Phylogeography of the brown hare (Lepus europaeus) in Europe: a legacy of south‐eastern Mediterranean refugia?

Aim We analysed the population genetics of the brown hare (Lepus europaeus) in order to test the hypothesis that this species migrated into central Europe from a number of late glacial refugia, including some in Asia Minor. Location Thirty‐three localities in Greece, Bulgaria, Italy, Croatia, Serbia, Poland, Switzerland, Austria, France, Germany, the Netherlands, Spain, the United Kingdom, Turkey and Israel. Methods In total, 926 brown hares were analysed for mitochondrial DNA (mtDNA) variation by restriction fragment length polymorphism (RFLP) performed on polymerase chain reaction‐amplified products spanning cytochrome b (cyt b)/control region (CR), cytochrome oxidase I (COI) and 12S–16S rRNA. In addition, sequence analysis of the mtDNA CR‐I region was performed on 69 individuals, and the data were compared with 137 mtDNA CR‐I sequences retrieved from GenBank. Results The 112 haplotypes detected were partitioned into five phylogeographically well‐defined major haplogroups, namely the ‘south‐eastern European type haplogroup’ (SEEh), ‘Anatolian/Middle Eastern type haplogroup’ (AMh), ‘European type haplogroup, subgroup A’ (EUh‐A), ‘European type haplogroup, subgroup B’ (EUh‐B) and ‘Intermediate haplogroup’ (INTERh). Sequence data retrieved from GenBank were consistent with the haplogroups determined in this study. In Bulgaria and north‐eastern Greece numerous haplotypes of all five haplogroups were present, forming a large overlap zone. Main conclusions The mtDNA results allow us to infer post‐glacial colonization of large parts of Europe from a late glacial/early Holocene source population in the central or south‐central Balkans. The presence of Anatolian/Middle Eastern haplotypes in the large overlap zone in Bulgaria and north‐eastern Greece reveals gene flow from Anatolia to Europe across the late Pleistocene Bosporus land‐bridge. Although various restocking operations could be partly responsible for the presence of unexpected haplotypes in certain areas, we nevertheless trace a strong phylogeographic signal throughout all regions under study. Throughout Europe, mtDNA results indicate that brown hares are not separated into discernable phyletic groups.     

Sorbitol and sorbitol phosphate in brown seaweeds

Small amounts of D-sorbitol were extracted from 15 species of brown seaweeds representing four orders, independent of seasonal effects. Sorbitol phosphate was also isolated and identified.     

麦红吸浆虫幼虫不同滞育时期体内山梨醇含量及山梨醇脱氢酶基因表达水平变化

【目的】本研究旨在明确麦红吸浆虫Sitodiplosis mosellana(Géhin)滞育进程中山梨醇含量及山梨醇脱氢酶基因(SmSDH)mRNA表达水平的变化规律,探讨麦红吸浆虫滞育与山梨醇含量及SmSDH表达水平的关系。【方法】分别采用比色法和实时荧光定量PCR方法,对2013年5月-2014年3月不同时间陕西杨凌养虫圃内麦红吸浆虫滞育前后及滞育期幼虫体内山梨醇含量及SmSDH2a和SmSDH2b mRNA表达水平进行分析。【结果】麦红吸浆虫幼虫滞育前山梨醇含量最高(为9.41μg/mg);滞育期间,12月份含量最高(为8.50μg/mg);滞育解除后山梨醇含量为3.25~3.49μg/mg,显著低于滞育前和整个滞育期(P0.05)。山梨醇脱氢酶基因SmSDH2a及SmSDH2b在滞育进程中mRNA表达水平变化趋势相同,且与滞育强度递减趋势一致,即进入滞育后表达水平显著降低(P0.05)并一直稳定在较低水平,滞育解除后逐渐升高并达到最高。滞育期间,同期不同滞育状态幼虫之间山梨醇含量和SmSDH2a表达水平差异不明显,但裸露幼虫SmSDH2b表达水平始终高于结茧幼虫。【结论】山梨醇和SmSDH参与麦红吸浆虫滞育调控,与滞育维持和解除密切相关。     

