ThU dissertation has been microfilmed exactly as received _ _ - ,. 66-15,144

SWEENEY, Carol Reichert, 1937- BIOSYSTEMATIC STUDIES IN THE GENUS SILPHIUM L. (COMPOSITAE): SILPHIUM COMPOSITUM MICHAUX. The Ohio State University, Fh.D., 1966 Botany

University Microfilms, Inc., Ann Arbor, Michigan BIOSYSTEMATIC STUDIES IN THE GENUS SILPHIUM L. (COMPOSITAE):

SILPHIUM COMPOSITUM MICHAUX

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By

Carol Reichert Sweeney, A.B., M.S.

The Ohio State University

1966

Approved by

Department of Botany and Plant Pathology ACKNOWLEDGMENTS

I wish to express my sincere appreciation to my adviser,

Dr. T. Richard Fisher, for suggesting this problem and unselfishly aiding and advising in the work. Gratitude is also extended to

Drs. Emanuel D. Rudolph, John A. Schmitt and Ronald L. Stuckey who read this paper and offered many helpful criticisms. Special recognition is given to John M. Speer who provided several photo­ graphs and gave much help in photography, cytological methods and collection of specimens; to Dr. Dale A. Ray who aided in the statistical treatment; and to Mr. Joseph Harvey who did much of the art work. Many thanks are extended to Drs. J.W.A. Burley and

Jerry McClure for suggestions and help with the chromatographic work.

The curators of the following herbaria are gratefully thanked for their kindness in loaning specimens and photographs:

Chicago Natural History (Field Columbian) Museum (F) Florida State University (FSU) Gray Herbarium, Harvard University (G) Michigan State University (MSC) New York Botanical Garden (NY) Philadelphia Academy of Natural Sciences (PH) United States National Herbarium (US) University of Indiana (IND) University of Kansas (KANU) University of North Carolina (UNC) University of Tennessee (TENN) University of West Virginia (WVA) University of Wisconsin (WIS)

ii VITA

January 9» 1937 • • Born— Meadville, Pennsylvania

1939 ...... A.B., Antioch College, Yellow Springs, Ohio

1960-1962 ...... Kettering Research Laboratory Fellow, Department of Botany and Plant Pathology, The Ohio State University, Columbus, Ohio

1962 ...... M.S., The Ohio State University, Columbus, Ohio

1962-1965...... Teaching Assistant, Department of Botany and Plant Pathology, The Ohio State University, Columbus, Ohio

1965-1966...... Research Assistant, Department of Botany and Plant Pathology, The Ohio State University, Columbus, Ohio

FIELDS OF STUDY

Major Field: Botany

Studies in Experimental Plant Taxonomy. Professor T. Richard Fisher

Studies in Cytogenetics. Professor E. F. Paddock

Studies in Plant Physiology. Professor Carroll A. Swanson

iii c o n t e n t s Page

ACKNOWLEDGMENTS ...... ii

VITA ...... iii

TABLES ...... v

ILLUSTRATIONS ...... wii

INTRO DUG TI ON ...... 1

GiJ-i’ji': CijAT 'HAL i*ISTT RY ■ •*•#•••*••**•#*♦•• 5

GENERAI MORPHOLOGY OP q,:E GEL ITS ...... 10

TAXONOMY...... 13

ECOLOGY OF SILPHIUM COMPOSITUM ......

MORPHOLOGICAL ANALYSIS OF RESEARCH GARDEU CLONES AITD POPULATIONS COLLECTED IN THE F I E L D ...... ^6

BIG CHEMICAL S CoTEMATICS: BACKGROUND .V.: DBASIS ...... 70

METHODS AND RESULTS CP CHROMATOGRAPHIC AN A L Y S I S ...... ?6

CYTOLOGY OF THE SILPHIUM COMFOSITUM RESEARCH SARDS, CLONES . 101

INTERSPECIFIC AMD INTEASFSCIFIC HYBRIDISATION OF SILPHIUM COMPOS IT U M ...... 112

THE OCCURRENCE OF NATURAL IMTERSPLCIFIC AMD INTRASPECIFIC HYBRIDISATION ...... 123

DISCUSSION AMD CONCLUSIONS ...... 123

SUMMARY ...... 139

BI3LIC5RAPHY ...... l'+l

iv m ► ■ ;

Table Page

1 Diagnostic characters cf Silphium conpositum subspecies 40

2 numbers of 3. compositum subspecies associated with forest and soil types 42

3 humbers and. collection sites of research garden clones of iilnhiiuo comuositun . • ...... , be

4 Population analysis of £. c cropsiturn ssp. composittwi cO and ssp. renifcme ......

5 Population, analysis of jj. compositum ssp. conpositum, ssp. venosun, and ssp. ovatifolium ...... 62

6 Collection sites of gog: al~ tiers used in polygono- grapli analysis ...... 63

nI Chromatogram running tires ...... •• 34

3 Characterization cf spots found on two-dimensional paper chromatograus cf 3. compositun ...... 8s

9 Characterization spots found on two-dimensional thin-layer chromatograms of ii. compositun . . . • 87

10 Chromatographic profiles of 3. corpooitun sub­ species from paper cbr'rr.atograr.s ...... 38

11 Chromatographic profiles of 3. conpositum sub­ species from thin-layer chrcmatograns ...... 39

12 Paired affirity values cf paper c’.rcaatograns of 3, conpositum arranged in subspecific groups . . • 90

13 Paired affinity values cf tiir-layer chromatograms of 3. corrposituc arranged ir. subspecific groups . 91

14 Pesalts of Dancen’s hultigle Pr.r.ge Test for Signifi­ cant Differences between <,h*: sear, paired affinity values of the intrasubspecific comparisons for paper and thin-layer chrcuetcgrans ...... 93

v TABLES (continued) Table Page

15 Results of the analysis of variance of the paired affinity values cf intrasubspecific and inter- subspecific comparisons fro:.: thin-layer chroma bo grams ...» , , , 95

16 Results of the analysis of variance of the paired affinity values of intrasubspecific and inter­ subspecific comparisons from paper chromatograms • 96

17 Results of the analysis of variance of the paired affinity values of intraspecific and interspecific comparisons from paper chromatograms cf clone standards of £. conpositum and ntlicr oilphium species in "end arrangement" groups A,. 3, and C • 98

18 hean paired affinity values from thin-layer chromatograms of 7,'2159 tested for reproducibility of the m e t h o d s ...... 99

19 Cytological behavior of meiotic chromosomes in 3. compositnm ssp, compositun c l o n e s...... » 103

20 Cytological behavior of neiotic chromosomes in C. compositum esp, rer.iforme clones 104

21 Cytological behavior of meiotic chromosomes in 3. compositun ssp, venocmn c l o n e s ...... 105

22 Cytological behavior of meiotic chromosomes in S. compositum ssp, ovatifolium clones *«•,•• 106

23 Results of interspecific and intraspecific hybridization of S. compositum . • . 116

24 Crossability of S. compositun with Silphium taxa found in the same habitat, other sympatric taxa and allopatric taxa •«••*•*•• ...... 127

vi ILLUSTRATIONS Figure Page

1 Silphium compositum Kichx* (Copy of holotype photograph; I T~...... 15

2 Silphium orae Smell (Holotype)...... 17

5 Achenes of compositum ssp. compositun (#2162) • 18

k Achenes of S. compositum ssp. conpositum (#1309) * l3

5 Distribution map of S. compositum ssp. compositum and ssp. compositun-reni forme inter grades . . . 1$

6 S. compositum ssp. compositum (#131^* field collection) ...... 21

7 S. compositun ssp. conpositum (#1301, field CGllec tion) 21

3 compositum ssp. compositum-reniforne intergrade (#1327» field collection) ...... 23

9 3. compositum ssp. compositum-reniforme intergrade (#1317| field collection) • 23

10 Distribution map of S, compositum subspecies . . . ?M

11 Silphium. conpositum ssp. reniforme (liaf. e£ IJutt.) Sweeney & Fisher comb. nov. (Lectotype) .... 26

12 Achenes of S. compositum ssp. reniforme (#2165) • 26

13 Distribution map of compositum ssp. reniforme . 28

1^ S. compositum ssp. reniforme (#2 1 6 6 , garden collection) ...... 30

15 Silphium compositum ssp. venosum (Snail) Sweeney & Fisher c omb. nov. (Holotype) T ...... 30

16 Silphium lapsuum Small (Holotype) ...... 31

17 Achenes of S. compositum ssp. venosum (#1238) . • 31

vii ILLUSTRATIONS (continued) Page

Distribution map of S. compositun ssp. venosum and ssp. ovatifolium ..•••• 33

19 S, compositun ssp. venosum (,^123^, garden collection) ...... 35

20 Silphium conpositum ssp. ovatifolium (T. Sc G.) Gweeney Zt Fisher comb. nov. (holotype, Sheet 1) • 35

21 3. compositum ssp. ovatifolium (T. Sc 3.) Sweeney 8c Fisher comb. nov. (holotype, Sheet 2) ...... 37

22 Achenes of S, compositun S3]:.ovatlfoliun (,/-12^5) • 37

23 S. compositun ssp. ovatifolium (#123^. garden collection) ...... 3°

2k S* compositum ssp. ovatifolium (£l2*+5, field collection) ...... 38

23 Flowering periodicity in oilphium compositum subspecies ...o...... kk

26 Pictorialized scatter diagram of research garden clones of 3, compositum ssp. compositum and ssp. reniforme and 3. te rebin tliinaceum ...... 30

27 Pictorialized scatter diagram o' research garden clones of S. compositum ssp. compositum, ssp. venosun and ssp. ovatifolium...... 32

28 Graph of mean hybrid number against mean hybrid index for natural population samples of compositum ssp. compositum and ssp. reniforme • 36

29 Graph cf mean hybrid number against mean hybrid index for natural population samples of S. compositum ssp. compositurn, ssp. venosum and ssp. ovatifolium 63

30 Polygonogr^phs of 3. compositum population samples . £6

31 Micro-reflux extractor ...... 78

viii ILLUSTRATIONS (continued) Figure Page

32 Diagram of the extraction flash used on the aicro-reflux extractor...... 79

33 Diakinesis configuretion with 7 II in S. compositun ssp. compositum (#1273) *..»,, 10?

3** Diakinesis configuration with 7 II in S. compositum ssp, reniforme (,£95)...... 107

35 Diakinesis configuration with 7 II in 3. compositum ssp, venosum ( # 2 1 5 9 ) ...... 107

36 Diakinesis configuration with 7 II in 3, compositum ssp, ovatifclium (#123*0 •*••»• ...... *• 10?

37 Anaphase I configuration in S. compositum ssp* reniforme ( # 2 1 6 7 ; ...... * ...... 103

38 Anaphase II configuration in S. compositum ssp, compositum (#2162) 108

Macro- and micro-pollen grains in 3. compositum ssp, venosum (#2 1 5 9 ) ...... 103

*1-0 Anaphase I configura.tion with an inversion bridge and fragment in S, compositum sen, compositum ( ; # 9 3 ) ...... ' ...... 109

4l Anaphase II configuration with persistent anaphase I bridge in 3. compositum ssp, compositum (#177) • • 109

kZ Diakinesis configuration with 5 II a chain of four chromosomes in 3, compositum s„j, reniforme G A M f ) ...... 109

43 Iietaphase I configuration with 7 II o.nd 1 I in S. compositum ssp, reniforme (#2166) 109

kk Anaphase I configuration in cells with a univalent in S. compositum ssp, reniforme (7/2 1 6 6 ) . . , , , 111

ix II-LUSTKATIONS (continued) Figure Page

43 Anaphase II configuration with reductional division and lagging of the univalent in S. conuositum sen. compositum ( # 9 3 ) ...... Ill

46 Diakinesis configuration with 1 IV and 1?. II in S. conpositum ssp. venosum (#1293) ••••••• 111

47 Diakinesis configuration with 4 IV and 6 II in S. conpositum ssp. venosum (#1293) • ••*••• 111

43 Grossing relationships of some Silphium research garden clones ...... 113

49 S. compositun ssp. compositum (#177) garden collection) ...... 121

50 S. compositum ssp. reniforme (#92) garden collection) ...... 121

51 hybrid of S. compositum ssp. reniforme x ssp, compositum (#92 x #177» garden collection) . • 122

x INTRODUCTION

The genus Silphium L* is composed of twenty to twenty-five species of perennial herbs of the family Compositae, tribe

Heliantheae. The distribution of the genus is entirely North

American, ranging from north central United States east to the mid-Atlantic states, south to southern Florida, and west to Texas,

Oklahoma and the prairie states* Many of the species in the genus are found in somewhat disturbed situations such as old fields, dry

sand hills, river banks, woodland borders, and road-cuts. Several

species have rather restricted distributions*

All known species in the genus Silphium are diploid

(2n = l4) and reproduce sexually by out-crossing. Selfing occurs

rarely, and the plants are almost entirely self-sterile. There

have been no reports of apomixis in the genUB* Flowering occurs

over most of the summer and early fall. Vegetative reproduction

by means of shoot development from underground parte appears to be

common*

The genus Silphium has been placed in the tribe Heliantheae

because of the radiate head, chaffy receptacle and blunt anthers*

It is distinguished from other Heliantheae in having a combination

of monosporangiate (ovulate) ray flowers, monosporanglate (stand.-

nate) disc flowers and flattened achenes* The most closely related

genus in the tribe is probably Berlandiera DC. which differs from

1 2

Silphium in size, leaf shape, achene morphology and number of series of ray flowers#

Small (1933) described five sections within the genus on the basis of leaf position, grouping of inflorescences and pu­ bescence of the phyllaries* Section I, Perfoliata, included the perfoliate-leaved species; sections II and III, Laciniata and

Composita respectively, the basal-leaved taxa, and sections IV and

V, Dentata and Integrifolia respectively, the leafy-stemmed species.

Small included nine species in the section Composita, which have an open branching inflorescence arrangement rather than a racemose arrangement as is found in the section Laciniata. The species of the Composita, S. rumicifolium Small, S. terebinthin- aceum Jacq. and S. pinnatifidum Ell. are different from others in that section in having larger heads and longer ray flowers, and are therefore not considered in this treatment. The six remaining species are S. compositum Michx., S. reniforme Raf. ex Nutt., S. orae Small, S. lapsuum Small, S. venosum Small and S. ovatifolium

(T. St G.) Small. These will be treated as one species and referred to simply as S. compositum. In over-all appearance S. terebinthin- aceum resembles S. compositum, differing mainly in size of the head and texture of the leaves.

The species Silphium compositum Michx. is distributed widely in southeastern United States. It occurs naturally in the area from the Atlantic coast of southern Virginia to northern

Florida; from the Gulf coast of central to western Florida;

throughout eastern Alabama, Georgia, South Carolina, North Carolina, 3 eastern Tennessee and eastern West Virginia. The physiographic provinces over which S. compositum occurs include the Atlantic

Coastal Plain, the eastern part of the Gulf Coastal Plain, the

Piedmont, the Blue Ridge, the Ridge and Valley and the southern part of the Appalachian Plateau. Silphium compositum occurs frequently throughout this range, but it is usually not very abundant in any one area, it occurs as small populations generally composed of five to twenty scattered individuals along woodland borders. In many populations there are often several plants on which no flowering stalks are present.

Extreme variability in the morphology of S. compositum has been noted by several authors since the species was first described in 1803. Elliott (182*+) commented on the variation in the species, particularly in leaf shape and vesture. Curtis (1837) also noted the differences in degree of leaf dissection, but he commented that only one species was represented because of the similarity in leaf blade outline and inflorescence morphology. Torrey and Gray

(l8*+2) redescribed Silphium compositum as a species "well-marked in habit and character, although polymorphous in foliage." Because of the variability in the foliage, they described three varieties of 5. compositum. Perry (1937) also noted the morphological diversity in this species complex, but nevertheless, she included three distinct species, one with a variety and a form, in her treatment.

The objectives of the present study are (1) to assess the morphological variation throughout the range of the species; k

(2 ) to correlate morphological variation with chemical* cytological geographical, physiographical and ecological features; (3 ) to revise the nomenclature of thia species complex to reflect these findings; and (A-) to speculate on the manner in which the species has evolved. NOMENCLATURAL HISTORY

The first published name attributed to plants of Silphium compositum was S. laciniatum by Walter (1788). However, this

epithet was illegitimate, because it had been assigned previously by Linnaeus to a species of the mldwestern prairies of North

America, Walter’s plant, apparently from the Carolinas, was

described as having plnnately-sinuate leaves,

Michaux (1803) described a taxon under the name S, com­

positum, This species with sinuate-pinnatifid or even ternately

compound basal leaves extended from the Carolinas to Florida,

presumably on the coastal plain, Walter's S. laciniatum is listed

in synonomy by Michaux. Later that year Willdenow (1803) also

described S. compositum in the third edition of Species Plantarum

and cited S. compositum Michaux and S. laciniatum Walter as

synonyms.

Pursh (l8l*f) described S. elatum from a specimen in the

herbarium of Joseph Banks as a taxon with cordate-sinuate leaves

found in the Carolinas, It had first been by Banks as a manu­

script name. This taxon was later listed in synonomy under S.

compositum by Torrey and Gray (1842). Pursh, in addition, listed

S, compositum Willd. as a species of Virginia and the Carolinas,

As a synonym of S« compositum, Pursh included S. sinuatum from the

Banks collection. This name was also listed by Torrey and Gray in

synonomy under S* compositum Michx, Pursh was acquainted with

5 6

Willdenow’s work and quoted extensively the latter's description of S. compositum, but he (Pursh) did not mention Michaux's S. compositum directly*

Elliott (l82U) described S. terebinthinaceum characterized by reniform, slightly lobed leaves, from the mountains of North

Carolina to Alabama. The epithet terebinthinaceum had earlier been applied by Jacquin (1770) to a similar species of the wet prairies of North America. Elliott evidently mistook the rcniform-leaved

Silphium of the mountains for the robust ovate-leaved taxon of the prairies. He also recognized S. compositum Michx. as a sinuate, pinnatifid-leaved species of the pine barrens.

