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Bayer, Randall James

EVOLUTIONARY INVESTIGATIONS IN ANTENNARIA GAERTNER (ASTERACEAE: INULEAE)

The Ohio State University Ph.D. 1984

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University Microfilms International EVOLUTIONARY INVESTIGATIONS IN ANTENNARIA GAERTMIR (ASTERACEAEs INULEAE).

DISSERTATION

Presented in Partial Fullfillntenfc of the Requiranents for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University

by Randall James Bayer, B.S., M.S.

*****

The Ohio State University 1984

Reading Committees Approved bys Dr. Daniel J. Cra&rford Dr. Gary L. Floyd Dr. Katherine L. Gross Dr. Tod F. Stuessy idvisor Dept, of Botany Dedicated to

Prof o Go Ledyard Stebbins, who first introduced me to the genus Mtennaria and whose continued support and invaluable discussions have provided a constant source of inspiration. ACKNOHLEDGMENTS

Numerous people should bs acknowledged for their contributions during the tenure of this studio Dr. Daniel J. Crawford? ny major professor ? is gratefully acknowledged for his continued encouragenenfc and guidance for the past six years. Members of ray committee? Drs. Daniel J. Crawford, Gary L. Floyd. Katherine L. Gross? and Tod F. Stuessy are given special thanks for their comments and criticisms on this dissertation. This study was supported by grants from Sigma Xi? The American Alpine Club, and NSF doctoral dissertation improvenent grant #DEB~82fl0359. I am especially grateful to Joe L. Bruner and G. Ledyard Stebbins for their aid and companionship in securing many of the collections. The following people are also gratefully acknowledged for their collecting efforts from various regions: L. Bayer? P. Bierzychudek. R. Bittman? J. Canne? D. Crawford, M. Galligan, H. Iltis? J. LaDuke? R. Pilatcwski? R. Stebbins? K. Urbanska? R. Whitkus? and E. Williams. I also appreciate the invaluable discussions with my fella*? graduate students in systematica and ecology at Ohio State. Special thanks are extended to Dr. Robert Warmbrodt for his advise concerning illustrations contained in this dissertation. I also wish to thank my family for their continued support and encouraganent.

iii VITA.

July 13, 1955...... Born - Buffalo, N w York* 1978.oeeooooooooooooeooooooe.B.S., Cornell University, Ithaca, New York. 1978-1979...... Graduate Teaching Assistant, Hie Ohio State University, Columbus. 1980...... M.S., Hie Ohio State University, Columbus.

1979-1982...... Graduate Research Assistant, Hie Ohio State University, Columbus. 1982-1983...... Graduate Teaching Assistant, Hie Ohio State University, Columbus. 1980...... Sigma Xi Research Grant. 1980...... American Alpine Club Research Grant. 1982-1984 ...... NSF Doctoral Dissertation Improvement Grant. 1983-1984...... Presidential Fellow, Hie Ohio State University, Columbus.

PUBLICATIONS Bayer, R.J. and G.L. Stebbins. 1981. Chromosome numbers of North American species of Antennaria Gaertner (Asteraceae? Inuleae). Amer. J. Bot. 68:1342-49. Crawford, D.J. and R.J. Bayer. 1981. Allozyme divergence in Coreopsis cyclocarpa (Ccmpositae). Syst. Bot. 6:373-379. Bayer, R.J. and G.L. Stebbins. 1982. A revised classification of Antennaria (Asteraceae: Inuleae) of the eastern United States.

iv Sysfco Bofco 7:300-313. Bayer, R.J. and G.L. Stebbins. 1983. Distribution of sexual and asexual populations of Antennaria parlinii. Evolution. 37:555-561. Bayer, R.J. 1984. Chromosome numbers and taxonomic notes for North American species of M t e n m r i a (Asteraceae: Inuleae). Syst. Bot. 9:74-83. Price, Ho J., Crawford, D.J., and R.J. Bayer. In press, n contents of Caresegis. xmesensfiidea and Ck meseagis (Asteraceae), a progenitor-derivative species pair. Bot. Gaz. PAPERS READ AT EKQFESSIONAL MEETINGS: Bayer, R.J. and G.L. Stebbins. Population structure and asexual reproduction in Anfcsmaria Earlinii of Ohio. At ic-SEB II, Vancouver B.C., July 1980. Bayer, R.J. and G.L. Stebbins. Geographic distribution of chromosome numbers, sex ratios, and aponictic reproduction in Antennaria of the eastern United States. At IC-SEB II, Vancouver, B.C., July 1980. Stebbins. G.L. and R.J. Bayer. Sexual and asexual reproduction in Antennaria and the origin of sex. At IC-SEB II, Vancouver, B.C.. July 1980. Crawford, D.J. and R.J. Bayer. Allozyme variation in Coreopsis cyclocarpa (Ccmpositae; Heliantheae). At AIBS, Bloomington, Indiana. LaDuke, J., Nescm, G., Crawford, D., and R.J. Bayer. The biology of Trillium nivaie Riddell. At AIBS, Bloomington, Indiana. Bayer, R.J. On the possible origins of the heteroploid agamic Antennaria Parlinli complex of the eastern United States. At Hie Ohio Academy of Sciences meetings, April 19©. Bayer, R.J. The compilospecies Antennaria Eaglmli and its possible relationship with certain diploid species of the genus. At AIBS, Pennsylvania State University, August 19©. Bayer, R.J. The compilospecies Antennaria neodioisa and its possible relationship to diploid species of the genus. At AIBS, Grand Forks, North Dakota, August 19©. Bayer, R.J. Electrophoretic assessment of the genomic composition of the Antennaria Parlinii polyploid agamic complex. At AIBS, Fort Collins, Colorado, August 1984. FIELD OF STUDY

Major Fields Systenatic Botany

Evolutionary Studies in Mtennaria. Professor Daniel Cranford. TH3LE OF CONTENTS

page

v m . . •••O...... 0 0 0 0 .00 iv LIST OF TABLES...... ix LIST OF FIGURES...... x INTRODUCTION ...... 1 CHAPTER I. INVESTIGATIONS INTO THE EVOLUTIONARY HISTORY OF THE POLYPLOID COMPLEXES IN ANTENNARIA (ASTERACEAE; INULEAE). I. THE NEODXQICA COMPLEX...... 4 Introduction. .... 4 Materials and Methods...... 6 Results...... 9 Discussion ...... 17 Literature Cited...... 22 II. INVESTIGATIONS INTO THE EVOLUTIONARY HISTORY OF THE P0LYHO3ED COMPLEXES IN ANTENNARIA (ASTERACEAE; INULEAE) II. THE A. PARLINIICOMPLEX.,...... 40 Introduction...... 40 Materials and Methods .... 43 Results...... 45 Discussion. .... 49 Literature Cited...... 58 III. ALLOZYME DIVERGENCE AMONG FIVE DIPLOID SPECIES OF ANTENNARIA AND THEIR ALLOEOLYELOID DERIVATIVES 74 Introduction...... 74 Materials and Methods...... 76 Results ...... 79

vii page Discussion...... 82 Literature Cited...... 95 SUMMARY ...... 113 APMDIX A. Chromosome numbers and taxonomic notes for North American species of Antennaria (Asteraceae; Inuleae)...... 115 B. Data matrix for all OTUs used in phenetic studies of Aotannaria...... 125 C. List of 38 characters and states used in the numerical study of Antennaria...... 158 D. Table of allelic frequencies for 37 populations of Antennaria used in the allozyme study .... 160 E. Table of genetic distances and identities for all pairwise comparisons between the 37 populations of Antjaanaria used in the allozynte study 164

viii LIST OF TABLES

TABLE CHAPTER I page 1 o Mean similarity matrix of taxa ©f AnfcmnsdLa with each Other eo ...... 37 2 S Mean similarity matrix of taxa of Antennaria with numerous artificial hybrid anbinatdons<>.s.e.s 38 3. Table of posterior probabilities derived from a discriminant analysis...... 39 CHAPTER II 4. Mean similarity matrix of taxa of Antennaria with each other and with numerous artificial hybrid combinations...... 72 5. Table of posterior probabilities derived from a discriminant analysis...... 73 CHAPTER III 6. Population designations, number of plants examined per population, locality data, and voucher number for the populations of AoteSDioa...... 101 7. Allelic frequencies for species of Antennaria 105 8. Genetic variation for species of Antennaria...... 107 9. Gene diversity statistics for five species of A nfcegnaria...... 110 10. Mean genetic identities and distances within five species of Antennaria...... Ill 11. Genetic identities and distances for pairwise interspecific comparisons of six species of Antennaria ...... 112

ix LIST OF FIGURES

FIGURE ' CHAPTER I gage

1. Principle components analysis composed of 201 GTUs including five diploid taxa of Mteonaria»...... 25 2. Percentage average pollen stainability of diploid species of Antennaria and their interspecific hybrids. 27 3. Principle components analysis composed of 276 OTUs including four diploid species, interspecific hybrids f and the polyploid JL. neodioica complex 29 4. Principle components analysis group outlines with interspecific hybrid OTUs and 2 anomalous natural taxa. .*...... 31 5. Correlation phenogram (UFGM&) composed of 170 OTUs including four diploid Antennaria species, interspecific hybrids, and the polyploid A*. neodioica. 33

6. Line drawings of capitulescences and basal leaves of representative specimens among four diploid species of Anfcamria, their interspecific hybrids, and 5 individuals of A*. neodioica, s.l...... 35 CHMTER II

7. Principle components analysis composed of 149 OTUs including three diploid AnteDnacia species, interspecific hybrids, and the polyploid A*. Barlinii s.l .... 62

8. Correlation phenogram (UFGMA) composed of 153 OTUs including three diploid species of Antennaria. interspecific hybrids, and A*. Parlinii s.l...... 64 9. Principle components analysis composed of 117 OTUs of the A*. SSLlrnix and Aa. neodioica polyploid agamic complexes...... 66 page 10. Line drawings of capitulescences and basal leaves of representative specimens among three diploid species of Antennaria. their interspecific hybrids, and A. Parlinii (both subspecies) ...... 68 11. Proposed relationship of the Anfceonada neodioiga s.l. and A& Parlinii agamic complexes to their sexual diploid progenitors...... 70

CHAPTER HI

12. Distance phenogram (UPGMA) composed of 37 populations of 6 taxa of M t e m a r l a ...... 99

xi introduction

Antennaria Gaertner (Asteraceae: Inuleae) is a genus of dioecious®, entire-leaved® herbaceous perennials distributed throughout the tanperate and arctic regions of the northern hanisphere. Hie genus is taxonanically complex due to widespread hybridization® polyploidy® and apcmixis (agamospermic seed production) 0 Antennaria consists of about 26 sexual diploid taxa (2n = 28) and five large® polymorphic® agamic complexes, Hie sexual diploid taxa are well defined morphologically® but the agamic complexes, like those in other genera such as Grepis® fljgjasiuffl, £sa? Rubus, and Taraxacum, present taxonomic difficulty. The agamic complex is thought to arise through the processes of natural hybridization among sexual diploid species and agamospermy® leading to the formation of a large number of agamospecies. To sane degree® the agamospecies are morphologically distinct and the variation between then is partially discontinuous. Hie agamic complex forms a morphological continuum with the sexual diploids from which it is derived, thus partially obscuring the morphological distinctness of the diploid species themselves. Hie main objectives of this study are to investigate the sexual diploid species and in turn the relationships between then and the two polyploid agamic complexes. It was hoped that a better understanding of the origins, genomic composition* and reproductive modes of the agamic complexes* would in turn provide the basis for a tetter taxonomic treatment of the group. The a,. Parlinii sensu Bayer and Stebbins and A« neodioica sensu Bayer and Stebbins agamic complexes were chosen for study because they were already well known with respect to chromosome numbers and reproductive modes. They are also less complicated than other agamic complexes in the genus with regard to morphological variation and taxonomy. Three sets of investigations* two morphological (Chapters I and II) and one electrophoretic (Chapter III) * were initiated to study the problem of the origins of the polyploids. Five diploid species*

A. neglecta. A. plantaginifolia, A. jaesnosa* A. solitaria, and A. virctinica. are most similar morphologically to the two polyploids and thus were chosen as their most likely progenitors. Crosses were made among the five diploid species in all combinations so that the morphology of the F^ hybrids could be compared with that of the naturally occurring polyploids. The morphology of the diploids* and their artificial interspecific hybrids were compared to the A. rtaodioica and A« Baglinii complexes using phenetic methods (Chapters I and II). Enzyme electrophoresis* which has teen useful in elucidating the origins of polyploids in several plant genera* was used (Chapter III) to test the hypotheses of the multiple hybrid origins of A» Parlinii and A. neodioica as formulated on morphological data (Chapters I and II). Additionally* genetic divergence among the five diploid taxa was evaluated employing allozymes8 Chapter I INVESTIGATIONS INTO H E EVOLUTIONARY HISTORY OF H E R3LYEL0ID ®MHjE3CES IN MffiEWftRm (ASTERACEAE: INULEAE). I. H E A® NEODIOICA 0QM5LEC.

Introduction The Antennaria neodioica polyploid agamic complex is widely distributed across North America between 40° and 60° north latitude. Bayer and Stebbins (1982) circumscribed A. neodioica of the eastern United States as consisting of the three subspecies A® neodioica Greene ssp. canadensis (Greene) Bayer and Stebbins, ssp. neodioica. and ssp. netaloidea (Fern.) Bayer and Stebbins. More recently, A» Howellii Greene of western North America has been included as a fourth subspecies (Bayer, 1984a). The polymorphic A* neodioica s.l. is characterized by its relatively large single-nerved basal leaves and primarily white-tipped involucral bracts. Stebbins (1932) demonstrated that pistillate manbers of the A® neodioica complex reproduce agamospermously (diplospory followed by diploid parthenogensis). Rare staminate plants were found to have highly irregular meiosis and were likely to be sterile (Stebbins, 1932). Because these rare staininate clones of A« neodioica are sterile, their presence is reproductively inconsequential and the species as a whole 4 reproduces asexually. The secies consists of both tetraploids (2n = 56) and hexaploids (2a = 84) (Bayer and Stebbins? 1981; Bayer? 1984a) . •Hie agamic complex of A® neodioica can be considered to be in the early mature stage of development (terminology of Grant? 1981) ? because the sexual diploids are still abundant; the genus as a whole contains approximately 26 sexual diploids (Bayer? 1984a). In many cases agamospermous manbers of agamic complexes have arisen through hybridization among several sexual diploid species

(Grant, 1981) . Diploid Antennaria neglesta is found in prairies and pastures from Maine? southwest to Oklahoma? northwest to the Canadian Northwest Territories, and southeast to Quebec. The deciduous forests of the Appalachian Region? Piechtont? and Atlantic Coastal Plain from

Georgia to Maine is the range of diploid A® plantaginifolia. Another diploid, Antennaria racemosa? occurs in dry coniferous montane forests from British Columbia and Alberta? south to South Dakota? Wyoming, Idaho? and California® The shale barren endemic? A® virginica, occurs in a restricted area of Maryland? Pennsylvania? West Virginia, Virginia? and at one locality in Ohio. In this paper I will test the hypothesis that the A® neodioica complex is the product of hybridization among four predominantly diploid (2a = 28) species s A® neglggfca Greene? A. plantaginifolia (L.) Richardson? A. racemosa Hook.? and A. virginica Stebbins. The purposes of this paper are (1) to determine via phenetic methods if the polyploid A* neodioica complex is distinct from its presuned diploid progenitors, (2) to investigate whether the five partially sympatric, sexual diploids are morphologically distinct from one another? (3) to compare synthetic interspecific hybrids among the diploids with the A. neodioica as a means of testing whether the latter is of multiple hybrid or single

origin, (4) to anploy the results from phenetics and pollen viability of interspecific hybrids as a means of inferring relationships among the sexual diploids, and (5) to ascertain the mode of inheritance of

several morphological characters. MATERIALS MID ME3H0DS Field observations were made throughout the ranges of the taxa in the Spring of 1978-84. Over 400 populations of Antennaria were collected and subsequently cultivated in the greenhouse at OSU. Interspecific hybrids were obtained as outlined in Bayer and Stebbins (1982). Several backcrosses were also attempted to determine how the phenotype of the differed morphologically from that of the hybrids. While A. virginica exists as both sexual diploids and sexual tetraploids (Bayer 1984a) , only sexual diploids were used in the crossing experiments. Pollen viability was ascertained by the use of Alexander's differential staining method (Alexander, 1980). Three hundred grains/individual were scored for viability. Specimens were examined from CAN, CM, OH, MO, M3NTU, NDG, NY, OS, EAC, rat, SDU, US, VPX, M S , and W A for morphological studies. Herbarium specimens of field collections and the artificial interspecific hybrids are on deposit at OS. Initially, 38 characters (16 i^egetative and 22 reproductive) were measured on 274 OTUs (operational taxonomic units) to construct a basic data matrix for phene tic analysis. A list of characters used and the basic data matrix can be found in Bayer (1984b, appendix) or can be obtained from the author * Principal components analysis (PCA) , cluster analysis, and correlation coefficients were generated using the NT-SYS package of Rohlf, Kishpaugh, and Kirk (1974). The basic data matrix was standardized by the STAND subroutine of NT-SYS. The correlation and distance matrices were computed using the SIMINWL subroutine. The cluster analysis (UBGMA) was produced by the TOTDN subroutine and the PCA was computed by the FACTOR subroutine of NT-SYS. The BMDP package of Dixon (1981) was used to compute simple univariate statistics and perform the stepwise discriminant analysis. PC&, cluster analysis, and discriminant analysis were used to compare the diploids, hybrids, and polyploid complex. In addition to PCA, cluster analysis, and discriminant analysis, correlation coefficients (mean similarity matrix) using average values of characters for each taxon were employed to compare the natural taxa to each other and to artificial interspecific hybrids between the diploids. A discriminant analysis (which employs characters that best discriminate among taxa) was used to place the artificially synthesized hybrids in their most probable group of inclusion. Computations were carried out at the Instructional Research Computer Center of The Ohio State University. Initially five diploid species of Antenmria; i.e., A. negl&gfca,

A. Plantaginifolia, A. racemosa, A. solitaria. and A* aisginlca, were chosen for analysis with the A. neodioica complex because they were partially sympatric with and morphologically closest to members of the complex. All taxa occur within the eastern half of North America except the diploid A. racemosa and the polyploid A. neodioica Greene ssp. Howellii (Greene) Bayer which are found in the Rocky Mountains of western North America (for details of the ranges see Bayer and Stebbins, 1982 and Bayer, 1984), AQfcJ33m&ia vii^inica consists of diploids and sexual autotetraploids (Bayer, 1984a), but the two can not be separated consistently on the basis of any known morphological character. In this paper both diploids and their sexual autotetraploid derivatives were included in the analysis and will hereafter both be referred to under the inclusive term A, virginica. Several data matrices were used in the study: (1) a 274 OKI matrix consisting of representative specimens of the four diploid species of Antennaria (40 OTOs of A. neglecta, 48 of A. BtoxfcaginifQliay 34 of A. XiasesMSA, and 40 of A« virginica), the four subspecies of A, neodioica (20 OTOs of each) and interspecific hybrids among the diploid taxa (32 GTUs). The remaining data sets are subsets of data matrix 1: (2) a 202 OTO matrix consisting of the four diploid species (40 OTOs of A« neglecta, 48 of A. plantaginifolia, 34 of A. and 40 of A* virginica) plus a fifth diploid, A. solitaria Rydb.(40 OTOs), (3) a 170 OTO matrix (consisting of 20 OTOs of of each of the diploids, 30 OTOs of the interspecific hybrids, and 15 of each of the subspecies of A* neodioica), basically the same as the 274 OTO matrix, but with 104 OTOs eliminated as an aid in displaying the output, and (4) a 39 OTO matrix utilizing the interspecific hybrids (30 OTOs) plus average values for the nine natural taxa, TWo 3-dimensional plots of the first three principal components for matrices 1 and 2 were constructed. A discriminant analysis was performed using matrix number 1. Matrix number 3 was used to construct a phenogram by the unweighted pair-group method using arithmetic averages (UFGM&? Sneath and Sokal, 1973). Pearson product-iranent correlation coefficients (Sneath and Sokal, 1973) were used to compare OTOs of matrix number 4, Antennaria solitaria was eliminated from matrices 1 and 3 as were certain hybrids because they more closely resemble members of the A® Parlinii s.l. (Bayer and Stebbins? 1982) polyploid complex which is to be the subject of another paper.

RESULTS

Jhenetics o£ diploid ®g£iggr~Hie five diploids were subjected to a PC& (Fig. 1) to demonstrate the morphological distinctness of these taxa. Hie first three principal components account for 62.03 % of the variation. Hie first eight factors have eigenvalues greater than one? indicating the characters are not highly correlated. High loadings for component one are mostly vegetative characters? while those for component two are primarily reproductive features. Hie third principal component has high loadings for both vegetative and reproductive characters. Hie five diploid species appear distinct from one another. Hie apparent overlap of A® neglecta and A. plantacrinifolia due primarily to the orientation frcan which figure 1 is d r a m in 3-dimensional space. Indeed? close inspection shows that on the plane of factor 1 versus factor 2 the species are well separated (Fig. 1). Hie two most similar species morphologically, A. neglecta and A. JZiEgiQisa, are distinct and are separated primarily on the basis of reproductive characters along component 2 (Fig. 1). Hie same is true for the species pair of A. 10

PlaxtagjmfolianB. solitaria, Antganaria xasemoga is separated from A. olantaginifolia on the basis of both vegetative and reproductive characters, and while it appears very distinct due to its racemose arrangement of capitula, this analysis reveals its dose morphological similarity to &. plantaainifolia.

The similarity matrix (correlation coefficients) for the taxa (Table 1) reveals that plantaainifolia is most similar to A» racemosa and vice-versa, Mfcemaria neglecta. most closely resembles

the polyploid A. fflgsdioisgi ssp. canadensis, while A. virginica is more like ssp. neodioica indicating the probable predominance of these parental diploid genomes in their respective polyploid derivatives. The fifth diploid. A* solitaria, is most similar to diploid A* neglecta. M u l e the two taxa differ in their basal leaves and habitat preferences, they both have long lash-like stolons, a flag-like

structure in the upper cauline leaves1, and heads that are alike with respect to a number of features (esp. phyllary color and shape). All of the four subspecies of A* neodioica are most similar to each other (Table 1) except for ssp. canadensis which is dosest to the diploid A« neglecta from which it may be derived. Infcerfert.ility of d i s M d ^pgsig£~-Int@rfertility of the

^The flag-like structure is a flat or curled scarious tip at the aid of the upper cauline leaves below the aggregate of heads and excluding the bracteal leaves in the corymb or raceme. This character is accorded a great deal of taxonanic importance and is used as a key character in separating several taxa. diploids can be used as a measure of relationship. Bayer and Stebbins (1982) reported that the diploid species of M t e n m r i a are reproductively isolated because lew numbers of seeds were set in interspecific as compared with conspecific crosses. Hybrids among species isolated by habitat differences (i.e. A® neglecta and A. solitaria) displayed higher seed set? indicating that post-mating reproductive isolating mechanisms are not strongly developed. Pollen stainabilities of the hybrids were determined? but only 27 % of 55 interspecific hybrids were starainate, thus limiting the available data. Figure 2 presents the pollen stainabilities for these hybrids and five of each of the five diploid snecies. The average stainabilities of the hybrids ranges from 51.0 % to 80.0 % with individual ranges of 40.5 % to 85.3 % (Fig. 2). Stainable pollen in the naturally occurring diploids is almost always above 85.0 %. Several hybrids between A* nealecta and A* virginica. were weak, had small, reddish, deformed leaves, and eventually died before flowering. Fhenetics of fhs diplaidsu. hybrids^ a M £Qly&l£ids--3he first three factors of the PCA of four diploids, their interspecific hybrids, and A® neodioica s.l. accounted for 54.43 % of the variance (Fig. 3). The first ten factors have eigenvalues greater than 1.0 indicating that the characters are not highly correlated. The high loadings for the first three factors were similar to those given for the first PCA analysis. Inspection of the PCA (Fig. 3) shows that the four diploids surround the A* neodioica polyploid complex. Also, A. neglecta and A. virginica. which were well separated in the first PCA (Fig. 1). are drawn much closer together in this PCA (Fig. 3) which m y be attributable to the hybrid nature of the complex. The interspecific hybrids, for the most part, fall within the A. neodioica complex. Anfcennaria ngodjoica ssp. HowelLii, which is diagrammtically represented as being separate from the ranainder of A. neodioica s.l. (Pig. 3), is most similar to the diploid A. m c m m s . o with which it is largely sympatric (Bayer, 1984a). Although the ranaining three subspecies of A. neodioica s.l. are not depicted separately in the figure, they do arrange within definite areas of the A. grouping. AofcgjfflaEia fleodioica ssp. canadensis is alligned most closely to A* neglecta; ssp. neodioica closest to A* ^dxginisa? and ssp. petaloidea is more or less associated with A* &tglgct& and A. plantagjnifolia (Pig. 3). Discriminant analysis has been shown to be useful in the identification of hybrids, but only when hybrids of known parentage are included in the analysis (Neff and Smith, 1979). The posterior probabilities of group membership, in which the highest probability indicates to which group each ofthe artificial hybrids belongs, are presented in Table 3. Characters which best discriminate are pappus length, presence or absence of a scarious flag-like appendage on the upper cauline leaves, number of primary veins in the basal leaves, involucre height, and leaf pubescence. Missing data are not allowed in the discriminant analysis, thus staminate hybrids could not be analyzed because the members of the A. neodioica complex lack stamina te plants. The results are displayed pictorially in figure 4 where only the group outlines from PGA are shown along with the interspecific hybrids positioned according to PCS.. Probable group of 13 inclusion of CTOs, as indicated by discriminant analysis, is shown by an arrow (Fig. 4). These results demonstrate that many artificially synthesized hybrids are most similar to members of the A» neodioica complex. In cases where the hybrids were not placed in the A. neodioica complex it was always associated most closely with one of its parents. Mother measure of similarity useful in assessing the relationship of hybrids to natural fcaxa is a matrix of correlation coefficients (Jensen and Eshbaugh, 1976a,b). As previously mentioned, averages for all characters of each of the natural taxa were used to compare the natural taxa with the artificially synthesized hybrids. The results (Table 2) were similar to those obtained for the discriminant analysis, where the majority of hybrids were most similar to either the polyploids or one of their parents. Discriminant analysis, PGA (Fig. 4). and mean similarity matrices (Tables 1 and 2) demonstrate that hybrids having A* solitaria as one of their parents (e.g. HY-11, 13, 21, 24, 25, 26, 27, 30, 31, and 34) do not closely resemble members of the A. neodioica complex, thus supporting the earlier mentioned statements that A. solitaria is not involved in the parentage of the complex. The A« neglecfca x A® racenaosa and A. virginica x A® racemosa hybrids most closely resemble msnbers of the

A. neodioica complex. The table of correlation coefficients (Table 2) also shows that other hybrid combinations, while most resembling their parents, have second and third highest values with members of the A, nesdloka complex (e.g. HY-10 (A. jaeglasta x A. mxgiQisa) or HY-18 (A. Plantaginifolia x A. idrgAnica). Ttie cluster analysis (Fig. 5) has a oophenetic correlation coefficient of 0.794, indicating that the phenogram is an adequate representation of the original resemblance matrix. The number of OTUs was reduced to aid in the visual display of the results. The phenogram shows that each diploid (except A. racemosa) clusters in a uninterrupted group and that the subspecies of A. neodioica are interspersed among the diploids, although not in continuous groups (Fig. 5). The cluster analysis agrees with results for other analyses in showing a close morphological similarity between A* racemosa and A.

