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1971 A survey of tapetal types and tapetal characteristics in the Leguminosae Paul Adelbert Buss Jr. Iowa State University

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BUSS, Jr., Paiil Adalbert, 1936- A SURVEY OF TAPETAL TYPES AND TAPETAL CHARACTERISTICS IN THE LEGUMINOSAE.

Iowa State University, Ph.D., 1971 Botany

University Microfilms, A XEROX Company, Ann Arbor, Michigan

THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED A survey of tapetal types and tapetal

characteristics in the Leguminosae

by

Paul Adalbert Buss, Jr

A Dissertation Submitted to the

Graduate Faculty in Partial Fulfillment of

The Requirements for the Degree of

DOCTOR OF PHILOSOPHY

Major Subject; Plant Morphology

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D^^ rZrad^^e College

Iowa State University Of Science and Technology Ames, Iowa

1971 PLEASE NOTE:

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TABLE OF CONTENTS

Page

INTRODUCTION 1

LITERATURE REVIEW 5

Tapetal Classification 5

Tapetal Nuclear Number in the Leguminosae 9

Mimosoideae 10 Caesalpinioideae 11 Papilionoideae 12

Somatic Polyploidy 14

Crystals in Tapetal Cells 22

MATERIALS AND METHODS 24

Plant Materials 24

Microtechniques 25

Paraffin Preparation 25 Anther Squashes 26

Photographic Techniques 27

Developmental Studies 27

OBSERVATIONS 28

Tapetal Types in the Leguminosae 28

Tapetal Nuclear Number and Nuclear Behavior 29

Mimosoideae 30 Caesalpinioideae 33 Papilionoideae 40

Tapetal Crystals 49

DISCUSSION 55

SUMMARY 62 iii

TABLE OF CONTENTS (continued)

Page

LITERATURE CITED 64

SUPPLEMENTARY BIBLIOGRAPHY 78

ACKNOWLEDGEMENTS 85

APPENDIX A: SUMMARY OF TAPETAL CHARACTERISTICS 86

Chart 1. Mimosoideae 86

Chart 2. Caesalpinioideae 89

Chart 3. Papilionoideae 92

APPENDIX B: FIGURES 101

APPENDIX C; SOURCES FOR SPECIES STUDIED 147 1

INTRODUCTION

The tapeturn is the cell layer immediately surrounding the sporogenous

tissue and spores in many sporangia. Because of this location and the

morphological changes which occur during its development, it has received

considerable attention, particularly in relation to its presumed nutri­

tional role in spore and pollen development (Mascre, 1919, 1921; Py, 1932;

Cooper, 1952; Takats, 1959, 1962; Knox, 1962; Heslop-Harrison, 1963, 1964;

Moss and Heslop-Harrison, 1967; Vasil, 1967; Echlin and Godwin, 1968;

Heslop-Harrison and Dickinson, 1969; and others). According to Davis

(1966), however, the tapeturn has not received the attention it deserves as far as its potential systematic importance is concerned. Partial infor­ mation is available for only 231 of the 411 angiosperm families recog­ nized by Hutchinson (1959).

One aspect which may be of taxonomic significance in certain taxa is

the number of nuclei per tapetal cell. In most angiosperm families, ta- petal cells become binucleate or multinucleate. In 26 families, however, species are reported in which the tapetal cells remain uninucleate

(Wunderlich, 1954; Davis, 1966). This condition may result from lack of nuclear division (Bonnet, 1912; Cooper, 1933; Carniel, 1952, 1954), from endomitosis or other related phenomena (Carniel, 1952, 1954, 1963; Turala,

1958, 1963; and others), or secondarily from nuclear fusion in tapetal cells that were binucleate or multinucleate at an earlier stage (Bonnet,

1912; Mascr/ and Thomas, 1930; Cooper, 1933; Smith, 1933; Steil, 1935,

1950; Roever, 1935; Brown, 1949; Francini, 1951; Carniel, 1952, 1954,

1963; Wunderlich, 1954; Nair and Kahate, 1961). 2

Of the 26 families of angiosperms with uninucleate tapetal repre­

sentatives, 15 are reported to be always uninucleate, whereas the remaining

11 families contain some uninucleate taxa as well as some which are bi-

nucleate or multinucleate. Of the latter 11 families, three deserve

special mention; Boraginaceae, Orchidaceae and Leguminosae.

Svensson (1925), Strey (1931) and Millsaps (1940) reported that the

tapetal cells of Cynoglossum of the tribe Cynoglosseae (Boraginaceae) were

uninucleate, Millsaps further suggested that this nuclear condition might

be a tribal character, since in the other tribes there were two or more

nuclei per tapetal cell. Unfortunately, this conclusion was based on

the study of only about five species.

In the Orchidaceae, according to Swamy (1949), "The tape turn is of the

secretory type and its cells are uninucleate in all monandrous orchids.

A binucleate type is observed only in Paphiopedilum (Diandrae)," Francini

(1931) and Carniel (1952) also observed only multinucleate tapetal cells

in this genus. Again, there are too few observations for a convincing

generalization to be made.

In the Leguminosae, however, a significant tapetal pattern, based on

considerably more published observations, seemed to be indicated. After

examining 43 species from 24 families of angiosperms, Cooper (1933) pro­

posed three tapetal groups; 1) uninucleate cells; 2) binucleate cells;

3) plurinucleate cells. In group 1, Cooper listed the five species of

legumes he examined. All were in the subfamily Papilionoideae. Previously,

Maheshwari (1931) examined the mimosoid Albizia lebbek, which was 3

uninucleate at all stages. In the caesalpinloid Cassia didymobotrya,

however, Sethi (1930) reported tapetal cells with two to four nuclei.

Newman (1933, 1934) later reported a uninucleate tapeturn in Acacia

baileyana, an additional mimosoid.

Referring to these previous studies, particularly Cooper's classi­

fication of 1933, Newman (1934) commented, "It is interesting to find that

species of the Papilionaceae and Mimoseae are associated in group 1, while

a Caesalpineous species is in group 3, otherwise than might be expected

in view of the floral forms of these subfamilies."

A few other authors have commented specifically on the occurrence of

a constant tapetal nuclear number in the legume subfamilies. Dnyansagar

(1949, 1955) and Maheshwari (1950) both reported that the species of the

subfamily Mimosoideae which had been examined were uninucleate throughout

floral development. According to Pantulu (1942), who summarized some of

the available information on papilionoid legumes, "Uninucleate tapetal

cells, therefore, appear to be a common feature of the Papilionaceae."

The first author, however, to actually suggest that the tapetal nuclear number could be of systematic value was Tlxier (1965), who had

studied chromosome numbers and tapetal nuclear behavior in some legumes from Vietnam and Laos. He stated that, "According to the cytotaxonomic

plan, the number of nuclei in the tapetal cells and their change in nuclear

type and chromosome number may furnish a supplementary cytotaxonomic character."

In addition to the above references, there are several others which mention the tapetal nuclear number in legumes, usually very briefly and 4

superficially. In most studies, however, tapetal information is complete­ ly lacking. Of the approximately 600 genera and 13,500 species, less than ten percent (51) of the genera and one percent (88) of the species had been examined up to 1965. Although a small sample, it did indicate that the tapetal nuclear number might be of phylogenetic significance, particularly at the subfamily level.

This study was undertaken with the primary objective of extending observations on the tapetum in the three subfamilies. The hypothesis to be tested was that a multinucleate or binucleate tapetum is character­ istic of the Caesalpinioideae and a uninucleate tapetum of the Mimosoideae and Papilionoideae. 5

LITERATURE REVIEW

Tapetal Classification

Goebel (1901) first established tapetal types based on tapetal be­

havior during pollen formation. Although not the first to observe dif­ ferences in tapetal behavior, Goebel was the first to recognize the need

for classifying the different conditions which he and others had observed.

He defined two major tapetal types, plasmodial and secretion (secretory),

between which he admitted that there are a number of transitions.

According to him, in the plasmodial type (sometimes called the amoeboid

type—Pickett, 1916; Schnarf, 1923, 19 29; Davis, 1966; and many others) the cell walls are disrupted and the protoplasm oozes out among the sporocytes and developing spores to be utilized by the spores or pollen grains.

This type of tapetum, considered by Goebel to be typical of ferns,

Equisetum and the seed plants, had been observed in other plants by earlier workers (Strasburger, 1882; Campbell, 1897, 1900; Caldwell, 1899;

Duggar, 1900).

In contrast, Goebel described cells of the secretory tapetum (glandular type of some authors--Maheshwari, 1950; Hindmarsh, 1964; Davis, 1966; others), as remaining in place and continuing to "excrete" soluble sub­ stances into the (anther) fluid until final dissolution at the time of pollen maturity, Goebel felt that this type of tapetum was restricted to

Selaginella, Lycopodium and Isoetes. 6

Hannig (1911) reviewed the previous literature on the plasmodial

tapetum. Although he accepted the classification of Goebel, he intro­

duced the term "periplasmodium" to describe the actual protoplasmic mass.

It was not his intention to designate a new capetal type. After Hannig's review, however, many investigators adopted the term periplasmodium as a new type instead of merely applying it to the protoplasmic mass. Thus,

plasmodial tapetum and periplasmodial tapetum have become two different types when they should be synonymous.

Tischler (1915) described a periplasmodium in the Commelinaceae and also found it to be common in the Araceae and other families of the monocotyledonous order Helobiales. On the basis of these findings he suggested that this type of tapetum was of systematic value in the mono­ cotyledons. While he recognized that there were minor differences in the appearance of the periplasmodium in different plants, he did not classi­ fy these subtypes.

Clausen (1927), mainly on the basis of the work of Tischler and previous authors, who had examined a number of monocots, divided the plas­ modial tapetum, which he referred to as a periplasmodium, into four sub­ types, which differed from each other only in time of formation or number of nuclei,

Juel (1915) examined a number of dicotyledons and concluded that the secretory tapetum was not only more common in this group, but also in the angiosperms in general.

Most recently, in a comprehensive review on angiosperm embryology that also included information on tapetal types, Davis (1966) recognized 7

only the two original tapetal types of Goebel (1901), while admitting that there might be intergrading subtypes. She supported the conclusion of Juel (1915) that the secretory tapeturn was more common. Of the 231 families for which she had information, 181 (mostly dicotyledons) re­ portedly had only the secretory type, 29 families had only a plasmodial tapetum, and 21 fa'^tlies had both types represented.

A slightly different approach to tapetal classification was taken by

Cooper (1933). Based on his own observations, which included mostly dicotyledons, he proposed three groups of tapetal cells; 1) uninucleate;

2) binucleate; 3) plurinucleate. He did not indicate whether the tapeta were of the secretory or plasmodial type, but his observations were made on cells from all stages of anther development, suggesting that they re­ mained intact during their entire development (i.e., cellular). His classification, based on nuclear number, deviates from those of previous authors, but has merits that are discussed later.

Also based on tapetal nuclear characters is the classification of

Garrigues (1951). He believed that it was necessary to compare the ploidy level of tapetal cells rather than merely nuclear number. He argued that most tapetal cells were octoploid and could be divided into three groups;

1) cells with one 8n nucleus; 2) cells with two nuclei; 3) a tapetum composed of cells, some with an 8n nucleus, others with two 4n nuclei or with four-2n nuclei. This classification has not received much recogni­ tion and is frequently untenable because one species may show all three types, or various intermediates, depending on degree of endomitosis or nuclear fusion (Carniel, 1952, 1963; D'Amato, 1952b). 8

In two extensive investigations on the anther tapeturn of angio-

sperms, Carniel (1952, 1963) proposed a phyletic scheme of the origin

of the various tapetal types and subtypes (Figure 1). He recognized two

basic types, true periplasmodial and cellular. The true periplasmodial

was equivalent to the plasmodial tapeturn of Goebel (1901). A sharp dis­

tinction was made, however, between a true and a false periplasmodium.

The latter condition he did not consider a tapetal type, but rather a

tapetal stage resulting from the premature degeneration of a cellular

tapeturn.

Carniel considered his concept of a cellular tapeturn as equivalent

to Goebel's secretory tapeturn and divided it into two subtypes, uninucleate and multinucleate. These subtypes are comparable to groups 1 and 3 of

Cooper (1933). Carniel further subdivided the multinucleate category into two classes based on nuclear behavior. In one class, the tapetal cells remain in a permanent anaphase-like figure following raeiosis in the pollen mother cells (PMCs). In the other class, the tapetal nuclei remain in a prophase or raetaphase-like stage, referred to as "arrested prophase".

