Selection and Validation of Reference Genes in Sudan Grass (Sorghum sudanense (Piper) Stapf) under Various Abiotic Stresses by qRT-PCR.

Quantitative reverse transcription PCR (qRT-PCR) can screen applicable reference genes of species, and reference genes can be used to reduce experimental errors. Sudan grass (Sorghum sudanense (Piper) Stapf) is a high-yield, abiotic-tolerant annual high-quality forage with a wide range of uses. However, no studies have reported reference genes suitable for Sudan grass. Therefore, we found eight candidate reference genes, including UBQ10, HIS3, UBQ9, Isoform0012931, PP2A, ACP2, eIF4α, and Actin, under salt stress (NaCl), drought stress (DR), acid aluminum stress (AlCl3), and methyl jasmonate treatment (MeJA). By using geNorm, NormFinder, BestKeeper, and RefFinder, we ranked eight reference genes on the basis of their expression stabilities. The results indicated that the best reference gene was PP2A under all treatments. eIF4α can be used in CK, MeJA, NaCl, and DR. HIS3 can serve as the best reference gene in AlCl3. Two target genes (Isoform0007606 and Isoform0002387) belong to drought-stress-response genes, and they are highly expressed in Sudan grass according to transcriptome data. They were used to verify eight candidate reference genes under drought stress. The expression trends of the two most stable reference genes were similar, but the trend in expression for Actin showed a significant difference. The reference genes we screened provided valuable guidance for future research on Sudan grass.

Conventional and molecular pharmacognostic characters integrated with chemical profiles of five Piper plants in the Thai herbal pharmacopoeia and their admixture/adulteration/substitution situations in Thailand.

The morphological and microscopy were combined with DNA-barcoding, together with rapid TLC for the characterization of Piper betle (PB), P. nigrum (PN), P. retrofractum (PR), P. sarmentosum (PS), and P. wallichii (PW), five medicinal Piper plants announced in the Thai Herbal Pharmacopoeia (THP). The authentic plants collected from various locations and voucher Piper products bought from commercial sites in Thailand were studied. The reproductive parts of authentic plants were subjected to ensure their morphological characters. Using sequencing analysis and genetic divergence for analyzing discriminatory performance, ITS2 was selected from eight candidate DNA markers to authenticate the origin of Piper crude drugs together with microscopic and TLC profiles for examining their characters, admixtures, adulterants, and substituents. PB and PR exhibited unique characters of the species, with no admixture, adulteration, and substitution. PN showed no variable characters of morphology and genetics. However, the microscopy could illustrate some commercial products of PN sold in Thailand have been adulterated with rice starch and roasted rice. In the herbal trade, PS has been sold in the form of mixed leaf, root, and stem more than the isolated part, but there is no variable character of the species. PW has shown more than one character of species explained by microscopic, chemical components, and genetic data. In conclusion, the conventional and molecular pharmacognostic data combined with chemical profile of authentic five Piper plants could be applied to indicate the plant origin and clarify the situations of admixture, adulteration, and substitution of the commercial Piper products launched in Thailand.

Selection and validation of reference genes for measuring gene expression in Piper species at different life stages using RT-qPCR analysis.

The secondary metabolism of Piper species is known to produce a myriad of natural products from various biosynthetic pathways which, represent a rich source of previously uncharacterized chemical compounds. The determination of gene expression profiles in multiple tissue/organ samples could provide valuable clues towards understanding the potential biological functions of chemical changes in these plants. Studies on gene expression by RT-qPCR require particularly careful selection of suitable reference genes as a control for normalization. Here, we provide a study for the identification of reliable reference genes in P. arboreum, P. gaudichaudianum, P. malacophyllum, and P. tuberculatum, at two different life stages: 2-month-old seedlings and adult plants. To do this, annotated sequences were recovered from transcriptome datasets of the above listed Piper spp. These sequences were subjected to expression analysis using RT-qPCR, followed by analysis using the geNorm and NormFinder algorithms. A set of five genes were identified showing stable expression: ACT7 (Actin-7), Cyclophilin (Peptidyl-prolyl cis-trans isomerase), EF1α (Elongation factor 1-alpha), RNABP (RNA-binding protein), and UBCE (Ubiquitin conjugating enzyme). The universality of these genes was then validated using two target genes, ADC (arginine decarboxylase) and SAMDC (S-adenosylmethionine decarboxylase), which are involved in the biosynthesis of polyamines. We showed that normalization genes varied according to Piper spp., and we provide a list of recommended pairs of the best combination for each species. This study provides the first set of suitable candidate genes for gene expression studies in the four Piper spp. assayed, and the findings will facilitate subsequent transcriptomic and functional gene research.