Gluconobacter oxydans WD膜结合山梨醇脱氢酶基因在E.coli中的克隆表达

本研究构建了四株含有氧化葡萄糖酸杆菌山梨醇脱氢酶基因的重组大肠杆菌,并初步探究SldB和SldA亚基在山梨醇脱氢酶转化甘油反应中的作用。将pET28a、pETduet与PCR扩增的目的基因连接,构建单启动子调控重组质粒pET28a-sldB、pET28a-sldA、pET28a-sldBA和双启动子调控重组质粒pETduet-sldB'-sldA'。只有含pET28a-sldBA和pETduet-sldB'-sldA'的重组菌具有转化甘油的活性,表明G.oxydans WD的山梨醇脱氢酶催化甘油脱氢需要SldB和SldA亚基的共同作用。串联基因sldBA的蛋白表达结果与双启动子控制sldB和sldA基因蛋白表达结果基本相同,表明位于sldB基因末端的sldA的RBS序列可被E.coli C43的核糖体识别。     

Rutin and meloxicam attenuate paw inflammation in mice: Affecting sorbitol dehydrogenase activity

Rutin, naturally occurring flavonoid, has reported to cover interesting multiple pharmacological properties. This study evaluated rutin or/and meloxicam effects in paw inflammation induced by formalin in mice. Mice were divided into four groups: I‐Formalin group, II‐Rutin 60 mg/kg (p.o.), III‐Meloxicam 10 mg/kg (p.o.), plus IV‐Combined rutin and meloxicam. Therapies were administered once a day for 7 days. The curative effects were assessed on inflammatory, oxidative stress, and apoptosis. Both rutin and/or meloxicam induced marked improvement in paw licking time on the 1st day and by combined treatment only on the 3rd day as well reduction in paw edema% on the 3rd day. Moreover, noticeable progress in liver malondialdehyde content, superoxide dismutase, and sorbitol dehydrogenase activities as well decline in paw interleukin‐1β level and extent of apoptosis. The results spot light on the good influence of combined rutin and meloxicam in formalin‐induced mice paw inflammation to a better extent than either rutin or meloxicam lonely.     

Biochemical genetic variability in brown hares (Lepus europaeus) from Greece

Allozyme variability of 91 brown hares (Lepus europaeus) from seven regions in Greece was compared to existing data of Bulgarian populations to test the hypothesis of the occurrence of specific alleles in Greece, likely stemming from an isolated Late Pleistocene refugial population in the southern Balkans. This hypothesis is particularly suggested by some subfossil Late Pleistocene hare remains in Greece and the reported high mtDNA diversity in Greek hares. Allozymic diversity could be higher in Greek hares than in hares from neighboring regions as a result of the accumulation of variants in a long-lasting Pleistocene refugium. Conversely, Greek hares could exhibit reduced genetic diversity because of long-lasting low effective population sizes during the Late Glacial Maximum and a lower chance of postglacial gene flow from other populations into this rather marginal part in the southern Balkans. Horizontal starch gel electrophoresis of proteins from 35~loci revealed three alleles (Es-1 ^–162, Pep-2 ¹¹⁴, Mpi ⁸⁸) at low frequencies, which were not found in Bulgarian or any other brown hare population. In contrast, some alleles from the populations from Bulgaria and other regions of Europe were absent in the Greek samples. Population genetic statistics indicated only a slight tendency of increased gene pool diversity in Greek hares, little substructuring in Greek and Bulgarian populations, respectively, as well as an only slightly lower level of gene flow between the two neighboring regions, as compared to the gene flow within each region. The results conform to the hypothesis of a Late Pleistocene refugial population in the southern Balkans, with some few specific nuclear gene pool characteristics, but little effect on the overall genetic differentiation between Greek and Bulgarian hares.     

Major histocompatibility complex variation at class II DQA locus in the brown hare (Lepus europaeus)

The major histocompatability complex (MHC) is a multigene family of receptors that bind and present antigenic peptides to T-cells. Genes of the MHC are characterized by an outstanding genetic polymorphism, which is considered to be maintained by positive selection. Sites involved in peptide binding form binding pockets (P) that are collectively termed the peptide-binding region (PBR). In this study, we examined the level of MHC genetic diversity within and among natural populations of brown hare ( Lepus europaeus ) from Europe and Anatolia choosing for analysis of the second exon of the DQA locus, one of the most polymorphic class II loci. We aimed at an integrated population genetic analysis of L. europeaus by (i) correlating MHC polymorphism to genetic variability and phylogenetic status estimated previously from maternally (mtDNA) and biparentally (allozymes, microsatellites) inherited loci; and (ii) comparing full-length exon amino acid polymorphism with functional polymorphism in the PBR and the binding pockets P1, P6 and P9. A substantial level of DQA exon 2 polymorphism was detected with two completely different set of alleles between the Anatolian and European populations. However, the phylogeny of full-length exon 2 Leeu-DQA alleles did not show a strong phylogeographic signal. The presence of balancing selection was supported by a statistically significant excess of nonsynonymous substitutions over synonymous in the PBR and a trans-species pattern of evolution detected after phylogenetic reconstruction. The differentiating patterns detected between genetic and functional polymorphism, i.e. the number and the distribution of pocket variants within and among populations, indicated a hierarchical action of selection pressures.     