SlantiSinifQlia (cf. Figs, 3 and 5}. Mat surprisingly, sane of the ssp. Howellii segregates cluster within A. racemosas the two taxa are sympatric over much of their ranges. The hybrids cluster either within the polyploids or near one of their diploid parents, in agreenent with previous analyses. A single, field collected, specimen (Fig. 5; HY-08) interpreted as being a possible natural hybrid between A® pliotagiDilQjLia and A. neglesta is most similar to A. plsntaginifslia. Additionally, a specimen of ssp. jxeodioto (Fig. 5? HY-06) looks morphologically like a robust A® virginica. and clusters between A® virginica and ssp. neodioica. further indicating the close relationship of ssp. neodioica to A. virginica. Visual inspection of the basal leaves of the diploids (A* neglecta. A. plantaginifQlia. A- racaaosa, and A* viEaimsa), their interspecific hybrids, and the naturally occurring polyploids (A. neodioica s.l.) further reveals the resemblance of the interspecific hybrids to the naturally occurring polyploids (Fig. 6). Likewise with respect to number, size, and arrangement of heads the interspecific 15 hybrids closely resemble the naturally occurring polyploids (Fig. 6).

lateitasga of Mgbp-logjcal flfaarasfcera in totomaKM— Bie interspecific hybrids synthesized during this study provide an opportunity to study the inheritance of characters, sane of which are of taxonomic importance,, The first of the characters to fee considered is pubescence of the adaxial leaf surfaces. Antennaria racemosa is glabrous while the rattaining diploids are pubescent. In crosses involving A. racemosa with the other four diploids, the hybrids (total of 26 individuals observed) range from entirely glabrous to very sparingly pubescent » This suggests that while leaf pubescence m y be controlled by more than one gene, factors for glabrity are dominant to those for pubescence, TWo other characters unique to A. racemosa, namely presence of a citronella-like odor in the leaves and purple glands on the upper cauline stem, are inherited in the same manner as leaf pubescence. These two characters are hard to quantify, but their presence appears to be dominant to lack of the odor and absence of glands* Anfcennarla racemosa has an open racemose arrangement of capitula, while the other four diploid species have a cymose or solitary (in A. solitaria) arrangement. In the hybrids involving A* racemosa and the other diploids (total of 26 observed) the arrangement is usually cymose or cymose with a tendency toward being racemose in the lower heads (see Fig. 6) • This indicates, that as with nianber of heads, the arrangement of heads is controlled polygenically. The racemose head arrangement is apparently recessive to the cymose or solitary arrangement of heads in the capitulescence. 16 Mother character with similar pattern of inheritance is head number per capitulescence. Mien the monoeephalous A. solitaria is crossed to polycephalous species, the resultant hybrids (total of 28 observed) have an intermediate number of heads par capitulescence, bit many hybrids have one or two heads. Head number is probably a polygenic character with alleles for monocephaly being dominant to those for polycephaly. Number of veins in the basal leaves is another character which is likely to be polygenic, but displays dominance. Antennaria neglecta and A® virginica both have single-nerved basal leaves, while the other diploids have 3-5 nerves in the basal leaves. Mien A. neglecta or A. virginica are crossed with A.

A. xamsssa, or A. mlitaria the resultant hybrids (total of 45 observed) usually have a single nerve or occasionally a single nerve and two snail lateral ones. Hius number of nerves in the basal leaves is a polygenic character with the single-nerved condition being dominant to the many nerved condition. In most hybrids the stolon length is intermediate between the two parents. However, in hybrids between A- neglecta and A® solitaria (total of 19 observed), which both have very long, lash-like, stolons, the hybrids have stolons that surpass both parents in length. This suggests that transgressive segregation has occurred.

Presence or absence of the scarious flag-like apjpendage on the upper cauline leaves displays discontinuous variation (a character * accorded taxoncmic significance throughout the entire genus). AnfcsmSEM neglecta and A. solitaria possess the flag while the other three diploid species lack it. The flag is apparently a single gene 17 recessive trait because all hybrids (total of 21 observed) involving A. neglecta or A® solitaria with one of the other three diploids lack the flag. When A. neglecta and A. solitaria are crossed the flag is expressed in the hybrids (total of 19 observed). It will be interesting to see whether the feature is expressed again in later generation hybrids and in what proportions. Crosses between pubescent-leaved A® neglecta and glabrous-leaved A® lafianosa produced an that was glabrous like A. racemosa. but had leaves shaped more like A* neglecta. When one of these F^s (HY-33) was backcrossed to A. neglecta the morphology was altered such that the hybrids were now very similar to A. neglecta. the recurrent parent. Otoo of these B1 hybrids (labelled "nrn", Fig. 4) are included in the FCA analysis and their location in the ordination space is most easily discerned in Fig. 4.

DISCUSSION

It was Juel (1900) who first realized that the Antennaria agamospecies were probably of hybrid origin. He came to this conclusion after examining rare stamina te clones of the primarily agamospermous A. aloina , which he found to be sterile. He knew that many hybrids were sterile, thus he hypothesized that A« alpine was the result of hybridization between two diploid species A® dioica (L.) Gaertner and A. monocephala IX. Stebhins (1932) discussed the origin of several of the agamospermous species of Antennaria occurring in the eastern United States and suggested that A. gataloidea (= A. neodioica ssp. petaloidea) could have resulted from hybridization between A. mglgcta and A* plantaginlfolia . Antimnaria virginisa was likewise added to the list of possible diploid progenitors of the A. neodioica complex by Stebbins (1935). The crossability and interfertility data from this study support previous conclusions (Bayer and Stebbins, 1982) that the diploid species are isolated by both spatial and reproductive isolating mechanisns (in the terminology of Levin, 1978). Soma species such as A. neglecta and A. are isolated ecologically, while others such as A* planfcaqinifolia and A- neglecta occasionally occur sympatrically (Bayer and Stebbins, 1982) but are apparently isolated by cross inccmpatability mechanisms and hybrid sterility. The interspecific hybrids display meiotic irregularities (Bayer, 1984a) and reproductive isolation among these taxa may be due to one or more inversions or transactions causing hybrid sterility. Despite all of these isolating mechanisms, several putative hybrids were discovered in the field (Bayer and Stebbins, 1982). West Virginia is apparently an area where hybridization among all four of the eastern diploids is common. Numerous populations with mixed nianbers of diploids and putative hybrids were observed (Bayer and Stebbins, 1982) in this area. The Black Hills of South Dakota is also a potential area of hybridization because this is where A* neglecta and Ao i^moosa occur in sympatry (Bayer and Stebbins, unpubl. obs.). The close similarity of seme of the segregates of the A. hgodioica complex, diploid A. flgglfisfca, &. m i s s a > A. filapfcaginifQlia, and especially A. virginica is demonstrated by the cluster analysis (Fig. 5) and the PGA (Figs. 3 and 4). PGA. has been shown to be useful in the identification of hybrid swarms of mixed genetic origin (Whiff in, 1973; Jensen and Eshbaugh, 1976a). The high variability in the A. neodioica complex (Fig. 3) could be the result

of extensive backcrossing following initial hybrid formation, as has been demonstrated for other groups (Neff and Smith, 1979). The BC& demonstrates that A® neodioica. while consisting mainly of genes of A® neglecta and A® virginica also contains genes of both A® racemosa and to a lesser extent A® Plantaainifolia (Fig. 3). Discriminant analysis (Table 3) and correlation coefficients (Tables 1 and 2) show the close similarity of the interspecific hybrids to members of the A® neodioica complex. Studies on backcross hybrids demonstrate how a single backcross can make a significant difference in the morphologies between the and (Fig. 4). Unfortunately, polyploid derivatives of the interspecific diploid hybrids were not obtained although several attempts were made to polyploidize them by the use of colchicine. It is uncertain to what degree polyploidy would affect the morphology of the hybrids, bit evidence from other species indicates that the morphology would not be significantly changed. For example, four diploid species of

M m k r namely A® aromatlca Evert, A® media Greene, A® mforinella Rydb., and A® virginica have both diploid and tetraploid cytotypas that are indistinguishable morphologically (Bayer, 1984a) • The diploid species, A® neglecta. A® plantaoinifolla. A® racemosa. and A® virginica are the most likely progenitors of the A® nsodisisa complex, Anfcemaria neodioica ssp. neodioica apparently arose somewhere in the area of West Virginia, where it is sympatric with its closest diploid relative, A® virginica (Bayer, 1984a). In areas closest to A. virginica» A® neodioica ssp. neodioica occurs as a tetraploid. but on the edges of its range (i.e. Montana or New England) it is hexaploid (Bayer; 1984a) „ In natural systens the backcrossing of F^s to their parents could easily have produced all the phenotypes that are present in the A® neodioica complex. Therefore, introgression has probably been an important evolutionary mechanism in the evolution of the polyploid complexes in Antennaria» It is thought that continued introgression is more important than initial interspecific hybridization alone in the formation of polyploid complexes (Stebbins, 1950) * These findings support the contention of Bayer and Stebbins

(1982) that members of the polyploid complex, which were called A. neodioica. should be recognized as a distinct species from the sexual diploid A* nealecta. Cronguist's intuitive classification (1945) recognized these probable allopolyploid derivatives as varieties of A* neglecta. The polyploids are no doubt of varied hybrid origin involving several diploids, a compilospecies in the terminology of Harlan and DeNet (1963). The evolutionary history of the group seems test reflected by the classification of Bayer and Stebbins (1982)« Most of the characters studied displayed continuous variation. There were no apparent reciprocal differences in the inheritance of characters in the interspecific hybrids, indicating that maternal inheritance is not a factor in the characters studied. Most of the characters are no doubt polygenic, i.e. under the control of several genes with small additive effects. The mode of inheritance of such characters requires several generations (usually up to at least F^) in order to ascertain facw many genes are involved in the determination of the characters (Bachmann et al, 1982)• Unfortunately, partial

sterility in the Fjs and slow growth prohibit detailed analysis at this tine. The flag character is the only one studied that appears to be under single gene control (complete dominance). The glabrous adaxial leaf surfaces present in A® neodioica ssp. canadensis and ssp, Howellii may be due to genes contributed by A® racemosa. because the other diploids lack this feature. M l the polyploid subspecies have a cymose arrangeaent of heads? occasionally the racemose type arrangement is displayed by some individuals of ssp. UCMelliij, suggesting it is more closely related to diploid A® racemosa than the other diploid species. Genes for single-nerved basal leaves, present in the subspecies of A® neodioica. were apparently contributed by A. lagjglaata and A. virginica. Mtennaria neodloisa ssp. canadensis, is the only subspecies of A. neodioica to express the flag character, and this trait is probably due to genes contributed by A. neglecta. its apparent closest diploid progenitor. Thus the four subspecies of A. neodioica that are recognized by Bayer and Stebbins (1982) and Bayer (1984a) suggest the predominance of the genomes of one diploid in their genetic constitution. 22

LITERATURE CITED ALEXANDER, M. P. 1980, A versatile stain for pollen, fungi, yeast, and bacteria. Stain Technol. 55? 13-18. BACHMANN, K., K. CHAMBER, H. J. PRIGS, and A. KONIG. 1982. Four additive genes determining pappus part numbers in Microseris annual hybrid C34 (Asteraceae/ Lactuceae). PI. Syst. Evol. 141? 123-141. BAYER, R. j. 1984a. Chromosome numbers and taxonomic notes for North American species of Antennaria (Asteraceae? Inuleae). Syst. Bot. 9?74-83. ------1984b. Evolutionary investigations in Antennaria

Gaertner (Asteraceae? Inuleae). Ph.D. dissertation, Ohio State Univ., Columbus. BAYER, R. J. and G. L. STEBBINS. 1981. Chromosome numbers of North American species of Antennaria Gaertner (Asteraceae? Inuleae). Amer. J. Bot. 68? 1342-1349. ... — — — ----— — . 1982. A revised classification of Antennaria (Asteraceae? Inuleae) of the eastern United States. Syst. Bot. 7? 300-313. --- -—— --- . 1983. Distribution of sexual and

aponictic populations of paiJLinii. Evolution 37 ? 555-561. GRGNQUIST, A. J. 1945. Notes on the Gompositae of the northeastern United States. I. Inuleae® Rhodora 471 182-184; DIKON? N® J® 1981, BMDP statistical software, Berkeley? California; Univer, of California Press. GRANT? V. 1981, Plant Speciation (2nd ed.) New Yorks Columbia. HARLAN? J® R, and J, M. J® DEWET. 1963. Hie oompilospecies concept. Evolution 17s497-501® JENSEN? R. J. and W® Ho KIMADGH. 1976a. numerical taxoncsnic studies of hybridization in Ouercus. I. Populations of resticted areal distribution and low taxoncmic diversity, Syst. Bot. Is 1-9. — ------arai — ■—-— -— . 1976b. Numerical taxoncmic studies of hybridization in Ouercus. II. Populations with wide areal distributions and high taxonomic diversity. Syst. Bot. 1: 10-19. JUEL? H. 0. 1900. Vergleichende Untersuchungen uber typische und parthenogenetische Fartpflanzung bei der Gattung ApfcsnDagia. Rongl. Svenska Vetenskaps-akad. Handl. 33(5)s 1-59. LEVIN? D. A. 1978. The origins of isolating mechanisms in plants. Evol. Biol. 11; 185-317. NEFF? N. A. and G. R. SMITH. 1979. Multivariate analysis of hybrid

fishes. Syst. Zool. 28: 176-196. M3HLF? F. J.? J. KXSHFAUGH? and D. KIRK. 1974. Numerical taxonciry

system of multivariate statistical programs. Stony Brook? New York: State Univ. of New York. SNEATH? P. H. A. and R. R. SOKAL. 1973. Numerical Taxonomy. San Francisco: W. H. Freenan and Co. STEBBINS, G. L. 1932. Cytology of Antennaria. II. Barthenogenetic species. Bot. Gaz. (Crawfordsville) 94: 322-345. . 1935. A new species of Antennaria from the Appalachian region® Rhodora 37: 229-237. ———:---- — . 1950. Variation and evolution in plants. New Yorks Columbia. MJIPFlNf T. 1973. Analysis of a hybrid swarm between Heterocentron elegants and glandulosum (Melostcmaceae). Tfexon 22: 413-423. Fig. 1. K& ecsaposed of 202 CTOs including five diploid taxa of Antennaria. Group outlines are indicated by a line. + ngglgsfear * A. plantaginifolia. <> A. iasass§&f # A. aalifana* r\]VO

o v a 93 N

ft ft V l d

10S • - - o Fig e 2. Percent average pollen stainability of diploid species (5 of each) of M t a m a r i a and their interspecific hybrids. Taxa are labelled with the first three letters of the specific epithet. ? = in two cases staminate individuals were not obtained, i.e. only pistillate individuals were obtained. Standard deviations were less than 10.0 %, except in three cases (labelled with a star) in which they were less than 20.0 %.

27 28 Fig. 3. PCft. composed of 274 OTOs including four diploid

Mfcenmria species, interspecific hybrids, and the polyploid &. neodioica s.l. Group outlines are indicated by a line. * A. negi.ecM? * A» Plantaginifolia. « &, rassoosa, -«■ airginiga, ® interspecific hybrids, #■ Jk. neodioica ssp. .canadensis, ssp. nefldioica, ssp. oetaloidea. and A, nsodioiga ssp. HoweHii. Parentages of interspecific hybrids are given in figure 4.

29 30

CO 0

Fig. 4. PO l group outlines with interspecific hybrid OTUs.

Letters next to the OTUs refer to their parentage and is given by the first letter of the specific epithet of the parental taxa (i.e. vs = A® mLigioiea x A® solitaria). Backcross segregates (A. uegl^ta x A. rasemosa) x A. neglecta are indicated by tsnrnw. ® OTUs subjected to a discriminant analysis that most probably belong to the group that they are enclosed in. 4 Outlying OTUs subjected to discriminant analysis with arrow pointing toward most probable group of inclusion (cf. Table 3). Staminate OTUs that could not be included in the discriminant analysis.

31 32 Fig. 5 0 Correlation phenogram (OTGMA) composed of 170 OTUs including four diploid species of AntennariaB interspecific hybrids^ and A« neodioica s.l. Taxa are labelled with specific or subspecific epithets. Parentage of hybrids is given in parentheses and are denoted by the first three letters of the parental specific epithets. Two ananalous specimens are HY-06, A. jaeodiaifia ssp. neodioica which resenbles a robust specimen of A. virginica: HY-08 is a probable natural hybrid between A. neglggfca and A« plantaginifolia. Cophenetic correlation coefficient is 0.794.

33 34

CORRELATION m *n n o» o O o * d

VIRGINICA

IVI3 (PLA X VIA) HYG9 (MEG X YIP) HY2S (VIP X SOL) HY2T (VIS I SOL) HV3S (MEG X V!R) HYIO (MEG X VIR) MEOOIOICA HY05 (NC00I01CAT) MEOOIOICA

PETALOIOEA MY11 (MEG X SOI) HT29 (NEC X SOL) HY30 (MEG X SOL) HYJI (MEG X sou KV26 (NEC X SOI) NEOOIOICA HY21 (keg X SOI) IIY14 CKEG X SOI) HY24 (MEG X SOL) MEOOIOICA HY1S MEG X RAC PETALOIOEA HV1A (lt£G X RAC) HYl7 (PLA X MCG) HV22 (PLA X MEG) (MEOOIOICA PETALOIOEA IIY23 (PLA X KEG) PETALOIOEA RACEHOSA HOWELL 11 RACLHOSA

HOWElliI CAflAOEKSIS

RACEHOSA

PETALOIOEA

N Y U (HEG I RAC) MV33 (KEG 2 RAC) HOWELLI! HY'M HEG X RAC) HYOS (MEG X PLAT) HY2G (MEG X RAC) HY28 (MEG X RAC) MV32 (MEG X RAC)

8169 PLANTAGIHIFQLIA

ih rs o d o Fige 6„ Line drawings of capitulescences and basal leaves of representative specimens among four diploid species of Antennaria, their interspecific hybrids, and 5 invididuals of A. neodioica s.l. Pubescence is indicated by stippling. Variation among taxa is apparent with respect to leaf shape and pubescence, number of veins in basal leaves, and number, size, and arrangements of heads in the capitulescence. A. A.

Elantaginifoliaf b . a . neglestar c. a . virginica, d . a . racarosar and E. A« neodioica (all subspecies). Bar = 5 cnu

35 36 37

Table 1. Mean similarity matrix (correlation coefficients) of nine taxa of Antennarla with each other. Taxa are

labelled with the first three letters of the specific or subspeclftc epithet.

NE6 PLA RAC SOL VIR CANHOW NEO PE T

NEG t.000

PLA -0.411 1.000

RAC -0.233 0.559 1.000

SOL 0.418 -0.100 -0.154 1.000

VIR -0.091 0.125 -0.157 -0.311 1.000

CAN 0.619 -0.330 -0.123 0.064 -0.125 1.000 m u 0.383 -0.237 0.256 0.047 -0.302 0.590 1.000

NEO -0.116 0.398 0.220 -0.522 0.394 0.332 0.259 1.000

PET 0.323 0.059 0.102 -0.095 -0.036 0.595 0.473 0.609 1.000 38

Table 2. Mean similarity matrix of taxa of Antennarla with thirty artificial hybrid combinations (HY— ). Taxa ar®

label lad with the first thro® letters of the specific or subspeclflc epithet. Thirty artificial hybrids and thalr

parentage ar® presented along with their mean similarities to the naturally occurring taxa. *®*hlghest, ** second

highest, and * third highest similarity between each hybrid and the taxa are Indicated. 1 hybrids closely 2 3 resembling both parents, hybrids closely resembling one parent, hybrids resembling the polyploids (CAN, HOW,

NEO, PET), but not either parent.

NES PLA RAC SOLVIRCANHOWNEO PET

NE6 x VIR (HY09)' 0.174fls -0.348 • -0.449 0.012 0.446*** -0.012 -0.085 0.072* 0.050

NEO x SOL (HYII)1 0.311 os -0.426 -0.593 0.375*0* -0.015 0.186* -0.292 -0.366 -0.144

MEG x SOL (HY2I)1 0.497000 -0.567 -0.540 0.452*0 -0.024 0.103* -0.254 -0.471 -0.196

NEO x SOL (HY24)* 0.24000 -0.089 -0.138 0.386**0 -0.143 -0.105 -0.318 -0.422 -0.232

NEG x SOL (HY26)' 0.111 nos -0.526 -0.564 -0.027*0 -0.040 -0.059 -0.243 -0.354 -0.402

NEG x SOL (HV29)1 0.133*0 -0.278 -0.481 0.3040*0 0.085 -0.209 -0.329 -0.469 -0.592

NEG x SOL (HY30)’ 0.549** -0.253 -0.246 0.585»o* -0.224 0.120 -0.028 -0.434 -0.118

NEG x SOL (HY31)* 0.487**0 -0.394 -0.407 0.383** -0.042 0.006 -0.193 -0.388 -0.243

NEG x SOL (HY34)1 0.427** -0.447 -0.514 0.478*0* -0.528 0.079 -0.379 -0.568 -0.275

PLA x VIR t HY18)1 -0.149 0.072* -0.261 -0.198 0.704*** -0.233 -0.325 0.168** -0.047

PLA x VIR (HY19)1 -0.330 0.126«* 0.047 -0.246 0.477*** -0.356 -0.402 0.071* -0.234

VIR x SOL (HY23)1 -0.176 -0.285 -0.524 -0.008** 0.435*** -0.336 -0.436 -0,146 -0.335

VIR x SOL (HY27)’ 0.135*0 -0.546 -0.559 0.028 0.619*** -0.200 -0.130 -0.292 -0.344

NEG x RAC < HY14)2 -0.322 0.023 0.221** -0.533 0.018 0.042 0.147* 0.326*** 0.076

NEG x RAC (HYI5)2 -0.075 -0.092 0.299*** -0.358 -0.241 0.0660 0.240** -0.105 -0.031

NEG x RAC (HVI6)2 -0.277 -0.173 0.190*** -0.555 -0.176 0.004 0.151** -0.023 -0.111

NEG x RAC (HY20)2 -0.140 -0.102 0.188*** -0.209 -0.434 0.005 0.039 -0.170 0.070**

NEG x RAC (HY28)2 -0.256 -0.097 0.178*** -0.113 -0.415 -0.083 -0.032** -0.331 -0.168

NEG x RAC (HY32)2 -0.283 -0.002 0.053** -0.168 -0.261 0.080*** -0.015 -0.043 -0.042

NEG x VIR (HV10)2 -0.167 -0.108 -0,373 -0.300 0.531*** -0.279 -0.361 0.174** -0.283 NEG x VIR (HY35)2 -0.102 -0.337 -0.636 -0.205 0.444*** -0.130 -0.378 -0.002** -0.220

PLA x NEG (HYI7)2 -0.480 0.582*** 0.302** -0.103 -0.137 -0.522 -0.258 0.014* -0.265

PLA x NEG (HY22)2 -0.442 0.569*** 0.3380* -0.367 0.019 -0.317 -0.140 0.260* 0.050

PLA x NEG (HY23)2 -0.422 0.453** 0.459*** -0.258 -0.152 -0.297 •^0.039 0.151* 0.086

VIR x SOL (HYI3)2 -0.036 -0.528 -0.372 -0.042 0.691*** -0.281 -0.313 -0.285 -0.381

NEG x RAC (HYI2)3 -0.215 -0.161 0.051 -0.146 -0.364 0.257*0 0.3140*0 0.171 0.162

NEG x RAC (HY33)3 -0.232 -0.068 0.000 -0.241 -0.331 0.2410* 0.397**0 0.204* 0.153

VIR x RAC (HY43)3 0.095 -0.105 0.312 -0.288 0.096 0.460 0.708*** 0.571** 0.4700

VIR x RAC (HY44)3 -0.069 -0.194 0,136 -0.294 0.059 0.204* 0.356*** 0.191 0.270**

VIR x RAC (HY43)3 0.287 -0.184 0.286 -0.089 -0.068 0.544*s 0.645*** 0.316 0.523* 39

Table 3. Table of posterior probabilities derived from a discriminant analysis. Twenty-six artificial

Interspecific hyblrds of Antennarla (HY--) and their parentage along with the posterior probability to

which of nine naturally occurring taxa of Antennarla they should belong to. Taxa are labelled with the

first three letters of the specific or subspecific epithet. ' hybrids most closely resembling one of 2 their parents, hybrids most closely resembling one of the subspecies of A* neodlolca.

NEG PLA RAC SCI VIR CAN HOW NEOPET

NEG x SOL (HYII)1 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

NEG x SOL (HY21)1 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

NEG x SOL CHY24)1 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

NEG x SOL (HY26)1 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

NEG x SOL (HY29)1 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

NEG x SOL (HY30)1 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

NEG x SOL (HY3I)' 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

NEG x SOL CHY34J1 1.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000

NEG x VIR (HYIO)1 0.000 0.000 0.000 0.000 0.922 0.000 0.000 0.076 0.002

PLA x NEG (HY17)1 0.000 0.843 0.000 0.000 0.003 0.000 0.000 0.043 0.109

PLA x NEG (HY22)1 0.000 0.801 0.000 0.000 C.144 0.000 0.000 0.049 0.005

PLA x NEG (HY23)1 0.000 0.901 0.000 0.000 0.073 0.000 0.000 0.014 0.012

PLA x VIR (HY18)1 0.000 0.000 0.000 0.000 0.965 0.000 0.000 0.032 0.003

PLA x VIR (HY19)’ 0.000 0.000 0.000 0.000 0.997 0.000 0.000 0.003 0.000

VIR x RAC (HY43)1 0.000 0.000 0.000 0.000 1.000 0.000 0.000 0.000 0.000

VIR x SOL (HY25)1 0.000 0.000 0.000 0.000 0.S82 0.000 0.000 0.114 0.004

NEG x RAC (HY12)2 0.000 0.000 0.000 0.000 0.000 0.000 0.379 0.132 0.489

NEG x RAC (HYI4)2 0.000 0.000 0.000 0.000 0.000 0.000 0.047 0.521 0.432

NEG x RAC (HY16)2 0.000 0.000 0.260 0.000 0.000 0.740 0.000 0.000 0.000

NEG x RAC (HY20)2 0.000 0.000 0.000 0.000 0.000 0.000 0.002 0.025 0.973

NEG x RAC (HY20)2 0.000 0.000 0.000 0.000 0.000 0.000 0.003 0.001 0.994

NEG x RAC (HY32)2 0.000 0.000 0.000 0.000 0.000 0.000 0.031 0.020 0.949

NEG x RAC (HY33)2 0.000 0.000 0.000 0.000 0.000 0.000 0.338 0.242 0.420

NEG x VIR (HY33)2 0.000 0.000 0.000 0.000 0.016 0.000 0.000 0.437 0.527

VIR x RAC

VIR x RAC (HY44)2 0.000 0.000 0.000 0.000 0.000 0.000 0.980 0.011 0.009 Chapter II J W E S T I G M M S MDO THE EVOLUTIONARY HISTORY OP THE POLYPLOID OOMELEXES IN AMTENNRRIR (USTffiACEAEs IMJLEAE).

