ORMDL3 regulates NLRP3 inflammasome activation by maintaining ER-mitochondria contacts in human macrophages and dictates ulcerative colitis patient outcome.

Genome-wide association studies in inflammatory bowel disease have identified risk loci in the orosomucoid-like protein 3/ORMDL sphingolipid biosynthesis regulator 3 (ORMDL3) gene to confer susceptibility to ulcerative colitis (UC), but the underlying functional relevance remains unexplored. Here, we found that a subpopulation of the UC patients who had higher disease activity shows enhanced expression of ORMDL3 compared to the patients with lower disease activity and the non-UC controls. We also found that the patients showing high ORMDL3 mRNA expression have elevated interleukin-1ß cytokine levels indicating positive correlation. Further, knockdown of ORMDL3 in the human monocyte-derived macrophages resulted in significantly reduced interleukin-1ß release. Mechanistically, we report for the first time that ORMDL3 contributes to a mounting inflammatory response via modulating mitochondrial morphology and activation of the NLRP3 inflammasome. Specifically, we observed an increased fragmentation of mitochondria and enhanced contacts with the endoplasmic reticulum (ER) during ORMDL3 over-expression, enabling efficient NLRP3 inflammasome activation. We show that ORMDL3 that was previously known to be localized in the ER also becomes localized to mitochondria-associated membranes and mitochondria during inflammatory conditions. Additionally, ORMDL3 interacts with mitochondrial dynamic regulating protein Fis-1 present in the mitochondria-associated membrane. Accordingly, knockdown of ORMDL3 in a dextran sodium sulfate -induced colitis mouse model showed reduced colitis severity. Taken together, we have uncovered a functional role for ORMDL3 in mounting inflammation during UC pathogenesis by modulating ER-mitochondrial contact and dynamics.

Distinct orchid mycorrhizal fungal communities among co-occurring Vanilla species in Costa Rica: root substrate and population-based segregation.

Despite being the second largest family of flowering plants, orchids represent community structure variation in plant-microbial associations, contributes to niche partitioning in metacommunity assemblages. Yet, mycorrhizal communities and interactions remain unknown for orchids that are highly specialized or even obligated in their associations with their mycorrhizal partners. In this study, we sought to compare orchid mycorrhizal fungal (OMF) communities of three co-occurring hemiepiphytic Vanilla species (V. hartii, V. pompona, and V. trigonocarpa) in tropical forests of Costa Rica by addressing the identity of their OMF communities across species, root types, and populations, using high-throughput sequencing. Sequencing the nuclear ribosomal internal transcribed spacer (nrITS) yielded 299 fungal Operational Taxonomic Units (OTUs) from 193 root samples. We showed distinct segregation in the putative OMF (pOMF) communities of the three coexisting Vanilla hosts. We also found that mycorrhizal communities associated with the rare V. hartii varied among populations. Furthermore, we identified Tulasnellaceae and Ceratobasidiaceae as dominant pOMF families in terrestrial roots of the three Vanilla species. In contrast, the epiphytic roots were mainly dominated by OTUs belonging to the Atractiellales and Serendipitaceae. Furthermore, the pOMF communities differed significantly across populations of the widespread V. trigonocarpa and showed patterns of distance decay in similarity. This is the first report of different pOMF communities detected in roots of wild co-occurring Vanilla species using high-throughput sequencing, which provides evidence that three coexisting Vanilla species and their root types exhibited pOMF niche partitioning, and that the rare and widespread Vanilla hosts displayed diverse mycorrhizal preferences.

Congeneric temperate orchids recruit similar-yet differentially abundant-endophytic bacterial communities that are uncoupled from soil, but linked to host phenology and population size.

PREMISE: Besides the beneficial plant-fungus symbiosis in mycorrhizal plants, bacteria also enhance plant fitness via tripartite interactions. While bacterial associations are presumably just as important for the obligate mycorrhizal family Orchidaceae, little is known about orchid associating bacteria (OAB). METHODS: We examined the OAB communities of two, congeneric, terrestrial orchids, Platanthera cooperi and Platanthera praeclara, which represent widely disparate North American ecosystems. We tested whether they recruit distinct OAB communities, and whether variability in OAB communities can be linked to phenology, population size, or habitat soil. Genomic DNAs from roots of seedling, vegetative, and reproductive plants and from soil were subjected to Illumina sequencing of V4 and V5 regions of the 16S rRNA gene. RESULTS: We obtained 809 OAB Zero-radius Operational Taxonomic Units (ZOTUs). Despite an overlap of 209 ZOTUs that accounted for >75% relative abundances of their respective OAB communities, the overall community structures of the two orchids were distinct. Within each orchid, distinctions were detected in the OAB communities of large and small populations and the three phenological stages. The OAB ZOTUs were either absent or present with low abundances in soil associated with both orchids. CONCLUSIONS: The two orchids exhibited preferential recruitment of known growth-promoting OAB communities from soil. Their OAB communities also showed considerable overlap despite the large environmental and geographical separation of the two host taxa. Our results lend further support to the emerging evidence that not only the fungi, but root-associated bacteria also have functional importance for orchid ecology.

