Morphology, phylogeny and fatty acid profiles of Meyerella similis from freshwater ponds and Meyerella krienitzii sp. nov. from soil (Trebouxiophyceae, Chlorophyta)

The taxonomic diversity of the algal genus Meyerella is difficult to study because of its very simple morphology. Within the Chlorella-clade Meyerella members are distinguished from the others by the absence of the pyrenoid. However, it is not possible to identify them only on the basis of light microscopy data without the involvement of molecular genetic analysis methods. Some Meyerella representatives have high biotechnological potential, because they are able to accumulate valuable metabolites, including polyunsaturated fatty acids. As a rule, water bodies are the main habitats for these green microalgae. However, strains ACSSI 428 and ACSSI 429, which described in detail in this study, were isolated from peat cryozems (Sakha Republic, Russia). In this study, a detailed comparative analysis of the morphology, phylogeny and fatty acid profiles of these strains isolated from soil and representatives of other planktonic species, primarily Myerella similis, was carried out. Based on the results obtained, it was found that the studied strains are representatives of a new species with high biotechnological potential – Myerella krienitzii sp. nov.


Introduction
The genus Meyerella is considered unique within the Chlorella-clade because only its members lack the pyrenoid.This genus with the type species Meyerella planktonica was first described by Fawley et al. (2005) based on strains of planktonic microalgae from Lake Itasca (Minnesota, USA).After that, it was considered monotypic for quite a long time, although the results of analysis of individual 18S rRNA gene and internal transcribed spacers ITS1 and ITS2 indicated otherwise (Fučíková et al. 2014;Lanzoni et al. 2016).Since then another species Meyerella similis was described (Krivina et al. 2023).This species inhabits freshwater bodies and includes both free-living organisms and facultative ciliate endosymbionts.In general, the traditional habitat for Meyerella members was considered to be water bodies.Until recently, the only exception was the strain Meyerella sp.BCP-CNP1VF19, isolated from a soil biocrust of Virginia Park (Utah, USA) within the framework of The Biotic Crust Project.The identity of the genus Meyerella was determined by analysis of the 18S rRNA gene.However, it could not be identified as species (Fučíková et al. 2014).The Meyerella strains we found in tundra soils of the North of Yakutia (Russia) broaden our understanding of the lifestyle, ecology, and distribution of this genus.In this regard, the questions arise: are the identified soil inhabitants different from representatives of aquatic species and how does the habitat affect their morphology and metabolism?In addition to the search for morphological and molecular genetic differences, a comparative analysis of their biotechnological potential is of particular significance, especially their ability to produce fatty acids (FAs).It is well known that many representatives of the Chlorella-clade can accumulate these valuable metabolites, which are then used for the production of food additives, animal feed, agricultural fertilizers, as raw materials for the production of biofuels and soap, in cosmetics, and also wastewater treatment.Dietary polyunsaturated fatty acids (PUFAs) are considered extremely important in terms of therapeutic and physiological properties.However, the previous research concerned primarily members of the trebouxiophyte genera Chlorella and Micractinium (Mahboob et al. 2012;Abou-Shanab et al. 2014;Chae et al. 2021;Jahromi et al. 2022).The genus Meyerella is practically unstudied in this regard.To date, among the strains whose taxonomic status is not in doubt, studies of the fatty acid profile have been conducted only for the strain Meyerella sp.N4, which was isolated from a freshwater pond in India.The study showed that this strain could be considered as a source of palmitic and oleic acids and a promising candidate for biodiesel production (Karpagam et al. 2015).
This paper presents a detailed comparative analysis of the morphology, phylogeny, and fatty acid profiles of a new soil species of the genus Meyerella and other planktonic members, including M. similis.

