Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Science and Technology 924
Disentangling the Reticulate
History of Polyploids in Silene
(Caryophyllaceae)
BY
MAGNUS POPP
ACTA UNIVERSITATIS UPSALIENSIS
UPPSALA 2004
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Therefore, one should know that Perfect Understanding is a great mantra, is
the highest mantra, is the unequalled mantra, the destroyer of all suffering,
the incorruptible truth. A mantra of Prajñaparamita should therefore be
proclaimed. This is the mantra:
“Gate gate paragate parasamgate bodhi swaha”
(The Prajñaparamita Heart Sutra)
List of Papers
This thesis is based on the following papers, which will be referred to in the text by
their Roman numerals:
I
II
III
IV
Popp, M. and B. Oxelman. 2001. Inferring the History of the
Polyploid Silene aegaea (Caryophyllaceae) Using Plastid and
Homoeologous
Nuclear
DNA
Sequences.
Molecular
Phylogenetics and Evolution 20 (3): 474–481.
Popp, M. and B. Oxelman. Evolution of a RNA Polymerase
Gene Family in Silene (Caryophyllaceae) –Incomplete Concerted
Evolution and Topological Congruence among Paralogues.
Submitted to Systematic Biology.
Popp, M., P. Erixon, F. Eggens and B. Oxelman. Origin and
Evolution of a Circumpolar Polyploid Species Complex in Silene
(Caryophyllaceae). Manuscript.
Popp, M. and B. Oxelman. Origin and Evolution of North
American Polyploid Silene (Caryophyllaceae). Manuscript.
The first paper is reproduced with the publisher’s kind permission.
All papers included in this thesis are written by the first author, with
comments and suggestions given by the co-authors. The studies were
planned in cooperation with the co-authors. The first author is responsible
for all analyses and the major part of the laboratory work. The second author
of the third paper produced the psbE-petL dataset.
Contents
Introduction.....................................................................................................1
Polyploidy – a brief history.............................................................................3
RNAP introns – a useful history .....................................................................5
Silene L. – a complex history..........................................................................6
Polyploidy in Silene L. – a reticulate history ..................................................8
Objectives...................................................................................................8
Materials and Methods ...............................................................................8
Summary of results.....................................................................................9
Paper I....................................................................................................9
Paper II ................................................................................................10
Paper III ...............................................................................................10
Paper IV...............................................................................................12
Conclusions ..............................................................................................14
Sammanfattning (Swedish summary) ...........................................................15
Acknowledgements.......................................................................................17
References.....................................................................................................19
Introduction
The significance of polyploidization, i.e., the multiplication of entire
chromosomal complements (Stebbins, 1971), as an evolutionary process has
been widely acknowledged (e.g., Stebbins, 1971; Grant, 1981; Masterson,
1994). Due to its hybrid origin, an allopolyploid contains two or more
different nuclear genomes. This often leads to differences between species
phylogenies and gene phylogenies as depicted in Fig. 1. There are, however,
several other evolutionary important processes causing multiple variants in a
sequence region. Allele variation, gene duplication, recombination and
lateral gene transfer are examples of such processes (reviewed in e.g., Avise,
1989; Doyle, 1997; Wendel and Doyle, 1998). By analyzing several
independent biparentally inherited DNA regions, it is possible to distinguish
the effects of allopolyploidization from other processes.
Figure 1 Taxon B originates after hybridization between taxon A and C as depicted
in (a). Biparentally inherited nuclear DNA reflects the history of both parental
lineages (b). In a phylogenetic analysis, the B1 genome in taxon B obtained from
taxon A is revealed as most closely related to taxon A, whereas the B2 genome
obtained from taxon C is most closely related to taxon C.
The genus Silene L. comprises ca. 650 species (Oxelman et al., 2001), and
is most diverse in the Mediterranean and Middle East. The incidence of
polyploidy in Silene is generally low, but e.g., Silene aegaea (endemic to
Greece), a number of Arctic and subarctic taxa and the vast majority of taxa
endemic to North America, are polyploids (Kruckeberg, 1961; Oxelman,
1995, Elven et al., in prep.). Analyses of several putatively unlinked
biparentally inherited low copy nuclear DNA regions, in combination with
the ITS region and chloroplast regions, made it feasible to study the origin
and evolution of some of these polyploids and their close relatives. The low
1
copy regions are intron regions in four genes, RPA2, RPB2, RPD2a and
RPD2b, encoding the second largest subunit of three different RNA
polymerases. A duplication of RPD2 detected in (but not necessarily
restricted to) the tribe Sileneae is inferred to account for the paralogy.