Rafinesque (1830) mentioned a Silphium reniforme in his

Medical Flora, but did not describe it. Nut tall (l8*fl) later provided a description for Rafinesque's S* reniforme. Nuttall also suggested that S. reniforme was "allied to S. terebinthin­ aceum," bi’t he did not state whether he meant S. terebinthinaceum

Jacq. of the prairies or S. terebinthinaceum Ell. of the mountains.

Silphium pseudo-laciniatum is a name applied to a specimen in the herbarium of L'Heritier de Brutelle by Fraser. The name was published by DeCandolle in his prodromus (1836) as a synonym of

S. compositum Michx.

Curtis (1837) listed two species of Silphium from the vicinity of Wilmington, North Carolina. One of these, S. canadensis,

is listed in synonomy with S. compositum Ell. without further

description. The other, S. terebinthinaceum var. sinuatum, having

pinnatifid basal leaves and small inflorescences, was a plant found 7 in sandy open wooded areas in the Carolinas and Virginia. It differed from the typical "terebinthinaceum1* in that the latter had undivided leaves and was not near the coast. Whether Curtis meant S« terebinthinaceum Jacq. or Ell* is not clear*

The name S. nudicaulls (S. nudicaule in Index Kewensis,

Vol. 2, p* 912) is attributed to Curtis by Perry (1937)» but this name does not appear in Curtis' article. Curtis' description of

S. terebinthinaceum var. sinuatum, however, appears on the page cited for the description of S. nudicaulis and begins "stem naked,

• • •" It is suggested by this author that Curtis' description of

S. terebinthinaceum var. sinuatum may have been translated into

Latin and as a result someone mistook the first phrase as the name of a taxon. In any case, S. nudicaulis is a nomen nudum.

In the Flora of North America Torrey and Gray (1842)

described three varieties of S* compositum: °< michauxii, Z3

reniforme and r ovatifolium* These were distinguished by differ­

ences in leaf morphology. When Torrey and Gray reduced Michaux*s

S. compositum to varietal rank, it appears that they renamed it

as S. compositum michauxii. They list Elliott's S. terebinthin­

aceum and Rafinesque*s S* reniforme in synonomy under S. compositum

reniforme. The varietal epithet ^ ovatifolium was applied to

plants from Florida which were described for the first time by

Torrey and Gray*

Small (1898) described S* venosum from the St. Mary's River

valley in southeastern Georgia. He stated that this species was

apparently restricted to the pine barrens in the area around the 8

Okefenokee Swamp. He separated it from S. compositum on the basis

of both leaf and achene characters.

Greene (1899) described S. collinum from the southern

Appalachian mountains. Perry (1937) listed S. collinum as a

questionable synonym of S. compositum.

Small in the Flora of the Southeastern United States (1903)

raised S. compositum var. ovatifolium to specific status and applied

it to plants growing on the coastal plain of southern Georgia and

Florida. The variety reniforme was also given specific status,

and its range was given as the mountains of North Carolina. Both

were distinguished from S. compositum by leaf and achene characters.

Silphium compositum, in the sense of Small (1933)* is a species of

the "coastal plain and adjacent provinces" ranging from North

Carolina and Tennessee to Georgia and Alabama.

Silphium orae was described by Small (1933) as a species

of the coastal plain of North and South Carolina which differed

from S. compositum by leaf morphology. Small (1933) also described

S. lapsuum, a species occurring in the sandhills near the Fall Line

in Georgia and South Carolina. It was distinguished from other

similar species by the scabrousness of the leaves.

Perry in her monograph of the genus (1937) reduced S. orae

Small to a form of S. compositum, and S. reniforme to varietal

rank under £3. compositum. Silphium venosum Small and S. ovati­

folium (T. 8c G.) Small were retained with specific status. 9

Silphium lapsuum Small was listed in synonomy under S. compositum by Perry.

As a result of this study it is proposed that Silpnium compositum is composed of four subspecies: 6sp. compositum. ssp. reniforme. ssp. venosum. and ssp. ovatifolium. GENERAL MORPHOLOGY OF THE GENUS

Habit. The species of Silphium are coarse, erect, perennial herbs, 0.5-3.5 meters in height.

Underground parts. The perennial underground part is composed of woody tap roots, fibrous rhizomes or a combination of both.

Stems. The stem or stems arising from the underground parts may be terete or ^-angled. They may be hirsute, strigose, scabrous, glandular, or completely glabrous or glacous. Stems of some species contain a resinous juice, and hence the name "rosin- weed."

Leaves. In all species of Silphium there is a spring rosette of basal leaves. In some species the basal leaves are persistent throughout the season, while in others they are early deciduous. In species which retain the basal leaves the remainder of the flowering stalk may be completely leafless or have a few reduced cauline leaves. In other species in which the basal leaves are shed, the flowering stalks are usually quite leafy. The cauline leaves may be alternate, opposite or whorled. Leaves may be

petiolate, sessile or connate -perfoliate.

Leaf blades range in shape from lanceolate to ovate or

reniform. The margin of the leaf blades may be entire, crenate-

dentate or lobed, of the blade may be pinnatifid or completely

10 11 dissected. Surfaces of the blades may be scabrous, hirsute, strigose, glabrous or glaucous.

Inflorescence. The inflorescence is a head, which is borne upright, singly on a stalk or in racemose or cyxnose clusters at the ends of a modified dichasial branching system. The head is radiate, being composed of ligulate ray flowers and tubular disc flowers.

Phyllaries. The heads are surrounded by two or three series of imbricate phyllaries. The shape of the phyllaries ranges from lanceacute to ovate. They may be glabrous, strigose, hirsute, glandular, or glabrous with ciliate margins. The length of the phyllaries ranges from 0.5 cm in some species to b cm in others.

Ray flowers. The ligulate flowers are monosporangiate

(ovulate) and range in number from five in some species to more than thirty in others. The color of the corolla may be lemon- yellow, orange-yellow or white. The ovules are laterally com­ pressed and in two or three series.

Disc flowers. The disc flowers are tubular with pale yellow, orange-yellow or white corollas. They are numerous and apparently monosporangiate (staminate); the ovules, if present, abort early.

The stamens are nearly connate and exerted from the tube. The pollen grains are round and spiny.

Receptacle and chaff. The receptacle may be either convex or flat. The pales of the chaff are lanceolate-acute and are

sometimes persistent for a time after the achenes have abscised. 12

They are hyaline, sometimes with purple pigmentation at the tips*

The pales may be strigose, stipitate-glandular or glabrous.

Fruit. The mature fruit is a laterally compressed achene

(cypsela) which is obovate or orbicular in shape. It may be either hirsute or glabrous and is usually surrounded by a narrow to broad wing. The wing is visually extended at the apex forming a shallow

to deep U- or V-shaped sinus. In some species, where the wing is not extended, the apex of the achene is truncate. Often aristate awns are found at the margins of the sinus. In Silphium compositum an additional awn may arise from the adaxial surface near the sinus.

There is no morphological structure which can be termed a pappus. TAXONOMY

Silphium compositum Michaux, FI. Bor. Am. 2:145. 1803*

Perennial with woody taproot; 1-5 m in height; basal leaves petiolate, blades elliptic to reniform in outline, often with prominent red veins, glabrous on the upper surface, glabrous or scabrous on the lower; leaf blades entire to variously dis­ sected, and in some becoming compound, margin sometimes serrate or doubly toothed; cauline leaves few and reduced in size, petiolate, lobed but not compound. Flowering stalk few- to many-branched, branching mainly in the upper half of the stalk; involucre 1-5 cm in width, composed of 2 or 5 series of imbricated, ciliate-margined phyllaries, ray flowers 5-10, light yellow to lemon-yellow, 0.5-

1.5 cm long; disc flowers numerous, yellow; achenes obovate to orbicular in outline, 6-l4 mm long, hirsute on the adaxial surface, narrow- to wide-winged, with a V- or U-shaped sinus at the apex, the wing tips acuminate to obtuse.

Key to the Subspecies of Silphium compositum

Flower heads several to many, and dense; involucre with achenes at maturity usually less than 2 cm in diameter; achene wing less than 2 mm wide

Basal leaf blades ovate in outline, usually longer than wide; leaf blade palmately to pinnately sinuate to compound; phyllaries shorter than the mature achenes ..•••••• ...... S. compositum ssp. compositum • . 1

13 l*f

Basal leaf blades reniform in outline, frequently wider than long; leaf blade entire to lobed more than half-way to the midrib; phyllaries longer than the mature achenes . • • • ...... S. compositum ssp. reniforme . . 2

Flower heads few, and sparse; involucre with achenes at maturity usually 2 cm or more in diameter; achene wing 2 mm or more wide

Achene wing tip acute; leaf blade usually as long as wide; petiole equalling or longer than the midrib ..*•••• ...... S. compositum ssp. venosum . . 3

Achene wing tip obtuse; leaf blade usually longer than wide; petiole equalling or shorter than the midrib ...... S. compositum ssp. ovatifolium . . 4

1. Silphium compositum Michx. ssp. compositum comb. nov.

Silphium compositum Michx. FI. Bor. Am. 2:1^5* l803*

(Holotype: "In sylvis maritimis a Carolina ad Floridam."

s.d., Andre Michaux s.n., specimen identified by P

3728. P. photograph!) See Figure 1.

S. laciniatum Walt. FI. Carol. 217. 1788. non L. 1753*

■S. compositum Willd. Sp. PI. 2331. 1803.

S. sinuatum Banks ex Pursh, FI. Am. Sept. 2:577. l8l6.

pro syn.

S. elatum Pursh, FI. Am. Sept. 2:579* l8l6. pro syn.

S. pseudo-laciniaturn Fras. in herb. L'Her. ex DC, Prodr.

5 :512. 1836. pro syn.

S. canadensis Curtis, Boat. Jour. Nat. Hist. 1:103* 1837.

pro syn.

S. terebinthinaceum var. sinuatum Curtis, Bost. Jour. Nat.

Hist. 1:127. 1837. S. nudicaule Curtis, Bost. Jour. Nat. Hist. 1:127. 1837. nom. nud.

S. compositum ^ michauxii T. & G. FI. N. Am. 2:276. 18^2. ^ h I 14 J« v

C * h < i i » / i

Fig. 1. Silphium compositum Michx. Copy of holotype photograph (MSC), courtesy of J. H. Beaman. Scale: 0.39x. 16

S. collinum Greene, Pittonia 4:44. 1899*

S. orae Small, Man. Se. FI. l4ll. 1953*

(Holotype: North Carolina: CNew Hanowver Co.]: sandy

country about Wilmington, s.d., M.A. Curtis s.n., NY1)

See Figure 2.

S. composltum f. orae (Small) Perry, Rhodora 39:295* 1937*

(based on Curtis s.n.)

Leaf blades generally ovate in outline, much dissected, often palmately to pinnately compound, petioles as long as midrib, base of blade cordate to sagittate, margin entire to serrate, the upper surface of the blade glabrous, the lower, glabrous to slightly scabrous; flowering stalk much branched, with many heads, involucre

1 .0-1.5 cm wide, ray flowers 5-7 i 1 cm or less long; achenes 6-9 mm long, equalling or exceeding the phyllaries in length at maturity; achene wing 1 mm or less wide, with long acute to acuminate wing tips on either side of a V-shaped sinus, occasionally with an aristate awn near the sinus (see Fig. 3 and 4).

Habitat. In sandy soil along the borders of or in thin pine or pine-hardwood forests.

Distribution. Coastal plain and piedmont in southeastern

Virginia, eastern and central North and South Carolina, and central

Georgia and eastern Alabama (see Fig. 5)*

The typical specimens of subspecies compositum (as illus­ trated in Fig. 6 and 7) are found on the coastal plain of North and

South Carolina, and are characterized by very dissected or even

compound leaves and acuminate achene wing tips which extend a short 17

Fig* 2. Silphium orae Small* Holotype (NY). Scale: 0.28x. Fig. 3* Achenes of S. compositum ssp• compositum (#2162). Note aristate awn in sinus. Scale: *K)Ox.

1- 1 i —

k L. i k □a L Ell li i ji ■ L m !_ ___1___■11 “1 ! i L

Fig. 4. Achenes of S. compositum ssp. compositum (#1309)* Scale 3?5X * File. 5* Distribution map of S . compositum 6Sp. compositum and sep. compositum-r e m f o r m e intergrades•

19 2 0

figure 5 - r

/3/V

Fig. 6 . S. compositum ssp. Fig. 7. S. compositum ssp. compositum (/^131^» field collection). compositum (#1301* field collection) Scale: 0.20x. Scale: 0.20x. 22 distance beyond the phylXaries at maturity. Intergrades with ssp, reniforme are found mainly on the piedmont of South Carolina and

Georgia and on both the coastal plain and piedmont of North

Carolina, These are characterized by moderately dissected leaves and acute achene wing tips which usually do not exceed the phyl- laries in length at maturity (see Fig. 8 and 9), Both the typical plants and the intergrades are associated with the loblolly-

shortleaf pine forest type (see Fig. 10).

2. Silphium compositum Kichx. ssp. reniforme (Raf. ex Nutt.)

Sweeney & Fisher comb, nov.

S. reniforme Raf. Med. FI. 2:263. 1830 . noa. nud*

S. reniforme Raf. ex Nutt. Trans. Am. Philos. Soc, n. ser,

7:342. l84l.

(Lectotype: s.d. C.S. Rafinesque s.n. PHI (see Fig. 11).

S. terebinthinaceum Ell. Sk. 2:463. 1324. non Jacq. 1770.

S. compositum ^ reniforme (Raf. ex Nutt.) T. & G. FI. N.

Am. 2:276. 1842.

(based on Rafinesque s.n.)

Basal leaf blades generally reniform in outline, entire to

somewhat dissected (usually less than half-way to the midrib),

petioles often longer than the midrib, base of blade cordate to

sagittate, margin entire to serrate, the upper surface of the blade

glabrous to somewhat scabrous; flowering stalk usually less branched

than in ssp* compositum, with several to many heads; involucre 1 .5-

2.0 cm wide; ray flowers 6-8 , 1 cm or more long; achenes 6-9 mm

long, shorter than the phyllaries at maturity, achene wing 1 mm or Fig. 9» S. compositum ssp. Fig. 8 , S. compositum ssp. compositum-reni forme intergrade (#1517, compositum-reniforme intergrade (#1527, field collection). Scale: 0.20x. field collection). Scale: 0.20x.

ro v>i Fig. 10. Distribution map of S. compositum subspecies. Ecological zones redrawn "from map by Hedlund and Jassen (1963)* courtesy of USDA, U.S. Government Printing Office.

2k 25

,1 ^J'1^ A * Ml' K O iN B ilirtt'1 Oak-ptM and Mfc-hick*ry forvsts

□ lahhlly ■harHiif pin* an4 I

^ UnflMf-ilaili pin* fornt

A t e m p e i i f u m

• »»p. r # n l f * r m « * X *tp. vtnMUin

m * «p. tV fltlltltW IN

9CALC IN M L C S

**-•*»» CQU*L ***A HAOjCCTtON

Figure 10 26

Fig* 11 * Silphium compositum ssp* reniforme (Raf. ex Nutt.) Sweeney 8c Fisher comb. nov. Lectotype (PH). Scale: 0.28x.

1 Fig. 12. Achenes of Silphium compositum ssp* reniforme (#2165)• Scale; 3&5x. 27 less wide with short acute wing tips or merely short awns on either side of a wide and shallow V-shaped sinus (see Fig. 12), rarely

with an aristate awn near the sinus.

Habitat. On shale, sandy-clay or sandstone slopes at the

edge of hardwood, pine-hardwood or occasionally pine forests.

Distribution. Mountains of eastern Tennessee; mountains

and piedmont regions of southern Virginia, western and central

North Carolina, western South Carolina, and northern Georgia; and

dissected plateau of northeastern Alabama (see Fig. 13)*

The typical specimens of ssp. reniforme (as illustrated in

Fig. 1^) are found in the mountains of eastern Tennessee and

western North Carolina and Virginia. In the piedmont area ssp.

reniforme is sympatric with ssp. compositum, and plants from that

area approach the ssp. compositum-reniforme intergrades mentioned

above. The typical ssp. reniforme is associated with the oak-pine

and oak-hickory forest types (see Fig. 10).

3. Silphium compositum Kichx. ssp. venosum (Small) Sweeney &

Fisher comb, nov.

S. venosum Small, Bull. Torr. Bot. Club 25:^73. 1898.

(Holotype: Georgia: Charlton Co.: in the St. Mary's

River Swamp, below Trader's Hill, June 12-15, l895»

John K. Small s.n. NY I (see Fig. 15)* Isotype: F!

S. lapsuum Small, Man. Se. FI. l^fll. 1933*

(Holotype: Georgia: [Richmond Co.3: dry oak savannah,

Augusta, July 17, 1898 , A. Cuthbert s.n. NY! (Bee

Fig. 16). Isotype: NY! Fig. 13* Distribution map of fl. compositum ssp. reniforme.

28 4'K.VSI'K OP I'HK OP ltU(KK M L £ & 9C*L£ IN 9C*L£ ttp. t»Bifgrai SilohiMt eowBoiitui • • • • • • •• • • • •

Figure 13 Fig. ]A. S. compositum ssp. Fig. 15* Silphium compositum ssp. reniforme (#2166, garden collection). venosum (Small) Sweeney 8c Fisher comb, nov. Scale: 0.20x. Holotype (NY). Scale: 0.29x. 31

S 3 S P T 5

Small

Fig. 1 7 . Achenes of S. comt>os-i tm* ssp. venosum (#1 2 8 8 ). Scale,Sojg;p - 32

Basal leaf blades generally ovate in outline, deeply dis­ sected or pinnately compound, petioles usually equalling or shorter than the midrib, base of the blades attenuate to sagittate, margin entire to serrate, upper surface of the blade glabrous, lower, slightly to very scabrous; flowering stalk little to much branched, with several heads; involucre 1 .5-2.5 cm wide; ray flowers 7 -9 » about 1 cm long; achenes 8-12 mm long, longer than the phyllaries at maturity; achene wing 1-2 mm wide, with acute to acuminate wing tips, on either side of a V-shaped sinus (see Fig. 17), oc­ casionally with an aristate awn near the sinus.