Comparative cytogenetics of three economically important Piper L. species from the Brazilian Amazon.

The species Piper hispidinervum, Piper aduncum, and Piper affinis hispidinervum have essential oils with high levels of safrole, dillapiole, and sarisan, respectively. Safrole is important for pharmaceutical and chemical industries, while dillapiole and sarisan are promising compounds to control insects and fungi. These species are very similar morphologically and their taxonomy is controversial. Divergent hypotheses consider P. aduncum and P. hispidinervum either as a single species or as distinct taxa, while P. affinis hispidinervum is inferred to be a natural hybrid or a chemotype of P. hispidinervum. Delimiting the taxonomic boundaries would be helpful for germplasm conservation and breeding programs. This study aimed to undertake a detailed analysis of P. aduncum, P. hispidinervum, and P. affinis hispidinervum karyotype and rDNA sites. Genomic in situ hybridization (GISH) was used to establish genomic homology among species and to test the natural hybridization hypothesis for origin of P. affinis hispidinervum. Karyotype traits were similar for all three species: 2n = 26 small chromosomes, predominantly metacentric. All three species exhibited CMA+ bands on the secondary constriction of chromosome pair 4. A size-heteromorphic 35S rDNA site was co-localized with the CMA+ band. A 5S rDNA site was located in the proximal region of chromosome pair 7. The patterns of genomic hybridization revealed that the repetitive DNA fraction of the species is highly similar in terms of proportion of genome, sequence type, and distribution. Our findings did not allow us to differentiate the three species and point to the importance of deeper genomic studies to elucidate the taxonomic controversy.

Phylogenetics and comparative plastome genomics of two of the largest genera of angiosperms, Piper and Peperomia (Piperaceae).

Biological radiations provide unique opportunities to understand the evolution of biodiversity. One such radiation is the pepper plant family Piperaceae, an early-diverging and mega-diverse lineage that could serve as a model to study the diversification of angiosperms. However, traditional genetic markers lack sufficient variation for such studies, and testing hypotheses on poorly resolved phylogenetic frameworks becomes challenging. Limited genomic data is available for Piperaceae, which contains two of the largest genera of angiosperms, Piper (>2100 species) and Peperomia (>1300 species). To address this gap, we used genome skimming to assemble and annotate whole plastomes (152-161kbp) and >5kbp nuclear ribosomal DNA region from representatives of Piper and Peperomia. We conducted phylogenetic and comparative genomic analyses to study plastome evolution and investigate the role of hybridization in this group. Plastome phylogenetic trees were well resolved and highly supported, with a hard incongruence observed between plastome and nuclear phylogenetic trees suggesting hybridization in Piper. While all plastomes of Piper and Peperomia had the same gene content and order, there were informative structural differences between them. First, ycf1 was more variable and longer in Piper than Peperomia, extending well into the small single copy region by thousands of base pairs. We also discovered previously unknown structural variation in 14 out of 25 Piper taxa, tandem duplication of the trnH-GUG gene resulting in an expanded large single copy region. Other early-diverging angiosperms have a duplicated trnH-GUG, but the specific rearrangement we found is unique to Piper and serves to refine knowledge of relationships among early-diverging angiosperms. Our study demonstrates that genome skimming is an efficient approach to produce plastome assemblies for comparative genomics and robust phylogenies of species-rich plant genera.

Targeted 16S rRNA gene and ITS2 amplicon sequencing of leaf and spike tissues of Piper longum identifies new candidates for bioprospecting of bioactive compounds.

