Livia Lucentini

Geographical and seasonal evidence of cryptic diversity in the Baetis rhodani complex (Ephemeroptera, Baetidae) revealed by means of DNA taxonomy

Previous phylogenetic investigations on the mayfly Baetis rhodani Pictet from several European countries, excluding Italy, strongly suggested the presence of cryptic species. Our paper reports a DNA-taxonomy phylogenetic analysis of B. rhodani with additional populations coming from Italian and UK sites, and aims to identify potential cryptic species with a coalescent-based method (GMYC model) and to understand the mechanisms of local coexistence of cryptic species. Twenty-five haplotypes of Italian samples and five haplotypes of UK samples were identified and added to a large European dataset. A total of 11 potential cryptic species have been recognised, and three of them co-occured in one Italian area. Such cryptic species seem to be phylogenetically over-dispersed on the tree and temporally segregated, and the seasonal substitution pattern of cryptic species could explain the apparently widespread distribution of the B. rhodani complex and its ability to adapt to different temperatures and food resources, justifying some of the differences observed in the relationship between water temperature, growth rates and phenology documented from field studies.

A comparison of conservative DNA extraction methods from fins and scales of freshwater fish: A useful tool for conservation genetics

Key words: brown trout, conservative DNA extraction, fish fin clip, fish scales, northern pikeNon-destructive protocols for DNA isolationfrom fresh or preserved specimens are fundamen-tal to study endangered or elusive species, breedersor samples sibship and specimens derived frompublic or private collections (Nielsen et al. 1999;Wasko et al. 2003; Hansen and Jensen 2005;Lucentini et al. 2006). Because of the low yieldand poor quality DNA, particular laboratory careand standardizations were needed to allow repro-ducible results. We compared six DNA extractionprocedures from conservative samples: three arecommercial kits [Wizard Genomic DNA Purifi-cation Kit (WGDPK) (Promega); Wizard Mag-netic DNA Purification System for Food(WMDPF) (Promega); NucleoSpin Food (NSF)(Macheray–Nagel)] and three are largelyemployed methodologies [Trizol (Life Technolo-gies); Chelex (Sigma–Aldrich); C-TAB]. For eachmethod, the standard protocol (reported on datasheets or the first published one) and severalvariations were tested varying sample storage andpre-lysis conditions, homogenization procedures,buffer solutions and concentrations, incubationsand resuspension times and temperatures. DNAwas extracted from 200 northern pikes (Esoxlucius L.) and 100 brown trout (Salmo trutta farioL.) (Table 1) from small (£10 mg) fin pieces and5–10 scales stored dried or in absolute alcoholboth at )20 C and at room temperature. DNAextraction was carried out on fresh materials andrepeated within a period of three years (Table 1),and from 34 years old dried scales culled from amuseum collection. Furthermore, DNA wasextracted from liver and muscle of both species,from individuals that had naturally died and usedas controls. Spectrophotometric readings (260 and280 nm) and densitometric evaluations (Image J)of DNAs obtained through different methodolo-gies and protocols (conservative versus controlDNA) were analyzed by means of ANOVA andt-test. DNA suitability was assayed through PCR–RFLP and microsatellite analyses (Lucentini et al.(2006) (Table 2). Microsatellites were run onABI377 DNA sequencer whereas PCR–RFLPswere assayed on 2.5% agarose electrophoresis fortwo mtDNA NADH coding regions (ND-1 ND5/6) (Cronin et al. 1993). DNA stability andamplificability was controlled every three months,for three years, in parallel with control DNA. Toevaluate genotyping errors, null alleles presenceand allelic dropout, all the experiments were rep-licated and statistically treated as suggested(Hoffman and Amos 2005; Roon et al. 2005)through MICRO-CHECKER 2.2.3 (Vanoosterhout et al. 2004). Deviations from theHardy–Weinberg equilibrium were tested byArlequin 2000 and Genepop.Our results indicate that the quality and quan-tity of DNA extracted from fin clips and scalesvaried according to extraction methods (Table 2)and storage conditions. Alcohol preservation at)20 C is fundamental for fin specimens, althoughdried scales conservation at room temperatureallowed good DNA extraction. One percent

Molecular and Phenotypic Evidence of a New Species of Genus Esox (Esocidae, Esociformes, Actinopterygii): The Southern Pike, Esox flaviae