2
Polyploidy – a brief history
During the work with vegetative grafts and chimeras of Solanum, Winkler
(1916) discovered that some of the plants thus obtained had twice the
number of chromosomes compared to the parent plant (Grant, 1981). To
describe organisms with more than two complete sets of chromosomes,
Winkler introduced the term “polyploid”. Preceding Winkler with almost a
decade, Lutz and other workers demonstrated similar “sudden chromosome
doublings in Oenothera mutants” (in Digby, 1912).
The polyploid condition had been known for some years, though, and a
classical example of polyploidy is Primula kewensis (Digby, 1912). Primula
kewensis was discovered 1899 as a spontaneous diploid hybrid between the
closely related species P. floribunda and P. verticillata growing next to each
other in the Royal Botanic Gardens, Kew, UK. The hybrid was highly sterile
and thus propagated by cuttings. Half a decade later, one of the P. kewensis
plants set seed after the pollination of a single pin flower with pollen from a
thrum flower (pin and thrum referring to alternative stamen and style
arrangements in the flowers). The progeny had both pin and thrum flowers
and was fully fertile. Working with both sterile and fertile P. kewensis,
Digby (1912) discovered that the sterile plants had the same number of
chromosomes as the parental species, i.e., 2n = 2x = 18, whereas the fertile
plants had twice that number, i.e., 2n = 4x = 36.
A number of definitions and criteria have been used to distinguish
different types of polyploidy. Using the terminology introduced by Kihara
and Ono (1926), the Oenothera and Solanum polyploids presented above are
referred to as autopolyploids, whereas the Primula polyploid is an
allopolyploid. Since the introduction of the terms auto- and allopolyploidy,
the terminology has been extended to cope with the different meiotic
behaviors and/or taxonomical ranks often used to describe the type of
polyploidy and infer the mode of origin of polyploid organisms (e.g.,
Stebbins, 1971; Grant, 1981). In this thesis, the terms auto- and
allopolyploidy are used in a strictly phylogenetic context, disregarding
chromosomal behavior and taxonomical ranks.
The modes of origin of polyploids are mere hypotheses that can be tested
in a phylogenetic context. If H0 = “the polyploid is an autopolyploid” then
H1 = “the polyploid is an allopolyploid”. Autopolyploidy is the nullhypothesis, and as such cannot be “confirmed” even though the analysis may
reveal a sistergroup relationship of the paralogues. If the analysis reveal a
3
non-sistergroup relationship of the paralogues, then autopolyploidy is
rejected in favor of allopolyploidy. However, duplication of a DNA region
followed by lineage sorting is a potential source of Type I error, i.e., H0
(autopolyploidy) is wrongly rejected in favour of H1 (allopolyploidy). Type I
errors are minimized by analysing several independent biparentally inherited
DNA regions, because it is unlikely that the same pattern of lineage sorting
will be displayed in unlinked regions. Type II errors, i.e. cases where H0
(autopolyploidy) is not rejected when, in fact, H1 (allopolyploidy) is true, are
best adressed by carefully choosing DNA regions containing a phylogenetic
signal strong enough to separate the lineages included in the analysis.
4
RNAP introns – a useful history
The RNA polymerase family (RNAP) consists of three large nuclear DNA
dependent RNA polymerase holoenzymes in most eukaryotes. RNAP I and
III transcribe structural RNA such as rRNA and tRNA, respectively, whereas
RNAP II mainly transcribes mRNA. However, a fourth member, RNAP IV,
is found only in plants and its function is yet to be elucidated (The
Arabidopsis Genome Initiative, 2000). In Arabidopsis thaliana, three of the
genes (RPA2, RPB2, and RPC2), encoding the second largest subunits of
these holoenzymes, are single-copy and are located on chromosomes 1, 4,
and 5, respectively, whereas the fourth (RPD2) is present in two, presumably
recently diverged paralogues located on chromosome 3 (The Arabidopsis
Genome Initiative, 2000).
