Mitochondrial cytochrome b indicates the presence of two paraphyletic diverged lineages of the blue sheep Pseudois nayaur across the Indian Himalaya: conservation implications.

BACKGROUND: Populations exhibit signatures of local adaptive traits due to spatial and environmental heterogeneity resulting in microevolution. The blue sheep is widely distributed across the high Asian mountains and are the snow leopard's principal prey species. These mountains differ in their evolutionary history due to differential glaciation and deglaciation periods, orography, and rainfall patterns, and such factors causes diversification in species. METHODS AND RESULTS: Therefore, we assess the phylogeographic status of blue sheep using the mitochondrial cytochrome b gene (220 bp) across the Indian Himalayan region (IHR) and its relationship with other populations. Of the observed five haplotypes, two and three were from the western Himalayas (WH) and eastern Himalayas (EH) respectively. One of the haplotypes from WH was shared with the population of Pamir plateau, suggesting historical maternal connectivity between these areas. The phylogenetic analyses split the blue sheep into two paraphyletic clades, and western and eastern populations of IHR were within the Pamir and Tibetan plateau clades, respectively. We observed a relatively higher mean sequence divergence in the EH population than in the WH. CONCLUSION: We propose five 'Evolutionary Significant Units' across the blue sheep distribution range based on observed variation in the species' ecological requirements, orography, climatic conditions, and maternal lineages, viz.; Western Himalaya-Pamir plateau (WHPP); Eastern Himalaya-Tibetan plateau (EHTP); Qilian mountains; Helan mountains and Hengduan mountains population. Despite the small sample size, population divergence was observed across the IHR, therefore, we suggest a transboundary, collaborative study on comparative morphology, anatomy, ecology, behaviour, and population genetics using harmonized different genetic markers for identifying the overall taxonomic status of the blue sheep across its range for planning effective conservation strategies.

Generation of SNP datasets for orangutan population genomics using improved reduced-representation sequencing and direct comparisons of SNP calling algorithms.

BACKGROUND: High-throughput sequencing has opened up exciting possibilities in population and conservation genetics by enabling the assessment of genetic variation at genome-wide scales. One approach to reduce genome complexity, i.e. investigating only parts of the genome, is reduced-representation library (RRL) sequencing. Like similar approaches, RRL sequencing reduces ascertainment bias due to simultaneous discovery and genotyping of single-nucleotide polymorphisms (SNPs) and does not require reference genomes. Yet, generating such datasets remains challenging due to laboratory and bioinformatical issues. In the laboratory, current protocols require improvements with regards to sequencing homologous fragments to reduce the number of missing genotypes. From the bioinformatical perspective, the reliance of most studies on a single SNP caller disregards the possibility that different algorithms may produce disparate SNP datasets. RESULTS: We present an improved RRL (iRRL) protocol that maximizes the generation of homologous DNA sequences, thus achieving improved genotyping-by-sequencing efficiency. Our modifications facilitate generation of single-sample libraries, enabling individual genotype assignments instead of pooled-sample analysis. We sequenced ~1% of the orangutan genome with 41-fold median coverage in 31 wild-born individuals from two populations. SNPs and genotypes were called using three different algorithms. We obtained substantially different SNP datasets depending on the SNP caller. Genotype validations revealed that the Unified Genotyper of the Genome Analysis Toolkit and SAMtools performed significantly better than a caller from CLC Genomics Workbench (CLC). Of all conflicting genotype calls, CLC was only correct in 17% of the cases. Furthermore, conflicting genotypes between two algorithms showed a systematic bias in that one caller almost exclusively assigned heterozygotes, while the other one almost exclusively assigned homozygotes. CONCLUSIONS: Our enhanced iRRL approach greatly facilitates genotyping-by-sequencing and thus direct estimates of allele frequencies. Our direct comparison of three commonly used SNP callers emphasizes the need to question the accuracy of SNP and genotype calling, as we obtained considerably different SNP datasets depending on caller algorithms, sequencing depths and filtering criteria. These differences affected scans for signatures of natural selection, but will also exert undue influences on demographic inferences. This study presents the first effort to generate a population genomic dataset for wild-born orangutans with known population provenance.

