HIGH LEVELS OF GENETIC VARIABILITY IN ... - Wiley Online Library

Evolution, 43(5), 1989, pp, 1085-1096

HIGH LEVELS OF GENETIC VARIABILITY IN THE HAPLOID MOSS PLAGIOMNIUM elLIARE ANN STONEBURNER Department of Botany, University of Georgia, Athens. GA 30602

ROBERT WYATT, IRENEUSZ}, ODRZYKOSKl,' AND

Abstract, - Horizontal starch-gel electrophoresis was used to measure variability at 14 enzyme loci from 13 natural populations of the dioecious moss Plagiomnium ciliare. Overall levels of genetic polymorphism were unexpectedly high for a haploid organism. Using a 1% frequency criterion, 71% of the loci surveyed were polymorphic for the species as a whole. The number of alleles per polymorphic locus for the species as a whole was 2.82 ± 0.34 (mean ± standard error), and mean gene diversity per locus was 0.078 ± 0.035. While total gene diversity (HT = 0.178) was similar to that observed for highly outcrossed diploid plants such as pines, the variance within (Hs = 0.098 ± 0.027) and among (D sT = 0.080 ± 0.033) populations was more evenly distributed than that reported for populations of conifers. Genetic distances between populations ranged from 0.0002 to 0.2064, with mosses from the Piedmont region of the southeastern United States showing less differentiation among populations than did mosses from the Appalachian Mountains. Gene diversity was much reduced in populations from disturbed, secondary forests in the Piedmont (0.058 ± 0.018) relative to those from minimally disturbed, primary forests in the mountains (0.146 ± 0.048). Intensive sampling within populations revealed heterogeneity even within small (5 x 5 ern) clumps. The discovery of high levels of genetic variability in a plant with a dominant haploid life cycle challenges the traditional view ofbryophytes as a genetically depauperate group. Multipleniche selection is proposed as a possible explanation for this anomaly, but the data are also consistent with the view that allozyme polymorphisms are selectively neutral. Received February I. 1988. Accepted March 20, 1989

Traditional views of the genetic structure of bryophyte populations hold that mosses and liverworts are genetically depauperate organisms that underwent adaptive radiation long ago and today are limited to a modest role in natural communities. The view that bryophytes evolve more slowly than flowering plants and have remained relatively unchanged for millions of years has been expressed by many authors (Gemmell, 1950; Steere, 1954; Anderson, 1963, 1980; Schuster, 1966; Crum, 1972). Such thinking is based on the facts that most bryophytes are functionally haploid and that the genotype is, therefore, subjected directly to natural selection. The widespread occurrence of asexual reproduction and presumably high levels of self-fertilization are also expected to contribute to low levels of genetic variability. Rates of evolution have been assumed to be slow, because fossil bryophytes usually are morphologically similar to extant taxa. On the other hand, some researchers have argued that genetic variation in bryophyte populations may be

extensive (Khanna, 1964; Longton, 1976; Smith, 1978; Wyatt, 1982, 1985). Their view is based on recent discoveries of extreme diversity in biochemical, physiological, and ecological properties of mosses and liverworts. Few workers have attempted to assess levels of genetic variability in natural populations of bryophytes using the technique most commonly used in similar studies of other plants and animals: electrophoresis of proteins. Existing evidence suggests that levels of electrophoretically detectable genetic variation in mosses are much higher than predicted by the traditional view (Cummins and Wyatt, 1981; Daniels, 1982, 1985; de Vries et aI., 1983; Wyatt et aI., 1988). Similarly, electrophoretic analyses of liverwort populations have uncovered a surprising abundance of genetic variation (Krzakowa and Szweykowski, 1977a, 1977b, 1979; Szweykowski and Krzakowa, 1979; Szweykowski et aI., 1981a, 1981b; Odrzykoski and Szweykowski, 1981; Odrzykoski etaI., 1981; Yamazaki, 1981, 1984; Dewey, 1989). Wyatt et aI. (1989) have discussed the implications of these findings in terms I Permanent address: Department of Genetics, Institute of Biology, Adam Mickiewicz University, 165 of bryophyte population structure and evoDabrowskiego, Poznan 60-594, Poland. lution. 1085

1086

R. WYATT ET AL.

I------i IOOkm

FIG. I. Locations of the 13 populations of Plagiomnium ciliare sampled in the southeastern United States. Abbreviations for populations (indicated by dots) are given in Table 2. Abbreviations for physiographic provinces are: AP = Appalachian Plateaus, VR = Valley and Ridge, BR = Blue Ridge, P = Piedmont, and CP = Coastal Plain.

As part of a larger study of evolutionary relationships among haploid-polyploid species pairs in the Mniaceae, we assessed levels of electrophoretically detectable genetic variation in natural populations of the dioecious moss Plagiomnium ciliare (c. Muell.) Kop, Gametophytes of this species are haploid (n = 6) and occur in colonies consisting of plagiotropic sterile shoots and erect fertile shoots 0.5-2 em tall. An endemic North American species, P. ciliare grows abundantly in mesic woods in the eastern United States and adjacent Canada, with its center of distribution in the Appalachian Mountains (Koponen, 1971). MATERIALS AND METHODS

Population Samples. - We sampled a total of 13 populations from throughout the range of P. ciliare in the southeastern United States (Fig. 1). These populations were located in several physiographic provinces. At

each site, we collected 5-cm x 5-cm clumps from within discrete colonies, placing these samples into small plastic pots. We collected along stream banks until we had sampled a total of 36 discrete colonies or until we had covered a distance of approximately 1 km. Samples were returned to the lab, and a single shoot from each pot was selected for electrophoresis. Plants from the Botanical Garden (BG) population, which were monomorphic at nearly all loci, were used as "standards" for comparing enzyme mobilities. To test for the possibility of microscale genetic variation, we sampled the 36 clumps from the Morning Star (MS) population intensively. From each 5-cm x 5-cm clump, we removed five erect shoots, one from the center and one from each corner of the square pots, and analyzed each shoot by horizontal starch-gel electrophoresis. We then tabulated the percentage of clumps

