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Molecular identification of species is especially important where traditional taxonomic methods fail. The genus Calypogeia belongs to one of the tricky taxons. The simple morphology of these species and a tendency towards environmental plasticity make them complicated in identification. The finding of the universal single-locus DNA barcode in plants seems to be ‘the Holy Grail’; therefore, researchers are increasingly looking for multiloci DNA barcodes or super-barcoding. Since the mitochondrial genome has low sequence variation in plants, species delimitation is usually based on the chloroplast genome. Unexpectedly, our research shows that super-mitobarcoding can also work! However, our outcomes showed that a single method of molecular species delimitation should be avoided. Moreover, it is recommended to interpret the results of molecular species delimitation alongside other types of evidence, such as ecology, population genetics or comparative morphology. Here, we also presented genetic data supporting the view that C. suecica is not a homogeneous species.

The study focused on the verification of the preferential site-specific concept hypothesizing, that mineral elements could be playing an initiating role in the biological speciation within Aneura pinguis cryptic species. A. pinguis species A and soil materials were collected from three ecological sites of Poland. They underwent genetic (Aneura pinguis) and chemical analyses (soil materials) for pH, total and water soluble (active) forms of Ca, Mg, K, Na fractions. Data revealed trends in the site preference of three genetic lineages (A1, A2 and A3) of A. pinguis cryptic species A. Lineage adaptability index Ca/(Mg + K + Na) reflecting the dynamic character of site pH implied, that lineages A1 and A2 were both calciphilous. The A3 lineages were intrinsically acidophilous and this characteristics was also observed at some A1 lineages. Site concentrations of Ca and in some cases Mg too were crucial in shaping pH, but this process could have been controlled by each mineral element, individually. Calciphilous or acidophilous A. pinguis species may be “remotely” attracted by high or low Ca (or Mg) concentrations, for alkalinity or acidity emergence, respectively. Mineral richness at investigated ecological sites has possibly initiated opportunistic and specific site colonisation by A. pinguis lineages.

Aneura pinguis (L.) Dumort. is a representative of the simple thalloid liverworts, one of the three main types of liverwort gametophytes. According to classical taxonomy, A. pinguis represents one morphologically variable species, however, genetic data reveal that this species is a complex consisting of 10 cryptic species (named by letters from A to J), of which four are further subdivided into two or three evolutionary lineages. The objective of this work was to develop an efficient method for the characterisation of plant material using marker compounds. The volatile chemical constituents of cryptic species within the liverwort A. pinguis were analysed by GC-MS. The compounds were isolated from plant material using the HS-SPME technique. Of the 66 compounds examined, 40 were identified. Of these 40 compounds, nine were selected for use as marker compounds of individual cryptic species of A. pinguis. A guide was then developed that clarified how these markers could be used for the rapid identification of the genetic lineages of A. pinguis. Multivariate statistical analyses (principal component and cluster analysis) revealed that the chemical compounds in A. pinguis made it possible to distinguish individual cryptic species (including genetic lineages), with the exception of cryptic species G and H. The classification of samples based on the volatile compounds by cluster analysis reflected phylogenetic relationships between cryptic species and genetic lineages of A. pinguis revealed based on molecular data.

Background Molecular research revealed that some of the European Calypogeia species described on the basis of morphological criteria are genetically heterogeneous and, in fact, are species complexes. DNA barcoding is already commonly used for correct identification of difficult to determine species, to disclose cryptic species, or detecting new taxa. Among liverworts, some DNA fragments, recommend as universal plant DNA barcodes, cause problems in amplification. Super-barcoding based on genomic data, makes new opportunities in a species identification. Results On the basis of 22 individuals, representing 10 Calypogeia species, plastid genome was tested as a super-barcode. It is not effective in 100%, nonetheless its success of species discrimination (95.45%) is still conspicuous. It is not excluded that the above outcome may have been upset by cryptic speciation in C. suecica, as our results indicate. Having the sequences of entire plastomes of European Calypogeia species, we also discovered that the ndhB and ndhH genes and the trnT-trnL spacer identify species in 100%. Conclusions This study shows that even if a super-barcoding is not effective in 100%, this method does not close the door to a traditional single- or multi-locus barcoding. Moreover, it avoids many complication resulting from the need to amplify selected DNA fragments. It seems that a good solution for species discrimination is a development of so-called “specific barcodes” for a given taxonomic group, based on plastome data.

