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(American Journal of Botany. 1999;86:753-757.)
© 1999 Botanical Society of America, Inc.

Genetic variation of Usnea filipendula (Parmeliaceae) populations in western Germany investigated by RAPDs suggests reinvasion from various sources1

Esther Heibel, H. Thorsten Lumbsch2 and Imke Schmitt

Botanisches Institut, Universität Essen, D-45117 Essen, Germany

Received for publication October 8, 1998. Accepted for publication January 22, 1999.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 LITERATURE CITED
 
Random amplified polymorphic DNA (RAPD) markers were characterized for 25 specimens of Usnea filipendula to evaluate the genetic diversity of populations reinvading formerly uninhabited regions in Northrhine-Westphalia due to decreasing sulfur dioxide levels. With six 10-mer randomly amplified polymorphic DNA (RAPD) primers, a 66 character by 25 specimens matrix was generated. Phenetic analysis (UPGMA) showed no obvious groupings. The reinvading populations are distributed over the phenogram and are not genetically closely related. The results suggest that the reinvading populations of this usually sterile species are derived from different sources and do not consist of a particular clone capable of re-entering the area.

Key Words: air pollution • bioindication • genetic variation • lichens • Parmeliaceae • random amplified polymorphic DNA (RAPD) • Usnea.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 LITERATURE CITED
 
The sensitivity of lichens to air pollution, especially sulfur dioxide, is well known and has been discussed in numerous review papers (e.g., Skye and Hallberg, 1969 ; Ferry, Baddeley and Hawksworth, 1973 ; LeBlanc and Rao, 1975 ; Nash and Wirth, 1988 ; Nash and Gries, 1991 ). Lichens have been used extensively in active and passive biomonitoring to evaluate air quality. Countless studies, listed in the series "Literature on air pollution and lichens" started by Hawksworth (1974) give evidence of this. Subsequent to recent decreases in sulfur dioxide levels, especially in Europe and North America, a reinvasion of lichens in formerly heavily polluted areas has been documented (e.g., Henderson-Sellers and Seaward, 1979 ; Hafellner and Grill, 1981 ; Rose and Hawksworth, 1981 ; Brightman and Seaward, 1983 ; Rabe and Wiegel, 1985 ; Gilbert, 1992 ; Showman, 1997 ). However, no data are available on the genetic diversity of these reinvading populations. It is not known, for instance, whether only specific clones of the species are capable of reinvading urban areas.

During a study by E. Heibel on the biodiversity of lichenized fungi in Northrhine Westphalia in northwestern Germany, the question arose of how the genetic diversity of the reinvading populations could be assessed. The formerly heavily industrialized Ruhr area lies within Northrhine Westphalia and data of the poor lichen flora of 30 yr ago are available (Domrös, 1966 ). Further, a reinvasion of different groups of macro- and microlichens in the area has been observed (Rabe and Wiegel, 1985 ; Heibel, unpublished data). Thus, this region appears to be an ideal place for investigating the genetic diversity of reinvading lichen populations using molecular methods.

Molecular studies to date in lichenology are primarily concerned with phylogenetic problems (e.g., Armaleo and Clerc, 1991 ; DePriest, 1993 , 1995 ; Gargas et al., 1995 ; Lutzoni and Vilgalys, 1995a , b ; Tehler, 1995 ; Groner and LaGreca, 1997 ). These methods have not yet been applied in lichen ecology. We chose a "randomly amplified polymorphic DNA-polymerase chain reaction" RAPD-PCR method since we were interested in an overall picture of the genetic variability of the invading populations. RAPD analysis has not been used extensively in lichenized fungi, mainly because RAPD primers are not fungal-specific and thus amplify all kinds of DNA. An examination of lichen thalli or apothecia containing symbiotic algae could lead to uninformative results, since the DNA of both bionts would be amplified. To avoid these complications we used Usnea filipendula as the object of investigation, the most common beard lichen in the area studied. In the genus Usnea, the algal-free central axis is easily prepared and can be used for RAPD-PCR without the risk of being contaminated by algal DNA.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSION
 LITERATURE CITED
 
