New ring-A modified cycloartane triterpenoids from Dysoxylum malabaricum bark: Isolation, structure elucidation and their cytotoxicity.

The Genus Dysoxylum (Meliaceae) consists of approximately 80 species that are abundant in structurally diverse triterpenoids. The present study focused on isolating new triterpenoids from the bark of Dysoxylum malabaricum, one of the predominant species of Dysoxylum present in India. The methanol-dichloromethane bark extract was subjected to LCMS profiling followed by silica gel column chromatography and HPLC analysis to target new compounds. Two new ring A-modified cycloartane-type triterpenoids (1 and 2) were isolated from the bark extract. Spectroscopic methods like NMR, HRESIMS data, and electronic circular dichroism calculations elucidated the structuresandabsolute configurations of the isolated compounds. These compounds were evaluated for their cytotoxic potential against breast cancer cells and displayed notable cytotoxicity. Compound 1 exhibited the highest cytotoxicity against the MDA-MB-231 cells and induced apoptotic cell death. Also, it was able to inhibit glucose uptake and increase nitric oxide production in breast cancer cells.

Opening the floodgates for invasion-modelling the distribution dynamics of invasive alien fishes in India.

Invasive alien species have become the second major threat to biodiversity affecting all three major ecosystems (terrestrial, marine, and freshwater). Increasing drivers such as habitat destruction, expanding horticulture and aquaculture industries, and global pet and food trade have created pathways for exotic species to be introduced leading to severe impacts on recipient ecosystems. Although relatively less studied than terrestrial ecosystems, freshwater ecosystems are highly susceptible to biological invasions. In India, there has been a noticeable increase in the introduction of alien fish species in freshwater environments. In the current study, we aimed to understand how climate change can affect the dynamics of the biological invasion of invasive alien fishes in India. We also evaluated the river-linking project's impact on the homogenization of biota in Indian freshwater bodies. We used species occurrence records with selected environmental variables to assess vulnerable locations for current and future biological invasion using species distribution models. Our study has identified and mapped the vulnerable regions to invasion in India. Our research indicates that the interlinking of rivers connects susceptible regions housing endangered fish species with invasive hotspots. Invasive alien fishes from the source basin may invade vulnerable basins and compete with the native species. Based on the results, we discuss some of the key areas for the management of these invasive alien species in the freshwater ecosystems.

New cycloartane triterpenoids from Dysoxylum malabaricum and their cytotoxic evaluation.

The cytotoxic dichloromethane-methanol bark extract of Dysoxylum malabaricum was subjected to bioassay-guided fractionation, followed by systematic dereplication to focus on the identification of new compounds. From the bark of Dysoxylum malabaricum, two new cycloartane-type triterpenoids were isolated in addition to two previously known triterpenoids. The structures and absolute configurations of the isolated compounds were elucidated unambiguously via NMR, HRESIMS data, and electronic circular dichroism calculations. The isolated compounds were tested for their cytotoxic potential against the panel of breast, lung, and hypopharynx cancer cell lines and displayed notable cytotoxicity against breast cancer cell lines. Compound 3 exhibited the most potent cytotoxic effect with an IC50 14 µM against MCF-7 cell lines and induced cell cycle arrest. Through western blot and cell cycle analysis, it was revealed that compound 3 halts the G0/G1 phase of the cell cycle by inhibiting CDC20 and CDC25 enzymes.

Endemicity and radiation in waterfalls of the Western Ghats: The genus Cremnoconchus (Gastropoda: Littorinidae).

