Evolution. 43(5), 1989, pp. 1085-1096
HIGH LEVELS OF GENETIC VARIABILITY IN THE
HAPLOID MOSS PLAGIOMNIUM CILIARE
ROBERT WYATT, IRENEUSZ J. ODRZYKOSKI,' AND ANN STONEBURNER
Department of Botany, University of Georgia, Athens, GA 30602
I
Abstract. —Horizontal starch-gel electrophoresis was used to measure variability at 14 enzyme loci
from 13 natural populations of the dioecious moss Plagiomniwn 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 (//T = 0.178) was similar
to that observed for highly outcrossed diploid plants such as pines, the variance within (/fs = 0.098
± 0.027) and among (DST = 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
cm) clumps. The discovery of high levels of genetic variability in a plant with a dominant haploid
life cycle challenges the traditional view of bryophytes 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 1, 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 al., 1983; Wyatt et al.,
1988). Similarly, electrophoretic analyses of
liverwort populations have uncovered a
surprising abundance of genetic variation
(KrzakowaandSzweykowski, \977a, 1977b,
1979; Szweykowski and Krzakowa, 1979;
Szweykowski et al., 198la, 1981*; Odrzykoski and Szweykowski, 1981; Odrzykoski
etal., 1981;Yamazaki, 1981, 1984;Dewey,
1989). Wyatt et al. (1989) have discussed
1
the
implications of these findings in terms
Permanent address: Department of Genetics, Institute of Biology, Adam Mickiewicz University, 165 of bryophyte population structure and evolution.
Dabrowskiego, Poznan 60-594, Poland.
1085
1086
R. WYATT ET AL.
100 km
FIG. 1. 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 cm 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
within which two or m
phoretic phenotypes oc
Electrophoretic Proa
dures for horizontal st.
resis were similar to tho
zykoski and Gottlieb (
snoots were homogeniz
traction buffer (0.1 M
containing 10 mM K(
6H2O, 1 mM EDTA (N
X-fOD, and [added jus
42 mM 2-mercaptoetha
then filtered through a
acloth onto 4-mm x 8per wicks. All steps of hi
done over crushed ice.
Saturated wicks were
tical slot (the origin) cut
gel, and enzymes were
three buffer systems. B
solved malate dehydrogi
phosphate isomerase (1
glucomutase (PGM), cc
citric acid, titrated to
(3-aminopropyl) morpru
was prepared by diluting
buffer with 964 ml wat
zymes also can be separ
(43 mM trisodium citra:
pH 7.0 with citric acid)
this system consisted of
HC1 titrated to pH 7.0
buffer gave similar pher
but yielded slightly bt
PGM. Buffer S was use(
mate oxaloacetate trans;
dolase (ALD), esterase (
coisomerase (PGI), mali<
peptidase (PEP). The e
this system was 190 m]\
mM LiOH-H 2 O (pH 8
was a mixture of 900 m
mM citric acid, (pH 8.
electrode buffer. After n
of the gel buffer decreas
Gels were run in a re
(4°C) for four hours in 1
a constant amperage of
hours in buffer S at a co
45 mA. By the end of the
phenol blue marker hac
in buffers M and H, and
front" had migrated 80 i
ter separation, enzymes
GENETIC VARIABILITY IN A HAPLOID MOSS
IB southeastern United States,
is for physiographic provinces
Piedmont, and CP = Coastal
ed 5-cm x 5-cm clumps
e colonies, placing these
plastic pots. We collectiks until we had sampled
:te colonies or until we
ince of approximately 1
returned to the lab, and
i each pot was selected
Plants from the Botanpopulation, which were
early all loci, were used
comparing enzyme motossibility of microscale
e sampled the 36 clumps
Star (MS) population in';h 5-cm x 5-cm clump,
pet shoots, one from the
;om each corner of the
malyzed each shoot by
jel electrophoresis. We
j percentage of clumps
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 jul extraction buffer (0.1 M Tris HC1, pH 7.5,
containing 10 mM KC1, 10 mM MgCl26H2O, 1 mM EDTA (Na2 salt), 0.1% Triton
X-100, 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 of homogenization 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 • 2H2O, titrated to
pH 7.0 with citric acid). The gel buffer for
this system consisted of 5 mM DL-histidine
HC1 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-H2O (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 mA and for five
hours in buffer S at a constant amperage of
45 mA. By the end of these 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°-40°C.
