A Phylogenomic Approach to Vertebrate Phylogeny Supports a Turtle-Archosaur Affinity and a Possible Paraphyletic Lissamphibia

In resolving the vertebrate tree of life, two fundamental questions remain: 1) what is the phylogenetic position of turtles within amniotes, and 2) what are the relationships between the three major lissamphibian (extant amphibian) groups? These relationships have historically been difficult to resolve, with five different hypotheses proposed for turtle placement, and four proposed branching patterns within Lissamphibia. We compiled a large cDNA/EST dataset for vertebrates (75 genes for 129 taxa) to address these outstanding questions. Gene-specific phylogenetic analyses revealed a great deal of variation in preferred topology, resulting in topologically ambiguous conclusions from the combined dataset. Due to consistent preferences for the same divergent topologies across genes, we suspected systematic phylogenetic error as a cause of some variation. Accordingly, we developed and tested a novel statistical method that identifies sites that have a high probability of containing biased signal for a specific phylogenetic relationship. After removing putatively biased sites, support emerged for a sister relationship between turtles and either crocodilians or archosaurs, as well as for a caecilian-salamander sister relationship within Lissamphibia, with Lissamphibia potentially paraphyletic.     

Turtle origins: insights from phylogenetic retrofitting and molecular scaffolds

Adding new taxa to morphological phylogenetic analyses without substantially revising the set of included characters is a common practice, with drawbacks (undersampling of relevant characters) and potential benefits (character selection is not biased by preconceptions over the affinities of the ‘retrofitted’ taxon). Retrofitting turtles (Testudines) and other taxa to recent reptile phylogenies consistently places turtles with anapsid‐grade parareptiles (especially Eunotosaurus and/or pareiasauromorphs), under both Bayesian and parsimony analyses. This morphological evidence for turtle–parareptile affinities appears to contradict the robust genomic evidence that extant (living) turtles are nested within diapsids as sister to extant archosaurs (birds and crocodilians). However, the morphological data are almost equally consistent with a turtle–archosaur clade: enforcing this molecular scaffold onto the morphological data does not greatly increase tree length (parsimony) or reduce likelihood (Bayesian inference). Moreover, under certain analytic conditions, Eunotosaurus groups with turtles and thus also falls within the turtle–archosaur clade. This result raises the possibility that turtles could simultaneously be most closely related to a taxon traditionally considered a parareptile (Eunotosaurus) and still have archosaurs as their closest extant sister group.     

Reptile phylogeny and the interrelationships of turtles

A comprehensive analysis of amniote interrelationships is presented in an attempt to test turtle interrelationships. The results refute earlier hypotheses that turtles are related to parareptiles, i.e. to procolophonids or pareiasaurs. Instead, turtles are shown to be the sister-group of Sauropterygia, the two clades being nested within Sauria as sister-group of Lepidosauriformes. This scenario is also supported by several developmental and soft tissue characters which are shown to be congruent with the current phylogeny. The analysis strongly supports a monophyletic Parareptilia, sister-group of a monophylctic Eurcptilia. The Diapsida, however, is paraphyletic unless it includes turtles and sauropterygians. Additionally, the position of turtles within Diapsida has major implications for the evolutionary history and/or significance of many characters, i.e. temporal fenestration.     

Complete mitochondrial DNA sequences of the green turtle and blue-tailed mole skink: statistical evidence for archosaurian affinity of turtles.

Turtles have highly specialized morphological characteristics, and their phylogenetic position has been under intensive debate. Previous molecular studies have not established a consistent and statistically well supported conclusion on this issue. In order to address this, complete mitochondrial DNA sequences were determined for the green turtle and the blue-tailed mole skink. These genomes possess an organization of genes which is typical of most other vertebrates, such as placental mammals, a frog, and bony fishes, but distinct from organizations of alligators and snakes. Molecular evolutionary rates of mitochondrial protein sequences appear to vary considerably among major reptilian lineages, with relatively rapid rates for snake and crocodilian lineages but slow rates for turtle and lizard lineages. In spite of this rate heterogeneity, phylogenetic analyses using amino acid sequences of 12 mitochondrial proteins reliably established the Archosauria (birds and crocodilians) and Lepidosauria (lizards and snakes) clades postulated from previous morphological studies. The phylogenetic analyses further suggested that turtles are a sister group of the archosaurs, and this untraditional relationship was provided with strong statistical evidence by both the bootstrap and the Kishino-Hasegawa tests. This is the first statistically significant molecular phylogeny on the placement of turtles relative to the archosaurs and lepidosaurs. It is therefore likely that turtles originated from a Permian-Triassic archosauromorph ancestor with two pairs of temporal fenestrae behind the skull orbit that were subsequently lost. The traditional classification of turtles in the Anapsida may thus need to be reconsidered.     

