Hybridization and reticulate evolution in Diphasiastrum (flat-branched clubmosses, Lycopodiaceae) - New data from the island of Taiwan and Vietnam.

In the species groups related to Diphasiastrum multispicatum and D. veitchii, hybridization was investigated in samples from northern and southern Vietnam and the island of Taiwan, including available herbarium specimens from southeast Asia. The accessions were analyzed using flow cytometry (living material only), Sanger sequencing and multiplexed inter-simple sequence repeat genotyping by sequencing. We detected two cases of ancient hybridization involving different combinations of parental species; both led via subsequent duplication to tetraploid taxa. A cross D. multispicatum × D. veitchii from Malaysia represents D. wightianum, a tetraploid taxon according to reported DNA content measurements of dried material (genome formulas MM, VV and MMVV, respectively). The second case involves D. veitchii and an unknown diploid parent (genome formula XX). Three hybridogenous taxa (genome formulas VVX, VVXX, VVVX) were discernable by a combination of flow cytometry and molecular data. Taxon I (VVX, three clones found on Taiwan island) is apparently triploid. Taxon II represents another genetically diverse and sexual tetraploid species (VVXX) and can be assigned to D. yueshanense, described from Taiwan island but occurring as well in mainland China and Vietnam. Taxon III is as well most likely tetraploid (VVVX) and represented by at least one, more likely two, clones from Taiwan island. Taxa I and III are presumably asexual and new to science. Two independently inherited nuclear markers recombine only within, not between these hybrids, pointing towards reproductive isolation. We present an evolutionary scheme which explains the origin of the hybrids and the evolution of new and fully sexual species by hybridization and subsequent allopolyploidization in flat-branched clubmosses.

Club-mosses (Diphasiastrum, Lycopodiaceae) from the Far East - Introgression and possible cryptic speciation.

Hybridization occurs often in the genus Diphasiastrum (Lycopodiaceae), which corroborates reports for the two other recognized lycophyte families, Isoëtaceae and Selaginellaceae. Here we investigate the case of D. alpinum and D. sitchense from the Russian Far East (Kamchatka). Their hybrid, D. × takedae, was morphologically recognizable in 16 out of 22 accessions showing molecular signatures of hybridization; the remaining accessions displayed the morphology of either D. alpinum (3) or D. sitchense (3). We sequenced markers for chloroplast microsatellites (cp, 175 accessions from Kamchatka) and for the two nuclear markers RPB and LFY (175 and 152 accessions). A selection of 42 accessions, including all hybrid accessions, was analysed via genotyping by sequencing (GBS). We found multiple, but apparently uniparental hybridization, clearly characterized by a deviating group of haplotypes for D. sitchense and all hybrids. All accessions showing molecular signatures of hybridization in nuclear markers revealed the parental haplotype of D. sitchense, however only the LFY marker differentiated between the parent species. GBS, including 69,819 quality-filtered single nucleotid polymorphisms, unambiguously identified the hybrids and revealed introgression to occur. Most of the hybrids were F1, but three turned out to be backcrosses with D. alpinum (one) and with D. sitchense (two). These observations are in contrast to prior findings on three European species and their intermediates where all three hybrids turned out to be independent F1 crosses without evidence of recent backcrossing. In this study, backcrossing was detected, which indicates a limited fertility of the hybrid taxon D. × takedae. A comparison of accessions of Kamchatkian D. alpinum with plants from Europe indicated possible cryptic speciation. Accessions from the Far East had (i) a lower DNA content (7.0 vs. 7.5 pg/2C), (ii) different prevailing cp haplotypes, and (iii) RPB genotypes, and (iv) a clearly different SNP pattern in GBS. Diphasiastrum sitchense and the similar D. nikoënse, for the latter additional accessions from Japan were investigated, appeared as forms of one diverse species, sharing genotypes in both nuclear markers, although chloroplast haplotypes and DNA content show slight variations.

Lycophyte plastid genomics: extreme variation in GC, gene and intron content and multiple inversions between a direct and inverted orientation of the rRNA repeat.

Lycophytes are a key group for understanding vascular plant evolution. Lycophyte plastomes are highly distinct, indicating a dynamic evolutionary history, but detailed evaluation is hindered by the limited availability of sequences. Eight diverse plastomes were sequenced to assess variation in structure and functional content across lycophytes. Lycopodiaceae plastomes have remained largely unchanged compared with the common ancestor of land plants, whereas plastome evolution in Isoetes and especially Selaginella is highly dynamic. Selaginella plastomes have the highest GC content and fewest genes and introns of any photosynthetic land plant. Uniquely, the canonical inverted repeat was converted into a direct repeat (DR) via large-scale inversion in some Selaginella species. Ancestral reconstruction identified additional putative transitions between an inverted and DR orientation in Selaginella and Isoetes plastomes. A DR orientation does not disrupt the activity of copy-dependent repair to suppress substitution rates within repeats. Lycophyte plastomes include the most archaic examples among vascular plants and the most reconfigured among land plants. These evolutionary trends correlate with the mitochondrial genome, suggesting shared underlying mechanisms. Copy-dependent repair for DR-localized genes indicates that recombination and gene conversion are not inhibited by the DR orientation. Gene relocation in lycophyte plastomes occurs via overlapping inversions rather than transposase/recombinase-mediated processes.

