Internal Repetition and Intraindividual Variation in the rDNA ITS1 of the <Emphasis Type="Italic">Anopheles punctulatus</Emphasis> Group (Diptera: Culicidae): Multiple Units and Rates of Turnover

The rapid divergence of repetitive sequences makes them desirable markers for phylogenetic studies of closely related groups, provided that a high level of sequence homogeneity has been maintained within species. Intraspecific polymorphisms are found in an increasing number of studies now, and this highlights the need to determine why these occur. In this study we examined intraindividual variation present in the first ribosomal internal transcribed spacer (ITS1) from a group of cryptic mosquito species. Individuals of the Anopheles punctulatus group contained multiple ITS1 length variants that ranged from 1.2 to 8.0 kb. Nucleotide and copy number variation for several homologous internal repeats is common, yet the intraspecific sequence divergence of cloned PCR isolates is comparable to that of other mosquito species (~0.2–1.5%). Most of the length variation is comprised of a 5′-ITS1 repeat that was identified as a duplication of a conserved ITS2 region. Secondary structure conservation for this repeat is pronounced and several repeat types that are highly homogenized have formed. Significant interspecific divergence indicates a high rate of evolutionary change for this spacer. A maximum likelihood tree constructed here was congruent with previous phylogenetic hypotheses and suggests that concerted evolution is also accompanied by interpopulation divergence. The lack of interindividual differences and the presence of homogenized internal repeats suggest that a high rate of turnover has reduced the overall level of variation. However, the intraindividual variation also appears to be maintained by the absence of a single turnover rate and the complex dynamics of ongoing recombination within the spacer. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Marked Intragenomic Heterogeneity and Geographical Differentiation of nrDNA ITS in <Emphasis Type="BoldItalic">Larix</Emphasis><Emphasis Type="BoldItalic">potaninii</Emphasis> (Pinaceae)

Nuclear ribosomal DNA (nrDNA) of gymnosperms, especially Pinaceae, is characterized by slow concerted evolution and exhibits substantial ITS-region length variation (975–3663 bp), in sharp contrast to the narrow range (565–700 bp) in angiosperms. Here we examined intra- and intergenomic heterogeneity of the nrDNA internal transcribed spacer (ITS) region in four varieties of Larix potaninii, a species from the mountainous areas of western China. Two clones with more than a 100-bp deletion in ITS1 were detected in L. potaninii var. chinensis and L. potaninii var. australis, respectively. The deletion resulted in the loss of most part, including the motif sequence, of subrepeat 1 (SR1). Sequence divergence and phylogenetic analyses showed that some clones would be pseudogenes given their low GC content, high substitution rates, unique positions in the phylogenetic trees, or significant length variation. These clones might represent orphons or paralogues at minor loci resulting from large-scale gene or chromosome reorganization. Some recombinants characterized by chimeric structure and discordant phylogenetic positions in partitioned sequence analyses indicate that unequal crossing-over plays an important role in the process of nrDNA concerted evolution. In addition, some varieties of L. potaninii might have experienced an nrDNA founder effect parallel to their geographical isolation.     

Evolution of the 5.8S nrDNA gene and internal transcribed spacers in Carapichea ipecacuanha (Rubiaceae) within a phylogeographic context

Nuclear ribosomal DNA (nrDNA) constitutes a multicopy gene family that is used widely to test evolutionary hypotheses across a broad range of organisms. It is presumed that, as a result of concerted evolution, tandem nrDNA repeats are homogeneous within species and different between species. We sampled 77 specimens of a disjunct species (Carapichea ipecacuanha) from throughout its three geographic ranges and obtained 266 nrDNA sequences, of which 26 were obtained by direct sequencing and 240 by cloning of PCR products. Complementary sequence analyses, which included analyses of secondary structure stability, the pattern of base substitutions, GC content, and the presence of conserved motifs, were used to characterize the internal transcribed spacer (ITS) region (ITS1-5.8S nrDNA-ITS2). Our results showed that concerted evolution of the ITS region was incomplete in C. ipecacuanha, particularly in the Atlantic range. In the highly polymorphic populations of the Atlantic range, intraindividual variation was observed and involved 56 functional paralogs and 15 pseudogenes from two highly divergent ribogroups. The Amazonian range (with 12 functional paralogs) and the Central-American range (with five functional paralogs) were genetically depauperate and exhibited no pseudogenes. In the two latter ranges, almost complete homogenization of the ITS sequences had occurred. We argue that it is important to consider past evolutionary history when making inferences about the efficiency with which concerted evolution homogenizes tandem nrDNA repeats a single sequence.     

