PHOSPHOGLUCOMUTASE AND ISOCITRATE DEHYDROGENASE GENE DUPLICATIONS IN LAYIA (COMPOSITAE)

Genetic analysis of isozyme segregation patterns in Layia (Compositae) showed that cytosolic phosphoglucomutase isozymes are encoded by duplicated genes, and that the cytosolic NADP-dependent isocitrate dehydrogenase isozymes are encoded by duplicated genes in species with haploid chromosome numbers of n = 7 and triplicated genes in those with n = 8. The duplicated genes specifying both isozymes assorted independently in all species tested. An electrophoretic survey of phosphoglucomutase in diploid species representing six additional genera of Madiinae, the subtribe to which Layia is assigned, revealed that Achyrachaena, Calycadenia, Hemizonia, Holocarpha, and Madia all possessed duplicated genes. In Lagophylla, one species also had duplicated genes for the isozyme but a second species did not, a loss probably resulting from mutation or chromosomal deletion. The phosphoglucomutase duplication characterizes nearly the entire subtribe and may prove useful to identify phylogenetic relationships between the Madiinae and other subtribes.     

CHLOROPLAST DNA EVOLUTION AND PHYLOGENETIC RELATIONSHIPS IN CLARKIA SECT. PERIPETASMA (ONAGRACEAE)

Restriction-site analysis of chloroplast DNA in Clarkia sect. Peripetasma (Onagraceae) was done to test previously proposed phylogenetic models. One hundred nineteen restriction-site mutations were identified among the nine species using 29 restriction enzymes, and these were used to construct rooted most parsimonious trees (Wagner and Dollo). A chloroplast DNA evolutionary clock could not be statistically rejected. Branch points of this tree were statistically tested by Felsenstein's bootstrap method. This tree 1) provided an unambiguous and detailed genealogical history for the section, 2) verified a previous partial phylogenetic model for the section based on gene duplications and differential silencing, 3) provided details of the phylogenetic model not inferred or expected based on morphology and reproductive isolation, and 4) indicated that morphology evolves at markedly different rates within and between lineages in the section.     

GENETIC VARIATION AND ESTIMATES OF GENE FLOW IN CLARKIA SPECIOSA SUBSP. POLYANTHA (ONAGRACEAE)

Estimates of interpopulational gene flow and the levels and distribution of genetic variation in Clarkia speciosa subsp. polyantha were obtained using enzyme electrophoresis. Eight enzymes encoded by 17 loci were analyzed. Nei's mean genetic identity was 0.96, indicating little genetic divergence among populations. Gene diversity statistics also suggest little heterogeneity among populations. Interpopulational gene flow, estimated according to Slatkin (1985), was fairly high, Nm = 3.9, probably accounting for the lack of differentiation among populations.     

PHYLOGENETIC IMPLICATIONS FROM CUTICULAR WAX ANALYSES IN EPILOBIUM SECT. ZAUSCHNERIA (ONAGRACEAE)

Analysis of n-alkane constitutents of the leaf cuticle have been utilized to elucidate phylogenetic relationships within Epilobium section Zauschneria. Specimens representing 136 populations were greenhouse-grown under the same conditions. Crosses were made between representatives of all taxa within the section. The resulting F₁ generations were also grown under the same conditions. Cuticle samples were removed in chloroform, purified with column chromatography and analyzed using gas chromatography and mass spectrometry. Resultant data were computer analyed using discriminant function analysis. Based on percent composition of 12 n-alkanes, each population and taxon can be characterized by a distinctive wax profile. Results suggest that the tetraploids E. canum subsp. canum and E. canum subsp. latifolium have been derived from the anastomosis of two distinct diploid genomes, E. canum subsp. canum “angustifolium” and E. septentrionale. Biochemical evidence from extant populations of E. canum are in agreement with geographic positions and ranges of the diploid species involved. Greenhouse hybridizations between the respective diploids, E. canum subsp. canum “canum” and E. septentrionale, result in F₁ progeny possessing phenotypes similar to naturally occurring tetraploid E. canum populations. Wax compositions of the natural tetraploid populations are intermediate between diploids and vary depending on geographical proximity to diploid species. Past taxonomic treatments of the genus have placed the tetraploid Zauschneria arizonica within the species Z. californica (now E. canum). Analysis of wax compositions now suggets that Z. arizonica had an independent origin which involved, at least in part, the diploid species E. canum subsp. garrettii from Utah.     

