biology teacher at the English Insti- sertation on the molecular systematics of Cac­ tute in Heidelberg, mentioned that he would taceae – Rhipsalideae. Soon after, Nadja and I like to graduate in addition to his professional found a highly motivated and dedicated student: activity. He started his dissertation in 1978 and Bernadette Große-Veldmann. She became in- could complete it already in 1981 thanks to the terested in the orchid seeds and we decided she extensive preliminary work and already avail- could continue the character reconstruction as able data. A particularly spectacular finding we her B.Sc. thesis, which we supervised. Berna- soon published was the extendable helical wall dette’s commitment for this project was incred- thickenings of Chiloschista (Fig. 5). A smaller ible and the fast progress is undoubtedly owed overview article followed and we had planned to her. the big orchid seed monograph for 1982. But Finally, I would like to convey my appreciation we never finished the manuscript – Bernhard’s to all of the many people who have contributed time was fully engaged by his profession as a toward the success of this project. In addition to teacher, and I myself was appointed professor at all the colleagues mentioned before, I would like the Freie Universität Berlin in 1981. to thank Friedrich G. Brieger (São Paulo), Guido The works were continued from 1985 at the J. F. Pabst (Rio de Janeiro), Clarence K. Horich University of Bonn where I was now director (Costa Rica), Mark Clements () and at the Botanical Institute and of the Botanical Friedhelm Butzin (curator of orchids in the Her- Gardens and had ideal working conditions. barium Berlin-Dahlem). But this list is far from High-resolution electron microscopy was avail- being complete – a glance at the list of the mate- able on-site and we also carried out an energy rial sources makes clear how many herbaria and dispersive X-ray microanalysis (EDX) of seeds. collectors have contributed. Our sincere thanks Through Karlheinz Senghas, I continuously re- are given to them all. We want to thank all the Preface and acknowledgements 11 many technical assistants and student assistants who assisted in scanning electron microscopy and image editing over the years. Robert L. Dressler and Peter K. Endress re- viewed an earlier draft of this monograph and provided useful comments. Bernd Haeseling and Alexandra Runge prepared the photographic im- ages and plates with great technical skill. We fi- nally acknowledge the financial support from the Academy of Sciences and Literature in Mainz in the framework of the long-term project “Biodi- versity in Change”. The basis of this monograph is the work in Heidelberg. The commitment of Werner Rauh (1913 – 2000) and Karlheinz Senghas (1928 – 2004) has made this work possible. This monograph is dedicated to them.

Bonn, June 2014 Wilhelm Barthlott 12 Introduction

Introduction

Current knowledge of orchid phylogeny vanilloids have been regarded as separate fami- lies besides Orchidaceae s.str. (see Pridgeon Orchidaceae is one of the two largest angiosperm & al. 2003b for an overview of former classi- families. At present some 22 000 species and 880 fication systems). These problems and uncer- genera are accepted. More than 23 000 species tainties in orchid classification result from the and 1600 genera are known in the Asteraceae, Orchidaceae being an extremely diverse and making it the largest family. species-rich family. Genera and other taxonomic Still, even larger species numbers have been esti- units were often established mainly based on mated for both families (information taken from floral characters, construction of the and the Angiosperm Phylogeny Website (Stevens pollinia morphology. These characters however 2001). Some authors regard Orchidaceae as be- are prone to homoplasy and especially the simi- ing the largest plant family and it is undoubtedly larities in morphology are often a result the largest family of . of co-evolution with pollinators. The Orchidaceae were always regarded as a natural group but were considered so unique that New insights into the evolution of flowering they used to be treated as their own order, Or­ plants have been gained in the last 20 years since chidales, in former classification systems, e.g. the application of DNA sequence data became by Takhtajan (1997) Nowadays they are with widely used in phylogenetic studies. Knowledge high confidence included into the monocot order of relationships within the Orchidaceae has been (APG 2003, 2009). The taxonomic much expanded by a large amount of molecular rank of certain groups within Orchidaceae has data (e.g. Cameron & al. 1999; Freudenstein & been discussed controversially in the past and Rasmussen 1999; Cameron 2004; Freudenstein groups such as cypripedioids, apostasioids, and & al. 2004; Cameron & Molina 2006). All these studies have found five well-supported major lineages within Orchidaceae and a new formal classification based on these clades was pro- posed by Chase & al. (2003); see also Chase (2005). To date, Orchidaceae are subdivided into the five subfamilies Apostasioideae, Vanilloide­ ae, Cypripedioideae, Orchidoideae and Epiden­ droideae. The positions and circumscriptions of many tribes and subtribes have also been clarified by molecular data. An overview of phylogenetic studies within Orchidaceae is given by Cameron (2007). In contrast to former classification sys- tems, there is now a large amount of data that provides a robust phylogenetic hypothesis mak- ing a consensus on the classification and phyl- ogeny of Orchidaceae conceivable. This knowl- edge on Orchidaceae phylogeny can now serve Fig. 1. – A: Green ripe fruit of aromat­ as a framework to study the evolution of mor- ica (Epidendroideae). The mature dust-seeds can be seen as whitish powder along the slit-like openings. – phological features or ecological adaptations. B: dry fruit of sp. (Epidendroideae). Morphological characters that could back up the findings of molecular studies are still desirable. Introduction 13

