STRUCTURAL, BIOCHEMICAL AND GEOGRAPHICAL RELATIONSHIPS IN

AUSTRALIAN c4 GRASSES (POACEAE) •

by

H.D.V. PRENDERGAST

A thesis submitted for the degree of Doctor of Philosophy

of the Australian National University.

January 1987.

Canberra, Australia. i

STATEMENT

This thesis describes my own work which included collaboration with Dr

N .. E. Stone (Taxonomy Unit, R .. S .. B.S .. ), whose expertise in enzyme assays enabled me to obtain comparative information on enzyme activities reported in Chapters 3, 5 and 7; and with Mr M.. Lazarides (Australian

National Herbarium, c .. s .. r .. R .. O .. ), whose as yet unpublished taxonomic views on Eragrostis form the basis of some of the discussion in Chapter 3. ii

This thesis describes the results of research work carried

out in the Taxonomy Unit, Research School of Biological

Sciences, The Australian National University during the

tenure of an A.N.U. Postgraduate Scholarship. iii

ACKNOWLEDGEMENTS

My time in the Taxonomy Unit has been a happy one: I could not have asked for better supervision for my project or for a more congenial atmosphere in which to work. To Dr. Paul Hattersley, for his help, advice, encouragement and friendship, I owe a lot more than can be said in just a few words: but, Paul, thanks very much! To Mr. Les Watson I owe as much for his own support and guidance, and for many discussions on things often psittacaceous as well as graminaceous!

Dr. Nancy Stone was a kind teacher in many days of enzyme assays and Chris Frylink a great help and friend both in and out of the lab ••

Further thanks go to Mike Lazarides (Australian National Herbarium, c.s.I.R.O.) for identifying many grass specimens and for unpublished data on infrageneric groups in Eragrostis; Dr. John Busby (Bureau of

Flora and Fauna, Canberra) for help and encouragement in the use of the

Bioclima te Prediction Sy stem; Drs. David Coates and David Shaw

(R.S.B.S.) for advice on cytological techniques; Drs. J.N. Burnell and

M.D. Hatch (Division of Plant Industry, C.S.I.R.o., Csnberra) for discussions on enzyme assays; H.A. Nix (Division of Water and Land

Resources, C.S.I.R.O., Csnberra) for discussions and permission to reproduce in part a published figure; Ross Cunningham (Statistics, The

Faculties) for statistical advice; Jo Pask (Taxonomy Unit) for help with electron microscopy; Vindhya Amarasinghe (Taxonomy Unit) for information on microhair types in Eragrostis; the staff of the Electron Microscopy

Unit and of the Plant Culture, Photography and Illustration Sections

(R.S.B.S.); Elizabeth Robertson and Gillian Hines (Taxonomy Unit) for a lot of demanding typing; and Barbara Piper for putting it all together.

I am grateful for the award of an A.N.U. Postgraduate Scholarship and thanks again to Paul, Les, Nancy and Chris. iv

ABSTRACT

I have studied aspects of the biology of Australian grasses

(Poaceae) in relation to photosynthetic pathway.

In a cytological investigation of interspecific relationships in

the photosynthetically variable tribe Neurachneae (subfamily

Panicoideae), the chromosomes of all species (C 3, c 3-c4 , c 4 l were found

to be small (x= 2 µm) and either metacentric or submetacentric. The base number (x= 9) is typical for panicoid grasses; tetraploidy is the most common of the three ploidy levels (2x, 4x, 6x). Using the

Bioclimate Prediction System, climatic profiles were compiled for each species anc: actual lnd predicted geographical distributions were mapped.

In conjunction Jith selective biochemical sampling, a survey of c.

370 c 4 species was then undertaken to document them for leaf blade structural ueed as predictors of the three C 4 acid decarboxylat~on types Jf the c 4 pathway. In the largest Australian c 4 grass genus, Eragro.9tis (Chloridoideae), some species have features previously associated with the PEP carboxykinase type (PCK), but their main decarbo;:ylation enzyme is NAD-malic enzyme (NAD-ME) as in species with typical ('classical') NAD-ME anatomy. An intermediate structure is also described.

Other NAD-ME species with broadly 'PCK-like' anatomy were found in

Enneapogon, Triodia, Triraphis (Chloridoideae) and Paniewn

(Panicoideaej. ALLoteropsis semialata (Panicoideae) is the first recorded PCK species with NADP-malic enzyme-like (NADP-ME) anatomy, and

Eriaehne and Pheidoehloa (Arundinoideae) contain the first known NADP-ME species with anatomy like that of 'classical' NAD-ME or PCK species. A schematic summary of all currently known structural/biochemical associations in grasses is presented, and possible physiological implications with regard to the maintenance of a high [C0 2 J in PCR v

(Photosynthetic Carbon Reduction, or Kranz) tissue are discussed.

Chlorophyll a:b ratios from all biochemical experiments were statistically analysed. Means (and standard errors of the mean) for each c4 type are: NADP-ME, 4.45 (±0.04); NAD-ME, 4.01 (±0.03); PCK,

3.45 (±0.02). The differences between the means are statistically significant, even in cornpari sons across major taxonomic and/or anatomical groups.

Cynoahloris m:iaivorii and C. reynoldsensis (Chloridoideae) are unusual examples of spontaneous intergeneric hybrids between parental species of different c4 types and leaf blade structures. Both were found to be structurally and biochemically intermediate between the 'iAD­

ME parent Cynodon daatylon and the PCK Chloris parent( s). Controlled, recriprocal crossing experiments between Cynodon and Chloris couC.,i explore genetic relationships between c4 types.

Es ti mates were made of the total number of native Australian r: 4 grass species of each c4 type: NADP-ME, 348 species; NAD-ME, ;95 species; PCK, 65 species. All three types are most numerous in the mega therm seasonal bioclima te of northern Q.leensland. NADP-ME species dominate, species number-wise, in 48 out of 73 State and Territory subdivisions, with NAD-ME species dominant in the remainder (and codominant in two). NAD-ME species are proportionally at their most numerous in the megatherm/mesotherm arid bioclimate. The extent of the mega therm seasonal (rainfall) bioclimate is paralleled by :.r:e di stri bu ti on of most PCK species, by Eragrostis species with centrifugal/peripheral PCR cell chloroplasts, and by a relatively high proportion of species of all c 4 types with a suberized lamella in their PCR cell walls. The physiological reasons for these correlations are unknown. Taxonomic, ecological and historical factors in relation to c 4 type distribution are discussed. vi

PRESENTATION OF THESIS

Chapters are presented in the order in which they were written during the course of my studentship. Chapter 1 is a discursive overview of the thesis which is meant to convey chronological and personal aspects of its development. Each chapter has been prepared in the format of a paper for a scientific journal, complete with its own introduction and discussion, the latter also incorporating ideas for future research. Chapters 2 and 3 have, in fact, already been published and Chapter 4 has been accepted for publication. Subsequent to original publication of Chapter 2, new data became available as a result of a field-trip and these are incorporated into the text, tables and figures of the version presented here. Chapters 3 and 4, however, remain virtually unchanged from their published or accepted versions. The size of many tables has meant that photoreduction (from original A3 page size) was necessary. Tables and figures (and appendices in Chapter 2) are pooled at the end of each chapter (in that order), and references for all chapters are pooled at the end of the thesis. The format of presentation follows that of the C.S.I.R.O. journals. CONTENTS

PAGE

CHAPTER 1 OVERVIEW

CHAPTER 2 DISTRIBUTION AND CYTOLOGY OF AUSTRALIAN NEURACHNE

AND ITS ALLIES (POACEAE), A GROUP CONTAINING , c3 c4

AND C3-C4 INTERMEDIATE SPECIES.

Abstract 10

Introduction 1 1

Materials and Methods 12

Results 14

Discussion 18

CHAPTER 3 ACID DECARBJXYIATION TYPE IN (POACEAE): c4 ERAGROS'rIS

PATTERNS OF VARIATION IN CIILOROPIAST POSITION,

l!]LTRASTRUCTURE AND GEDGRAPHICAL DISTRIBUTION.

Abstract 24

Introduction 25

Materials and Methods 28

Results and Discussion 31

Concluding Remarks 38

CHAPTER 4 AUSTRALIAN C4 GRASSES (POACEAE): LEAF BLADE ANATOMICAL

FEATURES IN RELATION TO ACID DECAROOXYIATION TYPES. c4

Abstract 42

Introduction 43

Materials and Methods 45

Results 46

Discussion 49 CHAPTER 5 NEii STRUCTURAL/BIOCHEMICAL ASSOCIATIONS IN LEAF BLADES OF

c4 GRASSES (POACEAE). Abstract 56

Introduction 57

Materials and Methods 58

Results 59

Discussion 63

CHAPTER 6 WHOLE LEAF BLADE CllLOROPHYLL A:B RATIOS IN C4 GRASSES

(POACEAE).

Abstract 72

Introduction 73

Materials and Methods 74

Results 76

Discussion 77

CHAPTER 7 LEAF BLADE STRUCTURE AND C4 ACID DECAROOXYIATION

ENZYMES IN CYNOCHWRIS SPP. (POACEAE), INTERGENERIC

HmRIDS BETWEEN PARENTS OF DIFFERENT c 4 TYPES.

Abstract 81

Introduction 82

Materials and Methods 83

Results 85

Discussion 87 CHAPTER 8 GIDGRAPHICAL DIS'rRIBOTION OF C ACID DECAROOXYLATION 4

TYPES AND ASSOCIATED S'rRcx::TORAL VARIANTS IN AUS'rRALIAN

GRASSES (POACEAE).

Abstract 89

Introduction 90

Methods and Sources of Data 92

Results 95

Discussion 107

REFERENCES 11 3 CHAPTER 1

OVERVIEW 1

I initially set out to address taxonomic and ecological aspects of

variation in photosynthetic pathways via a cytogeographical analysis of

the photosynthetically variable, endemic tribe Neurachneae (Chapter

2). I had hoped that cytological indications of the detailed

interrelationships between the very closely related c , c and c -c 3 4 3 4 intermediate species might emerge, but it soon became clear that there

was no future in this approach. The Neurachneae may well provide

important clues to the evolution of c 4 photosynthesis but this will

probably have to be pursued at the molecular level. I did, however,

contribute to the small body of information on karyotypes of Australian

grasses and, more interestingly, also highlighted the geographical

distributions of the species of Neurachneae which are unusual in

relation to photosynthetic pathways (see also Chapter 8) as well as

peculiar in a taxonomic context. Appendix 2. 2 includes probably the

most precise data yet available on the climatic range of any c 4 species and this phase of my work also represented one of the first practical

applications to plants of the Bioclima te Prediction System (BIOCLIM).

The distributions it predicted for species in terms of climatic

suitability may not always be very meaningful, however, since some of

the more recently discovered localities lie well outside the predicted

distributions. Two problems associated with the input data were the

difficulty in estimating the elevation of collection localities, because

these are often inexactly specified on herbarium specimens, and the lack of contours and spot heights on many large-scale maps, particularly

those of remote areas. Furthermore, the climate of the arid and semi-

arid zones, where most Neurachneae species grow, is relatively uniform

so the fact that a given species does not occupy large parts of these 2

zones suggests ipso faato that its distribution is not limited by present climatic parameters. Other factors, such as specific habitat requirements (which I have also documented), are probably involved.

BIOCLIM has been successfully applied to analyses of the geographic distribution of Nothofagus aunninghamii in south-eastern Australia

(Busby 1986), of Australian elapid snakes (Nix 1986) and as a guide to species selection by foresters (Booth 1985), but experience with the

Neurachneae suggests that it will not always be usefully informative.

Numerically, ecologically and economically c4 grass species are an important component of the Australian flora. There are about 600 native species alone (see Chapters 4 and 8) and they include some that collectively dominate the vegetation over about 25% of the entire continental area (see Chapter 8). Along with many introduced c 4 species, they support a livestock industry that still makes a major

1 contribution to Australia s economy. Other introduced c 4 species are valuable agricultural crops. Whilst some of these, including maize and sugarcane (in which the c 4 photosynthetic pathway was originally discovered), continue to be intensively studied, native c 4 species generally receive little attention.

By October 1984 my attention had shifted from the Neurachneae to other native grasses, especially c4 species in the arid and semi-arid zones. Mechanisms of survival in, and adaptation to, an area covering about 80% of Australia are important topics of applied research on a broad front. In trying to elucidate the adaptive significance and origins of variations in photosynthetic pathways, I undertook to study the interrelationships of diverse features including leaf blade structure, photosynthetic enzymes, climatic range and geographical di stribu ti on. A broad approach seemed from the start to offer several 3

benefits. It might be expected to unravel large-scale trends or correlations that would otherwise remain concealed, and in doing so may focus attention on features of adaptive significance and on the underlying mechanisms; it might pinpoint species particularly suitable as biochemical or physiological experimental material and might even provide clues concerning the evolution of the c 4 photosynthetic pathway of a kind that will possibly ultimately lead to the biotechnological transfer of the c syndrome to c species. 4 3

Biochemical variations of the c 4 pathway seemed likely to have adaptive significance. In contrast to the large number of studies on the distributions of and grasses (listed by Hattersley 1983), t.,e c3 c4 only equivalent analyses for c 4 types have been those of Ellis et al. ( 1980) and Vogel et al. ( 1986), which purported to show t.,at geographical distributions of c 4 types in i) South West i\frica/Namibia and ii) the Sinai, Negev and Judean Deserts respectively are correlated with differing amounts of annual rainfall. My project was therefore orientated towards finding out whether a similar situation exists in i\ustralia, and with the intention of pursuing the matter in more depth than I have finally done (Chapter 8) because of necessary diversions from this main theme as a result of the significant and unexpected findings reported in Chapters 3 and s.

In October and December 1984 I made field-trips to collect a wide range of arid and semi-arid zone grasses, which were then referred to c4 types on the basis of anatomy. The integrity and scope of the associations between leaf blade anatomy and c 4 type had not been critically investigated since their initial characterization in the mid

1970s, and the implications of this became apparent in connection with the first genus I examined, Eragrostis, the largest c 4 genus in 4

Australia (Chapter 3). Considering the identificatory and taxonomic problems posed, it was fortunate that Eragrostis was in the process of being revised at the Australian National Herbarium by M. Lazarides. The structural/biochemical associations which emerged for Eragrostis

(Chapter 3) showed that c 4 type is not always reliably predictable from the structural criteria previously employed and that in Eragrostis, at least, it is leaf blade structure rather than c 4 type which apparently correlates with climate and geographical distribution. These results also led to reassessments vis-a-vis the efficiency of the Kranz bundle sheath (including the presence there of a suberized lamella) in maintaining a high [C0 2 J at the site of ribulose-1,5-biphosphate carboxylase/oxygenase, a key physiological feature of the c syndrome 4 (also discussed in Chapter 5). Some Eragrostis species have structural features intermediate between those of the two main anatomical types found in the genus, a discovery which will be of interest in future studies on evolutionary and genetic relationships between c 4 grasses with different structural/biochemical suites, an aspect of c types that 4 has been almost neglected (but see Brown 1977). One previously unreported feature in grasses (but see Laetsch 1968 for comments on bundle sheath chloroplasts in c 4 dicotyledons), and whose significance is quite unknown, is the difference in shape between centripetal and centrifugal chloroplasts in Kranz cells. The first complete nucleotide sequences of chloroplast DNA (in a liverwort Marchantia poLymorpha and tobacco Nicotiana tabacum) have already been determined (Ohyama et aL.

1 986; Shinozaki et aL. 1986), so it may not be long, therefore, before the genetic relationships between these chloroplast variations (and, for that matter, those between the mesophyll and bundle sheath chloroplasts across the entire structural, biochemical and taxonomic spectrum of c4 5

grasses) can be fully explored.

By July 1985 I had examined and documented the leaf blade structure of most native c 4 species in the subfamilies Arundinoideae and Chloridoideae, and of considerable numbers from the panicoid supertribe

Panicanae (Chapter 4; all taxa sensu Watson et aZ. 1985). It was somewhat disappointing to find neither any intrageneric variation in photosynthetic pathway, as in Neuraahne (Hattersley et al.

1982) and southern African AUoteropsis and Eragrostis (Ellis 1974a,

1984), nor any intraspecific variation for structure or c 4 type (a possible exception being Miaroahloa indiaa (L. ) Beauv. ) • Originally I had also intended to look at the leaf blade structure of large

Australian genera such as Danthonia and Stipa, which are ostensibly totally c3 but have yet to be exhaustively examined. The structural survey was a prerequisite (along with biochemical studies) for my

'.'nalysis of the geographical distribution in Australia of the three c 4 types (Chapter 8) • The structural data may prove useful for experimentalists comparing the physiological significance of variations in the leaf blade structures of c 4 grasses and, as I point out (in

Chapter 4), they also seem to have some taxonomic value. For many species, particularly those in far ~orthern Australia, I had been unable to collect specimens in the course of my field-trips and so I made considerable use of herbarium material from the Australian National

Her barium. With increasing experience I was able to document the leaf blade structure of these species even when the material was in poor condition.

A problem left unsatisfactorily resolved by this structural survey concerns the possibility of movement of chloroplasts, particularly centripetal ones, within PCR (Photosynthetic Carbon Reduction, or Kranz) 6

cells. These chloroplasts are usually aligned perpendicular to the inner tangential walls, but how far they extend across the PCR cells towards the outer tangential walls is very variable. They could be described as 'evenly distributed' when they extend right across, but I suspect that this extension is largely a function of the amount of starch accumulated within them. Starch content in turn presumably depends on the quality and quantity of intercepted light, and may not follow a strictly diurnal rhythm. Starch would need to be depleted from

PCR cell chloroplasts prior to studies on their dimensions, which have yet to be systematically studied in c 4 grasses (see Chapter 4). An alternative explanation for chloroplast 'movement' which also needs study could involve water stress: Giles et ai. (1974) noted a centrifugal position for the bundle sheath chloroplasts in well-watered

Zea mays plants but a random distribution in plants from which water had been withheld for seven days. Such a change, however, was not apparent in a similar experiment with Sorghum biooior (Giles et ai. 1976).

In extending the structuraljbiochemi cal/phytogeographical approach from Eragrostis to a wider sample of the Australian c4 grass flora (Chapters 4, 5 and 8), I selected species for biochemical assay of activities of c4 acid decarboxylation enzymes on the basis of their 1 taxonomic position , leaf blade structure and the availability of seed

(mostly collected in the field or taken from field-collected specimens) • The careful selection paid dividends (Chapter 5). It revealed a number of new and totally unexpected structural/biochemical associations, which further reduce the reliability of using structural

The grass classification derived for the Taxonomy Unit's vast accumulation of data (held on the DELTA system's automated database; Watson et ai. 1985; see also Watson and Dallwitz 1985; Watson et ai. 1986) was invaluable as a guide to sampling. 7

features to predict c 4 type (see also Chapter 3), suggested that there is no relationship between activities (rather than just presence) of

specific c 4 acid decarboxylation enzymes and leaf blade structure, and provided more background concerning the role of the suberized lamella

inter alia in reducing co2 leakage out of Kranz cells. The large number of chlorophyll a:b ratio measurements (by far the most comprehensive yet

reported for c4 grasses) obtained during the course of enzyme assays

established the photochemical similarity of species of the same c 4 type irrespective of variation in leaf blade structure (Chapter 6). These

experiments also, incidentally, reconfirmed the reliability of using

structural criteria to distinguish between c3 and c4 grasses.

The structural diversity within c 4 types revealed by this part of my project, and the fact that some characteristics are common to two or

even three of them, raises questions concerning genetic linkage between

leaf blade structure and c 4 type. The two species of Cynochloris, each derived from intergeneric hybridisation between species (of Chloris and

Cynodon) of different structure and c 4 type (the only examples of which I am aware; see e.g. Knobloch 1968) are attractive material for genetic

study in this context. They both occur in southern Q.leensland, where I

collected specimens from their type localities in April 1986. Chapter 7

reports on the structural and biochemical characteristics of these two

hybrids and their parents, and indicates that Chloris and Cynodon could

provide very useful material for studying the inheritance of c 4 type,

leaf blade structure, and bundle sheath chloroplasts in c 4 grasses. These topics are at present completely nnexplored fields of research.

The last chapter (Chapter 8) describes and compares the geographical distributions of grasses of different c 4 types within

Australia. I compiled accurate and up-to-date species lists for all the 8

State and Territory subdivisions, these being the basis upon which most data are available (from herbarial. These data were supplemented with

records from other sources, notably from national parks, collecting expeditions, and ecological surveys. As a result, the number of

subdivisional records of native c4 grass species given here is overall

some 10% higher than in the only previous comparable study (by

Hattersley 1983 who analysed c3 and c4 grass distributions as a whole); for Western Australia the number is more than 40% higher. Distributions of grasses of different c4 types are compared with each other and related to the continent's bioclimatic zones as described by Nix

(1982). This bioclimatic classification, also referred to in the

Eragroatia study (Chapter 3), uses the same cL.:natic database as does the BIOCLIM program (applied to the Neurachneae; Chapter 2). There are interesting correlations of distributions wi t:..'1 the 'Jioclimatic zones involving not only the c4 types themselves but also structural variants within them.

The ecophysiological significance of these biochemical and structural variations seems to me a particularly promising line for future research, especially since there may be agricultural benefits.

Although c 4 plants have a higher water-use efficiency (ratio of transpiration to photosynthesis) than c 3 plants (e.g. Pearcy and

Ehleringer 1984), this is not necessarily related either to a greater drought tolerance or to an ability to with stand low water-potential

(e.g. Ludlow 1976). The phytogeographical data presented in Chapters 3 and 8, particularly the contrast between the c 4 grass flora of the arid and semi-arid zones with that of more humid areas, nonetheless suggest that there are consistent differences within c4 grasses in some aspect of water relations. It would be particularly interesting to have paired 9

physiological comparisons between closely related taxa, on the one hand of different c 4 type and leaf blade structure (e.g. in LeptoohLoa,

SporoboLus), on the other of the same c4 type but different structure (e.g. in 6ragrostis, Paniown).

In the event I have been obliged to put much more effort than anticipated into identifying and anatomically characterising c 4 types, and less into pursuing their adaptive significance. The mystery surrounding their origins remains and is intrinsically linked with that of the evolution of the c 4 syndrome as a whole within the grass family. In depth studies of intermediates, e.g. c 3-c4 Neuraohne minor (Chapter 2), PCK/NAD-ME Bouteloua ourtipendula (Chapter 5) and PCK/NAD­

ME CynoohLoris spp. (Chapter 7) may help resolve this. The outcome of the next logical step, the application of the biotechnology of gene transfer to the photosynthetic apparatus of grasses, remains to be seen. CHAPTER 2

DisrRIBUTION AND CYTOLOGY OF AUSTRALIAN NEURACHNE AND ITS ALLIES

(POACEA.E), A GROUP CONTAINING C3• c 4 AND c 3-c4 INTERMEDIATE SPECIES.

PUblished in Australian Journal of Botany, 1985, 33, 317-36.

H.D.v. Prendergast, P.W. Hattersley.

Manuscript received 21 August 1984, accepted 16 November 1984. 10

Abstraet

Cytological, phytogeog raphical and habitat data are presented for the

Neurachneae (Poaceae), a tribe endemic to Australia and containing seven c , two c and one c -c intermediate species. Chromosome counts for 34 3 4 3 4 accessions Australia-wide reveal a typical eu-panicoid base number (x =

9). Three species are diploid (Neuraehne tenuifolia c3 , Thyridolepis miteheLLiana c 3 and T. xerophiLa c 3 ); four species oidea and N. munroi ) are heteroploid. Aneuploidy was c3 c4 found in individuals of N. minor (2n = 4x + 1) and N. queensLandiea (2n

= 6x -1). Chromosomes are small {mean c.. 2 JJm) and metacentric or submetacentric. Using localities derived from all known collections in

Australian herbaria, actual and corrpu ter-predi cted distrih1 tions were mapped using the Bioclimate Prediction System (BIOCLIM) developed by

H .. A.. Nix and J. R.. Busby .. Species di stri:tu tions, habitats and chromosome counts are discussed in relation to photosynthetic pathway, present and past climates and evolutionary history.. The Neurachneae are mainly subtropical, arid and semi-arid zone plants .. However, the distrihl tion of their species contrasts with those of other eu­ c3 c3 panicoids and c3 grasses as a whole.. The temperate species N .. is the only native c eu-panicoid known from south-western aLopeeuroidea 3

Australia. It is suggested that phenotypic expression of c 4 photosynthesis in the Neurachneae occurred independently from other grasses and that they did not extend into arid and semi-arid regions from a mesic temperate zone .. 11

Introduction

The eu-panicoid grass tribe Neurachneae (Blake 1972) comprises

three genera. and 10 species (Table 2 .1), all of which are endemic to

Australia. Leaf anatomical studies indicated variation in

photosynthetic pathway not only within the tribe (Hattersley and Watson

1975; Brown 1977) but also within one of the genera, Neuraohne

(Hattersley et aL. 1982; see Table 2.1). c13c value determinations

(Hattersley and Roksandic 1983) and ultrastructural and -gas exchange cc 2 studies (Hattersley et aL. 1984, 1985) generally confirmed the predictions on photosynthetic pathway based on leaf anatomy, and also clearly demonstrated that is a intermediate species. N. minor c 3-c4 Among the angiosperms as a whole, at least 17 genera are now known to contain both and species (review: Raghavendra and Das 1978). c 3 c4 However, despite extensive surveys (Ha ttersley and Watson 1975; Brown

1977; Ellis 1977), Neuraohne is only the third such grass genus to be discovered; and of the other two, only Panioum has previously been shown to contain intermediate species (P. c3-c4 deoipiens, P. miLioides, P. sohenokii: Brown and Brown 1975; Kanai and Kashiwagi 1975; Brown et aL.

1983 and their preceding studies; and reviews by Apel and Peisker 1979,

Raqhavendra 198<1, and Ra thnam and Chollett 1980). Al though and c3 c4 subspecies of ALLoteropsis semial,,ata (Poaceae) are known (Ellis 1974;

Brown 1975; Barrett et al,,. 1983; Frean et al. 1983; Gibbs Russell 1983), no c 3-c4 intermediates have yet been discovered. also been demonstrated, however, or are suspected, in four dicotyledonous genera, viz. Fl,,averia (Asteraceae: Apel and Maas 1 981 ;

Ku et aL. 1983), MoLLugo (Aizoaceae: Kennedy et aL. 1980), Morioandia

( Brassicaceae: Apel et ai. 1978), and Parthenium (Asteraceae: Hegde and Patil 1981; Patil and Hegde 1983). 12

1'he existence of , and intermediate species within c3 c4 c3-c4 genera such as Neuraehne makes them fundamentally interesting with

respect to understanding aspects of the evolution of plants (cf. c4

Brown et al. 1983, and preceding work on Paniewn; Bouton et al. 1981).

Fur the nno re, any future attempts at genetic manipulation of

photosynthetic pathways involving gene exchange would seem more likely

to succeed using closely related species within genera such as Neurachne

and Paniaurn, than with more distantly related grasses.. The Neurachneae

also contain species palatable to livestock, making the group of

economic as well as fundamental interest.

Unlike the unwieldy genus Paniewn (with up to 500 species worldwide, and badly in need of taxonomic revision), NeUPaahne and its

allies Paraneur>aehne and Thyridolepie are a small grouping of indisputably closely related species, with a relatively restricted,

clearly defined geographical di strim ti on. They may therefore be more readily understandable in ecological and evolutionary terms. Detailed information is presented on the basic cytology and geographical distributions of all 10 species, and these data are considered alongside variation in photosynthetic pathway and in relation to past and present climates, in an attempt to uncover a pattern of evolution .. Detailed distrihltions of these closely related species are also discussed in relation to the general climatic regimes of c and c grasses in 3 4

Australia (cf. Hattersley 1983).

J!laterials and Methods

Plant Materials

Seeds and whole plants were collected from as many sites as 13

practicable in all mainland States1 (Table 2.1: the Neurachneae are not known from Tasmania). Attempts were made to sample across the entire range of widespread species, although plants were seldom setting seed at time of fieldwork.

Cytology

Root-tips were obtained either from seeds germinating in vermiculite in Petri dishes, from stolons rooting at the nodes, or from whole plants. Root-tips were immersed in a saturated solution of a-bromo-napthalene for 2-4 h at room temperature, fixed for at least 3 h in 1: 3 acetic acid: ethanol, hydrolysed in 1 N HCl at 60°C for 1 o min, then stained in Feulgen and squashed. Observation, counting and photography of chromosomes (Table 2.1, Fig. 2.1) was done with a Leitz orthoplan Microscope using oil immersion.

Geographioai Distribution

Latitudinal and longitudinal coordinates were recorded for all known specimens of the Neurachneae in Australian herbaria. Most pre-

1972 records were taken from Blake (1972); other pre-1972 and nearly all post-1972 collections were seen by me. The altitude at each collection site was estimated as accurately as possible from the Australian

1: 250,000 map series (Series R50a, Division of National Mapping). From these data, maps were produced using the Bioclima te Prediction System

(BIOCLIM) 2 developed by H. A. Nix and J. R. Busby (Nix and Busby, in preparation). This system maps known records of a species and, from a

1state and Territory abbreviations: w.A., Western Australia; N.T., Northern Territory; s.A., south Australia; Qld , <;:ueensland; N.s.w., New south Wales; Vic., Victoria; Tas., Tasmania. 2 user's manual available from Director, Bureau of Flora and Fauna, G.P.O. Bex 1383, Canberra, A.C.T. 2601. 14

climate profile constructed from them, indicates areas elsewhere which might be climatically suitable for it. For the purposes of this study the profiles ""'re based on 12 climate variables, thereby defining climates rigorously though somewhat arbitrarily via annual mean temperature, minimum temperature of coldest month, maximum temperature of hottest month, annual temperature range, mean temperature of wettest quarter, mean temperature of driest quarter, annual mean precipitation, precipitation of wettest month, precipitation of driest month, annual precipitation range, precipitation of wettest quarter, precipitation of driest quarter. Predicted species ranges were constructed using the full profile range for all 12 climate variables (see Appendix 2.2).

Results

Cytofogy

The base chromosome number of the Neurachneae is x = 9 (Table 2.1), a number typical of eu-panicoid grasses (e.g. Watson and Dallwitz

1 980) • At least three ploidy levels exist, with tetraploidy being most common, N. tenuifoiia, T. xerophifo, and T. mitcheliiana are exclusively diploid; N. minor (but see below) , P. nueHeri and T. mu.Uicuimis are tetraploid only and N. queensfondica is hexaploid only

(but see below) • Different ploidy levels are found within N. ianigera

(2x, 4x), N. aiopecuroidea (4x, 6x) and N. munroi (2x, 4x, 6x).

Aneuploidy was found in one individual of N. minor ( 4x + 1: Unit

856 accession), and in the only good spread obtained from N. queensiandica ( 6x - 1: Unit 920 accession). Secondary constrictions were occasionally observed in spreads of N. minor (Unit 856 accession) and T. mitcheiiiana (Unit 1064 accession), as shown in Fig. 2.1.

Preliminary investigation showed no correlation of ploidy level with 15

either stomatal size (guard cell length) or pollen grain diameter at

either intra- or interspecific level.

All species have asymmetrical karyotypes of metacentric or sub­

metacentric chromosomes (Fig, 2 .1), measurements of which are shown in

Table 2. 1 • The small size of the chromosomes did not facilitate

attempts at meiotic studies and Giemsa banding, Fig. 2 .1 apparently

represents the first illustration of karyotypes for any Australian

native eu-panicoid grass species.

Geographical Distribution

Nine of the 1 0 species of the Neurachneae occur in the arid and

semi-arid zones of Australia (Figs, 2 .3)' viz. in the

phytogeograhical Eremean zone and adjacent eastern interzones of

Burbidge (1960) or the bioclimatic Eyrean zone and Bassian/Eyrean

interzone of Nix (1982). Only N. queenslandica, T. mitchelLiana and T.

xerophila extend to the adjacent interzones in the east (Fig. 2.3), with

T. xerophiLa even occurring eastward of the Great Dividing Range in Qld.

(Fig. 2.3a: just in the Torresian zone of Nix 1982).

In sharp contrast, N. alopecuroidea is a mesic temperate zone

species, occurring in the Bassian zone and Bassian/Eyrean interzone of

Nix ( 1 982) of sou th-west w.A" sou th-east s ,A. and western vie. (Fig.

This distribution is slightly more extensive than the area of

true Mediterranean climate illustrated by Aschmann (1973), and corresponds very closely to seasonal rainfall zones where the May­

October/November-April rainfall ratio exceeds 1. 3: 1, and where median annual rainfall exceeds 250 mm. The species does not occur, however, in the extreme south-west corner of W.A., where annual median rainfall (50 percentile) exceeds 1000 mm (refer to 'Seasonal Rainfall Zones' map 16

1975, and 'Climatic Atlas of Australia' map set 5 1977, Bureau of

Meteorology, Department of sciences, Melbourne).

No member of the tribe has yet been found north of 17. 5° s, in the middle of the arid zone deserts, east of the Great Divide (except

reference to T. xerophiLa above), or in Tasmania. Nevertheless, the distributions of Neurachne, Paraneurachne and ThyridoLepia (Figs. 2.2,

2. 3) clearly show these to be among the most widespread of grass genera endemic to Australia, both geographically and in terms of the number of major plant communities in which their species are found (cf. Clifford and Simon 1981 ; Specht 1 981 ) • Only t"° species in the group have restricted ranges: N. queensLandica in the Great Divide near Tambe

(Qld.) and N. tenuifoUa in the Macdonnell Ranges west of Alice Springs

(N.T.) (Fig. 2.3a).

(C ), (C ), (C ) Neurachne minor 3-c 4 N. tenuifoLia 3 N. queensLandica 3 and N. Lanigera (C3 l were known from only five, seven, t"° and two localities respectively, when described as new species by Blake

(1972). As a result of the Taxonomy Unit's fieldwork and of other post-

1972 collections, N. minor is now known from >25 definite sites, with a longitudinal range of about 550 km (Fig. 2.3a). Furthermore, the known range of N. minor has not been markedly extended since 1972 (Fig. 2.3a; cf. Blake's maps). Neurachne tenuifolia is now known from 13 localities

(Fig. 2. 3a), but all within the previously known west-east range. The

Taxonomy Unit has found N. queensLandica at seven sites (Fig. 2.3a), two of which may correspond to the two original localities and the rest extending the species range by nearly 80 km to the south-east.

Neurachne Lanigera remains known from only three widely separated sites

(Fig. 2. 3a). The discovery of the first W.A. sites of N. rrrunroi (by

T.D. Macfarlane and the Taxonomy Unit), 1000 km west of the previous 17

westernmost site, emphasises inadequate collecting in these areas and/or

the sporadic, distribution of the species. Precise localities for the

rarer Neuraohne species are given in Appendix 2.1.

Species distributions predicted by BIOCLIM are extensive for the

widespread species (Thyridolepis spp. (Fig. 2.3b}, P. rrue'/..leri (Fig.

2.2a), N. munroi (Fig. 2.2b} and to a lesser extent N. alopeouroidea

(Fig. 2. 3a). If realised, they would markedly extend the range in some

cases, and also 'fill in' gaps where lack of collection may primarily

reflect inaccessibility due to lack of roads. That roads can influence

known distributions is clearly shown by the north-south line of

collection sites for P. muelleri along the Stuart Highway in the N.T.

(Fig. 2.2a}. Predicted sites for species of more limited range (N.

minor, N. tenuifoiia, N. queenslandioa: Fig. 2. 2a} are surprisingly

few, even within the known ranges.

Habitat

The habitat profiles given in Table 2.3 are based on all previously

published information, plus data derived from herbarium specimens. The widespread species occur in habitats characteristic of large areas of

arid and semi -arid Australia. Substrates of red earth or sand, often

with associated mulga (Aoaoia aneura), typify most collection sites of

N. munroi, P. muelleri and all three Thyridolepis spp., as well as a few

localities of the more restricted N. minor and the only characterised

site for N. lanigera. Species of Neu.raohne per se usually occur on

sandy or rocky substrates (Table 2.3) which could have very low nutrient

status and low water retention capacity.

Most Neurachneae species do not dominate the herbaceous flora,

although N. alopeou.roidea, N. queenslandioa and N. tenuifolia are 18

locally the dominant grasses in their respective habitats. All species

in the tribe except N. alopeeUI'oidea, which has a completely disjunct

distribution, have been found growing with other members of the group,

at least on occasion (Table 2.4).

Discussion

Small chromosome size (Table 2. 2) has precluded an assessment of

species relationships in the Neurachneae, and particularly of the origin of N. minor, on the basis of cytology alone. Nevertheless, certain possibilities can be explored by considering chromosome counts, in conjunction with known present geographic distributions, along side comparative morphology, photosynthetic pathway and the climatic and vegetational history of Australia. It is important to note at the outset that species of Neuraehne and Paraneuraehne (Blake 1972) are

satisfactorily delimited, to the extent that all existing collections are readily identifiable. There is no evidence of additional species nor of hybridization, in spite of a deliberate search in areas of sympatry (Table 2.4), nor is there marked morphological variation within a species, even for the disjunct populations of N. alopeeUI"oidea.

Cross-referencing of current data on o13c value (Hattersley and

Roksandic 1983), anatomy (Hattersley et al. 1982) and co -gas exchange 2 and ul trastructural data (Hattersley et aL 1984, 1985) indicates that each species of NeUI'aehne and ParaneUI'a,ehne is also conservative in its photosynthetic carbon metabolism and leaf structure. Current chromosome count data also tentatively suggest little intraspecific variation, with

JV. {_t;,."'i er-~ I ' onlyL N. munro-z- and N. alopeeuroidea exhibiting ploidy level variation, and with aneuploidy in hexaploid N. queenslandiea and tetraploid N. minor (Table 2 .1: in contrast to Australian Braehyiome,"' Smi t.'1-Whi te, 19

Carter and co-workers in Barlow 1982),

There are no obvious parallels between the Neurachneae and such

other Australian plants as have been studied cytogeographically (cf.

reviews by James 1981, Barlow 1982). The restriction of the only

exclusively diploid Neuraahne species (N. tenuifolia) to the Macdonnell

Ranges, however, is at least comparable wi~~ the existence there, and in

other mountainous, presumed refuge areas in arid Australia, of diploid

relict (?) Cassia spp. (Randell 1970). For Eremophila species, on the other hand, it is the 'ancient land surfaces of Western Australia which

appear to be a reservoir of relic diploid populations' (Barlow 1971),

In W.A. there are tetraploid as well as diploid accessions of N.

lanigeru and N, munroi; furthermore the diploid one of N. lanigera is

actually well to the east of the tetraploid one. The existence of N, minor (c -c ) in central W.A., however, is of interest if this species 3 4 is conceived as a surviving intermediary stage in the evolution of c 4 plants from c plants. 3 The updated distribution maps (Figs. 2.2, 2.3) have extended

species ranges and/or increased density of known sites within ranges.

Use of BIOCLIM suggests that most species ranges will be further extended in remoter areas. Future collecting will provide a critical test for the present capabilities of this system, which may as yet incorporate too few environmental factors of the kind likely to limit di stribu ti on. Meanwhile, it is w::irth noting that the recent w.A. collection sites for N. munroi would, indeed, conform with the BIOCLIM prediction based upon previously-known localities. The system does not predict many new localities for the species of relatively limited geographic range (Fig, 2.3a): it seems that the narrow climatic profiles for these species, as currently known, are not represented 20

elsewhere on the continent.

The geographic distributions of P. mueiLeri and N. munroi (both c 4: Fig. 2.2) and N. aZopeauroidea (c3: Fig. 2.3a) are not unusual for grasses of their respective photosynthetic types (cf. Hattersley

1983). The remaining six c 3 species in the group, however, and the c 3-c4 intermediate N. minor, occur in areas where >80%, and often >90%, of the native grass species are c 4 • For example, in the Central south subdivision of the N.T. only 10 out of c. 168 native grass species are c 3 (Hattersley 1983; see Figs. s.2, 8.3), and four of these belong to the Neurachneae. Similarly, of c. 103 native grasses recorded from the

Ashburton subdivision of W.A., only five are c 3 , including N. ianigera, T. multiaulmis and T. mitahelliana (N. minor also occurs here).

However, there is little about the microhabitat of c 3 (perennial) Neurachneae to explain their occurrence in grass zones, especially c4 for N. fonigera and Thyridolepis species (Table 2.3). Indeed, the latter have already attracted some interest as c 3 inhabitants of these areas (Christie 1975). N. queenslandiaa, however, is a forest species

(Table 2.3) and, like N. tenuifolia, may also derive extra run-off water from adjacent cliffs and slopes. I have yet to see N. tenuifoLia growing on north-facing slopes.

While the Neurachneae are unusual in being a conspicuous part of c 3 grass diversity in arid and semi-arid Australia, they are typically eu­ panicoid in having nine out of 1 o species in tropical and subtropical latitudes. There are, in fact, 33 other c 3 eu-panicoid grasses native to Australia, but their geographic di stribu ti on contrasts mao:kedly with that of the c Neurachneae (Fig. 2.4; cf. Fig. 2.3). They are primarily 3 shade-loving species in high summer-rainfall areas, or, less commonly, in warm temperate coastal areas. Occasionally, c3 eu-panicoids occur in 21

cooler temperate floras, where they are inhabitants of wet non-alpine

habitats, including river and creek margins ( e .g • I sachne globosa) •

Only five native non-Neurachneae species extend into the semi-arid zone

CAncistrachne uncinulata, Cleistochloa subjuncea, Dimorphochloa rigida,

Panioum prolutum, P. subxerophilum: D. rigida grows in similar habitats

to N. queenslandioa), and none extends into the arid zone, Considering

endemic species only, c 3 Neurachneae even outnumber C4 eu-panicoid species in arid and semi-arid Australia. Especially interesting is that

N. alopecuroidea is the only native c 3 eu-panicoid recorded from south­

western W.A. and much of southern S.A. (viz., temperate, winter-seasonal

rainfall areas: Fig. 2.3a; cf. Fig. 2.4).

The geographical distribution of the Neurachneae, and of the eu­

panicoids as a whole, suggests that the tribe did not extend into arid

zone communities from an adjacent temperate flora, as has been concluded

for certain other genera with arid zone representatives, e.g. Acacia,

Dampiera, Eucalyptus and Goodenia (see Carolin 1982). l\.rid phases in

Australia may have occurred as far back as the Eocene (Trusswell and

Harris 1982), but were certainly important biogeog raphical influences during and since the Pleistocene ( 1 O, 000 years - 1. 8 m. y. ago; e.g.

