Biogeographic and diversification patterns of Neotropical Troidini butterflies (Papilionidae) support a museum model of diversity dynamics for Amazonia

Background: The temporal and geographical diversification of Neotropical insects remains poorly understood because of the complex changes in geological and climatic conditions that occurred during the Cenozoic. To better understand extant patterns in Neotropical biodiversity, we investigated the evolutionary history of three Neotropical swallowtail Troidini genera (Papilionidae). First, DNA-based species delimitation analyses were conducted to assess species boundaries within Neotropical Troidini using an enlarged fragment of the standard barcode gene. Molecularly delineated species were then used to infer a time-calibrated species-level phylogeny based on a three-gene dataset and Bayesian dating analyses. The corresponding chronogram was used to explore their temporal and geographical diversification through distinct likelihood-based methods. Results: The phylogeny for Neotropical Troidini was well resolved and strongly supported. Molecular dating and biogeographic analyses indicate that the extant lineages of Neotropical Troidini have a late Eocene (33-42 Ma) origin in North America. Two independent lineages (Battus and Euryades + Parides) reached South America via the GAARlandia temporary connection, and later became extinct in North America. They only began substantive diversification during the early Miocene in Amazonia. Macroevolutionary analysis supports the "museum model" of diversification, rather than Pleistocene refugia, as the best explanation for the diversification of these lineages. Conclusions: This study demonstrates that: (i) current Neotropical biodiversity may have originated ex situ; (ii) the GAARlandia bridge was important in facilitating invasions of South America; (iii) colonization of Amazonia initiated the crown diversification of these swallowtails; and (iv) Amazonia is not only a species-rich region but also acted as a sanctuary for the dynamics of this diversity. In particular, Amazonia probably allowed the persistence of old lineages and contributed to the steady accumulation of diversity over time with constant net diversification rates, a result that contrasts with previous studies on other South American butterflies.

Multiple independent origins of mitochondrial control region duplications in the order Psittaciformes

mitochondrial genomes are generally thought to be under selection for compactness, due to their small size, consistent gene content, and a lack of introns or intergenic spacers. As more animal mitochondrial genomes are fully sequenced, rearrangements and partial duplications are being identified with increasing frequency, particularly in birds (Class Ayes). In this study, we investigate the evolutionary history of mitochondrial control region states within the avian order Psittaciformes (parrots and cockatoos). To this aim, we reconstructed a comprehensive multi-locus phylogeny of parrots, used PCR of three diagnostic fragments to classify the mitochondrial control region state as single or duplicated, and mapped these states onto the phylogeny. We further sequenced 44 selected species to validate these inferences of control region state. Ancestral state reconstruction using a range of weighting schemes identified six independent origins of mitochondrial control region duplications within Psittaciformes. Analysis of sequence data showed that varying levels of mitochondrial gene and tRNA homology and degradation were present within a given clade exhibiting duplications. Levels of divergence between control regions within an individual varied from 0-10.9% with the differences occurring mainly between 51 and 225 nucleotides 3' of the goose hairpin in domain I. Further investigations into the fates of duplicated mitochondrial genes, the potential costs and benefits of having a second control region, and the complex relationship between evolutionary rates, selection, and time since duplication are needed to fully explain these patterns in the mitochondrial genome. (C) 2012 Elsevier Inc. All rights reserved.

Parasites reveal movement of bats between the New and Old Worlds

The global distribution of bat taxa indicates that the Atlantic and Pacific Oceans are effective barriers to movement between the Old and New Worlds. For instance, one of the major suborders, Yinpterochiroptera, has an exclusively Old World distribution, and within the other, Yangochiroptera, no species and only five genera are common to both. However, as bats are sometimes blown out to sea, and have colonised isolated islands, occasional natural movement between the New and Old Worlds does appear to be possible. Here we identify new genotypes of a blood parasite, Trypanosoma dionisii, in Old World bats that are closely related to South American strains. Using highly conservative calibration points, divergence of Old and New World strains is estimated to have occurred 3.2-5.0 million years ago (MYA), depending on the method used (upper 95% CL for maximum time 11.4 MYA). The true date of divergence is likely to be considerably more recent. These results demonstrate that taxon-specific parasites can indicate historical movements of their hosts, even where their hosts may have left no lasting phylogenetic footprint. (C) 2012 Elsevier Inc. All rights reserved.