In contrast to various aspects of flower morphol- dendroideae–Neottieae), a species often found ogy, micromorphological characters such as seed growing next to streams. Hydrochory as the main coats or are usually barely influenced by dispersal mechanism has also been suggested environmental conditions. for some species of (Orchidoideae–Dis­ eae). The seeds of some Disa are very different compared to its related genera. They are unusu- Seeds of orchids ally large and also contain endosperm, which is unusual in orchids. This strikingly different Orchids produce capsules (Fig. 1) and typi- morphology has also been noted by Kurzweil cally form minute wind-dispersed seeds of only (1993). To explain this, Kurzweil discussed the 0.1 – 6 mm in size. They are characterized by a habitats and dispersal modes of Disa uniflora, thin balloon-like seed coat and (in the major- which occurs along the edges of perennial West- ity of cases) the absence of endosperm (Fig. 2). ern Cape streams where seeds must germinate The seed coat usually consists of uniform cells; quickly to prevent them being washed away by seed coats that consist of different cell types oc- rain. Kurzweil therefore assumed that the Disa cur only in genera with very specialized seeds seed is adapted for that habitat: the endosperm (see below). The embryo is much reduced and allows a quick growth of the seedling. consists usually of only a few cells, though some Some genera have large fruits with aromatic species may contain more than one embryo per pulp and sclerified black seeds, both suggesting seed. Polyembryony with up to 12 embryos per adaptations to zoochory. This is found mainly in seed (!) occurs in the seeds of Thecostele (Fig. the Vanilloideae (e.g. , and 3). ). Sclerified seeds are also found in Apostasia Blume and Neuwiedia Blume The structure of the orchid seed coat is rather (Apostasioideae), in Seleni­pedium (Cypripe­ conservative and supposed to be little prone to selective pressure since there is one predominant seed dispersal mechanism in the whole fam- ily. Orchids typically have tiny wind-dispersed seeds, often called “dust-seeds”. Most orchids are wind-dispersed, but there are exceptions and the dispersal mechanisms of orchids are more diverse than assumed earlier. A link of morphol- ogy and dispersal properties was already sug- gested by Arditti & al. (1980), and Healey & al. (1980) noted that “there is a functional correla- tion between orchid seed morphology, their wet- tability, and aerodynamics. These in turn affect the dispersion of the seeds”. A unique dispersal mechanism occurs in the Vanilloideae–Vanilleae: wind-dispersal by large sclerified winged seeds (Fig. 4). This type of anemochory is very different from the wind-dis- persal of the tiny “dust-seeds” of the rest of the family. Cameron & Chase (1998) assume dif- Fig. 2. Orchid seeds usually have no endosperm; the ferent dispersal mechanisms for the Vanilleae, seed coat embraces like a balloon the air-filled space including water dispersal for and between embryo and testa (). – zoochory not in only Vanilla, but also Galeola. A: fluorescence microscopic image of L. abortivum – B: SEM image of L. abortivum: due to the high ac- In addition to wind-dispersal, water-dispersal celerating voltage of 30 keV (method after Wolter & (hydrochory) could play a role in some species Barthlott 1991) the embryo becomes clearly visible. (Dressler 1981), e.g. in (Epi­ 14 Introduction

