Phylogenetic relationships in Interfilum and Klebsormidium (Klebsormidiophyceae, Streptophyta).

Members of the genus Klebsormidium have cosmopolitan distribution and occur in a very wide range of freshwater and terrestrial habitats. Due to its simple filamentous morphology, this genus represents a taxonomically and systematically complex taxon in which phylogenetic relationships are still poorly understood. The phylogeny of Klebsormidium and closely related taxa was investigated using new ITS rRNA and rbcL sequences generated from 75 strains (isolated from field samples or obtained from culture collections). These sequences were analyzed both as single-marker datasets and in a concatenated dataset. Seven main superclades were observed in the analyses, which included sixteen well-supported clades. Some species of Klebsormidium, including the type species Klebsormidium flaccidum, were polyphyletic. Interfilum was recovered with high statistical support as sister taxon to a clade of Klebsormidium formed mainly by strains identified as K. flaccidum. Whereas some clades could be easily associated with described species, this was not possible for other clades. A new lineage of Klebsormidium, isolated from arid soils in southern Africa and comprising undescribed species, was discovered. Several morphological characters traditionally used for taxonomic purposes were found to have no phylogenetic significance and in some cases showed intra-clade variation. The capacity to form packet-like aggregates (typical of Interfilum), features of the morphology of the chloroplast and the type of habitat were the main phylogenetically relevant characters. Overall, Klebsormidium and Interfilum formed a more diverse algal group than was previously appreciated, with some lineages apparently undergoing active evolutionary radiation; in these lineages the genetic variation observed did not match the morphological and ecological diversity.

Highly differentiated populations of the freshwater diatom Sellaphora capitata suggest limited dispersal and opportunities for allopatric speciation.

The diversities and distributions of diatoms are much more complex than was ever imagined. To understand the underlying mechanisms, research must focus on evolutionary processes occurring at a population level and employ sufficiently informative molecular markers. Using ten microsatellites and ITS rDNA sequence data, we investigated the genetic structure of populations of the benthic freshwater diatom Sellaphora capitata (until 2004 a cryptic entity within the S. pupula agg. species complex). This is the first time that microsatellites have been used to investigate the genetic structure of any freshwater or benthic microalga. Using an integrated approach (morphology, DNA barcoding and specificity of the microsatellite primers), we verified the identity of 70 S. capitata isolates obtained from lakes in the UK, Belgium and Australia. Standardized F'(ST) values were very high (>0.4) and in Bayesian analyses, isolates clustered according to their country of origin, with limited evidence of admixture. However, selected isolates from all countries were sexually compatible, a result consistent with limited ITS divergence. Considering the apparent absence of desiccation-resistant resting stages in most diatoms, we conclude that such levels of differentiation are likely to be a consequence of limited dispersal. With restricted dispersal, previously unacknowledged opportunities for allopatric speciation exist, which may help to explain the huge extant diversity of diatoms.

NEW STREPTOPHYTE GREEN ALGAE FROM TERRESTRIAL HABITATS AND AN ASSESSMENT OF THE GENUS INTERFILUM (KLEBSORMIDIOPHYCEAE, STREPTOPHYTA)(1).

Sarcinoid aeroterrestrial green algae were isolated from three arid locations in Ukraine and the Czech Republic. Although gross morphology suggested an affinity with Desmococcus (for taxonomic authorities, see Table S1 in the supplementary material), the cellular morphological characteristics were reminiscent of those of Geminella terricola. However, the presence of a complex of ultrastructural features indicated that these isolates were members of the streptophyte lineage in the green plants. 18S rDNA sequence phylogenies provided evidence of a close relationship with Klebsormidium in the Streptophyta, while the position of Desmococcus was within the Trebouxiophyceae. In the internal transcribed spacer (ITS) rDNA phylogeny, the sarcinoid isolates were closely related with strains of G. terricola and Interfilum paradoxum. Strains of that clade were morphologically united by a specific type of cell division that involves the association of persistent, cap-shaped remains of the mother cell wall with daughter cells. Consequently, these strains were assigned to a redefined genus Interfilum, and a new species, I. massjukiae, was described to accommodate one of the sarcinoid isolates. As the position of the genus Geminella was in the Trebouxiophyceae, the streptophyte G. terricola was transferred to Interfilum, as I. terricola comb. nov., but the ITS rDNA analyses proved inconclusive to resolve its affinities with other species of Interfilum due to intragenomic polymorphisms. The species of Interfilum had a closer relationship with K. flaccidum than with other species of Klebsormidium. The latter genus may not be monophyletic in its present circumscription.

ASSESSING PHYLOGENETIC AFFINITIES AND SPECIES DELIMITATIONS IN KLEBSORMIDIALES (STREPTOPHYTA): NUCLEAR-ENCODED rDNA PHYLOGENIES AND ITS SECONDARY STRUCTURE MODELS IN KLEBSORMIDIUM, HORMIDIELLA, AND ENTRANSIA(1).

Nuclear-encoded SSU, group I intron, and internal transcribed spacer (ITS) rDNA sequences were obtained for 16 strains of green algae representing species of Klebsormidium, Hormidiella attenuata, and Entransia fimbriata (for taxonomic authorities, see Table S1 in the supplementary material). The SSU phylogeny resolved a well-supported clade Klebsormidiales in the Streptophyta that comprised authentic Klebsormidium isolates described recently in a monograph by G. M. Lokhorst and various strains from culture collections. The H. attenuata and En. fimbriata pair was the sister group of Klebsormidium. Certain isolates from culture collections previously identified as "Klebsormidium" emerged as Trebouxiophyceae. Strains assigned to Koliella, Gloeotila, and Stichococcus previously allied with Klebsormidium because of shared morphological and ultrastructural characteristics also belonged to Trebouxiophyceae. Group I introns inserted at Escherichia coli position 516 were found in K. nitens and SAG strain 384-1, and at position 1506 in H. attenuata and En. fimbriata. Introns were not observed in other Klebsormidiales. Unambiguous alignment of ITS regions of Klebsormidiales was only possible after thermodynamic folding had predicted eight conserved helical domains. The ITS phylogeny provided support for five of the morphospecies recognized by Lokhorst (K. flaccidum, K. elegans, K. bilatum, K. crenulatum, K. mucosum), but the sequences of K. dissectum, K. fluitans, and K. nitens formed an unresolved clade. The species with the earliest origin in the Klebsormidium phylogeny was K. flaccidum. The incongruence between Lokhorst's morphology-based cladograms and the ITS phylogenies demonstrated the need for a critical reappraisal of the taxonomy and the morphological and molecular species concept in Klebsormidium on the basis of a more extensive taxonomic and geographic sampling strategy.