Construction and bootstrap analysis of DNA fingerprinting-based phylogenetic trees with a freeware program FreeTree: Applicati

SUMMARY The Win95/98/NT program " FreeTree " for computation of distance matrices and construction of phylogenetic or phenetic trees on the basis of random amplified polymorphic DNA (RAPD), restriction fragment length polymorphism (RFLP) and allozyme data is presented. In contrast to other similar software, the program FreeTree (available at topology by bootstrap, jackknife or OTU-jackknife analysis. Moreover, the program can be also used for an analysis of data obtained in several independent experiments performed with nonidentical subsets of taxa. The function of the program was demonstrated by an analysis of RAPD data from 42 strains of 10 species of trichomonads. On the phylogenetic tree constructed by the FreeTree the high bootstrap values and short terminal branches for Tritrichomonas foetus/suis 14-strains-branch suggested relatively recent and probably clonal radiation of this species. At the same time the relatively lower bootstrap values and long terminal branches for Trichomonas vaginalis 20-strains-branch suggested more ancient radiation of this species and possible existence of genetic recombination (sexual reproduction) in this human pathogen. The low bootstrap values and a star-like topology of the whole Trichomonadidae tree confirm that RAPD method is not suitable for phylogenetic analysis of protozoa on the level of higher taxa. We suggest that the repeated bootstrap analysis should be an obligatory part of any RAPD study. It makes possible to assess the reliability of the obtained tree and to adjust the amount of collected data (number of random primers) to the amount of phylogenetic signals in the RAPD data of the analysed taxon. The FreeTree program makes such analysis possible. 3 The advent of molecular taxonomy techniques offered a solution for many problems, which were out of reach of classical taxonomy methods and approaches. Currently, the methods of construction of phylogenetic trees on the basis of molecular data are widely used not only in systematic and comparative biology, but also in ecology, ethology, sociobiology and epidemiology. The methods of molecular taxonomy can be divided into two groups: single-locus methods and multi-locus methods. The result of single-locus methods such as DNA sequencing, microsatellite analysis and single-strand conformational polymorphism (SSCP) analysis is a so-called gene tree, the topology of which reflects the evolution of a particular gene. Very often, however, the aim of the analysis is to obtain a species tree, the phylogenetic tree of the taxa under the study or the genealogical tree of the individuals in the studied population. Under favourable conditions (long …