Wunderlich (1954), who accepted Carniel's scheme, reported further on the nuclear condition of tapetal cells. She also presented a list of those species in which the tapetal cells reportedly became secondarily uninucleate by fusion or incomplete mitosis (see also Bonnet, 1912;

Cooper, 1933; Steil, 1935, 1950; Carniel, 1952, 1954, 1963). 9

With regard to the type of tapeturn in the Leguminosae, Davis (1966) has said that most of the legumes have a secretory or cellular tapetum.

In the Caesalpinioideae, however, Venkatesh (1956b) reported and figured a plasmodial tapetum in three species of Cassia. Lens esculenta (Shukla,

1954) and Adenanthera pavonica (Dnyansagar, 1954b) were reported to have intermediate tapetal conditions. In Lens, the walls of the tapetal cells dissolved, but the cellular contents did not wander among the microspores.

They formed instead a peripheral Plasmodium (periplasmodium). In Adenan­ thera, the cells retained their walls, but were separated one from another and wandered among the microspores, which they continued to nourish.

Several investigators have mentioned, usually without figures or dis­ cussion, either a plasmodial or periplasmodial tapetum in other legumes

(Corti, 19461 Larson and Lewis, 1963; Chubirko, 1967). Most of these latter reports are subject to some doubt, since the same species which they describe as plasmodial or periplasmodial have been reported to be of the secretory (cellular) type by other researchers. In these cases, fur­ ther observations are necessary to clarify the matter, since as Carniel

(1952, 1963) suggested, a false periplasmodium can be formed by degenera­ tion of the cellular tapetum at any stage of development.

Tapetal Nuclear Number in the Leguminosae

As mentioned previously, tapetal classification based on nuclear number (Cooper, 1933) has merit, at least in a few plant groups. In the

Leguminosae, according to Wunderlich (1954), Davis (1966) and Dnyansagar 10

(1949, 1955), the tapeturn is uninucleate except for the subfamily Caesal-

pinioideae, which may be binucleate, multinucleate, or rarely uni­

nucleate.

Of the studies reported in the literature, most have been super­

ficial and unsupported by photographs, although some have shown drawings.

Only the more important papers concerned with the tapetum of legumes are

cited below. A more complete list of species and investigators is given

in Appendix A, Charts 1, 2, and 3.

Mimosoideae

Of the approximately 45 genera and 2,100 species recognized, in

only 21 species of 13 genera is there any information on the tapetum.

The first report on number of nuclei per tapetal cell was by Mahesh-

wari (1931), who found a consistently uninucleate tapetum throughout

anther development in Albizia lebbek. Newman (1933, 1934), who reported

the same condition in Acacia baileyana, was the first to comment on differ­

ences in tapetal nuclear number in the family (see quote on p. 3).

One investigator has contributed most of the observations on the

tapetum in this subfamily (Dnyansagar, 1949, 1951a,b, 1952, 1954a,b, 1955,

1957, 1958). In describing the embryology of 12 species in 11 genera

(Appendix A, Chart 1), he reported the tapetum to be uninucleate in all

cases. Similar reports have been given by Singh and Shivapuri (1935),

Narasimhachar (1948, 1951) and Mulay and Dhami (1955).

Tixier (1965) examined nine species of mimosoid legumes, but he noted

tapetal nuclear behavior in only four taxa; Leucaena glauca, Enterolobium 11

cyclocarputn. Mimosa invisa and Pithecolobium dulce. All were reported

to be uninucleate.

This large subfamily, with approximately 125 genera and 2,100

species, has received the least attention with regard to the tapeturn. In­

formation is available for only five genera and 22 species, of which 17

are in Cassia.

Sethi (1930), who examined Cassia didymobotrya, was the first in­

vestigator to report more than one nucleus per tapetal cell in a legume.

This species, as well as C, purpurea (Chose and Alagh, 1933) and C. tora

(Datta, 1934), contained two to four (occasionally six to seven) nuclei

per tapetal cell.

For almost twenty years there were no further reports, until Carniel

(1952) described tapetal cells in Cassia austrails and C. laevigata. These

were mainly binucleate, but some tapetal cells were uninucleate (with a

distinctly larger nucleus), while others contained three or four nuclei.

Most of the species in this subfamily for which information is avail­

able were studied by Venkatesh (1956a, b, 1957), who reported on Cassia,

subgenera Fistula, Lasiorhegma and Senna. Of the 13 species he examined

(Appendix A, Chart 2), all but those of the subgenus Lasiorhegma (C. absus,

C. kleinii, and C. mimosoides) were binucleate or trinucleate. These

three species, considered highly specialized on the basis of other charac­

teristics (Bentham, 1871; Irwin, 1964), were uninucleate and possessed

a plasmodial (amoeboid) tapeturn. 12

Another exception to binucleate tapetal cells was reported by Tixier

^ (1965), who found only uninucleate cells in Poin ci ana regia (Delonix

regia). He showed drawings in which tapetal nuclei resembled prophase-

like images, but he did not mention the type of tapetum present.

The first photograph (and the only one known to me in this subfamily)

to show tapetal cells and tapetal nuclei was by Francini (1951), who

examined Ceratonia siliqua. He reported binucleate tapetal cells, except

for a few rare uninucleate cells, which he said were the result of nuclear

fusion.

In Caesalpinia crista (Mukherjee and Thakur, 1959) and C. pulcherrima

(Tixier, 1965), the tapetal cells are reportedly binucleate. In the

latter species, Tixier also reported the appearance of raetaphase-like

images at the stage when the microsporocytes had completed meiosis. This

condition persisted until final dissolution of the tapetal cells,

Nair and Kahate (1961) examined Parkinsonia aculeata, which they

reported possessed a secretory tapetum with two to five nuclei per cell.

In a fine structure study on pollen wall development in this same species,

Larson and Lewis (1963) mentioned, but did not illustrate, a periplas-

modial tapetum. Neither did they mention the nuclear number of the

tapetal cells.

Papilionoideae

Although considerable research has been conducted in this largest

and most important subfamily (about 400 genera and 9,200 species), few

investigators have dealt with the tapetum. There are published records

for only 45 species from 33 genera (Appendix A, Chart 3), 13

The first report on tapetal nuclear number in a papilionoid legume, and also the first in any member of the Leguminosae, was by Castetter

(1925), who reported uninucleate tapetal cells in both annual and bi­ ennial varieties of Melilotus albus. Shortly after, Latter (1926) and

Weinstein (1926) independently reported the same condition in Lathyrus odoratus and Phaseolus vulgaris, respectively.

Of the 43 species of angiosperms examined by Cooper (1933), five were papilionoid legumes (four species of Medicago and two varieties of

Melilotus albus). The tapeturn was uninucleate in all during their de­ velopment.

There have been two somewhat dubious reports of binucleate tapetal cells in this subfamily. Cooper (1938) showed a drawing representing the tapeturn of Pisum sativum with a cell in nuclear division; however, only one division figure is present. Furthermore, it is uncertain whether this division would give rise to a binucleate tapetal cell or merely a second uninucleate tapetal cell. lijima (1962) supposedly showed a binucleate tapetal cell in Vicia faba, but the photograph is of such poor quality that no conclusion can be drawn from it. Carniel (1952) examined these same two species, as well as a number of other papilionoid legumes (Carniel,

1954, 1963), and reported only uninucleate tapetal cells.

In a study of microsporogenesis in Glycine max cv. Biloxi, Nielsen

(1942) showed what seems to be the first photograph of tapetal cells in a legume. Even at the tetrad stage, the tapetal cells had only a single nucleus per cell. 14

Pantulu (1942) reported uninucleate tapetal cells in Desmodium

gangeticum, Cyamopsis psoralioides and Tephrosia purpurea. He claimed

that this condition was "always" found in the Papilionoideae. In a more recent study, which included information on the tapetum in two species of Desmodium, Buss, Galen and Lersten (1969) described, with the aid of photographs, the development of a uninucleate cellular or secretory tapetum.

Somatic Polyploidy

Somatic polyploidy is the multiple replication of chromosomal

material within a normally diploid cell. Usually this is accomplished

by an increase in the number of nuclei, some or all of which may later

fuse, natural chromosome doubling or by an increase in the volume of

chromatin material within an intact nucleus of a nondividing cell. At

least in higher plants, cells and tissues with quite varied degrees of

differentiation and function are polyploid (D'Amato, 1952a,b; Chiarugi,

1954; Tschermak-Woess, 1956).

The anther tapetum, according to Ranquini (1928) and Brown and Bertke

(1969), is virtually always polyploid. Strasburger (1882) observed oc­

casional fusion of nuclei in the usually binucleate tapetal cells of

several members of the Liliaceae. Since that time, there have been many

additional reports of fusion; also other processes which lead to poly­

ploidy in this tissue have been described (D'Amato, 1952b). These

processes can be divided into three classes; 1) normal mitosis without

cytokinesis; 2) abortive mitosis and fusion of nuclei; 3) endoduplication

or supernumerary chromonemal reproduction. 15

Of these three classes, mitosis without cytokinesis, i.e., forma­

tion of binucleate or multinucleate cells, appears to be most common in

the tapetum of angiosperms (Wunderlich, 1954; Davis, 1966). Various in­

vestigators have attempted to account for continued mitoses in the tapetum.

Davis (1961) suggested that they might be the result of a "sympathetic"

reaction to the onset of meiosis. Garrigues (1951) postulated the oc­

currence of an "excito-mitotique" substance emitted by the mother cells

during the course of reduction division. So far, however, such a sub­

stance from the mother cells has not been found.

According to D'Amato (1952b, 1965), fusion of nuclei, lagging chromo­

somes and non-mitotic division of nuclei are frequent in ephemeral tissues

such as endosperm and tapetum. In the late nineteenth century, amitosis

or interphase fission was described as occurring in the tapetum.

Strasburger (1882), however, after reexamining his material, concluded

that the figures he had earlier interpreted as amitotic were merely closely appressed nuclei of regularly binucleate tapetal cells. Even­

tually some of the nuclei fused to form one, or occasionally more than

one, large nucleus. Tischler (1906) reported nuclear disturbances such

as fragmentation and lobing of tapetal nuclei in various Ribes hybrids.

He also concluded that amitosis did not occur in the tapetum. Still

later, other investigators found additional evidence of nuclear fusion and incomplete division in the tapetal cells of a number of plants

(Winkler, 1906; Bonnet, 1912; Mascre and Thomas, 1930; Cooper, 1933;

Smith, 1933; Roever, 1935; Steil, 1935, 1950; Davis, 1961). A special 16

type of incomplete division, chromosome "stickiness", was described by

D'Amato (1947) and Battaglia (1949). This often led to anaphase bridges

and eventually to restitution nuclei which were irregularly shaped and

polyploid.

The third class, endoduplication or supernumerary chromonemal reproduction (Lorz, 1947), i.e., intranuclear growth through chromonemal

or chromosomal multiplication without visible nuclear division, is sub­

divided into three subclasses; a) polyteny; b) polysomaty; c) endomitosis, including arrested mitosis. In each of these processes, the chromosomal material is somehow increased during interphase or early prophase without any nuclear division.

Polyteny, although not yet reported in the tapeturn, has been dis­ covered in the ephemeral embryo suspensor cells in Phaseolus coccineus and P. vulgaris (Nagl, 1962a, b, 1967, 1969). In this type of polyploidy the chromatin strands of each chromosome are repeatedly duplicated, but instead of separating they remain together, forming "giant" chromosomes like those found in the well known salivary gland cells of certain members of the insect order Diptera. The ploidy level in the suspensor cells may be as high as 4096. As our knowledge increases, polyteny will probably be found to occur in the tapeturn also.

The details of polysomaty are not understood, but it is believed that during interphase or early prophase the chromatids of each chromosome replicate twice for each mitotic cycle (Lorz, 1947; D'Amato, 1952b;

Brown and Bertke, 1969). This produces the so called "diplochromosomes" 17

or "quadruple chromosomes", four or eight chromatids joined side by side

(Witkus and Berger, 1947; Avanzi, 1950). According to D'Amato (1965) and

Brown and Bertke (1969), this type of endopolyploidy regularly occurs in

tapetal cells of Spinacia, Cannabis and Allium. Favarger (1944) also

reported paired chromosomes in tapetal cells of six members of the

Caryophyllaceae. Brown and Stack (1968) reported that in Haplopappus

gracilis (2n=4) tapetal cells have 2n=8 and 2n=16 metaphase chromosomes

as they undergo the last two regular nuclear divisions.

The third subtype, endomitosis, appears from the literature to be

more common in the tapeturn than the preceding two subtypes. This process

was first reported in water striders, the insect genus Gerris. Geitler

(1939) described a series of stages, endoprophase to endotelophase, within

an intact nuclear membrane. There were no strong metaphase contractions

and no anaphase spindles were formed to separate the chromosomes. Eventu­

ally at telophase the chromosomes, which were still relatively long,

separated simply by "falling apart" and the nucleus, now with twice as

many chromosomes, was reconstituted and reverted to interphase. With

each endomitotic division the chromosome number was doubled, but no

nuclear or cell division occurred.

