Molecular Characterization of Southern Rice Blacked‐Streaked Dwarf Virus (SRBSDV) from Vietnam

Southern rice black‐streaked dwarf virus (SRBSDV) is a novel putative member of the genus Fijivirus, family Reoviridae. We report here the genomic sequences of a Vietnamese isolate (SRBSDV‐V). The total genome of SRBSDV‐V has 29 115 nucleotides (nt), nine nt shorter than SRBSDV‐GD or ‐HN, but similar in organization to these two Chinese isolates. Nucleotide diversities among SRBSDV isolates were much lower than those among the corresponding ORFs of the available RBSDV isolates and there was a lower purifying selection pressure on SRBSDV than RBSDV, providing first molecular evidence for the view that SRBSDV is of recent origin. In studies of all available SRBSDV sequences, there was no obvious correlation between geographic distances and phylogenetic distribution. A high frequency of genetic recombination was found among both Chinese and Vietnam SRBSDV isolates, suggesting that recombination may play an important role in the molecular variation and evolution of SRBSDV.     

Selection pressure-driven evolution of the Epstein-Barr virus-encoded oncogene LMP1 in virus isolates from Southeast Asia

The geographically constrained distribution of Epstein-Barr virus (EBV)-associated nasopharyngeal carcinoma (NPC) in southeast Asian populations suggests that both viral and host genetics may influence disease risk. Although susceptibility loci have been mapped within the human genome, the role of viral genetics in the focal distribution of NPC remains an enigma. Here we report a molecular phylogenetic analysis of an NPC-associated viral oncogene, LMP1, in a large panel of EBV isolates from southeast Asia and from Papua New Guinea, Africa, and Australia, regions of the world where NPC is and is not endemic, respectively. This analysis revealed that LMP1 sequences show a distinct geographic structure, indicating that the southeast Asian isolates have evolved as a lineage distinct from those of Papua New Guinea, African, and Australian isolates. Furthermore, a likelihood ratio test revealed that the C termini of the LMP1 sequences of the southeast Asian lineage are under significant positive selection pressure, particularly at some sites within the C-terminal activator regions. We also present evidence that although the N terminus and transmembrane region of LMP1 have undergone recombination, the C-terminal region of the gene has evolved without any history of recombination. Based on these observations, we speculate that selection pressure may be driving the LMP1 sequences in virus isolates from southeast Asia towards a more malignant phenotype, thereby influencing the endemic distribution of NPC in this region.     

Variation in the Coat Protein Gene of Papaya ringspot Virus Isolates from Multiple Locations of China

The potyvirus Papaya ringspot virus （PRSV） is an important pathogen of papaya that causes severe losses in economic crops for papaya production globally. The coat protein （CP） genes of five PRSV isolates originating from different locations in China were cloned and sequenced. The CP-coding region varied in size from 864-873 nucleotides, encoding proteins of 288-291 amino acids. The five Chinese isolates of PRSV have been characterized as papaya-infecting （PRSV-P）. The CP sequences of the Chinese isolates were compared with those of previously published PRSV isolates originating from different countries at amino acid levels. A number of KE repeat boxes in the N terminus of the PRSV-CP were found in all Chinese isolates. The phylogenetic branching pattern revealed that there was certain extended grouping between geographic locations, and the Asian type probably represents the oldest population of PRSV. The information of CP genes will be useful in designing and developing durable virus resistant-PRSV transgenic papaya in China. Meanwhile broad-spectrum-virus resistant, strongly resistant-PRSV and good safe papaya lines are required.     

The marsupial MHC: The Tammar Wallaby,Macropus eugenii,contains an expressed DNA-like gene on chromosome 1

Isolates of cauliflower mosaic virus (CaMV) differ in host range and symptomatology. Knowledge of their sequence relationships should assist in identifying nucleotide sequences responsible for isolate-specific characters. Complete nucleotide sequences of the DNAs of eight isolates of CaMV were aligned and the aligned sequences were used to analyze phylogenetic relationships by maximum likelihood, bootstrapped parsimony, and distance methods. Isolates found in North America clustered separately from those isolated from other parts of the world. Additional isolates, for which partial sequences were available, were incorporated into phylogenetic analysis of the sequences of genome segments corresponding to individual protein coding regions or the large intergenic region of CaMV DNA. The analysis revealed several instances where the position of an isolate on a tree for one coding region did not agree with the position of the isolate on the tree for the complete genome or with its position on trees for other coding regions. Examination of the distribution of shared residue types of phylogenetically informative positions in anomalous regions suggested that most of the anomalies were due to recombination events during the evolution of the isolates. Application of an algorithm that searches for segments of significant length that are identical between pairs of isolates or contain a significantly high concentration of polymorphisms suggested two additional recombination events between progenitors of the isolates studied and an event between the XinJing isolate and a CaMV not represented in the data set. An earlier phylogenetic origin for CaMV than for carnation etched ring virus, the caulimovirus used as outgroup in these analyses, was deduced from the position of the outgroup with North American isolates in some trees, but with non-North American isolates in other trees. Correspondence to: U. Melcher     

