Pablo A. Martinez

Performance of human immunostimulating agents in the improvement of fish cytogenetic preparations.

Inoculation with bacterial or fungal antigens that stimulate cell proliferation has been widely used to obtain metaphases for cytogenetic studies of fish. We evaluated the potential of new pharmaceutical compounds as mitogenic agents in fish, testing the efficacy of Aminovac (mixed antigens and epsilon-acetamidocaproic acid), Broncho-Vaxom (bacterial lysate) and Estimoral (bacterial lysate) to increase the mitotic index in fingerlings of the Neotropical fish Prochilodus brevis (Prochilodontidae) and Hoplias malabaricus (Erythrinidae), which were obtained from an aquaculture facility. The animals were treated with intramuscular or intraperitoneal injections of 1 mL/50 g body weight of each compound. After 24 h, cytogenetic analyses were performed. All immunostimulants tested significantly stimulated cell division, although Aminovac proved to be the most efficient, leading to a 5-fold increase in the number of metaphase cells compared to the control group and to a 2-fold increase compared to conventional yeast inoculation. This compound facilitates fish cytogenetics analyses as it stimulates the proliferation of defense cells and reduces loss of samples. It will be especially useful for the study of specimens that either have a high commercial value or are fragile, small and/or rare.

Bergmann's rule across the equator: a case study in Cerdocyon thous (Canidae)

1. The variation in cranial size of the crab-eating fox Cerdocyon thous was analysed in relation to latitude and several environmental variables throughout its distribution in South America. 2. We tested the existence of clines to determine whether this canid follows Bergmanns rule to the north and south of the Equator. Also, using niche modelling, we analysed whether the climatic changes during the last glaciation could have influenced Bergmanns rule in this species. We quantified the size of the cranium of C. thous (n = 300). The data were divided into two groups: (i) south of the Equator (n = 163) and (ii) north of the Equator (n = 137). We performed correlations, OLS regressions and simultaneous autoregressions to analyse the relationship between the variation in size and different geographic and environmental variables. Data of occurrence (n = 594) together with ambient variables from the present and the last glacial maximum (LGM) were used to predict the occurrence of C. thous with the implementation of the maximum entropy method. Present-day and historical distribution maps were obtained. 3. The variation in the size of the cranium of C. thous showed two trends. In the south of Equator, we observed that the size of the skull shows an inverse relationship with temperature-related variables and a positive one with precipitation, while in north of the Equator, we observed the opposite relationship. Populations south of the Equator follow Bergmanns rule showing increasing size with increasing latitude. To the north of the Equator, a non-Bergmannian pattern occurs because size decreases with increasing latitude. 4. Niche modelling showed two present-day groupings in South America, one north of Amazonia and the other south. However, for the period of the LGM, four groups emerged, possibly related to the four subspecies presently described for C. thous. Therefore, it is possible that the observed pattern - southern populations following Bergmanns rule while northern populations reflecting the opposite - has been influenced by the events that occurred during the LGM that could have led to the differentiation of populations.

Evidence for meiotic drive as an explanation for karyotype changes in fishes

The process of preferential chromosome segregation during meiosis has been suggested to be responsible for the predominance of certain chromosome types in the karyotypes of mammals, birds and insects. We developed an extensive analysis of the fixation of mono- or bibrachial chromosomes in the karyotypes of the large Actinopterygii fish group, a key link in the evolution of terrestrial vertebrates, in order to investigate the generality of meiotic drive in determining karyotypic macrotrends. Unlike mammals, fishes have markedly undergone several types of preferential chromosomal rearrangements throughout evolution. Data from the analyzed orders indicate a prevalence of karyotypes with few (<33%) or many (>66%) acrocentric chromosomes and a low number of karyotypes with balanced numbers of mono- and bi-brachial elements. Parallel trends towards a higher number of karyotypes with prevalence of monobrachial chromosomes occurred in phylogenetically close orders (e.g. Perciformes and Tetraodontiformes, and in the order Mugiliformes) and in clades with prevalence of bibrachial elements (e.g. Characiformes, Gymnotiformes, Siluriformes, and Cypriniformes). Some orders where fewer species were available for study, such as Atheriniformes and Anguilliformes, showed karyotype assemblages where both trends were present. Our results strongly suggest a primary role of meiotic drive in karyotypic evolution as indicated by the accumulation of monobrachial chromosomes in Perciformes and Cypriniformes, or bibrachial chromosomes in Siluriformes and Characiformes. Further examinations of the interaction between life history traits, environmental characteristics, and the fixation of chromosomal rearrangements would be exceedingly valuable.

Preferential accumulation of sex and Bs chromosomes in biarmed karyotypes by meiotic drive and rates of chromosomal changes in fishes

Mechanisms of accumulation based on typical centromeric drive or of chromosomes carrying pericentric inversions are adjusted to the general karyotype differentiation in the principal Actinopterygii orders. Here, we show that meiotic drive in fish is also supported by preferential establishment of sex chromosome systems and B chromosomes in orders with predominantly bi-brachial chromosomes. The mosaic of trends acting at an infra-familiar level in fish could be explained as the interaction of the directional process of meiotic drive as background, modulated on a smaller scale by adaptive factors or specific karyotypic properties of each group, as proposed for the orthoselection model.

