Allopatric speciation is more prevalent than parapatric ecological divergence in a recent high-Andean diversification (Linochilus: Asteraceae).

Elucidating how species accumulate in diversity hotspots is an ongoing debate in evolutionary biology. The páramo, in the Northern Andes, has remarkably high indices of plant diversity, endemicity, and diversification rates. A hypothesis for explaining such indices is that allopatric speciation is high in the páramo given its island-like distribution. An alternative hypothesis is that the altitudinal gradient of the Andean topography provides a variety of niches that drive vertical parapatric ecological speciation. A formal test for evaluating the relative roles of allopatric and parapatric ecological speciation is lacking. The main aim of our study is to test which kind of speciation is more common in an endemic páramo genus. We developed a framework incorporating phylogenetics, species' distributions, and a morpho-ecological trait (leaf area) to compare sister species and infer whether allopatric or parapatric ecological divergence caused their speciation. We applied our framework to the species-rich genus Linochilus (63 spp.) and found that the majority of recent speciation events in it (12 events, 80%) have been driven by allopatric speciation, while a smaller fraction (one event, 6.7%) is attributed to parapatric ecological speciation; two pairs of sister species produced inconclusive results (13.3%). We conclude that páramo autochthonous (in-situ) diversification has been primarily driven by allopatric speciation.

The Origins of Coca: Museum Genomics Reveals Multiple Independent Domestications from Progenitor Erythroxylum gracilipes.

Coca is the natural source of cocaine as well as a sacred and medicinal plant farmed by South American Amerindians and mestizos. The coca crop comprises four closely related varieties classified into two species (Amazonian and Huánuco varieties within Erythroxylum coca Lam., and Colombian and Trujillo varieties within Erythroxylum novogranatense (D. Morris) Hieron.) but our understanding of the domestication and evolutionary history of these taxa is nominal. In this study, we use genomic data from natural history collections to estimate the geographic origins and genetic diversity of this economically and culturally important crop in the context of its wild relatives. Our phylogeographic analyses clearly demonstrate the four varieties of coca comprise two or three exclusive groups nested within the diverse lineages of the widespread, wild species Erythroxylum gracilipes; establishing a new and robust hypothesis of domestication wherein coca originated two or three times from this wild progenitor. The Colombian and Trujillo coca varieties are descended from a single, ancient domestication event in northwestern South America. Huánuco coca was domesticated more recently, possibly in southeastern Peru. Amazonian coca either shares a common domesticated ancestor with Huánuco coca, or it was the product of a third and most recent independent domestication event in the western Amazon basin. This chronology of coca domestication reveals different Holocene peoples in South America were able to independently transform the same natural resource to serve their needs; in this case, a workaday stimulant. [Erythroxylum; Erythroxylaceae; Holocene; Museomics; Neotropics; phylogeography; plant domestication; target-sequence capture.].

Corrigendum: Plastid Genomes of Five Species of Riverweeds (Podostemaceae): Structural Organization and Comparative Analysis in Malpighiales.

[This corrects the article DOI: 10.3389/fpls.2019.01035.].

Potent Antifungal Properties of Dimeric Acylphloroglucinols from Hypericum mexicanum and Mechanism of Action of a Highly Active 3'Prenyl Uliginosin B.

The success of antifungal therapies is often hindered by the limited number of available drugs. To close the gap in the antifungal pipeline, the search of novel leads is of primary importance, and here the exploration of neglected plants has great promise for the discovery of new principles. Through bioassay-guided isolation, uliginosin B and five new dimeric acylphloroglucinols (uliginosins C-D, and 3'prenyl uliginosins B-D), besides cembrenoids, have been isolated from the lipophilic extract of Hypericum mexicanum. Their structures were elucidated by a combination of Liquid Chromatography - Mass Spectrometry LC-MS and Nuclear Magnetic Resonance (NMR) measurements. The compounds showed strong anti-Candida activity, also against fluconazole-resistant strains, with fungal growth inhibition properties at concentrations ranging from 3 to 32 µM, and reduced or absent cytotoxicity against human cell lines. A chemogenomic screen of 3'prenyl uliginosin B revealed target genes that are important for cell cycle regulation and cytoskeleton assembly in fungi. Taken together, our study suggests dimeric acylphloroglucinols as potential candidates for the development of alternative antifungal therapies.

