Phylogeny and evolution of streptophyte algae.

The Streptophyta emerged about a billion years ago. Nowadays, this branch of the green lineage is most famous for one of its clades, the land plants (Embryophyta). Although Embryophyta make up the major share of species numbers in Streptophyta, there is a diversity of probably >5000 species of streptophyte algae that form a paraphyletic grade next to land plants. Here, we focus on the deep divergences that gave rise to the diversity of streptophytes, hence particularly on the streptophyte algae. Phylogenomic efforts have not only clarified the position of streptophyte algae relative to land plants, but recent efforts have also begun to unravel the relationships and major radiations within streptophyte algal diversity. We illustrate how new phylogenomic perspectives have changed our view on the evolutionary emergence of key traits, such as intricate signalling networks that are intertwined with multicellular growth and the chemodiverse hotbed from which they emerged. These traits are key for the biology of land plants but were bequeathed from their algal progenitors.

Numerical and Thermal Response of the Bacterivorous Ciliate Colpidium kleini, a Species Potentially at Risk of Extinction by Rising Water Temperatures.

We investigated the food-dependent growth and thermal response of the freshwater ciliate Colpidium kleini using numerical response (NR) experiments. This bacterivorous ciliate occurs in lotic water and the pelagial of lakes and ponds. The C. kleini strain used in this work was isolated from a small alpine lake and identified by combining detailed morphological inspections with molecular phylogeny. Specific growth rates (rmax) were measured from 5 to 21 °C. The ciliate did not survive at 22 °C. The threshold bacterial food levels (0.3 - 2.2 × 106 bacterial cells mL-1) matched the bacterial abundance in the alpine lake from which C. kleini was isolated. The food threshold was notably lower than previously reported for C. kleini and two other Colpidium species. The threshold was similar to levels reported for oligotrich and choreotrich ciliates if expressed in terms of bacterial biomass (0.05 - 0.43 mg C L-1). From the NR results, we calculated physiological mortality rates at zero food concentration. The mean mortality (0.55 ± 0.17 d-1) of C. kleini was close to the mean estimate obtained for other planktonic ciliates that do not encyst. We used the data obtained by the NR experiments to fit a thermal performance curve (TPC). The TPC yielded a temperature optimum at 17.3 °C for C. kleini, a maximum upper thermal tolerance limit of 21.9 °C, and a thermal safety margin of 4.6 °C. We demonstrated that combining NR with TPC analysis is a powerful tool to predict better a species' fitness in response to temperature and food.

Unexpected cryptic species among streptophyte algae most distant to land plants.

Streptophytes are one of the major groups of the green lineage (Chloroplastida or Viridiplantae). During one billion years of evolution, streptophytes have radiated into an astounding diversity of uni- and multicellular green algae as well as land plants. Most divergent from land plants is a clade formed by Mesostigmatophyceae, Spirotaenia spp. and Chlorokybophyceae. All three lineages are species-poor and the Chlorokybophyceae consist of a single described species, Chlorokybus atmophyticus. In this study, we used phylogenomic analyses to shed light into the diversity within Chlorokybus using a sampling of isolates across its known distribution. We uncovered a consistent deep genetic structure within the Chlorokybus isolates, which prompted us to formally extend the Chlorokybophyceae by describing four new species. Gene expression differences among Chlorokybus species suggest certain constitutive variability that might influence their response to environmental factors. Failure to account for this diversity can hamper comparative genomic studies aiming to understand the evolution of stress response across streptophytes. Our data highlight that future studies on the evolution of plant form and function can tap into an unknown diversity at key deep branches of the streptophytes.

Aquatic food webs in deep temperate lakes: Key species establish through their autecological versatility.

