Molecular markers and modern phylogenetics of mammals

Application of modern molecular techniques such as molecular cloning, sequences and polymerase chain reaction of DNA resulted in the increasing of resolution of the phylogenetic analysis and enhanced the role of molecular markers in the evolutionary and taxonomic studies. However, certain properties of the molecular markers are to be taken into consideration when results of the molecular phylogenetic analyses are discussed. This survey reviews the advantages and shortages of different molecular markers (mtDNA and nDNA genes, satellite sequences, long and short retroposons) at the various taxonomic levels. The most part of new phylogenetic reconstructions are established on the results of mtDNA analysis and must be interpreted cautiously because of non-mendelian inheritance of mitochondrial genome. The extremely rapid rate of nucleotide change in mtDNA as compared with nDNA reinforces the saturation in nucleotide sequence and screens the phylogenetic signal. The analysis of nuclear genomes in constrained by that only truly orthologous genes are suitable for the phylogeny reconstruction. So there is a problem to distinguish genes from pseudogenes. Besides, there are some general problems of gene reconstruction such as nucleotide and amino acid compositional shift, long branch attraction and the choice of outgroup. Short interspersed nuclear elements (SINEs) may provide the most valuable phylogenetic information. The markers of multilocus DNA analysis (RAPD-PCR, IS-PCR, RELP, ISSR-PCR), their advantages and shortages are also discussed. A brief survey of the recent studies of molecular phylogeny of mammals for the period of about ten years is presented. The results based on the combined analysis of the mitochondrial and nuclear genes reject the reliability of some previously recognized supraordinal Eutherian taxa in favour of independent range of four main super-order clades: Afrotheria, Xenarthra, Euarchontoglires, and Laurasiatheria. Within these clades, monophyly of some of traditionally recognized orders was proved by molecular data. The recent advances of molecular phylogenetics are very encouraging. However, its future developments are full off serious difficulties. The problem of accumulation of the data turns into the problem of their correct analysis that is more difficult from the methodological point of view. The careful analysis of the conformities and contradictions between different data sets and looking for congruent conclusions deduced from different characters are the most fruitful way of further phylogenetic development.     

Molecular phylogenetics of DNA 5mC-methyltransferases.

We have identified a total of 88 members of the DNA-(cytosine-5) methyltransferase (5mC MTase) family whose sequences have been deposited in the databases. The results of a comparison of these sequences is presented in the form of an alignment-based phylogenetic tree and sequence logos for 10 conserved motifs. Phylogenetic analysis showed that members of the family aggregate into subfamilies which are usually consistent with their target specificity. However, it was also shown that similar target specificity does not necessarily imply close homology of the catalytic domain of MTases, which strongly supports the hypothesis that target recognition evolved independently of catalytic properties. This analysis also indicate that the 5mC MTase was present in the cenancestor (last common ancestor) of eubacteria, archaebacteria, and eukaryotes. The phylogeny of the 5mC MTases catalytic domain provides the basis for establishing the patterns of evolutionary change that characterize this family of proteins with conserved structural core and variable and mobile modules not directly involved in formation of the active site.     

Molecular genetic analysis of the polymorphic apolipoprotein E in modern and ancient human DNA samples

In this report, methodical bases for the molecular genetic analysis of the three common apolipoprotein E alleles APOE*2, APOE*3 and APOE*4 in DNA isolated from ancient human skeletal remains are described. Considering that ancient DNA target regions for amplification are generally quite small, the detection method is based on short amplification products in the range from 71 bp to 75 bp. The applicability of the modified method for APOE genotyping was examined in modern human DNA samples.     

