16S rRNA GENE HETEROGENEITY IN THE FILAMENTOUS MARINE CYANOBACTERIAL GENUS LYNGBYA1

The SSU (16S) rRNA gene was used to investigate the phylogeny of the cyanobacterial genus Lyngbya as well as examined for its capacity to discriminate between different marine species of Lyngbya. We show that Lyngbya forms a polyphyletic genus composed of a marine lineage and a halophilic/brackish/freshwater lineage. In addition, we found morphological and genetic evidence that Lyngbya spp. often grow in association with other microorganisms, in particular smaller filamentous cyanobacteria such as Oscillatoria, and propose that these associated microorganisms have led to extensive phylogenetic confusion in identification of Lyngbya spp. At the species level, the phylogenetic diversity obtained from the comparison of 16S rRNA genes exceeded morphological diversity in Lyngbya. However, the expectation that this improved phylogeny would be useful to species and subspecies identification was eliminated by the fact that phylogenetic species did not correlate in any respect with the species obtained from current taxonomic systems. In addition, phylogenetic identification was adversely affected by the presence of multiple gene copies within individual Lyngbya colonies. Analysis of clonal Lyngbya cultures and multiple displacement amplified (MDA) single‐cell genomes revealed that Lyngbya genomes contain two 16S rRNA gene copies, and that these typically are of variable sequence. Furthermore, intragenomic and interspecies 16S rRNA gene heterogeneity was approximately of the same magnitude. Hence, the intragenomic heterogeneity of the 16S rRNA gene overestimates the microdiversity of different strains and does not accurately reflect speciation within cyanobacteria, including the genus Lyngbya.     

Underestimated biodiversity as a major explanation for the perceived rich secondary metabolite capacity of the cyanobacterial genus Lyngbya

Marine cyanobacteria are prolific producers of bioactive secondary metabolites responsible for harmful algal blooms as well as rich sources of promising biomedical lead compounds. The current study focused on obtaining a clearer understanding of the remarkable chemical richness of the cyanobacterial genus Lyngbya. Specimens of Lyngbya from various environmental habitats around Curaçao were analysed for their capacity to produce secondary metabolites by genetic screening of their biosynthetic pathways. The presence of biosynthetic pathways was compared with the production of corresponding metabolites by LC‐ESI‐MS² and MALDI‐TOF‐MS. The comparison of biosynthetic capacity and actual metabolite production revealed no evidence of genetic silencing in response to environmental conditions. On a cellular level, the metabolic origin of the detected metabolites was pinpointed to the cyanobacteria, rather than the sheath‐associated heterotrophic bacteria, by MALDI‐TOF‐MS and multiple displacement amplification of single cells. Finally, the traditional morphology‐based taxonomic identifications of these Lyngbya populations were combined with their phylogenetic relationships. As a result, polyphyly of morphologically similar cyanobacteria was identified as the major explanation for the perceived chemical richness of the genus Lyngbya, a result which further underscores the need to revise the taxonomy of this group of biomedically important cyanobacteria.     

Five chemically rich species of tropical marine cyanobacteria of the genus Okeania gen. nov. (Oscillatoriales,Cyanoprokaryota)

An adverse consequence of applying morphology‐based taxonomic systems to catalog cyanobacteria, which generally are limited in the number of available morphological characters, is a fundamental underestimation of natural biodiversity. In this study, we further dissect the polyphyletic cyanobacterial genus Lyngbya and delineate the new genus Okeania gen. nov. Okeania is a tropical and subtropical, globally distributed marine group abundant in the shallow‐water benthos. Members of Okeania are of considerable ecological and biomedical importance because specimens within this group biosynthesize biologically active secondary metabolites and are known to form blooms in coastal benthic environments. Herein, we describe five species of the genus Okeania: O. hirsuta (type species of the genus), O. plumata, O. lorea, O. erythroflocculosa, and O. comitata, under the provisions of the International Code of Nomenclature for Algae, Fungi, and Plants. All five Okeania species were morphologically, phylogenetically, and chemically distinct. This investigation provides a classification system that is able to identify Okeania spp. and predict their production of bioactive secondary metabolites.     

