Autopolyploidy in Dactylis glomerata L.: further evidence from studies of chloroplast DNA variation

Summary Chloroplast DNA variation has been used to examine some of the maternal lineages involved in the evolution of the intraspecific polyploid complex, Dactylis glomerata L. Diploid (2x) and tetraploid (4x) individuals were collected from natural populations of the subspecies glomerata (4x), marina (4x) and lusitanica (2x), as well as from sympatric 2x/4x populations of the Galician type. Digestion of their ctDNA with 11 restriction endonucleases revealed enough variation to characterise three ctDNA variants, designated MBMK, MBmK and mBMK. The distribution of these ctDNA variants reflects different stages in their spread among the populations. The MBMK ctDNA variant predominated at both ploidy levels in subspecies glomerata, lusitanica and marina, and in recent tetraploid Galician/glomerata hybrids. The MBmK variant was detected in a single tetraploid individual and probably results from a relatively recent mutation. Fixation of the mBMK minority variant in the diploid and tetraploid Galician populations adds to the evidence concerning the possible origin of the Galician tetraploids. It means that the Galician diploids were maternal ancestors of the tetraploids. This result complements evidence from earlier studies based on morphology or biochemical markers, and reduces the likelihood that the tetraploids arose by hybridisation between an ancient Galician diploid and an alien tetraploid. It is, however, consistent with a true autopolyploid origin of the tetraploids.     

Polyploidy and habitat differentiation in Dactylis glomerata L. from Galicia (Spain)

Summary The microdistribution of diploid and tetraploid plants of Dactylis glomerata L. was examined and related to their immediate environment in several sites in central Galicia, where morphologically indistinguishable individuals of both ploidies grow in sympatry. The two related cytotypes differed in habitat preference. Diploids were mainly confined to the low-density forest-floor habitat in woodlands of mostly ancient origin, whereas tetraploids were widespread in varied habitats but clearly predominant in open areas, particularly in disturbed anthropic sites. The in situ comparison of plant performance showed that where plants of each ploidy were more common they produced more tillers, panicles and seeds. This habitat preference closely reflected differences in life-history characteristics. The tetraploids had an early and short flowering time almost always completed before the aestival drought, whereas the diploids began to flower several weeks later and flowered throughout the drought. Comparisons along artificial gradients of soil water availability and light transmittance indicated that the cytotypes had distinct physiological requirements which probably originated in metabolic and more general genetic differentiation and could be directly attributable to ploidy. Habitat differentiation increases the species' colonizing ability. It also amplifies divergence in reproductive strategy between diploids and tetraploids, which reduces ineffective crossing between cytotypes and thereby permits them to coexist in sympatry. The effect of hybridization at the polyploid level on the differentiation between cytotypes was assessed from the recent introduction of a foreign tetraploid entity into the study area. Hybridization between the two distinct tetraploids was found to increase habitat differentiation between the diploids and the tetraploids, but the major part of this differentiation is probably attributable to ploidy itself.     

Successful crosses between Festuca arundinacea Schreb. and Dactylis glomerata L.

Summary Five F₁ plants have been obtained after extensive crossing between different ecotypes or varieties of Festuca arundinacea Schreb. and Dactylis glomerata L. The success did not appear to depend on specific treatments (spraying with -aminocaproic acid or gibberellic acid or pre-pollination with killed pollen from the seed parent), but the crossability is limited to exceptional plants.F₁ hybrids showed characteristics of both the parents. In four hybrids various developmental disturbances were observed (low viability, aneusomaty, absence of development of inflorescences). Only one hybrid consistently showed 2n=35 chromosomes, good viability and growth, however, it was sterile. After clonal propagation, attempts for polyploidization were started.     

Embryoid and plantlet formation from leaf segments of Dactylis glomerata L.

Summary The purpose of this investigation was to demonstrate callus induction and plantlet formation from cultured leaf segments of 12–15 week-old Dactylis glomerata L. (orchardgrass) plants. Flat half-leaf sections, approximately 2–3 mm square, from the three innermost (youngest) leaves were isolated and individually plated serially beginning at the leaf base on a solid SH medium containing 30 M of 3,6-dichloro-oanisic acid (dicamba). Callus formed on leaf sections from all 50 plants used in the study. After transfer to SH medium with 1 M dicamba, plantlets formed from leaf sections of 9 of the 50 plants. In most cases plantlets formed from embryogenic callus but in a few cases embryoids formed directly on the leaf surface without an intervening callus state. These developed into plantlets when transferred to low auxin medium. The response for both callus and plantlet formation decreased with increasing distance both spatially and temporally from the shoot apex. Histological examination of embryogenic callus revealed the presence of non-zygotic embryos in various stages of development. The results provide further support for compentency (if not totipotency) of Gramineae leaf cells.     

