Publications by authors named "Barbara Gravendeel"

54 Publications

DNA metabarcoding using nrITS2 provides highly qualitative and quantitative results for airborne pollen monitoring.

Sci Total Environ 2021 Sep 21;806(Pt 1):150468. Epub 2021 Sep 21.

Naturalis Biodiversity Center, Leiden, the Netherlands; Radboud Institute for Biological and Environmental Sciences, Nijmegen, the Netherlands.

Airborne pollen monitoring is of global socio-economic importance as it provides information on presence and prevalence of allergenic pollen in ambient air. Traditionally, this task has been performed by microscopic investigation, but novel techniques are being developed to automate this process. Among these, DNA metabarcoding has the highest potential of increasing the taxonomic resolution, but uncertainty exists about whether the results can be used to quantify pollen abundance. In this study, it is shown that DNA metabarcoding using trnL and nrITS2 provides highly improved taxonomic resolution for pollen from aerobiological samples from the Netherlands. A total of 168 species from 143 genera and 56 plant families were detected, while using a microscope only 23 genera and 22 plant families were identified. NrITS2 produced almost double the number of OTUs and a much higher percentage of identifications to species level (80.1%) than trnL (27.6%). Furthermore, regressing relative read abundances against the relative abundances of microscopically obtained pollen concentrations showed a better correlation for nrITS2 (R = 0.821) than for trnL (R = 0.620). Using three target taxa commonly encountered in early spring and fall in the Netherlands (Alnus sp., Cupressaceae/Taxaceae and Urticaceae) the nrITS2 results showed that all three taxa were dominated by one or two species (Alnus glutinosa/incana, Taxus baccata and Urtica dioica). Highly allergenic as well as artificial hybrid species were found using nrITS2 that could not be identified using trnL or microscopic investigation (Alnus × spaethii, Cupressus arizonica, Parietaria spp.). Furthermore, perMANOVA analysis indicated spatiotemporal patterns in airborne pollen trends that could be more clearly distinguished for all taxa using nrITS2 rather than trnL. All results indicate that nrITS2 should be the preferred marker of choice for molecular airborne pollen monitoring.
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http://dx.doi.org/10.1016/j.scitotenv.2021.150468DOI Listing
September 2021

Sepal Identity of the Pappus and Floral Organ Development in the Common Dandelion (; Asteraceae).

Plants (Basel) 2021 Aug 16;10(8). Epub 2021 Aug 16.

Evolutionary Ecology, Naturalis Biodiversity Center, Darwinweg 2, 2333 CR Leiden, The Netherlands.

The dry one-seeded fruits () of the Asteraceae are often crowned with a , an appendage of hairs or scales that assists in dispersal. It is generally assumed, but little investigated, that the pappus represents the outer floral whorl where the are usually located. We analysed pappus-sepal homology in dandelions using micromorphological and floral gene expression analyses. We show that the pappus initiates from a ring primordium at the base of the corolla, heterochronic to the petals. Pappus parts form from this ring, with those in the position usually being ahead in growth, referring to sepal identity. 1 expression increased during floret development and was highest in mature pappus. expression was high and confined to the floral tissues containing the petals and stamens, consistent with expectations for sepals. Apart from the pappus, the dispersal structure of dandelion consists of the upper part of the fruit, the , which originates from the inner floral whorl. Thus, our results support the homology of the pappus with the sepals, but show that it is highly derived. Together with our floral stage definitions and verified qPCR reference genes, they provide a basis for evolution and development studies in dandelions and possibly other Asteraceae.
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http://dx.doi.org/10.3390/plants10081682DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8398263PMC
August 2021

Hundreds of nuclear and plastid loci yield novel insights into orchid relationships.

Am J Bot 2021 07 11;108(7):1166-1180. Epub 2021 Jul 11.

Royal Botanic Gardens Kew, Richmond, TW9 3AE, UK.

Premise: The inference of evolutionary relationships in the species-rich family Orchidaceae has hitherto relied heavily on plastid DNA sequences and limited taxon sampling. Previous studies have provided a robust plastid phylogenetic framework, which was used to classify orchids and investigate the drivers of orchid diversification. However, the extent to which phylogenetic inference based on the plastid genome is congruent with the nuclear genome has been only poorly assessed.

Methods: We inferred higher-level phylogenetic relationships of orchids based on likelihood and ASTRAL analyses of 294 low-copy nuclear genes sequenced using the Angiosperms353 universal probe set for 75 species (representing 69 genera, 16 tribes, 24 subtribes) and a concatenated analysis of 78 plastid genes for 264 species (117 genera, 18 tribes, 28 subtribes). We compared phylogenetic informativeness and support for the nuclear and plastid phylogenetic hypotheses.

Results: Phylogenetic inference using nuclear data sets provides well-supported orchid relationships that are highly congruent between analyses. Comparisons of nuclear gene trees and a plastid supermatrix tree showed that the trees are mostly congruent, but revealed instances of strongly supported phylogenetic incongruence in both shallow and deep time. The phylogenetic informativeness of individual Angiosperms353 genes is in general better than that of most plastid genes.

Conclusions: Our study provides the first robust nuclear phylogenomic framework for Orchidaceae and an assessment of intragenomic nuclear discordance, plastid-nuclear tree incongruence, and phylogenetic informativeness across the family. Our results also demonstrate what has long been known but rarely thoroughly documented: nuclear and plastid phylogenetic trees can contain strongly supported discordances, and this incongruence must be reconciled prior to interpretation in evolutionary studies, such as taxonomy, biogeography, and character evolution.
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http://dx.doi.org/10.1002/ajb2.1702DOI Listing
July 2021

Molecular Clocks and Archeogenomics of a Late Period Egyptian Date Palm Leaf Reveal Introgression from Wild Relatives and Add Timestamps on the Domestication.

Mol Biol Evol 2021 Sep;38(10):4475-4492

Royal Botanic Gardens, Kew, Richmond, London, United Kingdom.

