Bmc Evolutionary Biology Phylogenetic Relationships and Taxonomic Position of Chlorella-like Isolates from Low Ph Environments (ph < 3.0)

Background: Little is known about phytoplankton communities inhabiting low pH environments such as volcanic and geothermal sites or acidic waters. Only specialised organisms are able to tolerate such extreme conditions. There is, thus, low species diversity. We have characterised the previously isolated acid tolerant Chlorella-like microalgae Viridiella fridericiana and Chlorella protothecoides var. acidicola by microscopical and biomolecular methods in order to assess their phylogenetic relationships.


Background
Very low pH environments can be found in volcanic and geothermal sites of Italy. They are generated by either sulphur springs emitting a continuous outflow of water at temperatures higher than 80°C, or fumaroles, which are mostly areas of small bubblers and steam vents. The in situ oxidation of H 2 S arising from the outlets of these geothermal systems causes the production of large amounts of H 2 SO 4 and, in turn, low pH on rocks and soil surrounding the offspring. Generally, the pH values remain below 2.5 for 10-25 square meters around the outflow of H 2 S. Temperature, on the other hand, declines from 60°C to 25-30°C within a few meters apart from the source of volcanic activity [1].
The distribution of algal flora on these soils is strictly dependent on the above two parameters. In the close vicinity of hot springs and fumaroles, soil temperature and pH are almost constant at 40°C and 1.5, respectively, thus enabling the exclusive presence of three thermoacidophilic red microalgae, Cyanidium caldarium, Galdieria sulphuraria and Cyanidioschyzon merolae [2,3]. At increasing distances, pH and temperature may dramatically change, also as a consequence of the season and rainfalls. At temperatures below 30°C and pH values between 1.8 and 2.5, coccoid green algae such as Chlorella and related genera represent a major component of algal populations [4].
The occurrence of Chlorella in low pH environments seems to be restriced to acidic soils, whereas in acidic waters it has been rarely recorded [5]. Here, the most widespread species belong to different diatom genera and to several Chlamydomonas, Ochromonas and Euglena species [6,7]. On the basis of a combination of morphological, biochemical and eco-physiological features [8], the Chlorella-like strains so far isolated from the acidic microsites have been attributed to three taxa; two of these, Viridiella fridericiana and C. protothecoides var. acidicola seem to be endemic to acidic soils.
V. fridericiana is a monospecific genus erected on the basis of the absence of a pyrenoid in the chloroplast and of sporopollenin in the cell wall [9]. It does not produce secondary carotenoids, and shows a pH limit for growth below pH 1.0 [10]. On the other hand, our knowledge on C. protothecoides var. acidicola is scanty; it has been only preliminarily investigated by light microscopy (LM), whereas neither Transmission Electron Microscopy (TEM) nor Scanning Electron Microscopy (SEM) studies have been carried out. C. protothecoides var. acidicola seems to be morphologically identical to Auxenochlorella (=Chlorella) protothecoides; the only diacritical character for separating this variety is a pH limit for growth at 2.0 compared to 3.5-4.0 for A. protothecoides [11]. The third taxon is represented by "C." saccharophila, a species which has been recently excluded together with "C." protothecoides from the genus Chlorella [12], and which is well known as the lowest pHresistant "Chlorella" species [13]. Although the type strain, SAG 211-9a, and other strains of "C." saccharophila available from Culture Collections were isolated from non-acidic environments such as tree sap, they show the same morphological features and the same growth limits of pH, salinity and temperature as found in our isolates from acidic soils (unpublished results).
The main goal of this paper is to assess the phylogenetic relationships of our acid tolerant isolates with members of the genus Chlorella sensu lato, and to ascertain if the colonisation of acidic soils of geothermal environments is a monophyletic event. Moreover, molecular data could provide evidence to validate the establishment of V. fridericiana and C. protothecoides var. acidicola. To investigate these aspects, 18S rRNA sequences of authentic strains of V. fridericiana and C. protothecoides var. acidicola were determined. 18S sequences are routinely used to assess the phylogenetic position of coccoid green algae and an increasing body of sequences is becoming available [14][15][16]. The first attempt to use 18S sequences to infer phylogenetic relationships within Chlorella dates back to the beginning of the last decade [17], whereas a comprehensive data set of 18S rRNA sequences of Chlorella sensu lato has recently been presented, leading to a systematisation of the taxonomy of this genus [12].
When more than one isolate from low pH soils was available, as in the case of "C." saccharophila and V. fridericiana, the Internal Transcribed Spacer 1 (ITS1) was also sequenced to reveal molecular differences within a population that have not been observed with usual morphological and biochemical methods. Data from ITS1 sequences have been a valuable guide to identify different isolates of Volvocacean species [18], and also within Chlorella, ITS1 comparisons became a useful tool to assign strains at the species level, although not sufficient to resolve phylogenetic relationships among species [19].
Finally, in the case of C. protothecoides var. acidicola, the need for a combined analysis of molecular and cytological parameters prompted us to investigate the morphology and ultrastructure of this strain by LM, TEM and SEM.