Plastid Genomes of Five Species of Riverweeds (Podostemaceae): Structural Organization and Comparative Analysis in Malpighiales.

With the advent of next-generation sequencing technologies, whole-plastome data can be obtained as a byproduct of low-coverage sequencing of the plant genomic DNA. This provides an opportunity to study plastid evolution across groups, as well as testing phylogenetic relationships among taxa. Within the order Malpighiales (∼16,000 spp.), the Podostemaceae (∼300 spp.) stand out for their unique habit, living attached to rocks in fast-flowing aquatic habitats, and displaying highly modified morphologies that confound our understanding of their classification, biology, and evolution. In this study, we used genome skimming data to assemble the full plastid genome of 5 species within Podostemaceae. We analyzed our data in a comparative framework within Malpighiales to determine the structure, gene content, and rearrangements in the plastomes of the family. The Podostemaceae have one of the smallest plastid genomes reported so far for the Malpighiales, possibly due to variation in length of inverted repeat (IR) regions, gene loss, and intergenic region variation. We also detected a major inversion in the large single-copy region unique to the family. The uncommon loss or pseudogenization of ycf1 and ycf2 in angiosperms and in land plants in general is also found to be characteristic of Podostemaceae, but the compensatory mechanisms and implications of this and of the pseudogenization of accD, rpl22, and clpP and loss of rps16 remain to be explained in this group. In addition, we estimated a phylogenetic tree among selected species in Malpighiales. Our findings indicate that the Podostemaceae are a distinct lineage with long branches that suggest faster rates of evolution in the plastome of the group, compared with other taxa in the order. This study lays the foundations for future phylogenomic studies in the family.

On the Causes of Rapid Diversification in the Páramos: Isolation by Ecology and Genomic Divergence in Espeletia.

How diversity arises and what is the relative role of allopatric and ecological divergence are among the most persistent questions in evolution and ecology. Here, we assessed whether ecological divergence has enhanced the diversification of the Neotropical alpine plant complex Espeletia, also known as frailejones. This genus has one of the highest diversification rates ever reported and is distributed in the world's fastest evolving biodiversity hotspot, the Páramo (Neotropical alpine grasslands at elevations of c. 2800-4700 m). Our goal was to determine whether ecology plays a role in divergence within the Espeletia complex by quantifying genome-wide patterns of ecological divergence. We characterized 162 samples of the three most common and contrasting ecotypes (distinct morphotypes occupying particular habitats) co-occurring in six localities in the northern Andes using Genotyping by Sequencing. Contrasting ecotypes were caulescent cloud forest populations, caulescent populations from wind-sheltered and well-irrigated depressions and acaulescent populations from wind-exposed drier slopes. We found high polymorphism with a total of 1,273 single nucleotide polymorphisms (SNPs) that defined the relationships among nine genetic clusters. We quantified allelic associations of these markers with localities and habitats using 18 different general and mixed-effects statistical models that accounted for phylogenetic distance. Despite that these models always yielded more SNPs associated with the localities, markers associated with the habitat types were recovered too. We found strong evidence for isolation-by-distance (IBD) across populations despite rampant gene flow, as expected for plant groups with limited seed dispersal. Contrasts between populations of different habitat types showed that an isolation-by-environment (IBE) trend emerged and masked the IBD signal. Maximum likelihood estimation of the number of migrants per generation (Nem) among ecotypes confirmed the IBE pattern. This result illustrates the importance of mountains' environmental variation at a local scale in generating rapid morphological radiations and maintaining multiple adaptations in a fast-evolving ecosystem like the Páramo.

Transcriptome mining for phylogenetic markers in a recently radiated genus of tropical plants (Renealmia L.f., Zingiberaceae).

