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Palynology and systematics of morinaceae
Review of Palaeobotany and Palynology, 40 ( 1 9 8 3 / 1 9 8 4 )
207-- 226 Elsevmr Sc-ence Publishers B.V , A m s t e r d a m -- Printed in The Netherlands
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PALYNOLOGY AND SYSTEMATICS OF MORINACEAE
S B L A C K M O R E and M.J. C A N N O N
Department of Botany, British Museum (Natural History), Cromwell Road, London SW7 5BD (Great Br~tatn) (Received April 12, 1983, revised and accepted August 9, 1983)
ABSTRACT Blackmore, S and Cannon, M J., 1983. Palynology and systematics of Morinaceae. Rev. Palaeobot. Palynol., 40 207--226 Pollen grains f r o m all thirteen species of the three genera to be recogn,sed in a forthcoming revis,on of the Morinaceae, Acanthocalyx, Cryptothladza and Morma, have been investigated by light and scanning electron m m r o s c o p y Fresh pollen of M long~folla has also been studied by hght, scanning electron and transmission electron microscopy. Three main pollen types axe described and a key provided for their ldenhflcation. Each pollen t y p e occurs t h r o u g h o u t a genus. F u r t h e r division of two of these pollen types enables groups of species to be recogmsed. F u n c t i o n a l aspects of the pollen are discussed, especmlly their unique pre-germinative processes. A cladistic analysis of pollen characters provides a hypothesis for their evolut,on and supports the classification proposed in the recent revision INTRODUCTION
The Morinaceae have, in the past, usually been regarded as a single genus,
Morina, belonging to the Dipsacaceae. The large and morphologically distinctive pollen grains of Morzna attracted the attention of the earliest palynologists (yon Mohl, 1835; Fritzsche, 1837; Edgeworth, 1877; Fischer, 1890) and, more recently, have been the subject of intensive light microscopical investigations b y Erdtman (1945, 1952, 1960a, 1962, 1963, 1965), and Vinokurova (1959). Vinokurova (1959) considered the Morinaceae to represent a distinct family rather than a genus, a conclusion reached b y Verlaque (1977) and Vljayaraghavan and Sarveshwari (1968) on palynological, embryological and other grounds. The present study was undertaken in conjunction with a monographic revision (Cannon and Cannon, in press) in which the Mormaceae are afforded familial status and three genera axe to be recognised. For the first time the palynology of all valid species has been studied, including that of one new species, and electron microscopy has been extensively employed. The names used in this paper are those which are currently correct but the genera to whmh the taxa will belong after publication of the revision have been indicated in parentheses. Some aspects of pollen function in Morinaceae were 0034-6667/83/$03.00
© 1983 Elsevier Science Publishers B V.
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investigated which drew attention to their unique pre-germinative processes first described by yon Mohl (1835). Using cladlstlc methods, now well estabhshed in botany (Funk and Stuessy, 1978; Funk and Wagner, 1982), an evolutmnary hypothesis for the Morinaceae and their pollen is presented. TERMINOLOGY
Pollen grains of Cryptothladia and Morina have ectoapertures situated in d o m e d or funnel-shaped sexinous protrusions termed "porus collaris" and "porus mfundlbuliformis" respectively b y Erdtman (1945). These complex structures will be referred to as apertural protrusions in this paper for the sake of linguistic simplicity. Strictly speaking, the apertures are aspidate (sensu Wodehouse, 1933) or aspldoporate (Erdtman, 1947). Each of the aspides contains a vestibulum and in Cryptothlad~a these are broad whilst in Morina they are narrow. Aspidate pollen grains occur scattered through a wide variety of living and fossil plants (Ueno, 1982) and their significance will be considered further in the discussion. MATERIALS AND METHODS
Pollen samples (listed below) were obtained from herbarium specimens and, in the case of Morina longifolia, from cultivated plants. For light and scanning electron microscope investigations of pollen morphology, the pollen was acetolysed (Erdtman, 1960b) and mounted in glycerine jelly or on SEM stubs. Sections of pollen were prepared b y freezing microtomy (Blackmore and Dmkinson, 1981). Ion beam etching (Blackmore and Claugher, 1983) was carried o u t on selected examples. SEM preparations were sputter-coated with gold palladium and examined in a Cambridge Stereoscan 180. For functional studies mature pollen was studied before and after dehiscence using the method of Payne (1972). Mature pollen grains from dehisced anthers and stigma surfaces were rehydrated in water or moisture-saturated air. These grains were studied b y light microscopy b y mounting in immersion oil or in air using a Schott fibre optic cold-light source or b y sputter coatmg and scanning electron microscopy without further treatment. Pollen for transmission electron microscopy was fixed with glutaraldehyde and osmium tetroxide, embedded in araldyte resin, sectioned with a Reichert ultrammrotome, post-stained with a saturated solution of uranyl acetate and Reynold's lead citrate and examined in an AEI 6B microscope. SPECIMENS EXAMINED
All specimens are from the herbarium of the British Museum (Natural History) except those marked (E) from the Royal Botanic Garden, Edinburgh and (PE) from the Institute of Botany, Peking. Collectors names are not available for some of the latter.
209 Morina (Acanthocalyx) alba Hand. Mazz. China: Forrest 28444, Tibet: Ludlow, Sherriff and Taylor 5017. Morina (Acanthocalyx) delavayi Franch. China: Maire 128, McLaren's native collectors 66, Rock 1898 (E) Morina (Acanthocalyx) nepalensis D. Don. China : McLaren's native collectors 73B, Nepal Dhwoj 167 (E), collector? 67 (PE), Polunin, Sykes and Williams 4536 Morina (Cryptothladia) chmensis Pal China: Hao 1002 (PE), Lment 4548, collector9 6129 (PE) Morma (Cryptothladia) chlorantha Diels China: Forrest 21255 (E), Forrest 5777, Forrest 10212 (E), collector? 5482 (PE). Morma (Cryptothladia) kokonorica Hao. China: collector9 6114 (PE), Tibet: Ludlow and Sherriff 8691, Richardson 124. Monna (Cryptothladia)polyphylla Wall. ex DC. Nepal. McCosh 381, Stainton 4325, Stainton, Sykes and Wilhams 2984 Morma (Cryptothladia) parviflora Kar et Kit. U.S.S.R. Konsevyanova s.n Morina (Cryptothladia) sp. nov. Bhutan Ludlow, Sherriff and Hicks 16903, Ludlow, Sherriff and Hicks 19050. Morina (Morina) coulterzana Royle. India: Ludlow and Sherriff 9123, Shernff 7350, Wendelbo 59; Tibet: Ludlow and Sherriff 1828, Ludlow, Shernff and Taylor 5481, U.S.S.R. Vassiljeva 5487. Morma (Mor,na) kokanlca Regl. China: Gnezdillo 186 (PE), U S.S.R : Tilchko 8539, Lmczevski and Rushkova 39. Morma (Monna) Iongifolia Wall. ex. DC. England: fresh material from cultivation; Pakmtan. Abel 78. Mo rma (Monna) pets,ca L. Greece : Guml 6771 ; India: Flemmlng 136, Marten 16, Presco ttDecie s n.; Turkey: Davis, Dodds and Cetik 19068 RESULTS General p o l l e n descrzption Pollen w e a k l y h e t e r o p o l a r , 3 - c o l p o r a t e or 3 - p o r o r a t e , in polar view triangular and in e q u a t o r i a l view a l m o s t cylindrical to r h o m b o i d a l s o m e t i m e s with 3 p r o m i n e n t apertural p r o t r u s i o n s at the e q u a t o r . C o l p o r a t e grains have b r o a d colpl with a c u t e l y t a p e r i n g ends and z o n o r a t e e n d o a p e r t u r e s . In the t w o p o r o r a t e pollen t y p e s the e c t o a p e r t u r e s are circular t o s u b o v a t e pores situated in d o m e d or f u n n e l - s h a p e d sexinous p r o t r u s i o n s . T h e e n d o a p e r t u r e s o f grains w i t h d o m e d p r o t r u s i o n s are z o n o r a t e whereas t h o s e with f u n n e l - s h a p e d protrusions are small, circular pores. All species have s m o o t h a p e r t u r e m e m branes and lack o p e r c u l a e . E x i n e very t h i c k , sexine and n e x i n e usually m o r e or less equal at poles w i t h n e x i n e either increasingly m a r k e d l y in thickness, or absent, t o w a r d s t h e e q u a t o r . A s m o o t h or, less c o m m o n l y , p a r t l y verrucate t e c t u m w i t h few to m a n y m i c r o p e r f o r a t i o n s overlays a s p o n g y or granular i n f r a t e c t u m . I n t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y it can be seen t h a t t h e r e is no f o o t layer a n d t h u s t h e n e x i n e is entirely e n d e x i n o u s . Pollen large, P 1 0 0 - 255 ~m, E 55--165 ~m. KEY TO POLLEN TYPES
1. a. C o l p o r a t e , w i t h o u t p r o m i n e n t apertural protrusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acanthocalyx type
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b. Pororate, with prominent apertural protrusions . . . . . . . . . . . . . . . . . . 2 2. a. Apertural protrusions domed, endoapertures zonorate, internally hourglass-shaped . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cryptothladia type b. Apertural protrusions funnel-shaped, endoapertures porate, internal shape similar to external . . . . . . . . . . . . . . . . . . . . . . . . . . . Morina type DESCRIPTIONS OF THE POLLEN TYPES
Acanthocalyx type (Plate II) Tricolporate. Triangular in polar view with the apertures slightly sunken in the rounded angles; elliptic to subrhomboidal in equatorial view. Weakly heteropolar, with one pole slightly narrower and more acutely rounded than the other. Ectoapertures colpate, one third as long as polar ares, broad, ends acutely tapered, margins distinct with wide margo. Endoapertures zonorate, widening greatly in the mesocolpia, margins distinct but less so in the mesocolpia. Exine thick, sexine equal to or as much as three times thicker than nexine, thickest along colpus margins. Tectum smooth with scattered microperforations increasing in number towards the equator; infratectum granular or spongy. Measurements: P 100-(116.8)-127 pm, E 55-(67.5)-75 um, exine 6 pm thick at poles, 12 ~m at colpus margins.
