Clonal Structure in the Moss, Climacium Americanum Brid
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Heredity 64 (1990) 233—238 The Genetical Society of Great Britain Received 25August1989 Clonal structure in the moss, Climacium americanum Brid. Thomas R. Meagher* and * Departmentof Biological Sciences, Rutgers University, P.O. Box 1059, Jonathan Shawt Piscataway, NJ 08855-1059. 1 Department of Biology, Ithaca College, Ithaca, NY 14850. The present study focussed on the moss species, Climacium americanum Brid., which typically grows in the form of mats or clumps of compactly spaced gametophores. In order to determine the clonal structure of these clumps, the distribution of genetic variation was analysed by an electrophoretic survey. Ten individual gametophores from each of ten clumps were sampled in two locations in the Piedmont of North Carolina. These samples were assayed for allelic variation at six electrophoretically distinguishable loci. For each clump, an explicit probability of a monomorphic versus a polymorphic sample was estimated based on allele frequencies in our overall sample. Although allelic variation was detected within clumps at both localities, our statistical results showed that the level of polymorphism within clumps was less than would be expected if genetic variation were distributed at random within the overall population. INTRODUCTION approximately 50 per cent of moss species that are hermaphroditic, self-fertilization of bisexual Ithas been recognized that in many taxa asexual gametophytes results in completely homozygous propagation as the predominant means of repro- sporophytes. Dispersal of such genetically iden- duction does not necessarily lead to genetic tical spores is functionally equivalent to asexual monomorphism (Bell, 1982; Silander, 1979; reproduction. Vrijenhoek, 1984). Indeed, localized patterns of Mosses are believed to be relatively depauper- allozymic variation have been useful tools for iden- ate in genetic variation (Crum, 1973), a circum- tifying the spatial extent of individual clones within stance which may reflect the predominance of plant populations (Silander, 1979) and for recog- asexual propagation as the most successful life nizing different clones in unisexual fish popula- history mode. Many bryophyte systematists believe tions (Vrijenhoek et a!., 1978). In order to under- that mosses evolve relatively slowly (Anderson, stand how genetic variation is maintained in 1963; Khanna, 1964), a view based largely on asexual populations, it is first necessary to deter- biogeographic observations. The geographic distri- mine the distribution of such genetic variation and butions of most species are relatively broad, and relate that distribution to the overall breeding intercontinentalrangedisjunctions without morphological differentiation are common. While system. Most species of mosses are believed to repro- some bryologists have assumed that almost all duce both sexually by spore formation, and moss reproduction is by means other than sexual asexually by a variety of methods (Longton, 1976; spores (e.g., Anderson, 1963), extremely little Wyatt, 1982; Wyatt and Anderson, 1984). Asexual information is available about how mosses actually reproduction in this group can occur by vegetative do reproduce under natural conditions. fragmentation, in which pieces of gametophytic Bryophytes in general, and mosses in par- plants are broken off and reestablish elsewhere, ticular, have been increasingly recognized as useful by the formation and dispersal of specialized organisms for studying a variety of concepts in reproductive structures such as axillary gemmae population biology (During and van Tooren, 1987; or rhizoidal tubers (Correns, 1899), or by the Wyatt, 1982). The reasons for this recent increase growth of localized clones. Moreover, within the in awareness of mosses as research tools are 234 T. R. MEAGHER AND J. SHAW several-fold. These plants exhibit unusual features creek crosses Old Erwin Road. The ecological for terrestrial plants, such as a conspicuous alterna- features of these sites were similar to those tion of generations with the haploid form perennial described for nearby populations of C. americanum and freeliving. Since the predominant growth form characterized in Shaw et al. (1987). In each site, is haploid, population genetic studies are greatly ten individual gametophores were sampled from simplified. Also, this group has been relatively each of ten distinct clumps. Clumps ranged in size overlooked so that virtually any population bio- from 13 cm to 100 cm in diameter, and logy study represents a novelty. gametophores were sampled evenly over a 13 cm Ironically, the fact that mosses have been rela- diameter portion of each clump. These tively understudied is a weakness as well as a gametophores