Molecular phylogenetics unveils the ancient evolutionary origins of the enigmatic fairy armadillos

Fairy armadillos or pichiciegos (Xenarthra, Dasypodidae) are among the most elusive mammals. Due to their subterranean and nocturnal lifestyle, their basic biology and evolutionary history remain virtually unknown. Two distinct species with allopatric distributions are recognized: Chlamyphorus truncatus is restricted to central Argentina, while Calyptophractus retusus occurs in the Gran Chaco of Argentina, Paraguay, and Bolivia. To test their monophyly and resolve their phylogenetic affinities within armadillos, we obtained sequence data from modern and museum specimens for two mitochondrial genes (12S RNA [MT-RNR1] and NADH dehydrogenase 1 [MT-ND1]) and two nuclear exons (breast cancer 1 early onset exon 11 [BRCA1] and von Willebrand factor exon 28 [VWF]). Phylogenetic analyses provided a reference phylogeny and timescale for living xenarthran genera. Our results reveal monophyletic pichiciegos as members of a major armadillo subfamily (Chlamyphorinae). Their strictly fossorial lifestyle probably evolved as a response to the Oligocene aridification that occurred in South America after their divergence from Tolypeutinae around 32 million years ago (Mya). The ancient divergence date (～17Mya) for separation between the two species supports their taxonomic classification into distinct genera. The synchronicity with Middle Miocene marine incursions along the Paraná river basin suggests a vicariant origin for pichiciegos by the disruption of their ancestral range. Their phylogenetic distinctiveness and rarity in the wild argue in favor of high conservation priority.     

Improving ancient DNA read mapping against modern reference genomes

ABSTRACT: BACKGROUND: Next-Generation Sequencing has revolutionized our approach to ancient DNA (aDNA) research, by providing complete genomic sequences of ancient individuals and extinct species. However, the recovery of genetic material from long-dead organisms is still complicated by a number of issues, including post-mortem DNA damage and high levels of environmental contamination. Together with error profiles specific to the type of sequencing platforms used, these specificities could limit our ability to map sequencing reads against modern reference genomes and therefore limit our ability to identify endogenous ancient reads, reducing the efficiency of shotgun sequencing aDNA. RESULTS: In this study, we compare different computational methods for improving the accuracy and sensitivity of aDNA sequence identification, based on shotgun sequencing reads recovered from Pleistocene horse extracts using Illumina GAIIx and Helicos Heliscope platforms. We show that the performance of the Burrows Wheeler Aligner (BWA), that has been developed for mapping of undamaged sequencing reads using platforms with low rates of indel-types of sequencing errors, can be employed at acceptable run-times by modifying default parameters in a platform-specific manner. We also examine if trimming likely damaged positions at read ends can increase the recovery of genuine aDNA fragments and if accurate identification of human contamination can be achieved using a strategy previously suggested based on best hit filtering. We show that combining our different mapping and filtering approaches can increase the number of high-quality endogenous hits recovered by up to 33%. CONCLUSIONS: We have shown that Illumina and Helicos sequences recovered from aDNA extracts could not be aligned to modern reference genomes with the same efficiency unless mapping parameters are optimized for the specific types of errors generated by these platforms and by post-mortem DNA damage. Our findings have important implications for future aDNA research, as we define mapping guidelines that improve our ability to identify genuine aDNA sequences, which in turn could improve the genotyping accuracy of ancient specimens. Our framework provides a significant improvement to the standard procedures used for characterizing ancient genomes, which is challenged by contamination and often low amounts of DNA material.     

Pathocoenoses ancient and modern

Problems concerning the concept of biocoenosis in ecology (the antecedent of the pathocoenosis concept) are discussed first of all. Six main problems are identified: the problem of emergent properties of ecological communities; the problem of ambiguity; the problem of heterogeneity; the boundary problem; the problem of retrospective differential diagnosis; and the problem of explaining change over time. The rest of the paper gives illustrations of these problems in relation to human pathogens drawn mainly from the interactions of malaria with other diseases, particularly but not exclusively in the Mediterranean world, from antiquity through to modern times.     

