PREMISE: The great variation of genome size (C-value) across land plants is linked to various adaptative features. Flow cytometry (FCM), the standard approach to estimating C-values, relies mostly on fresh materials, performing poorly when used with herbarium materials. No fern C-value reports have been derived from herbarium specimens; however, the herbarium spores of some ferns remain highly viable for decades and are thus promising for further investigation. To explore this possibility, we evaluated herbarium spore collections of Ophioglossaceae ferns using FCM. METHODS: Flow cytometry was conducted on 24 spore samples, representing eight of the 12 genera of the Ophioglossaceae, using specimens ranging in age from 2.6 to 111 years obtained from five herbaria. RESULTS: Regardless of the genus or the source herbarium, high-quality C-value data were generated from 17 samples, with the oldest being 26 years old. Estimates of the C-values from sporophytic tissues of known ploidy did not reveal any evidence of apomixis for the species surveyed here. We also detected a pronounced genome downsizing in Sceptridium polyploids. DISCUSSION: The recent success of FCM for C-value estimation using spores provides a much more convenient method of utilizing dry refrigerated materials. We demonstrate here that herbarium spores of some ferns are also promising for this use, even for older specimens.

Background

DNA barcoding will revolutionize our understanding of fern ecology, most especially because the accurate identification of the independent but cryptic gametophyte phase of the fern's life history--an endeavor previously impossible--will finally be feasible. In this study, we assess the discriminatory power of the core plant DNA barcode (rbcL and matK), as well as alternatively proposed fern barcodes (trnH-psbA and trnL-F), across all major fern lineages. We also present plastid barcode data for two genera in the hyperdiverse polypod clade--Deparia (Woodsiaceae) and the Cheilanthes marginata group (currently being segregated as a new genus of Pteridaceae)--to further evaluate the resolving power of these loci.

Principal findings

Our results clearly demonstrate the value of matK data, previously unavailable in ferns because of difficulties in amplification due to a major rearrangement of the plastid genome. With its high sequence variation, matK complements rbcL to provide a two-locus barcode with strong resolving power. With sequence variation comparable to matK, trnL-F appears to be a suitable alternative barcode region in ferns, and perhaps should be added to the core barcode region if universal primer development for matK fails. In contrast, trnH-psbA shows dramatically reduced sequence variation for the majority of ferns. This is likely due to the translocation of this segment of the plastid genome into the inverted repeat regions, which are known to have a highly constrained substitution rate.

Conclusions

Our study provides the first endorsement of the two-locus barcode (rbcL+matK) in ferns, and favors trnL-F over trnH-psbA as a potential back-up locus. Future work should focus on gathering more fern matK sequence data to facilitate universal primer development.