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Protoplasma (2015) 252:489–503 DOI 10.1007/s00709-014-0694-0

ORIGINAL ARTICLE

Similarity and diversity of the spp. microalgae isolated from associations with White Sea invertebrates

Olga A. Gorelova & Olga I. Baulina & Alexei E. Solovchenko & Konstantin A. Chekanov & Olga B. Chivkunova & Tatiana A. Fedorenko & Elena S. Lobakova

Received: 27 June 2014 /Accepted: 22 August 2014 /Published online: 5 September 2014 # Springer-Verlag Wien 2014

Abstract Similarity and diversity of the phenotype and nu- showed that all isolates studied differ from known classified cleotide sequences of certain genome loci among the single- representatives of Desmodesmus combining a deletion in the celled microalgae isolated from White Sea benthic inverte- conservative 5.8S rRNA gene and long AC-microsatellite brates were studied to extend the knowledge of oxygenic repeats in the ITS1 whereas 1Hp86E-2 represented a distinct photoautotrophs forming microbial communities associated branch within this group. with . We compared four Desmodesmus isolates (1Hp86E-2, 1Pm66B, 3Dp86E-1, 2Cl66E) from the sponge Keywords photosymbionts . Desmodesmus . Fatty Halichondria panicea, trochophore larvae of the acids . ITS1-5.8S rRNA-ITS2 . rbcL . Ultrastructure maculata, and the hydroids Dynamena pumila and Coryne lovenii, respectively. The microalgae appeared to be very similar featuring the phenotypic and genetic traits Introduction characteristics of unicellular representatives of the genus Desmodesmus. At the same time, isolates from different ani- It is well known that pro- and eukaryotic oxygenic mal displayed certain differences in (i) the epistructure phototrophic microorganisms (OPM) could exist in symbiotic morphology; (ii) type and number of the inclusions such as associations with diverse invertebrates. Endophotosymbionts interthylakoid starch grains and cytoplasmic oil bodies and (cyanobacteria, microalgae (MA) or their functional chloro- (iii) fatty acid composition; in Desmodesmus sp. 1Hp86E-2, plasts) which convey the symbiosis the capability of photo- these differences were most pronounced. Phylogenetic analy- synthesis were documented in certain representatives of the sis based on ITS1-5.8S rRNA-ITS2 and rbcL sequences phyla Mollusca (giant clams, nudibranchs), Porifera (sponges), Cnidaria (corals, sea anemones and hydra), Acoelomorpha (flatworms) and Chordata (ascidians) (Trench Handling Editor: Andreas Holzinger 1993; Lee et al. 2001; Carpenter and Foster 2003; Taylor et al. Electronic supplementary material The online version of this article 2007; Hooge and Tyler 2008;Usher2008;Vennetal.2008; (doi:10.1007/s00709-014-0694-0) contains supplementary material, which is available to authorized users. Rumpho et al. 2011). The existence of associations comprised : : : by colonial hydroids with microalgal and cyanobacterial O. A. Gorelova O. I. Baulina A. E. Solovchenko epibionts is also well established (Bavestrello et al. 1996; K. A. Chekanov : O. B. Chivkunova : T. A. Fedorenko : E. S. Lobakova Siqueiros-Beltrones et al. 2001; Di Camillo et al. 2005, Department of Bioengineering, Biological Faculty, Lomonosov 2006; Romagnoli et al. 2007;Gorelovaetal.2009, 2013). Moscow State University, Moscow 119234, Russia However, the metabolite exchange between the members of the association in this case remains to be proved. The studies A. E. Solovchenko K.A. Timiryazev Institute of Plant Physiology, Russian Academy of of these associations are currently limited to the investigation Sciences, Moscow 127276, Russia of their development, identification of epibiont species and description of structural foundation of spatial integration of * A. E. Solovchenko ( ) the animals and the microorganisms. Department of Bioengineering, Faculty of Biology, Moscow State – University, 119234GSP-1, Moscow, Russia The OPM invertebrate symbioses are unique biological e-mail: [email protected] systems combining auto- and heterotrophic components Author's personal copy

490 O.A. Gorelova et al. representing a promising source of a wide array of natural daylight fluorescent lamps. The samples were taken from products. Thus, the sponges and coelenterates as well as the stationary phase cultures. microorganisms including are the richest natural source of new bio-active compounds (Blunt et al. 2005). Light microscopy These products could be synthesized and accumulated by of the association members or synergistically by the host and its The intact or stained with Lugol’s solution or China ink microsymbionts. This is the case, e.g. for the symbioses of microalgae were studied under Leitz Laborlux D (Ernst Leitz sponges and certain microorganisms (Lee et al. 2001; Taylor Wetzlar GmbH, Germany) and Axioskop 40 FL microscope et al. 2007; Usher 2008). Attempts to isolate microsymbionts equipped with HBO-50 AC mercury lamp (Carl Zeiss, Ger- from their animal hosts are rarely successful. Only several many). The filtration of excitation light was made using an percent of microbial species comprising the diversity of interference filter with the transmission maximum at 365 and sponge symbionts were isolated and cultured (Hill 2004). 546 nm and the half width of transmission band of 25 and Still, it is a promising direction for obtaining microbial strains 12 nm, respectively. Recording of fluorescence emission was producing unique valuable compounds. through a boundary filter blocking the radiation with Animal hosts of photomicrosymbionts were documented wavelength less than 420 and 590 nm. predominantly in tropical and subtropical sea waters. By Vital staining of the microalgal cells containing oil bodies contrast, the symbiotic relationships between invertebrates with Nile Red and fluorescent microscopy of the stained cells and OPM of high latitude seas, especially of White Sea, are was carried out according to Elsey et al. (2007) using the insufficiently studied (Gorelova et al. 2009, 2012, 2013; Axioskop 40 FL microscope. Koksharova et al. 2013; Kravtsova et al. 2013). The same is true for isolation, cultivation, , morpho- Electron microscopy physiological and ultrastructural traits of OPM from associa- tions with invertebrates of White Sea. The microalgae samples for transmission electron microscopy To bridge this gap, at least in part, we aimed to obtain (TEM) were prepared with the standard protocol: fixed in 2 % comparative phenotypic characteristic of Desmodesmus spp. (w/v) glutaraldehyde solution in 0.1М sodium cacodylate isolated from the associations with different White Sea inver- buffer at room temperature for 0.5 h and then post-fixed for tebrate species. The present study was focused on the cell 4hin1%(w/v) ОsО4 in the same buffer. The samples, after ultrastructure, pigment and major fatty acid composition. We dehydration through graded ethanol series including anhy- also carried out the partial DNA sequence-based phylogenetic drous ethanol saturated with uranyl acetate, were embedded analysis involving (i) the fragment spanning ribosomal inter- in araldite. Ultrathin sections were made with an LKB-8800 nal transcribed spacer 1 (ITS1), 5.8S ribosomal RNA (rRNA) (LKB, Sweden) ultratome, stained with lead citrate according gene and internal transcribed spacer 2 (ITS2) and (ii) a partial to Reynolds (1963) and examined under JEM-100B or JEM- sequence of rbcLgene. 1011 (JEOL, Japan) microscopes. Forscanningelectronmicroscopy(SEM),thesamplesafter fixation and dehydration according to the procedures de- scribed above were transferred to anhydrous acetone and Material and methods critical-point dried on a Dryer HCP-2 (Hitachi, Japan), coated with Au–Pd alloy on a IB-3 Ion Coater (Eiko, Japan) and Algal culture examined under JSM-6380LA scanning electron microscope (JEOL, Japan) or without coating examined under ultra-high Four microalgae (MA) isolated from associations with the resolution scanning electron microscope SU8000 (Hitachi, benthic invertebrates obtained from Rugozerskaya Guba at Japan). Kandalaksha Bay of the White Sea (66° 34′ N, 33° 08′ E) were evaluated (Table 1). The microalgae spatially integrated with Spectral measurements the benthic animals were isolated from native animals or large fragments of the latter. To minimize possible contamination, The cells grown on solidified media were resuspended in the animals or their fragments were subjected to mild surface corresponding media prior to measurements. The absorption sterilization according to Gorelova et al. (2009) and were spectra of cell suspensions were recorded in standard 1-cm triple-rinsed in sterile water. The detailed description of the quartz cuvettes on a Hitachi 150–20 spectrophotometer initial culture obtaining and handling can be found elsewhere (Hitachi, Japan) equipped with an external integrating sphere (Gorelova et al. 2009). The microalgae were grown on solid- accessory (i.d. 150 mm, part 150–0901; Hitachi, Japan) ified or liquid BG-11 (Stanier et al. 1971)mediumatroom against a cuvette with corresponding medium. The temperature and constant illumination of 40–80 μEm−2 s−1 by scattering-compensated absorbance spectra were obtained as Author's personal copy