ends or elaborate hooks (Fig. 6). These exten- sions have so far been found only in those On­ cidiinae species that are twig epiphytes and they suggest to serve for attachment to the tree bark (Chase & Pippen 1988). The seeds of Sobralia dichotoma are unique within the Orchidaceae and are the only known example of an orchid seed truly adapted for wa- ter uptake (Prutsch & al. 2000). The seed coat of S. dichotoma consists of three different cell types and Prutsch & al. suggested that this is an Fig. 3. Thecostele alata is a unique case of obligate adaptation to protect the embryo from desicca- polyembryony with three to twelve (!) embryos per tion. The water uptake is possible through the seed, which may be a record in plants. Only very rare- ly do seeds with one embryo develop. development of tracheoidal idioblasts – special cells with secondary wall thickenings that re- semble the tracheids of the vascular tissue sys- tem but are not true vascular elements (Prutsch dioideae), Rhizanthella (Orchidoideae) and Pal­ & al. 2000). morchis (Epidendroideae). As far as is known, sclerified seeds are not homologous in all these genera (Freudenstein & Rasmussen 1999). All Earlier seed morphology studies in these genera also produce fleshy fruits, which Orchidaceae­ do not open. In combination with this fruit type, the sclerified seeds are most likely a convergent Orchid seeds have been known to science since adaptation to zoochory to protect the embryo in the middle of the 16th century from the works the digestive tract. and drawings of Conrad Gessner in his Historia plantarum. But almost all publication focused Many orchids are epiphytes and therefore ad- on the intriguing, complicated and aesthetically aptations of seeds to epiphytism have been sug- fascinating flowers and their functions. Charles gested. In Japanese Liparis (Epidendroideae), Darwin spent years researching these flowers – the seed volumes and air spaces were found to but in his admirable orchid book (Darwin 1862) be significantly different between epiphytes and he mentioned the tiny seeds only marginally in terrestrial species (Tsutsumi & al. 2007). Differ- the last chapter. The earliest truly comprehen- ences were found in embryo volumes, seed vol- sive publication on orchid seeds was the work umes, and air spaces, as well as in the lengths of Beer (1863), including beautiful and accurate and widths of the embryos. Tsutsumi & al. colour paintings, reproduced here (Fig. 7). Other (2007) therefore concluded that embryo size is studies did not follow until more than a century correlated with the evolution of epiphytism and later, Arditti & Ghani (2000) reviewed the early that larger embryos may be an advantage for the knowledge on orchid seed studies in detail. The epiphytic life form, though they did not exactly first study where the systematic value of orchid clarify which kind of advantage this would be. seed characters was pointed out was presented A very elaborate seed attachment mechanism by Clifford & Smith (1969). Their study was is known from a tropical Asian epiphytic orchid based on comparisons of 49 species mainly from Chiloschista lunifera. This species has special- the tribes Epidendreae and Neottieae and found ized seed-coat cells with helical wall thicken- “reasonable correlations” between suprageneric ings, which extend upon contact with water and groups and seed characters. Seeds of European produce long threads (Fig. 5). This mechanism orchids were studied by Wildhaber (1970, 1972, enables the seed to attach itself to moist tree bark 1974) based on light microscopy. Initial SEM (Barthlott & Ziegler 1980). Some Epidendroide­ studies of orchid seeds showed that they were ae form testa extensions in the form of capitate much more diverse than previously assumed Introduction 15 based on light microscopic surveys (Senghas & al. 1974). This was followed by structural analyses in which the fine balloon-shaped seed coats could be clarified as helical wall thicken- ing by oxygen ion etching. An overview of the functional morphology of the dust-like seeds appeared shortly after (Rauh & al. 1975). Bar- thlott (1976a) then undertook the first detailed SEM study and pointed out the systematic and taxonomic value of seed characters for orchid systematics. The only study covering the whole family is an unpublished dissertation of B. Ziegler (1981), which was supervised by W. Barthlott. Ziegler Fig. 4. Large winged seed (1300 µm) of Epistephium parviflorum (Vanilloideae). had summarized characters to define 20 seed types and discussed the correlation of single seed characters as well as the seed types with tematics. Wolter & Barthlott (1991) found that the Orchidaceae system of Dressler (1981). Zie- the composition of