A Polyphasic Characterisation of Tetradesmus almeriensis sp. nov. (Chlorophyta: Scenedesmaceae)

The microalga Tetradesmus almeriensis, previously known as Scenedesmus almeriensis, has been isolated and cultivated as a highly productive, fast-growing strain known as a natural source of different products of commercial interest, including bioactive compounds such as lutein. This strain produces up to 40 g·m−2·day−1 of lutein under optimal conditions and is highly recommendable for outdoor production in temperate and warm climates, showing maximal performance at temperatures up to 35 ◦C with no photo-inhibition taking place with irradiances greater than 1000 μE·m−2·s−1. Morphological and molecular data allow its assignment to the Chlorophycean genus Tetradesmus. The new species can be distinguished from similar Tetradesmus taxa due to its unique combination of features that are seen under light microscopy. We present herein a robust and comprehensive phylogenetic analysis of T. almeriensis, together with several additional Scenedesmaceae species, using a combination of maximum likelihood and Bayesian approaches. Our results confirm T. almeriensis as a distinct species consistently clustering with other Scenedesmaceae.


Introduction
The Scenedesmaceae family includes colonial coccal Chlorophyceae microalgae that form flat, curved, or three-dimensional coenobia with different cell shapes [1], and its most specious genus is Scenedesmus. Since this genus was first introduced by Meyen [2], a large number of taxa have been described morphologically [3], though their actual diversity and taxonomy are controversial [4] due to their large polymorphic, pleomorphic, and phenotypic variability [5]. The proliferation of numerous species, varieties, and forms can largely reflect morphological plasticity within the genus, which demands detailed investigations of the evolutionary diversity of the Scendesmaceae. Due to the large number of species contained in Scenedesmus, the genus was historically divided into subgenera and informal groups or subgroups [4,6,7].
Analysis of the small subunit (18S) of nuclear ribosomal RNA genes shows the phylogenetic position of Scenedesmus within the Chlorophyceae and its close relationship with coccoid green algae [8], but the sequence analysis of 18S rRNA suggests that the species of Scenedesmus sp. can be divided into at least two lineages corresponding to the subgenus Desmodesmus, and to another lineage containing the subgenera Scenedesmus and Acutudesmus [9,10]. Tsarenko and John [11] established taxonomic relationships between Acutodesmus and Desmodesmus, and they were later raised to the genus level.
Processes 2021, 9, 2006 2 of 11 Tetradesmus was originally described by Smith [12] based on the holotype T. wisconsinensis G.M. Smith. This species presents spindle-shaped, somewhat curved cells, convex in the centre with short cell tips, with a cosmopolite distribution in the plankton of oligotrophic freshwaters. Chodat [13] soon merged the genus into Scenedesmus, but West [14] maintained Tetradesmus as a distinct genus. Fott and Komárek [15] also recognised Tetradesmus and provided the first key to species and infraspecific taxa. Tetradesmus comprises ellipsoidal, spindle-shaped taxa with no or few longitudinal ridges, segregated from Scenedesmus s. str. by the presence of acute poles and no mucilage surrounding the coenobia, and unlike Desmodesmus, it lacks external spines [16,17]. Tetradesmus inhabits freshwater habitats, particularly in nutrient-rich conditions, constituting an important part of the biomass of green algae in many phytoplankton communities, and it is known for its high degree of phenologic plasticity [18]. By transferring Tetradesmus' holotype to Acutodesmus, Tsarenko and Petlevanny [19] synonymised both genera; however, the generic name Tetradesmus has nomenclatural priority over Acutodesmus, as noted by Wynne and Hallan [20], who combined existing Acutodesmus into Tetradesmus. It is noteworthy that in its actual sense, Tetradesmus is thought to be polyphyletic [16]. Currently, there are 21 validly published species names of Tetradesmus according to AlgaeBase, as well as three infraspecific names [21].
The name Scenedesmus almeriensis has been used informally in the literature since 2007 [22] to name a scenedesmoid species isolated from drying pools in greenhouses, and was first cultivated in the facilities of Las Palmerillas in La Mojonera (Almería, Southeast Spain) for industrial use. During the last decade, S. almeriensis has been commonly used in wastewater treatment studies, as well as in the production of compounds of commercial interest, such as biodiesel [23]. This name, which lacks a formal diagnosis and type designation, should be considered invalid according to International Code of Nomenclature (ICN) rules [3]. This work confirms this name under the most appropriate genus Tetradesmus, providing a differential diagnosis against closely related species through a morphological, phylogenetic, and autecological characterisation.