Bowler 1982). It is likely, therefore, that the distribution of N. alopeouroidea represents an example of the rare extension of a eu­ panicoid grass into the temperate zone from warmer climatic areas (and

the only example in the west of the continent), Whether at some time it actually migrated southwards to its present disjunct areas of distribution from central Australia, was able to cross the Great

Australian Bight at times of low sea-levels (thereby bypassing the

Nullarbor Plain: see Nelson 1981) or evolved in situ with changing climates, can only be speculated upon. Dating these possibilities is 22

also speculative since, although Bowler (1982) maintains that southern

Australia first came under the influence of the temperate zone

conditions of cool winter rains and westerly winds 2. 5 m. y. ago, Nix

( 1982) suggests that increasing seasonality of both thermal and water

regimes has been a trend throughout the Tertiary (1.8 - 65 m.y. ago).

The lack of representation of the Neurachneae in wet tropical, sub­

tropical and south-east temperate (including Tasmania) areas suggests

that they are especially adapted to arid and semi-arid conditions and/or

that their ancestors were not competitive in wetter areas. Perhaps the

occurrence of six species in the Wiluna area (W.A.) and five in and near

the M;icdonnell Ranges (N.T.) suggests a western and/or central

Australian centre of origin. Furthermore, the uniqueness of the group

within the eu-panicoids, both ecologically (for c3 species) and leaf-

anatomically (for c 4 and c 3-c4 species: Hattersley et ai. 1982) strongly suggests that phenotypic expression of c photosynthesis 4 evolved in the Neurachneae independently from its expression in other

eu-panicoid grasses, as implied by pathway variation per se. Even if,

for the Neurachneae, this event was known to be in response to an

increase in aridity, it w:iuld be impossible to date, since climatic

heterogeneity in Australia may have existed throughout the Tertiary, or

at least since the Miocene (Beard 1977; carolin 1982; Nix 1982;

Trusswell and Harris 1982) • Hattersley ( 1983) concluded that

heterogeneity in photosynthetic pathway for the Australian grass flora,

including different c types, existed before the main separation of 4 Australia from Antarctica (late Palaeocene: veevers and M<:Elhinny

1976).

For the ()laternary, Bowler (1982) has concluded that the arid belt on the Australian continent may have disappeared completely 30-50 23

thousand years ago. If so, the current distribution of the apparently

relict populations of N. Zanigera, N. tenuifoUa and N. queenslandica

(all c3 ) does not seem so bizarre. Perhaps they represent relicts of an older (but QUaternary?) sub-tropical species complex (like present­ c3 day Thyridolepis?), components of which subsequently became disjunct,

diverged and speciated, The widespread and common occurrence of

Thyridolepis spp. (C ) and the more northerly P. nueUeri (C ) may 3 4 result from their capacity for rapid vegetative propagation via long

stolons, and from their apparent self-fertility. Both these features may have permitted successful colonisation of vacant habitats during the rapid climatic changes of the Qlaternary. (C ) is also N. nunroi 4 widespread, but not common. This caespi tose perennial, like N. minor

(C ), is highly palatable (Table 2.3), and field observations suggest 3-c4 that both species may be adversely affected by current grazing regimes.

The origins of N. minor (C 3-c4 ) and N. nunroi (C 4 ) remain mysteries, Although, however, N. minor is in inflorescence morphology a diminutive form of N. alopecuroidea (Blake 1972), with respect to leaf anatomy (Hattersley et al. 1982) it is very similar to N. munroi (C 4 ), with which it has been found growing (Table 2. 4) • These observations still do not elucidate, however, whether N. minor ( tetraploid) represents an intermediary stage in the evolution of c4 Neurachne (munroi-like?) from (or c 3 Neurachne (alopecuroidea-like?) vice versa), whether it is of hybrid origin, or whether N. minor is an independent evolutionary line, Both N. munroi and N. alopecuroidea have tetraploid populations (Table 2.1) and it appears that Neuraahne species are almost totally self-sterile, so that at least an investigation of a hybrid origin for N. minor can be attempted. Table 2 .. 1 .. Cllro!Wlsome nu.bers and localities of Neurachneae apecles.

Abbreviations: W.A., Western Australia; N.T., Northern •rerrito1·y; S.A., South Australia 1 Qld., QUeensland 1 N.S,W., New South Wales; vie,, Victoria,

Photo- Chroma- Species synthetic some no. Locality Coo rdi na tes Accession1 Collector2 pathway 2n

llfUU'achn.8 Kenwick, Perth, 1l5°58'E 1058 aiopecuroUiea R.Br. c 3 54 W.A. 32°02' s TDM 54 Condinup Lookout, W.A, 33°46'S 116°30' E 818 JWW 36 7.8 km s of />bunt Hope, Eyre Peninsula, 34"08'S 135°22'E 1096 llOVP S.A, 36 4.0 km N of wangary, Eyre Peninsula, S,A. 34°34'$ 135°31 'E 1095 HDVP 36 6.1 km s of Ashbourne, S.A, 3S 0 2o•s 136°45' E 1091 HDVP 36 4.8 km s of Meningie, S.A. 35°44'S 139°20'E 1090 HDVP 36 Little Desert National Park, Vic. 36"28'5 142"02'E 1087 HOVP

Lanigera S.T. Blake 36 42 .9 km N of Wilona, W,A, 2t:i"12' s 120" l O' E 866 PWH 18 78 km NE of Wi'lrburton, new Giles road, W.A. 25°42' s 127°14'E 11 5'1 1984

minor S,T. Blake 36, 37 2.0 km E of Meekatherra, W.A. 26"35'S 118°32'E 856 PWH 36 20 km NW of Peak Hill, W.A. 25°31'5 118"35'E 854 PWH 36 4,3 km N of Lake Violet, W.A. 2& 0 32•s 120"42'E 1127 1984 36 46.2 km N of Ci'imel Well, W.A, 25°50' s 121°24' E 1136 1984 36 6 km w of Sydney Head Pass, W.A. 25°32'5 121 °44' E 1137 1984 36 Kaljahr Pinnacle, W.A. 25°34'8 122°25' E 1142 1984 36 3,4 km W of Carne

munroi (F. Muell.) 36 40 km N of Wiluna, W.A, '26" 16' s 120" 11' E BSB 'l'OM !'. Muell. 18 1 .6 km SW of Nl a

queenaZandica c 3 54 72 km N of 'l'ambo, Qld. 24°21'5 146"21 'E 925 PWll S.T. Blake 53' 54 45 km E of 'l'ambo, Ql.d. 24°54'5 146°40' E 920 PWll 54 75 km E of 'l'i'lmbo, Qld. 24<>4s•s l46°57'E 921 PWll

tenuifoiia S,T, Blake 1B Serpentine Gorlje, N • "r • 23°46'8 132°58'E 1080 HDVP 18 Ellery Gor!Je, N.T. 23°47' s 133"04'E 1079 HDVP 18 1 km W of ,Jay Creek:, N.'l', 23"49'S 133°28'E 1078 liDVP P(D."

Paynes Find, W.A. 29"00' s 117"46' E 852 PWll multioulmis (Pilger) c 3 36 33 km N of S.T. Blake 36 W.A. 3 852/7? PWU 36 31 km NE of Warburton, new Giles road, W.A, 25"56'S 126°49'E 1155 1984 36 17 km SE of Mt. Fanny, W,A, 25°54'5 128°4l'E 1160 1984 36 70 "'" SE of Piralyat)ara, W.A, 26"4 l's 129°23'E 1162 1984 xer>ophi1-a (Domin.) c 18 Mt. Nossi ter, W.A. 2s 0 25•s 123°47'E 1150 1984 3 S.T. Blake 18 Ayer's Lookout Rang<;:, N.'r. 24"32' s 132°34'£ 1062 IH>VP 18 8 km N of Orange Creek Ho1Uestead, N.T. 24°18'8 133°26' E 1063 llDVP 18 1 km w of Jay Creek, N.'r. 23°49'5 133°28'E 1077 HPVP 18 2 km E of Pine Gap, N.'r, 23"49'S 133°45'E 1081 HOVP 1 a 72 km N of Tambo, Qld. 24"21 'S l 46"21 'E 930 PWll 18 12 km s of Augathella, Qld. 25°51 •s 146°35'E 92"/ PWli

1 Taxonomy Unit accession number (Research school of Biol0

in Neurachneae.

Length measurements from spreads with

most contracted chromosomes. Where

data not given, highly contracted

chromosomes not obtained.

Chromosome length Species range mean µm

II euzoaaJme

afopeauroidea lanigera minor

munroi 1.0-3.0 1 • 9 queenalandica

tenuifolia 1.3-2.8 1 • 9

Pa:raneuraahne

muelleri 1.4-3.0

Thyridol.epia mitchelliana multiculmis

xerophila 1.1-2.4 Tlaible 2-3. Habitat. eail type, fire tolerance and palatability of Neurachneae species.

Where no source reference is qiven, information derived by direct observation or from herbarium specimens.

Species llabi tat and soil 'l'ype Fire tolerance Palatability

Nauranhwl aLopeauroidea W,A.: mallee woodland or heath; white sands, several from Probably tolerant: Not known. lateritic clay or gravel. regenerates from $.A., Western vie.: white sands predominant substrate. On burnt shoot bases. sandplains and sand-dunes widespread in a number of sclero­ phyllou s comuunities1 • Also on solonized and podsolised 'desert' soils in Eucal-yptua faaoiculosa-Xanthorrea aemiplana association2 • In E', diveraifolia-E. angulosa associations, limestone underlies shallow soil profile of sandhills3, likewise in west of Eyre Peninsula, but here usually red earths. In S Lofty Ranges found in E. odorata stands on various soils, and in 'the strinyybark edaphic complex' on soils low in nutrient and water retention capaci ty 4 •

l-anigera W.A.: one population on red sand spinifex plain, one on Probably tolerant: Not known. micaceous schist outcrop. regenerates from s.A.: site not described. burnt shoot bases.

minor W.A.: most collections from stony or rocky (e.g. quart.zite) Not known. Palatable: in grazed outcrops and hills or on red clayey sa.nd in flat open Acacia sites protected in scrub plains; also beside a creek, and disturbed

munroi W.A.: stony rises, of.ten quartzitic, with bar<'l surface Pr.obably intolerant; very palatable, grazed or red sandy clay in open Acao-ia shr-ubland. in N.'l' may be out (7); increases in S.A.; red soil on limestone rises. restricted to areas density in protected N.T.: in limestone areas and on stony tablelands5 , often which escape fire6, areas in good years 6 7 on mesas and buttes 1 also from Triodia sandplain, (N.s.w.) • N,S,W,; in NW usually on low gravelly ridges with red earths, associated with E,', popu'lnea, £', intertexta and Geijera parvifle>ra communities; sometimes on red calcat~eous earths7 • Qld,; In SW on loamy and sandy r-ed earths, and li tho sols in A. aneu1u and Ast1•ebla communities8 1 further north and west, from rocky or shaly hills. From E'uoalyptua woodlands, A. aneuro and A. oambagei woodlands and shrublands, and Troidia associations of the south i:lnd west9 , queenal-andioa Qld.: at base of and on slopes below sandstone cliffs or Not known. Not known. outcrops, in sandy soils, sometiines with humusi in eucalypt open-forest, often with Aoaoia shir'l-eyi.

tenuifo'L.ia N.T.: scree slopes, gullies and gorges; on scree, gravel Probably intolerant: Not known, or skeletal soil (often quartzitic). occurs in normally fire-free areas6. Paran'1U.raohn.8 mueLl.eri 'n\roughout rani.1e usually on red sand spinifex plains. Tolerant: in S N.'I'. Palatable to cattle N.T.: also skeletal soils on sandstone, quartzite and most common in early inSofN.'I'. 10 • limestone hills6 • seral stages after Producing little Qld.; also from Aoaoia open-forest and F.:uoa'L.yptua fire6, Collections fodder5 • woodland; additionally, floodplains, rocky hillsides, in W.A. and S.A. gravelly soil and disturbed ground e.g. road-sides. from recently burnt ground.

ThyridolBpia mitcheZ.l-iana 'l'hroughout ran<;Je mostly red ectcths or sands, usually with Not known, very palatable foe A. aneu1u. all livestock; W.A.: also on rocky outcrops. valualile fordge Qld.: also in Ca'L.l-itris open-forest, A. oambagei grass5 , able to comI1Unities, and eucalypt ;..uodland where 1'1•iodia spp. withstand moderate dominate understorey9 • gra:linq pressure7 N.s.w.: in NW also in Ca'Ll-itris co'Lume'L'Laris and E. popuZ.nea communities; on stony hillsides with E. deal-bata and A. bi.rr>rowii; in hilly areas sometimes the dominant grass but on red soil flats and ridges usually associated, or co-dominant, with Aristida spp, 7 ,

muiticulmis S.A., w.11.., N.'1'.: sandy ced earths with A. aneura, Not known. Not known. N.'l'.: sandy soils, usually in dune swales6 • xerophita W.A., N.'1'.: mostly quartzite O(" sandstone hills, someti111es Not known. Not known. with A. a11eura. S.A., Qld., N.S.W.: usually ced soils in Acacia sht:ublands or eucalypt forest. In Qld., also in A. hax•pophy'L.la and A. cambagei comnunities9 , and on white sandy soils.

1 Litchfield ( 1956). 2 Specht { 1951). 3 ,Jessup (1946). 4 Specht and Perry ( 1948), 5 Laza rides ( 1970). 6 P.K. Latz (in 'litt.). 7 Cunningham et a'L. (1981). 8 Roberts et al-. (1976). 9 'l'othill and Hacker (1983). lO Chippendale (1958). Table 2.4. Sy.pAtric associations within the Neurachneae.

Photosynthetic PhotosYnthetic State and Habitat Species state and tlahi tat Species pathway Pathway

W.A. N.T. Stony rise 1 N. muru•oi Red sandy loam T. mitohelliana N. minor between dunes2 T, tTUltioulmia :J.'. mitoheZ.Liana S.A. Granite mesa 2 N. munroi 1 Red sand plain T. mitoheZ.Z.iana T. mitoheZ.Liana T. nuZ.tioulmia Qld. 1 Micaceous schist P. mueLLeri White sandy soil N. queenalandioa outcrop 1 N, Lanigero T. xet•ophiZ.a T. mi toheL Liana Chert hi111 P. nue Z. t-eri N.T. N. munt•o·i Red sand plain 1 P. muellex•i T. mitoheLliana Loamy rod earth; N. //tl.lll'O-i sandy red ear·th; ']', mitohelLiana li thosol 3 stony slopes 1 N. tenuifolia c,

T, xerophiZ.a c 3

Pers. obs ••

2 Herbarium specimens.

3 Roberts et al. (1976). Fig. 2.1. Karyo types of Neurachneae species. Secondary constrictions are arrowed for N. minor and T. mitcheiiiana. scale bar = 2 µm.

Photographs taken using phase contrast and oil immersion. a,

Pa.raneurachne T1Ueiieri 2n = 4x = 361 b, Neurachne T1Unroi 2n = 4x = 36

(last pair of chromosomes overlapping; c, N. minor 2n = 4x + = 37; d.

N. tenuifoiia 2n = 2x = 18; e, N. queensiandica 2n = 6x - = 53; f,

N. aiopecuroidea 2n = 4x = 36 1 g, Thyridoiepis mitcheUiana 2n = 2x =

18 1 h, T. xerophiia 2n = 2x = 18. 11) 4- Olfll Ld 1111 ... llli • II i • .. 1111 • • ...... Ill .. • ,~ • ., II' • • • • • 0. • • • ¥ • - - 11111 - ... • • • tl/llll!I' • • • ii .... - • - • 1111 • ill - • .. • • 1111 • II • - • • • • 11111 ..- - -- ....- 1111 .... • -- • • 0 w • .. ·-• di 0 • • • .....- .. • • """' • illlill'll!llt Iii 11111111 • • • • ~ • -11111 •81 • -- • • -- - • u 0 _,Q "'ti ""' ..- ..- •- 11//11- ~ - - ..""" "" .. • .,,,,, .....- ... -~ •- .- -""' - - - -i/l!l!'P ...... - - • • .. • ~- --..... - c - -• • - - • '111111111> • 111111 • ..,. • - ""'"""' - -• - - - "" - - -- -~ .. - • 4 • -- - - - • --~ J/1111111111,- -...... - 11111111 --*" - - • ?~ -~ :Ma --IM - - • - - ~ Localities of a, Paraneuraahne 11UeLLeri ; b, Fig. 2. 2· c4

Neuraahne 11Unroi c4 • Dashes (-) indicate areas which are climatically similar to collection sites. Maps generated by the Bioclimate

Prediction System (Nix and Busby: see Materials and Methods) • Large symbols (closed circles) represent localities added after maps

(including dashes) were originally made. a r // r . '~ ~ .: ' '\..{ . .. • •... \ . " ...... ' ...... " ·.. I' ,. .. '. ~ ·"" ..

(/ ( 500km

b

•• . .,,• • • _.- .. .. I - . ' .. ' ' • ' • \ rl V]J\ C'")o.!.~\ \ 500km Fig. 2. 3. a, Localities of Neu:rachne alopecu:roidea c 3 (a),

(ml, (q), (t), and N. minor Crc4 N. queenslandica c3 N. tenuifolia c3

N. lanigera c 3 (1). b, Localities of ThyridoLepis mitcheLliana (•), T. muiticuLmis (0) and T. xerophila (V (all c 3 J. Dashes (-) indicate areas which are climatically similar to collection sites. In a dashes for each species do not overlap, and predictions were not made for N. Lanigera, for which only tm localities are knowg. In b dashes for the three species do overlap and have not been distinguished. Large symbols (closed circles) in a represent localities added after maps

(with dashes) were originally made. a ;

.t- .:. ~e -....:..... / ' '• ...... , • .. :.&!..... ·•• a .~·.~- -· ~- .. -·~-.-_-_._"' ·- .. I ' . :~-·~ :~-..

500km

b

\ . - . . - - - - . . . . .

;·.·•. ·.. ··~

SOOkm ·-. . ~'r . Fig. 2.4. "ltlmbers of native non-endemic and (after +) endemic

Neurachneae and Miaraira) (excluding c3 eu-panicoid species in geographic subdivisions of Australia. (See Hattersley 1983 for subdivision names and data sources.) ·------j-·-·-·1

r'~(_:,~--11+1 /-'.)~~--- 4+1 ),L---6

500km Appendix 2.1. Precise locali tie a: of rarer Neur:achneae species.

Neuraohna lanigera

47.2-49.2 km N of Wiluna on Cunyu road, E side, Grid ref: 308745 26°12•s 120"10'E 76 km NE of Warburton on new Palytjikarta and Giles road. On small micaceous outcrops, N of road, 2s 0 42•s 127"14'E Near Mt. wa tson, NW Sou th Australia, 21°1-•s 129Q4-'S

N. minor

Milgar Station. 7 km SSW of Mt. Labouchere, FDck outcrops on low hill. 25°06' s 118" 18'E 2 .a km from Meeka tharra P .o. on Wi luna Road, Disturbed ground on S side. 26"35'S 118"32' E 47.5-53.5 km N of junction with Gt. Northern Highway on Mt. Beasley road. rn scrub on E side, Grid ref: 134826 25"31 'S 118°35'E S0.5 km N of Meekatharra on Gt, Northern Highway, 200 m NW, on small hill on W side. Grid ref: l4T743 26" 12' s 118"4 l' E 55 km E of Meekatharra on Wiluna road. 26"27'S ll8"59'E 120 km NE of Sandstone on Wiluna road (via Mt. Merewether), Rock outcrop on E side. Grid rtif: 25 3648 27"01'S 119°39'E 133-134 km. NE of sandstone on Wiluna rodd (via Mt. Merewether). Rock outcrop Oil S side. Grid rt;;f: 277666 26"5 \' s 119"52' E Yeerlirrie Homestead, 27" 1 - 'S 120"0-'E 1.5 km N of turn-off to No. 2 Well Kukabubba on Wiluna-Cunyu road. 1-Dstly on E side. Grid ref: 31 07 39 26"15'S 120"1 l' E Mount Alice, and on both hill slopes and plains to 12.6 km N. 26"23'5 120°16'E Rotten granite outcrops. 26°39'S 120"34'E 4.3 km NE of Lake Violet. Open plain with spinifex. 26"32'S 120"42'E 25.6 km NE of Lake Violet, D:>minant grass species on hill-top. 26°26'5 120"54' E 32 km SW of Camel Well. Open plain, 26°17'5 121 "02' E 3.6 km SW of Cam.el Well. Open rocky rise, 26° l 0' s 121"16' E 2,0 km NW of Wonqaroo Homestesd, Hill slopes ou both sides of road, 27°07' s 121"19' B

9.2, 10.9, 24.1, 31.1 1 34.1, 46.2 km N of Camel well on Granite Peak road. 26"--' s 121°--'E 6.1 km SW of Sydney Head Pass. 25 ° 3 2' s 121 "44' E 194.7 km E of Wiluna on WJngawol Station road. 26" 18' s 12 J "55' E 1.4 km SE of Hootanvi Well on E sid.: of Bandya road. Open ground, lateritic clay. 27"34'5 122°00' f~ 1,8 km S of Swanson Well, near Bandya, W side of r·oad of stony hill. 27°49'5 122°17' E Kaljahr Pinnacle. Also 6.4, 11.5, 13.7, km to N and E along Glenayle road. 25"34'S 122"25'E 21,3 km w of Carnegie. Also 6.8, 11.s, 13.7, 1"1.4, 25.1, 31.3 km w of Can1eyie. 25"--'S 122"--' E Windidda Station, between Acacia Bore and Bilgarrie Cutarrie Uore on edye of voll Treutor 'l'ableland, On rock tops of breakaway. 26"J5'S 122"47'E Top of van Treuer Tableland. 11,6 km S of Prenti Downs, usually alon(] streilmS. 26"37'S 122"48'E Carneg.ie Station. 25"4-' s 122"5-'E 27 km W of Mt. Nossi ter, 25"28'S 12J 0 32'E N. queenaLandica

4.8 km N from turn-off 10.2 k111 W of Birkhead {= Myall Grove). In shallow sandstone gully. Grid ref: 431950 24"31 •s 146"17' E 2.9 km W of Birkhead {= Hyute to Springsure, 1.2 km E of Mt. playfair property turn-off. Sandstone spur on sou th side of track. Grid ref: 506917 24"48'S 146°57'E

N, tenu.ifoLia b.Ummit of Mt. Liebig. :t 1200 m 23°18'5 131"22' I:: Talipata East. W of Haast Bluff. Gravelly lower slopes of gully in quartzite hill. 23"22'5 131 "22' E Mt. Crawford. Haast Bluff Station. Slopes of steep qully in quartzite hill, 23"24'5 131 "34' E Mt. Sander. Base of cliff-face of quartz-schistose hill in skeletal soil. 23°35•s 132"33'E Mt. Sander. ± 1200 m. 23" 3-' s 132°3-'E Mt. Giles. Skeletal soil, high in gully, quartzite hills. 23"39'S 132"52'E Serpentine (brge, Amongst rocks on steep slope at southern entrance of gorge. Gr-id ref; 620039 23"45'S 132"57'E Ellery Gorge. Southern side of rocky hill in skeletal soil. Gr-id ref; 631036 23"47'$ 133"04'E Chewings Range, Skeletal soil, lower slopes of gully in quartzite hill. 23°43' s 133"19'E Standley Chasm. Steep west-facir~] slope. 20 m from S side of chasm, Grid ref: 676044 23"43'S 133°27'E 1 km W of Jay Creek Settlement. Steep south-facing slope, 50 m W of qrid on Hermannsbur<;J Rodd. Also 4.7 km W, on N side of road. Grid ref: 678035 23°49'5 133"38'E (Accession; Nelson 2519, N.T, Herbarium.) 23" 49' s l33"38'E Appendix 2.2. Climatic profiles of Neuraahrte and PaJ•aneuraahrte as evaluated by the Bioclimate Predi.ction System (see M.ater:l.als and Methods). l) ANTEMP, annual mean temperature; 2) MlNMIN, minimum temperature of the coldest month; 3) MAXMAX, maximum temperature of the hottest uionth; 4) TRANGE, annual temperature range (3) - 2))1 5) TWETQ, mean temperature of lhe wettest quarter (3 months); 6) TURYQ, mean temperature of the driest quarter; 7) MEANPRE, annual mean precipitation1 8) PRWETM, precipi.tation of the wettest month; 9) PRDRYM, precipitation of the driest month; 10) l'RERAN, annual precipitation range (8) -9)); 11) PRWETQ, precipitation of the wettest quarterj, 12) PRDRYQ, precipitation of the dri.est quarter. Temperature in QC, precipitation in mm. n "'number of geographical data poirlts where species were recorded, and from which climatic parameters were derived. Species Photo­ synthetic Climatic Parameters pathway ANT£MP MINMIN - MAXMAX TllANGE TWETQ TDRYQ MEANPRE PRWETM PRllRYM PRERAN PRWE'fQ l'RDRYQ

P. rrruelleri mean 23.74 7.38 38.16 30. 78 29.66 18.54 310.81 64.53 s. 2 7 59.26 166.45 21.48 (n•lS2) minimum 19, 94 2.86 34. 74 23.28 18. 77 13. 4S 1S8.40 24.22 0.63 18. l l 59.75 4.33 5 percentile 2 l .16 3.89 35.94 27.00 27.69 14. 73 204.0S 31. 90 1.32 2S.Jl 84.S6 7.84 95 percentile 26.26 11.04 39.75 33. 92 31.24 22. 80 461.67 108.82 10. 16 107.45 281.45 34.96 maximum 27.45 13. 67 40. 67 34. 92 32. 15 27.80 SS l. 39 125.92 16.92 118.71 321.0l 57. 19

N. munroi mean 21.79 5.33 37. 29 31.96 28.07 16.20 260. 77 41. 91 9.05 32. 86 111.32 34.00 (n•60) minimum 17.58 2.86 31.42 25.66 10.62 11.80 135.69 19.86 2.67 8.35 47. IO 14.32 5 percentile 18.42 3' l 7 33. 85 28.48 21. 99 13.37 151.30 21.06 3.28 J 0. 33 S0.26 16.01 95 percentile 24.96 9. 2 l 38.85 34. 10 30. 22 20.01 390.68 79. 05 17.55 76.38 216.34 S6.6S maximum 25. 79 10. so 39.SS 35. 08 31.21 23.S6 496.77 115.66 28.32 111.42 316.18 93.25

N. minor C3-C4 riiean 22.27 6. 10 38.34 32.24 29.50 20.85 208.10 35.02 2.94 32.08 95.45 14. 39 (n=l2) minimum 21.79 5.59 3 7. 79 31. 93 28.65 18. 35 20 l. 96 32.46 1.98 28.97 87 .60 11.62 5 percentile 21.79 S.59 37.79 31.93 28.65 18.35 201.96 32' 46 l '98 28.97 87. 60 11. 62 95 percentile 22.62 6.ld 38.63 32.23 29.9S 22.54 212.64 36.33 3. l+9 33.85 99 .12 l 5. 93 maximum 22.87 6.54 38.83 32. 35 30. l 7 22.57 212. 67 37.72 3.82 35. 01 lOS.47 15.94

N. queens Z.andica mean 20. 94 4.88 34.44 29. 56 26. 87 l 5. 31 560.58 90.42 20.00 70.43 243.65 67. 73 (n•7) minimum 20. 18 Li. 43 33. 5 7 29. 14 26. 16 l/i.60 517.99 86.09 17.24 67. 87 228.56 60.26 5 percentile 20,18 4. f.3 33.57 29" iii 26. 16 14.60 Sl7.99 86.09 17.24 6 7. 87 228.56 60. 26 95 percentile 21.20 S.02 34. 69 29.76 2 7. 2 7 15.57 586.09 9'1.62 20.70 74. 04 256. 76 70.47 maximum 21.84 5.40 35.44 30.04 2 7' 31 16. 16 602.96 94. 92 22.46 74.S4 261.05 74. 38

N. tenuifol.ia mean 21.24 J.67 37. 45 33.78 27.94 14. 84 254.01 36. 8 7 6.69 30. 18 102.04 28. 21 (n .. 12) min11nu111 21.07 3.52 Tl. 25 33.30 2 7. 64 14.66 226.61 3 t. 31 i1, Ii 2 2 5. 3 l 89.29 19.26 5 percentile 21,07 3.52 3 7. 25 33. J(J 27. 611 ll1" 66 226.61 Jl. JJ Li. 4 2 2 5. 3 l 89.29 19.26 95 percentile 21.56 3.97 37.57 34. 02 2!L 511 15.20 311.07 49. 98 10.02 39. 71 133.38 35.21 maximum 21.57 4.07 3'1.57 34 .03 28.54 IS. 2 ! 314.54 50. 30 10.27 40.t16 J 34. 16 36.38

N. alopecuroidea mean 16. 42 5.82 30. 12 24.30 11.53 21. S6 S83.39 99.25 15.01 84.25 270.31 52.09 (n .. 164) minimum 12.76 3.13 22.97 14.51 7.83 18.12 286.03 35. 15 4.08 14 '6 7 98. 98 18.30 5 percentile 14.04 3. 71 25.83 19.30 8.91 19.18 339.18 46.38 6. JS 27. 69 129.41 26.80 95 percentile 19.05 8.3S 3/i' 21 28' 51 l ). 48 23.64 1027.97 213.41 25.22 203.87 573.02 83. 7 7 max irnurn 20. 4 5 8.68 35.82 29.26 15. 99 25. 28 1250.68 251.38 3 7. 7 3 239.51 689.13 122. 92 1 l. W.A. localities only minirnum 15.04 4.57 25. 82 18. 7 5 10.18 1.9.18 286.03 35.30 4,08 28.81 98.98 18. 30 2. S.A., Vic. localities only maximum 20.45 8.68 35. 82 29.26 15.99 25.28 1250.68 251.38 23.50 239.51 689. 13 80.02 2 minimum 12.76 3. Li 22.97 14.51 7.83 18.12 33!.L06 35' 15 13.30 14. 6 7 99.24 46. 71 111<-iximum 16.88 8,47 31,62 2 7. 91 12.60 22.30 799.11 106. 4 l 3 7. 7 3 78. 4 9 309.21 122.92 CHAPTER 3

~. ACID DECAROOXYIATION TYPE IN ERNJROSTIS (POACEAE): mTTERNS OF

VARIATION IN CHI.OROPIAST POSITION, ULTRASTROCTURE AND GFDGRAPHICAL

DISTRIBUTION.

Published in PLant, CeLL and Envirorunent, 1986, 9, 333-344.

H.D.V. Prendergast, P.W. Hattersley, N.E. Stone and M. Lazarides.

Manuscript received 16 October 1985, accepted 23 January 1986. 24

Abstract

I investigated the activity of acid decarboxylating enzymes, the PCR c4 ('photosynthetic carbcn reduction', or 'Kranz') bundle sheath anatomy

and ultrastructure, activity of c4 acid decarboxylating enzymes, and the geographical distribution of Australian species of the grass genus c 4 Eragrostis. Species had either the even sheath outline and

centripetally located PCR cell chloroplasts characteristic of NAO-malic enzyme (NAO-ME) species (29 spp.,) or the uneven sheath outline and

centrifugal PCR cell chloroplasts characteristic of PEP carbcxykinase

(PCK) species (28 spp.), or were intermediate between these types (7

spp • ) • The suberized lamella was present in PCR cell walls of species with PCK-like and intermediate anatomy, and absent from those of species with NAO-ME-like anatomy. Biochemical determination of c type for 11 4 species, however, revealed only NAO-ME activity, irrespective of anatomical type; no PCK activity was detected. PCK-like species are most numerous in northern, high rainfall, tropical Australia and also predominate in relatively humid coastal and subcoastal areas. NAO-ME- like species are numerically and proportionally dominant where rainfall is < 30 cm y -1 • overall, as many species occur in high as in low rainfall areas. Results are discussed in relation to previously established anatomical/ultrastructural/ geographical/biochemical correlations and to infrageneric taxonomy. 25

Introduction

The c 4 pathway of photosynthesis has three biochemical types differing

in their c 4 acid decarboxylating systems (see reviews by Hatch and Osmond 1976; Edwards and Huber 1981). All three types (NADP-ME, NAD-ME

and PCK 1 ) occur in grasses (Poaceae), and each is consistently associated with specialised anatomical and ultrastructural features of

PCR2 (Kranz) leaf tissue, viz. position of the PCR sheath in major vascular bundles, the position and structure of PCR cell chloroplasts,

the frequency and distribution of mitochondria in PCR cells, and the presence or absence of the suberized lamella in PCR cell walls (see

Hattersley and Browning 1981 for references). These associations have been used to predict the biochemical types of c 4 grass species on anatomical grounds alone (Hattersley and Watson 1976; Brown 1977; Ellis

1977; Hattersley 1984), and confidently so, since there is considerable biochemical homogeneity within taxonomic groups at both subfamily (cf.

Brown 1977) and generic levels (e.g. G.ltierrez et ai. 1974; G.ltierrez

et ai. 1976; Hattersley and Watson 1976; Brown 1977; Ellis 1977).

Using anatomical features, Ellis (1977) assigned the chloridoid and panicoid grasses of southern Africa to c4 type and Ellis et aL ( 1980) used these data to analyse the distribution of c 4 types in south West

Africa/Namibia, where the grass flora is almost exclusively c 4 • The relative frequency of NADP-ME species increased with increasing rainfall, NAD-ME species dominated in the low rainfall areas, and PCK species were intermediate in this respect. I have also been examining c grass species anatomically with a view to completing a similar survey 4

1 NADP-malic enzyme type, NAD-malic enzyme type, PEP carboxykinase type ~Gutierrez et ai. 1974; Hatch et aL 1975). PCR tissue: 'photosynthetic carbon reduction' tissue, defined in Hattersley et ai. 1976; equivalent to Kranz tissue. PGA tissue: 'primary carbon assimilation tissue, usually equivalent to c 4 mesophyll. 26

of c 4 type di strim tions in Australia as an extension of the study by

Hattersley (1983).

It is already known that species of the Chloridoideae (Watson et

1985) are either NAD-ME of PCK (but see Ellis 1984) and, al. c 4 c 4 despite the small number of species biochemically typed, that at least three genera are known to be variable CBoutel,oua, Chloris and

Sporobolus; Gutierrez et al. 1974; Hatch et al. 1975). While examining the genus Eragrostis for this survey, I encountered not only species with NAD-ME and PCK anatomies, but also some with a leaf structure not classifiable as either, as previously found in 13 southern African

Eragrostis species (Ellis 1977). Such species were then typed biochemically in anticipation that they might represent NAD-ME/PCK biochemical intermediates. Boutel,oua c:urtipendula (Michx.) Torr. has already been suggested as a pcssible intermediate: although c4 acid decarboxylase activity and PCR cell chloroplast pcsition imply t.'1is species to be PCK (Gutierrez et al. 1974; my observations), its PCR cell walls lack a suberized lamella like those of NAD-ME species (Hattersley and Perry 1984; not possessing one, as previously reported by Hattersley and Browning 1981).

However there is now some evidence for doubting the reliability of predicting the type of aspartate forming (i.e. NAD-ME and PCK) c 4 species from anatomical features alone. In spite of centrifugal chloroplasts in their PCR cells (suggesting PCK activity), t.'1ree species in the Diahotomif/,ora group of the genus Paniaum have been simultaneously typed as biochemically NAD-ME, and also found to exhibit the sharp post-illumination burst characteristic of this group cc 2 (Ohsugi and Murata 1980 and 1981; Ohsug i et aJ,. 1982). Results were similar in species with •typical' NAD-ME chloroplast position 27

(centripetal). Among cultivars of a further species, P. aoZoratum L.,

both anatomical types were found, but again all were biochemically NAD­

ME. Ellis (1977) has also found anatomical variation within a single

species, the chloridoid MiaroahZoa aaffra l\lees, and Hattersley and

Browning (1981) have noted three species where anatomical/biochemical correlations may break down: Paniaum Zanipes Mez, P. bergii Arech., and

Eragrostia aurvuZa (Schrader) Nees. Ellis (1977) and Hattersley and

Browning ( 1981) were unable confidently to assign E. aurvuZa as NAD-ME or PCK using anatomical and ultrastructural criteria. It possesses a suberized lamella in its PCR cell walls (like PCK. species), yet it has consistently been shown to be NAD-ME biochemically (Gutierrez et ai.

1974; Hatch et aZ. 1975).

I decided to undertake a more detailed investigation of the genus

Eragrostis, simultaneously monitoring: i) PCR cell chloroplast position and the anatomy of PCR bundle sheaths; ii) activity of acid c4 decarboxylating enzymes; iii) presence or absence of the suberized lamella in !'CR cell walls; and iv) geographical distribution of anatomical/biochemical types in Australia. With >70 spp. (and c. 300 spp. worldwide) Eragrostis is the largest grass genus in Australia, is widely distributed geographically and climatically, and has recently been revised taxonomically (Lazarides in prep.). !he aim was to ascertain whether biochemical c4 type intermediates exist in Eragrostis, or whether the breakdown in established anatomical and ultrastructural/biochemical correlations is more extensive than previously realised. 28

!!!aterials and Methods

Leaf btade anatomy

55 native and 9 introduced Eragrostis species were examined. Fresh

leaf material was used for accessions of 11 species used

experimentally. For other accessions of these 11 species, and all

accessions of a further 18 species (indicated in Table 3 .1) fresh,

field-collected leaf samples were fixed in FAA (57% ethanol, 5% acetic acid, 1 • 5% formaldehyde) • For 35 species (Table 3 .1 ) , for which only herbarium material was available, samples were partially rehydrated in

Hp at room temperature to facilitate sectioning. Transverse sections were cut by hand from the mid-laminar portions of mature leaves, mounted in glycerol and examined and photographed in a Leitz Orthoplan light microscope using brightfield illumination.

For anatomical typing, all accessions were examined for: i) the

1 ffiaximum lateral cell count' and the 'maximum cells distant count' of

Hattersley and Watson (1975) to distinguish from species; ii) the c4 c3 presence (XyMS+), in FCK and NAD-ME species, or absence (XyMS-), in

NADP-ME species, of cells intervening between metaxylem vessel elements and laterally adjacent chlorenchymatous bundle sheath (PCR) cells of primary lateral vascular bundles (Hattersley and Watson 1976) and iii) the pcsi tion of chloroplasts in FCR cells: peripheral, evenly distributed or predominantly centrifugal in PCK species, centripetal in

NAD-ME species (Gutierrez et at. 1974; Hatch et at. 1975) and centrifugal in NADP-ME species (Downton et at. 1970). I also recorded the shape of the FCR l:undle sheath and its constituent cells in transection. In typical NAD-ME species the FCR cells have straight radial walls and the bundle sheath outline is even; in FCK species, 29

however, the radial walls are often curved and the very inflated, outer

tangential walls give the bundle sheath an uneven, 'bubbly' outline.

PCK species also often have bundle sheath extensions of PCR cells (Ellis

1977; referred to here as extension cells: Fig. 3.1).

Experimental plant material

Plants were grown from seed under natural light in glasshouses

(February-June 1985), maintained between 35°C (day maximum) and 1 s0 c

(night minimum), and regularly fertilized with Ruakura nutrient solution

(Smith et al. 1'l83). The same accessions of each species (Table 3. 3) were used in all aspects of the study. Identities of both original accessions and experimental plants were checked.

Electron microscopy

Leaf samples from biochemically typed accessions only were prepared fbr electron microscopy as described by Ha ttersley and Perry ( 1984) except: infiltration of Spurr' s resin began with a 50% (v /v) mixture with acetone, left overnight; staining was with 2% (w/v) Ba(Mno 4 J 2 for 15-30 min and specimens were examined and photographed either in a Jeol

JEM-100C or Hitachi H-500 transmission electron microscope. Except where cell walls are sectioned obliquely, the suberized lamella is readily visible as a pair of parallel electron-opaque lines separated by an electron transparent zone.

Geographical distribution

Sources of Australian distributional data were as listed by

Hattersley ( 1983) but with additional data from: computer print-au ts from QJ.eensland Herbarium ( 1 984) and lbrthern Territory Her barium 30

( 1985); computer database of the Bureau of Flora and Fauna, Canberra

(1985) for Western Australia and New south Wales data, and Jessop (1983)

and Beaug lehole ( 1 980) for South Australia and Victoria data

re spec ti vely. Species distributions were mapped accordiI'J:j to presence

in State and Territory subdivisions (see Fig. and Table in

Hattersley, 1983).

Assays of deoarboxyLating enzymes

hll extracts were assayed at 30°C in a Shimadzu Recording

Spectrophotometer ( W 240). For all extracts O. 5 g of young, fully

expanded leaf blades were cut into small segments and thoroughly ground

for several minutes in a chilled mortar containin:i extraction buffer and

25 mg polyvinylpolypyrrolidone.

NAD-maLio enzyme and NADP-maLio enzyme.

Extraction procedure closely followed that of Hatch et ai. (1982) and Edwards et ai. (1982). As extraction buffer we used a 2 ml solution 5 containing 50 mM HEPES-KOH (pH 7.;t>' 2 mM MnC12, 5 mM M:JCl2, 10 mM DTT1 and 1% (w/v) BSA. After removal of extract for chlorophyll determination, Tri ton X-100 was added to give a final concentration of o.5% (w/v). After 3 min at room temperature, the extract was spun for 1 min in a Beckmann Microfuge B. o.6 ml of the resulting supernatant was then run through a Sephadex G-25 column preequilibrated as in Edwards et ai. (1982). About 1. 6 ml of eluate, containing the who Le of the protein band, was collected, thus involving a dilution factor as part of the final calculation for enzyme activity. '.!his eluate was then gassed

1 Abbreviations: OTT, di thiothrei tol; BSA, bovine serum albumin; DTE, di thioerythri tol. 31

for 30 s with N2 • Assay procedures for both NAD-ME and NADP-ME were exactly as described by Edwards et aL. (1982) except that the NADP-ME assay mixture additionally contained 5 mM OTT and the reaction was initiated with

50 µl 0 .2 mM MgC1 • 2

PEP oarboxykinase

Extraction buffer was as described by KU et aL. (1983) except that

1% (w/v) BSA was added and OTT was used instead of DTE.