Within- and between-individual sequence variation among ITS1 copies in the meadow grasshopper Chorthippus parallelus indicates frequent intrachromosomal gene conversion

Sequencing multiple copies of the ITS1 region revealed the coexistence of two or more haplotypes within the genome of Chorthippus parallelus. Using a PCR-RFLP approach, the ITS1 numbers and frequencies of haplotypes present in each of 40 individuals were investigated, revealing a consistent lack of homogeneity. For each individual, the level of intra-individual variation was estimated from a sample of 20 ITS1 copies. The level of differentiation in haplotype frequency among individuals was then estimated by maximum likelihood using models based on the Dirichlet distribution. This confirmed the existence of significant levels of variation among individuals within each population studied. The most likely turnover mechanism that could generate this pattern of variation is gene conversion, operating at the intrachromosomal level. Furthermore, the discovery of linkage disequilibrium among the ITS1 haplotypes of C. parallelus suggests that intrachromosomal gene conversion occurs more frequently than interchromosomal recombination. Subspecies of C. parallelus showed significantly different haplotype distributions following about 0.5 Myr of divergence. With respect to the process of concerted evolution, we show that homogenization of repeats is slow relative to speciation, and the standing variation among individuals is sufficient for selection to operate.     

Molecular evidence of cryptic speciation, historical range expansion, and recent intraspecific hybridization in the Neotropical seasonal forest tree Cedrela fissilis (Meliaceae)

Molecular phylogeography can lead to a better understanding of the interaction between past climate events, large-scale vegetation shifts, and the evolutionary history of Neotropical seasonal forests. The endangered timber tree species Cedrela fissilis is associated with seasonal forests and occurs throughout South America. We sampled C. fissilis from 56 sites across the species' range in Brazil and Bolivia and obtained sequence data for nuclear and chloroplast DNA. Most specimens (149 out of 169) exhibited intraindividual polymorphism for the nuclear internal transcribed spacer (ITS). Cloning and an array of complementary sequence analyses indicated that the multiple copies of ITS were functional paralogs--concerted evolution in C. fissilis appeared to be incomplete. Independent Bayesian analyses using either ITS or cpDNA data revealed two separate phylogenetic lineages within C. fissilis that corresponded to populations located in separate geographic regions. The divergence occurred in the Early Pliocene and Late Miocene. We argue that climate-mediated events triggered dispersal events and split ancestral populations into at least two large refugial areas of seasonal forest that were located to the east and west of the present day Cerrado. Upon recent climate amelioration, formerly isolated lineages reconnected and intraspecific hybridization gave rise to intraindividual polymorphism and incomplete concerted evolution in C. fissilis.     

Comparisons of phylogenetic hypotheses among different data sets in dwarf dandelions (Krigia,Asteraceae): Additional information from internal transcribed spacer sequences of nuclear ribosomal DNA

The internal transcribed spacer (ITS) region of the 18 S–25 S nuclear ribosomal DNA repeat was sequenced from 19 populations of the tribeLactuceae, including all species of dwarf dandelion (Krigia) and five outgroup genera. The incidence of length changes and base substitutions was at least two times higher for ITS 1 than ITS 2. Interspecific sequence divergence withinKrigia averaged 9.62% (1.61%–15.19%) and 4.26% (0%–6.64%) in ITS 1 and ITS 2, respectively. Intergeneric sequence divergence ranged from 15.6% to 44.5% in ITS 1 and from 8.0% to 28.6% in ITS 2. High sequence divergence and homoplasy among genera of tribeLactuceae suggest that the phylogenetic utility of ITS sequence data is limited to interspecific studies or comparisons among closely related genera. Trees generated from ITS sequences are essentially identical to those from restriction site comparisons of the entire nuclear ribosomal (nr) DNA region. The degree of tree resolution differed depending on how gaps were treated in phylogenetic analyses. The ITS trees were congruent with the chloroplast DNA and morphological phylogenies in three major ways: 1) the sister group relationship betweenKrigia andPyrrhopappus; 2) the recognition of two monophyletic sections,Krigia andCymbia, in genusKrigia; and 3) the monophyly of theK. occidentalis-K. cespitosa clade in sect.Cymbia. However, the two nrDNA-based trees are not congruent with morphology/chloroplast DNA-based trees for the interspecific relationships in sect.Krigia. An average of 22.5% incongruence was observed among fourKrigia data sets. The relatively high degree of incongruence among data sets is due primarily to conflict between trees based on nrDNA and morphological/cpDNA data. The incongruence is probably due to the concerted evolution of nrDNA repeating units. The results fromKrigia and theLactuceae suggest that nrDNA data may have limited utility in phylogenetic studies of plants, especially in groups which exhibit high levels of sequence divergence. Our combined phylogenetic analysis as a total evidence shows the least conflict to each of the individual data sets.     