Microbial planktonic communities are the basis of food webs in aquatic ecosystems since they contribute substantially to primary production and nutrient recycling. Network analyses of DNA metabarcoding data sets emerged as a powerful tool to untangle the complex ecological relationships among the key players in food webs. In this study, we evaluated co-occurrence networks constructed from time-series metabarcoding data sets (12 months, biweekly sampling) of protistan plankton communities in surface layers (epilimnion) and bottom waters (hypolimnion) of two temperate deep lakes, Lake Mondsee (Austria) and Lake Zurich (Switzerland). Lake Zurich plankton communities were less tightly connected, more fragmented and had a higher susceptibility to a species extinction scenario compared to Lake Mondsee communities. We interpret these results as a lower robustness of Lake Zurich protistan plankton to environmental stressors, especially stressors resulting from climate change. In all networks, the phylum Ciliophora contributed the highest number of nodes, among them several in key positions of the networks. Associations in ciliate-specific subnetworks resembled autecological species-specific traits that indicate adaptions to specific environmental conditions. We demonstrate the strength of co-occurrence network analyses to deepen our understanding of plankton community dynamics in lakes and indicate biotic relationships, which resulted in new hypotheses that may guide future research in climate-stressed ecosystems.

Choricystis and Lewiniosphaera gen. nov. (Trebouxiophyceae Chlorophyta), two different green algal endosymbionts in freshwater sponges.

Associations of freshwater sponges with coccoid green algae have been known for a long time. Two types of coccoid green algae, which are commonly assigned as zoochlorellae, are recognized by morphology: small coccoids (< 3 µm) without pyrenoids and larger Chlorella-like algae (4-6 µm) with pyrenoids. Despite their wide distribution in some freshwater sponges, these green algae were never studied using a combined analysis of morphology and molecular phylogeny. We investigated several endosymbiotic strains isolated from different Spongilla species, which were available in culture collections. Phylogenetic analyses of SSU and ITS rDNA sequences revealed that the strain SAG 211-40a is a member of the Chlorellaceae and represents a new species of the newly erected genus Lewiniosphaera, L symbiontica. The phylogenetic position was confirmed by morphology and ITS-2 barcode. The endosymbionts without pyrenoid were identified as Choricystis parasitica by morphology and phylogenetic analyses. The comparison with free-living strains revealed the recognition of two new Choricystis species, C. krienitzii and C. limnetica, which were confirmed by molecular signatures in V9 region of SSU rDNA and ITS-2 barcode.

Pleurastrosarcina terriformae, a new species of a rare desert trebouxiophycean alga discovered by an integrative approach.

Biological soil crusts of extreme habitats (semi-deserts and deserts) are dominated by cyanobacteria and microalgae. The most abundant taxa are green algae belonging to the classes Chlorophyceae and Trebouxiophyceae. Specimens with sarcinoid-like morphology (cells arranged in packages) represent one group of these microalgae. The genus Pleurastrosarcina consists of two species, which were originally described as Chlorosarcina (P. brevispinosa and P. longispinosa). Both species are exclusively found from arid soils. However, these species were only reported few times and probably overlooked especially if no akinetes were present. During studying soil samples collected from different regions of the Atacama desert (Chile), we isolated two strains, which were morphologically similar to both Pleurastrosarcina species. The phylogenetic analyses confirmed that they belong to this genus. The ITS-2/CBC approach revealed that both new isolates represent a new species, P. terriformae. The comparison with other available strains demonstrated that this new species is not restricted to South America and was also found in coastal area in Europe. The six investigated strains showed a high phenotypic plasticity, which is reflected in the descriptions of several varieties.

Reevaluation and discovery of new species of the rare genus Watanabea and establishment of Massjukichlorella gen. nov. (Trebouxiophyceae, Chlorophyta) using an integrative approach.

Chlorella-like coccoid green algae are widely distributed in almost all terrestrial habitats and belong to different lineages of the Chlorophyceae and Trebouxiophyceae. The Watanabea clade of the Trebouxiophyceae shows a high genetic biodiversity. Re-investigation of the authentic strain of the rarely found W. reniformis showed several morphological differences compared to the original description. To clarify the taxonomic status of Watanabea, we compared several new isolates with similar morphology. Phylogenetic analyses of the SSU and SSU+ITS rDNA sequences revealed that all new isolates were distinct from W. reniformis. The ITS-2/CBC approach clearly demonstrated that the strains belonging to Watanabea represented species. We emended the generic diagnosis of Watanabea, and proposed four new species. One strain, SAG 2552, represented a separate lineage that we propose as a new genus Massjukichlorella with one species M. epiphytica.

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.

Solotvynia, a New Coccoid Lineage among the Ulvophyceae (Chlorophyta).