Piper longum (also known as Indian long pepper) is widely used in Ayurvedic, Siddha and Unani medicine systems. The principle bioactive compound of this plant is piperine, which mainly accumulates in the fruits called spikes. The report of piperine production by endophytic microbes isolated from Piper sp., motivated us to investigate the endophytic microbial diversity associated with the spikes vis-à-vis leaves (which contain negligible levels of piperine). This is the first report to use metagenomics approach to unravel the endophytic microbial diversity in P. longum. Our results indicate that 2, 56, 631 bacterial OTUs and 1090 fungal OTUs were picked cumulatively from both the tissues. Although bacterial and fungal endophytes occupy the same niche, remarkable differences exist in their diversity and abundance. For instance, the most abundant bacterial genera in spikes were Nocardioides and Pseudonocardia (Phylum Actinobacteria; reported to produce bioactive compounds); while, in leaves were Larkinella and Hymenobacter (Phylum Bacteriodetes). Likewise, the fungal endophytes, Periconia, Cladosporium and Coniothyrium (which have been earlier reported to produce commercially important metabolites including piperine), were also present in high abundance in spikes, in comparison to leaves. Further, the results of PICRUSt analysis reveal the high metabolic potential of spike-associated bacteria for secondary metabolism, namely biosynthesis of alkaloids (including pyridine/piperidine), terpenes, flavonoids and antibiotics. Therefore, our findings indicate that the endophytes abundant or unique in spikes could be explored for bioprospecting of novel/commercially important metabolites; an approach that has both ecological and economical benefits.

A systematic review of botany, phytochemicals and pharmacological properties of "Hoja sant a" (Piper auritum Kunth).

Hoja santa (Piper auritum) refers to an important presence in Mexican cuisine. The information of this review article was gathered from several electronic sources such as Scopus, Medline, Scielo, ScienceDirect, SciFinder, Web of Science, Google Scholar and Lilacs. Phytochemical studies have revealed the presence of benzoic acid derivatives, phenylpropanoids and triterpenoids, while the essential oils have shown its richness in safrole, hence it has several activities, such as antioxidant, toxicity, insecticidal, anti-diabetic and cytotoxic properties. This review is expected to draw the attention of medical professionals and the general public towards P. auritum as well as to open the door for detailed research in the future.

Evaluation of DNA markers for molecular identification of three Piper species from Brazilian Atlantic Rainforest.

Piper is one of two large genera in the Piperaceae, and with ca. 2600 species, is one of the largest plant genera in the world. Species delimitation and evaluation of genetic diversity among populations are important requisites for conservation and adequate exploitation of economically important species. DNA barcoding has been used as a powerful tool and a practical method for species characterization and delimitation. The present work aims to evaluate molecular markers for barcoding three Piper species native to Brazil: P. gaudichaudianum ("jaborandi" or "pariparoba"), P. malacophyllum ("pariparoba-murta") and P. regnellii ("caapeba" or "pariparoba"). A reference DNA barcode library was developed using sequences of three candidate regions: ITS2, trnH-psbA and rbcL. Transferability of the microsatellite (SSR) primers Psol 3, Psol 6 and Psol 10, designed originally for Piper solmsianum, to the three Piper species was also evaluated. The discriminatory power of the markers was based on the determination of inter- and intraspecific distances, phylogenetic reconstruction, and clustering analysis, as well as BLASTn comparison. Sequences of ITS2 enabled efficient species identification by means of the BLASTn procedure. Based on these sequences, intraspecific divergence was lower than interspecific variation. Maximum Parsimony analyses based on ITS2 sequences provided three resolved clades, each corresponding to one of the three analysed species. Sequences of trnH-psbA and rbcL had lower discriminatory value. Analyses combining sequences of these regions were less effective toward the attainment of resolved and strongly supported clades of all species. In summary, robustly supported clades of P. regnellii were obtained in most of the analyses, based either on isolated or combined sequences. The SSRs primers Psol 3, Psol 6 and Psol 10 were shown to be transferable to P. gaudichaudianum and P. regnellii, but not to P. malacophyllum. Preliminary cluster analyses based on the polymorphism of the amplified products suggested that Psol 3 has lower potential than Psol 6 and Psol 10 for discrimination of Piper species.

Transcriptomic analysis of pepper plants provides insights into host responses to Fusarium solani infestation.

Black pepper is an important commodity crop in Malaysia that generates millions of annual revenue for the country. However, black pepper yield is affected by slow decline disease caused by a soil-borne fungus Fusarium solani. RNA sequencing transcriptomics approach has been employed in this study to explore the differential gene expression in susceptible Piper nigrum L. and resistant Piper colubrinum Link. Gene expression comparative analysis of the two pepper species has yielded 2,361 differentially expressed genes (DEGs). Among them, higher expression of 1,426 DEGs was detected in resistant plant. These DEGs practically demonstrated the major branches of plant-pathogen interaction pathway (Path: ko04626). We selected five groups of defence-related DEGs for downstream qRT-PCR analysis. Cf-9, the gene responsible for recognizing fungal avirulence protein activity was found inexpressible in susceptible plant. However, this gene exhibited promising expression in resistant plant. Inactivation of Cf-9 could be the factor that causes susceptible plant fail in recognition of F. solani and subsequently delay activation of adaptive response to fungal invasion. This vital study advance the understanding of pepper plant defence in response to F. solani and aid in identifying potential solution to manage slow decline disease in black pepper cultivation.

