Intergenic Spacer Sequence Analysis Research Articles

Genotyping, antifungal susceptibility testing, and biofilm formation of Trichosporon spp. isolated from urine samples in a University Hospital in Bangkok, Thailand.

The basidiomycetes yeast Trichosporon is widespread in the natural environment, but can cause disease, mainly in immunocompromised patients. However, there have been only few studies about this infection in Thailand. In this study, we characterized 53 Trichosporon spp. isolated from urine samples from patients admitted to a single hospital in Bangkok, Thailand over a one-year period from 2019 to 2020. The strains were identified using colony morphology, microscopy, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and nucleotide sequence analysis of intergenic spacer 1 (IGS1). Fifty-one isolates were Trichosporon asahii, and the remaining isolates were Trichosporon inkin and other Trichosporon species. Three genotypes of IGS1-1, 3, and 7 were observed among T.asahii. The sensitivity of the yeasts to the antifungal drugs amphotericin B, fluconazole, and voriconazole ranged from 0.25 to >16 μg ml-1, 0.5-8 μg ml-1, and 0.01-0.25 μg ml-1, respectively. We investigated biofilm formation by the isolates, and no biofilm production was found in one isolate, low biofilm production in forty-four isolates, and medium biofilm production in six isolates. T. inkin produced biofilms at low levels, and Trichosporon spp. produced biofilms at medium levels. This research increases our understanding of the molecular epidemiology of Trichosporon spp. isolated from one university hospital in Bangkok, Thailand, and reveals their genetic diversity, antifungal susceptibility profiles, and capacity for in vitro biofilm production.

Morphological Characterization and DNA Barcoding of Duckweed Species in Saudi Arabia.

Duckweeds, or Lemnaceae, are widespread aquatic plants. Morphology-based identification of duckweed species is difficult because of their structural complexity. Hence, molecular tools provide significant advantages for characterizing and selecting species or clones for sustainable commercial use. In this study, we collected and characterized ten duckweed isolates from nine different regions in Saudi Arabia (SA). Based on the morphological characterization and phylogenetic analysis of intergenic spacer sequences of chloroplast DNA using six barcoding markers, the clones were classified into three genera, represented by seven species: Lemna gibba L., Lemna minor L., Lemna japonica Landolt, Lemna aequinoctialis Welw., Lemna perpusilla Torr., Spirodela polyryiza (L.) Schleid., and Landoltia punctate G. Mey. Lemna gibba was revealed to be a distinct dominant duckweed species in many regions of SA. Five barcoding markers showed that L. gibba, L. minor, and L. punctata were the most widely distributed species in the country. However, L. punctata, L. perpusilla, and S. polyryiza were the dominant species in the Al-Qassim, Madinah-1, and Madinah-2 regions, respectively. Moreover, the morphological traits revealed variations for these clones, relative to other studied duckweed clones. According to the results obtained in this study, three out of six plastid markers (trnH-psbA, matK, and atpF-atpH) helped to identify the dominant duckweed species in Saudi Arabia. Further evaluation based on adaptability, molecular genetic studies, and functional genomics is needed for these species to be used at the commercial level in Saudi Arabia.

Identification of thermo tolerant lactic acid bacteria isolated from silage prepared in the hot and humid climate of Southwestern Japan

To develop high-quality silage starters adapted to hot and humid weather, 12 LAB isolates from silage produced in Kyushu and Okinawa, Japan were characterized based on their morphological features, growth curves and sugar utilization. In addition, the nucleotide sequences of the V2-V3 region of their 16S rRNA genes and the 16S-23S rRNA intergenic spacer (ITS) regions were determined. The isolates were also subjected to RAPD-PCR analysis, DNA-DNA hybridization, G+C content analysis and immuno-identification using species-specific monoclonal antibodies and SDS-PAGE profiling. Nearly all of the isolates exhibited high thermotolerance and rapid growth. Combining ITS sequence analysis, RAPD-PCR and immuno-identification enabled rapid and accurate identification of closely related LAB strains that other methods failed to appropriately differentiate; for example, L. plantarum was distinguished from L. pentosus, and L. casei was distinguished from L. rhamonsus. Using the aforementioned techniques, the isolated strains were identified as L. plantarum, L. rhamonsus, L. rapi, Pediococcus pentosaceus and P. lolii. Our findings also showed that there is greater diversity among thermophilic LABs in silage prepared in a hot and humid environment.