GENETIC VARIABILITY IN A HAPLOID MOSS

within which two or more distinct electrophoretic phenotypes occurred. Electrophoretic Procedures. -Our procedures for horizontal starch-gel electrophoresis were similar to those described by Odrzykoski and Gottlieb (1984). Single moss shoots were homogenized in 50-100 JoLI extraction buffer (0.1 M Tris HCI, pH 7.5, containing 10 mM KCI, 10 mM MgCl2' 6H 20 , 1 mM EDTA (Na2 salt), 0.1% Triton X-lOO, and [added just before extraction] 42 mM 2-mercaptoethanol). The extract was then filtered through a small strip of Miracloth onto 4-mm x 8-mm Beckmann paper wicks. All steps ofhomogenization were done over crushed ice. Saturated wicks were placed into a vertical slot (the origin) cut across a 10% starch gel, and enzymes were separated in one of three buffer systems. Buffer M, which resolved malate dehydrogenase (MDH), triose phosphate isomerase (TPI), and phosphoglucomutase (PGM), consisted of 40 mM citric acid, titrated to pH 6.1 with N-3 (3-aminopropyl) morpholine. The gel buffer was prepared by diluting 36 ml of electrode buffer with 964 ml water. These three enzymes also can be separated using buffer H (43 mM trisodium citrate' 2H 20 , titrated to pH 7.0 with citric acid). The gel buffer for this system consisted of 5 mM DL-histidine HCI titrated to pH 7.0 with NaOH. This buffer gave similar phenotypes to buffer M but yielded slightly better resolution of PGM. Buffer S was used to separate glutamate oxaloacetate transaminase (GOT), aldolase (ALD), esterase (EST), phosphoglucoisomerase (PGI), malic enzyme (ME), and peptidase (PEP). The electrode buffer for this system was 190 mM boric acid and 60 mM LiOH· H 20 (pH 8.3). The gel buffer was a mixture of 900 ml of 50 mM Tris, 6 mM citric acid, (pH 8.3), and 100 ml of electrode buffer. After mixing, the final pH of the gel buffer decreased to 8.2. Gels were run in a refrigerated chamber (4°C) for four hours in buffers M and H at a constant amperage of 35 rnA and for five hours in buffer S at a constant amperage of 45 rnA. By the end ofthese runs, the bromophenol blue marker had migrated 90 mm in buffers M and H, and the brown "borate front" had migrated 80 mm in buffer S. After separation, enzymes were visualized us-

1087

ing standard colorimetric methods of staining (Shaw and Prasad, 1970; Harris and Hopkinson, 1976) with only slight modifications. Except for EST and GOT, which were stained in liquid assay, all enzymes were stained for 1-3 hours, using the agaroverlay method. Staining was done in an incubator at 37°-400C. Coded data for the 14 loci in the 13 populations were analyzed using BIOSYS-l (Swofford and Selander, 1981) and a program developed in the laboratory of J. L. Hamrick (Department of Botany, University of Georgia). REsULTS

Electrophoretic Patterns. - Two enzymes, ALD and ME, showed only one region of activity on the gels (see Appendix). ALD was monomorphic in all populations, while ME existed as two mobility variants. GOT and PGI showed one intensely staining region and another, less active isozyme, which we chose not to score. From 2-5 regions of EST activity, we scored the most intensely staining zone, which appeared to account for more than half of the total activity. This enzyme always appeared as a two-banded phenotype. Mobility differences invariably involved both bands changing in concert. A few plants showed no EST activity and were scored as carrying null alleles. We found three isozymes of PEP that could use DLleucyl-phenylalanine as a substrate. While PEP-l and PEP-3 used L-valyl-L-Ieucine as a substrate, only PEP-2 was able to use L-Ieucyl-glycyl-glycine. This substrate specificity convinced us that these enzymes should be treated as products of separate genes. PGM activity was detected in three regions, the fastest of which was monomorphic in all plants that expressed it. Many plants, however, lacked this isozyme or showed reduced PGM activity. Therefore, we did not include it in our analysis. The other PGM isozymes were consistently scoreable, each with three or four mobility variants. We found seven two-isozyme combinations of these variants, and therefore, we assumed that the enzymes are products of two separate genes. In three regions of the gel, we found bands ofMDH activity. The fastest single band, which was usually

1088

R. WYATT ET AL.

monomorphic, was unstable and, therefore, was omitted from our analysis. Since in all cases changes in mobility affected all three bands ofMDH-1 activity, this three-banded phenotype possibly represents posttranslational modification of a single gene. Phenotypes ofMDH-2 also were three-banded. We treated two variants detected in this region as two alleles of a single gene. TPI activity consisted offive isozymes in two separate regions, but only three of these had high activity. The first region contained three two-banded phenotypes, which we interpreted conservatively as allelic variants of a single gene (TPI -I). From the second region, only the slowest band had high activity and was found to exist as three allelic variants (TPI-2). Photographs and further discussion ofthese loci in closely related species of Plagiomnium section Rosulata are provided by Wyatt et al. (1988, 1989). Levels of Genetic Variation. - Of the 14 enzymes screened by electrophoresis, only three (GOT-I, ALD-l, and PGI-I) were monomorphic in all populations (Table 1). PEP-3 was polymorphic in only one population, while MDH-2 was polymorphic in only two. Using a 1% frequency criterion, 71% of the loci surveyed were polymorphic for the species as a whole. Even using the more stringent 5% frequency criterion, polymorphism in P. ciliare was 36%. On average, 31.1% ofthe loci were polymorphic per population, with a range from 0% for the Broad River population to 64% for the Coweeta and Morning Star populations. The mean number of alleles per locus ranged from 1.00 to 1.79, with a mean of 1.35. Considering only polymorphic loci, the number of alleles per locus for the species as a whole was 2.82 ± 0.34 (mean ± standard error). Mean intrapopulational gene diversity (Hs ; Nei, 1973) for all loci ranged from 0.000 to 0.138 with a weighted mean of 0.078 (Table 2). Total gene diversity (HT ; Nei, 1973, 1975) based on mean allelic frequencies of polymorphic loci over all populations was 0.178 (Table 3). For all polymorphic loci except MDH-I and ME-I, the largest proportion of this variance is due to diversity within populations (Hs ), rather than between populations (D ST ) ' Differences between the two measures were generally small, however,

