Complete mitochondrial genome of the Spanish toothcarp, Aphanius iberus (Valenciennes, 1846) (Actinopterygii, Aphaniidae) and its phylogenetic position within the Cyprinodontiformes order.

BACKGROUND: The Spanish toothcarp (Aphanius iberus Valenciennes, 1846) is a small fish endemic to the eastern coastline of the Iberian Peninsula and is currently listed as "Endangered" (category IUCN: EN). It mainly inhabits brackish waters which can exhibit large fluctuations in temperature and salinity throughout the year. The genetics of A. iberus are not well-known since most studies have only evaluated the genetic structure of the species under a conservation framework in order to identify its potential conservation units. Different phylogenetic relationships of Aphanius have been published based on some particular genes. In the present study, the entire mitochondrial genome of A. iberus was obtained for the first time in the context of an A. iberus reference genome and a hypothesis regarding its phylogenetic position was considered. METHODS AND RESULTS: The mitogenome (a circular doble-stranded DNA sequence of 16,708 bp) was reconstructed and aligned against 83 Cyprinodontiformes and two outgroup taxa to identify the phylogenetic position of A. iberus. PartitionFinder was first used to test for the best evolutionary model and the phylogenetic analyses were performed using two methods: Maximun-Likelihood Approximation (IQ-Tree) and Bayesian inference (MrBayes). Our results show that A. iberus forms a sister group with Orestias ascotanensis, a cyprinodontiform species native to South America. CONCLUSIONS: The results were congruent with the traditional morphometric reconstructed trees and with a geological vicariant hypothesis involving Cyprinodontiformes where Aphaniidae is shown as a monophyletic family separated from the family Cyprinodontidae. The information gathered from this study is not only valuable for improving our understanding of the evolutionary history of A. iberus, but for future genomic studies involving the species.

Cenozoic tectonic and climatic events in southern Iberian Peninsula: Implications for the evolutionary history of freshwater fish of the genus Squalius (Actinopterygii, Cyprinidae).

Southern Iberian freshwater ecosystems located at the border between the European and African plates represent a tectonically complex region spanning several geological ages, from the uplifting of the Betic Mountains in the Serravalian-Tortonian periods to the present. This area has also been subjected to the influence of changing climate conditions since the Middle-Upper Pliocene when seasonal weather patterns were established. Consequently, the ichthyofauna of southern Iberia is an interesting model system for analyzing the influence of Cenozoic tectonic and climatic events on its evolutionary history. The cyprinids Squalius malacitanus and Squalius pyrenaicus are allopatrically distributed in southern Iberia and their evolutionary history may have been defined by Cenozoic tectonic and climatic events. We analyzed MT-CYB (510 specimens) and RAG1 (140 specimens) genes of both species to reconstruct phylogenetic relationships and to estimate divergence times and ancestral distribution ranges of the species and their populations. We also assessed their levels of genetic structure and diversity as well as the amount of gene flow between populations. To investigate recent paleogeographical and climatic factors in southern Iberia, we modeled changes-through-time in sea level from the LGM to the present. Phylogenetic, geographic and population structure analyses revealed two well-supported species (S. malacitanus and S. pyrenaicus) in southern Iberia and two subclades (Atlantic and Mediterranean) within S. malacitanus. The origin of S. malacitanus and the separation of its Atlantic and Mediterranean populations occurred during the Serravalian-Tortonian and Miocene-Pliocene periods, respectively. These divergence events occurred in the Middle Pliocene and Pleistocene in S. pyrenaicus. In both species, Atlantic basins possessed populations with higher genetic diversity than Mediterranean, which may be explained by the Janda Lagoon. The isolation of S. malacitanus was earlier and related to the rising of the Betic Mountains. Divergence of its Atlantic and Mediterranean populations was associated with the creation of the freshwater systems of southern Iberia close to the Gibraltar Strait. The presence of S. pyrenaicus in southern Iberia may be the result of recent colonization associated with river capture, as demonstrated our biogeographic reconstruction.

New insights into the molecular phylogeny, biogeographical history, and diversification of Amblyomma ticks (Acari: Ixodidae) based on mitogenomes and nuclear sequences.