Alcohol dehydrogenase polymorphism in Apis mellifera

A polymorphic system of ADH isozymes is described in the honeybee Apis mellifera. Three and six different electrophoretic patterns were found, respectively, in drone and worker pupae analysis. The data indicate that the ADH isozymes are controlled by three alleles, Adh-1 ¹, Adh-1², and Adh-1 ³. The frequency of the Adh-1 alleles is different in two analyzed subspecies, Apis mellifera adansonii (African bees) and Apis mellifera ligustica (Italian bees). In the African bees, the frequencies are 0.256 and 0.697 for Adh-1 ¹ and Adh-1², respectively. In the Italian bees, these values are shown to be 0.902 and 0.098, respectively. The allele Adh-1 ³ was not detected in the Italian bee population. The effect of NAD on the resolution of this system was investigated, and only one region of ADH activity was obtained in drone pupae analysis when NAD was used in the gels. However, two different regions of activity were observed in the same samples, in the absence of the coenzyme. ADH activity was not detected in young larvae, but it increased to a maximum in prepupal and white-eyed pupal phases. It then declined progressively to total absence in the emerging bees.This work was supported by the Brazilian Research Council (CNPq) and S~ao Paulo State Research Foundation (FAPESP). This study was performed by the senior author in partial fulfillment of the requirements for a master's degree and was directed by Dr. M. A. Mestriner, Genetics Department, University of São Paulo at Ribeirão Preto.     

Genetic population structure of Norwegian brown trout

Biochemical genetic variation in populations of anadromous and resident brown trout, Salmo trutta L., was studied. Altogether 50 Norwegian populations were screened for 32 enzyme loci. Genetic polymorphism was found at the following 11 loci: AAT-4 * (E.C. 2.6.1.1), CK-1 * (E.C. 2.7.3.2), G3PDH-2 * (E.C. 1.1.1.8), IDHP-2 * (E.C. 1.1.1.42), LDH-5 * (E.C. 1.1.1.27), MDH-2 * (E.C. 1.1.1.37), MDH-3/4 * (E.C. 1.1.1.37), MEP-2 * (E.C. 1.1.1.40), GPI-2 * (E.C. 5.3.1.9). GPI-5 * (E.C. 5.3.1.9) and PGM-1 * (E.C. 5.4.2.2), giving an overall polymorphism of 34%, ranging from 3.7 to 29.6% among individual populations. The average calculated heterozygosity ranged from 1.4 to 10.2% among populations. Genetic heterogeneity was observed among anadromous populations, and significant differences in allelic frequencies were found between anadromous populations in neighbouring watercourses, among resident populations and between anadromous and resident populations inhabiting the same watercourses. Significant heterogeneity was also found among 12 populations from Lake Mjøsa, with a major division between the western and eastern populations of the lake. Differences in allelic frequencies were found between wild stocks and their hatchery derivatives, and between different hatchery derivatives originating from the same wild population. In some cases release of hatchery populations into wild stocks may have influenced the genetic characteristics of wild stocks. The data support the hypothesis of eastern as well as western postglacial colonization lines for Norwegian brown trout.     

Partial nucleotide sequences, and routine typing by polymerase chain reaction-restriction fragment length polymorphism, of the brown trout (Salmo trutta) lactate dehydrogenase, LDH-C1*90 and *100 alleles

The cDNA nucleotide sequences of the lactate dehydrogenase alleles LDH-C1*90 and *100 of brown trout (Salmo trutta) were found to differ at position 308 where an A is present in the *100 allele but a G is present in the *90 allele. This base substitution results in an amino acid change from aspartic acid at position 82 in the LDH-C1 100 allozyme to a glycine in the 90 allozyme. Since aspartic acid has a net negative charge whilst glycine is uncharged, this is consistent with the electrophoretic observation that the LDH-C1 100 allozyme has a more anodal mobility relative to the LDH-C1 90 allozyme. Based on alignment of the cDNA sequence with the mouse genomic sequence, a local primer set was designed, incorporating the variable position, and was found to give very good amplification with brown trout genomic DNA. Sequencing of this fragment confirmed the difference in both homozygous and heterozygous individuals. Digestion of the polymerase chain reaction products with BslI, a restriction enzyme specific for the site difference, gave one, two and three fragments for the two homozygotes and the heterozygote, respectively, following electrophoretic separation. This provides a DNA-based means of routine screening of the highly informative LDH-C1* polymorphism in brown trout population genetic studies. Primer sets presented could be used to sequence cDNA of other LDH* genes of brown trout and other species.     