Molecular phylogenetics of Thysanoptera

Analyses of morphological data on the higher-level phylogenetics of Thysanoptera have suggested two alternative hypotheses: (1) sister-taxon status of the monophyletic suborders Terebrantia and Tubulifera. and (2) paraphyly of Terebrantia with respect to Tubulifera. Analyses of nucleotide sequence data from the mitochondrial gene cytochrome oxidase I and the nuclear gene I8S rONA using maximum parsimony. neighbour joining. and maximum likelihood provide strong support for the hypothesis of a sister-taxon relationship between the Terebrantia and Tubulifera. These data resolve this long-standing controversy in thysanopteran phylogenetics and provide a framework for further studies of higher-level relationships in this order.     

Molecular phylogenetics: principles and practice

Phylogenies are important for addressing various biological questions such as relationships among species or genes, the origin and spread of viral infection and the demographic changes and migration patterns of species. The advancement of sequencing technologies has taken phylogenetic analysis to a new height. Phylogenies have permeated nearly every branch of biology, and the plethora of phylogenetic methods and software packages that are now available may seem daunting to an experimental biologist. Here, we review the major methods of phylogenetic analysis, including parsimony, distance, likelihood and Bayesian methods. We discuss their strengths and weaknesses and provide guidance for their use.     

Molecular phylogenetics of the exoneurine allodapine bees reveal an ancient and puzzling dispersal from Africa to Australia

Previous phylogenetic studies of the bee tribe Allodapini suggested a puzzling biogeographic problem: one of the key basal divergences involved separation of the southern African and southern Australian clades at a very early stage in allodapine evolution, but no taxa occur in the Palaearctic or Asian regions that might suggest a Laurasian dispersal route. However, these studies lacked sufficient sequence data and appropriate maximum likelihood partition models to provide reliable phylogenetic estimates and enable alternative biogeographic hypotheses to be distinguished. Using Bayesian and penalized likelihood approaches and an expanded sequence and taxon set we examine phylogenetic relationships between the Australian, African, and Malagasy groups and estimate divergence times for key nodes. We show that divergence of the three basal Australian clades (known as the exoneurines) occurred at least 25 Mya following a single colonization event, and that this group diverged from the African + Madagascan clade at least 30 Mya, but actual divergence dates are likely to be much older than these very conservative limits. The bifurcation order of the exoneurine clades was not resolved and analyses could not rule out the existence of a hard polytomy, suggesting rapid radiation after colonization of Australia. Their divergence involved major transitions in life history traits and these placed constraints on the kinds of social organization that subsequently evolved in each lineage. Early divergence between the African, Malagasy, and Australian clades presents a major puzzle for historical biogeography: node ages are too recent for Gondwanan vicariance hypotheses, but too early for Laurasian dispersal scenarios. We suggest a scenario involving island hopping across the Indian Ocean via a series of now largely submerged elements of the Kergulen Plateau and Broken Ridge provinces, both of which are known to have had subaerial formations during the Cenozoic. [Bayesian; biogeography; dispersal; Gondwana; Kerguelen Plateau; penalized likelihood.].     

Molecular phylogenetics of Candida albicans

We analyzed data on multilocus sequence typing (MLST), ABC typing, mating type-like locus (MAT) status, and antifungal susceptibility for a panel of 1,391 Candida albicans isolates. Almost all (96.7%) of the isolates could be assigned by MLST to one of 17 clades. eBURST analysis revealed 53 clonal clusters. Diploid sequence type 69 was the most common MLST strain type and the founder of the largest clonal cluster, and examples were found among isolates from all parts of the world. ABC types and geographical origins showed statistically significant variations among clades by univariate analysis of variance, but anatomical source and antifungal susceptibility data were not significantly associated. A separate analysis limited to European isolates, thereby minimizing geographical effects, showed significant differences in the proportions of isolates from blood, commensal carriage, and superficial infections among the five most populous clades. The proportion of isolates with low antifungal susceptibility was highest for MAT homozygous a/a types and then alpha/alpha types and was lowest for heterozygous a/alpha types. The tree of clades defined by MLST was not congruent with trees generated from the individual gene fragments sequenced, implying a separate evolutionary history for each fragment. Analysis of nucleic acid variation among loci and within loci supported recombination. Computational haplotype analysis showed a high frequency of recombination events, suggesting that isolates had mixed evolutionary histories resembling those of a sexually reproducing species.     

