Employing 454 amplicon pyrosequencing to reveal intragenomic divergence in the internal transcribed spacer rDNA region in fungi

The rDNA internal transcribed spacer (ITS) region has been accepted as a DNA barcoding marker for fungi and is widely used in phylogenetic studies; however, intragenomic ITS variability has been observed in a broad range of taxa, including prokaryotes, plants, animals, and fungi, and this variability has the potential to inflate species richness estimates in molecular investigations of environmental samples. In this study 454 amplicon pyrosequencing of the ITS1 region was applied to 99 phylogenetically diverse axenic single‐spore cultures of fungi (Dikarya: Ascomycota and Basidiomycota) to investigate levels of intragenomic variation. Three species (one Basidiomycota and two Ascomycota), in addition to a positive control species known to contain ITS paralogs, displayed levels of molecular variation indicative of intragenomic variation; taxon inflation due to presumed intragenomic variation was ≈9%. Intragenomic variability in the ITS region appears to be widespread but relatively rare in fungi (≈3–5% of species investigated in this study), suggesting this problem may have minor impacts on species richness estimates relative to PCR and/or pyrosequencing errors. Our results indicate that 454 amplicon pyrosequencing represents a powerful tool for investigating levels of ITS intragenomic variability across taxa, which may be valuable for better understanding the fundamental mechanisms underlying concerted evolution of repetitive DNA regions.     

Causes of Size Homoplasy Among Chloroplast Microsatellites in Closely Related <Emphasis Type="Italic">Clusia</Emphasis> Species

Chloroplast DNA sequences and microsatellites are useful tools for phylogenetic as well as population genetic analyses of plants. Chloroplast microsatellites tend to be less variable than nuclear microsatellites and therefore they may not be as powerful as nuclear microsatellites for within-species population analysis. However, chloroplast microsatellites may be useful for phylogenetic analysis between closely related taxa when more conventional loci, such as ITS or chloroplast sequence data, are not variable enough to resolve phylogenetic relationships in all clades. To determine the limits of chloroplast microsatellites as tools in phylogenetic analyses, we need to understand their evolution. Thus, we examined and compared phylogenetic relationships of species within the genus Clusia, using both chloroplast sequence data and variation at seven chloroplast microsatellite loci. Neither ITS nor chloroplast sequences were variable enough to resolve relationships within some sections of the genus, yet chloroplast microsatellite loci were too variable to provide any useful phylogenetic information. Size homoplasy was apparent, caused by base substitutions within the microsatellite, base substitutions in the flanking regions, indels in the flanking regions, multiple microsatellites within a fragment, and forward/reverse mutations of repeat length resulting in microsatellites of identical base composition that were not identical by descent.     

A multigene phylogeny of theGibberella fujikuroi species complex: Detection of additional phylogenetically distinct species

Phylogenetic relationships within theGibberella fujikuroi species complex were extended to newly discovered strains using nucleotide characters obtained by sequencing polymerase chain reaction (PCR) amplified DNA from 4 loci used in a previous study [nuclear large subunit 28S rDNA, nuclear ribosomal internal transcribed spacer (ITS) region, mitochondriaal small subunit (mtSSU) ribosomal DNA, and β-tubulin] together with two newly sampled protein-encoding nuclear genes, translation elongation factor EF-1α and calmodulin. Sequences from the ribosomal ITS region were analyzed separately and found to contain of two highly divergent, nonorthologous ITS2 types. Phylogenetic analysis of the individual and combined datasets identified 10 new phylogenetically distinct species distributed among the following three areas: 2 within Asia and 4 within both Africa and South America. Hypotheses of the monophyly ofFusarium subglutinans and its two formae speciales, f. sp.pini and f. sp.ananas, were strongly rejected by a likelihood analysis. Maximum parsimony results further indicate that the protein-encoding nuclear genes provide considerably more phylogenetic signal that the ribosomal genes sequenced. Relative apparent synapomorphy analysis was used to detect long-branch attraction taxa and to obtain a statistical measure of phylogenetic signal in the individual and combined datasets.     