Aluminium induced oxidative stress results in decreased mitochondrial biogenesis via modulation of PGC-1α expression.

The present investigation was carried out to elucidate a possible molecular mechanism related to the effects of aluminium-induced oxidative stress on various mitochondrial respiratory complex subunits with special emphasis on the role of Peroxisome proliferator activated receptor gamma co-activator 1α (PGC-1α) and its downstream targets i.e. Nuclear respiratory factor-1(NRF-1), Nuclear respiratory factor-2(NRF-2) and Mitochondrial transcription factor A (Tfam) in mitochondrial biogenesis. Aluminium lactate (10mg/kgb.wt./day) was administered intragastrically to rats for 12 weeks. After 12 weeks of exposure, we found an increase in ROS levels, mitochondrial DNA oxidation and decrease in citrate synthase activity in the Hippocampus (HC) and Corpus striatum (CS) regions of rat brain. On the other hand, there was a decrease in the mRNA levels of the mitochondrial encoded subunits-NADH dehydrogenase (ND) subunits i.e. ND1, ND2, ND3, Cytochrome b (Cytb), Cytochrome oxidase (COX) subunits i.e. COX1, COX3, ATP synthase (ATPase) subunit 6 along with reduced expression of nuclear encoded subunits COX4, COX5A, COX5B of Electron transport chain (ETC). Besides, a decrease in mitochondrial DNA copy number and mitochondrial content in both regions of rat brain was observed. The PGC-1α was down-regulated in aluminium treated rats along with NRF-1, NRF-2 and Tfam, which act downstream from PGC-1α in aluminium treated rats. Electron microscopy results revealed a significant increase in the mitochondrial swelling, loss of cristae, chromatin condensation and decreases in mitochondrial number in case of aluminium treated rats as compared to control. So, PGC-1α seems to be a potent target for aluminium neurotoxicity, which makes it an almost ideal target to control or limit the damage that has been associated with the defective mitochondrial function seen in neurodegenerative diseases.

MHC class I and MHC class II DRB gene variability in wild and captive Bengal tigers (Panthera tigris tigris).

Bengal tigers are highly endangered and knowledge on adaptive genetic variation can be essential for efficient conservation and management. Here we present the first assessment of allelic variation in major histocompatibility complex (MHC) class I and MHC class II DRB genes for wild and captive tigers from India. We amplified, cloned, and sequenced alpha-1 and alpha-2 domain of MHC class I and beta-1 domain of MHC class II DRB genes in 16 tiger specimens of different geographic origin. We detected high variability in peptide-binding sites, presumably resulting from positive selection. Tigers exhibit a low number of MHC DRB alleles, similar to other endangered big cats. Our initial assessment-admittedly with limited geographic coverage and sample size-did not reveal significant differences between captive and wild tigers with regard to MHC variability. In addition, we successfully amplified MHC DRB alleles from scat samples. Our characterization of tiger MHC alleles forms a basis for further in-depth analyses of MHC variability in this illustrative threatened mammal.

Revisiting the Woolly wolf (Canis lupus chanco) phylogeny in Himalaya: Addressing taxonomy, spatial extent and distribution of an ancient lineage in Asia.

Of the sub-species of Holarctic wolf, the Woolly wolf (Canis lupus chanco) is uniquely adapted to atmospheric hypoxia and widely distributed across the Himalaya, Qinghai Tibetan Plateau (QTP) and Mongolia. Taxonomic ambiguity still exists for this sub-species because of complex evolutionary history anduse of limited wild samples across its range in Himalaya. We document for the first time population genetic structure and taxonomic affinity of the wolves across western and eastern Himalayan regions from samples collected from the wild (n = 19) using mitochondrial control region (225bp). We found two haplotypes in our data, one widely distributed in the Himalaya that was shared with QTP and the other confined to Himachal Pradesh and Uttarakhand in the western Himalaya, India. After combining our data withpublished sequences (n = 83), we observed 15 haplotypes. Some of these were shared among different locations from India to QTP and a few were private to geographic locations. A phylogenetic tree indicated that Woolly wolves from India, Nepal, QTP and Mongolia are basal to other wolves with shallow divergence (K2P; 0.000-0.044) and high bootstrap values. Demographic analyses based on mismatch distribution and Bayesian skyline plots (BSP) suggested a stable population over a long time (~million years) with signs of recent declines. Regional dominance of private haplotypes across its distribution range may indicate allopatric divergence. This may be due to differences in habitat characteristics, availability of different wild prey species and differential deglaciation within the range of the Woolly wolf during historic time. Presence of basal and shallow divergence within-clade along with unique ecological requirements and adaptation to hypoxia, the Woolly wolf of Himalaya, QTP, and Mongolian regions may be considered as a distinct an Evolutionary Significant Unit (ESU). Identifying management units (MUs) is needed within its distribution range using harmonized multiple genetic data for effective conservation planning.