Chemical constituents of cryptic species detected within the liverwort Aneura pinguis were identified using headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS). Fibre coating with divinylbenzene/carboxen/polydimethylsiloxane (DVB/CAR/PDMS) was used. A total of 48 samples of A. pinguis were analysed. The studied plants were identified genetically based on barcode DNA sequences and represented three cryptic species (A, B and F) of A. pinguis. Cryptic species A and B are genetically diverse; both represent three evolutionary lineages: A1, A2, A3 and B1, B2, B3, respectively. The cryptic species F that was recently detected is not diverse. The most characteristic compounds in analysed samples were sesquiterpene hydrocarbons (up to 17.7% for A1; 15.7% for A2; 20.6% for A3; 7.7% for B1; 2.0% for B2; 3.7% for B3; 10.2% for F), oxygenated sesquiterpenoids (up to 68.0% for A1; 54.7% for A2; 52.6% for A3; 63.5% for B1; 88.7% for B2; 82.7% for B3; 78.8% for F), and linear aliphatic hydrocarbons (up to 14.8% for A1; 1.1% for A2; 12.1% for A3; 6.9 for B1; 5.2% for B2; 1.1% for B3; 7.0% for F). The dominant compound in the studied samples was pinguisone. The second dominant compound present in the tested plant material was deoxopinguisone, except for lineage B2, where only a small relative concentration of this compound was found. A high content of deoxopinguisone in cryptic species A (lineages A1, A2 and A3) was accompanied by the presence of isopinguisone and methyl norpinguisonate, whereas these two compounds were not detected in cryptic species B (lineages B1 and B3) and F. The chemical compounds detected in the studied samples of A. pinguis were subjected to multivariate statistical analysis. The results showed that the chemical composition depends mainly on the genotype of the plant and slightly on the habitat. However, there was no clear correlation between the volatile compounds and the date of collection of the studied plants.

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.

1. Amphidium lapponicum (Hedw.) Schimp.Contributor. R. OchyraPoland. Western Carpathians, Western Beskidy Mountains: Beskid Żywiecki Range, Babia Gora massif, on the northern slope of Diablak peak,...

Aneura pinguis and Aneura maxima belong to the simple thalloid liverworts. Previous isozyme studies revealed that A. pinguis is a complex of cryptic species difficult to distinguished based on morphology. In the present study four cryptic species of the A. pinguis complex and A. maxima were examined by means of ISSR method to assess genetic variation and to develop species-specific markers. Eight ISSR primers used generated 460 bands, of which 453 were polymorphic. The highest values of resolving power 28.4 and marker index 18.1 were noted for primer 835 (AG) 8 -YC, while polymorphism information content for primer 842 (GA) 7 -AYG. The total gene diversity (H T ) based on polymorphic loci was 0.284 for A. pinguis and 0.06 for A. maxima . ISSR markers supported existence of cryptic species in A. pinguis and showed genetic isolation between them. Species-specific bands were found for all studied cryptic species of A. pinguis and A. maxima , thus ISSRs can be used for their identification. A. maxima clearly differ from all the A. pinguis cryptic species in each amplified ISSR primer. The AMOVA conducted for the A. pinguis complex showed that most of genetic variation (Φ PT 0.586) was present among species.

