Where sociality and relatedness diverge: the genetic basis for hierarchical social organization in African elephants.

Hierarchical properties characterize elephant fission-fusion social organization whereby stable groups of individuals coalesce into higher order groups or split in a predictable manner. This hierarchical complexity is rare among animals and, as such, an examination of the factors driving its emergence offers unique insight into the evolution of social behaviour. Investigation of the genetic basis for such social affiliation demonstrates that while the majority of core social groups (second-tier affiliates) are significantly related, this is not exclusively the case. As such, direct benefits received through membership of these groups appear to be salient to their formation and maintenance. Further analysis revealed that the majority of groups in the two higher social echelons (third and fourth tiers) are typically not significantly related. The majority of third-tier members are matrilocal, carrying the same mtDNA control region haplotype, while matrilocality among fourth-tier groups was slightly less than expected at random. Comparison of results to those from a less disturbed population suggests that human depredation, leading to social disruption, altered the genetic underpinning of social relations in the study population. These results suggest that inclusive fitness benefits may crystallize elephant hierarchical social structuring along genetic lines when populations are undisturbed. However, indirect benefits are not critical to the formation and maintenance of second-, third- or fourth-tier level bonds, indicating the importance of direct benefits in the emergence of complex, hierarchical social relations among elephants. Future directions and conservation implications are discussed.

Genetic diversity and phylogenetic analysis of the attachment glycoprotein of phocine distemper viruses of the 2002 and 1988 epizootics.

To investigate the possible origin and spread of the dramatic re-emergent 2002 distemper epizootic observed among seals in Danish Waters, we have sequenced wild-type genes of the attachment (H) glycoproteins of viruses from both the 2002 and 1988 epizootics. Phylogenetic analysis of the H genes of phocine distemper virus (PDV) together with other morbilliviruses, suggests that the re-emergent 2002 PDV is more closely related to a putative recent ancestral PDV than the 1988 PDV isolates. Moreover, upsurges of distemper disease in land-living carnivores linked in time and locality to the 2002 seal epizootic in Danish Waters was investigated and determined to be caused by canine distemper virus, the closest relative of PDV, revealing no direct epidemiological link to the seal epizootics.

Population genetic structure of savannah elephants in Kenya: conservation and management implications.

We investigated population genetic structure and regional differentiation among African savannah elephants in Kenya using mitochondrial and microsatellite markers. We observed mitochondrial DNA (mtDNA) nucleotide diversity of 1.68% and microsatellite variation in terms of average number of alleles, expected and observed heterozygosities in the total study population of 10.20, 0.75, and 0.69, respectively. Hierarchical analysis of molecular variance of mtDNA variation revealed significant differentiation among the 3 geographical regions studied (F(CT) = 0.264; P < 0.05) and a relatively lower differentiation among populations within regions (F(SC) = 0.218; P < 0.0001). Microsatellite variation significantly differentiated among populations within regions (F(SC) = 0.019; P < 0.0001) but not at the regional levels (F(CT) = 0.000; P > 0.500). We attribute the high differentiation at the mitochondrial genome to the matrilineal social structure of elephant populations, female natal philopatry, and probably ancient vicariance. Lack of significant regional differentiation at the nuclear loci vis-a-vis strong differences at mtDNA loci between regions is likely the effect of subsequent homogenization through male-mediated gene flow. Our results depicting 3 broad regional mtDNA groups and the observed population genetic differentiation as well as connectivity patterns should be incorporated in the planning of future management activities such as translocations.

High variation and very low differentiation in wide ranging plains zebra (Equus quagga): insights from mtDNA and microsatellites.

Patterns of genetic differentiation in the plains zebra (Equus quagga) were analysed using mitochondrial DNA control region variation and seven microsatellites. The six morphologically defined subspecies of plains zebra lacked the population genetic structure indicative of distinct evolutionary units. Both marker sets showed high levels of genetic variation and very low levels of differentiation. There was no geographical structuring of mitochondrial DNA haplotypes in the phylogenetic tree, and the plains zebra showed the lowest overall differentiation recorded in any African ungulate studied so far. Arid-adapted African ungulates have shown significant regional genetic structuring in support of the Pleistocene refuge theory. This was not the case in the zebra, and the data are discussed in relation to the impact of Pleistocene climate change on a nonbovid member of the savannah ungulate community. The only other species showing a similar absence of genetic structuring is the African buffalo (Syncerus caffer), but this taxon lacks the high levels of morphological variation present in the plains zebra.