Within-stream variation in early life-history traits in brown trout

Significant additive genetic variance for most early life-history traits was found in brown trout Salmo trutta living in both allopatry above an impassable waterfall and sympatry (below the waterfall in the same stream) with alpine bullhead Cottus poecilopus. These traits included length, mass and yolk sac volume at hatching, and size at'button-up' (the time when yolk is enclosed within the body cavity). There were small differences in size at hatching and size at button-up among populations (adjusted for egg size). However, sympatric fry grew more rapidly and experienced lower mortality rates during the period of first feeding than allopatric fry. This might indicate behavioural differences between brown trout from the two populations. It is suggested that these phenotypic differences may be a result of adaptation to living in sympatry with alpine bullhead.     

Betaine aldehyde dehydrogenase polymorphism in spinach: Genetic and biochemical characterization

Spinach (Spinacia oleracea L.) has a major chloroplastic isozyme of betaine aldehyde dehydrogenase (BADH) and a minor cytosolic one. Among a diverse collection of spinach accessions, three electrophoretic banding patterns of chloroplastic BADH were found: two were single banded and one was triple banded. Genetic analysis of these patterns indicated that chloroplastic BADH is encoded by a single, nuclear gene with two alleles, designated slow (S) and fast (F), and that products of these alleles can hybridize to form either homodimers or a heterodimer. The S allele was by far the most common among the accessions examined. Native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the SS and FF homodimers differ in charge but not molecular weight.This research was supported by the U.S. DOE under Contract DE-AC02-76ERO-1338, by USDA-CRGO Grant 87-CRCR-1-2460, and by grants from the State of Michigan REED-Biotechnology Program, from CIBA-GEIGY Corporation, and from the Beet Sugar Development Foundation.     

Genetic structure of coastal and inland populations of the annual halophyte Suaeda maritima (L.) dumort. in Central Europe, inferred from amplified fragment length polymorphism markers

Naturally occurring inland salt habitats are highly threatened due to increasing fragmentation and area reduction, while the surroundings of former potash mining dumps have experienced a massive invasion by halophytes over the last 20 years. We reconstructed colonisation patterns of these purely anthropogenic inland salt sites using molecular markers in the obligate halophyte Suaeda maritima (L.) dumort. (Chenopodiaceae), a typical plant in such areas. In the present study, 120 individual plants from 40 coastal and inland populations in Central Europe were subjected to AFLP analysis with nine primer combinations. A total of 243 AFLP band positions were scored as presence/absence characters. Genetic diversity values were not significantly different in populations from natural and anthropogenic inland salt sites as compared to coastal habitats. Results from principal coordinate analysis, neighbour-joining analysis and analysis of molecular variance ( amova ) all indicated that most of the genetic variation is preserved within populations, while genetic differentiation among populations is comparatively low. We conclude that S. maritima has repeatedly and independently colonised the surroundings of former potash mining dumps in Central Germany. However, the absence of founder effects and the lack of phylogeographic structure prevented us from identifying putative donor populations.     

Protein polymorphism and genic variation in a population of the loach Cobitis delicata

Hemoglobin, non-enzymatic and enzymatic muscle proteins of the loach Cobitis delicata were examined electrophoretically. Eleven proteins were shown to be under control by 20 loci. Only two loci were defined as polymorphic and 14 loci were completely monomorphic. Estimated average heterozygosity over all 20 loci was 0.011 in the loach population.     

