Orchidinae-205: A new genome-wide custom bait set for studying the evolution, systematics, and trade of terrestrial orchids.

Terrestrial orchids are a group of genetically understudied, yet culturally and economically important plants. The Orchidinae tribe contains many species that produce edible tubers that are used for the production of traditional delicacies collectively called 'salep'. Overexploitation of wild orchids in the Eastern Mediterranean and Western Asia threatens to drive many of these species to extinction, but cost-effective tools for monitoring their trade are currently lacking. Here we present a custom bait kit for target enrichment and sequencing of 205 novel genetic markers that are tailored to phylogenomic applications in Orchidinae s.l. A subset of 31 markers capture genes putatively involved in the production of glucomannan, a water-soluble polysaccharide that gives salep its distinctive properties. We tested the kit on 73 taxa native to the area, demonstrating universally high locus recovery irrespective of species identity, that exceeds the total sequence length obtained with alternative kits currently available. Phylogenetic inference with concatenation and coalescent approaches was robust and showed high levels of support for most clades, including some which were previously unresolved. Resolution for hybridizing and recently radiated lineages remains difficult, but could be further improved by analysing multiple haplotypes and the non-exonic sequences captured by our kit, with the promise to shed new light on the evolution of enigmatic taxa with a complex speciation history. Offering a step-up from traditional barcoding and universal markers, the genome-wide custom loci targeted by Orchidinae-205 are a valuable new resource to study the evolution, systematics and trade of terrestrial orchids.

The origin and speciation of orchids.

Orchids constitute one of the most spectacular radiations of flowering plants. However, their origin, spread across the globe, and hotspots of speciation remain uncertain due to the lack of an up-to-date phylogeographic analysis. We present a new Orchidaceae phylogeny based on combined high-throughput and Sanger sequencing data, covering all five subfamilies, 17/22 tribes, 40/49 subtribes, 285/736 genera, and c. 7% (1921) of the 29 524 accepted species, and use it to infer geographic range evolution, diversity, and speciation patterns by adding curated geographical distributions from the World Checklist of Vascular Plants. The orchids' most recent common ancestor is inferred to have lived in Late Cretaceous Laurasia. The modern range of Apostasioideae, which comprises two genera with 16 species from India to northern Australia, is interpreted as relictual, similar to that of numerous other groups that went extinct at higher latitudes following the global climate cooling during the Oligocene. Despite their ancient origin, modern orchid species diversity mainly originated over the last 5 Ma, with the highest speciation rates in Panama and Costa Rica. These results alter our understanding of the geographic origin of orchids, previously proposed as Australian, and pinpoint Central America as a region of recent, explosive speciation.

Orchid fruit and root movement analyzed using 2D photographs and a bioinformatics pipeline for processing sequential 3D scans.

Premise: Most studies of the movement of orchid fruits and roots during plant development have focused on morphological observations; however, further genetic analysis is required to understand the molecular mechanisms underlying this phenomenon. A precise tool is required to observe these movements and harvest tissue at the correct position and time for transcriptomics research. Methods: We utilized three-dimensional (3D) micro-computed tomography (CT) scans to capture the movement of fast-growing Erycina pusilla roots, and built an integrated bioinformatics pipeline to process 3D images into 3D time-lapse videos. To record the movement of slowly developing E. pusilla and Phalaenopsis equestris fruits, two-dimensional (2D) photographs were used. Results: The E. pusilla roots twisted and resupinated multiple times from early development. The first period occurred in the early developmental stage (77-84 days after germination [DAG]) and the subsequent period occurred later in development (140-154 DAG). While E. pusilla fruits twisted 45° from 56-63 days after pollination (DAP), the fruits of P. equestris only began to resupinate a week before dehiscence (133 DAP) and ended a week after dehiscence (161 DAP). Discussion: Our methods revealed that each orchid root and fruit had an independent direction and degree of torsion from the initial to the final position. Our innovative approaches produced detailed spatial and temporal information on the resupination of roots and fruits during orchid development.

Female pond bats hunt in other areas than males and consume lighter prey when pregnant.

