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BackgroundThe need for enhancing the productivity of fisheries in Africa triggered the introduction of non-native fish, causing dramatic changes to local species. In East Africa, the extensive translocation of Nile tilapia (Oreochromis niloticus) is one of the major factors in this respect. Using 40 microsatellite loci with SSR-GBS techniques, we amplified a total of 664 individuals to investigate the genetic structure ofO. niloticusfrom East Africa in comparison to Ethiopian and Burkina Faso populations.ResultsAll three African regions were characterized by independent gene-pools, however, the Ethiopian population from Lake Tana was genetically more divergent (Fst = 2.1) than expected suggesting that it might be a different sub-species. In East Africa, the genetic structure was congruent with both geographical location and anthropogenic activities (Isolation By Distance for East Africa, R2 = 0.67 and Uganda, R2 = 0.24).O. niloticusfrom Lake Turkana (Kenya) was isolated, while in Uganda, despite populations being rather similar to each other, two main natural catchments were able to be defined. We show that these two groups contributed to the gene-pool of different non-native populations. Moreover, admixture and possible hybridization with other tilapiine species may have contributed to the genetic divergence found in some populations such as Lake Victoria. We detected other factors that might be affecting Nile tilapia genetic variation. For example, most of the populations have gone through a reduction in genetic diversity, which can be a consequence of bottleneck (G-W, ConclusionsThe anthropogenic activities particularly in the East AfricanO. niloticustranslocations, promoted artificial admixture among Nile Tilapia populations. Translocations may also have triggered hybridization with the native congenerics, which needs to be further studied. These events may contribute to outbreeding depression and hence compromising the sustainability of the species in the region.

Ailanthus altissima is a common tree species of the European non-native flora. In Eastern Austria, Ailanthus has both a long tradition, dating back to the late 18th century, and a high frequency of occurrence. Here, we apply molecular markers from the nuclear and chloroplast DNA in order to study the origin, as well as the clonal and genetic structure of the species in forest stands of this region. Our results indicate a single area of origin for all our study populations from the north-eastern part of the native range, in agreement with historical reports. Within populations, vegetative reproduction has resulted in extensive clonal structures at some sites. Long presence and repeated vegetative recruitment might have led to expansion of clones in areas of early introduction. In addition, limited mate availability may also have promoted vegetative reproduction. Tests for recent bottlenecks were significant in almost all stands, possibly reflecting founder effects since the introduction of Ailanthus in our study area. Among populations, we found a very pronounced and significant genetic structure, which did not follow a spatial pattern. We argue that founder effects due to population establishment with a low number of propagules – whether naturally or artificially – have caused genetic drift and a structured gene pool. The lack of spatial pattern or isolation-by-distance suggests that seed transfer by humans might have been a major factor shaping the genetic variation of Ailanthus in Eastern Austria.

Background Nile tilapia, Oreochromis niloticus (Linnaeus, 1758) is among the economically most important freshwater fish species in East Africa, and a major source of protein for local consumption. Human induced translocations of non-native stocks for aquaculture and fisheries have been found as a potential threat to the genetic diversity and integrity of local populations. In the present study, we investigate the genetic structure of O. niloticus from 16 waterbodies across Ethiopia using 37 microsatellite loci with SSR-GBAS techniques. Results The samples are structured into three main clusters shaped either by biogeographic factors or stocking activities. High FST values (Global FST = 0.438) between populations indicate a high level of genetic differentiation and may suggest long term isolation even within the same drainage systems. Natural populations of the Omo-Turkana system and the lakes in the Southern Main Ethiopian Rift showed the highest genetic variability while low variability was found in stocked populations of lakes Hora, Hashenge and Hayq. Conclusions The results presented herein, may provide an essential basis for the management and conservation of the unique genetic resources in northern East Africa, and advance our understanding of biodiversity, phylogeny, evolution and development towards phylogenetically more accurate taxonomic classifications.

Visuelle Merkmale gehören zu den wichtigsten biologischen Charakteristika um Organismen zu beschreiben. Farbe und Körperbau sind wichtige Merkmale zur Unterscheidung von Untergruppen, für die Beurteilung von Diversität und für Untersuchungen hinsichtlich Anpassung an lokale Gegebenheiten. In der klassischen Morphologie setzt man zur Charakterisierung auf vordefinierte Kartierungspunkte die für jede zu untersuchende Probe manuell in einer digitalen Abbildung markiert werden. Eine manuelle Zuordnung von Kartierungspunkten ist zeitaufwendig und fehleranfällig. Vordefinierte Kartierungspunkte haben darüber hinaus den Nachteil, dass Information, die für eine genaue Charakterisierung der betrachteten Organismen wichtig wäre gar nicht berücksichtigt wird. Dieses Problem tritt besonders dann auf, wenn Charakteristika komplexe Zusammenhänge aufweisen, die für menschliche Betrachter schwer zu erkennen sind. Im Bereich des maschinellen Lernens (ML) wurden in den vergangenen Jahren innovative Methoden zur Datenanalyse entwickelt, die insbesondere in der Bildanalyse maßgebliche Performanzsprünge erlauben. Um wissenschaftlich glaubwürdige Ergebnisse zu erhalten, ist es allerdings wichtig auf Interpretierbarkeit der Methoden zu achten und die black-box Charakteristik beim Einsatz von ML abzufedern. Um reproduzierbare visuelle Merkmale in Nile tilapia zu finden die auf eine Anpassung der Fischpopulationen an lokale Gegebenheiten hindeuten, setzt die vorliegende Arbeit auf komplementäre Analyseschritte und auf die Interpretation sogenannter "saliency maps". Als Datenbasis für die vorliegende Analyse dienen Nile tilapia Bilder, die von Fischen aufgenommen wurden, die aus sechs Äthiopischen Seen stammen. Ein Methodenvergleich, der zur Vorhersage des Ursprungs aus den Fischbildern auf deep learning bzw. auf eine klassische morphometrische Analyse setzt, deutet auf eine signifikante Verbesserung der Vorhersagegenauigkeit durch den Einsatz von deep learning hin. Eine kritische Analyse der saliency maps zeigt allerdings, dass die Vorhersagen mittels deep learning nicht nur auf authentischen biologischen Merkmalen, sondern auch auf technischen Fehlern basieren, die die Vorhersage des Ursprungs der Fische begünstigen. So werden zum Beispiel der unterschiedliche Bildhintergrund oder eine Nadel mit der Fische fixiert werden als Hinweis auf den Ursprung mancher Fische interpretiert. Vertrauenswürdigere Merkmale, die für den Ursprung der Nile tilapia Populationen charakteristisch sind, konnten mit einer Bayes'schen Methode automatisch aus den Fischbildern bestimmt werden. Die dabei entdeckten Merkmale überlappen teilweise mit den Kartierungspunkten der klassischen Morphologie, erweitern diese aber um Körperregionen, die bisher nicht berücksichtigt wurden. Aus methodischer Sicht kann aus der Analyse der Schluss gezogen werden, dass kritische hinterfragtes maschinelles Lernen geeignet ist, um morphologische Merkmale automatisch aus Bildern zu extrahieren, die es erlauben reproduzierbare biologische Erkenntnisse zu gewinnen. Hinsichtlich Fischbiologie kommt die vorliegende Analyse zum Schluss, dass Nile tilapia Populationen dazu tendieren, ortsspezifische Merkmale auszubilden. Visual characteristics are among the most important features for characterizing the phenotype of biological organisms. Color and geometric properties define population phenotype and allow assessing diversity and adaptation to environmental conditions. To analyze geometric properties classical morphometrics relies on biologically relevant landmarks which are manually assigned to digital images. Assigning landmarks is tedious and error prone. Predefined landmarks may in addition miss out on information which is not obvious to the human eye. The machine learning (ML) community has recently proposed new data analysis methods which by uncovering subtle features in images obtain excellent predictive accuracy. Scientific credibility demands however that results are interpretable and hence to mitigate the black-box nature of ML methods. To overcome the black-box nature of ML we apply complementary methods and investigate internal representations with saliency maps to reliably identify location specific characteristics in images of Nile tilapia populations. Analyzing fish images which were sampled from six Ethiopian lakes reveals that deep learning improves on a conventional morphometric analysis in predictive performance. A critical assessment of established saliency maps with a novel significance test reveals however that the improvement is aided by artifacts which have no biological interpretation. More interpretable results are obtained by a Bayesian approach which allows us to identify genuine Nile tilapia body features which differ in dependence of the animals habitat. We find that automatically inferred Nile tilapia body features corroborate and expand the results of a landmark based analysis that the anterior dorsum, the fish belly, the posterior dorsal region and the caudal fin show signs of adaptation to the fish habitat. We may thus conclude that Nile tilapia show habitat specific morphotypes and that a ML analysis allows inferring novel biological knowledge in a reproducible manner. (VLID)6040310