Coded data for the 14 loci in the 13 populations were analyzed using BIOSYS-1
(Swofford and Selander, 1981) and a program developed in the laboratory of ]. 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-1 and PEP-3 used L-valyl-L-leucine as
a substrate, only PEP-2 was able to use
L-leucyl-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 of MDH 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 of MDH-1 activity, this three-banded phenotype possibly represents posttranslational modification of a single gene. Phenotypes of MDH-2 also were three-banded.
We treated two variants detected in this region as two alleles of a single gene. TPI activity consisted of five 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-1). 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 of these 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-1, ALD-1, and PGI-1) 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% of the 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 (//s; 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-1 and ME-1, the largest proportion
of this variance is due to diversity within
populations (//s), rather than between populations CDST). Differences between the two
measures were generally small, however,
with Hs averaging 0.098 ± 0.027 and DST
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
of 0.086 ± 0.036 (Table 3). <7ST, which measures diversity between populations relative
to total diversity (Nei, 1973, 1975), averaged 0.248 ± 0.070, while JRsT, 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 of genetic 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-1, Pgm2, and Me-1) were far more variable in populations from the mountains than from the
Piedmont. On the other hand, Pgm-1
showed high levels of gene diversity in both
areas, although more of the variation in
Piedmont populations was due to differences between populations (GST = 0.548 for
the Piedmont, GST = 0.272 for the mountains). Overall gene differentiation, as measured by Nei's (1975) GST, 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; Dm = 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
TABLE 1. Allele frequencie
southeastern United States.
glutamate oxaloacetate trans
= malate dehydrogenase, PG
PEP = peptidase. Plants thai
Locus
Got-1
Ald-1
Pgi-1
Est-1
Mdh-1
Mdh-2
Pgm-1
Pgm-2
Tpi-1
Tpi-2
Me-1
Pep-1
Pep-2
Pep-3
Allele
a
a
a
a
b
c
null
a
b
c
a
b
a
b
c
a
b
c
d
a
b
c
a
b
c
a
b
a
b
c
a
b
a
b
BG
1.00
1.00
1.00
1.00
—
_
GMC
l.OC
l.OC
l.OC
0.87
0.13
_
—
1.00
—
—
1.00
—
0.93
0.07
—
1.00
—
_
—
1.00
—
—
1.00
—
0.31
0.69
_
—
1.00
—
—
1.00
—
—
1.00
—
1.00
—
—
1.00
—
1.00
—
0.94
0.06
_
—
1.00
—
—
1.00
—
—
1.00
—
0.84
0.16
—
0.81
0.19
1.00
—
these joined any of the
(Fig. 2). Populations fr<
were much less similar t(
were, however, no obvic
tween geographical dista
ulations and their geneti
Patterns of geographic
dividual loci generally n
from gene-diversity sta
distances. Me-lb is abse
populations of P. ciliare 1
as the most common alk
populations except Tan
3A). Piedmont populatii
GENETIC VARIABILITY IN A HAPLOID MOSS
.098 ± 0.027 and Dsr
033. Nei's (1973, 1975)
leasure of gene differaates the minimum net
:tween populations iniiversities within sub• moss populations, Dm
to 0.299, with a mean
ble 3). GST, which mea•en populations relative
fei, 1973, 1975), averwhile RST, the ratio of
i-population diversity,
.229 (Table 3). This inapproximately half as
een populations as there
is.
gene-diversity statistics
il populations of P. ciln levels of genetic polyions from the Piedmont
-norphic than those from
; provinces in terms of
olymorphic loci (16.5%
in number of alleles per
52), and gene diversity
hree loci (Mdh-1, Pgmar more variable in poplountains than from the
e other hand, Pgm-1
of gene diversity in both
ore of the variation in
ons was due to differilations (GST = 0.548 for
= 0.272 for the moundifferentiation, as mea5) OrST, is similar in Piedn populations (0.161 vs.
;, populations from the
nore similar genetically:
pairs of Piedmont popOS 2 for seven pairs of
ons.
