Proposal for Continued Support of Nsf Research Grant G13261

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PROPOSAL FOR CONTINUED SUPPORT OF NSF RESEARCH GRANT G13261

I. Name and Address of Participating Institution:

University of Louisville, Department of Biology, Louisville 8, Kentucky.

II. Endorsements:

Executive Vice President University of Louisville Woodrow M. Strickler

Head, Department of Biology University of Louisville William M. Clay, Ph. D.

Associate Professor, Biology University of Louisville Arland T. Hotchkiss, Ph. D.

III. Title of Project: A Cytotaxonomic Study of the Characeae.

IV, Desired Starting Date of the Research:

September 1, 1961

V. Time Period For Support: Three Years. (September 1, 1961 to September 1, 1964) VI. Resume of Progress to Date. A. Description of the Plant Groups Being Studied The charophytes are simple aquatic plants, worldwide in distribution, usually in fresh water, agreeing with the green algae in pigmentation, food reserves and cell morphology, but because of the distinctive complexity of organization in their stem nodes and internodes, and whorled branchlets, and especially their elaborate gametangial structures, they are placed in a separate class Charophyceae of the Chlorophyta by some workers as G. M. Smith, or still further removed from green algae into a separate Division Charophyta by others as Groves and Bullock-Webster, Zaneveld, following Sachs. Within the group of charophytes there is a single Order Chorales usually considered to have a single extant family, Characeee, subdivided into two tribes Chareae and Nitelleae. There is some evidence which might support raising the two tribes to the status of two or more subfamilies or even fami- lies but this has not been done formally. The Tribe Nitelleae contains two genera, one of which, Nitella with 153 species, is the largest in the entire group. The other genus, Toly- polls, has but 13 species. The Nitelleae are entirelyEnorti- cate and have two tiers of 5 crown cells surmounting the oogonium. In Nitella the branchlets are forked, stem branches two per node, the oospore is laterally compressed, the antheridium terminal in a branchlet furcation. In Tolypella the branchlets are monopodial, stem branches more than two per node, the oospore terete in section, the antheridium is lateral in position. Important specific characters in Nitella include the kinds of branchlets present, number of furcations in the branchlets, number of cells in the dactyls (ultimate rays), presence of heads, enveloping jelly, spore characters and monoecious or dioecious species. The Tribe Chareae contains four or five genera of which Chars with 116 species is the largest. The others have from one to four species apiece. The Charese are mostly corticated but a few are entirely ecorticate in the stem and branchlet internodes, as in the Nitelleae. In every case the oogonium is capped by a single tier of 5 crown cells, the branchlets monopodial and never forked. Important specific characters in the Genus Chars include the degree of cortication, number of whorls of TETDulodes, arrangement of gametangia on the branchlets, spore characters, monoecious and dioecious species. A Key to the Extant Genera A. Cells of the coronula in two superimposed rows of five cells each ...... NITELLEAE 1. Antheridia terminal in the furcations of the branchlets; oogonia lateral; oospores elliptic in transverse section ...... 1)Nitella 1. Antheridie and oogonia lateral at the branchlet- nodes; oospores terete in transverse section. . . 2)Tolypella -3- AA. Cells of the coronula in one single row of five cells ...... CHAREAE 2. Stipulodes and bracteoles absent. 3. Bract-cells also absent. Branchlets of 3-4 segments ...... 3)Protochara 3. Bract-cells 1-2, very long. Branchlets of 2-3 very long segments ...... 4)Nitellopsis 2. Stipulodes always present, sometimes rudimentary. Branchlets simple, of 4 or more segments. Bract-cells normally 4 or more, or rudimentary. Bracteoles also may be rudimentary or absent. 4. Oogonia and antheridia produced from separate peripheral cells of the node and situated side by side. Stem corticate ...... 5) Lychnothamnus Oogonia and antheridia produced from the same peripheral cell of the node. S. Oogonium normally situated below the antheridium. Stem ecorticate • • .6) Lamprothamnium 5. Oogonium situated above the antheridium Stem corticate or ecorticate ...... 7) Chars Fossil forms of the Charophytes are well known in paleo- botany and are described and figured in the standard texts by H. N. Andrews, C.A. Arnold, and J. Walton. Because many members of the charophytes secrete a limeshell around the oogonium, this portion of the plant is easily fossilized; other parts of the plant have also been found in the fossil state. Several genera are known from the Devonian, Paleo- chars with six spiral cells is from the Carboniferous, entire plants of the Jurassic genus Clavator were described by Harris from England. Although gyrogonites from the late Triassic up to recent times are well known, little has been done with recent Pleistocene forms which are the immediate ancestors of our present day species. B. Cytological Methods. Fruiting tips of plants are snipped off and either fixed immediately for examination later, or prepared for squashing directly in aceto-orcein. In the latter case, single antheridia of an appropriate size (age) are teased off the plant branchlets into drops of stain. The slide is warmed slightly and the material is squashed directly under a cover-slip with appropriate pressure. Warming and squashing continue until a proper degree of spread is achieved. The slides are then examined, chromosomes are counted, drawings and photographs are made as required. Slides with good preparations are made permanent by trans- ferring to 90% alcohol for a few hours and then in 100% alcohol for at least two hours. The cover-slip is allowed to float off the slide inverted in alcohol or it may be carefully pried off with a razor blade. Mounting in Euparal Is done directly from the absolute alcohol. Field collections which were fixed in either 3:1 (100 parts absolute alcohol: 33 parts glacial acetic acid) or Carney's -4- (100 parts absolute alcohol: 16 parts glacial acetic acid; 50 parts chloroform) for at least 12 hours, then transferred to 70% alcohol and kept preferably refrigerated (at least cool) have proved eminently satisfactory provided the fixative was made up fresh for each collection. Australian and Mexi- can materials and controls from all collecting in 1960 have been prepared in this way and show perfect preservation when later transferred to aceto-orcein stain for approximately an hour before squashing in the stain on a slide. C. In the following tables, are shown our own counts of chromosome numbers for the year 1960 and also, for comparison, the preceding years back to 1957. The perfect agreement with ourselves over the years reflects our practice of eliminating from the summary those results we later believed to be based on mistakes in counting or in identification of material. No attempt has been made here to make comparisons with numbers in the literature except our own. Other collectors' names and people who aided in our own collections are noted. In several instances in Tables 2 and 3, complete identifi- cations of the specimens collected by Tindall and Wood have not yet been made. -5- Table 1 Chromosome counts in the Characeae 1957-1961 Species Chromosome No. Location Dates Chareae 1. Chara aspera Deth. N = 14 Lake Wawasee, Ind. 1960 with Tindall ex. Willd 14 Mexic, 1961 Tindall (4-7-61-10) 2. Chars braunii Gmelin 14 Near Lake Gauvreau,1960 Quebec 14 Masson, Quebec 1957,58,59,60 14 Black Rapids,Ont. 1957,59,60 14 Picton,Ont. 1960 14 Keuka Lake, N.Y. 1959 14 Mecklenburg,N.Y. 1960 14 Cayuta Lake,N.Y. 1959,60 14 Lake Waramaug,Conn.1958 Wold 14 Texas; 1961 Tindall 4-1-61-4 var. schweinitzii 14 Starve Hollow 1960 with Tindall_ Hatchery,Ind. 3. Ohara brittonii 14 Cabin Creek Bog, 1960 Daily T.F. Allen ex Rob. Ind. 4. Chars contraria 28 Cayutaville,N.Y. 1960 Br. ex Kutz 28 Lansing, Mich. 1960 BLANKENBAKER 28 Lake Wawasee,Ind. 1960 with Tindall 28 Lake James, Pokagan 1960 with Tina Perk,Ind. 28 Cabin Creek Bog, 1960 with Tindall Ind. Daily 28 Richmond, Ind. 1960 with Daily 28 Starve Hollow 1960 with Tindall Hatchery,Ind. 28 Crystal Lake, 1957,58,59,60 Fort Knox, Ky. • Table 1 (continued)

Species Chromosome No. Location Dates 28 Grahampton Reser-: 1957 Weiss vation,Peade Co, Ky.

