Author Institution: Department of Botany, Ohio Wesleyan University, Delaware, Ohi

Long, Robert W.

The Ohio Journal of Science

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THE OHIO JOURNAL OF SCIENCEVOL. 57 MARCH, 1957 No. 2A CYTOLOGICAL ANALYSIS OF A NATURALLYOCCURRING HELIANTHUS HYBRIDROBERT W. LONG, JR.Department of Botany, Ohio Wesleyan University, Delaware, OhioOne of the most interesting examples of natural hybridization in the genusHelianthus (family Compositae) is that involving H. salicifolius A. Dietr. andH. grosseserratus Martens. The first species is a perennial sunflower with tall,smooth stems bearing many drooping, alternate lance-linear leaves; the diskcorollas may be either purple- or yellow-colored. It is found in dry prairies fromMissouri to Texas but is sometimes cultivated in northeastern United States forits unusual foliage and showy flowers. The second species is a common weedthat grows along roads and fencerows throughout much of the Midwest. Theplant produces tall, generally smooth stems with ascending, alternate, lanceolateor lance-ovate leaves; the disk corollas are yellow. The hybrid of these species isH. X Kellermani Britton pro sp. named for Dr. W. A. Kellerman who first col-lected the plant near Columbus, Ohio, in 1897 (Kellerman, 1902). Since thattime only a few collections have been made in widely separated places from Ohioto Wisconsin.In the course of a previous investigation, an artificial Fi hybrid was producedand compared morphologically and cytologically to a natural hybrid from Colum-bus, Ohio (Long, 1955). A preliminary study of microsporogenesis showed thatirregularities were present in both hybrids. This report deals with the results ofa detailed examination of microsporogenesis in a new collection (23) of putativeH. X Kellermani from near Leonardsburg, Ohio, made in 1954. The investiga-tion was undertaken primarily to obtain data with which to evaluate the taxonomicrelationship of H. salicifolius and H. grosseserratus.METHODS AND MATERIALSTransplants of H. salicifolius, H. grosseserratus, and the new collection ofH. X Kellermani were grown in an experimental garden during 1954. The cultureof H. salicifolius (115) was grown from seed originally obtained from the Pearce SeedCompany. Two cultures of H. grosseserratus (10 and 78) were kindly suppliedby Dr. C. B. Heiser of Indiana University; they were collected in New York andIllinois, respectively. Specimens of these plants were prepared and placed inthe Herbarium of Ohio Wesleyan University.In the summer of 1955 flower buds were collected from plants in the experi-mental garden, fixed in Carnoy's solution (3:1) for 12 hours, and then transferredto 70 percent ethyl alcohol. Pollen mother cells were examined using the aceto-carmine smear technique. Chromosome behavior was studied during diakinesis(late prophase I), metaphase I, anaphase I, and telophase II. Photomicrographswere made using a Leitz Makam camera. Estimates of pollen fertility are basedon differential staining of microspores with anilin blue in lactophenol. Fertilepollen stains dark blue; non-fertile pollen remains unstained. Approximately 200grains were examined from each plant.RESULTSThe data obtained from an examination of meiosis in the putative hybrid andTHE OHIO JOURNAL OF SCIENCE 57(2): 65, March, 1957.Helianthus X KellermaniRobert W. Long, Jr. PLATE IEXPLANATION OF FIGURES IN PLATE IPhotomicrographs of representative cells of H. X Kellermani during microsporogenesis(approximately X 1180).a. 17 bivalents, normal diakinesis.b. lagging bivalent, metaphase I.c. lagging chromosome (arrow), early telophase I.d. two chromatid bridges (fused chromatids), early telophase I.EXPLANATION OF FIGURES IN PLATE IICamera lucida drawings of representative cells during microsporogenesis of H. X Keller-mani (approximately X 1300).A. 15 bivalents and circle of four chromosomes ("figure eight"), diakinesis.B. Chromatid bridge and fragment, early telophase I.C. Chromatid bridge (fused chromatids), early telophase I.D. Lagging chromosome, early telophase I.E. Lagging chromosomes, early telophase I.F. Multiple chromatid bridges, early telophase I.G. Multiple chromatid bridges, early telophase I.H. Chromatid bridges (fused chromatids), anaphase III. Lagging chromosome, telophase II.66Helianthus X KellermaniRobert W. Long, Jr.PLATE IIspecies cultures are presented in table 1. The haploid chromosome number of17 reported for these sunflowers (Geisler, 1931; Heiser, 1950) is confirmed.The most common meiotic abnormality in the hybrid was the formation ofrings and chains of from 3 to 6 chromosomes during diakinesis (22 percent of cellsexamined). Usually this was a chain of four chromosomes. Chains were alsofound in species' cultures, especially in H. salicifolius (14.1 percent).6768 ROBERT W. LONG, JR. Vol. 57The second most commonly observed abnormality was lagging univalents andbivalents during metaphase (21.5 percent). Laggers occurred in all the othergarden plants (Plates I and II).During anaphase and early telophase I, chromatid bridges with fragments,bridges without fragments ("fused chromatids"), and lagging chromosomes wereobserved. Several cells contained multiple bridges. All of these aberrationswere rare in the other cultures.Telophase II • apparently proceeds normally in the hybrid as well as in theother plants. Regular tetrads are formed almost without exception. No signi-ficant number of abnormalities was observed although a few cells contained laggersand micronuclei.TABLE 1Meiosis in garden cultures and putative wild hybridPlant Diakinesis Metaphase I Anaphase I Telophase IIrings, with withno. 17II chains I's no. normal laggers no. normal bridges laggers no. normal abnormalgrosseserralus#78 56 98.2 1.8 0.0 216 97.2 2.8 305 100.0 0.0 0.0 209 98.6 1.4#10 44 100.0 0.0 0.0 204 92.0 8.0 308 98.4 0.8 0.8 206 99.3 0.7salicifolius#115 52 84.6 14.1 1.3 209 97.4 2.6 313 98.2 0.9 0.9 202 100.0 0.0X Kellermani#23 41 75.5 22.0 2.5 212 78.5 21.5 307 90.5 2.4 7.1 208 99.4 0.6Approximately 12 cells were studied during diakinesis in each plant to obtainchiasma frequencies. It was not possible to study meiosis at earlier stages inthis material. The chiasma frequency per chromosome pair in H. grosseserratus(78) was 1.41 ±.01; in H. salicifolius 1.48±.01; in the hybrid 1.62±.06. Mostof the chiasmata were terminal although X-shaped bivalents were numerous.The hybrid was vigorous and normal in every external respect during obser-vations that extended over two growing seasons. The morphological intermediacyof the new collection (23) was readily apparent when it was placed along sidethe putative parental species. Pollen fertility was high in both species culturesand hybrid (90 to 100 percent stainable pollen) although seed-set was poor inopen-pollinated heads.DISCUSSIONIn most hybrids between closely related species the chromosomes are eitherpartially or completely paired during prophase and metaphase. Any subsequentlagging, bridge-fragment configurations or other phenomena are associated withlack of chromosome homology. Generally, the chiasma-frequency is somewhatlower than that found in the parental species (Stebbins, 1945). Preliminarycounts gave a higher chiasma-frequency in the hybrid than in the putative species.However, the difference for the hybrid is not statistically significant. The putativehybrid exhibited regular chromosome pairing in approximately 75 percent of thecells studied. Lagging was observed during metaphase and early telophase.Bridge-fragments and "fused chromatids" were also formed.The presence of rings and chains of chromosomes during prophase suggeststhat chromosomal interchanges have taken place and the two species differ by atleast one translocation. It would appear that adjacent chromosomes usuallypass to opposite poles since pollen fertility is high.No. 2 HELIANTHUS X KELLERMANI 69Lagging bivalents were frequent in the hybrid and were also observed in allparental cultures. Most hybrids