Charles R. Werth

A MODEL FOR DIVERGENT, ALLOPATRIC SPECIATION OF POLYPLOID PTERIDOPHYTES RESULTING FROM SILENCING OF DUPLICATE-GENE EXPRESSION

We present a model for allopatric speciation at the polyploid level, based on different patterns of gene silencing that would occur in geographically isolated populations. Allopatric populations of a single allotetraploid species may experience silencing of the same gene but in different genomes (reciprocal silencing). Hybrids between such genotypes would experience a reduction in viability of gametophyte progeny to 0.75n, where n is the number of reciprocally silenced homoeologous gene pairs. A minimum value of n ≥ 8, representing a maximum hybrid fertility of 0.10, is shown to be virtually certain when the proportion of genes silenced reaches 0.2. Gene silencing would also lead to genetic divergence between the populations. Silencing of regulatory genes could lead to significant interpopulational differences in morphology and other complex traits. It is predicted that the combined effect of postzygotic hybrid sterility and genetic divergence resulting from gene silencing might lead to speciation of allopatric populations even in the absence of strong selection. It is also predicted that such speciation events would be relatively rapid, because they are driven by the most frequent class of mutations, and only a fraction of the genome needs to be silenced for speciation to occur. Criteria are provided for evaluating whether this mode of speciation has occurred in nature.

Electrophoretic evidence of reticulate evolution in the Appalachian Asplenium complex

The Appalachian Asplenium complex consists of six fertile species: three diploids and their three allotetraploid derivatives. This reticulate evolutionary pattern, originally proposed on the basis of morphological and cytological evidence, was later corroborated by analyses of flavonoid composition. Presented here are data from starch gel electrophoresis of 11 enzyme systems, coded by 15 interpretable loci, which are highly consistent with the proposed relationships in the Appalachian Asplenium complex. The diploids are strongly differentiated (genetic distance (D) = 0.67 to 1.30); each diploid possesses unique alleles at several loci. Each allotetraploid expresses the heterozygous, or less often homozygous, enzyme phenotypes expected for combinations of parental alleles at most loci. Exceptions are the expression of a novel PGI-2 allele and the loss of expression of parental IDH alleles in both allotetraploids. The importance of reticulate evolution in plants is well documented (Lewis 1980). This type of evolution involves hybridization of differentiated genomes resulting in high levels of heterozygosity. The application of the isozyme technique seems particularly relevant to gain additional insights into the evolution of polyploid complexes. This technique detects allelic variants (allozymes) at equivalent gene loci, thus facilitating analysis of genetic variation within and between species. Allopolyploids may be expected to exhibit heterozygous phenotypes at loci for which the presumed parental taxa possess electrophoretically different alleles. Indeed, early isozyme studies of plants often addressed the identification of diploid progenitors of allopolyploid crops (Cherry et al. 1972; Garber 1974; Reddy and Garber 1971; Sheen 1972). More recently Roose and Gottlieb (1976) demonstrated the utility of allozymes in confirming relationships and assessing heterozygosity in Tragopogon. However, given the preponderance of such complexes in nature and the general lack of allozyme studies on them, the technique is at present underexploited. Reticulate evolution is well known among most genera of temperate ferns (Walker 1979). The use of allozyme analysis to address problems of systematics and evolution in ferns has awaited development of techniques that allow extractions of active and electrophoretically resolvable enzymes from tissues noted for their high levels of phenolic compounds. Such techniques are now available (Soltis et al. 1980, 1983; Werth et al. 1982) and are in use in several laboratories. The evolutionary relationships among Asplenium species endemic to eastern North America were clarified by Wagner (1954) in a classic study of their morphology and meiotic chromosome behavior. Wagner showed that three diploid species, A. platyneuron, A. montanum, and A. rhizophyllum, gave rise to three allotetraploid derivatives, A. ebenoides (platyneuron x rhizophyllum), A. pinnatifidum (montanum x rhizophyllum), and A. bradleyi (montanum x platyneuron) (fig. 1). Studies on cytology (see Walker 1979 for review) and flavonoids (Smith and Levin 1963; Smith and Harborne 1971; Harborne et al. 1973) of the six fertile species as well as a number of sterile hybrids provided data consistent with the concept of relationships as originally hypothesized by Wagner (1954). The present paper reports allozymic evidence that further corroborates Wagners hypotheses, and demonstrates the potential, as well as some of the limitations, of allozyme data to elucidate parentages of allopolyploid taxa. MATERIALS AND METHODS Field collections from two or three populations of each fertile taxon as well as one sterile allodiploid A. ebenoides were obtained from isolated localities (table 1). Up to sixty individuals of each species were collected per population. However, collection of A. ebenoides from the unique tetraploid population in Alabama was limited to three juvenile sporophytes.

