An Evaluation of Sceptridium dissectum (Ophioglossaceae) with ISSR Markers: Implications for Sceptridium Systematics

19 Pages·2003·10.1 MB·English

by  Michael S Barker

American Fern
Journal 93(1):1-19
(2003)
An
Evaluation of Sceptridium
dissection
ISSR
(Ophioglossaceae) with Markers:
Implications
Sceptridium
for Systematics
Michael Barker 1 and Warren Hauk
S. D.
OH
Department Denison
of Biology, University, Granville, 43023
Abstract.—Sceptridium dissectum, the most variable North American grapefern species,
demonstrates variation in degree of blade dissection, blade color, pinnule shape, and pinnule
margins. Historically, various morphologies have been included within dissectum. For example,
S.
monograph
Clausen's recognized which
five infraspecific taxa in S. dissectum, of only the
morphologies of variety dissectum and obliquum are currently retained. However, the taxonomic
We
two
status of the varieties has been debated. used ISSR (Inter-Simple Sequence Repeat) markers
to assess the genetic distinctness of S. dissectum var. dissectum and var. obliquum in 17 Ohio
UPGMA
populations. Five ISSR primers generated 69 reproducible loci. In analyses and AMOVA,
dissectum dissectum
S. var. individuals did not cluster separately from var. obliquum individuals,
nor did individuals from the same population cluster together. ISSR markers revealed levels of
population genetic structure in S. dissectum similar to levels detected by previous isozyme
Our
investigations. results concur with recent treatments of S. dissectum that do not formally
recognize infraspecific taxa, and may bring into question current species circumscriptions
in
We
Sceptridium. illustrate the use of ISSR markers examining taxonomic boundaries
for in
Sceptridium.
common
Species of Sceptridium Lyon, the evergreen grapeferns, are
members
of temperate and north temperate habitats, though the genus has
a worldwide distribution (Wagner and Wagner, 1983). In North America, the
center of species diversity lies east of the Mississippi River to the Atlantic
Coast, and from the southern Gulf Coast to the northern coasts of the Great
Lakes (Wagner and Wagner, Within Wagner and Wagner
1993). this range,
uncommon
(1993) recognized seven species, and not more than one
it is to find
species at a single site (Wagner, 1960a). Most Sceptridium species inhabit
a variety of moderately disturbed habitats such secondary-growth woods,
as
and some may
old fields, grassy slopes, although species
undisturbed
habitats (Clausen, 1938).
members
Species of Sceptridium, like other of Ophioglossaceae, generally
produce one which
epigeal leaf per year, divided into a trophophore
is sterile
and a fertile sporophore (Clausen, 1938). Unlike some members of the family
Botrychium Wagner,
1990), Sceptridium species do not always
(e.g., s.s.;
produce sporophore, and under may
a stressful conditions not produce
a trophophore (Wagner, 1960b; Montgomery, 1990; Wagner and Wagner,
1993:
The
Kelly, 1994). leathery, photosynthetic trophophore through
persists the
winter, hence the moniker "evergreen" grapefern. Sceptridium
species, well
as
OH
1 Miami
Current address: Department of Botany, University, Oxford, 45056.
BOTANICAL
MISSOURI
WAY 1 9 2003
GARDEN LIBRARY
AMERICAN FERN JOURNAL: VOLUME NUMBER
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(2003)
1
members
as other of the family, possess subterranean, non-photosynthetic,
mycoparasitic gametophytes (Wagner Melan and
et al. 1985; Whittier, 1989).
9
The may
subterranean nature of the gametophytes be associated with high
self-
fertilization rates (Tryon and Tryon, 1982) as has been documented in some
Sceptridium (McCauley Watano and
species et aL, 1985; Sahashi, 1992).
—
Taxonomic History of Sceptridium and Dissectum
(Spreng.) Lyon.