Two highly conserved amino acid motifs (GDK and GEMERD, Fig. 2)
are present in all five genes, including the two RPD2 paralogues. By
targeting these amino acid motifs with degenerated primers in a nested PCR
approach, it is possible to amplify and separate the DNA regions between
GDK and GEMERD in all four subunits.
Figure 2 Structure of second largest subunits of the RNAP gene family in
Arabidopsis thaliana. Boxes represent exons and lines represent introns. Lengths are
proportional to scale bar. Arrows indicate the highly conserved amino acid regions
GDK and GEMERD, and also approximate primer sites for RNAP10F, RNAP10FF,
RNAP11R, and RNAP11bR used in paper II. Note that the two paralogous RPD2
sequences in A. thaliana are not orthologous to the two paralogues in Sileneae
(paper II).
5
Silene L. – a complex history
The genus Silene L. (Caryophyllaceae) is distributed mainly in the Northern
hemisphere and is most diverse in the Mediterranean and Middle East.
Together with the closely related Agrostemma, Cucubalus and Lychnis,
Silene was included by Linnaeus (1753) in Species Plantarum. Based on the
number of styles, Silene and Cucubalus (having three styles) were discerned
from Agrostemma and Lychnis (with five styles). Traditionally, most postLinnaean taxonomists have separated Cucubalus from Silene by fruit type (a
fruit with a fleshy mesocarp vs. a capsule), and Agrostemma from Lychnis
by its hairy styles and carpels alternating with the calyx segments (e.g.,
Chowdhuri, 1957). In 1812, Melandrium Röhl. was erected and discerned
from Lychnis by its usually inflated calyces and capsule teeth twice the
number of carpels, and from Silene by partial septation of the capsule.
Melandrium was recognized by both the major 19th century monographers
of Silene, Rohrbach (1869) and Williams (1896). Since Linnaeus, several
additional genera have been distinguished by different authors (see Oxelman
and Lidén, 1995, and Oxelman et al., 2001, for a more thorough review).
The following text discusses some of the groups where polyploidy is known
to occur.
In a revision of North American species of Silene, Hitchcock and Maguire
(1947) noted that the characters used to separate Melandrium from Silene
and Lychnis were too variable among American species to be of any
taxonomical value at the generic level. They chose to include the majority of
North American representatives of Melandrium in Silene, the revision thus
encompassing 54 species in total.
Chowdhuri (1957) dismissed the use of subgenera in his worldwide
revision of Silene, and instead erected 44 sections that he considered natural
groups. The native North American species, of which the majority was, or
had been, included in Melandrium, were included in Silene and dispersed
among several sections. Most of the species were included in the sections
Occidentales Chowdhuri, Graminifoliae Chowdhuri, and Quadrilobatae
Chowdhuri. Chowdhuri also brought back S. menziesii and its close allies
(=Anotites Greene) and placed them in section Rupifraga Otth in DC.
A number of Melandrium species not considered by Hitchcock and
Maguire (1947) were included in Silene section Physolychnis (Benth.)
Bocquet (=S. section Gastrolychnis (Fenzl.) Chowdhuri) by Bocquet (1969).
The taxonomy of some of the groups in S. section Physolychnis is somewhat
6
confused despite the revision by Bocquet (1969). The Arctic/subarctic taxa
have been referred to as Gastrolychnis (e.g., Cherepanov et al., 2000),
Lychnis (e.g., Polunin, 1959), Melandrium (e.g., Hultén, 1968; Komarov,
1970), or, more appropriately, included in Silene (e.g., Oxelman et al., 2001;
Elven et al., in prep.). Furthermore, recent phylogenetic studies (e.g.,
Oxelman and Liden, 1995; Oxelman et al., 1997; Oxelman et al., 2001)
based on molecular data have identified a well supported clade of
representatives from S. section Physolychnis, section Occidentales, section
Odontopetalae, the S. ajanensis (=Lychnis ajanensis) group, and several
other species.
Silene sedoides and its close allies have previously been classified in S.
section Dichasiosilene ser. Rigidulae by Rohrbach (1869) and in S. section
Atocion subsection Divaricatae by Chowdhuri (1957). Both morphological
and molecular data reject these groups as monophyletic (Oxelman, 1995;
Oxelman and Liden, 1995; Oxelman, 1996), and instead, Silene section
Sedoideae Oxelman and Greuter was erected (Oxelman, 1995). The
tetraploid S. aegaea was hypothesized by Oxelman (1995) to be an
allopolyploid, closely related to S. sedoides and S. pentelica.