Although Geitler is given credit for describing endomitosis, D'Amato

(1952b) pointed out that Winge (1917) had described much earlier a similar

series of mitotic stages in the tapetal nuclei of Humulus japonicus and

Atriplex littoralis.

Shortly after Geitler's discovery, two reports on endomitosis in

plants appeared (Witkus, 1945—Spinacia oleracea; Brown, 1949—Solanum 18

lycoperslcum). According to these authors, the first division in the

tapetal nucleus was usually a normal mitosis. This nuclear division,

however, was not followed by cytokinesis; therefore, the cells became

binucleate. Following this mitotic division, one or more endomitotic

divisions, described as of the Gerris type, occurred. These gave rise

to binucleate tapetal cells with 8n or 16n nuclei.

Other investigators later reported endomitosis in tapetal cells of

various plant families (Avanzi, 1950; Berger, Witkus and Joseph, 1951;

Mechelke, 1952; Scarascia, 1952; Carniel, 1952, 1954, 1963; Steffen and

Landmann, 1958; Turala, 1958, 1963; Turala and Urbaéska-Worytkiewicz,

1964; others).

Some of these reports have been challenged, Carniel (1952), Geitler

(1953) and Tschermak-Woess (1956) denied that Witkus (1945), Brown (1949),

Avanzi (1950), Berger et al. (1951) and Scarascia (1952) had described

true endomitosis, since a regular mitotic division usually preceded the

so called endomitotic divisions. Also, the chromosomes were more highly contracted than "typical" endomitotic chromosomes, and occasionally there were rudimentary, but defective, spindles. Furthermore, in Solanum lycopersicum (Brown, 1949), the nuclear membrane and nucleolus both dis­ appeared during endomitosis, Carniel, Geitler, and Tschermak-Woess inter­

preted the process instead as inhibited or arrested mitosis. Carniel

(1952) claimed that the chromatin material was inhibited at prophase of mitosis, and applied the term "prophasich gehemmte Mitose" (arrested prophase). 19

Mechelke (1952) studied the tapeturn in Antirrhinum majus and several mutant forms and described different degrees of inhibited mitosis. Prior

to meiosis in the pollen mother cells, some of the tapetal nuclei under­ went a normal mitosis with unusually contracted chromosomes, which re­ sulted in binucleate cells. During mitosis, these tapetal nuclei showed various stages of arrested mitosis, from prophase to telophase, within the intact nuclear membrane. This suggested endomitosis, but rudimentary non-functional spindles were present. He occasionally observed chromo­ somes and nuclei which, because of their large size, were considered highly polyploid. These he attributed to a "Polymermitose" in which the chromosomal material was replicated many times without any visible divi­ sion. Carniel (1963) considers the "Polymermitose" to be equivalent with his metaphase arrested mitosis, and not a case of polyteny.

In some plant tissues, including the tapetum, normal dividing chromo­ somes are invisible. Often these nuclei contain heterochromatic bodies which resemble diakinesis or metaphase chromosomes. These heterochromatic bodies, first described in roots, are usually referred to as "prochromo­ somes" (Overton, 1905) or "chromocenters" (Baccarini, 1908; Gregoire,

1931; Eichhorn, 1930, 1935), Often there are as many as there are chromosomes; however, they may be fewer than the somatic number (Turala,

1958, 1963), Occasionally as a result of fusion of nuclei or internal increase in the number of chromosomes, the number of chromocenters will be greater than the somatic number. Because of this number correlation, some investigators have suggested that each chromosome is represented by a heterochromatic region (Overton, 1905; Levan and Hauschka, 1953), 20

These two terms have been applied to the nuclear material of non-

dividing tapetal cells. Most authors use the term chromocenter (Carniel,

1952, 1963; Turala, 1958, 1963; Turala and Urbanska-Worytkiewicz, 1964;

others); however, according to Brown and Bertke (1969), the term pro­

chromosome should apply when the number of chromocenters is the same as

the number of somatic chromosomes. In Plantago ovata (Stack and Brown,

1969) the prochromosomes, like the regular chromosomes, increased in

number, but always in exact multiples of the base number.

An increase in size or number of prochromosomes or chromocenters is

usually taken as evidence for endopolyploidy, particularly that due to

endomitosis (Turala, 1958; Carniel, 1952).

There are still differences of opinion as to the precise meaning of

the term endomitosis and what intranuclear phenomena are. Even Geitler

(1953) admitted that endomitosis might be regarded as an arrested mitosis.

With respect to the processes leading to polyploidy in the tapetal cells of the Leguminosae, mitosis without cytokinesis and endopolyploidy, attributed to endomitosis, arrested prophase and polysoraaty, all have been reported. So far, polyploidy by means of continued mitosis is known only in the subfamily Caesalpinioideae, Here, binucleate and multi­ nucleate tapetal cells have been reported in almost all of the genera and species examined (Sethi, 1930; Chose and Alagh, 1933; Datta, 1934;

Francini, 1951; Carniel, 1952; Venkatesh, 1956a, 1957; Mukherjee and

Thakur, 1959; Nair and Kahate, 1961; Tixier, 1965), Except for two dubious reports (Cooper, 1938; lijima, 1962), the Mimosoideae and 21

Papilionoideae are always uninucleate (Pantulu, 1942; Dnyansager, 1949;

Maheshwari, 1950). In these two subfamilies polyploidy, when present, appears to be due to endomitosis, arrested prophase or polysomaty.

The only known reports of these types of endopolyploidy in legumes are those by Carniel (1952, 1954, 1963) and Tixier (1965). Carniel

(1952) described arrested prophase in the binucleate tapetal cells of

Cassia australis and C, laevigata. In both, the chromosomes were repre­ sented by large distinct chromocenters. These resembled late prophase or metaphase chromosomes. Carniel also reported significant differences in the size of tapetal cells and chromocenters. From cytological measure­ ments he was able to determine that some of the nuclei were 4n, others 8n and a few 16n.

Carniel (1952) reported endomitosis as occurring in the tapetal cells of two representatives of the uninucleate Papilionoideae (Lupinus polyphyllus and L. regalis). Later (Carniel, 1954, 1963), he extended observations on endomitosis to Lupinus angustifolius and Lathyrus vernus.

In all four species the nuclear volume was increased several times while the number of chromocenters, most of which were prominent, remained con­ stant. In Lupinus angustifolius, the nuclear material was not in discrete chromocenters. It more nearly resembled an early prophase nucleus with thin chromatin strands. Buss et al. (1969) showed similar tapetal cells in two species of Desmodium, In Vicia faba (Carniel, 1952) the tapetal nuclei contained distinct chromosomes which were like diplochromosomes or quadruple chromosomes resulting from polysomaty. 22

The only other report of endomitotic activity in the tapeturn of legumes is by Tixier (1965), who reported "...a tendency toward endomitosis or polysomaty..in Enterolobium cyclocarpum, Pithecolobj-um dulce, Mimosa invisa and Leucaena glauca (Mimosoideae). He did not elaborate, but did comment that this condition was not observed in other legumes. He briefly described the formation of lobed nuclei, claimed to be octoploid, in the binucleate tapetal cells of Caesalpinia pulcherrima.

He also showed drawings of tapetal nuclei of Delonix regia and £, pulcherrima with distinct chromocenters.

Crystals in Tapetal Cells

Crystals in tapetal cells have only rarely been reported in the literature, mostly in the Commelinaceae. Mascre (1925), Maheshwari and

Singh (1934), Carniel (1952) and Yasui (1952) reported calcium oxalate raphide crystals in the periplasmodial tapetum of Tradescantia virginiana.

Commelina benghalensis, Spironema fragrans and Tradescantia sp., re­ spectively. In two studies on microsporogenesis in Tradescantia paludosa.

Walker (1957) and Walker and Dietrich (1961) showed, without any mention or discussion, photographs (Figures 8-10, 12, 18, 24) of meiotic stages, in which large needle-like structures could be seen. These are probably raphide crystals from the periplasmodial tapetal cells. The only other reports of tapetal crystals are by Stejskal-Streit (1940), who described small calcium oxalate crystals in the form of tetragonal pyramids, oc­ curring two to four per cell, in Salvia verticillata (Labiatae); and by 23

Buss et al. (1969), who described and showed photographs of small discoid to rhomboid prismatic crystals in two species of Desmodium (Leguminosae).

Although not truly tapetal crystals, Heslop-Harrison (1963) reported the accumulation of crystalline particles, visible in the light micro­ scope, within plastids in Cannabis tapetal cells. These were thought to be storage proteins, since they were partially mobilized as the tapeturn degenerated. In addition to the above reports of crystals, there are a few descriptions of non-crystalline tapetal inclusions (Latter, 1926;

Wunderlich, 1954). 24

MATERIALS AND METHODS

Plant Materials

The Leguminosae is the third largest family of flowering plants, with approximately 600 genera and 13,500 specie^ divided into the sub­ families Mimosoideae, Caesalpinioideae and Papilionoideae. In this study

the classification of Taubert (1894) was followed, except that in the

Caesalpinioideae, the tribes Kramerieae and Tounateae have been removed, and in the Papilionoideae, the Swartzieae (Bentham and Hooker, 1865) and

Taubert*s subtribe Psoraliinae of the tribe Galegeae, now recognized as a distinct tribe, the Psoraleae, have been added.

In order to supplement the rather small number of species locally available, and to represent as many tribes and genera as possible, seeds of many species from various sources (Appendix C) were obtained. These were planted in plastic pots in a greenhouse of the Department of Botany and Plant Pathology at Iowa State University. No attempt was made to con­ trol the prevailing light and temperature conditions. Preserved flower buds generously supplied by various collectors (Appendix C) were also used.

Tapetal type, number of nuclei and other characteristics were observed from materials which were prepared in the following manner.

^Hutchinson (1964), in the introduction to the order Leguminales, gives the number of genera as about 570. However, the number of genera listed for each subfamily in the text adds up to 690 and the number of species approaches 18,000. These figures (Isely, 1969; personal com­ munication) are ..."probably moderately excessive." At the other extreme, Taubert (1894) gives a conservative number of about 450 genera and about 9,000 species. The figures given here are therefore a compromise. 25

Microtechniques

Paraffin preparation

As the plants began to flower, inflorescences or individual flower

buds were fixed in the field or brought to the laboratory. They were

placed in either CRAF III (Sass, 1958) or 10% acrolein (Feder and O'Brien,

1968), then aspirated until they sank.^ Following fixation for 24 hours,

the buds in CRAF III were dehydrated at room temperature using a tertiary-

butyl alcohol (TBA) dehydration series (Sass, 1958), gradually infiltrated

with 61°C Tissuemat (three changes) and embedded in fresh Tissuemat,

Although CRAF III was usually a satisfactory fixative, the best re­

sults were obtained using the 10% acrolein solution (24 hours at 4°C)

with a modified Feder and O'Brien (1968) dehydration schedule. Dehydra­

tion was carried through n-butyl alcohol, after which the material was

transferred to 100% TBA at room temperature and then processed as given

above.

Sections were cut at 7 - 10 pm on a Spencer.AO "828" rotary micro­

tome using a steel knife. The ribbons were floated on 1% formalin solution

over a thin layer of Haupt's adhesive (Johansen, 1940) on a glass slide.

The slide was then placed on a slide warmer until the ribbons were fully

expanded. The excess formalin was then removed and the slides allowed to

dry overnight prior to staining.

2 Flower buds collected by Richard W. Pohl, Gerrit Davidse and Richard Maxwell were preserved in Newcomer's fixative (Newcomer, 1953), Those fur­ nished by Duane Isely, Donald F. Galen, Nels R, Lersten, Don Windier and Roberta Waddle were preserved in FAA (Appendix C), These materials were fur­ ther processed in the same manner as described for the CRAF fixed material. 26

Of the several stain combinations used, including Heidenhain's

iron-hematoxylin (Johansen, 1940), Johansen's safranin (Johansen, 1940)

and self-mordanted safranin (Gray and Pickle, 1956), Gray and Pickle's

safranin followed by fast green counters tain was best.

Slides affixed with paraffin sections were mounted permanently in

piccolyte, A small lead weight was placed on the coverglass and the slide

was left on a slide drying plate for approximately 10 days or until dry.

