Magnus Lundberg

Systematics and polyploid evolution in Potentilleae
(Rosaceae)
Magnus Lundberg
Systematics and polyploid evolution
in Potentilleae (Rosaceae)
Magnus Lundberg
©Magnus Lundberg, Stockholm 2011
Cover illustration:
Fragaria virginiana, Bergianska trädgården (top)
Fragaria chiloensis, Oregon, USA (bottom)
Phylogeny of Fragariinae (right)
Photo: Magnus Lundberg
ISBN 978-91-7447-227-1
Printed in Sweden by Universitetsservice US-AB, Stockholm 2011
Distributor: Department of Botany, Stockholm University
Academic dissertation for the degree of Doctor of Philosophy in Plant Systematics presented
at Stockholm University 2011
Abstract
Lundberg, M., 2011. Systematics and polyploid evolution in Potentilleae
(Rosaceae).
This thesis comprises studies of the phylogenetic relationships in the flowering plant clade
Potentilleae in Rosaceae. The relationships were elucidated by using DNA sequence data from
the nuclear genome as well as from the plastid genome. In particular, the focus of the studies
was the investigation of allopolyploidy, i.e. speciation as a result of hybridization and
subsequent chromosome doubling. A phylogenetic method was used for identifying
allopolyploidy through comparison of trees resulting from the analyses of different DNA
sequences. Five sub-clades were investigated. First, both the sister clades that together contain
all of Potentilleae: Fragariinae and Potentilla. Secondly, three subclades of Fragariinae,
namely Alchemilla in wide sense, Sibbaldia and relatives, and Fragaria. The aim was to
unravel the phylogenetic relationships, including instances of allopolyploidy. Classification
issues were discussed in relation to the phylogenetic results. The split between Potentilla
(=Potentillinae) and Fragariinae received better support than in previous studies. The
phylogeny of Fragariinae was found to be consistent with classifying ten genera: Alchemilla
in wide sense (incl. Aphanes and Lachemilla), Comarum, Sibbaldia, Sibbaldianthe,
Sibbaldiopsis, Chamaerhodos, Drymocallis, Dasiphora, Potaninia, Fragaria, and also
including a few orphan Potentilla species. The segregated genera Ivesia, Horkelia,
Horkeliella and Duchesnea were found to be nested within Potentilla, corroborating earlier
studies, while the segregated genus Argentina (P. anserina and close relatives) showed an
ambiguous position. Plastid and nuclear (ribosomal) phylogenies were compared and
incongruences were detected as potential instances of allopolyploid speciation. Five strongly
supported incongruences were detected in Fragariinae and four of them were considered to be
potentially caused by allopolyploidy. In addition, five supported incongruences were found in
Potentilla. Alchemilla in the wide sense was found to contain four major clades, African
Alchemilla, Eurasian Alchemilla, Lachemilla and Aphanes. Both Lachemilla and Aphanes
were nested within Alchemilla and it was suggested that the name Alchemilla should be used
in the wide sense, i.e. including both the genera Lachemilla and Aphanes. The genus
Sibbaldia as commonly classified was shown to be polyphyletic in five different places in
Potentilleae. Three Sibbaldia clades ended up in Fragariinae and two in Potentilla. A
phylogeny of Fragaria, based on a nuclear low/single copy DNA region was estimated. The
gene copy phylogeny was used to construct a reticulate tree hypothesizing allopolyploid
speciation events. The evolution of Fragaria was shown to have been shaped by polyploidy.
Keywords: Potentilleae, Fragariinae, Potentilla, Sibbaldia, Fragaria, Alchemilla, systematics,
phylogeny, polyploidy, autopolyploidy, allopolyploidy, reticulate evolution.
Preface
This thesis is based on the following papers, referred to in the text by their Roman
numerals.
I
Lundberg, M., Töpel, M., Eriksen, B., Nylander, J. A. A., Eriksson, T.
2009. Allopolyploidy in Fragariinae (Rosaceae): Comparing four DNA
sequence regions, with comments on classification. Mol. Phylogenet. Evol.
51, 269-280.
II
Töpel, M., Lundberg, M., Eriksson, T., Eriksen, B. Molecular data and
ploidal levels indicate several putative allopolyploidization events in the
genus Potentilla (Rosaceae). Manuscript.
III
Gehrke, B., Bräuchler, C., Romoleroux, K., Lundberg, M., Heubl, G.,
Eriksson, T., 2008. Molecular phylogenetics of Alchemilla, Aphanes and
Lachemilla (Rosaceae) inferred from plastid and nuclear intron and spacer
DNA sequences, with comments on generic classification. Mol. Phylogenet.
Evol. 47, 1030–1044.
IV
Eriksson, T., Lundberg, M., Töpel, M., Östensson, P. Sibbaldia – a
molecular phylogenetic study of a polyphyletic genus in Rosaceae.
Manuscript.
V
Lundberg, M., Eriksson, T., Zhang, Q., Davis, T. M. New insights of
polyploid evolution in Fragaria (Rosaceae) based on the single/low copy
nuclear intergenic region of RGA1- Subtilase. Manuscript.
Paper I and Paper III is reprinted with the permission from the copyright holder, Elsevier.
