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SMITH, DALE M. and DONALD A. LEVIN. (U. Illinois, Urbana.) A chromatographic study of reticulate evolution in the Appalachian Asplenium complex. Amer. Jour. Bot. 50(9): 952-958. Illus. 1963.-The reticulate relationships of the members of the Appalachian Asplenium complex were studied by means of paper chromatography. Substances could be observed with ultraviolet light in the presence of ammonia vapor in all the taxa of the complex. The diploid species (A. montanum, A. platyneuron and A. rhizophyllum) had characteristic biochemical substances. Interspecific hybrids and/or their allotetraploid derivatives showed a complete complementation of all the substances of their known or presumed diploid ancestors. A combination of all the substances of the 3 diploids was found in the hybrids A. X kentuckiense and A. X gravesii. The results agree in all respects with the concept of reticulate evolution in the group which was advanced by Wagner on the basis of comparative morphology, hybridization and karyology. QUESTIONS of the origin and taxonomic treatment of polyploid taxa have received much attention, but the difficulties involved in carrying out detailed morphological comparisons, cytogenetic analyses, crossing experiments, and other pertinent investigations are so great that the ancestry of a polyploid taxon is rarely ever fully established. Furthermore, with respect to some polyploids, the data often do not allow one to differentiate between closely related members of a species group, any one of which might logically be considered ancestral to a particular polyploid. The use of paper chromatography of certain biochemical constituents to study interspecific hybridization may be extenided to an investigation of the problems associated with polyploidy, sinice polyploids are frequently of hybrid origin. An important generalization emerging from biochemical studies of hybridization is the idea of complementation of species-specific constituents of the parental species in interspecific hybrids (Alston and Turner, 1962); that is, the detectable substances of the parental species are found together in their hybrids. The implication of this fact for the study of polyploidy is that if one knows what substances occur in the diploid species which are presumed basic to a polyploid series, it should be possible to postulate the ancestry of a polyploid species on the basis of its complement of speciesspecific chemical constituents. The Appalachian Asplenium complex (Wherry, 1925; Wagner, 1954) offers an excellent opportunity to test the validity of a chemical approach 1 Received for publication March 20, 1963. 2 The authors wish to express their thanks to Professor Warren H. Wagner, Jr., of the University of Michigan, for many helpful comments and for supplying material of A. ebenoides, A. x wherryi, and A. x gravesii, and to Professor Ralph E. Alston, of the University of Texas, for many helpful suggestions and comments during an earlier phase of this work. Thanks are also due those collectors whose specimens were used in this study, especially Mr. T. R. Bryant and Mr. D. E. Tate. to the study of polyploidy. The interrelationships of these ferns have been worked out in great detail by more conventional studies. Wagner (1954) postulated that Asplenium montanum, A. platyneuron, and A. rhizophyllum (Camptosorus rhizophyllus) are the diploids basic to this complex, and genomic allotetraploids derived from these species are A. bradleyi (A. montanum-platyneuron), A. pinnatiJidum (A. montanum-rhizophyllum), and A. ebenoides (A. platyneuron-rhizophyllum). The latter taxon is known in the form of a sterile diploid hybrid as well as the fertile allotetraploid. Only tetraploids are known of A. bradleyi and A. pinnatifidum. In addition to the plants mentioned above, there are several natural and artificial hybrids which provide further insight into this complex (Darling, 1957; Smith, Bryant and Tate 1961a,bc; Wagner, 1954, 1956, 1958; Wagner and Boydston, 1958, 1961; Wagner and Darling, 1957; Wagner and Whitmire, 1957). Named hybrids include A. X gravesii (A. bradleyi X pinnatifidum), A. X kentuckiense (A. pinnatifidum X platyneuron), A. X trudellii (A. montanum X pinnatifidum), and A. X wherryi (A. bradleyi X montanum). Of this group, A. X kentuckiense is especially interesting since it combines equal numbers of chromosomes from each of the diploids (Smith et al., 1961c). Other known natural and/or artificial hybrids between members of this complex are A. bradleyi X platyneuron, A. ebenoides X platyneuron, A. ebenoides X rhizophyllum, and A. ebenoides X pinnatifidum. Most of these taxa were available to us, and the collection presented an unusual opportunity to test a number of facets of biochemical systematics as well as an opportunity to gain new insight into the problems of the interrelationships of the Appalachian spleenworts. First, it was essential to know whether or not these taxa could be identified by means of chromatography. Individual variation could be studied from population samples and by comparing individuals from one population to