:s between pairs of popm 0.0002 (between BoBroad River) to 0.2064
; Mill and Pond Drain)
ly, there was less differjpulations from the Piedig populations from the
logram summarizing geamong the populations
Piedmont samples plus
Alabama together before
1089
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.
Locus
Got-l
Ald-1
Pgi-1
Est-1
Mdh-1
Mdh-2
Pgm-1
Pgm-2
Tpi-1
Tpi-2
Me-]
Pep-1
Pep-2
Pep-3
Allele
a
a
a
a
uh
BG
1.00
1.00
1.00
1.00
OMC
1.00
1.00
1.00
0.87
n i ^j
\j,
null
a
b
c
a
b
a
b
C
a
b
c
ft14
a
b
c
a
b
c
a
b
a
b
c
a
b
a
b
—
1.00
—
—
—
1.00
—
—
1.00
—
0.93
0.07
1.00
—
0.31
0.69
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
—
—
1.00
—
—
1.00
—
0.84
0.16
—
0.81
0.19
1.00
—
1.00
—
—
1.00
—
—
1.00
—
1.00
—
—
1.00
—
1.00
1.00
—
—
1.00
—
—
1.00
—
1.00
_
WM
1.00
1.00
1.00
1.00
—
1.00
—
—
1.00
—
0.07
0.93
—
EM
1.00
1.00
1.00
1.00
—
0.92
0.08
—
0.81
0.19
—
1.00
—
1.00
—
1.00
—
ER
1.00
1.00
1.00
1.00
I'opulaliorI
COW
1.00
1.00
1.00
0.94
—
0.94
0.06
—
1.00
—
0.39
0.61
0.06
0.06
0.86
0.08
1.00
—
0.75
0.25
1.00
—
—
0.83
0.17
—
0.92
0.08
—
1.00
—
1.00
_
0.19
0.73
n —nx
0.78
0.22
—
0.97
0.03
—
0.94
0.06
0.97
—
0.03
BR
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
—
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 individual loci generally reinforce the picture
from gene-diversity statistics and genetic
distances. Me-lb 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
—
1.00
—
1.00
—
1.00
—
0.97
0.03
PC
1.00
1.00
1.00
1.00
—
0.45
0.55
—
1.00
—
0.32
0.68
0.91
0.09
—
1.00
—
—
1.00
—
—
0.45
0.55
1.00
_
—
1.00
—
1.00
—
OCS
1.00
1.00
1.00
1.00
TC
1.00
1.00
1.00
1.00
PD
1.00
1.00
1.00
1.00
MS
1.00
1.00
1.00
0.97
—
0.36
0.64
—
1.00
—
1.00
_
—
0.40
0.60
—
1.00
—
0.80
0.20
—
0.03
0.97
0.44
0.56
—
0.57
0.43
_
0.61
0.17
0.22
0.03
0.97
0.03
—
0.94
0.06
0.42
0.55
n\j.\jj
rn
0.72
0.25
0.03
0.86
0.11
0.03
1.00
—
—
0.19
0.81
1.00
—
—
1.00
—
1.00
—
0.70
0.30
—
1.00
—
—
1.00
—
—
0.94
—
0.06
1.00
—
0.93
—
0.07
0.11
0.89
1.00
—
—
0.97
0.03
1.00
—
1.00
—
1.00
—
0.81
0.19
—
1.00
—
HLD
1.00
1.00
1.00
0.92
n ns
u.uo
0.94
—
0.06
0.97
0.03
—
0.25
0.75
1.00
—
—
0.97
0.03
1.00
—
1.00
—
—
1.00
—
0.29
0.71
0.92
0.08
—
1.00
—
—
1.00
—
—
0.62
0.38
0.96
0.04
—
0.83
0.17
1.00
—
monomorphic for Mdh-1", while most
mountain populations have Mdh-lb as the
most common allele (Fig. 3B). Mountain
populations also are more variable among
themselves, and Mdh-lc is restricted to the
Coweeta population in the Blue Ridge
Mountains. Nearly all Piedmont populations are fixed for Pgm-2" (Fig. 3C). Pgm2b occurs in higher frequency in the mountains, and two rare alleles (Pgm-2c and
Pgm-2tf) are restricted to mountain populations. Finally, there is no clear pattern to
variation at Pgm-1 (Fig. 3D). Even closely
adjacent populations, such as Broad River
R. WYATT ET AL.
1090
TABLE 2. Sample sizes (N), gene diversities (Hs), and standard errors (SE) for 14 enzyme loci surveyed in 13
populations of Plagiomnium ciliare from the southeastern United States. The first six populations are from the
Piedmont.
