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.     

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.     

Phylogenic Diversity and Taxonomic Problems of the Dictyosphaerium Morphotype within the Parachlorella Clade (Chlorellaceae,Trebouxiophyceae)

The Parachlorella clade was put forward as a group within the family Chlorellaceae in 2004. Recent molecular analyses have revealed that Dictyosphaerium morphotype algae form several independent lineages within the Parachlorella clade, and new genera and species have been established. In this study, we focus on the diversity of Dictyosphaerium morphotype algae within the Parachlorella clade, based on 42 strains from China. We used combined analyses of morphology and molecular data based on SSU and internal transcribed spacer region (ITS) rDNA sequences to characterize these algae. In addition, the secondary structure of ITS2 was compared to delineate new lineages. Our results revealed high phylogenic diversity of Dictyosphaerium morphotype algae, and we describe five distinct lineages. We examined the morphological features of these five lineages, and morphological differences are difficult to find compared with other Dictyosphaerium morphotype algae. The five distinct lineages were not described as new genera currently. We lastly discuss the taxonomic problems regarding the Dictyosphaerium morphotype within the Parachlorella clade, and possible solutions are considered.     

New genus Koliellopsis (Trebouxiophyceae, Chlorophyta): its phylogenetic position inferred from ultrastructure and nuclear ribosomal DNA sequences

Koliellopsis inundata Lokhorst gen. & sp. nov. (Trebouxiophyceae) is described from periodically flooded agricultural fields in the borderland of Belgium and the Netherlands. This new, unbranched, filamentous alga is typified by relatively long vegetative cells, which have a bilobed, laminate chloroplast with a nucleus positioned in its median constriction. Its filaments lack a distinct basal‐distal differentiation and both ends terminate in about equally shaped, rounded or, more often (slightly) tapering cells. Despite its semiterres‐trial occurrence the new alga does not attach to hard substrate, presumably owing to the lack of end cells to produce mucilage and to function as a holdfast. The systematic position of Koliellopsis among the green algae is inferred from ultrastructural examinations of the cell division patterns and from phylogenetic analyses of partial 18S rRNA gene sequences.     

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.     

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.     

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.     

Heveochlorella (Trebouxiophyceae): a little‐known genus of unicellular green algae outside the Trebouxiales emerges unexpectedly as a major clade of lichen photobionts in foliicolous communities

Foliicolous lichens are formed by diverse, highly specialized fungi that establish themselves and complete their life cycle within the brief duration of their leaf substratum. Over half of these lichen‐forming fungi are members of either the Gomphillaceae or Pilocarpaceae, and associate with Trebouxia‐like green algae whose identities have never been positively determined. We investigated the phylogenetic affinities of these photobionts to better understand their role in lichen establishment on an ephemeral surface. Thallus samples of Gomphillaceae and Pilocarpaceae were collected from foliicolous communities in southwest Florida and processed for sequencing of photobiont marker genes, algal cultivation and/or TEM. Additional specimens from these families and also from Aspidothelium (Thelenellaceae) were collected from a variety of substrates globally. Sequences from rbcL and nuSSU regions were obtained and subjected to Maximum Likelihood and Bayesian analyses. Analysis of 37 rbcL and 7 nuSSU algal sequences placed all photobionts studied within the provisional trebouxiophycean assemblage known as the Watanabea clade. All but three of the sequences showed affinities within Heveochlorella, a genus recently described from tree trunks in East Asia. The photobiont chloroplast showed multiple thylakoid stacks penetrating the pyrenoid centripetally as tubules lined with pyrenoglobuli, similar to the two described species of Heveochlorella. We conclude that Heveochlorella includes algae of potentially major importance as lichen photobionts, particularly within (but not limited to) foliicolous communities in tropical and subtropical regions worldwide. The ease with which they may be cultivated on minimal media suggests their potential to thrive free‐living as well as in lichen symbiosis.     

