Phylogenetic species recognition reveals host-specific lineages among poplar rust fungi

Molecular Phylogenetics and Evolution 66 (2013) 628–644

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Molecular Phylogenetics and Evolution

journal homepage: www.elsevier .com/locate /ympev

Phylogenetic species recognition reveals host-specific lineages among poplarrust fungi

Agathe Vialle a,b,⇑, Nicolas Feau c, Pascal Frey d,e, Louis Bernier b, Richard C. Hamelin a,⇑a Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, 1055 du PEPS, P.O. Box 10380, Stn. Sainte-Foy, Québec, QC, Canada G1V 4C7b Centre d’étude de la forêt (CEF) and Institut de biologie intégrative et des systèmes (IBIS), Université Laval, Québec, QC, Canada G1V 0A6c INRA, UMR 1202 BIOGECO, INRA 69 Route d’Arcachon, 33612 Cestas Cedex, Franced INRA, UMR1136 Interactions Arbres – Microorganismes, F-54280 Champenoux, Francee Université de Lorraine, UMR1136 Interactions Arbres – Microorganismes, F-54280 Champenoux, France

a r t i c l e i n f o

Article history:Received 21 February 2012Revised 16 September 2012Accepted 11 October 2012Available online 10 November 2012

Keywords:MelampsoraHost-pathogen co-evolutionGenealogical concordance phylogeneticspecies recognitionBayesian concordance analysisPhylogenetic species

1055-7903/$ - see front matter Crown Copyright � 2http://dx.doi.org/10.1016/j.ympev.2012.10.021

⇑ Corresponding authors at: Natural Resources CanLaurentian Forestry Centre, 1055 du PEPS, P.O. Box 10QC, Canada G1V 4C7. Fax: +1 418 648 5849 (A. Vialle

E-mail addresses: [email protected] (A. Via(R.C. Hamelin).

a b s t r a c t

Fungal species belonging to the genus Melampsora (Basidiomycota, Pucciniales) comprise rust pathogens thatalternate between Salicaceae and other plant hosts. Species delineation and identification are difficult withinthis group due to the paucity of observable morphological features. Several Melampsora rusts are highly host-specific and this feature has been used for identification at the species level. However, this criterion is notalways reliable since different Melampsora rust species can overlap on one host but specialize on a differentone. To date, two different species recognition methods are used to recognize and define species within theMelampsora genus: (i) morphological species recognition, which is based solely on morphological criteria;and (ii) ecological species recognition, which combines morphological criteria with host range to recognizeand define species. In order to clarify species recognition within the Melampsora genus, we applied phyloge-netic species recognition to Melampsora poplar rusts by conducting molecular phylogenetic analyses on 15Melampsora taxa using six nuclear and mitochondrial loci. By assessing the genealogical concordance betweenphylogenies, we identified 12 lineages that evolved independently, corresponding to distinct phylogeneticspecies. All 12 lineages were concordant with host specialization, but only three belonged to strictly definedmorphological species. The estimation of the species tree obtained with Bayesian concordance analysis high-lighted a potential co-evolutionary history between Melampsora species and their reciprocal aecial hostplants. Within the Melampsora speciation process, aecial host may have had a strong effect on ancestral evo-lution, whereas telial host specificity seems to have evolved more recently. The morphological characters ini-tially used to define species boundaries in the Melampsora genus are not reflective of the evolutionary andgenetic relationships among poplar rusts. In order to construct a more meaningful taxonomy, host specificitymust be considered an important criterion for delineating and describing species within the genus Melamps-ora as previously suggested by ecological species recognition.

Crown Copyright � 2012 Published by Elsevier Inc. All rights reserved.

1. Introduction

Rust fungi (Order Pucciniales) are obligate biotrophs that com-prise some of the most important crop and forest pathogens. Manyspecies in the genus Melampsora cause poplar rust on different Pop-ulus species, a foliar disease responsible for important losses inpoplar plantations (Frey et al., 2005). Melampsora is the only genusin Melampsoraceae (Cummins and Hiratsuka, 2003). This genus dis-plays a great variety of life cycles and host relationships. Some spe-cies are heteroecious, i.e., species that need two different host

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ada, Canadian Forest Service,380, Stn. Sainte-Foy, Quebec,).lle), [email protected]

plants to complete their life cycle, alternating either between Pin-aceae and Salicaceae or between herbaceous plants and Salicaceae.Other Melampsora species are autoecious on various dicotyledon-ous plants, i.e., species that complete their life cycle on a singlehost plant (Gäumann, 1959). All the rust species known on poplarare considered macrocyclic, i.e., they produce five different sporestages, and all Melampsora species described on poplar with a com-plete life cycle are heteroecious rusts (Cummins and Hiratsuka,2003; Pei and Shang, 2005). However, several species are describedas hemicyclic (i.e., a short life cycle with only two different sporestages) and/or are suspected to overwinter in poplar buds (Savile,1973). The aecial hosts of poplar rusts include coniferous, dicotyle-donous and monocotyledonous plants, and some aecial host iden-tities (non-poplar host) remain unknown.

Species delineation and identification are difficult within thisgroup (Tian and Kakishima, 2005; Feau et al., 2009; Vialle et al.,

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2011). One of the most important criteria for species delineation inMelampsora taxonomy remains the morphology of the uredinialspore stage on poplar (spore stage II). Position of uredinia on pop-lar leaves (epiphyllous, hypophyllous or amphigenous), uredinio-spore size and wall thickness, paraphyse size and wall thickness,and especially urediniospore ornamentation are crucial traits fordifferentiating species (Arthur, 1903; Cummins and Hiratsuka,2003; Pei and Shang, 2005). Nevertheless, these traits have a ten-dency to overlap between different species and, initially, only threemorphological Melampsora species were identified based solely onurediniospore morphology (Schröter, 1886; Arthur, 1903). At theend of the 19th century, the discovery that a rust species could in-fect, at different periods, totally unlinked host plants modified andbroadened the concept of species in this group of fungi (DeBary,1866; Arthur, 1903). Based on extensive and empirical infectionstudies, rust species on poplars were then defined by combiningurediniospore morphology with telial and aecial host ranges(Klebahn, 1899, 1902, 1917; Arthur, 1903; Gäumann, 1959).However, the classification of morphologically similar rusts withdistinct aecial hosts remained problematic because theirconsideration as distinct species, even though morphologicallyindistinguishable, has not received total support (Peace, 1962;Wilson and Henderson, 1966; Boerema and Verhoeven, 1972;Bagyanarayana, 1998).

