Ecological Archives E095-129-A1

Leonie Färber, Knut Asbjørn Solhaug, Per-Anders Esseen, Wolfgang Bilger, Yngvar Gauslaa. 2014. Sunscreening fungal pigments influence the vertical gradient of pendulous lichens in boreal forest canopies. Ecology 95:1464–1471. http://dx.doi.org/10.1890/13-2319.1

Appendix A. Chlorophyll contents in pendulous lichens, including chlorophyll methods.

Mean initial chlorophyll content ± 1 SE (n = 10) for studied pendulous lichens.

Pigment

Species

Site

Total chlorophyll (mg g-1)

Usnic acid

Alectoria sarmentosa

Norway

0.59±0.10

 

Alectoria sarmentosa

Sweden

0.50±0.03

 

Usnea dasypoga

Norway

0.34±0.04

 

Usnea dasypoga

Sweden

0.83±0.10

 

Usnea longissima

Norway

0.33±0.04

Melanins

Bryoria capillaris

Sweden

1.11±0.06

 

Bryoria fremontii

Sweden

0.99±0.06

 

Bryoria fuscescens

Norway

0.58±0.10

 

Bryoria fuscescens

Sweden

0.95±0.09

Methods for chlorophyll quantification

Initial total chlorophyll was quantified in all species from all studied localities. Each thallus was ground in eppendorf tubes with a ball mill (Retsch MM 301, Germany). Ground material (15 mg) from each thallus was extracted in 1.5 mL MgCO3-saturated dimethyl sulphoxide (to avoid chlorophyll degradation in usnic lichens) at 60 °C for 40 min (Palmqvist and Sundberg 2002) and centrifuged. The Chl concentrations in the supernantant were quantified by absorbance measurements (649, 665, and 750 nm) in a Shimadzu UV-2101PC spectrophotometer using the formulae in Wellburn (1994).

However, a brown pigment was present in extracts from the three melanic lichens. To correct for this pigment, a new extraction was done from 5 new thalli of the melanic species as described above with additional filtration through a 45 µm filter. In this case, the extract was divided into two subsamples. One subsample (0.5 mL) was eluted through a Bond Elut (C18) column into a weighed eppendorf tube. Additional 1.5 mL DMSO were used to elute all brown pigment from the column, whereas chlorophylls and carotenoids were retained. The exact volume of DMSO was determined by weighing, and the absorbance of the brown extract measured at 649, 665, and 750 nm. These values were recalculated to a volume of 0.5 mL.

To validate this correction method, chlorophyll in the other subsamples was analyzed with HPLC (Niinemets et al. 1998). Here, the brown pigments did not interfere. Using the absorbance readings at 750 nm in the extracts, the species specific contribution of the brown pigment to the observed absorbances at 649 and 665 nm was calculated and subtracted. This procedure lead to chlorophyll contents which were very close to those determined by HPLC analysis. Therefore, the correction factors were used to adjust the earlier measured absorbances at 649 and 665 nm, and the chlorophyll content of the Bryoria species was computed again.

Literature Cited

Niinemets, Ü., W. Bilger, O. Kull, and J. D. Tenhunen. 1998. Acclimation to high irradiance in temperate deciduous trees in the field: changes in xanthophyll cycle pool size and in photosynthetic capacity along a canopy light gradient. Plant Cell and Environment 21:1205–1218.

Palmqvist, K., and B. Sundberg. 2002. Characterising photosynthesis and respiration in freshly isolated or cultured lichen photobionts. Pages 152–181 in I. Kranner, R. P. Beckett, and A. K. Varma, editors. Protocols in Lichenology. Culturing, Biochemistry, Ecophysiology and Use in Biomonitoring. Springer-Verlag, Berlin, Germany.

Wellburn, A. R. 1994. The spectral determination of chlorophyll a and chlorophhyll b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution. Journal of Plant Physiology 144:307–313.


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