Tomaso Patarnello

Strategies for microsatellite isolation: a review

In the last few years microsatellites have become one of the most popular molecular markers used with applications in many different fields. High polymorphism and the relative ease of scoring represent the two major features that make microsatellites of large interest for many genetic studies. The major drawback of microsatellites is that they need to be isolated de novo from species that are being examined for the first time. The aim of the present paper is to review the various methods of microsatellite isolation described in the literature with the purpose of providing useful guidelines in making appropriate choices among the large number of currently available options. In addition, we propose a fast and easy protocol which is a combination of different published methods.

Pillars of Hercules: is the Atlantic–Mediterranean transition a phylogeographical break?

The geological history of the Mediterranean Sea, its hydrography and connection with the Atlantic Ocean have been well documented. Despite a wealth of historical and oceanographic data, the Atlantic–Mediterranean transition remains controversial at the biological level as there are discordant results regarding the biogeographical separation between the Atlantic and Mediterranean biota. The opening of the Strait of Gibraltar at the end of the Messinian Salinity Crisis (some 5.33 million years ago), removed the land barrier that impeded the marine biota allowing it to disperse freely into the Mediterranean Sea. However, present day genetic patterns suggest a limitation to gene flow for some marine species, preventing population admixture. In the last few years, a large number of studies have challenged the hypothesis of the Strait of Gibraltar representing a phylogeographical break. A review of more than 70 papers reveals no obvious relationship between either dispersal ability or life history, and observed patterns of partial or complete genetic isolation between Atlantic and Mediterranean populations. We re‐analysed a selection of this large body of data (20 studies in total) in order to provide a homogeneous and coherent view on the generality of the phylogeographical patterns and the presence of a phylogeographical barrier. This offered the opportunity to summarize the state of the art on this matter and reach some general conclusions on the evolutionary history across the Atlantic–Mediterranean range. Geographically, some species in the transition zone showed step changes of allele frequencies associated with the Almeria‐Oran Front rather than with the Strait of Gibraltar itself. A major part of the data describe evolutionary events well within the time frame of the Quaternary age as very few taxa pre‐date closure of the Tethys Sea. Results point to a combined signature of vicariance, palaeoclimate fluctuation and life‐history traits on the Atlantic–Mediterranean phylogeographical patterns. Principal component analysis failed to show any particular association between biological traits and genetic variables. It would argue that organismal determinism may play a far less significant role than marine biogeographers have generally believed.

Mitochondrial cytochrome b: evolution and structure of the protein

Cytochrome b is the central redox catalytic subunit of the quinol: cytochrome c or plastocyanin oxidoreductases. It is involved in the binding of the quinone substrate and it is responsible for the transmembrane electron transfer by which redox energy is converted into a protonmotive force. Cytochrome b also contains the sites to which various inhibitors and quinone antagonists bind and, consequently, inhibit the oxidoreductase. Ten partial primary sequences of cytochrome b are presented here and they are compared with sequence data from over 800 species for a detailed analysis of the natural variation in the protein. This sequence information has been used to predict some aspects of the structure of the protein, in particular the folding of the transmembrane helices and the location of the quinone- and heme-binding pockets. We have observed that inhibitor sensitivity varies greatly among species. The comparison of inhibition titrations in combination with the analysis of the primary structures has enabled us to identify amino acid residues in cytochrome b that may be involved in the binding of the inhibitors and, by extrapolation, quinone/quinol. The information on the quinone-binding sites obtained in this way is expected to be both complementary and supplementary to that which will be obtained in the future by mutagenesis and X-ray crystallography.

Mitochondrial Phylogeny of Notothenioids: A Molecular Approach to Antarctic Fish Evolution and Biogeography

Antarctic waters represent a unique marine environment delimited by an oceanographic barrier, the Polar Front Zone, and characterized by constant subzero temperatures and presence of sea ice. A group of teleost fish, the Notothenioidei, have adapted to these challenging environmental conditions, undergoing a remarkable diversification. In the present study a total of 798 base pairs, generated from partial sequencing of 16S and 12S mitochondrial ribosomal RNA genes, were examined in 33 notothenioid species representative of all families included in the suborder Notothenioidei. Phylogenetic trees, reconstructed on the basis of sequence data by different methods, indicate that traditional hypotheses on notothenioid systematics and biogeography might be in need of reexamination. Molecular evidence suggests that vicariant speciation could be invoked to explain the early divergence of Eleginops maclovinus, a species previously included in the family Nototheniidae, which is now proposed as the closest sister group to all the rest of notothenioids apart from bovichtids. On the other hand, repeated, independent dispersal through the Polar Front is proposed for the divergence of other subantarctic notothenioid species. Likewise, multiple, independent transitions from benthic to pelagic habit are inferred from molecular data, at variance with the more conservative hypothesis based on cladograms reconstructed from morphological data.