7
Polyploidy in Silene L. – a reticulate history
Objectives
The objectives of paper I are to: 1) test the hypothesis of an allopolyploid
origin of Silene aegaea, 2) investigate whether evolutionary processes have
homogenized the putative paralogous DNA regions under study in S.
aegaea, and 3) evaluate whether the combination of RPB2, ITS, and rps16
sequence data may be a useful system to study the evolution of polyploids.
The objectives of paper II are to: 1) test the phylogenetic hypothesis based
on ITS and rps16 data in Sileneae (Oxelman et al., 2001), 2) provide future
studies of Sileneae with backbone information from several, presumably
unlinked regions, thus facilitating inferences of gene duplications and
allopolyploidizations, and 3) investigate the topological congruence among
the datasets.
The objectives of paper III are to: 1) test hypotheses of the origin and
evolution of a circumpolar polyploid species complex in Silene.
The objectives of paper IV are to: 1) test if the Silene native to North
America included in the study form a monophyletic group. 2) infer the mode
of origin (auto- or allopolyploid origin) and the relationships of the polyploid
taxa, and 3) investigate the relationship of S. section Physolychnis and the
native North American Silene.
Materials and Methods
All plant materials used are listed with voucher data in paper I – IV.
The approach here taken for studying the origin and evolution of
polyploid taxa in Silene, is using gene phylogenies inferred from biparentally
inherited, putatively independent, DNA sequences combined with
chloroplast DNA regions. The inferred gene phylogenies can explain the
phylogenetic network responsible for the historical relationships among
allopolyploids and their parental lineages.
In the polyploids, the biparentally inherited DNA regions are isolated and
characterized by cloning PCR products and sequencing secondary PCR
products obtained from individual clones. The chloroplast DNA is
8
sequenced from primary PCR products, as are the nuclear regions for the
majority of the diploids.
To make inferences of phylogenetic relationships, PAUP* (Swofford,
2002) is used. For the different optimality criteria and search strategies
employed, see paper I – IV.
Summary of results
Paper I
Phylogenetic analyses reveal two distinct types of biparentally inherited
nuclear RPB2 and ITS sequences in the two accessions of tetraploid (2n = 4x
= 48) Silene aegaea investigated. From the two datasets, respectively, one
sequence type is found in a clade formed by diploid (2n = 2x = 24) S.
pentelica and one in the clade formed by diploid (2n = 2x = 24) S. sedoides.
Analysis of the chloroplast rps16 dataset recovers both S. aegaea accessions
in the S. pentelica clade. Using the gene phylogenies of RPB2, ITS, and
rps16 to infer the history of tetraploid S. aegaea, there is strong support for
an allopolyploid origin of S. aegaea, with the maternal ancestor from the S.
pentelica lineage, and the paternal contributor from the S. sedoides lineage
(Fig 3).
Figure 3 The species phylogeny inferred from the gene phylogenies obtained from
analysis of RPB2, ITS and rps16 (paper I). Silene aegaea is inferred to be an
allopolyploid, originating from the S. sedoides and S. pentelica lineages.
Three of the 17 clones obtained from the ITS regions in S. aegaea were
inferred to be recombinants, i.e., chimerical sequences partly of sedoides
type and partly of pentelica type. A PCR and cloning experiment with S.
sedoides/S. pentelica template mixture demonstrates that under the
9
prevailing PCR conditions, PCR recombination cannot be ruled out as an
explanation for the recombinant S. aegaea clones. The conclusion is
therefore that no convincing sign of ongoing homogenization of
homoeologous DNA regions, such as in the ITS regions of Gossypium
(Wendel et al., 1995) and Saxifraga (Brochmann et al., 1996), is detected in
S. aegaea.
Paper II
A low stringency nested PCR approach is used to amplify the region
between the highly conserved amino acid regions GDK and GEMERD in the
low copy nuclear DNA genes RPA2, RPB2, RPD2a, and RPD2b (Fig. 2).
Used in concert with the ITS region and the rps16 intron from the
chloroplast, they resolve previously poorly known major relationships in
Sileneae (Fig. 4). Maximum parsimony analyses of the separate datasets
result in largely congruent phylogenetic trees for all regions.
