Towards modern classification of eustigmatophytes, including the description of Neomonodaceae, fam. nov. and three new genera

Abstract

The class Eustigmatophyceae includes mostly coccoid, freshwater algae, although some genera are common in terrestrial habitats and two are primarily marine. The formal classification of the class, developed decades ago, does not fit the diversity and phylogeny of the group as presently known and is in urgent need of revision. This study concerns a clade informally known as the Pseudellipsoidion group of the order Eustigmatales, which was initially known to comprise seven strains with oval to ellipsoidal cells, some bearing a stipe. We examined those strains as well as ten new ones and obtained 18S rDNA and rbcL gene sequences. The results from phylogenetic analyses of the sequence data were integrated with morphological data of vegetative and motile cells. Monophyly of the Pseudellipsoidion group is supported in both 18S rDNA and rbcL trees. The group is formalized as the new family Neomonodaceae comprising, in addition to Pseudellipsoidion, three newly erected genera. By establishing Neomonodus gen. nov. (with type species Neomonodus ovalis comb. nov.) we finally resolve the intricate taxonomic history of a species originally described as Monodus ovalis and later moved to the genera Characiopsis and Pseudocharaciopsis. Characiopsiella gen. nov. (with the type species Characiopsiella minima comb. nov.) and Munda gen. nov. (with the type species Munda aquilonaris) are established to accommodate additional representatives of the polyphyletic genus Characiopsis. A morphological feature common to all examined Neomonodaceae is the absence of a pyrenoid in the chloroplasts, which discriminates them from other morphologically similar yet unrelated eustigmatophytes (including other Characiopsis-like species).

INTRODUCTION

The Eustigmatophyceae constitute a well-defined clade of ochrophyte (heterokontophyte) algae that is considered a separate class related to Chrysophyceae, Synchromophyceae, and possibly Pinguiophyceae (Yang et al. 2012, Ševčíková et al. 2015, Eliáš et al. 2017). Eustigmatophytes are coccoid algae, solitary or in loose colonies, reproducing via autosporogenesis or, occasionally in some taxa, by zoospores with unique features (for a review see Eliáš et al. 2017). Eustigmatophytes occur primarily in freshwater and soil, but research on the class has been concentrated on the primarily marine genera Nannochloropsis and Microchloropsis (Fawley et al. 2015) which have shown potential for biotechnological exploitation (Ma et al. 2016).

The existence of eustigmatophytes as an independent group was realized in the early 1970’s upon investigation of the ultrastructure and pigment composition of several algae previously classified as Xanthophyceae (Hibberd and Leedale 1970, 1971). Since then, the class has been growing in diversity, both by recruiting additional traditional xanthophytes (Hibberd 1981, Santos 1990, Schnepf et al. 1996, Santos and Santos 2004, Přibyl et al. 2012) and description of brand new taxa (e.g., Preisig and Wilhelm 1989, Neustupa and Němcová 2001, Hegewald et al. 2007, Nakayama et al. 2015, Fawley et al. 2019). It is likely that this process of reassigning misclassified xanthophycean taxa will continue when other previously described yet poorly documented species are reinvestigated with modern methods, as demonstrated by the recent study of Tetraëdriella subglobosa, re-isolated from the original type locality and proved to be a eustigmatophyte by 18S rRNA and rbcL gene sequencing (Fawley and Fawley 2017). Hundreds of described xanthophytes have not been studied by transmission electron microscopy (TEM) or molecular approaches (see Ettl 1978), so they represent a particularly attractive target for investigation.

The need to clarify the diversity and phylogeny of the Eustigmatophyceae and to provide proper identifications of strains held in culture collections is now more urgent than ever before, given the rapid growth of interest in eustigmatophytes other than Nannochloropsis and Microchloropsis that has been stimulated by the fact that all studied Eustigmatophyceae produce valuable compounds such as lipids (Pal et al. 2013, Gao et al. 2018), carotenoids (Lubián 2000, Li et al. 2012) and antioxidants (Assunção et al. 2016). The first consolidated classification of eustigmatophytes developed by Hibberd (1981) recognized a single order Eustigmatales divided into four families. The growth of newly recognized or described eustigmatophytes and the advent of molecular phylogenetics quickly challenged Hibberd’s scheme. The inadequacy of the existing eustigmatophyte classification has become even more obvious with molecular characterization of new freshwater isolates (Prior et al. 2009, Fawley et al. 2014, 2017) and environmental DNA surveys (Lara et al. 2011, Nikouli et al. 2013, Villanueva et al. 2014), which revealed the existence of substantial undescribed phylogenetic diversity within this group.

Eustigmatophytes are thus now known to encompass two deeply separated principal lineages, one corresponding to the order Eustigmatales (Hibberd 1981) and the other comprised of eustigmatophytes recognized or described only after Hibberd’s seminal work (Eliáš et al. 2017). This second putative order not formalized under the International Code of Nomenclature for Algae, Fungi and Plants is presently referred to as the clade Goniochloridales and validated under the PhyloCode (Fawley et al. 2014). The same authors also described the existence of three robustly separated clades within the Eustigmatales. One clade corresponds to the Monodopsidaceae sensu Hibberd (1981), expanded by the addition of Pseudotetraëdriella kamillae, a species described and placed in the family Loboceae by Hegewald et al. (2007). The second clade referred to as the Eustigmataceae group comprises members of the families Eustigmataceae and Chlorobotrydaceae sensu Hibberd 1981, the strain Pseudocharaciopsis minuta UTEX 2113, an isolate identified as Characiopsis saccata plus several unidentified isolates (Fawley et al. 2014).

The present study concerns the third clade of the Eustigmatales, informally named the Pseudellipsoidion group by Fawley et al. (2014) according to its representative Pseudellipsoidion edaphicum, an organism described by Neustupa and Němcová (2001) as a eustigmatophyte but not formally classified into any family. Molecular characterization of several unidentified isolates showed one highly supported lineage comprising four unnamed strains positioned together with P. edaphicum (Fawley et al. 2014) and a second lineage within the Pseudellipsoidion group that included two Pseudocharaciopsis ovalis strains. These findings suggested that the genus Pseudocharaciopsis as circumscribed by Hibberd (1981) is polyphyletic. The taxonomy of the Pseudellipsoidion group is therefore in need of revision. To meet this objective, the present study provides morphological and molecular data (18S rRNA and rbcL gene sequences) for seven original Pseudellipsoidion group members and ten additional strains, nine previously assigned to the genus Characiopsis in the Xanthophyceae. The establishment of a new eustigmatophyte family, the Neomonodaceae, is proposed to include four genera, three of them newly described. In addition, clades at the species level are indicated for future further analysis.

