Molecular and pathogenic variation within Melampsora on Salix inwestern North America reveals numerous cryptic species

Chandalin Bennett1

Department of Forest Ecology and Biogeosciences,University of Idaho, Moscow, Idaho 83844-1133

M. Catherine Aime2,3

USDA-ARS, Systematic Botany and MycologyLaboratory, Beltsville, Maryland 20705

George NewcombeDepartment of Forest Ecology and Biogeosciences,University of Idaho, Moscow, Idaho 83844-1133

Abstract: In North America Melampsora rusts thatparasitize willows (Salix species) have never beenadequately studied and mostly have been referred to acollective species, Melampsora epitea (Kunze & Schm.)Thum, of European origin. Even taxa that arenominally distinct from M. epitea, such as M. abieti-caprearum and M. paradoxa, currently are consideredto be ‘‘races’’ of M. epitea. Within the range of ourfield surveys and collections in the Pacific Northwestand the Southwest only two species of Melampsorathus were expected: M. epitea (including its races)and M. ribesii-purpureae. In this study of Melampsoraon 19 species of Salix in the western United States 14phylogenetic species, or phylotypes, were apparentfrom nuclear rDNA sequencing of 140 collections orisolates. Our collections of the races of M. epitea, M.abieti-caprearum and M. epitea f. sp. tsugae belongedto one phylotype, termed lineage ‘N’. Assuming thatM. ribesii-purpureae represents one other phylotype,12 phylotypes still are unaccounted for by currenttaxonomy. Moreover Eurasian M. ribesii-purpureae wasnot closely related to any of the phylotypes reportedhere. Even more problematic was the resistance ofEurasian species of Salix, including the type host ofM. epitea, S. alba, to North American Melampsora,including phylotype ‘N’, in both the field and ininoculation experiments. These results suggest theneed for the description of many new species ofMelampsora on Salix in western North America.Additional analyses presented here might guidefurther research in this direction.

Key words: fungal diversity, Melampsoraceae,Pucciniales, rust resistance, Salicaceae, Uredinales

INTRODUCTION

Melampsora rusts are common fungal pathogens ofwillows (Salix spp.) in the northern temperate zone.Where willows have been introduced in the southernhemisphere Melampsora rusts frequently have fol-lowed (Spiers and Hopcroft 1996). Although Coleos-porium spp. have been reported on Salix in themountains of Central America (Arthur 1918) andMelampsorella and Puccinia spp. occur respectively onSalix in China and Japan (Chen 2002, Kobayashi2007), in North America only Melampsora spp. causewillow rust (Farr and Rossman 2011) with theuredinial/telial states occurring on willows and theaecial states on a variety of unrelated hosts.

Ten taxa have been recognized over the years onwillows in North America (Farr and Rossman 2011),and some scholars still recognize five (Sinclair andLyon 2005) associated with one of five aecial hosts(i.e. Abies, Tsuga, Saxifraga, Larix and Ribes).However others have simplified the task of diagnosisof the morphologically intergrading uredinial or telialstates on willows in North America by lumping almostall existing taxa as one collective species, Melampsoraepitea (Kunze & Schm.) Thum (Hylander, Jorstad etal. 1953, Wilson and Henderson 1966, Ziller 1974,Bagyanarayana 2005). In the latest global treatment ofMelampsora on Salix Pei (2005) recognized 33 taxaapart from the ‘‘M. epitea complex’’. Of the 33, M.abieti-caprearum Tubeuf, M. epitea f. sp. tsugae Ziller,M. paradoxa Dietel & Howl. and M. ribesii-purpureaeKleb. were the only ones that Pei (2005) listed forNorth America, in addition of course to M. epiteaitself. To this short list of five taxa might be addedUredo mckinleyensis Cummins in Alaska on Salixreticulata (Cummins and Stevenson 1956, Farr andRossman 2011), but we are not including it herebecause it is outside the range of this study. It must benoted that in North America M. abieti-caprearum, M.epitea f. sp. tsugae and M. paradoxa typically have beentreated as races of M. epitea (Ziller 1959, 1974).

Three of these taxa are thought to have nativeranges that include both North America and Eurasia:M. abieti-caprearum (aecial hosts, Abies spp.), M. epitea(full range of aecial hosts that might include any or allof Tsuga, Abies, Larix, Ribes, Saxifraga, Allium,

Submitted 21 Sep 2010; accepted for publication 9 Mar 2011.1 Current address: World Forest Institute, World Forestry Center,4033 SW Canyon Road, Portland, OR 97221.2 Current address: Louisiana State University Agricultural Center,Department of Plant Pathology and Crop Physiology, Baton Rouge,LA 70803.3 Corresponding author. E-mail: maime@agcenter.lsu.edu

Mycologia, 103(5), 2011, pp. 1004–1018. DOI: 10.3852/10-289# 2011 by The Mycological Society of America, Lawrence, KS 66044-8897

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Euonymus, Viola, Galanthus, Corydalis and somemembers of the Orchidaceae), and M. ribesii-purpur-eae (aecial hosts, Ribes spp.). The three taxa that arethought to be exclusively North American are M.epitea f. sp. tsugae (aecial hosts, Tsuga spp.), M.paradoxa (aecial hosts, Larix spp.) and Uredomckinleyensis (aecial host unknown).

However when rust is collected on Salix in NorthAmerica for routine diagnosis all too often the aecialhost is unknown. The fact that Thumen describedMelampsora epitea as a species with evenly echinulateurediniospores (Pei 2005) and all North Americanwillow rusts appear to share this feature, in additionto the common assumption that M. abieti-caprearum,M. epitea f. sp. tsugae and M. paradoxa are races of M.epitea (e.g. Ziller 1974), have contributed to the broadapplication of M. epitea in diagnoses especially whenthe aecial host is unknown.

However, as discussed by Pei (2005), M. epitea isactually an ambiguous taxon even when applied towillow rusts in Europe. Thumen’s type host for M.epitea was the Eurasian tree willow, Salix alba, butrusts on S. alba and other Eurasian tree willows (i.e.Salix subgenus Salix) are actually characterized inEurope as having urediniospores with smooth, non-echinulate apices instead of even echinulation. Notonly is M. epitea morphologically ambiguous, but thelesson from distributions of Melampsora on Populus,sister genus of Salix, is that North American species ofMelampsora are distinct from Eurasian species. Thus itis possible that M. epitea has been misapplied toNorth American species of Melampsora on Salix.

