Nikos Andreakis

Cryptic speciation in a model invertebrate chordate

We applied independent species concepts to clarify the phylogeographic structure of the ascidian Ciona intestinalis, a powerful model system in chordate biology and for comparative genomic studies. Intensive research with this marine invertebrate is based on the assumption that natural populations globally belong to a single species. Therefore, understanding the true taxonomic classification may have implications for experimental design and data management. Phylogenies inferred from mitochondrial and nuclear DNA markers accredit the existence of two cryptic species: C. intestinalis sp. A, genetically homogeneous, distributed in the Mediterranean, northeast Atlantic, and Pacific, and C. intestinalis sp. B, geographically structured and encountered in the North Atlantic. Species-level divergence is further entailed by cross-breeding estimates. C. intestinalis A and B from allopatric populations cross-fertilize, but hybrids remain infertile because of defective gametogenesis. Although anatomy illustrates an overall interspecific similarity lacking in diagnostic features, we provide consistent tools for in-field and in-laboratory species discrimination. Finding of two cryptic taxa in C. intestinalis raises interest in a new tunicate genome as a gateway to studies in speciation and ecological adaptation of chordates.

Phylogeography of the invasive seaweed Asparagopsis (Bonnemaisoniales, Rhodophyta) reveals cryptic diversity.

The rhodophyte seaweed Asparagopsis armata Harvey is distributed in the northern and southern temperate zones, and its congener Asparagopsis taxiformis (Delile) Trevisan abounds throughout the tropics and subtropics. Here, we determine intraspecific phylogeographic patterns to compare potential causes of the disjunctions in the distributions of both species. We obtained specimens throughout their ranges and inferred phylogenies from the hypervariable domains D1‐D3 of the nuclear rDNA LSU, the plastid spacer between the large and small subunits of RuBisCo and the mitochondrial cox 2–3 intergenic spacer. The cox spacer acquired base changes the fastest and the RuBisCo spacer the slowest. Median‐joining networks inferred from the sequences revealed the absence of phylogeographic structure in the introduced range of A. armata, corroborating the species’ reported recent introduction. A. taxiformis consisted of three nuclear, three plastid and four mitochondrial genetically distinct, lineages (1–4). Mitochondrial lineage 3 is found in the western Atlantic, the Canary Islands and the eastern Mediterranean. Mitochondrial lineages 1, 2, and 4 occur in the Indo‐Pacific, but one of them (lineage 2) is also found in the central Mediterranean and southern Portugal. Phylogeographic results suggest separation of Atlantic and Indo‐Pacific lineages resulted from the emergence of the Isthmus of Panama, as well as from dispersal events postdating the closure event, such as the invasion of the Mediterranean Sea by mitochondrial lineages 2 and 3. Molecular clock estimates using the Panama closure event as a calibration for the split of lineages 3 and 4 suggest that A. taxiformis diverged into two main cryptic species (1 + 2 and 3 + 4) about 3.2–5.5 million years ago (Ma), and that the separation of the mitochondrial lineages 1 and 2 occurred 1–2.3 Ma.

Assessing hidden species diversity in the coral Pocillopora damicornis from Eastern Australia

The incredible range of morphological plasticity present in scleractinian corals has confused the taxonomy of the group, prompting the introduction of “ecomorphs” to explain the observed correlation between local environmental conditions and phenotypic variation. Pocilloporadamicornis (Linnaeus, 1758) represents one of the best known examples of eco-phenotypic variation in scleractinian corals with a variety of forms and reproductive strategies reported across its global distribution range. Here, we reconstruct genealogical relationships of P. damicornis colonies collected from thirteen locations along the East Australian coast to examine the relationship between genetic and phenotypic diversity in this species. Haplotype networks computed from two mitochondrial DNA regions (CR, ORF) indicate that the range of morphotypes observed within this taxon fall into at least five genetically distinct mitochondrial lineages. Nuclear (HSP70, ITS2) haplowebs on the other hand recover sharp genetic discontinuities among three of the morphological groups. We conclude that P. damicornis from Eastern Australia constitutes a cryptic species complex. The misinterpretation of taxonomical units within P. damicornis may well explain its perceived variation in the ecology, biology and life history across its range.

