Community-level prevalence of a forest pathogen, not individual-level disease risk, declines with tree diversity.

Understanding why diversity sometimes limits disease is essential for managing outbreaks; however, mechanisms underlying this 'dilution effect' remain poorly understood. Negative diversity-disease relationships have previously been detected in plant communities impacted by an emerging forest disease, sudden oak death. We used this focal system to empirically evaluate whether these relationships were driven by dilution mechanisms that reduce transmission risk for individuals or from the fact that disease was averaged across the host community. We integrated laboratory competence measurements with plant community and symptom data from a large forest monitoring network. Richness increased disease risk for bay laurel trees, dismissing possible dilution mechanisms. Nonetheless, richness was negatively associated with community-level disease prevalence because the disease was aggregated among hosts that vary in disease susceptibility. Aggregating observations (which is surprisingly common in other dilution effect studies) can lead to misinterpretations of dilution mechanisms and bias towards a negative diversity-disease relationship.

Sporulation Potential of Phytophthora ramorum Differs Among Common California Plant Species in the Big Sur Region.

Sudden oak death (SOD), caused by the generalist pathogen Phytophthora ramorum, has profoundly impacted California coastal ecosystems. SOD has largely been treated as a two-host system, with Umbellularia californica as the most transmissive host, Notholithocarpus densiflorus less so, and remaining species as epidemiologically unimportant. However, this understanding of transmission potential primarily stems from observational field studies rather than direct measurements on the diverse assemblage of plant species. Here, we formally quantify the sporulation potential of common plant species inhabiting SOD-endemic ecosystems on the California coast in the Big Sur region. This study allows us to better understand the pathogen's basic biology, trajectory of SOD in a changing environment, and how the entire host community contributes to disease risk. Leaves were inoculated in a controlled laboratory environment and assessed for production of sporangia and chlamydospores, the infectious and resistant propagules, respectively. P. ramorum was capable of infecting every species in our study and almost all species produced spores to some extent. Sporangia production was greatest in N. densiflorus and U. californica and the difference was insignificant. Even though other species produced much less, quantities were nonzero. Thus, additional species may play a previously unrecognized role in local transmission. Chlamydospore production was highest in Acer macrophyllum and Ceanothus oliganthus, raising questions about the role they play in pathogen persistence. Lesion size did not consistently correlate with the production of either sporangia or chlamydospores. Overall, we achieved an empirical foundation to better understand how community composition affects transmission of P. ramorum.

Direct and indirect effects of forest microclimate on pathogen spillover.

Disease dynamics are governed by variation of individuals, species, and environmental conditions across space and time. In some cases, an alternate reservoir host amplifies pathogen loads and drives disease transmission to less competent hosts in a process called pathogen spillover. Spillover is frequently associated with multi-host disease systems where a single species is more tolerant of infection and more competent in pathogen transmission compared to other hosts. Pathogen spillover must be driven by biotic factors, including host and community characteristics, yet biotic factors interact with the abiotic environment (e.g., temperature) to create disease. Despite its fundamental role in disease dynamics, the influence of the abiotic environment on pathogen spillover has seldom been examined. Improving our understanding of disease processes such as pathogen spillover hinges on disentangling the effects of interrelated biotic and abiotic factors over space and time. We applied 10 yr of fine-scale microclimate, disease, and tree community data in a path analysis to investigate the relative influence of biotic and abiotic factors on pathogen spillover for the emerging infectious forest disease sudden oak death (SOD). Disease transmission in SOD is primarily driven by the reservoir host California bay laurel, which supports high foliar pathogen loads that spillover onto neighboring oak trees and create lethal canker infections. The foliar pathogen load and susceptibility of oaks is expected to be sensitive to forest microclimate conditions. We found that biotic factors of pathogen load and tree diversity had relatively stronger effects on pathogen spillover compared to abiotic microclimate factors, with pathogen load increasing oak infection and tree diversity reducing oak infection. Abiotic factors still had significant effects, with greater heat exposure during summer months reducing pathogen loads and optimal pathogen conditions during the wet season increasing oak infection. Our results offer clues to possible disease dynamics under future climate change where hotter and drier or warmer and wetter conditions could have opposing effects on pathogen spillover in the SOD system. Disentangling direct and indirect effects of biotic and abiotic factors affecting disease processes can provide key insights into disease dynamics including potential avenues for reducing disease spread and predicting future epidemics.