Overall model congruence is tested in a likelihood context using the
software PLATO. It is found that ITS, RPA2, and RPB2 deviates from the
maximum likelihood model for the combined data. The topology parameter
is isolated and topological congruence assessed by non-parametric
bootstrapping. No strong topological incongruence is found. Two paralogues
of RPD2 are found in –but not necessarily restricted to– Sileneae. Several
independent losses and incomplete concerted evolution are inferred in the
RPD2 gene phylogeny.
Paper III
Phylogenetic analyses of two chloroplast DNA regions and five putatively
unlinked nuclear DNA regions are used to explore the relationship of a
circumpolar Arctic/subarctic polyploid species complex in Silene. Gene
phylogenies inferred from introns in the low copy nuclear genes RPA2,
RPB2, RPD2a and RPD2b, and the ITS region from the nuclear ribosomal
DNA region, indicate two consecutive hybridization events. The inferred
species phylogeny is depicted in Fig. 5.
In general, two paralogous sequences are identified from the tetraploids
and three paralogues from the hexaploids in the low copy nuclear genes.
Interlocus concerted evolution appears to have homogenized the ITS
regions, and only the sequences inferred to correspond to the paternal
lineages were recovered in the polyploids.
Paralogous RPA2 introns sequences obtained from the diploid S.
ajanensis group were inferred to have originated by gene duplication
followed by extinction in the polyploid lineages, or by lineage sorting of an
ancient allele pool.
10
Figure 4 One of two most parsimonious trees from the analysis of the combined
datasets (paper II). Branch lengths are proportional to number of changes. Numbers
associated with nodes indicate parsimony bootstrap percentages. Nodes without
numbers have bootstrap percentages < 50.
11
Figure 5 Inferred species phylogeny of the polyploids S. involucrata (2n = 4x = 48)
and of S. sorensenis and S. ostenfeldii (2n = 6x = 72; paper III). The first
hybridization event (a) involved the diploid S. uralensis (2n = 2x = 24) lineage as
the cytoplasmic donor, and the diploid S. ajanensis (2n = 2x = 24) lineage as pollen
donor. The hybridization and polyploidization resulted in the tetraploid S.
involucrata lineage. A second hybridization and polyploidization with the S.
ajanensis lineage as pollen donor, and the tetraploid S. involucrata lineage as
cytoplasmic donor, resulted in the hexaploid lineages of S. sorensenis and S.
ostenfeldii.
Paper IV
DNA sequences from the rps16 intron and the psbE-petL spacer from the
chloroplast genome, combined with the ITS region and introns from the low
copy nuclear genes RPA2 and RPB2, are used to infer origins and
phylogenetic relationships of North American polyploid Silene and its close
relatives. Although the vast majority of North American Silene are polyploid
(2n = 4x, 6x, 8x), which contrasts to the diploid condition dominating in
other parts of the world, the phylogenetic analyses reject a single,
monophyletic origin of the North American polyploids. Two separate North
American lineages are revealed (Fig. 6). One of the lineages consists of
tetraploid Silene menziesii (2n = 4x = 48) and its diploid allies. The second
lineage leads to a clade consisting of Arctic, European and Asian taxa in
addition to the majority of the North American polyploids.
The tetraploid S. californica (2n = 4x = 48) and the hexaploid (2n = 6x =
72) S. hookeri are derived from separate allopolyploidization events between
the two main lineages. Despite extensive cloning, no trace of paralogy
indicating an allopolyploid origin is found in S. menziesii.
12
Figure 6 One of >32100 MP trees found in the RPB2 dataset analysis (paper IV).
Branches drawn in thin lines are collapsed in the strict consensus tree. Numbers
associated with nodes indicate maximum parsimony bootstrap frequencies. Branch
lengths are proportional to number of changes. Numbers of sequenced clones are
given after the taxon names. Arrows in bold indicate the positions of sequences
recovered from hexaploid S. hookeri. Groups with North American taxa are in
square brackets.
13
Due to sampling differences between the datasets, its it difficult to
distinguish patterns of allopolyploidy from e.g., lineage sorting in several
other taxa. There are, however, several more putatively separate
allopolyploid origins indicated by the failure of multiple sequences obtained
from individual accessions from both the RPA2 and the RPB2 intron to form
monophyletic groups.