Coccoid Ulvophyceae are often overlooked despite their wide distribution. They occur as epiphytes on marine seaweeds or grow on stones or on shells of mussels and corals. Most of the species are not easy to identify based solely on morphology. However, they form two groups based on the flagellated cells during asexual reproduction. The biflagellated coccoids are monophyletic and represent the genus Sykidion (Sykidiales). In contrast, the quadriflagellated taxa are polyphyletic and belong to different genera and orders. The newly investigated strains NIES-1838 and NIES-1839, originally identified as Halochlorococcum, belong to the genus Chlorocystis (C. john-westii) among the order Chlorocystidales. The unidentified strain CCMP 1293 had almost an identical SSU and ITS-2 sequence to Symbiochlorum hainanense (Ignatiales) but showed morphological differences (single chloroplast, quadriflagellated zoospores) compared with the original description of this species (multiple chloroplasts, aplanospores). Surprisingly, the strain SAG 2662 (= ULVO-129), together with the published sequence of MBIC 10461, formed a new monophyletic lineage among the Ulvophyceae, which is highly supported in all of the bootstrap and Bayesian analyses and approximately unbiased tests of user-defined trees. This strain is characterized by a spherical morphology and also form quadriflagellated zoospores, have a unique ITS-2 barcode, and can tolerate a high variation of salinities. Considering our results, we emend the diagnosis of Symbiochlorum and propose the new genus Solotvynia among the new order Solotvyniales.

Prasiolopsis wulf-kochii (Prasiolales, Trebouxiophyceae), a New Species Occurring in Hairs of the Sloth Bradypus tridactylus.

The monotypic genus Prasiolopsis has been known for a long time, but is often overlooked because of difficulties in identification and the morphological variability between uniseriate filaments and cell packages forming pseudoparenchymatic thalli depending on age. We investigated a strain (SAG 84.81) originally denoted as Trichophilus welckeri, which was isolated from the hairs of the sloth Bradypus tridactylus, and compared it with other available strains of Prasiolopsis and of the sister genus Pseudomarvania. Our investigations clearly showed that this strain differed in morphology, especially of the chloroplast, from those originally described for Trichophilus. Phylogenetic analyses of the SSU and ITS rDNA sequences revealed that the strain SAG 84.81 is sister to several strains of P. ramosa within the Prasiola clade (Trebouxiophyceae). Using the ITS-2/CBC approach, we clearly demonstrated that this strain represented a new species of Prasiolopsis, which we proposed here as P. wulf-kochii. In addition, we evaluated the ITS-2/CBC approach by comparing it with the two species of Pseudomarvania. All investigated strains showed CBCs and HCBCs, which support their species delimitation. The sequencing data of Trichophilus welckeri available in GenBank were phylogenetically re-evaluated by including all representatives of the Ulotrichales (Ulvophyceae). Our analyses showed that these sequences formed their own lineage within this order.

The New Genus Caulinema Revealed New Insights into the Generic Relationship of the Order Ulotrichales (Ulvophyceae, Chlorophyta).

Traditionally, the order Ulotrichales comprised green algae of an unbranched, uniseriate, filamentous morphology. However, since the establishment of ultrastructural features, the circumscription of this order has dramatically changed. Some genera and species have been excluded from this order and others with different morphologies (sarcinoid, branched filaments or even parenchymatous taxa) have been included. Phylogenetic analyses have confirmed the monophyly of this order, but its differentiation from the Ulvales and Acrosiphoniales remains difficult because of the lack of synapomorphies at every level (morphology, molecular signatures). To demonstrate the difficulties of placement into genera and orders, we investigated two sarcinoid taxa with the absence of zoospore formation. SSU and ITS rDNA tree topology and the ITS-2/CBC approach revealed that both strains SAG 2661 and CCAP 312/1 belong to Ulosarcina terrestrica and the newly erected genus Caulinema, respectively. The species conception using this approach was evaluated by sequencing the plastid-coding gene tufA, a commonly used barcode marker for green algae. All three molecular markers resulted in similar topologies at the generic and species levels, which is consistent with the ITS-2/CBC approach and tufA for barcoding. The reevaluation of the ultrastructural features revealed that the presence of organic scales on the surfaces of motile cells is characteristic for the order Ulotrichales and can be used for separation from the closely related orders. As a consequence of our study, we propose the new genus Caulinema for strain CCAP 312/1.