Nuclear DNA and salmonid phylogenetics

There are many unresolved problems in salmonid systematics, both at the interspecific and sub-specific levels. Some of the major systematic problems in the subfamily Salmoninae are briefly reviewed along with the available molecular methods for their analysis. Nuclear DNA markers available for use in molecular systematics include localized and dispersed highly repetitive DNA sequences, moderately repetitive sequences such as the ribosomal RNA genes (rDNA), and single copy DNA sequences. Both coding and non-coding sequences can be examined in the rDNA and single copy DNA. The rDNA is especially suitable for use in phylogenetic analysis, since different regions evolve at different rates and can be used for comparisons at different taxonomic levels. Comparison of restriction maps of the entire rDNA repeating unit in 17 salmonid species from Hucho. Sahelinus, Salmo and Oncorhynchus has shown that the transcribed spacer regions are the most informative for interspecific comparisons and that the intergenic spacer has potential for use in intraspecific comparisons. Our current approach is to amplify selected regions from each of these spacers for analysis by DNA sequencing. DNA sequence analysis of the internal transcribed spacers should be very informative in elucidating interspecific relationships in Salvelinus and Oncorhynchus . Analysis of a hypervariable region in the intergenic spacer has potential for identification of geographically separated stocks. The relative utility of different types of nuclear DNA sequences for identification of stocks and subspecies is examined.     

Molecular phylogenetics and evolution of turtles

Turtles are one of Earth's most instantly recognizable life forms, distinguished for over 200 million years in the fossil record. Even so, key nodes in the phylogeny of turtles remain uncertain. To address this issue, we sequenced >90% of the nuclear recombination activase gene 1 (RAG-1) for 24 species representing all modern turtle families. RAG-1 exhibited negligible saturation and base composition bias, and extensive base composition homogeneity. Most of the relationships suggested by prior phylogenetic analyses were also supported by RAG-1 and, for at least two critical nodes, with a much higher level of support. RAG-1 also indicates that the enigmatic Platysternidae and Chelydridae, often considered sister taxa based on morphological evidence, are not closely related, although their precise phylogenetic placement in the turtle tree is still unresolved. Although RAG-1 is phylogenetically informative, our research revealed fundamental conflicts among analytical methods for estimating phylogenetic hypotheses. Maximum parsimony analyses of RAG-1 alone and in combination with two mitochondrial genes suggest the earliest phylogenetic splits separating into three basal branches, the pig-nosed turtles (Carettochelyidae), the softshell turtles (Trionychidae), and a clade comprising all remaining extant turtles. Maximum likelihood and Bayesian analyses group Carettochelyidae and Trionychidae (=Trionychoidae) in their more traditional location as the sister taxon to all other hidden-necked turtles, collectively forming the Cryptodira. Our research highlights the utility of molecular data in identifying issues of character homology in morphological datasets, while shedding valuable light on the biodiversity of a globally imperiled taxon.     

Molecular phylogenetics of Hypoxidaceae--evidence from plastid DNA data and inferences on morphology and biogeography

