Fish diversification at the pace of geomorphological changes: evolutionary history of western Iberian Leuciscinae (Teleostei: Leuciscidae) inferred from multilocus sequence data.

The evolutionary history of western Iberian Leuciscinae, obligatory freshwater fish, is directly linked to the evolution of the hydrographic network of the Iberian Peninsula after its isolation from the rest of Europe, which involved dramatic rearrangements such as the transition from endorheic lakes to open basins draining to the Atlantic. Previous phylogenetic research on western Iberian leuciscines, using mainly mitochondrial DNA and more recently one or two nuclear genes, has found contradictory results and there remain many unresolved issues regarding species relationships, taxonomy, and evolutionary history. Moreover, there is a lack of integration between phylogenetic and divergence time estimates and information on the timing of geomorphological changes and paleobasin rearrangements in the Iberian Peninsula. This study presents the first comprehensive fossil-calibrated multilocus coalescent species tree of western Iberian Leuciscinae (including 14 species of Achondrostoma, Iberochondrostoma, Pseudochondrostoma and Squalius endemic to the Iberian Peninsula, seven of which endemic to Portugal) based on seven nuclear genes, and from which we infer their biogeographic history by comparing divergence time estimates to known dated geological events. The phylogenetic pattern suggests slow-paced evolution of leuciscines during the Early-Middle Miocene endorheic phase of the main Iberian river basins, with the shift to exorheism in the late Neogene-Quaternary allowing westward dispersals that resulted in many cladogenetic events and a high rate of endemism in western Iberia. The results of this study also: (i) confirm the paraphyly of S. pyrenaicus with respect to S. carolitertii, and thus the possible presence of a new taxon in the Portuguese Tagus currently assigned to S. pyrenaicus; (ii) support the taxonomic separation of the Guadiana and Sado populations of S. pyrenaicus; (iii) show the need for further population sampling and taxonomic research to clarify the phylogenetic status of A. arcasii from the Minho basin and of the I. lusitanicum populations in the Sado and Tagus basins; and (iv) indicate that A. occidentale, I. olisiponensis and P. duriensis are the most ancient lineages within their respective genera.

Effect of re-ensiling on the quality of sorghum silage.

The commercialization of silage in many countries, including Brazil, has increased in recent years. Re-ensiling of previously ensiled forage occurs when silage is relocated from one farm to another, where it will be compacted and sealed again. During this process, silage is exposed to oxygen before being ensiled, which may affect its quality. We exposed sorghum silage to air during the anaerobic storage phase to simulate the transportation of silages between farms. Experimental treatments included silage exposed to air for 0 or 12 h, with or without the use of an inoculant containing a mixture of Lactobacillus plantarum and the propionic bacteria Propionibacterium acidipropionici (1 × 106 cfu/g of forage; Biomax corn, Lallemand, Saint-Simon, France), totaling 4 treatments: conventional silage, conventional silage with inoculant use, re-ensilage after exposure to air, and re-ensilage after exposure to air with use of an inoculant. The sorghum was stored in experimental silos containing about 9.0 kg of fresh forage per replicate. Treatments were tested in a factorial 2 × 2 design with 5 replicates each. Chemical composition, in vitro dry matter digestibility, fermentative characteristics, losses (due to gas, effluents, and total dry matter), microorganism counts, and aerobic stability of sorghum silage were evaluated. Dry matter content of sorghum before ensiling was 273.12 g/kg. The 12-h re-ensiling process increased the effluent loss of the silage when compared with conventional silage (456.42 vs. 201.19 g/kg of FM, respectively). In addition, re-ensiled silages presented lower concentrations of lactic acid and higher concentrations of propionic acid than the silages that had not been opened during storage. The aerobic stability of silage was not affected by the re-ensiling process and the use of inoculant. The use of inoculant increased the pH and loss of dry matter of the silages (4.23 vs. 3.98 and 14.05 vs. 7.82%, respectively) and therefore did not provide any benefits in this study.

Natural pathways towards polyploidy in animals: the Squalius alburnoides fish complex as a model system to study genome size and genome reorganization in polyploids.

