Johannes C. Vogel

Three times out of Asia Minor: the phylogeography of Arabis alpina L. (Brassicaceae)

Arabis alpina is a characteristic plant in arctic‐alpine habitats and serves as a classical example to demonstrate biology, ecology and biogeography of arctic‐alpine disjuncts. It has a wider distribution than most other arctic‐alpine plants, covering all European mountain systems, the Canary Islands, North Africa, the high mountains of East Africa and Ethiopia, the Arabian Peninsula and mountain ranges of Central Asia in Iran and Iraq. Additionally it is found in the northern amphi‐Atlantic area including northeastern North America, Greenland, Iceland, Svalbard and northwestern Europe. We used markers from the nuclear (internal transcribed spacer of ribosomal DNA) and chloroplast genome (trnL‐F region) to reconstruct its phylogeographic history. Both markers revealed clear phylogeographic structure. We suggest that A. alpina originated in Asia Minor less than 2 million years ago based on synonymous mutation rates of different genes (plastidic matK, nuclear adh and chs). From the Asian ancestral stock one group migrated via the Arabian Peninsula to the East African high mountains. A second group gave rise to all European and northern populations, and also served as source for the northwest African populations. A third group, which is still centred in Asia, migrated independently southwards and came into secondary contact with the East African lineage in Ethiopia, resulting in high genetic diversity in this area. In the Mediterranean regions, the genetic diversity was relatively high with numerous unique haplotypes, but almost without geographic structure. In contrast, the populations in the northern amphi‐Atlantic area were extremely depauperate, suggesting very recent (postglacial) expansion into this vast area from the south.

Polyploidy, phylogeography and Pleistocene refugia of the rockfern Asplenium ceterach : evidence from chloroplast DNA

Chloroplast DNA sequences were obtained from 331 Asplenium ceterach plants representing 143 populations from throughout the range of the complex in Europe, plus outlying sites in North Africa and the near East. We identified nine distinct haplotypes from a 900 bp fragment of trnL‐trnF gene. Tetraploid populations were encountered throughout Europe and further afield, whereas diploid populations were scarcer and predominated in the Pannonian‐Balkan region. Hexaploids were encountered only in southern Mediterranean populations. Four haplotypes were found among diploid populations of the Pannonian‐Balkans indicating that this region formed a northern Pleistocene refugium. A separate polyploid complex centred on Greece, comprises diploid, tetraploid and hexaploid populations with two endemic haplotypes and suggests long‐term persistence of populations in the southern Mediterranean. Three chloroplast DNA (cpDNA) haplotypes were common among tetraploids in Spain and Italy, with diversity reducing northwards suggesting expansion from the south after the Pleistocene. Our cpDNA and ploidy data indicate at least six independent origins of polyploids.

Chloroplast Phylogeny of Asplenioid Ferns based on rbcL and trnL-F Spacer Sequences (Polypodiidae, Aspleniaceae) and its Implications for Biogeography

Abstract Molecular phylogenies have been generated to investigate relationships among species and putative segregates in Asplenium, one of the largest genera in ferns. Of the ∼700 described taxa, 71 are included in a phylogenetic analysis using the chloroplast rbcL gene and trnL-F spacer. Our results support Hymenasplenium as the sister lineage to all other asplenioid ferns, and all other putative satellite genera are nested within this asplenioid clade. Instead of the classical and well-recognized separation into Old and New World clades, asplenioid ferns reveal a separation of the deeper branches into tropical and temperate clades. Temperate clades have evolved from tropical, more-basal clades and the phylogeny indicates up to six shifts between temperate and tropical preferences in the evolution of this widespread genus. Implications for speciation processes and biogeographic aspects, including the re-colonization of temperate regions after the last glacial period, are discussed and we present a phylogenetic framework from which the historical biogeography of asplenioid ferns can be inferred for Europe and North America.

The importance of Anatolian mountains as the cradle of global diversity in Arabis alpina, a key arctic–alpine species