Lundberg's contribution to papers included in the thesis:
I - Lundberg is first author and has produced all the sequences new to this study. Conducted phylogenetic
analyses and has written the manuscript with comments and suggestions from co-authors. Convergence
diagnostics was performed and interpreted with help from co-authors.
II - Lundberg is co-author and has contributed by producing sequences for included outgroups, taken part
of writing the manuscript with additional comments and suggestions from co-authors.
III - Lundberg is co-author and has contributed by producing sequences for included outgroups, written
part of the method section and given comments of the manuscript.
IV - Lundberg is co-author and has produced a large set of the included sequences, taken part in analyzing
the data, as well as the writing of the manuscript.
V - Lundberg is first author and has contributed by producing the vast majority of the sequences,
conducted phylogenetic and reticulate analyses and written the manuscript with comments and
suggestions from co-authors.
Contents
Introduction.........................................................................................10
Aims....................................................................................................14
Material and Methods..........................................................................15
Results and Discussion........................................................................17
Conclusions.........................................................................................20
Svensk sammanfattning.......................................................................21
Tack/Thanks........................................................................................24
References...........................................................................................25
Abbreviations
bp
c.f.
DNA
et al.
ETS
ITS
pp.
rDNA
RGA1
s.l.
s.s.
ssp.
Subt
trn
var.
basepairs
confer, compare
deoxyribonucleic acid
et alii, and others
external transcribed spacers
internal transcribed spacers
posterior probability
ribosomal deoxyribonucleic acid
resistance gene analogue 1
sensu lato, in a broad sense
sensu stricto, in a strict sense
subspecies
subtilase
transfer ribonucleic acid
variety
Introduction
The research discipline of Systematic Biology involves studying various
groups of organisms and their relationships. Systematists are often interested
in natural groups (monophyletic clades), i.e. species groups that share a
common ancestor. Conclusions of what natural groups are, and the
relationships among organisms constituting the natural groups were
traditionally based on macromorphological characters, i.e. the appearance of
the organisms. As microscopes were improved, new possibilities emerged to
study micromorphological characters such as flower development and pollen
grains. As new techniques developed, information were also obtained from
e.g. chemical compounds and chromosome numbers. In the beginning of the
1980's, molecular methods were developed and DNA sequences from either
nuclear, plastid or mitochondrial genomes were a new source of information.
Along with this new source of data, phylogenetic methods were also
significantly developed. The results from DNA sequence analysis are usually
depicted as a phylogeny (phylogenetic tree with nodes and branches; e.g. see
Fig. 1) and used to make hypotheses about relationships among organisms,
based on synapomorphies (presence of shared derived character states) to
define natural groups. Information obtained from phylogenies can also be
used to make hypotheses about speciation processes, biogeography, character
evolution, co-evolution, biodiversity, gene evolution, nature conservation
and ecology.
In this thesis, the organisms that have been studied belong in the tribe
Potentilleae (Potentillineae [= Potentilla] + Fragariinae; see also Fig. 1),
belong in Rosoideae (Rosaceae; [Morgan et al., 1994; Eriksson et al., 2003;
Potter et al., 2002; Potter et al., 2007] ). Potentilleae includes at least 1000
species and most of them occur in northern temperate, arctic and alpine
regions but a few are found in the southern hemisphere. The classification
and circumscription of Potentilleae and closely related genera has been of
much debate for the last century and no taxonomic consensus has been
reached until recently with the aid of molecular sequence data. The various
taxonomic hypotheses are due to that many morphological characters have
been shown to be homoplasious, i.e. arisen or lost at several independent
occasions in the evolution of this clade. Most Potentilla species are perennial
or annual, rarely biannual herbs, or small shrubs. The leaves are pinnate or
palmately compound. Most Potentilla are yellow flowered, almost
consistently with 5 epicalyx segments, 5 sepals alternating with the epicalyx
segments and 5 petals. Other common characters are usually 20 stamens,
numerous free carpels and a basal chromosome number of x=7.
10
Figure 1. Phylogeny of Rosaceae. Phylogeny was redrawn based on Fig. 1
Potter et al. (2007). The clades of Fragariinae, Potentilleae and Rosoideae
among others are marked to the right.
11
Potentilla was found to be polyphyletic, with its species intermixed with
other genera based on nuclear ribosomal DNA data (Eriksson et al., 1998)
and several genera grouped in a clade of their own as sister to Potentilla in
the strict sense. This was the first support for the clade Fragariinae and
further support was received after combining the nuclear sequences with
plastid DNA sequences (Eriksson et al., 2003). The clade Fragariinae
comprise 10 genera, Alchemilla in the wide sense, Chamaerhodos,
Comarum, Dasiphora, Drymocallis, Fragaria, Potaninia, Sibbaldia,
Sibbaldiopsis and Sibbaldianthe and a few orphan Potentilla species.
Fragariinae share the morphological synapomorphy of anthers with confluent
thecae at apex (cf. Potentilla with anthers where the thecae are separated at
apex; Sojak, 2008), and possibly a second character based on lateral to basal
position of styles on achenes (but see discussion in Paper IV). Many genera
that belong in Fragariinae have according to earlier classifications been
placed either inside Potentilla or considered to be closely related as separate
genera, but Alchemilla and Potaninia have in many cases been placed
elsewhere in Rosaceae (Hutchinson, 1964; Kalkman, 1988; Kalkman, 2004).