Phylogenetic position of tetraodontiform fishes within the higher teleosts: Bayesian inferences based on 44 whole mitochondrial genome sequences

Tetraodontiformes includes approximately 350 species assigned to nine families, sharing several reduced morphological features of higher teleosts. The order has been accepted as a monophyletic group by many authors, although several alternative hypotheses exist regarding its phylogenetic position within the higher teleosts. To date, acanthuroids, zeiforms, and lophiiforms have been proposed as sister-groups of the tetraodontiforms. The monophyly and sister-group status was investigated using whole mitochondrial genome (mitogenome) sequences from 44 purposefully-chosen species (26 sequences newly-determined during the study) that fully represent the major tetraodontiform lineages plus all the groups that have been hypothesized as being close relatives. Partitioned Bayesian analyses were conducted with the three datasets that comprised concatenated nucleotide sequences from 13 protein-coding genes (with and without, or with RY-coding, 3rd codon positions), plus 22 transfer RNA and two ribosomal RNA genes. The resultant trees were well resolved and largely congruent, with most internal branches being supported by high posterior probabilities. Mitogenomic data strongly supported the monophyly of tetraodontiform fishes, placing them as a sister-group of either Lophiiformes plus Caproidei or Caproidei only. The sister-group relationship between Acanthuroidei and Tetraodontiformes was statistically rejected using Bayes factors. These results were confirmed by a reanalysis of the previously published nuclear RAG1 gene sequences using the Bayesian method. Within the Tetraodontiformes, however, monophylies of the three superfamilies were not recovered and further taxonomic sampling and subsequent efforts should clarify these relationships.     

Concordance analysis in mitogenomic phylogenetics

Here I advocate the utility of Bayesian concordance analysis as a mechanism for exploring the magnitude and source of phylogenetic signal in concatenated mitogenomic phylogenetic studies. While typically applied to the study of independently evolving gene trees, Bayesian concordance analysis can also be applied to linked, but individually analyzed, gene regions using a prior probability that reflects the expectation of similar phylogenetic reconstructions. For true branches in the mitogenomic tree, concordance factors should represent the number of gene regions that contain phylogenetic signal for a particular clade. As a demonstration of the application of Bayesian concordance analysis to empirical data, I analyzed two different salamander (Hynobiidae and Plethodontidae) mitogenomic data sets using a gene-based partitioning strategy. The results revealed many strongly supported clades in the concatenated trees that have high concordance factors, permitting the inference that these are robustly resolved through phylogenetic signal distributed across the mitogenome. In contrast, a number of strongly supported clades in the concatenated tree received low concordance factors, indicating that their reconstruction is either driven primarily by phylogenetic signal in a small number of gene regions, or that they are inconsistent reconstructions influenced by properties of the data that can produce inaccurate trees (e.g., compositional bias, selection, etc.). Exploration of the Bayesian joint posterior distribution of trees highlighted partitions that contribute phylogenetic information to similar clade reconstructions. This approach was particularly insightful in the hynobiid data, where different combinations of genes were identified that support alternative tree reconstructions. Concatenated analysis of these different subsets of genes highlighted through Bayesian concordance analysis produced strongly supported and contrasting trees, demonstrating the potential for inconsistency in concatenated mitogenomic phylogenetics. The overall results presented here suggest that Bayesian concordance analysis can serve as an effective exploration of the influence of different gene regions in mitogenomic (and other organellar genomic) phylogenetic studies.     

