title: Use of rbcL and trnL-F as a two-locus DNA barcode for identification of NW-European ferns: an ecological perspective.
abstract: Although consensus has now been reached on a general two-locus DNA barcode for land plants, the selected combination of markers (rbcL + matK) is not applicable for ferns at the moment. Yet especially for ferns, DNA barcoding is potentially of great value since fern gametophytes--while playing an essential role in fern colonization and reproduction--generally lack the morphological complexity for morphology-based identification and have therefore been underappreciated in ecological studies. We evaluated the potential of a combination of rbcL with a noncoding plastid marker, trnL-F, to obtain DNA-identifications for fern species. A regional approach was adopted, by creating a reference database of trusted rbcL and trnL-F sequences for the wild-occurring homosporous ferns of NW-Europe. A combination of parsimony analyses and distance-based analyses was performed to evaluate the discriminatory power of the two-region barcode. DNA was successfully extracted from 86 tiny fern gametophytes and was used as a test case for the performance of DNA-based identification. Primer universality proved high for both markers. Based on the combined rbcL + trnL-F dataset, all genera as well as all species with non-equal chloroplast genomes formed their own well supported monophyletic clade, indicating a high discriminatory power. Interspecific distances were larger than intraspecific distances for all tested taxa. Identification tests on gametophytes showed a comparable result. All test samples could be identified to genus level, species identification was well possible unless they belonged to a pair of Dryopteris species with completely identical chloroplast genomes. Our results suggest a high potential of the combined use of rbcL and trnL-F as a two-locus cpDNA barcode for identification of fern species. A regional approach may be preferred for ecological tests. We here offer such a ready-to-use barcoding approach for ferns, which opens the way for answering a whole range of questions previously unaddressed in fern gametophyte ecology.
authors.affiliation: Ecology and Biodiversity Group, Institute of Environmental Biology, Utrecht University, Utrecht, The Netherlands. G.A.deGroot@uu.nl
authors.forename: G Arjen
authors.initials: GA
authors.lastname: de Groot
authors.forename: Heinjo J
authors.initials: HJ
authors.lastname: During
authors.forename: Jan W
authors.initials: JW
authors.lastname: Maas
authors.forename: Harald
authors.initials: H
authors.lastname: Schneider
authors.forename: Johannes C
authors.initials: JC
authors.lastname: Vogel
authors.forename: Roy H J
authors.initials: RH
authors.lastname: Erkens
mesh_terms: D058893:DNA Barcoding, Taxonomic; D017753:Ecosystem; D005060:Europe; D029624:Ferns / Q000145:classification* / Q000235:genetics*; D005819:Genetic Markers; D012273:Ribulose-Bisphosphate Carboxylase / Q000235:genetics
chemical_list: D005819:Genetic Markers; C101625:RbcL protein, plastid; D012273:Ribulose-Bisphosphate Carboxylase