Recent origin and phylogenetic utility of divergent ITS putative pseudogenes: a case study from Naucleeae (Rubiaceae)

The internal transcribed spacer (ITS) of nuclear ribosomal DNA has been widely used by systematists for reconstructing phylogenies of closely related taxa. Although the occurrence of ITS putative pseudogenes is well documented for many groups of animals and plants, the potential utility of these pseudogenes in phylogenetic analyses has often been underestimated or even ignored in part because of deletions that make unambiguous alignment difficult. In addition, long branches often can lead to spurious relationships, particularly in parsimony analyses. We have discovered unusually high levels of ITS polymorphism (up to 30%, 40%, and 14%, respectively) in three tropical tree species of the coffee family (Rubiaceae), Adinauclea fagifolia, Haldina cordifolia, and Mitragyna rubrostipulata. Both secondary structure stability and patterns of nucleotide substitutions in a highly conserved region (5.8S gene) were used for distinguishing presumed functional sequences from putative pseudogenes. The combination of both criteria was the most powerful approach. The sequences from A. fagifolia appear to be a mix of functional genes and highly distinct putative pseudogenes, whereas those from H. cordifolia and M. rubrostipulata were identified as putative pseudogenes. We explored the potential utility of the identified putative pseudogenes in the phylogenetic analyses of Naucleeae sensu lato. Both Bayesian and parsimony trees identified the same monophyletic groups and indicated that the polymorphisms do not transcend species boundaries, implying that they do not predate the divergence of these three species. The resulting trees are similar to those produced by previous analyses of chloroplast genes. In contrast to results of previous studies therefore, divergent putative pseudogenes can be useful for phylogenetic analyses, especially when no sequences of their functional counterparts are available. Our studies clearly show that ITS polymorphism may not necessarily mislead phylogenetic inference. Despite using many different PCR conditions (different primers, higher denaturing temperatures, and absence or presence of DMSO and BSA-TMACl), we recovered only a few functional ITS copies from A. fagifolia and none from H. cordifolia and M. rubrostipulata, which suggests that PCR selection is occurring and/or the presumed functional alleles are located at minor loci (with few ribosomal DNA copies).     

Polyphyletic origin in <Emphasis Type="Italic">Pimpinella</Emphasis> (Apiaceae): evidence in Western Europe

The genus Pimpinella L. comprises about 150 species, being one of the largest genera within the family Apiaceae (subfamily Apioideae). Previous molecular phylogenetic studies have shown that Pimpinella is a taxonomically complex group. In this study, evolutionary relationships among representatives from Western Europe have been inferred from phylogenetic analyses of nuclear ribosomal DNA internal transcribed spacer (ITS 1 and ITS 2) and plastid sequences (trnL intron and the trnL-F spacer), with a representative sampling included (168 accessions in the ITS analysis, representing 158 species; and 42 accessions in the cpDNA analysis representing 35 taxa of Pimpinella and closely related species). All analyses resolved that Pimpinella is a non-monophyletic group, and Pimpinella’s taxa that grow in Western Europe are part of phylogenetically independent groups that correspond to three different tribes of the subfamily Apioideae: Pimpinelleae (core group), Pyramidoptereae and Smyrnieae.     

Comparative analysis of phylogenetic relationships of grain amaranths and their wild relatives (Amaranthus; Amaranthaceae) using internal transcribed spacer, amplified fragment length polymorphism, and double-primer fluorescent intersimple sequence repeat markers.

