Dating the evolution of the complex thalloid liverworts (Marchantiopsida): total-evidence dating analysis supports a Late Silurian-Early Devonian origin and post-Mesozoic morphological stasis.

Divergence times based on molecular clock analyses often differ from those derived from total-evidence dating (TED) approaches. For bryophytes, fossils have been excluded from previous assessments of divergence times, and thus, their utility in dating analyses remains unexplored. Here, we conduct the first TED analyses of the complex thalloid liverworts (Marchantiopsida) that include fossils and evaluate macroevolutionary trends in morphological 'diversity' (disparity) and rates. Phylogenetic analyses were performed on a combined dataset of 130 discrete characters and 11 molecular markers (sampled from nuclear, plastid and mitochondrial genomes). Taxon sampling spanned 56 extant species - representing all the orders within Marchantiophyta and extant genera within Marchantiales - and eight fossil taxa. Total-evidence dating analyses support the radiation of Marchantiopsida during Late Silurian-Early Devonian (or Middle Ordovician when the outgroup is excluded) and that of Ricciaceae in the Middle Jurassic. Morphological change rate was high early in the history of the group, but it barely increased after Late Cretaceous. Disparity-through-time analyses support a fast increase in diversity until the Middle Triassic (c. 250 Ma), after which phenotypic evolution slows down considerably. Incorporating fossils in analyses challenges previous assumptions on the affinities of extinct taxa and indicates that complex thalloid liverworts radiated c. 125 Ma earlier than previously inferred.

The role of paleontological data in bryophyte systematics.

Systematics reconstructs tempo and mode in biological evolution by resolving the phylogenetic fabric of biodiversity. The staggering duration and complexity of evolution, coupled with loss of information (extinction), render exhaustive reconstruction of the evolutionary history of life unattainable. Instead, we sample its products-phenotypes and genotypes-to generate phylogenetic hypotheses, which we sequentially reassess and update against new data. Current consensus in evolutionary biology emphasizes fossil integration in total-evidence analyses, requiring in-depth understanding of fossils-age, phenotypes, and systematic affinities-and a detailed morphological framework uniting fossil and extant taxa. Bryophytes present a special case: deep evolutionary history but sparse fossil record and phenotypic diversity encompassing small dimensional scales. We review how these peculiarities shape fossil inclusion in bryophyte systematics. Paucity of the bryophyte fossil record, driven primarily by phenotypic (small plant size) and ecological constraints (patchy substrate-hugging populations), and incomplete exploration, results in many morphologically isolated, taxonomically ambiguous fossil taxa. Nevertheless, instances of exquisite preservation and pioneering studies demonstrate the feasibility of including bryophyte fossils in evolutionary inference. Further progress will arise from developing extensive morphological matrices for bryophytes, continued exploration of the fossil record, re-evaluation of previously described fossils, and training specialists in identification and characterization of bryophyte fossils, and in bryophyte morphology.

Defying death: incorporating fossils into the phylogeny of the complex thalloid liverworts (Marchantiidae, Marchantiophyta) confirms high order clades but reveals discrepancies in family-level relationships.