Recent studies have provided new insights that the mode of speciation
known as allopolyploidy may be a major evolutionary force of speciation
and today allopolyploidy is considered much more common than previously
thought. For a review of hybridization and polyploidy see Soltis and Soltis
(2009). In allopolyploid speciation two species hybridize followed by a
chromosome doubling. If this hybrid is functional and survives, a new
species may have been formed. Another similar process involving
chromosome doubling is autopolyploidy, distinguished from allopolyploidy
in that the duplication of chromosomes occurs within a species
(hybridization is not involved). Early estimates of polyploid speciation
among angiosperms ranged from 30% (Stebbins, 1950) to 52% (Grant,
1981). Considerably higher estimates have been presented more recently. A
study based on guard cell sizes estimate that at least 70% of extant
angiosperms are the result of polyploid speciation (Masterson, 1994).
Several taxa in Potentilla and Fragariinae have members that are polyploid
(Goldblatt and Johnson, 2010) and both clades have been proposed to have
an evolution shaped by hybridization and polyploidy (Schulze-Menz, 1964).
One way to screen a larger clade (e.g. Fragariinae and Potentilla) for
potential allopolyploid speciation is to contrast phylogenies based on easily
amplified DNA sequence regions from the plastid and the nucleus (e.g.
Smedmark and Eriksson, 2002). Instances (nodes) in the separate
phylogenies that display well supported incongruent patterns are potentially
12
reflecting allopolyploid speciation because plastid DNA is maternally
inherited, hence reflecting maternal phylogenetic relationships while nuclear
DNA is biparentally inherited potentially reflecting the paternal phylogeny.
Based on primary results from the screening, further studies would focus on
the most promising incongruences and by cloning and sequencing nuclear
single/low copy DNA sequence regions it may be possible to trace the
parental species of the allopolyploids by obtaining all potential copies of
polyploid species. This method has been shown to be succesful in several
studies (e.g. Sang, 1999; Smedmark, 2003; Mason-Gamer, 2001; Popp et al.,
2005). A phylogeny based on all copies will depict a tree with allopolyploid
species having different copies positioned in different places in the tree, as
sisters to their respective parental species. When combining the multiple
copies from a single taxon into a reticulate tree, hypotheses about
allopolyploid speciation can be drawn. This method will also distinguish
allopolyploid speciation (where DNA copies from a taxon are distantly
related) from autopolyploidy (where DNA copies are sisters). With this
method ancient allopolyploid speciation may also be detected. The results of
studying nuclear single/low phylogenies will hopefully give better
knowledge and understanding of the role allopolyploid speciation have had
in larger clades such as Fragariinae and Potentilla. This will also give an
indication of the potential impact allopolyploid speciation may have played
in the evolution of other angiosperm groups.
The three Fragariinae genera, Alchemilla, Sibbaldia and Fragaria are of
special interest to us because, (1) Alchemilla and Sibbaldia have been
suspected to be polyphyletic due to complicated morphology, (2) intriguing
geographical distributions and (3) all three clades have taxa with established
polyploidy. Therefore we have performed more detailed investigations of
these three groups/genera.
The first group (“genus”) in Fragariinae we have focused on is Alchemilla in
the wide sense (i.e. Alchemilla, Lachemilla and Aphanes). Alchemilla is the
largest genus in Fragariinae and is characterized by its small apetalous
inconspicuous flowers and a basal chromosome number of x = 8. Alchemilla
in strict sense is mainly distributed in the northern hemisphere and East
Africa. While Lachemilla is restricted to montane South America and
Aphanes is scattered in different regions of the world but primarily found in
the Mediterranean area to central Europe, the west coast of South and North
America and southern parts of Australia. Alchemilla in the wide sense has by
earlier classifications been placed either in Sanguisorbeae ([Poterieae]
13
Hutchinson, 1964; Focke, 1894) or by on its own in the subtribe
Alchemillinae, belonging in Potentilleae (Schulze-Menz, 1964; Soják, 2008).
The second genus we have focused on is Sibbaldia. Sibbaldia has
consistently been placed in Rosoideae and often considered to be a separate
genus because of a small number of stamens 5 (-10). Other morphological
characters such as, trifoliate or pinnate leaves and relatively few carpels are
common in Sibbaldia. Otherwise, large variation regarding e.g. leaf form and
division, stamen placement, anther form, presence or absence of floral disc
and flower colour are observed in the genus. Sibbaldia has mainly a northern
temperate and alpine distribution and the centre of diversity is in the
Himalayas. More than 40 species names of Sibbaldia have been described,
but recent treatments lists 10-15 species (Dixit and Panigrahi 1981, Rajput et
al. 1997, Dikshit and Panigrahi 1998, Li et al. 2003) or as few as two (Soják,
2008).
The third genus we have focused on is Fragaria. Based on the results from
Lundberg et al. (2009; Paper I, in this thesis), Fragaria was considered one
of the most promising groups for further investigation of allopolyploidy.
Fragaria was suspected to have an evolution shaped by allopolyploidy due
to incongruent phylogenies and established polyploid species. About 24
Fragaria species are described (Staudt, 2008) and mainly distributed in the
northern hemisphere. Fragaria is distinguished from closely related genera
by having white flowers and a swollen receptacle (i.e. the 'strawberry') and
the ploidy level ranges from diploid (2x=2n=14) to decaploid (2x=10n=70).
