Molecular Ecology (1999) 8, 443Ð451

Clonal diversity in the rare Calamagrostis porteri ssp.

insperata (Poaceae): comparative results for allozymes and
random amplified polymorphic DNA (RAPD) and
intersimple sequence repeat (ISSR) markers
E . J . E S S E L M A N , * L . J I A N Q I A N G , D . J . C R AW F O R D , à J . L . W I N D U S ¤ a n d A . D . W O L F E à
*Department of Biology, Southern Illinois University at Edwardsville, IL 62026, USA, Wuhan Institute of Botany, Academica
Sinica, Wuhan 430074, China, àDepartment of Evolution, Ecology and Organismal Biology, The Ohio State University, Columbus,
OH 43210, USA, ¤Division of Natural Areas & Preserves, Ohio Department of Natural Resources 1889 Fountain Square Court,
Columbus, OH 43224, USA

Abstract

Four populations of the rare, highly clonal grass Calamagrostis porteri ssp. insperata were
examined using allozymes and the two polymerase chain reaction (PCR)-based markers,
random amplified polymorphic DNA (RAPD) and intersimple sequence repeat (ISSR)
bands. Only one of the 15 allozyme loci was variable and two alleles were detected, both
of which were found in two populations, while only one genotype was detected in the
other two populations. ISSR and RAPD markers detected more genotypes within popula-
tions than did allozymes. ISSR markers detected more diversity than RAPD markers in
three of the four populations examined. In one population, no RAPD diversity was found
whereas eight different genotypes were found among the 10 plants with ISSR markers.
This diversity is present despite rare flowering, no documented occurrence of seed set in
natural populations and very low seed set with experimental pollinations, all of which
suggest that sexual reproduction rarely occurs. The subspecies is self-compatible, but
seed initiation is lower in selfed ovules; also, there is high embryo abortion regardless of
pollen source. Variation detected by RAPD and ISSR primers may reflect higher levels of
sexual reproduction in the past, very rare sexual reproduction in extant populations,
somatic mutations, or a combination of the three. Although the PCR-based markers
identify several multilocus genotypes within populations, it is not known whether these
all represent distinct genets generated by sexual reproduction or result from somatic
mutations in the old, perennial and highly clonal plants.
Keywords: allozyme, clone, ISSR, RAPD, rare plant

Received 20 June 1998; revision received 22 October 1998; accepted 22 October 1998

Introduction be determined from counting the number of ramets

The conservation of plant populations and species must
be concerned with the number of genetic individuals
(genets) present in populations in order to assess factors
such as genetic drift, inbreeding depression and lack of
mates in self-incompatible species (Barrett & Kohn 1991;
Ellstrand & Elam 1993). For plants that reproduce clon-
ally, the genetically effective size of a population cannot
Correspondence: Daniel J. Crawford. Fax: 1-614-292-2030; E-mail:
crawford.13@osu.edu
present. What appears to be a ÔlargeÕ population may in
fact be ÔsmallÕ in terms of genotypes, and thus some of the
factors of concern in populations of sexually reproducing
plants with small numbers of individuals could also be
important in the conservation of clonal populations. It
follows therefore that any study of the conservation
biology of clonal plants should include an initial assess-
ment of the proportions of different genets found within
the ramets of a population (Sipes & Wolf 1997).
Determining the number of genets in populations of
clonal plants has been greatly facilitated by the availability

