Frank R. Blattner

Barley whole exome capture: a tool for genomic research in the genus Hordeum and beyond

Advanced resources for genome-assisted research in barley (Hordeum vulgare) including a whole-genome shotgun assembly and an integrated physical map have recently become available. These have made possible studies that aim to assess genetic diversity or to isolate single genes by whole-genome resequencing and in silico variant detection. However such an approach remains expensive given the 5 Gb size of the barley genome. Targeted sequencing of the mRNA-coding exome reduces barley genomic complexity more than 50-fold, thus dramatically reducing this heavy sequencing and analysis load. We have developed and employed an in-solution hybridization-based sequence capture platform to selectively enrich for a 61.6 megabase coding sequence target that includes predicted genes from the genome assembly of the cultivar Morex as well as publicly available full-length cDNAs and de novo assembled RNA-Seq consensus sequence contigs. The platform provides a highly specific capture with substantial and reproducible enrichment of targeted exons, both for cultivated barley and related species. We show that this exome capture platform provides a clear path towards a broader and deeper understanding of the natural variation residing in the mRNA-coding part of the barley genome and will thus constitute a valuable resource for applications such as mapping-by-sequencing and genetic diversity analyzes.

Identification and Dynamics of Two Classes of Aurora-Like Kinases in Arabidopsis and Other Plants

Aurora-like kinases play key roles in chromosome segregation and cytokinesis in yeast, plant, and animal systems. Here, we characterize three Arabidopsis thaliana protein kinases, designated AtAurora1, AtAurora2, and AtAurora3, which share high amino acid identities with the Ser/Thr kinase domain of yeast Ipl1 and animal Auroras. Structure and expression of AtAurora1 and AtAurora2 suggest that these genes arose by a recent gene duplication, whereas the diversification of plant α and β Aurora kinases predates the origin of land plants. The transcripts and proteins of all three kinases are most abundant in tissues containing dividing cells. Intracellular localization of green fluorescent protein–tagged AtAuroras revealed an AtAurora-type specific association mainly with dynamic mitotic structures, such as microtubule spindles and centromeres, and with the emerging cell plate of dividing tobacco (Nicotiana tabacum) BY-2 cells. Immunolabeling using AtAurora antibodies yielded specific signals at the centromeres that are coincident with histone H3 that is phosphorylated at Ser position10 during mitosis. An in vitro kinase assay demonstrated that AtAurora1 preferentially phosphorylates histone H3 at Ser 10 but not at Ser 28 or Thr 3, 11, and 32. The phylogenetic analysis of available Aurora sequences from different eukaryotic origins suggests that, although a plant Aurora gene has been duplicated early in the evolution of plants, the paralogs nevertheless maintained a role in cell cycle–related signal transduction pathways.

Nunatak survival of the high Alpine plant Eritrichium nanum (L.) Gaudin in the central Alps during the ice ages

Polymerase chain reaction–restriction fragment length polymorphisms (PCR‐RFLPs) and sequence analysis of noncoding regions of chloroplast DNA were used to investigate 37 populations of Eritrichium nanum covering its total distribution area, the European Alps. There was no haplotypic variation within the populations, and most haplotypes were restricted to single sites or to neighbouring populations, suggesting low levels of long distance gene flow via seeds. The present geographical distribution of haplotypes probably reflects an ancient geographical pattern within two regions in the intensely glaciated western and eastern central Alps identified as genetic hotspot areas. These two regions contained seven of the total of 11 haplotypes, including many of the most derived ones. The divergent haplotypes formed closely related groups, which supported a separate evolution of these haplotypes in these two regions and, more importantly, gave strong evidence for the in situ survival of these populations on nunataks within the western and eastern central Alps during Pleistocene glaciation. This result is in concordance with a previous study on E. nanum using nuclear markers. Only one haplotype was common and widespread throughout the distributional range of E. nanum. At the same time, it was the evolutionarily basal‐most and all other haplotypes were best described as its descendants. This haplotype is hypothesized to be genetically identical to a Tertiary Alpine colonizing ancestor, whose distribution was secondarily fragmented and infiltrated by derived haplotypes originating through local mutations.

