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This dissertation has been microfilmed exactly as received 69-4860

COOKE, Jr., John Francis, 1927- THE CHROMATOGRAPHY AND CYTOLOGY OF SOME CULTIVATED TAX A OF THE TRATT.

The Ohio State University, Ph.D., 1968

University Microfilms, Inc., Ann Arbor, Michigan THE CHROMATOGRAPHY AND CYTOLOGY OF SOME CULTIVATED

TAXA OF THE GENUS HOSTA TRATT.

DISSERTATION

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

by

John Francis Cooke, Jr., A.B., M.S.

The Ohio State University 1968

Approved by

Adviser Botany ACKNOWLEDGMENTS

I wish to express sincere gratitude to Dr* T. Richard

Fisher, my adviser, for his constant interest and guidance during my attendance at The Ohio State University* Also, thanks nre due to Drs* C. G. Weishaupt and E. D. Rudolph for reading this dis­ sertation and making many helpful criticisms and suggestions, and to Dr. E. F. Paddock for his help with cytological problems*

Special recognition is due to Dr. F. P. Lee of the United

States National Arboretum who so kindly supplied most of the

studied. Dr. Gl> un W. Blaydes of Botany and Mr. Richard J. Meyer

of Columbus also supplied plants. Thanks are extended also to

Mr. Goro Kuno of the Academic Faculty of Entomology for his

translation of papers written in Japanese.

ii VITA

August 7* 1927 • • • Born— Woodbury, New Jersey

1950 • ...... A.B., Cornell University, Ithaca, New York

1950-1960 . • • • • Several positions in the field of

1963 ...... M.S., Cornell University, Ithaca, New York

1963-1968 • • • • • Teaching Assistant, and Teaching Associate, Department of Botany (since January 1, 1968, Academic Faculty of Developmental and Organismic Biology), The Ohio State University

PUBLICATIONS

Cooke, John F., Jr. 1962. The chromosome number of woodii Morton. Baileya 10:53*

____ . 1963* The of Achimenes glabrata. Baileya 11:^7*

Cooke, John F., Jr. and R. E. Lee. 1966. Hybridization within and between Achimenes P. Br. and Smithiantha Kuntze () Baileya 1^:92-101.

Cooke, John F., Jr. 1966. A new tetraploid X Eucodonopsis. The African Violet Magazine 20 (2):^5-^6.

FIELDS OF STUDY

Major Field: Botany

Studies in Experimental . Professor T. Richard Fisher

Studies in Cytogenetics. Professor E. F. Paddock

iii CONTENTS

ACKNOWLEDGMENTS ......

VITA ......

TABLES ......

PLATES ......

FIGURES ......

INTRODUCTION ......

HISTORY OF THE GENUS ...... Familial relationships ...... Genus n a m e ...... • . Species names ......

MORPHOLOGY OF TAXA INCLUDED IN THIS STUDY

CHROMATOGRAPHY ...... The literature ...... Materials, methods, and results . . •

CYTOLOGY ...... The literature ...... Materials, methods, and results . . •

DISCUSSION ...... The genus ...... The valid species...... The species of questionable validity The hybrid taxa ......

CONCLUSIONS ......

BIBLIOGRAPHY TABLES Table Page

1 Taxa included in this study, with their names as received, sources, and names as identified . . 11

2 Chromatographic spots observed in this study, with their reactions to visible and ultra-violet light in absence of reagents and with two reagents . . . 27

3 List of chromatographic spots and taxa in which they were observed ..*•••• ...... 30

4 Published chromosome counts of members of Hosta Tratt...... 39

5 Summary of published viability data . • • • 44

6 Haploid chromosome numbers and pollen stainability percentages determined in this study . • • • • 48

v PLATES Plate Page

I Polygonal graphs of Paired Affinity Values of six Hosta taxa. Figures 1 through 6 ...... 32

II Polygonal graphs of Paired Affinity Values of six Hosta taxa. Figures 7 through 12 ..... • 3b

III Polygonal graphs of Paired Affinity Values of five Hosta taxa. Figures 13 through 17 ..... 36

vi FIGURES Figure Page

18 Camera lucida drawing of a microsporocyte of H. plantaginea . . • . • ...... k9

19 Camera lucida drawing of a microsporocyte of H a lancifolia ...... a...... ^f9

20 Photomicrograph of a microsporocyte of H. alb omarginat a a l b a ...... 51

21 Camera lucida drawing of the cell illustrated in Figure 2 0 * ...... • •••••• 31

22 Photomicrograph of a microsporocyte of H. alb omarginata alba 32

23 Camera lucida drawing of the cell illustrated in Figure 2 2 . • •••...... 32

2k Camera lucida drawing of a microsporocyte of H. alb omar gina ta a l b a ...... 33

25 Photomicrograph of a microsporocyte of H. sieboldii, in diakinesis ...... 5k

26 Camera lucida drawing of the cell illustrated in Figure 2 5 * ...... • 5k

27 Photomicrograph of a microsporocyte of H. sieboldii. in anaphase I ...... 36

28 Camera lucida drawing of the cell illustrated in Figure 27 ...... 5 6

29 Photomicrograph of a microsporocyte of H. undulata var. u n d u l a t a ...... 57

30 Photomicrograph of a microsporocyte of H. undulata var. univittata ...... 58

31 Camera lucida drawing of the cell illustrated in Figure 3 0...... 58

vii FIGURES (continued) Figure Page

32 Camera lucida drawing of a microsporocyte of H. sieboldiana, variegated form ...... 60

33 Photomicrograph of a microsporocyte of H. sieboldiana, variegated , in diplonema . . . 6l

34 Camera lucida drawing of the cell illustrated in Figure 3 3 ...... - 6l

35 Photomicrograph of a microsporocyte of H. ventricosa. in diplonema...... 63

36 Camera lucida drawing of the cell illustrated in Figure 35 .••••••••••*.••••• 63

37 Photograph of an open and the of H. tardiflora ••••••• 64

38 Camera lucida drawing of a microsporocyte of H. tardiflora »..•••*...... 67

39 Camera lucida drawing of a microsporocyte of H. tardiflora 67

40 Camera lucida drawing of a microsporocyte of H . decorata ...... 68

4-1 Photomicrograph of a microsporocyte of H. sieboldiana hybrid in metaphase I...... 70

42 Camera lucida drawing of the cell illustrated in Figure 4 l ...... 70

43 Photomicrograph of a microsporocyte of H. sieboldii in anaphase I...... 78

44 Camera lucida drawing of the cell illustrated in Figure 43 ...... 78

45 Diagram of a possible explanation of the chromosome configuration illustrated in Figures 43 and 44 ...... 79

46 Semi-diagrammatic drawing of a possible interpre­ tation of the multivalent illustrated in Figures 30 and 31 •••••»•• •••••• 85

viii FIGURES (continued) Figure Page

k7 Diagram of a possible interpretation of the multivalent illustrated in Figures 30 and 31 • 85

^8 Semi-diagrammatic drawing of a second possible interpretation of the multivalent illustrated in Figures 30 and 31 • ••••••...... * 87

Diagram of a second possible Interpretation of the multivalent illustrated in Figures 30 and 5 1 ...... 87

ix INTRODUCTION

Members of the genus Hosta Tratt* are among the few useful perennial herbs that attain their optimum growth in shaded areas of gardens* They are of horticultural interest because of the bold, neat, textural effects of the foliage* The vary from dark to light green, from glabrous and shining, almost coriaceous, to glaucous or pruinose, and membranous. There are also a number of clones with variegated leaves* Most herbaceous perennial species of the garden have foliage that becomes unsightly after flowering, but the leaves of those commonly called funkias remain attractive all summer. For these reasons, the genus has some horticultural importance*

Unlike most horticultural species, the are of minor value. They are usually small and lavender, and thus relatively inconspicuous, particularly in the shade. Exceptions to this are such white-flowered taxa as H. plantaginea (Lam.) Asch. and H* albomarginata (Hook.) Hyl* var. alba (Irving) Hylander. The former is a unique species of the genus in that the flowers are fragrant*

This desirable characteristic is evident in H. 'Honey Bells', a hybrid derived from it.

The first two species to be introduced to Europe were the two Chinese species, H. plantaginea and H. ventricosa Stearn. These were introduced to gardens before 1795* No more were cultivated ‘until von Siebold, a horticulturist of Ghent, Belgium, began introducing plants from Japan in about 1829* By 1863* there were

17 names listed in his catalogue. The uncertain status of these clones has been a problem beset by nomenclatural confusion since their introduction to Europe. Von Siebold's names are nomina nuda, and have been one source of nomenclatural confusion. Another source of the confusion that has complicated our understanding of the genus is the broad or narrow species concepts of various authors.

Perhaps the broadest was that of Miquel (1 8 6 9), who included in

H. ventricosa (as Funkia ovata Spreng.) the taxa Hylander (1951*) recognized as H. lancifolia Engl., H. albomarginata Hook., H. albomarginata var. alba, H. undulata (O.&D.) Bailey, and H. longissima Honda. Hylander has perhaps employed the narrowest species concept of all authors to deal with the genus to date. He agreed with Stearn (1931b), who called the funkias a "taxonomist's nightmare".

A third source of confusion is the plants themselves. There are few really good morphological characters that separate species, or at least the clones (including species and hybrids) in culti­ vation in the western hemisphere. All funkias have funnel-shaped , declinate anthers, racemose , and petiolate basal leaves. They differ in size, shape of the blade, number of

nerves per , the degree of invagination of the petioles, and a

few other characteristics such as the type of and color and

texture of leaves. These characteristics often vary considerably

within a single species. Hosta undulata, for instance, bears leaves 3 on the axis of the in gardens, but this character­ istic is absent in greenhouse-grown individuals. The number of nerves per leaf has been used as a diagnostic characteristic, but

I have observed that the number is dependent on the position of the leaf within the basal .

A fourth cause for the confusion in the genus may well lie with Japanese horticulturists. Maekawa (19*+0) states that the funkias have been garden subjects since "early Toku-Gawa times", the early 17th century. Undoubtedly, there have been hybridi­ zation and selection, although probably not to the extent that has been practiced in the group known as Chrysanthemum morifolium Bemat., the florists' mums. The morphological variation in chrysanthemums is considerable and quite probably the group has been derived from more than one species.

Similarly, many of the funkias of our gardens may be hybrids.

Von Siebold and his collectors may have sent to Europe plants col­ lected in the field in Japan, but it seems more probable that they were garden subjects. In fact, one of these, H. undulata, is un­ known in nature in Japan (Maekawa, 19^0).

To date, authors of taxonomic papers concerning the genus have dealt primarily with nomenclature and gross morphology.

Although several cytologists have presented chromosomal data, their main concern has been with the cytology and the relationship of the genus to other genera at the family level. I have elected to study the problem of the funkias using two approaches; namely, their cytology and an analysis of their methanolic leaf-extracts by two- dimensional paper chromatography. HISTORY OF THE GENUS

Familial relationship

Whittaker (193*0» and Suto (1936), and particularly Sato

(19**2) and Sharma and Chatterji (1958) have compared karyotypes of funkias with those of other monocotyledonous angiosperms. These investigators have suggested that Hosta may not belong in the

Hemerocallideae of the , although they proposed no tribal or familial changes. Traub (1953) transferred the funkias from the

Hemerocallideae to the Hosteae, a new tribe in the Agavaceae. How­ ever, his description of the new tribe is invalidly published, as there is no latin diagnosis. The name of the Hosteae is validly published in Hylander (195*0» where it is included in the Liliaceae.

Sharma and Chatterji (1958), Suto (1936), and Moran (19**9) have suggested that the funkias may belong in the Agavaceae.

Granik (19*t*0 has proposed that the Hosteae are offshoots of an

evolutionary "rhizomatous liliaceous stock", and are immediately below the Yucca-Agave group of genera in the Liliaceae, in a direct

line of descent. However, most authors, including Hutchinson (1959)1

retain Hosta in the Hemerocallideae of the Liliaceae. Cave (19*t8)

has proposed, contrary to Granik, that Hesperocallis A. Gray, of

the western United States, is the nearest relative of the funkias.

k 5

Genus name

Trattinick (1812) was the first to propose the generic name,

Hosta, for the funkias, not realizing that there was already such a name validly published in the . Hosta Jacq. is a synonym of Cornutia L. (Moldenke, 1956), in the Verbenaceae. Hosta Tratt. has been conserved in accordance with Article 24, IRBN (Briquet,

1955)* Funkia Sprengel (1817) has been a source of confusion in the nomenclature of the genus, since few authors used the earlier name.