In recent years, the use of extensive molecular and morphological datasets has clarified the phylogenetic relationships among the orders of complex thalloid liverworts (Marchantiidae). However, previous studies excluded extinct taxa; thereby, undersampling the actual taxonomic diversity of the group. Here, we conducted a total-evidence analysis of Marchantiidae incorporating fossils. The combined dataset consisted of 11 genes-sampled from the nuclear, mitochondrial and plastid genomes-and 128 morphological characters. Sixty-two species, representing all classes and orders within Marchantiophyta and genera within Marchantiidae were included in the analyses. Six fossils were scored from literature: two assigned to the outgroup (Metzgeriothallus sharonae and Pallaviciniites sandaolingensis) and four to the ingroup (Marchantites cyathodoides, M. huolinhensis, Ricciopsis ferganica and R. sandaolingensis). Tree searches were conducted using parsimony as the optimality criterion. Clade sensitivity was assessed across a wide range of weighting regimes. Also, we evaluated the influence of fossils on the inferred topologies and branch support. Our results were congruent with previously inferred clades above the order level: Neohodgsoniales was sister to a clade formed by Sphaerocarpales and Marchantiales. However, relationships among families within Marchantiales contradicted recent studies. For instance, a clade consisting of Monosoleniaceae, Wiesnerellaceae and Targioniaceae was sister to the morphologically simple taxa instead of being nested within them as in previous studies. Novel synapomorphies were found for several clades within Marchantiales. Outgroup fossils were more influential than Marchantiidae fossils on overall topologies and branch support values. Except for a single weighting scheme, sampling continuous characters and down-weighting characters improved fossil stability. Ultimately, our results challenge the widespread notion that bryophyte fossils are problematic for phylogenetic inference.

On the spore ornamentation of the microsoroid ferns (microsoroideae, polypodiaceae).

Microsoroideae is the third largest of the six subfamilies of Polypodiaceae, containing over 180 species. These ferns are widely distributed in the tropical and subtropical regions of the Old World and Oceania. We documented the spore ornamentation and integrated these data into the latest phylogenetic hypotheses, including a sampling of 100 taxa representing each of 17 major lineages of microsoroid ferns. This enabled us to reconstruct the ancestral states of the spore morphology. The results show verrucate ornamentation as an ancestral state for Goniophlebieae and Lecanoptereae, globular for Microsoreae, and rugulate surface for Lepisoreae. In addition, spore ornamentation can be used to distinguish certain clades of the microsoroid ferns. Among all five tribes, Lecanoptereae show most diversity in spore surface ornamentation.

Chloroplot: An Online Program for the Versatile Plotting of Organelle Genomes.

Understanding the complexity of genomic structures and their unique architecture is linked with the power of visualization tools used to represent these features. Such tools should be able to provide a realistic and scalable version of genomic content. Here, we present an online organelle plotting tool focused on chloroplasts, which were developed to visualize the exclusive structure of these genomes. The distinguished unique features of this program include its ability to represent the Single Short Copy (SSC) regions in reverse complement, which allows the depiction of the codon usage bias index for each gene, along with the possibility of the minor mismatches between inverted repeat (IR) regions and user-specified plotting layers. The versatile color schemes and diverse functionalities of the program are specifically designed to reflect the accurate scalable representation of the plastid genomes. We introduce a Shiny app website for easy use of the program; a more advanced application of the tool is possible by further development and modification of the downloadable source codes provided online. The software and its libraries are completely coded in R, available at https://irscope.shinyapps.io/chloroplot/.

Development of chloroplast microsatellite markers for giant ragweed (Ambrosia trifida).

PREMISE: Plant invasions are increasing globally, and extensive study of the genetic background of the source and invading populations is needed to understand such biological processes. For this reason, chloroplast microsatellite markers were identified to explore the genetic diversity of the noxious weed Ambrosia trifida (Asteraceae). METHODS AND RESULTS: The complete chloroplast genome of A. trifida was mined for microsatellite loci, and 15 novel chloroplast primers were identified to assess the genetic diversity of 49 Ambrosia samples. The number of alleles amplified ranged from two to six, with an average of 3.2 alleles per locus. Shannon's information index varied from 0.305 and 1.467, expected heterozygosity ranged from 0.178 to 0.645, and the polymorphism information content value ranged from 0.211 to 0.675 (average 0.428). The cross-species transferability of the 15 microsatellite loci was also evaluated in four related Ambrosia species (A. artemisiifolia, A. maritima, A. psilostachya, and A. tenuifolia). CONCLUSIONS: The novel chloroplast microsatellite markers developed in the current study demonstrate substantial cross-species transferability and will be helpful in future genetic diversity studies of A. trifida and related species.

Exploring the phylogeny of the marattialean ferns.