© 1999 Blackwell Science Ltd

444 E. J. ESSELMAN ET AL.
of a variety of molecular markers (Schaal et al. 1991; Avise
1994; Schaal & Leverich 1996; Smith & Wayne 1996; Wolfe
& Liston 1998). Ellstrand & Roose (1987) discussed geno-
typic variation in clonal plants, and over 65% of the stud-
ies they cited used allozymes as the sole method or one of
the methods for detecting different genotypes. This is par-
tially a reflection of the general use of allozymes in popu-
lation biology at the time of the review (Hamrick & Godt
1990). However, it is now evident that allozymes may
underestimate genetic diversity in plant populations and
species because additional diversity has been detected
when other molecular markers were employed. Examples
are available from genera such as Trifolium (Hickey et al.
1991; Hickey & Vincent 1992; Crawford et al. 1998), Lactoris
(Brauner et al. 1992; Crawford et al. 1994) and Penstemon
(Wolfe & Elisens 1993; Wolfe et al. 1998a,b). Ellstrand &
Roose (1987) concluded, from their survey of published
studies, that the greater number of characters used,
whether of the same type (such as number of allozyme
loci) or of different types, the more diversity encountered.
The molecular markers best suited for detecting geno-
typic diversity should be relatively easy and inexpensive
to use and should evolve rapidly enough to be variable
within populations. A number of polymerase chain reac-
tion (PCR)-based DNA markers have been found to meet
these criteria (Wolfe & Liston 1998), the most popular of
which, during the past several years, has been random
amplified polymorphic DNA (RAPD); a general discus-
sion of their use has been published by Hadrys et al. (1992).
A much less widely used PCR-based method, which
deserves increased attention as a molecular marker, is
intersimple sequence repeats (ISSR), where primers are
designed for the common sequences of simple sequence
repeats (SSR or microsatellites). Wherever in the genome a
particular SSR motif occurs on opposing strands within
distances short enough for amplification, bands will be
produced and they may be visualized in agarose or poly-
acrylamide gels (reviewed in Wolfe & Liston 1998). Two
plants with the same SSRs and flanking sequences will
produce the same (homologous) bands. Studies employ-
ing ISSR markers have focused on cultivated species
(Wolff et al. 1995; reviewed in Wolfe & Liston 1998; Wolfe
et al. 1998b). However, recent ISSR studies of natural popu-
lations have demonstrated the hypervariable nature of
these markers and their potential for population-level
studies (Robinson et al. 1997; Wolfe et al. 1998a,b).
Calamagrostis porteri A. Gray ssp. insperata (Swallen) C.
W. Greene (Poaceae) is an herbaceous, tufted perennial
grass with the common name of BartleyÕs reed bent
grass. The taxon was described by Swallen (1935) from
material collected in Jackson County, Ohio, and it has also
been reported from Missouri, Kentucky and Illinois; there
are historical but no recent records from Arkansas
(Schneider 1995). The subspecies is rare, occurring as
small populations, each consisting of distinct clumps or
patches. It has been variously treated as a distinct species
(Swallen 1935; Gleason & Cronquist 1991), not distinct
from C. porteri at any rank (Fernald 1950), or as a sub-
species of C. porteri (Greene 1984). It differs from typical C.
porteri in several minor morphological features, such as
stouter culms, leaf blades wider and less scabrous, and
collars of leaf sheaths glabrous rather than densely hairy.
Also, the chromosome number of ssp. insperata is octo-
ploid with 2n = 56 while the number for ssp. porteri ranges
from 2n = 88 to 2n = 100 (Greene 1984). Subspecies insper-
ata, like the other subspecies of C. porteri, is capable of sex-
ual reproduction (it is not agamospernous), yet flowering
is rare in natural populations (Greene 1984; Schneider
1995). Green (1984) reported C. porteri ssp. insperata as
self-incompatible, but a more recent study by Havens &
Holland (1998) demonstrated that it is self-compatible but
with reduced fruit initiation with selfed as compared to
outcrossed pollen. Regardless of pollen source, less than
half of all florets scored initiated fruits and there was very
high embryo abortion, resulting in only one viable seed
among the 2000 florets scored. Bittner & Gibson (1998)
studied the microhabitat ecology of the subspecies and
documented the production of tillers in spring and in late
to midsummer. They concluded that it is highly likely that
each population consists of a single fragmented genet
because it can spread vegetatively by tillers, but that sex-
ual reproduction must be rare given the infrequent flow-
ering. Havens & Holland (1998) indicated that the taxon
has never, to their knowledge, been observed to set
viable seed in natural populations.
The purpose of the present study was to use three dif-
ferent types of molecular markers to estimate the number
of genets (multilocus genotypes) in Ohio populations of
Calamagrostis porteri ssp. insperata. The subspecies is
known from three populations in Vinton County and one
small population in Jackson County (Fig. 1). The three
Vinton County populations are extensive, with numerous
patches scattered along ridgetops and roadways where
Fig. 1 Localities for populations of Calamagrostis porteri ssp.
insperata examined for genetic variation. Population designations
are the same as those given in Table 1.
© 1999 Blackwell Science Ltd, Molecular Ecology, 8, 443Ð451

