Christopher H. Haufler

Starch Gel Electrophoresis of Ferns: A Compilation of Grinding Buffers, Gel and Electrode Buffers, and Staining Schedules

The homosporous pteridophytes have been largely uninvestigated by electrophoresis, despite the fact that they offer many exciting research possibilities (Soltis et al., 1980). The paucity of electrophoretic studies of ferns and fern allies may be due in large part to the high concentrations of condensed tannins that many species contain (Cooper-Driver, 1976 and pers. comm.). These compounds render enzymes inactive by binding with them following cellular disruption, thereby frustrating researchers who have attempted electrophoretic analysis utilizing standard methods of sample preparation. The method of sample preparation developed by Kelley and Adams (1977a, b) in their analysis of enzyme variation in Juniperus was an important procedural breakthrough in overcoming the difficulties that result from the liberation of large amounts of phenolic compounds during tissue preparation. Recently, a simplified version of that method was applied by Soltis et al. (1980) to fern leaf tissue, facilitating rapid preparation of active enzyme samples and thereby making electrophoretic analyses of large numbers of individuals more feasible. In an attempt to improve methods of analysis of fern enzymes in starch gel electrophoresis, we have experimented with modifications of the method of sample preparation outlined by Soltis et al. (1980). We also have examined several different methods of sample preparation such as those of Gottlieb (1981a), Mitton et al. (1979), and Werth et al. (1982), and have evaluated the relative merits of each with fern tissue. Finally, during the course of our electrophoretic investigations of ferns we found that standard gel and electrode buffers and staining schedules, such as those of Brewer (1970) and Shaw and Prasad (1970), often provided unsatisfactory results when applied to ferns. We have determined gel and electrode buffers, as well as staining schedules, that provide clear starch gel enzyme banding for 22 enzyme systems in ferns. Requests for advice resulting from the recent surge of interest in fern enzyme electrophoresis have prompted us to compile our procedural data so that other researchers can take advantage of our experimentation. We hope that these data will stimulate more extensive electrophoretic investigation of pteridophytes and other electrophoretically difficult taxa. Gottlieb (1981b) recently reviewed aspects of enzyme electrophoresis primarily in gymnosperms and angiosperms. His discussion is equally relevant to understanding the potential applications and limitations of electrophoretic evidence in pteridophytes. Since homosporous pteridophytes have high chromosome numbers, it is tempting to invoke polyploidy in interpreting their enzyme band patterns. It is well

Enzyme Variability and Modes of Evolution in Bommeria (Pteridaceae)

As is true of other plants inhabiting xeric environments, desert ferns may be mor- phologically similar because of convergent evolution. Since phylogenetically important reproduc- tive features in these plants may be affected by vegetative modifications, electrophoretically de- tectable variability in 11 enzymes coding for 13 loci has been analyzed to provide an expanded, comparative data base for defining systematic relationships and population structure in the four species of Bommeria. Contrary to the ad hoc assumption that because Bommeria species have relatively high chromosome numbers they should be polyploid, enzyme studies suggest that all species are genetic diploids. These studies also demonstrate that genetic variability is partitioned among the individuals within populations and that the sexual species are outcrossing. Analyses of allelic frequencies indicate that the four species are genetically more distinct from one another than are angiosperm congeners. Bommeria subpaleacea and B. ehrenbergiana emerge as the most similar taxa with B. pedata and B. hispida being more distant. The enzyme data, combined with other characters, support the hypotheses that the mountains of west-central Mexico are the center of origin of Bommeria and that the triploid apomict B. pedata was not derived through hybridization among extant sexual species. Although the corroborative morphological, biogeographical, and enzymatic data present a reasonable phylogenetic hypothesis, the extraordinary genetic distance among these congeners suggests that Bommeria may be polyphyletic, composed of species that are grouped together because of superficial resemblance. Evolution in plants of warm deserts has been characterized by considerable vegetative and physiological convergence (Orians and Solbrig 1977). In assessing characters for phylogenetic analysis of these plants, it is necessary to con- sider carefully the role that the environment may have played in directing morphological modifications. Among angiosperms, xero- morphic, perennial members of the Cactaceae, Euphorbiaceae, and Asclepiadaceae share many vegetative features but may be unambiguously separated into distinct evolutionary lines based on floral characters. Since flowers are relatively ephemeral, their features are less affected by environmental pressures and can be used as reasonably accurate indicators of phylogenetic relationships. Among the ferns, there is a suite of genera commonly associated with xeric habitats. These share many of the features considered to be adaptations for survival in environments hav- ing frequent droughts, low humidity, and high insolation. Plants in genera such as Cheilanthes, Notholaena, Pellaea, and Bommeria are small, have highly dissected leaves, and are often clothed

