Raman temperature and density measurements in supersonic jets.

Prediction of flow-field properties in supersonic jets using computational fluid dynamics (CFD) code predictions has become routine; however, obtaining accurate solutions becomes more challenging when there is a significant temperature difference between the jet core and the ambient air and/or compressibility effects are significant. Benchmark sets of flow field property data are required in order to assess current CFD capabilities and develop better modeling approaches for these turbulent flow fields where accurate calculation of temperatures and turbulent heat flux is important. Particle Image Velocimetry, spontaneous rotational Raman scattering spectroscopy, and Background-Oriented Schlieren (BOS) have been previously used to acquire measurements of the mean and root-mean-square (rms) velocities, the mean and rms gas temperatures, and density gradients in subsonic jet flows and film cooling flows. In this work, the ability to measure density is added to the list of measurands available using the acquired Raman spectra. The suite of measurement techniques are now applied to supersonic jet flows. The computation of the local gas pressure in the potential core of an over-expanded jet is demonstrated using the Raman measured gas temperature and density. Additionally, a unique density feature in temperature matched, perfectly expanded jet flow shear layers identified using BOS was verified using the Raman measurement technique. These non-intrusive flow measurements are compared against RANS predictions of the supersonic jet flow properties as a means of assessing their prediction accuracy.

Loxodonta Localizer: A Software Tool for Inferring the Provenance of African Elephants and Their Ivory Using Mitochondrial DNA.

Illegal hunting is a major threat to the elephants of Africa, with more elephants killed by poachers than die from natural causes. DNA from tusks has been used to infer the source populations for confiscated ivory, relying on nuclear genetic markers. However, mitochondrial DNA (mtDNA) sequences can also provide information on the geographic origins of elephants due to female elephant philopatry. Here, we introduce the Loxodonta Localizer (LL; www.loxodontalocalizer.org), an interactive software tool that uses a database of mtDNA sequences compiled from previously published studies to provide information on the potential provenance of confiscated ivory. A 316 bp control region sequence, which can be readily generated from DNA extracted from ivory, is used as a query. The software generates a listing of haplotypes reported among 1917 African elephants in 24 range countries, sorted in order of similarity to the query sequence. The African locations from which haplotype sequences have been previously reported are shown on a map. We demonstrate examples of haplotypes reported from only a single locality or country, examine the utility of the program in identifying elephants from countries with varying degrees of sampling, and analyze batches of confiscated ivory. The LL allows for the source of confiscated ivory to be assessed within days, using widely available molecular methods that do not depend on a particular platform or laboratory. The program enables identification of potential regions or localities from which elephants are being poached, with capacity for rapid identification of populations newly or consistently targeted by poachers.

Evolutionary and demographic processes shaping geographic patterns of genetic diversity in a keystone species, the African forest elephant (Loxodonta cyclotis).

The past processes that have shaped geographic patterns of genetic diversity may be difficult to infer from current patterns. However, in species with sex differences in dispersal, differing phylogeographic patterns between mitochondrial (mt) and nuclear (nu) DNA may provide contrasting insights into past events. Forest elephants (Loxodonta cyclotis) were impacted by climate and habitat change during the Pleistocene, which likely shaped phylogeographic patterns in mitochondrial (mt) DNA that have persisted due to limited female dispersal. By contrast, the nuclear (nu) DNA phylogeography of forest elephants in Central Africa has not been determined. We therefore examined the population structure of Central African forest elephants by genotyping 94 individuals from six localities at 21 microsatellite loci. Between forest elephants in western and eastern Congolian forests, there was only modest genetic differentiation, a pattern highly discordant with that of mtDNA. Nuclear genetic patterns are consistent with isolation by distance. Alternatively, male-mediated gene flow may have reduced the previous regional differentiation in Central Africa suggested by mtDNA patterns, which likely reflect forest fragmentation during the Pleistocene. In species like elephants, male-mediated gene flow erases the nuclear genetic signatures of past climate and habitat changes, but these continue to persist as patterns in mtDNA because females do not disperse. Conservation implications of these results are discussed.

Genetic connectivity across marginal habitats: the elephants of the Namib Desert.