The reconstruction of relationships within species-rich groups that have recently evolved in biodiversity hotspots is hampered by a lack of phylogenetically informative markers. It is also made difficult by the lack of sampling necessary to reconstruct a species-level phylogeny. We use transcriptome mining to search for markers to reconstruct a phylogeny of the amphi-Atlantic genus Renealmia L. f. (Zingiberaceae). We recover seven introns from single copy genes and use them to reconstruct the phylogeny of the genus together with a commonly used phylogenetic marker, internal transcribed spacers of ribosomal DNA (ITS) that has previously been used to reconstruct the phylogeny of the genus. We targeted genes with low numbers of base pairs that improves sequencing success using highly degraded DNA from herbarium specimens. The use of herbarium specimens greatly increased the number of species in the study as these were readily available in historical collections. Data were obtained for 14 of the 17 African species and 54 of the 65 Neotropical species. The phylogeny was well-supported for a number of Renealmia subgroups although relationships among those clades remained poorly supported.

Lost crops of the Incas: Origins of domestication of the Andean pulse crop tarwi, Lupinus mutabilis.

PREMISE OF THE STUDY: The Andean highlands are a hotspot of domestication, yet our understanding of the origins of early Andean agriculture remains fragmentary. Key questions of where, when, how many times, and from what progenitors many Andean crops were domesticated remain unanswered. The Andean lupine crop tarwi (Lupinus mutabilis) is a regionally important pulse crop with exceptionally high seed protein and oil content and is the focus of modern breeding efforts, but its origins remain obscure. METHODS: A large genome-wide DNA polymorphism data set was generated using nextRADseq to infer relationships among more than 200 accessions of Andean Lupinus species, including 24 accessions of L. mutabilis and close relatives. Phylogenetic and demographic analyses were used to identify the likely progenitor of tarwi and elucidate the area and timing of domestication in combination with archaeological evidence. KEY RESULTS: We infer that tarwi was domesticated once in northern Peru, most likely in the Cajamarca region within, or adjacent to the extant distribution of L. piurensis, which is the most likely wild progenitor. Demographic analyses suggest that tarwi split from L. piurensis around 2600 BP and suffered a classical domestication bottleneck. The earliest unequivocal archaeological evidence of domesticated tarwi seeds is from the Mantaro Valley, central Peru ca. 1800 BP. CONCLUSIONS: A single origin of tarwi from L. piurensis in northern Peru provides a robust working hypothesis for the domestication of this regionally important crop and is one of the first clear-cut examples of a crop originating in the highlands of northern Peru.

Temperate radiations and dying embers of a tropical past: the diversification of Viburnum.

We used a near-complete phylogeny for the angiosperm clade Viburnum to assess lineage diversification rates, and to examine possible morphological and ecological factors driving radiations. Maximum-likelihood and Bayesian approaches identified shifts in diversification rate and possible links to character evolution. We inferred the ancestral environment for Viburnum and changes in diversification dynamics associated with subsequent biome shifts. Viburnum probably diversified in tropical forests of Southeast Asia in the Eocene, with three subsequent radiations in temperate clades during the Miocene. Four traits (purple fruits, extrafloral nectaries, bud scales and toothed leaves) were statistically associated with higher rates of diversification. However, we argue that these traits are unlikely to be driving diversification directly. Instead, two radiations were associated with the occupation of mountainous regions and a third with repeated shifts between colder and warmer temperate forests. Early-branching depauperate lineages imply that the rare lowland tropical species are 'dying embers' of once more diverse lineages; net diversification rates in Viburnum likely decreased in these tropical environments after the Oligocene. We suggest that 'taxon pulse' dynamics might characterize other temperate plant lineages.

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.

Páramo is the world's fastest evolving and coolest biodiversity hotspot.