Chromosomal diversity in tropical reef fishes is related to body size and depth range.

Tropical reef fishes show contrasting patterns of karyotypic diversity. Some families have a high chromosomal conservatism while others show wide variation in karyotypic macrostructure. However, the influence of life-history traits on karyotypic diversity is largely unknown. Using phylogenetic comparative methods, we assessed the effects of larval and adult species traits on chromosomal diversity rates of 280 reef species in 24 families. We employed a novel approach to account for trait variation within families as well as phylogenetic uncertainties. We found a strong negative relationship between karyotypic diversity rates and body size and depth range. These results suggest that lineages with higher dispersal potential and gene flow possess lower karyotypic diversity. Taken together, these results provide evidence that biological traits might modulate the rate of karyotypic diversity in tropical reef fishes.

Inter- and intra-basin phenotypic variation in two riverine cichlids from northeastern Brazil: potential eco-evolutionary damages of São Francisco interbasin water transfer

Intraspecific morphological variation can be generated by a set of historical and ecological processes, and can be induced by anthropogenic actions. One such activity that has large-scale environmental impacts in freshwater environments is interbasin water transfer. Brazil’s Mid-Northeastern Caatinga freshwater ecoregion is going through an interbasin diversion that will link waters from São Francisco ecoregion with the four major basins of Mid-Northeastern Caatinga. Here, we aimed to evaluate the morphological variation of two cichlids, Cichlasoma orientale and Crenicichla menezesi, from both ecoregions and test, whether this morphological variation is related to historical isolation among basins and/or to local water flow regimes, through mtDNA (cytb) and geometric morphometric approaches. Our results showed, for both species, significant morphological differences among basins. However, the comparisons between genetic and morphological differences among basins were not significant. Additionally, a significant shared morphological pattern between species (body depth and mouth position) among different habitats was found. These results reinforce a potential role of environmental pressures upon fish morphology, possibly through phenotypic plasticity. The evolutionary role of morphological responses in freshwater fish populations—especially in variable environments—is discussed, as well as the potential eco-evolutionary damages that might result from the artificial river connection.

Morphological and karyotypic differentiation in Caranx lugubris (Perciformes: Carangidae) in the St. Peter and St. Paul Archipelago, mid-Atlantic Ridge

Isolated oceanic islands constitute interesting model systems for the study of colonization processes, as several climatic and oceanographic phenomena have played an important role in the history of the marine ichthyofauna. The present study describes the presence of two morphotypes of Caranx lugubris, in the St. Peter and St. Paul Archipelago located in the mid-Atlantic. Morphotypes were compared in regard to their morphological and cytogenetic patterns, using C-banding, Ag-NORs, staining with CMA3/DAPI fluorochromes and chromosome mapping by dual-color FISH analysis with 5S rDNA and 18S rDNA probes. We found differences in chromosome patterns and marked divergence in body patterns which suggest that different populations of the Atlantic or other provinces can be found in the Archipelago of St. Peter and St. Paul.

Derived cytogenetic traits, multiple NORs and B chromosomes in the compact karyotype of Canthigaster figueiredoi (Tetraodontiformes).

The Tetraodontiformes are one of the main irradiation lineages of Teleosts representing a highly specialized group. Among its families, Tetraodontidae has the lowest DNA/cell content of the vertebrates, and for this reason has been used as a model group for genomic evolution studies. Seeking to widen the cytogenetic database of this family, we performed a chromosomal analysis of the species Canthigaster figueiredoi using conventional staining, Ag-NORs, C-banding, base-specific fluorochromes DAPI-CMA(3), and in situ hybridization with 18S rDNA probe. This species has 2n=36 (10 m+6sm+20a; FN=52). Multiple Ag-NORs (CMA(3)(+)) sites were detected on the four chromosome pairs. Heterochromatic blocks (CMA(3)(+)) were present in the centromeric regions of most of the pairs, extending on the short arm of some chromosomes. A GC-positive polymorphic heterochromatic region was identified in some of the individuals, in one or both of the homologs of the 6th pair. The presence of heteromorphic B microchromosomes was detected in the karyotype of one female, exhibiting intra-individual variation of 0-3 Bs. The occurrence of heterochromatic polymorphisms, multiple NORs, and B chromosomes in C. figueiredoi are very infrequent events in marine fish. They are probably associated to the accentuated restructuring and genomic reduction suffered by this family.