Following an exhaustive literature search, we retrieved recordsfor 20 poplar rust species established mainly on the basis of theirurediniospore morphology, aecial and telial host ranges (see Sup-plementary Data 1 for the species list with their respective taxo-nomic features). Some discrepancies were noticed regarding theidentification and taxonomic consideration of these 20 speciesdepending on the species recognition criterion used, i.e., morphol-ogy or host-specificity (Vialle et al., 2011). For instance, six taxa (M.aecidioides, M. magnusiana, M. larici-tremulae, M. pulcherrima, M.pinitorqua and M. rostrupii) are sometimes considered as a singlespecies, known as M. populnea, due to their similar urediniosporemorphology (Peace, 1962; Wilson and Henderson, 1966; Boeremaand Verhoeven, 1972; Bagyanarayana, 1998). Also, due to overlapin their aecial and telial host ranges (at least under laboratory con-ditions) and similar urediniospore morphology, some taxa, such asM. abietis-canadensis and M. medusae, have been considered synon-ymous (Bagyanarayana, 1998; Tian and Kakishima, 2005). How-ever, all of these taxa display aecial host specificity allowingspecies differentiation (see Vialle et al., 2011 for details). A recentDNA barcoding study highlighted genetic differences betweenthese taxa and emphasized the importance of revisiting the speciesconcept in this group of fungi (Feau et al., 2009). Other poplar rustshave been described as formae speciales, a mycological subspecifictaxonomic level defined by host specialization. This is the case forM. medusae, which was split into two different formae specialesbased on telial host specialization: M. medusae f. sp. deltoidae, pri-marily pathogenic on Populus deltoides, and M. medusae f. sp. trem-uloidae, primarily pathogenic on Populus tremuloides. As the telialhosts belong to two non-interfertile sections of the genus Populus,some doubt remains about the consideration of these two formaespeciales as two distinct species (Shain, 1988).

Another problem is that several species have been reported anddescribed only on their telial (poplar) host. Eight species, namelyM. castellana, M. ciliata, M. cumminsii, M. microspora, M. multa, M.nujiangensis, M. osmaniensis and M. pruinosae (Supplementary Data1), are rarely observed and were described only on the basis ofmorphological differences in their uredinial and telial stages. Thecomplete life cycle and aecial host identity of these rare species re-main unknown, and they may be occasional variants, which mayhave complicated poplar rust classification (Pei and Shang, 2005).

To date there is still no consensus on poplar rust fungal taxon-omy. On the one hand, taxonomists favoring the morphological

species recognition have proposed a number of ‘races’ (formaespeciales) that do not differ sufficiently in their morphology to beconsidered distinct species; on the other hand, a second taxonomicschool has used the ecological species recognition approach byconsidering aecial host specificity and pathogenicity, throughextensive experimental infection studies, as reliable criteria tocomplement morphological species recognition and has thus de-fined these formae speciales as distinct species (see Vialle et al.(2011) for details). Depending on the taxonomic school, 9 to 32Melampsora species can be considered as occurring on poplar inthe world.

In the case of a lack of traditional taxonomic features necessaryto recognize and delimit species, such as morphological speciesrecognition (Hawksworth et al., 1996), biological species recogni-tion (Mayr, 1942) or ecological species recognition (Van Valen,1976), species boundaries can be determined using the genealogi-cal concordance phylogenetic species recognition (GCPSR) methodwhich uses the concordance between the genealogies of severalunlinked genes to identify evolutionary-independent lineagesand delimit phylogenetic species (Taylor et al., 2000; Dettmanet al., 2003; Giraud et al., 2008). This method provides a strongbackground to infer phylogenetic and evolutionary relationshipsamong species (Taylor et al., 2000, 2006). Moreover, recent devel-opments in molecular phylogenetic analysis propose statistical ap-proaches to estimate the species tree from multiple single genetrees despite incongruence (Ané et al., 2007; Baum, 2007; Largetet al., 2010; Blair and Murphy, 2011; Leaché and Rannala, 2011).One of them, called Bayesian concordance analysis (BCA), esti-mates species relationships by integrating gene tree uncertainty(Baum, 2007; Chung and Ané, 2011).

In this study, we constructed multigene genealogies to investi-gate the genetic structure of Melampsora species retrieved on pop-lar and to provide a phylogenetic framework for understandingtheir evolutionary relationships. Using GCPSR, we aimed to clarifytaxonomical uncertainties by identifying independent evolution-ary lineages (i.e. phylogenetic species) among Melampsora poplarrust species. To complement GCPSR and infer phylogenetic rela-tionships among these phylogenetic species, we applied a Bayesianconcordance analysis. By identifying the phylogenetic species anddrawing an estimation of the species tree for the Melampsora rustsdescribed on poplar, we aimed to answer the following questions:(i) Do the genetically isolated lineages within the genus Melamps-ora retrieved on poplar correspond to the morphological Melamps-ora species described in the literature? (ii) Are these lineagesspecialized on aecial and/or telial host species, thus confirmingthe importance of host specificity in the speciation process amongpoplar rusts? Finally, we aimed to compare the two taxonomicschools of rust fungal taxonomy (morphological species recogni-tion and ecological species recognition) using a phylogenetic spe-cies recognition approach.

2. Materials and methods

2.1. Sample collection and herbarium specimen recovery

A total of 300 herbarium specimens were examined and we ob-tained DNA sequences for 92 Melampsora specimens (Table 1).Twenty-two specimens were retrieved from official herbariumsand assigned to species based on the determinator and curatoridentification. Thirty-two specimens were used and identified tothe species level in previous studies (Pei et al., 2005; Barrèset al., 2006; Feau et al., 2009; Desprez-Loustau, 1986). Speciesidentification of the remaining 38 specimens was based on sporemorphology in association with host specificity. These identifica-tions were confirmed with molecular data (ITS and 28S) whenthe barcode sequences provided species identification (Feau

Table 1Specimens included in this study.

