Nordic Journal of Botany
AUozyme variation in allotetraploid Saxifraga osloensis and its
diploid progenitors
Thomas Nilsson and Per Erik Jorde
Nilsson, T. & Jorde, P. E. 1998. Allozyme variation in allotetraploid Saxifraga
osloensis and its diploid progenitors. - Nord. J. Bot. 18: 425-430. Copenhagen. ISSN
0 107-055X.
The Scandinavian endemic plant Saxijruga osloensis is believed to be an allotetraploid derived from the diploids S. adscendens and S. tridactylites. We used gel electrophoresis of enzymes to characterize the genetic constitution of the three species.
The results support both the allotetraploid nature of S. osloensis and its ancestry
from the two assumed parental species. The pattern of genetic differences among local populations of the three species indicates that S. osloensis has arisen from hybridization in the western part of its present distribution area, possibly at a single occasion.
7: Nilsson. Department of Botany, Stockholm University, S-106 91 Stockholm,
Sweden.Present address: Swedish Environemntal Protection Agency, S-1 06 48 Stockholm, Sweden. - l? E. Jorde, Division of Population Genetics, Stockholm University,
S-106 91 Stockholm, Sweden. Present address: Department of Biology. Division of
Zoology, University of Oslo, P. 0.Box lOS0, N-0316 Oslo, Norway.
Introduction
Numerous examples of presumed allopolyploidy in
plants have accumulated since the beginning of the
twentieth century (Stebbins 1950, Crawford 1990).
Some studies in the mid-twentieth century identified
parentage of allopolyploids by cytological techniques,
i.e. by meiotic pairing behaviour or by karyotype morphology (Stebbins 1971). It is, however, only after the
development of molecular techniques, such as isozyme
electrophoresis and DNA sequence and restriction
analysis, that it has been possible to unambiguously
identify the progenitors of polyploids (Crawford 1989,
1990, Werth 1989, Soltis & Soltis 1990, Thompson &
Lumaret 1992). Roose & Gottlieb (1976) were the first
using isozyme electrophoresis for this purpose, and they
were able to confirm the allotetraploid genetical constitution of Tragopogon mirus and T. miscellus, as well as
identifying their parentage.
Because allozymes are normally codominantly ex-
pressed (but see Jorde & Ryman 1990), allozymes from
each of the parental species should be expressed in the
presumed allotetraploid hybrid if it is of recent origin.
When the two parental taxa have different alleles, a pattern of apparent “heterozygosity” is therefore observed
in the hybrid (Ranker et al. 1989). This pattern, which is
generally fixed within allotetraploids, does not reflect
genetic variation within loci on one of the sets of parental chromosomes, but rather fixed allelic differences
among homologous loci on the different sets of parental
chromosomes (Crawford 1989). Allotetraploids show
“fixed heterozygosity” because of exclusive pairing of
homologous chromosomes from each parental genome
(Crawford 1990).
A polyploid species may have arisen at one distinct
time and place ( i.e., single origin), or at several times,
possibly at different places, through repeated hybridization (i.e., multiple origins). In the latter case, and provided that polyploidization is recent, the genetic constitution of the resulting allotetraploid should reflect the
Accepted 2-12-1997
0NORDIC JOURNAL OF BOTANY
Nord. J. Bot. 18(4) 1998
425
Fig. 1. Geographic location of the sample sites (points) and current distribution of Saxifraga osloensis (encircled areas). Samples
of S. adscendens (A1 - A2) are indicated with triangles, S. osloensis (27 - 214) with dots, and S. tridac@lites (T1 - T4) with
squares. See also Tab. 1.
genes present in the local populations of each parental
species. Thus, multiple origin is inferred if populations
have different alleles derived from different diploid
populations of the progenitor(s). Multiple origins of
polyploids are reported in, e.g., Draba (Brochmann et
al. 1992 a,b) and Senecio (Ashton & Abbott 1992).
