Methods

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顕微結晶法による地衣成分の観察 Microcrystal Tests for Lichen Substances

Specimens

地衣類標本：採集と保管 Lichen specimens: collection and preservation [Ohmura (2014), PDF 3.6MB]

DNA experiments

Primer sets for mycobiont

Target	Forward	Reverse	Reference	Note
ITS rDNA	ITS1F CTTGGTCATTTAGAGGAAGTAA	LR1 GGTTGGTTTCTTTTCCT	Gardes & Bruns (1993) Vilgalys & Hester (1990)	This primer set was first applied in Ohmura (2002).
ITS rDNA	USITS1F ACCTGCGGAAGGATCATTAC	USITS2R CTGTTGGTTTCTTTTCCT	Ohmura (2008)	This primer set is useful when ITS1F/LR1 does not work because of a long insertion at the end of 18S rDNA.
nrLSU	LR0R ACCCGCTGAACTTAAGC	LR5 ATCCTGAGGGAAACTTC	Vilgalys (unpublished) Vilgalys & Hester (1990)
mtSSU	mrSSU1 AGCAGTGAGGAATATTGGTC	mrSSU3R ATGTGGCACGTCTATAGCCC	Zoller et al. (1999)
MCM7	X-Mcm7-F CGTACACYTGTGATCGATGTG	X-Mcm7-R GTCTCCACGTATTCGCATTCC	Leavitt et al. (2011)
RPB1	gRPB1-AF GADTGTCCDGGDCATTTTGG	fRPB1-Cr CNGCDATNTCRTTRTCCATRTA	Stiller & Hall (1997) Matheny et al. (2002)
RPB2	fRPB2-5F GAYGAYMGWGATCAYTTYGG	bRPB2-7.1R CCCATRGCYTGYTTMCCCATDGC	Liu et al. (1999) Matheny (2005)

Primer sets for photobiont

Target	Forward	Reverse	Reference	Note
ITS rDNA	ITS1T GGAAGGATCATTGAATCTATCGT	LR1 GGTTGGTTTCTTTTCCT	Kroken & Taylor (2000) Vilgalys & Hester (1990)	This primer set was first applied in Ohmura et al. (2006).
rbcL	Chlor1400F ATGTCTCCACAAACTGAAAC	Chlor1400R ATTTAATTTCTTTCCAAACTTCACA	Ohmura et al. (2019)	This primer set is useful for Trebouxiophyceae. Sequencing primers include rbcLT630F, rbcLT1035R, Chlor1400F and Chlor1400R. rbcLT630: ATTTATGCGTTGGAGAGA rbcLT1035R: CGTCACGCATTAAATCAACG

References

Gardes, M. & Bruns, T. D. 1993. ITS primers with enhanced specificity for Basidiomycetes—application to the identification of mycorrhizae and rusts. Molecular Ecology 2: 113–118.
Kroken, S. & Taylor, J. W. 2000. Phylogenetic species, reproductive mode, and specificity of the green alga Trebouxia forming lichens with the fungal genus Letharia. The Bryologist 103: 645–660.
Leavitt, S. D., Johnson, L. & St Clair, L. L. 2011. Species delimitation and evolution in morphologically and chemically diverse communities of the lichen-forming genus Xanthoparmelia (Parmeliaceae, Ascomycota) in western North America. American Journal of Botany 98: 175–188.
Liu, Y. L., Whelen, S. & Hall, B. D. (1999). Phylogenetic relationships among ascomycetes: evidence from an RNA polymerase II subunit. Molecular Biology and Evolution 16: 1799-1808.
Matheny, P. B. 2005. Improving phylogenetic inference of mushrooms with RPB1 and RPB2 nucleotide sequences (Inocybe, Agaricales). Molecular Phylogenetics and Evolution 35: 1-20.
Matheny, P. B., Liu, Y. J., Ammirati, J. F. & Hall, B. D. 2002. Using RPB1 sequences to improve phylogenetic inference among mushrooms (Inocybe, Agaricales). American Journal of Botany 89(4): 688–698.
Ohmura, Y., 2002. Phylogenetic evaluation of infrageneric groups of the genus Usnea based on ITS regions in rDNA. Journal of the Hattori Botanical Laboratory 92 :231–243.
Ohmura, Y. 2008. Taxonomy and molecular phylogeny of Usnea rubicunda and U. rubrotincta (Parmeliaceae, lichenized Ascomycotina). Journal of Japanese Botany, 83: 347-355.
Ohmura, Y. 2014. Lichen specimens: collection and preservation. BryoString 2: 29-38.
Ohmura, Y., Kawachi, M., Kasai, F., Watanabe, M. M. & Takeshita, S. 2006. Genetic combinations of symbionts in a vegetatively reproducing lichen, Parmotrema tinctorum, based on ITS rDNA sequences. The Bryologist 109: 43–59.
Ohmura, Y., Takeshita, S. & Kawachi, M. 2019. Photobiont diversity within populations of a vegetatively reproducing lichen, Parmotrema tinctorum, can be generated by photobiont switching. Symbiosis 77: 59-72.
Stiller, J. W. & Hall, B. D. 1997. The origin of red algae: implications for plastid evolution. Proceedings of the National Academy of Sciences USA 94: 4520–4525.
Vilgalys, R. & Hester, M. 1990. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. Journal of Bacteriology 172: 4238–4246.
Zoller, S., Scheidegger, C. & Sperisen, C. 1999. PCR primers for the amplification of mitochondrial small subunit ribosomal DNA of lichen-forming ascomycetes. Lichenologist 31(5): 511–516.