Viridiella fridericiana and "Chlorella" saccharophila
Molecular and phylogenetic studies ITS1 lengths were 289 and 261 bp for "C." saccharophila SAG 211-9a and V. fridericiana 237, respectively, and the GC content ranged from 61 mol% for the former species to 59 mol% for the latter. We found no sequence divergence of ITS1 among the strains of "C." saccharophila isolated from acidic soils and the authentic strain of the species, as well as among those belonging to V. fridericiana. On the other hand, the identity of ITS1 sequences (Clustal W alignment) between "C." saccharophila and V. fridericiana was only 35,7%. This indicates that the two taxa are clearly separated; a similar distance is found between the ITS sequences of "C." saccharophila and "C." protothecoides var. acidicola.
The 18S rRNA tree in Fig. 1 confirms that V. fridericiana and "C." saccharophila are not closely related. Although  both algae belong to the same subgroup within the trebouxiophytes, a close relationship on the genus level is excluded. The long branch of Viridiella, and also of "C." luteoviridis, hints to a higher mutation rate of their ribosomal RNA compared to most other algae included in Fig.  1. Limited taxon sampling as an alternative explanation can be largely ruled out as we have included all available trebouxiophycean and several chlorophycean 18S rRNA gene sequences in a first search for related algae resulting in the same long branch leading to V. fridericiana. For sake of clarity, only those sequences which appeared significant for demonstrating the phylogenetic position of our isolates within the trebouxiophytes, were included in Fig.  1. Moreover, the 18S rRNA gene of Viridiella, in addition to base substitutions in variable regions, contains several mutations at sites that are otherwise highly conserved in most green algae, which is a typical character of fast evolving genes. It is well known that long branches may generate artificial tree topologies [20]. However, considering the available data, it is obvious that V. fridericiana represents a distinct lineage justifying the establishment of a new genus Viridiella by [9] also on a molecular basis. Watanabea reniformis (formerly designated as Chlorella saccharophila strain SAG 211-9b, but excluded from the genus Chlorella [21]) was found to be most closely related in the maximum likelihood analysis, but this relationship is not significantly supported by either bootstrap analysis. Table 1 summarises biochemical and ecophysiological characters of "C." protothecoides var. acidicola compared with those of "C." minutissima and Auxenochlorella protothecoides, two of the most closely related algae for which these data are available. "C." protothecoides var. acidicola shares numerous features with A. protothecoides, such as the unability to grow on nitrate, the upper limit of temperature for growth, and the tolerance to high concentrations of NaCl. As far as the need for thiamine is concerned, "C." protothecoides var. acidicola does not strictly require thiamine for growth, even though growth is promoted by the presence of this vitamin.

