Using genetic profiles of African forest elephants to infer population structure, movements, and habitat use in a conservation and development landscape in Gabon.

Conservation of wide-ranging species, such as the African forest elephant (Loxodonta cyclotis), depends on fully protected areas and multiple-use areas (MUA) that provide habitat connectivity. In the Gamba Complex of Protected Areas in Gabon, which includes 2 national parks separated by a MUA containing energy and forestry concessions, we studied forest elephants to evaluate the importance of the MUA to wide-ranging species. We extracted DNA from elephant dung samples and used genetic information to identify over 500 individuals in the MUA and the parks. We then examined patterns of nuclear microsatellites and mitochondrial control-region sequences to infer population structure, movement patterns, and habitat use by age and sex. Population structure was weak but significant, and differentiation was more pronounced during the wet season. Within the MUA, males were more strongly associated with open habitats, such as wetlands and savannas, than females during the dry season. Many of the movements detected within and between seasons involved the wetlands and bordering lagoons. Our results suggest that the MUA provides year-round habitat for some elephants and additional habitat for others whose primary range is in the parks. With the continuing loss of roadless wilderness areas in Central Africa, well-managed MUAs will likely be important to the conservation of wide-ranging species.

Conservation outside protected areas and the effect of human-dominated landscapes on stress hormones in Savannah elephants.

Biodiversity conservation strategies are increasingly focused on regions outside national protected areas, where animals face numerous anthropogenic threats and must coexist with human settlements, livestock, and agriculture. The effects of these potential threats are not always clear, but they could have profound implications for population viability. We used savannah elephants (Loxodonta africana) as a case study to assess the physiological stress associated with living in a human-livestock-dominated landscape. We collected samples over two 3-month periods in 2007 and 2008. We used fecal DNA to identify 96 individual elephants in a community conservation area (CCA) and measured fecal glucocorticoid metabolite (FGM) concentrations as a proxy for stress. The CCA is community Maasai land managed for livestock and wildlife. We compared the FGM concentrations from the CCA to FGM concentrations of 40 elephants in Amboseli National Park and 32 elephants in the Maasai Mara National Reserve, where human settlements and intense livestock grazing were absent. In the CCA, we found no significant individual differences in FGM concentrations among the elephants in 2007 (p = 0.312) or 2008 (p = 0.412) and no difference between years (p = 0.616). The elephants in the CCA had similar FGM concentrations to the Maasai Mara population, but Amboseli elephants had significantly lower FGM concentrations than those in either Maasai Mara or the CCA (Tukey pairwise test, p < 0.001), due primarily to females excreting significantly lower FGM relative to males (p = 0.025). In the CCA, there was no relation among female group size, average pairwise group relatedness, and average group FGM concentration. We found no clear evidence of chronic stress in elephants living on CCA communal land, which is encouraging for conservation strategies promoting the protection of animals living outside protected areas.

High levels of polymorphism found through cross-amplification of microsatellite loci in a Ctenomys pearsoni (Rodentia, Ctenomyidae) population.

Ctenomys pearsoni (Pearson's tuco-tuco) is a subterranean rodent native to Uruguay. We tested the amplification pattern of 12 microsatellite loci, designed for C. sociabilis and C. haigi in a C. pearsoni population. DNA extractions were made from hair samples, and PCR amplification products were run on an ABI 3100 microcapillary gel. Eight loci were selected to form a highly polymorphic panel that could be used to efficiently screen populations of this species. In DNA from 35 tuco-tucos, the mean polymorphic information content value was 0.6536 and the mean expected heterozygosity was 0.7166. Paternity non-exclusion probabilities for seven independent loci were NE-1P = 0.0766 and NE-2P = 0.0108, and combined non-exclusion P(ID) was 6.2 x 10(-7). This panel of microsatellite loci has sufficient power to make inferences regarding group structure, mating strategies and evolutionary relationships among populations.

Cross-amplification tests of ungulate primers in the endangered Neotropical pampas deer (Ozotoceros bezoarticus).

In cross-species amplification tests of 15 ungulate primers in pampas deer, five were retained to form a small panel of highly polymorphic loci that could be used to efficiently screen populations of this endangered species. The polymerase chain reactions were performed incorporating the universal fluorescent labeled M13 (-21) primer. In 69 pampas deer, average allelic diversity was 15, expected heterozygosity was 0.869 and the mean polymorphic information content value was 0.847. Paternity exclusion probabilities over loci were NE-1P = 0.01336 and NE-2P = 0.00135, and combined non-exclusion probability of identity was P(ID) = 3 x 10(-8).