MATERIALS AND METHODS

Algal cultures

A total of seventeen strains of microalgae isolated from freshwater, soil, peat bogs and mines were studied (Table S1 in the Supporting Information). Seven strains are Portuguese isolates held at the Coimbra Collection of Algae (ACOI) (acoi.ci.uc.pt) maintained in liquid Desmideacean Medium (Schlösser 1994), pH 6.4 to 6.6, at 20 °C, under 12:12 h photoperiod and under 10 μmol · m⁻² · s⁻¹ light intensity provided by cool white fluorescent lamps. Four strains are isolates from Itasca State Park, Minnesota, U.S.A. and one strain was isolated from a small pond in Arkansas, USA; these strains are kept on agar slants of WH+ medium (Fawley et al. 2013). Three strains are soil isolates from the Czech Republic, held at the Culture Collection of Algae at Charles University (CAUP; botany.natur.cuni.cz/algo/caup.html) in Bold’s Basal Medium (BBM; Bischoff and Bold 1963). Two strains are isolates from inhospitable environments, namely coal and lignite mines in the Czech Republic and Germany; their cultures are kept at the Institute of Soil Biology in the Biology Centre Collection of Organisms (BCCO; www.soilalgae.cz) on BBM agar slants, pH 6 to 6.4, at 15°C, under continuous low light, and also cryopreserved under −150°C.

Light microscopy observations

Morphological evaluation of the cells was performed using a Leica DMRB either by light microscopy analysis or by DIC microscopy using 60x and 100x PLAN APO objectives. Micrographs were acquired with a Leica DFC420 digital camera. A Nikon Ni-U microscope equipped with a 100x Plan Apo objective and DIC was used for investigating the strains from the collection of Karen and Marvin Fawley. Observations and measurements were performed in young and old cultures (5 and 30 d). The presence of zoospores was recorded from one hour to two days after adding fresh culture medium to an old batch culture (more than one month). Drawings were obtained by digital tracing micrographs in Photoshop Elements using a Wacom Bamboo drawing tablet. Cell size was assessed using the digital image analysis software LAS V4.6 or Nikon Elements BR by measuring 5 cells of each strain, 5 and 30 d after sub-culturing.

Transmission Electron Microscopy

For TEM a suspension of cells was fixed for 2 h or 2.5 h with 2% or 2.5% glutaraldehyde in 0.05M phosphate buffer, pH 6.8 and then washed with the same buffer by centrifugation one to three times for 5 min at 2000 rpm. The cell suspension was embedded in 1.5% or 2% agar and post-fixed in 1% osmium tetroxide solution (prepared 1:1 v/v with the same phosphate buffer) for 2 h in the dark. The fixative was then washed out by centrifugation (2x buffer then 2x deionized water or 3x buffer, 5 min at 2000 rpm). Samples were dehydrated in an ethanol series (70%, 96% and 100% or 70%, 80%, 95% and 100%), each for 15 min and then embedded in Spurŕs resin with butanol or ethanol (5%, 10%, 25%, 50%, 75%, 95% and 100% or 33%, 50% and 66%) and kept overnight in a desiccator. Resin blocks were then cut with an ultramicrotome (Ultracut E, Reichert-Jung) and ultrathin sections were mounted on copper grids and stained with 1% or 2% uranyl acetate and 0.2% lead citrate. Samples were examined in a JEOL 1011 or a FEI-Tecnai G2 Spirit Bio Twin electron microscope. Direct preparations of zoospores were obtained by fixing a drop of zoospore suspension on a formvar/carbon-coated grid in 2% osmium tetroxide vapor, drying at room temperature and shadowcasting with gold/palladium.

PCR amplification and DNA sequencing

Cells were collected by centrifugation of 2 ml culture or harvested from agarized medium and disrupted using a mixer mill (MM200 Retsch, Haan, Germany) for 5 min. Genomic DNA was extracted using Spin Plant Mini Kit (Invisorb®, Invitek). PCR was performed with the MyTaq™ Red DNA Polymerase (Bioline, United Kingdom), under following conditions: denaturation 95°C for 2 min followed by 35 cycles of 95°C for 30 s, 52°C for 30 s, 72°C for 2.5 min and final extension at 72°C for 5 min. PCR products from amplification of the 18S rRNA and rbcL genes were purified using GenElute™ PCR Clean-Up Kit (SIGMA). Sequencing reactions were performed using BigDye® Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific) and analyzed using the 3130xl Genetic Analyzer in the DNA Sequencing Laboratory of the Faculty of Science, Charles University in Prague. Primers used for obtaining full sequences of the 18S rRNA gene included the amplification primers 18S-F and 18S-R and internal sequencing primers according to Katana et al. (2001). Primers used for amplification of rbcL were EU-rbcL-F1 (5´- ATGTTTCAATCTGTAGAAGAAAG-3’) and the reverse primer EU-rbcL-R1 (5´- CCTTGTGTTAATCTCACTCTTC-3’), which were newly designed based on a comparison of complete rbcL genes obtained as parts of fully sequenced eustigmatophyte plastid genome sequences (Ševčíková et al. 2015). They allow for highly efficient amplification of essentially a complete rbcL gene from diverse eustigmatophytes (see also Fawley et al. 2015, 2019, Fawley and Fawley 2017). For sequencing reactions, the amplification primers were used along with the newly designed sequencing primers (provided in Table S2 in the Supporting Information). Sequencing reads were assembled with SeqAssem (SequentiX, http://www.sequentix.de/software_seqassem.php), and manually edited by visual inspection of sequencing chromatograms. Sequence data from the strains from the collection of Karen and Marvin Fawley (the five “Pseudellipsoidion sp.” strains) were obtained using the procedures and primers described in Fawley and Fawley (2017). Sequences were trimmed to exclude primer regions and deposited at GenBank (accession numbers provided in Table S1).

Phylogenetic analyses

The complete dataset for analyses of the 18S rRNA gene sequences included in total 565 sequences and consisted of the 10 newly obtained sequences of the Neomonadaceae family, an exhaustive set of 539 non-redundant eustigmatophyte 18S rDNA sequences gathered from the GenBank database based on extensive blast searches and preliminary analyses (which also led us to exclude some low-quality and/or apparently chimeric sequences), and a selection of 14 sequences from phylogenetically diverse ochrophytes to provide an outgroup. The sequences were aligned with MAFFT 7.429 (Katoh and Frith 2012, Katoh and Standley 2013), using the “Add” option and a preexisting master alignment of ochrophyte 18S rRNA gene sequences manually curated to take into account the conserved secondary structure of 18S rRNA molecules (Eliáš et al. 2017). Redundant sequences were removed in BioEdit version 7.0.5 (Hall 1999) and the resulting final alignment was used in two different analyses. The first utilized a subset of 99 sequences (all Neomonodaceae sequences, 75 additional eustigmatophyte sequences representing all main lineages in the group, and the outgroup sequences). Trimming the alignment with GBlocks 0.91b (Castresana 2000) to remove unreliably aligned positions left 1614 positions in the final alignment. In the second analysis, the full alignment was trimmed with trimAl v1.4. rev6 using 0.02 similarity threshold (Capella-Gutiérrez 2009; https://www.genome.jp/tools/ete/), leaving 1756 positions for tree inference. For the rbcL gene analysis, a selection of 40 eustigmatophyte sequences available from GenBank (retaining only one sequence per described species for non-Neomonodaceae representatives) and the 13 newly obtained or updated sequences were aligned with MAFFT 7.429. The termini of the alignment were trimmed in GeneDoc (Nicholas and Nicholas 1997) to remove positions with a high percentage of missing data, leaving 1347 positions. Trees were inferred using the maximum likelihood (ML) method implemented in RAxML (8.2.12) at the Cyberinfrastructure for Phylogenetic Research (CIPRESS) Portal (http://www.phylo.org/sub_sections/portal; Miller et al. 2011) using the strategy of Stamatakis et al. (2008) for obtaining the highest likelihood tree. The evolutionary model used was the default GTR+Γ. In the case of the rbcL gene, two analyses were done, one considering the whole alignment as one partition and the other considering separate partitions for the three codon positions. Bootstrap analyses were performed with the rapid bootstrapping procedure, with the adequate number of replicates detected by the program itself (“halt bootstrapping automatically” option); the number of bootstrap replicated for each tree is specified in the respective figure legends. Trees were drawn with the aid of the iTOL tool (Letunic and Bork 2016; https://itol.embl.de/).