elements in orchid seeds is gler found that seed characters were of high sys- not influenced by soil and is therefore useful as tematic and taxonomic significance and could a character for comparative systematics. be used to support or define groups within the Kurzweil & al. (1991) studied the phyloge- family. Unfortunately, only some minor results netic relationships within the Pterygodium–Co­ of this work were published (Barthlott & Zie- rycium species group and made a first cladistic gler 1980, 1981) but the results nevertheless be- analysis including seed characters. Here, they came rather widely known (see below). found that the seed morphology was not inform- Many studies of orchid seed coat morphology ative but that only few groups showed synapo- have been published representing selected taxo- morphic seed characters. Kurzweil (1993) and nomic groups only or material from one geo- Linder & Kurzweil (1994) examined the seed graphic region. Arditti & al. (1979, 1980) and morphology of South African Orchidoideae, Healey & al. (1980) presented morphometric mainly the tribes Orchideae and Diseae, and studies and SEM images of Cypripedium and discussed the taxonomic value of the seed char- several Orchidoideae genera native to Califor- acters in that group. Kurzweil (1993) concluded nia and briefly discussed whether seed charac- that generally the characters he observed are not ters characterize genera. Chase & Pippen (1988) useful as taxonomic characters, as they appear analysed the seed morphology of the Oncidii­ to be not uniquely derived but to have evolved nae and related subtribes and Chase & Pippen independently in several genera. Nevertheless (1990) analysed the seeds of Catasetinae. These he suggested that seed characters could support studies were even the first detailed examination relationships between some species. of orchid seed morphology at the subtribal and Cameron & Chase (1998) studied seeds of va- generic level, besides the unpublished disserta- nilloid orchids (former tribe Vanilleae, currently tion by Ziegler (1981). The conclusions of both subfamily Vanilloideae). As stated by the au- these studies were that seed characters are well thors, seed characters may be highly significant suited for defining generic relationships and cir- for the systematics of this group as distinctive cumscribing subtribes in Orchidaceae. seed types were observed in vanilloids: typical Wolter & Barthlott (1991) tried yet another orchid “dust-seeds”, sclerotic seeds in Vanilla, approach by using energy-dispersive X-ray and seeds with wing-like testa extensions in spectroscopy (EDX) to study orchid seeds. Galeola, Erythrorchis and related genera. They analysed seeds of 98 predominantly Euro- Seed data play an important role in the most pean terrestrial species with the aim of provid- influential Orchidaceae system, namely in the ing additional characters for comparative sys- works of Dressler (1993). Dressler had obtained 16 Introduction the actual seed data and SEMs he presented in close collaboration with W. Barthlott and B. Zieg­ler. Dressler adapted the concept of seed- types as Ziegler (1981) had defined them; thus this concept became widely known. There are several seed studies using only ma- terial from one geographical origin or a country, for example Spain (Ortúñez & al. 2006), Turkey (Aybeke 2007; Akçin & al. 2009), the Western Ghats, (Krishna Swamy & al. 2004), the Himalaya (Verma & al. 2012) or (Tsut- sumi & al. 2007). Fig. 6. Corners of testa cells extended to hooks (Mi­ crocoelia obovata, Epidendroideae). Also statistical analyses of morphometric data have been used for orchid seeds. Molvray & Kores (1995) analysed seed characters of the The authors of the most recent studies on or- Diurideae and spiranthoid orchids. Their work chid seeds aimed at finding seed characters to is mainly an attempt to re-define some of Zie- support new generic circumscriptions based on gler’s seed types. Using a similar approach, the results of molecular phylogenetic studies in Chemisquy & al. (2009) analysed the seeds Orchideae (Gamarra & al. 2007, 2008, 2010, of Chloraeeae using traditional and geometric 2012). They found that seed ornamentation morphometrics with the aim of identifying di- patterns support the monophyly of Neotinea agnostic characters using a statistical approach (Gamarra & al. 2007), the splitting of Limnor­ instead of a merely descriptive one. The overall chis from Platanthera (Gamarra & al. 2008), results, however, were largely inconclusive and and the expanded Anacamptis (Gamarra & al. the authors could not find discrete characters to 2012). Gamarra & al. (2010) summarized their support generic circumscriptions. earlier findings in an additional overview article

