Heredity (2004) 93, 322–329 & 2004 Nature Publishing Group All rights reserved 0018-067X/04 $30.00 www.nature.com/hdy Recombination and clonal propagation in different populations of the lichen Lobaria pulmonaria J-C Walser1, F Gugerli1, R Holderegger1, D Kuonen2 and C Scheidegger1 1WSL Swiss Federal Research Institute, Zu¨rcherstrasse 111, CH-8903 Birmensdorf, Switzerland; 2Statoo Consulting, PO Box 107, CH-1015 Lausanne, Switzerland Propagation, dispersal, and establishment are fundamental evidence of recombination, from various tests, indicated that population processes, and are critical stages in the life cycle L. pulmonaria is a predominantly outcrossing species. of an organism. In symbiotic organisms such as lichens, Nevertheless, clonality occurred in all populations, but the consisting of a fungus and a population of photobionts, presence of recurring multilocus genotypes influenced the reproduction is a complex process. Although many lichens spatial genetic structure only within low-density populations. are able to reproduce both sexually and asexually, the extent This could be interpreted as indicative of genetic bottlenecks of vegetative propagation within local populations is un- owing to increased habitat loss and disturbance. Conse- known. We used six polymorphic microsatellite loci to quently, for a predominantly outcrossing lichen species, investigate whether recombination is common in natural exogenous factors might be substantially altering population populations, and to assess if and how clonal reproduction processes and hence genetic structure. influences the spatial genetic structure within populations of Heredity (2004) 93, 322–329. doi:10.1038/sj.hdy.6800505 the epiphytic lichen species Lobaria pulmonaria. High Published online 16 June 2004 genetic diversity within all 12 investigated populations and Keywords: asexual propagation; dispersal; lichenised ascomycete; outcrossing; sexual reproduction; spatial autocorrelation Introduction Recent developments of modern molecular markers allow the population genetic processes of lichens to be Propagation, whether sexual or asexual, is a fundamental investigated. However, the few studies available so far step in the life cycle of every single organism. Sexual suffer from various limitations (Walser et al, 2003). reproduction enhances the number of different geno- Some have focused on nuclear ribosomal DNA (nrDNA) types within populations and increases the probability amplified by fungus-specific primers (Bridge and of survival in a competitive and/or changing environ- Hawksworth, 1998) which, although sufficient for the ment (Maynard Smith, 1978; Balloux et al, 2000), whereas analysis of major fungal lineages (Lutzoni et al, 2001), asexual propagation, on the other hand, can be a have the disadvantage of detecting rather low levels of successful evolutionary strategy for well-adapted geno- intraspecific genetic variation (Bridge and Hawksworth, types in extreme but stable habitats (Murtagh et al, 2000). 1998; Zoller et al, 1999; Ho¨gberg et al, 2002). Greater While our knowledge of mating systems and patterns variation has been detected with random amplified in animals and plants is broad (eg, Jackson et al, 1996; polymorphic DNA markers (RAPDs), allowing studies Takebayashi and Morrell, 2001), we have only just on breeding systems (Murtagh et al, 2000) and on the started to investigate and understand them in symbiotic population structure of lichen-forming fungi (Dyer life forms such as lichens. Despite a few studies on et al, 2001). In these RAPD studies, DNA has been sexual reproduction in lichens (Murtagh et al, 2000; isolated either from axenic fungal cultures or from Kroken and Taylor, 2001; Ho¨gberg et al, 2002), nothing thallus tissue free of photobionts (Crespo and Cubero, is known about its importance as compared to 1998; Heibel et al, 1999). As it is not possible to obtain clonal reproduction (through thallus fragments, soredia, algae-free material in lichen species that are asexual or isidia) in natural populations. Therefore, the objec- and for which the mycobiont is not cultivatable tives of this study were (i) to investigate whether (Miao et al, 2001), anonymous DNA fingerprinting recombination, and hence outbreeding, is common in cannot provide specific results for lichenised fungi natural populations of lichens, and (ii) to determine and is thus not universally applicable in mutua- how clonal reproduction influences the spatial genetic listic endosymbiotic systems. Highly variable, fungus- structure within populations. specific microsatellites would circumvent these pro- blems. For Lobaria pulmonaria, several microsatellite loci have recently been developed (Walser et al, 2003), which suit the specific requirements of our study. Four Correspondence: J-C Walser, Department of Organismal Biology and of the 12 original primers used in this study were Anatomy, The University of Chicago, 1027 East 57th Street, Chicago, IL 60637, USA. E-mail: [email protected] re-designed in order to shorten allele sizes, to avoid Received 21 August 2003; accepted 23 March 2004; published online indels in