28 DeatsvillelKy. 1957

28 Californ . 1960 Wood

S. Chara delicatula Agardh 28 Masson,Quebec 1957,(cult.59,60)

28 Picton, Ontario 1960

28 Little Maschaug 1957 Wood Pond,Rhode Is.

28 Lake James, Ind. 1960 with Tindall 6. Chara globularis Thuill. 28 Mac,s Lake,Quebec 1960

28 Pictonr Ontario 1960 28 Lake James,Pokagan 1960 with Tindall Park, Ind.

28 DeatsvillelKy. 1957,(cult.58,59)

28 Carpenter's LakelKy.1957

28 Palo Alto, California 1960 Wood

7. Chara leptopitys Braun 14 Katanning llestern 1960 Wood Australia

8. Chara sejuncta Braun 14 Coston Pond,Apanaug,1957 with Wood Phode Island

14 Cayuta Lake outlet, 1959,60 N.Y.

14 Sinkhole Pond near 1957(cult.58,59) Otter Cr.,Ky.

14 Sinkhole Ponds near 1960 with Minckley Doe Run Ky.

14 Mexico 1960 Tindall

9.Chara vulgaris Vaillant 14 Virgil Creek, 1957,58,59,60 Dryden, N.Y.

14 Keuka Lake, N.Y. 1957 14 Jenkins Pond, 1960 Cayutaville, N.Y. -7- Table 1 (Continued)

Species Chromosome No. Location Dates

10. Chara zeylanica Klein 28. Kingstont R.I. 1957 Wood var. elegans_ 28 Cornell Greenhouse 1960 cult. Nitelleae 11. Nitella acuminata Braun ex Wall var. glomerulifera 18 Cornell greenhouse 1960 cult.

var. gubglomerata 18 South Park Pond 1957 Jefferson Co.,Ky. 18 Sinkhole Pond, Meade 1957 Co., KY, 12. Nitella axillaris Braun 18 Univer, Rhode Island 1957 cult. from S. 13. Nitella clavata Kutz 18 Masson, Quebec 1957, cult. to 1960

18 Palo Alto, California 1960 Wood

14, Nitella flexilis Agardh 12 Doe Run, Kentucky 1960 with Tindall

12 Poicagon St. Pk. Ind. 1960 with Tindall

15. Nitella gracilis Morioka 18 Berke1ey0a1ifornia 1960 Wood

16. Nitella intermedia Nord. 18 Paradox Lake, N.Y. 1957 Wood 17. Nitella microcarpa Braun asp. megacarpa 36 R.Lievre, Quebec 1957

18 Ottawa River, Quebec 1957

18. Nitella opaca Agardh 6 Richmond, Ind. 1960 with Daily

19. Nitella tenuissima 18 Paradox Lake, 1957 . Wood (Desv.) Katz Adirondacks, N.Y. 12 Kingston,Rhode Es. 1957 with Wood 20. Tolypella intertexta 12 Keuka Lake, N.Y. 1957 T.F.Allen

21. Tolypella prolifera 12 Pokagan State Park 1950 with Tindall (Ziz.) LEONH.ARDI Indiana

12 Syracuse Lake, Ind. 1960 with Tindall ll 8-23-60-1 1960 14 rt 8-20-60-2 1960

ex. Kutz. prox. 28 tt 8-20-60-1-B - 1960

28 11 8-21-60-1-A 1960 Willd.

Nitelleae

ft 9 3-29-61-1 1961 Tolypelleae 1. Tolypella sp. 12 It 3-29-61-4 1961 2. Tolypella sp. 9 it 3-28-61-1 1961

Uncounted Collections of Characeae, TindallTindall CollectionsCollections 12 cellections 1560 25 collections 1961 -9- Table 3. Chromosome counts in the Wood Collections of Characeae from Australia and New Zealand with Other Counts from the Literature as Noted

Species Chromosome No, Collection No. Year

Chareae 1. Protochara australis 14 Wood 60-10-13 1960 Woms.& Ophel 2. Protochara inflate (Fil) 14 Wood 60-10-1-12-5A 1960 Woms.& Ophel 3. Protochara sp. 14 Wood 60-10-10-11 1960 4. Protochara sp. 14 Wood 60-10-7-7 1960 5. Protochara sp. 9 Wood 60-10-16-13 1960 6. Protochara australis Woms.& Oph. 14 Macdonald & Hotchkiss 1955 7. Chara australis R. Brown 14 Macdonald & Hotchkiss 1955 8 Chars Leptopitys Braun 14 Wood 60-10-11-1-1B 1960 9, Chars sp. 14 Wood 60-10-11-1-1B 1960 10.Chara sp. 28 Wood 60-10-7-8-3C 1960 11.Chara sp. (Monoecious) 28 Wood 60-10-4-1-7A 1960 12.Chara sp. (Monoecious) 28 Wood 60-10-30-4 1960 13.Chara sp. (Monoecious) 28 Wood 60-11-4-1 1960 14.Chara sp. (dioecious) 14 Wood 60-10-1-7-3A 1960 15.Chara sp. (dioecious) 14 Wood 60-10-28-1 1960 16.Chera sp. (dioecious) 14 Wood60-10-7-13-4C 1960 17.Chara sp. (dioecious) 14 Wood 60-10-29-1 1960 18. Lamprothamnium macropogon (Braun) Ophel 301 s Wood 60-9-22-1 1960 19. Lamprothamnium macropogon 28 Wood 60-10-5-1-8A 1960 20.Lamprothamnium macropogon 28 Wood 60-10-1-5-1A 1960 21.Lamprothamnium macropogon 28 Wood 60-10-1-3-2A 1960 22. Lamprothamnium sp. 28 Wood 60-11-7-7 1960 (continued) -10- Table 1. (continued) Species Chromosome No. Collection No. Year Nitelleae 1. Nitella sp. 12 Wood 60-9-25-1 1960 2. Nitella sp. 9 Wood 60-9-18-3 1960 3. Nitella sp. 12 Wood 60-9-18-4 1960 4. Nitella sp. 18 Wood 60-10-16-6 1960 5. Nitella sp. 18 Wood 60-10-8-3-60 1960 6. Nitella sp. 9 Wood 60-10-7-3-9A 1960 7. Nitella sp. 18 Wood 60-10-9-4-8C 1960 8. Nitella sp. 12 Wood 60-10-16-5-9C 1960 9. Nitella sp. 9 Wood 60-11-8-13-D 1960 10. 9 60-11-22-10 1960

1-35 Characeae spp. Uncounted as yet (East and South Australia) Wood 1961 1-26 Characeae spp. Uncounted as yet (Tasmania) 1-3 Characeae spp. Uncounted as yet (N.W.T.) 1-20 Characeae spp. Uncounted as yet (New Zealand) -11- D. Polyploidy and general cytology in the Characeae.