After removal of a sample for chlorophyll determination, the extract was spun in a Beckmann Microfuge B for min. O .6 ml of supernatant was run through a Sephadex G-25 column preequilibrated with

50 mM HEPES-KOH (pH 8.0) containing 5 mM OTT, and c. 1.6 ml of eluate was collected (as described above for NAD-ME and NADP-ME).

The reaction mixture for the assay was exactly as described in

Hatch and Mau ( 1977) except that 2 uni ts of pyruva te kinase were used.

Preparation of OAA and calculation of PCK activity followed Hatch

(1973).

ChLorophyLL determination

Duplicates were taken from each sample extract for chlorophyll measurement in 80% acetone (Arnon 1949).

Re sul. ts and Discussion

PCR Bundle Sheath Anatomy

Table 3.1 lists the Eragrostis examined in currently recognised inf rag eneric groups (Laza rides in prep. ) , All have C 4 anatomy and are XyMS+ (see Fig. 3.1), and most can be unambiguously assigned as having 32

NAD-ME- or ECK-like leaf anatomy. In all field-collected and fixed

specimens, and even in some herbarium material, chloroplast position was

clearly either centripetal or centrifugal (Figs. 3.1 and 3.2), and

correlated perfectly with even and uneven vascular bundle outline

respectively. In those herbarium specimens where much of the tissue had

collapsed and the chloroplasts had become distorted or were not evident,

I found that the shape of the ECR bundle sheath and its cells was usually retained and anatomical 'determination' was made using this

feature alone. Species in which bundle sheath cell shape was atypical and/or did not completely correlate with chloroplast position and/or where chloroplast position was ambiguous, were described as 'anatomical

1 intermediates • These include species where field-collected and fixed material was examined, as well as species where only herbarium material was available.

29 species were found to have NAD-ME-like anatomy (23 native, 6 i'ntroduced), 28 species PCK-like anatomy ( 27 native, 1 introduced) and 7 species were classified as 'intermediate' (5 native, 2 introduced).

These compare with 28, 13 and 13 species respectively for southern

African Eragrostis (Ellis 1977). No species showed variation in major features of PCR bundle sheath anatomy (Table 3.1),

Most species with ECK-like anatomy had extension cells on at least a few of the smaller vascular bundles. A maximum of 13 such cells on one b.!ndle was observed (in E. elongata). Extension cells never numbered more than t,., per bundle in species wit.'1 NAD-ME-like anatomy; indeed, they were absent from most specimens of species in Groups 1-4 and from all species in Group 5 (Table 3 .1). When present, they were small and did not detract from the even, circular outline of the vascular bundles. 33

The varying extents to which chloroplasts filled PCR cells were ascribed to varying degrees of starch accumulation within them (perhaps associated with the time of day at which specimens were collected).

Although chloroplasts thus varied in size, a previously undocumented feature is the recognisably different appearance, in leaf blade transections, of the chloroplasts of each of the t;,u main anatomical types. In PCK-like species, chloroplasts were ovalish in shape but in

NAD-ME-like species they were conspicuously elongated, especially in material prepared for electron microscopy (Figs. 3.1 and 3.2).

In 'anatomically intermediate' species vascular bundle sheath outline was neither as even nor as uneven as in the two main types (Fig.

3 • 1 ) • Extension cells were found in specimens of all such species.

Though most chloroplasts were centripetal and resembled in shape those of species with NAD-ME-like anatomy, usually some were centrifugal in at least a few FCR cells of each vascular bundle (Fig. 3.2). E. eurvula, however, differed from other 'intermediates' in having exceptionally few centrifugal chloroplasts and noticeably more extension cells.

Anatomical types: infragenerie groups and evolutionary considerations.

Species of Eragrostis were assigned to their infrageneric groups quite independently of the collation of anatomical data. It is all t.'1e more remarkable, therefore, that each of these groups, defined primarily on spikelet characters, shows homogeneity with regard to vascular bundle sheath anatomy (Table 3.1). '!his is not the case for the sections of

Australian Eragrostis as understood by Bentham (1878). Only in Groups 4 and 12 (Table 3.1) is there anatomical variation, the three species involved all bein; 'intermediates', with one (E. eonfertifiora) being referred to DiandroehLoa (De Winter 1960) • Interestingly Group 11 is 34

composed only of 'anatomical intermediates' notwithstanding t.'1at two of

the species are of African origin. Also sugg es ti ve of the

'intermediate' nature of these species is evidence concerning ttNQ

distinctive categories of microhairs on the abaxial leaf epidermis of

Eragrostis species in general (Vindhya Amarasinghe pers. comm.). Each

category is restricted to species of one or other of the c anatomical 4 types but, in E. leptocarpa, E. parvif7,ora, and E. pilosa at least

( 1 intermediate' species: Table 3 • 1 ) ' the microhairs show

characteristics of both categories. curiously no microhairs at all have

yet been found in E. curvula.

Brown ( 1977) presented some speculation, but no evidence, for the

derivation of PCK species from NAD-ME species even though he also stated

that "the presence of both (types) within single genera su:igests that

perhaps they may be interconvertible during evolution". The existence

of the Eragrostis 'intermediates' lends some support to this idea, but

there is also a high degree of anatomical homogeneity within

infrageneric groups (Table 3.1) and no major intraspecific variation is known in Eragrostis (see above). Furthermore, since there appears to be

no evidence that 1 anatomically intermediate• species are intermediate in

terms of gross morphology, their origin, through hybridization or otherwise, remains unknown.

Ultrastructure: suberized lamella

A suberized lamella was present in the P::R cell walls of species with PCK-like and 'intermediate' anatomy and absent from those of

species with NAD-ME-like anatomy (Fig. 3.2, Table 3.3). In one species of the latter group, E. setifolia, striations were observed in PCR cell walls (Fig. 3.2b; see Hattersley and Browning 1981). 'these observations 35

confirm anatomical/ultrastructural correlations found by Hattersley and

Browning (1981). These authors cited three species in which

biochemical/ul trastructural correlations may break down (details given in Introduction), This study confirms that one of these, E, cUX'Vula, has a suberized lamella in its K:R cell walls, that its K:R cell chloroplasts are predominantly centripetal (some are centrifugal, making it an •anatomical intermediate': see above), but that it is biochemically NAD-ME (below).

Assays of c4 acid deca.rboxylating enzymes In all 11 species assayed, representing the full spectrum of anatomical variation in NAD-ME was found to be the main c Eragrostis, 4 acid decarboxylating enzyme (Table 3.2). No PCK and very little NADP-ME activity was detected. Only three Eragrostis species have previously been biochemically typed (E. cuz>vula, E. cilianensis (All.) Vign. ex

Janchen and E. superba Pe yr.), and all were likewise NAD-ME (Gutierrez et al. 1974, Hatch et al. 1975; Gutierrez and Edwards unpublished results in Brown 1977) • Eragrostis is the first chloridoid genus in which a breakdown of the correlation betwen PCR cell chloroplast position and decarboxylating enzyme has been reported.

The small sample size notwithstanding, species with NAD-ME-like anatomy showed considerably more activity of NAD-ME (x = 7.81 µmol mg

1 Chlor. min-1) than either 'intermediate' or PCK-like species (x = 5.24 and 5.36 µ mol mg-1 Chlor, min-1 respectively). There is no such pattern, however, in the anatomically variable NAD-ME Panicwn species studied by Ohsi;q i et al. ( 1982) • 36

Geographical distribution

'I.he occurrence in Australian state and Territory subdivisions of

native (only) Australian Eragroatia species and of the three anatomical

variants (NAO-ME-like, FCK-like and 'intermediate' ) is shown in Fig.

3.3. Total intra-subdivision species diversity ranges from 28 in the

Burke subdivision of Qleensland (Qld.) to 0 in the Stirling subdivision

in Western Australia (W.A.) and Kangaroo Island in south Australia

(S.A.). Diversity generally decreases southwards, consistent with the

trend for grasses as a whole (Hattersley 1983), whilst low numbers in c 4 central w.A. may also reflect a degree of under-collecting.

Species of each of the tw:> main anatomical types have markedly different distributions. PCK-like species reach their greatest diversity in northernmost Australia with 20 species occurrin:i in the

Darwin and Gllf sub-division in the Northern Territory (N.T.) and 19 in

the Cook subdivision ( Qld.) (Fig. 3. 3) • In these subdivisions they comprise 91% and 79% of total Eragroatia species respectively. High relative frequencies also occur in many coastal and near-coastal subdivisions (Fig. 3.4). NAD-ME-like species, by contrast, reach their greatest diversity in inland and central Australia, viz. 15 species occur in the Warrego subdivision (Qld.) and 14 in the Central North subdivision (N .T.) (Fig. 3. 3). The highest relative frequencies of these species are 100% and 89%, in N.!llarbor (S.A.) and Great Victoria

Desert (W.A.) subdivisions respectively.

Comparison of Figs. 3. 4 and 3. 5 clearly demonstrates correlations between anatomical type distributions and the main Australian bioclima tes recognised by Nix (1 982). Eragroatia species with NAD-ME- like anatomy dominate throughout the arid megatherm/mesotherm bioclimate 37

(B in Fig. 3.5) of central Australia, almost all of which has a mean

annual rainfall of <30 cm. Species with PCK-like anatomy are commonest

in the high (summer) rainfall, mega therm seasonal bioclima te (A in Fig.

3.5) of the northernmost subdivisions of Australia. Elsewhere, their

high (>50%) relative frequency closely matches: i) areas with a mean

annual rainfall of >60 cm1 ii) the extent of the mega therm bioclimate

(A) southwards along the east coast of Australia1 and iii) the extent of

the mesotherm/microtherm seasonal bioclimate (C) in southeastern and the

extreme southwestern parts of the continent (but here frequencies are

very low in these predominantly c3 zones). Though these marked overall trends are reflected in the distributions of the biochemically typed species (ignoring those of the introduced E. aurvuLa and E. minor), individual species of all anatomical types may nevertheless be very widespread. PCK-like E.

eZongata, for example, is recorded from 45 of the 73 subdivisions in

Australia, and from areas as climatically diverse as the temperate mesotherm/microtherm bioclimate of the south-west and south-east, the centre of the arid zone and the tropical far north of the continent.

The high diversity of Eragrostis in the Burke subdivision ( Qld. ) can be a ttri bu ted to the subdivision's straddling of t,,., of the three primary bicclimates (and their interzone) (A, AB and B in Fig. 3.5).

This subdivision is also listed as one of the world's regions with the highest percentage frequency of the sub-tribe Eragrostineae in the grass flora (21% 1 Hartley and Slater 1960). Eragrostis is by far the largest genus in the Eragrostineae. The "generally accepted view that the

Eragrostineae occur predominantly in regions of hot, dry climate"

(Hartley and Slater 1960) probably should be modified since as many species of Eragrostis have now been recorded from the tropical Cook 38

subdivision (Qld.) as, for exa~ple, from the arid central North

subdivision (N.T.).

Of the 31 native Eragrostis species occurring in South West

Africa/Namibia that have been anatomically typed (using data of Launert

1970 and Ellis 1977), 20 have typical NAD-ME -like anatomy, six are PCK- like and five are 1 intermediate'. Al though this sample size and the comparatively small rainfall range of South West Africa/Namibia (< 10 - c. 55 cm y-1 ; from Ellis et ai. 1980) do not permit full comparison with

Australian data, more species with NAD-ME-like anatomy occur in the lower rainfall districts (with < 30 cm y-1) than in the higher rainfall ones. Also repeating the Australian pattern, more (all six) PCK-like species occur in the higher rainfall districts but only two in the lower rainfall ones and none where rainfall is< 10 cm y-1 •

Concluding Remarks

The summarised results for species examined biochemically (Table

3, 3) clearly suggest that Eragrostia is a wholly NAD-ME-type grass genus, even though some of them exhibit PCK-like anatomical and ultrastructural features. There is no indication that the three anatomical NAD-ME/PCK 'intermediates' are biochemically intermediate.

These findings, together wit.'1 the results for Paniewn (Ohsugi and

Murata 1980 and 1981; Ohsugi et al. 1982), where four biochemically­ typed NAD-ME species have also been shown to have centrifugal PCR cell chloroplasts, cast doubt on the degree of reliability of using chloroplast position to distinguish between NAD-ME and PCK-type c 4 grasses. These correlations were first firmly established by G.ttierrez et ai. (1974; 1976) and Hatch et ai. (1975), Recognising the possibility of discovering exceptions, G.t tierrez et aL ( 197 4) refer to 39

Paniaum iaevifoliwn Hack. and P. diohotomi[ioI'Wn Michx. (amongst others)

as having centrifugal PCR chloroplasts but "decarboxylating enzymes

low". These are two of the four 'exceptional' Paniaum species identified by Ohsugi and Murata (1980) and Ohsugi et ai. (1982).

A further resemblance be~.reen Eragrostis and Paniown is possession of the suberized lamella by both P. foevifoiiwn and Eragrostis with

'intermediate' and l=CK-like anatomy, This means that, whilst the anatomioaL/ultrastructural correlations, established by Hattersley and

Browning (1981), between chloroplast position and presence or absence of a suberized lamella still hold in these genera, their bioohemioal/ul trastructural ones need modification. This finding of

NAD-ME species with a suberized lamella in their PCR cell walls also means that there is now no known structural criterion which distinguishes unequivocably between NAO-ME and PCK-type species.

Undoubtedly, therefore, at least some species have been mistyped in studies such as those of Ellis (1977) and Hattersley (1984), but mistyping is not expected to have been on a large scale.

The suberized lamella has been implicated in reducing apoplastic and/or symplastic co2/HC0 3 - leakage out of the PCR cell corrpartment in c grasses (references in Rattersley and Browning 1981 ), and PCR cell 4 chloroplast position variation among c 4 types has been postulated to represent "different compromises between the conflicting demands of maximizing rates of PCA-PCR metabolite transport on the one hand, and of reducing rates of co leakage on the other" (Hattersley and Browning 2 1981). If one accepts this, the existence of chloroplast position variation within a sing le biochemical type in Eragrostis and Paniown questions whether site of c 4 acid decarboxylation (i.e. c 4 type per ae) is an important contributing factor to co 2-leakiness of PCR tissue; NAD- 40

ME is located in mitochondria and JX:K in the cytoplasm (references in

Hattersley and Brownil>:j 1981).

The only direct physiological measure of ICR -leakiness may be co 2 the oxygen-insensitive post-illumination burst (P.I.B.), the co 2 transient of which (in grasses) exhibits three different forms (Downton

1970) apparently correlated (retrospectively) with c type (Hattersley 4 and Browning 1981). There was one exception, Paniawn virgatwn L., which

these authors report as PCK-type on the basis of anatomical and ultrastructural criteria, yet which has a type II transient typical of

NAD-ME species (Downton 1970). More recently (see Chapter 5), this

species has been found to be in fact another NAD-ME Paniawn which is

PCK-like anatomically. P. I .B. evidence therefore suggests that actual c type is important to co -leakage. supporting this idea is the fact 4 2 that the four NAD-ME Paniewn species with centrifugal PCR cell chloroplasts have been shown to have a similar P.I.B. to that of typical

NAD-ME species (Ohsugi and Murata 1980; Ohsugi et al., 1982). Since one of these is Paniawn LaevifoUwn, with a PCR cell suberized lamella, doubt could now be cast on the importance of this structure in maintaining a co 2-tight ICR corrpartment. It is not yet known, however, if other Paniewn 'exceptions' have a suberized lamella, if the P. laevifoliwn material used by Ohsugi and co-workers possessed a lamella, or indeed whether the latter really is the same species as used by

Hattersley and Browning (1981). Clearly more work is required on the

P.I.B., and of a quantitative nature; in any event, species variation in

Eragrostis and Paniawn should permit clarification of structure-function relationships in this regard.

There is clearly an intriguing differential geographical distribution of the tv.o main anatomical types in Eragrostis (Figs. 3.3 41

and 3,4), The predominance of NAD-ME-like species in arid areas of

Australia complements the findings of the only previous study to investigate c4 type/phytogeographical relationships {Ellis et al. 1980; for South West Africa/Namibia). Equally clearly distributions in

Eragrostis are related to anatomical type rather than to biochemical type per se. This is not to say, however, that biochemically determined

NAD-ME grasses in general will not still be found to dominate the c4 grass flora in arid Australia.

While the results of this study somewhat undermine previously established anatomical/biochemical correlations with respect to chloroplast posi tion/C type, they may reflect the existence of a more 4 di verse set of relationships than previously recognized, Perhaps they will eventually lead to a clearer, though more complex, appreciation of structure-function relationships, and to this end work is being extended to cover a much wider taxonomic range of c 4 grasses. Table 3.1, c4 types based on PCR bundle sheath anatomy in Australian E:t'a{JZ'oBtis species.

Introduced species are asterisked. Species arranged in ten ta ti ve infrageneric groups (Lazarides in preparation). Species assigned to

Lazarides are new, and descriptions are in preparation.

Group Anatomical type Species

NAO-ME-like •superba Pe yr. 1 (H3) 2

2 NAD-ME-like dielsii Pilger (F2), fafoata (Gaudich.)

Gaudich. ex Steudel (H2) , lacunaria

F ,Muell. ex Be nth. (F11) , lanioaulis

Lazarides (H1), longipedioeUata B.Simon

(H2), miorooarpa Vick. (F2), pergraoilis

S.T.Blake (F1), subtilis Lazarides (H1),

triquetra Lazarides (H2)

3 NAD-ME-like desertorum Domin (H2), eriopoda Ben th.

(F6) , lanif"lora Benth. (F2), lanipes

C.E.Hubb. (H2), olida Lazarides (H2),

setifolia Nees (F9), xerophila Domin (F4)

4 NAO-ME-like advena (Stapf) s.Phillips (H2),

australasioa (Steudel) C.E.Hubb. (F1),

infeounda J.Black (H2) , megalosperma

F .Muell. ex Benth. (H2), 'tenuifolia

(A.Rich.) Hochst. ex Steudel (F2) Intermediate (Schrader) Nees1 (F3) ,

traahyaa:rpa (Benth,) Domin (H1)

5 NAD-ME-like 'b arre.-ie1'1,~. . Daveau 1 ( F 1 ) , • ci Lianensis

(All.) Vign. ex Janchen 1 ( F 1 ) ,

Leptostaahya (R.Br.) Steudel (F3), ••rnnor

Host ( F 1 ) , moLybdea Vick. ( F 4) ,

•neomex-z-aana • Vasey (H3)

6 PCK-like 'paniaiformis (A.Br.) Steudel (H1 ) ,

unioLoides (Retz.) Nees ex Steudel (H2)

7 PCK-like benthamii Mattei (F1 ), 'bLoxsomei' 3 (FS),

brouinii (Kunth) Nees ex Steudel (F4),

eLongata (Willd.) Jacq. f. (F9), sororia

Domin ( FS)

8 PCK-like conainna (R.Br.) steudel (H2), faLLax

Lazarides (H3), fiLiaauLis Lazarides (H2)

hirtiaauLis Lazarides (H1 ) , petraea

Lazarides (H2), potamophiLa Lazarides

(H2), rigidiuscuLa Domin (H2), schuLtzii

Benth. (H1), spartinoides steudel (H3),

stenostaahya (R.Br.) Steudel (H1)

9 PCK-like basedowii Jedwabn. ( F 1 ) , aumingii S teudel

(H3), teneLLa (L.) P.Beauv. ex Roemer &

Schultes (H3) 1 0 PCK-like interrupta P.Beauv. ( F2} , pubescens

(R.Br.} Steudel (H1), speciosa (Roemer &

Schultes) Steudel (F2), stagnalis

Lazarides (H2}, sterilis Domin (H2}

1 1 Intermediate leptoaarpa Ben th. (F2} , parvifl,ora

( R. Br • } Tri n. ( F 1 9} , •pi losa ( L. } P •

Beauv. 1 (F1}, *tef (Zucc.} Trotter4 (-)

1 2 Intermediate aonfertiflora J.Black (H2)

PCK-like tenellula (Kun th} Steudel (F1 }

Species not assigned to infrageneric groups

PCK-like cassa Lazarides (H1), eaarinata Lazarides

(H2)

NAO-ME-like kennedyae F.Turner (F2)

Anatomical type for this species agrees with that given by, or

inferred from, Ellis (1977).

2 H: her barium specimen ( s) only. F: field-collected and -fixed

specimen ( s) • Figures refer to numbers of different accessions for

which anatomical determination was made.

3 The status of this taxon is currently undetermined.

4 Anatomical type inferred from Ellis (1977) only. Table 3. 2. Activities of c 4 acid decarboxylating eru.:ymes in 11.uatralian Eragroatia and control\ spec1es.

Introduced EiugrootiB species are asterisked. Three series of experiments were done for each enzyme, with the numbered PCK series being performed on separate extracts from those of t'IA.0-ME and NADP-M.E which were assayed using the sa111e extract for .:ach series. &lch b'ragl'oatia species was extracted at least twice (except E. aetifolia and E. parv·'flo:ria for PCK, and £'. auroul.a for NAO-ME and NA.OP-ME). Each number represents the mean of two

replicate assays on a sinqle extract, NA.D-M.E, NAD-malic enzyme; fCK, PEP carboxykinas~) 1 NADP-M.E, NADP-mctlic enzyme •

mg-1 1 C4 Type Species Ac ti vi ty (µ11\0l Chlor. min- NAO-ME PCK NA DP-ME 2 3 x 2 3 x 2 3 x

NAO-ME-like E. die'laii 7 .13 9.38 8.26 0 0 0 0.21 0,49 0.35

(anatomically) E. 'laounaria 8.0) 6,96 7,50 0 0 0 0.29 0.25 0.21

E. minoz•>i a. 39 tL 1 3 8.26 0 0 0 0.21 0.18 0.20

E. setifolia 6-49 7.95 7.22 0 0 0.15 0.22 0.19

PCK-like E. benthamii 2.56 7.27 4 .92 0 0 0 0,25 0.25 0.25

3 (anatomically) E. brouwii 4.05 4.57 4.22 0 0 0 0.103 0.10 0.10

E. eiongata 4.78 4.39

E. Bpeoiosa 7,94 7.32 7.63 0 0 0 0.30 0.24 0.27

. () Intermediate E. curvul.a 5.99 5.99 0 0 o. 20 o. 20

(anatomically) E. 'leptooarpa 5. 31 7.34 6.33 0 0 0 0.15 0.20 0.18

E. parvifl-ora 2.52 5.06 3.80 0 0 u.15 0.20 0.18

2 NAO-ME controls Bleusine oor>acana ( J,. } Gaertn. 3.01 3.01

E. indica (L.) Gaertn. 0 0

Paniau.m mil-iaoewn L. 0 0 NAO-ME !'CK NA DP-ME 2 3 x 2 3 x 2 3 x

PCK controls P. llllximwn Ja.cq. 1o.1 3 10.13

Ch Loria gayana Kun th 0.61 0.61 0 .14 0.14

c. truncata R. Br. 11.59 10.10 10.85

NADP-ME contra 1 Cymbopogcm oitratus (DC) Stapf l o06 0.69 0.74 0.83 0.22 5.61 5.ao 6.54

control T~itiown L. 0.15 0,28 0.17 0.09 0.15 0.07 0.10 c3 aeativwn o.o9

c control species have previously been biochemically typed: (Rathnam and Chollett 1978) /<.'. {Gutierrez 1974); 4 Eleu8ine aoraaana 1 indica. et al.

Panoiwrt miliaoewn, P. uuximwnJ Chloris gayana (Gutierrez et at. 1974; natch and Kayawa 1?74; Hatch et at, 1975); C, trunoata, Cymbopogon oitratuB

('l'axonomy Unit unpublished results),

2 J{l.eusine ooraoana used as a control in Series 1, Er>agrostis aurvu1.a in Series 2 (previously typed as Nl\O-M£ by OJtierrez et al. 1974 and Hatch et al.. 1975) and for Series J ten species had already been determined

3 values are means of two separate extracts: NAO-ME: values 3.80 and 4. 30 <.1ud both NADP-ME values 0.1 O. Table 3.3. S.ua.ar.ary of anato.U.c.:tl_. ultrastructural and f~t.ures of biocheaically-typed l!.'ragroati-a species.

1 4 Species Accession Dec a rboxy la ting enzyme Chloroplast position2 Suberized lamella 3 PCH bundle sheath outline

die-1.aii G14 NAD-ME cp even

lacunal'ia G212 NAD-ME cp even

nrinol' G'277 NAO-MB cp even

aetifol-ia X40 NAD-ME cp even

aUPVU'la. X206 NAO-ME cp (cf) + intermediate

'leptoca:rpa G293 NAO-ME cp (cf) + intermediate

parvifl-ora G270 NAD-ME cp (cf) + intermediate

benthamii X200 NAO-ME cf (cp) + uneven

brwnii X202 NAO-ME cf (cp) + unev~n

eZongata G307 NAD-ME cf (cpl + uneven

apeoioaa G273 NAO-MB cf (cp) + ufleven

Accession numbers of Taxonomy Unit.

2 In PCR cells. Abbreviations: cp centripetal, cf centrifU<]al. Brackets denote position of a minority of chloroplasts.

3 Present (+) or absent (-) in radial and outer tangential walls of PCR cells.

4 See Materials and Methods, and Fig. J.1. Fig. 3.1 • Light micrographs of vascular bundles from transverse

sections of Eragrostis leaf blades. a and e, hand-cut sections of fresh

leaf material fixed in FAA; e, hand-cut section of fresh leaf material; b, d and f, mi crotomed sections of fresh leaf material fixed and

embedded as for electron microscopy (see Materials and Methods), and

stained in toluidine blue. Abbreviations (in hand-cut sections only):

PCR, Photosynthetic Carbon Reduction ('Kranz') cells; ms, mestome

sheath; c, chloroplasts; ec, extension cell; x, rnetaxylem vessel element. Bar = 25 µm. a, E. australiense. One major and t;,o minor vascular bundles. NAD-ME-like anatomy with even K:R bundle sheath outline and PCR cells with straight radial walls and centripetal chloroplasts. b, E. iaeunaria. Two minor vascular bundles. NAD-ME- like anatomy as in a. Chloroplasts very elongated. e, E. pilosa. One major and one minor vascular bundle. 'Intermediate 1 anatomy with uneven

PCR sheath outline and mainly centripetal chloroplasts in PCR cells. d, E. parvifiora. Two minor vascular bundles. 'Intermediate' anatomy as in a. PCR. cell chloroplasts very elongated as in b, mainly centripetal but some clearly centrifugal. e, E. elongata. One major and t;,o minor vascular bundles. PCK-like anatomy with uneven PCR sheath outline, extension cells and mainly centrifugal chloroplasts in K:R cells. f, E. elongata. One major and one minor vascular bundle. PCK­ like anatomy as in e. Chloroplasts not elongated.

Electron micrographs of PCR cells and PCR cell walls from

transverse sections of Eragrostis leaf blades. Abbreviations: PCR,

Photosynthetic Carbon Reduction ('Kranz') cells; OTW, outer tangential

wall; ITW, inner tangential wall; RW, radial wall( s); c,

chloroplast; SL, suberized lamella. a, E. setifoLia. PCR cell. NAD­

ME-like anatomy with centripetal, elongated chloroplasts and straight

radial walls. Striations were visible in cell walls. Bar = 5 µm. b,

E. setifoiia. Two radial walls of adjacent PCR cells. No suberized

lamella present. Bar = o .4 µ m. E. parvifLora. PCR cell.

'Intermediate' anatomy with elongated chloroplasts in both centripetal

and centrifugal positions and relatively inflated outer tangential wall. Bar = 5 µm. d, E. parvifLora. J\lnction of outer tangential walls of adjacent PCR cells, each with a suberized lamella. Bar =

0.4 µm. e, E. speoiosa. PCR cell. PCK-like anatomy with predominantly centrifugal, non-elongated (ovalish) chloroplasts and very i:nflated outer tangential wall. Bar = 5 µ m. f, E. speoiosa. Outer tangential wall of PCR cell with suberized lamella. Bar = O .2 wn. PCR

0 Fig. 3.3. N..!mbers of native Eragrostis species in 1mstralian State and

Territory subdivisions grouped according to bundle sheath anatomy.

Upper integer (left): number of species with NAD-ME-like anatomy. Upper

integer (right, underlined): number of species with PCK-like anatomy.

Lower integer: number of species with 'intermediate' anatomy. State

and Territory abbreviations: W.A., Western Australia; N.T., Northern

Territory; S.A., Sout.'1 Australia; Qld,, QUeensland; N.s.w., New South

Wales; Vic., Victoria; Tas., Tasmania. subdivision abbreviations: SG,

Stirling; gvd, Great Victoria Desert; DG, Darwin & Gulf; CN, Central

Australia N:Jrth; NU, N..!llarbor; KI, Kangaroo Island; CK, Cook; BK,

Burke; WO, Warrego. CK

4 19 l - 4 ,- 8 •,- • BK • 16 3-

v

10 5 1 -

0 500 km ~ Tas.~v Fig. 3.4. Relative proportions of numbers of native Eragrostis species

with NAD-ME-like and ECK-like anatomies in Australian State and

Territory subdivisions. Species with 'intermediate' anatomy omitted.

Oblique shading: > 50% of species in subdivision have ECK-like

anatomy. Cross hatching: equal numbers of s_pecies in subdivision with

PCK-like and NAD-ME-like anatomy, Unshaded! > 50% of species in

subdivision have NAD-ME-like anatomy. N .B.: Stirling (W .A.) and

Kangaroo Island (S.A.) subdivisions have no recorded Eragrostis species and many southerly subdivisions have only 1-6 species (refer to Fig.

3. 3) •

Fig. 3.5. Main Australian bioclimates of Nix (1982), with 30 cm and 60 cm isohyets (median annual rainfall, 50 percentile; adapted from

'Rainfall' Map, Atlas of Australian Resources Second Series, 1970,

Geographic Section, Department of National Development, Ganberra),

Bioclimate abbreviations: A, megatherm seasonal; B, megatherm/mesotherm arid; c, mesotherm/microtherm seasonal. AB and BC are major interzones of semi-arid environments linking bioclimates A and B across northern

Australia and bioclimates B and C across southern Australia. .... ··············· ······ .. ····· ····· ········

B 30•• ··· ... .. ···...... ········· . .. CHAPTER 4

AUSTRALIAN c4 GRASSES (POACEAE): LEAF BLADE ANATOMICAL

FEATURES IN REIATION TO ACID DECAROOXYLATION TYPES. c4

To be published in Australian Journal of Botany, 1987, 35 (3).

H.D.V. Prendergast, P.W. Hattersley.

Manuscript received 16 June 1986, accepted 26 September 1986. 42

Using fresh, preserved and herbarium material, I have exarni ned the leaf blade anatomy of the Australian grass flora (Poaceae) in c4 relation to acid decarboxylation type (NADP-malic enzyme, NADP-ME; c4

NAD-malic enzyme, NAD-ME; phosphoenolpyruvate carboxykinase, PCK). The survey included alrrost all species of the subfamilies Arundinoideae and

Chloridoideae and of the panicoid genus Paniawn. For the Panicoideae I sampled all genera, but for many of these only representative species were examined. The data are cross-referenced with available anatomical and biochemical data on types, in particular with my own, which show c4 a number of somewhat taxonomically predictable exceptions to established anatomical/biochemical correlations. c4 types are briefly considered in relation to geographical distribution, and it is pointed out that NAD-ME type species are poorly represented in agronomical research on the arid and semi-arid zone, where many of these occur. 43

Introduction

As in nany other parts of the world, grasses (Poaceae) are of

enormous economic and ecological importance in Australia. They form the

basis of the grazing industry, not only in the extensive natural

grasslands which cover about 25% of the continent (estimate from

vegetation nap of Moore and Perry 1970), rut also in the induced

grasslands formed as a result of tree clearance, and in the understories

of the nany types of woodland which cover a further 25% of the

continental area (see Moore 1970; Beadle 1981; Gillison and Walker 1981;

Groves and Williams 1981). They are, furthermore, the third largest

plant family in 1\ustralia (see Morley and Toelken 1983) with some 220 genera (Watson and Dallwitz 1985) and >1200 species.

Grasses have long been distinguished as either 'cool season' or

•warm season' species depending on their time of growth (see Moore

1970). This fundamental difference largely mirrors, and results from,

their respective differentiation into and grasses (for reviews of c3 c4 c photosynthesis see Hatch and Osmond 1976 and Edwards and Buber 4 1 981) • The two photosynthetic pathways are not randomly distributed

among grasses taxonomically. Of the five subfamilies recognised by

Watson (1985), the Pooideae and Bambusoideae are exclusively , et ai. c3

the Chloridoideae are (although Ellis 1984 has reported one c4 c 3 species) whilst the Arundinoideae and Panicoideae contain both and c3 c4 species (Hattersley and Watson 1975, 1976; Brown 1977; Ellis 1977).

There is also some predictability in the taxonomic distribution of the

deca::boxyla ti on types of the c pathway: NADP-malic three c 4 acid 4 enz yrne type (NADP-ME), NAD-malic enzyme type (NAD-ME) and

phosphoenolpyruvate carbcxykinase type ( PCK). The only biocherni cally

typed species in the Arundinoideae are NADP-ME, while in the 44

Chloridoideae they are either NAD-ME or PCK. The panicoid supertribe

Andropogonanae contains only NADP-ME species but all three types are c4 found in the other panicoid supertribe, the Panicanae (Gutierrez et ai.

1974; Hatch et aL 1975; Hattersley and Watson 1976; Brown 1977; Ellis

1977; see Chapters 3 and 5). There is also some biochemical variation within genera in beth the Panicanae and the Chloridoideae.

Relatively few species have been biochemically typed conpared wi tJ1 determinations of type predicted from leaf blade anatomy (e.g. Ellis c4 1977; Ha ttersley 1984) • The latter are based on established

1 correlations between chloroplast position in PCR cells and C4 type

(Gutierrez et ai. 1974; Hatch et al. 1975) and between the XyMS character and C4 type (Hattersley and Watson 1976; Brown 1977) (see

Table 4. 1 ) • It is now known, however, that exceptions to beth these correlations exist viz. in Paniawn (Ohsugi and Murata 1980, 1981; Ohsugi et al. 1982) and in Alloteropsis, Enneapogon, Eragrostis, Eriaahne,

PheidoahLoa, Triodia and Triraphis (Chapters 3 and 5).

From an agronomic point of view it is clearly of importance to understand what factors influence the geographical distributions of grasses. To this end correlations between grass taxonomy and phytogeography have been investigated by Hartley ( 1958a, 1958b) and

Hartley and Slater (1960). subsequent to the discovery of c4 photosynthesis and its biochemical variants, Ellis et al. ( 1980) used anatomical predictions of type in grasses as the basis for an c4 analysis of type distributions in south West Africa/Namibia. The c 4 exceptions to anatomical/biochemical correlations mentioned above, however, clearly reduce the feasibility of an analysis of

Photosynthetic carbon Reduction cells: equivalent to Kranz cells, defined in Hattersley et al. (1976). PCA cells, Primary Carbon Assimilation cells; usually equivalent to c mesophyll. 4 45

biochemical/phytogeographical relationships relying only on anatomical predictions of c 4 type. Nonetheless, the work of Ellis et aL. ( 1980) showed that species with 'classical' NAO-ME type leaf anatomy become an increasingly prevalent part of the c grass flora (in terms of species 4 numbers) as mean annual rainfall decreases. Furthermore, there are similiar structural/phytogeographical correlations in Australia within

NAO-ME Eragrostis (Chapter 3), so it is these correlations which may be of agronomic significance. In order to analyse such correlations in the

Australian c 4 grass flora as a whole, I have documented the leaf blade anatomy, especially the PCR (Kranz) sheath characteristics, of a large number of Australian c4 grasses, and set the results alongside previous anatomical and biochemical data.

Materials and Methods

Complete hand-cut transverse sections from the central third of mature leaf blades were made from fresh material, from herbarium material (Australian National Herbarium, canberra) or from specimens collected in the field and preserved in FAA. Species were scored for currently recognised anatomical features which distinguish c types (see 4 Table 4. 1 ) •

Simon's (1978) check list of Australian grass species was used as the basis for sampling, supplemented by recent taxonomic work (Kakudidi

1984; I

Simon 1982, 1984a, 1984b, 1984c; Webster 1983). For generic compositions of subf2milies and supertribes, the classification of

Watson et ai. (1985) is used. 46

Results

Subfamily Chloridoideae

All 180 species examined, from 32 genera, are XyMS+ (Table 4. 2),

Sixteen genera ( 32 species) have only even PCR bundle sheath outlines and, where discernible, centripetal PCR cell chloroplasts (e .g, Figs,

This is the 'classical' NAD-ME leaf blade structure. Of the species sampled, ten have in fact been biochemically assayed and all are NAD-ME. By contrast, seven genera have only uneven outlines and, where discernible, centrifugal/peripheral chloroplasts (e.g. Figs. 4.1 a,

4.3a, 4.3a). This is the 'classical' PCK leaf structure. Seven of the

32 species examined have also been biochemically assayed and all are

PCK.

Twc genera, Leptoahloa and Sporobolus (Figs. 4.1 a, 4.1 b), contain species of both the preceding anatomical types. The eight species which have been biochemically assayed show 'classical' associations between

Anatomical variation in Eragrostis, on the other anatomy and c4 type. hand, is not mirrored by biochemical heterogeneity, 1 3 species, comprising both the above anatomical types as well as three species with intermediate anatomy, have all been biochemically assayed as NAO-ME

(Chapter 3). Lepturus is also anatomically variable but no biochemical data are available.

Triraphis and Enneapogon have uneven outlines (Figs. 4.2a, 4.2b).

Whilst Triraphis mollis has centrifugal/peripheral chloroplasts, species of Enneapogon have either mainly centrifugal, centrifugal/peripheral or centripetal/peripheral PCR cell chloroplasts. Triraphis mo His and seven species of Enneapogon have been biochemically determined as NAO-

ME.

The 12 species of Pleatraahne, Sympleatrodia (but see Comments on 47

S. graailis) and Triodia that were examined exhibit the unusual combination of even sheath outlines and mainly centrifugal chloroplasts. T. saariosa, the only species examined biochemically, is

NAD-ME,

Subfamily Arundinoideae

All 17 species of Aristida that were examined are XyMS- and have a double chlorenchymatous sheath (Table 4.3). In the inner sheath ( the

PCR sheath according to Ha ttersley and Browning 1981) chloroplasts are centrifugal/peripheral, while in the outer sheath they are centripetal. The outlines of both sheaths are neither clearly uneven nor even (i.e. intermediate), T'M'.> species, A. big Landu Losa and A. latifolia, have been biochemically typed as NADP-ME.

Pheidoahloa and Eriaahne are XyMS+ (Table 4.3; Figs. 4.2a, 4.2dl.

Pheidoahloa graailis and 33 species of Eriaahne (Fig. 4.2a) have uneven

PCR sheath outlines and, where discernible, centrifugal/peripheral chloroplasts. Of these species, P. graailis, E. aristidea, E. glabrata and E. ovata are known tc be NADP-ME. The four remaining Eriaahne species have centripetal chloroplasts; in three species, E. dominii, E. fastigiata and E. pulahella, (Fig. 4.2d) the sheath outline is even but in E. obtusa it is intermediate and there are also some peripheral and centrifugal chloroplasts. Nevertheless, both this species and E. pulahella have also been shown to be NADP-ME.

Subfamily Paniaoideae supertribe Panicanae (excluding Paniaum)

Sampling of the 33 Australian c 4 genera (all species of 19 small genera and representative species only of larger genera) indicates that 48

there is 1i ttle intrageneric variation with regard tc the XyMS character, PCR sheath outline and PCR cell chloroplast position (Table

4. 4) • Neurachne, however, also contains c 3 and c 3-c4 species (Hattersley et al. 1982; Hattersley and Roksandic 1983; Hattersley and stone 1 986) •

Twenty genera are XyMS- and have uneven FCR sheath outlines; chloroplast position, wherever discernible, is centrifugal/peripheral.

This is the 'classical' NADP-ME leaf structure. Neuraohne rrunroi and

Paraneuraohne mueLLeri differ in having an intermediate and even outline respectively. 23 species (including 6 non-Australian species not listed in Table 4.4) of nine genera have been biochemically typed as NADP-ME.

In four genera there are differing degrees of variation for the

XyMS character. In ALLoteropais this variation is interspecific: A. oimioina is XyMS+ and A. aemiaLata is XyMS-. surprisingly' the latter is PCK biochemically. In monotypic PaeudoohaetoohLoa and in XeroohLoa imberbia (two other XeroohLoa species are XyMS-), individual veins of the specimens I examined are XyMS- or are variable (see Table 4.4). In

Spinifex the leaf of one plant of S. aerioeus is entirely XyMS+, that of another has four XyMS+ veins and one variable one, whilst in the only examined leaf of a specimen of S. hirsutua three veins are XyMS- and five are variable.

seven genera are XyMS+, have uneven FCR sheath outlines and centrifugal/peripheral PCR cell chloroplasts i.e. 'classical' PCK leaf structure. 24 species (including twelve non-Australian species not listed in Table -A· 4) -of Tive-genera- have --been biochemically --typed--as

PCK. No species of Arthragrostis or Thuarea has been typed.

Yakirra is the only panicoid genus (apart from Panioum) containing species which are XyMS+ and have even K:R sheath outlines and 49

centripetal cell chloroplasts i.e. 'classical 1 NAD-ME leaf structure, None of the five species has been biochemically typed,

Panieum

Of the 21 species examined (Table 4.5), two, P. antidotaie and P. bulbosum, are XyMS- and have uneven PCR sheath outlines and centrifugal/peripheral PCR cell chloroplasts, Both species are NADP-ME.

Of the seven XyMS+ species with uneven sheath outlines and centrifugal/peripheral chloroplasts, two have been biochemically typed:

P, maxirrrwn as PCK and P. sahinzii as NAD-ME. The remaining 1 2 XyMS+ species have even PCR sheath outlines and, where discernible, centripetal chloroplasts. Of these P. aapiUare, P. aoloratum, p, deaompositum, P. effusum and P. miLiaaeum have been biochemically determined as NAD-ME.