Species Morina (Acanthocalyx ) alba, M. (Acanthocalyx ) delavayt, M. (Acanthocalyx ) nepalensls. Comments Thls pollen type most closely resembles some members of the Dipsacaceae (Erdtman, 1952; Clarke and Jones, 1981) in shape and ectoapertures but differs in exine stratification, endoapertures and the absence of operculae. Erdtman (1945) subdivided the type on the differences in exine thlckness and structure. In the present study these characters were found to be remarkably uniform throughout the three species and no subdividion of the pollen type is considered possible, even with scanning electron microscopy.
Cryptothladia type (Plates III--IV) Tripororate. Triangular at the equator in polar view becoming circular towards the poles. Prolate in equatorial view with domed apertural protrusions ("porus collaris" of Erdtman, 1945). Very weakly to quite distinctly heteropolar, with the apertures of heteropolar grains nearer to one pole than the other and the two poles of different diameters. Internal outline hourglass-shaped in equatorial view. Ectoapertuxes circular to subovate pores, in the domed sexinous protrusions, opening into narrow or wide vestibula; margins distinct. Endoapertures zonorate (Plate I, 2) narrow and parallelsided, margins distinct with very pronounced costae. Exine thick, sexine thicker than nexine or roughly equal at poles, sexine sometimes slightly thickened towards the equator, nexine very markedly thickened in the
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costae. Tectum smooth with scattered m~croperforatlons, more numerous towards the equator. Infratectum spongy or granular. Measurements: P 93(115.6)-125 tam, E 55-(64.5)-74 tam incluswe of apertural protrusions, E 45-58 tam wxthout protrusions; exme 3--5 tam think at poles, 8--11 pm at equator.
Spectes" Morma (Crypto thladta ) chinensts, M. ( Crypto thladta ) chlorantha, M. ( Cryp tothladza) kokonorica, M. (Cryptothladia) parvlflora, M. (Cryptothladza) polyphylla, M (Cryptothladza) sp. nov. Comments This highly dxstmctive pollen type is readily recognised b y its shape, endoapertures and exine stratification. It may be further subdivided into two groups on details of the apertural protrusions. In Morina (Cryptothladia) chznenszs, M (C. ) chlorantha, M. (C.) parviflora and M. (C. ) kokonorica the sides of the protrusions are rather angular m outline, uniformly thickened along their length but not at their apices, the pores are rounded and relatively large (Plate III). Whereas in M. (Cryptothladta) polyphylla and in a new specms (Plate IV) to be described in the forthcoming revision (Cannon and Cannon, m press) the protrusmns are rounded in outhne and less prominent, the thickening of the sides of the protrusions is reduced from the base towards the apices which are themselves slightly thickened and the pores are relatively small. There are slight differences in exine stratification of the polar areas which appear less granular in light microscopy in M. (Cryptothladia) polyphylla and M. (Cryptothlad~) sp, nov. than in the other specms.
Morina type (Plate V) Tripororate. Circular to subtrlangular in polar view with three large, funnelshaped apertural protrusions at the equator. Prolate to almost cylindrical with protrusions at the equator in equatorial view. Ectoapertures wide pores of uniform or slightly decreasing diameter through the sexinous protrusions. Endoapertures pores, contiguous with the ectoapertures and not zonorate although in SEM sections shallow depressions in the inner face of the nexme are suggestive of vestigial connections between the endopores (Plate I, 3, 4). Exine thick; sexine equal to nexine or slightly thinker than nexine at the poles, reduced abruptly in a distinct subpolar ring in some species; sexine thinner towards equator except for massive development in the apertural protrusions. The nexine is greatly thickened towards the equator and forms costae around the endoapertures. Ornamentation variable, sometimes even within a specimen, tectum smooth with few or many verrucae, sometimes of two distinct sizes and with few to many microperforations which are generally more numerous towards the equator. The largest verrucae are often arranged in t w o parallel rows between the bases of pairs of apertural protrusions. Infratectum granular or spongy. Measurements: P 159-(198)-255/am, E 120-(152)-165 tam inclusive of protrusions (79--120 tam without protrusions). Exine 6 tam thick at poles, 12--15 tam at equator.
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PLATE I (p. 213) Electron micrographs of Morinaceae exines and apertures (scale lines 20 u m except where stated otherwise) 1 M (Acanthocalyx) alba (Ludlow, Sherriff and Taylor 5017), section showing endoapertures. Note broad zones below mesocolpla 2. M (Cryptothladia) polyphylla (Stamton 4325), section showing narrowly zonorate endoapertures. 3, 4. M (Morma) longzfolia (from fresh material) 3. Section showing porate endoaperture and faint depression interpreted as a veshge of the zonorate condition. 4. Section through an apertural protrusion in equatorial plane showing much thmkened nexine. 5 M_ (Acanthocalyx) alba (collection as 1), section in equatorial plane across a colpus showing granular infratectum. 6, 7, M. (Morma) persica (Davis, Dodds and Cetik 19068). 6. Granular mfratectum exposed by ion beam etching to remove tectum (scale line 2 urn). 7. Unacetolysed TEM section of exine in mesocolpium (scale line 2 urn). PLATE II (p 214)
Acanthocalyx-type pollen (scale lines 20 urn). M. (Acanthocalyx) nepalensis (Polunin, Sykes and Williams 4536) 1. Polar view in high focus. 2. Polar view in optical sechon. 3. Equatorial view at two levels of focus 4. Equatorial view at two levels of focus. 5. Whole pollen grain (SEM). 6. View of mesocolpium (SEM). PLATE III (p. 215)
Cryptothladla-type pollen (scale lines 20 urn) M. (Cryptothladia) kokonor~ca (Ludlow and Sherriff 8691) 1. Equatorial view in high focus 2. Equatorial view in optical section. M (Cryptothladza) chmenszs (Lment 4548). 3. Equatorial view (SEM). 4. Detail of apertural protrusion and ornamentation (SEM). PLATE IV (p 216)
Cryptothlad~a.type pollen (scale lines 20 um) M. (Cryptothladia) sp. nov. (Ludlow, Sherriff and Hicks 19050) 1. Equatorial view at two levels of focus. 2. Equatorial view in optical section. 3. Equatorial view (SEM). 4. Detail of apertural protrusions and ornamentation (SEM) PLATE V (p 217)
Morma-type pollen (scale lines 20 urn): M. (Morina)perszca (Davis, Dodds and Cetik 19068) 1. Equatorial view at two levels of focus. 2. Equatorial view in optical section. M (Morma) coulteriana (Vassiljeva 5478) 3. Equatorial view (SEM). 4. Detail of apertural protrusion and ornamentation (SEM).
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D e s c r i p t i o n on p. 212
PLATE I
E
[
214
PLATE II
D e s c r i p t i o n o n p 212.
I
1
I
f
215
PLATE III
D e s c r i p t i o n o n p_ 212.
i
J
2
216
PLATE IV
D e s c r i p t i o n o n p 212.
I
217 PLATE
V
D e s c r i p t i o n o n p_ 212 |
218 PLATE
VI
D e s c r i p t i o n on p_ 219
i
3
4
219 PLATE VI (p 218) Fresh M (Morina) longifoha pollen (cultivated origin). 1 Reflected light micrograph of dissected flower showing sttcky stigma and a dehisced anther (scale line 500 t~m). 2. Pollen from undehisced anther mounted in immersion oll (scale hne 50 urn). 3. Pollen mounted in water with pollen-tube-like structures (scale hne 50 urn). 4 Pollen-tube-hke structure produced by pressure exserted on pollen mounted in immersion od (scale line 50 t~m). 5 Stigma with pollen grains (SEM, scale line 100 urn). 6. Pollen-tube-like structure produced by pressure (SEM, scale line 50 urn).