Phylogenetic relationships of the monocot family Hypoxidaceae (Asparagales), which occurs mainly in the Southern Hemisphere, were reconstructed using four plastid DNA regions (rbcL, trnL intron, trnL-F intergenic spacer, and trnS-G intergenic spacer) for 56 ingroup taxa including all currently accepted genera and seven species of the closely related families Asteliaceae, Blandfordiaceae, and Lanariaceae. Data were analyzed by applying parsimony, maximum likelihood and Bayesian methods. The intergenic spacer trnS-G--only rarely used in monocot research--contributed a substantial number of potentially parsimony informative characters. Hypoxidaceae consist of three well-supported major clades, but their interrelationships remain unresolved. Our data indicate that in the Pauridia clade one long-distance dispersal event occurred from southern Africa to Australia. Long-distance dispersal scenarios may also be likely for the current distribution of Hypoxis, which occurs on four continents. In the Curculigo clade, the present distribution of Curculigo s.s. on four continents could support a Gondwanan origin, but the level of divergence is too low for this hypothesis to be likely. The main clades correspond well with some floral characters, habit and palynological data, whereas chromosomal data exhibit plasticity and probably result from polyploidization and subsequent dysploidy and/or aneuploidy. Evolutionary flexibility is also suggested by the number of reported pollination syndromes: melittophily, myophily, sapromyophily, and cantharophily. Based on our phylogenetic results, we suggest cautious nomenclatural reorganization to generate monophyly at the generic level.     

Molecular breeding of polymerases for amplification of ancient DNA

In the absence of repair, lesions accumulate in DNA. Thus, DNA persisting in specimens of paleontological, archaeological or forensic interest is inevitably damaged. We describe a strategy for the recovery of genetic information from damaged DNA. By molecular breeding of polymerase genes from the genus Thermus (Taq (Thermus aquaticus), Tth (Thermus thermophilus) and Tfl (Thermus flavus)) and compartmentalized self-replication selection, we have evolved polymerases that can extend single, double and even quadruple mismatches, process non-canonical primer-template duplexes and bypass lesions found in ancient DNA, such as hydantoins and abasic sites. Applied to the PCR amplification of 47,000-60,000-year-old cave bear DNA, these outperformed Taq DNA polymerase by up to 150% and yielded amplification products at sample dilutions at which Taq did not. Our results demonstrate that engineered polymerases can expand the recovery of genetic information from Pleistocene specimens and may benefit genetic analysis in paleontology, archeology and forensic medicine.     

Molecular phylogenetics of representative Paramecium species

The genus Paramecium has been known to science for 250 years and contains some of the most widely studied species of ciliates. At present, the basic research object for phylogenetic studies is the genome of various paramecia. One of the most widely used markers are genes coding for various rRNA's. Comparative analyses of sequences coding rRNA were applied for resolving the systematic position of some paramecia species and also for the establishment of an accurate taxonomy of Paramecium. Paramecia were also model organisms for their systematic group in more general studies in a comparative analysis among ciliates, fungi, plants and multicellular animals, illustrating the evolutionary relationships between Archaebacteria and Eucaryota. A new, revolutionary genealogy proposed the shifting of presumptively advanced groups towards more primitive ones, and traditionally primitive forms were located closer to highly specialized taxa, but rRNA analysis did not unambiguously resolve associations within the studied groups. Because of the aforementioned concerns, the number of molecular markers used for alternative studies is growing, such as genes coding proteins from the Hsp family or histone proteins. Other promising candidate markers may be hemoglobin genes or genes coding á-tubulins. In case of comparative analyses ofnucleotide sequences, the outcome of the research usually depends upon a subjective choice of DNA. One of the directions of research in molecular phylogenetics include indirect methods that allow for an estimation of entire genomes, for example RAPD-PCR-fingerprinting.     

Molecular phylogenetics and biogeography of Lepus in Eastern Asia based on mitochondrial DNA sequences

In spite of several classification attempts among taxa of the genus Lepus, phylogenetic relationships still remain poorly understood. Here, we present molecular genetic evidence that may resolve some of the current incongruities in the phylogeny of the leporids. The complete mitochondrial cytb, 12S genes, and parts of ND4 and control region fragments were sequenced to examine phylogenetic relationships among Chinese hare taxa and other leporids throughout the World using maximum parsimony, maximum likelihood, and Bayesian phylogenetic reconstruction approaches. Using reconstructed phylogenies, we observed that the Chinese hare is not a single monophyletic group as originally thought. Instead, the data infers that the genus Lepus is monophyletic with three unique species groups: North American, Eurasian, and African. Ancestral area analysis indicated that ancestral Lepus arose in North America and then dispersed into Eurasia via the Bering Land Bridge eventually extending to Africa. Brooks Parsimony analysis showed that dispersal events followed by subsequent speciation have occurred in other geographic areas as well and resulted in the rapid radiation and speciation of Lepus. A Bayesian relaxed molecular clock approach based on the continuous autocorrelation of evolutionary rates along branches estimated the divergence time between the three major groups within Lepus. The genus appears to have arisen approximately 10.76 MYA (+/-0.86 MYA), with most speciation events occurring during the Pliocene epoch (5.65+/-1.15 MYA approximately 1.12 +/- 0.47 MYA).     

