Ostreopsis fattorussoi sp. nov. (Dinophyceae), a new benthic toxic Ostreopsis species from the eastern Mediterranean Sea

The new benthic toxic dinoflagellate, Ostreopsis fattorussoi sp. nov., is described from the Eastern Mediterranean Sea, Lebanon and Cyprus coasts, and is supported by morphological and molecular data. The plate formula, Po, 3′, 7″, 6c, 7s, 5?, 2′′′′, is typical for the Ostreopsis genus. It differs from all other Ostreopsis species in that (i) the curved suture between plates 1′ and 3′ makes them approximately hexagonal, (ii) the 1′ plate lies in the left half of the epitheca and is obliquely orientated leading to a characteristic shape of plate 6″. The round thecal pores are bigger than the other two Mediterranean species (O. cf. ovata and O. cf. siamensis). O. fattorussoi is among the smallest species of the genus (DV: 60.07 ± 5.63 μm, AP: 25.66 ± 2.97 μm, W: 39.81 ± 5.05 μm) along with O. ovata. Phylogenetic analyses based on the LSU and internal transcribed spacer rDNA shows that O. fattorussoi belongs to the Atlantic/Mediterranean Ostreopsis spp. clade separated from the other Ostreopsis species. Ostreopsis fattorussoi produces OVTX‐a and structural isomers OVTX‐d and ‐e, O. cf. ovata is the only other species of this genus known to produce these toxins. The Lebanese O. fattorussoi did not produce the new palytoxin‐like compounds (ovatoxin‐i, ovatoxin‐j₁, ovatoxin‐j₂, and ovatoxin‐k) that were previously found in O. fattorussoi from Cyprus. The toxin content was in the range of 0.28–0.94 pg · cell^?1. On the Lebanon coast, O. fattorussoi was recorded throughout the year 2015 (temperature range 18°C–31.5°C), with peaks in June and August.     

Phylogenetic relationships,morphological variation,and toxin patterns in the Alexandrium ostenfeldii (Dinophyceae) complex: implications for species boundaries and identities

Alexandrium ostenfeldii (Paulsen) Balech and Tangen and A. peruvianum (Balech and B.R. Mendiola) Balech and Tangen are morphologically closely related dinoflagellates known to produce potent neurotoxins. Together with Gonyaulax dimorpha Biecheler, they constitute the A. ostenfeldii species complex. Due to the subtle differences in the morphological characters used to differentiate these species, unambiguous species identification has proven problematic. To better understand the species boundaries within the A. ostenfeldii complex we compared rDNA data, morphometric characters and toxin profiles of multiple cultured isolates from different geographic regions. Phylogenetic analysis of rDNA sequences from cultures characterized as A. ostenfeldii or A. peruvianum formed a monophyletic clade consisting of six distinct groups. Each group examined contained strains morphologically identified as either A. ostenfeldii or A. peruvianum. Though key morphological characters were generally found to be highly variable and not consistently distributed, selected plate features and toxin profiles differed significantly among phylogenetic clusters. Additional sequence analyses revealed a lack of compensatory base changes in ITS2 rRNA structure, low to intermediate ITS/5.8S uncorrected genetic distances, and evidence of reticulation. Together these data (criteria currently used for species delineation in dinoflagellates) imply that the A. ostenfeldii complex should be regarded a single genetically structured species until more material and alternative criteria for species delimitation are available. Consequently, we propose that A. peruvianum is a heterotypic synonym of A. ostenfeldii and this taxon name should be discontinued.     