16S-23S Intergenic Spacer (ITS) Region Sequence Analysis: Applicability and Usefulness in Identifying Genera and Species Resembling Non- Hemolytic Streptococci

The species that are catalase-negative, gram-positive cocci and not belonging to streptococci or enterococci have become increasingly well characterized and the number of taxonomic entities steadily growing based on molecular taxonomic studies. This complicates their identification. 16S-23S Intergenic Spacer (ITS) region sequence analysis has proven to be a useful tool for species identification of the genera Streptococcus and Enterococcus. This study investigated the possibility of using ITS sequence analysis as a common tool for species identification within the genera Aerococcus, Abiotrophia, Alloiococcus, Dolosicoccus, Dolosigranulum, Facklamia, Granulicatella, Gemella, Ignavigranum, Leuconostoc, and Vagococcus. ITS sequences of 29 type strains and 103 well-characterized clinical strains were determined and BLAST analysis performed for species identification. All clinical strains were convincingly identified to the species level. Phylogenetic analysis showed distinct clustering of strains with the allocated species and the respective type strains. Thus, ITS sequence analysis was useful for species identification of bacteria belonging to the genera that are catalase-negative and gram-positive cocci. Potentially, ITS could be considered as the first line identification tool for the group of catalase-negative, gram-positive cocci, including nonhemolytic streptococci, enterococci and the taxons examined in this study.

Molecular Detection of Bartonella Sp. in Wild Ruminants and Analysis of its Genetic Diversity on the Basis of 16S–23S Rrna Intergenic Spacer (ITS)

Abstract The aim of this study was to describe the state of infection of roe deer (Capreolus capreolus) and red deer (Cervus elaphus) by Bartonella sp. in North-Western Poland through PCR detection of Bartonella 16S-23S rRNA ITS region in isolates of animal tissues, and also to describe the genetic diversity of detected Bartonella species based on molecular analysis of ITS. The multiple alignment analysis of ITS sequences was carried out, and homology matrices and phylogenetic trees were constructed. The DNA of Bartonella sp. was detected in tissues of 45.6% (36/79) C. capreolus and of 50% (15/30) C. elaphus. Products of two different sizes were detected: 317 bp, characteristic for B. schoenbuchensis, and 198 bp, characteristic for B. bovis. The obtained results suggest that roe and red deer are potential reservoirs of Bartonella sp. Most of the analysed ITS sequences was not specific for one host species. In constructed phylogenetic trees, sequences obtained from roe and red deer clustered together. These results suggest a lack of host specificity of most detected B. schoenbuchensis and B. bovis intraspecies strains

Development of species-specific primers for identifying Auricularia auricula-judae using intergenic spacer 1 (IGS1) sequences

Auricularia auricula-judae, an important edible and medicinal mushroom was first cultivated in China more than one thousand years ago. In this study, we developed a simple and rapid polymerase chain reaction (PCR)-based diagnostic tool for identifying A. auricula-judae from other morphologically similar Auricularia species.Based on the sequence analysis of intergenic spacer 1 (IGS1) in genus Auricularia, two species-specific primer combinations were designed from IGS1 sequence of A. auricula-judae, both of them can generate DNA fragments of different size unique toA. auricula-judae. The validity of this two primer combinations were confirmed by correctly amplifying species-specific fragments from genomic DNA of another 32 A. auricula-judae strains. To our knowledge, this was the first study on development of species-specific primers via IGS1 sequence in jelly fungi and also the first application of IGS1 sequence in genus Auricularia. Key words: Auricularia, Auricularia auricula-judae, intergenic spacer, species-specific primer.