Morphological variation and distribution of cytotypes in the diploid-tetraploid complex of the genus Dactylis L. (Poaceae) from Algeria

Morphological and cytological characters were analysed among thirty populations of the genus Dactylis L. from Algeria, to understand its infraspecific diversity. Principal Component Analysis using quantitative characters allowed discriminating the tetraploid populations into two subspecies, marina (Borrill) Greuter, localised on sea cliffs, and hispanica (Roth) Nyman, very widespread. Some individuals of these latest populations formed a distinct group, identified as subsp. glomerata Hayek. Three diploid taxa are described in the literature: subspecies santai Stebbins & Zohary, castellata Parker & Borrill and mairei Stebbins & Zohary that are considered as prevalent in Algeria, distributed in Tellian Atlas, in forest ecosystems within mesophytic habitats. Canonical Discriminant Analysis on natural populations and on experimental cultures showed two main groups: the first group corresponds to subspecies mairei, with a narrow distribution; the second one exhibits a wide morphological variability and belongs to santai type. Based on this study, a key to aid in identification of the subspecies is presented.     

Genetic variation and putative hybridization inSalix alba andS. fragilis (Salicaceae): Evidence from allozyme data

Genetic variability was estimated by enzyme electrophoresis in 239 Belgian clones from theSalix alba-S. fragilis complex. This morphological complex suggested a high frequency of hybrids. To test this hypothesis, the clones were pooled as a single co-adapted species complex and secondly as belonging to either species, i.e. beingS. alba-like orS. fragilis-like. The standard genetic variability measures showed higher values for the complex than for the separate species. However, the observed mean heterozygosity was lower in the putative hybrid complex than for each of the species separately. The fixation indices were more variable at the species level and indicated that mostS. fragilis locations appeared fixed forlap-1, whereas no fixation occured inS. alba locations. Averaged at the regional (i.e. catchment) level, this difference between the two species remained and values ranged from 0.457 to 0.617 inS. alba and were much higher, fixed homozygous or monomorph inS. fragilis. Hierarchical F-statistics revealed that most of the differentiation occured at the lower levels of localities and tributaries and that there was no further differentiation between catchments. Tributaries which are 10–25km in length were proposed as the most likely entities for further examination of putative hybridization and events of allelic fixations. By considering the two abovementioned approaches of data input, it could be suggested that most of the allozyme differentiation was between the species and less between the regions.     

Changes in endogenous polyamines during the formation of somatic embryos from isogenic lines of Dactylis glomerata L. with different regenerative capacities

The endogenous levels of polyamines (PAs) in leaf-base explants isolated from plants of two isogenic lines of Dactylis glomerata L., differing in their competence for somatic embryogenesis, were compared. Leaf-bases isolated from plants with a high level of competence for somatic embryogenesis (HEC) contained four times the level of polyamines compared to those isolated from plants with a low level of competence for somatic embryogenesis (LEC). When the levels of individual polyamines in the HEC and LEC lines were compared, leaf-bases from plants of the HEC line had much lower PUT/SPD ratios than those from the LEC line. When changes in the levels of PAs were monitored during the first 28 d of culture, on a medium which promotes initiation of somatic embryogenesis, leaf-base cultures from plants of the HEC line showed a 50% increase in the levels of PAs during the first 7 d of culture, after which time levels began to decline. By day 21, levels had dropped below those found in freshly isolated leaf bases. While PUT and SPM levels increased by about 30%, the greatest increase was shown by SPD, which increased by more than 100% during the first 7 d of culture, before declining. In contrast much smaller changes in PA levels were found when leaf-bases from plants of the LEC line were cultured.     

Anticytokinin effects on in vitro response of embryogenic and nonembryogenic genotypes of Dactylis glomerata L.

The effect of triazine and carbamate type of anticytokinins on in vitro response of embryogenic and nonembryogenic genotypes of orchardgrass (Dactylis glomerata L.) was studied. Both compounds stimulated callus production. Anticytokinins influenced both the efficiency of somatic embryogenesis and frequency of embryoid formation.     