Characterization of freshwater benthic biofilm‐forming Hydrocoryne (Cyanobacteria) isolates from Antarctica

The aims of this work were to study cyanobacterial isolates resembling the genus Hydrocoryne using a combination of morphology and phylogeny of 16S rRNA and nifH sequences and to investigate genes involved in cyanotoxin and protease inhibitor production. Four new cyanobacterial strains, isolated from biofilm samples collected from King George Island, Antarctica, were studied. In terms of morphology, these new strains share traits similar to true Anabaena morphotypes (benthic ones), whereas phylogenetic analysis of their 16S rRNA gene sequences grouped them with the sequence of the type species Hydrocoryne spongiosa (H. Schwabe ex Bornet and Flahault 1886–1888), but not with sequences of the type species from the genus Anabaena. This cluster is the sister group of Anabaena morphotypes isolated only from the Gulf of Finland. In addition, this cluster is related to two other clusters formed by sequences of Anabaena isolated from different sites. Partial nifH genes were sequenced from two strains and the phylogenetic tree revealed that the Antarctic nifH sequences clustered with sequences from Anabaena. Furthermore, two strains were tested, using PCR with specific primers, for the presence of genes involved in cyanotoxins (microcystin and saxitoxin) and protease inhibitor (aeruginosin, and cyanopeptolin). Only cyanopeptolin was amplified using PCR. These four Hydrocoryne strains are the first to be isolated and sequenced from Antarctica, which improves our knowledge on this poorly defined cyanobacterial genus.     

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.     

Phylogenetic position of the Sphaeropleaceae (Chlorophyta)

 The complete 18S rRNA gene sequences of four Sphaeroplea C.A. Agardh strains (Sphaeropleales, Sphaeropleaceae), two Atractomorpha Hoffman strains (Sphaeropleales, Sphaeropleaceae) and two Ankyra Fott strains (Chlorococcales, Characiaceae) were determined and subjected to phylogenetic analyses. The analyses indicated that all these taxa belong to a monophyletic lineage (Sphaeropleaceae) and are related to a group of chlorophycean algae comprising autosporic taxa and taxa that reproduce by zoospores which are characterized by directly opposed basal bodies. The taxonomic assignment of the Sphaeropleaceae as a family within the Sphaeropleales (Chlorophyta, Chlorophyceae) is discussed. Received December 22, 2000 Accepted September 25, 2001     

Phylogenetic Reappraisal of the Genus Monomorphina (Euglenophyceae) Based on Molecular and Morphological Data

A morphological and molecular examination of the genus Monomorphina was conducted on 46 strains isolated mainly from Korea. The strains were divided into two types based on morphological data: Monomorphina aenigmatica and M. pyrum ‐ like species. Phylogenetic analysis based on a combined data set of nuclear SSU and LSU and plastid SSU and LSU rDNA showed that the strains could be divided into eight clades: Clade A of M. aenigmatica, Clade B of the isolates (M. pyropsis) from Michigan, USA, Clade C of M. pseudopyrum, Clade D of the isolates (M. pyroria) from Bremen, Germany, Clade E of M. soropyrum, Clade F of M. pyriformis, Clade G of M. parapyrum, and Clade H of M. pyrum. Six of these clades came from strains that would be considered M. pyrum sensu Kosmala et Zakry?, one of which could be recognized as a traditional species (M. pyrum) and five were designated as new species; each species had unique molecular signatures at nr SSU rDNA helix 17 and 17′ and spacer E23_14′‐E23_15. The species of Monomorphina had a wide range of genetic diversity with interspecies sequence similarity of 85.6%–97.1% and intraspecies similarity of 96.4%–99.9%. Our results suggested that genetic diversity found in the M. pyrum complex justifies the recognition of a minimum of eight species within this genus, based on specific molecular signatures and gene divergence of the nr SSU rDNA sequences.     