Inter-annual Persistence of Canopy Fungi Driven by Abundance Despite High Spatial Turnover.

While it is now well established that fungal community composition varies spatially at a variety of scales, temporal turnover of fungi is less well understood. Here we studied inter-annual community compositional changes of fungi in a rainforest tree canopy environment. We tracked fungal community shifts over 3 years in three substrate types (live bryophytes, dead bryophytes, and host tree bark) and compared these changes to amounts of community turnover seen at small spatial scales in the same system. The effect of substrate type on fungal community composition was stronger than that of sampling year, which was very small but significant. Although levels of temporal turnover varied among substrates, with greater turnover in live bryophytes than other substrates, the amount of turnover from year to year was comparable to what is seen at spatial distances between 5 and 9 cm for the same substrate. Stability of communities was largely driven by a few fungi with high relative abundances. A majority of fungal occurrences were at low relative abundances (≤ 0.1%). These fungi tended to be short lived and persisted to following years ≤ 50% of the time, depending on substrate. Their presence and persistence are likely impacted by stochastic processes like dispersal limitation and disturbance. Most samples contained only one or a few fungi at high relative abundance (≥ 10%) that persisted half or more of the time. These more abundant and persistent fungi are expected to have sustained functional interactions within the canopy ecosystem.

Feature extraction techniques for noisy distributed acoustic sensor data acquired in a wellbore.

The distributed acoustic sensor (DAS) is a promising technology for real-time monitoring of wellbores and other infrastructures. However, the desired signals are often overwhelmed by background and environmental noise inherent in field applications. We present a suite of computationally inexpensive techniques for the real-time extraction of the gas signatures from noisy DAS data acquired in a 5163 ft. deep wellbore. The techniques are implemented on three well-scale DAS datasets, each representing multiphase flow conditions with different gas injection volumes, fluid circulation rates, and injection methods. The proposed denoising techniques not only helped in optimizing the gas slug signature despite the high background noise, but also reduced the DAS data size without compromising the signal quality.

Co-occurring epiphytic orchids have specialized mycorrhizal fungal niches that are also linked to ontogeny.

Mycorrhizal symbiosis has been related to the coexistence and community assembly of coexisting orchids in few studies despite their obligate dependence on mycorrhizal partners to establish and survive. In hyper-diverse environments like tropical rain forests, coexistence of epiphytic orchids may be facilitated through mycorrhizal fungal specialization (i.e., sets of unique and dominant mycorrhizal fungi associated with a particular host species). However, information on the role of orchid mycorrhizal fungi (OMF) in niche differentiation and coexistence of epiphytic orchids is still scarce. In this study, we sought to identify the variation in fungal preferences of four co-occurring epiphytic orchids in a tropical rainforest in Costa Rica by addressing the identity and composition of their endophytic fungal and OMF communities across species and life stages. We show that the endophytic fungal communities are formed mainly of previously recognized OMF taxa, and that the four coexisting orchid species have both a set of shared mycorrhizal fungi and a group of fungi unique to an orchid species. We also found that adult plants keep the OMF of the juvenile stage while adding new mycobionts over time. This study provides evidence for the utilization of specific OMF that may be involved in niche segregation, and for an aggregation mechanism where adult orchids keep initial fungal mycobionts of the juvenile stage while adding others.