Pangenome Analysis of Burkholderia pseudomallei: Genome Evolution Preserves Gene Order despite High Recombination Rates

The pangenomic diversity in Burkholderia pseudomallei is high, with approximately 5.8% of the genome consisting of genomic islands. Genomic islands are known hotspots for recombination driven primarily by site-specific recombination associated with tRNAs. However, recombination rates in other portions of the genome are also high, a feature we expected to disrupt gene order. We analyzed the pangenome of 37 isolates of B. pseudomallei and demonstrate that the pangenome is ‘open’, with approximately 136 new genes identified with each new genome sequenced, and that the global core genome consists of 4568±16 homologs. Genes associated with metabolism were statistically overrepresented in the core genome, and genes associated with mobile elements, disease, and motility were primarily associated with accessory portions of the pangenome. The frequency distribution of genes present in between 1 and 37 of the genomes analyzed matches well with a model of genome evolution in which 96% of the genome has very low recombination rates but 4% of the genome recombines readily. Using homologous genes among pairs of genomes, we found that gene order was highly conserved among strains, despite the high recombination rates previously observed. High rates of gene transfer and recombination are incompatible with retaining gene order unless these processes are either highly localized to specific sites within the genome, or are characterized by symmetrical gene gain and loss. Our results demonstrate that both processes occur: localized recombination introduces many new genes at relatively few sites, and recombination throughout the genome generates the novel multi-locus sequence types previously observed while preserving gene order.     

Role of recombination in the evolution of the model plant pathogen Pseudomonas syringae pv. tomato DC3000, a very atypical tomato strain

Pseudomonas syringae pv. tomato strain DC3000 (PtoDC3000) is one of the most intensively studied bacterial plant pathogens today. Here we report a thorough investigation into PtoDC3000 and close relatives isolated from Antirrhinum majus (snapdragon), Apium graveolens (celery), and Solanaceae and Brassicaceae species. Multilocus sequence typing (MLST) was used to resolve the precise phylogenetic relationship between isolates and to determine the importance of recombination in their evolution. MLST data were correlated with an analysis of the locus coding for the type III secreted (T3S) effector AvrPto1 to investigate the role of recombination in the evolution of effector repertoires. Host range tests were performed to determine if closely related isolates from different plants have different host ranges. It was found that PtoDC3000 is located in the same phylogenetic cluster as isolates from several Brassicaceae and Solanaceae species and that these isolates have a relatively wide host range that includes tomato, Arabidopsis thaliana, and cauliflower. All other analyzed tomato isolates from three different continents form a distinct cluster and are pathogenic only on tomato. Therefore, PtoDC3000 is a very unusual tomato isolate. Several recombination breakpoints were detected within sequenced gene fragments, and population genetic tests indicate that recombination contributed more than mutation to the variation between isolates. Moreover, recombination may play an important role in the reassortment of T3S effectors between strains. The data are finally discussed from a taxonomic standpoint, and P. syringae pv. tomato is proposed to be divided into two pathovars.     

Inferring the evolutionary history of rice yellow mottle virus from genomic, phylogenetic, and phylogeographic studies

Fourteen isolates of Rice yellow mottle virus (RYMV) were selected as representative of the genetic variability of the virus in Africa from a total set of 320 isolates serologically typed or partially sequenced. The 14 isolates were fully sequenced and analyzed together with two previously reported sequences. RYMV had a genomic organization similar to that of Cocksfoot mottle sobemovirus. The average nucleotide diversity among the 16 isolates of RYMV was 7%, and the maximum diversity between any two isolates was 10%. A strong conservative selection was apparent on both synonymous and nonsynonymous substitutions, through the amino acid replacement pattern, on the genome size, and through the limited number of indel events. Furthermore, there was a lack of positive selection on single amino acid sites and no evidence of recombination events. RYMV diversity had a pronounced and characteristic geographic structure. The branching order of the clades correlated with the geographic origin of the isolates along an east-to-west transect across Africa, and there was a marked decrease in nucleotide diversity moving westward across the continent. The insertion-deletion polymorphism was related to virus phylogeny. There was a partial phylogenetic incongruence between the coat protein gene and the rest of the genome. Overall, our results support the hypothesis that RYMV originated in East Africa and then dispersed and differentiated gradually from the east to the west of the continent.     

