Internal Transcribed Spacer Data Research Articles

Phylogenetic analysis of Piscirickettsia salmonis by 16S, internal transcribed spacer (ITS) and 23S ribosomal DNA sequencing.

Piscirickettsia salmonis, the etiologic agent of piscirickettsiosis, is a systemic disease of salmonid fish. Variations in virulence and mortality have been observed during epizootics at different geographical regions and in laboratory experiments with isolates from these different locations. This raises the possibility that biogeographical patterns of genetic variation might be a significant factor with this disease. To assess the genetic variability the 16S ribosomal DNA, the internal transcribed spacer (ITS) and the 23S ribosomal DNA of isolates from 3 different hosts and 3 geographic origins were amplified using the polymerase chain reaction (PCR). Results of this analysis confirm that P. salmonis is a member of the gamma subgroup of the Proteobacteria and show that the isolates form a tight monophyletic cluster with 16S rDNA similarities ranging from 99.7 to 98.5%. The ITS regions were 309 base pairs (bp), did not contain tRNA genes, and varied between isolates (95.2 to 99.7% similarity). Two-thirds of the 23S rRNA gene was sequenced from 5 of the isolates, yielding similarities ranging from 97.9 to 99.8%. Phylogenetic trees were constructed based on the 16S rDNA, ITS and 23S rDNA sequence data and compared. The trees were topologically similar, suggesting that the 3 types of molecules provided similar phylogenetic information. Five of the isolates are closely related (> 99.4% 16S rDNA similarity, 99.1% to 99.7% ITS and 99.3 to 99.8% 23S rDNA similarities). The sequence of one Chilean isolate, EM-90, was unique, with 16S rDNA similarities to the other isolates ranging from 98.5 to 98.9%, the ITS from 95.2 to 96.9% and the 23S rDNA from 97.6 to 98.5%.

Intraspecific Phylogeny Analysis Using ITS Sequences: Insights from Studies of the Streptanthus glandulosus Complex (Cruciferae)

Within many lineages diversity of the internal transcribed spacer (ITS) region of nuclear ribosomal RNA genes has proven comparable to that of restriction sites of chloroplast DNA (cpDNA). Al- though cpDNA polymorphism has proven useful in many phylogenetic studies at the intraspecific level, the utility of ITS sequences in this application has been subjected only to limited testing. The Streptanthus glan- dulosus complex is an ideal candidate for this evaluation, given the substantial variation in cpDNA restriction site and enzyme electrophoretic markers present among populations. ITS sequences were obtained for 31 populations within the complex; phylogenetic patterns derived from these data were compared to a cpDNA phylogeny based on restriction site data from a previous study. No population exhibited the same ITS se- quence, and average divergence among populations was 2.57%. The ITS strict consensus tree bears clades that are geographically distinct, but lacking in internal resolution; we suspect that some combination of gene flow, lineage sorting, and recombination has led to low resolution within these major clades. The ITS tree shares many features with the cpDNA tree, but the two also display substantial incongruities, including one that may have arisen via cytoplasmic introgression. Greater consistency is exhibited by the cpDNA tree, most likely due to the haploid and non-recombining nature of the chloroplast genome. Combining the ITS and cpDNA data results in a more highly resolved tree than those from either of the separate analyses. Despite the pitfalls of using a diploid, recombining data source like ITS at the intraspecific level, potential benefits include tracking of maternal and paternal lineages as well as insight into patterns and levels of gene flow among populations. Furthermore, the joint use of ITS and cpDNA markers facilitates independent assessment of intraspecific phylogenetic hypotheses and can expose cases in which introgression or phylogenetic sorting has caused incongruencies among haplotype trees.

Phylogenetic Utility of the External Transcribed Spacer (ETS) of 18S–26S rDNA: Congruence of ETS and ITS Trees ofCalycadenia(Compositae)

