CHROMOSOME MUTATIONS IN A FERN POPULATION GROWING IN A POLLUTED ENVIRONMENT: A BIOASSAY FOR MUTAGENS IN AQUATIC ENVIRONMENTS

A population of royal ferns, Osmunda regalis, which is periodically submersed by the waters of the Millers River was found to have a very high frequency of chromosome mutations. The Millers River is located in western Massachusetts and is heavily polluted with industrial wastes. Approximately 43 % of meiotic samples collected in 1973 from the Millers River population were heterozygous for mutations such as paracentric inversions or reciprocal translocations, whereas less than 1 % of meiotic samples from nearby non-polluted control populations were such heterozygotes. Cytological analysis within royal fern clones indicate that practically all of the chromosome mutations were post-zygotically induced and that at least 64 % were induced since 1969. In the course of this study over 26,000 spore mother cells were analyzed for chromosome aberrations.     

POPULATION GENETIC STRUCTURE IN CHEILANTHES GRACILLIMA

Population genetic structure was examined in five populations of the xerically adapted homosporous fern Cheilanthes gracillima. F statistics using allozymic data indicated substantial genetic structure in all populations. To determine the factors responsible for genetic structure, we calculated levels of intragametophytic selfing and the fixation index for each subpopulation of each population and estimated levels of intrapopulational gene flow in each population. These analyses indicated that each subpopulation was a panmictic unit; thus, population genetic structure is not due to family structure, arising via matings between relatives. Intrapopulational gene flow was surprisingly low, given the typically high dispersibility of fern spores. However, it seems unlikely that spore dispersal in C. gracillima is significantly reduced relative to other homosporous ferns. Instead, we propose that the low rates of intrapopulational gene flow reflect limited availability of safesites for spore germination and gametophyte establishment. This ecological factor may play a primary role in generating and/or maintaining population genetic structure in C. gracillima.     

POPULATION STRUCTURE AND ESTIMATES OF GENE FLOW IN THE HOMOSPOROUS FERN POLYSTICHUM MUNITUM

Levels and distribution of genetic variation were investigated in the homosporous fern, Polystichum munitum. Homosporous ferns differ from higher vascular plants in that they possess potentially bisexual gametophytes which can produce a completely homozygous sporophyte in a single generation. Because of this, it has long been maintained that ferns possess an inbreeding mating system, resulting in low levels of genetic variation and high levels of homozygosity within populations. The four populations sampled maintain high levels of genetic variation (P? = 0.542; H? = 0.111; ā = 2.23), comparable to that maintained by populations of outcrossing seed plants. The mean fixation index, F, for the four populations was 0.052, indicating no significant deviations from Hardy-Weinberg genotypic expectations. Polystichum munitum distributes most of its genetic variation within rather than among populations. Population-genetic structure was assessed by subdividing each of two large populations into 10 × 10-m subpopulations. Comparisons of genetic variation within and among subpopulations indicated little genetic substructure within either of the artificially subdivided populations. Estimates of interpopulational gene flow (Nm) are extremely high, comparable to those reported for gymnosperms. Statistical estimates of intragametophytic selling are very low, ranging from 0 to 3%. This study suggests that Polystichum munitum is an outcrossing species. Evidence from this and other investigations indicates that fern species do not typically self-fertilize and that mating systems in ferns vary as they do among species of seed plants.     

The Dehalococcoides population in sediment-free mixed cultures metabolically dechlorinates the commercial polychlorinated biphenyl mixture aroclor 1260