Investigations of the morphological variation ofwillow rusts (Hiratsuka and Kaneko 1982, Helfer1992) sometimes have been accompanied by studiesof host range (Pei, Royle et al. 1993, 1996; Spiers andHopcroft 1996) or of molecular variation (Pei,Whelan et al. 1997, Pei and Ruiz 2000, Samils,Lagercrantz et al. 2002, Smith, Blanchette et al.2004). In addition to these three elements studies ofwillow rusts ideally would include determinations ofthe aecial hosts of isolates, but this often is difficult(Pei 2005) and therefore is neglected frequently.Compounding the problem is the fact that rusts fromseveral aecial hosts may occur on the same Salixspecies (Arthur 1924, Ziller 1959). Furthermore theextent to which multiple species of Melampsoraparasitize the same Salix host is largely unknown.

A fresh look at the willow rusts of North America isoverdue. In particular the willow rusts of the PacificNorthwest (PNW) are under researched. More than50 native species of Salix occur in the region(Hitchcock and Cronquist 1973) in a wide variety ofnatural habitats from low riparian zones to alpinemeadows. Five subgenera of Salix (i.e. Salix, Protitea,

Longifoliae, Chamaetia and Vetrix) are present in thePNW (Argus 2010), and Melampsora rust is a commonoccurrence on willows native to the region (Farr andRossman 2011).

For this exploratory study of willow rusts in thePNW field surveys of Melampsora on four subgenera(i.e. Salix, Protitea, Longifoliae and Vetrix) werecarried out. Host ranges of isolates from Salix speciesfrom each of three subgenera (i.e. Protitea, Longifoliaeand Vetrix) also were determined experimentally viacontrolled inoculation experiments. The extent ofvariation in morphology of the uredinial state and innuclear rDNA sequences of the 28S large ribosomalsubunit and the internal transcribed spacer region-2was determined. A phylogenetic analysis was per-formed that included European taxa of Melampsoraon Salix for comparative purposes.

MATERIALS AND METHODS

Field surveys.—Incidence surveys for rust in willow popula-tions in the Pacific Northwest were conducted Sep–Nov2003, 2004, except for S. alba, the type host of M. epitea (Pei2005), which was surveyed for 10 y in Moscow, Idaho (1999–2008), inclusive. The surveys of S. alba might have includedhybrids of S. alba and S. fragilis that are common in riparianzones in Idaho (Johnson 1995) and are difficult todistinguish (de Cock, Lybeer et al. 2003). In all cases thecensus time for the presence or absence of rust on eachplant was 30 s. Up to 25 plants in each of 107 willowpopulations were surveyed. Fifty-four of the 69 populationsof the inoculation experiments were surveyed; an additional53 surveyed populations did not correspond to theinoculation experiments (SUPPLEMENTARY TABLE I).

Inoculation experiments.—Inoculations were conducted withrust isolates representing phylotypes ‘M’ (Melampsora ex S.amygdaloides from the Clearwater River), ‘B’ (Melampsora exS. melanopsis from the St Joe River) and ‘N’ (Melampsora exS. sitchensis from the St Joe River). Phylotype designationsare introduced in this paper and are explained inMATERIALS AND METHODS and RESULTS. Four green-house experiments were set up to test the host ranges ofisolates ‘M’, ‘B’ and ‘N’. Inoculations with ‘M’ wererepeated in two successive years, and results have beencombined here for both years. Test plants in general werecollected as seeds or cuttings from 16 native Salix speciesSep–Nov 2003 and Mar–Jun 2004 from 59 different willowpopulations in PNW. Seeds of two additional species andother populations also were received from outside theregion (i.e. Arizona, New Mexico, Colorado and Minne-sota). Cuttings of eight Eurasian species of Salix also werepropagated from both naturalized and cultivated popula-tions in Idaho, resulting in a total of 69 different willowpopulations in the inoculation experiments (SUPPLEMENTA-

RY TABLE I). After some losses in propagation 24 speciesultimately were inoculated with ‘M’ and 25 species with eachof ‘B’ and ‘N’. Inoculations were conducted in separate,non-adjacent greenhouse rooms for each of ‘M’, ‘B’ and ‘N’

BENNETT ET AL.: CRYPTIC SPECIES OF MELAMPSORA 1005

on plastic-lined greenhouse benches with 16 h lightprogrammed for each room.

Inoculations were performed with field-collected, uredin-iospore inoculum from each of the three source popula-tions. Inoculum of this kind may be more variable thanmonouredinial inoculum. A study in Japan showed thatmulti-uredinial inoculum was able to infect six Salix species,but inoculum propagated from a single uredinium fromthis same collection was able to infect only two of thosesame six Salix species (Nakamura, Kaneko et al. 2003). Thusthe host ranges reported here represent conservativeestimates. In each case spores were swabbed from theinfected leaves onto the abaxial side of 3–5 young leaves ofeach test plant. All inoculated plants were misted andplaced overnight in a plastic enclosure to ensure 100%

humidity for at least 15 h after inoculation.To ensure that there were no disease escapes additional

inoculations were carried out during each experiment.From nine to thirteen days after the initial inoculation allsets were heavily misted and covered once again to ensurean overnight period with 100% humidity that would beconducive to secondary infection. A third inoculation of thiskind was carried out a month after the second one. To makecertain that rust-free plants were resistant and not simplydisease-escapes a fourth and final swab inoculation wasperformed in each experiment within 30 d of finalinoculation.

Plants were monitored for uredinia every other day from5 d post inoculation until 13 d post inoculation. Thereaftermonitoring was weekly for 2 mo. Infection types were scored(as in FIG. 1) on a scale of 0–4 (i.e. R0 5 no uredinia,chlorosis or necrosis; R1 5 partially resistant, with somechlorosis and/or necrosis, but no uredinia; S2 5 marginallysusceptible with few, small uredinia and accompanyingchlorosis and/or necrosis (, one-third of leaf showing

rust); S3 5 moderately susceptible with small- to medium-sized uredinia and accompanying chlorosis and/or necrosis(one-third to two-thirds of leaf showing rust); S4 5 fullysusceptible with large uredinia and no accompanyingchlorosis or necrosis (. two-thirds of leaf showing rust).S2 and S3 were interpreted as susceptible phenotypes onlybecause the emphasis here was on host range instead of oninferring the presence of a gene for resistance, which ismore common whether in Populus (Newcombe, Stirling etal. 2001) or in Salix (Pei, Royle et al. 1996) .

Light microscopy of urediniospores.—Rust samples werecollected from wild populations throughout Washington,Idaho and Oregon, USA, as well as British Columbia,Canada, Sep–Nov 2003, 2004 from a total of 18 Salix species.Rust samples also were received from the USDA PlantMaterials Center in Los Lunas, New Mexico. In total 147different rust collections were obtained in western NorthAmerica (SUPPLEMENTARY TABLE I), all of which arevouchered in the Stillinger Herbarium of the University ofIdaho. Slide mounts of several uredinia from each weremade, and digital images were taken of the urediniosporeswith a Nikon CoolPix 950 digital camera mounted on aZeiss Axioskop2 compound microscope. Urediniosporesize, circularity and spine density were measured from theseimages with image-analysis software (SigmaScan Pro 5). Tenspores of each collection and 1470 spores in total weremeasured for each of the three variables.