Locally isolated populations in marginal habitats may be genetically distinctive and of heightened conservation concern. Elephants inhabiting the Namib Desert have been reported to show distinctive behavioral and phenotypic adaptations in that severely arid environment. The genetic distinctiveness of Namibian desert elephants relative to other African savanna elephant (Loxodonta africana) populations has not been established. To investigate the genetic structure of elephants in Namibia, we determined the mitochondrial (mt) DNA control region sequences and genotyped 17 microsatellite loci in desert elephants (n = 8) from the Hoanib River catchment and the Hoarusib River catchment. We compared these to the genotypes of elephants (n = 77) from other localities in Namibia. The mtDNA haplotype sequences and frequencies among desert elephants were similar to those of elephants in Etosha National Park, the Huab River catchment, the Ugab River catchment, and central Kunene, although the geographically distant Caprivi Strip had different mtDNA haplotypes. Likewise, analysis of the microsatellite genotypes of desert-dwelling elephants revealed that they were not genetically distinctive from Etosha elephants, and there was no evidence for isolation by distance across the Etosha region. These results, and a review of the historical record, suggest that a high learning capacity and long-distance migrations allowed Namibian elephants to regularly shift their ranges to survive in the face of high variability in climate and in hunting pressure.

Development and characterization of microsatellite markers in the African forest elephant (Loxodonta cyclotis).

BACKGROUND: African elephants comprise two species, the savanna elephant (Loxodonta africana) and the forest elephant (L. cyclotis), which are distinct morphologically and genetically. Forest elephants are seriously threatened by poaching for meat and ivory, and by habitat destruction. However, microsatellite markers have thus far been developed only in African savanna elephants and Asian elephants, Elephas maximus. The application of microsatellite markers across deeply divergent lineages may produce irregular patterns such as large indels or null alleles. Thus we developed novel microsatellite markers using DNA from two African forest elephants. FINDINGS: One hundred microsatellite loci were identified in next generation shotgun sequences from two African forest elephants, of which 53 were considered suitable for testing. Twenty-three microsatellite markers successfully amplified elephant DNA without amplifying human DNA; these were further characterized in 15 individuals from Lope National Park, Gabon. Three of the markers were monomorphic and four of them carried only two alleles. The remaining sixteen polymorphic loci carried from 3 to 8 alleles, with observed heterozygosity ranging from 0.27 to 0.87, expected heterozygosity from 0.40 to 0.86, and the Shannon diversity index from 0.73 to 1.86. Linkage disequilibrium was not detected between loci, and no locus deviated from Hardy-Weinberg equilibrium. CONCLUSIONS: The markers developed in this study will be useful for genetic analyses of the African forest elephant and contribute to their conservation and management.

Elephant natural history: a genomic perspective.

We review DNA-based studies of elephants and recently extinct proboscideans. The evidence indicates that little or no nuclear gene flow occurs between African savanna elephants (Loxodonta africana) and African forest elephants (Loxodonta cyclotis), establishing that they comprise separate species. In all elephant species, males disperse, whereas females remain with their natal social group, leading to discordance in the phylogeography of nuclear and mitochondrial DNA patterns. Improvements in ancient DNA methods have permitted sequences to be generated from an increasing number of proboscidean fossils and have definitively established that the Asian elephant (Elephas maximus) is the closest living relative of the extinct woolly mammoth (Mammuthus primigenius). DNA-based methods have been developed to determine the geographic provenance of confiscated ivory in an effort to aid the conservation of elephants.

The elephants of Gash-Barka, Eritrea: nuclear and mitochondrial genetic patterns.