Light microscopy
In exponentially grown cultures, "C." protothecoides var. acidicola consists of single cells, spherical to oval, 4.5-6.7 µm in diameter, with a thin cell wall. No mucilage surrounds the cells which are filled with a single parietal chloroplast, bright green, cup shaped, and without pyrenoid. Colourless oily droplets may occur in the cytoplasm. The propagation mode is by autosporulation. Sporangia are 8-10 µm in diameter and contain 4 (rarely 8) autospores, spherical to oval, 3-4 µm in diameter, with a single cup shaped chloroplast, which are released through rupture of the mother cell wall (Fig. 2a). Residues of sporangial cell walls are visible and keep the autospores together for a certain time (not shown).
Electron microscopy SEM micrographs reveal a cell wall which is smooth or covered by an irregular network of subtle ribs (Figs. 2b,2c). Smooth and "rough" cell walls were present in samples of the same culture, and apparently, they can be observed in cells of similar size. TEM micrographs show that sporangia of "C." protothecoides var. acidicola may contain autospores of different size (Fig. 3a). The autospores have a central nucleus and a single parietal chloroplast lacking a pyrenoid, with some interthylacoidal starch grains. A mitochondrion is also visible near the concave side of the chloroplast. Moreover, TEM indicates that the cell wall is composed of an inner granulo-fibrillar polysaccharide layer and an outer trilaminar (TL) layer (Fig. 3b).
As reported for Auxenochlorella protothecoides [22], the external surface of the TL-layer after fixation with glutaraldehyde-OsO 4 is slightly undulated and the polysaccharide layer is not clearly visible. After fixation with glutaraldehyde-OsO 4 -tannic acid, a film of electron dense material external to the TL-layer is recognisable, probably due to a precipitation of tannic acid; moreover, the inner polysaccharide layer becomes more evident.

Phylogenetic position
In spite of the various features shared between "C." protothecoides var. acidicola and A. protothecoides (see above), molecular evidence suggests that both are not closely related. The phylogenetic tree in Fig. 1 places "C." protothecoides var. acidicola inside a cluster that contains two Nannochloris strains as well as "Chlorella" spec. Yanaqocha and Koliella spiculiformis. Our analyses show a weak tendency, although not significantly supported by bootstrap values, to affiliate "C." protothecoides var. acidicola with the latter two algae which are also known to colonize extreme environments. "C." spec. Yanaqocha is a UV-B tolerant alga isolated from the Andean lake Yanaqocha (3980 m above sea level; [23]), and Koliella species, although polyphyletic [24], have been isolated e.g. from the Himalayan Yala Glacier (5100-5700 m above sea level; [25]) and also from acidic waters [http://www.npsumava.cz/ gabreta/hejzlar.html]. Interestingly, a microalga isolated from the German Lake Lugteich (Lusatia, Saxony) and designated as "Chlorella" spec. Pi98/29 turned out to be very closely related to "C." protothecoides var. acidicola with just one difference in their 18S rRNA genes (Hepperle, pers. commun.). Lake Lugteich is a highly eutrophic and extremely acidic mining lake (pH 2.6) which was characterised with respect to its phytoplankton communities [7].