A nested mixed-effects ANOVA was performed todetermine whether spore size, shape and spine density asdependent variables were significantly affected by hostspecies or host subgenus. This analysis was performed withSystat 13 software with measurements of dependentvariables nested within each collection. Discriminantanalysis (DA) also was performed to determine to what

FIG. 1. Photographs of resistant and susceptible reactions as characterized in the inoculation study. R0, no uredinia,chlorosis or necrosis; R1, partially resistant, with some chlorosis and/or necrosis, but no uredinia; S2, marginally susceptiblewith few, small uredinia and accompanying chlorosis and/or necrosis (, one-third of leaf showing rust); S3, moderatelysusceptible with small- to medium-sized uredinia and accompanying chlorosis and/or necrosis (one-third to two-thirds leafshowing rust); S4, fully susceptible with large uredinia and no accompanying chlorosis or necrosis (. two-thirds leafshowing rust).

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extent spore size, shape and spine density allowed forcorrect classification of host species or to host the subgenus.Salix species were assigned to subgenus according to theclassification system of Argus (2010).

Scanning electron microscopy of urediniospores and paraphy-ses.—Urediniospores and uredinial paraphyses of rustsamples from 16 Salix species also were observed with aLEO variable pressure field emission scanning electronmicroscope (SEM) to determine whether new and poten-tially diagnostic characters were present. A seven-nanometersputter coating of gold palladium was applied to 1 cm2 leafsections bearing uredinia.

DNA extraction, polymerase chain reactions (PCR) cyclesequencing and phylogenetic analyses of the 28S and ITS-2rDNA regions.—One hundred thirty-seven Melampsoracollections were sequenced from 19 Salix species (TABLE I).Collections were of one to several uredinia from one leaf ofeach sampled plant. Individual uredinia were removed withsterile forceps and placed in 400 mL AP1 buffer solutionfrom the QIAGEN DNeasy plant kit and extracted permanufacturer’s instructions (QIAGEN Inc., Valencia, Cali-fornia). DNA extractions were diluted 1 : 9 in sterile waterand amplified with rust-specific primer Rust2inv (Aime2006) and LR6 (Vilgalys and Hester 1990) in 25 mL reactionvolumes with 12.5 mL PCR Master Mix (Promega Corp.,Madison, Wisconsin), 1.25 mL of each 10 mM primer and10 mL diluted DNA template with PCR parameters describedin Aime (2006), amplifying approximately 1400 bp of aregion of the ribosomal repeat spanning the 5.8S subunit,the internal transcribed spacer region 2 (ITS-2) and thelarge subunit (28S). PCR products were cleaned withMontage PCR Centrifugal Filter Devices (Millipore Corp.,Billerica, Massachusetts) according to the manufacturer’sprotocol and sequenced with BigDye Terminator sequenc-ing enzyme 3.1 (Applied Biosystems, Foster City, California)in the reaction: 2 mL diluted BigDye in a 1 : 3 dilution ofBigDye : dilution buffer (400 mM Tris pH8.0, 10 mMMgCl2); 0.3 mL 10 mM primer; 10–20 ng cleaned PCRtemplate; and H2O to 5 mL total reaction volume with LR0R(Moncalvo et al. 1995) and/or LR3 (Vilgalys and Hester1990) in addition to amplification primers with parametersdescribed in Aime (2006). Sequencing reactions werecleaned by ethanol precipitation and sequenced on anABI 3100 Genetic Analyzer (Applied Biosystems, Foster City,California).

Contiguous sequences were assembled and edited inSequencher 4.1.4 (Gene Codes Corp., Ann Arbor, Michi-gan). Sequences were confirmed as belonging to Melamp-sora by BLASTN (http://blast.ncbi.nlm.nih.gov), and adataset of Melampsora sequences was assembled from thenewly generated sequences and all available 28S sequencesfrom the GenBank database (http://www.ncbi.nlm.nih.gov). Sequence alignments were constructed by eye in Se-Al 2.0a11 (Andrew Rambaut, Dept. Zoology, Univ. Oxford,UK; http://evolve.zoo.ox.ac.uk/). Initial phylogenetic anal-yses with maximum parsimony (MP) and neighbor joining(NJ) were conducted in PAUP* 4.0b10 (Swofford 2002)following Aime (2006). From these preliminary analysessequences were assigned to phylotypes. A phylotype was

defined as a population of 28S sequences (spanning ca. thefirst 1000 bp of the 59 end of the gene) sharing $ 99.8%

sequence similarity. The ITS-2 region was excluded fromconsideration in assigning phylotypes because more thanhalf of the PNW sequences showed evidence of multiple ITScopies in direct sequencing, although such a phenomenonwas not evident in the adjacent 28S region. All sequencesassigned to a given phylotype were re-examined inSequencher as batches to eliminate the possibility of editingerror. The dataset was pruned to contain a singlerepresentative of each of the 14 phylotypes recovered inaddition to a single representative for each phylogeneticspecies in GenBank for a total of 38 Melampsora sequencesin the final analyses. Naohidemyces vaccinii was chosen as anoutgroup for rooting purposes because studies showed thisto be a sister taxon of Melampsora (Aime 2006). Final MPanalyses were conducted in PAUP* as heuristic searcheswith 100 random addition replicates and TBR branchswapping. Support for MP branching topologies wasevaluated by bootstrap analysis derived from 10 000 repli-cates with 10 random addition replicates each. Maximumlikelihood (ML) analyses were conducted by quartetpuzzling in PAUP* with 10 000 puzzling steps; transition/transversion ratio 5 2. DNA sequences for each uniquerecovered phylotype were deposited in GenBank, accessionsnumbers EF192193–192211 (TABLE I).

RESULTS

Field surveys and observations.—Ninety of the 107surveyed populations were positive for incidence ofrust (SUPPLEMENTARY TABLE I). Of the 54 surveyedpopulations that also were included in the inocula-tion experiment 40 supported rust in the field.Seventy percent of these rusted populations (28/40)were susceptible to some degree to one of the threerust isolates of the inoculation experiments. Of the 26other populations surveyed and included in the hostrange inoculations, 12 that had supported rust in thefield were resistant to all three experimental isolates.A further seven of these 26 populations did notsupport rust in the field, but they were susceptible toat least one of the experimental isolates. Theremaining seven populations were Eurasian speciessampled in a common garden (i.e. University ofIdaho Arboretum, Moscow); rust was not observed onthese species and they did not prove susceptible tothe three experimental isolates, ‘M’, ‘B’ and ‘N’, asreported below.