Genetic diversity and hybrid formation in Central European club-mosses (Diphasiastrum, Lycopodiaceae) - New insights from cp microsatellites, two nuclear markers and AFLP.

In Europe, the genus Diphasiastrum (Lycopodiophyta) forms a reticulate network of six diploid taxa, including three parent species (D. alpinum, D. complanatum and D. tristachyum) and three hybrids (D. × issleri, D. × oellgaardii and D. × zeilleri). It was not clear if the hybrids arose once or repeatedly, if they have reproductive competence and if backcrossing occurs. We addressed these questions by analysing 209 accessions for chloroplast microsatellites (cp), two nuclear markers (introns of the RPB and LFY genes) and AFLP. For D. complanatum we show a sexual life cycle with alternation of generations: the gametophytic DNA amount is half of that of the sporophyte. With the exception of a single accession all hybrids display one of the two parental cp haplotypes; their frequencies do not differ significantly from a 1:1 ratio. Genotypes of nuclear markers are species-specific, displaying 2/4/1 (RPB) and 1/8/1 alleles (LFY) for the three parents mentioned above; all hybrids have one allele from each parent. All three hybrid taxa apparently represent independent F1 crosses. Hybridisation occurs bidirectional; no evidence for recent backcrossing was found. Asexual reproduction via agamospory is at least rare, since AFLP showed all hybrid plants to be different.

Cloning, molecular characterization, and phylogeny of two evolutionary distinct glutamine synthetase isoforms in the green microalga Haematococcus pluvialis (Chlorophyceae).

Haematococcus pluvialis (Chlorophyta) is a widely used microalga of great economic potential, yet its molecular genetics and evolution are largely unknown. We present new detailed molecular and phylogenetic analysis of two glutamine synthetase (GS) enzymes and genes (gln) under the Astaxanthin-inducing conditions of light- and nitrogen-stress. Structure analysis identified key residues and confirmed two decameric GS2 holoenzymes, a cytoplasmic enzyme, termed GS2c , and a plastidic form, termed GS2p , due to chloroplast-transit peptides at its N-terminus. Gene expression analysis showed dissociation of mRNA, protein, and enzyme activity levels for both GS2 under different growth conditions, indicating the strong post-transcriptional regulation. Data-mining identified novel and specified published gln genes from Prasinophyceae, Chlorophyta, Trebouxiophyceae, Charophyceae, Bryophyta, Lycopodiophyta, Spermatophyta, and Rhodophyta. Phylogenetic analysis found homologues to the cytosolic GS2c of H. pluvialis in all other photo- and non-photosynthetic Eukaryota. The chloroplastic GS2p was restricted to Chlorophyta, Bryophyta, some Proteobacteria and Fungii; no homologues were identified in Spermatophyta or other Eukaryota. This indicates two independent prokaryotic donors for these two gln genes in H. pluvialis. Combined phylogenetic analysis of GS, chl-b synthase, elongation factor, and light harvesting complex homologues project a newly refined model of Viridiplantae evolution. Herein, a GS1 evolved into the cytosolic GS2c and was passed on to all Eukaryota. Later, the chloroplastic GS2p entered the Archaeplastida lineage via a horizontal gene transfer at the divergence of Chlorophyta and Rhodophyta lineages. GS2p persisted in Chlorophyta and Bryophyta, but was lost during Spermatophyta evolution. These data suggest the revision of GS classification and nomenclature, and extend our understanding of the photosynthetic Eukaryota evolution.

Vascular plant taxa occurrences in exotic woodland and in natural and production forests on the Islands of São Miguel, Terceira and Pico (Azores).

Background: The data presented here originated from field expeditions carried out between 2017 and 2018, within the framework of Forest-Eco2 project: "Towards an Ecological and Economic valorisation of the Azorean Forest". The project aimed to quantify the ecological value of the Azorean forests, including carbon accumulation and to design and propose measures that could further enhance forest sustainability. For that, 90 forest plots were sampled on three Azores islands - São Miguel, Terceira and Pico - equally distributed into natural forest, exotic woodland and production forest. The aim of this report is to further expand knowledge on biodiversity trends enclosed in the different forest types present in the Azores, by providing a list of the occurrences of the 105 different vascular plant taxa together with a brief characterisation of their origin and life-form. New information: We provide an inventory of indigenous and non-indigenous vascular plant taxa from 90 forest stands. A total of 105 taxa were identified and registered, belonging to 60 families, 91 genera, 101 species and four subspecies. A total of 35% of the taxa were endemic, 27% native and 38% non-indigenous, including 19% of invasive taxa. Endangered and vulnerable taxa were registered, including Elaphoglossumhirtum (Sw.) C.Chr., Lactucawatsoniana Trel. and others which were considered by the authors a priority for conservation (e.g. Arceuthobiumazoricum Wiens & Hawksw., Bellisazorica Hochst. ex Seub., Saniculaazorica Guthnick ex Seub., Platantheramicrantha (Hochst. ex Seub.) Schltr.). Our records provide detailed and updated knowledge of Azorean Forest flora and highlight the role of natural forests as indigenous plant diversity hotspots and exotic woodland as a source of invasive taxa within the Archipelago.

