Characterization of novel gliotoxin biosynthesis-related genes from deep-sea-derived fungus Geosmithia pallida FS140.

Gliotoxins are epipolythiodioxopiperazine toxins produced by the filamentous fungi, which show great potential in the treatment of liver and lung cancer because of its cytotoxicity. In this study, three novel genes related to gliotoxin biosynthesis, gliT, gliM and gliK encoding thioredoxin reductase, O-methyltransferase and gamma-glutamyl cyclotransferase, respectively, from the deep-sea-derived fungus Geosmithia pallida were cloned from G. pallida and expressed in Escherichia coli. The recombinant GliT, GliM and GliK proteins were expressed and purified by Ni affinity column, which was demonstrated by SDS-PAGE and Western blot analysis. The inclusion bodies of GliT were renatured and the corresponding enzymatic properties of the two enzymes were further investigated. Using DTNB as a substrate, GliT showed the highest enzymatic activity of 11041 mU/L at pH 7.0, and the optimal reaction temperature was 40 °C. Using EGCG as a substrate, GliM showed the highest enzymatic activity of 239.19 mU/mg at pH 7.0, the optimum temperature was 35 °C. GliK from G. pallida was firstly reported to show bi-function of glutymal cyclotransferase and acetyltransfearse actvity with highest enzymatic activity of 615.5 U/mg in this study. The results suggested the important enzymatic function of GliT, GliM and GliK in the gliotoxin biosynthesis in G. pallida, which would lay a foundation for the mechanism elucidation of the gliotoxin biosynthesis in G. pallida and the exploitation of novel gliotoxin derivaties.

Ecotoxicity of losartan potassium in aquatic organisms of different trophic levels.

The intensive use of the antihypertensive losartan potassium (LOS) has culminated in its high occurrence in aquatic environments. However, insufficient studies had investigated its effects in non-target organisms. In this study, ecotoxicity of LOS was assessed in aquatic organisms from distinct trophic levels (Desmodesmus subspicatus, Daphnia magna, and Astyanax altiparanae). Genotoxicity was assessed by the comet assay in D. magna and A. altiparanae, and biochemical biomarkers for the fish. LOS was more toxic to D. subspicatus (EC50(72h) = 27.93 mg L-1) than D. magna (EC50 = 303.69 mg L-1). Subsequently, this drug showed to induce more DNA damage in D. magna than A. altiparanae, when exposed to 2.5 mg L-1. No significant stress responses were observed by the fish biomarkers, suggesting that higher trophic levels organisms are more tolerant to LOS toxicity. LOS showed relatively low toxic potential for a short period of exposure, but with different patterns of toxicity for the organisms from distinct trophic levels, contributing to further risk assessment of LOS.

Development of the first microsatellite markers using high-throughput sequencing for a hexaploid coastal species, Tournefortia argentea L. f. (Boraginaceae).

BACKGROUD: Tournefortia argentea L. f. is a hexaploid shrub or tree species with ecological and evolutionary significances, which forms the fringe of vegetation closest to the sea on tropical coral islands. Previous studies have never addressed on genetic information, and thus genomic resources remain scarce. METHODS AND RESULTS: We used nine individuals from different islands to identify polymorphic microsatellites of T. argentea by Illumina high-throughput sequencing. Thirty-five polymorphic microsatellite markers were developed. Characteristics of each locus were tested using 48 individuals collected from three populations of T. argentea. A total of 320 alleles were found across the 35 microsatellite loci. The number of alleles per locus ranged from 5 to 15, with an average of 9.1. Observed and expected heterozygosities in each locus per population varied from 0.000 to 1.000 and from 0.000 to 0.893, respectively. CONCLUSIONS: In this study, we report the development of 35 polymorphic microsatellite markers based on Illumina high-throughput sequencing. These markers will facilitate the investigations of genetic diversity, population structures and evolutionary history of T. argentea.

