Xudong LIU , Ran ZHANG , Jia FENG , Fabio RINDI , Shulian XIE ,**

1 School of Life Science, Shanxi Key Laboratory for Research and Development of Regional Plants, Shanxi University, Taiyuan 030006, China

2 Dipartimento di Scienze della Vita e dell’Ambiente, Università Politecnica delle Marche, Ancona 60131, Italy

Abstract The genus Neglectella Vodenicarov & Benderliev has a morphology similar to the well-known genus Oocystis, except for the numerous chloroplasts stacked in the surface layer of the cell. Neglectella subsequently experienced complex taxonomy changes with members being transferred in and out, and f ive species are now accepted. In this study, a new alga with morphology corresponding to this genus was collected in China and successfully cultured in laboratory. Morphological observations in light microscopy and transmission electronic microscopy (TEM) and phylogenetic analyses revealed that it is a new species,described here as Neglectella glomerata sp. nov. It diff ered from other Neglectella species due to a morphological diff erence in its unique mucilaginous colony with a large number of cells, multiple pyrenoids in each chloroplast, smaller cell size, and more elongated cell shape. Phylogenetic analyses placed this alga in the subfamily Eremosphaeroideae of the family Oocystaceae and showed its close relationship with Neglectella peisonis. Further morphological comparisons combined with phylogenetic results revealed three diff erent morphological types in Neglectella. The new type represented by our new species may represent a transitional morphology in intermediate taxonomic position between the other two types. The diversity of morphological types in Neglectella suggests that further unknown species of this genus are likely to be discovered in the future. More collections and DNA sequence data will be needed in the future for this genus.

Keyword: colony organization; Eremosphaeroideae; molecular phylogeny; Neglectella; Oocystaceae;pyrenoid

1 INTRODUCTION

The genusNeglectellaVodenicarov & Benderliev consists of small coccoid green algae and is commonly documented in freshwater environments in lakes,ponds, and swamps (Vodenicarov and Benderliev,1971; Vodeni?arov, 1989; Schagerl, 1993). Some species of this genus were also described from marine littoral environments, or even from Antarctica, and a few were reported to grow mixed with mosses or lichens (Komárek and Fott, 1983; ?tenclová et al.,2017; Faluaburu et al., 2019).

Members ofNeglectellaare morphologically similar to the well-known genusOocystisN?geli A.Braun, except for the diff erent arrangement of their chloroplasts. Based on the unique arrangement of the plastids placed radially and peripherically in the cell,Vodenicarov and Benderliev (1971) f irst describedNeglectellaas a distinct genus within the family Oocystaceae, withN.eremosphaerophilaVodenicarov& Benderliev as the type species. Then, in a comprehensive work focusing on the subfamily Oocystoideae, Fott (1976) transferred two species fromOocystistoNeglectella—Neglectellapermagna(Behre) Fott andNeglectellaasterifera(Skuja) Fott—and concluded thatOocystisgigasvar.incrassataW.West & G. S. West sensu Skuja, was conspecif ic with the type species. However, Vodeni?arov (1989), not taking into account the lack of pyrenoids, cell size,and ecology, did not accept this synonymy and renamed this algaNeglectellopsisskujaeVodenicarov.Furthermore, Vodeni?arov (1989) removedNeglectellaasterifera(Skuja) Fott fromNeglectellabecause of its diff erent plastid structure and renamed itSkujasterasterifera(Skuja) Vodenicarov. Two new combinations,Neglectellarotula(Playfair)Vodenicarov andNeglectellaovalis(W. B. Turner)Vodenicarov, were also proposed by Vodeni?arov(1989), andNeglectellapermagna(Behre) Fott was considered by this author a synonym of the latter(Vodeni?arov, 1989). In 1993, a new species,NeglectellapeisonisSchagerl, was described based on diff erences in ecology and cell size, pyrenoid position, and pole thickening from the similarNeglectellaeremosphaerophila.