Phylogeography, hybridization and Pleistocene refugia of the kob antelope (Kobus kob).

Mitochondrial DNA control region sequences and seven microsatellites were used to estimate the genetic structuring, evolutionary history and historic migration patterns of the kob antelope (Kobus kob). Ten populations were analysed, representing the three recognized K. kob subspecies: K. k. kob in west Africa, K. k. thomasi in Uganda and K. k. leucotis in Sudan and Ethiopia. Despite being classified as K. k. thomasi and being phenotypically identical to the kob in Queen Elizabeth National Park (NP), the Murchison Falls population in Uganda showed high genetic similarity with the phenotypically distinct K. k. leucotis populations in Sudan and Ethiopia. This was regardless of marker type. Pairwise comparisons and genetic distances between populations grouped Murchison with K. k. leucotis, as did the Bayesian analysis, which failed to find any genetic structuring within the group. We propose that the divergent phenotype and life-history adaptations of K. k. leucotis reflect the isolation of kob populations in refugia in west and east Africa during the Pleistocene. Subsequent dispersal has led to secondary contact and hybridization in northern Uganda between lineages, which was supported by high levels of genetic diversity in Murchison. The reduced variability observed in Queen Elizabeth NP reflects a small founder population from west Africa and in part the decimation of Uganda's wildlife during the country's political turmoil in the 1970s. Due to similarities in phenotype and ecology, and the joint evolutionary history of their mtDNA sequences, the taxonomic status of K. k. kob and K. k. thomasi as separate subspecies is called into question.

High qualitative and quantitative conservation of alternative splicing in Caenorhabditis elegans and Caenorhabditis briggsae.

Alternative splicing (AS) is an important contributor to proteome diversity and is regarded as an explanatory factor for the relatively low number of human genes compared with less complex animals. To assess the evolutionary conservation of AS and its developmental regulation, we have investigated the qualitative and quantitative expression of 21 orthologous alternative splice events through the development of 2 nematode species separated by 85-110 Myr of evolutionary time. We demonstrate that most of these alternative splice events present in Caenorhabditis elegans are conserved in Caenorhabditis briggsae. Moreover, we find that relative isoform expression levels vary significantly during development for 78% of the AS events and that this quantitative variation is highly conserved between the 2 species. Our results suggest that AS is generally tightly regulated through development and that the regulatory mechanisms controlling AS are to a large extent conserved during the evolution of Caenorhabditis. This strong conservation indicates that both major and minor splice forms have important functional roles and that the relative quantities in which they are expressed are crucial. Our results therefore suggest that the quantitative regulation of isoform expression levels is an intrinsic part of most AS events. Moreover, our results indicate that AS contributes little to transcript variation in Caenorhabditis genes and that gene duplication may be the major evolutionary mechanism for the origin of novel transcripts in these 2 species.

Hybridization between subspecies of waterbuck (Kobus ellipsiprymnus) in zones of overlap with limited introgression.

Two subspecies of waterbuck (Kobus ellipsiprymnus), common (Kobus ellipsiprymnus ellipsiprymnus) and defassa (Kobus ellipsiprymnus defassa), are recognized based on differences in rump pattern, coat colour and geographical distribution. These forms are parapatrically distributed with an area of range overlap in East Africa, where phenotypically intermediate populations occur. Variation in 478 bp of the mitochondrial DNA control region and 14 polymorphic microsatellite loci were used to describe the genetic structure and phylogeographical pattern of the species, and to assess if the intermediate populations are the results of hybridization. In total, 186 individuals from 11 localities were analysed. A high degree of genetic differentiation was found between subspecies, although this was most evident from the microsatellite data. Hybridization was suggested in the phenotypically and geographically intermediate Nairobi NP population in Kenya. A neighbour-joining (NJ) tree based on microsatellite population genetic distances grouped Nairobi between the common and defassa populations, and a Bayesian analysis clearly showed introgression. Individuals sampled in Samburu NP, Kenya, had a common waterbuck phenotype, but introgression was suggested by both markers. Although a high degree of maternal defassa input was indicated from the sequence data, the Samburu population grouped with the common waterbuck in the microsatellite population genetic distance tree, with high support. Analyses of linkage disequilibrium and maximum-likelihood estimates of genetic drift suggested that admixture between subspecies is a recent event. The fact that introgression is limited between subspecies could be caused by chromosomal differences, hindering gene flow between common and defassa waterbuck.

