Spawning habitat enhancement in the European bullhead (Cottus gobio), an endangered freshwater fish in degraded lowland rivers

Due to river regulation, the natural habitat of the European bullhead (Cottus gobio) has been degraded and often there is an apparent lack of suitable spawning substrates (hard objects like stones). Addition of artificial structures to degraded habitats may be a very promising tool for habitat enhancement. In this study, we evaluated the use of ceramic tiles as artificial spawning substrates in canalised and (remaining) meandering parts of anthropogenically perturbated lowland rivers in Flanders (northern part of Belgium). Furthermore, we examined whether water depth and velocity were important determinants for the choice of tiles in these different river trajectories. Tiles were successfully used by the bullhead as spawning substrates. In the meandering parts of the river, the number of egg deposits was significantly positively correlated with water depth, while in canalised river parts, water depth and velocity were of no importance for tile usage. In general, supplementation of rivers with artificial substrates like tiles may be a promising, inexpensive and easy-to-use enhancement technique for degraded bullhead spawning habitat.     

Population structure of North American beluga whales (Delphinapterus leucas) based on nuclear DNA microsatellite variation and contrasted with the population structure revealed by mitochondrial DNA variation

Beluga whales ( Delphinapterus leucas ) in North American waters migrate seasonally between wintering areas in broken pack ice and summering locations in estuaries and other open water areas in the Arctic and sub-Arctic. Results from our previous investigation of beluga whale mitochondrial DNA (mtDNA) revealed genetic heterogeneity among beluga from different summering locations that was interpreted as representing a high degree of summering site philopatry. However, mtDNA is maternally inherited and does not reflect mating that may occur among beluga from different summering locations in wintering areas or during annual migrations. To test the possibility that breeding occurs among beluga from different summering locations, genetic variability at five nuclear DNA (nDNA) microsatellite loci was examined in the same animals tested in the mtDNA study. Beluga samples ( n = 640) were collected between 1984 and 1994 from 24 sites across North America, mostly during the summer. Whales from the various sites were categorized into eight summering locations as identified by mtDNA analysis, as well as four hypothesized wintering areas: Bering Sea, Hudson Strait (Hudson Strait, Labrador Sea, southwest Davis Strait), Baffin Bay (North Water, east Davis Strait), and St Lawrence River. Microsatellite allele frequencies indicated genetic homogeneity among animals from summering sites believed to winter together but differentiation among whales from some of the wintering areas. In particular, beluga from western North America (Bering Sea) were clearly distinguished from beluga from eastern North America (Hudson Strait, Baffin Bay, and St Lawrence River). Based upon the combined data set, the population of North American beluga whales was divided into two evolutionarily significant units. However, the population may be further subdivided into management units to reflect distinct groups of beluga at summering locations.     

Population structure of North American beluga whales (Delphinapterus leucas) based on nuclear DNA microsatellite variation and contrasted with the population structure revealed by mitochondrial DNA variation

Beluga whales ( Delphinapterus leucas ) in North American waters migrate seasonally between wintering areas in broken pack ice and summering locations in estuaries and other open water areas in the Arctic and sub-Arctic. Results from our previous investigation of beluga whale mitochondrial DNA (mtDNA) revealed genetic heterogeneity among beluga from different summering locations that was interpreted as representing a high degree of summering site philopatry. However, mtDNA is maternally inherited and does not reflect mating that may occur among beluga from different summering locations in wintering areas or during annual migrations. To test the possibility that breeding occurs among beluga from different summering locations, genetic variability at five nuclear DNA (nDNA) microsatellite loci was examined in the same animals tested in the mtDNA study. Beluga samples ( n = 640) were collected between 1984 and 1994 from 24 sites across North America, mostly during the summer. Whales from the various sites were categorized into eight summering locations as identified by mtDNA analysis, as well as four hypothesized wintering areas: Bering Sea, Hudson Strait (Hudson Strait, Labrador Sea, southwest Davis Strait), Baffin Bay (North Water, east Davis Strait), and St Lawrence River. Microsatellite allele frequencies indicated genetic homogeneity among animals from summering sites believed to winter together but differentiation among whales from some of the wintering areas. In particular, beluga from western North America (Bering Sea) were clearly distinguished from beluga from eastern North America (Hudson Strait, Baffin Bay, and St Lawrence River). Based upon the combined data set, the population of North American beluga whales was divided into two evolutionarily significant units. However, the population may be further subdivided into management units to reflect distinct groups of beluga at summering locations.     