Somatic embryogenesis in suspension and suspension-derived callus cultures ofDactylis glomerata

Summary Suspension cultures were initiated from somatic embryos and embryogenic callus ofDactylis glomerata L. in SH-30 liquid medium [Schenk andHildebrandt (1972) containing 30 M 3,6-dichloro-o-anisic acid (dicamba)] with or without 1.5 gl^–1 casein hydrolysate. Established suspension cultures maintained in SH-30 without casein hydrolysate proliferated when cell masses underwent cell division and enlargement. These cultures contained numerous root primordia and increased in volume when the cell masses continued to grow and fragment. Embryos developed only when cell masses were plated on solidified SH-30 medium. Cultures maintained in SH-30 liquid medium with casein hydrolysate also proliferated by the growth and fragmentation of cell masses. However, these cell masses contained numerous developing embryos and possessed few or no root primordia. Embryos were either attached to cell masses by a suspensor-like structure or were free and became fully developed in the liquid medium. Newly formed embryos became callused and produced embryogenic cell masses. Embryos germinated either in liquid or on solid SH medium without dicamba. The resulting plantlets possessed green shoots and well developed roots. Plants from suspension and suspension-derived callus cultures have been established in soil and grown to maturity.     

Clone identification and clinal allozyme variation in populations ofAbies alba from the Eastern Alps (Austria)

Allelic frequencies at the polymorphic enzyme loci IDH-1, IDH-2, LAP-1, and 6-PGDH-1 were determined in population samples ofAbies alba from 5 different areas in Austria. The results reveal clear genetic differentiation between the eastern and the western provenances. Gradual frequency clines are observed with all four loci. The amount of variation, measured as average heterozygosity and genotypic diversity, is significantly higher in the west. The multilocus genotypes proved to be useful markers for the identification of clones. Functional differences with respect to substrate specifity were found for the two allozymes coded by the coexisting alleles of the IDH-2 locus.     

Asymmetric introgression between fishes in the Red River basin of Texas is associated with variation in water quality

When ecologically divergent taxa encounter one another, hybrid zones can form when reproductive isolation is incomplete. The location of such hybrid zones can be influenced by environmental variables, and an ecological context can provide unique insights into the mechanisms by which species diverge and are maintained. Two ecologically differentiated species of small benthic fishes, the endemic and imperiled prairie chub, Macrhybopsis australis, and the shoal chub, Macrhybopsis hyostoma, are locally sympatric within the upper Red River Basin of Texas. We integrated population genomic data and environmental data to investigate species divergence and the maintenance of species boundaries in these two species. We found evidence of advanced‐generation asymmetric hybridization and introgression, with shoal chub alleles introgressing more frequently into prairie chubs than the reciprocal. Using a Bayesian Genomic Cline framework, patterns of genomic introgression were revealed to be quite heterogeneous, yet shoal chub alleles were found to have likely selectively introgressed across species boundaries significantly more often than prairie chub alleles, potentially explaining some of the observed asymmetry in hybridization. These patterns were remarkably consistent across two sampled geographic regions of hybridization. Several environmental variables were found to significantly predict individual admixture, suggesting ecological isolation might maintain species boundaries.     

A histological study of tissue proliferation,embryogenesis, and organogenesis from tissue cultures ofDactylis glomerata L.

Summary Histological information is presented on the origin of initial tissue proliferation and on embryogenesis and organogenesis in sub-cultured tissue derived from mature orchardgrass (Dactylis glomerata L.) embryos. Embryos were plated on an LS agar medium containing 20 M 2,4-D. Examination of cultures between 96 and 144 hours after plating showed parenchyma proliferation originating primarily from the coleorhiza, especially the basal portion. Within 28 days after plating, the tissue showed various degrees and kinds of organized structures including lobed meristematic regions with vascular tissue. These are thought to develop ultimately into aerial roots which are common in grass tissue cultures. Subcultured tissue on 1.0 M 2,4-D medium showed somatic embryos completely isolated from the tissue mass suggestingde novo embryogenesis. Organogenesis was evident by shoot apical meristems existing on the surface of the tissue mass without attached roots and root meristems without shoots within the tissue. The observations are discussed in relation to the anatomy of the grass embryo.This research was supported in part by the Competitive Research Grants Office of the U.S. Department of Agriculture under Agreement No. 5901-0410-9-0331-0.     

Genetic differentiation,polyploidization and hybridization in northern EuropeanDactylorhiza (Orchidaceae): Evidence from allozyme markers

Taxa endemic to North-western Europe are rare, but the orchid genusDactylorhiza contains several species restricted to this area. Evidence from morphological and cytological studies have indicated that some species may have arisen recently and may be of hybrid origin. In the present report, I use allozymes to characterize the genomes in various species ofDactylorhiza and evaluate the possibilities for rapid evolutionary change in the genus. Allotetraploid species have evolved repeatedly from two principal diploid ancestral lineages. These lineages include extant diploid and autotetraploid species, from which allotetraploid derivatives may still arise. It is suggested that allotetraploidization dominates over introgression as speciation mechanism in the genus. The more common and widespread allotetraploid species could be characterized by their allozyme characters over considerable distances, indicating that each of them may have a unique origin and that they have spread from their ancestral populations to the present distribution areas. However, it is also possible that some allotetraploid species contain local populations that have been independently derived from the ancestral lineages.     

