Genetic structure of the killifish Aphanius fasciatus, Nardo 1827 (Teleostei, Cyprinodontidae), results of mitochondrial DNA analysis

Aphanius fasciatus is a cyprinodont distributed in the salty coastal water of the central and eastern Mediterranean Sea and occasionally in internal fresh water. In this work, the authors have investigated the genetic structure of eight populations of the killifish A. fasciatus from Sardinia and Sicily. The comparison of the mtDNA control region of 237 individuals revealed a total of 49 haplotypes. Several unique haplotypes were present in each population, and no common haplotype was found among Sicilian and Sardinian populations. Almost all Sardinian populations shared a common haplotype, and indeed the four Sicilian populations examined did not share any as determined by the parsimony network analysis. The analysis of molecular variance showed that the percentage of variation among populations is much higher than within each population of A. fasciatus . The overall F _ST value is very high (0·78) and supports an extensive genetic structure of the populations. The observed genetic differentiations of A. fasciatus populations were discussed taking into account the palaeogeographic and palaeoclimatic events that interested the Mediterranean area from Miocenic to Pleistocenic age. The results provide new insight into the knowledge of the pattern of genetic structure and of evolutionary processes occurring in this species.     

The role of habitat choice in micro‐evolutionary dynamics: An experimental study on the Mediterranean killifish Aphanius fasciatus (Cyprinodontidae)

Habitat choice is defined as a nonrandom distribution of genotypes in different microhabitats. Therefore, it could exert a great impact on the genetic variance of natural populations by promoting genetic divergence, local adaptation, and may even lead to sympatric speciation. Despite this potential role in micro‐ and macro‐evolutionary processes, there is little empirical evidence that the various genotypes within a population may differ in habitat choice‐related behaviors. Here, we tested whether habitat choice may have contributed to genetic divergence within a local population of the Mediterranean killifish Aphanius fasciatus, which emerged between groups inhabiting microhabitats with different oxygen concentrations during previous field studies. In a first experiment, we studied the distribution of individuals in conditions of hypoxia and normoxia to test whether they had a different ability to shy away from a hypoxic environment; in a second experiment, we analyzed the individual behavior of fish separately in the two conditions, to verify whether they showed peculiar behavioral responses linked to a possible differential distribution. We then analyzed the six allozyme loci, whose allelic and genotypic frequencies were significantly divergent in the previous studies. In the first test, we found that the distribution of the two homozygote genotypes of the glucose‐6‐phosphate isomerase‐1 locus (GPI‐1) was significantly different between the hypoxic and the normoxic conditions. During the second test, all individuals were more active in hypoxic conditions, but the two GPI‐1 homozygotes showed a significant difference in time spent performing surface breathing, which was consistent with their distribution observed in the first experiment. These results provide evidence that individual behavioral traits, related to genetic features, may lead to a nonrandom distribution of genotypes in heterogeneous although contiguous microhabitats and, consequently, that habitat choice can play a significant role in driving the micro‐evolutionary dynamics of this species.     

New inland sites for the Mediterranean killifish (Aphanius fasciatus Valenciennes, 1821) in the Sahara Desert of Algeria

The current study aims to update the range of Aphanius fasciatus occurring in inland aquatic systems, and to characterise the length–weight relationship of specimens caught in inland waters of the Northern Sahara of Algeria. Although the known distribution of A. fasciatus is restricted to coastal waters including islands of the Mediterranean region, visits to 39 hydrosystems in Northeast Algeria revealed the presence of the species at two sites: Oued Still and Lake Temacine. Both sites are in the Sahara Desert of Algeria, where the climate is hot and hyperarid. The surveyed populations perform their entire life cycle at the same sites. Analysis of a sample composed of 27 individuals captured in Oued Still allowed determination of the length–weight relationship. On average, females measured 4.2 ± 0.6 cm in length and weighed 1.1 ± 0.5 g; males were 3.2 ± 0.6 cm and 0.5 ± 0.2 g. Growth parameters of the von Bertalanffy equation for A. fasciatus were estimated at Lt = 5.5145 (1?exp (?0.341(t?0.0049))), Wt = 2.6692 (1?exp (?0.271t))³.     