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Table 1 Microalgae (single-celled green coccoids lacking rigid shell) associated with White Sea invertebrates

Host animals Localizationa Isolated microalgae

Systematic category Species Surface Internal

Sponge Halichondria panicea (Pallas, 1766) − +1Hp86E-2 Polychaeta Phyllodoce maculata (L., 1767)b, c + − 1Pm66B Hydroids Dynamena pumila (L., 1758) + + 3Dp86E-1 Coryne lovenii (M. Sars, 1846) + − 2Cl66E a According to bright-field, fluorescent optical and electron microscopy b The trochophore larvae, excised from the clutch attached to the sand sea bed c No EM data available described previously (Merzlyak and Naqvi 2000;Merzlyak cycles; final elongation 72 °C for 5 min. The PCR mixture et al. 2008). contained 10 ng of the algae genomic DNA in 25 μlof1× PCR Buffer for Tersus (Evrogen, Russia) containing 200 μM μ μ Lipid extraction and pigment analysis each of dNTPs, 0.2 M of each primer and 0.5 lof50× Tersus Taq polymerase (Evrogen, Russia). The fragment of ′ Cells were pelleted by centrifugation, transferred to a glass– rbcL gene was amplified with rbcL-DesF (5 -TCCGTATGAC ′ ′ glass homogenizer with a chloroform/methanol (10 mL, 2:1, TCCACAACCAG-3 ) and rbcL-DesR (5 -TGTAGTCATC ′ v/v) mixture and extracted to remove all pigment. The lipid ACGCATTAAGTC-3 ) primers (Fawley et al. 2011)accord- fraction including chlorophylls (Chl) and carotenoids (Car) ing to Fawley et al. (2005). The PCR products were purified was separated according to Folch et al. (1957). The chloro- using Cleanup Standard PCR purification kit (Evrogen, Rus- form phase was used for further pigment and lipid analysis sia) and sequenced on ABI Prizm 3730 (Applied Biosystems) according to earlier published protocols (Solovchenko et al. in both directions. The new sequences were submitted to 2014b). GenBank under accession numbers specified in Table 2.

DNA sequencing Sequence data analysis

To destroy the tough cell wall, 5–10 mg samples of Sequences were searched against NCBI GenBank (nucleotide microalgae biomass collected for DNA isolation were sub- collection nr/nt database) using BLAST (http://blast.ncbi.nlm. jected to three liquid nitrogen freeze–thaw cycles. For ampli- nih.gov/). Twenty-five sequences from the classified represen- fication of ITS1–5.8S rRNA–ITS2 sequence, genomic DNA tatives of the genus Desmodesmus were selected from was isolated using ‘Miniprep’ DNA isolation kit (Silex, Rus- GenBank for ITS1-5.8S rRNA-ITS2 data analysis. Twenty- sia). For amplification of the plastid rbcLgene,thesamples four sequences from the classified representatives of the were incubated for 1 h in 300 μlcitrate–phosphate buffer (pH Desmodesmus and (currently Acutodesmus) 5.0) containing 10 mg ml−1 cellulase (Fermentas, Lithuania), genera were selected from GenBank for rbcL sequence data 10 mg ml−1 pectinase (Fermentas, Lithuania) and 1 mM analysis. Multiple alignments of the datasets were carried out EDTA at 37 °C. The samples were then incubated with 2 % using ClustalW2 online tool (http://www.ebi.ac.uk/Tools/msa/ sodium dodecyl sulphate for 1 h at 40 °C and, after addition of clustalw2/) (Larkin et al. 2007). There were a total of 533 400 μl of 1 M NaCl, were left overnight at room temperature for protein salting. Then, standard phenol–chloroform extrac- Table 2 Microalgae isolates investigated in this study with their tion procedure (Sambrook and Russell 2006) was performed. GenBank accession numbers for rbcL and ITS1-5.8S rRNA-ITS2 DNA sequences The quality of the DNA samples was evaluated by electro- phoresis in 1.5 % agarose gel stained with ethidium bromide. Isolate GenBank accession number Aprimer(5′-ATGCAAACCACAACACGCAC-3′,forward; 5′-TTCCTGCTTGGCTCCTAACC-3′, reverse) designed rbcL ITS1-5.8S rRNA-ITS2 using Gene Runner 4.0.9.68 (http://www.generunner.net) 3Dp86E-1 KJ463405 JQ313132.1 was used for amplification of the ITS1-5.8S rRNA-ITS2 2Cl66E KJ463407 JQ313131.1 DNA fragment on Bio-Rad DNA engine (PTC 200) using 1Pm66B KJ463406 JQ313134.1 the following amplification profile: 94 °C for 3-min initial 1Hp86E-2 KJ463408 JQ313133.1 denaturation, 94 °C for 20 s, 60 °C for 25 s, 72 °C for 35 s, 30 Author's personal copy

