Jukes-Cantor Distance Research Articles

The number of k-mer matches between two DNA sequences as a function of k and applications to estimate phylogenetic distances.

We study the number Nk of length-k word matches between pairs of evolutionarily related DNA sequences, as a function of k. We show that the Jukes-Cantor distance between two genome sequences-i.e. the number of substitutions per site that occurred since they evolved from their last common ancestor-can be estimated from the slope of a function F that depends on Nk and that is affine-linear within a certain range of k. Integers kmin and kmax can be calculated depending on the length of the input sequences, such that the slope of F in the relevant range can be estimated from the values F(kmin) and F(kmax). This approach can be generalized to so-called Spaced-word Matches (SpaM), where mismatches are allowed at positions specified by a user-defined binary pattern. Based on these theoretical results, we implemented a prototype software program for alignment-free sequence comparison called Slope-SpaM. Test runs on real and simulated sequence data show that Slope-SpaM can accurately estimate phylogenetic distances for distances up to around 0.5 substitutions per position. The statistical stability of our results is improved if spaced words are used instead of contiguous words. Unlike previous alignment-free methods that are based on the number of (spaced) word matches, Slope-SpaM produces accurate results, even if sequences share only local homologies.

Genealogical and niche modeling analyses reveal recent expansion and limited genetic divergence in the Formicivora serrana complex (Passeriformes: Thamnophilidae)

The principal aim of this study was to infer the phylogeographic pattern of the antwrens of the Formicivora serrana complex, an endemic faunal component of the semi-deciduous forests and restinga formations of the Atlantic Forest of southeast Brazil. We combined niche-based modeling and phylogeographic approaches in describing the genetic spatial structure of the taxon, inferring the phylogenetic hypothesis of lineage diversification, and providing estimates of population growth, timing of coalescence, and gene flow. We also modeled the species potential distribution to examine the putative consequences of the Last Glacial Maximum (LGM) (21,000 years ago) and Holocene climate variability. To perform this, we analyzed a dataset composed of two mitochondrial (cytochrome oxidase I and NADH dehydrogenase subunit 2) and four nuclear (anonymous loci) DNA regions from 35 specimens. Genealogical analyses were performed using phylogenetic maximum likelihood and coalescent Bayesian approaches; genetic diversity was assessed using analysis of molecular variance and network analysis; ages of coalescent events were estimated using a relaxed molecular clock model. Ecological niche modeling of current and LGM ranges were performed using six algorithms (Bioclim, general additive model, general linear model, Maxent, support vector machine and Random forest) based on 178 occurrence localities and 19 bioclimatic variables. Limited genetic divergence among recognized forms of the F. serrana complex was evidenced in the low mitochondrial nucleotide divergence among populations (Jukes–Cantor distance between subspecies varied from 0.17 to 0.21%), unresolved phylogenetic trees, star-shaped haplotype networks, and overall lack of population genetic structure. Fixation index analyses, however, suggested an incipient geographic structure. Ecological niche modeling suggested expansion of the distribution range since the LGM. Our results provide the first evidence that bird taxa of xerophytic and semi-deciduous habitats of the AF were also affected by Quaternary climatic fluctuations. The recovered genealogical pattern indicates that F. serrana should be regarded as a single evolutionary unit, corroborating current taxonomic hypotheses.

DNA Repeats Detection Using a Dedicated Dot-Plot Analysis

DNA repeats are believed to play significant roles in genome evolution and diseases. Many of the methods for finding repeated sequences are part of the digital signal processing (DSP) field and most of these methods use distances, similarities and consensus sequences to generate candidate sequences. This paper presents results obtained using a dedicated numerical representation with a mapping algorithm (using DNA distances and consensus types) and a custom dot-plot analysis (using similarities to represent DNA patterns) combined with image processing techniques, to visual isolate the position of DNA patterns with different lengths. The final images that best put in evidence the presence of repeated sequences were obtained using weighted cosine cross-correlation, Jukes-Cantor distance and Motyka similarity.

Genome-wide gene order distances support clustering the gram-positive bacteria.