Conclusions
Polyploidy in general, and allopolyploidy in particular, has played, and most
likely, still plays an important role in the evolution of Silene. It occurs rather
isolated in the allotetraploid Silene aegaea, only known from a steep slope,
overlooking a stony beach on the Aegean island of Ikaria, Greece. It also
occurs in a complex pattern of reticulations, connecting Asia, Northern
Scandinavia, the Arctic and North America down to South America, and
gives the impression of gaining momentum from both diploid and polyploid
genomes.
The DNA regions used to infer the phylogenetic relationships all have
their share of advantages and disadvantages. Chloroplast regions, such as the
rps16 intron and the psbE-petL spacer, are usually easy to amplify and
sequence. They are useful for discerning the cytoplasmic lineage in a
phylogenetic network. Since the chloroplast is uniparentally inherited, it
cannot reveal reticulations.
The nuclear ribosomal regions, such as the ITS region, are biparentally
inherited and easy to amplify, too. The homogenizing effect of interlocus
concerted evolution of the paralogues, however, can virtually hide the
history of one or more parental genome in allopolyploids if standard PCR
methods are used. This is not always the case, though, as seen in S. aegaea.
Low copy nuclear DNA (lcnDNA), a group to which the RNA
polymerase gene family RPA2, RPB2 and RPD2 belongs, is the most
promising candidate in the toolbox. It is assumed to be less vulnerable to
interlocus concerted evolution than the ITS region. Furthermore, the number
of potentially useful low copy regions in the nucleus is practically
inexhaustible. The evolution of lcnDNA regions is largely unknown, but
seem to be rather dynamic with fluctuating copy numbers, differences in
chromosomal locations, and recombination events. Another drawback is that
few, if any, protocols for lcnDNA regions can be applied to a given plant
group without optimization and redesign of PCR primers and of PCR
parameters. However, by using degenerated primers and nested PCR, and an
increasing understanding of the dynamics of lcnDNA regions, they may
prove very well suited for inferring polyploid evolution.
14
Sammanfattning (Swedish summary)
När genmodifierade grödors vara eller icke vara diskuteras, är det sällan
naturens egen “genmodifiering” nämns. Som alltid är naturen mer
storslagen. Där människan nöjer sig med att ändra en egenskap här och en
där, slår naturen till och fördubblar nästan hela arvsmassan, cellkärnans
kromosomer, i ett slag! Ofta sker fördubblingen i samband med en
hybridisering, det vill säga en korsning mellan två olika arter.
Kromosomtalsfördubblingen gör ofta den vanligtvis sterila hybriden fertil,
samtidigt som den innebär en barriär mot återkorsningar med de båda
föräldraarterna. På så sätt har en ny utvecklingslinje, eller en ny art om man
så vill, bildats.
Kromsomtalsfördubblade hybrider är både vanliga och viktiga. Vanliga
på så sätt att uppskattningsvis 35 – 50% av alla nu levande blomväxtarter är
kromosomtalsfördubblade och av dem är troligen en stor del av
hybridursprung. I ett mer historiskt perspektiv kan man säga att de flesta
nulevande arter är härstammar från kromsomtalsfördubblade hybridföräldrar.
Viktiga är de så tillvida att mycket av det vi äter varje dag, är naturligt
kromsomtalsfördubblade. Potatis, majs, ris, vete, kaffe, äpple, banan,
sockerrör, sojabönor, de flesta av våra kålsorter – listan kan göras väldigt
lång! Två andra ekonomiskt viktiga kromosomtalsfördubblade växter är
tobak och bomull.
Genom att “läsa av” arvsmassan hos olika arter går det att analysera
släktskapsförhållandena mellan dem. Kromomsomtalsfördubblade hybrider
har kvar en stor del av sitt genetiska arv från de olika föräldra-arterna. Om
man “delar upp” arvsmassan kan man genom en släktskapsanalys se var de
olika delarnas närmaste släktingar finns, och sedan göra tolkningar om
föräldra-arterna utifrån det släktträdet.
Jag har studerat uppkomst och utveckling av kromsomtalsfördubblade
hybrider hos släktet Silene, bläror och glimmar, i nejlikfamiljen. Släktet
omfattar ca 650 arter och finns nästan uteslutande på norra halvklotet.
Arbetet har dels rört en liten, väl avgränsad grupp i medelhavsområdet, och
dels en betydligt större och mer komplex grupp som verkar sträcka sig över
Asien, norra Skandinavien, Arktis, Nordamerika och Sydamerika.