Understanding the processes that cause speciation is a key aim of evolutionary biology. Lineages or biomes that exhibit recent and rapid diversification are ideal model systems for determining these processes. Species rich biomes reported to be of relatively recent origin, i.e., since the beginning of the Miocene, include Mediterranean ecosystems such as the California Floristic Province, oceanic islands such as the Hawaiian archipelago and the Neotropical high elevation ecosystem of the Páramos. Páramos constitute grasslands above the forest tree-line (at elevations of c. 2800-4700 m) with high species endemism. Organisms that occupy this ecosystem are a likely product of unique adaptations to an extreme environment that evolved during the last three to five million years when the Andes reached an altitude that was capable of sustaining this type of vegetation. We compared net diversification rates of lineages in fast evolving biomes using 73 dated molecular phylogenies. Based on our sample, we demonstrate that average net diversification rates of Páramo plant lineages are faster than those of other reportedly fast evolving hotspots and that the faster evolving lineages are more likely to be found in Páramos than the other hotspots. Páramos therefore represent the ideal model system for studying diversification processes. Most of the speciation events that we observed in the Páramos (144 out of 177) occurred during the Pleistocene possibly due to the effects of species range contraction and expansion that may have resulted from the well-documented climatic changes during that period. Understanding these effects will assist with efforts to determine how future climatic changes will impact plant populations.

Explosive radiation in high Andean Hypericum-rates of diversification among New World lineages.

The páramos, high-elevation Andean grasslands ranging from ca. 2800 m to the snow line, harbor one of the fastest evolving biomes worldwide since their appearance in the northern Andes 3-5 million years (Ma) ago. Hypericum (St. John's wort), with over 65% of its Neotropical species, has a center of diversity in these high Mountain ecosystems. Using nuclear rDNA internal transcribed spacer (ITS) sequences of a broad sample of New World Hypericum species we investigate phylogenetic patterns, estimate divergence times, and provide the first insights into diversification rates within the genus in the Neotropics. Two lineages appear to have independently dispersed into South America around 3.5 Ma ago, one of which has radiated in the páramos (Brathys). We find strong support for the polyphyly of section Trigynobrathys, several species of which group within Brathys, while others are found in temperate lowland South America (Trigynobrathys s.str.). All páramo species of Hypericum group in one clade. Within these páramo Hypericum species enormous phenotypic evolution has taken place (life forms from arborescent to prostrate shrubs) evidently in a short time frame. We hypothesize multiple mechanisms to be responsible for the low differentiation in the ITS region contrary to the high morphological diversity found in Hypericum in the páramos. Amongst these may be ongoing hybridization and incomplete lineage sorting, as well as the putative adaptive radiation, which can explain the contrast between phenotypic diversity and the close phylogenetic relationships.

Drought tolerance in wild plant populations: the case of common beans (Phaseolus vulgaris L.).

Reliable estimations of drought tolerance in wild plant populations have proved to be challenging and more accessible alternatives are desirable. With that in mind, an ecological diversity study was conducted based on the geographical origin of 104 wild common bean accessions to estimate drought tolerance in their natural habitats. Our wild population sample covered a range of mesic to very dry habitats from Mexico to Argentina. Two potential evapotranspiration models that considered the effects of temperature and radiation were coupled with the precipitation regimes of the last fifty years for each collection site based on geographical information system analysis. We found that wild accessions were distributed among different precipitation regimes following a latitudinal gradient and that habitat ecological diversity of the collection sites was associated with natural sub-populations. We also detected a broader geographic distribution of wild beans across ecologies compared to cultivated common beans in a reference collection of 297 cultivars. Habitat drought stress index based on the Thornthwaite potential evapotranspiration model was equivalent to the Hamon estimator. Both ecological drought stress indexes would be useful together with population structure for the genealogical analysis of gene families in common bean, for genome-wide genetic-environmental associations, and for postulating the evolutionary history and diversification processes that have occurred for the species. Finally, we propose that wild common bean should be taken into account to exploit variation for drought tolerance in cultivated common bean which is generally considered susceptible as a crop to drought stress.

Molecular phylogenetics and morphological evolution of St. John's wort (Hypericum; Hypericaceae).