Sample ID Species identification Host Collectiondate

Geographicallocalization

Herbarium/collection ID

BOLD ID ITS LSU CO1 Nad6 MS277 MS208

Melampsora abietis-canadensis1399MEA-POG-USA Melampsora abietis-canadensis Populus grandidentata 1959-12-08 United States, Wisconsin PUR 56615 MPITS160-09 JN881733 JN934918 JQ011188 JN985832 JQ011008 JQ0110981400MEA-POG-USA Melampsora abietis-canadensis Populus grandidentata 1959-09-21 United States, Wisconsin PUR 61512 MPITS161-09 JN881734 JN934919 JQ011189 JN985833 JQ011009 JQ011099668X-TSC-SH13 Melampsora abietis-canadensis Tsuga canadensis 2007-07-01 Canada, Québec QFB 25031 MPITS044-08 GQ479826 JN934921 JQ011191 JN985835 JQ011012 JQ011102669X-TSC-SH-14 Melampsora abietis-canadensis Tsuga canadensis 2007-07-01 Canada, Québec QFB 25032 MPITS045-08 GQ479827 JN934922 JQ011192 JN985836 JQ011013 JQ011103968X-TSC-BE Melampsora abietis-canadensis Tsuga canadensis 2008-06-24 Canada, Québec QFB 25061 MPITS079-08 GQ479829 JN934924 JQ011194 JN985838 JQ011015 JQ011105967X-TSC-BE Melampsora abietis-canadensis Tsuga canadensis 2008-06-24 Canada, Québec QFB 25062 MPITS078-08 GQ479830 JN934923 JQ011193 JN985837 JQ011014 JQ011104667X-TSC-SH12a Melampsora abietis-canadensis Tsuga canadensis 2007-07-01 Canada, Québec MAITS008-08 EU808019 FJ666513 EU702378 FJ666498 JQ011011 JQ0111011451MEA-POG-QC Melampsora abietis-canadensis Populus grandidentata 2009-09-26 Canada, Québec QFB 25063 JN881735 JN934920 JQ011190 JN985834 JQ011010 JQ011100Melampsora aecidioides664ME-POA-BC45a Melampsora aecidioides Populus alba 2007-06-19 Canada, British Columbia MAITS005-08 EU808021 FJ666510 EU702383 FJ666495 JQ011022 JQ011112899MEI-POA-VC1a Melampsora aecidioides Populus hybrid P39 2006-11-01 Canada, British Columbia QFB 25040 MAITS016-08 EU808024 FJ666518 EU702382 FJ666503 JQ011023 JQ011113900MEI-POA-VC2a Melampsora aecidioides Populus hybrid P39 2006-11-01 Canada, British Columbia QFB 25041 MAITS017-08 EU808023 FJ666519 EU702381 FJ666504 JQ011024 JQ011111380ME-PO-BC7a Melampsora aecidioides Populus sp. 2007-10-13 Canada, British Columbia MAITS002-08 EU808041 JN934930 EU702405 JN985844 JQ011021 JQ0111141396MPO-POA-BRA Melampsora aecidioides Populus alba 1989-01-01 Brazil, Rio Grande do Sul PUR N4108 MPITS157-09 JN881748 JN934975 JQ011240 JN985889 JQ011073 JQ0111631448MPO-POA-UKc Melampsora aecidioides Populus

grandidentata � P. alba2000-09-07 United Kingdom, South

EastGxAAH00-1 JN881749 JN934976 JQ011241 JN985890 JQ011074 JQ011164

1449MPO-POA-UKc Melampsora aecidioides Populus alba 2000-09-08 United Kingdom, SouthEast

AAH00-1 JN881758 JN934977 JQ011242 JN985891 JQ011075 JQ011165

1450MPO-PAT-UK Melampsora aecidioides Populus alba � P. tremula 2008-01-01 United Kingdom,Hertfordshire

ATNWC08-1 JN881757 JN934978 JQ011243 JN985892 JQ011076 JQ011166

PFH-08-4 Melampsora aecidioides Populus sp. 2008-07-01 France, Beauvoir-sur-Mer (85)

MEFRA077-09 JN881753 JN934982 JQ011247 JN985896 JQ011080 JQ011170

PFH-04-8 Melampsora aecidioides Populus alba 2004-09-08 France, Mirabeau (84) MEFRA020-09 JN881751 JN934980 JQ011245 JN985894 JQ011078 JQ011168PFH-04-4 Melampsora aecidioides Populus alba 2004-04-01 France, Mirabeau (84) MEFRA018-09 JN881750 JN934979 JQ011244 JN985893 JQ011077 JQ011167PFH-09-10 Melampsora aecidioides Populus alba 2009-06-01 France, La Tremblade

(17)JN881754 JN934983 JQ011248 JN985897 JQ011081 JQ011171

1387MPO-POA-CHL Melampsora aecidioides Populus alba 1965-02-25 Chile PUR F17322 MPITS148-09 JN881747 JN934974 JQ011239 JN985888 JQ011072 JQ011162

Melampsora allii-populina1260MEAP-POC-HU Melampsora allii-populina Populus canadensis 1988-10-20 Hungary DAOM

216857MPITS112-08 JN881728 JN934902 JQ011172 JN985816 JQ010992 JQ011082

90C2B Melampsora allii-populina Allium cepa 1990 France, Champenoux(54)

MEFRA042-09 GQ479276 JN934906 JQ011176 JN985820 JQ010996 JQ011086

98Z3b Melampsora allii-populina Allium sp. 1998 France, Le Martinet (30) MEFRA027-09 GQ479289 JN934913 JQ011183 JN985827 JQ011003 JQ01109397M1 Melampsora allii-populina Allium sphaerocephalum 1997 France, Champenoux

(54)MEFRA036-09 GQ479282 JN934910 JQ011180 JN985824 JQ011000 JQ011090

97N1b Melampsora allii-populina Arum maculatum 1997 France, Champenoux(54)


98Z1bb Melampsora allii-populina Arum sp. 1998 France, Ramières (26) MEFRA026-09 GQ479290 JN934912 JQ011182 JN985826 JQ011002 JQ01109294IV7b Melampsora allii-populina Populus nigra 1994 France, Les Taillades (84) MEFRA039-09 GQ479278 JN934908 JQ011178 JN985822 JQ010998 JQ01108894IP1 Melampsora allii-populina Populus nigra 1994 France, Brésis (30) MEFRA038-09 GQ479287 JN934907 JQ011177 JN985821 JQ010997 JQ01108796M24.2b Melampsora allii-populina Muscari comosum 1996 France, Champenoux

