Rhizospheric bacteria from the Atacama Desert hyper-arid core: cultured community dynamics and plant growth promotion.

The Atacama Desert is the oldest and driest desert on Earth, encompassing great temperature variations, high ultraviolet radiation, drought, and high salinity, making it ideal for studying the limits of life and resistance strategies. It is also known for harboring a great biodiversity of adapted life forms. While desertification is increasing as a result of climate change and human activities, it is necessary to optimize soil and water usage, where stress-resistant crops are possible solutions. As many studies have revealed the great impact of the rhizobiome on plant growth efficiency and resistance to abiotic stress, we set up to explore the rhizospheric soils of Suaeda foliosa and Distichlis spicata desert plants. By culturing these soils and using 16S rRNA amplicon sequencing, we address community taxonomy composition dynamics, stability through time, and the ability to promote lettuce plant growth. The rhizospheric soil communities were dominated by the families Pseudomonadaceae, Bacillaceae, and Planococcaceae for S. foliosa and Porphyromonadaceae and Haloferacaceae for D. spicata. Nonetheless, the cultures were completely dominated by the Enterobacteriaceae family (up to 98%). Effectively, lettuce plants supplemented with the cultures showed greater size and biomass accumulation. We identified 12 candidates that could be responsible for these outcomes, of which 5 (Enterococcus, Pseudomonas, Klebsiella, Paenisporosarcina, and Ammoniphilus) were part of the built co-occurrence network. We aim to contribute to the efforts to characterize the microbial communities as key for the plant's survival in extreme environments and as a possible source of consortia with plant growth promotion traits aimed at agricultural applications.IMPORTANCEThe current scenario of climate change and desertification represents a series of incoming challenges for all living organisms. As the human population grows rapidly, so does the rising demand for food and natural resources; thus, it is necessary to make agriculture more efficient by optimizing soil and water usage, thus ensuring future food supplies. Particularly, the Atacama Desert (northern Chile) is considered the most arid place on Earth as a consequence of geological and climatic characteristics, such as the naturally low precipitation patterns and high temperatures, which makes it an ideal place to carry out research that seeks to aid agriculture in future conditions that are predicted to resemble these scenarios. Our main interest lies in utilizing microorganism consortia from plants thriving under extreme conditions, aiming to promote plant growth, improve crops, and render "unsuitable" soils farmable.

[The model transferability of AI in digital pathology : Potential and reality]. / Die Modelltransferierbarkeit von KI in der digitalen Pathologie : Potenzial und Realität.

OBJECTIVE: Artificial intelligence (AI) holds the potential to make significant advancements in pathology. However, its actual implementation and certification for practical use are currently limited, often due to challenges related to model transferability. In this context, we investigate the factors influencing transferability and present methods aimed at enhancing the utilization of AI algorithms in pathology. MATERIALS AND METHODS: Various convolutional neural networks (CNNs) and vision transformers (ViTs) were trained using datasets from two institutions, along with the publicly available TCGA-MIBC dataset. These networks conducted predictions in urothelial tissue and intrahepatic cholangiocarcinoma (iCCA). The objective was to illustrate the impact of stain normalization, the influence of various artifacts during both training and testing, as well as the effects of the NoisyEnsemble method. RESULTS: We were able to demonstrate that stain normalization of slides from different institutions has a significant positive effect on the inter-institutional transferability of CNNs and ViTs (respectively +13% and +10%). In addition, ViTs usually achieve a higher accuracy in the external test (here +1.5%). Similarly, we showcased how artifacts in test data can negatively affect CNN predictions and how incorporating these artifacts during training leads to improvements. Lastly, NoisyEnsembles of CNNs (better than ViTs) were shown to enhance transferability across different tissues and research questions (+7% Bladder, +15% iCCA). DISCUSSION: It is crucial to be aware of the transferability challenge: achieving good performance during development does not necessarily translate to good performance in real-world applications. The inclusion of existing methods to enhance transferability, such as stain normalization and NoisyEnsemble, and their ongoing refinement, is of importance.

[Histomolecular classification of urothelial carcinoma of the urinary bladder : From histological phenotype to genotype and back]. / Histomolekulare Klassifikation des Urothelkarzinoms der Harnblase : Vom histologischen Phänotyp zum Genotyp und zurück.

