, Volume 77, Issue 1, pp 59–72 | Cite as

Photobiont diversity within populations of a vegetatively reproducing lichen, Parmotrema tinctorum, can be generated by photobiont switching

  • Yoshihito OhmuraEmail author
  • Shunji Takeshita
  • Masanobu Kawachi


Photobiont diversity within populations of a vegetatively reproducing lichen can be generated by photobiont switching between the original lichen photobiont and the compatible algal partners on its surrounding substrate. The hypothesis was tested using Denaturing Gradient Gel Electrophoresis (DGGE) with a partial rbcL sequence amplified from thalli of Parmotrema tinctorum and from the substrate immediately adjacent to each thallus. On the surface of tombstones where P. tinctorum was growing, only various haplotypes of Trebouxia corticola (s. lat.) that is characterized by having distinct starch sheaths surrounding the pyrenoid were detected. DGGE could detect one to five bands of T. corticola (s. lat.) haplotypes on each substrate, and one (or rarely two) of them was often identical with the photobiont haplotype of P. tinctorum growing on the same tombstone. Through PCR screening directed at a fungal rDNA fragment, many substrate samples were found to be free of microscopic contamination from P. tinctorum. Individual algal haplotypes from the substrate were identified by sequencing of the DGGE rbcL bands and compared to the corresponding sequences of the P. tinctorum photobionts. The presence of compatible algae on the lichen substrate and the genetic identity between some of the substrate algae and those in the lichen suggest the possibility of photobiont switching in vegetatively reproducing lichens like P. tinctorum. The following observations also support the phenomenon of photobiont switching: 1) high genetic diversity of photobiont in small populations; 2) multiple photobionts in a single thallus; 3) incomplete correspondence in co-phylogenetic analyses between mycobiont and photobiont; and 4) clear selectivity for photobiont in diverse lichens.


Lichenized fungi Microalgae rbcTrebouxia corticola Selectivity Vegetative reproduction 



We would like to express our sincere gratitude to Larry St. Clair (Brigham Young University) and anonymous reviewers for their valuable constructive comments and linguistically revising this manuscript. We also wish to thank S. Handa (Hiroshima Environment and Health Association) for providing valuable information about aerial algae.


