J. Hattori Bot. Lab. No. 76: 159- 172 (Oct. 1994)

REPRODUCTIVE BIOLOGY IN THE CHALLENGE AND THE OPPORTUNITIES

R. E. LONGTON1

ABSTRACT. Current evidence suggests the occurrence of major differences among bryophytes in features such as frequency of production, facility for establishment from spores, and mating patterns, as well as in levels of ploidy and of intraspecific genetic variability. It is not clear from the limited evidence so far available how these features correlate among themselves, with habitat characteristics, and with other life history parameters such as longev­ ity, reproductive effort, and spore size in controlling microevolutionary patterns. Further empirical evidence is required about reproductive biology in specific groups of bryophytes, and could best be obtained from studies combining traditional observations and experiments with cytological and molecular analyses, in which electrophoresis and genetic fingerprinting are viewed as complementary approaches. A rigorous conceptual framework is also required, drawing on principles already established in the population biology of other organisms.

THE HISTORICAL BACKGROUND Students of reproductive biology seek answers to several fundamental questions. Among the most important are: how are populations of organisms maintained given the continuing death of individuals; how are new populations established, often at some distance from existing ones; and how, and to what extent, do reproductive processes influence ability to survive and compete through adaptation to existing and changing environments? There are taxonomic implications, for it is known that different modes of reproduction result in different patterns of variation within and between populations. Ecological questions are also raised. It is important to determine how environmental factors, and interaction with vegetative growth, affect different stages in the reproduc­ tive process (Imura 1994 - this Symposium; Longton 1990), and to understand the extent to which different reproductive strategies have evolved in response to the selection pressures of contrasting environments. Indeed, considerations of reproductive biology and of evolution appear to be inextricably linked. As a bryologist, I was brought up to believe that and liverworts are marked by slow evolution compared with other organisms. The fact that many early Tertiary fossils are referable to extant genera and even species, a view maintained in modern reviews (Miller 1984 ), was cited as evidence. So too was the fact that many species show patterns of disjunction more familiar at the generic level among angio­ sperms, e.g. between the deciduous forests of the Appalachian region and of south-east Asia (lwatsuki 1958) implying, in this case, that speciation had occurred among the angiosperms, but not among the bryophytes, since disruption of the formerly circumpol-

1 Department of Botany, School of Sciences, The University of Reading, Reading RG6 2AS, U.K. 160 J. Hattori Bot. Lab. No. 76 I 9 9 4 ar Arcto-Tertiary forest. Reproductive biology figured prominently in discussions seeking to explain slow evolution among bryophytes (Anderson 1963, Crum 1972). In particular, it was contended that asexual reproduction predominates over sexual reproduction culminat­ ing in establishment from spores. It was pointed out that of many species are rare or unknown, that establishment from spores is seldom observed in the field and that most species are able to reproduce by fragmentation of the gametophyte, with a minority also producing specialized asexual propagules. A predominance of self­ fertilization in monoecious species, which comprise about 50% of mosses and 20% of liverworts, was assumed, and it was pointed out that self-fertilization in haploid would lead to all spores within each being genetically identical to each other and to the parent gametophyte. Thus lack of effective genetic recombination through meiosis, resulting from a predominance of asexual reproduction accentuated by habitual self-fertilization in monoecious taxa, was viewed as crucial in robbing bryophyte species of evolutionary flexibility. It was suggested that the wide degree of phenotypic plasticity exhibited by many bryophytes represents an alternative strategy to genetic differentiation in enabling individual species to grow in a range of environments. Moreover, the normal fertiliza­ tion range was shown to be only a few centimetres, increasing to about one metre where splash-cups are involved (Longton & Schuster 1983, Wyatt & Anderson 1984 ), while spore trapping experiments revealed a strong decrease in catch per unit area with increasing distance from the parent colony over the first few metres suggesting that there was little potential for gene flow between populations (Wyatt & Anderson 1984, McQueen 1985). The concept that extant bryophyte species are of ancient origin, and are strongly isolated from each other genetically, was supported by the apparent rarity of interspecific hybrids, with only first generation hybrid sporophytes known and those generally sterile, and with autoploidy thought to predominate over alloploidy in cases where chromosome duplication had occurred (Anderson 1980). The dominance of the haploid gametophyte within the life-history was also considered to reduce genetic variation among bryophytes, since the range of genotypes possible among gametophytes would be reduced by the presence of only one allele of each gene, and there would be strong selection against any allele that might be deleterious in a given environment, but possibly advantageous under other conditions, or in new combinations, as each allele would be expressed during every generation (Anderson 1963). Bryophytes therefore came to be regarded as "evolutionary failures" (Crum 1972). However, this view was questioned on theoretical grounds (Longton 1976, Whitehouse 1985), and it was pointed out that organisms that had been in existence since the Devonian and yet remain the second largest group of land are "some failures!" (Longton 1985).