RESULTS

Expanded phylogenetic diversity of the family Neomonodaceae (Pseudellipsoidion group)

The phylogenetic tree inferred from 18S rRNA gene sequences (Fig. 1) shows the deep separation of eustigmatophyte into two clades, Goniochloridales and Eustigmatales. The latter is further resolved into three strongly to fully supported subclades, the Monodopsidaceae, the Eustigmataceae group, and the Neomonodaceae (i.e., Pseudellipsoidion group), plus a deep lineage represented solely by the recently described Paraeustigmatos columelliferus (Fawley et al. 2019). The Neomonodaceae is expanded by ten newly characterized strains. The strain Beav 4/26 T-6w proved to be closely allied with P. edaphicum and previously reported unidentified strains Tow 8/18 T-12d, WTwin 8/18 T-5d, Tow 9/21 P-2w and Mary 8/18 T-3d. The clade comprising these six strains, further referred to as the genus Pseudellipsoidion, is supported by a bootstrap value of 89% and separated from other lineages in the Neomonodaceae. Another clade, which we later formally describe as the new genus Neomonodus, is maximally supported and includes five strains previously identified as Pseudocharaciopsis ovalis or Characiopsis ovalis, three of them newly characterized here. Specifically, the strains BCCO_30_2917 and BCCO_30_2918 have the same 18S rRNA gene sequence as P. ovalis CAUP Q 302, whereas the 18S rRNA gene sequence of the strain Neomonodus sp. ACOI 2437 exhibited two and one nucleotide differences from P. ovalis CAUP Q 301 and P. ovalis CAUP Q 302, respectively.

Fig. 1.

Phylogeny of Eustigmatophyceae based on sequences of the 18S rRNA gene. The phylogeny shown was inferred using the maximum likelihood method implemented in RAxML (employing the GTR+Γ substitution model) with bootstrap analysis followed by thorough search for the ML tree. Bootstrap values correspond to the percentage calculated from 300 replicates and are shown when higher than 50. For simplicity, the outgroup (a selection of diverse ochrophyte 18S rRNA sequences) is omitted from the figure. Terminal leaves are labeled with the species/strain name (sometimes different from the name in the respective GenBank record to reflect recent taxonomic changes) and the GenBank accession number of the sequence. New sequences are highlighted in boldface.

The remaining six Neomonodaceae strains constitute two separate novel lineages (Fig. 1). One lineage comprises two strains from the ACOI culture collection (ACOI 2426 and ACOI 2423A) identified by us as Characiopsis minima. These two strains had identical 18S rRNA gene sequences and are described below as the new genus Characiopsiella. The second new lineage included strains identified as Characiopsis aquilonaris (ACOI 2424, 2424A, 2424B) and Characiopsis sp. (ACOI 2428); it is described below as the new genus Munda. The 18S rRNA gene sequences of the strains in this new lineage were also identical. The phylogenetic analysis of the 18S rRNA gene suggested that Neomonodus and Munda are sister lineages and that Characiopsiella is sister to the Neomonodus-Munda clade, but bootstrap support for the latter relationship is weak (61%). The family Neomonodaceae is strongly supported (bootstrap value of 99%) as monophyletic and clearly separated as one of the four main clades in the order Eustigmatales.

We performed a second phylogenetic analysis of eustigmatophyte 18S rRNA gene sequences that also included partial sequences (~500 to ~600 bp) obtained by surveying environmental DNA from an east African freshwater lake (Villanueva et al. 2014) and a tropical coastal lagoon (Alves-de-Souza et al. 2017). The former study reported the existence of five clades comprising sequences from uncultivated eustigmatophytes, denoted Group 1 to Group 5. Our analysis, which benefited from a substantial improvement of the sampling of cultured eustigmatophytes and employing a more sophisticated method of phylogenetic inference, enabled us to more precisely place these five groups within eustigmatophytes (Figs. 2 and S1 in the Supporting Information). Group 1 is confirmed as a cluster within the Goniochloridales clade, Group 2 is now revealed to correspond to the basal Eustigmatales lineage typified by Paraeustigmatos columelliferus, and Group 4 and Group 5 constitute a larger clade branching off basally in the Eustigmataceae group. Most significantly for our main focus here, the Group 3 of Villanueva et al. (2014) represents a novel, apparently diverse lineage within Neomonodaceae, potentially sister to the genus Characiopsiella. The partial sequences from the coastal lagoon (Alves-de-Souza et al. 2017) all fall within the genus Microchloropsis (Fig. S1).

Fig. 2.

Phylogeny of Eustigmatophyceae based on sequences of the 18S rRNA gene including partial sequences from environmental DNA surveys. The tree was inferred using the same procedure as the tree shown in Figure 1. For simplicity, the outgroup (a selection of diverse ochrophyte 18S rRNA sequences) is omitted from the figure and the main eustigmatophyte branches are collapsed as triangles, except for the family Neomonodaceae. Bootstrap values were calculated from 354 replicates and are shown when higher than 50. The positions of the five groups of partial sequences from uncultured eustigmatophytes obtained by Villanueva et al. (2014) are indicated. The full version of the tree is provided as Figure S1.

A phylogenetic analysis of rbcL sequences confirmed with maximal support the monophyly of the Neomonodaceae and its placement in the order Eustigmatales. Within the Eustigmatales, the Neomonodaceae was sister to the Eustigmataceae group, although with low bootstrap support (<50%; Fig. 3). All four clades treated here as separate Neomonodaceae genera were each resolved as monophyletic with maximal support and clearly separated from each other. However, their mutual relationships differed from the inferred tree topology that resulted from analysis of 18S rRNA gene sequence data (Figs. 1 and 2). Specifically, Pseudellipsoidion and Characiopsiella appeared as sister to each other, with Munda and Neomonodus branching off as more basal lineages (Fig. 3). An analysis considering the three codon positions of the rbcL gene as separate partitions yielded a tree with the same topology as the with no partitioning employed, with minor differences in bootstrap support values only (Fig. 3). The rbcL sequences revealed a degree of genetic diversity within each of the four main clades that was not apparent from the 18S rRNA gene. Thus, within Munda, the strain ACOI 2428 differed from the remaining three strains by four nucleotides whereas the 18S rRNA sequences were identical.

Fig. 3.