Genetic analyses favour an ancient and natural origin of elephants on Borneo.

The origin of the elephant on the island of Borneo remains elusive. Research has suggested two alternative hypotheses: the Bornean elephant stems either from a recent introduction in the 17th century or from an ancient colonization several hundreds of thousands years ago. Lack of elephant fossils has been interpreted as evidence for a very recent introduction, whereas mtDNA divergence from other Asian elephants has been argued to favor an ancient colonization. We investigated the demographic history of Bornean elephants using full-likelihood and approximate Bayesian computation analyses. Our results are at odds with both the recent and ancient colonization hypotheses, and favour a third intermediate scenario. We find that genetic data favour a scenario in which Bornean elephants experienced a bottleneck during the last glacial period, possibly as a consequence of the colonization of Borneo, and from which it has slowly recovered since. Altogether the data support a natural colonization of Bornean elephants at a time when large terrestrial mammals could colonise from the Sunda shelf when sea levels were much lower. Our results are important not only in understanding the unique history of the colonization of Borneo by elephants, but also for their long-term conservation.

Fine-scale population genetic structure of the Bengal tiger (Panthera tigris tigris) in a human-dominated western Terai Arc Landscape, India.

Despite massive global conservation strategies, tiger populations continued to decline until recently, mainly due to habitat loss, human-animal conflicts, and poaching. These factors are known to affect the genetic characteristics of tiger populations and decrease local effective population sizes. The Terai Arc Landscape (TAL) at the foothills of the Himalaya is one of the 42 source sites of tigers around the globe. Therefore, information on how landscape features and anthropogenic factors affect the fine-scale spatial genetic structure and variation of tigers in TAL is needed to develop proper management strategies for achieving long-term conservation goals. We document, for the first time, the genetic characteristics of this tiger population by genotyping 71 tiger samples using 13 microsatellite markers from the western region of TAL (WTAL) of 1800 km2. Specifically, we aimed to estimate the genetic variability, population structure, and gene flow. The microsatellite markers indicated that the levels of allelic diversity (MNA = 6.6) and genetic variation (Ho = 0.50, HE = 0.64) were slightly lower than those reported previously in other Bengal tiger populations. We observed moderate gene flow and significant genetic differentiation (FST= 0.060) and identified the presence of cryptic genetic structure using Bayesian and non-Bayesian approaches. There was low and significantly asymmetric migration between the two main subpopulations of the Rajaji Tiger Reserve and the Corbett Tiger Reserve in WTAL. Sibship relationships indicated that the functionality of the corridor between these subpopulations may be retained if the quality of the habitat does not deteriorate. However, we found that gene flow is not adequate in view of changing land use matrices. We discuss the need to maintain connectivity by implementing the measures that have been suggested previously to minimize the level of human disturbance, including relocation of villages and industries, prevention of encroachment, and banning sand and boulder mining in the corridors.

Quercetin protects against aluminium induced oxidative stress and promotes mitochondrial biogenesis via activation of the PGC-1α signaling pathway.