Leptothoe,a new genus of marine cyanobacteria (Synechococcales) and three new species associated with sponges from the Aegean Sea

Cyanobacterial diversity associated with sponges remains underestimated, though it is of great scientific interest in order to understand the ecology and evolutionary history of the symbiotic relationships between the two groups. Of the filamentous cyanobacteria, the genus Leptolyngbya is the most frequently found in association with sponges as well as the largest and obviously polyphyletic group. In this study, five Leptolyngbya‐like sponge‐associated isolates were investigated using a combination of molecular, chemical, and morphological approach and revealed a novel marine genus herein designated Leptothoe gen. nov. In addition, three new species of Leptothoe, Le. sithoniana, Le. kymatousa, and Le. spongobia, are described based on a suite of distinct characters compared to other marine Leptolyngbyaceae species/strains. The three new species, hosted by four sponge species, showed different degrees of host specificity. Leptothoe sithoniana and Le. kymatousa hosted by the sponges Petrosia ficiformis and Chondrilla nucula, respectively, seem to be more specialized than Le. spongobia, which was hosted by the sponges Dysidea avara and Acanthella acuta. All three species contained nitrogen‐fixing genes and may contribute to the nitrogen budget of sponges. Leptothoe spongobia TAU‐MAC 1115 isolated from Acanthella acuta was shown to produce microcystin‐RR indicating that microcystin production among marine cyanobacteria could be more widespread than previously determined.     

Benthic cyanobacterial bloom impacts the reefs of South Florida (Broward County, USA)

Benthic cyanobacteria of the genus Lyngbya can form prominent mats and blooms in tropical and subtropical coral reef and seagrass habitats worldwide. A Lyngbya bloom on the reef tract offshore of Broward County, Florida, was first noted in 2002, and although it is seasonally variable in its distribution and abundance, it has persisted and spread over the past 3 years. In this study, the most abundant species of Lyngbya found in the blooms have been identified and compared to other species of Lyngbya by morphological and molecular methods. The most common species of Lyngbya is consistent with the properties of Lyngbya confervoides C. Agardh. The 16S ribosomal DNA sequence shares 88–92% identity with other known Lyngbya sequences, suggesting that this bloom consists primarily of a new, previously unsequenced species of Lyngbya. The second most common Lyngbya in the bloom is consistent with Lyngbya polychroa. This persistent bloom is a concern because it smothers octocorals and other invertebrates and negatively impacts these southeastern Florida reefs.     

Uptake and localization of fluorescently‐labeled Karenia brevis metabolites in non‐toxic marine microbial taxa

Brevetoxin (PbTx) is a neurotoxic secondary metabolite of the dinoflagellate Karenia brevis. We used a novel, fluorescent BODIPY‐labeled conjugate of brevetoxin congener PbTx‐2 (B‐PbTx) to track absorption of the metabolite into a variety of marine microbes. The labeled toxin was taken up and brightly fluoresced in lipid‐rich regions of several marine microbes including diatoms and coccolithophores. The microzooplankton (20–200 μm) tintinnid ciliate Favella sp. and the rotifer Brachionus rotundiformis also took up B‐PbTx. Uptake and intracellular fluorescence of B‐PbTx was weak or undetectable in phytoplankton species representative of dinoflagellates, cryptophytes, and cyanobacteria over the same (4 h) time course. The cellular fate of two additional BODIPY‐conjugated K. brevis associated secondary metabolites, brevenal (B‐Bn) and brevisin (B‐Bs), were examined in all the species tested. All taxa exhibited minimal or undetectable fluorescence when exposed to the former conjugate, while most brightly fluoresced when treated with the latter. This is the first study to observe the uptake of fluorescently‐tagged brevetoxin conjugates in non‐toxic phytoplankton and zooplankton taxa, demonstrating their potential in investigating whether marine microbes can serve as a significant biological sink for algal toxins. The highly variable uptake of B‐PbTx observed among taxa suggests some may play a more significant role than others in vectoring lipophilic toxins in the marine environment.     

A new type of 3‐D peripheral ultrastructure in Glaucocystis (Glaucocystales,Glaucophyta) as revealed by ultra‐high voltage electron microscopy

The coccoid glaucophyte genus Glaucocystis is characterized by having a thick cell wall, which has to date prohibited examination of the native ultrastructural features of the protoplast periphery. Recently, however, the three‐dimensional (3‐D) ultrastructure of the protoplast periphery was revealed in two divergent Glaucocystis species, with the world's most powerful ultra‐high voltage electron microscope (UHVEM). The two species exhibit morphological diversity in terms of their 3‐D ultrastructural features. However, these two types do not seem to encompass actual ultrastructural diversity in the genetically diverse genus Glaucocystis. Here, we report a new type of peripheral 3‐D ultrastructure resolved in “G. incrassata” SAG 229‐2 cells by 3‐D modeling based on UHVEM tomography using high‐pressure freezing and freeze‐substitution fixation. The plasma membrane and underlying flattened vesicles in “G. incrassata” SAG 229‐2 exhibited grooves at intervals of 200–600 nm, and the flattened vesicles often overlapped one another at the protoplast periphery. This 3‐D ultrastructure differs from those of the two types previously reported in other species of Glaucocystis. The possibility of classification of Glaucocystis species based on the 3‐D ultrastructure of the protoplast periphery is discussed.     