Presence of a short repeat sequence in internal transcribed spacer (ITS) 1 of the rRNA gene of <Emphasis Type="Italic">Sogatella furcifera</Emphasis> (Hemiptera: Delphacidae) from geographically different populations in Asia

Nucleotide sequences of the internal transcribed spacer 1 (ITS1)–5.8S–ITS2 region of the nuclear ribosomal RNA gene were determined in the white-backed planthopper (WBPH) Sogatella furcifera (Horváth) to detect molecular variation among regional populations in Asia. We analyzed 932 sequences from 172 individuals (4–9 clones per individual) of 33 populations collected in 1987–2008 from six countries, Japan, China, Taiwan, Vietnam, Philippines, and Papua New Guinea. WBPH showed intra-individual variation in ITS1, which is mainly attributable to the frequency (0–10) of the 66-bp repeat sequence in ITS1. Among the examined clones, the sequences of 5.8S were mostly identical and those of ITS2 were similar. A single planthopper had a maximum of 6 different variants in the number of ITS1 repeats, suggesting highly varied repeat numbers in individual planthoppers. The ITS1 with four repeats was the most frequently (64%) detected. Such a repeat was not observed in two other economically important planthopper species, Nilaparvata lugens (Stål) and Laodelphax striatellus (Fallén). The ITS nucleotide sequences in the WBPH populations in Asia were genetically close and some variations in the sequences were not related to regional populations, indicating that the nucleotide sequences of the ITS region are not useful for geographical discrimination of the WBPH. This closeness seems to be caused by long distance migration and genetic exchange among populations.     

The heterochromatin of grasshoppers from the Caledia captiva species complex. I. Sequence evolution and conservation in a highly repeated DNA family

The restriction enzyme TaqI digests 0.2% of the genomic DNA from the grasshopper Caledia captiva to a family of sequences 168 bp in length (length of consensus sequence). The sequence variation of this "Taq family" of repeat units was examined among four races from C. captiva to assay the pattern of evolution within this highly repeated DNA. The Taq-family repeats are located in C-banded heterochromatin on at least one member of each homologous pair of chromosomes; the locations range from centromeric to telomeric. Thirty-nine cloned repeats isolated from two population 1A individuals along with 11 clones from seven populations taken from three of the races demonstrated sequence variation at 72 positions. Pairwise comparisons of the cloned repeats, both within an individual and between different races, indicate that levels of intraspecific divergence, as measured by reproductive incompatibility, do not correlate with sequence divergence among the 168-bp repeats. A number of subsequences within the repeat remain unchanged among all 50 clones; the longest of these is 18 bp. That the same 18-bp subsequence is present in all clones examined is a finding that departs significantly (P less than 0.01) from what would be expected to occur at random. Two other cloned repeats, from a reproductively isolated race of C. captiva, have sequences that show 56% identity with this 18-bp conserved region. An analysis showed that the frequency of occurrence of an RsaI recognition site within the 168- bp repeat in the entire Taq family agreed with that found in the cloned sequences. These data, along with a partial sequence for the entire Taq family obtained by sequencing uncloned repeats, suggest that the consensus sequence from the cloned copies is representative of this highly repeated family and is not a biased sample resulting from the cloning procedure. The 18-bp conserved sequence is part of a 42-bp sequence that possesses dyad symmetry typical of protein-binding sites. We speculate that this may be significant in the evolution of the Taq family of sequences.      