The impact of COVID-19 pandemic on access to treatment for children with cancer in India and treating center practices.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic led the Indian government to announce a nationwide lockdown on March 23, 2020. This study aimed to explore the impact of the pandemic on the accessibility of care for children with cancer and to view strategies adopted by hospitals for service delivery. METHODS: Weekly average of childhood cancer (≤18 years) patient registrations during pre-lockdown period (January 1 to March 23, 2020) were compared with post-lockdown period (March 24 to May 31, 2020). The effect on the scheduled treatment was investigated for post-lockdown period. A survey of health care providers was conducted to determine centers' adopted strategies. RESULTS: In 30 participating centers, 1146 patients with childhood cancer (797 pre-lockdown period and 349 post-lockdown period) were registered. The weekly average registration was 67.3 and 35.5 patients during pre-lockdown and post-lockdown respectively (decline of 47.9%). Although most centers experienced this decline, there were 4 that saw an increase in patient registrations. The distribution of patients registered post-lockdown was found significantly different by age (lesser older age, P = .010) and distance (lesser travel distance, P = .001). 36.1% of patients, who were scheduled for any of the treatment modalities (chemotherapy, surgery, radiotherapy, and hematopoietic stem cell transplantation) during the post-lockdown period, experienced delays. Centers adopted several strategies including modifications to treatment protocols, increased use of growth factors, and increased support from social organizations. CONCLUSIONS: This multicenter study from India suggests that the COVID-19 pandemic and the lockdown impacted 2 out of 3 children with cancer. The effect of this on survival is yet to be established.

Colonization with non-mycorrhizal culturable endophytic fungi enhances orchid growth and indole acetic acid production.

BACKGROUND: Symbiotic associations of endophytic fungi have been proved by possessing an ability to produce hormones and metabolites for their host plant. Members of the Orchidaceae are obligate mycorrhizal species but a non-mycorrhizal association needs more investigation for their ability to promote plant growth and produce plant growth hormones. In the present study, endophytic fungi were isolated from the roots of Dendrobium longicornu Lindl., to investigate the root colonizing activity and role in plant growth and development. RESULTS: Among 23 fungal isolates were identified both by morphological and molecular technique as Penicillium sp., Fusarium sp., Coniochaeta sp., Alternaria sp., and Cladosporium sp. The dominate species were Coniochaeta sp. and Cladosporium sp. The dominant species as per the isolation was Coniochaeta sp. These fungal strains were screened for growth-promoting activity of Cymbidium aloifolium (plantlet) consider as cross genus interaction and Dendrobium longicornu (protocorms) as a host plant in in-vitro condition. Importantly, Cladosporium sp., and Coniochaeta sp. showed successful colonization and peloton formation with roots of C. aloifolium. Moreover, it also enhanced acclimatization of plantlets. Fungal elicitors from nine fungal isolates enhanced the growth of the in vitro grown protocorms of D. longicornu. Key bioactive compounds detected in the fungal colonized plant extract were 2H-pyran-2-one, Cyclopropanecarboxylic acid, Oleic Acid and d-Mannitol, which may have a potential role in plant-microbe interaction. All fungal endophytes were able to synthesize the indole acetic acid (IAA) in presence of tryptophan. Moreover, fungal extract DLCCR7 treated with DL-tryptophan yielded a greater IAA concentration of 43 µg per ml than the other extracts. The iaaM gene involved in IAA synthesis pathway was amplified using iaaM gene primers successfully from Alternaria sp., Cladosporium sp., and Coniochaeta sp. CONCLUSIONS: Hence, this study confirms the production of IAA by endophytes and demonstrated their host as well as cross-genus plant growth-promoting potential by producing metabolites required for the growth of the plant.

Epiphytic fungal communities vary by substrate type and at submetre spatial scales.

Fungal species have numerous important environmental functions. Where these functions occur will depend on how fungi are spatially distributed, but the spatial structures of fungal communities are largely unknown, especially in understudied hyperdiverse tropical tree canopy systems. Here we explore fungal communities in a Costa Rican tropical rainforest canopy, with a focus on local-scale spatial structure and substrate specificity of fungi. Samples of ~1 cm3 were collected from 135 points along five adjacent tree branches, with intersample distances from 1 to 800 cm, and dissected into four substrates: outer host tree bark, inner bark, dead bryophytes and living bryophytes. We sequenced the ITS2 region to characterize total fungal communities. Fungal community composition and diversity varied among substrate types, even when multiple substrates were in direct contact. Fungi were most diverse in living bryophytes, with 39% of all operational taxonomic units (OTUs) found exclusively in this substrate, and the least diverse in inner bark. Fungal communities had significant positive spatial autocorrelation and distance decay of similarity only at distances less than 1 m. Similarity among samples declined by half in less than 10 cm, and even at these short distances, similarities were low with few OTUs shared among samples. These results indicate that community turnover is high and occurs at very small spatial scales, with any two locations sharing very few fungi in common. High heterogeneity of fungal communities in space and among substrates may have implications for the distributions, population dynamics and diversity of other tree canopy organisms, including epiphytic plants.