The date palm, Phoenix dactylifera, has been a cornerstone of Middle Eastern and North African agriculture for millennia. It was first domesticated in the Persian Gulf, and its evolution appears to have been influenced by gene flow from two wild relatives, P. theophrasti, currently restricted to Crete and Turkey, and P. sylvestris, widespread from Bangladesh to the West Himalayas. Genomes of ancient date palm seeds show that gene flow from P. theophrasti to P. dactylifera may have occurred by ∼2,200 years ago, but traces of P. sylvestris could not be detected. We here integrate archeogenomics of a ∼2,100-year-old P. dactylifera leaf from Saqqara (Egypt), molecular-clock dating, and coalescence approaches with population genomic tests, to probe the hybridization between the date palm and its two closest relatives and provide minimum and maximum timestamps for its reticulated evolution. The Saqqara date palm shares a close genetic affinity with North African date palm populations, and we find clear genomic admixture from both P. theophrasti, and P. sylvestris, indicating that both had contributed to the date palm genome by 2,100 years ago. Molecular-clocks placed the divergence of P. theophrasti from P. dactylifera/P. sylvestris and that of P. dactylifera from P. sylvestris in the Upper Miocene, but strongly supported, conflicting topologies point to older gene flow between P. theophrasti and P. dactylifera, and P. sylvestris and P. dactylifera. Our work highlights the ancient hybrid origin of the date palms, and prompts the investigation of the functional significance of genetic material introgressed from both close relatives, which in turn could prove useful for modern date palm breeding.
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http://dx.doi.org/10.1093/molbev/msab188DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8476131PMC
September 2021

Neural networks for increased accuracy of allergenic pollen monitoring.

Sci Rep 2021 05 31;11(1):11357. Epub 2021 May 31.

Naturalis Biodiversity Center, Leiden, The Netherlands.

Monitoring of airborne pollen concentrations provides an important source of information for the globally increasing number of hay fever patients. Airborne pollen is traditionally counted under the microscope, but with the latest developments in image recognition methods, automating this process has become feasible. A challenge that persists, however, is that many pollen grains cannot be distinguished beyond the genus or family level using a microscope. Here, we assess the use of Convolutional Neural Networks (CNNs) to increase taxonomic accuracy for airborne pollen. As a case study we use the nettle family (Urticaceae), which contains two main genera (Urtica and Parietaria) common in European landscapes which pollen cannot be separated by trained specialists. While pollen from Urtica species has very low allergenic relevance, pollen from several species of Parietaria is severely allergenic. We collect pollen from both fresh as well as from herbarium specimens and use these without the often used acetolysis step to train the CNN model. The models show that unacetolyzed Urticaceae pollen grains can be distinguished with > 98% accuracy. We then apply our model on before unseen Urticaceae pollen collected from aerobiological samples and show that the genera can be confidently distinguished, despite the more challenging input images that are often overlain by debris. Our method can also be applied to other pollen families in the future and will thus help to make allergenic pollen monitoring more specific.
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http://dx.doi.org/10.1038/s41598-021-90433-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8166864PMC
May 2021

Antimicrobial Activity of Necklace Orchids is Phylogenetically Clustered and can be Predicted With a Biological Response Method.

Front Pharmacol 2020 12;11:586345. Epub 2021 Mar 12.

Naturalis Biodiversity Center, Endless Forms Group, Leiden, Netherlands.

Necklace orchids (Coelogyninae, Epidendroideae) have been used in traditional medicine practices for centuries. Previous studies on a subset of unrelated orchid species utilized in these traditional practices revealed they possessed antimicrobial, anti-inflammatory, and anti-oxidant activity, providing experimental proof for their medicinal properties. To date however none of these species have been investigated ethno-botanically in a phylogenetic context. This study carried out comparative bioprospecting for a group of wild orchids using EBDCS (the Economic Botany Data Collection Standards) organ targeted and biological response methods. The traditional medicinal use of necklace orchids was recorded from books and journals published between 1984 and 2016. Two orchids, and , were selected, cultivated both indoors and outdoors, and the antimicrobial properties on extracts from their leaves and pseudobulbs tested against a selection of human pathogens. A molecular phylogeny of Coelogyninae based on nuclear ribosomal ITS and plastid DNA sequences obtained from 148 species was reconstructed with Maximum Likelihood (ML) using RAxML, Maximum Parsimony (MP) using PAUP and Bayesian Inference using MrBayes. Bioprospecting comparison of EBDCS and biological response was carried out using customized R scripts. Ethanolic extracts obtained from leaves of inhibited growth of , , and confirming the antimicrobial properties of these extracts. Leaf extracts were found to have slightly stronger antimicrobial properties for plants cultivated outdoors than indoors. These differences were not found to be statistically significant though. Three hot nodes with high potency for antimicrobial activities were detected with the EBDCS organ targeted classification method, and eight hot nodes were detected with the biological response classification method. The biological response classification method is thus a more effective tool in finding hot nodes amongst clades of species with high medicinal potential.
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http://dx.doi.org/10.3389/fphar.2020.586345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7994927PMC
March 2021

Asymmetric contributions of seed and pollen to gene dispersal in the marsh orchid Dactylorhiza umbrosa in Asia Minor.

Mol Ecol 2021 04 13;30(8):1791-1805. Epub 2021 Mar 13.

Department of Plant Sciences, School of Biology, College of Science, University of Tehran, Tehran, Iran.

Orchids differ from other plants in their extremely small and partly air-filled seeds that can be transported long distances by wind. Seed dispersal in orchids is expected to contribute strongly to overall gene flow, and orchids generally express low levels of genetic differentiation between populations and low pollen to seed flow ratios. However, studies in orchids distributed in northern Europe have often found a poor geographic structuring of genetic variation. Here, we studied geographic differentiation in the marsh orchid Dactylorhiza umbrosa, which is widely distributed in upland regions from Asia Minor to Central Asia. These areas were less affected by Pleistocene ice ages than northern Europe and the orchid should have been able to survive the last ice age in local refugia. In the plastid genome, which is dispersed by seeds, populations at close distance were clearly divergent, but the differentiation still increased with geographic distance, and a significant phylogeographic structure had developed. In the nuclear genome, which is dispersed by both seeds and pollen, populations showed an even stronger correlation between genetic and geographic distance, but average levels of differentiation were lower than in the plastid genome, and no phylogeographic structure was evident. Combining plastid and nuclear data, we found that the ratio of pollen to seed dispersal (mp/ms) decreases with physical distance. Comparison with orchids that grow in parts of Europe that were glaciated during the last ice suggests that a balanced structure of genetic diversity develops only slowly in many terrestrial orchids, despite efficient seed dispersal.
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http://dx.doi.org/10.1111/mec.15848DOI Listing
April 2021

Pitfall Flower Development and Organ Identity of (Apocynaceae-Asclepiadoideae).

Plants (Basel) 2020 Dec 14;9(12). Epub 2020 Dec 14.

Naturalis Biodiversity Center, Darwinweg 2, 2333 CR Leiden, The Netherlands.