The most economically important group of species in the genus Amaranthus is the A. hybridus species complex, including three cultivated grain amaranths, A. cruentus, A. caudatus, and A. hypochondriacus, and their putative wild progenitors, A. hybridus, A. quitensis, and A. powellii. Taxonomic confusion exists among these closely related taxa. Internal transcribed spacer (ITS) of nuclear ribosomal DNA, amplified fragment length polymorphism (AFLP), and double-primer fluorescent intersimple sequence repeat (ISSR) were employed to reexamine the taxonomic status and phylogenetic relationships of grain amaranths and their wild relatives. Low ITS divergence in these taxa resulted in poorly resolved phylogeny. However, extensive polymorphisms exist at AFLP and ISSR loci both within and among species. In phylogenetic trees based on either AFLP or ISSR or the combined data sets, nearly all intraspecific accessions can be placed in their corresponding species clades, indicating that these taxa are well-separated species. The AFLP trees share many features in common with the ISSR trees, both showing a close relationship between A. caudatus and A. quitensis, placing A. hybridus in the same clade as all grain amaranths, and indicating that A. powellii is the most divergent taxon in the A. hybridus species complex. This study has demonstrated that both AFLP and double-primer fluorescent ISSR have a great potential for generating a large number of informative characters for phylogenetic analysis of closely related species, especially when ITS diversity is insufficient.     

Comparative analysis of a translocated copy of the trnK intron in carnivorous family Nepenthaceae

During phylogenetic analysis of the Nepenthaceae cpDNA trnK intron, it became apparent that a second non-functional copy of the locus was present in most of the investigated taxa. The translocation event was older than the radiation of all recent Nepenthaceae, and the translocated pseudogenized copy was conserved in nearly all members of the plant family. Using single chloroplast PCR and inverse PCR, we could exclude a plastom location for the second copy. Although translocation into the nucleus is possible, mitochondrial localization seems more likely based on these data. In total, the translocated sequence contained at least 3525 base pairs (bp) that were homologous to the Spinacia oleracea chloroplast genome. Comparative phylogenetic analysis of the non-functional copy revealed a high amount of homoplasies compared to topologies from the cpDNA trnK intron phylogenetic reconstruction. Therefore, this copy proved to be insufficient for phylogenetic reconstruction of the family. Since two different paralogs of the non-functional copy were found in one species, it is feasible that different paralogs were conserved in different groups and that paralogous sequences were included in the data matrix. These data demonstrate that phylogenetic analyses of pseudogenized copies of phylogenetically relevant loci should be performed with great caution. In addition, pseudogenized copies can exist in nearly every member of a plant family, and can be PCR-amplified at levels comparable to the specific copy. In this case, the inclusion of such copies can easily remain unnoticed, thus leading to faulty hypotheses.     

Molecular analyses of Pythium irregulare isolates from grapevines in South Africa suggest a single variable species

The Pythium irregulare species complex is the most common and widespread Pythium spp. associated with grapevines in South Africa. This species complex has been subdivided into several morphological and phylogenetic species that are all highly similar at the sequence level [internal transcribed spacer (ITS) and cytochrome c oxidase (cox) regions]. The complex includes Pythium regulare and Pythium cylindrosporum, which are morphologically distinct, and P. irregulare sensu stricto (s.s.) and Pythium cryptoirregulare, which are morphologically similar. The aim of the current study was to determine whether 50 South African grapevine P. irregulare isolates represented more than one phylogenetically distinct species. The isolates were characterised using nuclear (ITS and β-tubulin) and mitochondrial (cox1 and cox2) gene region phylogenies and two isozyme loci [glucose-6-phosphate isomerase (Gpi) and malate dehydrogenase (Mdh-1)]. Some of the gene sequence data were difficult to interpret phylogenetically, since some isolates contained two or more polymorphic ITS copies within the same isolate (intra-isolate variation) that clustered into different ITS sub-clades, i.e. the P. irregulare s.s. and P. cryptoirregulare sub-clades. The molecular data furthermore only revealed the presence of one phylogenetic species, P. irregulare. Morphological analyses of a subset of the isolates confirmed that the isolates were P. irregulare, and further showed that the P. cylindrosporum ex-type strain formed typical P. irregulare oogonia, and not the previously reported distinct elongated oogonia. Some of the molecular analyses suggested the occurrence of outcrossing events and possibly the formation of aneuploids or polyploids since (i) the nuclear and mitochondrial gene data sets were incongruent, (ii) polymorphic ITS copies were present within the same isolate, (iii) heterozygosities were observed in the β-tubulin gene and Gpi and Mdh-1 loci in some isolates and (iv) more than two β-tubulin alleles were detected in some isolates. Altogether, the data suggest that P. irregulare, P. cryptoirregulare, P. cylindrosporum, and possibly P. regulare should be synonimised under the name P. irregulare.     