Microbial reductive dechlorination of commercial polychlorinated biphenyl (PCB) mixtures (e.g., Aroclors) in aquatic sediments is crucial to achieve detoxification. Despite extensive efforts over nearly two decades, the microorganisms responsible for Aroclor dechlorination remained elusive. Here we demonstrate that anaerobic bacteria of the Dehalococcoides group derived from sediment of the Housatonic River (Lenox, MA) simultaneously dechlorinate 64 PCB congeners carrying four to nine chlorines in Aroclor 1260 in the sediment-free JN cultures. Quantitative real-time PCR showed that the Dehalococcoides cell titer in JN cultures amended with acetate and hydrogen increased from 7.07 x 10(6) +/- 0.42 x 10(6) to 1.67 x 10(8) +/- 0.04 x 10(8) cells/ml, concomitant with a 64.2% decrease of the PCBs with six or more chlorines in Aroclor 1260. No Dehalococcoides growth occurred in parallel cultures without PCBs. Aroclor 1260 dechlorination supported the growth of 9.25 x 10(8) +/- 0.04 x 10(8) Dehalococcoides cells per mumol of chlorine removed. 16S rRNA gene-targeted PCR analysis of known dechlorinators (i.e., Desulfitobacterium, Dehalobacter, Desulfuromonas, Sulfurospirillum, Anaeromyxobacter, Geobacter, and o-17/DF-1-type Chloroflexi organisms) ruled out any involvement of these bacterial groups in the dechlorination. Our results suggest that the Dehalococcoides population present in the JN cultures also catalyzes in situ dechlorination of Aroclor 1260 in the Housatonic River. The identification of Dehalococcoides organisms as catalysts of extensive Aroclor 1260 dechlorination and our ability to propagate the JN cultures under defined conditions offer opportunities to study the organisms' ecophysiology, elucidate nutritional requirements, identify reductive dehalogenase genes involved in PCB dechlorination, and design molecular tools required for bioremediation applications.     

INBREEDING AND THE RATE OF SELF-FERTILIZATION IN A GRAPE FERN,BOTRYCHIUM DISSECTUM

Botrychium dissectum is a homosporous fern with bisexual, subterranean gametophytes. Because of these features, B. dissectum would be suspected of displaying a very high frequency of self-fertilization. Sporophytes collected from three populations of this species were assayed for heterozygosity by determining the electrophoretic mobility patterns displayed by two polymorphic enzymes. Extreme deviations from Hardy-Weinberg expectations were observed in each population and analyzed by means of F-statistics. The average inbreeding coefficient was found to be 0.951. A population genetic model is derived that demonstrates that the rate of intragametophytic self-fertilization in homosporous ferns is equal to the inbreeding coefficient calculated from deviations from Hardy-Weinberg expectations. It is therefore concluded that B. dissectum outcrosses about 5% of the time.     

NONHOMOLOGOUS CHROMOSOME PAIRING IN THE FERN CERATOPTERIS

The homosporous fern Ceratopteris thalictroides forms completely homozygous offspring sporophytes in one generation of selfing. Such homozygotes do not breed true and can exhibit structural heterozygosity for chromosomal mutations in some meiocytes. This is due to occasional nonhomologous (presumably homoeologous) chromosome pairing at meiosis in these polyploid ferns.     

CELL DIVISION KINETICS IN THE SHOOT APICAL MERISTEM OF CERATOPTERIS THALICTROIDES BRONG. WITH SPECIAL REFERENCE TO THE APICAL CELL

The duration of mitosis and the cell cycle were determined for defined cell populations of the shoot apical meristem of Ceratopteris thalictroides Brong. by using the colchicine-induced metaphase accumulation technique. The results indicate that the apical cell is mitotically active and cycles at an apparently greater frequency than the cells of subjacent populations. Duration of mitosis was similar for all cells of the meristem. These results are correlated with mitotic indices of control apices, the geometry of the apex, and the mean number of cells in the meristem. Shoot apices from adult plants were examined to determine mitotic indices within the meristem; mitotic activity was again noted for the apical cell. These results contradict recent proposals that the pteridophyte apical cell serves as a unicellular quiescent center which lacks histogenic potential and offer experimental support for the classical concept of apical cell function in those fern shoot meristems which terminate in a single apical cell.     

GENETIC VARIATION IN THE HOMOSPOROUS FERN PELLAEA ANDROMEDIFOLIA

Genetic variability was examined in nine sexual and three apogamous natural populations of the homosporous fern Pellaea andromedifolia by electrophoretic analysis of enzymes specified by eight loci. Genetic interpretations of heterozygous banding patterns were determined by segregational analysis of gametophytes. High levels of segregating heterozygosity characterized the sexually reproducing populations, and genotype frequencies at the five polymorphic loci were consistent with those expected under conditions of random intergametophytic mating. Multiple-banded patterns in the apogamously reproducing populations resembled those of heterozygous sexual individuals, but did not segregate. The results suggest that genetic variation in sexual homosporous vascular plants is produced by cross fertilization of genetically different gametes and may not result from pairing between homoeologous chromosomes carrying duplicated loci as previously thought.     

QUANTITATIVE VARIATION IN FINITE PARTHENOGENETIC POPULATIONS: WHAT STOPS MULLER'S RATCHET IN THE ABSENCE OF RECOMBINATION?