the soils are thin over sandstone bedrock. The popula-
tions are threatened by heavy, woody canopy, which sup-
presses flowering. The small Jackson County population
is located on a powerline right-of-way. As with the other
three populations, this one occurs on thin soils over sand-
stone and is threatened by the invasion of woody species.
All Ohio populations grow in the unglaciated Allegheny
Plateau on thin acidic soils derived from Pennsylvanian
sandstones and shales, which are very old habitats in
Ohio and the eastern United States (King 1979). Thus, it is
possible that the clumps present in populations represent
old individuals that have persisted for centuries.
Materials and methods
Plant material
Plant material was collected from four populations (Table 1;
Fig. 1). A total of 89 individuals were sampled for allozymes
with 20 from Watch Rock, Vinton County (WR), 20 from
Arch Rock, Vinton County (AR), 20 from Headquarters site,
Vinton County (HQ) and 29 from Jackson County (JA).
Fifty-one of the same individuals were examined for RAPD
and ISSR markers: 11 from WR, 11 from AR, 19 from HQ
and 10 from JA. Leaves of plants from AR and WR were col-
lected from different patches along transects through the
populations. All samples from JA were from individuals in
the single patch comprising the population, and plant mate-
rial was collected from ostensibly different individuals
within all six patches of HQ (Table 1).
Allozyme analysis
Leaf tissue was ground in a cold extracting buffer of 0.1 M
Tris-HCl, pH 7.5, 14 mM 2-mercaptoethanol, 1 mM EDTA
Designation Location
JA Jackson County: Jackson Quad,
Liberty Twp. Sec. 1
WR Vinton County: Watch Rock,
Vinton Experimental Forest,
Mc Arthur Quad.
Vinton Twp. Sec. 19, 24, 30
AR Vinton County: Arch Rock,
Vinton Experimental Forest,
Mc Arthur and Vales Mills Quad, 9
Vinton Twp. Sec. 23, 2
HQ Vinton County, Headquarters site,
Vinton Experimental Forest,
Mc Arthur-Vales Mills Quad,
Vinton Twp., Sec. 30
© 1999 Blackwell Science Ltd, Molecular Ecology, 8, 443Ð451
CALAMAGROSTIS MOLECULAR MARKERS 445
(tetrasodium salt), 10 mM KCl and 10 mM MgCl2 (Gottlieb
1981). Two buffer systems were employed to resolve
enzymes in 12% starch gels, with a TrisÐEDTAÐborate sys-
tem (Crawford et al. 1992) used for aminopeptidase
(AMP), glucose-6-phosphate isomerase (GPI) and phos-
phoglucomutase (PGM). The enzymes isocitrate dehydro-
genase ([NADP] IDH) and shikimate dehydrogenase
(SKD) were separated with a morpholine citrate buffer
system (Crawford et al. 1992). Polyacrylamide gels with
the buffer system described by Crawford et al. (1987) were
used to resolve alcohol dehydrogenase (ADH), asparate
aminotransferase (AAT), glutamate dehydrogenase
(GDH) and superoxide dismutase (SOD). Enzyme nomen-
clature and staining methods used were according to those
described previously by Wendel & Weeden (1989).
Because Calamagrostis porteri ssp. insperata is an octo-
ploid, it might be assumed that extensive duplication has
occurred for allozyme loci and that more than the Ômini-
malÕ conserved number for diploid plants (Gottlieb 1982;
Weeden & Wendel 1989) would be expressed for most
enzymes. However, in all instances simple patterns were
seen in which the number of zones of activity, represented
by single bands (except for ADH in certain individuals),
corresponded to the minimal number usually expressed
in diploid plants. These patterns could be the result of
extensive gene silencing, particularly if the polyploid is of
ancient origin. Another possibility is that duplicate loci
are expressed but they encode the same forms of the
enzymes, that is, the same alleles are present. In the pre-
sent study, it makes little difference which genetic situa-
tion exists because either way there is essentially no
variation at any of the loci, regardless of how many are
expressed. We assume the more conservative situation
because we know at least as many loci are expressed as
there are zones of activity but cannot document that more
Table 1 Locations and sizes of
Population size populations of Calamagrostis porteri ssp.
insperata examined for allozymes, random
One patch 7.0 × 0.5 m amplified polymorphic DNA markers and
intersimple sequence repeat markers
Approximately 40 patches
along the northÐsouth ridge
Approximately 32 patches
along two eastÐwest ridges
Approximately six patches
446 E. J. ESSELMAN ET AL.
are expressed. The number of loci scored were: AAT,
three; ADH, two; AMP, one; GDH, one; GPI, one; IDH,
one; PGD, one; PGM, two; SKDH, one; and SOD, one.
GPI, IDH, PGD and SOD each had one additional zone
of faint, poorly resolved activity, which was not included
in the analyses.
DNA analysis
Extraction. Total DNA was extracted from ≈ 0.5 g of fresh
leaf material using a modification of the method of Doyle
& Doyle (1987). The tissue was ground in 0.7 mL of 2×
CTAB isolation buffer (100 mM Tris-HCl, pH 8.1, 1.4 M
NaCl, 20 mM EDTA, 2% hexadecyltrimethyl-ammonium
bromide (CTAB), 1% Nabisulfite, 0.2% 2-mercap-
toethanol). Ground tissue was incubated at 60 ¡C for
30Ð60 min, extracted with chloroformÐisoamylalcohol
(24:1), centrifuged at high speed in a microcentrifuge for
2 min and then the supernatant was collected and placed
in a clean tube. Nucleic acids were precipitated by the
addition of 0.46 mL ice-cold isopropanol, pelleted by
high-speed centrifugation for 2 min, washed in 0.8 mL of
76% EtOH/0.01 M NH4OAc, and resuspended in 0.1 mM
NH4OAc/25 mM EDTA.
RAPD amplification. Reactions were carried out in vol-
umes of 25 µL consisting of 1.5 mM of MgCl2, 100 mM
each of dATP, dCTP, dGTP and dTTP, 0.2 mM of primer,
1.5 µL of genomic DNA, 0.75 units of Taq DNA poly-
merase (Gibco BRL) and 1× Taq DNA polymerase buffer.
PCR amplifications were performed in a Perkin-Elmer
Cetus DNA Thermal Cycler programmed for 40 cycles of
1 min at 94 ¡C, 2 min at 35 ¡C and 2 min at 72 ¡C, and 4 ¡C
soak. The three primers A4, A8 and A9 from Operon
Technology were chosen for amplification because of the
variation detected with them. The bands were resolved
by electrophoresis in a 2% agarose gel in 1× TBE
(TrisÐEDTAÐborate) buffer and stained with ethidium
bromide. Most plants were amplified twice with the
same primer. All variant banding patterns were run
together in the same gel.
ISSR amplification. Reactions were carried out in a volume
of 25 µL consisting of 3 mM MgCl2, 0.2 mM dNTPs, 0.4 µL
of primer, 0.5 µL of DNA, 0.25Ð0.5 units of Taq DNA poly-
merase and 1× Taq DNA polymerase buffer. A Stratagene
Robocycler was used with the thermocycle programme
set at: 1.5 min at 94 ¡C; 35 × 40 s at 94 ¡C, 45 s at 44 ¡C,
1.5 min at 72 ¡C; 45 s at 94 ¡C, 5 min at 72 ¡C; 6 ¡C soak.
The three ISSR primers employed were based on SSR
motifs reported for flowering plants (reviewed in Wolfe &
Liston 1998; Wolfe et al. 1998a,b). Anchoring sequences
were added to prevent artefacts resulting from strand
slippage (Wolfe & Liston 1998; Wolfe et al. 1998a,b). The
primer designations and composition were: 17 898
(CA)6RY; 17 899 (CA)6RG; and 17 901 (GT)6ÐYR. Bands
amplified by PCR were characterized on 1.5% agarose gels
in 1× TrisÐacetateÐEDTA buffer. Gels were stained with
ethidium bromide. The majority of plantÐprimer combin-
ations were run more than once to ensure reproducibility.
Number of primers used
Studies employing ISSR primers in natural populations
have shown that three primers are generally sufficient to
genotype every individual included in the study (Wolfe
et al. 1998a,b; Wolfe et al. unpublished) Thus we selected
three ISSR primers and three RAPD primers that yielded
polymorphisms in a preliminary analysis. Our intent was
to make a comparison among RAPD and ISSR primers
without the expense and time involved in screening many
primers on limited DNA stocks.
RAPD and ISSR analysis
RAPD and ISSR bands were visualized on a UV transillu-
minator and were recorded digitally using an Alpha
Innotech Imaging System. The digital imaging files were
transferred in a TIFF format to a PowerMac 7500, and ana-
lysed with the BioMax one-dimensional image analysis
software (Eastman Kodak Company). Fragment sizes
were estimated based on 1000 MW ladder size standards
(Gibco BRL) according to the algorithm provided in the
BioMax one-dimensional software. Fragment sizes were
designated as loci and bands shared as diallelic characters
(present = 1; absent = 0). The Jaccard coefficient was
employed to calculate pairwise band similarities for all 51
samples using a program written by Vera Ford,
University of California, Davis (unpublished). The pro-
gram then calculated average similarities between sam-
ples from the same and different populations.
In addition to overall similarity, the number of different
banding patterns (multilocus genotypes) was determined
for the samples from each population, and the number of
genotypes was divided by the number of samples to give
the proportion of distinguishable genotypes within popu-
lations (Ellstrand & Roose 1987). The percentage of vari-
able bands was also determined for each population.
Results
Allozymes
Only one of the 15 allozyme loci, Adh-2, was polymor-
phic. The allele Adh-2b was detected in populations WR
and JA where all samples were heterozygous Adh-2ab,
suggesting fixed heterozygosity. Populations AR and
HQ only had allele AdhÐ2a.
© 1999 Blackwell Science Ltd, Molecular Ecology, 8, 443Ð451
 CALAMAGROSTIS MOLECULAR MARKERS 447