We address the taxonomic position of the southern European individuals of pike, performing a series of tests and comparisons from morphology, DNA taxonomy and population genetics parameters, in order to support the hypothesis that two species of pike, and not only one, exist in Europe. A strong relationship emerged between a northern genotype supported by COI, Cytb, AFLP and specific fragments, and a phenotype with round spot skin colour pattern and a large number of scales in the lateral line, clearly separated from a southern genotype with other skin colour pattern and a low number of scales in the lateral line. DNA taxonomy, based on a coalescent approach (GMYC) from phylogenetic reconstructions on COI and Cytb together with AFLP admixture analysis, supported the existence of two independently evolving entities. Such differences are not simply due to geographic distances, as northern European samples are more similar to Canadian and Chinese samples than the southern Europe ones. Thus, given that the differences between the two groups of European pike are significant at the phenotypic, genotypic and geographical levels, we propose the identification of two pike species: the already known northern pike (Esox lucius) and the southern pike (E. flaviae n.sp.). The correct identification of these two lineages as independent species should give rise to a ban on the introduction of northern pikes in southern Europe for recreational fishing, due to potential problems of hybridisation.

Genetic characterization of a putative indigenous brown trout (Salmo trutta fario) population in a secondary stream of the Nera River Basin (Central Italy) assessed by means of three molecular markers

Genetic diversity was analysed in a putative autochthonous brown trout (Salmo trutta fario L.) population (Monterivoso Stream, Tyrrhenian Apennine Slope) by means of seven microsatellite loci and PCR‐RFLP of two mitochondrial (ND1 and ND5/6) and one nuclear (LDH‐C1*) locus. Monterivoso data were compared to those obtained analysing populations of the same basin (Nera River) and of the Po basin; Irish populations were used as a source of Atlantic strain brown trout. Haplotypes distributions, heterozygosity, F‐statistic and UPGMA analyses indicated a genetic diversification between these populations, suggesting a widespread alteration of the genetic structure due to repeated stocking with allochthonous material, mainly of Atlantic origin, that has partially polluted the Monterivoso population. This population appeared to be constituted of Mediterranean strain samples and might represent a residue of an indigenous pool: it shows high specificity characteristics and it is genetically separate from the others observed and from populations of the Adriatic slope of the Apennines. This population should be employed in managing and breeding programmes finalised to an eco‐sustainable restocking of brown trout in the Nera River Basin, with periodic monitoring by genetic analyses.

Low molecular weight phosphotyrosine protein phosphatase from PC12 cells Purification, some properties and expression during neurogenesis in vitro and in vivo

The purification and partial characterization of low molecular weight phosphotyrosine phosphatase (LMW-PTP) was reported for the first time in PC12 cells. In addition, the expression levels during neuronal phenotype induction by nerve growth factor (NGF) and during neurogenesis in chick embryos were investigated. LMW-PTP was purified to homogeneity and showed a single band of about 18 kDa with sodium dodecyl sulfate polyacrylamide gel electrophoresis. A native molecular mass of 20.1 kDa was determined by gel filtration on Sephadex G-75 column. The LMW-PTP from PC12 cells displays structural and biochemical characteristics similar to the enzyme isolated for normal tissues. It was specifically immunoprecipitated by an affinity purified antibody directed against the bovine liver enzyme. The enzyme is present in the cytosolic and cytoskeletal cell compartment where is tyrosine phosphorylated. Time course expression of LMW-PTP in PC12 cells was investigated after NGF treatment and showed an increase of about 30% in the basal level of LMW-PTP from 0 to 72 h. These changes were related to the appearance in PC12 cells of neuronal processes and to a decrease in cell proliferation. An increase of the LMW-PTP expression was also observed in vivo during chick embryo neurogenesis from 8-day-old embryos to adult chicks. The protein level, assayed by immunoblotting, increases from 14-day-old embryos to the hatched chicks reaching the adult levels within the first week after birth. These data indicate that the neurogenesis process is accompanied by a physiological increment of LMW-PTP expression in vitro and in vivo.

Muscle actin isoforms are differentially expressed in human satellite cells isolated from donors of different ages.

Myogenesis is mainly sustained by a subpopulation of myogenic cells known as satellite cells (SC). In this paper we studied α‐smooth muscle (αSMA) and α‐sarcomeric muscle (αSRA) actin isoform expression in cultures of human satellite cells (HSC) isolated from skeletal muscle biopsies from a 5‐day‐old newborn, a 34‐year‐old young adult and a 71‐year‐old donor. Myogenicity of cultures was assessed using immunocytochemical detection of desmin and myosin heavy chain. Time‐course expression of αSRA and αSMA were studied with both immunocytochemistry and western blotting procedures. Although αSMA was never detected in whole skeletal muscle, both αSMA and αSRA were detected in proliferating and differentiating HSC derived from donors of all examined ages. The expression level experiments showed that αSRA was gradually up‐regulated during HSC differentiation, but no significant differences were observed between newborn, young, and elderly HSC cultures. Our data demonstrated that HSC, isolated from subjects of different ages, re‐expressed αSMA, but its levels and expression pattern varied considerably in the newborn with respect to the young adult and elderly donors. These results are discussed in relation to the myogenic differentiation capability of HSC during human muscle senescence.