Cross-amplification tests of ungulate primers in the endangered Neotropical pampas deer (Ozotoceros bezoarticus)

In cross-species amplification tests of 15 ungulate primers in pampas deer, five were retained to form a small panel of highly polymorphic loci that could be used to efficiently screen populations of this endangered species. The polymerase chain reactions were performed incorporating the universal fluorescent labeled M13 (-21) primer. In 69 pampas deer, average allelic diversity was 15, expected heterozygosity was 0.869 and the mean polymorphic information content value was 0.847. Paternity exclusion probabilities over loci were NE-1P = 0.01336 and NE-2P = 0.00135, and combined non-exclusion probability of identity was P(ID) = 3 × 10-8.

Assessing reliability of microsatellite genotypes from kit fox faecal samples using genetic and GIS analyses.

Noninvasive faecal DNA sampling has the potential to provide a wealth of information necessary for monitoring and managing endangered species while eliminating the need to capture, handle or observe rare individuals. However, scoring problems, and subsequent genotyping errors, associated with this monitoring method remain a great concern as they can lead to misidentification of individuals and biased estimates. We examined a kit fox scat data set (353 scats; 80 genotypes) for genotyping errors using both genetic and GIS analyses, and evaluated the feasibility of combining both approaches to assess reliability of the faecal DNA results. We further checked the appropriateness of using faecal genotypes to study kit fox populations by describing information about foxes that we could deduce from the 'acceptable' scat genotypes, and comparing it to information gathered with traditional field techniques. Overall, genetic tests indicated that our data set had a low rate of genotyping error. Furthermore, examination of distributions of scat locations confirmed our data set was relatively error free. We found that analysing information on sex primer consistency and scat locations provided a useful assessment of scat genotype error, and greatly limited the amount of additional laboratory work that was needed to identify potentially 'false' scores. 'Acceptable' scat genotypes revealed information on sex ratio, relatedness, fox movement patterns, latrine use, and size of home range. Results from genetic and field data were consistent, supporting the conclusion that our data set had a very low rate of genotyping error and that this noninvasive method is a reliable approach for monitoring kit foxes.

Tripartite genetic subdivisions in the ornate shrew (Sorex ornatus).

We examined cytochrome b sequence variation in 251 ornate shrews (Sorex ornatus) from 20 localities distributed throughout their geographical range. Additionally, vagrant (S. vagrans) and montane (S. monticolus) shrews from four localities were used as outgroups. We found 24 haplotypes in ornate shrews from California (USA) and Baja California (Mexico) that differed by 1-31 substitutions in 392 bp of mitochondrial DNA (mtDNA) sequence. In a subset of individuals, we sequenced 699 bp of cytochrome b to better resolve the phylogeographic relationships of populations. The ornate shrew is phylogeographically structured into three haplotype clades representing southern, central and northern localities. Analysis of allozyme variation reveals a similar pattern of variation. Several other small California vertebrates have a similar tripartite pattern of genetic subdivision. We suggest that topographic barriers and expansion and contraction of wetland habitats in the central valley during Pleistocene glacial cycles account for these patterns of genetic variation. Remarkably, the northern ornate shrew clade is phylogenetically clustered with another species of shrew suggesting that it may be a unique lowland form of the vagrant shrew that evolved in parallel to their southern California counterparts.

Phylogenetic relationships, evolution, and genetic diversity of the domestic dog.

The spectacular diversity in size, conformation, and pelage that characterizes the domestic dog reflects not only the intensity of artificial selection but ultimately the genetic variability of founding populations. Here we review past molecular genetic data that are relevant to understanding the origin and phylogenetic relationships of the dog. DNA-DNA hybridization data show that the dog family Canidae diverged about 50 million years ago from other carnivore families. In contrast, the extant canids are very closely related and diverged from a common ancestor about 10 million years ago. The evidence supporting a close relationship of dogs with gray wolves is overwhelming. However, dogs are remarkably diverse in mitochondrial and nuclear genes. Mitochondrial DNA analysis suggests a more ancient origin of dogs than has been indicated by the fossil record. In addition, dogs have originated from or interbred with wolves throughout their history at different times and different places. We test the possibility of an independent domestication event in North America by analysis of mtDNA variation in the Xoloitzcuintli. This unusual breed is believed to have been kept isolated for thousands of years and may be one of the most ancient breeds in North America. Our results do not support a New World domestication of dogs nor a close association of the Xoloitzcuintli with other hair-less breeds of dogs. Despite their phenotypic uniformity, the Xoloitzcuintli has a surprisingly high level of mtDNA sequence variation. Other breeds are also genetically diverse, suggesting that dog breeds were often founded with a large number of dogs from outbred populations.

