Ecologically relevant choanoflagellates collected from hypoxic water masses of the Baltic Sea have untypical mitochondrial cristae

Wylezich et al. BMC Microbiology 2012, 12:271 http://www.biomedcentral.com/1471-2180/12/271 RESEARCH ARTICLE Open Access Ecologically relevant choanoflagellates collected from hypoxic water masses of the Baltic Sea have untypical mitochondrial cristae Claudia Wylezich1*, Sergey A Karpov2*, Alexander P Mylnikov3, Ruth Anderson1 and Klaus Jürgens1 Abstract Background: Protist communities inhabiting oxygen depleted waters have so far been characterized through both microscopical observations and sequence based techniques. However, the lack of cultures for abundant taxa severely hampers our knowledge on the morphology, ecology and energy metabolism of hypoxic protists. Cultivation of such protists has been unsuccessful in most cases, and has never yet succeeded for choanoflagellates, even though these small bacterivorous flagellates are known to be ecologically relevant components of aquatic protist communities. Results: Quantitative data for choanoflagellates and the vertical distribution of Codosiga spp. at Gotland and Landsort Deep (Baltic Sea) indicate its preference for oxygen-depleted zones. Strains isolated and cultivated from these habitats revealed ultrastructural peculiarities such as mitochondria showing tubular cristae never seen before for choanoflagellates, and the first observation of intracellular prokaryotes in choanoflagellates. Analysis of their partial 28S rRNA gene sequence complements the description of two new species, Codosiga minima n. sp. and C. balthica n. sp. These are closely related with but well separated from C. gracilis (C. balthica and C. minima p-distance to C. gracilis 4.8% and 11.6%, respectively). In phylogenetic analyses the 18S rRNA gene sequences branch off together with environmental sequences from hypoxic habitats resulting in a wide cluster of hypoxic Codosiga relatives so far only known from environmental sequencing approaches. Conclusions: Here, we establish the morphological and ultrastructural identity of an environmental choanoflagellate lineage. Data from microscopical observations, supplemented by findings from previous culture-independent methods, indicate that C. balthica is likely an ecologically relevant player of Baltic Sea hypoxic waters. The possession of derived mitochondria could be an adaptation to life in hypoxic environments periodically influenced by small-scale mixing events and changing oxygen content allowing the reduction of oxygen consuming components. In view of the intricacy of isolating and cultivating choanoflagellates, the two new cultured species represent an important advance to the understanding of the ecology of this group, and mechanisms of adaptations to hypoxia in protists in general. Background Choanoflagellates are colourless, free-living, exclusively heterotrophic protists that are characterized by a single anterior flagellum surrounded by a collar of microvilli; and flat cristae in the mitochondria [1]. These unikont flagellates form the sister taxon of metazoans as seen by * Correspondence: ; 1 IOW-Leibniz Institute for Baltic Sea Research Warnemünde, Rostock, Germany 2 Zoological Institute RAS and St. Petersburg State University, St. Petersburg, Russia Full list of author information is available at the end of the article sequence analyses [2-4]. Within the choanoflagellates, three families were originally distinguished based on morphology: Acanthoecidae Norris, 1965; Salpingoecidae Kent, 1880; and Codonosigidae Kent, 1880 (synonym Monosigidae Zhukov et Karpov, 1985). Recent taxonomic revision based on multigene analysis states that the class Choanoflagellatea Kent, 1880 comprises two orders: 1) Craspedida, with a single family Salpingoecidae (including the aloricate choanoflagellates of the former Codonosigidae and Salpingoecidae families); and 2) Acanthoecida, with the families Acanthoecidae and Stephanoecidae [5,6]. Choanoflagellates normally constitute 5 to 40% of the average heterotrophic nanoflagellates (HNF) biomass in © 2012 Wylezich et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Wylezich et al. BMC Microbiology 2012, 12:271 http://www.biomedcentral.com/1471-2180/12/271 oxygenated pelagic habitats [7,8]. They have also been detected in hypoxic (oxygen-deficient) water masses [9] and can constitute a significant proportion of total HNF biomass, reaching for example 10–40% in hypoxic water masses of the Baltic Sea [10]. Especially in Gotland Deep, the biomass of exclusively aloricate choanoflagellates can clearly exceed 40% [10]. However, to date, few choanoflagellate species have been successfully cultured [5], and none for hypoxic environments, limiting knowledge on the ecology of this ecologically relevant protist group. Clone library based approaches have produced many novel sequence types during the last decade, enhancing our knowledge of protist species richness and diversity [11,12]. However, morphological and quantitative data of microscopical life observations and cell counts are often hard to match with such environmental sequences. In some recent cases it has been possible to assign new described species to novel protistan lineages only known from culture-independent sequence investigations [13-15]. Many environmental sequences (18S rRNA) in public databases cluster within the choanoflagellates. A recent re-analysis of published environmental sequences belonging to this group [16,17] provided evidence for only a low correspondence between these sequences and sequences obtained from cultures. Clonal sequences from hypoxic environments (here referring to suboxic to anoxic/sulfidic conditions) have also been found to often cluster within the choanoflagellates. For instance, sequences from the anoxic Framvaren Fjord [18] branch off near Diaphanoeca grandis (Stephanoecidae); and clonal sequences found in the hypersaline Mediterranean L’Atalante Basin constitute the novel cluster F within the Acanthoecidae [16,19]. Stock et al. [20] also detected novel sequences in the redoxcline of the periodically anoxic Gotland Deep (central Baltic Sea), which branched within the Craspedida cluster A [16]. However, only a small fraction of choanoflagellates known at a sequence level have been isolated and maintained in culture to date, and none so far was derived from hypoxic marine environments. Thus, the morphology, ultrastructure and physiological strategies of these choanoflagellates from hypoxic environments remain unexplored. The Baltic Sea is one of the largest brackish water basins in the world. A stable halocline separates the water column into an upper oxygenated layer and underlying oxygen deficient and anoxic/sulfidic layers in the deeper basins (e.g., Gotland and Landsort Deep). Protist communities inhabiti (...truncated)