Genetic Diversity in Chrysophytes: Comparison of Different Gene Markers
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Fottea, Olomouc, 17(2): 209–221, 2017 209 DOI: 10.5507/fot.2017.005 Genetic diversity in chrysophytes: Comparison of different gene markers Christina BOCK1, Antonis CHATZINOTAS2,3 & Jens BOENIGK1,4 1 Department of Biodiversity, University Duisburg–Essen, Universitaetsstrasse 5, D–45117 Essen, Germany 2Department of Environmental Microbiology, Helmholtz Centre for Environmental Research – UFZ, Permos- erstrasse 15, D–04318 Leipzig, Germany 3German Centre for Integrative Biodiversity Research (iDiv) Halle–Jena–Leipzig, Deutscher Platz 5e, 04103 Leipzig, Germany 4Centre for Water and Environmental Research (ZWU), University of Duisburg–Essen, Universitaetsstr. 2, 45117 Essen, Germany Abstract: Chrysophytes are a diverse group of protists belonging to the Stramenopiles. They encompass a broad range of nutrition strategies (phototrophy, mixotrophy and heterotrophy) and morphological forms (unicellular, colonial, branched, non–scaled to silica–scaled). However, taxonomy, identification and delineation of genera and species are hampered by the polyphyletic origin of several morphotypes and the low resolution of morphological features; moreover, description of these features requires in many cases special equipment like TEM or SEM. To test the congruent phylogeny, genetic resolution and to reveal the genetic diversity of chrysophytes, we evaluated in this study different gene markers, in particular SSU and LSU rRNA genes, ITS, and COI in terms of phylogenetic clustering, separation of phylogenetic lineages and genetic distances within and between clades. Our results showed that the general topology of the SSU rRNA gene phylogeny is similar to that of the LSU rRNA gene phylogeny, with a higher genetic divergence for the latter. The topology of the COI phylogeny differed from the ribosomal data, and showed the highest genetic divergence within groups and between groups for all compared markers. The 5.8S rRNA gene offered only a limited phylogenetic resolution with low genetic divergence. ITS showed a high genetic divergence, but phylogenetic analyses were hampered by high variance in sequence lengths between clades. Key words: Chrysophyceae, SSU, LSU, COI, ITS INTRODUCTION belong to this diverse group of protists (ŠKALOUD et al. 2013; ŠKALOUDOVÁ & ŠKALOUD 2013). They encompass Chrysophyceae sensu lato (chrysophytes), the so called varying nutritional strategies, including phototrophy, “golden algae”, comprise the groups of Synurales, mixotrophy and heterotrophy with both phagotrophy Ochromonadales, Chromulinales and others within the by ingesting bacteria (THINGSTAD et al.1996; KAMJUNKE et al. 2007; UNREIN et al. 2007) or osmotrophy (PRING- Stramenopiles (ANDERSEN et al. 1999; Kristiansen & SHEIM 1952). Despite the broad range of morphological PREISIG 2007; JORDAN & IWATAKI 2012). They represent forms, identification and thus distinction of members an abundant and in some environments even domina- belonging to the same genus or even to closely related ting group of planktonic protists (ROTT 1988; TOLOTTI genera is often problematic. Species delineations are et al. 2003, 2006; KAMMERLANDER et al. 2015). They usually based on small morphological differences. occur in a broad range of ecotypes and comprise het- These features as for example the scale morphology in erokont biflagellate eukaryotes, which received their Synura or Paraphysomonas can often only be detect- common name due to the golden–brown color of the ed with special equipment like transmission electron accessory pigment fucoxanthin present in some of microscopy (TEM) or scanning electron microscopy their members (BELLINGER & SIGEE 2013). The mor- (SEM) (ŠKALOUD et al. 2012; SCOBLE & CAVALIER– phology of chrysophytes ranges from unicellular (e.g. SMITH 2014). The fact that morphology may change Ochromonas) to colonial (e.g. Synura) or branching under different environmental conditions because of forms (e.g. Hydrurus). Non–scaled as well as silica– phenotypic plasticity adds to the difficulties of species scaled taxa (belonging either to the Synurales [or Synu- delimitation (LUO et al. 2006). rophyceae], Paraphysomonadida or Chrysosphaerella) With the advent of marker–based phylogenetic ap- 210 BOCK et al.: Genetic diversity in chrysophytes proaches, new light was shed into the taxonomy and stable topology and phylogenetic lineage distinction. phylogenetic relationship of this group (ANDERSEN et The main aim of this paper was to find a suitable mark- al. 1999). Molecular analyses revealed that some mor- er region for a stable phylogeny, which further allows a photypes are in fact polyphyletic and form separate good separation between closely related lineages. clusters within the chrysophytes, showing