II. TOE A. PMLTOXI COMPLEX.

Introduction The Mtennaria Parlinii polyploid agamic complex occurs throughout the deciduous forests from Georgia, west to Texas, north to Minnesota, and the Thunder Bay District of Ontario, east to Nova Scotia (cf. Bayer and Stebbins, 1982 their fig. 7). Populations of A. Parlinii are either sexually reproducing (with sex ratios near 1:1) or are asexual, and composed entirely of pistillate clones (Bayer and Stebbins, 1983). These sexual and asexual populations have distinct geographic distributions (Bayer and Stebbins, 1983), with the former occurring mainly in the southwestern portions of the range. Recently, Bayer and Stebbins (1982) defined A* Parlinii as consisting of two subspecies, A. Parlinii Fern. ssp. .Parlinii and A. M r l l m i Fern. ssp. fallax (Greene) Bayer and Stebbins. They differ in that the former has glabrous adaxial leaf surfaces and glands on the upper portion of the cauline sten while the latter has pubescent adaxial leaf surfaces and is glandless. Both are found over the entire range of the species, but subspecies Parlinii is rare in the southwestern portion 40 41

of the range and more cannon in the northeast* Agamospermy in the A® Parlinii complex occurs via diplospory followed by diploid parthenogensis, as was first danonstrated by Stebbins (1932b) * Sexual members reproduce via the Folygoram-fcype embryo sac (Stebbins? 1932a) , as do sexual diploid members of the genus® Antennarla Parlinii has basal leaves with 3-5 primary veins and capitulescences composed of 2-15 (median of 6) relatively large capitula. Chromosome numbers in the complex include tetraploids (2n =

56), pentaploids (2d “ 70), hexaploids (2a = 84), and octoploids (2n = 112)(Bayer and Stebbins, 1981; Bayer, 1984a). Hexaploids are by far the predominant cytotype (Bayer, 1984a) with pentaploids and octoploids only encountered rarely* Sexual tetraploids are frequent (Bayer, 1984a) having been found at one site in Oklahoma, once in Missouri, and at five sites in the Driftless Area of Wisconsin. The ccsranon occurrence of the tetraploid cytotypes in the western portions of the range supports the hypothesis that the species originated in this area and spread eastward where it occurs both as sexual and agamosparxnous hexaploids (Bayer, 1984a) * Workers on agamic complexes have often postulated that apcmicts arise through hybridization between and among sexual diploid soecies (Gustafsson, 1947). Harlan and DeWet (1963) have termed polyploid complexes of multiple hybrid origin a conpilospecies. Hie question can be asked: What were the most probable origins of the A. Parlinii agamic complex? With this question in mind, crosses were made among five diploid species of Antennarla, viz., A* neglecta Greene, A. plantaglnifolia, A. A. sslifcaKia? and A. vimlnlca Stebbins to see whether any of the interspecific hybrids resenbled members of the A. Parlinii complex (see Bayer, 1984b, chapter I, for details). These five diploid species were chosen for comparison with A. Parlinii because they are morphologically most similar to and largely sympatric with A» Parlinii. The five diploid secies can be divided into two groups, the snail-leaved section Pioicae with basal leaves having a single primary vein, and the large-leaved section Planfcaginlfoliae which have 3-5 veins per basal leaf. Like the A® Parlinii complex,

the diploids A. planfcaginjfolia, A. xassniBfi&r and A. gfilifcaoa are of the large-leaved group and their hybrids most closely resemble menbers of A* Parlinii. Hybrids from crosses between diploids of the small- and large-leaved groups usually produce single-nerved basal leaves (Bayer, 1984b, chapter I). Consequently, the three diploid (2n = 28) species of Antennarla chosen for analysis along with A. .^rlmil were

A. jalantaginifolla (L.) Richardson, A. rasamsa Hook,, and A. solitaria Rydb. (i.e., M t e n m r i a neglesta and A. mxgloifia were eliminated from further analysis). Several crosses were also attempted between sexual clones of A. Parlinii and two of the putative diploid parents to test whether introgression could still be occurring under natural conditions. The purposes of this paper are (1) to discuss the relationship of the A. Parlinii polyploid complex to several sexual diploids, (2) to test the hypothesis that the A. Parlinii complex is of multiple hybrid origin, (3) to discuss the relationship A. Parlinii to the A. neodioica polyploid agamic complex. 43 and (4) to comment on the mode of inheritance of some important morphological features.

MATERIALS AND MEffiODS Field observations were made in the springs of 1978-83 and over 400 collections of Antennaria were cultivated in the greenhouse. Interspecific hybrids and other crosses were made as outlined previously (Bayer and Stebbins, 1982; 1983). Pollen viability of certain hybrids was ascertained through the use of Alexanders differential staining method (Alexander, 19®)). Three hundred grains/ individual were scored for viability. Morphological studies were aided by the use of specimens borrowed from the following herbaria; CAN, 05, ® , MO, MONTU, NDG, NY, OS, PAG, BM, SBU, US, VPI, WIS, and WA . Herbarium vouchers of field collections and artificial hybrids produced during this study are on deposit at OS. Numerical methods are explained in detail in Bayer (1984b, chapter I), but will be discussed here briefly. Thirty-eight characters (16 vegetative and 22 reproductive) were used to construct the initial basic data matrix. A list of characters used and the basic data matrix may be found in Bayer (1984b; appendix). The NT-SYS program of Rohlf, Kishpaugh, and Kirk (1974) was used to compute the principal components analysis (PGA), cluster analysis (UFGMA), and a table of correlation coefficients. A stepwise discriminant analysis and univariate statistics were generated by the 44

BMDP program of Dixon (1981). Computations were carried out at Hie Instruction Research Computing Center at Hie Ohio State University. Several data matrices were used for this study: (1) a 149 OTO (operational taxonomic unit) matrix consisting of manbers of the A« Parlinii (39 OTOs) complex* three related sexual diploids (34 OTUs of A® Plantaainifolia. 34 of A. racemosa. and 35 of A® solitar.ia) * and their interspecific hybrids (7 CTOs), (2) a 117 OTO data matrix composed of manbers of the A® neodioica (72 OTOs) and 4® Parlinii (45 OTOs) polyploid complexesr (3) a 153 OTO data matrix, basically the same as matrix number 1 (41 OTOs of A® Parlinii s.l., 35 of A® plantaginifolia* 35 of A® mLtolla# 35 of A. rasgms&r and 7 interspecific hybrids)» and (4) a 11 OTO matrix utilizing character averages for the three diploids and the A- Parlinii polyploid complex, plus the individual interspecific hybrids (7 OTOs) among the three diploids. A three dimensional plot of FCA computed from matrix number 1 is presented. Matrix number 2 was used to compute a FCA and the results are presented as two 2-dimensional plots of the first three factors. A cluster analysis was performed using matrix number 3 and the results are displayed as a ptienogram. Matrix number one was used to execute a discriminant analysis. Pearson product-mcment correlation coefficients (Snaath and Sokalf 1973) were used to compare OTOs of matrix number 4. RESULTS

Phenetiga of ^ diploids^. terid&L and polyploids— The first three factors of the PCA composed of the three diploids, their interspecific hybrids, and the A« Parlinii complex (Fig. 7) accounted for 54.6 % of the variation. The first 11 factors have eigenvalues greater than 1.0, indicating that the characters are not highly correlated. High loadings for factor 1 are reproductive characters such as number of heads par capitulescence, flowering stan height, and width and length of the cauline leaves. A mixture of reproductive and vegetative characters such as degree of pubescence on the upper surface of the basal leaves, stolon length, and absence or presence of the scarious flag-like structure on the upper cauline leaves, have high loadings for factor 2. Factor 3 has high loadings for vegetative characters such as the degree of pubescence of the upper leaf surfaces, width of the basal leaves, and number of stolons per basal rosette. Inspection of Fig. 7 shows that A« Parlinii is surrounded by the three diploids, A« piantaginifQiia, A. xasasssa, and Ao solitaria. Tie diploid species are separated mainly by reproductive characters along component number 1. All of the interspecific hybrid combinations fall within the A* Parlinii group. Although the two subspecies of A. Parlinii are not shown as distinct in Fig. 7, they do form somewhat distinct subgroups within A.

B a r l M l S.l. As expected, A. M s I i a H ssp. Parliflii with its glabrous basal leaves and gland-bearing upper cauline stens, groups more closely to A* racemosa. with which it shares these characters. Subspecies fallax forms a subgroup in the upper portions of the A- 46

Parlinii sal. group and is most similar to A® plantaginifolia and A® gQlitaria. Several specimens referable to A® fialfltoUa Greene (= A® Parlinii ssp. fallax) and A* munda Fern. (= A® Parlinii ssp. f&Hax) occupy positions at the upper end of the complex closest to A° solitaria. The cluster analysis (Fig. 8) has a cophenetic correlation coefficient of 0.857* and shows that the three diploids plus the A® Parlinii polyploid complex each form a distinct group. The interspecific hybrids are either grouped within the A® Parlinii complex or with one of their parents. The two subspecies within A® Parlinii are scattered in small groups throughout the A. Parlinii complex. Antennarla munda Fern. (= A® Parlinii ssp. faUas) is a seldomly recognized agamospecies, which we have included within A® Parlinii s.l. (Bayer and Stebbins* 1982). In many respects it closely resembles A® solitaria. which is the reason it is grouped more closely to A® solitaria than the renainder of A® Parlinii. Another seldomly recognized agamospecies* A® Brainerdii Fern. * which we (Bayer and Stebbins,, 1982) have included within A® Parlinii. represents another extraie in the morphological variation of A® Parlinii. With its relatively small* glabrous* bright green leaves* A® Brainerdii* closely resembles hybrids between A® racemosa and the other two diploids (Fig. 8). Jensen and Eshbaugh (1976a,b) have demonstrated the utility of mean similarity matrices (correlation coefficients) for identifying hybrid taxa. A matrix of mean similarities (Table 4) shows that A® plantaginifalia is most similar to A® £assm§a and vice-versa. Antennarla jgfilifcacia is most similar to A® ggiliniiy while A® B a J i m i is about equally similar to A® plantigijnifQlia and A. rasgn&ga. This analysis points to the overall similarity between the A® Farlinii polyploid complex and the three diploids, The interspecific hybrids are in all cases either most similar to one of their parents (e.g. HY02, HY03, and HY04) or to the A® Parlinii complex (e.g. HY01, HY40, HY41 and HY42). The method always associates the hybrid with one of its parents thereby showing the sensitivity of the technique, A discriminant analysis (Table 5), based on characters which best discriminate among the taxa, also demonstrates that these interspecific hybrids are always associated most closely with the polyploid complex or one of the diploid parents, A table of posterior probabilities derived from the discriminant analysis (Table 5) demonstrates that hybrids are identified as most probably belonging to either one of their parents (e.g, HY03 and HY42) or members of A® Parlinii (e.g. HY01, HY02, HY04, and HY41).

Belatdanahiff of the A. jgeadlolca. sxaaHssL M .the k*l qsiJlgg— The PCA (Fig. 9) of members of A. neodioica (72 OTOs) and A® Parlinii (45 OTOs) was computed to demonstrate the morphological distinctness of the two complexes. The first three principal components account for 51.5 % of the variation. High loadings for factors one and three are vegetative characters, while those for factor two were reproductive characters. The A. Parlinii and A® neodioica complexes are separated chiefly on the basis of basal leaf 48

characters along factor one* The first eight factors have eigenvalues greater than 1.0, indicating that the characters are not highly correlated. The PCA (Pig. 9) demonstrates that the two complexes do not overlap. Specimens of A® neodioica Greene ssp. Howellii (Greene) Bayer are morphologically closest to members of the A® Parlinii complexo Anfcennaria Earwellii Greene (= A® Parlinii ssp. fallax) is an agamospecies which has relatively small leaves, when compared with the rest of A® Parlinii s.l. and is similar to members of the A.

nsssUmsa complex. Itdtecifcanss of morphological flhaEacfceESr-Most of the morphological characters that I’ve considered in Anfcennaria are inherited polygenically (Bayer, 1984b® chapter I) and are probably controlled by a few (4-5) genes. Additional supportive feta are now available for the inheritance of leaf pubescence, which was reported (Bayer, 1984b, chapter I) as being a polygenic character with alleles for glabrity being dominant to those for pubescence. Antennaria Parlinii ssp. Parlinii has glabrous adaxial leaf surfaces while subspecies fallax has leaves that are pubescent afexially. Mien representative clones of each subspecies are crossed the hybrids (total of 37 observed) range from glabrous to slightly pubescent, thus substantiating previous results that leaf pubescence is controlled by several genes with glabrity being dominant to pubescence. The genus Antennarla is ordinarily dioecious, hat occasionally hermaphroditic or monoecious clones can be found. A monoecious clone of A. Parlinii ssp. fallax from Hocking County, Ohio, has heads which 49

are composed of pistillate florets in the inner whorls and staminate in the outer whorls. Mien self pollinated the progeny produced were all monoecious (7 progeny observed). When the monoecious clone was backcrossed to a normal dioecious clone the progeny (total of 11 observed) were all strictly dioecious. Another sexual anomaly observed was a hermaphroditic clone that was the result of an interspecific cross between A® Planfcaainifolia and A® solitaria.

DISCUSSION Previously, it was demonstrated by the use of PCA that the three diploids examined as progenitors of A® Parlinii are morphologically distinct from one another (Bayer, 1984b, chapter I). It has also been shown (Bayer and Stebbins, 1982; Bayer, 1984b, chapter I) that the diploids are isolated by both spatial and reproductive factors. It has also been noted that these isolating mechanisms (Bayer, 1984b, chapter I) occasionally breakdown and allow hybridization among the diploid species. Populations of A. Plantaoinif olia. A® solitaria. and * polyploid A® Parlinii growing side by side have been recently observed in Kentucky and in West Virginia (Bayer, unpubl. obs.; G. L. Stebbins, pars, comm.)• Field observations and crossing studies indicate ample opportunity for hybridization and introgression between the two eastern diploids, A. plantaginifolia and A® SPUfcaria, and the polyploid A® Parlinii s.l. Hie existance of tetraploid, pentaploids, and octoploids, in addition to the predominant hexaploid cytotypes of 50

A* Parlinii. could be cited as evidence of hybridization and intregression between polyploids and diploids. In the A. neodioica complex (2& = 56,84), which is totally apcmictic, there are only two cytotypes and this could be attributed to a reduction in crossing between the apcsnicfcic polyploids and diploids. Juel (1900) first concluded that the polyploid agamic species of Antennarla were probably the result of hybridization between sexual diploid species. It has been rather well documented that most agamospermous groups arise in this way (Grant, 1981). With regard to the origins of A* Parlinii. Stebbins (1932b) suggested that while A. fallax Greene (= A* Parlinii ssp. fallax) was most closely related to A. pJ^ntaginifQlia other cytological features indicated A. solitaria 4 or A. naglfigfea were also involved. Antennarla Parlinii Fern. (= A.

Bacltoil ssp. M U i n i i ) * although similar to A. s3imt§gini£Qliay had several morphological features such as bright green adaxially glabrous leaves, not present in A* plantaainifolia and was undoubtedly of allopolyploid origin (Stebbins, 1932b). With respect to this character in ssp. Parlinii. Beals and Peters (1966) state that wthe very characteristic glabrous upper leaf surface and occasional purple glands on the sfcan suggest that intr egression may have occurred in the past with seme unknown, now extinct species.*8. Ihey ware undoubtedly unaware of A. raceroosa, the only sexual diploid Anteonaria with adaxially glabrous leaves that are as large (3-5 veined) as those of A. B&slinii ssp. Bi&U M i . Mtemmria jafiiaroaa also has the purple glands found in ssp. Parlinii. Both A. alantmgioifolia and A° solitaria are sympatric with the A. Parlinii complex throughout their entire ranges in the eastern United States (see Bayer and Stebbins, 1982? their figs. 2,3, and 7). Anfcenmria .solitaria occurs in moist woodlands in the eastern United States mainly south of the Wisconsin glacial margin while Jko piantagioifollei is found throughout the Appalachian region, Atlantic Coastal Plain, north into New York. Anfcamaria plantaginifolia also is known as disjunct populations in the Driftless Area of Wisconsin, as has been confirmed recently (Bayer, 1984a? G.L. Stebbins, unpubl. obs.). A single specimen of A. solitaria from the Driftless Area of Jackson Co., Wisconsin, has been seen in the collection of WIS and thus it apparently occurs there. Antannaria r^ c m o m is found in the coniferous forests of British Columbia and Alberta, south to Washington, Oregon, northern California, ton tana, Idaho, and Wyoming. It has teen reported as far east as the Black Hills of South Dakota

(McIntosh, 1931? Dorn, 1977). Thus although A* racemose is not sympatric with A- plantaginifolia or A« solitaria at the present, they may have teen sympatric during the last glacial period, 10,000 years ago. when forests probably extended across what is now prairie (Wells, 1970). It is likely that many Cordilieran species migrated eastward during the last glacial period (Wells, 1970? Whitehead 1972? Marquis and Voss, 1981). For example, A. rosea Greene, a chiefly Cordilieran species which occurs sympatrically with A. racemose, has disjunct populations on the north shore of Lake Superior (Hyypio, 1952). If A. rosea once migrated or dispersed eastwards, it is possible that A. racemosa could also have done so.

The overlap of A® M t U m l and A® in the m (Pig. 7) indicates that A® racemosa was probably involved in the ancestry of the complex despite its present day geographic isolation. Hie position of the diploid species also indicates that no single one of than is predominantly responsible for the genetic composition of the polyploids (Fig. 7). In his original description? E. L. Greene made the perceptive observation that A® ealophvlla (= A® Parlinii ssp. faLLax) was ”related to the exclusively southern A® solitaria.68 and based on the FCA (Fig. 7) he was apparently correct. Likewise

AntemaSM mrnda (= A® Jterlinii ssp. fallax) has basal leaves which are shaped like those of A. solitaria. Thus, these agamospecies (A® calophylla and A. munda) appear to have a predominance of genes from A. golltaoju and this is portrayed by the KA (Fig. 7) in that the agamospecies occupy positions closest to A® solitaria. Hie extension of certain OTOs of A® Parlinii s.l. beyond the morphological types found in A® plantaginifolia or A® solitaria (Fig. 7) could be due to either the effects of polyploidy or trangressive segregation.

TTansgressive segregation was earlier noted (Bayer? 1984b? chapter I) in hybrids between A® solitaria and A® necrlecta. Hiesy and Mobs (1982) have also reported this phenanenon in diploid and polyploid agamic complexes in Poa. Hie cluster analysis (Fig. 8) shews that the two subspecies within A® Parlinii s.l. are scattered in snail groups throughout the A® Parlinii complex. Hiis is probably the result of segregation of genes from the genomes of several diploids and also 53

because ssp. Parlinii and ssp® fallax are separated mainly on the basis of only one character, pubescence of the adaxial surface of the

basal leaves® The PC'A (Fig. 7) and the cluster analysis (Fig. 8) demonstrate that while many segregates of A® Parlinii, ssp. JallaE. morphologically resemble the diploid A® planfcagMifoiia, other segregates of the complex referable to ssp® fallax (including A® and A® munda) and ssp. Parlinii (including A® BminerdiD have characteristics suggesting the presence of genes from both A® solitaria and A. XiSSDasa® Both A® Mrlinii s.l. and the A® soiUfeldLa X A® Plantaginifolia hybrids are intermediate between A® ssJJJada and A® plantaginifolia with respect to number of heads and number of florets per head (Fig. 10), Inspection of the basal leaves and capitulescences of the three diploid species, hybrids, and naturally occurring polyploids (Fig. 10) demonstrate the close resemblance of all the artificial interspecific hybrids with A® Parlinii segregates® The resemblance is evident not only with respect to the shape of the basal leaves, but also the number and arrangement of the heads (Fig. 10), and on closer inspection the number of florets per head. The discriminant analysis (Table 5) and mean similarity matrix (Table 4) also point to the similarity of most of the interspecific hybrids to members of the A. Parlinii complex. Many hybrids having A® solitaria as one of their parents frequently have high affinities with A® solitaria and this could be from the of expression in these hybrids of several dominant traits present in A® solitaria (See Bayer, 1984b, 54

chapter I, for fetalis of mode of inheritance of several morphological characters). AntennaEia mrlinii s.l. and A. ngfidiato s.l, both occur syrapafcrically in the deciduous forests of the eastern United States and adjacent Canada* but A. neodioica s.l. also occurs as far west as British Columbia and as far north as Hudson Bay and the Canadian

Northwest Territories (Bayer and Stebbins, 1982? Bayer, 1984b* chapter I). The two polyploids are most easily separated taxoncmically by the number of primary veins in the basal leaves, A- JBarlinii having 3-5 and A. neodioica having only a single nerve. Beals and Peters (1966) and Beals (1968). in studying Antennarla of Wisconsin, came to the conclusion that members of these two complexes form a continuum and that species names are convenient labels when applied to them. They postulated that this was due to hybridization, and polyploidy. Although some segregates of the A. Parlinii complex do resemble menbers of the A® neodioica complex and vice-versa, they can always be absolutely identified on the basis of number of veins in the basal leaves. In addition, A. neodioica consists entirely of asexual populations (Bayer, 1984b, chapter I), while A. Parlinii s.l. is composed of both sexual and asexual populations (Bayer and Stebbins, 1983). The FCA (Fig. 9) danonstrates that the two complexes are morphologically separable and their evolutionary history, reproductive mode differences, and chromosome number distinctions (Bayer, 1984a) support this argument. The two complexes are morphologically similar at the extrsne limits of their variation because they probably share the two sexual diploid progenitors A. ^ntaginifQlia and A. mcemosa (Fig. 11). The A® Parlinii complex has one unique diploid progenitor

A. solitaria, while A. Jiafidloiga has two, A. flggiesfea and A® m & i n i g i (Fig. 11). Figure 11 portrays the proposed relationdiip of A® Parlinii. A* neodioica. and their extant sexual diploid progenitors. Antennaria plantaginifolia and A® xacfimaaa can be viewed as pivotal genomes because they are shared by both polyploids. The diploids, unique to the ancestry of each complex, are responsible for their morphological distinctness. The contention (Bayer and Stebbins, 1982) that polyploid agamic complexes should be retained as distinct from their diploid relatives at the specific level is viewed as the most desirable solution to the species problem in Antennaria. Because the polyploids are of multiple hybrid origin, it is erroneous to include them simply as varieties of one of the diploid relatives as Cronquist (1945) did in including members of A. Parlinii as varieties of A. plantaginifolia. Aatemaria JfeuLlinii is certainly of hybrid origin because it has characteristics not found in diploid A. plantaginifolia, Although one can not rule out the possibility that diploid progenitors have become extinct, the most single and plausible explanation is that which has bean presented. Antennaria Parlinii s.l. is often found growing in sympatry with its two probable diploid progenitors, A» plantaginifolia and A. solitaria. The question arises as to whether introgression is still possible between the polyploid and its diploid progenitors. Many populations exist in which asexual clones of A® garlinii s.l. are found growing with sexual clones of A. plantaginifolia (Bayer and Stebbins, 1981) and in such cases the species are obviously reproductively isolated. In papulations where sexually reproducing clones of the polyploids (2a ~ 84) and diploids (2a = 28) are sympatric intr egression may still fee occurring. In crosses conducted

in the greenhouse between A® Parlinii s.l. aid the diploids A® Plantaginifolia and A- aolifcagia# the A® .BajUnii s.l. X A® Manfcaginifolia crosses set 1.0 % of their seeds (Bayer and Stebbins,

1982) while the A. Parlinii s.l. X A. BQlifcaria hybrids set 0.5 % (Bayer, unpubl.). As a result of the crosses, a single hybrid frcm each was obtained. Both were expected to be and were confirmed as tetraploids (2n - 56; Bayer unpubl.). The A. Parlinii X A® alanfcaginifolia hybrid was pistillate and was not analyzed further with respect to fertility, but the A® &3Elinil X A. Bfilifcacia hybrid had a pollen stainability of 77.3 %. The stainability is high, especially when compared with results obtained for interspecific crosses among diploid species (Bayer, 1984b, chapter I). Diploid species of Antennaria have pollen stainabilities greater than 85.0 % (Bayer, 1984b, chapter I). The A® Barlinii X A® hybrid should be quite fertile and thus it is likely that introgression is still occurring among the polyploid and diploids. Obviously, because of limited data, little can be said about the inheritance of gender or breeding systen in Antennaria. but the occasional appearance of monoecious and hermaphroditic clones in 57 Antennaria supports the hypothesis (Webb, 1979? Lloyd, 1980? Bawa, 1980) that dioecy evolved fran henaaphroditian via monoecy. Juel (1900) called these hermaphroditic clones atavisms or threw backs (Ruckschalagsfomen) and postulated that dioecious Antennarias evolved fran monoecious or hermaphroditic relatives such as Gnaohalium L. ®ie fact that hennaphroditisn rappeared in a clone that was the result of a fairly wide interspecific cross indicates that although the genes for hermaphroditic florets still exist, unusual gene combinations are required for their presence to be expressed., 58

LITERATURE CITED ALEXANDER? M.P. 1980. A versatile stain for pollen? fungi? yeast? and bacteria0 Stain Technol. 55$13-18.

MSA? KoSo 1980 e Evolution of dioacy in flowering plants8 Annual Revo Ecol. Syst. 11s15-39* BAYER? R.J. 1984a. Chrcmoscine numbers and taxonomic notes for North American species of Antennarla (Asteraceaes Inuleae). Syst. Bot. 9s74-83. — -- -- . 1984b. Evolutionary investigations in Antennaria (Asteraceaes Inuleae). Ph.D. dissertation? Hie Ohio State University? Columbus. BAYER? R.J. and G.L. STEBBINS. 1981. Chromosome numbers of North American species of Antennaria Gaertner (Asteraceaes Inuleae). Amer. J. Bot. 68sl342-1349. ------. 1982. A revised classification of Antennaria (Asteraceaes Inuleae) of the eastern United States. Syst. Bot. 7 s300-313. --- — ------— ---- . 1983. Distribution of sexual and apomictic populations of Antennaria parlinii. Evolution

37s555-561. BEALS? E.W. 1968. A taxoncmic continuum in the genus Antennaria in Wisconsin. Amer. Midi. Naturalist 79$31-47. ——— -- . and R.F. PETERS. 1966. Preliminary reports on the flora of Wisconsin no. 56. Gmpositae V- Ccmpositae family, tribe

Inuleae (MfcamaEM? SoaBhaliuiBr ^sasphalig, Inula)« Trans. Wisconsin Acad. Sci. 55s223-242. GOQUIST, A.J. 1945. Notes on the Gompositae of the northeastern United States. I. Inuleae. Ehodora 47:182-184.