Habitat. In sand or sandy-clay soil, along the borders of or in thin pine or pine-hardwood forests.

Distribution. Coastal plain of southeastern North Carolina,

South Carolina, southern Georgia and northern Florida (see Fig. 18).

Subspecies venosum is sympatric with ssp. compositum in the northern part of its range and with ssp. ovatifolium in the southern part. In morphological characters ssp. venosum is inter­ mediate between these two taxa, having the large heads and achenes of ssp. ovatifolium, and the acute wing tips and dissected leaves of ssp. compositum (see Fig. 19). However, it appears to be

distinct enough in its morphology and distribution to warrant sub­

specific status. Typical ssp. venosum is mainly associated with

the longleaf-slash pine forest type. Fig. l8. Distribution map of S. compositum ssp. venosum and ssp. ovatifolium.

33 n - k w -: < < -: hi vi ni vi i im Hi Hi m a t i l o l m m up. UftOHim - L'S llB h lu m COBIBOtltMIB L u p «o — ° •v

Figure 18 N l

Fig. 19* S. compositum ssp. Fig. 20. Silphium compositum ssp< venosum (#125^i garden collection). ovatifolium (T. 8c G . ) Sweeney 8: Fisher Scale: 0.29x. comb, nov. Holotype, sheet #1 (NY). Scale: 0.28x. 36

4. Silphium compositum Michx. ssp. ovatifolium (T. & G.) Sweeney

& Fisher comb, nov.

S. compositum ovatifolium T. & G. FI. N. Am. 2:277* 1842.

(Holotype: Florida: s.d., Dr. Chapman 138* NYl (see

Fig. 20 and 21).

S. ovatifolium (T. & G.) Small, FI. Se. U. S. 1242. 1903*

(Based on Chapman 138)

Basal leaf blades generally ovate to elliptic in outline, entire to lobed, occasionally dissected, petioles usually shorter than the midrib, base of the blade usually attenuate to truncate, rarely cordate, margin entire, occasionally serrate, both upper and lower surfaces of the blade, glabrous; flower stalk, little branched, with few to several heads, involucre 2.0-3.0 cm wide, ray flowers 7-8, 1.0-1.5 cm long; achenes 9-14 mm long, longer than the phyllaries at maturity; achene wing 1.5-2.0 mm wide, with obtuse or slightly acute wing tips on either side of a narrow U- shaped sinus (see Fig. 22).

Habitat. In sand along the edge of pine or pine-hardwood forests.

Distribution. Coastal plain in southern South Carolina, southeastern Georgia and central and northern Florida (see Fig. 17)*

In the southern extreme of its range ssp. ovatifolium is very distinct (see Fig. 23 and 24) and does not show any inter­ gradation with ssp. venosum. In Georgia and South Carolina, how­ ever, ssp. compositum, ssp. venosum and ssp. ovatifolium are all 37

Fig. 21. £. compositum ssp. ovatifolium (T. & G.) Sweeney g* Fisher comb, nov. Holotype, sheet #2 (NY). Scale: 0.28x.

i M A k. i i l a w

M I 1 I 1 I 1 11 1 Li Fig. 22. Achenes of S. compositum ssp. ovatifolium (#12^). scale: 375*. Fig. 23. S. compositum ssp. Fig. 2^. S. compositum ssp. ovatifolium (#123\ garden collection) ovatifolium (#12^5* field collection). Scale: 0.2&x. Scale: 0.29x. V* 00 39 found within close proximity of each other, and intergrades can be found.

In Table 1 the diagnostic characters of the four sub­ species are presented for comparative purposes. TABLE 1* Diagnostic characters of Silphium compoaitum subspecies

Character ssp. compositum ssp. reniforme ssp. michauxii ssp. ovatifolium leaf blade base cordate to cordate to attenuate to attenuate to sagittate sagittate sagittate truncate leaf blade shape ovate reniform ovate ovate to elliptic petiole length: > or = 1 > 1 = or < 1 < 1 midrib length relative number numerous several to several few to several of heads many achene length: = or > 1 < 1 > 1 > 1 phyllary length involucre width 1.0-1.5 cm 1*5-2.0 cm 1.5-2.5 cm 2.0-3.0 cm achene length 6—9 mm 6—9 mm 8-12 mm 9-1 k mm achene wing 0.5-1.0 mm 0.5-1.0 mm 1.0-2.0 mm 1.5-2.0 mm width achene wing- acuminate to acute or acute obtuse or tip shape acute absent slightly acute

-f o ECOLOGY OF SILPHIUM COMPOSITUM

Data on associated forest and soil characters from herbarium labels were tabulated for the four subspecies and the ssp. com- positum-reniforme intergrades. Only those specimens for which both forest and soil data were available were used. The data were taken as the collector wrote them on the label. Three categories for forest and soil characters were established. The three forest types were pine, pine-hardwood and hardwood; the three soil types were sand, sand-clay or sandy-loam, and clay or shale.

There was a tendency for all subspecies to be associated mainly with sandy soil, with very few specimens recorded on clay or shale (Table 2). Several specimens of ssp. reniforme were found on shale slopes, but were not included here because the corresponding forest data were lacking. Too few specimens of ssp. renfforme could be used to ascertain the associated forest type, but from the forest type map (Fig. 10), it appears to occur mainly in the hardwood and pine-hardwood forest regions. The ssp. com- positum-reniforme intergrades were found most often near pine- hardwood forest. Subspecies compositum and ssp. venosum were most often associated with pine and least often with hardwood forests,

while ssp. ovatifolium was most often found with pine-hardwood and

least often with hardwood forests.

*fl 42

TABLE 2. Numbers of specimens of S. compositum subspecies associ­ ated with forest and soil types

Forest Type

Soil Type Pine Pine-hardwood Hardwood Total ssp* compositum

sand 8 7 3 18 sandy-clay 2 o 0 2 clay or shale 0 0 1 1 total 10 7 4 21 ssp. compositum- reniforme intergrades

sand 7 9 2 18 sandy-clay 1 3 0 4 clay or shale 1 2 1 4 total 9 14 3 26 ssp. reniforme

sand 4 2 0 6 sandy-clay 1 2 1 4 clay or shale 0 0 0 0 total 5 4 1 10

ssp. venosum

sand 7 4 2 13 sandy-clay 3 1 0 4 clay or shale 0 1 0 1 total 10 6 2 18

ssp. ovatifolium

sand 7 14 2 23 sandy-clay 0 o 0 0 clay or shale 0 o 0 0 total 7 14 2 23 **3

The flowering periods of S. compositum subspecies are concurrent throughout most of the summer* The collection dates

(by month) of specimens with ray flowers attached to the receptacle were recorded for each subspecies. The flowering period for the species in all parts of its natural range was from early May to mid-Cctober (see Fig. 25) • The earliest date of flowering recorded for S. compositum ssp. reniforme was mid-June and the peak blooming time was August. The other three subspecies were found in flower in May, but the time of peak flowering varied.

For ssp. compositum the greatest number of flowering specimens were collected in September, while for ssp. ovatifolium, the peak period was June. The morphological and geographical intermediate, ssp. venosum, had two peak flowering periods, June and September. Fig. 2 % Flowering periodicity in Silphium compositum subspecies.

t

bk Figure 25. Flowering periodicity in Silphium compositum subspecies

40-

30-

c 1 v o £20- o. i , 1 1 , i "I i

10- ^5 ^ ^ , 1 1 1 1 l l i l l | H I

0. M illIIs ssp.ill!. reniforme ssp. compositum ssp. venosum ssp. ovatifolium Earliest date: Earliest date: Earliest date: Earliest date: June 10, Burke Co. , May 12, Edgefield May 13, Allen­ May 7, Alachua Co., N. C. Co., S.C. dale Co. , S.C. Florida Latest date: Latest date: Latest date: Latest date: Oct. 10, Alamance Oct. 4, Jones Co. , Oct. 19, Horry Oct. 7, Bay Co. , Co. , N.C» N.C. Co. , S.C. Florida

Number of specimens: Number of specimens: Number of specimens: Number of specimens: 123 124 31 34 MORPHOLOGICAL ANALYSIS OF RESEARCH GARDEN CLONES AND POPULATIONS COLLECTED IN TEE FIELD

Analysis of research garden clones

Clones representing populations of Silphium compositum from throughout its range have been placed in culture in the research garden at The Ohio State University. Because root stalks failed to survive transplanting, all clones are from seed col­ lections. The collection numbers and sites are listed in Table 3»

TABLE 3« Numbers and collection sites of research garden clones of Silphium compositum

Subspecies Number Collection Site reniforme 91 Dinwiddie Co., Virginia 92 Madison Co., North Carolina 95 Rowan Co., North Carolina ikk Caldwell Co., North Carolina 1139 Marshall Co., Alabama 1271 Muscogee Co., Georgia 128l Heard Co., Georgia 132^ Granville Co., North Carolina 2138 Walker Co., Georgia 2139 Etowah Co., Alabama 21^9 Calhoun Co., Alabama 2150 Haralson Co., Georgia 2151 Haralson Co., Georgia 2155 Baldwin Co., Georgia 2165 Catawba Co., North Carolina 2166 Catawba Co., North Carolina 2167 Mecklenberg Co., North Carolina

compositum 93 Stanly Co., North Carolina 177 Aiken Co., South Carolina ll^fO Marshall Co., Alabama 1263 Peach Co., Georgia 1265 Taylor Co., Georgia 1278 Troup Co., Georgia 1301 Charleston Co., South Carolina kf> 4 ?

TABLE 3 (continued)

Subspecies Number Collection Site compositum 1302 Georgetown Co*, South Carolina 1306 Horry Co., South Carolina 1309 Brunswick Co., North Carolina 1311 New Hanover Co., North Carolina 1314 Lenoir Co., North Carolina 1315 Pitt Co., North Carolina 1316 Greene Co., North Carolina 1313 Wake Co., North Carolina 1319 Wake Co., North Carolina 1320 Wake Co., North Carolina 1321 Wake Co., North Carolina 2162 Saluda Co., South Carolina venosum 1248 Appling Co., Georgia 1249 Appling Co., Georgia 1250 Appling Co., Georgia 1252 Toombs Co., Georgia 1254 Montgomery Co., Georgia 1255 Laurens Co., Georgia 1258 Twiggs Co., Georgia

1288 Richmond Co., Georgia - 1289 Aiken Co., South Carolina 1290 Aiken Co., South Carolina 1295 Orangeburg Co., South Carolina 1298 Dorchester Co., South Carolina 2159

ovatifolium 1207 Okaloosa Co., Florida 1209 Walton Co., Florida 1213 Walton Co., Florida 1234 Putnam Co., Florida 1237 St. Johns Co., Florida 1242 Nassau Co., Florida 1245 Charlton Co., Georgia

Specimens of S. compositum from the research garden were

collected for morphological analysis* Only specimens which included

mature leaves and inflorescences with achenes were used in this

analysis. The following characters were measured and recorded: (1)

width of the head (when pressed) with mature achenes, in mm; (2 ) * f 8 length of the achene in mm; (3 ) width of the achene wing half-way down the side of the achene in mm; and (4) length of the achene sinus in mm. Other characters were scored subjectively: (1) relative density of the inflorescence-sparse, intermediate or dense; (2 ) achene sinus shape— deep, medium or shallow notched;

(3 ) wing tip shape— acuminate, acute or obtuse; and (A-) leaf base shape— cordate to sagittate, truncate, or attenuate.

Other characters were expressed in terms of a ratio: (1) achene length/phyllary length; (2 ) petiole length/midrib length;

(3 ) leaf blade length/leaf blade width; and (4) leaf dissection

index. The leaf dissection index is computed in the following

manner: p Leaf dissection index = =--- — L + W

where P is the perimeter of the blade measured with a map distance

measurer, and L and W are the length and width at the longest and

widest place respectively. This index has the advantage of

allowing blade dissection to be estimated more or less independ­

ently of the sise and shape of the blade.

In a preliminary study, many other characters, such as ray

flower length, number of ray flowers, phyllary length and width,

achene sinus width, achene shape and achene width, were also

measured and scored. These were all rejected as usable characters

for one or more reasons: (1 ) very little measurable variability

throughout the species; (2 ) lacking in many specimens or in immature

condition at the time of collection; and (3 ) directly dependent on ^9 another character. An example of the latter is the dependence of achene Bhape on the length and width of the achene.

Pictorialized scatter diagrams according to the method of

Anderson (19^9) were constructed for the comparison of the sub­ species of Silphium compositum. Figure 26 is a plot of represent­ atives of ssp. reniforme and ssp. compositum and Silphium terebin­

thinaceum, the species most closely resembling S. compositum.

Silphium compositum ssp. compositum clusters at one extreme of the graph, and S. terebinthinaceum at the other end. Silphium com­ positum ssp. reniforme, though somewhat variable, clusters near

and slightly intergrades with ssp. compositum. However, it is

intermediate between 3. compositum ssp. compositum and S* terebin­

thinaceum in several characters. An artificial hybrid of S. com­ positum ssp. reniforme x S. terebinthinaceum (#92 x #146) is also

plotted on the scatter diagram. It occupies an intermediate

position between the two parental species and closely resembles a

specimen of S. compositum ssp. reniforme from West Virginia.

Figure 27 is a pictorialized scatter diagram derived from

representatives of ssp. compositum did not include those believed

to be intergrading with ssp. reniforme. In this instance ssp.

compositum ssp. ovatifolium cluster at opposite ends of the graph,

and ssp. venosum lies in an intermediate position.

Such a diagram is a graphic representation of genetic

linkage and recombination of polygenic characters. With the ad­

dition of glyphs, the association of more than two characters can

be shown in one diagram (Anderson, 19^9)• Introgressive Fig. 26. Pictorialized scatter diagrams of research garden clones of 3. compositum ssp. compositum and ssp. reniforme and S. Terebinthinaceum.

50 Leaf Dissection Achene Achene Head Index Wing Width Sinus Length Number

O 1.00-1.99 O 0.0m m O < I mm O few a 2.00-2.99 0 0.5m m 0 1.0-1.9mm O' intermediate ( \ 3.00- 3.99 p 1.0 mm vO 2.0-2.9mm c f many 1.5 < \ > 4 .0 0

f t O S. compositum ssp. compositum £ § compositum ssp. reniforme • §. terebinthinaceum a* TO <9 Artificial F, hybrid of 5 S. compositum X £. terebinthinaceum ro w>* On O

a.-C 1.0 o> c <

0.5 J _____ I----- 1----- 1----- 1----- L. 10 14 18 22 26 30 34 38 42 46 Head Diameter in mm Fig. 27. Pictorialized scatter diagram of research garden clones of S. compositum ssp. compositum, ssp. venosum and ssp. ovatifolium.

52 1 - r M _i Achene Sinus 3.0 E E 4.0 3.5 . q—p — q i.o 2.5 2.0 1.5 2 14 12 Midrib L Rel. Head Number e b m u N d a e H . l e R L b i r d i M : L e l o i l e P e p a h S e s a B f o e L x n xx & Cf attenuate * 0 sagittate O ---- cordate cordate truncate p S Or 16 P o 1.00-1.49 1.00-1.49 > 1.49 1.49 > < I< 00 18 & m m n i r e t e m a i D d a e H J* 20 O many many O Q. intermediate intermediate Q. few X 22 j. e p a h S p i t - g n i W P P O acuminate acuminate O X X X X _ 2 4 acute i _ obtuse j ---- s. compositum ssp. • ssp. O ® ssp. ssp. ® 6 8 2 26 1 ---- 1 ---- venosum ovotifolium 1 Vn 10 5 ^ hybridization is indicated by the occasional association of the characters typical of one taxon with that of the other taxon.

A definite association of head width and achene sinus length is indicated in Figure 27* In addition the glyphs indicate the association of other characters, such as leaf base shape and relative inflorescence density. Introgression is also indicated by the presence of some representatives having characters usually associated with the other subspecies. The scatter diagram can be interpreted as a recombination spindle, with ssp. venosum occupying the position of a theoretical hybrid. The representatives of ssp. compositum and ssp. ovatifoliiun also show some variability, which may be the result of continued backcrossing of hybrids to both parents. The variation indicated here appears to be con­ tinuous, particularly between ssp. venosum and ssp. ovatifolium.

Association is also indicated in Figure 26 between head diameter and achene length/phyllary length. Subspecies reniforme does not occupy the position of the theoretical F^ of S. compositum ssp. compositum x S. terebinthinaceum, but rather that of deriv­ atives of repeated backcrosses to S. compositum ssp. compositum.

The variation between ssp. compositum and ssp. reniforme is con­ tinuous.

Analysis of populations

Populations of Silphium compositum were collected and analyzed morphologically. Hybrid indexes (Anderson, 19^9) were computed for each specimen, and the mean hybrid index value for each population was obtained. In addition the hybrid number for 55

each specimen and the mean hybrid number for each population was calculated according to Gay (i960). For S. compositum ssp. com­ positum and ssp. reniforme populations the mean hybrid index values and mean hybrid numbers are listed in Table k . The plotting

of the mean hybrid index value against the mean hybrid number for

each population in the diagram presented in Figure 28.

Gay's hybrid number is calculated from the hybrid index

value for each specimen. If the hybrid index runs from 0 to 12,

as it does in Figure 28, then by definition the hybrid number

ranges from 0 to 6 . For any specimen whose hybrid index value

lies between 0 and 6 , the hybrid number is the same. For any

specimen whose hybrid index value lies from 6 to 12, the hybrid

number is 12 minus the hybrid index value. As a result an indi­

vidual specimen of S. compositum ssp. reniforme might have a

hybrid index score of 1 , and thus would also have a hybrid number

score of 1. A specimen of S. compositum ssp. compositum, on the

other hand, might have a hybrid index score of 10, and thus would

have a hybrid number score of 12 minus 10, or 2. The mean hybrid

number is computed in the same manner as the mean hybrid index.