A new aerial alga, Stichococcus ampulliformis sp. nov. (Trebouxiophyceae, Chlorophyta) from Japan

A new aerial alga, Stichococcus ampulliformis S. Handa sp. nov. (Trebouxiophyceae, Chlorophyta) is described based on a clone isolated from the bark of Cephalotaxus harringtonia (Knight ex Forbes) K. Koch collected from Taishaku‐kyo Gorge, Hiroshima Prefecture, south‐west Japan. This alga was examined by light microscopy and transmission electron microscopy and subjected to molecular phylogenetic analysis. Based on its morphological features and life‐cycle, especially short filament formation, the alga was assigned to the genus Stichococcus Nägeli. However, this alga differs from other described Stichococcus species in that it reproduces by a form of ‘budding’, producing two daughter cells of different sizes. The larger cell, enclosed within the mother cell wall, soon reaches the size of a normal vegetative cell. The smaller cell is extruded and takes longer to reach full vegetative size. A phylogenetic tree constructed using 18S rRNA sequences indicated that, within the Trebouxiophyceae, S. ampulliformis is closely related to S. bacillaris Nägeli and some species of Prasiola Meneghini.     

Ulva (Chlorophyta,Ulvales) Biodiversity in the North Adriatic Sea (Mediterranean,Italy): Cryptic Species and New Introductions

Ulva Linnaeus (Ulvophyceae, Ulvales) is a genus of green algae widespread in different aquatic environments. Members of this genus show a very simple morphology and a certain degree of phenotypic plasticity, heavily influenced by environmental conditions, making difficult the delineation of species by morphological features alone. Most studies dealing with Ulva biodiversity in Mediterranean waters have been based only on morphological characters and a modern taxonomic revision of this genus in the Mediterranean is not available. We report here the results of an investigation on the diversity of Ulva in the North Adriatic Sea based on molecular analyses. Collections from three areas, two of which subject to intense shipping traffic, were examined, as well as historical collections of Ulva stored in the Herbarium Patavinum of the University of Padova, Italy. Molecular analyses based on partial sequences of the rbcL and tufA genes revealed the presence of six different species, often with overlapping morphologies: U. californica Wille, U. flexuosa Wulfen, U. rigida C. Agardh, U. compressa Linnaeus, U. pertusa Kjellman, and one probable new taxon. U. californica is a new record for the Mediterranean and U. pertusa is a new record for the Adriatic. Partial sequences obtained from historical collections show that most of the old specimens are referable to U. rigida. No specimens referable to the two alien species were found among the old herbarium specimens. The results indicate that the number of introduced seaweed species and their impact on Mediterranean communities have been underestimated, due to the difficulties in species identification of morphologically simple taxa as Ulva.     

A molecular investigation of the genus Ecklonia (Phaeophyceae,Laminariales) with special focus on the Southern Hemisphere

Brown algae of the order Laminariales, commonly referred to as kelps, are the largest and most productive primary producers in the coastal inshore environment. The genus Ecklonia (Lessoniaceae, Phaeophyceae) consists of seven species with four species in the Northern Hemisphere and three in the Southern Hemisphere. It was recently transferred to the family Lessoniaceae based on phylogenetic analyses of nuclear and chloroplastic markers, though the type of the genus was not included and its relationship with allied genera Eckloniopsis and Eisenia remained unresolved. The present study is the first to produce a phylogeny focussed on the genus Ecklonia. It included sequences from nuclear, mitochondrial, and chloroplastic DNA, for most of the distribution range of the three current Southern Hemisphere species (Ecklonia radiata, Ecklonia maxima, and a sample of a putative Ecklonia brevipes specimen), sequences for East Asiatic species (Ecklonia cava, Ecklonia kurome, and Ecklonia stolonifera), as well as the closely related genera Eckloniopsis and Eisenia. Results confirmed E. radiata and E. maxima as two distinct species in South Africa, E. radiata as a single species throughout the Southern Hemisphere (in South Africa, Australia, and New Zealand) and East Asiatic species as a distinct lineage from the Southern Hemisphere clade. Results further pointed out a close sister relationship between Eckloniopsis radicosa and two Eisenia species (including the type species: Eisenia arborea) to the genus Ecklonia suggesting that the genera Eckloniopsis and Eisenia are superfluous.     

