Morphological, genetic and ecological divergence in near-cryptic bryophyte species widespread in the Holarctic: the Dicranum acutifolium complex (Dicranales) revisited in the Alps

There is mounting evidence that reproductively isolated, but morphologically weakly differentiated species (so-called cryptic species) represent a substantial part of biological diversity, especially in bryophytes. We assessed the evolutionary history and ecological differentiation of a species pair, Dicranum brevifolium and D. septentrionale, which have overlapping ranges in the Holarctic. Despite their morphological similarity, we found similar genetic differentiation as between morphologically well-differentiated Dicranum species. Moreover, we detected gene tree discordance between plastid and nuclear markers, but neither of the two datasets resolved the two as sister species. The signal in trnL–trnF better reflects the morphological and ecological affinities and indicates a close relationship while ITS sequence data resolved the two taxa as phylogenetically distantly related. The discordance is probably unrelated to the ecological differentiation of D. septentrionale to colonise subneutral to alkaline substrates (vs. acidic in D. brevifolium), because this ability is rare in the genus and shared with D. acutifolium. This taxon is the closest relative of D. septentrionale according to the trnL–trnF data and does not share the discordance in ITS. We furthermore demonstrate that beside D. acutifolium, both D. septentrionale and D. brevifolium occur in the Alps but D. brevifolium is most likely rarer. Based on morphological analyses including factor analysis for mixed data of 45 traits we suggest treating the latter two as near-cryptic species and we recommend verifying morphological determinations molecularly

Challenging the term symbiosis in plant-microbe associations to create an understanding across sciences

Scientific progress relies on clear and consistent definitions for effective communication and collaboration. The term "symbiosis" in the context of plant-microbe associations suffers from diverse interpretations, leading to ambiguity in classification of these associations. This review elaborates on the issue, proposing an inclusive definition as well as a keyword

Population genetic consequences of the reproductive system in the liverwort Mannia fragrans

Ecological factors affecting reproduction and dispersal are particularly important in determining genetic structure of plant populations. Polyoicous reproductive system is not rare in bryophytes; however, to date, nothing is known about its functioning and possible population genetic effects. Using the liverwort Mannia fragrans as a model species, the main aims of this study were to separate the relative importance of the components of the polyoicous reproductive system and to assess its consequences on the genetic structure of populations. High sex expression rates increasing with patch size and strongly female-biased sex ratios were detected. Additional input into clonal growth after production of sex organs was found in males compared to females. Similar clonal traits of the rare bisexual and asexual plants and preference toward newly colonized patches suggest that selection prefers colonizers that first develop organs of both sexes, hence ensuring sexual reproduction when no partner is present. Despite frequent spore production, ISSR markers revealed low genetic diversity, probably resulting from the effective clonal propagation of the species and frequent crossing between genetically identical plants. The presence of numerous rare alleles and unique recombinant haplotypes indicates occasional recombination and mutation. Effective spreading of new haplotypes is probably hampered however by large spore size. Since populations are small and isolated, such haplotypes are probably continuously eliminated by genetic drift. These results suggest that although both sexual and asexual reproductions seem to be effective, asexual components of the reproductive system play a greater role in shaping the genetic composition of the population

Genetikai polimorfizmus vizsgálata rendszertani és ökológiai szempontokból moha-populációkon = Taxonomic and ecological aspects of genetic variability in moss populations

