Monika Szczecińska

Evaluation of 11 single-locus and seven multilocus DNA barcodes in Lamium L. (Lamiaceae)

The aim of this work was to evaluate the suitability of selected DNA regions in the barcoding of plants, based on the species belonging to the genus Lamium (Lamiaceae). For this purpose, nine chloroplast barcodes, that is, accD, matK, rbcL, rpoA, rpoB, rpoC1, rpoC2, trnH‐psbA, trnL‐trnF, as well as ITS nuclear region, and intron of mitochondrial nad5 gene were tested. Among the single‐locus barcodes, most effective in the identification of Lamium species was the trnH‐psbA spacer and matK gene. The high level of variability and resolving power was also observed in the case of rpoA and rpoC2 genes. Despite the high interspecies variability of ITS region, it turned out to be inapplicable in Lamium identification. An important disadvantage of ITS as a barcode is a limitation of its use in polyploid plants, samples contaminated with fungal material or samples with partially degraded DNA. We have also evaluated five‐two‐locus and two‐three‐locus barcode regions created from a combination of most effective single loci. The best‐performing barcode combinations were matK + trnH‐psbA and matK + rpoA. Both of them had equally high discriminative power to identify Lamium species.

Preliminary Studies on the Phylogeny of Orthotrichum (Bryophyta) Inferred from Nuclear ITS Sequences

The study presents a phylogenetic analysis of species of the moss genus Orthotrichum. ITS1 and ITS2 for 30 species were sequenced. The results do not fully reflect the current division of Orthotrichum into subgenera and sections. Molecular data divide the genus into two groups of species with superficial and immersed stomata, and indicate a clear distinctness of dioecious species. This suggests that a previous concept postulating that the subgenus Orthophyllum should be excluded from the genus Orthotrichum might be justified.

Genetic diversity and population structure of the rare and endangered plant species Pulsatilla patens (L.) Mill in East Central Europe

Pulsatilla patens s.s. is a one of the most endangered plant species in Europe. The present range of this species in Europe is highly fragmented and the size of the populations has been dramatically reduced in the past 50 years. The rapid disappearance of P. patens localities in Europe has prompted the European Commission to initiate active protection of this critically endangered species. The aim of this study was to estimate the degree and distribution of genetic diversity within European populations of this endangered species. We screened 29 populations of P. patens using a set of six microsatellite primers. The results of our study indicate that the analyzed populations are characterized by low levels of genetic diversity (Ho = 0.005) and very high levels of inbreeding (FIS = 0.90). These results suggest that genetic erosion could be partially responsible for the lower fitness in smaller populations of this species. Private allelic richness was very low, being as low as 0.00 for most populations. Average genetic diversity over loci and mean number of alleles in P. patens populations were significantly correlated with population size, suggesting severe genetic drift. The results of AMOVA point to higher levels of variation within populations than between populations.The results of Structure and PCoA analyses suggest that the genetic structure of the studied P. patens populations fall into three clusters corresponding to geographical regions. The most isolated populations (mostly from Romania) formed a separate group with a homogeneous gene pool located at the southern, steppic part of the distribution range. Baltic, mostly Polish, populations fall into two genetic groups which were not fully compatible with their geographic distribution.Our results indicate the serious genetic depauperation of P. patens in the western part of its range, even hinting at an ongoing extinction vortex. Therefore, special conservation attention is required to maintain the populations of this highly endangered species of European Community interest.

Molecular evidence does not support the current division of Orthotrichum subgenus Gymnoporus

Eight Orthotrichum species of subgenus Gymnoporus were compared using the internally transcribed spacer regions-1 and -2 and the chloroplast trnH-psbA region. A phylogenetic analysis did not reflect the current division of this subgenus into sections Affinia and Leiocarpa. The investigated sequences revealed a close relationship between O. striatum, a typical species of section Leiocarpa and O. affine, a typical species of section Affinia. An easily distinguishable group was formed by samples of the dioecious O. lyellii, placed into section Leiocarpa. A large number of fixed differences between O. lyellii and other species of subgenus Gymnoporus raises doubts concerning its position within this subgenus. No marker mutations enabling to differentiate O. fastigiatum from O. affine have been found. In absence of such mutations for O. affine and O. striatum, the status of O. fastigiatum cannot be determined unambiguously.

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.

