The DNA barcode reference library for the mosses (Bryophyta) of Canada KUZMINA MARIA L. , JENNIFER C. DOUBT, CATHERINE LA FARGE, JUAN CARLOS VILLARREAL, AND PAUL N. D. HEBERT

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Abstract Background: Mosses (Bryophyta) are sensitive indicators of environmental quality and play a key role in the nutrient cycle in boreal and arctic ecosystems (Turetsky et al., 2012). However, their subtle morphology often makes species determinations challenging, even for specialists. To advance the identification of mosses a comprehensive and verified reference DNA barcode reference library is required. As mosses were not a key group in driving selection of the standard DNA barcodes (rbcL, matK) for land plants (CBOL, 2009), a search for a replacement marker was required (Liu et al., 2010; Stech and Quandt, 2010). Results: The plastid-encoded trnL(UAA) intron, a widely-adopted marker for mosses due to its universality and high variability, is a promising second DNA barcode for mosses. We tested sequence recovery and species resolution with rbcL and trnL(UAA) for 770 Canadian moss species, about three quarters of the Canadian flora. Samples of nearly 2000 specimens from the herbarium at the Canadian Museum of Nature (CANM) were analyzed at the Center for Biodiversity Genomics (CBG). Standard protocols recovered 94% of the species for both markers. The complementary resolution power for rbcL and trnL(UAA), allowed the discrimination of all genera and about 60% of the species examined, with some variation among orders (Sphagnales 32%, Splachnales 100%). Nearly 5% of the specimens were re-identified after reexaminations provoked by the barcode results. The barcode data revealed 15 cases of deep intraspecific variation, suggesting the presence of cryptic species. Significance: DNA barcoding will provide critical insight into the alpha and beta taxonomy of Canadian mosses and improve evaluation of both herbarium specimen determinations and species variation. The current reference sequence library is ready for use in the identification of bulk moss samples gathered in ecological surveys, for eDNA detection, ancient DNA and as a baseline resource for the molecular identification of Canadian mosses.

2000 specimens

770 species

~ 3 records per species

Species resolution (green and orange bars) with two DNA markers (overall 60%)

rbcL trnL(UAA)

DNA marker Specimens Species

rbcL 84% 94%

trnL(UAA) 85% 94%

Source location of the specimens and sequence recovery

Age of specimens

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Overall sequence recovery

Species resolution in the species-rich orders

References CBOL WORKING GROUP. 2009. A DNA barcode for land plants. Proceedings of the National Academy of Sciences of the United States of America 106: 12794–12797. doi: 10.1073/pnas.0905845106. LIU Y., H-F. YAN, T. CAO, AND X-J. GE. 2010. Evaluation of 10 plant barcodes in Bryophyta (Mosses). Journal of Systematics and Evolution. 48: 36–46. STECH, M., AND D. QUANDT. 2010. 20,000 species and five key markers: the status of molecular bryophyte phylogenetics. Phytotaxa. 9: 196–228. TURETSKY, M. R., B. BOND-LAMBERTY, E. EUSKIRCHEN, J. TALBOT, S. FROLKING, A. D. MCGUIRE, AND E-S. TUITTILA. 2012. The resilience and functional role of moss in boreal and arctic ecosystems. New Phytologist. 196: 49–67.

rbcL trnL(UAA) rbcL & trnL(UAA)

rbcL trnL(UAA)