Different genomic composition models involving allopolyploidy have been
proposed for the octoploid Fragaria (Fedorova, 1946; Senanayake and
Bringhurst, 1967; Bringhurst, 1990) and molecular studies trying to elucidate
relationships (Potter et al., 2000) and polyploidy (Rousseau-Gueutin et al.,
2009) have been published but never with any strong support for the
evolution of the polyploid Fragaria.
Aims
The overall aim for this thesis was to elucidate the systematics and evolution
of the clade Potentilleae, with focus on allopolyploid speciation. Earlier
studies have shown that polyploidy is widespread and recent molecular
studies have indicated that both inter- and intrageneric relationships among
Fragariinae genera/taxa and the sister clade Potentilla are intriguing and less
than well understood. In this doctorate thesis, five different studies are
14
presented, together giving new insights towards understanding speciation,
polyploidy and evolution in Potentilleae.
In paper I, the main aim was to identify potential allopolyploid speciation
events in the clade Fragariinae and also discuss taxonomic implications of
included genera/taxa.
The purpose of paper II was to identify potential allopolyploid speciation
events in the clade Potentillineae. Identify major clades in Potentilla and
evaluate the monophyly including phylogenetic position of segregated
genera Argentina as well as Ivesia, Horkelia and associated genera.
In paper III we tested the relationships of segregated genera in the clade
Alchemilla in the wide sense and presented a comprehensive phylogeny
based on molecular sequence data.
Paper IV focused on testing the monophyly of genus Sibbaldia and the
relationships of Sibbaldia species in the clade Potentilleae.
Paper V, the main purpose was to elucidate polyploid speciation in Fragaria.
Differentiate between auto- and allopolyploids, and if relevant, erect a well
supported and detailed hypothesis of the reticulate evolution in Fragaria.
Material and Methods
In Paper I, the sampling was based on the study of Eriksson et al. (2003)
where Fragariinae was established to be monophyletic. Additional sampling
intended to generally increase the number of taxa from each genus in
Fragariinae, and taxa with established polyploidy were particularly sampled.
In addition, Potentilla miyabei and P. cuneata, traditionally classified in
Potentilla in strict sense were also sampled. These two species have been
classified close to Potentilla tridentata, currently Sibbaldiopsis tridentata, a
genus in Fragariinae (Eriksson et al., 2003). In total, 40 taxa were sampled of
which 34 were part of the ingroup. Two nuclear ribosomal (ITS, ETS) and
two plastid regions (trnL/F, trnS/G) were amplified and sequenced. Analyses
using Bayesian inference (Huelsenbeck and Ronquist, 2001; Ronquist and
Huelsenbeck, 2003) were conducted. Topologies were compared in order to
detect incongruences, and three criteria (see paper I for details) had to be
fulfilled in order for the incongruences to be considered as potential
allopolyploid speciation events.
15
In paper II, we extended the Potentilla data sets used in the studies of
Eriksson et al. (1998 and 2003) with emphasis on sampling the major groups
proposed by Wolf (1908). In addition, taxa proposed to be close relatives to
Horkelia and Ivesia were sampled. Five representatives of the clade
Fragariinae and two taxa outside Potentilleae served as outgroup. In total
were 64 ingroup taxa and seven outgroup taxa sequenced for the two nuclear
ribosomal regions, ETS and ITS, as well as the two plastid regions, trnL/F
and trnS/G. Phylogenetic analyses were performed using MrBayes and
topologies were compared by eye to detect incongruencies.
In paper III, four criteria were applied for sampling (see Paper III for details)
reaching a total of 100 taxa. The ingroup consisted of 89 taxa: 71 Alchemilla,
6 Aphanes and 12 Lachemilla. The outgroup consisted of 11 taxa, sampled to
cover a large part of the Fragariinae clade, with representatives from
Potentilla in the strict sense and Rosa majalis. Amplification and sequencing
were performed of the plastid trnL/F region and nuclear ribosomal ITS
region. Parsimony and non-parametric bootstrap analyses to obtain support
values, as well as Bayesian inference analyses were performed.
In paper IV, the intention was to sample as many taxa traditionally described
in Sibbaldia as possible. In addition, at least two species from each genus of
Potentilleae along with three outgroup taxa that represented the two clades
most close to Potentilleae (Eriksson et al., 2003) were sampled. Diploids
were chosen before polyploids and taxa shown to be resolved in early splits
of each “genus” sampled were favoured whenever possible. Sibbaldia
trullifolia and S. tenuis were not sampled because of no available material
for DNA extraction. In total 43 ingroup and 3 outgroup taxa were sampled.
Amplification and sequencing of nuclear ribosomal DNA and plastid trnL/F
DNA regions were performed. DNA sequence data were analyzed by
Bayesian inference and evaluated by convergence diagnostics.
The aim in paper V was to sample all 24 Fragaria species (Staudt, 2008)
described and several closely related taxa in the clade Fragariinae (Lundberg
et al., 2009) as outgroups. In addition were several subspecies sampled for F.
vesca, F. virginiana and F. chiloensis (Staudt, 1999). For the first time was
the nuclear low copy intergenic region between the two genes, Resistance
Gene Analogue 1 (RGA1) and Subtilase (Subt) used to conduct phylogenetic
analyses. Diploid species were directly sequenced while polyploid species
were first cloned and subsequently sequenced in a sufficient number to
obtain a 95% confidence to acquire all potential copies of each ploidy level.