the flanking regions, and to amplify loci in 16 June 2004 multiplex PCR. Sexual and clonal propagation in lichen populations J-C Walser et al 323 Materials and methods ered a population as a patch of trees colonised by L. pulmonaria. Beyond the patch perimeter, the species did Species not occur locally. The samples from British Columbia The foliose epiphytic lichen L. pulmonaria (L.) Hoffm. is were collected within three different zones (hypermari widely distributed in circumboreal regions of the North- time, maritime, and intermontane; Goward, 1999) on a ern Hemisphere and in South Africa (Yoshimura, 1971, transect from the south coast of Vancouver Island to the 1998a, b). While L. pulmonaria has a large range and is interior of the province. These populations either still common in North America (Brodo et al, 2001), it is occurred in old growth forests (eg, CS, PR, DC, CL, BL, considered endangered in many parts of Europe (Wirth or OC) or in dense undisturbed forests (AY, TO, and BC). et al, 1996). Although it is described as both a sexually The samples from Switzerland were collected in com- and clonally reproducing species, apothecia (fruit bodies paratively large, but sparse populations in the Pre-Alps with sexual propagules) are formed infrequently and and the Jura Mountains, where anthropogenic distur- populations with only a limited number of fungal bance has been minimal. Across an entire population, genotypes appear to be nonsexual (Zoller et al, 1999). one thallus per tree was randomly taken from selected This correlation between genotypic diversity and fertility nearest neighbor trees. The distance between the host has been interpreted as evidence that L. pulmonaria might trees was measured, the area of the local population was be self-sterile (heterothallic; Zoller et al, 1999). calculated (Table 2), and the number of samples with apothecia was determined. Sampling In total, 562 thalli of L. pulmonaria were sampled from Microsatellite analysis nine populations in British Columbia, Canada, and from DNA was isolated according to Walser et al (2003) from three populations in Switzerland (Table 1). We consid- approximately 20 mg of cleaned marginal lobes per Table 1 Locations and their geographic circumscriptions of the 12 studied populations of Lobaria pulmonaria Location Code Life zones/area Altitude (m) Latitude Longitude Canada, British Columbia Ayum Creek, Vancouver Island AY Hypermaritimea 15 4812302900N 12313903700W Chesterman Beach, Tofino TO Hypermaritimea 54910604700N 12515303100W Cape Scott Provincial Park CS Hypermaritimea 55014002700N 12811603200W Lakelse Lake Provincial Park PR Maritimea 105 5412205400N 12813105200W Date Creek, Kispiox DC Maritimea 550 5512405200N 12714805200W Clayton Falls, Bella Coola BC Maritimea 55212201200N 12614804900W Carp Lake Provincial Park CL Intermontanea 860 5415200800N 12311503900W Bowron Lake Provincial Park BL Intermontanea 910 5311501900N 12112100300W Oregana Creek, Tumtum Lake OC Intermontanea 723 5115900800N11910502100W Switzerland Taaren Wald, Toggenburg TW Pre-Alps 1350 4711005000N911801500E Murgtal, Walensee MT Pre-Alps 1280 4710305200N911105100E Marchairuz, Jura Mountains UZ Jura Mountains 1200 4612905700N611002100E aLichen distribution classification system after Goward (1999). Table 2 Number of samples (N), area in acres of the investigated populations of Lobaria pulmonaria, local population density (N/area), mean number of alleles (A), effective mean number of alleles (Ae), and percentage of different genotypes (M), and of fertile samples (samples with apothecia) Population N Area (acres) N/Area AAe M (%) Apothecia (%) AY 39 30 1.30 7.8 5.1 79 13 TO 47 80 0.59 7.2 4.3 62 19 CS 50 30 1.67 7.7 4.8 52 42 PR 50 10 5.00 8.7 4.4 90 42 DC 52 10 5.20 7.3 3.4 92 25 BC 50 10 5.00 10.5 5.7 90 10 CL 51 50 1.02 6.3 3.1 53 4 BL 49 10 4.90 7.2 3.3 90 2 OC 52 40 1.30 8.7 4.2 92 10 TW 52 380 0.14 7.8 4.4 52 8 MT 38 680 0.06 5.3 3.6 47 11 UZ 32 80 0.40 6.8 3.8 53 0 Mean7SE CA 48.9 30 2.8970.68 7.970.4 4.370.3 78761975 Mean7SE CH 40.7 380 0.2070.10 6.670.7 3.970.2 5172673 Mean7SE total 46.8 120 2.2170.62 7.670.4 4.270.2 71761674 Means and standard errors are given for British Columbia in Canada (CA), for Switzerland (CH), and for the total sample set (total). For population codes, see Table 1. Heredity Sexual and clonal propagation in lichen populations J-C Walser et al 324 thallus. Six microsatellite loci were studied (LPu03, We also used the standardized index of association (IA) LPu09, LPu15, LPu16, LPu20, and LPu27; Walser et al, (Maynard Smith et al, 1993) to check for recombination or 2003), and four of the original primers were re-designed clonality within populations. From the distribution of in order to shorten allele sizes or to avoid indels in the mismatch values, an observed variance (VD) was flanking regions (Table 3). The loci were amplified in two calculated and compared with the expected variance multiplex polymerase chain reactions (PCRs) with each (Ve) under linkage equilibrium (Maynard Smith et al, containing three primer pairs (LPu03/LPu09/LPu15 and 1993; Haubold et al, 1998; Haubold and Hudson, 2000).