Locality
Population
BG
Botanical Garden, Athens, GA
Goldmine Creek, Braselton, GA
GMC
Watson's Mill State Park, GA
WM
EM
Echol's Mill, Lexington, GA
BR
Broad River, Elberton, GA
ER
Eno River, Durham, NC
COW
Coweeta Hydrologic Lab, NC
PC
Panther Creek, Toccoa, GA
Oconee Station Cove, SC
OCS
Tamassee Creek, Walhalla, SC
TC
PD
Pond Drain, Mt. Lake, VA
Morning Star, Basye, VA
MS
HLD
Holt Lock and Dam, Holt, AL
Grand weighted mean:
and Watson's Mill from the Piedmont of
Georgia, frequently have very different allele frequencies. Again, the rare allele Pgmlc 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-
N
72
32
29
26
15
36
36
22
36
30
36
36
24
430
US ± SE
0.009 ± 0.009
0.097 ± 0.040
0.023 ± 0.016
0.033 ± 0.024
0.000 ± 0.000
0.073 ± 0.039
0.127 ± 0.041
0.116 ± 0.055
0.110 ± 0.051
0.138 ± 0.054
0.088 ± 0.044
0.124 ± 0.047
0.113 ± 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 (Hi) and gene diversities within (Hs) and between (Z>ST) populations of Plagiomnium 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 (Gsr)- Enzyme codes are given in Table 1.
Locus
Est-1
Mdh-1
Mdh-2
Pgm-1
Pgm-2
Tpi-1
Tpi-2
Me- 1
Pep- 1
Pep-2
Pep-3
Mean:
SE:
H-f
0.0413
0.4203
0.0320
0.4573
0.3553
0.1307
0.0321
0.3830
0.0412
0.0543
0.0046
0.1775
0.0554
US
0.0381
0.1447
0.0276
0.2706
0.2309
0.1108
0.0306
0.1345
0.0373
0.0472
0.0045
0.0979
0.0268
DST
0.0032
0.2756
0.0045
0.1868
0.1244
0.0198
0.0015
0.2485
0.0039
0.0071
0.0001
0.0796
0.0329
Dm
0.0034
0.2985
0.0048
0.2023
0.1348
0.0215
0.0016
0.2692
0.0042
0.0076
0.0001
0.0862
0.0356
GST
0.0764
0.6557
0.1392
0.4084
0.3502
0.1519
0.0472
0.6489
0.0946
0.1301
0.0255
0.2480
0.0701
*ST
0.0896
2.0629
0.1752
0.7478
0.5839
0.1940
0.0536
2:0021
0.1132
0.1621
0.0283
0.5648
0.2291
at a single locus. It is po
these three-banded p
represent fixed heteroz
enzyme. Additional we
termine which of the
correct.
We found three isoz
ciliare and five isozyme
from the usual two ((
creases in isozyme nungene duplication or fr
genomes via polyploid}
viewed the evidence
zymes in diploid plant
duplication have been
in any bryophyte. Like^
gested that any moss sj
likely to be polyploid.
gested that repeated cycl
gene silencing have occ
rous ferns, yielding iso
ical of diploid plants (1
Soltis [1986] and Solti
for alternative explanat
therefore, that isozyme
ing from an ancient pol
ancestors of P. ciliare r
all but a few loci. Becat
detected at least three
been duplicated, it seei
the genome of P. ciliare
plicated by polyploidy a
lenced at the majority c
ently gathering add
including studies of ist
other species ofPfagiom
allow us to reject eithe
single-gene duplication
tion hypothesis.