The gene transformer-2 of Anastrepha fruit flies (Diptera, Tephritidae) and its evolution in insects

Abstract Background In the tephritids Ceratitis, Bactrocera and Anastrepha, the gene transformer provides the memory device for sex determination via its auto-regulation; only in females is functional Tra protein produced. To date, the isolation and characterisation of the gene transformer-2 in the tephritids has only been undertaken in Ceratitis, and it has been shown that its function is required for the female-specific splicing of doublesex and transformer pre-mRNA. It therefore participates in transformer auto-regulatory function. In this work, the characterisation of this gene in eleven tephritid species belonging to the less extensively analysed genus Anastrepha was undertaken in order to throw light on the evolution of transformer-2. Results The gene transformer-2 produces a protein of 249 amino acids in both sexes, which shows the features of the SR protein family. No significant partially spliced mRNA isoform specific to the male germ line was detected, unlike in Drosophila. It is transcribed in both sexes during development and in adult life, in both the soma and germ line. The injection of Anastrepha transformer-2 dsRNA into Anastrepha embryos caused a change in the splicing pattern of the endogenous transformer and doublesex pre-mRNA of XX females from the female to the male mode. Consequently, these XX females were transformed into pseudomales. The comparison of the eleven Anastrepha Transformer-2 proteins among themselves, and with the Transformer-2 proteins of other insects, suggests the existence of negative selection acting at the protein level to maintain Transformer-2 structural features. Conclusions These results indicate that transformer-2 is required for sex determination in Anastrepha through its participation in the female-specific splicing of transformer and doublesex pre-mRNAs. It is therefore needed for the auto-regulation of the gene transformer. Thus, the transformer/transfomer-2 > doublesex elements at the bottom of the cascade, and their relationships, probably represent the ancestral state (which still exists in the Tephritidae, Calliphoridae and Muscidae lineages) of the extant cascade found in the Drosophilidae lineage (in which tra is just another component of the sex determination gene cascade regulated by Sex-lethal). In the phylogenetic lineage that gave rise to the drosophilids, evolution co-opted for Sex-lethal, modified it, and converted it into the key gene controlling sex determination.

Shape differences rather than size differences between castes in the Neotropical swarm-founding wasp Metapolybia docilis (Hymenoptera: Vespidae, Epiponini)

Abstract Background Swarm-founding epiponine wasps are an intriguing group of social insects in which colonies are polygynic (several queens share reproduction) and differentiation between castes is often not obvious. However, caste differences in some may be more pronounced in later phases of the colony cycle. Results Using morphometric analyses and multivariate statistics, it was found that caste differences in Metapolybia docilis are slight but more distinct in latter stages of the colony cycle. Conclusions Because differences in body parts are so slight, it is proposed that such variation may be due to differential growth rates of body parts rather than to queens being larger in size, similar to other previously observed epiponines.

Different patterns of evolution for duplicated DNA repair genes in bacteria of the Xanthomonadales group

Abstract Background DNA repair genes encode proteins that protect organisms against genetic damage generated by environmental agents and by-products of cell metabolism. The importance of these genes in life maintenance is supported by their high conservation, and the presence of duplications of such genes may be easily traced, especially in prokaryotic genomes. Results The genome sequences of two Xanthomonas species were used as the basis for phylogenetic analyses of genes related to DNA repair that were found duplicated. Although 16S rRNA phylogenetic analyses confirm their classification at the basis of the gamma proteobacteria subdivision, differences were found in the origin of the various genes investigated. Except for lexA, detected as a recent duplication, most of the genes in more than one copy are represented by two highly divergent orthologs. Basically, one of such duplications is frequently positioned close to other gamma proteobacteria, but the second is often positioned close to unrelated bacteria. These orthologs may have occurred from old duplication events, followed by extensive gene loss, or were originated from lateral gene transfer (LGT), as is the case of the uvrD homolog. Conclusions Duplications of DNA repair related genes may result in redundancy and also improve the organisms' responses to environmental challenges. Most of such duplications, in Xanthomonas, seem to have arisen from old events and possibly enlarge both functional and evolutionary genome potentiality.