A prospectus of plant growth promoting endophytic bacterium from orchid (Vanda cristata).

BACKGROUND: A plant growth-promoting endophytic bacterium PVL1 isolated from the leaf of Vanda cristata has the ability to colonize with roots of plants and protect the plant. PVL1 was isolated using laboratory synthetic media. 16S rRNA gene sequencing method has been employed for identification before and after root colonization ability. RESULTS: Original isolated and remunerated strain from colonized roots were identified as Bacillus spp. as per EzBiocloud database. The presence of bacteria in the root section of the plantlet was confirmed through Epifluorescence microscopy of colonized roots. The in-vitro plantlet colonized by PVL1 as well as DLMB attained higher growth than the control. PVL1 capable of producing plant beneficial phytohormone under in vitro cultivation. HPLC and GC-MS analysis suggest that colonized plants contain Indole Acetic Acid (IAA). The methanol extract of Bacillus spp., contains 0.015 µg in 1 µl concentration of IAA. PVL1 has the ability to produce antimicrobial compounds such as ethyl iso-allocholate, which exhibits immune restoring property. One-way ANOVA shows that results were statistically significant at P ≤ 0.05 level. CONCLUSIONS: Hence, it has been concluded that Bacillus spp. PVL1 can promote plant growth through secretion of IAA during root colonization and ethyl iso-allocholate to protect plants from foreign infections. Thus, this study supports to support Koch's postulates of bacteria establishment.

Host population size is linked to orchid mycorrhizal fungal communities in roots and soil, which are shaped by microenvironment.

Interaction with orchid mycorrhizal fungi (OMF) is essential to all members of the Orchidaceae, yet we know little about whether or how OMF abundances in substrates shape orchid populations. While root-associated OMF diversity is catalogued frequently, technological constraints have impeded the assessments of OMF communities in substrates until recently, thereby limiting the ability to link OMF communities in a habitat to population responses. Furthermore, there is some evidence that edaphic and microclimatic conditions impact OMF in soil, yet we lack an understanding of the coupled influences of abiotic environment and OMF structure on orchid population dynamics. To discover the linkages between abiotic environment, OMF community structure, and population size, we characterized the microclimatic conditions, soil physicochemistry, and OMF communities hosted by roots and soil across large and small populations of a terrestrial orchid endemic to California Floristic Province in North America. By using high-throughput sequencing of the ITS2 region of nrDNA amplified from root and soil DNAs, we determined that both roots and soil of larger populations, which were high in phosphorus but low in zinc, organic matter, and silt, were dominated by Tulasnellaceae OTUs. In comparison, roots and soil from smaller populations of the orchid hosted higher relative abundances of the Ceratobasidiaceae. In this multiyear, range-wide study that simultaneously measured habitat environmental conditions, and soil and root OMF communities, our results suggest that soil chemistry is clearly linked to soil and root OMF communities, which then likely alter and shape orchid populations.

Well-scale multiphase flow characterization and validation using distributed fiber-optic sensors for gas kick monitoring.

Early detection of a gas kick is crucial for preventing uncontrolled blowout that could cause loss of life, loss of assets, and environmental damage. Multiphase flow experiments conducted in this research demonstrate the capability of downhole fiber optic sensors to detect a potential gas influx in real-time in a 5000 ft deep wellbore. Gas rise velocities estimated independently using fiber optic distributed acoustic sensor (DAS), distributed temperature sensor (DTS), downhole gauges, surface measurements, and multiphase flow correlations show good agreement in each case, demonstrating reliability in the assessment. Real-time data visualization was implemented on a secure cloud-based platform to improve computational efficiency. This study provides novel insights on the effect of circulation rates, gas kick volumes, backpressure, and injection methods on gas rise dynamics in a full-scale wellbore.

Integrating Fiber Optic Data in Numerical Reservoir Simulation Using Intelligent Optimization Workflow.