Animals with large energy requirements are forced to optimize their hunting strategy, which may result in differentiation of the diet between sexes and across seasons. Here, we examined spatiotemporal variation in the diet of both sexes of the Pond Bat Myotis dasycneme, a species known to have spatial segregation of sexes when the young are born and lactating. Fecal pellets were collected from live animals for a period of 15 years at various locations in the Netherlands. A total of 535 pellets were successfully analyzed by microscopy and an additional 160 pellets by DNA metabarcoding. Morphological and molecular analyses showed that the diet of pregnant and lactating pond bats differed significantly from the diet of females with no reproductive investment. Further analyses of the data showed that pregnant female pond bats are highly dependent on small prey and pupae, mainly nonbiting midges and mosquitoes (Diptera: Chironomidae and Culicidae). These insects can be found in large quantities in peatlands intersected with shallow waterways, the habitat type in which female pond bats were observed more often than males. Our results suggest that during pregnancy the spatial segregation of sexes coincides with sex-specific diets, which might reflect habitat selection based on energy requirements, in addition to lowered intraspecific competition.

Evolution and development of fruits of Erycina pusilla and other orchid species.

Fruits play a crucial role in seed dispersal. They open along dehiscence zones. Fruit dehiscence zone formation has been intensively studied in Arabidopsis thaliana. However, little is known about the mechanisms and genes involved in the formation of fruit dehiscence zones in species outside the Brassicaceae. The dehiscence zone of A. thaliana contains a lignified layer, while dehiscence zone tissues of the emerging orchid model Erycina pusilla include a lipid layer. Here we present an analysis of evolution and development of fruit dehiscence zones in orchids. We performed ancestral state reconstructions across the five orchid subfamilies to study the evolution of selected fruit traits and explored dehiscence zone developmental genes using RNA-seq and qPCR. We found that erect dehiscent fruits with non-lignified dehiscence zones and a short ripening period are ancestral characters in orchids. Lignified dehiscence zones in orchid fruits evolved multiple times from non-lignified zones. Furthermore, we carried out gene expression analysis of tissues from different developmental stages of E. pusilla fruits. We found that fruit dehiscence genes from the MADS-box gene family and other important regulators in E. pusilla differed in their expression pattern from their homologs in A. thaliana. This suggests that the current A. thaliana fruit dehiscence model requires adjustment for orchids. Additionally, we discovered that homologs of A. thaliana genes involved in the development of carpel, gynoecium and ovules, and genes involved in lipid biosynthesis were expressed in the fruit valves of E. pusilla, implying that these genes may play a novel role in formation of dehiscence zone tissues in orchids. Future functional analysis of developmental regulators, lipid identification and quantification can shed more light on lipid-layer based dehiscence of orchid fruits.

Orchids reduce attachment of herbivorous snails with leaf trichomes.

Protective structures in the epidermis are essential for land plants to defend themselves against herbivores. In this study, we investigated the effect of different types of trichomes of three orchids, Calanthe triplicata, Dendrochilum pallidiflavens and Trichotosia ferox, on attachment of herbivorous land snails, using histochemistry and centrifuge experiments. Size, ornamentation and histochemistry of epicuticular trichomes on the orchid leaves were assessed with light microscopy, scanning electron microscopy and transmission electron microscopy. Total forces needed to detach two differently shaped snail species, Subulina octona and Pleurodonte isabella, were measured using a turntable equipped with a synchronized strobe. Snails were placed in two positions, either perpendicular or parallel to the main veins on the orchid leaves, both on the adaxial (= upper) or abaxial (= lower) side. The results obtained provided three new insights. First, a perpendicular or parallel position of the snails to the main veins did not significantly affect the attachment performance of either species tested. Secondly, snails detached significantly easier on leaf sides covered with a high density of lignin filled epicuticular trichomes. Thirdly, the removal of glandular trichomes did not affect the attachment forces; however, the absence of lignified trichomes increased the attachment of the snails. Our study highlights the importance of studying micro-ornamentation in combination with performance for obtaining a better understanding of the defense mechanisms employed by different species of orchids to deter herbivorous snails.

