-1- 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 dis- tribution, 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 es- pecially 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 anther- idium 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 lat- eral 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 inter- nodes, 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 rudi- mentary. Branchlets simple, of 4 or more segments. Bract-cells normally 4 or more, or rudimentary. Bracteoles also may be rudi- mentary 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, en- tire 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 an- theridia 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 chromo- some 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 num- bers 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.