Roy H. J. Erkens

Genomic Treasure Troves: Complete Genome Sequencing of Herbarium and Insect Museum Specimens

Unlocking the vast genomic diversity stored in natural history collections would create unprecedented opportunities for genome-scale evolutionary, phylogenetic, domestication and population genomic studies. Many researchers have been discouraged from using historical specimens in molecular studies because of both generally limited success of DNA extraction and the challenges associated with PCR-amplifying highly degraded DNA. In todays next-generation sequencing (NGS) world, opportunities and prospects for historical DNA have changed dramatically, as most NGS methods are actually designed for taking short fragmented DNA molecules as templates. Here we show that using a standard multiplex and paired-end Illumina sequencing approach, genome-scale sequence data can be generated reliably from dry-preserved plant, fungal and insect specimens collected up to 115 years ago, and with minimal destructive sampling. Using a reference-based assembly approach, we were able to produce the entire nuclear genome of a 43-year-old Arabidopsis thaliana (Brassicaceae) herbarium specimen with high and uniform sequence coverage. Nuclear genome sequences of three fungal specimens of 22–82 years of age (Agaricus bisporus, Laccaria bicolor, Pleurotus ostreatus) were generated with 81.4–97.9% exome coverage. Complete organellar genome sequences were assembled for all specimens. Using de novo assembly we retrieved between 16.2–71.0% of coding sequence regions, and hence remain somewhat cautious about prospects for de novo genome assembly from historical specimens. Non-target sequence contaminations were observed in 2 of our insect museum specimens. We anticipate that future museum genomics projects will perhaps not generate entire genome sequences in all cases (our specimens contained relatively small and low-complexity genomes), but at least generating vital comparative genomic data for testing (phylo)genetic, demographic and genetic hypotheses, that become increasingly more horizontal. Furthermore, NGS of historical DNA enables recovering crucial genetic information from old type specimens that to date have remained mostly unutilized and, thus, opens up a new frontier for taxonomic research as well.

Use of rbcL and trnL-F as a Two-Locus DNA Barcode for Identification of NW-European Ferns: An Ecological Perspective

Although consensus has now been reached on a general two-locus DNA barcode for land plants, the selected combination of markers (rbcL + matK) is not applicable for ferns at the moment. Yet especially for ferns, DNA barcoding is potentially of great value since fern gametophytes—while playing an essential role in fern colonization and reproduction—generally lack the morphological complexity for morphology-based identification and have therefore been underappreciated in ecological studies. We evaluated the potential of a combination of rbcL with a noncoding plastid marker, trnL-F, to obtain DNA-identifications for fern species. A regional approach was adopted, by creating a reference database of trusted rbcL and trnL-F sequences for the wild-occurring homosporous ferns of NW-Europe. A combination of parsimony analyses and distance-based analyses was performed to evaluate the discriminatory power of the two-region barcode. DNA was successfully extracted from 86 tiny fern gametophytes and was used as a test case for the performance of DNA-based identification. Primer universality proved high for both markers. Based on the combined rbcL + trnL-F dataset, all genera as well as all species with non-equal chloroplast genomes formed their own well supported monophyletic clade, indicating a high discriminatory power. Interspecific distances were larger than intraspecific distances for all tested taxa. Identification tests on gametophytes showed a comparable result. All test samples could be identified to genus level, species identification was well possible unless they belonged to a pair of Dryopteris species with completely identical chloroplast genomes. Our results suggest a high potential of the combined use of rbcL and trnL-F as a two-locus cpDNA barcode for identification of fern species. A regional approach may be preferred for ecological tests. We here offer such a ready-to-use barcoding approach for ferns, which opens the way for answering a whole range of questions previously unaddressed in fern gametophyte ecology.

Evolution of syncarpy and other morphological characters in African Annonaceae : A posterior mapping approach

The congenital fusion of carpels, or syncarpy, is considered a key innovation as it is found in more than 80% of angiosperms. Within the magnoliids however, syncarpy has rarely evolved. Two alternative evolutionary origins of syncarpy were suggested in order to explain the evolution of this feature: multiplication of a single carpel vs. fusion of a moderate number of carpels. The magnoliid family Annonaceae provides an ideal situation to test these hypotheses as two African genera, Isolona and Monodora, are syncarpous in an otherwise apocarpous family with multicarpellate and unicarpellate genera. In addition to syncarpy, the evolution of six other morphological characters was studied. Well-supported phylogenetic relationships of African Annonaceae and in particular those of Isolona and Monodora were reconstructed. Six plastid regions were sequenced and analyzed using maximum parsimony and Bayesian inference methods. The Bayesian posterior mapping approach to study character evolution was used as it accounts for both mapping and phylogenetic uncertainty, and also allows multiple state changes along the branches. Our phylogenetic analyses recovered a fully resolved clade comprising twelve genera endemic to Africa, including Isolona and Monodora, which was nested within the so-called long-branch clade. This is the largest and most species-rich clade of African genera identified to date within Annonaceae. The two syncarpous genera were inferred with maximum support to be sister to a clade characterized by genera with multicarpellate apocarpous gynoecia, supporting the hypothesis that syncarpy arose by fusion of a moderate number of carpels. This hypothesis was also favoured when studying the floral anatomy of both genera. Annonaceae provide the only case of a clear evolution of syncarpy within an otherwise apocarpous magnoliid family. The results presented here offer a better understanding of the evolution of syncarpy in Annonaceae and within angiosperms in general.