The eusporangiate marattialean ferns represent an ancient radiation with a rich fossil record but limited modern diversity in the tropics. The long evolutionary history without close extant relatives has confounded studies of the phylogenetic origin, rooting and timing of marattialean ferns. Here we present new complete plastid genomes of six marattialean species and compiled a plastid genome dataset representing all of the currently accepted marattialean genera. We further supplemented this dataset by compiling a large dataset of mitochondrial genes and a phenotypic data matrix covering both extant and extinct representatives of the lineage. Our phylogenomic and total-evidence analyses corroborated the postulated position of marattialean ferns as the sister to leptosporangiate ferns, and the position of Danaea as the sister to the remaining extant marattialean genera. However, our results provide new evidence that Christensenia is sister to Marattia and that M. cicutifolia actually belongs to Eupodium. The apparently highly reduced rate of molecular evolution in marattialean ferns provides a challenge for dating the key phylogenetic events with molecular clock approaches. We instead applied a parsimony-based total-evidence dating approach, which suggested a Triassic age for the extant crown group. The modern distribution can best be explained as mainly resulting from vicariance following the breakup of Pangaea and Gondwana. We resolved the fossil genera Marattiopsis, Danaeopsis and Qasimia as members of the monophyletic family Marattiaceae, and the Carboniferous genera Sydneia and Radstockia as the monophyletic sister of all other marattialean ferns.

Reassessing the role of morphology in bryophyte phylogenetics: Combined data improves phylogenetic inference despite character conflict.

Morphological data has gained renewed attention and has been shown to be crucial in clarifying the phylogenetic relationship in a wide range of taxa. In the last decades, phylogenetic analyses of sequence-level data have radically modified the systematic schemes within bryophytes (early non-vascular land plants) and have revealed a widespread pattern of conflict with morphology-based classifications. Yet, a comprehensive evaluation of character conflict has not yet been performed in the context of combined matrices. In this study, we evaluate the impact of morphology on bryophyte phylogeny following a total-evidence approach across 10 published matrices. The analysed matrices span a wide range of bryophytes, taxonomic levels, gene sampling and number of morphological characters and taxa. Data conflict was addressed by measuring: (i) the topological congruence between individual partitions, (ii) changes in support values of the combined data relative to the molecular partition and (iii) clade stability. The association between these measures and the number of morphological characters per taxon (Nc/T ratio) and the proportion of non-fixed characters (i.e., inapplicable, polymorphic and missing data) was explored. In the individual partition analyses, the Nc/T ratio correlated positively with the topological congruence in six to seven datasets depending on the weighting scheme. The proportion of non-fixed cells had a minor influence on congruence between data partitions. The number of characters and proportion of non-fixed data varied significantly between morphological datasets that improved congruence between data types. This variation suggests that morphological datasets affect the results of combined analyses in different ways, depending on the taxa studied. Combined analyses revealed that, despite the low congruence values between partitions, integrating data types improves support values and stability. However, while non-fixed data had no negative effect on support values, stability was reduced as the proportion of non-fixed cells increased. Nc/T ratio was negatively associated with support values and it showed ambiguous responses in stability evaluations. Overall, the results indicate that adding morphology may contribute to the inference of phylogenetic relationships of bryophytes despite character conflict. Our findings suggest that merely comparing (a) morphology-based classifications with molecular phylogenies or (b) the outcome from individual data partitions can misestimate data conflict. These findings imply that analyses of combined data may provide conservative assessments of data conflict and, eventually, lead to an improved sampling of morphological characters in large-scale analyses of bryophytes.

Exploring phylogeny of the microsoroid ferns (Polypodiaceae) based on six plastid DNA markers.