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 genetic diversity of five populations of Liparis loeselii from two regions of Poland, Podlachia and Silesia, was compared with respect to minisatellite markers. Moreover, the standard 2-loci barcode DNA regions, rbcL and matK genes, as well as an additional region trnL-F from chloroplast and ITS2 from nuclear genome were studied. The total genetic diversity at the species level amounted to HT = 0.356. The analysis of molecular variance revealed that 58% of genetic variation was distributed within populations, 2% among populations, and 40% between regions from NE Poland (Podlachia), and S Poland (Silesia). Nei's genetic distances indicated that specimens from the Podlachia population were genetically isolated from Silesian ones. Two genetic barriers among the studied populations were found: one barrier separated the Podlachian population from all Silesian populations and another barrier divided Kuźnica Warezynska population from other Silesian populations. Based on barcode sequences it was found that a...

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.

1652 individuals of Aneura pinguis from Poland were surveyed for variation in 12 putative gene loci. Based on isozyme data, we distinguished three cryptic species. No evidence for gene flow between these species was found. To date, no qualitative morphological characters are available, which would allow delimitation of the cryptic species of A. pinguis. Hence, these species are not formally described, but assigned as cryptic species A, B, and C. The mean genetic distance (D) between them is 1.3393. The highest genetic variation within populations (Hs ) was found in species A, and the lowest in species B. Individual species of A. pinguis differ in their habitat preferences. Species A is the most common, it occurs mostly in the Western Carpathians, grows mainly on calcareous rocks and humus. Species B is the most frequent in the Eastern Carpathians on clay soil. Species C is the rarest, it can be found both in lowlands and mountains, but mainly in lowlands and on various substrata. All studied cryp...

Oil bodies are the unique feature of most liverworts. Their shape, color and distribution pattern in leaf and underleaf cells are important taxonomic features of the genus Calypogeia. Most species of the genus Calypogeia have pellucid and colorless oil bodies, whereas colored, including gray to pale brown, purple-brown or blue oil bodies, are rare. To date, C. azurea was the only species with blue oil bodies to have been considered as a species of the Holarctic range. This species has been noted in various parts of the northern hemisphere-from North America, through Europe to the Far East. The aim of this study was to determine the genetic diversity of C. azurea from different parts of its distribution range and to ascertain whether blue oil bodies appeared once or several times in the evolution of the genus Calypogeia. The phylogenetic analyses based on four plastid regions (rbcL, trnG, trnL, trnH-psbA) and one nuclear region (ITS2) revealed that C. azurea is presently a paraphyletic taxon, with other Calypogeia species nested among C. azurea accessions that were clustered into four different clades. Based on the level of genetic divergence (1.03-2.17%) and the observed morphological, ecological and geographical differences, the evaluated clades could be regarded as previously unrecognized species. Four species were identified: C. azurea Stotler & Crotz (a European species corresponding to the holotype), two new species from Pacific Asia-C. orientalis Buczkowska & Bakalin and C. sinensis Bakalin & Buczkowska, and a North American species which, due to the lack of identifiable morphological features, must be regarded as the cryptic species of C. azurea with a provisional name of C. azurea species NA.

Liverwort mitogenomes are considered to be evolutionarily stable. A comparative analysis of four Calypogeia species revealed differences compared to previously sequenced liverwort mitogenomes. Such differences involve unexpected structural changes in the two genes, cox1 and atp1, which have lost three and two introns, respectively. The group I introns in the cox1 gene are proposed to have been lost by two-step localized retroprocessing, whereas one-step retroprocessing could be responsible for the disappearance of the group II introns in the atp1 gene. These cases represent the first identified losses of introns in mitogenomes of leafy liverworts (Jungermanniopsida) contrasting the stability of mitochondrial gene order with certain changes in the gene content and intron set in liverworts.