Deceptive pitfall flowers are an outstanding example of synorganized morphological complexity. Floral organs functionally synergise to trap fly-pollinators inside the fused corolla. Successful pollination requires precise positioning of flies headfirst into cavities at the gynostegium. These cavities are formed by the corona, a specialized organ of corolline and/or staminal origin. The interplay of floral organs to achieve pollination is well studied but their evolutionary origin is still unclear. We aimed to obtain more insight in the homology of the corona and therefore investigated floral anatomy, ontogeny, vascularization, and differential MADS-box gene expression in using X-ray microtomography, Light and Scanning Electronic Microscopy, and RT-PCR. During 10 defined developmental phases, the corona appears in phase 7 at the base of the stamens and was not found to be vascularized. A floral reference transcriptome was generated and 14 MADS-box gene homologs, representing all major MADS-box gene classes, were identified. B- and C-class gene expression was found in mature coronas. Our results indicate staminal origin of the corona, and we propose a first ABCDE-model for floral organ identity in to lay the foundation for a better understanding of the molecular background of pitfall flower evolution in Apocynaceae.
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http://dx.doi.org/10.3390/plants9121767DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7764971PMC
December 2020

Ontogeny and Anatomy of the Dimorphic Pitchers of Jack.

Plants (Basel) 2020 Nov 18;9(11). Epub 2020 Nov 18.

Naturalis Biodiversity Center, Darwinweg 2, 2333 CR Leiden, The Netherlands.

An enigmatic feature of tropical pitcher plants belonging to the genus is their dimorphic prey-capturing pitfall traps. In many species, the conspicuously shaped upper and lower pitchers grow from a swollen leaf tendril tip until finally opening as insect-alluring devices. Few have studied the ontogeny of these traps from an anatomical and quantitative morphological perspective. We investigated whether the anatomy and development of lower and upper type pitchers of differ or overlap in terms of 3D geometric morphology and microstructure progression and presence. We hypothesized that there is an overlap in the initial, but not all, developmental stages of the two pitcher types and that one pitcher type is suspended in development. We identified four important morphological changes of pitcher ontogeny and defined these as curvation, elongation, inflation and maturation phases. Pitcher length indicated progress through developmental phases, and we propose to use it as a tool for indication of developmental stage. Microstructure development coincided with the developmental phases defined. Additionally, we discovered a new anatomical feature of extrafloral nectariferous peristomal glands between the inner peristome ridges of upper and lower pitchers being hollow and analyze the chemistry of the sugars on the outside of these glands. Ontogenetic shape analysis indicated that upper and lower pitcher types develop with similar phase progression but have no directly overlapping morphology. This means that upper pitchers are not a derived state from lower pitchers. Independent developmental programs evolved to produce distinctly shaped upper and lower pitchers in likely to exploit different food sources.
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http://dx.doi.org/10.3390/plants9111603DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7698951PMC
November 2020

Evolution and development of three highly specialized floral structures of bee-pollinated species.

Evodevo 2020 10;11:16. Epub 2020 Aug 10.

Naturalis Biodiversity Center, Endless Forms Group, Darwinweg 2, 2333 CR Leiden, The Netherlands.

Background: Variation in shape and size of many floral organs is related to pollinators. Evolution of such organs is driven by duplication and modification of MADS-box and MYB transcription factors. We applied a combination of micro-morphological (SEM and micro 3D-CT scanning) and molecular techniques (transcriptome and RT-PCR analysis) to understand the evolution and development of the callus, stelidia and mentum, three highly specialized floral structures of orchids involved in pollination. Early stage and mature tissues were collected from flowers of the bee-pollinated and , two species that differ in floral morphology: has a large callus but short stelidia and no mentum, whereas has a small callus, but long stelidia and a pronounced mentum.

Results: Our results show the stelidia develop from early primordial stages, whereas the callus and mentum develop later. In combination, the micro 3D-CT scan analysis and gene expression analyses show that the callus is of mixed petaloid-staminodial origin, the stelidia of staminodial origin, and the mentum of mixed sepaloid-petaloid-staminodial origin. clade 1 copies are expressed in the larger callus of , whereas clade 1 and clade 1 copies are expressed in the pronounced mentum and long stelidia of clade 4, -, clade 2 and clade 1 copies might have a balancing role in callus and gynostemium development. There appears to be a trade-off between clade 2 expression with clade 1 expression in the callus, on the one hand, and with clade 1 and clade 1 expression in the stelidia and mentum on the other.

Conclusions: We detected differential growth and expression of MADS box -like, 6-like and -like, and MYB -like gene copies in the callus, stelidia and mentum of two species of of which these floral structures are very differently shaped and sized. Our study provides a first glimpse of the evolutionary developmental mechanisms driving adaptation of flowers to different pollinators by providing combined micro-morphological and molecular evidence for a possible sepaloid-petaloid-staminodial origin of the orchid mentum.
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http://dx.doi.org/10.1186/s13227-020-00160-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7418404PMC
August 2020

Mushroom DNA barcoding project: Sequencing a segment of the 28S rRNA gene.

Biochem Mol Biol Educ 2020 07 25;48(4):404-410. Epub 2020 Jun 25.

University of Applied Sciences Leiden, Faculty of Science and Technology, Leiden, The Netherlands.

DNA barcoding is an important molecular methodology for species identification that was developed over the last two decades and it should be covered in the biology bachelor curriculum. Here, we present an example of DNA barcoding by sequencing a segment of the 28S nuclear ribosomal large subunit rRNA gene of wild mushrooms and framing the education in a project form for undergraduate students in biology. Students perform this project in 6-8 weeks, which also includes preparing a poster, writing a report and presenting a paper related to the work in a journal club format. First, fieldwork in the Netherlands was carried out, during which students collected mushrooms under supervision of a professional mycologist with the goal to (a) verify morphologically based identifications with a molecular method and (b) assess phylogenetic relationships of the different species collected. Next, DNA extractions and quantitation were performed, PCR amplification was done, and samples were sent out for Sanger sequencing. Students aligned and analyzed the sequences using BLAST and Geneious and subsequently created a phylogenetic tree. In case of collecting DNA barcodes of an earlier sequenced species, students could upload the data to a repository established for facilitation of future research projects. The method described is very robust, reagents and equipment are readily available, and costs are relatively low. In addition, the results can be compared to published fungal phylogenetic trees.
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http://dx.doi.org/10.1002/bmb.21388DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7497104PMC
July 2020

Organic matter reduces the amount of detectable environmental DNA in freshwater.

Ecol Evol 2020 Apr 21;10(8):3647-3654. Epub 2020 Mar 21.

Naturalis Biodiversity Center Leiden The Netherlands.

Environmental DNA (eDNA) is used for monitoring the occurrence of freshwater organisms. Various studies show a relation between the amount of eDNA detected and target organism abundance, thus providing a potential proxy for reconstructing population densities. However, environmental factors such as water temperature and microbial activity are known to affect the amount of eDNA present as well. In this study, we use controlled aquarium experiments using L. (Amphipoda) to investigate the relationship between the amount of detectable eDNA through time, pH, and levels of organic material. We found eDNA to degrade faster when organic material was added to the aquarium water, but that pH had no significant effect. We infer that eDNA contained inside cells and mitochondria is extra resilient against degradation, though this may not reflect actual presence of target species. These results indicate that, although estimation of population density might be possible using eDNA, measured eDNA concentration could, in the future, be corrected for local environmental conditions in order to ensure accurate comparisons.
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http://dx.doi.org/10.1002/ece3.6123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7160167PMC
April 2020

DNA barcoding augments conventional methods for identification of medicinal plant species traded at Tanzanian markets.