Molecular characterization of Indian Sugarcane streak mosaic virus isolates reveals recombination and negative selection in the P1 gene

Sugarcane streak mosaic virus (SCSMV), a member of the genus Poacevirus is an important viral pathogen affecting sugarcane production in India. The P1 gene of ten Indian isolates was sequenced and compared with previously reported SCSMV isolates. Comparative sequence analysis revealed a high level of diversity in the P1 gene (83–98% nucleotide sequence identity; 87–100% amino acid sequence identity), and the Indian SCSMV isolates were found to be the most variable (up to 9% diversity at the amino acid level). Phylogenetic tree analysis showed clustering of 17 SCSMV isolates into two groups: group I included isolates from India (except SCSMV-TPT) and Pakistan, and group II consisted of isolates from Japan, Indonesia, Thailand and SCSMV-TPT. The results obtained from phylogenetic study were further supported by the different in silico analysis viz. SNPs (single nucleotide polymorphism), INDELs (insertion and deletion) and evolutionary distance analysis. A significant proportion of recombination sites were observed at the N terminal region of P1 gene. Analysis of selection pressure indicated that the P1 gene of the Indian SCSMV isolates is under strong negative or purifying selection. It is likely that recombination identified in Indian SCSMV isolates, along with strong purifying selection, enhances the speed of elimination of deleterious mutations in the P1 gene. The evolutionary processes (recombination and selection pressure) together contributed to the observed genetic diversity and population structure of Indian SCSMV isolates.     

A phylogeographical study of the Turnip mosaic virus population in East Asia reveals an 'emergent' lineage in Japan

The genetic structure of populations of Turnip mosaic virus (TuMV) in East Asia was assessed by making host range and gene sequence comparisons of 118 isolates utilizing a population genetic approach. Most, but not all, isolates collected from Brassica plants in China infected only Brassica plants, whereas those from Japan infected both Brassica and Raphanus (BR) plants. Analyses of the positions of recombination sites in five regions of the genomes (one third of the full sequence) of the many recombinant isolates were fully congruent with the results of phylogenetic analysis, and at least one recombination type pattern was shared between Chinese and Japanese populations. One lineage of nonrecombinant isolates from the basal-BR lineage was found in 2000 in Kyushu, Japan but none in China, and have since been found over the whole island. The sudden expansion of this basal-BR population was strongly supported by calculations showing the deviations from the neutral equilibrium model for the individual geographical lineages with overall lack of nucleotide diversity, and by analysis of mismatch distribution. Our study shows that the recent Chinese and Japanese TuMV isolates are part of the same population but are discrete lineages.     

中国H5N1禽流感病毒HA基因的分子进化分析

为了阐明中国H5N1禽流感病毒血凝素(HA)基因的分子进化规律,从GenBank下载中国近年来分离的27株禽流感H5N1病毒的HA基因,利用LaserGene软件包中的EditSeq程序剪切共同序列并翻译成氨基酸序列,与DNAMAN软件进行比对,并以LaserGene软件包中的MegAlign程序构建分子进化树,结合背景资料分析其传播流行规律。结果显示:①在HA裂解位点上游毗邻的位置,gdch04、gddu04、gxch0405、gxdu05、gxqu0502、hkcph05、stch05、stqu05、ynch0502株都缺失了三个碱基;②27株毒株在HA裂解位点上游毗邻的位置存在多个连续的碱性氨基酸;③来自广西的gxch0405、gxdu05、gxqu0502和来自香港的hkcph05、hkgh04的序列同源性很高,很可能与苍鹭的迁移有关;④来自广西的gxqu0502和来自香港的hkcph05亲源关系很近,很可能来自同一祖先。     

Recombination between Clonal Lineages of the Asexual Fungus Verticillium dahliae Detected by Genotyping by Sequencing

