Internal Transcribed Spacer Clade Research Articles

First Report of Phytophthora inundata Associated with Decline and Death of Walnut (Juglans regia L.) in Italy.

Persian walnut (Juglans regia L.) is an important nut crop in Italy. In recent years, incidence of walnut decline and death has increased in many Italian commercial orchards. In early summer 2020, we observed a serious decline in approximately 5% of trees in a waterlogged area of a Veneto-region walnut orchard (J. regia, cv Lara). Symptoms included extensive foliar wilt and canopy decline associated with collar and root rot. Symptomatic tissues excised from larger roots of affected trees were surface disinfested for 1 min in a 1% NaOCl solution, rinsed for 5 min in sterile distilled water, and placed onto P5ARPH selective medium (Jeffers and Martin 1986). A Phytophthora-like organism was consistently isolated. Pure cultures were obtained by single-hyphal transfers onto potato dextrose agar (PDA). Isolates were identified as Phytophthora inundata based on morphological characteristics (Brasier et al. 2003), sequences of internal transcribed spacer (ITS) amplicons from universal primers ITS6 (Cooke et al. 2000) and ITS4 (White et al. 1990) and sequences of cytochrome c oxidase, subunit II (Cox II) from Fm75 and Fm78 primer pair (Martin and Tooley 2003). On carrot agar (CA), colonies had a characteristic stellate to broad lobed patterns. On this medium, optimal growth was at 28-30 °C (7,3 mm/day) and the upper temperature limit for mycelial growth was 37°C. Mycelial disks of isolate CREADC-Om306, grown on CA, were floated in Petri plates with soil extract solution and incubated under continuous fluorescent light at room temperature (25+/-2 °C). Within 48 to 72 h, sporangia were produced that were persistent, non-papillate, ovoid or ovoid-obpyriform, measuring 55.0 to 80.7 (length) x 41.3 to 65.2 (width) µm (averages 64.3+/-10.2 x 47.9+/-9.7 µm). Oospores and chlamydospores were absent. BLAST analysis of the amplicons from CREADC-Om306 revealed ITS sequences (854-bp; GenBank accession no. OK342200) and Cox II sequences (568-bp; GenBank accession no. OK349677) that shared 100% identity with published P. inundata sequences available in GenBank (acc. n. AF266791 for ITS; MT458994 for Cox II). Pathogenicity tests were conducted in the greenhouse on six 2-year-old walnut (J. regia, cv Lara) plants. Four of the plants were inoculated with CREADC-Om306 on two opposite sides of each plant's stem at 1-2 cm above soil line. A cork borer was used to remove a 5-mm disk of bark that was replaced by a 5-mm diameter mycelial plug from 10-day-old cultures of the pathogen on PDA. Two control plants were treated in the same way except the bark wounds were inoculated with sterile PDA plugs. Plants were kept in greenhouse at 24 ± 2°C. After 3 months, lesions had developed from all points of inoculation with. P. inundata (mean lesion length 55,25+/-6,22 mm) and the pathogen was reisolated from the lesion margins of all inoculated plants. The control plants remained symptomless and did not yield the pathogen. P. inundata is widely distributed across the world as a plant pathogen on several native as well as horticultural crops, especially in riparian or other areas subject to flooding or waterlogging. This report is the first to document P. inundata as a pathogen on Persian walnut and adds it to the diverse list of known susceptible perennial native, ornamental, and agricultural hosts of this organism. In addition to P. inundata, which belongs to the major Phytophthora ITS Clade 6, other members of the clade including P. megasperma (Belisario et al. 2012) and P. gonapodyides (Belisario et al. 2016) have been described as walnut pathogens.

Diversity of Phytophthora Communities across Different Types of Mediterranean Vegetation in a Nature Reserve Area