THE PRESENT SITUATION Continuing speculation, combined with evaluation of the limited empirical data that have become available, has led to a partial reappraisal of the position outlined R. E. LONGT ON: Reproductive biology: challenge and opportunities 161 above. This has become clear in several of the contributions to the present Symposium. Polyploidy Thus it is by no means clear that bryophyte gametophytes universally function as haploids. In mosses the base chromosome numbers appear to be x = 6- 7, but 85- 90% of extant species have n = 10-14 or higher suggesting the widespread occurrence of ancestral polyploidy (Newton 1984), possibly resulting from selection to overcome the disadvantages of haploidy. The situation in liverworts, where most species have n = 8-10, is less clear. Newton ( 1984) regarded the weight of evidence as favouring the view that these complements also originated by past chromosome duplication, although the recent demonstration that Takakia ( n = 4- 5) is a (Smith & Davison 1993) may tip the balance in favour of the alternative view that x = 9 (Smith 1978). Even in mosses, there may have been extensive gene deletion and other structural changes since chromosome duplication, because there is some evidence that crosses in apparently polyploid taxa give 1: 1 ratios among the progeny, as would be expected in haploids (Cove 1983, Hofman, van Delden & van Zanten 1992), while electrophoretic studies have not yet revealed heterozygous banding patterns in presumed polyploid gameto­ phytes (Hofman 1988, Wyatt 1994 - this Symposium). Determining the extent to which the gametophytes of extant bryophytes function as haploids is of crucial importance in understanding current evolutionary processes. Mating Patterns Little is yet known about mating patterns. The short fertilization range combined with the clustering of genetically uniform, presumably clonal individuals that has been reported in some species (e.g. Meagher & Shaw 1990) might indicate a tendency towards inbreeding among groups of individuals with a relatively narrow range of genotypes. However, Innes ( 1990) found that sporophytes in populations of Polytri­ chum juniperinum , a dioecious moss with male inflorescences in the form of splash cups, showed almost the full range of electrophoretically defined genotypes predictable from random mating among the gametophytic genotypes present. Recent evidence has provided little support for the view that self-fertilization is essentially obligate in monoecious taxa, although self-compatibility in single spore cultures has been demonstrated in most of the few species so far investigated (Shaw 1991 a). It has been shown that most monoecious mosses are autoecious, with antheridia and archegonia borne on separate branches of an individual, rather than synoecious or paroecious where they occur in closer proximity (Longton & Schuster 1983). This could have the selective advantage of combining the regular sporophyte production that accompanies moneocy (see below) with some potential for cross-fertilization. Phenological observations such as those of Imura (1994 - this Symposium), which describe seasonal reproductive cycles, have been important in paving the way for more detailed case studies. In several monoecious species (e.g. Atrichum undulatum and Tortu/a muralis; Longton & Miles 1982) such work has shown that the majority of shoots fail to produce both male and female gametangia during a given cycle, with most young sporophytes occurring on functionally female shoots, so that cross-fertilization 162 J. Hattori Bot. Lab. No. 76 I 9 9 4 between physiologically independent individuals clearly takes place. Stark ( 1983) suggested that both cross- and self-fertilization occur in Entodon cladorrhizans, with the latter predominating, as sporophytes are produced by a higher proportion of function­ ally bisexual than of functionally female shoots. A low frequency of cross-fertilization was confirmed in Weissia controversa by Anderson and Lemmon (1974) in cushions artificially created by combining parts of colonies differing in the number of accessory chromosomes. However, it was considered that cross-fertilization between genotypes rarely occurs in nature since the naturally occurring cushions at the study site proved internally uniform cytologically, suggesting that each represented a single clone. Two populations of hygrometrica studied by Shaw ( 1991 b) also appeared to be uniclonal. However there is electrophoretic evidence that individual colonies of Atrichum undulatum may include more than one genotype (Miles & Longton 1987), as is clearly the case in many dioecious taxa where males and females regularly grow together. Studies of peroxidase alleles in sporophytes suggested that the frequency of cross-fertilization was at least 25 % in Pe/lia epiphylla, but much lower, although detectable, in P. borealis (Zielinski 1984, 1986). Such situations may be advantageous in terms of environmental adaptation since, at least in haploids, a predominance of self-fertilization maintains any favourable gene combina­ tions produced by occasional crossing (Longton l 988a). Sexual and Asexual Reproduction As regards the frequency of sexual reproduction, recent analyses have confirmed that almost all monoecious mosses, and the majority of dioecious species, produce sporophytes freely. Conversely, fruiting is rare or absent in a substantial proportion of dioecious species, in parts of or throughout their range, through failure of males and females to grow side by side (Longton & Schuster 1983). There remains little evidence of establishment from spores in the field, but it is not clear whether this is due to its rarity or to the technical difficulty of confirming its occurrence (Miles & Longton 1990, Newton & Mishler 1994 - this Symposium). Experiments designed to compare ease of establishment from spores as opposed to fragments of gametophytes at field sites have demonstrated important differences between species. At one extreme establishment of Funaria hygrometrica occurred more readily from spores than from gametophytes fragments. At the other, new colonies of Polytrichum alpestre developed from fragments, but no evidence of spore germination at field sites was obtained during several years of observations and experiment (Miles & Longton 1990). Chemical inhibition, as discussed by Newton and Mishler (this Symposium), was suspected as spores of P. alpestre failed to germinate when planted in their native blanket bog but did so in cultivation following recovery from the bog twelve months later. Intermediate results were given by Atrichum undulatum and Bryum argenteum , in which establishment occurred rarely from spores and freely from gametophyte fragments. Observations Archidium alternifolium suggested a similar pattern (Miles & Longton 1992a), as did Kimmere's (1991) experiments on pellucida in which establishment occurred more freely from gemmae than from spores. R. E. L ONGTON: Reproductive biology: challenge and opportunities 163