A pályázat 3 alprojektből állt. A Tortula ruralis gyepek genetikai struktúrájának feltárása során kimutattuk, hogy a távoli populációknál alig, az állományokon belül jelentős a foltok genetikai differenciációja. Az eltérés (egy mohapárna nem egyetlen genetikai klón) a populációk ivaros szaporodásának jelentőségét sejteti. Ez ellentmond a korábbi elképzelésnek, hogy a száraz termőhelyi stressz a klonális növekedésnek kedvez, és egyben azt is sugallja, hogy a modellfaj generatív képletei is jelentős kiszáradástűréssel rendelkeznek. A Hypnum cupressiforme reproduktív sajátságainak szubsztrátfüggését és ferdült ivararányokat találtunk. A hím klónok dominanciája a kidőlt fákon erőteljesebb vegetatív növekedésükkel magyarázható. A két nem koegzisztenciáját az magyarázza, hogy a hím és női gametofitonok térben ugyan nem, de időben szeparáltak eltérő szezonális növekedési ritmusuknak köszönhetően. Az ivari dimorfizmus nem feltétlenül morfológiai sajátságokban jelentkezik, hanem inkább a nemekre jellemző reakciónormaként. A Mannia fragrans példáján úttörőként megmutattuk, hogy a moha-diaspórabank rendelkezik "genetikai memória" funkcióval és jelentős szerepet játszik a genetikai variabilitás generációkon keresztül megőrzésében, ami különösen az időszakos élőhelyek kitartó propagulumokkal rendelkező mohafajainak esetében fontos. A vizsgálat azt is kimutatta, hogy a populációk genetikai struktúráját a tágabb értelemben vett aszexuális szaporodás is alakítja. | The support has contributed to the realization of three subprojects. The genetic structure of Tortula ruralis at remote populations was not different, but fine-scale genetic differentiation of individual patches was significant. The difference (several genetic clones within the same mat) suggests the presence of sexual reproduction. This contradicts the literature stating that abiotic stress would select for clonal reproduction and suggests that even the generative structures of the model species are highly drought tolerant. Substrate dependence of the reproductive characteristics and skewed sex-ratios were found at Hypnum cupressiforme. The dominance of male clones on decaying logs is related to their more dynamic growth. Sexes are not separated in space, their coexistence can be explained by separation in time due to differences in their seasonal growth rhythm. Due to sex-specific differences in growth, sexual dimorphism is not necessarily present in average characteristics but more as a reaction norm. The results at Mannia fragrans suggest 'genetic memory', that bryophyte diaspore bank has an important role in accumulating and storing genetic variability over generations and seasons. This may be especially important in species of temporarily available habitats that have long-lived spores and genetically variable populations. Furthermore, the results suggest that both sexual and asexual reproduction play role in shaping the genetic composition of the populations

A metabarcoding protocol targeting two DNA regions to analyze root‐associated fungal communities in ferns and lycophytes

Premise: Detailed studies of the fungi associated with lycophytes and ferns provide crucial insights into the early evolution of land plants. However, most investigations to date have assessed fern–fungus interactions based only on visual root inspection. In the present research, we establish and evaluate a metabarcoding protocol to analyze the fungal communities associated with fern and lycophyte roots. Methods:We used two primer pairs focused on the ITS rRNA region to screen the general fungal communities, and the 18S rRNA to target Glomeromycota fungi (i.e., arbuscular mycorrhizal fungi). To test these approaches, we collected and processed roots from 12 phylogenetically distant fern and lycophyte species. Results:We found marked compositional differences between the ITS and 18S data sets. While the ITS data set demonstrated the dominance of orders Glomerales (phylum Glomeromycota), Pleosporales, and Helotiales (both in phylum Ascomycota), the 18S data set revealed the greatest diversity of Glomeromycota. Non-metric multidimensional scaling (NMDS) ordination suggested an important geographical effect in sample similarities. Discussion: The ITS-based approach is a reliable and effective method to analyze the fungal communities associated with fern and lycophyte roots. The 18S approach is more appropriate for studies focused on the detailed screening of arbuscular mycorrhizal fungi

Comparative transcriptomics reveals commonalities and differences in the genetic underpinnings of a floral dimorphism

Distyly, a floral dimorphism associated with heteromorphic self-incompatibility and controlled by the S-locus supergene, evolved independently multiple times. Comparative analyses of the first transcriptome atlas for the main distyly model, Primula veris, with other distylous species produced the following findings. A set of 53 constitutively expressed genes in P. veris did not include any of the housekeeping genes commonly used to normalize gene expression in qPCR experiments. The S-locus gene CYP$^{T}$ acquired its role in controlling style elongation via a change in expression profile. Comparison of genes differentially expressed between floral morphs revealed that brassinosteroids and auxin are the main hormones controlling style elongation in P. veris and Fagopyrum esculentum, respectively. Furthermore, shared biochemical pathways might underlie the expression of distyly in the distantly related P. veris, F. esculentum and Turnera subulata, suggesting a degree of correspondence between evolutionary convergence at phenotypic and molecular levels. Finally, we provide the first evidence supporting the previously proposed hypothesis that distyly supergenes of distantly related species evolved via the recruitment of genes related to the phytochrome-interacting factor (PIF) signaling network. To conclude, this is the first study that discovered homologous genes involved in the control of distyly in distantly related taxa