Initially using 143 genomes, we developed a method for calculating the pair-wise distance between prokaryotic genomes using a Monte Carlo method to estimate the conservation of gene order. The method was based on repeatedly selecting five or six non-adjacent random orthologs from each of two genomes and determining if the chosen orthologs were in the same order. The raw distances were then corrected for gene order convergence using an adaptation of the Jukes-Cantor model, as well as using the common distance correction D′ = −ln(1-D). First, we compared the distances found via the order of six orthologs to distances found based on ortholog gene content and small subunit rRNA sequences. The Jukes-Cantor gene order distances are reasonably well correlated with the divergence of rRNA (R2 = 0.24), especially at rRNA Jukes-Cantor distances of less than 0.2 (R2 = 0.52). Gene content is only weakly correlated with rRNA divergence (R2 = 0.04) over all distances, however, it is especially strongly correlated at rRNA Jukes-Cantor distances of less than 0.1 (R2 = 0.67). This initial work suggests that gene order may be useful in conjunction with other methods to help understand the relatedness of genomes. Using the gene order distances in 143 genomes, the relations of prokaryotes were studied using neighbor joining and agreement subtrees. We then repeated our study of the relations of prokaryotes using gene order in 172 complete genomes better representing a wider-diversity of prokaryotes. Consistently, our trees show the Actinobacteria as a sister group to the bulk of the Firmicutes. In fact, the robustness of gene order support was found to be considerably greater for uniting these two phyla than for uniting any of the proteobacterial classes together. The results are supportive of the idea that Actinobacteria and Firmicutes are closely related, which in turn implies a single origin for the gram-positive cell.

Genetic distinctiveness of the greater long-tailed hamster, Tscherskia triton nestor (Rodentia: Mammalia), from Jeju Island, Korea: cytochrome oxidase I and cytochrome b sequence analyses

To examine the taxonomic status of Tscherskia triton nestor, which inhabits in Korea, we first obtained partial cytochrome oxidase I (COI) (657 bp) and cytochrome b (426 bp) sequences of T. t. nestor from the Korean Jeju Island and Tscherskia triton fuscipes from northeastern China, and these sequences were compared with the corresponding sequences of Tscherskia triton, available from GenBank. In the COI analysis, T. t. nestor from Jeju was distinct from T. t. fuscipe from northeastern China, with the average Jukes–Cantor distance of 3.45%, and in the cytochrome b analysis, T. t. nestor from Jeju was divergent from T. t. fuscipes from northeastern China and T. t. triton from the North China Plain, with the average distance of 3.88%. We conclude that these sequencing results on the distinctiveness of T. t. nestor from Jeju support the current classification of T. t. nestor from Korea, although further analyses with mitochondrial sequences of T. t. nestor from the mainland Korea and nuclear sequences of T. triton from Korea and China are necessary to confirm our findings. In addition, we considered genetically distinct T. t. triton from Jeju as an allopatric phylogroup, which needs special protection for its conservation. Additionally, we found the lack of cytochrome b sequences between T. t. fuscipes from northeastern China and T. t. triton in the North China Plain, although further systematic analyses to clarify the taxonomic status of T. t. fuscipes are necessary.

Stochastic errors vs. modeling errors in distance based phylogenetic reconstructions.

BackgroundDistance-based phylogenetic reconstruction methods use evolutionary distances between species in order to reconstruct the phylogenetic tree spanning them. There are many different methods for estimating distances from sequence data. These methods assume different substitution models and have different statistical properties. Since the true substitution model is typically unknown, it is important to consider the effect of model misspecification on the performance of a distance estimation method.ResultsThis paper continues the line of research which attempts to adjust to each given set of input sequences a distance function which maximizes the expected topological accuracy of the reconstructed tree. We focus here on the effect of systematic error caused by assuming an inadequate model, but consider also the stochastic error caused by using short sequences. We introduce a theoretical framework for analyzing both sources of error based on the notion of deviation from additivity, which quantifies the contribution of model misspecification to the estimation error. We demonstrate this framework by studying the behavior of the Jukes-Cantor distance function when applied to data generated according to Kimura’s two-parameter model with a transition-transversion bias. We provide both a theoretical derivation for this case, and a detailed simulation study on quartet trees.ConclusionsWe demonstrate both analytically and experimentally that by deliberately assuming an oversimplified evolutionary model, it is possible to increase the topological accuracy of reconstruction. Our theoretical framework provides new insights into the mechanisms that enables statistically inconsistent reconstruction methods to outperform consistent methods.

