Species concept and nomenclatural changes within the genera Elliptochloris and Pseudochlorella (Trebouxiophyceae) based on an integrative approach

The genera Elliptochloris and Pseudochlorella were erected for Chlorella‐like green algae producing two types of autospores and cell packages, respectively. Both genera are widely distributed in different soil habitats, either as free living or as photobionts of lichens. The species of these genera are often difficult to identify because of the high phenotypic plasticity and occasional lack of characteristic features. The taxonomic and nomenclatural status of these species, therefore, remains unclear. In this study, 34 strains were investigated using an integrative approach. Phylogenetic analyses demonstrated that the isolates belong to two independent lineages of the Trebouxiophyceae (Elliptochloris and Prasiola clades) and confirmed that the genera are not closely related. The comparison of morphology, molecular phylogeny, and analyses of secondary structures of SSU and ITS rDNA sequences revealed that all of the strains belong to three genera: Elliptochloris, Pseudochlorella, and Edaphochlorella. As a consequence of the taxonomic revisions, we propose two new combinations (Elliptochloris antarctica and Pseudochlorella signiensis) and validate Elliptochloris reniformis, which is invalidly described according to the International Code for Nomenclature (ICN), by designating a holotype. To reflect the high phenotypic plasticity of P. signiensis, two new varieties were described: P. signiensis var. magna and P. signiensis var. communis. Chlorella mirabilis was not closely related to any of these genera and was, therefore, transferred to the new genus Edaphochlorella. All of the taxonomic changes were highly supported by all phylogenetic analyses and were confirmed by the ITS‐2 Barcodes using the ITS‐2/CBC approach.     

Genetic variability and taxonomic revision of the genus Auxenochlorella (Shihira et Krauss) Kalina et Puncocharova (Trebouxiophyceae,Chlorophyta)

The monotypic genus Auxenochlorella with its type species A. protothecoides is so far only known from specific habitats such as the sap of several tree species. Several varieties were described according to physiological performances in culture on different organic substrates. However, two strains designated as Auxenochlorella were isolated from other habitats (an endosymbiont of Hydra viridis and an aquatic strain from an acidic volcano stream). We studied those isolates and compared them with six strains of Auxenochlorella belonging to different varieties. The integrative approach used in this study revealed that all strains showed similar morphology but differed in their SSU and ITS rDNA sequences. The Hydra endosymbiont formed a sister taxon to A. protothecoides, which included the varieties protothecoides, galactophila, and communis. The variety acidicola is not closely related to Auxenochlorella and represented its own lineage within the Trebouxiophyceae. In view of these results, we propose a new species of Auxenochlorella, A. symbiontica, for the Hydra symbiont, and a new genus Pumiliosphaera, with its type species, P. acidophila, for acidophilic strain. These results are supported by several compensatory base changes in the conserved region of ITS‐2 and ITS‐2 DNA barcodes.     

Four New Members of Foliicolous Green Algae Within the Watanabea Clade (Trebouxiophyceae,Chlorophyta) from China

Members of the Watanabea clade of Trebouxiophyceae are genetically diverse and widely distributed in all kinds of habitats, especially in most terrestrial habitats. Ten new strains of terrestrial algae isolated from the tropical rainforest in China, and four published strains were investigated in this study. Morphological observation and molecular phylogenetic analyses based on the 18S, ITS, rbcL, and tufA genes were used to identify the new strains. Four previously described species were reinvestigated to supplement molecular data and autospores’ morphological photographs. The phylogenetic analyses based on 18S only, the concatenated dataset of 18S and ITS, as well as the concatenated dataset of rbcL and tufA, showed the same phylogenetic positions and relationships of these new strains. According to the phylogenetic analysis and morphological comparisons results, we described these 10 strains as four new members within the Watanabea clade, Polulichloris yunnanensis sp. nov., Polulichloris ovale sp. nov., Massjukichlorella orientale sp. nov., and Massjukichlorella minus sp. nov., and two known species, Massjukichlorella epiphytica, and Mysteriochloris nanningensis. Additionally, we provide strong evidence proving that Phyllosiphon, Mysteriochloris, Polulichloris, and Desertella all reproduce through unequal sized autospores.     