A Bioinformatic Pipeline to Estimate Ploidy Level from Target Capture Sequence Data Obtained from Herbarium Specimens.

Whole genome duplications (WGD) are frequent in many plant lineages; however, ploidy level variation is unknown in most species. The most widely used methods to estimate ploidy levels in plants are chromosome counts, which require living specimens, and flow cytometry estimates, which necessitate living or relatively recently collected samples. Newly described bioinformatic methods have been developed to estimate ploidy levels using high-throughput sequencing data, and these have been optimized in plants by calculating allelic ratio values from target capture data. This method relies on the maintenance of allelic ratios from the genome to the sequence data. For example, diploid organisms will generate allelic data in a 1:1 proportion, with an increasing number of possible allelic ratio combinations occurring in individuals with higher ploidy levels. In this chapter, we explain step-by-step this bioinformatic approach for the estimation of ploidy level.

Genotyping by sequencing for estimating relative abundances of diatom taxa in mock communities.

BACKGROUND: Diatoms are present in all waters and are highly sensitive to pollution gradients. Therefore, they are ideal bioindicators for water quality assessment. Current indices used in these applications are based on identifying diatom species and counting their abundances using traditional light microscopy. Several molecular techniques have been developed to help automate different steps of this process, but obtaining reliable estimates of diatom community composition and species abundance remains challenging. RESULTS: Here, we evaluated a recently developed quantification method based on Genotyping by Sequencing (GBS) for the first time in diatoms to estimate the relative abundances within a species complex. For this purpose, a reference database comprised of thousands of genomic DNA clusters was generated from cultures of Nitzschia palea. The sequencing reads from calibration and mock samples were mapped against this database for parallel quantification. We sequenced 25 mock diatom communities containing up to five taxa per sample in different abundances. Taxon abundances in these communities were also quantified by a diatom expert using manual counting of cells on light microscopic slides. The relative abundances of strains across mock samples were over- or under-estimated by the manual counting method, and a majority of mock samples had stronger correlations using GBS. Moreover, one previously recognized putative hybrid had the largest number of false positive detections demonstrating the limitation of the manual counting method when morphologically similar and/or phylogenetically close taxa are analyzed. CONCLUSIONS: Our results suggest that GBS is a reliable method to estimate the relative abundances of the N. palea taxa analyzed in this study and outperformed traditional light microscopy in terms of accuracy. GBS provides increased taxonomic resolution compared to currently available quantitative molecular approaches, and it is more scalable in the number of species that can be analyzed in a single run. Hence, this is a significant step forward in developing automated, high-throughput molecular methods specifically designed for the quantification of [diatom] communities for freshwater quality assessments.

Genomic, spatial and morphometric data for discrimination of four species in the Mediterranean Tamus clade of yams (Dioscorea, Dioscoreaceae).

BACKGROUND AND AIMS: Among the numerous pantropical species of the yam genus, Dioscorea, only a small group occurs in the Mediterranean basin, including two narrow Pyrenean endemics (Borderea clade) and two Mediterranean-wide species (D. communis and D. orientalis, Tamus clade). However, several currently unrecognized species and infraspecific taxa have been described in the Tamus clade due to significant morphological variation associated with D. communis. Our overarching aim was to investigate taxon delimitation in the Tamus clade using an integrative approach combining phylogenomic, spatial and morphological data. METHODS: We analysed 76 herbarium samples using Hyb-Seq genomic capture to sequence 260 low-copy nuclear genes and plastomes, together with morphometric and environmental modelling approaches. KEY RESULTS: Phylogenomic reconstructions confirmed that the two previously accepted species of the Tamus clade, D. communis and D. orientalis, are monophyletic and form sister clades. Three subclades showing distinctive geographic patterns were identified within D. communis. These subclades were also identifiable from morphometric and climatic data, and introgression patterns were inferred between subclades in the eastern part of the distribution of D. communis. CONCLUSIONS: We propose a taxonomy that maintains D. orientalis, endemic to the eastern Mediterranean region, and splits D. communis sensu lato into three species: D. edulis, endemic to Macaronesia (Canary Islands and Madeira); D. cretica, endemic to the eastern Mediterranean region; and D. communis sensu stricto, widespread across western and central Europe. Introgression inferred between D. communis s.s. and D. cretica is likely to be explained by their relatively recent speciation at the end of the Miocene, disjunct isolation in eastern and western Mediterranean glacial refugia and a subsequent westward recolonization of D. communis s.s. Our study shows that the use of integrated genomic, spatial and morphological approaches allows a more robust definition of species boundaries and the identification of species that previous systematic studies failed to uncover.