There seems little doubt, therefore, that asexual reproduction predominates in colony maintenance in many bryophytes, with sexual reproduction possibly more effective in establishing new colonies (Kimmerer 1993, Newton & Mishler 1994 - this Symposium). It is thus significant that recent trapping experiments with several stegocarpous species have suggested that although the density of spore deposition rapidly decreases with distance from the parental gametophytes (page 160), the majority of spores are nevertheless transported to distances beyond the trapping areas investigated (Miles & Longton 1987, 1992b, Stoneburner et al. 1992, SOderstrom 1994 - this Symposium). This supports van Zanten's view, based on studies of spore viability following exposure to conditions experienced during aerial transport, that long-distance dispersal is a real possibility for many species (van Zanten 1976, van Zanten & Gradstein 1989). In contrast, the unusually large spores of the cleistocarpous moss Archidium a/ternifo/ium tend to remain, and may even germinate, in the perichaetia where the parent sporophytes develop (Miles & Longton 1992b). Thus one can visualise sexual reproduction producing a wide range of genotypes among the spores, with those proving successful in new, and possibly remote habitats being maintained by asexual propagation, and the resulting populations subsequently being enriched by occasional establishment of incoming spores. Newton and Mishler (1994 - this Symposium) argue that asexual reproduction can itself lead to genetic variation among bryophytes, since any mutation occurring in the single apical cell of a young individual, or of a branch, can subsequently be transmitted throughout a new plant. This is undoubtedly true, but I remain unconvinced that such a mechanism alone is as effective as recombination at meiosis in producing the widest possible rang~ of gene combinations upon which natural selection can act. Reproduction and Rarity While recorded cases of successful establishment from spores remain few, circum­ stantial evidence that sexual reproduction is effective in species that produce spores is provided by the wider geographical range and greater morphological variability that characterises fruiting compared with non-fruiting species (Gemmel 1950). This was clearly demonstrated by Inoue (1974) in the hepatic genus Plagiochila. More recently it has been shown that the majority of British moss species unknown to produce sporophytes anywhere in their range are marked by narrow or highly disjunctive distribution patterns and low variability, at least as assessed by the number of recognised infraspecific taxa (Longton & Schuster, 1983). Many such species produce specialized asexual propagules, but these appear to be even more strongly associated with dioecy as opposed to monoecy than with absence of sporophytes. Possible reasons for this curious relationship are discussed by Longton (1992). In a comparison of rare versus common British mosses it was established that rarity is positively associated with both monoecy and failure to produce sporophytes. This appears anomalous given that failure to fruit is associated with dioecy. The explanation lies in the fact that a substantially higher proportion ofmonoecious fruiting species is considered rare (29% ) than of dioecious fruiting species (9% ). 164 J . Hattori Bot. Lab. No. 76 I 9 9 4