According to Hylander (1954), much of the confusion in the genus would have been avoided had the name Hosta Tratt. not been conserved over Funkia Sprengel.

Salisbury (1807) suggested calling the funkias Saussurea, but it is a nomen nudum, a "provisional name". Later, he proposed

Niobe and Bryocles for three members of the genus, but these are also nomina nuda, and thus illegitimate.

The funkias are a distinct, natural group whose correct genus name is Hosta Trattinick. The problems lie within the group, not with its delimitation. Only one species, H. plantaginea, differs enough from the others to be possibly considered in a separate genus. It would be of doubtful uBe to recognize two genera*

Species names

The nomenclatural problems within the genus began with the

first species to be given a binary name. Before Trattinick separated

the funkias from Hemerocallis L., Thnnberg (1 7 8 0) described Aletris

japonica from a plate in Kaempfer's "Amoenitatum Exoticarum . •

The precise date of publication was not known to Hylander (1954). Four years later, Thunberg realized that the species was not an

Aletris, and transferred A. .japonica to Hemerocallis. In 17911

Banks published an engraving of a drawing in Kaempfer's work, under the name Hemerocallis .japonica, apparently using Thunberg's name, although he did not cite the author or describe the plant. Three years later, Thunberg used the name for Bank's plant, giving at the same time a new description of the species and a new name, Hemero­ callis lancifolia, a procedure illegitimate under the present

International Code of . The confusion arises in part because there are two drawings of different funkias among

Kaempfer's collection, Bank's illustration being one, the other being the one that agrees with pressed specimens in Thunberg's . Hylander (195*0 feels that Thunberg's species concept was broad, and that he included his collections and both of

Kaempfer's drawings as representing one species. The problem is the identity of Hemerocallis .japonica.

According to Hylander, this is not the only difficulty with the epithet japonica. Houttuyn (1 7 8 0) also described an Aletris

.japonica which is a Hosta. The exact date of publication is July 5»

I78O (Stafleu, 1967), not mentioned by Hylander. The species was interpreted by Ker-Gawler Cl8l2) and Miquel (1 8 6 9) as what is now known as Hosta plantaginea. The type illustration of Houttuyn's plant, reproduced in Hylander (195*0* is unlike any funkia I have seen. There is no indication of the declination of all funkia , and the within the inflorescence are far larger than

those of any Hosta that I have seen. Even so, the flowers are shaded, and it is difficult to see how Ker-Gawler and Miquel could have mistaken it for Hosta plantaginea, which always has white flowers*

Hylander suggested that it is a form of H. lancifolia because of a collection from a Swedish garden of an unusual form of that species having expanded bracts subtending the flowers. He appears to be correct, because the photograph he presents of the form agrees with H. lancifolia in other respects.

When the genus Hosta was described by Trattinick, he used the names Hemerocallis japonica Houtt. ( = possibly H. japonica Thunb ex Houtt.), H. lancifolia Thunb. (nom. illegit. for Hosta lancifolia

Engl. + (?) H. sieboldiana (Hook.) Engl.), and H. coerulea Andrews

(= Hosta ventricosa Steam) as the segregates from Hemerocallis L. of the new genus. Trattinick's Illustration of the species he called Hosta japonica Houtt. is undoubtedly of the species known today as Hosta plantaginea (Lam.) Ascherson. He may have chosen to

follow Ker-Gawler, the expert on the Liliaceae at that time, or he may have been unaware of Hemerocallis plantaginea Lamarck (1789)*

Because of this long, involved tangle, Hylander suggested that the

epithet japonica be rejected as a nomen ambiguum, according to

Article 6 9, ICBN (Lanjouw, 1 9 6 1).

The preceding summary, taken mostly from Hylander (195*0,

is not the only case of nomenclatural confusion in Hosta, but is perhaps the worst. All revisions to date suffer from differences in species concepts, as well as in taxonomic judgment. Some of the

earlier revisions are confusing today because of a lack of system­

atized procedures in the earlier taxonomy. 8

There have been several nomenclatural treatments of the genus. In Europe, the first was by Miquel (1 8 6 9), followed by

-Baker (1 8 7 0), Regel (1 8 7 6), Silva Tarouca (1910), and Wehrhan

(193^)• Mottet (1897) and de Noter (1905)* in French horticultural magazines, published short summaries of garden clones and species.

In the United States, Nash (1911) interpreted the genus under the

American Code, placing all the funkias in the genus Niobe Salisbury, and Lee (1937) summarized the garden species and clones^ grown in

America. Three important articles concerning the nomenclature of a few species appeared in the early thirties (Bailey, 1930 and 1932;

Steam, 1931b).

The first world monograph is that of Maekawa (19^0). There are many errors in this work that appear to be typographical, and others that appear to result from unfamiliarity with the English and Latin languages. There are other mistakes that are not so simple to explain. In one place (p. klk, the description of section

Eubryocles) he states that the bracts are green ("bractae . . . virides"). On page ^16, in the description of the sole species of the section, H. ventricosa, he states that the bracts are "rubro- suffusi" (purplish-suffused) at the base. In a third place, in the key to sections.and subsections, page 330, he states that the bracts of section Eubryocles are whitish, "h.-actae albidae". With in­ consistencies such as this, it is difficult to give credence to any part of the monograph. Because of these variations, I have elected to follow the nomenclature of other authors where possible. MORPHOLOGY OF THE TAXA CONSIDERED IN THIS STUDY

Hosta Trattiniek

Archlv der Gewachskunde I 2: 55* l8l2*

Perennial herbs* Rhizome short or rarely horizontally elongated^ often with fibrous remains surrounding the crown, fibrous* Leaves petiolate, spiralled, convolute, nearly all basal; petioles usually invaginated and furrowed on the adaxial surface; blades expanded, rarely very narrow, parallel nerved, entire, sometimes undulate, rarely erose-crispulate. Scape terminal, rarely with sterile foliose bracts; the inflorescence a usually extended above the foliage, simple* Flowers ­ late, each subtended by a , rarely two, horizontal, rarely pendulous; parts fused for 2/3 the length of the segments, funnel-shaped, the tube of two parts, narrow and dilated, segments imbricate in two series, herbaceous to thick in texture, white to purple* Stamens six, free (inserted on the perianth in one species), declined, the apexeB ascending. Anthers small, oblong, versatile, bilocular, the parallel, ictrorse* of three fused carpels, superior, the style as long as the fila­ ments, the stigma obscurely three-lobed, papillate. Fruit a cylindrical, pendulous to sub-pendulous, loculicidal *

Seeds many per , thin, black, papery, winged*

9 10

The genus Hosta as represented In the United States Is a homogeneous group. There are few good morphological characters that separate species. The differences are subtle; they tend to be of degree. All species have basalt petiolate, prominently nerved leaves with expanded blades. They differ among themselves in the number of nerves, width of blades, and color. Continuous gradation exists from taxon to taxon in certain characteristics. Hosta plantaginea is one of the few species well marked by "key" characters. It is the sole species of the genus that has fragrant flowers, two bracts immediately subtending the lowest on the raceme, and filaments inserted on the perianth.

Since all the older taxonomic treatments of the genus have been based on comparative morphology of pressed specimens (Stearn,

Bailey, and Maekawa excepted) and several characters are destroyed by pressing, I have elected to restrict this study to live plants only.

Table 1 contains the list of taxa included in this study,

their sources and their names as they were received. I have

elected to follow the nomenclature of Hylander (195*0 where ap­ plicable, with the exception that I agree with Ingram (196?) that

H. albomarginata (Hook.) Hyl. should be named H. sieboldii (Paxt.)

J. Ingram. A full review of the nomenclature of clones cultivated

in Europe and America will be found in these papers. TABLE 1. Taxa included in this study, with their names as received, sources, and names as identified

Received as Source Identified as

1. Funkia subcordata Wayside Gardens, Mentor, Ohio Hosta plantaginea grand! 2. Hosta lanceolata Sky-Cleft Gardens, Barre, Vt. H. lancifolia 3. H. minor alba U. S. National Arboretum H. albomarginata alba 4. H. lancifolia U. S. National Arboretum H. seiboldii albomarginata 5. Funkia variegata Wayside Gardens, Mentor, Ohio H. undulata undulata 6. Hosta coerulea G. W. Blaydes, The Ohio State H. u. univittata University 7. H. erromena U. S. National Arboretum H. u. erromena 8. H. 'Yellow-edged* Fairmount Gardens, Lowell , Mass. H. sieboldiana, sieboldiana variegated form 9. H. fortune! albopicta U. S. National Arboretum H. fortune! 'Viridis' viridis 10. H. ventricosa U. S. National Arboretum H. ventricosa, aureomaculata variegated form 11. H. ventricosa U. S. National Arboretum H. ventricosa 12. H. aurea U. S. National Arboretum H. ventricosa 13. H. lancifolia U. S. National Arboretum H. tardiflora tardiflora 14. H. albomarginata U. S. National Arboretum H. crispula 15. H. 'Thomas Hogg' Sky-Cleft Gardens, Barre, Vt. H. decorata f. decorata 16. H . --- Self of No. 15 H. decorata f. normalis 17. H. albopicta aurea U. S. National Arboretum £• sieboldiana hybrid 12

The descriptions of the 17 taxa Included In this study are as follows:

1. Hosta plantaginea (Lam.) Asch.

Bot. Ztg. 21: 63. 1863.

LEAVES: light yellowish green, glabrous; blades glossy, ovate-cordate, short acuminate, with 7-9 pairs of lateral veins; petioles narrow, with a deep furrow, invagination thin, narrow.

AXIS OF BACEME: extended shortly above the foliage, stiff, nearly erect, with one to several leaves gradually passing to bracts; the flowers crowded at the summit. FLOWEBS: each subtended by a large, nearly shovel-shaped bract, the lowermost flower often subtended by two bracts, the smaller hidden within the larger; perianth always white, trumpet-shaped; stamens barely exserted, barely declined, the filaments adnate to the tube to their length; anthers yellow with yellow pollen.

2. Hosta lancifolia Engl.

in Engler & Prantl. Die NatUrlichen Pflanzenfamilien II

5: 40. 1 8 8 8 .

LEAVES: medium dark green above, lighter below, glabrous, glossy; blades lanceolate-ovate, with 3-^ pairs of lateral veins; petioles with slight invagination, reddish-spotted towards the base. AXIS OF BACEME: extended to twice the height of the foliage,

stiff, sub-erect, basally red-spotted, few flowered. FLOWEBS:

each subtended by a solitary, purple-suffused, boat-shaped bract; perianth lavender, the inner segments pale, broadly darker-edged, 13 the outer pale, evenly colored; hyaline lines present from base of sinus to apex of narrow part of tube; stamens barely exserted; anthers lavender-gray, pollen yellow*

3* Hosta albomarginata (Hook.) Hyl* var* alba (Irving) Hyl*

Acta Hort* Berg, 16: ^10* 195^ •

LEAVES: medium green, slightly glossy above, glossy below, glabrous; blades ovate-lanceolate, acute, with 3-** pairs of lateral veins; petioles shallowly furrowed, with moderate invagination, unspotted* AXIS OF RACEME: extended to twice the height of the foliage, stiffly erect, moderately thin, flowers not crowded*

FLOWERS: each subtended by a small concave green bract that withers soon after anthesiB; stamens shortly exserted; anthers yellow with yellow pollen.

if* Hosta sieboldii (Paxt.) J. Ingram

Baileya 15s 29* 1967*

LEAVES: medium green with narrow white margin, dull on both surfaces, glabrous; blades elliptic-lanceolate, short-acute, with 6-8 pairs of lateral veins; petioles narrow, distinctly in- vaginated, unspotted. AXIS OF RACEME: extended well above the foliage, erect, thin, flowers widely spaced* FLOWERS: each sub­ tended by a concave blunt small green bract that withers soon after anthesis; perianth lavender, streaked darker, evenly funnel-shaped, the segments strongly recurved, narrow, acute; stamens shortly exserted; anthers yellow with yellow pollen* 5* Hosta undulata (0. & D.) Bailey

Gentes Herb* Is 33• 1923*

LEAVES: grass-green, irregularly and imperfectly streaked centrally with white and yellow, becoming incompletely green in light as season advances, dull above, shining below, glabrous; blades undulate, acuminate, base decurrant; petiole broad with more than moderate invagination, basally reddish-spotted* AXIS OF

RACEME: extended to 3 times the height of the foliage, lax, green, red-spotted basally, terete but - angled towards the summit, 1-3 folioBe bracts on lower portion, the flowers widely spaced.