Soltis & Soltis (1990) concluded that polyploids in most
cases have multiple origins.
Allotetraploid species of recent origin often show
low levels of allozyme variation, particularly within
populations, as reported for, e.g., Tragopogon spp.
(Roose & Gottlieb 1976) and Spartina anglica (Gray et
al. 1991, Raybould et al. 1991). These species are estimated to be no more than 100 years old. In contrast,
Brown & Marshall (1981) found substantial amounts of
variation within populations of the much older tetraploid Bromus mollis.
Herein we report the amounts and distribution of
allozyme variation for an allotetraploid plant, the
Scandinavian endemic Saxifraga osloensis Knaben, and
its assumed diploid progenitors S. tridactylites L. and S.
adscendens L. Our main objective with this study is to
explore whether the assumed ancestry is correct, and if
so, whether S. osloensis has one single origin or if different populations of the species may have arisen independently (multiple origins).
The three species of Saxifraga examined in this study
are all very similar to each other, both in morphology
426
and in life history characteristics. They are all shortlived: S. tridactylites and S. osloensis are winter-annuals (Nilsson 1995a), whereas S. adscendens is a biennial. All three species are self-fertilizing (Knaben 1954,
Nilsson 1995b).
SaxiJraga osloensis is distributed from southern Norway in a narrow zone eastwards to the coast of the Baltic Sea in Sweden (Fig. l). In the south, S. osloensis is
replaced by S. tridactylites, and in the north, in the
mountain range, by S. adscendens. Both parental species have wider distribution ranges extending throughout Europe and western Asia. The distribution of S.
tridactylites extends into northern Africa, whereas that
of S. adscendens includes North America. The distribution pattern of the endemic S. osloensis indicates a
postglacial origin of the species (Knaben 1954, Hult
ghrd 1987).
Saxifraga osloensis was first described by Knaben
(1954). She distinguished S. osloensis from the closely
related S. tridactylites and S. adscendens, mainly because of the double number of chromosomes in S.
osloensis (2n=44) compared to the other two species
(both 2n=22). Knaben also gave convincing evidence,
based on various characteristics, that S. tridactylites and
S. adscendens were the parental species of S. osloensis.
Some evidence came from the work of Melchers
(1935) and Drygalsky (1935). Melchers made crosses
between S. adscendens and S. tridactyiites, in order to
Nord. J. Bol. 18(4) 1998
Tab. I . Description of populations of Saxifraga used in the analyses. The populations of S. osloensis are numbered in accordance
with Nilsson (1995b).
Population
code
Al
A2
27
68
86
97
I04
108
21 1
214
T1
T2
T3
T4
Species
S. adscendens
S. adscendens
S. osloensis
S. osloensis
S. osloensis
S. osloensis
S. osloensis
S. osloensis
S. osloensis
S. osloensis
S. tridactylites
S. tridactylites
S. tridactylites
S. tridactylites
Sample Geographic location
size
12
12
50
30
10
50
50
50
2
47
50
30
12
12
Sweden, Vastmanland, Viker: I km NE of Bengtstorp
Norway, Buskerud, Hole: Vik
Sweden, Dalsland, Vlrvik: Gunnebyn
Sweden, Narke, Axberg: 400 m NE of Sveaborg
Sweden, Narke, Skollersta: Omhalla
Sweden, Sodermanland, 0. Vinglker: Glopphalla
Sweden, Uppland, Borstil: 1 km NE of Llngalma
Sweden, Uppland, Harg: Hargshamn
Norway, Akershus (Oslo), Barum: Haug gird
Norway, Akershus (Oslo), Barum: Fornebu, Rolfstangen
Sweden, Sodermanland, Holo: 300 m W of Skogstorp
Sweden, Sodermanland, Trosa-Vagnharad: 500 m NW of Furholmen
Norway, Akershus (Oslo), Baerum: Kalveya
Norway, Buskerud, Hole: Vik
study the genetics of the species’ different life cycles.