(54)MEFRA034-09 GQ479285 JN934909 JQ011179 JN985823 JQ010999 JQ011089

PFH-07-6 Melampsora allii-populina Populus � euramericana 2007-10-27 Italy, Perugia JN881730 JN934915 JQ011185 JN985829 JQ011005 JQ011095PFH-04-9 Melampsora allii-populina Populus nigra 2004-10-18 Spain, Madrid MEFRA021-09 JN881729 JN934914 JQ011184 JN985828 JQ011004 JQ011094PFH-03-33 Melampsora allii-populina Populus nigra 2003-07-20 Turkey, Erzincan QFB 25065 MEFRA016-09 JN881732 JN934917 JQ011187 JN985831 JQ011007 JQ011097PFH-03-23 Melampsora allii-populina Populus nigra 2003-11-17 Bulgaria, Pazardjik QFB 25064 JN881731 JN934916 JQ011186 JN985830 JQ011006 JQ0110961349MEAP-POL-CHI Melampsora allii-populina Populus laurifolia 1986-08-31 China, Xinjiang Uygur

ZizhiquBPI 746008/HMAS52892

MPITS155-09 JN881756 JN934904 JQ011174 JN985818 JQ010994 JQ011084

1362MEAP-PON-IS Melampsora allii-populina Populus nigra 1934-12-10 Israel BPI 0020976 MPITS136-09 GQ479834 JN934905 JQ011175 JN985819 JQ010995 JQ0110851335MEAP-PON-IS Melampsora allii-populina Populus nigra 1950-12-26 Israel BPI 0020977 MPITS120-09 JN881755 JN934903 JQ011173 JN985817 JQ010993 JQ011083

630A

.Vialle

etal./M

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Melampsora larici-populina880MLP-LAD-QC Melampsora larici-populina Larix decidua 2007 Canada, Québec MPITS075-08 GQ479844 JN934946 JQ011213 JN985860 JQ011040 JQ011130837MLP-POP-VI18 Melampsora larici-populina Populus balsamifera 2007-08-28 Canada, Québec QFB 25043 MPITS067-08 GQ479841 JN934945 JQ011212 JN985859 JQ011039 JQ0111291420MLP-POH-CHI Melampsora larici-populina Populus cathayana 2000-08-19 China, Gansu TSH-R16927 MPITS181-09 GQ479835 JN934944 JQ011211 JN985858 JQ011038 JQ01112897A3b Melampsora larici-populina Populus nigra 1997 New Zealand, Wanganui MEFRA045-09 GQ479295 JN934949 JQ011216 JN985863 JQ011043 JQ01113395US1b Melampsora larici-populina Populus nigra ‘italica‘ 1995 United States,

WashingtonMEFRA053-09 GQ479300 JN934947 JQ011214 JN985861 JQ011041 JQ011131

97EA2b Melampsora larici-populina Populus sp. 1997 China MEFRA047-09 GQ479294 JN934950 JQ011217 JN985864 JQ011044 JQ01113498AE3b Melampsora larici-populina Populus sp. 1998 Finland MEFRA049-09 GQ479298 JN934952 JQ011219 JN985866 JQ011046 JQ01113699D1b Melampsora larici-populina Populus trichocarpa 1999-08-08 Iceland MEFRA050-09 GQ479299 JN934954 JQ011221 JN985868 JQ011048 JQ01113898AR11b Melampsora larici-populina Populus � interamericana 1998 Belgium, Geraardsbergen MEFRA052-09 GQ479291 JN934953 JQ011220 JN985867 JQ011047 JQ01113797A11b Melampsora larici-populina Populus � euramericana 1997 Morocco MEFRA044-09 GQ479296 JN934948 JQ011215 JN985862 JQ011042 JQ01113297J10b Melampsora larici-populina Populus � euramericana 1997 South Africa, Natal MEFRA046-09 GQ479293 JN934951 JQ011218 JN985865 JQ011045 JQ01113500A19b Melampsora larici-populina Populus � euramericana 2000-04-13 Chile, Talca MEFRA051-09 GQ479297 JN934943 JQ011210 JN985857 JQ011037 JQ011127

Melampsora larici-tremulaePFH-04-5 Melampsora larici-tremulae Populus tremula 2004-08-01 France, Champenoux

(54)MEFRA019-09 JN881744 JN934956 JQ011223 JN985870 JQ011051 JQ011141

MLT01F2 Melampsora larici-tremulae Populus tremula 2001-06-05 France, Champenoux(54)

MEFRA011-09 JN881743 JN934955 JQ011222 JN985869 JQ011050 JQ011140

PFH-99-1 Melampsora larici-tremulae Populus tremula 1999-01-01 France, La Clusaz (74) QFB 25068 JN881745 JN934957 JQ011224 JN985871 JQ011052 JQ0111421461MLT-PTA-FRd Melampsora larici-tremulae Populus tremula 1986-10-24 France, Champenoux

(54)JQ664559 JQ664561 JQ664563 JQ664565 JQ664567 JQ664569

MLT01F1b Melampsora larici-tremulae Populus tremula 2001-06-05 France, Champenoux(54)

MAITS021-08 EU808026 FJ666509 EU702384 FJ666494 JQ011049 JQ011139

Melampsora magnusiana1426MEG-CJ-DSD Melampsora magnusiana Chelidonium majus 2008-05-01 Germany, Sachsen GLM 81495 MPITS187-09 GQ479845 JN934927 JQ011196 JN985841 JQ011018 JQ0111081424MEG-CORV-

DSDMelampsora magnusiana Corydalis cava 2005-04-24 Germany, Sachsen GLM 77297 MPITS185-09 GQ479846 JN934926 JQ011195 JN985840 JQ011017 JQ011107

1328MEG-CORV-AT Melampsora magnusiana Corydalis cava 2000-04-19 Austria BPI 878595 JN881736 JN934925 GQ501094 JN985839 JQ011016 JQ0111061428MEG-CORV-DSD Melampsora magnusiana Corydalis cava 1997-06-05 Germany, Sachsen GLM 58747 MPITS189-09 GQ479847 JN934928 JQ011197 JN985842 JQ011019 JQ0111091429MEG-CORV-

DSDMelampsora magnusiana Corydalis cava 2005-04-04 Germany, Sachsen GLM 77294 MPITS190-09 GQ479849 JN934929 JQ011198 JN985843 JQ011020 JQ011110

Melampsora medusae98D10 Melampsora medusae

f.sp. deltoidaePopulus � euramericana 1998 South Africa, Natal MEFRA059-09 GQ479307 JN934962 JQ011229 JN985876 JQ011057 JQ011147