When comparing the known picture of polyploidy in animals and in plants, it is possible to recognize some similarities, namely: (i) multiple and recurrent origins in several well-established taxonomic groups; (ii) a strong and regular association with hybridization events; (iii) the production of genotypic diversity; (iv) a rapid genomic reshuffling; (v) a very active role of transposable elements in allopolyploids; (vi) a comparatively privileged occurrence in harsher environments when compared with their diploid relatives, and (vii) gene silencing and divergence of duplicated genes without disruption of duplicated loci. Research on polyploidy was highly biased towards plants during the last century because polyploidy in animals was for long time considered rare, occasional and irrelevant from an evolutionary perspective. However, as empirically observed in plants, genome rediploidization starts in polyploid organisms immediately after the polyploid shock. Given the speed and dynamicity of this process, evidence of genome multiplication is completely erased over time, and hence, only the most recent events are likely to be acknowledged. Although varying in expression between and within taxonomic groups, polyploidy and hybridization are ubiquitous in animals and may be recurrent, fostering evolution. Since evolutionary allopolyploid genomes behave as biologically diploid, zoologists have to challenge the old paradigm of an irrelevant evolutionary role in animals using current genomic and cytogenomic tools. These methods are most likely to reveal the role of polyploid mechanisms in producing evolutionary novelties. Nonsexual complexes are the perfect models to bridge the gap between empirical and theoretical research, while the evolutionary process is in action. Such animal complexes represent a transient stage that, in general, moves towards a polyploid stage, where bisexuality might be recovered, ultimately giving rise to a new gonochoric species. These pathways are herein illustrated by the Iberian allopolyploid Squalius alburnoides. Some general aspects on this fish's complex are updated and reviewed, namely the reproductive modes of the distinct genomotypes, since variable ploidies and genomic combinations occur in natural populations. Most recent data on the mechanisms of gene expression regulation and the importance of the genomic context in driving allelic expression are also included. It was first demonstrated that a regulatory mechanism involving dosage compensation by gene-copy silencing exists in allotriploid females and that allelic expression patterns differed either between genomically equivalent individuals or within the same individual (between tissues and genes). Thus, instead of a whole haplome inactivation, a biased silencing towards repression of a specific allele was observed as well as a reduction of the transcript levels to the diploid state. See also sister article focusing on plants by Tayalé and Parisod in this themed issue.

Influences of past climatic changes on historical population structure and demography of a cosmopolitan marine predator, the common dolphin (genus Delphinus).

Climatic oscillations during the Pleistocene have greatly influenced the distribution and connectivity of many organisms, leading to extinctions but also generating biodiversity. While the effects of such changes have been extensively studied in the terrestrial environment, studies focusing on the marine realm are still scarce. Here we used sequence data from one mitochondrial and five nuclear loci to assess the potential influence of Pleistocene climatic changes on the phylogeography and demographic history of a cosmopolitan marine predator, the common dolphin (genus Delphinus). Population samples representing the three major morphotypes of Delphinus were obtained from 10 oceanic regions. Our results suggest that short-beaked common dolphins are likely to have originated in the eastern Indo-Pacific Ocean during the Pleistocene and expanded into the Atlantic Ocean through the Indian Ocean. On the other hand, long-beaked common dolphins appear to have evolved more recently and independently in several oceans. Our results also suggest that short-beaked common dolphins had recurrent demographic expansions concomitant with changes in sea surface temperature during the Pleistocene and its associated increases in resource availability, which differed between the North Atlantic and Pacific Ocean basins. By proposing how past environmental changes had an effect on the demography and speciation of a widely distributed marine mammal, we highlight the impacts that climate change may have on the distribution and abundance of marine predators and its ecological consequences for marine ecosystems.

Population divergence with or without admixture: selecting models using an ABC approach.

Genetic data have been widely used to reconstruct the demographic history of populations, including the estimation of migration rates, divergence times and relative admixture contribution from different populations. Recently, increasing interest has been given to the ability of genetic data to distinguish alternative models. One of the issues that has plagued this kind of inference is that ancestral shared polymorphism is often difficult to separate from admixture or gene flow. Here, we applied an approximate Bayesian computation (ABC) approach to select the model that best fits microsatellite data among alternative splitting and admixture models. We performed a simulation study and showed that with reasonably large data sets (20 loci) it is possible to identify with a high level of accuracy the model that generated the data. This suggests that it is possible to distinguish genetic patterns due to past admixture events from those due to shared polymorphism (population split without admixture). We then apply this approach to microsatellite data from an endangered and endemic Iberian freshwater fish species, in which a clustering analysis suggested that one of the populations could be admixed. In contrast, our results suggest that the observed genetic patterns are better explained by a population split model without admixture.

Living in endemic area for infectious diseases accelerates epigenetic age.