DHQN, N.J. 1981. B ® P statistical software. Berkeley, Californias Univer. of California Press. K3RN, R.D. 1977. Flora of the Black Hills. Authors. 377p. GRANT, V. 1981. Plant Speciation (2nd ed.) New Yorks Columbia. GUSTAFSSGN, A. 1947. Apcsnixis in higher plants. Acta Univ. Lund. Kungl. Bysiogr. Sallsk. Handl. N. R. Avd. 2. 42-44:1-370. HARLAN, J.R. and J.M.J. DEWET. 1963. Hie oompilospecies concept. Evolution 17:497-501. EIESEY, w.M. and M.A. MBS. 1982. Experimental studies on the nature of species. VI. Interspecific hybrid derivatives between facultatively apomictic species of bluegrasses and their responses to contrasting envirormenfs. Publ. Carnegie Inst. Wash. 636:1-119. HYYPIO, P.A. 1952. Antennaria rosea Greene in the Lake Superior region. Rhodora 54:291. JENSEN, R. J. and W. H. E3HBAUGH. 1976a. Numerical taxonomic studies of hybridization in Ouercus. I. Populations of restricted areal distribution and low taxoncsnic diversity. Syst. Bot. 1:1-9. ------a n d ------. 1976b. Numerical taxonomic studies of hybridization in Onerous. II. Populations with wide areal distributions and high taxonomic diversity. Syst. Bot. Is10-19. JtJEL, H.O. 1900. Vergleichende Untersuchungen uber typische und parthenogenetische Fortpflanzung bei der Gattung Antennaria. Kongl. Svenska Vetenskaps-akad. Handl. 33(5)s1-59. UDYQS D.G. 1980. Hie distributions of gender in four Angiospasm species illustrating two evolutionary pathways to dioecy. Evolution 34:123-134. McIMDSH, A.C. 1931. A botanical survey of the Black Hills of South Dakota. Black Hills Engineer. 19:159-276. MARQUIS, R.J. and E.G. VOSS. 1981. Distributions of some western North American plants disjunct in the Great Lakes region. Michigan Bot. 20:53-82. RCHLF, F. J., J. KISHEADGH, and D. KIRK. 1974. Numerical taxonomy system of multivariate statistical programs. Stony Brook, New York: State Univ. of New York. SNEA3H, P. H. A. and R. R. SOKAL. 1973. Numerical Taxonomy. San Francisco: W. H. Freenan and Co. STEBBINS, G.L. 1932a. Cytology of Antepnaria. I. Normal species. Bot. Gaz. (Crawfordsville) 94:134-151. ------. 1932b. Cytology of Antennaria. II. Parthemgenefcic species. Bot. Gaz. (Crawfordsville) 94:322-345. WEBB, C.J. 1979. Breeding systens and the evolution of dioecy in New Zealand apioid Umbelliferae. Evolution 33:662-672. WELLS, P.V. 1970. Postglacial vegetational history of the Great 61

Plains. Science 167s1574-1582. WHITEHEAD, D.R. 1972. Approaches to disjunct populationss the contribution of palynology. Ann. Missouri Bot. Gard. 59sl25-137. Pig. 7. PCA coursed of 149 OTJs including three diploid Bnfcsomna species? interspecific hybrids, and the polyploid A. Parlinii s.l. Group outlines are indicated by a line. ® A* plantmjajfojiaF A. I3SSSSQS&, -$-A. solitaria. # A. £ s & U m i r and # interspecific hybrids. Interspecific hybrid identifications are given as follows from uppermost to lowermost s HY41, H2T01, HY42, HY02r HY03, HY04, HY40. Parentage of the hybrids is given in Table 4.

62 63 Fig., 8® Correlation phonogram (UPfflA) composed of 153 OTOs including three diploid species of Antennaria. interspecific hybrids, and A. Mrlinii s.l. Taxa are labelled with specific epithets. Otoo agamopecieSj, A. munda and A® Brainerdii. are presented separately from the renainder of A® Parlinii s.l. Parentage of hybrids is given in parentheses and are denoted by the first two letters of the parental specific epithets. Cophenetic correlation coefficient is 0.857.

64 r-G.14 HV41PLxRA) A R PI) s A x R L 1 2 P 4 1 Y H • 4 V H • '• IpARLRm; i O RA) 9 I 4 0 • V H BRAINERDII . HY40(RA I HY40(RA f & t R A » PLANTAQINtFOUA 3 L & si. PI J N0I1V13UH00 r £ £ UUNDA 1Px ) 0 3 01(PLx Y H SOUTARIA HY03IPL 8 HY02(PLSO) i SO) RACEMOSA 8 Fig 9. PC& composed of 117 OTOs of the A. Barlinii (<>) and A. neodioica {#) s.l. polyploid agamic complexes. Group outlines are indicated by a line. Presented are factor 1 vs. 2 and factor 2 vs. 3.

#A. neodjoica A.

66 67 ©

O®,

O 0 9 Fig* 10. Line drawings of capitulescences and basal leaves of representative specimens among three diploid species of M t e n m r l a . their interspecific hybrids, and A. Parlinii (both subspecies). Pubescence is indicated by stippling. Variation among fcaxa is apparent with respect to leaf shape and pubescence, number of veins in basal leaves, and number, size, and arrangement of heads in the

capitulescence. A. A. Planfcaginiiolia. B. A. KJLQSMmr C. A. solltaria# and D. A. M s l i m l (both subspecies). Bar = 5.0 cm.

68 69

5 cm Fig® 11. Proposed relationship of the Mtermaria neodioica s.l. (NED)

and Barlinii s.l. (PM) agamic complexes to the sexual diploids A. nsglefita (NES), A. Plantaainifolia (PM), A. (RAC), A. golitaria (SQL), and A. ^dminlca (VIR). Degree or relatedness is indicated by proximity of the circles to each other. See text for detailed explanation.

70 11 PLA

NEO

RAC 72

Table 4. Mean similarity matrix of Antennaria with each other and with six artificial hybrid combinations. Taxa are labelled with the first three letters of the specific or subspecific epithet. Six artificial hybrids (HY— -) and their parentage are presented along with their mean similarities to naturally occurring taxa. ** highest and * second highest similarity between each hybrid and the taxa are indicated.

PAR PLA RACSQL PAR 1.000 0.578 0.502 0.114

HA 0.578 1.000 0.599 -0.100 RAC 0.502 0.599 1.000 -0.154 SQL 0.114 -0.100 -0.154 1.000 HY01 (PLA x SQL) 0.547** 0.352 -0.010 0.489 HY02 (PLA x SQL) 0.485* 0.312 -0.122 0.678**

HY03 (PLA x SOL) 0.228 0.322* 0.005 0.482** HY04 (SOL x RAC) 0.502* 0.416 0.581**-0.116

HY4Q (RAC x PLA) 0.691** 0.449 0.591* -0.042

HY41 (PLA x RAC) 0.644** 0.563* 0.513 0.084

HY42 (RAC x HA) 0.650** 0.514* 0.474 0.155 73

Table 5. Table of posterior probabilities derived from a discriminant analysis. Six artificial interspecific hybrids of Anfceonaria (HY— ) and their parentage are presented along with the posterior probability to which of four naturally occurring groups of M f a m a o a they should belong to. Taxa are labelled with the first three letters of the specific or subspecific epithet.

PM HARAC SQL

HY01 ( H A x SOL) 1.000 0.000 0.000 0.000

HY02 ( H A x SQL) 0.998 0.002 0.000 0.000

HYQ3 ( H A x SOL) 0.001 0.000 0.000 0.999

HY04 (SQL x RAC) 0.962 0.000 0.038 0.000

HY41 ( H A x RAC) 0.997 0.003 0.000 0.000

HY42 (RAC x HA) 0.329 0.671 0.000 0.000 Chapter III

m i m i m d w i s g e i o m s s s g e w e b ie lo id s p e c ie s o f m c t o t a r i a

(ASTERACEAE: IHULEftE) MB TOEIR MJi0K3LYFL0ID DERWATWES.

Introduction

The M t e n m r l a JBaclinii s.l. and A* asodi&isa polyploid agamic complexes are two large polymorphic species occurring in North America. Anfcennaria Parlinii occurs in the deciduous forests of the eastern United States and adjacent Canada? while A. neodioica s.l. is found across North America between 40° and 60° N latitude primarily north of the terminal margin of the Wisconsin glaciation. Anfcennaria Gaertner consists of dioecious, perennial, entire-leaved herbs with sexual populations consisting of equal proportions of staminate and pistillate clones (Bayer and Stebbins 1983). Each polyploid agamic complex in Anfcennaria is of multiple hybrid origin involving several sexual diploid (2a = 28) species. AnfcennaEia

Parlinii. which was recently circumscribed to consist of too subspecies a. Parlinii Fern. ssp. Parlinii and A. feEliaii ssp. jEallas. (Greene) Bayer and Stebbins, has both sexual and agamospennous populations and these have distinct geographic distributions (Bayer and Stebbins 1983). Phene tic analysis of the sexual diploids A*

(Lo) Richardson, A* racemosa Hook., and A. jgfiJifca&ia 74 75

Kydb. has sha m then to be the probable diploid progenitors of A. Parlinii s.l® (Bayer 1984b® chapter II). Anfcennaria ngfldioioa s.l. was likewise shown (Bayer 1984b® chapter I) to be of multiple hybrid origin and to include the geneses of A. nealecfca Greene, A®

planfcaginifolia , A. racemose. and A. vixginica Stebbins. Bayer and

Stebbins (1982) and Bayer (1984a) have described A* neodioica s.l. as consisting of the four variable subspecies, A® neodioica Greene ssp® canadensis (Greene) Bayer and Stebbins, A. neodioica ssp. Ecwellll (Greene) Bayer, A* necdioica ssp. ngcdioisa, and A. nesdioiga ssp. pefcaloidea (Fern.) Bayer and Stebbins. Unlike A. Parlinii s.l., A. neodioica s.l. is totally agamospermous and therefore composed almost entirely of pistillate clones. Anfcennaria neodi.Qi.ga s.l. consists of fcefcraploids (2a = 56) and hexaploids (2a = 84) » while A. Parlinii s.l. has tefcraploid. pentaploid (2a s 70) . hexaploid. and octoploid (2a = 112) cytotypes (Bayer and Stebbins 1981; Bayer 1984a).

She relationship of the diploids A. negiecta, A. slaafcagiaifQlia, A. xacanosa® A. aolitaria, and A. virginLca have been assessed through gross morphology (Bayer and Stebbins 1982) and phenetic analysis

(Bayer 1984b, chapter I). The origin of the A® neodioica and A®

Parlinii agamic complexes is thought to have been from among these diploids (Bayer 1984b, chapters I and II). The present study employs enzyme electrophoretic techniques to evaluate the relationship of the five diploids and to determine whether the hybrid origin of the agamic complexes is substantiated by use of isozymes. 76

MATERIALS AMD METHODS Portions of clones (raraets) for isozyme studies were gathered at randan fran papulations throughout the United States and adjacent Canadao An effort was made to collect 30 individuals per population with the intention of electrophoresing approximately 20. Individuals

for the analysis were collected fran 76 populations composed of A.

n^lesta (6 populations), A. plantaginifplia (12 populations), A* x a s a m a (6 populations), a. solifcaua (4 populations) * a. sztomisa (4 diploid and 4 tetraploid populations)r A* neodioica s.l. (19 populations), and A« Parlinii s.l. (21 populations). Chromosome counts were determined for all populations enploying a previously described methodology (Bayer 1984a). Clones fran the populations were planted in flats and were placed in the greenhouse until they had become established and had developed flower buds. Hie flats were then placed in a cold roan (1° C ± 0.5° C) under lights for a period of two to three months. Following vernalization individual flats were pit into the greenhouse or growth chamber where they retained for two weeks until the plants flowered* at which time they were in best condition for electrophoresis because allozyme activity was highest in young flowering heads. Young leaves were used when flowering heads were not available.

Hie extracting buffer employed was 0.1 M tris-HCl, eH 7.5, 114 mM 2-mercaptoethanol, 1 mM EDTA (tetrasodium salt), 10 mM KC1, and 10 mM

MgCl2 (Gottlieb, 1981b). About 20 mg of W P were added to each sample at the time of grinding. Hie buffer was made up in 20 % sucrose to obtain the necessary density for loading the acrylamide gel* Hie extracts were then centrifuged and the supernatant was then pipetted into wells in the acrylamide and also was soaked onto wicks to be inserted into 12.25 % starch gels. T&o buffer systems were employed for starch gels. Phosphoglucose isomerase (PGI), leucine amino peptidase (LAP), and triose phosphate iscmerase (TPI), were resolved on starch using a gel buffer of nine parts tris-citrate (0.05

M tris, 0.007 M citric acid • H20, pH 8.3) and one part lithium-borate (0.038 M lithium hydroxide, 0.188 M boric acid, pH 8.3). The electrode reservoirs contained only the lithium-borate buffer. A gel buffer consisting of 0.016 M L-histidine (free base) and 0.002 M citric acid • l^O (pH 6.5) and an electrode buffer of 0.065 M L-histidine (free base); 0.007 M citric acid • h2o, pH 6.5, (Cardy, Stuber, and Goodman, 1981) were used to resolve shikimate dehydrogenase (SKEH), malate dehydrogenase (MDH), 6-phosphogluconate dehydrogenase (6-PGDH), and acid phosphatase (AGP). An acrylamide system described by Maurer and Allen (1972) was used for phosphoglucomutase (PGM), glutamate dehydrogenase (GEH), and glyceraldehyde-3- phosphate dehydrogenase ([MADP] dependant) ([NADP] G-3-HH). Hie separating gel was 0.38 M tris - H Q , pH 8.9 in 5.5 % acrylamide and the spacer gel (3.0 % acrylamide) was 0.08 M tris - HC1, pH 6.9. The electrode buffer was 0.05 M tris- 0.38 M glycine, pH 8.3. The acrylamide gel was run at 200 volts constant for about four hours. A second acrylamide system (Maurer and Allen 1972) was employed to resolve PGI-1 and -2 and TPI-1 and -2, which are the plastid forms of these enzymes. This used a separating gel buffer (8 7 8

% acrylamide) of 0.67 M imidazole - HC1, pH 7.8, spacer gel (3 % acrylamide) of 0.07 M imidazole - HCL, pH 5.9, and an electrode buffer of 0.096 M arginine- 0.007 M imidazole, pH 7.3. The gel was run at 10 watts constant power. Gels were stained according to the procedures of Soltis et al. (1983). Intact chloroplasts were isolated fran leaves enpGLoying the

methodology of Gastony and Barrow (1983). The final chloroplast pellet was resuspended in the extracting buffer described previously. Pollen was soaked to leach enzymes (Weeden aid Gottlieb 1980a) which were then used to identify cytoplasmic isozymes. The locus specifying the most anodally migrating form of each enzyme was designated 1, the next 2, and so on. Likewise, the most anodally migrating allozymes were designated as A, the next as B, and so on. Standard genetic identity and distance statistics were calculated utilizing the methodology of Nei (1972). The methods of Nei (1973) were also employed to compute gene diversity statistics. All statistics were calculated using the GENESTAT program written by Richard Whitkus and executed at the Instructional Research Computer Center of The Ohio State University. The genetic distance matrix was used to construct a phenogran by the unweighted pair-group method using arithmetic averages (UFGMA? Sneath and Sokal, 1973). The cluster analysis was generated by the T M Q N subroutine of the OT-SYS program of Rohlf, Kishpaugh, and Kirk (1974). 79

RESULTS Locality data? chromosome numbers? maximum number of individuals surveyed* and voucher numbers for each population are presented in Table 6 e Allelic frequencies for the sexual diploid populations (including one tetraploid population of Ac ffissdLoisi ssp. neodioica) can be found in Bayer (1984b) or are available from the author. Hereafter diploid and tetraploid populations of A® virginica. for the sake of conciseness* will be referred to voider the inclusive term A. virmnica. Allelic frequencies for each fcaxon are given in Table 7. Allelic frequencies for most populations of A* neodioica s.l. and A* JBarlinii s.l. could not be obtained because of the high ploidy levels of the plants. Surveyed were 362 individuals of A. Parlinii s.l.? 53 of A® ngodifiica ssp. nesdioisa? 19 of subspecies jsgmdsnsis? 9 of subspecies ffiazeUii, and 62 of subspecies pstaloidea. The allozymes detected at each locus are summarized in Table 7. Ten enzyme systems coded by 20 genetic loci were surveyed. They are as follows (subunit composition given in parentheses) s ACP-1 (dimeric) ? <330 (tetramaric) ? [MDP] G-3-HSI-1 (tetrameric ?), [NM)P] G-3-H3H-2 (tetrameric)? LAP-1 (monomeric) ? MDH-1?-2?-3?-4 (undetermined? but probably dimeric based on other studies: Gottlieb?

1981? Goodman? Stuber? Lee? and Johnson? 1980? El-Kassaby? 1981)? BGI-l?-2?-3 (dimeric) ? BGM-1,-2 (monomeric) ? SKEH-1 (monomeric) ? SOD-1 (dimeric)» SOD-2 (dimeric ?)? and TPl-1,-2,-3 (dimeric). The subunit composition of [NftDP] G-3-HH-1 and SOD-2 could not be determined because the loci are monomorphic* but were inferred respectively as tetrameric (based on [NADP] G-3-HH-2) and dimeric (based on SOD-1). MDB isozymes were monomorphic in all species and thus their subunit apposition could not be ascertained- 6-phosphogluconate dehydrogenase (6-PGEH) was scored for most runs, but could not be readily interpreted because of isozyme overlap- Production of additional isozymes for AGP, LAP, and SKDS were occasionally visualized in the gels, but were not included in the analyses because of their sporadic occurrence. Pour enzyme systems ([MfiDP] G-3-MH, BGI, PGM, and TPI) are part of the glycolytic pathway and have 2 or 3 isozymes - Isozymes of certain glycolytic enzymes are localized in the chloroplast, while the others are confined to the cytoplasm (Gottlieb, 1981a; Gastony and Darrow, 1983). Pollen leachate reveals the cytosolic forms while isolation of chloroplasts produces the piastid forms- Soaked pollen produced [NADPJ G-3-PDH-1, FGI-3, BGM-2, and TPI-3 suggesting these are the cytosolic isozymes. Isolated chloroplasts gave [NftDP] G-3-PES-2, PGI-1,-2, BGM-1, and TPI-1,-2 indicating that these were the piastid isozymes. BGI and TPI are somewhat unusual in that each system is composed of three loci. Although no genetic analyses of these loci could be performed because of lack of variation, the third locus is apparently due to a duplication in the gene coding die chloroplast form of the enzyme- Interlocus heterodimers are formed between the two chloroplastic isozymes of both TPI and PGI. Duplicated piastid TPI loci have been previously reported in Clarkia (Onagraceae) (Pichersky and Gottlieb, 1983). PGI duplications in the piastid form of the enzyme have been tentatively identified in 81

(Cranford and Smith, 1982, 1984). Die numbers of isozymes reported (in parentheses) here for GEE (1), MDH (4), FGM (2), and G-3-FDH (2) are consistant with that which has been reported for diploid plants in other genera (Gottlieb, 1982? Gastony and Darrow, 1983). Unbalanced heterozygotes, synthesized by crossing A. Parlinii (6js) and ho solltaria (2s) were used as a standard to compare heterozygotes in tetraploid populations of A. virginica and A. neodioica ssp. neodioica. Proportionate (2:2-dosage) and disproportionate (3; 1-dosage) heterozygotes are readily discernible in tetraploid individuals. In hexaploid cytotypes of A. Parlinii and A. neodioica. it is impossible to distinguish the 5:1 from the 4:2-dosage types with any degree of confidence. Ten isozymes were found to be monortorphic for all the species surveyed: [NADP] G-3-PES-1, MDH-1,-2,-3,-4, PGI-1,-2, SOD-2, and TPI-1,-2. Percent of loci polymorphic, proportion of loci heterozygous, average number of alleles per locus, and proportion of loci heterozygous were determined for populations of the five diploid taxa and polyploids and are presented in Table 8.

Total gene diversity (Hj), gene diversity within (Hs), and between (DgT) papulations, and degree of genetic differentiation between populations (Gg^) for each diploid taxon are given in (Table 9). These statistics are presented as a mean including both variant and invariant loci. Lap=l (J^ = 0.704, Hs = 0.209, % T = 0.495) and £girl ( ^ = 0.697, Hs = 0.183, DgT = 0.514) are the most diverse loci, and this genetic diversity is partitioned more between 82

populations of these species than within populations* Genetic identities and distances are presented as means between populations within each species (Table 10) , between each taxon (Table 11) , and as a phenogram based on the genetic distance matrix of the distances of pairwise comparisons between the individual populations of all of the species (fig* 12)*

DISCUSSION

d m s g s a s s m g the dJslfiM tssa— Bie diploid species examined in this investigation have been studied previously using phenetic analyses (Bayer, 1984b, chapter I). The most widely distributed species is A. neolecta. which is distributed in prairies and pastures from New England, southwest to Oklahoma, northwest to South Dakota, and north to the Northwest Territories of Canada* Antemacia plantaginifolia occurs in dry forest margins in the Appalachian region and New England with disjunct populations in the driftless area of Wisconsin and the upper Mississippi watershed. Moist forest slopes of the Appalachian Region south of the terminal margin of the Wisconsin glaciation are the habitat of A® solitaria® Mteonarla rassemoaa, which is found in the dry coniferous forests of British Columbia, Alberta, south to Washington, Oregon, northern California, Montana, Idaho, and Wyoming. The shale barren endemic, A® virqinica has the most narrow distribution of the five, occurring on shale barrens in West Virginia, western Virginia, western Maryland, extreme south-central Pennsylvania, and a few localities in extrene eastern Ohio. All species are strictly diploid (2a = 28) except A* 83

virqinica which has sane tetraploid (2a = 56) populations (Bayer 1984a). Phenetic analysis indicates that the five diploids are morphologically distinct (Bayer, 1984b, chapter I), with A. plantaginlfolia and A. JBfigQQSa the most similar of the five species (Bayer, 1984b, chapter I). Crossing data indicate that although the species are isolated by sane genic mechanisms (primarily chromosomal rearrangements), the primary factors are habitat differences (Bayer and Stebbins, 1982; Bayer 1984a). The percent of polymorphic loci in these species (ranging fran 30

% in A. rafigrosa to 45 % in A. virginica and A. plihlmmlfQliii), and average number of alleles/locus (ranging fran 1.45 ± 0.67 in A* golitaria to 1.85 ± 1.06 in A. Plantaginifolla) and proportion of loci heterozygous (ranging from 3.7 % in A. racemosa to 10.1 % in A. nealectas Table 8) are within the range of, although somewhat lower than, many of the values obtained for other outcrossing perennials (Gottlieb, 1981a). Antennaria is apparently the first dioecious genus to be investigated electrophor etically consequently no data are available for comparison. When all populations of all taxa are considered (Table 9), the gene diversities within populations (Hg = 0.071) and between populations (Dg^ = 0.065) are about equal. The value of GgT (0.478) for all taxa indicates that about 47.8 % of the genetic variation in all species as a whole is due to between population gene differences. Surprisingly, the most restricted taxon, A* yixgjjaiga, has the greatest amount of gene diversity (HT = 0.107), while one of the most widespread taxa, A. plantaoinifolia. has the least (H^ = 0 *066 ? Table 9). Hie gene diversity statistics demonstrate that the majority of the gene diversity in these species is due to variation

within (Hg) the populations than between than (DgT) . The greatest amount of between population differentiation is seen in A® neglecta

(GST = 0®255) and the least amount is between populations of A® (GgT = 0 ei06; i.e. 10.6 % of the genetic variation in A® plantaginifolia is the result of between population gene differences) ® The gene diversity in populations of the individual species of Anfcennaria is less than that which has been reported in other outcrossing perennials such as Coreopsis grandiflora Hogg ex Sweet (Crawford and Smith, 1984), Pseudotsuga menxiesii (Yeh and O ’Malley, 1980), Pinus contorta (Yeh and Layton, 1979) * and Gilia achilleifolia (Schoen, 1982). Genetic identities for all pairwise comparisons of populations within each species range fran 0.967 in A® neglecta to 0.994 in A.

Blantaginifolia (Table 10) indicating that populations within a species show a lack of differentiation. The mean genetic identity for A. viroinca (0.976) represents the value for four diploid and four tetraploid (2n = 56) populations. This supports the hypothesis (Bayer and Stebbins, 1981, 1982? Bayer, 1984a) that the tetraploids are autopolyploid (non-fcybrid) derivatives of diploid A* yixginica. They contain the same alleles at all genes, except that one population of tetraploid A* virginica contained Pgi-lh at a frequency of 0.17 (Table 7) • The cytotypes of A. virginica cannot be separated confidently by any known morphological character and also do not segregate in the phenetic analysis (Bayer, 1984b, chapter I). 85

Similarly, Crawford and Smith (1984) demonstrated using isozymes that a polyploid variety of fiacfiopBia grandiflora is of nonrhybrid (autopolyploid) origin fran diploid cytotypes of £&. grandiflora. Genetic identities and distances between species (Table 11) disclose that neglecta. Ju Plantaainifolia. and A. racemosa are the

most similar to each other, with plantaainifolia least similar to

ho vircdnica and A, soliferia. Morphologically, A. rdantaginifolia

and ho racemosa are most similar (Bayer, 1984b. chapters I and II) and the electrophoretic data are concordant with a close relationship

because they share most frequent alleles at all loci except Fai-3 (Table 7). Compared with studies of other congeners, the identities are somewhat high and the distances are lower than might be expected (Gottlieb, 1981a; Crawford, 1983). Phenetic studies denonstrate that these diploid species are diverse morphologically, but electrophoretic data shew that they are not nearly as diverse with respect to allozymes. The lack of correlation between morphological and allozymic divergence has been noted in Sulllvantia (Saxifragaceae;

Soltis, 1982), Capsicum (Solanaceae; Jensen, McLeod, Eshbaugh, and Guttman, 1979), among others.

A cluster analysis (UBGMA) of the inter populations! genetic distance matrix (see appendix of Bayer, 1984b) summarizes the data (fig. 12). The cophenetic correlation coefficient was 0.883 indicating that the phenogram is a reasonably good portrayal of the original distance matrix. Populations of each of the five diploid taxa form distinct groups (fig. 12). Anfcenmria plaatagjhifQlii. and ho raceaasa are closely associated. Anfcennaria neglecta is also 86

allied to both A. jalanfcaglnifQlia and A. racemosa (fig. 12) . The shale barren endemic* A. virginica is widely separated fran A. nealecta (fig. 12). Bayer and Stebbins (1982) have recognized the differences between these two taxa with regard to habitat, crossability, distribution, and morphology. These differences are supported by this study and it supports the taxonomic judgement that

A. virginica Es A. naalesfca Greene var. asgilltola (Stebb.) Cronq.3 should be retained as a distinct species from A® neglecta. Antennaria solitaria is most distant fran the ranaining four diploid species as is also disclosed by the morphology (Bayer and Stebbins 1982; Bayer, 1984b, chapter I). Gene diversity statistics indicate that populations of A. plantaainifolia have the least amount of differentiation between the populations (GgT = 0.106; Table 9) and this is also portrayed by the tight linkage of the populations of A® plantaginifolia in the cluster analysis based on genetic distances (fig. 12). Two populations of A. racemosa (19 and 24) fran the southern part of the range are genetically differentiated fran the retaining four populations surveyed (20, 21, 22,and 23) from more northern localities and thus there nay be geographic differentiation in A. jacemosa (fig. 12). Populations 29, 30, 31, and 32 of A. virginica are diploids (2n = 28)» while 33, 34, 35, and 36 are tetraploid. The two cytotypes are interspersed among each other in a single group (fig. 12), showing that they are not genetically distinct and that the tetraploids are probably of non-hybrid (autopolyploid) origin. Again, the between population genetic distances show that populations of A. virginica are 87

as distant from each other as are populations of the widespread species demonstrating this geographically restricted species maintains as much variability as edaphically widespread species • Origin fif iha polyploid ®ie use of enzyme electrophoresis as data for confirming the origin of polyploids has been successfully employed in several groups such as Traqooogon (Roose

and Gottlieb? 1976)? BteAancsneria (Gottliebs 1973) ? Coreopsis

(Crawford and Smith? 1984)? and several cultivated taxa (See Crawford?