Development of species-specific primers for identifying Auricularia auricula-judae using intergenic spacer 1 (IGS1) sequences

Auricularia auricula-judae , an important edible and medicinal mushroom was first cultivated in China more than one thousand years ago. In this study, we developed a simple and rapid polymerase chain reaction (PCR)-based diagnostic tool for identifying A. auricula-judae from other morphologically similar Auricularia species. Based on the sequence analysis of intergenic spacer 1 (IGS1) in genus Auricularia , two species-specific primer combinations were designed from IGS1 sequence of A. auricula-judae , both of them can generate DNA fragments of different size unique to A. auricula-judae . The validity of this two primer combinations were confirmed by correctly amplifying species-specific fragments from genomic DNA of another 32 A. auricula-judae strains. To our knowledge, this was the first study on development of species-specific primers via IGS1 sequence in jelly fungi and also the first application of IGS1 sequence in genus Auricularia .

Molecular characterization through IGS sequencing of formae speciales of Fusarium oxysporum pathogenic on lamb's lettuce.

Twenty-nine strains of Fusarium oxysporum isolated from wilted lamb’s lettuce plants ( Valerianella olitoria ) along with eight ATCC reference strains were examined for differences in the nucleotide sequences of the ribosomal DNA (rDNA) intergenic spacer (IGS) region, which is about 2.5 kb long in these strains. A phylogenetic (neighbour-joining) analysis was performed on the strains and identified four phylogenetic groups, I, II, III, and IV. Most F. oxysporum isolates recovered from wilted lamb’s lettuce plants cultivated in northern Italy clustered in group I and were very similar to F. oxysporum f. sp. raphani . All the isolates, including the 8 control strains, were tested for pathogenicity on lamb’s lettuce cv. Trophy cultivated in the glasshouse. Most isolates from northern Italy were pathogenic on lamb’s lettuce, displaying varying degrees of virulence, and their IGS sequence were similar to the forma specialis raphani . However, four isolates were not pathogenic on lamb’s lettuce. Among the control strains, six showed moderate virulence; these belonged to the formae speciales raphani and conglutinans ; and two, belonging to the forma specialis matthioli (ATCC16602 and ATCC16603) were not pathogenic on lamb’s lettuce. In conclusion, analysis of the IGS sequences indicated that the isolates studied had different origins, and that phylogeny and pathogenicity were related; non-pathogenic isolates differed genetically from isolates that were poorly, moderately or highly virulent. To the best of our knowledge, this is the first report on the differences between formae speciales of F. oxysporum on lamb’s lettuce plants as determined by IGS sequence analysis.

Rhizobium mesosinicum sp. nov., isolated from root nodules of three different legumes

Thirteen novel strains were isolated from root nodules of three different leguminous plants of the genera Albizia, Kummerowia and Dalbergia grown in China. Cells of these strains were Gram-negative, strictly aerobic, non-spore-forming, motile rods. Phylogenetic analysis of 16S rRNA gene sequences revealed that they belong to the genus Rhizobium. A representative strain, CCBAU 25010T, showed 98.8% 16S rRNA gene sequence similarity to its closest phylogenetic relative, Rhizobium sullae IS123T. The 16S-23S intergenic spacer (ITS) sequence of CCBAU 25010T shared 91.5 and 87.2% sequence similarity, respectively, with those of Rhizobium etli CFN 42T and Rhizobium leguminosarum LMG 14904T. Analysis of the sequences of the housekeeping genes atpD and recA was in agreement with the results of ITS sequence analysis. The nodC gene sequence of CCBAU 25010T was identical to that of Rhizobium tropici CFN 299. DNA-DNA hybridization values for strain CCBAU 25010T ranged from 20.7% (with Rhizobium mongolense USDA 1844T) to 34.4% (with R. leguminosarum USDA 2370T). Cell protein SDS-PAGE, BOX-PCR and several phenotypic characteristics, such as use of sole carbon sources and antibiotic resistance, could differentiate the novel strains from defined Rhizobium species. We therefore propose that the novel strains reported in this study form a novel species, Rhizobium mesosinicum sp. nov., with the type strain CCBAU 25010T (=LMG 24135T =JCM 14777T).