Eritrea has one of the northernmost populations of African elephants. Only about 100 elephants persist in the Gash-Barka administrative zone. Elephants in Eritrea have become completely isolated, with no gene flow from other elephant populations. The conservation of Eritrean elephants would benefit from an understanding of their genetic affinities to elephants elsewhere on the continent and the degree to which genetic variation persists in the population. Using dung samples from Eritrean elephants, we examined 18 species-diagnostic single nucleotide polymorphisms in 3 nuclear genes, sequences of mitochondrial HVR1 and ND5, and genotyped 11 microsatellite loci. The sampled Eritrean elephants carried nuclear and mitochondrial DNA markers establishing them as savanna elephants, with closer genetic affinity to Eastern than to North Central savanna elephant populations, and contrary to speculation by some scholars that forest elephants were found in Eritrea. Mitochondrial DNA diversity was relatively low, with 2 haplotypes unique to Eritrea predominating. Microsatellite genotypes could only be determined for a small number of elephants but suggested that the population suffers from low genetic diversity. Conservation efforts should aim to protect Eritrean elephants and their habitat in the short run, with restoration of habitat connectivity and genetic diversity as long-term goals.

Triangulating the provenance of African elephants using mitochondrial DNA.

African elephant mitochondrial (mt) DNA follows a distinctive evolutionary trajectory. As females do not migrate between elephant herds, mtDNA exhibits low geographic dispersal. We therefore examined the effectiveness of mtDNA for assigning the provenance of African elephants (or their ivory). For 653 savanna and forest elephants from 22 localities in 13 countries, 4258 bp of mtDNA was sequenced. We detected eight mtDNA subclades, of which seven had regionally restricted distributions. Among 108 unique haplotypes identified, 72% were found at only one locality and 84% were country specific, while 44% of individuals carried a haplotype detected only at their sampling locality. We combined 316 bp of our control region sequences with those generated by previous trans-national surveys of African elephants. Among 101 unique control region haplotypes detected in African elephants across 81 locations in 22 countries, 62% were present in only a single country. Applying our mtDNA results to a previous microsatellite-based assignment study would improve estimates of the provenance of elephants in 115 of 122 mis-assigned cases. Nuclear partitioning followed species boundaries and not mtDNA subclade boundaries. For taxa such as elephants in which nuclear and mtDNA markers differ in phylogeography, combining the two markers can triangulate the origins of confiscated wildlife products.

Forest elephant mitochondrial genomes reveal that elephantid diversification in Africa tracked climate transitions.

Among elephants, the phylogeographic patterns of mitochondrial (mt) and nuclear markers are often incongruent. One hypothesis attributes this to sex differences in dispersal and in the variance of reproductive success. We tested this hypothesis by examining the coalescent dates of genetic markers within elephantid lineages, predicting that lower dispersal and lower variance in reproductive success among females would have increased mtDNA relative to nuclear coalescent dates. We sequenced the mitochondrial genomes of two forest elephants, aligning them to mitogenomes of African savanna and Asian elephants, and of woolly mammoths, including the most divergent mitogenomes within each lineage. Using fossil calibrations, the divergence between African elephant F and S clade mitochondrial genomes (originating in forest and savanna elephant lineages, respectively) was estimated as 5.5 Ma. We estimated that the (African) ancestor of the mammoth and Asian elephant lineages diverged 6.0 Ma, indicating that four elephantid lineages had differentiated in Africa by the Miocene-Pliocene transition, concurrent with drier climates. The coalescent date for forest elephant mtDNAs was c. 2.4 Ma, suggesting that the decrease in tropical forest cover during the Pleistocene isolated distinct African forest elephant lineages. For all elephantid lineages, the ratio of mtDNA to nuclear coalescent dates was much greater than 0.25. This is consistent with the expectation that sex differences in dispersal and in variance of reproductive success would have increased the effective population size of mtDNA relative to nuclear markers in elephantids, contributing to the persistence of incongruent mtDNA phylogeographic patterns.

Distinguishing forest and savanna African elephants using short nuclear DNA sequences.

A more complete description of African elephant phylogeography would require a method that distinguishes forest and savanna elephants using DNA from low-quality samples. Although mitochondrial DNA is often the marker of choice for species identification, the unusual cytonuclear patterns in African elephants make nuclear markers more reliable. We therefore designed and utilized genetic markers for short nuclear DNA regions that contain fixed nucleotide differences between forest and savanna elephants. We used M13 forward and reverse sequences to increase the total length of PCR amplicons and to improve the quality of sequences for the target DNA. We successfully sequenced fragments of nuclear genes from dung samples of known savanna and forest elephants in the Democratic Republic of Congo, Ethiopia, and Namibia. Elephants at previously unexamined locations were found to have nucleotide character states consistent with their status as savanna or forest elephants. Using these and results from previous studies, we estimated that the short-amplicon nuclear markers could distinguish forest from savanna African elephants with more than 99% accuracy. Nuclear genotyping of museum, dung, or ivory samples will provide better-informed conservation management of Africa's elephants.