Simple Summary The gut microbiota (all microbes in the intestine) of fishes is known to play an essential role in diverse aspects of their life. The gut microbiota of fish is affected by various environmental parameters, including temperature changes, salinity and diet. This study characterised the microbial composition in gut samples of Nile Tilapia collected from Lake Tana and the Bahir Dar aquaculture facility centre applying modern molecular techniques. The results show clear differences in the gut microbiota in fish from the Lake Tana and the ones from aquaculture. Further, also significant differences were observed on the composition of the gut microbiota across sampling months. Samples from the aquaculture centre displayed a higher diversity than the wild catch Nile tilapia from Lake Tana even though there is also an overlapping of the detected microbial groups. Overall, this is the first study on the effects of sampling season and catching site on the gut microbiota of Nile tilapia in Ethiopia. Future work will help to precisely explain the causes of these changes and their influence of the health and growth of Nile tilapia in Ethiopian lakes as well as under aquaculture conditions. Abstract The gut microbiota of fishes is known to play an essential role in diverse aspects of host biology. The gut microbiota of fish is affected by various environmental parameters, including temperature changes, salinity and diet. Studies of effect of environment on gut microbiota enables to have a further understanding of what comprises a healthy microbiota under different environmental conditions. However, there is insufficient understanding regarding the effects of sampling season and catching site (wild and aquaculture) on the gut microbiota of Nile tilapia. This study characterised gut microbial composition and diversity from samples collected from Lake Tana and the Bahir Dar aquaculture facility centre using 16S rDNA Illumina MiSeq platform sequencing. Firmicutes and Fusobacteria were the most dominant phyla in the Lake Tana samples, while Proteobacteria was the most dominant in the aquaculture samples. The results of differential abundance testing clearly indicated significant differences for Firmicutes, Fusobacteria, Bacteroidetes and Cyanobacteria across sampling months. However, Proteobacteria, Chloroflexi, Fusobacteria and Cyanobacteria were significantly enriched in the comparison of samples from the Lake Tana and aquaculture centre. Significant differences were observed in microbial diversity across sampling months and between wild and captive Nile tilapia. The alpha diversity clearly showed that samples from the aquaculture centre (captive) had a higher diversity than the wild Nile tilapia samples from Lake Tana. The core gut microbiota of all samples of Nile tilapia used in our study comprised Firmicutes, Proteobacteria and Fusobacteria. This study clearly showed the impact of sampling season and catching site (wild and aquaculture) on the diversity and composition of bacterial communities associated with the gut of Nile tilapia. Overall, this is the first study on the effects of sampling season and catching site on the gut microbiota of Nile tilapia in Ethiopia. Future work is recommended to precisely explain the causes of these changes using large representative samples of Nile tilapia from different lakes and aquaculture farms.

BackgroundInvasive species are increasingly driving biodiversity decline, and knowledge of colonization dynamics, including both drivers and dispersal modes, are important to prevent future invasions. The bee speciesMegachile sculpturalis(Hymenoptera: Megachilidae), native to East-Asia, was first recognized in Southeast-France in 2008, and has since spread throughout much of Europe. The spread is very fast, and colonization may result from multiple fronts.ResultTo track the history of this invasion, codominant markers were genotyped using Illumina sequencing and the invasion history and degree of connectivity between populations across the European invasion axis were investigated. Distinctive genetic clusters were detected with east–west differentiations in Middle-Europe.ConclusionWe hypothesize that the observed cluster formation resulted from multiple, independent introductions of the species to the European continent. This study draws a first picture of an early invasion stage of this wild bee and forms a foundation for further investigations, including studies of the species in their native Asian range and in the invaded range in North America.

The Nile tilapia (Oreochromis niloticus) gut harbors a diverse microbial community, however, their variation across gut regions, lumen and mucosa is not fully elucidated. In this study, gut microbiota of all samples across gut regions and sample types (luminal content and mucosa) were analyzed and compared from two Ethiopian lakes. Microbiota were characterized using 16S rRNA Illumina MiSeq platform sequencing. A total of 2061 operational taxonomic units (OTUs) were obtained and the results indicated that Nile tilapia from Lake Chamo harbored a much more diversified gut microbiota than Lake Awassa. In addition, the gut microbiota diversity varied significantly across the gut region based on the Chao1, Shannon and Simpson index. The microbiome analyses of all samples in the midgut region showed significantly higher values for alpha diversity (Chao 1, Shannon and Simpson). Beta diversity analysis revealed a clear separation of samples according to sampling areas and gut regions. The most abundant genera were Clostridium_sensu_stricto and Clostridium_XI genera across all samples. Between the two sampling lakes, two phyla, Phylum Fusobacteria and Cyanobacteria, were found to be significantly different. On the other hand, six phyla (Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Proteobacteria and Cyanobacteria) were significantly different across gut regions. In this study, we found that all samples shared a large core microbiota, comprising a relatively large number of OTUs, which was dominated by Proteobacteria, Firmicutes, Cyanobacteria, Fusobacteria and Actinobacteria. This study has established the bases for future large-scale investigations of gut microbiota of fishes in Ethiopian lakes.

By applying second‐generation sequencing technologies to microsatellite genotyping, sequence information is produced which can result in high‐resolution population genetics analysis populations and increased replicability between runs and laboratories. In the present study, we establish an approach to study the genetic structure patterns of two European hedgehog species Erinaceaus europaeus and E. roumanicus. These species are usually associated with human settlements and are good models to study anthropogenic impacts on the genetic diversity of wild populations. The short sequence repeats genotyping by sequence (SSR‐GBS) method presented uses amplicon sequences to determine genotypes for which allelic variants can be defined according to both length and single nucleotide polymorphisms (SNPs). To evaluate whether complete sequence information improved genetic structure definition, we compared this information with datasets based solely on length information. We identified a total of 42 markers which were successfully amplified in both species. Overall, genotyping based on complete sequence information resulted in a higher number of alleles, as well as greater genetic diversity and differentiation between species. Additionally, the structure patterns were slightly clearer with a division between both species and some potential hybrids. There was some degree of genetic structure within species, although only in E. roumanicus was this related to geographical distance. The statistically significant results obtained by SSR‐GBS demonstrate that it is superior to electrophoresis‐based methods for SSR genotyping. Moreover, the greater reproducibility and throughput with lower effort which can be obtained with SSR‐GBS and the possibility to include degraded DNA into the analysis, allow for continued relevance of SSR markers during the genomic era.

PREMISE OF THE STUDY Qat (Catha edulis, Celastraceae) is a woody plant species cultivated for its stimulant alkaloids. Qat is important to the economy and culture in large regions of Ethiopia, Kenya, and Yemen. Despite the importance of this species, the wild origins and dispersal of cultivars have only been described in often contradictory historical documents. We examined the wild origins, human-mediated dispersal, and genetic divergence of cultivated qat compared to wild qat. METHODS We sampled 17 SSR markers and 1561 wild and cultivated individuals across the historical areas of qat cultivation. KEY RESULTS On the basis of genetic structure inferred using Bayesian and nonparametric methods, two centers of origin in Kenya and one in Ethiopia were found for cultivated qat. The centers of origin in Ethiopia and northeast of Mt. Kenya are the primary sources of cultivated qat genotypes. Qat cultivated in Yemen is derived from Ethiopian genotypes rather than Yemeni wild populations. Cultivated qat with a wild Kenyan origin has not spread to Ethiopia or Yemen, whereas a small minority of qat cultivated in Kenya originated in Ethiopia. Hybrid genotypes with both Ethiopian and Kenyan parentage are present in northern Kenya. CONCLUSIONS Ethiopian cultivars have diverged from their wild relatives, whereas Kenyan qat has diverged less. This pattern of divergence could be caused by the extinction of the wild-source qat populations in Ethiopia due to deforestation, undersampling, and/or artificial selection for agronomically important traits.