1983? for a review?) * As was discussed earlier? the A® Barlinii and A® neodioica complexes are of multiple hybrid origin involving three and four diploid species? respectively® Although allelic frequencies could only be determined for one population of A® neodioica ssp. neodioica (population 51; Table 6) the allozyrces present at each polymorphic locus for the rest of the populations are indicated in Table 7. The average proportion of loci heterozygous for A® Parlinii s.l, and A® neodioica (Table 7) is higher than that found in the diploid species and this nay be the result of fixed heterozygosity at seme loci in the asexual populations of the polyploids® The moncmorphic loci in the diploids are also moncmorphic in the polyploidso Of the polymorphic loci? Lap-1 and Bcd-I are the most diverse and are the most useful for examining the origin of the polyploids o The five diploids each have unique allozymes in the highest frequency at one or? in most cases? both of these loci (Table 7) o Considering other polymorphic genes (AcprOL? Gdh? [NADP] fi=a=Edh=2., £gHfcL*r£? and M f e l ) these species all share the same common allozymes? but nay possess different less frequent alleles 88

(Table 7) . Bins while these loci are variable they are not very useful in diagnosing the origins of the polyploids . Birough phenetic analysis, the A® Parlinii polyploid complex has been shewn to be of hybrid origin among the diploids A®

A® xsGsnog&r and A® m L L k a n ,a (Bayer, 1984b, chapter t I). Analysis of 21 populations (total of 362 individuals) of A® Pag.lin.ii s.l. (including both ssp. Barlinil and ssp. fallax) supports this hypothesiso Inspection of Table 7 shows that both subspecies have primarily Xs§e=ic and , as does A® plmfcaginifQlia. Members of A® Parlinii should have alleles of A® pianfcaginlfoiia because morphologically they most closely resemble this diploid (Bayer and Stebbins, 1982; Bayer, 1984b, chapter II). lap-1e and Pgi-3a are allozymes that are almost exclusively present in A. solitaria, but have also been found in populations of A® Parlinii ssp, Parlinii and ssp. fallax (Table 7). Artificial interspecific hybrids among A* jalantaginiffllia. A. racanoBa, and A. jjplifcaria have identical patterns when compared to sane of the A® Parlinii polyploids. One population of A® Parlinii (Table 6; population 72) from Highland Co., Virginia consists of individuals that were unusual in having had only one, two, or three heads per capitulescence (typically there are 6-8 heads). Therefore these plants morphologically expressed the presence of genes from the monocephalous A® solitaria. Interestingly, the two alleles characteristic of A® solitaria. Lap~Ie and Pai-3a . were detected. Antennaria raceroosa has the two allozymes LAP-l-A and PGI-3-G that are not found in A® SlaotaainifQlia or A. solitaria. but they were detected in A® Parlinii s.l. IAP-1-A has only been found in A® Parlinii ssp. Parlinii. this being the only instance in which the two subspecies were not identical with respect to allozymes (Table 7). This situation represents another example of the concordance of morphology and isozymes because A. Parlinii ssp. Parlinii has the adaxially glabrous basal leaves and purple glands (Bayer, 1984b. chapter II) found only in A. racemosa. It should be pointed out that A. neglecta also possesses Fgi-39 in a low frequency, but based on morphology this species is not a likely progenitor of A. Parlinii (Bayer, 1984b. chapter II). In summary, A. Parlinii s.l. is composed

primarily of allozymes of A. Plantagjnifolia. but many clones also possess alleles that are indicative of the genomes of A* solitaria and A. racemosa. One individual was discovered that was heterozygous for three alleles Pgi-3a >^'9, indicating the presence of the genomes of all three diploid species. These results are concordant with those obtained from phenetic analyses and crossing experiments (Bayer, 1984b, chapter I). The situation in A. neodioica is slightly more complicated than that found in A. Parlinii. Aotemria ngffldioto has four widely distributed subspecies (See Bayer, 1984b, chapter I) and it has been proposed that they are the result of hybridization among four diploid species. As in A. Parlinii. the diagnostic loci in A. neodioica are Lap-1 and Pci-3 . Results from three larger populations of A. neodioica (i.e. populations 37, 40, and 51) indicate that they are comprised of one (or two) genotypes and therefore small sample sizes are probably adequate in the case of this obligately agamospermous species. Nineteen populations (143 individuals) of A. neodioica s.l. were used in this study. One large population (Table 6? population 51) of A, neodioica ssp. neodioifia was tetraploid so that allelic frequencies could be determined. Genetic identities and distances (Table 11? fig. 12) allign this polyploid most closely with A« vixflinica, but also close to A* Manfcaginifolia and A. r a s m s a . Rienetic analysis demonstrated that each of the four recognizable subspecies in A® neodioica could be the result of the predominance of genes from a specific diploid (Bayer, 1984b, chapter I) and this is partially substantiated by the allozyme data. Subspecies neodioica

possesses J2gi=lc (most frequently), L§BrIf (most frequently), and Lap-lc. which are indicative of the genomes

of A. nggtefe&y A. M m t m M M L M e A. and A. mmniga (Table 7). M t a m a r l a virglnica mist frequently has La©=Lf and Pqj-3C and is the predominant genome in subspecies neodioica (Table 7). Antennaria neodioica ssp. has (most frequently), Lap-1 Pai-3^ (most frequently), and Pg.i-3C and this suggests the presence of A. neglecta, A. plantaginifolia. and A. virqinica (Table 7) • Morphologically subsides canadensis, most closely resanbles A° nealecta (Bayer, 1984b, chapter I) and the enzyme profiles confirm this in that Lao-l9 and Pgi-3^ are most frequently present in both taxa (Table 7). M t e n n a o a virginica and A. piantaqjmfolia are also implicated in the parentage of subspecies

bagel9, Pai-3^. and Pai-3C have teen detected in subsides Howellii, a western taxon (Table 7). Lap-lg predominant in A» JOgglssfea, JEgirl9 in A. racemosa (most frequently) and A. neglsst&r and Sgidic exclusively in A® m g M s a (Table 7). Interspecific hybrids between A® XiSSSmSA and A® neglesta and A® racemosa and A.

sissiniea are morphologically most like A. neodioica ssp. Bm m l l i i (Bayer, 1984b, chapter I) • Thus there is good correlation between morphological and electrophoretic data with regard to the origin of subspecies Hswellii. Mtermarla neodioica ssp. loafcaloidea is morphologically most similar to both A, Plantaainifolia and A® jaealficta (Bayer, 1984b, chapter I) . Enzyme profiles demonstrate that subspecies pefcaloidea assesses £.ap"lc „ Lap-l£. Lap-19.

Pgi-3cg and Pgi-39 (Table 7). These alleles are also present in

A* neglects, A> planfcaglnilolAa, A. racemosa, and A. Subspecies oetaloidea is the most variable of the four subspecies of A. neodioica (Bayer, 1984b, chapter I) and this could be due to the common occurence of these four diploids genomes within these polyploids. In summary, while all the subspecies possess allozymes from several or all of the four diploid species that have been implicated in their origin, in several instances the predominance of the genome of one of these diploids in a specific subspecies is indicated. Many of the polyploids have enzyme profiles that are identical to those of artificial interspecific hybrids among the four diploids. The gencme of A. virginica is predominant in subspecies aeodioica, bit is also present in the other three subspecies (Table 7) • Subspecies canadensis primarily has the allozymes of A® neglecta, which is also a component in the genetic composition of the other three subspecies. Antennaria racemosaf whose range overlaps with subspecies Howellii. is 92

present in the genane of this subspecies, Bgi-3g has been detected only in A. raeanosa and subspecies Howellii. bit other less diagnostic alleles common to both A. plantaginifolia and A® racemosa are resent in the other three subspecies. Anfcennacia neodioica ssp. ps&aloMea contains allozymes commonly found in all diploidsy but morphologically most often varies in the direction of A« Planfcagloifolia and ho

neglecta. aaammmte. sod Evolutionary ^Qpsif e a fcions.— Both phenetic analysis (Bayer , 1984b, chapters I and IX) and enzyme electrophoresis have demonstrated that the A. Parlinii and A. neodioica agamic complexes are of multiple hybrid origin. In terms of genome composition A* Parlinii could be defined genetically as those polyploids which have varying dosages of genes from A. plantaglnifoJLla, A. and A* solitaria. Similarly, A. neodioica can be thought of as those polyploids which contain the genomes of A» neglecta. A. PlantaginifQlia. A. racemosa. and A. viminica in their genetic composition. The A. neodioica segregates consistently contain the genome of the snail-leaved diploid species (A® neglecta and A® v virginica) in their genome, bit not always that of A® planfcasdLnifolia and ho racemosa [the reader is referred to Bayer (1984b. chapters I and II) for full explanation of the morphology]. Bayer and Stebbins (1982) and Bayer (1984b. chapters I and II) have reiterated that it is most desirable to retain these polyploids as specifically distinct from their diploid progenitors. The rationale in doing this is that the polyploids have several diploid genomes in their genetic structure and it is therefore most reasonable to treat the polyploids as being 93

specifically distinct from their diploid progenitors. Cronquist (1945) treated the A® Barlinii segregates as varieties of A. plantaainifolia. which is considered to be an inaccurate portrayal of the relationship of these two fcaxa because this disregards the contributions by A® m c m m a and A® solitaria to the genaaes of A® Parlinii. Likewise A® neodioica s.l. was treated as varieties of ho neolecfca (Cronquist, 1945). Allozymes indicate that in the case of A. neodioica. ssp. neodioica Is A. neglfida Greene var. attenuata (Fern.) Cronq.], the genome of A® virginica is more prevalent in its genetic composition than is ho neolecfca. It is true

that the ho neodioica agamospecies do contain genes from h* neglecta, but recognizing the® as varieties of A® neglecta ignores contributions by h» plantaginifolia, A. xas s m m t and A. m s m i s a to their genetic composition® Hie five diploids each occur, in different habitatss A* neolecta. xeric, grasslands; A® Plantaoinifolia. xeric, deciduous forest margins; A. racemosa xeric, coniferous forest margins; A. SOHtsxM, mesic forests; and A® virginica. xeric, open, shale barrens. Hie polyploids occur in a wider variety of habitats and are more widely distributed than the diploid fcaxa (see Bayer and Stebbins, 1982 their figs. 2-7 for distribution of the species). Hie presence of several diploid genanes in the polyploids, as has been confirmed by electrophoresis, produces "novel" hetercmeric allozymes. H e heteromers in the polyploids nay have different dosages of monomers, which produces multiple novel heteromers. These novel heteraners can act as a buffer in the event of envirorsnental changes (Roose and Gottlieb, 1976 j Levin, 1983), because the various novel allozymes may have different substrate affinities or new regulatory control patterns. It is possible that this buffering effect allows the polyploids to occupy many habitats in addition to those occupied by the parental diploids. Additionally, multiple enzyme profiles in the form of fixed heterozygotes may be able to eictend the range of habitats in which the polyploids can survive. The polyploids should be able to occupy many different habitats because the habitats where the diploids occur are quite diverse (Roose and Gottlieb, 1976). Thus the Antennaria polyploid agamic complexes nay be successful because of the various combinations of diverse diploid genomes in their genetic composition. 95

LITERATURE CITED BfflER; R.J. 1984a. Chromosome numbers and taxonomic notes for North American species of Antennaria (Asteraceae: Inuleae). Syst. Bot. 9174-83. —-- — — . 1984b. Evolutionary investigations in Anfceamacia Gaertner (Asteraceaes Inuleae). Ph.D. dissertation; Ohio State Univ.; Colianbus. BAYER; R.J. and G.L. STEBBINS. 1981. Chromosome numbers of North American species of Antennaria Gaertner (Asteraceaes Inuleae). Amer. J. Bot. 68:1342-1349. — ------— --- . 1982. A' revised classification of Antennaria (Asteraceaes Inuleae) of the eastern United States. Syst. Bot. 7:300-313. ------. 1983. Distribution of sexual and apomictic populations of Antennaria parlinii. Evolution 37:555-561. GARDY; B.J., C.W. S T U B ® ? and M.M. GOODMAN. 1981. Techniques for starch gel electrophoresis of enzymes from maize [%,m m y a L.). Institute of Statistics Mimeograph series No. 1317. North Carolina State University; Raleigh; North Carolina, CRAWFORD, D.J. 1983. Phylogenetic and systsnatic inferences from electrophoretic studies. la Isozymes in Plant Genetics and Breeding. Part A. Tanksley and Orton Eds. Elsevier Publishers: 96

Amsterdam, The Netherlands. pgs. 257-287. CRANFORD, DeJe and E.B. SMIQE* 1982. Allozyme variation in Coreopsis and C*_ maficsogia, a progenitor-derivative species pair. Evolution 36:379-386. — ---- _ ------— — o 1984. ALlozyme divergence and interspecific variation in Coreopsis m r m d l f l o m (Compositae).

Syst. Bot. 9:219-225. CRONQUIST, A.J. 1945. Notes on the Compositae of the northeastern United States. I. Inuleae. Rhodora 47:182-184. ED-KASSABY* Y.A. 1981. Genetic interpretation of xnalate dehydrogenase isozymes in sane conifer species. J. Hered. 72:451-2. GAS0X3NY, G.J. and D.C. DARRCM. 1983. Chloroplastic and cytosolic isozymes of the hanosporous fern Atbyrium filix-femina. Amer. J. Bot. 70:1409-1415. GOODMAN, M.M., C.W. STUBER, C.-N. LEE, and P.M. JOHNSON. 1980. Genetic control of malate dehydrogenase in maize. Genetics 94:153-68. GOTTLIEB L.D. 1973. Genetic control of glutamate oxaloacetate transaminase isozymes in the diploid plant Shephanomeria and its allopolyploid derivative. Biochen. Genet. 9:97-107. *------. 1981a. Electrophoretic evidence and plant populations. Prog. Phytochau. 7:1-46. ------. 1981b. Gene number in species of Astereae that have different chromosome numbers. Proc. Natl. Acad. Sci. U.S.A. 78:3726-3729. — — — "— . 1982* Conservation and duplication of isozymes in plants. Science• 216 %373-380. JENSEN, R.J. f M.J. McLEODp W,H. ESHBADGH, and S.l. GDIDRN. 1979. Numerical taxoncmic analysis of allozyme variation in Capsicum (Solanaceae). Taxon 28:315-327. LETIN, DoAo 1983. Polyploidy and novelty in flowering plants, itaer. Naturalist 122:1-25. MAURER, H.R. and R.C. ALLEN. 1972. Useful buffer and gel systems for polyacrylamide gel electrophoresis. Z. Klin. Chen. Klin. Biochsn. 10:220-225. NEI, M. 1972. Genetic distance between populations. Amer. Naturalist 106:283-292. — --- . 1973. Analysis of gene diversity in subdivided populations. Rroc. Nat. Acad. Sci. U.S.A. 70:3321-3323. PIGHERSKY, E. and L.D. GOTTLIEB. 1983. Evidence for duplication of the structural genes coding plastid and cytosolic isozymes of triose phosphate isomerase in diploid species of Clarkia. Genetics 105:421-436. RCHLF, P. J., J. KESHFADGH? and D. KIRK. 1974. Numerical taxonomy syst©n of multivariate statistical programs. Stony Brook. New York: State Univ. of New York. ROOSE, M.L. and L.D. GJHGL.IEB. 1976. Genetic and biochemical

consequences of polyploidy in Tracropogon. Evolution 30:818-830. SCHOENr D.J. 1982. Genetic variation and the breeding systen of Gilia achilleifolia. Evolution 36:361-370. SNEA3H, P.H.A. and R.R. SOKAL. 1973. Numerical Taxonomy. San 98

Franciscos W.H. Fresnan and Coa SCLTIS, D.E. 1982. Allozymic variability in Sullivantia (Saxifragaceae) a Syst. Bot. 7:26-34. --- -- , C.H. HADFLER, D.C. D M R C M P and G.J. GA8T0NY. 1983. Starch gel electrophoresis of ferns: A compilation of grinding buffers, gel and electrode buffers, and staining schedules. Auer. Fern J. 73s9-27. WEEDEN N.F. and L.D. (XOTLIEB. 1980a. Isolation of cytoplasmic ensyir.se from pollen. PI. Physiol. 66s400-403.

------. a n d ------. 1980b. The genetics of chloroplast enzymes. J. Hered. 71s392-396. YEH, F.C. and C. LAYTON. 1979. The organization of genetic variability in central and marginal populations of lodgepole pine Pinus contorts ssp. latifolia. Canad. J. Genet. Cytol.

21s487-503. and D. O'MALLEY. 1980. Enzyme variations in natural populations of Douglas-fir, Pseudotsuoa menziesii (Mirb.) Franco, from British Columbia. I. Genetic variation patterns in coastal

populations. Silvae Genet. 29:83-92. Figure 12 „ Distance phenogran (UEGM&) composed of JL. (populations 1-6), Jk plantaglnilolia (populations 7-18) £ M S 3 i m & (populations 19-24), ha. solitaria (populations 29-36)» h* virginica (populations 29-36), and h ^ neodioica ssp. neacUflifia (population 51). Population designations are those given in Table 6.

99 1 .360- 1.360

1.160- 1.160

0.960-- 0.960

0.760* 0.760

0.560- 0.560

0.360- 0.360

0.160- 0.160

-0.040- m iThi h -0.040 IXJwrorofowwoj I IV IV W U l u jwc*jcowu)hororoi\)(\3f\)HHHHHMHOOMHHOOOOOOo CO W (. p>03--JCrjfJpiCTiCOf\iCo u> t—* o ^ crv co cn co ro wo co i—* ro 014^ cn co >-g cK 3 5" o m m 1— o o CD 100 101

Table 6. Population designations* number of plants examined per population (in parentheses)* locality data* and voucher number for populations of Antennaria, Agamosparmous populations are indicated by an asterix. Subspecies identifications for manbers of JL&. neodioica s,l, are given by the first three letters of the subspecific epithet (followed by their chromosome number) in parentheses after the voucher number. Collection numbers are of the author and vouchers are at OS.

iL. nealecfca Greene (2a = 28)— USAs 1.(17) IL; Vermillion Co., Rickapoo State Park* KP-10. 2.(20) INs Bartholomew Co., 1.5 miles W. of Hartsville, W-216. 3.(14) IN? Brown Co.* Grounds of Mount Zion Church* MZ-218. 4.(18) IN; Dearborn Co.* 3.5 miles E. of Dearborn/Ripley Co. line on rte. 46* EB-219. 5.(18) OH: Delaware Co.* 1.2 miles W. of Bellepcir.te* BEN-56. 6.(17) OH; Fairfield Co.* 0.7 miles W. of Tarleton, CP-06.

A m. planfcsoinifslia (L.) Richardson (2a = 28)— USAs 7.(17) KYs Johnson Co.* just S. of Sitka* SK-206. 8.(10) KYs Lawrence Co.* Cherokee* CK-207. 9.(21) KYs Lawrence Co.* 3 miles S. of Blaine* COW-278. 10.(20) KYs Magoffin Co.* 1 mile N of jet. 580 and 40* EV-282. 11.(28) KYs Magoffin Co.* 2 miles N. of Salyersville* S?-279. 12.(18) KYs Magoffin Co., Staffordville, MA-280. 13.(21) KYs Morgan

Co.* 1 mile W. of Grassy Creek* GC-281. 14.(20) VA: Bath Co.* N. of Hot Springs, HS-293. 15.(18) VA; Bath Go.*N. of Warm Springs* BA-294. 16.(17) W ; Greenbrier Co.*W. of Ronceverte, GB-291. 17.(19) 102 Table 6 con’t.

W s Raleigh Co.? just E. of Shady Springs? SS-289. 18.(18) W s Sumners Co.? 11 miles W. of Sumners/Greenbrier Co. line? SU-290. A®. racemosa Hook. (2a = 28)— USAs 19.(20) MTs Carton Co.? Below Beartooth Plateau? BTP-214. 20.(20) M s Granite Co.? W. of Skalkahoe Pass? M-350. 21.(18) M s Granite Co.? Anaconda-Pintlar Wilderness? 1-330. 22.(17) MTs Ravalli Co.? below St. Mary’s Peak? M-336. 23.(17) MTs Ravalli Co.? Bitterroot River near Sula? M-337. 24.(20) W s Park Co. ? Yellowstone River at Fox Creek Campground? FC-213. h*. solitaria Rydb. (2& = 28)— USAs 25.(11) KYs Bath Co., 6 miles N. of Frenchburg? MBS-44. 26.(11) KYs Magoffin Co.? 2 miles N. of Salyersville? SV-279. 27.(18) VAs Smyth Co.? Hungry Mother State Park. GS-238. 28.(21) W s Boone Co.? just S. of Danville? MJ-286.

A il virginica Stebb. (2a = 28)— USAs 29.(23) OHs Columbiana Co.? 2 miles N. of Salineville? AS7-78. 30.(17) W s Hampshire Co.? 1.3 miles S. of Ruda? RA-250. 31.(18) W s Pendleton Co.? 0.6 miles S. of Grant/Pendleton Co. line on 220? W-298. 32.(18) W s Pendleton Co.? 3 miles N. of Brushy Rim? BR-109. A. gjgginlca Steto. (2a - 56)— -!KAs 33.(18) W s Grant Co.? 2 miles N. of Petersburg? GR-110. 34.(10) W s Hampshire Co.? just E. of Hanging Rock? HR-110. 35.(17) W s Hampshire Co.? 2.5 miles N. of Purgitville? FV-300. 36.(20) W s Hardy Co.? 1.7 miles S. of Moorefield? HY-299. A. neodioica s.l. (2tt = 56,84? or 140)— CANADAs 37.(20) CNTs 103

Table 6 oon't

Frontenac Co., Barrie Twp., S. shore of Mississagagon Lake, MS-247(PET,84)*. 38. (3) ONTs Peterborough Co., Burleigh Falls,

BF-87(PET,8 4 ) 3 9 . ( 3 ) ONTs Peterborough Co., Harvey T/up., KL-182(CAN,84)*. 40.(24) ONTj Peterborough Co., N. shore of Pigeon Lake, BO-220 (CBN, EET,84) *.--USAs 41.(3) CHs Delaware Co., E. side of Musi Creek Lake, ALC-53(NB0,56)*. 42.(10) (Ms Fairfield Go., Chestnut Ridge, CR-82(NEO,PET,84) *. 43.(5) CBs Guernsey Co., 5.5 miles E. of

Cambridge, CM-19(NE0,84)*. 44.(1) OH; Jefferson Co., Anapolis, AN^-79(CfiN,84) *. 45.(3) MTs Meagher Co., W. base of Neihart Baldy, 8109(NED,84)*. 46.(15) MTs Ravalli Co., M o n g east fork ofBitterroot River, 0.5 miles NW of Jennings Campground, M-348(HCW,140)*, M-349(PET,84)*. 47.(2) NX’s Erie Co., Aurora Ttop. Fish and Game Club. AFG-61(PET,84)*. 48.(3) NYs Erie Co., Aurora Ttop. 0.9 miles W. of East Aurora, EA-58(NEO,56)*. 49.(3) NYs Erie Co., banks of Casanovia Creek, W. of E. Aurora, EA-63(CAN,84) *, 50.(6) NYs Erie Co., Aurora Fish and Game Club. AFG-62(PET,84)*. 51.(18) PAs Greene Co., 2 miles

W. of Rogersville, RO-301(NE0,56)*. 52.(5) VAs Highland Co., 4.6 miles S. of V A / W border on 220, ED-295(NEO,PET,84)*. 53.(6) W s Hampshire Co., 1.3 miles W. of Hanging Rock, HR-162-A(PET,84)*. 54.(4) W s Pendleton Co., 0.6 miles S. of Randolph Co. line, QN-111(NEQ,56)*. 55.(9) W s Randolph Co., 14.3 miles W. of Elkins, EEC-252 (NEO,84) *. A.. Parlinii s.l. (2a = 84)— USA: 56.(28) IN: Brown Co., 2 mil.es 104 Table 6 con't.

W. of Nashville, NA-217*.57. (7) INs Brown Co®f Grounds of Mount Zion Church, MZ-218*. 58.(11) KYs Bath Co., 6 miles N. of Frenchburg. MB-41. 59.(17) KYs Greenup Co., Kenoe, KE-277. 60. (17) KYs Greenup Co®, S. of Beston, CH-275. 61.(21) KYs Greenup Co., S. of Letitia, GR-276. 62.(10) KYs Lawrence 03., Cherokee, CK-207. 63.(18) CHs Fairfield Co., Bloom 3top., Section 9, PS-202*. 64.(19) CMs Fairfield Co., 1 mile W. of Bar bydale, EE-12. 65.(35) O s Fairfield Co., Beck8 s Knob, BK-88. 66.(18) O s Hocking Co., N. of rte. 56 along Salt Creek, SC-192 . 67.(17) O s Hocking Co., Salt Creek Ttop., sections 3/4, BC-198. 68.(13) O s Ferry Co., 0.8 miles S. of Maxville, MX-68. 69.(17) O s Vinton Co., 1.5 mile S. of New Plymouth, NP-29. 70.(18) PAs Centre Co., 1.5 miles W. of Unionville, CE-255. 71.(19) VA: Allegheny Co., 16 miles S. of Hot Springs, VA-292. 72.(5) VAs Highland Co., 4.6 miles S. of VA / W border on 220, SO-296. 73.(19) W s Boone Co., 10 miles S. of Bald Knob, BN-287. 74.(18) W s Lincoln Co., S. of Hamlin, HM-285 . 75.(17) W : Pendleton Co., 1 mile S. of Franklin, FR-297. 76.(18) W s Raleigh Co., 2.7 miles W. of Eccles, BK-288. 105

Table 7® Allelic frequencies for species of Antennaria. Presented are locus names and allelic designations for polymorphic loci. Taxa are labeled with the first three letters of their specific or subspecific epithets. Allelic frequencies for h*. neglecta (populations 1-6)? JL_ Planfcaginifolia (7-18). JL>. racgnosa (19-24) * &, solitaria (25-28). ik® Mrginica (29-36) * and A*. necd±oLca ssp. nsodiolca (51) are presented as combined frequencies of the individual frequencies. Allelic frequencies for the remainder of the polyploid fcaxa could not be determined? but the presence of absence of particular allozymes is so indicated (+) = present? (-) ■ absent? (+*) = overwhelmingly most frequent allele based on visual inspection of enzyme profiles. Population designations are those given in Table 6.