In some instances observations in the field sup-ported inferences of host specificity. For example andas just mentioned, in the cultivated landscape of theUI Arboretum three native Salix species (S. bebbiana,S. scouleriana and S. geyeriana) were severely infectedwith rust whereas the 18 interspersed willow speciesfrom Eurasia were rust free. Among the wildpopulations of willows in the PNW there were also

BENNETT ET AL.: CRYPTIC SPECIES OF MELAMPSORA 1007

TABLE I. Collection locations, host species, and GenBank reference numbers for Melampsora isolates used for rDNAsequencing arranged by phylotype as presented in FIG. 6

Phylotype Salix species Salix subgenus Collection locationYear

collectedGenBank

accession No.

A S. arizonica Vetrix Arizona 2004 EF192193S. amygdaloides Protitea Arizona 2004S. bebbiana Vetrix Lick Creek, ID 2004S. bebbiana Vetrix Puyallup, WA 2003S. boothii Vetrix Radium, B.C. 2004S. drummondiana Vetrix Lake Fork, ID 2004S. eastwoodii Vetrix Lake Fork, ID 2004S. eriocephala Vetrix Troy, ID 2004S. eriocephala Vetrix Little Salmon River, ID 2004S. exigua Longifoliae N. F. Salmon River, ID 2003S. geyeriana Vetrix Goose Creek, ID 2003S. geyeriana Vetrix Moscow, ID 2004S. hookeriana Vetrix Bellevue, WA 2004S. lasiandra Salix Bellevue, WA 2004S. lemmonii Vetrix Deadwood River, ID 2004S. melanopsis Longifoliae St. Joe River, ID - Boulder Creek 2003S. melanopsis Longifoliae Payette River, ID 2004S. melanopsis Longifoliae St. Joe River, ID - Marble Creek 2003S. melanopsis Longifoliae Salmon River, ID 2003S. melanopsis Longifoliae Payette River, ID 2003S. melanopsis Longifoliae Locksa River, ID 2003S. melanopsis Longifoliae Bitteroot River, MT 2003S. melanopsis Longifoliae Lake Fork, ID 2003S. piperi Vetrix Bellevue, WA 2004S. piperi Vetrix Bellevue, WA 2004S. piperi Vetrix Bellevue, WA 2004S. scouleriana Vetrix Enterprise, WA 2003S. scouleriana Vetrix Enterprise, WA 2003S. scouleriana Vetrix Arizona 2004S. scouleriana Vetrix Mt. Rainier, WA 2003S. scouleriana Vetrix Tacoma, WA 2003S. scouleriana Vetrix Deadwood Campground, ID 2003S. scouleriana Vetrix McCrosky State Park, ID 2003S. scouleriana Vetrix Lick Creek, ID 2004S. scouleriana Vetrix Puyallup, WA 2003S. scouleriana Vetrix McCrosky State Park, ID 2004S. scouleriana Vetrix Winchester Lake, ID 2004S. sitchensis Vetrix Skykomish River, WA 2004S. sitchensis Vetrix Skykomish River, WA 2004S. sitchensis Vetrix Skagit River, WA 2004S. sitchensis Vetrix Priest Lake, ID 2003S. sitchensis Vetrix Bellevue, WA 2004S. sitchensis Vetrix Creston, B.C. 2004S. sitchensis Vetrix Snoqualmie River, WA 2004S. wolfii Vetrix Stanley Basin, ID 2004S. wolfii Vetrix Stanley Basin, ID 2003

B S. melanopsis Longifoliae St. Joe River, ID - Huck 2004 EF192194S. drummondiana Vetrix Bitteroot River, MT 2004S. exigua Longifoliae Winchester Lake, ID 2004S. exigua Longifoliae Lake Fork, ID 2004S. exigua Longifoliae Grande Rhonde, OR 2003S. exigua Longifoliae Lick Creek, ID 2004S. exigua Longifoliae Arizona 2004S. exigua Longifoliae N. F. Salmon River, ID 2003

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TABLE I. Continued

Phylotype Salix species Salix subgenus Collection locationYear

collectedGenBank

accession No.

S. exigua Longifoliae Little Salmon River, ID 2003S. exigua Longifoliae Little Salmon River, ID 2003S. exigua Longifoliae Little Salmon River, ID 2004S. lasiandra Salix Carbon River Drainage, WA 2004S. lasiandra Salix Bellevue, WA 2004S. lasiandra Salix St. Joe River, ID - Huck 2003S. lasiandra Salix Warm Lake, ID 2003S. lasiandra Salix Lick Creek, ID 2003S. lasiandra Salix Bellevue, WA 2004S. lasiandra Salix Lake Fork, ID 2004S. melanopsis Longifoliae St. Joe River, ID - Huck 2004S. melanopsis Longifoliae Lick Creek, ID 2004S. melanopsis Longifoliae Salmon River, ID 2004S. melanopsis Longifoliae Salmon River, ID 2004S. melanopsis Longifoliae Salmon River, ID 2004S. melanopsis Longifoliae Yakima, WA 2004S. melanopsis Longifoliae Hurricane Creek 2003S. melanopsis Longifoliae Lick Creek, ID 2003S. melanopsis Longifoliae Lake Fork, ID 2003S. melanopsis Longifoliae Little Salmon River, ID 2003S. melanopsis Longifoliae Payette River, ID 2003S. melanopsis Longifoliae St. Joe River, ID - Huck 2003S. melanopsis Longifoliae St. Joe River, ID - Huck 2004S. melanopsis Longifoliae Kettle River, WA 2004S. melanopsis Longifoliae Locksa River, ID 2003S. melanopsis Longifoliae Little Salmon River, ID 2003S. melanopsis Longifoliae Payette River, ID 2003S. melanopsis Longifoliae Payette River, ID 2003S. sitchensis Vetrix Mt. Rainier, WA 2003S. sitchensis Vetrix Stevens Pass, WA 2003S. sitchensis Vetrix Skykomish River, WA 2003S. sitchensis Vetrix Merry Creek, ID 2003S. sitchensis Vetrix St. Joe River, ID - Marble Creek 2004S. sitchensis Vetrix St. Joe River, ID - Huck 2004S. sitchensis Vetrix Puyallup, WA 2003S. sitchensis Vetrix Kootney Pass, B.C 2004S. sitchensis Vetrix Bellevue, WA 2004S. sitchensis Vetrix Locksa River, ID 2004

C S. bebbiana Vetrix Santa, ID 2004 EF192195S. bebbiana Vetrix Lick Creek, ID 2004S. bebbiana Vetrix Kelowna, B.C. 2004S. scouleriana Vetrix Emida, ID 2004S. scouleriana Vetrix Deadwood Campground, ID 2004

D S. geyeriana Vetrix Lolo Pass, MT 2004 EF192196

E S. bebbiana Vetrix Payette River, ID 2004 EF192197S. bebbiana Vetrix Goose Creek, ID 2003S. bebbiana Vetrix Moscow, ID 2004S. scouleriana Vetrix Lick Creek, ID 2004S. scouleriana Vetrix Moscow, ID 2004

F S. boothii Vetrix Bear Lake, ID 2004 EF192198S. sitchensis Vetrix Radium, B.C. 2004

G S. sitchensis Vetrix Bellevue, WA 2003 EF192200

H S. lasiandra Salix Little Salmon River, ID 2003 EF192201

BENNETT ET AL.: CRYPTIC SPECIES OF MELAMPSORA 1009

indicators of some degree of host specificity. Forexample populations of S. melanopsis and S. sitchensiswere interspersed along the Skykomish River, Wash-ington. Whereas S. sitchensis was severely infectedwith rust in both survey years, no rust was observed onSkykomish S. melanopsis.