Spectes: Morina (Morina) coulteriana, M. (Morina) kokanica, M. (Morina) longifolia, M. (Monna) persica. Comments A distinctive pollen t y p e similar in general f o r m to t h e Cryptothladia t y p e b u t differing in its m u c h larger size, discrete e n d o a p e r t u r e s and t h e shape o f t h e e q u a t o r i a l protrusions. T h e pollen t y p e can be subdivided into t w o groups o n t h e basis o f o r n a m e n t a t i o n and shape. M. (Morina) coulteriana, M. (Morina) kokanica and M. (Morina) persica have d o u b l e rows or bands of p r o m i n e n t verrucae and are m o r e or less cylindrical in shape, w i t h r o u n d e d ends. M. (Morina) longifolia (Plate VI) is a l m o s t d e v o i d o f v e r r u c a e and is s u b r h o m b o i d a l in shape. It was n o t e d , however, t h a t t h e d e g r e e o f developm e n t o f t h e verrucate o r n a m e n t a t i o n varied considerably f r o m o n e s p e c i m e n t o a n o t h e r , a n d s o m e t i m e s w i t h i n a specimen. T h u s it is s o m e t i m e s difficult t o distinguish b e t w e e n t h e t w o groups. OBSERVATIONS ON FRESH POLLEN Fresh pollen o f M. (Morina) longifolia (Plate VI), t h e o n l y m e m b e r o f t h e f a m i l y f o r which a few live plants were available, was e x a m i n e d using t h e m e t h o d o f P a y n e ( 1 9 7 2 ) to d e t e r m i n e its m e a n s o f h a r m o m e g a t h y . A comparison o f pollen, m o u n t e d in i m m e r s i o n oil, f r o m dehisced and u n d e h i s c e d anthers was made. No differences in shape were d e t e c t e d b e t w e e n the two and n o obvious h a r m o m e g a t h i c m e c h a n i s m s n o t e d . Pollen grains f r o m dehisced anthers and f r o m stigmatic surfaces were r e h y d r a t e d in water o r waters a t u r a t e d air. R e h y d r a t i o n in w a t e r resulted in s u d d e n e x t r u s i o n o f pollentube-like s t r u c t u r e s f r o m each a p e r t u r e . These were p r o d u c e d m o r e slowly, and in a smaller p e r c e n t a g e o f grains, w h e n pollen was placed in a moisturesaturated a t m o s p h e r e . T h e t u b e s q u i c k l y a t t a i n e d a length o f a b o u t 8 0 / a m and t h e n ceased e x t e n d i n g . Similar t u b e s could also b e p r o d u c e d b y light pressure being applied t o individual grains with a fine needle. When grains had b e e n in w a t e r f o r a n u m b e r o f m i n u t e s m a n y o f t h e m r u p t u r e d and t h e entire p o l l e n wall was t h e n seen to c o n t r a c t u m f o r m l y to a b o u t 70% o f its f o r m e r size. Pollen.tube-like structures were n o t p r o d u c e d b y pollen grains o f m a t u r e b u t u n d e h i s c e d anthers n o r f r o m dehisced pollen grains after
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h x a t m n with glutaraldehyde. Essentially similar structures were, however, produced b y pollen taken from herbarium specimens of M. (Acanthocalyx) alba and M. (Cryptothladia)polyphylla and placed in water. Examination of the pollen-tube-like structures b y SEM proved quite possible and even without specml preparation the structures could be examined without their collapsing. DISCUSSION
Functional interpretatzon The observations on fresh pollen provide interesting information concerning two important aspects of pollen function, namely harmomegathy and germination. The term harmomegathy was introduced b y Wodehouse (1935) for the processes b y which pollen grains are able to adapt to the changes in volume which result from differences in hydration. Morphological features which permitted the accommodation of volume changes he called harmomegathm. Payne (1972, 1981) and Muller (1979) have drawn attention to numerous harmomegathic mechanisms in a wide varmty of flowering plants. In the Lactuceae (Blackmore, 1982a) harmomegathlc mechamsms were shown to be of considerable adaptive significance and presumed to be an Important aspect of pollen-morphological evolution. Harmomegathic mechamsms fall into three main categorms. Firstly, the apertures frequently act as harmomegathia in addition to providing a site for the exit of the pollen tube. Secondly, regions of the pollen surface other than the apertures may be capable of flexibility, often because they axe thinner than the surrounding, more rigid, parts of the wall. Thirdly, sporopollenin has an inherent elasticity which allows exines to accommodate hmited changes in volume. However, as Muller (1979) pointed out, th~s elasticity is generally insufficient to accommodate all the volume changes necessitated and thus harmomegathia of the other two kinds are also usually present. Muller drew attention to the important relationship between surface area and volume in a discussion of what he termed the "Wodehouse effect" (Muller, 1979, p.613). He pointed out that distinctly cylindrical or prolate pollen grains have an increased surface area compared to spherical pollen grains of the same volume. This enables them to achieve a given percentage volume reduction b y means of a smaller change in equatorial diameter than is necessary in the case of spherical pollen. The markedly prolate shape of pollen of the Cryptothladia-type and Monna-type reduces the requirement for harmomegathic structures, such as colpi, which would enable drastm changes in equatorial diameter. Imtial observations on M. (Morina) longzfolia pollen, using the method of Payne (1972), failed to reveal any obvious harmomegathia. However, it was observed that if the pollen wall is ruptured either by the uptake of excess water or physically, it shrinks dramatically over its entire surface to about 70% of its original size. This marked shrinkage appears to be the key to
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harmomegathy in Morinaceae and is interpreted as implying sufficient elasticity for the wall to accommodate substantial changes in volume simply b y shrinkage or stretching. A comparison was made with pollen from five species of D,psacus, Knautia and Scabiosa from the Dipsacaceae. When treated m the same way these ruptured b u t the pollen walls did not shrink. This suggests that Morinaceae pollen walls are in some way different from those of the Dipsacaceae and, m fact, most other flowering plants. The main morphological difference between the walls of Morinaceae and Dipsacaceae pollen is the granular rather than columellate infratectum which seems unlikely to account for the differences in physical properties. It may be speculated that a difference in the chemical nature of the walls would provide an explanation although no attempt has been made to demonstrate such a difference. Exines do vary in their physmal properties, including elasticity. Clarke and Jones (1981) noted that acetolysed Dlpsacaceae exines increased considerably in size over a period of months and years when m o u n t e d in glycerine jelly. No comparable data are yet available for the Monnaceae but the phenomena of unusual elasticity and a gradual swelling in mounting medm may be related and indicate that the pollen walls of at least some members of the Dipsacales are in some way unusual. An interesting feature of this method of harmomegathy is that the overall shape of the pollen grains remains stable. Various other specialised systems of harmomegathy share this attribute including those of the lophate pollen of Compositae (Bolick, 1978; Blackmore, 1982a) and of many pantoporate pollen grains (Muller, 1979). Systems of harmomegathy in which the overall shape remains stable may possess the adaptive advantage of being better suited to enduring the successive dehydration and rehydration which may occur during polhnation (Heslop-Harrison, 1979), perhaps because they are not subjected to repeated flexing. That the walls of the pollen-tube-like structures are composed of ratine may be seen b y transmission electron microscopy and had been illustrated by Erdtman (1960a, 1962, 1963) using ultraviolet microscopy. Linskens and Mulleneers (1967) reported the production of "instant pollen tubes" in pollen of Petunia and other plants. The p h e n o m e n o n found in Morinaceae pollen appears significantly different in that the pollen-tube-like structures may be produced simply b y physical pressure or b y hydration of the pollen. It is presumed that at least one of the tubes may elongate to become a normal pollen tube and effect fertilization. They do not, however, only appear during the germination of the pollen and their production is consequently regarded here as a pre-germinative process. Several possible functions may be suggested for this pre-germmative process which await experimental investigation. They may, for example, be exserted as a final part of the harmomegathic process to prevent rupture of the pollen wall. Alternatively t h e y may increase the surface area of ratine exposed on the stigma surface and either achieve closer adhesion or permit some form of chemical interaction between pollen and stigma.
222
Palynology and classification Erdtman (1945) pointed out that in Monna sensu lato the two sections A canthocalyx and Diotocalyx could be distinguished by their different pollen morphologies. He suggested that further study of the plants themselves might support the exclusion of Morina sensu lato from the Dipsacaceae. This was indeed the conclusion reached by Vinokurova (1959) and, more recently, Verlaque (1977). In a recent monograph (Cannon and Cannon, in press), the Mormaceae are recognised as a distinct family with three genera and thirteen species. In the monograph section Acanthocalyx is given generic status and section Dtotocalyx is divided into the two genera Cryptothladia and Morina. These decisions were based on a close accord between data from floral morphology, embryology, palynology and other characters. The three pollen types are each found throughout all members of a single genus. The subdivisions of the pollen types suggest infrageneric relationships but these are not borne out by the other lines of evidence. For example, two groups of species within Cryptothladia are indicated by pollen morphology: M. (Cryptothladia)polyphylla and M. (Cryptothladia) sp. nov. in one and M. (Cryptothladia) chinensis, M. (Cryptothladia) chlorantha, M. (Cryptothladta) parvzflora and M (Cryptothladia) kokonor~ca in the other. However, at the macromorpholog~cal level there is no evidence for the existence of two such groups and no suggestion that M (Cryptothladm) polyphylla and the new specms are more closely related to each other than to any of the other specxes. Fossil pollen from the form genus Aquilapollenites has been associated with the Dlpsacaceae (Rouse, 1957) because it also has distinctive apertural protrusions which Rouse interpreted as being air bladders. The dipsacaceous pollen implicated can only be that of the Cryptothladia-type or the Morinatype, now of the Morinaceae. It is perhaps not surprising t h a t the occurrence of such morphologically distinctive pollen grains should be interpreted as an indication of affinity. However, as Stanley (1961), Erdtman (1971) and Jarzen (1977) pointed out, Aqudapollenites more closely resembles certain genera of the Santalales and, whatever their true affinities, a relationship with the Morinaceae can be discounted. In this respect it should also be noted that Aquilapollenites does not have a granular infratectum. Ueno (1982) has postulated that the basic tetrahedral shape of the developing microspores when united in tetrads has been modified to produce pollen grains with apertural protrusmns in such diverse living genera as Medusagyne and Protea and in the fossil form genera of the Triprojectacites group, including AquilapoUenites. Ueno implies, in effect, that the occurrence of apertural protrusions in such diverse taxa is a consequence of similarities in ontogeny rather than of relationship. Rowley (1981) has demonstrated in the origin of apertural protrusions of certain Proteaceae a mechanism which m a y also occur in other taxa. Rowley f o u n d that protrusions are present in the tetrad stages of virtually all Proteaceae but are retained at m a t u r i t y only in those cases where oncoid plugs prevent the extension of the developing cytoplasm into the
223
protrusion. It may be that all pollen grains with apertural protrusions have a similar ontogeny and perhaps that functional advantages have led to their convergent evolution in a variety of taxa.