The advantage in using the mean hybrid number in addition

to the mean hybrid index is that the composition in terms of

hybridity of a population can be much better demonstrated. If a

hypothetical population, a, were found in which there were equal

numbers of two parental species and only one or two F^ hybrids,

the mean hybrid index score using the scale in Figure 26, would be

about 6. If, on the other hand, another population, b, composed Fig* 28. Graph of mean hybrid number against mean hybrid index for natural population samples of S. compositum ssp. compositum and ssp. reniforme.

56 Mean Hybrid Number 4 6 0 2 3 5 0 2 . opstm s. compositum ssp. reniforme compositum ssp. S. O compositum S. # Figure 28 Figure 4 en yrd Index Hybrid Mean 8 6 012 10 57 58

TABLE 4. Population analysis of S. compositum ssp* compositum and ssp. reniforme

Mean Hybrid Mean No* of Index Hybrid Population Individuals Value Number Location

Subspecies compositum

2430 7 6.1 5.7 Bertie Co., N. C. 2431 4 6.0 4.5 Edgecombe Co., II. C. 2432-3 2 10.0 2.0 Wayne Co., N • C. 2436 8 9.3 2.6 New Hanover Co., N. C. 2437 8 3.5 3.5 Columbus Co., N . C. 2438 6 6.8 5.1 Marion Co., S. C. 2^39 12 9.7 2.3 Florence Co., S. C. 2463 5 8.4 3.6 Muscogee Co., Ga. 2464 6 8.0 4.0 Harris Co., Ga. 2466 3 8.3 3.6 Jasper Co., Ga. 2467 4 9.0 . 3.0 Abbeville Cc., S. C. 2468 6 5.8 5.5 Laurens Co., S. C. 2470 3 8.6 3.3 Chester Co., S. C. 2473 4 8.0 4.0 Randolph Co., N. C. SC-1 3 8.0 3.3 Chesterfield Co., S. C. SC-2 3 9.6 2.6 Berkeley Co., S. C. sc-3 2 7.5 4.5 Darlington Co., S. C. V-2. 2 9.5 2.5 Sussex Co., Va.

Subspecies reniforme

2427 6 4.6 4.6 Dinwiddie Co., Va. 2440 5 4.0 4.0 Richland Co., S. C. 2469 7 6.0 5.1 Union Co., S. C. 2474 2 1.5 1.5 Pittsylvania Co., Va. A 7 1.2 1.2 Bath Co., Va* NC-1 3 4.6 4.6 Buncombe Co., N. C. mostly of hybrids were found, the mean hybrid index score would also be about 6. It would be very clear to the observer that these

two populations are not nearly as similar as the mean hybrid index values indicate. The difference between these two populations 59 could be graphically shown only by including the frequency diagrams for each population.

By using in addition the mean hybrid number this difference between these two and many other populations can be shown in a single graph. Populations similar to the hypothetical a with a mean hybrid index score of 6 would have a mean hybrid number of near 0 and would plot in the middle of the base line of the tri­ angle. Populations, like b, with a mean hybrid index of 6, would also have a mean hybrid number of about 6 and would plot near the apex of the triangle, populations with equal numbers of parents and hybrids would be found near the middle of the triangle. Popu­ lations consisting of hybrids and only one parent or populations consisting of hybrid derivatives which backcrossed to only one parent would be plotted on or near the sides of the triangle.

Populations containing mostly specimens of one parent with only a few hybrids and many specimens of the other parent would be found near the bottom of the triangle towards one side of the center.

Gay (I960) points out that variation among the scores on one side of the central value which does not alter the total score of the population cannot be distinguished by this method. For instance the hypothetical populations mentioned above consisting of specimens of one parent and hybrids could not be distinguished

from a population consisting of specimens of one-directional back-

cross hybrid derivatives. 60

The characters used in the analysis of the populations of

S. compositum ssp. compositum and ssp. reniforme were the following:

Range Score

Achene length/phyllary length <1 0 1 1 >1 2

Relative inflorescence density sparse 0 intermediate 1 dense 2

Head width 19-21 mm 0 16-18 mm 1 13-15 mm 2 10-12 mm 3

Achene sinus shape wide and 0 shallow intermediate 1 deep and 2 narrow

Leaf dissection index 1.00-1.99 0 2.00-2.99 1 3.00-3.99 2 4.00 or more 3

The position of the dots on or near the sides of the tri­ angle in Figure 28 indicates that the populations are rather homoge­

neous. The lack of any populations along the base line bears out

the observation that mixed populations of two subspecies were not

found. The lack of dots in the lower right-hand corner of the

triangle indicates that pure populations of "good" ssp. compositum

were not obtained. The dots at the apex of the triangle represent

populations whose members have the phenotype of the theoretical

hybrid of ssp. compositum x ssp. reniforme. Thus the same kind of

variation noted in Figure 26 when individuals were analyzed is also 61 apparent when whole populations are examined. There is continuous variation between populations of ssp. reniforme with those of ssp. compositumt but each population by itself is quite homogeneous*

Mean hybrid index values and mean hybrid numbers of popu­ lations of "good" ssp. compositum (intergrading populations with

ssp. reniforme not used), ssp. venosum, and ssp. ovatifolium are listed with their locations in Table 5 and plotted in Figure 29.

The hybrid index values were calculated using the following

characters:

Range Scoi

Achene length 8.5 mm or less 0 9.0-10.5 1 11*0 or more 2

Achene wing width less than 1 mm 0 1 .0-1 .5 mm 1 2 mm or more 2

Achene sinus shape broad V 0 narrow V 1 very narrow U 2

Head diameter 10-15 mm 0 16-20 mm 1 21 mm or more 2

Relative inflorescence density dense 0 intermediate 1 sparse 2

Leaf base shape cordate or 0 sagittate truncate 1 attenuate 2

Petiole length:midrib length 1.50 or more 0 1 .00-1.^9 1 less than 1.00 2 TABLE 5* Population analysis of S. compositum ssp. compositum, ssp. venosum, and ssp. ovatifolium

Mean Hybrid Mean No. of Index Hybrid Population Individuals Value Number Location

Subspecies compositum

2431 4 2.0 2.0 Edgecombe Co., N. C. 2k 36 8 1.3 1.3 New Hanover Co., N, C. 2437 10 2.0 2.0 Columbus Co., N. C. 2433 5 1.6 1.6 Marion Co., S. C. 2439 12 2.6 2.6 Florence Co., S. C. 2b 6? 8 l.l 1.1 Abbeville 0 0 ., S, C.

Subspecies venosum

2bb 1 9 6.6 6.0 Charleston Co., S. C. 2bb2 3 -/e • *3C. 5*6 Barnwell Co., S. C. 2bb 5 10 7.9 6.1 Jenkins Co., Ga. 2445 5 7.0 6.3 Treutlen Co., Ga. 2461 6 3.6 3.5 Randolph Co., Ga.

Subspecies ovatifolium

2bb7 9 1 0 . 7 3.2 Appling Co., Ga. 2448 3 10.6 3.3 Appling Co., Ga. 2449 3 12.0 2.0 Pierce Co., Ga. 245 0 13 11.3 2.6 Brantley Co., Ga. 2453 8 12.7 1.2 Clay Co., Fla. 2454 4 12.7 1.2 Clay Co., Fla. 2456 3 12.0 2.0 St. Johns Co., Fla. 2457 8 1 3. 0 1.0 Sumpter Co., Fla. 2460 8 9.7 4.2 Holmes Co., Fla.

The data from the population samples suggest a distinct delimitation of ssp* compositum, ssp. venosum and ssp. ovatifolium which was not so evident from the analysis of the research garden representatives. Subspecies compositum and ssp. ovatifolium fall toward the two extremes of the hybrid index, and the position of Fig. 29. Graph of mean hybrid number against mean hybrid index for natural population samples of 3. compositum ssp. compositum. ssp. venosum and ssp. ovatifolium.

65 Mean Hybrid Number 7 6 4 5 3 2 0 0 2 O S. compositum ssp. ssp. compositum compositum S. O # S. compositum ssp. ovotifolium ssp. compositum S. # < 9 S. compositum ssp. venosum ssp. compositum S. <9 Figure 29 Figure 4 en yrd Index Hybrid Mean 6 8 0 1 12 64 14 65 the populations along the sides of the triangle suggests some homogeneity. No populations were found which contained both ssp* composinum and ssp. ovatifolium. The apparent morphological separation between ssp. compositum and ssp, venosum is greater than that between ssp. ovatifolium and ssp. venosum. These data agree with those obtained from the analysis of the research garden clones.

When a hybrid index is computed for a specimen, the vari­ ability of any one character from one population to another is lost. This variability is very important in terms of indicating character clines from one part of the range to another. This can best be shown by the use of polygonographs. Six characters believed

to have the least intra-populational variability were chosen, and averages were computed for each population listed in Table 6 .

Except for four populations (B, D, F and J) there were at least six

specimens averaged together for each polygonograph. The six

characters were: (1 ) achene length/phyllary length; (2 ) head

diameter; (3 ) leaf blade length/leaf blade width; (*0 leaf dis­

section index; (5 ) achene length; and (6 ) achene wing width.

The polygonographs were plotted on a map shown in Figure

30. From this diagram several character clines can be detected.

The ratio of achene length/phyllary length is highest in the

mountains of Tennessee and Virginia, and lowest in the eastern

coastal plain from North Carolina to Florida. Both head diameter

and achene length are greatest in the Florida populations and

decrease gradually northward. In the two Virginia mountain popu­

lations an increase in head diameter and achene length is noted. Fig. 30. Polygonographs of 3. compositum population samples (see explanation in the text)•

66 SCALE II wo fe-i* JU-M** COUAL - U (« OftOJtCTION

O s -si

Ifl HKAC

TABLE 6. Polygonograph populations and their locations

Number of Population Specimens Subspecies Location

A 11 reniforme Bath Co,, Virginia B 2 reniforme Pittsylvania Co., Virginia C 6 reniforme Dinwiddle Co., Virginia D 2 reniforme Forsyth Co., North Carolina E 7 compositum Bertie Co., North Carolina F compositum Randolph Co., North Carolina G 8 compositum Columbus Co., North Carolina H 8 compositum New Hanover Co., North Carolina J 3 reniforme Cocke Co., Tennessee K 7 reniforme Union Co., South Carolina L 9 compositum Laurens Co., South Carolina M 8 compositum Abbeville Co., South Carolina N 10 compositum Florence Co., South Carolina 0 6 compositum Marion Co., South Carolina P 9 venosum Charleston Co., South Carolina Q 6 compositum Jasper Co., Georgia R 10 venosum Jenkins Co., Georgia S 6 compositum Muscogee Co., Georgia T 7 compositum Harris Co., Georgia U 7 venosum Randolph Co., Georgia V 9 ovatifolium Appling Co., Georgia w 13 ovatifolium Brantley Co., Georgia X 8 ovatifolium Holmes Co., Florida Y 8 ovatifolium Clay Co., Florida Z 8 ovatifolium Sumpter Co., Florida

The length/width ratio of the leaf blade is lowest in the piedmont

populations and increases toward the south and east, reaching its

greatest value in the Florida populations. Leaf dissection is

quite variable, sometimes even within populations. However, certain

trends can also be detected with regard to this character. The

lowest values are found in the mountain populations of Virginia and

Tennessee and in the southeastern coastal plain of Florida and

Georgia. There is a gradual increase in leaf dissection to the

east of the mountains and north of Florida, culminating in the 69 greatest dissection in populations of the North and South Carolina coast, particularly the New Hanover Co., N. C., population. Achene wing width is lowest in the Carolinas and increases slightly to the north and greatly to the south.

Populations A and B are good examples of ssp. reniforme with its characteristic low leaf dissection index, rather large heads and achenes, and low achene length/phyllary length ratio.

Populations F, G, H f M, N and 0 are typical ssp. compositum populations with very dissected leaves, small heads and achenes, high achene length/phyllary length ratio and very narrow achene wing. In the piedmont area between the populations of ssp. reniforme and ssp. compositum are some intergrading populations,

C, D, E, K and L, having some of the characters of ssp. reniforme and some of ssp. compositum. The polygons made for populations in

southern Georgia and South Carolina are of still a different shape,

and these (P, B and U) characterize populations of ssp. venosum

with a high achene length/phyllary length ratio, quite dissected

leaves, large heads and achenes, but with a narrow achene wing.

Finally in Florida populations Y and Z best characterize ssp.

ovatifolium with its achene length/phyllary length ratio, large

heads and achenes with wide wings, high leaf length/width ratio

and low dissection index. BIOCHEMICAL SYSTEMATICS: BACKGROUND AND BASIS

There have been many recent taxonomic investigations in which biochemical data, either crude or refined, have been used as supporting evidence of certain relationships previously sus­ pected on other grounds or as an indication of relationships and differences not previously noted. The practice of associating chemical characters with species or larger groups of plants dates from the earliest times when plants were used for dyes, medicines and foods. Several early botanists, among them Nehemiah Grew,

James Petiver, and Rudolph Jacob Camerarius, referred to the medicinal properties of various groups of plants and the distinctive tastes and odors peculiar to them (Gibbs, 1963)* A. P. DeCandolle was one of the first taxonomists to use chemical characters in making taxonomic judgments, such as the separation of Jasmineae from Oleineae (Gibbs, 1963)0

Helen C. de S. Abbott in 1886 stressed the correlation between morphology and chemistry and introduced the idea that evolution of chemical constituents may parallel that of morpho­ logical characters. She suggested that classification based on comparative chemistry might be an even better indicator of the degree to which a plant has evolved than one based on comparative morphology.

70 71

The present day concept stems from the idea of the evolution of biochemical pathways rather than the evolution of chemicals or morphological forms per se. Both morphological forms and the presence of certain chemicals in organisms are the result or end- products of the action of these metabolic pathways* Whether or not all the "end-products" are correlated depends on the organi­ zation of the genetic material conditioning their formation.

For many years chemical characteristics have been the basis of classification in such plant groups as bacteria, lichens and algae where, for one reason or another, morphological characters cannot be used with any degree of confidence. The presence of certain kinds of crystalline inclusions in cells of higher plants has been used for taxonomic purposes in some groups of plants.

Amino acid analysis of proteins by paper chromatography and electro­ phoresis has been used to ascertain taxonomic and genetic relation­ ships of protozoa (Lolfer and Scherbaum, 1962), molluscs (Kirk

et_al., 195^, Wright, 1959)t insects (Robertson, 1957)) echinoderms

(Chen and Baltzer, 1958)) fishes (Viswanathan and Krishna, 1956),

reptiles and amphibians (Dessauer and Fox, 1956), birds (McCabe

and Deutsch, 1952), and mammals (Johnson and Wicks, 1959)*

The most recent use of biochemical data in higher plant

taxonomy has been to verify relationships previously suspected from

morphological and cytological data. Generally this biochemical

data is obtained by two dimensional chromatography of plant extracts

usually containing phenolic and flavanoid compounds. The resulting

array of spots on the chromatogram is referred to as the chromato­

graphic pattern. Identification of the compounds, while necessary 72 for the complete understanding of the evolution of the metabolic pathway, is not essential for the simple comparison of chromato­ graphic patterns. Such a comparison could indicate the degree of present relatedness of the plants (just as much as morphological comparisons), but not tell us anything of the primitive or ad­ vanced nature of the plants.

The results of these studies can be summarized as follows:

Relationships between taxa above the species level

The group of dicotyledon families included in the Centro- spermae, for morphological reasons, have been shown to contain a group of distinctive compounds, the betaxanthins and betacyanins, not found in other plants. These compounds are chemically similar to flavanoids which are absent from the Centrospermae (Mabry et al.,

1963; Mabry and Turner, 196*0-

In the Iridaceae, species of the genus Watsonia were shown to be closely related biochemically and at the same time chemically distinct from other genera in the family (Riley and Bryant, 1961).

Sections within the genus Papaver, as defined morphologically, were found to be chemically distinct (Acheson et al., 1962). Gas chromatographic data obtained from species of Allium (Saghir and

Mann, 1961) and Carya (Stone, 196*0 supported to some extent the morphological groupings previoxisly described within the genus.

Species-specific patterns were found in Asplenium (Smith and Levin,

1963)t Pinus (Seikel et al., 1965), Artemisia (Holba and Mozingo,

1965)i Gossypium (Parks, 1965a, 1965b), Fragaria (Bringhurst et al.. 73

196*0* Baptisia (Alston and Turner, 1961), and Collinsia (Garber and Strjrfmnaes, 1964; Strjrfmnaes and Garber, 1963)*

Relationships between species and their interspecific hybrids

In some groups, the chromatographic pattern has been found to have all or nearly all the compounds found in both parents.

Exrjnples of this have been found in Tragopogon (Brehm and Ownbey,

1964, 1965)» Lotus (Harney and Grant, 1964), Zinnia (Torres and

Levin, 1964), Papaver (Kawatani and Asakina, 1959), and Gossypium

(Parks, 1965b). In such cases the parentage of putative hybrids can be predicted with a fair degree of accuracy. The parentage of certain diploid hybrids of Gossypium could be extrapolated when the chromatographic pattern of the hybrid and one parent was known

(Parks, 1965b). Hybrid Baptisia populations involving from 2 to 4 species have been subjected to chromatographic analysis, and in this way hybrids have been detected which were morphologically unidentifiable (Alston and Turner, 1963)* Identification of the parental species of the hybrids can also be determined (Alston and

Turner, 1961, 1962; Alston and McHale, 1963; Turner and Alston,

1939a, 1959b).