Phylogeneny of Eustigmatophyceae based on sequences of the rbcL gene. The phylogeny shown was inferred using maximum likelihood method implemented in RAxML (employing the GTR+Γ substitution model) with bootstrap analysis followed by thorough search for the ML tree. The topology of the tree reflects a result obtained without defining separate partitions for different codon positions of the rbcL gene. Two sets of bootstrap support values (calculated from 354 replicates) are given, one from the non-partitioned analysis, the other (separated by a slash) an analysis with partitions. Only values higher than 50 are shown. Labels at terminal leaves comprise the strain updated taxonomic name followed by the collection reference number when applicable and the GenBank accession number. New sequences highlighted in boldface. The root of the tree is placed between the order Eustigmatales (including Paraeustigmatos columelliferus) and the clade Goniochloridales, following results of phylogenetic analyses of the 18S rRNA gene (see also Fig. 1) and multiple plastid-encoded proteins (Ševčíková et al. 2019).

Morphological and ultrastructural characterization of the Neomonodaceae

The main morphological characters showing variation among different Neomonodaceae representatives are summarized in Table S1. Vegetative cells of the Neomonodaceae (Figs. 4–7) are light green with different oval, ellipsoidal or elongated shapes simultaneously found in the same culture. Cell size is also quite variable, 8–11 × 4–5 μm (without stipe) with much smaller (6 × 3 μm) or larger cells (up to 30 × 10 μm) occasionally observed. Generally, the cells widen and sometimes round up when the cultures age. Many are free-floating cells with the anterior end rounded (Figs. 4C and 6, A and C), acute (Figs. 6A, 7D) or with a papilla (Fig. 4, A and B). Sometimes these morphologies are seen in different cells in the same culture. Sessile cells with a marked polarity were also observed (Figs. 4A, 6E, 7B). An attaching stipe and/or a disc was positioned at the posterior end of the cells of some species providing cell polarity. The stipe is always short (usually ≤ 1 μm) and consists of an extension of the cell wall with cell content (Figs. 6B, 7C). Substances from the surrounding medium may adhere to the stipe, causing it to become dark orange-brown. In a morphologically similar yet not directly related eustigmatophyte Pseudocharaciopsis minuta, this coloration was shown to be due to the accumulation of metals such as Mn (Wujek 2012). Vegetative cells of Neomonodus (Fig. 4), Characiopsiella (Fig. 6) and Munda (Fig. 7) are mainly populated with stipitate cells, whereas those of Pseudellipsoidion (Fig. 5) are exclusively free-floating. Due to their resemblance to the genera Characiopsis and Monodus, the cells with a stipe have been referred to as Characiopsis-like and those without a stipe as Monodus-like (Lee and Bold 1973, Neustupa and Nĕmcová 2001).

Fig. 4.

Vegetative cells of Neomonodus ovalis BCCO_30_2918 (A, B), Neomonodus sp. ACOI 2437 (C, D) and Neomonodus ovalis CAUP Q 302 (E - G) observed under light and electron microscopy. Apical papilla (p), chloroplast (chl), lamellate vesicles (lv), mitochondrion (m), oil droplets (oil), osmiophilic vesicles (ov), reddish globule (rg). Light micrographs with DIC, bar 10 μm; TEM micrographs, bar 1 μm.

Fig. 7.

Vegetative cells of Munda aquilonaris ACOI 2424A (A), ACOI 2424B (B), ACOI 2424 (C, E, F) and Munda sp. ACOI 2428 (D), observed under light and electron microscopy. Chloroplast (chl), chloroplast membrane (chl m), Golgi body (gb), lamellate vesicles (lv), mitochondrion (m), nucleolus (nu), nucleus (n), osmiophilic vesicles (ov), reddish globule (rg), stipe (s), connection between the chloroplast endoplasmic reticulum and the nuclear envelope (arrowheads). Light micrographs with DIC, bar 10 μm; TEM micrographs, bar 1 μm.

Fig. 6.

Vegetative cells of Characiopsiella minima ACOI 2426 (A, B, C) and Characiopsiella minima ACOI 2423A (D and E) observed under light and electron microscopy. Lamellate vesicle (lv), oil droplets (oil), osmiophilic vesicles (ov), reddish globule (rg), stipe (s). Light micrographs with DIC, bar 10 μm; TEM micrographs, bar 1 μm.

Fig. 5.

Vegetative cells of Pseudellipsoidion sp. WTwin 8/18 T-5d (A), Pseudellipsoidion sp. Mary 8/18 T-3d (B) and Pseudellipsoidion edaphicum CAUP Q 401 (C, D, E), observed under light and electron microscopy. Chloroplast (chl), lamellate vesicles (lv), nucleolus (nu), nucleus (n), osmiophilic vesicles (ov), reddish globule (r g). Light photographs with DIC, bar 10 μm; TEM micrographs, bar 1 μm.

Neomonodaceae cells display a cell wall in one piece, usually smooth. One to several chloroplasts are present in the cells (Fig. 4A) with a typical eustigmatophycean lamellate structure with a few evenly spaced thylakoids not bounded by a girdle lamella (Fig. 7E). No pyrenoid was observed under the light microscope and its absence was further noted in TEM sections of all genera (Figs. 4E, 5C, 6B and 7C). Special attention was paid to the clarification of the presence of a pyrenoid in Pseudellipsoidion edaphicum CAUP Q 401, where no pyrenoid was found (see the emended diagnosis of Pseudellipsoidion edaphicum below). One or more nearly spherical nuclei may be found in the cell (Fig. 7C) with a central nucleolus often observed (Fig. 7E). An apparent connection between the chloroplast endoplasmic reticulum and the nuclear envelope was observed in some cells of Neomonodaceae (Fig. 7F, arrowheads), although in some sections the nucleus and the chloroplasts stay quite apart (Fig. 7C).

Although it may be small or undetected in young cells, a very conspicuous orange-reddish globule is usually found in vegetative cells observed under light microscopy (Figs. 4C, 5D, 7A); sometimes more than one. Sections show this red body composed of many adjacent droplets and not bounded by a membrane (Fig. 4G). Oil droplets are frequently observed in old cells of the Neomonodaceae (Figs. 4D and 6A). Lamellate vesicles with refractive properties under light microscopy (Figs. 4A and 5A) are scattered throughout the cytoplasm (Figs. 4E, 6B and 7C) and display a finely lamellate structure (Fig. 4F). Other structures and organelles common in eukaryotic cells can be found in the cytoplasm, such as tubular mitochondria (Figs. 4D and 7E) or a small Golgi body lying next to the nucleus (Fig. 7E).

Regarding reproductive cells, the formation of autospores was observed, followed by their release after mother cell wall disruption (Fig. 8A) and rounded or elongated flask-shaped zoospores were observed in liquid cultures of all Neomonodaceae strains examined (examples shown in Fig. 8, B-D). Zoospore movement was observed under the light microscope, with a visible long flagellum (Fig. 8, B and C). Shadowcast preparations in Pseudellipsoidion sp. WTwin 8/18 T-5d and in Munda aquilonaris ACOI 2424 revealed a second shorter and thinner emergent flagellum (Fig. 8, D and E). An extra-plastidial eyespot, associated with a swelling at the base of the anterior, long and mastigoneme-bearing flagellum has been detected in TEM sections of zoospores (Fig. 8, F and G).

Fig. 8.