Isolation and cultivation of algal strains
The two strains were isolated from peat cryozems, Yedoma near Panteleikha River, Pleistocene Park, Sakha Republic, Russia (N 68.511° E 161.495°), in 2020.The study area belongs to the northern taiga zone and is characterized by continental climate with mean annual air temperature -10.3 °C and mean annual precipitation 236 mm (according to meteorological records of the Cherskiy Weather Station).The active seasonally thawing layer thickness varied from 30 to 50 cm and was underlain by continuous permafrost.The study area is located on a drained, slightly convex slope occupied by sparse larch (Larix cajanderi Mayr.) forest with a predominance of dead-cover parcels (larch needle litter) and partially moss-lichen ground cover.Samples of soil, including peat and litter, were taken from the upper 6-cm layer of peat cryozem located above a 30 cm deep interpolygonal cryogenic crack.The material was collected in the period of maximal seasonal thawing (in September).The samples were placed in hermetic sterile bags, kept and transported at low positive temperatures (2-4 °C) and field moisture.A small amount of the soil samples was re-wetted with sterile milliQ water and plated in Petri dishes with solid BG-11 medium (1% agar) (Rippka et al. 1979).The cultures were grown at 23-25 °C with a 12:12 h light: dark (L:D) cycle, at a light intensity of 60-75 μmol photons m -2 s -1 provided by cool white fluorescent lamps.Both strains were deposited at the Algal Collection of the Soil Science Institute (ACSSI, Pushchino, Russia) under numbers ACSSI 428 and ACSSI 429.

Microscopy
The morphology and life cycle of the strains were studied using light microscopy with Leica DM750 and Carl Zeiss Axio Scope A1 microscopes (Germany) in the Collective Use Center, Institute of Physicochemical and Biological Problems in Soil Science, Russian Academy of Sciences.The results were documented using photographs obtained with color digital cameras Leica Flexacam C3 (Germany) and Carl Zeiss MRc 5 (Germany).The observations of the strains were performed for 2 weeks to 6 months.Leica Application Suite X software was used for morphometric measurements of algae.One hundred cells of the strain were measured for size comparison.
Phylogenetic analyses were performed on a concatenated dataset of the 18S, ITS1, 5.8S, and ITS2 sequences.All of the sequences were searched using the BLASTn algorithm in GenBank (https:// blast.ncbi.nlm.nih.gov) and selected based on the criteria of maximum affinity (similarity ≥ 95%), reading quality (without degenerate and unknown nucleotides), reading length (sequences including 18S-ITS1-5.8S-ITS2 and measuring at least 2300 bp), and belonging to the type species and collection authentic strains.Thus, the dataset contained 77 sequences of representatives of Trebouxiophyceae using Parachlorella-clade as the outgroup.If there were introns in the 18S rRNA gene, they were removed from the alignment.Taxon names are listed according to AlgaeBase (Guiry and Guiry 2023).To compare the topology of trees, we used data from articles by Krienitz et al. (2004), Luo et al. (2006), Hoshina et al. (2010, 2017, 2021), Pröschold et al. (2010Pröschold et al. ( , 2011Pröschold et al. ( , 2020Pröschold et al. ( , 2021)), Bock et al. (2010Bock et al. ( , 2011)), Hoshina and Fujiwara (2013), Hoshina and Nakada (2018), Pröschold and Darienko (2020), Krivina et al. (2023).Multiple alignment was performed in BioEdit 7.2.5 using the ClustalW algorithm (Hall 1999).Based on the AIC in jModelTest (Darriba et al. 2012), the GTR + I + G nucleotide substitution model was selected as the optimal model for ML and BI.ML was performed using PhyML (Guindon et al. 2010) with 1000 bootstrap replicates.BI was performed using BEAST v. 1.8.4 (Drummond et al. 2012) with 100,000,000 generations of Markov chain Monte Carlo iterations, and parameters were saved every 10,000 th tree, discarding the first 25% as burn-in.The calculation of genetic distances was performed in the MEGA 6.0 program (Tamura et al. 2013).When calculating the genetic distances for ITS1-5.8S-ITS2fragment, the alignment was carried out taking into account the secondary structure of ITS1 and ITS2.Only representatives of the genus Meyerella were included in the matrix for alignment to exclude distortions.