Anther squashes

The preparation of stained paraffin sections is time consuming, and

considerable detail of tapetal cells is lost. To determine tapeta1 nuclear

number and nuclear behavior in a large number of species, squashes were

made of whole anthers. Individual anthers were removed from buds which had

been fixed in 10% acrolein and dehydrated in two changes ©f methyl cello- solve, They were placed in a drop of 45% propio-carmine stain on a slide,

A coverglass was added and the anther carefully "squashed" by gentle tap­

ping using the tip of a dissecting needle. To intensify the stain, the

slide was passed through the flame of an alcohol lamp (taking care not to

boil the stain) and then further "squashed" between two paper towels, making certain that the pressure was applied uniformly on the coverglass.

Slides which contained suitable material were photographed immediately, or made permanent by the freezing technique described by Bowen (1956), For comparative reasons buds at or near the tetrad stage were selected. 27

Photographie Techniques

Photomicrographs were made on Kodak Panatomic-X 35mm film using a

Leitz Ortholux microscope fitted with an Orthomat camera. Contrast was enhanced by using a green (Wratten 58) photographic filter. For the photographic identification of crystals, both analyzer and polarizer discs were used.

Developmental Studies

To follow tapetal cell ontogeny, representative species from each subfamily were selected for developmental studies. Those selected were;

Desmanthus depressus (Mimosoideae), Cercis canadensis and Ghamaecrista fasciculata (Caesalpinioideae), Desmodium glutinosum, D. illinoense and

Psoralea tenax (Papilionoideae).

Desmanthus depressus was selected because it lacked compound pollen grains or pollinia and because, unlike most other mimosoids examined, it had tapetal cells that remained in arrested prophase. Cercis canadensis, a locally abundant perennial tree, showed a typical tapeturn (mostly binucleate with only occasional multinucleate cells) and Ghamaecrista fasciculata, a common herbaceous annual, showed an advanced character, a uninucleate plasmodial tapeturn, Psoralea tenax, a herbaceous perennial, and the two species of Desmodium, both herbaceous perennials, were selected to show the extremes of tapetal nuclear activity in this sub­ family and for the development of crystals in the tapetal cells. 28

OBSERVATIONS

3 Observations on tapetal type (cellular or plasmodial), number oi nuclei per cell, nuclear behavior and distribution, and morphology of crystals in the tapetum were made on 167 species in 89 genera distributed among the three subfamilies. For comparison, all observations were made

on tapetal cells at the late tetrad or microspore stage. Earlier

(pretetrad) and later (pollen grain) stages, in some instances, are also included. These observations, with similar information from the litera­ ture where available, are summarized in Appendix A, Charts 1, 2, and 3.

Tapetal Types in the Leguminosae

All species examined in the Mimosoideae and Papilionoideae, and all but six species in the Caesalpinioideae, possessed a cellular (secretory or glandular tapetum which remained intact until the microspore stage, or sometimes even until after pollen formation. After that time the tapetum rapidly degenerated. In the remaining six caesalpinioid species

(four of Cassia and two of Chamaecrista^) the tapetal cells appeared to lose their walls sometime during the sporogenous or early sporocyte stage and separated from each other to form a Plasmodium (periplasmodium) or amoeboid mass, which oozed among the developing microspores (Figures

52-60).

^Based on Carniel (1952), I consider the cellular tapetum to be syn­ onymous with the secretory tapetum. This term also indicates the major difference between this type of tapetum and the plasmodial tapetum,

^Chamaecrista is sometimes considered merely a section within Cassia. I consider it as a separate genus, based on a treatment by Isely (1958). 29

Tapetal Nuclear Number and Nuclear Behavior

With regard to number of nuclei in tapetal cells, the species of

Mimosoideae and Papilionoideae examined were all uninucleate, while the

Caesalpinioideae, with some exceptions discussed below, were always bi-

nucleate or multinucleate. Concerning the nuclear behavior, I observed

that some tapetal cells had nuclei which were sometimes two or more times

larger than the rest. Also, in some species, particularly those with uni­ nucleate tapetal cells, the nuclear material was represented by chromo- centers instead of chromosomes. In other species, the nuclei lacked chromocenters. Such cytological phenomena, according to some investi­ gators (Carniel, 1952; Turala, 1958, 1963), are often associated with endopolyploidy. Because these were incidental to the main study and be­ cause I did not conduct spectrophotometrie or other volumetric measure­ ments on the nuclei, the interpretations given here are based strictly on visual observations.

Because there is disagreement among cytologists on what nuclear phenomena properly constitute the endomitotic processes, I have chosen to recognize three levels of activity based upon the appearance of the tapetal nuclei: interphase, early prophase and late prophase.

Interphase nuclei—the nucleus, which may be either lightly or darkly stained, does not show any sign of organized chromocenters or chromonemal strands (Figures 95, 96, 114, 122).

Early prophase nuclei--the nucleus shows evident chromonemal strands interconnecting prophase-like chromatin, which is usually associated with the nuclear membrane (Figures 34, 35, 46, 77, 124, 153). 30

Late prophase nuclei—the nucleus exhibits distinct chromocenters which resemble diakinesis or raetaphase chromosomes. Nucleoli may or may not be visi­ ble (Figures 5, 6, 8, 9, 31, 105, 144-151, 186, others).

Mimosoideae

This subfamily, with about 45 genera and 2,100 species, is dis­

tributed mainly in the tropics and subtropics of the southern hemisphere.

This limited my investigation to seven genera and 16 species. These

represented, however, four of the six tribes recognized by Taubert (1894).

The tapeturn is always uniseriate and no differences between the inner and

outer tapeturn, like those described by Periasamy and Swamy (1966), were observed. In all taxa examined (Appendix A, Chart 1), there was only a single nucleus per tapetal cell. This agreed with all published reports.

To determine if a uninucleate tapeturn was constant throughout de­ velopment or merely the result of nuclear fusion, I traced the critical stages in Desmanthus depressus, an herbaceous annual. I was unable to determine the ontogenetic origin of the wall layers, but by the micro- sporocyte stage there are three wall layers external to the tapetum, which has been enlarging (Figure 2). Two rows of six to 12 pollen mother cells (PMCs) are produced in each locule (Figures 2, 3). In sectioned locules, the tapetal nuclei often stain so densely that much of the detail is obscured. In smear preparations at the sporocyte stage (Figures 4, 5), however, tapetal cells with a single nucleus and prominent chromocenters are obvious. The chromocenters remain until after the tetrads and in­ dividual microspores are released from their surrounding callose sheaths 31

(Figures 6, 8, 9). This peculiar nuclear behavior, interpreted as late

prophase, is similar to the "arrested prophase" described by Carniel

(1952) for Cassia australls and C, laevigata or the chromocenters of the

endomitotic nuclei of Lupinus po'lyphyllus, L. regalis and Lathyrus vernus

(Carniel, 1952, 1954), Microspore nuclei did not display similar chromo­

somal behavior.

Following meiosis II the microspores separated, became vacuolate

and underwent mitosis to form two-celled pollen grains (Figure 10). The

tapetum, although near collapse, remained until after pollen was fully

engorged with food reserves (Figure 11). Finally it disappeared completely.

The development of pollen and tapetum in Desmanthus depressus, with the

exception of chromocenters in the nuclei, was similar to that described

in the uninucleate papilionoid genus Desmodium (Buss et al., 1969).

The tapetum in Desmanthus is, therefore, a good example of a cellular

type of tapetum.

Although a uninucleate tapetum was constant in this subfamily,

other differences in the tapetum and in microspore formation were noted.

One point of interest was the occurrence in Prosopis juliflora of occasional

tapetal septa which bisected the locule (Figure 12), Similar tapetal septa in other Leguminosae have previously been described only in one other mimosoid, Dichrostachys cinerea (Dnyansagar, 1954a), and in three

papilionoid species, Lathyrus odoratus (Latter, 1926), Desmodium glutinosum and D, illinoenae (Buss et al., 1969).

With regard to nuclear behavior in tapetal cells, Desmanthus depressus,

D. illinoensis and Schrankia uncinata were the only species which had 32

tapetal nuclei at late prophase. Except for Calliandra confusa and Mimosa

sp., the remaining taxa had tapetal nuclei which remained at an early

prophase stage (Figures 12, 14-17). Other than the late prophase nuclei,

no evidence for endopolyploidy was observed.

Another difference observed was the occurrence of separate pollen

grains in Desmanthus depressus (Figure 11), D. illinoensis and Prosopis

juliflora (Figure 13). All of the remaining species examined had com­

pound pollen grains or pollinia made up of varying numbers of microspores

(four, eight or 16). Schrankia uncinata. Mimosa pigra, M. pudica and M. had pollinia with only four microspores (tetrads). In M. pudica, in addi­

tion to only four microspores, the tapetal cells were substantially larger

than individual microspores or entire pollinia and contained large prom­ inent vacuoles (Figure 14). Acacia, Albizia and Calliandra pollinia con­ tained either eight or 16 microspores, depending on the number of PMC s included in the locule or the number grouped together prior to meiosis.

In Albizia julibrissin the PMCs are in tetrads (Figure 15); following meiosis a polyad of 16 microspores was produced. Ten of these are shown in Figure 16. Similar polyads were observed in three species of Calliandra, while a fourth species, C. eriophylla, produced only eight microspores

(octad). In Albizia and Calliandra, the tapetal cells usually were smaller than the adjacent microspores. In the three species of Acacia, all with polyads, the tapetal cells varied in size, but in general were about the same size as the microspores. Two tapetal cells and two of the 16 micro­ spores (these were separated by squashing) are shown in Acacia sp. 33

(Figure 17), The most striking characteristic of this plant was the oc­ currence of crystals in the tapetal cells, the only mimosoid known to

possess them, either from personal observation or from the literature,

Caesalpinioideae

This subfamily, with about 125 genera and 2,100 species, also occurs mainly in the tropics and subtropics. Representatives of a few genera

(Cercis, Ghamaecrista, Gleditsia and Gymnocladus), however, occur in Iowa,

A total of 12 genera and 25 species were examined (Appendix A, Chart 2),

As noted previously (p, 28), both cellular and plasmodial tapeta occur in this subfamily. The cellular tapetum, however, is by far the most frequent. Only six highly advanced herbaceous species had a plasmodial

tapetum; these are discussed on pp. 38-39, Based on the,most frequent . number of nuclei per tapetal cell, three levels of the cellular tapetum were recognized; binucleate, multinucleate and uninucleate.

The tapetal cells in most members of this subfamily are binucleate, but occasional multinucleate or uninucleate cells occur along with them.

To determine when the tapetal cells became binucleate and whether they continued to divide during the meiotic cycle, I traced tapetal development in redbud, Cercis canadensis. Although I examined many anthers, I was not able to determine the exact time of division. However, shortly after tapetal cells and sporocytes could be distinguished, many tapetal cells were already binucleate (Figure 18), At this time there were three or four wall layers external to the tapetum (Figures 21, 23),

The anthers of Cercis are many times larger than those of Desmanthus 34

and contain numerous rows of PMCs (Figures 18, 21-23). In a smear

preparation at diakinesis (Figure 19), a tapetal cell is seen with two

relatively large nuclei, each containing several darkly stained prominent

nucleoli, not chromocenters, which persist throughout tapetal cell dif­

ferentiation. As the PMCs undergo meiosis, some tapetal cells also at­

tempt further mitotic division (Figure 20). No further divisions in the

tapetal cells occurred after meiosis II. Cytokinesis in these cells is

inhibited so that the tapetal cells contain two or occasionally more nuclei.

Sections of anthers at different stages of meiosis II (Figures 21-23)

do not reveal any changes in the tapetum. At the tetrad stage (Figure 23)

the endothecium is filled with dark staining material, possibly tannin.

In a smear preparation at about this same stage (Figure 24), the tapetal cells are densely granular and appear to be uninucleate. This condition is brought about by the peculiar crowding of the nuclei, which show only

light staining threads of chromonemata and large nucleoli, some of which may be the product of earlier nucleolar fusions. Each tapetal cell, how­ ever, contains two or more nuclei (Figure 25, arrows) which also may later fuse. Even at the late microspore stage (Figure 26), the tapetal cells with four to nine nucleoli are intact. These nuclei appear to be in a very early prophase stage, which is maintained until the tapetum finally

disappears.

Similar tapetal cells, that is, bi- to multinucleate with early prophase nuclei, were observed in Bauhinia variegata, Gleditsia triacanthos,

Gymnocladus dioica and Parkinsonia aculeata. Larson and Lewis (1963) 35

reported a periplasmodial tapeturn in Parkinsonia aculeata. Contrary to

this report, I observed throughout anther development, a cellular bi-

to multinucleate tapetum.

In Gleditsia triacanthos many, if not all, of the tapetal cells were

binucleate by early sporocyte stage (Figure 27), Occasional cells had more than two nuclei (Figure 27, arrows). Throughout the remainder of meiosis and until after microspore release (Figures 28-30), the tapetal cells showed distinct nuclei. At the microspore stage (Figure 30) there are two nuclei per cell, but the number of nucleoli has been reduced and only minimal evidence of chromonemata exists. This stage corresponds then to early prophase.