EXPERIMENTAL NON-STIGMATIC POLLINATIONS IN CLARKIA UNGUICULATA LINDL. (ONAGRACEAE)

Within angiosperms, the stigma-style apparatus provides a barrier to direct contact between ovules and the external environment. In traversing this barrier, male gametophytes presumably are subject to intense competition and natural selection. Successful fertilizations and resultant seed set can be accomplished following surgical decapitation of the stigma and style, provided that these normally receptive sites are replaced with an optimal environment suitable for pollen germination and growth. Unlike other methodologies, these in situ-in vitro pollinations are comparatively easily accomplished, and provide a means for circumventing natural fertility barriers; additionally, they may provide a simple means of artificially selecting pollen genomes. In Clarkia unguiculata 23.0% of attempted non-stigmatic pollinations produced outcrossed F₁ progeny.     

GENETIC AND ENVIRONMENTAL VARIATION IN FLORAL TRAITS AFFECTING OUTCROSSING RATE IN CLARKIA TEMBLORIENSIS (ONAGRACEAE)

Clarkia tembloriensis exhibits a wide range of variation among its natural populations in outcrossing rate and in separation of male and female function in space (anther-stigma separation or herkogamy) and in time (protandry). Here we show that outcrossing rate is highly correlated with protandry and anther-stigma separation. Both genetic and environmental variation contribute to inter- and intrapopulation variation in protandry and anther-stigma separation. Interpopulation differentiation for protandry and anther-stigma separation was found to be polygenic. Genetic variation for protandry and anther-stigma separation within populations was demonstrated by a significant among-family variance in two populations with contrasting breeding systems. Environmental effects on the expression of mating system traits were manifested in two ways. First, significant variation among lathhouse benches suggests that small-scale environmental heterogeneity may affect the development of floral traits. Second, protandry was shortened under hot summer conditions. Hence, hotter and drier habitats, typical of the more self-pollinating populations of C. tembloriensis, can promote self-pollination purely through environmental effects.     

EXTENSIVE GENE DUPLICATIONS IN DIPLOID EUPATORIUM (ASTERACEAE)

An electrophoretic study of isozyme number for seven soluble enzymes revealed extensive gene duplications in eight diploid species of American Eupatorium belonging to three morphological groups. The enzymes isocitrate dehydrogenase, phosphoglucomutase, phosphoglucose isomerase, 6-phosphogluconate dehydrogenase, and shikimate dehydrogenase occur as three to six isozymes in all species, whereas the minimal conserved number typical of diploid plants is two isozymes for each. Fructose 1, 6-biphosphate aldolase is expressed as multibanded pattern suggesting fixed heterozygosity in all examined species. It was not possible to document gene duplication for triosephosphate isomerase from the electrophoretic patterns. All species examined have a chromosome number of 2n = 20, which has been regarded as the basic diploid number for Eupatorium. However, the detection of extensive duplications suggests that 2n = 10 may be the original diploid chromosome number in Eupatorium and that plants with 2n = 20 are of polyploid origin. This hypothesis would mean that extensive duplications at isozyme gene loci have been maintained since the origin of the genus, despite chromosomal diploidization having occurred.     

STABILITY OF A CHROMOSOMAL HYBRID ZONE IN THE CLARKIA NITENS AND CLARKIA SPECIOSA SSP. POLYANTHA COMPLEX (ONAGRACEAE)

Clarkia nitens and Clarkia speciosa polyantha (Onagraceae) are distinct chromosomal taxa differing by at least six reciprocal translocations. Where the taxa have come into contact, a chromosome boundary zone exists characterized by high levels of translocation heterozygosity due to at least 12 new chromosome arrangements which have evolved there. Previous studies have shown that these boundary arrangements are distributed such that they provide for full interfertility between adjacent populations. It was hypothesized that the geographic distributions of each of these arrangements will remain generally stable due largely to the very adverse effect that major changes would have on fertility. Evidence is presented here that over a ten year period the frequencies and geographic distributions of the chromosome arrangements within this boundary region have remained stable. The frequencies of the various chromosomal configurations (nine pairs, ring of four, two rings of four, ring of six, ring of four + ring of six, and ring of eight) from surveys in 1968 and 1978 have been analyzed statistically. In general, the analysis indicates that there have been no detectable changes over the 10-yr period.     

CRYPTIC SELF-INCOMPATIBILITY AND THE BREEDING SYSTEM OF CLARKIA UNGUICULATA (ONAGRACEAE)

Analysis of 2,117 segregating progeny from competitive pollinations involving self and foreign pollen reveals that Clarkia unguiculata possesses a cryptic self-incompatibility mechanism. This mechanism promotes outcrossing when foreign pollen is available, yet allows for high fecundity through selling in marginal or catastrophically reduced populations. Competitively based self-incompatibility in the species suggests that discrimination against genetically similar siblings may also be possible. The preferential self-exclusion system insures that a seed crop of maximal size and genetic heterogeneity will be produced despite unpredictable environmental perturbations.     