Snails of the genus Cremnoconchus - the only freshwater members of the gastropod family Littorinidae - are endemic to the spray zones of numerous waterfalls in the Western Ghats of India. Cremnoconchus consists of nine described and possibly numerous undescribed species as many of these appear to be restricted to specific waterfalls. This is the first attempt at resolving the relationships between the various species in this genus and establishing its monophyly in the family. Further, we also undertake species delimitation analysis to characterize cryptic diversity in this group. Phylogenetic analyses based on nuclear and mitochondrial genes support the monophyly of Cremnoconchus within the family. A fossil-calibrated Bayesian time tree suggests that this freshwater lineage diverged from its marine counterparts around 90.40 million years ago. The separation of Cremnoconchus from its marine ancestors might have been facilitated by the break-up of Gondwana or fluctuating sea levels during this period. Species delimitation analysis retrieved 12 potentially undescribed species in this group. These species formed two distinct clades in the phylogeny, one largely confined to the northern Western Ghats and the other to the central Western Ghats. Species belonging to the northern and central Western Ghats seem to have separated around 56.11 mya, i.e. after the northern Western Ghats were formed. Additionally, spatial isolation due to the patchiness of suitable habitats (waterfalls) and low mobility might have facilitated their diversification.

Can species distribution models and molecular tools help unravel disjunct distribution of Rhododendron arboreum?

The apparent absence of Himalayan low-elevation taxa in the central Indian region and resumption of their distribution in the high elevation of Western Ghats has puzzled biogeographers for several decades. Many theories have been proposed to explain this but attempts remain futile owing to insufficient empirical support. Here, we have employed a montane tree species, Rhododendron arboreum to investigate this pattern by integrating past ecological niche modelling with molecular signatures. Reconstruction of paleo-ecological niche from interglacial to Last Glacial Maxima (LGM) portrayed a gradual depletion of vegetation cover with extreme impoverishment in the Holocene. A similar pattern was also reflected from genetic signatures; population history revealed a very recent split between the Himalayas and Western Ghats in the late Quaternary. A few other tree species exhibiting the same disjunction demonstrated a similar modification of paleo-ecological niche from last interglacial. The study clearly indicated that the populations in the Western Ghats to be a relictual remnants of a once continuous distribution of R. arboreum.

Inhibition of plant pathogenic fungi by endophytic Trichoderma spp. through mycoparasitism and volatile organic compounds.

Antagonism of plant pathogenic fungi by endophytic fungi is a well-known phenomenon. In plate assays, the antagonism could be due to mycoparasitism, competition for space or antibiosis, involving a chemical diffusate, or a volatile organic compound (VOC). In this study, we demonstrate that besides mycoparasitism, VOCs play a major role in antagonism of pathogenic fungi by four endophytic fungi belonging to the genus Trichoderma. Using a double-plate assay, we show that all the four endophytic Trichoderma species significantly inhibited mycelial growth of three of the four pathogens, (Sclerotinia sclerotiorum-TSS, Sclerotium rolfsii-CSR and Fusarium oxysporum-CFO), while that of Macrophomina phaseolina-CMP was not affected. GC-MS analysis of the pure cultures of one of the endophytic fungi studied, namely, Trichoderma longibrachiatum strain 2 (Acc. No. MK751758) and the pathogens, F. oxysporum-CFO and M. phaseolina-CMP revealed the presence of several VOCs including hydrocarbons, alcohols, ketones, aldehydes, esters, acids, ethers and different classes of terpenes. In mixed double plates, where the endophyte was grown along with either of the two plant pathogens, F. oxysporum-CFO or M. phaseolina-CMP, there was an induction of a number of new VOCs that were not detected in the pure cultures of either the endophyte or the pathogens. Several of these new VOCs are reported to possess antifungal and cytotoxic activity. We discuss these results and highlight the importance of such interactions in endophyte-pathogen interactions.

An endophyte from salt-adapted Pokkali rice confers salt-tolerance to a salt-sensitive rice variety and targets a unique pattern of genes in its new host.