Identification of miRNA targets with stable isotope labeling by amino acids in cell culture.

miRNAs are small noncoding RNAs that regulate gene expression. We have used stable isotope labeling by amino acids in cell culture (SILAC) to investigate the effect of miRNA-1 on the HeLa cell proteome. Expression of 12 out of 504 investigated proteins was repressed by miRNA-1 transfection. This repressed set of genes significantly overlaps with miRNA-1 regulated genes that have been identified with DNA array technology and are predicted by computational methods. Moreover, we find that the 3'-untranslated region for the repressed set are enriched in miRNA-1 complementary sites. Our findings demonstrate that SILAC can be used for miRNA target identification and that one highly expressed miRNA can regulate the levels of many different proteins.

A hidden Markov model approach for determining expression from genomic tiling micro arrays.

BACKGROUND: Genomic tiling micro arrays have great potential for identifying previously undiscovered coding as well as non-coding transcription. To-date, however, analyses of these data have been performed in an ad hoc fashion. RESULTS: We present a probabilistic procedure, ExpressHMM, that adaptively models tiling data prior to predicting expression on genomic sequence. A hidden Markov model (HMM) is used to model the distributions of tiling array probe scores in expressed and non-expressed regions. The HMM is trained on sets of probes mapped to regions of annotated expression and non-expression. Subsequently, prediction of transcribed fragments is made on tiled genomic sequence. The prediction is accompanied by an expression probability curve for visual inspection of the supporting evidence. We test ExpressHMM on data from the Cheng et al. (2005) tiling array experiments on ten Human chromosomes. Results can be downloaded and viewed from our web site. CONCLUSION: The value of adaptive modelling of fluorescence scores prior to categorisation into expressed and non-expressed probes is demonstrated. Our results indicate that our adaptive approach is superior to the previous analysis in terms of nucleotide sensitivity and transfrag specificity.

Regional genetic structuring and evolutionary history of the impala Aepyceros melampus.

Samples of 162 impala antelope (Aepyceros melampus) from throughout its distribution range in sub-Saharan Africa were surveyed using eight polymorphic microsatellite loci. Furthermore, 155 previously published mitochondrial DNA (mtDNA) sequences from the same localities were reanalyzed. Two subspecies of impala are presently recognized--the isolated black-faced impala (Aepyceros melampus petersi) in southwest Africa and the common impala (Aepyceros melampus melampus) abundant in southern and east Africa. All tests performed indicated significant genetic differentiation at the subspecific level. Furthermore, individual-based analyses split the common impala subspecies into two distinct genetic groups, conforming with regional geographic affiliation to southern or east Africa. This was supported by assignment tests, genetic distance measures, pairwise theta values, and analysis of molecular variance. We suggest that the presence of such previously unknown regional structuring within the subspecies reflects a pattern of colonization from a formerly large panmictic population in southern Africa toward east Africa. This scenario was supported by a progressive decline in population diversity indices toward east Africa and a significant increase in the quantity theta/(1 - theta). Both microsatellite and mtDNA data indicated a genetic distinctiveness of the Samburu population in Kenya.

Beringian paleoecology inferred from permafrost-preserved fungal DNA.

The diversity of fungi in permanently frozen soil from northeastern Siberia was studied by culture-independent PCR amplification of diverse environmental 18S rRNA genes. Elaborate protocols to avoid contamination during drilling, sampling, and amplification were used. A broad diversity of eukaryotic DNA sequences that were 510 bp long, including sequences of various fungi, plants, and invertebrates, could be obtained reproducibly from samples that were up to 300,000 to 400,000 years old. The sequences revealed that ancient fungal communities included a diversity of cold-adapted yeasts, dark-pigmented fungi, plant-parasitic fungi, and lichen mycobionts. DNA traces of tree-associated macrofungi in a modern tundra sample indicated that there was a shift in fungal diversity following the last ice age and supported recent results showing that there was a severe change in the plant composition in northeastern Siberia during this period. Interestingly, DNA sequences with high homology to sequences of coprophilic and keratinophilic fungi indicated that feces, hair, skin, and nails could have been sources of ancient megafauna DNA recently reported to be present in small amounts of Siberian permafrost sediments.

Direct isolation of poly(A)+ RNA from 4 M guanidine thiocyanate-lysed cell extracts using locked nucleic acid-oligo(T) capture.

