Heredity 59 (1987) 337—343 The Genetical Society of Great Britain Received 17 December 1986

Electrophoretic evidence of reproductive isolation between two varieties of the , americanum

Jonathan Shaw, Department of Botany, Duke University, Durham, Thomas R. Meagher and North Carolina 27706, U.S.A. Pamela Harley

Twohundred and twenty individuals of the moss, Climacium americanum, from three localities in the Piedmont of North Carolina were sampled for a study of electrophoretically detectable genetic variation. Eight enzymes (ACP, ADH, ESTfl, GOT, PER, PGI, PGM, and SOD) were assayed and only two (GOT, SOD) were monomorphic at all three localities, in spite of the extreme rarity of sexual reproduction in this species. Although the var. kindbergii has generally been considered a form of C. americanum induced by submersion in water, there was a strong correlation between enzyme phenotypes and morphological characters distinguishing the two varieties of C. americanum. These data suggest that there is linkage disequilibrium among the loci coding for ACP, PGM, and PGI, and between these loci and those controlling several morphological characters that distinguish the two varieties of C. americanum.

INTRODUCTiON However, the ecological correlation is far from absolute and both forms sometimes occur in what TheClimaciaceae is a small family of con- appears to be the same habitat. sisting of four species in a single genus, Climacium. The dimorphism in growth habit in Climacium In North America, the genus is represented by two americanum has resulted in various systematic species, C. americanum Brid. and C. dendroides treatments of the species. Renauld and Cardot (Hedw.) Web. and Mohr. (1890) described C. americanum var. kindbergii has a circumboreal range and is widespread in based on from Louisiana and Massachusetts north-temperate North America, whereas C. having more distant leaves and shorter, oblong leaf americanum is endemic to the eastern United cells as compared to the var. americanum. Although States. Species of Climacium are among the most Renauld and Cardot did not discuss growth form robust and recognizable of temperate mosses, when they described the var. kindbergii, the varietal characterised by below-ground rhizomatous stems epithet has since become associated with forms of and erect, branched, dendroid secondary branches. C. americanum characterized by irregular, non- The common name, tree moss, is descriptive of the dendroid branching. Grout (1901) raised the var. dendroid growth form typical of species in the kindbergii to specific rank, arguing that C. kindber- genus. The production of sporophytes is very rare gii difiers in geographic distribution as well as in both species of Climacium, apparently a result morphology. In 1904, Cardot and Theriot, of most natural populations being unisexual, pro- however, described C. americanum var. pseudo- ducing only female gametangia (Bedford, 1938). kindbergii from Missouri, remarking that it has the Climacium americanum varies in growth form growth form of C. kindbergii but the areolation of from erect and strongly dendroid to elongate and C. americanum, and argued against considering trailing with an irregular branching pattern (fig. them two species. On the basis of additional 1). The variation in growth form of C. americanum observations, Cardot and Theriot (1906) argued has been observed to be dimorphic and is corre- that C. americanum and C. kindbergii intergrade lated with habitat: plants are generally dendroid to a significant extent. in mesic forests and are trailing with irregular More recently, C. kindbergii has been con- branching on rocks in small streams or in swamps. sidered to be an environmentally induced variant 338 J. SHAW, T. R. MEAGHER AND P. HARLEY