Taxonomic diversity, origin, and conservation status of Bermuda killifishes (Fundulus) based on mitochondrial cytochrome b phylogenies

Sequence variation in the mitochondrialCytochrome b (Cytb) gene was assayed infour of six extant Bermuda killifishpopulations, representing two endemic species,to test taxonomic and phylogenetic hypothesesand reconstruct colonization history. Twodivergent (4.6%) haplotypes were detected; oneis identical to the Georgia 2c haplotype ofF. heteroclitus and is fixed in threeeastern populations: Lover's Lake (F.relictus), Mangrove Lake (F. bermudae),and Walsingham Pond. The second is fixed andrestricted to a western population, Evan'sPond. Likelihood and parsimony cytochrome btrees recognize a Bermuda Fundulus / F. heteroclitus clade in which the Evan's Pondhaplotype is basal. Phylogenies and haplotypedivergence indicate at least two Bermudacolonizations, the more recent involvingtransfer of the Georgia 2c haplotype. Enforcing Bermuda killifish monophyly, aspredicted from a single colonization event,does not increase tree length significantly;i.e., the trees also are consistent with asingle colonization. However, divergencebetween the Evan's Pond haplotype and the F. bermudae / F. relictus / Georgia 2chaplotype (4.6%) far exceeds the maximumdivergence among all F. heteroclitus,F. bermudae, and F. heteroclitushaplotypes (1.2%) and argues for independentcolonizations. Alternatively, recentintroduction of F. heteroclitus couldaccount for occurrence of the GA2c haplotype inBermuda but does not explain the presence ofthe genetically divergent Evans' Pondhaplotype. Cytb sequences areuninformative of the taxonomic status ofBermuda endemics, F. bermudae and F.relictus, but support recognition of theEvan's Pond population as an evolutionarilysignificant unit within the F. heteroclitus group.     

Microsatellite variation in Bavarian populations of European grayling (Thymallus thymallus): Implications for conservation

European grayling populations in Bavaria have shown steady declines during the last 10–20years. In order to provide guidelines for conservation strategies and future management programs, we investigated the genetic structure of 15 grayling populations originating from three major Central European drainages (the Danube, the Elbe and the Rhine/Main) using 20 microsatellite loci. Genetic divergence between the three drainage systems was substantial as illustrated by highly significant heterogeneity of genotype frequencies, high number of drainage-specific private alleles, high between-drainage F _ST values, high assignment success of individuals to their drainage of origin and the high bootstrap support for the genetic distance based drainage-specific population clusters. In agreement with earlier studies, microsatellites revealed relatively low levels of intrapopulational genetic diversity in comparison to the overall level of variation across populations. Maximum likelihood methods using the coalescent approach revealed that the proportion of common ancestors was generally high in native populations and that the estimates of N _e were correlated with the genetic diversity parameters in all drainages. The number of effective immigrants per generation (N _e m) was less than one for all pairwise comparisons of populations within the drainages, indicating restricted interpopulational gene flow. Based on these findings we recommend a drainage and sub-drainage specific conservation of grayling populations in order to preserve their overall genetic diversity and integrity. For large-scale stocking actions to supplement declining or to restore extinct populations, creation of separate broodstocks for major conservation units (ESUs and MUs) is warranted. This revised version was published online in July 2006 with corrections to the Cover Date.     

Phylogeographic differentiation in the mitochondrial control region in the koala, Phascolarctos cinereus (Goldfuss 1817)

The koala, Phascolarctos cinereus, is a geographically widespread species endemic to Australia, with three currently recognized subspecies: P.c. adustus, P.c. cinereus, and P.c. victor. Intraspecific variation in the mitochondrial DNA (mtDNA) control region was examined in over 200 animals from 16 representative populations throughout the species’ range. Eighteen different haplotypes were defined in the ≈ 860 bp mtDNA control region, as determined by heteroduplex analysis/temperature gradient gel electrophoresis (HDA/TGGE). Any single population typically possessed only one or two haplotypes yielding an average within-population haplotypic diversity of 0.180 ± 0.003, and nucleotide diversity of 0.16%. Overall, mtDNA control region sequence diversity between populations averaged 0.67%, and ranged from 0% to 1.56%. Nucleotide divergence between populations averaged 0.51%, and ranged from 0% to 1.53%. Neighbour-joining methods revealed limited phylogenetic distinction between geographically distant populations of koalas, and tentative support for a single evolutionarily significant unit (ESU). This is consistent with previous suggestions that the morphological differences formalized by subspecific taxonomy may be interpreted as clinal variation. Significant differentiation in mtDNA-haplotype frequencies between localities suggested that little gene flow currently exists among populations. When combined with microsatellite analysis, which has revealed substantial differentiation among koala populations, we conclude that the appropriate short-term management unit (MU) for koalas is the local population.     