Fig. 5. The unique seeds of Chiloschista lunifera (Epidendroideae). This species has extendable helical wall thickenings that serve as an attachment mechanism. – A: drawing reproduced from Barthlott & Ziegler (1980) – B: SEM-image; see also Fig. 88 G, H for further SEM images. 20 Descriptors and terminology

Seed size (length of seed) Seeds of orchids range from 100 µm (Oberonia similis) to 6000 µm (). The size used here refers to the length of the seed and can be classified into five categories. Medium-sized represents the average. very small 100 – 200 µm small 200 – 500 µm medium-sized 500 – 900 µm large 900 – 2000 µm very large 2000 – 6000 µm

Seed colour Fresh orchid seeds appear in many different col- ours. Most often it is whitish, brownish or dark brown, but can also be beige, yellow, reddish, orange, greenish, yellowish brown, or black Fig. 10. Variation in the number of cells per seed – A: (Fig. 9). The colour is determined by the testa few cells in arcuata – B: many cells in So­ bralia dichotoma (Epidendroideae). and especially by the embryo, which can be an intense yellow, orange or red-orange colour, or greenish in seeds that contain chlorophyll. 2003), this pattern probably results from a slow Number of testa cells (along longitudinal axis or stopped cell division in the integuments after of seed) fertilization. In other genera, where cell division The number of cells that form the testa in an in- continues, the seed coats are composed of nu- dividual seed coat varies highly between genera merous small cells (Fig. 10 B). but is almost constant within a . In some genera, seeds are composed of only few (about Shape of individual testa cells five) cells or fewer along the longitudinal axis of Most commonly the testa cells are tetragonal, the seed; in the most extreme case of only two hexagonal or polygonal; sometimes the cell cells (Fig. 10 A). Since the number of testa cells shape is irregular. We distinguish three kinds of is coupled to cell division (Molvray & Chase testa cell shape: (1) all cells are more or less iso- diametric (regardless of their actual shape); (2) the cells are elongate in the longitudinal axis of the seed (prosenchymatic) and rectangular; and (3) the cells are elongate but rounded at the ends (Fig. 11 A). Only the testa cells located in the middle part of the seed are used for description. This is be- cause the shape of cells at the poles usually dif- fers from those in the middle.

Testa cell pattern Within a single seed coat, all cells are either equal in size (regardless of their shape) or the medial cells are highly elongate in comparison to the cells at the poles (Fig. 11 B). Both these Fig. 9. Colour variation in orchid seeds – From top: patterns result from cell division in the outer in- Vanilla, Angraecum, Calanthe, Dendrobium (twice), tegument. . – Tube diameter: 6.5 mm. Descriptors and terminology 21

Anticlinal wall curvature Transverse anticlinal walls The anticlinal cell walls are usually straight but The transverse anticlines can also show modi- may also be curved or undulate. Undulations can fications. Many Epidendroideae have elevated, be S-like or V-like (Fig. 11 C). The thickness of arch-like transverse anticlines (Fig. 11 D). Zieg­ the anticlinal cell walls can vary; sometimes the ler (1981) suggested that seeds with such el- apices have thicker walls. evated anticlines fall more slowly thus can better attach to the substrate.