Reproductive cells of the Neomonodaceae, observed under light and electron microscopy. Autospore release in Pseudellipsoidion sp. Tow 8/18 T-12d (A), biflagellate zoospores of Pseudellipsoidion spp. strains Tow 8/18 T-12d, Mary 8/18 T-3d and WTwin 8/18 T-5d (B), Pseudellipsoidion sp. WTwin 8/18 T-5d (C), Pseudellipsoidion sp. WTwin 8/18 T-5d (D) and Munda aquilonaris ACOI 2424 (E - G). Chloroplast (chl), eyespot (eye), long flagellum (lf), mitochondrion (m), short flagellum (sf).

DISCUSSION

Phylogeny of the Neomonodaceae

The overall structure of the phylogenetic tree inferred from 18S rRNA gene sequences (Fig. 1) agrees well with previous similar analyses (Fawley et al. 2014, 2019, Nakayama et al. 2014, Eliáš et al. 2017, Fawley and Fawley 2017, Kryvenda et al. 2018). The chief difference compared to previous studies is the expansion of the Pseudellipsoidion group – here formalized as the family Neomonodaceae – by ten newly characterized strains. Three of them belong to the lineage here described as the genus Neomonodus and one joins a group of five previously characterized strains that we here classify as the genus Pseudellipsoidion. The remaining six new strains added much more phylogenetic novelty to the Neomonodaceae by constituting two novel lineages deeply separated from the genera Pseudellipsoidion and Neomonodus, here established as new genera Characiopsiella and Munda. The monophyly of Neomonodaceae as a whole and of each of its four genera recognized here is independently supported by an analysis rbcL sequences, although the two phylogenetic markers suggest a different branching order among the genera. Future studies, ideally employing genome-scale sequence data (such as complete organellar genomes), will help resolve the internal phylogeny of this group. The degree of genetic diversity within the individual Neomonodaceae genera found using the rbcL gene is higher than that apparent from 18S rRNA gene sequence comparisons, in agreement with the known higher evolutionary rate of rbcL in comparison to the 18S rRNA gene (e.g., Patwardhan et al. 2014).

The separate status of the Neomonodaceae is also supported by the recent phylogenomic analysis of multiple plastid genes including one representative of the group, Pseudellipsoidion edaphicum CAUP Q 404 (Ševčíková et al. 2019). The latter analysis placed P. edaphicum with maximal support as a sister lineage to the family Monodopsidaceae and the Eustigmataceae group combined, in agreement with our 18S rRNA gene phylogenies (Figs. 1 and 2) but not the rbcL gene phylogeny, which shows, albeit without support, Neomonodaceae as a sister lineage of the Eustigmataceae group (Fig. 3). Although the branching order of the main Eustigmatales lineages certainly needs to be corroborated by further investigations including multigene phylogenetic analyses of nuclear and mitochondrial sequences, the status of the Neomonodaceae as a family-level lineage separated from all other previously described eustigmatophyte families is firmly established.

Interestingly, inclusion of partial gene sequences from a previous environmental DNA survey (Villanueva et al. 2014) in the analysis of 18S rDNA sequence data revealed that the phylogenetic diversity of the Neomonodaceae is not limited to the four recognized genera. A diverse cluster of environmental DNA sequences is nested within the Neomonodaceae clade as a lineage that seems to correspond to a hitherto unknown separate genus, if not multiple separate genera (Fig. 2). This cluster was previously referred to as the Group 3 (to distinguish it from four additional clusters represented solely by sequences from uncultivated eustigmatophytes; Villanueva et al. 2014) and the authors could not recognize its actual phylogenetic position within Eustigmatales because of the lack of 18S rRNA sequences from characterized members of the Neomonodaceae at that time. The number of different yet related genotypes constituting the Group 3, which all come from a single lake, is surprising. It may reflect a true genetic (and presumably taxonomic) diversity of this novel clade, but the presence of multiple different copies of the 18S rRNA gene in the same genome (i.e., its intragenomic heterogeneity) might also partly account for this apparent diversity (e.g., Alverson and Kolnick 2005). Isolation of the algae representing the Group 3 and their careful investigation is crucial for proper interpretation of the results of the environmental DNA survey.

Interestingly, we could now also illuminate the identity of the Group 2 defined by Villanueva et al. (2014; see Fig. S1). From our results, the Group 2 lineage includes the recently described Paraeustigmatos columelliferus (Fawley et al. 2019), which represents a novel separate lineage sister to all the previously known Eustigmatales including Neomonodaceae (see also the position of P. columelliferus, referred to as strain Mont 10/10–1w, in the plastid phylogenomic analysis by Ševčíková et al. 2019). Thus, P. columelliferus may be the first encountered representative of a diverse eustigmatophyte clade for which a new formal taxon – perhaps a new family – may be established in the future. Interpretation of the family status of two more clusters of eustigmatophyte environmental DNA sequences (i.e., Groups 4 and 5) will also depend on direct characterization of the organisms behind the sequences and on the eventual formal taxonomic treatment of the phylogenetically adjacent Eustigmataceae group.

Morphology and ultrastructure of the Neomonodaceae

The cytology of all studied members of the Neomonodaceae (Figs. 4–8) conforms to the diagnostic features used to segregate the Eustigmatophyceae from the Xanthophyceae. The most distinctive features are the presence of a reddish globule (sometimes more than one) in the vegetative cell and the exclusively eustigmatophycean lamellate vesicles, also present in the zoospores. The zoospores have a unique eyespot composition of extraplastidial droplets positioned near the long flagellum (Hibberd and Leedale 1970, 1971, 1972, Hibberd 1980). Although an absence of a connection between the chloroplast endoplasmic reticulum and the nuclear envelope has been considered a general eustigmatophyte characteristic separating them from other ochrophytes (Hibberd and Leedale 1970, 1972), a connection was observed in TEM preparations of the Neomonodaceae member Munda aquilonaris. The preservation of the continuity of those membranes has previously been documented for Monodopsis and Nannochloropsis species (Antia et al. 1975, Lubián 1982, Maruyama et al. 1986, Santos and Leedale 1995). This suggests that a more detailed investigation by employing electron tomography is needed to rule out that the nucleus-chloroplast connection has simply been overlooked in the majority of eustigmatophytes.

One of the most conspicuous characteristics of eustigmatophytes is an orange-reddish globule usually found in vegetative cells, often more than one in larger or older cells. It is present in all Neomonodaceae, seen in light microscopy (Figs. 4C, 5D, 7A). It has a typical structure composed of many adjacent droplets not bound by a membrane (Fig. 4G), as previously reported for other members of the eustigmatophyte class (Hibberd and Leedale 1972, Santos and Leedale 1995, Santos 1996, Eliáš et al. 2017). Its lipidic nature was hypothesized by Hibberd (1980), and a possible relation with lipid globules released from the chloroplast in Trachydiscus minutus has been considered (Přibyl et al. 2012). Lipids are the most acknowledged reserve material found in eustigmatophytes and are of biotechnological importance, especially for biofuel and food purposes (Gao et al. 2018). The accumulation of lipid droplets in the cytoplasm often has been reported (Schnepf et al. 1996, Přibyl et al. 2012). Lipid droplets were frequently observed in old cells of the Neomonodaceae (Figs. 4D and 6A). Lamellate vesicles have been described as another typical feature of eustigmatophytes (Hibberd and Leedale 1972, Santos and Leedale 1995), so far consistently found in all analyzed eustigmatophytes (Santos 1996), including the Neomonodaceae (Figs. 4, A and E, F, 5 A, 6 B, 7 C). The origin, composition and function of these structures remains unclear, but a polysaccharide nature, possibly paramylon-like, has been suggested (Schnepf et al. 1996).