16
Bayesian inference was used to obtain the complete phylogeny and the
reticulate tree was obtained by hand.
Results and Discussion
Paper I: Phylogenies based on two regions of nuclear and two regions of
plastid DNA sequence data, respectively, were compared and revealed five
instances of conflict. In order to classify detected incongruences as potential
allopolyploid speciation events, three criteria were defined and that had to be
fulfilled: incongruences had to be (1) supported by a clade credibility value
above 0.95, (2) strength of conflict should be at least 0.90 (see Paper I for
explanation) and (3) convergence diagnostics were assessed to rule out the
possibility that the incongruences were the result of inadequate Bayesian
inference analysis. The five incongruences that fulfilled the defined criteria
involved Aphanes arvensis, Potentilla cuneata, Potentilla miyabei, Fragaria
vesca/moschata and the Drymocallis clade. Incongruences were evaluated
and allopolyploid speciation appear to be a probable hypothesis for all
conflicts except the last, involving the Drymocallis clade (see discussion
Paper I). Several genera in Fragariinae were found to be monophyletic,
Alchemilla in the wide sense, Sibbaldianthe, Chamaerhodos, Drymocallis
and Fragaria. The two orphan Potentilla species, P. miyabei and P. cuneata
were placed in a clade comprising Sibbaldia and close relatives, and both
were involved in incongruences between topologies. The two monotypic
genera Sibbaldiopsis and Potaninia were nested within Sibbaldia and
Dasiphora respectively. The incongruences detected in Sibbaldia and
Fragaria were considered to be of main interest and Fragaria were further
studied using single/low copy sequence region (see Paper V).
Paper II: Six major clades, the Anserina-, Alba-, Fragarioides-, Reptans-,
ivesioid Potentilleae and the Argentea clade were found in both phylogenies
based on nuclear or plastid data. In addition were three supported backbone
clades identified. When nuclear and plastid phylogenies were compared, five
strongly supported incongruencies were found in Potentilla. The
incongruencies were: 1) within the Anserina clade, P. peduncularis and P.
microphylla are changing places. 2) Within the Alba clade, P. articulata is
changing position. 3) The Fragarioides and Reptans clades are switching
positions. 4) P. dickinsii is part of the Fragarioides clade in the nuclear
phylogeny while positioned as sister to P. fragarioides/stolonifera, ivesioid
Potentilleae and Argentea clades in the chloroplast phylogeny. 5) P.
norvegica is part of the Argentea clade in the chloroplast phylogeny but
sister to the ivesioid Potentilleae in the nuclear phylogeny. Four of the
17
incongruences are considered to be caused by potential allopolyploid
evolution. The ivesioid Potentilleae clade was nested within Potentilla and
should therefore be merged with Potentilla in order for Potentilla to stay
monophyletic. Potentilla lignosa that have earlier been placed elsewhere
(Wolf, 1908; Botschantzev, 1952; Soják 2008), were shown to be part of the
Anserina clade according to both nuclear and chloroplast data. Two
morphological characters were tested for systematic information but only
style morphology was considered valuable.
Paper III: Analyses of plastid and nuclear phylogenies were largely
congruent and four distinct clades were resolved. These four clades were
African Alchemilla, Eurasian Alchemilla, South American Lachemilla and
annual Aphanes, where each of the clades corresponds to either distinct
geographical areas or life forms. Within the clade of African Alchemilla
moderate support for some patterns of mainly geographical distribution
could be found (in at least the combined; plastid + nuclear DNA sequence
data) analysis: (1) Southern African taxa form a clade, (2) taxa from
Madagascar group together and (3) A. kiwunensis and A. volkensii are sister
taxa, otherwise the clade was poorly resolved. The Eurasian Alchemilla clade
comprise two major clades that correspond to the morphology of leaves,
dissected (or nearly so) and lobed (at least not entirely dissected), with a few
taxa as exceptions. The Lachemilla clade displayed incongruent pattern
between plastid and nuclear topology, but not well supported. Lachemilla
was sister to the African Alchemilla clade in the plastid phylogeny while
sister to a clade including both Aphanes and Eurasian Alchemilla in the
nuclear phylogeny. The topology of the nuclear analysis was also reflected
the combined phylogeny. The resolution within the Lachemilla clade was
better than the other major clades. The Aphanes clade is strongly supported
and sister to the Eurasian Alchemilla clade in all analyses. All analyses show
that Alchemilla in the wide sense is monophyletic but in the more restricted
sense (excluding Aphanes and Lachemilla), Alchemilla is paraphyletic.
Based on three factors involving priority of monophyly, morphological
synapomorphies and nomenclatural stability, it was suggested that the name
Alchemilla should be used in the wide sense, i.e. including both the genera
Aphanes and Lachemilla.