Research Highlights: Protected natural areas are a reservoir of Phytophthora species and represent the most suitable sites to study their ecology, being less disturbed by human activities than other environments. Background and Objectives: The specific objective of this study was to correlate the diversity and distribution of Phytophthora species with the vegetation in aquatic, riparian and terrestrial habitats within a protected area in Eastern Sicily, Southern Italy. Materials and Methods: Environmental samples (water and soil) were sourced from two streams running through the reserve and six different types of vegetation, including Platano-Salicetum pedicellatae, the Sarcopoterium spinosum community, Myrto communis-Pistacietum lentisci, Pistacio-Quercetum ilicis,Oleo-Quercetum virgilianae and a gallery forest dominated by Nerium oleander (Natura 2000 classification of habitats). Phytophthora species were recovered from samples using leaf baiting and were classified on the basis of morphological characteristics and sequencing of internal transcribed spacer (ITS) regions of ribosomal DNA (rDNA). Results: As many as 11 Phytophthora species, within five different ITS clades, were identified, including P. asparagi, P. bilorbang, P. cryptogea, P. gonapodyides, P. lacustris, P. multivora, P. nicotianae, P. oleae, P. parvispora, P. plurivora and P. syringae. No Phytophthora species were found in the Sarcopoterium spinosum comm. Phytophthora asparagi, P. lacustris and P. plurivora were the prevalent species in the other five plant communities, but only P. plurivora was present in all of them. Overall aquatic species from clade 6 (100 out of 228 isolates) were the most common; they were recovered from all five types of vegetation, streams and riparian habitats. Phytophthora populations found in the Platano-Salicetum pedicellatae and Oleo-Quercetum virgilianae show the highest diversity, while no correlation was found with the physicochemical characteristics of the soil. Conclusions: The vegetation type and the aquatic or terrestrial habitat were identified as major environmental factors correlated with the diversity of Phytophthora communities in this reserve.

Ecology and pathology of Phytophthora ITS clade 3 species in forests in western Oregon, USA

ABSTRACTPhytophthora species are widespread and diverse in forest ecosystems, but little is known about their ecology. We explore ecological attributes of the closely related clade 3 species that occur sympatrically in western North American forests. We address the population structure, pathology, and epidemiology of P. ilicis, P. nemorosa, P. pluvialis, P. pseudosyringae, and P. psychrophila. Phytophthora species were isolated from plant tissues, rainwater falling through the forest canopy, streams, and soils in forests in western Oregon. Species identifications were based on morphology in culture with molecular confirmation using COX spacer and internal transcribed spacer (ITS) sequences. All five clade 3 Phytophthora species are present in western Oregon forests, although P. ilicis (only 1 forest isolate) and P. psychrophila (only 12 isolates) are apparently rare. P. ilicis is known only from holly in horticultural situations and once from a naturalized seedling in an urban forest. The known distribution of P. nemorosa in forest settings coincides with the ranges of its principle hosts, tanoak and myrtlewood, in Oregon and California. Although it is regularly identified from streams within the tanoak range, it has not been recovered from streams beyond that range. P. pluvialis is primarily associated with Douglas-fir canopies. It was identified from scattered locations throughout western Oregon in rain traps beneath Douglas-fir plantations and from diseased needles. P. pseudosyringae is also isolated from tanoak and myrtlewood in southwest Oregon and California, but its distribution, in streams at least, extends throughout much of western Oregon. P. psychrophila in Oregon is known only from rain traps beneath tanoak trees. Little intraspecific variation was detected in the nuclear rDNA ITS of clade 3 species. Variation in the mitochondrial COX spacer region was more frequent, with 2 to 10 haplotypes identified in the clade 3 species, for which we had multiple isolates.

Multiple Phytophthora species associated with a single riparian ecosystem in South Africa

The diversity of Phytophthora spp. in rivers and riparian ecosystems has received considerable international attention, although little such research has been conducted in South Africa. This study determined the diversity of Phytophthora spp. within a single river in Gauteng province of South Africa. Samples were collected over 1 y including biweekly river baiting with Rhododendron indicum leaves. Phytophthora isolates were identified with phylogenetic analyses of sequences for the internal transcribed spacer (ITS) region of the ribosomal DNA and the mitochondrial cytochrome oxidase c subunit I (coxI) gene. Eight Phytophthora spp. were identified, including a new taxon, P. taxon Sisulu-river, and two hybrid species from Cooke’s ITS clade 6. Of these, species from Clade 6 were the most abundant, including P. chlamydospora and P. lacustris. Species residing in Clade 2 also were encountered, including P. multivora, P. plurivora and P. citrophthora. The detection of eight species in this investigation of Phytophthora diversity in a single riparian river ecosystem in northern South Africa adds to the known diversity of this genus in South Africa and globally.