  1. Ahmadjian V (1960) Some new and interesting species of Trebouxia, a genus of lichenized algae. Am J Bot 47:677–683CrossRefGoogle Scholar
  2. Ahmadjian V (1987) Coevolution in lichens. Ann N Y Acad Sci 503:307–315CrossRefGoogle Scholar
  3. Ahmadjian V (1988) The lichen alga Trebouxia: does it exist free living? Plant Syst Evol 158:243–247CrossRefGoogle Scholar
  4. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucl Acids Res 25:3389–3402CrossRefGoogle Scholar
  5. Archibald PA (1975) Trebouxia de Puymaly (Chlorophyceae, Chlorococcales) and Pseudotrebouxia gen. Nov. (Chlorophyceae, Chlorosarcinales). Phycologia 14:125–137CrossRefGoogle Scholar
  6. Beck A (1999) Photobiont inventory of a lichen community growing on heavy-metal-rich rock. Lichenologist 31:501–510CrossRefGoogle Scholar
  7. Beck A, Friedl T, Rambold G (1998) Selectivity of photobiont choice in a defined lichen community: inferences from cultural and molecular studies. New Phytol 139:709–720CrossRefGoogle Scholar
  8. Beck A, Kasalicky T, Rambold G (2002) Myco-photobiontal selection in a Mediterranean cryptogam community with Fulgensia fulgida. New Phytol 153:317–326CrossRefGoogle Scholar
  9. Bischoff H, Bold HC (1963) Some soil algae from enchanted rock and related algal species. Phycological studies IV. Univ Texas Pub 6318:1–95Google Scholar
  10. Blaha J, Baloch E, Grube M (2006) High photobiont diversity associated with the euryoecious lichen-forming ascomycete Lecanora rupicola (Lecanoraceae, Ascomycota). Biol J Linnean Soc 99:283–293CrossRefGoogle Scholar
  11. Bubrick P, Galun M, Frensdorff A (1984) Observations on free-living Trebouxia DePuymaly and Pseudotrebouxia Archibald, and evidence that both symbionts from Xanthoria parietina (L.) Th. Fr. Can be found free-living in nature. New Phytol 97:455–462CrossRefGoogle Scholar
  12. del Campo EM, Grimeno J, De Nova JPG, Barreno LE (2010) South European populations of Ramalina farinacea (L.) ach. Share different Trebouxia algae. Bibl Lichenol 105:247–256Google Scholar
  13. Dahlkild Å, Källersjö M, Lohtander K, Tehler A (2001) Photobiont diversity in the Physciaceae (Lecanorales). Bryologist 104:527–536CrossRefGoogle Scholar
  14. Dal Grande F, Alors D, Divakar PK, Balint M, Crespo A, Schmitt I (2014) Insights into intrathalline genetic diversity of the cosmopolitan lichen symbiotic green algae Trebouxia decolorans Ahmadjian using microsatellites markers. Molec Phylogen Evol 72:54–60CrossRefGoogle Scholar
  15. DePriest PT (2004) Early molecular investigations of lichen-forming symbionts: 1986-2001. Annual Rev Microbiol 58:273–301CrossRefGoogle Scholar
  16. Fischer SG, Lerman LS (1983) DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels: correspondence with melting theory. Proc Natl Acad Sci U S A 80:1579–1583CrossRefGoogle Scholar
  17. Friedl T (1987) Thallus development and phycobionts of the parasitic lichen Diploschistes muscorum. Lichenologist 19:183–191CrossRefGoogle Scholar
  18. Friedl T (1989) Systematik und Biologie von Trebouxia (Microthamniales, Chlorophyta) als Phycobiont der Parmeliaceae (lichenisierte Ascomyceten). Universität Bayreuth, Bayreuth, GermanyGoogle Scholar
  19. Friedl T, Gärtner G (1988) Trebouxia (Pleurastrales, Chlorophyta) as a phycobiont in the lichen genus Diploschistes. Arch Protistenkd 135:147–158CrossRefGoogle Scholar
  20. Gardes M, Bruns TD (1993) ITS primers with enhanced specificity for basidiomycetes –application to the identification of mycorrhizae and rusts. Molec Ecol 2:113–118CrossRefGoogle Scholar
  21. Gärtner G (1985) Die Gattung Trebouxia Puymaly (Chlorellales, Chlorophyceae). Arch Hydrobiol Suppl 74:495–548Google Scholar
  22. Handa S, Nakahara M, Nakano T, Itskovich VB, Masuda Y (2001) Aerial algae from southwestern area of Lake Baikal. Hikobia 13:463–472Google Scholar
  23. Handa S, Nakano T, Takeshita S (1991) Some corticolous algae from Shibetsu, Hokkaido, northern Japan. J Jpn Bot 66:211–223Google Scholar
  24. Handa S, Ohmura Y, Nakano T, Nakahara-Tsubota M (2007) Airborne micro green algae (Chlorophyta) in snowfall. Hikobia 15:109–120Google Scholar