The microsoroid ferns are one of the largest subfamilies of the Polypodiaceae with over 180 species mainly found in the humid forests of tropical Australasia. The phylogenetic relationships are still unclear, especially the delimitation of the genus Microsorum which has been recognized to be non-monophyletic. We analysed the microsoroid ferns using six chloroplast DNA regions (rbcL, rps4+rps4-trnS, trnL+trnL-trnF, atpA, atpB and matK) in order to present a robust hypothesis of their phylogeny. Our results suggest that they comprise up to 17 genera; of them, 12 agree with a previously accepted generic classification. Five tribes are proposed based on the phylogenetic relationships. Most of the species traditionally included in the genus Microsorum are found in six genera belonging to two tribes. In addition to the commonly used DNA markers, the additional atpA and matK are helpful to provide information about the phylogenetic relationships of the microsoroid ferns.

Sequencing the Plastid Genome of Giant Ragweed (Ambrosia trifida, Asteraceae) From a Herbarium Specimen.

We report the first plastome sequence of giant ragweed (Ambrosia trifida); with this new genome information, we assessed the phylogeny of Asteraceae and the transcriptional profiling against glyphosate resistance in giant ragweed. Assembly and genic features show a normal angiosperm quadripartite plastome structure with no signatures of deviation in gene directionality. Comparative analysis revealed large inversions across the plastome of giant ragweed and the previously sequenced members of the plant family. Asteraceae plastid genomes contain two inversions of 22.8 and 3.3 kb; the former is located between trnS-GCU and trnG-UCC genes, and the latter between trnE-UUC and trnT-GGU genes. The plastid genome sequences of A. trifida and the related species, Ambrosia artemisiifolia, are identical in gene content and arrangement, but they differ in length. The phylogeny is well-resolved and congruent with previous hypotheses about the phylogenetic relationship of Asteraceae. Transcriptomic analysis revealed divergence in the relative expressions at the exonic and intronic levels, providing hints toward the ecological adaptation of the genus. Giant ragweed shows various levels of glyphosate resistance, with introns displaying higher expression patterns at resistant time points after the assumed herbicide treatment.

Phylogeny of the family Cladoniaceae (Lecanoromycetes, Ascomycota) based on sequences of multiple loci.

Cladoniaceae is a family of lichenized fungi that belongs to the Lecanorales, Ascomycota. The family is distributed widely, although several genera are restricted to the Southern Hemisphere. The circumscriptions of the genera and species in the family have traditionally been based on thallus morphology, the type of vegetative propagules and the secondary metabolites. However, numerous species are highly variable phenotypically, making their delimitation problematic. In the present study a new phylogeny of Cladoniaceae is constructed using five loci (ITS rDNA, IGS rDNA, RPB2, RPB1, EF-1a) from a worldwide sample of 643 specimens representing 304 species. Cladoniaceae was resolved as a monophyletic group. The circumscription of the genera and the relationships among them are discussed. Pycnothelia, Carassea and Metus are closely related, forming a sister clade to the larger genus Cladonia. Cladia in its recent wide sense turned out to be paraphyletic, including species that have been recognized in Thysanothecium and Notocladonia. Cladonia was resolved as monophyletic, with C. wainioi as the earliest diverging lineage. Eleven major clades were resolved in Cladonia. No synapomorphies were found for most of them. We propose the new genera Pulchrocladia and Rexia, as segregates of Cladia, five new combinations, and the resurrection of the genus Heterodea.

iMEC: Online Marker Efficiency Calculator.

PREMISE OF THE STUDY: To accurately design plant genetic studies, the information content of utilized markers and primers must be calculated. Plant genotyping studies should take into account the efficiency of each marker system by calculating different parameters to find the optimal combination of primers. This can be problematic because there are currently no easily accessible applications that can be used to calculate multiple indices together. METHODS AND RESULTS: The program Online Marker Efficiency Calculator (iMEC) was developed using R for the simple computation of seven polymorphism indices (heterozygosity index, polymorphism information content, discriminating power, effective multiplex ratio, marker index, arithmetic mean heterozygosity, and resolving power). These indices are based on dominant and codominant DNA fingerprinting markers, thus allowing comparison and selection of optimal genetic markers for a given data set. CONCLUSIONS: iMEC simplifies the calculation of diverse indices for the marker of choice to better enable researchers to measure polymorphism information for individual markers. The program is available at https://irscope.shinyapps.io/iMEC/.