Among the most important characteristics identifying individual chromosomes in mitoses are their number, form, size and relative behavior. The group of characteristics which may be taken into consideration in identifying a particular chromosomal complex, the karyotype, is common to, or characteristic of, an individual, race, species, genus or larger grouping. Work in the Characeae is directed at establishing as many facts as possible concerning the karyo- types of the members of the group. phylogenetic relationships among themselves as well as comparison with other plant groups.

As far as number is concerned we are dealing, so far, simply with antheridial mitoses which are easily accessible in squashes of the male gametangia. Meioses occur in the in- terior of heavily walled oospores at an unpredictable moment in their germination into haploid plants bearing the male and female gametangia. Thus the gametangia with their filaments, male and female, are haploid in constitution containing, one would expect, a complement (a group of varying chromosomes) designated N, the lowest haploid number or actual X. However, initial cytological work revealing such gametic numbers as 14 and 28 in the genus Chars; 12, 18 in the genus Nitella, led us to suggest that ploidy was at work in the group with hypothetical (if no longer in existence) or real base numbers of 7 and 6 respectively to be found as the bases of lines of auto- or allopolyploidy. Other workers, for example, Gillett, have been impressed by the Same reasoning and additional numbers have strengthened this hypothesis (for example, a 6 in Nitella opaca). In an initial analysis of the numbers, there appear to be definite 1428 series in the genus Chara correlated with either a dioecious or monoecious distribution of game- tangle; another (?) series in the ecorticate Chars australis, Chars braunii group which is uniformly 14 whether dioecious or monoecious in sex organs; and a third 6, 12, 18 series in the Nitelleae. Finally, the 9 count forms in Nitella may be partly an additional 9:18 series, or a possible indication that the entire group is based on a 3 multiple. It is apparent, therefore, that if ploidy has occurred in the "haploid" sets, we are actually dealing with sets of homologues. On the whole, cytologically homologous chromosomes in mitoses are scattered at random in the cells of most organisms, but there are groups, for example, dipteran Insects, where homologous chromosomes are found close to each other - somatic pairing - and the phenomenon is now known to be more common than once believed. Frequently in slide preparations of the Characeae, we have seen some Indication of more than chance positioning of equally sized chromosomes. A full knowledge of the entire process of meiosis of polyploids is essential in determining the origin of individual genomes which have formed the species. Slides showing zygotene pairing and formation of chiasmata at dip- lotene may furnish keys to phylogeny of the individual as to whether auto- or alloploidy are involved, for example, 3 or more chromosomes COMB together to form multivalent chromosomes. As soon as it is possible to germinate spores of Characeae at will in the laboratory, and studies are -12- in progress on this problem, we can begin studies of meiosis which is almost undescribed in the literature on the Characeae.

Lacking meiotic proof, we are setting up the chromosomal configurations of various species, comparing sizes of individual chromosomes to show the existence of pairs and relying on the odd occurrence of a true somatic pairing picture to strengthen this. In this connection, the work fells interest- ingly in line with discoveries by Berger, Grell, and Huskins in plants Where at least a few cases of "somatic pairing" were observed in ordinary untreated material showing varying degrees of somatic reduction leading to reduced nuclei. The incidence of this was greatly increased by an abundance of RNA and various chemicals from extra'-cellular sources.

A series of modern works (1947-) has revealed that the concept that all cells of an organism contain the same number of chromosomes (a pillar of cytology) must be reviewed in a different light. The Huskins school was possibly respon- sible for the initial spark in this direction, indicating that not only are polytene and polyploid cells common in differentiating tissues but in ordinary diploid tissues of plants for instance there was a fairly high degree of endopolyploidy present. Once again, although chemicals could increase the incidence, there is a degree of polyploidy or at least polysomy (some chromosomes doubled) naturally present. The Belgian school was struck by this condition as seen in radiated material where it was difficult to deter- mine numbers following radiation. In untreated Characeae these conditions are the exception, not the rule, but we have found a striking example of endopolyploidy in Chars leptopitys. In this species (from Wood's collection 60-10- - 11-1) the fertile branchlets are arranged in whorls in loose heads. The antheridia are mostly about the same size but here and there some are strikingly larger (and differentiating toward maturity?). The number 14 was found constantly in most antheridia but in the larger ones, mixed with filaments containing mature sperm, were filaments of dividing cells of 35, 42, etc., in paired and connected groups of 3-4 chromosomes. Similar conditions have been seen in material recovering from the "shock" of shipping, and in N. flexilis treated with X-radiation we have observed induced pairing.

From observations so far, it is possible to see in some groups a "typical" cytological picture, Nitella can be dis- tinguished from Chars, and within the genus Chars such species as C. braunii are quitedistinct from C. vuiTigFrs and C. contraria. Different chromosomes vary considerably in size and configuration within a nucleus but homologous chromosomes appear to be relatively constant in form from cell to cell.within a species. -13- VI. E. Special Studies

1. Antheridial mitoses studied with X-radiation treatment. The bearing of cytological data on the taxonomy and morphology of extant Characeae, on the interpretation of the evolution and speciation of this group, and on an understanding of the potentialities of Cheraceee speciation, coupled with an almost complete lack of information in this field has been the original motivation for research in this field. Following the initial work, directed at establishing as many facts as possible concerning the karyo- types of members of the group, separate and special study seemed indicated. Here was a group of plants whose constitution seemed basically ploid; the homologues showed a certain normal tendency towards pairing; on occasion the genomes do continue to divide; and, under adverse conditions supplied by application of chemicals, and radiation), these tendencies may increase to a striking degree. In the spring of 1960, work was begun on radiation studies using primarily the large and constantly available population of Nitella flexilis from Doe Run, Meade County, Ken- tucky. Comparisons were begun with irradiated N. opace from Richmond, Indiana, and the naturally occurring and cytologically variable Chara contraria from the Fort Knox area, and the similarly aberrant C. sejuncta from a sinkhole pond near Doe Run. The work was carried out through the generous cooperation of Mr. Jan Balling, using the facilities of the Fort Knox Radiation Laboratory.

