Juncaceae
Phylogeny Group
Phylogeny Conclusion
Firstly we used for our phylogeny plastome data – rbcL
(see Fig.
1), trnL intron, trnL-F
intergenic spacer and matK. The
analysis of cp DNA sequences indicates a non-monophyletic
status of Juncus
and monophyly of Luzula.
We found very unexpected but
well-supported position of the South African J.
lomatophyllus and J.
capensis in the ´South Hemisphere Clade´.
The
subg. Agathryon is represented
by one well-defined clade,
generally corresponding to the accepted taxonomic circumscription of
the
subgenus based on shared inflorescence characters. However, there is
one species
traditionally included in the subgenus that occupies isolated position.
Juncus
trifidus, is generally
considered to form a taxonomically
distinct group together with J. monanthos Jacq.
The group is
characterized by a number of distinct morphological characters.
Additionally, J.
trifidus also has a unique habit with terminal
inflorescence, reduced to
1–3 (4) subsessile, congested flowers conspicuously
overtopped by leaves,
auricles ±lacerate, and serrulate leaves. This
very unusual character combination supports its separate position on
the tree,
as a sister group to Luzula or both - Luzula
and Juncus.
The traditional subdivision of the subgenus (sect. Tenageia,
sect. Steirochloa,
and sect. Juncotypus) is not well
supported by the tree, mainly because of the position of J.
balticus
within the Steirochloa clade (Fig. 1), and because
of J. squarrosus (sect.
Steirochloa) is sister to the whole clade.
The
subg. Juncus clade is well
supported. However, a few species are
scattered within the overall tree topology. Sect. Juncus,
which is
very well supported, is morphologically unique
within subg. Juncus,
as it comprises species with terete, leafless, pungent, non-septate
stems with
vascular bundles scattered over the most of transverse section, and
lower bracts
apparently forming a prolongation of the stem. The separate status of
the sect. Juncus
is obviously well founded. The section Iridifolii,
with its centre
of diversity in W North America and Eastern Asia, is not resolved in rbcL
analysis, better results offered non-coding plastome data. Five species
were
included, but their relationships remain entirely unresolved within the
main
clade of subg. Juncus. Relationships within the sect.
Ozophyllum
also remain unclear. The South African J. oxycarpus
is sister to the
other taxa of the subg. Juncus clade, whereas the
other species of the
section are scattered among species belonging to three other sections.
The well-supported
monophyly of the morphologically heterogeneous sect. Stygiopsis
was unexpected. The species included in this investigation were
selected because
they were morphologically highly divergent (J. triglumis, J.
castaneus, J.
himalensis, and J. stygius) and they
appear to be more closely
related.
One of the most important results of our study is the establishment of Luzula as a monophyletic taxon. The moderately well-supported clade within Luzula is the one comprising species belonging to section Luzula. The clade is comprised of seven representatives of the section from Europe, South Africa, Australia, and New Zealand. The only unusual member of this clade is L. arcuata, a northern species treated as a member of the sect. Luzula (another two members of the sect. Thyrsanochlamydeae included in the analysis are placed outside the sect. Luzula clade; see L. subcongesta and L. kjellmaniana). The phylogenetic relationships and the origin of members of this section need to be more thoroughly studied, especially as both morphological and karyological data suggest a hybrid origin of the section or a hybrid origin of several of its members.
Fig.1 Strict consensus tree based on rbcL data (Drábková et al., 2003)
Structural mutations of trnL-F intergenic spacer and trnL intron define major clades within Juncus and Luzula (Drábková et al., 2004)
Totally,
we sequenced regions from 332-654 bp in length of the trnL
intron and
from 147-540 bp in length of the trnL-trnF
intergenic spacer of
the plastome DNA of 55 taxa representing most of subgenera and sections
of Luzula
and Juncus and also Rostkovia magellanica and
two species of Oxychloë,
namely O. andina and O. bisexualis.
Structural
mutations in the trnL intron
The
trnL intron length range was detected in both Luzula
and Juncus.