Phylogenomic insights into the first multicellular streptophyte.

Streptophytes are best known as the clade containing the teeming diversity of embryophytes (land plants).1,2,3,4 Next to embryophytes are however a range of freshwater and terrestrial algae that bear important information on the emergence of key traits of land plants. Among these, the Klebsormidiophyceae stand out. Thriving in diverse environments-from mundane (ubiquitous occurrence on tree barks and rocks) to extreme (from the Atacama Desert to the Antarctic)-Klebsormidiophyceae can exhibit filamentous body plans and display remarkable resilience as colonizers of terrestrial habitats.5,6 Currently, the lack of a robust phylogenetic framework for the Klebsormidiophyceae hampers our understanding of the evolutionary history of these key traits. Here, we conducted a phylogenomic analysis utilizing advanced models that can counteract systematic biases. We sequenced 24 new transcriptomes of Klebsormidiophyceae and combined them with 14 previously published genomic and transcriptomic datasets. Using an analysis built on 845 loci and sophisticated mixture models, we establish a phylogenomic framework, dividing the six distinct genera of Klebsormidiophyceae in a novel three-order system, with a deep divergence more than 830 million years ago. Our reconstructions of ancestral states suggest (1) an evolutionary history of multiple transitions between terrestrial-aquatic habitats, with stem Klebsormidiales having conquered land earlier than embryophytes, and (2) that the body plan of the last common ancestor of Klebsormidiophyceae was multicellular, with a high probability that it was filamentous whereas the sarcinoids and unicells in Klebsormidiophyceae are likely derived states. We provide evidence that the first multicellular streptophytes likely lived about a billion years ago.

Hidden genetic diversity in the green alga Spirogyra (Zygnematophyceae, Streptophyta).

BACKGROUND: The unbranched filamentous green alga Spirogyra (Streptophyta, Zygnemataceae) is easily recognizable based on its vegetative morphology, which shows one to several spiral chloroplasts. This simple structure falsely points to a low genetic diversity: Spirogyra is commonly excluded from phylogenetic analyses because the genus is known as a long-branch taxon caused by a high evolutionary rate. RESULTS: We focused on this genetic diversity and sequenced 130 Spirogyra small subunit nuclear ribosomal DNA (SSU rDNA) strands of different origin. The resulting SSU rDNA sequences were used for phylogenetic analyses using complex evolutionary models (posterior probability, maximum likelihood, neighbor joining, and maximum parsimony methods). The sequences were between 1672 and 1779 nucleotides long. Sequence comparisons revealed 53 individual clones, but our results still support monophyly of the genus. Our data set did not contain a single slow-evolving taxon that would have been placed on a shorter branch compared to the remaining sequences. Out of 130 accessions analyzed, 72 showed a secondary loss of the 1506 group I intron, which formed a long-branched group within the genus. The phylogenetic relationship to the genus Spirotaenia was not resolved satisfactorily. The genetic distance within the genus Spirogyra exceeded the distances measured within any other genus of the remaining Zygnemataceae included in this study. CONCLUSION: Overall, we define eight distinct clades of Spirogyra, one of them including the genus Sirogonium. A large number of non-homoplasious synapomorphies (NHS; 114 NHS in total) was found for Spirogyra (41 NHS) and for each clade (totaling 73 NHS). This emphasizes the high genetic diversity of this genus and the distance to the remaining Zygnematophyceae.

Morphological diversity and molecular phylogeny of five Paramecium bursaria (Alveolata, Ciliophora, Oligohymenophorea) syngens and the identification of their green algal endosymbionts.