Two novel species representing a new clade and cluster of Phytophthora

Phytophthora stricta sp. nov. and Phytophthora macilentosa sp. nov. are described based on morphological, physiological and molecular characters in this study. Phytophthora stricta represents a previously unknown clade in the rRNA internal transcribed spacer (ITS)-based phylogeny. Phytophthora macilentosa, along with nine other species, consistently forms a high temperature-tolerant cluster within ITS clade 9. These observations are supported by the sequence analysis of the mitochondrial cytochrome c oxidase 1 gene. Both species are heterothallic and all examined isolates are A1 mating type. Phytophthora stricta produces nonpapillate and slightly caducous sporangia. This species is named after its characteristic constrictions on sporangiophores. Phytophthora macilentosa produces nonpapillate and noncaducous sporangia, which are mostly elongated obpyriform with a high length to breadth ratio. Both species were recovered from irrigation water of an ornamental plant nursery in Mississippi, USA and P. stricta was also recovered from stream water in Virginia, USA.

Development of New Polymorphic Microsatellite Markers for Three Closely Related Plant-Pathogenic Phytophthora Species Using 454-Pyrosequencing and Their Potential Applications

Phytophthora spp. (oomycetes) are causal agents of devastating diseases on a high number of crops, ornamentals, and native plants worldwide. Neutral molecular markers are increasingly being used to investigate the genetic population structure and possible pathways of spread of different plant pathogens, including Phytophthora spp. In this study, polymorphic microsatellite markers were developed for three species of the former Phytophthora citricola species complex-namely, P. multivora, P. plurivora, and P. pini (P. citricola I)-using the 454-pyrosequencing technique. In total, 35 polymorphic microsatellite loci were found and further characterized: 11 for P. plurivora, 16 for P. multivora, and 8 for P. pini. Microsatellites with dinucleotide motifs repeated 6 to 10 times were the most common for all three species. On average, 65 alleles per species and 5.3 alleles per locus were detected. Most loci were characterized by a low observed heterozygosity, which might be due to the homothallic mating system of the three Phytophthora spp. targeted. Cross amplification of the newly developed markers was tested on 17 Phytophthora spp. belonging to five different internal transcribed spacer clades. Transferability success was generally low and decreased with increasing genetic distance from the species to the three target species. A set of four loci was selected to easily discriminate P. plurivora, P. multivora, and P. pini on the basis of presence or absence of a polymerase chain reaction amplicon on an agarose gel.

A taxonomic re‐evaluation reveals that Phytophthora cinnamomi and P. cinnamomi var. parvispora are separate species

SummaryBetween 2008 and 2011, severe dieback associated with root and collar rot was reported on Arbutus unedo in several sites in Sardinia, Italy. Isolations from infected tissues and rhizosphere soil samples consistently yielded a Phytophthora species. It was initially identified as Phytophthora cinnamomi var. parvispora Kröber and Marwitz by comparing morphological features with the original description and the internal transcribed spacer (ITS) sequences with those present in GenBank. A multigene phylogeny based on DNA sequence data from two nuclear (ITS and β‐tubulin) and two mitochondrial (cox1 and cox2) gene regions combined with extensive morphological and physiological properties of these isolates, including the ex‐type culture of P. cinnamomi var. parvispora, demonstrates that this taxon is unique and it is redesignated here as Phytophthora parvispora sp. nov. Although morphologically similar to P. cinnamomi, P. parvispora differs by its smaller‐sized sporangia, chlamydospores, oogonia and oospores, higher oospore wall index, single‐celled antheridia, higher minimum and maximum temperatures for growth and faster growth at optimum temperature. In the phylogeny, P. parvispora falls within Phytophthora ITS clade 7a, grouped in a well‐supported clade sister to P. cinnamomi. In pathogenicity tests, P. parvispora and P. cinnamomi were equally aggressive towards A. unedo seedlings. The possible geographic origin of P. parvispora is also discussed.

Characterization of Phytophthora hybrids from ITS clade 6 associated with riparian ecosystems in South Africa and Australia

Surveys of Australian and South African rivers revealed numerous Phytophthora isolates residing in clade 6 of the genus, with internal transcribed spacer (ITS) gene regions that were either highly polymorphic or unsequenceable. These isolates were suspected to be hybrids. Three nuclear loci, the ITS region, two single copy loci (antisilencing factor (ASF) and G protein alpha subunit (GPA)), and one mitochondrial locus (cytochrome oxidase c subunit I (coxI)) were amplified and sequenced to test this hypothesis. Abundant recombination within the ITS region was observed. This, combined with phylogenetic comparisons of the other three loci, confirmed the presence of four different hybrid types involving the three described parent species Phytophthora amnicola, Phytophthora thermophila, and Phytophthora taxon PgChlamydo. In all cases, only a single coxI allele was detected, suggesting that hybrids arose from sexual recombination. All the hybrid isolates were sterile in culture and all their physiological traits tended to resemble those of the maternal parents. Nothing is known regarding their host range or pathogenicity. Nonetheless, as several isolates from Western Australia were obtained from the rhizosphere soil of dying plants, they should be regarded as potential threats to plant health. The frequent occurrence of the hybrids and their parent species in Australia strongly suggests an Australian origin and a subsequent introduction into South Africa.