These relationships are consistent with the view that loss of evolutionary flexibility consequent upon bypassing of meiosis is important in leading some species to become rare, that dioecious species therefore tend to become rare if they fail to produce sporophytes and that monoecious species become rare if self-fertilization becomes essentially obligate. However, there are other explanations. Thus it is possible that onset of rarity is unrelated to frequency of meiosis, with increasing rarity restricting sporophyte production in dioecious, but not in monoecious taxa by reducing the chance of male and female plants growing side by side(Longton 1992). Life-History Strategies It is readily apparent that different modes of reproduction are loosely associated with different habitats. On this basis During (1979, 1992) proposed a series of life-history strategies among bryophytes (Table 1), and Longton ( l 998b) suggested an evolutionary framework. The perennial stayer, involving long-lived, typically dioecious perennials in stable habitats, was visualised as the most primitive strategy, marked by low reproductive effort and with many non-fruiting, dioecious species represented. Where sporophytes are produced they generally show features thought to be associated with effective dispersal, e.g. long setae, small spores and, in mosses, well developed peristomes. From that starting point, two principal evolutionary trends were envisaged, with the changes thought to have occurred independently in several phyletic lines in each case. Both trends involve reduced duration of habitat availability, associated wjth tendencies towards reduced life span of gametophytes, early and prolific production of spores and/or asexual propagules, and monoecy in sexually reproducing species. The first trend, from the perennial stayer through the colonist to the fugitive strategy, characterises situations where a habitat, once disrupted, does not predictably become available again at the same point. Thus colonists and fugitives retain small spores and other features associated with dispersal (During 1992). It is interesting to note that establishment from spores was achieved readily in a fugitive species (Funaria hygrometrica ), occasionally in colonists (e.g. Bryum argenteum ), and not at all in a perennial stayer (Polytrichum alpestre), in the experiments considered earlier. It is noteworthy also that the ratio of gametophyte: sporophyte biomass in the perennial stayer Tortellajlavovirens in Spanish pine woodland exceeded that of F. hygrometrica on nearby burnt ground by a factor of more than 60 : 1, confirming a substantially higher reproductive effort in the fugitive (Longton 1988a).

