Striking genetic homogeneity in the widespread South American bracken.

PREMISE: Bracken (Pteridium, Dennstaedtiaceae) is a cosmopolitan genus of aggressive disturbance colonizers that are toxic to agricultural livestock. The taxonomy of Pteridium has been treated in multiple schemes, ranging from one to six species worldwide, with numerous subspecies and varieties. Recent work has focused on the worldwide distribution and systematics of the bracken fern, but South America has been poorly represented. We present the first continent-wide sampling and analysis of Pteridium esculentum, a Southern Hemisphere diploid species. METHODS: Within South America, P. esculentum has several morphotypes, distinguished into subspecies by variation in indument and lamina architecture. We used double digest restriction site-associated DNA sequencing (ddRADSeq) to assess the phylogenetic relationships of P. esculentum subspecies. RESULTS: We found a striking genetic homogeneity in the species, being able to support only two morphotypes from molecular data: P. e. arachnoideum and P. e. campestre. We had high confidence for shallow and deep phylogenetic relationships, but less support for relationships among crown groups. CONCLUSIONS: We describe an east-west geographic pattern that would explain the relationships between populations; and, in contrast to previous studies, we detected differences with P. esculentum from Australia. These results will lay the foundations for studying variations in this species' behavior as a weed, as well as its impact on the production of agricultural livestock in South America.

De novo characterization of the gametophyte transcriptome in bracken fern, Pteridium aquilinum.

BACKGROUND: Because of their phylogenetic position and unique characteristics of their biology and life cycle, ferns represent an important lineage for studying the evolution of land plants. Large and complex genomes in ferns combined with the absence of economically important species have been a barrier to the development of genomic resources. However, high throughput sequencing technologies are now being widely applied to non-model species. We leveraged the Roche 454 GS-FLX Titanium pyrosequencing platform in sequencing the gametophyte transcriptome of bracken fern (Pteridium aquilinum) to develop genomic resources for evolutionary studies. RESULTS: 681,722 quality and adapter trimmed reads totaling 254 Mbp were assembled de novo into 56,256 unique sequences (i.e. unigenes) with a mean length of 547.2 bp and a total assembly size of 30.8 Mbp with an average read-depth coverage of 7.0×. We estimate that 87% of the complete transcriptome has been sequenced and that all transcripts have been tagged. 61.8% of the unigenes had blastx hits in the NCBI nr protein database, representing 22,596 unique best hits. The longest open reading frame in 52.2% of the unigenes had positive domain matches in InterProScan searches. We assigned 46.2% of the unigenes with a GO functional annotation and 16.0% with an enzyme code annotation. Enzyme codes were used to retrieve and color KEGG pathway maps. A comparative genomics approach revealed a substantial proportion of genes expressed in bracken gametophytes to be shared across the genomes of Arabidopsis, Selaginella and Physcomitrella, and identified a substantial number of potentially novel fern genes. By comparing the list of Arabidopsis genes identified by blast with a list of gametophyte-specific Arabidopsis genes taken from the literature, we identified a set of potentially conserved gametophyte specific genes. We screened unigenes for repetitive sequences to identify 548 potentially-amplifiable simple sequence repeat loci and 689 expressed transposable elements. CONCLUSIONS: This study is the first comprehensive transcriptome analysis for a fern and represents an important scientific resource for comparative evolutionary and functional genomics studies in land plants. We demonstrate the utility of high-throughput sequencing of a normalized cDNA library for de novo transcriptome characterization and gene discovery in a non-model plant.

LysM domains from Pteris ryukyuensis chitinase-A: a stability study and characterization of the chitin-binding site.

The LysM domain probably binds peptidoglycans, but how it does so has yet to be described. For this report, we measured the thermal stabilities of recombinant LysM domains derived from Pteris ryukyuensis chitinase-A (PrChi-A) and monitored their binding to N-acetylglucosamine oligomers ((GlcNAc)n) using differential scanning calorimetry, isothermal titration calorimetry, and NMR spectroscopy. We thereby characterized certain of the domains' functional and structural features. We observed that the domains are very resistant to thermal denaturation and that this resistance depends on the presence of disulfide bonds. We also show that the stoichiometry of (GlcNAc)n/LysM domain binding is 1:1. (GlcNAc)5 titration experiments, monitored by NMR spectroscopy, allowed us to identify the domain residues that are critical for (GlcNAc)5 binding. The binding site is a shallow groove formed by the N-terminal part of helix 1, the loop between strand 1 and helix 1, the C-terminal part of helix 2, and the loop between helix 2 and strand 2. Furthermore, mutagenesis experiments reiterate the critical involvement of Tyr72 in (GlcNAc)n/LysM domain binding. Ours is the first report describing the physical structure of a LysM oligosaccharide-binding site based on experimental data.

Kinetics of the natural evolution of hydrogen cyanide in plants in neotropical Pteridium arachnoideum and its ecological significance.

The time-dependent natural release of hydrogen cyanide (HCN) was studied quantitatively using young croziers of the neotropical bracken fern Pteridium arachnoideum. HCN production was quantified in crushed tissue using a flow reactor at 30.0 +/- 0.1 degrees C. Released HCN was carried into appropriate traps with a moist air flow. Aliquots were drawn from the traps at fixed time intervals, and the HCN concentration was evaluated spectroscopically. All available prunasin (Pru), the only cyanogenic glycoside present, underwent decomposition into HCN in less than 1200 min. Fiddleheads (N = 76) contained 1.84-107.70 mg Pru g(1) dw in a continuous fashion suggesting genetic polymorphism. Acyanogenic morphs were rare (1/77). From the kinetics of the samples with Pru content near the median histographic distribution (N = 46), accumulated HCN formation as a function of time, initial velocities, average HCN production rate, and corresponding rate equations were obtained. Initial and average velocities correlated well with total Pru content. The yield of cyanide liberation varied widely between 0.51 and 47.86 microg HCN min(-1) g(-1) dw and was a linear function of [Pru]t. However, the beta-glucosidase enzyme involved in this reaction was not rate limiting and occurs in excess in the natural system. Enzyme activity was found to be independent of [Pru]t. The contribution of HCN as an allomone-upon-request against herbivores was assessed quantitatively. Bracken fiddleheads produced a pulse of HCN soon after tissue injury that waned rapidly, leaving a large portion of intact prunasin to decompose more slowly in the herbivore's lumen. The balance between the external and internal courses was found to depend on the concentration of prunasin in the plant, the amount of crozier eaten, and the time used to consume it.