A novel workflow is presented for integrating fiber optic Distributed Temperature Sensor (DTS) data in numerical simulation model for the Cyclic Steam Stimulation (CSS) process, using an intelligent optimization routine that automatically learns and improves from experience. As the steam-oil relationship is the main driver for forecasting and decision-making in thermal recovery operations, knowledge of downhole steam distribution across the well over time can optimize injection and production. This study uses actual field data from a CSS operation in a heavy oil field in California, and the value of integrating DTS in the history matching process is illustrated as it allows the steam distribution to be accurately estimated along the entire length of the well. The workflow enables the simultaneous history match of water, oil, and temperature profiles, while capturing the reservoir heterogeneity and the actual physics of the injection process, and ultimately reducing the uncertainty in the predictive models. A novel stepwise grid-refinement approach coupled with an evolutionary optimization algorithm was implemented to improve computational efficiency and predictive accuracy. DTS surveillance also made it possible to detect a thermal communication event due to steam channeling in real-time, and even assess the effectiveness of the remedial workover to resolve it, demonstrating the value of continuous fiber optic monitoring.

Distributed Fiber Optic Sensing for Real-Time Monitoring of Gas in Riser during Offshore Drilling.

Effective well control depends on the drilling teams' knowledge of wellbore flow dynamics and their ability to predict and control influx. Unfortunately, detection of a gas influx in an offshore environment is particularly challenging, and there are no existing datasets that have been verified and validated for gas kick migration at full-scale annular conditions. This study bridges this gap and presents pioneering research in the application of fiber optic sensing for monitoring gas in riser. The proposed sensing paradigm was validated through well-scale experiments conducted at Petroleum Engineering Research & Technology Transfer lab (PERTT) facility at Louisiana State University (LSU), simulating an offshore marine riser environment with its larger than average annular space and mud circulation capability. The experimental setup instrumented with distributed fiber optic sensors and pressure/temperature gauges provides a physical model to study the dynamic gas migration in full-scale annular conditions. Current kick detection methods primarily utilize surface measurements and do not always reliably detect a gas influx. The proposed application of distributed fiber optic sensing overcomes this key limitation of conventional kick detection methods, by providing real-time distributed downhole data for accurate and reliable monitoring. The two-phase flow experiments conducted in this research provide critical insights for understanding the flow dynamics in offshore drilling riser conditions, and the results provide an indication of how quickly gas can migrate in a marine riser scenario, warranting further investigation for the sake of effective well control.

SNP genotyping reveals major QTLs for plant architectural traits between A-genome peanut wild species.

KEY MESSAGE: QTL mapping of important architectural traits was successfully applied to an A-genome diploid population using gene-specific variations. Peanut wild species are an important source of resistance to biotic and possibly abiotic stress; because these species differ from the cultigen in many traits, we have undertaken to identify QTLs for several plant architecture-related traits. In this study, we took recently identified SNPs, converted them into markers, and identified QTLs for architectural traits. SNPs from RNASeq data distinguishing two parents, A. duranensis (KSSc38901) and A. cardenasii (GKP10017), of a mapping population were identified using three references-A. duranensis V14167 genome sequence, and transcriptome sequences of A. duranensis KSSc38901 and OLin. More than 49,000 SNPs differentiated the parents, and 87.9% of the 190 SNP calls tested were validated. SNPs were then genotyped on 91 F2 lines using KASP chemistry on a Roche LightCycler 480 and a Fluidigm Biomark HD, and using SNPType chemistry on the Fluidigm Biomark HD. A linkage map was constructed having ten linkage groups, with 144 loci spanning a total map distance of 1040 cM. Comparison of the A-genome map to the A. duranensis genome sequence revealed a high degree of synteny. QTL analysis was also performed on the mapping population for important architectural traits. Fifteen definitive and 16 putative QTLs for petiole length, leaflet length and width, leaflet area, leaflet length/width ratio, main stem height, presence of flowers on the main stem, and seed mass were identified. Results demonstrate that SNPs identified from transcriptome sequencing could be converted to KASP or SNPType markers with a high success rate, and used to identify alleles with significant phenotypic effects, These could serve as information useful for introgression of alleles into cultivated peanut from wild species and have the potential to allow breeders to more easily fix these alleles using a marker-assisted backcrossing approach.

Babesia divergens-infected red blood cells take up glutamate via an EAAT3 independent mechanism.

Human red blood cells infected with the malaria parasite Plasmodium falciparum show an increased permeability to a number of solutes. We have previously demonstrated that such infected cells take up glutamate via a member of the excitatory amino acid transporter protein family (EAAT), namely EAAT3. Babesia divergens is a parasite that also infects human erythrocytes, and also induces increased solute permeability, including for glutamate. Here we have investigated whether glutamate uptake in B. divergens infected human red blood cells is also dependent on EAAT3 activity. We find that, although B. divergens infected cells do take up glutamate, this uptake is independent on EAAT3. Thus, though infecting the same host cell, two related parasites have developed distinct pathways to obtain access to nutrients from the extracellular milieu.