Reproduction of eustigmatophytes is usually autosporic and production of zoospores can occur in some genera; however, sexual reproduction has not been reported (Eliáš et al. 2017). In the Neomonodaceae reproduction is achieved by both the formation of autospores and the formation of rounded or elongated flask-shaped zoospores (Fig. 8). The presence of a unique type of extra-plastidial eyespot in zoospores, associated with a swelling at the base of the anterior, long and mastigoneme-bearing flagellum is one of the most typical features of the Eustigmatophyceae and is the basis of its name (Hibberd and Leedale 1972). This structure has been detected in TEM sections of the studied strains, as expected. Zoosporic eustigmatophytes are characterized by an emerging long mastigoneme-bearing flagellum and a second flagellum that may be reduced to the basal body or emerge from the cell as a second shorter and thinner flagellum (Hibberd 1970, Santos 1996). Two emergent flagella were detected in representatives of the Neomonodaceae, with shadowcast preparations in Pseudellipsoidion sp. WTwin 8/18 T-5d and in Munda aquilonaris ACOI 2424 revealing a shorter flagellum (Fig. 5, D and E). The previous report on P. edaphicum CAUP Q 401 indicated only one, long emerging flagellum (Neustupa and Nĕmcová 2001) but the presence of a second smaller flagellum may be interpreted from the published shadowcast photo (fig. 16 in Neustupa and Nĕmcová 2001); the description of the species has been emended below to reflect this. Zoospores with two flagella were previously reported for Pseudocharaciopsis ovalis CAUP Q 301 (Neustupa and Nĕmcová 2001), here classified in the genus Neomonodus. Hence, zoospores with two flagella are probably a common characteristic of Neomonodaceae, although this needs to be confirmed for the genus Characiopsiella.

No morphological character stands out as potentially synapomorphic for Neomonodaceae, but two traits – the absence of a pyrenoid and zoospores having two flagella – are noteworthy, as their combination may be unique for this family. The consistent absence of a pyrenoid in the Neomonodaceae constitutes a distinctive morphological character separating the members of this family from Characiopsis-like eustigmatophytes belonging to the Eustigmataceae group (Amaral et al. 2011, R. Amaral, T. Ševčíková, M. Eliáš and L.M.A. Santos, unpub. data). Note that a pyrenoid was originally reported by light microscopy in some cells of Pseudellipsoidion edaphicum CAUP Q 401 (Neustupa and Němcová 2001). However, the strain was re-evaluated in the present study and no pyrenoid was found, so the absence of a pyrenoid is considered a common feature for all studied members of the family.

A polyhedral pyrenoid in the vegetative cells was originally listed as one of the characteristics of the Eustigmatophyceae, with a possible exception noted for Ellipsoidion acuminatum CCAP 822/1 (Hibberd and Leedale 1970, 1971) that was subsequently confirmed by the authors (Hibberd and Leedale 1972). The strain was later re-identified as Monodus ovalis and reclassified as Pseudocharaciopsis ovalis by Hibberd (1981). The author pointed to the fact that the absence of a pyrenoid in P. ovalis contrasts with the presence of a spherical stalked pyrenoid in P. minutum, but considered this difference not substantial enough to place the two species in different genera. The other morphological and ultrastructural characters of both vegetative cells and zoospores of the CCAP 822/1 strain as reported by Hibberd are indeed consistent with his species identification, implying the strain might be Neomonodus ovalis or a closely allied species. It is impossible now to verify this identity by molecular data, because the culture CCAP 822/1 maintained in the Culture Collection of Algae and Protozoa now represents a scenedesmacean alga (data not shown, but see also images of the strain provided by the CCAP collection, https://www.ccap.ac.uk/strain_info.php?Strain_No=822/1) and the original alga has most likely been lost.

Additional pyrenoid-less eustigmatophytes, unrelated to Neomonodaceae, are now known (Eliáš et al. 2017, Fawley et al. 2019). It remains to be determined whether the distribution of pyrenoid-less taxa in the eustigmatophyte phylogeny reflects multiple independent origins or multiple independent losses of the pyrenoid. In contrast, biflagellated zoospores are clearly a plesiomorphic character in eustigmatophytes, retained by Neomonodaceae and at least one independent lineage represented by Pseudocharaciopsis minuta (=P. texensis) in the Eustigmataceae group (Lee and Bold 1973). In addition, biflagellated zoospores were documented from Botryochloropsis similis (Preisig and Wilhelm 1989), a colonial eustigmatophyte with a phylogenetic position that remains undetermined because of the lack of molecular data (the culture is no longer available). Since B. similis also lacks a pyrenoid, it may in fact belong to the Neomonodaceae. The other eustigmatophytes without a pyrenoid are known to produce uniflagellate zoospores, as is the case of Pseudostaurastrum limneticum and Trachydiscus minutus (Schnepf et al. 1996, Přibyl et al. 2012) or do not produce zoospores (at least at conditions tested), which is the case of the genera Nannochloropsis and Microchloropsis (Eliáš et al. 2017) and the recently described Paraeustigmatos columelliferus (Fawley et al. 2019).

There are no striking morphologic characters distinguishing the organisms belonging to the four Neomonodaceae genera, so molecular data is crucial for distinguishing the genera in this family. There are nevertheless some differences which may indicate the genus when molecular data is not yet available. Pseudellipsoidion stands out of the other three genera because its cells are devoid of a stipe. The stipitate genera Neomonodus, Characiopsiella and Munda present narrow morphological differences, with Characiopsiella having on average smaller cells than those found in the cultures of Neomonodus and Munda although some small cells may also be seen in the latter, so this characteristic must be examined and used carefully. For these reasons the genera here presented are delimited based on molecular clades defined by 18S rRNA and rbcL gene phylogenies.

Taxonomic considerations

Three genera of Neomonodaceae are established here to accommodate species that have been placed in the genus Characiopsis since their description (Characiopsiella, Munda) or at least for a transient period of time (Neomonodus). They form a weakly supported clade excluding Pseudellipsoidion in the 18S rRNA gene tree (Figs. 1 and 2), but the rbcL tree suggests (with stronger bootstrap support) that they are paraphyletic with respect to Pseudellipsoidion (Fig. 3). Regardless of the uncertain branching order within Neomonodaceae, the molecular data justify the description of these three Characiopsis-like lineages as separate genera, since in both 18S rRNA and rbcL gene trees they are resolved as lineages just as deeply diverged from each other and from the Pseudellipsoidion lineage as are various other pairs of eustigmatophyte taxa classified is separate genera (e.g., Nannochloropsis and Microchloropsis; Figs. 1 and 3).

Another question is whether three new genera are needed for the three Characiopsis-like Neomonodaceae lineages or whether any of them could retain its current generic assignment or be placed into another existing genus. An obvious possibility is that one of the lineages equates to the genus Characiopsis. The identity of this genus and its actual phylogenetic provenance (Eustigmatophyceae versus Xanthophyceae) have remained unclear, partly because of the uncertainties concerning the type of the genus discussed by Hibberd (1981). However, as we will discuss in detail elsewhere (R. Amaral, T. Ševčíková, M. Eliáš and L.M.A. Santos, unpub. data), there is now little doubt that Characiopsis is a eustigmatophyte typified by the species Characiopsis minuta, presently referred to as Pseudocharaciopsis minuta and belonging to the Eustigmataceae group (Fig. 1). Hence, the name Characiopsis is not applicable to any of the lineages of the Neomonodaceae family.