Sexual dimorphism of vertical bar patterning in the South European toothcarp Aphanius fasciatus

Sexual dimorphism of vertical bar patterning along the ontogenetic gradient was investigated in the South European toothcarp Aphanius fasciatus through the analysis of field samples collected from four different Venice Lagoon sites. Results suggested that, after controlling for site effect, vertical bar number and density were more correlated with size in females than in males, yielding a higher number of less spaced bars in females than in males. Results are discussed with regard to the interplay between natural and sexual selection acting on vertical bar dimorphism, suggesting a role of disruptive colouration in females and a more pronounced role of sexual selection in males.     

First record of Aphanius almiriensis from Italy and notes on the distribution of Aphanius fasciatus (Teleostei: Aphaniidae)

Aphanius almiriensis is the first time reported from Italy, where it occurs in the Palude del Capitano. The newly found population agree with Greek A. almiriensis in molecular (COI) and morphological characters. Aphanius almiriensis might be native to the the Palude del Capitano, which is outside from the species known range in the Aegean Sea basin. It might also have been imported with goods from the eastern Aegean in the late Republican age and the late Imperial period. The diagnostic morphological characters as well as the distribution and conservation status of A. almiriensis is discussed. A distribution map and associated, georeferenced database of A. almiriensis as well as of A. fasciatus are presented.     

Genetic and environmental variation in the visual properties of bluefin killifish, Lucania goodei

Animals use their sensory systems to detect information about the external environment in order to find mates, locate food and habitat and avoid predators. Yet, there is little understanding of the relative amounts of genetic and/or environmental variation in sensory system properties. In this paper, we demonstrate genetic and environmental variation in opsin expression in a population of bluefin killifish. We measured expression of five opsins (which correlates with relative frequency of corresponding cones) using quantitative, real-time polymerase chain reaction for offspring from a breeding study where offspring were raised under different lighting conditions. Sire (i.e. genetic) effects were present for opsin found in yellow photopigment. Dam effects were present for opsins that create violet, blue and red photopigment. Lighting conditions affected expression of all opsins except SWS2A and mimicked the pattern found among populations. These results highlight the fact that sensory systems are both plastic and yet readily evolvable traits.     

Comparative pattern of genetic structure in two Mediterranean killifishes Aphanius fasciatus and Aphanius iberus inferred from both mitochondrial and nuclear data

In this study, genetic variation was assessed in Aphanius fasciatus and Aphanius iberus characterized by similar ecological traits but with very different distribution ranges in the Mediterranean area. Five populations of A. iberus and five of A. fasciatus were analysed using five polymorphic microsatellite loci and partial mitochondrial control region (D‐loop) sequences. Congruent results were found with both nuclear and mitochondrial molecular markers. The results showed that similar levels of genetic divergence, based on mitochondrial control region sequences, are present among populations of A. iberus and among populations of A. fasciatus despite the very different geographic distance existing among the examined populations of the two species (low geographic distance in A. iberus and high in A. fasciatus). A possible explanation could be that the populations of A. iberus were isolated for a longer time than the populations of A. fasciatus supporting the hypothesis that the split in the lineage leading to A. iberus is older than the split in the lineage leading to A. fasciatus. The possibility that the wide circum‐Mediterranean distribution of A. fasciatus ensures the high connectivity of its populations, preventing, in some cases, local differentiation, however, cannot be ruled out.     

Life history of the Mediterranean killifish <Emphasis Type="Italic">Aphanius fasciatus</Emphasis> in brackish water habitat of Algerian low Sahara

The Mediterranean tooth carp Aphanius fasciatus, a strongly eurythermic and euryhaline species, is an important component of the ichthyofauna in the valley of Oued Righ, situated in Algerian low Sahara. This study aims to understand the life history of this species in arid environment. A total of 1868 individuals (16.7–60.2 mm TL, 0.04–26.8 g TW) were collected between November 2010 and November 2011 from Ayata Lake (Algerian low Sahara). Individual age was determined by scalimetry. Some aspects of the sexual cycle are studied. The population was composed of 6 age classes in both sexes. Back-calculations of total length-at-age were suitably adjusted to von Bertalanffy growth model with: Lt?=?80.00 [1?e^{?0.172 (t + 1.377)}] for females and Lt?=?77.58 [1?e^{?0.138 (t + 2.236)}] for males. The fitted total length-total weight relationship was W?=?0.013 L^2.848 for females, W?=?0.013 L^2.809 for males. Spawning occurs once a year between February and July with peak activity in May for Females and in April for Males. Gonado-somatic index reaches a maximum of 4.16% in males and 15.73% in females. The length at the first sexual maturity is 45 mm for both sexes.     