Genetic markers distinguishing between the two subspecies of the rosy bitterling,Rhodeus ocellatus (Cyprinidae)

Eleven populations of the rosy bitterling,Rhodeus ocellatus, from different localities in Japan, were genetically compared at 16 protein-coding loci using starch-gel electrophoresis. Two loci,Ldh-2 andPgdh, were demonstrated as diagnostic markers for the identification of two subspecies;R. ocellatus kurumeus, which is native to Japan, andR. ocellatus ocellatus, which was introduced from China. The two subspecies were distinguished by the complete substitution of different alleles between them. Population ofR. ocellatus kurumeus occurring in Yao City, Osaka, and in Kanzaki, Saga Prefecture were genetically closely related to each other (genetic distance: D=0.056) but distantly so toR. ocellatus ocellatus from Saitama Prefecture (D=0.202 or 0.265). Electrophoretic analyses also elucidated the existence of hybrid populations of the two subspecies. The populations ofR. ocellatus kurumeus in Yao City had lower genetic variability and a lower incidence of white coloration on the ventral fins than populations of the same in Saga Prefecture. The present study strongly implies that the introduction of the foreign freshwater fishes with subspecific differentiation, into the original range of indigenous subspecies, should be averted not to bring the genetic pollution.     

Genetic variation in sympatric populations of diploid and polyploid brine shrimp (Artemia parthenogenetica)

We examined genetic variation in sympatric diploid and polyploid brine shrimp Artemia parthenogenetica from each of three populations (China, Italy and Spain). Italian and Spanish tetraploids are closely related (I=0.964). Diploids and tetraploids within each of the two European populations are also closely related (mean I=0.905). Most alleles found in diploids also exist in sympatric polyploids. In contrast, the asexual Artemia (2N, 4N and 5N) in our study share few alleles with their close sexual relative, A. tunisiana (mean I=0.002). These results, as well as the work of other authors, strongly suggest that at least the tetraploid Artemia in our study have an autopolyploid origin.Clonal diversity of polyploid Artemia can be very high at least in some population. Both diploids and polyploids had low clonal diversities in the populations dominated by polyploids and high clonal diversities in the population dominated by diploids.The most common genotypes of sympatric diploid and polyploid Artemia frequently differed. Some alleles occurred only in diploids, while others were restricted to polyploids. These results suggest that polyploidy in Artemia has led to genetic divergence from diploid progenitors, and that ploidy-level variation must also be considered in developing an understanding of spatial and temporal allozyme polymorphism in asexual populations.     

Influence of dicamba and casein hydrolysate on somatic embryo number and culture quality in cell suspensions of Dactylis glomerata (Gramineae)

The effects of various concentrations and combinations of dicamba (3,6-dichloro-o-anisic acid) and casein hydrolysate on growth, mucilage accumulation, somatic embryo and root development in suspension cultures of Dactylis glomerata L. (orchardgrass) were examined. Fresh weight of culture tissue was increased with 20 M but not with 80 or 160 M dicamba in treatments with 1–4 g/l casein hydrolysate. Different casein hydrolysate concentrations did not alter the amount of mucilage (measured by viscosity) in the supernatant in the absence of dicamba. However, the addition of dicamba increased viscosity with 80 M giving the maximum response. Casein hydrolysate produced the greatest viscosity at 1–3 g/l in treatments where dicamba was present. Both dicamba and casein hydrolysate were required for development of somatic embryos. Dicamba at 40 M with 3–4 g/l casein hydrolysate produced approximately 2000 embryos/35 ml of suspension. Root development was inhibited by dicamba and stimulated by the presence of casein hydrolysate. The usefulness of medium component manipulations for influencing somatic embryogenesis and culture quality is discussed.     

Contrasting allozyme and RAPD variation in spider populations from patchy forest habitats

During the last 1000 years, massive deforestation events have occurred in Flanders (the northern part of Belgium) and the remaining forests have become very isolated patches. It is expected that organisms bound to these patchy forest habitats and with limited dispersal capacities will likely experience strong effects of genetic drift. One such organism is the spider Coelotes terrestris. Allozyme data suggested that 10 Flemish populations of this spider showed little genetic variation, as only one out of 20 loci was polymorphic (phosphoglucose isomerase). In view of this result, we used random amplified polymorphic DNA (RAPD) markers to test whether this lack of allozyme diversity is an inherent feature of the populations and/or species studied or whether it rather reflects a characteristic of the markers and/or methods used. Since the RAPD data revealed a substantial amount of genetic diversity in the same 10 populations, our results suggest that the latter is true. Furthermore, the RAPD data agree with the expectations for an organism with low dispersal capacities that has lived in isolated forest patches for at least 200 generations. Supplemented with the results of other techniques and studies, these findings might be of importance for the future conservation of this spider species in Flanders.     