Classification of crucigenoid algae: phylogenetic position of the reinstated genus Lemmermannia,Tetrastrum spp. Crucigenia tetrapedia,and C. lauterbornii (Trebouxiophyceae,Chlorophyta)1

The subfamily Crucigenioideae was traditionally classified within the well‐characterized family Scenedesmaceae (Chlorophyceae). Several morpho‐logical revisions and questionable taxonomic changes hampered the correct classification of crucigenoid species resulting in a high number of synonymous genera. We used a molecular approach to determine the phylogenetic position of several Tetrastrum and Crucigenia species. The molecular results were correlated with morphological and ontogenetic characters. Phylogenetic analyses of the SSU rDNA gene resolved the position of Tetrastrum heteracanthum and T. staurogeniaeforme as a new lineage within the Oocystis clade of the Trebouxiophyceae. Crucigenia tetrapedia, T. triangulare, T. punctatum, and T. komarekii were shown to be closely related to Botryococcus (Trebouxiophyceae) and were transferred to Lemmer‐mannia. Crucigenia lauterbornii was not closely related to the other Crucigenia strains, but was recovered within the Chlorella clade of the Trebouxiophyceae.     

Euchlorocystis gen. nov. and Densicystis gen. nov., Two New Genera of Oocystaceae Algae from High‐altitude Semi‐saline Habitat (Trebouxiophyceae,Chlorophyta)

The Oocystaceae family is generally considered to contain common freshwater eukaryotic microalgae, and few are reported living in semi‐saline habitats. Our latest ecological survey in Qinghai Lake and Angzicuo Lake, both large, closed, high‐altitude, semi‐saline lakes located on the Qinghai‐Tibet plateau in China, revealed Oocystaceae species as a dominant group among plankton. Since limited knowledge exists about semi‐saline species in the Oocystaceae family, a taxonomical study was carried out using morphological and phylogenetic methods. Using this approach, four new strains of Oocystaceae were identified and successfully cultured in the lab. Molecular results correlated with morphological characters and resolved these species into at least three genera. A new genus, Euchlorocystis, with type species Euchlorocystis subsalina, is described here as having the distinctive morphology of multiple pyrenoids per chloroplast among Oocystaceae, and an independent phylogenetic position at the base of the Oocystaceae. Similarly, the genus Densicystis, with type species Densicystis glomerata, is newly proposed here as having a unique colony morphology of dozens or hundreds of little cells tightly embedded in ellipsoid to round mucilage masses. Oocystis marina, originally described from the Baltic Sea, was also identified in Qinghai Lake and Angzicuo Lake and phylogenetically positioned in the semi‐saline clade of the Oocystaceae. The result that a marine species was detected in the closed inland lakes implies a further need to reevaluate the origins of these species.     

Reconsideration of the taxonomy of ellipsoidal species of Chlorella (Trebouxiophyceae, Chlorophyta), with establishment of Watanabea sen. nov.

Subcultures of SAG 211–9b and 1AM C-211, ultimately derived from CCAP 211/9b, a strain isolated by Pringsheim in 1939 and identified as Chlorella sac-charophila (Kruger) Migula were observed using light and electron microscopy. Their morphology proved to be basically identical. Both have two forms of cells, one (E-form) narrowly to broadly ellipsoidal, the other (S-form) ovoid to spheroidal. The cell wall of both forms is composed of a single smooth layer. The chloroplast of young cells is trough-like or saucer-shaped with a smooth margin, while that of mature cells is band- or cup-shaped with deep incisions. The thylakoid lamellae are loosely stacked and neither form has a pyrenoid. Both types of cells are capable of producing autospores: eight to 16 in E-form cells, two to four in S-form cells. These morphological features are different from those of C. saccharophila, which has a pyrenoid and produces only one form of autospores. In the absence of any existing genus that includes Chlorella-like algae with a simple cell wall, no pyrenoid, and two forms of mature cells and autospores, a new genus, Watanabea, is proposed with the type species W. reniformis.     

Taxonomic revision of Chlamydomonas subg. Amphichloris (Volvocales,Chlorophyceae), with resurrection of the genus Dangeardinia and descriptions of Ixipapillifera gen. nov. and Rhysamphichloris gen. nov.