99W3 Melampsora medusaef.sp. deltoidae

Populus � euramericana 1999-09-24 France, Champenoux(54)


97CN5 Melampsora medusaef.sp. deltoidae

Populus � interamericana‘ 1997 France, Deyme (31) MEFRA057-09 GQ479302 JN934961 JQ011228 JN985875 JQ011056 JQ011146

583ME-LAL-MT5 Melampsora medusaef.sp. deltoidae

Larix laricina 2007-06-06 Canada, Québec MPITS025-08 GQ479854 JN934958 JQ011225 JN985872 JQ011053 JQ011143

761MMD-POD-BA2 Melampsora medusaef.sp. deltoidae

Populus deltoides 2007-08-02 Canada, Québec QFB 25044 MPITS053-08 GQ479857 JN934960 JQ011227 JN985874 JQ011055 JQ011145

760MMD-POD-BA1 Melampsora medusaef.sp. deltoidae

Populus deltoides 2007-08-02 Canada, Québec MPITS052-08 GQ479858 JN934959 JQ011226 JN985873 JQ011054 JQ011144

610ME-LAL-LE7 Melampsora medusaef.sp. tremuloidae

Larix laricina 2007-06-15 Canada, Québec MPITS040-08 GQ479865 JN934967 JQ011234 JN985881 JQ011062 JQ011152

1028ME-LAL-LJ Melampsora medusaef.sp. tremuloidae

Larix laricina 2008-06-29 Canada, Québec QFB 25066 MPITS095-08 GQ479883 JN934965 JQ011232 JN985879 JQ011060 JQ011150

414MMT-POT-VA11 Melampsora medusaef.sp. tremuloidae

Populus tremuloides 2005 Canada, Québec MPITS020-08 GQ479860 JN934966 JQ011233 JN985880 JQ011061 JQ011151

1017MMT-POT-NB Melampsora medusaef.sp. tremuloidae

Populus tremuloides 2008-06-19 Canada, New Brunswick MPITS090-08 GQ479882 JN934964 JQ011231 JN985878 JQ011059 JQ011149

897MMT-POT-QC12a Melampsora medusaef.sp. tremuloidae

Populus tremuloides 2005-07-30 Canada, Québec JN881746 JN934969 EU702387 JN985883 JQ011064 JQ011154

796ME-POT-LI2 Melampsora medusaef.sp. tremuloidae

Populus tremuloides 2007-08-05 Canada, Québec QFB 25067 MPITS055-08 GQ479871 JN934968 JQ011235 JN985882 JQ011063 JQ011153

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Table 1 (continued)

Sample ID Species identification Host Collectiondate

Geographicallocalization

Herbarium/collection ID

BOLD ID ITS LSU CO1 Nad6 MS277 MS208

Melampsora microspora1407MEMI-PON-IRQ Melampsora microspora Populus nigra 1950-01-10 Iraq PUR F17540 MPITS168-09 JN881737 JN934931 JQ011199 JN985845 JQ011025 JQ011115

Melampsora nujiangensis1423MEN-POY-CHI* Melampsora nujiangensis Populus yunnanensis 1998-09-29 China, Yunnan TSH-R20042 MPITS184-09 JN881739 JN934933 JQ011201 JN985847 JQ011027 JQ0111171418MEN-POY-CHI Melampsora nujiangensis Populus yunnanensis 1998-09-29 China, Yunnan TSH-R20046 MPITS179-09 JN881738 JN934932 JQ011200 JN985846 JQ011026 JQ011116

Melampsora occidentalis411MEO-PO-BC13 Melampsora occidentalis Populus trichocarpa 2006-09-01 Canada, British Columbia MPITS019-08 GQ479885 JN934937 JQ011205 JN985851 JQ011031 JQ0111211452MO-PTC-USA Melampsora occidentalis Populus trichocarpa 2002-09-01 United States, Idaho MO96H JN881740 JN934934 JQ011202 JN985848 JQ011028 JQ0111181454MO-PTC-US Melampsora occidentalis Populus trichocarpa 1996-08-20 United States,

WashingtonMOWA1 JN881741 JN934935 JQ011203 JN985849 JQ011029 JQ011119

1455MO-PTC-USA Melampsora occidentalis Populus trichocarpa 1996-08-20 United States,Washington

MOWA6 JN881742 JN934936 JQ011204 JN985850 JQ011030 JQ011120

Melampsora pinitorqua1367MPI-PNI-FI Melampsora pinitorqua Pinus sylvestris 1962-07-17 Finland BPI 0025026 MPITS141-09 GQ479897 JN934973 JQ011238 JN985887 JQ011068 JQ0111581462MPI-PTA-FRd Melampsora pinitorqua Populus tremula 1986-10-22 France, Pindères (47) JQ664560 JQ664562 JQ664564 JQ664566 JQ664568 JQ66457097MP08a Melampsora pinitorqua Pinus sylvestris 1997-06-01 France, Charmes (88) MAITS024-08 EU808034 FJ666521 EU702393 FJ666506 JQ011069 JQ01115997MP10a Melampsora pinitorqua Pinus sylvestris 1997-06-01 France, Charmes (88) MAITS023-08 EU808035 FJ666523 EU702394 FJ666508 JQ011071 JQ01116197MP09a Melampsora pinitorqua Pinus sylvestris 1997-06-01 France, Charmes (88) MAITS025-08 EU808033 FJ666522 EU702392 FJ666507 JQ011070 JQ01116000S1b Melampsora pinitorqua Populus tremula 2000 France, Cestas (33) MAITS026-08 EU808032 JN934970 EU702391 JN985884 JQ011065 JQ01115500S2b Melampsora pinitorqua Populus tremula 2000 France, Vabres (15) MEFRA072-09 GQ479318 JN934971 JQ011236 JN985885 JQ011066 JQ01115600S3b Melampsora pinitorqua Populus tremula 2000 France, Brinon-sur-

Sauldre (18)MEFRA073-09 GQ479319 JN934972 JQ011237 JN985886 JQ011067 JQ011157

Melampsora pruinosae1343MEPR-POR-CHI Melampsora pruinosae Populus diversifolia 1959-06-24 China, Xinjiang Uygur

ZizhiquBPI 1109446 MPITS128-09 GQ479899 JN934938 JQ011206 JN985852 JQ011032 JQ011122

1366MEPR-POPR-UR*

Melampsora pruinosae Populus pruinosae 1910-08-12 Uzbekistan, Boukhara BPI 0031207 MPITS140-09 GQ479898 JN934939 JQ011207 JN985853 JQ011033 JQ011123