Lichen material
Twenty-five specimens of Usnea filipendula were studied. All specimens examined were chemically studied using gradient elution high-performance liquid chromatography (Feige et al., 1993 ) to verify the determination of the sometimes small samples. A list of specimens examined is found on Table 1; the positions of the German localities are shown in Fig. 1. The material was collected in the formerly heavily polluted areas of the Ruhr area (G, H), the Niederrhein plain (A-F), and the surroundings of Cologne (U). In these three areas only very small specimens were found and due to the absence of this species in previous mapping studies, these specimens can be regarded as recent recolonizations. Material from the mountain areas of the Eifel (V) and the Sauerland (I-T) near to the Ruhr area consisted of small to medium large specimens and may belong to both recently reinvading and relictal persisting populations. Three samples (specimens W-Y) from the Austrian Alps were included in this study as comparative material from an undisturbed habitat.


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  		Table 1. List of specimens examined.

 


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  		Fig. 1. Map showing the origin of the German samples examined.

 
RAPD analysis
The algal-free central axis of Usnea filipendula specimens was carefully prepared and used for the examination. Transverse sections of the central axis (20–30 µm thick) were obtained using a freezing microtome and transferred directly into PCR tubes, as described in Wolinski, Blanz, and Grube (1997) . No isolation of the DNA was performed. We compared parallel experiments with and without using microwaves as proposed by Ferreira and Glass (1996) or Wolinski, Blanz, and Grube (1997) , but since no differences were found, the application of microwaves was skipped. Ready-to-Go RAPD Analysis Beads (Pharmacia Biotech, Freiburg) were used in which, beside the microtome slices of the central axis, only the corresponding primer and sterile distilled water needed to be added. Six 10-mer RAPD primers were used (Table 2). Amplification in a total volume of 25 µL was performed in a robocycler (Stratagene, La Jolla, California) as follows: initial denaturation for 6 min at 95°C, then 40 cycles of 1.3 min at 95°C, 1.5 min at 35°C, and 2.3 min at 72°C. Amplification products were separated by electrophoresis on agarose gels in 1 x Tris-acetic acid—EDTA (TAE) buffer, stained with ethidium bromide and visualized under UV light. For each gel, a 250-bp ladder (Pharmacia, Freiburg) was also run to serve as a size marker. Gels were photographed over a UV transilluminator using a Polaroid camera.


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  		Table 2. Sequences of RAPD primers used in this study and numbers of bands observable.

 
Data analysis
For the analysis the relative density of bands was not considered and the presence or absence of bands was coded in binary (0, 1) form in a data matrix (Table 3). Relationships among individuals were determined by a distance matrix method. The approach involved calculation of the Nei and Li (1979) index of genetic similarity. This coefficient scores the number of common bands divided by the total number of bands found in the two samples being compared. Distance values were subjected to phenetic analyses using the unweighted pair group method with arithmetic mean (UPGMA) (Michener and Sokal, 1957 ) as implemented in the UPGMA program of the PAUP* software package, which was used in a test version 4.0d64 written by David L. Swofford.


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  		Table 3. The data matrix used in the analysis.

 

   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
LITERATURE CITED
 
All six 10-mer primers used gave interpretable and repeatable results and a total of 66 different RAPD markers were generated from the 25 Usnea filipendula specimens (Table 3). The numbers of amplified RAPD markers varied, depending on the primer and specimen combination. The numbers of bands observable in the specimens examined with each primer are indicated on Table 2.

Usnea filipendula is usually a sterile species, which is dispersed by vegetative propagules. The species develops apothecia and reproduces sexually only in well-developed thalli from undisturbed areas. All specimens collected in this study were sterile. Thus it could be expected that the genetic variability of the specimens may be low among the reinvading populations, if they were derived from a single source. However, a phenogram generated from distance values using the UPGMA method (Fig. 2) showed no obvious clustering of the reinvading specimens. A similar phenogram topology was also obtained using the neighbor-joining method (Saitou and Nei, 1987 ) (phenogram not shown).



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  		Fig. 2. Phenogram clustered by the UPGMA method showing the genetic similarity of Nei and Li of 25 Usnea filipendula specimens (Aus = Austrian sample; Col = Cologne sample; Eif = Eifel sample; NiP = Niederrhein plain sample; RuA = Ruhr area sample; SL = Sauerland sample).