However these observations were uncommon. Moreoften rust was present on all willow species in asampling site, typically a riparian zone. In these casesspecificity was revealed only by the inoculationexperiments. Significantly rust was never observedon S. alba, the Eurasian-type host of Melampsora epitea(Pei 2005), during the 10 y of the Moscow surveys,even though rust was common on native species ofSalix in the UI Arboretum and in surrounding areas.

Inoculation experiments.—Of the three isolates usedto determine host range ‘M’ was able to infect thegreatest number of species of Salix tested (i.e. 11 of24). However ‘M’ was fully virulent (i.e. infection typeS4, FIG. 1) on only two of these 11 species, including99% (480/485 plants) of seedlings of its sourcepopulation of S. amygdaloides along the ClearwaterRiver in Idaho, 96% of S. amygdaloides seedlings fromanother population from the Arkansas River inColorado and 96% (434/450 plants) of S. arizonica,subgenus Vetrix. The phenotypes associated withpartial resistance and reduced sporulation, S2 andS3, were produced on an additional nine species ofSalix (SUPPLEMENTAL TABLE I): S. eastwoodii (3/3plants, or 100%), S. pentandra (1/1 plant, or

TABLE I. Continued

Phylotype Salix species Salix subgenus Collection locationYear

collectedGenBank

accession No.

I S. sitchensis Vetrix Snoqualmie River, WA 2003 EF192202

J S. geyeriana Vetrix Stanley Creek, ID 2004 EF192203S. bebbiana Vetrix Grand Forks, B.C. 2004S. drummondiana Vetrix Stanley Basin, ID 2004S. drummondiana Vetrix Stanley Creek, ID 2004S. drummondiana Vetrix Payette River, ID 2004S. drummondiana Vetrix Stanley Basin, ID 2004S. drummondiana Vetrix Stanley Creek, ID 2004S. lemmonii Vetrix Payette River, ID 2004S. planifolia Vetrix Payette River, ID 2004

K S. geyeriana Vetrix Bellevue, WA 2004 EF192204

L S. amygdaloides Protitea Clearwater River, ID 2004 EF192205

M S. amygdaloides Protitea Clearwater River, ID 2004 EF192206

N S. sitchensis Vetrix St. Joe River, ID - Marble Creek 2004 EF192211S. bebbiana Vetrix Winchester Lake, ID 2004S. hookeriana Vetrix Cannon Beach, OR 2004S. piperi Vetrix Puyallup, WA 2004S. piperi Vetrix Puyallup, WA 2004S. piperi Vetrix Bellevue, WA 2004S. piperi Vetrix Bellevue, WA 2004S. piperi Vetrix Bellevue, WA 2004S. piperi Vetrix Bellevue, WA 2004S. piperi Vetrix Bellevue, WA 2004S. piperi Vetrix Tacoma, WA 2003S. piperi Vetrix Bellevue, WA 2004S. piperi Vetrix Bellevue, WA 2004S. piperi Vetrix Puyallup, WA 2004S. purpurea Vetrix St. Joe River, ID - Marble Creek 2004S. purpurea Vetrix Bellevue, WA 2004S. sitchensis Vetrix Skagit River, WA 2003S. sitchensis Vetrix Merry Creek, ID 2003Abies grandis — St. Joe River, ID - Marble Creek 2003Tsuga heterophylla — St. Joe River, ID - Marble Creek 2003

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100%), S. wolfii (35/36 plants, or 97%), S. scouleriana(7/17 plants, or 41%), S. lasiandra (6/17 plants, or35%), S. bebbiana (42/301 plants, or 14%), S. piperi(1/31 plants, or 3%), S. sitchensis (2/64 plants, or3%) and S. melanopsis (2/160 plants, or 1%). Ofthese nine species six are from the subgenus Vetrix, S.pentandra and S. lasiandra are from the subgenusSalix and S. melanopsis is from the subgenus Long-ifoliae. No infection was recorded for the other 14inoculated species, eight of which are Eurasianspecies. ‘M’ was unable to reproduce at all on 49/49plants of S. exigua, 20/20 plants of S. eriocephala, 5/5plants of S. interior, 4/4 plants of S. drummondiana,3/3 plants of S. hookeriana, and 2/2 plants of S.lemmonii.

Overall the frequency of susceptibility as definedhere (i.e. S2, S3 or S4) was greatest in those species ofthe same subgenus, Protitea, as that of the sourcespecies of ‘M’, S. amygdaloides. This was supported bya chi-squared analysis showing that the phenotypicreaction of the test plants to ‘M’ was highlydependent on host subgenus (x2 5 183.43, P ,

0.0001).The experimentally determined host ranges of ‘B’

and ‘N’ were more restricted than that of ‘M’. Eachwas able to reproduce to some extent on only fivespecies, the source species of each and four others(SUPPLEMENTAL TABLE I). Each therefore failed toreproduce on 20 species of Salix. Even on S.melanopsis, the source of ‘B’, overall incidence ofsusceptibility to ‘B’ (either S2, S3 or S4) was only66/169, or 39%. Outside S. melanopsis, but still withinits subgenus Longifoliae, limited susceptibility wasobserved among PNW populations of S. exigua; 3/50plants, or 6%, were S2 phenotypes. Although the Salixinterior seedlings from Minnesota also belong to thesubgenus Longifoliae, they were entirely resistant to‘B’. The only other susceptibility of any kind that wasobserved in response to ‘B’ involved three speciesfrom the subgenus Vetrix: five seedlings of S.scouleriana (5/11, or 45%) from the isolated Sangrede Cristo Mountains of New Mexico and singleseedlings of each of S. piperi from the PNW (1/32plants, or 3%) and S. wolfii (1/1 plants), all withlimited S2 infection types. Overall the reaction of thetest plants to ‘B’ was dependent on host subgenus (x2

5 54.7, P , 0.0001), as one would expect giveninfection of 30.8% of the plants in the subgenusLongifoliae, limited infection of the subgenus Vetrixand no reproduction whatsoever of ‘B’ on subgeneraSalix and Protitea.