Aneura pinguis is known as a species complex with several morphologically indiscernible species, which are often reproductively isolated from each other and show distinguishable genetic differences. Genetic dissimilarity of cryptic species may be detected by genomes comparison. This study presents the first complete sequences of chloroplast and mitochondrial genomes of six cryptic species of A. pinguis complex: A. pinguis A, B, C, E, F, J. These genomes have been compared to each other in order to reconstruct phylogenetic relationships and to gain better understanding of the evolutionary process of cryptic speciation in this complex. The chloroplast genome with the nucleotide diversity 0.05111 and 1537 indels is by far more variable than mitogenome with π value 0.00233 and number of indels 1526. Tests of selection evidenced that on about 36% of chloroplast genes and on 10% of mitochondrial genes of A. pinguis acts positive selection. It suggests an advanced speciation of species. The phylogenetic analyses based on genomes show that A. pinguis is differentiated and forms three distinct clades. Moreover, on the cpDNA trees, Aneura mirabilis is nested among the cryptic species of A. pinguis. This indicates that the A. pinguis cryptic species do not derive directly from one common ancestor.

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.

Aneura pinguis is a thalloid liverwort species with broad geographical distribution. It is composed of cryptic species, however, the number of cryptic species within A. pinguis is not known. Five cpDNA regions (matK, rbcL, rpoC1, trnH-psbA and trnL-trnF) and the entire nuclear ITS region were studied in 130 samples of A. pinguis from different geographical regions. The relationships between the cryptic species of A. pinguis, A. maxima and A. mirabilis were analyzed. All of the examined samples were clustered into 10 clades corresponding to 10 cryptic species of A. pinguis (marked A to J). Aneura mirabilis and A. maxima were nested among different cryptic species of A. pinguis, which indicates that A. pinguis is a paraphyletic taxon. Subgroups were found in cryptic species A, B, C and E. As single barcodes, all tested DNA regions had 100% discriminant power and fulfilled DNA barcode criteria for species identification; however, the only combination detected in all subgroups was trnL-trnF with trnH-psbA or ITS2. The distances between cryptic species were 11- to 35-fold higher than intraspecific distances. In all analyzed DNA regions, the distances between most pairs of cryptic A. pinguis species were higher than between A. maxima and A. mirabilis. All cryptic species of A. pinguis clearly differed in their habitat preferences, which suggests that habitat adaptation could be the main driving force behind cryptic speciation within this taxon.

The study presented in this research paper is the first taxonomic investigation focusing on Uropodina (Acari: Mesostigmata) mites with a brief discussion of the genetic differences of two very closely related species from the genus Oodinychus Berlese, 1917, i.e. O. ovalis (C.L. Koch, 1839) and O . karawaiewi (Berlese, 1903). These two morphologically similar species are quite common and they have a wide range of occurrence in Europe. They also live in almost the same types of habitat. However, O. ovalis usually exhibits higher abundance and frequency of occurrence. The major aim of the study was to carry out a comparative analysis of the systematic position, morphological and biological differences, as well as habitat preferences and distribution of O. ovalis and O. karawaiewi . The next aim was to ascertain whether the differences in number and frequency of these species may stem from the genetic differences at the molecular level (16S rDNA and COI). The study shows that O. ovalis , which is a more abundant species than O. karawaiewi , turned out to be genetically more polymorphic.