Locus/allele NEG PLA RACSOLVIR NEO CAN' HOW NEOPET FAL PAR

ACP-1 A 0.021 0.014 0.000 0.000 0.064 0.000 --- - +

B 0.979 0.986 1.000 1.000 0.936 1.000 + + + + +

GDH A 0.000 0.000 0.006 0.085 0.000 0.000 ---- +

B 0.928 0.986 0.983 0.915 1.000 1.000 + + + + +

C 0.072 0.014 0.011 0.000 0.000 0.000 ---- +

G-3-PDH-2 A 0.087 0.013 0.213 0.000 0.069 0.500 ---- +

B 0.913 0.833 0.787 0.720 0.866 0.000 + + + + +

C 0.000 0.154 0.000 0.280 0.065 0.500 ---- +

LAP-1 A 0.000 0.000 0.170 0.000 0.000 0.000 -----

B 0.000 0.007 0.005 0.000 0.000 0.000 - -- - -f*

C 0.000 0.944 0.735 0.000 0.065 0.500 -- + + +*

D 0.000 0.049 0.000 0.000 0.010 0.000 ---- +

E 0.000 0.000 0.090 0.671 0.000 0.000 ---- +

F 0.000 0.000 0.000 0.000 0.925 0.500 + - + -

G 0.644 0.000 0.000 0.329 0.000 0.000 +* + - + -

H 0.314 0.000 0.000 0.000 0.000 0.000 -- --

I 0.042 0.000 0.000 0.000 0.000 0.000 __ 106 Table 7 o o n ' t

Locus/allele NEG PLA RAC SOLVIR NEO CAN HOW . NEO PET FAL PAR

PGI-3 A 0.000 0.000 0.000 0.990 0.000 0.000 - -- - + +

B 0.000 0.000 0.000 0.010 0.000 0.000 - - » - - -

C 0.000 0.000 0.000 0.000 0.761 0.500 + + +

0 0.038 0.000 0.000 0.000 0.000 0.000 - - -- -

E .0.000 0.024 0.000 0.000 0.217 0.000 -- - - + F 0.842 0.935 0.206 0.000 0.000 0.000 +* - + +* + *

' G 0.120 0.000 0.782 0.000 0.000 0.000 - + - - + +

H 0.000 0.000 0.000 0.000 0.022 0.000 - -- - -

I 0.000 0.041 0.012 0.000 0.000 0.500 - - + - + +

PGM-1 A 0.000 0.020 0.000 0.020 0.024 0.000 - - - - + +

B 1.000 0.952 1.000 0.980 0.948 1.000 + + + + + +

C 0.000 0.028 0.000 0.000 0.028 0.000 - --- + +

PGM-2 A 0.000 0.003 0.044 0.198 0.000 0.000 - - - - -

B 0.144 0.073 0.156 0.091 0.324 0.500 - - + + +

C 0.726 0.891 0.752 0.711 0.676 0.500 + + + + + +

D 0.130 0.033 0.048 0.000 0.000 0.000 - --- + + SKDH-1 A 0.000 0.069 0.031 0.169 0.061 0.000 - -- + + +

B 0.021 0.003 0.000 0.000 0.223 0.000 - - + + + +

C 0.860 0;824 0.969 0.713 • 0.686 1.000 + + + + + +

D 0.119 0.104 0.000 0.118 0.028 0.000 - - - + + +

E 0.000 0.000 0.000 0.000 0.002 0.000 - - ---

SOD-1 A 0.000 0.000 0.000 0.000 0.017 0.000 - - - -_

B 1.000 1.000 1.000 1.000 0.983 1.000 + + + + +

TPI-3 A 0.895 0.921 1.000 1.000 0.874 1.000 + + + +

B 0.105 0.079 0.000 0.000 0.126 0.000 + 107

Table 80 Genetic variation for species of Anfcenmria. Presented are percent of loci polymorphic, average number of alleles per locus, and proportion of loci heterozygous* The frequency of the least camion allele is greater than 0,01. Population designations are as given in Table 6®

Percent Average Proportion of loci number of of loci

poiy- alleles/ hetero­ morphic locus zygous

1. 25.00 1.25 ± .433 12.7 2. 30.77 1.38 ± .624 18.1 3. 14.29 1.14 ± .350 8.7 4. 14.29 1.15 ± .360 9.4 5. 15.00 1.20 ± .510 3.5 6, 20.00 1.20 ± .040 9.5 average 40.00 1.60 ± .800 10.1

• » 1 » ^ « a A m.. 7. 25.00 1.28 ± .450 5.0 8 . 26.67 1.40 ± .710 2.0 9. 17.65 1.29 ± .670 2.3 10. 25.00 1.33 ± .590 4.5 108 Table 8 con’fc.

11. 30.00 1.35 ± .570 6.6 12. 30.00 1.50 ± .810 6.8 13 o 43.75 1.56 ± .700 5.9 14. 25.00 1.35 ± .650 5.3 15. 25.00 1.40 ± .800 5.9 16. 20.00 1.20 ± .400 4.8 17. 15.00 1.20 ± .510 7.2 18. 16.67 1.22 ± .530 5.1 average 45.00 1.85 ± 1.06 5.3

Aft. 19. 21.43 1.29 ± .590 5.1 20. 22.22 1.22 ± .420 1.7 21. 20.00 1.30 ± .690 3.6 22. 16.67 1.28 ± .650 2.0 23. 20.00 1.33 ± .870 5.6 24. 17.65 1.24 ± .550 5.0 average 30.00 1.60 ± 1.02 3.7

25. 18.75 1.25 ± .560 7.4 26 • 12.50 1.13 ± .330 2.3 27. 14.29 1.14 ± .350 4.4 28. 30.00 1.35 ± .570 8.7 average 35.00 1.45 ± .670 6.3 109

Table 8 con8t.

29. 17.65 1.18 ± .380 5.9 30. 25.00 1.25 + .430 15.3 31 Q 35.00 1.50 ± .920 32. 23.53 1.24 i .420 4.7

33. 15.00 1.20 + .510 9.4 34. 6.67 1.07 ± .250 4.0 35. 35.00 1.35 ± .480 16.5 36. 25.00 1.35 ± .730 7.3 average 45.00 1.80 ± 1.08 9.6 grand mean 22.04 1.28 ± .110 6.9 ikfl. neodioica s.l.

3 7 f4 0 , 51

average 1 3 . 7

&j> Parlirai s»l«

56-76

average 1 2 , 7 8 110

Table 9. Gene diversity statistics for five species of Antennaria.

Hsj, = total gene diversity within a species. Hg = gene diversity within a population of the species. DgT = gene diversity between populations of the species = G^, = degree of genetic differentiation between populations of the species.

Tsaion % d s t gST 0.098 0.073 0.025 0.255 A*. 0.066 0.059 0.007 0.106 A*. xasgffiosa 0.080 0.061 0.019 0.238 A. solitaria 0.098 0.082 0.016 0.163 0.107 0.087 0.020 0.187 All taxa 0.136 0.071 0.065 0.478 Ill

Table 10. Mean genetic identities and distances within five species of Antennaria. Ranges of values are given in parentheses® Means for each taxon were computed using populations cited in Table 6.

Identity Distance

A« neglecta 0.967 (0.923-0.996) 0.033 (0.004-0.080) A® plantaainifolia 0.994 (0.980-0.999) 0.006 (0.001-0.021)

A®, rasanosa 0.976 (0.951-0.998) 0.024 (0.002-0.050) Asl asLLifcsoa 0.983 (0.964-0.995) 0.017 (0.005-0.037) A*. ^assinisa 0.976 (0.948-0.993) 0.024 (0.008-0.054) 112

Table 11. Genetic indentities (upper) and distances (lower triangle) for pairwise interspecific comparisons of six species of Antennaria. Taxa are labeled with the first three letters of their specific or subspecific epithet.

MEG ELARACSCLVIR NED NEG 0.000 0.958 0.944 0.927 0.920 0.896 ELA 0.042 0.000 0.963 0.906 0.909 0.912 RAC 0.057 0.038 0.000 0.920 0.921 0.928 SQL 0.075 0.099 0.083 0.000 0.908 0.896 VIR 0.084 0.095 0.082 0.097 0.000 0.941 NED 0.110 0.092 0.075 0.110 0.061 0.000 SUMMARY

In tills study* til® origins of two of the agamic complexes were danonstrated to be from among five sexual diploid species* which are similar morphologically to the polyploids 0 Five sexual diploid (2n = 28) taxa (A*. negleefca* As.

planfcaglMfolia* As. m a a a > A* ASliteEia* and JL. vixgmL

isolating mechanisms are spatial ones such as different habitat distributions. Allozyme studies indicate that gene diversities are sequestered primarily within populations of the diploids and not between than. The tetraploid individuals of Aa. virginlca appear to be of autopolyploid origin from the diploid cyfcotypes because they are morphologically indistinguishable from than aid have the same alleles

at all loci. The origins of the Antennaria MclinAi and 1L. neodioifia agamic complexes were investigated anploying phenetic (morphological) and electrophoretic techniques. Phenetic analyses disclose the morphological similarity of the polyploid complexes to several diploids. Principal components suggests that the agamic complexes are 113 1 14

of hybrid origin because they are surrounded by the diploid taxa in the ordination space*, Additionally, artificially synthesized interspecific hybrids cluster with the polyploids in PGA and cluster analyses. Electrophoresis demonstrates that the polyploids contain the alleles of several diploids at many genetic loci* thus corroborating the results of the morphological studies. MteooadLa Paglinii is the result of multiple hybrid origin including the diploids JL*. ^lantaginifalia, A* m m , and h&. jaalitacia. Mtiroasia s m Issfca, ha. Plaixtacpnif0liag A*, xsssnssa? and A* yaxgihiga are the diploid progenitors of the ha. neodioica agamic complex. Sane members of h*. neodioica resanble sane of the ha. Parlinii segregates because both complexes share the diploid genomes of ha. plantaginifolia and h*. racemosa. The ha. Parlinii agamic complex has both sexual and asexual populations and is primarily hexaploid, while h*. neodioica is entirely asexual and has tetraploid and hexaploid populations in equal frequencies. Antennaria Parlinii is in the early nature stage of development because it has both sexual and asexual populations and its sexual diploid progenitors are extant. Because the ha. neodioica complex lacks sexual populations and its diploid relatives exist, it is in the nature stage of development. The agamic conplexes are recognized taxononically as distinct species from their diploid progenitors because they contain several diploid genomes. APPENDIX A

Systematic Botany (1984), 9(1): pp. 74-83 © Copyright 1984 by the American Society of Plant Taxonomists

Chromosome Numbers and Taxonomic Notes for North American Species of A n t e n n a r i a (Asteraceae: Inuleae)

R a n d a l l J. Ba y e r Department of Botany, Ohio State University, Columbus, Ohio 43210

A b s tr a c t. Chromosome num bers are presented for 132 populations of 27 taxa and three natu­ rally occurring hybrids of Antennaria. O f t h e 27 ta x a , 15 apparently have not been counted before and two counts are new num bers for species previously determ ined. Twenty taxa consist entirely or partially of sexual diploids (2n = 28): A . arcuata, A. argentea, "aromatica," A. corymbosa, A. dimorpha, A. dioica, A . flagellaris, A . Ceyeri, A . lanata, A. luzuloides, A. media, A . microcephala, A. microphylla, A. neglecta, A. plantaginifolia, A . racemosa, A. solitaria, A. suffrutescens, A. umbrinella, a n d A . virginica. S ix other species for w hich counts are not available are thought to exist as sexual diploids, bringing t o 26 the total num ber of sexual diploids in the genus as a whole. Chromosome num bers are also presented for the five heteroploid complexes: A. alpina s.l. (including A. media), A. neodioica s .l., A . parlinii s .l., A . paroifolia s.l., and A. rosea s.l. W hile A. alpina s .l., A . rosea s.l., and A. neodioica s.l. each consist of one or two euploid chrom osom e levels, A. parlinii a n d A . paroifolia each have at least four different levels. Antennaria paroifolia has the highest chromosome number known in the genus, 2n =» 140 (decaploid). B-chrom osom es w ere found in A. suffrutescens. Based on the counts presented and karyotypic inform ation, it is hypothesized that the original base num ber in Antennaria w a s x = 14. A fourth subspecies of the A. neodioica complex, A ntennaria neodioicr. subsp. how ellii, is form ally named.

Antennaria Gaertner comprises 25-30 sexual in populations that have staminate members, diploid species and several large polyploid the maturation of the meiocytes is completed agamic complexes distributed throughout the over a very short period of time, which makes temperate to arctic regions of the northern field collection of suitable meiotic material very hemisphere. It has long been thought that the difficult. For most counts, pieces of several in­ base num ber of Antennaria is x — 7 even though dividuals were uprooted from each population, n — 14 was the lowest number known (Gus- potted, and grown in the greenhouse or growth tafsson 1947). Bayer and Stebbins (1981) indi­ chamber for several weeks until new roots had cated that the base number could not be ascer­ regenerated. Cuttings of some specimens were tained until more species had been counted. suspended in an aerated water bath to promote Chromosome reports now available for species fast, active, root growth. Root tips were col­ viewed as primitive on morphological grounds, lected in the morning and placed in a solution along with additional cytological information, of 0.015% colchicine (w /v ) for 8-10 hours at suggest that x = 14 is the base number in A n­ 4°C. The root tips were then fixed overnight in tennaria. The objectives of this paper are 1) to Ostergren and Heneen's fluid (Ostergren and delimit the number and ranges of sexual dip­ Heneen 1962), which provides excellent visu­ loid (n — 14) species of North American Anten­ alization of primary and secondary constric­ naria, 2) to report the chromosome numbers tions for karyotypic studies. Staining employed of various members of the polyploid com­ the Feulgen reaction (Feulgen and Rossenbeck plexes, 3) to present evidence for a base num­ 1924) according to the procedure of Ostergren ber of x — 14 in Antennaria, and 4) to discuss and Heneen (1962). The root tips were hydro­ several karyotypic features of the species. lyzed in 1 N HC1 at 60°C for 8 minutes and washed briefly in distilled water. They were then transferred to Schiff's reagent (leuco-basic M a t e r i a l s a n d M e t h o d s fuchsin) for 2 hours and finally to a 5% pecti- Root tips were used for chromosome counts nase solution for 1 hour. The root tips may be for most collections owing to the lack of sta- stored in 45% acetic acid at -20°C. In prepara­ minate plants in agamospermous species. Even tion for examination, root tips were macerated 115 116 BAYER: ANTENNARIA

in a drop of 45% acetic acid and pressure Was T a b le 1. Chromosome numbers determined for applied to the coverslip with a new pencil eras­ North American species of Antennaria Gaertner. er. Slides were then observed with phase con­ Voucher specimens are on deposit at DAV and OS. trast optics and photographed with Kodak Presented are state (province), county, and voucher designation for each collection (Bayer's voucher technical pan film #2415. To make slides per­ num bers consist of letters and num bers w hile Bayer manent, they were frozen according to the dry and Stebbins' num bers are 80— a n d 81—). T h e f r e ­ ice method (Conger and Fairchild 1953), air- quency of staminate clones, when determined, is dried on a warming plate overnight, and made presented in parentheses after voucher designations. permanent with the addition of one drop of * = first count for the species, ** = new num ber for Euparal and a new coverslip. the species; *** = count from m eiotic m aterial. Meiotic material was fixed in 3:1 (v/v) 95% ethanol: glacial acetic acid and stored in 70% A . arcuata Cronq. 2n = 28’'. Nevada, Elko Co., NE- ethanol at -20°C. Individual florets were 258, NE-259. squashed and stained in acetocarmine stain A. argentea Benth. 2n = 28. California, Sierra Co., (Love and Love 1975) and heated with an al­ 8001. "aromatica" Evert in press. 2 n = 28*. M ontana, cohol lamp before examination. Carbon Co., 8092 (0.64). 2n = 56*. M ontana, Gallatin C o ., 8105 (0 .4 7 ). R e s u l t s A. brainerdii Fern, s.str. (=A. pariinii Bayer & Stebb. Table 1 lists the 132 populations of 27 taxa s.l.). 2n = 56*. W est Virginia, Pendleton Co., SR-172. and three natural hybrids of Antennaria col­ A. corymbosa E. N elson. In = 28. California, M ono C o ., C-218; Eldorado Co., ENF-269. W yoming, John­ lected in 28 U.S. states, two Canadian prov­ s o n C o ., 8051 (0 .4 3 ). inces, and Switzerland. As far as can be deter­ A. dimorpha T o r r e y & A. Gray. 2 n = 28*. M ontana, mined, 15 taxa had not been counted previously Carbon Co., 8066. W ashington, Spokane Co., 8141. and two new numbers have been obtained for A. dioica (L.) G aertner. In = 28. Sw itzerland, Da­ taxa previously determined. Each entry repre­ v o s , K. Urbanska- Worytkiewicz s.n. sents an individual population. Voucher spec­ A. farwellii E. Greene s.str. (=A. parlinii B a y e r & imens are on deposit at DAV and OS, with du­ Stebb. s.l.). 2 n =* 56*. M ichigan, W exford Co., MI- plicates of the WI- numbers at WIS. Figures 1- 8001. 9 are photographs of some of the Antennaria A. flagellaris (A. Gray) A. Gray. In = 28*. Califor­ nia, Lassen Co., AF-227. chromosomes discussed. Photographs of chro­ A. geyeri A . G r a y . In = 28*. California, Siskiyou mosomes of other species of Antennaria may be C o ., 8030. W ashington, Spokane Co., 8139. seen in Bayer and Stebbins (1981). A. lanata (Hook.) E. Greene. 2n = 28*. W yoming, Park Co., 3095***. D i s c u s s i o n A. luzuloides Torrey & A. Gray. 2n = 28*. Mon­ tana, Sanders Co., 8138. W ashington, Spokane Co., In order to present the results in an evolu­ 8140. tionary perspective the species will be dis­ A. media E. Greene. 2n = 28**. California, Inyo Co., cussed as three separate groups: 1) diploid C-235; Tulare Co., C-245. 2n = 56. California, Inyo species from sections not containing polyploid C o ., C-227, C-232, C-233, C-238, C-239, 8185; M ono Co., apomicts, 2) diploid species from sections C -2 2 3 , C-217, C-219,8188; Siskiyou Co., MS-266 (0 .0 0 ); containing polyploid apomicts, and 3) poly­ Trinity Co., JT-232; Tulare Co., C-246. Oregon, Des­ ploid agamospermic and amphimictic species. chutes Co., SIN-222 (0 .5 1 ), DE-221 (0 .4 7 ), GL-229; It is probable that many of the polyploid aga- Douglas Co., DO-224 (0.00); K lam ath Co., CL-225 (0 .0 0 ); mospecies are of hybrid origin between two or L a n e C o ., DPW-223 (0 .0 0 ). more diploid members of the genus (Juel 1900; A. microcephala A . G r a y . In = 28*. California, Sier­ r a C o ., 8003. Bayer and Stebbins 1982). A. microphylla R y d b . In = 28*. M ontana, M eagher In the following discussion a few taxa are C o ., 8108 (0.24). N orth Dakota, G rand Forks Co., JL- interpreted narrowly (sensu stricto) to indicate 212. W yoming, Johnson Co., 8048. 2n = 5 6 * . M o n ­ the amount of variation present in the species. tana, Carbon Co., 8088. W yoming, Johnson Co., 8056. Inclusion or probable inclusion of these taxa A. neglecta E. Greene. 2 n = 28. Colorado, Boulder within another species is indicated in paren­ C o ., 8020. Ohio, Fairfield Co., CE-201. Iowa, Johnson theses after a name (table 1). For example, col­ C o ., Ul-211. Indiana, Bartholom ew Co., MZ-218, HV- lections referable to the type of A. brainerdii 216; Dearborn Co., DB-219. Kansas, Cherokee Co., KA- 117

SYSTEMATIC BOTANY

Table 1. C o n tin u e d . Table 1. C o n tin u e d .

186. New Jersey, Sommerset Co., NJ-240. S o u t h D a ­ A. racemosa x A. umbrinella. 2 n = 28*. M ontana, kota, C uster Co., 8044. W est V irginia, H am pshire Co., Carbon Co., 8086. PV-166. A. media x A. umbrinella. 2 n = 56*. California, A. neodioica Bayer & Stebb. s.l. 2 n = 84. O ntario, M ono Co., C-222. Peterborough Co., KB-257. M ontana, Cascade Co., 8109 A. media x A. rosea x A. umbrinella. 2n = 56*. Cal­ (0.00). South Dakota, Pennington Co., 8037 ( 0 .0 0 ), ifornia, Tulare Co., C-243. 8045. W isconsin, Iowa Co., WI-109. A. neodioica E. Greene subsp. Howellii (E. Greene) B a y e r . In = 56". South Dakota, Custer Co., 8046- B. 2n = 84*. South Dakota, Custer Co., 8040, 8040-A. Fern, were considered by us to belong to a more A. parlinii Bayer & Stebb. s.l. In = 56. M issouri, broadly circumscribed taxon A. parlinii s.l. (Bay­ Jasper Co., 8012-C. W isconsin, D ane Co., DC-194, WI- er and Stebbins 1982). 204, Wl-206; Iowa Co., WI-207, WI-212 ( 0 .0 0 ). In = Diploid species from sections not containing 70. M issouri, Jasper Co., 8012-A, 8012-D. In = 8 4 . apomicts. Species in this group are all dip­ Ontario, G uelph, GU-185; Peterborough Co., KL-183. loids, but tetraploids have been reported pre­ Arkansas, W ashington Co., AR-189. Indiana, Brown C o ., MZ-218. Kentucky, Greenup Co., GP-208. M i n ­ viously, e.g., A. eucosma (2n = 56 + 4B) of the nesota, Rice Co., MN-191. Ohio, Adams Co., LX-253, maritime provinces of Canada (Morton 1981). BR-254; W ashington Co., MG-215. Oklahoma, Payne As far as is known, members of this group are C o ., 8010. N ew York, Cattaraugus Co., ASP-234, AL- strictly sexual. These species include A. arcuata, 235. W isconsin, Dane Co., WI-203, WI-205; Iowa Co., A. microcephala, A. luzuloides, A. geyeri, and A. W /-2 0 7 , WI-208, WI-210; M onroe Co., LA-186; R o c k argentea, all of which have retained a number C o ., Wl-211, WI-213 (0 .5 0 ). o fcharacteristics considered primitive for A n ­ A. paroifolia N u t t . 2n = 56*. M ontana, Carbon Co., tennaria, and they resemble members of Gna- 8067 (0.00). 2n = 112*. Colorado, Larim er Co., 8018. phalium L. (n = 7), the genus from which A n ­ N orth Dakota, K idder Co., La Duke 504. South Dakota, tennaria is thought to be derived (Stebbins 1974). Custer Co., 8019. W ashington, Spokane Co., 8142. Antennaria arcuata, which is known from only W yoming, Johnson Co., 8047; Park Co., PC-213. 2 n = 140*. Colorado, Gunnison Co., WR-271; L a r i m e r a few localities in northern Nevada, southern C o ., 8019. Idaho, and Wyoming, has been determined as diploid (table 1; fig. 1). It is unique in the genus A. plantaginifolia (L.) Richardson. 2 n = 28. Geor­ gia, Chattooga Co., GA-267. Kentucky, Johnson Co., in having long, arching stolons and ephemeral SK-206; Lawrence Co., CK-207. N orth Carolina, Hal­ basal leaves and in occurring in seasonally moist i f a x C o ., HX-230. Virginia, W ythe Co., RP-272. W is ­ meadows. Its basal leaves somewhat resemble consin, Colum bia Co., WI-201; D ane Co., Wl-202. those of A. microcephala. Another narrowly dis­ A. racemosa H o o k . 2n = 28. M ontana, G allatin Co., tributed species, A. argentea, occurs in dry open 8106; M issoula Co., 8133 ( 0 .4 3 ), 8134 (0.36). W yom ing, woods and hillsides in southern Oregon, ex­ P a r k C o ., FC-214. treme northern California, and western Neva­ A. rosea (D. C. Eaton) E. Greene. 2 n = 56. Alberta, da. The count of 2n = 28 agrees with one made B a n f f, BNP-266. California, Eldorado Co., ENF-268. by Strother (1972) for A. argentea, although W yoming, Johnson Co., 8049. 2n= 70**. W yoming, B-chromosomes were not observed in my mi­ Johnson Co., 8054. A. solitaria R y d b . 2 n = 28. O hio, Fairfield Co., M G- totic material, as was seen by Strother in meiot­ 200; Hocking Co., SC-192, NE-197. North Carolina, ic preparations. M cDowell Co., MD-195. Antennaria dimorpha and A. flagellaris occur on A. suffrutescens E. Greene. 2n = 28 + 2B*. Oregon, dry, open, stony slopes and are among the most Josephine Co., 0-201. xerophytic of all Antennaria species. Antennaria A. umbrinella R y d b . 2n = 28*. W yoming, Johnson dimorpha, characterized by having a single large C o ., 8052. 2 n => 56*. M ontana, M eagher Co. (type lo­ capitulum on a peduncle and closely cespitose c a li ty ) , 8111 (0.65). W yoming, Johnson Co., 8058. habit, occurs from British Columbia and Alber­ A. virginica S t e b b . 2 n ** 28. W est Virginia, H am p­ ta south to Washington, Oregon, and Califor­ shire Co., RA-250. 2 n = 56. M aryland, A llegheny Co., nia, and east to Wyoming, Nebraska, and Mon­ MY-236. Pennsylvania, Bedford Co., EV-180 ( 0 .6 8 ), WE-181 (0.56). W est Virginia, Ham pshire Co., HR- tana. Long filiform stolons and solitary capitulae 162***. are characteristic of A. flagellaris, which is known from eastern Washington and Oregon 118

BAYER: ANTENNARIA

Figs. 1-4. Chromosomes of Antennaria (cf. table 1). 1. M itotic chrom osom es (2 n = 2 8 ) o f A. arcuata (NE- 258) with nucleolus organizing regions indicated by arrows. 2. M itotic chromosomes (2 n = 28 + 2B) of A. suffrutescens (0-201) with two B-chromosomes (arrows). 3. M itotic chromosomes (2 n = 56) o f A. media (C- 227). 4. Meiocyte at diakinesis of A. plantaginifolia x A. solitaria (JC-52 x TE-38-1) showing multivalent (arrow). Scales = 10 iim.

east to Idaho and northern Wyoming, with a habiting dry hillsides and meadows. The for­ single locality in northeastern California. Both mer is distributed from British Columbia south A. dimorpha and A. flagellaris were counted as to eastern Oregon and Lassen County, Califor­ diploid (table 1). nia, east to Montana, Wyoming, and Colorado, A very distinctive species, A . geyeri is found while the latter is more narrowly distributed on dry hills and open woods from eastern from southern Oregon to Lassen and Nevada Washington south to northern California and counties, California, and adjacent western Ne­ west to western Nevada. Typical of A . geyeri vada. Both species have a non-stoloniferous are its woody basal stolons, upright ascending habit and numerous small capitulae but differ stems with tomentose leaves, and often roseate in the three-nerved leaves of A. luzuloides ver­ phyllaries. In its lack of prostrate stolons and sus the one-nerved leaves of A. microcephala. basal rosettes, this diploid resembles certain Lastly, A. suffrutescens occurs on dry serpen­ species of the closely related Anaphalis DC. A n ­ tine ridges from Josephine and Curry counties, tennaria lanata occurs in alpine regions of Brit­ Oregon, and Del Norte and Humboldt coun­ ish Columbia and Alberta, south to northeast­ ties, California. This very distinct species has ern Oregon, and east to Montana and Wyoming. small, coriaceous, bright green leaves and sol­ As the epithet implies, it is characterized by itary heads and is diploid with two supernu­ densely pubescent leaves and has been counted merary or B-chromosomes (In = 28 + 2B). The as diploid from one site in Wyoming (table 1). two B-chromosomes (fig. 2, arrows) were ob­ Antennaria luzuloides and A. microcephala are served in most mitotic cells. B-chromosomes two closely related diploid (2 n = 28) species in­ have been previously reported in A. argentea SYSTEMATIC BOTANY

Figs. 5-9. Chromosomes of Antennaria (cf. table 1). 5. M eiocyte at diakinesis of A. plantaginifolia x A. solitaria (JC-52 x TE-38-1) showing probable m ultivaients (arrows) and a single bivalent with nucleolus organizing region associated w ith the nucleolus. 6. M eiocyte at anaphase I of A. plantaginifolia x A. solitaria (JC-52 x TE-38-1) show ing an anaphase bridge betw een the two groups of chromosomes. 7. Same as figure 6, but show ing a fragm ent lagging at the m etaphase plate. 8. Karyogram of A. neglecta (UI-211) s h o w i n g 14 pairs of prim arily m etacentric chromosomes. 9. Karyogram of A. plantaginifolia (CO-102) s h o w i n g 14 p a i r s of prim arily subm etacentric chromosomes. Scales = 10 pm.