J Ethnopharmacol 2020 Mar 23;250:112495. Epub 2019 Dec 23.

Department of Systematic Biology, Evolutionary Biology Center, Uppsala University, Norbyvägen 18D, SE-75236, Uppsala, Sweden; Naturalis Biodiversity Center, P.O. Box 9517, 2300 RA, Leiden, the Netherlands; Natural History Museum, University of Oslo, Norway.

Ethnopharmalogical Relevance: In Africa, traditional medicine is important for local healthcare and plants used for these purposes are commonly traded. Identifying medicinal plants sold on markets is challenging, as leaves, barks and roots are often fragmented or powdered. Vernacular names are often homonymic, and identification of material lacking sufficient morphological characters is time-consuming, season-dependent and might lead to incorrect assessments of commercialised species diversity.

Aim Of The Study: In this study, we identified cases of vernacular heterogeneity of medicinal plants using a tiered approach of literature research, morphology and DNA barcoding.

Material And Methods: A total of 870 single ingredient medicinal plant samples corresponding to 452 local names were purchased from herbal markets in Dar-es-Salaam and Tanga, Tanzania, and identified using conventional methods as well as DNA barcoding using rbcL, matK and nrITS.

Results: Using conventional methods, we could identify 70% of samples to at least family level, while 62% yielded a DNA barcode for at least one of the three markers. Combining conventional methods and DNA barcoding, 76% of the samples could be identified to species level, revealing a diversity of at least 175 species in 65 plant families. Analysis of the market samples revealed 80 cases of multilingualism and over- and under-differentiation. Afzelia quanzensis Welw., Zanthoxylum spp., Allophylus spp. and Albizia anthelmintica Brongn. were the most evident cases of multilingualism and over-differentiation, as they were traded under 8-12 vernacular names in up to five local languages. The most obvious case of under-differentiation was mwingajini (Swahili), which matched to eight scientific species in five different plant families.

Conclusions: Use of a tiered approach increases the identification success of medicinal plants sold in local market and corroborates findings that DNA barcoding can elucidate the identity of material that is unidentifiable based on morphology and literature as well as verify or disqualify these identifications. Results of this study can be used as a basis for quantitative market surveys of fragmented herbal medicine and to investigate conservation issues associated with this trade.
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http://dx.doi.org/10.1016/j.jep.2019.112495DOI Listing
March 2020

Phylogenetic comparative methods improve the selection of characters for generic delimitations in a hyperdiverse Neotropical orchid clade.

Sci Rep 2019 10 22;9(1):15098. Epub 2019 Oct 22.

Naturalis Biodiversity Center, Endless Forms group, Leiden, The Netherlands.

Taxonomic delimitations are challenging because of the convergent and variable nature of phenotypic traits. This is evident in species-rich lineages, where the ancestral and derived states and their gains and losses are difficult to assess. Phylogenetic comparative methods help to evaluate the convergent evolution of a given morphological character, thus enabling the discovery of traits useful for classifications. In this study, we investigate the evolution of selected traits to test for their suitability for generic delimitations in the clade Lepanthes, one of the Neotropical species-richest groups. We evaluated every generic name proposed in the Lepanthes clade producing densely sampled phylogenies with Maximum Parsimony, Maximum Likelihood, and Bayesian approaches. Using Ancestral State Reconstructions, we then assessed 18 phenotypic characters that have been traditionally employed to diagnose genera. We propose the recognition of 14 genera based on solid morphological delimitations. Among the characters assessed, we identified 16 plesiomorphies, 12 homoplastic characters, and seven synapomorphies, the latter of which are reproductive features mostly related to the pollination by pseudocopulation and possibly correlated with rapid diversifications in Lepanthes. Furthermore, the ancestral states of some reproductive characters suggest that these traits are associated with pollination mechanisms alike promoting homoplasy. Our methodological approach enables the discovery of useful traits for generic delimitations in the Lepanthes clade and offers various other testable hypotheses on trait evolution for future research on Pleurothallidinae orchids because the phenotypic variation of some characters evaluated here also occurs in other diverse genera.
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http://dx.doi.org/10.1038/s41598-019-51360-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6805863PMC
October 2019

A Target Capture-Based Method to Estimate Ploidy From Herbarium Specimens.

Front Plant Sci 2019 24;10:937. Epub 2019 Jul 24.

Royal Botanic Gardens, Kew, Richmond, United Kingdom.

Whole genome duplication (WGD) events are common in many plant lineages, but the ploidy status and possible occurrence of intraspecific ploidy variation are unknown for most species. Standard methods for ploidy determination are chromosome counting and flow cytometry approaches. While flow cytometry approaches typically use fresh tissue, an increasing number of studies have shown that recently dried specimens can be used to yield ploidy data. Recent studies have started to explore whether high-throughput sequencing (HTS) data can be used to assess ploidy levels by analyzing allelic frequencies from single copy nuclear genes. Here, we compare different approaches using a range of yam () tissues of varying ages, drying methods and quality, including herbarium tissue. Our aims were to: (1) explore the limits of flow cytometry in estimating ploidy level from dried samples, including herbarium vouchers collected between 1831 and 2011, and (2) optimize a HTS-based method to estimate ploidy by considering allelic frequencies from nuclear genes obtained using a target-capture method. We show that, although flow cytometry can be used to estimate ploidy levels from herbarium specimens collected up to fifteen years ago, success rate is low (5.9%). We validated our HTS-based estimates of ploidy using 260 genes by benchmarking with dried samples of species of known ploidy (, , and ). Subsequently, we successfully applied the method to the 85 herbarium samples analyzed with flow cytometry, and successfully provided results for 91.7% of them, comprising species across the phylogenetic tree of . We also explored the limits of using this HTS-based approach for identifying high ploidy levels in herbarium material and the effects of heterozygosity and sequence coverage. Overall, we demonstrated that ploidy diversity within and between species may be ascertained from historical collections, allowing the determination of polyploidization events from samples collected up to two centuries ago. This approach has the potential to provide insights into the drivers and dynamics of ploidy level changes during plant evolution and crop domestication.
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http://dx.doi.org/10.3389/fpls.2019.00937DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6667659PMC
July 2019

A customized nuclear target enrichment approach for developing a phylogenomic baseline for yams (Dioscoreaceae).