FLOWERS: each subtended by a membranous green bract that withers by anthesis, pale lavender, basally whitish inside, the segments recurved; stamens exserted; anthers lavender-gray with yellow pollen*

6* Hosta undulata (0* & D.) Bailey var. univittata (Miq.)

Hyl.

Acta Hort. Berg* 16: 399* 195^ •

Differs from the typical variety in that the leaves are darker green, a little larger and less undulate, and the varie­ gation is limited to a better defined area about 1/3 the linear area of the leaf*

7* Hosta undulata (0. & D.) Bailey var. erromena (Stearn)

Maekawa

J* Jap* Bot. 3: 506• 1936*

Differs from the typical variety only in that the plants are entirely green and a little larger* 15

Hosta sieboldiana (Hook.) Engl*— clone with yellow- margined leaves.

Hosta sieboldiana. sensu strictu, was described in Engler and Prantl. Die Naturlichen Pflanzenfamilien II 5s ^0. Hosta sieboldiana (Hook.) Engl. var. aureo-marginata Makino (J. Jap.

Bot. 5* 22. 1928) has been described as having "leaves green and deeply marginated yellow," but the plant described here does not fit Engler's description. Engler's plant has bluish-green foliage with pale lavender flowers that have narrow, thin perianth segments.

They are held at or just above the level of the foliage at anthesis.

LEAVES: bluish-green, glaucous, with heavy pruina on upper surface, less below, widely margined yellow; blades cordate, acuminate, with 9-11 pairs of lateral veins; petioles deeply furrowed with slight invaginations. AXIS OF RACEME: extended to

1 1/3 the height of the foliage at anthesis, stiffly erect, the

flowers condensed. FLOWERS: each subtended by a boat-shaped bluish-green bract; perianth white outside, the inside of each segment flushed linearly pale lavender; hyaline lines from the base

of the sinus to almost 1/2 the length of expanded part of tube,

constricted part obscurely 6-angled; stamens exserted; anthers lavender-gray with yellow pollen.

9. Hosta fortune! (Bak.) Bailey var. albopicta (Miq.) Hyl.

f. viridis Hyl.

Acta Hort. Berg. 16: 391. 195^« 16

LEAVES: bluish-green, glaucous below, slightly above; blades cordate to cordate-ovate, acuminate, slightly undulate, with

7-9 pairs of lateral veins; petioles deeply furrowed with distinct invaginations. AXIS OF RACEME: extended well above the foliage, stout, the flowers close together but equally spaced. FLOWERS: each subtended by a concave obtuse bract that withers soon after anthesis; perianth lavender, segments wide-spreading and recurved; stamens shortly exserted; anthers lavender-gray with yellow pollen.

10. Hosta ventricosa Stearn (variegated form)

Gard. Chron. Ser. Ill 90: 27. 1931*

Differs from the typical form (numbers 11 and 12) only in that the margins of the leaves are darker green than the central portions. This character disappears as the season progresses.

There is a description in the literature of a variegated form

(Funkia latifolia lusus aureo-maculata Miquel) of this species in which the leaves are smaller than typical and streaked with pale whitish-yellow, but the form I have has no yellow variegation.

11 and 12. Hosta ventricosa Steam

Gard. Chron. Ser. III. 90: 27. 1931*

Two clones, morphologically the same.

LEAVES: dark green, slightly glossy above, strongly below, glabrous; blades short-acuminate, sub-cordate to sub-orbicular, with 6-9 pairs of lateral veins; petioles broad, short, narrowly invaginated, the furrow broad and open. AXIS OF RACEME: extended well above the foliage, the flowers evenly spaced. FLOWERS: each 17 subtended by a broad obtuse thin green bract that withers by anthesis; perianth dark violet with darker lines, distinctive, the tube widening abruptly above the narrow part, the limb erect, not at all recurved; stamens included; anthers dark violet with yellow pollen*

13* Hosta tardiflora (Irving) Stearn

ap. Grey, Hardy Ills 303• 1938*

LEAVES: very dark green, sub-coriaceous, glossy; blades lanceolate, long acuminate, with *f-5 pairs of partially obscured veins; petiole not invaginated although slightly flattened on the adaxial side, spotted and suffused purplish-brown. AXIS OF RACEME: extended slightly above the leaves, stiffly erect, purplish-brown- spotted, the flowers usually crowded. FLOWERS: each subtended by a membranous boat-shaped, whitish, purple-suffused bract; perianth pale lavender, narrowly funnel-shaped, the segments widely expanded and slightly recurved; stamens exserted; anthers yellowish with yellow pollen.

l*f. Hosta crispula Maekawa

J. Fac. Sci. Univ. Tokyo, Sect. 3» Bot. 55 J>Sk, 19^0.

LEAVES: gras6-green with narrow - white margin, dull above, glossy below, glabrous; blades sub-cordate to ovate-lanceolate, long acuminate and characteristically twisted, base sub-cordate to slightly decurrent; petioles of outer leaves broadly flattened and broad-margined, of inner, distinctly channeled with narrow wings*

AXIS OF RACEME: extended to 3 times the height of the foliage, lax 18 and curving upward towards the apex, the flowers evenly spaced.

FLOWERS: each subtended by a small short spreading pale yellow- green bract; perianth pale lavender, small, broadly funnel-shaped, the outer segments - glossy; stamens exserted; anthers gray- lavender with orange-yellow pollen.

15* Hosta decorata Bailey

Qentes Herb. 2: l*fl. 1930.

LEAVES: medium green with white margins, dull above, glossy below; blades ovate with obtuse apex, decurrent bases; petioles shallowly, broadly furrowed with rather wide invaginations. AXIS OF

RACEME: extended over twice the height of the foliage, stiffly erect, stout, the flowers evenly spaced. FLOWERS: each subtended by a dark -green, shovel-like, erect bract; perianth pale lavender, heavily lined darker, the narrow part of tube expanding rather abruptly into a sub-campanulate limb, the segments slightly recurved; stamens included; anthers light yellow with light yellow pollen.

16. Hosta decorata Bailey, selfed

The same as the typical form, with the exception of the

entirely green leaves.

17* Hosta sieboldiana (Hook.) Engl.

The description of this clone does not agree with the

description of H. sieboldiana. but is closer to that of this species

than any other. LEAVES: medium green, dull, upper surface with no pruina, lower, with slight pruina; blades broadly lanceolate, shortly acuminate; petioles furrowed with moderate invagination. AXIS OF

RACEME: extended to 1 1/3 the height of the foliage at anthesis, moderately stiff, the flowers condensed. FLOWERS: each subtended by a boat-shaped erect purple-suffused bract that is persistent after anthesis; perianth funnel-shaped, lavender, the inside

streaked darker; hyaline lines present from base of sinus to top

of terete narrow part of tube, segments narrow, rather thick;

stamens slightly exserted; anthers lavender-gray with yellow pollen. CHROMATOGRAPHY

The literature

Various constituents and products of plants have been the concern of man for longer than recorded history. Plants have been used as sources of food, clothing, and shelter for an unknown period

of time. In the course of a never-ending search for economic products and the cures of man's various ills, social as well as

constitutional, numerous more or less important uses for the various by-products of plants have been found. Indeed, the Doctrine of

Signatures attributed to each plant species a capacity that was for

the use of man and the solving of his many problems. There have

been enough elements of truth in many folk medicine uses and tales

about values of plants that pharmacologists have consistently

followed such leads in the search for new drugs.

Extraction of these chemical constituents has been by various

methods, but when compounds are present in minute amounts, the

expense and labor of the conventional methods of extraction, as well

as the danger of the loss of the important fraction, has been great

enough to prevent the isolation of many biologically important

compounds. Fowden (1962) presents a graph indicating that the

number of new non-protein amino acids isolated from plants between

1912 and 19Mt was less than 10. However, between 19^^ and I960,

over 60 more were identified.

20 21

The Impetus was largely due to paper partition chroma­ tography, first put to practical use by Cons den et al. in 1944.

This simple method has been applied to many fields, wherever separation of a small amount of a mixture into its components is important*

One of the first applications of the method to taxonomy was by Buzzati Traverso and Rechnitzer (1953)* who compared chromatographic patterns of fish muscle protein hydrolysates from different species* In regard to plant systematica, the most im­ portant early paper is by Alston and Turner (1959), who studied 1% hydrochloric acid in ethanol extracts of Baptisia species and hybrid swarms, and predicted that the technique might be of vital

importance to the field of plant systematica* This paper was the

first of a series, now classical, on Baptisia» In this genus, the

technique has been most useful— to the extent of enabling the

discovery of morphologically indistinguishable chemical races

(Brehm and Alston, 1964). Similarly, Tragopogon (Brehm and Ownbey,

1964, 1 9 6 5), Lotus (Harney and Grant, 1964), and Zinnia (Torres and

Levin, 1964), among others, have been analysed by chromatography*

One of the latest groups to which the technique has been applied is

Dicentra (Fahselt and Ownbey, 1968)* These workers were able to

confirm relationships within the genus determined by more conven­

tional methods* However, they found "hybrid substances", compounds

missing in either parent but present in the hybrid. They also found

substances that were present in the parents but absent in the hybrids* 22

Occasionally, the technique has been less successful* Smith and Abashian (196?) found that artificial hybrids of Nicotiana species usually did not have all the alkaloids found in both parents, even though species-specific patterns were present in the parents* Also, McClure and Alston (1 9 6 6) in their thorough and generally successful analysis of the Lemnaceae, found that oc­

casionally there were spots on chromatograms from different species that reacted the same to the reagents used and were in the same place on the chromatograms, but were not the same compounds.

Chromatography can yield significant data for systematica

just as the study of morphology, distribution, and cytology can*

However, it should not be the only criterion for the separation or union of taxa. Chromatographic data should be integrated with other

data*

There have been no data reported that have been derived from

chromatography in connection with the systemstics of the funkias*

However, there are reports concerning the of members

of the genus. Villar Palasi (in Hegnauer, 1 9 6 3) found sapogenins

in seeds of H. ventricosa* Takeda, Okanishi, and Shimaoka (1993)

found that gitogenin was the main sapoganin in H. plantaginea* H.

sieboldiana (Hook*) Engl*, and H* longipes Matsumura* Takeda et al*

(196*0 identified seven sapogenins in H. montana var. liliiflora

Maekawa* Pi&tte and Parvanch&re (I960) studied the serology of 32

members of the Liliaceae, among which were "Funkia coerulea Sweet

x Sieboldiana Hook., F. fortunei Baker var. robusta [and] F. ovata

Spreng. aureo-marginata11» The horse sera derived from these caused 23 instant hemolysis of both rabbit and human blood. Fowden and

Steward (1957) were concerned with alcohol-soluble nitrogen com­ pounds of members of the Liliaceae. They stated that a certain amino acid, pipecolic acid, could be present in seeds but not in leaves of Hosta species. They worked with leaves of H. lancifolia and seeds of H. sieboldiana. Perhaps it would have been better to work with leaves and seeds of the same clone.

In the present study, 17 clones of the genus Hosta were analyzed for the chromatographic pattern of the acidulated methanol- extractable materials, principally flavenoids and phenolic acids.

Materials, methods, and results

Leaves were collected from mature plants of each of the taxa analyzed. Since the plants were grown in the greenhouse and were in two groups as to the cold period, the actual time of collecting varied considerably. However, all leaves were collected when the plants had ceased forming young leaves. There were about 3 to 13 leaves, depending on the size of the plant, chosen for analysis from the central portion of the rosettes. They were placed in either paper bags or a plant press and allowed to dry at room tem­ perature. When completely dry, the leaves were reduced to a powder with a mortar and pestle.