Recurring Origins of Allopolyploid Species in Asplenium

A large proportion of plant species has originated through allopolyploidy: interspecific hybridization followed by chromosome doubling. Heterozygosity remains fixed in allopolyploids because of nonsegregation of parental chromosomes. Two allotetraploid species of the fern genus Asplenium show allozyme polymorphisms at loci that are polymorphic in their diploid progenitors, indicating that each has originated more than once and implicating continued gene flow from diploids to tetraploids.

The use of isozyme data for inferring ancestry of polyploid pteridophytes

Abstract Numerous pteridophyte species are of polyploid origin, but the identity of diploid ancestors is well documented in few cases. Recent studies of pteridophyte polyploid complexes using isozyme electrophoresis have demonstrated the value of this technique for elucidating the ancestry of polyploids. This paper reviews these studies and considers various aspects of the isozyme technique and accompanying data analysis germane to polyploid pteridophytes. The relatively high speed and low cost of obtaining isozyme data are pointed out. Interpretation of banding patterns in polyploids is discussed, including different approaches used for locus and allele designations. The use of isozyme data in discriminating between autopolyploids and allopolyploids, and (more appropriately for pteridophytes) between intraspecific and interspecific polyploids, is addressed. Detailed attention is given to the use of isozyme data to test between competing hypotheses of ancestry for interspecific polyploids. Complicating factors resulting from evolution of both progenitor diploids and polyploid derivatives are discussed. A method is proposed for evaluating seemingly equivocal results on the ancestry of interspecific polyploids using statistical likelihood, as in paternity analysis. It is argued that isozyme electrophoresis will continue to be an important analytical tool in studying the evolution of polyploids and, in the future, will be used in conjunction with studies on nucleic acids, as well as morphology and cytology.

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

Clonal population structure and genetic variation in sand-shinnery oak,Quercus havardii (Fagaceae)

We investigated clonal population structure and genetic variation in Quercus havardii (sand-shinnery oak), a deciduous rhizomatous shrub that dominates vegetation by forming uninterrupted expanses of ground cover over sandy deposits on the plains of western Texas, western Oklahoma, and eastern New Mexico. Isozyme electrophoresis (15 loci coding 11 enzymes) was used to recognize and map clones arrayed in a 2000-m transect (50-m sample intervals) and a 200 × 190 m grid (10-m sample intervals). Ninety-four clones were discovered, 38 in the transect and 56 in the grid, resulting in an estimated density of ∼15 clones per hectare. Clones varied greatly in size (∼100-7000 m), shape, and degree of fragmentation. The larger clones possessed massive interiors free of intergrowth by other clones, while the smaller clones varied in degree of intergrowth. The population maintained substantial levels of genetic variation (P = 60%, A = 2.5, H(exp) = 0.289) comparable to values obtained for other Quercus spp. and for other long-lived perennials. The population was outcrossing as evidenced by conformance of most loci to Hardy-Weinberg expected genotype proportions, although exceptions indicated a limited degree of population substructuring. These data indicate that despite apparent reproduction primarily through vegetative means, Q. havardii possesses conventional attributes of a sexual population.

Levels and patterns of genetic variation in the endangered species Abronia macrocarpa (Nyctaginaceae)

Genetic variation was evaluated in the federally endangered species Abronia macrocarpa (large-fruited sand-verbena), an herbaceous perennial restricted to deep sandy soils and endemic to three counties of east-central Texas. Seven of the ten known populations were sampled and analyzed using starch gel electrophoresis of eight enzymes coded by 18 interpretable loci. Duplicate gene expression was observed for four loci, suggesting polyploid ancestry for the lineage that includes A. macrocarpa. Values for estimators of genetic polymorphism within populations (ranges: P = 38.9%-61.1%, A = 1.7-2.1, H = 0.122-0.279) exceeded average values for seed plants (P = 34.2%, A = 1.53, H = 0.113). Genotype proportions at most loci in most populations were in Hardy-Weinberg equilibrium, consistent with obligate outcrossing previously documented for this species; exceptions could be attributed to population substructure. Values of F(ST) tended to be high, ranging from 0.021 to 0.481 for individual loci (mean F(ST) = 0.272), indicating substantial divergence and limited gene flow among populations, despite their close geographic proximity. Pairwise values of Neis genetic identity between populations ranged from 0.799 to 0.975 and tended to be influenced by geographic proximity of population pairs. Collectively, these data suggest a long history of isolation among populations that have not been subjected to bottlenecks. Isolation of A. macrocarpa populations apparently results from the disjunct occurrence of suitable habitat and perhaps has been accentuated by human disturbance.

