molecular evidence for limited dispersal of vegetative propagules in ...

American Journal of Botany 91(8): 1273–1276. 2004.

BRIEF COMMUNICATION

MOLECULAR

EVIDENCE FOR LIMITED DISPERSAL OF

VEGETATIVE PROPAGULES IN THE EPIPHYTIC LICHEN

LOBARIA PULMONARIA JEAN-CLAUDE WALSER2 Swiss Federal Research Institute WSL, Zu¨rcherstrasse 111, CH-8903 Birmensdorf, Switzerland Propagation, whether sexual or asexual, is a fundamental step in the life cycle of every organism. In lichenized fungi, a great variety of vegetative propagules have evolved in order for the symbiotic partners to disperse simultaneously. For lichens with the ability of sexual and asexual reproduction, the relative contribution of vegetative dispersal is unknown but could, nonetheless, be inferred by studying genotype distribution. The genetic structure of three Lobaria pulmonaria (Lobariaceae) populations from Switzerland was investigated based on the observed variation at six microsatellite loci. All three populations had a clustered distribution of identical genotypes at small spatial scales. The maximum distance between identical genotypes was 230 m. At a distance of 350 m from a source tree, seemingly suitable habitat patches were too far apart to be colonized. Some multilocus genotypes were frequent within local populations but no genotypes were shared among populations. The restricted occurrences of common genotypes as well as the clustered distributions are evidence for a limited dispersal of vegetative propagules in L. pulmonaria. Gene flow among isolated populations will ultimately depend on the capacity of long-distance dispersal and thus probably depend on sexual reproduction. Key words: clonal propagules; dispersal range; fragmented habitat, lichen-forming ascomycetes; Lobaria pulmonaria (Lobariaceae); microsatellites.

Dispersal and establishment are fundamental parameters of population processes and are critical stages in the life cycle of any organism (Clobert et al., 2001). In symbiotic organisms such as lichens, with a fungal partner (mycobiont) and a population of algae and/or cyanobacteria (photobiont), reproduction is a complex process (Honegger, 1998). Aside from the sexually derived fungal spores, lichens have developed different kinds of asexual dispersal units including thallus fragments (Bu¨del and Scheidegger, 1996). The distribution patterns and local population sizes of many epiphytic lichens may be influenced by their low dispersal capacities (Dettki et al., 2000; Zoller et al., 2000). On one hand, sexually derived fungal spores are believed to be important for long-distance dispersal (Bailey, 1976), but they require a suitable photobiont partner in order to reestablish the symbiosis after dispersal. On the other hand, vegetative propagules (e.g., thallus fragments, soredia, isidia) have an advantage in that both symbionts are simultaneously dispersed but the dispersal range might be lower because of their greater mass (e.g., Heinken, 1999). The objectives of this study were (1) to investigate the proportion of different types of propagation in local populations and (2) to determine if dispersal is somehow spatially limited. The foliose lichen Lobaria pulmonaria was used as a model Manuscript received 24 July 2003; revision accepted 9 April 2004. The author thanks Christian Ginzler, Michael Ga¨hwiler, Miriam Ru¨themann, and Silke Werth for field assistance; Urs Groner, Christine Keller, and Christoph Scheidegger for providing helpful discussions; and Ariel Bergamini, Felix Gugerli, Susan Hoebee, Rolf Holderegger, and Gary Walker for their constructive comments on the manuscript. This research was supported by the Swiss National Science Foundation (SNF grant No. 31-59241.99) and is associated with its research program NCCR Plant Survival, PS 6. 2 Present address: J.-C. Walser, Department of Organismal Biology and Anatomy, The University of Chicago, 1027 East 57th Street, Chicago, IL 60637 USA. Tel.: (773) 834-0467; Fax: (773) 702-0037; e-mail: jwalser@ uchicago.edu). 1

species. Although it is described as using both sexual and asexual reproduction, fruiting bodies (apothecia) are formed infrequently and local populations with only a limited number of fungal genotypes appear to be nonsexual (Zoller et al., 1999). While many lichen species are still common in North America, L. pulmonaria (like many others) has suffered a substantial decline in Europe during the 20th century (e.g., Wirth, 1976; Scheidegger et al., 2002). It has vanished almost completely from the Swiss Plateau, and the remnant populations in the Pre-Alps and the Jura Mountain have become increasingly fragmented. It was proposed that under the present environmental forest management conditions, the dispersal of propagules and subsequent establishment of new thalli are insufficient to maintain local populations (Rose, 1992; Zoller et al., 2000). Furthermore, the degree of isolation of the populations is unknown, and it is not clear whether propagules are exchanged among them (Zoller et al., 1999). A recent study detected a local dispersal range of up to 50 m within a period of 5 d (Walser et al., 2001). However, information about the effective distance over longer periods is important for developing efficient strategies to conserve populations of endangered lichen species. Studies of disturbance events, e.g., forest fires (Romagni and Gries, 2000) or clear cuts (Coxson et al., 2003), were used to investigate dispersal dynamics and colonization in pioneer lichen species. Together these studies have improved the understanding of the dispersal abilities of lichens, but until now, it was not possible to discriminate between sexual and asexual propagation. Recently developed, highly polymorphic genetic markers were used in this study to investigate the spatial genetic structure of three L. pulmonaria populations from Switzerland. The multilocus genotype distributions among and within these populations were compared on different geographic scales to estimate the relative extent of asexual vs. sexual reproduction.