Most of these structural mutations are Juncus
specific (indels B-G; Fig. 2) and none of them is shared by
two genera.
One insertion type (A; Fig.2) is identical in several Luzula
species and Rostkovia
magellanica. All Juncus species are
characterized by a 7 bp deletion.
Carex species differ by only one substitution (AAAGATA).
Insertion
type A is typical for Luzula sect. Luzula,
Alpinae, Thyrsanochlamydeae,
Nodulosae and Anthelaea (Fig.
2). J. trifidus is unique
among the other species by a 322 bp deletion (C; Fig. 2). Another
long deletion
(D, 334 bp) was found in the ´Southern Hemisphere
Clade´ that consists of Juncus
species of the sect. Graminifolii (J.
lomatophyllus, J. capensis),
Rostkovia magellanica and two Oxychloë
(O. andina and O.
bisexualis). Insertion E (22
bp) is typical of Juncus sect. Ozophyllum,
Iridifolii and one representative of Graminifolii.
For both subg. Juncus
and subg. Agathryon the 4 bp insertion is typical.
On the other hand Juncus
subg. Agathryon has an additional 5 bp
deletion close to the 3´end
of the intron.
Structural
mutations in the trnL-trnF intergenic spacer
Six main structural
mutations (H-M) were investigated in the trnL-trnF
region and in
addition, three trnF pseudogenes were found (Fig.
2). Compared to the trnL
intron the trnL-trnF spacer
contains shorter indels (from
3 to 85 bp). All of them are genus specific: deletions H, K and M are
typical of
the genus Luzula, deletions I, L and insertion J
are Juncus specific
(Fig. 2). The first five bp deletion (H) was found in all Luzula
species
studied (Fig. 2). The 9 bp deletion (I) is typical of subg. Juncus
only.
Insertion J is typical of J. trifidus of the
section Steirochloa. This
insertion consists of 84 bp repetition of (TATATAAT)6
motif in
combination with “A” duplication. The
5 bp deletion K was found in
several Luzula species from the sections Anthelea,
Luzula, Alpinae,
Thyrsanochlamydeae and Nodulosae.
It was not found in subg. Marlenia,
Pterodes and sect. Diprophylateae.
The 8 bp deletion (L) was
typical of the entire Iridifolii, Ozophyllum
sections and one
taxon of the Graminifolii. The 10 bp indel M was
found in all Luzula representatives
(22 species). Indels occurred with prevalence in A/T rich stem regions
of
hairpins (Fig. 2). It was used for understanding of position homology
in an
aligment (especially in difficult taxa, e.g. J. trifidus).
TrnF gene of one
representative of the Juncaceae (J.
articulatus) demonstrating that there is a duplication of
“G” and two
transitions (A®G,
T®C)
in Juncaceae when compared to the homologous region of Nicotiana
tabacum.
Our study shows the trnF pseudogene
of differing length in
the Juncus species occur only to subg. Juncus.
This diversity was
not observed in subg. Agathryon and Luzula.
No variation was found
in 5´sequence of the acceptor stem of the trnF
pseudogene in Juncus
and Luzula species.
There
is an insertion of 8 bp between acceptor stem and D-domain in trnF
pseudogene
and in all examined species of subg. Juncus. The
T-domain and the
acceptor stem at the 3´end of the gene were not found in the
pseudogenes. The trnF
pseudogene varies in length in different species. Sections Ozophyllum
and Iridifolii contain at least part of
D-domain and anticodon domain
of trnF pseudogene structures (y1).
Juncus ensifolius (sect. Iridifolii)
belongs to the
separate group (y2)
with lack of most of the anticodon domain. Section Stygiopsis
(J.
biglumis, J. triglumis, J. castaneus and J. stygius)
contains the
shortest pseudogene (y3),
where lacks the anticodon and most of the D-domain. Two mutations were
found
among the Juncus trnF pseudogenes: A®C,
C®T
substitutions are found in the D-domains and anticodon domain,
respectively. A
few substitutions in comparison with trnF gene
contains acceptor
stem, D-domain and anticodon domain too.