were placed in plastic vials and strength. Even basic information about population stored at —70°C for several weeks until they were structure and dynamics are poorly understood for subjected to electrophoretic analysis. many species, hindering detailed investigations. Enzyme extraction was performed by grinding Many moss species occur as compact clumps of each sample in liquid nitrogen and then suspend- gametophores. The genotypic composition of such ing them in an extraction buffer of 0.1 M tris-HCI clumps, however, is generally not known. Each (pH 7.2) buffer with 0.5 per cent mercaptoethanol. clump could represent a collection of clonal ramets Extracts were absorbed onto filter paper wicks of the same genotype (as is commonly assumed in which were loaded directly into 12 per cent many bryological studies) or, alternatively, they horizontal starch gels. Following electrophoresis, could represent an aggregation of co-occurring enzyme assays were conducted for six systems that distinct genotypes. The differences between these had been found in our previous investigation to possibilitieshave significantand obvious be polymorphic: alcohol dehydrogenase (ADH), ramifications for microevolutionary processes in acid phosphatase (ACP), fluorescent esterase moss populations. (EST5), peroxidase (PER), phosphoglucomutase The present study was focussed on the moss (PGM), and superoxide dismutase (SOD). The species, Climacium americanum Brid., commonly electrophoretic and staining protocols for C. amer- known as the "tree moss" because of its dendroid icanum are outlined in detail in Shaw eta!. (1987). growth form. Sexual reproduction, as evidenced For each clump, an explicit probability of by sporophyte formation, is extremely rare in C. observing a monomorphic versus a polymorphic americanum (Shaw et a!., 1987). The species sample was estimated based on allele frequencies occupies a broad geographic range in the eastern in our overall sample. For a haploid organism, United States, from Nova Scotia and Wisconsin these probabilities for a given sample size can be south to Florida and Kansas (Crum and Anderson, calculated by use of a binomial expansion series 1981). The typical growth habit for this species is (table 1; Feller, 1968). Thus for a sample of n the formation of discreet mats or clumps along gametophores, and a diallelic locus with allele stream margins and on flood plains. These clumps frequencies Pi and P2, the probability of are generally small (Ca. <60 cm2), but are occasion- monomorphism would be ally more extensive. In order to determine the genetic structure of these clumps in C. americanum, P, (monomorphism) =(p'+p°) the distribution of genetic variation was analysed for the ith locus. A combined probability across by an electrophoretic survey of two natural popula- multiple loci can be obtained by multiplying tions. Table 1 Probability distribution of a sample of 10 haploid MATERIALSAND METHODS genotypes within clumps based on population allele frequencies Pt and P2 Clumpsof C. americanum were sampled in two Ratio of Ratio of locations in the Piedmont of North Carolina. The g1 to g2 Probability g1 to g2 Probability first sample site was on the flood plain of the Eno River 50 m downstream from its intersection with 10:0 p° 4:6 210pp Guess Road in the city of Durham (Durham 9:1 10p'p2 3:7 120pp County, NC). The second sample site was located 8:2 45pp 2:8 45pp in the Korstian Division of the Duke Forest 7:3 120p,p 1:9 10pp 6:4 210pp 0:10 (Orange County, NC) along New Hope Creek 5:5 approximately 200 m upstream from where the 252pp CLONAL STRUCTURE IN CLIMACIUM AMER/CANUM 235 together all of the P,assumingthe loci are variation was predominantly due to variation unlinked. A combined probability that a clump between clumps rather than to variation within will be polymorphic for at least one locus can be clumps (tables 3—4). obtained by subtracting the combined probability In our previous results (Shaw et a!., 1987) of monomorphism from 1. Within each site, demonstrated that two varieties of C.americanum explicit probabilities of monomorphism versus which differ in morphology can also be distin- polymorphism were calculated for each clump guished by their enzyme phenotypes coded by sampled using the cumulative allele frequencies several loci. In the Eno River sample, nine of observed for that site. Clumps varied in these prob- the clumps showed a PGM enzyme pattern abilities due to small differences in sample size. characteristic of Climacium americanum var. americanum and the remaining clump was clearly var. kindbergii. Within clump allelic variation RESULTS was restricted to the PER locus, although allelic variation among clumps was observed for ACP, Fourof the six enzymes assayed showed poly- PGM, and SOD as well (table 3). morphism in the Eno River collection,