  25. Hecker KH, Roux KH (1996) High and low annealing temperatures increase both specificity and yield in touchdown and stepdown PCR. BioTechniques 20:478–485CrossRefGoogle Scholar
  26. Helms G, Friedl T, Rambold G, Mayrhofer H (2001) Identification of photobionts from the lichen family Physciaceae using algal-specific ITS rDNA sequencing. Lichenologist 33:73–86CrossRefGoogle Scholar
  27. Hildreth KC, Ahmadjian V (1981) A study of Trebouxia and Pseudotrebouxia isolates from different lichens. Lichenologist 13:65–86CrossRefGoogle Scholar
  28. Huelsenbeck JP, Ronquist F (2001) MrBayes: Bayesian inference of phylogenetic trees. Bioinformatics 17:754–755CrossRefGoogle Scholar
  29. Jensen MA, Straus N (1993) Effect of PCR conditions on the formation of heteroduplex and single-stranded DNA products in the amplification of bacterial ribosomal DNA spacer regions. PCR Meth Appl 3:186–194CrossRefGoogle Scholar
  30. Kasai F, Kawachi M, Erata M, Watanabe MM (2004) NIES-Collection. List of strains, seventh edition, 2004, microalgae and protozoa. Tsukuba, Japan: National Institute for Environmental StudiesGoogle Scholar
  31. Kon Y, Kashiwadani H (2005) Lobule formation from isidia in Parmotrema tinctorum. Bull. Natl. Sci. Mus., Ser. B 31:127–131Google Scholar
  32. Kopczynski ED, Bateson MM, Ward DM (1994) Recognition of chimeric small-subunit ribosomal DNAs composed of genes from uncultivated microorganisms. Appl Environm Microbiol 60:746–748Google Scholar
  33. Kroken S, Taylor JW (2000) Phylogenetic species, reproductive mode, and specificity of the green alga Trebouxia forming lichens with the fungal genus Letharia. Bryologist 103:645–660CrossRefGoogle Scholar
  34. Larget B, Simon D (1999) Markov chain Monte Carlo algorithms for the Bayesian analysis of phylogenetic trees. Molec Biol Evol 16:750–759CrossRefGoogle Scholar
  35. Mansournia MR, Wu B, Matsushita M, Hogetsu T (2012) Genotypic analysis of the foliose lichen Parmotrema tinctorum using microsatellite markers: association of mycobiont and photobiont, and their reproductive modes. Lichenologist 44:419–440CrossRefGoogle Scholar
  36. Moya P, Molins A, Martinez-Alberola F, Muggia L, Barreno E (2017) Unexpected associated microalgal diversity in the lichen Ramalina farinacea is uncovered by pyrosequencing analyses. PLoS One 12:e0175091CrossRefGoogle Scholar
  37. Muggia L, Vancurova L, Skaloud P, Peksa O, Wedin M, Grube M (2013) The symbiotic playground of lichen thalli – a highly flexible photobiont association in rock-inhabiting lichens. FEMS Microbiol Ecol 85:313–323CrossRefGoogle Scholar
  38. Mukhtar A, Garty J, Galun M (1994) Does the lichen alga Trebouxia occur free-living in nature: further immunological evidence. Symbiosis 17:247–253Google Scholar
  39. Muyzer G, de Waal EC, Uitterlinden AG (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environm Microbiol 59:695–700Google Scholar
  40. Nagamine CM, Chan K, Lau YF (1989) A PCR artifact: generation of heteroduplexes. Am J Hum Genet 45:337–339Google Scholar
  41. Nash TH (2008) Lichen biology, 2nd edn. Cambridge University Press, New YorkCrossRefGoogle Scholar
  42. Nelsen MP, Gargas A (2009) Symbiont flexibility in Thamnolia vermicularis (Pertusariales: Icmadophilaceae). Bryologist 112:404–417CrossRefGoogle Scholar
  43. Ohmura Y, Kawachi M, Kasai F, Watanabe MM, Takeshita S (2006) Genetic combinations of symbionts in a vegetatively reproducing lichen, Parmotrema tinctorum, based on ITS rDNA sequences. Bryologist 109:43–59CrossRefGoogle Scholar
  44. Opanowicz M, Grube M (2004) Photobiont genetic variation in Flavocetraria nivalis from Poland (Parmeliaceae, lichenized Ascomycota). Lichenologist 36:125–131CrossRefGoogle Scholar
  45. Orita M, Iwahana H, Kanazawa H, Hayashi K, Sekiya T (1989) Detection of polymorphisms of human DNA by gel electrophoresis as single-strand conformation polymorphisms. Proc Natl Acad Sci U S A 86:2766–2770CrossRefGoogle Scholar
  46. Ott S (1987) Sexual reproduction and developmental adaptations in Xanthoria parietina. Nordic J Bot 7:219–228CrossRefGoogle Scholar