Inflammaging is a low-grade inflammatory state generated by the aging process that can contribute to frailty and age-related diseases in the elderly. However, it can have distinct effects in the elderly living in endemic areas for infectious diseases. An increased inflammatory response may confer protection against infectious agents in these areas, although this advantage can cause accelerating epigenetic aging. In this study, we evaluated the inflammatory profile and the epigenetic age of infected and noninfected individuals from an endemic area in Brazil. The profile of cytokines, chemokines and growth factors analyzed in the sera of the two groups of individuals showed similarities, although infected individuals had a higher concentration of these mediators. A significant increase in IL-1ra, CXCL8, CCL2, CCL3 and CCL4 production was associated with leprosy infection. Notably, elderly individuals displayed distinct immune responses associated with their infection status when compared to adults suggesting an adaptive remodelling of their immune responses. Epigenetic analysis also showed that there was no difference in epigenetic age between the two groups of individuals. However, individuals from the endemic area had a significant accelerated aging when compared to individuals from São Paulo, a non-endemic area in Brazil. Moreover, the latter cohort was also epigenetically aged in relation to an Italian cohort. Our data shows that living in endemic areas for chronic infectious diseases results in remodelling of inflammaging and acceleration of epigenetic aging in individuals regardless of their infectious status. It also highlights that geographical, genetic and environmental factors influence aging and immunosenescence in their pace and profile.

The evolutionary history of the allopolyploid Squalius alburnoides (Cyprinidae) complex in the northern Iberian Peninsula.

Understanding the population structure, population dynamics and processes that give rise to polyploidy and helps to maintain it is central to our knowledge of the evolution of asexual vertebrates. Previous studies revealed high genetic diversity and several reproductive pathways in the southern populations of the Squalius alburnoides hybrid complex. In contrast, lower genetic variability and the associated limited chance of introducing new genetic combinations may threaten the survival of the northern Mondego populations. We analysed the genetic diversity and structure of nine populations of S. alburnoides in the Iberian Peninsula using microsatellite loci to provide further insights on the evolutionary history of this complex. Special attention was given to the less-studied northern populations (Mondego and Douro basins). Marked population structure, a high frequency of private alleles and a high diversity of some biotypes in the Douro basin indicate that some northern populations may not be at high risk of extinction, contrary to what was expected. The genetic diversity found in the northern Douro populations contradicts the general trend of remarkable genetic impoverishment northwards that occurs in other species and regions. The results indicate the possible existence of a glacial refugium in the Rabaçal River, corroborating findings in other species of this region. Historical events seem to have affected the geographical patterns of genetic variability found among and within the northern and southern populations of this complex and contributed to different patterns of genome composition. Therefore, historical events might have a major role in the long-term persistence of some polyploid hybrid taxa.

Introgressive hybridization between two Iberian endemic cyprinid fish: a comparison between two independent hybrid zones.

Pseudochondrostoma duriense and Achondrostoma oligolepis are two Iberian endemic cyprinid fish species that occur in sympatry over most of their distribution range and that are suspected to hybridize in nature. Here, we employed a combination of mitochondrial and microsatellite markers to explore the extent of introgressive hybridization between these fishes. Two natural hybrid zones were identified in different river basins. Introgression was bi-directional and both hybrid zones consisted mostly of parental genotypes/phenotypes (i.e. bimodal hybrid zones). Yet, they appeared to differ in the extent and direction of introgression, which supports the view that they constitute independent outcomes of different hybridization processes probably influenced by environmental features. Several discordances were found between mtDNA and microsatellite results, suggesting that this hybridization process has complex consequences and illustrating the importance of using independent markers to define accurately the hybrid status of individuals in the presence of high levels of backcrossing.

Reconfirming the hybrid origin and generic status of the Iberian cyprinid complex Squalius alburnoides.

The recent allocation of the cyprinid complex Squalius alburnoides to the genus Iberocypris in the Handbook of European Freshwater Fishes is refuted by confirming evidence of a close relationship with the genus Squalius. Squalius alburnoides is here defined as a complex based on the existence of a hybrid form with distinct genome combinations and ploidies and of a reconstituted nuclear non-hybrid male form also with hybrid origin.

An easier method to identify the individual genomic composition of allopolyploid complexes.

A new method for the fast identification of the genomic composition of the cyprinid Squalius alburnoides is presented. The method is based on a length polymorphism detected in the beta-actin gene, which serves as the basis for the development of a semi-quantitative PCR.

Body shape evolution among ploidy levels of the Squalius alburnoides hybrid complex (Teleostei, Cyprinidae).