Symbiodinium (Dinophyceae) community patterns in invertebrate hosts from inshore marginal reefs of the southern Great Barrier Reef,Australia

The broad range in physiological variation displayed by Symbiodinium spp. has proven imperative during periods of environmental change and contribute to the survival of their coral host. Characterizing how host and Symbiodinium community assemblages differ across environmentally distinct habitats provides useful information to predict how corals will respond to major environmental change. Despite the extensive characterizations of Symbiodinium diversity found amongst reef cnidarians on the Great Barrier Reef (GBR) substantial biogeographic gaps exist, especially across inshore habitats. Here, we investigate Symbiodinium community patterns in invertebrates from inshore and mid‐shelf reefs on the southern GBR, Australia. Dominant Symbiodinium types were characterized using denaturing gradient gel electrophoresis fingerprinting and sequencing of the ITS2 region of the ribosomal DNA. Twenty one genetically distinct Symbiodinium types including four novel types were identified from 321 reef‐invertebrate samples comprising three sub‐generic clades (A, C, and D). A range of host genera harbored C22a, which is normally rare or absent from inshore or low latitude reefs in the GBR. Multivariate analysis showed that host identity and sea surface temperature best explained the variation in symbiont communities across sites. Patterns of changes in Symbiodinium community assemblage over small geographic distances (100s of kilometers or less) indicate the likelihood that shifts in Symbiodinium distributions and associated host populations, may occur in response to future climate change impacting the GBR.     

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.     

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.     

Photosynthetic activity and proteomic analysis highlights the utilization of atmospheric CO2 by Ulva prolifera (Chlorophyta) for rapid growth

Free‐floating Ulva prolifera is one of the causative species of green tides. When green tides occur, massive mats of floating U. prolifera thalli accumulate rapidly in surface waters with daily growth rates as high as 56%. The upper thalli of the mats experience environmental changes such as the change in carbon source, high salinity, and desiccation. In this study, the photosynthetic performances of PSI and PSII in U. prolifera thalli exposed to different atmospheric carbon dioxide (CO₂) levels were measured. Changes in photosynthesis within salinity treatments and dehydration under different CO₂ concentrations were also analyzed. The results showed that PSII activity was enhanced as CO₂ increased, suggesting that CO₂ assimilation was enhanced and U. prolifera thalli can utilize CO₂ in the atmosphere directly, even when under moderate stress. In addition, changes in the proteome of U. prolifera in response to salt stress were investigated. Stress‐tolerance proteins appeared to have an important role in the response to salinity stress, whereas the abundance of proteins related to metabolism showed no significant change under low salinity treatments. These findings may be one of the main reasons for the extremely high growth rate of free‐floating U. prolifera when green tides occur.     

Kraftionema allantoideum,a new genus and family of Ulotrichales (Chlorophyta) adapted for survival in high intertidal pools

The marine, sand‐dwelling green alga Kraftionema allantoideum gen. et sp. nov. is described from clonal cultures established from samples collected in coastal, high intertidal pools from south eastern Australia. The species forms microscopic, uniseriate, unbranched, 6–8 μm wide filaments surrounded by a gelatinous capsule of varying thickness. Filaments are twisted, knotted, and variable in length from 4 to 50 cells in field samples but straighter and much longer in culture, up to 1.5 mm in length. Cell division occurs in several planes, resulting in daughter cells of varying shape, from square to rectangular to triangular, giving rise to gnarled filaments. Mature cells become allantoid, elongate with rounded ends, before dividing one time to form bicells comprised of two domed cells. Adjacent bicells separate from one another and mature filaments appeared as a string of loosely arranged sausages. A massive, single, banded chloroplast covered 3/4 of the wall circumference, and contained a single large pyrenoid encased in a starch envelope that measures 1.5–2.5 μm. Filaments were not adhesive nor did they produce specialized adhesive cells or structures. Reproduction was by fragmentation with all cells capable of producing a new filament. No motile or reproductive cells were observed. Filaments in culture grew equally well in freshwater or marine media, as well as at high salinity, and cells quickly recovered from desiccation. Phylogenetic analysis based on the nuclear‐encoded small subunit ribosomal RNA (18S) shows the early branching nature of the Kraftionema lineage among Ulotrichales, warranting its recognition as a family (Kraftionemaceae).     