The host range of ‘N’ was narrower still. ‘N’reproduced only on species of the subgenus Vetrixto which S. sitchensis, the source species of ‘N’,belongs. No susceptible plants (i.e. not even S2)

were found in the subgenus Salix (i.e. 0/17), thesubgenus Protitea (0/5) or the subgenus Long-ifoliae (i.e. 0/222). All 20 seedlings of the sourcepopulation of S. sitchensis were susceptible, ofwhich 15 (75%) were S4. Some resistance wasobserved among other, non-source populations ofS. sitchensis, but of 43/46 seedlings on which ‘N’reproduced only 11 (24%) were fully susceptible(S4). Four other species of the subgenus Vetrix, S.arizonica, S. bebbiana, S. piperi and S. scouleriana,exhibited limited susceptibility. Given that suscep-tibility to ‘N’ was limited to the subgenus Vetrix(98/197 plants), it was not surprising to find thatits incidence was highly dependent on thesubgenus of the test plants (x2 5 158.79, P ,

0.0001).

Light microscopy of urediniospores.—With host speciesas the independent variable, significant differences inurediniospore area, shape and spine density were notobserved. Of 459 pairwise comparisons of uredinio-spores, only 14 pairs of host species differedsignificantly (P , 0.05) in spore area, two pairs inspine density and three pairs in spore shape(SUPPLEMENTAL TABLE II). However with host subge-nus as the independent variable differences inurediniospore morphology were significant. Rustcollections from species in the subgenus Salix hadsignificantly larger spores (P , 0.05), fewer spines persurface area (P , 0.05) and less circular spores (P ,

0.0001) than rust collections on willows in thesubgenus Vetrix (FIG. 2). The spore morphology ofthe rust collections on willows in the subgenusLongifoliae was intermediate; spores on the subgenusLongifoliae differed significantly in spore area (P ,

0.0001) and spine density (P 5 0.0124) from those inthe subgenus Vetrix and in spore shape (P 5 0.0292)from those in the subgenus Salix. Spore morphologyon the subgenus Protitea differed from that on thesubgenus Salix only in spore area.

Discriminant analysis resulted in only 16% correctclassification of host species when the basis for theirclassification was the combination of urediniosporearea, shape and spine density. However correctclassification to subgenus was obtained for 70% ofthe collections overall and 94% of those from thesubgenus Vetrix, even at the modest samplingintensity of this study.

Scanning electron microscopy of urediniosporesand paraphyses.—SEM examination of uredinio-spores and paraphyses revealed some potentiallyuseful characters for further research. Echinulationwas always even but below the spines urediniosporesurfaces varied from rugose (FIG. 3A) to stronglyreticulate (FIG. 3B). The surfaces of paraphyses also

BENNETT ET AL.: CRYPTIC SPECIES OF MELAMPSORA 1011

varied from smooth (FIG. 3C) to reticulate (FIG. 3D).In some uredinia the surfaces of urediniospores andparaphyses were quite similar (FIG. 4A), whereas inother uredinia they were distinct (FIG. 4B). Shallowpits were observed at the base of spines of uredinio-spores in some cases (FIG. 4C). Ear-shaped paraphy-ses (FIG. 4D), which were often more peripheral thaninterspersed in uredinia, were evident in somecollections, particularly from S. piperi. However moretypically paraphyses were pear- or lollipop-shaped(FIG. 5A) and interspersed throughout uredinia.Some uredinia from leaves of S. sitchensis and S.boothii were characterized by a relatively high ratio ofparaphyses to urediniospores. To our knowledge

aeciospores of M. epitea f. sp. tsugae have not beenexamined previously under SEM; we illustrated(FIG. 5B) the unevenly verrucose aeciospores that weobserved from a collection from Tsuga heterophyllathat subsequently was used successfully to infect S.sitchensis.

Phylogenetic analysis.—Phylogenetic diversity of 140rust collections (TABLE I) from 92 willow populationsfrom 19 species of Salix was determined, based onnuclear rDNA sequences. Analyses indicated 14 phylo-types, ‘A’ through ‘N’, that must be the result of

FIG. 2. Mean values (6 standard deviation) of uredin-iospore area, spine density and circularity of rust collectionswithin three host Salix subgenera. Letters denote signifi-cantly different means (P , 0.05) among subgenera.

FIG. 3. Scanning electron micrographs of Melampsoraurediniospores and uredinial paraphyses. Below the spinesof urediniospores in A and B, are seen rugose and reticulatesurfaces, respectively. Smooth and reticulate surfaces ofparaphyses are seen in C and D, respectively.

FIG. 4. Scanning electron micrographs of Melampsoraurediniospores and uredinial paraphyses. Within a uredini-um, surfaces of urediniospores and paraphyses could be thesame (A) or different (B). Pits at the base of spines werepresent (C) or absent. Paraphyses were sometimes ear-shaped (D).

1012 MYCOLOGIA

extensive diversification involving host specialization(FIG. 6, TABLE I). Each of the 14 phylotypes sharedmore recent common ancestors with rusts thatspecialize on other hosts, particularly Populus, thanwith most of the other phylotypes of Melampsora fromwestern Salix. Of the 14 phylotypes only ‘A’ wascommon to both the Pacific Northwest and Southwestand found on the four subgenera of Salix of thisstudy (i.e. Vetrix, Longifoliae, Salix and Protitea). ‘B’was found on three subgenera (i.e. Vetrix, Longifoliaeand Salix). Each of the remaining 12 phylotypes wasrestricted to a single subgenus. Phylotype ‘A’ wasfound on 17 of the 19 species of Salix from whichrust was collected for sequencing in this study; ‘A’ wasnot found on S. planifolia, which was sampled onlyonce, and S. purpurea, a Eurasian species that wassampled only twice. Overall ‘A’ and ‘B’ were the mostabundant phylotypes in that 46 collections belongedto each. ‘N’ was of intermediate abundance with 20rust collections. Seven phylotypes (i.e. ‘D’, ‘G’, ‘H’, ‘I’,‘K’, ‘L’ and ‘M’) were singletons represented by asingle rust collection each.

Some host specificity was demonstrated by rusts onspecies of Salix subgenus Longifoliae. Salix exigua andS. melanopsis were sampled intensively with 10 (fromriparian areas in Idaho and Arizona) and 27collections (Idaho, Montana, Oregon and Washing-ton) respectively. Yet all belonged to the twoabundant and closely related phylotypes, ‘A’ and‘B’, with 28 of the 37 in ‘B’.

None of the 14 phylotypes reported here wasclosely related to Eurasian taxa of Melampsora onSalix (FIG. 6). Phylotypes ‘A’ and ‘B’ were clearlydivergent from their nearest Eurasian relative, M.laricis-epitea Kleb. from S. viminalis (i.e. AY444790,FIG. 6) in the UK (Pei, Bayon et al. 2005). Eurasian M.salicis-albae Kleb., M. ribesii-purpureae and M. laricis-pentandrae Kleb. were not closely related to any of theNorth American phylotypes reported here.