Sister group relationship of turtles to the bird-crocodilian clade revealed by nuclear DNA-coded proteins

The phylogenetic position of turtles is a currently controversial issue. Recent molecular studies rejected a traditional view that turtles are basal living reptiles (Hedges, S. B., and L. L. Poling. 1999. A molecular phylogeny. Science 83:998-1001; Kumazawa, Y., and M. Nishida. 1999. Complete mitochondrial DNA sequences of the green turtle and blue-tailed mole skink, statistical evidence for archosaurian affinity of turtles. Mol. Biol. Evol. 16:784-792). Instead, these studies grouped turtles with birds and crocodiles. The relationship among turtles, birds, and crocodiles remained unclear to date. To resolve this issue, we have cloned and sequenced two nuclear genes encoding the catalytic subunit of DNA polymerase alpha and glycinamide ribonucleotide synthetase-aminoimidazole ribonucleotide synthetase-glycinamide ribonucleotide formyltransferase from amniotes and an amphibian. The amino acid sequences of these proteins were subjected to a phylogenetic analysis based on the maximum likelihood method. The resulting tree showed that turtles are the sister group to a monophyletic cluster of archosaurs (birds and crocodiles). All other possible tree topologies were significantly rejected.     

Turtle isochore structure is intermediate between amphibians and other amniotes

Vertebrate genomes are comprised of isochores that are relatively long (>100 kb) regions with a relatively homogenous (either GC-rich or AT-rich) base composition and with rather sharp boundaries with neighboring isochores. Mammals and living archosaurs (birds and crocodilians) have heterogeneous genomes that include very GC-rich isochores. In sharp contrast, the genomes of amphibians and fishes are more homogeneous and they have a lower overall GC content. Because DNA with higher GC content is more thermostable, the elevated GC content of mammalian and archosaurian DNA has been hypothesized to be an adaptation to higher body temperatures. This hypothesis can be tested by examining structure of isochores across the reptilian clade, which includes the archosaurs, testudines (turtles), and lepidosaurs (lizards and snakes), because reptiles exhibit diverse body sizes, metabolic rates, and patterns of thermoregulation. This study focuses on a comparative analysis of a new set of expressed genes of the red-eared slider turtle and orthologs of the turtle genes in mammalian (human, mouse, dog, and opossum), archosaurian (chicken and alligator), and amphibian (western clawed frog) genomes. EST (expressed sequence tag) data from a turtle cDNA library enriched for genes that have specialized functions (developmental genes) revealed using the GC content of the third-codon-position to examine isochore structure requires careful consideration of the types of genes examined. The more highly expressed genes (e.g., housekeeping genes) are more likely to be GC-rich than are genes with specialized functions. However, the set of highly expressed turtle genes demonstrated that the turtle genome has a GC content that is intermediate between the GC-poor amphibians and the GC-rich mammals and archosaurs. There was a strong correlation between the GC content of all turtle genes and the GC content of other vertebrate genes, with the slope of the line describing this relationship also indicating that the isochore structure of turtles is intermediate between that of amphibians and other amniotes. These data are consistent with some thermal hypotheses of isochore evolution, but we believe that the credible set of models for isochore evolution still includes a variety of models. These data expand the amount of genomic data available from reptiles upon which future studies of reptilian genomics can build.     

A whole-genome phylogeny of the family Pasteurellaceae

A phylogenomic approach was used to generate an amino acid phylogeny for 12 whole genomes representing 10 species in the family Pasteurellaceae. Orthology of genes was determined using an approach similar to OrthologID (http://nypg.bio.nyu.edu/orthologid/about.html) and resulted in the generation of a matrix with 3130 genes with 1,194,615 aligned amino acid characters of which 239,504 characters are phylogenetically informative. Phylogenetic analysis of the concatenated matrix using all standard approaches (maximum parsimony, maximum likelihood, and Bayesian analysis) results in a single extremely robust phylogenetic hypothesis for the species examined in this study. Remarkably, no single gene partition gives the same tree as the concatenated analysis. By analyzing partitioned support in the data matrix, we show that there is very little negative support emanating from individual gene partitions to suggest that the concatenated hypothesis is not tenable. The large number of characters in the matrix allows us to test hypotheses concerning missing data and character number in phylogenomic studies, and we conclude that matrices constructed using genome level information are very robust to missing data. We show that a very large number of concatenated gene sequences (>160) are needed to reliably obtain the same topology as the overall analysis.     

The western painted turtle genome,a model for the evolution of extreme physiological adaptations in a slowly evolving lineage

Background

We describe the genome of the western painted turtle, Chrysemys picta bellii, one of the most widespread, abundant, and well-studied turtles. We place the genome into a comparative evolutionary context, and focus on genomic features associated with tooth loss, immune function, longevity, sex differentiation and determination, and the species'' physiological capacities to withstand extreme anoxia and tissue freezing.