Polyphyly of the hawk genera Leucopternis and Buteogallus (Aves, Accipitridae): multiple habitat shifts during the Neotropical buteonine diversification

Abstract Background The family Accipitridae (hawks, eagles and Old World vultures) represents a large radiation of predatory birds with an almost global distribution, although most species of this family occur in the Neotropics. Despite great morphological and ecological diversity, the evolutionary relationships in the family have been poorly explored at all taxonomic levels. Using sequences from four mitochondrial genes (12S, ATP8, ATP6, and ND6), we reconstructed the phylogeny of the Neotropical forest hawk genus Leucopternis and most of the allied genera of Neotropical buteonines. Our goals were to infer the evolutionary relationships among species of Leucopternis, estimate their relationships to other buteonine genera, evaluate the phylogenetic significance of the white and black plumage patterns common to most Leucopternis species, and assess general patterns of diversification of the group with respect to species' affiliations with Neotropical regions and habitats. Results Our molecular phylogeny for the genus Leucopternis and its allies disagrees sharply with traditional taxonomic arrangements for the group, and we present new hypotheses of relationships for a number of species. The mtDNA phylogenetic trees derived from analysis of the combined data posit a polyphyletic relationship among species of Leucopternis, Buteogallus and Buteo. Three highly supported clades containing Leucopternis species were recovered in our phylogenetic reconstructions. The first clade consisted of the sister pairs L. lacernulatus and Buteogallus meridionalis, and Buteogallus urubitinga and Harpyhaliaetus coronatus, in addition to L. schistaceus and L. plumbeus. The second clade included the sister pair Leucopternis albicollis and L. occidentalis as well as L. polionotus. The third lineage comprised the sister pair L. melanops and L. kuhli, in addition to L. semiplumbeus and Buteo buteo. According to our results, the white and black plumage patterns have evolved at least twice in the group. Furthermore, species found to the east and west of the Andes (cis-Andean and trans-Andean, respectively) are not reciprocally monophyletic, nor are forest and non-forest species. Conclusion The polyphyly of Leucopternis, Buteogallus and Buteo establishes a lack of concordance of current Accipitridae taxonomy with the mtDNA phylogeny for the group, and points to the need for further phylogenetic analysis at all taxonomic levels in the family as also suggested by other recent analyses. Habitat shifts, as well as cis- and trans-Andean disjunctions, took place more than once during buteonine diversification in the Neotropical region. Overemphasis of the black and white plumage patterns has led to questionable conclusions regarding the relationships of Leucopternis species, and suggests more generally that plumage characters should be used with considerable caution in the taxonomic evaluation of the Accipitridae.

SmTRC1, a novel Schistosoma mansoni DNA transposon, discloses new families of animal and fungi transposons belonging to the CACTA superfamily

Abstract Background The CACTA (also called En/Spm) superfamily of DNA-only transposons contain the core sequence CACTA in their Terminal Inverted Repeats (TIRs) and so far have only been described in plants. Large transcriptome and genome sequence data have recently become publicly available for Schistosoma mansoni, a digenetic blood fluke that is a major causative agent of schistosomiasis in humans, and have provided a comprehensive repository for the discovery of novel genes and repetitive elements. Despite the extensive description of retroelements in S. mansoni, just a single DNA-only transposon belonging to the Merlin family has so far been reported in this organism. Results We describe a novel S. mansoni transposon named SmTRC1, for S. mansoni Transposon Related to CACTA 1, an element that shares several characteristics with plant CACTA transposons. Southern blotting indicates approximately 30–300 copies of SmTRC1 in the S. mansoni genome. Using genomic PCR followed by cloning and sequencing, we amplified and characterized a full-length and a truncated copy of this element. RT-PCR using S. mansoni mRNA followed by cloning and sequencing revealed several alternatively spliced transcripts of this transposon, resulting in distinct ORFs coding for different proteins. Interestingly, a survey of complete genomes from animals and fungi revealed several other novel TRC elements, indicating new families of DNA transposons belonging to the CACTA superfamily that have not previously been reported in these kingdoms. The first three bases in the S. mansoni TIR are CCC and they are identical to those in the TIRs of the insects Aedes aegypti and Tribolium castaneum, suggesting that animal TRCs may display a CCC core sequence. Conclusion The DNA-only transposable element SmTRC1 from S. mansoni exhibits various characteristics, such as generation of multiple alternatively-spliced transcripts, the presence of terminal inverted repeats at the extremities of the elements flanked by direct repeats and the presence of a Transposase_21 domain, that suggest a distant relationship to CACTA transposons from Magnoliophyta. Several sequences from other Metazoa and Fungi code for proteins similar to those encoded by SmTRC1, suggesting that such elements have a common ancestry, and indicating inheritance through vertical transmission before separation of the Eumetazoa, Fungi and Plants.