Discussion

Herbarium material has not previously been used in a large scale survey of leaf blade anatomical features in relation to c4 type. In such material of alrrost all the species I examined PCR cell shape and

PCR sheath outline are reasonably well retained. Even when this is not the case, as in Heteraahne gulliveri (Fig. 4.3a), the mere presence of extension cells permits outline to be characterised as uneven (Table

4.1; but cf. Astrebfo squarrosa, Table 4.2). Chloroplast position, however, is often impossible to describe al though, for some reason, the centripetal chloroplasts in species with an even sheath outline are more often, and better, preserved in herbarium material than are centrifugal/peripheral chloroplasts in species with an uneven outline.

Al though characterisation of PCR sheath outline is based on purely 50

subjective assessment, this usually presents few problems in small veins, especially in XyMS+ species. An exception, Lepturus repens, is illustrated in Fig, 4.3d: t,'1e outline is generally even but because t;;o cells in each vein clearly have inflated outer tangential walls, I describe this outline as uneven. In other species, such as Aristida spp. (Table 4,3), Eriaahne obtusa (Table 4, 3), Neuraahne munroi (Table

4,4) and SympLeatrodia graaiLis (Table 4,2) the outlines appear genuinely intermediate.

In sections of fresh leaf material, as pointed out for Eragrostis

(Chapter 3), centripetal PCR cell chloroplasts often look distinctly elongated conpared with the more oval shape of centrifugal/peripheral chloroplasts. Ellis ( 1977) described centrifugal chloroplasts of PCK- type species as "disc-like" but did not mention the shape of centripetal chloroplasts in NAD-ME type species. Chloroplast shape is best defined in material fixed for electron microscopy (see Fig. 4. 1 ) ; it may, however, be variably obscured and affected by preservation of leaf blades in FAA and usually, rut not always, by the desiccation of herbarium specimens. As far as I know, only NAD-ME Enneapogon species

(Fig, 4.2b) have the combination of uneven PCR sheath outline and elongated chloroplasts, some of which are centripetal. In this respect they are fairly similiar to Eragrostis species with intermediate anatomy

(Chapter 3) • Unlike elongated chloroplasts in all other genera, those of Enneapogon are not aligned perpendicular to the inner tangential walls but tend instead to lie close to, and often parallel with, all PCR cell walls, PCR cell chloroplast dimensions are available for only four c grass species (NAD-ME ELeusine aoraaana, Rathnam and Das 1975; PCK 4 ChLoris gayana, and NADP-ME Sorghum biaoLor L. and Zea m:i.ys, W:>o et ai.

1971). 51

The detailed analysis of Eragrostis (Chapter 3) revealed no

intraspecific variation in leaf blade anatomy, and this study found

evidence of only a little. In southern A.frica, ALLoteropsis semiaLata

has both and subspecies (Ellis 197 4a, 197 4b; Gibbs Russell 1983) c 3 c 4

and in MiaroahLoa aaffra some specimens have uneven PCR sheath outlines

and centrifugal PCR cell chloroplasts whilst others have even outlines

and centripetal chloroplasts (Ellis 1977). Although Ellis (1977) stated

that "this finding may be superficial and is probably due to

identificatory or classificatory inadequacies", it is noteworthy that

the even outline of the one herbarium specimen of M. indiaa that I

examined contrasts with the uneven outline (and centrifugal

chloroplasts) of southern African specimens (one of which is illustrated

in Ellis 1977). Miaroahloa species may then be genuinely variable in

their PCR sheath anatomy (and C type?) • Hattersley ( 1976) recorded 4 that Spinifex seriaeus is variable for the XyMS character, although most

veins are XyMS-. In this study I found one specimen to be variable in

one vein but XyMS+ in all others, and the other specimen to be entirely

XyMS+. It would obviously be of interest to know if such anatomical

variation is mirrored by biochemical variation as well (see Chapter 5,

which shows that one plant, at least, of S. seriaeus is NADP-ME).

One important conponent of the A.ustralian c 4 grass flora excluded from this survey is the panicoid super tribe Andropogonanae. i\ll 67 genera, w:>rldwide, which have been recorded for the XyMS character, are

XyMS- (Watson et ai. 1986: incorporating original observations as well

as data from Metcalfe 1960; Hattersley and Watson 1976; Brown 1977 and

Ellis 1977) • Furthermore, since 12 species of eight of these genera

(Andropogon, Cymbopogon, Euahlaena, Miarostegium, Saaaharwn,

Sorghastrwn, Sorghum and Zea) have been biochemically determined as 52

NADP-ME (Kanai and Black 1972; G.ltierrez et aZ. 1974; Hatch and Kagawa

1974a, b; Hatch et ai. 1975; Usada et aZ. 1984; Chapter 3), all

available evidence to date suggests anatomical and biochemical

homogeneity within this supertribe.

The Arundinoideae are biochemically homogeneous despite anatomical

variation. The finding that two biochemically assayed species of XyMS­

Ar,ietida are NADP-ME confirms the only previous determination of c 4 type in this genus (by Gutierrez et ai. 1974). F:riaohne and PheidoahZoa

species are also NADP-ME but are extraordinary in being the only known

XyMS+ species of this type in the Poaceae. The only known non-NADP-ME

xyMS- species is the panicoid AUoteropsie eemiafota, which is PCK

(Chapter 5; but cf. Frean et aZ. 1983).

The most diverse genus, anatomically and biochemically, is Paniaum

( Panicoideae) , As it is presently constituted taxonomically, Panioum

contains not only species of all three c 4 types but also PCK and NAD-ME

species which appear to be anatomically indistinguishable from one

another. some at least of the biochemical heterogeneity may be

attributable to taxonomic confusion: R.D. Webster (in prep,), in

transferring P. maximum to UroahZoa, has removed the only Australian

(but introduced) species which has actually been biochemically

determined as PCK. The five other untyped xyMS+ Panioum species with uneven PCR sheath outlines (Table 4. 5) could either be PCK, like P.

maximum, or be NAD-ME like P. eohinzii (Table 4.5). The anatomical observations reported here support the validity of other recent

nomenclatural changes in Australian c 4 grasses. Examples include

OxyahZorie soariosa which, prior to revision (Lazarides 1985), had been

the only Chloris with an even sheath outline and centripetal

chloroplasts; and all five species of the new genus Yakirra (Lazarides 53

and Webster 1985) which are the only Australian panicoids outside of

Paniown to have an even outline and centripetal chloroplasts. My

observations confirm the anatomical heterogeneity within MioroohLoa and

SporoboLus reported by Ellis ( 1977) as well as indicate variation on a

worldwide level within Braohyaohne, Eustaohys and LeptoohLoa (cf. Table

4.2 and Ellis 1977).

The anatomical and biochemical investigations presented here and in

Chapter 5 have led to a considerable increase in the known number of

anatomical/biochemical combinations in c 4 grasses. The corollary of this is that anatomical predictions of biochemical type are less

reliable than previously realised. A good example concerns the suite of

characters which comprises XyMS+, uneven PCR sheath outline and

centrifugal/peripheral PCR cell chloroplasts. 1Ihis combination occurs

not only in 'classical' PCK type species (e.g. Paniaum maximum,

Panicoideae), but also in some NAD-ME species (e.g. Triraphis moLLis,

Chloridoideae) and some NADP-ME species (e.g. EI'iaohne aristidea,

Arundinoideae) • The selective sampling of species for biochemical

investigation ensured wide taxonomic coverage and it is apparent that

some of the correlation breakdowns occur in genera which are either

difficult to place taxonomically (e.g. the 'chloridoids with arundinoid

affinities' of Watson et ai. 1985), are of particular interest in other

respects (e.g. AUoteropsis: Ellis 1974a, 1974b) or belong to outlying,

non-mainstream groups (e.g. the tribe Eriachneae comprising Eriaohne and

PheidoohLoa). Whilst the anatomical/biochemical variation in Panimmt

may partly arise as a result of taxonomic confusion, there is no

taxonomic reason why Eragrostis, uniquely (as far as is known) among

'mainstream' chloridoid genera, should be biochemically homogeneous

irrespective of variation in leaf blade structure. In the subfamilies 54

Chloridoideae and Panicoideae there is no known exception yet to the correlation between centripetal PCR cell chloroplast position and NAD-ME activity. The data as a whole indicate that only one anatomical/biochemical combination has not yet been found: a XyMS­ species which is NAD-ME. If such a species does occur, it is likely to be in some outlying taxon.

In this study I have paid particular attention to chloridoid and arundinoid grasses. This is because more genera in the Chloridoideae

(viz. Bouteloua, Braahyaahne, Chloris, Eragrostis, Eustaahys,

Leptoahloa, Miaroahloa, Sporobolus) are variable with respect to those features of relevance to c 4 photosynthesis than in other subfamilies (excepting panicoid Paniawn; see Gutierrez et ai. 1974; Brown 1977;

Ellis 1977; Chapter 3). Predictions of c 4 type at generic level, therefore, cannot be made in these subfamilies. Concerning the arundinoids, three of the world's five c 4 genera occur in Australia (see Watson et al. 1986), including by far the largest one, Aristida. Only three of the c. 330 species of this genus w:orldwide have been biochemically typed. Both subfamilies are a significant component of the grass flora of the arid and semi-arid zones which cover about 75% of

Australia. Research on such grasses is particularly appropriate in view of !\'bore' s (1970) observation that, in t.11ese areas, 11 animal production continues to depend almost entirely on the native vegetation ••• more needs to be known to ensure continued economic utilisation of arid lands for comparatively low cost production of wool and beef and for conservation of wildlife. 11

For Australian native grasses the relationships between leaf blade anatomy, c type and geographical distribution have been investigated 4 only in the chloridoid genus Eragrostis. In this apparently exclusively 55

NAD-ME genus, species with centripetal PCR cell chloroplasts outnumber those with centrifugal/peripheral chloroplasts in all State and

Territory subdivisions of arid and semi-arid Australia where rainfall is

<30 cm y-1 (Chapter 3). In areas with higher rainfall the reverse is the case. The former anatomical type among grasses is also the c4 dominant one (species number-wise) in areas of very low rainfall in

Namibia/South West Africa (Ellis et aL 1980) It is all the more noteworthy, therefore, that such species are generally poorly represented in arid and semi-arid grassland research programmes. This paper provides not only a sampling guide to the anatomical and biochemical variation within Australian c4 grasses but also some basic data needed for a broader appreciation of their biology and phytogeography. Table 4.1. Suuuuacy of leaf blade anat.o.ica:J- characteristics in grasses of the three c 4 acid decarl>oxylation types.

The XyMS character refers to the presence (XyMS+) or absence 1XyMS-) of cells intervening between n1etaxylem vessel

elements and laterally adjacent PCR (Photosynthetic Carl:x>n Reduction or Kranz:) bundle sheath cells of primary

lateral vascular bundles (Hattersley and Watson 1976). PCR cell chloroplast position is either c..intrifugal

{towards outer tangential walls) and/or peripheral (adjacent to all cell walls) or centripetal (towards inner

tangential walls) (G.ltierrez: et al. 1974; Hatch et al. 19751 Ellis 19771 this study), PCR cells with

centrifugal/peripheral chlorvplasts always have chloroplasts adjacent to the outer tangential walls and variably

less of them along the radial and, especially, the inner tanqential walls. centripetal PCR cell chloroplasts are

usually aligned perpendicular to the inner tangential wall. FCR l:lheath outline (in transection) is recorded as

even (r{.qUlar) or uneven {irregular; applied by Ellis 1977 to Nl\D-ME type and PCK type species respectively but

extended here t.o NADP-ME species a.s well). Sht;>atli outline of small veins is easier to classify than that of large

veins. J?CK species commonly have extensions to the 1-CR b.lndle sheath {Ellis 1977). I refer to these as

•extension cells• and record thei.r presence in all species examined (see Figs. 3.1-3.3 in Chapter 3). 'l'hey dl'.'e

entirely separated from vascular tissue, usually by other PCR cells, and they are especially conspicuous in XyMS+

species havio:3 centrifugal/peripheral chloroplasts and an uneven sheath outline.

type XYMS character PCR cell p(:R bundle c4 chlorop.Ltst position sheath outline

NADP-ME XyMS-A centri fu.J a 1/periphera l 8 unevenc NAO-ME XyMS+ i) centripetal even ii) centrifu:ial/peripheral uneven° F PCK XyMS+E cen trifuyal/peripheral uneven

A Eriaohne and Pheidoohloa (Arundinoideae) are XyMS+. B E:riachne puloheZ.la has centripetal chloroplasts, Eriachne obtusa has mainly centripetal chloroplasts but some are also centrifugal/peripheral. c Neuraohne munroi and Paraneuraohne rm.eller-i have even/uneven and even ontlines respectively. D E T[iodia ao4r-iosa ,hQs an even outline. LoteropB~B eem~alata (C accessions; Panicoideae) is XyMS-. A 4 F Bouteloua ourtipendula (Michx.) Torr. has an even outline. Tablo 4.2. Anatollical features of the l'CR 6heath of leaf blddes of Australian species of the subfamily O'i).oridoideae (excludln<:j Kragroatia; uee O'iapter 3).

PCR (Photosynthetic Carbon Reduction or Kranz) cell chloroplast position recorded as ei thee centrifugal/peripheral {cf 1 most, or at least some, aligned towards outer tangential wall) or centripetal (cpi aligned along inner tangential wall and extending into the centre of the cell, sometimes as far as the outer t.:tngential wall). PCR sheath Qlltline recorded as

even (regular), uneven {irregular) or, as mentioned for },'pagroatia, as i11termediate (int.) between the two. Extension cells (PCR cells not adjacent to vascular tlssue) are recorded

as the inaximum number observed on any one vein of all specimens examined. !'land-cut sections were made either of fresh or pickled leaf blades (F) or of partially rehydrated leat

blades from herbarium material (R). Data in parentheses taken or deduced from sources for anatomy in Comments. Biochemical determlnatton of c types given where known, with source 4 i-eferences. All species are XyMS+ (see Table 4.1). o=neric conposition of the Chloridoideae according to Watson et a1.. (1985). Asterisks denote species not native to Australia. ?

indicates not recorded (usually for chloroplast position in herbarium material), Ill.: illustration. n.a.: not applicable.

Genus PCR cell PCR sheath Maximum no. c type No. specimens in which Condi ti on o-f Comments 4 Species chloroplast outline extension leaf blade anatomy leaf blades position cells/vein examined examined

Aorachna Wright & Arn. ex Chiov.

A. raoemo8a (Heyne ex Roemer & Schultes) Ohwi (cp) (even) 7 Anatomy; Ellis (1977).

Astreb1.a F. Muell. A. 61.ymoidea F. Muell. cp even 0 NAO-ME"" 2 ' A. 1.appaoea (Lindi.) Domin cp CV<.:ll Nfl.D-Mlf f

A. pectinata (Lind!.) F. Muell. ex Ben th, cp eveB 0 NAD-Mtf'

A. Bquarrosa c. E. Hubbard cp even 7 NAD-MiJ' 2 f' Although their sheath outline is even, both specimens have maximum of seven extension cells/vein, an unusually high number for an NAO-MB species with centcipetal chloroplasts. Extension cells usually occur in species wi d1 an uneven outline, Genus pCR Cell PCR sheath Maximum no. C4 type No. specimens in which Condition of Comments Species chloroplast outline- extension leaf blade anatomy leaf blades position cells/vein examined examined

Auatroohioria Lazarides

A. diohanthioidea (Everist) r.azarides cp even 0 2 II

Broohyaohrw (Ben th.) Stapf The only specie's examined by Ellis { 1977) has uneven outline and centrifugal chloro- plasts in contrast to the species examined here.

B. ambigua ohwi cp even 0 2 II

B. oi'liai>ia (Ben ti<.) c. E. Hubbard cp even 0 3 II

B. convergerw (F. Muell.) Stapf cp even 0 f

B. proetrata Girdner & Hubbard cp eve11 0 H

8. tenella (R. Br.) c. E. Hubbat"d cp even 0 H

Chloris o. Swartz ,. ' c. barbata Swartz cf uneven 0 P

PCKA,B,C,D c. gayana ICunth cf uneven 2 2 f Anatomy also examined by ' Gutierrez et a!. ( 1974), !latch et a!. ( 19751 ill.) arid Ellis ( 19771 ill.). c. Lobata Lazarides cf uneven 0 3 II

c. pectinata Ben th. cf uneven· 0 PCKA 3 F c. puntilio R. "'· cf uneven 0 2 " c. truncata R. Be. cf uneven 0 PCKA 3 F c. ventricoaa R, B<. uneven 0 2 " • c. vir9ata S>.l'artz cf uneven 0 PCKA 2 ,. Anatomy also examined by Ellis ( 1977). Genus PCR cell !'CR sheath Maximum no. c. type No. specimens in which Condition of Comments Species chloroplast outline extension le<1.f blade anatomy leaf blades position cells/vein examined examined

Cynodon Rich. c. arcuatua J. s. Presl ex C. B, Presl (cp) ? ' NAO-MEG ,D Anatomy: Hatch et al. ( 1975). c. daotylon (L.) Pers. cp even 0 NAD-MEA,H ,- Ana to my also examined by Ellis (1977). Ill; llattersley'and Watson (1975).

•c. nlemfiu.uusis vanderyst cp even 0 F Anatomy •l~ examined by Ellis ( 1977).

DaotylooteniU!lJ willd, " D. aegyptiwn (I,.) Beauv. (cf) (uneven) ' Anatomy: Ellis ( 1977). •D. auatrale steudel {cf) (uneven) Anatomy and i 11: Ellis ' (1977).

* D. gigantewn Fisher & Schweikerdt (cf) (uneven) 1

D. radu1-ana (R. Br.) Beauv. cf uneven F Anatomy: Ellis ( 1977).

Dinebra Jacq,

•D. retrofiexa. (Vahl.) Panz ? uneven 0 Anatomy also exami. neJ by " Ellis ( 1977).

Dipl-aohne Beauv.

D. fusca (L.) Beauv. (cpl even 0 2 Ana to my also examined by " Ellis ( 1977).

D. parviflor~ (R. Br.) Benth, cp even 0 4 l f, 311

Distiohtia F.afin D. distiohop11y1-1-a (I,abill.) Fassett cp even " Genus PCR cell PCR sheath Maximum no. No. specimens in which Condition of Comments Spec1$s chloroplast outline extension leaf blade anatomy leaf blades position cells/vein examined examined

Eotl'oaia R. Br.

E. agroatoidoa Benth. ? uneven 3 II

E. anomaia c. E. Hubbard ? uneven 2 2 II

E. appl'6B8a s. T. Blake ? uneven 3 II

E. btakei c. E. Hubbard ? uneven 4 2 II Ill: Fig. 4.3a.

E. oonfuaa C. E. Hubbard ? uneven 3 3 II

E. daneaii Domin ? uneven 4 II

... gutlivari F. Muell • ? uneven 2 II E. taaiootoda (Merr.) s. "· Blake ·1 uneven 2 2 II E. taxa S. T. Blake ? uneven 2 2 II

E. 1.aporina R, Br, ·1 uneven 4 II

E. acabrida c. E. Hubbard uneven 6 II E. aohuttzii Benth. ' unevl:ln 2 II

"'E. ooraoana (L.) Gaertn. cp even 0 NAD-Mif f Anatomy also examined by oenqler et at. 11905).

"' E. indica ( L.) Gaertn. cp even 0 f Anatomy aloo eicamlned by Ellis (1977).

• E'. triataohya (Lam.) Lam. cp even () 2 II

Ennaapogon Desv. ex Beauv. In this genus position of the chloroplasts is variable. Whether centrifugal or: centr:ipetal, however, they tend to be peripheral. Genus PCR cell pCR sheath Maximum no. c, type No. specimens in which Condi ti on of Comments species chloroplast outline extension leaf blade anatomy leaf blades position cells/vein examined examined

E. aaperatus c. E. ttubbard ? uneven 2 H

E. avenaoeua (Lindl.) c. E. Hubbard cp/cf uneven NAD-M!f 2 F In one specimen cl-\loroplasts equally centripetdl and centrifugal, in Other primarily centrifugal.

E. oae:ruleaoena (Gaudich) N. T. Burbidg{i cp/cf uneven NAD-MgA F Chloroplasts more centripetal than centrifugal.

E. clelandii N. T. Burbidge cp/cf uneven F Chlorolasts mainly centrifuJal.

E. oylindricua N. T. Burbidge uneven H

E. deoipiena Kakudidi ? uneven 2 H

E. eremophilua Kakudidi ? uneven 3 H E. graoilia (R. Br.) Beauv. ? uneven " E. intermediua N. T. Burbidge cp/cf uneven NAD-Mrf 2 Chloroplasts mostly " centripetal in one specimen but in other equally centripetal and centrifugal.

E. 1-indlayanue (Domin) c. E. Hubbard cp/cf uneven NAD-M£" F

E. nigrioana (R. Br.) Beauv. cp/cf uneven 2 NAO-M'f!'- 3 2F, lli E. oblongua "· T. Burbidr1e ? uneven 2 Ii E. pallidua (R. Br.) Beauv. ? uneven II

E. polyphyllua (Domin) N. T. Burbidge cf/cp uneven NAD-Mi" 4 F In three specimens chloroplasts are ITT4i nl y centrifuqal t~t in other (fig. 4. 2b} m

E. pubasoena (Domin) N. T. Burbidge ? uneven H

E. pu.rpuPaeoena (R. Sr.) Beauv. cf/cp uneven F Chloropla.sts slightly ooce centrifugal than centripetal.

E. robuatiaaimua (Domin) N. T. Burbidge cp/cf uneven NA.D-Mif F

Ente:ropogon Nees

E. aciaularia (Lindl.) Lazar ides cp even 2 9 F

E. doliohoataohyus (Lag,) Keng ex 1.azarides cp even 0 2 H

E. minutua Laza rides cp even 0 3 l!

E. uniapioeua (F • Muell.) w. D. Clayton ? even () 3 II

E. !'alll08U8 B. K. Simon ? even 2 II

Eragrostiel-la Bor

E. bifaria (Vahl) Bar ., even 2 2 II Ill: fig. 4.Jb.

Eragrostia N. M. Wolf cp even n.a. NAl)-MEI<: See Chapter 3 for (23 s!_)p.) (29 spp.) (6 sp~,) full treatment of cf unevt!n n.a. NAO-ME this genus. ( 11 spp.) ( 28 spp.) (4 tip~.) int. int. n.a. NAD-ME (4 spp. l (? l:!pp.) (3 spp.)

Eustachya oesf. 'l'he only species examined by Ellis ( 1977) hdSI uneven outlint:;! •nd centrifugal chloropla1:1t!:1 in contrdst to the specl.es exilmined here. 13 • E. diatichophyl-l-a (Lag.) Nees ? even 0 NAD-ME II Genus PCR cell PCR sheath Maximum no. No. specimens in which Condition of Comments Species chloroplast outline· extension leaf blade anatomy leaf blades position cells/vein examined examined

Heteraohne Benth.

H. abortiva (R. Br.) Druce ? uneven 5 4 II

H. gulliveri Benth. ? uneven 4 2 H Ill: fiq. 4.Ja.

L6ptoohloa Deauv.

L. ciliolata (Jedw.) S .T. Blake cf uneven 0 PCKA 2 F

L. deoipiena (R. Br.) Stapf ex Maiden ? uneven II

L. digitata (R. Br.) Domin cp even 0 NAD-110 4 F L. neeaii ('rhwaites) eenth. cp even ' II

Lepturu.B R. Br.

L. repena (l•~orst.) R. Br. ? uneven 0 II Ill: Fig. 4. Jd. ' Some PCR cells with very rounded outer tangential walls, others with less rounded ones. L. xerophilua Domin ? eveu 0 2 "

Mioroohl.oa R. Br.

M. indioa (L. f.) Beauv. ? even 0 II The sheath outline of this specimen is mart like thdt of the illustrated specimen of M. oaffra Nees, which Ellis ( 1977) predicted to be NAO-ME, rather than that of the illustrated specimen of M. indica which he predicted to be PCK. In southern Africa M. oaffPa is andtOmically variablei perhaps M. indica 1$ similarly variable, Genus PCR cell PCR sheath Maximum no. c, type No. specimens in which Condition of Comments Species chloroplast outline" extension leaf blade anatomy leaf blades position cells/vein examined examined oxyohtoria Lazarides

o. aoa.rioaa (F. Muell.) Lazar ides cp even NAD-M~ F outline less even than that of most species with centripetal chloroplasts.

Porotia Aiton

F Ill: Vickery ( 1935). P. rara •· Br. cp even 0 2

Pleotrachno Henrard Chloroplasts are centrifugal in PCR cells adjacent to vascular tissue and adjacent to PCA tissue in those cells entirely separate from vascular tissue. In Pieotroohrie and the allil;!d genera Sympieotrodia and Triodia, most PCR cells form distinctive strands which I do not record and count hero;; ., extension cells.

P. meivillei c. E. Hubbard cf even n.a. 5 F

P. achinaii Henrard cf even n.a. F

Spartina Ewart

• s. angl.ioa c. E. Hubbard ined. (cf) (uneven} en PCK~· Anatomy and ill: Mallott et a!. ( 1975).

• s. X townaendii H • & J. Groves (cf) {unev"'nl (8) Anatomy and il 1: Long et al.. { 1975). Genus pCR cell pCR sheath Maximum no. c, type No. specimens in which Condi ti on of Comments Species chloroplast outline extension leaf blade anatomy leaf blades position cells/vein examined examined

Sporobolua R. Br.

s. aotinooZ.adua (F • Muell.) F. Muell. cp even 2 5 F • s. afrioanua (Pair.) Robyns • Tournay cf ur1even 5 PCKA f I 11: Ellis ( 1977). s. auat.ral-aaioua Domin cp even 0 3 ti

s. oa.roli Mez cp even 0 NAD-Mi0- 4 f Ill: Fiy. 4.1b.

s. oontiguua s. T· Blake ? even 0 H

s. orebeP De Nardi cf unev

s. diander (Retz.) Beauv. cf uneven 2 f

s. til.ongatus R. Br. cf uneven 5 PCKA F Ill• nengler et a!. ( 1985).

• s. jaaquemontii KUnth cf uneven 4 PCKA 2 F

s. laxua B. K. Simon ? uneven 2 2 H

s. 1-enticularia s. T. Blake ? uneven ? 2 H

s. mitohelli (Trin.) c. E. ttubbard ex s. T· Blake cp even 0 5 .-

s. puZ.ohellus R. Br. cp even 0 .NAP-Mlf 2 F

• s. pyrimidaZU Beauv. (cf) (uneven) (1) Anatoniy and i 11• Ellis (1977).

s. aoabridua s. T. Blake ? even 0 II

s. virginioua (L,) IO.lnth cp even 0 ,. Anatomy also examined and illustrated by Ellis (1977).

Sympleatrodia Lazarides Soe Comments under Pleotraohne.

s. graoilis La~arides ? even/unev.;n n.a. 2 Ii Much more uneven PCR sheath outline thdn S. lanoaa.

s. lanoaa Lazarid~s even n.a, 2 II Genus PCR cell pCR sheath Maximum no. c, type No. specimens in which Condition of Comments Species chloroplast outline extension leaf blade anatomy leaf blade a position cells/vein examined examined

Tragua Haller

T. auatralianus s. T. Blake cp even 2 F Ill: Vickery (19)5).

T.riodia R. Br. See comments under Pieotl'aohne.

T. baaedowli Pritzel cf even n.a. 7 F

T. conoinna N. T. Burbidge cf even n.a. 2 ..

1'. hubbardii N. T. Burbidge cf even n.a. f

T. irritans Br. cf ev'"u n.a. 4 f Anatomy also examined and •·. illustrdted by Craig and Goodchild ( 1977).

T. Longioepa J. M. Black cf even 11.a. 3 F

T. narginata N. T. Butbidqe cf eveu n.a. F

T. pungena R· Br. (cf) (even) n.a. Anatomy and il 11 Uattersley at al. (1977).

T. aca:r>ioaa N· T. Burbidge cf even n.a. NAD-MJf f

Tl'ipogon Roemer & Schultes

T. J,oliiformi8 (F. Muell.) c. E. Hubbard cp even F I 11; fig. 4.1d.

Triraphia R. Br.

T. moilia Br. cf un<':lven NAO-Mif' •• 2 2 •. Ill; Fi

Zoyaia Willd,

z. rracrantha Desvaux cf Ul).,Vt:!.n 6 F I 11: Fiy. 4. lC df\d Vickery { 1935). z. matl'elta (L.) Hern ·1 uneven 4 " A Chapter s •

• G.ltierrez at ai. ( 1974).

c Hatch and Kagawa ( 1974a).

D Hatch (Jt al. (1975).

• Chapter J. F smith et al. ( 1902).

G aoag ( 1981).

H Hatch and Kagaw(:l {1974b). Table 4.3. Anatollical features of t:he PCR ~heath in leaf blades of Australian species of the aubfd!ll.ily Arundinoideae.

All Ariatida species examined have typical Ariatida anatomy viz. XYMS-, cent"ripetal chlot"oplasts in

Genua PCR cell PCR Maximum no. c. No. specimens Condition comments Species chloroplast sheath extension type in which leaf of leaf positions outline cells/vein blade anatomy blades examined examined

A:ria·tida L. Brown {1977) li~t~ 25 species with the characteristic double chlorenchym<1tous she

A. annua a. K. Si men ----(see Table leqend )---- F

A. arida B. K. Simon

A. benthamii Henrard f A. bigtandutoaa J. "· Black NADP-ME I 11: Dengler et al. (1985). A. browniana Henrard 12 F

A. caiycina R. Br. F

A. caput-meduaae Domin 2 F

A. oontorta F. Muell. 8

A. inaequigtumis Domin 4 i• Genus pCR cell PCR Max:1m;;mno-.---c:---No. specimens Condition Comments 4 species chloroplast sheath extension type in which leaf of leaf positions outline cells/vein blade anatomy blades ex:amined ex:amined

A. J°sriahoenai-B (Domin) Henrard ----(see 'l'able legend)---- 6 F

A, iatifoZ.ia Domin NA DP-ME 3 F

A. Z.eptopoda Ben th. 3 F

A. 'lignoaa B. K. Simon

A. nitidu'la (HenrQ.rd) s. T. Blake ex: J. Black

A. pruinoaa Domin 2 F A. :ramoaa R. Br, " Ill: Hattersley et al. (1977)• A. vagana Cav.

Eriachne R. Br. Tateoka (1961) recorded two bundle sheaths in examined species i ,e. as XyMS-t-. Chloroplast position was not recorded and illustrations show only cell outlines. 'rwo further species t·ecorded by Bi-own (t9Tl) as PS i.e. XyMSt. E. agroatoidea F. Muell. ? uneven 0 3 " E. anomaZ.a Hartley ? uneven 0 !l Anatomy also examined by Tdleoka (1961).

E, ariatid?a F. Muell. cf uneven 0 NA DP-ME 4 3F, 11l Ill: Fig. 4.20. E. armittii F. Mllell. ex Bent:h, 7 uneven 3 " Anatomy also ex:...1mined and illustrated by 'l'ateoka (1961). E. avenaoea •• Hr, cf uneven 0 3 H Anatomy al~ examined by Tateoka ( 1961). E. baaedoLJii Hartley cf lll\t!V<:Hl 2 " E. benthamii Hartley cf Ul\'2!V

E. bleeaeri Pilger cf uneven 0 2 II Genuo !'CR cell PCR Maximum no. c, No. specimens Condt tion Comments Species chloroplast Shea.th extension type in which leaf of leaf positions outline cells/vein blade anatomy blades exarni ned examined

;;. Wrkittii Jansen ? uneven 0 3 II E. oapil.'laria •• Br. ? uneven 0 3 H E, oi'liata •• Br. ? uneven 3 II Anatomy also examined by Ta teak.a (1961). E. dominii Hartley cp even 0 3 H

E, fastigiata Lazarides cp even 0 2 II

E. festucaoea. F. Muell. cf uneven 3 3 ll Anatomy also examined and illustrated by 'l'a teoka ( 1961).

E. fitiformis Ha~tley ? uneven 0 2 ll

E. flacoida Hartley ? uneven 2 II

E. gtabrata (Maiden) Bartley cf uneven NADP-ME 2 f

E. glauca R. Br. cf uneven 3 H Anatomy also exaini ned by 'l'a teokd (1961).

E. hetmsii Domin cf uneven ? f Anatomy ulsu examin.::d by 'l'ateoka ( 1961).

E. humitis Hartley cf uneven 0 II

E. insul.aris Domin cf unev

E. mel.icaoea F, Huell. ? uneven 0 II Ana toiny also tixami ned by Ta teoka (1961).

E. muo.ronata R. Sr. cf uneven 6 ? F Maximum extension cell number::> occur in large veins where they farm t"" separate strands ext:endi ng tOWdCdti ddaxial leaf surface. Anatomy a loo eKamined by Tateoko. ( 1961 l. E, ne.rvooa Ewart • Cookson ' uneven 5 2 II Anatomy also examined by Tateoka ( 1961) • E. obtuaa R. Br. cp/cf even/uneven 4 NADP-ME 3 F Most chloroplasts are centripetal tru t they also oc<.."tl~ alor¥J the radial walls of most cells and less commonly in a Ct!ntrifugal position. Sheath outlinti somewhat intennt!diate i" evenne!HI. Maxi muui extension cell Genus PCR cell PCR Maximum no. c, No. specimens Condition Comments Species chloroplast sheath extension type in which leaf of lthl.f positions outline cells/vein blade anatomy blades examined examined

numbers occur in the midrib in strands of l'CR cells connecting a complex of t'"" or three separate vascular bundles.

E. ovata Nees cf uneven 0 NADI)-ME F Anatomy also examined .;:ind illustrated by Tateoka ( 1961).

E. palloacena R. Br. ? uneven 3 4 H Anatomy .;:ilso eiw.mi ned by Tdteoka (1961).

E. pauoiflora Fitzgerald cf unev<:>n 3 H

E. pulohella lbm.in cp even 0 NADP-ME 2 F Anatomy also exetmi oed by Ta teok.;:i {1961). Ill; Fig. '· ?4.

E. raro R. Br. cf unevt:!n 3 H Anatomy also examined by Ta teoka ( 1961).

E. achu1.tBiana F • Muell. ? uneven 3 II

E. acZ.eranthoi

E. squarroaa R. Br. ? uneven 2 ti Anatomy also examined and ilu::>trattld by Ta teok.a (1961).

E'. atipaaaa F. Muell, ? uney(;Hl 2 2 H

E. au1.aata Hartley cf uneven H Anatomy also examined by Ta t

E. t.riodioi

E. t.riaata Nees ? uneven 15 3 II !'CR eel l~ of lai:-

Phaidoohloa s. ,.. Blake

P. gracil,.is s. T. Blake cf uneven 0 NADP-ME F Table 4.4.. AnatoMi.cal features of leaf blades of Auut.rali

XYKS character recorded as+ or - for each species (see Table 4,1), PCR cell chloroplast position, pCR sheath outline and extension cells recorded as in Table 4,2. Representative species only were examined for larger genera. Data in parentheHes taken or deduced from sources for anatomy under Comments. Biochemical deteru

known, ..,ith source refecences. See Tabh: 4.2 foe abbreviations and symbols.

XyMS PCR cell PCR Maximum no. No. specimens cOnctition of Comments c 4 chloroplast sheath extension type in which leaf leaf blades position outline cells/vein blade anatomy examined examined

ALZ.oteropaia Presl 4 0 A. cimicina (Retz~ Stapf + ? ? ) " A. aemialata (R. Br.) Hi tchc. cf even/uneven ) ) F For full accounts of the anatomy of this species see Ellis { l974a, 1974b), Ill: Ellis ( 1974a, 1974b) and Hattersley etai. (1977),

Arthrag:roatis Lazarides n.a. n.a.

A. desohampsioidea Lazarides ? uneven 0 H

Arundineiia Raddi 9

A. nepa.1.-erwis Trio. cf unevBn NADP-Mh.A 2 'Distinctive' PCR cells scattered throughout sections (see Ga1•notia and Paapalwn aonju.gatwn). Jl.nat:omy also examined by Tateoka ( 1958), flattersley and Watson (1976), Brown (1977) and Ellis (19771 ill,). Genus XYMS ['CR cell PCR Maximum no. c, No. specimens Condition of Commt!nts No. non-Australian Species chloroplast sheath extenSion type in which leaf leaf blades species of each position outline cells/vein blade anatoiny examined genus examined examined anatomically bioche1ni cal ly

Axonopua Beauv. 4 0

• A. affinia chase ? uneven 0 II Anatomy also examined by ttattersley and Watson ( 1976) and Brown ( 1977).

• A. oompr11aaua (Swartz) Beauv. (-) ? ? ? NADP-Mf~G Anatomy; Ellis ( 1977).

Braohia.ria Griseb. R.O. Webster (in prep.) has 20 8 transferred all Australian species of Brachiaria, except 8. eruciformis, to Uroahtoa.

• B. advena Vickery (+) (cf) (uneven) ? Anatomy: Ellis ( 1977).

• B. bri~antha (Hochst ex A. (+) (cf) (uneven) (0) PCKB Anatomy: 0.1tierre~ et al. Rich.) Stapf ( 1976), Brown (1977) and };} li B { 1977; i 11. ) •

• 8. decwnben.a s tapf + cf' uneven F Anato111Y also examined by Brown { 1977).

13 • B. eruoiforrJtia (Smith) Gri seb (+) (cf) (uneven) ? PCK Anatomy, 0.1 ti<;±rrez et al. (I 976), llrown {1977) and Ellis ( 19'/7).

B. fol,ioaa (R. Br.) !-lug hes + cf uneven () F Anatomy al »eJ examined by H1;ttersley aud Wd tson ( 1976) oind Brown { 1 977).

B. hotoaeriaa (R. ur.) Hughes + cf uneven 0 F

B. mitiiformia (presl) Chase + cf u11eve.n 0 F

11 B. mutica {Forssk,) Stapf (+) (cf) I "/ PCK A1i<"tomy' Guti•~rrez e' al. ( 1 976) and Brown ( 1977). Genus xyHS PCR PCR Maximum flO • c, No, specimens Condition of Comments No. non-Australian Species chloroplast sheath extension type in which leaf leaf blades species of each position outline cells/vein blade anatomy examined genus examined exdmi ned anatomically biochemically

8. piZ.ige:ra (F. Muell.) Hughes + cf uneven 0 F

B. romoaa (L.) Stapf ? (cf) ., ., Anatomy: Gutierrez et at. (1976).

B. :reptana (L.) Gardner & Hubbard (+) ? ? ? Anatomy: tlattersley and Watson (1976} and Brown (1977).

Cenohrua L. J 0

• c. bifl.orua Roxb. ? ? ? Andtomy: 11attersley and Watson { 1976).

• c. oiZ.ia:ria L· (-) (cf) ? ? Anatomy: nattersley and Watson (1976), Brown (1977) and J<;llis (1977),

*C. eohinatua L. ? uneven 0 II Anatorny also examined by !iatter;;;ley a11d wat;;;on (1976).

• c. inoertua M· A. Curtis (-) (cf) ? ? NADP-MEC Anatomy: {ntierrez et at. (1974) and Brown (1977).

"' C. aetiger vahl cf uneven 0 F

Chamae:raphia R. Br. n.d. n.d.

C. hordeao?a R. Br. ·1 uneven II A.nato1ny also examined by llattersl<1y and Wdtson (1976) and Brown ( 1977), Genus XyMS PCR cell PCR Maximum no, C4 No. specimens Condition of Comments No. non-Australian Species chloroplast sheath extensfon type in which leaf leaf blades species of each position outline cells/vein blade anatomy examined genus examined examined anatomically l>iochemically

Digi taria Haller 38

D. bicornia (Lam.) Roemer & cf uneven 0 Schultes

D. braviglwnia (Domin) Henrard cf uneven 0 Some large and small veins with a partial double chlorenchyma tous sheath.

D. broMnii (Roemer & Schultes) cf uueven 0 2 F Some large and small veins of both Hughes specimens with partial double chlorenchymatous sheath. Anatomy also examined by Hattersley and Watson (1976), Ill: Webster (1983).

D. coenicola (F. Muell.) Hughes cf uneven 0 ) f Some larye veins of all three specimens with partial double ch lorenchyn1a taus sh ea th,

• D. eriantha steudel cf uneven 0 F

D. longiflora (Retz.) Pers. F Anatomy; Ellis {1977).

D. nematoataohya (F. M. Bailey) cf uneven 0 Sorne lan;1e awl ;;ma.11 veins with Henrard partidl double chlorenchymatous sh ca th.

" D. aanguina1-is (I.. ) Scop. (cf) uneven 0 NADP-MEC,D,E,ll II Anatomy also examined by OJ.tierrez et al. (1974), Hdtch et al, (1975), flattersley and Wd.tson (1976), Brown (1977) and ~~llis (1977). Ill: Webster (1983). • D. ternata (A. Rich.) Stapf (-) (cf) ' ' Anatorny: ~:llis (1977).

Echinoch1-oa Beauv. 0

• E. colona (L.) Link (cf) uneven 0 II Anatomy also l:!:x.amined by Genus XYMS PCR cell PCR Maximum oo. C4 NO. specimens Condition of Comments No, non-Australian Species chloroplast sheath extension type in which leaf leaf blades species of each position outline cells/vein blade anatomy examined genus examined examined anatomically biochemically

Gltierrez et at. {1974),

Hattersley and Watson {1976),

Brown (1977) and Ellit> (1977),

* E. crua-galli (L.) Beauv. (cf) uneven 0 2 II Anatomy also examined by Gutierrez et at. (1974), Hattersley and Watson (19761 ill.), Brown (1977) and f<;ilis (1977.)

• E. oiou.a-pavonis {Kunth) 7 ? ? II Anatomy: Brown (1977). J. A. Schultes

• E. fru.mentaoea (Roxb.) Link ? uneven 0 NADP-MEC II Anatomy ali:>o examined by !lattersley and Watson (1976).

• E. pyrirrridalis (Lam.) (-) ? ? ? Anatomy: Brown ( 1977) and Hi tchc. & Chase Ellis {1977).