The extract was derived by adding a boiling solution of

10 ml methyl alcohol with two drops of concentrated hydrochloric acid to a vial containing powdered leaves to a depth of twice the powder. The vial was then tightly closed and allowed to remain at 24- room temperature in the dark for 24- hours. The extract was then carefully decanted and used within three or four hours. Chromato­ grams derived from extracts that had been decanted over four hours previous to use did not yield comparable results.

Whatman no. 3 MM chromatography paper was used for two- dimensional chromatograms. Paper was cut in such a way that the solvent front moved 13 inches in each direction, and the solvent plus extract front moved 12 inches in each direction. One hundred lambdas of the extract were spotted in the lower right hand corner with 5 lambda disposable micro-pipettes (Drummond Scientific Co.,

Broomall, Pa.). The size of the spot was not allowed to exceed a diameter of 7 to 8 millimeters. Drying time was reduced by using cool air from a portable hair dryer. The chromatograms were rolled into a cylinder when the spot was dry, stapled with stainless steel staples at the top and bottom, edges abutting. The solvent was added to the jars and then the chromatograms were carefully in­ serted. The jars were covered with Saran Wrap securely sealed with rubber bands.

Several solvent systems were investigated. The most satisfactory was tertiary butyl alcohol:glacial acetic acid:water

(1:1:1, v/v) for the first direction, and glacial acetic acid:water

(3s7, v/v) for the second. All chromatograms for this study were

run in these systems.

The reagents for the solvent systems were always added in

the same order and thoroughly mixed immediately before use. Two hundred ml of solvent per jar were used. Each jar was lined with 25 filter paper to increase the saturation of the atmosphere within the jar and lessen the running time*

All chromatograms were run at room temperature, which varied between 2**-° and 29°C* The approximate running time in the first solvent system was 19 to 21 hours; in the second, k to k 1/2 hours*

Two to three hours' drying time with the aid of an exhaust fan was allowed between directions. After the second run, at least one hour was allowed for drying before the chromatograms were developed*

The chromatograms were developed under the following con­ ditions: in visible and ultra-violet light (short wave, Mineralight,

Ultra-Violet Products, Inc., San Gabriel, Calif.); NH^OH, in visible and ultra-violet light; and 2% phospho-molybdic acid in 50# acetone

(C. E. Sweeney, 1966), in visible and ultra-violet light. All spots were marked on the chromatogram with pencil and their re­ actions recorded* The phenol indicator and the ammonia were

sprayed with an aerosol sprayer (Sprayon Products, Inc., Cleveland,

Ohio; Nutritional Biochemicals Corp., Cleveland, Ohio). The chro­ matograms were dried and kept as vouchers* sjs- No attempts were made to elute material from any of the

spots, or to identify specific compounds present. Some of the

spots gave a characteristic reaction to phospho-molybdic acid and

could be identified generally as phenolic compounds*

There were three replicates of each chromatogram run at

the same time with the same extract. After the three replicate

chromatograms of each plant extract were developed and the spots

recorded, the replicates were compared* Spots that were common to 26 all three were designated by letters— A, B, C, and so forth* If a spot on the chromatograms of a second taxon occurred at the same place with the same reactions as did spot A, it was labeled A* A summary of the observed spots and their reactions is presented in

Table 2*

When chromatograms derived from extracts of all clones included in this study had been observed in this manner, a chro­ matographic profile was constructed* For instance, no* 1 had spots

A through I, lacked spots J and K, had spot L, and so forth.

Seventeen representatives of Hosta were selected for chromatography, and profiles for each are included in Table 3* (la all Plates,

Tables, and Photographs, various taxa may be referred to by number*

These may be identified by reference to Table 1.)

Paired Affinity Values were calculated between pairs of

chromatographic profiles in all possible combinations by a method

similar to that proposed by Ellison, Alston, and Turner (1962).

These authors present the following formula for Paired Affinity

Values: „ . „ Spots in common for species A & B , p - A< V - * ----Total spots in A * B------* 100

However, I have used for this B t u d y the following formula:

p A V - No* of sP°ts 3-n common for taxa A 8c B ___ “ No. of possible spots in common for taxa A & B x

Thus when no* 1 is compared with no* 2, the paired affinity value

is 7/18 x 100, or 39* When a chromatographic profile is compared

with itself, or when chromatographic profiles are identical, the

Paired Affinity Value is 100. If the two profiles have no spots in TABLE 2* Chromatographic spots observed in this study, with their reactions to visible and ultra-violet light in absence of reagents and with two reagents

Phospho-molybdic Acid Reagent: None NH^OH Then NH^OH

Light: Visible UV Visible UV Visible UV

Spot A Pale green Red Pale green Red Pale green Red B - Purple - Pale purple - Purple C - Pale bright - Bright blue - Bright blue blue D - Dirty Yellow Dirty Yellow Dirty E Tan * Dirty Tan to Dirty Pale Dirty orange orange-tan F - Pale blue - Purple - Pale purple G - Dirty Pale yellow Dirty Pale yellow Pale, pale yellow H - Pale blue - Pale blue - Bright pale blue I Nothing until after last ammonia spray dries, then grey-yellow J - Pale - K - Quench - - - _ L - Blue Yellow Greenish Yellow Greenish M - Pale dirty Yellow Dirty Yellow Dirty grey when dry N Nothing until after last ammonia spray dried, then yellowish 0 - Pale - - — _ TABLE 2 (continued)

Phospho-molybdic Acid Reagent: None NH^OH Then NH^OH

Light: Visible UV Visible UV Visible UV

Spot p - Pale -- mm

Q Pale - mm - R Pale tan Dirty Pale tan Dirty Grey-larger s Pale tan Dirty Pale tan Dirty Pale tan

T - Blue - Pale yellow mm Pale yellow U Pale tan Dirty yellow? Pale tan Dirty Pale tan Dirty V - Pale - Dirty - Dirty yellowish W - Pale - Pale - X - - - Quench - Dirty

y - - mm Pale - Pale z - Bright blue Pale yellow Yellow Pale yellow AA - Bright blue Pale yellow Pale blue Pale yellow BB - - - Yellow Pale yellow CC - - - Yellow - DD - - - Pale blue - Pale blue EE - - - Pale, pale - Pale, pale blue blue FF Pale tan Dirty Pale tan Dirty Pale tan Dirty GG - Quench Yellow Quench Yellow Dirty yellow TABLE 2 (continued)

Phospho-molybdic Acid Reagent: None NH^OH Then NH^OH

Light: Visible UV Visible UV Visible UV

Snot HH Pale, pale Pale ** Pale dirty Pale yellow tan II - Pale blue - Pale - Pale JJ Pale tan Dirty yellow Tan Yellow Pale tan Dirty yellow KK Pale tan Dirty yellow Tan Brownish Pale tan Dirty brown yellow LL - - - Pale - Pale MM Light yellow — Light purple - Light - purple NN Pale purple - Pale blue - Pale blue - 00 Nothing until after last ammonia spray dried, then clean grey at the same place as I PP - Pale - Pale, pale - Pale blue QQ Pale olive Dark brown- Olive Dark Olive Dark brownish yellow brownish yellow yellow

ro vo 50

TABLE 3* List of chromatographic spots and taxa in which they were observed* The absence of a spot is indicated by a period* (See Table 1 for key to names of taxa)

Taxon: 1 2 5 4 5 6 7 8 9 10 11 12 13 14 15 16 17

Spot: A + + + + + + + + + + + + + + + B + • ••••• 4 4 • 4 • • • • • • C + • + + + + + + + + + + + + ffiJa D + • • • ••• •• • • •• • + to • E ♦ + + + + + + + + ■f + + + F + G + + + • + • 4 4 4 4 •• •• + 4 H + ••• + • 4 4 + + 4 + 4 + + I + 4 + • • 4 4 + + 4 4 4 4 • • 4

J • + • •• • 4 4 + 4 4 4 4 4 + • 4 K « « + + + to to 4 + + + 4

L + + + + • • 4 4 4 + + 4 + • 4 M • + N * + • + + • 4 t + + + 4 + 4 + + 4 0 + •• + • 4 4 4 4 4 + 4 + 4 4 P • + Q ■f + + + + 4 4 + 4 4 4 4 + + 4 R S • • a + •• 4 4 4 + + + + 4 + + 4 T ••• + • + 4 + + + + + + U « •• + •• + 4 4 + + + + + 4

V • •*• • • 4 4 4 + + 4 4 4 4 + 4

W • • ••• a 4 4 4 + 4 + 4 4 4 4 4 X + • + + 4 + • + + y * 4 • •• • 4 4 4 + + + 4 4 4 4 4 z «•••• • + 4 4 4 4 + • 4 4 4 4 AA • • •• • • 4 4 4 4 + 4 • 4 + 4

BB « • a • 4 4 4 4 4 + 4 4 4 4

CC • •••• + 4 4 4 4 4 4 4 4 4 4 DD + •• • • + 4 4 4 4 4 4 to 4 + 4 EE • • • •• 4 4 4 4 4 4 4 • 4 + 4 FF • •• • • 4 + 4 4 4 + + +

GG • •• • •• 4 4 4 4 4 4 + 4 4 4 4

HH • • •• •• 4 4 4 4 4 + 4 II • ♦ • • • m 4 4 4 4 4 4 4 + • 4 4 JJ •• 4 ••• 4 4 4 4 4 • 4 + 4 4 4 KK • ••• s • 4 + 4 4 4 4 + 4 4 4 LL •• • ♦ a • + + 4 4 4 4 4 4 4 •

MM • 4 • • •• 4 + 4 4 4 4 4 • 4 4 4 NN ♦ • • • • • + + 4 4 4 4 4 • 4 4 • 00 •• • •• 4 + + + 4 4 4 4 + 4 + PP ••• • • 4 4 4 + 4 4 4 4 4 4 4 4 QQ • •• • • 4 4 4 4 4 4 4 4 4 4 4 + Total each: 14 ll 8 14 12 10 14 13 16 13 11 13 17 14 16 16 12 31 common, the Paired Affinity Value is 0. Thus these values were used as a measure of similarity between any two chromatographic profiles.

The calculated Paired Affinity Values are summarized in

Plates I, II, and III, according to a method proposed by Ellison,

Alston, and Turner (1962). In each graph, the radius of the outer circle represents Paired Affinity Values from 0 at the center to

100 at the perimeter. The inner circle in each graph represents the 50% level. For instance, in Plate I, Figure 1, H. plantaginea

(no. 1) compared with itself, has a Paired Affinity Value of 100.

When it is'compared with no. 2, H. lane: folia, H. plantaginea has a

Paired Affinity Value of 39» and so forth. Plate I. Polygonal graphs of Paired Affinity Values of six Hosta taxa.

1. H. plantaginea

2. H. lancifolia

3. H. X alba

H. sieboldii

5. H. X undulata 'Undulata'

6. H. X undulata 'Univittata'

32 I. H. 2. H. plantaginea lancifolia

lO 17

16 16 12 14 3. H. 4. H. X a lb a sieboldii

lO 17

16s 12 14 14 5. H. 6. H. X undulata 'Undulata' X undulata ’Univittata' Plate II* Polygonal graphs of Paired Affinity Values of six Hosta taxa.

Fig. 7. H. X undulata 'Erromena'

8-. H. sieboldiana, variegated

9. H. fortunei 'Viridis' O H • H. ventricosa, variegated

11. H. ventricosa H C\J • H. ventricosa

3^ 7 H. 8.H. Xundulata Erromena' sieboldiana, variegated

4

lO 17

16 12 15 14 >3 14 13 9. H. 10. H. fortunei 'Viridis' ventricosa, variegated

lO

12

11. H. 12. H ventricosa ventricosa Plate III. Polygonal graphs of Paired Affinity Values of five Hosta taxa.

Fig. 13. H. tardiflora

l*f. H. crispula

15. H. decorata

1 6. H. decorata* selfed

17. H. sieboldiana, hybrid

36 37

10 17

14 13. H. 14. H. tardiflora crispula

16 12 14 15. H. 16. H. decorata, selfed decorata

siebodiana hybrid CYTOLOGY

The literature

There have been about 50 chromosome counts of funkias published by various authors, and these are summarized in Table 4.