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TABLE 1. Locations and codes of the three Swiss Lobaria pulmonaria populations investigated and for the 30 juvenile samples taken from a single tree at location UZ. Number of different genotypes (nGT) and number of samples (n) per population or subpopulation, respectively, as well as altitude and geographic position are given. Location

Codes

nGT/n

Taarenwald Subpopulation A Subpopulation B Murgtal Marchairuz Juvenile samples

TW

27/52 7/16 20/36 18/38 17/32 3/30

MT UZ

MATERIALS AND METHODS Lichen material—Tissue samples of 122 L. pulmonaria thalli were collected from two populations in the Pre-Alps and one population in the Jura Mountains in Switzerland (Table 1). The sampled populations are large (more than 30 colonized trees) as compared to other Swiss populations and have been only marginally disturbed by cultivation (Scheidegger et al., 2002). For the genetic analysis, between 32 and 52 fragments (depending on population size) of mature thalli per population were collected, each randomly chosen

Altitude (m a.s.l.)

Latitude

Longitude

1250

478109500 N

98189150 E

1280 1200

478039520 N 468299570 N

98119510 E 68109210 E

from a different tree trunk. The investigated populations reflected L. pulmonaria stands with different structures. Population Taarenwald (TW) consisted of two subpopulations, spatially separated by approximately 350 m (Fig. 1a). Population Murgtal (MT) was located in a low-density mixed forest spanning a distance of 900 m along a creek (Fig. 1b). Both populations were located in different valleys, but the distance between the two populations was only about 15 km. On a smaller scale, samples from a denser population in Marchairuz (UZ) were collected over an area of about 100 3 200 m (Fig. 1c). This population in the Jura Mountains is located in a more open landscape and is geographically separated from the two populations in the Pre-Alps by about 240 km. In order to examine small-scale dispersal and establishment, 30 juvenile thalli (,1 cm2) were also sampled in an area of 20 3 60 cm from a single tree in population UZ. Laboratory procedures—DNA was isolated from one or two cleaned, marginal lobes (approximately 20 mg) per thallus fragment, following the protocols of Walser et al. (2003). The six fungus-specific microsatellite loci used in this study were LPu03, LPu09, LPu15, LPu16, LPu20, and LPu27 (Walser et al., 2003, in press). Polymerase chain reaction (PCR) conditions and genotyping of the haploid genotypes were performed as described in Walser et al. (2003). Genetic measurements—As a measurement of genetic variation, we used the unbiased gene diversity (unbiased estimate of the expected heterozygosity over all loci): H 5 [n/(n 2 1)](1 2 Ski51x2i ), where n is the number of samples, k is the number of different multilocus genotypes observed in a population, and xi is the frequency of the ith genotype (Nei and Roychoudhury, 1974; Nei, 1987). The value of H can range from 0 (no diversity, monomorphic population) to 1 (where all individuals within a population are unique). The probability that two individuals are different at the jth locus is 1 2 Sp2 (Maynard Smith et al., 1993), and the probability that two samples have the same multilocus genotype if all individuals were the result of independent recombination was calculated by m pequal 5 PLj51(Si51 pij2),

where pij is the frequency of the ith allele at the jth locus, m is the number different alleles at locus j, and L is the number of loci. The genetic differentiation among populations and among the subpopulations within population TW was investigated using a hierarchical analysis of molecular variance (AMOVA; Excoffier et al., 1992). This was achieved using the stepwise-mutation model based on the genetic distances estimator RST (Slatkin, 1995) with 1000 permutations implemented in ARLEQUIN, version 2.001 (Schneider et al., 2000). Fig. 1. Spatial patterns of genotypes in three Lobaria pulmonaria populations in the Pre-Alps or the Jura Mountains, Switzerland. Genotypes are consecutively numbered per population; recurring genotypes received the same number. Unique genotypes, i.e., those only occurring once, are marked with a plus sign (1). For population codes see Table 1. Note the different scales of the axes.