S.
was
Sceptridium recognized genus by Lyon
first as a (1905) after observing
Botrychium dissectum obliquum (Muhl
var.
from
embryo
the of B. virginianum (L.) Swartz by possessing a long suspensor that
lacked a pronounced cotyledon and emerges from
lateral a root that the basal
Lyon
side of the gametophyte. Further, noted most Underwood's
(1905) that of
Botrychium which
(1898) ternate species, of dissectum obliquum was
B. var.
had
included, a sporophyll that divided into a trophophore and a sporophore
On
near the rhizome. Lyon placed most Underwood's
this basis, of
(1898)
Botrychium
Most
Sceptridium subgenus Botrychium
as a of (Clausen, 1938; Lellinger, 1985;
Wagner
and Wagner,
However,
1993). other authors have maintained
Sceptridium
as a separate genus within Ophioglossaceae
(Sahashi, 1979; Kato,
Watano and
1987; Sahashi, Hauk,
1992; 1996).
Of
the seven currently recognized North American
Sceptridium
species,
S.
Wa
(Wagner,
1960b;
Wa
Botrych
variable that has apparently misled
it botanists in delimiting other, closely
more
related, but uniform, The
species
correctly..." diversity of blade
morphologies encompassed
by
dissectum
has taxonomic
S. led to disagree-
ment what
over range of variation should be included within and
dissectum,
S.
what
putative segregates deserve
recognition
as distinct species (Clausen,
Wagner,
1938; 1960a; Wagner, Wagner,
1960b;
1961).
Sceptridium dissectum was
described by Sprengel 1804 Botrychium
in as
dissectum, and
only sporophytes more
of the dissected morphology were
included
in the species by
early authors Underwood,
(Sprengel, 1804; 1898).
Sporophytes
possessing
relatively less dissected and a more broadly laminated
blade morphology were
ascribed to B. obliquum Muhl. (Underwood,
1898).
Variations on these names
existed, example,
for Prantl recognized
(1884) B.
obliquum and
obliquum
B. dissectum
var. (Spreng.) Prantl. Until Clausen's
mono
limits of B. dissectum complete synonyms
(for a of
list see Clausen, 1938).
monograph
In his
of the Ophioglossaceae, Clausen
(1938) treated B.
dissectum
as four and one
varieties subspecies: B. dissectum typicum
var.
(Muhl
ifoli
(Mart
BARKER
3
elevated to species or subsumed into other taxa. Only varieties dissectum and
obliquum remain
(Fig. designated as varieties (Clausen, 1938), forms
1)
McCauley
(Wagner, 1960a; et aL, 1985), or not officially recognized but their
morphologies mentioned Wagner and Wagner, To have
(Lellinger, 1985; 1993).
we
working and
taxa for analyses discussion, followed the nomenclature of
Clausen (1938) and considered the two morphologies as varieties.
—
DNA
ISSR PCR. Multilocus markers have become examining
a useful tool for
among
relationships closely related taxa and Abbott, 1998; Kardolus
(Gillies
et aL, 1998; Parker etal., 1998; Campbell etal., 1999; Nkongolo, 1999; Crawford,
Huang
2000; and Sun, 2000; Wolfe and Randle, 2001) because they provide
numerous genome
characters derived from multiple within the (Wolfe
sites
PCR
and Liston, 1998). ISSR (inter-simple sequence repeat polymerase chain
DNA
reaction) a multilocus marker system that has successfully examined
is
among Huang and
relationships closely related taxa (Wolfe et aL, 1998; Sun,
2000; Culley and Wolfe, 2001; Wolfe and Randle, 2001). Highly variable
PCR
regions flanking microsatellites are amplified by ISSR primers and minute
When
amounts of genetic variation can be detected (Wolfe and Liston, 1998).
RAPD
compared
similar techniques such (random amplified polymorphic
to as
DNA)
PCR, ISSR loci are more polymorphic (Kojima et aL, 1998; Esselman
et aL, 1999; McGregor et aL, 2000) and reproducible, presumably because of
longer primer length and higher annealing temperatures (Nagaoka and
Ogihara, 1997; Wolfe and Liston, 1998; Wolfe et aL, 1998).
we
In the present study, present an investigation of taxonomic boundaries
within Sceptridium dissectum by comparing inter-simple sequence repeat
We
(ISSR) marker patterns of dissectum dissectum and obliquum.