Identification of Acinetobacter Genomic Species 13TU by Sequence Analysis of the 16S-23S rRNA Gene Spacer Region

The genus Acinetobacter currently contains up to 32 described named and unnamed (genomic) species (3). Acinetobacter baumannii, genomic species 3, and 13TU, three of the most clinically relevant species, are genetically and phenotypically very similar to an environmental species, Acinetobacter calcoaceticus, and are therefore grouped together into the so-called A. calcoaceticus-A. baumannii (Acb) complex (3). Because phenotypic identification of Acinetobacter isolates to the species level has proven to be insufficient, several genotypic methods have been developed for genomic species identification (1, 3, 4). In the last 3 years, our laboratory has routinely used the 16S-23S rRNA gene intergenic spacer (ITS) sequence-based method described by Chang et al. (1) to delineate isolates belonging to the Acb complex to the genomic species level. We found the procedure accurate and easy to perform. However, we have recently encountered a discrepancy during the characterization of Acinetobacter strain 74510 isolated originally from a clinical source at the Prince of Wales Hospital, Hong Kong, in 1995 (2). Strain 74510 was previously identified as genomic species 13TU by restriction analysis of amplified ribosomal DNA (2, 4). However, examination of the single PCR amplicon of the ITS region of strain 74510 (GenBank accession no. {type:entrez-nucleotide,attrs:{text:FJ360743,term_id:210063372,term_text:FJ360743}}FJ360743) revealed an ITS sequence length of 607 bp, as opposed to the 615 bp reported by Chang et al. for the genomic species 13TU reference strain BCRC 15417 (1). On the other hand, DNA sequence analysis of this 607-bp ITS of strain 74510 showed an identity of 0.9561 to that of strain BCRC 15417 (Table ​(Table1).1). Upon further analysis of nucleotide sequences in GenBank, the strain 74510 ITS sequence was also found to share increasing identity (0.9950 to 0.9967) with three other genomic species 13TU strains, namely, v104-2, 00574, and DR25612/96 (Table ​(Table1).1). Interestingly, the ITS length (607 bp) of these three genomic species 13TU strains was also 607 bp, identical to that of strain 74510 (Table ​(Table1).1). In addition, the ITS sequences of these genomic species 13TU strains 74510, v104-2, 00574, and DR25612/96 appeared to be more closely related to the A. baumannii (genomic species 2) strain BCRC 10591T than to the 13TU reference strain BCRC 15417 (0.9538 to 0.9588 versus 0.9171 identity) (Table ​(Table1),1), which reinforces the notion that genetic similarity among A. baumannii and genomic species 13TU is high (3) and perhaps suggests that BCRC 15417 may not be as representative for genomic species 13TU. TABLE 1. ITS sequence similarity between A. baumannii and genomic species 13TUa Acinetobacter genomic species 13TU is the second most clinically relevant species in the Acb complex (3, 5, 6). Therefore, we believe that it is important to extend the number of Acinetobacter genomic species 13TU ITS sequences in the ITS sequence database for more-accurate genomic species identification. Also, we would suggest that the reference ITS sequence length for genomic species 13TU be 607 bp long and that strain 74510 could be considered as the “type” strain for genomic species 13TU.