Table 2 Diversity of banding patterns of random amplified polymorphic DNA (RAPD) markers and intersimple sequence repeat (ISSR) markers within populations of Calamagrostis
RAPD

distinguishable
ISSR & RAPD
proportion of
The three RAPD primers generated a total of 40 consis-

genotypes
tently scorable bands, nine (22.5%) of which were vari-

0.909
0.818
1.000
0.800
able. No variation was detected within populations using
primer A8. Primer A4 produced 17 bands, 24% of which
were variable, while primer A9 generated 12 bands of

No. of ISSR & RAPD

which 42% were variable. RAPD markers detected diver-

banding patterns
sity within three of the four populations; no variation was
found in population JA. (Table 2). The proportion of dis-
tinguishable genotypes in populations varied between
0.000 and 0.789, and the percentage of variable bands

10
9
19
8
within populations ranged from 0.0 to 17.5 (Table 2). The

RAPD bands
mean similarity of banding patterns for individuals

% Variable
within populations varied from 0.935 to 1.000, and the
average value for plants from different populations

5.0
2.5
17.5
0.0
ranged from 0.609 to 0.809 (Table 3).

of distinguishable
RAPD proportion
ISSR

genotypes
A total of 67 bands were scored for the three ISSR primers,
with 16 (24%) of them variable. All three primers detected

0.364
0.182
0.789
0.000
variation in two or more populations. Primer 17 898 pro-
duced 15 bands, 20% of which were variable. Primers

banding patterns
17 899 and 17 901 generated 29 bands (35% variable) and
23 (26% variable) bands, respectively. The number of dif-

No. of RAPD
ferent banding patterns, proportion of distinguishable
genotypes and percentage of variable bands for each pop-
ulation are shown in Table 2. The proportion of distin-

4
2
15
1
guishable genotypes ranged from 0.684 to 0.818 and the
ISSR bands
% Variable
variable bands ranged from about 10.4 to 20.8% for the
four populations (Table 2). The mean similarities of band-
13.4
20.8
10.4
17.9
ing patterns for individuals within populations ranged
from 0.965 to 0.971, and average values varied from 0.712
of distinguishable

to 0.757 between populations (Table 3).

ISSR proportion

Population designations are the same as those given in Table 1.

genotypes

RAPD and ISSR combined

0.818
0.818
0.684
0.800

When ISSR and RAPD markers were combined, one addi-

tional multilocus genotype was detected in population
WR and the proportion of distinguishable genotypes
banding patterns

increased to 0.909 for this population (Table 2). With both

No. of ISSR

sets of markers, all samples from population HQ could be

distinguished; the combination of markers did not
increase the proportion of distinguishable genotypes for
9
9
13
8

populations AR and JA (Table 2).