Genetic Distinctness of Sorex caecutiens hallamontanus (Soricomorpha: Mammalia) from Jeju Island in Korea: Cytochrome Oxidase I and Cytochrome b Sequence Analyses

To examine genetic divergences of two endemic Sorex caecutiens subspecies from Korea (S. c. hallamontanus in Korean Jeju Island and S. c. annexus in the mainland Korean Peninsula), we obtained partial cytochrome oxidase I (COI) sequences (429 bp) and complete cytochrome b sequences (1,140 bp) from the two Korean subspecies, and we compared these sequences to the corresponding sequences of S. caecutiens, obtained from GenBank. We found that Jeju S. c. hallamontanus is one of three clades within S. caecutiens, with an average Jukes-Cantor distance of 1.57% in the COI sequences and the distance of 2.07% and 11 fixed site differences in the cytochrome b sequences, indicating that Jeju S. c. hallamontanus is one endemic subspecies with concordant genetic distinctness, although further analyses with nuclear DNA sequences are necessary to confirm these findings. However, S. c. annexus from the mainland Korean Peninsula was not divergent from S. c. macropygmaeus from northeastern China and adjacent Russia, indicating that S. c. annexus from the mainland Korean Peninsula is another endemic subspecies with only morphological differences, although it is necessary to reexamine the subspecies status of S. c. annexus.

Genetic Identification of Subspecies and Stocks of Minke Whale Meats Purchased from Korean Markets during 2005-2007

To identify subspecies and stocks of minke whale meats purchased from Korean markets during 2005-2007, we first obtained their complete sequences of mitochondrial DNA cytochrome b and control region sequences, and compared these sequences to the corresponding sequences of the common minke whale (Balaenoptera acutorostrata), obtained from GenBank. From analyses with partial cytochrome b sequences (383 bp) and non-coding, partial control region sequences (463 bp), Korean mink whale meats are identified as the products from the North Pacific minke whale (B. a. scammoni). In addition, the sequences of the partial control region from these meats showed G at site no. 298 and G or A at site no. 463, and the meats appear to be originated from the J stock within this subspecies. Thus, it seems to be urgent to fulfill strict regulation measures to reduce their accidental fisheries bycatch because the J stock has been protected since 1986. Additionally, B. a. scammoni is distinct from B. a. acutorostrata, with average Jukes-Cantor distance of 2.21% in the complete control region sequence analysis (935 bp) and 1.31% distance in the complete cytochrome b gene sequence analysis, and these present results support the present subspecies classification, although further sequencing analyses with nuclear genes are necessary.

Pyrosequencing of the 16S rRNA gene to reveal bacterial pathogen diversity in biosolids

Given the potential for a variety of bacterial pathogens to occur in variably stabilized sewage sludge (biosolids), an understanding of pathogen diversity and abundance is necessary for accurate assessment of infective risk when these products are land applied. 16S rDNA was PCR amplified from genomic DNA extracted from municipal wastewater residuals (mesophilic- and thermophilic-phased anaerobic digestion (MAD and TPAD), composting (COM)), and agricultural soil (SOIL), and these amplicons were sequenced using massively parallel pyrosequencing technology. Resulting libraries contained an average of 30,893 16S rDNA sequences per sample with an average length of 392 bases. FASTUNIFRAC-based comparisons of population phylogenetic distance demonstrated similarities between the populations of different treatment plants performing the same stabilization method (e.g. different MAD samples), and population differences among samples from different biosolids stabilization methods (COM, MAD, and TPAD). Based on a 0.03 Jukes-Cantor distance to 80 potential bacterial pathogens, all samples contained pathogens and enrichment ranged from 0.02% to 0.1% of sequences. Most (61%) species identified were opportunistic pathogens of the genera Clostridium and Mycobacterium. As risk sciences continue to evolve to address scenarios that include multiple pathogen exposure, the analysis described here can be used to determine the diversity of pathogens in an environmental sample. This work provides guidance for prioritizing subsequent culturable and quantitative analysis, and for the first time, ensuring that potentially significant pathogens are not left out of risk estimations.