Reconciling apparent conflicts between mitochondrial and nuclear phylogenies in African elephants.

Conservation strategies for African elephants would be advanced by resolution of conflicting claims that they comprise one, two, three or four taxonomic groups, and by development of genetic markers that establish more incisively the provenance of confiscated ivory. We addressed these related issues by genotyping 555 elephants from across Africa with microsatellite markers, developing a method to identify those loci most effective at geographic assignment of elephants (or their ivory), and conducting novel analyses of continent-wide datasets of mitochondrial DNA. Results showed that nuclear genetic diversity was partitioned into two clusters, corresponding to African forest elephants (99.5% Cluster-1) and African savanna elephants (99.4% Cluster-2). Hybrid individuals were rare. In a comparison of basal forest "F" and savanna "S" mtDNA clade distributions to nuclear DNA partitions, forest elephant nuclear genotypes occurred only in populations in which S clade mtDNA was absent, suggesting that nuclear partitioning corresponds to the presence or absence of S clade mtDNA. We reanalyzed African elephant mtDNA sequences from 81 locales spanning the continent and discovered that S clade mtDNA was completely absent among elephants at all 30 sampled tropical forest locales. The distribution of savanna nuclear DNA and S clade mtDNA corresponded closely to range boundaries traditionally ascribed to the savanna elephant species based on habitat and morphology. Further, a reanalysis of nuclear genetic assignment results suggested that West African elephants do not comprise a distinct third species. Finally, we show that some DNA markers will be more useful than others for determining the geographic origins of illegal ivory. These findings resolve the apparent incongruence between mtDNA and nuclear genetic patterns that has confounded the taxonomy of African elephants, affirm the limitations of using mtDNA patterns to infer elephant systematics or population structure, and strongly support the existence of two elephant species in Africa.

Genomic DNA sequences from mastodon and woolly mammoth reveal deep speciation of forest and savanna elephants.

To elucidate the history of living and extinct elephantids, we generated 39,763 bp of aligned nuclear DNA sequence across 375 loci for African savanna elephant, African forest elephant, Asian elephant, the extinct American mastodon, and the woolly mammoth. Our data establish that the Asian elephant is the closest living relative of the extinct mammoth in the nuclear genome, extending previous findings from mitochondrial DNA analyses. We also find that savanna and forest elephants, which some have argued are the same species, are as or more divergent in the nuclear genome as mammoths and Asian elephants, which are considered to be distinct genera, thus resolving a long-standing debate about the appropriate taxonomic classification of the African elephants. Finally, we document a much larger effective population size in forest elephants compared with the other elephantid taxa, likely reflecting species differences in ancient geographic structure and range and differences in life history traits such as variance in male reproductive success.

Extensive population genetic structure in the giraffe.

BACKGROUND: A central question in the evolutionary diversification of large, widespread, mobile mammals is how substantial differentiation can arise, particularly in the absence of topographic or habitat barriers to dispersal. All extant giraffes (Giraffa camelopardalis) are currently considered to represent a single species classified into multiple subspecies. However, geographic variation in traits such as pelage pattern is clearly evident across the range in sub-Saharan Africa and abrupt transition zones between different pelage types are typically not associated with extrinsic barriers to gene flow, suggesting reproductive isolation. RESULTS: By analyzing mitochondrial DNA sequences and nuclear microsatellite loci, we show that there are at least six genealogically distinct lineages of giraffe in Africa, with little evidence of interbreeding between them. Some of these lineages appear to be maintained in the absence of contemporary barriers to gene flow, possibly by differences in reproductive timing or pelage-based assortative mating, suggesting that populations usually recognized as subspecies have a long history of reproductive isolation. Further, five of the six putative lineages also contain genetically discrete populations, yielding at least 11 genetically distinct populations. CONCLUSION: Such extreme genetic subdivision within a large vertebrate with high dispersal capabilities is unprecedented and exceeds that of any other large African mammal. Our results have significant implications for giraffe conservation, and imply separate in situ and ex situ management, not only of pelage morphs, but also of local populations.