BACKGROUND AND AIMS Anatolia is a biologically diverse, but phylogeographically under-explored region. It is described as either a centre of origin and long-term Pleistocene refugium, or as a centre for genetic amalgamation, fed from distinct neighbouring refugia. These contrasting hypotheses are tested through a global phylogeographic analysis of the arctic-alpine herb, Arabis alpina. METHODS Herbarium and field collections were used to sample comprehensively the entire global range, with special focus on Anatolia and Levant. Sequence variation in the chloroplast DNA trnL-trnF region was examined in 483 accessions. A haplotype genealogy was constructed and phylogeographic methods, demographic analysis and divergence time estimations were used to identify the centres of diversity and to infer colonization history. KEY RESULTS Fifty-seven haplotypes were recovered, belonging to three haplogroups with non-overlapping distributions in (1) North America/Europe/northern Africa, (2) the Caucuses/Iranian Plateau/Arabian Peninsula and (3) Ethiopia-eastern Africa. All haplogroups occur within Anatolia, and all intermediate haplotypes linking the three haplogroups are endemic to central Anatolia and Levant, where haplotypic and nucleotide diversities exceeded all other regions. The local pattern of haplotype distribution strongly resembles the global pattern, and the haplotypes began to diverge approx. 2·7 Mya, coinciding with the climate cooling of the early Middle Pleistocene. CONCLUSIONS The phylogeographic structure of Arabis alpina is consistent with Anatolia being the cradle of origin for global genetic diversification. The highly structured landscape in combination with the Pleistocene climate fluctuations has created a network of mountain refugia and the accumulation of spatially arranged genotypes. This local Pleistocene population history has subsequently left a genetic imprint at the global scale, through four range expansions from the Anatolian diversity centre into Europe, the Near East, Arabia and Africa. Hence this study also illustrates the importance of sampling and scaling effects when translating global from local diversity patterns during phylogeographic analyses.

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

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

Genetic discontinuity, breeding‐system change and population history of Arabis alpina in the Italian Peninsula and adjacent Alps

Arabis alpina is a widespread plant of European arctic and alpine environments and belongs to the same family as Arabidopsis thaliana. It grows in all major mountain ranges within the Italian glacial refugia and populations were sampled over a 1300 km transect from Sicily to the Alps. Diversity was studied in nuclear and chloroplast genome markers, combining phylogeographical and population genetic approaches. Alpine populations had significantly lower levels of nuclear genetic variation compared to those in the Italian Peninsula, and this is associated with a pronounced change in within‐population inbreeding. Alpine populations were significantly inbred (FIS = 0.553), possibly reflecting a change to the self‐incompatibility system during leading edge colonization. The Italian Peninsula populations were approaching Hardy–Weinberg equilibrium (outbreeding, FIS = 0.076) and genetic variation was highly structured, consistent with independent local ‘refugia within refugia’ and the fragmentation of an established population by Quaternary climate oscillations. There is very little evidence of genetic exchange between the Alps and the Italian Peninsula main distribution ranges. The Alps functioned as a glacial sink for A. alpina, while the Italian Peninsula remains a distinct and separate long‐term refugium. Comparative analysis indicated that inbreeding populations probably recolonized the Alps twice: (i) during a recent postglacial colonization of the western Alps from a Maritime Alps refugium; and (ii) separately into the central Alps from a source outside the sampling range. The pronounced geographical structure and inbreeding discontinuities are significant for the future development of A. alpina as a model species.

Diversity arrays technology (DArT) for pan-genomic evolutionary studies of non-model organisms.

Background High-throughput tools for pan-genomic study, especially the DNA microarray platform, have sparked a remarkable increase in data production and enabled a shift in the scale at which biological investigation is possible. The use of microarrays to examine evolutionary relationships and processes, however, is predominantly restricted to model or near-model organisms. Methodology/Principal Findings This study explores the utility of Diversity Arrays Technology (DArT) in evolutionary studies of non-model organisms. DArT is a hybridization-based genotyping method that uses microarray technology to identify and type DNA polymorphism. Theoretically applicable to any organism (even one for which no prior genetic data are available), DArT has not yet been explored in exclusively wild sample sets, nor extensively examined in a phylogenetic framework. DArT recovered 1349 markers of largely low copy-number loci in two lineages of seed-free land plants: the diploid fern Asplenium viride and the haploid moss Garovaglia elegans. Direct sequencing of 148 of these DArT markers identified 30 putative loci including four routinely sequenced for evolutionary studies in plants. Phylogenetic analyses of DArT genotypes reveal phylogeographic and substrate specificity patterns in A. viride, a lack of phylogeographic pattern in Australian G. elegans, and additive variation in hybrid or mixed samples. Conclusions/Significance These results enable methodological recommendations including procedures for detecting and analysing DArT markers tailored specifically to evolutionary investigations and practical factors informing the decision to use DArT, and raise evolutionary hypotheses concerning substrate specificity and biogeographic patterns. Thus DArT is a demonstrably valuable addition to the set of existing molecular approaches used to infer biological phenomena such as adaptive radiations, population dynamics, hybridization, introgression, ecological differentiation and phylogeography.