 
Only two groupings have a bootstrap support over 50%. These are Usnea P and S collected in the Sauerland. However, the bootstrap support is not high with only 59%. The only group with a high bootstrap support (86%) includes three specimens from the Austrian Alps (Usnea W, X, Y), which were included in the study for comparison with samples from an undisturbed area. The Austrian specimens were collected in close localities at different altitude and show that natural populations of this only facultatively fertile species are genetically close. It is interesting to note that this group is very similar to a reinvading specimen (E) collected in the Niederrhein plains.

The collections made in the Sauerland may consist of a mixture of old persisting populations and recently reinvaded material as suggested by the different size of the thalli, as mentioned above. This is also reflected in the genetic variability of the Sauerland collections. While some (I–N) samples form a cluster of genetically related specimens, others, such as samples Q, R, or T can be found isolated from each other in the phenogram and may represent recently invaded populations.

The recently invading specimens do not cluster together, but are distributed over the phenogram. Material collected in the Niederrhein plains can be found at five different positions. While two samples (D, F) seem to be related to specimens collected in the Sauerland (I–N, R) and the Eifel (V), another (E) clusters together with the Austrian material. The other Niederrhein specimens group together with other reinvading populations (A–C) collected in the surroundings of Cologne (U) or the Ruhr area (G). One sample (H) collected in the Ruhr area does not seem to be genetically similar to any other of the specimens examined.

The results of this examination indicate that the formerly heavily polluted areas in the area studied are reinvaded by genetically heterogeneous populations. This suggests, especially in a usually sterile species, that the reinvading populations are derived from different sources and do not consist of particular clones capable of reinvading urban areas. More data are necessary to trace probable locations of the sources of the reinvading populations.

The present investigation shows that the RAPD-PCR technique can be used in ecological studies on lichen-forming fungi and is eligible for a wider application considering its low costs and convenient execution. We now wish to extend our examinations including the genetic variability of toxitolerant lichens, such as Amandinea punctata. In this manner we hope to reach a better understanding of the influence of air pollution on the genetic diversity of lichenized ascomycetes, which are model organisms for assessing the influence of air pollution on biodiversity.


   FOOTNOTES
 
1 The authors thank Ullrich Abts (Krefeld), Dr Bruno Mies (Düsseldorf), Uwe Raabe (Marl), and Professor Roman Türk (Salzburg) for providing us with fresh lichen material. We are grateful to Professor Benno Feige (Essen) for comments on an earlier draft of this manuscript. Permission by David L. Swofford (Washington, D.C.) to use a test version of the computer program PAUP* is gratefully acknowledged. This study was supported by the Deutsche Forschungsgemeinschaft (DFG) by a ‘Heisenbergstipendium’ to H.T. Lumbsch for which we are appreciative. Back

2 Author for correspondence. Back


   LITERATURE CITED
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
 LITERATURE CITED
 
Armaleo, D., and P. Clerc. 1991 Lichen chimeras: DNA analysis suggests that one fungus forms two morphotypes. Experimental Mycology 15: 1–10.

Brightman, F. H., and M. R. D. Seaward. 1983 Notes on the bryophytes and lichens: Ruxley Gravel Pit. Transactions of the Kent Field Club 9: 101–102.

DePriest, P. T. 1993 Molecular innovations in lichen systematics: the use of ribosomal and intron nucleotide sequences in the Cladonia chlorophaea complex. Bryologist 96: 314–325.[CrossRef][ISI]

———. 1995 Phylogenetic analyses of the variable ribosomal DNA of the Cladonia chlorophaea complex. Cryptogamic Botany 5: 60–70.

Domrös, M. 1966 Luftverunreinigung und Stadtklima im Rheinisch-Westfälischen Industriegebiet und ihre Auswirkung auf den Flechtenbewuchs der Bäume. Arbeiten zur Rheinischen Landeskunde [Geographisches Institut der Universitat Bonn] 23: 1–132.

Feige, G. B., H. T. Lumbsch, S. Huneck, and J. A. Elix. 1993 Identification of lichen substances by a standardized high-performance liquid chromatographic method. Journal of Chromatography 646: 417–427.[CrossRef]

Ferreira, A. V. B., and N. L. Glass. 1996 PCR from fungal spores after microwave treatment. Fungal Genetics Newsletter 43: 25–26.