Genetic diversity of riverine reed stands indicating the water regime of the habitat

Although genetic diversity of reed stands developing in habitats with unchanged water levels have been frequently investigated, little is known about reed propagation along rivers, where fluctuating water levels may provide various conditions for germination and seedling development.The present paper evaluates genetic diversity of reed stands grown in (i) an oxbow with significant water level fluctuations related to the flood events in the main river; (ii) a separated oxbow which has lost connection to the main channel; and (iii) artificially controlled running water with low water level fluctuation. At each site, reed stands were investigated along transects parallel to the shore and multilocus phenotypes were determined by microsatellite analysis.The results demonstrated that genetic diversity of riverine reed stands can indicate the water regime of the habitat. Reed colonizes mostly by vegetative propagation where regular inundation can impede germination and seedling development and, therefore, genetic diversity is low in the whole stand. If a former oxbow becomes practically a lake, the clonal diversity and colonization processes are similar to those observed in regular lakes; clone number decreases toward the open water. When reed forms floating mats (i.e. the effect of water level fluctuation is excluded), generative reproduction prevails in the entire stand, resulting in high genetic diversity even at the open water edge of the stand.     

Temporal Genetic Variation in a Population of Aphanius fasciatus (Cyprinodontidae) from a Brackish-water Habitat at Elba Island (Italy)

Allozyme electrophoresis was used to assess temporal genetic variation in three successive generations of the Mediterranean killifish, Aphanius fasciatus. Samplings were carried out in 1995, 1996 and 1997 in a brackish-water habitat at Elba Island, Italy and a total of 212 specimens were collected. The five loci for which polymorphism has been detected in a previous study were assayed. Mean expected heterozygosity values [H=0.397 (SE 0.077), H=0.336 (SE 0.092) and H=0.313 (SE 0.092) in 1995, 1996 and 1997, respectively] were not significantly different by ANOVA test. Deviations from Hardy–Weinberg equilibrium were minimal, with only one out of the 15 probability tests showing a significant departure from the equilibrium; whereas genotypic linkage disequilibrium was not detected. Values of Nei's genetic distance were lower than 0.04. Temporal genetic variation in the A. fasciatus population at Elba Island was observed, with F-statistics indicating significant genetic divergence among samples (=0.035, SE 0.027, p<0.001). Genetic drift acting on two loci (GPD-1 ^* and LDH-3 ^*) is presumably the main force determining the temporal genetic heterogeneity observed; however, the occurrence of selection on individual loci and/or sampling error cannot be excluded. The observed allelic variation among generations in a single population of A. fasciatus is much less than levels observed among geographically discrete samples in previous studies.     

The diversity of culturable organotrophic bacteria from local solar salterns

We isolated and cultured bacteria inhabiting solar saltern ponds in Taean-Gun, Chungnam Province, Korea. All of the isolated 64 strains were found to be moderately halophilic bacteria, growing in a salt range of 2-20 %, with an optimal concentration of 5% salt. Bacterial diversity among the isolated halophiles was evaluated via RFLP analyses of PCR-amplified 16S rDNAs, followed by phylogenetic analysis of the partial 16S rDNA sequences. The combination of restriction enzyme digestions with HaeIII, CfoI, MspI and RsaI generated 54 distinct patterns. A neighbor-joining tree of the partial 16S rDNA sequences resulted in the division of the 64 strains into 2 major groups, 45 strains of gamma-Proteobacteria (70.3%) and 19 strains of Firmicutes (29.7%). The alpha-Proteobacteria and Cytophaga-Flavobacterium-Bacterioides groups, which were repeatedly found to exist in thalassohaline environments, were not represented in our isolates. The gamma-Proteobacteria group consisted of several subgroups of the Vibrionaceae (37.5%), Pseudoalteromonadaceae (10.9%), Halomonadaceae (7.8%), Alteromonadaceae (7.8%), and Idiomarinaceae (6.3%). Members of Salinivibrio costicola (29.7%) were the most predominant species among all of the isolates, followed by Halobacillus treperi (12.5%). Additionally, three new species candidates were found, based on similarities of the 16S rDNA sequences to those of previously published species.     