Evolution of the Nitric Oxide Synthase Family in Metazoans

Nitric oxide (NO) is essential to many physiological functions and operates in several signaling pathways. It is not understood how and when the different isoforms of nitric oxide synthase (NOS), the enzyme responsible for NO production, evolved in metazoans. This study investigates the number and structure of metazoan NOS enzymes by genome data mining and direct cloning of Nos genes from the lamprey. In total, 181 NOS proteins are analyzed from 33 invertebrate and 63 vertebrate species. Comparisons among protein and gene structures, combined with phylogenetic and syntenic studies, provide novel insights into how NOS isoforms arose and diverged. Protein domains and gene organization--that is, intron positions and phases--of animal NOS are remarkably conserved across all lineages, even in fast-evolving species. Phylogenetic and syntenic analyses support the view that a proto-NOS isoform was recurrently duplicated in different lineages, acquiring new structural configurations through gains and losses of protein motifs. We propose that in vertebrates a first duplication took place after the agnathan-gnathostome split followed by a paralog loss. A second duplication occurred during early tetrapod evolution, giving rise to the three isoforms--I, II, and III--in current mammals. Overall, NOS family evolution was the result of multiple gene and genome duplication events together with changes in protein architecture.

Asparagopsis taxiformis and Asparagopsis armata (Bonnemaisoniales, Rhodophyta): genetic and morphological identification of Mediterranean populations

The tropical-subtropical red seaweed Asparagopsis Montagne (Bonnemaisoniales) constitutes the haploid, gametophytic phase in a heteromorphic diplo-haplontic life cycle. The diploid tetrasporophyte is known as the ‘Falkenbergia’ stage. The genus contains two species, A. armata and A. taxiformis, both present in the Mediterranean Sea where they are regarded as introduced. A. armata is morphologically distinct from A. taxiformis in that it possesses long stolons bearing harpoon-like hooks. The seemingly morphologically identical ‘Falkenbergia’ stages of the two Asparagopsis species and phenotypic variation within these species have caused taxonomic confusion. We defined species boundaries in the Mediterranean Sea by inferring phylogenies from sequence data from a variable region in the nuclear LSU rDNA gene, the plastid RuBisCo spacer, and the mitochondrial cox2 – 3 spacer of specimens from the Mediterranean, western Europe and the Canary Islands. Results indicate that A. armata and its ‘Falkenbergia’ tetrasporophyte are genetically distinct from A. taxiformis and its ‘Falkenbergia’ phase. No phylogeographic structure was detected within A. armata, whereas A. taxiformis seems to consist of at least two genetically distinct but morphologically cryptic species, an Atlantic one (from the Canary Islands) and a Mediterranean one. Hypothetical distribution patterns of the two species as reconstructed from critical temperature limits to growth, survival and reproduction and from the summer and winter isotherms in the Mediterranean Sea agree with the actual Mediterranean distribution patterns as gleaned from our data.

Cryptic diversity in flathead fishes (Scorpaeniformes: Platycephalidae) across the Indo-West Pacific uncovered by DNA barcoding.

Identification of taxonomical units underpins most biological endeavours ranging from accurate biodiversity estimates to the effective management of sustainably harvested, protected or endangered species. Successful species identification is now frequently based on a combination of approaches including morphometrics and DNA markers. Sequencing of the mitochondrial COI gene is an established methodology with an international campaign directed at barcoding all fishes. We employed COI sequencing alongside traditional taxonomic identification methods and uncovered instances of deep intraspecific genetic divergences among flathead species. Sixty‐five operational taxonomic units (OTUs) were observed across the Indo‐West Pacific from just 48 currently recognized species. The most comprehensively sampled taxon, Platycephalus indicus, exhibited the highest levels of genetic diversity with eight lineages separated by up to 16.37% genetic distance. Our results clearly indicate a thorough reappraisal of the current taxonomy of P. indicus (and its three junior synonyms) is warranted in conjunction with detailed taxonomic work on the other additional Platycephalidae OTUs detected by DNA barcoding.

Differential regulation of the zebrafish orthopedia1 gene during fate determination of diencephalic neurons