In other cases even when there were species-specific patterns for the parental species, parentage of hybrids cannot be clearly shown. When chromatograms were made of the Nicotiana artificial F^ hybrids, it was found that they usually did not have all the alkaloids found in both parents (Smith and Abashian, 1963).

In chromatograms of F^ hybrids of Collinsia (Garber and Str^mnaes, 74

1964; StrjSmnaes and Garber, 1963) and Zinnia (Torres and Levin,

1964), spots were found which were not present in either parent*

Relationships between species where polyploidy occurs

Tii groups where hybridization followed by changes in ploidy level is a mechanism of speciation, correlative chemical relationships have been most clearly demonstrated. In the genus

Asplenium the interspecific relationships are well understood cytogenetically, and the chromatographic analysis supports un­ equivocally the conclusions derived from morphological and cyto- logical data (Smith and Levin, 1963)* In other genera where amphidiploid origin of a species was suspected, such as Viola

(Stebbins et al., 1963)» Tragopogon (Brehm and Ownbey, 1964,

1965)» Nicotiana (Smith and Abashian, 1963)* Zinnia (Torres and

Levin, 1964), and Gossypium (Parks, 1965b), chromatographic analysis further supported this assumption.

Colchicine-induced autotetraploids of Lotus were found to

have slightly different chromatographic patterns than the diploids

from which they were derived (Harney and Grant, 1964). While

hybrids are often rather variable in their chromatographic patterns,

polyploids derived from them, with few exceptions, have the ad­

ditive pattern of both parents.

Relationships below the species level

Extensive studies within one species have revealed the

presence of chemical races in certain species of Eucalyptus (Penfold 75 and Morrison, 1927) and Acorus (Wullf and Stahl, i960). In the latter case the differences were principally quantitative and were associated with ploidy levels.

One of the most thorough chemotaxonomic studies was made by Brehm and Alston (1964) on Baptisia leucophaea var, laevicaulis.

In their study inter- and intra-populational variation in phenolic content was noted, and two chemical races with distinct geographical distributions were found. There was no correlation of the chemical races with morphological features. Analysis of free amino acids indicated little interspecific variation, while alkaloid content varied greatly within and between populations and with age of the plant.

In this study of Silphium compositum representatives of populations of the 4 subspecies were analysed for the chromato­ graphic pattern of the methanol-extractable materials, principally flavanoids and phenolic acids, Intra-populational variability was not studied. METHODS AND RESULTS OF CHROMATOGRAPHIC ANALYSIS

Basal leaves were collected from each of the clones of

Silphium compositum in the research garden on July 17 and

September 8, 1965* between 9:00 and 11:00 A.M. For several clones which did not survive the 196^-65 winter in the garden, leaves collected in 196^+ were used. Two to four healthy deep green mature leaves were chosen frcm each clone.

The leaves were placed in paper bags and returned to the laboratory where the bags were opened to facilitate drying. The mid-summer collection (July 17) was placed on a plant drier to promote quick drying and prevent molding of the leaves. Drying of both collections was done in a large room with frequent gentle air movement, but there was no attempt to critically control the tem­ perature. At no time during the drying period were the leaves

subjected to temperatures higher than about 55°C.

After a few days to a week of drying in this manner the leaves were transferred to 5 x 711 envelopes and stored in a dry

warm room until used. The leaves were judged dry when they

crumbled easily when handled. Most of the leaves had retained an

emerald green color through the drying process. Leaves which

turned brown or black during the drying process were not used for

chromatography.

76 77

When leaf material was selected for chromatography, the petiole and thick midrib portions were removed. The blade was

ground to a fine powder with mortar and pestle. A small quantity

was weighed out on a "Dial-O-Gram" balance, Model 310 (Ohuas Scale

Corporation, Union, N. J.) for extraction.

Preparation of paper chromatograms

Extraction was done with the modified micro-reflux system

(designed by J.V/.A. 3urley, unpublished) seen in Figure 31 (photo

of extractor). Cold tap water was circulated to provide cooling

for condensation at the top of the extraction container. Heating

of the extraction solution was accomplished by running an electric

current, volts, 2 amps, through nichrome wire wrapped around the

extraction flask (as seen in Fig. 32). The voltage was regulated

by means of a "powerstat" (The Superior Electric Company, Bristol,

Conn.) so that the extraction solution was kept just at the boiling

point.

Glass extraction cones were made by drawing out glass

tubing (O.D. 9 mm, I.D. 7 mm) and breaking it off so that the cone

was about 1 inch long. The upper part of the cone had the dimen­

sions of the original tube, while the lower end was narrowed to an

opening of 3-k mm. The lower end was stoppered with a small wad

of glass wool loosely enough to allow the solvent to drip through

freely.

The leaf material, 0.015 gm, was placed in the extraction

cone. A boiling chip was put in the bottom of the extraction i i. t L o ^j

& (jo j

Fig. 31* Kicro-reflux extractor (designed by J.W.A. Burley). 7 9

OVER-FLOW

GROUND GLASS FITTING

WATER COOLED ELEMENT NICHROME - WIRE

EXTRACTING CONE

LEAF MATERIAL

GLASS WOOL

SOLVENT BOILING CHIP

FIGURE _32* Diagram of the extraotlon flask used on the mioro-reflux extractor. (Actual size) 8o flask, and then the cone was carefully lowered into place. Care was taken so that no leaf material fell to the bottom of the flask. An extraction solution of methanol with 0»k% cone. HC1 was used. One ml was added slowly to the extraction flask by dripping it through the leaf material* When the leaf material was thoroughly wetted, the remaining part of the 1 ml was allowed to run down the side of the extraction flask. The flask was then attached to the extractor by means of a rubber band*

The extraction time was usually about 1 hour. Difficulties in regulating the boiling rate in 6 flasks at one time prevented equal rates of extraction on come occasions. In these cases extraction was stopped when the remaining leaf material was a gray color. The extract was green or brownish green when the extraction was completed*

The extract solutions were poured into small vials and stoppered with corks. They were either used immediately for chromatography or stored over night at room temperature. Extracts more than 2k hours old were not used. It was found that refriger­ ating or freezing occasionally caused precipitation of some material in the extract.

Whatman if3 MM filter paper cut to 15" x 15" was used for the paper chromatograms. The paper was not pre-treated or pre­ washed. Spotting of the chromatograms was facilitated by using a stream of cool air from a portable hair drier. One hundred lambda of the extract was spotted with a 5- ^ microcap tube (Drummond

Scientific Company, Broomall, Pa.) in the lower right hand corner 8l of the chromatogram 1" from either edge. The size of the spot was kept to about 1 cm in diameter. When the spot felt dry to the touch, it was ready to be placed in the chromatographic jar*

The jars used for ascending paper chromatography were ob­ tained from the State fish Hatchery and had originally been used for raising hatchlings. The jars were covered tightly with a layer of Saran wrap which was held in place with a rubber band.

Preparation of thin-layer chromatograms

Extracts used in the thin-layer chromatography were pre­ pared by allowing 1 gm of leaf material to stand in 10 ml of 09b%

HC1 in methanol at room temperature for 2b hours. At the end of

this period the extract solutions were poured into vials and stoppered until used. Extract solutions prepared in this manner were less uniform than those prepared with the extractor. As a result many of the extracts could not be used. Only those which were deep green or brown-green in color were used.

Thin-layer plates were prepared with a hand-controlled

spreading device (Research Specialties Company, Hodel 200-11,

Richmond, Calif•)• The 8" x 8" plates were cleaned with Alconox

detergent and rinsed thoroughly with de-ionized water. Just prior

to pouring the plates were wiped with 95% ethanol. Six plates

were placed on the plate aligner and a slurry of silica gel was

prepared by mixing and shaking 25 gm of silica gel G (Stahl

formula, E. Merck Ag., Darmstadt, Germany) with 50 gm of water for

30 seconds. The slurry was poured into the tray of the spreader, 32 and the spreader was moved rather quickly and evenly down the aligner. This amount of slurry was sufficient for 5 plates having a layer of 2^0 microns in thickness; the sixth plate merely acted as a stopping place for the spreader at a level even with the rest

of the plates. This prevented formation of a thicker layer near

the edge of the fifth plate.

The plates were allowed to dry at least 2*t hours at room

temperature before they were used. Plates could be kept in this manner for 2 weeks or so without apparent change. The plates were

not activated by heating or treated in any manner before being

used. It was felt that adequate separation was effected without

these treatments.

Spotting of thin-layer chromatograms was done in a similar

manner to that of the paper except that only 12 lambda of extract

were used, and the size of the spot was kept to about }£ cm in

diameter. It was found that a stream of air blowing across the

spot was not necessary to dry it sufficiently.

The thin-layer plates were placed on aluminum racks and

developed in battery jars. Saturation could be maintained in the

jars by lining the walls with filter paper, but this was found to

be undesirable for maximum separation. These jars were sealed with

a vasoline seal and a glass plate placed on the top.

Solvent systems for paper and thin-layer chromatograms

Various solvent systems were tried for both one and two

directional paper and thin-layer chromatography. The ones chosen for the two dimensional chromatograms were butanol:2-7% glacial acetic acid (1:1) for the first direction and ethylacetate:butanone: formic acid:water (5:3:1:1) for the second direction. Both are one-phase systems although considerable stirring and shaking was sometimes necessary for the butane 1:acetic acid system.

Solvents were nixed immediately before use. The reagents were always added in the same order and mixed thoroughly. For thin-layer chromatograms 250 ml of solvent was required for each

2 plates, and for paper chromatograms 100 ml for each jar. The paper chromatograms were rolled into a cylinder and stapled at the top and bottom. After the solvents were in the containers, the chromatograms were lowered carefully into the solvent. Approximate running times for both kinds of chromatograms and both solvent systems are seen in Table ?• The chromatograms were all run at

room temperature which ranged from 25 to 29°C. The paper chro­ matograms were developed to within lfl of the top of the paper before being removed (total developing distance 13")* The thin-

layer plates were marked off with pencil in such a way that the

solvent stopped at a line drawn 1#" from the top (total developing

distance of 6").

The chromatograms were dried at room temperature with the

aid of an exhaust fan until they felt dry to the touch. For the

paper chromatograms, a period of 1)4 hours after the first solvent

was sufficient, but overnight drying for the thin-layer plates was

found to be necessary. After the second solvent a period of 1 hour

was sufficient drying time for both kinds of chromatograms. TABLE 7« Chromatogram running times

Time for Thin-layer Time for Paper Solvent System Chromatograms Chromatograms

Butanol:acetic acid Zft-b hours 18-20 hours

Ethyl acetate:butanone: 1 hour b- 5 hours formic acid:water

Analysis of the chromatograms

The chromatograms were read and marked under the following conditions: no reagent, in visible and UV light (short wave,

Mineralight, Ultra-violet Products Inc., San Gabriel, Calif.)}

NH^OH, in visible and UV light; and 2% phosphomolybdic acid in 50% acetone (Dr. Jack Beal, personal communication), in visible light.

In addition the thin-layer chromatograms were sprayed with aqueous

5% NaCOj, in visible and UV light and with aqueous FeCl^ in visible light.

All spots, except chlorophyll, were marked on the chro­ matogram with pencil and their color reactions recorded. The ammonia and phenol indicator were sprayed with an aerosol sprayer

(Sprayon Products, Inc., Cleveland, Ohio, Nutritional Biochemicals

Corp., Cleveland, Ohio),

The paper chromatograms were dried and kept as vouchers.

The thin-layer plates were traced on to graph paper with the aid of a light table, and the tracings act as the vouchers.

No attempts were made to elute material from any of the spots or to identify the compounds present. Some of the spots were 35 found to give a characteristic reaction to phosphomolybdic acid and could be identified in a general manner as phenolic compounds*

After all visible and fluorescing spots on the chromato­ grams were marked and recorded, each spot was characterized by its location and cclor reactions, and was given a letter— A, B, C, etc.— as presented in Tables 8 and 9» Whenever a spot on a chromatogram appeared in the positirn of spot A and had the appropriate color reaction, it was labeled A. When the chromatograms were marked in this manner, a chromatographic profile was drawn up* For instance

#91 (Table 10) had spots A, E, C, end D; was missing E and F; had

G, K, I, etc. Six representatives of each subspecies were selected and profiles such as this were constructed. These profiles are listed in Tables 10 and 11.

Chromatographic profiles then were compared with one another by use of the paired affinity test (.bllison, Turner Alston, 1962)*

In this test each chromatographic profile is compared with every

other, and a score of 0 to 100 results. The formula is:

^ ^ soots in common for plant #1 and #2 Paired Affinity =* —»--- -——— ------:----- ^ , --- x 100 17 total spots in #1 and #2 jz

For example, when #91 is compared to #92, it is found that the two

profiles have 15 spots in common. A total of 22 different spots

are represented by the two chromatographic profiles. Therefore, the

paired affinity value for #91:#92 is 15/22 x 100, or 71* If two

chromatographic profiles are identical, the paired affinity value

is 100. If the two chromatographic profiles have no spots in

common, the paired affinity value is 0. Thus these values were 86

TABLE 8* Characterization of spots found on two-dimensional paper chromatograms of £. compositum

n h 4OH No Reagent

Ultra- Ultra­ Phospho- Spot Visible violet Visible violet molybdic Acid Designation Light Light Ligilt Light Visible Light

A Blfl Blfl B - Blfl - Blfl - C - Blfl — Blfl + D - Blfl - Grnfl + E - Yfl Y + F - Yfl Y —— G - Blfl Y Grnfl + H - Q Y Yfl Y+ I — Pkfl — YPkfl — J — Blfl - Pur fl - K - Purfl - Blfl - L - Blfl - Blfl - M - Q Y Yfl T + N - Q Y Yfl Y+ 0 - Blfl — Blfl - P IBr YOrfl Br Q + Q IBr YOrfl Br Q + H - Q Y Yfl + S - Blfl — Blfl — T m m — - Blfl - U — Orfl Pk ti? - V - - Pk —— w - Blfl Y Grnfl + Y — Blfl - -- Z - —- Blfl - AA - BlGrnfl - BlGrnfl - EB - Blfl - Blfl - CC - Purfl - Blfl DD ——- - + EE — -— Yfl + ? HH — Blfl — Blfl - JJ - Q — Q +

B1 - blue Or - orange Y+ - positive reaction Br - brown Pk - pink (yellow-gray) fl - fluorescent (light) Pur - purple + - positive reaction Grn - green Q - quench (dark) (blue-gray) 1 - light X - yellow - - invisible or negative reaction 87

TABLE 9* Characterization of spots found on two-dimensional thin- layer chromatograms of S. compositum

Ho Reagent NHU0H HaC03 FeC13

Ultra- Ultra- Ultra- Visible violet Visible violet Visible violet Visible Spot Light Light Light Light Light Light Light

A OrBr - OrBr ft OrBr ft + B OrBr - OrBr ft OrBr ft + C Gry - GryPur ft Pur ft + D - Blfl ** Blfl - Blfl -

E YGrn - YBr ft YBr Q + - fl fl - fl E2 - -

F Y ft Y ft Y ft + F2 - fl - fl - fl - G GryGrn — YBr ft YBr ft + G2 - fl - fl - fl - II YBr ft YBr ft YBr ft + I - Pkfl Pkfl - Pkfl - JY fl? Y ft Y ft? +? K GryGrn fl Br ft Br ft + - fl - — - K2 ft ft L Y Yfl Y Yfl Y Yfl - GryGrn 3r Br ft + L 3 ft ft M _ fl? — fl - fl - N — fl? - fl - fl - 0 - fl — fl - fl - P —-- —- Pkfl - - - - ft fl fl fl - R fl — fl _— —

B1 - blue Gry - gray Q - quench (dark) Br - brown Or - orange Y - yellow fl - fluorescent Pk - pink + - positive reaction (light) Pur - purple (black or brown) Grn - green - - invisible or no reaction 88

TABLE 10. Chromatographic profiles of S. compositum subspecies from paper chromatograms

ssp, ssp. ssp. ssp. reniforme compositum venosum ovatifolium

IA VO OO OJ OJ O n NO O n O ON LfN ON C\J -d- A- rA J- A- OJ on 4- VD vo KN A-NO O O H H UN IA ON CO IA IA O H A A d ' O rH (M -d - H H r | A - H KN KN KN KN fVJ r l

A X X X X X X XXX X X XX X X X 3 XX XXX X X X X X XX X XXX C XX X XXX XX X X X XX X XX X X XX XX D XXX XX X XX XXX X XX XX XX XX XX X X E X XX X X XX XX XX XX X V XXXX X X X F X XX X X X X X X X X X X XXX X X G XXX X X X X XXX X XX X XX XX XX XXX X H XXXX X X XX XX V X XX XXX X X X XXXX I XX XXX X X X X x X X XXXXXX J X X XXX X XX XXX X XXXX XX X XX XX K XX X X X XXX X X X XX X XX L XXX X XX X X XX XX V X X x X XXX M XX X X X X XX X X XXX X X XXXX XX N X X X X X X X XXX X XX XX X XXX XXX X 0 X XX XX X XX X X X XX X XX XXXXX XX P X X XXX X XX XXX X X X XX XX X XX XX Q XXX X X XX X X X XX X X X X X X X XXXX H XX XXXX X X XXX X X X X XX X XX S XX XXXX X X X X X XX X XX XX T X U X X X X XX XX V X X X XX X w XXX XX X XXX Y X XX X X X XX a XXX AA X XX X BB XX X X X X X X X cc X XXX X X X X X X XXX DD X XX X XX XXX EE X X X HH JJ X TAELE 11. Chromatographic profiles of S. compositum subspecies from thin-layer chromatograms

ssp . ssp. ssp. ssp. reniforme compositum venosum ovatifolium

ITsKC LT\ OVCC OJ C

A V XX X X ■■r X y X XXX X X X X y X 3 X XX X XX X X XXXXX x XX X X XXX X X C X X V XXX X X x X X X X -•r X XX XXX X X D X XX X *• X X X ■v X X X X X X X X X X VJ VX X X X EXX X X X X jr *v X V X X XX V X X X X E2 F XX X X x V X :c XX ■V X X X X X jr X X F X 2 XX X X X XXX X X X X X X X XX X X X XX X X X <3„ X X XXX y XX X X V "7 x V X X XX V XX XX X XX XX X X XXX X X I XX X X X X X X 3 : J X XX X XX K X X XXX X X X X XXX X X XXX X X X XXX X X K 2 '■* X X X L X X X XXXX XX XXX X X X X X X X X X X X X X XXX *v X X b M XXXX XX XX X X XX XX N X X X y_ X XX X XXXX X X xr X X X X 0 X X X XX X XX X X X XX y XXX X XX P XX X XXX Q X V XX X X X XX XX R X X V X

used as r. measure of similarity between any two chromatographic

profiles.