Finite parthenogenetic populations with high genomic mutation rates accumulate deleterious mutations if back mutations are rare. This mechanism, known as Muller's ratchet, can explain the rarity of parthenogenetic species among so called higher organisms. However, estimates of genomic mutation rates for deleterious alleles and their average effect in the diploid condition in Drosophila suggest that Muller's ratchet should eliminate parthenogenetic insect populations within several hundred generations, provided all mutations are unconditionally deleterious. This fact is inconsistent with the existence of obligatory parthenogenetic insect species. In this paper an analysis of the extent to which compensatory mutations can counter Muller's ratchet is presented. Compensatory mutations are defined as all mutations that compensate for the phenotypic effects of a deleterious mutation. In the case of quantitative traits under stabilizing selection, the rate of compensatory mutations is easily predicted. It is shown that there is a strong analogy between the Muller's ratchet model of Felsenstein (1974) and the quantitative genetic model considered here, except for the frequency of compensatory mutations. If the intensity of stabilizing selection is too small or the mutation rate too high, the optimal genotype becomes extinct and the population mean drifts from the optimum but still reaches a stationary distribution. This distance is essentially the same as predicted for sexually reproducing populations under the same circumstances. Hence, at least in the short run, compensatory mutations for quantitative characters are as effective as recombination in halting the decline of mean fitness otherwise caused by Muller's ratchet. However, it is questionable whether compensatory mutations can prevent Muller's ratchet in the long run because there might be a limit to the capacity of the genome to provide compensatory mutations without eliminating deleterious mutations at least during occasional episodes of sex.     

ALLOZYMIC VARIATION IN LOCAL APOMICTIC POPULATIONS OF LEMNA MINOR (LEMNACEAE)

Flowering occurrence and allozymic variation were studied in eight local populations of Lemna minor in eastern Ontario, Canada. After 2 years of survey, not a single flower was observed. This absence of flowering suggests the possibility of loss of sexual reproduction. This may have had no net adverse effect on fitness given the simple life history and prolific vegetative propagation in L. minor. However, the absence of sexual reproduction may limit genotypic diversity. The allozymic analysis detected 18 loci from 13 enzyme systems. Large deviations from Hardy-Weinberg equilibrium were common because of an excess of homozygotes for several enzyme systems. The genotypic diversity within these eight populations had a mean D value of 0.973 with an average number of genotypes per population of 19.6. These results suggest that genotypic diversity within these populations is not severely limited by the rarity of sexual reproduction. The mean genotypic distance index (D₁₄ = 0.801) suggests a high degree of differentiation between populations. The mean number of populations per genotype was 1.78. Using a Mantel test, the genotypic distance matrix was not significantly related to the population-to-population distance matrix (t = -0.161, P = 0.413). Although rare events of sexual reproduction may help maintain genetic variation, somatic mutations and multiple origins of clones may be important factors maintaining genetic diversity both within and between populations of L. minor.     

INTERPOPULATIONAL VARIATION IN THE GAMETOPHYTES OF PELLAEA ANDROMEDAEFOLIA

The development and mature morphology of the gametophytes from both sexual and apogamous populations of the fern Pellaea andromedaefolia were investigated. While most sexual examples were indistinguishable, some differences were noted. An insular collection was distinctive in its variability and irregularity of form. Although the latter was a representative of var. pubescens, other collections of the variety could not be distinguished from var. andromedaefolia on the basis of gametophytic characteristics. The apogamous gametophytes were decidedly more variable in development and often very different from sexual thalli. The mature asexual thalli tended to be more irregular in form and usually sharply divergent from the typical cordate type characteristic of the sexual populations. Each of the five apogamous samples was unique with respect to gametophyte development. The differences among the gametophytes of the various populations do not correlate with the sporophytic characteristics which differentiate the two varieties of the species.     