The 3′ region of the external transcribed spacer (ETS) of 18S–26S nuclear ribosomal DNA was sequenced in 19 representatives ofCalycadenia/Osmadeniaand two outgroup species (Compositae) to assess its utility for phylogeny reconstruction compared to rDNA internal transcribed spacer (ITS) data. Universal primers based on plant, fungal, and animal sequences were designed to amplify the intergenic spacer (IGS) and an angiosperm primer was constructed to sequence the 3′ end of the ETS in members of tribe Heliantheae. Based on these sequences, an internal ETS primer useful across Heliantheaesensu latowas designed to amplify and sequence directly the 3′ ETS region in the study taxa, which were the subjects of an earlier phylogenetic investigation based on ITS sequences. Size variation in the amplified ETS region varied across taxa of Heliantheaesensu latofrom approximately 350 to 700 bp, in part attributable to an approximately 200-bp tandem duplication in a common ancestor ofCalycadenia/Osmadenia.Phylogenetic analysis of the 200-bp subrepeats and examination of apomorphic changes in the duplicated region demonstrate that the subrepeats inCalycadenia/Osmadeniahave evolved divergently. Phylogenetic analyses of the entire amplified ETS region yielded a highly resolved strict consensus tree that is nearly identical in topology to the ITS tree, with strong bootstrap and decay support on most branches. Parsimony analyses of combined ETS and ITS data yielded a strict consensus tree that is better resolved and generally better supported than trees based on either data set analyzed separately. We calculated an approximately 1.3- to 2.4-fold higher rate of sequence evolution by nucleotide substitution in the ETS region studied than in ITS-1 + ITS-2. A similar disparity in the proportion of variable (1.3 ETS:1 ITS) and potentially informative (1.5 ETS:1 ITS) sites was observed for the ingroup. Levels of homoplasy are similar in the ETS and ITS data. We conclude that the ETS holds great promise for augmenting ITS data for phylogenetic studies of young lineages.

Phylogenetic analysis of Artemisia section Tridentatae (Asteraceae) based on sequences from the internal transcribed spacers (ITS) of nuclear ribosomal DNA

Artemisia sect. Tridentatae is composed of 11 species of xerophytic shrubs, which dominate much of western North America. Sequences of the internal transcribed spacers (ITS) of nuclear ribosomal DNA were used to construct a phylogeny, examine circumscription of the section, resolve interspecific relationships, and test competing hypotheses on the origin of the section. The data support the monophyly of sect. Tridentatae, with the exclusion of A. bigelovii and A. palmeri-two historically, anomolous species. However, the ITS data provide insufficient variation to fully resolve interspecific relationships or to support major lineages within the Tridentata clade. Nuclear and chloroplast DNA phylogenies are discordant, which may be a result of interspecific gene flow and subsequent chloroplast capture, particularly related to the placement of A. filifolia and A. californica, in addition to A. bigelovii. Furthermore, the ITS data are in conflict with cpDNA data, providing equivocal evidence for competing hypotheses on the Old World vs. New World origin for the section and do not provide support for definitive subgeneric placement.

Shrinking the Violets: Phylogenetic Relationships of Infrageneric Groups in Viola (Violaceae) Based on Internal Transcribed Spacer DNA Sequences

A phylogenetic study of the genus Viola used internal transcribed spacer (ITS) DNA sequences for 44 taxa representing many infrageneric groups in Viola plus outgroups Hybanthus concolor and Noisettia orchidiflora. Parsimony and maximum likelihood approaches place Latin American sections basal in Viola, sup- porting an Andean origin for the genus. Groups of sect. Chamaemelanium, mostly stemmed and yellow-flow- ered with x = 6 chromosomes, intermingle with groups of sect. Nomimium that are stemless and white- or blue- flowered with x = 12 or an aneuploid number. Neither section is monophyletic, and the assemblage forms a weak clade or grade, depending on the analysis. The remaining sect. Nomimium groups with primarily blue flowers and x = 10 and aneuploid or polyploid numbers form a clade including Hawaiian sect. Nosphinium, with pansies of sect. Melanium (typically stemmed with multicolored flowers and x = 5 to 17) at the base. Phy- logenetic relationships from ITS data herald the need for drastic remodeling of Viola. Proposed changes include splitting sect. Chamaemelanium and some of sect. Nomimium into several sections, transferring the remainder of Nomimium to the segregate sect. Viola, and merging Hawaiian sect. Nosphinium with the amphi- Beringian V. langsdorffii complex under the Langsdorffianae in sect. Viola.