Most asexual species of fungi have either lost sexuality recently, or they experience recombination by cryptic sexual reproduction. Verticillium dahliae is a plant-pathogenic, ascomycete fungus with no known sexual stage, even though related genera have well-described sexual reproduction. V. dahliae reproduces mitotically and its population structure is highly clonal. However, previously described discrepancies in phylogenetic relationships among clonal lineages may be explained more parsimoniously by recombination than mutation; therefore, we looked for evidence of recombination within and between clonal lineages. Genotyping by sequencing was performed on 141 V. dahliae isolates from diverse geographic and host origins, resulting in 26,748 single-nucleotide polymorphisms (SNPs). We found a strongly clonal population structure with the same lineages as described previously by vegetative compatibility groups (VCGs) and molecular markers. We detected 443 recombination events, evenly distributed throughout the genome. Most recombination events detected were between clonal lineages, with relatively few recombinant haplotypes detected within lineages. The only three isolates with mating type MAT1-1 had recombinant SNP haplotypes; all other isolates had mating type MAT1-2. We found homologs of eight meiosis-specific genes in the V. dahliae genome, all with conserved or partially conserved protein domains. The extent of recombination and molecular signs of sex in (mating-type and meiosis-specific genes) suggest that V. dahliae clonal lineages arose by recombination, even though the current population structure is markedly clonal. Moreover, the detection of new lineages may be evidence that sexual reproduction has occurred recently and may potentially occur under some circumstances. We speculate that the current clonal population structure, despite the sexual origin of lineages, has arisen, in part, as a consequence of agriculture and selection for adaptation to agricultural cropping systems.     

以P型PRSV-VPg为模板多位点突变获得W型PRSV-VPg的研究

以P型脉PRSV-VPg（Papaya Ringspot Virus,VPggene）基因为模板利用高保真酶通过两对引物（突变4个位点）．PCR扩增获得一条276bp和一条141bp的两个小片段．以此为基础,利用长引物延伸和套叠PCR的方法,定点突变剩余5个位点,最后拼接获得形型PRSV—V段基因．结果表明通过上述方案可以成功改造P型PRSV-VPg基因,获得与W型PRSV-VPg同源的目的基因,成功率为100％．该技术可以绕开传统的通过寻找毒源进行RT－PCR方法和人工合成方法获得病毒基因,其技术操作简单,且节约时间和成本,并在人工合成基因、多位点基因定点突变等方面具有很好的借鉴作用和应用价值．     

Identification,genotyping, and molecular evolution analysis of duck circovirus

Duck circovirus (DuCV) is a contagious immunosuppressive virus affecting many duck species, which is responsible for multiple outbreaks in poultry industries worldwide. In this study, the first DuCV isolate GH01 was identified in Sichuan by PCR, which shared a high level of nucleotide identity (81.8–99.4%) with sequences of other DuCV isolates available in GenBank. Comparative phylogenetic and pairwise sequence comparison analyses indicated that DuCV could be divided into two genotypes (DuCV-1 and DuCV-2) and six subtypes (1a, 1b, 1c, 2a, 2b and 2c) based on the complete genome sequence. The results revealed that both DuCV-1 and DuCV-2 had evolved from the same ancestor but undergone divergent evolution. Interestingly, phylogenetic analyses indicated that three isolates were classified into a cluster DuCV-2a using complete DuCV genome sequence and cap gene, except rep gene. Recombination analyses revealed that DuCV-2a arose from recombination between DuCV-1a and DuCV-2b isolates within the rep genes, and the recombination events mainly occur both in non-structural protein coding region and structural protein coding region. In addition, the mechanisms of recombination supporting the genetic variability in DuCV isolates were investigated. Likewise, selective pressure indicated that purifying selection had been a major driving force in maintaining diversity among the DuCV isolates. Because eradicating the virus from commercial ducks is impossible, it is necessary to take effective control measures and implement them throughout the world.     

Virulence evolution of the human pathogen Neisseria meningitidis by recombination in the core and accessory genome

Background

Neisseria meningitidis is a naturally transformable, facultative pathogen colonizing the human nasopharynx. Here, we analyze on a genome-wide level the impact of recombination on gene-complement diversity and virulence evolution in N. meningitidis. We combined comparative genome hybridization using microarrays (mCGH) and multilocus sequence typing (MLST) of 29 meningococcal isolates with computational comparison of a subset of seven meningococcal genome sequences.