Results

Our phylogenetic analyses confirm that turtles are the sister group to living archosaurs, and demonstrate an extraordinarily slow rate of sequence evolution in the painted turtle. The ability of the painted turtle to withstand complete anoxia and partial freezing appears to be associated with common vertebrate gene networks, and we identify candidate genes for future functional analyses. Tooth loss shares a common pattern of pseudogenization and degradation of tooth-specific genes with birds, although the rate of accumulation of mutations is much slower in the painted turtle. Genes associated with sex differentiation generally reflect phylogeny rather than convergence in sex determination functionality. Among gene families that demonstrate exceptional expansions or show signatures of strong natural selection, immune function and musculoskeletal patterning genes are consistently over-represented.

Conclusions

Our comparative genomic analyses indicate that common vertebrate regulatory networks, some of which have analogs in human diseases, are often involved in the western painted turtle''s extraordinary physiological capacities. As these regulatory pathways are analyzed at the functional level, the painted turtle may offer important insights into the management of a number of human health disorders.     

Inferring species phylogenies from multiple genes: concatenated sequence tree versus consensus gene tree

Phylogenetic trees from multiple genes can be obtained in two fundamentally different ways. In one, gene sequences are concatenated into a super-gene alignment, which is then analyzed to generate the species tree. In the other, phylogenies are inferred separately from each gene, and a consensus of these gene phylogenies is used to represent the species tree. Here, we have compared these two approaches by means of computer simulation, using 448 parameter sets, including evolutionary rate, sequence length, base composition, and transition/transversion rate bias. In these simulations, we emphasized a worst-case scenario analysis in which 100 replicate datasets for each evolutionary parameter set (gene) were generated, and the replicate dataset that produced a tree topology showing the largest number of phylogenetic errors was selected to represent that parameter set. Both randomly selected and worst-case replicates were utilized to compare the consensus and concatenation approaches primarily using the neighbor-joining (NJ) method. We find that the concatenation approach yields more accurate trees, even when the sequences concatenated have evolved with very different substitution patterns and no attempts are made to accommodate these differences while inferring phylogenies. These results appear to hold true for parsimony and likelihood methods as well. The concatenation approach shows >95% accuracy with only 10 genes. However, this gain in accuracy is sometimes accompanied by reinforcement of certain systematic biases, resulting in spuriously high bootstrap support for incorrect partitions, whether we employ site, gene, or a combined bootstrap resampling approach. Therefore, it will be prudent to report the number of individual genes supporting an inferred clade in the concatenated sequence tree, in addition to the bootstrap support.     

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.     

Phylogenies from genetic and morphological characters do not support a revision of Gigasporaceae (Glomeromycota) into four families and five genera

The family Gigasporaceae consisted of the two genera Gigaspora and Scutellospora when first erected. In a recent revision of this classification, Scutellospora was divided into three families and four genera based on two main lines of evidence: (1) phylogenetic patterns of coevolving small and large rRNA genes and (2) morphology of spore germination shields. The rRNA trees were assumed to accurately reflect species evolution, and shield characters were selected because they correlated with gene trees. These characters then were used selectively to support gene trees and validate the classification. To test this new classification, a phylogenetic tree was reconstructed from concatenated 25S rRNA and β-tubulin gene sequences using 35% of known species in Gigasporaceae. A tree also was reconstructed from 23 morphological characters represented in 71% of known species. Results from both datasets showed that the revised classification was untenable. The classification also failed to accurately represent sister group relationships amongst higher taxa. Only two clades were fully resolved and congruent among datasets: Gigaspora and Racocetra (a clade consisting of species with spores having one inner germinal wall). Other clades were unresolved, which was attributed in part to undersampling of species. Topology of the morphology-based phylogeny was incongruent with gene evolution. Five shield characters were reduced to three, of which two were phylogenetically uninformative because they were homoplastic. Therefore, most taxa erected in the new classification are rejected. The classification is revised to restore the family Gigasporaceae, within which are the three genera Gigaspora, Racocetra, and Scutellospora. This classification does not reflect strict topology of either gene or morphological evolution. Further revisions must await sampling of additional characters and taxa to better ascertain congruence between datasets and infer a more accurate phylogeny of this important group of fungi.     