Appl Plant Sci 2019 Jun 13;7(6):e11254. Epub 2019 Jun 13.

Royal Botanic Gardens Kew, Richmond Surrey TW9 3DS United Kingdom.

Premise: We developed a target enrichment panel for phylogenomic studies of , an economically important genus with incompletely resolved relationships.

Methods: Our bait panel comprises 260 low- to single-copy nuclear genes targeted to work in , assessed here using a preliminary taxon sampling that includes both distantly and closely related taxa, including several yam crops and potential crop wild relatives. We applied coalescent-based and maximum likelihood phylogenomic inference approaches to the pilot taxon set, incorporating new and published transcriptome data from additional species.

Results: The custom panel retrieved ~94% of targets and >80% of full gene length from 88% and 68% of samples, respectively. It has minimal gene overlap with existing panels designed for angiosperm-wide studies and generally recovers longer and more variable targets. Pilot phylogenomic analyses consistently resolve most deep and recent relationships with strong support across analyses and point to revised relationships between the crop species and candidate crop wild relatives.

Discussion: Our customized panel reliably retrieves targeted loci from , is informative for resolving relationships in denser samplings, and is suitable for refining our understanding of the independent origins of cultivated yam species; the panel likely has broader promise for phylogenomic studies across Dioscoreales.
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http://dx.doi.org/10.1002/aps3.11254DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580989PMC
June 2019

Morphological and Molecular Characterization of Orchid Fruit Development.

Front Plant Sci 2019 19;10:137. Epub 2019 Feb 19.

Endless Forms Group, Naturalis Biodiversity Center, Leiden, Netherlands.

Efficient seed dispersal in flowering plants is enabled by the development of fruits, which can be either dehiscent or indehiscent. Dehiscent fruits open at maturity to shatter the seeds, while indehiscent fruits do not open and the seeds are dispersed in various ways. The diversity in fruit morphology and seed shattering mechanisms is enormous within the flowering plants. How these different fruit types develop and which molecular networks are driving fruit diversification is still largely unknown, despite progress in eudicot model species. The orchid family, known for its astonishing floral diversity, displays a huge variation in fruit dehiscence types, which have been poorly investigated. We undertook a combined approach to understand fruit morphology and dehiscence in different orchid species to get more insight into the molecular network that underlies orchid fruit development. We describe fruit development in detail for the epiphytic orchid species and compare it to two terrestrial orchid species: and . Our anatomical analysis provides further evidence for the split carpel model, which explains the presence of three fertile and three sterile valves in most orchid species. Interesting differences were observed in the lignification patterns of the dehiscence zones. While and develop a lignified layer at the valve boundaries, fruits did not lignify at these boundaries, but formed a cuticle-like layer instead. We characterized orthologs of fruit-associated MADS-domain transcription factors and of the Arabidopsis dehiscence-related genes and in , and found that the key players of the eudicot fruit regulatory network appear well-conserved in monocots. Protein-protein interaction studies revealed that MADS-domain complexes comprised of FRUITFULL (FUL), SEPALLATA (SEP) and AGAMOUS (AG) /SHATTERPROOF (SHP) orthologs can also be formed in , and that the expression of , and can be associated with dehiscence zone development similar to Arabidopsis. Our expression analysis also indicates differences, however, which may underlie fruit divergence.
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http://dx.doi.org/10.3389/fpls.2019.00137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6390509PMC
February 2019

Trade in Zambian Edible Orchids-DNA Barcoding Reveals the Use of Unexpected Orchid Taxa for .

Genes (Basel) 2018 Nov 30;9(12). Epub 2018 Nov 30.

Department of Organismal Biology, Systematic Biology, Uppsala University, Norbyvägen 18D, 75236 Uppsala, Sweden.

In Zambia, wild edible terrestrial orchids are used to produce a local delicacy called , which has become increasingly popular throughout the country. Commercialization puts orchid populations in Zambia and neighbouring countries at risk of overharvesting. Hitherto, no study has documented which orchid species are traded on local markets, as orchid tubers are difficult to identify morphologically. In this study, the core land-plant DNA barcoding markers and were used in combination with nrITS to determine which species were sold in Zambian markets. Eighty-two interviews were conducted to determine harvesting areas, as well as possible sustainability concerns. By using nrITS DNA barcoding, a total of 16 orchid species in six different genera could be identified. Both and proved suitable to identify the tubers up to the genus or family level. , and were identified most frequently and three previously undocumented species were encountered on the market. Few orchid species are currently listed on the global International Union for the Conservation of Nature (IUCN) Red List. Local orchid populations and endemic species could be at risk of overharvesting due to the intensive and indiscriminate harvesting of orchids, and we therefore encourage increased conservation assessment of terrestrial African orchids.
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http://dx.doi.org/10.3390/genes9120595DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6315803PMC
November 2018

A Linnaeus NG interactive key to the species of (Orchidaceae, Coelogyninae) from Southeast Asia.

PhytoKeys 2018 26(110):9-22. Epub 2018 Oct 26.

Naturalis Biodiversity Center, Endless Forms group, P.O. Box 9517, Leiden, The Netherlands.

We present a multilingual interactive key available online (http://glomera.linnaeus.naturalis.nl) that can be used on any web browser without the need for installing additional software. The key includes 169 species of , a genus within the necklace orchids (Coelogyninae) not yet comprehensively treated in any recent field guide or web-based survey. With this key, plants can be identified using a combination of vegetative and floristic characters in addition to distribution and ecology as a first step to further taxonomic revisions. We urge anyone with an interest in wild orchids in Southeast Asia to contribute new observations to update current information on the distribution of these overlooked plants as a first step for a taxonomic revision and to gain more insight into their conservation status.
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http://dx.doi.org/10.3897/phytokeys.110.28435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6215026PMC
October 2018

Satellite DNA in Paphiopedilum subgenus Parvisepalum as revealed by high-throughput sequencing and fluorescent in situ hybridization.

BMC Genomics 2018 Aug 2;19(1):578. Epub 2018 Aug 2.

School of Biological and Chemical Sciences, Queen Mary University of London, London, E1 4NS, UK.

Background: Satellite DNA is a rapidly diverging, largely repetitive DNA component of many eukaryotic genomes. Here we analyse the evolutionary dynamics of a satellite DNA repeat in the genomes of a group of Asian subtropical lady slipper orchids (Paphiopedilum subgenus Parvisepalum and representative species in the other subgenera/sections across the genus). A new satellite repeat in Paphiopedilum subgenus Parvisepalum, SatA, was identified and characterized using the RepeatExplorer pipeline in HiSeq Illumina reads from P. armeniacum (2n = 26). Reconstructed monomers were used to design a satellite-specific fluorescent in situ hybridization (FISH) probe. The data were also analysed within a phylogenetic framework built using the internal transcribed spacer (ITS) sequences of 45S nuclear ribosomal DNA.