Phylogenetic path analysis reveals the importance of niche-related biological traits on geographic range size in mammals

MIGUELA. OLALLA-T ARRAGA, ERIK JOAQU IN TORRES-ROMERO,TALITA FERREIRA AMADO andPABLO A. MARTINEZDepartment of Biology and Geology, Physics and Inorganic Chemistry, Rey Juan Carlos University, 28933-Mostoles, Madrid,SpainThe geographic range of a species is arguably the basicunit in biogeography and macroecology (Brown et al.,1996). In particular, there has been a long-standinginterest in understanding the mechanisms that shapethe immense interspecific variation in geographic rangesize, a question often framed around Rapoport’s rule(Whitton et al., 2012). As an emergent species-level trait,range sizes reflect the interplay of ecological and evolu-tionary processes and are of utmost importance for pre-dicting speciation-extinction dynamics (Jablonski,2008). Species tend to have a higher risk of extinction ifthey occupy a small geographic range (Purvis et al.,2000), which also places the investigation of patternsand processes in the variation of species’ range sizes asa central question in applied conservation science.In a recent paper, Di Marco & Santini (2015, and here-after DM&S) analysed which are the determinants ofrange size variation in extant terrestrial mammals glob-ally. They concluded that extrinsic factors (climate andhuman impacts), not intrinsic biological traits, are themost influential variables. This study brings to the tablethe importance of considering anthropogenic effects inmacroecological research. Surprisingly, even for thebest-studied taxa in macroecology such as mammalsand birds, workers have traditionally overlooked theinfluence of human pressures on the observed patterns.We ourselves have called the attention on the need toincorporate human impact metrics, such as humanfootprint or accessibility, to better understand the spa-tial distribution of extant mammal species in some bio-geographical realms (Torres-Romero & Olalla-Tarraga,2015). However, we do not agree that human effectsprevail over biological traits in determining the rangesizes of mammalian species and would like to call theattention on a few conceptual and methodologicalaspects of DM&S’s analyses and interpretation that arenot at all correct to our view.First, DM&S neglect the relevance of phylogeneticrelatedness on the geographic range sizes of species.Intrinsic autoecological features of mammals oftenreflect shared ancestry, so that closely related specieswill tend to share similar biological traits and possiblysimilar range sizes (Brown et al., 1996, Jablonski, 2008).A number of comparative methods have been designedto address potential phylogenetic correlation issues inmodel residuals (as long as there is an underlyingrobust phylogenetic hypothesis, as is the case for mam-mals). Second, DM&S also ignore the importance of therelationship niche breadth-range size as a biologicalexplanation (Slatyer et al., 2013). The concept of ecologi-cal niche was indeed formalized to describe the set ofbiotic and abiotic conditions where a species can persistand maintain stable population sizes. Its projection ontogeographic space (i.e. the duality niche-biotope) is inex-tricably linked to the spatial distribution of a speciesand is highly relevant to analyse biogeographical pat-terns (Colwell & Rangel, 2009). DM&S only incorporatea simplistic diet category variable that classifies mam-mals in terms of trophic position (i.e. carnivores, herbi-vores or omnivores), but do not consider diet breadth.Perhaps more importantly, DM&S characterized andincluded in the analyses the realized climatic niches ofeach mammal species (see also Olalla-Tarraga et al.,2011 for a similar estimation method), but erroneouslyreferred to them as extrinsic variables. These measuresof environmental tolerance breadth typically have astrong positive relationship with range size (Slatyeret al., 2013). Third, DM&S use random forest regressionmodels that allow estimating direct effects betweeneach predictor and the response variable, but cannotcalculate indirect effects via other dependent variables.We have used a recently developed phylogeneticconfirmatory path analysis (von Hardenberg & Gonz-alez-Voyer, 2013) and included previously untestedvariables to provide a reassessment on which are themain determinants of range size in mammals globally(for methodological details see supplementary infor-mation). We find that the range sizes of both nonvo-lant mammals and chiropterans strongly depend ontheir thermal and hydric niches, an intrinsic biologicalproperty, followed by a secondary extrinsic effect of

Climatic suitability, isolation by distance and river resistance explain genetic variation in a Brazilian whiptail lizard

Spatial patterns of genetic variation can help understand how environmental factors either permit or restrict gene flow and create opportunities for regional adaptations. Organisms from harsh environments such as the Brazilian semiarid Caatinga biome may reveal how severe climate conditions may affect patterns of genetic variation. Herein we combine information from mitochondrial DNA with physical and environmental features to study the association between different aspects of the Caatinga landscape and spatial genetic variation in the whiptail lizard Ameivula ocellifera. We investigated which of the climatic, environmental, geographical and/or historical components best predict: (1) the spatial distribution of genetic diversity, and (2) the genetic differentiation among populations. We found that genetic variation in A. ocellifera has been influenced mainly by temperature variability, which modulates connectivity among populations. Past climate conditions were important for shaping current genetic diversity, suggesting a time lag in genetic responses. Population structure in A. ocellifera was best explained by both isolation by distance and isolation by resistance (main rivers). Our findings indicate that both physical and climatic features are important for explaining the observed patterns of genetic variation across the xeric Caatinga biome.