with H s averaging 0.098 ± 0.027 and D ST averaging 0.080 ± 0.033. Nei's (1973, 1975) Dm is an absolute measure of gene differentiation which estimates the minimum net codon differences between populations independent of gene diversities within subpopulations. For our moss populations, Dm ranged from 0.000 to 0.299, with a mean of0.086 ± 0.036 (Table 3). GST ' which measures diversity between populations relative to total diversity (Nei, 1973, 1975), averaged 0.248 ± 0.070, while Rsr. the ratio of between- to within-population diversity, averaged 0.565 ± 0.229 (Table 3). This indicates that there is approximately half as much variation between populations as there is within populations. As indicated by gene-diversity statistics (Table 4), individual populations of P. ciliare differ strongly in levels ofgenetic polymorphism. Populations from the Piedmont are clearly less polymorphic than those from other physiographic provinces in terms of the proportion of polymorphic loci (16.5% vs. 44.9%), the mean number of alleles per locus (1.17 vs. 1.52), and gene diversity (0.058 vs. 0.146). Three loci (Mdh-I, Pgm2, and Me-I) were far more variable in populations from the mountains than from the Piedmont. On the other hand, Pgm-I showed high levels ofgene diversity in both areas, although more of the variation in Piedmont populations was due to differences between populations (G ST = 0.548 for the Piedmont, GST = 0.272 for the mountains). Overall gene differentiation, as measured by Nei's (1975) Gsr. is similar in Piedmont and mountain populations (0.161 vs. 0.171). On average, populations from the Piedmont are also more similar genetically: Dm = 0.039 for six pairs of Piedmont populations; ti; = 0.082 for seven pairs of mountain populations. Genetic distances between pairs of populations ranged from 0.0002 (between Botanical Garden and Broad River) to 0.2064 (between Watson's Mill and Pond Drain) (Table 5). Generally, there was less differentiation among populations from the Piedmont than among populations from the mountains. A phenogram summarizing genetic similarities among the populations grouped all of the Piedmont samples plus the sample from Alabama together before

1089

GENETIC VARIABILITY IN A HAPLOID MOSS

TABLE 1. Allele frequencies for 14 enzyme loci sampled in 13 populations of Plagiomnium ciliare in the southeastern United States. Abbreviations for populations are given in Table 2. Codes for enzymes: GOT = glutamate oxaloacetate transaminase, ALD = aldolase, PGI = phosphoglucoisomerase, EST = esterase, MDH = malate dehydrogenase, PGM = phosphoglucomutase, TPI = triose phosphate isomerase, ME = malic enzyme, PEP = peptidase. Plants that showed no EST activity were scored as carrying null alleles. Population

Locus

Got-l Ald-l Pgi-l Est-l

Allele

a a a a

BO

OMC

WM

EM

BR

ER

COW

PC

OCS

TC

PO

MS

HLO

1.00 1.00 1.00 1.00

1.00 1.00 1.00 0.87

1.00 1.00 1.00 1.00

1.00 1.00 1.00 1.00

1.00 1.00 1.00 1.00

1.00 1.00 1.00 1.00

1.00 1.00 1.00 0.94

1.00 1.00 1.00 1.00

1.00 1.00 1.00 1.00

1.00 1.00 1.00 1.00

1.00 1.00 1.00 1.00

1.00 1.00 1.00 0.97

1.00 1.00 1.00 0.92

b

Mdh-l

c null a b

0.13

0.08 0.06 1.00

1.00

1.00

0.92

1.00

0.94 0.06

0.45 0.55

0.36 0.64

0.40 0.60

0.03 0.97

0.97 0.03

1.00

1.00

0.06 0.86 0.08 1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

0.39 0.61

0.75 0.25

0.32 0.68

1.00

0.80 0.20

0.81 0.19

0.19 0.73

0.91 0.09

0.44 0.56

0.57 0.43

0.61 0.17 0.22

0.94 0.06 0.42 0.55 0.03 0.72 0.25 0.03

1.00

0.08

c Mdh-2

a

1.00

1.00

1.00

b

Pgm-I

a b

0.81

0.03

0.19

0.93 0.07

0.31 0.69

0.07 0.93

1.00

0.94 0.06

0.90 0.10

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

0.83 0.17

0.08 0.78 0.22

1.00

1.00

1.00

1.00

1.00

0.92 0.08

0.97 0.03

1.00

0.86 0.11 0.03 1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

0.84 0.16

1.00

1.00

1.00

1.00

0.94 0.06 0.97

0.45 0.55 1.00

0.19 0.81 1.00

1.00

0.81 0.19 1.00

1.00

1.00

1.00

1.00

0.03 1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

1.00

c Pgm-2

Tpi-l

a b c d a b

c Tpi-2

Me-l Pep-I

Pep-2 Pep-3

a b c a b a b c a b a b

1.00

these joined any of the mountain samples (Fig. 2). Populations from the mountains were much less similar to each other. There were, however, no obvious correlations between geographical distances between populations and their genetic distances. Patterns of geographical variation for individualloci generally reinforce the picture from gene-diversity statistics and genetic distances. Me-L" is absent from Piedmont populations of P. ciliare but is present, often as the most common allele, in all mountain populations except Tamassee Creek (Fig. 3A). Piedmont populations also are nearly