In Cassia bonariensis (Figure 31) the tapetal cells are mostly bi­ nucleate and each nucleus contains many small chromocenters (late pro­ phase), Occasionally a tapetal cell has only one nucleus, but with a volume of nuclear material comparable to those with two nuclei (Figure

31, lower right). This may be due to fusion of two nuclei, although no actual fusions were observed, or to inhibited division of this nucleus.

The upper cell resembles the so called endotelophase of Brown (1949),

Somewhat similar nuclei were observed in Cassia artemisioides (Fig­ ures 32-34) and C, 11225 (Figure 35), except that the nuclear material was not organized into distinct chromocenters. Rather, it was loosely arranged, like the chromatin of an early prophase nucleus. An enlargement of a single tapetal cell of C, artemisioides (Figure 34) reveals two nuclei which are slightly irregular and which may eventually "bud off" 36

to become three or more nuclei. The nuclei of C. s£. 11225 (Figure 35)

are even more extremely lobed; although each cell is basically "bi-

nucleate" the nuclei are in contact with each other. It is not known

whether these nuclei are fusing or whether they are separating. This

nuclear configuration resembles the fusion nucleus figured by Mechelke

(1952, Figure 39) in Antirrhinum.

The tapetal cells of Caesalpinia pulcherrima were unusual in their

early development. Instead of dividing prior to meiosis, the tapetal

cells remained uninucleate until approximately the beginning of meiosis

or even until later (Figures 36, 37), They rapidly completed one or more

nuclear divisions to become binucleate or multinucleate (Figures 38-39).

By the microspore stage, the tapetal nuclei appeared completely normal,

i.e., in prophase with small chromocenters (Figure 40). Two other

species, C. gilliesii and C. eriostachys, were similar in tapetal charac­

teristics.

Some species examined had tapetal cells which were mainly multi­

nucleate. The most interesting of these was Hoffmanseggia jamesii, a common weedy herbaceous species of the deserts of the southwestern United

States. Although I was not able to determine when the first division oc­ curred in the tapetal cells, many of them were already binucleate or even multinucleate by the PMC stage (Figure 41, arrows). This figure also shows a tapetal nucleus which has only recently undergone karyokinesis.

In an enlargement of tapetal cells similar to those of Figure 41, the nuclei appear to be "budding" or "pinching" off (Figure 42). Between 37

the sporocyte stage and microspore stage, additional divisions or

"nuclear buds" may occur, resulting in a large number of nuclei in some

tapetal cells (Figures 43, 45), while adjacent tapetal cells have only two or three nuclei (Figure 44). The tapetal cells still are intact even at the late microspore or early pollen grain stage. The maximum number of nuclei observed in this species was eight, but seven was more common (Figures 43, 45).

In Brownea sp., a tropical species, from two to eight .nuclei were also observed. Figure 46 shows a tapetal cell with five small nuclei and an adjacent tapetal cell with only one large nucleus. These two species so far represent the maximum number of nuclei known in the sub­ family.

Although most frequently the tapetal cells in the cellular Caesal- pinioideae are bi- to multinucleate, occasional uninucleate tapetal cells occur. Thus far, however, there has been only one report in which the tapetum was cellular throughout development and entirely uninucleate.

Tixier (1965), who examined Delonix regia, noted this seemingly rare condition. He did not elaborate, except to mention that this differed from Caesalpinia pulcherrima, which was constantly binucleate, I re­ examined Delonix regia to determine if the tapetum was uninucleate and if so, whether it might be associated with a plasmodial tapetum,

A portion of a longitudinal section of a locule of Delonix regia at the microsporocyte stage (Figure 47) shows a cellular tapetum with rather long, rectangular uninucleate tapetal cells at right angles to the locule. At the end of each locule and along the outer walls, particularly 38

at the center, two or occasionally three rows of tapetal cells, which vary in size, are present (Figure 48). In smear preparations at the tetrad stage (Figures 49, 50) mainly uninucleate tapetal cells are present.

The capacity of tapetal cells to undergo further division has not been entirely lost, because occasional cells show t\:o nuclei (Figure 49, small arrows). In a cross section .(Figure 51), differences in tapetum thickness and cell size are shown. This figure also shows callose be­ ginning to disperse. No nuclear divisions occurred after this stage.

The only report of a plasmodial uninucleate^ tapetum in the

Caesalpinioideae (Venkatesh, 1956b) was in three species of Cassia, sub­ genus Laslorhegma (£, absus, £, kleinii and £. mimosoides), Prior to meiosis the tapetum was cellular and uninucleate, but as the pollen mother cells underwent meiosis, the tapetum became plasmodial and surrounded the tetrads. The Plasmodium persisted until the pollen grains had rounded off, then was completely absorbed.

I observed a similar plasmodial tapetum in the following species;

Cassia leptadenia, C, mimosoides, C. s£, 1353, C. sp, 11222, Chamaecrista fasciculata and £. stenocarpa. Of these six species, Chamaecrista fasciculata (Figures 52-57) and Cassia mimosoides (Figures 58-60) re­ ceived the most attention.

The time of plasmodial formation in £, fasciculata differed from that reported by Venkatesh (1956b). When the tapetum first became distinguish-

Plasmodium by definition infers a coenocytic or multinucleate con­ dition. As applied here, the tapetum was uninucleate prior to structural modification. Since divisions were not observed, it is assumed that the cellular fragments remain uninucleate. 39

able it was cellular and uninucleate. Later, as the tapetal cells en­

larged, the walls seemingly became modified so that by the sporocyte

stage the tape turn lost its shape and formed a Plasmodium which surrounded

the sporocytes (Figure 52). At this stage the Plasmodium was very vacu­

olate. In sectioned material, individual cells were not distinguishable,

so the tapetum appeared coenocytic. However, in a smear preparation just

prior to diakinesis, cells with only one nucleus were seen (Figure 53).

As meiosis continued, the tapetum became even more fragile and the nuclei

became scattered throughout the tapetal mass (Figure 54). In a cross

section of a locule at the tetrad stage, the Plasmodium can be seen both

externally and internally to the microspore tetrads (Figure 55). Even

after this stage, the Plasmodium occasionally showed cellular fragments

(cells), some of which contained nuclei (Figure 56). Following the re­

lease of the microspores, the Plasmodium was still present, but showed

signs of degeneration. The nuclei were prominent and exhibited chromo- centers similar to those shown in Desmanthus depressus (Figures 6, 8, 9).

No divisions in the tapetal nuclei of either species of Chamaecrista were observed.

The plasmodial tapetum in Cassia mimosoides was more fragile than the

tapetum in Chamaecrista fasciculata. By the tetrad stage, the Plasmodium was more of a "soup" (Figure 58). Portions of the Plasmodium occasionally showed distinct but scattered nuclei, some of which contained smàll chromocenters (Figure 59). Finally, at the pollen grain stage, there were only remnants, mainly nuclei of the Plasmodium (Figure 60). 40

Except for a single very inconclusive electron micrograph of a re­ portedly periplasmodial tapetum in Parkinsonia aculeata (Larson and Lewis

1963), Figures 52-60 are the first photographs of a Plasmodium in a legume. From the squash technique it appears that until the tetrad stage the Plasmodium is more or less cellular and not jusL a free protoplasmic mass,

Papilionoideae

This subfamily, with about 400 genera and 9,200 species, is world wide in distribution. Moreover, many genera and species occur in the tem­ perate regions of the United States, thus they are readily available.

Seventy genera and 126 species were examined (Appendix A, Chart 3). They represented 10 of the 12 generally recognized tribes. Only the Dalbergieae and Swartzieae were unavailable.

The tapetum in all species was of the cellular type, uniseriate and constantly uninucleate. No differences between the inner and outer tapetal cells were observed. The major variable characteristics were tapetal cell size, nuclear behavior and the presence or absence of crystals. These differences, however, were not restricted to any particular taxon.

To avoid too frequent repetition of information, only selected mem­ bers of each tribe, beginning with the Hedysareae, are described here.

More complete tapetal information on these and the remaining species, in­ cluding citation of all photographic figures, is listed in Appendix A,

Chart 3. 41

Hedysareae Desmodium glutinosum and D, illinoense were selected

as representative of cellular tapetal development for the entire sub­

family. The development of the tape turn and pollen as previously re­

ported by Buss et al. (1969) is here repeated with only minor modifica­

tions.

Although I examined many anthers, I was unable to determine the

ontogenetic origin of the cell layers surrounding the sporogenous tissue.

When sporogenous cells became defined, there were three external cell

layers present (Figure 61), After the tapetum began to enlarge, two or

occasionally three, parietal layers were present between the tapetum and

the epidermis (Figure 62). As in some mimosoids and most caesalpinioids

described previously, the tapetal cells were often irregular in shape and

of many sizes (Figures 63, 64, 77). Only 12-24 PMCs, occurring in two

rows of 6-12 cells, are produced in each of the anther locules (Figures

61-64, 187, 188).

Occasional locules were bisected by a transverse tapetal septum

(Figures 64, 65), similar to the septum described in Prosopis juliflora

(Figure 12) and several other legumes,

Callose is secreted by each PMC beginning prior to meiotic prophase.

However, it is not secreted uniformly, so that many, perhaps all, PMCs remain in physical contact during meiosis I. During early prophase I

(Figure 65) the PMCs all appear to be connected, except where the locule is bisected by a transverse septum (Figure 65). By metaphase I more callose appears to have been secreted, although all PMCs are still 42

associated through broad connections (Figure 66). By anaphase I (Figure

67, right locule) contacts between PMCs are less noticeable. After meiosis I contact is restricted to pairs or trios of PMCs (Figure 68), and following meiosis II (Figure 69) each young tetrad is completely isolated by its callose sheath.

Cytokinesis within tetrads is by furrowing (Figures 70-72). Occasion­ al delicate cytoplasmic strands still join the separating microspores

(Figure 71), suggesting that the microspores are actually "pulled apart".

After cytokinesis each microspore and each tetrad is surrounded by callose (Figures 72, 73), identified from anther squashes and paraffin sections according to the callose-aniline blue-fluorescence method (Jensen,

1962), Usually callose of the tetrad and of the individual microspores fluoresces, but under partial fluorescence (Figure 82) the walls of the adjacent tapetal cells also appear bright. However, under complete fluorescence only the callose surrounding the tetrads and microspores ap­ pears bright (Figure 83),

Microspores are released into the locule by dissolution and dispersal

(Figure 74) and final loss of the callose (Figure 75), After callose dissolution, tapetal cells were still intact, but separated readily from each other (Figure 76), A squash preparation at this stage showed that the tapetal cells were still quite variable in size and shape; they ap­ peared to have no cell wall (Figure 77), The loss of the wall, however, has not been confirmed.

At the vacuolate pollen grain (2-celled) stage the tapeturn, while still present, appeared to be near collapse (Figure 78), A paraffin 43

section from another anther at a comparable stage shows intact tapetal

cells and three adjacent wall layers (Figure 81). Even after the pollen

became engorged with food reserves (Figure 79) some tapetal cells remained.

Finally, as the endothscium developed its characteristic thickenings,

the tapeturn disappeared (Figure 80). Like Desmanthus, the tapetum in

Desmodium is another good example of a cellular (secretory or glandular)

type of tapetum.

Besides these two species of Desmodium, I observed tapetal charac­

teristics in another 12 genera and 24 species, some of which are repre­

sented in Figures 84-92. This included the common peanut, Arachis hypogaea,

which has been studied by many cytologists, all of whom have failed to

mention the tapetum. The tapetal cells in Arachis (Figure 89) and Adesmia muricata (Figure 90) appear to be filled with food reserves which eventual­

ly disappear as the pollen grains are filled. These two species, together with Adesmia tenella, Alysicarpus longifolius (Figure 91) and Alysicarpus

vaginalis (Figure 92), possessed distinct chromocenters in their nuclei.

The remaining species were about equally divided between those having

interphase nuclei and those having early prophase nuclei. In general,

the tapetal cells in this tribe are as large as, or larger than, the microspores. They remain intact until after the pollen grains become filled with food reserves (Figure 92).

Galegeae Previously, only eight genera and 10 species of this very large tribe had been examined. I examined 10 genera and 20 species,

Tapetal cells of some of these species are shown in Figures 93-105. Stored 44

food was readily apparent in the tapetal cells of Astragalus cicer

(Figures 99-101), Galega officinalis (Figure 97), Indigofera pilosa

(Figure 104), Sesbania arabica (Figure 95) and Tephrosia incana (Figure

94). With regard to nuclear behavior, most of the species remained at early prophase (Figures 99, 102). Only two of the species, Biserrula

pelecinus (Figures 105, 193) and Indigofera pilosa (Figure 104) possessed

'chromocenters. Occasional tapetal cells contained very large nuclei, sug­ gesting polyploidy as a result of intranuclear doubling (Figure 99).