A community-derived classification for extant lycophytes and ferns

Phylogeny has long informed pteridophyte classification. As our ability to infer evolutionary trees has improved, classifications aimed at recognizing natural groups have become increasingly predictive and stable. Here, we provide a modern, comprehensive classification for lycophytes and ferns, down to the genus level, utilizing a community‐based approach. We use monophyly as the primary criterion for the recognition of taxa, but also aim to preserve existing taxa and circumscriptions that are both widely accepted and consistent with our understanding of pteridophyte phylogeny. In total, this classification treats an estimated 11 916 species in 337 genera, 51 families, 14 orders, and two classes. This classification is not intended as the final word on lycophyte and fern taxonomy, but rather a summary statement of current hypotheses, derived from the best available data and shaped by those most familiar with the plants in question. We hope that it will serve as a resource for those wanting references to the recent literature on pteridophyte phylogeny and classification, a framework for guiding future investigations, and a stimulus to further discourse.

Phylogeny and evolution of grammitid ferns (Grammitidaceae): a case of rampant morphological homoplasy

We conducted phylogenetic analyses of the fern family Grammitidaceae using sequences from two cpDNA genes and from morphological characters. Data were obtained for 73 species from most recognized genera in the family. Thegenera Adenophorus, Ceradenia, Calymmodon, Cochlidium, Enterosora, and Melpomene were each strongly supported as being monophyletic. Other recognized genera that were not supported as monophyletic included Ctenopteris, Grammitis, Lellingeria, Micropolypodium, Prosaptia, and Terpsichore. Several previously unrecognized clades were identified, some of which are characterized by distinctive morphological features. Analyses of the distribution of morphological character states on our inferred phylogeny showed extremely high levels of homoplastic evolution for many different characters. Homoplasy for morphological characters was considerably greater than for molecular characters. Many of the characters that exhibited high levels of convergent or parallel evolution across the phylogeny are features that have been commonly used to circumscribe genera in this group (e.g., leaf blade dissection, various rhizome scale characters, and glandular paraphyses). Conversely, some of the characters that exhibited relatively low levels of homoplasy have either not been regarded as having taxonomic value or have been ignored (e.g., root insertion, rhizome scale sheen). Our data support a New World origin of Grammitidaceae, with Old World taxa generally being more evolutionarily derived. Several clades are either primarily Neotropical or primarily Paleotropical but also have a few members distributed in the opposite hemisphere. Thus, we postulate multiple, independent dispersal and colonization events in several lineages.