Analysis of phylogenetic relationships in some species of the genus Lens using ITS sequences

SummaryWe compared sequence variations in the internal transcribed spacer (ITS) region of five Lens species, including cultivated lentil (Lens culinaris subsp. culinaris) and its four wild relatives distributed in Iran and Turkey, in order to clarify their genetic relationships. Cluster analysis based on the neighbour-joining algorithm and Jukes-Cantor distances revealed a phenetic tree with a topology congruent with patterns derived by previous studies using ITS sequence analysis. Phylogenetic analysis confirmed the divergence of L. nigricans from all other species. In this study, we found that some accessions of L. culinaris subsp. orientalis were very similar to L. culinaris subsp. culinaris, the cultivated lentil, but several others were not.

FastTree: Computing Large Minimum Evolution Trees with Profiles instead of a Distance Matrix

Gene families are growing rapidly, but standard methods for inferring phylogenies do not scale to alignments with over 10,000 sequences. We present FastTree, a method for constructing large phylogenies and for estimating their reliability. Instead of storing a distance matrix, FastTree stores sequence profiles of internal nodes in the tree. FastTree uses these profiles to implement Neighbor-Joining and uses heuristics to quickly identify candidate joins. FastTree then uses nearest neighbor interchanges to reduce the length of the tree. For an alignment with N sequences, L sites, and a different characters, a distance matrix requires O(N2) space and O(N2L) time, but FastTree requires just O(NLa + N) memory and O(Nlog (N)La) time. To estimate the tree's reliability, FastTree uses local bootstrapping, which gives another 100-fold speedup over a distance matrix. For example, FastTree computed a tree and support values for 158,022 distinct 16S ribosomal RNAs in 17 h and 2.4 GB of memory. Just computing pairwise Jukes–Cantor distances and storing them, without inferring a tree or bootstrapping, would require 17 h and 50 GB of memory. In simulations, FastTree was slightly more accurate than Neighbor-Joining, BIONJ, or FastME; on genuine alignments, FastTree's topologies had higher likelihoods. FastTree is available at http://microbesonline.org/fasttree.

Assessment of Protein Distance Measures and Tree-Building Methods for Phylogenetic Tree Reconstruction

Distance-based methods are popular for reconstructing evolutionary trees of protein sequences, mainly because of their speed and generality. A number of variants of the classical neighbor-joining (NJ) algorithm have been proposed, as well as a number of methods to estimate protein distances. We here present a large-scale assessment of performance in reconstructing the correct tree topology for the most popular algorithms. The programs BIONJ, FastME, Weighbor, and standard NJ were run using 12 distance estimators, producing 48 tree-building/distance estimation method combinations. These were evaluated on a test set based on real trees taken from 100 Pfam families. Each tree was used to generate multiple sequence alignments with the ROSE program using three evolutionary models. The accuracy of each method was analyzed as a function of both sequence divergence and location in the tree. We found that BIONJ produced the overall best results, although the average accuracy differed little between the tree-building methods (normally less than 1%). A noticeable trend was that FastME performed poorer than the rest on long branches. Weighbor was several orders of magnitude slower than the other programs. Larger differences were observed when using different distance estimators. Protein-adapted Jukes-Cantor and Kimura distance correction produced clearly poorer results than the other methods, even worse than uncorrected distances. We also assessed the recently developed Scoredist measure, which performed equally well as more complex methods.

MamMiBase: a mitochondrial genome database for mammalian phylogenetic studies

MamMiBase, the mammalian mitochondrial genome database, is a relational database of complete mitochondrial genome sequences of mammalian species. The database is useful for phylogenetic analysis, since it allows a ready retrieval of nucleotide and aminoacid individual alignments, in three different formats (NEXUS for PAUP program, for MEGA program and for PHYLIP program) of the 13 protein coding mitochondrial genes. The user may download the sequences that are useful for him/her based on their parameters values, such as sequence length, p-distances, base content, transition transversion ratio, gamma, which are also given by MamMiBase. A simple phylogenetic tree (neighbor-joining tree with Jukes Cantor distance) is also available for download, useful for parameter calculations and other simple tasks. MamMiBase is available at http://www.mammibase.lncc.br