When size makes a difference: allometry, life-history and morphological evolution of capuchins (Cebus) and squirrels (Saimiri) monkeys (Cebinae, Platyrrhini)

Abstract Background How are morphological evolution and developmental changes related? This rather old and intriguing question had a substantial boost after the 70s within the framework of heterochrony (changes in rates or timing of development) and nowadays has the potential to make another major leap forward through the combination of approaches: molecular biology, developmental experimentation, comparative systematic studies, geometric morphometrics and quantitative genetics. Here I take an integrated approach combining life-history comparative analyses, classical and geometric morphometrics applied to ontogenetic series to understand changes in size and shape which happen during the evolution of two New World Monkeys (NWM) sister genera. Results Cebus and Saimiri share the same basic allometric patterns in skull traits, a result robust to sexual and ontogenetic variation. If adults of both genera are compared in the same scale (discounting size differences) most differences are small and not statistically significant. These results are consistent using both approaches, classical and geometric Morphometrics. Cebus is a genus characterized by a number of peramorphic traits (adult-like) while Saimiri is a genus with paedomorphic (child like) traits. Yet, the whole clade Cebinae is characterized by a unique combination of very high pre-natal growth rates and relatively slow post-natal growth rates when compared to the rest of the NWM. Morphologically Cebinae can be considered paedomorphic in relation to the other NWM. Geometric morphometrics allows the precise separation of absolute size, shape variation associated with size (allometry), and shape variation non-associated with size. Interestingly, and despite the fact that they were extracted as independent factors (principal components), evolutionary allometry (those differences in allometric shape associated with intergeneric differences) and ontogenetic allometry (differences in allometric shape associated with ontogenetic variation within genus) are correlated within these two genera. Furthermore, morphological differences produced along these two axes are quite similar. Cebus and Saimiri are aligned along the same evolutionary allometry and have parallel ontogenetic allometry trajectories. Conclusion The evolution of these two Platyrrhini monkeys is basically due to a size differentiation (and consequently to shape changes associated with size). Many life-history changes are correlated or may be the causal agents in such evolution, such as delayed on-set of reproduction in Cebus and larger neonates in Saimiri.

Radiation of the Tnt1 retrotransposon superfamily in three Solanaceae genera

Abstract Background Tnt1 was the first active plant retrotransposon identified in tobacco after nitrate reductase gene disruption. The Tnt1 superfamily comprises elements from Nicotiana (Tnt1 and Tto1) and Lycopersicon (Retrolyc1 and Tlc1) species. The study presented here was conducted to characterise Tnt1-related sequences in 20 wild species of Solanum and five cultivars of Solanum tuberosum. Results Tnt1-related sequences were amplified from total genomic DNA using a PCR-based approach. Purified fragments were cloned and sequenced, and clustering analysis revealed three groups that differ in their U3 region. Using a network approach with a total of 453 non-redundant sequences isolated from Solanum (197), Nicotiana (140) and Lycopersicon (116) species, it is demonstrated that the Tnt1 superfamily can be treated as a population to resolve previous phylogenetic multifurcations. The resulting RNAseH network revealed that sequences group according to the Solanaceae genus, supporting a strong association with the host genome, whereas tracing the U3 region sequence association characterises the modular evolutionary pattern within the Tnt1 superfamily. Within each genus, and irrespective of species, nearly 20% of Tnt1 sequences analysed are identical, indicative of being part of an active copy. The network approach enabled the identification of putative "master" sequences and provided evidence that within a genus these master sequences are associated with distinct U3 regions. Conclusion The results presented here support the hypothesis that the Tnt1 superfamily was present early in the evolution of Solanaceae. The evidence also suggests that the RNAseH region of Tnt1 became fixed at the host genus level whereas, within each genus, propagation was ensured by the diversification of the U3 region. Different selection pressures seemed to have acted on the U3 and RNAseH modules of ancestral Tnt1 elements, probably due to the distinct functions of these regions in the retrotransposon life cycle, resulting in both co evolution and adaptation of the element population with its host.