BACKGROUND: Of all urothelial carcinomas (UCs), 25% are muscle invasive and associated with a 5-year overall survival rate of 50%. Findings regarding the molecular classification of muscle-invasive urothelial carcinomas (MIUCs) have not yet found their way into clinical practice. OBJECTIVES: Prediction of molecular consensus subtypes in MIUCs with artificial intelligence (AI) based on histologic hematoxylin-eosin (HE) sections. METHODS: Pathologic review and annotation of The Cancer Genome Atlas (TCGA) Bladder Cancer (BLCA) Cohort (N = 412) and the Dr. Senckenberg Institute of Pathology (SIP) BLCA Cohort (N = 181). An AI model for the prediction of molecular subtypes based on annotated histomorphology was trained. RESULTS: For a five-fold cross-validation with TCGA cases (N = 274), an internal TCGA test set (N = 18) and an external SIP test set (N = 27), we reached mean area under the receiver operating characteristic curve (AUROC) scores of 0.73, 0.8 and 0.75 for the classification of the used molecular subtypes "luminal", "basal/squamous" and "stroma-rich". By training on correlations to individual molecular subtypes, rather than training on one subtype assignment per case, the AI prediction of subtypes could be significantly improved. DISCUSSION: Follow-up studies with RNA extraction from various areas of AI-predicted molecular heterogeneity may improve molecular classifications and thereby AI algorithms trained on these classifications.

Tillandsia landbeckii phyllosphere and laimosphere as refugia for bacterial life in a hyperarid desert environment.

BACKGROUND: The lack of water is a major constraint for microbial life in hyperarid deserts. Consequently, the abundance and diversity of microorganisms in common habitats such as soil are strongly reduced, and colonization occurs primarily by specifically adapted microorganisms that thrive in particular refugia to escape the harsh conditions that prevail in these deserts. We suggest that plants provide another refugium for microbial life in hyperarid deserts. We studied the bacterial colonization of Tillandsia landbeckii (Bromeliaceae) plants, which occur in the hyperarid regions of the Atacama Desert in Chile, one of the driest and oldest deserts on Earth. RESULTS: We detected clear differences between the bacterial communities being plant associated to those of the bare soil surface (PERMANOVA, R2 = 0.187, p = 0.001), indicating that Tillandsia plants host a specific bacterial community, not only dust-deposited cells. Moreover, the bacterial communities in the phyllosphere were distinct from those in the laimosphere, i.e., on buried shoots (R2 = 0.108, p = 0.001), indicating further habitat differentiation within plant individuals. The bacterial taxa detected in the phyllosphere are partly well-known phyllosphere colonizers, but in addition, some rather unusual taxa (subgroup2 Acidobacteriae, Acidiphilum) and insect endosymbionts (Wolbachia, "Candidatus Uzinura") were found. The laimosphere hosted phyllosphere-associated as well as soil-derived taxa. The phyllosphere bacterial communities showed biogeographic patterns across the desert (R2 = 0.331, p = 0.001). These patterns were different and even more pronounced in the laimosphere (R2 = 0.467, p = 0.001), indicating that different factors determine community assembly in the two plant compartments. Furthermore, the phyllosphere microbiota underwent temporal changes (R2 = 0.064, p = 0.001). CONCLUSIONS: Our data demonstrate that T. landbeckii plants host specific bacterial communities in the phyllosphere as well as in the laimosphere. Therewith, these plants provide compartment-specific refugia for microbial life in hyperarid desert environments. The bacterial communities show biogeographic patterns and temporal variation, as known from other plant microbiomes, demonstrating environmental responsiveness and suggesting that bacteria inhabit these plants as viable microorganisms. Video Abstract.

Challenges for Plant Growth Promoting Microorganism Transfer from Science to Industry: A Case Study from Chile.

