Rolland and Dujon, 2011
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C. R. Biologies 334 (2011) 620–628 Contents lists available at ScienceDirect Comptes Rendus Biologies www.sciencedirect.com Evolution/E´ volution Yeasty clocks: Dating genomic changes in yeasts Horloges tremblantes : datation des changements ge´nomiques chez les levures Thomas Rolland, Bernard Dujon * Unite´ de ge´ne´tique mole´culaire des levures (CNRS URA2171 and University P.-M.-Curie UFR927), Institut Pasteur, 25, rue du Docteur-Roux, 75724 Paris cedex 15, France ARTICLE INFO ABSTRACT Article history: Calibration of clocks to date evolutionary changes is of primary importance for Received 12 November 2010 comparative genomics. In the absence of fossil records, the dating of changes during Accepted after revision 17 March 2011 yeast genome evolution can only rely on the properties of the genomes themselves, given Available online 1 July 2011 the uncertainty of extrapolations using clocks from other organisms. In this work, we use the experimentally determined mutational rate of Saccharomyces cerevisiae to calculate Keywords: the numbers of successive generations corresponding to observed sequence polymor- Evolution phism between strains or species of other yeasts. We then examine synteny conservation Mutational rate Polymorphism across the entire subphylum of Saccharomycotina yeasts, and compare this second clock Divergence times based on chromosomal rearrangements with the first one based on sequence divergence. A Synteny conservation non-linear relationship is observed, that interestingly also applies to insects although, for equivalent sequence divergence, their rate of chromosomal rearrangements is higher than that of yeasts. ß 2011 Acade´mie des sciences. Published by Elsevier Masson SAS. All rights reserved. RE´ SUME´ Mots cle´s: L’e´talonnage d’horloges mole´culaires pour dater les changements e´volutifs a une grande E´ volution importance pour la ge´nomique comparative. En l’absence de fossiles, la datation des Taux de mutation changements durant l’e´volution des ge´nomes de levures ne peut se baser que sur les Polymorphisme proprie´te´s des ge´nomes eux-meˆmes, e´tant donne´e l’incertitude des extrapolations a` partir Temps de divergence d’horloges d’autres organismes. Dans ce travail, nous utilisons le taux de mutation Conservation de synte´nie expe´rimentalement de´termine´ chez Saccharomyces cerevisiae pour calculer les nombres de ge´ne´rations successives correspondant aux degre´s de polymorphisme de se´quences observe´s entre souches ou espe`ces d’autres levures. Nous examinons ensuite la conservation de synte´nie a` travers tout le sous-embranchement des levures Sacchar- omycotina, et comparons cette seconde horloge base´e sur les re´arrangements chromo- somiques avec la premie`re base´e sur la divergence de se´quence. Une relation non-line´aire est observe´e, qui s’applique e´galement aux insectes bien que, pour une divergence de se´quence e´quivalente, leur taux de re´arrangements chromosomiques soit plus e´leve´ que celui des levures. ß 2011 Acade´mie des sciences. Publie´ par Elsevier Masson SAS. Tous droits re´serve´s. * Corresponding author. E-mail address: [email protected] (B. Dujon). 1631-0691/$ – see front matter ß 2011 Acade´mie des sciences. Published by Elsevier Masson SAS. All rights reserved. doi:10.1016/j.crvi.2011.05.010 T. Rolland, B. Dujon / C. R. Biologies 334 (2011) 620–628 621 1. Introduction experimental approaches [16]. Most yeasts whose gen- omes have been fully sequenced so far belong to the The concept of molecular evolutionary clocks is central Saccharomycotina (also called hemiascomycetes), a large to modern comparative genomics. From the pioneering subphylum of Ascomycota that includes Saccharomyces work of Zuckerlandl and Pauling [1], it is commonly cerevisiae. Despite the conservation of their unicellular admitted that amino-acid substitutions between ortholo- mode of life with bud formation, these yeasts cover a very gous proteins accumulate with the time separating them broad evolutionary range, and very important degrees of from their common ancestor, and differences between sequence divergence exist between orthologous genes of aligned sequences are, therefore, used to build phyloge- distinct yeast species, even those belonging to the same netic trees and to estimate the dates of separation between clade [17,18]. Dating major evolutionary changes in yeast living species (or groups of species). With the increasing genomes, such as the change of codon assignation in the availability of genome sequence data, it became clear, CTG group [19], the triplication of mating cassettes in however, that the rate at which protein sequences evolve Saccharomycetaceae [13], or the whole-genome duplica- varies among lineages [2], leading to