The present investigation was carried out to elucidate a possible molecular mechanism related to the protective effect of quercetin administration against aluminium-induced oxidative stress on various mitochondrial respiratory complex subunits with special emphasis on the role of PGC-1α and its downstream targets, i.e. NRF-1, NRF-2 and Tfam in mitochondrial biogenesis. Aluminium lactate (10mg/kg b.wt./day) was administered intragastrically to rats, which were pre-treated with quercetin 6h before aluminium (10mg/kg b.wt./day, intragastrically) for 12 weeks. We found a decrease in ROS levels, mitochondrial DNA oxidation and citrate synthase activity in the hippocampus (HC) and corpus striatum (CS) regions of rat brain treated with quercetin. Besides this an increase in the mRNA levels of the mitochondrial encoded subunits - ND1, ND2, ND3, Cyt b, COX1, COX3 and ATPase6 along with increased expression of nuclear encoded subunits COX4, COX5A and COX5B of electron transport chain (ETC). In quercetin treated group an increase in the mitochondrial DNA copy number and mitochondrial content in both the regions of rat brain was observed. The PGC-1α was up regulated in quercetin treated rats along with NRF-1, NRF-2 and Tfam, which act downstream from PGC-1α. Electron microscopy results revealed a significant decrease in the mitochondrial cross-section area, mitochondrial perimeter length and increase in mitochondrial number in case of quercetin treated rats as compared to aluminium treated ones. Therefore it seems quercetin increases mitochondrial biogenesis and makes it an almost ideal flavanoid to control or limit the damage that has been associated with the defective mitochondrial function seen in many neurodegenerative diseases.

Tigers of Sundarbans in India: is the population a separate conservation unit?

The Sundarbans tiger inhabits a unique mangrove habitat and are morphologically distinct from the recognized tiger subspecies in terms of skull morphometrics and body size. Thus, there is an urgent need to assess their ecological and genetic distinctiveness and determine if Sundarbans tigers should be defined and managed as separate conservation unit. We utilized nine microsatellites and 3 kb from four mitochondrial DNA (mtDNA) genes to estimate genetic variability, population structure, demographic parameters and visualize historic and contemporary connectivity among tiger populations from Sundarbans and mainland India. We also evaluated the traits that determine exchangeability or adaptive differences among tiger populations. Data from both markers suggest that Sundarbans tiger is not a separate tiger subspecies and should be regarded as Bengal tiger (P. t. tigris) subspecies. Maximum likelihood phylogenetic analyses of the mtDNA data revealed reciprocal monophyly. Genetic differentiation was found stronger for mtDNA than nuclear DNA. Microsatellite markers indicated low genetic variation in Sundarbans tigers (He= 0.58) as compared to other mainland populations, such as northern and Peninsular (Hebetween 0.67- 0.70). Molecular data supports migration between mainland and Sundarbans populations until very recent times. We attribute this reduction in gene flow to accelerated fragmentation and habitat alteration in the landscape over the past few centuries. Demographic analyses suggest that Sundarbans tigers have diverged recently from peninsular tiger population within last 2000 years. Sundarbans tigers are the most divergent group of Bengal tigers, and ecologically non-exchangeable with other tiger populations, and thus should be managed as a separate "evolutionarily significant unit" (ESU) following the adaptive evolutionary conservation (AEC) concept.

Effective population size dynamics and the demographic collapse of Bornean orang-utans.

Bornean orang-utans experienced a major demographic decline and local extirpations during the Pleistocene and Holocene due to climate change, the arrival of modern humans, of farmers and recent commercially-driven habitat loss and fragmentation. The recent loss of habitat and its dramatic fragmentation has affected the patterns of genetic variability and differentiation among the remaining populations and increased the extinction risk of the most isolated ones. However, the contribution of recent demographic events to such genetic patterns is still not fully clear. Indeed, it can be difficult to separate the effects of recent anthropogenic fragmentation from the genetic signature of prehistoric demographic events. Here, we investigated the genetic structure and population size dynamics of orang-utans from different sites. Altogether 126 individuals were analyzed and a full-likelihood Bayesian approach was applied. All sites exhibited clear signals of population decline. Population structure is known to generate spurious bottleneck signals and we found that it does indeed contribute to the signals observed. However, population structure alone does not easily explain the observed patterns. The dating of the population decline varied across sites but was always within the 200-2000 years period. This suggests that in some sites at least, orang-utan populations were affected by demographic events that started before the recent anthropogenic effects that occurred in Borneo. These results do not mean that the recent forest exploitation did not leave its genetic mark on orang-utans but suggests that the genetic pool of orang-utans is also impacted by more ancient events. While we cannot identify the main cause for this decline, our results suggests that the decline may be related to the arrival of the first farmers or climatic events, and that more theoretical work is needed to understand how multiple demographic events impact the genome of species and how we can assess their relative contributions.

