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.

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.

Two extinct diploid progenitors were involved in allopolyploid formation in the Hordeum murinum (Poaceae: Triticeae) taxon complex.

Wall barley (Hordeum murinum) occurs with three subspecies, naturally distributed from southern Central Asia through the Mediterranean region to northwestern Europe, but now is an invasive weed in many parts of the world. Subspecies glaucum is diploid, while subspp. murinum and leporinum are tetraploids, the latter also occurring with a hexaploid cytotype. Earlier analyses were inconclusive regarding auto- or allopolyploid origins of subspp. murinum and leporinum. We analyzed the phylogeny of the taxon group using amplified fragment length polymorphisms (AFLP), sequences of cloned PCR products of the nuclear ribosomal DNA internal transcribed spacer region (ITS), a part of the nuclear single-copy gene topoisomerase 6 (Topo6) spanning two introns, and sequences of the chloroplast trnL-F region together with length variation at six chloroplast microsatellite loci, including multiple individuals of each subspecies and cytotype, covering the entire natural distribution area of the species. Phylogenetic analyses with all used markers differentiate diploid and polyploids. Sequences of both nuclear regions indicated that diploid subsp. glaucum was involved in tetraploid formation together with a now extinct species belonging to the same Hordeum genome group (Xu). Furthermore, AFLP and ITS analyses suggest that a third, though closely related extinct taxon contributed to hexaploid formation. No method was able to discern tetraploid subspp. murinum and leporinum, which we attribute to the young age of subsp. murinum. None of the used molecular markers revealed a strong geographic pattern of genetic variation that would allow comprehensive phylogeographic analysis, most probably due to the very effective seed dispersal of the taxa.

Evolutionary history of wild barley (Hordeum vulgare subsp. spontaneum) analyzed using multilocus sequence data and paleodistribution modeling.

Studies of Hordeum vulgare subsp. spontaneum, the wild progenitor of cultivated barley, have mostly relied on materials collected decades ago and maintained since then ex situ in germplasm repositories. We analyzed spatial genetic variation in wild barley populations collected rather recently, exploring sequence variations at seven single-copy nuclear loci, and inferred the relationships among these populations and toward the genepool of the crop. The wild barley collection covers the whole natural distribution area from the Mediterranean to Middle Asia. In contrast to earlier studies, Bayesian assignment analyses revealed three population clusters, in the Levant, Turkey, and east of Turkey, respectively. Genetic diversity was exceptionally high in the Levant, while eastern populations were depleted of private alleles. Species distribution modeling based on climate parameters and extant occurrence points of the taxon inferred suitable habitat conditions during the ice-age, particularly in the Levant and Turkey. Together with the ecologically wide range of habitats, they might contribute to structured but long-term stable populations in this region and their high genetic diversity. For recently collected individuals, Bayesian assignment to geographic clusters was generally unambiguous, but materials from genebanks often showed accessions that were not placed according to their assumed geographic origin or showed traces of introgression from cultivated barley. We assign this to gene flow among accessions during ex situ maintenance. Evolutionary studies based on such materials might therefore result in wrong conclusions regarding the history of the species or the origin and mode of domestication of the crop, depending on the accessions included.

Progenitor-derivative relationships of Hordeum polyploids (Poaceae, Triticeae) inferred from sequences of TOPO6, a nuclear low-copy gene region.

