Comprehensive phylogenomic time tree of bryophytes reveals deep relationships and uncovers gene incongruences in the last 500 million years of diversification.

PREMISE: Bryophytes form a major component of terrestrial plant biomass, structuring ecological communities in all biomes. Our understanding of the evolutionary history of hornworts, liverworts, and mosses has been significantly reshaped by inferences from molecular data, which have highlighted extensive homoplasy in various traits and repeated bursts of diversification. However, the timing of key events in the phylogeny, patterns, and processes of diversification across bryophytes remain unclear. METHODS: Using the GoFlag probe set, we sequenced 405 exons representing 228 nuclear genes for 531 species from 52 of the 54 orders of bryophytes. We inferred the species phylogeny from gene tree analyses using concatenated and coalescence approaches, assessed gene conflict, and estimated the timing of divergences based on 29 fossil calibrations. RESULTS: The phylogeny resolves many relationships across the bryophytes, enabling us to resurrect five liverwort orders and recognize three more and propose 10 new orders of mosses. Most orders originated in the Jurassic and diversified in the Cretaceous or later. The phylogenomic data also highlight topological conflict in parts of the tree, suggesting complex processes of diversification that cannot be adequately captured in a single gene-tree topology. CONCLUSIONS: We sampled hundreds of loci across a broad phylogenetic spectrum spanning at least 450 Ma of evolution; these data resolved many of the critical nodes of the diversification of bryophytes. The data also highlight the need to explore the mechanisms underlying the phylogenetic ambiguity at specific nodes. The phylogenomic data provide an expandable framework toward reconstructing a comprehensive phylogeny of this important group of plants.

Gene-rich UV sex chromosomes harbor conserved regulators of sexual development.

Nonrecombining sex chromosomes, like the mammalian Y, often lose genes and accumulate transposable elements, a process termed degeneration. The correlation between suppressed recombination and degeneration is clear in animal XY systems, but the absence of recombination is confounded with other asymmetries between the X and Y. In contrast, UV sex chromosomes, like those found in bryophytes, experience symmetrical population genetic conditions. Here, we generate nearly gapless female and male chromosome-scale reference genomes of the moss Ceratodon purpureus to test for degeneration in the bryophyte UV sex chromosomes. We show that the moss sex chromosomes evolved over 300 million years ago and expanded via two chromosomal fusions. Although the sex chromosomes exhibit weaker purifying selection than autosomes, we find that suppressed recombination alone is insufficient to drive degeneration. Instead, the U and V sex chromosomes harbor thousands of broadly expressed genes, including numerous key regulators of sexual development across land plants.

Novel anti-infective potential of salvianolic acid B against human serious pathogen Neisseria meningitidis.

BACKGROUND: Epidemics of meningococcal meningitis cause significant health problems especially in Sub-Saharan Africa. Novel anti-infective candidates are needed. In modern anti-adhesion therapy initial attachment of bacteria to host cells is prevented. Our unique studies have revealed anti-adhesive candidates from natural products, namely milk and berries, against Neisseria meningitidis adhesion. In the present study against N. meningitidis adhesion, a novel binding inhibitor was found; salvianolic acid B (SA-B), a polyphenol from the radix Salviae miltiorrhizae, an important part of Chinese folk medicine. METHODS: In order to test inhibition of meningococcal pili binding and anti-adhesion activity of SA-B, bovine thyroglobulin, a reference glycoprotein for meningococcal receptor was used in a microtiter plate assay. Inhibitory activity was tested by using serial dilutions of SA-B extracts of 98 and 70% purity. Results were confirmed in a HEC-1B cell dot assay and antimicrobial activity was measured by using a microbroth dilution assay. RESULTS: Almost total (93%) inhibition of pili binding, anti-adhesion, was achieved with the 70% extract of SA-B at the concentration of 0.3 mg/mL in the bovine thyroglobulin reference model. 50% binding inhibition activity was achieved with 0.6 µg/mL of the SA-B extract. Total inhibition of the pili binding to HEC-1B cells was found at the tested concentration of 0.5 mg/mL. The 98% pure SA-B resulted in weaker inhibition. At the concentration of 0.3 mg/mL 78% inhibition was achieved in the thyroglobulin model. For 50% inhibition 2.4 µg/mL of pure SA-B was needed. The difference between the binding inhibition activities (70 and 98% pure SA-B) was statistically significant (P = 0.03). Antimicrobial activity of 70% SA-B, when investigated against N. meningitidis, was detected only in relatively high concentrations. CONCLUSIONS: Our results indicate that plant SA-B may prevent meningococcal infections by inhibiting meningococcal binding and may thus have an impact on the amount of nasopharyngeal carriers of N. meningitidis. This may prevent the spreading of meningococcal infections between humans. One could conclude that SA-B and its source dried radix S. miltiorrhizae, which is an important part of Chinese folk medicine, could be valuable candidates for further research in meningococcal disease prevention.