Retama species growing in different ecological–climatic areas of northeastern Algeria have a narrow range of rhizobia that form a novel phylogenetic clade within the Bradyrhizobium genus

Sixty-seven isolates were isolated from nodules collected on roots of Mediterranean shrubby legumes Retama raetam and Retama sphaerocarpa growing in seven ecological–climatic areas of northeastern Algeria. Genetic diversity of the Retama isolates was analyzed based on genotyping by restriction fragment length polymorphism of PCR-amplified fragments of the 16S rRNA gene, the intergenic spacer (IGS) region between the 16S and 23S rRNA genes (IGS), and the symbiotic genes nifH and nodC. Eleven haplotypes assigned to the Bradyrhizobium genus were identified. Significant biogeographical differentiation of the rhizobial populations was found, but one haplotype was predominant and conserved across the sites. All isolates were able to cross-nodulate the two Retama species. Accordingly, no significant genetic differentiation of the rhizobial populations was found in relation to the host species of origin. Sequence analysis of the 16S rRNA gene grouped the isolates with Bradyrhizobium elkanii, but sequence analyses of IGS, the housekeeping genes ( dnaK, glnII, recA), nifH, and nodC yielded convergent results showing that the Retama nodule isolates from the northeast of Algeria formed a single evolutionary lineage, which was well differentiated from the currently named species or well-delineated unnamed genospecies of bradyrhizobia. Therefore, this study showed that the Retama species native to northeastern Algeria were associated with a specific clade of bradyrhizobia. The Retama isolates formed three sub-groups based on IGS and housekeeping gene phylogenies, which might form three sister species within a novel bradyrhizobial clade.

Identification of Clinically Relevant Nonhemolytic Streptococci on the Basis of Sequence Analysis of 16S-23S Intergenic Spacer Region and Partial gdh Gene

Nonhemolytic streptococci (NHS) cause serious infections, such as endocarditis and septicemia. Many conventional phenotypic methods are insufficient for the identification of bacteria in this group to the species level. Genetic analysis has revealed that single-gene analysis is insufficient for the identification of all species in this group of bacteria. The aim of the present study was to establish a method based on sequence analysis of the 16S-23S intergenic spacer (ITS) region and the partial gdh gene to identify clinical relevant NHS to the species level. Sequence analysis of the ITS region was performed with 57 NHS reference or clinical strains. Satisfactory identification to the species level was achieved for 14/19 NHS species included in this study on the basis of sequence analysis of the ITS region. Streptococcus salivarius and Streptococcus vestibularis obtained the expected taxon as the best taxon match, but there was a short maximum score distance to the next best match (distance, <10). Streptococcus mitis, Streptococcus oralis, and Streptococcus pneumoniae could not be unambiguously discriminated by sequence analysis of the ITS region, as was also proven by phylogenetic analysis. These five species could be identified to the group level only by ITS sequence analysis. Partial gdh sequence analysis was applied to the 11 S. oralis strains, the 11 S. mitis strains, and the 17 S. pneumoniae strains. All except one strain achieved a satisfactory identification to the species level. A phylogenetic algorithm based on the analysis of partial gdh gene sequences revealed three distinct clusters. We suggest that sequence analysis of the combination of the ITS region and the partial gdh gene can be used in the reference laboratory for the species-level identification of NHS.

Molecular identification, genotyping, and drug susceptibility of the basidiomycetous yeast pathogenTrichosporonisolated from Turkish patients

Deep-seated infections due to Trichosporon species are emerging mycoses that have a very poor prognosis in patients with persistent neutropenia. This study elucidated the mycological characteristics of Trichosporon strains obtained from deep-seated infections in Turkish patients and identified by DNA sequence analysis of intergenic spacer (IGS) region 1 of the rDNA locus. In addition, we genotyped the major causative agent, T. asahii, and evaluated the in vitro drug susceptibility of the isolates. While 87 (81.3%) of the 107 isolates were T. asahii, the remaining 20 were T. faecale (14.0%), T. asteroids (0.9%), T. coremiiforme (0.9%), T. japonicum, (0.9%), T. lactis (0.9%), and a new species (0.9%). In addition to the eight known T. asahii genotypes, one novel genotype was identified. The distribution of the T. asahii genotypes in this study were genotype 1 (79.3%), followed by 5 (8.0%), 3 (6.9%), 6 (3.4%), 4 (1.1%), and 9 (1.1%). Turkish isolates showed low susceptibility to amphotericin B, 5-flucytosine, and fluconazole. Although relatively low minimum inhibitory concentrations (MICs) were found with all drugs, voriconazole appeared to be the most active. The MICs of the non-Trichosporon asahiiTrichosporon species were similar to those of the T. asahii strains. Our findings suggest that Trichosporon species isolated from Turkish patients are more diverse than those reported from other countries.