No. of plants
examined

Discussion
porteri ssp. insperata

Method comparisons
19
10
11
11

A taxon such as Calamagrostis porteri ssp. insperata, which

Population

rarely reproduces sexually and perpetuates itself clonally

by rhizomes (Greene 1984), presents difficulties for assess-
WR

HQ
AR

JA

ing population size because the ratio of ramets to genets is

© 1999 Blackwell Science Ltd, Molecular Ecology, 8, 443Ð451

448 E. J. ESSELMAN ET AL.
Table 3 Intersimple sequence repeat marker and random ampli-
fied polymorphic DNA marker (in parentheses) similarity within
and among populations of Calamagrostis porteri ssp. insperata
WR AR HQ JA
WR (0.967
(0.981)
AR (0.730 (0.965
(0.685) (0.991)
HQ (0.728 (0.748 (0.971
(0.609) (0.680) (0.935)
JA (0.712 (0.720 (0.757 (0.967
(0.809) (0.696) (0.680) (1.000)
Population designations are the same as those given in Table 1.
unknown and may be quite high. This means that certain
genetic processes important in small populations, such as
inbreeding and genetic drift, may be factors in what
appear to be large populations (many ramets) of clonal
plants (Barrett & Kohn 1991; Ellstrand & Elam 1993).
Thus, obtaining an estimate of the number of genets
within a population is an important early step in the
study of a clonal plant, particularly a rare taxon. A review
of the literature by Ellstrand & Roose (1987), up to about a
decade ago, showed that the number of ÔclonesÕ (genets,
multilocus genotypes) detected in a population often
depends on the number of characters used. Molecular
markers have become commonplace for assessing diver-
sity within plant populations (Avise 1994; Swensen et al.
1995; Smith & Wayne 1996; Ayres & Ryan 1997; Sydes &
Peakall 1998; Whitkus et al. 1998; Wolfe & Liston 1998;
Wolfe et al. 1998a,b), and the purpose of the present study
was to compare the utility of three different molecular
markers for assessing the number of genets present in a
highly clonal taxon.
Allozymes, which have been the molecular markers
used most frequently (Hamrick & Godt 1990), identified
the fewest number of genets within four different popula-
tions of Calamagrostis porteri ssp. insperata, with the only
variation being two different alleles at one locus among
the 89 samples examined. Thus, if clonal diversity in pop-
ulations was assessed using only allozymes it might be
concluded that this taxon is indeed highly clonal with
vegetative reproduction via rhizomes influencing the
structure of populations. In this study, allozyme vs. PCR-
generated markers gave quite different pictures of popu-
lation variation in C. porteri ssp. insperata.
Wolfe et al. (1998b) reviewed the advantage of using
ISSR markers compared with other available PCR-based
techniques. The major advantages cited included the
hypervariability of banding patterns and low cost per
polymorphism. Other researchers who have compared
RAPD and ISSR methods have found that ISSR markers
exhibit higher levels of polymorphism and/or repro-
ducibility compared with RAPD markers (Yang et al. 1996;
Nagaoka & Ogihara 1997; Parsons et al. 1997). When a
direct cost comparison was made among restriction frag-
ment length polymorphism (RFLP), RAPD and ISSR tech-
niques, the latter was found to be the most economical per
polymorphism observed (Yang et al. 1996). In addition,
the primer sequences of ISSR loci are readily available
from the literature. Information on ISSR primer sequences
used in studies of natural populations can be obtained
from the ISSR Resource Website (http://www.biosci.ohio-
state.edu/~awolfe/ISSR/ISSR.html).
Genetic diversity and reproductive biology of
Calamagrostis porteri ssp. inseperata
Field observations indicate that flowering culms of
Calamagrostis porteri ssp. inseperata are very rare in all popu-
lations, with flowering more common in open areas
because the woody canopy suppresses flowering (Greene
1984; Schneider 1995; <28>Bittner & Gibson 1998; DM
Spooner, unpublished). Green (1984) reported 29Ð66%
pollen viability (estimated by staining with Cotton Blue in
lactophenol); Havens & Holland (1998) found a range of
pollen viability from 0 to 99% (employing AlexanderÕs
stain) for plants from Ohio populations of C. porteri ssp.
insperata, but all plants studied except two produced
95Ð99% stainable pollen. It does not appear that low pollen
viability is a limiting factor in sexual reproduction. The low
rate of seed initiation and high rate of embryo abortion
combined with infrequent flowering no doubt account for
the lack of a single report of a viable seed produced in natu-
ral populations (Havens & Holland 1998). Havens &
Holland (1998) suggested that C. porteri ssp. inseperata may
be a very old polyploid, and that low seed initiation in
selfed ovules and high embryo abortion are reflections of
high genetic load accumulated over time. High genetic
load has been suggested as the cause of low seed produc-
tion in other, presumably old, long-lived taxa (Wiens et al.
1989; Krebs & Hancock 1991). As stated above, all Ohio
populations occur in ancient habitats of the unglaciated
Allegheny Plateau; therefore the habitat in which the sub-
species occurs is concordant with the hypothesis that it is
an old, relictual polyploid. Bittner & Gibson (1998) pre-
sented data indicating that populations maintain them-
selves primarily, if not exclusively, by the production of
tillers, and that expansion to new sites by vegetative means
is highly unlikely. The apparent lack of seed production
likewise argues against population expansion or founding
of new populations by sexual means.
If populations of C. porteri ssp. insperata reproduce
totally (or nearly so) by vegetative means, as available
data indicate, it may seem surprising that so many dif-
ferent genotypes were detected with RAPD and ISSR
© 1999 Blackwell Science Ltd, Molecular Ecology, 8, 443Ð451