Tie paired affinity values from both the thin-layer and

paper chromatograms were calculated. The results are presented in

Tables 12 and 1JS. Intrasubspecific comparisons are those paired

affinity values which were obtained by comparing two chromatographic TAELE 12. Paired affinity values of paper chromatograms of S. compositum arranged in subspecific groups

ssp. reniforme ssp. compositum ssp. venosum ssp. ovatifolium

LA VO CO f\J ov \£> Ov O ON IA on (M A IA 4 - N AJ Ov VO IA O O r-t H IA IA ON oo LA lA O H K\ K\ -4" O r\i H H H IA K \ OJ H <\J C\J OJ A) cvj r\i oj oj oj oj 2162 ON H A] AJ <\i 177 H r-i r-i H H <\J H H H r ) H H H H H H 66 65 80 66 58 50 50 68 4) 91 71 61 80 66 51 50 55 65 68 65 53 56 66 50 54 s 92 70 80 75 72 70 66 58 56 57 56 61 62 68 60 81 60 44 54 • h 75 65 65 63 ft o 144 70 73 73 56 62 65 58 32 44 61 53 63 54 59 56 46 70 65 66 41 52 10 2166 78 60 66 69 60 59 48 59 57 68 58 63 60 56 66 69 65 52 57 2138 68 73 77 68 75 53 75 6k 60 58 63 68 56 66 77 63 45 57

177 65 61 60 65 59 72 62 60 64 62 60 68 58 90 69 46 56 i +> 2162 80 72 64 76 6k 80 65 62 75 86 73 70 73 68 57 62 • 'H f t CO 1302 61 66 60 66 6k 68 59 6k 75 62 73 69 70 60 65 CO o CO f t 1309 65 65 58 76 66 70 69 81 68 58 68 62 58 63 ao 1316 51 80 68 52 50 48 58 53 56 66 48 38 47 o 1319 51 67 59 72 61 82 66 57 60 55 64 62

1250 61 52 50 3k 58 60 63 73 60 38 47 •H •H 2159 57 66 59 76 70 68 70 53 50 53 • X ft 0 1295 78 69 66 61 65 68 69 65 71 co oj to 1289 60 70 72 69 72 62 69 68 o 1252 64 54 70 72 *TJ n_1 77 54 59 a 1254 75 72 68 69 65 71

I 1207 66 76 57 53 58 r t rH 1213 66 68 56 70 • O ft *H 1234 70 48 58 CO *r» 1237 54 Uii n +j j 1 67 <0 1209 78 o> TABLE 13. Paired affinity values of thin-layer chromatograms of compositum arranged in subspecific groups

ssp. reniforme ssp. compositum ssp. venosum ssp. ovatifolium

lA VO LA ON o o OJ O <\J -d" O n IA ON N ^ N (\1 IA -4- NO vT> A- NO VO A- NO IA LA LA CO O n LA O H CA fA -4" - 4 O J I A - 4 H H pa A- oj oj oj h m m i\ i n n h r\j oj oj oj oj oj O n O n H OJ OJ O n H H H H OJ r-C H H H H OJ H H H H H H

91 50 61 55 63 66 59 55 55 50 55 57 47 55 63 52 55 55 57 60 50 45 45 4o 1) S 92 47 60 45 55 52 58 50 45 42 47 60 58 58 55 50 50 73 64 62 56 56 60 • u f t o 95 43 55 50 56 36 44 33 36 36 43 44 50 50 44 44 56 58 56 50 50 53 CO

93 58 80 61 58 63 71 80 80 55 80 80 73 64 73 73 78 71

+i 177 75 57 64 68 56 75 75 61 64 64 50 61 58 62 62 56 • *H ft U 1265 57 64 77 78 86 75 61 86 86 68 61 80 85 85 78 CO O 1269 42 to p j 76 70 50 50 55 57 57 45 55 45 47 47 42 a 1278 68 47 55 64 61 64 64 50 61 50 52 52 47 o o 2162 61 77 68 65 77 77 55 57 63 66 66 61

•rl 1250 78 66 62 78 78 84 52 84 91 91 83 •H * X 1252 86 61 86 86 68 61 80 85 85 78 P i 0 1254 co « 61 75 75 68 70 68 73 73 66 CO J 3 1289 70 70 64 76 68 68 62 o 75 •H 1295 100 68 70 80 85 85 78 a 2159 68 70 80 85 85 78

•Hs 1207 64 85 78 78 71 H 121} • O 64 58 58 62 ft«H 1234 92 84 CO *H 92 CQ -P 1237 100 91 OS > 124-5 91 O 92 profiles of the oame subspecies, i.e., ssp. reniforme:ssp. reniforme, ssp. compositum;sep. compositum, etc. The number of different comparisons is where n is the number of repre­ sentatives. Thus, with 6 representatives for each subspecies, there would be 15 different intrasubspecific comparisons for each subspecies.

Intersubspecific comparisons are those paired affinity values obtained by comparing two chromatographic, profiles of different subspecies, i.e., ssp. compositum;ssp. venosum, ssp. reniforcte:ssp. ovatifolium, etc. The number of different comparisons that can be made in this way is n x n ’ where n and n' are the number of representatives in the two subspecies. There­

fore, for each two-subspecific combination, there are 6x6, or 36 different paired affinity values.

A one-way analysis of variance test was run on the four intraeubspecific comparison groups. The purpose was to test

whether these groups had equal mean paired affinity values; that

is, whether they were equally homogeneous. The null hypothesis

was that there were no significant differences in the means of the

four intrasubspecific comparison groups. This hypothesis was

rejected if the F values obtained were higher than the critical F

value at the .05 level of significance. Duncan's Multiple Range

Test was applied to determine which means were significantly

different from each other.

The results of this test for paper and thin-layer chro­

matograms are presented in Table 13* With the paper chromatogram 93 technique, the six representatives of ssp. reniforme are signifi­ cantly more homogeneous than the representatives of the other three subspecies. The representatives of ssp. compositum, ssp. venosum and ssp. ovatifolium are all about equally homogeneous. With thin-layer chromatography the results were nearly opposite. The representatives of ssp. venosum and ssp. ovatifolium were about equally homogeneous, and significantly more homogeneous than either of the other two groups. The representatives of ssp, compositum, however, are significantly more homogeneous than those of ssp. reniforme.

TABLE 14. Results of Duncan's Multiple Bange Test for Significant Differences between the mean paired affinity values of the intra­ subspecific comparisons for paper and thin-layer chromatography. (The line connecting the means indicates that there is no signifi­ cant difference between those means)

Subspecies Subspecies Subspecies Subspecies reniforme compositum venosum ovatifolium

Thin-layer 55.22 66.40 75.46 77.86 chromatograms

I'aper chro­ 73.13 6^.66 63.53 63.00 matograms

To test the degree of similarity of the four subspecies one-way analysis of variance tests were run on the intrasubspecific comparison group and the three corresponding intersubspecific comparison groups. For instance, the mean paired affinity values

from intrasubspecific comparisons of ssp. compositum were compared to the mean paired affinity values from intersubspecific comparisons of ssp. compos!turn:ssp. reniforme, ssp. compositum;ssp. venosum, and ssp. compositum;ssp. ovatifolima. 9^

The null hypothesis was that the paired affinity values obtained from intrasubspecific comparisons were not higher than those obtained from intersubspecific comparisons. F values were obtained for a one-way analysis of variance, and the hypothesis was rejected if the F value was greater than the critical value at the .05 level of significance.

When the hypothesis was rejected, further tests were neces­ sary to determine which means were different from each other. In terms of the null hypothesis, the paired affinity values obtained from intrasubspecific comparisons were designated as the control group, while those obtained from intersubspecific comparisons were considered the treatment groups. This made possible the use of

Dunnett's test of significant difference. A method of using this test with samples of unequal sample size was devised, but its validity has not been verified (Steel & Torrie, i960).

The most valid comparisons between subspecies would be between those which are about equally homogeneous. Therefore, with the thin-layer chromatographic data (Table IS) ssp. venosum and ssp. ovatifoliuifl can be compared, and with the paper chromatographic data (Table 16) ssp. compositum, ssp. venosum and ssp* ovatifolium can be compared. In none of these instances are there any signifi­ cant differences between the mean intrasubspecific and intersub­ specific paired affinity values. The significant differences in mean paired affinity values observed in Tables 15 and 16 all occur between subspecies with differing amounts of homogeneity (Table 1*0 •

For example, ssp. venosum, ssp. compositum and ssp. ovatifolium 95

TABLE 15* Kesults of the analysis of variance of the paired af­ finity values of introsubspecific and intersubspecific comparisons from thin-layer chromatograms

Intrasubspecific Intersubspecific Comparison Comparisons ssp. compositum ssp. venosum ssp. ovatifolium ssp. reniforme

X 66.40 66.77 61.22 55.63** s 7.80 11.88 11.90 13.24 ssp. reniforme ssp. venosum ssp. ovatifolium ssp. compositum

X 55.20 58.30 58.22 53.63

6 3,78 9.50 7.57 13.24 ssp. venosum ssp. ovatifolium ssp. compositum ssp. renifori

X 75.^6 75.15 66.77* * 58.30**

5 11.05 9.20 11.88 9.50 ssp. ovatifolium ssp. venosum ssp. compositum ssp. reniforme

X 77.86 75.13 61.22** 5 8 .22** s 14.13 9.20 11.90 7.57 • * Significant at the ,01 level. 96

TABLE 16. Results of the analysis of variance of the paired af­ finity values of intrasubspecific and intersubspecific comparisons from paper chromatograms

Intrasubspecific Intersubspecific Comparison Comparisons ssp. compositum ssp. venosum ssp. reniforme ssp. ovatifolium

X 64.66 66.00 62.33 62.16 s 8.50 9.30 7.12 9.45 ssp. reniforme ssp. venosum ssp. compositum ssp. ovatifolium

X 73.13 62.36** 62.33** 60.86** s 6.09 6*80 8.30 9.^5 ssp. venosum ssp. compositum ssp. ovatifolium ssp, reniform<

X 63.33 66.00 64,13 62.36 s 9.06 9.30 S.46 6.80 ssp. ovatifolium ssp. venosum ssp. compositum ssp. reniforme

X 63 .OO 64.13 62.16 60.86 s 8.33 3.46 9.43 9.45 * * Significant at the *01 level* 97

(Table 15) appear to be significantly different from ssp. reniforme, but this may be the result of the significantly greater homogeneity of the ssp. reniforme representatives (Table 1*0. Thus the dif­ ferences revealed, although statistically significant, are really only the result of comparing a homogeneous group with a hetero­ geneous one.

Intr&specific and interspecific comparisons of the standard

Silphium clones from the research garden were made with the end arrangement groups as the basic categories (see Cytology section).

Intraspecific comparisons of the S* compositum standard clones and intra-group comparisons of the other species were made. The mean paired affinity values for these comparisons are listed vertically in Table 17. The mean paired affinity values of group A and group

C of the same leaf were also found to be identical (see Table l8 ).

When different leaves from the same plant were chromatographed, slight differences in the chromatographic profiles resulted, and

the mean paired affinity value was 86.3» This can easily be ex­ plained if one or more of the leaves was immature. Leaves col­ lected from the same plant at different times during the summer also resulted in some variability, and the paired affinity value

in this case was 70*5. Therefore, care was taken to choose only mature leaves, and to collect them at the same time.

The spots were distinct in their position in relation to

each other and in their color reactions, thus in most cases there

was no question about the identification of the spots. Because 98

TABLE 17. Results of the analysis of variance of the paired af­ finity values of intraspecific and interspecific comparisons from paper chromatograms of clone standards of S. compositum and other Silphiua species in end arrangement groups A, B, and C

Intraspecific Comparisons Interspecific Comparisons

S. compositum ^ Group A Group B Group Cg Clone Standards Species Species Species

X 66.10 5 2 .60** 54.86** 59.08* s 6.43 8.60 8,79 9.66

Group A Species

X 54.33* s 6.71

Group B Species

X 62.00 s 8.26

Group C Species^

X 54.30** s 10.81

S. compositum Clone Standards used were: #2159* ssp. venosum # 1771 ssp. compositum #2163. ssp. reniforme #2139, ssp, reniforme #1237, ssp. ovatifolium 2 Other than £. compositum. 99 the spots were not eluted and co-chroraatographed, there may have been chemical composition differences present which were not detected. Future chromatographic studies in this species will have to involve not only co-chromatography, but also identification of the compounds.

TABLE 18. Mean paired affinity values from thin-layer chromatograms of jy'2159 tested for reproducibility of the methods

Mean Paired Conditions Affinity Value same leaf, same extract 100.0 same leaf, four different extracts 100.0 same plant, extracts from three leaves 86.3 collected on same date same plant, extracts from leaves collected 70.3 on five different dates

Though probably not a source of error, the paired affinity value as a method of analyzing chromatographic data has some dis­ advantages. Because the value is the result of a comparison of two chromatograms, there is twice the chance of error. Mo other method of analysis could be discovered which would avoid this problem. In addition when one or both of the chromatograms contain a low number of spots, the presence or absence of just one spot causes considerable differences in paired affinity values. A low number of spots on one chromatogram compared to another with many spots may result in some very low scores. In other instances a moderate number of spots could result in. a series of very high scores, Eoth instances may distort the cver-all relationships, and suggest similarities or differences which do not really exist* CYTOLOGY Oj? THE 5ILPHIUM COMPOSITUM HE3EAECE GAPDEH CLONES

Kerrell (1900) first reported chromosome counts of n = 8 in Silphinm per folia t urn, S. integrifolium, S. terebinthinaceumf

S. 1-nciniatma and 3. trifoliatum, a3 though Taylor (1928) repeatedly found 2n = 14 in S. perfoliatum. Fisher and Cruden (1962) reported chromosome counts for 15 species in the genus. All were n = 7 except for one instance of an extra unpaired chromosome in several plants of 3. trifoliatum. Settle (unpubl.) studied the morphology of root tip chromosomes of 18 species and found that all were diploid with 2n = 14. He found that the chromosome complements were very similar morphologically in all species, but different significantly in length in some caseo. Several clones of S. compositum were included in these studies (Fisher and Cruder*,

1962; Gettie, unpubl.).

Buds were collected from the research garden clones of S. compositum and fixed in alcohol;glacial acetic acid solutions (5:1) for 2h hours. The buds were stored in 70% alcohol solutions and refrigerated until used. If the buds were large, the outer phyllaries were removed or the buds split to facilitate rapid

fixation.

The anthers were removed from the disc flowers and smeared in iron aceto-carmine. The slides were made permanent by freezing

101 102 on dry ice until the cover slip was well frosted over. The cover slip was removed quickly with a razor blade, and the slide placed in 93% alcohol solution for several minutes. The anther material remained on the slide almost entirely, and very little destaining was noted. The slide was next placed in 100% alcohol for several minutes. A clean cover slip was mounted on the slide with euparal, and the slide was placed on a slide warming table (p8°C) for several days to allow the euparal to set comp'letely.

Flower heads with open disc flowers were collected from the research garden plants. Pollen viability was estimated by staining with cotton blue. It has been shown by Fisher (1959) that this test agrees well vrith pollen tube growth in Silphium.

Cells in diakinesis, anaphase I and anaphase II were

examined in most of the clones. The results were recorded in

Tables 19-22. The majority of the clones in all four subspecific categories had seven bivalents at diakinesis (Fig. 55-56) and had good separation at anaphase I (Fig. 57) and anaphase II (Fig. 5 8 ).

Pollen stainability in the 7-paired clones was generally above 90%•

There were two plants (#92 and #2159) with good pairing

followed by apparently normal anaphase I and II in which pollen

stainability was unaccountably low. In addition to normal sized

pollen grains macro- and micro-pollen grains were found in #2159

(Fig. 59)• In another plant (#95)» although pairing was good at

diakinesis, a bridge and fragment were observed at anaphase I

(Fig. ^0), indicating the presence of a heterozygous inversion.

As expected, this resulted in about 50% reduction in pollen 103

TABLE 19* Cytological behavior of meiotic chromosomes in S. com­ positum ssp. compositum clorres. (IT = normal meiotic behavior, L ~ lagging, IBF = inversion bridge and fragment)

Stainable Clone Diakinesis Anaphase I Anaphase II Pollen

93 5 II, ch 4- & 1 I N L 4-8.5^

177 7 XI IBF IBF 93 56

1263 7 II N t; 99 %

1265 7 II & 1 I M IT 4-9 %

1269 7 II N N 98 %

1278 7 II - - 78 %

1301 7 II N - 97 #

1302 7 II - N 100 %

1306 7 II & 1 I N - 75.5%

1309 7 II N - -

1311 7 II N IT 100 %

131^ 7 II IT N 97 %

1316 7 II N IT 100 %

1318 7 II f: 1 I N IT 73 % OJ rl r*^ 0 7 II N N 98 %

1321 7 II N II 99 %

2162 7 II IT w 100 % 104

TABLS 20. Cytological behavior of meiotic chromosomes in S. com- gositum ssp. reniforme clones. (N = normal meiotic behavior, IBF = inversion bridge and fragment)

Stainable Clone Diakinesis Anaphase I Anaphase II Pollen

91 7 11 NN 100 % O O - H 92 7 11 l\ IA

95 7 11 IBF - 46.7?i

144 5 II & ch 4 N N 43.9%

1139 7 II II N 95 %

1324 7 II II II 82 %

2139 7 II *T N 93 %

2149 7 II II II IOC %

2150 7 II NN 100 %

2151 7 II; 5 II & ch 4; N N 100 % 6 II Se 2 I

2155 7 ii; 5 11 & ch 4 N M 98 %

2165 5 11 8: ch 4 N N 100 %

2166 7 11 i l l II N 100 %

2167 7 II 1:N 100 % 105

TABLE 21. Cytological behavior of meiotic chromosomes in S. com- positum ssp. venosum (N = normal meiotic behavior)

Pollen Clone Diakinesis Anaphase I Anaphase II Stainability

1248 7 II N _ 100 %

1249 7 II N ;t 99 % 1250 - - - 100 %

1252 7 II - - 100 % 1254 7 II II - 99 %

1255 7 II - - 99 %

1258 7 II N - 100 %

1238 7 II N - 100 %

1289 7 II IT - 87 %

1290 7 II; 5 II + N - 98 % c’n 4?