Paramecium bursaria is a mixotrophic ciliate species, which is common in stagnant and slow-flowing, nutrient-rich waters. It is usually found living in symbiosis with zoochlorellae (green algae) of the genera Chlorella or Micractinium. We investigated P. bursaria isolates from around the world, some of which have already been extensively studied in various laboratories, but whose morphological and genetic identity has not yet been completely clarified. Phylogenetic analyses of the SSU and ITS rDNA sequences revealed five highly supported lineages, which corresponded to the syngen and most likely to the biological species assignment. These syngens R1-R5 could also be distinguished by unique synapomorphies in the secondary structures of the SSU and the ITS. Considering these synapomorphies, we could clearly assign the existing GenBank entries of P. bursaria to specific syngens. In addition, we discovered synapomorphies at amino acids of the COI gene for the identification of the syngens. Using the metadata of these entries, most syngens showed a worldwide distribution, however, the syngens R1 and R5 were only found in Europe. From morphology, the syngens did not show any significant deviations. The investigated strains had either Chlorella variabilis, Chlorella vulgaris or Micractinium conductrix as endosymbionts.

The plastomes of Hyalomonas oviformis and Hyalogonium fusiforme evolved dissimilar architectures after the loss of photosynthesis.

The loss of photosynthesis in land plants and algae is typically associated with parasitism but can also occur in free-living species, including chlamydomonadalean green algae. The plastid genomes (ptDNAs) of colorless chlamydomonadaleans are surprisingly diverse in architecture, including highly expanded forms (Polytoma uvella and Leontynka pallida) as well as outright genome loss (Polytomella species). Here, we explore the ptDNAs of Hyalomonas (Hm.) oviformis (SAG 62-27; formerly known as Polytoma oviforme) and Hyalogonium (Hg.) fusiforme (SAG 62-1c), each representing independent losses of photosynthesis within the Chlamydophyceae. The Hm. oviformis ptDNA is moderately sized (132 kb) with a reduced gene complement (but still encoding the ATPase subunits) and is in fact smaller than that of its photosynthetic relative Hyalomonas chlamydogama SAG 11-48b (198.3 kb). The Hg. fusiforme plastome, however, is the largest yet observed in nonphotosynthetic plants or algae (~463 kb) and has a coding repertoire that is almost identical to that of its photosynthetic relatives in the genus Chlorogonium. Furthermore, the ptDNA of Hg. fusiforme shows no clear evidence of pseudogenization, which is consistent with our analyses showing that Hg. fusiforme is the nonphotosynthetic lineage of most recent origin among known colorless Chlamydophyceae. Together, these new ptDNAs clearly show that, in contrast to parasitic algae, plastid genome compaction is not an obligatory route following the loss of photosynthesis in free-living algae, and that certain chlamydomonadalean algae have a remarkable propensity for genomic expansion, which can persist regardless of the trophic strategy.

The systematics of Zoochlorella revisited employing an integrative approach.

Symbiosis of green algae with protozoa and invertebrates has been studied for more than 100 years. Endosymbiotic green algae are widely distributed in ciliates (e.g. Paramecium, Stentor, Climacostomum, Coleps, Euplotes), heliozoa (e.g. Acanthocystis) and invertebrates (e.g. Hydra, Spongilla), and have traditionally been identified as named or unnamed species of Chlorella Beij. or Zoochlorella K. Brandt or referred to as Chlorella-like algae or zoochlorellae. We studied 17 strains of endosymbionts isolated from various hosts and geographical localities using an integrative approach (nuclear encoded small subunit and internal transcribed spacer regions of rRNA gene sequences including their secondary structures, morphology, physiology and virus sensitivity). Phylogenetic analyses have revealed them to be polyphyletic. The strains examined belong to five independent clades within the Trebouxiophyceae (Choricystis-, Elliptochloris-, Auxenochlorella- and Chlorella-clades) and Chlorophyceae (Scenedesmus-clade). The most studied host organism, Paramecium bursaria, harbours endosymbionts representing at least five different species. On the basis of our results, we propose a taxonomic revision of endosymbiotic 'Chlorella'-like green algae. Zoochlorella conductrix K. Brandt is transferred to Micractinium Fresen. and Zoochlorella parasitica K. Brandt to Choricystis (Skuja) Fott. It was shown that Choricystis minor (Skuja) Fott, the generitype, is a later heterotypic synonym of Choricystis parasitica (K. Brandt) comb. nov. A new species, Chlorella heliozoae, is proposed to accommodate the endosymbiont of Acanthocystis turfacea.

UPDATING THE GENUS DICTYOSPHAERIUM AND DESCRIPTION OF MUCIDOSPHAERIUM GEN. NOV. (TREBOUXIOPHYCEAE) BASED ON MORPHOLOGICAL AND MOLECULAR DATA1.

