Background: The mitogenomes of vascular plants are one of the most structurally diverse molecules. In the present study we characterize mitogenomes of a rare and endangered speciesPulsatilla patens. We investigated the gene content and its RNA editing potential, repeats distribution and plastid derived sequences.Results: The mitogenome structure of early divergent eudicot, endangeredPulsatilla patensdoes not support the master chromosome hypothesis, revealing the presence of three linear chromosomes of total length 986 613 bp. The molecules are shaped by the presence of extremely long, exceeding 87 kbp repeats and multiple chloroplast-derived regions including nearly complete inverted repeat. Since the plastid IR content of Ranunculales is very characteristic, the incorporation into mitogenome could be explained rather by intracellular transfer than mitochondrial HGT. The mitogenome contains almost a complete set of genes known from other vascular plants with exception ofrps10 andsdh3, the latter being present but pseudogenized. Analysis of long ORFs enabled the identification of genes which are rarely present in plant mitogenomes, including RNA and DNA polymerases, albeit their presence even at species level is variable. Mitochondrial transcripts ofP. patenswere edited with a high frequency, which exceeded the level known in other analyzed angiosperms, despite the strict qualification criteria of counting the editing events and taking into analysis generally less frequently edited leaf transcriptome. The total number of edited sites was 902 andnad4 was identified as the most heavily edited gene with 65 C to U changes. Non-canonical, reverse U to C editing was not detected. Comparative analysis of mitochondrial genes of threePulsatillaspecies revealed a level of variation comparable to chloroplast CDS dataset and much higher infrageneric differentiation than in other known angiosperm genera. The variation found in CDS of mitochondrial genes is comparable to values found amongPulsatillaplastomes. Despite the complicated mitogenome structure, 14 single copy regions of 329 kbp, not splitted by repeats or plastid-derived sequences (MTPT), revealed the potential for phylogenetic, phylogeographic and population genetics studies by revealing intra- and interspecific collinearity.Conclusions: This study provides valuable new information about mitochondrial genome of early divergent eudicots,Pulsatilla patens, revealed multi-chromosomal structure and shed new light on mitogenomics of early eudicots.

The simple thalloid liverwort Apopellia endiviifolia is a widespread Holarctic species belonging to the family Pelliaceae. European populations of this species comprise two distinct evolutionary lineages named “species A”, known also as water form, and typical, mainly terrestrial forms named “species B”. Newly sequenced, assembled and annotated chloroplast genomes of six European specimens belonging to the two cryptic lineages occupying different microhabitats, revealed the structure typical for liverworts and previously sequenced reference. The plastomes of A. endiviifolia are 120,537–120,947 bp long with a structure typical for most plants, including a pair of IR regions (each of 9,092–9,207 bp) separated by LSC (82,506–82,609 bp) and SSC (19,854–19,924 bp) regions and consist of 121 unique genes, including 81 protein-coding genes, 6 genes of unknown function (ycf genes), 4 ribosomal RNAs and 30 transfer RNAs. Comparative analysis of typical, terrestrial and water forms revealed 4971 molecular diagnostic characters (MDCs), which exceeds numbers found in many well recognized liverworts taxa. Moreover, beside the presence of evolutionary hotspots like ycf1 and ycf2 genes and several intergenic spacer like ndhB-psbM, rps4-ndhJ and ndhC-atpE, the molecular identification of Apopellia cryptic species was possible by almost 98% of 500 bp long frames simulating mini barcodes. The different ecological niches can be driven by different pressures of positive selection, which was detected in nine genes including ccsA, ndhD, ndhF, petA, psbB, psbC, rpoB, ycf1 and ycf2. Despite clearly genetic differences and ecological preferences, the current observation of morphological differentiation does not no allow to separate terrestrial and water forms into taxonomic species.

