Manuel Curto

Development of phylogenetic markers from single-copy nuclear genes for multi locus, species level analyses in the mint family (Lamiaceae).

The use of single copy nuclear markers is of increasing importance in plant phylogenetics. The generally higher level of variability compared to chloroplast DNA and the ability to use incongruence in a multilocus analysis to determine reticulation patterns makes these kinds of sequence based markers especially useful for species level investigations. However, the prevalence of gene duplication that results from the high frequency of polyploidization events during the evolution of higher plants can impede marker development especially for groups lacking model organisms. Here, we present the strategy and results of marker development for phylogenetic analysis in Micromeria, using publicly available DNA sequences and ESTs from related genera from Lamiaceae, subfamily Nepetoideae. By eliminating markers with signatures of duplication during four steps of marker development, we were able to select 19 primer pairs that resulted in orthologous products for all the species studied. This corresponds to 23% of the initial 84 primer pairs designed. Using an initial sampling of eight individuals, we tested the markers for support of phylogenetic hypotheses related to the evolution of Micromeria on the Canary Islands. While some hypotheses were supported by all markers, an east west split, with a closer relationship between the species of Tenerife and Madeira on one hand and the ones from Gran Canaria and the eastern islands on the other is supported by 12 markers but contradicted by the remaining seven. This indicates that reticulation and inter-island gene flow played a role in the evolution of Micromeria.

Molecular phylogenetics of Micromeria (Lamiaceae) in the Canary Islands, diversification and inter-island colonization patterns inferred from nuclear genes☆

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.

A new amplicon based approach of whole mitogenome sequencing for phylogenetic and phylogeographic analysis: An example of East African white-eyes (Aves, Zosteropidae)

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.

Genetic structure of Micromeria (Lamiaceae) in Tenerife, the imprint of geological history and hybridization on within-island diversification

Abstract 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 habitats specific conditions causing postzygotic isolation and preserving morphological differentiation.

New microsatellite markers for two sympatric tinamou species, the Ornate Tinamou (Nothoprocta ornata) and Darwin's Nothura (Nothura darwinii)

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 Darwins 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 Darwins Nothura, respectively. In the Ornate Tinamou, 25 loci were polymorphic and in the Darwins 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 Darwins 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.

Erratum to: Development and characterization of 16 microsatellite markers for Micromeria (Lamiaceae) from Tenerife (Canary Islands, Spain) using 454 sequencing

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.

Phylogeography of the wild and cultivated stimulant plant qat (Catha edulis, Celastraceae) in areas of historical cultivation

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.

Influence of past agricultural fragmentation to the genetic structure of Juniperus oxycedrus in a Mediterranean landscape

Habitat fragmentation can have a profound effect on the genetic diversity of forest species. These effects 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 colonized by individuals from a few older J. oxycedrus populations. Thus, the genetic structure pattern found may best be explained by this recent expansion. This expansion may be currently influenced by the construction of two hydroelectric dams that will flood areas with older individuals of the species.

Analysis of microsatellite loci in tree of heaven (Ailanthus altissima (Mill.) Swingle) using SSR-GBS

Microsatellite markers are still the marker of choice for many research questions in the field of forest genetics. However, the number of available markers is often low for species that have not been studied intensively like the tree of heaven (Ailanthus altissima). During the last decade, next-generation sequencing (NGS) has offered advanced techniques for efficiently identifying microsatellite markers and accurately genotyping samples. Here, we identify new microsatellite markers for the tree of heaven by applying an NGS-based method using the Illumina MiSeq platform. NGS technology was proved to be an effective method for fast and cost-efficient identification of microsatellite markers by implementing a genotyping-by-sequencing approach based on Illumina amplicon sequencing (SSR-GBS). We screened three populations from Eastern Austria for genetic variation at 19 newly identified microsatellite loci. We tested two different genotyping approaches: (1) considering only allele lengths (forming a so-called “allele length dataset”), (2) taking also single nucleotide polymorphisms (SNPs) within the amplified fragments into account (forming a so-called “SNP dataset”). The results revealed higher values for all genetic diversity parameters, as well as a better resolution of genetic assignment, when the latter approach was followed. Thus, by taking advantage of sequence information which is provided by SSR-GBS, one may achieve considerable gains in performance using the same marker set. The developed markers provide a cost-efficient tool for genotyping populations of tree of heaven and the approach presented here promises to be of high value for medium throughput genotyping applications in non-model forest tree species. We will use this method to widen the perspectives for further population genetic investigations of the tree of heaven.

Using a new RAD-sequencing approach to study the evolution of Micromeria in the Canary islands

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.