ATP Drives Efficient Terpene Biosynthesis in Marine Thraustochytrids.

Understanding carbon flux controlling mechanisms in a tangled metabolic network is an essential question of cell metabolism. Secondary metabolism, such as terpene biosynthesis, has evolved with low carbon flux due to inherent pathway constraints. Thraustochytrids are a group of heterotrophic marine unicellular protists and can accumulate terpenoids under the high-salt conditions in their natural environment. However, the mechanism behind terpene accumulation is not well understood. Here, we show that terpene biosynthesis in Thraustochytrium sp. ATCC 26185 is constrained by local thermodynamics in the mevalonate pathway. Thermodynamic analysis reveals metabolite limitation in the nondecarboxylative Claisen condensation of acetyl-coenzyme A (CoA) to the acetoacetyl-CoA step, catalyzed by the acetyl-CoA acetyltransferase (ACAT). Through a sodium-elicited mechanism, higher respiration leads to increased ATP investment into the mevalonate pathway, providing a strong thermodynamic driving force for enhanced terpene biosynthesis. Proteomic and metabolomic analyses further show that the increased ATP demands are fulfilled by shifting energy generation from carbohydrate to lipid oxidation. This study demonstrates a unique strategy in nature that uses ATP to drive a low-flux metabolic pathway, providing an alternative solution for efficient terpene metabolic engineering. IMPORTANCE Terpenoids are a large class of lipid molecules with important biological functions and diverse industrial and medicinal applications. Metabolic engineering for terpene production has been hindered by the low-flux distribution to its biosynthesis pathway. In practice, a high substrate load is generally required to reach high product titers. Here, we show that mevalonate-derived terpene biosynthesis is constrained by local pathway thermodynamics, which can only be partially relieved by increasing substrate levels. Through comparative omics and biochemical analyses, we discovered a unique mechanism for high terpene accumulation in marine protist thraustochytrids. Through a sodium-induced mechanism, thraustochytrids shift their energy metabolism from carbohydrate to lipid oxidation for enhanced ATP production, providing a strong thermodynamic driving force for efficient terpene biosynthesis. This study reveals an important mechanism in eukaryotes to overcome the thermodynamic constraint in low-flux pathways by increased ATP consumption. Engineering energy metabolism thus provides an important alternative to relieve flux constraints in low-flux and energy-consuming pathways.

An Analysis of Biosynthesis Gene Clusters and Bioactivity of Marine Bacterial Symbionts.

Symbiotic marine bacteria have a pivotal role in drug discovery due to the synthesis of diverse biologically potential compounds. The marine bacterial phyla proteobacteria, actinobacteria and firmicutes are commonly associated with marine macro organisms and frequently reported as dominant bioactive compound producers. They can produce biologically active compounds that possess antimicrobial, antiviral, antitumor, antibiofilm and antifouling properties. Synthesis of these bioactive compounds is controlled by a set of genes of their genomes that is known as biosynthesis gene clusters (BGCs). The development in the field of biotechnology and bioinformatics has uncovered the potential BGCs of the bacterial genome and its functions. Now-a-days researchers have focused their attention on the identification of potential BGCs for the discovery of novel bioactive compounds using advanced technology. This review highlights the marine bacterial symbionts and their BGCs which are responsible for the synthesis of bioactive compounds.

Untangling filamentous marine cyanobacterial diversity from the coast of South Florida with the description of Vermifilaceae fam. nov. and three new genera: Leptochromothrix gen. nov., Ophiophycus gen. nov., and Vermifilum gen. nov.