Genetic Evidence for Allopolyploidy in the Neotropical Fern Hemionitis pinnatifida (Adiantaceae) and the Reconstruction of an Ancestral Genome

Genetic evidence from enzyme electrophoresis and restriction site analyses of cpDNA was employed to explore the origin of the tetraploid Hemionitis pinnatifida by determining the role of diploid species of Hemionitis in contributing to the tetraploid genome. The results clearly supported the hypothesis that H. pinnatifida is an allotetraploid with H. palmata as one of its diploid progenitors. The other diploid parent was not identified among the existing species of Hemionitis and is either extinct or exceedingly rare. Genetic identity analyses compared the extant diploid genomes with the divergent diploid genomes combined in tetraploids. One of these divergent genomes was shown to be a good approximation of that of H. palmata, and the other is a reconstruction of that of the missing diploid parent. These analyses demonstrated that the missing ancestor was genetically more similar to H. palmata than either of these species is to the other diploid species surveyed. Such greater genetic similarity may have been important in allowing hybridization between the two diploid progenitors of H. pinnatifida. The data also suggest that tetraploid individuals may have been synthesized de novo at least five times among the three populations surveyed.

Antheridiogen and Natural Gametophyte Populations

could control the sex expression of fern gametophytes was made in the laboratory. In 1950, Dopp published a paper in which he showed that old media or aquatic extractions from media containing female gametophytes of bracken (Pteridium aquilinum) induced antheridia formation in young gametophytes of its own species or those of the male fern (Dryopteris filix-mas). This pioneer work was supplemented by two further publications in 1959 and 1962. In the United States, it was mainly Naf (for review see Nif, 1979) who started studies on antheridiogen in the late 1950s. Some of the most interesting questions that arose were: Does a selected fern taxon produce or react to antheridiogen? Are antheridiogen-systems widely distributed? What is the sex expression of isolated gametophytes? How much antheridiogen is required to induce antheridia? What happens to a culture of young sterile gametophytes when antheridiogen of a female or a meristic gametophyte is added? Do meristic gametophytes become insensitive to antheridiogen? Since D6pps initial work, a large number of publications have appeared. Nearly all of these have been based on laboratory investigations and our knowledge of antheridiogen has increased strikingly. It has been shown that at least three main classes of antheridiogens occur (Nif, 1979), antheridiogen A (bracken-antheridiogen), antheridiogen B (produced by members of the family Schizaeaceae) and antheridiogen C (produced by species of the genus Ceratopteris). Although the chemistry of only antheridiogen B has been studied in detail (Corey & Myers, 1985; Nakanishi et al., 1971; Yamane et al., 1979) many similarities among the various types of antheridiogens can be seen from the standpoint of physiology. Antheridiogens are all water soluble, are active in very low concentrations, and are formed by large, meristic gametophytes. At present, the term pheromone is preferred over the term hormone when speaking of antheridiogens (Nif, 1979). Although much has been accomplished, many questions may still be pursued through laboratory experiments. In contrast to the multitude of laboratory-oriented studies, only a few publications have focused on the occurrence and importance of antheridiogen in natural fern populations. The first publication giving evidence for the natural role of antheridiogen was that of Tryon & Vitale (1977). By mapping gametophytes on natural sites Tryon & Vitale (1977) could show that there was

Hybridization, Reticulation, and Species Concepts in the Ferns

Hybrids and hybrid species are common among ferns, and they account for many of the problems in species definition in the group. Most systematic inquiry into the evolutionary process in ferns has addressed hybrid species, because meaningful explanations of their origins are feasible (Manton, 1950). As a result, complexes of hybrids, hybrid species, and their progenitor species have been popular subjects for experimental work. Here, we address the definition and changing perception of these hybrid species in the light of improvements in the data available to systematists. Once we have established basic definitions, we demonstrate the utility of recent advances in defining hybrid species of ferns. With this orientation, we investigate the status of hybrid species in the context of reigning species concepts. Renewed reproductive interaction between populations or species following a period of isolation characterizes all hybrids; hence hybrids are often spoken of as the products of secondary contact. Hybrids are unique in that they arise when isolating mechanisms fail; thus they are evolutionarily a consequence of the disruption of the divergence process that leads to ordinary (primary) species. Consequently, the hybrid is at once a novelty and a rehash: it is a novel combination of genetic and morphological features already present in its progenitors. These features need not be intermediate: see Grant (1975) on transgressive segregation and Barrington, 1986a. Fern hybrids are predominantly sterile (Knobloch, 1976), though there is a small, disparate set of variously fertile hybrids (in Pteris, Walker, 1958; in Dryopteris, Whittier & Wagner, 1961; in the Cyatheaceae, Conant & Cooper-Driver, 1980). The origin and evolutionary significance of sterile hybrids have been the subject of most

Electrophoretic Evidence for Genetic Diploidy in the Bracken Fern (Pteridium aquilinum).