Conservation genetics of the endangered Pampas deer (Ozotoceros bezoarticus).

The Pampas deer (Ozotoceros bezoarticus L. 1758) is the most endangered neotropical cervid, and in the past occupied a wide range of open habitats including grassland, pampas, savanna, and cerrado (Brazil) from 5 degrees to 41 degrees S. To better understand the effect of habitat fragmentation on gene flow and genetic variation, and to uncover genetic units for conservation, we examined DNA sequences from the mitochondrial control region of 54 individuals from six localities distributed throughout the present geographical range of the Pampas deer. Our results suggest that the control region of the Pampas deer is one of the most polymorphic of any mammal. This remarkably high variability probably reflects large historic population sizes of millions of individuals in contrast to numbers of fewer than 80,000 today. Gene flow between populations is generally close to one migrant per generation and, with the exception of two populations from Argentina, all populations are significantly differentiated. The degree of gene flow was correlated with geographical distance between populations, a result consistent with limited dispersal being the primary determinant of genetic differentiation between populations. The molecular genetic results provide a mandate for habitat restoration and reintroduction of Pampas deer so that levels of genetic variation can be preserved and historic patterns of abundance can be reconstructed. However, the source of individuals for reintroduction generally should be from populations geographically closest to those now in danger of extinction.

Multiple and ancient origins of the domestic dog.

Mitochondrial DNA control region sequences were analyzed from 162 wolves at 27 localities worldwide and from 140 domestic dogs representing 67 breeds. Sequences from both dogs and wolves showed considerable diversity and supported the hypothesis that wolves were the ancestors of dogs. Most dog sequences belonged to a divergent monophyletic clade sharing no sequences with wolves. The sequence divergence within this clade suggested that dogs originated more than 100,000 years before the present. Associations of dog haplotypes with other wolf lineages indicated episodes of admixture between wolves and dogs. Repeated genetic exchange between dog and wolf populations may have been an important source of variation for artificial selection.

Endomyocardiopathy with eosinophilia.

Five patients were seen at the Mayo Clinic over an 8-year period with the following complex of clinical and morphologic features; striking eosinophilia, cardiomyopathy, hepatosplenomegaly, and either a rapidly fatal or a prolonged, debilitating illness. In recent years, controversy has raged over the precise designation of this syndrome, with proposals ranging from eosinophilic leukemia to hypereosinophilic syndromes. To focus on the major target organ of the disease, we have favored the term endomyocardiopathy with eosinophilia. Experience with these five patients showed that (1) eosinophilia can persist for many years before symptoms appear; (2) progressive restrictive cardiac disease was the major cause of death and debility; (3) osmiophilic cytoplasmic inclusions are present in eosinophils of these patients and also in cells from other patients with marked eosinophilia; and (4) echocardiography may prove to be a useful noninvasive tool to diagnose and follow the progress of cardiac involvement. Although none of these patients was thought to have leukemia, intensive therapy with steroids or cytotoxic agents, or both, is considered necessary to control the progression of the disease.

Ultrastructural cytochemistry of platelets and megakaryocytes in the carcinoid syndrome.

Platelets and megakaryocytes from 11 patients with the carcinoid syndrome have been studied by transmission electron microscopy. Cells fixed in phosphate-buffered glutaraldehyde are oval to discoid, with pseudopods, a dilated open-channel system, and a prominent dense tubular system as defined by peroxidase activity and alkaline bismuth stain. Atypical with hexagonal lattices and treaded substructures and large (diameter greater than 0.5 mum), phosphatase-positive, debris-containing vacuoles are four times more numerous than in normal platelets. Incubation of platelets in a 0.05% suspension of latex results in particle incorporation into phagosomes and the debris-containing vacuoles. Molybdate-dichromate stain reveals two classes of dense bodies, one of which (with a reticular core) is 20 times more numerous than in normal platelets. Bone marrow megakaryocytes lack both dense bodies and debris vacuoles analogous to those found in circulating platelets. These results suggest autophagy or endocytosis abnormalities and provide evidence for multiple types of dense bodies in carcinoid platelets.

Platelet granulopathy: a new morphologic feature in preleukemia and myelomonocytic leukemia: light microscopy and ultrastructural morphology and cytochemistry.