Characterization of phenotypic variation and genome aberrations observed among Phytophthora ramorum isolates from diverse hosts.

BACKGROUND: Accumulating evidence suggests that genome plasticity allows filamentous plant pathogens to adapt to changing environments. Recently, the generalist plant pathogen Phytophthora ramorum has been documented to undergo irreversible phenotypic alterations accompanied by chromosomal aberrations when infecting trunks of mature oak trees (genus Quercus). In contrast, genomes and phenotypes of the pathogen derived from the foliage of California bay (Umbellularia californica) are usually stable. We define this phenomenon as host-induced phenotypic diversification (HIPD). P. ramorum also causes a severe foliar blight in some ornamental plants such as Rhododendron spp. and Viburnum spp., and isolates from these hosts occasionally show phenotypes resembling those from oak trunks that carry chromosomal aberrations. The aim of this study was to investigate variations in phenotypes and genomes of P. ramorum isolates from non-oak hosts and substrates to determine whether HIPD changes may be equivalent to those among isolates from oaks. RESULTS: We analyzed genomes of diverse non-oak isolates including those taken from foliage of Rhododendron and other ornamental plants, as well as from natural host species, soil, and water. Isolates recovered from artificially inoculated oak logs were also examined. We identified diverse chromosomal aberrations including copy neutral loss of heterozygosity (cnLOH) and aneuploidy in isolates from non-oak hosts. Most identified aberrations in non-oak hosts were also common among oak isolates; however, trisomy, a frequent type of chromosomal aberration in oak isolates was not observed in isolates from Rhododendron. CONCLUSION: This work cross-examined phenotypic variation and chromosomal aberrations in P. ramorum isolates from oak and non-oak hosts and substrates. The results suggest that HIPD comparable to that occurring in oak hosts occurs in non-oak environments such as in Rhododendron leaves. Rhododendron leaves are more easily available than mature oak stems and thus can potentially serve as a model host for the investigation of HIPD, the newly described plant-pathogen interaction.

Distinct Trophic Specializations Affect How Phytophthora ramorum and Clade 6 Phytophthora spp. Colonize and Persist on Umbellularia californica Leaves in Streams.

Phytophthora spp. are regularly recovered from streams but their ecology in aquatic environments is not well understood. Phytophthora ramorum, invasive in California forests, persists in streams at times when sporulation in the canopy is absent, suggesting that it reproduces in the water. Streams are also inhabited by resident, clade 6 Phytophthora spp., believed to be primarily saprotrophic. We conducted experiments to determine whether differences of trophic specialization exist between these two taxa, and investigated how this may affect their survival and competition on stream leaf litter. P. ramorum effectively colonized fresh (live) rhododendron leaves but not those killed by freezing or drying, whereas clade 6 species colonized all leaf types. However, both taxa were recovered from naturally occurring California bay leaf litter in streams. In stream experiments, P. ramorum colonized bay leaves rapidly at the onset; however, colonization was quickly succeeded by clade 6 species. Nevertheless, both taxa persisted in leaves over 16 weeks. Our results confirm that clade 6 Phytophthora spp. are competent saprotrophs and, though P. ramorum could not colonize dead tissue, early colonization of suitable litter allowed it to survive at a low level in decomposing leaves.

Structural Insight into Acyl-ACP Thioesterase toward Substrate Specificity Design.

Acyl-ACP thioesterase (TE) catalyzes the hydrolysis of thioester bonds during type II fatty acid synthesis and directly determines fatty acid chain length. Most TEs are responsible for recognition of 16:0 and 18:1 substrates, while specific TEs interrupt acyl-ACP elongation at C8-C14. However, the acyl selection mechanism of TE has not been thoroughly elucidated to date. In this study, the crystal structure of the C12-specific thioesterase FatB from Umbellularia californica, which consists of two independent hotdog domains, was determined. An uncanonical Asp-His-Glu catalytic network was identified on the C-terminal hotdog domain, whereas the substrate binding pocket was determined to be on the N-terminal hotdog domain. Moreover, we elucidated UcFatB's substrate selection mechanism, which is accommodated by several unconservative amino acids on the ß5, ß2, and ß4 sheets and enclosed by T137 on the α1 helix. On this basis, the C12-specific TE was rationally redesigned toward C14 selectivity by tuning the substrate binding pocket capacity. The T137G mutant demonstrated comparative relative activity on C14 substrates compared to C12 substrates in vitro. Furthermore, the reconstructed UcFatB_T137G achieved C14 fatty acid content up to 40% in contrast to 10% C14 from the wild type in engineered E. coli cells. The unraveled substrate selection mechanism of TE provides a new strategy for tailoring fatty acid synthesis.