Research on the plant growth promoting microorganisms (PGPM) is increasing strongly due to the biotechnological potential for the agricultural, forestry, and food industry. The benefits of using PGPM in crop production are well proven; however, their incorporation in agricultural management is still limited. Therefore, we wanted to explore the gaps and challenges for the transfer of biotechnological innovations based on PGPM to the agricultural sector. Our systematic review of the state of the art of PGPM research and knowledge transfer takes Chile as an example. Several transfer limiting aspects are identified and discussed. Our two main conclusions are: neither academia nor industry can meet unfounded expectations during technology transfer, but mutually clarifying their needs, capabilities, and limitations is the starting point for successful collaborations; the generation of a collaborative innovation environment, where academia as well as public and private stakeholders (including the local community) take part, is crucial to enhance the acceptance and integration of PGPM on the way to sustainable agriculture.

New Insights into Bacillus-Primed Plant Responses to a Necrotrophic Pathogen Derived from the Tomato-Botrytis Pathosystem.

Induced systemic resistance (ISR) is one of the most studied mechanisms of plant−microbe interaction and is considered a very promising alternative for integrated pest management programs. In our study, we explored the plant defense response induced by Bacillus velezensis BBC047 in relation to its application before or after Botrytis cinerea infection of tomato plants. The inoculation of BBC047 did not considerably alter the gene expression of the tomato tissues, whereas infection with B. cinerea in BBC047-primed plants induced expression of LRR and NBS-LRR receptors, which are highly related to the ISR response. As expected, B. cinerea infection generated molecular patterns typical of a defense response to pathogen infection as the overexpression of pathogenesis-related proteins (PRs) in leaflets distant to the point of infection. The curative treatment (P + F + B) allowed us to gain insights into plant response to an inverted priming. In this treatment, B. cinerea caused the m tissue damage, extending nearly entirely across the entire infected leaves. Additionally, genes generally associated with early SAR response (<16 h) were overexpressed, and apparently, the beneficial strain was not perceived as such. Therefore, we infer that the plant defense to the curative treatment represents a higher degree of biological stress triggered by the incorporation of strain BBC047 as second arriving microorganism. We highlight the importance the phytosanitary status of plants prior to inoculation of beneficial microorganism for the biocontrol of pathogens.

Plant migration under long-lasting hyperaridity - phylogenomics unravels recent biogeographic history in one of the oldest deserts on Earth.

The post-Miocene climatic histories of arid environments have been identified as key drivers of dispersal and diversification. Here, we investigate how climatic history correlates with the historical biogeography of the Atacama Desert genus Cristaria (Malvaceae). We analyze phylogenetic relationships and historical biogeography by using next-generation sequencing (NGS), molecular clock dating, Dispersal Extinction Cladogenesis and Bayesian sampling approaches. We employ a novel way to identify biogeographically meaningful regions as well as a rarely utilized program permitting the use of dozens of ancestral areas. Partial incongruence between the established taxonomy and our phylogenetic data argue for a complex historical biogeography with repeated introgression and incomplete lineage sorting. Cristaria originated in the central southern part of the Atacama Desert, from there the genus colonized other areas from the late Miocene onwards. The more recently diverged lineages appear to have colonized different habitats in the Atacama Desert during pluvial phases of the Pliocene and early Pleistocene. We show that NGS combined with near-comprehensive sampling can provide an unprecedented degree of phylogenetic resolution and help to correlate the historical biogeography of plant communities with cycles of arid and pluvial phases.

The Role of Surfactin Production by Bacillus velezensis on Colonization, Biofilm Formation on Tomato Root and Leaf Surfaces and Subsequent Protection (ISR) against Botrytis cinerea.

Many aspects regarding the role of lipopeptides (LPs) in bacterial interaction with plants are not clear yet. Of particular interest is the LP family of surfactin, immunogenic molecules involved in induced systemic resistance (ISR) and the bacterial colonization of plant surfaces. We hypothesize that the concentration of surfactin produced by a strain correlates directly with its ability to colonize and persist on different plant surfaces, which conditions its capacity to trigger ISR. We used two Bacillus velezensis strains (BBC023 and BBC047), whose antagonistic potential in vitro is practically identical, but not on plant surfaces. The surfactin production of BBC047 is 1/3 higher than that of BBC023. Population density and SEM images revealed stable biofilms of BBC047 on leaves and roots, activating ISR on both plant surfaces. Despite its lower surfactin production, strain BBC023 assembled stable biofilms on roots and activated ISR. However, on leaves only isolated, unstructured populations were observed, which could not activate ISR. Thus, the ability of a strain to effectively colonize a plant surface is not only determined through its production of surfactin. Multiple aspects, such as environmental stressors or compensation mechanisms may influence the process. Finally, the importance of surfactin lies in its impacts on biofilm formation and stable colonization, which finally enables its activity as an elicitor of ISR.