Characterization of Prorocentrum elegans and Prorocentrum levis (Dinophyceae) from the southeastern Bay of Biscay by morphology and molecular phylogeny

Benthic Prorocentrum species can produce toxins that adversely affect animals and human health. They are known to co‐occur with other bloom‐forming, potentially toxic, benthic dinoflagellates of the genera Ostreopsis, Coolia, and Gambierdiscus. In this study, we report on the presence of P. elegans M.Faust and P. levis M.A.Faust, Kibler, Vandersea, P.A. Tester & Litaker from the southeastern Bay of Biscay. Sampling was carried out in the Summer‐Autumn 2010–2012 along the Atlantic coast of the Iberian Peninsula, but these two species were only found in the northeastern part of the Peninsula. Strains were isolated from macroalgae collected from rocky‐shore areas bordering accessible beaches. Morphological traits of isolated strains were analyzed by LM and SEM, whereas molecular analyses were performed using the LSU and internal transcribed spacer (ITS)1‐5.8S‐ITS2 regions of the rDNA. A bioassay with Artemia fransciscana and liquid chromatography–high‐resolution mass spectrometry analyses were used to check the toxicity of the species, whose results were negative. The strains mostly corresponded to their species original morphological characterization, which is supported by the phylogenetic analyses in the case of P. levis, whereas for P. elegans, this is the first known molecular characterization. This is also the second known report of P. elegans.     

Synergistic effects of HSE and LTR elements from hsp70 gene promoter of Ulva prolifera (Ulvophyceae,Chlorophyta) upon temperature induction1

Besides heat stress, the 70 kDa heat shock proteins (HSP70s) have been shown to respond to cold stress. However, the involved cis‐acting elements remain unknown. The hsp70 gene from the green macroalga Ulva prolifera (Uphsp70) has been cloned, from which one heat shock element HSE and one low‐temperature‐responsive element LTR were found in the promoter. Using the established transient expression system and quantitative GUS assay, a series of element deletion experiments were performed to determine the functions of HSE and LTR in response to temperature stress. The results showed that under cold stress, both HSE and LTR were indispensable, since deletion leads to complete loss of promoter activity. Under heat stress, although the HSE could respond independently, coexistence with LTR was essential for high induced activity of the Uphsp70 promoter. Therefore, synergistic effects exist between HSE and LTR elements in response to temperature stress in Ulva, and extensive bioinformatics analysis showed that the mechanism is widespread in algae and plants, since LTR coexists widely with HSE in the promoter region of hsp70. Our findings provide important supplements to the knowledge of algal and plant HSP70s response to temperature stress. We speculated that for algal domestication and artificial breeding, HSE and LTR elements might serve as potential molecular targets to temperature acclimation.     

Phylogenetic Relationships and Morphological Character Evolution of Photosynthetic Euglenids (Excavata) Inferred from Taxon‐rich Analyses of Five Genes

Photosynthetic euglenids acquired chloroplasts by secondary endosymbiosis, which resulted in changes to their mode of nutrition and affected the evolution of their morphological characters. Mapping morphological characters onto a reliable molecular tree could elucidate major trends of those changes. We analyzed nucleotide sequence data from regions of three nuclear‐encoded genes (nSSU, nLSU, hsp90), one chloroplast‐encoded gene (cpSSU) and one nuclear‐encoded chloroplast gene (psbO) to estimate phylogenetic relationships among 59 photosynthetic euglenid species. Our results were consistent with previous works; most genera were monophyletic, except for the polyphyletic genus Euglena, and the paraphyletic genus Phacus. We also analyzed character evolution in photosynthetic euglenids using our phylogenetic tree and eight morphological traits commonly used for generic and species diagnoses, including: characters corresponding to well‐defined clades, apomorphies like presence of lorica and mucilaginous stalks, and homoplastic characters like rigid cells and presence of large paramylon grains. This research indicated that pyrenoids were lost twice during the evolution of phototrophic euglenids, and that mucocysts, which only occur in the genus Euglena, evolved independently at least twice. In contrast, the evolution of cell shape and chloroplast morphology was difficult to elucidate, and could not be unambiguously reconstructed in our analyses.     