Results: SatA comprises c. 14.5% of the P. armeniacum genome and is specific to subgenus Parvisepalum. It is composed of four primary monomers that range from 230 to 359 bp and contains multiple inverted repeat regions with hairpin-loop motifs. A new karyotype of P. vietnamense (2n = 28) is presented and shows that the chromosome number in subgenus Parvisepalum is not conserved at 2n = 26, as previously reported. The physical locations of SatA sequences were visualised on the chromosomes of all seven Paphiopedilum species of subgenus Parvisepalum (2n = 26-28), together with the 5S and 45S rDNA loci using FISH. The SatA repeats were predominantly localisedin the centromeric, peri-centromeric and sub-telocentric chromosome regions, but the exact distribution pattern was species-specific.

Conclusions: We conclude that the newly discovered, highly abundant and rapidly evolving satellite sequence SatA is specific to Paphiopedilum subgenus Parvisepalum. SatA and rDNA chromosomal distributions are characteristic of species, and comparisons between species reveal that the distribution patterns generate a strong phylogenetic signal. We also conclude that the ancestral chromosome number of subgenus Parvisepalum and indeed of all Paphiopedilum could be either 2n = 26 or 28, if P. vietnamense is sister to all species in the subgenus as suggested by the ITS data.
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http://dx.doi.org/10.1186/s12864-018-4956-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6090851PMC
August 2018

Anchored hybrid enrichment generated nuclear, plastid and mitochondrial markers resolve the Lepanthes horrida (Orchidaceae: Pleurothallidinae) species complex.

Mol Phylogenet Evol 2018 12 21;129:27-47. Epub 2018 Jul 21.

Naturalis Biodiversity Center, Endless Forms Group, Sylviusweg 72, Leiden 2333 BE, The Netherlands; University of Applied Sciences Leiden, Faculty of Science and Technology, Leiden, The Netherlands; Institute Biology Leiden, Leiden University, Sylviusweg 72, Leiden 2333 BE, The Netherlands.

Phylogenetic relationships in species complexes and lineages derived from rapid diversifications are often challenging to resolve using morphology or standard DNA barcoding markers. The hyper-diverse genus Lepanthes from Neotropical cloud forest includes over 1200 species and many recent, explosive diversifications that have resulted in poorly supported nodes and morphological convergence across clades. Here, we assess the performance of 446 nuclear-plastid-mitochondrial markers derived from an anchored hybrid enrichment approach (AHE) coupled with coalescence- and species network-based inferences to resolve phylogenetic relationships and improve species recognition in the Lepanthes horrida species group. In addition to using orchid-specific probes to increase enrichment efficiency, we improved gene tree resolution by extending standard angiosperm targets into adjacent exons. We found high topological discordance among individual gene trees, suggesting that hybridization/polyploidy may have promoted speciation in the lineage via formation of new hybrid taxa. In addition, we identified ten loci with the highest phylogenetic informativeness values from these genomes. Most previous phylogenetic sampling in the Pleurothallidinae relies on two regions (ITS and matK), therefore, the evaluation of other markers such as those shown here may be useful in future phylogenetic studies in the orchid family. Coalescent-based species tree estimation methods resolved the phylogenetic relationships of the L. horrida species group. The resolution of the phylogenetic estimations was improved with the inclusion of extended anchor targets. This approach produced longer loci with higher discriminative power. These analyses also disclosed two undescribed species, L. amicitiae and L. genetoapophantica, formally described here, which are also supported by morphology. Our study demonstrates the utility of combined genomic evidence to disentangle phylogenetic relationships at very shallow levels of the tree of life, and in clades showing convergent trait evolution. With a fully resolved phylogeny, is it possible to disentangle traits evolving in parallel or convergently across these orchid lineages such as flower color and size from diagnostic traits such as the shape and orientation of the lobes of the petals and lip.
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http://dx.doi.org/10.1016/j.ympev.2018.07.014DOI Listing
December 2018

DNA metabarcoding of orchid-derived products reveals widespread illegal orchid trade.

Proc Biol Sci 2017 Sep;284(1863)

Naturalis Biodiversity Center, Leiden, The Netherlands.

In eastern Mediterranean countries orchids continue to be collected from the wild for the production of salep, a beverage made of dried orchid tubers. In this study we used nrITS1 and nrITS2 DNA metabarcoding to identify orchid and other plant species present in 55 commercial salep products purchased in Iran, Turkey, Greece and Germany. Thirty samples yielded a total of 161 plant taxa, and 13 products (43%) contained orchid species and these belonged to 10 terrestrial species with tuberous roots. Another 70% contained the substitute ingredient (Guar). DNA metabarcoding using the barcoding markers nrITS1 and nrITS2 shows the potential of these markers and approach for identification of species used in salep products. The analysis of interspecific genetic distances between sequences of these markers for the most common salep orchid genera shows that species level identifications can be made with a high level of confidence. Understanding the species diversity and provenance of salep orchid tubers will enable the chain of commercialization of endangered species to be traced back to the harvesters and their natural habitats, and thus allow for targeted efforts to protect or sustainably use wild populations of these orchids.
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http://dx.doi.org/10.1098/rspb.2017.1182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5627200PMC
September 2017

Evolution of wood anatomical characters in Nepenthes and close relatives of Caryophyllales.

Ann Bot 2017 05;119(7):1179-1193

Naturalis Biodiversity Center, PO Box 9517, 2300 RA Leiden, The Netherlands.

Background And Aims: Nepenthes attracts wide attention with its spectacularly shaped carnivorous pitchers, cultural value and horticultural curiosity. Despite the plant's iconic fascination, surprisingly little anatomical detail is known about the genus beyond its modified leaf tip traps. Here, the wood anatomical diversity of Nepenthes is explored. This diversity is further assessed with a phylogenetic framework to investigate whether the wood characters within the genus are relevant from an evolutionary or ecological perspective, or rather depend on differences in developmental stages, growth habits, substrates or precipitation.

Methods: Observations were performed using light microscopy and scanning electron microscopy. Ancestral states of selected wood and pith characters were reconstructed using an existing molecular phylogeny for Nepenthes and a broader Caryophyllales framework. Pairwise comparisons were assessed for possible relationships between wood anatomy and developmental stages, growth habits, substrates and ecology.

Key Results: Wood anatomy of Nepenthes is diffuse porous, with mainly solitary vessels showing simple, bordered perforation plates and alternate intervessel pits, fibres with distinctly bordered pits (occasionally septate), apotracheal axial parenchyma and co-occurring uni- and multiseriate rays often including silica bodies. Precipitation and growth habit (stem length) are linked with vessel density and multiseriate ray height, while soil type correlates with vessel diameter, vessel element length and maximum ray width. For Caryophyllales as a whole, silica grains, successive cambia and bordered perforation plates are the result of convergent evolution. Peculiar helical sculpturing patterns within various cell types occur uniquely within the insectivorous clade of non-core Caryophyllales.