Antimicrobial activity of different Finnish monofloral honeys against human pathogenic bacteria.

The antimicrobial activity and phenolic compounds of five Finnish honey products against important human pathogens Streptococcus pneumoniae, S. pyogenes, Staphylococcus aureus, and methicillin-resistant S. aureus were analyzed. Microbroth dilution method and HPLC-DAD were used in antimicrobial testing and phenolic compound determination, respectively. Significant antimicrobial activity (p < 0.01) against all the tested pathogens was found from willow herb (Epilobium angustifolium), heather (Calluna vulgaris), and buckwheat (Fagopyrum esculentum) honeys. This is the first report on antimicrobial activity of Finnish monofloral honeys against streptococcal and staphylococcal bacteria. To our knowledge this is also the first report on the antimicrobial effect of honey against S. pneumoniae.

Phylogeny-based comparative methods question the adaptive nature of sporophytic specializations in mosses.

Adaptive evolution has often been proposed to explain correlations between habitats and certain phenotypes. In mosses, a high frequency of species with specialized sporophytic traits in exposed or epiphytic habitats was, already 100 years ago, suggested as due to adaptation. We tested this hypothesis by contrasting phylogenetic and morphological data from two moss families, Neckeraceae and Lembophyllaceae, both of which show parallel shifts to a specialized morphology and to exposed epiphytic or epilithic habitats. Phylogeny-based tests for correlated evolution revealed that evolution of four sporophytic traits is correlated with a habitat shift. For three of them, evolutionary rates of dual character-state changes suggest that habitat shifts appear prior to changes in morphology. This suggests that they could have evolved as adaptations to new habitats. Regarding the fourth correlated trait the specialized morphology had already evolved before the habitat shift. In addition, several other specialized "epiphytic" traits show no correlation with a habitat shift. Besides adaptive diversification, other processes thus also affect the match between phenotype and environment. Several potential factors such as complex genetic and developmental pathways yielding the same phenotypes, differences in strength of selection, or constraints in phenotypic evolution may lead to an inability of phylogeny-based comparative methods to detect potential adaptations.

Morphology informed by phylogeny reveals unexpected patterns of species differentiation in the aquatic moss Rhynchostegium riparioides s.l.

Bryophyte floras typically exhibit extremely low levels of endemism. The interpretation, that this might reflect taxonomic shortcomings, is tested here for the Macaronesian flora, using the moss species complex of Rhynchostegium riparioides as a model. The deep polyphyly of R. riparioides across its distribution range reveals active differentiation that better corresponds to geographic than morphological differences. Morphometric analyses are, in fact, blurred by a size gradient that accounts for 80% of the variation observed among gametophytic traits. The lack of endemic diversification observed in R. riparioides in Macaronesia weakens the idea that the low rates of endemism observed in the Macaronesian bryophyte flora might solely be explained by taxonomic shortcomings. To the reverse, the striking polyphyly of North American and European lineages of R. riparioides suggests that the similarity between the floras of these continents has been over-emphasized. Discriminant analyses point to the existence of morphological discontinuities among the lineages resolved by the molecular phylogeny. The global rate of error associated to species identification based on morphology (0.23) indicates, however, that intergradation of shape and size characters among species in the group challenges their identification.

Inhibition of adhesion of Neisseria meningitidis to human epithelial cells by berry juice polyphenolic fractions.

The adhesion of pathogens to host tissues is the requirement for the initiation of the majority of infectious diseases. It was shown recently that the binding of Neisseria meningitidis pili to immobilized human epithelial cells is inhibited by molecular size fractions (10-100 kDa) of berry juices. Additionally, the isolated meningococcal pili bound to polyphenolic fractions of berry juices. The present study investigated the antiadhesive effects of berry juice polyphenolics against living meningococcal bacteria in a human epithelial cell culture model. The ability of bilberry, cranberry, crowberry and lingonberry juice polyphenolic fractions to inhibit the attachment of N. meningitidis bacteria to HEC-1B human epithelial cells in a cell culture model was examined. The antibacterial effect of the fractions was tested using a microtiter broth microdilution assay. The most effective adhesion inhibition of 75% was achieved with cranberry juice polyphenolic fraction followed by crowberry (63%), bilberry (63%) and lingonberry (57%) juice polyphenolic fractions. Bacterial survival rates after incubation with the fractions varied between 75-100%. The present results suggest berry juice polyphenols as inhibitors of adherence of N. meningitidis. Thus the binding of meningococci to berry juice polyphenols might be protective for the host against the infection.

Inhibition activity of wild berry juice fractions against Streptococcus pneumoniae binding to human bronchial cells.