Stimulated rejuvenation of dormant Aulacoseira granulata (Bacillariophyta) by Gloeocystis planctonica (Chlorophyta) in a eutrophic river

The abundance of Aulacoseira granulata (Ehrenburg) Simonsen and Gloeocystis planctonica (West & G.S.West) Lemmermann was assessed during the summers of 2005 and 2010 in the eutrophic Fox River, Wisconsin, USA. In both years, a mid‐summer bloom of G. planctonica was followed by the rapid growth of A. granulata. Laboratory experiments in which A. granulata was grown in cell‐free filtrate of a G. planctonica culture revealed that the growth of A. granulata was stimulated in the G. planctonica‐treated medium relative to controls. This effect was detected when dormant A. granulata cells were used as the source culture for the experiment but not when actively growing cells were used. Dormant A. granulata also grew more rapidly in river water collected after the 2010 G. planctonica bloom relative to river water collected before the bloom. These results suggest that the summer bloom of A. granulata in the river was stimulated by G. planctonica. This relationship can be described as stimulated rejuvenation, an interaction where the transition of an algal resting stage into active growth is triggered by exposure to another species.     

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.     

A survey of Polytomella (Chlorophyceae,Chlorophyta) strains in public culture collections

Polytomella is a genus of colorless green algae in the Reinhardtinia clade of the Chlamydomonadales, which has proven useful for a broad range of studies particularly those exploring the evolutionary loss of photosynthesis and mitochondrial genomics/biochemistry. Although 13 Polytomella strain accessions are currently available from public culture collections, the taxonomic status and redundancy of many of these strains is not clear because of possible mix‐ups, deficient historical records, and incomplete molecular data. This study therefore considers previously available and/or new cox1 and mitochondrial DNA telomere sequences from all 13 Polytomella strain accessions. Among four of these, namely P. parva SAG 63‐3, P. piriformis SAG 63‐10, P. capuana SAG 63‐5, and P. magna SAG 63‐9, cox1 and mitochondrial telomere regions are both highly divergent between strains. All of the remaining nine Polytomella strain accessions have cox1 sequences that are identical to that of P. parva SAG 63‐3 and although five of these have a mitochondrial telomere haplotype that is identical to that of P. parva SAG 63‐3, the remaining four have one of three different haplotypes. Among the 10 strains with identical cox1 sequences, we suggest that three of the telomere haplotypes are associated with distinct geographical isolates of Polytomella and the fourth evolved from one of these isolates during 50 years of active culture.     

A new Eocene fossil of the genus Phycopeltis (Ulvophyceae,Chlorophyta)

A fossil of the aerophytic green algal genus Phycopeltis (Trentepohliaes, Ulvophyceae) dated to 35 Ma, is reported from the Pikopiko Fossil Forest, Southland, New Zealand. Previous reports of fossilized Phycopeltis have been subsequently synonymized with fungi by other authors; however, our specimen is not vulnerable to their criticisms. Inflated cells present in two approximately concentric rings are interpreted as gametangia, with irregular structures resembling the gametangial pores of modern material; sporophytic material is absent. The fossil resembles the modern disc‐forming species P. novae‐zelandiae, P. expansa, and P. arundinacea. The limited material available prevents the assignation of a specific epithet, but the habit and dimensions of the fossil clearly fall within those of modern representatives of the genus. Its single cell thickness throughout, absence of distinct melanization, and larger size demonstrate that it is not a fungal shield. The specimen constitutes arguably the most convincing fossil belonging to Trentepohliales, and the first unambiguously for the genus Phycopeltis. It is consistent in age with other known fossils of the order that, when combined with molecular evidence, suggests a terrestrial radiation far more recent than that of land plants.