## 3 Results and Discussion  
### Identification  
Microscopic observations of _Dunaliella_ sp. TAV01 cells revealed spherical to pear-shaped cells with flagella (Figure 1) and the presence of carotenoid pigments (Figure 1A,B). The identification based on 188 rDNA did not provide sufficient information to differentiate this isolate from other _Dunaliella_ species (results not shown). Therefore, for identification down to the species level, phylogenetic analysis using sequences of the ITS1-5.8S-ITS2 rDNA region were used. Upon Maximum Likelihood (ML) and Bayesian phylogenetic analyses, congruent trees were obtained (Figure 2). TAV01 ITS1-5.8S-ITS2 nucleotide sequence clustered together with those of _Dunaliella viridis_, _D. polymorpha_ and _D. parva_ with an ML branch support value of 0.96 and maximum Bayesian posterior probability (PP) support (100%). This cluster was named as \"Clade Viridis\".  
Figure 1: Microscopic images of Dunaliella sp. TAV01. (**A**) The differential interference contrast (DIC) on bright field. (**B**) Cell excitation on natural fluorescence on Ex 470/40; Em 525/50. (**C**) The same picture as in **B** but using differential interference contrast (DIC).  
The remaining sequences clustered into two additional clades, namely \"Clade Primoleta\", containing sequences of \(D\). _primoleta, D. bioculata, D. quartolecta, D. polymorpha_ and \(D\). _salina_; and \"Clade Salina\", containing several \(D\). _salina_ sequences and one sequence of \(D\). _pseudosalina_ (MZ079594). As with clade Viridis, clades Primoleta and Salina displayed high to maximum branch support values (ML \(>\) 0.94/PP = 100%). A few sequences did not fall into of these three clades, namely that of \(D\). _acidophila_ and a sequence classified as belonging to \(D\). _pseudosalina_ (KU641615). As previously described by other authors [(43)], classifying a novel _Dunaliella_ strain down to the species level is notoriously difficult. However, our results do support the idea that the genus _Dunaliella_ has been subdivided into more species than the molecular data suggests [(24)]. This has caused confusion and mismaming of strains and species, which may explain why sequences classified as \(D\). _salina_ and \(D\). _pseudosalina_ appear in different branches of the consensus tree (Figure 2). The division of the genus _Dunaliella_ into three major clades as suggested here is further supported by recent phylogenetic data [(43; 44; 24)]. Thus, it is here proposed that not only TAV01 should be classified as \(D\). _viridis_, but also the strains previously classified as \(D\). _polymorpha_ (MN167111 and KJ756825) should be reclassified as \(D\). _viridis_. Such a conclusion seems to be supported by the fact that internal branches within the clade Viridis have lower support except the branch containing \(D\). _parva_ sequences (ML = 0.97/PP = 100%). Moreover, the secondary structure modelling of TAV01 ITS2 showed a 99% similarity with \(D\). _viridis_ ITS2 structure (Figure 3), again strongly suggesting that TAV01 should be classified as \(D\). _viridis_. Indeed, the inclusion of RNA secondary structures is known to improve the accuracy and robustness of strain assignment to specific taxa using phylogenetics [(25)].  
Figure 2: Maximum likelihood (ML) and Bayesian phylogenetic consensus tree where the TAV01 appears in a cluster together with Dunaliella viridis, D. polymorpha and D. parva ITS1-5.8S-ITS2 sequences. The latter sequence cluster was named as “Clade Viridis”. Other sequences clustered together into two major clades: “Clade Salina” and “Clade Primoleta”, which seem to be sister clades of each other. ML and Bayesian branch support values are given on the left (\(\geq\)0.43) and on the right (\(\geq\)63%), respectively. Lower support values were omitted.