Folding of ITS1 and ITS2 was performed using the RNAfold web server (http:// rna.tbi.univie.ac.at/ cgi-bin/ RNAWe bSuite/ RNAfo ld.cgi) in accordance with the principle of minimum energy.When assessing the correctness of the prediction of the secondary structure, ITS1 and ITS2 were guided by Coleman (2015) and Caisová et al. (2013), respectively.The comparison of the secondary structure of ITS1 and ITS2 between strains, the search for conservative motives, and compensatory base changes (CBCs) was carried out in the 4SALE program (Seibel et al. 2006).In the analysis of ITS2 for the species distinguishing, special attention is paid to the approach of sensu Coleman (2009Coleman ( , 2015Coleman ( , 2000Coleman ( , 2007)), according to which the presence of even one CBC in conservative regions of ITS2 (5 bp of helix I, 10 bp of helix II, all helix III) in two microalgae correlates with their sexual incompatibility.The secondary structures of spacers are visualized in the PseudoViewer3 program (https:// mybio softw are.com/ pseud oview er-3-0-visua lizat ionrna-pseud oknots-type.html) (Byun and Han 2009).When analyzing genetic distances of ITS1, the ITS1 nucleotide sequences were aligned taking into account the secondary structure in the 4SALE program.After that the genetic distances were calculated in the MEGA 6.0 program (using the Kimura 2-parameter model).The analysis of the genetic differences of ITS2 was carried out by 2 methods.According to the first method, the nucleotide sequences of ITS2 were aligned taking into account the secondary structure in the 4SALE program.Then the genetic distances were calculated in the MEGA 6.0 program (using the Kimura 2-parameter model) (classical method).As the second method, the method proposed by Hoshina and Fujiwara (2013) was used, when gaps counted as the fifth character.

Fatty acid composition analysis
For fatty acid analysis, the modified Bligh and Dyer (1959) method was used.Frozen algae samples were homogenized in 1 mL chloroform/methanol (1:2) solution, using a glass bead-beating procedure (the samples were mixed with glass beads (diameter 1 mm) (Merck, Germany) and vortexed for 1 min), and centrifuged at 3000 rpm for 5 min.The supernatant was transferred to a glass vial and 1 mL chloroform/methanol (1:2) solution and 270 µL of 1% KCl were added to the pellet, vortexed again with glass beads, and centrifuged at 3000 rpm for 5 min.The supernatant was also transferred to the glass vial, and 670 µL of chloroform and 0.4 mL of 1% KCl were added to the collected supernatant, vortexed well, and centrifuged at 3000 rpm for 5 min.The lower phase was transferred to a glass tube and dried under a nitrogen stream.Fatty acid methyl esters (FAMEs) were prepared by the addition of 1 mL of 8% (w/v) sulfuric acid in methanol and incubation at 90 °C for 90 min.One mL of l0% sodium chloride in water and 250 µL of hexane were added to the vial, and in 10 min upper phase containing methyl esters was transferred to a vial for gas chromatography-mass spectrometry (GC-MS) analysis.FAMEs were analyzed by GC-MS using a Chromatec-Crystal 5000 gas chromatograph equipped with a Chromatec mass selective detector operating in electron ionization mode.A glass capillary column CR-5 ms (Chromatec) (30 m*0.25 mm, coated with 0.25 µm 5%-phenyl-95%-dimethylpolysiloxane) was used as a separation column.The column temperature was programmed as follows: held at 40 °C for 1 min, then to 150 °C at the rate of 15 °C per min, to 245 °C at the rate of 10 °C min, and held at 245 °C for 15 min.The injector and detector temperatures were set at 250 °C.One µL of the sample has been injected in splitless mode.Helium was used as carrier gas at a flow rate of 0.5 mL min −1 .Data were acquired in the electron impact mode (70 eV), scanning from 10 to 500 mass units at 0.1 s per scan.Identification of the peaks from total ion chromatograms was carried out through Wiley Registry of Mass Spectral Data, 11 th Edition search, and confirmed by comparison with the fatty acid standard (Supelco 37 component FAME mix, Sigma Aldrich, USA).The areas of the peaks were established by a computerized integration.FA unsaturation Ind ex es were calculated as described earlier (Sidorov et al. 2014).

Comparative analyses of the V4 and V9 regions of the 18S rRNA gene
The V4 region length of studied strains ACSSI 428 and ACSSI 429 was 375 nucleotides as in all Meyerella members.Our strains also had the molecular signature of the genus Meyerella (AA in 152-153 bp, A in 190 bp, T in 242 bp) (Fig. 3), which distinguishes this genus from others within the Chlorella-clade (Krivina et al. 2023).The studied ACSSI strains, unlike other Meyerella strains, had a specific signature -CC (88-89 bp), G (205 bp).The V9 region length of all studied Meyerella members was 106 bp.Within this region, a unique combination was found for ACSSI strains, distinguishing them from other Meyerella strains, -(G in 41 bp, G in 46 bp, C in 62 bp, C in 66 bp, G in 74 bp) (Fig. 3).It should be noted that the molecular signatures in V4 and V9 regions, previously proposed for M. similis, were still unique.