Assessment of age and greenness of herbarium specimens as predictors for successful extraction and amplification of DNA

Age and the greenness of leaves have been frequently used as indicators for selecting herbarium specimens for molecular studies. Although plant DNA extraction and amplification have been common lab procedures for the past 20 years, no studies specifically investigated the success of these indicators. Here the predictive value of age and the greenness for extraction and amplification success is assessed, using a large number of herbarium specimens from different plant groups. The investigation of these indicators is important because herbarium material is a precious commodity, and is often the only remaining floral record of now extinct ecosystems. In cases where little leaf material is available, most researchers still attempt to extract DNA. This study shows that age and greenness of leaves are unreliable indicators of extraction and amplification success, although together they can have limited usefulness. Furthermore, we found that the amount of extracted DNA from herbarium specimens decreases with c. 1% per year in age of the specimens. Therefore, researchers sometimes should refrain from using old rare specimens because chances of success are unpredictable and precious herbarium material might be wasted. The uncritical use of indicators such as age or leaf colour is therefore not recommendable. Furthermore, botanists should annotate how specimens were collected and dried because this information is essential for successful DNA extraction. Hopefully, similar studies will be reported in order to identify the best approaches to extract DNA from herbarium specimens.

Classification of a large and widespread genus of Neotropical trees, Guatteria (Annonaceae) and its three satellite genera Guatteriella, Guatteriopsis and Heteropetalum

A phylogenetic study of the genus Polycarpon was made using DNA sequence data from the chloroplast rpsl6 intron and nuclear RPB2 regions. Polycarpon as currently recognised is shown to be polyphyletic. The South American species P. coquimbense and P. suffruticosum are more closely related to Haya obovata (Socotra) and Polycarpaea spicata (paleotropical) than to the remaining species of Polycarpon, and the widespread tropical species P. prostratum is nested with Macaronesian species of Polycarpaea. All three should therefore be excluded from Polycarpon. The remaining species, the P. tetraphyllum group, has its main diversity in the Mediterranean region. It is a polyploid complex with morphologically very similar taxa mostly separated only by partially overlapping characters. The members of this group form a strongly supported, but largely unresolved clade. It is proposed that they are all included in a widely circumscribed P. tetraphyllum. The dehiscence of the capsules in P. tetraphyllum, by valves that elastically roll inwards and finally throw out the seeds, is unique in Caryophyllaceae and provides a strong synapomorphy for Polycarpon in this new restricted sense.

Sampling bias in geographic and environmental space and its effect on the predictive power of species distribution models

Despite ever-growing popularity of species distribution models (SDM), their performance under conditions of spatially biased data has rarely been studied in detail. Here we explore the effect of a known spatial bias on the predictive ability of Maxent models, using five species of the genus Asplenium with variable reproductive modes. The models were trained and tested on western and central European presence-only distributional data, first with random background and then with target-group background. Then we tested the models on an independent Ukrainian dataset of the same species, using the area under the curve (AUC) value as test statistic. We carried out a principal components analysis (PCA) on the collection localities of the individual species to explore the properties of their ecological niches. In all but one species, spatial bias in the distributional data resulted in poor performance of the Maxent models (trained on the European dataset and tested on the Ukrainian dataset). In all species correction for sampling bias resulted in significantly wider predicted climatic niches. Based on the results of the PCA, spatial bias resulted in environmental bias of variable degree. We argue that species reproductive biology should be taken into account when distributional data are analysed in terms of their suitability for species distribution modelling. The reported results will inform biodiversity conservation assessments, particularly those using data from natural history collections.