exhibit the dendroid growth form. However, the species also occurs on rocks in a small stream in a narrow ravine that drains into the Eno River. All plants observed in the latter habitat are of the non-dendroid, or "kindbergii", form. The small stream varies seasonally in flow rate but some water is apparently always present and many of the clumps of Climacium were at least partially sub- merged. Sixty plants were collected along a transect in the floodplain parallel to the Eno River and sixty were collected along a transect in the LI seasonal stream. Only a single was collected kind berg ii from each clump encountered along the transects. americanum At the remaining two localities, plants con- Figure 1 Morphologicaldifferences between Climacium forming to the vars. americanum and kindbergii americanum var. americanum (left) and var. kindbergii grew in closer proximity to one another although (right). any particular clump exhibited either dendroid or nondenroid growth, i.e., the two forms did not of C. americanum (e.g., Crum and Anderson, grow intermixed in the same discreet clump. Forty 1981). Horton and Vitt (1976) and Crum and plants were collected in the floodplain of New Anderson (1981) interpreted C. kindbergii as a Hope Creek, a small river in the Duke Forest about phenotypic expression of C. americanum induced 3 miles southeast of Durham in Durham County. by submersion in water during all or part of the Again, collections were made at least one meter growing season. Shaw (in press) grew forms refer- apart to avoid multiple collections from the same able to C. americanum and C. kindbergii under clump. Plants characterised by dendroid versus controlled environmental conditions and found nondendroid growth were sampled as they were only weak evidence for discontinuity between encountered along the transect. Sixty plants were them. The degree of tolerance to desiccation varied collected from a similar floodplain habitat along among populations but did not correlate with habi- the Flat River, approximately 18 miles north of tat or morphology. Shaw presented experimental Durham in Person Co. Plants of the two contrasting evidence suggesting that the degree of desiccation growth forms occurred as discreet clumps and were tolerance was more closely related to environ- sampled as encountered along the transect. At both mental pretreatment than to genetic differences New Hope Creek and along the Flat River, plants among plants. were subjectively classified as var. kindbergii or This study of electrophoretically detectable var. americanum as they were collected. Intermedi- variation was initiated in order to assess the level ate forms were noted as such. of genetic variation in C. americanum, a species in Plants were refrigerated in a moistened state which sexual reproduction appears to be extremely for 2-3 days prior to enzyme extraction. Several rare, and to determine whether variation in enzyme extraction buffers were tried in a preliminary study, phenotypes correlates with growth form variation including those described by Kelley and Adams corresponding to the vars. kindbergii and (1977) for sequestering phenolic compounds americanum. released by Juniperus leaves, and by Soltis et a!. (1983) for extracting enzymes from gametophytes of ferns and fern allies. These extraction buffers MATERIALSAND METHODS did not improve the results obtained by extraction in a simpler 01 M tris-HC1 (pH 7.2) buffer with Collectionswere made at three localities in the 05 per cent mercaptoethanol so that the latter was Piedmont of North Carolina near the city of used exclusively in this study. Extractions were Durham. The first locality was along the Eno River tried with and without freezing the plant material in Durham County where the river crosses U.S. in liquid nitrogen prior to grinding; better results Route 70, about 12 miles northwest of Durham. were obtained from frozen material, probably due Climacium americanum is extremely common in to more effective fractionation of the tissue. After the floodplain between two and 15 feet from the the enzymes were extracted and absorbed onto river bank, and forms discreet clumps of irregular paper wicks, the latter were stored in a deep-freeze size and shape. Floodplain plants invariably at —70°C until use. Electrophoretic separation of REPRODUCTIVE ISOLATION IN A MOSS 339

enzyme variants was accomplished on 12 per cent TabJe 1 Summary of electrophoretic variability in C. horizontal starch gels in either a discontinuous americanumby enzyme system and locality. M = monomorphic;P =polymorphic.Numbers in parentheses 02 M tris-citrate/lithium borate buffer system at indicate the number of different bands present among pH 83 (ADH, ACP, GOT, MOH, PGI) or a con- individuals in populations for a given enzyme system tinuous 0065 M histidine buffer atpH 57 (ESTfl, PER, PGM, SOD) (Cardy et a!., 1983). Eno River New Hope Creek Flat River Approximately 20 enzyme systems were tested ADH P(2) M P(2) for activity, and scorably distinct bands were ACP M P(2) P(2) obtained for 8: acid phosphatase (ACP), alcohol GOT M M M dehydrogenase (ADH), fluorescent esterase PGI P(8) P(8) P(8)