In the under taxon, I have listed the names exactly as published* In the last column, I have placed, wherever possible, the name of the taxon that is currently accepted. References to the authors are in the Bibliography. I have used the nomenclature of Hylander (195*0 primarily, although I have followed later authors in some instances. Those taxa that are not known to be cultivated in Europe or the United States are named as they are in Maekawa

(1940).

None of the authors of the published chromosome counts state that they have kept voucher specimens of species counted, and one cannot be certain they were correct in their identifications.

Only one author (Kaneko, 1 9 6 6) mentions even consulting a tax­ onomist; Kaneko received a personal communication from F. Maekawa, confirming that hybridization occurs in the genus. Also, "Funkia aromatica11, counted by Sharma and Chatterji (1958), is not a Hosta.

Chromosome morphology and number of this taxon are entirely different from that of other published chromosomal data on Hosta species. Also, Sharma and Chatterji state that the rootstock is a . All funkias are rhizomatous. I believe the species they 38 TABLE k» Published chromosome counts of members of Hosta Tratt«

Taxon n 2n Author Currently Accepted Name

Hosta japonica 50 — Akemine, 1935 H, lancifolia Engl,? H. japonica, var. aestivalis 30 — H ? H. japonica, var. albomarginata 30 — ii H, sieboldii (Paxt.) J. Ingram H. japonica, var. angustifolia(?) 30 — ii H. longissima Honda ?? H. rectifolia 30 — H. rectifolia Nakai H. ruprifraga 30 H. ruprifraga Nakai H. sieboldiana 30 — H. sieboldiana (Hook.) Engl* H. sieboldiana, var. nigricans 30 — H. nigrescens (Makino) Maekawa? H. undulata 30 60 H. undulata (0. & D.) Bailey ? H. ventricosa 30 — H. ventricosa Stearn ? H. sp. 30 — ? H. sp. 30 60 ? H. sp. 30 — ?

H. coerulea Tratt. (Funkia 12 2k Imai & Kanna, 193^ H. ventricosa Stearn ovata Spreng.) H. coerulea, cultivated forms 36 ti H. coerulea, tetraploid 2k n H. sieboldiana Engl. 2k n H. sieboldiana (Hook.) Engl* H. japonica Voss 2k i i H. lancifolia Engl.

H. japonica Aschers. & Graeb. 30 — Matsuura & Suto, 1935 H. lancifolia Engl.?? If H. lancifolia Engl. — 60 H. lancifolia Engl. Hi sp. — 60 If

Funkia ovata Spreng. 20 kO Mookerjea, 1956 H. ventricosa Stearn Hosta subcordata Spreng. var. - - 60- " H. plantaginea (Lam.) Asch.variety? cordifolia also 58,65, 70,125 so H. atropurpurea Nakai — Ca. Sakai, 193^ H. atropurpurea Nakai k2 TABLE 4 (continued)

Taxon n 2n Author Currently Accepted Name

H. coerulea (Andrews) Tratt. 30 — Whittaker, 193*f E. ventricosa Stearn

H. foruni, var. gigantea Bailey 30 60 Yasui, 1935 H. elata Hyl. ? H. clausa Nakai 90 ti H . clausa Nakai fi. sieboldiana Bosk. 30 60 it H. sieboldiana (Hook.) Engl. H. venusta F. Maekawa 30 60 it H. venusta Naekawa H lancifolia Stern, var. 60 it H. capitata ? *long±folia Nakai I. plantaginea Asch. 30 60 i i E. plantaginea (Lam.) Asch. H. lancifolia Stern (H. japonica) 30 60 i i H. sieboldii (Paxt.) J. Ingram ?? f. albomarginata Makino & its derivatives H. undulata Bailey and its green — 60 E. undulata (0. & D.) Bailey variation and albomarginata forms E. sp. — 60 ?

Funkia aromatica — 20 Sharma & Cardiocronum Lindley ??? Chatterji, i 1958 F. lancifolia 30(35) 60 11 H. lancifolia Engl. ? (also noted 2 0,3 0,^0,5 0,5^- in tips)

Hosta montana — 60 Kaneko, 1966 H. elata Hyl. ? E. lancifolia — 60 11 H. lancifolia Engl. (3 clones that differed karyotypically) H. chibai — 60 " H. capitata — 60 " H. capitata Nakai (or H. capitata (2 clones that differed karyotypically) Nakai and H. nakaiana Maekawa ?) i TABLE 4 (continued)

Taxon n 2n Author Currently Accepted Name 1 H. sieboldiana 24 48 Strasburger, 1900 H. sieboldiana (Hook.) Engl* Miyake, 1905 Sykes, 1908 Inariyama, 1928

H* coerulea 24 48 Sykes, 1908 H. ventricosa, Stearn

H. ventricosa 24 48 Sato, 1955 (in H. ventricosa Stearn Akemine, 1935)

H. sieboldiana Ca. Lewitzky, 1931 H. sieboldiana (Hook.) Engl* 62 (in Akemine, 1935)

Funkia ovata 24 -- Sykes, 1908 H. ventricosa Stearn

Hosta ventricosa 6o Sato, 19^2 H. ventricosa Stearn H. lancifolia — 60 u H. lancifolia Engl.

H. ventricosa Stearn 60 — Kaneko, 1968 H. ventricosa Stearn (in litt*)

i 42 investigated may be a member of Cardiocrinum Lindley. This genus is native to the Himalayas, vaguely resembles a Hosta when not in bloom, and, like Hosta, was once included in Hemerocallis L.

(Stearn, 1931a)•

According to Akemine (1935)* Strasburger (1882, 1900, and

1905) appears to have been the first to publish chromosome counts

of . At that time anthers were fixed, stained, embedded in

paraffin, and sectioned prior to study. Intact nuclei at metaphase

I were chosen, and the mean number of chromosomes per nucleus was

inferred to be the haploid number (Sykes, 1908). With the advent

of smear techniques, chromosome counting has become more accurate

and possible, even in species with high numbers.

It may be that the early accounts of numbers are inaccurate,

as Akemine (1935) suggests. He states that those authors who have

counted 24 meiotic or 48 somatic configurations have miscounted two

associated bivalents as one. For instance, at mitotic metaphase,

the largest chromosome in H. lancifolia is 7.3 microns long, and

the shortest is 1.3 microns long (Kaneko, 1966). There is a great

possibility that a small one may be hidden by a large one, as well

as the possibility that two small chromosomes in juxtaposition may

be mistaken for one.

On the basis of counts of 13 species, Akemine (1935) states

that the base number within the genus is x = 30, and that polyploidy

does not exist. He discounted the report of Imai and JCanna (1934)

concerning a polyploid series in H. ventricosa (as H. coerulea

Tratt.). He was possibly unaware of a paper by Yasui (1935)t in which was reported the triploidy of H. clausa Nakai. Yasui's report 43 is the only paper dealing with meiosis in any detail: she included a camera lucida drawing of a pollen mother cell of H. clausa in metaphase I, indicating JO trivalents. She also observed univalents, bivalents, and trivalents, plus bridges, fragments, laggards, and other evidence of faulty meiosis in other microsporocytes. However, other authors (Akemine, 1933; Imai and Kanna, 1934; and Hylander,

195*0, whose reports are summarized in Table 5, mention pollen fertility as measured by pollen stainability or morphology.

Although authors of several of the papers mentioned in

Table 4 include camera lucida drawings or photomicrographs, only

Kaneko (1966) presents karyotypes. He reported on karyotypes of four species of Hosta. All had 2n = 60 chromosomes, but two, H. lancifolia and H. capitata Nakai, apparently had different chromo­ some races within each species. The former was represented by three clones, growing wild in three different places in Japan. Two were apparently typical in that cells in the root-tips had pairs of each chromosome, but they differed in that the six small of the eight large chromosomes had a longer short arm in one collection than the other. The third collection was considered a possible hybrid because two pairs of the medium chromosomes and one of the long were heteromorphic•

Similarly, H. capitata was represented by two differing

clonus. One had eight large, four medium, and 48 small medium

grading to small chromosomes. The chromosomes were in pairs. The

other clone had 8 large, 8 medium, and 44 small medium grading to

small chromosomes. One of the pairs of medium chromosomes was

heteromorphic. 44 TABLE 5 . Summary of published pollen viability data

Taxon % Stainable Pollen Author

H. japonica (H. lanceolata 96.9 Akemine, 1935 Engl. ?) H. japonica var. anguetifolia 98.? It (?) (H. longissima Honda ??) - H. rectifolia (H. H. rectifolia 98.9 If Nakai) H. ruprifraga (H. ruprifraga 94.3 fl Nakai) H. sieboldiana (H. sieboldiana 95.8 tt (Hook.) Engl.) H. sieboldiana var. nigricans 95.2 ft (H. nigrescens (Makino) Maekawa) H. undulata— all clones* 10.4 If (H. undulata (0. & D.) Bailey) H. " 26.5 11 H. " 21.0 ft H. sp. (H. sp.) 97.8 ft H. ventricosa (n = 30)* 17.9 ft (H. ventricosa Stearn) H. coerulea Tratt. (= H. ovata High Imai & Kaiina* 193** Spreng.), n = 12 (H. ventricosa Stearn) it H. » , 2n = 48 High [?] it H. " , 2n = J6 "sterile" H. sieboldiana, var. typica 95-100% Hylander, 195^* (H. sieboldiana, var. sieboldiana) H. sieboldiana, var. elegans 95-100% it H. albomarginata (H. sieboldii 95-100% n (Paxt• ) J . Ingram) H. decorata Bailey 95-100% it 3. crisp Vila (J plants— same 70- 90% it clone?) H. crispula, "spotted sport" H. elata (6 plants: J - ca. 70% it 1 — 90% tl H. fortune!, var. albopicta ca. 5% tl H. undulata types 5% (some 10%) ft H. fortune! var. stenantha 30% fl H. fortune! var. hyacinthina fl 3 clones: 1. 20% 2. 30% 11 3. 4o% tl H. fortune! var. rugosa 40-50% ft H^ lancifolia 2 0-30% »t H. ventricosa to 100% tl * The author says: "faulty meiosis present in these." **5

As in other previous cytological research, Kaneko apparently kept no voucher specimens. He gives Japanese common names for the four species. The common name he gives for H. capitata is 'Kanzashi- gibosi1. According to Maekawa (19^0), cited in Kaneko's paper, this is the Japanese common name for H. nakaiana Maekawa. It is not clearly stated in the paper, but apparently Maekawa has decided that H. capitata and H. nakaiana are conspecific. Since the epithet capitata predates nakaiana, the species becomes H. capitata. This information is as yet unpublished (Kenichiro Kaneko, in litt.). The name H. chibai, also as yet unpublished by Maekawa, is used for another taxon by Kaneko, with a karyotype and some discussion of its chromosome morphology. This last mentioned species has eight large, four medium, and *f6 medium small grading to small chromosomes,

2n = 58* according to his Figure 5» although he states that 2n = 60 in the text as well as the legend.

Kaneko should have made clear in his paper that he was using unpublished nomenclatural changes of another author in connection with his own cytological data. It is this sort of confusion that has been characteristic of much of the published material on the genus, from Houttuyn (l?80) to date.

Materials, methods, and results

All chromosome preparations were made from microsporocytes of anthers removed from fresh immediately before squashing.

The anthers were placed in a drop of stain, cut in half, and gently pressed with the flattened point of a dissecting needle to squeeze ke out the pollen mother cells. Before a cover slip was placed on the slide, as much as possible of the remains of the anther sacs was removed. After the cover slip was in place, the slide was in­ spected. If there were stages of meiosi6 visible, gentle pressure was applied to the cover slip to spread the cytoplasm. The tem­ porary mount was sealed with a mixture of equal parts of gum mastic and paraffin.

Slides were made permanent no more than a week after stain­ ing by freezing on dry ice, removing the cover slip, and passing both the slide and cover slip through 95% ethanol followed by 100% ethanol, then mounting in Diaphane (Will Scientific, Inc., Columbus,

Ohio). The old cover slip was placed on a new slide and a new cover slip was placed on the old slide to avoid overlapping of squashed material. The slides were then placed and weighted on a warming plate at k^>°C for one week.