1295 7 II; 12 II + IT N 99 % IV; 6 II + 4 iv

1298 7 II; 5 II + ch 4 IT - 9^. 8 %

2159 7 II II N 12. 7* * Macro-pollen (20.2%) and nicro-pollen grains (26.4%) were present and not stained. 5^% were normal sized. 106

TABLE 22. Cytological "behavior of meiotic chromosomes in S. com- positum ssp. ovatifolium. (N = normal meiotic "behavior)

Pollen Clone Diakinesis Anaphase I Anaphase II Stainability

120? 7 II N II 90

1213 7 II N - 80 %

123^ 7 II - - 99 %

1237 7 II H - -

12 k2 7 II N - 100 %

12^5 7 II NN 97. stainability. In #177, an anaphase I bridge persisting into anaphase II was seen (Fig. ^1). Pollen stainability was high, but many of the pollen grains were very lightly stained.

In all subspecies of 3. compositum except ssp. ovatifolium, plants were found in which five bivalents and a ring or chain of four chromosomes formed at diakinesis indicating a reciprocal translocation in the heterozygous condition (Fig. ^-2). In all instances but two (#93 and #lV+), when the remainder of the meiotic sequence was normal, pollen stainability was high. In a few cases cells with 7 II and 5 II and a chain of ^ were observed all in the same anther. This can be explained if the translocated segment is very short, and no chiasmata form in the translocated arms.

’/Then diplotene repulsion occurs, the quadrivalent comes apart and two bivalents with flared ends are seen instead.

Small univalents were found in some plants of ssp. reniforme and ssp. compositum. In polar view of metaphase I the univalent Fig-*. 33- Diakinesis Fig. 3^* Diakinesis configuration with 7 II in 3. configuration with 7 II in S. compositum ssp. compositum compositum ssp. reniforme (#1278). Scale: ll?5x. Scale: 1125x.

£

Fig. 35- Diakinesis Fig. 3^. Diakinesis configuration with 7 II in S. configuration with 7 II in S* compositum ssp. venosum comoositum ssp. ovatifolium (#2159). Scale: 1125x. T35-2 3M . Scale: 1125x. 108 *

4

Fig. 37. Anaphase I Fig. 38* Anaphase II configuration in S. compositurn configuration in S. compositum ssp. reniforme (#2167). Scale: ssp. compositum (^2lfi2). Scale: 1125*. 1125x.

Fig. 39* Macro- and micro-pollen grains in S. compositum ssp. venosum (#2159). Scale: 59^x. Fig. ^0. Anaphase I Fig. ^1* Anaphase II configuration with an inversion configuration with persistent bridge and fragment in S. cora- anaphase I bridge In S. con- positum ssp* compositum (#93)• positum ssp, compositum (#177)• Scale: 1125x. Scale: 1125x.

f t

*

k

Fig* ^2, Diakinesis Fig. Metaphase I configuration with 5 II and a configuration with 7 II and 1 I chain of four chromosomes in in S^. compositum ssp. reniforme 3* compositum ssp. reniforme (#2166). Scale: 1125x. " D l W . Scale: 1125x. 110 usually remained off to one side and was probably not attached to the spindle (Fig. 43). At anaphase I the univalent was incorpo­ rated into either one group or the other (Fig. 44), but lagging was not generally noted at either anaphase I or IX. In one instance (Fig. 45) precocious equational division of the univalent evidently occurred at anaphase I, and was followed by reductional division and lagging at anaphase II. Pollen stainability of those plants having a univalent ranged from slightly below 50% to 100%, but in general reduced stainability was associated with the uni­ valent condition. One plant (£93) had both a univalent and a heterozygous tranalocation.

In one plant (£1295) cells v.'ere found which contained various combinations of bivalents and quadrivalents (Fig. 46 and

4?). The meiotic behavior at anaphase I and II was normal.

Examination of another bud collected from a different plant con­ tained cells with 7 II at diakinesis.

The low pollen stainability found in some representatives of ssp. reniforme, ssp. compositum and ssp. venosum is not altogether explainable on the basis of structural anomalies. In the case of £2159 uo chromosomal abnormalities accompanied the abnormal pollen formation. On the other hand, high pollen stain­ ability is found in spite of heterozygous translocations (£2151*

#2I53)i heterozygous inversions (£177)» tetraploidy (£1295), or univalents (£2166). Ill

*

.t

#* * •’* t

fig. 44. Anaphase I Fig* 45. Anaphase II configuration in cells with a configuration with reductional univalent in S . compositum ssp. division and lagging of the reniforme (#2l66yi Scale: univalent in S. compositum ssp. 1125x. compositum (#93)• Scale: 1125x,

Fig. 46. Diakinesis Fig. 4?. Diakinesis configuration with 1 IV and 12 II configuration with 4 IV and 6 II in S • compositum ssp. venosum in S. compositum ssp. venosum (#1295)* Scale: 1125x. (#129577Scale: 1125x. INTERSPECIFIC AND INTRASPECIFIC HYBRIDIZATION OF SILPHIUM COMPOSITUM

Hybridization and cytologic studies in the genus Silphium have disclosed several established reciprocal translocations*

These are evident when found in the heterozygous condition in interspecific hybrids. Because of the low number, large size and distinctive morphology of the chromosomes, it has been possible to ascertain which chromosomes are involved in the translocation.

In each case the translocation has been found to involve arms of chromosomes number 1 and 7* the longest and the shortest re­ spectively. The results of this study 'nave led to the discovery of three groups of species in the genus, each with a different

"end arrangement" with regard to the arms of chromosomes 1 and 7«

Group A (Fig. 43) which includes S. reverchonii Bush (#90), was chosen arbitrarily as the reference to which others were com­ pared and was designated as having the standard end arrangement

1*2, 13*14. When F^ hybrids are synthesized with #90 as one parent, one genome in the hybrid will have the 1*2, 13*14 end arrangement. If the second genome contributed by the other parent has the same end arrangement as #90, pairing at diakinesis in the

F^ will be good, and 7 II will be seen. If, however, the second genome has a different end arrangement, such as 1*14, 13*2 (Group

3) or 1*13* 14*2 (Group C), a quadrivalent will be formed at diakinesis in place of two bivalents in the F^ hybrid. Thus the 112 Fig. 43. Grossing relationships of some Silphium research garden clones.

113 93 95 Group c ,77 91 113 14 2 1.77 / ' / \ ' ' Group A 144 /92---v._ 12 1314 'N ' 251— 146^200-V - 90 \ N \ / \ N \ / / ' \ ' / / \ \ ' / 7 n \ \ \ 5 II + ch 4 \ ' \ \ 7 n + i i \ \ /

\ heterozygous 105 translocation \ present in clone Group B trTT 116 1 , 4 , 3 '2 115 end arrangement of chromosomes 1 and 7 of any plant can be as­ certained by crossing it to a plant from each of these three groups*

Interspecific hybrids of compositum and the other species indicate that the S. compositum clones tested so far all belong to the end arrangement Group C along with S. laciniatum

(#200), S. pinnatifidum (#251) and S. terebinthinaceum (#1^6).

Insufficient crosses have been made with plants of the other end arrangement groups to identify the other end arrangement involved in those clones of 3. compositum which have a heterozygous trans- location .

In addition to determining the end arrangement of the clones, interspecific crosses in the genus Silphium may disclose some degree of relatedness among the species* The results of inter- and intraspecific hybridization are presented in Table 25*

When plants of Silphium compositum clones are crossed with other species of end arrangement Group C, there is a slightly lower chance of successful crossing and a lower crossability index, than when crosses are made to plants of either Group A or Group B*

Although not many plants could be tested, differences in pollen stainability between these three groups in interspecific hybrids appear to be rather slight*

Intraspecific crosses of Silphium compositum resulted in a much higher rate of successful crosses (nearly 80%) and higher crossability index (27***%) than interspecific crosses. The average pollen 6tainability of intraspecific hybrids is higher (nearly 90%) than that of the interspecific hybrids. It should be pointed out in TABLE 23* Results of interspecific and intraspecific crosses of S* compositum (reciprocal crosses included)

No. of % Avg. Number Crosses Successful Crossability Pollen Hybrids Attempted Crosses Index-*- Stain. Tested

Interspecific Crosses S. compositum x Group A spp. 28 42.8 8.8 77.6 2 S. compositum x Group B spp. 71 38.0 5.2 71.7 6 S. compositum x Group C spp0 78 34.6 3.3 72.4 9

Intraspecific Crosses ssp. comp, x ssp. comp. 2 100.0 14.9 99.0 2 ssp. reni. x ssp. reni. 44 79.5 26.8 100.0 1 ssp. ven* x ssp. ven. 0 - - - - ssp. ovat. x ssp. ovat. 2 100.0 32.1 0 Total intrasubspecific crosses 48 81.2 26.5 99.3 3

ssp. comp, x ssp. reni. 18 50.0 21.2 32.3 4 ssp. comp, x ssp. ven. 2 50.0 12.5 - 0 ssp. com£. x ssp. ovat. 8 100.0 33.6 - 0 ssp* reni. x ssp. ven. 10 9 0.0 26.5 - 0 ssn. reni. x ssp. ovat. 11 100.0 41.6 - 0 ssp. ven. x ssp. ovat. 0 - - - - Total intersubspecific crosses k? 76.5 23.3 82.3 4

Total 95 79.6 27.4 89.6 7

^"Crossability index = seed germination % x seed set % (Vasek, 1964) * 116 117 the case of pollen stainability that very few plants were tested.

The majority of these hybrids are in their first year of growth at the present time and as yet have not flowered.

Morphology of the interspecific hybrids

The species which most closely resembles S. compositum in general morphology is £. terebinthinaceum. The artificial hybrid of 3, compositum ssp, reniforme x 5, terebinthinaceum (#92 x #1^6) was intermediate in many characters including leaf shape, head and achene size and number of ray flowers. The prominent bases of the scabrous hairs on the leaves of 5, terebinthinaceum were lacking in the hybrid, and the achenes were narrow-winged as in

S, compositum. On the scatter diagram (Fig, 26) it lies about half-way between 3, compositum ssp, compositum and S. terebinthin­ aceum, and resembles a specimen collected in VJest Virginia at the northern extreme of the 5, compositum range,

A plant of another taxon placed in the same section by

Small, S. pinnatifidum, was also crossed with S. compositum ssp, reniforme. Three specimens of hybrids (#91 x #251, #92 x ,#251, and #95 x #251) were examined. In leaf outline and pubescence

these hybrids resembled ii. pinnatifidum, but the leaf blades were not as deeply dissected. The head size and number of ray flowers of the hybrids were intermediate between the two parental species.

The inflorescences were slightly more dense than that of S. piu-

natifidum. The achenes were notched and narrow-winged as in both parents. In a cross between S. compositum and a natural hybrid of 118

S. terebinthinaceum x S. leciniatum L. (#92 x #2129) the leaves were very slightly dissected, but otherwise the plants resembled the hybrids of S. compositum x S. pinnatifidum.

Two specimens of S. compositum ssp. reniforme x S. laciniatum

(#200 x #92, and #l^k x #2-00) were examined. Head size and number of ray flowers were intermediate between the two parental species.

The leaves of the hybrid were somewhat dissected and resembled those of £>. pinnatifidum in out?."- ae. The heads were in an open cymosely branched arrangement rather ‘'han the raceme-like arrange­ ment of S. laciniatum. The phyllaries were acute to acuminate and very scabrous resembling S . laciniatum.

A hybrid of S. compositum ssp. reniforme x S. reverchonii

(#92 x #90) of end arrangement group A was intermediate between the two parents in several ways. The phyllaries had acute tips but were not ciliate-margined. There were cauline leaves present in the hybrid, but the branched inflorescence was less leafy than in 5. reverchonii. The leaves of the stem were larger and longer- petioled than those of S. reverchonii. The achenes were larger and broader-winged than those of S. compositum ssp. reniforme, end the heads were nearly as large as those of #90.

Another hybrid, S* compositum x £3. gracile. Gray (end arrangement group A species), is #lVt- x #2211. This hybrid resembled S. gracile more than S. compositum ssp. reniforme in stem, leaf and inflorescence characters. The ray flowers were deep lemon-yellow as in S. gracile, but were intermediate in length between S. gracile and S. compositum. The branches of the inflorescence were somewhat less leafy than in S. gracile. 119

The hybrids of S. compositum and species of end arrange­ ment group B were also examined. The hybrid between S. compositum ssp. reniforme and #52, an unnamed taxon from Kogers Co., Okla., was intermediate in head size and phyllary length, but otherwise

resembled #52 much more than the S. compositum parent, #91* The

hybrid, like #52, had many cauline leaves extending up into the

inflorescence region, although the upper leaves in the hybrid

were smaller. The lower stem leaves of the hybrid were somewhat

larger and longer-petioled than #52. Both the hybrid and #52 had

strigosely hirsute stems and scabrous leaves.

The other hybrid of S. compositum with a member of end

arrangement group 3 was #91 x #2071 (S. perfoliatum ssp. connatum

(L.) Cruden). The heads and achenes were intermediate, but the

ray flowers were the size of those of S. perfoliatum. The leaves

were opposite, scabrous on both surfaces and ccnnately attached

as in ssp. connatum, but the stem was glabrous as in S. compositum.

The leafy bracts in the region of the inflorescence were much

smaller than in the hybrid.

Morphology of the intraspecific hybrids

At present few intraspecific hybrids of Silphium compositum

have grown to maturity. Of those which have, all but a few are

either intrasubspecific hybrids of ssp. compositum or ssp. reniforme.

The otherB are intersubepecific hybrids of ssp. compositum x ssp.

reniforme. The hybrids are morphologically intermediate in leaf 120 shape and dissection, relative inflorescence density and head size* The achene is rather deeply notched and resembles ssp* compositum more closely. The two parental clones and their F^ are presented in Figures ^9» 50, and 51* Fig. ^9. S. compositum ssp. Fig. S. compositum sep* compositum (#177, garden collection). reniforme (#92, garden collection), Scale: 0.28x. ocale; 0.2Sx. 121 Fig. 51* hybrid of a* compositum ssp. reni forms x ssp. compositun (^92 x ,^177, garden collection). .Scale: 0 . 28x. THE OCCURRENCE OF NATURAL INTER- AND INTRASPSCIFIC HYBRIDISATION

The occurrence of natural hybridization can be tested by making morphological comparisons between artificially produced hybrids and naturally occurring putative hybrids. In the natural situation when a plant fitting the criteria of the artificial F^ is found, both parental species are likely to be found in the vicinity. When hybrid derivatives of backcross generations are found only one parent, or perhaps neither, may be found in the area.

Naturally occurring hybridization in Silphium has been reported previously by Fisher (1959)* Mixed populations of S. terebinthinaceum and S. laciniatum are commonly encountered in

Illinois and Indiana. Frequently hybrids occur with subsequent backcrossing to one or both parents. Fisher and Speer (1965)

discussed the probable origin of S* pinnatifidum from a hybrid

derivative of ^ terebinthinaceum and S. laciniatum and presented

morphological, cytological and chromatographical evidence in

support of this hypothesis.

Silphium compositum is sympatric vrith several other species

of Silphium in various parts of its range. In northwest Georgia

both S. aeperrimum (Hook.) and S. pinnatifidum occur, while S_.

asteriscus L. ssp. dentatum (Ell.) Speer P< Fisher (unpubl.) is

found on the piedmont of Georgia and piedmont and coastal plain of

123 12k

North and South Carolina. Silphium asteriscus L. sap. asteriscus is found in the mountains of Tennessee and on the piedmont of

North Carolina, while S. asteriscus L. ssp. angustatum (Gray)

Speer £; Fisher (unpubl.) occurs on the coastal plain from South

Carolina to Florida. Silphium perfoliatum ssp. connatum is found in the mountains of western Ncrtn. Carolina and Virginia and southern l.Test Virginia. The range of S* terebinthinaceum, while primarily midwestern, does extend into the piedmont of North and

South Carolina. Although the ranges of S. trifoliatum L., S. mohrii Small, S. laevigatum Pursh, S. scaberrimuro Sll., S. brachiatum Gattinger, S. gracile Gray and £, simpsonii Greene are not yet known accurately, it is likely that they also occur within the range of 3. compositum in some area.

Of the species of Cilphiura v/hich are sympatric ’with S. compositurn, only u few have been found occurring in the same populations. Populations of S. asteriscus ssp. angustatum were found growing with S. compositum ssp. venosum in Randolph Co.,

Georgia, ar.d with S. compositum. ssp. ovatifolium in Jackson Co.,

Florida. In Talbot Co. ar.d Troup Co., Georgia, asteriscus ssp. dentatum was found in the same site as 3. compositum ssp* compositum. Silphium asteriscus ssp. asteriscus and S. compositum ssp. compositum were found in close proximity in Person Co., North

Carolina. Silphium asperrimum was collected in Marshall Co.,

Alabama, from the same location as S. compositum ssp. compositum and in Twiggs Co., Georgia, along with specimens of S. compositum 125 ssp, venoaum. In none of these populations were any hybrids between S, compositum and the other species detected.