Phylogenetic origin of <Emphasis Type="Italic">Salmo trutta</Emphasis> L 1758 from Sicily,based on mitochondrial and nuclear DNA analyses

The phylogenetic status of brown trout Salmo trutta L 1758 in Sicily is uncertain as some reports describe these trout as S. macrostigma or S. cettii on one hand while other, contradictory reports imply a hatchery origin on the other. In order to clarify this situation, we performed sequence analysis of the mtDNA control region and restriction fragment analysis of the nuclear lactate dehydrogenase (LDH-C1*) gene. A single mitochondrial haplotype (At-s6) found previously in brown trout in Morocco, and two alleles at LDH-C1* (the ancestral*100, at a high frequency, and *90) were revealed. Our results suggest that Sicilian brown trout are native and that they probably colonized Sicily from west to east in an expansion, from the Atlantic Ocean basin, along the North-West African coast. Handling editor: C. Sturmbauer     

DNA sequence variation of the mitochondrial control region among geographically and morphologically remote European brown trout Saltno trutta populations

Throughout its natural range, the brown trout Salmo trutta L. exhibits a complex pattern of morphological and life-history variation. This has led to considerable taxonomic confusion, hampering the understanding of the evolutionary history of the species. To document the phylogenetic relationships among morphologically and geographically remote brown trout populations across western Europe, we determined the DNA sequence variation in segments of the mitochondrial control region for 151 individuals representing 24 populations. DNA was prepared for double-stranded sequencing by the polymerase chain reaction (PCR). Twenty-one variable nucleotide positions within a 640-bp fragment surveyed defined 12 genotypes differing by a mean of 7 nucleotide substitutions (range 1-12). Five major phylogenetic assemblages differing by mean sequence divergence estimates of 0.96 to 1.44% were identified. These groupings exhibited a strong spatial partitioning but lacked congruence with either ecological or morphological differentiation. Complete mitochondrial DNA (mtDNA) monomorphism across all Atlantic basin populations contrasted with the high interdrainage genetic diversity observed in more southerly populations. This study exemplified the usefulness of mitochondrial DNA sequence analysis for estimating phylogenetic relationships within S. trutta populations.     

Population genetic structure of North Atlantic, Mediterranean Sea and Sea of Cortez fin whales, Balaenoptera physalus (Linnaeus 1758): analysis of mitochondrial and nuclear loci

Samples were collected from 407 fin whales, Balaenoptera physalus , at four North Atlantic and one Mediterranean Sea summer feeding area as well as the Sea of Cortez in the Pacific Ocean. For each sample, the sex, the sequence of the first 288 nucleotides of the mitochondrial (mt) control region and the genotype at six microsatellite loci were determined. A significant degree of divergence was detected at all nuclear and mt loci between North Atlantic/Mediterranean Sea and the Sea of Cortez. However, the divergence time estimated from the mt sequences was substantially lower than the time elapsed since the rise of the Panama Isthmus, suggesting occasional gene flow between the North Pacific and North Atlantic ocean after the separation of the two oceans. Within the North Atlantic and Mediterranean Sea, significant levels of heterogeneity were observed in the mtDNA between the Mediterranean Sea, the eastern (Spain) and the western (the Gulf of Maine and the Gulf of St Lawrence) North Atlantic. Samples collected off West Greenland and Iceland could not be unequivocally assigned to either of the two areas. The homogeneity tests performed using the nuclear data revealed significant levels of divergence only between the Mediterranean Sea and the Gulf of St Lawrence or West Greenland. In conclusion, our results suggest the existence of several recently diverged populations in the North Atlantic and Mediterranean Sea, possibly with some limited gene flow between adjacent populations, a population structure which is consistent with earlier population models proposed by Kellogg, Ingebrigtsen, and Sergeant.     

Intraspecific variation in nuclear DNA content among world populations of a mosquito,Aedes albopictus (Skuse)

Summary Aedes albopictus is commonly distributed in most parts of the Oriental region and on many islands in the Indian and the Pacific Oceans. The species was recently introduced into the United States and Brazil. Feulgen cytophotometric quantitation of haploid nuclear DNA content was carried out for 37 populations of Ae. albopictus to determine the extent of intraspecific variation in nuclear DNA content and whether the range expansion of the species has coincided with an increase in DNA content. The haploid nuclear DNA content varied nearly three-fold. The minimum DNA content was 0.62 pg in Koh Samui from Thailand, and the maximum DNA content was 1.66 pg in Houston-61 from the United States. Statistical comparisons of populations revealed significant differences in DNA contents. No geographic clustering of populations was noted with respect to DNA content. In general, populations from the United States and Brazil had higher DNA contents, but there was no indication that the range expansion had occurred hand in hand with an increase in DNA content. Each population had a specific amount of DNA that is probably imposed by the microenvironment.     