Grape phylloxera is one of the most dangerous insect pests for worldwide viticulture. The leaf- and root-galling phylloxerid has been managed by grafting European grapevines onto American rootstock hybrids. Recent reports pinpoint the appearance of host-adapted biotypes, but information about the biomolecular characteristics underlying grape phylloxera biotypisation and its role in host performance is scarce. Using RNA-sequencing, we sequenced the transcriptome of two larval stages: L1 (probing) and L2-3 (feeding) larvae of two root-feeding grape phylloxera lineages feeding on the rootstock Teleki 5C (biotype C) and V. vinifera Riesling (biotype A). In total, 7501 differentially expressed genes (DEGs) were commonly modulated by the two biotypes. For the probing larvae, we found an increased number of DEGs functionally associated with insect chemoreception traits, such as odorant-binding proteins, chemosensory proteins, ionotropic, odorant, and gustatory receptors. The transcriptomic profile of feeding larvae was enriched with DEGs associated with the primary metabolism. Larvae feeding on the tolerant rootstock Teleki 5C exhibited higher numbers of plant defense suppression-associated DEGs than larvae feeding on the susceptible host. Based on the identified DEGs, we discuss their potential role for the compatible grape phylloxera&ndash, Vitis interaction belowground. This study was the first to compare the transcriptomes of two grape phylloxera lineages feeding on a tolerant and susceptible host, respectively, and to identify DEGs involved in the molecular interaction with these hosts. Our data provide a source for future studies on host adaptation mechanisms of grape phylloxera and help to elucidate grape phylloxera resistance further.

Biodiversity monitoring programs are the baseline of species abundancy studies, which in case of introduced species are especially critical. Megachile sculpturalis Smith, 1853 native to Eastern-Asia, constitutes the first ever recorded wild bee species accidently introduced in Europe. Since its first discovery in 2008, M. sculpturalis has been spreading across the continent. By initiating a citizen science monitoring program, we aimed to investigate the occurrence pattern of M. sculpturalis. Within only two years after starting the project, 111 new reports from Switzerland, Liechtenstein and Austria were recorded. Comparably to other European countries, the population progressed remarkably fast from year to year expanding its area geographically but also ecologically by increasing its altitudinal range. The distribution pattern indicates human assisted jump-dispersal travelling on the major traffic routes of central Europe.

Cinchona officinalisL., a tree species endemic to the cloud forests of the northern Tropical Andes, has suffered from historical bark harvesting for extraction of antimalarial compounds and has also experienced recent demographic losses from high rates of deforestation. Most remnant populations are found in severely degraded habitat on the edges of pastures while a minority are protected in private reserves. The goals of our research were to assess the genetic diversities of fragmented populations ofC. officinalisin the Loja province of southern Ecuador, characterize their phylogeographic distribution with respect to the region’s complex topography, and identify priority populations for conservation. Five nuclear microsatellite loci and the chloroplastrps16intron were used to analyze six populations. Moderate levels of genetic diversity were found in all populations although the more remote southern population (Angashcola) had slightly higher heterozygosity and allelic richness. There were no indications of recent genetic bottlenecks although anrps16intron haplotype was fixed in four populations. Genetic distance analysis based on microsatellite data placed the four easternmost populations in the same clade while the Angashcola population was the most divergent. Also, the most frequentrps16intron haplotype in Angashcola was not found in any other population. Although each of the studied populations should be protected from further deforestation and agricultural expansion, the Angashcola population deserves highest conservation priority.

International audience; Gene Drives are regarded as future tools with a high potential for population control. Due to their inherent ability to overcome the rules of Mendelian inheritance, gene drives (GD) may spread genes rapidly through populations of sexually reproducing organisms. A release of organisms carrying a GD would constitute a paradigm shift in the handling of genetically modified organisms because gene drive organisms (GDO) are designed to drive their transgenes into wild populations and thereby increase the number of GDOs. The rapid development in this field and its focus on wild populations demand a prospective risk assessment with a focus on exposure related aspects. Presently, it is unclear how adequate risk management could be guaranteed to limit the spread of GDs in time and space, in order to avoid potential adverse effects in socio-ecological systems. The recent workshop on the “Evaluation of Spatial and Temporal Control of Gene Drives” hosted by the Institute of Safety/Security and Risk Sciences (ISR) in Vienna aimed at gaining some insight into the potential population dynamic behavior of GDs and appropriate measures of control. Scientists from France, Germany, England, and the USA discussed both topics in this meeting on April 4–5, 2019. This article summarizes results of the workshop.

The increasing demand for ornamental, cosmetic and pharmaceutical products is driving exploitation of plant species globally. Sub-Saharan Africa harbours unique and valuable plant resources and is now a target of plant resource depletion. African Sandalwood (Osyris lanceolata), a multi-purpose and drought-tolerant species, has seen increased exploitation for the last thirty years and is now declared endangered. Initiatives to conserve O. lanceolata are not yet successful in Africa due to poor understanding of the species. This review surveys relevant research on the ecology, taxonomy, population dynamics, genetic diversity and ethnobotany of O. lanceolata, and highlights gaps in the literature for further research. A scoping review of grey literature, scholarly papers and reports was applied with pre-determined criteria to screen relevant information. Review findings indicate O. lanceolata is a globally distributed species with no identified center of origin. In Africa, it ranges from Algeria to Ethiopia and south to South Africa; in Europe it occurs in the Iberian Peninsula and Balearic Islands; in Asia from India to China, and also on Socotra. The species has a confusing taxonomy, with unresolved issues in nomenclature, country range distribution, extensive synonymisation and variation in growth form (shrub or tree). The species population is reported to be declining in Africa, but information on population dynamics across its entire range of distribution is anecdotal. Additionally, ecological factors influencing spatial distribution and survival of the species remain unknown. A variety of uses are reported for O. lanceolata globally, including: cultural; medicinal and food; dye; perfumery; timber; ethnoveterinary and phytoremediation. Key research areas and implications for conservation of O. lanceolata in Sub-Saharan Africa are proposed.

The introduction of new genetic lineages into a population promotes interbreeding or hybridization and can be associated with the loss of genetic adaptation. This may contribute to financial losses for fish farmers following investments into fish strains that do not perform to their expectations. Oreochromis niloticus is one of the tilapiine species that has been widely distributed in areas far from its native range mainly for aquaculture purposes. This is principally due to its fast growth rates, adaptability to new habitats in addition to attractive market prices. Oreochromis niloticus has also been previously associated with the loss of native tilapiine species such as O. variabilis and O. esculentus in Lake Victoria and other lakes in Africa. To understand the impacts of O. niloticus translocation on native fish stocks, its species borders need to be delimited. Here we use microsatellite genotyping by amplicon sequencing (SSR-GBAS) for species delimitation of O. niloticus and seven related species from different regions in East and West Africa obtained from natural and farm environments. In total, 47 markers previously developed for O. niloticus were tested for cross-species amplification resulting in thirty-eight working markers. Most species were represented by different genetic units with little admixture between them even if ocuring in the same waterbodies. Exceptions involved the farmed populations from O. niloticus and O. mossambicus, which may be indicative of hybrid introgression. Our approach did also detect lower genetic diversity on the critically endangered species O. variabilis and O. esculentus. The possibility of introgressive hybridisation between O. niloticus and some of its congenerics may affect the sustainability of its exploitation. Moreover, long-term projects to monitor the response of genetic diversity and species boundaries to anthropogenic activities are required. In this context, SSR-GBAS can be a powerful tool given its high sample throughput and higher reproducibility compared to other methods.