Selaginella guihaia (Selaginellaceae): A new spikemoss species from southern China and northern Vietnam around the Gulf of Tonkin.

Selaginella guihaiasp. nov. (Selaginellaceae), a new species of spikemoss from southern China and northern Vietnam around the Gulf of Tonkin (Beibu Gulf), is described and illustrated. Morphological and molecular comparisons of the new species with other similar species (S. doederleinii, S. ornata and S. trachyphylla) are provided. The morphological and molecular evidence clearly indicates S. guihaia is a distinct species. Morphologically S. guihaia differs from other species by its obviously white-margined leaves, the ventral leaves scabrous on upper surfaces throughout the basiscopic or also rarely present on upper halves, and the ovate axillary leaves.

The Huperzia selago Shoot Tip Transcriptome Sheds New Light on the Evolution of Leaves.

Lycopodiophyta-consisting of three orders, Lycopodiales, Isoetales and Selaginellales, with different types of shoot apical meristems (SAMs)-form the earliest branch among the extant vascular plants. They represent a sister group to all other vascular plants, from which they differ in that their leaves are microphylls-that is, leaves with a single, unbranched vein, emerging from the protostele without a leaf gap-not megaphylls. All leaves represent determinate organs originating on the flanks of indeterminate SAMs. Thus, leaf formation requires the suppression of indeterminacy, that is, of KNOX transcription factors. In seed plants, this is mediated by different groups of transcription factors including ARP and YABBY.We generated a shoot tip transcriptome of Huperzia selago (Lycopodiales) to examine the genes involved in leaf formation. Our H. selago transcriptome does not contain any ARP homolog, although transcriptomes of Selaginella spp. do. Surprisingly, we discovered a YABBY homolog, although these transcription factors were assumed to have evolved only in seed plants.The existence of a YABBY homolog in H. selago suggests that YABBY evolved already in the common ancestor of the vascular plants, and subsequently was lost in some lineages like Selaginellales, whereas ARP may have been lost in Lycopodiales. The presence of YABBY in the common ancestor of vascular plants would also support the hypothesis that this common ancestor had a simplex SAM. Furthermore, a comparison of the expression patterns of ARP in shoot tips of Selaginella kraussiana (Harrison CJ, etal. 2005. Independent recruitment of a conserved developmental mechanism during leaf evolution. Nature 434(7032):509-514.) and YABBY in shoot tips of H. selago implies that the development of microphylls, unlike megaphylls, does not seem to depend on the combined activities of ARP and YABBY. Altogether, our data show that Lycopodiophyta are a diverse group; so, in order to understand the role of Lycopodiophyta in evolution, representatives of Lycopodiales, Selaginellales, as well as of Isoetales, have to be examined.

Evolutionary aspects of non-cell-autonomous regulation in vascular plants: structural background and models to study.

Plasmodesmata (PD) serve for the exchange of information in form of miRNA, proteins, and mRNA between adjacent cells in the course of plant development. This fundamental role of PD is well established in angiosperms but has not yet been traced back to the evolutionary ancient plant taxa where functional studies lag behind studies of PD structure and ontogenetic origin. There is convincing evidence that the ability to form secondary (post-cytokinesis) PD, which can connect any adjacent cells, contrary to primary PD which form during cytokinesis and link only cells of the same lineage, appeared in the evolution of higher plants at least twice: in seed plants and in some representatives of the Lycopodiophyta. The (in)ability to form secondary PD is manifested in the symplasmic organization of the shoot apical meristem (SAM) which in most taxa of seedless vascular plants differs dramatically from that in seed plants. Lycopodiophyta appear to be suitable models to analyze the transport of developmental regulators via PD in SAMs with symplasmic organization both different from, as well as analogous to, that in angiosperms, and to understand the evolutionary aspects of the role of this transport in the morphogenesis of vascular plant taxa.

New and existing combinations in Palaeotropical Phlegmariurus (Lycopodiaceae) and lectotypification of the type species Phlegmariurus phlegmaria (L.) T.Sen & U.Sen.

The genus Phlegmariurus Holubis recognised more widely than originally proposed and is circumscribed to include both Neotropic and Palaeotropic epiphytic and terrestrial species of Huperzioid Lycopodiaceae that have isotomous shoots, lack bulbils in their sporophyllous shoots and have spores with convex lateral margins and foveolate-fossulate sculpture restricted to their distal surfaces. New combinations with Phlegmariurus are proposed for 81 species and existing combinations identified for 33 species originating from the Palaeotropics. This installs a generic circumscription that is consistent between the Neotropics and Palaeotropics. A lectotype is designated for the type species of the genus, Phlegmariurus phlegmaria (L.) T.Sen & U.Sen.