Single colony genetic analysis of epilithic stream algae of the genus Chamaesiphon spp.

Hydrobiologia, Aug 2017

In order to understand Chamaesiphon spp. evolution and ecological diversification, we investigated the phylogenetic differentiation of three morphospecies from field samples by means of single colony genetics. Individual colonies of three different morphospecies (C. starmachii, C. polonicus, C. geitleri,) were isolated from lotic gravel streams and their 16S rDNA nucleotide variability was analyzed. For a number of individual colonies, microscopical and ultrastructural analysis was also performed. A phylogenetic tree of all major lineages of the phylum of Cyanobacteria assigned all Chamaesiphon genotypes (1149–1176 bp) most closely with the family of Gomontiellaceae of the order Oscillatoriales. The sequences obtained from colonies assigned to C. starmachii (n = 21), C. polonicus (n = 9), and C. geitleri (n = 17) were found to reveal high average (3.5%) nucleotide diversity. No phylogenetic sub-branching in correspondence with morphology was observed suggesting that the three Chamaesiphon morphospecies did not represent monophyletic taxa. We could not attribute specific thylakoid ultrastructure to phylogenetic sub-branches; however, the observed parietally and loosely arranged thylakoids indicate that for the genus Chamaesiphon, the variability in thylakoid ultrastructure might have been underestimated. In summary, the high nucleotide diversity of the 16S rDNA gene implies phylogenetic diversity that corresponds little to morphological classification.

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Single colony genetic analysis of epilithic stream algae of the genus Chamaesiphon spp.

Single colony genetic analysis of epilithic stream algae of the genus Chamaesiphon spp. . Guntram Christiansen . Andreas Holzinger . 0 1 0 A. Holzinger E. Rott Institute of Botany, University of Innsbruck , Sternwartestraße 15, 6020 Innsbruck , Austria 1 R. Kurmayer (&) G. Christiansen Research Institute for Limnology, University of Innsbruck , Mondseestraße 9, 5310 Mondsee , Austria In order to understand Chamaesiphon spp. evolution and ecological diversification, we investigated the phylogenetic differentiation of three morphospecies from field samples by means of single colony genetics. Individual colonies of three different morphospecies (C. starmachii, C. polonicus, C. geitleri,) were isolated from lotic gravel streams and their 16S rDNA nucleotide variability was analyzed. For a number of individual colonies, microscopical and ultrastructural analysis was also performed. A phylogenetic tree of all major lineages of the phylum of Cyanobacteria assigned all Chamaesiphon genotypes (1149-1176 bp) most closely with the family of Gomontiellaceae of the order Oscillatoriales. The sequences obtained from colonies assigned to C. starmachii (n = 21), C. polonicus (n = 9), and C. Gravel streams; Heteropolar cyanobacteria; Bioindication; Single colony PCR; 16S rDNA gene sequencing - Guest editors: Eugen Rott, Allan Pentecost & Jan Mares / Aspects of cyanobacterial biogeography, molecular ecology, functional ecology and systematics Introduction The cyanobacterial morphogenus Chamaesiphon represents one of the most widespread taxa forming thin biofilms in mountain rivers worldwide (e.g., Bu¨rgi et al., 2003; Rott et al., 2006; Rott, 2008; Sant’Anna et al., 2011; Scott & Marcarelli, 2012; Gutowski et al., 2015) . Several Chamaesiphon species can cover up to [70% of the wetted perimeter in clear mountain streams (Rott & Wehr, 2016) . The genus Chamaesiphon was recorded over a broad range of environmental situations in respect to (i) light (i.e., from shaded to highly light (UV) exposed sites), (ii) pH (i.e., from acid to alkaline, Cantonati et al., 2007) and (iii) nutrients (i.e., from ultra-oligotrophic to eutrophic conditions, Rott et al., 1999; Rott & Schneider, 2014) . Up to date, 33 species of the morphogenus Chamaesiphon have been validly described under the rules of the Botanical Nomenclatoric Code (ICBN, Koma´rek & Anagnostidis, 1999, and Cyano Database: http://www.cyanodb.cz, February, 2017). These cyanobacterial species are distinguished based on morphological characters, and although some morphological characters are less stable than others, several of the morphospecies of the genus Chamaesiphon have been recorded repeatedly from different regions in the world (e.g., Koma´rek & Anagnostidis, 1999; Rott & Wehr, 2016) . Microbiological typification defines the morphogenus Chamaesiphon as a simple unicellular cyanobacterium with typical asymmetrical binary fission (called budding or exocyte or exospore formation) produced at one pole of the mother cell, where typically one single or a consecutive large series of small exospores (or small buds) is formed (Herdman et al., 2001) . Colony forming cyanobacteria can reach a macroscopically visible size from clonal cellular growth. This facilitates the isolation of single individuals directly from field samples for analyses of their genetics, cyanotoxins or other bioactive secondary metabolites (e.g., Kurmayer et al., 2002) . Advanced molecular techniques have increased the sensitivity of PCR, resulting in the possibility to amplify DNA fragments from just picograms of template DNA, such as from single colonies of cyanobacteria. This makes it possible to perform dozens of PCR experiments amplifying large DNA fragments ([1000 bp) from a single specimen (e.g. Chen et al., 2016) . Combining the field-based techniques with advanced molecular biological methods holds great potential to analyze the ecological, as well as phylogenetic, diversification of cyanobacteria, particularly if they are difficult to cultivate (e.g., Maresˇ & Cantonati, 2016) . In general, a detailed molecular resolution of Chamaesiphon morphospecies might allow a better characterization of running water habitats that are under environmental stress induced by regional climate change or by anthropogenic influence (e.g., Loza et al., 2013). The aim of this study was to characterize Chamaesiphon morphospecies using molecular biological techniques on the individual (single colony) scale, which would allow to relate morphological descriptors to genotype variability. We speculated that morphological differentiation might correlate with phylogeny, even between morphospecies, because of selective environmental pressure resulting in phylogenetically distinct ecotypes. For example, high mechanical pressure in lotic ecosystems may lead to morphospecies forming a multilayered cell wall and growing in colonies (assigned to Chamaesiphon subgenus Godlewskia, Koma´rek & Kasˇtovsky´ , 2003 (...truncated)


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Rainer Kurmayer, Guntram Christiansen, Andreas Holzinger, Eugen Rott. Single colony genetic analysis of epilithic stream algae of the genus Chamaesiphon spp., Hydrobiologia, 2017, pp. 1-15, DOI: 10.1007/s10750-017-3295-z