markers. In fact, the almost total lack of variation at
allozyme loci is more concordant with the hypothesis of
highly clonal populations resulting from reproduction by
tillers. It is possible, however, that sexual reproduction
may have once been more common, and the RAPD and
ISSR diversity now detected is largely relictual. Allozyme
diversity may have always been lower than PCR-based
variation, and drift and inbreeding in small populations
combined with vegetative reproduction may be responsi-
ble for the nearly nonexistent diversity now present.
Under this scenario, the multilocus genotypes detected
with PCR-based markers may provide a more accurate
assessment of genets present in a population. The differ-
ences in number of genets estimated by allozymes and
PCR-based markers in this study are similar to the results
obtained by Waycott (1998) in allozyme and RAPD stud-
ies of the seagrass Posidonia australis and contrast with the
results of Swenson et al. (1995) showing no diversity in
two populations of the rare Malacothamnus fasciculatus
var. nesioticus at allozyme or RAPD loci. The lack of varia-
tion in Malacothamnus was attributed to drift or extensive
vegetative reproduction by rhizomes. Sydes & Peakall
(1998) were able to detect very few multilocus genotypes
in populations of the rare clonal species Haloragodendron
lucasii using allozymes and RAPD markers; both data sets
gave a nearly identical picture of population structure
and clonality. As discussed above, one might expect more
diversity from PCR-based markers than from allozymes,
yet the two studies cited above demonstrate that similar
conclusions regarding genetic diversity may sometimes
be drawn from the two data sets.
Although sexual reproduction is apparently quite rare
in populations of C. porteri ssp. insperata as a result of the
combined effects of rare flowering, low seed initiation
and high embryo abortion (Havens & Holland 1998), it
cannot be dismissed totally as a means of generating
diversity. The study by Havens & Holland (1998) demon-
strated that it is possible for viable seed to be produced
and that it is more likely for seed to result from outcrossed
than selfed pollen, which could generate novel genotypes
by crossing between genets.
Another possible explanation for the greater diversity
found in the PCR-based markers as compared to
allozymes is somatic mutations (Gill et al. 1995). This is
especially plausible given that the plants may be very old
and some of the diversity detected with the PCR-based
markers represents segments of DNA within noncoding
regions (Begun & Aquadro 1993; Baruffi et al. 1995; Ayres
& Ryan 1997). Of the two PCR-based markers used in this
study, there is greater ISSR diversity than RAPD diversity
within populations (higher percentage of variable bands,
more multilocus genotypes) while RAPD bands have
lower similarity among populations. These differences
may reflect the nature of the primer sequences and the
© 1999 Blackwell Science Ltd, Molecular Ecology, 8, 443Ð451
CALAMAGROSTIS MOLECULAR MARKERS 449
assumptions made as to what parts of the genome are
being surveyed. For example, RAPD primers are based
on anonymous sequences and there is little or no infor-
mation regarding whether RAPD bands are in function-
ally important regions. The possibility exists that
conserved banding patterns reflect functional con-
straints on the genome. Indeed, quantitative trait loci
(QTL) mapping studies have shown that RAPD markers
are found throughout the genome and may be associated
with functionally important loci (Penner 1996). Recent
studies have shown that microsatellites are also
widespread in the genome (Condit & Hubbell 1991; Wu
& Tanksley 1993), but there is little information regard-
ing whether or not they are functionally important. If
tandemly repeated DNA sequences are not under func-
tional constraints, then one could expect ISSR bands to
represent neutral markers. If so, they should evolve at a
fast evolutionary rate and the hypervariability may pro-
vide higher estimates of genetic diversity in a popula-
tion compared to RAPD or other markers where there is
some functional constraint.
When various molecular markers provide different
estimates of genet number in populations, it is usually
tacitly assumed that the marker or combination of mark-
ers showing the highest number also provides the most
accurate estimate of genet number. In most instances this
is probably true because molecular marker variation is
one reflection of genetic diversity generated by sexual
reproduction. However, with a long-lived highly clonal
organism such as Calamagrostis porteri ssp. insperata the
situation may not be so clear because of the possibility of
somatic mutations in largely neutral, hypervariable
regions, such as those amplified with ISSR primers. It is
also possible that the variation detected results from a
combination of somatic mutations and sexual reproduc-
tion. If this is true, it means that when different molecular
markers give very different estimates of genet number
within populations then both the molecular markers
employed and the reproductive biology of the organisms
must be considered when interpreting the results.
Acknowledgements
We would like to thank Todd Hutchinson (U. S. Forest Service)
and Roger Barber (Division of Natural Areas and Preserves) for
their assistance in collecting plants. Louis Iverson (U. S. Forest
Service), Marilyn Ortt (Division of Natural Areas and Preserves)
and Steve Sutherland (The Nature Conservancy) provided valu-
able information. Allison Snow provided valuable comments on
the manuscript.
References
Avise JA (1994) Molecular Markers, Natural History and Evolution.
Sinauer, Sunderland, MA.
450 E. J. ESSELMAN ET AL.
Ayres DR, Ryan RJ (1997) The clonal and population structure of
a rare endemic plant, Wyethia reticulata (Asteraceae): allozyme
and RAPD analysis. Molecular Ecology, 6, 761Ð772.
Barrett SCH, Kohn JR (1991) Genetic and evolutionary conse-
quences of small population size in plants: Implications for
conservation. In: Genetics and Conservation of Rare Plants