In early June of 196**, all plants of the H. undulata clones and several others bloomed at a time that was inconvenient for making squashes. I therefore placed all buds in a modified Farmer's solution (3 parts 100% ethanol to one part glacial acetic acid, plus a few drops of chloroform) for 2k hours. The killed and fixed buds were then transferred to 70% ethanol for storage. All squashes resulting from these preserved buds were useless. The cytoplasm stained so much that the chromosomes were obscured.

Aceto- and propiono-carmine and aceto- and propiono-orcein stains were tried, with mediocre results. The best stain for Hosta material I found is lacto-propionic orcein (Dyer, 1 9 6 3)- The cytoplasm is nearly colorless, the nucleoli are very pale, and the chromosomes are darkly colored when this stain is vised with Hosta microsporocytes. However, the preparations were difficult to make permanent. The cover slip droze tightly to the slide, or, particularly on humid days, the stain became thick and sticky, not

freezing. But since results were excellent when the freezing

technique was successful, this was the staining schedule I elected

to follow. I modified Dyer's technique to the extent of adding a small amount of fast green to the first alcohol of the dehydration

series to stain the cytoplasm light green, thus increasing contrast.

Some of the early preparations were not made permanent and photographed, although camera lucida drawings were made (Figs. l8,

19, 24, 32, 3 8, 391 40). However, there are camera lucida drawings

and photographs of the same cell of the later squash preparations.

These are presented in Figures 20-23, 25-28, 30, 31* 33-36, 41-44.

The haploid chromosome numbers of taxa counted are presented

in Table 6. Percentage of stainable pollen was estimated by de­

riving the percent of pollen grains that were plump and evenly

stained after 24 hours in aniline blue in lacto-phenol. Those

grains that were unevenly stained or shrunken or not stained were

judged inviable. Over 500 pollen grains were counted for each

percentage. These data, also, are presented in Table 6.

Hosta plantaginea (Fig. 1 8 ) has been reported to have typical

meiosis, with n = 30 (Mookerjea, 1956). This study confirms

Mookerjea's report. However, in the past three years I have been

unable to repeat the observation. Although I have always found 48

TABLE 6* Haploid chromosome numbers and pollen stainablllty per­ centages determined in this study

n Stainability

1. H. plantaginea 3° — 2. H. lancifolia 30 37% 3* H. albomarginata alba 31 31% 4. H. sieboldii 31 98% 5. H. undulata undulata ca. 34+1 4#

6. Ho undulata univittata - 20% 7. H. undulata erromena - 19% 8 . H. sieboldiana, variegated 30 94% 9. H. fortunei viridis - 2496 H O • H. ventricosa, variegated - 60 90% 1 1. H. ventricosa - 60 92% 1 2. H. ventricosa - 60 84% 1 3. H. tardiflora 30 68% 14. H. crispula 30 84% 15. H. decorata 30 97% 16. H. decorata, selfed 30 94% 17* H. sieboldiana 30 28% tetrads of , the anthers from the bud immediately above the one yielding tetrads have contained only degenerated pollen mother cells* It has been long known that meiosis can be affected by environment (Stow, 1927)* The explanation may lie in the hot dry weather of many Columbus summers. Potted plants in the greenhouse have never bloomed; the buds for anther squashes came from nearby

gardens* Meiosis occurs when or shortly after the inflorescence

has appeared above ground*

On several occasions, H. lancifolia has been reported to

have n = 30 and 2n = 60 chromosomes (Matsuura and Suto, 1935; I

Fig, l8 . Camera lucida drawing of a Fig* 19* Camera lucida drawing of a microsporocyte of H. plantaginea. n = }0. microsporocyte of H. lancifolia* n = 30

•P" VO 50

Akemine, 1935; Sharma and Chatterji, 1958; Kaneko, 1966), and the present study confirms the meiotic counts of n = 30. However, meiosis is irregular. Figure 19 is a camera lucida drawing of a microsporocyte that contained JO bivalents. One of the long bi- valents has no interstitial chiasmata, which is an unusual situation. The long bivalent next to it is more typical of the long bivalents of the meiotically regular taxa during diakinesis.

Pollen stainability percentage is low, and the species has never been reported to set fruit. Meiosis occurs after the inflorescence is above the foliage.

Hosta albomarginata alba had not been studied cytologically previously. The haploid number is 31, and meiosis is irregular.

In all pollen mother cells that were decipherable, there was a quadrivalent consisting of two long and two short chromosomes

(Figs. 20, 21, 22, 23, 2*0. The free arms of the short members of the configuration are very seldom synapsed; I have seen weak synapsis in them only once, in an otherwise poor preparation of a

cell. There also may be two to four univalents present, and pos­

sibly an occasional trivalent (Figs.(_22 and 23). Meiosis occurs

when the inflorescence is above the foliage.

Hosta sieboldii (Paxt.) J. Ingram had not been studied

before, unless the report by Miss Tasui (1929) is about this taxon.

The haploid number of chromosomes is 31, and meiosis appears typical

(Figs. 25 and 26). The pollen stainability percentage is very high,

and the taxon is reported to set fruit well filled with

(Hylander, 195*0* a fact which I also have observed. However, while %

Fig. 20. Photomicrograph of a micro­ Fig. 21. Camera lucida drawing of sporocyte of H. albomarginata alba. the cell illustrated in Figure 20. Long arrows indicate univalents. Broad arrow indicates a quadrivalent.

v j i H Fig. 22. Photomicrograph of a micro­ Fig. 23. Camera lucida drawing of the sporocyte of H. albomarginata alba, in cell illustrated in Figure 22. One long arrow diakinesis. points to a univalent; the other points to what may be either a trivalent or a univalent and a bivalent. The short, wide arrow indicates a quadrivalent. VJ1to

4 e

11.5>

Fig. 2k. Camera lucida drawing of a misrisporocyte of H. albomarginata alba. The two long arrows indicate univalents; the broad arrow indicates a chain of four. 6 * *

* « <7 s 5

o

* * * I I n.s>, *

Fig. 25. Photomicrograph of a micro­ Fig. 26. Camera lucida drawing of the sporocyte of H. sieboldii, in diakinesis. cell illustrated in Figure 25. There are 31 bivalents.

V j l -P- 55 scanning a permanent slide for a cell suitably squashed for pho­ tography, I noted the bridge-fragment configuration illustrated in

Figures 27 and 28. Meiosis occurs after the inflorescence is above the foliage.

Hosta undulata has been reported as having n = 30 and

2n = 60 chromosomes, with faulty meiosis (Akemine, 1935)* This study indicates that meiosis is irregular, but the chromosome number of var. univittata is probably higher than has been reported for the species (Akemine, 1955)• Figure 29 is an illustration of a pollen mother cell of variety undulata in which there is a long univalent and a long arm of a synapsed configuration in which there is a typical inversion loop. The pollen stainability of the clone is k%, Meiosis in all H. undulata clones occurs when the inflo­ rescence is well above the foliage.

Figures 30 and 31 are illustrations of a microsporocyte of variety univittata in diplonema. There are 27 ring or rod bivalents, one heteromorphic pair, one pair in which de-synapsis has occurred,

five univalents, and a multivalent which may involve six chromo­ somes. If these are valid observations, the haploid number of this

clone is about 3** + 1* This is contrary to published data. The pollen stainability of the clone is 2.0%,

A third variety of H. undulata. variety erromena, also has

a low pollen stainability of 19%» This variety differs from the

typical variety in having no variegation. Green-leaved vegetative

segregates have been observed by Hylander (195*0» as well as by me,

from the variegated clones. US*

Fig. 27. Photomicrograph of a micro­ Fig. 28. Camera lucida drawing of the sporocyte of H. sieboldii. in anaphase I. cell illustrated in Figure 2 7. The configuration between the daughter groups of chromosomes is a side-arm bridge.

vn ON 57

r x

* *

Fig. 29 • Photomicrograph of a micro­ sporocyte of H. undulata var. undulata in diplonema. There is a long univalent and an inversion con­ figuration in the lower right part of the cell. Fig. JO. Photomicrograph of a micro­ Fig. 31. Camera lucida drawing of the sporocyte of H. undulata var. undulata. in cell illustrated in Figure 30* Long arrows early diplonema. indicate univalents; short, open arrows indi­ cate a heteromorphic pair and a desynapsed pair; short, solid arrow indicates a multi- valent. 59 Hosta sieboldiana, variegated form, has 30 bivalents at diakinesis (Fig. 32) in 91% of the cells that were countable. In the other 9%» there was a quadrivalent present among the large chromosomes (Figs. 33 and 3*0 • A total of 121 cells were examined.

The plant had 90% stainable pollen grains. Meiosis occurs when the inflorescence is about half the height of the foliage.

v Hosta fortunei is represented in this study by its variety albopicta f. viridis. This species is quite variable and there are

a number of named varieties. The typical variety was included by

Maekawa (19^0) as a variety of H. sieboldiana. He did not report

on the several sub-specific clones.

This was one of the first clones with which I used lacto-

propionic orcein, and the series of slides I have are not good.

Neither could I find suitable cells for photography, nor can I

state with any certainty a meiotic chromosome number, although it

is probably 30. However, meiosis is quite irregular, with bridges

and fragments at anaphase I and laggard univalents. In one cell,

there are two bridges and three centromeres in one configuration.

The pollen stainability- is 2*f%. Meiosis occurs when the inflo­

rescence is well above the foliage.

Hosta ventricosa is represented in my collection by three

clones, two of which are indistinguishable morphologically. The

third differs in that the central portion of the leaves is lighter

green than the margins. Otherwise, it is the same as the other two.

The cytology of this Chinese species has been investigated on

several occasions, the first time by Sykes (1908), who reported 60

Fig. 32. Camera lucida drawing 'of a microsporocyte of H. sieboldiana. variegated. of the microsporocytes observed had n = 30 chromosomes, with typical meiosis. i

• * JdL

% i\

11.5,*

Fig. 33» Photomicrograph of a micro­ Fig. 3^> Camera lucida drawing of the sporocyte of H. sieboldiana variegated, in cell illustrated in Figure 33* The arrow diplonema. Although most of the observed indicates the quadrivalent. cells were as in Figure 32, 7% contained a ON quadrivalent* H 62 that n = 24 and 2n = 48. This and three other reports of these numbers (Miyake, 1905; Inariyama, 1928; Sato, 1935) may be questioned, the first two because of the method employed— the squash technique had not come into use. Sato apparently retracted his account (Akemine, 1935)» later agreeing that n = 3° and 2n = 60.

Another report (Imai and Kanna, 1934)• mentions a polyploid series,

2n = 24, 3 6, and 48 in three clones. Whittaker (1934) reported that n = 30* Most reports are n = 3°» 2n = 60 for various taxa in the genus, but the present study has revealed that H. ventricosa has a haploid count of - 60 (confirmed by Kenichiro Kaneko, 1968, in litt.), and two other taxa that have n = 31*

Figures 35 and 36 are illustrations of a pollen mother cell of the variegated clone of H. ventricosa that is the best squashed cell of the three individuals of that species I have examined.

There are univalents present, but the most striking configurations in the cell are the quadrivalents. A small ring and a chain of four are marked by arrows in the picture, but there are several other quadrivalents visible, the most conspicuous being a large one, lower center. All three clones are probably n = - 60.

Meiosis occurs when the inflorescence is a little above the foliage.

Hosta tardiflora (Irving) Hyl. has never been reported to set fruit, but a made during the course of this study resulted in the fruit illustrated in Figure 37* Both parents had been in a cold storage room at 33°F for about three months. They were removed to the greenhouse about March 25* where flowering occurred in due course. After pollination, the fruit was allowed 1 1 . > A

fig* 35* Photomicrograph of a micro Fig* 3 6. Camera lucida drawing of the sporocyte of H. ventricosa, in diplonema* cell illustrated in Figure 35* Arrows indicate univalents and quadriv&lehts*

o\ v > i I

Fig. 37. Photograph of the open fruit and seeds of H. tardiflora. The scale is a centimeter ruler.

6^

66 to remain on the plant until dehiscence occurred naturally, in early December, It would appear that have never been re­ ported because there is too little time after anthesis for the ripening of fruit before killing frosts occur. In Columbus, H. tardiflora flowers in middle to late September. The average date of the last killing frost is October 23 (Anonymous, 19^1).