Multiple invasions of Gypsy and Micropia retroelements in genus Zaprionus and melanogaster subgroup of the genus Drosophila

Abstract Background The Zaprionus genus shares evolutionary features with the melanogaster subgroup, such as space and time of origin. Although little information about the transposable element content in the Zaprionus genus had been accumulated, some of their elements appear to be more closely related with those of the melanogaster subgroup, indicating that these two groups of species were involved in horizontal transfer events during their evolution. Among these elements, the Gypsy and the Micropia retroelements were chosen for screening in seven species of the two Zaprionus subgenera, Anaprionus and Zaprionus. Results Screening allowed the identification of diverse Gypsy and Micropia retroelements only in species of the Zaprionus subgenus, showing that they are transcriptionally active in the sampled species. The sequences of each retroelement were closely related to those of the melanogaster species subgroup, and the most parsimonious hypothesis would be that 15 horizontal transfer events shaped their evolution. The Gypsy retroelement of the melanogaster subgroup probably invaded the Zaprionus genomes about 11 MYA. In contrast, the Micropia retroelement may have been introduced into the Zaprionus subgenus and the melanogaster subgroup from an unknown donor more recently (~3 MYA). Conclusion Gypsy and Micropia of Zaprionus and melanogaster species share similar evolutionary patterns. The sharing of evolutionary, ecological and ethological features probably allowed these species to pass through a permissive period of transposable element invasion, explaining the proposed waves of horizontal transfers.

Endosymbiosis in trypanosomatids: the genomic cooperation between bacterium and host in the synthesis of essential amino acids is heavily influenced by multiple horizontal gene transfers

Background Trypanosomatids of the genera Angomonas and Strigomonas live in a mutualistic association characterized by extensive metabolic cooperation with obligate endosymbiotic Betaproteobacteria. However, the role played by the symbiont has been more guessed by indirect means than evidenced. Symbiont-harboring trypanosomatids, in contrast to their counterparts lacking symbionts, exhibit lower nutritional requirements and are autotrophic for essential amino acids. To evidence the symbiont’s contributions to this autotrophy, entire genomes of symbionts and trypanosomatids with and without symbionts were sequenced here. Results Analyses of the essential amino acid pathways revealed that most biosynthetic routes are in the symbiont genome. By contrast, the host trypanosomatid genome contains fewer genes, about half of which originated from different bacterial groups, perhaps only one of which (ornithine cyclodeaminase, EC:4.3.1.12) derived from the symbiont. Nutritional, enzymatic, and genomic data were jointly analyzed to construct an integrated view of essential amino acid metabolism in symbiont-harboring trypanosomatids. This comprehensive analysis showed perfect concordance among all these data, and revealed that the symbiont contains genes for enzymes that complete essential biosynthetic routes for the host amino acid production, thus explaining the low requirement for these elements in symbiont-harboring trypanosomatids. Phylogenetic analyses show that the cooperation between symbionts and their hosts is complemented by multiple horizontal gene transfers, from bacterial lineages to trypanosomatids, that occurred several times in the course of their evolution. Transfers occur preferentially in parts of the pathways that are missing from other eukaryotes. Conclusion We have herein uncovered the genetic and evolutionary bases of essential amino acid biosynthesis in several trypanosomatids with and without endosymbionts, explaining and complementing decades of experimental results. We uncovered the remarkable plasticity in essential amino acid biosynthesis pathway evolution in these protozoans, demonstrating heavy influence of horizontal gene transfer events, from Bacteria to trypanosomatid nuclei, in the evolution of these pathways.

Convergent evolution of [D-Leucine1] microcystin-LR in taxonomically disparate cyanobacteria