Genetic structure, reproductive biology and ecology of isolated populations of Asplenium csikii (Aspleniaceae, Pteridophyta)

The potential for environmental heterogeneity to generate spatial structuring of genotypes in seed-plant populations that occupy patchy habitats has been demonstrated by several studies, but little is known about the population structure of pteridophytes occupying patchy environments. In this study we have examined the genetic structure of isolated populations of the rock fern Asplenium csikii, an ecological specialist, growing almost exclusively on perpendicular walls of natural rock outcrops. All genetic variation observed in this taxon was partitioned between localities; no allozyme variation was found within a site and each site was colonized by a single multilocus phenotype (MLP). In total, five different MLPs were recorded from the nine localities, with two MLPs present at more than one site. Previous examination of population structure and genetic diversity in another rock fern, A. ruta-muraria, showed that the genetic diversity increases through multiple colonization over time. However, we cannot find any such correlation for A. csikii. All populations are genetically uniform, despite the probably considerable age of the populations and sites. Earlier studies concluded that the ample production of wind-borne propagules would lead to multiple colonization of sites and that reproductive features, such as single-spore colonization and subsequent intragametophytic selfing, would lead to very little genetic structuring of fern populations. In contrast to this prediction, it appears that ecological specialization and the scarcity of the narrowly defined niche contribute strongly to the pronounced partitioning of genetic variability observed in populations of A. csikii.

Schizaeaceae: a phylogenetic approach

Schizaeaceae fossils have been documented throughout Mesozoic and Cenozoic deposits, but our understanding of this fossil record is hampered by uncertainties with respect to the relationships of living species. To start building a phylogenetic framework for the family, an initial phylogenetic analysis of living species using plastid rbcL nucleotide sequence data is conducted. The analysis supports Schizaea s. lat. and Lygodium monophyly, but Anemia is resolved as paraphyletic to Mohria. In the Anemia/Mohria clade, monophyly of subgenus Anemiorrhiza is supported, but Coptophyllum is resolved as paraphyletic to subgenus Anemia. In Schizaea s. lat., both Schizaea s. str. and Actinostachys are well supported and Microschizaea is grouped with Schizaea s. str., although only one Microschizaea species (Schizaea pusilla) was included. These results are largely congruent with previous morphology-based analyses. In Lygodium however, results presented contrast with recent morphological analyses highlighting the problems of identifying Lygodium subgeneric groups. Using the resulting phylogeny as a framework, putative relationships of fossil species are discussed, tentative minimum age estimates for generic crown group diversifications are made, and possible conclusions with respect to the origins of habit and habitat preferences are discussed. The fossil evidence indicates that subgeneric groups within the Anemia/Mohria clade are comparatively ancient, originating during the Early Cretaceous, and the putative placement of fossil Anemia within the crown group of living subgenus Anemiorrhiza would indicate that their calcareous habitat preference may be a relic feature that has persisted ever since the Early Cretaceous. Lygodium on the other hand appears to have passed through a diversity bottleneck. Modern species diversity probably originated in the Neogene, and the earliest fossil evidence for the origin of their vining and trailing habit comes from the placement of Miocene fossil Lygodium within the crown group of living species.

Mixed mating system in the fern Asplenium scolopendrium: implications for colonization potential

BACKGROUND AND AIMS Human-mediated environmental change is increasing selection pressure for the capacity in plants to colonize new areas. Habitat fragmentation combined with climate change, in general, forces species to colonize areas over longer distances. Mating systems and genetic load are important determinants of the establishment and long-term survival of new populations. Here, the mating system of Asplenium scolopendrium, a diploid homosporous fern species, is examined in relation to colonization processes. METHODS A common environment experiment was conducted with 13 pairs of sporophytes, each from a different site. Together they constitute at least nine distinct genotypes, representing an estimated approx. 95 % of the non-private intraspecific genetic variation in Europe. Sporophyte production was recorded for gametophytes derived from each parent sporophyte. Gametophytes were grown in vitro in three different ways: (I) in isolation, (II) with a gametophyte from a different sporophyte within the same site or (III) with a partner from a different site. KEY RESULTS Sporophyte production was highest in among-site crosses (III), intermediate in within-site crosses (II) and was lowest in isolated gametophytes (I), strongly indicating inbreeding depression. However, intragametophytic selfing was observed in most of the genotypes tested (eight out of nine). CONCLUSIONS The results imply a mixed mating system in A. scolopendrium, with outcrossing when possible and occasional selfing when needed. Occasional intragametophytic selfing facilitates the successful colonization of new sites from a single spore. The resulting sporophyte, which will be completely homozygous, will shed large amounts of spores over time. Each year this creates a bed of gametophytes in the vicinity of the parent. Any unrelated spore which arrives is then selectively favoured to reproduce and contribute its genes to the new population. Thus, while selfing facilitates initial colonization success, inbreeding depression promotes genetically diverse populations through outcrossing. The results provide further evidence against the overly simple dichotomous distinction of fern species as either selfing or outcrossing.