Hybridization, ploidy level and genomic constitution may be important to respond to different environments, by producing different phenotypes and thus reducing competitive interaction. Through geometric morphometrics, we examined variation in body size and shape among biotypes of the Squalius alburnoides hybrid complex and their sperm donor (Squalius carolitertii). Results showed that S. carolitertii is significantly larger in size than the biotypes of the complex. No significant relationship was observed between ploidy and body size among S. alburnoides biotypes. Significant variation in body shape was found between S. carolitertii and S. alburnoides, and between tetraploids and the other biotypes. These differences in biotypes may reduce resource competition, highlighting the potential importance of resource availability favouring one biotype over another. In S. alburnoides, the adaptation to different trophic niches through modification of trophic morphology, body shapes, and feeding behaviour, may result from an increase in ploidy and genomic constitution. This adaptation may account also for the formation and maintenance of this nonsexual complex.

Redistribution of the geographical ranges of the Iberian cyprinid genus Pseudochondrostoma based on a phylogenetic analysis: implications for the historical rearrangements of the north-western Iberian drainages.

A new distribution range is proposed for Pseudochondrostoma polylepis and Pseudochondrostoma duriense based on the phylogeny of two nuclear and one mitochondrial gene sequences, with implications on the geographic history of the north-western Iberian drainages.

Expression pattern of anti-Müllerian hormone (amh) in the hybrid fish complex of Squalius alburnoides.

In fish of the Squalius alburnoides complex, hybridisation and polyploidy have affected sex ratios, resulting in strong correlations between sex and genotype. The preponderance of females among triploids and the occurrence of an all male lineage among diploids seem to imply that sex ratio deviations should have a strong genetic basis. Until now, no information has been gathered regarding the molecular basis of sex determination in this intricate hybrid system. Thus, putative regulatory elements of the cascade that potentially are involved in sex determination in S. alburnoides have to be investigated. Being reported to have an important role in teleost sex determination, and more particularly in male gonad development, the anti-Müllerian hormone, amh was a good initial candidate. Here we report the isolation, cloning and characterization of the amh ortholog in S. alburnoides and the ancestral species S. pyrenaicus. In adult S. alburnoides and S. pyrenaicus of both sexes, amh shows a gonad specific expression pattern, restricted to the Sertoli cell lineage in testis and to granulosa cells in ovaries. During development, it plays an early role in male gonad differentiation in S. alburnoides. Overall the observed patterns are similar to what has been reported in other teleost species. This suggests a conserved role of amh and implies that its expression dynamics cannot be directly responsible for the sex ratio deviations reported in S. alburnoides. It is possible that a conjunction of other factors could be contributing for sex ratio imbalance. The present results constitute the starting point in the characterization of the S. alburnoides sex determination cascade, a process that we expect to shed some light on the molecular basis of sex distribution, within the context of hybrid system evolution.

Contrasting views over a hybrid complex: Between speciation and evolutionary "dead-end".

The Squalius alburnoides complex, of hybrid origin, comprises diploid, triploid and tetraploid forms and has a widespread distribution in the Iberian waters. The southern populations of this complex, sympatric with S. pyrenaicus, show high genetic variability, diversity of forms and reproductive modes which create pathways that may allow for the establishment of a new species in the future. Here we report a contrasting view over the S. alburnoides complex: in the Mondego River basin (northern Portugal), nuclear "non-hybrid" and tetraploid forms are absent and a clearly impoverished genetic diversity is observed, contributing to a general scarcity of possibilities of generating novel genetic material. Moreover, the bisexual species involved in the maintenance of the complex in this basin (S. carolitertii) exhibits a considerably lower genetic variability, when compared with S. pyrenaicus. The observed differences suggest that, despite being originated by similar hybridization events and maintained by analogous reproductive mechanisms, different populations of the complex were exposed to distinct evolutionary constrains, which in some cases resulted in diversification and speciation while in others led to a compromising situation in terms of evolutionary potential. Additionally, and for the first time all forms were used in the calculation of genetic distances and diversity indices, widening the possibilities of analysis of the complex by allowing the inclusion of a large part of the available data, irrespective of ploidy level.

Production of fertile unreduced sperm by hybrid males of the Rutilus alburnoides complex (Teleostei, cyprinidae). An alternative route to genome tetraploidization in unisexuals.