First Detection of Human Metapneumovirus in Children with Acute Respiratory Infection in India: a Preliminary Report

In 2001, van den Hoogen et al. first reported the discovery of human metapneumovirus (HMPV) in respiratory patients from The Netherlands. The disease spectrum ranged from mild upper respiratory tract disease to severe bronchiolitis and pneumonia. Serological studies documented circulation of HMPV for at least 50 years (8). Subsequently, this virus has been recognized as a significant cause of acute respiratory illness (ARI) in infants, children, and adults worldwide (1, 2, 5). Though respiratory diseases are highly prevalent in India (7), the role of HMPV has not been examined so far. We analyzed 17 nasopharyngeal aspirate specimens collected from the pediatric inpatient department of Sassoon General Hospital, Pune (Maharashtra state), India, and 12 throat swab and 15 nasal swab specimens from outpatient departments during July-August 2003 for detection of HMPV by reverse transcription-PCR. These were stored in transport medium at −70°C till being tested. Total RNA was extracted from 250 μl of the respiratory specimens using the TRIZOL (LS) reagent (GIBCO BRL, Life Technologies) according to the manufacturer's instructions and used for nested reverse transcription-PCR according to the published protocol and primer sequences (6). Stringent precautions were taken to avoid false positivity in the PCR. Negative controls were included between two samples and subjected to the entire PCR protocol. Pre- and postamplification procedures were performed on the different floors of the laboratory. All five HMPV-RNA-positive PCR products were sequenced directly on both strands. The phylogenetic status of HMPV-positive specimens was assessed by employing the software MEGA (3). For analysis in MEGA, the Jukes-Cantor distance was utilized, employing the neighbor-joining algorithm. The reliability of different phylogenetic groupings was evaluated by using the bootstrap test (1,000 bootstrap replications) available in MEGA. Patients with severe ARI (17 children) admitted in the inpatient department had fever and severe lower respiratory tract infection and were diagnosed as having pneumonia or bronchopneumonia. Patients with mild ARI attending outpatient departments had symptoms of common cold, pharyngitis, laryngitis, tracheitis, bronchitis, body ache, fever, and weakness in different combinations. These included 9 children and 18 adults. HMPV RNA was detected in 19.2% (5 of 26) of the children and 0 of 18 adults. Importantly, four out of five patients in whom HMPV RNA was detected were children below the age of 1 year, and one was from the age group 1 to 5 years. The GenBank accession numbers for the sequences generated during this study are {type:entrez-nucleotide,attrs:{text:AY532377,term_id:46488852,term_text:AY532377}}AY532377 to {type:entrez-nucleotide,attrs:{text:AY532381,term_id:46488860,term_text:AY532381}}AY532381. Phylogenetic analysis based on a 216-nucleotide fragment of the M gene (Figure ​(Figure1)1) demonstrated genetic heterogeneity of the Indian HMPV viral isolates with 100% bootstrap support. Four isolates (INDHMP1 and INDHMP2, both from mild cases, and INDHMP4 and INDHMP5, both from severe cases) clustered with several Canadian strains isolated in 1997, 1998, and 2000. Isolate INDHMP3 (from a severe case) belonged to a distinct second lineage comprising isolates from The Netherlands (2000) and Canada (1997, 1998, 1999, and 2002). In the second lineage, the Indian isolate (INDHMPV3) was separated from other isolates, forming a separate subgroup. FIG. 1. Phylogenetic analysis of partial M gene (216-base fragment) sequences of 24 HMPV isolates. See the text for details of the isolates and abbreviations used. Percent bootstrap support is indicated by the values at each node. The results of this preliminary study showed that during the 2-month study period, HMPV was detected in nearly five children with respiratory infections. Though none of the 18 adults were positive, unless a larger sample size is screened, infrequent infection of adults with HMPV cannot be concluded. The fact that four of the five PCR-positive patients were infants suggests importance of HMPV in this vulnerable age group. Importantly, both severe and mild cases yielded PCR positivity, demonstrating the clinical spectrum of the disease. The presence of viral RNA could be demonstrated in nasopharyngeal aspirates (n = 3) as well as nasal swabs (n = 2). The latter specimens are easy to collect. Reports from Italy have shown similar observations (4). M-gene-based phylogenetic analysis demonstrated cocirculation of distinctly different HMPV strains in Pune in 2003. The four isolates belonging to a lineage represented both mild and severe cases, whereas the only isolate belonging to the other lineage came from a severe case. In summary, the present study suggests the importance of HMPV in causing mild and severe respiratory infections among children, especially infants in India. Multicentric, long-term studies from different parts of India are needed to assess the disease burden.