(Strother 1972) and A. eucosma Fern. & Wieg. portant because they appear to be closely re­ (Morton 1981). lated to the polyploid, agamic complexes. They In summary, the aforementioned species have occur primarily as diploids, but tetraploids all been determined to be diploids and they (presumably autopolyploids based on mor­ probably reproduce via monosporic sexual em­ phology) are known in four of them. It is likely bryo sacs of the Polygonum-type, as has been that hybridization between and among these described for other sexual species of Antennaria species has led to the formation of many of the by Juel (1900) and Stebbins (1932a). The pres­ polyploid apomictic complexes (Bayer and ence of equal numbers of staminate and pistil­ Stebbins 1982). Sexual embryo sacs occur in A. late clones in populations of Antennaria, as is neglecta and A. plantaginifolia (Stebbins 1932a) typical of these species (Bayer unpubl.), is a and in A. dioica (Juel 1900). Sex ratios close to good indicator of the presence of an amphi- 1:1 suggest that the remaining species are also mictic reproductive mode (Bayer and Stebbins sexual (Bayer and Stebbins in press). in press). These sexual diploid species, like oth­ Antennaria corymbosa occurs in wet, alpine to ers in Antennaria (Bayer and Stebbins 1981), are subalpine meadows from Colorado and Utah, often quite narrowly distributed as compared north to Montana and Idaho, west to Oregon, to polyploid agamospermic species. and sparingly in California and Nevada. It is Sexual diploids and tetraploids from sections con­ probably the most mesophytic species of A n ­ taining apomictic species. These species are im­ tennaria, and it characteristically has a large dark 120

BAYER: ANTENNARIA

brown spot at the base of each white phyilary. A. pulchella E. Greene, a species that further re­ It was first counted by us (Bayer and Stebbins search may reveal is distinct from the tetra­ 1981) and has been further verified as a dip­ ploid A . media (pers. comm. G. L. Stebbins, who loid. Antennaria corymbosa appears to be related is currently revising sect. Alpinae). A newly dis­ to the A . rosea agamic complex. covered species of the A lpinae group (Evert in Antennaria dioica occurs from the British Isles press) is "aromatica" E. F. Evert, which is char­ eastward to the Aleutian Islands of Alaska, thus acterized by its dark brownish green phyllaries it occurs, albeit sparingly, in North America. It and pungently aromatic, cuneiform leaves. It has been confirmed as a diploid by numerous occurs on limestone or talus in xeric subalpine European workers since the first count by Juel areas of the mountains of Wyoming and Mon­ (1900) and is reported here also as a diploid tana and has been counted as a diploid from from one locality in Switzerland (table 1). Mor­ the Beartooth Mountains of Montana and a tet­ phologically, A . dioica closely resembles mem­ raploid from the Bridger Mountains, east of Sa- bers of the A. parvifolia polyploid complex, cagawea Peak, Montana. which is common throughout the western Diploid A. racemosa, with its glabrous, bright United States. green leaves and raceme of capitula, is found Antennaria microphylla occurs as a sexual dip­ in open woods and subalpine mountain slopes loid (table 1) from eastern Washington east to from Alberta and British Columbia, south and western Minnesota and southward to Colo­ east to Washington, Oregon, northern Califor­ rado. It has small, often spathulate, leaves and nia, Montana, Idaho, and Wyoming. The species white phyllaries. A putatively (based on sex ra­ appears to be closely related to the eastern dip­ tios) agamospermous form of A. microphylla is loids, A. plantaginifolia and A. solitaria, and the tetraploid [In = 56) and is often found growing polyploid agamic complex of A. parlinii s.l. in sympatry with the tetraploid, agamosper­ (Bayer and Stebbins 1982). A putative hybrid mous A . rosea (table 1). It differs from A . rosea between two partially sympatric populations of mainly by its white phyllaries as opposed to A. racemosa and A. umbrinella is diploid (table the pink or red phyllaries of A . rosea, and this 1). Hybridization between sympatric diploid situation will be discussed further in the next species of Antennaria is not uncommon and is section. Sexual A. microphylla is probably closely probably an important evolutionary factor related to the A. rosea agamic complex and also leading to the production of the large, variable, the A. gaspensis Fern. [=A. neglecta var. gaspensis taxonomically difficult, agamic complexes found (Fern.) Cronq.] agamic complex of eastern Can­ in Antennaria (Juel 1900; Bayer and Stebbins ada. 1982). Another sexual species of Antennaria similar The final four sexual species to be consid­ to A. microphylla is the brown-phyllaried A . um ­ ered, A. neglecta, A. plantaginifolia, A. solitaria, brinella. Antennaria umbrinella occurs as a diploid and A. virginica, all occur in the eastern United and a tetraploid and is distributed on dry, sub- States and have been discussed by us both tax­ alpine slopes, meadows, and ridges from Al­ onomically (Bayer and Stebbins 1982) and cy- berta and British Columbia south through tologically (Bayer and Stebbins 1981). A n te n ­ Washington, Oregon, California, and Montana naria neglecta has been verified as diploid from to Wyoming, Colorado, and Arizona. A fre­ ten localities across its range, confirming the quency of staminate clones of 0.68 (table 1) from previous counts of Stebbins (1932a) and Bayer the approximate topotype locality in Meagher and Stebbins (1981). The seven diploid counts Co., Montana, suggests that the tetraploid (pre­ of A. plantaginifolia presented here confirm the sumed to be an autopolyploid) is sexual (Bayer previous reports by Stebbins (1932a), Bayer and unpubl.). Like A. microphylla, A. umbrinella ap­ Stebbins (1981), and Love and Love (1982). pears most closely related to the A . rosea com­ Monocephalous A. solitaria has been verified as plex based on the often brownish phyllaries diploid, thus confirming previously published seen in many of the A. rosea segregates. counts (Stebbins 1932a; Bayer and Stebbins Two diploid counts (table 1; listed under A. 1981). media) have been obtained for plants that re­ The shale barren endemic A. virginica occurs sem ble A . media and occur in the Sierra Neva­ in West Virginia and Virginia and in limited da. These plants closely resemble the type of areas in Maryland, Pennsylvania, and Ohio (one 121

SYSTEMATIC BOTANY

locality). Originally suspected to be a diploid now known cytologically. Other probable sex­ (Stebbins 1935), it was confirmed to occur both ual diploids or tetraploids in need of investi­ as a diploid and a tetraploid (Bayer and Steb­ gation are: A. marginata E. Greene (SW U.S.A.), bins 1981). One additional diploid and four tet­ A. rosulata E. Greene (SW U.S.A.), A. stenophylla raploids are reported here (table 1). The fre­ A. Gray (NW U.S.A.), A. alaskana Malte (Alas­ quencies of staminate clones (table 1) from two ka), A. monocephala DC. (Alaska), and A. ana- of these tetraploid populations indicate a sex­ phaloides Rydb. (NW U.S.A.). ual mode of reproduction. The situation of dip­ Polyploid sexual and agamospermous species com­ loids and autotetraploids occurring in A. virgin­ plexes. Antennaria appears to contain five large ica parallels the condition previously mentioned polyploid species complexes namely, A. alpina for A. umbrinella, A. media, and "aromatica." Many s.l., A. paroifolia s.l., A . neodioica s.l., A. parlinii more determinations will be needed before s.l., and A. rosea s.l. Antennaria rosea and A. neo­ meaningful statements can be made about geo­ dioica are composed entirely of polyploid aga­ graphic distributions of diploids and tetra­ mospermous populations that are completely ploids of these species. pistillate. By contrast, A. alpina, A. paroifolia, and As far as relationships are concerned, A. A. parlinii (Bayer and Stebbins in press) contain plantaginifolia and A. solitaria are most closely some sexual populations with both staminate allied to the A. parlinii s.l. polyploid complex and pistillate clones and some that are agamo­ (Bayer and Stebbins 1982; Bayer unpubl.). Mor­ spermous. The sexual populations in each phologically, A. neglecta, A. plantaginifolia, and species generally occur in the southern parts of A. virginica are most similar to the A. neodioica the respective ranges of the species complexes. s.l. polyploid agamic complex (Bayer and Steb­ Agamospermous seed production occurs via bins 1982; Bayer unpubl.). Interspecific hybrids diplospory followed by diploid parthenogen­ among the four eastern diploids (Bayer and esis, as was first demonstrated for A. alpina (Juel Stebbins 1982) indicate that the diploid species 1900) and later for members of the A. parlinii differ primarily by a few structural rearrange­ and A. neodioica complexes by Stebbins (1932b). ments of the chromosomes. Meiosis in hybrids Included within sect. Alpinae is A. media (in­ betw een A. plantaginifolia and A. solitaria typi­ cluding A. scabra E. Greene and A. pulchella E. cally exhibits irregularities such as multiva- Greene), which occurs on alpine tundra from lents (figs. 4-5), but generally 12 to 14 II are Alberta and British Columbia south to the Sier­ observed. Meiotic irregularities such as bridge ra Nevada of California and east to Montana, chromosomes (fig. 6) at anaphase I and lagging Wyoming, and Colorado. Antennaria media is fragments (fig. 7) at telophase I suggest that A. known as both sexual and asexual populations; plantaginifolia and A. solitaria differ by one or it has been confirmed as tetraploid (fig. 3). At more inversions. two localities (table 1; 8185 and 8188) the spec­ In summary, it is probable that all the above- imens match the type of A. scabra, which is con­ mentioned species have a sexual mode of re­ sidered a synonym for A. media. Greenish production and are for the most part diploid brown phyllaries are a distinctive characteristic (2n = 28) with some sexual tetraploids known of tetraploid A. media. This species is viewed as in four of the species. Morphologically, all the the southern relative of the circumboreal, fully species are characterized by having prostrate agamospermous, primarily hexaploid {In — 84; stolons, considered to be an advanced charac­ Fedorov 1969) A. alpina (L.) Gaertner s.str. Two teristic in Antennaria (Stebbins 1974). As is true unusual collections of A. media from California of almost all of the sexual diploids (and tetra­ may represent introgressant segregates (G. L. ploids) these species are morphologically well- Stebbins pers. comm.). The first of these, C-222 defined and represent the sexual relatives of (table 1), while being very similar to A. media, the widespread polyploid, agamospermous also bears characteristics of A. umbrinella. species complexes to be discussed in the next Another collection, C-243 (table 1), is also mor­ section. While some of them (e.g., A. neglecta phologically most like A. media, but probably and A. dioica) are broadly distributed, most are also has genes from A. corymbosa and A. rosea. not as widespread geographically as are their Antennaria rosea is a widespread polymorphic derived polyploid agamic complexes. group of fully agamospermous populations, Nearly all the sexual species of Antennaria are which was first reported as a tetraploid by Love 122

BAYER: ANTENNARIA and Love (in Fedorov 1969) and confirmed by 84). Thus, A. paroifolia s.l. occurs as a polyploid us (Bayer and Stebbins 1981). Morton (1981) at four euploid levels ranging from tetraploid reported A. rosea from the Yukon as 2 n = ca. 68 to decaploid and contains populations with the (approximately pentaploid). It occurs in mon­ highest chromosome numbers known in A n ­ tane subalpine zones from Alaska, Yukon, and tennaria.’ Northwest Territories, east to Hudson Bay, Antennaria neodioica s.l. (Bayer and Stebbins south to Alberta, British Columbia, and Cali­ 1982) is composed of obligately agamosper­ fornia, and east to Colorado, Wyoming, and mous populations (Stebbins 1932b; Bayer and Montana. I have verified A. rosea s.str. (with Stebbins 1982) that are widely distributed (see pink or red phyllaries) as being tetraploid (ta­ Bayer and Stebbins 1982, fig. 6). It is distin­ ble 1). A white-bracted form of A. rosea s.l., guished by its single-nerved often spathulate which can be referred to as A. microphylla s.str., basal leaves and has been determined previ­ has also been counted as tetraploid. Frequently ously as a tetraploid (Stebbins 1932b; Bayer and the white-bracted form (A. microphylla s.str.) and Stebbins 1981) and a hexaploid (Bayer and the rose-bracted form ( A . rosea s.str.) are found Stebbins 1981). Antennaria neodioica appears to growing in sympatry. An extremely rare sta­ be tetraploid primarily in regions where it is minate clone, discovered in Wyoming, was de­ not too distant from its closest diploid progen­ termined as pentaploid (2n = 70; table 1). Such itor, A. oirginica (Bayer and Stebbins 1981). In clones could have arisen from the fertilization regions more distant from A. oirginica, w hich of an occasional sexual embryo sac produced occurs mainly in Virginia and West Virginia, by tetraploid A . rosea by pollen from an un­ A. neodioica is primarily hexaploid and this is known hexaploid to produce a pentaploid hy­ confirmed by additional hexaploid counts ob­ brid. tained from localities in Ontario, Wisconsin, Another widespread complex, A. paroifolia s.l., South Dakota, and Montana (table 1). occurs in dry plains and slopes in arid areas Antennaria howellii, a western relative of A. from Manitoba south to Oklahoma and west to neodioica s.l., is characterized by one- to three- New Mexico, Arizona, Utah, Washington, and nerved glabrous basal leaves with heads in cor- British Columbia. As previously mentioned, it ymbiform cymes. It occurs in montane woods appears to be primarily sexual in the southern from Alberta and British Columbia, south to part of its distribution and predominantly aga­ Oregon, Washington, northern California, Ida­ mospermous in the northern part of its range, ho, Wyoming, Montana, and South Dakota and from which most of my determinations have is here reported as a tetraploid and a hexaploid. been made. It has rather large capitula that usu­ It seems to consist entirely of obligate apomicts ally have pink or white phyllaries except for with staminate plants being rare. The results of the brownish phyllaries found in A. aprica E. a numerical analysis using 38 vegetative and Greene s.str., which is here included in the A. reproductive characters (Bayer unpubl.) sug­ paroifolia complex. It is quite polymorphic gest A. howellii be recognized as a subspecies chromosomally with tetraploids, octoploids of the polymorphic A. neodioica s.l. complex (see (2n = 112), and decaploids (2 n — ca. 140) known Bayer and Stebbins 1982 for details). Subspe­ for the complex. The count of 2 n = ca. 140 (ap­ cies howellii can be distinguished from A . neo­ proximate decaploid) represents the highest re­ dioica E. Greene subsp. neodioica and subsp. pe- corded in the genus. One of the decaploid taloidea (Fern.) Bayer & Stebb. by its glabrous counts, WR-271 (table 1) is the only count made adaxial basal leaf surfaces. It is separable from from a sexual population of A. paroifolia. This subsp. canadensis (E. Greene) Bayer & Stebbins sexual decaploid is noteworthy for Antennaria by its lack of a flag-like appendage on the up­ because the polyploids are usually agamosper­ per cauline leaves and basal leaves usually mous, or if they are amphimictic they are gen­ greater than 9 mm wide. erally at the lower tetraploid or hexaploid levels. Sexuality was known previously as high Antennaria neodioica E. Greene subsp. how­ as the hexaploid level in A. parlinii. Recently ellii (E. Greene) Bayer, comb, etstat. nov.— Love and Love (1982) counted A. paroifolia Nutt, Antennaria howellii E. Greene, Pittonia 3: (listed by them both as A . aprica and incorrectly 174. 1897.— Antennaria neglecta E. Greene named as A. paroiflora Nutt.) as a hexaploid {In = var. howellii (E. Greene) Cronq., Leafl. W. 123

SYSTEMATIC BOTANY

Bot. 6:43. 1950.— L e c t o t y p e (here desig­ are predominantly one ploidy level while oth­ nated): U.S.A., Oregon, St. Helens, 20 May er complexes such as A. parlinii s.l. and A . par- 1887, Thomas Howell s.n. (NDG!; isolecto- vifolia s.l. each contain four euploid levels. type: MO!). E. L. Greene erred in this orig­

inal description by stating that the locality Ka r y o t y p e M o r p h o l o g y from Howell's specimen was "Oregon, Mt. St. Helens" because obviously Mt. St. Hel­ As discussed by Bayer and Stebbins (1982), a ens is in Washington. The specimen (ac­ number of European workers (e.g., Gustafsson tually from near the town of St. Helens, 1947) have suggested that the base number in Oregon) is actually from a locality that is Antennaria is x = 7 even though the lowest nearer to the typical habitat of the species. number found is x — 14. The reasons for sug­ gesting a base number of x — 7 in Antennaria include the following: 1) Some counts of 2 n = Antennaria Parlinii s.l. (Bayer and Stebbins 63 have been reported (Fedorov 1969), and these 1982) is distributed throughout the deciduous could be interpreted as nine-ploid based on x = forests of the eastern United States and adja­ 7; and 2) the presence of x = 7 in the related cent areas in southern Ontario (see Bayer and Gnaphalium suggests n - 14 was originally de­ Stebbins 1982, fig. 7). It has been discussed in rived from x - 7. Although the 2 n = 63 deter­ detail with respect to cytology (Bayer and Steb­ minations can be neither discounted nor easily bins 1981), taxonomy (Bayer and Stebbins 1982), explained, all the counts reported here are and populational reproductive modes (Bayer multiples of 14 with several of them being pen­ and Stebbins in press). Both sexual and aga­ taploid (odd-ploid) based on n = 14 (2n = 70). mospermous populations are known (Bayer and Species such as A. arcuata, A. geyeri, and A . mi­ Stebbins in press) and it has been previously crocephala are regarded as having retained reported (Bayer and Stebbins 1981) as a tetra­ primitive characters found in Gnaphalium, and ploid, pentaploid, hexaploid, and octoploid. these are all 2 n = 28. I f an n = 7 Antennaria were Tetraploids, previously known from only one to be found it would most likely have been site (in Oklahoma), have now been found to among these species of Antennaria, yet it was occur at one locality in Missouri and five lo­ not. calities in the Wisconsin driftless area. The Additional evidence supporting an x = 14 presence of sexual tetraploids at the western base number in Antennaria comes from karyo­ limits of the range of the species strongly sup­ typic studies. Chromosomes can be most par­ ports the hypothesis that they arose in this area simoniously paired into 14 groups of two (figs. (the Midwest) and spread eastward and north­ 8-9), not into seven groups of four. The pres­ ward, where they occur primarily as sexual and ence of one pair with nucleolus organizing re- agamospermic hexaploids. Pentaploids (2 n = 70) » gions ( N O R ; cf. fig. 1) indicates the presence of have been verified at one locality in Missouri two genomes in these cells. The presence of a and the common hexaploids have been verified single paired chromosome attached to the nu­ at 18 localities throughout its range. Antennaria cleolus in early prophase of meiosis I , similar farwellii s.str. and A. brainerdii s.str. are two to those seen in figure 5, indicates that in this small-leaved agamospecies that Bayer and Steb­ n — 14 individual there are two genomes pres­ bins (1982) included in synonymy under A. ent. Thus karyotypic data also support an x = parlinii s.l. Specimens collected in the field are 14 base number in Antennaria. These prelimi­ referable to the types of A. brainerdii and A. nary karyologic data demonstrate karyotypic farwellii and are so referred here (table 1). Each differences between certain species in the ge­ agamospecies has been determined as tetra­ nus. For example, chromosomes of A. neglecta ploid (table 1). are primarily metacentric (fig. 8) while those of In summary, the polyploid sexual and agam­ A. plantaginifolia are mostly submetacentric (fig. ic complexes have much wider distributions 9). than their diploid relatives. In Antennaria agamospermy is always associated with poly­ Acknowledgments. This paper is p a r t of a d i s ­ ploidy, but polyploidy is not necessarily al­ sertation done under the direction of Daniel J. Craw ­ ways associated with agamospermy. Two of the ford and to be subm itted as partial fulfillm ent for the polyploid complexes, i.e., A. media and A. rosea. degree of Doctor of Philosophy at Ohio State U ni­ 124

BAYER: ANTENNARIA versity. This study was supported by grants from Sig­ pish-chemisher Nachweis einer Nucleinsaure ma Xi, The American Alpine Club, and in part by vom Typus der Thym onucleinsaure und die dar- NSF grant #DEB-8200359.1 am especially grateful to auf beruhende elektive Farbung von Zellkernen G. Ledyard Stebbins for his continued encourage­ in m ikroskopischen Praparatin. Hoppe-Seyler's m ent, invaluable discussions, and aid and compan­ Z. Physiol. Chem. 135:203-252. ionship in securing m any of the collections. The fol­ Juel, H. O. 1900. Vergleichende Untersuchungen lowing people are also gratefully acknowledged for uber typische und parthenogenetische Fort- their collecting efforts from various regions: P. Bier- pflanzung bei der Gattung Antennaria. K o n g l. zychudek, R. Bittman, J. Bruner, J. Canne, D. Craw­ Svenska Vetenskapsakad. Handl. 33(5):l-59. ford, M. G alligan, H. Iltis, J. La Duke, R. Pilatow ski, G u s t a f s s o n , A. 1947. Apomixis in higher plants. M. Roberts, R. Stebbins, K. Urbanska-W orytkiewicz, Acta Univ. Lund. 42-44:1-370. R. W hitkus, and E. W illiams. LSve, A. and D. L6ve. 1975. Plant chromosomes. L e u - tershausen, Germany: J. Cramer.

L it e r a t u r e C it e d and . 1982. IOPB chromosome num­ ber reports LXXV. Taxon 31:356. Bayer, R. J. a n d G . L. Stebbins. 1981. Chromosome M o r t o n , J. K. 1981. Chromosome num bers in Com­ num bers of North American species of Anten­ positae from Canada and the U.S.A. J. Linn. Soc., naria G aertner (Asteraceae: Inuleae). Am er. J. B o t. Bot. 82:357-368. 68:1342-1349. O s t e r g r e n , G. and W. H e n e e n . 1962. A squash tech­ a n d ------. 1982. A revised classification of nique for chromosome morphological studies. Antennaria (Asteraceae: Inuleae) of the eastern H ereditas 48:332-341. U nited States. Syst. Bot. 7:300-313. Stebbins, G. L. 1932a. Cytology of Antennaria. I. a n d ------. In press. Distribution of sexual Norm al species. Bot. Gaz. (Crawfordsville) 94: and apom ictic populations of Antennaria parlinii. 1 3 4 - 1 5 1 . Evolution 37. . 1932b. Cytology of Antennaria. II. Parthe- C o n g e r, A . D . a n d L. M. Fairchild. 1953. A q u i c k - nogenetic species. Bot. Gaz. (Craw fordsville) 94: freeze m ethod for m aking smear slides perm a­ 3 2 2 - 3 4 5 . nent. Stain Technol. 28:281-283. . 1935. A new species of Antennaria f r o m t h e Evert, E. F. In press. A new species of Antennaria Appalachian region. Rhodora 37:229-237. (Compositae) from M ontana and W yoming. Ma­ . 1 9 7 4 . Flowering plants: Evolution above the d r o n o . species level. Cambridge, Mass.: Belknap Press, Fedorov, A. A. 1969. Chromosome numbers of flower­ H arvard Univ. Press. ing plants. Acad. Sci. USSR. V. L. Kom arov Botan­ S t r o t h e r , J. L. 1972. Chromosome studies in west­ ical Institute. ern N orth American Compositae. Amer. J. Bot. F e u l g e n , R. and H. R o s s e n b e c k . 1924. Mikrosko- 59:242-247. APPENDIX Be

Data matrix for all GTUs used in phenetic studies of Antennaria.<»

Character codes are presented along the top margin and explanation of them is given in Appendix C. OTOs are labelled as follows § NE— = JL, jraglecfca# VI— = m s i D i s a ^ NO— = A*, neodioica sspQ flgsdaste, GA— = Ab. neodlolsa ssp. cana.deag.isy PE— --A*. neodioica ssp.

^fcaloid^y ho— = neodioiea ssp. ESESlHi^ B>— = A,

PlintagMiQlMf ba— ■ A*. xasatea? ea— = A*. JSarlmii s.l., so— = A, solitaria. and HY— = interspecific hybrids.

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9ST 157 000*65 030*66 030*65 000*6 000*66 030*65 000*66 032*5 030*66 030*5 030*5 030*5 335*5 300*5 300*5 030*9 030*6 030*2 030*66 030*66 00£*9 031*5 330*66 3* 003*5 033*6 030*9 000*66 001*5 036*5 330*5 ddVdWVIS 0*5000*66 000*55 003*55 0*5000*66 003*55 003*65 000*65 005*5 030*5 S 1 ' » 5 S B 000*65 002*5 000*66 005*5 003*65 003*65 000*5 000*65 033*55 303*55 o o 003*5 0* 002*0 000*55 005*5 002*5 ODB " 030 £ *£ ODE ODE *5 000*5 2* e 000*65 001*1 000*65 000*65 000*1 00£*1 006*0 008*0 000*65 006*1 000*65 000*1 881*1 88?:|6 OOE * 005*1 1 000*6b 006*0 005*1 002*0 MHdN9 0 3 N0!lVftMIlN03 APEENDIX C. List of 38 characters and characters states used in the numerical analysis of Antennaria. Abbreviations that are used for the characters in the data matrix (Appendix B) are given in parentheses. Numbers or letters following each state indicate the scale used or the qualitative state measurements o

lasal rosette .Qhir.acfee.ra; 1. Length of entire basal leaf (LEML0K3) , ran. 2. Length of the petiole (PET3LENG), non. 3. Maximum width of the basal leaves (MAXWID3H), ran. 4, Length, along the mid-vein, from leaf tip to maximum width (WIETOTOP), ran. 5. Shape of the anterior margin, i.e. length from tip to widest point in the leaf (MSTEMAR3) , mm. 6. Number of principle veins in the leaf (NUMBVEIN). 7. Presence of a crinkled margin on the leaf (CRINKMAR), 0.0 = absent, 1.0 = present. 8. Adaxial leaf surface pubescence (HARIOOVR), 0.0 = glabrous, 0.1 = glabrous-villous, 0.2 = villous, 0.3 = pilose, 0.4 = lanate, 0.5 = tcmentose, 1.0 = canescenfc. 9. Number of leaves per basal rosette (MJMBASLV). a s t o flharafitsca: 10. Number of leaves per stolon (MJMLESTL). 11. Length of the largest leaf (STCLENEN), ran. 12. Width of the largest leaf

158 159

Appendix C con't. (STOLMID) * ran. 13 e Length of the smallest leaf (LENSMSTO) , ran. 14®

Width of the anallest leaf (WIBSM8TO) , rcm® 15. Stolon length (STQLONLN), ran® 16. Number of stolons per basal rosette (NUM8TOLN).

gaulioa XflszmaaL ftan chasasfegms 17® Flowering stem height (FLW81HGT), mm® 18® Number of leaf nodes per cauline stan (MMKODES). 19® Width of the longest leaf

(WDLQNCAU), ran® 20 ® Length of the longest leaf (LNDONCHJ), ran® 21. Width of the shortest leaf (WDSHGAHL) , ran® 22. Length of the shortest leaf (LNSHOCAU), ran. 23. Presence of a scarious flag-like structure on upper leaves (ERE&FLAG), 0.0 = absent, 1.0 = present. flLsfcillafce itoasfagsas 24. Height of the involucre (INVQLGST), nan. 25. Number of heads per capitulescence (HEADSINF). 26. Phyllary length (PHYLLLEN), ran. 27. Ehyllary width (FHYLLWDD), ran. 28. Corolla length (CDRGLLAL), ran. 29® Pappas length (PAPHJSLN), ran. 30. Athene length (ACHENELN), ran, 31. Ehyllary colors (PHYLLCDL), 1.0 = green base, white tips, 2.0 = green base, purple or brown middle, white tips, 3.0 = brown or purple base, white tig©, and 4.0 = green base, light brown tig©. 32. Number of florets par head (ELQHHEAD).