(ESTfl), glutamate oxaloacetate transaminase Esifi M P(2) P(3) (GOT), peroxidase (PER), phosphoglucosiso- PER M P(2) M merase (PGI), phosphoglucomutase (PGM), and PGM P(3) P(4) P(3) superoxidedismutase (SOD). Scorable bands were SOD M M M obtained from some individuals for malate dehy- drogenase (MDH) but the amount of activity was The single ESTfl band present at Eno River was too inconsistent to provide scorable reSults. Stain- also present at the other two localities. A second ing of enzymes was according to recipes slightly ESTfl band was present at both Flat River and modified from Shaw and Prasad (1970) except for New Hope Creek localities, and a third, much ESTfl, for which the staining schedule of Soltis et faster, band was expressed in three individuals al. (1983) was followed. (out of 60) from the Flat River. ADH was monomorphic at New Hope Creek but was poly- morphic at the Eno and Flat River localities. Plants RESULTS at the latter two localities expressed the same band as was present at New Hope Creek, plus a second Sixout of the eight enzyme systems studied were band in common between the two localities where polymorphic in at least one of the three collection ADH was polymorphic. Any given individual localities (table 1; table 2). Only GOT and SOD expressed only one band for these enzymes. were monomorphic within each site and were fixed PGI and PGM were both polymorphic at all for the same allele at all three sites. The same PER three sites. Up to eight PGI bands were expressed band was present in all of the 220 individuals in individuals from the three sites although all were sampled except one from New Hope Creek which not scorable in every individual. Variation in PGI expressed a slower band. This individual was also was noted in two sets of bands on the gel. A slowly unique with regard to its PGM phenotype, express- migrating pair of bands exhibited variation, but ing a band not found elsewhere. Two enzymes were too indistinct to be scored consistently; (ACP and ESTfl) were monomorphic at the Eno among faster migrating bands, individual plants River locality but were polymorphic at New Hope expressed either band 3 or band 5, but never both. Creek and Flat River. The ACP band fixed at the In the present analysis, bands 3 and 5 of PGI were Eno River locality was also present at New Hope interpreted as allelic variation at a single locus. Creek and Flat River, and a second ACP band PGM consistently expressed two bands per present at the latter sites was the same at both sites. individual (fig. 2), one of which was monomorphic

Table 2Allele frequencies for polymorphic enzyme loci in Climacium americanum. Sample sizes (N) vary due to uneven scorability of gels

Eno River New Hope Creek Flat River enzymeN alleles N alleles N alleles

ADH 40 0'58 042 40 100 — 37 035 065 *ACp 37 046' 0.542 39 0.741 0.262 ESTJ, 120 — 1.00 — 17 — 041 059 59 005 024 071 *pGI 100 0.582 042' 20 0.602 040' 58 0.292 O•71' *PGM 120 050' — 0.502 40 0.351 005 0.602 60 040' 0.602

* Allelicvariation at these loci correspond to morphological differentiation between C.americanumvar. americanum (1) and var. kindbergii (2) (see table 3). 340 J. SHAW, T. R. MEAGHER AND P. HARLEY

— — — — - a — —

B 2;', a••• 4 ' •

D

Figure 2Phosphoglucomutase banding patterns in the var. americanumandvar. kindbergii.Thelanes correspond to consecutive collections along transects. A. Eno River; all var. kindbergii. B.EnoRiver; all var. americanum. C. Flat River. D.NewHope Creek. At the last two localities both varieties were encountered along the same transects.

across all three populations and the other showed can range from 0 for no association to a maximum three variants which were interpreted as alleles at value of 025 for complete association between a single locus. alleles at different loci. The strongest associations Linkage disequilibrium coefficients (Rough- between observed morph and enzyme variation garden, 1979) among the enzyme loci scored were shown by PGI and PGM. For both of these showed striking genetic differentiation between enzymes, there was almost a complete correspon- Climacium americanum var. kindbergii and var. dence between enzyme banding pattern and americanum (table 3). In the estimation of linkage morphological variation (fig. 2). There was also a disequilibrium (D), the following assumptions and strong association betweeen ACP alleles and adjustments were made: allelic variants that oc- morph; and, where there was sufficient sampling curred in fewer than 3 individuals (ESTfl allele 1 and variation to estimate D for ADH (Flat River), and PGM allele 2) were not included and morpho- a weaker still significant association between ADH logical variants were assigned numerical allelic alleles and morph type. The only enzyme for which scores of 1 for dendroid (var. americanurn) and 2 variation was observed that did not show allelic for nondendroid (var. kindbergii). Estimates of D association with morphological types was ESTfl.