Abstract Background Many important toxins and antibiotics are produced by non-ribosomal biosynthetic pathways. Microcystins are a chemically diverse family of potent peptide toxins and the end-products of a hybrid NRPS and PKS secondary metabolic pathway. They are produced by a variety of cyanobacteria and are responsible for the poisoning of humans as well as the deaths of wild and domestic animals around the world. The chemical diversity of the microcystin family is attributed to a number of genetic events that have resulted in the diversification of the pathway for microcystin assembly. Results Here, we show that independent evolutionary events affecting the substrate specificity of the microcystin biosynthetic pathway have resulted in convergence on a rare [D-Leu1] microcystin-LR chemical variant. We detected this rare microcystin variant from strains of the distantly related genera Microcystis, Nostoc, and Phormidium. Phylogenetic analysis performed using sequences of the catalytic domains within the mcy gene cluster demonstrated a clear recombination pattern in the adenylation domain phylogenetic tree. We found evidence for conversion of the gene encoding the McyA2 adenylation domain in strains of the genera Nostoc and Phormidium. However, point mutations affecting the substrate-binding sequence motifs of the McyA2 adenylation domain were associated with the change in substrate specificity in two strains of Microcystis. In addition to the main [D-Leu1] microcystin-LR variant, these two strains produced a new microcystin that was identified as [Met1] microcystin-LR. Conclusions Phylogenetic analysis demonstrated that both point mutations and gene conversion result in functional mcy gene clusters that produce the same rare [D-Leu1] variant of microcystin in strains of the genera Microcystis, Nostoc, and Phormidium. Engineering pathways to produce recombinant non-ribosomal peptides could provide new natural products or increase the activity of known compounds. Our results suggest that the replacement of entire adenylation domains could be a more successful strategy to obtain higher specificity in the modification of the non-ribosomal peptides than point mutations.

Phylogeny and population dynamics in European Reticulitermes and Kalotermes genera (Insecta, Isoptera)

The aim of this thesis is to detect the phylogeny and the population dynamics of the European termites of the genera Reticulitermes and Kalotermes, by the use of different mitochondrial (16S, COI/tRNA/COII, CR) and nuclear (microsatellites and Inter-SINE) molecular markers. In the phylogenetic analyses, the obtained results have well defined the cladogenetic events that generated the nowadays species biodiversity of the genus Reticulitermes, while the analysis of the Kalotermes flavicollis taxon showed the presence of at least four genetic clades, defined on the basis of the geographical distance. The second part of the thesis is centred on the population dynamics of two species: Reticulitermes urbis and Kalotermes flavicollis. The first species, native of the Balkans, is known to be present in some cities of Italy and France. I’ve analyzed the colony genetic structure of the introduced population of Bagnacavallo (RA, Italy), using nine microsatellite loci. The obtained results are in accordance with those obtained from another population in France: this species in fact confirms its invasive and infestation capacities. The analysis of the natural population of K. flavicollis, performed with a combination of mitochondrial (control region) and nuclear (microsatellites and I-SINE) markers, clearly evidenced the presence of two genetic lineages that coexist in the same area. Moreover, results clearly indicate that the cross-breeding is allowed. Finally, the whole results are discussed in a comparative view to better understand the differences in ecology, evolutionary dynamics and colony social structure between these two genera.

The impact of intraclonal heterogeneity on the outcomes of Multiple Myeloma patients treated with new drugs

Understanding the biology of Multiple Myeloma (MM) is of primary importance in the struggle to achieve a cure for this yet incurable neoplasm. A better knowledge of the mechanism underlying the development of MM can guide us in the development of new treatment strategies. Studies both on solid and haematological tumours have shown that cancer comprises a collection of related but subtly different clones, a feature that has been termed “intra-clonal heterogeneity”. This intra-clonal heterogeneity is likely, from a “Darwinian” natural selection perspective, to be the essential substrate for cancer evolution, disease progression and relapse. In this context the critical mechanism for tumour progression is competition between individual clones (and cancer stem cells) for the same microenvironmental “niche”, combined with the process of adaptation and natural selection. The Darwinian behavioural characteristics of cancer stem cells are applicable to MM. The knowledge that intra-clonal heterogeneity is an important feature of tumours’ biology has changed our way to addressing cancer, now considered as a composite mixture of clones and not as a linear evolving disease. In this variable therapeutic landscape it is important for clinicians and researchers to consider the impact that evolutionary biology and intra-clonal heterogeneity have on the treatment of myeloma and the emergence of treatment resistance. It is clear that if we want to effectively cure myeloma it is of primarily importance to understand disease biology and evolution. Only by doing so will we be able to effectively use all of the new tools we have at our disposal to cure myeloma and to use treatment in the most effective way possible. The aim of the present research project was to investigate at different levels the presence of intra-clonal heterogeneity in MM patients, and to evaluate the impact of treatment on clonal evolution and on patients’ outcomes.