Chlamydomonas (Cd.) is one of the largest but most polyphyletic genera of freshwater unicellular green algae. It consists of 400–600 morphological species and requires taxonomic revision. Toward reclassification, each morphologically defined classical subgenus (or subgroup) should be examined using culture strains. Chlamydomonas subg. Amphichloris is characterized by a central nucleus between two axial pyrenoids, however, the phylogenetic structure of this subgenus has yet to be examined using molecular data. Here, we examined 12 strains including six newly isolated strains, morphologically identified as Chlamydomonas subg. Amphichloris, using 18S rRNA gene phylogeny, light microscopy, and mitochondria fluorescent microscopy. Molecular phylogenetic analyses revealed three independent lineages of the subgenus, separated from the type species of Chlamydomonas, Cd. reinhardtii. These three lineages were further distinguished from each other by light and fluorescent microscopy—in particular by the morphology of the papillae, chloroplast surface, stigmata, and mitochondria—and are here assigned to three genera: Dangeardinia emend., Ixipapillifera gen. nov., and Rhysamphichloris gen. nov. Based on the molecular and morphological data, two to three species were recognized in each genus, including one new species, I. pauromitos. In addition, Cd. deasonii, which was previously assigned to subgroup “Pleiochloris,” was included in the genus Ixipapillifera as I. deasonii comb. nov.     

Mixotrophy in the terrestrial green alga Apatococcus lobatus (Trebouxiophyceae,Chlorophyta)

The green microalga Apatococcus lobatus is widely distributed in terrestrial habitats throughout many climatic zones. It dominates green biofilms on natural and artificial substrata in temperate latitudes and is regarded as a key genus of obligate terrestrial consortia. Until now, its isolation, cultivation and application as a terrestrial model organism has been hampered by slow growth rates and low growth capacities. A mixotrophic culturing approach clearly enhanced the accumulation of biomass, thereby permitting the future application of A. lobatus in different types of bio‐assays necessary for material and biofilm research. The ability of A. lobatus to grow mixotrophically is assumed as a competitive advantage in terrestrial habitats.     

Molecular phylogeny and taxonomy of the genus Chaetophora (Chlorophyceae,Chlorophyta), including descriptions of Chaetophoropsis aershanensis gen. et sp. nov.

The broadly defined genus Chaetophora consisted of species with minute, uniseriate branching filaments enveloped in soft or firm mucilage forming macroscopic growths that are spherical, hemispherical, and tubercular or arbuscular, growing epiphytically on freshwater aquatic plants and other submerged surfaces in standing or fast‐flowing water. Recent molecular analyses clearly showed that this genus was polyphyletic. In this study, eight strains of Chaetophora and three strains of Stigeoclonium were identified and successfully cultured. In combination with the morphological data, a concatenated data set of four markers (18S + 5.8S + ITS2+ partial 28S rDNA) was also used to determine their taxonomic relationships and phylogenetic positions. The molecular analysis resolved the broadly defined Chaetophora to at least two genera. Species with a globose thallus of genus Chaetophora formed a separate monophyletic clade, which clearly separated from, a type of lobe‐form Chaetophora species. Therefore, we propose to erect a new genus, Chaetophoropsis, which includes all globose species of the Chaetophora. Chaetophoropsis aershanensis was determined to be a new species, based on its special characteristic of profuse long rhizoids. Stigeoclonium polyrhizum, as the closest relative to Chaetophoropsis, revealed its distant relationships to other species of Stigeoclonium. A globose thallus with a thick, soft mucilage matrix, and special rhizoidal branches lent further support to the placement of S. polyrhizum in the genus Chaetophoropsis and had the closest relationship to C. aershanensis. Taxonomic diversity was proven by distinctive morphological differences and by phylogenetic divergence in the broadly defined Chaetophora identified herein.     

Molecular phylogeny of the Cladophoraceae (Cladophorales,Ulvophyceae), with the resurrection of Acrocladus Nägeli and Willeella Børgesen,and the description of Lurbica gen. nov. and Pseudorhizoclonium gen. nov.