Paper IV: Based on molecular nuclear and plastid DNA data, Sibbaldia was
shown to be polyphyletic. A conglomerate of taxa primarily based on a
reduced number of stamens 5 (-10) and often with trifoliate leaves end up
scattered in five separate clades of Potentilleae. Three clades ended up in
Fragariinae, (1) Sibbaldia in the strict sense comprising four taxa including
18
the type species Sibbaldia procumbens, (2) S. adpressa is placed in
Sibbaldianthe and (3) S. perpusilloides is considered to represent a new
undescribed genus which is nested in a clade with the genera Chamaerhodos,
Drymocallis, Dasiphora and Potaninia. The two remaining Sibbaldia clades
are placed within Potentilla (Potentillinae sensu Soják [2008]), (4) a clade
comprising four Himalayan species end up in a basal position and (5)
Sibbaldia micropetala is nested within the Potentilla anserina clade. The
monospecific genus Sibbaldiopsis is close to or congeneric with Sibbaldia
(see discussion in paper IV) but the species should be named Sibbaldiopsis
retusa. Our results are mostly congruent with the findings of Soják (2008)
which separate Fragariinae from Potentilla (Potentillinae) based on anther
structure.
Paper V: The low copy nuclear intergenic region between the two genes,
Resistance Gene Analogue 1 (RGA1) and Subtilase (Subt) was used for the
first time for phylogenetic studies. The octoploid Fragaria virginiana and
chiloensis had copies positioned in three defined main clades. One of the
main clades also consisted of the diploid species F. vesca and mandshurica,
the tetraploid F. orientalis and hexaploid F. moschata along with the
octoploids. The second main clade constituted the octoploids, decaploid F.
iturupensis and copies of the hexaploid F. moschata while the last main
clade only had gene copies of the octoploids and decaploids. In addition, a
clade with diploid F. viridis and gene copies of F. moschata and a clade
consisting of the Asian diploids F. pentaphylla and nipponica and tetraploids
F. gracilis, tibetica and corymbosa were also found. When gene copies of the
polyploid species were found in more than one clade, the process of
allopolyploidization was considered likely to have occurred and by
combining the copies found in multiple clades from a single taxon we
obtained a reticulate tree. Our favoured scenario to explain the reticulate
evolution of genus Fragaria was obtained by inferring six events of
allopolyploidy: 1) between the diploids F. vesca and viridis, resulting in an
undescribed tetraploid. 2) Between this undescribed tetraploid and a diploid
of the F. iinumae lineage gave rise to the hexaploid F. moschata. 3) Between
the hexaploid F. moschata and a diploid of the F. iinumae lineage again,
creating the origin of the octoploid lineage. 4) Between the octoploid lineage
and an unknown diploid close to the base of the Fragaria phylogeny (but
close to F. daltoniana) resulting in the potentially new decaploid species
earlier known as F. virginiana ssp. platypetala. 5) Between the octoploid
lineage and a diploid of the F. iinumae lineage that gave rise to the decaploid
F. iturupensis and 6) in the Asian Fragaria clade, between a diploid of the F.
nipponica lineage and a unknown diploid sister to the F. nipponica lineage
19
resulting in the tetraploid F. corymbosa. The other tetraploid Fragaria
sampled were considered to be of autopolyploid origin because all gene
copies of each tetraploid formed a separate clade in the phylogeny. The
diploid progenitor of autotetraploid F. orientalis was F. mandshurica, the
diploid F. nipponica was progenitor of autotetraploid F. tibetica and the
tetraploid F. gracilis was the result of autopolyploidization of a diploid of the
F. nipponica lineage. The octoploid Fragaria species have one origin and the
speciation event resulting in F. virginiana and chiloensis took place after the
octoploid lineage was formed. It should be noted that the Fragaria root is
differently placed in our study compared with the study of Rousseau-Gueutin
et al. (2009).
Conclusions
New knowledge of the evolution and relationships of taxa in Potentilleae
have emerged based on the results of the five studies presented in this thesis
and will be a good framework for further studies. Our results reinforces the
split in Potentilleae, into the clades Fragariinae and Potentilla
(=Potentillineae). The only morphological character known to coincide with
this split is the anthers with confluent thecae at apex in Fragariinae while
Potentilla species have anthers with two distinct thecae.
Several genera in Fragariinae received further support of being monophyletic
while others were shown to be polyphyletic. Two “orphan” Potentilla
species, P. miyabei and P. cuneata was placed in Fragariinae and five events
of potential allopolyploid speciation were detected.
Six major clades were found within Potentilla. The ivesioid Potentilleae
were shown to be an ingroup in Potentilla. Four instances of potential
allopolyploid speciation were found in Potentilla. Style morphology was the
single most important morphological character in Potentilla.
Alchemilla in the wide sense is monophyletic but in the more restricted sense
(excluding Aphanes and Lachemilla), Alchemilla is paraphyletic. Four
distinct clades were resolved, African Alchemilla, Eurasian Alchemilla,
South American Lachemilla and annual Aphanes, where each of the clades
corresponds to either distinct geographical areas or life forms.
Sibbaldia was shown to be polyphyletic and scattered in five different clades
in Potentilleae and S. perpusilloides was considered to represent a new
undescribed genus.
20
The evolution of Fragaria was shaped by polyploidization. The hexaploid F.
moschata is the result of two allopolyploidy events, first involving F. vesca
and F. viridis, secondly by a diploid of the F. iinumae lineage. The octoploid
lineage emerged after an allopolyploidization event between F. moschata and
a diploid of the F. iinumae lineage. The decaploid F. iturupensis was the
result of allopolyploidization between the octoploid lineage and a diploid of
the F. iinumae lineage. The potentially new decaploid species known as F.
virginiana ssp. platypetala originated after an allopolyploidization event
between the octoploid lineage and an unknown diploid close to the base of
the Fragaria phylogeny. The tetraploid Fragaria species mainly originated
from autopolyploidization.