  47. Piercey-Normore MD (2006) The lichen-forming ascomycete Evernia mesomorpha associates with multiple haplotypes of Trebouxia jamesii. New Phytol 169:331–344CrossRefGoogle Scholar
  48. Piercey-Normore MD (2009) Vegetatively reproducing fungi in three genera of the Parmeliaceae show divergent algal partners. Bryologist 112:773–785CrossRefGoogle Scholar
  49. Piercey-Normore MD, DePriest PT (2001) Algal switching among lichen symbionts. Am J Bot 88:1490–1498CrossRefGoogle Scholar
  50. Posada D, Crandall KA (1998) Modeltest: testing the model of DNA substitution. Bioinformatics 14:817–818CrossRefGoogle Scholar
  51. Puymaly A (1924) Le Chlorococcum humicola (Näg.) Rabenh. Rev Algol 1:107–114Google Scholar
  52. Rambold G, Friedl T, Beck A (1998) Photobionts in lichens: possible indicators of phylogenetic relationships? Bryologist 101:392–397CrossRefGoogle Scholar
  53. Romeike J, Friedl T, Hems G, Ott S (2002) Genetic diversity of algal and fungal partners in four species of Umbilicaria (lichenized ascomycetes) along a transect of the Antarctic peninsula. Molec Biol Evol 19:1209–1217CrossRefGoogle Scholar
  54. Rozen S, Skaletsky HJ (2000) “Primer3 on the WWW for general users and for biologist programmers” in Bioinformatics Methods and Protocols: Methods in Molecular Biology, eds Krawetz, S, and Misener, S. (Totowa, NJ, USA: Humana press), pp 365–386Google Scholar
  55. Škaloud P, Moya P, Molins A, Peska O, Santos-Guerra A, Barreno E (2018) Untangling the hidden intrathalline microalgal diversity in Parmotrema pseudotinctorum: Trebouxia crespoana sp. nov. Lichenologist 50:357–369CrossRefGoogle Scholar
  56. Speksnijder AGCL, Kowalchuk GA, de Jong S, Kline E, Stephen JR, Laanbroek HJ (2001) Microvariation artifacts introduced by PCR and cloning of closely related 16S rRNA gene sequences. Appl Environm Microbiol 67:469–472CrossRefGoogle Scholar
  57. Swofford DL (2002) PAUP*: phylogenetic analysis using parsimony (*and other methods). Version 4.0b10. Sunderland, MA, USA: Sinauer associatesGoogle Scholar
  58. Takeshita S (2001) A taxonomic revision of the genus Trebouxia (Trebouxiophyceae, Chlorophyta). Hikobia 13:425–455Google Scholar
  59. Tamura K, Nei M (1993) Estimation of the number of nucleotide substitutions in the control region of mitochondrial DNA in humans and chimpanzees. Molec Biol Evol 10:512–526Google Scholar
  60. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucl Acids Res 22:4673–4680CrossRefGoogle Scholar
  61. Thompson JR, Marcelino LA, Polz MF (2002) Heteroduplexes in mixed-template amplifications: formation, consequence and elimination by ‘reconditioning PCR’. Nucl Acids Res 30:2083–2088CrossRefGoogle Scholar
  62. Tomar RS (2010) Molecular markers and plant biotechnology. New India PublishingGoogle Scholar
  63. Tschermak-Woess E (1978) Myrmecia reticulate as a phycobionts and free-living Trebouxia – the problem of Stenocybe septata. Lichenologist 10:69–79CrossRefGoogle Scholar
  64. Vilgalys R, Hester M (1990) Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. J Bacteriol 172:4238–4246CrossRefGoogle Scholar
  65. Wornik S, Grube M (2010) Joint dispersal does not imply maintenance of partnerships in lichen symbioses. Microb Ecol 59:150–157CrossRefGoogle Scholar
  66. Yahr R, Vilgalys R, Depriest PT (2004) Strong fungal specificity and selectivity for algal symbionts in Florida scrub Cladonia lichens. Molec Ecol 13:3367–3378CrossRefGoogle Scholar
  67. Yoshinaga K, Ohta T, Suzuki Y, Sugiura M (1988) Chlorella chloroplast DNA sequence containing a gene for the large subunit of ribulose-1,5-biphosphate carboxylase/oxygenase and a part of a possible gene for the beta′ subunit of RNA polymerase. Pl Molec Biol 10:245–250CrossRefGoogle Scholar
  68. Zoller S, Lutzoni F (2003) Slow algae, fast fungi: exceptionally high nucleotide substitution rate differences between lichenized fungi Omphalina and their symbiotic green algae Coccomyxa. Molec Phylogen Evol 29:629–640CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.National Museum of Nature and ScienceIbarakiJapan
  2. 2.Hiroshima UniversityHiroshimaJapan
  3. 3.National Institute for Environmental StudiesIbarakiJapan

Personalised recommendations