Sophoreae and Podalyrieae There are no previous reports from

these two tribes. In the Sophoreae I examined Sophora japonica (Figures

106-108) and Cladrastis lutea (Figures 109-111). The tapetal cells were very similar in all respects. In the Podalyrieae I examined Baptisia leucantha, B. leucophaea leucophaea (Figures 112-114) and Thermopsis montana. Except for minor differences in nuclear behavior, the tapetal cells of these two tribes were alike. In the Sophoreae the nuclear material is associated with the nuclear membrane and is not organized into chromocenters (early prophase nuclei). In the Podalyrieae, although the nucleus is granular, there are no chromocenters and no chromonemata. The nuclei in this tribe correspond to interphase nuclei (Figure 114). In both tribes the tapetal cells were many times larger than the microspores

(Figures 108, 110, 111, 114).

Genisteae Only five genera and eight species had previously been examined. Corti (1946) reported a plasraodial tape turn in Genista monosperma. 45

All of my observations in this tribe, eight species and six genera, in­

dicate only a cellular uninucleate tapetum (Figures 115-123). Most of

the species examined had nuclei which corresponded to interphase of ac­

tivity (Figures 117, 121, 122). In Lupinus elegans (Figure 119) there

were chromocenters similar to those reported by Carniel (1952, 1954).

Trifolieae Previously, only three genera and seven species,

mainly in the genus Medicago, had been studied. I examined six genera

and 10 species, including some of the same species studied by previous

investigators. My observations on the tapetal cells confirmed previous

reports of a cellular uninucleate tapetum in this tribe (Figures 124-132).

I examined for the first time species of Ononis, Parochetus and Trigonella.

The tapetal nuclei in most species remained at early prophase (Figures

124, 126, 131). Tapetal nuclei in Ononis pubescens (Figure 129) approach activity similar to late prophase. Interphase nuclei were seen only in

Trifolium incarnatum (Figure 132). Occasional nuclei in Parochetus communis

(Figure 131, lower right) were approximately twice as large as the sur­ rounding nuclei.

A most unusual tapetal characteristic observed in this tribe was the occurrence of a yellow carotenoid substance, Pollenkitt (Pankow, 1958), found throughout the cytoplasm in Ononis pubescens (Figures 127, 128) and

0. speciosa. It gave a granular appearance to the cytoplasm. Pollenkitt was observed only in freshly fixed material and disappeared when treated with alcohol. It also disappeared as the pollen grains became engorged and the exine thickened, suggesting that it is in some way associated with the pollen grains. 46

Loteae Only two species had been examined previously in this

tribe. Hansen (1953) mentioned a cellular tapeturn in Lotus corniculatus,

but did not give the number of nuclei. According to Chubirko (1967), the

tapeturn in Lotus corniculatus is uninucleate, but "pseudoplasmodial". The

tapetal cells reportedly become separated from the adjacent wall layers

and form a periplasmodium around the developing microspores, I found, on

the contrary, that Lotus corniculatus had a typical cellular uninucleate

tapeturn which did not break down to form a Plasmodium (Figures 139, 140).

Besides L. corniculatus, I examined L, requieni, Anthyllis tetraphylla

(Figures 133, 134), Coronilla varia (Figures 135-138) and Securigera securidaca (Figures 141, 196), The tapetal cells were comparatively larger than the microspores with nuclei mainly at early prophase (Figures

136-138).

Psoraleae There are no known reports in this tribe dealing with the tapetum. I examined five genera and 15 species (Figures 142-159).

I traced, by means of anther squashes, tapetal nuclear development in

Psoralea tenax. At the time when the PMCs are at zygotene of meiosis

(Figure 142), each tapetal nucleus shows heterochromatin appressed to the nuclear membrane. This gradually condenses during the early stages of meiosis (Figure 143) so that by the tetrad stage (Figure 144) there are distinct chromocenters. These remain until the final dissolution of the tapetal cells (Figures 145, 146). Many other species in this tribe also exhibited chromocenters (Figures 147-149, 150, 151). Exceptions were Dalea argyraea (Figures 154, 155), D, lagopus, Eysenhardtia polystachya,

Petalostemon candidum, P. purpureum (Figures 156, 157), Psoralea adscendens 47

and P. argophylla (Figures 152, 153). In the latter species, the nuclear

material with interconnecting chromonemata is organized like a prophase

nucleus. Tapetal nuclei in Amorpha canescens (Figures 158, 159). A

fruticosa and Dalea alopecuroides apparently remain at interphase.

Vicieae Previous reports mentioning the tapeturn in members of

this tribe are available only from five genera and eight species. Two

investigators, Cooper (1938) and lijima (1962), reported the occurrence

of binucleate tapetal cells in Pisum sativum and Vicia faba. respectively.

In my material, Cicer arietinum (Figure 165), Lathyrus nissolia (Figures

163, 164), L. odoratus. Lens culinaris, Vicia americana (Figure 162),

V. faba and V. monantha (Figures 160, 161), I did not find any evidence for binucleate tapetal cells. However, when tapetal cells were ruptured, free nuclei were found next to other nuclei. Such a situation may then resemble a binucleate cell (Figure 164). According to Shukla (1954), the tapeturn in Lens esculenta is periplasmodial, and although the cells lost their walls they did not wander among the microspores. In Lens culinaris, as well as the other species cited, the tapeturn was cellular and always intact. I did not observe any abnormal tapetal cells. The nuclei in this tribe are usually large and densely staining, and, with the exception of Cicer arietinum (Figure 165), the tapetal nuclei remain at interphase.

Phaseoleae Previous investigators had examined only seven genera and seven species. I examined 18 genera and 30 species (Figures 166-186).

The size of the tapetal cells varied, but in general they were about the 48

same size as the microspores or pollen grains. With regard to nuclear

behavior in this tribe, Centrosema pubescens (Figures 185, 186), C,

virginiana, Glycine max (Figure 184), Rhynchosia sp, (Figure 183), R.

tomentosa and Vigna unguiculata all possessed nuclei with prominent

chromocenters. Abrus precatorius (Figure 171), Amphicarpa bracteata,

Apios americana (Figure 176), Cajanus cajan (Figures 169, 170), Clitoria

ternatea (Figure 166), Vigna cylindrica, V. racemosa and V, sesquipedalis

(Figure 173) had nuclei which remained at interphase. The remainder of

the species examined exhibited early prophase nuclei (Figures 168, 174,

175, 177-182, 191). In Erythrina sp. there were marked differences in

size between several of the tapetal nuclei (Figures 177, 178). Even

the nucleolus was approximately twice the regular size (Figure 178). In

Glycine max some tapetal cells contained enlarged nuclei with approxi­

mately twice as many chromocenters. As stated previously, no volumetric

analyses were made on these nuclei, but visual observation suggests that

these enlarged nuclei are polyploid.

Dalbergieae and Swartzieae No representatives of these tribes

have previously been studied and none were available for this investigation.

As a general point of observation, although I examined a large number

of tapetal cells, many under the oil immersion objective, I did not ob­

serve any Ubisch bodies or sporopollenin granules. These structures, how­

ever, have been observed in other families (Davis, 1966) at the light

microscope level. They either are too minute to be seen with the light 49

microscope or else they do not occur in this family.^

Tapetal Crystals

Except for occasional large distinctive prismatic crystals observed

in the anther connective of a few species, calcium oxalate crystals were restricted to the tapetum. Here they were observed in 84 out of 167 species and 52 out of 89 genera (Appendix A, Charts 1, 2, and 3).

The development of tapetal crystals was not followed in all species.

However, based on observations from representatives of each subfamily, I was able to determine that the stages in crystal formation are essentially the same in all three and are nearly identical to those described in

Desmodium (Buss et al., 1969). Therefore, the crystal development as given for Desmodium is repeated here with only minor modifications.

Sections of young anthers of D. glutinosum and D. illinoense, before meiosis and tapetal cell enlargement, were examined under polarized light.

At this stage (Figure 187) no crystals were seen. As the tapetal cells enlarged, small oval crystals appeared (Figure 188, arrows). The crystals reached maximum size (about 9 jum in these two species) during prophase I

(Figure 189), thereafter remaining prominent until tapetal dissolution

(Figure 190). The mature crystals were flat and prismatic with oval to rhomboid or variously shaped faces (Figures 77, 189, 190). In sections only certain tapetal cells seemed to have crystals (Figures 189, 190),

6 Observations from several electron micrographs of tapetal cells of Desmodium glutinosum also reveal no Ubisch bodies. 50

but in squash preparations most tapetal cells, unless ruptured, contained

one or occasionally more crystals (Figure 77), These crystals disappeared

when treated with concentrated nitric acid or concentrated hydrochloric

acid, but were unaffected by acetic acid.

Crystals are usually found in cytoplasmic vacuoles, but in my ma­

terial, due to the harsh treatment associated with fixation and the

preparation of tapetal squashes and sections, it was not always possible

to determine if the crystals were located in vacuoles or whether they were

found free in the cytoplasm.

Not all genera had the same size, shape or number of crystals per

tapetal cell. In addition to the flat rhomboid or variously sided pris­

matic crystals (Figures 189-191), some were lens-shaped (Figures 138, 193),

others were large tetragonal prismatic crystals (Figure 194). Occasion­

ally, tiny prismatic crystals were clustered together in two's or three's

(Figure 192) or scattered throughout the tapetal cell like crystal sand

(Figure 196). One species, in addition to rhomboid crystals, had styloid

•prismatic crystals (Figures 49, 50, 195).

The distribution of tapetal crystals in the Leguminosae is sporadic

and is not confined to any tax on. Only one of the 16 mimosoid legumes examined. Acacia sp., had crystals. They were small prismatic crystals and occurred one or two per cell (Figure 17, tip of arrows). It is not known whether the lack of crystals is characteristic of this subfamily or, as is more likely, the infrequent occurrence is due strictly to the limited sample. 51

In the Caesalpinioideae, tapetal crystals occurred more frequently.

Of the 25 species examined, 13 contained crystals. They included

Gleditsia triacanthos (Figures 27, 28, 30, arrows), Cassia artemisioides,

C. leptadenia, C. mimosoides (Figure 58), C. 11225 (Figure 35), C. sp. 11244, Chamaecrista fasciculata (Figures 53, 54, 56, 57), £. stenocarpa,

Caesalpinia eriostachys, C. gilliesii, C. pulcherrima (Figure 39),

Cercidium microphyllum and Delonix regia (Figure 49-51), In Gleditsia both lens-shaped and flattened rhomboid prismatic crystals occurred

(Figures 27, 28, 30). In others, Caesalpinia pulcherrima (Figure 39, arrow), Chamaecrista fasciculata (Figure 54, 57, arrows) and Cassia mimosoides (Figure 58, arrows), there were also tetragonal crystals.

Delonix regia, in addition to rhomboid crystals, had styloid prismatic crystals (Figures 49, 50, 195). This is the first report of styloid crystals in this subfamily (Metcalfe and Chalk, 1950).

In the Papilionoideae I observed crystals in 70 species from 45 genera (Appendix A, Chart 3). All ten of the tribes examined are repre­ sented.

In the Hedysareae, in addition to Desmodium glutinosum and D. illinoense (Figures 77, 81, 188-190), crystals also were found in four other species of this genus. Each tapetal cell in Aeschynomene indiea had one or more small clusters of prismatic crystals (Figure 152). Similar crystals occurred in Arachis hypogaea, Onobrychis crista-galli and

Stylosanthes sp.

In the Galegeae, crystals were found in eight of 10 genera and 13 of 20 species. These included Astragalus cicer (Figures 100, 194), 52

A. crassicarpus, A. inflexus, Biserrula pelecinus (Figures 106, 193),

Galega officinalis, Indigofera echinata, subulata, Olneya tesota

(Figure 103), Robinia pseudo-acacia, Sesbania arabica (Figure 95),

£. sesban, sp. and Wisteria sinensis. The most striking crystal shape

in this tribe was the tetragonal prismatic crystal. This type was ob­

served in three species each of Astragalus and Sesbania and in Olneya

tesota. The other members of this tribe had lens-shaped, irregular

prismatic or flattened rhomboid-shaped crystals.