Two different high throughput sequencing approaches identify thousands of de novo genomic markers for the genetically depleted Bornean elephant.

High throughput sequencing technologies are being applied to an increasing number of model species with a high-quality reference genome. The application and analyses of whole-genome sequence data in non-model species with no prior genomic information are currently under way. Recent sequencing technologies provide new opportunities for gathering genomic data in natural populations, laying the empirical foundation for future research in the field of conservation and population genomics. Here we present the case study of the Bornean elephant, which is the most endangered subspecies of Asian elephant and exhibits very low genetic diversity. We used two different sequencing platforms, the Roche 454 FLX (shotgun) and Illumina, GAIIx (Restriction site associated DNA, RAD) to evaluate the feasibility of the two methodologies for the discovery of de novo markers (single nucleotide polymorphism, SNPs and microsatellites) using low coverage data. Approximately, 6,683 (shotgun) and 14,724 (RAD) SNPs were detected within our elephant sequence dataset. Genotyping of a representative sample of 194 SNPs resulted in a SNP validation rate of ~83 to 94% and 17% of the loci were polymorphic with a low diversity (H(o)=0.057). Different numbers of microsatellites were identified through shotgun (27,226) and RAD (868) techniques. Out of all di-, tri-, and tetra-microsatellite loci, 1,706 loci had sufficient flanking regions (shotgun) while only 7 were found with RAD. All microsatellites were monomorphic in the Bornean but polymorphic in another elephant subspecies. Despite using different sample sizes, and the well known differences in the two platforms used regarding sequence length and throughput, the two approaches showed high validation rate. The approaches used here for marker development in a threatened species demonstrate the utility of high throughput sequencing technologies as a starting point for the development of genomic tools in a non-model species and in particular for a species with low genetic diversity.

Solid lipid microparticles (SLM) containing juniper oil as anti-acne topical carriers: preliminary studies.

Solid lipid microparticles (SLM) were used as carriers of juniper oil and proposed for the topical treatment of acne vulgare. The formulations were obtained by the o/w emulsification method. Compritol and Precirol were employed as lipidic materials. Emulsions containing 1.5% (w/w) of lipophilic phase (lipid and oil) and two different lipid to oil ratios (1:1 and 2:1) were prepared. Blank particles were also prepared, as a comparison. The SLM were characterized in terms of encapsulation efficiency, size, and morphology. The particle size stability in aqueous dispersions was monitored over one month. Evaporation of volatile compounds of oil from microparticles by weight loss was investigated. The qualitative composition of Juniper oil before and after the encapsulation process was determined by gas chromatography (GC) and gas chromatography/mass spectrum (GC/MS) analyses. The antimicrobial activity of the oil encapsulated into the lipid microparticles against P. acnes was studied as contact time assay and compared to the activity of the oil not encapsulated. The emulsification method here described was a good technique for the encapsulation of essential oils. Percentage yields of production and encapsulation efficiencies were higher for Compritol preparations than for these prepared using Precirol. All preparations were characterized by similar particle size distributions (dvs about 3-4 microm) regardless of lipid type and lipid to oil ratios. Microscopy observations showed that the microparticles in aqueous dispersions had almost spherical shape, independently from their composition. The scanning electron microscopy (SEM) analyses showed that when the particles were dried, they had an irregular shape and a rough surface. The SLM dispersions based on Compritol revealed particle size stability over the investigated period of 30 days. In contrast, an increase of the mean dimensions in the preparations containing Precirol was observed. A low loss of volatile oil compounds owing to evaporation from dry particles was found in all preparations. This indicated that the microparticles were able to substantially maintain the oil loaded inside their lipidic structure, reducing its volatility. Some modifications of composition were found in the oil encapsulated in SLM with respect to the juniper oil raw material, but these modifications did not decrease the antibacterial activity of the oil. The SLM here described are promising carriers for the development of anti-acne topical formulations containing Juniper oil.