Benthic cyanobacterial mats are an integral component of aquatic communities in tropical marine waters. These mats can develop into massive nuisances at risk of expansion due to climate change. The extent of diversity occurring within these mats, still remains largely unexplored, especially in Florida. To reveal this diversity, coastal environments of South Florida were sampled and subsequently processed for isolation and systematic identification. Three new genera are proposed based on the molecular phylogeny, morphology, and ecology. These new genera are characterized by discoid cells and homocytous, unbranched filaments without sheaths. Individual genus morphological differences include either rounded bent, rounded, or conical rounded apical cells. A unique molecular fingerprint including a base pair insert within the 16S rRNA gene sequence and genetic similarities facilitates the delimitation of a novel family Vermifilaceae. Using the polyphasic approach, our research presents three new genera and four new species of marine cyanobacteria inhabiting coastal ecosystems of South Florida.

Exposure of microalgae Euglena gracilis to polystyrene microbeads and cadmium: Perspective from the physiological and transcriptional responses.

Micro(nano)plastics (MPs/NPs) are already present as contaminants in the natural environment globally and have been shown to be difficult to degrade, resulting in the potential for ecological damage and public health concerns. However, the adverse effects of exposure to MPs/NPs by aquatic organisms, especially freshwater microalgae, remains unclear. In the present study, the growth, physiology and transcriptome of the freshwater microalgae Euglena gracilis were comprehensively analyzed following exposure to 1 mg/L of polystyrene (PS) microbeads (5 µm PS-MPs and 100 nm PS-NPs), 0.5 mg/L cadmium (Cd), or a mixture of PS microbeads and Cd for 96 h. Results showed that the toxicity of PS-MPs to microalgae was greater than PS-NPs, inducing increased growth inhibition, oxidative damage and decreased photosynthesis pigment concentrations. PS-MPs alone or in combination with Cd caused cavitation within microalgal cells, as well as increasing the number and volume of vacuoles. The combined exposure toxicity test showed that a combination of Cd + PS-NPs was more toxic than Cd + PS-MPs, which may be explained by the transcriptomic analysis results. Differentially expressed genes (DEGs) in the Cd + PS-NPs group were mainly enriched in metabolism-related pathways, suggesting that algal metabolism was hindered, resulting in aggravation of toxicity. The reduced toxicity induced by Cd + PS-MPs may indicate a response to resist external stress processes. In addition, no adsorption of 0.5 mg/L Cd to 1 mg/L PS microbeads was observed, suggesting that adsorption of MPs/NPs and Cd was not the key factor determining the combined toxicity effects in this study.

Ancestral Reconstructions Decipher Major Adaptations of Ammonia-Oxidizing Archaea upon Radiation into Moderate Terrestrial and Marine Environments.

Unlike all other archaeal lineages, ammonia-oxidizing archaea (AOA) of the phylum Thaumarchaeota are widespread and abundant in all moderate and oxic environments on Earth. The evolutionary adaptations that led to such unprecedented ecological success of a microbial clade characterized by highly conserved energy and carbon metabolisms have, however, remained underexplored. Here, we reconstructed the genomic content and growth temperature of the ancestor of all AOA, as well as the ancestors of the marine and soil lineages, based on 39 available complete or nearly complete genomes of AOA. Our evolutionary scenario depicts an extremely thermophilic, autotrophic, aerobic ancestor from which three independent lineages of a marine and two terrestrial groups radiated into moderate environments. Their emergence was paralleled by (i) a continuous acquisition of an extensive collection of stress tolerance genes mostly involved in redox maintenance and oxygen detoxification, (ii) an expansion of regulatory capacities in transcription and central metabolic functions, and (iii) an extended repertoire of cell appendages and modifications related to adherence and interactions with the environment. Our analysis provides insights into the evolutionary transitions and key processes that enabled the conquest of the diverse environments in which contemporary AOA are found.

METATRYP v 2.0: Metaproteomic Least Common Ancestor Analysis for Taxonomic Inference Using Specialized Sequence Assemblies-Standalone Software and Web Servers for Marine Microorganisms and Coronaviruses.