Phytophthora elongatasp. nov., a novel pathogen from theEucalyptus marginataforest of Western Australia

A novel homothallic species of Phytophthora producing semipapillate sporangia on sympodially branching sporangiophores, thick-walled oospores in smooth-walled oogonia, and paragynous antheridia is described here as Phytophthora elongata sp. nov. DNA sequencing of the internal transcribed spacer (ITS) DNA and coxI gene confirm P. elongata as a distinct species within ITS clade 2. It has been isolated in the northern jarrah forest of Western Australia (WA) from the roots and collars of dead and dying Eucalyptus marginata and occasionally Corymbia calophylla in rehabilitated bauxite mine pits. It has also been associated with dead and dying plants of several mid- and understorey species in the northern and southern jarrah forest—Banksia grandis, Leucopogon propinquus, Dryandra squarrosa and an Andersonia sp., as well as the monocotyledonous Xanthorrhoea preissii, X. gracilis and Patersonia xanthina. P. elongata has also been isolated from sandy soils and loams in Victoria in eastern Australia. The pathogenicity of P. elongata to E. marginata and Banksia spp. has been shown in this and earlier studies. Due to the uniformity of the ITS DNA and cox 1 sequence data in WA, P. elongata may be the result of a recent clonal introduction. More pathogenicity tests on a wider range of native plant species are needed to assess the host range of P. elongata and its invasive potential in WA.

Vegetatively reproducing fungi in three genera of the Parmeliaceae share divergent algal partners

The taxonomic pattern of algal association with lichen fungi may be explained by the fungal selection of algae that are available in the habitat, genetic compatibility between symbionts or mode of reproduction of the lichen. This study examined algal selection among species in the genera Evernia, Pseudevernia and Imshaugia of the Parmeliaceae using nucleotide sequences of the algal Internal Transcribed Spacer (ITS) of the nuclear ribosomal DNA (rDNA), the algal actin gene, the 1512 group I intron and secondary RNA structures. The hypothesis of high selectivity within one species of Evernia examined, E. mesomorpha, was supported suggesting that E. mesomorpha exhibits high selectivity toward its photobiont, which is not shared with other species of lichenized fungi in the same habitat. The eight strongly supported ITS clades correspond with two newly described algal species broadly known as Trebouxia jamesii s.l. and one existing species, T. simplex. The possibility of lateral algal transfer is discussed with reference to algal availability and mode of reproduction of the lichen.

Functional and genetic diversity of mycorrhizal fungi from single plants of Caladenia formosa (Orchidaceae).

Mycorrhizal associations are essential to the plant kingdom. The largest flowering plant family, the Orchidaceae, relies on mycorrhizal fungi for germination, growth and survival. Evidence suggests varying degrees of fungal-host specificity based on a single fungal isolate from a single plant. This paper shows for the first time the diversity of endophytes colonizing in a single plant over consecutive years and the functional significance of this diversity. Stem-collars of Caladenia formosa were collected in different seasons and years. Mycorrhizal fungi isolated were tested for their efficacy to induce leafing and genetically determined using ITS-RFLP and sequencing. Multiple mycorrhizal fungi were repeatedly isolated from a single collar that displayed varying effectiveness in germination percentages and adult leaf length. Additional factors contributed to the isolation of effective mycorrhizal fungi; fungal collection season, year of collection and individual isolates. Surface sterilization only improved the number of isolated mycorrhizal fungi. Dual inoculation did not increase germination. All 59 mycorrhizal fungi effective in germinating seed belonged to one clearly defined ITS (internal transcribed spacer) clade and clustered close to Sebacina vermifera (79-89 % homology). Isolates resulting in the greatest germination were not necessarily those resulting in the greatest survival and growth 1 year after germination. Single orchid plants contained multiple mycorrhizal fungal strains of one species that had diverse functional differences. These results suggest that our current knowledge of fungal-host specificity may be incomplete due to experimental and analytical limitations. It also suggests that the long-term effectiveness of a mycorrhizal fungus or fungi could only be found by germination and longer-term growth tests rather than genetically.