The ITS1 secondary structures of strains ACSSI 428 and ACSSI 429 corresponded to the generally accepted model of eukaryotic organisms: four unbranched helices, helices I − III were located next to each other, helix IV was separated from the rest by unpaired nucleotides, there was a single-stranded A-rich region adjacent to the 5.8S rRNA gene (Coleman 2007(Coleman , 2015)).The ITS1 lengths of the strain ACSSI 428 and ACSSI 429 were 273 nt.The ITS1 lengths of M. planktonica and M. similis strains were shorter (259 nt and 238 nt, respectively).One CBC was found between the studied strains and M. planktonica strains, and four CBCs -between the strains ACSSI 428, ACSSI 429, and M. similis strains (Fig. 4).In addition, all M. similis strains are distinguished by the structure of helix I from other Meyerella members.They had a short helix I (only 12 nt), which is the smallest in genus Meyerella and all Chlorella-clade.Also, in the present study, the "molecular signature" of M. similis strains was confirmed (Krivina et al. 2023).Combination A-U in 4 bp of helix I remains unique within the genus Meyerella.The genetic differences between the studied strains were 0%.Genetic distances (with secondary structure) between studied strains and M. planktonica -15.8%, our strains and M. similis -16.9%,M. planktonica and M. similis -22.6%.
The ITS2 secondary structures of studied ACSSI strains under consideration had common features specific for green microalgae (Chlorophyta): four unbranched helices, a pyrimidine-pyrimidine mismatch of the helix II, and conservative motif GGU AGG on the 5′-side of helix III (Coleman 2009(Coleman , 2015)).The ITS2 length of ACSSI 428 and ACSSI 429 were 242 nt, whereas ITS2 length of M. planktonica was 242 nt, M. similis -252 nt.Four CBCs were revealed in ITS2 secondary structure of strains ACSSI 428 and ACSSI 429 compared to M. planktonica, and three CBCs -compared to M. similis (Fig. 5).Furthermore, they were all located in conservative ITS2 regions.The classic analysis of ITS2 aligned sequences genetic distances (with secondary structure) showed that the genetic differences between our strain and strains of other sister species are-20.3-20.4%, between M. planktonica and M. similis -7.9%.Analysis by the Hoshina et al. method showed that the level of the differences between the studied ACSSI strain and other Meyerella members are 24.9-25.8%,between M. planktonica and M. similis -14.39%.
Ecology Both studied ACSSI strains were isolated from cryogenic crack soil.At the same time, at the time of sampling, the habitat conditions in this biotope could be characterized as humid (100% and higher) and relatively cool (7.2-11.1 °C).All representatives of the related species were inhabitants of freshwater lakes (Table 1).At the same time, members of M. planctonica were only free-living organisms, whereas M. similis representatives were free-living planktonic organisms or endosymbionts of ciliates.

Fatty acid composition
Results of the GC-MS analysis of fatty acid composition are shown in Fig. 6.The fatty acid profiles of both species are mainly represented by 16-and 18-carbon chain compounds, which make up more than 90% of total fatty acids.The most abundant fatty acid in both species is linoleic acid.The relative content of linoleic acid is higher in ACSSI 428, while hexadecatrienoic and octadecenoic acids are less abundant in this alga than in M. similis ACSSI 346.The unsaturation index of ACSSI 428 fatty acids is equal to 1.9, M. similis ACSSI 346-1.8.Several minor fatty acids were detected in the samples of both studied algae.Among them tetradecanoic acid, 2'-hexyl-[1,1'-Bicyclopropyl]-2-octanoic acid, and several branched-chain fatty acids, such as 16-methylheptadecanoic, 14-methylhexadecanoic, and 12-methyltetradecanoic acids found in both species.Odd chain pentadecanoic and heptadecanoic acids were found in M. similis ACSSI 346, while 15-methylhexadecanoic and 17-methyloctadecanoic acids -in ACSSI 428.