Moutschen and Dhamen (1956) had subjected Chars vulgaris (there is reason to believe this species may have been con- fused with a closely similar form of C. contraria) anther- idle to (soft?) X-radiation doses of 1000, 5000, 10,000, 20,000, 50,000 and 100,000 roentgens and had studied premitotic, mitotic, telophase stages and the completed or incompleted sperm. They noted most aberrations -- such as fragmentation, fusion of chromocenters, amitosis, etc., as exaggerations of ordinary, naturally occurring phenomena; they noted again that spermatogenesis could proceed to com- pletion after doses up to 10,000 roentgens and noted a tendency in treated material for the 28-chromosome nucleus to fragment into four equal groups, indicating a basic genome of 7? In another study (1956) these workers reported that Chars vulgaris, treated with varying concentrations of maleic and isonicotinic hydrazides, produced interesting radiomimetic effects. Little information was given as to radiation equipment, standards used and other data necessary for repetition of this work. In applying radiation techniques to the present project our own standards were established and a search made for radiation tolerance and limits of the material at hand. Results were compared with the apparently similar stages in Moutschen and Dhaments work, compared with their results with radiomimetic chemicals, and with standard works on the action of radiation on living cells. (Lea, 1959). -14- Radiation Techniques Employed. Material to be irradiated was selected in the morning from cultures grown in the University greenhouse, and transported to the Army Medical Research Laboratory at Fort Knox, where irradiation was accomplished as soon as possible (around 9 a. m 1 Shortly before irradiation, a convenient aliquot of the alga was weighed and transferred to a lucite petri dish. Thereupon, 40 cc of the water in which the alga had been transported was added to the petri dish, and its cover fitted into place. The petri dish dimensions were: top, 12.4 cm outer diameter and 12.3 inner diameter; bottom, 12.0 cm outer diameter and 11.9 cm inner diameter. The wet weight of the alga that would readily fit into the petri dish was approximately 15 g. Following irradiation at room temperature (22 degrees centigrade) the algae were re- turned to their container for the return to the University Laboratory around 5 p.m. This trip required an hour each way. Irradiation was accomplished by means of a 250 kilovolt potential General Electric Maxitron x-ray machine operated at 250 kilovolts and 30 milliamperes with mm Cu plus 1 mm Al added filtration. Target distance, as measured from the top of the lucite petri dish to the x-ray tube, was 30 centimeters in most experiments, 45 cm and 56 cm in others. Air dose was determined by means of a Victoreen roentgen- meter and its 250 roentgen thimble chamber. For purposes of dosimetry, the thimble chamber was placed on top of the lucite petri dish containing only 40 cc of water. The lucite petri dish rested upon a lucite plate (18,8 cm in diameter and 0.7 cm thick) that in turn rested upon a stain- less steel stand. These items and the thimble chamber were centered with respect to the radiation cone, and three separate dose rate measurements were taken that gave an average dose rate of 400 r per minute. The total dose delivered to the aliquot of algae was varied by altering the time of radiation. An explanation of the full dose, half dose, and eighth dose, etc., is given in the following table. These fractions of doses are made with reference to irradiation time and not always to the same total dose received. For example, an eighth dose means the specimen was irradiated for 94 seconds. In order to find the total dose delivered, one must multiply this time in minutes (1.57 minutes) by the dose rate for a particular experiment. Most of our data are based on the 30 cm target distance and assumed dose rate of 400 r per minute. Full Dose 752 seconds 12.53 minutes iDose 376 seconds 6.26 minutes 4 Dose 188 seconds 3.13 minutes 1/8 Dose 94 seconds 1.57 minutes 1/16 Dose 47 seconds 0.78 minutes 1/32 Dose 24 seconds 0.40 minutes 1/64 Dose 12 seconds 0.20 minutes 1/128 Dose 6 seconds 0.10 minutes 1/256 Dose 3 seconds 0.05 minutes -15- It is to be noted that these experiments are qualitative and the dosimetry a relative matter. Since the dosimetry was done in the air, the roentgen values of dose are relative to sir and cannot be stated absolutely for the tissues. More importantly though, the endpoints of these experiments can be duplicated by anyone with the information provided here. A total of 17 separate experiments were carried out on Nitella flexilis, and 2 on N. opace. Work was discontinued during the summer because or the growing maturity of the antheridial filaments, and the warmer weather was contributing adverse effects during the transportation of the material the considerable distance for irradiation. (This adverse factor will now be overcome by the installation of X-ray equipment in the laboratory in the Department of Biology as noted below.) Variations in length of time and dosage of radiation, produced the classical stages of biological damage, from a complete destruction of the cell through various stages of physiological clumping, physical breaks and bridges in chromosomes, to A complete absence of an effect on the cell - even the expected temporary inhibition of division with the lesser dosages. Material of the original experiment receiving a dosage of 5000 roentgens survived in some instances for nine days. Cells died steadily. Sub- sequent experiments at a half and a quarter of original dose showed the beginning stages of recovery after a couple of days with lagging chromosomes, clusters of metaphase chromosomes, anaphase bridges, and a very few successful telo- phases, accompanied always by occasional dead cells or filaments of dead cells. The change down to 1/8 strength was of great interest to the investigators and in this range of irradiation much more detailed work will be carried on. For three days all the cells that had been irradiated in the resting stage or early prophase remained resting with no evident attempted recovery. This period was followed by a long slow recovery time of over six days. Divisions showed again the various aberrations and anaphase bridges were especially prominent. By the sixth day several cells showed an interesting pairing of like chromosomes and the beginning separation into two like masses. Repeats of this experiment with the Wattle dosage gave such e close approximation of results in most cases that it is believed that at least the rough limits of irradiation and range of behavior have been established for N. flexilis. From this experience with "hard" X-rays, it is concluded that the larger dosages utilized by Moutschen and Dhamen (1956) were of the "soft" X-ray type. Nitella °par was subjected to two irradiations which, unfortunately because of filter changes), were not exactly comparable to dosages applied to N. flexilis. One dosage Inhibited division of cells in younger antheridia for 48 hours but they recovered with no apparent ill effects. Older cells continued normal divisions. Irradiation results in a particularly striking spreading of the chromosomes seen in squashes and some figures revealed the 6-chromosome complement of N. opacn with 5 long chromosomes and one short one. -16- No indication of two completely balanced sets of (3 plus 3) homologues was apparent here. The other dosage scrambled all divisions in the cells and dying cells were soon in the majority. In two instances naturally occurring material of the Chara- cene has been examined and found to have an appreciable proportion of mitotic divisions in antheridial nuclei. In Chars sejuncta collected at Station 6, Doe Run area, normal divisions were present but most filaments showed masses of resting nuclei multibudded, nuclei divided in prophases, and late prophases divided into two equal masses. There were bridges present and an increase in the numbers of extra large cells. No known chemical or radiation effects have been postulated for the area. The population is to be investigated further.

Chara contraria from the Fort Knox area has always shown cytological radiomimetic effects as widely aberrant numbers ranging from rare 14's up to 42's, as well as normal filaments with the expected 28's. These plants were collected from a small lake frequented by fishermen and known to be annually treated with doses of sodium arsenate for the control of aquatic vegetation. C. contraria from other collecting sites shows a lower (but still unusual) proportion of lobulate nuclei and this species seems particularly suscept- ible to extracellular influences as the Belgian workers have found. This species will be studied further under natural conditions and after treatments.

Preliminary results will be published in a short intro- ductory paper in which the Characeae species thus far examined will be shown to be sensitive to relatively small dosages of X-radiation.

E2. A Study of Chars braunii Gmelin.