Collections from Abies grandis and from Tsugaheterophylla were assumed to be M. abieti-caprearumand M. epitea f. sp. tsugae respectively (Hylander,Jorstad et al. 1953, Ziller 1959). These two taxa cannotbe distinguished morphologically on Salix, but theydiffer in aecial hosts (Ziller 1959). In this study theyproved to be phylogenetically indistinguishable be-cause both belonged to phylotype ‘N’.

DISCUSSION

We observed variation within Melampsora on Salix inwestern North America that will necessitate thedescription of many new taxa in coming years.Although new taxa must be described, the task willnot be easy because the life cycles of these rusts mustbe determined. Ideally monouredinial isolates will beused, due to the possibility of mixed infections ofsingle plants. It is possible that some of the distinctphylotypes (i.e. putative new species) reported herewill possess indistinguishable uredinial states, butother spore states that we have not yet studied mightpossess distinctive characteristics. It is worth notingthat Melampsora on the subgenus Longifoliae has notbeen studied in Europe or Asia because its eightspecies are restricted to western North America andMexico (Argus 2010). We sampled Melampsora onsubgenus Longifoliae for the first time here, and all 37collections belonged to the closely related phylotypes‘A’ and ‘B’ with 28 of the 37 in ‘B’. Thus ‘A’ and ‘B’in particular are good candidates for description asnew species.

The range of aecial hosts for willow rusts in NorthAmerica is limited apparently to Tsuga, Abies, Larix,Ribes and Saxifraga. In Eurasia willow rusts also caninfect Allium, Euonymus, Viola, Galanthus, Corydalisand some members of Orchidaceae, in addition toAbies, Larix, Ribes and Saxifraga (Pei 2005). Thisdifference is relevant to the question of the extent towhich Eurasian taxa of Melampsora on Salix can beapplied to North American species; reproductiveisolation and eventually speciation are likely in theabsence of a common aecial host on which matingcan occur. Therefore it is instructive to consider theextent to which the willow rusts of North Americainfect European aecial hosts when the latter areintroduced and grown in North America. A search ofthe USDA Systematic Mycology and MicrobiologyLaboratory (SMML) databases for example revealedthat all 49 records of willow-associated Melampsora onAllium are from Europe (Farr and Rossman 2011). Inother words Allium is never infected with Melampsorain North America, despite the fact that Eurasianspecies of Allium are widely cultivated in NorthAmerica and there are a further 96 species of this

FIG. 5. Scanning electron micrographs of Melampsorauredinial paraphyses and aeciospores. The most commonshape for uredinial paraphyses is seen in A. In B areunevenly verrucose aeciospores of Melampsora phylotype‘N’ from Tsuga heterophylla.

BENNETT ET AL.: CRYPTIC SPECIES OF MELAMPSORA 1013

FIG. 6. First of eight most parsimonious trees derived by maximum parsimony (MP) analyses of combined internaltranscribed spacer region-2 (ITS-2) and large ribosomal subunit (28S) nuclear rDNA sequences of 38 Melampsora isolates.Naohidemyces vaccinii was chosen as an outgroup for rooting purposes. Each lineage (phylotypes A–N) represents 1–46individuals based on preliminary analyses (not shown) of sequences obtained from 140 Melampsora isolates from westernNorth America and sequences from 22 different Melampsora species obtained from GenBank (accession numbers areindicated for each terminal branch and TABLE I). MP bootstrapping values were obtained by 1000 random addition sequencereplicates. Maximum likelihood (ML) analyses were conducted by the quartet puzzling method with 10 000 puzzling steps.

1014 MYCOLOGIA

genus that are native to North America (Mabberley2008). Thus Allium-alternating M. allii-fragilis and M.salicis-albae must be exclusively Eurasian. In this lightit is not surprising that M. salicis-albae (i.e. AY444788,FIG. 6) from S. alba in the UK (Pei, Bayon et al. 2005)was not closely related to any of the phylotypesreported here.

Similarly all records of willow-associated Melamp-sora on Euonymus are from Eurasia, even though onceagain Eurasian species of Euonymus are widelycultivated in North America alongside four nativespecies. Thus Euonymus-alternating M. euonymi-caprearum must be native to Eurasia alone.

Eight genera of Orchidaceae are aecial hosts forfour taxa of willow-associated Melampsora repentisPlowr. in Eurasia, but not a single orchid species inNorth America hosts Melampsora, so M. repentis mustbe exclusively Eurasian. The same pattern is revealedwith a SMML search for Melampsora on Viola,Galanthus and Corydalis; thus Viola-alternating M.lapponum Lindf., Galanthus-alternating M. galanthi-fragilis Kleb., and Corydalis-alternating M. chelidonii-pierotii Matsumoto and M. yezoensis Miyabe & T.Matsumoto also must be limited in their native rangesto Eurasia.

Ribes species are hosts of willow-associated Mel-ampsora in both Eurasia and North America, so atfirst glance this appears to present a differentpattern. But because some Eurasian species of Ribesare also widely cultivated in North America (i.e.Ribes nigrum and Ribes rubrum, black and redcurrants respectively) it is clear that the samepattern continues with Ribes: willow rusts are hostedby black and red currants in Eurasia but not inNorth America. Instead North American willow rustsparasitize only species of Ribes that are native toNorth America; thus M. ribesii-epitea Kleb., M.ribesii-viminalis Kleb. and M. ribesii-purpureae, whichinfect red and black currants in Eurasia, may beexclusively Eurasian. M. ribesii-purpureae has beenconsidered tentatively to be native to both Eurasiaand North America (Pei 2005). However all 14phylotypes reported here were distinct from M.ribesii-purpureae (i.e. AY 444791, FIG. 6) from S.purpurea in the UK (Pei, Bayon et al. 2005). Ziller(1974) had said that M. ribesii-purpureae is ‘‘rare inwestern Canada, at least on Ribes spp.’’

Saxifraga species have a distribution similar to thatof Ribes species as aecial hosts, although a fewcircumpolar species host willow rusts in both Eurasiaand North America. Even Abies yields this patterninasmuch as the only Eurasian firs that are grown inNorth America, A. alba and A. homolepis, are attackedby willow-associated Melampsora only in Eurasia (Farrand Rossman 2011). Larix decidua, European larch,

provides what appears to be the only exception to thisrule in that it appears to host willow-associatedMelampsora wherever it is planted (Farr and Rossman2011).