Genetic population structure of the deep-sea whelk Buccinum tsubai in the Japan Sea

: The purpose of the present paper was to examine the genetic population structure of Buccinum tsubai in the Japan Sea. Mitochondrial 16SrRNA gene sequence analysis was performed with specimens from various stations in the Japan Sea. Mitochondrial lineages were clearly patterned geographically in four separate areas: the Hokkaido area, the Yamagata–Toyama area, the Yamato Bank area, and the San’in area. The main distribution depth range of B. tsubai is between 200 m and 1000 m isodepths, and the horizontal distance between the 200 m and 1000 m isodepth lines represents the specific spatial scale of the habitat (SSSH). These four areas were separated either by the complete discontinuity of the SSSH area or by its narrow spatial extension. Genetic distances between the main haplotypes of each area were calculated as Jukes–Cantor distances, the value of which ranged between 0.012 and 0.017. This value seemed to be unrelated to the geographic distance. There was no tendency for clustering according to depth. In future, the morphological characters of the four lineages of B. tsubai should be compared in detail in order to elucidate significant genetic differences among them.

Genomic divergence between human and chimpanzee estimated from large-scale alignments of genomic sequences.

To study the genomic divergence between human and chimpanzee, large-scale genomic sequence alignments were performed. The genomic sequences of human and chimpanzee were first masked with the RepeatMasker and the repeats were excluded before alignments. The repeats were then reinserted into the alignments of nonrepetitive segments and entire sequences were aligned again. A total of 2.3 million base pairs (Mb) of genomic sequences, including repeats, were aligned and the average nucleotide divergence was estimated to be 1.22%. The Jukes-Cantor (JC) distances (nucleotide divergences) in nonrepetitive (1.44 Mb) and repetitive sequences (0.86 Mb) are 1.14% and 1.34%, respectively, suggesting a slightly higher average rate in repetitive sequences. Annotated coding and noncoding regions of homologous chimpanzee genes were also retrieved from GenBank and compared. The average synonymous and nonsynonymous divergences in 88 coding genes are 1.48% and 0.55%, respectively. The JC distances in intron, 5' flanking, 3' flanking, promoter, and pseudogene regions are 1.47%, 1.41%, 1.68%, 0.75%, and 1.39%, respectively. It is not clear why the genetic distances in most of these regions are somewhat higher than those in genomic sequences. One possible explanation is that some of the genes may be located in regions with higher mutation rates.

Branch length heterogeneity leads to nonindependent branch length estimates and can decrease the efficiency of methods of phylogenetic inference.

Branch length estimates play a central role in maximum-likelihood (ML) and minimum-evolution (ME) methods of phylogenetic inference. For various reasons, branch length estimates are not statistically independent under ML or ME. We studied the response of correlations among branch length estimates to the degree of among-branch length heterogeneity (BLH) in the model (true) tree. The frequency and magnitude of (especially negative) correlations among branch length estimates were both shown to increase as BLH increases under simulation and analytically. For ML, we used the correct model (Jukes-Cantor). For ME, we employed ordinary least-squares (OLS) branch lengths estimated under both simple p-distances and Jukes-Cantor distances, analyzed with and without an among-site rate heterogeneity parameter. The efficiency of ME and ML was also shown to decrease in response to increased BLH. We note that the shape of the true tree will in part determine BLH and represents a critical factor in the probability of recovering the correct topology. An important finding suggests that researchers cannot expect that different branches that were in fact the same length will have the same probability of being accurately reconstructed when BLH exists in the overall tree. We conclude that methods designed to minimize the interdependencies of branch length estimates (BLEs) may (1) reduce both the variance and the covariance associated with the estimates and (2) increase the efficiency of model-based optimality criteria. We speculate on possible ways to reduce the nonindependence of BLEs under OLS and ML.