BACKGROUND: Amblyomma is the third most diversified genus of Ixodidae that is distributed across the Indomalayan, Afrotropical, Australasian (IAA), Nearctic and Neotropical biogeographic ecoregions, reaching in the Neotropic its highest diversity. There have been hints in previously published phylogenetic trees from mitochondrial genome, nuclear rRNA, from combinations of both and morphology that the Australasian Amblyomma or the Australasian Amblyomma plus the Amblyomma species from the southern cone of South America, might be sister-group to the Amblyomma of the rest of the world. However, a stable phylogenetic framework of Amblyomma for a better understanding of the biogeographic patterns underpinning its diversification is lacking. METHODS: We used genomic techniques to sequence complete and nearly complete mitochondrial genomes -ca. 15 kbp- as well as the nuclear ribosomal cluster -ca. 8 kbp- for 17 Amblyomma ticks in order to study the phylogeny and biogeographic pattern of the genus Amblyomma, with particular emphasis on the Neotropical region. The new genomic information generated here together with genomic information available on 43 ticks (22 other Amblyomma species and 21 other hard ticks-as outgroup-) were used to perform probabilistic methods of phylogenetic and biogeographic inferences and time-tree estimation using biogeographic dates. RESULTS: In the present paper, we present the strongest evidence yet that Australasian Amblyomma may indeed be the sister-group to the Amblyomma of the rest of the world (species that occur mainly in the Neotropical and Afrotropical zoogeographic regions). Our results showed that all Amblyomma subgenera (Cernyomma, Anastosiella, Xiphiastor, Adenopleura, Aponomma and Dermiomma) are not monophyletic, except for Walkeriana and Amblyomma. Likewise, our best biogeographic scenario supports the origin of Amblyomma and its posterior diversification in the southern hemisphere at 47.8 and 36.8 Mya, respectively. This diversification could be associated with the end of the connection of Australasia and Neotropical ecoregions by the Antarctic land bridge. Also, the biogeographic analyses let us see the colonization patterns of some neotropical Amblyomma species to the Nearctic. CONCLUSIONS: We found strong evidence that the main theater of diversification of Amblyomma was the southern hemisphere, potentially driven by the Antarctic Bridge's intermittent connection in the late Eocene. In addition, the subgeneric classification of Amblyomma lacks evolutionary support. Future studies using denser taxonomic sampling may lead to new findings on the phylogenetic relationships and biogeographic history of Amblyomma genus.

Mitochondrial phylogeography and molecular evolution of the rhodopsin visual pigment in troglobitic populations of Astyanax mexicanus (De Filippi, 1853).

Cave-adapted animals provide a unique opportunity to study the evolutionary mechanisms underlying phenotypic, metabolic, behavioral, and genetic evolution in response to cave environments. The Mexican tetra ( Astyanax mexicanus) is considered a unique model system as it shows both surface and cave-dwelling morphs. To date, at least 33 different cave populations have been identified, with phylogenetic studies suggesting an origin from at least two independent surface lineages, thereby providing a unique opportunity to study parallel evolution. In the present study, we carried out the most exhaustive phylogeographic study of A. mexicanus to date, including cave and surface localities, using two mitochondrial markers (cytochrome b (cyt b) and cytochrome c oxidase subunit I ( COI)) and nuclear rhodopsin visual pigment ( rho). Additionally, we inferred the molecular evolution of rho within the two contrasting environments (cave and surface) and across three geographic regions (Sierra de El Abra, Sierra de Guatemala, and Micos). In total, 267 individuals were sequenced for the two mitochondrial fragments and 268 individuals were sequenced for the rho visual pigment from 22 cave and 46 surface populations. Phylogeographic results based on the mitochondrial data supported the two-lineage hypothesis, except for the Pachón and Chica caves, whose introgression has been largely documented. The Sierra de El Abra region depicted the largest genetic diversity, followed by the Sierra de Guatemala region. Regarding the phylogeographic patterns of rho, we recovered exclusive haplogroups for the Sierra de El Abra (Haplogroup I) and Sierra de Guatemala regions (Haplogroup IV). Moreover, a 544 bp deletion in the rho gene was observed in the Escondido cave population from Sierra de Guatemala, reducing the protein from seven to three intramembrane domains. This change may produce a loss-of-function (LOF) but requires further investigation. Regarding nonsynonymous ( dN) and synonymous ( dS) substitution rates (omega values ω), our results revealed the prevailing influence of purifying selection upon the rho pigment for both cave and surface populations (ω<1), but relaxation at the El Abra region. Notably, in contrast to the other two regions, we observed an increase in the number of dN mutations for Sierra de El Abra. However, given that a LOF was exclusively identified in the Sierra de Guatemala region, we cannot dismiss the possibility of a pleiotropic effect on the Rho protein.