Macrochaete gen. nov. (Nostocales,Cyanobacteria), a taxon morphologically and molecularly distinct from Calothrix

Historically, the genus Calothrix included all noncolonial, tapered, heterocytous filaments within the cyanobacteria. However, recent molecular phylogenies show that “Calothrix” defined in this sense represents five distinct clades. The type species of Calothrix is marine, with solitary basal heterocytes, no akinetes, and distal ends tapering abruptly into short hairs. We examined the morphology and phylogeny of 45 tapering cyanobacteria in the Rivulariaceae, including freshwater and marine representatives of both Calothrix (35 strains) and its sister taxon Rivularia (10 strains). The marine Calothrix fall into two lineages, but we lack the generitype and so cannot identify the clade corresponding to the type species. The freshwater and soil Calothrix fall into the C. parietina clade and are characterized by having a basal heterocyte, no akinetes, and gradual tapering—but not into a long hyaline hair. Macrochaete gen. nov. is a freshwater taxon sister to the Calothrix lineages but clearly separated from Rivularia. The species in this genus differ morphologically from Calothrix by their ability to produce two heteromorphic basal heterocytes and specific secondary structures of the 16S–23S ITS. An additional feature present in most species is the presence of a distal, long hyaline hair, but this character has incomplete penetrance due to its expression only under specific environmental conditions (low phosphate), and in one species appears to be lost. We recognize three species: M. psychrophila (type species) from cold environments (high mountains, Antarctica), M. santannae from wet walls of subtropical South America, and M. lichenoides, a phycobiont of lichens from Europe.     

Two new species in the Chaetoceros socialis complex (Bacillariophyta): C. sporotruncatus and C. dichatoensis,and characterization of its relatives,C. radicans and C. cinctus

The diatom genus Chaetoceros is one of the most abundant and diverse phytoplankton in marine and brackish waters worldwide. Within this genus, Chaetoceros socialis has been cited as one of the most common species. However, recent studies from different geographic areas have shown the presence of pseudo‐cryptic diversity within the C. socialis complex. Members of this complex are characterized by curved chains (primary colonies) aggregating into globular clusters, where one of the four setae of each cell curves toward the center of the cluster and the other three orient outwards. New light and electron microscopy observations as well as molecular data on marine planktonic diatoms from the coastal waters off Chile revealed the presence of two new species, Chaetoceros sporotruncatus sp. nov. and C. dichatoensis. sp. nov. belonging to the C. socialis complex. The two new species are similar to other members of the complex (i.e., C. socialis and C. gelidus) in the primary and secondary structure of the colony, the orientation pattern of the setae, and the valve ultrastructure. The only morphological characters that can be used to differentiate the species of this complex are aspects related to resting spore morphology. The two newly described species are closely related to each other and form a sister clade to C. gelidus in molecular phylogenies. We also provide a phylogenetic status along with the morphological characterization of C. radicans and C. cintus, which are genetically related to the C. socialis complex.     

On Sea Turtle‐associated Craspedostauros (Bacillariophyta), with Description of Three Novel Species

The current study focuses on four species from the primarily marine diatom genus Craspedostauros that were observed growing attached to numerous sea turtles and sea turtle‐associated barnacles from Croatia and South Africa. Three of the examined taxa, C. danayanus sp. nov., C. legouvelloanus sp. nov., and C. macewanii sp. nov., are described based on morphological and, whenever possible, molecular characteristics. The new taxa exhibit characters not previously observed in other members of the genus, such as the presence of more than two rows of cribrate areolae on the girdle bands, shallow perforated septa, and a complete reduction of the stauros. The fourth species, C. alatus, itself recently described from museum sea turtle specimens, is reported for the first time from loggerhead sea turtles rescued in Europe. A 3‐gene phylogenetic analysis including DNA sequence data for three sea turtle‐associated Craspedostauros species and other marine and epizoic diatom taxa indicated that Craspedostauros is monophyletic and sister to Achnanthes. This study, being based on a large number of samples and animal specimens analyzed and using different preservation and processing methods, provides new insights into the ecology and biogeography of the genus and sheds light on the level of intimacy and permanency in the host–epibiont interaction within the epizoic Craspedostauros species.     

A new surface gliding species of Chironomidae: An independent invasion of marine environments and its evolutionary implications

Insects have invaded marine habitats only rarely and secondarily. Recently, we discovered a flightless dipteran species skating rapidly on the surface of seawater ponds at the Pacific coast of eastern China. Morphological analyses initially suggested an isolated position of the non‐biting midge, suggesting the erection of a new genus within Chironomini (Diptera: Chironomidae). However, an analysis of molecular data revealed that the marine species is in fact nested within the species‐rich genus Dicrotendipes. The apparent conflict between molecular and morphological data can be easily explained. It is likely that the new species has evolved a series of autapomorphic adaptations. These traits clearly distinguish the taxon from other species of the genus but do not justify the erection of a new supraspecific taxon, which would render Dicrotendipes paraphyletic. The switch to marine environments was likely a trigger for various morphological modifications resulting from increased selective pressure. Molecular data suggest that the potential speciation event occurred around 19–29 Ma, linked with a migration from freshwater to seawater ponds along the Pacific Ocean. Considering the results of our analysis, we place the flightless marine skater in the genus Dicrotendipes. All life stages of Dicrotendipes sinicus Qi & Lin sp. n. are described and illustrated, associated with larvae obtained by rearing or confirmed through association with DNA barcodes. The biology and ecology of the species are outlined based on collection data and in situ observations. Evolutionary patterns linked with repeated invasions of marine habitats are discussed.     