Phylogeny of Bacterial and Archaeal Genomes Using Conserved Genes: Supertrees and Supermatrices

Over 3000 microbial (bacterial and archaeal) genomes have been made publically available to date, providing an unprecedented opportunity to examine evolutionary genomic trends and offering valuable reference data for a variety of other studies such as metagenomics. The utility of these genome sequences is greatly enhanced when we have an understanding of how they are phylogenetically related to each other. Therefore, we here describe our efforts to reconstruct the phylogeny of all available bacterial and archaeal genomes. We identified 24, single-copy, ubiquitous genes suitable for this phylogenetic analysis. We used two approaches to combine the data for the 24 genes. First, we concatenated alignments of all genes into a single alignment from which a Maximum Likelihood (ML) tree was inferred using RAxML. Second, we used a relatively new approach to combining gene data, Bayesian Concordance Analysis (BCA), as implemented in the BUCKy software, in which the results of 24 single-gene phylogenetic analyses are used to generate a “primary concordance” tree. A comparison of the concatenated ML tree and the primary concordance (BUCKy) tree reveals that the two approaches give similar results, relative to a phylogenetic tree inferred from the 16S rRNA gene. After comparing the results and the methods used, we conclude that the current best approach for generating a single phylogenetic tree, suitable for use as a reference phylogeny for comparative analyses, is to perform a maximum likelihood analysis of a concatenated alignment of conserved, single-copy genes.     

The Impact of Outgroup Choice and Missing Data on Major Seed Plant Phylogenetics Using Genome-Wide EST Data

Background

Genome level analyses have enhanced our view of phylogenetics in many areas of the tree of life. With the production of whole genome DNA sequences of hundreds of organisms and large-scale EST databases a large number of candidate genes for inclusion into phylogenetic analysis have become available. In this work, we exploit the burgeoning genomic data being generated for plant genomes to address one of the more important plant phylogenetic questions concerning the hierarchical relationships of the several major seed plant lineages (angiosperms, Cycadales, Gingkoales, Gnetales, and Coniferales), which continues to be a work in progress, despite numerous studies using single, few or several genes and morphology datasets. Although most recent studies support the notion that gymnosperms and angiosperms are monophyletic and sister groups, they differ on the topological arrangements within each major group.

Methodology

We exploited the EST database to construct a supermatrix of DNA sequences (over 1,200 concatenated orthologous gene partitions for 17 taxa) to examine non-flowering seed plant relationships. This analysis employed programs that offer rapid and robust orthology determination of novel, short sequences from plant ESTs based on reference seed plant genomes. Our phylogenetic analysis retrieved an unbiased (with respect to gene choice), well-resolved and highly supported phylogenetic hypothesis that was robust to various outgroup combinations.

Conclusions

We evaluated character support and the relative contribution of numerous variables (e.g. gene number, missing data, partitioning schemes, taxon sampling and outgroup choice) on tree topology, stability and support metrics. Our results indicate that while missing characters and order of addition of genes to an analysis do not influence branch support, inadequate taxon sampling and limited choice of outgroup(s) can lead to spurious inference of phylogeny when dealing with phylogenomic scale data sets. As expected, support and resolution increases significantly as more informative characters are added, until reaching a threshold, beyond which support metrics stabilize, and the effect of adding conflicting characters is minimized.     

Congruence,non‐homology,and the phylogeny of basal turtles

Joyce, W.G. and Sterli J. 2010. Congruence, non‐homology, and the phylogeny of basal turtles.–Acta Zoologica (Stockholm) Modern cladistic analysis is characterized by the assembly of increasingly larger data sets coupled with the use of congruence as the final test of homology. Some critics of this development have recently called for a return to more detailed primary homology analysis while questioning the utility of congruence. This discussion appears to be central to the debate regarding the phylogenetic relationships of basal turtles, as the large data sets developed by us have been criticized recently for utilizing poorly constructed characters and including too many homoplasy‐prone characters. Our analysis of this critique reveals that (1) new information regarding poorly understood taxa has a greater impact on the outcome of turtle phylogenies than the characters under dispute; (2) most current turtle phylogenies differ in taxon sampling, not character sampling, and so it appears illogical to condemn a particular analysis for its character sampling; (3) even evolutionary taxonomists should agree that key characters utilized to resolve basal turtle relationships cannot be thought to be ‘infallible’; (4) whereas various criteria provide positive evidence for homology, only congruence provides positive evidence for non‐homology; and (5) a stalemate between conflicting camps within a congruence frame work is preferable to the ad hoc dismissal of data sets, because authoritative statements are untestable.     