Polyploidization is a major mechanism of speciation in plants. Within the barley genus Hordeum, approximately half of the taxa are polyploids. While for diploid species a good hypothesis of phylogenetic relationships exists, there is little information available for the polyploids (4×, 6×) of Hordeum. Relationships among all 33 diploid and polyploid Hordeum species were analyzed with the low-copy nuclear marker region TOPO6 for 341 Hordeum individuals and eight outgroup species. PCR products were either directly sequenced or cloned and on average 12 clones per individual were included in phylogenetic analyses. In most diploid Hordeum species TOPO6 is probably a single-copy locus. Most sequences found in polyploid individuals phylogenetically cluster together with sequences derived from diploid species and thus allow the identification of parental taxa of polyploids. Four groups of sequences occurring only in polyploid taxa are interpreted as footprints of extinct diploid taxa, which contributed to allopolyploid evolution. Our analysis identifies three key species involved in the evolution of the American polyploids of the genus. (i) All but one of the American tetraploids have a TOPO6 copy originating from the Central Asian diploid H. roshevitzii, the second copy clustering with different American diploid species. (ii) All hexaploid species from the New World have a copy of an extinct close relative of H. californicum and (iii) possess the TOPO6 sequence pattern of tetraploid H. jubatum, each with an additional copy derived from different American diploids. Tetraploid H. bulbosum is an autopolyploid, while the assumed autopolyploid H. brevisubulatum (4×, 6×) was identified as allopolyploid throughout most of its distribution area. The use of a proof-reading DNA polymerase in PCR reduced the proportion of chimerical sequences in polyploids in comparison to Taq polymerase.

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 (2x) co-occur throughout the Mediterranean basin, while the autotetraploid cytotype of H. gussoneanum (4x) 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 2x in the Mediterranean region is interpreted as a result of secondary contact after fast Holocene range expansion out of different ice age refugia.

A chloroplast genealogy of hordeum (poaceae): Long-term persisting haplotypes, incomplete lineage sorting, regional extinction, and the consequences for phylogenetic inference.

To analyze reasons for inconclusive results of earlier chloroplast phylogenies in the grass genus Hordeum, we established a genealogy of chloroplast haplotypes by sequencing the trnL-trnF region in 875 individuals, covering all 31 species of the genus. Although the outcomes of phenetic and parsimony analyses of 88 haplotypes were ambiguous, a network approach showed that in Hordeum ancient chloroplast types co-occur with their descendants. Moreover, we found up to 18 different chloroplast haplotypes within a single species and up to 6 species sharing single haplotypes. Persisting polymorphisms together with incomplete lineage sorting occurred preferentially in the rapidly speciating New World taxa of the genus, where ancient chloroplast types have survived for at least 4 Myr. Lineages-through-time plots and a high number of missing chloroplast haplotypes indicated far-reaching extinction of chloroplast lineages in Europe and particularly the Mediterranean. Survival of these lineages in East Asia and North America resulted in chloroplast relationships that markedly differed from nuclear estimations of species relationships. Thus, even for the deepest splits in the genus, reaching back more than 9 Myr, no safe phylogenetic inference from chloroplast data is possible in Hordeum. The chloroplast genealogy, however, revealed biogeographic patterns and indicated processes involved in speciation in Hordeum. We conclude that the described phenomena are not restricted to Hordeum and that the knowledge of the chloroplast relationships within a genus is indispensable to prevent misinterpretation of phylogeographic data within single species.

The considerable genome size variation of Hordeum species (poaceae) is linked to phylogeny, life form, ecology, and speciation rates.

Genome size variation in plants is thought to be correlated with cytological, physiological, or ecological characters. However, conclusions drawn in several studies were often contradictory. To analyze nuclear genome size evolution in a phylogenetic framework, DNA contents of 134 accessions, representing all but one species of the barley genus Hordeum L., were measured by flow cytometry. The 2C DNA contents were in a range from 6.85 to 10.67 pg in diploids (2n = 14) and reached up to 29.85 pg in hexaploid species (2n = 42). The smallest genomes were found in taxa from the New World, which became secondarily annual, whereas the largest diploid genomes occur in Eurasian annuals. Genome sizes of polyploid taxa equaled mostly the added sizes of their proposed progenitors or were slightly (1% to 5%) smaller. The analysis of ancestral genome sizes on the base of the phylogeny of the genus revealed lineages with decreasing and with increasing genome sizes. Correlations of intraspecific genome size variation with the length of vegetation period were found in H. marinum populations from Western Europe but were not significant within two species from South America. On a higher taxonomical level (i.e., for species groups or the entire genus), environmental correlations were absent. This could mostly be attributed to the superimposition of life-form changes and phylogenetic constraints, which conceal ecogeographical correlations.