E. atagnina (Retz.) Beauv. ( -) (cf) ·1 ? /\natomy: ~;l.lis (19"/7).

Eriochloa Kunth 16 4

E. auatra.lienaie Stapf ex 'l'hell. uneven 0 Anaturny also examined by Jiattersley and Watson (1976).

E. creb:ra s. T. Blak~ 7 (cf) ? ·1 Anatomy: Gutlei:-rez et ai. (!976).

E. psaudoaorot:rioha + (cf') uneven 0 H Anatomy also examined by (Stapf. ex '.rhell,) J, M, Black OJtierrez et at. {1976),

Ga:rnotia Brongn 3 0

G. atriota Brongn ? uneven 0 II Many •distinctive' pCR cello; thi:-ouc1hout J:>ect1on (see Al't

HygPoohloa Lazarides n,a. n • .t. H. aquatica Lazarides ' uneven 0 " H. cravenii r.azarides ., uneven 0 II

MetiniB Beauv. 0

minutif1,ora Bea,uv. + cf uneven pCKA F AniltOmY also examined by . "· Hatt.ersley and Wat.son ( 1976).

Neurachne R. B<. n.a, n.a.

N. nunroi (F. Muell.) F. Muell. cf even/uneven 4 Nl\Ol'-MEF 14 Moot vt:ins have an unuS11ally even outline for an NA.DP-ME species althouyh there ace some mostly adaxially located extension cells. For full account and illustration see Hattersley et aL (1982).

Paraneuraahne s. T. Blake n.a. n.a.

P. nuelleri (Hackel) s. ·r. Blake cf even NADP-MJ::F 4 F The even outline i' unu !;:Ua 1 foe an NA OP-ME :,;pecies. For full account and illustration see Battecsley et a!. ( 1982).

Pareoteniwrt aeauv. n.a. n.d.

P. novae-hollandiae Beauv. cf uneven F

Paapalidiwn Stapf 4 0

P. aonatriatwn (Domin) (-) ? ·1 AnatGmy: Brown ( 1977). C.E. Hubbard Genua XyMS PCR cell PCR Maxiu;u~n no. c, No. specimens Candi tian of Coinments No. non-Australian species chloroplast she a th extensi"on type in which leaf leaf blades species of each pasi tion outline cells/vein blade anatomy examined genus examined examined anatomically biochemically

P. fiavidwn (Ret1lJ A. camus 1-l ? ? ? Anatomy: Brown (1977).

P. inequal.e (F • Muell.) Hughes cf uneven 0 F

P. jubiflorwn (•rein.) Hughes ? uneven 0 II Anatomy also examined by Hattersley and Watson ( 1976).

p, (R, Br,) Hughes ? uneven 0 F Anatomy al;;;o examined by """""' Hattersley and Watson {1976).

PaspaLwn L. 17 0

• P. oonju.gatwn Berqius cf uneven 0 F Specimen has three 'distinctive• PCR cells reminiscent of AruruiineLLa and Garnotia. Anatomy al;;;o examined by uattersley and Watson ( 1976).

•P, diLatatwn Poiret cf uneven 0 NADP-MEH 2 II Anatomy also examined by tla ttersley and Watson ( 1976) and Ellis ( 19·111 ill.).

P. di8tiohwn L. ? uneven 0 Anatomy a 1 so examined by " Ha tter::iley and Watson. (1976) and Brown. ( 1977).

• p, notatwn Fluegge (..::f) uneven 0 NADP-M!~C, I! II Anatomy also examined by G.l tierr.;,z et al. ( 1974)' f\attersley and Watson ( 19./6). Brown ( 1977) and Ellis ( 1977).

• P. panicu 'l-atwn L. cf uneven 0 Anatomy also exa111ined by ' OJ tierrez et al. ( 1974).

P. paspaZ.odea (Michaux) cf uneven 0 Anat?111Y also ex<:1.minerl and Lams-Scribn. ' illustrated by Ellis ( 1977).

P. aorobiouZ.atwn !,. (-) (cf J ? ? Anatomy1 Hattersley and Wat::i.on ( 1976). Genus XyMS PCR cell PCR Maximum _no. c. No. specimens Condition of Comments No. non-Australian Species chloroplast sheath extension type in which leaf leaf blades species of each position outline cells/vein blade anatomy exaiuined genus examined examined anatomically biochemically

• P. urviLLei Steudel (-) (cf) 7 7 Anatomy: Br:own (1977) and Ellis (1977).

Pennisetwn Rich 4

P. aLopeouroides (L.) Sprengel cf uneven 0 II Anatomy also examined by Hattersley and Watson (1976).

• P. amerioanwn (L.) K· Schum (cf) ? 0 Jj Anatomy; Hatch et at. (1975), Hattersley and Watson {1976) and 1-:llis {1977).

'" P. otandeatinwn lJochst. ex Chiov. (Cf) uneven 0 Anatomy also examined by Hatter;;ley and Watson (1976).

• P. maorourwn Trin. (-) (cfl ? Anatomy: ti

*P. potystaahion (L.) cf uneven 0 f J. A. Schultes

• P. purpurewn Schumacher (-) (cf) ? ? A.nat()my; ().Jtier-i-ez et al. 1974), l\att~csley ond Watson (1976), Brown (l':f17) and r-:llis (1977).

P. sataaeum {forrsk.) Chiov. (-) (cf) ? ? AniltO!!ly: Ellis ( 1977).

• P. vitloswn R. Br. ex fresen. cf uneven 0 II Ani:ttomy also ex<.1111int!d by !!attersley and wat,;on {1976). IllUBtrations: !lattersley and Watson (19.,':>) and Hattecsl<::y et al. (197'1).

Ptagioaetwn Benth. n.a. n.a.

P. refraotWT/ (F. Muell.) Benth. cf uneven 0 f Anatomy also 1~xa1ni ned by Brown (1'J"l7). Genus XyMS PCR cell PCR Maximum no. C4 No. specimens Condition of Comments NO. non-Australian Species chloroplast sheath extension type in which leaf leaf blades species of each position outline cells/vein blade anatomy examined genus examined examined anatomically biochemically

PoewloohaGtochtoa Hi tchc. n.a. n.a.

P. auatratienaia Hitchc. +/- ? uneven H XyMS feature variable in one large vein, but consistently XyMS- in four others.

Pf1eudoraphia Griff. n.a. n,a.

P. spineaccns (R. Br.) vi ckery '/ ? H

Rhynohetytrwn Nees 5 0

* R. repena {Willd.) c. E. Hubbard + cf uneven 0 PCKA f Anat0111y also examined by Hattersley and Watson ( 1976). I 11; f;llis et a!. ( 1980).

Seta.Fla Beauv. 20 2

• s. anoepa Stapf cf uneven 0 F

s. baPbata (Lam.) KUnth (-) ? ? ? Anatomy; Hattersley and Wati>on (1976) and Bi:- own ( 1977).

• s. ganiautata (Lam.) Beauv. cf uneven 0 Anatomy also examined by llattersley and W<.1 tson (1976).

• s. gtauoa (L.) Beauv. (-) ? ? ? NADP-MEH Anatomy also examined by Ha ttersley and wa tson ( 1976}.

• s. italioo (L.) Beauv, (cf) ? () NADP-·M~;C P.n.atomy also exa.mined by " Ql tierre:.: et al. ( 1974). Hattersley and Watson (1976) and Brown {1977).

• s. patlide-fuaoa (Schumacher) (-) (cf) ? 0 Anatomy: Ellis ( 1977). Stapf & Hubbard

• s. pa1-mif otia (Koenig) sta[.if cf uneven 0 F

• s. vertioit lata (L.) Beauv. (-) {cf) ? '/ NADP-MEC Anatomy: Hattei:-sley and Watson ( 1976) and Ellis ( 1977). • s. viridia (L,) Beauv. ? uneven ? NAOP-MEC Anatomy al"'° examined by " Ila ttet"sley and Wat.son ( 1976). Genus XyHS JX:R cell PCR Maximum n9, c. No, specimens Condition of Comments No, non-Australian Species chloroplast sheath extension type in which leaf leaf blades species of each position outline cells/vein blade anatomy examined genus examined examined anatomically biochemically

Spini.fex L. 2

s. hirautua Labill. +/- cf uneven c.10 f Five veins ace variable for XyMS character, three are entirely XyMS-, Most extension cells contain fewer choroplasts than other l'CR cells,

s. serioaua R. Br. +/- cf uneven 3 NA.DP-MeA 2 f One specimen has seven XyMS+ veins, thtJ o thee has four XyMS+ veins and one which is variable. A.natomy al~ examined by HattBrsley { 1976).

Stenotaphrwn Trin. 0

s. micranthwn (Desvaux) ct uneven 0 F c. E, l-lubbard

• s. seoundatwn (Walter) Kuntze (-) (cf) (uneven) ? Anatomy: fla ttei:-sley and Watson ( 1976), Brown ( 1977) and Ellis ( 1977; i 11. ) •

Thuarea Pers. 0 0

T. involuta (Forster f.) R. Br. + ct unevt:n 0 2 F Anatomy al!:iu exd111i ned by ex Roemer • Schultes llattersley and Watson ( 1976) and Brown ( 1977).

Uranthoeoiwn Stapf n,a. n.a,

u. tru.noatwn (Maiden • 7 ? ? Anat.omy dlso ""xc11qined by Brown Betche) Stapf " ( 1977). Genus XyMS !'CR cell PCR Maximum no. c4 No. specimens Condition of Comments No. non-Australian Species chloroplast sheath extension type in which leaf leaf blades species of each position outline cells/vein blade anatomy examined genus examined exai11i ned anatomically biochemically

Uroohloa Beauv. See Braohia1·~a. 0

• u. moaambicenaia (Hackel) + cf uneven 0 PCKB 2 11', 1H Anatomy also examined by Dandy Gltierrez. et al. { 1976)' Brown (1977) and Ellis ( t 977).

• u. 07,igotricha (Fig • De Not.) (+) (cf) (uneven J ? PCKB Anatomy: Gutierrez et al. • Henrard ( 19 76}' Brown ( 1977) and Ellis (1977).

• u. paniooidea Beauv. (+) (cf) (uneven) ? PCKC Anatomy: Gutierrez et a!. ( 1974)' Hattersley and Watson ( 1976), Brown (1977) and Ellis ( 1977; i 11.).

• u. pufLulana stapf (+) (cf) {uneven) ·1 PCKB Anatomy; Gutierrez. et a!. (1976)' Ha ttersley and Wd tson ( 1976}. Brown ( 1977} and Ellis ( 19"!7) •

Whiteoohloa c. E. Hubbard 0 0

w. oapillipea {Ben th.) ., uneven 0 ? Laza rides "

w. aemitonaa (f. Muell. ex ? uneven () I:lenth.) c. E. Hubbard "

Xe:rochloa R. Br. 0

x. barbata R. Br. ? uneven 0 Anatomy a loo examint:ld by " Hattersley and wa tson (1976) and Brown { l'J77).

x. imberbia R. Br. +/- ? uneven 0 II Three l

x. laniflora Benth. ? ? ? II Anatomy also e·xamined by Hattersley and Watson ( 1976) and Brown ( 1977).

Yakirra Lazarides & R. Webster n.a. n.a,

Y. auatra-Z.ienaia (Domin) + cp even 0 2 1f' lll Anatomy also exam1 ned by Lazarides & R. Webster Brown ( 1977).

Y. rrujuacula (F • Muell. •• + cp even 0 3 II Anatomy also examined by Benth.) Lazarides Brown ( 1977). R. Webster •

Y. mu.el Leri (R. llr. ) Laza rides + cp even 0 2 II Anatomy also examined by R. Webster Hattersley and Watson (1976) • and Brown (1977). Y. nuL La Lazarides • R • Webster + cp even 2 II 1. pauoifLora (R. Br,} + cp eve11 0 2 II Anatomy also examined by Lazarides & R· Webster Brown (1977).

Zygoohloa s. T. Blake n.a. n.a.

z. paradoxa (R. Br,) s. T. Blake ·1 uneven ? II Specimen d ~ plant. Anatomy <1l<10 examined by Brown (1977).

A Chapter 5. B Qititirrez et al. {1976). c Gutierrez et al. (1974}. D Hatch and Kagawa (1974 a~ b). E Hatch et al. (1975). F Ha ttersley and Stone ( J 986). G Gutierrez and Edwards (unpubl.) in Brown (1977). II usada et al. (1984). Table 4.5. Anato•ical feat.urea Qf leaf Llades of Australian species of PaniCWJt (l'anicoldeae1 Panicanae).

XyMS character recorded aa +or - for each species (see Table 4.1), fCR cell chloroplast position, fCR sheath outline and extension cells recorded as in Table 4.2. Data in parentheses taken or deduced from sources for anatomy in Comments. Biochemical determinations of c type given where known, with source references. See Table 4.2 for 4 abbreviations and symbols,

Genus XyMS fCR cell PCR Maximum no, c 4 No. specimens Condition of Comments Species chloroplast sheath extension type in which leaf leaf blades position outline cells/vein blade anatomy exarni ned examined

Paniewn L. N.B. taxononlic revisions by Lazarides and Webster (1985) and Lazarides (1985).

' P. antidotale Retz cf uneven 0 NAOP-MEA ,. Anatomy also exainin..:d by Gutierrez et al. (1974; ill.), Hattersley and Wats1Jn (1976; ill.) and Brown (1977).

* P. buLbOBWTl Kunth cf uneven 0 NADP-MEB ,. Anatomy also examined by Hattersley and Watson (1976), Brown (1977) and Dengler et al. (1965; ill.).

P. buncei F. Muell. ex Ben th, + cp even 0 F

* P. capiLLare L. + (cpl even 0 NAD-MJ!' Anatoniy also examined by OJtierre.z et al. " (1974). dlld Hattersley and Watson (1976).

• P. coloratwn L· + even NAD-Mtf ,C F Anatomy also examined by tlattersley and Watson {1976). variable anatomy found by Ohsugi et al. (1962; ill.), some accessions with centripetal PCR cell chlot"oplasts and even PCR sheath outline and othecs with centrifugal PCR cell chloroplasts and uneven PCR sheath outline. All accessions were biochemically NAP-ME,

P. decompoBitwn R. Bi, (<) (cpl (even) 'I NAD-Mb!' Anato1ny: Gutierrez et al. ( 1974) and Hatt..:rsley

P. effUBWn R. Br. + cp 0 NAD-Mrf F Anatomy also examined by Dengler et al. (1985; ill.), Genus XyM.S PCR cell PCR Maximum no. c, No. specimens Condition of Comments Species chloroplast sheath extension type in which leaf leaf blades position outline cells/vein blade anatomy examined examined

P. Laroomianwn HUqhes + even 0 2 ll

P. maximum Jacq. + cf uneven F Anatomy also examined by Gutierrez ot al.. ( 19'/4), Hattersley and Watson (19'161 ill.) and Ellis (19'171 ill.). R. o. Webster (in prep.) has transferred this species to Uroch!oa.

• P. mi l.iaoeum L. (-t-) (cp) (even) ? NAD-M~,E Anatomy also examined by Gutierrez at aL. (1974; ill.) Hatch et aL. (1975) and !lattersley and Watson (19'/6).

P. mindanaense Merr. + "! uneven 0 2 ll

P. mitchelLii Ben th. + ? 11neven 0 2 ii ' P. novenmerve Stapf + ? even 0 ll P. obaeptum Tcin. + ? uneven ? ll

P. pa1.udoaum Roxb. cf uneven 0 ii

P. queenal.andicwr1 Domin + cp even 0 lf, 2H Outline less even than that of most species with centripetal chloroplasts.

P. achinzii Hackel + cf uneven 0 Nl\D-MEC,F F

P. seminudwn Domin + ? uneven 0 2 ll

P. simile Domin + ? even 0 2 ll

P. traohyrhaohis Ben th. + cp even 0 2 H

P. whitei ,J. M. Black + cp even 0 2 H

A Gutierrez et a!. ( 1974). H Downton (1970). c Ohsugi et al. ( 1982). D Kanai and Black (l 972}. E Hatch et al. ( 1975). F Chapter 5. Fig. 4.1 • Light micrographs of vascular l:undles from transverse sections of chloridoid leaf blades. Adaxial surface is uppermost. a and b, microtomed sections of fresh leaf material fixed and embedded as for electron microscopy and stained in toluidine blue. This treatment provides better definition for chloroplasts than that used in o and d. o and d, hand-cut sections of fresh leaf material fixed in FAA.

Abbreviations: PCR, Photosynthetic Carbon Reduction (Kranz) cells; ec, extension cells; c, chloroplasts; x, metaxylem vessel element. All bars: a, Sporobolus areber. Tl'.O small vascular bundles.

Biochemically typed as PCK. uneven PCR bundle outline, and inflated PCR cells containing centrifugal/peripheral chloroplasts. One extension cell. b, S. oaroU. One large and one small vascular l:undle.

Biochemically typed as NAD-ME. Even PCR bundle sheath outline and centripetal chloroplasts. The large vascular l:undle shows this species to be XyMS+, o, Zoysia maorantha. Three small vascular bundles. Not biochemically typed, but outline and chloroplast position are similiar to those of PCK S. oreber. d, Tripogon loUiformis. One large and one small vascular bundle. Not biochemically typed, but outline is similiar to that of NAD-ME S. oaroU. In this micrograph PCR cell chloroplasts are swollen and fill cells but in other material of this species they are clearly centripetal. The large vascular bundle shows this species to be XyMS+. \ r " \ Light mi crog raphs of vascular rundles from transverse sections of chloridoid and arundinoid leaf blades. Adaxial surface is uppermost. a and d, hand-cut sections of fresh leaf material fixed in

FAA. b and o, mi crotomed sections of fresh leaf material fixed and embedded as for electron microscopy and stained in toluidine blue. All bars: 25 µ m. a, Triraphis moUis. Three small vascular rundles.

Biochemically typed as NAD-ME al though PCR sheath outline and PCR cell chloroplast position are sirni liar to those of P'.:K Sporobol.us oreber

(Fig. 4.1a). b, Enneapogon pol.yphyUus. One large and one small vascular bundle. Biochemically typed as NAD-ME although chloroplast position is centripetal/peripheral and sheath outline is uneven. o,

Eriaohne aristidea. One small and one large vascular bundle.

Biochemically typed as NADP-ME although species is XyMS+, has quite an uneven sh ea th outline and centrifu:i al/peripheral chloroplasts (in this micrograph mainly centrifugal). d, E. pul.ohena. One small and one l'arge vascular bundle. Biochemically typed as NADP-ME although species is XyMS+, has an even sheath outline and centripetal chloroplasts. '

..('',/ ! 1' Fig. 4.3. Light micrographs of vascular l::undles from transverse sections of chloridoid leaf blades. Adaxial surface is uppermost.

Hand-cut sections of herbarium material partially rehydrated in H 2o. PCR cell chloroplast position not identifiable. Abbreviations: as for

Fig. 4 .1. All scales; 25 µm, a, Heterachne guLLiveri. One small and one large vascular l::undle. Not biochemically typed. PCR cells distorted but 'inflated' outer tangential walls, especially of the large

'corner' cells near abaxial leaf surface in the small bundle, and presence of extension cells are indicative of uneven outline, similar to that of PCK Sporobolus creber (Fig. 4.1a). 'Ihe large vascular l::undle shows this species to be XyMS+. b, EragrostieHa bifaria. XyMS+. One sma 11 and one large vascular bundle. Nat biochemically typed, Even bundle sheath outline like that of NAD-ME SporoboLus caroLi (Fig.

4.1b). c, Ectrosia bLakei. Two small vascular l::undles. Not biochemically typed. Uneven bundle sheath outline, extension cells and

•l.nflated' PCR cells (especially the large 'corner' cells near abaxial leaf surface) are similiar to those of Zoysia macrantha (Fig. 4.1c), d,

Lepturus repens. Two small vascular bundles. Not biochemically typed. Bundle sheath outline mostly even but 'corner• cells have

'inflated' outlines, N.B. Cells at adaxial apex of vascular bundles do not appear to contain chloroplasts and so do not rank as PCR extension cells.

CHAPTER 5

liEW STRUCTURAL/BIOCHEMICAL ASSOCIATIONS IN LEAF BLADES OF

C4 GRASSES (POACEAE). 56

Abstraat

Forty-three previously uninvestigated, mainly Australian grass

species (Poaceae) were assayed for c 4 acid decarboxylation enzyme activity (NADP-malic enzyme, NADP-ME; NAD-malic enzyme, NAD-ME; PEP

carboxykinase, PCK). Twenty-five species exhibit long-established

('classical') associations between c 4 type and structural features of leaf blade vascular bundles. However, Paniaum virgatum and Triraphis moUis are NAD-ME species with structure like that of 'classical' l?CK

species. Seven Enneapogon species and Triodia saariosa are NAD-ME but

are structurally intermediate between 'classical' NAD-ME and l?CK

species. AUoteropsis semialata (R. Br.) Hitch. is PCK, the first

recorded non-NADP-ME XyMS- species, and Pheidoahloa graailis s. T. Blake and five Eriaahne species are the first known XyMS+ NADP-ME species, with either centripetal or centrifugal/peripheral l?CR (Photosynthetic

Carbon Reduction, or Kranz) cell chloroplasts. A suberized lamella is absent from the l?CR cell walls of all species with an even l?CR bundle

sheath outline, irrespective of c 4 type, as well as from NADP-ME Aristida, Eriaahne and Pheidoahloa; it is present in all other species with an uneven outline and centrifugal/peripheral chloroplasts.

Pheidoahloa graaiUs and Eriaahne spp. have unusually well-developed grana in PCR cell chloroplasts for NADP-ME species. This new-found structural/biochemical diversity is discussed in relation to high (co 2 J maintenance in PCR cells. 57

Jtntrodlction

All three biochemical types (NADP-ME 1 , NAD-ME, PCK) of the c4 pathway of photosynthesis are found in grasses (Poaceae). The diagnostic structural features of each c4 type were described by Gutierrez et al. (1974) and Hatch et al. (1975), with later additions by

Hattersley and Watson (1976) and Ellis (1977). Thus NADP-ME species are expected to be XyMS- 2 and to have centrifugal PCR 3 cell chloroplasts;

NAD-ME species to be XyMS+, to have centripetal chloroplasts and an even

PCR bundle sheath outline; PCK species also to be XyMS+, but to have centrifugal chloroplasts and an uneven outline (see Fig. 5.1). The chloroplast position of 'classical' NADP-ME and PCK species was redefined as centrifugal/peripheral in Chapter 4. This redefinition also applies to the more recently discovered ('non-classical') structural/biochemical suite of NAD-ME species in Paniaum and Eragrostis whose leaf blade structure is, nevertheless, apparently identical to that of 'classical' PCK species (Ohsugi and Murata 1980, 1981; Ohsugi et al. 1982; Chapter 4; see the latter for full introduction to topic).

Such latter species are described here as 'PCK-like'.

Subsequent to a structural, biochemical and phytogeographical study of the c genus (Chapter 3), and in parallel with a large- 4 Eragrostis scale leaf structural survey of Australian grasses in relation to c 4 type (Chapter 4), a biochemical and ultrastructural investigation of a selection of species was undertaken with two aims: i) to assess the

NADP-ME, NADP-malic enzyme; NAD-ME, NAD-malic enzyme; PCK, PEP carboxykinase. 2 XyMS+/-, presence/absence of cells intervening between metaxylem vessel elements and laterally adjacent chlorenchymatous bundle sheath PCR cells of primary lateral bundles (Hattersley and Watson 1976). 3 PCR tissue, photosynthetic carbon reduction tissue, equivalent to Kranz tissue; PCA tissue, primary carbon assimi la ti on tissue, usually equivalent to c mesophyll (definitions by Hattersley 1976). 4 et al. 58

extent and taxonomic distribution of exceptions to the 'classical'

structural/biochemical correlations and ii) to establish whether

exceptions of other kinds occur in previously uninvestigated taxa.

Apart from assessing the reliability of previously established

structural indicators of c4 type, such data might elucidate the

contribution made by each of the various structural features of FCR

tissue in maintaining a high a key feature of

photosynthesis. They are also a prerequisite for phytogeographical and ecophysiological analyses of leaf blade structure and c 4 types (cf. Ellis et al. 1980; Chapter 8).

Materials and Methods

Experimental plant material

Plants were grown either in glasshouses subject to natural lighting conditions and maintained between 35°C (day maximum) and 15°c (night minimum), or in growth cabinets with a 1 6 hour day at 30°C and an 8 hour night at 15 °c. Plants were regularly fertilised with Ruakura nutrient solution (Smith et al. 1983). The same accessions of each species were used for all aspects of the study. Iden ti ties of both original accessions and experimental plants were checked.

Leaf blade anatomy and ultrastruature (suberized lamella)

Examination of fresh leaf material for light and electron microscopy was as described in Chapter 3.

Assays of c4 aaid deaarboxylation enzymes Details are given in Chapter 3. Extraction procedure for NAD-malic enzyme and NADP-malic enzyme closely followed that of Hatch et ai. 59

( 1982) and Edwards et al. (1982), the latter also describing the assay

procedures for both enzymes. For PEP carboxykinase, extraction buffer

was mainly as described by Ku et aL. (1983) and the reaction mixture for

the assay mainly as described by Hatch and Mau (1977). For experiments

11-15, differences from the procedure for the PCK assay as described in

Chapter 3 were as follows: 0.5mM Mnc1 2 was used (instead of 2mM), 0.3mM oxaloacetic acid (OAA) (instead of 0.6mM), and 1 unit of pyruvate kinase

(instead of 2). Chlorophyll determination followed Arnon (1949).

Previously uninvestigated species were mostly assayed at least

twice for each enzyme, each figure in the table representing the mean

activity of duplicates from one extract. NAD-ME (NAD-malic enzyme) and

NADP-ME (NADP-malic enzyme) assays were done on different eluates of the

same extract, PCK (PEP carboxykinase) assays on a separate extract.

Results

Activities of c 4 acid decarboxylation activities are shown in Table

5.1. Table 5.2 lists species scored according to biochemical, anatomical and ultrastructural characteristics and Fig. 5.2 summarises this diversity schematically.

Main c4 aoid deoarboxyLating enzyme in relation to leaf blade anatomy In four NADP-ME, nine NAD-ME and 12 PCK species of the 43 species not previously examined biochemically, the established structural predictors of c 4 type remain valid (see Table 5.2 and Fig. 5.1). Three of the four NADP-ME species (Aristida biglandulosa, A. latifolia and

ArundineUa nepalensis) have 'non-classical' leaf blade structure but are typically NADP-ME species in that they are XyMS- and have centrifugal/peripheral PCR cell chloroplasts. 60

Paniawn bergii, P. virgatwn and Triraphis mo l Lis 1 are further examples of NAD-ME species whose anatomy is apparently indistinguishable from that of 'classical' PCK species (i.e. it is PCK-like). Tridens brasiLiensis also has PCK-like anatomy and lacks PCK activity, but its

NAD-ME activity is also exceptionally low; in most experiments to date, indeed, NADP-ME activity is greater (Table 5.1 and unpublished results). seven species of Enneapogon (Fig. 5.1) have an uneven PCR bundle sheath outline and are NAD-ME; many, often most, of their PCR cell chloroplasts are centripetal rather than centrifugal/peripheral (Fig. 5.1bJ.

Furthermore, these chloroplasts are elongated rather than ovalish in shape, a feature otherwise alrrost exclusively characteristic of

'classical' NAD-ME species (Fig. 5.2). Triodia seariosa, with an even outline and centrifugal/peripheral chloroplasts, is also NAD-ME.

ALLoteropsis semiaLata is PCK, and as such is the first known XyMS­ species that is not NADP-ME. Pheidoehloa graeiiis1 and five Eriaehne species are the first recorded XyMS+/NADP-ME species. On the basis of established structural/biochemical correlations and light microscope examination, P. graeiLis, E. aristidea1, E. glabrata and E. ovata were expected to be PCK and E. puLeheUa 1 to be NAD-ME (but see

Ultrastrueture: suberized fomeUa). E. obtusa has mostly centripetal chloroplasts, but since it also has chloroplasts in a centrifugal/peripheral position and a PCR bundle sheath outline that is somewhat intermediate (even/uneven), initial assignment to c4 type on the basis of leaf anatomy was rather equivocal. E. obtusa and E. puLehella are the only known NADP-ME species with (mainly) centripetal chloroplasts. These chloroplasts, however, are ovalish in shape as in other Eriaehne species, in contrast to the elongated centripetal

1 Leaf blade structure illustrated in Chapter 4. 61

chloroplasts of 'classical' NAD-ME species (and of Enneapogon).

NAD-ME/PCK Bouteloua aurtipendula and NAD-ME Triodia saariosa are

the only species of ~~eir respective c4 types known to combine centrifugal/peripheral chloroplasts and even PCR bundle sheath outlines.

Ultrastxn~ature: suberized lamella

In the species examined ultrastructurally examined by me, a

suberized lamella is absent from the PCR cell walls of the 'classical'

NAD-ME species Leptoahloa digitata, Oxyahloris saariosa, Sporobolus aaroli and S. pulaheZlus, of NAD-ME Triodia saariosa and of NADP-ME

Pheidoahloa graailis and five Eriaahne species (Table 5.2). Apart from

Aristida spp., P. graailis and Eriaahne spp. are the only known NADP-ME

species without a suberized lame lla. It is present in those XyMS+ species examined by me with an uneven PCR bundle sheath outline and at least some centrifugal/peripheral chloroplasts: NAD-ME Enneapogon intermedius, E. lindleyanus, E. polyphyllus, and PCK-like Triraphis moUis and 'classical' FCK Chloris divariaata, C. virgata, Leptoahloa ailiolata and Sporobolus areber.

Primary veins of the arundinoid species Pheidoahloa graailis and

Eriaahne aristidea and E. glabrata (E. obtusa, E. ovata and E. pulahella were not examined) and the chloridoid Triraphis mollis apparently lack a suberized lamella in the walls of the mestome sheath cells, a characteristic previously unrecorded in c4 XyMS+ species. A further unusual ultrastructural feature of Eriaahne and Pheidoahloa spp. is that granal stacking in their FCR cell chloroplasts is more extensive than has been recorded for any other NADP-ME species. 62

Activities of c4 acid decarboxylating enzymes

At least some NAD-ME and NADP-ME activity was detected in all species (Table 5.1 ) • Except where PCK was the main c acid 4 decarboxylating enzyme, PCK activity was otherwise only apparent in four

NAD-ME species (maximum: 0.33 µmol mg-1 Chlor. min-1 in Sporobolus puLcheLLus) and in five NADP-ME species (maximum: 1 .83 p mol mg-1 Chlor. min-1 in Spinifex sericeus).

There is no evidence here of any quantitative difference in the

NAD-ME activities of NAD-ME species with contrasting PCR cell chloroplast positions and PCR bundle sheath outlines. Among chloridoid species with 'classical' NAD-ME structure, NAO-ME activities ranged from as low as 0.97 and 1.07 µmol mg- 1 Chlor. min-1 in Sporobolus puLcheLLus up tc 7.01 and 7.87 µ mol mg-1 Chlor. min-1 in Leptoahfoa digitata.

Within ('non-classical') Enneapogon, minimum and maximum activities were

0.88 (E. nigricans) and 6.96 (E. Undleyanus) µmol mg- 1 Chlor. min-1 •

Likewise, no trends are apparent in activities of the smaller samples of

'classical' and 'non-classical' NAD-ME Panicwn (Panicoideae) species.

PCK activities in PCK species were usually higher than were NAD-ME activities in NAD-ME species. NAD-ME activities in PCK species were also often higher than in NAD-ME species e.g. the 3. 79 µmol mg-1 Chlor. min- 1 recorded for Bouteloua curtipendula. This value actually exceeded the PCK activity in the same eXPeriment al though it was considerably less than that in the preceding one (Table 5.1 ). PCK Leptoohfoa eiliofota consistently had a higher NAD-ME activity than some NAD-ME species. Prior tc the modification of the PCK reaction mixture (i.e. in experiments 1-10; see Materials and Methods), high rates of non-enzymic

OAA degradation were obtained in a number of NAD-ME and NADP-ME species.

Al though XyMS- like all biochemically determined NADP-ME species, 63

PCK AHoteropsis semiafota had as little NADP-ME activity as other PCK

(XyMS+) species. XyMS+ Pheidoahloa graailis and Eriaahne spp. had no

PCK activity at all (with the apparent exception of E. pulahelia) and

only very small activities of NAD-ME, Their NADP-ME activities,

however, were as high as those of other (all XyMS-) NADP-ME species;

that of E. pulahella in experiment 8, indeed, (19.50 µmol mg-1 Chlor.

1 ) min- being the highest of any c4 acid decarbcxylating enzyme recorded

in these experiments.

Enzyme activities of species assayed twice in the same experiment

(i.e. two sets of duplicate measurements) generally showed good

reproducibility e.g. Aristida biglandulosa (NADP-ME), Paniawn

diahotomiflorwn (NAD-ME) and Sporobolus elongatus (PCK). A notable

exception, perhaps the result of experimental error, was the very

different NAD-ME activities recorded for P. lanipes in experiment 11

(1.74 and 8.34 µmol mg-1 Chlor. min-1 ). The activities of the main

enzyme for a species differed to varying extents between experiments cf.

the fairly consistent PCK activities of Leptoahloa ailiofota and the

very different ones of Bouteloua aurtipendufo. B. aurtipenduia and

Tridens brasiliensis were the only two species in which assignments of

type on the basis of the main acid decarbcxylating enzyme activity c4 c4 were at all equivocal.

Di sOJ. ssion

In sampling most of the taxonomic and structural diversity of the

Australian grass flora, this study has not only considerably c4 increased the number of grass species biochemically assayed for c4 acid decarboxyla ting enzymes, but has also uncovered several new ana tomi cal/u l trastructural/biochemi cal combinations. It has, 64

furthermore, confirmed the reliability of using structural para.meters to distinguish between c 3 and c 4 species, as well as mostly substantiated the typing of species in the sample that have been previously c4 investigated for activities of c 4 acid decarboxylating enzymes. Among species of the same c4 type, there is no evidence that either str~cture or taxonomic position are associated with constant differences in activities of the main c4 acid decarbcxylation enzyme (as is broadly true in chlorophyll a:b ratios as well; see Chapter 6). It is unknown whether such differences would emerge even under the most standardised and/or optimal conditions for all species.

For three species, however, the structural/biochemical results are apparently inconsistent with those of previous studies. Paniown lanipes is NADP-ME according to Gutierrez et al. (1974) but both Ohsugi et al.

(1982) and this study find it to be NAD-ME. P. bergii has been found to be NAD-ME (this study; and Gutierrez et al. 1974) but its PCR cell

~hloroplasts are described here as centrifugal/peripheral and as centripetal by Gutierrez et al. (1974), Either there are identification problems (not unlikely considering the taxonomic difficulty of the genus), or else P. bergii really is structurally variable (like P. ooloratwn L.; Ohsugi et al. 1982). The third species, AUoteropsis semialata, may well be biochemically variable within representatives. The unexpected, but considerable, PC!< activity found in the Australian accession is at variance with the apparently high

NADP-ME activity, and negligible PCK activity, recorded in glasshouse­ g rown South African plants (Frean et al. 1983). In South African plants grown outside, NADP-ME activity was very considerably reduced and was actually just exceeded by PCK activity (also very low). Frean et al.

(1980) and Hattersley and Browning (1981) observed well stacked grana in 65

the PCR ~ell chloroplasts of A. semiaLata (South African and Australian accessions respectively), a feature not then recorded for any biochemically determined NADP-ME species; cf. results for PheidochLoa graci Us and Eriachne spp. in this study and for Neurachne munroi and

Paraneurachne 111J.eUeri (Hattersley et ai. 1986). If South African plants really are NADP-ME, in contrast to Australian plants, or if they are plastic for c 4 type, this would add greatly to the interest of a species already remarkable for containing both c 3 and c 4 forms (Ellis

1974a, 1974b).

The ultrastructural results for NAD-ME and PCK species are largely consistent with the structural/biochemical correlations found by

Hattersley and Browning (1981) and the studies of Chapter 3; namely, that the suberized lamella is absent from the PCR cell walls of NAD-ME species with 'classical' NAD-ME structure, but present in species of both c types with centrifugal/peripheral chloroplasts. The absence of 4 a suberized lamella from NAD-ME Triodia scariosa (substantiating previous reports for the genus by Carolin et ai. 1973 and Craig and

Goodchild 1977) and from PCK BouteLoua curtipenduLa (Hattersley and

Perry 1984), all species with centrifugal/peripheral chloroplasts but an even PCR sheath outline, means, however, that the presence of a lamella in NAD-ME and PCK species is more highly correlated with uneven outline than with centrifugal/peripheral chloroplasts. The absence of a lamella in the PCR cell walls of NADP-ME Eriachne spp. studied here complements a similar observation for E. lllJ.cronata by Carolin et ai. (1973) (and for the other NADP-ME arundinoid genus Aristida). PheidochLoa graciUs,

Eriachne aristidea and E. gLabrata (arundinoids) and Triraphis moLLis (a chloridoid), unlike all other XyMS+ species surveyed to date, lack a lamella in the mestome sheath also. In T. moUis the lamella was 66

clearly visible in the walls of the PCR bundle sheath cells in the same

leaf blade transverse section, this species being unique among grasses,

therefore, in the distribution of the suberized lamella in peri vascular

sheaths.

The suberized lamella is absent from the PCR cell walls of all

species with even PCR bundle sheath outlines (except Paraneuraohne mueUeri s.T. Blake: Chapter 4), irrespective of c4 type, PCR cell chloroplast position and taxonomic pcsition, as well as from arundinoid

NADP-ME species with uneven or intermediate sheath outlines and centrifugal/peripheral chloroplasts. It is present in all other species, irrespective of c 4 type, with uneven PCR sheath outlines and centrifugal/peripheral chloroplasts. Apart from P. muelleri, no species is known to have both an even outline (i.e. perhaps relatively limited

PCR/PCA tissue contact and/or a low PCR tissue surface area/volume ratio, for co2 leakage) and a suberized lamella, even when PCR cell chloroplasts are centrifugal/peripheral and thus largely lie alongside this interface, as in Bouteloua aurtipendula and Triodia soariosa. An uneven outline, with perhaps its larger PCA/PCR tissue interface and higher PCR tissue surface area/volume ratio (and concomitant potential for higher co 2 leakage rates) may 'need' to develop a suberized lamella to create an adequately •co2-tight' compartment. This idea is supported by the apparently identical co 2 post-illumin.ttion"' burst (a measure of co2 leakage) shown by 'classical' and PCK-like NAD-ME Panioum spp. (Ohsugi and Murata 1980; Ohsugi et al. 1982): i.e. by species with either an even outline and no suberized lamella or an uneven one with a suberized lamella.

The structural characteristics of NADP-ME Aristida may represent a

The OU tline of its PCR unique suite for dealing with CO 2 leakage• 67

tissue (as identified by Hattersley and BrowniIKJ 1981) is somewhat

intermediate (i.e. even/uneven) and there is no suberized lamella

(Carolin et ai. 1973; Hattersley and BrowniIKJ 1981). The significant

feature, characteristic of the genus, could be the second, outer

chlorenchymatous sheath which closely envelopes the inner sheath and whose chloroplasts are centripetal. This positioning of both sheath and

chloroplasts ('compensatory' features) may permit refixation via the

Calvin cycle of cc 2 leaking from the inner PCR sheath (see Hattersley

and BrowniIKJ 1 981 ) •

The most notable known exceptions to the combination of uneven PCR

sheath outline and presence of a suberized lamella are NADP-ME

Pheidoahfoa graaiUs and Eriaahne aristidea, E. gfobrata and E. ovata.

Again, however, there are structural features in these species which may have 'compensatory' roles. In the centrifugal/peripheral PCR cell chloroplasts of these species, and also in those of E. obtusa and E. pulohella (which are mainly centripetal), there are well-developed grana

(Fig. 5. 1d, e, f; up to 21 appressed thylakoids visible in latter 1 ).

The PCR cell chloroplasts of all other NADP-ME species which have been investigated (see e.g. Laetsch 1969, Downton 1971 and Woo et ai. 1971 for species now known to be NADP-ME) are either agranal or have very few

(<6) appressed thylakoids per granum (exceptionally more in the double- sheathed species Aristida bigfondulosa, Neuraahne munroi and

Paraneuraahne muelleri; Hattersley and BrowniIKJ 1981; Hattersley et ai.

1986), structural conditions associated with a lack of non-cyclic electron flow (Downton et ai. 1970), a high chlorophyll a/b ratio (Woo

Only mature leaf blades were used for ultrastructural analysis. Laetsch ( 1969) showed that the absence of grana in mature chloroplasts of NADP-ME Saoaha1'W/I offioinQ1'W/I L. is the result of "regression"; immature chloroplasts have grana with up to nine thylakoids each. 68

et ai. 1971 l, and a deficiency in photo system II (Woo et al. 1970;

Anderson et al. 1971; Ku et al. 1974; Mayne et al. 1974). Al though

malate decarboxylation via NADP-ME provides half the NADPH required for

the reduction of 3-phosphoglyceric acid (PGA) in the photosynthetic

carbon reduction cycle (Downton 1970; see Edwards and Huber 1981), the

above studies have indicated that there is still insufficient

photochemical production of NADPH in PCR tissue for complete reduction of PGl\., some of which is therefore transported to PCA (C 4 mesophyll) tissue. Pheidoehloa graeilis and Eriaehne spp., with well developed grana, perhaps have a greater capacity for NADPH production in K:R tissue than other NADP-ME species and this in turn would presumably

lessen the necessity for PGA transport to PCA tissue. The relationship between such an enhanced reducing capacity and the rate of co 2 leakage is not known, rut certainly the potential for the latter may be particularly high given the centrifugal/peripheral position of the PCR cell chloroplasts (where decarboxylation in this c 4 type takes place; Slack et al. 1969), the uneven PCR sheath outline (i.e. an extensive

PCR/PCA tissue interface) and the lack of a suberized lamella in the cell walls of both mestome and PCR sheaths.

In E. obtusa, at least (Fig. 5.1 d), the outer tangential walls have striations simi liar to those noted in 'classical' NAD-ME species which also lack a suberized lamella (Hattersley and Browning 1981; Chapter

3). The composition of these striations is not known, but perhaps they fulfil a similiar role to the suberized lamella. The Eriachneae may be additionally unusual in their mesophyll: bundle sheath area ratio; the ratio for E. ovata (2.2), at least, is considerably lower than those of all other NADP-ME species examined (x= 3.7; Hattersley 1984) and lies within the range characterising NAD-ME and PCK species. 69

Fig. 5.2 summarises schematically the presently known diversity of

structural/biochemical suites in c 4 grasses. The discovery of this diversity has come about through a deliberate taxonomic approach to

sampling, which ensured the inclusion of outlying groups such as the

tribe Eriachneae (comprising Eriaahne and Pheidoahloa), and of others, such as Enneapogon (one of a number of genera described by Watson et al.