Importance of crop phenological stages for the efficient use of PGPR inoculants.

During the last decades, the incorporation of beneficial microorganisms in agriculture crop management has become a common practice. Seed coating of these microorganisms still faces technical issues, which limit its implementation in conventional agriculture. An adaption to widely established agricultural practices, e.g. fertigation, could help to overcome these issues. Here, using Bacillus velezensis strain BBC047, we show the influence of the crop phenological stages on the efficiency and success of microbial inoculation under agricultural conditions. In the commercial nursery, strain BBC047 improved growth in a variety of horticulture crops like basil, cabbage, tomato and bell pepper, the latter with the strongest effects in strengthening and accelerating the seedling growth (root and aerial biomass). For a field trial under productive conditions, different application strategies were compared, using bell pepper (Capsicum annuum L.) as crop under fertigation: conventional management (T1), application to the seedling (only nursery, T2), only post-transplant application (field, T3) and a combination of both (T4). In T2 and T4, the post-transplantation survival rate (p < 0.05) improved and the productivity of the plants increased (> 100%). Applications of BBC047 post-transplantation (T3) caused a lower increase in productivity (25%). Fruits from all three application strategies contained significantly more Vitamin C. We conclude that in conventional agriculture, the applications of PGPR inoculants to early crop phenological stages like nurseries are a viable alternative for the efficient use of PGPR inoculants. In comparison, a late introduction of a PGPR reduces its beneficial effect on crop productivity. We highlight that an appropriate timing in the use of PGPR inoculants is crucial for product development and success in sustainable agriculture.

Effect of Metabolites Produced by Bacillus atrophaeus and Brevibacterium frigoritolerans Strains on Postharvest Biocontrol of Alternaria alternata in Tomato (Solanum lycopersicum L.).

In the present study, the antifungal activity of metabolites produced by Bacillus atrophaeus B5 and a new Brevibacterium strain against Alternaria alternata was evaluated. Assays in vitro and in vivo on tomato fruit during postharvest were made. Based on the 16S rDNA gene sequence analysis, the new strain (strain B7) was identified as Brevibacterium frigoritolerans. Metabolites produced by both bacterial strains reduced the spore germination of A. alternata in vitro and decreased the severity of the alternaria rot disease on tomato fruit during postharvest. This is the first report that demonstrates the potential of B. frigoritolerans B7 as a biocontrol agent against this fungal phytopathogen. The use of metabolites produced by B. atrophaeus B5 and B. frigoritolerans B7 represents a new approach to reduce the use of chemical pesticides and control fungal decay during the postharvest stage.

Plant life at the dry limit-Spatial patterns of floristic diversity and composition around the hyperarid core of the Atacama Desert.

Extreme arid conditions in the Atacama Desert in northern Chile have created a unique vegetation almost entirely restricted to the desert margins along the coast of the Pacific Ocean and the Andean range. In this study we provide data on the desert vegetation along elevational gradients at four localities from the western Andean slopes, between 19° and 21° S. Additionally, zonation of floristic data was explored. Three altitudinal zones could be classified and described in detail for each locality. Conspicuously divergent floras in the Atacama Desert have been recorded in the coastal 'lomas formations' and in the Andean desert vegetation, separated by a narrow band of absolute desert. In this study, we investigate the floristic relationships between both regions by implementing similarity analyses for 21 localities from the coastal and Andean deserts in northern Chile. Our results show a drastic east-west divergence in pairwise floristic similarity, which is in stark contrast to a weaker north-south divergence. A biotic barrier, preventing plant exchange from east to west and vice versa, imposed by the hyperarid conditions of the absolute desert, is one possible explanation for this finding. Moreover, the coastal and Andean deserts likely represent ecologically divergent habitats, e.g., in rainfall seasonality. Essential differences in factors determining plant life between both regions have probably contributed to a divergent evolution of the floras. Both explanations-ecological divergence and ecogeographical isolation-are not mutually exclusive, but likely complementary. We also combined floristic data from northern Chile and southern Peru. Similarity analyses of this combined dataset provide first floristic evidence for the existence of a biotic north-south corridor along the western slope of the Andes. Sub-Andean distributions of several species are discussed in the light of floristic connectivity between the Peruvian and Chilean Andean floristic clusters.