0.97 0.03

0.29 0.71 0.92 0.08

1.00

1.00

1.00 0.93 0.07 0.97 0.03 1.00

0.06 0.11 0.89 1.00

1.00 1.00

0.25 0.75 1.00

0.62 0.38 0.96 0.04

0.97 0.03 1.00

0.83 0.17 1.00

monomorphic for Mdh-L", while most mountain populations have Mdh-I" as the most common allele (Fig. 3B). Mountain populations also are more variable among themselves, and Mdh-I: is restricted to the Coweeta population in the Blue Ridge Mountains. Nearly all Piedmont populations are fixed for Pgm-Z" (Fig. 3C). Pgm2b occurs in higher frequency in the mountains, and two rare alleles (Pgm- 2c and Pgm-2d) are restricted to mountain populations. Finally, there is no clear pattern to variation at Pgm-l (Fig. 3D). Even closely adjacent populations, such as Broad River

1090

R. WYATT ET AL.

TABLE 2. Sample sizes (N), gene diversities (Hs), and standard errors (SE) for 14 enzyme loci surveyed in 13 populations of Plagiornniurn ciliare from the southeastern United States. The first six populations are from the Piedmont. Population

locality

BG GMC WM EM BR ER COW PC OCS TC PD MS HLD

Hs

N

Botanical Garden, Athens, GA Goldmine Creek, Braselton, GA Watson's Mill State Park, GA Echol's Mill, Lexington, GA Broad River, Elberton, GA Eno River, Durham, NC Coweeta Hydrologic Lab, NC Panther Creek, Toccoa, GA Oconee Station Cove, SC Tamassee Creek, Walhalla, SC Pond Drain, Mt. Lake, V A Morning Star, Basye, V A Holt Lock and Dam, Holt, AL

72 32 29 26 15 36 36 22 36 30 36 36 24

Grand weighted mean:

430

and Watson's Mill from the Piedmont of Georgia, frequently have very different allele frequencies. Again, the rare allele PgmIe is found only in one mountain population. Intensive sampling within the 36 clumps of P. ciliare from the Morning Star population detected five clumps that were genetically heterogeneous (i.e., consisting of two or more plants that differed in multilocus electrophoretic phenotypes). Three of these five clumps showed variability for more than two enzyme loci. DISCUSSION

Gottlieb (1982) proposed a basic model for plant isozymes that suggests that num-

0.009 0.097 0.023 0.033 0.000 0.073 0.127 0.116 0.110 0.138 0.088 0.124 0.113

±

SE

± ± ± ± ± ± ± ± ± ± ± ± ±

0.009 0.040 0.016 0.024 0.000 0.039 0.041 0.055 0.051 0.054 0.044 0.047 0.045

0.078 ± 0.035

bers and subcellular locations of isozymes are highly conserved in diploid flowering plants. Most plants have two isozymes for each specific enzyme, one of which is active in organelles and one of which catalyzes the same reaction in the cytosol. Most of the enzymes we surveyed in the moss P. ciliare are consistent with this model. Some nonspecific enzymes, such as esterases and peptidases, showed additional isozymes, as did MDH, which often exists as 3-4 different isozymes in flowering plants (Gottlieb, 1982). MDH-2 displayed an unusual phenotype consisting of three bands of equal activity. We treated the two variants that we detected as corresponding to two alleles

TABLE 3. Total gene diversity (HT) and gene diversities within (Hs) and between (DsT) populations of Plagiornniurn ciliare for the polymorphic loci. Also represented are indexes of gene differentiation between populations (Dm ), between-population diversity relative to within-population diversity (RST), and between-population diversity relative to total diversity (GST). Enzyme codes are given in Table I. Locus

Est-l Mdh-l Mdh-2 Pgrn-l Pgm-Z

Tpi-l Tpi-2 Me-l Pep-l Pep-Z

Pep-3 Mean: SE:

HT

Hs

DsT

Dm

0.0413 0.4203 0.0320 0.4573 0.3553 0.1307 0.0321 0.3830 0.0412 0.0543 0.0046

0.0381 0.1447 0.0276 0.2706 0.2309 0.1108 0.0306 0.1345 0.0373 0.0472 0.0045

0.0032 0.2756 0.0045 0.1868 0.1244 0.0198 0.0015 0.2485 0.0039 0.0071 0.0001

0.1775 0.0554

0.0979 0.0268

0.0796 0.0329

0.0034 0.2985 0.0048 0.2023 0.1348 0.0215 0.0016 0.2692 0.0042 0.0076 0.0001

GsT 0.0764 0.6557 0.1392 0.4084 0.3502 0.1519 0.0472 0.6489 0.0946 0.1301 0.0255

0.0896 2.0629 0.1752 0.7478 0.5839 0.1940 0.0536 2.0021 0.1132 0.1621 0.0283

0.0862 0.0356

0.2480 0.0701

0.5648 0.2291

RST

1091

GENETIC VARIABILITY IN A HAPLOID MOSS

at a single locus. It is possible, however, that these three-banded phenotypes actually represent fixed heterozygosity of a dimeric enzyme. Additional work is necessary to determine which of the two explanations is correct. We found three isozymes of PGM in P. ciliare and five isozymes ofTPI, an increase from the usual two (Gottlieb, 1982). Increases in isozyme numbers can result from gene duplication or from the addition of genomes via polyploidy. Gottlieb (1982) reviewed the evidence for duplicated isozymes in diploid plants. No cases of gene duplication have been reported previously in any bryophyte. Likewise, no one has suggested that any moss species with n = 6 is likely to be polyploid. Haufter (1987) suggested that repeated cycles of polyploidy and gene silencing have occurred in homosporous ferns, yielding isozyme numbers typical of diploid plants (but see Haufter and Soltis [1986] and Soltis and Soltis [1989] for alternative explanations). It is possible, therefore, that isozyme multiplicity resulting from an ancient polyploidization in the ancestors of P. ciliare has been silenced at all but a few loci. Because we have already detected at least three loci that may have been duplicated, it seems quite likely that the genome of P. ciliare may have been duplicated by polyploidy and subsequently silenced at the majority ofloci. We are presently gathering additional evidence, including studies of isozyme numbers in other species of Plagiomnium, which should allow us to reject either the hypothesis of single-gene duplication or the polyploidization hypothesis. The most important conclusions to be drawn from our analyses of genetic variation in P. ciliare are that this haploid moss maintains an unexpectedly high amount of

