Variability in ITS1 and ITS2 sequences of historic herbaria and extant (fresh) Phalaris species (Poaceae)

Allison L. Graper, Andrzej Noyszewski, Neil O. Anderson, Alan G. Smith

Research output: Contribution to journalArticlepeer-review

4 Scopus citations


Background: Phalaris 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. Results: We 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. Conclusions: While 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.

Original languageEnglish (US)
Article number515
JournalBMC plant biology
Issue number1
StatePublished - Dec 2021

Bibliographical note

Funding Information:
Funding for this research was from a University of Minnesota Undergraduate Research Opportunities Program Grant which paid for Allison Graper’s salary, limited lab supplies and the Minnesota Agricultural Experiment Station, MAES21–0045, which funded sequencing and greenhouse charges.

Funding Information:
We sincerely thank Drs. Timothy Whitfeld and Deborah Lewis for their time and work spent in preserving and providing the availability of the herbaria specimens. Furthermore, we thank the University of Minnesota Bell Museum Herbarium and Iowa State University Ada Hayden Herbarium for the institutional lending services provided.

Publisher Copyright:
© 2021, The Author(s).


  • DNA barcoding
  • Diagnostic SNPs
  • Herbarium specimens
  • PCR amplification


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