Genetic diversity and structure of a rare endemic cactus and an assessment of its genetic relationship with a more common congener.

Endemic, obligate outcrossing plant species with narrow geographic distributions and disjunct populations are prone to loss of genetic diversity. Simultaneously, delineating clear species boundaries is important for targeted conservation efforts. The rare and endemic cactus, Sclerocactus brevihamatus subsp. tobuschii (SBT), has a parapatric relationship with Sclerocactus brevihamatus subsp. brevihamatus (SBB) but genetic distance between the two taxa is unknown. We: (1) developed taxon-specific polymorphic microsatellites, (2) assessed genetic diversity within and among nine populations of SBT, and within one population of SBB, and (3) estimated the genetic relationship between the two subspecies. Within-population genetic diversity of SBT was moderate to high (mean Ho = 0.37; mean He = 0.59). Indirect estimate of inbreeding corrected for null alleles (Fis-INEst) was low for SBT, ranging from 0.03 to 0.14 (mean Fis-INEst = 0.07). Genetic differentiation among populations of SBT was low based on Fst (0.08) and AMOVA (ФPT = 0.10). Lack of genetic and spatial correlation in SBT populations coupled with the presence of private alleles and bottleneck events in several populations suggests that reproductive isolation is occurring but that sufficient time may not have yet passed to manifest strong differentiation. Cluster analyses segregated the 10 populations into three distinct groups, and separated SBB genotypes clearly. Results suggest that while hybridization between the two subspecies may occur, SBT is clearly differentiated genetically from SBB to retain its current taxonomic status.

The evolutionary dynamics of variant antigen genes in Babesia reveal a history of genomic innovation underlying host-parasite interaction.

Babesia spp. are tick-borne, intraerythrocytic hemoparasites that use antigenic variation to resist host immunity, through sequential modification of the parasite-derived variant erythrocyte surface antigen (VESA) expressed on the infected red blood cell surface. We identified the genomic processes driving antigenic diversity in genes encoding VESA (ves1) through comparative analysis within and between three Babesia species, (B. bigemina, B. divergens and B. bovis). Ves1 structure diverges rapidly after speciation, notably through the evolution of shortened forms (ves2) from 5' ends of canonical ves1 genes. Phylogenetic analyses show that ves1 genes are transposed between loci routinely, whereas ves2 genes are not. Similarly, analysis of sequence mosaicism shows that recombination drives variation in ves1 sequences, but less so for ves2, indicating the adoption of different mechanisms for variation of the two families. Proteomic analysis of the B. bigemina PR isolate shows that two dominant VESA1 proteins are expressed in the population, whereas numerous VESA2 proteins are co-expressed, consistent with differential transcriptional regulation of each family. Hence, VESA2 proteins are abundant and previously unrecognized elements of Babesia biology, with evolutionary dynamics consistently different to those of VESA1, suggesting that their functions are distinct.

Microsatellite-based genetic diversity patterns in disjunct populations of a rare orchid.

We investigated the patterns of genetic diversity and structure in seven disjunct populations of a rare North American orchid, Cypripedium kentuckiense by including populations that represented the periphery and the center of the its range. Eight nuclear and two chloroplast microsatellites were used. Genetic diversity was low across the sampled populations of C. kentuckiense based on both nuclear (average An = 4.0, Ho = 0.436, He = 0.448) and cpDNA microsatellites (average An = 1.57, Nh = 1.57 and H = 0.133). The number of private alleles ranged from one to four per population with a total of 17 private alleles detected at five nuclear microsatellites. One private allele at one cpDNA microsatellite was also observed. Although the absolute values for nuclear microsatellite based population differentiation were low (Fst = 0.075; ϕPT = 0.24), they were statistically significant. Pairwise Fst values ranged from 0.038 to 0.123 and each comparison was significant. We also detected isolation by distance with nDNA microsatellites based on the Mantel test (r(2) = 0.209, P = 0.05). STRUCTURE analysis and the neighbor joining trees grouped the populations similarly whereby the geographically proximal populations were genetically similar. Our data indicate that the species is genetically depauperate but the diversity is distributed more or less equally across its range. Population differentiation and isolation by distance were detectable, which indicates that genetic isolation is beginning to manifest itself across the range in this rare species.