BG BR EM ,-----GMC L..-. HLO ,------WM L..-.---ER

....-------pc

----[===== C ===== 6~s

~------MS

r

cow

L

L..-.----i ,

0.80

PO

i i i

083

0.87

0.90

I

I

i

0.93

0.97

1.00

Genetic Similarity FIG. 2. Phenogram expressing overall levels of genetic similarity among 13 populations of P/agiomnium ciliare based on Rogers's (1972) coefficient of genetic similarity using 14 putative gene loci. Abbreviations for populations are given in Table 2.

vanation and that populations from the southeastern United States display strong population differentiation. Also of major importance is the discovery that genetic variability is severely reduced in the disturbed Piedmont region versus the relatively undisturbed mountain regions. Our results agree with those of most previous electrophoretic studies of bryophyte populations: more genetic variation exists than is predicted by the traditional view of bryophyte variation and evolution. High levels of polymorphism and mean numbers ofalleles per locus were detected by de Vries et at. (1983) in two species of Racopilum. In fact, average gene diversities within populations of R. spectabile and R. cuspidigerum were closely comparable to those for wind-pollinated, highly outcrossed pines (Guries and Ledig, 1982; Loveless and Hamrick, 1984). With the exception ofYamazaki's (1981, 1984) studies, genetic variation reported in liverworts appears to be less than that in mosses, as predicted by Khanna (1964).

TABLE 4. Estimates of genetic variation in populations of Plagiomnium ciliare from the Piedmont and the mountains. Npop = number of population samples; Nenz = number of enzymes screened; Nloci = number of putative loci; PLP = average percentage of loci polymorphic; k = mean number of alleles per locus. HT. Hg, GST, and Dm are defined in Table 3. N enz

Nloci

PLP

k

HT

Hs

GST

Dm

Piedmont Mountain

6 7

9 9

14 14

16.5 44.9

1.17 1.52

0.091 0.216

0.058 0.146

0.161 0.1 71

0.039 0.082

Total:

13

9

14

31.1

1.35

0.178

0.098

0.248

0.086

Population

Npo p

A) ME-I

~

B) MOH-I

b

,, I

:n " "J.l~} 0) PGM-I c~a

~

c

b

,-------

, , I

I

I I I

,, I

I

, I

I

I I

, I

~ ~tl

-,

'.:~.:,J

FIG. 3. Geographic patterns of variation in allele frequencies at four loci of P/agiomnium ciliare: A) Me-J; B) Mdh-J; C) Pgm-2; D) Pgm-J. See Figure I for more information regarding the sample locations.

TABLE 5. Nei's (1972) genetic distances (below the diagonal) and genetic identities (above the diagonal) for 13 populations of P/agiomnium ciliare in the southeastern United States. Abbreviations for populations are given in Table 2. Geographical locations of the populations are shown in Figure I. Population

BG

BG GMC WM EM BR ER COW PC OCS

TC PD

MS HLD

0.0419 0.0633 0.0024 0.0002 0.0310 0.1336 0.0924 0.1262 0.0550 0.1502 0.0808 0.0530

GMC

WM

EM

BR

ER

COW

PC

0.9590

0.9387 0.9918

0.9976 0.9520 0.9312

0.9998 0.9542 0.9334 0.9978

0.9695 0.9936 0.9892 0.9633 0.9651

0.8749 0.8550 0.8409 0.8774 0.8734 0.8677

0.9118 0.9448 0.9439 0.9095 0.9070 0.9491 0.8919

0.0083 0.0491 0.0469 0.0064 0.1566 0.0568 0.1748 0.0833 0.1847 0.0585 0.0121

0.0713 0.0689 0.0108 0.1732 0.0577 0.1965 0.1020 0.2064 0.0643 0.0156

0.0022 0.0374 0.1308 0.0949 0.1225 0.0532 0.1468 0.0865 0.0604

0.0355 0.1353 0.0976 0.1260 0.0562 0.1514 0.0853 0.0581

0.1419 0.0522 0.1599 0.0669 0.1696 0.0565 0.0152

0.1144 0.0716 0.0265 0.1051 0.1710 0.1640

0.0746 0.0717 0.0603 0.0335 0.0320

GENETIC VARIABILITY IN A HAPLOID MOSS

Plagiomnium ciliare, despite its haploiddominant life cycle, falls close to the mean values of dicots for the percentage of polymorphic loci per population, the mean number of alleles per locus, and gene diversity (Hamrick et al., 1979). Comparing P. ciliare with diploid seed plants on the basis of polymorphic loci only, this haploid moss is above average for among-population diversity and below average for withinpopulation diversity (see Loveless and Hamrick, 1984). Overall levels of gene diversity in P. ciliare are closely comparable to those measured in pitch pine (Pinus rigida) by Guries and Ledig (1982). In pitch pine, however, there is almost no differentiation among populations (mean GST = 0.023). Populations ofP. ciliare show values an order of magnitude larger for these statistics, reflecting stronger dissimilarity among localities. This pattern is reinforced by close examination of geographical patterns in allele frequencies at particular loci, which differ sharply even between closely adjacent populations. Pairwise genetic distances among the 13 populations of P. ciliare in the southeastern United States are generally within the range observed for conspecific populations ofdiploid plants (e.g., mean = 0.0954 for Clarkia species tabulated by Ayala [1975]). Piedmont populations of P. ciliare were more similar to each other than were the populations from the Appalachian Mountains. This may be explained by the fact that our Piedmont samples came almost entirely from Georgia, while the Appalachian