Table 3 Linkage disequilibrium coefficients.(D) among loci assayed (sample sizes in parentheses). G2 statistics (Bishop et al., 1975) for tests of significance of D are also shown. All G2 values are based on 2-way tabulations of allelic variation between pairs of loci and hence have 1 degree of freedom

Eno River New HopeCreek Flat River

PGM PGI ACP EST PGM PGI ACP ADH EST PGM PGJ

ADHD2 016(17) G 100' EST D2 017(17) — 010(37) G 12.2*** 7.7** PGM 021 (36)024 (17) 015 (39)009 (37)004 (56) G 32.3*** 23.0*** 21.4*** 9.3*** 16 PGI D2 0.23(106) 018(19)017(17)019(19) 009(38)006(37)002(55) 013(58) G 118.5*** 12.4*** 12.2*** 15.5*** 9.9*** 3.5 fl.5. 08 fl.S. 18.9*** morphD2 025 (120) 023 (100) 023 (36)020 (16)022 (37)021 (19) 011 (39)008 (37)002 (56) 017 (60)008 (58) G 1664*** 1185*** 414*** 145*** 37.Q*** 19.0*** 11.4*** 81*** 04n.s. 33.6*** 83*** REPRODUCTIVE ISOLATION IN A MOSS 341

DISCUSSION The rarity of sexual reproduction in C. americanum raises the question of the origin of Enzymevariability demonstrated in this study by the genetic variation found in this study. There starch gel electrophoresis provides strong evidence have been a number of recent studies of genetic for the presence of genetic variation in Climacium variation in asexually reproducing plants with the americanum both within and among localities. surprising result that such species contain sig- Variability in enzyme expression can include nificant levels of variation. Jeffries and Gottlieb differences among alleles at a single locus (1983) found 39 enzyme phenotypes among clones (allozymic variability) and differences in the loci of the sterile triploid Puccinellia x phyganodes in that are expressed (isozymic variability). To distin- arctic North America. Christensen eta!. (1982) and guish these sources of variation requires a genetic Elistrand and Levin (1982) likewise demonstrated analysis involving electrophoretic studies of considerable variation in Oenothera laciniata, a parents and their offspring. Climacium rarely permanent translocation heterozygote. Clones of undergoes sexual reproduction, so that genetic Taraxacum officinale have been shown by several analyses of this type are not feasible. Con- workers to have variable enzyme phenotypes. sequently, tentative genetic interpretations of Usberti and Jam (1978) compared asexual and banding patterns were based on observed variation sexual populations of Panicum maximum and in natural populations. found that sexual lines had higher coefficients of In spite of ambiguities concerning the genetic variation for morphological characters and higher basis of observed enzyme variation in Climacium, levels of allozyme polymorphism, but they found our study has demonstrated a surprising degree of no significant differences in the levels of within- variation within and among localities. Sexual population variation. Estimates of heritability reproduction, as indicated by the production of (based on parent-offspring regression) were not sporophytes, appears to be extremely rare in higher for asexual species. Krzakowa (1977) and Climacium americanum throughout its range Krzakowa and Szweykowski (1979) found sig- (Crum and Anderson, 1981). Bedford (1938) found nificant levels of peroxidase variation in the liver- that in C. dendroides, where sporophyte production wort, Plagiochila asp!enioides, a species that is is also very rare, most natural populations express asexual over much of its range. Daniels (1982) also exclusively female sexuality and fertilisation found higher levels of variation in three enzymes occurs only in those infrequent populations which (ACP, EST, PER) within and among British popu- are functionally bisexual. Bedford experimentally lations of Sphagnum pulchrum, in spite of an induced sexual reproduction by transplanting male apparent absence of sexual reproduction in the gametophytes into a population of female populations. Most explanations for the presence gametophytes. Whether such unisexual popula- of such variation in asexually reproducing plants tions contain genetically male plants that produce involves hypothesizing multiple colonisation of no gametangia, has not been determined. sites by different genotypes. Elistrand and Levin Our field surveys have revealed few