the idea of relaxed tion in the ancestry of Saccharomyces sensu stricto and molecular clocks [3–5], and raising the question of related clades [20], remains, therefore, highly imprecise. appropriate calibration to date major phylogenetic separa- Phylogenetic interpolation within the fungal tree of life has tions. In fungi, for example, this problem was remarkably been attempted [21–23], but the specific mode of illustrated by the work of Taylor and Berbee [6]: depending propagation of yeasts with rapid clonal expansions raises upon the reference used to calibrate the clock, the the question of the validity of the comparisons with separation date between Ascomycota and Basidiomycota multicellular organisms having obligate sexual reproduc- varies between 400 and 1800 Myr. Similarly, the origin of tion and possibly distinct evolutionary rates. A specific Saccharomycotina (budding yeasts) is dated, according to calibration of the molecular clock of yeasts is, therefore, calibrations, at 250 Myr ago or 900 Myr ago, i.e. a range of desirable. But, besides the genomic changes themselves, no uncertainty linking the Permian-Trias transition to deep independent piece of information such as fossils records, is precambrian times. Even when calibration is properly set, available to cover their very large evolutionary range. extrapolation of molecular clocks to large evolutionary In this work, we have addressed this question from two scales can only give seemingly precise results if one takes different viewpoints. Starting from the mutation rates that the statistical limits of confidence into proper consider- have been precisely measured by experiments in ation [7]. Greater precision would require independent S. cerevisiae [24–26], we have computed the minimal calibration points within short evolutionary timescales number of successive generations separating distinct using increased taxon sampling or continuous fossil lineages in this yeast, and extrapolated similar calculations records, two conditions not always readily accessible. to the separation of species within clades. This clock is The identification of Paleopyrenomycites devonicus as the appropriate for short evolutionary timescales but gradual- oldest fossil ascomycete dated to 400 Myr [8] played an ly loses precision with increasing evolutionary range. We important role to calibrate the fungal tree of life, but such have, therefore, looked for a second clock more appropriate fossils remain rare in fungi. Also, they are non-existent in to larger evolutionary timescales by examining the yeasts, if one excepts amber inclusions which have relationship between sequence divergence and degrees received only limited attention so far [9,10] and are, of chromosomal rearrangements. This relationship has anyway, too recent for setting clocks over long evolution- been quantitatively established over the entire evolution- ary times. Increasing taxon sampling is not easier for ary range of Saccharomycotina, and compared to a similar yeasts, since it is unlikely that living intermediates exist, relationship established for insects. given their very mode of propagation that creates constant bottlenecks. 2. Calibrating sequence divergence in terms of the Another important problem for dating using molecular minimal number of successive generations data is that substitution rates also vary between the different genes of a same organism. In yeasts, for example, The spontaneous mutation rate has recently been a dispersion of nearly three orders of magnitude exists in determined with precision in S. cerevisiae by three the rate of non-synonymous substitutions per site (dN) independent approaches. A per-base-pair mutation rate between the fastest and the slowest evolving proteins [11]. (m) was established for two genes using the classical Luria- 10 The dispersion is lower in organisms with smaller Delbru¨ ck fluctuation assays [24]. Figures of 3.80 10À 10  genetically effective population sizes such as Drosophila and 6.44 10À mutations per nucleotide per generation  and mammals [12], hence the necessity to compare were obtained for the URA3 and the CAN1 genes, homogeneous groups of organisms sharing similar life respectively, indicating that, even if not entirely uniform style and mode of propagation to properly date evolution- across the genome, the mutation rate shows a limited ary changes. Yeasts offer such a case with more than three variation range (ca. two times). An independent estimation dozens of species fully sequenced [13] and population of the per-base-pair mutation rate (m) along the entire genomic studies now available for a few of them [14,15]. genome was obtained using novel sequencing technology These fungi proved particularly meaningful to elucidate in mutation-accumulation experiments [25]. Partial rese- the