Nuclear Ribosomal ITS Functional Paralogs Resolve the Phylogenetic Relationships of a Late-Miocene Radiation Cycad Cycas (Cycadaceae)

Cycas is the most widespread and diverse genus among the ancient cycads, but the extant species could be the product of late Miocene rapid radiations. Taxonomic treatments to date for this genus are quite controversial, which makes it difficult to elucidate its evolutionary history. We cloned 161 genomic ITS sequences from 31 species representing all sections of Cycas. The divergent ITS paralogs were examined within each species and identified as putative pseudogenes, recombinants and functional paralogs. Functional paralogs were used to reconstruct phylogenetic relationships with pseudogene sequences as molecular outgroups, since an unambiguous ITS sequence alignment with their closest relatives, the Zamiaceae, is unachievable. A fully resolved and highly supported tree topology was obtained at the section level, with two major clades including six minor clades. The results fully supported the classification scheme proposed by Hill (2004) at the section level, with the minor clades representing his six sections. The two major clades could be recognised as two subgenera. The obtained pattern of phylogenetic relationships, combined with the different seed dispersal capabilities and paleogeography, allowed us to propose a late Miocene rapid radiation of Cycas that might have been promoted by vicariant events associated with the complex topography and orogeny of South China and adjacent regions. In contrast, transoceanic dispersals might have played an important role in the rapid diversification of sect. Cycas, whose members have evolved a spongy layer in their seeds aiding water dispersals.     

The internal transcribed spacer of nuclear ribosomal DNA in the gymnosperm Gnetum

We analyze the structure of the internal transcribed spacers ITS1 and ITS2 of the nuclear ribosomal DNA in the gymnosperm Gnetum, using a phylogenetic framework derived mainly from an intron in the nuclear low-copy LEAFY gene. Gnetum comprises 25-35 species in South America, Africa, and Asia, of which we sampled 16, each with two to six clones. Criteria used to assess ITS functionality were highly divergent nucleotide substitution, GC content, secondary structure, and incongruent phylogenetic placement of presumed paralogs. The length of ITS1 ranged from 225 to 986 bp and that of ITS2 from 259 to 305 bp, the largest ranges so far reported from seed plants. Gnetum ITS1 contains two informative sequence motifs, but different from other gymnosperms, there are only few and short (7-13 bp) tandem repeats. Gnetum ITS2 contains two structural motifs, modified in different clades by shortening of stems and loops. Conspecific sequences grouped together except for two recombinant pseudogenes that had ITS1 of one clade and ITS2 of another. Most of the pseudogenic ITS copies, paralogs, and putative chimeras occurred in a clade that according to a fossil-calibrated chloroplast-DNA clock has an age of a few million years. Based on morphology and chromosome numbers, the most plausible causes of the observed high levels of ITS polymorphism are hybridization, allopolyploidy, and introgression.     

Evaluation of the internal transcribed spacer 2 (ITS2) as a molecular marker for phylogenetic inference using sequence and secondary structure information in blow flies (Diptera: Calliphoridae)

The internal transcribed spacer 2 (ITS2) is a small non-coding region located inside the nuclear ribosomal DNA cluster. ITS2 sequence variability is thought to be appropriate to differentiate species and for phylogenetic reconstructions analyses, which can be further improved if structural information is considered. We evaluated the potential of ITS2 as a molecular marker for phylogenetic inference in Calliphoridae (Diptera: Brachycera) using a broad range of inference methods and different substitution models, accounting or not for structural information. Sequence analyses revealed a hierarchically organized pattern of sequence variation and a small level of nucleotide substitution saturation. Intragenomic variation due to small sequence repeats was found mainly in the most variable domain (IV), but it has no significant impact on the phylogenetic signal at the species level. Inferred secondary structures revealed that GC pairs are more frequently found flanking bulges and loops regions in more conserved domains, thus ensuring structure stability. In the phylogenetic analyses, the use of substitution models accounting for structural information significantly improves phylogenetic inference in both neighbour-joining and Bayesian analyses, although the former provides limited resolution for dealing with highly divergent sequences. For Bayesian analyses, a significant improvement in likelihood was observed when considering structure information, although with small changes in topology and overall support, probably reflecting better evolutionary rates estimates. Based on these findings, ITS2 is a suitable molecular marker for phylogenetic analyses in Calliphoridae, at both species and generic level.     