Table 1. A revised system of bryophyte life-history strategies (During 1992). Potential life span Spores numerous, small Spores few, large Reproductive (yr) ( < 20µm) ( > 20µm) effect < 1 Fugitives Annual shuttle High Few Colonists (ephemeral colonists, Short-lived shuttle and Medium pioneers, colonists ss) long-lived shuttle Many Perennial stayers Dominants Low R. E. L ONGTON: Reproductive bi ology: challenge and opportunities 165

The second trend is towards the annual shuttle strategy, characterising situations were favourable conditions are of short duration, but do recur predictably at a given site. The annual shuttle strategy, as represented by annual or ephemeral species of, for example, Eccremidium and Phascum growing in deserts or arable fields, is marked by the production of relatively few, large spores in cleistocarpous capsules lacking disper­ sal mechanisms. The significance of large size is thought to lie in facilitating spore establishment, but the normal mode of reproduction remains to be confirmed. These strategies were proposed on a subjective basis (During 1979). However, Hedderson and Longton (in press) have analysed data on life-history characteristics of members of the , Polytrichales and Pottiales by principal component and cluster analyses, the results indicating six clusters of species with characteristics broadly corresponding to strategies in Table I. Intraspecific Variability Recent studies, reviewed by Shaw (199la), have confirmed the occurrence of substantial genetic variability within a number of bryophyte species. However, the extent and patterns of variation differ between species in ways that remain difficult to interpret. The lack of differentiation between populations of many bryophyte species believed to have been isolated on different continents for tens of millions of years is well known, and supports the traditional view of slow evolution among bryophytes. Indeed, Shaw's ( 1991a) own data suggest greater levels of phenotypic variation in morphology among populations of Scope/ophila cataractae within geographical regions as different from each other central Asia and tropical America than between the regions. Studies of clones in collateral cultivation have confirmed the occurrence of substantial genetic variation in several species. Thus in Bryum argenteum individual isolates from many populations remained distinct in axenic culture for several years, and in this case material from continental Antarctica proved distinctive in its leaves having rounded apices and short midribs, in the field and in cultivation (Longton 1981 ). More recent experiments have shown that British plants of B. argenteum var. /anatum remain distinct, in cultures initiated from field-collected gametophytes and from spores, by developing leaves with a hyaline hair point (Longton, unpublished data). Similar hair points have been shown to be effective in reducing water loss in other mosses (Proctor 1980), and could be adaptive in the dry habitats typically occupied by var. lanatum. There are numerous other cases where genecological differentiation in morphological and physiological characters in bryophytes, thought to reflect selection in different environments, has been confirmed by collateral cultivation or reciprocal transplantation. These include topoclinal variation in Polytrichum alepestre that results in short, compact turfs in cool, dry, polar environments (Longton 1974, 1994), and the low maxima for net assimilation rate reported in polar, as compared with temperate populations of several species (Kallio & Saarnio 1986, Sveinjornsson & Oechel 1983). In Funaria hygrometrica, greater tolerance of heavy metals was shown by populations from contaminated as compared with normal soils (Shaw 1991a), and inherent physiological differences have been reported between provenances from different lati- 166 J. Hattori Bot. Lab. No. 76 I 9 9 4 tudes (Dietert 1990, Weitz & Heyn 1981). In several of these studies the variation appeared to result from a combination of complementary genetic and environmental effects and, in general, it appears that phenotypic plasticity is of greater importance in permitting species to survive in a range of environments in bryophytes than in angiosperms. Thus no enhanced tolerance was shown by plants of Bryum argenteum from