Phylotranscriptomics reveals the reticulate evolutionary history of a widespread diatom species complex.

In contrast to surveys based on a few genes that often provide limited taxonomic resolution, transcriptomes provide a wealth of genomic loci that can resolve relationships among taxonomically challenging lineages. Diatoms are a diverse group of aquatic microalgae that includes important bioindicator species and many such lineages. One example is Nitzschia palea, a widespread species complex with several morphologically defined taxonomic varieties, some of which are critical pollution indicators. Morphological differences among the varieties are subtle and phylogenetic studies based on a few genes fail to resolve their evolutionary relationships. We conducted morphometric and transcriptome analyses of 10 Nitzschia palea strains to resolve the relationships among strains and taxonomic varieties. Nitzschia palea was resolved into three clades, one of which corresponds to a group of strains with narrow linear-lanceolate valves. The other morphological group recovered in the shape outline analysis was not monophyletic and consisted of two clades. Gene-tree concordance analyses and phylogenetic network estimations revealed patterns of incomplete lineage sorting and gene flow between intraspecific lineages. We detected reticulated evolutionary patterns among lineages with different morphologies, resulting in a putative recent hybrid. Our study shows that phylogenomic analyses of unlinked nuclear loci, complemented with morphometrics, can resolve complex evolutionary histories of recently diverged species complexes.

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

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 (R2 = 0.821) than for trnL (R2 = 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.

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

The dry one-seeded fruits (cypselae) of the Asteraceae are often crowned with a pappus, 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 sepals 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 alternipetalaous position usually being ahead in growth, referring to sepal identity. Tof-APETALLA1 expression increased during floret development and was highest in mature pappus. Tof-PISTILLATA 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 beak, 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.

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

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.

Neural networks for increased accuracy of allergenic pollen monitoring.

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.

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

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.

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

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.

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

Deceptive Ceropegia 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 Ceropegia sandersonii 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 Ceropegia to lay the foundation for a better understanding of the molecular background of pitfall flower evolution in Apocynaceae.

Ontogeny and Anatomy of the Dimorphic Pitchers of Nepenthes rafflesiana Jack.

An enigmatic feature of tropical pitcher plants belonging to the genus Nepenthes 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 N. rafflesiana 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 Nepenthes, likely to exploit different food sources.

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

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 Phalaenopsis equestris and Phalaenopsis pulcherrima, two species that differ in floral morphology: P. equestris has a large callus but short stelidia and no mentum, whereas P. pulcherrima 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. SEP clade 1 copies are expressed in the larger callus of P. equestris, whereas AP3 clade 1 and AGL6 clade 1 copies are expressed in the pronounced mentum and long stelidia of P. pulcherrima. AP3 clade 4, PI-, AGL6 clade 2 and PCF clade 1 copies might have a balancing role in callus and gynostemium development. There appears to be a trade-off between DIV clade 2 expression with SEP clade 1 expression in the callus, on the one hand, and with AP3 clade 1 and AGL6 clade 1 expression in the stelidia and mentum on the other. CONCLUSIONS: We detected differential growth and expression of MADS box AP3/PI-like, AGL6-like and SEP-like, and MYB DIV-like gene copies in the callus, stelidia and mentum of two species of Phalaenopsis, 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 Phalaenopsis flowers to different pollinators by providing combined micro-morphological and molecular evidence for a possible sepaloid-petaloid-staminodial origin of the orchid mentum.

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

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.