In a recent taxonomic reassessment of the Oocystaceae, ?tenclová et al. (2017) transferredOocystissolitariaWittrock into the genusNegletellaasN.solitaria(Wittrock) ?tenclová & Ka?tovsky, and assessed the phylogenetic position ofNeglectellain the subfamily Eremosphaeroideae (Oocystaceae)using molecular phylogenetic analyses. Diff erent from the other members ofNeglectella,N.solitariais very common and has been widely studied (Komárek and Fott, 1983; Quader et al., 1983; Quader, 1986; Turmel et al., 2009; Liu et al., 2010). The close relationship ofN.solitariaandNeglectellapeisoniswas proposed by Schagerl (1993), due to the similar larger cell size and higher number of chloroplasts than for other members of Oocystaceae (Hepperle et al., 2000; ?tenclová et al.,2017). At the same time, however, Schagerl (1993)pointed out the diff erent arrangement of the chloroplasts inN.solitaria, which are loosely distributed and not radially arranged. Therefore, the concept ofNeglectella-like chloroplast characteristic was expanded to numerous chloroplasts stacked in the surface layer of the cell, which can be diff erent fromEremosphaera, the other genus in the Eremosphaeroideae (?tenclová et al., 2017).Chloroplasts ofEremosphaeraare irregularly scattered and radiating from the central nucleus to the plasma membrane (De Bary, 1858; ?tenclová et al., 2017).

Currently, f ive taxonomically accepted species ofNeglectellaare recognized in AlgaeBase (Guiry,2020). In this work, a new strain withNeglectella-like morphology was collected in China. Morphological and phylogenetic analyses identif ied this strain as a new species,Neglectellaglomeratasp. nov.,belonging to the Eremosphaeroideae (Oocystaceae,Trebouxiophyceae, Chlorophyta).

2 MATERIAL AND METHOD

Neglectellaglomeratasamples were obtained from a pond in the Luoping County in the Yunnan Province of China in April 2019 (24°51′53″N, 104°18′25″E,alt. 1 475.3 m, water temperature=18.6 °C, pH=8.05).The sample was f ixed in Formaldehyde and subsequently incorporated into the Herbarium of Shanxi University (SXU). Water samples were examined under an inverted microscope and isolated into single cells using the serial dilution pipetting technique (Hoshaw and Rosowski, 1973). Liquid BG11 medium was used to culture the strains for a better observation of mucilage covers (Stanier et al.,1971). The standard culture conditions were: constant temperature of 25 °C and cool-white f luorescent light source of 30-50 μmol photons/(m2·s) on a light?dark cycle as 12 h?12 h.

Morphological observation was conducted using diff erential interference contrast by an Olympus BX53 light microscope (Olympus Corp., Tokyo,Japan). Micrographs were captured using an Olympus DP80 camera with software. Negative staining by India ink was used to show the mucilage envelope.For transmission electronic microscopy (TEM), algal samples were collected in exponential growth phase and f ixed in 2.5% glutaraldehyde in phosphate buff er overnight at 4 °C. Then, following washing in 0.05-mol/L phosphate buff er, algal cells were then f ixed in 0.1-mol/L cacodylate buff er with 1% aqueous OsO4for 2 h. After repeated washing by phosphate buff er saline (PBS) and stepwise dehydration with isopropanol, samples were embedded in Spurr’s resin(Spurr, 1969). Uranyl acetate and lead citrate were used to stain the f inal ultrathin sections (Reynolds,1963).

A Universal DNA Isolation Kit (AxyPrep) was used to extract the DNA following the manufacturer’s instructions. The 18S rDNA andrbcL cpDNA genes were selected for phylogenetic inference for comparability with the results of previous studies(?tenclová et al., 2017). Primers selection and procedures for PCR amplif ication followed Xia et al.(2013).

All informative 18S rDNA andrbcL sequences of Oocystaceae were downloaded from GenBank(https://www.ncbi.nlm.nih.gov/genbank/) after BLAST searches. The sequences for concatenated analyses were selected based on previous studies by ?tenclová et al. (2017) and Liu et al. (2017, 2018,2020). The accession numbers of the new species are MT752941 for 18S rDNA and MT757669 forrbcL.The concatenated 18S rRNA andrbcL data set included sequences of Oocystaceae, and three Chlorellaceae were selected as outgroups to root the tree according to ?tenclová et al. (2017).Ankistrodesmusfusiformiswas selected as outgroup for the 18S rRNA analyses and members of Chlorellaceae (includingChlorellavariabilisand aChlorellasp.,MicractiniumpusillumandAuxenochlorellaprotothecoides) were chosen forrbcL (Liu et al., 2020). All data sets were aligned using ClustalW (Larkin et al., 2007) and, after visual inspection, edited manually in Mega 5.2.2 (Tamura et al., 2011). Phylogenetic trees were inferred for three datasets (18S rRNA,rbcL, and concatenated 18S rRNA+rbcL) by Randomized Axelerated Maximum Likelihood using RAxML v.8.0(Stamatakis, 2014), applying the GTRGAMMA model. Nonparametric bootstrap support (1 000 replicates) was calculated to determine the maximum likelihood (ML) branch support. Bayesian analyses(BI) were conducted in MrBayes 3.1.2 (Huelsenbeck and Ronquist, 2001). Four Markov chains (three heated, one cold) were run for 3×106generations for Markov chain Monte Carlo (MCMC) analyses. Trees were sampled every 1 000 generations and the f irst 25% was discarded as burn-in.