1985 as 'chloridoids with arundinoid affinities') which are difficult to place taxonomically. Species whose leaf blade structure was already known to be unusual, e.g. Triodia saariosa (Burbidge 1946; Craig and

Goodchild 1977) and Alloteropsis semialata (Ellis 1974a, 1974b), have also proved to be particularly interesting in the structural/biochemical context. The commonest 'non-classical 1 structural/biochemical combination appears to be that found in those chloridoid and Paniaum

NAD-ME species whose leaf blade structure (including the shape of the

PCR cell chloroplasts) is apparently indistinguishable from that of

;classical' PCK species. Despite the diversity of suites now known, it is worth emphasising that the original ('classical') suites described by

Gutierrez et al. (1974) and Hatch et al. (1975) may still apply to the majority of c 4 grasses. Nothing is known about the genetic and evolutionary relationships between c 4 types and their structural variations. Brown (1977) pointed out that in chloridoid genera "most species seem to be NAD-ME, so that the few PEP-ck (PCK) species seem to be derived from some recent or extant NAD-ME ancestors. 11 Perhaps this hypothesis is supported by the fact that, in contrast to NADP-ME and NAD-ME, PCK is generally only detectable in those species where it is the main c 4 acid decarboxylation enzyme (Table 5.1). Intraspecific structural variation seems to be unusual. It does occur, however, in NAD-ME Paniaum aoloratwn, 70

accessions of which can either have 'classical' NAD-ME or PCK-like

structure, although there are no quantitative differences in their

enzyme activities (Ohsugi et aL. 1982). Intraspecif ic biochemical

variation has yet to be definitely established for any species (cf. discussion on ALLoteropsis semiaLata).

There are several instances of intermediate structural suites. In

NAD-ME Enneapogon and intermediate Eragrostis spp. (Chapter 3) the shape and position of the PCR cell chloroplasts are more like those of structurally 'classical' NAD-ME species, but the outline of the bundle sheath more approaches that of 'classical' PCK (or PCK-like NAD-ME) species; as in the latter there is also a suberized lamella in the PCR cell walls. This structural intermediacy, however, is not matched in biochemical terms since very few NAD-ME species, whatever their structure, have any PCK activity at all. The term 1 intermediate' could also be applied to NAD-ME Triodia saariosa and PCK BouteLoua aurtipenduLa, but intermediacy in the PCR sheath structures of these species is the reverse of that in Enneapogon and Eragrostis: here the shape and position of the chloroplasts are more like those of

'classical' PCK (or PCK-like NAD-ME) species, whereas the (even) sheath outline and the absence of a PCR cell suberized lamella is more

'classical' NAD-ME-like. In B. aurtipenduLa either NAD-ME or PCK activity may predominate (Gutierrez et aL. 1974; this study), indicating that this species may well be intermediate biochemically as well as structurally. It also exhibited the lowest (most NAD-ME-like) value of any of the PCK species studied by Hattersley ( 1982). Eriaahne obtusa, with an even/uneven outline, and centrifugal/peripheral as well as centripetal chloroplasts, also looks structurally intermediate between other species in the genus. 71

There have been no large-scale, comparative, physiological studies

of species (now known to be) of different c 4 type, apart from those

concerning the co2 post-illumination burst (PIB) (viz. Downton 1970; 13 Brown and Gracen 1972; Murata and Ohsugi 1984), 0 c values (Hattersley

1982) and to a lesser extent quantum yield (Ehleringer and Pearcy

1983). Ohsugi and Murata ( 1985) compared PIB and growth characteri sties

of NAD-ME Paniown spp. with 'classical' and PCK-like leaf blade

structure, claiming that the latter show relatively higher "productivity

in early growth, particularly high rate of leaf development" and that

they "may be adapted particularly to high moisture conditions." There

is support for the latter statement in the finding that NAD-ME

Eragrostis spp. with PCK-like structure are 11 most numerous in northern,

high rainfall, tropical Australia and also predominate in relatively humid coastal and subcoastal areas 11 in contrast to 'classical 1 species which are numerically and proportionally dominant in arid areas (Chapter

3). In terms of species numbers, species with 'classical' NAD-ME

structure are also apparently the dominant structural/biochemical type

in the most arid areas of South West Africa/Namibia (Ellis et ai.

1 980) • The available evidence, therefore, suggests that it wuuld be worthwhile conducting experiments to pin-point the ecophysiological

significance of the structural/biochemical diversity of c 4 grasses highlighted by this study. These would need to employ samples of

species chosen to observe the effects of variations in c 4 type and in structural features independently of one another. Table 5.1. Activities of acid decarboxylating enzymes in (mainly) Australian grasses. c4

·rhere were 15 experiments (numbered), each in vol vi1¥J up to 12 species includiaJ c and appropriate C 3 4 controls. C denotes enzyme activity of control species in relevant experiment. controls c4 have been previously typed: Bouteloua graailis (Gutierrez et al. 1974); Chloris gayana

(Gutierrez et al. 1974; Hatch and Kagawa 1974; Hatch et al. 1975); Cynodon daatylon (Hatch and Kagawa

1974); Chloris trunaata, Cymbopogon aitratus (Chapter 3); Paniaum bulbosum (Downton 1970);

P. sahinzii (= P. laevifolium Hackel) (Ohsugi et al. 1982); Zea mays (e.g. Gutierrez et al. 1974; Hatch

et al. 1975). Other c 4 control species were assayed earlier in the study. Rhynahelytrum repens was used

errcneously as a PCK control in experiment 1 3 as this species had not in fact been previously assayed;

was originally misidentified and used as an NAO-ME control in experiment 9. A control Paniewn maximwn c 3

was omitted in experiment 1. N::> NAD-ME or NADP-ME assays were done in experiments 1 and 2, and no PCK

assays in experi1nents 5 and 6. Asterisks denote sr)ecies introduced to Australia; crosses denote

non-Australian species; dashes denote enzyme activities not measured. G, growth cabinet-grown; L ,

glasshouse-grown; O, cultivated outside; w, wild plant outside. Subfamily classification after

Watson et al. (1985). -1 . -1 ) Subfamily Experiment Growth Ac ti vi ty (µmo l IUI) Chlor. min Species number en vi ronme n t NAD-ME PCK NADP-ME ·------c4 species

Chloridoideae

Astrebla elymoides F. Muell. 1 2 G 3.26 0 0.79 12 G 3.59 0 0.69

A. lappaoea (Lindl.) Domin 1 3 G 1. 68 0 0.42 13 G 1.93 0 0.40

A. peotinata (Lindl.) 12 G 2. 1 7 0 0.42 F. Muell. ex Benth. 12 G 2.38 0 0.56

A. squarrosa c. E. Hubbard 12 G 2.10 0 o. 32 12 G 2.93 0 0.38

+ Bouteloua ourtipendula 1 L 11. 08 (Michx.) Torr. 4 L 3.70 2.45 0.42 6 L 0.89 0.16 15 G 3.75 1 • 99 0.46

+ B. graoilis (H.B. K.) Lag. 2 [, 0 3 L 0 0.29 5 L 0.29

Chloris divarioata R. Br. L 11.29 3 L 0.78 16.50 0.24 5 L 0.98 0. 17 1 4 G 0.90 0.21

* C. gayana Kunth L 9.61c 2 L 9. 23c 15 L 0.31 s .nc 0.03 ------Subfamily Experiment Growth Activity (µmol mg -1 Chlor. min -1 ) Species number environment NAO-ME PCK NADP-ME

c. peatinata Benth. 1 L 12.05 3 L 0.95 15.35 0.18 5 L 0.82 0.17

c. trunaata R. Br. 4 L 1.28 4.00C 0.25 6 L 1. 05 0.28 9 L 1 • 21 9.42c 0.28 1 2 L 0.97 6.1F 0.41

• c. virgata Swartz 2 L 10.33 3 L o. 31 1 O. 1 3C 0.17 5 L 0.41 o. 16

Cynodon daotylon (L.) Pers. 14 G 3.29c 0 o. 34 15 L 1.96c 0 0.31

Enneapogon avenaoeus (Lindl. ) 7 L 4.68 0.14 0.46 c. E. Hubbard 8 L 4.53 0 0.31

E. caerulesaens (Gaudich.) 7 L 3.74 0 o.97 N. ·r. Burbidge 8 I, 3,74 0 0.72

E. intermedius N. T. Burbidge 2 L 0 3 L 6.82 0 0.43 5 L 5.92 0.61 1 Subfamily Experime-nt Growth Activity (µmol mg-l Chlor. min- ) Species number environment NAD-ME PCK NADP-ME ------

E. UndZ.eyanus (Domin) c.e. "'~••••"'- 2 L 0 4 L 6.96 0 0.49 6 L 6.23 0.56 11 L 5.24c 0 0.48

E. nigrieans (R. Br. ) Beauv. 7 L 3.55 0.28 o. 35 8 L 0.88 0 0.18

E. poZ.yphyZ.Z.us (Domin) 2 L 0 N. T. Burbidge 4 L 4.73 0 0.78 6 L 4.67 o.53

E. robustissimus (Domin) 9 L 0 0.63 N. T. Burbidge 9 L 0 0.43 12 L 0 o.69

Leptoehl.oa eiliofota (Jedw.) 2 L 3.39 s. T. Blake 3 L 3.02 5.30 0.45 5 L 3.23 0.29 7 L 1.70 4.34c 0.18 8 L 1. 93 4. 76c 0.17

L. digitata (R. Br.) Domin L 0 2 L 0 4 L 7.87 0 0.32 6 L 7.01 0.41 -1 -1 Subfamily Experiment Growth Activity (µ mol mg Chlor. 1nin ) Species number environment NAD-ME PCK NADP-ME

Oxyahloris saariosa (F • Muell. ) 1 L 0 Lazar ides 3 I, 4.1 5 Q 0.35 5 L 5.70 0.28

• Sporobolus afriaanus (Pair.) 1 L 16.79 Robyns & Tournay 3 L 0.96 1 3. 76 0.14 5 L 1.57 0.18 1 1 L o.74 12. 54C 0.24

s. aaroli Mez L 0 3 L 1 • 61 0 0.22 5 L 1.67 0.43

s. ereber De Nardi 1 L 18.27 2 L 13.34 3 L 1. 06 14.25 0.09 5 L 1. 30 0.09

s. elongatus R. Br. 10 L 0.93 11. 44 0.11 10 L o. 71 12.34 0. 1 3

• s. jaaquemontii Kunth 7 G 0.52 1 3. 99 0.08 8 G 0.49 8.93 0.08

s. pulaheUus R. Br. 7 L 0.97 0.33 0.32 8 L 1. 07 0.15 0.27

+ Tridens brasiliensis 13 L 0.22 0 0.36 Nees ex Steud. 13 L 0.26 0 0.47 ------1 Subfamily Experiment Growth Activity (µ mol mg Chlor. min. -1) Species number environment NAD-ME PCK NADP-ME ------·------·------14 L 0.70 0 0.60 14 L 0.61 0 0.41

Triodia seariosa N. T. Burbidge 4 0 3.07 0 0.63 6 0 1.54 o.52

Triraphis moUis R. Br. 7 L 5.74 0 0.29 8 L 4.11 0 0.25

Arundinoideae

Aristida bigLandu Losa 10 G 0.25 0 10.63 J. M. Black 10 G 0.22 0 10.00

A. Latifoiia Domin 10 G 0.24 0 11.28 10 G 0.21 0 9.20

Eriaehne aristidea F .. Muell. 7 G 0.58 0 1 o. 70 8 G 0.40 0 4.65

g. glabrata (Maiden) Hartley 9 G 0.07 0 0.97 9 G 0.09 0 3.02

E. obtusa R. Br. 7 G 0.26 0 6.34 8 G 0.27 0 6.76 9 L o.32 0 4.59c 10 G 0.22 0 7. 1 OC

E. ova ta Nees 2 L 0 4 L 0.82 0 13.19 6 L 0.62 10.02 ------Subfamily Experiment Growth Activity (µmol mg-1 Chlor. min-l) Species nu1nber environment NAD-ME PCK NADP-ME

E. puleheHa Domin 2 L 0 4 L o.56 0 14.00 6 L 0.62 10.00 7 L,G o.39 0.52 9.14c 8 L,G 0.92 0 19. 5oc

Pheidoahloa graailis 12 G 0.22 0 8.83 s. T. Blake 12 G 0.17 0 7.87

Panicoideae

Ailoteropsis semiafota (R. Br.) 9 G o. 30 10.55 0.24 Hitchc. 9 G o.38 13.04 0.34 1 0 G 0.20 5.5oc 0.16

Arundineiia nepalensis Trin. 11 L o.53 0.98 4. 1 2 1 1 L o.51 o. 73 3.17

+ Cymbopogon aitratus (DC.) Stapf. 3 L 0.84 0 7.86c 4 L 1. 55 0 1O.43C 5 L 1. 30 11 • ooc 6 L 1. 35 s.o3c 14 L 1. 76 0.31 1S.74C

* Melinis minutiflora Beauv. 1 1 L 1. 53 8.63 0.15 11 L 1. 21 9.44 0.08

+ Paniaum bergi-i Arech. 10 G 3.47 0 0.31 10 G 3.94 0 0.33

* P. bulboswn Kunth 11 L 0.44 0.90 12. 60c 1 Subfamily Exper:lment Growth l\c ti vi ty ( 1,mol Chlor. min- ) Species number en vi ron1nent Nl\D-ME Nl\DP-ME , ______-__ , ______, ______--- --

+ P. diahotomiflorwn Michx. 1 1 G 6.40 0 o.56 11 G 6.44 0 0.68

P. effuswn R. Br. 1 3 L 2.90 0 0.37

13 L 2.96 0 0.31

+ P. lanipes Mez 1 1 J, 1. 74 0 0.47 11 L 8.34 0 0.43 1 3 L S.75C 0 0.40

• P. maximwn Jacq. 9 G 0.80c 14.88 0.15

• P. sahinzii Hackel L 0 4 L 3. 78C 0 0.46 6 L 3.98c 0.17 7 G 2.37C 0.06 0.20 8 G o.88c 0 0.18

+ P. stapfianwn Fourc 9 G 1.35 0 0.33 9 G 0 0.46 10 G O. 75C 0 0.29

+ P. virgatwn L. 4 L 4.07 0 0.22 6 L 5.29 0.32 + Pseudobraehiaria deflexa 13 L 1. 22 4.35 0.37 ( Sch111oach.) I.aunert 13 L 1.28 5.41 0.36

• Rhynahelytrurn repens (Willd.) 13 L 1. 48 3. 82c o. 1 3 c. E. Hubbard ------1 Subfamily Experiment Growth Ac ti vi ty ( µmol mg Chlor. min-l) Species number environment NAO-ME PCK NADP-ME ------Spinifex seriaeus R. Br. 14 G 1.45 1. 63 15.60 14 G 1.54 1.83 18.69

* Zea mays L. 12 L 1. 85 1. 76 11. 8 3C 13 L 1.63 1 • 03 12 .15C

controls c 3 Pooideae

* Anthoxanthum odoratum L. 1 3 L 0.20 0 o. 19

* Tritiawn aestivum L. 2 L 0 3 L 0.18 0 0 4 L 0.31 0 0.08 5 L 0.37 0.08 6 L 0.19 0.07 7 L 0.18 0.08 0.09 8 L 0.24 0 0.08 1 1 L 0.31 0 0.13 12 L 0.22 0 0.11

Arundinoideae

Chionoahloa paUida (R. Br.) 9 w o.67 0 0.32 Zotov 10 w 0.20 0 0.13 Table 5.2. Leaf blade structural-features of (mainly) Australian grasses assayed for acid c4 decarboxylating enzymes.

Assignment of species to type is based on the enzyme with the highest activity (see Table 5.1 ). Abbreviations: c4 XyMS +/-, presence/absence of cells intervening between metaxylem vessel elements and laterally adjacent chlorenchymatous bundle sheath PCR (Photosynthetic Carbon Reduction, Kranz) cells of primary lateral bundles (Hattersley and Watson 1976); cp, centripetal; cf, centrifugal/peripheral; cp/cf, evenly distributed; elong., elongated; oval., ovalish; devel., well developed; rudi., rudimentary. Dashes indicate not recorded. Species authorities are given in •rable 5.1. Subfamily

classification after Watson et al. (1985).

Subfamily XyMS PCR cell PCR cell PCR Suberized Grana in PCR chloroplast chloroplast bundle lamella in cell Species position shape sheath PCR cell chloroplasts outline walls

Chloridoideae

Astrebla elymoides NAD-ME + cp elong. even

A A. lappaeea NAD-ME + cp elong. even absentA devel.

A. peotinata NAO-ME + cp elong. even Subfamily c4 XyMS PCR PCR cell PCR Suberized Grana in PCR type chloroplast chloroplast bundle lamella in cell Species position shape sheath PCR cell chloroplasts outline walls

A. squarrosa NAD-ME + cp elong. even

8 Bouteloua aurtipendula PCK + cp/cf oval. even absent devel. A,C

B. graaiUs NAD-ME + cp elong. even absentA devel. A

Chloris divariaata PCK + cf oval. uneven present devel.

c. gayana PCK + cf oval. uneven presentA devel.A,D

c. peatinata PCK + cf oval. uneven

c. trunaata PCK + cf oval. uneven

c. virgata PCK + cf oval. uneven present devel.

Cynodon daatylon NAD-ME + cp elong. even absentE devel.F ,G

Enneapogon avenaaeus NAD-ME + cp/cf elong. uneven E. aaerulesaens NAD-ME + cp/cf elong. uneven Subfamily C4 XyMS PCR cell PCR cell PCR Suberized Grana in PCR type chloroplast chloroplast bundle lamella in cell Species position shape sheath PCR cell chloroplasts outline walls ---- E. interrnedius NAO-ME + cp/cf elong. uneven present devel.

E. Undleyanus NAO-ME + cp/cf elong. uneven present devel.

E. n-lgriaans NAO-ME + cp/cf elong. uneven presentA devel. A

E. polyphyllus NAO-ME + cp/cfH elong. II uneven present devel.

E. robustissirnus NAO-ME + cp/cf elong. uneven

Leptoahloa ailiolata PCK + cf oval. uneven present devel. 11 L. digitata NAO-ME + elong • even absent devel.

Oxyahloris saariosa NAO-ME + cp elong. even absent devel.

Sporobolus afriaanus PCK + cf oval. uneven

S. aaroU NAO-ME + cp elong. even absent devel. Subfamily C4 XyMS PCR cell PCR cell PCR Suberized Grana in PCR type chloroplast chloroplast bundle lamella in cell Species position shape sheath PCR cell chloroplasts outline walls ------s. areber PCK + cfH oval. H uneven present deve 1.

s. efongatua PCK + cf oval. uneven presentA devel. A

s. jaaquemontii PCK + cf oval. uneven

s. pufoheliua NAD-ME + cp elong. even absent devel.

Tridena braaitienaia NAD-ME?I + cf oval. uneven presen0 devel. A

Triodia aaariosa NAD-ME + cf oval. even absent devel.

Triraphia molUs NAD-ME + cf oval. uneven present devel.

Arundinoideae

Aristida biglandulosa NADP-ME cf oval. even/uneven dbsen0 rudi.A cell PCR Subfamily c 4 XyMS PCR PCR cell Suberized Grana in PCR type chloroplast chloroplast bundle lamella in cell Species position shape sheath PCR cell chloroplasts outline walls

A. latifoiia NADP-ME cf oval. even/uneven

Eriaehne aristidea NADP-ME + cf oval. uneven absent devel.

E. glabrata NADP-ME + cf oval. uneven absent devel.H

H fl E. obtusa NA DP-ME + cp/cf oval. even/uneven absent devel.

E. ova ta NADP-ME + cf oval. uneven absent devel.

E. puleheUa NADP-ME + cp oval. even absent devel.

Pheidoeh[oa graeiLis NADP-ME + cf oval. uneven absentfl devel.H

Panicoideae

A[[oteropsis semialata PCK cf oval. uneven presentA devel.A,J

A Arundinella nepaLensis NA DP-ME cf oval. uneven present rudi.A ----

Subfamily C4 XyMS PCR cell PCR cell PCR Suberized Grana in PCR type chloroplast chloroplast bundle lamella in cell Species position shape sheath PCR cell chloroplasts outline walls

Cymbopogon oitratus NAOP-ME cf oval. uneven

MeUnis minutiflora PCK + cf oval. uneven

Panioum bergii NAO-ME + cf oval. uneven presentA devel. A ,G

P. bulbosum NA DP-ME cf oval. uneven presentA rudi. A

P. ooloratum NAO-ME + cp elong. even absen0 deve 1. A

P. diohotomiflorwrf NAO-ME + cf oval. uneven

P. effusum NAO-ME + cp elong. even absen0 devel. A

P. lanipes NAD-ME + cp elong. even absentA devel. A

P. maximum PCK + cf oval. uneven presen0 devel.A,G

P. sohinzii NAO-ME + cf oval. uneven presentA devel. A

P. stapfianum NAO-ME + cp elong. even absen0 devel.A P. virgatum NAO-ME + cf oval. uneven ------Subfamily C4 XyMS PCR cell PCR cell PCR Suberized Grana in PCR type chloroplast chloroplast bundle lamella in cell Species position shape sheath PCR cell chloroplasts outline walls ------Pseudobraahiaria deflexa PCK + cf oval. uneven

E Rhynahelytrwn repens PCK + cf oval. uneven devel.

Spinifex serieeus NADP-ME cf oval. uneven

A . M Zea mays NADP-ME cf oval. uneven present ru d 1.

A Hattersley and Browning (1981). B Hattersley and Perry (1984). C Laetsch (1971). D Woo et al. (1971). E Carolin et al. (1973). F Black and Mollenhauer (1971). G Brown and Gracen (1972). H Illustrated in Fig. 5.1. I See Results °'"et. ,,,• .._,...n;o"· J Frean et al. (1983). K Species listed as this by Hattersley and Browning (1981) is in fact P. buneei F. Muell. ex Benth •• ~ One leaf Plade of the plant biochemically assayed has four XyMS+ veins and one which is variable. Many studies. Fig. 5.1. Electron micrographs of !'CR cells, chloroplasts and cell

walls from transverse sections of leaf blades. Abbreviations: !'CR

cells, Photosynthetic Carbon Reduction (Kranz) cells; otw, outer

tangential wall; rw, radial wall; c, chloroplast. Bar= 5 µ m in a, b and

a and 0.5 µm in d, e and f. a, LeptoahLoa digitata (NAD-ME). PCR cell

with centripetal, elongated chloroplasts. b, Enneapogon poLyphyLLus

(NAD-ME). PCR cell. Chloroplasts are peripheral, although are slightly

more centripetal than centrifugal. Chloroplast shape unclear.

Suberized lamella (arrowed) just visible in outer tangential wall. a,

SporoboLus areber (PCK), !'CR cell with mainly centrifugal

chloroplasts. Chloroplasts relatively much broader than in L. digitata

(1a). Suberized lamella (arrowed) visible in outer tangential wall and outer parts of adjacent radial walls. Outer tangential wall much more

'inflated' than those in a and b. d, Eriaahne obtusa (NADP-ME). !'CR cell chloroplast with g rana (arrowed). Outer tangential wall has striations but no suberized lamella. This chloroplast is centrifugally positioned but most chloroplasts in this species are centripetal. e,

PheidoahLoa graaiLis (NADP-ME). Junction of radial and outer tangential walls of adjacent PCR cells. No suberized lamella. Chloroplasts have g rana (arrowed). f, Eriaahne gfobrata (NADP-ME). Part of !'CR cell chloroplast with grana. The arrowed grana contain 21 and 17 appressed thylakoids. ,£~ f/Z)~ ,..- Fig. 5.2. Schematic representation of currently known structural suites in leaf blade vascular b.rndles of grasses (Poaceae) and their associations with c 4 acid decarboxylation types. Cells of bundle

sheaths are shown as in transverse section. Vascular tissue would be to

the left and PCA (Primary Carbon Assimilation, or c 4 mesophyll) tissue to the right (neither shown), Species with suites 1-3 are XyMS- (see footnote in Materials and Methods): the sole (suite 1 ) or inner she a th

(suites 2 and 3) is PCR (Photosynthetic Carbon Reduction, Kranz) tissue; in 2 and 3 the outer sheath contains chloroplasts but exact function is not known. Species with suites 4-9 are XyMS+: the inner (mes tome) sheath (left) is achlorenchymatous and the outer sheath (right) is PCR tissue. The presence or absence of chloroplasts in each cell is indicated: these are either elongated in shape as in 5 and 7, or are ovalish as in all other suites. Chloroplasts, where dotted, contain well developed grana; where dotting is light, as in the inner bundle sheath chloroplasts of 2 and 3, grana are present but are not well developed i.e. they have

(including inner sheath of 3). The presence of a suberized lamella in cell walls is indicated by a line; where this is dashed the lamella is either incomplete or not always found. The outline of the PCR sheath, comprising the outer tangential (here the right) walls of PCR cells, is uneven as in and 4-6, intermediate (even/uneven) as in 2 and 3, or even as in 7-9. All known genera and species with 'non-classical' structural/biochemical sui t:es are mentioned in the appropriate row and column; taxa in the subfamilies Chloridoideae and Panicoideae with

'classical' suites (i.e. those originally described by Gutierrez et al.

1974 and Hatch et ai. 1975) are not listed individually. NA DP-ME PCK NAD-ME

Panicoideae1

2 Neurachne munroi ALLoteropsis semiaLata2 Paraneurachne mueLLeri3

3 Aristida bigLanduLosa A. fotifoUa

4 Chloridoideae Eragrostis4 spp. Panicoideae Panicwn4 spp. Triraphis moLLisS

5 Enneapogon spp. Eragrostis6 spp.

6 Eriachne aristidea E. gLabrata E. ovata PheidochLoa graciLis

7 Chloridoideae Panicum

8 BouteLoua curtipenduia7 Triodia scariosa7

9 Eriachne obtusa8 E. puLcheUa

ArundineLLa nepaLensis additionally has 'distinctive cells' (see e.g. Hattersley and Watson 1975). 2 Has well developed grana in the (PCR) inner bundle sheath cells. 3 Has even PCR sheath outline. 4 Other NAD-ME Eragrostis species have 'classical' NAD-ME structure; other Panicum species have 'classical' NAD-ME or NADP-ME structure. 5 No suberized lamella in the inner (mestome) sheath was observed in the specimen examined. 6 Species with 'intermediate' anatomy. 7 Chloroplasts of T. scariosa are centrifugal/peripheral; those of B. curtipenduLa are evenly distributed. 8 E. obtusa has structure somewhat intermediate between those of suites 6 and 9. 1

2

3

4 s DO

6

7 DIJ 8 Dti 9 CHAPTER 6

WHOLE LEAF BLADE CHLOROPHYLL A:B RATIOS IN C4 ACID OECl\RBOX'lLATION TYPES AND THEIR STRUCTURAL VARIANTS IN GRASSES (POACEAE). 72

Abstraat

Whole leaf blade chlorophyll a:b ratios of a taxonomically diverse sample of 73 species of c 4 grasses (Poaceae) are shown to differ significantly between c4 acid decarbcxylation types. Mean ratios (and standard errors) are 4. 45 ( ±0. 04), NADP-malic enzyme type (NADP-ME);

4,01 (±0.03), NAD-malic enzyme type (NAD-ME); and 3.45 (±0.02), PEP carboxykinase type (PCK). These results, which closely agree with available data for a small sample of species in earlier studies, include ratios of a large number of species with newly-discovered, 'non- classical' associations between leaf blade PCR (Photosynthetic Carbon

Reduction, or Kranz) bundle sheath anatomy and c 4 type. There is no significant difference between the mean ratios of NAD-ME species with

'classical' anatomy, and of NAD-ME species in Enneapogon, Eragrostis,

Panic:um, Triodia and Triraphis with 'non-classical' anatomy. Likewise,

PCK AUoteropsis semialata (R. Br.) Hitchc. and NADP-ME PheidoahJ,oa graailis s. T. Blake and Eriaahne spp., with anatomies very atypical of their types, have ratio ranges commensurate with those of 1 classical' c4 PCK and NADP-ME species re spec ti vely • Chlorophyll a:b ratios, therefore, are apparently unaffected by variations in PCR bundle sheath anatomy. NADP-ME species in the subfamily Arundinoideae and 'classical'

NAD-ME Paniaum species have high ratios for their c 4 types. 73

Introduction

The differential involvement of chlorophylls a and b in

photosystems I and II (e.g. Anderson 1980) complements the photochemical

variation, and probably the structural dimorphism, in chloroplasts of c 4 grasses ( Poaceae) ( Laetsch 1968; Hatch and Slack 1970; Downton et al.

1970; Anderson et al. 1971; Woo et al. 1971; Mayne et al. 1974; Rathnam

and Das 1975). In the leaf blades of c4 grasses the ratio of chlorophyll a:b varies with tissue (PCA 1 and PCR: Woo et al. 1971;

Kanai and Edwards 1971; Chang and Troughton 1972; Ku et al. 1974) and/or

ti 2 with c4 acid decarboxyl a on type The most comprehensive data on ratios in grasses are in Holden ( 1973) and Mayne et al. ( 1974). In both

studies, each comprising data for 19 c4 species, there are clear

differences between the whole leaf ratios of species of the three c4 types, the uneven sample size notwithstanding (Table 6.1), and there is

also close agreement between the ratios of five species common to both

The reasons for the ratio differences between the types studies. c4 (and between tissues within each type) have been fully discussed by

Mayne et al. (1974; see also Edwards and Walker 1983).

In grasses each c4 type has been associated with a particular

1 c1assical 1 suite of structural features in leaf blade vascular

bundles. All 24 species studied by Holden ( 1973) and Mayne et al.

(1974), which have been biochemically assayed for c4 type, exhibit these

'classical' structural/biochemical correlations i.e. NADP-ME species are

PCA tissue, "primary carbon assimilation" tissue, usually equivalent to mesophyll; PCR tissue, "photosynthetic carbon reduction" (Kranz) c 4 tissue, usually bundle sheath in c4 plant leaves (Hattersley et al. 1976). 2 NADP-malic enzyme, NADP-ME; NAD-malic enzyme, NAO-ME; PEP carboxykinase, PCK (Gutierrez et al. 1974; Hatch et al. 1975). 74

XyMS- 1 and have centrifugal PCR cell chloroplasts; NAD-ME species are

XyMS+ and have centripetal c,hloroplasts and an even PCR bundle sheath

outline; and ECK species are also XyMS+, but have centrifugal chloroplasts and an uneven outline (Gutierrez et al. 1974; Hatch et al.

1975; Hattersley and Watson 1976; Brown 1977; Ellis 1977). 'There are now known, however, to be a number of exceptions to these correlations;

in two of the world's largest grass genera, Panicwn and Eragrostis,

there are NAD-ME species which seem to be structurally indistinguishable from ECK species (Ohsugi & Murata 1980, 1981; Ohsugi et al. 1982;

Chapter 3). During a continuing survey of the activities of c acid 4 decarboxyl a ti on enzymes in (1 argely) Australian grasses, sirnil ar examples have been encountered in other genera and also further

'discrepancies' involving NADP-ME and PCK species (Chapter 5). During this and other work (Hattersley and Stone 1986; Chapter 3), chlorophyll a:b ratios have been obtained for a large and taxonomically diverse array of c 4 grasses. These data show that chlorophyll a:b ratios in grasses are correlated with c 4 acid decarboxyl a ti on types irrespective of intra-c4 type variations in leaf blade structure.

Haterials and methods

Experimental plants

Plants were grown either in a growth cabinet with a 16 hour day

(photon flux density of about 500 µmol m- 2 s-1 ) at 30°C and an 8 hour night at 20°C, or in glasshouses subject to the natural light regimes of

Canberra in January-October 1985. Glasshouse temperatures were maintained between approximately 30°C (day maximum) and 15°C (night

XyMS+/-, presence/absence of cells intervening between metaxylem vessel elements and laterally adjacent PCR bundle sheath cells of primary lateral vascular bundles (Hattersley and Watson 1976). 75

minimum). Plants were regularly fertilised with Ruak.ura nutrient

solution (Smith et al. 1983).

Preparation of Leaf sampLes

Preparation of leaves was for experiments measuring activities of

c 4 acid decarboxylation enzymes reported in Hattersley and Stone (1986), and in Chapters 3 and 5. 0.5 g of young, fully expanded leaf blades of

each species were cut into small segments and vigorously ground for several minutes in a chilled mortar containing 25 mg polyvinylpolypyrrolidone and 2.0 ml extraction buffer. Extraction buffers for the NAD-ME and NADP-ME assays were based on those of Edwards

et aL. (1982) and for the PCK assays on that of Ku et ai. (1983).

A possible source of error in obtaining whole leaf blade chlorophyll a:b ratios in c 4 grasses is the differential effect of inadequate grinding on the extraction of chlorophyll from PCA (usually mesophyll) and PCR (Kranz) tissue. Since PCR cells of NADP-ME species have a higher ratio than the PCA cells, and viae versa for NAD-ME species (e.g. Ku et ai. 1974; Mayne et aL. 1974), it is essential that grinding is vigorous enough to rupture all cells. (In PCK species both cell types have nearly similar chlorophyll a:b ratios e.g. Ku et ai.

1974; Mayne et ai. 1974),

ChLorophylL a:b ratio aaLauLation

Chlorophyll a:b ratios were calculated from Arnon's (1949) equations for the determination of total leaf chlorophyll content,

Absorption of 50 µl tissue extract in 4. 95 ml 80% acetone was measured wi t.'1 a Shimadzu Recording Spectrophotometer (UV 240). Each species datum (Ta bl es 6. 4-6. 7; Fig, 6.1 ) is the mean of du pl ica te measurements 76

from a single extract. Holden (1965) gives equations and wavelengths for the most commonly used sol vents, and reassessments of equations are given by Jeffrey and Humphrey (1975) and Wellburn and Lichenthaler

(1984).

Results

All 242 chlorophyll ratios obtained for 73 c 4 grass species are shown in Fig. 6.1 and listed in Tables 6.4-6.7. Mean values, with standard errors, of c4 types and subfamilies are shown in Table 6.2 and statistical analyses in Table 6.3. Ratios for Panicum and Eragrostis species are sha.m in Tables 6.4 and 6.5 respectively where comparisons can be made of ratios within and between experiments. Ratios of other species exhibiting 'non-classical' structuraljbiochemi cal associations are shown in Table 6.6, and those of all other species in Table 6.7.

For the three c4 types the mean chlorophyll a:b ratios (and standard errors of the mean) are: NADP-ME ( 14 species), 4. 45 ±o. 04;

NAD-ME (42 species), 4.01 ±o.03; PCK (17 species) 3.45 ±0.02 (see Table

6. 2). There are significant differences between these ratios (p=

<0.001; Table 6.3), and also between all species groups of different c 4 types (p= <0.001) except NADP-ME panicoids and 'classical' NAD-ME panicoids (the latter all in Panicum). PCK species (inclusive or exclusive of Attoteropsis semiatata, the only species with 'non­ classical' anatomy; see Tables 6.4, 6.6., 6. 7) have a significantly lower mean ratio (p= <0.001) than those NAD-ME chloridoid or panicoid species which would have been predicted as PCK on the basis of

1 classical' structural/biochemical associ a ti ans (in Enneapogon,

Eragrostis, Panicum, Triodia, Triraphis and possibly Tridens; Tables

6.4, 6.5, 6.6). 77

Within c 4 types significant differences in ratios exist between

NADP-ME arundinoids and panicoids (p= <0.001; Table 6.3), and also berNeen 'classical' NAD-ME panicoids and 'non-classical 1 chloridoids and panicoids (p= <0.01). Within NAD-ME Eragrostis (Chloridoideae) there are no significant differences (at p= <0.05; not shown) between the mean ratios of species of three anatomical variants ('classical', PCK-like and intermediate; Table 6.5). Species exhibiting other exceptional

'non-classical• structural/biochemical associations e.g. PCK

Ailoteropsis semia7,ata, and NADP-ME Pheidoahioa graaiUs and Eriaahne spp. (Table 6.6), also have chlorophyll a:b ratio ranges commensurate with their biochemical rather than their structural characteristics (the ratios of the arundinoid genera Pheidoah7,oa and Eriaahne being at the high end of the range for NADP-ME species).

Illi. scussion

The data on chlorophyll a:b ratios are by-products of a series of experiments designed to measure the activities of acid decarboxylation enzymes in c 4 grasses. Over a ten month period the glasshouse-grown experimental plants were exposed to a wide variation of

1 igh t quantity and quality, two environmental conditions known to affect chloroplast ultrastructure and chlorophyll a:b ratios (e.g. Aro et ai.

1985, for study of changes in a c 3 grass). Despite such environmental variation, and despite the (often wide) range of ratio values obtained from what were, for most species, the same individual plants (see, for example, Pani= sahinzii; Table 6.4), the differences in mean ratios between the biochemical types are statistically significant, even when comparisons are made across major taxonomic and/or anatomical groups.

The results closely agree, furthermore, with the ratios of four species 78

also studied by Holden (1973) and of another six, out of seven, by Mayne

et ai. (1974). The exception is Eteusine indica, one of whose ratios

(3.43; Table 6.7) is the third lowest recorded among all NAD-ME species

(Mayne et aZ.., using data from Ku et aZ.. 1974, reported its ratio as

4.25). For some unknown reason the first experiment with Eragrostis

resulted uniformly in the lowest ratios of any obtained for the seven

species concerned (Table 6.5).

Among NADP-ME species the significant difference between the mean ratios of arundinoids and panicoids (Table 6.3) is largely due to the high ratios of PheidoohZ.oa graoiZ.is and Eriaohne aristidea, E. gZ.abrata and E. puZ.oheZ.Z.a (Table 6.6). Species of these genera are the only ones known to be NADP-ME and XyMS+ (see Hattersley and Watson 1976; Brown

1977; Chapter 4), and they are al so very unusual in a number of other structural features (Chapter 5; cf. Hattersley and Browning 1981). Why, however, the ratios of these particular species are so high is not known. Aristida big1.andu1.osa and A. 7..atifo1.ia (also arundinoids) and the panicoids, Arundineiia nepaZ.ensis, Neuraohne munroi and

Paraneuraohne mueZ.Z.eri, also have unusual anatomy (illustrated in

Hattersley et aZ.. 1976, 1982, 1986; Dengler et ai. 1985), although the

XyMS- condition and the centrifugal/peripheral position of the PCR cell chloroplasts are the same as in 'classical' NADP-ME species. In contrast to the ratios of PheidoohZ.oa and Eriaohne spp., their ratios are neither distinctly high nor low compared with those of the three panicoid species with 'classical' NADP-ME anatomy (Tables 6.4, 6.7).

Among NAD-ME species particularly high chlorophyll a:b ratios were recorded in Triodia soariosa (Table 6.6) and Panioum Z.anipes (Table

6. 4) • Whilst Triodia has overall extremely unusual 1 eaf blade anatomy

(e.g. Burbidge 1946; Craig and Goodchild 1977), P. Z.anipes has 79

'classical' NAD-ME anatomy. Whatever the reason for them , the high

ratios of this species (Table 6.4) contribute largely to the significant

difference between the mean ratios of 'classical 1 NAD-ME panicoids and

those of other NAD-ME species groups (Table 6.3).

The two highest ratios among PCK species (3.85 and 3.93; Table 6.6)

are both of Chloris trunoata, another species with 'classical' anatomy.

One species which might be expected to have a high ratio is Bouteloua

aurtipendula. It is not only structurally somewhat intermediate between

'classical' NAD-ME and 'classical' PCK species (Hattersley and Perry

1984; Chapter 5), but its main c 4 acid decarboxylation enzyme is equivocally NAD-ME or PCK (Gutierrez et al. 1974; Chapter 5). The

ratios are well within the range of PCK species, however, and below

those of all but a few NAD-ME species; indeed one ratio (3.13; see Fig.

6.1) is the second lowest of all.

There are no quantitative differences between the activities of c 4 a~id decarboxylation enzymes of 'classical' and 'non-classical' species

of the same c 4 type (Chapters 3 and 5). The chl orophyl 1 a: b ratios indicate that these species are also photochemically similar; this is particularly apparent in the low ratios of PCK Alloteropsis semialata,

predicted to be NADP-ME on the basis of anatomical criteria (al though

Frean et al. 1980 and Hattersley and Browning 1981 noted granal PCR cell chloroplasts), and in the high ratios of NADP-ME Pheidoahloa graaiUs and some Eriaahne spp. which were expected to be PCK. The c 4 types of these species are clearly reflected by chlorophyll a:b ratios; in retrospect indeed, they might have been predicted by them since the ratios of NADP-ME and PCK species as a whole scarcely overlap (Fig.

6. 1 ) • Also in a predictive context, any ratios >4.0 would appear from the data to distinguish NAD-ME species with PCK-like anatomy (Tables 80

6.4, 6.5, 6.6} from 1 classical 1 PCK species.