Fig. 11. Overview of various seed characters – A: testa cells elongated and rounded at ends (Zygopetalum mackayi, Epidendroideae) – B: seed with elongated medial cells (Anacamptis pyramidalis, Orchidoideae) – C: undulated anticlinal walls (Disa uniflora, Orchidoideae) – D: transverse anticlinal walls elevated (Arundina graminifolia, Epidendroideae) – E: intercellular gaps at cell corners (Vryda­ gzynea sp., Orchidoideae) – F: inter- cellular gaps along anticlinal walls (Cyclopogon sp., Orchidoideae). 78 Epidendroideae – Vandeae

Aeridinae Pfitzer A very elaborate seed attachment mecha- nism is known from Chiloschista lunifera J. Lindl. J. Sm. This species has specialized seed-coat Seed shape: scobiform, grain-shaped, A. mom­ cells with helical wall thickenings, which ex- bassensis slightly twisted; seed colour: yellow- tend upon contact with water and produce long brown; fewer than 5 testa cells along the long- threads. This mechanism enables the seed to axis; shape of testa cells: elongate, irregular, attach itself to moist tree bark (Barthlott & rounded at the end; anticlinal walls: straight; cell Ziegler 1980). corners: often extended, with trichomes; pericli- Fig 5 A, B; 88 G, H. nal walls: not visible. Fig. 88 A, B. D. Don Seed shape: longish scobiform; seed colour: Blume ochre; fewer than 5 testa cells along the long- Seed shape: scobiform, tapered apical end; seed axis; shape of testa cells: elongate, irregular, size: very small, length: 130 µm; seed colour: rounded at the end; anticlinal walls: straight, yellowish; fewer than 5 testa cells along the marginal ridges strong and twisted; cell corners: long-axis; shape of testa cells: strongly elongate smooth; periclinal walls: not visible, at the out- and irregular; anticlinal walls: straight; cell cor- er periclinal walls twisted marginal ridges of a ners: smooth; periclinal walls: not visible, ver- deeper testa cell layer are visible. rucosities; other features: seed surface is covered Fig. 89 E. with fine verrucose sculptures. Fig. 88 C, D. Schltr. Seed shape: scobiform, grain-shaped; seed Aerides Lour. size: small, length: 300 µm; seed colour: strong Seed shape: scobiform; seed size: very small, brown; fewer than 5 testa cells along the long- length: 250 µm; seed colour: brownish; fewer axis; shape of testa cells: elongate, irregular, than 5 testa cells along the long-axis; shape of rounded at the end; anticlinal walls: straight; testa cells: elongate, irregular, rounded at the transverse anticlinal walls: plain and dense mar- end; all cells about the same size; anticlinal ginal ridges; cell corners: smooth; periclinal walls: straight; transverse anticlinal walls: el- walls: not visible. evated, arch-like; cell corners: extended: small Fig. 89 A, B. trichomes on the basal and apical poles; pericli- nal walls: sometimes ridges of deeper testa cell Kingidium P. F. Hunt layers visible. Seed shape: longish scobiform; seed size: small, Fig. 88 E, F. length: 400 µm; seed colour: light brown; fewer than 5 testa cells along the long-axis; shape of Blume testa cells: elongate, irregular; transverse an- No data and material available. ticlinal walls: elevated, arch-like; cell corners: extended, trichomes developed only apical and Chiloschista Lindl. basal; periclinal walls: not visible. Seed shape: scobiform; seed size: small, length: Fig. 89 C, D. 400 – 650 µm; seed colour: yellowish; fewer than 5 testa cells along the long-axis; shape of testa Gaudich. cells: strongly elongate and irregular, rounded at No data and material available. the end, secondary helical wall thickenings, well visible at the basal part of the seed, barely vis- Schltr. ible at the apical part; anticlinal walls: straight, Seed shape: scobiform; shape of testa cells: marginal ridges with fine striated longitudinal elongate, irregular, rounded at the end; anticlinal structures; cell corners: extended, micropapillae; walls: straight; cell corners: smooth; periclinal periclinal walls: not visible. walls: verrucosities; other features: verrucose 98 Vanilloideae – Vanilleae

A B

C D

E F

G H

Fig. 18. Vanilloideae – Vanilleae – A, B: Galeola nudifolia, length: 1500 µm – C, D: Galeola septentrionalis, length: 1500 µm – E, F: multiflora, length: 2400 µm – G, H: , length: 500 µm. – H: longitudinal section through a mature seed showing the thick sclerified testa. 132 Epidendroideae – Cymbidieae