Ancient climate change, antifreeze, and the evolutionary diversification of Antarctic fishes

The Southern Ocean around Antarctica is among the most rapidly warming regions on Earth, but has experienced episodic climate change during the past 40 million years. It remains unclear how ancient periods of climate change have shaped Antarctic biodiversity. The origin of antifreeze glycoproteins (AFGPs) in Antarctic notothenioid fishes has become a classic example of how the evolution of a key innovation in response to climate change can drive adaptive radiation. By using a time-calibrated molecular phylogeny of notothenioids and reconstructed paleoclimate, we demonstrate that the origin of AFGP occurred between 42 and 22 Ma, which includes a period of global cooling approximately 35 Ma. However, the most species-rich lineages diversified and evolved significant ecological differences at least 10 million years after the origin of AFGPs, during a second cooling event in the Late Miocene (11.6–5.3 Ma). This pattern indicates that AFGP was not the sole trigger of the notothenioid adaptive radiation. Instead, the bulk of the species richness and ecological diversity originated during the Late Miocene and into the Early Pliocene, a time coincident with the origin of polar conditions and increased ice activity in the Southern Ocean. Our results challenge the current understanding of the evolution of Antarctic notothenioids suggesting that the ecological opportunity that underlies this adaptive radiation is not linked to a single trait, but rather to a combination of freeze avoidance offered by AFGPs and subsequent exploitation of new habitats and open niches created by increased glacial and ice sheet activity.

Discord in the family Sparidae (Teleostei): divergent phylogeographical patterns across the Atlantic–Mediterranean divide

The Strait of Gibraltar has been proposed to be the divide between two marine biogeographical regions, the Mediterranean Sea and the Northeast Atlantic. Intraspecific studies have shown, for several of the examined species, a reduction of gene flow between the two basins. The present study examines genetic variation at nuclear and mitochondrial loci in five marine teleost species belonging to the family Sparidae. Four samples for each species were analysed spanning the Northeast Atlantic and the Mediterranean. For all individuals 17 allozyme loci were scored and a combined single strand conformation polymorphism‐sequencing approach was used to survey approximately 190 bp of the mitochondrial DNA (mtDNA) D‐loop region. All five species share similar biological features. For three species, namely Lithognathus mormyrus, Spondyliosoma cantharus, and Dentex dentex, large mtDNA divergence was observed between Atlantic and Mediterranean samples. Little or no mtDNA differentiation was found in the other two species, Pagrus pagrus and Pagellus bogaraveo. Allozyme data revealed strong differentiation when comparing Atlantic and Mediterranean samples of L. mormyrus and D. dentex, moderate for P. pagrus, and no differentiation for P. bogaraveo and S. cantharus. These results provide evidence for a sharp phylogeographical break (sensu Avise) between the Atlantic and the Mediterranean for two (or possibly three) sparid species of the five investigated. At the same time, the obtained results for the other two species raise the question on which ecological/historical factors might have caused the observed discrepancy in the geographical distribution of genetic variation among otherwise biologically similar species.

Differential population structuring of two closely related fish species, the mackerel (Scomber scombrus) and the chub mackerel (Scomber japonicus), in the Mediterranean Sea

Population genetic structures of the mackerel (Scomber scombrus) and chub mackerel (Scomber japonicus) were studied in the Mediterranean Sea. Fragments of 272 bp (S. scomber) and 387 bp (S. japonicus) of the 5′‐end of the mitochondrial control region were sequenced from spawning individuals collected off the coasts of Greece, Italy, Spain, and Portugal. High levels of mitochondrial control region haplotypic diversity (> 0.98) were found for both Scomber species. Nucleotide diversity was higher in the mackerel (0.022) than in the chub mackerel (0.017). Global FST values were also higher and significant in the mackerel (0.024, P < 0.0001) as opposed to the chub mackerel (0.003, P > 0.05). Molecular variance analyses showed differential genetic structuring for these two closely related species. There is extensive gene flow between Mediterranean Sea and Atlantic Ocean populations of chub mackerel, which are organized into a larger panmictic unit. In contrast, Mediterranean Sea populations of mackerel show some degree of genetic differentiation and are structured along an east–west axis. The analysed eastern Mediterranean Sea mackerel populations (Greece, Italy) are clearly separated from that of the western Mediterranean Sea (Barcelona), which forms a panmictic unit with eastern Atlantic Ocean populations. The genetic structures of both species showed asymmetric migration patterns and indicated population expansion.