I det första arbetet (paper I) visar jag att den mycket sällsynta grekiska
Silene aegaea från ön Ikaria är en kromsomtalsfördubblad hybrid mellan de
närbesläktade arterna S. sedoides och S. pentelica som båda finns på Ikaria.
Det andra arbetet (paper II) innebär en utveckling av de metoder jag använde
15
i det det första arbetet. Fler delar av arvsmassan används för att få mer
tillförlitliga resultat i släktskapsanalyserna. Jag tar också fram ett mer
omfattande släktskapsträd för släktet Silene och några närbesläktade
växtsläkten. Det tredje arbetet (paper III) visar att den arktiska S. involucrata
(polarblära) uppkommit som en kromsomtalsfördubblad hybrid mellan
förfäder till en grupp växter (representerad av S. ajanensis och S. linnaeana)
från Sibirien och nordöstra Asien och en annan arktisk grupp (representerad
av S. uralensis, fjällblära). Genom en återkorsning och ytterligare en
kromosomtalsfördubbling mellan S. involucrata och S. ajanensis-gruppen
uppkom sedan bland annat den grönländska S. sorensenis. Det fjärde och
sista arbetet (paper IV) visar att de nordamerikanska kromsomtalsfördubblade hybriderna inom Silene inte bildar en isolerad grupp, utan ingår
i en större grupp där växter från Asien, Europa (studerade också i paper II
och III) och Sydamerika också ingår. De olika geografiskt vitt skilda
växtarterna hålls samman av ett komplext nätverk av hybridiseringar och
kromsomtalsfördubblingar.
16
Acknowledgements
Det har varit lite pyssel att få ihop den här avhandlingen... Den hjälp jag
fått av er, mina vänner, har inte varit en tillgång utan själva förutsättningen
för att lyckas. Jag hoppas att ni förstår det.
En som kanske tycker att det här har varit lika pyssligt som jag, är min
huvudhandledare Bengt Oxelman. Bengan, du har dragit ett enormt lass,
framförallt nu när slutet närmat sig med överraskande snabba steg. Jag fick
en känsla av samförstånd redan när du var i Seattle i början av min
doktorandperiod. Jag skulle köpa en dator och mailade ett förslag till dig.
Ditt motförslag var att skippa datorn och i stället investera de ca. 20000
kronorna i Single Malt av god kvalitet. Du erbjöd dig till och med att ansvara
för lejonparten av inköpen under din hemresa från Seattle! Det erbjudandet,
tillsammans med din förmåga att lyssna, diskutera och engagera, har gjort att
jag förstått att ingen rimligtvis kan önska sig en bättre handledare. Tack
Bengt.
Min biträdande handledare, Magnus Lidén, var den som fick mig
intresserad av botanik från början. Visserligen lurade du mig på fältarbete i
Kina, men det kompenserades mer än väl genom att vi åkte till Iran där vi
samlade Dionysia. Det var också i Iran som du gav mig tipset att söka den
här doktorandtjänsten. Magnus, jag har mycket att tacka dig för. Inte att
undra över att jag alltid blir väldigt glad av att se dig!
Ett stort tack vill jag ge till Birgitta Bremer, Kåre Bremer, Leif Tibell och
Mats Thulin, dels för stöd och uppmuntran under åren som gått, och dels för
värdefulla kommentarer av manuskript och avhandling nu mot slutet.
Sylvain Razafimandimbison have spent quite some time discussing the
peculiarities of ITS with me, and I greatly acknowledge you for that, and
also for your valuable comments on the manuscripts. Jag vill också tacka
Ulla Hedenquist, inte för det administrativa arbetet du utför och som jag
tackar dig för i tid och otid. Nej, istället vill jag tacka dig för dina oerhört
träffsäkra cynismer, dräpande kommentarer och små underfundigheter med
vilka du förgyllt min vardag under fem år. Utan Reija Dufva, Inga Hallin och
Nahid Heidari hade det inte blivit mycket till avhandling. Ett stort tack till er
för allt arbete med mina sekvenser. Ett speciellt tack till Nahid för ditt stora
personliga engagemang, alla uppmuntrande ord och allt godis!