Tinamous (Tinamidae) represent one of the most ancient living avian lineages but their life history traits are relatively unstudied. Here we identified microsatellite loci for two sympatric tinamou species, the Ornate Tinamou ( Nothoprocta ornata) and the Darwin's Nothura ( Nothura darwinii) from low coverage Illumina sequencing of genomic DNA. The experiment yielded a large number of candidate loci. We designed primers and tested them for successful amplification in 1 to 2 populations of the target species, tested for deviation from the Hardy–Weinberg equilibrium and the presence of null alleles, the levels of polymorphism and potential cross-amplification. All 30 and 24 loci amplified consistently, in the Ornate Tinamou and in Darwin's Nothura, respectively. In the Ornate Tinamou, 25 loci were polymorphic and in the Darwin's Nothura 12, with 2 to 14 alleles per locus in both species. The expected heterozygosity ranged from 0.054 to 0.917 in the Ornate Tinamou and from 0.044 to 0.908 in the Darwin's Nothura. 23 (40%) of 54 loci were successfully cross-amplified. These newly discovered, polymorphic microsatellite loci represent a valuable tool for future studies on social behaviour, parentage and genetic population structure in tinamous.

Geological history of oceanic islands can have a profound effect on the evolutionary history of insular flora, especially in complex islands such as Tenerife in the Canary Islands. Tenerife results from the secondary connection of three paleo-islands by a central volcano, and other geological events that further shaped it. This geological history has been shown to influence the phylogenetic history of several taxa, including genus Micromeria (Lamiaceae). Screening 15 microsatellite markers in 289 individuals representing the eight species of Micromeria present in Tenerife, this study aims to assess the genetic diversity and structure of these species and its relation with the geological events on the island. In addition, we evaluate the extent of hybridization among species and discuss its influence on the speciation process. We found that the species restricted to the paleo-islands present lower levels of genetic diversity but the highest levels of genetic differentiation suggesting that their ranges might have contracted over time. The two most widespread species in the island, M. hyssopifolia and M. varia, present the highest genetic diversity levels and a genetic structure that seems correlated with the geological composition of the island. Samples from M. hyssopifolia from the oldest paleo-island, Adeje, appear as distinct while samples from M. varia segregate into two main clusters corresponding to the paleo-islands of Anaga and Teno. Evidence of hybridization and intraspecific migration between species was found. We argue that species boundaries would be retained despite hybridization in response to the habitat's specific conditions causing postzygotic isolation and preserving morphological differentiation.

Invasive species are increasingly driving biodiversity decline, and knowledge of colonization dynamics, including both drivers and dispersal modes, are important to prevent future invasions. The bee species Megachile sculpturalis (Hymenoptera: Megachilidae), native to East-Asia, was first recognized in Southeast-France in 2008, and has since spread throughout much of Europe. The spread is very fast, and colonization may result from multiple fronts.To track the history of this invasion, codominant markers were genotyped using Illumina sequencing and the invasion history and degree of connectivity between populations across the European invasion axis were investigated. Distinctive genetic clusters were detected with east-west differentiations in Middle-Europe.We hypothesize that the observed cluster formation resulted from multiple, independent introductions of the species to the European continent. This study draws a first picture of an early invasion stage of this wild bee and forms a foundation for further investigations, including studies of the species in their native Asian range and in the invaded range in North America.

DNA barcoding constitutes a supplemental genetically based characterization tool for the identification of species. Traditionally, the barcodes are generated with a length of 650 bp using standardized Sanger sequencing, but with the introduction of high-throughput sequencing (HTS) methods new opportunities for sequencing are available. To use HTS for barcode collection and identification, the amplification of shorter fragments is preferred. Reference DNA midi-barcodes of wild bees were produced using the Illumina MiSeq as well as the Sanger method. Although DNA midi-barcodes derived from Illumina were comparatively shorter (418 bp), their sequences were coherent to the morphological assignment of species. The Illumina barcodes proved to be effective and dealt better with some general limitations of DNA barcoding.

Micromeria presents eight species in Tenerife, and from these, three are endangered or critically endangered. In this paper we use a 454 run from M. hyssopifolia to develop and characterize 16 new microsatellite markers and test them using 19 individuals from two populations of this species. The number of alleles per locus varied from 2 to 10 and observed heterozygosity ranged from 0.125 to 0.900. Most loci successfully amplified in the threatened species M. densiflora, M. glomerata and M. rivas-martinezii and were also polymorphic. These loci will be valuable tools for assessing the genetic diversity of these endangered species.

Cultivars are selected for advantageous traits, which may enable them to have a greater competitive effect (CE), particularly under high nutrient conditions. Sites with high nutrient availability may favor the development of dominant grasses rather than subordinate species, especially in nutrient-limited systems, such as calcareous grasslands. The aim of this experiment was to determine whether seed source influences the CE of a dominant grass on a subordinate species, and whether this relationship is mediated by nutrient availability. A greenhouse experiment with three nutrient levels was established; Dianthus carthusianorum was chosen as the subordinate “phytometer” to detect variation in the CE of different Festuca rubra seed sources. The grass species was sourced from 13 cultivar and 12 commercially propagated, but not selected, wild sources. When CE was calculated from biomass, propagated wild seed sources of F. rubra had a greater CE on the subordinate species than cultivars for medium and high nutrient levels. Based on phytometer height, propagated wild seed sources of F. rubra had a greater CE under all three nutrient levels. Our findings do not support the general notion that cultivars are more competitive than wild genotypes. Thus, the cultivar F. rubra may facilitate the establishment of other species during grassland restoration, particularly under elevated nutrient conditions.

According to the cultivar vigour hypothesis, cultivars are frequently selected for traits such as increased biomass and resource capture, which may hinder the establishment of non-cultivars. Despite this concern, cultivar seed material is still used in grassland restoration. An alternative is wild plant material, which is commercially produced and used in the respective region (‘regional seed’). We established a greenhouse experiment to examine the performance of cultivar and regional forb species with and without competition from cultivar and regional dominant grass species. This was conducted in order to understand the potential effects of sowing cultivar seed in grassland restoration projects. Three calcareous grassland forb species (Buphthalmum salicifolium, Dianthus carthusianorum and Linum perenne) and one dominant grass species (Festuca rubra ssp. commutata) were selected. One forb species was sown in the centre of each pot and either zero, two or four F. rubra ssp. commutata individuals were planted around it. Above-ground biomass, plant height, leaf length, leaf width, number of leaves and stems were measured. When grown without competitors, each forb species performed differently with only L. perenne exhibiting cultivar vigour. Cultivar forbs were less negatively affected by competition with F. rubra ssp. commutata than their regional counterparts, thus supporting the cultivar vigour hypothesis. Overall, regional F. rubra ssp. commutata suppressed the above-ground biomass of the forbs species more than the cultivar F. rubra ssp. commutata, thus highlighting the potential competitiveness of regional seed. Ultimately, the goals of the restoration project will determine the source of seed material to be used in the project. Due to the complex nature of the issues involved in restoration, it should be considered that the reliance on simple guidelines in relation to the source of seed material used in grassland restoration has its limitations.

Aim Using phylogenetic and morphometric approaches, our study aims tounderstand the diversification process of the two groups of Micromeria speciesin Tenerife: the species restricted to the palaeoislands, and the species widelydistributed in the younger part of the island.Location Tenerife, Canary Islands.Methods We calculated a calibrated phylogeny and a Neighbor-Net networkbased on eight nuclear loci from 37 samples: 22 of the 8 species currently rec-ognized in Tenerife, and 15 of their closest relatives occurring in neighbouringislands and continental populations. We performed a principal componentsanalysis (PCA) of 27 morphological characters from 54 specimens sampledfrom Tenerife.Results Our phylogeny showed that the species from Tenerife can be subdi-vided into three main clades: one composed of the species inhabiting the palaeo-island of Anaga (M. teneriffae, M. glomerata and M. rivas-martinezii); anothercomposed of the species present in the palaeoisland of Teno (M. densiflora);and a third group that includes all the central species (M. hyssopifolia, M. varia,M. lachnophylla and M. lasiophylla). Morphometric analyses indicated twomain groups corresponding to the palaeoisland species and the central ones.Main conclusions Our study points to a relationship between the diversifica-tion in Micromeria and the geological history of Tenerife. We conclude thatMicromeria first arrived in Anaga where it diversified, subsequently colonizedTeno and from there occupied the central part, presumably after the formationof the Teide volcano. The species of Micromeria in Tenerife constitute an inter-esting example of how species diversification on oceanic islands can be shapedby the island’s geological history, which probably contributed to the high levelsof endemism on Tenerife.KeywordsDiversification, endemism, island biogeography, island evolution, Lamiaceae,Macaronesia, Micromeria, oceanic islands, palaeoislands, Tenerife.INTRODUCTIONThe biodiversity of oceanic islands is characterized by a highrate of endemism and by groups of closely related speciesthat can be highly differentiated morphologically, as an adap-tive response to empty ecological niches (e.g. Crawford et al.,1987; Weigelt et al., 2013). Oceanic islands by definitionhave never had a connection to the mainland, so the diver-sity of their biota is a result of colonization events, relatedgenetic-drift effects and subsequent evolutionary processes(e.g. Francisco-Ortega et al., 1996; Silvertown, 2004). Therate of diversification after colonization may also depend onthe geological history of the islands. The ontogeny of volca-nic archipelagos is composed of different phases, beginning

