Alina Bączkiewicz

Allopolyploid speciation of Calypogeia sphagnicola (Jungermanniopsida, Calypogeiaceae) based on isozyme and DNA markers

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.

Volatile compounds in cryptic species of the Aneura pinguis complex and Aneura maxima (Marchantiophyta, Metzgeriidae).

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.

Genetic Diversity of Leafy Liverwort Species (Jungermanniidae, Marchantiophyta) in Poland: Diversity of Leafy Liverwort Species with Various Reproductive Modes

Abstract 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.

The extraordinary variation of the organellar genomes of the Aneura pinguis revealed advanced cryptic speciation of the early land plants

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.

Differentiation and Genetic Variability of Three Cryptic Species within the Aneura pinguis Complex (Jungermanniidae, Marchantiophyta)

ABSTRACT 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 cryptic species occur partly sympatrically.

Two morphologically distinct groups of the Calypogeia fissa complex were found in Europe

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.

DNA barcoding, ecology and geography of the cryptic species of Aneura pinguis and their relationships with Aneura maxima and Aneura mirabilis (Metzgeriales, Marchantiophyta)

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 complete mitochondrial genome of the cryptic species C of Aneura pinguis

Abstract 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.

Genetic structure and barcode identification of an endangered orchid species, Liparis loeselii, in Poland

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). Neis 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 Warężyńska population from other Silesian populations. Based on barcode sequences it was found that all the examined samples were identical with respect to all the studied DNA regions. Liparis loeselii trnL-F region sequences were detected for the first time and they can be useful as complementary barcodes for this species. The analysed sequences of L. loeselii are fully consistent with sequences of specimens originating from the UK, Canada, Czech Republic, Hungary, Sweden, Italy, and Russia.

Genetic Diversity of Leafy Liverwort Species (Jungermanniidae, Marchantiophyta) in Poland: Regional Genetic Differentiation of Leafy Liverworts

Abstract For each of 8 species of leafy liverworts, 9-10 populations were sampled in 2-3 regions of Poland. In total, 5 regions were taken into account: the Tatra National Park, Bieszczady Mts., Białowieża Forest, Pomeranian Lake District, and Suwałki Lake District. Populations of most of the studied species did not show any correlation between genetic differentiation and geographic distances. Clear differences between regional groups of populations were found in only 2 species. The other species showed a complete or partial lack of genetic differentiation between groups of populations from various geographic regions. Generally, however, mountain populations had greater genetic diversity (HT, HS) and coefficient of genetic differentiation (GST) than lowland populations. In the Tatra National Park all the studied liverworts turned out to be more diverse than in the Bieszczady Mts. Białowieża Forest created a uniform group, standing out markedly from mountainous populations but population in this region had slightly smaller genetic diversity, then in the mountains. In the Pomeranian and Suwałki Lake Districts, genetic diversity of liverworts was significantly lower than in mountains. The decrease in diversity in these regions is a likely consequence of habitat fragmentation (causing population depletion) combined with negative effects of urban development. Habitat fragmentation results in genetic drift and inbreeding depression, which cause a decrease in genetic diversity. In the Pomeranian Lake District the level of total diversity (HT) and intra-population diversity (HS) was markedly higher than in the Suwałki Lake District. It may be linked to differences in climate, in the Suwałki Lake District climate is stronger.