Cladisttc analys~s The cladistlc methods originated b y Hennig (1965, 1966) have now been used in numerous botanical studies. Funk and Stuessy (1978) have reviewed the methods and Funk and Wagner (1982) the growing literature of the subject. Blackmore (1982b) used a cladlstic analysis of palynological characters to provide an evolutionary hypothesis for one subtribe of the Compositae. Here a cladistic analysis of the pollen characters of the Mormaceae is used to present a branching diagram, or cladogram, showing the evolutionary relationships within the family as indicated b y their pollen. In essence the method consists of interpreting as either primitive or derived the character states of the taxon and its outgroup. A cladogram is then constructed which shows the distribution of derived characters in the simplest way, that is with the least invocation of parallelism, convergence or reversal of trends. Table I shows the primitive and derived character states of the pollen characters used and their distribution in the genera of Morinaceae and their closest relatives, the Dipsacaceae. Some of these characters and their interpretation require discussion whilst others are self ewdent. The nuclear condition of the pollen at anthesis (character 1) is a useful, although often neglected (Blackmore, in press), character. Neither the Dipsacaceae nor Morinaceae have been extensively surveyed and only the limited data given b y Brewbaker (1967) and Vijayaraghavan and Sarveshwari (1968) are presented here. In accordance with the conclusions of Schfirhoff (1926) and Brewbaker (1967), trinucleate pollen is interpreted as being derived. TABLE
I
Primitive (--) and derived (+) character states used in the construction of the cladogram (Fig. I ), and their distribution in the Dipsacaceae (D), A canthocalyx (A), Crypto thladia (C) and
Morma
(M)
Primitive state
state
D
A
C
M
trinucleate
+
--
--
--
present
+
--
--
--
granular structures present
---
+ +
+ +
+ +
Derived
1
binucleate
2
operculae
3 4
infratectum columellate no pollen-tube-hke structures
absent
5
endoapertures
free
zonorate
6
endoaperLures
zonorate
secondarily
7
isopolar
8
narrow
9
apertural
10
--
+
+
--
--
-
--
+
heteropolar
--
+
+
+
in mesocolpla
--
+
--
--
--
--
+
+
--
--
+
+
constricted
--
--
+
--
present
--
--
--
+
weakly endoapertures protrusions
subspheroidal
11
internally
12
verrucae absent
unconstricted
broad absent
free
present prolate
to perprolate
224
The difference b e t w e e n granular and columellate infratectum (character 3) is very distinct b u t it is difficult to ascertain which is the derived state. Granular infratecta may be primitive in certain families with thin-walled, winddispersed pollen grains (Doyle, 1978 ; Muller, 1979). In other taxa including, for example, tribe Indigofereae of the Papilionoideae (Ferguson and Strachan, 1982) granular sexines are clearly a derived condition and they are interpreted as being so in the Morinaceae. However, as with characters 2 and 4, the opposite interpretation of the trend would not, in fact, alter the branching of the cladogram. The evolution of endoapertures in the Morinaceae (characters 5 and 6) is more complex. The hypothesis adopted here is that in the primitive condition the endoapertures are unfused, as in the Dlpsacaceae, that the zonorate condition In pollen of Acanthocalyx and Cryptothladia is derived but that the genus Morina has secondarily unfused endoapertures. The presence of vestiges of the zonorate condition, in the form of slight thinnings in the nexine b e t w e e n the endoapertures, in Morina, is taken as evidence in support of this. These thinnings are most clearly seen in sectioned pollen examined by scanning electron microscopy (Plate I). The very broad endoapertures in the mesocolpia of Acanthocalyx pollen (character 8) are interpreted as being derived from the narrow zonorate endoapertures which would presumably result if the lalongate endocolpi of the Dipsacaceae were extended until they anastomosed. Heteropolar pollen (character 9) is interpreted as being derived in the Morinaceae and it is thought that it may have arisen as a consequence of the large size of the pollen grains and the complexities, due to shape, of their arrangement in tetrads during development. Heteropolarity is discernible in all Morinaceae b u t is generally more marked in pollen grains of Cryptothladia and Morina where the apertures are nearer to one pole than the other and one pole, presumably the proximal pole in the tetrad, is more acutely tapered than the other. The cladogram (Fig.l) shows that the pollen characters used support the familial status of the Morinaceae. Acanthocalyx is shown to be more closely related to the Dipsacaceae than are either Cryptothladia or Morina and the latter two genera to be more closely related to each other than to Acanthocalyx. CONCLUSIONS
Morinaceae pollen is morphologically distinct from that of the Dipsacaceae m its lack of columellae and echinulate opeculae and in the presence of zonorate endoapertures in some of the pollen types. The Morinaceae also differ in having unusually elastic pollen walls and in producing pollen-tubelike structures as a unique pre-germinative process. Each genus has a single pollen t y p e and these exhibit relatively little variation. Acanthocalyx pollen shows the closest relationship to the Dipsacaceae. Cryptothladia and Morina, which b o t h have apertural protrusions, are closely related to each other and are more distinct from the Dipsacaceae than Acanthocalyx is.
225
D
A
C
M
v~3,4,5,7 ~
9,10
Fig.1. Cladogram showing the evolutionary relationships of the Dipsacaceae (D), Acanthocalyx (A), Cryptothladia (C) and Morina (M) based on the palynological data in Table I. ACKNOWLEDGEMENTS We are g r a t e f u l t o J . F . M . C a n n o n a n d J.M. P e t t l t t f o r r e a d i n g a n d c o m m e n t ing on the manuscript. REFERENCES Blackmore, S., 1982a. A functional interpretation of Lactuceae (Compositae) pollen. Plant Syst Evol., 141. 153--168. Blackmore, S., 1982b. Palynology of subtribe Scorzonerinae (Compositae Lactuceae) and its taxonomic implications. Grana, 21: 153--168. Blackmore, S., in press. Pollen characters and plant systematics. In'. V.H. Heywood and D.M. Moore (Editors), Current Topics in Plant Taxonomy. Academic Press, London. Blackmore, S. and Dickinson, H.G., 1981. A simple technique for sectioning pollen grains. Pollen Spores, 23 : 281--285. Blackmore, S and Claugher, D., 1983. Ion beam etching in palynology. Grana, 22 (in press) Bolick, M.R., 1978. Taxonomic, evolutionary and functional considerations of Compositae pollen ultrastructure and sculpture. Plant Syst. Evol., 130: 209--218. Brewbaker, J.L., 1967. The distribution and phylogenetic significance of binucleate and trinucleate pollen grains m the Angiosperms. Am. J. Bot,, 54: 1069--1083. Cannon, M.J. and Cannon, J.F.M., A revision of the family Morinaceae (Magnoliophyta/ Dipsacales). Bull. Br. Mus. (Nat. Hist.), Bot Ser., in press. Clarke, G.C.S. and Jones, M.R., 1981. The Northwest European Pollen Flora, 21. Dipsacaceae. Rev. Palaeobot. Palynol., 33 1--26. Doyle, J.A., 1978. Origin of Angiosperms. Annu. Rev. Ecol. Syst., 9. 365--392. Edgeworth, M.P , 1877. Pollen Hardwicke and Bogue, London, 92 pp. Erdtman, G., 1945. Pollen morphology and plant taxonomy. III. Sven. Bot. Tidskr , 39. 187--191. Erdtman, G , 1947. Suggestions for the classification of fosml and recent pollen grams and spores. Sven. Bot Tldskr., 41. 104--114. Erdtman, G., 1952. Pollen Morphology and Plant Taxonomy Angiosperms. Almqwst and Wiksell, Stockholm, 539 pp Erdtman, G., 1960a. Notes on the finer structure of some pollen grains. Bot. Not., 113. 285--288.