No alternative generic placements have been previously proposed for the species presently known as Characiopsis aquilonaris and Characiopsis minima, so new genera need to be established for them. Our proposal to establish the third new genus, Neomonodus, requires a more elaborate justification. Chodat (1913) described Monodus ovalis as a species of the new genus Monodus he erected in the same study. Subsequently, Chodat (in Poulton 1925) transferred the species to the genus Characiopsis as Characiopsis ovalis, but Hibberd (1981) later moved the species to still another genus, Pseudocharaciopsis. Molecular phylogenetic evidence from multiple isolates that morphologically fit the description of Monodus ovalis clearly shows it cannot be placed in the genus Pseudocharaciopsis, since the 18S rRNA gene sequence from the authentic strain of the type species of the genus (P. texensis UTEX 2113, now referred to as P. minuta UTEX 2113) places it robustly in the Eustigmataceae group (Fig. 1). The species also cannot remain in the Characiopsis (see above), so its original placement in the genus Monodus must be revisited.

Indeed, as discussed by Silva (1980), Monodus ovalis should be regarded as the original type of the genus Monodopsis. However, the transfer of M. ovalis to the genus Characiopsis by Chodat made the status of other described Monodus species uncertain, which motivated Silva to propose conservation of the genus Monodus with a different type, Monodus acuminatus. This proposal was later approved by the Committee for Algae of the International Association for Plant Taxonomy (Silva 1994). Hence, accepting Monodus ovalis as a member of the genus Monodus would imply that it is specifically related to M. acuminatus, at present usually referred with a changed orthography as M. acuminata (see the respective AlgaeBase record at http://www.algaebase.org/search/species/detail/?species_id=62247; Guiry and Guiry 2019). However, the description of M. acuminata differs from the morphological characteristics of the members of the “P. ovalis” clade in important details, namely in the shape of the cells being always round at one end and sharply acute at the opposite, the presence of a single chloroplast lying only on one side of a cell, and in the absence of an attaching stipe (Ettl 1978). Hence, erecting the new genus Neomonodus for Monodus ovalis and its allies appears to be the best way to finally settle the taxonomic status of this species.

Because authentic strains of the species of the newly established genera Neomonodus, Characiopsiella and Munda no longer exist, we below designate epitypes for the species to stabilize their definition for the future. Each epitype is derived from an existing culture that could be identified without reasonable doubts to the species level.

New and emended taxonomic diagnoses

Neomonodaceae R.Amaral, K.P.Fawley, Nĕmcová, T.Ševčíková, Lukešová, M.W.Fawley, L.M.A.Santos et M.Eliáš, fam. nov.

Unicellular with oval, ellipsoidal or slightly curved elongated cells, sometimes simultaneously present in culture. Free cells without polarity or cells possessing a posterior short attaching stipe and an anterior end rounded, acute or with a papilla. Vegetative cells with one to several chloroplasts, no pyrenoid detected, with a reddish globule and lamellate vesicles. Reproduction by formation of autospores and biflagellate zoospores. Found in ponds, lakes, soil, peat bog soil and metal mine tailings.

TYPE GENUS:

Neomonodus gen. nov.

REMARKS:

The family as delimited here presently includes four genera (together with four formally described species) confirmed as belonging to the family by DNA sequences. Botryochloropsis similis, currently classified as a eustigmatophyte incertae sedis (Eliáš et al. 2017), may be an additional member of Neomonodaceae based on its morphological characteristics.

Neomonodus R.Amaral, K.P.Fawley, Nĕmcová, T.Ševčíková, Lukešová, M.W.Fawley, L.M.A.Santos et M.Eliáš, gen. nov.

Very diverse cell morphologies and sizes in culture (8–11 × 4–5 μm). Most cells with a short stipe (0.2–1.5 μm) and anterior end acute or with a papilla. Usually more than two parietal chloroplasts. The genus is distinguished from other genera with a similar morphology on the basis of 18S rRNA and rbcL sequences.

TYPE SPECIES:

Neomonodus ovalis (Chodat) R.Amaral, K.P.Fawley, Nĕmcová, T.Ševčíková, Lukešová, M.W.Fawley, L.M.A.Santos et M.Eliáš gen. et comb. nov.

BASIONYM:

Monodus ovalis Chodat 1913, In Materiaux pour la Flore Cryptogamique Suisse 4 (2). Berne: 182.

HOLOTYPE:

figs. 156–159 in Chodat 1913.

HOMOTYPIC SYNONYMS:

Characiopsis ovalis (Chodat) Chodat ex Poulton 1925, In Étude sur les Hétérokontes, thèse no. 777, Université de Genève. Geneva: 32. Pseudocharaciopsis ovalis (Chodat) Hibberd 1981, In Notes on the taxonomy and nomenclature of the algal classes Eustigmatophyceae and Tribophyceae (synonym Xanthophyceae). Botanical Journal of the Linnean Society of London 82: 110.

ETYMOLOGY:

the ancient Greek prefix neo meaning new plus the original name Monodus. The genus name was proposed by the late Prof. Paul C. Silva.

EPITYPE (designated here to support holotype): strain CAUP Q 302 permanently preserved in a metabolically inactive state (cryopreserved in liquid nitrogen), deposited at Coimbra Collection of Algae (ACOI), University of Coimbra.

REMARKS:

DNA sequence data revealed the separation of the five Neomonodus strains into two internal groups (Fig. 2), although the strains do not exhibit striking morphological differences. One group includes three strains with the morphological characteristics of Monodus (Pseudocharaciopsis) ovalis (CAUP Q 302, BCCO_30_2917, and BCCO_30_2918). The second clade comprises strains CAUP Q 301 and ACOI 2437 previously identified as Pseudocharaciopsis ovalis and Characiopsis anabaenae, respectively. Most cells of ACOI 2437 resemble C. ovalis; however, narrower cells are similar to those of Characiopsis anabaenae Pascher 1938. A rigorous comparative morphological study of the Neomonodus clade combined with data from multiple genetic markers are required to decide whether all five strains represent one or multiple separate species. For this reason, we cautiously recommend the strains CAUP Q 301 and ACOI 2437 be considered as unidentified Neomonodus species.

REFERENCE MOLECULAR DATA (GenBank accession numbers): 18S rRNA gene – KF848932, rbcL gene – MN401200.

Pseudellipsoidion (Neustupa et Němcová) Němcová, emend.

Oval to ellipsoidal cells without a stipe. Cells without a pyrenoid. Biflagellate zoospore production observed.

TYPE SPECIES:

Pseudellipsoidion edaphicum Neustupa et Němcová

Vegetative cell shape globular, oval or ellipsoidal. The cell does not possess a pyrenoid. Production of biflagellate zoospores.