Instead of the expected segregation in the second generation (F2), most plants in this generation were found
to be spontaneously arisen tetraploids. Drygalsky
showed that irregularities at meiosis caused the formation of diploid pollen grains in the original hybrid (FI).
It is now clear that this is the common mechanism of
polyploid evolution in plants (Harlan & DeWet 1975,
Thompson & Lumaret 1992). Knaben succeeded in producing only the first generation hybrid (Fl) between the
parental species. She compared the artificially produced
hybrid to the natural S. osloensis, and she found many
similarities between them, both in morphological traits
and in life history. Many of the features of the hybrid
between S. adscendens and S. tridactylites, as well as of
S. osloensis, were intermediate between the two parental species.
Further evidence for the parentage of S. osloensis
came from cytological studies. Knaben (1954) showed
that a group of small chromosomes in S. osloensis appears to have originated from S. tridactylites, and that
another group of larger chromosomes appears to have
originated from S. adscendens. The occurrence of two
satellite chromosomes in S. osloensis, that are also
present in S. adscendens, strengthened the assumed relationship between these two species.
Recent analyses by Brochmann et al. (1996), using
RAPD markers and nucleotide sequences, strongly supported a hybrid origin of S. osloensis. Sequencing of
chloroplast DNA showed that S. adscendens probably is
the maternal parent and S. tridactylites the paternal parent.
Nord. J. Bot. I8(4) 1998
Altitude
(m)
135
110
110
55
90
40
5
15
130
10
30
5
5
110
Materials and methods
Whole plants for electrophoretic screening of isozymes
were collected in the field in the spring of 1994 and
1996. The plants were deep-frozen in carbon dioxide
snow (-79” C) in the field immediately after collection,
and stored in an ultra-freezer until electrophoretic
analysis (storage for a period not exceeding 3 months).
Sampling was performed within the main distribution
areas of Saxifraga osloensis, and in nearby populations
of S. adscendens and S. tridactylites (Fig. 1). Altogether
eight populations of S. osloensis, four populations of S.
tridactylites, and two populations of S. adscendens were
sampled (Tab. 1). Populations 27, 104, 108, 21 1 and
214 of S. osloensis are referred to as “coastal”, and
populations 68, 86 and 97 as “inland’ in the following.
Between 2 and 50 individuals were collected from each
population (Tab. 1).
The whole plants were ground with extraction buffer
(recipe in Coulhart & Denford 1982), and centrifuged to
remove debris. The homogenates were absorbed on filter paper wicks, used in the subsequent electrophoresis.
Horizontal starch gel electrophoresis of isozymes was
performed by routine procedures, described in e.g.
Wendel & Weeden (1989). A number of commonly
used enzymes in electrophoretic studies were tested on
several different buffer systems, of which three were selected for routine screening (Tab. 2). Several enzymes
did not give sufficient resolution or activity with any
buffer system, and were excluded from further consideration. The following enzymes were screened in all individuals and species: aldolase (ALD; E.C. 4.1.2.13),
diaphorase (DIA; E.C. 1.6.99.-) and phosphogluco-
427
Tab. 2. Buffer systems used in the electrophoretic screening.
No.
Electrode
Gel
Reference
1
2
3
Lithium-borate, pH 8.5
Lithium-borate, pH 8.1
Tris-citrate, pH 7.0
Tris-citrate, pH 8.1
Tris-citrate, pH 8.5
Histidine-EDTA, pH 7.0
Ashton & Braden 1961
Ridgway et al. 1970
Cheliak & Pitel 1984
mutase (PGM; E.C. 5.4.2.2) were resolved with buffer
system no. 1, phosphogluconate dehydrogenase (PGD;
E.C. 1.1.1.44) and triose-phosphate isomerase (TPI;
5.3.1.1) with system no. 2, and glyceraldehyde-3-phosphate dehydrogenase (G3PDH; E.C. 1.2.1.12 + 1.2.1.9),
glucose-6-phosphate isomerase (GPI; E.C. 5.3.1.9),
malate dehydrogenase (MDH; E.C. 1.1.1.37) and
shikimate dehydrogenase (SKD; E.C. 1.1.1.25) with
system no. 3.
