Molecular, Biochemical and Morphological Data Suggest an Affiliation of Spongiochrysis Hawaiiensis with the Trentepohliales (Ulvophyceae, Chlorophyta)
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bs_bs_banner Phycological Research 2013; 61: 133–144 Molecular, biochemical and morphological data suggest an affiliation of Spongiochrysis hawaiiensis with the Trentepohliales (Ulvophyceae, Chlorophyta) Christian Boedeker,1* Ulf Karsten,2 Frederik Leliaert3 and Giuseppe C. Zuccarello1 1School of Biological Sciences, Victoria University of Wellington, Wellington, New Zealand, 2Applied Ecology, Institute of Biological Sciences, University of Rostock, Rostock, Germany, and 3Phycology Research Group, Biology Department, Ghent University, Ghent, Belgium a peculiar unicellular alga growing on the bark of Cas- SUMMARY uarina trees on Hawai’i (Rindi et al. 2006). The genus was firmly placed in the Cladophorales based on The aeroterrestrial, unicellular green alga Spongiochry- phylogenetic analyses of small subunit (SSU) rDNA sis hawaiiensis had been included in the ulvophycean sequences. Spongiochrysis is an atypical member of order Cladophorales based on small subunit (SSU) the Cladophorales as it represents the only strictly ter- rDNA sequence data, and represents so far the only restrial members of the order Cladophorales with a fully terrestrial member of this order. Other character- number of unusual features, such as a unicellular istics of S. hawaiiensis that are atypical for Cladopho- growth form (but occasionally producing short fila- rales include the presence of large amounts of ments) and a specialized form of budding-like cell carotenoids and a budding-like mode of cell division. As division. Initial phylogenetic reconstructions placed the position of this terrestrial, unicellular alga in an S. hawaiiensis in the Aegagropila clade (Rindi et al. order of aquatic, multicellular green algae is unusual, 2006), a clade of the Cladophorales consisting mainly we re-evaluated the phylogenetic relationships of this of freshwater and brackish water species (presently enigmatic organism based on supplementary SSU rDNA split into the Pithophoraceae and the Pseudocla- sequences as well as novel large ribosomal subunit dophoraceae, Boedeker et al. 2012). Based on this (LSU) rDNA and internal transcribed spacer (ITS rDNA) phylogenetic association, it was hypothesized that sequences. Additionally, we examined several morpho- S. hawaiiensis might have evolved from a marine ances- logical characters of S. hawaiiensis, as well as low tor by the gradual reduction of the thallus and the molecular weight carbohydrate (LMWC) patterns of production of carotenoid pigments as an adaptation to S. hawaiiensis and members of the Cladophorales and terrestrial habitats (Rindi et al. 2006). Trentepohliales as potential chemotaxonomic markers. Common adaptations of algae to the terrestrial realm We found S. hawaiiensis to be uninucleate. The analy- include a simple morphology, such as coccoid cells; sis of the LMWC content detected the presence of the thick cell walls; the production of mucilage to retain polyol erythritol in S. hawaiiensis and in the Trente- moisture (Broady 1977; Shephard 1987; Rindi 2011); pohliales, while this compound was missing in the restriction of metabolic activity to periods of favorable Cladophorales. The phylogenetic analyses of the novel conditions (e.g. Friedman et al. 1987); and the synthe- sequences placed S. hawaiiensis in the terrestrial Tren- sis of metabolites that protect against high irradiation tepohliales. This placement is supported by the aero- (carotenoids, e.g. Bidigare et al. 1993; Abe et al. terrestrial habitat, the presence of large amounts of 1998) and desiccation/water loss (polyols such as carotenoids, the uninucleate cells, and the presence of ribitol, sorbitol, glycerol, erythritol, e.g. Feige & Kremer the polyol erythritol as a protective compound against 1980; Darienko et al. 2009; Gustavs et al. 2010). water loss. Terrestrial algae are found in several of the major groups of green algae: in the charophyte lineage they Key words: aeroterrestrial algae, Chlorophyta, Clado- occur in the Klebsormidiophyceae, and among the core phorales, erythritol, molecular phylogenetics, Spongi- ochrysis hawaiiensis, systematics, Trentepohliales, Ulvophyceae. *To whom correspondence should be addressed. INTRODUCTION Email: [email protected] Communicating editor: M. Kamiya. Spongiochrysis hawaiiensis Rindi, López-Bautista, Received 6 August 2012; accepted 20 December 2012. Sherwood & Guiry has recently been described based on doi: 10.1111/pre.12011 © 2013 Japanese Society of Phycology 134 C. Boedeker et al. chlorophytes they have been found in the Trebouxi- nuclear DNA and analysis of the low molecular weight ophyceae, in several lineages of the Chlorophyceae, and carbohydrates (LMWC). within the Ulvophyceae (see Leliaert et al. 2012). Within the ulvophyte lineage, aeroterrestrial algae are MATERIALS AND METHODS mainly found in the strictly terrestrial order Trente- pohliales (e.g. López-Bautista et al. 2002), but several Studied organisms other members of the Ulvophyceae such as Desmo- chloris mollenhaueri Darienko, Friedl et Pröschold The material of Spongiochrysis hawaiiensis investigated (Darienko et al. 2009) and Trichophilus welckeri in this study (sample number G89, deposited as Weber-van Bosse, which grows on the hair of sloths voucher WELT A031452 (National Museum of New (Suutari et al. 2010), are also terrestrial. Some fresh- Zealand Te Papa Tongarewa)) was collected at the type water species of Ulothrix (Ulotrichales) are also found location (on the bark of Casuarina equisetifolia Lin- on soil (Metting 1981). naeus, Waimanalo Beach Park, Waimanalo, Oahu, The placement of the unicellular S. hawaiiensis in Hawai’i) on 4 February 2006 by Alison Sherwood. The the Cladophorales is exceptional, as this order is dis- cells of S. hawaiiensis formed a thick, powdery layer of tributed in marine, brackish or freshwater environments intense yellow color on the bark surface. For the and is characterized by a siphonocladous organization, molecular and biochemical analyses, the surface of which means that multicellular thalli are composed especially thick parts of the yellow layer was scraped off of multinucleate cells (van den Hoek et al. 1995). with a scalpel into Petri dishes (approximately 10 mm2 However, several taxa are known to occur in semi- each), and the powder was transferred after micro- terrestrial environments such as on moist stone and soil scopic inspection to 1.5-mL microfuge tubes. surfaces, for example Wittrockiella calcicola (F.E. To check for the presence of other (algal) organisms Fritsch) Boedeker, W. fritschii (A.K. Islam) Boedeker on the tree bark that could possibly lead to contami- and W. sunderbanensis (A.K. Islam) Boedeker (Fritsch nating signals in the molecular and biochemical analy- 1944; Islam 1964). Furthermore, some members of sis, the surface of the bark was scraped with a scalpel Arnoldiella (former Basicladia, see Boedeker et al. (approximately 10 cm2). The resulting material was 2012) that occur preferably on freshwater turtles can fixed in 5% formaldehyde-freshwater solution and withstand extended periods of desiccation (Proctor allowed to settle overnight. The sediment was then 1958). distributed with a pipette into three plastic Petri dishes Spongiochrysis hawaiiensis was initially believed to (diameter 8 cm) with counting grids and the dishes be a member of the Trentepohliales based on its subsequently checked using an inverted microscope crustose occurrence on tree barks and the supposed (Wild M40, Wild Heerbrugg, Switzerland). All filamen- production of carotenoids (Rindi et al. 2006). The Tren- tous and biological structures were photographed with a tepohliales are characterized by filamentous thalli com- digital camera (ColorView Illu, Olympus Soft Imaging posed of uninucleate cells, and several other unique Systems, Münster, Germany). features such as the formation of sessile zoosporangia that detach and are dispersed, cell division by phrag- Staining of nuclei moplast formation, transverse cell walls containing plasmodesmata, a multilayered structure in the flagellar Cells of S. hawaiiensis were fixed in 5% formaldehyde- root system, the presence of b-carotene and astaxan- freshwater solution and stained using the fluorescent, thin, and the synthesis of uncommon low molecular DNA-binding dye Hoechst 33258 (Molecular Probes, weight carbohydrates such as polyols (Feige & Kremer Invitrogen, Eugene, OR, USA) and were photographed 1980; van den Hoek et al. 1995; Thompson & Wujek under exciting ultraviolet radiation (340–380 nm, 1997; Chapman et al. 2001; López-Bautista et al. XF06 filterset) using a Zeiss Axioplan2 Imager micro- 2002). Furthermore, several species of the Trente- scope (Carl Zeiss) with a Zeiss Axiocam MRc5 digital pohliales are common photobionts in lichens. About camera (Carl Zeiss) and Zeiss Axiovision software (Carl 20–30% of lichen-forming fungi associate with trente- Zeiss MicroImaging GmbH, Germany). pohlialean algae (Ahmadjian 1993; Nelsen et al. 2011). Low molecular weight Because of the unusual placement of S. hawaiiensis carbohydrate content in the Cladophorales, a group with which it apparently shares few characters, and because some features such The LMWC content of members of the Cladophorales as the high pigment content and the terrestrial habitat and Trentepohliales as well as S. hawaiiensis was exam- are strikingly reminiscent of the Trentepohliales, we ined (Table 1, see Appendix S1 in Supporting Informa- reinvestigated this alga using novel sequences and tion for complete dataset). LMWCs were extracted other approaches including fluorescent