Abstract
BackgroundPhalaris species (Poaceae) occupy diverse environments throughout all continents except Antarctica. Phalaris arundinacea is an important forage, ornamental, wetland restoration and biofuel crop grown globally as well as being a wetland invasive. The nuclear ribosomal internal transcribed spacer (ITS) region has been used for Phalaris barcoding as a DNA region with high nucleotide diversity for Phalaris species identification. Recent findings that P. arundinacea populations in Minnesota USA are most likely native and not European prompted this analysis to determine whether Eurasian vs. native North American P. arundinacea differed in ITS regions. Our objectives were to amplify and compare ITS regions (ITS1 and ITS2) of historic herbaria (1882–2001) and extant (fresh) Phalaris specimens; analyze ITS regions for species-specific polymorphisms (diagnostic SNPs) and compare ITS regions of historic Phalaris specimens with known, extant Phalaris species.ResultsWe obtained complete ITS1 and ITS2 sequences from 31 Phalaris historic (herbaria samples, 1908 to 2001) and five extant (fresh) specimens. Herbaria Phalaris specimens did not produce new SNPs (single nucleotide polymorphisms) not present in extant specimens. Diagnostic SNPs were identified in 8/12 (66.6%) Phalaris species. This study demonstrates the use of herbaria tissue for barcoding as a means for improved species identification of Phalaris herbaria specimens. No significant correlation between specimen age and genomic DNA concentration was found. Phalaris arundinacea showed high SNP variation within its clade, with the North American being distinctly different than other USA and most Eurasian types, potentially allowing for future identification of specific SNPs to geographic origin.ConclusionsWhile not as efficient as extant specimens to obtain DNA, Phalaris herbaria specimens can produce high quality ITS sequences to evaluate historic genetic resources and facilitate identification of new species-specific barcodes. No correlation between DNA concentration and age of historic samples (119 year range) occurred. Considerable polymorphism was exhibited in the P. arundinacea clade with several N. American accessions being distinct from Eurasian types. Further development of within species- and genus-specific barcodes could contribute to designing PCR primers for efficient and accurate identification of N. American P. arundinacea. Our finding of misidentified Phalaris species indicates the need to exercise stringent quality control measures on newly generated sequence data and to approach public sequence databases in a critical way.
Highlights
Phalaris species (Poaceae) occupy diverse environments throughout all continents except Antarctica
The objectives of this study were to: 1) amplify and compare internal transcribed spacer (ITS) regions (ITS1 and ITS2) of historic herbaria and extant Phalaris specimens; 2) analyze ITS regions for species-specific polymorphisms and 3) compare ITS regions of historic Phalaris specimens with known extant Phalaris species
For the majority of herbaria samples, the Optical Density (OD)260/280 values were within the expected range for high quality DNA (2.10 ± 0.79, mean ± standard deviation; Table 1) and the second measure of DNA purity (OD 260/230) had higher variation (1.64 ± 1.41; Table 1)
Summary
Phalaris species (Poaceae) occupy diverse environments throughout all continents except Antarctica. The nuclear ribosomal internal transcribed spacer (ITS) region has been used for Phalaris barcoding as a DNA region with high nucleotide diversity for Phalaris species identification. Numerous plant families, genera and species have been analyzed for variance in ITS sequence differences, for phylogenetic studies, species identification (barcoding) as well as ascertaining cultivar or genotype identities. Such is the case with the genus Phalaris L. which is an important forage, ornamental, birdseed, wetland remediation/restoration and biofuel crop grown across the globe as well as being recognized as an invasive wetland species [7,8,9]. A diploid ancestor of P. arundinacea came across on the land bridge of the Bering Strait into the present-day State of Alaska, USA [20] during the late Tertiary period [21]
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