Histochemical staining of gels followed the recipes
of Wendel & Weeden (1989), sometimes with small
modifications of amounts of chemicals to increase
staining intensity. Enzyme loci were numbered
sequentially with the most anodally migrating isozyme
designated 1, encoded by locus 1, with additional labels
A and T denoting homologous loci in S. adscendens and
S. triductylites, respectively. Allozymes were denoted
alphabetically with the most anodally migrating designated a. A total of 14 different loci were analysed in all
three species.
Results
All 14 loci were found to be monomorphic within
populations. Six of these loci (Ald, Dia-1, Diu-2,
G3pdh-1, G3pdh-2, and Skd) were firther found to be
fixed for the same electromorph (allele) in all three species. Five loci (Mdh, Pgd-1, Pgd-2, Tpi-1, and Tpi-3)
were fixed for different alleles in the two parental species, and Saxifraga osloensis displayed both alleles (Lee,
“fixed heterozygotes”) in all individuals and
populations (Tab. 3). These results are in agreement
with the notion that S. adscendens and S. tridactylites
represent the parental species of S. osloensis.
The remaining three loci (Pgm, Gpi, and Tpi-2; Tab.
3) displayed variation within one species only. First, a
deviant allele (b) was found in Pgm in one population
(T2) of S. triductylites that did not occur in any of the
other populations of this and the other two species,
which were all fixed for the same allele (a). Second,
two different alleles were found in Gpi in different
populations of S. triductylites (a in populations T3 and
T4 and b in T1 and T2; Tab. 3), whereas only one of
these ( a ) was found in S. osloensis. At the same locus
428
all S. adscendens populations had a third allele (c) that
was also present in all S. osloensis populations. Thus,
the two eastern S. triductylites populations (T1 and T2)
both contain alleles that appear not to occur in S.
osloensis. Finally, the three inland populations (nos. 68,
86, and 97; Tab. 3) of S. osloensis all lacked a Tpi-2
allozyme that was found in all other populations of this
species, as well as in both of the S. adscendens
populations. The implications of these findings with regards to possible speciation patterns in S. osloensis are
discussed below.
Discussion
The pattern of “fixed heterozygosity” in several duplicated loci, with one allele originating from each of the
parental species, is in agreement with the hypothesis of
Knaben (1954) of Saxifraga osloensis being a polyploid
hybrid between S. tridactylites and S. adscendens. Our
results are also concordant with the results of
Brochmann et al. (1996). Their analysis of RAPD markers showed a clear pattern of additivity of parental
markers in S. osloensis, similar to the pattern of “fixed
heterozygosity” revealed in this study. Furthermore,
Brochmann et al. found a closer resemblance of
chloroplast genes between S. osloensis and S.
adscendens, than between S. osloensis and S.
tridactylites. Since chloroplast genes are mainly inherited maternally, this indicates that S. adscendens is the
maternal parent.
The fact that the eastern populations of S.
triductylites have unique alleles at some loci not appearing in S. osloensis suggests that these populations have
not taken part in the formation of S. osloensis. Eastern
Sweden should then be excluded as an area of origination of S. osloensis. However, only a limited number of
populations of S. tridactylites from this part of Sweden
were examined in this study, and other genotypes might
exist within the area. In the western populations, there
was a total allelic correspondence between the diploid
populations of S. adscendens and S. tridactylites, and
the tetraploid populations of S. osloensis. For each locus
in S. osloensis each allele could be traced either to S.
adscendens or to S. tridacwlites.
