Dynamic range expansion leads to establishment of a new, genetically distinct wolf population in Central Europe.

Local extinction and recolonization events can shape genetic structure of subdivided animal populations. The gray wolf (Canis lupus) was extirpated from most of Europe, but recently recolonized big part of its historical range. An exceptionally dynamic expansion of wolf population is observed in the western part of the Great European Plain. Nonetheless, genetic consequences of this process have not yet been fully understood. We aimed to assess genetic diversity of this recently established wolf population in Western Poland (WPL), determine its origin and provide novel data regarding the population genetic structure of the grey wolf in Central Europe. We utilized both spatially explicit and non-explicit Bayesian clustering approaches, as well as a model-independent, multivariate method DAPC, to infer genetic structure in large dataset (881 identified individuals) of wolf microsatellite genotypes. To put the patterns observed in studied population into a broader biogeographic context we also analyzed a mtDNA control region fragment widely used in previous studies. In comparison to a source population, we found slightly reduced allelic richness and heterozygosity in the newly recolonized areas west of the Vistula river. We discovered relatively strong west-east structuring in lowland wolves, probably reflecting founder-flush and allele surfing during range expansion, resulting in clear distinction of WPL, eastern lowland and Carpathian genetic groups. Interestingly, wolves from recently recolonized mountainous areas (Sudetes Mts, SW Poland) clustered together with lowland, but not Carpathian wolf populations. We also identified an area in Central Poland that seems to be a melting pot of western, lowland eastern and Carpathian wolves. We conclude that the process of dynamic recolonization of Central European lowlands lead to the formation of a new, genetically distinct wolf population. Together with the settlement and establishment of packs in mountains by lowland wolves and vice versa, it suggests that demographic dynamics and possibly anthropogenic barriers rather than ecological factors (e.g. natal habitat-biased dispersal patterns) shape the current wolf genetic structure in Central Europe.

Deficiencies in Natura 2000 for protecting recovering large carnivores: A spotlight on the wolf Canis lupus in Poland.

If protected areas are to remain relevant in our dynamic world they must be adapted to changes in species ranges. In the EU one of the most notable such changes is the recent recovery of large carnivores, which are protected by Natura 2000 at the national and population levels. However, the Natura 2000 network was designed prior to their recent recovery, which raises the question whether the network is sufficient to protect the contemporary ranges of large carnivores. To investigate this question we evaluated Natura 2000 coverage of the three wolf Canis lupus populations in Poland. Wolf tracking data showed that wolves have recolonised almost all suitable habitat in Poland (as determined by a recent habitat suitability model), so we calculated the overlap between the Natura 2000 network and all wolf habitat in Poland. On the basis of published Natura 2000 criteria, we used 20% as the minimum required coverage. At the national level, wolves are sufficiently protected (22% coverage), but at the population level, the Baltic and Carpathian populations are far better protected (28 and 47%, respectively) than the endangered Central European Lowland population (12%). As Natura 2000 insufficiently protects the most endangered wolf population in Poland, we recommend expansion of Natura 2000 to protect at least an additional 8% of wolf habitat in western Poland, and discuss which specific forests are most in need of additional coverage. Implementation of these actions will have positive conservation implications and help Poland to fulfil its Habitats Directive obligations. As it is likely that similar gaps in Natura 2000 are arising in other EU member states experiencing large carnivore recoveries, particularly in Central Europe, we make the case for a flexible approach to Natura 2000 and suggest that such coverage evaluations may be beneficial elsewhere.

NAD-preferring malic enzyme: localization, regulation and its potential role in herring (Clupea harengus) sperm cells.

Herring spermatozoa exhibit a high activity of NAD-preferring malic enzyme (NAD-ME). This enzyme is involved in the generation of NADH or NADPH in the decarboxylation of malate to form pyruvate and requires some divalent cations to express its activity. In order to confirm that NAD-ME isolated from herring sperm cells is localized in mitochondria, we performed immunofluorescent analysis and assayed spectrophotometrically the malic enzyme reaction. Production of polyclonal rabbit antibodies against NAD-ME from herring spermatozoa enabled identification of mitochondrial localization of this enzyme inside herring spermatozoa. The kinetic studies revealed that NAD-ME was competitively inhibited by ATP up to tenfold. Addition of fumarate reversed ATP-dependent inhibition of NAD-ME to 55 % of its maximum activity. The pH-dependent regulation of malic enzyme activity was also examined. Malic enzyme showed maximum activity at pH near 7.0 in all studied conditions. Finally, the role of malic enzyme activity regulation in mitochondria of herring sperm cells was discussed.

Purification and stability of octameric mitochondrial creatine kinase isoform from herring (Clupea harengus) organ of vision.

Creatine kinases (CKs) constitute a large family of isoenzymes that are involved in intracellular energy homeostasis. In cells with high and fluctuating energy requirements ATP level is maintained via phosphocreatine hydrolysis catalyzed by creatine kinase. In contrast to invertebrates and higher vertebrates, in poikilothermic vertebrates the adaptations for the regulation of energy metabolism by changes in the oligomeric state of CK isoforms are not well known. The present study aimed at identification of herring eye CK isoforms and focuses on factors affecting the CK-octamer stability. In addition to the CK octamer, three different dimeric isoforms of CK were detected by cellulose acetate native electrophoresis. Destabilization of octamer was studied in the presence of TSAC substrates and about 50% of octamers dissociated into dimers within 24h. Moreover, we found that the increase of temperature from 4 °C to 30 °C caused rapid inactivation of dimers in TSAC-treated samples but did not affect octameric structures. In a thermostability assay we demonstrated that octamers retain their activity even at 50 °C. Our results indicate that destabilization of the octameric structure can lead to loss of enzyme activity at higher temperatures (above 30 °C). Furthermore, our results based on N-terminal sequence analysis suggest that probably the mitochondrial s-type CK, rather than u-type, is predominantly expressed in herring eye. In conclusion the existence of four various CK isoforms in one organ may reflect complex regulation of energy metabolism in the phototransduction process in teleost fishes.

Purification and properties of malic enzyme from herring Clupea harengus spermatozoa.

Herring spermatozoa exhibit higher activity of malic enzyme (ME) than Atlantic salmon (Salmo salar), brown trout (Salmo trutta), carp (Cyprinus carpio) and African catfish (Clarias gariepinus) spermatozoa. Two molecular forms of ME are present in herring spermatozoa: an NAD-preferring malic enzyme with very high activity and an NADP-specific malic enzyme with much lower activity (ratio about 33:1). NAD-preferring ME was purified by chromatography on DEAE-Sepharose, Red Agarose and Sephadex G-200 to a specific activity of 36 µmol/min/mg protein and NADP-specific ME on DEAE-Sepharose and 2'5'-ADP Sepharose. The molecular mass for NAD-preferring and NADP-specific ME determined by SDS-PAGE was equal to 61 and 64 kDa, respectively. High activity of ME suggests adaptation of herring spermatozoa to metabolism at high oxygen tension for herring spawn.