The organellar genomes of Pellidae (Marchantiophyta): the evidence of cryptic speciation, conflicting phylogenies and extraordinary reduction of mitogenomes in simple thalloid liverwort lineage.

Organellar genomes of liverworts are considered as one of the most stable among plants, with rare events of gene loss and structural rearrangements. However, not all lineages of liverworts are equally explored in the field of organellar genomics, and subclass Pellidae is one of the less known. Hybrid assembly, using both short- and long-read technologies enabled the assembly of repeat-rich mitogenomes of Pellia and Apopellia revealing extraordinary reduction of length in the latter which impacts only intergenic spacers. The mitogenomes of Apopellia were revealed to be the smallest among all known liverworts-109 k bp, despite retaining all introns. The study also showed the loss of one tRNA gene in Apopellia mitogenome, although it had no impact on the codon usage pattern of mitochondrial protein coding genes. Moreover, it was revealed that Apopellia and Pellia differ in codon usage by plastome CDSs, despite identical tRNA gene content. Molecular identification of species is especially important where traditional taxonomic methods fail, especially within Pellidae where cryptic speciation is well recognized. The simple morphology of these species and a tendency towards environmental plasticity make them complicated in identification. Application of super-barcodes, based on complete mitochondrial or plastid genomes sequences enable identification of all cryptic lineages within Apopellia and Pellia genera, however in some particular cases, mitogenomes were more efficient in species delimitation than plastomes.

Super-Mitobarcoding in Plant Species Identification? It Can Work! The Case of Leafy Liverworts Belonging to the Genus Calypogeia.

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.

Breaking the limits - multichromosomal structure of an early eudicot Pulsatilla patens mitogenome reveals extensive RNA-editing, longest repeats and chloroplast derived regions among sequenced land plant mitogenomes.

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 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 almost a complete set of genes known from other vascular plants with exception of rps10 and sdh3, 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 of P. patens were 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 and nad4 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 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 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.

Molecular delimitation of European leafy liverworts of the genus Calypogeia based on plastid super-barcodes.

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.

The Increase of Simple Sequence Repeats during Diversification of Marchantiidae, An Early Land Plant Lineage, Leads to the First Known Expansion of Inverted Repeats in the Evolutionarily-Stable Structure of Liverwort Plastomes.

The chloroplast genomes of liverworts, an early land plant lineage, exhibit stable structure and gene content, however the known resources are very limited. The newly sequenced plastomes of Conocephalum, Riccia and Sphaerocarpos species revealed an increase of simple sequence repeats during the diversification of complex thalloid liverwort lineage. The presence of long TA motifs forced applying the long-read nanopore sequencing method for proper and dependable plastome assembly, since the length of dinucleotide repeats overcome the length of Illumina short reads. The accumulation of SSRs (simple sequence repeats) enabled the expansion of inverted repeats by the incorporation of rps12 and rps7 genes, which were part of large single copy (LSC) regions in the previously sequenced plastomes. The expansion of inverted repeat (IR) at the genus level is reported for the first time for non-flowering plants. Moreover, comparative analyses with remaining liverwort lineages revealed that the presence of SSR in plastomes is specific for simple thalloid species. Phylogenomic analysis resulted in trees confirming monophyly of Marchantiidae and partially congruent with previous studies, due to dataset-dependent results of Dumortiera-Reboulia relationships. Despite the lower evolutionary rate of Marchantiales plastomes, significant barcoding gap was detected, even for recently divergent holarctic Conocephalum species. The sliding window analyses revealed the presence of 18 optimal (500 bp long) barcodes that enable the molecular identification of all studied species.

Potential of Transcript Editing Across Mitogenomes of Early Land Plants Shows Novel and Familiar Trends.

RNA editing alters the identity of nucleotides in an RNA sequence so that the mature transcript differs from the template defined in the genome. This process has been observed in chloroplasts and mitochondria of both seed and early land plants. However, the frequency of RNA editing in plant mitochondria ranges from zero to thousands of editing sites. To date, analyses of RNA editing in mitochondria of early land plants have been conducted on a small number of genes or mitochondrial genomes of a single species. This study provides an overview of the mitogenomic RNA editing potential of the main lineages of these two groups of early land plants by predicting the RNA editing sites of 33 mitochondrial genes of 37 species of liverworts and mosses. For the purpose of the research, we newly assembled seven mitochondrial genomes of liverworts. The total number of liverwort genera with known complete mitogenome sequences has doubled and, as a result, the available complete mitogenome sequences now span almost all orders of liverworts. The RNA editing site predictions revealed that C-to-U RNA editing in liverworts and mosses is group-specific. This is especially evident in the case of liverwort lineages. The average level of C-to-U RNA editing appears to be over three times higher in liverworts than in mosses, while the C-to-U editing frequency of the majority of genes seems to be consistent for each gene across bryophytes.