Pulsatilla patens is a rare and endangered species in Europe and its population resources have significantly decreased over the past decades. Previous genetic studies of this species made it possible to estimate the genetic diversity of the European population and to describe the structure of chloroplast and mitochondrial genomes. The main aim of these studies was to characterize the variability of chloroplast and mitochondrial genomes in more detail at the intra-population and inter-population levels. Our study presents new organelle genome reference sequences that allow the design of novel markers that can be the starting point for testing hypotheses, past and modern biogeography of rare and endangered species P. patens, and adaptive responses of this species to changing environments. The study included sixteen individuals from five populations located in Northeastern Poland. Comparative analysis of 16 P. patens plastomes from 5 populations enabled us to identify 160 point mutations, including 64 substitutions and 96 InDels. The most numerous detected SNPs and Indels (75%) were accumulated in three intergenic spacers: ndhD—ccsA, rps4—rps16, and trnL(UAG)—ndhF. The mitogenome dataset, which was more than twice as large as the plastome (331 kbp vs. 151 kbp), revealed eight times fewer SNPs (8 vs. 64) and six times fewer InDels (16 vs. 96). Both chloroplast and mitochondrial genome identified the same number of haplotypes—11 out of 16 individuals, but both organellar genomes slightly differ in haplotype clustering. Despite the much lower variation, mitogenomic data provide additional resolution in the haplotype detection of P. patens, enabling molecular identification of individuals, which were unrecognizable based on the plastome dataset.

We identify a large number of microsatellites from Galium trfidum, a plant species considered rare and endangered in Central and Western Europe. Using a combination of a total enriched genomic library and small-scale 454 pyrosequencing, we determined 9755 contigs with a length of 100 to 6192 bp. Within this dataset, we identified 153 SSR motifs in 144 contigs. Here, we tested 14 microsatellite loci in 2 populations of G. trifidum. The number of alleles and expected heterozygosity were 1–8 (mean 3.2) and 0.00–0.876 (0.549 on average), respectively. The markers described in this study will be useful for evaluating genetic diversity within and between populations, and gene flow between G. trifidum populations. These markers could also be applied to investigate the biological aspects of G. trifidum, such as the population dynamics and clonal structure, and to develop effective conservation programs for the Central European populations of this species.

Background: The mitogenomes of vascular plants are one of the most structurally diverse molecules. In the present study we characterize mitogenome of a rare and endangered species Pulsatilla patens. We investigated the gene content and its RNA editing potential, repeats distribution and plastid derived sequences.Results: The mitogenome structure of early divergent eudicot, endangered Pulsatilla patens does not support the master chromosome hypothesis, revealing the presence of three linear chromosomes of total length 986 613 bp. The molecules are shaped by the presence of extremely long, exceeding 87 kbp, repeats and multiple chloroplast derived regions including nearly complete inverted repeat. Since the plastid IR content of Ranunculales is very characteristic, the incorporation into mitogenome could be explained rather by intracellular transfer than mitochondrial HGT. The mitogenome contains an almost complete set of genes known from other vascular plants with exception of rps10 and sdh3, the latter being present but pseudogenised. Analysis of long ORFs enabled the identification of genes which are rarely present in plant mitogenomes, including RNA and DNA polymerases, albeit their presence even at species level is variable. Mitochondrial transcripts of P. patens were edited with a high frequency, exceeding the level known in other analyzed angiosperms, despite strict qualification criteria of editing event’s count and analysis of generally less frequently edited leaf transcriptome. The total number of edited sites was 902 and nad4 was identified as the most edited gene with 65 C to U changes. Non-canonical, reverse U to C editing was not detected. Comparative analyses of mitochondrial genes of three Pulsatilla species revealed a level of variation comparable to chloroplast CDS dataset and much higher infrageneric differentiation than in other known angiosperm genera. The variation found in CDS of mitochondrial genes is comparable to values found among Pulsatilla plastomes. Despite a complicated mitogenome structure, 14 single copy regions not splitted by repeats or MTPT of 329 kbp revealed potential for phylogenetic, phylogeographic and population genetics studies by revealing intra- and interspecific collinearity.Conclusions: This studies provides valuable new information about mitochondrial genome of early divergent eudicots, Pulsatilla patens, revealed multi-chromosomal structure and shed new light on mitogenomics of early eudicots.