instances (1982) suggested that Qenothera !aciniata may be of sexual reproduction in C. americanum in North polyphyletic. Daniels (1982) suggested that vari- Carolina. Although the species is common, ation in Sphagnum pulchrum has been retained sporophytes have been found at only five localities, from some hypothetical time in the past when and in each case only two to five sporophytes sexual reproduction was more common. occurred in a given population. Moreover, sexual The correlation between enzyme phenotypes reproduction was restricted to the non-dendroid and morphology indicates the presence of a strong morphological type occurring on rocks in small linkage disequilibrium between the genes coding streams. We have not seen any sporophytes on for these enzymes and the genes that control the plants corresponding to the var. americanum in mode of branching, leaf shape, and leaf areolation North Carolina. During the fall of 1984, sporo- in C. americanum. Linkage disequilibrium, the phytes occurred on a few plants in one clump of nonrandom association of alleles at different loci var. kindbergii from our Eno River collection local- (technically in the gametes, but in bryophytes, in ity, but sporophytes have not been observed at the the gametophyte generation) can have several two other sites included in this study. We found causes. Disequilibrium may be the result of phys- no more variation among plants at the Eno River ical linkage among the genes, the founding of a locality where sexual reproduction is known to population by a few colonisers, associative over- have occurred recently than at other localities. dominance, or by selection acting on favourable 342 J. SHAW, T. R. MEAGHER AND P. HARLEY epistatic gene interactions (Lewontin, 1974). Link- more related to plants of var. kindbergii at another age disequilibrium resulting from physical linkage locality than they are to plants of the var. or a founder event is expected to decrease over americanum at the same locality. This suggests that time as a result of recombination, but selection, the kindbergii populations sampled in this study either directly on the loci under consideration, or are monophyletic and the results are not consistent by associative overdominance, can maintain the with the view that the non-dendroid (kindbergii) disequilibrium in a stable state over long time morphology is an induced modification of the den- periods. The infrequency of sexual reproduction droid morphology influenced by the environment. in general, and in particular the apparent extreme Nor are our results consistent with a view that the rarity of recombination between the vars. kindber- var. kindbergii is a genetically differentiated gii and americanum, indicate that linkage disequi- ecotype of C. americanum that has arisen poly- librium between enzyme loci and those controlling phyletically in wet, stream habitats. Whether our morphology are maintained by a combination of results hold over a broader part of the geographic a founder effect and a lack of inter-varietal re- range of C. americanum is yet to be determined. combination. Those cases in which the disequilib- Although published studies are still relatively rium appeared to break down (i.e., when there was few, there is reason to believe that electrophoretic recombination between morphological and data provide valuable insights into systematic enzyme phenotypes) may be due either to sexual problems in bryophytes. Szweykowski and recombination between the two varieties of C. Krzakowa (1979) studied variation in PER, EST, americanum, or to an incorrect field determination GOT, and GDH in 20 Polish populations of the of the variety made on the basis of morphology. liverwort Conocephalum conicum and found nine When the three enzyme systems of a particular patterns of banding for the four enzymes. As a individual fit the common pattern of disequilib- result of linkage disequilibrium, they could reduce rium but are out of phase with our morphological the variation to two or three recurring enzyme determination, the second explanation is sug- phenotypes. Szweykowski and Krzakowa found gested. Other cases may be the result of sexual that enzyme phenotypes correlated with morpho- recombination. logical races of C. conicum