Conclusions: The wood anatomical variation in Nepenthes displays variation for some characters dependent on soil type, precipitation and stem length, but is largely conservative. The helical-banded fibre-sclereids that mainly occur idioblastically in pith and cortex are synapomorphic for Nepenthes , while other typical Nepenthes characters evolved convergently in different Caryophyllales lineages.
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http://dx.doi.org/10.1093/aob/mcx010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5604564PMC
May 2017

Exploring the evolutionary origin of floral organs of Erycina pusilla, an emerging orchid model system.

BMC Evol Biol 2017 03 23;17(1):89. Epub 2017 Mar 23.

Endless Forms group, Naturalis Biodiversity Center, Vondellaan 55, 2332 AA, Leiden, The Netherlands.

Background: Thousands of flowering plant species attract pollinators without offering rewards, but the evolution of this deceit is poorly understood. Rewardless flowers of the orchid Erycina pusilla have an enlarged median sepal and incised median petal ('lip') to attract oil-collecting bees. These bees also forage on similar looking but rewarding Malpighiaceae flowers that have five unequally sized petals and gland-carrying sepals. The lip of E. pusilla has a 'callus' that, together with winged 'stelidia', mimics these glands. Different hypotheses exist about the evolutionary origin of the median sepal, callus and stelidia of orchid flowers.

Results: The evolutionary origin of these organs was investigated using a combination of morphological, molecular and phylogenetic techniques to a developmental series of floral buds of E. pusilla. The vascular bundle of the median sepal indicates it is a first whorl organ but its convex epidermal cells reflect convergence of petaloid features. Expression of AGL6 EpMADS4 and APETALA3 EpMADS14 is low in the median sepal, possibly correlating with its petaloid appearance. A vascular bundle indicating second whorl derivation leads to the lip. AGL6 EpMADS5 and APETALA3 EpMADS13 are most highly expressed in lip and callus, consistent with current models for lip identity. Six vascular bundles, indicating a stamen-derived origin, lead to the callus, stelidia and stamen. AGAMOUS is not expressed in the callus, consistent with its sterilization. Out of three copies of AGAMOUS and four copies of SEPALLATA, EpMADS22 and EpMADS6 are most highly expressed in the stamen. Another copy of AGAMOUS, EpMADS20, and the single copy of SEEDSTICK, EpMADS23, are most highly expressed in the stelidia, suggesting EpMADS22 may be required for fertile stamens.

Conclusions: The median sepal, callus and stelidia of E. pusilla appear to be derived from a sepal, a stamen that gained petal identity, and stamens, respectively. Duplications, diversifying selection and changes in spatial expression of different MADS-box genes shaped these organs, enabling the rewardless flowers of E. pusilla to mimic an unrelated rewarding flower for pollinator attraction. These genetic changes are not incorporated in current models and urge for a rethinking of the evolution of deceptive flowers.
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http://dx.doi.org/10.1186/s12862-017-0938-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5364718PMC
March 2017

You are what you get from your fungi: nitrogen stable isotope patterns in Epipactis species.

Ann Bot 2017 05;119(7):1085-1095

Laboratory of Isotope Biogeochemistry, Bayreuth Center of Ecology and Environmental Research (BayCEER), University of Bayreuth, 95440 Bayreuth, Germany.

Background And Aims: Partially mycoheterotrophic plants are enriched in 13 C and 15 N compared to autotrophic plants. Here, it is hypothesized that the type of mycorrhizal fungi found in orchid roots is responsible for variation in 15 N enrichment of leaf tissue in partially mycoheterotrophic orchids.

Methods: The genus Epipactis was used as a case study and carbon and nitrogen isotope abundances of eight Epipactis species, fungal sporocarps of four Tuber species and autotrophic references were measured. Mycorrhizal fungi were identified using molecular methods. Stable isotope data of six additional Epipactis taxa and ectomycorrhizal and saprotrophic basidiomycetes were compiled from the literature.

Key Results: The 15 N enrichment of Epipactis species varied between 3·2 ± 0·8 ‰ ( E. gigantea ; rhizoctonia-associated) and 24·6 ± 1·6 ‰ ( E. neglecta ; associated with ectomycorrhizal ascomycetes). Sporocarps of ectomycorrhizal ascomycetes (10·7 ± 2·2 ‰) were significantly more enriched in 15 N than ectomycorrhizal (5·2 ± 4·0 ‰) and saprotrophic basidiomycetes (3·3 ± 2·1 ‰).

Conclusions: As hypothesized, it is suggested that the observed gradient in 15 N enrichment of Epipactis species is strongly driven by 15 N abundance of their mycorrhizal fungi; i.e. ɛ 15 N in Epipactis spp. associated with rhizoctonias < ɛ 15 N in Epipactis spp. with ectomycorrhizal basidiomycetes < ɛ 15 N in Epipactis spp. with ectomycorrhizal ascomycetes and basidiomycetes < ɛ 15 N in Epipactis spp. with ectomycorrhizal ascomycetes.
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http://dx.doi.org/10.1093/aob/mcw265DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5604585PMC
May 2017

DNA barcoding of tuberous Orchidoideae: a resource for identification of orchids used in Salep.

Mol Ecol Resour 2017 Mar 28;17(2):342-352. Epub 2016 Nov 28.

Department of Organismal Biology, Evolutionary Biology Centre, Uppsala University, Norbyvägen 18D, SE-75236, Uppsala, Sweden.

Tubers of terrestrial orchids are harvested and traded from the eastern Mediterranean to the Caspian Sea for the traditional product Salep. Overexploitation of wild populations and increased middle-class prosperity have escalated prices for Salep, causing overharvesting, depletion of native populations and providing an incentive to expand harvesting to untapped areas in Iran. Limited morphological distinctiveness among traded Salep tubers renders species identification impossible, making it difficult to establish which species are targeted and affected the most. In this study, a reference database of 490 nrITS, trnL-F spacer and matK sequences of 133 taxa was used to identify 150 individual tubers from 31 batches purchased in 12 cities in Iran to assess species diversity in commerce. The sequence reference database consisted of 211 nrITS, 158 trnL-F and 121 matK sequences, including 238 new sequences from collections made for this study. The markers enabled unambiguous species identification with tree-based methods for nrITS in 67% of the tested tubers, 58% for trnL-F and 59% for matK. Species in the genera Orchis (34%), Anacamptis (27%) and Dactylorhiza (19%) were the most common in Salep. Our study shows that all tuberous orchid species in this area are threatened by this trade, and further stresses the urgency of controlling illegal harvesting and cross-border trade of Salep tubers.
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http://dx.doi.org/10.1111/1755-0998.12615DOI Listing
March 2017

Incorporation of an invasive plant into a native insect herbivore food web.