The cosmopolitan species, Chars braunii, which belongs to the ecorticate haplostephanous section of the genus, includes several varieties and forms certain of which are treated as of specific rank by some workers. Our own collections which in- elude several more minor variations as well as the var, schweinitizii, sometimes maintained as a species (as Prescott, 1950) were made in the area ranging from southwestern Quebec to southern Indiana, over a period of years from 1957-1960. This has permitted the checking of nearly all our collections on more than one occasion. In our counts for this species, we have found invariably a count of 14 chromosomes. This lends additional weight to the concept of a single large species for these plants (if more were needed). On the other hand, although most ChPrologists are in essential agreement on what forms are to be included in the species, the placing of the species in the genus Chars has been questioned in the recent paper (1959) of the Belgian worker, Claude Gillet, who takes up the old combination, Charopsis braunii for this species. This aspect of the systematics of the Characeae, depends on conclusions based largely on cytological data. -17- It may well he determined finally on the basis of additional cyto-taxonomic evidence. The species, Chara braunii, was proposed by Gmelin in 1826. Bischoff redesignated the species Chara coronata in 1828. Despite its lack of priority, the use of the latter name was continued until recent years because it was des- criptive. In 1843, Kiltzing erected the genus Charopsis, distinct from the Section Chareae, and in it placed the species Chara braunii Gmelin as Charopsis braunii (Gmelin). This separation was based on the following TETITEFT 1) the absence in Charopsis braunii of all cortication resembling that in corticated species of Chara, 2) the haplostephanous character of the ring of stipulodes, 3) the disposition of the branchlet segments, and 4) the arrangement of the repro- ductive organs. After listing these points from KUtzing, Gillet (1959) dismisses the characters in 3 and L. with the comment that they do not present anything essentially different from Chars. He makes considerable use of the haplostephanous condition in building his system of classification of the Chareae. In his study of the cytology of Charopsis braunii, Gillet examined the various phases of antheridial mitoses. He was impressed particulmrly by the 2-3 large and several smaller chromocenters conspicuously displayed at interphase, and by his count of 12 chromosomes at metaphase. Since 12 is a number characteristic of the Nitelleae, Gillet concluded that the totality of evidence from cytology, morphology and phyto- sociology was sufficient to remove Charopsis from the tribe Chareae and place it close to Nitella in the Nitelleae. In considering his findings in the cytology of Charopsis braunii, we are not prepared to state that Gillet is mis- taken in making n count of 12 which differs from our consis- tent 14. It is to be noted though that a previous count from European material (Karling, 1928) agrees with our 14 in American materials. As for the reestablishment of the genus Charopsis to include Charopsis braunii, we feel the change may have considerable merit but it can scarcely be based on a count of 12 chromosomes, nor, for the same reason, can the genus be regarded as having an affinity with the Nitelleae. There is, however, another line of evidence which tends to distinguish C. braunii, and the remaining members of the Ecorticatae from the corticated species of Chars. This is the degree of correlation between polyploidy and sex as expressed in the arrangement of gametangia whether monoecious, dioecious or disjunct. In nil the corticate species, with but one exception so far, this correlation consists of haploid species being dioecious (or disjunct) whereas the diploid species are invariably monoecious. This positive correlation seems to indicate that in the genotype of the Corticatae there is a common mechanism controlling sex expression in haploid species which produces n common but different effect on species of the Corticatae concomitant with change to the higher level of ploidy. In the Ecorti- catae, all species whether dioecious or monoecious are invariably haploid (N = 14) thus indicating that there is no ploidy in this group (beyond this level). Moreover, because monoecious species have evolved without benefit of -18- ploidy, the mechanism controlling sex expression in this group, apparently differs from that in the Corticatae where the correlation (or direct relationship between sex and ploidy does not exist.) Such a conclusion as this may serve to substantiate the separation of Charopsis braunii from the corticate species of Chars. At the same time, it also has the effect of greatly enhancing the relative importance of corticate vs. ecorticate characters in the systems of classification of the Chereae and somewhat diminishing the importance of haplo- vs diplostephanous characters, because it is apparent that the other ecorticate members of the genus Chars must also be involved by the same cytological considerations which pertain to Chars breunii whether the species are haplostephanous or diplostephanous. Thus the species Chars britonii is monoecious, diplostephanous, - critical in an evaluation of the relaTIVT importance of the stipulodes in classifying the Ecorticatae. With a complement of 14 chromosomes, Chars brittonii should be included with the haplostephsnous species Chars braunii, Chars australis and other ecorticate siDTTITF which must remain together as a group, which may well be regarded as generically distinct from Chars. Our results and conclusions as to the Chareae Ecorticatse are to be discussed in a forthcoming paper which will include the following. Corticatae (Section of Chars) 1) Ploidy does exist in the Corticatae to the 28 chromosome level.

2) The monoecious (homothnllic) condition with 28 chromosomes has evolved from the dioecious (heterothallic) condition with 14 chromosomes.

3) The monoecious disposition of sex organs is directly related to the higher level of ploidy.

4) Changes in ploidy and disposition of sex organs are independent of the condition or number of stipulodes which are variable within this group. Ecorticatne (Section of Chars) 1) Ploidy beyond the 14 chromosome level probably does not exist inthe Ecorticatae.

2) The monoecious condition with 14 chromosomes probably arose from the dioecious level with 14 (or ancestral 7) chromosomes. 3) Monoecious disposition of sex organs not a concomitant of ploidy (beyond the 14 chromosome level). 4) Changes in ploidy and disposition of sex organs are independent of condition or number of stipulodes which are variable in this group. -19- E3. Studies in the Char° contraria-Chara vulgaris Problem

In 1956, Wood and Muenscher united the two controver- sial species Chara vulgaris and Chara contraria as C. vulgaris L. They mentioned the fact that this had been done previously (Cosson and Germain, 1882) in a combination of the two as C. foetida var. contraria, and that on the basis of studies of populations of the two forms in New York concluded that such overlapping variation was involved that the plants could only constitute one species. The type plant for the grouping was described as possessing a main axis which was diplostichous and tylancanthous (with primary cortical cells more prominent than a single series of secondary cells); branchlets with 7-9 corticated segments, typically with a terminal, ecorticate end cell. Any attempts to separate these two groupings -- such as that of Migula on the basis of oospore size could be shown usually to break down under wider population analysis. Other workers, (for example, Daily) continue to separate the groups on the basis of a broad grouping of characters.

From a study of the chromosome numbers it was early seen that the group recognized by many as "C. contraria n possessed (and consistently so) 28 chromosomes, Irc. vulgaris" had 14. As seen in Table 1, "contraria" sampled at 3 collecting sites in Kentucky, 5 in Indiana, 1 in California and 1 in New York all showed 28; the "vulgaris" sampled at 5 sites in New York, 1 in Rhode Island had the 14. In working with the plants, although a degree of character overlap was apparent, it became increasingly apparent that a working separation was possible. This conclusion was reinforced in the summer of 1960 by finding a small farm pond (Jenkins' Pond, near Cayutaville, New York) in which easily separable populations of C. contraria and C. vulgaris were growing. Each population had free access to the territory of the other but remained separated apparently by the conditions of a microhabitat formed by a cold spring entering one edge of the pond. Jenkins Pond was constructed in 1947-48 over an area with several springs, to a depth of nine feet. The pond has been stocked with trout and used experimentally by fish hatchery workers thus providing ample opportunity for the introduction of Chars species. Forming a dense bed near a spring at the edge of the pond up to 1 ft. depth, were short, slender plants of C. vulgaris (N equals 14) all of which were definitely aulacanthous, doubly corticate and the smaller primaries represented by deep furrows in the cortex, broader secondaries as higher ridges. Bract cells were long, branchlets corticated up to the last fertile node but not beyond, branchlet cells somewhat barrel-shaped. In the limited area, occupied by C. vulgeris,. the temperature ranged 10 degrees F. cooler than the other surface waters of the pond.

Growing in deeper water, 2-3 feet, at some distance from the C. vulgaris, was a more extensive population of C. contraria. N equals 28). These plants were all definitely tylacanthous (particularly clear on younger internodes), the stipulodes were well developed end branchlets corticated above the last fertile node. The plants were larger, more -20-

robust, longer and more spreading bronchlets, longer bracts and internodes than in the C. vulgaris in this pond. Jenkins Pond would seem to be a clear-cut ecological control for the study of the two groups and will be investigated further. The Belgian workers (n.b. Gillet, 1959) have noted 2 chromosome numbers, 14 and 28, in populations of C. vulgaris L. This they interpret as indicative of the presence of polyploids within the population of a single species. We interpret this as evidence for the presence of two separate species.