New Combinations in Malva (Malvaceae: Malveae)

Four new combinations in Malva, M. assurgentiflora, M. lindsayi, M. occidentalis, and M. wigandii, and five replacement names, M. australiana, M. canariensis, M. dendromorpha, M. linnaei, and M. pacifica, are proposed to accommodate species of Lavatera that are more closely related to the type species of Malva than they are to the type species of Lavatera. These species are in the discussed by Ray in an earlier paper. One other Lavatera species that should also be placed in Malva is discussed but not formally transferred. Some lectotypes and neotypes are designated. Elsewhere (Ray, 1995), I analyzed and discussed the relationships among species of Malva L., Lavatera L., and six outgroups. I described an analysis of morphological character data that initially appeared to have little value. That analysis was followed by an extensive molecular study and a reinterpretation of morphological characters that brought to light a new way to view these genera and problems associated with them. Ray (1994) and Ray (1995) included extensive discussions of morphological characters and evolutionary trends, generic relationships, relevant literature, chromosome numbers, biogeography, and dispersal issues and should be consulted for a better understanding of the results and conclusions briefly described here. Based on my analyses of nuclear rDNA Internal Transcribed Spacer (ITS) sequence data and morphological characters (Ray, 1994, 1995), I concluded that the species now in Malva and Lavatera are all closely related in comparison to outgroups in Alcea, Althaea, Anisodontea, Callirhoe, and Hibiscus. Among Malva and Lavatera species, at least one well-supported group, consisting of a mixture of species from the two genera, could be defined on the basis of fruit differences and the ITS-based phylogenetic tree. A phylogenetic tree from Ray (1995) showing the results of analysis of ITS is shown in Figure 1. This figure also shows the major species that can be defined with the additional support of fruit characters, and includes a listing of species that can be assigned to the on this basis alone. Another more tentative grouping is also shown. Interesting geographic disjunctions in the distribution of species in Malva and Lavatera are indicated. Malva L. and Lavatera L. have been increasingly problematic since Linnaeus (1753) reinterpreted Tournefort's (1706) generic concept for Lavatera and expanded Lavatera to cover a much wider range of species than Tournefort intended. Tournefort originally described the genus to segregate Lavatera trimestris (a species with relatively unusual characteristics) from Malva on the basis of fruitaxis characters. Linnaeus disregarded this and used epicalyx characters to distinguish the two genera. Lavatera trimestris L. has an unusual epicalyx that may have led Linnaeus to use this character, but the epicalyx of L. trimestris is atypical in comparison to those of nearly all other species traditionally included in Lavatera and Malva. These genera have long been separated by most workers on the unsatisfactory (Fernandes, 1968a) basis of fusion or non-fusion of epicalyx bracts. I found the use of epicalyx characters for the generic distinction to be untenable based on extensive morphological and molecular analyses (Ray, 1994, 1995). Several species presently in Lavatera are closely related to species in Malva, in particular to the type species Malva sylvestris L. In fact, some species currently included in Lavatera are more closely related to Malva sylvestris than are some other species traditionally included in Malva; they are among the core Malva species. My group (see Fig. 1) includes some Malva species plus those species currently included in Lavatera that are very closely related to Malva sylvestris L. These close relationships among Lavatera and Malva species are strongly supported by the analyses of ITS data and by fruit characters (Ray, 1995). When the analysis is forced to retain the traditional division between the genera, the trees become significantly longer. Maximum likelihood testing using the forced tree also shows that, given the ITS data, the traditional generic arrangement (based on the epicalyx) is significantly less likely than the tree found. Species in the Malvoid (so named because it includes the type species of Malva) have true mericarps that: (a) are rounded in only the axial direction on the abaxial side, (b) have lateral angles NovoN 8: 288-295. 1998. This content downloaded from 207.46.13.180 on Thu, 08 Sep 2016 04:49:22 UTC All use subject to http://about.jstor.org/terms Volume 8, Number 3 Ray 289 1998 New Combinations in Malva

Molecular evidence for polyploid origins in Saxifraga (Saxifragaceae): the narrow arctic endemic S. svalbardensis and its widespread allies

The recently described polyploid Saxifraga svalbardensis is endemic to the arctic archipelago of Svalbard. We investigated relationships among four closely related species of Saxifraga in Svalbard and tested three previously proposed hypotheses for the origin of S. svalbardensis: (1) differentiation from the morphologically and chromosomally variable polyploid S. cernua; (2) hybridization between the diploid S. hyperborea and S. cernua; and (3) hybridization between the tetraploid S. rivularis and S. cernua. Fifteen populations were analyzed using random amplified polymorphic DNAs (RAPDs) and nucleotide sequences of the chloroplast gene matK and the internal transcribed spacers (ITS) of nuclear ribosomal DNA (rDNA). RAPD and matK data suggest that S. svalbardensis has originated from a hybrid with S. rivularis as the maternal parent and S. cernua as the paternal parent, possibly a single time, whereas ITS data could not be used to discriminate among the hypotheses. The data also suggest that the diploid S. hyperborea is a progenitor of the tetraploid S. rivularis. The four populations examined of S. svalbardensis were virtually identical for RAPD and ITS markers, whereas S. cernua showed high levels of variation, suggesting that the latter polyploid either has formed recurrently or has undergone considerable differentiation since its origin.

