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.

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.

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.

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.

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.