Species of Hepatozoon Miller, 1908 are blood parasites most commonly found in snakes but some have been described from all tetrapod groups and a wide variety of hematophagous invertebrates. Previous studies have suggested possible associations between Hepatozoon spp. found in predators and prey. Particularly, some saurophagous snakes from North Africa and the Mediterranean region have been found to be infected with Hepatozoon spp. similar to those of various sympatric lizard hosts. In this study, we have screened tissue samples of 111 North African and Mediterranean snakes, using specific primers for the 18S rRNA gene. In the phylogenetic analysis, the newly-generated Hepatozoon spp. sequences grouped separately into five main clusters. Three of these clusters were composed by Hepatozoon spp. also found in snakes and other reptiles from the Mediterranean Basin and North Africa. In the other two clusters, the new sequences were not closely related to geographically proximate known sequences. The phylogeny of Hepatozoon spp. inferred here was not associated with intermediate host taxonomy or geographical distribution. From the other factors that could explain these evolutionary patterns, the most likely seems series of intermediate hosts providing similar ribotypes of Hepatozoon and a high prevalence of host shifts for Hepatozoon spp. This is indicated by ribotypes of high similarity found in different reptile families, as well as by divergent ribotypes found in the same host species. This potentially low host specificity has profound implications for the systematics of Hepatozoon spp. © 2014 Springer Science+Business Media Dordrecht., Sampling in Turkey and the Caucasus was supported by the project “Preserving Armenian biodiversity: Joint Portuguese – Armenian program for training in modern conservation biology” funded by Gulbenkian Foundation. Sampling in North-West Africa was partially supported by the National Geographic Society (CRE-7629-04, CRE-8412-08) and by Fundação para a Ciência e Tecnologia (FCT: PTDC/BIA-BEC/099934/2008) through the EU Programme COMPETE, and by the Percy Sladen fund. JPM is supported by a Fundação para a Ciência e a Tecnologia (FCT) PhD grant (SFRH/BD/74305/2010) and co-financed by FSE and POPH and EU. DJH and JCB are supported by “Genomics and evolutionary biology” and “Biodiversity, Ecology and Global Change”, respectively, co-financed by the North Portugal Regional Operational Programme 2007/2013 (ON.2 - O Novo Norte), under the National Strategic Reference Framework (NSRF), through the European Regional Development Fund (ERDF). Snakes were captured and handled under permits from the Italian Ministry of the Environment (DPN/2D/2003/2267). DS is supported by a FCT post-doctoral fellowship SFRH/BPD/66592/2009 and AP by a FCT IF contract IF/01257/2012.

The use of Next Generation Sequencing (NGS) techniques to identify microsatellite markers has replaced more time intensive methods such as molecular cloning. The main advantage of NGS over traditional methods of identifying microsatellite markers is the generation of many more sequences with less effort. It is possible to design primers from unenriched DNA, thereby further reducing the workload and also allowing the use of SSRs that are difficult to enrich (e.g., TA/AT and TAA/ATT). We present microsatellite primer pairs that may be used for phylogeographic analysis as well as to infer the geographical origin of traded material of Catha edulis, which contains two amphetamines that are controlled substances in many counties. We used data from two partial 454 pyrosequencing runs that generated about 2000 sequences containing microsatellites (3% of all sequences) as well as flanking regions sufficient for primer design. Using 23 samples of C. edulis we identified 27 single-copy markers that were broadly amplified across the sampled individuals; 18 showed polymorphism information content (PIC) higher than 0.5. The genetic structure in wild individuals is concordant with their geographic origins; wild samples from northern Kenya are more closely related to Ethiopian samples than are other wild samples from Kenya. The geographic differences in allele frequencies indicate that microsatellite analysis can be used to determine the geographic source of cultivated and wild collected material.

As found in other oceanic islands, the Canary Islands include a large number of single island endemic species, some of which form clades that are broadly distributed within the archipelago. The genus Micromeria (Lamiaceae), for instance, includes groups of morphologically similar but ecologically diverse species on each island, representing a great model to investigate niche shifts and adaptation within the Canary Archipelago. Previous attempts to reconstruct phylogenetic relationships within the genus did not lead to robust phylogenies, presumably due to introgression and/or incomplete lineage sorting. In this study, we use a newly developed RAD-sequencing method to improve phylogenetic resolution and to better understand relationships among the Canary Island endemic Micromeria. Overall, we obtained 3571 loci that were genotyped for a total of 46 individuals of Micromeria. Our data reconstructed a highly resolved phylogeny, and corroborated the latest species reclassification of the M. varia s.l. species complex, the taxonomically most complicated group within the genus. Furthermore, taxa occupying similar ecological conditions in different islands, were shown to be closely related. This is the case of taxa from the laurel forest from La Gomera and Gran Canaria, suggesting that the laurel forest likely worked as a filter, only allowing the establishment of colonizers already pre-adapted to these conditions. We also found introgression between these species so it is also possible that the genes that facilitated the adaptation to laurel forest were swapped between Gran Canaria and La Gomera. The observations obtained in this study also allowed us to explain the role of introgression in the origin of M. varia s.l. species complex.

Background Especially on islands closer to the mainland, such as the Canary Islands, different lineages that originated by multiple colonization events could have merged by hybridization, which then could have promoted radiation events (Herben et al., J Ecol 93: 572–575, 2005; Saunders and Gibson, J Ecol 93: 649–652, 2005; Caujapé-Castells, Jesters, red queens, boomerangs and surfers: a molecular outlook on the diversity of the Canarian endemic flora, 2011). This is an alternative to the scenario where evolution is mostly driven by drift (Silvertown, J Ecol 92: 168–173, 2004; Silvertown et al., J Ecol 93: 653–657, 2005). In the former case hybridization should be reflected in the genetic structure and diversity patterns of island species. In the present work we investigate Micromeria from the Canary Islands by extensively studying their phylogeographic pattern based on 15 microsatellite loci and 945 samples. These results are interpreted according to the hypotheses outlined above. Results Genetic structure assessment allowed us to genetically differentiate most Micromeria species and supported their current classification. We found that populations on younger islands were significantly more genetically diverse and less differentiated than those on older islands. Moreover, we found that genetic distance on younger islands was in accordance with an isolation-by-distance pattern, while on the older islands this was not the case. We also found evidence of introgression among species and islands. Conclusions These results are congruent with a scenario of multiple colonizations during the expansion onto new islands. Hybridization contributes to the grouping of multiple lineages into highly diverse populations. Thus, in our case, islands receive several colonization events from different sources, which are combined into sink populations. This mechanism is in accordance with the surfing syngameon hypothesis. Contrary to the surfing syngameon current form, our results may reflect a slightly different effect: hybridization might always be related to colonization within the archipelago as well, making initial genetic diversity to be high to begin with. Thus the emergence of new islands promotes multiple colonization events, contributing to the establishment of hybrid swarms that may enhance adaptive ability and radiation events. With time, population sizes grow and niches start to fill. Consequently, gene-flow is not as effective at maintaining the species syngameon, which allows genetic differentiation and reproductive isolation to be established between species. This process contributes to an even further decrease in gene-flow between species. Electronic supplementary material The online version of this article (doi:10.1186/s12862-017-1031-y) contains supplementary material, which is available to authorized users.