Phylogeny of the Hamamelidaceae based on the ITS sequences of nuclear ribosomal DNA

The phylogeny of Hamamelidaceae was constructed based on sequences of the internal transcribed spacers (ITS) and the 5.8S coding region of nuclear ribosomal DNA. The monophyly of both the Altingioideae and the Hamamelidoideae was strongly supported. The tribe Hamamelideae was, however, shown to be polyphyletic. Loropetalum of the tribe Hamamelideae formed a monophyletic group with Corylopsis of the tribe Coryopsideae. Hamamelis of the tribe Hamamelideae was closely related to Distylium, Fothergilla and Shaniodendron of the tribe Fothergilleae. The ITS data supported Endress’ treatment of merging the tribes Distylieae and Fothergilleae. Fortunearia of the tribe Eustigmateae was phylogenetically basal to the Hamamelis–Fothergilleae monophyletic group. The subfamily Altingioideae was cladistically basal within Hamamelidaceae. Liquidambar of the Altingioideae was paraphyletic and Altingia and Semiliquidambar formed a sister group relationship. Disanthus was highly divergent from other taxa and its subfamilial status was supported.

Molecular phylogenetic relationships inAveneae (Poaceae) species and other grasses as inferred from ITS1 and ITS2 rDNA sequences

A phylogenetic analysis was conducted on sequences of the internal transcribed spacer (ITS) region of nuclear ribosomal DNA in 23 species ofAveneae (Poaceae subfam.Pooideaae). These sequences ofHelictotrichon spp.,Arrhenatherum elatius, Avena spp.,Trisetum spp.,Koeleria spp.,Holcus lanatus, Alopecurus vaginatus together with published ITS sequences of furtherAveneae, Poeae, Triticeae, andBromeae were analysed by the neighbor-joining distance method to assess the molecular phylogenetic relationship in perennial and annualAveneae. The results suggest unexpectedly close affinities of the agronomically important genusAvena to comparatively small-flowered taxa ofAveneae. GenusArrhenatherum and small-flowered subgenera ofHelictotrichon are close extant relatives. The large genusHelictotrichon is para- if not polyphyletic, only its subgenera are monophyletic.Trisetum is clearly separated fromHelictotrichon and forms together withKoeleria and perhaps others a monophyletic lineage which is characterised by a conspicuous 9-bp deletion in ITS1. The impact of the ITS data on the delineation of some genera and subtribes ofAveneae and on the recognition of their biogeographical and ecological patterns is outlined.

Molecular evidence for a Mediterranean origin of the Macaronesian endemic genus Argyranthemum (Asteraceae)

The internal transcribed spacers (ITS) of nuclear ribosomal DNA were sequenced for 52 species from 32 genera and eight subtribes of Anthemideae. Phylogenetic analyses of ITS data generated trees that are largely incongruent with the recent classification of Anthemideae; most of the subtribes examined are not resolved as monophyletic. However, ITS trees are congruent with morphological, isozyme, phytochemical, and chloroplast DNA (cpDNA) restriction site data in supporting a Mediterranean origin for Argyranthemum, the largest endemic genus of the Atlantic oceanic islands. A combined analysis of ITS sequences and cpDNA restriction sites indicates that Argyranthemum is sister to the other three genera of Chrysantheminae (i.e., Chrysanthemum, Heteranthemis, and Ismelia). Times of divergence of Argyranthemum inferred from the ITS sequences ranged between 0.26 and 2.1 million years ago (mya) and are lower than values previously reported from isozyme and cpDNA data (1.5-3.0 mya). It is likely that rate heterogeneity of the ITS sequences in the Anthemideae accounts for the low divergence-time estimates. Comparison of data for 20 species in Argyranthemum and Chrysantheminae indicates that the cpDNA restriction site approach provided much more phylogenetic information than ITS sequences. Thus, restriction site analyses of the entire chloroplast genome remain a valuable approach for studying recently derived island plants.