Other species of Silphium, although their general distri­ butions are sympatric, do not grow in similar habitats. Silphium terebinthinaceum and 3, perfoliatum ssp. connatum are usually found in rather moist areas such as wet prairies or along river banks, where 3. compositum does not usually grow. Silphium pinnatifidum has a very restricted distribution which coincides with that of 3. compositum only in northwest Georgia where S* pinnatifidum is found in undisturbed open wonted areas. Silphium compositum has not been found in the same habitat.

In spite of the occurrence of S. compositum in the same populations with other species of dilphium, interspecific hybrids of 3. compositum in nature have not been detected, with one possible exception. The exception is that specimen of S. com­ positum ssp, reniforme from West Virginia (Fig. 26) which appears to be intermediate between 3. compositum and 3. terebinthinaceum and nearly identical to the artificial F^ hybrid of these species.

The apparent lack of interspecific natural hybrids of S. compositum can be explained in several ways. The morphological analysis of artificial interspecific hybrids indicated that hybrids with S. perfoliatum ssp. connatum, S, gracile, and S_, reverchonii and the unnamed taxon from Hogers Co., Oklahoma, resembled the other parent more than the S. compositum parent, and in nature they might go undetected. Naturally occurring hybrids

of 3. laciniatum and S. pinnatifidum with 3. compositum might be 126 classified as slightly aberrant forms of the non-compositum parent, whereas hybrids of S. compositum with S. terebinthinaceum might be classified as unusual forms of compositum ssp. reni­ forme . If, in succeeding generations, backcrossing to the non- compositum parent occurred in any of the above mentioned instances, the hybrids would not be detected at all or be considered as variants of the other parent.

Intraspecific hybridization, on the other hand, appears to be or to have been at one time rather common, at least between some S. compositum subspecies. The presence of intergrading forms between ssp. reniforme and ssp. compositum on the piedmont where they are sympatric, suggests hybridization of the two subspecies.

In general, the hybrid of these two subspecies from the research garden (Fig. 51) resembles the intergrade forms of ssp. compositum tFig. 8 and 9). It is unlikely, however, that these naturally occurring forms are F^ hybrids in most cases, because the two parental subspecies are not found in the same populations.

Intergradation between ssp. compositum and ssp. venosum, and between ssp. ovatifolium and ssp. venosum has also been en­ countered where tney arc sympatric. However, the variability in any one population is rather low, and populations containing both parents and hybrids were not found.

Of the five taxa which have been found in the same popu­ lation with S. compositum, four have produced hybrids with S.

compositum in the research garden. The results are presented in

Table 2^-. 127

TABLE 2k, Crossability of S. compositum with Silphium taxa found in the same habitat, other sympatric taxa and non-sympatric taxa

Number Average % Average Crossing Successful Crossability Taxon Attempts Crosses Index

Taxa found in same habitat:

S. asperrimum 6 35.3 1 . 6 % S. asteriscus ssp. 2 50.0 k,l% asteriscus S. asteriscus ssp. 6 0.0 0.0 % dentatum S. asteriscus ssp. 1 100.0 3.0% angustatum S. asteriscus ssp. 1 100.0 5 . 1 % asteriscus x ssp. dentatum Average - 56.0 2.7%

Average for other - ^5.3 5.5% sympatric taxa

Average for taxa allopatric - 37.2 7.°%

Crossability of S. compositum with S. asperrimum and S. asteriscus is very low even under research garden conditions, thus the chances of finding hybrids in the natural habitat would probably be even lower. The percentage of successful crosses is highest for those taxa found in the same habitat, and lowest for the allopatric species, while, at the same time, crossability is slightly higher for the allopatric species than for the sympatric taxa. DISCUSSION AND CONCLUSIONS

It is clear from the results discussed previously that the subspecies of S. compositum, while distinct enough to warrant taxonomic recognition, are not completely separated entities.

Morphologically there is considerable intergradation between sub­ species, particularly between ssp. compositum and ssp. reniforme in the piedmont region. Variation of most morphological characters is discontinuous, and several clinec appear to exist when popu­ lation studies are made. The morphological variability observed in the field has a genetic basis as is shown by the retention of these characters in the research garden clones. Cytologically the four subspecies appear similar although more information is needed about chromosome arm end arrangement, for example. Hybridisation studies to date indicate that the subspecies are interfertile.

With the methods used in this study significant chromatographic differences cannot be detected between any of the subspecieE.

Geographically ssp. reniforme and ssp. compositum are sympatric on the piedmont, while ssp. compositum, ssp. venosum and ssp. ovatifolium are sympatric on the coastal plain. The flowering

Xjeriods of the subspecies are also coincident. Ecologically the four subspecies occupy rather similar habitats.

No populations were found in which plants of two subspecies were present. The morphological analysis of both the research

128 129 garden clones and population samples indicated intermediates between subspecies exist, however. This suggests that hybridi­ zations have occurred sometime in the past and that the present- day populations are segregates of these hybridizations.

In areas where there is sympatry of two or more sub­

species, the morphological variability in any one subspecies

appears to be greater. The coincidence of the flowering periods,

the similarity of habitats in which the subspecies are found,

and the apparent irtereubspecific fertility all suggest that

hybridization should be commonly encountered in parts of the

coastal plain and piedmont.

The important factors for understanding variability in

this species are the population structure, the nature of cross

pollination, and seed development, dispersal and germination of

S. compositum. Like other members of the genus this species is

insect-pollinated, primarily by honey bees. Because of the small,

scattered populations In which S. compositum occurs, and because

many bees travel no farther than two or three miles from their

hive, cross pollination berween any two plants is apt to be

limited to a distance of only a few miles. If cross pollination

by a vector occurs over a distance of four miles, and if one

allows a minimum of 2 years for the resultant hybrid to reach

maturity and flower, then introgression of genetic material could

advance four miles in two years time. Experimental evidence

indicates, however, that even in the relatively ideal conditions

of the greenhouse and research garden, the average crossability 130 index of all intraspecific crossing attempts is slightly more than.

25#. Therefore, the tirae in which introgression could occur over the same distance is increased by a factor of four, and thus the rate would tlien be four miles in eight years, or one mile every tv.ro years.

This low crossability index is probably the result of both genetic and cytologic factors. Within the fifty research garden clones examined, twelve were found to have either a heterozygous trar.slocation or inversion, or an extra univalent. These cyto- logical abnormalities coupled with genetic factors caused eleven of the fifty clones to have pollen stainability percentages under

90#. The existence of lethal gene action was not demonstrated directly, but was suspected as a result of low pollen stainability without accompanying cytologies! phenomena.

The fruits of J. compositum in the research garden fall to the ground near the base of the parent plant. It is not likely that the fruits stick to the coats of animals or are transported by the wind over any great distance, though birds eating the fruits may provide some degree of dispersal in the natural situation.

It appears from observations made of germinating seeds in the greenhouse that light is a necessary factor in Silphium seed germination. Cold-treated seeds planted too deeply do not germi­ nate, but when they are brought to the surface of the soil, they will sometimes germinate within a few days. In an established natural habitat light penetration through a thick growth of vege­ tation might well be insufficient to effect germination of most 131 seed3. Seedlings were never seen in the natural habitats in the numbers that they were found in the research garden on the cleared

soil surface.

The establishment of new colonies of 3. compositum on

newly-created road-cuts and other cleared areas would afford an

opportunity for more seedlings to develop. Several areas of this

sort were seen along roadsides, but S. compositum did not appear

to be much more numerous here than in more established communities.

Seed production in natural populations appears to be good,

in that the fruits are fully formed in most population collections.

Subsequent germination failure, however, often occurs in the green­

house in spite of cold treatment and apparent sufficient light

penetration. This failure of germination in the greenhouse and

lack, of seedlings in natural populations on cleared areas suggests

that some gemination failure might possibly be the result of

genetic conditions within the seed, as well as unfavorable environ­

mental factors.

The combination of all these factors greatly impedes the

rate of introgression. Added to this must be the possibility that

there are not populations of b. compositum every three or four

miles in many areas, and until such time as a new colony is estab­

lished near by, there can be no introgressicn. An additional

factor which must be considered is that during some years, because

of severe climatic conditions, roadside cutting and spraying and

other catastrophic events, a population will not contain any

flowering plants. When all these are considered, the suggested 132 introgression rate of one mile every two years is very unlikely, and a more realistic estimate ’would be about one mile every fifty or one hundred years. A long period of time must be considered in accounting for the present geographical variation in 3. compositum.

Once introgression occurs, genetic material could be passed slowly from population to population until a gradual clinsl situation exists between the subspecies. Selection of the segre­ gates then would result in a series of rather homogeneous popu­ lations, each slightly different from the next, almost imperceptibly changing from one subspecies to another. This kind of hybridi­ zation is probably occurring at the present time, but it is very difficult to detect. Complete swamping is prevented because of the rate at which this occurs.

In spite of all the odds against introgression of genetic material from one subspecies to another, morphological data at the present time indicate that this is what has happened. The chromatographic data, while not showing any conclusive evidence about the relationship of the subspecies, do support the contention that they are all quite closely related, i'uture cytological ar.d hybridization studies will undoubtedly aid greatly in unraveling this relationship further.

Any hypothesis of the evolution of the four subspecies must take into account the physiographic history of the area in which this occurred and an estimate of the amount of time required*

Much of the present range of S. compositum has been continuously available for colonization since the late Cretaceous period. 133

Following the recession of the Cretaceous seas, herbaceous flowering plants were probably colonizing the newly obtained habitats. The climate of the late Cretaceous and early Tertiary in what is now southeastern United States was more subtropical than at present, and the forests of this time were composed of many present-day genera, including Firms, ^uercue ard Kyssa. The constant erosion following recession of the seas resulted in an increase in lowland and decrease in upland habitats (Braun, 1950),

The subtropical nature of the early Tertiary forest was replaced by a more temperate vegetation migrating from the north beginning in the Cligocene, The northern er.d of the present range

of ssp. compositum has not beet submerged since the Cretaceous,

and it seems probable that this subspecies evolved in place on the

coastal plain and piedmont prior to the Pleistocene glaciation.

Curing the southerly migration of northern species during the

glacial advances, a segment of a S. terebinthinaceum-like taxon

from the midwest may have come in contact with ssp. compositum.

Through hybridization and subsequent backcrossing the present

array of intergrading forms from the coastal plain to the mountains

has resulted. The most likely area for the contact of these two

elements would probably have been along the fall line in the

Carolinae where present-day remnant populations of S, terebinthin­

aceum are found. Segregates remaining in the mountains and back-

crossing to the terebinthinaceum-like parent would have resulted

in ssp. reniforme. Presently S. terebinthinaceum and £. compositum

ssp. reniforme are ecologically isolated from each other. 13^

Continued southward shifting; of the vegetation may have resulted in the migration of ssp. reniforme along the mountains and ssp. compositum along the piedmont and coastal plain. Habitats on the coastal plain of western Florida became available with each glacial advance as the sea level receded. Subspecies ovatifolium may have evolved at this time from a segment of ssp. reni forme by hybridization with an element which has since disappeared from the area. Following the establishment of ssp, ovatifolium in Florida and migration of ssp. compositum southward, ssp* venosum undoubt­ edly arose from hybridization of these two subspecies in the area betv/een their centers of distribution.

Under this hypothesis gene exchange and introgression between ssp. compositum and ssp. reniforme has been occurring for a longer period of time than it has between ssp* compositum and ssp. ovatifolium. This may account for the lach of clearly de­ limited intermedia tec between ssp. reniforme and ssp. compositum, while ssp. venosum is still a distinct intermediate between ssp. compositum and ssp. ovatifolium. This agrees with the idea that the youngest taxa are found on the youngest land surfaces.

Using the previous estimate of one mile in one hundred years as a reasonable rate of introgression, it is apparent that the over one million years since the first ice advance of the

Pleistocene is more than sufficient to account for the present

state of introgression of ssp. compositum and ssp. reniforme.

The origin of ssp. venosum from hybridization of ssp, compositum 135 and ssp. ovatifoliuu could have occurred well toward the end of the Pleistocene at the estimated rate of introgression.

This hypothesized mode of subspeciation in S. compositum is in agreement v.'ith that cf Vernonia angustifolia as stated by-

Jones (196^). Vernonia angusti folia, like 3. compositum, is diploid, insect-polline ted ar.d self-incompatible, ar.d is a species of the southeastern coasta1 plain composed of three varieties having in general the sane distribution as 3. compositum ssp. compositum, ssp. venosum and ssp. ovatifolium. Jones stated that the morphological variation between these varieties of V. angusti- folia is ir.ter grading, and the varieties appear to hybridize wherever thev come into contact with each other. he suggested a combination of isolation of three segments during the Pleistocene follov:ed by subsequent hybridization, backcrossing, mutation, recombination and natural selection as a possible mode of origin of the varieties and the occurrence of morphological clines between them. In this case the morphological intermediate, V. angustifolia ver. angustifolia, did not occupy an intermediate range between the other two varieties, but extended farther north than either of the other two varieties.

Several important differences between these two species,

3. compositum and V. angustifolia, can be noted. There are other closely related species of Vernonia or. the coastal plain, and wherever these come into contact with V. angustifolia, hybridi­ zation is apt to occur. The frequency of this natural hybridi­ zation is indicated by the tanonoraic recognition given several 136 interspecifi.c hybrids* Hybridization studies indicated that the taxa were all quite interfertile, and Jones suggests that the most important isolating mechanisms between these closely related

species are ecological. None of the above phenomena are true of

5. compositum*

In sharp contrast to V. angustifolia is the subspeciation problem in the western species, Clarkia biloba, which is a diploid

out-crossing, insect-pollinated annual occurring in large popu­

lations separated by short distances (Roberts and lewis, 1935)*

Lnlike Vernonia angustifolia, Clarkia biloba is self-compatible,

and self-pollination occurs whenever the pollinating insect visits more than one flower on the same plant. Seed production is high,

and dispersal is only in the immediate area around the parent plant.

Morphological variation was more noticeable in the flowers than in

other parts of the i>lant, and three distinct morphological races

with adjacent but allopatric ranges were found. The transition

from one race to another is apparently more abrupt than that found

among the subspecies of S. compositum. Roberts and Lewie (1955)

concluded that "subspeciation ir> G. biloba appears to be the result

of barriers to gene exchange other than those imposed by ecological

differentiation" (p. *+52). The barriers which do appear to be

responsible for subspeciation in Clarkia biloba are the colonial

habit and the presence of a chromosomal structural rearrangement

in one of the subspecies, Roberts and Lewis also concluded that

interspecific hybridization was not a factor in the intraspecific

variation of C. biloba. 137

It is somewhat doubtful whether distinct ecotypes of £, compositurn could be demonstrated. Ecotypes are detected by ob­ serving differential responses to varying environmental conditions, and this has not been studied in Silphium compositum. The im­ portant, differences are physiologic in nature, related directly to the plants' ability to survive in a particular habitat. These may or may not be accompanied by morphological differences of

taxonomic value. In their studies of Potentilla glandulosa

Clausen, Keck and Heisey (19^0) found that in many instances there

was genetic linkage of taxonomic characters with those of

physiological and ecological importance. Even so, some ecological

races were found which were not marked by diagnostic morphological

characters. These authors suggested that "greater differentiation

into regional races nay therefore be expected in areas with more

varied topography and climate, as, for example, in the Facific

Coast states." (p. ^09). The changes in topography and climate

of the southeastern united otates are less abrupt than those on

the Pacific Coast, and great differences in habitat from subspecies

to subspecies of 3, compositum are not noticeable.

Isolation cf segments, accumulation of mutations, re­

combination and introgressive hybridization appears to be re­

sponsible for the origin of several of the subspecies of S* com-

positum as it is in Vernonia angustifolia. The maintenance of the

subspecies as distinct entities, as with Clarkia biloba, results

from (1 ) cytological and genetic factors leading to reduced viability, (2) the nature of the population structure, and (2) the rate at which out-crossing occurs. The isolation of £• compositum from other species in the genus seems to be the result of the cytological, geographical and ecological factors mentioned previously. SUKMAHY

The diploid perennial species Silphium compositum Kichx. was studied morphologically, chemically and cytologically by use of representative clones in the research garden and population samples collected in the field. The nomenclature was revised to include four subspecies: ssp. compositum, scp. reni for .ie, ssp. venosum and ssp. ovatifolium. The distribution, habitat and flowering period of each subspecies was discussed, Morphologically it v.’as shown that compositum ssp. reniforme is intermediate between a. compositum ssp. compositum and »j. terebinthinaceum, though, more similar to the former, and that o. compositum ssp. venosum is intermediate between ssp. compositum and ssp. ovatifolium.

Populations of S. compositum are snail, scattered and rather homogeneous, but the inter-populational variability results in

character clines extending from the mountains eastward to the

coastal plain, and then south into Florida. ho significant

differences in chromatographic profiles of the four subspecies

could be ascertained. Cytological examination revealed that

macro- and micro-pollen grains, univalents, ard structural

aberrations such as heterozygous translocations and inversions

are present in several clones of three of the subspecies. In

addition there is some reduction in fertility evidenced by pollen

stainability which appears to be genic rather than structural,

139 1^0

The morphological intergradation between subspecies suggests that intraspecific hybridization lias occurred or is occurring. The rate at which introgressicn could occur in relation to the population structure, crossability, seed dispersal and seed germination is discussed, V/ith this rate of introgression as a basis, a hypothesis of the origin of the subspecies is presented which accounts for the present variation pattern. The

isolation between the subspecies appears to b ? due to the rate at

which gene exchange can occur, as experimental studies indicate

that crossability is moderately high. Crossability of S. com-

positum with other species, however, is very low, ar.d natural

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