Discussion
The complete identity of ITS1 sequences of "Chlorella" saccharophila isolates from acidic soils with that of the authentic strain confirms the widespread distribution of this species and its ability to occupy extreme environments. Although it has been suggested that "C." saccharophila could be a component of littoral microflora [26], this species is mainly a "luftalga", occurring on bark of trees and bare rocks, and, less frequently, on soils of temperate regions [27]. However, this alga might be distributed worldwide: in 1982, a "C." saccharophila strain (labelled CCAP 211/57), isolated from Mt. Erebus soils, Antarctica, was deposited by P. Broady in the Culture Collection of Algae and Protozoa. There are no available molecular and cytological results confirming this attribution. Molecular studies have confirmed that "C." saccharophila does not belong to the group of "true" Chlorella, and that it is distant from "C." ellipsoidea [12] once considered a variety of "C." saccharophila [28,29]. Moreover, the erection of Watanabea reniformis has recently been proposed [21], which in- cludes strains of "C." saccharophila with a low sequence identity with the authentic strain CCAP 211/9a, and a reduced tolerance to low pH values and high NaCl concentrations.
The taxonomy of Chlorellaceae sensu Komarek & Fott [27] is largely based on chloroplast morphology, and at the generic level on the presence or absence of a pyrenoid. Recently, studies on 18S rRNA sequences of coccal green algae belonging to Selenastraceae have demonstrated the inconsistency of several "small" genera separated on the basis of a single-character difference such as presence or absence of a pyrenoid [16]. In the case of Viridiella, which is morphologically separated from "C." saccharophila only by the absence of a pyrenoid, the establishment of the genus seems to be well supported by molecular analyses (Fig. 1).
On the other hand, the comparative approach attempted to clarify the taxonomic position and the phylogenetic relationships of "C." protothecoides var. acidicola did not lead to unequivocal conclusions. From a morphological point of view, this organism showed a number of features similar to Auxenochlorella protothecoides ranging from shape and dimension of cells, lack of pyrenoid and absence of growth on nitrate. TEM observations, along with the confirmation of the absence of a pyrenoid, indicated the occurrence in the cell wall of a trilaminar layer, another characteristic shared with A. protothecoides. In contrast, the occurrence of autospores of different dimensions in the same sporangium is a feature observed in other "Chlorella" species such as "C." luteoviridis [30] and, most important, the genus Auxenochlorella is defined primarily on the strict need of thiamine for growth [22], which was not observed in our strain. Analyses of 18S rRNA gene sequences rule out a close relationship of "C." protothecoides var. acidicola with A. protothecoides and indicate that our isolate is related, although weakly, to other algae apparently not related from a morphological point of view (Fig. 1). "C". minutissima and Nannochloris are characterized by their small size of about 2 µm [12], whereas Koliella is represented either by two-celled filaments or by spindle shaped unicells, very similar to Raphidonema [24]. The availability of increasing information on phylogenetic relationships among coccoid green algae is evidencing the difficulties arising when a traditional system of classification is compared with molecular data. In unicellular organisms lacking a sexual life cycle, species and genera boundaries often remain uncertain, even after comprehensive data sets of morphological, biochemical, ecophysiological and molecular features have been collected, probably because limitations and the way how to use this multimethod approach are not clear. At the present time, with no consensual species concept in organisms in which sexual phenomena are not existent, more and more conflicting results are expected to be found.
It is evident from the phylogenetic tree in Fig. 1 that acid tolerant strains such as V. fridericiana, "C." saccharophila, and "C." protothecoides var. acidicola are distributed among strains that are less tolerant or even sensitive to conditions of high acidity or salinity such as "C." minutissima ( Table  1). This indicates that acquisition of acid tolerance per se in this group of algae is unlikely to be a monophyletic event. Rather, different strains have apparently adapted to the specific environmental conditions they were exposed to. Once the resistance to such conditions was genetically fixed, they might have been able to spread and colonise other extreme environments eventually giving rise to monophyletic "miniclusters" of extremotolerant taxa as represented by "C." protothecoides var. acidicola, "C." spec. Yanaqocha, and K. spiculiformis. An example for such a spreading is demonstrated by the occurrence of "C." protothecoides var. acidicola in volcanic sites in Italy and of the genetically closely related "Chlorella" spec. Pi98/29 isolated from an acidic mining lake in Germany (see Results). It has to be kept in mind, however, that the loss of a charac- teristic such as acid tolerance is at least as likely to occur as its acquisition thus obscuring monophyletic events in gene trees.
The number of algal species inhabiting low pH environments is generally considered to be very low [31]. Our results suggest that a comprehensive list of coccoid green algae occurring in low pH habitats is far from complete. Collecting algae from acidic soils is not simple, due to the variations of pH, light intensity, and ion concentrations which can remarkably change within few centimetres, allowing the establishment of different algal populations. Moreover, the identification of coccoid green microalgae through sampling and fixation can be misleading, because morphological characters are often not sufficient for a correct taxonomic determination. On the other hand, isolation and establishment of pure cultures is frequently unsuccessful. However, acidic soils could represent an unsuspected source of biological diversity, and the isolation and characterisation of acid and salt tolerant strains could open a promising avenue of research on their potential biotechnological utilisations.

Conclusions
The present study demonstrates that the taxonomic treatment proposed for Viridiella fridericiana seems to be well supported by molecular data. On the other hand, our results indicate that "C." protothecoides var. acidicola can not be incorporated into the genus Chlorella sensu stricto and can not be regarded as a variety of Auxenochlorella protothecoides as previously assumed from morphological comparisons. We suggest that this taxon should be treated as a distinct species to be included in a new genus. However, this treatment also needs reconsideration of the taxonomic status of other closely related microalgae, namely "C." spec. Yanaqocha and Koliella spiculiformis. Moreover, our data show that acquisition of acid tolerance was unlikely a monophyletic event in green microalgae. Several strains of different genetical equipment have apparently independently adapted to acidic environments making them interesting candidates for various biotechnological applications. Acidic soils of volcanic and geothermal sites from which our strains have been isolated may provide an unsuspected source of biological diversity for such extremely acid tolerant organisms.