Tinamous have male-only parental care. For their mating system, simultaneous polygyny and sequential polyandry is proposed as an optimal model and is confirmed in all species according to the existing literature, except in the Ornate Tinamou. For the Ornate Tinamou a monogamous mating system is reported. However, this knowledge is based only on few direct observations in the field. Social monogamy in association with promiscuity would probably expose males to cuckoldry and, at the same time, constrain additional mating opportunities for them. To understand the evolutionary context for monogamy in the Ornate Tinamou, the present study aimed to determine whether this species is genetically monogamous or not and whether males are subjected to cuckoldry. We used polymorphic microsatellite markers to perform a sib-ship and paternity analysis based on samples from eggs and feathers of incubating males from four clutches. Promiscuity was found in all nests and the observed rate of cuckoldry ranged from 0% to 33.3%. These results rebut the consideration of monogamy for the Ornate Tinamou. Thus, the optimal model with polygynous males and polyandrous females prevails as an evolutionary stable strategy among tinamous, even in different social mating systems and despite high levels of cuckoldry.

This study presents the results of the identification and quantification of 12 isoflavones (prunetin, irilone, pseudobaptigenin, glycitein, daidzin, genistin, daidzein, pratensein, puerarin, biochanin A, formononetin and genistein) in 23 species of Trifolium (T. arvense, T. pratense, T. ligusticum, T. striatum, T. lappaceum, T. angustifolium, T. hirtum, T. subterraneum, T. isthmocarpum, T. stellatum, T. mutabile, T. strictum, T. fragiferum, T. alexandrinum, T. tomentosum. T. nigrescens subsp. petrisavii, T. nigrescens, T. glomeratum, T. subterraneum subsp. brachycalycinum, T. cherleri, T. resupinatum, T. campestre and T. repens). Isoflavones were extracted by an MSPD method and analyzed with HPLC coupled with a diode-array detector. The evaluation of molecular phylogeny of the IFS gene and the relation with isoflavone content was also performed. Five species (T. subterraneum subsp. brachycalycinum, T. alexandrinum, T. pratense, T. subterraneum and T. lappaceum) were identified with high levels of biochanin A (431–83 mg/kg), formononetin (72–365 mg/kg) and genistein (9–509 mg/kg), which could be utilized as alternative sources for the nutraceutical industry. Genetic phylogeny for the IFS gene was found in the species studied, with 20 out of 23 species having been divided into two clades, while the remaining three were genetically distant. Based on our results, we confirm the direct correlation between IFS gene polymorphism and isoflavones content in species of Trifolium particularly noted for formononetin. Therefore, the IFS gene can be utilized for screening Trifolium genotypes for formononetin. The relation of the three isoflavones' contents and the molecular phylogeny of plants determined by the IFS sequences, as a screening marker for plants with high isoflavone contents in Trifolium species, are to the best of our knowledge described for the first time.

Multiple introductions can play a prominent role in explaining the success of biological invasions. One often cited mechanism is that multiple introductions of invasive species prevent genetic bottlenecks by parallel introductions of several distinct genotypes that, in turn, provide heritable variation necessary for local adaptation. Here, we show that the invasion of Aegilops triuncialis into California, USA, involved multiple introductions that may have facilitated invasion into serpentine habitats. Using microsatellite markers, we compared the polymorphism and genetic structure of populations of Ae. triuncialis invading serpentine soils in California to that of accessions from its native range. In a glasshouse study, we also compared phenotypic variation in phenological and fitness traits between invasive and native populations grown on loam soil and under serpentine edaphic conditions. Molecular analysis of invasive populations revealed that Californian populations cluster into three independent introductions (i.e. invasive lineages). Our glasshouse common garden experiment found that all Californian populations exhibited higher fitness under serpentine conditions. However, the three invasive lineages appear to represent independent pathways of adaptation to serpentine soil. Our results suggest that the rapid invasion of serpentine habitats in California may have been facilitated by the existence of colonizing Eurasian genotypes pre-adapted to serpentine soils.

High-impact biological invasions often involve establishment and spread in disturbed, high-resource patches followed by establishment and spread in biotically or abiotically stressful areas. Evolutionary change may be required for the second phase of invasion (establishment and spread in stressful areas) to occur. When species have low genetic diversity and short selection history, withingeneration phenotypic plasticity is often cited as the mechanism through which spread across multiple habitat types can occur. We show that trans-generational plasticity (TGP) can result in pre-adapted progeny that exhibit traits associated with increased fitness both in high-resource patches and in stressful conditions. In the invasive sedge, Cyperus esculentus, maternal plants growing in nutrient-poor patches can place disproportional number of propagules into nutrient-rich patches. Using the invasive annual grass, Aegilops triuncialis ,w e show that maternal response to soil conditions can confer greater stress tolerance in seedlings in the form of greater photosynthetic efficiency. We also show TGP for a phenological shift in a low resource environment that results in greater stress tolerance in progeny. These lines of evidence suggest that the maternal environment can have profound effects on offspring success and that TGP may play a significant role in some plant invasions.

Chloroplast DNA sequences and recently established morphological characters were used to confirm the widespread invasion of California's vernal pools by European low mannagrass. Morphological similarities between low mannagrass and western mannagrass have led to different taxonomic treatments, depending on the geographical extent of a particular flora. When California's flora was last revised, the two species were combined as western mannagrass, which was then considered to be a native species. Unfortunately, the revised flora was published just as low mannagrass began to rapidly expand its range within the state and, because it was considered to be a native species in the new flora, no actions were initiated to limit the invasion. Our data show that low mannagrass was present at all localities in the Central Valley of California that were investigated, indicating a widespread and undetected invasion. The invasion has led to a degradation of the vernal pool ecosystems, which are the habitat of many federal and state protected endangered and threatened species.

Classical Sanger sequencing is still frequently used to generate sequence data for phylogenetic and phylogeographic inference. In this contribution we present a novel approach to genotype whole mitogenomic haplotypes using Illumina MiSeq reads from indexed amplicons. Our new approach reduces preparation time by multiplexing loci within a single or few PCR reactions and by plate format library construction. The use of paired-end reads allows covering amplicons of about 0.5kb and thus no nebulisation and assembly are necessary. We tested the power and effectiveness of this technique by analysing the mitogenomic diversity of East African white-eye bird species (Zosteropidae), a taxonomically highly diverse and complex species flock found in various ecosystems spread across major parts of Africa. We compare the newly generated mitogenomic data set with published data of three mitochondrial genes for a similar set of populations and taxa. The comparison demonstrates that our new procedure represents a cost effective use of NGS for medium throughput phylogenetic analyses. Using this method, we were able to increase the amount of phylogenetic information significantly, while reducing the costs and effort in the laboratory. The mitogenomic data show a higher resolution than previous studies providing higher support and new insights in the relationships of Zosterops species. Our data suggest to split Z. poliogaster into four distinct species, three of which had previously been proposed: Z. silvanus, Z. mbulensis, Z. kikyuensis and Z. kulalensis. Our approach allows the genotyping of whole mitogenomes for a large number of individuals and thus allows more reliable reconstruction of phylogenetic and phylogeographic relationships - also for non-model organisms.

The Resedaceae, containing 6 genera and ca. 85 species, are widely distributed in the Old World, with a major center of species diversity in the Mediterranean basin. Phylogenetic analyses of ITS and plastid trnL-trnF sequences of 66 species from all genera of the Resedaceae reveal (1) monophyly of the family, in congruence with preliminary phylogenetic studies; (2) molecular support for the traditional morphological subdivision of the Resedaceae into three tribes according to ovary and placentation types, and carpel number; (3) two monophyletic genera (Caylusea, Sesamoides), and one natural group (core Reseda), which includes the remaining four genera of the family (Ochradenus, Oligomeris, Randonia, Reseda); (4) a monophyletic origin for four of the six taxonomic sections recognized within Reseda (Leucoreseda, Luteola, Glaucoreseda, Phyteuma). Our results lead us to interpret an increment of the basic chromosome number in the family from x=5 to x=6 in at least two independent instances, and a broad representation of polyploids in multiple lineages across phylogenies, including association between octoploids and alien invasion in many parts of the world. Species diversity, endemism number, phylogenetic relationships and sequence divergence in Resedaceae suggest two major centers of differentiation, one in the western Mediterranean, and the other in the eastern Mediterranean and SW Asia. Two independent colonization events to the Canary Islands from Africa are indicated for the two Canarian Reseda endemics.