A B

C D

E F

G H

Fig. 52. Epidendroideae – Cymbidieae – Catasetinae – A, B: Galeandra devoniana, length: 500 µm – C, D: Gro­ bya amherstiae, length: 800 µm – E, F: sp. (BG Heidelberg: 12314), length: 900 µm – G, H: guttata, length: 800 µm. 146 Epidendroideae – Cymbidieae

A B

C D

E F

G H

Fig. 66. Epidendroideae – Cymbidieae – Zygopetalinae – A, B: pulchella, length: 400 µm – C, D: Ben­ zingia estradae, length: 250 µm – E, F: Cochleanthes aromatica, length: 250 µm – G, H: lunata, length: 250 µm. Phylogenetic trees: Shape of testa cells 195

Shape of testa cells Gomesa Leochilus elongated, rectangular Notylia elongated, rounded at the end Ornithocephalus Pachyphyllum Warrea Zygopetalum Houlletia Maxillaria Cymbidieae Anguloa Cyrtopodium Dressleria Catasetum Galeandra Graphorkis Dipodium Cymbidium Calypso Corallorhiza Calypsoeae Govenia Epidendrum Cattleya Meiracyllium Encyclia Arpophyllum Epidendreae Isochilus Ponera Chysis Coelia Bletia Earina Agrostophyllinae Dendrobium Cadetia Dendrobiinae Epigeneium Bulbophyllum Malaxis Malaxideae Epidendroideae Liparis Bromheadia Cymbidieae Vanda Aerides Phalaenopsis Vandeae Angraecum Campylocentrum Polystachya Neobenthamia Sirhookera Agrostophyllinae Eria Podochileae Mediocalcar Collabium Plocoglottis Spathoglottis Collabieae Calanthe Phaius Arundina Dendrochilum Thunia Arethuseae Glomera Coelogyne Bletilla Tropidia Tropidieae Corymborkis Elleanthus Sobralieae Sobralia Palmorchis Neottia Limodorum Neottieae Aphyllorchis Epipactis Gastrodia Gastrodieae Monophyllorchis Triphoreae Wullschlaegelia Calypsoeae Xerorchis Xerorchideae Nervilia Nervilieae

Fig. 110 B. Character reconstruction for shape of testa cells: Epidendroideae. Phylogenetic trees: Seed types 211

Seed types Gomesa Leochilus Bletia-type Notylia Dendrobium-type Ornithocephalus -type Pachyphyllum Epidendrum-type Warrea Epidendrum-type: E. secundum variant Zygopetalum -type Houlletia Gastrodia-type Maxillaria Cymbidieae Limodorum-type Anguloa Goodyera-type Cyrtopodium Cymbidium-type Dressleria Maxillaria-type Catasetum Vanda-type: Maxillaria transition variant Galeandra Graphorkis Vanda-type: Gomesa variant Dipodium Vanda-type Cymbidium -type Calypso Corallorhiza Calypsoeae Govenia Epidendrum Cattleya Meiracyllium Encyclia Arpophyllum Epidendreae Isochilus Ponera Chysis Coelia Bletia Earina Agrostophyllinae Dendrobium Cadetia Dendrobiinae Epigeneium Bulbophyllum Malaxis Malaxideae Epidendroideae Liparis Bromheadia Cymbidieae Vanda Aerides Phalaenopsis Angraecum Vandeae Campylocentrum Polystachya Neobenthamia Sirhookera Agrostophyllinae Eria Podochileae Mediocalcar Collabium Plocoglottis Spathoglottis Collabieae Calanthe Phaius Arundina Dendrochilum Thunia Arethuseae Glomera Coelogyne Bletilla Tropidia Tropidieae Corymborkis Elleanthus Sobralieae Sobralia Palmorchis Neottia Limodorum Neottieae Aphyllorchis Epipactis Gastrodia Gastrodieae Monophyllorchis Triphoreae Wullschlaegelia Calypsoeae Xerorchis Xerorchideae Nervilia Nervilieae

Fig. 121. A combination of several characters defines the 17 seed types named after representative genera. Twelve of the seed types and their variants present in the Epidendroideae are plotted on the tree and indicate the usefulness of the seed types to circumscribe clades.