Genetic diversity of trout (genus Salmo) from its most eastern native range based on mitochondrial DNA and nuclear gene variation

Russia and western Asia harbour trout populations that have been classified as distinct species and subspecies, most often on the basis of morphological and ecological variation. In order to assess their origins and to verify whether traditional taxonomy reflects their evolutionary distinctiveness, we documented their genetic relationships on the basis of mitochondrial DNA (mtDNA) RFLP, mtDNA sequence analysis, and allozyme variation. Both mtDNA and nuclear gene variation defined two ancient phylogenetic assemblages of populations distributed among northern (Baltic, White, Barents), and southern (Black, Caspian, Aral) sea basins, between which gene flow has been possible but limited in postglacial times. These results supported the traditional taxonomic differentiation between populations of these two regions. They provided weak support for the taxonomic distinction of southern brown trout (Salmo trutta) populations based on their basin of origin. They also refuted the hypothesis that L. Sevan trout (Salmo ischchan) diverged from a primitive brown trout ancestor. Nevertheless, all trout populations from southern sea basins possessed private alleles or mtDNA genotypes and were genetically distinct Therefore, they represent unique gene pools that warrant individual recognition for conservation and management.     

A survey of equid mitochondrial DNA: Implications for the evolution, genetic diversity and conservation of Equus

The evolution, taxonomy and conservation of the genus Equuswere investigated by examining the mitochondrial DNA sequences of thecontrol region and 12S rRNA gene. The phylogenetic analysis of thesesequences provides further evidence that the deepest node in thephylogeny of the extant species is a divergence between twolineages; one leading to the ancestor of modern horses (E.ferus, domestic and przewalskii) and the other to thezebra and ass ancestor, with the later speciation events of the zebrasand asses occurring either as one or more rapid radiations, or withextensive secondary contact after speciation. Examination of the geneticdiversity within species suggested that two of the E. hemionussubspecies (E. h. onager and E. h. kulan) onlyrecently diverged, and perhaps, are insufficiently different to beclassified as separate subspecies. The genetic divergence betweendomestic and wild forms of E. ferus (horse) and E.africanus (African ass) was no greater than expected within anequid species. In E. burchelli (plains zebra) there was anindication of mtDNA divergence between populations increasing withdistance. The implications of these results for equid conservation arediscussed and recommendations are made for conservation action.     

Molecular,quantitative and abiotic variables for the delineation of evolutionary significant units: case of sandalwood (<Emphasis Type="Italic">Santalum austrocaledonicum</Emphasis> Vieillard) in New Caledonia

Various approaches have been developed to define conservation units for plant and animal species. In this study we combined nuclear microsatellites (from a previous published study) and chloroplast microsatellites (assessed in the present study), leaf and seed morphology traits and abiotic variables (climate and soil) to define evolutionary significant units (ESU) of Santalum austrocaledonicum, a tree species growing in New Caledonia. Results for chloroplast microsatellites showed that the total population heterozygosity was␣high, (H _cp = 0.84) but varied between islands. Differentiation was strong in the total population (F _stcp = 0.66) but also within the main island Grande Terre (F _stcp = 0.73) and within Iles Loyauté (F _stcp = 0.52), highlighting a limited gene flow between populations. These results confirmed those obtained with nuclear microsatellites. The cluster analysis on molecular markers discriminated two main groups constituted by the populations of Grande Terre and the populations of Iles Loyauté. A principal component analysis of leaf and seed morphology traits singled out the populations of Iles Loyauté and the western populations of Grande Terre. Quantitative genetic analyses showed that the variation between populations was under genetic control (broad sense heritability close to 80%). A high correlation between rainfall and morphological traits suggested an impact of climate on this variation. The integration of these results allows to define two ESUs, one corresponding to Grande Terre and Ile des Pins and the other the Iles Loyauté archipelago. This study stresses the need to restore some populations of Grande Terre that are currently threatened by their small size.     