Different molecular changes underlie the same phenotypic transition: Origins and consequences of independent shifts to homostyly within species

The repeated transition from outcrossing to selfing is a key topic in evolutionary biology. However, the molecular basis of such shifts has been rarely examined due to lack of knowledge of the genes controlling these transitions. A classic example of mating system transition is the repeated shift from heterostyly to homostyly. Occurring in 28 angiosperm families, heterostyly is characterized by the reciprocal position of male and female sexual organs in two (or three) distinct, usually self-incompatible floral morphs. Conversely, homostyly is characterized by a single, self-compatible floral morph with reduced separation of male and female organs, facilitating selfing. Here, we investigate the origins of homostyly in Primula vulgaris and its microevolutionary consequences by integrating surveys of the frequency of homostyles in natural populations, DNA sequence analyses of the gene controlling the position of female sexual organs (CYPᵀ), and microsatellite genotyping of both progeny arrays and natural populations characterized by varying frequencies of homostyles. As expected, we found that homostyles displace short-styled individuals, but long-style morphs are maintained at low frequencies within populations. We also demonstrated that homostyles repeatedly evolved from short-styled individuals in association with different types of loss-of- function mutations in CYPᵀ. Additionally, homostyly triggers a shift to selfing, promoting increased inbreeding within and genetic differentiation among populations. Our results elucidate the causes and consequences of repeated transitions to homostyly within species, and the putative mechanisms precluding its fixation in P. vulgaris. This study represents a benchmark for future analyses of losses of heterostyly in other angiosperms

An optimised transformation protocol for Anthoceros agrestis and three more hornwort species

Land plants comprise two large monophyletic lineages, the vascular plants and the bryophytes, which diverged from their most recent common ancestor approximately 480 million years ago. Of the three lineages of bryophytes, only the mosses and the liverworts are systematically investigated, while the hornworts are understudied. Despite their importance for understanding fundamental questions of land plant evolution, they only recently became amenable to experimental investigation, with Anthoceros agrestis being developed as a hornwort model system. Availability of a high-quality genome assembly and a recently developed genetic transformation technique makes A. agrestis an attractive model species for hornworts. Here we describe an updated and optimised transformation protocol for A. agrestis which can be successfully used to genetically modify one more strain of A. agrestis and three more hornwort species, Anthoceros punctatus, Leiosporoceros dussi and Phaeoceros carolinianus. The new transformation method is less laborious, faster and results in the generation of greatly increased numbers of transformants compared to the previous method. We have also developed a new selection marker for transformation. Finally, we report the development of a set of different cellular localisation signal peptides for hornworts providing new tools to better understand hornwort cell biology

An Agrobacterium-mediated stable transformation technique for the hornwort model Anthoceros agrestis

Despite their key phylogenetic position and their unique biology, hornworts have been widely overlooked. Until recently there was no hornwort model species amenable to systematic experimental investigation. Anthoceros agrestis has been proposed as the model species to study hornwort biology. We have developed an Agrobacterium-mediated method for the stable transformation of A. agrestis, a hornwort model species for which a genetic manipulation technique was not yet available. High transformation efficiency was achieved by using thallus tissue grown under low light conditions. We generated a total of 274 transgenic A. agrestis lines expressing the β-glucuronidase (GUS), cyan, green, and yellow fluorescent proteins under control of the CaMV 35S promoter and several endogenous promoters. Nuclear and plasma membrane localization with multiple color fluorescent proteins was also confirmed. The transformation technique described here should pave the way for detailed molecular and genetic studies of hornwort biology, providing much needed insight into the molecular mechanisms underlying symbiosis, carbon-concentrating mechanism, RNA editing and land plant evolution in general. Keywords: Anthoceros; development; evolution; hornworts; transformatio