The chloroplast genome sequence of bittersweet (Solanum dulcamara): Plastid genome structure evolution in Solanaceae.

Bittersweet (Solanum dulcamara) is a native Old World member of the nightshade family. This European diploid species can be found from marshlands to high mountainous regions and it is a common weed that serves as an alternative host and source of resistance genes against plant pathogens such as late blight (Phytophthora infestans). We sequenced the complete chloroplast genome of bittersweet, which is 155,580 bp in length and it is characterized by a typical quadripartite structure composed of a large (85,901 bp) and small (18,449 bp) single-copy region interspersed by two identical inverted repeats (25,615 bp). It consists of 112 unique genes from which 81 are protein-coding, 27 tRNA and four rRNA genes. All bittersweet plastid genes including non-functional ones and even intergenic spacer regions are transcribed in primary plastid transcripts covering 95.22% of the genome. These are later substantially edited in a post-transcriptional phase to activate gene functions. By comparing the bittersweet plastid genome with all available Solanaceae sequences we found that gene content and synteny are highly conserved across the family. During genome comparison we have identified several annotation errors, which we have corrected in a manual curation process then we have identified the major plastid genome structural changes in Solanaceae. Interpreted in a phylogenetic context they seem to provide additional support for larger clades. The plastid genome sequence of bittersweet could help to benchmark Solanaceae plastid genome annotations and could be used as a reference for further studies. Such reliable annotations are important for gene diversity calculations, synteny map constructions and assigning partitions for phylogenetic analysis with de novo sequenced plastomes of Solanaceae.

IRscope: an online program to visualize the junction sites of chloroplast genomes.

Motivation: Genome plotting is performed using a wide range of visualizations tools each with emphasis on a different informative dimension of the genome. These tools can provide a deeper insight into the genomic structure of the organism. Results: Here, we announce a new visualization tool that is specifically designed for chloroplast genomes. It allows the users to depict the genetic architecture of up to ten chloroplast genomes in the vicinity of the sites connecting the inverted repeats to the short and long single copy regions. The software and its dependent libraries are fully coded in R and the reflected plot is scaled up to realistic size of nucleotide base pairs in the vicinity of the junction sites. We introduce a website for easier use of the program and R source code of the software to be used in case of preferences to be changed and integrated into personal pipelines. The input of the program is an annotation GenBank (.gb) file, the accession or GI number of the sequence or a DOGMA output file. The software was tested using over a 100 embryophyte chloroplast genomes and in all cases a reliable output was obtained. Availability and implementation: Source codes and the online suit available at https://irscope.shinyapps.io/irapp/ or https://github.com/Limpfrog/irscope.

The complete chloroplast genome sequence of the CAM epiphyte Spanish moss (Tillandsia usneoides, Bromeliaceae) and its comparative analysis.

Spanish moss (Tillandsia usneoides) is an epiphytic bromeliad widely distributed throughout tropical and warm temperate America. This plant is highly adapted to extreme environmental conditions. Striking features of this species include specialized trichomes (scales) covering the surface of its shoots aiding the absorption of water and nutrients directly from the atmosphere and a specific photosynthesis using crassulacean acid metabolism (CAM). Here we report the plastid genome of Spanish moss and present the comparison of genome organization and sequence evolution within Poales. The plastome of Spanish moss has a quadripartite structure consisting of a large single copy (LSC, 87,439 bp), two inverted regions (IRa and IRb, 26,803 bp) and short single copy (SSC, 18,612 bp) region. The plastid genome had 37.2% GC content and 134 genes with 88 being unique protein-coding genes and 20 of these are duplicated in the IR, similar to other reported bromeliads. Our study shows that early diverging lineages of Poales do not have high substitution rates as compared to grasses, and plastid genomes of bromeliads show structural features considered to be ancestral in graminids. These include the loss of the introns in the clpP and rpoC1 genes and the complete loss or partial degradation of accD and ycf genes in the Graminid clade. Further structural rearrangements appeared in the graminids lacking in Spanish moss, which include a 28-kb inversion between the trnG-UCC-rps14 region and 6-kb in the trnG-UCC-psbD, followed by a third <1kb inversion in the trnT sequence.