TABLE

5. Extended. Population

OCS

TC

PD

MS

HLD

0.8814 0.8396 0.8216 0.8847 0.8816 0.8523 0.9309 0.9282

0.9465 0.9201 0.9031 0.9482 0.9454 0.9353 0.9738 0.9308 0.9379

0.8606 0.8314 0.8135 0.8635 0.8595 0.8440 0.9003 0.9415 0.9716 0.9114

0.9223 0.9432 0.9377 0.9172 0.9182 0.9451 0.8428 0.9670 0.9247 0.8951 0.9110

0.9484 0.9880 0.9845 0.9414 0.9435 0.9849 0.8487 0.9685 0.8738 0.9107 0.8668 0.9766

0.0641 0.0288 0.0782 0.1349

0.0928 0.1108 0.0935

0.0932 0.1429

0.0236

1093

Mountain populations were sampled from five states and were spread over a much wider geographical area. Plagiomnium ciliare is a dioecious moss that reproduces regularly by sexual means. Sporophytes mature in late summer and release approximately 1-2 x 105 wind-dispersed spores. No specialized asexual propagules are produced, but like most mosses, P. ciliare is capable ofregeneration from leaf or stem fragments. This moss is a common constituent of the bryoflora of mesic deciduous forests in eastern North America, with a continuous range across various physiographic provinces. Populations generally consist of millions of individual gametophores. The discovery of considerable differentiation among populations of P. ciliare suggests that gene flow may be more restricted than one might expect from the large numbers of wind-dispersed propagules produced. Alternatively, it is possible that selection pressures for the loci we scored differ strongly among populations. Support for this view comes from the observation of large differences in statistics of gene diversity for different loci. It is also very likely, ofcourse, that both selection and genetic drift in isolated populations act in concert to produce the observed pattern. Intensive sampling ofclumps of P. ciliare revealed microscale genetic differentiation. Similarly, Cummins and Wyatt (1981) found genetic variation within small patches ofthe moss Atrichum angustatum. Given the limited range of gene flow in this species and most other bryophytes, such differentiation is to be expected (Wyatt, 1977, 1982, 1985; Wyatt and Anderson, 1984). Certainly, if such differentiation exists within populations, it is also to be expected among populations. One of the most clear-cut differences among populations of P. ciliare is the significantly reduced genetic variation in the Piedmont. In the Appalachian Mountains, P. ciliare occurs in primary forests consisting ofa highly diverse mixture of hardwood trees. Most of our sampling sites were in forests that had been minimally disturbed. On the other hand, populations in the Piedmont occur mainly along streams in secondgrowth oak-hickory-pine forests. Most of

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R. WYATT ET AL.

these areas were cleared in the 1800's for cultivation ofcrops and have had only about 100 years in which to recover. Therefore, although the present abundance of P. ciliare in Piedmont forests appears to be similar to that in the Appalachian Mountains, the genetic diversity of Piedmont populations is strikingly reduced. This impoverishment ofgenetic stocks may have occurred because ofthe bottlenecks in population size to which Piedmont populations were subjected. To test this prediction, we sampled Piedmont populations of P. ciliare from two sites that historical records suggested had never been cleared or heavily logged: Gold Mine Creek in the University of Georgia Arboretum and Eno River State Park in North Carolina. These sites showed gene diversities much higher than other Piedmont sites. In fact, their values were more similar to those for sites from undisturbed forests in the Appalachian Mountains. It appears likely, therefore, that the reduction in genetic diversity in the Piedmont of Georgia is due to recent habitat destruction, which reduced population sizes and forced colonies to reestablish from a limited number of surviving sources. Piedmont populations are completely or nearly fixed at all loci and totally lack rare or unique alleles, a pattern to be expected in extreme cases of genetic drift. The implications ofour discovery oflarge amounts ofgenetic variability in the haploid moss P. ciliare are wide-ranging. Assuming that the plants are truly haploid and that the genotype is therefore subjected directly to natural selection, most models of genetic population structure would predict reduced levels of genetic variation (Ennos, 1983). Effectsdue to dominance or overdominance cannot be invoked to explain the maintenance of this variability in haploid organisms. Furthermore, models of temporally varying selection suggest that genetic variation cannot be maintained in haploid populations by temporal heterogeneity (Ennos, 1983). Rather, it appears most likely that some form of spatial heterogeneity, such as multiple-niche selection, must be involved if selection is indeed responsible for the genetic variation observed in P. ciliare. Genetic heterogeneity is most likely to be maintained when there is restricted gene flow and when there are large differences in se-