Life-history variations of killifish (Aphanius sophiae) populations in two environmentally different habitats in central Iran

Two populations of Aphanius sophiae from two different habitats (one in a spring with stable environmental conditions and the other in a river with variable conditions) were examined to determine whether these conditions elicit differences in life history traits. Age composition revealed that females from the spring live longer. Von Bertalanffy growth parameters showed that L(∞) in the river population was higher than in the spring population. GSI showed a shorter reproduction period in A. sophiae from the river system. Egg diameter was significantly larger in the spring population. These results confirm the hypothesis of the response of killifish to local selective pressures in different habitats through adaptations via changes in the duration of the reproductive period, egg diameter, size changes, age composition and growth rate.     

Morphological variation among populations of Aphanius fasciatus Nardo, 1827 (Teleostei, Cyprinodontidae) from the Mediterranean

The amount of osteological variation among 11 Italian killifish Aphanius fasciatus populations was examined by the univariate and multivariate analysis of 40 morphometric and meristic variables of the skull and vertebral column. Populations were sampled in three geographically distinct areas (the Adriatic, Sardinia and Sicily). The statistical analysis confirmed that several populations were well differentiated. In particular, discriminant analysis revealed a strong discriminating power of the morphometric variables. Morphometrics of the vertebrae, bony elements of the pharyngeal jaws, supraoccipital and parasphenoid were the most important in discriminating populations. The dendrogram obtained by UPGMA cluster analysis shows the separation of the south-eastern Sicilian populations, that of the Sardinian populations and that of the central-northern Sicilian plus Adriatic populations, as well as the isolation of the Sicilian population from Pantano Viruca and of the Sardinian populations from Pauli Figu from all the others. The significance of the observed differentiation pattern is discussed.     

Genetic diversity changes in Indian lentils over the times

The genetic diversity of 96 genotypes of lentil comprising 34 cultivars, 46 advanced breeding lines, and 16 germplasm lines were studied using 260 SSR markers. These markers generated a total of 749 alleles. The alleles/locus ranged from 2 to 16 with an average value of 2.87. Polymorphic information content varied from 0.02 to 0.91 with a mean of 0.30. Major allelic frequency ranged from 0.14 to 0.99 with a mean of 0.77. Studied genotypes were clustered into two groups according to their breeding history. Advanced breeding lines derived from exotic lines were clustered in one group, while another group accommodated most of the cultivars and advanced breeding lines with common cultivars in parentage. The germplasm lines were sub-clustered within first group. Cumulatively, first three principal components contributed 21.2% to the total variability. Advanced breeding lines showed higher number of alleles/locus and gene diversity (He) than other sets of genetic materials. In present study, no significant differences were observed between cultivars developed in different decadal groups for both NA and He. Moreover, genetic diversity changes between small and large seeded lentil cultivars were also found non-significant in this study. These findings showed that the use of alien genes can help to diversify active gene pool for developing improved new cultivars in lentil.     

Isolation and characterization of eight polymorphic microsatellite markers in Aphanius fasciatus (Teleostei: Cyprinodontidae)

The first set of eight polymorphic microsatellites markers was successfully optimized from a partial genomic library enriched for an AC motif of the killifish Aphanius fasciatus Nardo, 1827 (Teleostei, Cyprinodontidae), a brackish‐water fish widely distributed along the coasts of the Mediterranean Sea. The developed loci proved to be polymorphic, displaying from two to six alleles per locus with expected heterozygosity ranging from 0.277 to 0.752. All loci were under Hardy–Weinberg equilibrium, except Af8 that showed significant heterozygotes deficiency, and there was no evidence of linkage associations between any pair of loci. Markers reported here will be potentially useful in monitoring of genetic population structure in this species.     

Microbial diversity in Maras salterns, a hypersaline environment in the Peruvian Andes

Maras salterns are located 3,380 m above sea level in the Peruvian Andes. These salterns consist of more than 3,000 little ponds which are not interconnected and act as crystallizers where salt precipitates. These ponds are fed by hypersaline spring water rich in sodium and chloride. The microbiota inhabiting these salterns was examined by fluorescence in situ hybridization (FISH), 16S rRNA gene clone library analysis, and cultivation techniques. The total counts per milliliter in the ponds were around 2 x 10(6) to 3 x 10(6) cells/ml, while the spring water contained less than 100 cells/ml and did not yield any detectable FISH signal. The microbiota inhabiting the ponds was dominated (80 to 86% of the total counts) by Archaea, while Bacteria accounted for 10 to 13% of the 4',6'-diamidino-2-phenylindole (DAPI) counts. A total of 239 16S rRNA gene clones were analyzed (132 Archaea clones and 107 Bacteria clones). According to the clone libraries, the archaeal assemblage was dominated by microorganisms related to the cosmopolitan square archaeon "Haloquadra walsbyi," although a substantial number of the sequences in the libraries (31% of the 16S rRNA gene archaeal clones) were related to Halobacterium sp., which is not normally found in clone libraries from solar salterns. All the bacterial clones were closely related to each other and to the gamma-proteobacterium "Pseudomonas halophila" DSM 3050. FISH analysis with a probe specific for this bacterial assemblage revealed that it accounted for 69 to 76% of the total bacterial counts detected with a Bacteria-specific probe. When pond water was used to inoculate solid media containing 25% total salts, both extremely halophilic Archaea and Bacteria were isolated. Archaeal isolates were not related to the isolates in clone libraries, although several bacterial isolates were very closely related to the "P. halophila" cluster found in the libraries. As observed for other hypersaline environments, extremely halophilic bacteria that had ecological relevance seemed to be easier to culture than their archaeal counterparts.     