CaLecRK-S.5, a pepper L-type lectin receptor kinase gene, accelerates Phytophthora elicitin-mediated defense response.

Innate immunity in plants relies on the recognition of pathogen-associated molecular patterns (PAMPs) by pattern-recognition receptors (PRRs) located on the plant cell surface. CaLecRK-S.5, a pepper L-type lectin receptor kinase, has been shown to confer broad-spectrum resistance through priming activation. To further elucidate the molecular mechanism of CaLecRK-S.5, transgenic tobacco plants were generated in this study. Interestingly, hemizygous transgenic plants exhibited a high accumulation of CaLecRK-S.5, but this accumulation was completely abolished in homozygous transgenic plants by a cosuppression mechanism. Gain-of-function and loss-of-function analyses revealed that CaLecRK-S.5 plays a positive role in Phytophthora elicitin-mediated defense responses.

Origin and evolution of the genus Piper in Peninsular India.

The evolution of Peninsular Indian biodiversity has been a fascinating topic of research due to historical connections of this region to the ancient Gondwanaland. We investigated the phylogeny and historical biogeography of nearly all extant species of the genus Piper reported from the region to assess the biogeographical origins and test mechanisms of lineage diversification (dispersal, vicariance and in situ radiation) of this highly diverse genus of angiosperms commonly found in the understory of evergreen forests. The phylogeny of 21 species of Piper reported from Peninsular India was reconstructed for the first time, which included three new putative species from the Western Ghats. We used BEAST for the divergence time estimations (using three constraints), and ancestral range estimations were performed with the dated phylogenetic tree using BIOGEOBEARS. Divergence dating analysis revealed that the genus Piper originated during lower Cretaceous around 110 Ma [95% highest posterior density (HPD): 116-105 Ma] and colonized Peninsular India five times independently, from Southeast Asia starting from the Oligocene. The two major dispersals into India occurred during the periods of 27.3 Ma (95% HPD: 35.8-19.9.) and 15.5 Ma (95% HPD: 24.9-7.11). This was followed by rapid radiations in some lineages with subsequent back dispersals to Southeast Asia. Our study indicates that dispersals from Southeast Asia led to the arrival of Piper to Indian subcontinent following the Indo-Eurasian collision. Members of Piper have colonized and diversified within the climatically stable habitats of Peninsular India. Furthermore, the present study provides evidence for the Miocene overland dispersal of Piper species to Africa from South Asia.

Gene isolation using degenerate primers targeting protein motif: A laboratory exercise.

Structures and functions of protein motifs are widely included in many biology-based course syllabi. However, little emphasis is placed to link this knowledge to applications in biotechnology to enhance the learning experience. Here, the conserved motifs of nucleotide binding site-leucine rich repeats (NBS-LRR) proteins, successfully used for the isolation and characterization of many plant resistance gene analogues (RGAs), is featured in the development of a series of laboratory experiments using important molecular biology techniques. A set of previously isolated RGA sequences is used as the model for performing sequence alignment and visualising 3D protein structure using current bioinformatics programs (Clustal Omega and Argusdock software). A pair of established degenerate primer sequences is provided for the prediction of targeted amino acids sequences in the RGAs. Reverse transcription-polymerase chain reaction (RT-PCR) is used to amplify RGAs from total RNA samples extracted from the tropical wild relative of black pepper, Piper colubrinum (Piperaceae). This laboratory exercise enables students to correlate specific DNA sequences with respective amino acid codes and the interaction between conserved motifs of resistance genes with putatively targeted proteins. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(1):47-53, 2018.

Resistance Genes in Piper colubrinum: In Silico Survey From Leaf Transcriptome and Expression Studies Upon Challenge Inoculation with Phytophthora capsici.