Variation in the nrDNA ITS of Pinus subsection Cembroides: implications for molecular systematic studies of pine species complexes.

The pinyon pines (Pinus subsection Cembroides), distributed in semiarid regions of the western United States and Mexico, include a mixture of relictual and more recently evolved taxa. To investigate relationships among the pinyons, we screened and partially sequenced 3000-bp clones of the nuclear ribosomal DNA internal transcribed spacer (ITS) region for 16 taxa from subsect. Cembroides and nine representatives from four other subsections of subgenus Strobus. Restriction digests of clones reveal within-individual heterogeneity, suggesting that concerted evolution is operating slowly on the ITS in pine species. Two ITS clones were identified as pseudogenes. Tandem subrepeats in the ITS1 form stem loops comparable to those in other genera of Pinaceae and may be promoting recombination between rDNA repeats, resulting in ITS1 chimeras. Within the pinyon clade, phylogenetic structure is present, but different clones from the same (or different) individuals of a species are polyphyletic, indicating that coalescence of ITS copies within individual genomes predates evolutionary divergence in the group. At the level of subsection and above, the ITS region corresponds well with morphological and cpDNA evidence. Except for P. nelsonii, the pinyons are monophyletic, with both subsect. Cembroides and P. nelsonii forming a clade with the foxtail and bristlecone pines (subsect. Balfourianae) of western North America.     

A Phylogeographic Split in Buxus balearica (Buxaceae) as Evidenced by Nuclear Ribosomal Markers: When ITS Paralogues Are Welcome

Sequences from the ribosomal nuclear internal transcribed spacers (ITS) have been widely used to infer evolutionary hypotheses across a broad range of living organisms. Intraspecific sequence variation is assumed to be absent or negliable in most species, but few detailed studies have been conducted to assess the apportionment of ITS sequence variation within and between plant populations. Buxus balearica was chosen as a model species to assess the levels of infraspecific and intragenomic ITS variation in rare and endangered species occurring in disjunct populations around the Mediterranean basin. Intragenomic polymorphic sites were detected for western and eastern accessions of B. balearica and in two accessions of the sister species B. sempervirens. Overall, 19 different ribotypes were found in B. balearica after sequencing 48 clones, whereas 15 ribotypes were detected in 19 clones of B. sempervirens. The integrity and secondary structure stability of the ribosomal sequences suggest that they are not pseudogenes. The high number of ribotypes recovered through cloning suggested that some sequences could be chimeric or generated in vivo by partial homogenization through gene conversion or unequal crossing-over. Average sequence divergence among B. balearica clones was 0.768%, and the most divergent sequences differed by 1.62%. Available evidence does not suggest that B. balearica paralogues have been obtained from other extant Buxus species through interspecific hybridization. The presence of several ribosomal sequences in box implies that the molecular forces driving the concerted evolution of this multigene family are not fully operational in this genus. Phylogenetic analyses of cloned ITS sequences from B. balearica displayed very poor resolution and only two clades received moderate bootstrap support. Despite the marked intragenomic sequence divergence found, ribosomal data suggest a clear phylogeographic split in B. balearica between western and eastern accessions. The distinct, nonchimeric sequences that are postulated as being present in each biogeographic group suggest that box populations from Anatolia (eastern Mediterranean) are relict. [Reviewing Editor: Dr. Rafael Zardoya]     

Intragenomic Variation and Evolution of the Internal Transcribed Spacer of the rRNA Operon in Bacteria