Phylogenetic relationships of the algae scraping cyprinid genus Capoeta (Teleostei: Cyprinidae).

We reconstructed the matrilineal phylogeny of Asian algae-eating fishes of the genus Capoeta based on complete mitochondrial gene for cytochrome b sequences obtained from 20 species sampled from the majority of the range and 44 species of closely related barbs of the genera Barbus s. str. and Luciobarbus. The results of this study show that Capoeta forms a strongly supported monophyletic subclade nested within the Luciobarbus clade, suggesting that specialized scraping morphology appeared once in the evolutionary history of the genus. We detected three main groups of Capoeta: the Mesopotamian group, which includes three species from the Tigris-Euphrates system and adjacent water bodies, the Anatolian-Iranian group, which has the most diversified structure and encompasses many species distributed throughout Anatolian and Iranian inland waters, and the Aralo-Caspian group, which consists of species distributed in basins of the Caspian and Aral Seas, including many dead-end rivers in Central Asia and Northern Iran. The most probable origination pathway of the genus Capoeta is hypothesized to occur as a result of allopolyploidization. The origin of Capoeta was found around the Langhian-Serravallian boundary according to our molecular clock. The diversification within the genus occurred along Middle Miocene-Late Pliocene periods.

Quaternary geomorphological and climatic changes associated with the diversification of Iberian freshwater fishes: The case of the genus Cobitis (Cypriniformes, Cobitidae).

We studied the population genetic structure of Cobitis vettonica, an endangered freshwater fish species endemic to the Iberian Peninsula, in order to propose a biogeographic model of the responses of species to the multiple changes that occurred in the Iberian hydrological system during the Quaternary period. We also deciphered the relationship of C. vettonica with its sister species C. paludica, particularly in sympatric areas, and provide genetic information for conservation purposes. To achieve this goal, we analyzed both mitochondrial and nuclear data (the cytochrome b and the nuclear recombination activating 1 genes) and a battery of single-nucleotide polymorphisms (SNPs) of 248 individuals of C. vettonica or C. paludica from 38 localities, including some sympatric ones, covering the entire distribution area of C. vettonica. We highlight the important role played by the hydrogeomorphological processes and climatic changes that occurred in the Iberian Peninsula during the Quaternary on both the population structure of C. vettonica and its relationship with its sister species C. paludica. Our results support the genetic introgression of populations at the eastern limit of the distribution of C. vettonica. Furthermore, we postulate genetic introgression in sympatric areas. Finally, we propose the establishment or expansion of four Operational Conservation Units (OCUs) for C. vettonica, and highlight the threat faced by its populations due to the low level of genetic diversity detected for some of its populations and genetic introgression with C. paludica, which could eventually displace C. vettonica, resulting in a loss of diversity in this species.

Phylogenetic relationships and biogeographical patterns in Circum-Mediterranean subfamily Leuciscinae (Teleostei, Cyprinidae) inferred from both mitochondrial and nuclear data.