lection coefficients between niches (Ennos, 1983), a situation likely to be common in bryophyte populations (Wyatt, 1982; Wyatt and Anderson, 1984). On the other hand, Yamazaki (1981, 1984) interpreted his discovery ofextensive genetic variation in Japanese populations of the liverwort Conocephalum conicum as a clear demonstration that allozyme polymorphisms are selectively neutral. He argued that "only under the model ofselective neutrality ofgenetic variability do we expect the equality of polymorphisms between haploid and diploid organisms" (Yamazaki, 1981 p, 374). Yamazaki's (1981, 1984) data, however, are open to question. He chose to use a large number of nonspecific enzymes known to be unusually variable in other species, including five esterases and three oxidases. This may have artifically elevated his estimates of genetic variability. Furthermore, his results conflict strongly with other studies of C. conicum (e.g., Szweykowski and Krzakowa, 1979; Szweykowski etal., 1981a; Odrzykoski, unpubl.), in which little polymorphism was found within different races of this liverwort species. All studies do agree, however, that there is little differentiation among populations, a finding at odds with our results for P. ciliare. We have, therefore, two contrasting pictures of genetic population structure within bryophyte species: l) the "Conocephalum model," in which there are low levels of variation within races (which probably represent separate biological species), weak interpopulation differentiation, and no microscale heterogeneity; and 2) the "Plagiomnium model," in which there are high levels of genetic variation, strong interpopulation differentiation, and microscale heterogeneity. Wyatt (1985) has discussed the ecological and evolutionary implications of these differing population structures. In any event, it is clear that at least some bryophytes, despite their status as "phylogenetic relicts," maintain significant stores of genetic variability. Perhaps further study will reveal that these organisms are as diverse in terms ofgenetic population structure as are angiosperms or various groups of animals. Certainly, their genetic systems appear to be similar in kind to those of diploid plants and do not ob-

GENETIC VARIABILITY IN A HAPLOID MOSS

viously constrain their potential for variation and evolution. ACKNOWLEDGMENTS

This research was supported by NSF Grant BSR-8408931. The Palfrey Fund of the Department ofBotany at the University ofGeorgia and the Jessup Fund ofthe Academy of Natural Sciences in Philadelphia helped to make I. J. Odrzykoski's visit to the U.S. possible. We thank G. E. Wyatt for field assistance and J. L. Hamrick for comments on the manuscript. LITERATURE CITED ANDERSON, L. E. 1963. Modem species concepts: Mosses. Bryologist 66: 107-119. - - . 1980. Cytology and reproductive biology of mosses, pp. 37-76. In R. J. Taylor and A. E. Leviton (eds.), The Mosses of North America. Amer. Assoc. Adv. Sci., San Francisco, CA. AYAlA, F. J. 1975. Genetic differentiation during the speciation process. Evol. BioI. 8:1-78. CRUM, H. 1972. The geographic origins of the mosses of North America's eastern deciduous forest. J. Hattori Bot. Lab. 35:269-298. CuMMINS, H., AND R. WYATT. 1981. Genetic variability in natural populations of the moss Atrichum angustatum. Bryologist 84:30-38. DANIELS, R. E. 1982. Isozyme variation in British populations of Sphagnum pulchrum (Braithw.) Warnst. J. Bryol. 12:65-76. - - - . 1985. Isozyme variation in populations of Sphagnum recurvum var. mucronatum from Britain and Finland. J. Bryol. 13:563-570. DE VRIES, A., B. O. VAN ZANrEN, AND H. VAN DUK. 1983. Genetic variability within and between populations oftwo species of Racopilum (Racopilaceae, Bryopsida). Lindbergia 9:73-80. DEWEY, R. 1989. Genetic variation in the liverwort Riccia dictyospora (Ricciaceae, Hepaticopsida). Syst. Bot. 14:155-167. ENNos,R. A. 1983. Maintenance ofgenetic variation in plant populations. Evol. BioI. 16:129-155. GEMMELL, A. R. 1950. Studies in the Bryophyta. I. The influence of sexual mechanism in varietal production and distribution of British Musci. New Phytol. 49:64-71. GOTTUEB, L. D. 1982. Conservation and duplication ofisozymes in plants. Science 216:373-380. GURIES, R. P., AND F. T. LEOIG. 1982. Genetic diversity and population structure in pitch pine iPinus rigida Mill.). Evolution 36:387-402. HAMRICK, J. L., Y. B. LINHART, AND J. B. MITTON. 1979. Relationships between life history characteristics and electrophoretically detectable genetic variation in plants. Ann. Rev. Ecol. Syst, 10:173200. HARRIS, H., AND D. A. HOPKINSON. 1976. Handbook of Enzyme Electrophoresis in Human Genetics. North-Holland, Amsterdam, Neth. HAUFLER, C. H. 1987. Electrophoresis is modifying

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our concepts of evolution in homosporous pteridophytes. Arner. J. Bot. 74:953-966. HAUFLER, C. H., AND D. E. SoLTIS. 1986. Genetic evidence suggests that homosporous ferns with high chromosome numbers are diploid. Proc. Nat. Acad. Sci. USA 83:4389-4393. KHANNA, K. R. 1964. Differential evolutionary activity in the bryophytes. Evolution 18:642--670. KOPONEN, T. 1971. A monograph of Plagiomnium Sect. Rosulata (Mniaceae). Ann. Bot. Fenn. 8:305367. KRzAKOWA, M., AND J. SZWEYKOWSKI. I 977a. Peroxidases as taxonomic markers in two critical Pellia taxa (Hepaticae, Pelliaceae). Bull. Acad. Polon. Sci. Ser. Sci. BioI. 25:203-204. - - - . 1977b. Peroxidases as taxonomic characters. II. Plagiochila asplenioides (L.) Dum. sensu Grolle (= P. maior S. Amell) and Plagiochila porelloides (= P. asplenioides aucti non Grolle; Hepaticae, Plagiochilaceae). Bull. Soc. Sci. Lett. Poznan Ser. D 17:33-36. . 1979. Isozyme polymorphism in natural populations ofa liverwort, Plagiochila asplenioides. Genetics 93:711-719. LoNGTON, R. E. 1976. Reproductive biology and evolutionary potential in bryophytes. J. Hattori Bot. Lab. 41:205-223. LoVELESS, M. D., AND J. L. HAMRICK. 1984. Ecological determinants of genetic structure in plant populations. Ann. Rev. Ecol. Syst. 15:65-95. NEI,M. 1972. Genetic distance between populations. Amer. Natur. 106:283-292. - - - . 1973. Analysis of gene diversity in subdivided populations. Proc. Nat. Acad. Sci. USA 70:33213323. - - . 1975. Molecular Population Genetics and Evolution. North-Holland, Amsterdam, Neth. ODRZYKOSKI, I. J., M. A. BOBOWICZ, AND M. KRzAKOWA. 1981. Variation in Conocephalum conicum- The existence oftwo genetically different forms in Europe, pp. 519-542. In J. Szweykowski (ed.), New Perspectives in Bryotaxonomy and Bryogeography. Adam Mickiewicz Univ., Poznan, Poland. ODRZYKOSKI, I. J., AND L. D. GOTTUEB. 1984. Duplications of genes coding 6-phosphogiuconate dehydrogenase in Clarkia (Onagraceae) and their phylogenetic implications. Syst. Bot. 9:479-489. ODRZYKOSKl, I. J., AND J. SZWEYKOWSKI. 1981. An interesting enzymatic polymorphism in some European populations of the liverwort Mannia Iragrans (Balbis) Frye and Clark, pp. 33-37. In J. Szweykowski (ed.), New Perspectives in Bryotaxonomy and Bryogeography. Adam Mickiewicz Univ., Poznan, Poland. ROGERS, J. S. 1972. Measures of genetic similarity and genetic distance. Univ. Texas Publ. Stud. Genet. 7213:145-153. SCHUSTER, R. M. 1966. The Hepaticae and Anthocerotae of North America, Vol. I. Columbia Univ. Press, N.Y. SHAW, C. R., AND R. PRAsAD. 1970. Starch gel electrophoresis of enzymes: A compilation of recipes. Biochem. Genet. 4:297-320. SMITH, A. J. E. 1978. Cytogenetics, biosystematics