Botryosphaeriaceae species associated with dieback and canker disease of bay laurel in northern California with the description of Dothiorella californica sp. nov.

Members of the Botryosphaeriaceae are cosmopolitan fungi that may exist as seemingly innocuous endophytes or as destructive pathogens of numerous woody hosts, including fruit and nut crops, urban ornamental trees and shrubs, and forest trees. Surveys of bay laurel in northern California have revealed symptoms of dieback and branch canker of unknown aetiology. The goals of this study were to identify and clarify the species of Botryosphaeriaceae associated with these symptoms and to confirm their pathogenicity. To understand the role of members of the Botryosphaeriaceae in the dieback and canker disease of bay laurel, 23 isolates were isolated from symptomatic wood. Phylogenetic analyses of ITS, translation elongation factor 1-α, and beta-tubulin revealed three species: Botryosphaeria dothidea, Neofusicoccum nonquaesitum, and the newly described and typified species Dothiorella californica sp. nov. When select isolates were inoculated to 2- to 3-year-old branches of Umbellularia californica in a natural forest, both B. dothidea and N. nonquaesitum were pathogenic with N. nonquaesitum producing the largest lesions at 12- and 18-months post inoculation, respectively, while Do. californica did not cause wood lesions significantly greater than the mock-inoculated controls. This study represents the first attempt to identify and test the pathogenicity of Botryosphaeriaceae species associated with dieback and canker disease of bay laurel in a northern California forest.

Which Bay Leaf is in Your Spice Rack? - A Quality Control Study.

The accurate identification of bay leaf in natural products commerce may often be confusing as the name is applied to several different species of aromatic plants. The true "bay leaf", also known as "bay laurel" or "sweet bay", is sourced from the tree Laurus nobilis, a native of the Mediterranean region. Nevertheless, the leaves of several other species including Cinnamomum tamala, Litsea glaucescens, Pimenta racemosa, Syzygium polyanthum, and Umbellularia californica are commonly substituted or mistaken for true bay leaves due to their similarity in the leaf morphology, aroma, and flavor. Substitute species are, however, often sold as "bay leaves". As such, the name "bay leaf" in literature and herbal commerce may refer to any of these botanicals. The odor and flavor of these leaves are, however, not the same as the true bay leaf, and for that reason they should not be used in cooking as a substitute for L. nobilis. Some of the bay leaf substitutes can also cause potential health problems. Therefore, the correct identification of the true bay leaf is important. The present work provides a detailed comparative study of the leaf morphological and anatomical features of L. nobilis and its common surrogates to allow for correct identification.

Accumulation of medium-chain, saturated fatty acyl moieties in seed oils of transgenic Camelina sativa.

With its high seed oil content, the mustard family plant Camelina sativa has gained attention as a potential biofuel source. As a bioenergy crop, camelina has many advantages. It grows on marginal land with low demand for water and fertilizer, has a relatively short life cycle, and is stress tolerant. As most other crop seed oils, camelina seed triacylglycerols (TAGs) consist of mostly long, unsaturated fatty acyl moieties, which is not desirable for biofuel processing. In our efforts to produce shorter, saturated chain fatty acyl moieties in camelina seed oil for conversion to jet fuel, a 12:0-acyl-carrier thioesterase gene, UcFATB1, from California bay (Umbellularia californica Nutt.) was expressed in camelina seeds. Up to 40% of short chain laurate (C12:0) and myristate (C14:0) were present in TAGs of the seed oil of the transgenics. The total oil content and germination rate of the transgenic seeds were not affected. Analysis of positions of these two fatty acyl moieties in TAGs indicated that they were present at the sn-1 and sn-3 positions, but not sn-2, on the TAGs. Suppression of the camelina KASII genes by RNAi constructs led to higher accumulation of palmitate (C16:0), from 7.5% up to 28.5%, and further reduction of longer, unsaturated fatty acids in seed TAGs. Co-transformation of camelina with both constructs resulted in enhanced accumulation of all three medium-chain, saturated fatty acids in camelina seed oils. Our results show that a California bay gene can be successfully used to modify the oil composition in camelina seed and present a new biological alternative for jet fuel production.