Camera lucida drawings of microsporocytes of H. tardiflora are presented in Figures 38 and 39* The haploid chromosome number is 30 bivalents; the pollen stainability is 68%. Meiosis appears to be regular in over half the pollen mother cells, but there is a lack of homology as evidenced by the univalents, indicated in

Figure 3 8. Meiosis occurs before the inflorescence reaches the top of the foliage.

Hosta decorata Bailey had not been investigated cytologically before this study. Figure *f0 is a camera lucida drawing of a cell of the typical form, with variegated leaves. There sire 30 bivalents, and meiosis appears to be regular, occurring when the inflorescence is above the foliage. I have observed no sort of reproductive irregularity. The pollen stainability was 97%»

Fruits are rarely produced naturally in the greenhouse, but in the fall of 196^ I harvested several fruits resulting from controlled self . Some seeds were planted immediately and did not grow. Others were sown later, but only four seedlings were raised to maturity. They bloomed three years after germi­ nation. These plants are indistinguishable from each other, and differ from the parent only in that the leaves are entirely green. ) 41

“ V /* f / j S / * * 41 ft * " % ’ • i I I U.5A 11.5A

Fig, 3 8. Camera lucida drawing of a micro- Fig. 3 9, Camera lucida drawing of a sporocyte of H. tardiflora. The arrows indicate microsporocyte of H. tardiflora, n = 30 univalents.

CT\ ~o 68

Fig. A-0. Camera lucida drawing of a microsporocyte of H. decorata. n = 30 69

In these plants, pollen stainability averaged 9*4$, there are 30 bivalents at diakinesis, and meiosis appears to be typical*

Hosta sieboldiana hybrid was received from the United States

National Arboretum as H. albopicta aurea. It has grown poorly, but has flowered the past two years. Although the leaves do not re­ semble those of H. sieboldiana, the flowers and flowering do, and meiosis occurs at generally the same time as it does in others of the sieboldiana group, well before the inflorescence reaches the top of the foliage. It keys neither to H. fortunei nor to

H. sieboldiana, but resembles both. The haploid number of chro­ mosomes is 30, and the pollen stainability is 28%. Figures *41 and

*42 are illustrations of a pollen mother cell at metaphase I that has two univalents and one pair in which de-synapsis has occurred.

In other cells I have observed micronuclei and anaphase I bridges and fragments. Fig. 4l. Photomicrograph of a Fig. kZ, Camera lucida drawing of the microsporocyte of H. sieboldiana hybrid cell illustrated in Figure 4l. Arrows indicate in metaphase !• univalents. There is also a small pair in which de-synapsis has occurred, n = 30

-o o DISCUSSION

The genus

Salisbury (1807) considered the funkias as comprising two genera, Niobe and Bryocles. He included in the former only H. plantaginea. and included in the latter H. ventricosa and H. lancifolia. His basis for the separation was the morphological isolation of H. plantaginea. In this species, the filaments are adnate to the base of the perianth, and two bracts, one smaller and hidden by the larger, subtend the lowest flower in the inflo­ rescence. These two characters are absent from the two other species known to Salisbury. This separation has not been supported by later workers. In spite of the morphological characters that separate H. plantaginea from other species in the genus, it has so many characters in common with other Hostas that it logically belongs in the same genus.

If the Paired Affinity Values as presented in Plates I, II, and III are compared in an over-all fashion, it can be seen that

H. plantaginea (Plate I, Fig. 1) does not differ markedly from any of the others. Indeed, together the taxa present a homogeneous appearance biochemically. Similarly, the chromosome morphology is alike in them. With the exception of the tetraploid H. ventricosa. there are four or five large and two to four medium bivalents, the rest of the complement consisting of small medium to small bivalents

71 at metaphase I* Genetic barriers may not be present between H. plantaginea and other species in the genus, for it has been the parent of at least one hybrid, 'Honey Bells' (with H. lancifolia,

Lee, 1957)* There seems to be no valid reason for the recognition of two genera. Thus this study supports the single genus concept for the funkias.

The valid species

There are four entities that are probably valid species among the taxa considered in this study. These are H. plantaginea,

H. decorata, H. crispula, and H. ventricosa. They will be dis­ cussed in that order.

Hosta plantaginea. Although I have not been able to repeat my observation (Fig. 18), H. plantaginea appears to have regular meiosis as reported (Yasui, 1935)* The chromosome morphology of this species is similar to that of other species in the genus. The chromatographic data (Plate I, Fig. 1) indicate similarity to the other taxa considered in this study because of the relatively large size of the dark area in the graph. There is closer biochemical affinity to H. undulata var. undulata, no. 5» but this is a member of a hybrid group, to be discussed later.

Although this species is a member of the genus Hosta, the clear morphological separation from other species in the genus supports Maekawa (19^0), who placed H. plantaginea apart from all others as the sole member of the subgenus Niobe. 73

Hoeta decorata.— The typical form of this,species, with variegated leaves, is frequently sold as H. 'Thomas Hogg1* It has regular meiosis and a high pollen stainability percentage, and fruits are often observed in gardens* The plants that resulted from the selfing of a plant of the typical form are very similar to each other and to the parent morphologically. They have a high pollen stainability percentage* The polygraphs resulting from the plotted Paired Affinity Values of the parent and the F^ plants

(Plate III, Figs. 15 and 1 6) are not so similar as might be eucpected.

However, the leaves of the offspring are totally green, unlike the variegated-leaved parent. This may account for the differences in the Paired Affinity Values*

Hosta crispula.— This species has 30 bivalents at metaphase

I and a pollen stainability of 84%. When its chromatographic profile is compared with other taxa (Plate III, Fig. 14), only two hybrid- ogenous clones (to be discussed later) have 50% or more spots in

common with it* I have seen a form of the species in the garden

of Mrs. V. R. Frederick, Urbana, Ohio, that differs from the typical

form only in that it has no variegation in the leaves. It appears

to be a valid species.

Hosta ventricosa.— This is the species (as Funkia ovata

Spreng.) in which Strasburger (in Maheshwari, 1950) found evidence

of apomixis. It sets fruit in gardens, and its seedlings resemble

the parent in a selfing cross. There are conflicting reports about

the chromosome number of the species. Imai and Kanna (1935) re­

ported a polyploid series of n = 12, 1 8 , and 24, but 30 is the most 74 frequently reported meiotic number. Although I cannot report with certainty the melotic number of bivalents of plants considered in this Btudy, there probably are about 60 at metaphase I* Because of the high number of quadrivalents in the microsporocytes (Fig.

36), it seems probable that my clones are tetraploid. If this is so, then the report of Imai and Kanna may be in error. Their work should be repeated. If there really are lower chromosome numbers within the genus, the base number may not be x = 30 as Akemine and others have suggested.

The Paired Affinity Values of the variegated clone of H. ventricosa (Plate II, Fig. 10) and of one of the two morphologically alike clones (Plate II, Fig. 11) are similar, but those of the third clone of the species (Plate II, Fig. 12) differ. The latter may possibly differ as a result of the nucellar embryony noted by

Strasburger. If any mutations occurred that resulted in different biochemical pathways, the compounds in the leaves would differ.

The vegetative reproduction by seeds would perpetuate the mutants.

Of course, there may also be chemical races within the species.

Regardless of how the biochemical differences originated, these races might be detected chromatographically, even though there are essentially no morphological differences.

The pollen stainability percentages of these three clones are not what one would expect from tetraploid individuals. Because of the high incidence of quadrivalents, one would expect to find that meiotic irregularities would result in abortive pollen. How­

ever, pollen stainability percentages are 90, 92, and 84. There are three possible reasons for the high stainability. First, all percentages of stainable pollen were made from freshly Opened

flowers, by dipping the whole anther in the dye solution. It may be that by the time of anthesis, the abortive grains had already

degenerated, leaving mostly stainable grains which then were

counted. Secondly, the abortive grains might have been lighter and

dry by anthesis, and were lost during the process of picking the

anthers. However, I believe the most probable explanation for the

high pollen stainability percentage is the third possibility: most

microspores received at least one genome, in whatever way the chro­

mosomes segregated. The extra genetic material may not have been

detrimental to pollen formation.

As the percentage of pollen stainability is high, the pollen

is presumably functional. Aneuploid hybrids might then be derived

from the tetraploid H. ventricosa.

The species of questionable validity

There are three named taxa involved in this study that may

be hybrids. They are H. sieboldii, H. lancifolia, and H. tardi-

flora. The first two were among those plants imported to Europe

by von Siebold. The last was introduced to European gardens in

1895 by Mr. Max Leichtlin. According to Hylander (195^)* all

plants in Europe and the United States of this named variety are

’fevidently of one clone".

Hosta sieboldii.— Plants of this taxon bear fruit regularly

and abundantly in gardens, and the plants in the greenhouse set

fruit sparsely. The pollen stainability percentage is high, and 76 meiosis appears regular. There are 31 bivalents at metaphase I.

It might be concluded that H. sieboldii is a valid species.

However, when the Paired Affinity Values with other taxa are plotted and compared (Plate I, Fig. k), the shaded area is large, and the biochemical affinities with H. ventricosa (no. 1),

H. tardiflora (no. 13)» and H. decorata (no. 15) are over the 5096 level. This, plus the meiotic chromosome count of n = 31, unusual in the genus and possibly aneuploid, suggest that H. sieboldii is of hybrid origin. Also, Yasui (1929) worked with a taxon she

called "H. lancifolia Stern (H. .japonica) f. albomarginata Makino"

that may have been H. sieboldii. She studied the inheritance of leaf-variegation, and concluded that maternal inheritance of pro-

was the explanation of the random appearance of different

variegation patterns in leaves of the progeny. The report contains

pictures of leaves of different plants of the F^ generation. These

vary considerably, from leaves resembling those of the parent to

those much narrower and those with undulate margins that resemble

leaves of H. undulata.

I think that H. sieboldii may be a meiotically stable

hybrid, possibly involving H. ventricosa in the parentage, with

H. decorata and either H. tardiflora or one of the parents of H.

tardiflora. The chromatographic profiles and the unusual chromo­

some number are not proof of hybridity, but they do suggest it.

Final proof would be the reconstitution of H. sieboldii from the

putative parents. 77

While searching for a microsporocyte suitable for pho­ tography, I found the cell in anaphase I illustrated in Figure 43*

I observed one other cell, later in anaphase I, that had a similar bridge-fragment configuration. Perhaps a dozen cells in telophase

I and II had visible fragments. There were over 100 pollen mother cells observed. When one finds a bridge-fragment configuration, the first explanation that comes to mind is that it is the result of an inversion phenomenon. The fragment illustrated is large, several times the length of the smallest chromosome in the cell*

If the plant were heterozygous with respect to an inversion of that length, it would be visible more often, and the pollen staina­ bility would be lower than the observed 9

It seems more probable that the configuration illustrated in Figures 43 and 44 is the result of a failure in the usual meiotic process. If an eight-stranded bivalent has a typical chiasma near the centromere, and a non-sister sub-chromatid chiasma farther from the centromere in one member of one of the arms in­ volved in the typical chiasma, as illustrated at the top of Figure

4-5, then the configuration as illustrated at the bottom of Figure

45 will result at anaphase I. Homologous chromosomes separate at anaphase I and chromatids do not. The bridge then, will not extend from centromere to centromere with a free (though possibly associ­ ated) fragment, but will be included in what is to become the

fragment. In the photomicrograph taken of the cell with an oil immersion lens (Fig. 43), one can see a hole in the middle of the

fragment* On the slide, one can see that the bridge is not free from the fragment, but is involved with it. * 3ft ft

* •*. * - V v /

n.s

Fig* kj>. Photomicrograph (under oil) Fig. kh. Camera lucida drawing of the of a microsporocyte of H. sieboldii in cell illustrated in Figure kj. anaphase I*

-o 00 Fig. 4-5* Diagram of a possible explanation of the chromosome configu­ ration illustrated in Figures and 8o

Such phenomena have been called side-arm bridges by Wilson,

Sparrow, and Pond (1959)» who noted this type of configuration in

X-irradiated pollen mother cells of Trillium erectum. Lewis and

John (1 9 6 6) noted the same phenomena in untreated Tradescantia,

Lilium, and Paeonia pollen mother cells, as well as in untreated primary spermatocytes of all the grasshoppers and locusts with which they have worked, though infrequently. They caution cyto- taxonomists that the automatic association of bridge-fragment con­ figurations with inversions may be a source of error in bio- systematic studies.