Phylogeographic analysis of the nocturnal velvet ant genus Dilophotopsis (Hymenoptera: Mutillidae) provides insights into diversification in the Nearctic deserts

Understanding the history of diversification in the North American deserts has long been a goal of biogeographers and evolutionary biologists. Although it appears that a consensus is forming regarding the patterns of diversification in the Nearctic deserts in vertebrate taxa, little work has been done exploring the historical biogeography of widespread invertebrate taxa. Before a robust model of geobiotic change in the North American deserts can be proposed, it needs to be determined whether the same historical events affected vertebrate and invertebrate taxa in the same way. We explore the phylogeographic patterns in a widespread nocturnal wasp genus Dilophotopsis using two rDNA loci, the internal transcribed spacer regions 1 and 2 (ITS1 and ITS2). We use Bayesian phylogenetic analysis and haplotype network analysis to determine whether a consistent geographic pattern exists among species and populations within Dilophotopsis. We also used molecular dating techniques to estimate divergence dates of the major phylogenetic clades. Our analyses indicates that the species‐level divergences in Dilophotopsis occurred in the Neogene, and likely were driven by mountain building during the Miocene–Pliocene boundary (approximately 5 Mya) similar to the divergences in many vertebrate taxa. The population‐level divergences within species occurred during the Pleistocene (0.1–1.8 Mya). The present study shows that similar patterns of diversification exist in vertebrate and invertebrate taxa. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 101 , 360–375.     

Examination of five nuclear markers for phylogenetic study of Hologalegina (Leguminosae)

Most legume phylogenies have relied heavily on plastid gene datasets, with or without nuclear ribosomal DNA ITS data, but the sequences of nuclear genes and gene-spanning regions offer certain advantages. We tested the phylogenetic utility of five nuclear loci across the species-rich legume clade Hologalegina: PGDH, TRPT, HRIP, RNAR, and CNGC4 (CNGC4-like protein). Our objective was to determine whether any of these nuclear loci could be beneficial at resolving lower-level phylogenetic relationships in this clade, with a particular interest in finding markers that might work at the species level. While the phylogenetic utility of these nuclear loci is unknown outside of Hologalegina, we determined that two of the loci, PGDH and TRPT, are useful for phylogenetic analyses within Hologalegina, depending upon the desired scale of resolution.     

Lower level relationships in the mushroom genus Cortinarius (Basidiomycota, Agaricales): a comparison of RPB1, RPB2, and ITS phylogenies

We sampled and analyzed approximately 2900bp across the three loci from 54 taxa belonging to a taxonomically difficult group of Cortinarius subgenus Phlegmacium. The combined analyses of ITS and variable regions of RPB1 and RPB2 greatly increase the resolution and nodal support for phylogenies of these closely related species belonging to clades that until now have proven very difficult to resolve with the ribosomal markers, nLSU and ITS. We present the first study of the utility of variable regions of the genes encoding the two largest subunits of RNA polymerase II (RPB1 and RPB2) for inferring the phylogeny of mushroom-forming fungi in combination with and compared to the widely used ribosomal marker ITS. The studied region of RPB1 contains an intron of the size and variability of ITS along with many variable positions in coding regions. Though almost entirely coding, the studied region of RPB2 is more variable than ITS. Both RNA polymerase II genes were alignable across all taxa. Our results indicate that several sections of Cortinarius need redefinition, and that several taxa treated at subspecific and varietal level should be treated at specific level. We suggest a new section for the two species, C. caesiocortinatus and C. prasinocyaneus, which constitute a well-supported separate lineage. We speculate that sequence information from RNA polymerase II genes have the potential for resolving phylogenetic problems at several levels of the diverse and taxonomically very challenging genus Cortinarius.     