ALLOZYME DIVERGENCE WITHIN THE BOVIDAE.

We describe a phylogeny of the Bovidae based on 40 allozyme loci in 27 species, representing 10 of the 14 bovid tribes described by Vrba (1985). Giraffe represented a related family (Giraffidae). A phenogram was derived using the unweighted pair-group method with arithmetic means (UPGMA), based on Nei's genetic distances (ND) between species. A tree was also derived using the neighbor-joining technique, also based on ND. To provide a cladistic interpretation, the data were analyzed by a maximum parsimony method (phylogenetic analysis using parsimony, PAUP). We found marked divergence within the Bovidae, consistent with the appearance of the family in the early Miocene. Unexpectedly, the most divergent species was the impala, which occupied a basal position in all trees. Species in the tribe Alcelaphini were the most derived taxa in all trees. These patterns conflict strongly with the previous taxonomic alliance, based on immuno-distance and anatomical evidence, of the impala as a sister group of the Alcelaphini. All trees agreed that tribes described by Vrba (1985) are monophyletic, except the Neotragini, which was polyphyletic, with suni occupying a long branch by itself. The dikdik and klipspringer were consistently placed as sister taxa to species in the Antilopini. Three tribes (Aepycerotini, Tragelaphini and Cephalophini), whose fossils have not been found outside Africa, were basal in all trees, suggesting that bovids originated in Africa. Nodes connecting the remaining tribes were closely clustered, a pattern that agrees with fossil evidence of rapid divergence within the Bovidae in the mid-Miocene (about 15 mybp). The allozyme data suggested a second phase of rapid divergence within tribes during the Plio-Pleistocene, a pattern that also agrees with fossil evidence. Rates of bovid divergence have therefore been far from constant. However, the clustering of nodes imparts considerable uncertainty to the branching order leading to the derived tribes, and to a lesser extent, species within tribes. The classical division of the Bovidae into the Boodontia and Aegeodontia does not agree with the phylogenetic grouping of tribes presented in this analysis. However, the maximum parsimony tree derived using 'local' branch swapping clustered all grazing species into a derived, monophyletic group, suggesting that grazing may have evolved only once in bovid evolution.

Ecological conditions that determine when grazing stimulates grass production.

We report the results of a pot experiment that examined the effects of three ecologically important factors controlling plant growth rates in savanna grasslands: defoliation, soil nitrogen and soil water availability. The experiment was conducted in the Amboseli region in east Africa, and was designed to simulate natural conditions as far as possible, using local soils and a grass species that is heavily grazed by abundant large herbivores. Productivity by different plant components was reduced, stimulated or unchanged by defoliation, depending on specific watering and fertilization treatments. Total above-ground production was stimulated by defoliation and was maximized at moderate clipping intensities, but this was statistically significant only when plants were watered infrequently (every 8 days), and most important, periods between clipping events were extended (at least 24 days). Under these conditions, plant growth rates were limited by water availability at the time of clipping, and soil water conserved in clipped, compared to unclipped plants. Within a given fertilization treatment, whole-plant production was never stimulated by defoliation because root growth was unaffected or inhibited by clipping. However, when fertilization was coupled to defoliation, as they are in the field, whole-plant production by fertilized and moderately clipped plants exceeded production by infertilized, unclipped plants. Under this interpretation, maximum whole-plant production coincided with optimum conditions for herbivores (maximum nitrogen concentration in grass leaves) when watering was frequent, and plants were moderately defoliated. However, these conditions were not the same as those that maximized relative above-ground stimulation of growth (infrequent watering and clipping).The results indicate that above-ground grass production can be stimulated by grazing, and when that is likely to occur. However, the results emphasize that plant production responses to defoliation can vary widely, contigent upon a complex interaction of ecological factors.