THE ROLE OF INTROGRESSIVE HYBRIDIZATION IN THE EVOLUTION OF THE GILA ROBUSTA COMPLEX (TELEOSTEI: CYPRINIDAE)

Abstract The extent and impact of introgressive hybridization was examined in the Gila robusta complex of cyprinid fishes using mitochondrial DNA (mtDNA) sequence variation. Lower Colorado River basin populations of G. robusta, G. elegans, and G. cypha exhibited distinct mtDNAs, with only limited introgression of G. elegans into G. cypha. The impact of hybridization was significant in upper Colorado River basin populations; most upper basin fishes sampled exhibited only G. cypha mtDNA haplotypes, with some individuals exhibiting mtDNA from G. elegans. The complete absence of G. robusta mtDNA, even in populations of morphologically pure G. robusta, indicates extensive introgression that predates human influence. Analysis of the geographic distribution of variation identified two distinctive G. elegans lineages; however, the small number of individuals and localities sampled precluded a comprehensive analysis. Analysis of haplotype and population networks for G. cypha mtDNAs from 15 localities revealed low divergence among haplotypes; however, significant frequency differences among populations within and among drainages were found, largely attributable to samples in the Little Colorado River region. This structure was not associated with G. cypha and G. robusta, as morphotypes from the same location are more similar than conspecific forms in other locations. This indicates that morphological and mtDNA variation are affected by different evolutionary forces in Colorado River Gila and illustrates how both hybridization and local adaptation can play important roles in evolution.     

DIFFERENTIAL ANTHERIDIOGEN RESPONSE AND EVOLUTIONARY MECHANISMS IN CYSTOPTERIS

It has been demonstrated that antheridiogens determine gametangial initiation in cultures of some homosporous fern gametophytes. Studies of natural populations have implicated these compounds as breeding system controllers. In Cystopteris, response to antheridiogen varied significantly among species. While Cystopteris protrusa responded to exogenous antheridiogen, C. bulbifera did not. The allopolyploid derivative of these two species, C. tennesseensis, had an intermediate level of response. This marked physiological difference among morphologically similar taxa is valuable in delineating species and suggests a mechanism that may be associated with interspecific hybridization.     

Network-based cancer genomic data integration for pattern discovery

Background

Next-generation sequencing (NGS) has profoundly changed the approach to genetic/genomic research. Particularly, the clinical utility of NGS in detecting mutations associated with disease risk has contributed to the development of effective therapeutic strategies. Recently, comprehensive analysis of somatic genetic mutations by NGS has also been used as a new approach for controlling the quality of cell substrates for manufacturing biopharmaceuticals. However, the quality evaluation of cell substrates by NGS largely depends on the limit of detection (LOD) for rare somatic mutations. The purpose of this study was to develop a simple method for evaluating the ability of whole-exome sequencing (WES) by NGS to detect mutations with low allele frequency. To estimate the LOD of WES for low-frequency somatic mutations, we repeatedly and independently performed WES of a reference genomic DNA using the same NGS platform and assay design. LOD was defined as the allele frequency with a relative standard deviation (RSD) value of 30% and was estimated by a moving average curve of the relation between RSD and allele frequency.

Results

Allele frequencies of 20 mutations in the reference material that had been pre-validated by droplet digital PCR (ddPCR) were obtained from 5, 15, 30, or 40 G base pair (Gbp) sequencing data per run. There was a significant association between the allele frequencies measured by WES and those pre-validated by ddPCR, whose p-value decreased as the sequencing data size increased. By this method, the LOD of allele frequency in WES with the sequencing data of 15 Gbp or more was estimated to be between 5 and 10%.

Conclusions

For properly interpreting the WES data of somatic genetic mutations, it is necessary to have a cutoff threshold of low allele frequencies. The in-house LOD estimated by the simple method shown in this study provides a rationale for setting the cutoff.

     

MULTIPLE EVOLUTIONARY PATHWAYS TO DECREASED LATERAL PLATE COVERAGE IN FRESHWATER THREESPINE STICKLEBACKS

Adaptation to novel environments can be based either on standing genetic variation or variation attributable to new mutations. When standing genetic variation for a functional adaptation is lacking, and variation due to new mutations is not yet available, adaptation is possible only through alternative functional solutions. Reduction in the number of bony lateral plates as an adaptation to freshwater colonization by marine threespine sticklebacks (Gasterosteus aculeatus) has occurred in numerous independent cases through allelic substitution in the ectodysplasin‐a (Eda) gene. Studying the phenotypic and genetic variation in plate number and size in five marine and six freshwater threespine stickleback populations, we found that when variation in Eda was limiting (i.e., alleles associated with the low‐plate morph were missing or in extremely low frequency), plate number reduction did not take place in freshwater populations, but reduced lateral plate coverage was achieved by a reduction in the size of lateral plates. Our results suggest that this phenotypically and genetically discrete "small‐plated" threespine stickleback—which is the dominant form in three northern European freshwater populations—may be functionally equivalent to the low‐plated morph and hence, serve as an example of convergent evolution toward functional similarity in the face of genetic constraints.     