Sequencing of organellar genomes of Gymnomitrion concinnatum (Jungermanniales) revealed the first exception in the structure and gene order of evolutionary stable liverworts mitogenomes.

BACKGROUND: Comparative analyses of chloroplast and mitochondrial genomes have shown that organelle genomes in bryophytes evolve slowly. However, in contrast to seed plants, the organellar genomes are yet poorly explored in bryophytes, especially among liverworts. Discovering another organellar genomes of liverwort species by sequencing provides new conclusions on evolution of bryophytes. RESULTS: In this work, the organellar genomes of Gymnomitrion concinnatum liverwort were sequenced, assembled and annotated for the first time. The chloroplast genome displays, typical for most plants, quadripartite structure containing large single copy region (81,701 bp), two inverted repeat regions (8704 bp each) and small single copy region (20,179 bp). The gene order and content of chloroplast are very similar to other liverworts with minor differences observed. A total number of 739 and 222 RNA editing sites were predicted in chloroplast and mitochondrial genes of G. concinnatum. The mitochondrial genome gene content is also in accordance with liverworts except few alterations such as: intron loss in cox1 and atp1 genes. Nonetheless the analysis revealed that G. concinnatum mitogenome structure and gene order are rearranged in comparison with other mitogenomes of liverworts. The causes underlying such mitogenomic rearrangement were investigated and the probable model of recombination was proposed. CONCLUSIONS: This study provide the overview of mitochondrial and chloroplast genome structure and gene order diversity of Gymnomitrion concinnatum against the background of known organellar genomes of liverworts. The obtained results cast doubt on the idea that mitogenome structure of early land plants is highly conserved as previous studies suggested. In fact is the very first case of recombination within, evolutionary stable, mitogenomes of liverworts.

Does Calypogeia azurea (Calypogeiaceae, Marchantiophyta) occur outside Europe? Molecular and morphological evidence.

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.

Development and characterization of microsatellite markers for endangered species Stipa pennata (Poaceae) and their usefulness in intraspecific delimitation.

Stipa pennata (Poaceae), has become a rare and endangered species in Central Europe due habitat loss and fragmentation. This species is characterized by high morphological variability, which has resulted in the description of numerous intraspecific taxa. The aim of present work is to develop microsatellite markers useful in population genetics studies as well as in intraspecific taxonomy of S. pennata s.l. We developed ten microsatellite markers using Illumina high-throughput. Polymorphism at each marker was evaluated using 4-15 individuals from four morphotypes of S. pennata s.l. Seven markers showed polymorphism while three were monomorphic. The number of alleles per locus ranged from 7 to 12, and the observed and expected heterozygosity varied from 0.000 to 1.000 and 0.000 to 0.8670, respectively. Our results confirm that three of four studied morphotypes are genetically distinct. The microsatellite markers developed here will be useful for evaluating levels of genetic diversity and differentiation, to study gene flow, population dynamics and in future conservation studies as well as for intraspecific delimitation of morphologically similar taxa within S. pennata s.l.

Comparative Analysis of Four Calypogeia Species Revealed Unexpected Change in Evolutionarily-Stable Liverwort Mitogenomes.

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.

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.

Mitogenomic analyses support the recent division of the genus Orthotrichum (Orthotrichaceae, Bryophyta).

A recently presented taxonomical arrangement of the moss genus Orthotrichum Hedw. s.l. substantially changed the traditional view of the taxon that had been accepted throughout the twentieth century. This paper provides the results of mitogenomic studies that strongly support the new taxonomical concept. Comparative analyses presented in this study confirmed the stable structure of moss mitogenomes. Moreover, 17 complete mitogenome sequences were used to identify the major evolutionary groups, including 11 newly sequenced ones, for this study. The analysis of mitochondrial hotspots revealed intron 4 of the cox1 gene to be the most variable non-coding region. The most variable protein-coding genes in the tribe Orthotricheae were ccmFC and tatC. The intergenic and intronic hotspots of Orthotrichum s.l. identified in the present study do not correspond to those described in vascular plant mitogenomes.

The complete mitochondrial genome of the rare and endangered Orthotrichum rogeri (Orthotrichaceae, Bryophyta).

The mitogenome of the Orthotrichum rogeri (GenBank accession number KM873610) has a total length of 106,634 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. A complete mitochondrial genome sequence of O. rogeri will help the development of primers for examining mitochondrial variation across bryophytes.