Ell.. Life Cycle Studies. We propose to make a special study of the biology and ecology and entire life cycle of Nitella flexilis (L.) Ag. as a subproject of the overall cytotaxonomic survey of the Characeae. Nitella flexilis is widely regarded as a primitive member of the genus. Its size, and particularly its large and spreading branchlets, not condensed into heads in the early stages, mark it as an unspecialized species. The entire life history, including the cytological details, are well worth studying in this species. Comparison will be made especially with the related and dioecious species, N. opaca. Such a study is being made by Mr. Donald R. Tindal]. andthe results will be presented as partial fulfillment for the requirements for the degree of Master of Science to be completed in the first half of 1962. A large and constant population of Nitella flexilis is available for study in a nearby stream (DoeRun, Meade County) with an aquatic environment which promises to yield some interesting ecological data. Ecological studies, already made, indicate that the quality and flow of water, the constancy of water temperature (56 deg. F.), are such as to yield stable conditions permitting a study of season variations as well as developmental stages under natural conditions that one could scarcely hope to duplicate in the laboratory. It is possible to assume from observations, already made, that a form of N. flexilis, which has been given specific rank, is merely the protnndrous winter form of the species which is to be succeeded in late spring by the usual monoecious conditions. Developmental stages of proembryo and young gametophytes of N. flexilis and N. opaca are now in cultivation.

E5. A special point should be made of our intention to study intensively the Characeous flora of northern Mexico and adjacent areas in the southwestern United States. From the literature, and from our own sampling of the North American Characene we have concluded that this area is the most promis- ing, in our immediate reach, for yielding new and interesting data on the group. The study will be as broadly based as resources permit and will include not only cytotaxonomic but also floristic and ecological development as well. The need for further study in this area is very great. Many species were collected by Charles Wright, botanist of the -21- Mexican Boundary survey (1851-1852). These passed through the hands of the European charologists Engelman and Braun, and the American, T. F. Allen. Some species, although often cited, have not been taken since the original collection. This study is in progress largely through the efforts of Mr. Donald R. Tindall, the results will be presented as partial fulfillment for the requirements for the degree of Doctor of Philosophy. Two expeditions to Mexico have now been completed and others are projected during the next three years. Collections are noted in the accompanying tables. -22- VII. Description of Proposed Research

A. Abstract.

The further work on the cytology and morphology of the Chars- ceae will be aimed at extending and continuing the preliminary study along the lines outlined above. In addition to the accumulation of more cytological data from eastern North America, extensive collections now available and yet to be collected from Mexico, Australasia, and other new areas will be examined. More extended analyses involving the use of radiation, chemicals, breeding culturing, and ecological data will be employed in the program.

B. Thd Objectives of the study may be stated briefly as follows:

1. To continue the studies in the cyto:taxonomy of the Characese. 2. To analyze the presumed polyploids by treatments designed to show artificial reduction and other indications of chromosome homology by means of a. Radiation treatments b. Chemical treatments c. Elucidation of the meiotic divisions

To attempt the synthesis of polyploid chromosome comple- ments by means of

a. Chemical treatments. b. Breeding experiments 4. Life cycle studies, correlating changes in form related to seasonal and environmental factors.

C. Significance and Relationships of the Study Relationship to previous work.

Our previous work on this project is summed up in the resume of progress to date as given above. It has been an attempt to amass information concerning the cytology of the Characeae from as broad a base of understanding as possible. Most of the information is found in the chromosome counts of many new species of Chars and Nitella, new genera Tolypella and Lamprothamnium, and new populations of those few species whose accounts have appeared In the scanty literature. There has been a beginning made in drawing cytotaxonomic conclusions as to relationships in the group. Future work is planned to continue the same type of study with a scope much broader in geographical area, and with the use of a wider range of study methods. Similar and Related Work.

Until the present study, there has been little cytological investigation of the Characeae in the United States. -23- The earlier European workers, studying life cycles in cryptogamic plants, became interested in the Characeae. In 1897, Debski, who was searching for the reduction division in Chars fragilis, found 23, 24 and 25 chromosomes in mitotic divisions of the antheridial filaments. He interpreted the 23 as "hidden", the 25 as a probable knife dissection, and arrived at 24 as the chromosome number for this species. The difficulties encountered in working with sections are summarized in Debskils conclusion that : "In Mittel ergehen sich aber 24, und ich habe nie weniger als 20 und nie mehr als 28 gefunden." Oehlkers (1916) by microtome sectioning at 30u found 24 chromosomes in Chara fragilis. Lindenbein (1927) and Telezynski (1929) made lists, including their own counts, of a few species of Characeae but neither of these workers had enough information to critically evalu- ate the earlier published records. Because of the differ- ence of opinion among the older workers, it is apparent that verification of chromosome numbers by use of newer methods becomes necessary. Material of many species, and material from far-ranging sources for each cosmopolitan species is needed before full use be made of the cytological data to be found in the Characeae. Because of a paucity of information, there was little attempt by the older workers to use chromosome number as a basis for evaluating the importance of the classical morphological and taxonomic conclusions regarding the Characeae. However, Telezynski (1929) suggested that Chars fragilis with the then accepted chromosome count of 2)4 wasan autopolyploid. In 1955, MacDonald and Hotchkiss made use of a common chromosome number (1)4) in combining the two Australian species Protochara australis and Chara australis. Basing their work on X-radiated material, Moutschen and Dhamen (1956) suggested the tetraploid nature of Chara vulgaris. A relationship between ploidy and sex in the genus Chnra was suggested (Hotchkiss, 1958) as well as a polyploid series with 14 and 28 chromosomes in that genus. Gillet (1959a, 1960) presents chromosome numbers for several species from Belgium and adjacent France, and, more dramatically (1959b), maintains the genus Charopsis Ktitzing (for Chore braunii) which is placed close to the Nitellene on the basis of cytological evidence.

Floristic and taxonomic work on the Characeae is being pursued Vigorously in various countries by recent workers: the Americas (Daily, 19)44, 1945, 1946, 1950, 1953, (wood, 19)47, 1948, 1952, 1956), (Allen, 195)4), Horn af Rantzien, 1950); Australia, (Williams, 1959); Belgium (Gillet, 1959a, 1960); France (Corillion, 1957), and others reported in progress elsewhere. These serve to supplement the older works, particularly in areas where the characeous flora is only partially known. In botanically well-known areas, as the British Isles, not much remains to be added to such works as Groves and Bullock-Webster (1920,192)4) but in North America the works of Allen (1888-1896) and Robin- son (1906) need extensive additions and revision for com- pletion. Other areas of the world- as Australasia will -24- continue to yield much new information on this group of plants. In a ddition to the usual taxonomic procedures, the techniques of palynology have been applied with great success to the spores of living as well as fossil members of the Characeae by Horn af Rantzien (1959a,1959b) and Daily (manuscript in preparation), thus adding a new facet of information bearing on the systematics of the group. Added to the external morphological featurespalready well-known for many species of the Characeae, cytological information will provide yet another basis for the work of the systematic botanist dealing with the Characeae. We ourselves have already noted several avenues of enquiry along this line, some of which have been noted above. In some cases cytological information bears on the relation between two species, while in others, the significance may be much broader and relate to the nature of a genus or larger taxon. It is our intention to make all our information available, as soon as it can be deemed reasonably precise, to taxonomists, monographers and others through early publication of the details.

D. Plans and Procedures

1. Methods for counting chromosomes will continue much as outlined above. This work includes the making of permanent slide mounts, preservation of material in fluid and dried herbarium specimens. Graduate assistants will be trained and guided in these procedures.