We present METATRYP version 2 software that identifies shared peptides across the predicted proteomes of organisms within environmental metaproteomics studies to enable accurate taxonomic attribution of peptides during protein inference. Improvements include ingestion of complex sequence assembly data categories (metagenomic and metatranscriptomic assemblies, single cell amplified genomes, and metagenome assembled genomes), prediction of the least common ancestor (LCA) for a peptide shared across multiple organisms, increased performance through updates to the backend architecture, and development of a web portal (https://metatryp.whoi.edu). Major expansion of the marine METATRYP database with predicted proteomes from environmental sequencing confirms a low occurrence of shared tryptic peptides among disparate marine microorganisms, implying tractability for targeted metaproteomics. METATRYP was designed to facilitate ocean metaproteomics and has been integrated into the Ocean Protein Portal (https://oceanproteinportal.org); however, it can be readily applied to other domains. We describe the rapid deployment of a coronavirus-specific web portal (https://metatryp-coronavirus.whoi.edu/) to aid in use of proteomics on coronavirus research during the ongoing pandemic. A coronavirus-focused METATRYP database identified potential SARS-CoV-2 peptide biomarkers and indicated very few shared tryptic peptides between SARS-CoV-2 and other disparate taxa analyzed, sharing <1% peptides with taxa outside of the betacoronavirus group, establishing that taxonomic specificity is achievable using tryptic peptide-based proteomic diagnostic approaches.

Antimicrobial Activity of Small Synthetic Peptides Based on the Marine Peptide Turgencin A: Prediction of Antimicrobial Peptide Sequences in a Natural Peptide and Strategy for Optimization of Potency.

Turgencin A, a potent antimicrobial peptide isolated from the Arctic sea squirt Synoicum turgens, consists of 36 amino acid residues and three disulfide bridges, making it challenging to synthesize. The aim of the present study was to develop a truncated peptide with an antimicrobial drug lead potential based on turgencin A. The experiments consisted of: (1) sequence analysis and prediction of antimicrobial potential of truncated 10-mer sequences; (2) synthesis and antimicrobial screening of a lead peptide devoid of the cysteine residues; (3) optimization of in vitro antimicrobial activity of the lead peptide using an amino acid replacement strategy; and (4) screening the synthesized peptides for cytotoxic activities. In silico analysis of turgencin A using various prediction software indicated an internal, cationic 10-mer sequence to be putatively antimicrobial. The synthesized truncated lead peptide displayed weak antimicrobial activity. However, by following a systematic amino acid replacement strategy, a modified peptide was developed that retained the potency of the original peptide. The optimized peptide StAMP-9 displayed bactericidal activity, with minimal inhibitory concentrations of 7.8 µg/mL against Staphylococcus aureus and 3.9 µg/mL against Escherichia coli, and no cytotoxic effects against mammalian cells. Preliminary experiments indicate the bacterial membranes as immediate and primary targets.

The bacterial potassium transporter gene MbtrkH improves K+ uptake in yeast and tobacco.

K+ is an essential nutrient for plant growth and is responsible for many important physiological processes. K+ deficiency leads to crop yield losses, and overexpression of K+ transporter genes has been proven to be an effective way to resolve this problem. However, current research on the overexpression of K+ transporter genes is limited to plant sources. TrkH is a bacterial K+ transporter whose function generally depends on the regulation of TrkA. To date, whether TrkH can improve K+ uptake in eukaryotic organisms is still unknown. In this study, a novel MbtrkH gene was cloned from marine microbial metagenomic DNA. Functional complementation and K+-depletion analyses revealed that MbTrkH functions in K+ uptake in the K+-deficient yeast strain CY162. Moreover, K+-depletion assays revealed that MbtrkH overexpression improves plant K+ uptake. K+ hydroponic culture experiments showed that, compared with WT tobacco lines, MbtrkH transgenic tobacco lines had significantly greater fresh weights, dry weights and K+ contents. These results indicate that MbTrkH promotes K+ uptake independently of TrkA in eukaryotes and provide a new strategy for improving K+-use efficiency in plants.

Marine bacterial community analysis on 316L stainless steel coupons by Illumina MiSeq sequencing.