Pseudoparamoeba garorimi n. sp., with Notes on Species Distinctions within the Genus

A new marine species of naked lobose amoebae Pseudoparamoeba garorimi n. sp. (Amoebozoa, Dactylopodida) isolated from intertidal marine sediments of Garorim Bay, Korea was studied with light and transmission electron microscopy. This species has a typical set of morphological characters for a genus including the shape of the locomotive form, type of subpseudopodia and the tendency to form the single long waving pseudopodium in locomotion. Furthermore, it has the same cell surface structures as were described for the type species, Pseudoparamoeba pagei: blister‐like glycostyles with hexagonal base and dome‐shaped apex; besides, cell surface bears hair‐like outgrowths. The new species described here lacks clear morphological distinctions from the two other Pseudoparamoeba species, but has considerable differences in the 18S rDNA and COX1 gene sequences. Phylogenetic analysis based on 18S rDNA placed P. garorimi n. sp. at the base of the Pseudoparamoeba clade with high PP/BS support. The level of COX1 sequence divergence was 22% between P. garorimi n. sp. and P. pagei and 25% between P. garorimi n. sp. and P. microlepis. Pseudoparamoeba species are hardly distinguishable by morphology alone, but display clear differences in 18S rDNA and COX1 gene sequences.     

Molecular phylogeny of the New World gecko genus Homonota (Squamata: Phyllodactylidae)

The genus Homonota was described by Gray (1845) and currently includes 10 species: Homonota andicola, H. borellii, H. darwinii, H. fasciata, H. rupicola, H. taragui, H. underwoodi, H. uruguayensis, H. williamsii & H. whitii and one subspecies of H. darwinii (H. darwinii macrocephala). It is distributed from 15° latitude south in southern Brazil, through much of Bolivia, Paraguay, Uruguay and Argentina to 54° south in Patagonia and across multiple different habitats. Several morphological taxonomic studies on a subset of these species have been published, but no molecular phylogenetic hypotheses are available for the genus. The objective of this study is to present a molecular phylogenetic hypothesis for all the described species in the genus. We sequenced two mitochondrial genes (cyt‐b & 12S: 1745 bp), seven nuclear protein coding (RBMX, DMLX, NKTR, PLRL, SINCAIP, MXRA5, ACA4: 5804 bp) and two anonymous nuclear loci (30Hb, 19Hb: 1306 bp) and implemented traditional concatenated analyses (MP, ML, BI) as well as species‐tree (*beast ) approaches. All methods recovered almost the same topology. We recovered the genus Homonota as monophyletic with strong statistical support. Within Homonota, there are three strongly supported clades (whitii, borellii and fasciata), which differ from those previously proposed based on scale shape, osteology, myology and quantitative characters. Detailed morphological analyses based on this highly resolved and well‐supported phylogeny will provide a framework for understanding morphological evolution and historical biogeography of this phenotypically conservative genus. We hypothesize that extensive marine transgressions during Middle and Late Miocene most probably isolated the ancestors of the three main clades in eastern Uruguay (borellii group), north‐western Argentina‐southern Bolivia (fasciata group), and central‐western Argentina (whitii group). Phylogeographic and morphological/morphometric analyses coupled with paleo‐niche modelling are needed to better understand its biogeographical history.     

First report of three benthic dinoflagellates,Gambierdiscus pacificus,G. australes and G. caribaeus (Dinophyceae), from Hainan Island,South China Sea

Species of the marine benthic dinoflagellate genus Gambierdiscus are the principal cause of Ciguatera fish poisoning. This genus has been recorded from tropical to temperate oceans, although Gambierdiscus species have rarely been found in Chinese waters. Our work revealed the morphological and genetic characteristics of three potentially toxic Gambierdiscus species observed in the temperate to subtropical waters of China. The fine thecal morphology was determined based on light microscopy and scanning electron microscopy analyses, and these species were also characterized by sequencing the D1–D3 and D8–D10 regions of the LSU rDNA. The morphological and genetic data indicated that these three Gambierdiscus species were G. pacificus, G. australes and G. caribaeus. This work provides the first report of these species in Chinese waters, which increases the known species distribution of this genus.     

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.