and thus their results An alternative explanation for the linkage dis- are much like those reported here. equilibrium we observed is that the enzyme Krzakowa (1977) studied peroxidase variation variants we assayed are not selectively neutral (or in four species of the liverwort genus Pellia and are closely linked to genes that affect fitness). It found that each species could be characterised by might be argued that the two forms of C. its enzyme phenotype. In addition, Krzakowa americanum occupy distinct habitats and that re- found three populations that were morphologically combinant genotypes are strongly selected against. similar to P. endivifo1ia but which expressed a However, we consider this explanation to be unique enzyme phenotype. DeVries et a!. (1983) extremely unlikely. Although in general, the non- investigated eight enzyme systems of Racopilum dendroid plants occupy wetter sites than dendroid spectabile and R. cuspidatum in the only electro- plants, in many cases the two occur in sites that phoretic study to date on a tropical bryophyte. appear indistinguishable. This is reflected in our They estimated a genetic distance of 0672 between collection of both forms along transects at the New species but the estimate was based on a total of Hope Creek and Flat River localities. There was only 59 plants. There have also been a number of no obvious ecological differentiation between the other recent electrophoretic investigations of infra- two growth forms at these sites. Moreover, selec- specific variation in bryophytes (e.g., Cummins tion against recombinants would have to be and Wyatt, 1981; Daniels, 1982; Krzakowa, 1977; extraordinarily intense to explain the levels of link- Zielinski, 1984a, b, 1986; Zielinski et a!., 1985). age disequilibrium we observed. If favourable epi- The most recent checklist of the mosses of static gene interactions were the cause of the strong North America (Crum et a!., 1973) listed disequilibrium (e.g., Mitton and Koehn, 1973), it Climacium kindbergii as a synonym of C. would be very surprising that precisely the same americanum. Our results, however, coupled with nonrandom association of alleles was observed at morphological data, suggest that C. kindbergii is three localities, each a part of a different drainage distinct and should probably be disinguished either system. at the varietal or specific level. Shaw (in press) The systematic significance of these results is found that when transplanted into a greenhouse, that the genetic data suggest that plants corre- the vars. kindbergii and americanum exhibit sponding to the var. kindbergii in one locality are different modes of growth. Erect, dendroid REPRODUCTIVE ISOLATION IN A MOSS 343 branches of var. americanum appear to be deter- ELLSTRAND, N. C. AND LEVIN, D. A. 1982. Genotypic diversity minate in growth and new growth of plants trans- in Oenothera laciniata (Onagraceae), a permanent translo- cation heterozygote. Evolution, 36, 63-69. planted into a greenhouse occurs by the origin of GROUT, A. .i. 1901. Climacium Web. & Mohr., Iter. Suec. 96. new dendroid branches from the underground 1804. Bryologist, 4, 54. rhizomatous main stems. That is, already formed HORTON, D. G. AND VITT, D. H. 1976. Morphological charac- branches do not exhibit renewed growth. In con- ters, relative to distribution, and taxonomic considerations trast, when C. is transplanted into a of the genus Climacium. Can. .1. Bot., 54, 1872-1883. kindbergii JEFFRIES, R. J. AND GOTTLIEB, L. D. 1983. Genetic variation greenhouse, existing branches produce new growth within and between populations of the asexual plant Puc- from their apices and from the apices of their cinellia x phyganodes. Can. I Bot., 61, 774—779. lateral branches. When grown under non-aquatic KELLEY, W. A. AND ADAMS, R. P. 1977. Preparation of extracts conditions, new growth produced by these plants from Juniper leaves for electrophoresis. Phytochemistrv, 16, is often more dendroid in form than field-grown 513—5 16. KRZAKOWA, M. 1977. Isoenzymes as markers of inter- and portions of the same plants. However, they rarely intraspecific differentiation in hepatics. Bryophyt. Biblio., become as dendroid as plants of the var. 13, 427-434. americanum grown in the field (Shaw, in