In order to evaluate the corrosive action of microorganisms on 316L metal exposed directly to a marine environment, a system was designed to immerse coupons in seawater. After periods of 30, 60 and 90 days, the coupons were recovered, the corrosion rates evaluated and the biofilm samples on their surface were analyzed by 16S rRNA gene sequencing. The results of the corrosion rate showed an acceleration over the entire experimental period. Alpha diversity measurements showed higher rates after 60 days of the experiment, while abundance measurements showed higher rates after 90 days of exposure to the marine environment. The beta-diversity results showed a clear separation between the three conditions and proximity in the indices between replicates of the same experimental condition. The results of 16S rRNA gene sequencing showed that after 30 days of exposure to seawater, there was massive representativeness of the pioneer bacteria, Gamma and Alphaproteobacteria, with emphasis on the genera Alcanivorax, Oceanospirillum and Shewanella. At the 60-day analysis, the Gammaproteobacteria class remained dominant, followed by Alphaproteobacteria and Flavobacteria, and the main representatives were Flexibacter and Pseudoalteromonas. In the last analysis, after 90 days, a change in the described bacterial community profile was observed. The Gammaproteobacteria class was still the largest in diversity and OTUs. The most predominant genera in number of OTUs were Alteromonas, Bacteriovorax and, Nautella. Our results describe a change in the microbial community over coupons directly exposed to the marine environment, suggesting a redirection to the formation of a mature biofilm. The conditions created by the biofilm structure suggest said condition favor biocorrosion on the analyzed coupons.

Exploring Diversity, Taxonomy and Phylogeny of Diatoms (Bacillariophyta) from Marine Habitats. Novel Taxa with Internal Costae.

In many marine littoral and sublittoral benthic habitats, we find small diatoms with few features resolvable with light microscopy (LM) other than internal costae across their valves. While classically those internal costae have defined their identification and classification, the use of electron microscopy and of molecular data have started to reveal the true diversity of unrelated forms and genera (e.g., Anaulus, Eunotogramma, Hustedtiella, or Plagiogramma) which possess these structures. Here we describe the new genus Ambo, in an attempt to clarify some of the polyphyly of taxa with internal costa by formally transferring Anaulus balticus, Anaulus simonsenii, and Plagiogramma tenuissimum as well as Ambo gallaeciae, described here. Related to this, we attempt to document and characterize the genus Anaulus itself, which was formally described by Ehrenberg with an illustration. A search by LM of mica designated by Ehrenberg as the holotype of Anaulus scalaris, the generitype of Anaulus, failed to recover a specimen which adequately describes the genus to the exclusion of other genera with internal costa. We also present morphological and molecular data for Anaulus creticus and suggest a new genus-Ceratanaulus-to reflect the distinct morphological and molecular characters we documented.

Ecophysiological traits of highly mobile large marine predators inferred from nucleic acid derived indices.

Nucleic acid-derived indices such as RNA/DNA ratios have been successfully applied as ecophysiological indicators to assess growth, nutritional condition and health status in marine organisms given that they provide a measure of tissue protein reserves, which is known to vary depending on changes in the environment. Yet, the use of these biochemical indices on highly mobile large predators is scarce. In this study, we tested the applicability of using nucleic acids to provide insights on the ecophysiological traits of two marine mammal species (common bottlenose dolphins and short-finned pilot whales) and explored potential related factors (species, sex, season, and residency pattern), using skin tissue (obtained from biopsy darts) of apparently healthy and adult free-ranging animals. Significantly higher RNA/DNA ratios were obtained for bottlenose dolphins (p < 0.001), and for visitor pilot whales when compared with resident pilot whales (p = 0.001). No significant changes were found between the sexes. Based on the percentile approach, the samples contain individuals in a general good condition (as the 10th percentile is not closer to the mean than the 75th percentile), suggesting that the studied region of Macaronesia may be considered an adequate habitat. The combination of this effective tool with genetic sexing and photographic-identification provided an overall picture of ecosystem health, and although with some limitations and still being a first approach, it has the applicability to be used in other top predators and ecosystems.