Phylogenetic hypotheses for the large cosmopolitan genus Hypericum (St. John's wort) have previously been based on morphology, and molecular studies have thus far included only a few species. In this study, we used 360 sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) for 206 species representing Hypericum (incl. Triadenum and Thornea) and three other genera of Hypericaceae to generate an explicit phylogenetic hypothesis for the genus using parsimony and model-based methods. The results indicate that the small genus Triadenum is nested in a clade within Hypericum containing most of the New World species. Sister to Hypericum is Thornea from Central America. Within Hypericum, three large clades and two smaller grades were found; these are based on their general morphology, especially characters used previously in taxonomy of the genus. Relative to the most recent classification, around 60% of the sections of Hypericum were monophyletic. We used a Bayesian approach to reconstruct ancestral states of selected morphological characters, which resulted in recognition of characters that support major clades within the genus and a revised interpretation of morphological evolution in Hypericum. The shrubby habit represents the plesiomorphic state from which herbs evolved several times. Arborescent species have radiated convergently in high-elevation habitats in tropical Africa and South America.

Nucleotide diversity patterns at the drought-related DREB2 encoding genes in wild and cultivated common bean (Phaseolus vulgaris L.).

Common beans are an important food legume faced with a series of abiotic stresses the most severe of which is drought. The crop is interesting as a model for the analysis of gene phylogenies due to its domestication process, race structure, and origins in a group of wild common beans found along the South American Andes and the region of Mesoamerica. Meanwhile, the DREB2 transcription factors have been implicated in controlling non-ABA dependent responses to drought stress. With this in mind our objective was to study in depth the genetic diversity for two DREB2 genes as possible candidates for association with drought tolerance through a gene phylogenetic analysis. In this genetic diversity assessment, we analyzed nucleotide diversity at the two candidate genes Dreb2A and Dreb2B, in partial core collections of 104 wild and 297 cultivated common beans with a total of 401 common bean genotypes from world-wide germplasm analyzed. Our wild population sample covered a range of semi-mesic to very dry habitats, while our cultivated samples presented a wide spectrum of low to high drought tolerance. Both genes showed very different patterns of nucleotide variation. Dreb2B exhibited very low nucleotide diversity relative to neutral reference loci previously surveyed in these populations. This suggests that strong purifying selection has been acting on this gene. In contrast, Dreb2A exhibited higher levels of nucleotide diversity, which is indicative of adaptive selection and population expansion. These patterns were more distinct in wild compared to cultivated common beans. These approximations suggested the importance of Dreb2 genes in the context of drought tolerance, and constitute the first steps towards an association study between genetic polymorphism of this gene family and variation in drought tolerance traits. We discuss the utility of allele mining in the DREB gene family for the discovery of new drought tolerance traits from wild common bean.

Relationships between the litter colonization by saprotrophic and arbuscular mycorrhizal fungi with depth in a tropical forest.

Fungal colonization of litter has been described mostly in terms of fructification succession in the decomposition process or the process of fungal ligninolysis. No studies have been conducted on litter colonization by arbuscular mycorrhizal fungi (AMF) and their relationship with the presence of saprotrophic fungi. The aim of the present study was to evaluate the relationships that exist in simultaneous leaf litter colonization by AMF and saprotrophic fungi and the relationships between rates of litter and associated root colonization by AMF at different soil depths. We selected Eugenia sp. and Syzygium sp. in a riparian tropical forest, with an abundant production of litter (O horizon), we evaluated litter and root colonization at different depths, its C:N ratios, and the edaphic physico-chemical parameters of the A horizon immediately below the litter layer. Litter colonization by saprotrophic fungi and AMF increased with depth, but the saprotrophic fungal colonization of some litter fragments decreased in the lowermost level of the litter while AMF litter colonization continued to increase. Plant roots were present only in the middle and bottom layers, but their mycorrhizal colonization did not correlate with litter colonization. The external hyphae length of AMF is abundant (ca. 20 m g(-1) sample) and, in common with sample humidity, remained constant with increasing depth. We conclude that in zones of riparian tropical forest with abundant sufficient litter accumulation and abundant AMF external hyphae, the increase in litter colonization by AMF with depth correlates to the colonization by saprotrophic fungi, but their presence in the deepest layers is independent of both litter colonization by saprotrophic fungi and root colonization by AMF.

Molecular ecology and selection in the drought-related Asr gene polymorphisms in wild and cultivated common bean (Phaseolus vulgaris L.).