The oomycetes, Phytophthora capsici, cause foot rot disease in black pepper. Piper colubrinum Link, a distant relative of cultivated black pepper, is highly resistant to this destructive pathogen. Identification of resistance (R) genes in P. colubrinum and the study of its expression profile during interaction with the pathogen can help in understanding the resistance mechanism involved. In the present study, 1289 R gene-related transcripts were mined from P. colubrinum transcriptome, clustered, and classified according to the conserved motifs and domains. Transcripts belonging to four major R gene classes were identified in P. colubrinum, but TIR-NBS-LRR-type R genes were absent. The relative expression of 12 selected R genes was studied using two virulent isolates of P. capsici, and these were found to be upregulated in the initial hours of plant pathogen interaction. The R genes studied were expressed even in aseptically maintained tissue-cultured plants and uninoculated greenhouse-grown plants at basal level suggesting that the plants are geared up with the R gene all the time and are under continuous surveillance for the pathogen and basal level of R gene expression do not require a pathogen trigger. ACT, ATUB, and EIF3E were identified as the most stable reference genes that can be used for real-time PCR study. The present study identified promising R genes in P. colubrinum which can be used in developing Phytophthora-resistant black pepper.

Phylogenomics of 2,4-Diacetylphloroglucinol-Producing Pseudomonas and Novel Antiglycation Endophytes from Piper auritum.

2,4-Diacetylphloroglucinol (DAPG) (1) is a phenolic polyketide produced by some plant-associated Pseudomonas species, with many biological activities and ecological functions. Here, we aimed at reconstructing the natural history of DAPG using phylogenomics focused at its biosynthetic gene cluster or phl genes. In addition to around 1500 publically available genomes, we obtained and analyzed the sequences of nine novel Pseudomonas endophytes isolated from the antidiabetic medicinal plant Piper auritum. We found that 29 organisms belonging to six Pseudomonas species contain the phl genes at different frequencies depending on the species. The evolution of the phl genes was then reconstructed, leading to at least two clades postulated to correlate with the known chemical diversity surrounding DAPG biosynthesis. Moreover, two of the newly obtained Pseudomonas endophytes with high antiglycation activity were shown to exert their inhibitory activity against the formation of advanced glycation end-products via DAPG and related congeners. Its isomer, 5-hydroxyferulic acid (2), detected during bioactivity-guided fractionation, together with other DAPG congeners, were found to enhance the detected inhibitory activity. This report provides evidence of a link between the evolution and chemical diversity of DAPG and congeners.

Construction of a cDNA library and preliminary analysis of expressed sequence tags in Piper hainanense.

Black pepper is a perennial climbing vine. It is widely cultivated because its berries can be utilized not only as a spice in food but also for medicinal use. This study aimed to construct a standardized, high-quality cDNA library to facilitated identification of new Piper hainanense transcripts. For this, 262 unigenes were used to generate raw reads. The average length of these 262 unigenes was 774.8 bp. Of these, 94 genes (35.9%) were newly identified, according to the NCBI protein database. Thus, identification of new genes may broaden the molecular knowledge of P. hainanense on the basis of Clusters of Orthologous Groups and Gene Ontology categories. In addition, certain basic genes linked to physiological processes, which can contribute to disease resistance and thereby to the breeding of black pepper. A total of 26 unigenes were found to be SSR markers. Dinucleotide SSR was the main repeat motif, accounting for 61.54%, followed by trinucleotide SSR (23.07%). Eight primer pairs successfully amplified DNA fragments and detected significant amounts of polymorphism among twenty-one piper germplasm. These results present a novel sequence information of P. hainanense, which can serve as the foundation for further genetic research on this species.

Down-regulation of osmotin (PR5) gene by virus-induced gene silencing (VIGS) leads to susceptibility of resistant Piper colubrinum Link. to the oomycete pathogen Phytophthora capsici Leonian.

Piper colubrinum Link., a distant relative of Piper nigrum L., is immune to the oomycete pathogen Phytophthora capsici Leonian that causes 'quick wilt' in cultivated black pepper (P. nigrum). The osmotin, PR5 gene homologue, earlier identified from P. colubrinum, showed significant overexpression in response to pathogen and defense signalling molecules. The present study focuses on the functional validation of P. colubrinum osmotin (PcOSM) by virus induced gene silencing (VIGS) using Tobacco Rattle Virus (TRV)-based vector. P. colubrinum plants maintained under controlled growth conditions in a growth chamber were infiltrated with Agrobacterium carrying TRV empty vector (control) and TRV vector carrying PcOSM. Three weeks post infiltration, viral movement was confirmed in newly emerged leaves of infiltrated plants by RT-PCR using TRV RNA1 and TRV RNA2 primers. Semi-quantitative RT-PCR confirmed significant down-regulation of PcOSM gene in TRV-PcOSM infiltrated plant compared with the control plants. The control and silenced plants were challenged with Phytophthora capsici which demonstrated that knock-down of PcOSM in P. colubrinum leads to increased fungal mycelial growth in silenced plants compared to control plants, which was accompanied by decreased accumulation of H2O2 as indicated by 3,3'-diaminobenzidine (DAB) staining. Thus, in this study, we demonstrated that Piper colubrinum osmotin gene is required for resisting P. capsici infection and has possible role in hypersensitive cell death response and oxidative burst signaling during infection.