226 Erdtman, G , 1960b. The acetolysis method. A revised description. Sven. Bot. Tldskr., 54 561--564. Erdtman, G., 1962. Palynologiska aspekter. Sven Naturvetensk., 1 5 219--217. Erdtman, G., 1963 Sporomorphology and phytomorphology. J Indian Bot. Soc., 42A 35--38. Erdtrnan, G , 1965 Pollenkorner und Sporen als Teilchen lm Getreibe der Botanik. Sven Bot. Tidskr, 5 9 49--58. Erdtman, G., 1971. On the affimties Aqutlapollenltes Rouse Grana, 11 15--17 Ferguson, I.K and Strachan, R., 1982 Pollen morphology and taxonomy of the tribe Indigofereae (Leguminosae- Papilionoideae). Pollen Spores, 24 171--210. Fischer, H , 1890. Beitrage zur vergleichenden Morphologie der Pollenkorner Thesis, Breslau, 72 pp. Funk, V.A and Stuessy, T., 1978. Cladistics for the practicing plant taxonomist. Syst_ B o t , 3 159--178 Funk, V.A. and Wagner, W.H., 1982 A bibliography of botanical cladlstics I 1981. Brittonia, 34. 118--124. Fntzsche, J., 1837. Ueber den pollen. M~moires des Savants Etrangers, St Petersburg, 3 649--769 Henmg, W., 1965. Phylogenetm Systematms. Annu. Rev Entomol., 10 97--116. Hennig, W_, 1966. Phylogenetic Systematics. Urbana, Chicago and London, 263 pp Heslop-Harrison, J., 1979. An interpretation of the hydrodynamics of pollen. Am. J. Bot.,66 737--743. Jarzen, D.M., 1977. Aquilapollenztes and some Santalalean genera. Grana, 16" 29--39 Linskens, H.F. and Mulleneers, J M.L., 1967. Formation of instant pollen tubes. Acta Bot Neerl., 1 6 132--142. Mohl, H yon, 1835. Sur la structure et les formes des grains de pollen. Ann. Scl Nat., Ser. 2, 3 148--180. Muller, J., 1979. Form and function in angiosperm pollen grains. Ann. Mo Bot. G a r d , 66 593--632 Payne, W.W , 1972. Observations of harmomegathy in pollen of Anthophyta Grana, 12. 93--98. Payne, W.W., 1981 Structure and function m angiosperm pollen wall evolution. Rev Palaeobot Palynol., 35 39--59. Rouse, G , 1957. The apphcation of a new nomenclatural system to upper Cretaceous plant microfossils from Western Canada Can. J. Bot., 3 5 349--375 Rowley, J.R., 1981. Pollen wall characters with emphasis upon applicability. Nordic J B o t , 1 357--380. Schtirhoff, P N , 1926. Die Zytologie der Blutenpflanzen. Enke-Verlag, Stuttgart Stanley, E.A, 1961. The fossil pollen genus Aquzlapollen~tes Pollen Spores, 3- 329--352 Verlaque, R , 1977. Rapports entre les Valerianaceae, les Morlnaceae et les Dipsacaceae. Bull. Soc. Bot. Fr,, 124' 475--482. Vlnokurova, L.V., 1959. Palynological data on the systematic position of Dipsacaceae and Morinaceae. Probl. Bot., 4 51--67 (in Russian). Vijayaraghavan, M.R. and Sarveshwarl, G S , 1968. Embryology and systematic position of Morzna longifolia Wall. Bot. N o t , 121: 383--402. Ueno, J , 1982 Mathematmal analysis of pollen morphology III. Tetrahedron and its metamorphosis, Triprojectacites. Jap. J. Palynol., 2 8 13--21 (m Japanese) Wodehouse, R.P., 1933. Tertiary pollen. II The oil shales of the Eocene Green River formation. Bull. Torrey Bot. Club, 60 479--524. Wodehouse, R.P., 1935. Pollen Grains, Their Structure, Identifmation and Slgmficance in Medicine McGraw-Hill, New York, N.Y, 514 pp
207-- 226 Elsevmr Sc-ence Publishers B.V , A m s t e r d a m -- Printed in The Netherlands
207
PALYNOLOGY AND SYSTEMATICS OF MORINACEAE
S B L A C K M O R E and M.J. C A N N O N
Department of Botany, British Museum (Natural History), Cromwell Road, London SW7 5BD (Great Br~tatn) (Received April 12, 1983, revised and accepted August 9, 1983)
ABSTRACT Blackmore, S and Cannon, M J., 1983. Palynology and systematics of Morinaceae. Rev. Palaeobot. Palynol., 40 207--226 Pollen grains f r o m all thirteen species of the three genera to be recogn,sed in a forthcoming revis,on of the Morinaceae, Acanthocalyx, Cryptothladza and Morma, have been investigated by light and scanning electron m m r o s c o p y Fresh pollen of M long~folla has also been studied by hght, scanning electron and transmission electron microscopy. Three main pollen types axe described and a key provided for their ldenhflcation. Each pollen t y p e occurs t h r o u g h o u t a genus. F u r t h e r division of two of these pollen types enables groups of species to be recogmsed. F u n c t i o n a l aspects of the pollen are discussed, especmlly their unique pre-germinative processes. A cladistic analysis of pollen characters provides a hypothesis for their evolut,on and supports the classification proposed in the recent revision INTRODUCTION
The Morinaceae have, in the past, usually been regarded as a single genus,
Morina, belonging to the Dipsacaceae. The large and morphologically distinctive pollen grains of Morzna attracted the attention of the earliest palynologists (yon Mohl, 1835; Fritzsche, 1837; Edgeworth, 1877; Fischer, 1890) and, more recently, have been the subject of intensive light microscopical investigations b y Erdtman (1945, 1952, 1960a, 1962, 1963, 1965), and Vinokurova (1959). Vinokurova (1959) considered the Morinaceae to represent a distinct family rather than a genus, a conclusion reached b y Verlaque (1977) and Vljayaraghavan and Sarveshwari (1968) on palynological, embryological and other grounds. The present study was undertaken in conjunction with a monographic revision (Cannon and Cannon, in press) in which the Mormaceae are afforded familial status and three genera axe to be recognised. For the first time the palynology of all valid species has been studied, including that of one new species, and electron microscopy has been extensively employed. The names used in this paper are those which are currently correct but the genera to whmh the taxa will belong after publication of the revision have been indicated in parentheses. Some aspects of pollen function in Morinaceae were 0034-6667/83/$03.00
© 1983 Elsevier Science Publishers B V.
208
investigated which drew attention to their unique pre-germinative processes first described by yon Mohl (1835). Using cladlstlc methods, now well estabhshed in botany (Funk and Stuessy, 1978; Funk and Wagner, 1982), an evolutmnary hypothesis for the Morinaceae and their pollen is presented. TERMINOLOGY
Pollen grains of Cryptothladia and Morina have ectoapertures situated in d o m e d or funnel-shaped sexinous protrusions termed "porus collaris" and "porus mfundlbuliformis" respectively b y Erdtman (1945). These complex structures will be referred to as apertural protrusions in this paper for the sake of linguistic simplicity. Strictly speaking, the apertures are aspidate (sensu Wodehouse, 1933) or aspldoporate (Erdtman, 1947). Each of the aspides contains a vestibulum and in Cryptothlad~a these are broad whilst in Morina they are narrow. Aspidate pollen grains occur scattered through a wide variety of living and fossil plants (Ueno, 1982) and their significance will be considered further in the discussion. MATERIALS AND METHODS
Pollen samples (listed below) were obtained from herbarium specimens and, in the case of Morina longifolia, from cultivated plants. For light and scanning electron microscope investigations of pollen morphology, the pollen was acetolysed (Erdtman, 1960b) and mounted in glycerine jelly or on SEM stubs. Sections of pollen were prepared b y freezing microtomy (Blackmore and Dmkinson, 1981). Ion beam etching (Blackmore and Claugher, 1983) was carried o u t on selected examples. SEM preparations were sputter-coated with gold palladium and examined in a Cambridge Stereoscan 180. For functional studies mature pollen was studied before and after dehiscence using the method of Payne (1972). Mature pollen grains from dehisced anthers and stigma surfaces were rehydrated in water or moisture-saturated air. These grains were studied b y light microscopy b y mounting in immersion oil or in air using a Schott fibre optic cold-light source or b y sputter coatmg and scanning electron microscopy without further treatment. Pollen for transmission electron microscopy was fixed with glutaraldehyde and osmium tetroxide, embedded in araldyte resin, sectioned with a Reichert ultrammrotome, post-stained with a saturated solution of uranyl acetate and Reynold's lead citrate and examined in an AEI 6B microscope. SPECIMENS EXAMINED
All specimens are from the herbarium of the British Museum (Natural History) except those marked (E) from the Royal Botanic Garden, Edinburgh and (PE) from the Institute of Botany, Peking. Collectors names are not available for some of the latter.
209 Morina (Acanthocalyx) alba Hand. Mazz. China: Forrest 28444, Tibet: Ludlow, Sherriff and Taylor 5017. Morina (Acanthocalyx) delavayi Franch. China: Maire 128, McLaren's native collectors 66, Rock 1898 (E) Morina (Acanthocalyx) nepalensis D. Don. China : McLaren's native collectors 73B, Nepal Dhwoj 167 (E), collector? 67 (PE), Polunin, Sykes and Williams 4536 Morina (Cryptothladia) chmensis Pal China: Hao 1002 (PE), Lment 4548, collector9 6129 (PE) Morma (Cryptothladia) chlorantha Diels China: Forrest 21255 (E), Forrest 5777, Forrest 10212 (E), collector? 5482 (PE). Morma (Cryptothladia) kokonorica Hao. China: collector9 6114 (PE), Tibet: Ludlow and Sherriff 8691, Richardson 124. Monna (Cryptothladia)polyphylla Wall. ex DC. Nepal. McCosh 381, Stainton 4325, Stainton, Sykes and Wilhams 2984 Morma (Cryptothladia) parviflora Kar et Kit. U.S.S.R. Konsevyanova s.n Morina (Cryptothladia) sp. nov. Bhutan Ludlow, Sherriff and Hicks 16903, Ludlow, Sherriff and Hicks 19050. Morina (Morina) coulterzana Royle. India: Ludlow and Sherriff 9123, Shernff 7350, Wendelbo 59; Tibet: Ludlow and Sherriff 1828, Ludlow, Shernff and Taylor 5481, U.S.S.R. Vassiljeva 5487. Morma (Mor,na) kokanlca Regl. China: Gnezdillo 186 (PE), U S.S.R : Tilchko 8539, Lmczevski and Rushkova 39. Morma (Monna) Iongifolia Wall. ex. DC. England: fresh material from cultivation; Pakmtan. Abel 78. Mo rma (Monna) pets,ca L. Greece : Guml 6771 ; India: Flemmlng 136, Marten 16, Presco ttDecie s n.; Turkey: Davis, Dodds and Cetik 19068 RESULTS General p o l l e n descrzption Pollen w e a k l y h e t e r o p o l a r , 3 - c o l p o r a t e or 3 - p o r o r a t e , in polar view triangular and in e q u a t o r i a l view a l m o s t cylindrical to r h o m b o i d a l s o m e t i m e s with 3 p r o m i n e n t apertural p r o t r u s i o n s at the e q u a t o r . C o l p o r a t e grains have b r o a d colpl with a c u t e l y t a p e r i n g ends and z o n o r a t e e n d o a p e r t u r e s . In the t w o p o r o r a t e pollen t y p e s the e c t o a p e r t u r e s are circular t o s u b o v a t e pores situated in d o m e d or f u n n e l - s h a p e d sexinous p r o t r u s i o n s . T h e e n d o a p e r t u r e s o f grains w i t h d o m e d p r o t r u s i o n s are z o n o r a t e whereas t h o s e with f u n n e l - s h a p e d protrusions are small, circular pores. All species have s m o o t h a p e r t u r e m e m branes and lack o p e r c u l a e . E x i n e very t h i c k , sexine and n e x i n e usually m o r e or less equal at poles w i t h n e x i n e either increasingly m a r k e d l y in thickness, or absent, t o w a r d s t h e e q u a t o r . A s m o o t h or, less c o m m o n l y , p a r t l y verrucate t e c t u m w i t h few to m a n y m i c r o p e r f o r a t i o n s overlays a s p o n g y or granular i n f r a t e c t u m . I n t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y it can be seen t h a t t h e r e is no f o o t layer a n d t h u s t h e n e x i n e is entirely e n d e x i n o u s . Pollen large, P 1 0 0 - 255 ~m, E 55--165 ~m. KEY TO POLLEN TYPES
1. a. C o l p o r a t e , w i t h o u t p r o m i n e n t apertural protrusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acanthocalyx type
210
b. Pororate, with prominent apertural protrusions . . . . . . . . . . . . . . . . . . 2 2. a. Apertural protrusions domed, endoapertures zonorate, internally hourglass-shaped . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Cryptothladia type b. Apertural protrusions funnel-shaped, endoapertures porate, internal shape similar to external . . . . . . . . . . . . . . . . . . . . . . . . . . . Morina type DESCRIPTIONS OF THE POLLEN TYPES
Acanthocalyx type (Plate II) Tricolporate. Triangular in polar view with the apertures slightly sunken in the rounded angles; elliptic to subrhomboidal in equatorial view. Weakly heteropolar, with one pole slightly narrower and more acutely rounded than the other. Ectoapertures colpate, one third as long as polar ares, broad, ends acutely tapered, margins distinct with wide margo. Endoapertures zonorate, widening greatly in the mesocolpia, margins distinct but less so in the mesocolpia. Exine thick, sexine equal to or as much as three times thicker than nexine, thickest along colpus margins. Tectum smooth with scattered microperforations increasing in number towards the equator; infratectum granular or spongy. Measurements: P 100-(116.8)-127 pm, E 55-(67.5)-75 um, exine 6 pm thick at poles, 12 ~m at colpus margins.