3 RESULT

3.1 Morphological observations

The alga consisted of single cells (Fig.1a-b), small colonies formed by 2-4 cells (Fig.1c-d), and large mucilaginous colonies formed by dozens to hundreds of cells (Fig.1e-i). The cells in the colonies were embedded in mucilage, which was released from the mother cell wall; the external mucilage envelope surrounding the cell was mostly not thick (Fig.1h).The mother cell wall was not expanded while the daughter cells gradually matured, but eventually it ruptured and released them from one pole (Fig.1e).The inner daughter cells that were not in proximity of the wall opening often were retained within the wall,and the released daughter cells were often still adhering to the inner ones or stuck to the mother cell wall remnant, then they divided forming new generations (Fig.1e-f). This made the small colonies more or less irregularly dendroid (Fig.1h). The opening direction was variable, and sometimes the daughter cells were released back into the old mother cell wall (Fig.1c), which made the morphology of the mucilaginous colonies amorphous (Fig.1i). Cells were elongated, elliptical in shape, 18.46-30.24-μm long and 10.71-18.58-μm wide, with round ends(Fig.1a). Their walls formed polar thickenings when they became old (Fig.1b). Multiple parietal chloroplasts occurred peripherally below the plasma membrane and were arranged more or less radially(Fig.1a). Each chloroplast contained 1-3 pyrenoids,which were often indistinct and not easily observed in light microscopy (Fig.1d & g). Numerous oil droplets and other brown particles occurred commonly in fully developed cells (Fig.1b-c). Propagation took place by formation of 2-4-8 autospores (Fig.1g). The autospores were tightly surrounded by the mother cell wall before to be released (Fig.1g). The mother cell wall remnants expanded after the rupture occurred,and could be easily observed in the mucilaginous colonies before the f inal gelatinization (Fig.1e & h).Sexual reproduction and f lagellated stages were not observed.

In TEM observation, multiple wedge-shaped chloroplasts surrounding the central nucleus were obvious (Fig.2a & d). Several pyrenoids and numerous starch grains occurred within each chloroplast(Fig.2a). Each globular pyrenoid was surrounded by a thick starch sheath (Fig.2c & e). Many tubular thylakoids were tightly stacked and some penetrated the pyrenoid (Fig.2c & e). The ultrastructure of the cell wall showed the typicalOocystis-like multilayered cellulose arrangement with perpendicularly oriented adjacent layers (Fig.2f). Cellulose layers broke at the thickened pole of cells (Fig.2b).

3.2 Phylogenetic analyses

The 18S rDNA andrbcL sequences were obtained for the strain. Introns were not found in the sequences of either marker. The f inal dataset contained 1 463 characters for 18S rDNA, 1 037 characters forrbcL cpDNA, and 2 514 characters for the concatenated dataset. ML and BI yielded similar topologies; the ML tree for the concatenated dataset is presented in Fig.3.

Fig.1 Light microscopy of Neglectella glomerata sp. nov.

All three phylogenies consistently recovered the newly isolated strain in the lineage corresponding to the genusNeglectella(Eremosphaeroidea,Oocystaceae) with high statistical support (Fig.3,Supplementary Figs.S1 & S2). Two well-supported clades were formed withinNeglectella. One included strains ofNeglectellasolitaria; in the other,Neglectellaglomeratawas sister toNeglectellapeisoniswith robust support.

4 DISCUSSION

Fig.2 Transmission electronic microscopy (TEM) of Neglectella glomerata sp. nov.

Recently, a high diversity of Oocystaceae has been discovered in the Yunnan Province (China),which has led to the description of many new taxa(Liu et al., 2017, 2018, 2020). In this work,Neglectellaglomerata, a new species of the genusNeglectella(Eremosphaeroidea, Oocystaceae), is presented.