492 O.A. Gorelova et al. positions and 29 taxa in the final ITS1-5.8S rRNA-ITS2 The cell wall structure of the studied microalgae was typ- dataset and 832 positions and 28 taxa in the final rbcLdataset. ical of the genus Desmodesmus, it consisted of a thick inner The parameters of pairwise alignment are given below. Align- polysaccharide (cellulose- and/or hemicellulose-containing) ment type: slow, DNA weight matrix: International Union of layer and a thin outer sporopolleninic layer (Figs. 2 and 3a– Biochemistry (IUB), gap open: 100 and gap extinction: 10.0. c, e, h). The inner layer varied in thickness (60–220 nm) and Theparametersofmultiplealignmentswereasfollows:DNA osmiophilicity and was comprised by fine granules and short weight matrix: IUB, gap open: 100, gap extinction: 10.0, gap microfibrils. The sporopolleninic layer (22–35 nm thick) re- distances: 10, no end gaps, iteration type: “tree” and number sembled the trilaminar layer common in certain microalgae of iterations: 10. Phylogenetic trees for multiple alignments (Atkinson et al. 1972). The structure of this layer was mark- were designed with using maximum likelihood (ML) method edly asymmetric: the inner electron-dense lamina was thinner (Aldrich 1997)inPhyML3.0(Guindonetal.2010)(http:// than the outer one; the latter carried diverse epistructures www.atgc-montpellier.fr/phyml/), maximum parsimony (MP) detectable only by electron microscopy (Figs. 1, 2 and 3). method (Fitch 1971) in TNT 1.1 (Goloboff et al. 2000), and According to Hegewald (1997), the epistructures were desig- neighbour-joining method (NJ) (Saitou and Nei 1987)in nated as short spines, simple elongated tubes, warts or ro- BioNJ (Gascuel 1997)(http://www.phylogeny.fr/,Dereeper settes. Moreover, thin villi, various fibrous structures and a et al. 2008, 2010). The best of nearest neighbour interchange few barely visible fine ribs were also observed; the latter could (NNI) and subtree pruning and regrafting (SPR) results were represent rupture lines without accompanying warts (Fig. 1c, used for tree improvement in PhyML 3.0. HKY85 model of e, f). Short spines (bundle of tubes of different length) and DNA evolution (Hasegawa et al. 1985) was used. For the single elongate tubes were the most abundant epistructures. other parameters, default values were accepted. The trees were Short spines were formed by 2–4(rarely5–7) elongate tubes, rendered in TreeDyn 198.3 (Chevenet et al. 2006)(http:// parallel to each other or spirally twisted (Fig. 2e, f). Other www.phylogeny.fr/version2_cgi/index.cgi). The accuracy of prominent epistructure type, as evidenced by SEM images, the tree topology was tested using 1,000-replicate bootstrap was represented by randomly located warts. The warts of analysis (Felsenstein 1985). different morphology were encountered in the ultrathin sec- The probability of rejecting the null hypothesis that se- tions: (i) conic warts with an osmiophilic knob at the top quences of rbcL gene fragment and ITS1-5.8SrRNA-ITS2 (diameter at the base 35–60 nm, height 75–100 nm; have evolved with the same pattern of substitution was tested Fig. 2b); (ii) simple round warts 60 nm in diameter at the base by estimation of the extent of differences in base composition andupto40nmhigh(Fig.2c) and (iii) elongate warts 45– biases between the sequences (Kumar and Gadagkar 2001). A 60 nm in diameter (at the base) and 40–60 nm high (Fig. 2d). Monte Carlo test (1,000 replicates) was used to estimate the P The different patterns of epistructures characteristic of the value (Kumar and Gadagkar 2001). The analysis was per- studied microalgae are elaborated on below. formed using MEGA4 software with default parameters Desmodesmus sp. 1Pm66B presented on its cell surface (Tamura et al. 2007). elongated tubes (single tubes or tube bundles forming short spines) 500–900 nm long and 70–100 nm thick at the base. The tubes were filled with the electron-transparent matter Results comprising the middle lamina of the trilaminar layer with small osmiophilic pellets. The tube apexes were conic or The morphology and ultrastructure of the isolated microalgae oblique, carrying one or two branches at a 30–90 ° angle to the base of the tube (Figs. 1c and 2e). Judging from SEM All the investigated Desmodesmus spp. isolates (1Hp86E-2, images, the randomly located warts were not ubiquitous. On 1Pm66B, 3Dp86E-1 and 2Cl66E) obtained from different the ultrathin sections, simple round warts and conic warts with animal species (Table 1) possessed a number of common osmiophilic knob at the top were found. The rosettes with traits. They were round-shaped, non-motile, single-celled coc- rectangular cross-section ca. 210 nm wide and 115 nm high coid microalgae lacking flagellae and rigid shells (Fig. 1a–d). (Fig. 3a) and thin villi 50–70 nm long perpendicular to and Their cells possessed no epistructures resembling teeth or evenly distributed on the cell wall surface (Figs. 2a, c and 3a) spines discernible under light microscope. Under our cultiva- were also observed in addition to the thin fibrillar matter or tion conditions, the microalgae multiplied by producing 2–8 mucous strands (Fig. 2a, e). The rosettes and conic warts were autospores (more in the case of 3Dp86E-1) in a single spo- rarely encountered. rangium. Based on their cell size, all isolates belonged to the Desmodesmus sp. 2Cl66E possessed diverse epistructures group small green microalgae: the cells of 1Pm66B varied generally similar to that of Desmodesmus sp. 1Pm66B. At the widely in diameter (2.9–8.1 μm); for the other isolates same time, the following differences were found: (i) the conic (3Dp86E-1, 2Cl66E and 1Hp86E-2) the cell diameter was in warts topped with osmiophilic knob were predominant the range of 3–5 μm. (Figs. 1a, 2b and 4a); (ii) the simple round warts were rarely Author's personal copy