Combined ecological niche modelling and molecular phylogeography revealed the evolutionary history of Hordeum marinum (Poaceae) — niche differentiation, loss of genetic diversity, and speciation in Mediterranean Quaternary refugia

The Hordeum marinum species group consists of two annual grasses of western Eurasian saline meadows or marshes. The two grasses split in the Quaternary about two million years ago. Hordeum marinum and the diploid of Hordeum gussoneanum (2×) co‐occur throughout the Mediterranean basin, while the autotetraploid cytotype of H. gussoneanum (4×) overlaps with its diploid progenitor geographically only in the utmost Eastern Mediterranean, extending from there eastwards into Asia. Using chloroplast sequences of the trnL‐F region, six newly developed chloroplast microsatellite loci, ecological predictive models based on climate data, and the present geographical distribution of the two species we analysed differentiation processes in the H. marinum group. The chloroplast data indicated clear differences in the history of both species. For H. marinum we found a subdivision between genetically variable populations from the Iberian Peninsula and the more uniform populations from the remaining Mediterranean. As an explanation, we assume Pleistocene fragmentation of an earlier widespread population and survival in an Iberian and a Central Mediterranean glacial refuge. Chloroplast variation was completely absent within the cytotypes of H. gussoneanum, indicating a severe and recent genetic bottleneck. Due to this lack of chloroplast variation only the combination of ecological habitat modelling with molecular data analyses allowed conclusions about the history of this taxon. The distribution areas of the two cytotypes of H. gussoneanum overlap today in parts of Turkey, indicating an area with similar climate conditions during polyploid formation. However, after its origin the polyploid cytotype underwent a pronounced ecological shift, compared to its diploid progenitor, allowing it to colonize mountainous inland habitats between the Mediterranean basin and Afghanistan. The extant sympatric occurrence of H. marinum and H. gussoneanum 2× in the Mediterranean region is interpreted as a result of secondary contact after fast Holocene range expansion out of different ice age refugia.

Selfish supernumerary chromosome reveals its origin as a mosaic of host genome and organellar sequences

Supernumerary B chromosomes are optional additions to the basic set of A chromosomes, and occur in all eukaryotic groups. They differ from the basic complement in morphology, pairing behavior, and inheritance and are not required for normal growth and development. The current view is that B chromosomes are parasitic elements comparable to selfish DNA, like transposons. In contrast to transposons, they are autonomously inherited independent of the host genome and have their own mechanisms of mitotic or meiotic drive. Although B chromosomes were first described a century ago, little is known about their origin and molecular makeup. The widely accepted view is that they are derived from fragments of A chromosomes and/or generated in response to interspecific hybridization. Through next-generation sequencing of sorted A and B chromosomes, we show that B chromosomes of rye are rich in gene-derived sequences, allowing us to trace their origin to fragments of A chromosomes, with the largest parts corresponding to rye chromosomes 3R and 7R. Compared with A chromosomes, B chromosomes were also found to accumulate large amounts of specific repeats and insertions of organellar DNA. The origin of rye B chromosomes occurred an estimated ∼1.1–1.3 Mya, overlapping in time with the onset of the genus Secale (1.7 Mya). We propose a comprehensive model of B chromosome evolution, including its origin by recombination of several A chromosomes followed by capturing of additional A-derived and organellar sequences and amplification of B-specific repeats.

Phylogeographic Analyses and Paleodistribution Modeling Indicate Pleistocene In Situ Survival of Hordeum Species (Poaceae) in Southern Patagonia without Genetic or Spatial Restriction