Phylogenetic position of Salinibacter ruber based on concatenated protein alignments

A total of 22 genes from the genome of Salinibacter ruber strain M31 were selected in order to study the phylogenetic position of this species based on protein alignments. The selection of the genes was based on their essential function for the organism, dispersion within the genome, and sufficient informative length of the final alignment. For each gene, an individual phylogenetic analysis was performed and compared with the resulting tree based on the concatenation of the 22 genes, which rendered a single alignment of 10,757 homologous positions. In addition to the manually chosen genes, an automatically selected data set of 74 orthologous genes was used to reconstruct a tree based on 17,149 homologous positions. Although single genes supported different topologies, the tree topology of both concatenated data sets was shown to be identical to that previously observed based on small subunit (SSU) rRNA gene analysis, in which S. ruber was placed together with Bacteroidetes. In both concatenated data sets the bootstrap was very high, but an analysis with a gradually lower number of genes indicated that the bootstrap was greatly reduced with less than 12 genes. The results indicate that tree reconstructions based on concatenating large numbers of protein coding genes seem to produce tree topologies with similar resolution to that of the single 16S rRNA gene trees. For classification purposes, 16S rRNA gene analysis may remain as the most pragmatic approach to infer genealogic relationships.     

TaxMan: a taxonomic database manager

Background  

Phylogenetic analysis of large, multiple-gene datasets, assembled from public sequence databases, is rapidly becoming a popular way to approach difficult phylogenetic problems. Supermatrices (concatenated multiple sequence alignments of multiple genes) can yield more phylogenetic signal than individual genes. However, manually assembling such datasets for a large taxonomic group is time-consuming and error-prone. Additionally, sequence curation, alignment and assessment of the results of phylogenetic analysis are made particularly difficult by the potential for a given gene in a given species to be unrepresented, or to be represented by multiple or partial sequences. We have developed a software package, TaxMan, that largely automates the processes of sequence acquisition, consensus building, alignment and taxon selection to facilitate this type of phylogenetic study.     

马铃薯Y病毒多基因系统发育分析及其在株系鉴定中的应用

为实现马铃薯Y病毒(Potato virus Y, PVY)常见株系的快速鉴定,本文以PVY的P1、HC-pro、VPg和CP 4个基因为研究对象建立了快速准确的多基因联合体系。根据基因的不同组合建立5个不同数据集,分别进行系统发育分析,并通过贝叶斯标签关联显著性(Bayesian tip-association significance, BaTS)分析各数据集中代表分离物与株系的关联性,以确定实现PVY快速鉴定的最佳组合。不同数据集的系统发育及BaTS分析结果显示,除了联合P1、VPg和CP 3个基因数据集外,其他4个数据集均无法实现PVY常见株系的准确鉴定。采用不同建树方法对联合P1、VPg和CP 3个基因数据集比较分析显示,基于ML法和NJ法的系统发育树在拓扑结构上基本一致,均优于基于贝叶斯算法的最大分支置信(maximum clade credibility, MCC)树。同时,以HLJ26分离物为研究对象,对建立的多基因联合体系进行实际应用,结果显示该分离物与PVY^NTN-NW株系的3个分离物SYR-Ⅱ-2-8、SYR-Ⅱ-Be1和SYR-Ⅱ-DrH以高置信值聚为一亚簇,表明该分离物可能属于PVY^NTN-NW株系(SYR-Ⅱ型)。重组分析显示,HLJ26基因组存在4个潜在的重组信号,分别位于P1、HC-pro/P3、VPg和CP的5°-末端,与PVY^NTN-NW株系(SYR-Ⅱ型)的重组位点相一致,表明其属于PVY^NTN-NW株系(SYR-Ⅱ型)。同时,应用多重RT-PCR成功扩增出约为1000 bp和400 bp的2个特异性片段,与PVY^NTN-NW株系(SYR-Ⅱ型)的特异条带大小相一致。这些结果进一步支持了多基因联合体系的鉴定结果。联合P1、VPg和CP 3个基因数据集系统发育分析,可以实现PVY常见株系的准确鉴定。