Composition, Predicted Functions and Co-occurrence Networks of Rhizobacterial Communities Impacting Flowering Desert Events in the Atacama Desert, Chile.

Flowering desert (FD) events consist of the rapid flowering of a wide variety of native plants in the Atacama Desert of Chile, which is categorized as the driest desert in the world. While ephemeral plants are an integral part of the desert ecosystem, there is little knowledge on plant-microbe interactions that occur during FD events. Consequently, the overall goals of this present study were to investigate changes in the composition and potential functions of rhizobacterial community of Cistanthe longiscapa (Montiaceae) during the 2014 and 2015 FD events and determine the composition, potential functions, and co-occurrence networks of rhizobacterial community associated with the root zone of C. longiscapa during pre- (PF) and full-flowering (FF) phenological stages. Results of this study showed that the Proteobacteria and Actinobacteria were the dominant taxa in rhizosphere soils during the three FD events (2014, 2015, and 2017) examined. In general, greater microbial richness and diversity were observed in rhizosphere soils during the 2015-, compared with the 2014-FD event. Similarly, predicted functional analyses indicated that a larger number of sequences were assigned to information processing (e.g., ion channel, transporters and ribosome) and metabolism (e.g., lipids, nitrogen, and sulfur) during 2015 compared with 2014. Despite the lack of significant differences in diversity among PF and FF stages, the combined analysis of rhizobacterial community data, along with data concerning rhizosphere soil properties, evidenced differences among both phenological stages and suggested that sodium is a relevant abiotic factor shaping the rhizosphere. In general, no significant differences in predicted functions (most of them assigned to chemoheterotrophy, magnesium metabolisms, and fermentation) were observed among PF and FF. Co-occurrence analysis revealed the complex rhizobacterial interactions that occur in C. longiscapa during FD, highlighting to Kouleothrixaceae family as keystone taxa. Taken together this study shows that the composition and function of rhizobacteria vary among and during FD events, where some bacterial groups and their activity may influence the growth and flowering of native plants, and therefore, the ecology and trophic webs in Atacama Desert.

Activation of the phenylpropanoid biosynthesis pathway reveals a novel action mechanism of the elicitor effect of chitosan on avocado fruit epicarp.

Secondary metabolites play an important role in the avocado fruit defense system. Phenolic compounds are the main biosynthesized metabolites of this system response. Our objective in this investigation was to evaluate the induction of specific metabolic pathways using chitosan as an elicitor. Extracts obtained from avocado in intermediate and consumption maturity stages treated with chitosan exhibited an increase in antifungal activity, which caused inhibition of mycelial growth and a decrease in sporulation as well as spore germination of Colletotrichum gloeosporioides. Additionally, RNA from epicarp of the fruits treated and untreated with chitosan was obtained in order to evaluate the expression of genes related to phenylpropanoids and the antifungal compound 1-acetoxy-2-hydroxy-4-oxo-heneicosa-12,15-diene biosynthesis. An increased in gene expression of genes that participates in the phenylpropanoids route was observed during the stage of physiological fruit maturity, others genes such as Flavonol synthase (Fls), increased only in samples obtained from fruit treated with chitosan at consumption maturity. Our results reveal a new molecular mechanism where chitosan induces a specific accumulation of phenylpropanoids and antifungal diene; this partially explains avocado's resistance against fungal pathogens. Finally, we discuss the molecular connections between chitosan induction and gene expression to explain the biological events that orchestrate the resistance pathways in fruits.

Root inoculation of green bell pepper (Capsicum annum) with Bacillus amyloliquefaciens BBC047: effect on biochemical composition and antioxidant capacity.