Microscopy Examination
Scenedesmus almeriensis strains were provided by the Department of Chemical Engineering at the University of Almeria, Spain. The sample used for the characterisation of the species belongs to a pure culture that came from an inoculate containing 10 mL of the sample, which was preserved in cold, dark conditions throughout the study. For the morphological characterisation, the sample was observed under light (LM), epifluorescence (EM), and scanning electron (SEM) microscopy. LM examination was carried out with a Leica DMR microscope (Leica, UK) equipped with differential interference contrast (DIC) and an Optika digital camera. For this purpose, a subsample was set to sedimentation in distilled water, previously fixed with 5% formaldehyde. Biometry and ultrastructural features were analysed using a total of 50 cells. For the study of the internal structure, a subsample was concentrated, fixed with albumin glycerol, and stained with safranin. The SEM examination was carried out with a high-resolution field emission scanning electron microscope (FESEM; Carl Zeiss MERLIN, Germany), with energy/wavelength dispersive X-ray (EDX/WDX) analytical capability. For this purpose, a subsample was previously fixed with 5% formaldehyde, dehydrated in an increasing ethanol/acetone dilution series, dried at the critical point, and then gold-metallised.

Sequence Data and Phylogenetic Analysis
We collected ITS (ITS1/ITS4) and rbcL-like homologous sequences from a set of 27 species representing the main lineages of the Scenedesmaceae family plus Chlorella vulgaris, belonging to the closely related Chlorellaceae family and conveniently used as the outgroup sequence in our analysis, by scanning the NIH genetic sequence data bank (GenBank) through BLAST using the ITS (Sequence ID MF977406) or rbcL (Sequence ID MG257492) Processes 2021, 9,2006 3 of 11 sequences from T. almeriensis, respectively, as queries (Supplementary Tables S1 and S2). Among the set of sequences obtained after the search, different selection criteria were taken into account. Firstly, the sequences used in previously published phylogenetic analyses were selected. In addition, it was considered that the chosen species was representative of the family, and, when possible, the most complete sequences were chosen.
Nucleotide sequence alignments were constructed using MUSCLE, with settings left at default [24]. Phylogenetic analyses were performed on the basis of the resulting multiple nucleotide sequence alignments using two alternative tree reconstruction methods: maximum likelihood (ML) and Bayesian analysis (BA). Prior to the phylogenetic analysis, the best fit nucleotide substitution model and the associated relevant parameters were inferred for each multiple sequence alignment using jModelTest v2.1 under the AIC test, and further considered to reconstruct ML phylogenies [25]. For rbcL sequences, the best fit selected model included +G, i.e., modelling heterogeneity in nucleotide substitution rates across positions in the alignment by means of a Gamma distribution with eight categories and an alpha shape parameter of 0.4810, +I, i.e., with the proportion of invariant sites fixed at 0.45. For ITS sequences, the best fit model returned associated parameters as +G, correcting heterogeneity [26] across substitution rates with a gamma distribution with eight categories and an alpha shape parameter of 0.8790, +F, i.e., estimating nucleotide substitution frequencies empirically from the alignment. ML trees were reconstructed by means of PhyMLv3.1 software [26], using the General Time Reversible (GTR) nucleotide substitution model and associated parameters returned for each alignment together with their best fit models. The search for the most likely tree was optimized by selecting "best of NNI & SPR" (nearest-neighbour interchange (NNI) and subtree pruning and regrafting (SPR)) [26], and statistical significance on the retrieved topology was assessed by means of a bootstrap analysis with 1000 replicates [26]. BA trees were obtained using MrBayes v3.2.5 [27], with the setting lset nst = mixed rates = gamma to sample across the GTR substitution model space in the Bayesian MCMC analysis, together with the parameters associated to the best fit models returned in each case by jModelTest v2.1. Searches of the tree space were performed through two independent runs with four Markov chains each, sampling every 100th tree during 1,000,000 generations until convergence between the two runs was reached, determined by the average standard deviation of split sequences below 0.05 (i.e., approaching 0), which reflects the independent tree samples becoming increasingly similar. Finally, the first 25% of the trees were discarded as burn-in, and a majority-rule consensus tree was then constructed to evaluate Bayesian posterior probabilities on the clades. The resulting trees were represented and edited using FigTree v1.4.2.