Bacterial adhesion to the cell surface is a crucial step before infection can take place. Inhibition of bacterial binding offers a novel preventive approach against infections. Cranberry (Vaccinium macrocarpon Ait.) juice has been found to have antiadhesive activity against different bacteria. Streptococcus pneumoniae is an important pathogen and the most common cause for pneumonia, meningitis, and otitis media. In this study the inhibitory activity of cranberry (Vaccinium oxycoccos L.), bilberry (Vaccinium myrtillus L.) and crowberry (Empetrum nigrum and Empetrum hermaphroditum L.) juice fractions against pneumococcal binding was tested using human bronchial cells (Calu-3) as an adhesion model. In addition, the antimicrobial activity of the berry juice fractions was tested. It was found that the studied berry juice fractions had antiadhesion activity and cranberry juice was the most active. The adhesion inhibition activity of cranberry juice was nearly 90% at a concentration of 8.7 mg/g of soluble solids. The antimicrobial activity of the studied berry juice fractions was found to be remarkable; pneumococcal growth was inhibited totally at a concentration of ∼86 mg/g. Both antiadhesion and antimicrobial activities were reduced after solid-phase extraction of the berry juices, which may suggest molecular synergistic effects of the berry juice molecules against S. pneumoniae. The findings indicate that cranberry, bilberry and crowberry juices have potential against pneumococcal infections.

Screening of binding activity of Streptococcus pneumoniae, Streptococcus agalactiae and Streptococcus suis to berries and juices.

Antiadhesion therapy is a promising approach to the fight against pathogens. Antibiotic resistance and the lack of effective vaccines have increased the search for new methods to prevent infectious diseases. Previous studies have shown the antiadhesion activity of juice from cultivated cranberries (Vaccinium macrocarpon Ait.) against bacteria, especially E. coli. In this study, the binding of two streptococcal strains, Streptococcus pneumoniae and Streptococcus agalactiae, to molecular size fractions (FI, FII and FIII, <10 kDa, 10-100 kDa, and >100 kDa, respectively) of berries and berry and fruit juices from 12 plant species were studied using a microtiter well assay. For Streptococcus suis a hemagglutination inhibition assay was used. In general, binding activity was detected especially to wild cranberry (Vaccinium oxycoccos L.) and to other Vaccinium species. S. pneumoniae cells bound most to cranberry juice fraction FI and S. agalactiae cells to cranberry fraction FIII. Hemagglutination induced by S. suis was most effectively inhibited by cranberry fraction FII. NMR spectra of some characteristic active and non-active fractions were also measured. They indicate that fractions FII and FIII contained proanthocyanidins and/or other phenolic compounds. The results suggest Vaccinium berries as possible sources of antiadhesives against bacterial infections.

And if Engler was not completely wrong? Evidence for multiple evolutionary origins in the moss flora of Macaronesia.

The Macaronesian endemic flora has traditionally been interpreted as a relict of a subtropical element that spanned across Europe in the Tertiary. This hypothesis is revisited in the moss subfamily Helicodontioideae based on molecular divergence estimates derived from two independent calibration techniques either employing fossil evidence or using an Monte Carlo Markov Chain (MCMC) to sample absolute rates of nucleotide substitution from a prior distribution encompassing a wide range of rates documented across land plants. Both analyses suggest that the monotypic Madeiran endemic genus Hedenasiastrum diverged of other Helicodontioideae about 40 million years, that is, well before Macaronesian archipelagos actually emerged, in agreement with the relict hypothesis. Hedenasiastrum is characterized by a plesiomorphic morphology, which is suggestive of a complete morphological stasis over 40 million years. Macaronesian endemic Rhynchostegiella species, whose polyphyletic origin involves multiple colonization events, evolved much more recently, and yet accumulated many more morphological novelties than H. percurrens. The Macaronesian moss flora thus appears as a complex mix of ancient relicts and more recently dispersed, fast-evolving taxa.

Binding of Neisseria meningitidis pili to berry polyphenolic fractions.

Blocking bacterial adhesion to host surfaces provides novel potential to control infections. The present study was directed to binding and inhibitory activity of different fresh berries and berry and fruit juices against Neisseria meningitidis . Berries and juices were fractionated according to their molecular size into three fractions. A microtiter well assay for binding of N. meningitidis pili to berry and juice fractions was constructed. In addition, adhesion inhibition to human epithelial cells (HEC-1B) was tested. The active fractions were then subfractionated by employing solid-phase extraction. Subfractions were characterized by RP-HPLC-DAD, and the pili binding was evaluated by using microtiter well binding assay. Binding and inhibitory activity were detected to bilberry, cranberry, lingonberry, and crowberry fractions, which contained anthocyanins or a mixture of proanthocyanidins and flavonols. Thus, the findings identify several previously unknown binding and inhibitory activities and may suggest Vaccinium berries and crowberry as promising sources against meningococcal adherence.