BACKGROUND: Consumption of bell peppers is recommended because of their bioactive compound content and their positive effects on health. Growth-promoting rhizobacteria are popular because of their ability to promote plant growth by improving the fixation of nutrients or by inducing a systemic response. Green bell pepper (Capsicum annum) roots were inoculated with an autochthonous strain of Bacillus amyloliquefaciens, at different stages of development: T1, inoculation in the seedbed before transplant; T2, inoculation at and after transplant; T3, inoculation in the seedbed, at and after transplant. Bell pepper plants without inoculation were considered as control. Physicochemical composition and antioxidant activity of the fruits were measured to select the best treatment. RESULTS: T1 increased crude proteins, fat, Ca, Fe, vitamin C, total phenolic content, antioxidant capacity by DPPH and by ORAC. On the other hand, T1 decreased reducing sugars, K and Cu content. No significant differences for total carbohydrates, ash and photosynthetic pigments were found. CONCLUSION: Inoculated green bell peppers have enhanced its functional value and could be considered as an important source of bioactive compounds with elevated antioxidant activity. © 2019 Society of Chemical Industry.

Microbiome Dynamics Associated With the Atacama Flowering Desert.

In a desert, plants as holobionts quickly respond to resource pulses like precipitation. However, little is known on how environment and plants modulate the rhizosphere-associated microbiome. As a model species to represent the Atacama Desert bloom, Cistanthe longiscapa (Montiaceae family) was selected to study the influence of abiotic and biotic environment on the diversity and structure of the microbiota associated to its rhizosphere. We analyzed the rhizosphere and soil microbiome along a North-South precipitation gradient and between a dry and rainy year by using Illumina high-throughput sequencing of 16S rRNA gene fragments and ITS2 regions for prokaryotes and fungi, respectively. In the rhizosphere of C. longiscapa the microbiota clearly differs in composition and structure from the surrounding bulk soil. The fungal and bacterial communities respond differently to environmental conditions. The diversity and richness of fungal OTUs were negatively correlated with aridity, as predicted. The community structure was predominantly influenced by other soil characteristics (pH, organic matter content) but not by aridity. In contrast, diversity, composition, and structure of the bacterial community were not influenced by aridity or any other evaluated soil parameter. These findings coincide with the identification of mainly site-specific microbial communities, not shared along the sites. These local communities contain a group of OTUs, which are exclusive to the rhizosphere of each site and presumably vertically inherited as seed endophytes. Their ecological functions and dispersal mechanisms remain unclear. The analysis of co-occurrence patterns highlights the strong effect of the desert habitat over the soil- and rhizosphere-microbiome. The site-independent enrichment of only a small bacterial cluster consistently associated with the rhizosphere of C. longiscapa further supports this conclusion. In a rainy year, the rhizosphere microbiota significantly differed from bulk and bare soil, whereas in a dry year, the community structure of the former rhizosphere approximates to the one found in the bulk. In the context of plant-microbe interactions in desert environments, our study contributes new insights into the importance of aridity in microbial community structure and composition, discovering the influence of other soil parameters in this complex dynamic network, which needs further to be investigated.

Identification and characterization of a new Bacillus atrophaeus strain B5 as biocontrol agent of postharvest anthracnose disease in soursop (Annona muricata) and avocado (Persea americana).

Anthracnose is a fungal disease caused by Colletotrichum species that is detrimental to numerous fruit, including soursop and avocado. The use of fungicides to maintain the high quality of fruit creates a potential health risk. One alternative to this problem is the biological control, which has been applied successfully during postharvest. The Bacillus species are one of the most studied biological agents against postharvest pathogens because accomplish their biocontrol performance by producing a variety of metabolites. In this study, we evaluated the activity of metabolites contained in the cell free supernatant, obtained from Bacillus strain B5 culture, against micelial growth and spore germination of two virulent strains of C. gloeosporioides isolated from soursop and avocado. On the basis of 16S rDNA gene sequence analysis, this strain was identified as Bacillus atrophaeus. A preventive treatment using cell free supernatant, reduced severity and incidence of anthracnose disease on harvested soursop and avocado fruit. B. atrophaeus strain B5 harbors genes involved in the production of antibiotics such as surfactin, bacillomycin and iturin, which could be contributing to the efficiency of the preventive treatment during postharvest. The antagonistic role of metabolites contained in the cell free supernatant against anthracnose disease, provide a new approach by which to attack this problem and can help reduce the use of chemical pesticides, environmental pollution, leading to the safer fruit preservation.

