Clonality, local population structure and gametophyte sex ratios in cryptic species of the Sphagnum magellanicum complex.

BACKGROUND AND AIMS: Sphagnum (peatmoss) comprises a moss (Bryophyta) clade with ~300-500 species. The genus has unparalleled ecological importance because Sphagnum-dominated peatlands store almost a third of the terrestrial carbon pool and peatmosses engineer the formation and microtopography of peatlands. Genomic resources for Sphagnum are being actively expanded, but many aspects of their biology are still poorly known. Among these are the degree to which Sphagnum species reproduce asexually, and the relative frequencies of male and female gametophytes in these haploid-dominant plants. We assess clonality and gametophyte sex ratios and test hypotheses about the local-scale distribution of clones and sexes in four North American species of the S. magellanicum complex. These four species are difficult to distinguish morphologically and are very closely related. We also assess microbial communities associated with Sphagnum host plant clones and sexes at two sites. METHODS: Four hundred and five samples of the four species, representing 57 populations, were subjected to restriction site-associated DNA sequencing (RADseq). Analyses of population structure and clonality based on the molecular data utilized both phylogenetic and phenetic approaches. Multi-locus genotypes (genets) were identified using the RADseq data. Sexes of sampled ramets were determined using a molecular approach that utilized coverage of loci on the sex chromosomes after the method was validated using a sample of plants that expressed sex phenotypically. Sex ratios were estimated for each species, and populations within species. Difference in fitness between genets was estimated as the numbers of ramets each genet comprised. Degrees of clonality [numbers of genets/numbers of ramets (samples)] within species, among sites, and between gametophyte sexes were estimated. Sex ratios were estimated for each species, and populations within species. Sphagnum-associated microbial communities were assessed at two sites in relation to Sphagnum clonality and sex. KEY RESULTS: All four species appear to engage in a mixture of sexual and asexual (clonal) reproduction. A single ramet represents most genets but two to eight ramets were dsumbers ansd text etected for some genets. Only one genet is represented by ramets in multiple populations; all other genets are restricted to a single population. Within populations ramets of individual genets are spatially clustered, suggesting limited dispersal even within peatlands. Sex ratios are male-biased in S. diabolicum but female-biased in the other three species, although significantly so only in S. divinum. Neither species nor males/females differ in levels of clonal propagation. At St Regis Lake (NY) and Franklin Bog (VT), microbial community composition is strongly differentiated between the sites, but differences between species, genets and sexes were not detected. Within S. divinum, however, female gametophytes harboured two to three times the number of microbial taxa as males. CONCLUSIONS: These four Sphagnum species all exhibit similar reproductive patterns that result from a mixture of sexual and asexual reproduction. The spatial patterns of clonally replicated ramets of genets suggest that these species fall between the so-called phalanx patterns, where genets abut one another but do not extensively mix because of limited ramet fragmentation, and the guerrilla patterns, where extensive genet fragmentation and dispersal result in greater mixing of different genets. Although sex ratios in bryophytes are most often female-biased, both male and female biases occur in this complex of closely related species. The association of far greater microbial diversity for female gametophytes in S. divinum, which has a female-biased sex ratio, suggests additional research to determine if levels of microbial diversity are consistently correlated with differing patterns of sex ratio biases.

Predicted protein-protein interactions in the moss Physcomitrella patens: a new bioinformatic resource.

BACKGROUND: Physcomitrella patens, a haploid dominant plant, is fast becoming a useful molecular genetics and bioinformatics tool due to its key phylogenetic position as a bryophyte in the post-genomic era. Genome sequences from select reference species were compared bioinformatically to Physcomitrella patens using reciprocal blasts with the InParanoid software package. A reference protein interaction database assembled using MySQL by compiling BioGrid, BIND, DIP, and Intact databases was queried for moss orthologs existing for both interacting partners. This method has been used to successfully predict interactions for a number of angiosperm plants. RESULTS: The first predicted protein-protein interactome for a bryophyte based on the interolog method contains 67,740 unique interactions from 5,695 different Physcomitrella patens proteins. Most conserved interactions among proteins were those associated with metabolic processes. Over-represented Gene Ontology categories are reported here. CONCLUSION: Addition of moss, a plant representative 200 million years diverged from angiosperms to interactomic research greatly expands the possibility of conducting comparative analyses giving tremendous insight into network evolution of land plants. This work helps demonstrate the utility of "guilt-by-association" models for predicting protein interactions, providing provisional roadmaps that can be explored using experimental approaches. Included with this dataset is a method for characterizing subnetworks and investigating specific processes, such as the Calvin-Benson-Bassham cycle.