S. var. var.
PCR
chose ISSR to 1) assess the genetic distinctness of S. dissectum var. dis-
sectum and obliquum, examine dissectum population genetic
var.
2) S.
PCR
structure, and evaluate the utility of ISSR for studying Sceptridium taxa.
3)
Materials and Methods
Individual sporophytes were sampled from 17 Sceptridium dissectum
populations Ohio Table Ten dissectum dissectum and
in var.
(Fig. 2, 1). S.
52 dissectum obliquum sporophytes were collected. Individuals were
S. var.
selected to represent the range of morphological variation present at each site.
Five sporophytes were collected for nine populations, whereas all sporophytes
(<5) were collected from eight smaller populations (Table Leaf material
1).
DNA
was and
from each individual dried in silica gel for extraction, the
remaining laminar material was pressed. Vouchers were deposited at the
Miami
Willard Sherman Turrell Herbarium at University (MU).
DNA mg
Total genomic was extracted from approximately 100 of silica gel
DNeasy Mini
dried material using Qiagen's Plant Kit (Qiagen Inc.,
leaf
DNA
was
Valencia, CA). Genomic from each sporophyte quantitated fluoro-
dsDNA
metrically using the PicoGreen quantitation reagent (Molecular Probes,
OR) and TD-360 mini-fluorometer (Turner Designs, Sunnyvale,
Eugene,
a
Inc.,
4 AMERICAN FERN JOURNAL: VOLUME NUMBER
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Pinnae
Fig. of dissectum
1. S. var. obliquuni (top) and S. dissectum var. dissectum (bottom).
CA).
Quantitations were
performed
according
to the manufacturer's
protocol
(Molecular DNA
Probes, Eugene, Each
Inc., OR). sporophyte's was
quantitated
and mean
twice,
the
concentration was
calculated.
PCR
ISSR
primers were
selected from
the University Columbia
of British
Biotechnology
Laboratory
(IJBC) primer #9
(Vancouver,
set BC, Canada:
http://
www.biotech.ubc.ca).
Ninety ISSR UBC
primers
in the were
set screened using
DNA
from two
Sceptridium
dissectum We
sporophytes &
(O-lc
O-ld).
selected
the primers
five that produced most
the robust and
clear amplification
profiles
during
primer
screening
(Table
2).
The PCR
ISSR
reaction mixture DNA
included one Taq
unit of
polvmerase,
mM
mM
MgCl
0.2
2
,
uM
BARKER
r>
Fig. 2. Distribution of 17 Ohio Sceptridium dissection populations. Numbers correspond
to
population codes Table
in
1.
DNA
10 ng of template in a total volume of 25 ^L. Reactions were performed in
Eppendorf
Mastercycler Personal thermalcyclers (Eppendorf AG, Hamburg,
Germany)
using the following temperature regime: 94°C 60 seconds, then
for
35 cycles consisting of 45 seconds 94°C, 45 seconds and 90 seconds
at at 55°C,
72°C followed by
at a final 5 minute, 72°C extension.
PCR
Each ISSR was
reaction repeated with
twice, appropriate
controls, to
DNA
ensure ISSR
consistent profiles. Using a 1 kb Plus ladder size standard
(Gibco-BRL, PCR
Life Technologies, Rockville, MD), products were
Inc.,
2%
separated electrophoretically 80 seven hours on
at volts for agarose gels in
TBE UV
ng/mL
buffer with 0.2 EtBr. Bands were visualized on a trans-
MP
MA).
from
I
temperature helped ensure ISSR bands were among
(55°C) that reproducible
Only and
reactions. clear consistently reproducible bands were scored. Bands
of indistinguishable mobility between lanes were assumed be homologous,
to
and
to represent a single ISSR locus. For each sporophyte, each locus was
= "0" =
("1"
scored as present or absent locus present, locus Data
absent).
were compiled into a Nexus data matrix using MacClade (Maddison and
4.0
Maddison,
2000).