(eds Falk DA, Holsinger KE), pp. 3Ð30. Oxford University
Press, New York.
Baruffi L, Daniani G, Gugliemino CR et al. (1995) Polymorphism
within and between populations of Ceratitis capitata: compari-
son between RAPD and multilocus electrophoretic data.
Heredity, 74, 425Ð443.
Begun DJ, Aquadro CF (1993) African and North American pop-
ulations of Drosophila melanogaster are very different at the
DNA level. Nature, 365, 548Ð549.
Bittner RT, Gibson DJ (1998) Microhabitat relations of the rare
reed bent grass, Calamagrostis porteri ssp. insperata (Poaceae),
with implications for conservation. Annals of the Missouri
Botanical Garden, 85, 69Ð80.
Brauner S, Crawford DJ, Stuessy TF (1992) Ribosomal DNA and
RAPD variation in the rare plant family Lactoridaceae.
American Journal of Botany, 79, 1436Ð1439.
Condit R, Hubbell SP (1991) Abundance and DNA sequences of
two-base repeat regions in tropical tree genomes. Genome,
34, 66Ð71.
Crawford DJ, Esselman EJ, Windus JL et al. (1998) Genetic varia-
tion in running buffalo clover (Trifolium stoloniferum:
Fabaceae) using random amplified polymorphic DNA mark-
ers (RAPDs). Annals of the Missouri Botanical Garden, 85, 81Ð89.
Crawford DJ, Stuessy TF, Cosner MB et al. (1994) Lactoris fernan-
deziana (Lactoridaceae) on the Juan Fernandez Islands:
allozyme uniformity and field observations. Conservation
Biology, 8, 277Ð280.
Crawford DJ, Stuessy TF, Haines DW et al. (1992) Allozyme
diversity within and divergence among four species of
Robinsonia (Asteraceae: Senecioneae), a genus endemic to
the Juan Fernandez Islands, Chile. American Journal of
Botany, 79, 962Ð966.
Crawford DJ, Stuessy TF, Silva OM (1987) Allozyme diver-
gence and the evolution of Dendroseris (Compositae:
Lactuceae) on the Juan Fernandez Islands. Systematic
Botany, 12, 435Ð443.
Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for
small amounts of fresh leaf tissue. Phytochemical Bulletin, 19,
11Ð15.
Ellstrand NC, Elam DR (1993) Population genetic consequences
of small population size: implications for plant conservation.
Annual Review of Ecology and Systematics, 24, 219Ð242.
Ellstrand NC, Roose ML (1987) Patterns of genotypic diversity
in clonal plant species. American Journal of Botany, 74,
123Ð131.
Fernald ML (1950) GrayÕs Manual of Botany, 8th edn. American
Book Company, New York.
Gill DE, Chao L, Perkins SL, Wolf JB (1995) Genetic mosaicism in
plants and clonal animals. Annual Review of Ecology and
Systematics, 26, 423Ð444.
Gleason HA, Cronquist A (1991) Manual of the Vascular Plants of
the Northeastern United States and Canada, 2nd edn. The New
York Botanical Garden, Bronx, New York.
Gottlieb LD (1981) Gene number in species of Astereae that have
different chromosome numbers. Proceedings of the National
Academy of Sciences of the USA, 78, 3726Ð3729.
Gottlieb LD (1982) Conservation and duplication of isozymes in
plants. Science, 216, 373Ð380.
Greene CW (1984) Sexual and apomictic reproduction in
Calamagrostis (Gramineae) from eastern North America.
American Journal of Botany, 71, 285Ð293.
Hadrys H, Balick M, Schierwater B (1992) Applications of ran-
dom amplified polymorphic DNA (RAPD) in molecular ecol-
ogy. Molecular Ecology, 1, 55Ð63.
Hamrick JL, Godt MJW (1990) Allozyme diversity in plant
species. In: Plant Population Genetics, Breeding, and Genetic
Resources (eds Brown AHD, Clegg MT, Kahler AL, Weir BS),
pp. 43Ð63. Sinauer Associates, Sunderland, MA.
Havens K, Holland DL (1998) Factors effecting reproductive suc-
cess in a rare grass Calamagrostis porteri ssp. Insperata. Annals
of the Missouri Botanical Gardens, 85, 64Ð68.
Hickey RJ, Vincent MA (1992) Application of isozyme techniques
in native North American T. stoloniferum species. In:
Proceedings of the Twelfth Trifolium Stoloniferum Conference (eds
Woffard DS, Quesenberry KH), pp. 15Ð17. University of
Florida, Gainesville.
Hickey RJ, Vincent MA, Guttman SI (1991) Genetic variation in
running buffalo clover (T. stoloniferum, Fabaceae).
Conservation Biology, 5, 309Ð316.
King CC (1979) Hill country. In: OhioÕs Natural Heritage (ed.
Lafferty MB), pp. 160Ð181. Ohio Academy of Science,
Columbus, OH.
Krebs S, Hancock JF (1991) Embryonic genetic load in the high
bush blueberry Vaccinium corymbosum (Ericaceae). American
Journal of Botany, 78, 1427Ð1437.
Nagaoka T, Ogihara Y (1997) Applicability of inter-simple
sequence repeat polymorphisms in wheat for use as DNA
markers in comparison to RFLP and RAPD markers.
Theoretical and Applied Genetics, 94, 597Ð602.
Parsons BJ, Newbury HJ, Jackson MT, Ford-Lloyd BV (1997)
Contrasting genetic diversity relationships are revealed in
rice (Oryza sativa L) using different marker types. Molecular
Breeding, 3, 115Ð125.
Penner GA (1996) RAPD analysis of plant genomes. In: Methods of
Genome Analysis in Plants (ed. Jauhar PP), pp. 251Ð268. CRC
Press, Boca Raton.
Robinson WA, Liston A, Doescher PS, Svejcar T (1997) Using
ISSR markers to quantify clonal vs. sexual reproduction in
Festuca idahoensis (Poaceae). American Journal of Botany, 84, 89
(abstract).
Schaal BA, Leverich WJ (1996) Molecular variation in isolated
plant populations. Plant Species Biology, 11, 33Ð40.
Schaal BA, Leverich WJ, Rogstad SH (1991) A comparison of
methods for assessing genetic variation in plant conservation
biology. In: Genetics and Conservation Biology of Rare Plants (eds
Falk DA, Holsinger KE), pp. 123Ð134. Oxford University
Press, New York.
Schneider GJ (1995) Survey for Ofer Hollow reedgrass
Calamagrostis porteri A. Gray ssp. insperata C. W. Greene in
Ohio. A Report for Region 3. U.S. Fish and Wildlife Services,
Twin Cities, MN.
Sipes SD, Wolf PG (1997) Clonal structure and patterns of
allozyme diversity in the rare endemic Cycladenia humilis var.
jonesii (Apocynaceae). American Journal of Botany, 84, 401Ð409.
Smith TB, Wayne RK (1996) Molecular Genetic Approaches in
Conservation. Oxford University Press, New York.
Swallen JR (1935) Two new grasses from the United States and
Mexico. Journal of the Washington Academy of Science, 25, 413Ð414.
© 1999 Blackwell Science Ltd, Molecular Ecology, 8, 443Ð451