Analysis of isozyme variability demonstrates that bracken fern (Pteridium aquilinum) has a diploid genetic system and expresses solely disomic inheritance patterns. Electrophoretic data indicate that genetically variable progeny are produced in natural populations after intergametophytic mating rather than by a process involving recombination between duplicated unlinked loci. Although some enzymes are encoded by more than one locus, this has resulted from subcellular compartmentalization of isozymes, and there is no evidence of extensive gene duplication resulting from polyploidy. The conclusions reached in this report differ from those which propose polyploidy as an adaptive mechanism for maintaining genetic variability in Pteridium and other homosporous pteridophytes.

A Revised Circumscription of the Genera of the Fern Family Vittariaceae

Based on phylogenetic analysis of rbcL gene sequences from species of Vittariaceae, Vittaria and Antrophyum are, respectively, polyphyletic and paraphyletic. New circumscriptions are developed with the goal of organizing the species into genera that are strictly monophyletic. Species of Vittaria are placed in three genera, Vittaria, Haplopteris, and Radiovittaria, reflecting new hypotheses of their phylogenetic relation- ships. Species with leaves 4 mm wide and paraphyses bearing narrow apical cells are retained in Vittaria. Old World species with funnelform terminal cells on paraphyses and distichous phyllotaxy are placed in Haplopteris, whereas those with funnelform terminal cells on paraphyses and spiral phyllotaxy are placed in the new genus Radiovittaria. Species of Antrophyum are divided between the monophyletic genera Antrophyumn and Scoliosorus. Antrophyum is restricted to paleotropical species with pluriseriate venation and globose- tetrahedral spores. Those species with pluriseriate venation (neotropical and African) and bilateral spores are assigned to Scoliosorus. The genera Ananthacorus, Anetium, Hecistopteris, Monogramma, and Polytaenium are retained and given clarified circumscriptions. A description of the Vittariaceae and a key to the genera of the family are presented; all genera are described and new combinations are made for a number of species.

Genetic diversity and breeding system in a group of neotropical epiphytic ferns (Pleopeltis; Polypodiaceae).

Epiphytes are ecologically important components of tropical forests worldwide and yet they have been underrepresented in studies of reproductive biology. Given the presumed ephemeral nature of their substrates, and the importance of dispersal and colonization, epiphytes might be expected to undergo substantial inbreeding to ensure reproductive success, as in weedy terrestrial plants. While there is some evidence for inbreeding in epiphytic angiosperms, the only previous studies of fern epiphytes indicate that they are predominantly outcrossing. The present study reports on the genetic diversity and breeding system of six members of the Neotropical epiphytic fern genus Pleopeltis (Polypodiaceae). A survey of isozyme variability using starch gel electrophoresis revealed high population levels of polymorphism (P = 0.62), allelic diversity (A = 2.3), and individual heterozygosity (Ho = 0.181), but little differentiation among conspecific populations (I ³ 0.98; Gst = 0.048), and high interpopulational gene flow rates (Nm > 1). In addition, there was no indication of homozygote excess within populations that might indicate a history of selfing in these ferns: populations generally conformed to Hardy-Weinberg expected genotype frequencies, and both Wrights inbreeding coefficient (Fis) and Holsingers intragametophytic selfing rates approached zero. Possible mechanisms limiting inbreeding in these ferns include antheridiogen activity and high levels of genetic load that would lead to inbreeding depression upon selfing.