ISSR were examined with
loci data three types of analyses: primer
1)
UPGMA
banding (Unweighted Group Method
profiles, Pair using Arithme-
2)
AMOVA
and
tic averages) cluster analyses, (Analysis of MOlecular
3)
we
VAriance; Excoffier et 1992). For all analyses assumed ISSR locus
a/.,
was
variation representative of overall genetic variation.
AMERICAN FERN VOLUME NUMBER
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Table 1. Populaition codes locations, sample sizes, an-d vouc:her numbers tor Ohio Sceptridium
»,
I
dissectum collections. Note that four populations contained both varieties Vouchers deposited
at
.
MU. D = dissectum dissectum and O = dissectum obliquum.
S. var. S. var.
:
Population code Location (Coun ample Voucher
ty) Si size
O-l Licking 5 Barker #70
0-2 Coshocton Barker #85
5
D-3 Franklin Barker #86
1
0-3 Franklin 4 Barker #129
0-4 Perry 5 Barker #103
D-5 Lucas 2 Barker #107
0-5 Lucas Barker #130
3
D-6 Richland 2 Barker #109
0-6
Richland Barker #131
2
0-7 Morgan 4 Barker #112
0-8
Hocking #113
2 Barker
0-9 Logan #115
Barker
3
O-10 Ross Barker #121
5
O-ll Pike Barker #123
5
0-12
Scioto Barker #127
5
Adams
D-13 Barker #128
1
0-13 Adams
4 Barker #136
D-14 Licking 1 Barker #84
D-15 Licking Barker #95
1
D-16 Richland Barker #108
1
D-17 Athens #110
Barker
1
Primer ban ding were analyzed ISSR PCR
profiles to assess the util ity of in
Sceptridium, and to examine the eh on<>hip between the two varieties.
r
iti.
\
Banding
generated by each primer were examined
profiles the following
for
>25%
parameters: 1) variety-specific markers (loci present in of one variety,
but in only a few individuals of the other variety; Wolfe et 1998), percent
al., 2)
polymorphic number
of and number
of per primer, unique
loci, loci of
3) 4)
multilocus genotypes per primer. Mean loci and mean multilocus genotypes
were
also calculated.
UPGMA
was
cluster analysis used
to investigate the distinctness of
S.
dissectum var. dissectum and obliquum, and examine dissectum
var. to
S.
A
population
genetic structure. phenetic rather than parsimony-based method
was
used we
for cluster analyses because did not verify that co-migrating
all
were homologous
loci
or that they sorted independently. Distance matrices
were
constructed from Dice and
(1945) Jaccard (1908) similarity coefficients for
UPGMA
Taxonomy
cluster analysis (Numerical System (NTSYSpc)
ver. 2. It;
Rohlf, and were
2000), based only on the shared presence The absence
of
loci.
of an ISSR
locus not informative because any number non-homologous
is of
may
mutations
result in the loss of a band. Coefficients that calculate distance
from both presence and
absence
of ISSR
loci are, generally, not appropriate
for
UPGMA
data analyses (Wolfe and
Liston, Support was
1998).
for clusters
calculated (WinBoot; Yap and
Nelson, 1996) with 1000 bootstrap iterations of
the data
(Felsenstein,
1985).
BARKER HAUK: MARKERS SCEPTRIDIUM DISSECTUM
& ISSR IN
7
Table Sequences of the ISSR primers used in this study.
2.
Primer Sequence (5 '-3') Length (bp)
CAC ACA CAC ACA CAC AG
UBC-818
17
TCT CTC TCT CTC TCT CG
UBC-824
17
AGA GAG AGA GAG AGA GYC
UBC-835
18
CAC ACA CAC ACA CAC ART
UBC-846
18
GGA GAG GAG AGG AGA
UBC-880
15
AMOVAs
were conducted an assessment
as alternative of the relationship
between the S. dissectum varieties, and of S. dissectum population genetic
AMOVA
were
structure. Distance matrices for generated (Arlequin 2.001,
Schneider et ah, 2001) as described by Huff et (1993). The statistical
al.