Synonym: "Scenedesmus almeriensis", nom. nud. Etymology: Named after Almeria, the region where the type locality is placed. The original strain was isolated in the Department of Chemical Engineering at the University of Almeria and patented by the University of Almeria and the Cajamar Foundation [28].
Description: Colonies of (2)-4-(8) cells, usually isolated under exponential growth conditions, as seen in other Tetradesmus taxa [29]; the individual cells of each coenobium are fusiform-cylindrical with small, polar, conoid-shaped protuberances ( Figure 2B); terminal cells equal to central cells ( Figure 1A). Cells coplanar, usually displaced vertically from each other ( Figure 1). External membrane with a smooth surface, somewhat coarse at high magnifications ( Figure 2B), without epistructures such as thorns or spines (Figure 2A) [30]. Under LM, the cytoplasm has a granular structure and cells exhibit a more or less central plastid. The nucleus is located in the centre of the cell and the pyrenoid starch layer stained blue reacts with the combination of albumin and Lugol's solution, turning bright red. The pyrenoid is conchoidal, homogeneous, and constituted by 2-3 large starch grains ( Figure 1B,C). Autospores, flagellate cells, and sexual reproduction were not observed.
Differential diagnosis: Morphologically, the new species can be placed in the genus Tetradesmus based on the criteria proposed by Krienitz and Bock [17], that is, the presence of ellipsoidal cells with acute poles, with no surrounding mucilage and no external spines. Within the group of Tetradesmus species with straight cells, T. almeriensis exhibits a unique combination of features. The cells in T. crocinii are smaller (up to 5.5 µm wide) but larger in T. cumbricus (11.0-13.5 µm wide) [15]. Mature cells of T. adustus are almost spherical in shape [29]. The similar T. bajacalifornicus and T. deserticola never constitute colonies, and these taxa show apical protrusions larger than those seen in T. almeriensis (compare Figure 2B with Lewis and Flechner [31]). Contrary to what occurs in T. incrassatulus, T. almeriensis never forms two-row colonies. Finally, the external cells in the colonies of T. obliquus are often lunate, a character that is not seen in the species described here. Available SEM images of T. obliquus (He et al. [32], Figure 4E) show that this taxon lacks the characteristic apical cone-shaped protuberances that are seen in T. almeriensis ( Figure 2B).
Synonym: "Scenedesmus almeriensis", nom. nud. Etymology: Named after Almeria, the region where the type locality is placed. The original strain was isolated in the Department of Chemical Engineering at the University of Almeria and patented by the University of Almeria and the Cajamar Foundation [28].
Description: Colonies of (2)-4-(8) cells, usually isolated under exponential growth conditions, as seen in other Tetradesmus taxa [29]; the individual cells of each coenobium are fusiform-cylindrical with small, polar, conoid-shaped protuberances ( Figure 2B); terminal cells equal to central cells ( Figure 1A). Cells coplanar, usually displaced vertically from each other (Figure 1). External membrane with a smooth surface, somewhat coarse at high magnifications ( Figure 2B), without epistructures such as thorns or spines ( Figure  2A)  [30]. Under LM, the cytoplasm has a granular structure and cells exhibit a more or less central plastid. The nucleus is located in the centre of the cell and the pyrenoid starch layer stained blue reacts with the combination of albumin and Lugol's solution, turning bright red. The pyrenoid is conchoidal, homogeneous, and constituted by 2-3 large starch grains ( Figure 1B,C). Autospores, flagellate cells, and sexual reproduction were not observed.
Differential diagnosis: Morphologically, the new species can be placed in the genus Tetradesmus based on the criteria proposed by Krienitz and Bock [17], that is, the presence of ellipsoidal cells with acute poles, with no surrounding mucilage and no external spines. Within the group of Tetradesmus species with straight cells, T. almeriensis exhibits a unique combination of features. The cells in T. crocinii are smaller (up to 5.5 µ m wide) but larger in T. cumbricus (11.0-13.5 µ m wide) [15]. Mature cells of T. adustus are almost spherical in shape [29]. The similar T. bajacalifornicus and T. deserticola never constitute colonies, and these taxa show apical protrusions larger than those seen in T. almeriensis (compare Figure  2B with Lewis and Flechner [31]). Contrary to what occurs in T. incrassatulus, T. almeriensis never forms two-row colonies. Finally, the external cells in the colonies of T. obliquus are often lunate, a character that is not seen in the species described here. Available SEM images of T. obliquus (He et al. [32], Figure 4E) show that this taxon lacks the characteristic apical cone-shaped protuberances that are seen in T. almeriensis ( Figure 2B).