Species Morina (Acanthocalyx ) alba, M. (Acanthocalyx ) delavayt, M. (Acanthocalyx ) nepalensls. Comments Thls pollen type most closely resembles some members of the Dipsacaceae (Erdtman, 1952; Clarke and Jones, 1981) in shape and ectoapertures but differs in exine stratification, endoapertures and the absence of operculae. Erdtman (1945) subdivided the type on the differences in exine thlckness and structure. In the present study these characters were found to be remarkably uniform throughout the three species and no subdividion of the pollen type is considered possible, even with scanning electron microscopy.
Cryptothladia type (Plates III--IV) Tripororate. Triangular at the equator in polar view becoming circular towards the poles. Prolate in equatorial view with domed apertural protrusions ("porus collaris" of Erdtman, 1945). Very weakly to quite distinctly heteropolar, with the apertures of heteropolar grains nearer to one pole than the other and the two poles of different diameters. Internal outline hourglass-shaped in equatorial view. Ectoapertuxes circular to subovate pores, in the domed sexinous protrusions, opening into narrow or wide vestibula; margins distinct. Endoapertures zonorate (Plate I, 2) narrow and parallelsided, margins distinct with very pronounced costae. Exine thick, sexine thicker than nexine or roughly equal at poles, sexine sometimes slightly thickened towards the equator, nexine very markedly thickened in the
211
costae. Tectum smooth with scattered m~croperforatlons, more numerous towards the equator. Infratectum spongy or granular. Measurements: P 93(115.6)-125 tam, E 55-(64.5)-74 tam incluswe of apertural protrusions, E 45-58 tam wxthout protrusions; exme 3--5 tam think at poles, 8--11 pm at equator.
Spectes" Morma (Crypto thladta ) chinensts, M. ( Crypto thladta ) chlorantha, M. ( Cryp tothladza) kokonorica, M. (Cryptothladia) parvlflora, M. (Cryptothladza) polyphylla, M (Cryptothladza) sp. nov. Comments This highly dxstmctive pollen type is readily recognised b y its shape, endoapertures and exine stratification. It may be further subdivided into two groups on details of the apertural protrusions. In Morina (Cryptothladia) chznenszs, M (C. ) chlorantha, M. (C.) parviflora and M. (C. ) kokonorica the sides of the protrusions are rather angular m outline, uniformly thickened along their length but not at their apices, the pores are rounded and relatively large (Plate III). Whereas in M. (Cryptothladta) polyphylla and in a new specms (Plate IV) to be described in the forthcoming revision (Cannon and Cannon, m press) the protrusmns are rounded in outhne and less prominent, the thickening of the sides of the protrusions is reduced from the base towards the apices which are themselves slightly thickened and the pores are relatively small. There are slight differences in exine stratification of the polar areas which appear less granular in light microscopy in M. (Cryptothladia) polyphylla and M. (Cryptothlad~) sp, nov. than in the other specms.
Morina type (Plate V) Tripororate. Circular to subtrlangular in polar view with three large, funnelshaped apertural protrusions at the equator. Prolate to almost cylindrical with protrusions at the equator in equatorial view. Ectoapertures wide pores of uniform or slightly decreasing diameter through the sexinous protrusions. Endoapertures pores, contiguous with the ectoapertures and not zonorate although in SEM sections shallow depressions in the inner face of the nexme are suggestive of vestigial connections between the endopores (Plate I, 3, 4). Exine thick; sexine equal to nexine or slightly thinker than nexine at the poles, reduced abruptly in a distinct subpolar ring in some species; sexine thinner towards equator except for massive development in the apertural protrusions. The nexine is greatly thickened towards the equator and forms costae around the endoapertures. Ornamentation variable, sometimes even within a specimen, tectum smooth with few or many verrucae, sometimes of two distinct sizes and with few to many microperforations which are generally more numerous towards the equator. The largest verrucae are often arranged in t w o parallel rows between the bases of pairs of apertural protrusions. Infratectum granular or spongy. Measurements: P 159-(198)-255/am, E 120-(152)-165 tam inclusive of protrusions (79--120 tam without protrusions). Exine 6 tam thick at poles, 12--15 tam at equator.
212
PLATE I (p. 213) Electron micrographs of Morinaceae exines and apertures (scale lines 20 u m except where stated otherwise) 1 M (Acanthocalyx) alba (Ludlow, Sherriff and Taylor 5017), section showing endoapertures. Note broad zones below mesocolpla 2. M (Cryptothladia) polyphylla (Stamton 4325), section showing narrowly zonorate endoapertures. 3, 4. M (Morma) longzfolia (from fresh material) 3. Section showing porate endoaperture and faint depression interpreted as a veshge of the zonorate condition. 4. Section through an apertural protrusion in equatorial plane showing much thmkened nexine. 5 M_ (Acanthocalyx) alba (collection as 1), section in equatorial plane across a colpus showing granular infratectum. 6, 7, M. (Morma) persica (Davis, Dodds and Cetik 19068). 6. Granular mfratectum exposed by ion beam etching to remove tectum (scale line 2 urn). 7. Unacetolysed TEM section of exine in mesocolpium (scale line 2 urn). PLATE II (p 214)
Acanthocalyx-type pollen (scale lines 20 urn). M. (Acanthocalyx) nepalensis (Polunin, Sykes and Williams 4536) 1. Polar view in high focus. 2. Polar view in optical sechon. 3. Equatorial view at two levels of focus 4. Equatorial view at two levels of focus. 5. Whole pollen grain (SEM). 6. View of mesocolpium (SEM). PLATE III (p. 215)
Cryptothladla-type pollen (scale lines 20 urn) M. (Cryptothladia) kokonor~ca (Ludlow and Sherriff 8691) 1. Equatorial view in high focus 2. Equatorial view in optical section. M (Cryptothladza) chmenszs (Lment 4548). 3. Equatorial view (SEM). 4. Detail of apertural protrusion and ornamentation (SEM). PLATE IV (p 216)
Cryptothlad~a.type pollen (scale lines 20 um) M. (Cryptothladia) sp. nov. (Ludlow, Sherriff and Hicks 19050) 1. Equatorial view at two levels of focus. 2. Equatorial view in optical section. 3. Equatorial view (SEM). 4. Detail of apertural protrusions and ornamentation (SEM) PLATE V (p 217)
Morma-type pollen (scale lines 20 urn): M. (Morina)perszca (Davis, Dodds and Cetik 19068) 1. Equatorial view at two levels of focus. 2. Equatorial view in optical section. M (Morma) coulteriana (Vassiljeva 5478) 3. Equatorial view (SEM). 4. Detail of apertural protrusion and ornamentation (SEM).
213
D e s c r i p t i o n on p. 212
PLATE I
E
[
214
PLATE II
D e s c r i p t i o n o n p 212.
I
1
I
f
215
PLATE III
D e s c r i p t i o n o n p_ 212.
i
J
2
216
PLATE IV
D e s c r i p t i o n o n p 212.