Presence of a novel 16S–23S rRNA gene intergenic spacer insert in Bradyrhizobium canariense strains

Seven slow-growing bacterial strains isolated from root nodules of yellow serradella (Ornithopus compressus) that originated from Asinara Island on North Western Sardinia in Italy were characterized by partial 16S rRNA gene and intergenic spacer (ITS) sequencing as well as amplified fragment length polymorphism (AFLP) genomic fingerprinting. The results indicated that the O. compressus isolates belong to the Bradyrhizobium canariense species. The analysis of ITS sequences divided the branch of B. canariense strains into two statistically separated groups (ITS clusters I and II). All the strains in ITS cluster I showed the presence of unique oligonucleotide insert TTAGAGACTTAGGTTTCTK. This insert was neither found in other described species of the family Rhizobiaceae nor in any other bacterial families and can be used as a natural and high selective genetic marker for ITS cluster I of B. canariense strains. ITS grouping of O. compressus isolates was supported by the unweighted pair group method with arithmetic averages cluster analysis of their AFLP patterns, suggesting that the strains of ITS cluster II were genetically closer to each other than to isolates from the ITS cluster I. A taxonomic importance is supposed of the revealed 19 bp ITS insert for an intraspecific division within high heterogeneous B. canariense species.

Species-Level Identification of Isolates of the Acinetobacter calcoaceticus - Acinetobacter baumannii Complex by Sequence Analysis of the 16S-23S rRNA Gene Spacer Region

The species Acinetobacter calcoaceticus, A. baumannii, genomic species 3, and genomic species 13TU included in the Acinetobacter calcoaceticus-Acinetobacter baumannii complex are genetically highly related and difficult to distinguish phenotypically. Except for A. calcoaceticus, they are all important nosocomial species. In the present study, the usefulness of the 16S-23S rRNA gene intergenic spacer (ITS) sequence for the differentiation of (genomic) species in the A. calcoaceticus-A. baumannii complex was evaluated. The ITSs of 11 reference strains of the complex and 17 strains of other (genomic) species of Acinetobacter were sequenced. The ITS lengths (607 to 638 bp) and sequences were highly conserved for strains within the A. calcoaceticus-A. baumannii complex. Intraspecies ITS sequence similarities ranged from 0.99 to 1.0, whereas interspecies similarities varied from 0.86 to 0.92. By using these criteria, 79 clinical isolates identified as A. calcoaceticus (18 isolates) or A. baumannii (61 isolates) with the API 20 NE system (bioMerieux Vitek, Marcy l'Etoile, France) were identified as A. baumannii (46 isolates), genomic species 3 (19 isolates), and genomic species 13TU (11 isolates) by ITS sequencing. An identification rate of 96.2% (76 of 79 isolates) was obtained by using ITS sequence analysis for identification of isolates in the A. calcoaceticus-A. baumannii complex, and the accuracy of the method was confirmed for a subset of strains by amplified rRNA gene restriction analysis and genomic DNA analysis by AFLP analysis by using libraries of profiles of reference strains. In conclusion, ITS sequence-based identification is reliable and provides a promising tool for elucidation of the clinical significance of the different species of the A. calcoaceticus-A. baumannii complex.

Specific detection of Bradyrhizobium and Rhizobium strains colonizing rice (Oryza sativa) roots by 16S-23S ribosomal DNA intergenic spacer-targeted PCR.