REMARKS:

Pseudellipsoidion was erected by Neustupa and Němcová (2001) in order to accommodate P. edaphicum CAUP Q 401. Present reinvestigation of this strain revealed, contrary to the initial report, the absence of a pyrenoid and zoospores being biflagellate rather than having only one flagellum, necessitating emendation of the original diagnosis. Sequences of the rbcL gene show the existence of five substantially different internal lineages within Pseudellipsoidion, which may be interpreted at the species level once more morphological and molecular data are available. Additional strains of Pseudellipsoidion spp. may also be required to fully assess the species-level taxonomy.

REFERENCE MOLECULAR DATA (GenBank accession numbers): 18S rRNA gene – KF848933, rbcL gene – MK281457.

Characiopsiella R.Amaral, K.P.Fawley, Nĕmcová, T.Ševčíková, Lukešová, M.W.Fawley, L.M.A.Santos et M.Eliáš, gen. nov.

Small cells 5–8 × 3–4 μm, oval or ellipsoidal, with a short attaching stipe, producing zoospores. The genus is distinguished from other genera with a similar morphology on the basis of 18S rRNA and rbcL sequences.

TYPE SPECIES:

Characiopsiella minima (Pascher) R.Amaral, K.P.Fawley, Nĕmcová, T.Ševčíková, Lukešová, M.W.Fawley, L.M.A.Santos et M.Eliáš, gen. et comb. nov.

BASIONYM:

Characiopsis minima Pascher 1938, In Heterokonten, Kryptogamen-Flora von Deutschland, Österreich und der Schweiz. (Rabenhorst, L. Eds) Vol. 11, Teil 5, Akademische Verlagsgesellschaft, Leipzig: 731–732.

HOLOTYPE:

fig. 582 in Pascher 1938.

EPITYPE (designated here to support holotype): strain ACOI 2426 permanently preserved in a metabolically inactive state (cryopreserved in liquid nitrogen), deposited at Coimbra Collection of Algae (ACOI), University of Coimbra.

ETYMOLOGY:

The name is derived from Characiopsis and the Latin diminutive suffix –ella in reference to the morphological resemblance to Characiopsis species and the small size of the cells.

REMARKS:

The genus is presently considered monotypic, as the two strains representing it (ACOI 2426 and ACOI 2423A) are morphologically highly similar and exhibit identical 18S rRNA and rbcL gene sequences.

REFERENCE MOLECULAR DATA (GenBank accession numbers): 18S rRNA gene – MN389511, rbcL gene – MN401194.

Munda R. Amaral, K.P. Fawley, Y. Nĕmcová, T. Ševčíková, A. Lukešová, M.W. Fawley, L.M.A. Santos et M. Eliáš, gen. nov.

Cells 9–11 × 3–4 μm, elliptical to cylindrical with a short stipe and fewer than five large parietal chloroplasts. Production of biflagellate zoospores. The genus is distinguished from other genera with a similar morphology on the basis of 18S rRNA and rbcL sequences.

TYPE SPECIES:

Munda aquilonaris (Skuja) R.Amaral, K.P.Fawley, Nĕmcová, T.Ševčíková, A. Lukešová, M.W.Fawley, L.M.A.Santos et M.Eliáš, gen. et comb. nov.

BASIONYM:

Characiopsis aquilonaris Skuja 1964, In Grundzüge der Algenflora und Algenvegetation der Fjeldgegenden um Abisko in Schwedisch-Lappland. Nova Acta Regiae Societatis Scientiarum Upsaliensis, Series 4, 18(3): 333.

HOLOTYPE:

Tab. LXV, fig. 12–13 in Skuja 1964.

EPITYPE (designated here to support holotype): strain ACOI 2424 permanently preserved in a metabolically inactive state (cryopreserved in liquid nitrogen), deposited at Coimbra Collection of Algae (ACOI), University of Coimbra.

ETYMOLOGY:

The genus name is a tribute to the Mondego river, the largest entirely Portuguese river that runs through the city of Coimbra, since all strains were isolated from its basin. Munda is a Roman name for Mondego, meaning clarity and purity.

REMARKS:

Some genetic diversity among the four strains assigned to Munda is apparent from rbcL gene sequences. Three of them (ACOI 2424, ACOI 2424A, ACOI 2424B) have identical rbcL sequences and can be unambiguously identified as Characiopsis aquilonaris, whereas the forth does not fit the description of this species that well and differs from the other three strains at four positions of the rbcL gene, so it may represent a separate species and is hence cautiously identified as Munda sp. ACOI 2428.

REFERENCE MOLECULAR DATA (GenBank accession numbers): 18S rRNA gene – MN389513, rbcL gene – MN401191.

CONCLUSIONS

Our expanded sampling and the analysis of rbcL gene sequences in addition to 18S rRNA gene sequences corroborate the former Pseudellipsoidion group as a robustly monophyletic familial lineage within the Eustigmatales, here formalized as the family Neomonodaceae. We established a new genus, Neomonodus, to hopefully provide a final taxonomic home for the species introduced to science as Monodus ovalis and subsequently moved to different genera, the most recent being the polyphyletic genus Pseudocharaciopsis. By obtaining the first ultrastructural and molecular data from Characiopsis minima and Characiopsis aquilonaris we demonstrated that these algae are eustigmatophytes, further enriching the diversity of this class at the expense of xanthophytes. At the same time, we show that the genus Characiopsis, as presently conceived, is polyphyletic, which we partly solve by erecting two new genera, Characiopsiella and Munda in the Neomonodaceae to include Characiopsiella minima and Munda aquilonaris. Our study thus takes an important step towards modern classification of eustigmatophytes. Further work on the Neomonodaceae has to be done to clarify the taxonomic significance of the genetic diversity apparent within individual genera and a comprehensive reassessment of the large genus Characiopsis is needed to resolve its identity and scope.

Supplementary Material

Supp FigS1

Phylogeny of Eustigmatophyceae based on sequences of the 18S rRNA gene including partial sequences from environmental DNA surveys. The tree was inferred using RAxML (GTR+Γ model). A selection of representative non-eustigmatophyte ochrophytes is used as an outgroup. Bootstrap values (based on 354 rapid bootstrap replicates) are shown when higher than 50. The main eustigmatophyte clades are highlighted by different colour background. The five groups of partial sequences from uncultivated eustigmatophytes obtained by Villanueva et al. (2014) are labelled accordingly as Group 1 to Group 5.

Supp TableS1

Morphological characters, collection sites, former and new taxonomic assignment and GenBank accession numbers for 18S rRNA and rbcL gene sequences of the Neomonodaceae strains. ACOI - Coimbra Collection of Algae, Portugal; CAUP - Culture Collection of Algae at Charles University in Prague, Czech Republic, Tow, WTwin and Mary strains are kept in the laboratory of K. and M. Fawley, University of the Ozarks, U.S.A; BCCO strains as well as CAUP Q 302 are kept in the Culture Collection of Soil Algae and Cyanobacteria of the Institute of Soil Biology BC CAS, member of BCCO (Biology Centre Collection of Organisms).

Supp TableS2

Primers used for the amplification and sequencing of the 18S and rbcL genes of the studied strains.

Abbreviations:

ACOI

Coimbra Collection of Algae

BCCO

Institute of Soil Biology in the Biology Centre Collection of Organisms

CAUP

Culture Collection of Algae at Charles University

DIC

differential interference contrast