Strains and culture conditions
The algal strains used in this study were: "Chlorella"

Ecophysiological and biochemical tests on C. protothecoides var. acidicola
The synthesis of secondary carotenoids under nitrogen deficiency was carried out according to Kessler et al. [32]. All tests were carried out in 100 ml Erlenmeyer flasks containing 50 ml of modified Allen medium [33] at pH 3.0, except in the different pH tests, at the same culture conditions as specified above. For the salt tolerance tests, the algae were grown at different concentrations of NaCl (2,4,6, and 8%). For the tests dealing with pH limits for growth, H 2 SO 4 was added at different concentrations in modified Allen medium to obtain final pH values of 1.5, 2.0, 2.5, 3.0, 5.0, 6.0 and 7.0. During the experiments, the pH value was daily monitored in each flask. For growth tests on nitrate as sole nitrogen source, algal cultures, previously centrifuged (5000 rpm × 10 min) and washed twice with nitrogen-free medium, were inoculated in Allen medium containing 3 mM NaNO 3 . In all experiments a control with C. protothecoides var. acidicola containing only modified BBM medium at pH 3.0 was also tested. After sterilisation by autoclaving, the flasks were inoculated with several drops of enrichment cultures in exponential growth phase to have a cell density of 0.003 units (corresponding to 100.000 cells/ml). Growth of each flask was followed daily by measuring the cell density as described above. Ecophysiological tests were carried out in triplicate for each strain and were repeated three times. Specific growth rates were calculated for each individual flask by linear regression of logarithmic cell density data obtained during the experiments. The results were evaluated on the basis of the average of three tests and the relative standard error was never higher than 5%.

Biomolecular studies
Cells of each Chlorella or Viridiella strain were harvested by centrifugation (6000 rpm × 5 min) and total DNA was extracted from liquid cultures following the procedure described in Doyle & Doyle [34]. 18S rRNA genes and ITS1 regions were amplified by PCR. For 18S, PCR conditions and sequencing primers were the same as previously described [12]; the ITS1 region was amplified using primers annealing with the 3' region of the 18S rRNA (5'-GGA-GAAGTCGTAACAAGGTTTCCG-3') and with the 5' region of the 5.8S rRNA (5'-ATCCTGCAATTCACACCAAG-TATCG-3'), respectively. PCR products were directly sequenced in an ABI Prism 310 Genetic Analyzer (Perkin-Elmer Cetus, Foster City, CA, U.S.A.). The 18S rRNA gene sequences of Chlorella protothecoides var. acidicola strain 124 and Viridiella fridericiana strain 237 were deposited in the EMBL database under the accession numbers AJ439399 and AJ439401, and the ITS1 sequences of "Chlorella" saccharophila SAG 211-9a and Viridiella fridericiana 237 under the accession numbers AJ439400 and AJ439402, respectively.

Phylogenetic analyses
For the analysis of ITS1 regions, sequences were reduced to only ITS1 by comparing them with the 3' terminus of 18S rRNA and of 5.8S rRNA of Chlorella species available from the literature [19]. ITS1 sequences were aligned using Clustal W ver. 1.6 [35]. 18S rRNA sequences were manually aligned on a MicroVAX computer with the sequence editor program distributed by G. Olsen [36]. External primer sequences and highly variable regions that could not be aligned unambiguously were excluded from the analyses resulting in a total of 1726 positions. Phylogenetic trees were inferred from the aligned sequence data by the neighbor-joining (NJ), the maximum parsimony (MP), and the maximum likelihood (ML) method. For all methods, heuristic bootstrap analyses [37] with 1000 (NJ, MP) or 100 replicates (ML) were conducted with the PAUP program package 4.0b8a [38] on a Power Macintosh G3 computer. For the NJ analysis, the HKY85 correction was used to convert pairwise sequence similarities into evolutionary distances, starting trees were obtained via neighbor-joining, and the TBR branch-swapping algorithm was selected. In the MP analysis, starting trees were obtained via random stepwise addition of taxa repeated 10 times, gaps were treated as "fifth base", and TBR was selected. In the ML bootstrap analysis, empirical base frequencies were used, the transition/transversion (ti/tv) ratio was set to 2, a gamma distribution of 0.5 was assumed for variable sites, and addition of taxa was by neighbor joining. In contrast, the tree topology shown in Fig. 1 was obtained by a ML analysis with ti/tv, base frequencies, proportion of invariable sites and gamma shape parameter estimated via ML, and with addition of taxa by the "as-is" mode.