Hosta lancifolia.— The thousands of plants of this taxon in America may possibly be one clone, for this is one of the taxa that has not been reported to set fruit. The pollen stainability is low, 37#* Chromatographic evidence (Plate I, Fig. 2) does not indicate particular affinity to any other taxon in this study.

The haploid number is 30, and meiosis is irregular (Fig. 19)* One might conclude that it is a hybrid of taxa not included in this study. However, Kaneko (1 9 6 7) has reported on three wild clones of H. lancifolia, all with different karyotypes. One has three heteromorphic pairs; the other two have chromosomes present in pairs, but the chromosome morphology differs between them. All are 2n = 60. If his taxonomic identification of the clones he worked with is correct, there may be chromosome races within the species. If so, then the clone so common in our gardens may be a hybrid plant of differing chromosomal race, not of taxa not included in this study. Thus, Hosta lancifolia may or may not be a valid species. 81

Hosta tardiflora.— The last funkia to bloom each summer is aptly naried, for inflorescences form in mid-September in Columbus.

Meiosis is irregular, and the pollen stainability percentage is low. As indicated in Plate III, Figure 13, there is a close bio­ chemical affinity with H. sieboldii (#*t, H. decorata (#15), and

H. sieboldiana hybrid (#17)• The haploid number of chromosomes is 30. If it is involved genetically with H. sieboldii, n = 31, it is most probably in the parentage because of the difference in chromosome number between the two. As to be mentioned later, it may be involved with H. sieboldiana in hybrid forms. However, though H. tardiflora probably is a hybrid, it is difficult to account for the late flowering habit when compared to other taxa in this study. The situation may be similar to that of H. lanci­ folia; that is, the clone of H. tardiflora in Europe and America may be a hybrid of chromosomal races within the same species, or a hybrid of taxa not grown in the western hemisphere.

The hybrid taxa

There are among the 12 taxa (17 clones) considered in this study four valid species: H. plantaginea, H, decorata, H. crispula, and H. ventricosa. There are also three possibly valid species:

H. sieboldii, H. lancifolia, and H. tardiflora. The remaining taxa are most probably of hybrid origin.

Among these are two clones that resemble H. sieboldiana

(Hook.) Engler. They are members of a group of hybrids and species that has been collectively called the sieboldiana group in the 82 literature. Maekawa (19**0) included five species in the group, and

Hylander (195*0 discussed only H* sieboldiana. including in it two varieties, describing the species as "polymorphic". The typical form of the species has large, cordate-ovate, pruinose, bluish- green leaves. The flowers are pale lavender, held stiffly erect with the inflorescence at or barely exceeding the height of the leaves at flowering time. The two clones of the sieboldiana group included in this study resemble the central species of the group, but are not the same taxon.

One of them was received as "yellow-edged sieboldiana". I have called it here H. sieboldiana, variegated, to indicate its morphological affinity, although I do not believe it is a member of

H. sieboldiana sensu stricta. It differs from the typical form in that the inflorescence is well above the yellow-margined leaves, and in that the flowers are pure white outside, with a slight central flush of lavender on the inner perianth segments. The perianth segments are less reflexed, wider, and thicker than in the typical variety. Over 90% of the pollen mother cells contained

30 bivalents, the remainder contained a quadrivalent consisting of only large chromosomes (Figs. 32, 33, 3*0• Since the description of the clone does not agree with the descriptions of various species of the sieboldiana group in the literature and is atypical mei- otically, it is probably a hybrid. The chromatographic data

(Plate II, Fig. 8) indicate that it shares over 50% of detectable compounds with H. crispula. The latter species may be involved in the parentage, with H. sieboldiana. There is also a biochemical 83 affinity to H. undulata var. erromena, no. 7 in Plate II, Figure 8, a member of a hybrid group.

Hosta sieboldiana hybrid.--A second clone of the sieboldiana group included in this study also differs from the typical H. sieboldiana in both leaves and flowers. The leaves are more lanceolate and less glaucous, and the flowers, although clOBely resembling the typical variety, are borne above the foliage.

Because of the similarity of the flowers and the time of meiosis to H. sieboldiana types on hand, I have called it a H. sieboldiana hybrid, although it resembles the H. fortune! group of clones nearly as much as it does H. sieboldiana.

However, I believe that the resemblance to H. fortune! is superficial, arising because one of the parents of this hybrid is

H. tardiflora. This latter taxon has lanceolate leaves with no pruina. They are narrower than H. sieboldiana hybrid, which is approximately intermediate between H. tardiflora and H. sieboldiana.

Also, the chromatographic data indicate a close biochemical affinity with H, tardiflora. Hosta crispula might be involved, since there

is some indication of biochemical affinity, but this is doubtful,

since the two have very little morphological similarity.

Hosta fortunei, var. albopicta f. viridis is a member of a

group that Hylander (195^) and Hensen (1963) have assumed to be of

hybrid origin. It is the only clone of the group included in this

study. The chromatographic data (Plate II, Figure 9) indicate no

particular biochemical relationship with any of the taxa considered.

However, the pollen stainability percentage is Zh% , and meiosis is irregular. This particular clone is hybridogenous, but it is possible that the more nearly typical forms are not. The re­ semblance to H. fortunei could be because of the combination of dominant and recessive parental genes. Hensen (19&3) bas accepted

H. fortunei as a species but has listed the variations as horti­ cultural clones, and has treated the names of the varieties and forms as clonal names. This clone he calls H. fortunei 'Viridis'.

In this case, I would agree. Further work may very well indicate that H. fortunei is a hybrid group. However, I would be more con­ servative if I were treating all fortunei clones. Until there is positive proof that the species is in reality an assemblage of hybrids, I would follow Hylander's nomenclature.

Hosta undulata.— None of the several varieties of this variable taxon are known in the wild in Japan, although they were imported to Europe by von Siebold’s collectors over 100 years ago.

Three of the varieties are included in this study. Pollen staina­ bility percentages are low in all three, and two are complex hybrids as evidenced by the involved multivalent configurations in

Figures 29 and 30.

Hosta undulata var. undulata is heterozygous with respect to an inversion and has a long univalent (Fig. 29). Hosta undulata var. univittata is also cytologically complex. Figure 30 is an illustration of a cell in diplonema. The multivalent may be a hexavalent, as presented semi-diagrammatically in Figure 46.

Figure 47 is a diagrammatic representation of the same configuration.

There are two terminal and two interstitial translocations and a Fig. 46. Semi-diagrammatic drawing Fig. 47. Diagram of a possible intar- of a possible interpretation of the multi- pretation of the multivalent illustrated in valent illustrated in Figures 30 and 31• Figures 30 and 31*

vn00 86 large inversion. Figures kS and **9 are similar drawings of another possible interpretation of the hexavalent, also with two terminal and two interstitial translocations, and one large inversion. The two interpretations differ in the 6' and chromosome ends, and in the Jf' and 5' ends. Because of the twiBted synapsed segments, I have not been able to decide exactly what the chromosome end arrangement is.

Also, whenever I have seen a bivalent associated with the nucleolus in any Hosta, it has always been a small medium pair.

In the drawing of a pollen mother cell of H. undulata var. univittata.

Figure 31» the nucleolus is next to the multivalent in such a way that the bivalent associated with it is not distinguishable. If the pair associated with the nucleolus is involved with the multi­ valent, the configuration is an octovalent. However, the bivalent with the nucleolar organizer may merely be hidden by the multi­ valent.

Although all three cloneB of H. undulata are similar morphologically, the chromatographic data indicate little affinity

(Plates I and II, Figs. 5» 6, and 7). The species is known only from cultivation in Japan, and has never been reported to set fruit.

The variegated forms were among those taxa imported to Europe by von Siebold, and variety erromena was described as a species by

Stearn (in Bailey, 1932) from a garden form. The group is un­ doubtedly of hybrid origin, and probably not from the species in­ cluded in this study. Hosta undulata is the only taxon in this study in which the scape is obscurely angled towards the summit. All four 1

Fig. 48. Semi-diagrammatic drawing Fig. 4-9. Diagram of a second possible of a second possible interpretation of the interpretation of the multivalent illustrated multivalent illustrated in Figures 30 and 31. in Figures 30 and 31*

00 •vJ species in Section Lamellatae (Maekawa, 19^0) have angled scapes*

All other species in the genus have terete ones. At least one of the funkias of this section probably is involved in the parentage of the hybrid group, H. undulata. None of the species of Lamellatae is cultivated in the United States or Europe as far as I know.

Hosta albomarginata var. alba, given the provisional name

H. X alba in Plate I, Figure 3, is known as H. minor alba in the horticultural trade of the United States. It is of hybrid origin.

There was a quadrivalent in all the microsporocytes that I observed in metaphase I and earlier stages of meiosis. It consists of two long and two short chromosomes. The configuration is most often visible as an open ring, the short members each having a free arm.

The haploid number is 31.

Hylander (195^+) allied this hybrid as a variety of H. sieboldii (H. albomarginata, sensu Hylander), but admitted that it was possibly an artificial arrangement. The chromatographic data

(Plate I, Fig. 3) indicate that there is little biochemical affinity between the two, even though both are n = 31* There is a weak relation with H. undulata var. erromena, a member of a hybrid group.

Hosta albomarginata var. alba is a hybrid of unknown parentage, possibly not closely related to any of the other taxa included in this study. CONCLUSIONS

1. The genus Hosta Tratt. is a homogeneous group of species closely related morphologically, biochemically, and cytologically.

There is no reason to consider them as members of two genera*

2. Using the criteria of this study, Hosta plantaginea

(Lam.) Asch., H. crispula Maekawa, H. decorata Bailey, and H. ventricosa Stearn are all valid species*

3. Hosta sieboldii (Paxt.) J. Ingram appears to be a valid morphological and cytological species. However, it may be a meiotically stable hybrid, possibly aneuploid, derived from H* ventricosa and others as suggested by chromatographic data.

if. Hosta lancifolia Engl, of our gardens is hybridogenous, but may be a hybrid of clones of different chromosomal races within

the same species, rather than of different species. Similarly,

tardiflora is of hybrid origin with unknown parents.

5* Hosta fortunei (Bak.) Bailey var. albopicta (Miq.) Hyl.

f. viridis Hyl. is the only member of a group of clones of uncertain

origin considered here. Hensen (19&3) has treated them all as

of the species, using the final epithet of each as a

clonal name. This particular clone is a hybrid of unknown parentage.

I agree with the designation of it as H* fortunei 'Viridis1, but I

do not feel that the procedure should be extended to sill members of

H. fortunei without further study.

89 90

6. Hosta undulata (0. & D.) Bailey is a group of morpho­ logically similar hybrids, distinguished among themselves by the presence or absence of leaf variegation, and the patterning of the variegation when present. The name of the collective hybrid taxon should be written Hosta X undulata (0. & D.) Bailey, emend. The varietal epithets are best treated as clonal names as follows:

H. X undulata ’Undulata'

Syn: H. undulata (0. & D.) Bailey var. undulata

H. X undulata 'Univittata*

Syn: H. undulata (0. & D.) Bailey var.

univittata (Miq.) Hyl.

H. X undulata 'Erromena'

Syn: H. undulata (0. 8c D.) Bailey var. erromena

(Stearn) Maekawa

7. The small, white flowered funkia sold in the horticultural trade as H. minor alba is a hybrid of unknown parentage. It was validly described as a variety of Funkia lancifolia (Thunb.) Spreng.

(= H. lancifolia Engl.), but is not closely related to that taxon or

to H. sieboldii, to which it has also been allied. A solution to

the problem of a for it might be to elevate the varietal

epithet given to it by Irving to specific status and give an emended

description. However, this is impossible, as Andrews (1801) has

used that epithet in a description of a member of Hemerocallis

Linnaeus. The name is a synonym of Hemerocallis plantaginea 91

Lamarck 179^« Therefore, I propose the hybrid be given a popular name in accordance with the International Code of Nomenclature for

Cultivated Plants#

Hosca 'Little Whitey'

Syn: Hosta albomarginata (Hook.) Hyl. var. alba

(Irving) Hyl.

Hosta (vel Funkia) minor alba Hort. BIBLIOGRAPHY

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