Phylogenomics at the tips: inferring lineages and their demographic history in a tropical lizard,Carlia amax

High‐throughput sequencing approaches offer opportunities to better understand the evolutionary processes driving diversification, particularly in nonmodel organisms. In particular, the 100–1000's of loci that can now be sequenced are providing unprecedented power in population, speciation and phylogenetic studies. Here, we apply an exon capture approach to generate >99% complete sequence and SNP data across >2000 loci from a tropical skink, Carlia amax, and exploit these data to identify divergent lineages and infer their relationships and demographic histories. This is especially relevant to low‐dispersal tropical taxa that often have cryptic diversity and spatially dynamic histories. For C. amax, clustering of nuclear SNPs and coalescent‐based species delimitation analyses identify four divergent lineages, one fewer than predicted based on geographically coherent mtDNA clades (>9.4% sequence divergence). Three of these lineages are widespread and parapatric on the mainland, whereas the most divergent is restricted to islands off the northeast Northern Territory. Tests for population expansion reject an equilibrium isolation‐by‐distance model for two of the three widespread lineages and infer refugial expansion sources in the relatively mesic northeast Top End and northwest Kimberley. The latter is already recognized as a hotspot of endemism, but our results also suggest that a stronger focus on the northeast Top End, and adjacent islands is warranted. More generally, our results show how genome‐reduction methods such as exon capture can yield insights into the pattern and dynamics of biodiversity across complex landscapes with as yet poorly understood biogeographic history and how exon data can link between population and phylogenetic questions.     

Phylogenetic relationships and genetic diversity of the Salicornieae (Chenopodiaceae) native to the Atlantic coasts of France

Phylogenetic relationships of members of the tribe Salicornieae, native to the Atlantic coasts of France, were assessed by three molecular markers: the nuclear ribosomal internal transcribed spacer (ITS), the chloroplast trnL-F and the chloroplast matK sequences. In parallel to the phylogenetic studies, a population genetic study was carried out based on randomly amplified polymorphic DNAs (RAPD). Neither the MP/ML analyses of the sequences, nor the AMOVA and NJ analyses of RAPD fingerprints confirmed the morphology-based classification at the specific level within Salicornia. Instead, our investigations are in favour of the species aggregate concept. Two sister groups were revealed in the genus: one is composed of the diploid taxa, while the other clusters the tetraploid taxa. Conflicting nuclear versus plastid phylogenetic positions of some tetraploid samples, referred to as S. fragilis, indicate that they most likely derive from a reticulate evolution.     

Sequence variation in mitochondrial ATP synthase subunit 6 & 8 and nuclear genes ITS1 and ITS2 in 17 cyprinid species

Sequence variation for the mitochondrial ATP synthase subunit 6 & 8 and the nuclear ITS1 marker was investigated for 17 cyprinid fish species in order to increase the number of possible loci for character sampling. The mitochondrial locus provided appropriate information for further phylogenetic studies, which can be readily applied to other taxa; the nuclear ITS1, however, cannot be recommended for inference of relationships among different taxa. No congruence was obtained between the trees resulting from the mitochondrial and nuclear gene using Bayesian Inference.     

Coding of intraspecific nucleotide polymorphisms: A tool to resolve reticulate evolutionary relationships in the ITS of beech trees (Fagus L., Fagaceae)

The internal transcribed spacers ITS1 and ITS2 of the nuclear ribosomal DNA (rDNA) have recently been found to display remarkable intraspecific polymorphism, a feature suggested as limiting their value for phylogenetic reconstructions. A comparative study of oligonucleotide motives and intraindividual nucleotide variability across all species of the tree genus Fagus (beech) shows, however, that this intraspecific ITS polymorphism follows a particular pattern, which can be used to detect reticulation and ancient polymorphism within the genus. Coding ITS polymorphisms as phylogenetically informative characters, moreover, resulted in better‐resolved phylogenies than traditional ‘base‐per‐base’ maximum parsimony and maximum likelihood analyses.