Morphological differentiation of Calypogeia muelleriana (Jungermanniales, Hepaticae) in Poland
Morphological and anatomical characters and oil bodies were studied in 52 samples from Poland: 31 samples of typical Calypogeia muelleriana and 21 of a newly detected taxon with the use of isozyme markers. Plants of the new taxon morphologically resemble C. muelleriana, but differ from it significantly in 16 morphological traits as well as oil body characters. The greatest morphometric differences were found in the features connected with underleaves. The Mahalanobis distance based on the 47 quantitative traits, between the newly detected taxon and C. muelleriana was larger than that between C. muelleriana and C. azurea - a generally accepted species. The forward stepwise method of discriminant analysis showed that the set of diagnostic characters could be limited to five. A dendrogram constructed on the basis of the Euclidean distance, using the set of diagnostic characters, divided the examined samples into two groups that correlated with groups detected by genetic markers. Results of a multivariate analysis showed that five morphological traits are sufficient for a proper classification of plants to these two taxa.
Currently, two subspecies are formally recognized within Calypogeia fissa: C. fissa subsp. fissa occurring in Europe and C. fissa subsp. neogea known from North America. Genetic studies have revealed a complex structure of this species. Within the European part of distribution, three genetically distinct groups PS, PB and G are distinguished. The combination of the SCAR marker Cal04 and PCR-RFLP markers with three restriction enzymes (SmaI, TaqI and TspGWI) allowed the recognition of all groups within the C. fissa complex. The TaqI enzyme recognizing the restriction sites in the PCR product of SCAR marker Ca104 turned out to be the best marker
The genus Aneura is represented in Poland by two species - A. pinguis and A. maxima. A. pinguis in contrast to A. maxima is a complex of cryptic species temporarily named A. pinguis species: A, B, C, and E. All species of the A. pinguis complex and A. maxima differ in their geographic distribution and habitat preferences. A. pinguis species A grows mainly on humus over limestone rocks in the Western Carpathians, A. pinguis species B occurs mainly on clay soil in Bieszczady Mts. and in clayish areas of lowlands, A. pinguis species C grows both in lowlands and mountains and it occupies mostly wet sandy soils, on the shores of oligotrophic lakes and river and mountain stream banks, A. pinguis species E is connected with calcareous rocks in flowing water in mountains. A. maxima grows over the country - both in lowlands and mountains, in marshes situated on the river banks.
Calypogeia fissa is a suboceanic-mediterrean and amphiatlantic species, which comprises two subspecies: C. fissa subsp. fissa occurring in Europe and C. fissa subsp. neogea Schust. known from North America. Recently, within the European part of distribution, three groups (PS, PB and G) were distinguished with the aid of genetic and molecular markers. The flow cytometry results revealed that two of the detected groups of the European C. fissa, which are frequent in Poland (PS and PB), differ in ploidy level: the PS group is haploid, whereas the PB group is diploid. Isozyme pattern at two loci may suggest an allopolyploid origin of the diploid PB group.
Within Calypogeia fissa, two subspecies connected with geographic distribution are formally recognized: C. fissa subsp.fissa in Europe and C. fissa subsp.neogea in North America. Isoenzyme studies have shown that the European subspecies is genetically differentiated and composed of three genetically distinct groups PS, PB and G. The PS group has the most distinctive morphological features, but no morphological diagnostic traits have been found for groups PB and G. The sequence characterized amplified region (SCAR) markers developed on the basis of ISSR markers, applied in the study, allowed the delimitation of all groups distinguished in Europe within the C. fissa complex (PS, PB and G). The markers also revealed genetic differences between the European and American subspecies. Five primer pairs (Cal01, Cal03-Cal06) of the six pairs studied are useful as the diagnostic tool for the identification of particular groups from the C.fissa complex. The examined SCAR markers showed that the PS group of C.fissa subsp.fissa was the most distinct; it differed from both groups PB and G as well as from C.fissa subsp.neogea. All plants determined on the basis of diagnostic isozyme loci as the PS group amplified a longer product (380 bp) of the Cal04 primer pair than the rest of studied groups and yielded no amplification products in Cal03, Cal05 and Cal06 primers. The primer pair Cal03 distinguished the plants of the PB group from the remaining groups, since only the PB group generated a PCR product of about 290 bp. The genetic differences between all four studied groups of the C.fissa complex were supported by DNA sequences of the SCAR marker Cal04.
Progeny from 19 family lines of silver fir (Abies alba Mill.) from a small, native and isolated population from the Tisovik Reserve (Belarusian part of Białowieża Primeval Forest) growing in an experimental plot near Hajnówka (Polish part of Białowieża Primeval Forest) were analysed in terms of 4 nuclear microsatellite DNA loci and 9 isozyme systems with 14 loci. The aim of this study was to determine the genetic variation within and between progeny lines. Analysis of isozyme loci showed that all progeny lines, except the progeny lines T6 and T16, were characterised by an excess of heterozygotes and 20% of the detected variation occurred between progeny. Progeny formed two groups. Microsatellite loci showed that 6 progeny lines demonstrated an excess of heterozygotes and 12 an excess of homozygotes. On an average, the population was in the Hardy-Weinberg equilibrium. Analysis of molecular variance (AMOVA) showed that 14% of the detected variation occurred between offspring and the remaining 86% within progeny lines. The most distinct progeny line was Tl, where the highest number of alleles per locus was detected. Generally, progeny of Tisovik is characterised by high level of differentiation as the offspring of isolated population that have limited number of individuals to crossing (only 20). In some progeny line, the private alleles that are detected may be the result of pollination from Polish part of Białowieża Forest where in 1920s and 1930s of XX century had planted the seedling of silver fir of unknown origin. The substructuring of population is observed, and the detected deficiency of heterozygotes may be ostensible as a result of the Wahlund effect. Such pattern of genetic structure could also be an effect of harsh environmental conditions exerting selection pressure and modifying the genetic composition of this population.
