Paleobiogeography of two Iberian endemic cyprinid fishes (Chondrostoma arcasii-Chondrostoma macrolepidotus) inferred from mitochondrial DNA sequence data

We tested different hypotheses related to the origin and evolution of the endemic Iberian fishes Chondrostoma arcasii and Chondrostoma macrolepidotus from northern and central regions of the Iberian Peninsula. We evaluated the monophyly of the populations within each species and sought to determine if diversification of the populations coincided in time with the formation of the Iberian drainages dating back to the upper Pliocene (2.5-1.8 million years ago). A molecular phylogenetic analysis of the mitochondrial cytochrome b gene showed that the different populations of the northern Iberian Peninsula are clustered into five phylogroups and do not fit into the dichotomy C. arcasii-C. macrolepidotus. We propose that species differentiation occurred prior to the upper Pliocene formation of the present hydrographic basins and that endorheic basins, a system of inland lakes found in Spain during the Mio-Pliocene, played an important role in this diversification and differentiation process.     

Symbiosome-like intracellular colonization of cereals and other crop plants by nitrogen-fixing bacteria for reduced inputs of synthetic nitrogen fertilizers

It has been forecast that the challenge of meeting increased food demand and protecting environmental quality will be won or lost in maize, rice and wheat cropping systems, and that the problem of environmental nitrogen enrichment is most likely to be solved by substituting synthetic nitrogen fertilizers by the creation of cereal crops that are able to fix nitrogen symbiotically as legumes do. In legumes, rhizobia present intracellularly in membrane-bound vesicular compartments in the cytoplasm of nodule cells fix nitrogen endosymbiotically. Within these symbiosomes, membrane-bound vesicular compartments, rhizobia are supplied with energy derived from plant photosynthates and in return supply the plant with biologically fixed nitrogen, usually as ammonia. This minimizes or eliminates the need for inputs of synthetic nitrogen fertilizers. Recently we have demonstrated, using novel inoculation conditions with very low numbers of bacteria, that cells of root meristems of maize, rice, wheat and other major non-legume crops, such as oilseed rape and tomato, can be intracellularly colonized by the non-rhizobial, non-nodulating, nitrogen fixing bacterium, Gluconacetobacter diazotrophicus that naturally occurs in sugarcane. G. diazotrophicus expressing nitrogen fixing (nifH) genes is present in symbiosome-like compartments in the cytoplasm of cells of the root meristems of the target cereals and non-legume crop species, somewhat similar to the intracellular symbiosome colonization of legume nodule cells by rhizobia. To obtain an indication of the likelihood of adequate growth and yield, of maize for example, with reduced inputs of synthetic nitrogen fertilizers, we are currently determining the extent to which nitrogen fixation, as assessed using various methods, is correlated with the extent of systemic intracellular colonization by G. diazotrophicus, with minimal or zero inputs.     

Genotyping of pantophysin I (Pan I) of Atlantic cod (Gadus morhua L.) by allele‐specific PCR

The two main allelic variants of the Atlantic cod (Gadus morhua L.) pantophysin I (Pan I) locus have different frequencies within different cod stocks. The Dra I polymorphism which distinguishes the two alleles can thus be used for discrimination of coastal and offshore cod populations. We present a new method for Pan I genotyping using fluorescent allele‐specific duplex polymerase chain reaction (PCR). This method is more rapid, reliable and cost‐effective than the previously published method and it is not affected by DNA source and quality. This improvement is important for studies demanding high throughput and accuracy of Pan I genotyping     

Diabetes induces changes of catecholamines in primary mesangial cells

Diabetes mellitus is a frequent cause of kidney function damage with diabetic nephropathy being predominantly related to glomerular dysfunction. Diabetes is capable of interfering with distinct hormonal systems, as well as catecholamine metabolism. Since mesangial cells, the major constituent of renal glomerulus, constitute a potential site for catecholamine production, the present study was carried out to investigate alterations in catecholamine metabolism in cultured mesangial cells from the nonobese diabetic mouse, a well-established model for type I diabetes. We evaluated mesangial cells from normoglycemic and hyperglycemic nonobese diabetic mice, as well as cells from normoglycemic Swiss mice as control. Mesangial cells from normoglycemic mice presented similar profiles concerning all determinations. However, cells isolated from hyperglycemic animals presented increased dopamine and norepinephrine production/secretion. Among the studied mechanisms, we observed an upregulation of tyrosine hydroxylase expression accompanied by increased tetrahydrobiopterin consumption, the tyrosine hydroxylase enzymatic cofactor. However, this increase in synthetic pathways was followed by decreased monoamine oxidase activity, which corresponds to the major metabolic pathway of catecholamines in mesangial cells. In addition, whole kidney homogenates from diabetic animals also presented increased dopamine and norepinephrine levels when compared to normoglycemic animals. Thus, our results suggest that diabetes alters catecholamine production by interfering with both synthesizing and degrading enzymes, suggesting a possible role of catecholamine in the pathogenesis of acute and chronic renal complications of diabetes mellitus.     