Development of microsatellite markers and assembly of the plastid genome in Cistanthe longiscapa (Montiaceae) based on low-coverage whole genome sequencing.

Cistanthe longiscapa is an endemic annual herb and characteristic element of the Chilean Atacama Desert. Principal threats are the destruction of its seed deposits by human activities and reduced germination rates due to the decreasing occurrence of precipitation events. To enable population genetic and phylogeographic analyses in this species we performed paired-end shotgun sequencing (2x100 bp) of genomic DNA on the Illumina HiSeq platform and identified microsatellite (SSR) loci in the resulting sequences. From 29 million quality-filtered read pairs we obtained 549,174 contigs (average length 614 bp; N50 = 904). Searching for SSRs revealed 10,336 loci with microsatellite motifs. Initially, we designed primers for 96 loci, which were tested for PCR amplification on three C. longiscapa individuals. Successfully amplifying loci were further tested on eight individuals to screen for length variation in the resulting amplicons, and the alleles were exemplarily sequenced to infer the basis for the observed length variation. Finally we arrived at 26 validated SSR loci for population studies in C. longiscapa, which resulted in 146 bi-allelic SSR markers in our test sample of eight individuals. The genomic sequences were also used to assemble the plastid genome of C. longiscapa, which provides an additional set of maternally inherited genetic markers.

Isolation and characterization of 12 microsatellite loci in soapbark, Quillaja saponaria (Quillajaceae).

PREMISE OF THE STUDY: Microsatellite primers were developed for the endemic Chilean tree Quillaja saponaria (Quillajaceae), a common member of the sclerophyllous Mediterranean forest, to investigate intraspecific patterns of genetic diversity and structure. METHODS AND RESULTS: Using an enriched library, 12 polymorphic microsatellite loci were developed in Q. saponaria. All loci consisted of dinucleotide repeats. The average number of alleles per locus was 5.3 (2-13), with a total of 64 alleles recorded in 39 individuals from three populations. CONCLUSIONS: The microsatellite markers described here are the first characterized for Q. saponaria. The polymorphic loci will be useful in studies of genetic diversity and genetic population differentiation in natural populations of this species.

Verhuellia is a segregate lineage in Piperaceae: more evidence from flower, fruit and pollen morphology, anatomy and development.

BACKGROUND AND AIMS: The perianthless Piperales, i.e. Saururaceae and Piperaceae, have simple reduced flowers strikingly different from the other families of the order (e.g. Aristolochiaceae). Recent molecular phylogenies proved Verhuellia to be the first branch in Piperaceae, making it a promising subject to study the detailed structure and development of the flowers. Based on recently collected material, the first detailed study since 1872 was conducted with respect to morphology, anatomy and development of the inflorescence, pollen ultrastructure and fruit anatomy. METHODS: Original scanning electron microscopy (SEM), transmission electron microscopy (TEM) and light microscopy (LM) observations on Verhuellia lunaria were compared with those of Piperaceae, Saururaceae and fossils. KEY RESULTS: The inflorescence is an indeterminate spike with sessile flowers, each in the axil of a bract, developing in acropetal, helical succession. Flowers consist of two (occasionally three) stamens with basifixed tetrasporangiate anthers and latrorse dehiscence by a longitudinal slit. The gynoecium lacks a style but has 3-4 stigma branches and a single, basal orthotropous and unitegmic ovule. The fruit is a drupe with large multicellular epidermal protuberances. The pollen is very small, inaperturate and areolate, with hemispherical microechinate exine elements. CONCLUSIONS: Despite the superficial similarities with different genera of Piperaceae and Saururaceae, the segregate position of Verhuellia revealed by molecular phylogenetics is supported by morphological, developmental and anatomical data presented here. Unitegmic ovules and inaperturate pollen, which are synapomorphies for the genus Peperomia, are also present in Verhuellia.