On the evolutionary origins of the cacti

SummaryHershkovitz, M. A. & Zimmer, E. A.: On the evolutionary origins of the cacti. – Taxon 46: 217‐232. 1997. – ISSN 0040‐0262.Understanding evolutionary responses of plants to desert environments depends upon phylogenetic knowledge of desert plants. The diverse American desert family Cactaceae has been presumed, on the basis of distinctiveness, to be phylogenetically isolated and relatively ancient (> 65 million years old). Using maximum likelihood and parsimony analyses of the rapidly evolving internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA (nrDNA), we show that the cacti are phylogenetically nested among other aridity‐adapted lineages of the angiosperm family Portulacaceae. The ITS divergence between pereskioid cacti and the genus Talinum (Portulacaceae) is less than that between many Portulacaceae genera. Synthesis of the ITS data with morphological and chloroplast DNA evidence suggests an origin of cacti in mid‐Tertiary, C. 30 million years ago, and a later Tertiary diversification coincident with development of the American desert. This, in turn, implies that the diversification rate in cacti was much higher than in their nearest relatives. The present results illustrate the central role of phylogenetic reconstruction in ecological and evolutionary theory.

Molecular phylogenetics ofThlaspis.l. (Brassicaceae) based on chloroplast DNA restriction site variation and sequences of the internal transcribed spacers of nuclear ribosomal DNA

Systematics of the genus Thlaspi s.l. is difficult and controversial. Previous hypotheses have been based on morphological and anatomical data. We have analyzed sequence variation of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (nrDNA) among 13 Thlaspi s.l. taxa, representing all sections of the genus. Phylogenetic relationships among ITS sequences of the Thlaspi s.l. taxa studied are in general concordance with a previously published chloroplast DNA based phylogeny of this group. Most-parsimonious trees from ITS and chloroplast DNA data support three groups that are congruent with lineages (Thlaspi s.str., Noccaea–Raparia, Microthlaspi) previously described by Meyer on the basis of seed anatomy. The ITS data grouped Microthlaspi granatense outside the Microthlaspi clade and, therefore, Microthlaspi appeared paraphyletic on the ITS tree, in contrast with the chloroplast DNA phylogeny. We speculate that concerted evolutionary forces have acted among different nrDNA arrays (brought together in M. granatense by hybridization with a related taxon), resulting in the fixation of the alien species nrDNA type in M. granatense, which, however, maintains a Microthlaspi chloroplast genome type. Both molecular data sets detected intraspecific variation among Microthlaspi perfoliatum accessions of different geographic origin and different ploidy levels. Our molecular evidence would suggest the hybrid origin of polyploid M. perfoliatum from diploid M. perfoliatum and M. natolicum. Key words: chloroplast DNA, restriction site variation, sequence analysis of nuclear ribosomal DNA ITS regions, Thlaspi s.l. (Brassicaceae), molecular phylogeny, congruence.

Subdivision and genetic structure of four populations of Venturia inaequalis in Switzerland

Analyses of four populations of Venturia inaequalis in Switzerland were performed to obtain information about migration and to predict the probable speed of the spread of new pathotypes able to overcome resistance, e.g. Vf-resistance, of new cultivars. Genetic and haplotype diversity was calculated based on allele frequencies of random amplified polymorphic DNA (RAPD) markers and the internal transcribed spacer (ITS)-region of ribosomal DNA, which are regarded to be neutral, and the β-tubulin locus which may be under selection pressure. Within-population diversity was found to be quite similar over all four populations. Normalised haplotype diversity based on RAPD and ITS data was very high with a mean of 0.95. Diversity among populations (GST) was consistent over all neutral loci with a low mean of 0.04, but reached the high value of 0.26 for the selected β-tubulin locus. Low GST based on neutral loci may suggest a high level of gene flow. Considering these results, new pathotypes would be expected soon outside their place of identification. But actual gene flow is easily overestimated because of effects of gene flow in the past. However, naturally occurring gene flow could be increased by human activity. Therefore, it is very difficult to predict durability of the Vf-resitance in Switzerland.