The hybrid minnow Rutilus alburnoides comprises diploid and polyploid females and males. Previous studies revealed that diploid and triploid females exhibit altered oogenesis that does not involve random segregation and recombination of the genomes of the two ancestors, constituting unisexual lineages. In the present study, we investigated the reproductive mode of hybrid males from the Tejo basin, using experimental crosses and flow cytometric analysis of blood and sperm. The results suggest that diploid hybrids produced fertile unreduced sperm, transmitting their hybrid genome intact to offspring. Triploid hybrids also produced unreduced sperm, but it was not possible to obtain data concerning their fertility. Finally, tetraploid hybrids produced fertile diploid sperm, which exhibited Mendelian segregation. Tetraploid R. alburnoides may reestablish biparental reproduction, as individuals of both sexes with the appropriate constitution for normal meiosis (two haploid genomes from each parental species) are likely to occur in natural populations. Tetraploids probably have arisen from syngamy of diploid eggs and diploid sperm produced by diploid hybrid males. Diploid hybrid males may therefore play a significant role in the dynamics of the complex, starting the evolutionary process that may ultimately lead to a new sexually reproducing species.

Gene expression dosage regulation in an allopolyploid fish.

How allopolyploids are able not only to cope but profit from their condition is a question that remains elusive, but is of great importance within the context of successful allopolyploid evolution. One outstanding example of successful allopolyploidy is the endemic Iberian cyprinid Squalius alburnoides. Previously, based on the evaluation of a few genes, it was reported that the transcription levels between diploid and triploid S. alburnoides were similar. If this phenomenon occurs on a full genomic scale, a wide functional ''diploidization'' could be related to the success of these polyploids. We generated RNA-seq data from whole juvenile fish and from adult livers, to perform the first comparative quantitative transcriptomic analysis between diploid and triploid individuals of a vertebrate allopolyploid. Together with an assay to estimate relative expression per cell, it was possible to infer the relative sizes of transcriptomes. This showed that diploid and triploid S. alburnoides hybrids have similar liver transcriptome sizes. This in turn made it valid to directly compare the S. alburnoides RNA-seq transcript data sets and obtain a profile of dosage responses across the S. alburnoides transcriptome. We found that 64% of transcripts in juveniles' samples and 44% in liver samples differed less than twofold between diploid and triploid hybrids (similar expression). Yet, respectively 29% and 15% of transcripts presented accurate dosage compensation (PAA/PA expression ratio of 1 instead of 1.5). Therefore, an exact functional diploidization of the triploid genome does not occur, but a significant down regulation of gene expression in triploids was observed. However, for those genes with similar expression levels between diploids and triploids, expression is not globally strictly proportional to gene dosage nor is it set to a perfect diploid level. This quantitative expression flexibility may be a strong contributor to overcome the genomic shock, and be an immediate evolutionary advantage of allopolyploids.

Central involvement of kinin B1 and B2 receptors in the febrile response induced by endotoxin in rats.

1. The effect of central injection of selective kinin B1 and B2 receptor antagonists on the febrile response induced by endotoxin (E. coli lipopolysaccharide, LPS) in rats was investigated. 2. Intracerebroventricular (i.c.v.) injection of a selective B2 receptor antagonist (Hoe-140, 8 nmol) reduced the early (0-2 h), but increased the late phase (4-6 h) of the febrile response induced by intravenous (i.v.) injection of LPS (0.5 microgram kg-1). 3. Co-administration of Hoe-140 (8 nmol, i.c.v.) with LPS (0.5 microgram kg-1, i.v.), followed 2.5 h later by the i.c.v. injection of a selective B1 receptor antagonist [des-Arg9-Leu8]-bradykinin (BK, 8 nmol), significantly reduced the febrile response induced by LPS throughout the whole experimental period. 4. Intravenous injection of Hoe-140 (1 mg kg-1) significantly reduced the febrile response induced by LPS (0.5 microgram kg-1, i.p.). 5. Pretreatment (24 h) with LPS (0.5 microgram kg-1, i.v.) reduced the febrile response induced by BK or [Tyr8]-BK (both, 5 nmol, i.c.v.), but markedly increased the febrile response induced by [des-Arg9]-BK (5 nmol, i.c.v.). The response induced by [des-Arg9]-BK in LPS-pretreated rats was significantly inhibited by co-injection of [des-Arg9-Leu8]-BK (15 nmol, i.c.v.). 6. The results suggest that kinins are involved in the induction of LPS-induced fever and that central B2 and B1 receptors are activated during the initial and late phase of this response, respectively. The results also suggest that downregulation and/or desensitization of B2 receptors and induction and/or upregulation of B1 receptors in LPS-pretreated animals may have a significant pathophysiological role in the induction and maintenance of fever. These observations may be specifically important in the case of chronic inflammatory conditions, because the BK metabolite [des-Arg9]-BK, so far considered an inactive metabolite, acquires an active and relevant role with the progressive expression of B1 receptors that occurs in such states.