Ecology
According to the literature that describes the past occurrences of T. almeriensis, this is a euryoic microalga that is able to grow under a wide range of temperatures (from 10 to 48 °C) and pH conditions (from 7.0 to 9.5) and shows strong tolerance to high Cu concentrations (up to 1 ppm) [28], which makes it especially suitable for external production [33], the most suitable growth conditions being a temperature of 30 °C and a pH of 8.0, with no vitamin addition. Regarding conductivity, it is considered to be a freshwater species [34], but it shows a high tolerance to medium concentrations of salt (0.1 M NaCl) [35]. Tetradesmus almeriensis shows a very unusual carotenoid content, especially lutein (up to 1% in d.w.) and β-carotene (0.75 mg/g) [32,36]; the starch and fatty acid content are also unusually high, with a yield of 16.7% (similar to that found in Neochloris oleoabundans) and 6% (higher than in Arthrospira platensis), respectively [33,[37][38][39].
Strains of T. almeriensis are distributed worldwide, but no natural occurrences have been reported to date outside the type locality. This Tetradesmus has been proven to be a common contaminant in industrial-scale microalgal cultures due to its ability to flourish under a wide range of environmental conditions [40].

Phylogenetic Analysis and Systematic Position
A preliminary analysis using ribosomal DNA sequencing and subsequent comparison with available similar sequences revealed that the original strain is related to other Scenedesmus/Desmodesmus species within the class Chlorophyceae. It deviates from the most closely related species such as S. hindakii or S. obtusus; however, both species are closely related in all published studies (e.g., [16] ). Subsequent sequence comparison analyses using rbcL sequences confirmed this preliminary analysis. Both sequences were

Ecology
According to the literature that describes the past occurrences of T. almeriensis, this is a euryoic microalga that is able to grow under a wide range of temperatures (from 10 to 48 • C) and pH conditions (from 7.0 to 9.5) and shows strong tolerance to high Cu concentrations (up to 1 ppm) [28], which makes it especially suitable for external production [33], the most suitable growth conditions being a temperature of 30 • C and a pH of 8.0, with no vitamin addition. Regarding conductivity, it is considered to be a freshwater species [34], but it shows a high tolerance to medium concentrations of salt (0.1 M NaCl) [35]. Tetradesmus almeriensis shows a very unusual carotenoid content, especially lutein (up to 1% in d.w.) and β-carotene (0.75 mg/g) [32,36]; the starch and fatty acid content are also unusually high, with a yield of 16.7% (similar to that found in Neochloris oleoabundans) and 6% (higher than in Arthrospira platensis), respectively [33,[37][38][39].
Strains of T. almeriensis are distributed worldwide, but no natural occurrences have been reported to date outside the type locality. This Tetradesmus has been proven to be a common contaminant in industrial-scale microalgal cultures due to its ability to flourish under a wide range of environmental conditions [40].

Phylogenetic Analysis and Systematic Position
A preliminary analysis using ribosomal DNA sequencing and subsequent comparison with available similar sequences revealed that the original strain is related to other Scenedesmus/Desmodesmus species within the class Chlorophyceae. It deviates from the most closely related species such as S. hindakii or S. obtusus; however, both species are closely related in all published studies (e.g., [16]). Subsequent sequence comparison analyses using rbcL sequences confirmed this preliminary analysis. Both sequences were deposited in GenBank under the following accession numbers: MF977406 (ITS1-5.8S-ITS2) and MG257492 (rbcL) [40].
In order to reconstruct the evolutionary relationships of T. almeriensis with its close relatives from the Scenedesmaceae family, we compiled two datasets of rbcL and ITS nucleotide sequences putatively homologous to T. almeriensis from 27 Scenedesmaceae species and performed independent ML and BA phylogenetic analyses. For both rbcL and ITS sequences, the ML and BA trees showed nearly identical topologies, with T. almeriensis forming a robustly supported clade together with S. obtusus and S. hindakii (shown in green, Figures 3 and 4), both belonging to the Scenedesmus group of species [41]. In rbcL-based trees, both ML and BA trees grouped the three-species clade including T. almeriensis together with an heterogeneous group integrated by Scenedesmus, Desmodesmus, and Tetradesmus taxa. However, according to the ITS trees, the species that are more closely related to the T. almeriensis cluster belong to the genus Tetradesmus, which evidences the close relationship of the species described here, together with S. obtusus and S. hindakii, with this genus. Notably, according to Hegewald and Wolf [16], S. arcuatus, morphologically identical to S. hindakii, clusters with high bootstrap support along with other Tetradesmus taxa.