Characterization of two Perkinsus spp. from the softshell clam, Mya arenaria using the small subunit ribosomal RNA gene.

Sequence analysis and riboprinting of the small subunit ribosomal RNA genes were used to characterize two morphologically different Perkinsus species isolates from the gill (G117) and the hemolymph (H49) of the softshell clam, Mya arenaria. Sequence data of the polymerase chain reaction amplified ribosomal RNA loci of G117 and H49 indicated that these genes are 1803 and 1806 base-pair long, respectively. A sequence similarity of > 98.9% was calculated among ribosomal RNA sequences of the two isolates of this study and the published sequences of Perkinsus marinus from the American eastern oyster, Crassostrea virginica, and Perkinsus sp. from the blood cockle of the Australian mollusc, Anadara trapezia. From a phylogenetic tree obtained from Jukes-Cantor distances of the aligned ribosomal RNA gene sequences of 13 eukaryotic taxa using the Neighbor-Joining method, we showed that G117 and H49 clustered within the genus Perkinsus. Guided by the sequence data of Perkinsus marinus (accession # X75762) and Perkinsus sp. (accession # L07375), restriction endonucleases were selected for restriction fragment analysis of polymerase chain reaction products of the small subunit ribosomal RNA genes (riboprinting). Riboprinting was used to distinguish the four members of the genus Perkinsus from each other.

Polymorphism and divergence at a Drosophila pseudogene locus.

The larval cuticle protein (Lcp) cluster in Drosophila melanogaster contains four functional genes and a closely related pseudogene. A 630-bp fragment including the larval cuticle pseudogene locus (Lcp psi) was nucleotide sequenced in 10 strains of D. melanogaster and a 458-bp Lcp psi fragment from D. simulans was also sequenced. We used these data to test the hypotheses that the rates of synonymous and nonsynonymous substitution are equal, that the absolute levels of variation are higher than in functional genes, and that intraspecific polymorphism is correlated with interspecific divergence. As predicted, synonymous and nonsynonymous substitution rates were equivalent, and overall nucleotide divergence between D. melanogaster and D. simulans (Jukes-Cantor distance = 0.149 +/- 0.150) was extremely high. However, within-species DNA sequence comparisons at Lcp psi revealed lower levels of polymorphism (theta = 0.001 +/- 0.001) than at many functional loci in D. melanogaster. Using the HUDSON, KREITMAN, and AGUADE (HKA) test, we show that the level of polymorphism in Lcp psi within D. melanogaster is lower than expected given the amount of divergence between D. melanogaster and D. simulans when the pseudogene data are compared to the Adh 5' flanking region. Because the Lcp psi lies in a region of relatively infrequent recombination, we suggest that the low level of within-species polymorphism is the result of background selection.

ITS1 sequences of nuclear ribosome DNA in rice species from China and their phylogenetic implications

This study assessed the capability of using internal transcribed spacer (ITS1) sequences for reconstructing the phylogeny of rice species from China. Three accessions of wild and two accessions of cultivated rice were used (Table 1). Total DNA was extracted from leaf samples following the procedures described by Tai and Tanksley (1990). Polymerase chain reaction (PCR) amplifications were performed in 50 μL volumes containing 100 ng DNA template and 2.5 μL Taq polymerase. A PE-480 thermal cycler was used under the following conditions: 35 cycles of 94°C for 1 min, 57°C for 1 min, and 72°C for 1 min. PCR-amplified DNA products were recovered, complemented, and then cloned into the vector pUC 118 with standard protocols. Sequencing of ITS1 was conducted on an AB1 373A automated sequencer using a TaqDye Primer Cycle Sequencing Kit (ABI). The ITS1 sequences of the five taxa together with a published rice sequence (Takaiwa et al 1985) were aligned with the Clustal V program. The data matrix was analyzed using UPGMA with Jukes-Cantor distance and Neighbor-Joining with Kimura 2-parameter distance of the MEGA version 1.02 (Kura et al 1993).