RESULTS The number of alleles per locus and population ranged from 2 to a maximum of 14 (Walser, 2003). The patterns of allelic variation were not very different and the alleles size range was

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EVIDENCE FOR DISPERSAL IN

overlapping among the three populations (Walser, 2003). Furthermore, the total genetic diversity (H) and the proportion of unique genotypes (approximately 30%) were about the same in all three populations (see below). Only subpopulation A in population TW and the cohort of juvenile individuals taken from a single tree in population UZ had a lower genetic diversity (Table 1). The pairwise genetic differentiation between populations was low (RST , 0.01), and the AMOVA showed that none of the total genetic variation was accounted by the differences among populations (RST 5 20.006; P 5 0.56). However, the three populations did not share a single genotype, although some genotypes were frequent within populations. Population TW—The 16 thalli from the smaller subpopulation (A) and the 36 thalli from the larger subpopulation (B) consisted of seven and 20 different multilocus genotypes respectively, totaling 27 genotypes (H 5 0.943; Table 1). In subpopulation A, six genotypes were unique and one was common (Fig. 1a), resulting in an unbiased gene diversity of H 5 0.625. In subpopulation B, 12 genotypes were unique and eight genotypes occurred more than once (Fig. 1a), resulting in a higher unbiased gene diversity (H 5 0.951) than in subpopulation A. No genotype was shared between the two subpopulations, and the AMOVA (including all samples) revealed significant differentiation (RST 5 0.266; P , 0.001) between them. To test whether clonal reproduction might be responsible for the subpopulation differentiation observed, an AMOVA without recurring genotypes (clone-corrected samples) was performed. This resulted in a nonsignificant RST value of 20.028 (P 5 0.57). The greatest geographic distance between two identical genotypes was 230 m in subpopulation A and 100 m in subpopulation B. Population MT—In the second population of the Pre-Alps, 18 different multilocus genotypes were found in the 38 samples investigated, resulting in an unbiased gene diversity of H 5 0.923. The genotypes were clustered in four groups along the creek and the maximum geographic distance between two identical genotypes was 115 m (Fig. 1b). Population UZ—The 32 sampled thallus fragments revealed a total of 17 different multilocus genotypes with an unbiased gene diversity of H 5 0.925. The most common genotype was found seven times, and 11 genotypes were unique. As in population MT, the genotypes were also spatially clustered, and the maximum geographic distance between two identical genotypes was 140 m (Fig. 1c). The 30 juvenile samples taken from a single tree exhibited only three different genotypes. Most of the young thalli had the same genotype (N 5 27), resulting in low genetic variation on a small spatial scale (H 5 0.191). DISCUSSION All three populations revealed a clear spatial clustering of identical multilocus genotypes. If all individuals were the result of independent recombination, the probability that two samples have the same multilocus genotype was between 1024 and 1025. Therefore, we may assume that identical genotypes are the product of clonal propagation. Based on this assumption, the results presented here showed that clonal propagation dominates on a small geographic scale, and it can be suggested

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that dispersal of vegetative propagules is spatially limited in all three L. pulmonaria populations investigated. Aside from being minute, lichen propagules do not have morphological adaptations for any specific mode of dispersal, and it is commonly assumed that wind, water, and/or animals are the likely vectors (Bailey, 1976; Ahmadjian, 1993; Heinken, 1999). While water could play an important role for local dispersal (e.g., run-off water on a tree; Armstrong, 1981), longer distances might be covered by wind dispersal. Investigations on dispersal range (Armstrong, 1990; Walser et al., 2001) confirmed the dispersal limitation hypothesis (Bailey, 1976; Hawksworth and Hill, 1984; Armstrong, 1987), which predicts leptokurtic dispersal of propagules. Several studies focusing on colonization came to the same conclusion, namely that lichens are limited by local dispersal (Dettki et al., 2000; Sillett et al., 2000). However, it is commonly accepted that extreme events (like wind storms) or long-distance vectors (e.g., birds; Bailey and James, 1979) may carry not only the light sexual propagules, but also the heavier vegetative propagules of lichens, several hundreds of meters. Ho¨gberg et al. (2002) suggest that Letharia vulpine (L.) Hue., another epiphytic lichen species, originated in western North America and migrated to Europe by long-distance dispersal with lightweight symbiotic propagules (soredia). In contrast, the data presented here suggested a geographically restricted distribution of vegetative propagation. Neither the three populations nor the two subpopulations at location TW contained common multilocus genotypes. The observed maximum distances between identical genotypes within populations varied from 115 m to 230 m, and yet other seemingly suitable habitats at distances between 100 m and 350 m from the source tree were not colonized. Limited dispersal of vegetative propagules might be an important factor in explaining the clustered distribution of identical genotypes. Nevertheless, other aspects, such as the distribution and density of potential trees and the degree of disturbance or differences in site qualities (e.g., microclimatic conditions, prevailing wind direction, or site history), may also contribute to local population structure and restrict colonization of new habitat patches (Hilmo and Sastad, 2001; Walser et al., 2001). Sexual propagules are believed to be important for longdistance dispersal, but sexual reproduction also enhances the number of different genotypes in local populations and increases the probability of survival in a competitive and/or changing environment (Maynard Smith, 1978). On the other hand, clonal propagation can be a successful evolutionary strategy for well-adapted genotypes in extreme, yet stable habitats (Murtagh et al., 2000). Therefore, vegetative propagules help to maintain and promote colonization of suitable habitat (Hansson et al., 1992). This could explain the low diversity within the cohort of juvenile thalli on a single tree. Clonal growth and dispersal on a small scale (e.g., on a tree) can be a suitable strategy for a single individual to reach a high density in a local population (Scheidegger, 1995). This may later lead to a higher production of both sexual and asexual propagules, which might compensate for low dispersal range and high mortality of young individuals and help the lichen to persist in a habitat following colonization (Scheidegger et al., 1995). The multilocus genetic structure of all three investigated Swiss populations presented in this study supports the assumptions that (1) the proportion of clonal propagation is high and (2) that dispersal of symbiotic propagules is spatially lim-

