Juncaceae Phylogeny Group

Phylogeny Conclusion

Main Juncaceae phylogeny features obtained so far

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 (Záveská Drábková et Vlček, 2008)

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)


Published Work and List of samples

Main topics of interest and Onging Projects

Phylogeny Conclusion

Group Participants

Juncaceae Literature