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Fig. 1 The morphology (SEM images) of the Desmodesmus spp. isolated from benthic invertebrates. They were visibly round in shape and possess diverse epistructures on their cell surfaces: warts, spines (bundle of tubes of different length) or single elongated tubes, and rosettes. The rupture line was visible without accompanying warts. a Desmodesmus sp. 2Cl66E from the hydroid Coryne lovenii. b Desmodesmus sp. 1Hp86E-2 from the sponge Halichondria panicea. c Desmodesmus sp. 1Pm66B from trochophore larvae of the polychaete Phyllodoce maculata. d–f Desmodesmus sp. 3Dp86E-1 from the hydroid Dynamena pumila. d and e SEM images the samples without coating. As autospore, FM fibrillar matter, R rosette, S short spines, Sp sporangium and W warts. The rupture line is indicated by an arrow.Thetube branches are indicated by an arrowhead. Scale bars 2 μm(a, c, d), 1 μm(b, e, f)

encountered; (iii) the villi and mucous strands were not ob- possessed larger rosettes in comparison with 1Pm66B (up to served at all and (iv) the short spines formed of twisted tubes 420 nm wide at the base, featuring perforations ca.46nmin were also absent. diameter; Fig. 3b). Thin bristles perpendicular to the cell In Desmodesmus sp. 3Dp86E-1, the epistructure types surface were regularly encountered near the rosettes. were similar to those present in Desmodesmus sp. 1Pm66B Desmodesmus sp. 1Hp86E-2 differed most significantly excepting the conic warts with osmiophilic knobs and the from the abovementioned microalgae in epistructure morphol- evenly distributed villi (Figs. 1d–f and 3e). At the same time, ogy. It featured: (1) the short spines formed by 2–4tubes fibrous matter forming glomeruli or stochastically scattered (spirally twisted, 65–120 nm in diameter at the base) with chords of various lengths were observed. The elongate tubes conic or oblique apexes without branches (Figs. 1b and 2f); (single and bundled in the short spines) were longer (up to (2) a dense 20–30 nm layer of thin villi encrusted by small 2 μm) and arrow-headed due to the presence of branches grains carpeting the surface of the cell (Fig. 2d, f); (3) elon- (Figs. 1e, f and 2g). Additional branches were observed on gated warts (Fig. 2d); (4) round rosettes with a convex outer the cylindrical part of tubes as well; the basal cylindrical part ridge (external diameter of 315 nm, 110–120 nm high) and a of a tube could be thinner than its main part. This microalga rise in the centre (195 nm in diameter) with a thick bundle of Author's personal copy

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Fig. 2 The ultrastructure of the cell wall of the microalgae isolated from associations with the benthic invertebrates (TEM images). The cell wall was composed of a thick inner polysaccharide layer and a thin outer sporopolleninic layer that carried diverse epistructures. Represented cell wall fragments with different morphology of warts, short spines and villi. a, c, e Desmodesmus sp. 1Pm66B from trochophore larvae of the polychaete Phyllodoce maculata. b Desmodesmus sp. 2Cl66E from the hydroid Coryne lovenii. d, f Desmodesmus sp. 1Hp86E-2 from the sponge Halichondria panicea. g Desmodesmus sp. 3Dp86E-1 from the hydroid Dynamena pumila. Ch , ChE chloroplast envelope, CM cytoplasmic membrane, CW cell wall, IL inner layer of cell wall, N nucleus, OB oil body, S short spines, SG starch granule, TL trilaminar layer of cell wall, V villi, VIM vacuole with granules constituted by a matter of inhomogeneous electron density and W wart. The tube branches are indicated by an arrow. The fibrillar matter is indicated by an asterisk. Scale bars 2 μm

villi ca. 100 nm high (Fig. 3c, d) and (5) ball- or pyramid-like pyrenoid was surrounded by two or (less frequently) three– excrescences formed by grains and micelles of outer lamina of four bean-shaped starch grains. Cross-sections of the the sporopolleninic layer carrying the villi (Fig. 3f–h). The 1Hp86E-2 cells often revealed the pyrenoid with two asym- excrescences of neighbouring cells frequently merged metrically arranged RuBisCO grains surrounded by 2–5irreg- forming bulk bridges between the cells. The conic warts with ularly shaped starch grains (Fig. S1). The enve- osmiophilic knob at the top and simple round warts were not lope was highly osmiophilic (Figs. 4a, e, S1 and S2). observed at all. In all microalgae studied, a high degree of thylakoid stack- All of the microalgae studied were uninucleate. They fea- ing was revealed. Number of thylakoids per stack varied tured simple chloroplasts with two-membrane envelope and between cells as well as between isolates. In the 1Pm66B, identical pigment composition characteristic of green algae 3Dp86E-1 and 1Hp86E-2 cells the thylakoids formed elon- (see below). The lobe-shaped chloroplasts occupied the most gated stacks (2–20 thylakoids per stack). The thylakoids of of the cell volume and contained a pyrenoid (Fig. 4a–d)with grana were hardly discernible from these of stroma since only fine-grained stroma lacking intrapyrenoidal thylakoids. The a few short thylakoids (single or paired) emerged from the Author's personal copy

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Fig. 3 The ultrastructure of the rosettes (a–d) and fibrous structures (e–h) on the cell surface of the microalgae isolated from associations with the benthic invertebrates. a TEM image of Desmodesmus sp. 1Pm66B (from the trochophore larvae of the polychaete Phyllodoce maculata). b, e TEM images of Desmodesmus sp. 3Dp86E-1 (from the hydroid Dynamena pumila. c, d, f–h TEM images (c, h) and SEM images (d, f, g)of Desmodesmus sp. 1Hp86E-2 (from the sponge Halichondria panicea). CM cytoplasmic membrane, CW cell wall, ETL excrescences of the outer lamina matter of the trilaminar layer of cell, FM fibrous matter, IL inner layer of cell wall, R rosette, S short spines, TL trilaminar layer of cell wall, V villi and VIM vacuole with granules constituted by a matter of inhomogeneous electron density. Scale bars 0.2 μm(a–e), 0.5 μm(f–h)