Phylogeny and Biogeography ofPanaxL. (the Ginseng Genus, Araliaceae): Inferences from ITS Sequences of Nuclear Ribosomal DNA

Panax,the ginseng genus, is one of the most medicinally important genera in the Orient and demonstrates a classical eastern Asian and eastern North American disjunct distributional pattern. Sequences of the internal transcribed spacers (ITS) and the 5.8S coding region of the nuclear ribosomal DNA repeat were obtained for the 12 species ofPanaxto reconstruct phylogenetic relationships. Of the 2 eastern North American species,P. quinquefoliusandP. trifolius, P. quinquefoliuswas suggested to be more closely related to the eastern Asian species in the ITS tree, whileP. trifoliuswas phylogenetically isolated. Monophyly of the three medicinally most important species,P. ginseng, P. notoginseng,andP. quinquefolius,suggested by previous workers, was not supported by the ITS data. A close phylogenetic relationship betweenPanaxandAraliawas supported. Several biogeographical implications were inferred: (1) two divergence events have produced the eastern Asian and eastern North American disjunct distribution inPanax;(2) no intercontinental species pairs are found inPanax;(3) a discrepancy between the sequence divergence pattern and the phylogenetic pattern was observed inPanax,suggesting the need for caution in using sequence divergence data alone in inferring biogeographical patterns; (4) the Himalayas and central and western China are the current centers of diversity of the ginseng genus; and (5) the low ITS sequence divergence and a close relationship among species in that region suggest that rapid evolutionary radiation may have created such a diversity ofPanaxin the Himalayas and in central and western China.

Zea systematics: ribosomal ITS evidence.

Ribosomal internal transcribed spacer (ITS) sequences were used to evaluate the phylogenetics of Zea and Tripsacum. Maximum likelihood and polymorphism parsimony were used for phylogenetic reconstructions. Zea ITS nucleotide diversity was high compared to other plant species, but approximately equivalent to other maize loci. Coalescence of ITS alleles was rapid relative to other nuclear loci; however, there was still much diversity within populations. Zea and Tripsacum form a clade clearly differentiated from all other Poaceae. Four Zea ITS pseudogenes were identified by phylogenetic position and nucleotide composition. The phylogenetic position of Z. mays ssp. huehuetenangensis was clearly established as basal to the other Z. mays. The ITS phylogeny disfavored a Z. luxurians and Z. diploperennis clade, which conflicted with some previous studies. The introgression of Z. mays alleles into Z. perennis and Z. diploperennis was also established. The ITS data indicated a near contemporary divergence of domesticated maize and its two closest wild relatives.

Phylogeny of Polemoniaceae Based on Nuclear Ribosomal Internal Transcribed Spacer DNA Sequences

Nuclear ribosomal internal transcribed spacer (ITS) DNA sequences are used to estimate the phylogeny of 53 members of Polemoniaceae, representing all but two genera of the family. Fitch parsimony analysis of equal-weighted nucleotide sites result in 1080 minimal-length trees. However, when alignment-ambiguous positions are removed and an II: 10 transition to transversion weighting is imposed only eight trees are found. These data are used to address two issues: I) patterns of diversification in Polemoniaceae, and 2) the circumscription and monophyly of the genus Gilia. Although the monophyly of Polemoniaceae is well supported, relationships inferred among the earliest diverging lineages are altered by character weighting, treatment of indels, and taxon inclusion. In spite of the lack of reliable resolution at the basal nodes, ITS data provide evidence that Gilia, as currently interpreted, is polyphyletic and comprises at least five independent lineages.

Phylogenetic utility of the internal transcribed spacers of nuclear ribosomal DNA in plants: An example from the compositae

The internal transcribed spacer (ITS) region of 18–26S nuclear ribosomal DNA was sequenced in 12 representatives of the Compositae subtribe Madiinae and two outgroup species to assess its utility for phylogeny reconstruction. High sequence alignability and minimal length variation among ITS 1, 5.8S, and ITS 2 sequences facilitated determination of positional homology of nucleotide sites. In pairwise comparisons among Madiinae DNAs, sequence divergence at unambiguously aligned sites ranged from 0.4 to 19.2% of nucleotides in ITS 1 and from 0 to 12.9% of nucleotides in ITS 2. Phylogenetic relationships among ITS sequences of Hawaiian silversword alliance species ( Argyroxiphium, Dubautia, and Wilkesia) and California tarweed taxa in Adenothamnus, Madia, Raillardella, and Raillardiopsis are highly concordant with a chloroplast DNA-based phylogeny of this group. Maximally parsimonious trees from ITS and chloroplast DNA data all suggest (a) origin of the monophyletic Hawaiian silversword alliance from a California tarweed ancestor, (b) closer relationship of the Hawaiian species to Madia and Raillardiopsis than to Adenothamnus or Raillardella, (c) paraphyly of Raillardiopsis, a segregate of Raillardella, and (d) closer relationship of Raillardiopsis to Madia and the silversword alliance than to Raillardella. These findings indicate that the ITS region in plants should be further explored as a promising source of nuclear phylogenetic markers.