Journal of Plant Research

, Volume 130, Issue 2, pp 407–416 | Cite as

Detection of chlorophylls in spores of seven ferns

  • Mei-Hwei Tseng
  • Kuei-Huei Lin
  • Yi-Jia Huang
  • Ya-Lan Chang
  • Sheng-Cih Huang
  • Li-Yaung Kuo
  • Yao-Moan Huang
Regular Paper


Fern spores were traditionally classified into chlorophyllous (green) and nonchlorophyllous (nongreen) types based on the color visible to the naked eye. Recently, a third type, “cryptochlorophyllous spores”, is recognized, and these spores are nongreen under white light but contain chlorophylls. Epifluorescence microscopy was previously used to detect chlorophylls in cryptochlorophyllous spores. In addition to epifluorescence microscopy, current study performed some other approaches, including spore-squash epifluorescence, absorption spectra, laser-induced fluorescence emission spectra, thin layer chromatography (TLC), and ultra-high performance liquid chromatography with ultraviolet and mass spectrometric detection (UHPLC-UV-MS) in order to detect chlorophylls of spores of seven ferns (Sphaeropteris lepifera, Ceratopteris thalictroides, Leptochilus wrightii, Leptochilus pothifolius, Lepidomicrosorum buergerianum, Osmunda banksiifolia, and Platycerium grande). Destructive methods, such as TLC and UHPLC-UV-MS, successfully detected chlorophylls inside the spores when their signals of red fluorescence under epifluorescence microscope were masked by spore wall. Although UHPLC-UV-MS analysis was the most sensitive and reliable for determining the chlorophylls of spores, spore-squash epifluorescence is not only reliable but also cost- and time-effective one among our study methods. In addition, we first confirmed that Lepidomicrosorium buergerianum, Leptochilus pothifolius, Leptochilus wrightii, and Platycerium grande, produce cryptochlorophyllous spores.


Chlorophyll Cryptochlorophyllous spore Spore-squash epifluorescence UHPLC-UV-MS 



We thank Dr. Wen-Liang Chiou, Tzu-Tong Kao, and Daniel P. Chamberlin for fruitful discussions and English editing assistance, respectively. We also thank Hajime Ikeda and two anonymous reviews for providing constructive comments on the manuscript. This study was supported by the Ministry of Science and Technology, Taiwan (MOST 102-2313-B-845-001-MY3).

Supplementary material

10265_2016_901_MOESM1_ESM.pdf (72 kb)
Online resource 1 Spectrum of laser-induced fluorescence of Sphaeropteris lepifera spores (PDF 71 KB)
10265_2016_901_MOESM2_ESM.pdf (2.2 mb)
Online resource 2 LC UV ESI MS fragment ions of spore acetone extracts (PDF 2205 KB)


  1. Farrar DR (1976) Spore retention and release from overwintering fern fronds. Am Fern J 66:49–52. doi: 10.2307/1546518 CrossRefGoogle Scholar
  2. Farrar DR, Dassler CL, Watkins JEJ, Skelton C (2008) Gametophyte ecology. In: Ranker TA, Haufler CH (eds) Biology and Evolution of Ferns and Lycophytes. Cambridge University Press, Cambridge, pp 222–256CrossRefGoogle Scholar
  3. Hennipman E, Veldhoen P, Kramer KU, Price MG (1990) Polypodiaceae. In: Kramer KU, Green PS (eds) The families and genera of vascular plants. vol I: pteridophytes and gymnosperms. Springer, Berlin, pp 203–230Google Scholar
  4. Hill RH, Wagner WHJ (1974) Seasonality and spore type of the pteridophytes of Michigan. Mich Bot 13:40–44Google Scholar
  5. Hoff AJ, Amesz J (1991) Visible absorption spectroscopy of chlorophylls. In: Scheer H. (ed) Chlorophylls. CRC Press, Florida, pp 723–738Google Scholar
  6. Huang YM, Wong SL, Chiou WL (2003) The collection and storage of pteridophyte spores. Taiwan J For Sci 18:75–79Google Scholar
  7. Huang YM, Amoroso VB, Coritico FP, Ko CW, Kao TT, Callado JR, Chiou WL (2015) Reproductive biology of Aglaomorpha cornucopia (Copel.) M.C. Roos, A rare and endemic fern from the Philippines. Am Fern J 105:31–44CrossRefGoogle Scholar
  8. Jeffrey SW, Mantoura RFC, Wright SW (1997) Phytoplankton pigments in oceanography: guidelines to modern methods. UNESCO, ParisGoogle Scholar
  9. Lichtenthaler HK (1997) Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Meth Enzymol 148:350–382CrossRefGoogle Scholar
  10. Lichtenthaler HK, Buschmann C (2001) Chlorophylls and carotenoids: measurement and characterization by UV–VIS spectroscopy. In: Wrolstad RE, Acree TE, An H, Decker EA, Penner MH, Reid DS, Schwartz SJ, Shoemaker CF, Sporns P (eds) Current protocols in food analytical chemistry (CPFA). John Wiley and Sons, New York (F4.3.1–F4.3.8)Google Scholar
  11. Lichtenthaler HK, Buschmann C, Rinderle U, Schmuck G (1986) Application of chlorophyll fluorescence in ecophysiology. Radiat Environ Biophys 25:297–308CrossRefPubMedGoogle Scholar
  12. Lichtenthaler HK, Rinderle U (1988) The role of chlorophyll fluorescence in the detection of stress conditions in plants. Cri Rev Anal Chem 19:29–85CrossRefGoogle Scholar
  13. Lloyd RM, Klekowski EJ (1970) Spores germination and viability in pteridophyta: evolutionary significance of chlorophyllous spores. Biotropica 2:129–137CrossRefGoogle Scholar
  14. Maciel-Silva AS, da Silva FCL, Válio IFM (2014) All green, but equal? Morphological traits and ecological implications on spores of three species of mosses in the Brazilian Atlantic forest. Ann Brazilian Acad Sci 86:1249–1262CrossRefGoogle Scholar
  15. Raghavan V (1989) Developmental biology of fern gametophyte. Cambridge University Press, New YorkCrossRefGoogle Scholar
  16. Stokey AG (1951) Duration of viability of spores of the Osmundaceae. Am Fern J 41:111–115CrossRefGoogle Scholar
  17. Sundue M, Vasco A, Moran RC (2011) Cryptochlorophyllous spores in ferns: nongreen spores that contain chlorophyll. Int J Plant Sci 172:1110–1119CrossRefGoogle Scholar
  18. Talamond P, Verdeil JL, Conéjéro G (2015) Secondary metabolite localization by autofluorescence in living plant cells. Molecules 20:5024–5037CrossRefPubMedGoogle Scholar

Copyright information

© The Botanical Society of Japan and Springer Japan 2016

Authors and Affiliations

  • Mei-Hwei Tseng
    • 1
  • Kuei-Huei Lin
    • 1
  • Yi-Jia Huang
    • 1
  • Ya-Lan Chang
    • 2
  • Sheng-Cih Huang
    • 3
  • Li-Yaung Kuo
    • 4
  • Yao-Moan Huang
    • 4
  1. 1.Department of Applied Physics and ChemistryUniversity of TaipeiTaipeiTaiwan
  2. 2.Dr. Cecilia Koo Botanic Conservation CenterPingtungTaiwan
  3. 3.Department of Applied ChemistryNational Chiao Tung UniversityHsinchuTaiwan
  4. 4.Division of SilvicultureTaiwan Forestry Research InstituteTaipeiTaiwan

Personalised recommendations