stacks to the stroma. The lumen was narrow and contained Vacuoles (up to 1.8×0.8 μm in size) were sometimes osmiophilic matter (Figs. 4e and S2). There were plastoglobuli encountered in the cells of the studied microalgae. These of different osmiophilicity and starch grains between the vacuoles contained round granules 0.35–0.91 μmindiameter thylakoids of the chloroplasts. The structure of the 2Cl66E constituted by a matter of inhomogeneous electron density. chloroplasts was generally similar to that of the three previous These inclusions often contained irregular holes formed dur- isolates though the stacks were less prolonged and more ing the sample preparation (Fig. 4a, c, d). Similar granules similar to grana of higher plants (Figs. 4a and S2). were described in green microalgae and identified as The studied microalgae differed in starch accumulation. polyphosphate granules (Matusiak-Mikulin et al. 2006). The stroma of the chloroplasts in most of 1Pm66B cells contained numerous large starch grains, whereas in 3Dp86E- Suspension absorption spectra, fatty acid and pigment 1, 2Cl66E and, especially, in 1Hp86E-2 the number of stromal composition starch grains was considerably smaller (Fig. 4a–d). In all investigated microalgae cytoplasmic oil bodies (OB) In the red region of the spectrum, the absorption spectra of cell were found (Figs. 4b, c and S3).Theisolates3Dp86E-1and suspensions of all isolates studied exhibited a strong maxi- 2Cl66E featured a limited number of OB of medium electron mum at 678 nm and a shoulder near 651 nm, characteristic of density and moderate size (0.278±0.024 and 0.369± Chl a and b,respectively(Fig.S5). In the blue-green region, 0.021 μm, respectively). The OB of 1Hp86E-2, even those the spectra contained maxima and shoulders attributable to in the same cell, differed considerably in osmiophilicity strong combined absorption by Chl and Car. The quantitative (Figs. 4c and S3b–d). The average diameter of the OB was analysis revealed significant diversity of Chl a/b ratio in the 0.422±0.032 μm. In 1Pm66B, OB comprised the dominant studied isolates (1.71–2.03). This strongly suggests significant type of cytoplasmic inclusions reaching 1.163 μmindiameter changes in Chl a and b spectral features and their contributions (the average diameter 0.620±0.059 μm) (Figs. 2a and 4b). into absorption between 550 and 750 nm. The HPLC analysis The OB were readily apparent under luminescent microscope showed that the major Car profile of the isolates studied after vital staining with Nile Red as round structures with contained lutein, violaxanthin, antheraxanthin and β- bright green-yellow fluorescence (Fig. S4). carotene (Fig. S6). Notably, the xanthophylls participated in Author's personal copy

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Fig. 4 The ultrastructure (TEM images) of the Desmodesmus spp. isolated from benthic invertebrates. The chloroplasts possessed a lobed shape, occupied most of the cell volume and contained a pyrenoid. The thylakoids were stacking. The microalga displayed certain differences in the content of the starch grains and intracytoplasmic oil bodies. a Desmodesmus sp. 2Cl66E from the hydroid Coryne lovenii. b Desmodesmus sp. 1Pm66B from trochophore larvae of the polychaete Phyllodoce maculata. c Desmodesmus sp. 1Hp86E-2 from the sponge Halichondria panicea. d, e Desmodesmus sp. 3Dp86E-1 from the hydroid Dynamena pumila. Ch chloroplast, ChE chloroplast envelope, CW cell wall, N nucleus, OB oil body, P pyrenoid, Pg plastoglobule, S short spines, SG starch granule, T thylakoids, VIM vacuole with granules constituted by a matter of inhomogeneous electron density and W wart. An osmiophilic matter in the narrow lumen is indicated by an arrowhead. Scale bars 1 μm (a–d), 0.2 μm(e)

the violaxanthin cycle comprised a considerable part (58– USA, Mexico, Germany, Hungary, Czech Republic, New 62 %) of total Car. At the same time, the extent of violaxanthin Zealand and Fiji (Table S1), in particular with Desmodesmus de-epoxidation was uniformly low (0.26–0.27) among the multivariabilis var. turskensis isolate Mary 8/18T-1W studied isolates under cultivation conditions employed in the (DQ417525.1) (identity 95 %). The partial rbcL sequence work. displayed the highest homology with same isolate of Generally, the isolates under investigation featured similar Desmodesmus multivariabilis var. turskensis (Mary 8/18T- fatty acid (FA) composition (Fig. 5). In all cases, the FA 1W (GU192431.1); identity: 92 %) and Desmodesmus hystrix profile was dominated by the saturated FA C16:0 together (strain NDEM 9/21T-9w (GU192433.1); identity 93 %). with three representatives of C18 family of unsaturated FA The multiple alignment analysis data showed a number of (C18:1, C18:2 and C18:3). Other FA (C14–C20 with different specific features of 3Dp86E-1, 1Pm66B, 2Cl66E and extent of saturation, 14 different FA species in total) com- 1Hp86E-2 isolates, namely the extended AC-microsatellite prised 12–18 % of total FA. The isolate 1Hp86E-2 as well as repeats in the ITS1 region (Fig. S7a). These repeats were the microalgal isolate from the hydroid (2Cl66E) contained longer than in other classified Desmodesmus representatives: C20:1 (Fig. 5b, d) in addition to C20:0. 6 copies in 1Pm66B and 2Cl66E, 7 in 3Dp86E-1, and 10 in 1Hp86E-2. Furthermore, these isolates featured a deletion in Molecular phylogenetic analysis the conservative 5.8S rRNA gene region (position 151 in the alignment) lacking in other known sequences excluding The homology search using BLAST showed maximal identity Desmodesmus multivariabilis var. turskensis isolate Mary of the ITS1-5.8S rRNA-ITS2 fragment of the studied isolates 8/18T-1W (DQ417525.1) (Fig. S7b). Multiple alignment of with the sequences of freshwater representatives of the genus rbcL sequences revealed neither deletions nor insertions. Most Desmodesmus from natural and artificial water bodies of the of the changes in rbcL gene fragment were point mutations Author's personal copy