Ferry, B., M. Baddeley, and D. L. Hawksworth [eds.]. 1973 Air pollution and lichens. University of Toronto Press, Toronto.

Gargas, A., P. T. DePriest, M. Grube, and A. Tehler. 1995 Multiple origins of lichen symbioses in fungi suggested by SSU rDNA phylogeny. Science 268: 1492–1495.[Abstract/Free Full Text]

Gilbert, O. L. 1992 Lichen reinvasion with declining air pollution. In J.W. Bates and A. M. Farmer [eds.], Bryophytes and lichens in a changing environment, 159–177. Clarendon, Oxford.

Groner, U., and S. LaGreca. 1997 The ‘Mediterranean’ Ramalina panizzei north of the Alps: morphological, chemical and rDNA sequence data. Lichenologist 29: 441–454.[ISI]

Hafellner, J., and D. Grill. 1981 Der Einfluss der Stillegung einer Zellstoffabrik auf die Vegetation der Umgebung. Phyton [Austria] 21: 25–38.

Hawksworth, D. L. 1974 Literature on air pollution and lichens I. Lichenologist 6: 122–125.[CrossRef]

Henderson-Sellers, A., and M. R. D. Seaward. 1979 Monitoring lichen reinvasion of ameliorating environments. Environmental Pollution 19: 207–213.[CrossRef]

Hillis, D. M., C. Moritz, and B. K. Mable [eds.]. 1996 Molecular systematics, 2nd ed. Sinauer, Sunderland, MA.

LeBlanc, F., and D. N. Rao. 1975 Effects of air pollutants on lichens and bryophytes. In J. B. Mudd and T. T. Kozlowski [eds.], Responses of plants to air pollution, 237–272. Academic Press, New York, NY.

Lutzoni, F., and R. Vilgalys. 1995a Integration of morphological and molecular data sets in estimating fungal phylogenies. Canadian Journal of Botany 73 (Supplement 1): S649–S659.

———, and R. Vilgalys. 1995b Omphalina (Basidiomycota, Agaricales) as a model system for the study of coevolution in lichens. Cryptogamic Botany 5: 71–81.

Michener, C. D., and R. R. Sokal. 1957 A quantitative approach to a problem in classification. Evolution 11: 130–162.[CrossRef][ISI]

Nash, T. H., III, and C. Gries. 1991 Lichens as indicators of air pollution. In O. Hutzinger [ed.], The handbook of environmental chemistry, vol. 4, part C, 1–29. Springer, Heidelberg.

———, and V. Wirth [eds.]. 1988 Lichens, bryophytes and air quality. J. Cramer, Berlin-Stuttgart.

Nei, M., and W. H. Li. 1979 Mathematical model for studying gene variation in terms of restriction endonucleases. Proceedings of the National Academy of Sciences, USA 76: 5269–5273.[Abstract/Free Full Text]

Rabe, R., and H. Wiegel. 1985 Wiederbesiedlung des Ruhrgebiets durch Flechten zeigt Verbesserung der Luftqualität an. Staub Reinhaltung der Luft 45: 124–126.

Rose, C. I., and D. L. Hawksworth. 1981 Lichen recolonization in London's cleaner air. Nature 289: 289–292.[CrossRef]

Saitou, N., and M. Nei. 1987 The neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution 4: 406–425.[Abstract]

Showman, R. E. 1997 Continuing lichen recolonization in the upper Ohio river valley. Bryologist 100: 478–481.[ISI]

Skye, E., and I. Hallberg. 1969 Changes in the lichen flora following air pollution. Oikos 20: 547–552.[CrossRef][ISI]

Tehler, A. 1995 Morphological data, molecular data, and total evidence in phylogenetic analysis. Canadian Journal of Botany 73 (Supplement 1): S667-S676.

Wolinski, H., P. Blanz, and M. Grube. 1997 Slide PCR als Alternative zu DNA-Isolationstechniken bei Pilzen. Tagung der Gesellschaft für Mykologie und Lichenologie, Regensburg, Abstract der Poster: 62.




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