FERTILITY SELECTION ON A DISCRETE FLORAL POLYMORPHISM IN CLARKIA (ONAGRACEAE)

This study investigated fertility selection on a flower petal pigmentation polymorphism in Clarkia gracilis ssp. sonomensis. Natural populations are typically composed of nearly 100% spotted-petal plants, although rare populations contain a majority of unspotted plants. I compared fitness values for the two morphs using a simple fertility model to estimate selection for experimental arrays of plants placed into existing populations of different phenotypic frequencies. Both male and female reproductive success were estimated as well as the pattern of mating among phenotypes. Although the separate fitness components varied from no differences to a strong advantage for spotted plants, for every situation the selection calculations predicted an increase in the frequency of the spotted allele. Pollinator behavior and postpollination mechanisms may be responsible for the fitness differences. The apparent inability of the unspotted allele to spread though most natural populations is consistent with its selective disadvantage in this study.     

KARYOTYPIC DATA ON NORTH AND CENTRAL AMERICAN ZAMIACEAE (CYCADALES) AND THEIR PHYLOGENETIC IMPLICATIONS

Chromosome numbers and karyotypes of species from four American Zamiaceae (Cycadales) are reported. Zamia shows interspecific and intraspecific chromosome variation, whereas Microcycas, Ceratozamia, and Dioon have constant karyotypes within each genus. In Zamia, all karyotypes have the same number of submetacentric and acrocentric chromosomes, but they differ in the number of metacentric and telocentric chromosomes. Centric fission of metacentric chromosomes is proposed to explain the karyotypic variation in this genus. Zamia shows karyological relationships with Microcycas and Ceratozamia, whereas Dioon appears very distinct from the other American cycad genera. Affinity among Zamia, Ceratozamia, and Microcycas karyotypes and distinctiveness of Dioon karyotypes are supported by comparative analysis of phenotypic characters in the four genera.     

INBREEDING EFFECTS IN CLARKIA TEMBLORIENSIS (ONAGRACEAE) POPULATIONS WITH DIFFERENT NATURAL OUTCROSSING RATES

Inbreeding depression is commonly observed in natural populations. The deleterious effects of forced inbreeding are often thought to be less pronounced in populations with self-pollinating mating systems than in primarily outcrossing populations. We tested this hypothesis by comparing the performance of plants produced by artificial self- and cross-pollination from three populations whose outcrossing rate estimates were 0.03, 0.26, and 0.58. Outcrossing rates and inbreeding coefficients were estimated using isozyme polymorphisms as genetic markers. Analysis of F statistics suggests that biparental inbreeding as well as self-fertilization contribute to the level of homozygosity in the seed crop. Biparental inbreeding will reduce the heterozygosity of progeny produced by outcrossing, relative to random outcrossing expectations, and hence will reduce the effects of outcrossing versus self-fertilization. Heterotic selection may increase the average heterozygosity during the life history. Selfed and outcrossed seeds from all three populations were equally likely to germinate and survive to reproduce. However, inbreeding depression was observed in fecundity traits of plants surviving to reproduction in all three populations. Even in the population whose natural self-fertilization rate was 97%, plants grown from seed produced by self-pollination produced fewer fruits and less total seed weight than plants grown from outcrossed seed. There was no detectable inbreeding depression in estimated lifetime fitness. Inbreeding effects for all reproductive yield characters were most severe in the accession from the most outcrossing population and least severe in the accession from the most self-fertilizing population.     

DEVELOPMENTAL STABILITY IN LEAVES OF CLARKIA TEMBLORIENSIS (ONAGRACEAE) AS RELATED TO POPULATION OUTCROSSING RATES AND HETEROZYGOSITY

Four natural populations of Clarkia tembloriensis, whose levels of heterozygosity and rates of outcrossing were previously found to be correlated, are examined for developmental instability in their leaves. From the northern end of the species range, we compare a predominantly selfing population (t? = 0.26) with a more outcrossed population (t? = 0.84), which is genetically similar. From the southern end of the range, we compare a highly selfing population (t? = 0.03) with a more outcrossed population (t? = 0.58). We measured developmental stability in the populations using two measures of within-plant variation in leaf length as well as calculations of fluctuating asymmetry (FA) for several leaf traits. Growth-chamber experiments show that selfing populations are significantly more variable in leaf length than more outcrossed populations. Developmental instability can contribute to this difference in population-level variance. Plants from more homozygous populations tend to have greater within-plant variance over developmentally comparable nodes than plants from more heterozygous populations, but the difference is not significant. At the upper nodes of the plant, mature leaf length declines steadily with plant age, allowing for a regression of leaf length on node. On average, the plants from more homozygous populations showed higher variance about the regression (MSE) and lower R² values, suggesting that the decline in leaf length with plant age is less stable in plants from selfing populations than in plants from outcrossing populations. Fluctuating asymmetry (FA) was calculated for four traits within single leaves at up to five nodes per plant. At the early nodes of the plant where leaf arrangement is opposite, FA was also calculated for the same traits between opposite leaves at a node. Fluctuating asymmetry is significantly greater in the southern selfing population than in the neighboring outcrossed population. Northern populations do not differ in FA. Fluctuating asymmetry can vary significantly between nodes. The FA values of different leaf traits were not correlated. We show that developmental stability can be measured in plants using FA and within-plant variance. Our data suggest that large differences in breeding system are associated with differences in stability, with more inbred populations being the least stable.     