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Fig. 5 Representative data on a b major fatty acid composition of 20:0 20:1 – 18:3 20:0 the Desmodesmus isolates (a d) 3% 4% 16:0 10% 16:0 4% 3Dp86E1-1, 2Cl66E, 1Pm66B 23% 18% and 1Hp86E-2 from White Sea 16:1 invertebrates (see also Table 1) 18:3 4% 18% 18:2 16:3 19% 5% 16:1 18:0 7% 1% 16:3 6% 18:2 18:0 18% 18:1 2% 18:1 30% 28%

c d 20:0 20:1 20:0 16:0 4% 3% 16:0 4% 15% 18:3 18% 16:1 18:3 20% 16:1 3% 15% 6% 16:3 16:3 5% 4% 18:0 18:0 1% 2% 18:2 18:2 17% 21% 18:1 18:1 33% 29%

predominantly in the third base positions of the codons significant incongruence between sequences and the se- effecting no change in the encoded protein amino acid quences have evolved with the same pattern of substitution. sequence. This finding made it possible to analyze the combined se- In all phylogenetic trees inferred with NJ, ML and MP quence dataset. The results of this analysis (Fig. 8)confirmed algorithms, the studied isolates formed a distinct cluster with that the microalgal isolates from White Sea invertebrates form high bootstrap value (92 % and more). The other a monophyletic group with bootstrap of 100 regardless of the Desmodesmus from the dataset weren’t included in this group algorithm used for construction of the tree. As was shown by (Figs. 6, 7 and S8). In the trees based on rbcLsequences separate analyses of partial rbcL (NJ, MP and ML methods) (Fig. 6) and in ITS1-5.8S rRNA-ITS2 tree inferred with ML and ITS1-5.8S rRNA-ITS2 sequences (using ML and MP and MP algorithms, the isolates 3Dp86E-1, 1Pm66B and methods), the isolates 3Dp86E-1, 1Pm66B and 2Cl66E 2Cl66E formed a subcluster and 1Hp86E-2 comprised an formed a distinct subcluster whereas 1Hp86E-2 comprised outstanding branch (insert in Fig. 7). By contrast, in the an outstanding branch. ITS1-5.8S rRNA-ITS2 tree inferred with NJ (Fig. S8) the microalgae isolated from hydroids (2Cl66E, 3Dp86E-1) and those from a sponge (1Hp86E-2) and a polychaete (1Pm66B) formed separate subgroups. At the same time, 3Dp86E-1, Discussion 1Pm66B, 2Cl66E formed a single subcluster in the trees inferred (with NJ algorithm) from ITS1 or ITS2 sequences The microalgae of the genus Desmodesmus in symbioses only (data not shown). with White Sea invertebrates The homogeneity test of the sequences of rbcLgenefrag- ment and ITS1-5.8S rRNA-ITS2 of the isolates under inves- The findings reported in the present paper extend current tigation yielded a P value of 0.310 meaning that there was no understanding of biodiversity of oxygenic phototrophic Author's personal copy

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microorganisms (OPM) associated with White Sea inverte- brates. To date, 19 cultures of eukaryotic unicellular algae lacking rigid shells were isolated from animal samples of five hydroid, two sponge species and one polychaete (Gorelova et al. 2012), whereas cyanobacteria were isolated from the hydroids only (Gorelova et al. 2009; Koksharova et al. 2013). It should be stressed that all these animals were associated with the microbial community of host-specific structure; sym- bioses of an animal with a single cyanobacterial or microalgal species were not discovered; eukaryotic unicellular algae lacking rigid shells were frequently encountered within these associations but never dominated it (Gorelova et al. 2009). The eukaryotic phototroph part of the epibiont microbial community of the hydroid Dynamena pumila was dominated by diatoms (Gorelova et al. 2009, 2013) as in Mediterranean hydroid Eudendrium racemosum (Romagnoli et al. 2007). As evidenced by electron microscopy, bacteria and microalgae Fig. 6 Evolution relationship of the Desmodesmus spp. (2Cl66E, from the epibiont biofilm formed intrusions into the perisarc 3Dp86E-1, 1Pm66B, 1Hp86E-2) isolated from White Sea invertebrates and coenosarc of D. pumila, but the diatom cells were never based on the partial rbcL sequence (inferred with NJ, ML and MP found inside the animal perisarc or coenosarc; only shell algorithms). HKY85 model of DNA evolution were used. NNI and SPR were used for tree improvement. The optimal tree is shown. The fragments without the protoplast were noticed within the percentage of replicate trees in which the associated taxa clustered to- perisarc matrix under the biofilm and in the vacuoles inside gether in the bootstrap test (1,000 replicates) are shown under the the hydroid coenosarc. By contrast, structurally intact branches for NJ/ML/MP method, respectively. All positions containing microalgae lacking rigid shells, including those similar to gaps and missing data were eliminated from the dataset. The branches with bootstrap value less than 70 % were collapsed. There were a total of Desmodesmus sp. 3Dp86E-1, were found in the matrix of 832 positions and 28 taxa in the final dataset. Phylogenic analyses were the perisarc (Fig. S9, see also Gorelova et al. 2009, 2013). conducted in PhyML 3.0, in BioNJ and TNT 1.1. Tree was rendered The sponge Halichondria panicea carried no OPM on its using TreeDyn 198.3 software surface; the OPM association in the sponge body was

Fig. 7 Evolution relationship of the Desmodesmus spp. (2Cl66E, bootstrap test (1,000 replicates) are shown under the branches 3Dp86E-1, 1Pm66B, 1Hp86E-2) isolated from White Sea invertebrates for ML/MP methods, respectively. All positions containing gaps based on the ITS1- 5.8SrRNA- ITS2 sequence (inferred using ML and and missing data were eliminated from the dataset. There were a MP algorithms) and (insert) the cladogram of the studied microalga total of 533 positions and 29 taxa in the final dataset. Phylogenic cluster. HKY85 model of DNA evolution were used. NNI and SPR were analyses were conducted in PhyML 3.0 and TNT 1.1. Tree was used for tree improvement. The optimal tree is shown. The percentage of rendered using TreeDyn 198.3 software replicate trees in which the associated taxa clustered together in the Author's personal copy

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Fig. 8 Evolution relationship of the Desmodesmus spp. (2Cl66E, respectively; identical bootstrap values yielded by all algorithms repre- 3Dp86E-1, 1Pm66B, 1Hp86E-2) isolated from White Sea invertebrates sented by a single number. All positions containing gaps and missing data based on the combined rbcL and ITS1- 5.8SrRNA- ITS2 dataset. HKY85 were eliminated from the dataset. There were a total of 1,308 positions model of DNA evolution were used. NNI and SPR were used for tree and 15 taxa in the final dataset. Phylogeny analysis was conducted in improvement. The optimal tree is shown. The percentage of replicate PhyML 3.0, BioNJ and TNT 1.1. Tree was rendered using TreeDyn 198.3 trees in which the associated taxa clustered together in the bootstrap test software (1,000 replicates) are shown under the branches for NJ/ML/MP method,