AMOVA
significance of results were calculated by a non-parametric
permutational analysis of a null distribution for the variance component. To
assemble component,
the null distribution of a variance individuals are
randomly assigned populations while the number of populations and
to
population from main
sizes are retained the analysis (Excoffier et 1992).
al.,
The
P-value calculated from the null distribution represents the probability of
obtaining a larger variance component than the observed values by chance
low
alone. In biological terms, a small P-value indicates a probability of
identifying more genetic structure than measured in the observed distribution
of individuals, and a high probability of recording less genetic structure. Thus,
AMOVA
more
P-values only reflect the probability of finding genetic structure,
and do not indicate the biological significance of the observed quantities of
AMOVAs,
genetic structure. In our null distributions were generated with
1023 permutations of the data (Arlequin 2.001, Schneider et al, 2001).
AMOVA
F
was
also used to calculate an t value for the distribution of S.
S
dissectum population genetic variation. For dominant marker data ISSR
(e.g.,
AMOVA
F
or RAPD), the value calculated by a correlation of genotypes
t is
S
rather than individual co-dominant as in isozymes. Further, identical
sites,
breeding mechanisms were assumed dissectum populations. Thus, an
for all S.
F may
value calculated from dominant marker data not be directly
t
S
F
comparable values generated from co-dominant marker
to data.
t
S
Results
=
94%
Five ISSR primers produced 69 (mean 13.8/primer) with of the
loci
polymorphic Primer UBC-818 produced most
(Table the
loci loci
3). (16),
whereas primer UBC-846 produced the fewest The mean number of
(12).
was No
unique multilocus genotypes distinguished per primer 38.2 (Table
4).
markers were For each primer surveyed, some
variety-specific identified.
individuals of Sceptridium dissectum var. dissectum possessed banding
some
obliquum
profiles identical to those of var. individuals. All individuals
were distinguished as unique multilocus genotypes using a combination of any
NUMBER
8
Table Sample number and
3. size, total of loci, percent polymorphic loci generated by
five
primers from the ISSR survey of S. dissectum var. dissectum and var. obliquum.
%
Taxon
Sample
size Total # Polymorphic
loci loci
S. dissectum var dissectum 10 50 82%
dissectum obliquum 92%
S. var. 52 62
Total 94%
62 69
and
three primers, using
all five primers, the genotypic diversity genotypes/
(#
# individuals) each taxon was
for
1.0.
Our
investigation of dissectum population
S. genetic structure revealed
that
individuals from the same S. dissectum population did not cluster closely in
UPGMA
&
analyses Most
(Figs. 3 clusters consisted of individuals from
4).
and
sometimes,
different, distant populations. For example,
0-9a, collected in
Logan County
west
in central Ohio, clustered with 0-6d, specimen from
a
Richland County
in north central Ohio, approximately 120 miles away. This
was
pattern repeated
for other individuals from geographically
distant
sites,
but for the clusters supported by bootstrap values >50%, were
individuals
all
from the same population & Seven
two
(Figs. 3 4). clusters consisted of
individuals from the same population, and, of these, only O-lc + O-ld + O-le
and 0-12a + 0-12b were >50%
supported by bootstrap values &
ABA (Figs. 3 In
4).
fl 1 1 A*_— . A,
(MP)
MP
UPGMA
levels of bootstrap support.
AMOVA
Of
tions. the total genetic variation detected, among-population
genetic
was A
variation 8.49%, whereas
within-population was 91.51%. low
level of
genetic structure for the dissectum populations was
S. by
also indicated the
~"*
P<
F ~"
""
value
ST (0.085, 0.0001, Table The highly
5)'. " significant P-value suggests
that our observed
distribution of individuals in populations produces
nearly
amount
the
largest of genetic structure possible our
in data
set.
In a comparison of the two dissectum
S. varieties, neither dissectum nor
var.