Molecular phylogenetics of Meliaceae (Sapindales) based on nuclear and plastid DNA sequences

Phylogenetic analyses of Meliaceae, including representatives of all four currently recognized subfamilies and all but two tribes (32 genera and 35 species, respectively), were carried out using DNA sequence data from three regions: plastid genes rbcL, matK (partial), and nuclear 26S rDNA (partial). Individual and combined phylogenetic analyses were performed for the rbcL, matK, and 26S rDNA data sets. Although the percentage of informative characters is highest in the segment of matK sequenced, rbcL provides the greatest number of informative characters of the three regions, resulting in the best resolved trees. Results of parsimony analyses support the recognition of only two subfamilies (Melioideae and Swietenioideae), which are sister groups. Melieae are the only tribe recognized previously that are strongly supported as monophyletic. The members of the two small monogeneric subfamilies, Quivisianthe and Capuronianthus, fall within Melioideae and Swietenioideae, respectively, supporting their taxonomic inclusion in these groups. Furthermore, the data indicate a close relationship between Aglaieae and Guareeae and a possible monophyletic origin of Cedreleae of Swietenioideae. For Trichilieae (Melioideae) and Swietenieae (Swietenioideae) lack of monophyly is indicated.     

Classical catalase: ancient and modern

This review describes the historical difficulties in devising a kinetically satisfactory mechanism for the classical catalase after its identification as a unique catalytic entity in 1902 and prior to the breakthrough 1947 analysis by Chance and co-workers which led to the identification of peroxide compounds I and II. The role of protons in the formation of these two ferryl complexes is discussed and current problems of inhibitory ligand and hydrogen donor binding at the active site are outlined, especially the multiple roles involving formate or formic acid. A previous mechanism of NADPH-dependent catalase protection against substrate inhibition is defended. A revised model linking the catalytic ('catalatic') action and the one-electron side reactions involving compound II is suggested. And it is concluded that, contrary to an idea proposed in 1963 that eukaryotic catalase might be a 'fossil enzyme', current thinking gives it a central role in the redox protective processes of long term importance for human and other eukaryotic and prokaryotic life.     

Molecular phylogenetics of Stenodermatini bat genera: congruence of data from nuclear and mitochondrial DNA

Within the tribe Stenodermatini the systematics of the complex of species allied with the genus Artibeus has generated several alternative phylogenetic hypotheses. The most recent treatment recognized four genera (Artibeus, Dermanura, Enchisthenes, and Koopmania) and suggested that the most recent common ancestor of these four genera would include the common ancestor of all other currently recognized Stenodermatini genera except Sturnira. To test this hypothesis, we examined an EcoRI-defined nuclear satellite DNA repeat and 402 bp of DNA sequence variation from the mitochondrial cytochrome b gene. Phylogenetic conclusions based on Southern blot analyses, in situ hybridization, and mitochondrial DNA sequence data indicate that Enchisthenes is not closely related to Dermanura, Artibeus, or Koopmania and that Dermanura, Artibeus, and Koopmania shared a common ancestor after diverging from the remainder of the Stenodermatini. If our conclusions are correct, then justification for recognizing Dermanura and Koopmania as generically distinct from Artibeus must be based on the magnitude of difference that distinguishes each rather than on the conclusion that to place them as congeneric with Artibeus creates a paraphyletic taxon.