Evolutionary history of freshwater sculpins, genus Cottus (Teleostei; Cottidae) and related taxa, as inferred from mitochondrial DNA phylogeny

The freshwater sculpins, genus Cottus (Teleostei; Cottidae), comprise bottom-dwelling fishes that exhibit various life-history styles, having radiated throughout Northern Hemisphere freshwater habitats. The phylogenetic relationships among Cottus and related taxa were estimated from mitochondrial DNA 12S rRNA and control region (CR) sequences, the freshwater sculpins examined falling into five lineages (A-E). Lineage A consisted of Trachidermus fasciatus and C. kazika, both having a catadromous life-history. The remaining species (lineages B-E) spawn in freshwater habitats regardless of life-history (amphidromous, lacustrine or fluvial), suggesting that the various life-history types post-dated a common ancestor of lineages B-E. Molecular clock estimates suggested a Pliocene-Pleistocene radiation (or Miocene-Pliocene from the alternative clock) of lineages B-E. In eastern Eurasia, speciation with life-history changes to amphidromous or fluvial styles has apparently occurred independently in some lineages, as a general pattern. Mitochondrial DNA CR phylogeny showed the monophyletic Baikalian cottoids (Cottoidei) to be nested within Cottus and Trachidermus, suggesting that the former ecologically and morphologically divergent cottoids may have originated from a single lineage which invaded the ancient lake.     

Microsatellite variation within and among recently fragmented populations of the golden lion tamarin (Leontopithecus rosalia)

Four variable microsatellite loci were used toexamine the genetic diversity and differentiation of golden lion tamarins (Leontopithecus rosalia) in four populations recently isolated by habitat fragmentation. Using Rst estimates of genetic differentiation, a considerable genetic divergence was detected among these populations, with an averagedifferentiation of 31%. Significant differences in allele number among these populations were found. However, the heterozygosity among these populations was not statistically different. These results suggestthat loss in allele diversity was faster than loss in heterozygosity. Conservation implications, particularly for golden lion tamarins, are then discussed. Loss of allelic diversity might be as serious a concern to endangered species as heterozygosity or inbreeding.     

The origin of Indian Star tortoises (Geochelone elegans) based on nuclear and mitochondrial DNA analysis: A story of rescue and repatriation

The Indian Star tortoise (Geochelone elegans) belongs to the family Testunidae and is distributed in southwest India and Sri Lanka. In addition to facing loss of its natural habitat, the species is also illegally traded as food and as an exotic pet internationally. Here we report DNA-based analyses for identification and repatriation of these tortoises into their natural habitat. We have attempted to establish the geographical origin of these tortoises rescued from smugglers, by comparing the microsatellite and mitochondrial markers of rescued animals with animals of known provenance. Star tortoises exhibited strong genetic structure in India. The populations from western India were genetically distinct at microsatellite and mitochondrial loci from southern populations. The rescued individuals had similar multilocus genotypes and mitochondrial DNA haplotypes as the reference individuals from south India. However, the precise geographic origin of many of the rescued samples remains unresolved, because we could not assign them to southern populations and the Neighbor-Joining cluster analysis indicated that some of rescued tortoises formed distinct clusters. These data strongly suggest that the rescued group of tortoises is composed of a mix of individuals from differentiated source populations that are probably located in southern India and possibly Sri Lanka. Our study provides valuable information based on molecular markers for the assessment of genetic diversity in Indian Star tortoises.     

Phylogeographic study of brown trout from Serbia,based on mitochondrial DNA control region analysis

In order to illuminate the phylogeography of brown trout (Salmo trutta) populations in the Balkan state of Serbia, the 561 bp 5''-end of mtDNA control region of 101 individuals originating from upland tributaries of the Danubian, Aegean and Adriatic drainages were sequenced and compared to corresponding brown trout sequences obtained in previous studies. Among 15 haplotypes found, 14 were considered native, representing the Danubian and Adriatic lineages of the brown trout, while one haplotype (ATcs1), found only in two individuals originating from two stocked rivers, corresponded to the Atlantic lineage and was considered introduced. Native haplotypes exhibited a strong geographic pattern of distribution: the Danubian haplotypes were strictly confined to the Danubian drainage, while the Adriatic haplotypes dominated in the Aegean and Adriatic drainages; most of the total molecular variance (69%) was attributed to differences among the drainages. Phylogenetic reconstruction, supplemented with seven haplotypes newly described in this study, suggested a sister position of the Atlantic-Danubian and Adriatic-Mediterranean-marmoratus ("southern") phylogenetic group, and pointed to the existence of a distinct clade, detected within the "southern" group. The data obtained confirmed our expectation of the existence of high genetic diversity in Balkan trout populations, and we recommend more widespread surveys covering trout stocks from the region.