The plastid genome of Vanillon (Vanilla pompona, Orchidaceae).

This study presents the complete sequence of Vanilla pompona chloroplast genome. This 148,009 bp long genome consist of 107 genes out of which 30 of them are tRNA, 4 rRNA. Fairly long inverted repeat regions (IR) and large single-copy (LSC) of length 29,807 and 86,358 bp, respectively, were detected. This means an exceptionally short single-copy (SSC) region with only 2037 bp. This truncation of the SSC is due to multiple translocation of ndh genes to the mitochondrion as in majority of the Orchidaceae and especially in the genus Vanilla. The phylogeny presented here meaningfully places Vanillon within the orchid family.

The plastid genome sequence of the invasive plant common Ragweed (Ambrosia artemisiifolia, Asteraceae).

In the current study, we present the complete chloroplast genome sequence of Ambrosia artemisiifolia. The genome is 152,223 bp long and consist of 83 protein coding genes, 38 tRNAs, and four rRNAs duplicated in the inverted repeat. Detected large single-copy (LSC) and small single-copy (SSC) regions separated with two inverted repeat regions (IR) of length 25,098. The phylogenetic hypotheses obtained based on the analyses of 18 cp genomes places common ragweed within the tribe Heliantheae of the Asteraceae.

Complete chloroplast genome sequence of Coyote tobacco (Nicotiana attenuata, Solanaceae).

In this study, we announce the complete chloroplast genome sequence of Nicotiana attenuata. The genome sequence of 155,941 bp consists of two inverted repeat (IRa and IRb) regions of 25,438 bp each, a large single-copy (LSC) region of 86,513 bp and a small single-copy (SSC) region of 18,524 bp. The overall GC content is 37.9% and the GC contents of LSC, IRs, and SSC are 36%, 43.2%, and 32.1%, respectively. The plastome with 129 annotated unique genes includes 84 protein-coding genes, 37 tRNA genes, and 8 rRNA genes. Using the whole chloroplast genome sequences alignment of 16 Solanaceae species a phylogenetic hypothesis is presented validating the position of N. attenuata within Nicotianeae.

Internal transcribed spacer (ITS) evolution in populations of the hyperparasitic European mistletoe pathogen fungus, Sphaeropsis visci (Botryosphaeriaceae): The utility of ITS2 secondary structures.

We investigated patterns of nucleotide polymorphism in the internal transcribed spacer (ITS) region for Sphaeropsis visci, a hyperparasitic fungus that causes the leaf spot disease of the hemiparasite European mistletoe (Viscum album). Samples of S. visci were obtained from Hungary covering all major infected forest areas. For obtaining PCR products we used a fast and efficient direct PCR approach based on a high fidelity DNA polymerase. A total of 140 ITS sequences were subjected to an array of complementary sequence analyses, which included analyses of secondary structure stability, nucleotide polymorphism patterns, GC content, and presence of conserved motifs. Analysed sequences exhibited features of functional rRNAs. Overall, polymorphism was observed within less conserved motifs, such as loops and bulges, or, alternatively, as non-canonical G-U pairs within conserved regions of double stranded helices. The secondary structure of ITS2 provides new opportunities for obtaining further valuable information, which could be used in phylogenetic analyses, or at population level as demonstrated in our study. This is due to additional information provided by secondary structures and their models. The combined score matrix was used with the methods implemented in the programme 4SALE. Besides the pseudoprotein coding method of 4SALE, the molecular morphometric character coding also has potential for gaining further information for phylogenetic analyses based on the geometric features of the sub-structural elements of the ITS2 RNA transcript.