Discussion
Representatives of the Chlorella clade in general, and the genus Meyerella in particular, are characterized by an extremely simple morphology.As mentioned above, a distinctive feature of the genus Meyerella is the absence of a pyrenoid.To date, the pyrenoid has not been detected in any of the strains belonging to the genus Meyerella, which belonging to this genus was confirmed by the results of molecular genetic analysis (Fawley et al. 2005;Fučíková et al. 2014;Karpagam et al. 2015;Lanzoni et al. 2016; ) and other similar 'little green balls' that also lack the pyrenoid.Therefore, it is very difficult to determine whether strains belong to the genus Meyerella based on the light microscopy data alone, without the use of molecular genetic analysis.The morphology of the studied strains ACSSI 428 and ACSSI 429 was typical of Meyerella members despite the atypical soil habitat (Table 1).Although we would most likely expect Edaphochloris algae in this habitat.Their morphological characteristics were closer to M. planktonica strains.Representatives of M. planktonica and studied ACSSI strains had spherical-shaped cells, but ACSSI strains had broad-shaped and oval cells in addition to globular cells, rather than cylindrical with rounded cell shape ends as of M. planktonica.Cell sizes of our strains and M. planktonica were similar, although the range of size characteristics was slightly wider in ACSSI strains.In addition, a cup-shaped chloroplast was found in the spherical cells of ACSSI 428 and ACSSI 429.The studied ACSSI strains differed from M. similis in cell shape (the oval shape of the cells was not typical for M. similis), size (adult cells of ACSSI 428 and ACSSI 429 could be almost twice larger), and chloroplast type (ACSSI 428 and ACSSI 429 sometimes had mostly lateral in young cells and trough-shaped in adult cells).Phylogenetic analysis of the 18S-ITS1-5.8S-ITS2fragment showed that the studied strains ACSSI 428 and ACSSI 429 formed an independent phylogenetic lineage within the genus Meyerella.The genetic distances between strains ACSSI 428 and ACSSI 429 were less than 0.1% and  (Krivina and Temraleeva 2020;Krivina et al. 2023).The genetic difference between the studied soil ACSSI strains and aquatic Meyerella species (3.1-3.4%) was significantly higher than between the actual aquatic species (2.4%) and corresponded to the interspecific level.So, the genus Meyerella is characterized by the high interspecific genetic distances within the Chlorella-clade.For example, interspecific genetic differences within the genus Micractinium varied from 0.4% to 4.4%, within the genus Hindakia -0.6-0.9%,genus Heynigia -1.7%, genus Didymogenes -1.4-1.7%.The genetic distances between the studied strains and other Meyerella members, calculated separately for the 18S rRNA gene, also corresponded to the interspecific level within Chlorellaclade (0.6%).Although they were somewhat lower than between M. planktonica and M. similis (0.7%).For comparison, in the genus Micractinium, the level of genetic differences between representatives of different species varies from 0% to 1.7%.Although the 18S rRNA gene, which is considered the main phylogenetic marker for green microalgae, the use of this gene alone in phylogenetic analysis of members of the Chlorella-clade does not allow reliable distinction between species and genera Usually only two genera (Hegewaldia and Meyerella) are clustered in such an analysis into independent groups with high statistical support whereas members of other genera are chaotically arranged (Heeg and Wolf 2015;Krivina and Temraleeva 2020).This is also consistent with our results (Online Resource 1).This is why most work uses 18S-ITS1-5.8S-ITS2fragment which includes a combination of conserved genes and more variable spacers (Bock et al. 2010(Bock et al. , 2011;;Hoshina et al. 2010Hoshina et al. , 2017Hoshina et al. , 2021;;Pröschold et al. 2010Pröschold et al. , 2011Pröschold et al. , 2020Pröschold et al. , 2021;;Hoshina and Fujiwara 2013;Heeg and Wolf 2015;Hoshina and Nakada 2018;Krivina and Temraleeva 2020;Pröschold and Darienko 2020;Krivina et al. 2023).Additional analysis of a more variable fragment ITS1-5.8S-ITS2also confirmed the interspecific level of genetic differences between our strains and other representatives of the genus Meyrella (13.9-14.5%).For example, among members of the genus Micractinium, genetic distances vary from 2.4% to 14.6%.