between closely related species have normalspindle behavior. Lagging bivalents in these sunflowers may be caused by cer-tain internal factors that upset the spindle mechanism and terminalizations'timing relations (Stebbins, 1945). Certain environmental factors, such as tem-perature, might also tend to interfere with normal spindle behavior. This laggingdid not seem to affect the later course of meiosis, however, at least to any extent.During anaphase and early telophase chromosome behavior was normal exceptfor an occasional cell with bridge-fragment configurations, "fused chromatids,"and laggers. Cross-overs in regions of inversions were assumed to be the causeof bridge-fragments, and probably some of the fused chromatids were formed inthe same manner. In addition, fused chromatids may be caused by non-disjunctionof one or more pairs of homologues and the persistent association of distal ends ofhomologous chromatids (Sax, 1932). Restitution nuclei were not seen at telo-phase I nor were characteristic "dumbbell" shaped nuclei of persistent chromatidbridges observed. The low frequency of bridges and absence of any detectableeffect on later stages of meiosis argue against considering them of much importancecontributing to genetic isolation. It would appear that inversions, along withtranslocations, have occurred in H. salicifolius and H. grosseserratus but thatthese chromosomal alterations have not been directly important in the evolutionof races of the species.The degree of chromosome homology, vigor, and pollen-fertility in H. XKellermani demonstrate that H. salicifolius is closely related, cytogenetically,to H. grosseserratus. Morphological differences between the two species are quitestriking, chiefly because of the unusually narrow leaves of H. salicifolius. Bothspecies, however, possess alternate leaves and loose, attenuate phyllaries, andthese are probably more important from the taxonomic standpoint. It seemsadvisable, from the evidence at hand, to include H. salicifolius in the "Giganteus"section of the genus (Long, 1954). This will necessitate expansion of the morpho-logical description of the section to include species with disk-corollas red to purplish-brown, as well as those with disk-corollas yellow.SUMMARYA cytological analysis of a new collection of Helianthus X Kellermani hasbeen undertaken to obtain data for evaluating the taxonomic relationship of H.salicifolius and H. grosseserratus. Approximately 75 percent of the pollen mothercells examined from the hybrid showed regular chromosome-paring. Structuraldifferences were evidenced by the formation of rings and chains of chromosomesand chromatid bridges. Disturbances at metaphase I and anaphase I were alsonoted, but none of these phenomena interfere with the formation of large amountsand proportions of fertile pollen.It was concluded that H. salicifolius and H. grosseserratus are closely related.Helianthus salicifolius should be included in the "Giganteus" section of the genus.LITERATURE CITEDGeisler, F. 1931. Chromosome numbers in certain species of Helianthus. Butler Univ.Bot. Studies 11: 53-62.Heiser, C. B., Jr. 1950. Report of progress. Yearbook Amer. Philos. Soc. 1949: 181-183.Kellerman, W. A. 1902. Minor plant notes, No. 4. Ohio Nat. 2: 179-181.Long, R. W., Jr. 1954. A biosystematic investigation of Helianthus giganteus L. and relatedspecies. Ph. D. thesis. University Microfilms A54-1659. Ann Arbor, Michigan.. 1955. Hybridization between the perennial sunflowers Helianthus salicifolius andH. grosseserratus. Amer. Midi. Nat. 54: 61-64.Sax, K. 1932. Meiosis and chiasma formation in Paeonia suffruticosa. Jour. Arnold Arboretum13: 375-384.Stebbins, G. L. 1945. The cytological analysis of species hybrids. Bot. Rev. 11: 463-486.

A Cytological Analysis of a Naturally Occurring Helianthus Hybrid

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A Cytological Analysis of a Naturally Occurring Helianthus Hybrid

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