The most important
drawn from our analys
tion in P. ciliare are thz
maintains an unexpecte
TABLE 4. Estimates of gene
mountains. TVpop = number •
putative loci; PLP = average
GST, and £>m are denned in 1
Population
Piedmont
Mountain
Total:
A'enz
6
7
13
GENETIC VARIABILITY IN A HAPLOID MOSS
or 14 enzyme loci surveyed in 13
; first six populations are from the
)
)
j
4
0
HS ± SE
0.009 ± 0.009
0.097 ± 0.040
0.023 ± 0.016
0.033 ± 0.024
0.000 ± 0.000
0.073 ± 0.039
0.127 ± 0.041
0.116 ± 0.055
0.110 ± 0.051
0.138 ± 0.054
0.088 ± 0.044
0.124 ± 0.047
0.113 ± 0.045
0.078 ± 0.035
illular locations of isozymes
iserved in diploid flowering
lants have two isozymes for
izyme, one of which is active
nd one of which catalyzes the
in the cytosol. Most of the
irveyed in the moss P. ciliare
with this model. Some nonics, such as esterases and pep•d additional isozymes, as did
often exists as 3-4 different
flowering plants (Gottlieb,
2 displayed an unusual pheting of three bands of equal
treated the two variants that
s corresponding to two alleles
I between (£>ST) populations of Plaf gene differentiation between popuersity C/?ST), and between-population
e 1.
\
\
>
:j
j
;t
•1 2
' '•>
:\
2
3
GST
0.0764
0.6557
0.1392
0.4084
0.3502
0.1519
0.0472
0.6489
0.0946
0.1301
0.0255
0.2480
0.0701
KST
0.0896
2.0629
0.1752
0.7478
0.5839
0.1940
0.0536
2.0021
0.1132
0.1621
0.0283
0.5648
0.2291
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 of TPI, 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. Haufler (1987) suggested that repeated cycles of polyploidy and
gene silencing have occurred in homosporous ferns, yielding isozyme numbers typical of diploid plants (but see Haufler 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 of loci. 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
1091
1
EM
1
1
| 1
0.80
HLD
WM
0.83 0.87 0.90 0.93 0.97 1.00
Genetic Similarity
FIG. 2. Phenogram expressing overall levels of genetic similarity among 13 populations of Plagiomnium
ciliare based on Rogers's (1972) coefficient of genetic
similarity using 14 putative gene loci. Abbreviations
for populations are given in Table 2.
variation 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
of alleles per locus were detected by de Vries
et al. (1983) in two species of Racopilum.
In fact, average gene diversities within populations of/?, 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 of Yamazaki'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. A^p = number of population samples; Nen2 = number of enzymes screened; A^i — number of
putative loci; are
PLP = average percentage of loci polymorphic; k = mean number of alleles per locus. HI, HS,
GST, and An
denned in Table 3.
Population
Piedmont
Mountain
Total:
Nfov
6
1
13
Ncal
9
9
9
Moci
14
14
14
PLP
16.5
44.9
31.1
k
1.17
1.52
1.35
H-r
0.091
0.216
0.178
US
0.058
0.146
0.098
GST
0.161
0.171
0.248
Dm
0.039
0.082
0.086
FIG. 3. Geographic patterns of variation in allele frequencies at four loci of Plagiomnium ciliare: A) Me-1;
B) Mdh-1; Q Pgm-2; D) Pgm-1. See Figure 1 for more information regarding the sample locations.
Plagiomnium ciliare
dominant life cycle, fa:
values of dicots for the
morphic loci per poi
number of alleles per
versity (Hamrick et al
P. ciliare with diploid
basis of polymorphic Ic
moss is above average
tion diversity and be^
population diversity
Hamrick, 1984). Over;
versity in P. ciliare are
to those measured in p
ida) by Guries and Le
pine, however, there is
tiation among popula
0.023). Populations off
an order of magnitude
tistics, reflecting strc
among localities. This ;
by close examination <
terns in allele frequence
which differ sharply e^
adjacent populations.
Pairwise genetic dist;
populations of P. cilian
United States are gener,
observed for conspecific
loid plants (e.g., mean =
species tabulated by A
mont populations of F
similar to each other t"
lations from the Appa
This may be explained
Piedmont samples cai
from Georgia, while
TABLE 5. Nei's (1972) genetic distances (below the diagonal) and genetic identities (above the diagonal) for 13
populations of Plagiomnium ciliare in the southeastern United States. Abbreviations for populations are given
in Table 2. Geographical locations of the populations are shown in Figure 1.