BackgroundThe homeodomain transcription factor Orthopedia (Otp) is essential in restricting the fate of multiple classes of secreting neurons in the neuroendocrine hypothalamus of vertebrates. However, there is little information on the intercellular factors that regulate Otp expression during development.ResultsHere, we identified two otp orthologues in zebrafish (otp1 and otp2) and explored otp1 in the context of the morphogenetic pathways that specify neuroectodermal regions. During forebrain development, otp1 is expressed in anterior groups of diencephalic cells, positioned in the preoptic area (PO) (anterior alar plate) and the posterior tuberculum (PT) (posterior basal plate). The latter structure is characterized by Tyrosine Hydroxylase (TH)-positive cells, suggesting a role for otp1 in the lineage restriction of catecholaminergic (CA) neurons. Disruptions of Hedgehog (HH) and Fibroblast Growth Factor (FGF) pathways point to the ability of SHH protein to trigger otp1 expression in PO presumptive neuroblasts, with the attenuating effect of Dzip1 and FGF8. In addition, our data disclose otp1 as a determinant of CA neurons in the PT, where otp1 activity is strictly dependent on Nodal signaling and it is not responsive to SHH and FGF.ConclusionIn this study, we pinpoint the evolutionary importance of otp1 transcription factor in cell states of the diencephalon anlage and early neuronal progenitors. Furthermore, our data indicate that morphogenetic mechanisms differentially regulate otp1 expression in alar and basal plates.

Proteomic characterisation of toxins isolated from nematocysts of the South Atlantic jellyfish Olindias sambaquiensis

Surprisingly little is known of the toxic arsenal of cnidarian nematocysts compared to other venomous animals. Here we investigate the toxins of nematocysts isolated from the jellyfish Olindias sambaquiensis. A total of 29 unique ms/ms events were annotated as potential toxins homologous to the toxic proteins from diverse animal phyla, including cone-snails, snakes, spiders, scorpions, wasp, bee, parasitic worm and other Cnidaria. Biological activities of these potential toxins include cytolysins, neurotoxins, phospholipases and toxic peptidases. The presence of several toxic enzymes is intriguing, such as sphingomyelin phosphodiesterase B (SMase B) that has only been described in certain spider venoms, and a prepro-haystatin P-IIId snake venom metalloproteinase (SVMP) that activates coagulation factor X, which is very rare even in snake venoms. Our annotation reveals sequence orthologs to many representatives of the most important superfamilies of peptide venoms suggesting that their origins in higher organisms arise from deep eumetazoan innovations. Accordingly, cnidarian venoms may possess unique biological properties that might generate new leads in the discovery of novel pharmacologically active drugs.

Genomic DNA isolation from green and brown algae (Caulerpales and Fucales) for microsatellite library construction

A method for isolating high‐quality DNA is presented for the green algae Caulerpa sp. (C. racemosa, C. prolifera, and C. taxifolia) and the brown alga Sargassum muticum. These are introduced, and invasive species in Europe, except for the native C. prolifera. Previous methods of extraction, using cetyl trimethyl ammonium bromide or various commercial kits, were used to isolate genomic DNA but either no DNA or DNA of very low quality was obtained. Genomic libraries were attempted with Caulerpa sp. on three occasions and either the restriction enzyme, the Taq polymerase, or the T4 ligase was inhibited, probably by the large amount of polysaccharides in these algae. The method presented here consists of the rapid isolation of stable nuclei, followed by DNA extraction. Yields of 6–10 μg genomic DNA from 1 g fresh blades were obtained. After genomic DNA was isolated from fresh material, the quality was checked by agarose gel. Quantification of DNA concentration was performed using UV spectrophotometric measurement of the A260/A280 ratio. The DNA was suitable for PCR, cloning, and hybridization. The DNA isolated using this method allowed successful construction of microsatellite libraries for Caulerpa species and S. muticum. The technique is inexpensive and appropriate for the isolation of multiple samples of DNA from a small amount of fresh material.

A complex life cycle in a warming planet: gene expression in thermally stressed sponges

Sponges are abundant, diverse and functionally important components of aquatic biotopes with crucial associations for many reef fish and invertebrates. Sponges have strict temperature optima, and mass mortality events have occurred after unusually high temperatures. To assess how sponges may adapt to thermal stress associated with a changing climate, we applied gene expression profiling to both stages of their bipartite life cycles. Adult Rhopaloeides odorabile are highly sensitive to thermal stress (32 °C), yet their larvae can withstand temperatures up to 36 °C. Here, we reveal the molecular mechanisms that underpin these contrasting thermal tolerances, which may provide sponges with a means to successfully disperse into cooler waters. Heat shock protein 70 was induced by increasing temperature in adult sponges, and genes involved in important biological functions including cytoskeleton rearrangement, signal transduction, protein synthesis/degradation, oxidative stress and detoxification were all negatively correlated with temperature. Conversely, gene expression in larvae was not significantly affected until 36 °C when a stress response involving extremely rapid activation of heat shock proteins occurred. This study provides the first transcriptomic assessment of thermal stress on both life history stages of a marine invertebrate facilitating better predictions of the long‐term consequences of climate change for sponge population dynamics.