Post-glacial colonization of brown trout, Salmo trutta L.: Ldh-5 as a phylogeographic marker locus

The Ldh-5 locus, which codes for the eye-specific lactate dehydrogenase in brown trout, has been shown to be polymorphic for two codominant alleles, Ldh-5 (100) and Ldh-5 (90). The Ldh-5 (100 ) allele is present in 11 other salmonid species and is therefore likely to be the ancestral one, whereas the unique brown trout Ldh-5 (90 ) allele would seem to be the result of a mutation in that lineage. The Ldh-5 (90 ) allele appears to have arisen in north-west Europe during or after the last glaciation, with allelic substitution taking place under the action of natural selection. The Ldh-5 (90 ) allele can be used as a phylogeographic marker to trace the post-glacial spread of the populations possessing it. Examination of the current distribution of the two alleles suggests that, in the formerly glaciated area of north-west Europe, there have been two post-glacial colonizations by brown trout. The first was by an 'ancestral' race fixed for the Ldh-5(100 ) allele. This was later replaced by, or introgressed with, the later-arriving 'modern' race characterized by the Ldh-5 (90 ) allele, except where physical barriers prevented colonization by this latter form. Artificial stocking has resulted in 'genetic contamination' of many populations of the ancestral race and there is an urgent need to conserve the remaining pristine populations, especially in view of the likely genetic propensity for longevity and ultimate large size exhibited by this race.     

Latitudinal variation for two enzyme loci and an inversion polymorphism in Drosophila melanogaster from Central and South America

Abstract.— Many organisms show latitudinal variation for various genetically determined traits. Such clines may involve neutral variation and originate from historical events or their maintenance may be explained by selection. For Drosophila melanogaster , latitudinal variation for allozymes, inversions, and quantitative traits has been found on several continents. We sampled D. melanogaster populations in Panama and along a transect of 40 latitudinal degrees on the west coast of South America. Negative correlations with latitude were found for Adh^s and _αGpdh^F allele frequencies and for the frequency of the cosmopolitan inversion In(2L)t in Adh^sαpdh^F chromosomes. A positive correlation existed between wing length and latitude. Significant correlations were found between these traits and climatic variables like temperature and rainfall. The observed clines show considerable resemblance to those found on other continents. Gametic disequilibrium between Adh^s and αGpdh^F occurred predominantly at higher latitudes and was caused by the presence of In(2L)t . The reasons for the clinal distributions are discussed and it is argued that selection is the most likely explanation. However, the exact nature of the selective force and the interactions of allozymes with each other and with In(2L)t are complex and not fully understood. In tropical regions In(2L)t -containing genotypes have higher fitness than ST/ST and Adh and αGpdh hitchhike with the inversion, but there is also evidence for balancing selection at the Adh locus.     

Isolation and characterization of 19 polymorphic microsatellite DNA markers in the Japanese brown frog (Rana japonica)

We report the isolation and characterization of 19 polymorphic microsatellite DNA markers in the Japanese brown frog (Rana japonica). These markers were tested in 24 individuals each collected from three distinct populations in Ichikai-machi, Tochigi Prefecture. The number of observed alleles per locus ranged from 3 to 24 across all populations, and the values of observed and expected heterozygosities ranged from 0.130 to 1 and from 0.125 to 0.941, respectively. These microsatellite loci will be useful for investigating the intraspecific genetic variation and population structure of this species.     

Genetic variation of some aldehyde-oxidizing enzymes in the mouse

• 1 Twenty-six strains of mice were surveyed by starch gel electrophoresis for genetic variation of four liver enzymes; aldehyde dehydrogenase, aldehyde oxidase, xanthine oxidase and formaldehyde dehydrogenase.
• 2 A variant of aldehyde dehydrogenase was found in strains ICFW, IS/Cam, NZB, NZW, Simpson and Schneider. A variant of aldehyde oxidase was found in CE. A possible variant of xanthine oxidase was found in SF/Cam.
• 3 The gene determining the electrophoretic variant of aldehyde oxidase is either the same as, or very closely linked to, the Aox gene which determines aldehyde oxidase activity.

     

An inherited variant of mouse sn-glycerol-3-phosphate dehydrogenase detected by isoelectric focusing: Genetical and biochemical analyses

An electrophoretically detectable mutant of sn-glycerol-3-phosphate dehydrogenase (GPDH) has been found in the offspring of 1-ethyl-1-nitrosourea-treated mice. The banding alteration was detected by isoelectric focusing (IEF) of crude liver extract on polyacrylamide gels. The GPDH alteration is not organ specific. The mutant protein is more positively charged than the wild type. The mutation is codominantly expressed. Heterozygous and homozygous mutants have distinguishable IEF banding patterns. The specific activity of GPDH is not altered by the mutation. The mutated allele causes a greater heat stability to the GPDH protein. Enzymes extracted from the three genotypes are indistinguishable in terms of their pH optima. Gdc-1 ^e is proposed as the allele symbol for the new mutation.