BG
GMC
WM
EM
BR
ER
COW
PC
OCS
TC
PD
MS
HLD
BG
_
GMC
0.9590
0.0419
0.0633
0.0024
0.0002
0.0310
0.1336
0.0924
0.1262
0.0550
0.1502
0.0808
0.0530
0.0083
0.0491
0.0469
0.0064
0.1566
0.0568
0.1748
0.0833
0.1847
0.0585
0.0121
WM
0.9387
0.9918
—
0.0713
0.0689
0.0108
0.1732
0.0577
0.1965
0.1020
0.2064
0.0643
0.0156
Population
EM
BR
0.9976
0.9998
0.9542
0.9520
0.9312
0.9334
0.9978
_
—
0.0022
0.0374
0.0355
0.1308
0.1353
0.0949
0.0976
0.1260
0.1225
0.0532
0.0562
0.1514
0.1468
0.0865
0.0853
0.0604
0.0581
ER
0.9695
0.9936
0.9892
0.9633
0.9651
—
0.1419
0.0522
0.1599
0.0669
0.1696
0.0565
0.0152
cow
0.8749
0.8550
0.8409
0.8774
0.8734
0.8677
—
0.1144
0.0716
0.0265
0.1051
0.1710
0.1640
PC
0.9118
0.9448
0.9439
0.9095
0.9070
0.9491
.0.8919
—
0.0746
0.0717
0.0603
0.0335
0.0320
TABLE 5. E
OCS
0.8814
0.8396
0.8216
0.8847
0.8816
0.8523
0.9309
0.9282
—
0.0641
0.0288
0.0782
0.1349
TC
0.9465
0.9201
0.9031
0.9482
0.9454
0.9353
0.9738
0.9308
0.9379
Populal
PD
0.86C
0.831
0.8i:
0.86;
0.85S
0.84^
0.90C
0.941
0.971
0.91)
0.0928
0.1108
0.0935
0.09;
0.14:
GENETIC VARIABILITY IN A HAPLOID MOSS
B)MDH-
iloci of Plagiomnium ciliare: A) Me-1;
irding the sample locations.
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 rigidd) by Guries and Ledig (1982). In pitch
pine, however, there is almost no differentiation among populations (mean GST =
0.023). Populations of P. 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 of diploid 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
c identities (above the diagonal) for 13
.bbreviations for populations are given
re 1.
'
ER
0.9695
0.9936
0.9892
0.9633
0.9651
__
cow
0.8749
0.8550
0.8409
0.8774
0.8734
0.8677
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
PC
0.9118
0.9448
0.9439
0.9095
0.9070
0.9491
0.8919
—
0.0746
0.0717
0.0603
0.0335
0.0320
TABLE 5. Extended.
OCS
0.8814
0.8396
0.8216
0.8847
0.8816
0.8523
0.9309
_
0.9282
0.0641
0.0288
0.0782
0.1349
Population
PD
0.8606
0.8314
0.8135
0.8635
0.8595
0.8440
0.9003
0.9415
0.9716
0.9114
0.0928
—
0.1108 0.0932
0.0935 0.1429
TC
0.9465
0.9201
0.9031
0.9482
0.9454
0.9353
0.9738
0.9308
0.9379
MS
0.9223
0.9432
0.9377
0.9172
0.9182
0.9451
0.8428
0.9670
0.9247
0.8951
0.9110
—
0.0236
HLD
0.9484
0.9880
0.9845
0.9414
0.9435
0.9849
0.8487
0.9685
0.8738
0.9107
0.8668
0.9766
—
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 of regeneration 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, of course,
that both selection and genetic drift in isolated populations act in concert to produce
the observed pattern.
Intensive sampling of clumps of P. ciliare
revealed microscale genetic differentiation.
Similarly, Cummins and Wyatt (1981) found
genetic variation within small patches of the
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 of a 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
1094
R. WYATT ET AL.
these areas were cleared in the 1800's for
cultivation of crops 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
of genetic stocks may have occurred because
of the 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 of our discovery of large
amounts of genetic 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).
Effects due 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 of extensive
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 of selective
neutrality of genetic 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
et al., 198 la; 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: 1) 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 of genetic 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-
viously constrain th
tion and evolution.
