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Open Access Research article

A close-up view on ITS2 evolution and speciation - a case study in the Ulvophyceae (Chlorophyta, Viridiplantae)

Lenka Caisová123*, Birger Marin1 and Michael Melkonian1

Author Affiliations

1 Universität zu Köln, Biozentrum Köln, Botanisches Institut, Zülpicher Str. 47b, 50674 Köln, Germany

2 Current Address: Institute of Botany v.v.i., Academy of Sciences of the Czech Republic, Dukelská 135, 379 82 Třeboň, Czech Republic, CZ

3 Current Address: University of South Bohemia, Faculty of Science, Branišovská 31, 370 05 České Budějovice, Czech Republic, CZ

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BMC Evolutionary Biology 2011, 11:262  doi:10.1186/1471-2148-11-262

Published: 20 September 2011

Additional files

Additional file 1:

Selected ITS2 'template' structures of Ulva spp. from the ITS2 Database III, showing artificial folding. All Ulva spp. are characterized by (1) the ITS2 Database III identification number, and (2) the accession number of the sequence entry, and (3) the method used for folding in the ITS2 Database III [Method 1 (M1) - direct folding (e.i. derived from e.g. MFold, RNAstructure, Method 2 (M2) - homology modeling].

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Additional file 2:

18S rDNA maximum likelihood phylogeny of the Ulvales (74 taxa) based upon 1702 aligned characters. Habitat preferences as well as presence/absence of scales on zoospores (aplanospores)/gametes are emphasized in the same way as in Figure 2. The branch separating the Capsosiphonaceae, Gomontiaceae and Pseudoneochloris marina from the remaining Ulvales was designated as root of the tree. Significances at branches as in Figure 2; bold branches have maximal support by all methods. Note that Pseudoneochloris marina diverged as an independent branch, in contrast to the ITS2 phylogeny.

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Additional file 3:

Evolution of synapomorphic CBCs (Compensatory Base Changes)/hCBCs (hemi-Compensatory Base Changes) in ITS2 of the Ulvales. Branch lengths (L = apomorphic evolutionary changes of the basal branch in Figure 3) referred to the common branch of the clade. Base pairs were labeled by the nucleotide numbering system introduced in Figure 1 (e.g. as 72/108). Information on hCBCs was indicated by [brackets]. 15 H2+3_CBCs (CBCs discovered in the conserved regions of ITS2) were indicated in gray boxes. Unique synapomorphies were flagged as NHS (Non-Homoplasious Synapomorphy), whereas Homoplasious Synapomorphies are designated as HS. Only Homoplasious Synapomorphies were further characterized as (1) parallel CBCs (PAR), (2) parallel hCBCs (hPAR), (3) convergent CBCs (CONV), (4) reversals of CBCs (REV), or (5) reversals of hCBCs (hREV).

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Additional file 4:

List of all substitutions of ITS2 base pairs during the evolution of the Ulvales. For nucleotide numbers, see Figure 1A. CBCs (blue) and hCBCs (red) were classified into non-homoplasious (NHS) and homoplasious character changes (HS, categorized into parallelisms, convergences, and reversals; or further explanation, see Additional file 3). For every pair, the likely plesiomorphic character status within the Ulvales is given. Moreover, non-compensating base changes are listed here, that involve a pair ⇔ unpair conversion. The conserved regions of helices 2 and 3 were depicted in gray shades.

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Additional file 5:

Compensatory base changes distributed over conserved regions of helices 2 and 3 of ITS2 in the Ulvales. All 15 compensatory base changes found in conserved regions of helices 2 and 3 (H2+3_CBCs) were mapped on the consensus secondary structure model of ITS2 in the Ulvales. Comments refer either to their non-homoplasious (NHS) or to homoplasious (HS) status. For further information on universal/non-universal positions see Figure 1.

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Additional file 6:

Numbers of compensating changes in ITS2 helices diagrammed against branch lengths in the ITS2 phylogeny. A) The number of CBCs appeared weakly correlated with the length of branches where the CBCs occurred (brown squares with numbers indicating the frequency of CBCs versus evolutionary steps). For branches with > 0 CBCs, the CBC vs. branch length ratio was calculated (CBC_R = 2xCBC/evolutionary steps, blue squares), showing negative correlation with branch lengths. B) Hemi-CBCs were not strictly correlated with branch lengths (brown squares with numbers showing the frequency of hCBCs versus evolutionary steps)), but the hCBC vs. branch length ratio (hCBC_R = hCBC/evolutionary steps, blue squares) again clearly showed negative correlation. For both diagrams, branch length calculation was restricted to double-stranded ITS2 positions. Note that the gray colour in the diagrams indicates the area in which no CBCs (A) and hCBCs (B) occur.

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Additional file 7:

Strain designations, origins and accession-numbers of nuclear-encoded ITS2 rRNA 86 strains of the Ulvales. Newly determined sequences are in bold. An asterisk (*) indicates authentic cultures.

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Additional file 8:

Primers used for PCR amplification/sequencing of ITS2 in the nuclear-encoded rRNA operon of the Ulvales. Since a few cultures were contaminated with fungi, PCR reactions were performed with specific reverse primers that mismatched with fungal rDNA sequences (labelled 'exFungi'; specific 3'-positions

    underlined
).

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