Nord. J. Bot. 18(4) 1998
Tab. 3. Genotypes at 8 loci in diploid Suxijiruga triductylites (tri) and S. udscendens (ads), and tetraploid S. osloensis (osl). Six
other loci were monomorphic across all populations and species, and are not included in the table. As all loci were homozygous
within populations, the genotype is for simplicity denoted with a single letter (a, b or c). The loci of S. udscendens are denoted by
A, and the homologous loci of S. triductylites by T.In S.osloensis, the loci supposedly originating from S. adscendens are placed
in the A-columns, and the loci from S. triductylites in the T-columns. The population codes refer to Tab. 1 and Fig. I .
Species
Population
Gpi
AT
Mdh
AT
Pgd-1
AT
Pgd-2
AT
Pgm
AT
Tpi-1
AT
Tpi-2
AT
Tpi-3
AT
ads
ads
A1
A2
C
C
a
a
a
a
b
b
a
a
a
a
a
a
b
b
osl
osl
osl
osl
osl
osl
osl
27
68
86
97
104
108
21 1
214
ca
ca
ca
ca
ca
ca
ca
ca
ab
ab
ab
ab
ab
ab
ab
ab
ab
ab
ab
ab
ab
ab
ab
ab
ba
ba
ba
ba
ba
ba
ba
ba
aa
aa
aa
aa
aa
aa
aa
aa
ab
ab
ab
ab
ab
ab
ab
ab
ab
b
b
b
ab
ab
ab
ab
ba
ba
ba
ba
ba
ba
ba
ba
tri
tri
tri
tri
T1
T2
T3
T4
b
b
a
a
b
b
b
b
b
b
b
b
a
a
a
a
a
b
a
a
b
b
b
b
b
b
b
b
a
a
a
a
0.4
The inland populations (nos. 68, 86 and 97) of S.
osloensis deviated from the other populations in Tpi-2,
where they displayed only one allele, identical to the
one in S. tridactylites. One explanation for this pattern
could be independent origin of the deviating
populations. However, an independent origin implies
that individuals of S. adscendens involved in the
speciation should have had the same allele as presently
occurring in S. tridactylites. This seems unlikely since
the local population (Al) of S. adscendens in the area
does not display this allele. A more reasonable explanation is that differentiation of populations of S. osloensis
has occurred after the species formation. If the allele in
Tpi-2 originating from S. adscendens is not expressed or
has subsequently been lost, the observed pattern of “homozygosity” would be expected. Loss of duplicate
gene expression has been documented in tetraploid
Chenopodium (Wilson et al. 1983).
Our results are thus in accordance with a single origin of S. osloensis, probably in the western part of the
present distribution range. Multiple origins cannot be
ruled out completely on the basis of present data, however, as there was little genetic differentiation within
the parental species. It is thus possible that different
populations of S. osloensis may have originated independently from different progenitor populations that are
Nord. J. Bol. 18(4) 1998
indistinguishable from each other at the loci investigated herein.
There is a rather extensive literature about the reasons for small amounts of genetic variation in plants.
Such a discussion goes far beyond the scope of this
study, but it should be mentioned that self-fertilized
short-lived plants, such as these species, often show
very low degrees of genetic variation, especially within
populations (Hamrick & Godt 1990). Isozyme studies
of other self-fertilized species in the Scandinavian flora
have also demonstrated a pattern of no variation within
and very little variation among populations (e.g., in species of Draba, Brochmann 1992, and in Petrorhagia
prolifera, Lonn & Prentice 1990).
Acknowledgements - This work was carried out at the
Division of Population Genetics, Stockholm University,
with analysis of additional material at the Division of
Botany, University of Oslo. We thank professors N.
Ryman (Stockholm) and I. Nordal (Oslo) for support
and use of laboratory and other facilities. This study
was financed by the Swedish Environmental Protection
Agency and “Stiftelsen Oscar och Lili Lamms Minne”
through grants to T. N., and by the Research Council of
Norway through a grant to P. E. J.
429
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