dominated by obviously intact diatoms; the chlorophyte-like not yet described in White Sea except in the associations with were revealed as well (Table 1). Evidently, sponges could the benthic invertebrates. This circumstance could stem from contain phototrophic symbionts from different taxa. Most of the complexity of phenotypic identification and morphologi- the eukaryote phototrophs encountered in these animals are cal plasticity of the microalgae as well as from scarce appli- represented by dinoflagellates and diatoms dominating the cation of molecular identification techniques. On the other associations with Antarctic sponges (Bavestrello et al. 2000; hand, the environment of Kandalaksha Bay in White Sea Webster et al. 2004; Taylor et al. 2007). To the best of our including salinity is extremely volatile (Pesciaroli et al. knowledge, there were no reports on the isolation of symbiotic 2012) and, according to our data, could be as high as 26–27 representative of from sponges. Still, the ‰. Under laboratory conditions, the studied microalgae could studies of the White Sea sponges in this context are at its grow at this salinity level but at a lower rate in comparison beginning. The different morphotypes of green microalgae with the cultures grown in BG-11 medium (Selyakh, were distinguished in the enrichment cultures of the OPM Semenova, personal communication). One may suggest that, isolated from the Halichondria panicea sample and under the adverse conditions, the Desmodesmus microalgae Desmodesmus sp. was identified among them (Gorelova have better chances to survive in the association with et al. 2012; Kravtsova et al. 2013). the animal host. One may also speculate that the evo- Previously, the microscopic study of the trochophore lution in symbioses with different animal species could larvae of the polychaete Phyllodoce maculata yielded be one of the factors leading to the phenotypic and no evidence of the intrusion of diatoms into the clutch genetic diversity of the Desmodesmus isolates studied or the colonization of the mucous matrix of the clutch in this work. or the larvae by the diatoms. At the same time, green coccoid bodies with red fluorescence were revealed on the larval surface (Table 1). The similarity and the diversity of the symbiotic It is known that Desmodesmus spp. is among the most Desmodesmus sp. isolates as apparent from their phenotype abundant and ubiquitous coccoid green algae in fresh and brackish water bodies of the world. Surprisingly, no represen- We compared a wide array of ultrastructural (cell mor- tatives of Desmodesmus were found in the biota of White Sea phology, structure of the cell wall, epistructure types, at Biological Research Station of Moscow State University organization of chloroplasts, their envelope and thyla- (66° 34′ N, 33° 08′ E) either in brackish water of supralittoral koids, pyrenoid structure) and biochemical (suspension pools or in open sea (Chesunov et al. 2008). Still, microalgae absorbance, fatty acid and pigment composition) traits from this genus are encountered in Baltic Sea (Matusiak- of the four Desmodesmus spp. obtained from different Mikulin et al. 2006). Representatives of Desmodesmus are benthic invertebrates from the White Sea. Author's personal copy

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All the isolates studied were morphologically similar The epistructures are important for the taxonomy of the new coccoid single-celled autospore-producing microalgae. symbiotic microalgae They possessed common ultrastructural features including the cell wall with trilaminar and polysaccharide layers, Establishing the presence and obtaining the detailed descrip- diverse epistructures, lobed chloroplasts surrounded by tion of the epistructures, especially those discernible only two membranes, and a pyrenoid without intrapyrenoidal under EM is essential, together with DNA sequence data, for thylakoids. All the isolates studied were characterized by the taxonomy of the microalgae of the genus Desmodesmus the presence of starch grains and plastoglobuli in the and their close relatives (see e.g. Fawley et al. 2011). The chloroplast and OB and vacuoles filled with a matter of overwhelming majority of such data was obtained using SEM inhomogeneous electron density in the cytoplasm. The whereas in the present work, we documented the diversity and structure of cell wall, chloroplasts and pyrenoids as well fine structure of the epistructures with TEM. These as the presence and localization of the inclusions were epistructures were generally similar to those of the microalgae typical of microalgae from the family Scenedesmaceae belonging to the family Scenedesmaceae, genus Scenedesmus (Bisalputra and Weier 1964; Nilshammar and Walles (Hegewald 1997). It should be stressed that, among the three 1974; Matusiak-Mikulin et al. 2006;Eliáš et al. 2010). groups denoted in the work of Hegewald (1997)assub-genera At the same time, the isolates studied differed in abun- (Acutodesmus, Scenedesmus, Desmodesmus), only the latter dance and predominant type of the inclusions under stan- featured diverse epistructures. According to the sequence dardized cultivation conditions suggesting the difference analysis, the non-spiny and spiny forms were divided into of their biosynthetic potential. Indeed, our pilot experi- two sections—Scenedesmus and Desmodesmus,respectively ments revealed differences in their ability to accumulate (An et al. 1999). Currently, the genus Desmodesmus is includ- starch and/or neutral lipids in these microalgae (in prepa- ed with the subfamily Desmodesmoideae of the family ration, see also Solovchenko et al. 2013, 2014a). Scenedesmaceae (see, e.g. Guiry and Guiry 2011). The The similarity of the Desmodesmus spp. from different microalgae described here and representatives of the genus invertebrates was confirmed by comparative investigation of Desmodesmus both have the short spines and single elongated spectral absorption of the cell suspension, fatty acid and tubes, simple round and conic warts with osmiophilic knob at pigment composition. All of these microalgae possessed pig- the top and the structures with the profile resembling rosettes. ment profile characteristic of green microalgae (Goss and Both types of warts are characteristic of microalgae earlier Jakob 2010): the analysis of absorption spectra and pigment identified as Scenedesmus armatus var. subalternans. The composition did not revealed the presence of phycobilins, Chl rosettes and warts were also found on the cell surface of other than Chl a and b or Car species unusual for green Desmodesmus armatus (R. Chod.) Hegew (according to the microalgae. Interestingly, an unusually high proportion of modern systematics (Guiry and Guiry 2011), this species is violaxanthin cycle xanthophylls have been revealed identical to the former one). Larger rosettes similar to that (Fig. S6). One could speculate that these pigments could be, found in 3Dp86E-1 are common for Desmodesmus to a certain extent, represented by secondary Car which is not subspicatus (Elliot 2011). Still, it is pointed out (see, e.g. involved in the functioning of violaxanthin cycle Hegewald 1997; Lürling 2003; Ellio 2011; Fawley et al. (Solovchenko 2010). Further investigations are needed to 2011)thatDesmodesmus exert a certain degree of phenotypic clarify this issue. plasticity and includes spineless morphotypes. Notably, the The FA composition of the studied microalgae appeared microalgae isolated in this work possessed no spines discern- to be similar to that characteristic of green algae ible under light microscope. (Siegenthaller et al. 2004; Guschina and Harwood 2006, The results of SEM showed that the cell surface of the 2009) with a considerable proportion of unsaturated FA microalgae studied in this work featured a few barely visible (>65 %) which could reflect an adaptation to the perma- fine ribs, which probably represent a rupture line without nently cold environment (Morgan-Kiss et al. 2006)ofthe accompanying warts. These structures were similar to merid- White Sea. Moreover, the FA composition of the studied ional ribs that was found using the same method in microalgae resembled that of closely related representatives Hylodesmus singaporensis identified basing on the sequence of the genus Desmodesmus (see e.g. Pan et al. 2011;Do data analysis among the representatives of the sister branch of Nascimento et al. 2012;Hoetal.2013). Specifically, the the genus Desmodesmus (Eliáš et al. 2010). The tubes with major FA included C16:0, C16:1, C16:3, C18:0, C18:1, branched apexes characteristic of 1Pm66B, 2Cl66E and espe- C18:2 and C18:3 but essentially lacked C16:4. At the same cially of 3Dp86E-1 short spines resembled that of the spines time, all of the microalgae displayed certain differences in of Desmodesmus santosii (Fawley et al. 2011). However, the FA composition under cultivation conditions employed in short spines or the single tubes were shorter than this work, e.g. the presence of C20:1 FA was characteristic Desmodesmus santosii spines. In addition, cell surface of of 2Cl66E and 1Hp86E-2 (Fig. 5). 3Dp86E-1 and 1Pm66B possessed mucous strands whereas Author's personal copy