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and evolution in the Bryophyta. Adv. Bot. Res. 6: 195-276. SoLTIS, D. E., AND P. S. SoLTIS. 1989. Polyploidy, breeding systems, and genetic differentiation in homosporous pteridophytes. In D. E. Soltis and P. S. Soltis (eds.), Plant Isozymes. Dioscorides Press, Portland, OR. In press. STEERE, W. C. 1954. Bryophytes. Bot. Rev. (Lancaster) 20:425-450. SWOFFORD, D. L., AND R. B. SELANDER. 1981. BIOSYS-I: A Fortran program for the comprehensive analysis of electrophoretic data in population genetics and systematics. J. Hered. 72:281-283. SZWEYKOWSKI, J., AND M. KRzAKOWA. 1979. Variation of four enzyme systems in Polish populations of Conocephalum conicum (L.) Dum. (Hepaticae, Marchantiales). Bull. Acad. Polon. Sci. Ser. Sci. Biol. 27:37-41. SZWEYKOWSKI, J., I. J. ODRZYKOSKl, AND R. ZIEUNSKI. 1981a. Further data on the geographic distribution of two genetically different forms of the liverwort Conocephalum conicum (L.) Dum: The sympatric and allopatric regions. Bull. Acad. Polon. Sci. Ser. Sci. BioI. 28:437-449. SZWEYKOWSKI, J., R. ZIEUNSKI, AND M. MENDELAK. 1981b. Variation of peroxidase isoenzymes in central European taxa of the liverwort genus Pellia. Bull. Acad. Polon. Sci. Ser. Sci. BioI. 29:9-19. WYATT, R. 1977. Spatial pattern and gamete dispersal distances in Atrichum angustatum, a dioicous moss. Bryologist 80:284-291.

1982. Population ecology of bryophytes. J. Hattori Bot. Lab. 52: 179-198. - - . 1985. Species concepts in bryophytes: Input from population biology. Bryologist 88:182-189. WYATT, R., AND L. E. ANDERSON. 1984. Breeding systems of bryophytes, pp. 39-64. In A. F. Dyer and J. G. Duckett (eds.), The Experimental Biology of Bryophytes. Academic Press, London, U.K. WYATT, R., I. J. ODRZYKOSKl, A. STONEBURNER, H. W. BASS, AND G. A. GALAU. 1988. Allopolyploidy in bryophytes: Multiple origins of Plagiomnium medium. Proc. Nat. Acad. Sci. USA 85:5601-5604. WYATT, R., A. STONEBURNER, AND I. J. ODRZYKOSKI. 1989. Bryophyte isozymes: Systematic and evolutionary implications. In D. E. Soltis and P. S. Soltis (eds.), Plant Isozymes. Dioscorides Press, Portland, OR. In press. YAMAZAKI, T. 1981. Genic variabilities in natural population of haploid plant, Conocephalum conicum. I. The amount of heterozygosity. Jap. J. Genet. 56:373-383. - - - . 1984. The amount of polymorphism and genetic differentiation in natural populations of the haploid liverwort Conocephalum conicum. Jap. J. Genet. 59:133-139. Corresponding Editor: P. W. Hedrick

ApPENDIX The table below shows migration distances for electrophoretic variants of Plagiomnium ciliare under standard conditions (see text). All distances are expressed in mm from the origin. Enzyme codes are given in Table I, and buffer compositions are described in the text. Code for phenotypes: I = single band; 2 = doublet of bands which do not segregate within populations; and 3 = triplet of bands which do not segregate within populations. Alleles encoding electrophoretic variants are represented by a-d. Migration distance Enzyme

Buffer

Phenotype

a

b

GOT-I ALD-I PGI-I EST-I MDH-I MDH-2 PGM-I PGM-2 TPI-I TPI-2 ME-I PEP-I PEP-2 PEP-3

S S S S M(H) M(H) H(M) H(M) M(H) M(H) S S S S

I I 1 2 2 (3) 3 1 I 2 1 I 1 I I

45 28 33 38,40 48,46 38,35,32 37 31 45,48 30 17 47 38 25

33, 30 49,47 38,29,22 34 34 47, 50 35 15 49 35 23

d

44,42 45,43 31 27 44,40 39 38

35