There has been no -comparative, ultrastructural analysis of PCA and

PCR tissue chloroplasts in species of all three c4 types, let alone one that samples species with the new anatomical/biochemical associations it1hose ratios are presented here. In Chapters 3 and 4 I have al ready pointed out, however, that 1 classical 1 and PCK-like NAD-ME species possess quite different PCR cell chloroplasts, even though these species are apparently similar both biochemically and photochemically. In PCK- like species (as in 'classical' PCK species) they appear ovalish in shape in leaf blade transections, whereas in 'classical' NAD-ME species they are distinctly elongated (as they also are in 'non-classical'

Enneapogon and 'intermediate' Eragrostis species). The significance of this structural variation of chloroplasts in the same tissue and c4 type is quite unknown. Table 6.1. Means and ranges of vhole leaf blade chlorophyll a:b ratios for c acid decarboxylation types in grasses {Poaceae}, 4 taken from Holden {1973) and H

NADP-malic enzyme type, NADP-ME; NAD-malic enzyme type, NAO-ME; PEP carboxykinase type, PCK, c types given in Mayne et al., 4

(1974); for Uolden (1973) types taken from the literature, determined biochemically (see L."'hapter 5), or deduced an the basis of c 4 leaf anatomy and/or taxonomic affinity (see Chapter·s 3 and 4). In Holden (1973) leaf extracts were in 80% acetone and ratios were calculated as in Arnon (1949); in Mayne et ai. (1974) the methods of Wintermans and DeMots (1965) were followed. n: number of species, one ratio far each species except far n"' 18 far the NADP-ME rnean in Holden {1973), which includes seven cultivars and

hybrids of two species (i.e. 13 spE!cies in total),

Source chlorophyll a:b ratio NADP-ME specJ.es NAD-ME species PCK species ii range n ' range n ' ram1e "

Mayne et a'l. (1974) 4, 45 4.12-4.76 7 4.05 3.60-4.38 5 3.36 3.13-3.62 ,,

Holden {1973) 4 .1 3.6-4.7 18 3.8 ],4-4.0 4 3. 4 3 .4 2

Bouteloua r::urtipendul.a, included as PCK, mi:ly be a PCK/NAD-ME intermediate (Gutierrez et al. 1974; Chapter 5; see Discussion). 't'able 6.2. Means, standard errors of tl1e .eans and canqes for whole leaf blade extract chlorophyll a:b ratios in

c4 acid decarboxylation types and sub-families (Arundinoideae, Chloridoideae, Panicoideae) in grasses (Poaceae).

NAD-ME species are further grouped according to variation in leaf blade PCR {Photosynthetic Carbon Reduction, or

Kranz) bundle sheath anatomy viz. 'classical' species (with centi:-ipetal PCR cell chloroplasts and even bundle sheath outline) or 'non-classical', mainly PCK-like species (with centi:-ifugal/periphecal chloroplasts aod uneven outline), as well as Triodia aoarioaa, seven Enneapogon species and three intermediate l!'ragr>oatia species. Generic

composition of subfamilies follows classification of W~tson et al. {1985). n.a.o not applicable,

Species group Chloi:-ophyl 1 a:b i::-atios

c 4 type Subfamily PCH bundle sheath No. anatomy (NAD-ME species No. species only) measurements ii S.E. Ranqe

------· ·------·----~------· NADP-ME Arundinoideae n.a. 8 22 4.67 0,06 4.12-s.02

2 NA DP-ME Panicoideae n.a. 6 37 4. 33 o.os 3,68-5.26 3 NADP-ME Total n.a. " 59 4. 45 0.04 3.68-5.26 4 PCK Chloridoidede n.a. 12 39 ] • 45 0.09 3.04-].9]

5 <'CK Panicoideae n.a. 5 15 ]. 45 0,03 3.20-3.63

6 PCK Tot.al n.a. JU 54 ),45 0.02 ] .0.1-3 .93

Nl\D-ME Chloridoi deae 'Classical' 16 43 4.04 0.04 3.43-4.SJ 8 NAD-ME Panicoideae 5 '" 4.20 o.oa 3.56-4.B2 9 NAO-ME Chloridoidei'le 'Non-classical' 17 55 3.95 o.04 3.39-4.91

10 NAD-ME Panicoideae 4 13 3 ,91 0, 10 ].36-4.54

11 NAO-ME 'l'otal n.a. 42 129 4 .()1 0.03 J.36-4.91 Table 6.3. Statistical analyses (Student's t) of differences between the inean whole leaf blade eictract chlorophyll a:b ratios, in

paired comparisons, of species groups of different c 4 acid decarbaxylation types and subfaallies.

NAD-M.E species also grouped according to PCR bundle sheath anatomy as in Table 6.2. Species groups numbered as in Table 6.2. Values are probabilities that differences between mean ratios are not statistically significant. n.s.: not significant, No analyses {indicated by

dashes) were done between mean ratios of each c4 type {'l'otal) and specii;:s 9roups within the sc1me c 4 type.

Species qroup 2 3 4 5 6 7 8 9 10 11

NA.DP-ME Arund i noideae <0.001 <0.001 <0.001 <0.001 <0.001 <0,001 (0.001 <0.001 <0-001

2 NADP-ME Pa ni coideai;: <0.001 <0.001 <0.001 <0.001 <0.001 n.s. <0.001 <0.001 <0.001

3 NADP-ME Total <0.001 <0.001 <0,001 <0.001 <0.001 <0.001 <0.001 <0.001

4 PCK Chloridoideae <0. 001 <0.001 <0.001 n.s. <0.001 <0,001 <0.DOI <0.001 <0.001

5 PCK Panicoideae <0.001 <0.001 <0.001 n.s. <0.001 <0.001 <0-001 <0.001 <0.001

6 PCK 'l'otal <0.001 <0.001 <0.001 <0.001 <0,001 <0.001 <0.001 <0.001

7 NAD-ME Chloridoideae <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 n.s. n,s. n.s, 'Classical'

8 NAD-ME Panicoideae

9 NAD-ME Chloridoideae <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 n.s. <0.01 n.s. 'Non-clao>sical'

10 NAO-ME Panicoideae <0.001 <0.001 <0.001 <0.001 <0.001 n.s. <0 ,0 J n.s. 'Non-classical'

11 NAO-ME Total <0,001 <0.001 <0,001 <0 .001 <0.001 <0.001 Table 6.4. Chlorophyll a:b ratios (vi th aeans) of whoie leaf blade extracts of Panicwn species of different c 4 acid decarboxylation type.

NA.D-ME species are of t\.IO anatomical types (see Table 6.2). Ratios werl:l: obtained from 17 experiments (for other purpo~es)1 all are included in t"ig. 6.1.

C4 8pecies Chlorophyll a;b ratios type

NADP-ME

P. bul.boBwn H. B. K. 4.46 4.24 4.11 3. 86 4.36

PCK

P. nuximwn Jacq. 3. 20 3. 40 J.56 3.51 3.55 3.46 3.48 3.45

NAD-ME

•Classical'

P. coloratwn L. '3,91 4. 1 3 4. 2'J

P. effuswn R. Br. 4.00 3.98 3.96

P. 1-anipeB Mez 4.66 4,38 4.53 4.55 P. miliacewn L. 3.56 4.66 4 .42 ] .87 4.22 ) ,86 3.97 4.08

P. stapfianwn Foore. 4.27 3.94 4.18 4.32

PCK-like

P. bei'gii Arechav. J. "/] 3.50

P. sehinzii Hack. 4.29 3.36 3.61 4.54 3.95

P. virgatwn L. 3.87 4.02 4.34 4.02 4.06

P. dichotomiflorwn Michx. - ] • 7 "/ 3.79 3. Bl Table 6.5. 01lorophyll a:b ratios (with means) of whole leaf blade extracts of species in the NAO-HE 9enus Krag:roBtis.

Ther:-e are thr:-ee anatomical types: 'classical', PCK-like and intermediate. Ratios were obtained in eight experiments {foe other purposes). All are

includerl in ~'ig. 6.1.

Anatomical type Species Chlorophyll a;b ratios Anatomical Species type

'Classical'

E. citianenais (Al 1. ) Vigo. ., Janchen 3.91 3.91

E. dielaii Pilger 3.76 4.07 4.04 4.48 4.07 4.01 3.9"1

E. 1-acunar-ia F. Muell. e' Ben th. 4. 1] 4.32 4.23 4.23

E. minor Host 3. 55 3.71 3.71 3,88 3. 71

E. aetifotia Nees 4.09 3 .81 3.81 3.90

Intermediate

E. cur-vu la {Schrader) Nees 4.11 4. 1 3 3.88 3.71 J.')6

E. 1-eptocarpa Ben th. J.72 3.8U 3.87 3.77 3.81 3.08

E. parviftor•a (R. Br-.) Tr-in. 3.70 3.85 4.05 3.89 3. 87

PCK-like

E. benthamii Mattei 3. 96 4.08 3. 7 2 3. 75 3.88

E. brownii (Kun th) Nees e' Steudel 3.39 3.90 3.63 3.67 3.65 3.65 3.82

E. e1-on9ata (Will d.) ,Jacq. f. 3.59 3 ."18 3 .69 3.87 ] • "J 3

•• specioaa (Roemer & Schultes) Steudel 3. 59 4.34 4.23 4.01 4.05 Table 6.6. ~ans and ranqes of whole leaf blade extract chlorophyll a:b ratios of specieu exhibitinq •non-classical' associations between PCR bundle sheath anatomy <.tnd biochemically detennined c type. 4 Eragroatia and Pan.icwn SPP• are excluded (!'lee 'l'ables 6.4 and 6.5). C4 type el!:pected on the basis of leaf blade anatomy and 'classical' anatomical/biochemical associations is given. n: number of extracts measured for chlorophyll a:b ratio. Generic composition of subfamilies follows classification of Watson et a'l. (1985).

Chl o::-ophyl 1 a:b ratio Subfamily Species Expected C4 type Actual C4 type x range n

Arundinoideae E'xiiachne a:riatidea "· Muell. PCK NADP-ME 4.96 4.92-5.00 2 E. glabrota (Maiden) Hartley PCK NA.DP-ME 4.82 4.81-4.85 2 E. ovata Nees PCK NADP-ME 4.28 4.15-4,39 3 E. obtusa R. Br. NAD-ME NA DP-ME 4.54 4.12-4.75 4 E. pulcheZ.l.a Doudn NAD-ME NADP-ME 4.81 4.52-5.02 5

Pheidoch1.oa gPaai1.ia s. T. Blake PCK Nl\DP-ME 4.89 4.85-4.93 2 Chloridoideae Enneapogon avenaoeus {Lindl.) c. E. Hubbard PCK l NAD-ME 3. 71 3.66-3. 77 2 E. oae.r>u1.esoens (Gaudich.) N. T. Burbidge PCK NAD-ME 4.40 4.37-4.44 2

E. intermedius N. T. Burbidge PCK NAD-ME 3,91 3.85-4.00 3

E. lindleyanua (Domin) c. E. Hubbard PCK NAD-ME 3.87 3.67-4.01 4

E. nigricans (R. Br.) Beauv. PCK NAD-ME 3.91 3.66-3.95 2 E. po!yphyHua (Domin) N. T. Burbidge PCK NAO-ME 4.01 3.89-4.19 3 E. robuatisaimu.a (Domin) N. T. Burbidge PCK NAD-ME 4.11 ).98-4.33

2 Txiidena brasiliensia Nees ex. Steud. PCK NAD-ME 3.85 3.83-3.87 2

Triodia soarioaa N. T. Burbidge PCK 3 NAO-ME 4. 76 4.52-4.91 3

Tri1•aphis moll.is R. Br. PCK Nl\D-ME 4 .oo 4 .00-4 .01 2

Panicoideae

A'ltoteropais aemialata (R. Be, ) Hi tchc. NADP-ME PCK 3. 48 ).36-3.63

Enneapogon species have PCR sheath outline like that of PCK species, but chloroplast shdpe and position is more like tho~if~ of 'claso;ical' NAO-ME species. 2 NAD-ME activity was exceptionally low. 3 PCR sheath outline is even like that of 'classical' NAO-Ml~ species, but cilloroplast shape and position is like that of 'clas;;ical' PC" species. Ta.ble 6. 7. ~ans and ranqes of whole 1eaf b1ade extract chlorophyll a: b ratios of species ..ost.ly ex:hlbi tiog

'classical• associations between PCR b.lndle sheath anatomy and c 4 type (but see Footnote 1).

Paniown and Eragroatis spp. excluded (see Tables 6.4 and 6.5). n: number of extracts measured for chlorophyll

a:b ratio. Generic composition of subfamilies follows classification of Watson et a"l. (1985).

C Subfamily 4 Chlorophyll a:b ratios Species type x range n

Arundi noi<:leae 1 Ariatida big'landu'lo8a a. M. Black N.ll,DP-ME 4.56 4.55-4.58 2 1 A. 'latifoLia Domin NA DP-ME 4. 56 4.46-4.65 2 Ch loridoideae

Ast.rebla elymoides F. Muell. NAO-ME 4. 19 4.19-4.19 2

A. lappaoea (Lindl. J Doinin NAO-ME 4.2) 4.17-4.)0 2

A. peotinata (Lindl.) F, Muell. ex nenth, NAO-ME 4. 42 4,32-4.52 2

A. aquarroaa c. fL ll

Boute'loua our•tipendula (Mictix.) Torr. 3. 36 3.1 3-3. 59 3

B. graoi'lia {H. 13. K.) Lag. ex Steud. NAO-ME 4. l 2 3.97-4,30 3

ChLo.ria divai•ioata R, Br. FCK 3.52 3.45-3,61

C. gayana Kun th PCK 3.43 3.34-3.50 c. peotinata Benth. OCK 3.35 3,23-3.52

C. trunoata R. Br. l'CK 3.56 3.34-3.93 6

C. virgata Swartz 3.42 3. )9-3, 43 3

Eleuaine ooraaana ( L.} Gde r Ln. NAD-MJ~ 4 .17 4.04-4.30 2 {<,', indioa (L.) Gaertn. NAO-ME 3. 61 3.43-3. 7U

Leptooh'loa oiliolatu. (J.;,,dw,) s. •r. Blake PCK 3.49 3 .41-3 .62 5

L. digitata (R, !:Ir.) Domin NAO-Ml~ 4. 10 3.97-4.26 4

Oxyohloris 8oario

I Subfamily c, Chlorophyll a:b ratios Species type x range n

SporoboZ.ua afr•ioanu.a {Pair.) Robyns & •rournay PCK 3.46 3.28-3.69 4 s. oaroli MeZ NA.0-ME 3.82 3. 75-3. 89 3 s. o:rebe:r De Nardi FCK 3.47 3. 34-3. 64 4 s. elongatua R. er. PCK 3.54 3.53-3.54 2 s. fimbriatua Nees "2K 3,04 s. dacquemontii Kun th PCK 3.33 3.30-3.36 2 s. puZ.cheLZ.u.s R. Hr. NAD-MF: 3.69 3.64-3.74 2 Panicoideae 1 Ai>undineZ.Z.a nepalensia T:i:·in. NADP-ME 4.48 4.45-4.51 2

Cymbopogon cit1'atua {DC) Stapf NA DP-ME 4 .42 3 .92-5 .26 10

MeZ.inis minutifZ.ora Beauv, PCK 3.39 3.31-3.46 2

1 Neu.raehne mu.nroi (F • Muell.) F, Muell. NA DP-MR 4,45 4. 06-4. 97 6

1 Paraneurachne rru.eLLeri (Hackel) s. ". Blake NADP-ME 4.08 3,68-4.40 B

l'aeudobrachiaria deflexa (Schumacher) Launert PCK 3.40 3.36-J.43 2

Rhynchelytr>um re pens (Willd.) c. E. Hubbard PCK 3.63

Zea mays L. NA DP-ME 4.37 4.37-4.37 2

1 Although leaf hlade anatomy is 'non-classicdl' {st;e Disc"tlSSion for references), expected and actual c 4 tyr:ie is NADP-ME in all cases (see headin'l to 'Pable 6.6). 2 Bou.teloua curtipendula may he an NAD-ME/PCK intermediate (see Pi scussion). Fig. 6.1. Whole leaf chlorophyll a:b ratios in c4 types (NADP-ME, NADP­ rnalic enzyme tvpe; NAO-ME, NAD-malic enzyme type; ?CK, PEP carboxykinase

type) in grasses (Poaceae). NAD-ME species are subdivided according to leaf blade PCR (Photosynthetic Carbon Reduction, or Kranz) bundle sheath anatomy: 'classical' species, with centripetal PCR cell chloroplasts and an ev~n PCR bundle sheath outline; 'non-classical' species, including

PCK-1 ike species with centrifugal /peripheral chloroplasts and an uneven outline, as well as Triodia saariosa, seven Enneapogon species and three intermediate Eragrostis species. All 242 ratios (of 73 species) listed in Tables 6.4-6.7 are shown. Each bar represents the 95% confidence interval of the 'true' population mean. 5.3 • 5.2 5 . 1 • 5.0 • 4.9 • • • • 4.8 • • • 4.7 r 4.6 .. • 4.5 ..• • ..• • 0 " • • 4.4 • CCl I .. • I -.... 4.3 c I: .Cl • • • • • «ll 4.2 • • • • ... >. ..• • .J::. • • 4. 1 • • c. • • ....0 -• .,. 0 4.0 I • .,• .J::. • • (.) 3.9 • • .: I • • • 3.8 • • • • • I "'I• 3.7 • • .. • • • 3.6 I .. • • 3.5 • t • !" 3.4 I • or 3.3 • • 3.2 '• 3. 1 • • 3.0 'Classical' 'Non-classical' NADP-ME NAO-ME PCK CHAPTER 7

LEM" BLADE STRUCTURE AND C ACID DECAROOXYIATION ENzn!ES IN 4

CYNOCHLORIS SPP. (POACEAE), INTERGENERIC HYBRIDS BETWEEN

SPECIES OF DIFFERENT c4 TYPE. 81

Abstract

Cynoohloris maoivorii Clifford and Everist and C. reynoldsensis

B. K. Simon (Poaceae) are intergeneric c4 hybrids between Cynodon daotylon (L.) Pers., an NAD-malic enzyme (NAD-ME) species, and two different PEP carboxykinase (PCK) species of Chloris. Parental species of each hybrid species have the 'classical' leaf blade structure of their respective c4 acid decarboxylation types. The outline of the PCR

(Photosynthetic Carbon Reduction, or Kranz) bundle sheath and the position of the PCR cell chloroplasts in Cynoohloris are intermediate between those of the parental species, C. maoivorii being more like

Cynodon daotylon and C. reynoldsensis more like Chloris spp.. PCR chloroplast shape in C. maoivorii and C. reynoldsensis is like that of

Cynodon daotylon and Chloris spp. respectively. Differences between the hybrids in their enzyme activities complement these structural differences: C. maoivorii has more NAD-ME and less PCK activity than C. t>eynoldsensis although in both species PCK activity is the greater.

Both hybrids, however, have a suberized lamella in PCR cell walls as do

Ch loris spp •• The close taxonomic re la ti on ship between Cynodon and

Chloris make these genera especially suitable for reciprocal crossing experiments aimed at increasing understanding of the genetic relationships between subtypes of the c 4 photosynthetic pathway. 82

Introduction

The genus Cynoehloris (Poaceae) comprises two species, C. maeivorii

Clifford and Everist (Clifford and Everist 1964) and C. reynoldsensis B,

K. Simon (Simon 1982). On morphological grounds both species were postulated be intergeneric hybrids between c (L.) to 4 Cynodon daetylon Pers. (Berlltt.lda Grass) and a species of c Chloris. The Chloris parent 4 of Cynoehloris maeivorii was presumed to be C. divarieata R. Br., which grew at the type locality (Clifford and Everist 1964), as indeed it still does (pers. obs. 1986). The identity of the Chloris parent species of Cynoehloris reynoldsensis, on the other hand, is in some doubt (B. K. Simon pers. comm.). Simon ( 1982) originally considered

Chloris ventrieosa R. Br. to be the parent, but no plants of this species were found at the type locality in 1975. Another candidate is

C. gayana Kun th which, in 1986 at least, was very common there (pers. obs.) ..

A particularly interesting feature of Cynoehloris, which I note, is that each of the parental species is known to be of a different c 4 acid decarboxyla tion type. Three such c4 types are known (Gutierrez et al. 1974; Hatch et al. 1975) 1, each of them characterised by what was thought to be a diagnostic suite of leaf blade structural features (see

Chapters 3 and 5 for recently discovered structural/biochemical suites). The distinguishing features involve the position of the chloroplasts in the cells of the PCR 2 bundle sheath and whether or not they contain conspicuous grana (Gutierrez et al. 1974; Hatch et al.

1975), the outline of the outer tangential walls of these cells in

1 NADP-malic enzyme type, NADP-ME; NAD-malic enzyme type, NAD-ME; PEP carboxykinase type, PCK. 2 PCR tissue, photosynthetic carbon reduction (Kranz) tissue; PCA tissue, primary carbon assimilation tissue, usually equivalent to c4 mesophyll (definitions by Hattersley et al. 1976). 83

transverse section (Ellis 1977) and the XyMS 1 character of Ha ttersley

and Watson (1976).

Cynodon daatylon is an NAD-ME-type species (Hatch and Kagawa 1974;

Chapter 5) with 'classical' NAD-ME leaf blade structure (Ellis 1977;

Chapter 4; see Fig. 7 .1 a and Table 7 .1). By contrast all Australian

Chloris spp. (including C. gayana and C. divariaata) that have been been

biochemically typed are PCK-type (e.g. Gutierrez et aL 197 4; Hatch et

ai. 1975; Chapter 5) and they all have 'classical' PCK leaf blade

structure (Chapter 4; see Fig. 7.1d and Table 7.1). Unlike NAD-ME species such as Cynodon daatyion, PCK species have a suberized lamella in the walls of their PCR cells.

A topic that has hitherto received no attention is the genetic inheritance of, and relationships between, the structural and biochemical variations of the c4 pathway. CynoahLoris maaivorii and C. reynoldsensis, as naturally-occurring hybrid species, will clearly be of interest here in the future. This chapter reports on their leaf blade structure and acid decarboxylation enzyme activities. c4

Materials and Methods

Experimental Plant Material

C. macivorii is now known from only one of its type localities, on a very small stretch of a roadside verge between a croquet green and

Finimore Avenue, Q..leens Park, Ipswich (27°37'S., 152°46'E.; grid ref,

591942 on the Ipswich 1:250,000 map, sheet SG-14 (Edition 1), Series

R502). Several plants (of a single clone?), growing within a few metres of each other, were collected on 3 April 1986 (Taxonomy Unit collection

XyMS+/-, presence/absence of cells intervening between metaxylem vessel elements and laterally adjacent chlorenchymatous bundle sheath cells of primary lateral vascular bundles (Hattersley and Watson 1976), 84

no. V7; Simon et ai. collection no. 3803).

C. reynoldsensis is abundant at its type locality, along at least

parts of a c. 500 m stretch of the wooded south bank of Reynold's Creek,

near Mount Greville, south-east Queensland (28°05'S., 152°31 'E.; grid

ref. MU528933 on the Mount Lindesay 1: 100,000 map, sheet 9441 (Edition

1), Series R631). Plants from three disjunct areas (but still of the

same original clone?) were collected on 3 April 1986 (Taxonomy Unit

collection nos. V10-12; Simon et al. collection no. 3804).

The Cynodon daotylon plant used in this study was collected from

the A.N.U. campus (35°17'S, 149°08'E) in April 1986. Chloris divarioata was grown from seed from an accession originally collected 62 km east of

Longreach, Queensland (23°32'S, 144°53'E; Taxonomy Unit collection no.

G266). No other Chloris species was investigated since available evidence indicates that the genus is structurally and biochemically uniform (see Chapters 4 and 5).

Experimental plants were grown for fourteen weeks in a growth cabinet with a 16 hour day at 30°C and an eight hour night at 20°c.

They were regularly fertilised with Ruakura solution (Smith et al.

1983).

Herbariwn material

An anatomical examination was made of t:w'o type specimens: CANB

199074 of C. maoivorii, collected in 1935, and BRI 227424 of C. reynoldsensis, collected in 1975.

Leaf blade anatomy and ultrastruoture

Transverse sections of fresh leaf blade material were examined by light microscopy for: i) the XyMS character of Ha ttersley and Watson 85

(1976); ii) the shape of the PCR bundle sheath cells; iii) the position

and shape of the PCR cell chloroplasts (see Table 7.1; Fig. 7.1; Chapter

4). PCR cell walls were investigated by electron microscopy for the

presence or absence of a suberized lamella (see Ha ttersley and Perry

1 984 and Chapter 3).

Assays of c4 acid decarboxylation enzymes

Details are given in Chapter 3. Extraction procedure for NAD-malic

enzyme and NADP-malic enzyme closely followed that of Hatch et al.

(1982) and Edwards et al. (1982), the latter also providing the assay procedures for both enzymes. For PEP carboxykinase, extraction buffer was mainly as described by Ku et al. (1983) and the reaction mixture for

the assay mainly as described by Hatch and Mau (1977). The procedure for the PCK assay differed from that described in Chapter 3 as follows: O.SmM MnC1 2 was used (instead of 2mM), 0.3mM oxalacetic acid

(instead of 0.6mM), and unit of pyruvate kinase (instead of 2).

Chlorophyll determination followed Arnon (1949).

NAD-ME and NADP-ME assays were done on different eluates of the same extract, PCK assays on a separate extract. Each figure in Table

7.2 represents the mean activity of duplicates from one extract.

Results

Leaf blade anatomy and ultrastructure

The leaf anatomies of both Cynochloris spp. are intermediate between the 'classical' NAD-ME structure of Cynodon dactylon and the

'classical' PCK structure exemplified by Chloris divaricata (see Table

7.1; Figs. 7.1 and 7.2). They are, however, clearly different from each other: Cynochloris macivorii is structurally closer to Cynodon dactylon 86

whereas Cynochloris reynoldsensis is more like Chloris divaricata.

Leaves of C. reynoldsensis plants from three disjunct parts of its only

locality are structurally identical to one other, perhaps indicating

that these plants are all of the same clone.

In both Cynoohloris species the cells at the (adaxial) apex of the

PCR bundle sheaths, when present (Fig. 7.1b, c), are colourless (i.e. with no chloroplasts) as in Cynodon daotyfon (Fig. 7.1a). In

Cynoohloris maoivorii the PCA (mesophyll) tissue associated with adjacent veins is only just contiguous laterally; this and the radiate appearance of the PC.n. tissue are similar to those of Cynodon daotylon.

PCA tissue in Cynoohforis reynoldsensis is more like that of Chloris divaricata, as are the shape and number of the interveinal bulliform and

colour less cells. These cell-types are not clear in the illustrated section of Cynoohloris maoivorii but in other sections they look more like those of Cynodon dactylon than Chloris divaricata.

Both Cynochloris spp. have a suberized lamella in the walls of all

PCR bundle sheath cells, as in Chloris.

Activities of c4 acid decarboxylation enzymes In two series of measurements (Table 7. 2) Cynochloris m:wivorii showed more NAD-ME activity and less PCK activity than C. reynoldsensis; in both species, however, B:K activity exceeded NAD-ME activity.

Cynodon dactyl on had high NAD-ME activity and no PCK activity, whilst

Chloris divarioata had high PCK activity and low NAD-ME activity. Very low NADP-ME activity was detected in all non-NADP-ME species. 87

Discussion

Whilst both CynoahLoris spp. are structurally and biochemically intermediate between their parent species, C. maeivorii is more like the

'classical' NAD-ME-type parent Cynodon daatylon and C. reynoldsensis is more like a 'classical' PCK-type Chloris species, both in leaf blade

structure and c 4 acid decarboxyla ti on enzyme activities. On the basis of a number of morphological characters, Simon (1982) also considered C. reynoldsensis to be closer to Chloris than to Cynodon. The PCR tissue structure of C. maaivorii is very like that of some ('intermediate')

Eragrostis species and all Australian species of Enneapogon so far examined; in neither of these (perhaps totally) NAD-ME genera, however, is there any evidence for a hybrid origin (Chapters 3, 4 and 5).

Both Cynoahloris species may be infertile clones comprising the original F1 hybrids. In both, the leaf blade structures of the specimens examined here are very similar to those of herbarium specimens collected in the type localities in 1935 (C. maaivorii) and in 1975 (C. reynoldsensis), although the structure of the c. reynoldsensis specimen was poorly preserved. Furthermore, the persistence of the two species may owe much to their very vigorous stoloniferous growth habits because no seed-set has yet been recorded, either in C. maaivorii by Clifford and Everist ( 1964) or in the specimens of both species grown in cabinets and glasshouses for this study. Perhaps it is not appropriate to give these taxa specific status.

This study raises questions about the inheritance of PCR l::undle

Results of work on the maternal sheath chloroplasts in c 4 plants. inheritance of chlorophyll deficiency in three c 4 NADP-ME grasses, Coix laaryma-jobi L. (Rao 1975), Sorghum vulgare (L.) Pers. (Karper 1934) and

Zea rruys L. (Jenkins 1924; Shumway and Weier 1967), suggest that there 88

is no difference in the inheritance of chloroplasts of PCA and PCR

tissue. Only Shumway and Weier ( 1967), however, examined the two

tissues separately, finding similar chloroplast mutations in both. If chloroplast inheritance is maternal in Cynodon daotylon and Chloris

spp., as is reasonable to expect (see Kirk and Tilney-Bassett 1978), and it is assumed that the two Cynoohloris species are F 1 hybrids, then the difference in the shape of the latters' PCR cell chloroplasts as observed here might reflect the direction of pollen flow in the original crosses: i.e. Cynodon daotylon as the pollen donor in the case of C. reynoldsensis and Chloris divarioata in the case of C. maoivorii.

Cynoohloris spp. are the only spontaneous hybrids known to me between species of differing c 4 type (see e .g • Knobloch 1968). Any discussion, however, on the inheritance of parental characteristics in the specimens examined here would be purely speculative. Cynodon and

Chloris are closely related genera (usually put in the same tribe; see e.g. Hitchcock 1950; Tsvelev 1976), which may hybridise far more frequently than has been reported hitherto, and they should provide suitable material for controlled, reciprocal crossing experiments. Such experiments could examine the inheritance of characteristics noted in the structural/biochemical suites of Cynoahloris, which resemble those in other genera (viz. Enneapogon and Eragrostis) where there is as yet no evidence of hybridisation. Table 7.1. Structural features of PCR (photosynthetic carbon reduction, or Kranz) tissue in leaf blade transections of CynochLoris spp., 'classical' NAD-ME Cynodon dactyLon and

'classical' PCK Chloris divaricata.

Species PCR bundle PCR cell PCR cell Suberized

sheath chloroplast chloroplast lamella in

outline position shape PCR cell walls

Cynodon dactyLon even centripetal elongated absent

CynochLoris macivorii even {uneven) centripetal/ elongated present peripheral

Cynochloris reynoldsensis uneven (even) evenly ovalish present distributed

Chloris divariaata uneven centrifugal/ ovalish present peripheral Table 7.2. Activities of acid decarbo:xylation enz}'llles in c4 CynochLoris 1Tl'.1ciVorii,

C. reynoldsensis, Cynodon dactylon, Chloris divaricata and control species.

Cynodon dactylon has previously been typed as NAD-malic enzyme (NAD-ME) (Hatch and Kagawa

1974; Chapter 5), Chloris divarioata as PEP carboxykinase (PCK) and Cymbopogon oi·tratus

(DC) Stapf as NADP-malic enzyme (NADP-ME) (Chapter 3). Tritiown aestivum L. is Cr

Species Leaf blade Activity (µmol mg- 1 Chlor. min-1

structure NAD-ME PCK NADP-ME

Cynodon daotylon 'Classical' NAD-ME 3.29 0 0.34

Cynoohloris maoivorii Intermediate 2.78 3.98 0.19 1. 93 3.01 0.23

Cynoohloris reynoldsensis Intermediate 1 • 30 4.45 0.22 1. 49 4.18 0.23

Chloris divar•ioata 'Classical' PCK 0.90 9.78 0. 21

Cymbopogon oitratus 'Classical' NADP-ME 1. 76 o. 31 15.74

Tritiown aestivum C3 0.30 0 0.04 Fig. 7.1. Light micrographs of vascular bundles from leaf blade

transverse sections. Adaxial leaf surface uppermost. a, hand-cut

section of fresh material; b, c, d, microtomed sections of fresh leaf

material fixed and embedded as for electron microscopy, and stained in

toluidine blue. Star: 'apical' cell, either 1 colourless 1 or with PCR

chloroplasts (i.e. 'extension cell 1 ) • PCR, Photosynthetic carbon

Reduction (Kranz). Bars= 25 µm. a, Cynodon dactyLon. NAD-ME, with

'classical' NAD-ME leaf blade structure. One non-primary and one

primary vein, each with two colourless cells (with no chloroplasts) at

apex. PCR bundle sheath outline is 'even' i.e. outer tangential walls

of PCR cells are not 'inflated'. PCR cell chloroplasts are centripetal

(towards inner tangential wall). Chloroplast shape unclear in

micrograph, but is elongated. b, CynochLoris macivorii. Two non- primary and one primary vein, central vein with colourless 'apical'

cell. Sheath outline is less even than in a. Chloroplasts are mostly

centripetal, but some are centrifugal or peripheral (i.e. adjacent to outer tangential or radial walls). Chloroplasts elongated compared to

those in c and d. c, c. reynoldsensis. Three non-primary veins, the central one (and the left one?) with colourless 'apical' cell. Bundle

sheath outline more uneven than in a and b but less so than in d.

Chloroplasts distributed throughout PCR cells; shape unclear in micrograph, but is ovalish. d, Chloris divaricata. PCK, with

'classical' PCK leaf blade structure. One primary and one non-primary vein, the latter with an extension cell (asterisked) i.e. a PCR cell

(with chloroplasts) separated from vascular tissue by other PCR cells.

Bundle sheath outline very uneven, particularly in non-primary vein.

Chloroplasts mostly centrifugal, but some are centripetal; often none in cell centres. Chloroplast shape, where apparent (e.g. in arrowed cell of non-primary vein), much more oval than in b.

Fig. 7.2. Electron micrographs of PCR cells and PCR cell walls from

transverse sections of leaf blades of CynochLoris spp. Abbreviations:

PCR, Photosynthetic Carbon Reduction (Kranz); otw, outer tangential wall; rw, radial wall(s); c, chloroplast; sl, suberized lamella. a, C.

maaivorii. PCR cell. Outer tangential wall not nearly as inflated as

in a. Chloroplasts mainly centripetal, but t~o are also (partly) adjacent to outer tangential wall. Chloroplasts elongated compared to

those in a. Bar= 5 µm. b, C. macivorii. Suberized lamellae (arrowed) in the radial walls of adjacent PCR cells. Bar= 0.5 µm. c, c. reynoLdsensis. PCR cell. outer tangential wall rruch more 'inflated'

than in a. Chloroplasts evenly distributed round periphery of cell.

Chloroplasts more ovalish and less elongated than in a. Bar= 5 µm. d,

C. reynoLdsensis. Suberized lamella in the outer tangential wall of PCR

cell (arrowed). Bar= O. 5 µm.

CHAPTER 8

GEOGRAPHICAL DISTRIBUTION OF C4 ACID DECARIDXYIATION TYPES

AND ASSOCIATED STROCTORAL VARIANTS IN NATIVE AUSTRALIAN c4

GRASSES (POACEAE). 89

Abstraet

The native Australian c 4 grass (Poaceae) flora is estimated, on the basis of extensive leaf blade structural and biochemical examination, to

"I comprise 34fol' (57% of total) NADP-ME (NADP-malic enzyme) type species, 3 19,l? (32%) NAD-ME (NAD-malic enzyme) type species and 65 (11%) PCK (PEP carboxykinase) type species. Species of all c 4 types are most numerous in northern tropical Queensland, within the mega therm seasonal (summer) rainfall bioclimate of Nix (1982). NADP-ME species are numerically dominant in 48 out of 73 State and Territory subdivisions, including 23 wholly or partly within the mega therm/me so therm arid bioclima te which closely corresponds to the arid and semi-arid zones covering c. 80% of

Australia. NAO-ME species diversity is proportionately at its highest in this bioclimate; PCK species may be the most dependent there on high soil moisture availability. The extent of the megatherm seasonal bioclimate is parallelled by the distribution of most PCK species and of a high proportion of species of all c 4 types with a suberized lamella in their PCR cell walls. The physiological reasons for these correlations are unknown. Taxonomic, ecological and historical factors in relation to c type distribution are discussed. 4 90

Introcbction

Distributions of grasses (Poaceae) in Australia have been studied from a number of standpoints. Hartley (e.g. 1950, 1958a, 1958b) and

Hartley and Slater ( 1960) included Australia in their worldwide surveys of the origin, evolution and distributions of different tribes with partiailar reference to the influence of climatic factors. Clifford and

Simon (1981) divided Australian genera into geographic elements based on worldwide distributions, and analysed distributions within Australia in terms of habitat and taxonomic groupings. The distribution of dominant grass genera in Australian savannas and grasslands as a whole was presented by Walker and Gillison ( 1982), whilst Johnson and Tothill

(1985) provided a more detailed and diagrammatic scheme, incorporating rainfall and soil-water relations, for northern and eastern tropical and subtropical areas. Simon (1981) compared State and Territory grass floras. Cytogeographical analyses have been made for Themeda austra.Lis

R. Br. (Hayman 1960), Heteropogon aontortus (L.) Beauv. ex Roemer &

Schultes (Tothill and Hacker 1976) and the three genera of the tribe

Neurachneae (Chapter 2). The latter study also compared distributions in relation to variation in photosynthetic pathway (see Hattersley et al. 1982; Hattersley and Stone 1986). This theme had already been examined for c 3 and c 4 grasses as a whole by Hattersley ( 1983) who showed that c4 species predominate over some 80-85% of the continental area of Australia and are most numerous where the summer is hot and wet, their subdivisional number correlating most highly with "October average minimum temperature ( +ve) and February median rainfall ( +ve)."

Within the c4 photosynthetic pathway there are three biochemical variants (types). Using a number of leaf blade structural features as predictors of c4 type (see Gutierrez et al. 1974; Hatch et al. 1975; 91

Hattersley and Watson 1976; Ellis 1977), Ellis et al. (1980) analysed the geographical distributions of the three c4 types in the grasses of

South West Africa/Namibia. In a grass flora that is 91% , they found c4 predicted NAD-ME species to be numerically dominant in arid coastal and subcoastal areas (especially where rainfall is <20 cm y-1 ), predicted

NADP-ME species to be dominant in the furthest inland and wettest areas, and predicted PCK species to be at their most numerous, as a percentage of the total number of c4 species, in intermediate areas. Vogel et al.

(1986), analysing the distribution of (predicted) c4 types in grasses in the Sinai, Negev and Judean deserts, concluded that 11 NADP-ME grasses grow where water stress is not a dominating factor, while the aspartate­ forrning grasses are more successful under xeric conditions. The PEP-ck species are not important and form an intermediate group between the

NADP-me and NAD-me subtypes."

Findings of bbth the above studies, however, may now need extensive reviewing in the light of recently discovered exceptions to established structural/biochemical correlations, for example instances of NAD-ME species with PCK-like anatomy (found in Panicwn by Ohsugi and Murata

1980, 1981 and Ohsugi et ai. 1982; in Eragrostis, Chapter 3; and in

Enneapogon, Triodia, and Triraphis, Chapter 5; see Chapter 5 for full details). As a result of an extensive structural survey and of biochemical determination of c4 type in a taxonomically diverse species sample, the c4 types of Australian grasses are now reasonably well known

(Chapters 3, 4 and 5). Using species distribution data for State and

Territory subdivisions compared alongside the bioclima tic classification of Australia by Nix ( 1982), this chapter examines the geographical 92

distribution of c4 types, and their structural variants, in native

Australian grasses. Taxonomic, historical and ecological aspects of c 4 type distributions are also considered.

Methods and Sources of Ila ta

c4 aoid deoa.:t'boxyLation types

c 4 typing of species is based on the structural and biochemical

data on native Australian c 4 grasses presented in Chapters 3, 4 and s. Using these data I have made the following generalisations and

predictions concerning the taxonomic distribution of each c type (which 4

may not be wholly applicable to c 4 grass floras of other parts of the 1 world): i) all (C 4 ) species in the Arundinoideae and Andropogonanae and all XyMs-2 Panicanae species (except PCK ALLoteropsis semiaLata R.

Br.) are NADP-ME; ii) all XyMS+ species in the Chloridoideae and the

Panicoideae which have centripetal PCR3 cell chloroplasts are NAD-ME;

iii) all species in the chloridoid genera Enneapogon, Eragrostis,

PLeotraohne, SympLeotrodia, Triodia and Triraphis are NAD-ME even though

they have at least some centrifugal/peripheral PCR cell chloroplasts;

iv) all other species in the Chloridoideae and all XyMS+ species in the

Panicoideae with centrifugal/peripheral chloroplasts are PCK. This

latter group may be the least reliably predicted (especially for five

such Panioum spp. ) •

Generic cornposi ti on of subfamilies (Arundinoideae, Chloridoideae, Panicoideae) and supertribes (Andropogonanae, Panicanae; Panicoideae) after Watson et aL (1985). 2 XyMS+/-, presence/absence of cells intervening between metaxylem vessel elements and laterally adjacent chlorenchymatous bundle sheath (PCR) cells of primary lateral vascular bundles (Ha ttersley and Watson 1976). 3 PCR tissue, 'photosynthetic carbon reduction' (or Kranz) tissue; PCA, c mesophyll 'primary carbon assimilation' tissue, usually equivalent to 4 (definitions by Hattersley et ai. 1976). 93

PCR cell chloroplast position

PCR cell chloroplast position was determined in Chapters 3 and 4.

Most species in i), iii) and iv) (above) have centrifugal/peripheral PCR cell chloroplasts; in Enneapogon and some Eragrostis spp. (in iii) above) most chloroplasts are centripetal but some are centrifu:ial/peripheral; in four Eriachne spp. (in i) above) chloroplasts are all or mainly centripetal. All species in ii) have centripetal chloroplasts.

Suberized lamella

The following generalisations and predictions concerning the occurrence of the suberized lamella in PCR cell walls of native

Australian grasses are based on the data of carolin et aL. (1973),

Hattersley and Browning (1981), and Chapters 3 and 5: i) it is present in NADP-ME Panicoideae but absent from NADP-ME Arundinoideae; ii) it is present in all PCK species; iii) it is usually absent from NAD-ME species but it is present in Enneapogon and Triraphis and in those

Eragrostis and Panicum spp. with at least some centrifugal/peripheral

PCR cell chloroplasts.

Geographical distribution and bioclimatic classification

Records of species occurrence within State and Territory subdivisions were obtained mainly from State and Territory herbaria (as indicated in Chapter 3) and also via numerous check lists from national parks, collecting expeditions and vegetation surveys . Distributional data for newly described taxa were taken from lists of examined specimens in the relevant publications (listed in Chapter 4). Full names of Australian State and Territory subdivisions are in Hattersley 94

(1983).

The geographical data are corrpared alongside the bioclimatic classification of Australia by Nix (1982; see Fig. 8.1 for details).