Svensk sammanfattning
I den här avhandlingen behandlas gruppen (kladen) Potentilleae som är en
del av rosväxtfamiljen (Rosaceae) och gruppen består av ett tusental arter.
De är vanligast förekommande på norra halvklotet i tempererade, alpina eller
arktiska områden men återfinns också på södra halvklotet. Under de senaste
hundra åren har det föreslagits ett antal olika hypoteser om vilka arter som
ingår i Potentilleae och hur dessa arters släktskapsrelationer (systematik) ser
ut. Traditionellt har utseendekaraktärer (morfologi) använts som grund för
hur växterna är besläktade med varandra men eftersom det har visat sig att
flertalet av de morfologiska karaktärer som använts (t. ex. blomfärg, antalet
ståndare, behåring på frukterna, bladutseende etc.) har uppkommit eller
förlorats vid flera tillfällen under evolutionen i Potentilleae har det varit svårt
att tolka systematiken enbart baserat på morfologiska karaktärer. Det har
resulterat i att inget konsensus nåtts.
När molekylära metoder utvecklades och framförallt när ”Polymerase Chain
Reaction-tekniken” (PCR) slog igenom på 1980-talet, blev det möjligt att ta
fram nya och stora mängder data i form av DNA-sekvenser. DNAsekvenserna som används vid systematisk forskning kommer ursprungligen
från växtens cellkärna (kärn-DNA) eller från plastiden (samlingsnamn för de
strukturer i varje cell vars primära uppgift är att fotosyntetisera; kloroplasten
är en plastid; plastid-DNA). Parallellt med de nya molekylära metoderna
utvecklades också nya analysmetoder. Oftast presenteras resultatet från
analyserna i form av ett släktträd (fylogeni) som består av grenar och noder
(de punkter där grenar möts i trädet). Fylogenin är en hypotes om hur
växterna är besläktade.
21
Baserat på DNA-sekvenser från plastiden och cellkärnan har tidigare studier
funnit att Potentilleae består av två evolutionära grenar (klader), nämligen
Fragariinae respektive Potentilla (=Potentillineae). Den här avhandlingen
behandlar främst Fragariinae (artikel I) och delar av denna klad (artikel III,
IV och V) men även Potentilla (artikel II).
Förutom släktskapsrelationerna i Potentilleae har den artbildningsprocess
som har att göra med kromosomtalsfördubblingar studerats. Arter som är
kromosomtalsfördubblade kallas gemensamt för polyploider och de ssa delas
in i två huvudkategorier, allopolyploider och autopolyploider. Allopolyploida
arter bildas genom att två olika arter hybridiserar (reproduktion mellan
individer av olika arter) och därefter kromosomtalsfördubblas, med an
autopolyploider kromosomtalsfördubblas inom arten (involverar inte
hybridisering). Man kan spåra föräldraarterna vid allopolyploidisering eller
föräldraarten vid autopolyploidisering genom att ta fram sekvensen från en
kärn-DNA region som endast finns i en eller ett fåtal kopior och undersöka
en fylogeni baserad på denna sekvens. Eftersom en autopolyploid art endast
har en förälder kommer samtliga genkopior vara placerade tillsammans på
samma gren som föräldraarten (närmast besläktad), medan en allopolyploid
art med två olika föräldrar kommer ha genkopior placerade på två olika
grenar i fylogenin, tillsammans med respektive föräldraart.
Den första studien (artikel I) i avhandlingen gav ytterligare stöd för
delningen av Potentilleae i de två kladerna Fragariinae och Potentilla.
Fragariinae är monofyletisk (ett gemensamt evolutionärt ursprung) och
består av tio släkten, Alchemilla (t.ex. daggkåpa) i vid bemärkelse,
Comarum (t.ex. kråkklöver), Sibbaldia (t.ex. dvärgfingerört), Sibbaldianthe
(spetsfingerört), Sibbaldiopsis (tretandsfingerört), Drymocallis (t.ex.
trollsmultron), Chamaerhodos, Dasiphora (t.ex. tok), Potaninia och
Fragaria (t.ex. smultron). Dessutom visas att två arter som för tillfället är
klassificerade i Potentilla, P. cuneata och P. miyabei tillhör Fragariinae och
är nära besläktade med Sibbaldia. Genom att jämföra och identifiera
olikheter mellan den fylogeni som var baserad på plastidsekvenser med den
fylogeni som var baserad på kärn-sekvenser, togs hypoteser om möjliga
allopolyploidihändelser fram. Resultaten från denna studie låg till grund för
övriga studier i denna avhandling. Fem välstödda olikheter (inkongruenser)
identifierades
och
fyra
av
dem
betraktades
som
troliga
allopolyploidiseringar. De två mest intressanta inkongruenserna fanns i
Fragaria (grunden för artikel V) och Sibbaldia (pågående projekt, men inte
inkluderat i denna avhandling).