Principal Findings

We found that lateral gene transfer of minimal mobile elements as well as prophages are major forces shaping meningococcal population structure. Extensive gene content comparison revealed novel associations of virulence with genetic elements besides the recently discovered meningococcal disease associated (MDA) island. In particular, we identified an association of virulence with a recently described canonical genomic island termed IHT-E and a differential distribution of genes encoding RTX toxin- and two-partner secretion systems among hyperinvasive and non-hyperinvasive lineages. By computationally screening also the core genome for signs of recombination, we provided evidence that about 40% of the meningococcal core genes are affected by recombination primarily within metabolic genes as well as genes involved in DNA replication and repair. By comparison with the results of previous mCGH studies, our data indicated that genetic structuring as revealed by mCGH is stable over time and highly similar for isolates from different geographic origins.

Conclusions

Recombination comprising lateral transfer of entire genes as well as homologous intragenic recombination has a profound impact on meningococcal population structure and genome composition. Our data support the hypothesis that meningococcal virulence is polygenic in nature and that differences in metabolism might contribute to virulence.     

Discordant mtDNA and cpDNA phylogenies indicate geographic speciation and reticulation as driving factors for the diversification of the genus Picea

The phylogeny of the genus Picea was investigated by sequencing three loci from the paternally inherited chloroplast genome (trnK, rbcL and trnTLF) and the intron 2 of the maternally transmitted mitochondrial gene nad1 for 35 species. Significant topological differences were found between the trnK tree and the rbcL and trnTLF phylogenetic trees, and between cpDNA and mtDNA phylogenies. None of the phylogenies matched morphological classifications. The mtDNA phylogeny was geographically more structured than cpDNA phylogenies, reflecting the different inheritance of the two cytoplasmic genomes in the Pinaceae and their differential dispersion by seed only and seed and pollen, respectively. Most North American taxa formed a monophyletic group on the mtDNA tree, with topological patterns suggesting geographic speciation by range fragmentation or by dispersal and isolation. Similar patterns were also found among Asian taxa. Such a trend towards geographic speciation is anticipated in other Pinaceae genera with similar life history, autecology and reproductive system. Incongruences between organelle phylogenies suggested the occurrence of mtDNA capture by invading cpDNA. Incongruences between cpDNA partitions further suggested heterologous recombination presumably also linked to ancient reticulate evolution. Whilst cpDNA appears potentially valuable for molecular taxonomy and systematics purposes, these results emphasize the reduced value of cpDNA to infer vertical descent and the speciation history for plants with paternal transmission and high dispersal of their chloroplast genome.     

Molecular characterisation of papaya ringspot potyvirus isolates from India

Papaya ringspot potyvirus (PRSV) causes major diseases of papaya and cucurbits in the Indian subcontinent. Based on biological properties, PRSV isolates are classified as either papaya infecting (P), or non-papaya infecting (W) types. To characterise the P and W isolates from India at the molecular level, c. 1.7 Kb of the 3′-terminal regions comprising a part of the nuclear inclusion b (Nib) gene, the complete capsid protein (CP) gene and the untranslated region (UTR) of both the P and W isolates were cloned and sequenced. Comparative sequence analyses showed that the 3′-UTRs in isolates P and W were 209 nucleotides in length excluding the poly (A) tail, and shared 96% identity. The CP genes of the two isolates were also similar, with 87% nucleotide identity and 93% amino acid identity. The amino acid differences between the CP genes were mostly confined to the amino terminus. The DAG triplet associated with aphid transmissibility was present in the CP of isolate W, but it was replaced by DAD in the P isolate. The partially sequenced Nib genes were also 90% identical, but isolate W contained an additional amino acid (threonine) just upstream of the cleavage site (Q/S) between Nib and CP. This is the first reported comparison of the molecular characterisation of PRSV-P and W isolates from the Indian subcontinent.     

Intraspecies comparison of Streptomyces pratensis genomes reveals high levels of recombination and gene conservation between strains of disparate geographic origin

Background

Streptomyces are widespread bacteria that contribute to the terrestrial carbon cycle and produce the majority of clinically useful antibiotics. While interspecific genomic diversity has been investigated among Streptomyces, information is lacking on intraspecific genomic diversity. Streptomyces pratensis has high rates of homologous recombination but the impact of such gene exchange on genome evolution and the evolution of natural product gene clusters remains uncharacterized.