BACKGROUND: Leuciscinae is a subfamily belonging to the Cyprinidae fish family that is widely distributed in Circum-Mediterranean region. Many efforts have been carried out to deciphering the evolutionary history of this group. Thus, different biogeographical scenarios have tried to explain the colonization of Europe and Mediterranean area by cyprinids, such as the "north dispersal" or the "Lago Mare dispersal" models. Most recently, Pleistocene glaciations influenced the distribution of leuciscins, especially in North and Central Europe. Weighing up these biogeographical scenarios, this paper constitutes not only the first attempt at deciphering the mitochondrial and nuclear relationships of Mediterranean leuciscins but also a test of biogeographical hypotheses that could have determined the current distribution of Circum-Mediterranean leuciscins. RESULTS: A total of 4439 characters (mitochondrial + nuclear) from 321 individuals of 176 leuciscine species rendered a well-supported phylogeny, showing fourteen main lineages. Analyses of independent mitochondrial and nuclear markers supported the same main lineages, but basal relationships were not concordant. Moreover, some incongruence was found among independent mitochondrial and nuclear phylogenies. The monophyly of some poorly known genera such as Pseudophoxinus and Petroleuciscus was rejected. Representatives of both genera belong to different evolutionary lineages. Timing of cladogenetic events among the main leuciscine lineages was gained using mitochondrial and all genes data set. CONCLUSIONS: Adaptations to a predatory lifestyle or miniaturization have superimposed the morphology of some species. These species have been separated into different genera, which are not supported by a phylogenetic framework. Such is the case of the genera Pseudophoxinus and Petroleuciscus, which real taxonomy is not well known. The diversification of leuciscine lineages has been determined by intense vicariant events following the paleoclimatological and hydrogeological history of Mediterranean region. We propose different colonization models of Mediterranean region during the early Oligocene. Later vicariance events promoted Leuciscinae diversification during Oligocene and Miocene periods. Our data corroborate the presence of leuciscins in North Africa before the Messinian salinity crisis. Indeed, Messinian period appears as a stage of gradually Leuciscinae diversification. The rise of humidity at the beginning of the Pliocene promoted the colonization and posterior isolation of newly established freshwater populations. Finally, Pleistocene glaciations determined the current European distribution of some leuciscine species.

Molecular phylogeny and biogeography of the Cuban genus Girardinus Poey, 1854 and relationships within the tribe Girardinini (Actinopterygii, Poeciliidae).

Phylogenetic relationships among members of the freshwater fish tribe Girardinini were inferred to test existing colonization and diversification hypotheses for this group in the Caribbean. The genetic material examined was mitochondrial (cytochrome b, 1140 bp) and nuclear (RAG-1 and beta-actin, 2450 bp) DNA from 161 specimens representing 44 ingroup and three outgroup taxa. Our mtDNA and combined data matrix (mtDNA+nuclear DNA) results rendered a well-supported phylogeny for the tribe Girardinini and suggest the need to review the group's current taxonomy. From the data presented here, it may be inferred that the Girardinini diverged from other poeciliid fishes approximately 62 Mya ago in the Palaeocene period. This estimate, however, conflicts with the hypothesis that today's vertebrate fauna is the result of the more recent colonization of the Antillean islands during the Early Oligocene (35-33 Mya ago). The isolation of western, central and eastern Cuba during the Miocene and that of the Juventud Island and Guanahacabibes Peninsula during the Pliocene, are the main geologic events that could have promoted speciation in this group.

The complete mitogenome of the Moroccan Luciobarbus rifensis Doadrio, Casal-López & Yahyaoui, 2015 (Actinopterygii: Cyprinidae).

The Rifian barbel (Luciobarbus rifensis) is a tetraploid cyprinid species from North Africa. The aim of this work is to characterize the mitogenome of Luciobarbus rifensis in order to contribute in the future exploration of regions. The circular mitogenome of the Rifian barbel (16 607bp) consists of 37 genes (13 protein-coding genes, 2 rRNA genes, and 22 tRNA genes). The cyprinid mitogenome is most related to L. rifensis available is L. capito.

Phylogeography and Conservation Genetics of the Ibero-Balearic Three-Spined Stickleback (Gasterosteus aculeatus).

Genetic isolation and drift may imperil peripheral populations of wide-ranging species more than central ones. Therefore, information about species genetic variability and population structure is invaluable for conservation managers. The Iberian populations of three-spined stickleback lie at the southwestern periphery of the European distribution of Gasterosteus aculeatus. This teleost is a protected species in Portugal and Spain and local extinctions have been reported in both countries during the last decades. Our objectives were (i) to determine whether the Iberian populations of G. aculeatus are unique or composed of any of the major evolutionary lineages previously identified and (ii) to assess the evolutionary potential of these peripheral populations. We genotyped 478 individuals from 17 sites at 10 polymorphic microsatellite loci to evaluate the genetic variability and differentiation of the Ibero-Balearic populations. We also sequenced 1,165 bp of the mitochondrial genome in 331 of those individuals in order to complement the estimates of genetic diversity in the Ibero-Balearic region. We predicted the evolutionary potential of the different sites analysed based on the contribution of each of them to total allelic/mitochondrial diversity. An intraspecific phylogeny at European level was reconstructed using our data from the mitochondrial cytochrome b gene (755 bp) and published sequences. The so-called Transatlantic, European and Mediterranean mitochondrial lineages were found to be present in the Ibero-Balearic region. Their phylogeography suggests a history of multiple colonisations. The nuclear results show, however, a strong correlation between population structure and drainage system. The following basins should be prioritised by conservation policies in order to preserve those populations with the highest evolutionary potential: the Portuguese Vouga and Tagus as well as the Spanish Majorca and Limia. Maintenance of their connectivity, control of exotic species and monitoring of habitat properties are strongly recommended in those areas. Genetic variation alone cannot, however, ensure the persistence of these peripheral southern populations of G. aculeatus. On the one hand, the analysis of a historical sample from Eastern Spain (Penyscola) revealed no genetic erosion, which suggests a fairly sudden extinction of that population. On the other hand, the reintroduction program implemented in the Valencian Community has mostly failed despite our finding of similar level of genetic diversity between the wild source and the captive-bred released individuals.