I
217 PLATE
V
D e s c r i p t i o n o n p_ 212 |
218 PLATE
VI
D e s c r i p t i o n on p_ 219
i
3
4
219 PLATE VI (p 218) Fresh M (Morina) longifoha pollen (cultivated origin). 1 Reflected light micrograph of dissected flower showing sttcky stigma and a dehisced anther (scale line 500 t~m). 2. Pollen from undehisced anther mounted in immersion oll (scale hne 50 urn). 3. Pollen mounted in water with pollen-tube-like structures (scale hne 50 urn). 4 Pollen-tube-hke structure produced by pressure exserted on pollen mounted in immersion od (scale line 50 t~m). 5 Stigma with pollen grains (SEM, scale line 100 urn). 6. Pollen-tube-like structure produced by pressure (SEM, scale line 50 urn).
Spectes: Morina (Morina) coulteriana, M. (Morina) kokanica, M. (Morina) longifolia, M. (Monna) persica. Comments A distinctive pollen t y p e similar in general f o r m to t h e Cryptothladia t y p e b u t differing in its m u c h larger size, discrete e n d o a p e r t u r e s and t h e shape o f t h e e q u a t o r i a l protrusions. T h e pollen t y p e can be subdivided into t w o groups o n t h e basis o f o r n a m e n t a t i o n and shape. M. (Morina) coulteriana, M. (Morina) kokanica and M. (Morina) persica have d o u b l e rows or bands of p r o m i n e n t verrucae and are m o r e or less cylindrical in shape, w i t h r o u n d e d ends. M. (Morina) longifolia (Plate VI) is a l m o s t d e v o i d o f v e r r u c a e and is s u b r h o m b o i d a l in shape. It was n o t e d , however, t h a t t h e d e g r e e o f developm e n t o f t h e verrucate o r n a m e n t a t i o n varied considerably f r o m o n e s p e c i m e n t o a n o t h e r , a n d s o m e t i m e s w i t h i n a specimen. T h u s it is s o m e t i m e s difficult t o distinguish b e t w e e n t h e t w o groups. OBSERVATIONS ON FRESH POLLEN Fresh pollen o f M. (Morina) longifolia (Plate VI), t h e o n l y m e m b e r o f t h e f a m i l y f o r which a few live plants were available, was e x a m i n e d using t h e m e t h o d o f P a y n e ( 1 9 7 2 ) to d e t e r m i n e its m e a n s o f h a r m o m e g a t h y . A comparison o f pollen, m o u n t e d in i m m e r s i o n oil, f r o m dehisced and u n d e h i s c e d anthers was made. No differences in shape were d e t e c t e d b e t w e e n the two and n o obvious h a r m o m e g a t h i c m e c h a n i s m s n o t e d . Pollen grains f r o m dehisced anthers and f r o m stigmatic surfaces were r e h y d r a t e d in water o r waters a t u r a t e d air. R e h y d r a t i o n in w a t e r resulted in s u d d e n e x t r u s i o n o f pollentube-like s t r u c t u r e s f r o m each a p e r t u r e . These were p r o d u c e d m o r e slowly, and in a smaller p e r c e n t a g e o f grains, w h e n pollen was placed in a moisturesaturated a t m o s p h e r e . T h e t u b e s q u i c k l y a t t a i n e d a length o f a b o u t 8 0 / a m and t h e n ceased e x t e n d i n g . Similar t u b e s could also b e p r o d u c e d b y light pressure being applied t o individual grains with a fine needle. When grains had b e e n in w a t e r f o r a n u m b e r o f m i n u t e s m a n y o f t h e m r u p t u r e d and t h e entire p o l l e n wall was t h e n seen to c o n t r a c t u m f o r m l y to a b o u t 70% o f its f o r m e r size. Pollen.tube-like structures were n o t p r o d u c e d b y pollen grains o f m a t u r e b u t u n d e h i s c e d anthers n o r f r o m dehisced pollen grains after
220
h x a t m n with glutaraldehyde. Essentially similar structures were, however, produced b y pollen taken from herbarium specimens of M. (Acanthocalyx) alba and M. (Cryptothladia)polyphylla and placed in water. Examination of the pollen-tube-like structures b y SEM proved quite possible and even without specml preparation the structures could be examined without their collapsing. DISCUSSION
Functional interpretatzon The observations on fresh pollen provide interesting information concerning two important aspects of pollen function, namely harmomegathy and germination. The term harmomegathy was introduced b y Wodehouse (1935) for the processes b y which pollen grains are able to adapt to the changes in volume which result from differences in hydration. Morphological features which permitted the accommodation of volume changes he called harmomegathm. Payne (1972, 1981) and Muller (1979) have drawn attention to numerous harmomegathic mechanisms in a wide varmty of flowering plants. In the Lactuceae (Blackmore, 1982a) harmomegathlc mechamsms were shown to be of considerable adaptive significance and presumed to be an Important aspect of pollen-morphological evolution. Harmomegathic mechamsms fall into three main categorms. Firstly, the apertures frequently act as harmomegathia in addition to providing a site for the exit of the pollen tube. Secondly, regions of the pollen surface other than the apertures may be capable of flexibility, often because they axe thinner than the surrounding, more rigid, parts of the wall. Thirdly, sporopollenin has an inherent elasticity which allows exines to accommodate hmited changes in volume. However, as Muller (1979) pointed out, th~s elasticity is generally insufficient to accommodate all the volume changes necessitated and thus harmomegathia of the other two kinds are also usually present. Muller drew attention to the important relationship between surface area and volume in a discussion of what he termed the "Wodehouse effect" (Muller, 1979, p.613). He pointed out that distinctly cylindrical or prolate pollen grains have an increased surface area compared to spherical pollen grains of the same volume. This enables them to achieve a given percentage volume reduction b y means of a smaller change in equatorial diameter than is necessary in the case of spherical pollen. The markedly prolate shape of pollen of the Cryptothladia-type and Monna-type reduces the requirement for harmomegathic structures, such as colpi, which would enable drastm changes in equatorial diameter. Imtial observations on M. (Morina) longzfolia pollen, using the method of Payne (1972), failed to reveal any obvious harmomegathia. However, it was observed that if the pollen wall is ruptured either by the uptake of excess water or physically, it shrinks dramatically over its entire surface to about 70% of its original size. This marked shrinkage appears to be the key to
221
harmomegathy in Morinaceae and is interpreted as implying sufficient elasticity for the wall to accommodate substantial changes in volume simply b y shrinkage or stretching. A comparison was made with pollen from five species of D,psacus, Knautia and Scabiosa from the Dipsacaceae. When treated m the same way these ruptured b u t the pollen walls did not shrink. This suggests that Morinaceae pollen walls are in some way different from those of the Dipsacaceae and, m fact, most other flowering plants. The main morphological difference between the walls of Morinaceae and Dipsacaceae pollen is the granular rather than columellate infratectum which seems unlikely to account for the differences in physical properties. It may be speculated that a difference in the chemical nature of the walls would provide an explanation although no attempt has been made to demonstrate such a difference. Exines do vary in their physmal properties, including elasticity. Clarke and Jones (1981) noted that acetolysed Dlpsacaceae exines increased considerably in size over a period of months and years when m o u n t e d in glycerine jelly. No comparable data are yet available for the Monnaceae but the phenomena of unusual elasticity and a gradual swelling in mounting medm may be related and indicate that the pollen walls of at least some members of the Dipsacales are in some way unusual. An interesting feature of this method of harmomegathy is that the overall shape of the pollen grains remains stable. Various other specialised systems of harmomegathy share this attribute including those of the lophate pollen of Compositae (Bolick, 1978; Blackmore, 1982a) and of many pantoporate pollen grains (Muller, 1979). Systems of harmomegathy in which the overall shape remains stable may possess the adaptive advantage of being better suited to enduring the successive dehydration and rehydration which may occur during polhnation (Heslop-Harrison, 1979), perhaps because they are not subjected to repeated flexing. That the walls of the pollen-tube-like structures are composed of ratine may be seen b y transmission electron microscopy and had been illustrated by Erdtman (1960a, 1962, 1963) using ultraviolet microscopy. Linskens and Mulleneers (1967) reported the production of "instant pollen tubes" in pollen of Petunia and other plants. The p h e n o m e n o n found in Morinaceae pollen appears significantly different in that the pollen-tube-like structures may be produced simply b y physical pressure or b y hydration of the pollen. It is presumed that at least one of the tubes may elongate to become a normal pollen tube and effect fertilization. They do not, however, only appear during the germination of the pollen and their production is consequently regarded here as a pre-germinative process. Several possible functions may be suggested for this pre-germmative process which await experimental investigation. They may, for example, be exserted as a final part of the harmomegathic process to prevent rupture of the pollen wall. Alternatively t h e y may increase the surface area of ratine exposed on the stigma surface and either achieve closer adhesion or permit some form of chemical interaction between pollen and stigma.