Characterization of the myostatin gene in the gilthead seabream (Sparus aurata): sequence, genomic structure, and expression pattern.

Abstract: We report on the sequence and expression analysis of the myostatin gene (MSTN) in the gilthead seabream Sparus aurata. A 2189-bp transcript was isolated, encoding an open reading frame (385 amino acids) that showed 74% to 60% protein similarity with other vertebrate myostatins. Phylogenetic analysis of MSTN and other related genes confirmed the evolutionary relationships of the isolated sequence. The complete sequences of two introns were also determined. Intron-exon boundaries were conserved when compared with those of mammalian MSTN genes, whereas intron size was smaller. Reverse transcriptase polymerase chain reaction on total RNA extracted from different tissues and developmental stages revealed MSTN expression in the skeletal muscle, but also in other tissues. The observed expression profile differed from that in mammals, suggesting possible additional functions of myostatin in the teleost fish.

A Genetic Linkage Map of the Hermaphrodite Teleost Fish Sparus aurata L.

The gilthead sea bream (Sparus aurata L.) is a marine fish of great importance for fisheries and aquaculture. It has also a peculiar sex-determination system, being a protandrous hermaphrodite. Here we report the construction of a first-generation genetic linkage map for S. aurata, based on 204 microsatellite markers. Twenty-six linkage groups (LG) were found. The total map length was 1241.9 cM. The ratio between sex-specific map lengths was 1:1.2 (male:female). Comparison with a preliminary radiation hybrid (RH) map reveals a good concordance, as all markers located in a single LG are located in a single RH group, except for Ad-25 and CId-31. Comparison with the Tetraodon nigroviridis genome revealed a considerable number of evolutionary conserved regions (ECRs) between the two species. The mean size of ECRs was 182 bp (sequence identity 60–90%). Forty-one ECRs have a known chromosomal location in the pufferfish genome. Despite the limited number of anchoring points, significant syntenic relationships were found. The linkage map presented here provides a robust comparative framework for QTL analysis in S. aurata and is a step toward the identification of genetic loci involved both in the determination of economically important traits and in the individual timing of sex reversal.

Transcriptome sequencing and microarray development for the Manila clam, Ruditapes philippinarum: genomic tools for environmental monitoring

BackgroundThe Manila clam, Ruditapes philippinarum, is one of the major aquaculture species in the world and a potential sentinel organism for monitoring the status of marine ecosystems. However, genomic resources for R. philippinarum are still extremely limited. Global analysis of gene expression profiles is increasingly used to evaluate the biological effects of various environmental stressors on aquatic animals under either artificial conditions or in the wild. Here, we report on the development of a transcriptomic platform for global gene expression profiling in the Manila clam.ResultsA normalized cDNA library representing a mixture of adult tissues was sequenced using a ultra high-throughput sequencing technology (Roche 454). A database consisting of 32,606 unique transcripts was constructed, 9,747 (30%) of which could be annotated by similarity. An oligo-DNA microarray platform was designed and applied to profile gene expression of digestive gland and gills. Functional annotation of differentially expressed genes between different tissues was performed by enrichment analysis. Expression of Natural Antisense Transcripts (NAT) analysis was also performed and bi-directional transcription appears a common phenomenon in the R. philippinarum transcriptome. A preliminary study on clam samples collected in a highly polluted area of the Venice Lagoon demonstrated the applicability of genomic tools to environmental monitoring.ConclusionsThe transcriptomic platform developed for the Manila clam confirmed the high level of reproducibility of current microarray technology. Next-generation sequencing provided a good representation of the clam transcriptome. Despite the known limitations in transcript annotation and sequence coverage for non model species, sufficient information was obtained to identify a large set of genes potentially involved in cellular response to environmental stress.