Results

We report draft genome sequences of four S. pratensis strains and compare to the complete genome of Streptomyces flavogriseus IAF-45-CD (=ATCC 33331), a strain recently reclassified to S. pratensis. Despite disparate geographic origins, the genomes are highly similar with 85.9% of genes present in the core genome and conservation of all natural product gene clusters. Natural products include a novel combination of carbapenem and beta-lactamase inhibitor gene clusters. While high intraspecies recombination rates abolish the phylogenetic signal across the genome, intraspecies recombination is suppressed in two genomic regions. The first region is centered on an insertion/deletion polymorphism and the second on a hybrid NRPS-PKS gene. Finally, two gene families accounted for over 25% of the divergent genes in the core genome. The first includes homologs of bldB (required for spore development and antibiotic production) while the second includes homologs of an uncharacterized protein with a helix-turn-helix motif (hpb). Genes from these families co-occur with fifteen pairs spread across the genome. These genes have evidence for co-evolution of co-localized pairs, supporting previous assertions that these genes may function akin to a toxin-antitoxin system.

Conclusions

S. pratensis genomes are highly similar with exceptional levels of recombination which erase phylogenetic signal among strains of the species. This species has a large core genome and variable terminal regions that are smaller than those found in interspecies comparisons. There is no geographic differentiation between these strains, but there is evidence for local linkage disequilibrium affecting two genomic regions. We have also shown further observational evidence that the DUF397-HTH (bldB and hpb) are a novel toxin-antitoxin pair.     

Complete mtDNA sequence of the North American freshwater mussel,Lampsilis ornata (Unionidae): an examination of the evolution and phylogenetic utility of mitochondrial genome organization in Bivalvia (Mollusca)

Molluscs in general, and bivalves in particular, exhibit an extraordinary degree of mitochondrial gene order variation when compared with other metazoans. Two factors inhibiting our understanding the evolution of gene rearrangement in bivalves are inadequate taxonomic sampling and failure to examine gene order in a phylogenetic framework. Here, we report the first complete nucleotide sequence (16,060 bp) of the mitochondrial (mt) genome of a North American freshwater bivalve, Lampsilis ornata (Mollusca: Paleoheterodonta: Unionidae). Gene order and mt genome content is examined in a comparative phylogenetic framework for Lampsilis and five other bivalves, representing five families. Mitochondrial genome content is shown to vary by gene duplication and loss among taxa and between male and female mitotypes within a species. Although mt gene arrangement is highly variable among bivalves, when optimized on an independently derived phylogenetic hypothesis, it allows for the reconstruction of ancestral gene order states and indicates the potential phylogenetic utility of the data. However, the interpretation of reconstructed ancestral gene order states must take in to account both the accuracy of the phylogenetic estimation and the probability of character state change across the topology, such as the presence/absence of atp8 in bivalve lineages. We discuss what role, if any, doubly uniparental inheritance (DUI) and recombination between sexual mitotypes may play in influencing gene rearrangement of the mt genome in some bivalve lineages.     

Frequency and dynamics of recombination within different species of human enteroviruses

Enteroviruses are members of the family Picornaviridae that cause widespread infections in human and other mammalian populations. Enteroviruses are genetically and antigenically highly variable, and recombination within and between serotypes contributes to their genetic diversity. To investigate the dynamics of the recombination process, sequence phylogenies between three regions of the genome (VP4, VP1, and 3Dpol) were compared among species A and B enterovirus variants detected in a human population-based survey in Scotland between 2000 and 2001, along with contemporary virus isolates collected in the same geographical region. This analysis used novel bioinformatic methods to quantify phylogenetic compatibility and correlations with serotype assignments of evolutionary trees constructed for different regions of the enterovirus genome. Species B enteroviruses showed much more frequent, time-correlated recombination events than those found for species A, despite the equivalence in population sampling, concordant with a linkage analysis of previously characterized enterovirus sequences obtained over longer collection periods. An analysis of recombination among complete genome sequences by computation of a phylogenetic compatibility matrix (PCM) demonstrated sharply defined boundaries between the VP2/VP3/VP1 block and sequences to either side in phylogenetic compatibility. The PCM also revealed equivalent or frequently greater degrees of incompatibility between different parts within the nonstructural region (2A-3D), indicating the occurrence of extensive recombination events in the past evolution of this part of the genome. Together, these findings provide new insights into the dynamics of species A and B enterovirus recombination and evolution and into the contribution of structured sampling to documenting reservoirs, emergence, and spread of novel recombinant forms in human populations.