Host Phenology and Leaf Effects on Susceptibility of California Bay Laurel to Phytophthora ramorum.

Spread of the plant pathogen Phytophthora ramorum, causal agent of the forest disease sudden oak death, is driven by a few competent hosts that support spore production from foliar lesions. The relationship between traits of a principal foliar host, California bay laurel (Umbellularia californica), and susceptibility to P. ramorum infection were investigated with multiple P. ramorum isolates and leaves collected from multiple trees in leaf-droplet assays. We examined whether susceptibility varies with season, leaf age, or inoculum position. Bay laurel susceptibility was highest during spring and summer and lowest in winter. Older leaves (>1 year) were more susceptible than younger ones (8 to 11 months). Susceptibility was greater at leaf tips and edges than the middle of the leaf. Leaf surfaces wiped with 70% ethanol were more susceptible to P. ramorum infection than untreated leaf surfaces. Our results indicate that seasonal changes in susceptibility of U. californica significantly influence P. ramorum infection levels. Thus, in addition to environmental variables such as temperature and moisture, variability in host plant susceptibility contributes to disease establishment of P. ramorum.

Antibacterial activity of native California medicinal plant extracts isolated from Rhamnus californica and Umbellularia californica.

BACKGROUND: Antimicrobial resistance (AMR) is a major threat to global public health. Medicinal plants have long been used as remedies for infectious diseases by native cultures around the world and have the potential for providing effective treatments for antibiotic-resistant infections. Rhamnus californica (Rhamnaceae) and Umbellularia californica (Lauraceae) are two indigenous California plant species historically used by Native Americans to treat skin, respiratory and gastrointestinal infections. This study aimed to assess the in vitro antimicrobial activity of methanolic extracts of leaves and bark of R. and U. californica against methicillin-resistant Staphylococcus aureus (MRSA) and other Gram-positive and Gram-negative bacteria. METHODS: Methanolic extracts of leaves and bark of R. and U. californica were prepared by soxhlet extraction and evaluated for their antimicrobial activity against Bacillus cereus, Streptococcus pyogenes, Mycobacterium smegmatis, Staphylococcus aureus, MRSA, Escherichia coli and Pseudomonas aeruginosa using disc diffusion and minimal inhibitory concentration (MIC) assays. Chemical profiling of the extracts was performed using standard methods. RESULTS: All extracts inhibited the growth of MRSA and other Gram-positive bacteria with MICs of 3.3-6.0 mg/ml. Gram-negative organisms were unaffected by these extracts. U. californica extracts (leaves and bark) had the lowest MIC values. Chemical profiling detected the presence of quinones, alkaloids, flavonoids, cardenolides, tannins and saponins in these extracts. Our study is the first to report the antimicrobial properties of R. and U. californica and illustrates their promising anti-MRSA potential. CONCLUSIONS: Our results give scientific credence to the traditional medicinal uses of these plants by the indigenous peoples of California. Further investigation of the secondary metabolites responsible for the antimicrobial activity of these extracts against MRSA is warranted.

Microclimate impacts survival and prevalence of Phytophthora ramorum in Umbellularia californica, a key reservoir host of sudden oak death in Northern California forests.

Phytophthora ramorum, an invasive pathogen and the causal agent of Sudden Oak Death, has become established in mixed-evergreen and redwood forests in coastal northern California. While oak and tanoak mortality is the most visible indication of P. ramorum's presence, epidemics are largely driven by the presence of bay laurel (Umbellularia californica), a reservoir host that supports both prolific sporulation in the winter wet season and survival during the summer dry season. In order to better understand how over-summer survival of the pathogen contributes to variability in the severity of annual epidemics, we monitored the viability of P. ramorum leaf infections over three years along with coincident microclimate. The proportion of symptomatic bay laurel leaves that contained viable infections decreased during the first summer dry season and remained low for the following two years, likely due to the absence of conducive wet season weather during the study period. Over-summer survival of P. ramorum was positively correlated with high percent canopy cover, less negative bay leaf water potential and few days exceeding 30°C but was not significantly different between mixed-evergreen and redwood forest ecosystems. Decreased summer survival of P. ramorum in exposed locations and during unusually hot summers likely contributes to the observed spatiotemporal heterogeneity of P. ramorum epidemics.