Melampsora pulcherrimaO8ZK4 Melampsora pulcherrima Mercurialis annua 2008-04-01 Italy, Monticiano MEFRA015-09 GQ479320 JN934941 JQ011209 JN985855 JQ011035 JQ011125O8ZK2 Melampsora pulcherrima Mercurialis annua 2008-04-01 Italy, Monticiano MEFRA013-09 GQ479321 JN934940 JQ011208 JN985854 JQ011034 JQ011124

Melampsora rostrupii01G1b Melampsora rostrupii Mercurialis perennis 2001-05-12 France, Gorze (57) MAITS022-08 EU808038 JN934942 EU702397 JN985856 JQ011036 JQ011126PFH-08-3 Melampsora rostrupii Populus alba 2008-10-23 France, Châteauroux-les-

Alpes (05)MEFRA025-09 JN881752 JN934981 JQ011246 JN985895 JQ011079 JQ011169

Bold specimens ID correspond to specimens included in the BCA reduced dataset.* Type or part of the holotype.

a Species identification previously published in Feau et al. (2009).b Species identification previously published in Barrès et al. (2006).c Species identification previously published in Pei et al. (2005).d Species identification previously published in Desprez-Loustau (1986).

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et al., 2009). Our final molecular dataset comprised 14 Melampsoraspecies including five macrocyclic species (M. abietis-canadensis, M.allii-populina, M. larici-populina, M. medusae [including the two for-mae speciales M. medusae f. sp. deltoidae and M. medusae f. sp. trem-uloidae], and M. occidentalis), six species considered as belonging tothe M. populnea species complex (M. aecidioides, M. magnusiana, M.larici-tremulae, M. rostrupii, M. pinitorqua, and M. pulcherrima), andthree hemicyclic rare species (M. microspora, M. nujiangensis, andM. pruinosae). Despite numerous attempts, we were unable to am-plify DNA from the herbarium specimens identified as M. castell-ana, M. ciliata, and M. abietis-populi. Finally, three rare species, M.cumminsii, M. multa and M. osmaniensis, were excluded since theywere considered to be nomina dubia, in the zoological code sense,because type material could not be found and no other collectionshave been reported (Vialle et al., 2011).

2.2. Selection of loci

Six loci were amplified and sequenced. The first two belong to thenuclear ribosomal RNA gene region, i.e., the internal transcribedspacer regions 1 and 2 with the 5.8S ribosomal subunit (ITS) andthe large ribosomal subunit (28S). We also retrieved two mitochon-drial genes, the cytochrome oxidase subunit 1 and the dehydroge-nase subunit 6 (CO1 and Nad6), to infer the specific mitochondrialDNA evolutionary history among Melampsora species. The tworemaining loci correspond to single-copy protein coding genesMS277 and MS208 (Aguileta et al., 2008). Within the M. larici-popu-lina genome (Duplessis et al., 2011), MS277 is homologous to theTsr1 gene, one of the best performing genes for phylogenetic studiesof fungi retrieved by Aguileta et al. (2008). This gene is required forrRNA accumulation during biogenesis of the ribosome and localizedon scaffold 7 in the M. larici-populina genome V1.0 (Protein ID711889 in the Filtered Gene Models Database). The MS208 sequenceis homologous to the Mcm5 gene, a DNA replication-licensing factorrequired for DNA replication initiation and cell proliferation local-ized on scaffold 3 in the M. larici-populina genome V1.0 (Protein ID74054 in the Filtered Gene Models Database).

2.3. Molecular data generation

Total genomic DNA was extracted using a modified protocol ofthe DNeasy Plant Mini Kit from QIAGEN (Qiagen, Mississauga, ON).For rust samples collected on Pinaceae, three infected needles were

Table 2Primers sequences and PCR conditions used.

Loci Amplicon name andlength

Primer (sequences or references) PCR therconditio

ITS ITS1-5.8s-ITS2800 bp

FUN18SF (Pitkäranta et al., 2008)ITS4BR (Vialle et al., 2009)

40 cycleAnnealin

28S 28S740 bp

ITS4-BRf (Vialle et al., 2009)LR5 primers (Vigalys and Hester, 1990)

40 cycleAnnealin

CO1 CO1 Exon1260 bp

Cox1MlpAF (Vialle et al., 2009)Cox1MlpAR (Vialle et al., 2009)

40 cycleAnnealin

CO1 CO1 Exon2560 bp

Cox1MlpB2F (Vialle et al., 2009)Cox1MlpCR (Vialle et al., 2009)

40 cycleAnnealin

Nad6 Nad6530 bp

Nad6PucciF1: 50-TTCGATAATAAGTAGCCTAATAGTG-30

Nad6PucciR1: 50-AAATACAATAGGGCCAATCAT-30

40 cycleAnnealin

MS277 MS277530 bp

MS277_EX1F2: 50-GCAGATGATCTGGTCTCCGAGAA-30

MS277_DR: 50-TTCCCATACTCCGCAGGTAG-30

50 cycleAnnealin

MS208 MS208 Exon1700 bp

MS208_EX1F: 50-ATGCCTGGAATCGTCATCTC-30

MS208_EX1R: 50-TTTAGCCGTTCCTGGATCAC-30

50 cycleAnnealin

used for DNA extraction. For all other samples collected on angio-sperm hosts, aecia or uredinia were excised from infected leaf tis-sues. Infected tissues were placed into 2.0 mL tubes with liquidnitrogen and ground with pestles. Then, 10 lL of Proteinase K(10 mg/mL) and 400 lL of extraction buffer (AP1) were added toeach sample. The homogenized mixture was incubated for120 min at 60 �C and mixed by inversion two or three times duringincubation. The remaining protocol followed the manufacturer’sinstructions (Mini protocol) from step 9 onward. DNA extractswere resuspended in 30 lL of elution buffer and stored at �20 �C.