Variation in the internal transcribed spacer (ITS) of the rRNA (rrn) operon is increasingly used to infer population-level diversity in bacterial communities. However, intragenomic ITS variation may skew diversity estimates that do not correct for multiple rrn operons within a genome. This study characterizes variation in ITS length, tRNA composition, and intragenomic nucleotide divergence across 155 Bacteria genomes. On average, these genomes encode 4.8 rrn operons (range: 2–15) and contain 2.4 unique ITS length variants (range: 1–12) and 2.8 unique sequence variants (range: 1–12). ITS variation stems primarily from differences in tRNA gene composition, with ITS regions containing tRNA-Ala + tRNA-Ile (48% of sequences), tRNA-Ala or tRNA-Ile (10%), tRNA-Glu (11%), other tRNAs (3%), or no tRNA genes (27%). Intragenomic divergence among paralogous ITS sequences grouped by tRNA composition ranges from 0% to 12.11% (mean: 0.94%). Low divergence values indicate extensive homogenization among ITS copies. In 78% of alignments, divergence is <1%, with 54% showing zero variation and 81% containing at least two identical sequences. ITS homogenization occurs over relatively long sequence tracts, frequently spanning the entire ITS, and is largely independent of the distance (basepairs) between operons. This study underscores the potential contribution of interoperon ITS variation to bacterial microdiversity studies, as well as unequivocally demonstrates the pervasiveness of concerted evolution in the rrn gene family. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Reviewing Editor: Dr. Margaret Riley     

Discordance in Variation of the ITS Region and the Mitochondrial COI Gene in the Subterranean Amphipod Crangonyx islandicus

The amphipod Crangonyx islandicus is a recently discovered species endemic to Iceland. Populations of C. islandicus are highly structured geographically and genetically. The COI and 16S mitochondrial genes confine six monophyletic groups which have diverged for up to 5 million years within Iceland, and may present two cryptic species. To investigate the potential cryptic species status we analyse here the internal transcribed spacers (ITS1 and ITS2) and compare its variation with the patterns obtained with the mtDNA. The ITS regions present much less divergence among the geographic regions in comparison with the mtDNA, distances based on ITS1 are correlated with the COI distances as well as with geographic distances, but most of the variation is observed within individuals. The variation in the ITS region appears to have been shaped both by homogenization effect of concerted evolution and divergent evolution. A duplication of 269 base pairs is found in the ITS1 of all individuals from the southern populations, its divergence from its paralog appears to predate the split of the different groups within Iceland but some evidence point to rapid diversification after the split. This duplication does not affect the secondary structures found in the 3' and 5' ends of the sequence, suggested to have a role in the excision of the ITS1. Compensatory base changes within the ITS2 sequences which have been suggested to be a species indicator were not detected.     

Structure and Conservation of Tandem Repeats in the Mitochondrial DNA Control Region of the Least Brook Lamprey (<Emphasis Type="Italic">Lampetra aepyptera</Emphasis>)

Tandemly repeated sequences are a common feature of vertebrate mitochondrial DNA control regions. However, questions still remain about their mode of evolution, function, and phylogenetic distribution. We report phylogenetic and geographic patterns of variation of control region repeat sequence and number in a nonparasitic lamprey, Lampetra aepyptera. A survey of populations from throughout the species’ range revealed remarkably low repeat sequence polymorphism but some interpopulation variation in repeat number. The high sequence similarity extended to repeats observed in other species in the genus Lampetra and other lamprey genera. The very low levels of variation suggest a high copy turnover. Our data are consistent with the illegitimate elongation model of repeat gain and loss and further suggest that repeat change occurs at internal copies. However, the limited variation across some species of lamprey suggests that functional constraints may further limit variation.     

The evolution pattern of rDNA ITS in Avena and phylogenetic relationship of the Avena species (Poaceae: Aveneae)

Ribosomal ITS sequences are commonly used for phylogenetic reconstruction because they are included in rDNA repeats, and these repeats often undergo rapid concerted evolution within and between arrays. Therefore, the rDNA ITS copies appear to be virtually identical and can sometimes be treated as a single gene. In this paper we examined ITS polymorphism within and among 13 diploid (A and C genomes), seven tetraploid (AB, AC and CC genomes) and four hexaploid (ACD genome) to infer the extent and direction of concerted evolution, and to reveal the phylogenetic and genome relationship among species of Avena. A total of 170 clones of the ITS1-5.8S-ITS2 fragment were sequenced to carry out haplotype and phylogenetic analysis. In addition, 111 Avena ITS sequences retrieved from GenBank were combined with 170 clones to construct a phylogeny and a network. We demonstrate the major divergence between the A and C genomes whereas the distinction among the A and B/D genomes was generally not possible. High affinity among the A(d) genome species A. damascena and the ACD genome species A. fatua was found, whereas the rest of the ACD genome hexaploids and the AACC tetraploids were highly affiliated with the A(l) genome diploid A. longiglumis. One of the AACC species A. murphyi showed the closest relationship with most of the hexaploid species. Both C(v) and C(p) genome species have been proposed as paternal donors of the C-genome carrying polyploids. Incomplete concerted evolution is responsible for the observed differences among different clones of a single Avena individual. The elimination of C-genome rRNA sequences and the resulting evolutionary inference of hexaploid species are discussed.     