Ancient Mitochondrial Capture as Factor Promoting Mitonuclear Discordance in Freshwater Fishes: A Case Study in the Genus Squalius (Actinopterygii, Cyprinidae) in Greece.

Hybridization and incomplete lineage sorting are common confounding factors in phylogeny and speciation resulting in mitonuclear disparity. Mitochondrial introgression, a particular case of hybridization, may, in extreme cases, lead to replacement of the mitochondrial genome of one species with that of another (mitochondrial capture). We investigated mitochondrial introgression involving two species of the cyprinid genus Squalius in the western Peloponnese region of Greece using molecular and morphological data. We found evidence of complete mitochondrial introgression of Squalius keadicus into two populations recognized as Squalius peloponensis from the Miras and Pamissos River basins and a divergence of mitochondrial genomes of S. keadicus from the Evrotas basin from that of the introgressed populations dating from the Pleistocene. Secondary contact among basins is a possible factor in connection of the species and the introgression event. Morphological analyses support the hypothesis of mitochondrial introgression, as S. keadicus was different from the other three populations recognized as S. peloponensis, although significant differences were found among the four populations. Isolation by geographical barriers arose during Pleistocene in the western Peloponnese were the source of the evolution of the two reciprocally monophyletic subclades found in the S. keadicus mitochondrial clade, and the morphological differences found among the four populations. Along with the lack of structure in the nuclear genome in the three populations ascribed to S. peloponensis, this suggests an incipient speciation process occurring in these Squalius species in the western Peloponnese.

Glutathione transferase pi (GSTpi) expression in breast cancer: an immunohistochemical and molecular study.

Breast cancer is the most frequent cancer in women worldwide. Prognostic markers are important for diagnosis, allowing therapeutic strategies to be defined more efficiently. The expression of the glutathione S-transferase pi isoenzyme (GSTpi) in tumor cells has been evaluated as a predictor of prognosis and in response to cytotoxic treatments. Its immunoexpression was assessed in 63 women diagnosed with invasive ductal carcinoma in a retrospective study. The results were statistically correlated with clinicopathological parameters of patients. The results showed that high GSTpi expression was related to p53-positive tumors, grade III histology, large tumor size and death (p<0.05). The 37 patients who received adjuvant treatment, checked separately, showed high expression of GSTpi in relation to local recurrence, metastasis and death (p<0.05). In addition, high levels of GSTpi expression were significantly associated with a shorter overall survival (p<0.05). To confirm this suspicion, GSTpi gene expression was checked by Real-time PCR in neoplastic mammary cells cultured and subjected to treatment with doxorubicin. Our results suggest that high levels of GSTpi may be related to the development of resistance to chemotherapy in these tumors, the response of these tumors to treatment and the clinical course of the patients involved.

Linfoma não-hodgkin primário de mama simulando mastite / Primary breast non-hodgkin lymphoma mimiching mastitis

Os autores apresentam o caso de uma paciente de 78 anos com linfoma não-hodgkin (LÑH) primário de mama simulando mastite sendo que, com a piora progressiva do quadro, procurou nossos serviços, onde foi diagnosticado, por biópsia, linfoma maligno não-hodgkin. O objetivo do trabalho é atentar para o diagnóstico diferencial de mastite, carcinoma inflamatório e LÑH de mama quando há envolvimento linfomatoso da pele