Nine species of the genus Calypogeia Raddi are currently known from Europe: C. azurea, C. integristipula, C. neesiana,C. suecica, C. muelleriana, C. sphagnicola, C. fissa, C. arguta, and C. azorica. Recently, another species, morphologically resembling C. muelleriana but genetically distinct from it, was detected using isozyme markers. In the present study, relationships between the newly detected species (C. sp. nov.) and typical C. muelleriana were analyzed using the DNA sequencesdata of three regions from the chloroplast genome: introns of trnG and trnL genes and intergenic spacer trnH-psbA. Calypogeia sp. nov. differs from C. muelleriana s. str. (typical form) in all examined chloroplast regions. It differs as well from C. azurea, which was used as a reference species. The number of fixed nucleotide differences between C. muelleriana s. str. and C. sp. nov. is almost the same as between C. muelleriana s. str. and C. azurea. The results of the present study suggest a closer affinity of C. sp. nov. to C. azurea than to C. muelleriana s. str. in Europe, C. muelleriana s. str. was noted in Poland, Germany, Holland, United Kingdom and Azores. Samples determined as C. sp. nov., besides Poland, were so far detected also in North America
Two morphologically distinct groups of the Calypogeia fissa complex were found in Europe
Two genetically distinct groups (PS and PB) detected previously within the C. fissa complex in Europe were studied with respect to 47 morphometric characters. The two examined groups differed statistically significantly with respect to 34 morphological traits. The forward stepwise method of discriminant analysis showed that the set of diagnostic characters could be limited to nine. The best diagnostic features were morphological characters describing the shape of leaf: length and width of leaf, height of dorsal part and distance from the apex to the ventral base of the leaf, length of the 3rd coordinate of the leaf, and underleaf width as well as characters of the stem: length of internodes and size of internode cells. Plants of the PS group were smaller (shoot width range from 922-1780 μm) than plants of the PB group (1600-3900 μm). Based on genetically identified samples, classification functions for each group were computed and the derived functions were used for the classification of samples from the herbarium collections. The principal component analysis and dendrogram constructed on the basis of Euclidean distance, using the set of diagnostic characters, divided the examined samples into two groups that correlated with groups detected by isozyme markers. Results of multivariable analysis showed that it is possible to satisfactorily characterise morphologically both genetically distinct groups of the C. fissa complex.
Calypogeia azurea, a widespread, subboreal-montane liverwort species, is one of a few representatives of the Calypogeia genus that are characterized by the occurrence of blue oil bodies. The aim of the study was to investigate the genetic variation and population structure of C. azurea originating from different parts of its distribution range (Europe and North America). Plants of C. azurea were compared with C. peruviana, another Calypogeia species with blue oil bodies. In general, 339 gametophytes from 15 populations of C. azurea were examined. Total gene diversity (HT) estimated on the basis of nine isozyme loci of C. azurea at the species level was 0.201. The mean Nei’s genetic distance between European populations was equal to 0.083, whereas the mean genetic distance between populations originating from Europe and North America was 0.413. The analysis of molecular variance (AMOVA) showed that 69% of C. azurea genetic variation was distributed among regions (Europe and North America), 15% - among populations within regions, and 16% - within populations. Our study revealed that C. azurea showed genetic diversity within its geographic distribution. All examined samples classified as C. azurea differed in respect of isozyme patterns from C. peruviana.
The main aim of this study was to describe the variation between the populations of the dwarf mountain pine Pinus mugo Turra based on the morphological and anatomical traits of their needles, and to investigate the relationship between the observed variation and environmental conditions (altitude and substrate). Two-year-old needles were collected from 180 individuals of six populations of P. mugo growing in the Tatra Mts. Two populations were classified as dense, located at 1360–1450 m altitude, and the remaining four formed loose clusters and were situated at 1500–1650 m altitude. Four of the populations are growing on granite and two on a limestone substrate. The natural variation of 10 morpho-anatomical and 3 synthetic needle traits was measured. In addition to descriptive statistics, the analyses of variance (ANOVA) with a Tukey test and principal component analysis were computed. We also estimated Pearson correlation coefficients for the examined needle traits and altitude as well as substrate. Our results indicate that the P. mugo populations differ significantly with regard to the investigated traits for which the Trzydniowiański Wierch population was the most distinct. The observed pattern of variability is largely caused by differences in stomatal traits and these features are positive correlated with altitude. Additionally, populations growing on granite have larger values for most of the examined traits compared to populations growing on limestone.