Gibberellin precursor is involved in spore germination in the moss Physcomitrella patens.

Gibberellins are ent-kaurene derived phytohormones that are involved in seed germination, stem elongation, and flower induction in seed plants, as well as in antheridia formation and spore germination in ferns. Although ubiquitous in vascular plants, the occurrence and potential function(s) of gibberellins in bryophytes have not yet been resolved. To determine the potential role of gibberellin and/or gibberellin-like compounds in mosses, the effect of AMO-1618 on spores of Physcomitrella patens (Hedw.) B.S.G. was tested. AMO-1618, which inhibited ent-kaurene and gibberellin biosynthesis in angiosperms, also inhibited the bifunctional copalyl diphosphate synthase (E.C. 5.5.1.13)/ent-kaurene synthase (E.C. 4.2.3.19) of P. patens. AMO-1618 also caused a decrease in spore germination rates of P. patens, and this inhibitory effect was less pronounced in the presence of ent-kaurene. These results suggest that ent-kaurene biosynthesis is required by P. patens spores to germinate, implying the presence of gibberellin-like phytohormones in mosses.

Novel localization of callose in the spores of Physcomitrella patens and phylogenomics of the callose synthase gene family.

BACKGROUND AND AIMS: Callose involvement in spore development is a plesiomorphic feature of land plants. Correlated light, fluorescence and immuno-electron microscopy was conducted on the developing spores of Physcomitrella patens to probe for callose. Using a bioinformatic approach, the callose synthase (PpCalS) genes were annotated and PpCalS and AtCalS gene families compared, testing the hypothesis that an exine development orthologue is present in P. patens based on deduced polypeptide similarity with AtCalS5, a known exine development gene. METHODS: Spores were stained with aniline blue fluorescent dye. Capsules were prepared for immuno-light and immuno-electron microscopy by gold labelling callose epitopes with monoclonal antibody. BLAST searches were conducted using the AtCalS5 sequence as a query against the P. patens genome. Phylogenomic analysis of the CalS gene family was conducted using PAUP (v.4.1b10). KEY RESULTS: Callose is briefly present in the aperture of developing P. patens spores. The PpCalS gene family consists of 12 copies that fall into three distinct clades with AtCalS genes. PpCalS5 is an orthologue to AtCalS5 with highly conserved domains and 64 % similarity of their deduced polypeptides. CONCLUSIONS: This is the first study to identify the presence of callose in moss spores. AtCalS5 was previously shown to be involved in pollen exine development, thus making PpCalS5 a suspect gene involved in moss spore exine development.

The hidden Heuchera: How science Twitter uncovered a globally imperiled species in Pennsylvania, USA.

The genus Heuchera is recognized as one of the most diverse endemic radiations of Saxifragaceae in North America, yet species delimitation and geographic distribution within the group remain controversial. Many species remain difficult to identify, including Heuchera alba, a narrow Appalachian endemic and globally imperiled (G2) taxon recorded only from West Virginia and Virginia that occurs in sympatry with H. pubescens and H. americana. A recent survey of the cliffside flora of the Shikellamy Bluffs, PA recorded dozens of Heuchera individuals that, through the use of social media, were positively identified as H. alba. Aided by examination of historical herbarium records, subsequent searches of similar habitats in Pennsylvania led to the discovery of seven more populations and established a significant range expansion for this rare species. The uncovering of H. alba in Pennsylvania is an exciting conservation outcome and an example of what can happen when botanists embrace a combination of modern and classical approaches to discovery and collaboration.