Discovery of Bioactive Indole-Diketopiperazines from the Marine-Derived Fungus Penicillium brasilianum Aided by Genomic Information.

Identification and analysis of the whole genome of the marine-derived fungus Penicillium brasilianum HBU-136 revealed the presence of an interesting biosynthetic gene cluster (BGC) for non-ribosomal peptide synthetases (NRPS), highly homologous to the BGCs of indole-diketopiperazine derivatives. With the aid of genomic analysis, eight indole-diketopiperazines (1-8), including three new compounds, spirotryprostatin G (1), and cyclotryprostatins F and G (2 and 3), were obtained by large-scale cultivation of the fungal strain HBU-136 using rice medium with 1.0% MgCl2. The absolute configurations of 1-3 were determined by comparison of their experimental electronic circular dichroism (ECD) with calculated ECD spectra. Selective cytotoxicities were observed for compounds 1 and 4 against HL-60 cell line with the IC50 values of 6.0 and 7.9 µM, respectively, whereas 2, 3, and 5 against MCF-7 cell line with the IC50 values of 7.6, 10.8, and 5.1 µM, respectively.

Marine Proteobacteria as a source of natural products: advances in molecular tools and strategies.

Covering: up to 2019Humanity is in dire need for novel medicinal compounds with biological activities ranging from antibiotic to anticancer and anti-dementia effects. Recent developments in genome sequencing and mining have revealed an unappreciated potential for bioactive molecule production in marine Proteobacteria. Also, novel bioactive compounds have been discovered through molecular manipulations of either the original marine host bacteria or in heterologous hosts. Nevertheless, in contrast to the large repertoire of such molecules as predicted by in silico analysis, few marine bioactive compounds have been reported. This review summarizes the recent advances in the study of natural products from marine Proteobacteria. Here we present successful examples on genetic engineering of biosynthetic gene clusters of natural products from marine Proteobacteria. We also discuss the future prospects of discovering novel bioactive molecules via both heterologous production methodology and the development of marine Proteobacteria as new cell factories.

Transcriptomic Analysis of Marine Gastropod Hemifusus tuba Provides Novel Insights into Conotoxin Genes.

The marine gastropod Hemifusus tuba is served as a luxury food in Asian countries and used in traditional Chinese medicine to treat lumbago and deafness. The lack of genomic data on H. tuba is a barrier to aquaculture development and functional characteristics of potential bioactive molecules are poorly understood. In the present study, we used high-throughput sequencing technologies to generate the first transcriptomic database of H. tuba. A total of 41 unique conopeptides were retrieved from 44 unigenes, containing 6-cysteine frameworks belonging to four superfamilies. Duplication of mature regions and alternative splicing were also found in some of the conopeptides, and the de novo assembly identified a total of 76,306 transcripts with an average length of 824.6 nt, of which including 75,620 (99.1%) were annotated. In addition, simple sequence repeats (SSRs) detection identified 14,000 unigenes containing 20,735 SSRs, among which, 23 polymorphic SSRs were screened. Thirteen of these markers could be amplified in Hemifusus ternatanus and seven in Rapana venosa. This study provides reports of conopeptide genes in Buccinidae for the first time as well as genomic resources for further drug development, gene discovery and population resource studies of this species.

Marine Bacteria, A Source for Alginolytic Enzyme to Disrupt Pseudomonas aeruginosa Biofilms.