Phylogenetic conclusion
We
constructed a phylogenetic tree (simplified in Fig. 2) based on
substitutions
and indels. We included in our study geographically diverse species and
concluded that the large indels are autapomorphies for some species
(e.g. 322 bp
deletion for J. trifidus) and synapomorphies for
others (e.g. 334 bp
insertion for Southern Hemisphere Clade) and that small structural
rearrangements seem to be more often homoplasious (e.g. 7 bp insertion
for Luzula
and Rostkovia).
The
monophyly of Luzula suggests
that deletions in the trnL-trnF
spacer occurred in an ancestor of all Luzula species
(three <10 bp
indels). This is satisfactorily confirmed by morphology
(Fig. 2). On the other
hand Juncus is non-monophyletic as shown by the
analysis of both, trnL-trnF
and rbcL regions (Drábková et
al., 2003). The subg. Juncus (and Agathryon)
clades are amply supported by specific indels. Additionally,
it seems that
tRNA pseudogene in Juncaceae evolved more recently since they occur in
the
youngest part of the tree (Fig. 5) in subg. Juncus.
Generally genus Juncus
(excl. Juncus trifidus) forms recently
evolved clade. When the tree
is based on both, substitutions and indels in whole trnL-trnF
region, the results are better (CI=0.61, RI=0.87, RC=0.54, HI=0.39)
than when
only substitutions are used (CI=0.55, RI=0.82, RC=0.45, HI=0,45). The
inclusion
of indels clearly improved statistical support of the phylogenetic tree
(nevertheless
the tree topology of the main clades remain unchanged).
J.
trifidus forms a
sister group of the whole genus Luzula and,
according to the trnL-trnF
spacer analysis, also a sister group of the main Juncus
clade (for more
details see Drábková et al, 2003). J.
trifidus is characterized by two
main autapomorphic indels, one in the trnL intron
(322 bp deletion) and
the other in the trnL-trnF
spacer (84 bp insertion that forms a
hairpin). These two structural events are unique among the species
of Juncus and Luzula.
´Southern Hemisphere Clade´ that was
inferred from the rbcL data
(Drábková et al., 2003) is supported by
autapomorphic substitutions in the trnL-F region.
It is also
characterized by a unique 334 bp deletion. In the small South American
genus Oxychloë
(represented by O. andina and O.
bisexualis), the deletion is
even 144 bp longer, the longest indel within this region in the whole
Juncaceae.
This separates it from other species of this clade (not shown).
Fig. 2 Simplified phylogenetic tree of Juncaceae based on substitutions and indels from trnL-F region (for details see also Drábková et al., 2004)
Fig. 3 Strict consensus tree of Juncaceae based on plastome sequence data from rbcL gene and trnL-F region (for details see also Drábková et al., 2006)
Mitochondrial
DNA variation within Juncaceae: Comparison of impact of organelles
regions to the phylogeny
The main aim of this work was to produce a robust phylogeny of the Juncaceae validated by data from both organelles. Our data confirm the monophyly of the genus Luzula, but do not provide support for monophyly of the genus Juncus. The majority of taxa clustered within two subgenera, Agathryon and Juncus, morphologically supported by the presence or absence of bracteoles and cymose or racemose inflorescences respectively. The subgenus Juncus is divided into two separate clades, the first closely related to the subgenus Agathryon and the second in the most basal part of the tree. Moreover, small South American genera clustered together with Juncus sect. Graminifolii and also with Juncus sect. Juncus. In fact, comparison of results from separate analyses of mitochondrial and plastome genes demonstrates that the general resolution of main topology of the atp1 tree is similar to the separate rbcL tree; the genus Juncus is better resolved, but the genus Luzula remains mainly polytomic.
Fig. 4 Strict consensus tree of the Juncaceae based on chondriome sequence data from atp1 gene (for details see also Záveská Drábková et Vlček, 2008)
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