Endophytes, both of bacterial and fungal origin, are ubiquitously present in all plants. While their origin and evolution are enigmatic, there is burgeoning literature on their role in promoting growth and stress responses in their hosts. We demonstrate that a salt-tolerant endophyte isolated from salt-adapted Pokkali rice, a Fusarium sp., colonizes the salt-sensitive rice variety IR-64, promotes its growth under salt stress and confers salinity stress tolerance to its host. Physiological parameters, such as assimilation rate and chlorophyll stability index were higher in the colonized plants. Comparative transcriptome analysis revealed 1348 up-regulated and 1078 down-regulated genes in plants colonized by the endophyte. Analysis of the regulated genes by MapMan and interaction network programs showed that they are involved in both abiotic and biotic stress tolerance, and code for proteins involved in signal perception (leucine-rich repeat proteins, receptor-like kinases) and transduction (Ca2+ and calmodulin-binding proteins), transcription factors, secondary metabolism and oxidative stress scavenging. For nine genes, the data were validated by qPCR analysis in both roots and shoots. Taken together, these results show that salt-adapted Pokkali rice varieties are powerful sources for the identification of novel endophytes, which can be used to confer salinity tolerance to agriculturally important, but salt-sensitive rice varieties.

A review on the conservation genetic studies of Indian amphibians and their implications on developing strategies for conservation.

Amphibians show a very high level of diversity and endemism and are facing global declines from the past few decades. Studies have shown that the molecular tools can be helpful in their conservation efforts. In India, more than 80% of amphibians are endemic and most show a narrow range of distribution. Most of the Indian amphibians lack information on their genetic diversity. In this study, were view the overall trend on amphibian studies in India with the specific focus on conservation genetics. Overall, of the 173 studies, only 14 dealt with the conservation of amphibians through genetic tools and five studies estimated the genetic diversity or gene structure. Here, we discuss the gaps and provide future directions on how genetic studies can be helpful in Indian amphibian conservation.

Development and characterization of microsatellite markers for Phyllanthus emblica Linn., important nontimber forest product species.

Phyllanthus emblica and P. indofischeri, commonly known as the Indian gooseberry, are important nontimber forest product (NTFP) species widely distributed across the Indian subcontinent. The fruits of these species are rich in vitamin C and are used in the preparation of a number of herbal medicines for treating a wide range of disorders. Due to the increased demand, they have been harvested extensively and form a major source of income for the forest-dwelling communities living in southern India. There are limited studies to understand the impact of harvesting on the genetic structure of these species. In this study, 15 polymorphic microsatellite markers have been developed for P. emblica and were characterized by screening 20 individuals each of P. emblica and P. indofischeri. The number of alleles per locus ranged 2-9 for P. emblica and 2-11 for P. indofischeri. The observed and expected heterozygosity of P. emblica ranged 0-1 and 0.401-0.825, respectively. Similarly, the observed and expected heterozygosity of P. indofischeri ranged 0.5-1 and 0.366-0.842, respectively. Cross-amplification of the designed primers was assessed with seven related Phyllanthus species. The microsatellite markers developed can be used for studying the population genetic structure, gene flow and genetic diversity of P. emblica and P. indofischeri.

Mechanism of Resistance to Camptothecin, a Cytotoxic Plant Secondary Metabolite, by Lymantria sp. Larvae.

Camptothecin (CPT), a monoterpene indole alkaloid, is a potent inhibitor of eukaryotic topoisomerase I (Top 1). Because of this property, several derivatives of CPT are widely used as chemotherapeutic agents. The compound is produced by several plant species, including Nothapodytes nimmoniana (Family: Icacinaceae) presumably as a deterrent to insect pests. Here, we report, a lepidopteran larva, Lymantria sp. of Lymantriidae family which feeds voraciously on the leaves of N. nimmoniana, without any adverse consequences. Larval body weight and molting period were unaffected despite captive feeding of the larva with CPT enriched leaves. Mass spectrometric analysis indicated that nearly 46% of the ingested CPT was excreted while the rest was sequestered predominantly in the exuviae and setae (~35%). Although most of the CPT was in the parental form as found in the plant, traces of inactive, sulfated forms of CPT were recovered from the larva. Compared to that in plant, there were no critical mutations at the CPT binding domain of the insect's Top 1. The gut pH of the larva was alkaline (pH 10.0). The alkaline gut environment converts CPT from its active, lactone form to inactive, carboxylate form. It is likely that such conversion might help the larva to reduce the overall burden of CPT in its gut. We discuss the results in the context of the mechanisms of resistance adapted by insects to plant toxins.