metal-contaminated soils (Shaw 199la), while polar, temperate and tropical provenances of several mosses have proved remark­ ably similar in relationships between temperature and net assimilation rate (e.g. Rhacomitrium lanuginosum : Kallio & Heinonen 1975) or growth (e.g. B. argenteum : Longton 1981, 1988a). Shaw (199la) has demonstrated substantial variability in growth rate of sporelings and protonema, and in tolerance to copper, among single-spore cultures derived from a single clone of Funaria hygrometrica. Variation was recorded between spores in individual capsules and between families of spores from different capsules, although it was assumed that all the sporophytes had developed following self-fertilization within the clone. Noting that clonal variation is well known in other organisms, Shaw attributed the result to either a high rate of mutation or to microenvironmental effects, of which competition for nutrients among developing spores seems a likely factor. However, the possibility that the clone was polyploid, and that some of the variation arose through meiotic recombination, can not be excluded as a polyploid series with chromosome numbers up ton = 56 (8 X ) has been reported in F hygrometrica (Ander­ son 1980). Electrophoretic studies have recently provided instructive data on genetic variabil­ ity in bryophyte species without resolving all the controversial issues, as discussed in this Symposium by Wyatt ( 1994). The results indicate that the range of means for such parameters as percentage of loci polymorphic and number of alleles per locus is similar in mosses and in vascular plants, even in undoubtedly haploid mosses such as Plagio­ mnium ellipticum (n = 6). The corresponding values are typically lower in liverworts, but still higher than would be expected in haploid-dominant organisms. As Wyatt ( 1994) notes, these data suggest that isozyme polymorphisms are selectively neutral, or that balancing selection is operating. Variation of this type arises by mutation independently of meiosis, and informa­ tion relevant to the effects of any regular bypassing of meiosis requires data on the frequency and distribution of multilocus genotypes. Here, it may be significant that gametophytes in populations of Polytrichum commune were found to be more uniform in this respect than would be predicted for a dioecious, fruiting species (Derda & Wyatt 1990). Similarly, Innes (1990) reported that the range of genoptypes among gameto­ phytes in a population of P. juniperinum was less than would be expected given the range present in the sporophytes, suggesting restricted establishment by spores. In contrast, Hofman ( 1991) reported random distribution of genotypes in fruiting populations, compared with local clustering of individuals sharing the same genotype in non-fruiting populations of P/agiothecium undulatum, suggesting successful spore establishment in the former. Moreover, the data on genetic differentiation between R. E. L ONGTON: Reproductive biology: challenge and opportunities 167 populations, reviewed by Wyatt ( 1994 ), suggest that values for Nei's genetic identity are generally similar in bryophytes and in vascular plants, except in the case of intercontinental disjuncts. Here, the disjunct populations of bryophytes generally show less differentiation in terms of isozyme polymorphism, as in morphology, than do those of vascular plants. This could be interpreted as implying gene flow through spore dispersal, but Vitt ( 1982) has suggested that bryophyte species are held together less by gene flow than by adaptation to particular environments by strong selective forces. One particularly significant finding by Wyatt and his colleagues is that alloploidy has played a greater part in speciation among mosses that was previously assumed, at least in the Mniaceae. This implies also that interspecific hybridisation may be more frequent than so far demonstrated by morphological studies, especially as the large number of multilocus genotypes discovered in the allopolyploid Plagiomnium medium suggest that this species has originated on several occasions (Wyatt et al. 1988). In this Symposium Wyatt ( 1994) comments on the potential benefits of alloploidy in permitt­ ing both heterozygosity in the free-living gametophyte generation and recombination across loci.