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

This monograph presents results of research on genetic diversity of 8 leafy liverwort species differing in reproductive mode. The frst 4 species in Poland are regarded as sterile and reproduce only vegetatively: Bazzania trilobata, Trichoc-olea tomentella, Lophozia hatcheri, and Mylia anomala. The next 4 are fertile, including the monoecious Lepidozia reptans and Calypogeia integristipula as well as the dioecious Mylia taylorii and Tritomaria quinquedentata. For each species, 9-10 populations were sampled. In total, 4744 gametophytes from 73 populations were examined by isozyme analysis. The level of their genetic diversity (total, HT, and within populations, HS) was high, higher than in thallose liverworts, but comparable to the genetic diversity of mosses or even some species of vascular plants. Thus the traditional opinion that the entire group of liverworts has a much lower level of genetic diversity than mosses is erroneous, as it holds true only for thallose liverworts (Metzgeriidae and Marchantiopsida). My results indicate that the effect of reproductive mode on genetic diversity in leafy liverworts is lower than in vascular plants. Sterile and fertile species of liverworts exhibited similar levels of genetic diversity. Moreover, both groups included species that had both high and low levels of HT and HS. In fertile species, monoecious and dioecious species also did not differ signifcantly in genetic diversity, but dioecious liverworts had slightly higher total diversity (HT) than monoecious species. In most of the studied leafy liverworts, the share of genetic diversity within populations in the total genetic diversity of species is greater than between populations. The percentage share of variation among populations (ΦPT) in the total genetic variation was correlated with the total genetic diversity of the species (HT). In species with high HT, differences between populations tended to be rather small. By contrast, in species with lower HT, the percentage share of differentiation among populations in the total diversity of species was much higher. My results confrm theory, based on studies by Kimura, that the main causes of genetic diversity of bryophytes are neutral somatic mutations developing in various vegetative parts of plants. The separation of branches or other plant sections with somatic mutations, followed by the growth of new shoots, can increase the level of genetic diversity. The high level of genetic diversity in sterile liverworts indicates that vegetative reproduction has a greater infuence on the level of genetic diversity than recombination. My results suggest also that mutation rates are similar in closely related species, but species with a wider ecological range exhibit higher genetic diversity because the variability of habitats can infuence the rate and type of somatic mutations. Accordingly, species inhabiting more diverse environments may be more genetically diverse. Patches of the studied species generally consisted of several genotypes (MLGs). Two types of distribution of genotypes in patches were noticed. Patches of species with low total diversity (HT), were often dominated by 1-2 genotypes, which constituted the major part of a patch. In patches of species with higher HT, there was no tendency to form patches with predomination of a single genotype. Different genotypes constituted similar proportions of a patch. In all the studied leafy liverwort species there was a high degree of repeatability of the same genotypes (MLGs) in plants from various patches within the same population or in various populations. Probably the main cause of this is the independent repeatability of the same mutations in different specimens.

Calypogeia sphagnicola is one of nine species of the genus Calypogeia known in Europe. Occurrence of the species is closely connected with peat bogs. Nowadays, two forms of this species are distinguished—C. sphagnicola f. sphagnicola and C. sphagnicola f. paludosa. The results of the present study, based on two classes of markers—isozymes and sequences of chloroplast genom (trnH-psbA, rpoC1)—unanimously support the genetic differentiation within the taxon and show that the present-day forms represent genetically distinct species. Phylogenetic analysis resolved two lineages that correspond with the present-day forms with high bootstrap support, which differ in ploidy level: C. sphagnicola f. sphagnicola is haploid, whereas C. sphagnicola f. paludosa is a diploid form. Allopolyploid origin of the diploid form was revealed by the isozyme pattern. Nei’s genetic distance between the two present-day forms of C. sphagnicola was 0.472. The forms in Poland have an allopatric pattern of geographic distribution: C. sphagnicola f. sphagnicola occurs exclusively in the lowlands of the northern part of the country on raised peat bogs, whereas C. sphagnicola f. paludosa is found only in the mountains of southern Poland, mainly in the subalpine zone, where it grows on Sphagnum-Polytrichum hummocks on the upper part of north-facing slopes. Plants regarded in this study as C. sphagnicola f. sphagnicola morphologically correspond to the syntype specimen of C. sphagnicola.

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.