Identification and Expression Analysis of Candidate Genes Associated with Defense Responses to Phytophthora capsici in Pepper Line "PI 201234".

Phytophthora capsici (Leonian), classified as an oomycete, seriously threatens the production of pepper (Capsicum annuum). Current understanding of the defense responses in pepper to P. capsici is limited. In this study, RNA-sequencing analysis was utilized to identify differentially expressed genes in the resistant line "PI 201234", with 1220 differentially expressed genes detected. Of those genes, 480 were up-regulated and 740 were down-regulated, with 211 candidate genes found to be involved in defense responses based on the gene annotations. Furthermore, the expression patterns of 12 candidate genes were further validated via quantitative real-time PCR (qPCR). These genes were found to be significantly up-regulated at different time points post-inoculation (6 hpi, 24 hpi, and 5 dpi) in the resistant line "PI 201234" and susceptible line "Qiemen". Seven genes were found to be involved in cell wall modification, phytoalexin biosynthesis, symptom development, and phytohormone signaling pathways, thus possibly playing important roles in combating exogenous pathogens. The genes identified herein will provide a basis for further gene cloning and functional verification studies and will aid in an understanding of the regulatory mechanism of pepper resistance to P. capsici.

A Late Cretaceous Piper (Piperaceae) from Colombia and diversification patterns for the genus.

PREMISE OF THE STUDY: Documented fossil floras in the neotropics are sparse, yet their records provide evidence on the spatial and temporal occurrence of taxa, allowing for testing of biogeographical and diversification scenarios on individual lineages. A new fossil Piper from the Late Cretaceous of Colombia is described here, and its importance for assessing diversification patterns in the genus is addressed. METHODS: Leaf architecture of 32 fossil leaf compressions from the Guaduas Formation was compared with that of 294 extant angiosperm species. The phylogenetic position of the fossil named Piper margaritae sp. nov. was established based on leaf traits and a molecular scaffold of Piper. The age of the fossil was independently used as a calibration point for divergence time estimations. KEY RESULTS: Natural affinities of P. margaritae to the Schilleria clade of Piper indicate that the genus occurred in tropical America by the Late Cretaceous. Estimates of age divergence and lineage accumulation reveal that most of the extant diversity of the genus accrued during the last ∼30 Myr. CONCLUSIONS: The recent radiation of Piper is coeval with both the Andean uplift and the emergence of Central America, which have been proposed as important drivers of diversity. This pattern could exemplify a recurrent theme among many neotropical plant lineages.

Analysis of genetic diversity of certain species of Piper using RAPD-based molecular markers.

The utility of RAPD markers in assessing genetic diversity and phenetic relationships of six different species of Piper from Northeast India was investigated. Polymerase chain reaction (PCR) with four arbitrary 10-mer oligonucleotide primers applied to the six species produced a total of 195 marker bands, of which, 159 were polymorphic. On average, six RAPD fragments were amplified per reaction. In the UPGMA phenetic dendrogram based on Jaccard's coefficient, the different accessions of Piper showed a high level of genetic variation. This study may be useful in identifying diverse genetic stocks of Piper, which may then be conserved on a priority basis.

Host conservatism, host shifts and diversification across three trophic levels in two Neotropical forests.

Host-parasite systems have been models for understanding the connection between shifts in resource use and diversification. Despite theoretical expectations, ambiguity remains regarding the frequency and importance of host switches as drivers of speciation in herbivorous insects and their parasitoids. We examine phylogenetic patterns with multiple genetic markers across three trophic levels using a diverse lineage of geometrid moths (Eois), specialist braconid parasitoids (Parapanteles) and plants in the genus Piper. Host-parasite associations are mapped onto phylogenies, and levels of cospeciation are assessed. We find nonrandom patterns of host use within both the moth and wasp phylogenies. The moth-plant associations in particular are characterized by small radiations of moths associated with unique host plants in the same geographic area (i.e. closely related moths using the same host plant species). We suggest a model of diversification that emphasizes an interplay of factors including host shifts, vicariance and adaptation to intraspecific variation within hosts.