Improving the tolerance of Escherichia coli to medium-chain fatty acid production.

Microbial fatty acids are an attractive source of precursors for a variety of renewable commodity chemicals such as alkanes, alcohols, and biofuels. Rerouting lipid biosynthesis into free fatty acid production can be toxic, however, due to alterations of membrane lipid composition. Here we find that membrane lipid composition can be altered by the direct incorporation of medium-chain fatty acids into lipids via the Aas pathway in cells expressing the medium-chain thioesterase from Umbellularia californica (BTE). We find that deletion of the aas gene and sequestering exported fatty acids reduces medium-chain fatty acid toxicity, partially restores normal lipid composition, and improves medium-chain fatty acid yields.

Efficient odd straight medium chain free fatty acid production by metabolically engineered Escherichia coli.

Free fatty acids (FFAs) can be used as precursors for the production of biofuels or chemicals. Different composition of FFAs will be useful for further modification of the biofuel/biochemical quality. Microbial biosynthesis of even chain FFAs can be achieved by introducing an acyl-acyl carrier protein thioesterase gene into E. coli. In this study, odd straight medium chain FFAs production was investigated by using metabolic engineered E. coli carrying acyl-ACP thioesterase (TE, Ricinus communis), propionyl-CoA synthase (Salmonella enterica), and ß-ketoacyl-acyl carrier protein synthase III (four different sources) with supplement of extracellular propionate. By using these metabolically engineered E. coli, significant quantity of C13 and C15 odd straight-chain FFAs could be produced from glucose and propionate. The highest concentration of total odd straight chain FFAs attained was 1205 mg/L by the strain HWK201 (pXZ18, pBHE2), and 85% of the odd straight chain FFAs was C15. However, the highest percentage of odd straight chain FFAs was achieved by the strain HWK201 (pXZ18, pBHE3) of 83.2% at 48 h. This strategy was also applied successfully in strains carrying different TE, such as the medium length acyl-ACP thioesterase gene from Umbellularia californica. C11 and C13 became the major odd straight-chain FFAs.

Comparative investigation of Umbellularia californica and Laurus nobilis leaf essential oils and identification of constituents active against Aedes aegypti.

Umbellularia californica (California bay laurel) and Laurus nobilis (Mediterranean bay laurel) leaves may be mistaken or used as a substitute on the market due to their morphological similarity. In this study, a comparison of anatomical and chemical features and biological activity of both plants is presented. L. nobilis essential oil biting deterrent and larvicidal activity were negligible. On the other hand, U. californica leaf oil showed biting deterrent activity against Aedes aegypti . The identified active repellents was thymol, along with (-)-umbellulone, 1,8-cineole, and (-)-α-terpineol. U. californica essential oil also demonstrated good larvicidal activity against 1-day-old Ae. aegypti larvae with a LD50 value of 52.6 ppm. Thymol (LD50 = 17.6 ppm), p-cymene, (-)-umbellulone, and methyleugenol were the primary larvicidal in this oil. Umbellulone was found as the principal compound (37%) of U. californica essential oil, but was not present in L. nobilis essential oil. Umbellulone mosquito activity is here reported for the first time.

Mortality and community changes drive sudden oak death impacts on litterfall and soil nitrogen cycling.

Few studies have quantified pathogen impacts to ecosystem processes, despite the fact that pathogens cause or contribute to regional-scale tree mortality. We measured litterfall mass, litterfall chemistry, and soil nitrogen (N) cycling associated with multiple hosts along a gradient of mortality caused by Phytophthora ramorum, the cause of sudden oak death. In redwood forests, the epidemiological and ecological characteristics of the major overstory species determine disease patterns and the magnitude and nature of ecosystem change. Bay laurel (Umbellularia californica) has high litterfall N (0.992%), greater soil extractable NO3 -N, and transmits infection without suffering mortality. Tanoak (Notholithocarpus densiflorus) has moderate litterfall N (0.723%) and transmits infection while suffering extensive mortality that leads to higher extractable soil NO3 -N. Redwood (Sequoia sempervirens) has relatively low litterfall N (0.519%), does not suffer mortality or transmit the pathogen, but dominates forest biomass. The strongest impact of pathogen-caused mortality was the potential shift in species composition, which will alter litterfall chemistry, patterns and dynamics of litterfall mass, and increase soil NO3 -N availability. Patterns of P. ramorum spread and consequent mortality are closely associated with bay laurel abundances, suggesting this species will drive both disease emergence and subsequent ecosystem function.