Hybrids in artificial cultures have contributed to the understanding of the systematic morphology of ferns. Since the classic work of Margaret Slosson (1902), which first proved the origin of the natural hybrid x Asplenosorus ebenoides (R. R. Scott) Wherry (=Asplenium platyneuron (L.) B.S.P. x Camptosorus rhizophyllus (L.) Link), the techniques of growing fern gametophytes and producing crosses have improved, and many new developments have ensued. In 1957, Wagner and Whitmire provided the first demonstration of the conversion of a sterile allodiploid fern to a fertile allotetraploid. In 1968, Lovis reconstructed a fertile hybrid species of fern (Asplenium ( x )adulterinum Milde). In this article, all taxa, fertile and sterile, of interspecific origin will be referred to as "nothospecies," and indicated by the use of the times sign if sterile and with parentheses around the sign if fertile-i.e., (x), a convention proposed by C. Werth (pers. comm.); divergent species will be referred to as "orthospecies" and will lack the multiplication sign. Many important experiments on Aspleniaceae were accomplished in European laboratories especially, as discussed and summarized by Reichstein (1981). In almost all cases, such experimental hybridizations were carried out to test some hypothesis of the origin of a given nothospecies. By comparison, many fewer hybridizations have been undertaken simply to find out what a cross between taxon A and taxon B might look like, and what combining, for example, a creeping rhizome with an upright caudex, or hairs with scales, or discrete sori with acrostichoid sori, might yield morphologically. Yet such questions may bear upon our understanding of the determinants of structure and form; we may be able to gain insights that would otherwise be unavailable. The plants involved in this report are all members of the spleenwort family, Aspleniaceae, always popular objects for culture work and hybridization experiments because of their conveniently small size, ease of culture, rapid growth, and often very distinct forms. As a matter of fact, the bizarre hybrid that we briefly describe below was formed by accident. A terrarium containing numerous gametophytes of the lobed spleenwort, (x )Asplenosorus pinnatifidus (Muhl.) Mickel (= mountain spleenwort, Asplenium montanum Willd. x walking fern, Camptosorus rhizophyllus (L.) Link) from south of Shoals, Martin Co., Indiana (kindly provided by Warren P. Stoutamire) was opened at the same time spores of the

On the Shawangunk Mountain ridge in Ulster County, New York, on September 18, 1973, I discovered 60 fronds of an unusual Asplenium growing in a fissure on a vertical cliff of Silurian conglomerate. Asplenium montanum Willd. is abundant on these cliffs. I have observed numerous living plants of Asplenium montanum in Ulster County, New York, in seven counties in North Carolina, and I have seen herbarium specimens at the New York Botanical Garden (NY). All specimens were found to be uniform in morphology, and more than 99 per cent of the fronds bore sori. There are no described forms or varieties of this species. The new form (Fig. 1) differs from the typical form in the following characters: (1) blades light yellow-green instead of dark blue-green, (2) tip of frond not tapered to a lobed apex (Fig. 2A versus 3A), (3) pinnae and ultimate divisions more dissected (Fig. 2B versus 3B), (4) frond length averages 2.3 cm shorter than typical New York specimens, and (5) the absence of sori. The possibility of ecological variation is minimal, as plants of the typical species grow on the same cliffs and even on both sides of the new taxon in the same rock fissure. These differences warrant naming this a new form of Asplenium montanum. Asplenium montanum f. shawangunkense Reeves, f. nov. Ab Asplenio montano f. montano lamina flavo-virenti sterili, apice non lobato, et pinnis lobisque incisioribus differt. HOLOTYPE: Between Lake Minnewaska and Mohonk Lake in the Shawan