222
Palynology and classification Erdtman (1945) pointed out that in Monna sensu lato the two sections A canthocalyx and Diotocalyx could be distinguished by their different pollen morphologies. He suggested that further study of the plants themselves might support the exclusion of Morina sensu lato from the Dipsacaceae. This was indeed the conclusion reached by Vinokurova (1959) and, more recently, Verlaque (1977). In a recent monograph (Cannon and Cannon, in press), the Mormaceae are recognised as a distinct family with three genera and thirteen species. In the monograph section Acanthocalyx is given generic status and section Dtotocalyx is divided into the two genera Cryptothladia and Morina. These decisions were based on a close accord between data from floral morphology, embryology, palynology and other characters. The three pollen types are each found throughout all members of a single genus. The subdivisions of the pollen types suggest infrageneric relationships but these are not borne out by the other lines of evidence. For example, two groups of species within Cryptothladia are indicated by pollen morphology: M. (Cryptothladia)polyphylla and M. (Cryptothladia) sp. nov. in one and M. (Cryptothladia) chinensis, M. (Cryptothladia) chlorantha, M. (Cryptothladta) parvzflora and M (Cryptothladia) kokonor~ca in the other. However, at the macromorpholog~cal level there is no evidence for the existence of two such groups and no suggestion that M (Cryptothladm) polyphylla and the new specms are more closely related to each other than to any of the other specxes. Fossil pollen from the form genus Aquilapollenites has been associated with the Dlpsacaceae (Rouse, 1957) because it also has distinctive apertural protrusions which Rouse interpreted as being air bladders. The dipsacaceous pollen implicated can only be that of the Cryptothladia-type or the Morinatype, now of the Morinaceae. It is perhaps not surprising t h a t the occurrence of such morphologically distinctive pollen grains should be interpreted as an indication of affinity. However, as Stanley (1961), Erdtman (1971) and Jarzen (1977) pointed out, Aqudapollenites more closely resembles certain genera of the Santalales and, whatever their true affinities, a relationship with the Morinaceae can be discounted. In this respect it should also be noted that Aquilapollenites does not have a granular infratectum. Ueno (1982) has postulated that the basic tetrahedral shape of the developing microspores when united in tetrads has been modified to produce pollen grains with apertural protrusmns in such diverse living genera as Medusagyne and Protea and in the fossil form genera of the Triprojectacites group, including AquilapoUenites. Ueno implies, in effect, that the occurrence of apertural protrusions in such diverse taxa is a consequence of similarities in ontogeny rather than of relationship. Rowley (1981) has demonstrated in the origin of apertural protrusions of certain Proteaceae a mechanism which m a y also occur in other taxa. Rowley f o u n d that protrusions are present in the tetrad stages of virtually all Proteaceae but are retained at m a t u r i t y only in those cases where oncoid plugs prevent the extension of the developing cytoplasm into the
223
protrusion. It may be that all pollen grains with apertural protrusions have a similar ontogeny and perhaps that functional advantages have led to their convergent evolution in a variety of taxa.
Cladisttc analys~s The cladistlc methods originated b y Hennig (1965, 1966) have now been used in numerous botanical studies. Funk and Stuessy (1978) have reviewed the methods and Funk and Wagner (1982) the growing literature of the subject. Blackmore (1982b) used a cladlstic analysis of palynological characters to provide an evolutionary hypothesis for one subtribe of the Compositae. Here a cladistic analysis of the pollen characters of the Mormaceae is used to present a branching diagram, or cladogram, showing the evolutionary relationships within the family as indicated b y their pollen. In essence the method consists of interpreting as either primitive or derived the character states of the taxon and its outgroup. A cladogram is then constructed which shows the distribution of derived characters in the simplest way, that is with the least invocation of parallelism, convergence or reversal of trends. Table I shows the primitive and derived character states of the pollen characters used and their distribution in the genera of Morinaceae and their closest relatives, the Dipsacaceae. Some of these characters and their interpretation require discussion whilst others are self ewdent. The nuclear condition of the pollen at anthesis (character 1) is a useful, although often neglected (Blackmore, in press), character. Neither the Dipsacaceae nor Morinaceae have been extensively surveyed and only the limited data given b y Brewbaker (1967) and Vijayaraghavan and Sarveshwari (1968) are presented here. In accordance with the conclusions of Schfirhoff (1926) and Brewbaker (1967), trinucleate pollen is interpreted as being derived. TABLE
I
Primitive (--) and derived (+) character states used in the construction of the cladogram (Fig. I ), and their distribution in the Dipsacaceae (D), A canthocalyx (A), Crypto thladia (C) and
Morma
(M)
Primitive state
state
D
A
C
M
trinucleate
+
--
--
--
present
+
--
--
--
granular structures present
---
+ +
+ +
+ +
Derived
1
binucleate
2
operculae
3 4
infratectum columellate no pollen-tube-hke structures
absent
5
endoapertures
free
zonorate
6
endoaperLures
zonorate
secondarily
7
isopolar
8
narrow
9
apertural
10
--
+
+
--
--
-
--
+
heteropolar
--
+
+
+
in mesocolpla
--
+
--
--
--
--
+
+
--
--
+
+
constricted
--
--
+
--
present
--
--
--
+
weakly endoapertures protrusions
subspheroidal
11
internally
12
verrucae absent
unconstricted
broad absent
free
present prolate
to perprolate
224
The difference b e t w e e n granular and columellate infratectum (character 3) is very distinct b u t it is difficult to ascertain which is the derived state. Granular infratecta may be primitive in certain families with thin-walled, winddispersed pollen grains (Doyle, 1978 ; Muller, 1979). In other taxa including, for example, tribe Indigofereae of the Papilionoideae (Ferguson and Strachan, 1982) granular sexines are clearly a derived condition and they are interpreted as being so in the Morinaceae. However, as with characters 2 and 4, the opposite interpretation of the trend would not, in fact, alter the branching of the cladogram. The evolution of endoapertures in the Morinaceae (characters 5 and 6) is more complex. The hypothesis adopted here is that in the primitive condition the endoapertures are unfused, as in the Dlpsacaceae, that the zonorate condition In pollen of Acanthocalyx and Cryptothladia is derived but that the genus Morina has secondarily unfused endoapertures. The presence of vestiges of the zonorate condition, in the form of slight thinnings in the nexine b e t w e e n the endoapertures, in Morina, is taken as evidence in support of this. These thinnings are most clearly seen in sectioned pollen examined by scanning electron microscopy (Plate I). The very broad endoapertures in the mesocolpia of Acanthocalyx pollen (character 8) are interpreted as being derived from the narrow zonorate endoapertures which would presumably result if the lalongate endocolpi of the Dipsacaceae were extended until they anastomosed. Heteropolar pollen (character 9) is interpreted as being derived in the Morinaceae and it is thought that it may have arisen as a consequence of the large size of the pollen grains and the complexities, due to shape, of their arrangement in tetrads during development. Heteropolarity is discernible in all Morinaceae b u t is generally more marked in pollen grains of Cryptothladia and Morina where the apertures are nearer to one pole than the other and one pole, presumably the proximal pole in the tetrad, is more acutely tapered than the other. The cladogram (Fig.l) shows that the pollen characters used support the familial status of the Morinaceae. Acanthocalyx is shown to be more closely related to the Dipsacaceae than are either Cryptothladia or Morina and the latter two genera to be more closely related to each other than to Acanthocalyx. CONCLUSIONS
Morinaceae pollen is morphologically distinct from that of the Dipsacaceae m its lack of columellae and echinulate opeculae and in the presence of zonorate endoapertures in some of the pollen types. The Morinaceae also differ in having unusually elastic pollen walls and in producing pollen-tubelike structures as a unique pre-germinative process. Each genus has a single pollen t y p e and these exhibit relatively little variation. Acanthocalyx pollen shows the closest relationship to the Dipsacaceae. Cryptothladia and Morina, which b o t h have apertural protrusions, are closely related to each other and are more distinct from the Dipsacaceae than Acanthocalyx is.
225
D
A
C
M
v~3,4,5,7 ~
9,10
Fig.1. Cladogram showing the evolutionary relationships of the Dipsacaceae (D), Acanthocalyx (A), Cryptothladia (C) and Morina (M) based on the palynological data in Table I. ACKNOWLEDGEMENTS We are g r a t e f u l t o J . F . M . C a n n o n a n d J.M. P e t t l t t f o r r e a d i n g a n d c o m m e n t ing on the manuscript. REFERENCES Blackmore, S., 1982a. A functional interpretation of Lactuceae (Compositae) pollen. Plant Syst Evol., 141. 153--168. Blackmore, S., 1982b. Palynology of subtribe Scorzonerinae (Compositae Lactuceae) and its taxonomic implications. Grana, 21: 153--168. Blackmore, S., in press. Pollen characters and plant systematics. In'. V.H. Heywood and D.M. Moore (Editors), Current Topics in Plant Taxonomy. Academic Press, London. Blackmore, S. and Dickinson, H.G., 1981. A simple technique for sectioning pollen grains. Pollen Spores, 23 : 281--285. Blackmore, S and Claugher, D., 1983. Ion beam etching in palynology. Grana, 22 (in press) Bolick, M.R., 1978. Taxonomic, evolutionary and functional considerations of Compositae pollen ultrastructure and sculpture. Plant Syst. Evol., 130: 209--218. Brewbaker, J.L., 1967. The distribution and phylogenetic significance of binucleate and trinucleate pollen grains m the Angiosperms. Am. J. Bot,, 54: 1069--1083. Cannon, M.J. and Cannon, J.F.M., A revision of the family Morinaceae (Magnoliophyta/ Dipsacales). Bull. Br. Mus. (Nat. Hist.), Bot Ser., in press. Clarke, G.C.S. and Jones, M.R., 1981. The Northwest European Pollen Flora, 21. Dipsacaceae. Rev. Palaeobot. Palynol., 33 1--26. Doyle, J.A., 1978. Origin of Angiosperms. Annu. Rev. Ecol. Syst., 9. 365--392. Edgeworth, M.P , 1877. Pollen Hardwicke and Bogue, London, 92 pp. Erdtman, G., 1945. Pollen morphology and plant taxonomy. III. Sven. Bot. Tidskr , 39. 187--191. Erdtman, G , 1947. Suggestions for the classification of fosml and recent pollen grams and spores. Sven. Bot Tldskr., 41. 104--114. Erdtman, G., 1952. Pollen Morphology and Plant Taxonomy Angiosperms. Almqwst and Wiksell, Stockholm, 539 pp Erdtman, G., 1960a. Notes on the finer structure of some pollen grains. Bot. Not., 113. 285--288.
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