The six loci were amplified through seven PCR reactions persample (the two CO1 exons were amplified separately). Each locuswas amplified with thermocycling conditions as follows: denatur-ation for 3 min at 95 �C, a locus-specific number of cycles at 95 �Cfor 45 s, 30 s at annealing locus-specific temperature, and 1 min30 s at 72 �C with a final extension of 10 min at 72 �C. Primer se-quences for the ribosomal, mitochondrial and nuclear loci, as wellas PCR locus-specific conditions (number of cycles and annealingtemperature), are provided in Table 2. Both strands of the purifiedPCR products were sequenced with the appropriate amplificationprimers using an ABI 377 automated DNA sequencer at the CHULResearch Centre Sequencing and Genotyping Facility (Québec, QC,Canada). Forward and reverse sequences were assembled andtrimmed using SeqMan from the DNASTAR Lasergene 7.1 softwarepackage (DNASTAR, Inc., Madison, WI). For the ribosomal and nu-clear genes, heterozygous sites were coded using ambiguity codes.All sequences associated with specimen information were depos-ited in GenBank and Barcode of Life Database (BOLD) (Table 1).Multiple sequence alignments were generated using MUSCLEv3.6 with default parameters (Edgar, 2004) and then visually ad-justed using BioEdit v.7.0.9 (Hall, 1999).

2.4. Phylogenetic analysis for recovering single gene region trees andidentifying phylogenetic species

To identify evolutionary independent lineages, concordancewas evaluated between unlinked gene genealogies (Taylor et al.,2000; Le Gac et al., 2007). However, ribosomal loci (ITS and 28S)cannot be considered as unlinked given that they are contiguous,implying that both loci have likely evolved according to the similarunderlying history. We also considered both mtDNA loci (CO1 andNad6) as a single linkage group given the uniparental inheritanceand haploidy of mtDNA (Baum, 2007). The two remaining loci

mocycling locus-specificns

PCR reaction mix 20 lL reaction volume

sg temperature 50 �C

3 lL of undiluted DNA template0.2 lm of each primer0.15 m m of each dNTPs (GE Healthcare)1.5 mm MgCl22 U of Platinum Taq DNA polymerase (Invitrogen)1� Taq DNA polymerase buffer (20 mm Tris–HCl, pH8, 50 mm KCl)







634 A. Vialle et al. / Molecular Phylogenetics and Evolution 66 (2013) 628–644

(MS277 and MS208) were considered as unlinked genes since theyoccur on two different scaffolds in the M. larici-populina genomeV1.0 (Duplessis et al., 2011). We thus decided to reconstruct andcompare four genealogies to evaluate their concordance: (1) con-catenated ribosomal regions ITS and 28S; (2) concatenated mtDNACO1 and Nad6; (3) MS277; and (4) MS208 (Supplementary Data 2).Nucleotide substitution models were selected for each data parti-tion using the Akaike information criterion (AIC criterion) inMrModeltest v2.2 (Nylander, 2004) (Supplementary Data 2). Phy-logenetic relationships were inferred using maximum parsimony,maximum likelihood and Bayesian inference. Partitioned Bayesianphylogenetic analyses were conducted using MrBayes v3.1.2(Ronquist and Huelsenbeck, 2003). Priors were constrainedaccording to the MrModelTest results on each partition considered(Table 2). Gaps and indels were coded with FastGap 1.2(Borchsenius, 2009) based on the method developed by Simmonsand Ochoterena (2000). Each analysis used four heated Markovchains (using default heating values) that were run for 20 milliongenerations, and trees were sampled every 1000 generation,resulting in an overall sampling of 20,000 trees. Bayesian posteriorprobability values (PP) were obtained after the process hadreached stationarity, by summarizing the posterior distributionof trees (25% post-burn-in trees), with a 50% majority rule consen-sus tree. Maximum likelihood (ML) analyses were performed usingMEGA 5 (Tamura et al., 2011), with the initial NJ tree generatedautomatically. Evolutionary models were applied for each gene re-gion with no partition according to the MrModeltest results usingthe AIC criterion (Supplementary Data 2). Tree topologies wereestimated with the nearest-neighbor interchanging (NNI) ap-proach. Maximum parsimony (MP) trees were generated throughsectorial searches with tree fusing and driven search options usingNew Technology search algorithms implemented in TNT version1.1 (Giribet, 2005; Goloboff et al., 2008).

In both MP and ML analyses, gaps were treated as missing dataand 50% majority rule consensus tree statistical supports wereevaluated with 1000 non-parametric bootstrap replicates (BS)(Felsenstein, 1985). For all analyses, trees were rooted with homol-ogous sequences retrieved from the Puccinia graminis genome se-quence (Duplessis et al., 2011). Monophylies supported byBS P 70% and PP P 90% were considered significant (Dettmanet al., 2003; Le Gac et al., 2007). A node was considered stronglysupported when it was significant using two of the three recon-struction methods.

To detect independent evolutionary units within Melampsoraspecies retrieved on poplar, we used phylogenetic species recogni-tion adapted from Dettman et al. (2003). A clade was recognized asan independent evolutionary lineage if it satisfied either one of twocriteria: (i) genealogical concordance or (ii) genealogical non-dis-cordance (Dettman et al., 2003; Le Gac et al., 2007).

2.5. Detection of incongruence between gene region phylogenies

Incongruence between gene region phylogenies was first de-tected by visual inspection of topologies. During visual inspection,we concluded there was incongruence between gene region phy-logenies when conflicting nodes were retrieved and supported bysignificant statistical values using two of the three reconstructionmethods (BS P 70% and PP P 90%). An approximately unbiased(AU) test, as implemented in CONSEL (Shimodaira and Hasegawa,2001), was conducted for each incongruence detected by compar-ing, for each gene region, the likelihood of the ML topology ob-tained for the focal gene with the likelihood of the conflictingtopology obtained for another gene region. Likelihoods were ob-tained in PAUP� version 4 (Swofford, 2003) using the sequence evo-lution model selected according to the MrModelTest results. The

null hypothesis of congruence was rejected when the P-value ob-tained for the AU test was 60.05.