Habitat fragmentation can have a profound ef- fect on the genetic diversity of forest species. These ef- fects are especially interesting when forests previously fragmented by agriculture start to reconnect due to land abandonment. In this study, we investigate the genetic structure and diversity patterns of Juniperus oxycedrus populations from the Sabor river valley in Northeast Portugal. We developed 17 microsatellite markers using pyrosequencing technology as implemented in the 454 platform. As expected, among population differentiation was low with high variability within populations. There was no strong pattern of genetic structure in our analyses (FST=0.018) suggesting that the individuals analyzed here belong to one population. The genetic structure seems to be equally explained by locality and by tree age. We hypothesize that this is a consequence of the land use history from the region. After the abandonment of cultivated fields, these terrains were probably colo- nized by individuals from a few older J. oxycedrus pop- ulations. Thus, the genetic structure pattern found may best be explained by this recent expansion. This expan- sion may be currently influenced by the construction of two hydroelectric dams that will flood areas with older individuals of the species.

The Canary Islands have been a focus for phylogeographic studies on the colonization and diversification of endemic angiosperm taxa. Based on phylogeographic patterns, both inter island colonization and adaptive radiation seem to be the driving forces for speciation in most taxa. Here, we investigated the diversification of Micromeria on the Canary Islands and Madeira at the inter- and infraspecific level using inter simple sequence repeat PCR (ISSR), the trnK-Intron and the trnT-trnL-spacer of the cpDNA and a low copy nuclear gene. The genus Micromeria (Lamiaceae, Mentheae) includes 16 species and 13 subspecies in Macaronesia. Most taxa are restricted endemics, or grow in similar ecological conditions on two islands. An exception is M. varia, a widespread species inhabits the lowland scrub on each island of the archipelago and could represent an ancestral taxon from which radiation started on the different islands. Our analyses support a split between the "eastern" islands Fuerteventura, Lanzarote and Gran Canaria and the "western" islands Tenerife, La Palma and El Hierro. The colonization of Madeira started from the western Islands, probably from Tenerife as indicated by the sequence data. We identified two lineages of Micromeria on Gomera but all other islands appear to be colonized by a single lineage, supporting adaptive radiation as the major evolutionary force for the diversification of Micromeria. We also discuss the possible role of gene flow between lineages of different Micromeria species on one island after multiple colonizations.

Micromeria has recently been revealed polyphyletic by molecular analyses, with members of sect. Pseudomelissa placed in close relationship to Clinopodium vulgare. Morphological data suport transferring the section to Clinopodium. A list of names under Clinopodium (including 15 new combinations) is provided for the taxa hitherto included in Micromeria sect. Pseudomelissa. Three species names are typified.

Nepenthaceae, the pitcher plants of the Old World tropics show a remarkable diversity in SE Asia, especially on the islands of Borneo and Sumatra. This region is considered as a secondary center of diversity. Sequence analysis of the cpDNA trnK intron supports this hypothesis showing the species of the Malay Archipelago as neighbour group to the isolated species from Sri Lanka, the Seychelles, and Madagascar. Based on phylogenetic reconstructions an origin of recent Nepenthaceae in the Indian subcontinent is assumed. A recent investigation focused on a non-plastid, translocated copy of the trnK intron has revealed an incongruence to tree topology based on the cpDNA trnK intron. Although the translocated copy emerged as insufficient for phylogenetic reconstruction of Nepenthaceae some taxa showed, contrary to the cpDNA dataset, relatively high distances to the rest of the taxa. These results indicated that the phylogeny of the trnK intron could not reflect true phylogenetic relationships. We investigated the peptide transferase 1 (PTR1), to develop a phylogenetic marker that is based on a nuclear low copy gene in Nepenthes. All sequences obtained were probably functional, indicated by the ratio of point mutations of the single codon positions in exon and intron regions. Comparative analysis showed that this locus is of similar variability as the cpDNA trnK intron and, contrary to the translocated copy of trnK, potential useful for phylogenetic reconstruction. While in parts congruent to the plastid trnK intron phylogeny, a higher divergence of some sequences in PRT1 and in the previously reported, non cpDNA dataset indicates that remnants of an older species stock persisted east of Wallace's line and on the Sunda Shelf. This suggests that plastid haplotypes existing today in the main distribution center of the Nepenthaceae could be descendants of more recently dispersed lineages that had been transmitted to an old species stock.

During phylogenetic analysis of the Nepenthaceae cpDNA trnK intron, it became apparent that a second non-functional copy of the locus was present in most of the investigated taxa. The translocation event was older than the radiation of all recent Nepenthaceae, and the translocated pseudogenized copy was conserved in nearly all members of the plant family. Using single chloroplast PCR and inverse PCR, we could exclude a plastom location for the second copy. Although translocation into the nucleus is possible, mitochondrial localization seems more likely based on these data. In total, the translocated sequence contained at least 3525 base pairs (bp) that were homologous to the Spinacia oleracea chloroplast genome. Comparative phylogenetic analysis of the non-functional copy revealed a high amount of homoplasies compared to topologies from the cpDNA trnK intron phylogenetic reconstruction. Therefore, this copy proved to be insufficient for phylogenetic reconstruction of the family. Since two different paralogs of the non-functional copy were found in one species, it is feasible that different paralogs were conserved in different groups and that paralogous sequences were included in the data matrix. These data demonstrate that phylogenetic analyses of pseudogenized copies of phylogenetically relevant loci should be performed with great caution. In addition, pseudogenized copies can exist in nearly every member of a plant family, and can be PCR-amplified at levels comparable to the specific copy. In this case, the inclusion of such copies can easily remain unnoticed, thus leading to faulty hypotheses.

Although exotic species cause tremendous economic and ecological loss, we know relatively little about the post-introduction evolutionary dynamics of the invasive species themselves. Barbed goatgrass, Aegilops triuncialis L., is a cleistogamous annual grass with a native range throughout the Mediterranean Basin and Asia and introduced to California during the last century. It is considered a serious noxious range weed and is one of the few exotic plant species that is invading serpentine soil habitats. We examined whether patterns of molecular variation are consistent with a single or multiple introduction events into California and further, if individual populations show evidence for a genetic bottleneck during introduction. Fingerprinting patterns, using microsatellite loci derived from Triticum aestivum, were investigated for 57 Eurasian accessions, broadly spanning the native range and for 108 individuals from 11 localities in California. There is strong evidence for an extreme bottleneck in this species as it colonizes its new range because we detected only three multilocus genotypes occurring in California and 36 genotypes in Eurasia. In California one of the genotypes differs from one other by only one fragment and only occurs in one individual. This suggests two separate introductions. Each population is composed of highly uniform individuals and the two main genotypes are geographically separated. A. triuncialis is still expanding its range in California despite genomic uniformity after a strong bottleneck and its recently increased rate of spread is not correlated to a high within-population variability created by multiple introductions.

Micromeria Benth. (Lamiaceae, Nepetoideae) is a very common genus in the Mediterranean region. To test the monophyly of the genus and to elucidate its phylogenetic placement within subtribe Menthinae (Dumort) Endl. of tribe Mentheae Dumort we performed parsimony analysis of trnK intron sequence data of 51 accessions representing 15 genera of Nepetoideae and two genera of subfamily Ajugoideae. Tree topology reveals a well-supported "core group" indicating four distinct lineages. The first one comprises three species of Satureja L. s.str., the second one includes taxa of the genus Clinopodium L. from both the Old and the New World, paraphyletic with respect to Monarda L. and two species of Micromeria section Pseudomelissa Benth. A third group contains all samples of the remaining Micromeria species. Within this monophyly, a western lineage including taxa from NW Africa, the Balearic, and the Canary Islands, is sister to an eastern lineage with species distributed from SE Asia to the western Mediterranean. In a further clade the genera Thymbra L., Thymus L., and Origanum L. are grouped together. Combined analysis using a reduced dataset of trnK/trnL-F sequences increased support for the infrageneric resolution within Micromeria. Based on the phylogenetic reconstructions there is evidence that the genus as currently circumscribed is polyphyletic. Results are discussed in the context of morphology, karyology, and biogeography, outlining the necessity of removing section Pseudomelissa from Micromeria.