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ited. The restricted occurrences of common genotypes as well as their spatially clustered distribution are evidence for a limited dispersal of vegetative propagules in the epiphytic lichen L. pulmonaria. Gene flow among the remnant populations from the Pre-Alps and the Jura Mountains will ultimately depend on the capacity of long-distance dispersal and therefore depend on sexual reproduction. LITERATURE CITED AHMADJIAN, V. 1993. The lichen symbiosis. John Wiley & Sons, New York, New York, USA. ARMSTRONG, R. A. 1981. Field experiments on the dispersal, establishment, and colonization of lichens on a slate rock surface. Environmental and Experimental Botany 21: 115–120. ARMSTRONG, R. A. 1987. Dispersal in a population of the lichen Hypogymnia physodes. Environmental and Experimental Botany 27: 357–363. ARMSTRONG, R. A. 1990. Dispersal, establishment and survival of soredia and fragments of the lichen, Hypogymnia physodes (L.) Nyl. New Phytologist 114: 239–245. BAILEY, R., AND P. JAMES. 1979. Birds and dispersal of lichen propagules. Lichenologist 11: 105–106. BAILEY, R. H. 1976. Ecological aspects of dispersal and establishment in lichens. In D. H. Brown, D. L. Hawksworth, and R. H. Bailey [eds.], Lichenology: progress and problems, 215–247. Academic Press, London, UK. BU¨DEL, B., AND C. SCHEIDEGGER. 1996. Thallus morphology and anatomy. In T. Nash [ed.], Lichen biology, 37–64. University Press, Cambridge, UK. CLOBERT, J., F. DANCHIN, A. A. DHONDT, AND J. D. NICHOLS. 2001. Dispersal. Oxford University Press, New York, New York, USA. COXSON, D., S. STEVENSON, AND J. CAMPBELL. 2003. Short-term impacts of partial cutting on lichen retention and canopy microclimate in an Engelmann spruce–subalpine fir forest in north-central British Columbia. Canadian Journal of Forest Research 33: 830–841. DETTKI, H., P. KLINTBERG, AND P. A. ESSEEN. 2000. Are epiphytic lichens in young forests limited by local dispersal? Ecoscience 7: 317–325. EXCOFFIER, L., P. SMOUSE, AND J. QUATTRO. 1992. Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics 131: 479– 491. HANSSON, L., L. SODERSTROM, AND C. SOLBRECK. 1992. The ecology of dispersal in relation to conservation. In L. Hansson [ed.], Ecological principles of nature conservation. Applications in temperate and boreal environments, 162–200. Conservation Ecology Series: Principles, Practices and Management. Elsevier Applied Science, London, UK. HAWKSWORTH, D. L., AND D. J. HILL. 1984. The lichen-forming fungi. Chapman & Hall, London, UK. HEINKEN, T. 1999. Dispersal patterns of terricolous lichens by thallus fragments. Lichenologist 31: 603–612. HILMO, O., AND S. M. SASTAD. 2001. Colonization of old-forest lichens in a young and an old boreal Picea abies forest: an experimental approach. Biological Conservation 102: 251–259. HO¨GBERG, N., S. KROKEN, G. THOR, AND J. W. TAYLOR. 2002. Reproductive mode and genetic variation suggest a North American origin of European Letharia vulpina. Molecular Ecology 11: 1191–1196. HONEGGER, R. 1998. The lichen symbiosis: what is so spectacular about it? Lichenologist 30: 193–212. MAYNARD SMITH, J. 1978. The evolution of sex. Cambridge University Press, Cambridge, UK.

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