Rhizome tips (about 2 cm long) of diploid Davallia fejeensis were collected in the Enid A. Haupt Conservatory of the New York Botanical Garden, cleaned, rinsed in tap water and further trimmed. Voucher specimens are at NY. Explants (6-8 mm long) were cultured on 1% agar-gelled sterile nutrient medium of Knudson as modified by Steeves et al. (1955) and supplemented with 2% sucrose in order to provide control diploid sporophytes growing in axenic culture. Roots, leaves, and rhizomes of the resulting plants were excised and induced to differentiate into aposporous diploid gametophytes. Stock cultures of diploid gametophytes were multiplied and further grown on liquid media supplemented with 0.6% agar to effect fertilization and raise tetraploid sporophytes. Tetraploid gametophytes were isolated from this tissue culture-induced tetraploid material by repeating the procedure followed at the diploid level. In this way, both diploid and tetraploid sporophytes and gametophytes were made available for this study. Phenolic compounds were isolated by two-dimensional paper chromatography of an 80% methanol extract of green material in t-BuOH-HOAc-H20, 3:1:1 (TBA) and 15% aqueous acetic acid (HOAc), followed by one-dimensional paper chromatography in water. C-glycosylxanthones appeared as orange compounds in ultraviolet light and turned yellow when fumed with ammonia. Both purified compounds were co-chromatographed with mangiferin and isomangiferin isolated from Asplenium montanum Willd. (Bozeman & Radford 11552, NY). Rf values in TBA, BAW, HOAc, and H20 were: mangiferin, 0.31, 0.43, 0.43, 0.12; isomangiferin, 0.19, 0.29, 0.23, 0.04; Asplenium mangiferin, 0.34, 0.42, 0.42, 0.11; Asplenium isomangiferin, 0.21, 0.32, 0.23, 0.03; and rutin standard, 0.43, 0.45, 0.56, 0.25. The BAW and HOAc values are very similar to those of Smith and Harborne (1971), but the TBA and HOAc values differ from those of Markham and Wallace (1980). Absorption spectra (MeOH, nm) were: mangiferin, 232, 260, 270sh, 310, 364; isomangiferin, 240, 258, 270sh, 312, 365. The compounds were unaffected by acid hydrolysis (1 hr, 2N HC1, 100?C).

At Crab Orchard, Tennessee, which is in the Cumberland Mts., I found the largest plants of Pellaea atropurpurea I have ever seen. These plants, which seemed to be so thrifty in growth, were found on limestone cliffs and abutments of a railroad bridge. Some of them were more than 18 inches high. Along the highway just before reaching Monterey, Tennessee, I found a large number of Asplenium montanum growing on sandstone cliffs shaded by trees. I soon left the mountain region, and this brought my fern collecting to an end. BENTONSPORT, IOWA.

The Scott's Spleenwort, Asplenium ebenoides, was demonstrated long ago to be a cross between A. platyneuron and Camptosorus rhizophyllus by Miss Slosson.1 Later, another cross, A. cryptolepis Fernald [A. Rutamuraria var. cryptolepis Wherry, as I prefer to call it] with Camptosorus, was described by Dr. E. Lucy Braun. It has recently been rather conclusively demonstrated by Dr. Warren H. Wagner, Jr.,2 that the well-known Asplenium pinnatifidum is a fertile tetraploid (allopolyploid) derived by the crossing of Asplenium montanum and Camptosorus rhizophyllus. This being so, it is quite likely that this crossing is taking place de novo occasionally, and that some plants of A. pinnatifidum found in the wild may be sterile diploid hybrids, the result of recent hybridization and not the descendants of preexisting plants of A. pinnatifidum. A condition similar to this has been shown by Dr. Wagner to exist in A. ebenoides, for the Alabama population of this is a fullfledged "species," being a fertile tetraploid, whereas plants occurring elsewhere are newly formed, sterile diploid hybrids. Dr. Wagner has indicated that other Aspleniums also, such as A. Trudellii, A. Gravesii, and A. kentuckiense, have some Camptosorus "blood" in them, being crosses of pinnatifidum. As I pointed out in my review of Wagner's paper,3 these facts point up a serious nomenclatural difficulty. The International Code of Botanical Nomenclature provides that intergeneric hybrids (if designated by more than a formula, i.e. Asplenium platyneuron x Campto