Swensen SM, Allen GJ, Howe M, Elisens WJ, Junak SA,
Rieseberg LH (1995) Genetic analysis of the endangered
island endemic Malacothamnus fasciculatus (Nutt.) Greene
var. nesioticus (Rob.) Kearn (Malvaceae). Conservation
Biology, 9, 404Ð415.
Sydes MA, Peakall R (1998) Extensive clonality in the endan-
gered shrub Haloragodendron lucasii (Haloragaceae) revealed
by allozymes and RAPDs. Molecular Ecology, 7, 87Ð93.
Waycott M (1998) Genetic variation, its assessment and implica-
tions to the conservation of seagrasses. Molecular Ecology, 7,
793Ð800.
Weeden NF, Wendel JF (1989) Genetics of plant isozymes. In:
Isozymes in Plant Biology (eds Soltis DE, Soltis PS), pp. 46Ð72.
Dioscorides Press, Portland, OR.
Wendel JF, Weeden NF (1989) Visualization and interpretation of
plant isozymes. In: Isozymes in Plant Biology (eds Soltis DE,
Soltis PS), pp. 4Ð45. Dioscorides Press, Portland, OR.
Whitkus R, la Cruz M, Mota-Bravo L, Gomez-Pompa A (1998)
Genetic diversity and relationships of cacao. (Theobroma
cacao L.) in Southern Mexico. Theoretical and Applied Genetics,
96, 621Ð627.
Wiens D, Nickrent DL, Davern CI, Calvin CL, Vivrette NJ (1989)
Developmental failure and loss of reproductive capacity in
the rare palaeoendemic shrub Dedeckera eurkensis. Nature,
338, 65Ð67.
Wolfe AD, Elisens WJ (1993) Diploid hybrid speciation in
Penstemon (Scrophulariaceae) revisited. American Journal of
Botany, 80, 1082Ð1094.
© 1999 Blackwell Science Ltd, Molecular Ecology, 8, 443Ð451
CALAMAGROSTIS MOLECULAR MARKERS 451
Wolfe AD, Liston A (1998) Contributions of the polymerase chain
reaction to plant systematics. In: Molecular Systematics of
Plants II: DNA Sequencing (eds Soltis DE, Soltis PS, Doyle JJ),
pp. 43Ð86. Kluwer, New York.
Wolfe AD, Xiang Q-Y, Kephart SR (1998a) Diploid hybrid specia-
tion in Penstemon (Scrophulariaceae). Proceedings of the
National Academy of Sciences of the USA, 95, 5112Ð5115.
Wolfe AD, Xiang Q-Y, Kephart S (1998b) Assessing hybridization
in natural populations of Penstemon (Scrophulariaceae) using
hypervariable inter-simple sequence repeat markers.
Molecular Ecology, 7, 1107Ð1125.
Wolff K, Zietkiewicz E, Hofstra H (1995) Identification of
chrysanthemum cultivars and stability of DNA fingerprint
patterns. Theoretical and Applied Genetics, 91, 439Ð447.
Wu K-S, Tanksley SD (1993) Abundance, polymorphism and
genetic mapping of microsatellites in rice. Molecular and
General Genetics, 241, 222Ð235.
Yang W, de Oliveira AC, Godwin I, Schertz K, Bennetzen JL
(1996) Comparison of DNA marker technologies in character-
izing plant genome diversity: variability in Chinese
sorghums. Crop Science, 36, 1669Ð1676.
The authors are all interested in the biology and conservation of
rare plant species, with emphasis on the use of molecular mark-
ers to examine the apportionment of genetic diversity within and
between populations.