Pseudomonas aeruginosa biofilms are typically associated with the chronic lung infection of cystic fibrosis (CF) patients and represent a major challenge for treatment. This opportunistic bacterial pathogen secretes alginate, a polysaccharide that is one of the main components of its biofilm. Targeting this major biofilm component has emerged as a tempting therapeutic strategy for tackling biofilm-associated bacterial infections. The enormous potential in genetic diversity of the marine microbial community make it a valuable resource for mining activities responsible for a broad range of metabolic processes, including the alginolytic activity responsible for degrading alginate. A collection of 36 bacterial isolates were purified from marine water based on their alginolytic activity. These isolates were identified based on their 16S rRNA gene sequences. Pseudoalteromonas sp. 1400 showed the highest alginolytic activity and was further confirmed to produce the enzyme alginate lyase. The purified alginate lyase (AlyP1400) produced by Pseudoalteromonas sp. 1400 showed a band of 23 KDa on a protein electrophoresis gel and exhibited a bifunctional lyase activity for both poly-mannuronic acid and poly-glucuronic acid degradation. A tryptic digestion of this gel band analyzed by liquid chromatography-tandem mass spectrometry confirmed high similarity to the alginate lyases in polysaccharide lyase family 18. The purified alginate lyase showed a maximum relative activity at 30 °C at a slightly acidic condition. It decreased the sodium alginate viscosity by over 90% and reduced the P. aeruginosa (strain PA14) biofilms by 69% after 24 h of incubation. The combined activity of AlyP1400 with carbenicillin or ciprofloxacin reduced the P. aeruginosa biofilm thickness, biovolume and surface area in a flow cell system. The present data revealed that AlyP1400 combined with conventional antibiotics helped to disrupt the biofilms produced by P. aeruginosa and can be used as a promising combinational therapeutic strategy.

Increasing species sampling in chelicerate genomic-scale datasets provides support for monophyly of Acari and Arachnida.

Chelicerates are a diverse group of arthropods, represented by such forms as predatory spiders and scorpions, parasitic ticks, humic detritivores, and marine sea spiders (pycnogonids) and horseshoe crabs. Conflicting phylogenetic relationships have been proposed for chelicerates based on both morphological and molecular data, the latter usually not recovering arachnids as a clade and instead finding horseshoe crabs nested inside terrestrial Arachnida. Here, using genomic-scale datasets and analyses optimised for countering systematic error, we find strong support for monophyletic Acari (ticks and mites), which when considered as a single group represent the most biodiverse chelicerate lineage. In addition, our analysis recovers marine forms (sea spiders and horseshoe crabs) as the successive sister groups of a monophyletic lineage of terrestrial arachnids, suggesting a single colonisation of land within Chelicerata and the absence of wholly secondarily marine arachnid orders.

Molecular phylogenetic species delimitation in the aquatic genus Ottelia (Hydrocharitaceae) reveals cryptic diversity within a widespread species.

Ottelia, a pantropical genus of aquatic plants belonging to the family Hydrocharitaceae, includes several narrowly distributed taxa in Asia. Although the Asian species have received comparatively more research attention than congeners in other areas, various key taxonomic questions remain unaddressed, especially with regards to apparent cryptic diversity within O. alismoides, a widespread species complex native to Asia, northern Australia and tropical Africa. Here we test taxonomic concepts and evaluate species boundaries using a phylogenetic framework. We sampled five of the seven species of Ottelia in Asia as well as each species endemic to Africa and Australia; multiple samples of O. alismoides were obtained from across Asia. Phylogenetic trees based on five plastid DNA markers and the nuclear ITS region shared almost identical topologies. A Bayesian coalescent method of species delimitation using the multi-locus data set discerned one species in Africa, one in Australia and four in Asia with the highest probability. The results lead us to infer that a population sampled in Thailand represents a hitherto unrecognised cryptic taxon within the widespread species complex, although the apparent lack of unambiguous diagnostic characters currently precludes formal description. Conversely, no molecular evidence for distinguishing O. cordata and O. emersa was obtained, and so the latter is synonymised under the former. Two accessions that exhibit inconsistent positions among our phylogenetic trees may represent cases of chloroplast capture, however incomplete lineage sorting or polyploidy are alternative hypotheses that ought to be tested using other molecular markers.