The taxonomy of the Cladophoraceae, a large family of filamentous green algae, has been problematic for a long time due to morphological simplicity, parallel evolution, phenotypic plasticity, and unknown distribution ranges. Partial large subunit (LSU) rDNA sequences were generated for 362 isolates, and the analyses of a concatenated dataset consisting of unique LSU and small subunit (SSU) rDNA sequences of 95 specimens greatly clarified the phylogeny of the Cladophoraceae. The phylogenetic reconstructions showed that the three currently accepted genera Chaetomorpha, Cladophora, and Rhizoclonium are polyphyletic. The backbone of the phylogeny is robust and the relationships of the main lineages were inferred with high support, only the phylogenetic position of both Chaetomorpha melagonium and Cladophora rupestris could not be inferred unambiguously. There have been at least three independent switches between branched and unbranched morphologies within the Cladophoraceae. Freshwater environments have been colonized twice independently, namely by the freshwater Cladophora species as well as by several lineages of the Rhizoclonium riparium clade. In an effort to establish monophyletic genera, the genera Acrocladus and Willeella are resurrected and two new genera are described: Pseudorhizoclonium and Lurbica.     

The genus Pseudo-nitzschia (Bacillariophyceae) in a temperate estuary with description of two new species: Pseudo-nitzschia plurisecta sp. nov. and Pseudo-nitzschia abrensis sp. nov.

The genus Pseudo‐nitzschia contains potentially toxic species of problematic taxonomy, making it one of the most intensively studied diatom genera. The study of 35 clonal strains isolated from the Bilbao estuary, an area that experiences recurrent blooms of Pseudo‐nitzschia, revealed the presence of two new species, P. abrensis and P. plurisecta, differing from their congeners in both morphology and gene sequence. The morphological features were analyzed by LM and EM, whereas molecular analyses were based on the internal transcribed spacer (ITS) and large subunit (LSU) regions of the rDNA. P. plurisecta appears closely related to P. cuspidata/P. pseudodelicatissima in the phylogenetic tree, whereas P. abrensis forms a moderately supported clade with P. heimii/P. subpacifica and P. caciantha/P. circumpora. Comparison of the secondary structure of ITS2 regions reveals marked differences in the most highly conserved regions among related taxa. Morphologically, the new species differ from their closest congeners in the arrangement of the poroid sectors and the density of valve striae and fibulae. The two species share similar pigment composition, and belong to the group of Pseudo‐nitzschia species containing only chlorophyll c₂ and c₃.     

Coccomyxa actinabiotis sp. nov. (Trebouxiophyceae,Chlorophyta), a new green microalga living in the spent fuel cooling pool of a nuclear reactor

Life can thrive in extreme environments where inhospitable conditions prevail. Organisms which resist, for example, acidity, pressure, low or high temperature, have been found in harsh environments. Most of them are bacteria and archaea. The bacterium Deinococcus radiodurans is considered to be a champion among all living organisms, surviving extreme ionizing radiation levels. We have discovered a new extremophile eukaryotic organism that possesses a resistance to ionizing radiations similar to that of D. radiodurans. This microorganism, an autotrophic freshwater green microalga, lives in a peculiar environment, namely the cooling pool of a nuclear reactor containing spent nuclear fuels, where it is continuously submitted to nutritive, metallic, and radiative stress. We investigated its morphology and its ultrastructure by light, fluorescence and electron microscopy as well as its biochemical properties. Its resistance to UV and gamma radiation was assessed. When submitted to different dose rates of the order of some tens of mGy · h^?1 to several thousands of Gy · h^?1, the microalga revealed to be able to survive intense gamma‐rays irradiation, up to 2,000 times the dose lethal to human. The nuclear genome region spanning the genes for small subunit ribosomal RNA‐Internal Transcribed Spacer (ITS) 1‐5.8S rRNA‐ITS2‐28S rRNA (beginning) was sequenced (4,065 bp). The phylogenetic position of the microalga was inferred from the 18S rRNA gene. All the revealed characteristics make the alga a new species of the genus Coccomyxa in the class Trebouxiophyceae, which we name Coccomyxa actinabiotis sp. nov.     