LNA oligonucleotides constitute a class of bicyclic RNA analogues having an exceptionally high affinity for their complementary DNA and RNA target molecules. We here report a novel method for highly efficient isolation of intact poly(A)+ RNA using an LNA-substituted oligo(dT) affinity ligand, based on the increased affinity of LNA-T for complementary poly(A) tracts. Poly(A)+ RNA was isolated directly from 4 M guanidine thiocyanate-lysed Caenorhabditis elegans worm extracts as well as from lysed human K562 and vincristine-resistant K562/VCR leukemia cells using LNA_2.T oligonucleotide as an affinity probe, in which every second thymidine was substituted by LNA thymidine. In accordance with the significantly increased stability of the LNA_2.T-A duplexes in 4 M GuSCN, we obtained a 30- to 50-fold mRNA yield increase using the LNA-substituted oligo(T) affinity probe compared with DNA-oligo(dT)-selected mRNA samples. The LNA_2.T affinity probe was, furthermore, highly efficient in isolation of poly(A)+ RNA in a low salt concentration range of 50-100 mM NaCl in poly(A) binding buffer, as validated by selecting the mRNA pools from total RNA samples extracted from different Saccharomyces cerevisiae strains, followed by northern blot analysis. Finally, we demonstrated the utility of the LNA-oligo(T)-selected mRNA in quantitative real-time PCR by analysing the relative expression levels of the human mdr1 multidrug resistance gene in the two K562 cell lines employing pre-validated Taqman assays. Successful use of the NH2-modified LNA_2.T probe in isolation of human mRNA implies that the LNA-oligo(T) method could be automated for streamlined, high throughput expression profiling by real-time PCR by covalently coupling the LNA affinity probe to solid, pre-activated surfaces, such as microtiter plate wells or magnetic particles.

Differences in non-LTR retrotransposons within C. elegans and C. briggsae genomes.

An exhaustive study of the Sam/Frodo family of non-LTR retrotransposons in the Caenorhabditis elegans and Caenorhabditis briggsae genomes demonstrated that C. briggsae contains 60 Sam/Frodo elements including a new subfamily designated Merry, while at least 1000 elements are present in C. elegans. In contrast to C. elegans, C. briggsae does not contain any other non-LTR retrotransposons. The Sam/Frodo/Merry sequences in C. briggsae are shorter and less complete than the Sam/Frodo sequences in C. elegans probably because they all lack a functional first open reading frame (ORF1) and because the genome only encodes one functional reverse transcriptase gene of a non-LTR retrotransposon. Evidence of purifying selection for a functional reverse transcriptase sequence in master/leader elements was found in both nematodes in spite of low copy numbers in C. briggsae. Sam elements in C. elegans are the most abundant Sam/Frodo/Merry family members. They contain the only functional ORF1 copies and, unlike Frodo and Merry members, have a higher GC content than the genomic regions in which they reside. This may indicate a higher transcription rate within this subfamily.

An exceptional case of historical outbreeding in African sable antelope populations.

Empirical investigations of intraspecific outbreeding and subsequent introgressive hybridization in natural populations are rare, particularly among conspecific populations of large mammals. Using mitochondrial DNA data [partial control region (496 basepairs - bp) and cytochrome b gene (343 bp) sequences analysed from 95 individuals representing 17 sampling locations scattered through the African miombo (Brachystegia) woodland ecosystem] and phylogeographical statistical procedures (gene genealogy, nested cladistic and admixture proportion analyses), we (i) give a detailed dissection of the geographical genetic structure of Hippotragus niger; (ii) infer the processes and events potentially involved in the population history; and (iii) trace extensive introgressive hybridization in the species. The present-day sable antelope population shows a tripartite pattern of genetic subdivision representing West Tanzanian, Kenya/East Tanzanian and Southern Africa locations. Nested clade analysis revealed that past allopatric fragmentation, caused probably by habitat discontinuities associated with the East African Rift Valley system, together with intermediary episodic long-distance colonization and restricted, recurrent gene flow have played an predominant role in shaping the extent of maternal genetic diversity (10.4%) and population structure. An extensive (average rate of admixture = 20.0%), but geographically circumscribed and unidirectional hybridization event in the past was inferred, resulting in an extreme (the highest discovered so far in mammals) intraspecific difference of 18.2% among morphologically monotypic sable antelopes from West Tanzania. The results are used to provide an evolutionary framework within which taxonomic implications and conservation decisions can be evaluated.