Although many phylogeographic studies have been conducted to analyze the impact of the ice age on species history of Northern Hemisphere mountain plants, such studies are nearly absent for plants of the Southern Hemisphere, particularly for lowland vegetation units. These species should have been primarily influenced by climate cooling and changes in precipitation regime instead of glaciers covering their distribution areas. It is thought that New World lowland species generally evaded climate changes by equatorial migration during Pleistocene cold cycles and recolonized their habitats at higher latitudes when climate warmed up again. In contrast to Eurasia, latitudinal orientation of the major mountain ranges in the Americas made these migrations easily possible. In the huge steppe of the Patagonian plains and adjacent Andes of southern South America thrives a group of three sympatrically distributed diploid species of the barley genus Hordeum, which originated during the last 1.3 million years (My) from a common progenitor. To get insights into the speciation mode of the taxa and to test the hypothesis of longitudinal migration of steppe vegetation during the Pleistocene, we conducted population genetic and phylogeographic analyses based on sequences of the chloroplast trnL-F region from 922 individuals. We found a high number of chloroplast haplotypes shared among species, which indicate speciation through vicariance events. Analysis of the distribution of genetic diversity within and among species inferred an origin of Hordeum comosum in the Central Argentine Andes, whereas Hordeum patagonicum and Hordeum pubiflorum originated in southern Patagonia. The extant occurrence of H. comosum in southern Patagonia and H. pubiflorum northward along the Argentine Andes was caused by reciprocal migration after the origin of the species. Surprisingly, molecular data provided no evidence for range shifts toward the north during the last glacial maximum and recolonization of southerly habitats afterward, but indicated in situ survival of large populations of Hordeum species within their extant distribution ranges even in southernmost Patagonia and Tierra del Fuego. Ecoclimatic niche modeling used to reconstruct the potential paleodistribution areas of the species during the last glacial maximum shows that climate conditions were sufficient for the species to survive Pleistocene cold cycles in Patagonia without significant geographic restrictions. Molecular data together with ecological niche modeling indicate stable geographic distribution areas in two of the three species for at least the Holocene. As the Hordeum species are characteristic taxa of different steppe habitats, we speculate that the Patagonian steppe might be an old vegetation unit occurring for up to 4.5 My in southern South America.

Comparative Genome Analysis Reveals Divergent Genome Size Evolution in a Carnivorous Plant Genus

The C‐value paradox remains incompletely resolved after >40 yr and is exemplified by 2,350‐fold variation in genome sizes of flowering plants. The carnivorous Lentibulariaceae genus Genlisea, displaying a 25‐fold range of genome sizes, is a promising subject to study mechanisms and consequences of evolutionary genome size variation. Applying genomic, phylogenetic, and cytogenetic approaches, we uncovered bidirectional genome size evolution within the genus Genlisea. The Genlisea nigrocaulis Steyerm. genome (86 Mbp) has probably shrunk by retroelement silencing and deletion‐biased double‐strand break (DSB) repair, from an ancestral size of 400 to 800 Mbp to become one of the smallest among flowering plants. The G. hispidula Stapf genome has expanded by whole‐genome duplication (WGD) and retrotransposition to 1550 Mbp. Genlisea hispidula became allotetraploid after the split from the G. nigrocaulis clade ∼29 Ma. Genlisea pygmaea A. St.‐Hil. (179 Mbp), a close relative of G. nigrocaulis, proved to be a recent (auto)tetraploid. Our analyses suggest a common ancestor of the genus Genlisea with an intermediate 1C value (400–800 Mbp) and subsequent rapid genome size evolution in opposite directions. Many abundant repeats of the larger genome are absent in the smaller, casting doubt on their functionality for the organism, while recurrent WGD seems to safeguard against the loss of essential elements in the face of genome shrinkage. We cannot identify any consistent differences in habitat or life strategy that correlate with genome size changes, raising the possibility that these changes may be selectively neutral.

Data congruence and phylogeny of the Papaveraceae s.l. based on four data sets: atpB and rbcL sequences, trnK restriction sites, and morphological characters