Molecular and morphological delimitation and generic classification of the family Oocystaceae (Trebouxiophyceae,Chlorophyta)

The family Oocystaceae (Chlorophyta) is a group of morphologically and ultrastructurally distinct green algae that constitute a well‐supported clade in the class Trebouxiophyceae. Despite the family's clear delimitation, which is based on specific cell wall features, only a few members of the Oocystaceae have been examined using data other than morphological. In previous studies of Trebouxiophyceae, after the establishment of molecular phylogeny, the taxonomic status of the family was called into question. The genus Oocystis proved to be paraphyletic and some species were excluded from Oocystaceae, while a few other species were newly redefined as members of this family. We investigated 54 strains assigned to the Oocystaceae using morphological, ultrastructural and molecular data (SSU rRNA and rbcL genes) to clarify the monophyly of and diversity within Oocystaceae. Oonephris obesa and Nephrocytium agardhianum clustered within the Chlorophyceae and thus are no longer members of the Oocystaceae. On the other hand, we transferred the coenobial Willea vilhelmii to the Oocystaceae. Our findings combined with those of previous studies resulted in the most robust definition of the family to date. The division of the family into three subfamilies and five morphological clades was suggested. Taxonomical adjustments in the genera Neglectella, Oocystidium, Oocystis, and Ooplanctella were established based on congruent molecular and morphological data. We expect further taxonomical changes in the genera Crucigeniella, Eremosphaera, Franceia, Lagerheimia, Oocystis, and Willea in the future.     

Brandtia ciliaticola gen. et sp. nov. (Chlorellaceae,Trebouxiophyceae) a common symbiotic green coccoid of various ciliate species

Many freshwater protists harbor unicellular green algae within their cells and these host‐symbiont relationships slowly are becoming better understood. Recently, we reported that several ciliate species shared a single species of symbiotic algae. Nonetheless, the algae from different host ciliates were each distinguishable by their different genotypes, and these host‐algal genotype combinations remained unchanged throughout a 15‐month period of sampling from natural populations. The same algal species had been reported as the shared symbiont of several ciliates from a remote lake. Consequently, this alga appears to play a key role in ciliate‐algae symbioses. In the present study, we successfully isolated the algae from ciliate cells and established unialgal cultures. This species is herein named Brandtia ciliaticola gen. et sp. nov. and has typical ‘Chlorella‐like’ morphology, being a spherical autosporic coccoid with a single chloroplast containing a pyrenoid. The alga belongs to the Chlorella‐clade in Chlorellaceae (Trebouxiophyceae), but it is not strongly connected to any of the other genera in this group. In addition to this phylogenetic distinctiveness, a unique compensatory base change in the SSU rRNA gene is decisive in distinguishing this genus. Sequences of SSU‐ITS (internal transcribed spacer) rDNA for each isolate were compared to those obtained previously from the same host ciliate. Consistent algal genotypes were recovered from each host, which strongly suggests that B. ciliaticola has established a persistent symbiosis in each ciliate species.     

Molecular and morphological evidence for Sheathia gen. nov. (Batrachospermales,Rhodophyta) and three new species

The freshwater red algal genus Batrachospermum has been shown to be paraphyletic since the first molecular studies of the Batrachospermales. Previous research, along with this study, provides strong support for the clade Batrachospermum section Helminthoidea. This study has found that heterocortication, the presence of both cylindrical and bulbous cells on the main axis, is an underlying synapomorphy of this clade. Based on support from DNA sequences of the rbcL gene, the COI barcode region and the rDNA ITS 1 and 2, along with morphological studies, the new genus Sheathia is proposed. Seven heterocorticate species were recognized from the molecular clades. Sheathia boryana and S. exigua sp. nov. appear to be restricted to Europe, whereas S. confusa occurs in Europe and New Zealand. Sheathia involuta is widespread in the USA and reported for the first time from Europe. Sheathia americana sp. nov., has been collected in the USA and Canada, and S. heterocortica and S. grandis sp. nov. have been collected only in the USA. Sheathia confusa and S. grandis can be distinguished based on morphological characters, whereas DNA sequence data are required to conclusively distinguish the other species. Sheathia fluitans and S. carpoinvolucra also are placed within this genus based on the presence of heterocortication. These data also hint at greater diversity among non‐heterocorticate Sheathia than is recognized by the single species name S. arcuata.