Ref ere nee is of ten made to two north-sou th 1 transects' cornpri sing

subdivisions (see Table 8.1) which span most of the bioclimatic

variation of those parts of Australia where c 4 grasses numerically predominate over c 3 grasses (see Hattersley 1983). The east-west aligned subdivisional boundaries of N.T., which closely parallel the orientation of isohyets and isotherms (for climatic data see 'Rainfall' and 'Temperatures' Maps, Atlas of Australian Resources Second Series,

1970 and 1973, Geographic Section, Department of National Development,

Canberra), make the N.T. especially convenient for analysis. The northern part of the northernmost subdivision Darwin and Gulf (DG) lies within the megatherm seasonal rainfall bioclimate of Nix (1982; see Fig. s.1) (more or less synonymous here with the high seasonal rainfall tropics) and the southernmost Central Australia South (CS) subdivision lies in the middle of the megatherm/mesotherrn arid bioclimate (in the centre of the arid zone of Australia). There is declining rainfall southwards from >120 cm y-1 in the northern part of DG to <15 cm y-1 in

CS (median annual rainfall, 50 percentile); rainfall is shown by Nix

( 1982) as "highly seasonal" (summer) in DG and "mod era tely0 and

"slightly" seasonal (equally summer and winter) in CS; average annual temperature declines southwards from >27°C to 21°C. The second transect lies entirely within the megatherm seasonal bioclimate and encompasses subdivisions southwards along the east coast of Australia from Cook (CK) subdivision in northern Qld. to the Central Coast (CC) subdivision in 95

central N.S.W., where c3 and c 4 grass species occur in equal numbers

(Hattersley 1983). Median annual rainfall (50 percentile) is >c. 80 cm y-1 in at least parts of all subdivisions; rainfall is shown by Nix

(1982) as "highly seasonal" (summer) in most of CK and 0 moderately 11 or

"slightly seasonal" in most of the rest of eastern coastal Qld. (summer rainfall exceeding winter rainfall) and in coastal N.s.w. (in most parts winter rainfall exceeding summer rainfall). Average annual temperature is mostly from 24°C - >27°C at the northern end of the transect, and

>15°C <1 8°C at the southern end. This transect, therefore, is characterised more by a temperature gradient than a rainfall one.

li!esults

Comparative distributions of c4 types

Table 8.1 lists numbers of native species of each c 4 type in 73

Australian State and Territory subdivisions. Fig. 8. 2 maps these distributional data. 7 There are in Australia an estimated 341! NADP-ME species, 57% of the total native c 4 grass flora; this is the most numerous c 4 type in 48 3 (out of 73) State and Territory subdivisions. 19)0 species (32% of total) are known or predicted to be NAD-ME; this is the dominant c 4 type, species number-wise, in 23 subdivisions. These subdivisions are more southerly than those in which NADP-ME species dominate, especially in the east. NADP-ME and NAD-ME species are equally numerous in two subdivisions (Eyre Peninsula, S.A.; Grids H,L,M, Vic.). There are an estimated 65 PCK species (11% of total); in no subdivision is this the dominant c 4 type. All three types are at their most numerous, species number-wise, c4 96

in the northern subdivisions of tropical Australia: 190 NAOP-ME species

(55% of all NAOP-ME species) and 46 PCK species (71% of all PCK species)

are recorded from the CK subdivision (Qld. ), which lies in the megatherm

seasonal (rainfall) bioclimate; 83 NAO-ME species (44% of all NAO-ME

species) occur in neighbouring BK subdivision, which (uniquely)

straddles bot.'1 the megatherm seasonal and the megatherm/mesotherm arid

bioclimates. 51% of all native c4 grasses occur in CK (cf. 54% in

Hattersley 1983, data being more comprehensive now, especially for

W.A. ) •

In the N.T. transect overall species diversity declines southwards

from 280 in OG (mega therm seasonal bioclima te) to 158 in CS

(megatherm/mesotherm arid bioclimate). NADP-ME species are the rro st

numerous in all five subdivisions; their proportion of the total c 4 grass flora in each subdivision, however, declines from 59% (in OG) to

48% (in CS). PCK species are the least numerous in all subdivisions and their proportion also declines southwards, from 13% to 7%. By contrast,

NAD-ME species numbers remain the most constant in the transect: with

79 species in OG and 71 in CS, their proportion of the total c 4 grass flora increases from 28% (in OG) to 45% (in CS). NAO-ME species diversity is, therefore, the least affected by the bioclimatic variation. 38% of all NAD-ME species occur in cs compared with 22% of all NADP-ME species and 17% of all PCK species.

The relatively high proportion of NAO-ME species within the megatherm/mesotherm arid bioclimate characterising the CS subdivision is repeated elsewhere. Fig. 8.2b contrasts subdivisions with comparatively high percentages of NAO-ME species (unstippled) with those with cornparati vely low percentages (stippled). The unstippled area corresponds closely with the extent of the arid bioclimate, the stippled 97

area with the other two main bioclimates (see Fig. 8.1).

The coastal transect from CK (Qld.) to CC (N.S.W.) shows little

change in the relative proportions of each of the c 4 types (in CK and cc respectively: NADP-ME 63% and 61%; NAD-ME 22% and 24%; PCK 15% and 16%)

even though overall species diversity declines from 304 to 71.

The only three Australian genera known to be variable for c type 4 provide a good opportunity for corrparing correlations, relatively free of taxonomic influences, between c 4 type and distribution. Paniewn (Panicoideae) and Leptoehloa and Sporobolus (Chloridoideae) have NAD-ME as well as PCK species, the contrast between the distributions of which

is clearly shown in Fig. 8.3. Whereas the PCK species are most numerous in the megatherm seasonal bioclimate and generally are absent from the arid bioclimate, the NAD-ME species are more or less equally numerous in both (as with NAD-ME species as a whole. Note, however, the overall decline southwards in species numbers of all c 4 types; see Hattersley

1983). None of the NAD-ME species in these genera is restricted to the arid bioclima te. If the Panimun species here assumed to be PCK are in fact NAD-ME (see Introduction and Chapter 4), the contrast in the distributions of species of the same c 4 type, but with different FCR cell chloroplast positions, would resemble that within NAD-ME Eragrostis

(Chapter 3) •

Comparative distributions of taxa within eaeh c4 type NADP-ME

NADP-ME species of Australia are found in the subfamily

Arundinoideae ( 102 species) and in the two supertribes Andropogonanae and Panicanae (126 and 119 species, respectively) of the subfamily

Panicoideae. 98

Fig, 8. 4 shows the predominant NADP-ME subfamily or supertribe in

each State and Territory subdivision. Broadly speaking the

Andropogonanae dominate in northern i'

Arundinoideae through most of the megatherm/mesotherm arid bioclimate

(i.e. over most of the arid and semi-arid zone). The Panicanae

generally dominate only in S.A. and south-eastern Australia where

overall species diversity is low: e.g. only one species in EU (W.A.)

and KI ( s .A.) and <1 O species in nine others ..

All three groups have their maximum species diversity in the

megatherm seasonal bioclimate of northern and north-eastern tropical

Australia: viz. the CK subdivision, where 55% of all native NADP-ME

species have been recorded (Fig. g2a), contains 69% of all Andropogonanae

species, 47% of all Panicanae species and 46% of all Arundinoideae

species (Table 8.1).

In the N.T. transect total NADP-ME species diversity declines

southwards from 164 to 76, as do absolute numbers of each taxonomic group individually. Whilst the sub divisional percentage of NADP-ME

species which are Panicanae remains more or less constant at 26-31 %,

that for the Andropogonanae decreases from 45-46% (in DG, VR and BT) to

36% in CN and 30% in CS, and that for the Arundinoideae increases from

24%-28%, to 38% and 41% respectively. In CS (and slightly in CN), in

other words, the Arundinoideae outnumber the Andropogonanae (Table 8.1)

but in most subdivisions within the arid bioclimate absolute numbers of

the two groups are about the same (Fig. 8.4). In the coastal transect

total NADP-ME species diversity declines southwards from 190 to 43; the

main trend of relative numbers of each group is the declining proportion

southwards of the Andropogonanae from 46% to 28% and the increasing one

of the Panicanae from 29% to 53%. Proportions of the Arundinoideae 99

fluctuate slightly but remain low (between 18% and 27%).

NADP-M.E Arundinoideae comprise Aristida (65 species), Eriachne (36

species) and Pheidochloa (1 species) • Eriachne has its maximum

diversity (23 species) in DG (N.T.) and Aristida (32 species) in NK

(Qld.). In the N.T. transect Eriaahne numbers decline steeply

southwards (from 23 to 7 species) but those of Aristida increase from 19

to 24 species (DG to CS respectively). As with NAD-ME species in

general (see previous section) Aristida species diversity is fairly

uniform in both the megatherm seasonal and the megatherm/mesotherm arid

bioclimates of northern and central Australia and it is this genus,

therefore, one of the largest of all c 4 grass genera in Australia, that mostly accounts for the prominence of the Arundinoideae in areas within

this bioclima te. Four species of Eriachne have centripetal PCR cell

chloroplasts, in contrast to the observed or predicted (see Chapter 4)

centrifugal/peripheral chloroplasts of 32 other species in this genus,

nut no distributional difference between the two groups is apparent.

The supertribe Andropogonanae consists of 34 genera, only four of which (Cymbopogon, Dichanthium, Iseilema, Sorghum) have more than 1 O

species in Australia. These largest genera (with a total of 56 species) have wide distributions and they largely account for the species diversity of the Andropogonanae in the arid bioclima te. The 30 small genera are proportionately very much more concentrated within the mega therm seasonal bioclima te, some occurring only in sing le northern

subdivisions (e.g. Heterophol.is, Miorostegiwn and Polytrias). One

species, Themeda austral.is, roeri ts special mention. As pointed out by

Hayman ( 1960) "no other species in the Australian flora has a di stril:>l ti on more extensive than T. australis, and very few have one as great"; indeed this species seems to be absent from only seven State and 100

Territory subdivisions.

More than half of the species in the Pani.canae belong to Digita.ria

(37 species) and PaspaLidiwn (25 species). Both genera are most numerous in northeastern coastal Qld. with diversity decreasing sharply both westwards and southwards. In contrast to all other Australian c 4 genera, irrespective of c type and taxonomic position, (c 4 Neuraahne 4 species only), Pareateniwn, Paraneuraahne and PLagiosetwn have di striru tions entirely restricted to within the arid bioclima te (see

Chapter 2 for discussion on Neuraahne and Paraneuraahne).

PCK

There are an estimated 65 PCK species in Australia, 33 of six genera in the Panicoideae and 32 of eight genera in the Chloridoideae.

PCK species of both subfamilies are most numerous in CK (Qld.; in the megatherm seasonal bioclimate; Fig. 8.2). Of the 46 species here

(71% of all PCK species) 24 are panicoids and 22 are chloridoids. Only

1 O subdivisions have ).20 PCK species and 35 have <:5 species.

In both the N.T. and coastal transects there are steep declines southwards in total PCK species diversity. Throughout the arid bioclimate PCK panicoids usually outnumber PCK chloridoids (but numbers are small; Table 8.1). In the coastal transect panicoid species predominate in Qld. but chloridoids predominate in coastal (and also subcoastal) N.s.w ••

Distributions of (by far) the largest PCK genera of each subfamily differ markedly from each other. Al though both panicoid UroahLoa ( 19 species) and chloridoid Eatrosia (1 2 species) are most numerous in northern, tropical Australia (in DG, N.T. and CK, Qld.), the former genus is recorded from all Qld. subdivisions and many in S.A. and 101

N.s.w., but Eatroaia is recorded only from four Qld. subdivisions and

does not extend south of the Tropic of Capricorn.

NAD-ME

In Australia 13 NAD-ME species are in the subfamily Pani'coideae and

180 species in the Chloridoideae (including 64 that have been examined

in Eragroatia and which are not discussed in detail here; see Chapter

3). Species of both subfamilies are most numerous in the mega therm

bioclimate in northern Australia although chloridoid numbers are high

throughout Qld. with the exception of the extreme south-east.

Paniaum (8 species) and Yakirra (5 species) are the only panicoid genera with centripetal chloroplasts. Their distributions are very different, such Paniaum species being widespread and especially numerous in Qld. (Fig. 8. 3a) whilst Yakirra is essentially a genus of northern w.A. and N.T ••

In the Chloridoideae there are 17 NAD-ME genera (40 species) with centripetal PCR cell chloroplasts only. They reach their maximum diversity (26 species) in BK (Qld.). The N.T. transect shows only a slight decrease southwards in species diversity (from 23 to 20 species), whereas in the coastal transect there is a steep decline in south-east

Qld. and only 5 species occur in CC (N.s.w.). An extreme contrast in distribution is shown by Diatiahlia and EragrostieLLa (each with only a single Australian species): the former, unusually for a genus of any c 4 type, is restricted almost entirely to c 3 grass-dominated temperate areas of extreme south-eastern and sou th-western Australia, and

EragostieLLa is only recorded from tropical northern Qld.. (See

Comparative distributions of c4 types for discussion of Leptoahloa and Sporobo ius. ) 102

Five NAD-ME chloridoid genera (69 species, excluding Eragrostis;

see Chapter 3) have species with mainly or at least scme centrifugal/

peripheral chloroplasts. These are Enneapogon (19 species), PLeatraahne

( 15 species) , SympLeatrodia (two species) , Triodia ( 32 species) and

Triraphis (one species). Prior to findings presented in Chapter 5

species of these genera would have, or had, been classified as PCK.

They have their maximum species diversity (25 and 24 species

respectively) in DG and CS (N.T.); i.e. the N.T. transect reveals little

change in diversity, a vecy similar pattern to that shown by NAD-ME

species with centripetal chloroplasts. There are eight Triodia species

in both DG and cs, six and 11 Enneapogon species respectively, and six and four PLeatraahne species respectively. Whilst there are also high numbers (20 species) in BK (Qld. ), only 13 occur in neighbouring CK, an unusual contrast. Much of this 1 imbalance' is due to the western centre of diversity of PLectraahne (12/15 species in W.A., eight in N.T., three in Qld.) and T:riodia (25/32 species in w.A., 18 in N.T., 11 in Qld.), many species being highly localised (as are the two species of

SympLeatrodia, recorded only in DG, N.T.). Enneapogon is widely distributed but its area of highest species diversity is eastern rather than western (10/19 species in W.A., 13 in N.T., 16 in Qld.). Triraphis moLlis is widespread in all mainland states. The distinctive distributions of each of the five genera make it difficult to generalise about their combined distribution but they are nuch less diverse, species number-wise, in all eight subdivisions comprising the coastal transect than those NAD-ME chloridoid genera with centripetal chloroplasts (despite being overall more diverse: 69 versus 40 species). Both overall and individually the distributions of these genera alsc contrast with that of NAD-ME Eragrostis species wiG~ 103

centrifugal/ peripheral chloroplasts (Chapter 3).

Distribution in relation to presenae or absenae of the suberized lamella

in PCR aell walls

An estimated 402 native c4 species (66% of the total) of all three c types have a suberized lamella in their PCR cell walls. 206 species, 4 all either NAD-ME or NADP-ME, do not have a suberized lamella.

The three subdivisions with the greatest numbers of species without a suberized lamella are also those with the greatest number of c4 species as a whole: BK (Qld.) with 97 species, DG (N.T.) with 92 species and CK ( Qld. ) with 86 species (Fig. 8. 5) • Whilst all these subdivisions lie at least partially within the megatherm seasonal bioclimate, the subdivisions with the next highest number of species lie wholly in the megatherm/mesotherm arid bioclimate: 84 species occur in

CN (N.T.) and 80 in neighbouring CS (N.T.). In the N.T., therefore, there is little overall north-south decline in the diversity of species without a lamella, although the intervening VR and BT subdivisions have appreciably fewer species (especially of Aristida). In the coastal transect there is a continuous and steep decline in numbers (as in c4 species as whole) from 86 species in CK (Qld.; 28% of all c4 species in

CK) to 18 species in CC (N.S.W.; 25% of all c4 species in CC).

When relative frequencies, rather than absolute numbers, are considered, a different picture emerges of the geographical distribution of species without a suberized lamella. The distribution of the unstippled subdivisions (in Fig. 8.5), those where the percentage of species without a suberized lamella is >40%, closely resembles that of the arid bioclima te (see Fig. 8.1 ) ; the distribution of the stippled subdivisions, those where the percentage of species without a lamella is 104

~40%, is very like that of the other two main bioclimates, This overall

distribution pattern is reflected in both transects: in N.T. there is

an increase southwards in the proportion of species without a lamella,

from 33% in DG (partially in the mega therm seasonal bioclimate) to 51 %

in CS (in the arid bioclimate); in the coastal transect, however, there

is relatively little change in the relative frequencies from nort.'1 to

south (24% 36%). Comparison of the two transects indicates that

relative frequencies of species without a suberized lamella could be

influenced more by rainfall than by temperature; this is also apparent

from the low percentages of species without a lamella in extreme sout.'1-

eastern and sou th-western Australia. The highest re la ti ve frequencies

of all occur in subdivisions (AV, EU, (W.A.) 71%; KI (S.A.) 66%) where c 4 species diversity as a whole is low (<20 species), so these may not be very meaningful.

Ecological aspects of c4 type distribution By far the most extensive natural grassland formations (sensu Moore and Perry 1970) in Australia are dominated by NAD-ME genera in the

Chloridoideae. The arid hummock grasslands of Triodia and Plectrachne

spp. and the semi-arid tussock grasslands of Astrebla spp. together cover about 25% of the continental area (visual assessment from vegetation map of Australia by !bore and Perry 1970), most of it within

the megatherm/mesotherm arid bioclimate. The other extensive type of natural grassland recognised by !bore and Perry (1970), sub-humid grassland, is dominated by NADP-ME species of the Andropogonanae in both its tropical and temperate sub-formations (e,g. by Dichanthiwn and

Themeda spp. respectively). The dominance of the Andropogonanae in both northern tropical and tropical and subtropical eastern Australia (i.e. 105

mainly within the megatherm seasonal rainfall bioclimate) is emphasised

by Johnson and Tothill ( 1985), who state that these grasses provide "the bulk of the biomass of these pastures as we 11 as much of the floral diversity." They also consider tt1e dominance of grass genera and tribes in relation to rainfall and soil-water gradients: with drier conditions

the Andropogonanae give way to genera such as Astrebla, Enneapogon,

Eragrostis and Triodia ( Chloridoideae) and/or to Aristida

(Arundinoideae). I note that this represents a replacement of NADP-ME genera by NAD-ME ones, except that Aristida is also NADP-ME (and is also very unusual in other respects; see e.g. Chapters 4 and 5). Ecological dominance by one or other of the c4 types, however, is by no means always clear. One example is the coastal strand and foredune formation of southern QJ.eensland, an extreme environment where pioneering species are Spinife:r: hirsutus Labill. ( Panicoideae), predicted to be NADP-ME,

Zoysia m::iorantha Desvaux (Chloridoideae), predicted to be PCK, and

Eragrostis interrupta Beauv. and Sporobolus virginious (L.) Kunth

(Chloridoideae), both predicted to be NAD-ME (distribution data from

Tothill and Hacker 1983). A common component of the temperate salt- marshes of Victoria and Tasmania, DistiohUs distiohophyHa (Labill.)

Fassett (Love 1981), is predicted to be NAD-ME whereas its northern hemisphere salt-marsh counterpart, Spartina anglioa c. E. Hubbard ined,, has been biochemically determined as PCK (Smith et al. 1982). PCK species are not a particularly prominent part of native vegetation in

Australia but I note that several introduced species of this c 4 type (notably Braohiaria deoumbens Sta;.:if, B, rrutioa (Forssk.) Stapf, B. ruziziensis Germain & Evrard, Melinis minutiflora Beauv. and Panioum maximum Jacq. (Panicoideae), and Chloris gayana Kunth (Chloridoideae)) are among the most important tropical pasture grasses (see Davies and 106

Hutton 1970).

In the mega therm/seasonal arid bioclima te of Central Australia, in

parts of which median annual rainfall ( 50 percentile) can be <15 cm

yr-1 , NAD-ME and NADP-ME species are more or less equally "dominant" or

"common" in xeric land types (based on data of Lazarides 1970).

Floodplains, by comparison, have a higher diversity of species,

presumably because of increased moisture availability, but since there

are 11 11 dominant" or 11 common" species of each type, neither type c4 c4 seems to be favoured. Ten of the 12 PCK species present are restricted

to areas of increased moisture availability (e.g. drainage channels, depressions), a characteristic also noted by Ellis et aL, (1980) in areas of South West Africa/Namibia with <35 cm rainfall yr-1• The other

two species, Eraahiaria hoLoseriaa (R, Br.) Hughes and DaatyLoateniwn raduLans (R, Br.) Beauv., are the most widespread PCK species in terms of the number of land types in which they occur, and they belong to the group of ephemeral drought-evading plants whose life-cycle "is such that although they exist in an arid area, they are present vegetatively only under relatively non-arid conditions following rain. During long dry periods they exist only as seeds" (Lazarides 1970), Such temporal occurrence, also characteristic of certain NADP-ME and NAD-ME species, would obviously decrease the sensitivity of any climatic analysis of c 4 type distribution that is based on large-scale spatial occurrences of species. There are also both NADP-ME and NAD-ME species in the group described by Laza rides ( 1970) as perennial drought-evading plants "whose leaves die during drought periods, but which resume growth from vegetative buds with the onset of favourable conditions." Only NAD-ME

Triodia and PLeatraahne, however, are mentioned as belonging to the third group, the perennial drought-resisting plants which "remain in a 107

vegetative, though generally dormant, state throughout droughts and

resume growth wi t.'l the onset of favourable conditions" (Lazar ides 1970).

Historioai aspeets of c4 type distribution Australian grass genera were grouped by Clifford and Simon ( 1981)

into a number of 'elements' based on common patterns of geographical distributions outside Australia. The majority of genera, of all three c 4 types, are in either the Gondwanan or Old world Tropical geographical elements (Table 8. 2); the Indo-Malayan element contains only NADP-ME genera. Some endemic c 4 genera, notably NAD-ME Triodia and NADP-ME

Neuraehne (which also includes c 3 and c 3-c4 species; see Chapter 2), are widespread in a geographic sense but not in terms of the number of major native Australian plant communities (as defined by Clifford and Simon

1 981) in which they occur. The six most widespread genera in these

terms are in the Gondwanan element: Aristida ( NADP-ME) , Eragrostis

(NAD-ME), Panieum (NAD-ME and PCK), Enneapogon (NAD-ME), Sporoboius

(NAD-ME and PCK), and ChZoris (PCK). All three c 4 types, therefore, are represented in this list which includes, furthermore, NAD-ME genera with centripetal chloroplasts as well as those with centrifugal/peripheral chloroplasts. That Gondwanan genera outnumber Old world Tropical and

Indo-Malayan genera in almost all Australian plant communities "strongly

supports the suggestion that the Gondwanan element is an ancient component of the Australian flora" (Clifford and Simon 1981) and, by inference, that all c4 types may be equally ancient components.

lJ)i scu ssion

Several patterns of distribution in native Australian grasses have emerged which are similar to those of the bioclimates of Nix ( 1982; Fig. 108

8. 1 ) • They clearly suggest that c 4 type alone is neit,'ler the sole determinant of geographical distribution nor necessarily always an

adaptation to a particular bioclimatic regime. This conclusion was

first reached in relation to the structurally variable NAD-ME genus

Eragrostis, in which distribution is correlated with PCR cell chloroplast position rather than with c4 type per se (Chapter 3): the megatherm/mesotherm arid bioclimate, by far the rrost extensive in

Australia, is dominated by Eragrostis species with centripetal chloroplasts and the other two main (more humid) bioclimates by species with centrifugal/peripheral chloroplasts. The same

structural/bioclima tic correlation exists in Paniaum, Leptoohloa and

Sporobolus (Fig. 8. 3) but with the difference that those species with centrifugal/peripheral chloroplasts in at least the latter two genera are PCK rather than NAD-ME; it is therefore impossible in their cases to disassociate the significance of c 4 type from that of chloroplast position. Irrespective of chloroplast position, NAD-ME species as a whole are proportionately at their highest in subdivisions in the arid bioclima te (Fig. 8. 2b), without necessarily being numerically dominant in them. In this bioclimate, species of all c 4 types without a suberized lamella in their PCR cell walls also reach their highest proportions (Fig. s. 5), and only there are Arundi noideae species the dominant NADP-ME taxa (Fig. 8.4). All of these patterns clearly highlight the different composition of the grass flora of the megatherm mesotherm arid bioclimate compared with that of the surrounding humid bioclima tes, even in southern Australia where c4 species diversity is low. Despite its extent, however, only four c 4 genera (Neuraohne, Pareoteniwn, Paraneuraohne and Plagiosetwn, all NADP-ME panicoids) have distributions entirely restricted to the arid bioclimate. 109

Al though large areas within the mega therm seasonal bioclima te are ecologically dominated by NADP-ME species, and equally large ones wi t.'1in

the megatherm/mesotherm arid bioclimate by NAD-ME species, there is no degree of mutual exclusiveness between the geographical distributions of the three c 4 types. Extreme habitats, to which, if anywhere, one particular c4 type might be expected tD be best adapted, may instead be occupied by species of all three. Habitat preferences of PCK species in the arid bioclimate, and the avoidance of this bioclimate by PCK species in and spp., are perhaps indications that this c Leptoahioa Sporoboius 4 type may be the most dependent on high moisture availability.

The notions that "the NAD-ME type of species ••• dominate in areas of very low precipi ta ti on" (Ellis et ai. 1980) and that "NAD-ME species are more successful under xeric conditions" . (Vogel et ai. 1986) are clearly not particularly valid for Australia where NADP-ME species are more numerous in more subdivisions in the arid bioclima te than are NAD-ME species. NAD-ME species diversity neither increases nor substantially decreases with increasing aridity although their relatively high proportion in the c 4 grass flora of subdivisions in the arid bioclimate suggests some degree of tolerance for it. The same applies to the NADP­

ME genus Aristida which is just as prominent (species number-wise) in the Australian arid bioclimate as in the more humid bioclimates. The fact that Stipagrostis, another arundinoid and probably NADP-ME genus

(see below), is primarily restricted to the arid zone of south West

Africa/Namibia (R.P. Ellis pers. comm.) suggests that taxonomic affinity is often as well correlated with climate as c 4 type and that other taxonomically related characteristics are relevant tD determining geographic di stribu ti on. This notion may also be supported by the fact that the overwhelming majority of NAD-ME species in Australia are in the 110

subf arni ly Chloridoideae. Nevertheless, where there is variation in c4 type and/or PCR cell chloroplast position within the subfamily, NAD-ME

species with centripetal chloroplasts have a greater prominence in the

arid bioclimate than elsewhere (but are not necessarily dominant); PCK

species or NAD-ME species with centrifugal/peripheral chloroplasts (as

in Eragrostis) are more prominent in other bioclimates.

There is a strong possibility that the origin of the idea that NAD­

ME species as a whole are particularly adapted to an arid bioclimate may

lie in the erroneous assignment to c4 type of a number of genera by Ellis et ai. (1980) and Vogel et ai. (1986). The most significant genus

in this respect is Stipagrostis which was typed as NAD-ME on the basis of anatomical predictors of c 4 type (R.P. Ellis, pers. comm.; Vogel et al. 1986). Species of this genus, the second largest and largest c 4 genus (24 and 8 species respectively) in the study areas of both sets of authors, may be NADP-ME like all other c 4 arundinoids (sensu Watson et ai. 1985) that have been biochemically assayed for c 4 type (in Aristida, Eriaahne and Pheidoahloa; see Chapter 5). If Stipagrostis (and another arundinoid genus Asthenathel'Ull1, with two species) is NADP-ME then this c . type is the dominant one, species number-wise, in 18 (instead of 6) 4 out of 21 magisterial districts in South West Africa/Namibia for which there has been adequate plant-collecting (the exception being Lfideritz­

Nord; see Methods and Fig. 2 in Ellis et aL. 1980; Launert 1970; cf. following paragraph). Vogel et ai. (1986) list 16 species as NAD-ME but, since nine of them are in Stipagrostis and Asthenathern.JJ!l, the claim that "NAD-me types are thus better adjusted to extremely arid environments than either the malate forming NADP-ME type or the PEP-ck types" is perhaps invalid.

In South West Africa/Namibia there are a number of species (in 111

Enneapogon, Pani(JW!1, Triraphis and especially Eragrostis) which could be

NAD-ME but were classed as PCK by Ellis et a?,. ( 1980; see anatomical data and c 4 type predictions in Ellis 1977). To some extent they could balance the number of species 'removed' from the NAD-ME group if

Stipagrostis is NADP...ME rather than NAD-ME, but they could also invalidate the conclusion of Ellis et aL. (1980) that PCK species do not

"attain their maximum abundance in areas receiving rainfall of 350 mm/yr and decline when the precipitation departs from this level".

This phytogeographical analysis of the distribution of c types is 4 the first to be based on sufficient biochemical data. There are a number of features about such an analysis, however, which limit clear­ cut conclusions regarding the adaptive significance of c types. 4 Firstly, the geographical data themselves are imprecise in that they are entirely on a presence/absence basis which gives no indication of abundance; secondly, the State and Territory subdivisions seldom follow climatic boundaries, e.g. BK subdivision (Qld.) which spans both the megatherm (seasonal) and megatherm/mesotherm arid bioclimates; thirdly, no account can be made of microclimatic and edaphic heterogeneity within these subdivisions; and lastly, the very size of some Australian genera

(e.g. Aristida, Digitaria, Eragrostis, Eriaohne and Triodia, which together constitute 38% of the native c 4 grass flora) means that there is a considerable taxonomic influence on the results of the analysis.

Rather than a statistical analysis on correlations between climate and c type distributions, a more fruitful future approach towards 4 understanding the adaptive significance of c 4 types and their structural variants might be to seek directly for explanations of the patterns of distril:u tion that have emerged from this study and from Chapter 3.

Water-stress experiments, for example, on species within genera variable 112

for type (e.g. or PCR leaf blade anatomy c4 LeptochLoa, Sporobolus) alone (e.g. Eragrostis) might reveal growth response characteristics which rnigh t be linked with differences in bioclima tic tolerance and

Australia-wide geographic distributions. As stated in Chapter S, c4 types in grasses have been the subject of remarkably few comparative physiological studies. Because of the data presented in this thesis,

the Australian c4 grass flora presently offers the best experimental material for comparative physiological investigations of the full structural, biocherni cal and taxonorni c diversity of c4 grasses. Table 6.1. ~ti111.ated number of native Aut1tralian speci6S of grasses (Poaceae) of eacll c 4 acid decarboxylation type (NADP-malic eu:.i:yi:11e, NADP...KE; NAD-iaalic eozyae, NAD-H.81

PEP carboxykinase. PCK).

NAO-KE species are subdivided according to position of chloroplasts in PCR (Photosynthetic Carbon Reduction, or Kranz) bundle sheath cells. b'ra9roatia data from Chapter 31 species with intermediate anatomy have some centrifugal/peripheral chloroplasts. State and 'ferritory abbreviations, see Methods and Sources of Datai subdivision abbreviations,

see Hattersley (1963), Asterisked subdivisions (in N.'1',, Qld. and N,S.W.) constitute transects discussed in tex:t (see Methods and Soui:-ces of Data).

NADP-ME PCK NAO-ME

PANICOIOEA.E ARUNOINOIDEAE PANICOIDEAE CliWRIOOlDEAE PANIWIDEAE CliWRIOOIDEAE 'l'OTAL Andropogonanae Panicanae Non -E'1•agrostia E'ragroatia

Centripetal Centt·ipetal At least some Centripetal At least SO!llld chloroplasts chloroplasts centrifugal/ chloroplasts centrifugal./ State peripheral periphl:!r-al Subdivision chloroplasts chloroplasts

W.A. HA 46 19 24 10 10 5 19 1 3 3 9 150 OR 42 23 24 9 7 5 21 21 4 "/ 163 fZ 36 18 14 3 9 4 9 10 3 6 112 OA 16 7 11 6 7 3 9 10 4 5 78 fO 20 15 19 7 6 17 17 8 6 110 AB 16 10 18 2 3 2 17 14 11 5 98 CA 20 17 18 6 3 3 11 23 12 4 116 gad 15 10 8 3 1 2 6 14 6 3 69 td 8 6 8 2 3 2 8 4 43 lad 9 8 5 1 2 2 4 8 5 4 48 gd 6 3 9 0 0 3 6 6 0 34 wr 9 6 a 1 1 6 13 6 4 55 gvd 5 3 4 0 1 0 4 u 8 1 34 AU 10 7 12 2 2 2 10 13 u 4 70 IR 8 3 4 1 3 1 5 2 4 0 31 DR 5 3 4 0 0 5 0 0 19 AV 3 0 2 0 0 5 4 2 0 17 CG 6 2 5 2 2 2 5 7 4 0 35 EU 0 1 0 0 0 0 2 1 3 0 7 WN 5 2 0 0 0 3 0 0 1 12 SG 3 0 0 3 u 0 0 9 EY 2 0 0 4 u 3 2 " NA DP-ME PCK NAO-ME

PANICOIOEAE ARUNDIOOID£AE PANICOIDEAE CliLORIOOIDEAE PANIL'OIOEAE CHLORIOOIDEAE Andropogonanae Panicanae Non -E'ragroa tia

Centripetal Centripetal At least some Centripetal A.t least some chloroplasts chloroplasts centrifugal/ chloroplasts centrifugal/ State peripheral peripheral subdivision chloroplasts chloroplasts

N.T. •OG 75 47 42 21 16 9 23 25 1 21 280 'VR 41 29 22 6 7 4 21 21 4 10 165 •BT 40 23 24 9 7 6 20 16 6 8 161 'CN 30 22 32 8 6 5 22 22 14 10 171 •cs 23 22 31 8 3 5 20 24 12 10 158

S.A. NW 10 11 9 5 2 3 8 16 8 3 75 LE 12 15 ts 6 3 3 15 0 8 7 92 NU 4 9 1 0 0 3 6 6 0 30 m 6 7 4 5 2 4 8 10 3 50 fR 7 8 9 3 2 11 10 5 61 >.'N 4 7 4 4 2 2 6 7 5 '2 43 EP 6 7 1 0 1 2 5 5 2 0 29 yp 2 4 3 2 3 2 6 3 1 0 26 NL 3 7 2 0 2 5 3 3 1 27 6 9 3 1 2 I 5 5 4 43 "'KI 0 1 0 0 0 0 0 6 SL 7 7 6 2 2 8 2 1 2 39 SE 4 2 0 0 2 1 4 2 0 2 17

Qld. •cK 67 56 47 24 22 7 24 13 4 20 304 'NK 69 52 44 21 14 20 11 6 18 260 'SK 44 26 25 15 9 5 21 12 8 11 176 •pc 50 41 23 13 10 7 19 12 5 0 1013 'WB 35 34 15 9 8 4 9 2 2 I 125 35 38 26 11 11 6 9 5 3 8 152 '"'BK 61 50 47 16 19 9 26 20 9 19 276 GN 31 21 29 8 5 5 22 17 10 7 155 HL 30 30 28 10 0 8 22 16 11 10 173 LH 42 33 30 13 11 7 25 14 7 11 1Y3 GS 25 22 21 8 4 6 19 14 11 7 13"1 m 23 28 29 11 9 7 22 14 15 B 166 MA 20 23 28 8 8 5 20 15 12 6 145 OD 32 30 25 10 7 ') 19 12 8 7 157 BN 30 26 19 7 8 6 9 8 5 b 124 NADP-ME PCK NAO-ME

PANI())!DEAE ARUNDINOIDEAE PANIL'OIDE:AE CHWRIOOIDEAE PANIOJID£AE CHIDRIOOIDEA.E 'l'O'fAL Andropogonanae Panicanae Non -EPag1•oat·l11 b'NfJl'08tiB

Centripetal Centripetal At lt!aSt some Centripetal At least some chloroplasts chloroplasts centrifugal/ chloroplasts centrifugal/ State peripheral peripheral Subdi vi aion chloropl.asts chloroplasts

N.s.w. 'NC 22 24 12 5 9 4 5 2 6 90 •cc 12 23 8 3 8 3 5 2 6 71 FN 11 18 17 7 4 3 15 7 13 7 102 NP 21 26 24 9 8 5 16 9 12 6 136 NX 24 22 17 7 5 11 8 7 5 113 Nr 18 9 10 2 6 3 2 2 1 6 59 FS 2 4 5 2 3 l 8 5 7 38 SP 7 12 7 4 2 5 13 6 4 4 64 ex 17 15 12 2 6 5 10 8 3 5 83 Cl' 8 8 5 0 3 2 2 2 5 36 sx 6 9 4 2 3 5 2 3 36 ST 6 5 1 2 3 4 25 SC 7 11 3 5 2 2 2 35

Vic. AG 7 8 3 l 3 2 10 5 5 4 48 HM 7 5 2 2 2 6 2 2 3 32 QZ 9 10 3 2 3 3 4 5 41 DK 5 3 () 2 0 4 2 19 PT 5 3 1 2 2 5 3 24

Tas.

TA 4 4 0 0 0 3 0 14 Table a.2. &m.bers of native Australian c4 genera of

each c4 type in the geographical 'elements' of Clifford and Simon (1981).

Geographical Number of genera

element NADP-ME PCK NAD-ME

Co smopo li tan 0 0 0

Gondwanan 2d' 6B 8B

Old World Tropics 13A 4 7

Inda-Malayan 1 3C 0 0

Australasian 2C 0

Endemic D,E Tropical/subtropical 9 1D 3

Temperate 0 0 0

Both 0

1?i sjunct distributions

Temperate North and Sou th America 0 0

Africa 0 0

A BothrioohLoa (Kuntze) (Gondwanan element) and Diohanthiwn (Willem.) (Old World Tropics) are synonyms (see Watson et aL. 1985) and are included in both elements.

B Three genera with both PCK and NAD-ME species are included in both columns.

C Germainia (Bal. & Poi tr.) (Indo-Malayan) and SoLerandriwn (Stapf & c. E. Hubb.) (Australasian) are synonyms (see Watson et aL. 1985), and are included in both elements.

D CynoohLoris species are hybrids between NAD-ME Cynodon and PCK Chloris species; the genus is omitted (see Chapter 7). Psammagrostis, also omitted, has yet to be assigned to c 4 type but is predicted to be NAD-ME or PCK.

E TheLLungia is omitted because the only species (T. advena Stapf) has been transferred to Eragrostis (Phillips 1982). Fig. 8.1. Main Australian bioclimates of Nix (1982), with superimposed

30 cm and 60 cm isohyets (median annual rainfall, 50 percentile; see

Materials and Methods for map reference). The bioclimate classification

is based upon a generalised model of plant growth response to radiation,

temperature and water regimes, and an extensive climatic data base.

Bioclimate abbreviations: A, megatherm seasonal (with respect to

rainfall); B, megatherm/mesotherm arid; c, mesotherm/microtherm

seasonal. AB and BC are major interzones of semi-arid environments linking bioclimates A and B across the northern half of Australia and bioclimates B and C across the southern half of Australia. 30• ...... ·-. ··.. ·•·· ... B ·.:, ·1 l ! : a

b NAO-ME

"

c PCK Fig. 8.3. Distribution of NAO-ME and PCK species of Paniaum

(Panicoideae) and LeptochLoa and Sporobolus (Chloridoideae) in State and

Territory subdivisions. Asterisked species have been assayed for c 4 acid decarboxylation enzymes. a) Paniaum. N.B. No native XyMS+ species with centrifugal/peripheral PCR (Photosynthetic Carbon Reduction, Kranz) cell chloroplasts have been biochemically assayed for c4 type but it is assumed here that all five are PCK (see Introduction and Methods and

Sources of Data). NAO-ME species (centripetal chloroplasts) (top row/s, nos. 1-8): 1) P. buncei F. Muell. ex Benth.; 2) •P. decompositwn R.

Br. i 3) •P. effuswn R. Br.; 4) P. larcomianwn Hughes; 5) P. queensLandicwn Domin; 6) P. simile Domin; 7) P. trachyrhachis Benth.; 8)

P. whitei J. M. Black, PCK species (centrifugal/peripheral chloroplasts) (nos. 9-13, usually bottom row, in italics, underlined):

9) P. mindanaense Merr.; 1 O) P. mitaheUii Benth.; 11) P. obseptum

Trin.; 12) P. paLudoswn Roxb.; 1 3) P. seminudwn Domin. bl Sporobotus

(nos. 1-14», Leptoahloa (nos. 15-18). NAD-ME species (centripetal chloroplasts) (top row/s): 1) S. actinocLadus (F, Muell.) F. Muell.; 2)

S. virginicus (L.) Kunth; 3) •S. aaroLi Mez; 4) s. mitahellii(Trin.) c.

E. Hubbard ex s. T. Blake; 5) •S. pulaheLLus R, Br.; 6) S. aontiguus s.

T. Blake; 7) s. australasicus Domin; 8) S. scabridus s. T. Blake; 15) L. neesii (Thwaites) Benth.; 16) •L. digitata (R. Br.) Domin. PCK species

(centrifugal/peripheral chloroplasts) (bottom row/s in italics, underlined): 10) •S. areber De Nardi; 11) S. diander (Retz.) Beauv.;

12) •S. efongatus R. Br.; 13) S. LenticuLaris s. T. Blake; 14) s. laxus

B. K. Simon; 17) •L. cilioLata (Jedw.) s. T. Blake; 18) L. deaipiens (R.

Br.) Stapf ex Maiden. a Panietm

2) \ l.8 l 9.1-3'\ 2,3,S _,-9, 13 -1 l

b Leptochloa Sporobolus 1.2.~.5JlS 11 Fig. 8.4. :OOminance, species number-wise, in State and Territory subdivisions among NADP-ME taxa: Arundinoideae (arundinoids; vertical hatching), Andropogonanae (andropogonoids; diagonal hatching) and

Panicanae (panicoids; shading). Taxa were mapped as codominants if their respective species numbers are within 5% (of the total NADP-ME species present) of each other. Andropogonoids Fig. a.s. Relative (% of total native c4 grass flora) and absolute numbers, in State and Territory subdivisions, of species of all c4 types without a suberized lamella in PCR (Photosynthetic Carbon Reduction,

Kranz) cell walls. %, top or left integer; number, lower or right integer, in italics Stippled subdivisions, ~40% of species have no suberized lamella; unstippled subdivisions, >40% of species have no suberized lamella. AV (Avon, W.A.) represents subdivision not previously mentioned in text.

•

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