Three new fully dehydrogenated naphthylisoquinoline alkaloids, the 7,1‘-coupled ent-dioncophylleine A (3a), the likewise 7,1‘-coupled 5‘-O-demethyl-ent-dioncophylleine A (4), and the 7,8‘-linked dioncophylleine D (5), have been isolated from the leaves of the recently described Malaysian highland liana Ancistrocladus benomensis. All of them lack an oxygen function at C-6; this so-called Dioncophyllaceae-type structural subclass had previously been found only in naphthylisoquinoline alkaloids from West and Central African plants. Moreover, compounds 3a and 4 are the first fully dehydrogenated, i.e., only axially chiral, naphthylisoquinoline alkaloids of this type that are optically active; compound 5, by contrast, is fully racemic, due to its configurationally unstable biaryl axis. The structural elucidation was achieved by spectroscopic and chiroptical methods. Biological activities of these alkaloids against different protozoan parasites are described.

4-Coumarate:CoA ligase (4CL) is involved in the formation of coenzyme A thioesters of hydroxycinnamic acids that are central substrates for subsequent condensation, reduction, and transfer reactions in the biosynthesis of plant phenylpropanoids. Previous studies of 4CL appear to suggest that many isoenzymes are functionally equivalent in supplying substrates to various subsequent branches of phenylpropanoid biosyntheses. In contrast, divergent members of a 4CL gene family were identified in soybean (Glycine max L.). We isolated three structurally and functionally distinct 4CL cDNAs encoding 4CL1, 4CL2, and 4CL3 and the gene Gm4CL3. A fourth cDNA encoding 4CL4 had high similarity with 4CL3. The recombinant proteins expressed in Escherichia coli possessed highly divergent catalytic efficiency with various hydroxycinnamic acids. Remarkably, one isoenzyme (4CL1) was able to convert sinapate; thus the first cDNA encoding a 4CL that accepts highly substituted cinnamic acids is available for further studies on branches of phenylpropanoid metabolism that probably lead to the precursors of lignin. Surprisingly, the activity levels of the four isoenzymes and steady-state levels of their transcripts were differently affected after elicitor treatment of soybean cell cultures with a β-glucan elicitor of Phytophthora sojae, revealing the down-regulation of 4CL1 vs. up-regulation of 4CL3/4. A similar regulation of the transcript levels of the different 4CL isoforms was observed in soybean seedlings after infection with Phytophthora sojae zoospores. Thus, partitioning of cinnamic acid building units between phenylpropanoid branch pathways in soybean could be regulated at the level of catalytic specificity and the level of expression of the 4CL isoenzymes.

Recently, the authors presented a proposal to conserve the names Micromeria varia Bentham (1834: 374) with a conserved type and M. hyssopifolia Webb & Berthelot (1844: 72) against Thymus ericifolius Roth (1800: 50) (Puppo et al. 2014a) to avoid disruption of current usage of these two well established names. This proposal, however, was not recommended by the Nomenclature Committee (Applequist 2016) and subsequently turned down by the General Committee (Wilson 2017). While regretting this decision, we are prepared to accept the taxonomic and nomenclatural implications.

Premise of the study: Polymorphic microsatellite loci were developed and used to genotype individuals of Herbertia zebrina (Iridaceae) as a first step for assessment of intraspecific genetic diversity. Methods and Results: Primer pairs for 47 markers were developed: 20 from a microsatellite-enriched library and 27 from a next-generation sequencing run using the Illumina MiSeq platform. Of those, 15 loci were considered successful, of which 12 were polymorphic and three were monomorphic. The primers were tested in 50 individuals from three populations of H. zebrina. Two to 14 alleles per locus were identified, and observed and expected heterozygosity were 0.00–0.95 and 0.18–0.89, respectively. Tests of cross-amplification to evaluate the applicability of these markers showed positive results in one congeneric species, H. darwinii, and in a phylogenetically closely related species, Calydorea crocoides. Conclusions: These microsatellite markers can be used for studies of genetic variation and genetic populat...

© 2016 John Wiley & Sons Ltd. Gene flow may influence the formation of species range limits, and yet little is known about the patterns of gene flow with respect to environmental gradients or proximity to range limits. With rapid environmental change, it is especially important to understand patterns of gene flow to inform conservation efforts. Here we investigate the species range of the selfing, annual plant, Mimulus laciniatus, in the California Sierra Nevada. We assessed genetic variation, gene flow, and population abundance across the entire elevation-based climate range. Contrary to expectations, within-population plant density increased towards both climate limits. Mean genetic diversity of edge populations was equivalent to central populations; however, all edge populations exhibited less genetic diversity than neighbouring interior populations. Genetic differentiation was fairly consistent and moderate among all populations, and no directional signals of contemporary gene flow were detected between central and peripheral elevations. Elevation-driven gene flow (isolation by environment), but not isolation by distance, was found across the species range. These findings were the same towards high- and low-elevation range limits and were inconsistent with two common centre-edge hypotheses invoked for the formation of species range limits: (i) decreasing habitat quality and population size; (ii) swamping gene flow from large, central populations. This pattern demonstrates that climate, but not centre-edge dynamics, is an important range-wide factor structuring M. laciniatus populations. To our knowledge, this is the first empirical study to relate environmental patterns of gene flow to range limits hypotheses. Similar investigations across a wide variety of taxa and life histories are needed.

Here we reconstruct the evolutionary history of Micromeria in the Canary Islands using eight nuclear markers. Our results show two centers of diversification for Micromeria, one in the eastern islands Gran Canaria and Lanzarote, the other in the western islands, Tenerife, La Palma and El Hierro. Suggested directions of inter-island colonization are the following: Gran Canaria to Lanzarote and La Gomera; Tenerife to La Palma (from the paleoisland of Teno), to El Hierro (from the younger, central part), and to La Gomera and Madeira (from the paleoislands). Colonization of La Gomera probably occurred several times from Gran Canaria and Tenerife. The taxonomic implications of these results are discussed. Incongruence among the different markers was evaluated and, using next generation sequencing, we investigated if this incongruence is due to gene duplication.

Nepenthaceae are an exceptional family with regard to carnivo ry and the uniformity of characters. This makes it dif- ficult to resolve phylogenetic relationships due to conv ergent evolution of morphological features. Using comparative se- quencing of the chloroplast trnK intron, the monophyly of this complex family and hypoth eses of infrageneric relationships were tested. Sequences from 71 Nepenthes taxa, representing all groups and two taxa of the closely related Ancistrocladaceae and Dioncophyllaceae as outgroup, were determined and ana- lysed using maximum parsimony methods. Results of this analy- sis show that the isolated taxa N. distillatoria (Sri Lanka) and N. pervillei (Seychelles) are the most basal, clearly separated from the Madagascan taxa N. madagascariensis and N. masoalensis which are placed in a distinct subclade. This corresponds with some plesiomorphic character s shared by these taxa. N. khasi- ana (North India) has an intermediate position between these relic Western species and the remaining taxa. The species of the Malay Archipelago can be referred to three distinct lineages which indicate a correlation to biogeography . Thus the recent disjunct distribution of Nepenthes is interpreted as a result of an incisive extinction of progenitors, a process of migration and a subsequent diversification on the islands of Borneo, Sumatra, Sulawesi and New Guinea. Based on our molecular data, two in- terpretations concerning the origin of Nepenthes are possible: i) evolution in the Northern Tethys which is suppor ted by fossil pollen records from the European Focene, or , ii) a Gondwana- land origin at a time when the Indian plate was separated from Madagascar. Molecular data indicate that colonization of SE Asia started from an ancient Indian stock. Subsequently, in the Malay Archipelago a new secondary centre of diversity developed. Ma- dagascar, the Se ychelles and New Caledonia were probably reached by migration via land bridges, starting from widespead common ancestors with subsequent extinction leaving the cur- rent taxa. There is no evidence for long-distance dispersal. Cur- rent infragenic classification of Nepenthes is only partly in accordance with the phylogen y inferred from trnK intron data.