EXTINCT TRANSITIONAL FAGACEAE FROM THE OLIGOCENE AND THEIR PHYLOGENETIC IMPLICATIONS

Fruits, catkins, and associated leaves of at least two extinct trigonobalanoid taxa have been discovered at an Oligocene fossil plant locality rich in fagaceous remains. These fossils exhibit a mosaic of fruit and pollen characters found in the two extant subfamilies Castaneoideae and Fagoideae of Fagaceae. Comparison with cladograms based on modern taxa suggests that these extinct taxa were similar to the ancestors of subfamily Fagoideae and may have been intermediate between Fagus and the modern trigonobalanoid genera. Pollen types isolated from the fossil staminate catkins provide unique character states that are transitional between modern pollen types in Fagaceae and are important in understanding the evolution of exine micromorphology within the family. This analysis provides a striking example of the use of character data from fossils to determine character-state adjacency prior to polarization of characters using outgroup comparison. Because of the mosaic nature of their character complexes, these fossils support monophyly in both the family Fagaceae and the subfamily Fagoideae. In addition, the occurrence of trigonobalanoid fossils in the Oligocene of North America has interesting biogeographic implications and provides insights into the nature of North American Fagaceae during the Tertiary.     

HYBRIDS AND PHYLOGENETIC SYSTEMATICS I. PATTERNS OF CHARACTER EXPRESSION IN HYBRIDS AND THEIR IMPLICATIONS FOR CLADISTIC ANALYSIS

Interspecific hybridization is considered common among plants, but the methods of cladistic systematics produce only divergently branching phylogenetic hypotheses and thus cannot give the correct phylogeny if an analysis includes hybrids. Empirical studies of the impact of known hybrids on phylogenetic analysis are lacking, and are necessary to begin to understand the problems that we face if hybrids are often included in cladistic analysis. Examination of the implications of hybrids for cladistics must begin with patterns of character expression in hybrids. This study includes 17 hybrids and their nine parental taxa that are Central American species of Aphelandra (Acanthaceae), analyzed using a set of 50 morphological characters. The hybrids are overwhelmingly intermediate as quantitatively scored for phylogenetic analysis. They express maternal and paternal, and primitive and derived characters in equal frequencies, showing no evidence of predominant inheritance of derived character states as has been assumed by most cladists who have considered hybrids theoretically. Because of their known genetic constitution, hybrids were useful in homology assessment and ordering character states. The parental character set was generally robust, but some changes were made to reflect the special evidence offered by the hybrids. These hybrids suggest that the inclusion of hybrids in phylogenetic analysis will not lead to unresolved cladograms with rampant homoplasy, as has been predicted by other authors. Instead, the patterns of character inheritance in these hybrids lead to the prediction that a hybrid will be placed by phylogenetic analysis as a basal lineage to the clade that includes its most derived parent, with relatively little effect on homoplasy. These predictions will be evaluated by incorporation of the hybrids in phylogenetic analyses, to be reported in a subsequent paper.     

A COMPARISON OF FLORAL DEVELOPMENT IN WILD TYPE AND A HOMEOTIC SEPALOID PETAL MUTANT OF CLARKIA TEMBLORIENSIS (ONAGRACEAE)

The mature wild type petals of Clarkia tembloriensis consist of a long slender claw and an expanded deltoid-shaped limb. They are pink, with a maroon spot at the base of the limb. Their surface texture is smooth. A variant of petal form, crinkled petal, occurs commonly in several natural populations of C. tembloriensis. The mature crinkled petals are elongated, greenish pink, and possess trichomes. They resemble the mature sepals of C. tembloriensis in general shape, color, and surface texture. Organ initiation and subsequent patterns of development of wild type petals, wild type sepals, and crinkled petals were examined and compared using scanning electron microscopy and allometric growth analysis. Crinkled petals are similar to wild type petals in time and position of primordia initiation, and in size and shape at inception. Crinkled petals are similar to wild type sepals in pattern of allometric growth. The crinkled petal mutant fits the broad definition of a homeotic mutant in that the petal has assumed characteristics of the sepal.