Genetic variation in the liverwort Bazzania trilobata inferred from ISSR markers

Abstract The distribution of Bazzania trilobata in Poland coincides with two parts of the natural distribution range of Norway spruce: the mountains of southern Poland and the northeastern lowlands. The occurrence of this species is connected with primeval forest communities and it was recognized as a relict of them. Genetic variation of 10 populations of B. trilobata from two different regions of Poland (lowlands and mountains) was studied in order to establish the genetic structure of this species and to compare the level of genetic variation within and among populations originating from primeval and managed forests. An analysis based on polymorphic inter-simple sequence repeat (ISSR) loci revealed a high level of total gene diversity in B. trilobata (H T=0.308). A higher amount of polymorphism was found among than within populations. Genetic variation of populations from the primary forest (Tatras and Białowieza National Park) was higher than for those originating from disturbed habitats. The Bayesian method showed consistent grouping of samples corresponding with populations, with the best grouping in 12 different clusters reflecting a geographic pattern. The geographic pattern of genetic differentiation was supported by a neighbour-joining (NJ) dendrogram based on genetic distances. All mountain populations were grouped together in one subcluster, but lowland populations were more differentiated and did not form one clear group.

New taxonomical arrangement of the traditionally conceived genera Orthotrichum and Ulota (Orthotrichaceae, Bryophyta)

Abstract The traditionally conceived genera Orthotrichum Hedw. and Ulota F.Weber are here reclassified into six genera, Orthotrichum, Dorcadion Lindb., Nyholmiella Holmen & E.Warncke, Pulvigera Plášek, Sawicki & Ochyra, Plenogemma Plášek, Sawicki & Ochyra, and Ulota, based on morphological differences and partially on molecular evidence. The genus Pulvigera includes P. lyellii (Hook. & Taylor) Plášek, Sawicki & Ochyra (Orthotrichum lyellii Hook. & Taylor) which was selected as its generitype. The genus Plenogemma includes P. phyllantha (Brid.) Plášek, Sawicki & Ochyra (Ulota phyllantha Brid.) which was selected as its generitype.

Isolation and Characterization of Simple Sequence Repeats (SSR) Markers from the Moss Genus Orthotrichum Using a Small Throughput Pyrosequencing Machine

Here, we report the results of next-generation sequencing on the GS Junior system to identify a large number of microsatellites from the epiphytic moss Orthotrichum speciosum. Using a combination of a total (non-enrichment) genomic library and small-scale 454 pyrosequencing, we determined 5382 contigs whose length ranged from 103 to 5445 bp. In this dataset we identified 92 SSR (simple sequence repeats) motifs in 89 contigs. Forty-six of these had flanking regions suitable for primer design. We tested PCR amplification, reproducibility, and the level of polymorphism of 46 primer pairs for Orthotrichum speciosum using 40 individuals from two populations. As a result, the designed primers revealed 35 polymorphic loci with more than two alleles detected. This method is cost- and time-effective in comparison with traditional approaches involving cloning and sequencing.

New national and regional bryophyte records, 45

D. S. G. Henriques, O. V. Ilina, J. E. Gil-Novoa, M. E. Morales-Puentes, S. R. Gradstein, R. Gupta, V. Nath, A. K. Asthana, A. Koczur, M. Lebouvier, A. Mesterházy, F. Mogro, A. Mežaka, Cs. Németh, J. D. Orgaz, Y. Sakamoto, J. Paiva, F. Sales, N. Pande, M. S. Sabovljević, J. Pantivić, A. D. Sabovljević, A. Pérez-Haase, D. Pinheiro da Costa, V. Plášek, J. Sawicki, M. Szczecińska, J. Chmielewski , A. Potemkin , A. Scha 31 32 ̈fer-Verwimp , W. B. Schofield 33 , C. Sérgio, M. Sim-Sim, S. Sjögren, D. Spitale, A. Stebel, S. Ştefănuţ , G. M. Sua 40 ́rez , J 41 . R. Flores , L. Thouvenot , J. Va 41 42 ́ňa , 43

The complete mitochondrial genome of the epiphytic moss Orthotrichum speciosum

Abstract The mitogenome of the Orthotrichum speciousum (GenBank accession number KM288416) has a total length of 104,747 bp and consist of 40 protein-coding genes, 3 ribosomal RNA (rRNA) and 24 transfer RNA. The gene order is identical to other known moss mitogenomes.