Assessment of adulteration in raw herbal trade of important medicinal plants of India using DNA barcoding.

A number of studies have shown that there could be widespread substitution and/or adulteration (hereafter referred to as substitution) in raw herbal trade of medicinal plants. Substitution could potentially endanger the health and safety of the consumers. In this study, the extent of adulteration in raw herbal trade of 30 important medicinal plants in South India was analyzed. Biological reference material (BRM) consisting of taxonomically authenticated samples of each of the 30 species along with 14 other co-occurring and congeneric allied species that are likely to be used in adulteration was established. DNA barcode signatures of 124 BRM using two candidate regions, nr-ITS and psbA-trnH were identified. A total of 203 herbal trade samples representing the 30 medicinal plant species were collected from 34 locations in South India. Using the DNA barcode sequences of the BRM as reference, the analysis indicated that the substitution ranged from 20 to 100%. Overall, approximately 12% of the market samples were adulterated. Considering the potential health hazard that such adulteration can cause, the need for a national regulatory framework that can authenticate and regulate raw herbal trade in the country is discussed.

Camptothecin-producing endophytic bacteria from Pyrenacantha volubilis Hook. (Icacinaceae): A possible role of a plasmid in the production of camptothecin.

BACKGROUND: Camptothecin (CPT), a quinoline alkaloid, is a potent inhibitor of eukaryotic topoisomerase I. Because of this property, several derivatives of CPT are used as chemotherapeutic agents. CPT is produced by several plant species belonging to the Asterid clade as well as by a number of endophytic fungal associates of these plants. In this study, we report the production of CPT by four bacterial endophytes and show the possible role of a plasmid in the biosynthesis of CPT. METHODS: Endophytic bacteria were isolated from leaves, stems and fruits of Pyrenacantha volubilis Hook. (Icacinanceae). The bacterial isolates were purified and analyzed for production of CPT by ESI-MS/MS and NMR analysis. Bacterial identity was established based on the morphology and 16s rRNA sequence analysis. Crude extracts of the bacterial endophytes were evaluated for their cytotoxicity using colon cancer cell lines. The role of plasmid in the production of CPT was studied by purging the plasmid, using acriflavine, as well as reconstituting the bacteria with the plasmid. RESULTS: Four bacterial isolates, Bacillus sp. (KP125955 and KP125956), Bacillus subtilis (KY741853) and Bacillus amyloliquefaciens (KY741854) were found to produce CPT in culture. Both based on ESI-MS/MS and NMR analysis, the identity of CPT was found to be similar to that produced by the host plant. The CPT was biologically active as evident by its cytotoxicity against colon cancer cell line. The production of CPT by the endophyte (Bacillus subtilis, KY741853) attenuated with sub-culture. A likely role of a plasmid in the production of CPT was established. A 5 kbp plasmid was recovered from the bacteria. Bacterial isolate cured of plasmid failed to produce CPT. CONCLUSION: Our study implies a possible role of a plasmid in the production of CPT by the endophytic bacteria and opens up further work to unravel the exact mechanisms that might be involved.

Assigning conservation value and identifying hotspots of endemic rattan diversity in the Western Ghats, India.

Rattans, or canes, are one of the most important non-timber forest products supporting the livelihood of many forest-dwelling communities in South and North-eastern India. Due to increased demand for rattan products, rattans have been extracted indiscriminately from the Western Ghats, a 1600-km mountain chain running parallel to the west coast of India. Extensive harvesting, loss of habitat and poor regeneration has resulted in dwindling rattan populations, necessitating an urgent attempt to conserve existing rattan resources. In this study, using niche-modelling tools, an attempt has been made to identify areas of high species richness of rattans in the Western Ghats, one of the mega-diversity regions of the world. We have also developed conservation values for 21 economically important and endemic rattans of the Western Ghats. We identified at least two to three sites of extremely high species richness outside the existing protected area network that should be prioritized for in situ conservation. This study emphasizes the need to develop strategies for the long-term conservation of rattans in the Western Ghats, India.