Only representatives of M. similis had one group I intron (416 bp, S1367) compared with other genus members, including ACSSI 428 and ACSSI 429.This difference can be considered as one of the additional characteristics confirming the independent species status of M. similis and, consequently, the difference between our soil strains and strains of this species (Vorobyev et al. 2009;Hoshina et al. 2010;Gaonkar et al. 2018;Spanner et al. 2020;Pröschold et al. 2021;Krivina et al. 2023).
Regions V4 and V9 of gene 18S rRNA are frequently used as DNA barcodes for the study of eukaryotic microorganisms diversity in the environment using high-throughput sequencing technology (Sogin et al. 2006;Decelle et al. 2014;Sverdrup and Frolova 2020).Although these regions are among the most variable regions of the 18S rRNA gene, they are highly conserved in Chlorella-clade members.The proportion of conserved sites in region V4 was 89% and in region V9 -83%.From the above, the specific signatures specific to the genus or species found in these regions become even more significant.The strains ACSSI 428 and ACSSI 429 examined had the same molecular signature in the V4 region as the other Meyerella members.Also in the regions V4 and V9 stable motifs were found characteristic only of representatives of ACSSI strains.In our opinion, these molecular signatures can be used to identify ACSSI The analysis of the internal transcribed spacers ITS1 and ITS2 also confirmed that the studied ACSSI strains belong to new species.Thus, one CBC in ITS1 and four CBC in conservative ITS2 regions were found between strains ACSSI 428, ACSSI 429, and M. planktonica strains, four CBC -in ITS1, and three CBC -in conservative ITS2 regions between strains ACSSI 428, ACSSI 429, and M. similis strains.According to Coleman (2000), this indicates the independent species status of the studied soil strains compared to aquatic species.The genetic differences of ITS1 (with secondary structure) between the studied strains and representatives of the genus Meyrella were high (15.8-16.9%),although somewhat lower than between M. planktonica and M. similis (22.6%).However, within the Chlorella-clade, this is unquestionably an interspecific level.By comparison, interspecific genetic distances of the genus Hindakia were 1.6-3.7%, of the genus Heynigia -11.8%, of the genus Neochlorella -15.1%.This is consistent with the results of the ITS2 genetic distance analysiswhici was performed using both the classical algorithm and the method by Hoshina and Fujiwara (2013).In both cases, the level of genetic differences between soil strains ACSSI 428, ACSSI 429, and aquatic species of Meyerella was significantly higher than between taxonomically recognized M. planktonica and M. similis.In addition, they significantly higher than minimum species threshold (2%) proposed by Hoshina and Fujiwara (2013).
Among other things, differences in habitat and lifestyle were identified.As already mentioned, the studied strains ACSSI 428 and ACSSI 429 were free-living organisms that were isolated from soil of the cryogenic crack, with high humidity, but without stagnant water.The studied area is located on the yedoma surface, which is never flooded with water and has no direct connection with freshwater objects -rivers and lakes.However, in our opinion, it is early to state that these strains are true soil organisms.It must be borne in mind that the area is characterised by high soil moisture.The possibility of airborne transport of freshwater algae also can not be ruled out.Members of M. planktonica and M. similis were found only in freshwater lakes and reservoirs (Fawley et al. 2005;Sverdrup and Frolova 2020;Krivina et al. 2023).Moreover, some representatives of M. similis strains were facultative endosymbionts and were found both in symbiotic associations with Pseudoblepharisma and Holophrya ciliates (Krivina et al. 2023).The soil is not a typical habitat not for the genus Meyerella, nor for the Chlorellaclade as a whole (Fawley et al. 2005;Fučíková et al. 2014;Lanzoni et al. 2016;Hoshina and Nakada 2018;Krivina and Temraleeva 2020;Pröschold and Darienko 2020).To date, only one Meyerella strain was found outside of the aquatic habitat.This strain was Meyerella sp.BCP-CNP1VF19 isolated from a soil bio crust (Fučíková et al. 2014).Within the Chlorella-clade, soil inhabitants are represented by only Lobosphaeropsis lobophora (Andreeva 1998), C. lewinii, and C. volutis.However, in the last two cases, the strains were isolated from the soil near lakes (Bock et al. 2011).In addition, it should be noted that the studied strains grow in a cool environment, and M. planktonica was also isolated from winter and spring phytoplankton.Representatives of M. similis were isolated from the summer phytoplankton of small well-heated reservoirs when the water temperature reached + 23 °C.