Neglectellawas originally erected by Vodenicarov and Benderliev (1971) to includeOocystis-like algae of relatively large size, with numerous radially arranged chloroplasts (?tenclová et al., 2017).Subsequently, Fott (1976) emphasized the unique radial chloroplast arrangement as a distinguishing feature and transferred twoOocystisspecies to this genus. While some members ofNeglectellawas moved out to establish two new genera,NeglectellopsisandSkujaster, by Vodeni?arov (1989) and some new species were subsequently added (Vodeni?arov, 1989;Schagerl, 1993), all members of the genus share the same chloroplast arrangement. ?tenclová et al. (2017)expanded the circumscription of the genus based on molecular data and proposed the new combinationNeglectellasolitaria, transferring fromOocystissolitaria. This alga diff ers from the traditionalNeglectellain its smaller cells and arrangement of peripheral chloroplasts; inNeglectellasolitaria, the chloroplasts are irregularly arranged and not radially organized (?tenclová et al., 2017). The new species presented here,Neglectellaglomerata, shows an intermediate chloroplast morphology betweenNeglectellasolitariaand otherNeglectellaspecies with chloroplasts smaller in number but regularly organized around the cell and more or less radially from the middle of the cell (Fig.1a). Also, the new species shares similarly thick cell walls, numerous assimilation products, and a central nucleus with theNeglectellaspecies. Most importantly, however, the phylogenetic analyses robustly support its placement in theNeglectellaclade (Fig.3).

Fig.3 Phylogenetic tree inferred using concatenated genes of 18S rDNA and rbc L cpDNA sequences from Oocystaceae species

Table 1 Morphological comparisons for all species in genus Neglectella

WithinNeglectella,Neglectellaglomeratacan be distinguished from other species by its unique colony organization. All other species are usually unicellular and rarely form 2-8-celled colonies (Schagerl, 1993).Composite colonies formed by 2-3 generations of cells were occasionally documented inNeglectellasolitaria(Komárek and Fott, 1983). InNeglectellaglomerata, however, dozens to hundreds of cells in mucilaginous colonies are common. The colony organization ofNeglectellaglomeratais mainly due to the mucilage, diff ering from other species ofNeglectellafor the expanded mother cell wall that encloses the daughter cells. When observed,Neglectellaglomeratashows a more or less dendroid organization, especially in small colonies; this arrangement cannot be found in otherNeglectellaspecies and resembles the type of colony morphology of the genusEcballocystisBohlin. However, as described by Iyengar (1932), the cells ofEcballocystisdisplay a marked degree of polarity, which leads to a more obvious thickening in the basal cell wall,invariable rupture in apical position for the mother cell wall, a mucilage pad formed only from the basal part, and f inally successive colony branching. InNeglectellaglomerata, however, this polarity was not observed. The ruptured pole of the mother cell wall is not def ined; therefore, some daughter cells are released back into the old mother cell wall (Fig.1c).The mucilage pad at the base of the colony, which allows theEcballocystisepiphytic or epilithic attachment (Iyengar, 1932; Komárek and Fott, 1983;Xia et al., 2013), is absent inNeglectellaglomerata.This species lives in freshwater in stationary water pools. Moreover, its more or less dendroid morphology is due to the random adhesion of a part of the daughter cells which are released together (Fig.1f & h). UnlikeEcballocystis, the remnant mother cell wall does not play an important role in the formation of colonies ofNeglectellaglomerata. When a larger mucilaginous colony is formed, the cell arrangement is often irregular.

Apart from the colony morphology,Neglectellaglomeratacan also be distinguished from other species ofNeglectellafor the higher number of pyrenoids per chloroplast, smaller cell size, and more elongated cell shape (Table 1).Neglectellaglomeratahas more than one pyrenoid in each chloroplast whereas the other species have only one. The pyrenoid number is considered an important taxonomic feature,especially in the Oocystaceae (Printz, 1913; Korsikov,1939). In this family, multiple pyrenoids in each chloroplast are documented inEremosphaera(Komárek and Fott, 1983), another genus of the Eremosphaeroideae, which further indicates the close phylogenetic relationship betweenNeglectellaandEremosphaera. Another genus with a similar pyrenoid structure isEuchlorocystis, a recently described genus also found in China (Liu et al., 2018). These three genera are all positioned at the base of the family Oocystaceae (Krienitz and Bock, 2011; ?tenclová et al., 2017), which might support a basal origin of multiple pyrenoids in the Oocystaceae as mentioned by Liu et al. (2018). Apart from the characteristic pyrenoid,Neglectellaglomeratais morphologically diff erent from congeners in terms of cell size and cell shape. The cell size of the otherNeglectellaspecies(except theNeglectellasolitaria) is almost twice as this new species (Schagerl, 1993). Considering the cell shape, the broadly ellipsoid cells with broad round end ofNeglectellaeremosphaerophila,Neglectellaovalis, andNeglectellapeisonis, as well as the round shape ofNeglectellarotula, are all diff erent from the elongated elliptical shape ofNeglectellaglomerata(Playfair, 1918; Komárek and Fott, 1983; Schagerl, 1993).

