Epitranscriptome insights into Riccia fluitans L. (Marchantiophyta) aquatic transition using nanopore direct RNA sequencing.

BACKGROUND: Riccia fluitans, an amphibious liverwort, exhibits a fascinating adaptation mechanism to transition between terrestrial and aquatic environments. Utilizing nanopore direct RNA sequencing, we try to capture the complex epitranscriptomic changes undergone in response to land-water transition. RESULTS: A significant finding is the identification of 45 differentially expressed genes (DEGs), with a split of 33 downregulated in terrestrial forms and 12 upregulated in aquatic forms, indicating a robust transcriptional response to environmental changes. Analysis of N6-methyladenosine (m6A) modifications revealed 173 m6A sites in aquatic and only 27 sites in the terrestrial forms, indicating a significant increase in methylation in the former, which could facilitate rapid adaptation to changing environments. The aquatic form showed a global elongation bias in poly(A) tails, which is associated with increased mRNA stability and efficient translation, enhancing the plant's resilience to water stress. Significant differences in polyadenylation signals were observed between the two forms, with nine transcripts showing notable changes in tail length, suggesting an adaptive mechanism to modulate mRNA stability and translational efficiency in response to environmental conditions. This differential methylation and polyadenylation underline a sophisticated layer of post-transcriptional regulation, enabling Riccia fluitans to fine-tune gene expression in response to its living conditions. CONCLUSIONS: These insights into transcriptome dynamics offer a deeper understanding of plant adaptation strategies at the molecular level, contributing to the broader knowledge of plant biology and evolution. These findings underscore the sophisticated post-transcriptional regulatory strategies Riccia fluitans employs to navigate the challenges of aquatic versus terrestrial living, highlighting the plant's dynamic adaptation to environmental stresses and its utility as a model for studying adaptation mechanisms in amphibious plants.

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.

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.

Are the Organellar Genomes Useful for Fine Scale Population Structure Analysis of Endangered Plants?-A Case Study of Pulsatilla patens (L.) Mill.

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.

Geochemical alkalinity and acidity as preferential site-specific for three lineages liverwort of Aneura pinguis cryptic species A.

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.

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.

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.

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.

Phylogenetic implications of nuclear rRNA IGS variation in Stipa L. (Poaceae).

The article takes up the problem of deficiency of molecular marker, which could illustrate molecular variability as well as phylogenetic relation within the genus of Stipa L. (Poaceae). Researches made so far hadn't delivered sufficient information about relationships between particular taxa from the genus of Stipa. In the present study, we analyzed variability and phylogenetic informativeness of nuclear ribosomal DNA in six species from the genus against five other species from Poaceae including a division of this region into functional elements and domains. Our results showed that the intergenic spacer region, and especially its part adjacent to 26 S nrDNA, is a molecular marker giving a real chance for a phylogeny reconstruction of Stipa. The region seems to be the most phylogenetically informative for Stipa from all the chloroplast and nuclear markers tested so far. Comparative analysis of nrDNA repeat units from Stipa to other representatives of Poaceae showed that their structure does not deviate from the general scheme. However, the rate of evolution within the inter-repeats in the IGS region is extremely high and therefore it predestines the region for phylogenetic analyses of Stipa at genus level or in shallower taxonomic scale.

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 cryptic species C of Aneura pinguis.

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.

Comparison of the effectiveness of ISJ and SSR markers and detection of outlier loci in conservation genetics of Pulsatilla patens populations.

BACKGROUND: Research into the protection of rare and endangered plant species involves genetic analyses to determine their genetic variation and genetic structure. Various categories of genetic markers are used for this purpose. Microsatellites, also known as simple sequence repeats (SSR), are the most popular category of markers in population genetics research. In most cases, microsatellites account for a large part of the noncoding DNA and exert a neutral effect on the genome. Neutrality is a desirable feature in evaluations of genetic differences between populations, but it does not support analyses of a population's ability to adapt to a given environment or its evolutionary potential. Despite the numerous advantages of microsatellites, non-neutral markers may supply important information in conservation genetics research. They are used to evaluate adaptation to specific environmental conditions and a population's adaptive potential. The aim of this study was to compare the level of genetic variation in Pulsatilla patens populations revealed by neutral SSR markers and putatively adaptive ISJ markers (intron-exon splice junction). METHODS: The experiment was conducted on 14 Polish populations of P. patens and three P. patens populations from the nearby region of Vitebsk in Belarus. A total of 345 individuals were examined. Analyses were performed with the use of eight SSR primers specific to P. patens and three ISJ primers. RESULTS: SSR markers revealed a higher level of genetic variation than ISJ markers (He = 0.609, He = 0.145, respectively). An analysis of molecular variance (AMOVA) revealed that, the overall genetic diversity between the analyzed populations defined by parameters FST and Φ PT for SSR (20%) and Φ PT for ISJ (21%) markers was similar. Analysis conducted in the Structure program divided analyzed populations into two groups (SSR loci) and three groups (ISJ markers). Mantel test revealed correlations between the geographic distance and genetic diversity of Polish populations of P. patens for ISJ markers, but not for SSR markers. CONCLUSIONS: The results of the present study suggest that ISJ markers can complement the analyses based on SSRs. However, neutral and adaptive markers should not be alternatively applied. Neutral microsatellite markers cannot depict the full range of genetic variation in a population because they do not enable to analyze functional variation. Although ISJ markers are less polymorphic, they can contribute to the reliability of analyses based on SSRs.

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.

The complete plastid genome of the middle Asian endemic of Stipa lipskyi (Poaceae).

The structure of the Stipa lipskyi (GenBank accession no. KT692644) plastid genome is similar to that of closely related Poaceae species: it has a total length of 137 755 bp, the base composition of the plastome is the following: A (30.7%), C (19.3%), G (19.4%) and T (30.5%). The S. lipskyi plastid genome contains 71 genes, excluding second IR region. A complete plastome sequence of S. lipskyi will help the development of primers for examining phylogeny and hybridization events in this taxonomically difficult genus.

The complete mitochondrial genome of Niphotrichum ericoides (Bryophyta, Grimmiaceae).

The mitogenome of Niphotrichum ericoides (GenBank accession number KP 233863) has a total length of 106,727 bp and consists of 40 protein-coding genes, three ribosomal RNAs (rRNAs), and 24 transfer RNAs. The gene order is identical to that known in other moss mitogenomes.

The complete mitochondrial genome of the South American endemic moss Codriophorus laevigatus (Grimmiaceae).

The mitogenome of the Codriophorus laevigatus (GenBank accesion number KM506905) has a total length of 106,809 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.