GC-MS analysis did not reveal significant qualitative differences between fatty acid profiles of studied algae (Fig. 6).While the amount of polyunsaturated fatty acids is similar in both species, the fatty acids with two double bonds, especially linolenic acid, prevail in studied strain ACSSI 428, and the Unsaturation Index was also slightly higher in this alga.Other fatty acids detected in the samples, particularly branched-chain fatty acids, were present in small amounts, but they could have a significant effect on the alga physiology and metabolism.Most of the detected compounds of this group were iso-methyl branched fatty acids, which had the branch point on the penultimate carbon (ω-1).The total content of branched fatty acids was slightly higher in M. similis ACSSI 346 (about 3% of total fatty acid content in comparison to 2.6% in ACSSI 428, but their diversity was higher in ACSSI 428 (5 different branched-chain fatty acids in comparison to 3 in M. similis ACSSI 346).Methyl branching tends to decrease the thickness of lipid bilayers and lower chain ordering, which modifies the membrane fluidity and facilitates adaptation to changing environmental conditions (Poger et al. 2014).Information about the fatty acid profile of other members of the genus Meyerella was very limited.So the strain Meyerella sp.N4, which was a member of a new invalid species, was characterised by a significantly higher content of saturated (~ 28%) and monounsaturated fatty acids (~ 46%) than the strains examined above (Karpagam et al. 2015).All of these Meyerella members had significant biotechnological potential.For example, they may be in demand as a raw material for the production of biodiesel.In biodiesel production, the selection of target strains depends on which biodiesel parameters are prioritised.If increasing energy yield and oxidative stability are the primary objectives, strains high in saturated and monounsaturated fatty acids, as in Meyerella sp.N4, are preferred.However, the main disadvantage of such products is their tendency to solidify at low temperatures, which requires additional adjustment.In contrast, products derived from strains high in polyunsaturated fatty acids, such as strains ACSSI 428 and M. similis ACSSI 346, have very good cold flow properties, but biodiesels with these properties are more vulnerable to oxidation (Musharraf et al. 2012).At the same time, strains ACSSI 428 and M. similis ACSSI 346 could be used as a valuable source of omega-6 and omega-3 polyunsaturated fatty acids.These acids are actively used in the production of dietary supplements for the prevention of cardiovascular diseases.In addition, they have found wide application in the cosmetic products industry, as they have anti-inflammatory and moisturizing properties (Ando et al. 1998;Letawe et al. 1998;Pan et al. 2012;Dąbrowski and Konopka 2022).
Thus, the comparison of morphological characteristics, habitat and lifestyle, analysis of tree topology, genetic distances, and secondary structures of spacers ITS1 and ITS2, and fatty acids analysis allowed us to establish that the studied strains ACSSI 428 and ACSSI 429 are representatives of new Meyerella species with high biotechnological potential.

Etymology
This species is named in honor of Dr. Lothar Krienitz (Germany), who made a great contribution to the study of green coccoid microalgae.GENBANK ACCESSION: ACSSI 428 (OQ650228).

Fig. 1
Fig. 1 Morphology of Meyerella krienitzii sp.nov.strains ACSSI 428 (A) and ACSSI 429 (B).Sporangia are represented on the inserts.The scale on the inserts coincides with the scale in the main figures.Scale bar: 10 μm

Fig. 2
Fig. 2 Bayesian phylogenetic tree of Chlorella-clade (Trebouxiophyceae, Chlorophyta) based on the comparison of the nucleotide sequences of the 18S-ITS1-5.8S-ITS2region (2574 bp).The support values are given for Bayesian inference and maximum likelihood (PP/BP).The cut-off values for BI and ML are 0.7 and 70%, respectively.The model

Table 1
Comparison of studied strains Meyerella krienitzii sp.nov.ACSSI 428 and ACSSI 429 with other Meyerella species Colony Mucilage Shape of young cells Shape of adult cells Adult cell size, μm Chloroplast