Among the six species of genusNeglectella,Neglectellarotulais obviously diff erent in morphology for having round cell shape and central pyrenoid. Its taxonomic position needs further verif ication. The remaining members can be subdivided into three types based on cell and colony morphology. The f irst and biggest types, includingNeglectellaeremosphaerophila,Neglectellaovalis,andNeglectellapeisonis, represent the traditionalNeglectellamorphology, with numerous radial chloroplasts, clearly bigger cell size, and broadly ellipsoid cell shape (Schagerl, 1993). The second type only includesNeglectellasolitariawith relatively smaller cell size and diff erent chloroplast organization and number (?tenclová et al., 2017). Compared to the two types mentioned, the third type represented by our new species shows diff erent colony morphology.Currently, three representative species of the three types inNeglectellahave been sequenced. Based on the molecular phylogenetic analyses,Neglectellaglomeratais resolved as sister toNeglectellapeisonis(Fig.3). However, the new species shows a more similar cell morphology withNeglectellasolitaria,especially obvious on the smaller cell size and not broad round cell poles. The chloroplast, with more or less radially arrangement which resembles the f irst type and the lower number which resembles the second type, may further imply theNeglectellaglomeratais in intermediate taxonomic position between the other two morphology types. The diverse morphology types in genusNeglectellamay also imply more hidden species that have not been discovered yet. More species collections and sequencing will be needed in the future for this genus.

Taxonomic assessment

NeglectellaglomerataLIU, ZHANG et XIE sp.nov. (Fig.1a-i)

Diagnosis: Dozens or hundreds of cells were embedded in the mucilage colony, sometimes it was the scattered colony with 2-4 cells or solitary cells observed. The cells in the colony connected via the mucilage and organized more or less via dendroid or amorphous. The cell mucilage envelopment was irregular and not thick. The cell was elongated elliptical, with the ends thickening when old, at 18.46-30.24-μm long and 10.71-18.58-μm wide.Four-multiple chloroplasts extended more or less peripherally and radially from the center of the cell,each with 1-3 pyrenoids. Assimilate particles and oil droplets were numerous. Propagation occurred via 2-4-8 autospores. The mother cell wall tightly enclosed the daughter cells initially, then released the latter at one pole. This species diff ered from other members of theNeglectellagenus due to its unique mucilage colony with a large number of cells, multiple pyrenoids in one chloroplast, smaller cell size, more elongated cell shape and the nucleotide sequence for 18S rDNA andrbcL cpDNA.

Type: Luoping County, Yunnan Province, China:sample collected in a pond, 24°51′53″N, 104°18′25″E,18/4/2019, Xudong LIU, (Holotype SXU-ZR83!).Population partially illustrated here in light microscopy (Fig.1).

Reference strain: the cultured strain was deposited as No. FACHB-2424 in Freshwater Algae Culture Collection at the Institute of Hydrobiology, Wuhan,China (FACHB).

Habitat: pond; planktonic; altitude 1 475.3 m;water temperature=18.6 ℃; pH=8.05.Etymology: the species was named for a mucilage colony with numerous embedded cells.

5 CONCLUSION

A new freshwater green algal species,Neglectellaglomeratasp. nov., is erected here based on morphological and ultrastructure comparison and molecular phylogenetic analysis. It is morphologically characterized by the diff erence in unique mucilage colony with a large number of cells, multiple pyrenoids in one chloroplast, smaller cell size and more elongated cell shape. Further morphology analysis is completed including all the species in genusNeglectellaand three types are summarized.Our new species may represent a new transitional morphology type and taxonomic position between the other two types.

6 DATA AVAILABILITY STATEMENT

The authors declare that the datasets in this study are available on reasonable request from the corresponding author.