Origin and evolution of the northern hemisphere disjunction in the moss genus Homalothecium (Brachytheciaceae).

Competing hypotheses that rely either on a stepping-stone dispersal via the North Atlantic or the Bering land bridges, or more recent transoceanic dispersal, have been proposed to explain the disjunct distribution of Mediterranean flora in southern Europe and western North America. These hypotheses were tested with molecular dating using a phylogeny of the moss genus Homalothecium based on ITS, atpB-rbcL, and rpl16 sequence data. The monophyly of two main lineages in Western Palearctic (Europe, central Asia and north Africa) and North America is consistent with the ancient vicariance hypothesis. The monophyly of Madeiran H. sericeum accessions supports the recognition of the Macaronesian endemic H. mandonii. A range of absolute rates of molecular evolution documented in land plants was used as probabilistic calibration prior by a Bayesian inference implementing a relaxed-clock model to derive ages for the nodes of interest. Our age estimates for the divergence of the American and Western Palearctic Homalothecium clade (5.7 Ma, IC 3.52-8.26) and the origin of H. mandonii (2.52 Myr IC 0.86-8.25) are not compatible with the ancient vicariance hypothesis. Age estimates suggests that species distributions result from rare instances of dispersal and subsequent sympatric diversification. The calibrated phylogeny indicates that Homalothecium has undergone a fast radiation during the last 4 Myr, which is consistent with the low levels of morphological divergence among sibling species.

Molecular phylogenetics of the Meteoriaceae s. str.: focusing on the genera Meteorium and Papillaria.

In order to delimit and understand the evolution of the Meteoriaceae, we provide phylogenetic analyses using the internal transcribed spacer 2 (ITS2) of nuclear ribosomal DNA in combination with two plastid markers, trnL-F and psbT-H. In contrast to the widely used trnL-F region, the psbT-H gene cluster, coding for proteins of photosystem II, has been rarely used to address systematic questions among the different land plant lineages. To overcome the problem of potential ambiguous alignments of non-coding DNA regions, the data were independently analyzed using direct optimization. The comparison and evaluation of the obtained results showed that the inferred cladograms based on the different phylogenetic approaches are very similar, with only minor differences. In combination with morphological characters, generic relationships as well as taxonomic and nomenclatural problems, especially regarding the key genera Meteorium and Papillaria are discussed in detail. New insights into generic relationships of the Meteoriaceae are provided, such as the exclusion of the monospecific southern South American genera Ancistrodes and Cryphaeophilum, which are subsequently transferred to the Hookeriaceae and Cryphaeaceae, respectively. Phylogenetic reconstructions using maximum likelihood as well as parsimony approaches reveal that at the familial level the Meteoriaceae s. l. are polyphyletic, if the formerly recognized "Trachypodaceae" are considered as a separate family. Based on our results we favor the synonym of the Trachypodaceae with the Meteoriaceae.

Phylogeny of the Brachytheciaceae (Bryophyta) based on morphology and sequence level data.

Brachytheciaceae is often considered a taxonomically difficult group of mosses. For example, morphological variation has led to difficulty in generic delimitation. We used DNA sequence data (chloroplast psbT-H and trnL-F and nuclear ITS2) together with morphology (63 characters) to examine the relationships within this family. The combined unaligned length of the DNA sequences used in the phylogenetic analyses varied between 1277 and 1343 bp. For phylogeny reconstruction we performed direct optimization, as implemented in POY. Analyses were performed with three different gap costs and the morphological data partition was weighted both: (1) equal to gap cost, and (2) with a weight of one. The utility of sensitivity analysis has recently been cast into doubt; hence in this study it was performed only to explore the effects of weighting on homology statements and topologies and to enable more detailed comparisons between earlier studies utilizing the direct optimization method. The wide sequence length variation of non-coding ITS2 sequences resulted in character optimizations (i.e., "alignments") of very different lengths when various gap costs were applied. Despite this variation, the topologies of equally parsimonious trees remained fairly stable. The inclusion of several outgroups, instead of only one, was observed to increase the congruence between data sets and to slightly increase the resolution. An inversion event in the 9 bp loop region in the chloroplast psbT-N spacer in mosses has been postulated to include only uninformative variation, thus possibly negatively impacting the phylogeny reconstruction. Despite this inversion, its variation within Brachytheciaceae was clearly congruent with information from other sources, but inclusion of these 9 bp in the analysis had only a minor effect on the phylogenetic results. In the most parsimonious topology, which was obtained with equal weighting of all data, Meteoriaceae and Brachytheciaceae were resolved as monophyletic sister groups, which had recently been suggested based on a few shared morphological characters. Our study revealed some new generic relationships within the Brachytheciaceae, which are discussed in light of the morphological characters traditionally used for generic delimitation.