22
I artikel II användes samma tillvägagångssätt som i den första studien, där
fylogenier baserade på plastid-DNA respektive kärn-DNA jämfördes och
inkongruenser identifierades. I detta fall var det Potentilla-kladen
(fingerörter m.fl.) som studerades mer i detalj och fem välstödda
inkongruenser identifierades. Några släkten som tidigare brutits ut ur
Potentilla, Horkelia, Ivesia, Horkeliella och Duchesnea visade sig vara delar
av Potentilla-kladen. Detta resultat gav ökat stöd till tidigare, mer
översiktliga studier. Det utbrutna släktet Argentina (P. anserina, gåsört; inkl.
nära besläktade arter) var väl stött som syster till övriga arter av Potentilla
enligt plastidfylogenin men svagt stött som syster till Fragariinae enligt kärnfylogenin. Sex välstödda klader identifierades: Anserina-, Alba-,
Fragarioides-, Reptans-, ivesioid Potentilleae och Argentea kladen.
Utseendet på blommans stift var den enda morfologiska karaktär som kunde
identifieras som innehållande relevant systematisk information inom
Potentilla.
Gruppen/släktet Alchemilla (daggkåpor) i vid bemärkelse vilket också
inkluderar Aphanes (jungfrukammar) och Lachemilla studerades i artikel III.
Fylogenier baserade på sekvenser från plastid- och kärn-DNA var i stort sett
kongruenta och fyra distinkta klader identifierades, afrikanska Alchemilla,
eurasiska Alchemilla, sydamerikanska Lachemilla och Aphanes. Analyserna
visar att släktet Alchemilla är monofyletiskt om det klassificeras i vid
bemärkelse inklusive alla fyra kladerna, men om det delas upp tre separata
släkten Alchemilla, Lachemilla och Aphanes– som ofta har skett – är släktet
parafyletiskt (En parafyletisk grupp är inte en hel gren på ett fylogenetiskt
träd utan utesluter delar av grenen.). Vi föreslog att namnet Alchemilla bör
användas i vid bemärkelse, och inkludera släktena Lachemilla och Aphanes.
Ett antal arter blivit förda till Sibbaldia huvudsakligen baserat på de
morfologiska karaktärerna, färre ståndare än Potentilla (5 eller 10 i stället
för 20) och oftast förekomst av trefingrade blad. Baserat på en kombination
av sekvenser från plastid-DNA och kärn-DNA visades att Sibbaldia är ett
polyfyletiskt (består av olika grenar i en fylogeni) släkte som hör hemma på
fem olika ställen inom Potentilleae ( artikel IV). Tre klader placerar sig i
Fragariinae; (1) Sibbaldia i strikt bemärkelse, som består av fyra arter (bland
annat dvärgfingerört), (2) Sibbaldia adpressa placerar sig i Sibbaldianthe
och (3) Sibbaldia perpusilloides som anses representera ett nytt obeskrivet
släkte, placerade sig i en klad med släktena Chamaerhodos, Drymocallis,
Dasiphora och Potaninia. De två återstående Sibbaldia-kladerna återfanns i
Potentilla varav en klad (4) som bestod av fyra arter från Himalaya och (5)
Sibbaldia micropetala som är en del av ”Potentilla anserina-kladen”. En
23
morfologisk karaktär skiljer Fragariinae från Potentilla: ståndarknapparnas
två pollensäckar flyter samman i toppen hos Fragariinae, medan hos
Potentilla är pollensäckarna skilda från varandra även i toppen.
En ingående studie av släktet Fragaria (smultron) utfördes för att försöka
spåra de polyploida arternas ursprung samt att försöka identifiera
föräldrarterna (artikel V). De polyploida arterna klonades och kolonier
sekvenserades i ett representativt antal för att med 95% säkerhet få med alla
potentiella genkopior. Resultaten visade att tre tetraploida (dubbel
kromosomuppsättning) Fragaria-arter var autotetraploider och deras
potentiella föräldraarter identifierades. En tetraploid art ansågs vara
allopolyploid och föräldraarterna föreslogs. Den hexaploida (trippel
kromosomuppsättning) F. moschata (parksmultron) föreslogs vara fullt
allopolyploid, med ursprung från F. vesca (smultron), F. viridis
(backsmultron) och F. iinumae. De två oktoploida (fyra kromosomuppsättningar) arterna F. virginiana och F. chiloensis visade sig ha ett
gemensamt ursprung och allopolyploida med föräldraarterna F. vesca, F.
viridis samt två separata allopolyploidiseringar med F. iinumae-linjen. Den
dekaploida (fem kromosomuppsättningar) F. iturupensis var allopolyploid
och arten bildades efter en hybridisering mellan den oktoploida linjen och F.
iinumae linjen, följt av polyploidisering.
Tack/Thanks
I am grateful to my supervisor Torsten Eriksson for good discussions,
support and feedback. I acknowledge my supervisor Jürg Schönenberger. I
would also like to thank all colleagues at the Department of Botany for help
and support. I acknowledge financial support from the Department of
Botany, SU (salary), the Swedish Research Council (VR, to T. Eriksson), and
FORMAS (to T. Eriksson) for lab work.
Thank you Tom Davis and all the people I met at UNH.
Mamma, Pappa, Syster, Ulla och Marielle.
Sist men inte minst, stort tack alla vänner för att jag har just er!
24
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