All species examined in the tribes Sophoreae and Podalyrieae had

tapetal crystals. In the Sophoreae solitary lens-shaped or irregular

prismatic crystals were observed in Sophora japonica (Figure 108, arrow) and Cladrastis lutea (Figures 110, 111, arrow). In the Podalyrieae,

Baptisia leucantha, B. leucophaea leucophaea (Figures 112, 113 arrows) and Thermopsis montana had one to several scattered small prismatic crystals per tapetal cell.

Only two of eight species in the Genisteae had tapetal crystals.

They were Crotalaria saltiana (Figure 116) and Lotononis bainesii (Fig­ ures 121, 123). This sample is too small to make any conclusions con­ cerning the over all occurrence of crystals in the tribe.

In the Trifolieae, six of ten species possessed crystals. Although a small sample, it appears that crystals are of common occurrence in this tribe. They were found in Medicago sativa (Figure 124) and Melilotus alba, two species that have been studied cytologically many times, without any reports of tapetal crystals. Besides these two species, crystals 53

were also found in Ononis mitissima, 0. pubescens (Figures 127, 128,

arrows), 0. speciosa and Parochetus communis (Figures 130, 131, arrows).

Most of these species had lens-shaped or small prismatic crystals.

Of the five species examined in the Loteae, only Coronilla varia

and Securigera securidaca had crystals. In Coronilla the crystals are

lens-shaped and occur one per cell (Figures 135, 136, 138). In Securigera

there are many small prismatic crystals scattered throughout the cell

(Figures 141, 196).

Crystals were present in all five genera and 11 of the 15 species in

the Psoraleae. They were found in Amorpha canescens (Figures 158, 159),

A. fruticosa, Dalea alopecuroides, D. argyraea (Figures 154, 155), D.

lagopus, Eysenhardtia polystachys, Petalostemon candidum, P. purpureum

(Figures 156, 157), Psoralea argophylla, P. dentata, P. tenax (Figure

146) and P. ££. The crystals were mainly rhomboid, although some lens-

shaped crystals were also observed. Figure 146 shows a side view of a

rhomboid crystal. Crystals occurred in most cells, but without polariza­

tion they did not always show up in photographs.

All four genera and seven species in the Vicieae had crystals.

These included Cicer arietinum, Lathyrus nissolia (Figures 163, 164),

L. odoratus. Lens culinaris. Vicia americana (Figure 162, arrows), V.

faba, and V. monantha (Figure 160 161). Both small prismatic and

tetragonal prismatic crystals occur. A tetragonal crystal, seen in

longitudinal view, is shown in Figure 162 (upper left).

The number of genera and species examined in the Phaseoleae was

1i 54

greater than for the other tribes. Of the 18 genera and 30 species

examined, 11 genera and 14 species had crystals. They included the

following; Amphicarpa bracteata, Cajanus cajan (Figures 169, 170, arrows), Canavalia gladiata (Figure 175, arrows), Centrosema pubescens,

Dolichos biflorus, D. lablab (Figure 179, 180, 191), Glycine max, Mucuna

pruriens, Phaseolus coccineus (Figure 182, arrow), Pueraria thunbergiana

(Figure 167, arrow), Rhynchosia tomentosa, R, usambarensis (Figure 181, arrow), R. S£, and Strophostyles umbellata (Figure 168, arrow). Lens- shaped (Figures 170, 175, 181), small prisms (Figures 167, 182) and rhomboid-shaped crystals (Figures 168, 169, 179, 180) were observed in this tribe.

My observations on crystal shape in this family agree with those of

Pobequin (1943), who reported lens-shaped and prismatic crystals of the trihydrate form in the vegetative tissues of this family, and Frey (1929), who described the various shapes which the trihydrate form of calcium oxalate can assume, Raphide and druse crystals were never observed in the tapetal cells. 55

DISCUSSION

The tapeturn in most angiosperm families has not been very extensively

investigated for taxonomic characteristics. In this study of the three

subfamilies of the Leguminosae, the tapetum was surveyed specifically to

determine if certain tapetal characteristics could be of systematic value.

One of these characteristics, perhaps the most unusual, was the occur­ rence of calcium oxalate crystals. Comparatively little is known concern­

ing the factors which regulate the rate and mode of crystallization,

although they are usually considered to be by-products of metabolic activ­

ity, Furthermore, crystals ordinarily occur in long-lived tissues of some roots, sterna and leaves. Their presence in tapetal cells, which although active metabolically are only ephemeral, is puzzling and not understood.

Although crystals seem to be rare in tapetal cells of most angio- sperms, they are rather frequent in legumes, especially in the Caesalpini- oideae and Papilionoideae. Their infrequent occurrence in the Mimosoideae may indicate that crystals are also rare in this subfamily. However, before valid conclusions can be made, a larger and more diverse sample will be necessary.

Of the two basic crystal types, lens-like and prismatic, the latter with its several shapes and sizes (tetragonal, flat rhomboid or with various numbers of sides, small prismatic, styloid) was the most abundant.

One or more shapes were observed in each of the three subfamilies.

The occurrence of two or more crystal shapes within a single tapetal cell or within a species suggests that crystal shape is not rigidly 56

determined. This is also supported by evidence that a specific crystal

shape is. usually not restricted to any one taxon. One possible exception

is the occurrence of styloid prismatic crystals in the tapetal cells of

Delonix regia. This represents the only known report of such crystals

from any tissue in any member of the subfamily Caesalpinioideae, although

they are occasionally found in vegetative tissues of the other two sub­ families. It also represents the only occurrence of styloid crystals in

this study.

Although not carried out in this study, shapes of crystals from the

tapetal cells need to be compared with those found in the rest of the

plant to determine if they are similar. From this information distinct lines of crystal evolution, similar to those reported for Allium (Chart- schenko, 1933), may be possible.

In the Leguminosae, septate microsporangia, when present, have gener­ ally been restricted to mimosoid species with pollinia (Rosanoff, 1865;

Engler, 1876; Dnyansagar, 1954a). In this subfamily the septa, which appear to be genetically fixed and linked with pollinia, may be of taxo- nomic value. My observations on species known to possess pollinia were mainly from squashes, therefore, I was not able to determine whether or not septa were present.

Because of their similarity to the tapeturn, septa have been regarded as nutritive (Periasamy and Swamy, 1959; others). Lersten (1971), who has reviewed the literature on septate microsporangia in vascular plants, challenges this nutritional view. He suggests that septa are usually derived from a sterilization of sporogenous tissue and are simply a means of reducing the number of sporogenous cells, Lersten predicts 57 that future investigations will reveal septa to be common, particularly in herbaceous animal-pollinated taxa. It may be that they will be common in highly specialized, small anthered papilionoid legumes.

At first it was thought that the patterns observed in tapetal nuclei, i.e., interphase, early prophase and late prophase, might be character­ istic of certain taxa, and therefore of systematic value. Delay (1940), who studied interphase nuclei in roots from approximately 40 species of legumes, proposed a phylogenetic scheme to the tribal level based on the type of nucleus. Although she did not use the same terms employed in this investigation, the behavior was about the same. The Mimosoideae, Caesal- pinioideae and the papilionoid tribes Genisteae and Phaseoleae were reported to have non-reticulate nuclei with distinct chromocenters (late prophase). The other tribes had different degrees of reticulate or netted nuclei with or without small chromocenters (interphase or early prophase).

Based on a much larger sample, my findings on behavior of tapetal nuclei do not support Delay's conclusion that the type of nucleus is dis­ crete enough to distinguish between the tribes. I found, on the contrary, that the three types of nuclear behavior were more or less randomly dis­ tributed throughout the three subfamilies and were represented in most tribes. Nuclear behavior, therefore, does not appear to be of systematic value.

Several additional points regarding nuclear behavior in tapetal cells might be mentioned. Brown and Bertke (1969) have suggested that all tapetal cells increase their chromosomal material one way or another.

Some investigators have attributed part or all of this increase to endo- polyploidy by endomltosis. However, there is inconsistency in description 58 and considerable disagreement as to what constitutes endomitosis. There­ fore, this entire area of nuclear behavior needs to be reviewed; and as

D'Amato (1954) pointed out, until the processes are better understood cytologists need to remain openminded so that dogmatic statements do not hinder the furthering of knowledge on endopolyploidy.

Before conclusions on intranuclear duplication, particularly in the uninucleate tapetal cells of legumes, can be made, accurate spectrophoto­ me trie measurements will be necessary.

As determined in this investigation, the characteristics of most taxo- nomic significance are type of tapeturn and number of nuclei per cell. In the Leguminosae, the tapetum is cellular or exceptionally plasmodial.

Most reports of a plasmodial tapetum have been of tapetal cells which, during meiosis or at the microspore stage, separated from the parietal layer, underwent wall changes, and formed a peripheral periplasmodium.

Carniel (1952, 1963) regards this type of tapetum as either a false peri- plasmodial or an amoeboid tapetum, and cautions that it may result from premature degeneration of a normally cellular tapetum. A true periplas- modial tapetum is produced prior to, or at the beginning of, meiosis and is not just an occasional event. My observations, some on species prev­ iously reported to be plasmodial, support Carniel and further indicate that a plasmodial condition may result from various techniques employed in fixation and preparation of the anther tissue. Furthermore, different environmental stresses may affect the condition of the anthers and therefore the tapetum.

The only examples of a plasmodial tapetum which seem to be valid and consistent are those by Venkatesh (1956b) and those described in this 59

investigation. Thus far, this type is limited to herbaceous members of

the genera Cassia and Chamaecrista (Caesalpinioideae). Some investigators

(Irwin and Turner, 1960; others) consider these two as a single genus.

Cassia. If so considered, those species with a plasmodial tapetum are in

the subgenus Lasiorhegma and mainly in section Chamaecrista. However,

regardless of whether these are considered as one genus or two (footnote

4, p. 28), the occurrence of a plasmodial tapetum, which is always uni­

nucleate, seems to be a supplementary taxonomic characteristic in a sec­

tion of this subfamily.

Because the plasmodial (amoeboid) tapetum is characteristic of a few

lower vascular plants and of some "lower" monocotyledons and dicotyledons,

Eames (1961) considered it to be primitive. However, evidence from

families such as the Magnoliaceae (Maneval, 1914; Reed, 1947), Winteraceae

(Sampson, 1963), Rosaceae (Cooper, 1933; others) and others (Wunderlich,

1954; Davis, 1966) considered by other characteristics to be among the

more primitive angiosperms, indicates that the cellular tapetum is more

common and primitive. The widespread occurrence of the cellular tapetum

in the Leguminosae and the restricted occurrence of the plasmodial type

support this conclusion.

In addition to a cellular tapetum, the primitive families cited above

have binucleate to multinucleate tapetal cells. In the Leguminosae, con­

sidered by most phylogenists, including Taubert (1894), Engler (1915),

Melchior (1964), Hutchinson (1964, 1969) and Leppik (1966), to be derived

from the Rosaceae, the tapetum is either constantly uninucleate or else

binucleate to multinucleate. Based on available information, the binucleate and multinucleate conditions occur only in the Caesalpinioideae. The other 60

two subfamilies and several caesalpinioid exceptions, namely those species with a plasmodial tapeturn and Delonix regia, are uninucleate. Therefore, with respect to nuclear number, the Caesalpinioideae is unique and seems

to be the most primitive of the three subfamilies.

This conclusion agrees with and seems to support evidence from floral

morphology. The presumed primitive members of the Caesalpinioideae have

flowers most like those in the Rosaceae, particularly the subfamily Chryso-

balanoideae which is unicarpellate and has both actinomorphic and zygo- morphic flowers. Although not as conclusive in indicating phylogeny, chromosome numbers and seed morphology also support the view that the caesalpinioids are the most primitive.

Based on these same criteria, there is general agreement that the

Caesalpinioideae and Papilionoideae are closely related, and that the latter subfamily is derived. Morphological connections between these two subfamilies may be inferred from the way the tribe Swartzieae has been treated, Bentham and Hooker (1865) placed this tribe in the Papilionoideae, while Taubert (1894) considered it to be allied with the Caesalpinioideae.

Turner and Fearing (1959), who studied chromosome numbers in these two subfamilies, have suggested the Swartzieae as a possible connecting link between them. They reported a base number of x=8 in Swartzia and in several of the older caesalpinioid tribes. According to these authors, this base number represents the original base number for the entire family.

The other base numbers x=7, 9, 10, 11, probably have arisen by aneuploid loss or gain.

Senn (1938), in his chromosomal review of this family, concluded that the Papilionoideae was derived from a primitive form or primitive forms of 61

8-chromosome legumes.

Corner (1951) and Isely (1955), in their investigations on seeds of

the Leguminosae, observed that there appeared to be only two basic seed

types: a mimosoid-caesalpinioid type and a papilionoid type. Corner (1951)

and Kopooshian and Isely (1966) each studied species of Swartzia. Accord­

ing to these authors the seeds were intermediate between the Caesalpini-

oideae and the Papilionoideae, and each had a different combination of

characters.