Redescription and Range Extension of Microhyla sholigari Dutta & Ray (Amphibia: Anura: Microhylidae) from South West India.

Microhyla sholigari is an endangered frog described in the year 2000. The original description was based on non-adult types and lacked information on several morphological characters, call structure, genetic material and photographs of the animal in life. The absence of such information posed challenges in field identification of this species. Since the original description, there is one other reported sighting of this species from Kerala in 2001. We encountered specimens that we confer to this species based on morphological similarity to the subadult holotype, from several new localities within and outside the Western Ghats of Karnataka. We here redescribe the species based on additional adult vouchers, provide molecular data, describe the advertisement call and report a range extension. Based on its current distribution, we assess the threat status of the species and suggest listing it as Least Concern according to IUCN Red List criteria. Our paper bridges an important gap in the knowledge of the genus Microhyla in India and highlights the importance of systematic surveys in documenting and understanding amphibian diversity in the region.

Modeling the impact of climate change on wild Piper nigrum (Black Pepper) in Western Ghats, India using ecological niche models.

The center of diversity of Piper nigrum L. (Black Pepper), one of the highly valued spice crops is reported to be from India. Black pepper is naturally distributed in India in the Western Ghats biodiversity hotspot and is the only known existing source of its wild germplasm in the world. We used ecological niche models to predict the potential distribution of wild P. nigrum in the present and two future climate change scenarios viz (A1B) and (A2A) for the year 2080. Three topographic and nine uncorrelated bioclim variables were used to develop the niche models. The environmental variables influencing the distribution of wild P. nigrum across different climate change scenarios were identified. We also assessed the direction and magnitude of the niche centroid shift and the change in niche breadth to estimate the impact of projected climate change on the distribution of P. nigrum. The study shows a niche centroid shift in the future climate scenarios. Both the projected future climate scenarios predicted a reduction in the habitat of P. nigrum in Southern Western Ghats, which harbors many wild accessions of P. nigrum. Our results highlight the impact of future climate change on P. nigrum and provide useful information for designing sound germplasm conservation strategies for P. nigrum.

Microhyla laterite sp. nov., A New Species of Microhyla Tschudi, 1838 (Amphibia: Anura: Microhylidae) from a Laterite Rock Formation in South West India.

In recent times, several new species of amphibians have been described from India. Many of these discoveries are from biodiversity hotspots or from within protected areas. We undertook amphibian surveys in human dominated landscapes outside of protected areas in south western region of India between years 2013-2015. We encountered a new species of Microhyla which is described here as Microhyla laterite sp. nov. It was delimited using molecular, morphometric and bioacoustics comparisons. Microhyla laterite sp. nov. appears to be restricted to areas of the West coast of India dominated by laterite rock formations. The laterite rock formations date as far back as the Cretaceous-Tertiary boundary and are considered to be wastelands in-spite of their intriguing geological history. We identify knowledge gaps in our understanding of the genus Microhyla from the Indian subcontinent and suggest ways to bridge them.

Integrative Taxonomic Approach for Describing a New Cryptic Species of Bush Frog (Raorchestes: Anura: Rhacophoridae) from the Western Ghats, India.

A new cryptic species of bush frog Raorchestes honnametti sp. nov. is described from the south-eastern part of the Western Ghats, India. This newly described species belongs to the Charius clade and is morphologically similar to other clade members--R. charius and R. griet. Therefore, an integrative taxonomic approach based on molecular and bioacoustic analysis along with morphology was used to delimit the new species. Raorchestes honnametti sp. nov., is currently known only from Biligiri Rangaswamy Temple Tiger Reserve, a part of Biligiri Rangaswamy horst mountain range (a mountain formed due movement of two faults) formed during the Late Quaternary period (1.8-2.58 Ma). Discovery of cryptic species from a highly speciose and well-studied genus Raorchestes hints at the possible existence of several more cryptic species in this genus. We discuss the possible reasons for crypsis and emphasize the need for continued systematic surveys of amphibians across the Western Ghats.