The same argument can be applied to telial hosts.In this study Eurasian species of Salix (i.e. S.acutifolia, S. alba, S. arenaria, S. babylonica, S. caprea,S. cinerea, S. daphnoides, S. elaeagnos, S. integra, S.matsudana, S. myrsinifolia, S. pentandra, S. purpurea,S. repens and S. yezoalpina) exhibited resistance toNorth American Melampsora in that they remainedrust-free while growing among native populations ofSalix that produced uredinia in 2003 and 2004 in acommon garden in the University of Idaho Arbore-tum. The inoculations reported here confirmed thisresistance of Eurasian Salix species to North Ameri-can Melampsora species. Records of rust-host distri-butions provide additional, albeit indirect, evidenceof this resistance. For example S. matsudana, thecorkscrew willow, and S. pentandra are Eurasianspecies affected respectively by six and eight speciesof Melampsora in their native ranges, yet they havebeen rust-free as widely planted ornamentals in NorthAmerica (Farr and Rossman 2011). However we didcollect rust in the field on S. purpurea, a Eurasianspecies that may possess exceptional susceptibility to astrain or strains of phylotype ‘N’.

Our combined results show evidence of pathogenicvariation within a phylotype. For example S. sitchensis,S. melanopsis and S. lasiandra grow together alongthe St Joe River in Idaho and all were positive for rustin our field surveys. When we inoculated populationsof these three species from the St Joe River withphylotype ‘B’ from S. melanopsis, only S. lasiandra wassusceptible, even though ‘B’ was found on the othertwo species in the field. Phylotype ‘A’ also mustharbor pathogenic variation because it was found inthe field survey on 17 species of Salix, but theexperimentally determined host ranges were muchnarrower. Pathogenic variation within a phylotypealso was evident in ‘N’; collections from Abies grandisand Tsuga heterophylla of M. abieti-caprearum and M.epitea f. sp. tsugae respectively are reputed to bespecific to these aecial hosts (Ziller 1959).

Both phylotypic and pathogenic diversity appearto be likely within any well sampled, riparian area orcollection site in western North America. Forexample in Bellevue, Washington, five different Salixspecies were found to co-occur in a natural slough.Sequences of rust from these five belonged to fourphylotypes (i.e. rusts of S. lasiandra and S.hookeriana in ‘A’, S. piperi in ‘A’ and ‘N’, S. geyerianain ‘K’ and S. sitchensis in ‘A’ and ‘G’). Pathogenicvariation within a site might be explained by thisphylotypic diversity but in any case was inferred from

BENNETT ET AL.: CRYPTIC SPECIES OF MELAMPSORA 1015

incongruities between inoculations and field surveys.For example 12 populations that were susceptible insitu were resistant in all inoculation experiments.Phylotype ‘N’ from S. sitchensis reproduced on itssource population from the St Joe River but not oneither S. melanopsis or S. lasiandra from the samecommunity.

Descriptions of new species of Melampsora on Salixin North America of course should include not onlythe uredinial state but the other four spore states andtherefore the aecial host(s) as well. In our compar-isons of the morphology of uredinia differences weremost apparent at host subgenus rank, but other sporestates might be more distinctive. It was interestingthat the tree willows of subgenera Salix and Protitea,which retain the ancestral characters of the genus(Argus 2010), supported rust with relatively largeurediniospores and low spine density. Phylotype ‘M’from the tree willow S. amygdaloides also possessedthe broadest host range with at least some infection ofthree Salix subgenera. In contrast the shrub willows ofthe subgenus Vetrix are characterized by the mostrecently evolved characters in the genus (Azuma,Kajita et al. 2000, Trybush, Jahodova et al. 2008), andurediniospores on Vetrix were relatively small withgreater spine densities than those on other subgen-era. Moreover the inoculations with rust from theshrub willow S. sitchensis showed this rust able toinfect only other shrub willows of the subgenus Vetrix.The subgenus Longifoliae, restricted to western NorthAmerica and Mexico (Brunsfeld, Soltis et al. 1991,1992; Dorn 1998; Argus 2010) supported rust that wasintermediate in morphology and host range. Theurediniospore morphology of Melampsora species ontree willows in Europe is also distinct from that ofspecies on shrub willows (Pei, Royle et al. 1993).Morphological characters that were visible only underSEM (FIGS. 3–5) also might be of value in speciesdescriptions.

Central to further efforts to segregate species ofMelampsora on Salix in North America is the questionof M. epitea. Here M. epitea was represented bycollections of two of its races, M. abieti-caprearum (viarust from Abies grandis) and M. epitea f. sp. tsugae(Tsuga heterophylla). Both of these collections provedto be phylotype ‘N’, which shared more recentcommon ancestors with species of Melampsora onAsian Populus (i.e. P. yunnanensis ‘AB116821’, P.pseudoglauca ‘AB116797’ and P. wilsonii ‘AB116799’in FIG. 6) than with accepted taxa of Melampsora onSalix (Tian, Shang et al. 2004).

However it is not possible to accept phylotype ‘N’ asM. epitea. First, with inoculum of ‘N’ we were unableto infect the type host of M. epitea, S. alba or anyother member of Salix subgenus Salix. Second, we

were unable to infect any other Eurasian species ofSalix in the inoculation experiments, although S.purpurea was infected with ‘N’ in the field. Melamp-sora epitea originally was described from Europe on S.alba (Pei 2005). Whatever M. epitea may be it cannotbe a species that is unable to reproduce on S. alba.Technically it might be that some other variant orisolate of ‘N’ could infect S. alba, but that seemsunlikely given the entirely negative results of the 10 ysurvey of S. alba in Moscow, Idaho, and the observedprevalence of ‘N’ in the vicinity and nearby on bothAbies and Tsuga. Relatedness of ‘N’ to Melampsorataxa on Asian Populus also argues against acceptanceof ‘N’ as M. epitea.

It is ironic that Arthur’s first instinct, uponencountering willow-associated Melampsora on Abiesin North America, was to describe M. americanaArthur, which he regarded as the ‘‘most common’’rust on Salix in North America. Only later did hesynonymize his M. americana with M. abieti-caprearum Tubeuf (Arthur 1962), a taxon thatHylander came to regard as a race of M. epitea(Hylander, Jorstad et al. 1953). Hylander in turninfluenced Ziller (Ziller 1959) who chose to describea Tsuga-alternating willow rust as M. epitea f. sp.tsugae. Because Ziller (1974) also asserted that M.paradoxa belonged to the ‘‘M. epitea complex’’ andthat M. ribesii-purpureae was rare in western NorthAmerica we are actually somewhat conservative inour conclusions here that only 12 phylotypes areunaccounted for by current taxonomy. It is possiblethat all 14 will require designation as new speciesand that additional new species await more extensiveand intensive sampling.

ACKNOWLEDGMENTS

We thank the M.J. Murdock Charitable Trust for use ofthe FESEM. We thank Dave Dreesen, Carl-Eric Granfeltand the late Steve Brunsfeld for helping us with Salixcollections and species identifications, Cindy Park fortechnical laboratory assistance and two reviewers forhelpful comments. Financial support was provided bythe Stillinger Foundation and the Center for Research onInvasive Species and Small Populations of the Universityof Idaho.

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