Phylogenetic analyses of various combinations of atpB and rbcL sequence data, trnK restriction sites, and morphological characters of taxa representing the Papaveraceae s.l. (Pteridophyllaceae, Fumari- aceae subfamilies Fumarioideae and Hypecoideae, Papaveraceae s. str. subfamilies Eschscholzioideae, Chelidonioideae, Papaveroideae, Platystemonoideae) are presented. Several measures of data set incongru- ency indicate considerable congruence between data sets. In order to correctly identify the root of the family, combined atpB and rbcL sequence data for the Papaveraceae s.l. and appropriate outgroups (Asarum, Illicium, Platanus, Euptelea, and representatives of the Ranunculaceae and Berberidaceae) were analyzed. Regardless of the outgroup used, all topologies obtained suggest that Pteridophyllum (Pteridophyllaceae) is sister group to Fumariaceae and Papaveraceae s. str. Data sets of the ingroup with Pteridophyllum used as the outgroup were analyzed separately and together, with results from the combined data sets showing increased bootstrap support. Contrary to recent claims, Papaveraceae s. str. remain monophyletic after the exclusion of Pteridophyl- lum and Fumariaceae. In the Papaveraceae s. str., Eschscholzioideae are sister group to Papaveroideae (including Platystemonoideae) and Chelidonioideae (including Glaucium/Dicranostigma). The evolution of floral morphology, geographical distribution, and ecology of Papaveraceae s.l. are briefly discussed.

A chloroplast genealogy of myrmecophytic Macaranga species (Euphorbiaceae) in Southeast Asia reveals hybridization, vicariance and long‐distance dispersals

Macaranga (Euphorbiaceae) includes about 280 species with a palaeotropic distribution. The genus not only comprises some of the most prominent pioneer tree species in Southeast Asian lowland dipterocarp forests, it also exhibits a substantial radiation of ant‐plants (myrmecophytes). Obligate ant‐plant mutualisms are formed by about 30 Macaranga species and 13 ant species of the genera Crematogaster or Camponotus. To improve our understanding of the co‐evolution of the ants and their host plants, we aim at reconstructing comparative organellar phylogeographies of both partners across their distributional range. Preliminary evidence indicated that chloroplast DNA introgression among closely related Macaranga species might occur. We therefore constructed a comprehensive chloroplast genealogy based on DNA sequence data from the noncoding ccmp2, ccmp6, and atpB‐rbcL regions for 144 individuals from 41 Macaranga species, covering all major evolutionary lineages within the three sections that contain myrmecophytes. A total of 88 chloroplast haplotypes were identified, and grouped into a statistical parsimony network that clearly distinguished sections and well‐defined subsectional groups. Within these groups, the arrangement of haplotypes followed geographical rather than taxonomical criteria. Thus, up to six chloroplast haplotypes were found within single species, and up to seven species shared a single haplotype. The spatial distribution of the chloroplast types revealed several dispersals between the Malay Peninsula and Borneo, and a deep split between Sabah and the remainder of Borneo. Our large‐scale chloroplast genealogy highlights the complex history of migration, hybridization, and speciation in the myrmecophytes of the genus Macaranga. It will serve as a guideline for adequate sampling and data interpretation in phylogeographic studies of individual Macaranga species and species groups.

Population demography influences climatic niche evolution: evidence from diploid American Hordeum species (Poaceae)

In this study, we explore the interplay of population demography with the evolution of ecological niches during or after speciation in Hordeum. While large populations maintain a high level of standing genetic diversity, gene flow and recombination buffers against fast alterations in ecological adaptation. Small populations harbour lower allele diversity but can more easily shift to new niches if they initially survive under changed conditions. Thus, large populations should be more conservative regarding niche changes in comparison to small populations. We used environmental niche modelling together with phylogenetic, phylogeographic and population genetic analyses to infer the correlation of population demography with changes in ecological niche dimensions in 12 diploid Hordeum species from the New World, forming four monophyletic groups. Our analyses found both shifts and conservatism in distinct niche dimensions within and among clades. Speciation due to vicariance resulted in three species with no pronounced climate niche differences, while species originating due to long‐distance dispersals or otherwise encountering genetic bottlenecks mostly revealed climate niche shifts. Niche convergence among clades indicates a niche‐filling pattern during the last 2 million years in South American Hordeum. We provide evidence that species, which did not encounter population reductions mainly showed ecoclimatic niche conservatism, while major niche shifts occurred in species which have undergone population bottlenecks. Our data allow the conclusion that population demography influences adaptation and niche shifts or conservatism in South American Hordeum species.