2. In addition to the radiation treatments outlined above, the use of radiomimetic chemicals, (following work already familiar to Mr. Sawa) will be initiated in cultivated plants. In addition, colchicine, following standard procedures, will be applied to antheridial mitoses. The present information concerning meiosis is only fragmentary simply because it has been impossible to demonstrate these important divisions within the thick, lime-filled walls of oospores whose germination time is entirely unpredictable. This will be a "tough nut to crack" and various leads involving pretreatment of zygotes, fixing, and squashing hate elreeidy been suggested by the team of investigators. Breeding experiments will utilize the approximately 15 different species of Characeae brought into cultivation largely through the efforts of Mr. Tindall. Techniques will follow those outlined in MacDonald and Hotchkiss (1957). Others will be added from time to time. Life cycle studies, which have been largely the responsibility of Mr. Tindall, also will depend on cultivated materials,

E. Bibliography A selected bibliography of pertinent literature is appended to this research proposal. -25-

VIII. Project Personnel and Cooperating Biologists.

The principal investigator will continue to be Dr. Arland T. Hotchkiss, Associate Professor of Biology, University of Louisville. Dr. Hotchkiss has published seven scientific papers which are directly concerned with the algae or cytotaxonomy. Dr. Hotchkiss received the degree of Doctor of Philosophy from Cornell University under the direction of Dr. Walter Muenscher in 1949. Graduate training in cytology was taken under Dr. L. W. Sharp and Dr. L. R. Randolph. Since leaving graduate school Dr. Hotchkiss has served as Assistant Professor and Associate Professor of Biology at the University of Louisville, and on leave of absence from Louisville, as Fulbright Lecturer in Biology at Robert College, Istanbul, Turkey. This was followed by four years as Lecturer in Botany at the University of Sydney, Sydney, N. S. W., Australia.

Interest in the Characeae was generated as a graduate student under the tutelage of Dr. Muenscher. Field work during this period included studies of the algal flora of Bergen Swamp, New York; a summer survey of the aquatic vegetation of eastern Lake Ontario and the upper St. Lawrence river with reference to mapping duck food plants for the New York State Department of Con- servation; a second summer surveying the aquatic vegetation of the Connecticut River from Springfield to Long Island Sound for the Connecticut Fish and Game Commission. The aquatic vegetation indluding charophytes were studied in Got- land and the Gulf of Bothnia (Chara baltica was encountered here) on field trips with Dr. B. Peterson and Dr. Mats Waern in connection with the International Botanical Congress in Sweden. A few charophytes were seen in Turkey, and in Australia a most valuable experience with new and endemic species, and genera of charophytes renewed interest in this group. In Australia, observations and experiments with Protochara australis and Chara australis led to the conclusion (with M. B. Macdonald) that these two species were not generically or specifi- cally distinct. Chromosome numbers and breeding experiuents were an important basis of this work. Dr. Hotchkiss will continue to devote two full time summer months to the project as well as considerable time during the remainder of the year. His principal duties will include active participation in the field work, the cytological investigations, as well as supervision of the graduate students doing research, and in the general programming and execution of the studies,

The efforts of the principal investigator in carrying out the aims and Objectives of the study of the Characeae were greatly enhanced and strengthened by the generous contributions of time, labor, and guidance expended by quite a long list of individuals.

Mr. Donald R. Tindall, graduate student, presently Instructor in Botany, University of Louisville, is studying the Characeae and is now completing the requirements for the degree of Master of Science. Mr. Tindall will begin full time work on the project in June 1961, other than time required to fulfill other requirements for the degrees of Master of Science (1962) and Doctor of Philosophy. Mr. Tindall's contributions to the project are noted under the lists of collections and special studies.

Mr. Takashi Sawa, currently research assistant to Professor Kozo Imahori, Professor of Botany, University of Osaka, Japan, has indicated a desire to -26-

further his studies in this laboratory along cytotaxonomic lines in the Charace- ae. He is strongly recommended by Professor Imahori who is the leading Japanese student of the Characeae. He is also highly recommended by Professor T. Sugiura, Emeritus Professor at the Botanical Institute, Osaka, with whom he formerly served as assistant, and co-authored certain papers (1957, 1958). His work has been cytotaxonomic in nature, training in the higher plants, and he appears to be keenly interested in karyoanalysis. He has been working on karyological studies of the influences of certain chemicals on root-tip chromosomes and seems to be well qualified to pursue general and experimental cytological work in the Characeae. Hc will be a welcome worker in the proposed studies. He is also recommended by Professor I. Sato, Osaka University .

Mr. Sawa will begin full time work on the project upon his arrival in Louis- ville during the summer of 1961 (Probably August), other than the time required to fulfill other requirements for the degree of Doctor of Philosophy at the University of Louisville.

Dr. Doreen Maxwell Hotchkiss, the project was very fortunate in having the part-time services of a competent cytologist in the person of my wife who is giving invaluable aid without pay, in the laborious task of preparing and analyzing chromosome counts in the home lab and on trips to the field.

Dr. Louis A. KruMholz, Professor of Biology, University of Louisville,(permanent member of the National Academy of Sciences - National Research Council Committee on the Effects of Atomic Radiation on Oceanography and Fisheries, Editor of Wildlife Monographs) is serving as consultant in radiation biology.

Mr. Jan Balling, Instructor in Biology, University of Louisville, is serving as consultant and technician in X-ray techniques.

Mrs. F. K. Daily, Butler University, guided and fed us in the field, and con- tributed valuable specimens, identification of specimens submitted, and information on collecting in Indiana.

Dr. Richard Wood, University of ii.hode Island, has made many contributions to the study and more recently sent several shipments of many valuable cytological specimens from Australia as noted under collections.

Specimens have also been received from the following:

Dr. V. Proctor, Texas Tech. Dr. G. Cole, Arizona StateUniversity Dr. L. A. Whitford, North Carolina State College Mr. Gene Blankenbaker, Michigan State University

IX. Materials, Equipment and Facilities.

The University of Louisville has the following equipment and facilities available for the project:

1 Leitz, ortholux research microscope and optics $2200.00 Laboratory and equipment for radio-assay of biological materials 5300.00 1 General Electric, 250 KV, X-ray machine Office, laboratory, greenhouse, herbarium, darkroom space for the project. X. Expenditures -27- A. Salaries At present, Dr. Hotchkiss receives a salary of S7500.00 for a nine-month period. It is expected that the project will continue to require two full months of his time in summer. Two graduate students will spend full time on the project. 1st Yr. 2nd Yr. 3rd Yr. Total Dr. Hotchkiss,(Sr. 1300.00 $ 1300.00 $1300.00 $ 3900.00 (Investigator) Mr. Tindall (grad. stu.) 3000.00 3000.00 3000.00 9000.00 Mr. Sawa (grad. stu.) 3000.00 3000.00 3000.00 9000.00 Total 7300.00 7300.00 7300.00 21900.00 B. Travel, and other items Mileage and gasoline for 300.00 300.00 300.00 900.00 short trips from Louisville Expeditions to northern 500.00 500.00 500.00 1500.00 Mexico (2-3 wks, 2 people) Travel for principal investi- 60.00 60.00 60.00 60.00 gator and grad. students to one national meeting each Typing, mimeographing, office 40.00 40.00 40.00 120.00 supplies

Total S900.00 4t 900.00 $900.00 $2700.00 C. Permanent Equipment 1 small boat trailer 180.00 180.00 Air cooling eopkoment for 400.00 400.00 greenhouse culture room Herbarium equipment 150.00 150.00 Culture containers 60.00 60.00 60.00 180.00 Photographic equipment 40.00 40.00 40.00 120.00 Microtome (ultra thin 160.00 160.00 sections) Total S840.00 $250.00 $100.00 31190.00