Culture Conditions
According to Sánchez et al. [33], the lutein production in T. almeriensis may vary depending on environmental parameters such as pH, temperature, culture medium, irradiation, and salinity. Over the last few years, several media have been used to cultivate this species, such as Bioprocess [42], Mann and Myers' medium [43], and Hemmerick [44], which were tested with the original T. almeriensis strain under standard culture conditions (Iomax: 1100 µE·m −2 ·s −1 , T: 20 • C, pH: 8, [NaCl]: 0 ppm). Brindley et al. [45] modelised the light regime, thus allowing an optimal growth of T. almeriensis in photobioreactors, and they found that a culture at 33 • C in a homogenously illuminated tubular PBR under Io = 3000 µE·m −2 ·s −1 gives optimal productivity.
Continuous mode experiments showed that both Bioprocess and Mann and Myers' media are suitable for this strain, resulting in biomass productivities of up to 0.55 g·L −1 ·day −1 , decreasing to 0.26 g·L −1 ·day −1 using Hemmerick medium. Cultures can also grow photoautotrophically in bubble column photobioreactors, with the growing conditions that were tested being air bubbling at 0.5 vol −1 ·min −1 , circulating heat-treated water through the jacket to maintain the set temperature, and the controlling of pH conditions by injecting CO 2 into the inflowing airstream. In this case, the photobioreactors require artificial illumination to simulate a solar cycle, the irradiance on the reactor surface being controlled by an automated system to provide maximum irradiation ranging from 625 to 1625 µE·m −2 ·s −1 at noon. The biomass productivity obtained under such conditions reaches 0.56 g·L −1 ·day −1 [33]. In a solid medium, T. almerensis was cultivated successfully in nutritive agar [46] at pH 8 and 26 • C with an 18:6 photoperiod, which led to a productivity of 0.144 g·L −1 ·day −1 [23]. Finally, tris-acetate-phosphate (TAP), a common medium for freshwater green algae, was also used to cultivate this species in a liquid medium. Recently, the influence of culture conditions introduced into the performance of this strain has been studied [36]. Optimal conditions maximising the photosynthesis rate were found to be an irradiance of 200-500 µE·m −2 ·s −1 , temperature up to 35 • C, pH 8.0, and dissolved oxygen concentration below 20 mg·L −1 , with no photo-inhibition taking place with irradiances greater than 1000 µE·m −2 ·s −1 . According to these optimal conditions, this strain is highly recommendable for outdoor production in temperate and warm climates, with biomass productivities greater than 40 g·m −2 ·day −1 being reported in thin-layer reactors, including using wastewaters as the source of nutrients [47].
with this genus. Notably, according to Hegewald and Wolf [16], S. arcuatus, morphologically identical to S. hindakii, clusters with high bootstrap support along with other Tetradesmus taxa.  resulting from an analysis with 1000 replicates or Bayesian posterior probabilities for ML and BA trees, respectively. The tree is drawn to scale, with branch lengths proportional to evolutionary distances between nodes. The scale bar indicates the estimated number of nucleotide substitutions per site. Figure 4. Molecular phylogeny of ITS sequences from T. almeriensis and 27 related species. ML and BA trees depicting the evolutionary relationships of ITS sequences from T. almeriensis and 27 related species (left and right panels, respectively). Both trees were rooted using the Chlorella vulgaris orthologous sequence. Values next to each node indicate statistical support on clades, i.e., bootstrap support values resulting from an analysis with 1000 replicates or Bayesian posterior probabilities for ML and BA trees, respectively. The tree is drawn to scale, with branch lengths proportional to evolutionary distances between nodes. The scale bar indicates the estimated number of nucleotide substitutions per site.

Conclusions
Microalgae growing on photobioreactors are often neither monitored nor controlled in terms of species composition and community structure [48]. Traditionally, in biotechnological studies on novel scenedesmoid microalgae, most isolates are identified as Scenedesmus sp. based only on LM [18]. In this regard, molecular taxonomy is particularly important for groups of micro-organisms lacking evident and diversified ultrastructural features [28]. Moreover, the phylogenetic significance of these morphological characters in scenedesmoid taxa is poorly understood [49]. Therefore, the need to investigate not only the phenotypic but also the genotypic characters in a polyphasic approach has been clearly demonstrated. Hence, to the best of our knowledge, researchers all over the world are adopting such multidisciplinary studies whereby knowledge obtained from morphological features is integrated with molecular and ecological data to resolve taxonomic problems within and between scenedesmoid taxa. In this study, we demonstrated Tetradesmus almeriensis as a distinct species that contributes to completing a robust phylogenetic dataset on the Tetradesmus species for addressing important evolutionary questions within the Scenedesmaceae [29], as well as the biotechnological importance of these taxa in the modern applied industry.
Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/pr9112006/s1, Table S1: ITS sequences extracted from GenBank, NCBI, and used in this study; Table S2: rbcL sequences extracted from GenBank, NCBI, and used in this study.