Desmodesmus from invertebrate associations 501 in Desmodesmus santosii no mucilage sheath was observed the isolates based on their rbcL sequence. On the contrary, this (Fawley et al. 2011). topology was not observed in ITS1-5.8S rRNA-ITS2-based The microalgae studied in this work possessed different tree inferred with the NJ algorithm. This difference in the types of the epistructures, the epistructures of the same type topology of the trees may be caused by the fact that this also differed in their morphology in different isolates in spite algorithm does not take into account the concept of molecular of standardized cultivation conditions. The presence of conic clock (Saitou and Nei 1987) and/or different mutation fre- warts with an osmiophilic knob only in 1Pm66B and 2Cl66E quencies are inherent to the different nucleotide positions in suggests the close relation between these microalgae whereas the sequence. One cannot also rule out the effects of different the presence of tubes with branched apexes (the single tubes number of plastidial and nuclear gene copies (Harris and as well as the short spine tubes) suggests the relationship Crandall 2000). between 1Pm66B, 2Cl66E and 3Dp86E-1; at the same time, 1Hp86E-2 did not possess the branched tubes at all. Isolates 3Dp86E-1 and 1Pm66B were also related since they had fibrous matter on the cell surface. It should be emphasized Conclusion that the isolate 1Hp86E-2 was distinct from the other isolates studied (1Pm66B, 2Cl66E and 3Dp86E-1) considering Before recent reports (Gorelova et al. 2012; Kravtsova the types and morphology of the epistructures including et al. 2013), microalgae of the genus Desmodesmus have warts, short spines, single tubes and ball- or pyramid- not been found in associations with invertebrates dwell- like excrescences formed by grains and micelles of ing in saline water bodies. In the present work, we outer lamina of sporopollenic layer carrying villi. This confirmed their presence in distant invertebrate taxa finding is consistent with the results of phylogenetic (such as sponges and hydroids) of the White Sea and analysis (see below). obtained detailed characteristics of these microalgae. Our findings, together with recent discovery of Desmodesmus The symbiotic Desmodesmus isolates belong komarekii in association with a marine fish (Koike et al. to a monophyletic group but are not identical 2013 ) and presence of a Desmodesmus closely related to D. subspicatus (Kovačević et al. 2010) in a freshwater Phylogenetic analysis of 3Dp86E-1, 1Pm66B, 2Cl66E and hydra, form a basis to believe that certain representatives 1Hp86E-2 based on ITS1-5.8S rRNA-ITS2 and rbcL se- of Desmodesmus capable of forming symbiotic associa- quences and the combined dataset showed that they are very tions could be considered widespread photosymbionts similar. The greatest homology with ITS1-5.8S rRNA-ITS2 since these microalgae can form symbioses with diverse fragment of all isolates had Desmodesmus multivariabilis var. and very distant taxa like Symbiodinium with marine turskensis isolate Mary 8/18T-1W (identity 95 %) among the sponges, corals, gastropods and bivalves (Venn et al. classified (in this paper) and Desmodesmus sp. FG (identity 2008)orNostoc with fungi, mosses, ferns, cycads or 99 %) and Desmodesmus sp. M1-20 (identity 99 %) among certain flower plants (Rai et al. 2002). Still, most of theunclassified(Kravtsovaetal.2013). The greatest homol- these symbioses are not obligatory and these microorgan- ogy for all microalgae studied with rbcL fragment had the isms may exist in aposymbiotic state. Future discoveries same Desmodesmus multivariabilis var. turskensis isolate of associations with Desmodesmus would further support (identity 92 %) and Desmodesmus hystrix strain NDEM the inclusion of Desmodesmus into ubiquitous symbiotic 9/21T-9w (identity 93 %). At the same time, all isolates microalgae. studied had a number of common features that distinguish All the investigated Desmodesmus spp. isolates ob- them from other classified Desmodesmus. In particular, they tained from different animal species, judging from their featured both a deletion in the conservative 5.8S rRNA gene phenotype and the studied genome loci belong to a region and long AC-microsatellite repeats in the ITS1 region. monophyletic group of organisms which are closely We believe that these repeats can serve as a kind of molecular related but are, by no means, identical. Further investi- clock (Sun et al. 2009) provided that a repeat length is pro- gations are needed to reveal whether the differences portional to the duration of the evolution of the organism or between these organisms stem from the association with the group. Accordingly, basing on a longer microsatellite different animal hosts. repeats in the ITS1 region, the studied isolates from the association with animals formed a distinct cluster including Acknowledgments The work was supported by the Russian a sub cluster comprised by the isolates 3Dp86E-1, 1Pm66B Foundation for Basic Research, projects no. 09-04-00675-a, and and 2Cl66E but not 1Hp86E-2 which possessed longer repeats by the Russian Ministry of Education and Science, project no. GK 16.512.11. 2182. The electron microscopy part of the work suggesting an earlier departure from the group of the former was carried out at the User Facilities Center of M.V. Lomonosov three isolates. This interpretation is supported by clustering of Moscow State University. Author's personal copy

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