Sfcsmimfea sp i t u t e s m s jdaracteca: 33. Heigth of the involucre (S3AMINVO), ran. 34. Number of heads per capitulescence (STAMHDNM). 35. Ehyllary length (STAMEHLN), ran, 36. Ehyllary width (STAMEHW), ran. 37. Corolla length (STAMCORO), ran.

38. Pappus length (STAMPAPP), ran. i\PEEM)IX Do Table of allelic frequencies for 37 populations of M t e n m r i a » used in the allosyme study. Presented are allelic frequencies for 10 ensyme systans coded by 20 genetic locio Loci and their allelic frequencies are indicated along the left margin, while population designations (See Chapter III, Table 6 for species, locality data, etc.) are presented along the top of the table. Number of individuals from each population sampled at each locus (N) is given at the top left corner of each locus.

160 L C C U S - 01 02 03 06 05 06 01 08 39 . 10 11 12 13 ALLELEM SN .6 if M N NN H N H P G 1-3 17 20 16 13 13 17 i 7 10 21 20 23 18 21 I 0.0 q.o 0.0 0.0 3.3 '3.3 0.3 O.CoO J.O 0.130 0.090 3 .063 3.020 H 0. 0 0.0 C.O 3.0 O.JO.C 0. 0 o.c 0.0 0.0 0.0 3.0 0.0 G 0.660 C. 250 0.0 0.0 0.9 0.0 0.0 O.C •0.0 0.0 0.0 3.0 0.0 F 0.560 C. 750 l.OCO 1.000 0.7 a J 1.030 1.330 J- 5.0 0.950 0.870 0.910 0.910 0. 980 E 0.0 0.0 0.0 C.O 0 . C 0.0 0.3 O.C 3.053 0.0 0.0 0.030 0.0 0 0. J c.o c.o J.O 3.220 J.O 0.3 O.C 3.0 0.0 0.0 O.J 0.0 C 0.0 c.o c.o 3.0 0.0 0.0 0.3 0.0 3.0 3.0 0.0 J.O 3.0 B 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 J.O 0.0 A 0.3 c.o 0.0 0.0 0.0 3.0 0.3 o.c 3.0 0.0 0.0 J.O 0.0 P G l - 2 17 20 16 18 13 17 1 7 10 21 20 23 18 21 A 1. 000 1.000 1.000 1.000 1 .000 1.000 1.000 1.C3J 1 .003 1.000 I.00(3 1.000 1.000 P G I - l 17 20 16 18 13 17 I 7 10 21 20 23 18 21 A 1.000 1.000 l.OCO 1.000 1 .OCO 1.000 1.030 I. 3 JO 1.003 1.000 1.000 1.300 1.300 L A P — I 17 20 16 IS 18 17 1 7 10 21 20 23 18 21 1 0.0 C. 220 C.O J.O 3.0 C.O 0.0 0.3 0.0 0.0 0.0 3.0 Q.O H 0.500 C.530 0.070 C.500 0.0 0.150 0.3 0.3 3.0 0.0 0.0 3.0 0.0 G 0.500 C.2C0 0.930 C.500 l.CCO 0. 850 0.0 0.3 3.0 0.0 O.J 3.0 0.0 F 0.0 0.0 0.0 0.0 0.0 0.0 0.3 0.0 0.0 0.0 0.0 3.0 0.0 E 0.0 C.O 0.0 0.0 0.0 0.3 0.3 0.3 3.0 0.0 0.0 J.O 0.0 0 0.0 0.0 0.0 J.O 3.0 V. . C C.OiO 0.3 3.0 0.0 0.053 0.090 0.090 C 0.0 3.0 0.0 0.0 3.0 0.0 0.970 1.330 1.030 1.000 0.950 3.910 0.910 B 0.0 C.O 0.0 0.0 0.0 0.0 O.J 0.3 3.0 0.0 J.O 0.0 0.0 A 0.0 C.O 0.0 0.0 0.0 Q.O 0.0 O.C 0.0 0.0 0.0 3.0 0.0 M O H - 6 17 20 16 18 IS 17 17 10 21 20 28 18 21 A 1.000 1.000 1.000 1.000 1.003 1.000 1.000 1.300 1.003 1.000 1.000 1.000 1.000 HO H - 3 17 20 16 18 18 17 1 7 10 21 20 28 18 21 A 1.000 1.000 1.000 1.000 1 .OCO 1.000 1.000 1.300 1.000 1.000 1.000 1.000 1.000 MO H - 2 17 20 16 ia 18 17 17 10 21 20 28 18 21 A 1.000 1.000 1.000 l.GOG 1 .OCO 1.000 1.300 l.COO l.OCO 1.000 1.000 1.000 1.000 H D H - l 17 20 16 18 18 17 17 10 21 20 2a 18 21 A 1.000 1.000 1.000 1.000 I . oco 1.000 1.000 1.300 1.000 1.000 1.000 1.000 1.000 TP I - 3 17 0 0 0 18 17 17 a 21 20 28 18 21 B 0.320 C.O 0.0 0.0 0.0 0.0 0.170 0.0 0.0 0. 130 0.090 0.0 0.090 A 0.630 c.o 0.0 J.O l.OCO 1.000 0.830 0.3 1.003 0.870 0.910 1.000 0.910 T PI— 2 17 0 0 0 18 17 17 0 21 20 28 18 21 A l.GOO 0.0 0.0 0.0 L.OCO 1.000 1.300 0.0 1.000 1.000 1.000 1.300 1.000 TPI-1 17 0 0 0 18 17 17 0 o o 21 20 28 18 21 A 1.000 0.0 0.0 0.0 1.000 1.000 1.300 • 1.003 1.000 1.000 1.000 1.000 AC P - 1 17 20 16 18 IS 17 17 10 21 20 28 18 21 8 1.000 1.000 1.000 0.8 30 1.030 1.000 1.000 1.300 l.COO 1.000 1.000 1.000 1.000 A 0.0 c.o 0.0 0.12C O.C 0.0 O.J O.J 3.0 0.0 0.0 0.0 0.0 GOHE-1 17 20 16 IS 16 16 17 7 21 0 28 16 17 C 0.0 C. 350 0.0 0.0 0.0 0.0 0.0 0 . 3 ’ 3.0 0.0 0.0 0.0 0. 150 8 1 .000 0.650 1.000 1.000 1 .cco 1.000 1.300 1. COO 1.000 0.0 1.000 1.000 ' 0. B50 A 0.0 C.0 0.0 0.0 0.0 3.0 0.0 0.3 3.0 0.0 0.0 3.0 0.0 P G M - 2 15 20 16 0 16 16 16 10 20 18 26 16 17 0 0.0 C.500 0.0 c.o 0.0 0.0 Q.J60 0.330 0 . 080 0.3 0.0 0.030 0.070 C 1.000 C. 500 0.500 0.0 l.OCO 0.710 0.960 0.750 0 . B6 3 0.920 0.810 0.790 3.910 a 0.0 C.O C.500 0.0 0.0 0.290 0.0 0.150 0.080 0.050 0. 190 0.180 3.020 A 0.0 0.0 0.0 C.O 0.0 0.0 0.0 0.3 3.0 0.030 0.0 0.0 0.0 P G M - l 15 0 16 18 16 16 16 10 23 18 26 16 17 C 0.0 0.0 0.0 0.0 0.0 0.0 0.110 0.360 0.030 0.080 0.060 0.0 0.0. 8 1.000 0.0 l.OCO l.COO l.OCO 1.000 o.aio 0.530 0.910 0.920 0.960 0.880 0.980 A 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.360 0.063 0.0 0.0 0.120 0.020 SKDH-1 17 0 0 0 18 17 17 10 0 13 28 18 21 E 0.0 0.0 0.0 c.o 0.0 0.0 0.3 0.0 0.0 0.0 0.0 0.0 0.0 0 0.090 c.o 0.0 0.0 O.C60 0.210 0.130 0.190 ■3.0 0.0 0.063 3.360 0.090 C 0.910 c.o 0.0 0.0 0.830 0.790 0.8 70 o.aio 0.0 1.000 0.910 0.760 Ov 760 B 0.0 c.o u.Q c.o 0.060 0.0 0.0 0.0 0.0 0.0 0.3 Q.O 0.0 A 0.0 c.o 0.0 0.0 0.0 0.0 0.0 0.3 3.0 0.0 0.050 0.180 0. 150 SOD-2 17 29 16 0 18 17 17 10 20 0 2d 18 0 a 1.000 1.000 l.CGO 3.0 l.CCO l.CCO 1.900 1.C30 1.033 0.0 1.000 1.000 0.0 A 0.0 c.o 0.0 J.O 0.0 0.0 0.0 O.C 0.0 0.0 0.0 3.0 0.0 SOD-1 17 20 16 0 Id 17 17 10 23 0 28 18 0 B 0.0 c.o 0.0 » 0.0 3.0 0.0 0.0 9.3 9.0 0.0 0.0 3.0 0.0 A 1.000 1.000 l.COO 0.0 1.030 1.000 1.000 1. .30 1.000 0.0 1.000 1.000 0.0 G3POHI 15 0 0 13 16 16 0 0 0 0 26 L 8 0 o o A 1.000 0.0 0.0 1.000 l.CCO 1.000 0.0 0.3 0.0 0.0 1.000 1.000 • G 3 P D H 2 15 0 0 18 16 16 0 0 3 0 26 13 0 A 0.120 0.0 0.0 0.0 0.113 0.160 O.J 0.3 0.0 . 0.0 3.0 0.090 0.0 161 3 J.ddO c.o 0.0 l.OCO C. & .0 C. 86 0 3.3 0.3 0.0 0.0 1.000 0.820 0.0 C 0.0 ~c,a 0.0 3.0 J.O 0.0. 3.9 0.3 0.0 0.0 0.0 0.090 0.0 LCCuS- 14 IS 16 17 Id 19 20 •21 22 23 24 25 26 ALLELE n ft N Af N N H H N N H N PGI-3 23 13 1 7 19 13 20 20 13 17 17 20 1 1 11 I 3. 130 i.. 0 0.0 3.0 3.0 0.3 0.0 3.0 0.030 0.0 3.340 3.0 J.O h j • J 2.0 ..0 V. . 0 3.3 C.O VJ • 0 o.o 9. J J. 3 J. 3 J.O 3.0 G 0.0 C. 0 C.O 0.0 0.0 0.42 3 0.90 3 0.940 0.970 1.000 0.540 J.O 3.0 F 0.330 C. 360 l.OCO l.CCO I.3- 3 C . 5 i J 3. 100 3.060 3.0 •J.J 0.420 J.O J.O E J. 070 C.14J C.O C.O 0.0 0. c 3.3 0.0 3.3 0.0 j.O J.O 0.0 3 0.0 :.o i.. 0 0.3 0.0 0.0 0.0 0.0 9.0 0.0 3.0 3.0 3.0 C 0.0 0.0 C.O C.O 3.0 0.0 3.0 3.3 J.O 0.0 0.0 0.3 J.O 8 u .0 0.0 C.O C.O 0.0 0.0 0.0 3.0 3.0 0.0 0.0 0.0 3.0 A 0.0 C. 0 C.O C.O 0.0 0.0 3. 3 3.0 3.0 0-0 3.0 1.030 1.000 PG1-2 20 13 17 19 l 9 20 20 18 17 17 23 1 I 11 A I .000 l.Oou 1 «ObO l.CCO 1 .COo 1 . 0 3 0 1 .Ouu 1.000 1.000 1.030 1.000 1.000 1.000 PGl-l 20 13 17 19 Id 20 2 3 13 17 I 7 20 11 11 A 1.000 1.000 1.000 I. COO 1 .000 l.CCO 1.300 1.000 1 .090 1.000 1.033 I .000 1.000 LAP-1 20 13 17 1 7 13 20 2 3 Id 1 7 17 20 1 1 11 I 0.0 G. 0 0.3 0.0 0.0 0.0 0.0 0.0 0.0 o.o 9.0 J.O 0.0 H 0.0 C.O 0.0 0.0 0.0 0.0 0.0 0.0 3.0 0.0 0.0 0.0 J.O G 0.0 0.0 C.O 0.0 0.0 C.C 0.0 3.0 0.0 0.3 0.0 ■J. 300 0.020 F 0.0 C.O 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 J.O J.O E 0.0 0.0 0.0 0.0 0.0 0.0 3.330 0. 1 JO 0.060 0.340 0.0 0.700 0.930 0 0.0 0.130 .«c C.O 0.190 0 . c 0.0 0.0 0.0 3.0 3.0 0.0 J.O C 1.000 C. 780 1 .000 l.COO 0.310 0.670 0. 7uO 0.370 0.910 0.960 0. JdO J.O 0.3 B 0,0 C. 090 0.0 0.0 0.0 C.O 0.0 0.0 3.030 0.0 0.0 J.O 0.0 A 0.0 0.0' 0.0 C.OO.U 0.330 J.O 0.0 0.0 0.0 0.620 •3.0 0.0 MOH-4 20 13 17 19 18 20 20 Id 17 17 20 11 11 A I.000 1.000 1.000 1.000 1.000 1.000 1.000 l.COO 1.000 1.000 1.000 1 .000 1.000 MDH-3 20 13 1 7 13 13 20 20 Id 17 17 20 11 11 A 1.000 1. 000 1.000 1.000 1.000 l.COO 1.000 1.000 1.000 1.000 1.000 1.000 1.000 HQH-2 20 13 17 19 13 20 20 Id 17 17 20 11 11 A l.OCO 1.000 1.000 l.CCO 1.300 1.000 i .000 l.COO 1.000 1.000 1.000 1.000 1.000 MDH— 1 20 13 17 19 18 20 20 13 17 17 20 11 11 A 1.000 1.000 1.000 1.000 1 .000 1.030 1.300 1.300 1.000 1.000 l.OCO 1 .000 1.000 rpj-3 20 13 1 7 19 16 0 20 18 17 17 20 11 11 a 0.120 C.O 0.0 0.260 0.0 0.0 0.0 0.0 0.0 0.0 0.0 9.0 0.0 A 0. 3dO 1.000 l.OCO C. 743 l.JOO C.O 1.000 1.000 1.000 1.000 1.003 1 . uCO 1.000 t P 1-2 20 13 17 I i 13 0 23 Id 17 17 29 11 11 A l.OCO 1.000 1.000 1.000 1 .000 0.0 1.300 1.000 1.000 1.000 1.000 l.OJO 1.000 TPl-l 20 13 17 13 18 0 20 11 17 17 20 11 11

A I.000 i.ooo 1.000 1. cco 1 .OCO o • o 1.000 1.000 1.000 1.300 1.000 1.000 1.000 A CP— I 20 13 17 19 Id 0 20 13 17 1 7 20 11 11 3 1.000 1.000 0.920 J.910 I .COO 0.3 I .000 I .000 I .003 1.000 1.000 1.000 1.000 A 0.0 C.O C.GoO 0.090 0.0 0.0 0.3 0.0 0.0 0.0 0.0 0.0 0.0 GOH-1 16 16“* 15 16 13 20 1 7 15 17 16 20 0 11 C 3.3 C.OC.O 0.0 0.0 0.0 0.0 0.0 0.0 0.070 0.0 0.0 0.0 3 1.000 1.030 1.000 l.JOO 1.000 l.COO l.COO 1.000 1.000 0.390 1.000 0.0 1.000 A 0.0 0.0 0.0 •3.0 0.0 o.a 0.0 3.0 0.0 0.340 0.0 0.0 0.0 PGM— 2 16 to 15 la 13 id 17 15 17 16 20 1 1 11 D o.o C. 120 0.0 0.0 0.0 C.O 3.0 0. 060 0.240 0.3 ■3.0 0.0 0.0 C 0.940 C. 380 o.aao l.COO 1.000 0.630 0.930 0.820 0. 7 JO 0.320 3.620 0.550 1.000 d O.ObO 0.0 0.120 C.O 0.0 0.330 0.0 3.060 3.0JO 0.070 0. 3dO 0.100 J.O A 0.0 0.0 0.0 o.a o.a 0.C40 3.070 0.G63 J.O 0.110 0.0 0. J50 0.0 PGM-1 16 16 15 16 18 13 17 15 17 16 20 11 11 C 0.030 C.O 0.0 C.O 0.0 0.0 0.3 3.0 3.0 0.0 0.0 J.O 0.0 3 0.970 1.000 1 • oco l.COO 1.000 1 • 0 vrf 0 1.000 1.003 1.000 1.300 1.000 0.900 1.000 A 0.0 0.0 C.O 0.0 0.0 0*0 0.0 0.0 9.0 0.3 3.0 0.100 0.0 SKOW-1 20 13 17 13 13 20 20 Id 1 7 0 0 0 11 E 0.0 C.O 0.0 0.0 0.0 0. 0 3.0 3.0 3.0 0.0 9.0 0.0 3.0 0 0.163 C . \j 3 0 .. 33 j 3.250 3.110 0 • tj 3.3 3.0 0.0 3.3 3.0 0.0 0.0 c 0. SCO C. 340 C.920 0.590 0.33 0 I.030 1 .900 3.870 1.003 0.3 0.0 0.0 0.910 e 0.0 v.. 330 C.O 0.0 0.0 a. a 0.0 0.0 3.0 0.3 0.0 0.0 3.0 A 0.O50 C. 050 0.0 C • 160 0.060 0.0 0.3 0.1J0 3.0 0.0 0.0 0.0 0.090 SCO-2 20 13 17 16 0 20 0 13 17 0 20 11 0 3 I. 000 1.000 1.300 1.330 0.0 I • C JO 3. 3 1.000 l.COO 0.0 1 .000 I .000 0.0 A 0.0 C.OC.O C.O 0.0 0*0 0.0 0.0 3.0 3.3 3.0 0.0 3.0 SCO— 1 20 13 17 16 0 20 w Id 17 0 20 1 1 0 3 0.0 C. J •. . 0 0.0 0.0 0.0 0.0 0.0 3.0 0.0 3.0 3.0 0.0 A l.ooo 1..00 1.000 1 • aCO u ■ 0 1.600 0.9 l.CCO 1.000 0.0 l.COO 1.000 0.0 G3POHI 16 13 1 7 16 14 0 17 15 0 0 3 0 A 1-000 1. JOO l.GCO l.OCO 1 .000 0.6 1.000 l.COO 0.0 3.0 J.O 9.0 3.0 G3POH2 16 13 1 7 16 14 0 17 15 0 0 0 0 0

A 0.0 162 C.O 0.0 3. C 0.0 0.04 .4 0.403 0.0 3.0 0.3 0.0 0.0 3.0 3 1 .000 :. 720 G.53J l.COO 3.690 U • vj 0.630 1.009 3.0 3. J J.O 3.0 0.0 C 0.0 •2.230 ;.4 7u 3.0 3.310 9.3 J.O 3.0 3.0 0.9 0.0 J.O J.O LCCUS- 23 29 30 31 32 33 34 35 36 51 ALLELE li N H N ,N N N . N NN N PGi-A 1 3 21 2 3 It 13 Id Id 10 17 20 Id 1 0.0 0.0 C.O C.O 0.0 0.0 0.0 0.0 o.u 3.0 C.500 H 0.0 o.O u . 0 0.0 0.0 0.0 0.170 O.C 0.0 3.0 0.0 o o.o w . 0 3.0 J. J J.O 3.0 0.0 0.0 0.0 0.0 0.0 F J.O :.o : .o 3.0 J.O o • w 0.0 3.0 0.3 0.0 3.0 E 0.0 3.0 C.O 0. 130 3.360 C. 250 9.0 0.0 0.620 0. 300 0.0 0 J.O C.O 3.0 0.0 0.0 J.O 0.3 0.0 3.0 0.0 0. 0 C 0.0 C.O 1 .000 0. 320 J.640 0.750 0.330 l.COO 0. 330 0. 700 0.500 8 0.0 J.030 o.O J.O 3.0 0.3 0.9 0.0 0.0 0.0 0.0 A 1. 00 0 C. 373 C.O J.O J.O 3.0 3.0 J.O 0.0 0.0 0.0 PGI-2 la 21 23 17 It. Id 13 10 17 20 18 A t .000 l.OJJ I. 300 1.G30 1.030 1. 300 1.000 l.COO 1.030 1.030 1.000 PGI-1 la 21 23 17 1 3 13 1 o 10 17 20 18 A 1 .000 1.000 I .OCO l.OuO l.OCO 1 • COO 1 .000 1 .000 I.000 1 «uuO 1 .000 LAP— I 13 21 23 17 IS 13 1 5 10 17 20 Id 1 0.0 C.O 3.0 0.0 0.0 0.0 0.0 0.0 0. J 0.0 9.0 H 0.0 C.O 3.0 3.0 0.0 C . c 0.0 0.0 0.0 0.0 0.0 G 0.5u0 C. 3o J 3.0 3.3 0.0 3.0 0.0 C.O 0.0 0.0 0.0 F 0.0 C.O 0.160 l.COO 0.920 0.920 1.000 I.000 0.940 0.920 0.500 E 0.500 C . o 4 . 3.0 C.O 0.0 0.0 0.0 o.c 0.0 0.0 3.0 0 0.0 C.OJ.O 0.0 0.0 o.cao 0.0 0.0 0.0 0.0 0.0 C 0.0 C.O 0.220 0.0 0.080 0.0 0.0 0.0 0.060 o.oao 0.500 8 0.0 C.O 0.0 0.0 O.C C.O 0.0 0.0 0.0 0.0 0.0 » & 0.0 0.0 L « 0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 IS ^1 23 17 1 3 13 ia 13 17 20 Id A 1.000 I.000 1.000 l.OCO 1.000 1 • COG 1.000 1.000 1.000 1.000 1.000 MDH-3 18 21 23 17 10 18 18 10 17 20 13 A 1.000 1.000 1.000 1.009 1.000 l.COO 1.000 l.OCO 1.000 l.JOO I. 000 HOH-2 13 21 23 17 18 Id ia 10 17 20 18 A l.OCO 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 1.000 MOH-1 13 21 23 17 18 18 Id 10 17 20 18 A 1. OCO 1.000 l.COO 1.000 1.000 1.000 1 .00 0 1 .000 1.000 1.030 1.000 TPI-3 0 21 23 17 1 3 18 IS 10 17 20 18 8 0.0 0.0 0.450 0.0 0.110 0.0 0.0 0.350 0.060 0.0 0.0 A 0.0 l.CCO 0.510 l.CCO 0.390 1.000 i.oco 0.650 0. 740 1.000 l.COO TP 1-2 0 21 23 17 ia IS Id 10 17 20 18 A 0.0 1.000 I.000 l.COO l.CCO l.COO 1.000 I.000 1.000 I.000 1.000 i p i -1 0 21 23 17 le IS Id 10 17 20 18 A 0.0 l.OCO 1 .000 1.003 1.000 I.000 1.000 l.COO l.COO 1.000 I.000 ACP-1 18 21 2) 17 18 13 13 10 17 20 18 a 1 .000 1.000 1 .000 0.630 0.d60 0. 340 1.000 1.000 l.COO I.000 I.000 A 0.0 0.0 -.0 0.320 0.140 0.060 0.0 0.0 0.0 0.0 0.9 GDH— 1 13 17 23 13 15 16 18 10 16 Id 18 C 0.0 0.0 3 .0 0.0 0.0 C.O 0.0 o.o 0-0 0.0 0.0 3 i. C. 773 l.JOO 1.003 1.000 1.000 I.000 1.000 1.000 1.000 1.000 A 0.0 C.230 3.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 PGM-2 16 17 13 13 15 16 Id 10 16 13 Id 0 0.0 0.0 J.O 3.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 C 0.560 C. 770 0.550 0.690 I .000 0.360 0.610 1.000 0.530 0.820 0.500 B 0.0 C.230 0.450 0.310 0.0 0.640 C. 390 C.O 0.470 O.ldO 0.500 A 0.440 C.O 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 PGM— 1 16 17 Id 13 15 16 Id 10 16 18 18 C 0.0 C.O C.O 0.0 0.0 0.0 0.0 0.0 0.220 0.0 0.0 B I.000 l.OCO 1.000 0. 770 1.000 1.000 1.000 1.000 0. 780 1.000 1.000 A J.O C.OC.O 0.230 0.0 0.0 0.0 0.0 0.0 0.0 0.0 SKOH-l 0 21 0 17 IS 0 18 0 17 20 Id E 0.0 C.O 0.0 0.0 9.010 0.0 0.0 C.O 0.0 0.0 0.0 0 0.0 C. ISO 0.0 0.0 0.030 0.0 0.100 0.0 0.0 0.010 0.0 C 0.0 C . 6 10 C.O C . 5CC 0.650 0.0 C. 780 0.0 0.620 0.850 1.000 0 0.0 C.O C.O J.5C0 0.120 0.0 0. 120 C.C 0.380 0.040 0.0 A 0.0 C.213 C.O 0.0 0 .190 0.0 0.0 0.0 0.0 0. 100 0.0 SGO-2 13 17 13 13 15 16 l a 0 16 Id 13 B l.OCO 1.000 1.000 l.COO 0.870 1.000 1.000 0.0 l.COO 1.000 1.000 A 0.0 0.0 0.0 C.O 0.130 0.0 0.0 0.0 0.0 0.0 0.0 SOO-l Id 17 Id 13 15 16 Id 0 16 18 18 a 0.0 C.OC.O o.c 0.0 0.0 0.0 o.c 0.0 0.0 0.0 A 1.000 l.CCO l.COO 1.000 1 .COO 1.000 1.000 0.0 l.COO 1 .coo 1 .OCO G3PDH1 0 17 0 13 15 u 18 0 16 18 18 A 0.0 1. Cud 0.0 l.CCO 1 .OCO 0.0 l.OCO u.G l.OCO 1.000 l.COO G3PDH2 0 17 ‘J 13 15 0 18 0 16 18 18 A 0.0 0.0 J.O C.O 0.370 0.0 0.0 0.0 0.0 0.0 0.500 a 0.0 C.72 3 0.0 l.CJO O.o 30 0.0 1.000 Q.O 0.320 0.370 0.0 C 0.0 C.230 C.O 0.0 0.0 0.0 0.0 0.0 0.180 0.130 0.500 APPENDIX E. Table of genetic distances (lower triangle) and genetic identities (upper triangle) for all pairwise comparisons among the 37 populations of Antennaria used in the allosyme study. Population designations given along the left and top margins are the same as those presented in Table 6 of Chapter III.

164 o 165

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