SUMMARYLittle is known about the mechanisms that control transcription of the mitochondrial and chloroplastic genomes, and their interplay within plant cells. Here, we describe the positional cloning of the Arabidopsis RUG2 gene, which encodes a protein that is dual-targeted to mitochondria and chloroplasts, and is homologous with the metazoan mitochondrial transcription termination factors (mTERFs). In the loss-offunction rug2 mutants, most organs were pale and showed reduced growth, and the leaves exhibited both green and pale sectors, with the latter containing sparsely packed mesophyll cells. Chloroplast and mitochondrion development were strongly perturbed in the rug2-1 mutant, particularly in pale leaf sectors, in which chloroplasts were abnormally shaped and reduced in number, thereby impairing photoautotrophic growth. As expected from the pleiotropic phenotypes caused by its loss-of-function alleles, the RUG2 gene was ubiquitously expressed. In a microarray analysis of the mitochondrial and chloroplastic genomes, 56 genes were differentially expressed between rug2-1 and the wild type: most mitochondrial genes were downregulated, whereas the majority of the chloroplastic genes were upregulated. Quantitative RT-PCR analyses showed that the rug2-1 mutation specifically increases expression of the RpoTp nuclear gene, which encodes chloroplastic RNA polymerase. Therefore, the RUG2 nuclear gene seems to be crucial for the maintenance of the correct levels of transcripts in the mitochondria and chloroplasts, which is essential for optimized functions of these organelles and proper plant development. Our results highlight the complexity of the functional interaction between these two organelles and the nucleus.

Aim Recent biogeographical studies have postulated a North African, Late Pleistocene, origin for some species of the Iberian Peninsula. However, a robust assessment of such range expansions requires high-resolution molecular tools to resolve overlapping biogeographical and cultural processes. Here we aim to determine whether the spur-thighed tortoise, Testudo graeca, arrived in southeastern Spain during historical or prehistoric times, and whether its dispersal to the Iberian Peninsula was human-mediated.Location The western Mediterranean Basin (south-eastern Spain, northern Algeria and north-western Morocco).Methods Using 428 samples from 19 sites in North Africa and 18 in southeastern Spain, we obtained mitochondrial sequences from the cytochrome b gene and genotypes derived from seven microsatellite loci. These data were employed to obtain population genetics descriptors, haplotype networks, Bayesian cluster analyses and isolation-by-distance patterns. Moreover, we used a Bayesian demographic approach to delimit the dates involved in the range expansion.Results We found lower levels of genetic variability and weak mitochondrial differentiation in the south-eastern Spanish tortoises compared with the North African ones. However, exclusive haplotypes occurred in the Iberian samples and microsatellite cluster analyses revealed moderate levels of admixture across both sides of the Mediterranean. A coastal area in the west of Algeria and the central-southern region in south-eastern Spain are suggested as the most probable founder and arrival places, respectively. Finally, we identified signatures of an ancient bottleneck event approximately 20,000-30,000 years ago. Main conclusionsThe spur-thighed tortoise probably arrived in south-eastern Spain during Late Pleistocene sea-level low stands. The role that humans may have played as dispersers across the Mediterranean remains unclear. Our results are in accordance with other recent findings of trans-Mediterranean expansions during this period and highlight the importance of employing precise methodological approaches before a species can be considered as historically introduced.

We investigated the mitochondrial phylogeography of spur-thighed tortoises (Testudo graeca) in the Western Mediterranean. In North Africa, four major lineages (A-D) occur that together constitute a well-supported clade corresponding to one of the six major clades within T. graeca; the North African clade is sister to a Caucasian clade representing the subspecies T. g. armeniaca. Phylogenetic relationships between the North African lineages are badly resolved. Lineage A is distributed in Tunisia and adjacent Algeria, lineage B in Algeria and northern Morocco, lineage C in the Libyan Cyrenaica Peninsula, and lineage D north of the High Atlas Mts. and in the Souss Valley (southern Morocco). Lineage B is subdivided into two subgroups, B1 (eastern Morocco and Algeria) and B2 (north-western Morocco). Italian tortoises harbour haplotypes of lineage A, Spanish tortoises of subgroup B1. Based on a relaxed molecular clock calibrated with fossil evidence, the six major mtDNA clades of T. graeca are estimated to have diverged approximately 4.2-1.8 Ma ago; the split between the clades representing the eastern subspecies T. g. ibera and T. g. terrestris is younger than the split between Western Mediterranean tortoises and T. g. armeniaca. The Western Mediterranean lineages A-D were dated to have diverged at least 1.4-1.1 Ma ago; B1 and B2 split approximately 0.7 Ma ago. Our results suggest that Italian and Spanish tortoises were either introduced or originated from trans-oceanic dispersal in historic or prehistoric times. Spur-thighed tortoises invaded North Africa probably across Near Eastern landbridges that emerged in the Late Tertiary. Their diversification in North Africa seems to be correlated with habitat aridization cycles during the Pleistocene. The ranges of the Western Mediterranean lineages largely correspond to the distribution of morphologically defined subspecies in North Africa, with exception of T. g. graeca and T. g. whitei, and of T. g. lamberti and T. g. marokkensis, which are not differentiated. We propose to lump the first two subspecies under the name of T. g. graeca and the latter under the name of T. g. marokkensis. The complex differentiation of spur-thighed tortoises in North Africa implies that the model of a bipartite east-west differentiation, as proposed for other Maghrebian amphibians and reptiles, may be too simplistic, reflecting incomplete locality sampling rather than actual phylogeographic differentiation.

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Much of our current knowledge about the genetic dynamics in range expansions originates from models, simulations and microcosm experiments that need to be corroborated by field data. Here, we report a neutral genetic pattern that matches the predictions of the genetic surfing theory. Genetic surfing occurs when repeated founding events and genetic drift act on the wave of advance of an expanding population, promoting strong spatial structure. In the range expansion of the tortoise Testudo graeca from North Africa to southeastern Spain, we found several genetic signatures consistent with surfing: a decrease of genetic diversity with distance from the initial founder area, clinal patterns in allele frequencies, rare African alleles which have become common at distal sites in the Spanish range, and stronger spatial differentiation in the expanded range than in the original one. Our results provide support for the theory that genetic drift can be an important force in shaping the genetic structure of expanding populations.