(continued) -28- D. Expendable equipment Collecting containers and $ 80.0o 80.00 80.00 $ 240.00 supplies Chemicals, stains 80.00 80.00 80.00 240.00 Slides, cover glasses 60.00 60.00 60.00 180.00 Miscellaneous glassware 200.00 50.00 50.00 300.00 Total 420.00 270.00 270.00 960.00 E. Publication Costs, reprints 50.00 50.00 100.00 F. Other direct costs (none) G. Total direct costs 9,460.00 8,770.00 8;620.00 26,850.00 H. Indirect Costs (20%) 5,370.00 TOTAL $ 32,220.00

X. There are no other sponsors suggested for this project and no funds from other sources are being requested.

Arland T. Hotchkiss Associate Professor of Biology -29-

BIBLIOGRAPHY

1. Allen, G. 0. 1954. An annotated key to the Nitelleae of North America. Bull. Torr. Bot, Club 81: 35-60. 2. Allen, T. F. 1888-1896. The Characeae of America. New York. 3. Corillion, R. 1957. Les Charophycees de France et d'Europe Occidentale. Tray. Lab. Bot. 'Univ. Libre d'Angers, 11 et 12, 1-499.

4. Cosson, E. 1882. Atlas de la flore des environs de Paris. Paris. avec E. Germain 5. Daily, F. K. 1944. The Characeae of Nebraska. Butler Univ. Bot. Stud. 6: 149-171. 6. -----. 1945. The Characeae of Indiana - A Preliminary Report. Butl r Univ. Bot. Stud. 7: 124-131. 7. - . 1946. Species of Tolypella in Nebraska. Butler Univ. Bot. Stud. 8: 113=117:;. 8. - . 1950. Tolypella prolifera Leonh. in Indiana. Butler Univ. Bot. Stud. 2: 273-276. 9. - . 1953. The Characeae of Indiana. Butler Univ. Bot. Stud. 11: 5-49. 10. Debski, B. 1897. Beobachtungen Uber die Kernteilung bei Chara fragilis. Jahrb. wiss. Bot" 32. U. Delay, C. 1949. Observations cytologiques sur les Characees. I. Llevolution du noyau pendant la spermiogenese de Chara vulgaris 1. Rev. Cyt. Biol. veg. 11: 315-332.

12. Geitler, 1. 1948. Nver die Teilungsrythmen in der spermatogenen Paden der Characeen. Coster. Bot. Zeit._25: 146-163.

13. Gillet, Cl. 1959a. Criteres cytologiques, anatomiques et ecologiques en faveur du maintien d'un genre Charopsis chez les Charophycees. Bull. Soc. Sci. de Bretagne 34: 65-73. avec Corillion et Guerlesquin.

14. - -. 1959b. Nombres chromosomiques de plusieurs especes de Charophycees (genres Nitella et Chara). Rev. Cvt. Biol. Veg. 20: 229-234. 15. - -.1960. Les Charophycees de l'Ardenne et des regions voisines. Bull. Soc. Roy. Bot. Belgique. 92% 197-228.

16. Horn af Rantzien, H. 1950. Charophyta reported frotheiLatin America. Arkiv fbr Bot., Ser. 2, 1: 355-411.

17. - - 1959a. Recent charophyte fructifications and their relations to fossil charophyte gyrogonites. K. Svenska Vet. Akad. Arkiv f. Bot., ser. 2, bd. 4: 165-332. 18. - . 1959b. Morphological types and organ-genera of Tertiary charophyte fructifications. Ltockholm Contr. Geol. 4: 45-197. 19. Hotchkiss, A. T. 1950. The vegetation of Bergen Swamp. IV. The Algae. Proc. andbester Ac ad. Sci. 9: 237-364. (continued) -30- 20. - -. 1954. A new species oZ Hibbertia Andr. from T4estern Australia. Proc. Linn. Soc. N.S.W. 79: 29-33. 21. - . 1955. Geographical distribution of the lupomntiaceao. Jour. Arnold Arb. 36: 385-396.

- 22. - 1957. Pollen and pollination in the '11pomatiaceac. Proc. linn. Soc. N.S.W. 83: 86-91.

23. - • Some chromosome numbers in Kentucky Characeae. Trans. Kentucky Acad. Sci. 19: 14-18.

24. Karling, J. S. 1928. Nuclear and cell division in Nitella and Chara. Bull. Torr. Bot. Club 53: 319-379.

25. Lea, D. 1956. Actions of radiations on living cells. 1-416. Cambridge. 26. Lindenbein, VT. 1927. Beitrag zur Cytologic der Charales. Planta 4: 437-466. 27. Macdonald, M. B. and A. T. Hotchkiss. 1955. An estipulodic form of Chara australis R. Br. Proc. Linn. Soc. N.S.W. 80: 274-284. - 28. Mendos, E. J. 1946. Mitosis in the spermatog mous threads of Chara vulgaris L. var. longibracteata Katz. Portugaliae Acta Biol. 1: 251-265. 29. Moutschen, J. and Dahmen, M. 1956. Sur les modifications de la spermiogenese de Chara vulgaris L. induites par los rayons X. Rev. Cytol. et Biol. Veg. 177733-450. 30. - , et Gillet, C. 1956. Sur lea modifications induites par les - hydrazides w maleique bt isonicotinique dans les antheridies de Chara vulgaris L. La Cellule 58: 65-78. 31. Oehlkers, F. BeitrRge zur Kenntnis der Kernteilungen bei den Charazeen. Ber. deutsch. Bot. Ges. 34: 223-227. 32. Prescott, G. 1950. Algae of the 7estern Great Lakes Area. 1-946. Cranbrook. 33. Robinson, C. 1906. The Chareae of North America. Bull. N.Y. Bot. Gard. 4: 244-308.

34. Sawa, T. 1957. On the influences of 8-ovquinoline, paradichlorobenzene and a-bromonaphthalene on the root-tip-chromosomes of Allium cepa. Osaka University Sci. Rep. 6: 133-138. 35. - , and T. Sugiura. 1958. On the influences of coumarine, sulfanilamide and esculin on the root-tip-chromosomes of Allium cepa. Osaka Univer. Sci. Rep. 7: 89-101.

36. Telezynski l H. 1929. Garnitures des chromosomes et synchronismebs divisions dans les filaments dlantherozoides chez certaines especes du genre Chara Vaill. Acta Soc. Bot. Poloniae 6: 230-247.

(continued) -31-

37. Williams, Mary B. 1959. A revision of Nitolla. cristata iraun (Characeae) AND its allies. Part II. l' xoncTiy. Proc. LINN. Soc. 84: 346-355.

38. Wood, R. D. 1947. The Characeae of the Put-in-Bay region, lake Erie (Ohio). Ohio Jour. Sci. 47: 240-258. 39. . 1948. A review of the genus Nitella (Characeae) of North America. Farlowia 3: 331-398.

4O. . 1949. The Characeae of the Woods Hole Region, Massachusetts. Biol. Bull. Woods Hole 96: 179-203.

41. . 1950. Stability and zonation of Characeae. Ecology 31: 642-647. 42. . 1952. The Characeae, 1951. Bot. Rev. 18: 317-353.

43. , and W. C. Muonschor. 1956. The Characeae of the State of Now York. Cornell Agri. Exp. Sta. Morn. 338. 1-77. Ithaca.