Genetic variation and hybridization in SwedishSchoenus (Cyperaceae)

Schoenus ferrugineus andS. nigricans have restricted distributions in Sweden and are almost exclusively confined to calcareous fen habitats. AtS. nigricans sites,S. ferrugineus is usually also present, and hybrids are frequently found. In this report, I used allozymes to estimate the amount of gene flow between the two species, and to compare the partitioning of genetic diversity in each of them. Thirteen loci were analysed at eight different enzyme systems. Seven loci were variable between or within the species. The two species had completely different alleles at two of the seven variable loci, whereas there was overlap at five loci. In all, 22 different alleles were found. Six of these alleles were confined toS. nigricans, and five alleles were confined toS. ferrugineus. Nei's genetic identity was 0.55.—InS. ferrugineus, three loci (23%) were polymorphic, and the average number of alleles per polymorphic locus was 2.0 (each polymorphic locus had two alleles). InS. nigricans, three loci (23%) were polymorphic, and the average number of alleles per polymorphic locus was 2.3.—The proportion of genetic diversity due to variation among sites (G _ST) was fairly similar in the two species, mean over loci = 0.12 inS. ferrugineus and 0.15 inS. nigricans. However, the proportion of genetic diversity due to variation among individuals within sites (G _IS) differed markedly between the two species, mean over loci = 0.54 inS. ferrugineus and 0.17 inS. nigricans. Accordingly, there was a much higher individual heterozygosity inS. nigricans than inS. ferrugineus. — Most hybrids were interpreted as F₁ hybrids. However, a small proportion, 0.5–1.6 %, were F_n hybrids or back-crosses.—On the Swedish mainland, all former occurrences ofS. nigricans are extinct, but viable hybrids are still present at a few sites in southernmost Sweden.     

Genetic introgression between native and introduced brown trout Salmo trutta L. populations in the Rhone River Basin

In the Doubs River (Rhône drainage) two distinct brown trout ( S. trutta ) phenotypes are observed. One phenotype is locally called Doubs trout and is characterized by four black stripes on the sides, similar to perch ( Perca fluviatilis L.) and the other is the common phenotype of the fluviatile ecotype of brown trout, Salmo trutta f. fario . Protein data for three samples from the Doubs show that the Doubs trout belongs to the Mediterranean population group of brown trout, whereas the fario phenotype originates from stocking with hatchery strains of Atlantic basin origin. The two forms, however, do not hybridize freely. This is indicated by considerable gametic phase disequilibrium between alleles of hatchery and Doubs trout at one sampling site, and by lack of intermediate genotypes and phenotypes at another sampling site. The introgression patterns observed at the two sites suggest that differences in local habitat conditions can affect the degree of hybridization and introgression.     

Allozyme variation in Carex sect. Digitatae – Evidence of introgression, genetic distinctiveness and evolution of taxa

 A subset of populations of the European taxa of Carex sect. Digitatae, including Carex digitata, C. pallens, C. ornithopoda, C. pediformis ssp. rhizodes and C. humilis have been investigated for allozyme variation. The (presumably) distantly related C. pendula was used as a reference taxon. Carex digitata, C. pallens and C. ornithopoda on the one hand, and C. humilis and C. pediformis on the other, shared the majority of the alleles. Cluster analyses based on genetic distances grouped populations of C. digitata and C. ornithopoda in a mixed subcluster whereas all populations of C. pallens formed a subcluster distinct from the digitata/ornithopoda cluster. This in spite of the fact that C. ornithopoda is morphologically clearly distinct from C. digitata whereas C. pallens is barely distinguishable from the latter. Carex pediformis and C. humilis appeared not more genetically similar to C. digitata than to the reference taxon. It is concluded that 1) C. digitata, C. pallens and C. ornithopoda are closely related and most probably forms a monophyletic group, 2) C. pallens is a genetically distinct species, 3) C. pediformis and C. humilis are not closely related to the rest of C. sect. Digitatae, 4) C. pediformis and C. humilis are relatively closely related, 5) introgression do occur between the investigated species but not to such an extent that it can explain major geographic patterns of variation in C. digitata. Received December 21, 2001; accepted November 14, 2002 Published online: March 24, 2003