Recent molecular analyses of Dictyosphaerium strains revealed a polyphyletic origin of this morphotype within the Chlorellaceae. The type species Dictyosphaerium ehrenbergianum Nägeli formed an independent lineage within the Parachlorella clade, assigning the genus to this clade. Our study focused on three different Dictyosphaerium species to resolve the phylogenetic position of remaining species. We used combined analyses of morphology; molecular data based on SSU and internally transcribed spacer region (ITS) rRNA sequences; and the comparison of the secondary structure of the SSU, ITS-1, and ITS-2 for species and generic delineation. The phylogenetic analyses revealed two lineages without generic assignment and two distinct clades of Dictyosphaerium-like strains within the Parachlorella clade. One clade comprises the lineages with the epitype strain of D. ehrenbergianum Nägeli and two additional lineages that are described as new species (Dictyosphaerium libertatis sp. nov. and Dictyosphaerium lacustre sp. nov.). An emendation of the genus Dictyosphaerium is proposed. The second clade comprises the species Dictyosphaerium sphagnale Hindák and Dictyosphaerium pulchellum H. C. Wood. On the basis of phylogenetic analyses, complementary base changes, and morphology, we describe Mucidosphaerium gen. nov with the four species Mucidosphaerium sphagnale comb. nov., Mucidosphaerium pulchellum comb. nov., Mucidosphaerium palustre sp. nov., and Mucidosphaerium planctonicum sp. nov.

Molecular Phylogeny of Unicellular Marine Coccoid Green Algae Revealed New Insights into the Systematics of the Ulvophyceae (Chlorophyta).

Most marine coccoid and sarcinoid green algal species have traditionally been placed within genera dominated by species from freshwater or soil habitats. For example, the genera Chlorocystis and Halochlorococcum contain exclusively marine species; however, their familial and ordinal affinities are unclear. They are characterized by a vegetative cell with lobated or reticulated chloroplast, formation of quadriflagellated zoospores and living epi- or endophytically within benthic macroalgae. They were integrated into the family Chlorochytriaceae which embraces all coccoid green algae with epi- or endophytic life phases. Later, they were excluded from the family of Chlorococcales based on studies of their life histories in culture, and transferred to their newly described order, Chlorocystidales of the Ulvophyceae. Both genera form a "Codiolum"-stage that serves as the unicellular sporophyte in their life cycles. Phylogenetic analyses of SSU and ITS rDNA sequences confirmed that these coccoid taxa belong to the Chlorocystidales, together with the sarcinoid genus Desmochloris. The biflagellated coccoid strains were members of the genus Sykidion, which represented its own order, Sykidiales, among the Ulvophyceae. Considering these results and the usage of the ITS-2/CBC approach revealed three species of Desmochloris, six of Chlorocystis, and three of Sykidion. Three new species and several new combinations were proposed.

Lake Ecosystem Robustness and Resilience Inferred from a Climate-Stressed Protistan Plankton Network.

Network analyses of biological communities allow for identifying potential consequences of climate change on the resilience of ecosystems and their robustness to resist stressors. Using DNA metabarcoding datasets from a three-year-sampling (73 samples), we constructed the protistan plankton co-occurrence network of Lake Zurich, a model lake ecosystem subjected to climate change. Despite several documentations of dramatic lake warming in Lake Zurich, our study provides an unprecedented perspective by linking changes in biotic association patterns to climate stress. Water temperature belonged to the strongest environmental parameters splitting the data into two distinct seasonal networks (October-April; May-September). The expected ecological niche of phytoplankton, weakened through nutrient depletion because of permanent thermal stratification and through parasitic fungi, was occupied by the cyanobacterium Planktothrix rubescens and mixotrophic nanoflagellates. Instead of phytoplankton, bacteria and nanoflagellates were the main prey organisms associated with key predators (ciliates), which contrasts traditional views of biological associations in lake plankton. In a species extinction scenario, the warm season network emerged as more vulnerable than the cold season network, indicating a time-lagged effect of warmer winter temperatures on the communities. We conclude that climate stressors compromise lake ecosystem robustness and resilience through species replacement, richness differences, and succession as indicated by key network properties.