Bryophytes are amongst the most ancestral terrestrial plants and often have large distribution ranges across continents. Recent biochemical and molecular studies have suggested that many worldwide morphological species of bryophyte may represent genetically divergent and reproductively isolated cryptic species. We tested the cryptic species hypothesis in the thalloid liverwort Aneura pinguis complex. We applied analyses of chloroplast DNA (cpDNA) sequence variation and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods to discriminate between specimens of A. pinguis derived from various habitats in five distant geographical regions in Poland. Of the 19 specimens sequence characterized for the cpDNA tRNALeu region, seven haplotypes were identified divided into three nonmonophyletic clusters. The application of developed PCR-RFLP markers confirmed the existence of three tRNALeu types of A. pinguis (A–C) within the specimens derived from 21 populations. Sympatric populations of different tRNALeu types were found in lowland and mountain regions. No clear correlation between stand type and the presence of two tRNALeu types (A, B) was observed, as both were growing on soil, humus, and rocks. The tRNALeu type C was found only on humus and its distribution was restricted to low-lying northern populations. The above results indicate that the A. pinguis complex is highly differentiated at the molecular level and may represent three cryptic species. © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society, 2007, 155, 273–282.

Aneura pinguis is one of the liverwort species complexes that consist of several cryptic species. Ten samples collected from different regions in Poland are in the focus of our research. Eight of the A. pinguis complex belonging to four cryptic species (A, B, C, E) and two samples of closely related species Aneura maxima were tested for the composition of volatile compounds. The HS-SPME technique coupled to GC/FID and GC/MS analysis has been applied. The fiber coated with DVB/CAR/PDMS has been used. The results of the present study, revealed the qualitative and quantitative differences in the composition of the volatile compounds between the studied species. Mainly they are from the group of sesquiterpenoids, oxygenated sesquiterpenoids and aliphatic hydrocarbons. The statistical methods (CA and PCA) showed that detected volatile compounds allow to distinguish cryptic species of A. pinguis. All examined cryptic species of the A. pinguis complex differ from A. maxima. Species A and E of A. pinguis, in CA and PCA, form separate clusters remote from two remaining cryptic species of A. pinguis (B and C) and A. maxima. Relationship between the cryptic species appeared from the chemical studies are in accordance with that revealed on the basis of DNA sequences.

The structure of the Aneura pinguis mitochondrial genome (GenBank accession no. NC_026901) is similar to that of closely related Metzgeriales species: it has a total length of 165 603 bp, the base composition of the mitogenome is the following: A (26.2%), C(23.6%), G(23.8%), and T(26.4%). The A. piguis mitochondrial genome contains 69 genes. A complete mitochondrial genome sequence of A. pinguis will help better to understand mitogenome structure and content among Metzgeriales order.

We estimated similarity and differences at the level of isozymes among populations of Malva alcea from various habitats and parts of Central Europe. Our analyses revealed the activity of 8 enzyme systems: 6 polymorphic (PGI, IDH, PX, DIA, PGM, SHD KB) and 2 monomorphic (GOT, ME). The mean number of alleles per locus is 2.12. Nei's genetic distances among populations are small, not exceeding 0.3. Mean values of Nei's genetic similarity are typical of populations of the same species. Grouping by UPGMA based on Nei's genetic distances showed that the distinguished groups are only partly correlated with geographic region.

Two closely related species of the genus Barbilophozia: B. lycopodioides and B. hatcheri were studied in populations from the Tatra Range (S Poland), where they are frequent and widely distributed. Both species play an important role in plant communities and grow here very often side by side. Typically developed plants are quite easy to distinguish (even in the field), however morphologically intermediate forms, difficult to recognize by using of classical taxonomic methods, sometimes are found. We found enzymatic markers, that allow to recognize the critical forms. Both studied species are different in enzymatic patterns of glutamate oxaloacetate transaminase (GOT) and peroxidases (PX). In GOT four different phenotypes were detected. The first two (GOT 1 and GOT 2) were characteristic for B. hatcheri and next two (GOT 3 and GOT 4) for B. lycopodioides. Peroxidase patterns, that were monomorphic and specific for each species, exhibit different mobility in anodal and cathodal parts of gel. Results of the studies allowed us to draw the conclusion, that PX and GOT are good isoenzymatic markers and they can have practical application for identification of Barbilophozia species.