THE FUTURE Recent studies have clearly challenged early views, at least in demonstrating that a wide range of genetic variability exists within many moss species. They have also demonstrated that it is dangerous to generalise about the topics under discuission. The extent and pattern of electrophoretic variation differs markedly between species. Some bryophytes fail to produce sporophytes: many others do so and often liberate spores in prodigeous numbers (Longton & Schuster 1983). Some species can be established experimentally from spores in the field: in others this has so far proved impossible to achieve. The extent of cross-fertilization in monoecious species, and of genecological differentiation in species occupying a diversity of habitats, also appear to vary. One suspects, too, that generalising about the age of extant bryophyte species is unwise. The fossil record clearly indicates the persistence of ancient species in some genera, but in other groups, as among epiphytic species of Orthotrichum, the variation patterns are suggestive of currently active speciation (Vitt 1982). A major challenge for the future lies in attempting to discover patterns among these various aspects of reproductive biology and genetics that may indicate the evolutionary processes operating in different groups of bryophytes. Allozyme diversity in Plagiothecium appears to be greatest in the monoecious species (Hofman 1991), but too few data are available to establish whether this is a general trend. Similarly, it is not yet clear whether there are correlations between, for example, the extent of elec­ trophoretic variation, ecotypic differentiation and mode of reproduction, or between degree of variation and chromosome number. Nor is it yet known whether these parameters vary in relation to life-history strategies of the type envisaged in Table 1, so that any discussion of the influence of such strategies on evolutionary processes can only be speculative. One relationship does seem clear. Most species that fail to produce sporophytes 168 J. Hattori Bot. Lab. No. 76 I 9 9 4 anywhere in their range are dioecious, and such species tend also to be rare. Even here, however, we have a chicken and egg situation that is difficult to interpret. Have such species become rare because of failure to reproduce by spores, through consequent lack of meiosis and/or reduction in dispersal ability, or did increasing rarity reduce sporo­ phyte production by limiting the occurrence of bisexual populations? This question has important implications in terms both of managing rare species, and assessing the significance of meiosis. Again, there may be no generally applicable answer, as indicated by the contrasting case studies discussed by Longton and Schuster (1983). Bryophytes are one of few successful groups of organisms whose life-history includes a functionally dominant, free-living haploid phase, and thus the evolutionary processes operating among these plants are of great general interest. Progress in elucidating these mechanisms is dependent on advances along two fronts. First, the wide diversity that is already evident among bryophytes in variation patterns and modes of reproduction implies that a large body of empirical data will be required on the topics discussed in this Symposium before patterns can be established and processes properly understood. At present our pool of information about such matters as inheritance patterns in presumed polyploids, the effectiveness of spore dispersal and establishment, and mating patterns is pitifully small. Advances along this front are most likely to accrue from detailed studies of individual species, or species groups, which combine traditional methods of observation and experiment (Kimmerer 1991, Miles & Longton 1990) with cytological studies of the type proposed by Newton ( 1988) and the molecular approach. It is clear from Wyatt's paper ( 1994 - this Symposium) that electrophoretic studies are already yield­ ing information of great interest. These techniques are capable of assessing diversity, both among alleles and among genotypes, in respect of a relatively narrow range of genes coding for the enzymes employed. Genetic fingerprinting, an approach as yet little applied to bryophytes, is more sensitive as it permits recognition of all individual genotypes based on direct analysis of DNA, but it gives no information about the alleles at individual loci. The two methodologies are therefore complementary, and their combined use offers the prospect of significant advances in studies of genetic diversity and its distribution within and between populations, temporal changes in these patterns, recruitment of new genotypes, fertilization range and related topics. Second, a more robust conceptual framework is required in which to evaluate the results of empirical studies. In this area bryologists have generally been slow to apply the ideas and techniques already successfully applied to other organisms (e.g. Futuyma 1986, Harper 1977, Maynard-Smith 1989). However, the metapopulation approach discussed in this Symposium by Herben ( 1994) and by SOderstrom ( 1994) illustrates the potential of these techniques, as do the earlier simulation models by these authors which attempt to relate population survival to factors such as habitat distribution and duration, and to the production, dispersal and establishment of propagules (Herben, Rydin & Soderstrom 1991). The study of reproductive biology and its relationship to evolutionary processes in bryophytes thus remains an intriguing and challenging area of investigation, and one R. E. LONGTON: Reproductive biology: challenge and opportunities 169 that has considerable biological significance. Exciting advances can be anticipated from studies of the type discussed in this Symposium and in the excellent review by Shaw (199la).

ACKNOWLEDGMENTS I am grateful to Hironori Deguchi and Lars Soderstrom for their cooperation and assistance as co-organisers of this Symposium, to all the participants for their contribu­ tions and stimulating discussion, and to The University of Reading, The Royal Society and the organisers of the XV International Botanical Congress for financial support.

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