BACKGROUND: The abscisic acid (ABA) pathway plays an important role in the plants' reaction to drought stress and ABA-stress response (Asr) genes are important in controlling this process. In this sense, we accessed nucleotide diversity at two candidate genes for drought tolerance (Asr1 and Asr2), involved in an ABA signaling pathway, in the reference collection of cultivated common bean (Phaseolus vulgaris L.) and a core collection of wild common bean accessions. RESULTS: Our wild population samples covered a range of mesic (semi-arid) to very dry (desert) habitats, while our cultivated samples presented a wide spectrum of drought tolerance. Both genes showed very different patterns of nucleotide variation. Asr1 exhibited very low nucleotide diversity relative to the neutral reference loci that were previously surveyed in these populations. This suggests that strong purifying selection has been acting on this gene. In contrast, Asr2 exhibited higher levels of nucleotide diversity, which is indicative of adaptive selection. These patterns were more notable in wild beans than in cultivated common beans indicting that natural selection has played a role over long time periods compared to farmer selection since domestication. CONCLUSIONS: Together these results suggested the importance of Asr1 in the context of drought tolerance, and constitute the first steps towards an association study between genetic polymorphism of this gene family and variation in drought tolerance traits. Furthermore, one of our major successes was to find that wild common bean is a reservoir of genetic variation and selection signatures at Asr genes, which may be useful for breeding drought tolerance in cultivated common bean.

The Great American Biotic Interchange in frogs: multiple and early colonization of Central America by the South American genus Pristimantis (Anura: Craugastoridae).

The completion of the land bridge between North and South America approximately 3.5-3.1 million years ago (Ma) initiated a tremendous biogeographic event called the Great American Biotic Interchange (GABI), described principally from the mammalian fossil record. The history of biotic interchange between continents for taxonomic groups with poor fossil records, however, is not well understood. Molecular and fossil data suggest that a number of plant and animal lineages crossed the Isthmus of Panama well before 3.5 Ma, leading biologists to speculate about trans-oceanic dispersal mechanisms. Here we present a molecular phylogenetic analysis of the frog genus Pristimantis based on 189 individuals of 137 species, including 71 individuals of 31 species from Panama and Colombia. DNA sequence data were obtained from three mitochondrial (COI, 12S, 16S) and two nuclear (RAG-1 and Tyr) genes, for a total of 4074 base pairs. The resulting phylogenetic hypothesis showed statistically significant conflict with most recognized taxonomic groups within Pristimantis, supporting only the rubicundus Species Series, and the Pristimantis myersi and Pristimantis pardalis Species Groups as monophyletic. Inference of ancestral areas based on a likelihood model of geographic range evolution via dispersal, local extinction, and cladogenesis (DEC) suggested that the colonization of Central America by South American Pristimantis involved at least 11 independent events. Relaxed-clock analyses of divergence times suggested that at least eight of these invasions into Central America took place prior to 4 Ma, mainly in the Miocene. These findings contribute to a growing list of molecular-based biogeographic studies presenting apparent temporal conflicts with the traditional GABI model.

Chemical composition and antibacterial activity of the essential oil of Drimys granadensis L.f. leaves from Colombia.

Drimys granadensis L.f., a native plant from Colombia, has been included in the Vademecum de Plantas Medicinales de Colombia by the Instituto de Investigación de Recursos Biológicos Alexander von Humboldt, due to its widespread use in the folk medicine for the treatment of gastrointestinal ailments. The chemical composition of the essential oil of Drimys granadensis obtained by hydrodistillation of the leaves was analyzed by GC and GC/MS analyses. A total of 85 components were identified, with the major compounds germacrene D (1, 14.7%), sclarene (9.5%), a-cadinol (7.3%), longiborneol acetate (2, 6.3%), drimenol (4.2%), (Z)-ß-ocimene (3, 4.2%), a-pinene (4, 3.2%), and ß-elemene (5, 2.7%). The essential oil was also tested against eight bacteria using the Kirby-Bauer disk-diffusion method. Most of the Gram-positive bacteria tested were susceptible to the D. granadensis essential oil, while the Gram-negative bacteria tested were not.