High intraindividual variation in internal transcibed spacer sequences in Aeschynanthus (Gesneriaceae): implications for phylogenetics

Aeschynanthus (Gesneriaceae) is a large genus of tropical epiphytes that is widely distributed from the Himalayas and China throughout South-East Asia to New Guinea and the Solomon Islands. Polymerase chain reaction (PCR) consensus sequences of the internal transcribed spacers (ITS) of Aeschynanthus nuclear ribosomal DNA showed sequence polymorphism that was difficult to interpret. Cloning individual sequences from the PCR product generated a phylogenetic tree of 23 Aeschynanthus species (two clones per species). The intraindividual clone pairs varied from 0 to 5.01%. We suggest that the high intraindividual sequence variation results from low molecular drive in the ITS of Aeschynanthus. However, this study shows that, despite the variation found within some individuals, it is still possible to use these data to reconstruct phylogenetic relationships of the species, suggesting that clone variation, although persistent, does not pre-date the divergence of Aeschynanthus species. The Aeschynanthus analysis revealed two major clades with different but overlapping geographic distributions and reflected classification based on morphology (particularly seed hair type).     

Molecular phylogenetics of subfamily Calamoideae (Palmae) based on nrDNA ITS and cpDNA rps16 intron sequence data

Phylogenetic relationships among the 22 genera of the palm subfamily Calamoideae were investigated using DNA sequence data from the nuclear ribosomal internal transcribed spacer (ITS) region and the chloroplast rps16 intron. The rps16 intron displayed low levels of variation, corroborating previous reports that the chloroplast genome of palms is highly conserved. High levels of within-individual polymorphism were identified in the ITS region, indicating that concerted evolution is not effectively homogenizing the ITS repeats. In the majority of cases, multiple clones from individuals resolved as monophyletic. However, the high levels of homoplasy in the ITS dataset, along with generally poor jackknife support for many clades, led to concerns that topologies obtained from these data might be unreliable. Nevertheless, congruence between trees based on ITS data alone and those based on rps16 intron data was high. Simultaneous analyses of both datasets yielded well-resolved topologies with high levels of jackknife support. A number of exciting groups emerged from the analyses: the African rattan clade comprising the endemic African rattan genera Laccosperma, Eremospatha, and Oncocalamus; the Lepidocaryeae-Raphia clade comprising the fan-leaved New World tribe Lepidocaryeae and the African genus Raphia; and the Asian clade comprising all Asian genera except Eugeissona. The position of Eugeissona was variable, although it did not resolve inside any of the three major clades mentioned above.     

ITS sequence variation and concerted evolution in the natural hybrid species Malus toringoides

The internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA) is one of the most used molecular characters in plant systematics. Our previous studies based on morphological analysis and ITS sequence variation suggested that Malus toringoides (Rehd.) Hughes is derived from hybridization between M. transitoria (Batal.) Schneid. and M. kansuensis (Batal.) Schneid. To further understand the variation pattern of ITS sequences in M. toringoides, and to elucidate the evolutionary processes that affect ITS sequence variation after hybridization, we sampled 99 accessions from multiple populations of the hybrid and parental species, and then obtained totally 254 ITS sequences by cloning and sequencing. Our ITS variation data demonstrates three outcomes of ITS repeats after hybrid speciation. ～ 27–41% of M. toringoides have only M. transitoria type ITS sequence, ～ 40–70% have M. transitoria type ITS sequence plus one or two chimeric ITS sequences generated by recombination between parental ITS sequences, and six accessions retain both parental type ITS sequences. The plausible evolutionary processes that created the observed ITS variations were inferred to be the joint actions of recombination, concerted evolution, pseudogenization and backcrossing. Our study provides further understandings of the variation model of ITS repeats after hybridization as well as the evolution of M. toringoides after its hybrid speciation.     