Effect of plant sterols and tannins on Phytophthora ramorum growth and sporulation.

Elicitin-mediated acquisition of plant sterols is required for growth and sporulation of Phytophthora spp. This study examined the interactions between elicitins, sterols, and tannins. Ground leaf tissue, sterols, and tannin-enriched extracts were obtained from three different plant species (California bay laurel, California black oak, and Oregon white oak) in order to evaluate the effect of differing sterol/tannin contents on Phytophthora ramorum growth. For all three species, high levels of foliage inhibited P. ramorum growth and sporulation, with a steeper concentration dependence for the two oak samples. Phytophthora ramorum growth and sporulation were inhibited by either phytosterols or tannin-enriched extracts. High levels of sterols diminished elicitin gene expression in P. ramorum; whereas the tannin-enriched extract decreased the amount of 'functional' or ELISA-detectable elicitin, but not gene expression. Across all treatment combinations, P. ramorum growth and sporulation correlated strongly with the amount of ELISA-detectable elicitin (R (2) = 0.791 and 0.961, respectively).

The key host for an invasive forest pathogen also facilitates the pathogen's survival of wildfire in California forests.

The first wildfires in sudden oak death-impacted forests occurred in 2008 in the Big Sur region of California, creating the rare opportunity to study the interaction between an invasive forest pathogen and a historically recurring disturbance. To determine whether and how the sudden oak death pathogen, Phytophthora ramorum, survived the wildfires, we completed intensive vegetation-based surveys in forest plots that were known to be infested before the wildfires. We then used 24 plot-based variables as predictors of P. ramorum recovery following the wildfires. The likelihood of recovering P. ramorum from burned plots was lower than in unburned plots both 1 and 2 yr following the fires. Post-fire recovery of P. ramorum in burned plots was positively correlated with the number of pre-fire symptomatic California bay laurel (Umbellularia californica), the key sporulating host for this pathogen, and negatively correlated with post-fire bay laurel mortality levels. Patchy burn patterns that left green, P. ramorum-infected bay laurel amidst the charred landscape may have allowed these trees to serve as inoculum reservoirs that could lead to the infection of newly sprouting vegetation, further highlighting the importance of bay laurel in the sudden oak death disease cycle.

Phenotypic diversification is associated with host-induced transposon derepression in the sudden oak death pathogen Phytophthora ramorum.

The oomycete pathogen Phytophthora ramorum is responsible for sudden oak death (SOD) in California coastal forests. P. ramorum is a generalist pathogen with over 100 known host species. Three or four closely related genotypes of P. ramorum (from a single lineage) were originally introduced in California forests and the pathogen reproduces clonally. Because of this the genetic diversity of P. ramorum is extremely low in Californian forests. However, P. ramorum shows diverse phenotypic variation in colony morphology, colony senescence, and virulence. In this study, we show that phenotypic variation among isolates is associated with the host species from which the microbe was originally cultured. Microarray global mRNA profiling detected derepression of transposable elements (TEs) and down-regulation of crinkler effector homologs (CRNs) in the majority of isolates originating from coast live oak (Quercus agrifolia), but this expression pattern was not observed in isolates from California bay laurel (Umbellularia californica). In some instances, oak and bay laurel isolates originating from the same geographic location had identical genotypes based on multilocus simples sequence repeat (SSR) marker analysis but had different phenotypes. Expression levels of the two marker genes analyzed by quantitative reverse transcription PCR were correlated with originating host species, but not with multilocus genotypes. Because oak is a nontransmissive dead-end host for P. ramorum, our observations are congruent with an epi-transposon hypothesis; that is, physiological stress is triggered on P. ramorum while colonizing oak stems and disrupts epigenetic silencing of TEs. This then results in TE reactivation and possibly genome diversification without significant epidemiological consequences. We propose the P. ramorum-oak host system in California forests as an ad hoc model for epi-transposon mediated diversification.