PeerJ 2016 10;4:e1954. Epub 2016 May 10.

Groningen Institute for Evolutionary Life Sciences, University of Groningen , Groningen , the Netherlands.

The integration of invasive species into native food webs represent multifarious dynamics of ecological and evolutionary processes. We document incorporation of Prunus serotina (black cherry) into native insect food webs. We find that P. serotina harbours a herbivore community less dense but more diverse than its native relative, P. padus (bird cherry), with similar proportions of specialists and generalists. While herbivory on P. padus remained stable over the past century, that on P. serotina gradually doubled. We show that P. serotina may have evolved changes in investment in cyanogenic glycosides compared with its native range. In the leaf beetle Gonioctena quinquepunctata, recently shifted from native Sorbus aucuparia to P. serotina, we find divergent host preferences on Sorbus- versus Prunus-derived populations, and weak host-specific differentiation among 380 individuals genotyped for 119 SNP loci. We conclude that evolutionary processes may generate a specialized herbivore community on an invasive plant, allowing prognoses of reduced invasiveness over time. On the basis of the results presented here, we would like to caution that manual control might have the adverse effect of a slowing down of processes of adaptation, and a delay in the decline of the invasive character of P. serotina.
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http://dx.doi.org/10.7717/peerj.1954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4867706PMC
May 2016

Advances in DNA metabarcoding for food and wildlife forensic species identification.

Anal Bioanal Chem 2016 Jul 13;408(17):4615-30. Epub 2016 May 13.

RIKILT Wageningen UR, P.O. Box 230, 6700 AE, Wageningen, The Netherlands.

Species identification using DNA barcodes has been widely adopted by forensic scientists as an effective molecular tool for tracking adulterations in food and for analysing samples from alleged wildlife crime incidents. DNA barcoding is an approach that involves sequencing of short DNA sequences from standardized regions and comparison to a reference database as a molecular diagnostic tool in species identification. In recent years, remarkable progress has been made towards developing DNA metabarcoding strategies, which involves next-generation sequencing of DNA barcodes for the simultaneous detection of multiple species in complex samples. Metabarcoding strategies can be used in processed materials containing highly degraded DNA e.g. for the identification of endangered and hazardous species in traditional medicine. This review aims to provide insight into advances of plant and animal DNA barcoding and highlights current practices and recent developments for DNA metabarcoding of food and wildlife forensic samples from a practical point of view. Special emphasis is placed on new developments for identifying species listed in the Convention on International Trade of Endangered Species (CITES) appendices for which reliable methods for species identification may signal and/or prevent illegal trade. Current technological developments and challenges of DNA metabarcoding for forensic scientists will be assessed in the light of stakeholders' needs.
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http://dx.doi.org/10.1007/s00216-016-9595-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4909793PMC
July 2016

Development of a multiplex Q-PCR to detect Trichoderma harzianum Rifai strain T22 in plant roots.

J Microbiol Methods 2016 Feb 31;121:44-9. Epub 2015 Dec 31.

University of Applied Sciences Leiden, Zernikedreef 11, 2333, CK, Leiden, The Netherlands; Naturalis Biodiversity Center, Darwinweg 2, 2333, CR Leiden, The Netherlands; Institute Biology Leiden, Leiden University, Sylviusweg 72, 2333, BE, Leiden, The Netherlands. Electronic address:

The fungal species Trichoderma harzianum is widely used as a biological agent in crop protection. To verify the continued presence of this fungus on plant roots manually inoculated with T. harzianum strain T22, a Q-PCR was designed using specific probes for this particular strain. To develop these molecular diagnostic tools, genome mining was first carried out to retrieve putative new regions by which different strains of T. harzianum could be distinguished. Subsequently, Sanger sequencing of the L-aminoacid oxidase gene (aox1) in T. harzianum was applied to determine the mutations differing between various strains isolated from the Trichoderma collection of Koppert Biological Systems. Based on the sequence information obtained, a set of hydrolysis probes was subsequently developed which discriminated T. harzianum T22 strains varying in only a single nucleotide. Probes designed for two strains uniquely recognized the respective strains in Q-PCR with a detection limit of 12,5ng DNA. Titration assays in which T. harzianum DNA from distinct strains was varied further underscored the specificity of the probes. Lastly, fungal DNA extracted from roots of greenhouse cultured tomato plants was analyzed using the probe-based assay. DNA from T. harzianum strain T22 could readily be identified on roots of greenhouse reared tomato plants inoculated with varying concentrations up to one week after treatment with a detection limit of 3e6 colony forming units of T. harzianum T22. We conclude that the Q-PCR method is a reliable and robust method for assessing the presence and quantity of T. harzianum strain T22 in manually inoculated plant material. Our method provides scope for the development of DNA based strain specific identification of additional strains of Trichoderma and other fungal biological control agents.
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http://dx.doi.org/10.1016/j.mimet.2015.12.014DOI Listing
February 2016

Resistance to Antibiotics and Antifungal Medicinal Products: Can Complementary and Alternative Medicine Help Solve the Problem in Common Infection Diseases? The Introduction of a Dutch Research Consortium.

Evid Based Complement Alternat Med 2015 11;2015:521584. Epub 2015 Oct 11.

University of Applied Sciences, 2333 CK Leiden, Netherlands ; Department of Healthcare and Nutrition, Louis Bolk Institute, 2972 LA Driebergen, Netherlands.

The increase of antibiotic resistance worldwide, rising numbers of deaths and costs associated with this, and the fact that hardly any new antimicrobial drugs have been developed during the last decade have increased the interest in Complementary and Alternative Medicine (CAM) therapeutic interventions, if proven safe and effective. Observational studies on clinical CAM practices demonstrate positive effects of treatment of infections with CAM therapies (clinical effects, patient satisfaction) in combination with small percentages of antibiotics prescription. However, Cochrane reviews and other studies demonstrate that in most instances the quality of clinical trials on CAM treatment of infections is currently too low to provide sufficient evidence. Therefore a Dutch consortium on (in vitro and clinical) scientific research on CAM and antibiotic resistance has been formed. The aim and objective of the consortium is to establish an enduring partnership and to develop expertise to further develop and investigate safe and effective CAM treatments for infectious diseases of humans (and animals). A first ongoing project on the development of safe and effective biobased CAM antimycotics in women with (recurrent) vaginal candidiasis infection is introduced.
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http://dx.doi.org/10.1155/2015/521584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4619906PMC
November 2015
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