Nuclear rDNA evidence for major lineages of helenioid Heliantheae (Compositae)

Abstract Results of phylogenetic analyses of nuclear 18S–26S rDNA internal transcribed spacer (ITS) region sequences for representatives of most genera of helenioid Heliantheae and various members of Heliantheae s.s. (sensu stricto) and Eupatorieae help to clarify major lineages and relationships in the clade corresponding to Heliantheae s.l. (sensu lato) and Eupatorieae. Most subtribes of helenioid Heliantheae as circumscribed by Robinson (1981) correspond closely with ITS clades. Polygeneric subtribes of helenioid Heliantheae that appear to be monophyletic based on ITS data include Flaveriinae sensu Turner and Powell (1977), Madiinae sensu Carlquist (1959), and Peritylinae sensu Robinson (1981). Chaenactidinae sensu Robinson (1981) is polyphyletic, but most members of the group are encompassed within only four ITS clades. Based on the ITS data and results of combined analyses of ITS variation plus previously published morphological and chloroplast DNA data, we conclude that Heliantheae s.s. and Eupatori...

Genetic Relationships Among Magnaporthe poae Isolates from Turfgrass Hosts and Relative Susceptibility of 'Penncross' and 'Penn A-4' Creeping Bentgrass.

Isolates of Magnaporthe poae from turfgrass hosts were analyzed for mating type, genetic relatedness according to ITS sequences, reaction to a previously developed species-specific poly-merase chain reaction (PCR) assay, and virulence on two creeping bentgrass cultivars in growth chamber experiments. Analysis of internal transcribed spacer (ITS) sequences revealed three clades, designated A, B, and C. Clade A contained isolates of both mating types from creeping bentgrass, annual bluegrass, and Kentucky bluegrass. Clade B contained only mating type 'A' isolates from annual bluegrass, whereas Clade C contained only mating type 'a' isolates from creeping bentgrass. The M. poae PCR assay failed to positively identify several North Carolina isolates from annual bluegrass and creeping bentgrass. M. poae isolates from Kentucky blue-grass were most virulent toward creeping bentgrass in growth chamber experiments. Although isolates of M. poae are not host specific, certain ITS clades may have a limited host or geographical range. The improved creeping bentgrass cv. Penn A-4 was more susceptible to summer patch than cv. Penncross. Additional research is needed to develop methods for accurate diagnosis of summer patch and other patch diseases in creeping bentgrass and to determine how creeping bentgrass cultivars vary in their susceptibility to these root pathogens.

Phytophthora pistaciae sp. nov. and P. melonis: the principal causes of pistachio gummosis in Iran

Two non-papillate species of Phytophthora are the principal cause of pistachio gummosis in Iran. Their previous description as P. megasperma and P. drechsleri was re-examined in the light of RFLPs and sequence comparisons of internal transcribed spacer (ITS) regions of rDNA. Both taxa were more closely related to a clade comprising P. sojae, P. cajani, P. vignae, P. melonis and P. sinensis than to the unrelated P. megasperma s. str. or P. drechsleri. The P. megasperma-like isolates from pistachio differed from all the above taxa in morphology, ITS sequence and AFLP patterns, and are described as a new species, P. pistaciae. The P. drechsleri-like isolates from pistachio had identical ITS sequences to those of P. melonis, P. sinensis and P. drechsleri-like isolates from cucurbits in Iran and the AFLP profiles of P. melonis, P. sinensis and the P. drechsleri-like isolates from pistachio were virtually identical. This and other published isozyme and mtDNA evidence suggests these taxa should be considered conspecific and all subsumed within P. melonis with a revised host range and geographical distribution. The relationships of P. melonis and P. pistaciae with other members belonging to the same ITS clade are considered.

MOLECULAR PHYLOGEOGRAPHY, RETICULATION, AND LINEAGE SORTING IN MEDITERRANEAN SENECIO SECT. SENECIO (ASTERACEAE)