Inter-and intraspecific variation in fern mating systems after long-distance colonization: the importance of selfing

BackgroundPrevious studies on the reproductive biology of ferns showed that mating strategies vary among species, and that polyploid species often show higher capacity for self-fertilization than diploid species. However, the amount of intraspecific variation in mating strategy and selfing capacity has only been assessed for a few species. Yet, such variation may have important consequences during colonization, as the establishment of any selfing genotypes may be favoured after long-distance dispersal (an idea known as Bakers law).ResultsWe examined intra-and interspecific variation in potential for self-fertilization among four rare fern species, of which two were diploids and two were tetraploids: Asplenium scolopendrium (2n), Asplenium trichomanes subsp. quadrivalens (4n), Polystichum setiferum (2n) and Polystichum aculeatum (4n). Sporophyte production was tested at different levels of inbreeding, by culturing gametophytes in isolation, as well as in paired cultures with a genetically different gametophyte. We tested gametophytes derived from various genetically different sporophytes from populations in a recently planted forest colonized through long-distance dispersal (Kuinderbos, the Netherlands), as well as from older, less disjunct populations.Sporophyte production in isolation was high for Kuinderbos genotypes of all four species. Selfing capacity did not differ significantly between diploids and polyploids, nor between species in general. Rather selfing capacity differed between genotypes within species. Intraspecific variation in mating system was found in all four species. In two species one genotype from the Kuinderbos showed enhanced sporophyte production in paired cultures. For the other species, including a renowned out crosser, selfing capacity was consistently high.ConclusionsOur results for four different species suggest that intraspecific variation in mating system may be common, at least among temperate calcicole ferns, and that genotypes with high selfing capacity may be present among polyploid as well as diploid ferns. The surprisingly high selfing capacity of all genotypes obtained from the Kuinderbos populations might be due to the isolated position of these populations. These populations may have established through single-spore colonization, which is only possible for genotypes capable of self-fertilization. Our results therewith support the idea that selection for selfing genotypes may occur during long-distance colonization, even in normally outcrossing, diploid ferns.

Diverse spore rains and limited local exchange shape fern genetic diversity in a recently created habitat colonized by long-distance dispersal.

BACKGROUND AND AIMS Populations established by long-distance colonization are expected to show low levels of genetic variation per population, but strong genetic differentiation among populations. Whether isolated populations indeed show this genetic signature of isolation depends on the amount and diversity of diaspores arriving by long-distance dispersal, and time since colonization. For ferns, however, reliable estimates of long-distance dispersal rates remain largely unknown, and previous studies on fern population genetics often sampled older or non-isolated populations. Young populations in recent, disjunct habitats form a useful study system to improve our understanding of the genetic impact of long-distance dispersal. METHODS Microsatellite markers were used to analyse the amount and distribution of genetic diversity in young populations of four widespread calcicole ferns (Asplenium scolopendrium, diploid; Asplenium trichomanes subsp. quadrivalens, tetraploid; Polystichum setiferum, diploid; and Polystichum aculeatum, tetraploid), which are rare in The Netherlands but established multiple populations in a forest (the Kuinderbos) on recently reclaimed Dutch polder land following long-distance dispersal. Reference samples from populations throughout Europe were used to assess how much of the existing variation was already present in the Kuinderbos. KEY RESULTS A large part of the Dutch and European genetic diversity in all four species was already found in the Kuinderbos. This diversity was strongly partitioned among populations. Most populations showed low genetic variation and high inbreeding coefficients, and were assigned to single, unique gene pools in cluster analyses. Evidence for interpopulational gene flow was low, except for the most abundant species. CONCLUSIONS The results show that all four species, diploids as well as polyploids, were capable of frequent long-distance colonization via single-spore establishment. This indicates that even isolated habitats receive dense and diverse spore rains, including genotypes capable of self-fertilization. Limited gene flow may conserve the genetic signature of multiple long-distance colonization events for several decades.

Increasing diversity in the species-rich genus Guatteria (Annonaceae)

During a taxonomic treatment of Annonaceae for the Flora of the Guianas project, an unusual new species of Guatteria Ruiz & Pav., G. anteridifera from French Guiana and Amapa in Brazil (Northern South America) was found and described herein.

Confronting a morphological nightmare: revision of the Neotropical genus Guatteria (Annonaceae)

A taxonomic revision of the genus Guatteria, including the former genera Guatteriella, Guatteriopsis and Heteropetalum is given. Within the genus Guatteria 177 species are recognized, 25 of which are new. Included are chapters on the history of the taxonomy of the genus, morphology, wood anatomy, karyology, palynology, chemistry, flower biology and pollination, dispersal, distribution and ecology, phylogeny and molecular studies, conservation, and uses. A synoptical key to all species is included, as well as two dichotomous keys, one for the species of Central America and Mexico, and one for the species of NE, E and SE Brazil. The species treatments include descriptions, full synonymy, geographical and ecological notes, vernacular names and taxonomic notes. For all species distribution maps are made. A complete identification list with all exsiccatae studied, an index to vernacular names and an index of scientific names is included at the end.