MUTATIONAL LOAD IN A FERN POPULATION GROWING IN A POLLUTED ENVIRONMENT

The Osmunda regalis sporophyte is well organized for the storage and perpetuation of post-zygotic mutations. Techniques have been developed to detect sporophytes in which such mutations have occurred. Two populations were screened for sporophytes which are chimeras for gametophytic or recessive sporophytic lethals. The populations had different ecologies; one was in a pristine lake side bog and the other was growing in the waters of a river heavily polluted with industrial wastes. Post-zygotic mutations were detected only in the polluted river population. The level of mutational damage approximated that previously documented from chromosome studies.     

THE EFFECTS OF KINETIN AND NAPHTHALENEACETIC ACID ON IN VITRO SHOOT MULTIPLICATION AND ROOTING IN THE FISHTAIL FERN

This study is concerned with the determination of the shoot and root inducing effects of kinetin (K) and naphthaleneacetic acid (NAA) on shoots ofthe fishtail fern (Nephrolepisfalcata formafurcans) in sterile tissue culture. The data shows that K is the major factor involved in maximal shoot production. The NAA is not essential. Specific concentrations ofNAA must be present with specific concentrations of K for maximal root production. At the same time, the data also demonstrate that shoots on media containing no NAA but with K concentrations of 5 × 10⁻⁷ and 10⁻⁶ M produced as many roots as with any other NAA concentration. The data can be used as a guide to rapid commercial propagation of fishtail fern, and demonstrate that media available commercially for Boston fern multiplication will induce maximal shoot production in fishtail fern.     

GENETIC ANALYSIS OF ANTHERIDIOGEN SENSITIVITY IN CERATOPTERIS RICHARDII

The pheromone antheridiogen promoted the production of males in gametophyte populations of the fern Ceratopteris richardii. Two homozygous strains of this species exhibited markedly different sensitivities to the pheromone, resulting in populations with either high or low frequencies of males. The two strains were characterized by dose responses to antheridiogen and by gametophyte population density studies to investigate the effect of antheridiogen produced within cultures. Analyses of Fl hybrids and an F2 generation indicated that differences in the antheridiogen responses of the two parental strains are associated with either one or two nuclear genes.     

Condition status of the endangered desert pupfish,Cyprinodon macularius Baird and Girard, 1853, in the Lower Colorado River Basin (Mexico)

The length‐weight relationship (LWR) was used to test differences in the fish somatic condition factor among four populations of the endangered desert pupfish, Cyprinodon macularius, from the Lower Colorado River Basin, Mexico. Bimonthly fish sampling and habitat assessments were carried out from September 1996 to August 1997 for four sites in Baja California and Sonora. Slope b of the WLR varied from 3.238 (Cerro Prieto population) to 3.613 (Welton‐Mohawk population), showing in all populations a positive allometric growth. Male b values were higher than those of females for Cerro Prieto (3.303 vs 3.071), Welton‐Mohawk (3.866 vs 3.579), and Flor del Desierto (3.357 vs 3.169) populations. The Cerro Prieto population showed the highest somatic condition (y‐intercept, a = ?11.759). Water depth and salinity were the ecological variables that better accounted for most of the variation in the somatic condition of this pupfish, with a negative and a positive effect, respectively.     

GENETIC VARIATION AND POPULATION STRUCTURE IN THE FERN BLECHNUM SPICANT (BLECHNACEAE) FROM WESTERN NORTH AMERICA

Population genetic structure in the homosporous fern Blechnum spicant was analyzed in six populations from western North America. Each population was divided into approximately 10 m by 10 m subpopulations, and genetic variation within and among subpopulations was compared using enzyme electrophoresis and F statistics. These analyses indicated that there was no evidence of genetic structure in four of the six populations examined. However, significant genetic heterogeneity among subpopulations was observed for the other two populations. The genetic structure of these populations may be attributable, in part, to family structure resulting from high rates of intragametophytic selling and/or spatial patchiness in the distribution of individuals due to limited habitat availability in these areas. Outcrossing populations of B. spicant generally lack genetic structure, whereas the most highly inbreeding population maintains significant genetic structure. The information obtained in this investigation of population genetic structure in Blechnum spicant is consistent with data for angiosperms and gymnosperms. It appears that the outcrossing mating system and effective mechanism of spore dispersal in B. spicant may account for the general lack of genetic structure within populations of this species.