Ett stort tack vill jag ge Per Kornhall, min rumskamrat sedan fem år. Jag
har trivts oerhört bra i ditt sällskap och du vet att jag inte skulle vilja byta dig
mot någon annan! Eller förresten, kanske mot Raveena Tandon, men annars
17
ingen. Eller kanske mot någon som vattnar sina blommor själv. Helst
Raveena dock. Jag vill också passa på att tacka för illustrationen som pryder
omslaget. Tack till Per Erixon för att du alltid sett till att jag verkligen fått
valuta för min arbetstid. Genom dig har jag alltid varit ordentligt uppdaterad
vad det gäller din senaste PCR, dina konstiga sekvenser och hur det gick för
dina studenter på förra tentan. Utan dig skulle doktorandtiden känts mycket
längre –fast i verkligheten skulle den antagligen ha varit mycket kortare! Få
har på samma sätt som Frida Eggens fått mig att känna fysisk och psykisk
smärta. Fysisk smärta genom att i svåra stunder med osviklig precision
pricka mig med jordnötter i pannan, och psykisk smärta genom att i andra
svåra stunder (men med samma osvikliga precision) annonsera sina
fältarbeten på Grönland och i Tibet. Johan Nylander har, bland mycket
annat, lärt mig att timing är allt. En av många konsekvenser av det är att man
måste kunna prata tydligt med mat i munnen. Alla vet (Kristina Articus inte
minst!) att jag är väldigt svag för Hege Vårdal och Annika Vinnersten. Utan
er skulle mitt liv vara väldigt torftigt. Ord kan inte beskriva hur glad jag är
att jag har er!
Det finns många fler att tacka på avdelningarna för systematisk botanik
och systematisk zoologi och även en dryg handfull människor på andra
avdelningar inom EBC, men jag kan inte få tiden att räcka till att klä mina
tack i ord och nöjer mig därför med ett stort och varmt kollektivt tack till er
alla –nya, gamla och blivande doktorander, personalen på Fytoteket, i
bibliotek och Botaniska trädgården– ja, alla. Tack så mycket!
Många och varma tack till Petra Korall, Jenny Smedmark och Torsten
Eriksson för allt stöd och all vänskap. Min bana började på Botan i Göteborg
och där finns en hoper människor som förtjänar många tack. Ett stort och
kärleksfullt tack går till Hanne Hegre Grundt i Oslo –jag hoppas du kommer
över till den rätta sidan (den som har växter med både morfologisk och
molekylär variation) och att Bengan välkomnar dig till Uppsala igen nu när
du har en egen korkskruv. Bernard Pfeil, thank you for your guidance with
the title and also the good company!
Så har turen kommit till mina familjer. Familj av blod och ingifte –jag vill
bara säga tack för att ni funnits medan jag varit borta! Och du far, inte gick
det väl så illa som du trodde, när jag åkte tillbaka till Indien istället för att
börja läsa kemi? Mitt glas har alltid fyllts på hos Harriet och Börje och jag
har alltid givits en rättvis chans att slåss om en filt eller en varm plats vid
kaminen. Tack också till Ulrika. Jag har alltid känt mig välkommen hos
Benny och Christina. Anders och Johanna, Sari och Strokarn –ord är
otillräckliga. Tack. Pelle –du har alltid haft en tumme ledig att peta mig i
ögat med. Ord hade varit tillräckligt! Jonsson och Krille, tack för att ni alltid
har ett mer än tillräckligt ont ord till övers för en vän i nöd. Algot, i alla
former och tider, i alla skepnader –med dig som måttstock kommer jag alltid
att framstå i mycket god dager. Det är väl min smala lycka.
18
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20
Acta Universitatis Upsaliensis
Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Science and Technology
Editor: The Dean of the Faculty of Science and Technology
A doctoral dissertation from the Faculty of Science and Technology, Uppsala
University, is usually a summary of a number of papers. A few copies of the
complete dissertation are kept at major Swedish research libraries, while the
summary alone is distributed internationally through the series Comprehensive
Summaries of Uppsala Dissertations from the Faculty of Science and Technology.
(Prior to October, 1993, the series was published under the title “Comprehensive
Summaries of Uppsala Dissertations from the Faculty of Science”.)
Distribution:
Uppsala University Library
Box 510, SE-751 20 Uppsala, Sweden
www.uu.se, [email protected]
ISSN 1104-232X
ISBN 91-554-5845-9