We have previously reported on the ultrastructure of platelets in preleukemia and myelomonocytic leukemia. We referred to an unusual and distinct anomaly of the platelet granules found in 15 of 16 patients. In the present communication we wish to describe and illustrate the light microscopic appearance of giant anomalous granules. Close scrutiny of the platelet morphology and a search for the aforementioned platelet granulopathy are important in the evaluation of patients with myeloproliferative diseases. In this paper we describe and illustrate in more detail the ultrastructure and ultrastructural histochemistry of the abnormal granules. In those patients with the platelet granulopathy, we have conducted in vitro platelet aggregation studies and carried out an electron microscopic evaluation of the aggregates. At least some of the giant granules remained morphologically intact in advanced stages of the aggregation phenomenon, and thus they are probably composed of elements that were not released during aggregation.

Gel-filtered human platelets. Ultrastructure, function, and role of proteins in inhibition of aggregation by aspirin.

Gel filtration of human platelet-rich plasma (PRP) on columns of Sepharose 2B removed at least 99.85% of the plasma proteins from platelets when a column 10 cm in height was used and a plasma volume 11 to 14% of the gel-bed volume was applied. ADP and ATP levels in gel-filtered platelets (GFP) were not significantly different from those in PRP. By transmission electron microscopy, GFP were indistinguishable from PRP. Gel filtration appears to be a highly satisfactory technique of separating platelets from plasma without modifying structure, function, or contents significantly. The roles of several crude protein fractions in platelet aggregation and aspirin's inhibition of aggregation were examined. Fraction I (mostly fibrinogen) enhanced collagen-induced aggregation of gel-filtered platelets; Fraction V (mostly albumin) was inhibitory. Fraction II (mostly gamma-globulin) or gelatin had no significant effect. Aspirin added to gel-filtered platelets inhibited aggregation by 80%. The addition of mixtures of plasma proteins containing albumin increased albumin's inhibitory effect. Incubation of gel-filtered platelets with aspirin labeled in the carboxyl position resulted in no uptake of the label. In contrast, incubation with acetyl-labeled aspirin was followed by uptake of more than 2 X 10(6) acetyl groups per platelet in 1 minute. Incubation for 30 minutes resulted in a five- to sixfold further increase in uptake of the label. Aspirin can acetylate platelets and inhibit aggregation directly. Plasma proteins, in particular albumin or a contaminant of the albumin fraction tested, enhance the inhibitory effect of aspirin on platelet aggregation.

Ultrastructure of platelet aggregation in refractory anemia and myelomonocytic leukemia. II. Individual platelet abnormalities: thrombasthenia-like platelets, surface defects, and dissociation phenomena.

Studies of in vitro platelet aggregation were done in five patients with refractory anemia and two with acute myelomonocytic leukemia. The macroscopic results as well as the general ultrastructural findings were reviewed in a companion paper. Electron microscopic analysis of changes in the individual platelets within aggregates revealed a striking heterogeneity, both in the degree of response of each platelet are in the ultrastructural characteristics of the platelet population. Many of the unaggregated platelets had reacted individually, resembling the platelets of patients with Glanzmann's thrombasthenia. There were other abnormalities suggesting the presence of surface defects, such as the presence of areas of obliteration of the interplatelet space (so-called tight connections). One of the most striking findings was a peculiar dissociation between the different components of the aggregation sequence.

Ultrastructure of platelet aggregation in refractory anemia and myelomonocytic leukemia. I. Ultrastructure of aggregation in normal controls and general defects in refractory anemia and myelomonocytic leukemia.

In vitro aggregation of the platelets from four patients with refractory anemia and two patients with acute myelomonocyctic leukemia revealed distinctive abnormalities. In five patients, there was deficient or minimal aggregation with adenosine diphosphate (ADP), epinephrine, or collagen and only one wave of aggregation could be elicited with ADP at any concentration. Ultrastructural studies revealed numerous isolated platelets, small aggregates with few platelet pseudopods, and the presence of a characteristic type of aggregate with heterogeneous platelet composition combining features of both the primary and the secondary waves of aggregation. These "mixed aggregates" were particularly abundant in the four patients who had refractory anemia and may constitute the structural basis of the single wave of aggregation observed.

Phagocytosis in human platelets: localization of acid phosphatase-positive phagosomes following latex uptake.

Incubation of human platelets in plasma containing a suspension of latex particles for 1-90 min resulted in progressive accumulation of particles in the open-channel system, followed by localization of latex in electron-opaque vacuoles. After 60 min, acid phosphatase was localized within latex-containing vacuoles. The periodate-alkaline-bismuth reaction intensely stained external membranes and membranes of the open-channel system. Membranes of latex-containing organelles were not stained. Latex phagocytosis was independent of both anticoagulant choice and aspirin effects. Our results indicate that the platelet can act as a true phagocyte, and we suggest that the phagocytic process is chronologically similar to that reported for polymorphonuclear leukocytes.