that classi- fication solely based on morphology is sometimes mis- leading (GROSSMANN et al. 2016). One good example ATERIAL AND ETHODS are colorless, nonscaled chrysophytes which are M M genetically far more diverse than morphology reflects Isolates and culture conditions. Most strains were isolated (BOENIGK et al. 2005; PFANDL et al. 2009; FINDENIG et during sampling campaigns in 2002 and 2006. Isolation and al. 2010). Several new genera were recently erected maintenance of strains were conducted as already described mainly based on molecular identification, since light in BOENIGK et al. (2005). For details on origin of the isolates microscopy could not distinguish between isolates be- see table S1. Isolates were discarded after the DNA–extrac- longing to different phylogenetic lineages (GROSSMANN tion. et al. 2016). Even TEM and SEM offer only a limited amount of information within this species complex DNA isolation, amplification and sequencing. For DNA (FINDENIG et al. 2010). A huge hidden diversity was isolation, cells were harvested by centrifugation at 14000× g also observed within species complexes of Paraphyso- and the resulting pellet was used for DNA extraction. DNA monas, which lead to the establishment of several new was extracted using the DNeasy Tissue kit (Qiagen Gmbh Hilden, Germany) and amplified separately for the different species; it is very likely, that even more species exist gene regions (details on used primers see table S2). since environmental surveys have detected additional undescribed phylogenetic lineages within the Paraphy- Phylogenetic analyses. Four different alignments were somonadidae (SCOBLE & CAVALIER–SMITH 2014). constructed for the phylogenetic analyses with the newly In the past, biodiversity and monitoring ques- obtained sequences and additional sequences obtained from tions in freshwater systems were mainly addressed NCBI (http://www.ncbi.nlm.nih.gov/). Reference sequences based on morphological data (e.g. PADISÁK et al. 1998). representing the different clades of the Chrysophyceae were With the new knowledge gained from molecular bio- selected on the basis of previous publications (e.g. MEDINGER et al. 2010; SILBERFELD et al. 2010; KLAVENESS et al. 2011; logical approaches, environmental surveys can over- BOCK et al 2014). The sequences were initially aligned us- come the risks associated with campaigns solely based ing ClustelW (THOMPSON et al. 1994) and adjusted by eye us- on morphology of organisms, namely to miss cryptic ing the SequentiX Alignment Editor (HEPPERLE 2004). Only species complexes and to lose information regarding highly ambitious regions were excluded from the analyses. their geographical and /or seasonal distribution. The phylogeny of the SSU rRNA gene (Fig1–2, FigS1) was Many studies base their results on SSU rRNA inferred from an aligned dataset including 238 sequences gene data. Even if the SSU rRNA gene offers a good based on 1206 characters. The sequences Synchroma grande backbone for the phylogeny within protist groups, it DQ788730, Leukarachnion sp. FJ356265 were chosen as was shown in several studies that this genetic marker outgroup according to BOCK et al. (2014). LSU rRNA gene phylogeny was inferred from 175 sequences with 615 char- might be too conserved to reveal the occurring genetic acters (Fig 3, FigS2). COI phylogeny was interfered from diversity within related lineages (BOCK et al. 2010; 139 sequences with 421 characters (Fig 4, FigS4). ITS phy- NASSONOVA et al. 2010; ŠKALOUD et al. 2012). Within logeny was interfered from 17 sequences with 902 characters the Synura petersenii complex, six genetic lineages (Fig 5). The 5.8S rRNA gene phylogeny was interfered with were revealed by multiple genetic markers (internal 95 sequences and 132 characters (Fig S3). All phylogenetic transcribed spacer rDNA, psaA, rbcL and COI) which trees (except for ITS) presented here were inferred by maxi- were further supported by morphological data based on mum likelihood settings with RAXML 8.2.9 (STAMATAKIS 2014)using the Cipres gateway (MILLER et al. 2010). Mod- TEM and SEM of silica scales (ŠKALOUD et al. 2010). els for ITS were used as proposed by Treefinder under AICc This combination of morphological and molecular data criteria: partitioned dataset (ITS1: HKY[Optimum,Empiri allowed the authors an additional interpretation on the cal]:G[Optimum]:5, 5.8S: HKY[{3,1,1,1,1,3},Empirical], diversity and distribution patters. These results dem- ITS2: TVM[Optimum,Empirical]:G[Optimum]:5}; 5.8S: onstrate the need of phylogenetic markers with high HKY[{3,1,1,1,1,3},Empirical). To test the confidence of resolution and reliable stable topology to distinguish the tree topologies, bootstrap analyses were carried out for between closely related species and thus to obtain re- distance (neighbor–joining, NJ; 1000 pseudoreplicates)