In the practice of molecular dating, substitution saturation will bias the results if not properly modeled. Date estimates based on commonly used mitochondrial DNA sequences likely suffer from this problem because of their high substitution rate. Nevertheless, the patterns and extent of such expected bias remain unknown for many major evolutionary lineages, which often differ in ages, available calibrations, and substitution rates of their mitochondrial genome. In this case study of salamanders, we used estimates based on multiple nuclear exons to assess the effects of saturation on dating divergences using mitochondrial genome sequences on a timescale of ~200-300 My. The results indicated that, due to saturation for older divergences and in the absence of younger effective calibration points, dates derived from the mitochondrial data were considerably overestimated and systematically biased toward the calibration point for the ingroup root. The overestimate might be as great as 3-10 times (about 20 My) older than actual divergence dates for recent splitting events and 40 My older for events that are more ancient. For deep divergences, dates estimated were strongly compressed together. Furthermore, excluding the third codon positions of protein-coding genes or only using the RNA genes or second codon positions did not considerably improve the performance. In the order Caudata, slowly evolving markers such as nuclear exons are preferred for dating a phylogeny covering a relatively wide time span. Dates estimated from these markers can be used as secondary calibrations for dating recent events based on rapidly evolving markers for which mitochondrial DNA sequences are attractive candidates due to their short coalescent time. In other groups, similar evaluation should be performed to facilitate the choice of markers for molecular dating and making inferences from the results.

The Arabidopsis flowering locus T (FT) gene encodes the mobile florigen essential for floral induction. While movement of the FT protein has been shown to occur within plants, systemic spread of FT mRNA remains to be unequivocally demonstrated. Utilizing novel RNA mobility assay vectors based on two distinct movementdefective viruses, Potato virus X and Turnip crinkle virus, and an agroinfiltration assay, we demonstrate that nontranslatable FT mRNA, independent of the FT protein, moves throughout Nicotiana benthamiana and mutant Arabidopsis plants and promotes systemic trafficking of viral and green fluorescence protein RNAs. Viral ectopic expression of FT induced flowering in the short-day N. tabacum Maryland Mammoth tobacco under long-day conditions. Recombinant Potato virus X bearing FT RNA spread and established systemic infection more quickly than the parental virus. The cis-acting element essential for RNA movement was mapped to the nucleotides 1 to 102 of the FT mRNA coding sequence. These data demonstrate that a plant self-mobile RNA molecule can mediate long-distance trafficking of heterologous RNAs and raise the possibility that FT RNA, along with the FT protein, may be involved in the spread of the floral stimulus throughout the plant.RNA trafficking plays an important role in systemic signaling that controls plant development and defense against pathogen infection (25). Hundreds of RNA transcripts have been recently identified in phloem, suggesting phloem-mobile RNAs may act as long-distance signaling molecules in plants (4,8). Indeed, systemic movement of a homeobox fusion transcript and gibberellic acid-insensitive RNA regulates leaf architecture (13, 19), a non-cell-autonomous mobile RNA represents a long-distance signal that modulates potato tuber formation (3), and small interfering RNAs are components of intercellular and systemic mobile signals for innate RNA silencing defense (9,12,14). RNA trafficking is also critical for plant viruses and viroids to establish systemic infection. It has been demonstrated that an RNA motif directs long-distance trafficking of a small naked RNA viroid (33,44,46). Moreover, a short RNA sequence is found to be involved in cell-to-cell movement of a plant viral RNA (24), and replication-independent viral RNA can move over long distances in plants (11).In floral induction, the mobile florigen is encoded by the Arabidopsis flowering locus T (FT) gene. FT transcribes mRNA in the leaf, but its encoded FT protein functions in the shoot apices where flowers develop (1, 2, 40). The Arabidopsis FT protein and its orthologues have been shown to be involved in long-distance signaling in floral induction (7,18,22,23,29,30,37). However, whether FT mRNA is also capable of systemic spread remains to be demonstrated. We describe novel approaches which show that not only does FT RNA move over long distances but, remarkably, also facilitates the systemic spread of heterologous green fluorescent protein (GFP) mRNA and different viral RNAs in plants. The FT RNA movement does not re...

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