Transcriptome analysis of stem wood of Nothapodytes nimmoniana (Graham) Mabb. identifies genes associated with biosynthesis of camptothecin, an anti-carcinogenic molecule.

Camptothecin (CPT), a monoterpene indole alkaloid, is a potent inhibitor of DNA topoisomerase I and has applications in treating ovarian, small lung and refractory ovarian cancers. Stem wood tissue of Nothapodytes nimmoniana (Graham) Mabb. (family Icacinaceae) is one of the richest sources of CPT. Since there is no genomic or transcriptome data available for the species, the present work sequenced and analysed transcriptome of stem wood tissue on an Illumina platform. From a total of 77,55,978 reads, 9,187 transcripts were assembled with an average length of 255 bp. Functional annotation and categorization of these assembled transcripts unraveled the transcriptome architecture and also a total of 13 genes associated with CPT biosynthetic pathway were identified in the stem wood tissue. Four genes of the pathway were cloned to full length by RACE to validate the transcriptome data. Expression analysis of 13 genes associated with CPT biosynthetic pathway in 11 different tissues vis-a-vis CPT content analysis suggested an important role of NnPG10H, NnPSLS and NnPSTR genes in the biosynthesis of CPT. These results indicated that CPT might be synthesized in the leaves and then perhaps exported to stem wood tissue for storage.

Restoration of camptothecine production in attenuated endophytic fungus on re-inoculation into host plant and treatment with DNA methyltransferase inhibitor.

Fungal endophytes inhabit living tissues of plants without any apparent symptoms and in many cases are known to produce secondary metabolites similar to those produced by their respective host plants. However on sub-culture, the endophytic fungi gradually attenuate their ability to produce the metabolites. Attenuation has been a major constraint in realizing the potential of endophytic fungi as an alternative source of plant secondary metabolites. In this study, we report attempts to restore camptothecine (CPT) production in attenuated endophytic fungi isolated from CPT producing plants, Nothapodytes nimmoniana and Miquelia dentata when they are passed through their host plant or plants that produce CPT and when treated with a DNA methyl transferase inhibitor. Attenuated endophytic fungi that traversed through their host tissue or plants capable of synthesizing CPT, produced significantly higher CPT compared to the attenuated fungi. Attenuated fungus cultured in the presence of 5-azacytidine, a DNA methyltransferase inhibitor, had an enhanced CPT content compared to untreated attenuated fungus. These results indicate that the attenuation of CPT production in endophytic fungi could in principle be reversed by eliciting some signals from plant tissue, most likely that which prevents the methylation or silencing of the genes responsible for CPT biosynthesis.

Ambient ionization mass spectrometry imaging of rohitukine, a chromone anti-cancer alkaloid, during seed development in Dysoxylum binectariferum Hook.f (Meliaceae).

Rohitukine, a chromone alkaloid, possesses anti-inflammatory, anti-cancer and immuno-modulatory properties. It has been reported from four species, belonging to the families, Meliaceae and Rubiaceae. Stem bark of Dysoxylum binectariferum (Meliaceae) accumulates the highest amount of rohitukine (3-7% by dry weight). In this study, we examine the spatial and temporal distribution of rohitukine and related compounds during various stages of seed development in D. binectariferum using desorption electrospray ionization mass spectrometry imaging (DESI MSI). Rohitukine (m/z 306.2) accumulation increased from early seed development to seed maturity stage. The spatial distribution of rohitukine was largely restricted to the cotyledonary tissue followed by the embryo and least in the seed coat. Besides rohitukine, rohitukine acetate (m/z 348.2) and glycosylated rohitukine (m/z 468.2) were also detected, both through mass fragmentation and exact mass analysis through Orbitrap mass spectrometry. These results indicate a dynamic pattern of chromane alkaloid accumulation through seed development in D. binectariferum.