The Mediterranean species complex of Senecio serves to illustrate evolutionary processes that are likely to confound phylogenetic inference, including rapid diversification, gene tree-species tree discordance, reticulation, interlocus concerted evolution, and lack of complete lineage sorting. Phylogeographic patterns of chloroplast DNA (cpDNA) haplotype variation were studied by sampling 156 populations (502 individuals) across 18 species of the complex, and a species phylogeny was reconstructed based on sequences from the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA. For a subset of species, randomly amplified polymorphic DNAs (RAPDs) provided reference points for comparison with the cpDNA and ITS datasets. Two classes of cpDNA haplotypes were identified, with each predominating in certain parts of the Mediterranean region. However, with the exception of S. gallicus, intraspecific phylogeographic structure is limited, and only a few haplotypes detected were species-specific. Nuclear sequence divergence is low, and several unresolved phylogenetic groupings are suggestive of near simultaneous diversification. Two well-supported ITS clades contain the majority of species, amongst which there is a pronounced sharing of cpDNA haplotypes. Our data are not capable of diagnosing the relative impact of reticulation versus insufficient lineage sorting for the entire complex. However, there is firm evidence that S. flavus subsp. breviflorus and S. rupestris have acquired cpDNA haplotypes and ITS sequences from co-occurring species by reticulation. In contrast, insufficient lineage sorting is a viable hypothesis for cpDNA haplotypes shared between S. gallicus and its close relatives. We estimated the minimum coalescent times for these haplotypes by utilizing the inferred species phylogeny and associated divergence times. Our data suggest that ancestral cpDNA polymorphisms may have survived for ca. 0.4-1.0 million years, depending on molecular clock calibrations.

MOLECULAR PHYLOGEOGRAPHY, RETICULATION, AND LINEAGE SORTING IN MEDITERRANEAN SENECIO SECT. SENECIO (ASTERACEAE)

The Mediterranean species complex of Senecio serves to illustrate evolutionary processes that are likely to confound phylogenetic inference, including rapid diversification, gene tree-species tree discordance, reticulation, interlocus concerted evolution, and lack of complete lineage sorting. Phylogeographic patterns of chloroplast DNA (cpDNA) haplotype variation were studied by sampling 156 populations (502 individuals) across 18 species of the complex, and a species phylogeny was reconstructed based on sequences from the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA. For a subset of species, randomly amplified polymorphic DNAs (RAPDs) provided reference points for comparison with the cpDNA and ITS datasets. Two classes of cpDNA haplotypes were identified, with each predominating in certain parts of the Mediterranean region. However, with the exception of S. gallicus, intraspecific phylogeographic structure is limited, and only a few haplotypes detected were species-specific. Nuclear sequence divergence is low, and several unresolved phylogenetic groupings are suggestive of near simultaneous diversification. Two well-supported ITS clades contain the majority of species, amongst which there is a pronounced sharing of cpDNA haplotypes. Our data are not capable of diagnosing the relative impact of reticulation versus insufficient lineage sorting for the entire complex. However, there is firm evidence that S. flavus subsp. breviflorus and S. rupestris have acquired cpDNA haplotypes and ITS sequences from co-occurring species by reticulation. In contrast, insufficient lineage sorting is a viable hypothesis for cpDNA haplotypes shared between S. gallicus and its close relatives. We estimated the minimum coalescent times for these haplotypes by utilizing the inferred species phylogeny and associated divergence times. Our data suggest that ancestral cpDNA polymorphisms may have survived for ca. 0.4-1.0 million years, depending on molecular clock calibrations.

Nuclear ribosomal DNA evidence for a western North American origin of Hawaiian and South American species of Sanicula (Apiaceae).

Results from phylogenetic analysis of nuclear rDNA internal transcribed spacer (ITS) sequences from a worldwide sample of Sanicula indicate that Hawaiian sanicles (Sanicula sect. Sandwicenses) constitute a monophyletic group that descended from a western North American ancestor in Sanicula sect. Sanicoria, a paraphyletic assemblage of mostly Californian species. A monophyletic group comprising representatives of all 15 species of S. sect. Sanicoria and the three sampled species of S. sect. Sandwicenses was resolved in all maximally parsimonious trees, rooted with sequences from species of Astrantia and Eryngium. All sequences sampled from eastern North American, European, and Asian species of Sanicula fell outside the ITS clade comprising S. sect. Sanicoria and S. sect. Sandwicenses. A lineage comprising the Hawaiian taxa and three species endemic to coastal or near-coastal habitats in western North America (Sanicula arctopoides, Sanicula arguta, and Sanicula laciniata) is diagnosed by nucleotide substitutions and a 24-bp deletion in ITS2. The hooked fruits in Sanicula lead us to conclude that the ancestor of Hawaiian sanicles arrived from North America by external bird dispersal; similar transport has been hypothesized for the North American tarweed ancestor of the Hawaiian silversword alliance (Asteraceae). Two additional long-distance dispersal events involving members of S. sect. Sanicoria can be concluded from the ITS phylogeny: dispersal of Sanicula crassicaulis and Sanicula graveolens from western North America to southern South America.