UPGMA
obliquum
formed
var.
discrete clusters in analyses &
(Figs. 3
4).
Individuals
of dissectum
var. frequently clustered more closely with members
of obliquum than with own
var. taxonomic
their group.
Bootstrap support
for
UPGMA
MP
and
NJ
which had
almost
identical and
tree topologies
similar
levels of bootstrap
support.
AMOVA
revealed
little genetic difference between dissectum
S. var.
dissectum and
obliquum. The two
var. were
varieties only 3.38%
genetically
amount
genetic difference between
identified the two was
varieties close to the largest
amount
0.0140, Table
6).
BARKER HAUK: MARKERS
& ISSR IN SCEPTRIDIUM
g
Number
Table 4. of loci and genotypes distinguished for each primer. Because some sporophytes
when
of each variety were indistinguishable examined with a single primer, # loci and genotype
values are the combined result for both S. dissectum varieties.
Primer
# Loci # Genotypes
UBC-818
16
55
UBC-824
14
51
UBC-835
13 36
UBC-846
12 27
UBC-880
14
22
Mean
13.8
38.2
Total 69
62
Discussion
Marker systems used examine among
to relationships species or subspecific
taxa should provide highly variable and the system should be
loci, able to
many
distinguish as individuals of a single species as possible, in concordance
with
the organism's breeding system ISSR
(Avise, 1994). In the present study,
markers
distinguished dissectum individuals by any combination
all S. of
three primers, a result similar to that observed in other studies (Wolfe et al,
1998; Esselman et al, 1999). Of the ISSR loci distinguished in our dissectum
S.
82% 92% 94%
taxa, dissectum), obliquum) and were
(var. (var. (species
total)
polymorphic when
(Table values well within the range of ISSR variability
3),
ISSR
markers have
successfully discriminated taxa at the species level and
lower (Wolfe et al, 1998; Culley and Wolfe, 2001; Wolfe and Randle, 2001).
For example, Wolfe et al (1998) demonstrated patterns of diploid hybrid
speciation in Penstemon using ISSR markers, and reported percent poly-
morphic 72-95%
loci values of for the seven taxa sampled. Wolfe and Randle
(2001) used ISSR markers examine taxonomic boundaries and relationships
to
Hyobanche and found 64-96% ISSR were polymorphic
in that of their loci in
four taxa. ISSR markers discriminated between two varieties of Viola
100%
pubescens with ISSR polymorphic and
of loci for the species (Culley
Wolfe, 2001). As values for ISSR variability in S. dissectum were within the
range reported from studies that have successfully used ISSR markers
to
examine taxonomic boundaries and relationships, ISSR markers appear to be
among
an appropriate examining taxonomic boundaries Sceptridium
tool for
and
subspecific taxa possibly species.
—
As by ISSR
Distribution of Genetic Variation. assessed genotypes, the
was
dissectum with
distribution of genetic variation in consistent results
S.
from studies of other pteridophyte species, where most genetic variation was
and
distributed within populations (Haufler Soltis, 1984; Holsinger, 1987;
and and
Kirkpatrick et al, 1990; Soltis Soltis, 1987; Soltis et al, 1988; Soltis
Watano and Using ISSR PCR, Camacho and
Soltis, 1988; Sahashi, 1992). Liston
(2001) found most genetic diversity partitioned within populations of
among
Botrychium pumicola, and population genetic
differentiation.
little
81%
Within Sceptridium, Watano and Sahashi (1992) reported that of isozyme
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062
80
0.89
0.98
Coefficient
UPGMA
Fig. 3. cluster analysis of 62 Sceptridium dissectum sporophytes based on 69 ISSR
loci
generated from five ISSR primers using a distance matrix generated with the Dice algorithm.
(1945)
>50%
Bootstrap values are reported above branches. The below dendogram
scale the the
refers to
coefficient of similarity represented by corresponding branch
lengths. Labels correspond to
population codes in Table 1, asterisks indicate var. dissectum individuals, and lowercase
letters
{i.e., a, b, c, d, e) distinguish individuals of the same population.