Genetic incompatibilities in killifish and the role of environment

Measuring reproductive isolation across multiple generations and environments is a key endeavor in speciation research because it indicates which isolating barriers currently prevent introgression and the extent to which they are intrinsic versus environmentally dependent. Here, I present data from several crosses (parental crosses, F1s, F2s, back-crosses) between two species of killifish (Lucania goodei and L. parva) that have diverged along a salinity gradient (L. goodei--freshwater, L. parva--euryhaline). Offspring were raised under high and low salinity to test for (1) extrinsic isolation, (2) intrinsic isolation manifested through genetic incompatibilities, and (3) environmentally dependent genetic incompatibilities. I found evidence for both intrinsic and extrinsic isolation, but no evidence for environmentally dependent genetic incompatibilities. The presence of extrinsic and intrinsic isolation varied among fitness measures, and all forms of reproductive isolation were asymmetric. Early egg survival was independent of salinity, but demonstrated pronounced intrinsic isolation. Both extrinsic and intrinsic isolation existed for egg hatching and survival of fry to the eating stage. Unfortunately, the order in which extrinsic and intrinsic isolation arose is unresolved. Understanding the extent to which adaptation to salinity creates multiple forms of reproductive isolation is critical for understanding diversification in many fish taxa.     

Effects of Cadmium and High Temperature on Some Parameters of Calcium Metabolism in the Killifish (Aphanius fasciatus)

This study aims to investigate the influence of high temperature on cadmium (Cd) toxicity in Aphanius fasciatus (Pisces: Cyprinodontidae). For this reason, Cd, mineral, and organic content in the vertebral column as well as the histological structure of gills and bone were compared in fishes exposed for 30 days to Cd (2 mg/L CdCl₂) and/or high temperature (26 °C). Cd exposure caused a negative correlation between Cd and Ca concentrations (r?=?0.98, p?<?0.05), as well as a significant decrease in inorganic components (p?<?0.05) and ash weight/dry weight ratio (p?<?0.05) in the vertebral column. These changes were accompanied by an increased frequency of histological alterations in gills and bone. Concomitant treatment with Cd and high temperature increases Cd accumulation and Ca depletion in the skeletal tissue and increases the frequency and the severity of histological alterations. These results confirm that temperature increases Cd toxicity and needs to be taken into account for the accurate prediction and assessment of Cd-induced spinal deformities in fish.     

Genetic diversity of Lotus corniculatus in relation to habitat type,species composition and species diversity

The genetic variability of Lotus corniculatus, a common and important fodder legume, was studied in relation to habitat type and to species diversity at a local level. The study was conducted in Cholomontas mountain, northern Greece, at altitudes of 760–870 m. Genetic material was selected from four forested, at the edge of forest, and four open grassland sites, and was studied with the aid of ISSR molecular markers. The plant cover at each study site was measured and the floristic composition was estimated. The percentage of graminoides increased in grassland sites with high grazing intensity, while the abundance of legumes, including L. corniculatus, forbs and woody species, increased in forested sites with low grazing intensity. Gene diversity H_E within the studied populations of L. corniculatus ranged from 0.167 to 0.213 and Shannon index (I) from 0.269 to 0.340. Genetic differentiation was detected between habitats as well as among the populations in each habitat, although it was low, 3% and 7% respectively. However, genetic differentiation was significant within the populations (90%). Genetic diversity of L. corniculatus was not correlated with species diversity (richness and evenness) either within or between populations, while was negatively correlated with L. corniculatus abundance.