press). KRZAKOWA, M. AND SZWEYKOWSKI, .s. 1979. Isoenzyme poly- In order to assess the taxonomic status of C. morphism in natural populations of a liverwort Plagiochila asplenioides. Genetics, 93, 711—719. americanum and C. kindbergii it would be desirable LEWONTIN, R. C. 1974. The Genetic Basis of Evolutionary to extend the electrophoretic study reported here Change, Columbia University Press, New York. over a broader geographic range, and to further MITTON, J. B. AND KOEHN, R. K. 1973. Population genetics of investigate the morphological variation by marine Pelecypods. III. Epistasis between functionally reciprocal transplant studies done in the field. related isozymes of Mytilus edulis. Genetics, 73, 489—496. RENAULD, F. AND CARDOT, .j.1890.New mosses of North America. IV. Bot. Gaz., 15, 57-62. ROUGHGARDEN,i. 1979. Theory of population genetics and ecology: an introduction, MacMillan Publishing Co., Inc.; REFERENCES New York. SHAw, .i. 1987. Growth form variation within and among popu- lations of Climacium americanum Brid. Acta Bot. Hung. BEDFORD,T. H. B. 1938. Sex distribution in colonies of (in pçess). Cli,nacium dendroides W. & M. and its relation to fruit SHAW, D. R. AND PRASAD, R. 1970. Starch gel electrophoresis bearing. NW Nat., 13, 213-221. of enzymes—a compilation of recipes. Biochem. Gen. 4, BISHOP, V. M. M., FEINBERG, S. E. AND HOLLAND, P. W. 1975. 297—320. Discrete multivariate analysis: theory and practice, MIT SOLTIS, D. E., HAUFLER, C. H., DARROW, D.C. AND GASTONY, Press, Cambridge, Mass. o. .t., 1983. Starch gel electrophoresis of ferns: a compilation CARDOT, J. AND THERIOT, I. 1904. New or unrecorded mosses of grinding buffers, gel and electrode buffers, and staining of North America, II. Bot. Gaz., 37, 363-382. schedules. Am. Fern. J., 73, 9—27. CARDOT,3. AND THERIOT, I. 1906. New or unrecorded mosses SZWEYKOWSKI, J. AND KR.ZAKOWA, M. 1979. Variation of four of North America. Bryologist, 9, 6—10. enzyme systems in Polish populations of Conocephalum CARDY, B. 3., STUBER, C. W. AND GOODMAN, M. M. 1983. conicum (L.) Dum. (Hepaticae, Marchantiales). Bull. Acad. Techniques for starch gel electrophoresis of enzymes from Polon. Sci., Ser. Sci. Biol., 27, 37—41. maize (Zea mays L.). Institute of Statistics Mimeo Series USBERTI, J. A. AND JAIN, S. K. 1978. Variation in Panicum No. 1317. North Carolina State University, Raleigh, NC. maximum: a comparison of sexual and asexual popula- 35pp. tions. Botanical Gazette, 139, 112—116. CHRISTENSEN, B., BERG, M. AND JELNES, .j.1982.Genotypic ZIELINSKI, R. 1984a. Genetic variation and evolution of the diversity in Oenothera laciniata (Onagraceae), a permanent liverwort genus Pellia with special reference to Central translocation heterozygote. Evolution, 36, 63-69. European territory. I. Electrophoretic evidence of cross- CRUM, H. A. AND ANDERSON, L. E. 1981. Mosses of Eastern fertilization in the monoecious Pellia epiphilla, n =9.J. North America, Columbia Univ. Press, New York. Hattori Bot. Lab., 56, 255-262. CRUM, H. A., W. C. STEERE AND ANDERSON, L. E. 1973. A new ZIELINSKI, R. 1984b. Genetic variation and evolution of the list of mosses of North America north of Mexico. Bryol- liverwort genus Pellia with special reference to Central ogist, 76, 85—130. European territory. II. Electrophoretic and cytological CUMMINS, H. AND WYATT, R. 1981. Genetic variability in study of the Pellia epiphylla and P. borealis complex. I natural populations of the moss Atrichum angustatum. Bry- Hattori Bot. Lab., 56, 263—269. ologist, 84, 30-38. ZIELINSKI, R. 1986. Cross-fertilization in the monoecius Pellia DANIELS, R. E. 1982. Isozyme variation in British populations borealis, n =18,and spatial distribution of two peroxidase of Sphagnum pulchrum (Braithw.) Warnst. J. Bryol., 12, genotypes. Heredity 56, 299-304. 1—11. ZIELINSKI, R., SZWEYKOWSKI, J. AND RUTKOWSKA, v. 1985. DEVRIES, A., VAN ZANTEN, B. 0. AND VAN DJJK, H. 1983. A further electrophoretic study of peroxidase isoenzyme Genetic variability within and between populations of two variation in Pellia epiphylla (L.) Dum. from Poland, with species of Racopilum (Racopilaceae, ). Lindber- special reference to the status of Pellia borealis Lorbeer. gia, 9, 73-80. Monogr. Syst. Bot. Missouri Bot. Gard., 11, 199-209.