Because the Swartzieae seems to be intermediate in many characters and primitive in others, an investigation of this tribe Would be desire-

able since it might reveal intermediates in nuclear number and help to

bridge the gap between the Caesalpinioideae and the Papilionoideae.

Tapetal characteristics have clearly been shown to be significant in delimiting the Caesalpinioideae. While the Papilionoideae and Mimo- soideae both have a uninucleate tapetum, it is not possible to use this at present to shed much light on the relationships between these two otherwise morphologically distinct taxa. 62

SUMMARY

1. The tape turn in 167 species from 89 genera representing the three

subfamilies was studied for tapetal type, number of nuclei, nuclear be­

havior and occurrence of calcium oxalate crystals.

2. Except for six caesalpinioid species (four of Cassia and two of

Chamaecrlsta), the tapetum in the Leguminosae is cellular throughout devel­

opment. In these species the tapetum is plasmodial.

3. The number of nuclei per tapetal cell will distinguish the Mimo-

soideae and Papilionoideae from the Caesalpinioideae. Tapetal cells in

the latter subfamily, except for Delonix regia and those species with a

plasmodial tapetum, are binucleate or multinucleate. The other two sub­

families are always uninucleate.

4. The cellular tapetal type and the binucleate or multinucleate

tapetal condition are considered primitive to the cellular uninucleate

and the plasmodial types.

5. Tapetal cells differ in size and shape; however, there are no significant differences between the tapetal cells on the epidermal side

of the microsporangium and those on the connective side.

6. Nuclei in the tapetal cells of many legumes contain chromocenters resembling diakinesis or metaphase chromosomes. In other species the nuclei lack chromocenters, but resemble early prophase or interphase nuclei. This nuclear behavior occurs in all three subfamilies and does not appear to have systematic value. This behavior may be associated with endopolyploidy.

7. Calcium oxalate lens-like or variously shaped prismatic crystals 63 occur in tapetal cells. They are more frequent in the Papilionoideae

(70 of 126 species) and the Caesalpinioideae (13 of 25 species). Only one species out of 16 of the Mimosoideae, Acacia s£., had tapetal crystals.

The presently known distribution and shape of crystals in this family does not permit their use in delimiting taxa.

8. Styloid crystals, which have not been reported previously from any cell type in the Caesalpinioideae, were found in Delonix regia.

9. Occasional tapetal septa bisecting anther locules were observed in the non-pollinial species, Prosopis juliflora, Desmodium glutinosum and D, illinoense.

10. Ubisch bodies were not observed.

11. The tapeturn as a source of taxonomic characteristics is worthy of continued investigation. 64

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Numerous other papers with nearly identical titles, but dealing with different species have been published by Soueges (see Davis, 1966). None of these contained any information on the tapeturn. 84

Veillet-Bartoszewska, M. 1956b. Recherches embryogéniques sur le Glycyrrhiza foetida. Bulletin de la Société Botanique de France 103; 439-443.

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ACKNOWLEDGEMENTS

I wish to express my appreciation to Dr. Nels R, Lersten for his assistance in the selection of the problem and for his advice, patience and encouragement during the investigation and preparation of this manu­ script, The technical advice and discussions with Dr. Harry T. Homer, Jr. and Dr. Duane Isely have been helpful and are appreciated.

Special thanks are extended to Dr, Willis H, Skrdla and Dr. Raymond

Lo Clark of the Regional Plant Introduction Station, Ames, Iowa, for their cooperation in obtaining seed of many species used in this study. I would also like to thank Dr, Richard W, Pohl and Mr, Gerrit Davidse for special collections of cytological material,

I am grateful to the Department of Botany and Plant Pathology for the facilities made available and for financial assistance during this study,

I also acknowledge financial support from a National Science Foundation summer fellowship,

I thank John T, Jones and family for their role in the completion of this manuscript.

Last but not least, I publicly thank my wife, Janet, and family for their patience, encouragement and sacrifices to make the completion of this work possible. 86

APPENDIX A: SUMMARY OF TAPETAL CHARACTERISTICS

The ordering of subfamilies and tribes in the following charts is not intended to reflect any particular phylogenetic scheme, but rather is merely a convenient way of arranging the material. Following each subfamily and tribe are the approximate number of genera and species.

The number of genera and species of each subfamily and each tribe that have been reported by others is listed next. Below these figures are my observations.

Chart 1. Mimosoideae Ie loielp ivnm IM> f 1 ! IS |:|g e 1S- »- f. w w o» g. M S ^ w u» o o o s 1 O O O 3 i S u.»§ ggg * # * yyo 7 7 7

#4 Iff £5gfir X II r ""II"m » •M^S r 00 IH « f-x f— g 8%

+ + + ++++++ + + ++++++ + + Cellular tapetun Plaamodlal tapeturn + + + + + ++++++ ++ Uninucleate Blnucleate Multinucleate + laterphaae nuclei + + ++ + + Early prophase nuclei + + Late prophase nuclei Cryatals

Figure 88

I 1 Hi I S I s I t Ttxoa Rtfmenc* ! 11 a1 i L#uc««na L. gl«uc« Day ana agar, 1949 + + TlKlar, 1965 + + +

Mtoe»» M. iMBâU Doyaoaagar, 1951a + + RT Invita Tlxlar, 1965 + + + Bust + + + M. pudlca Haratlnhachar,1951 + + Butt + + + 14 N. teaalana Butt + + + HT niblcaulla Mulay and Dhaml, + + 1955 Butt + + +

SeliTaiikla S. uncfaata Butt + +

4. Admaatbarlaaa 16 Ci 100 ap. 4 C: S ap. 1 Ci 1 ap.

Adwanthara A. pavwSTca Doyansagar, 1954b 4* + 1958 +• +

Dlchreataehva P. citwtâa Dnyanaagar, 1954a +

Haptunla *. ol«rac#a Singh and Shivaputii + + 19S5 K trifluatra Dnyanaagar, 1952 + +

Proaopla P. Wlflora Biiaa + • 12,13 T: tplclewa Duyaoaagar, 1957 + +

5. Plptadaaiaaa 5 Oi 50 sp. 0 Gt 0 fp. 0 Oi 0 ap.

6. Parklaaa 2 Gi 40 ap. 1 OI 1 ap. 0 Gi 0 ap.

Parkla P. blglanduloaa Dnyanaagar, 1954a +

*Datcribad aa intanaadlat* batwaan callular and plaamedial (aaoabotd), but mora Ilka tha plaaaodial tap#turn. 89

Chart 2. Caesalpinioideae Ipipio minin |n|rMotolnirï|otn|o|n|ntolSIL *** ••• ••••«••••«•«1^ 'pII I S & g g ff 1 1 5 k # i M» # 1 g # O 1 isNi I £ |l S 1 le oT 0 K) W O A O O M O m o o iS o o oC o a a CAO non n n n o n n •"* S M O % •"Ou* ooS ooSS KB8 SSS •5 5 -S "8 <3 <3 •S -S 5

g g g STîî £?®SÎÎÎÎ?ïy^ # rr» rt y y y y y &&&&% S- vO r- «o w r- ^ o O SSS S < ^ k» ^ ^ 51

+++ + ++ +++++++++ Cellular tapeturn

++ + + + Plaamodial tape tu* ++ + + + Uninucleate +++ + ++ +++++++++ + + Blnucleate

+ ++++++ + + Multinucleate

+ Interphase nuclei

+ + Early prophase nuclei

+ + + + Late prophase nuclei + + + Crystals

Figure H» io ipipip in I Iff» •rIS iplol.ololo ipio in: I 6 p. o Sli sr lal i«»s|p»si8 r 1 7 s 1 s o o o 1e II e o o o •X4WO # ^ ^ AAA cjSBI o o u* •S «s *0 «0*^8 •Sv*S

Iff s^g I, III I ning^ « II si Cl 5 H

• + + + + + + + + + + + ++ ++++ + Cellular tapecum + + + Plaaaodial tapeturn + + + + + Uninucleate + + + + + + + + + + ++ ++++ + Blnucleate + + + + + + + + + + + + Multinucleate Interphaae nuclei + + + + + Early prophase nuclei + + + &at« propha«« nucl#! + + + + + + ++ Cryatala S: w Figure 1> 2 r* 5 92

Chart 3. Papilionoideae 93

I g I I B. S i I S Taxon Reference II 111 3 1I Papilionoldeae 400 G: 9100 ap 33 G: 45 ap. 70 C: 126 ap.

14. Sopboreae 50 G: 350 sp. 0 G: 0 sp. 2 G: 2 sp, CleJraatl» Ç. lute» Buaa + + + + 109-111 Sophor» S. ieponlc» Buas + + + + 106-108

15. PodalyriCM 30 Ci 450 «p. 0 G: 0 ap. 2 Ci 3 sp. Baptiaia B. leucantha Buss + + + + 1* leucophaea Buss + + + + 112-114 ThetBopal» T. aontana Buaa + + + +

16. Geniateae 50 Ci 975 •P 5 C: 8 •P 6 C: 8 sp. Crotalarla C. incaôa Buss + + + 117 Ç. Intetaadla Buas + + + Ç, iuncea Banerjl and Samal, ? + 1936 Ç. aaltlan» Buaa + + + + 115,116 C. verrucoia Tlxiet, 1965 + +

Cytiaua . purpureu»put Camlel, 1952 + + §. acacoparlua Buaa + + +

Caniata C. noneapema Cortl, 1946 + J

Laburnum L. Mggroidea Camlel, 1952 + + Buaa + + + 120 Lotoaonl» L. balnesll Buaa + + + + 121-123 94

Taxoa R«f«tanc«

Luplnus anguatlfoltu» Carnlal, 1963 + + + L. «l«g«ns Buaa + + + 118 119 L, poly^lXu» Catnlal, 1932,1963 + + + Carnlal, 1932,1963 + + +

Ulex £. «urop—u» Buaa + + +

17. TrlfollM* 6 Gi 430 ap. 3 GI 1 «p. 6 GI 10 ip.

M»dlcaflO M. giutinoi* Cooper, 1933 + + Cooper, 1933 + + Cooper, 1933 + + M. —tlT« Raavea, 1930 + + Cooper, 1933 + + Cblldera, 1932 + + Buaa + + + + 124 Malllotu» M. «Ibû# Caatetter, 1935 + + Cooper, 1933 + + Buaa + + + + 123,126 M. »lbw cv, Radflald Cooper, 1933 + + Yallow M, «uavaolani Tixler, 1965 + + +

Onoola 0. ultlaalna Buaa + + + + Ô. pa^tegiu Buaa + + + + Ô. «pacloaa Buaa + + + +

Parochatua P. caawml* Bua* + 130,131

Trlfollaa T. incarnatu» Buaa + + 132 T. ptatiwaa " Hindnarah, 1964 + + Buaa + +

Tritonalla T. waSTca Buaa + + + T. fotniMHgrMcua Buaa + + + 18. Lotaaa 8 GI 130 ap. 2 Gi 2 ap. 4 GI 3 ap. ipipiog i». !î »r isilisli ISl SSHillI

riir ri r ? vO p Ln

+ + + + + + Cellular tapeturn +0. Plaaoodlal tapetua

+ + + +_ + + + Uninucleate Blnucleata Multinucleate Interphaae nuclei + + Early prophase nuclei

Late propbase nuclei Cxystala Figure 96

J 3 i I i M 3 g 3 . I I_ 11111 f KM T«xon Reference

GllrlclJU G. maeul'atJ Tixler, 1963 + + G, «aplaa Chandravadana,1965 + +

lodlgofera I. «chinata Buss + + + + J[. piloga Buss + + + IM I, psaudotlnctoria Buss + + + I. iubulata Buss + + + +

miUtla M. ovallfolia Pal, 1961 + +

OInava 0. tesoU Buss + + + + 103 Roblnla R. pgeudo-acacia Carniel, 1952 + + R. pgaudo-acacla Buss + + + + S«*b#nia S. arabica Buss + + + + 93 I* Buss + + + + S, «««ban Buss + + + + Taphrosta T. Incana Buss + + + 91,94 T. purpurea Pantulu, 1942 + + T. aubt^lora Buss + + + T, vlrglnlanâ Buss + + + Wigterla W. «Inensii Bugg + + + + 20. Psoraleae 10 C: 350 sp. 0 Ci 0 ip. 5 Ci 15 gp. teorpha A. canaiceng Bug g + + + 198,139 A, frutlCQga' Sugg + + + Dalea D. alepecuroldag Bug# + + D. argyraea Bug a + + + 154,155 £. ISriâ Bug g + + D. lagopui Bufg + + + Eyianhardtla »• polyitachva Bu««