ACKNOW
This research w;
Grant BSR-840893
the Department of B
of Georgia and the Je
emy of Natural Sc
helped to make I. J
the U.S. possible. W
field assistance and.
ments on the manu;
LlTERAl
ANDERSON, L. E. 1963.
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mosses, pp. 37-76. In
iton (eds.), The Mossi
Assoc. Adv. Sci., San
AYALA, F.J. 1975. Gen<
speciation process. Ev
CRUM, H. 1972. The gee
of North America's i
Hattori Bot. Lab. 35:1
CUMMINS, H., AND R. W
ability in natural popu
angustatum. Bryologh
DANIELS, R. E. 1982. I
populations of Spha
Warnst. J. Bryol. 12:6
. 1985. Isozyme
Sphagnum recurvum •*
ain and Finland. J. Br
DE VRIES, A., B. O. VAN
1983. Genetic variabi
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Bryopsida). Lindbergi;
DEWEY, R. 1989. Genet
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ENNOS, R. A. 1983. Mai
in plant populations. I
GEMMELL, A. R. 1950.
The influence of sexua
duction and distribut
Phytol. 49:64-71.
GOTTLIEB, L. D. 1982. C
of isozymes in plants.
CURIES, R. P., AND F. T.
versity and population
mis rigida Mill.). Evoli
HAMRICK, J. L., Y. B. Li
1979. Relationships 1
teristics and electroph
variation in plants. Ar
200.
HARRIS, H., AND D. A. Ho
of Enzyme Electroph<
North-Holland. Amstc
HAUFLER, C. H. 1987.
GENETIC VARIABILITY IN A HAPLOID MOSS
fncients between niches (Ennos,
tuation likely to be common in
populations (Wyatt, 1982;Wyatt
son, 1984).
other hand, Yamazaki (1981,
preted his discovery of extensive
iation in Japanese populations of
ort Conocephalum conicum as a
onstration that allozyme polys are selectively neutral. He aronly under the model of selective
3f genetic variability do we expect
ity of polymorphisms between
d diploid organisms" (Yamazaki,
4).Yamazaki's(1981, 1984) data,
are open to question. He chose to
3 number of nonspecific enzymes
be unusually variable in other
icluding five esterases and three
This may have artifically elevated
ates of genetic variability. Furhis results conflict strongly with
lies of C. conicum (e.g., Szweyd Krzakowa, 1979; Szweykowski
la; Odrzykoski, unpubl.), in which
morphism was found within dif:es of this liverwort species. All
> agree, however, that there is little
ition among populations, a finding
ith our results for P. ciliare.
r
er therefore, two contrasting picenetic population structure within
13 species: 1) the "Conocephalum
I in which there are low levels of
i within races (which probably repkarate biological species), weak intion differentiation, and no miheterogeneity; and 2) the
•mum model," in which there are
Is of genetic variation, strong in'.tion differentiation, and micro;rogeneity. Wyatt (1985) has dishe ecological and evolutionary
ons of these differing population
5. In any event, it is clear that at
ie bryophytes, despite their status
^genetic relicts," maintain signifies of genetic variability. Perhaps
|tudy will reveal that these organas diverse in terms of genetic pop:ructure as are angiosperms or varups of animals. Certainly, their
/stems appear to be similar in kind
of diploid plants and do not ob-
viously constrain their potential for variation and evolution.
ACKNOWLEDGMENTS
This research was supported by NSF
Grant BSR-8408931. The Palfrey Fund of
the Department of Botany at the University
of Georgia and the Jessup Fund of the 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.
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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 1,
and buffer compositions are described in the text. Code for phenotypes: 1 = 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.
Enzyme
GOT-l
ALD-1
PGI-1
EST-1
MDH-1
MDH-2
PGM-1
PGM-2
TPI-1
TPI-2
ME-1
PEP-1
PEP-2
PEP-3
Buffer
S
S
S
S
M(H)
M(H)
H(M)
H(M)
M(H)
M(H)
S
S
S
S
Phenotype
i
i
i
2
2(3)
3
1
1
2
1
1
1
1
1
a
45
28
33
38,40
48,46
38, 35, 32
37
31
45,48
30
17
47
38
25
Migration distance
b
33,30
49,47
38, 29, 22
34
34
47,50
35
15
49
35
23
C
d
44,42
45,43
31
27
44,40
39
38
35