Genetic differentiation among populations of Baetis rhodani (Ephemeroptera, Baetidae) in three Italian streams

Abstract Seven microsatellite loci were recently described for the mayfly Baetis rhodani, but, at present, no data are available on the population structure of this species using these loci. We used four of these microsatellite loci for a preliminary analysis of the genetic differentiation of the mayfly B. rhodani in six populations from three Italian streams belonging to the same or different, but geographically distant, drainage basins. From these data, genetic polymorphism was observed between the sampled populations and clear differences were identified between pairs of populations; however, no specific population pattern was observed. The high FST values suggest a limited dispersal of the species across the sampling sites. On the whole, the results obtained with microsatel‐lite loci, characterized by marked heterozygosity deficiency, confirm the hypothesis of the patchy recruitment from a small number of matings and limited in‐stream dispersal, as previously pointed out for some other baetid mayflies by genetic analyses using al‐lozymes and mtDNA. This is the first contribution to a knowledge of the genetic structure of Italian Baetis rhodani populations.

Effects of short‐ and long‐term thermal stress in perch (Perca fluviatilis) determined through fluctuating asymmetry and HSP70 expression

Abstract The effect of short‐ and long‐term exposure to temperature ranges near the optimal developmental values of perch (Perca fluviatilis) during pre‐ and post‐natal development was studied. A fertilized egg raft was divided and placed in four tanks kept at 10°, 17°, 20° C and variable temperature (10° to 20° C), respectively. Short‐time effects of temperature were investigated analyzing fluctuating asymmetry by measuring three meristic and seven morphometric bilateral characters. Perches reared below 17° C or at variable temperature were less asymmetric than those reared at 20° C. Perches reared at 10° C showed a non‐homogeneous trend of the characters analysed. Long‐term effects were investigated by studying the liver and skeletal muscle 70 kDa heat shock proteins (HSP/HSC70) three and six months after hatching. Two HSP isoforms, HSC73 (73 kDa) and HSP72 (72 kDa), were detected in perch liver by immunoblotting. HSC73 was present at the two developmental stages and at all temperature conditions, while HSP72 was detected only at the variable temperature and 20° C. The muscle tissue always showed the HSC73 form. Our data suggest that below 20° C only some morphological characters were affected, whereas the 20° C temperature and, presumably, higher ones, seemed to have both short‐ and long‐term effects.

Short-term cadmium exposure induces stress responses in frog (Pelophylax bergeri) skin organ culture

There have been a few studies on the negative effects of pollutants on amphibian skin, the first structural barrier that interacts with the environment and its potential contaminants. In this study an ex vivo skin organ culture from the amphibian Pelophylax bergeri was used to evaluate cell stress responses induced by short-term exposure to cadmium (Cd), a toxic heavy metal known to be an environmental hazard to both humans and wildlife. Histopathological studies were carried out on skin explants using light microscopy and changes in the expression of stress proteins, such as Metallothionein (MT) and Heat shock proteins (HSPs), were investigated by Real-time RT-PCR. Results revealed that amphibian skin reacts to Cd-induced stress by activating biological responses such as morphological alterations and dose- and time-dependent induction of Mt and Hsp70 mRNA expression, suggesting their potential role as biomarkers of exposure to Cd. This work provides a basis for a better understanding of the tissue-specific responses of amphibian skin as a target organ to Cd exposure and its in vitro use for testing potentially harmful substances present in the environment.

New Evidences of Mitochondrial DNA Heteroplasmy by Putative Paternal Leakage between the Rock Partridge (Alectoris graeca) and the Chukar Partridge (Alectoris chukar)

The rock partridge, Alectoris graeca, is a polytypic species declining in Italy mostly due to anthropogenic causes, including the massive releases of the closely related allochthonous chukar partridge Alectoris chukar which produced the formation of hybrids. Molecular approaches are fundamental for the identification of evolutionary units in the perspective of conservation and management, and to correctly select individuals to be used in restocking campaigns. We analyzed a Cytochrome oxidase I (COI) fragment of contemporary and historical A. graeca and A. chukar samples, using duplicated analyses to confirm results and nuclear DNA microsatellites to exclude possible sample cross-contamination. In two contemporary specimens of A. graeca, collected from an anthropogenic hybrid zone, we found evidence of the presence of mtDNA heteroplasmy possibly associated to paternal leakage and suggesting hybridization with captive-bred exotic A. chukar. These results underline significant limitations in the reliability of mtDNA barcoding-based species identification and could have relevant evolutionary and ecological implications that should be accounted for when interpreting data aimed to support conservation actions.