Harmonising Adult and Paediatric Reference Intervals in Australia and New Zealand: An Evidence-Based Approach for Establishing a First Panel of Chemistry Analytes

Scientific evidence supports the use of common reference intervals (RIs) for many general chemistry analytes, in particular those with sound calibration and traceability in place. Already the Nordic countries and United Kingdom have largely achieved harmonised RIs. Following a series of workshops organised by the Australasian Association of Clinical Biochemists (AACB) between 2012 and 2014 at which an evidence-based approach for determination of common intervals was developed, pathology organisations in Australia and New Zealand have reached a scientific consensus on what adult and paediatric intervals we should use across Australasia. The aim of this report is to describe the processes that the AACB and the Royal College of Pathologists of Australasia have taken towards recommending the implementation of a first panel of common RIs for use in Australasia.     

Heterozygous indels as useful tools in the reconstruction of DNA sequences and in the assessment of ploidy level and genomic constitution of hybrid organisms.

In this paper we describe a simple approach using double peaks in chromatograms generated as artefacts in the vicinity of heterozygous indels, to identify the specific sequences present in individual strands of a given DNA fragment. This method is useful to assign bases in individuals that are heterozygous at multiple sites. In addition, the relative sizes of the double peaks help to determine the ploidy level and the relative contribution of the parental genomes in hybrids. Our interpretation was confirmed with the analysis of artificial mixtures of DNA of two different species. Results were robust with varying PCR and sequencing conditions. The applicability of this method was demonstrated in hybrids of the Squalius alburnoides complex and in heterozygotes of Chondrostoma oligolepis. Far from being limited to these fish models and the gene where it was tested (beta-actin), this sequence reconstruction methodology is expected to have a broader application.     

Fertile triploid males: an uncommon case among hybrid vertebrates

The endemic Iberian minnow Squalius alburnoides is a complex of fishes of hybrid origin including both males and females with distinct ploidy levels and varying proportions of the parental genomes. In this paper we demonstrated that in contrast to many vertebrate hybrid lineages the sperm of triploid hybrid males of S. alburnoides is viable and fully functional. Flow cytometry and analysis of sequences of a fragment of the beta-actin nuclear gene applied to progenitors and offspring evidenced that these males produced their sperm clonally, as already described for diploid hybrids. The presence of different types of fertile males (nonhybrid diploids with normal meiosis and both diploid and triploid hybrids) coupled with hybridogenetic meiosis in females endows this vertebrate complex with a high level of independence from other species and contributes to maintain its genetic variability.     

Identification of ESUs in the critically endangered Portuguese minnow <Emphasis Type="Italic">Chondrostoma lusitanicum</Emphasis> Collares-Pereira 1980, based on a phylogeographical analysis

In this paper, we analysed the phylogeography of the Critically Endangered fish Chondrostoma lusitanicum (Cyprinidae) endemic to Portugal. A fragment of the mitochondrial cyt b gene and one from the nuclear beta-actin gene were sequenced. The results support recognition of 3 ESU's distributed as follows: 1) Sado River, 2) the Tagus River basin and 3 independent nearby coastal rivers (collectively named "West") and 3) Lagoa de Albufeira, a small coastal lagoon between Tagus and Sado basins.     

Establishment of a coastal fish in the Azores: recent colonisation or sudden expansion of an ancient relict population?

The processes and timescales associated with ocean-wide changes in the distribution of marine species have intrigued biologists since Darwin''s earliest insights into biogeography. The Azores, a mid-Atlantic volcanic archipelago located >1000 km off the European continental shelf, offers ideal opportunities to investigate phylogeographic colonisation scenarios. The benthopelagic sparid fish known as the common two-banded seabream (Diplodus vulgaris) is now relatively common along the coastline of the Azores archipelago, but was virtually absent before the 1990s. We employed a multiple genetic marker approach to test whether the successful establishment of the Azorean population derives from a recent colonisation from western continental/island populations or from the demographic explosion of an ancient relict population. Results from nuclear and mtDNA sequences show that all Atlantic and Mediterranean populations belong to the same phylogroup, though microsatellite data indicate significant genetic divergence between the Azorean sample and all other locations, as well as among Macaronesian, western Iberian and Mediterranean regions. The results from Approximate Bayesian Computation indicate that D. vulgaris has likely inhabited the Azores for ∼40 (95% confidence interval (CI): 5.5–83.6) to 52 (95% CI: 6.32–89.0) generations, corresponding to roughly 80–150 years, suggesting near-contemporary colonisation, followed by a more recent demographic expansion that could have been facilitated by changing climate conditions. Moreover, the lack of previous records of this species over the past century, together with the absence of lineage separation and the presence of relatively few private alleles, do not exclude the possibility of an even more recent colonisation event.