Molecular and morphological criteria for revision of the genus Microcoleus (Oscillatoriales,Cyanobacteria)

Ninety‐two strains of Microcoleus vaginatus (=nomenclatural‐type species of the genus Microcoleus Desmazières ex Gomont) and Phormidium autumnale Trevisan ex Gomont from a wide diversity of regions and biotopes were examined using a combination of morphological and molecular methods. Phylogenies based on the 16S rDNA and 16S‐23S ITS (partial) demonstrated that the 92 strains, together with a number of strains in GenBank, were members of a highly supported monophyletic clade of strains (Bayesian posterior probability = 1.0) distant from the species‐cluster containing the generitype of Phormidium. Similarity of the 16S rRNA gene exceeded 95.5% among all members of the Microcoleus clade, but was less than 95% between any Microcoleus strains and species outside of the clade (e.g., Phormidium sensu stricto). These findings, which are in agreement with earlier studies on these taxa, necessitate the revision of Microcoleus to include P. autumnale. Furthermore, the cluster of Phormidium species in the P. autumnale group (known as Group VII) must be moved into Microcoleus as well, and these nomenclatural transfers are included in this study. The main diacritical characters defining Microcoleus are related to the cytomorphology of trichomes, including: narrowed trichome ends, calyptra, cells shorter than wide up to more or less isodiametric, and facultative presence of sheaths. The majority of species are 4–10 μm in diameter. The possession of multiple trichomes in a common sheath is present facultatively in many but not all species.     

Phylogenetic relationships of green algae assigned to the genus Planophila (Chlorophyta): evidence from 18S rDNA sequence data and ultrastructure

Phylogenetic analyses, based upon nuclear small-subunit ribosomal RNA gene sequences, of four ‘chlorosarcinoid’ species referred to Planophila Gerneck show that the genus is polyphyletic. The type species, P. laetevirens Gerneck, is closely related to species in the Ulotrichales, Ulvophyceae. The monotypic sarcinoid genus Pseudendocloniopsis is the closest relative of Planophila; the two genera represent the addition of a new morphological type to the Ulotrichales. Planophila microcystis (Dangeard) Kornmann & Sahling forms a clade at the base of the Ulvophyceae with Oltmannsiellopsis, and thus belongs to the Oltmannsiellopsidales. This result is also supported by the Oltmannsiellopsis-like ultrastructure of P. microcystis zoospores. Planophila sp. B from Antarctica, which has Trebouxia-like pyrenoid structure, is a trebouxiophyte closely related to Chlorella-like unicellular coccoids, Stichococcus bacillaris and Prasiola species. This is the first robustly supported molecular phylogenetic analysis that places Prasiola in the Trebouxiophyceae. As shown previously, P. terrestris Groover & Hofstetter belongs to the Chaetopeltidales, Chlorophyceae. Dangemannia gen. nov. (type species : D. microcystis (Dangeard) comb. nov.), Floydiella gen. nov. (type species : F. terrestris (Groover & Hofstetter) comb. nov.) and Pabia gen. nov. (type: P. signiensis sp. nov.) are proposed.     

Sorting out the Snakebirds: The species status,phylogeny, and biogeography of the Darters (Aves: Anhingidae)

Although the avian family Anhingidae is unequivocally monophyletic, the number and relationships of the component species within the single genus (Anhinga) have long remained unclear. Here, we use extensive mitochondrial and nuclear DNA sequence data (8,878 bp) to show that four species should be recognized. Our fully resolved and well‐supported tree shows that the American Anhinga (Anhinga anhinga) is sister to the three Old World species, with the Oriental (A. melanogaster) and African (A. rufa) Darters sister within the Old World clade, which also includes the Australian Darter (A. novaehollandiae). We estimate that the divergence between the New World and Old World branches occurred 19–22 mya, with the Australian Darter separating from its Old World congeners 14–16 mya and the Oriental and African species splitting ~10 mya. The genus is yet another example of osteological conservatism in the Suliformes, which is comparable to that shown by the cormorants and shags. Nevertheless, the relationships we infer are congruent with recent plumage studies and are biogeographically plausible. We suggest that further investigation of the variation within the African and Australian Darters would be of interest.