In addition to forming symbiotic nodules on legumes, rhizobial strains are members of soil or rhizosphere communities or occur as endophytes, e.g., in rice. Two rhizobial strains which have been isolated from root nodules of the aquatic legumes Aeschynomene fluminensis (IRBG271) and Sesbania aculeata (IRBG74) were previously found to promote rice growth. In addition to analyzing their phylogenetic positions, we assessed the suitability of the 16S-23S ribosomal DNA (rDNA) intergenic spacer (IGS) sequences for the differentiation of closely related rhizobial taxa and for the development of PCR protocols allowing the specific detection of strains in the environment. 16S rDNA sequence analysis (sequence identity, 99%) and phylogenetic analysis of IGS sequences showed that strain IRBG271 was related to but distinct from Bradyrhizobium elkanii. Rhizobium sp. (Sesbania) strain IRBG74 was located in the Rhizobium-Agrobacterium cluster as a novel lineage according to phylogenetic 16S rDNA analysis (96.8 to 98.9% sequence identity with Agrobacterium tumefaciens; emended name, Rhizobium radiobacter). Strain IRBG74 harbored four copies of rRNA operons whose IGS sequences varied only slightly (2 to 9 nucleotides). The IGS sequence analyses allowed intraspecies differentiation, especially in the genus Bradyrhizobium, as illustrated here for strains of Bradyrhizobium japonicum, B. elkanii, Bradyrhizobium liaoningense, and Bradyrhizobium sp. (Chamaecytisus) strain BTA-1. It also clearly differentiated fast-growing rhizobial species and strains, albeit with lower statistical significance. Moreover, the high sequence variability allowed the development of highly specific IGS-targeted nested-PCR assays. Strains IRBG74 and IRBG271 were specifically detected in complex DNA mixtures of numerous related bacteria and in the DNA of roots of gnotobiotically cultured or even of soil-grown rice plants after inoculation. Thus, IGS sequence analysis is an attractive technique for both microbial ecology and systematics.

Evolutionary divergence of promoters and spacers in the rDNA family of four Drosophila species : Implications for molecular coevolution in multigene families

The organization and sequence of the rDNA multigene family of four Drosophila species ( melanogaster, orena, virilis and hydei) have been compared in order to understand the quality and quantity of the differences which are involved with interspecific divergence of promoters and the polymerase I complexes (molecular coevolution). Each species has an intergenic spacer (IGS) made up of subrepeats which contain duplications of the promoter. Major structural and point-mutational differences exist, most of which have been spread by unequal crossingover through the family and species. Structural differences involve the types, lengths and copy-number of the IGS subrepeats, and the lengths and position of “unique” regions between blocks of repeats. The 240 base-pair repeat array shared by D. melanogaster and D. orena has been replaced by a 220 base-pair repeat, and the 95 and 330 base-pair arrays are absent altogether in D. virilis and D. hydei. The length of the “unique” region between the 240/220 base-pair arrays and the start of transcription varies, with the unusual situation of the last of the 220 repeats ending at the external transcribed spacer (ETS) boundary in D. virilis. Other structural differences involve regions of high cryptic simplicity arising from slippage in D. virilis and D. hydei IGSs. Sequence analysis of IGS and the ETSs indicates that the rDNA is not uniformly divergent throughout its length. Apart from the genes, there are regions of relatively high conservation covering the promoter regions and at some but not all potential RNA processing sites. The conserved promoter regions are more extensive within each pair of species D. melanogaster versus D. orena and D. virilis versus D. hydei, in keeping with their phylogenetic distances. Slippage-like mechanisms are involved with large numbers of deletions/insertions that make up the ETS differences between the species. Patterns of shared mutations between IGS subrepeats indicate stages of transition during rDNA differentiation by continual homogenization. The simultaneous operation of different turnover mechanisms, at different periodicities and rates, generates a complex picture of reorganization, some of which would influence the process of molecular coevolution in the family.