Heterozygote advantage in the American chestnut, Castanea dentata (Fagaceae)

The American chestnut (Castanea dentata; Fagaceae) was a dominant canopy tree in the Appalachian Mountains of North America. Since the introduction of the chestnut blight fungus (Cryphonectria parasitica; Valsaceae) in America, the American chestnut has been reduced to a predominantly clonal, understory species. Our objective was to determine whether the ecological changes and absence of new recruits have influenced the population genetics of American chestnut. Leaf samples were collected from four populations in southwestern Virginia. Electrophoretic data from five polymorphic loci were used to determine the genetic diversity and population structure of the populations and subpopulations. Growth data and infection status were recorded for one of the populations to determine their relationship with heterozygosity. F statistics revealed a significant amount of differentiation among subpopulations and an excess of heterozygotes within subpopulations. Heterozygous individuals also had higher rates of vegetative growth. The superior performance and excess of heterozygotes suggests that selection favors heterozygous individuals. The prolonged absence of sexual reproduction in C. dentata has allowed subtle fitness differences to accumulate to the extent that they have had significant effects on the genetics of chestnut populations.

A study of genetic diversity among host-specific populations of the witchweed Striga hermonthica (Scrophulariaceae) in Africa.

Striga hermonthica is a root hemiparasite that attacks onlyGramineae, includingSorghum and millet for which it is a principal cause of lowered yield. Enzyme electrophoresis was used to investigate genetic diversity inStriga hermonthica and to determine the level of differentiation between host-specialized populations. Nine genetic loci coding eight enzymes were interpreted and data obtained from three populations: oneSorghum-adapted population from Sudan and two populations from Burkina Faso, oneSorghum-adapted and the other millet-adapted. Levels of polymorphism were similarly high in all three populations (P=0.625, A=2.6−2.8, H=0.293−0.401). Genotypic frequencies at most loci conformed to Hardy-Weinberg expectations in each population, consistent with outcrossing as predicted from previous studies of floral biology. Occasional heterozygote deficiencies were probably the result of Wahlund effect. The mean value of FST over the three populations was 0.068, indicating a slight to moderate level of genetic differentiation among the populations. The two Burkina Faso populations were more closely related (S=0.940, D=0.006) than either was to the Sudan population, suggesting that geographic separation is more important than host specialization in contributing to population differentiation. TheSorghum-adapted population was slightly closer to the Burkina FasoSorghum-adapted population (S=0.873, D=0.047) than to Burkina Faso millet-adapted population (S=0.851, D=0.074). The absence of substantial genetic divergence between host-specific populations ofStriga could result either from recent evolution of host-specialized strains or from strong selection for physiological specialization in the face of substantial gene flow between the populations.

Summary: the contributions of population studies on ferns

The preceding papers have presented a wide array of investigative approaches centered on the dynamics of population phenomena in ferns. Collectively, these studies have addressed all phases of the fern life cycle: spore production and dispersal, gametophyte establishment, mating mechanisms and their evolution, and both genetic and demographic attributes of sporophytes. Represented in these studies are some of the shifts in emphasis and approach that characterize recent trends in population ecology: attention to all stages of the life cycle (including the challenging and important study of gametophytes in nature), coordination of field data with laboratory studies (e.g. isozyme analysis, tests for antheridiogen response, estimation of genetic load), and development of theoretical models that predict outcomes of dynamic processes (e.g. evolution of mating systems) based on biological features of the organisms being modeled. Taken together, these studies begin to draw a picture of how ferns and other pteridophytes establish and maintain populations in nature, a picture that as yet is incomplete. While recent investigations have taken us strides forward in our understandings of fern populations, additional knowledge will emerge only after considerably more effort. With the goal of encouraging such effort, we would like in this symposium summary to point out the potential for additional research that will deepen our understanding of the biology of ferns as representatives of plants and of organisms in general. Below, as we order our thoughts principally along life history stages, we will attempt to bring together some of the concepts generated by the symposium papers, and also to identify areas of uncertainty that are of particular interest for future investigation.