REPRODUCTIVE COMPATIBILITY AND RDNA SEQUENCE ANALYSES IN THE SELLAPHORA PUPULA SPECIES COMPLEX (BACILLARIOPHYTA)1

We tested whether internal transcribed spacer (ITS) rDNA sequence differences are correlated with sexual compatibility in the Sellaphora pupula complex, a model system for investigations of the species concept and speciation in diatoms. The phylogenetic relationships among the demes and the systematic position of the genus within the raphid diatoms were also investigated. The division of clones of S. pupula and S. laevissima into groups, based on sequence similarities and phylogenetic analyses, resembled groupings based on sexual compatibility: A high ITS sequence divergence, making full alignment difficult or impossible, was found among clones whose gametangia do not interact, whereas there was little sequence divergence among interfertile clones. This is clearly consistent with the idea that “Z clades” exhibit less intraclade than interclade variation in ITS and, as comparisons of secondary structure models for the RECT and PSEUDOCAP clones showed, that there is an equivalence of “CBC” and Z clades in the rectangular and pseudocapitate demes of S. pupula, as earlier hypothesized for chlorophytes. Intraclonal, presumably intraindividual, variation in ITS was found in S. pupula, though with a degree of variation less than that found within a single Z clade; it was too minor to affect the interclonal relationships in the ITS phylogeny. Sellaphora, which appears monophyletic in 18S phylogenies, with Pinnularia and “Navicula”pelliculosa as its closest allies, may also include some species currently classified in Eolimna. The S. pupula–S. laevissima group began to diversify in or before the Miocene.     

Analysis of nrDNA polymorphism in closely related diploid sexual, tetraploid sexual and polyploid agamospermous species

Nuclear sequences of ITS1-5.8S-ITS2 region of rDNA may be an important source of phylogenetically informative data provided that nrDNA is cloned and the character of sequence variation of clones is properly analyzed. nrDNA of selected Taraxacum sections was studied to show sequence variation differences among diploid sexual, tetraploid sexual and polyploid agamospermous species. We examined nucleotide characteristics, substitution pattern, secondary structure, and the phylogenetic utility of ITS1-5.8S-ITS2 from 301 clones of 32 species representing 11 sections. The most divergent sequences of ITS1&2 differed by 17.1% and in 5.8S only by 3.7%. The ITS1-5.8S-ITS2 characteristics, integrity and also stability of secondary structures confirmed that pseudogenes are not responsible for the above variation. The within-individual polymorphism of clones implies that the concerted evolution of ITS cistron of agamospermous polyploid Taraxacum is remarkably suppressed. Sequences of ITS clones proved to be a useful tool for mapping pathways of complex reticulation (polyploid hybridity) in agamospermous Taraxacum.     

Variation in the nrDNA ITS of Pinus Subsection Cembroides: Implications for Molecular Systematic Studies of Pine Species Complexes

The pinyon pines (Pinus subsection Cembroides), distributed in semiarid regions of the western United States and Mexico, include a mixture of relictual and more recently evolved taxa. To investigate relationships among the pinyons, we screened and partially sequenced 3000-bp clones of the nuclear ribosomal DNA internal transcribed spacer (ITS) region for 16 taxa from subsect. Cembroides and nine representatives from four other subsections of subgenus Strobus. Restriction digests of clones reveal within-individual heterogeneity, suggesting that concerted evolution is operating slowly on the ITS in pine species. Two ITS clones were identified as pseudogenes. Tandem subrepeats in the ITS1 form stem loops comparable to those in other genera of Pinaceae and may be promoting recombination between rDNA repeats, resulting in ITS1 chimeras. Within the pinyon clade, phylogenetic structure is present, but different clones from the same (or different) individuals of a species are polyphyletic, indicating that coalescence of ITS copies within individual genomes predates evolutionary divergence in the group. At the level of subsection and above, the ITS region corresponds well with morphological and cpDNA evidence. Except for P. nelsonii, the pinyons are monophyletic, with both subsect. Cembroides and P. nelsonii forming a clade with the foxtail and bristlecone pines (subsect. Balfourianae) of western North America.