2.6. Estimation of the species tree

Bayesian posterior distributions over the four gene regiontopologies were estimated with MrBayes v3.1.2 as described insection 2.4. Then, concordance factor (CF) calculations and con-struction of the primary concordance tree were performed withBUCKy v1.4.0 (Ané et al., 2007), with 10 million generations (4 runsand 4 chains) and an a priori level of discordance among gene re-gions of a = 3. Since we included gene regions with different inher-itance patterns (mitochondrial and nuclear regions), CF of a cladewas inferred as the proportion of genes that have the clade (Baum,2007). To avoid computer memory saturation, the second step ofthe BCA was performed on a reduced dataset of 45 taxa (Table 1;sample ID in bold). Whenever possible, this reduced dataset in-cluded two specimens per described species or f. sp., one retrievedon the telial host (Populus spp.) and one on the aecial host, and ex-cluded specimens with strict identical sequences among the sixloci within the same species or f. sp. As the CF threshold to definespecies boundaries is still under debate (Baum, 2007; Allman et al.,2011), a group of taxa retrieved from the primary concordance treeby the BCA was recognized as an independent lineage if it satisfiedeither one of two criteria: (i) this group of taxa corresponded to anindependent evolutionary lineage as previously identified by theGCPSR; or (ii) this group of taxa clustered with CF > 50%, i.e., thisclade was truly present in more than half of the single gene regiontrees (Baum, 2007). BUCKy provided a fully resolved primary con-cordance tree that included low-probability clades (CF < 50%),which could result in an incorrect estimate of species relationships,especially when the CF of the clade was less than 33% (Allmanet al., 2011). Thus, among the phylogenetic species identified, wedecided to consider as truly resolved only the clades retrieved withCF > 33% (i.e., clades present in at least one third of the single generegion trees) with no other low-probability conflicting clade. Alow-probability conflicting clade is one that is not included inthe primary concordance tree with lower CF but that presents anoverlapping CF credibility interval (CI) with that of the clade repre-sented in the primary concordance tree.

3. Results

3.1. Phylogenies

Information on datasets, partitions and MP trees is shown inSupplementary Data 2. Except for the mitochondrial trees, Bayes-ian, ML and MP consensus trees revealed the same relationshipsbetween the significantly supported clades. Therefore, only Bayes-ian consensus trees of the ribosomal loci and the MS277 and MS208gene regions are shown in Figs. 1, 3 and 4, respectively. As ex-pected, MP and ML consensus trees had lower statistical supportthan Bayesian consensus trees.

Visual inspection of the nodes and statistical supports showedsome phylogenetic relationships to be congruent among generegions. All phylogenies retrieved one deep clade called A(Figs. 1–4), which was strongly supported in the ribosomal andmitochondrial phylogenies. Two other deep clades, called B andC, were retrieved in three other phylogenies (Figs. 1, 3 and 4) butnot in the mitochondrial phylogeny. A fourth clade, D, clusteredthe two M. nujiangensis specimens (1423MEN-POY-CHI and1418MEN-POY-CHI) and was also strongly supported in the ribo-somal, mitochondrial and MS277 phylogenies. These four deepclades A, B, C and D identified in single gene region phylogenieswere both retrieved and clearly distinct in the primary concordanttree (Fig. 5).

Fig. 1. Bayesian 50% majority-rule consensus tree based on the ribosomal (ITS + 28S) gene region (mean LogLikelihood = �3394.68). Nodes with PP 6 70% and BS 6 50% arerepresented as unresolved. Branch annotations indicate Bayesian posterior probabilities/maximum likelihood bootstraps/maximum parsimony bootstrap values. Onlysupports higher than 85/65/65 are indicated. Arrow indicates conflicting node between the different gene region phylogenies. Dotted lines indicate independent evolutionarylineages identified and braces indicate deep clades identified. Species names in brackets represent one unique evolutionary lineage. Orange and green colors indicate theaecial (non-poplar) host specificity of the taxa and their related clades (green: angiosperms host [pale green = monocotyledonous and dark green = dicotyledonous]; orange:coniferous host; black: unknown). Species abbreviations: [M. medusae trem.] M. medusae f. sp. tremuloidae; [M. medusae delt.] M. medusae f. sp. deltoidae; [M. abietis-can.] M.abietis-canadensis; [M. larici-trem.] M. larici-tremulae; [M. larici-pop.] M. larici-populina; [M. allii-pop.] M. allii-populina. (For interpretation of the references to color in thisfigure legend, the reader is referred to the web version of this article.)


Incongruence was visually detected between the ribosomal andMS208 phylogenies. Ribosomal phylogeny supported the mono-

phyly between clade D and clade A (Fig. 1), whereas MS208 sup-ported the monophyly between clades B, C and D (Fig. 4). AU

Fig. 2. Bayesian 50% majority-rule consensus tree based on the mitochondrial (CO1 + Nad6) gene regions (mean LogLikelihood = �2371.67). Nodes with PP 6 70% andBS 6 50% are represented as unresolved. Branch annotations indicate Bayesian posterior probabilities/maximum likelihood bootstraps/maximum parsimony bootstrapvalues. Only statistical supports higher than 85/65/65 are indicated. Dotted lines indicate independent evolutionary lineages identified and braces indicate deep cladesidentified. Orange and green colors indicate the aecial (non-poplar) host specificity of the taxa and their related clades (green: angiosperms host [pale green = monocot-yledonous and dark green = dicotyledonous]; orange: coniferous host; black: unknown). MP topology box represents alternative topology obtained with maximumparsimony analysis with bootstraps support and number of steps in brackets. Species abbreviations: [M. medusae trem.] M. medusae f. sp. tremuloidae; [M. medusae delt.] M.medusae f. sp. deltoidae; [M. abietis-can.] M. abietis-canadensis; [M. larici-trem.] M. larici-tremulae; [M. larici-pop.] M. larici-populina; [M. allii-pop.] M. allii-populina. (Forinterpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


tests were significant for this incongruence when enforcing the po-sition of clade D in the ribosomal phylogeny as in the MS208 phy-logeny (AU test: P = 0.009), and in the MS208 phylogeny as in theribosomal phylogeny (AU test: P = 0.001).

The phylogenetic relationships of M. nujiangensis (clade D) andM. larici-populina (clade C) could not be unambiguously inferred

with BCA analysis. In the primary concordance tree, clades C andD were retrieved as sister groups to clade B (CF = 0.33, CI [0.25–0.50]; data not shown). However, two other low-probability cladesconflicted with this result: in the first one, clades C and D were athird deep clade, independent from clades A and B (CF = 0.21, CI[0–0.50]), whereas the second one suggested close relationships

Fig. 3. Bayesian 50% majority-rule consensus tree based on the MS277 nuclear region (mean LogLikelihood = �1457.03). Nodes with PP 6 70% and BS 6 50% are representedas unresolved. Branch annotations indicate Bayesian posterior probabilities/maximum likelihood bootstraps/maximum parsimony bootstrap values. Only statistical supportshigher than 85/65/65 are indicated. Dotted lines indicate independent evolutionary lineages identified and braces indicate deep clades identified. Species names in bracketsrepresent one unique evolutionary lineage. Orange and green colors indicate the aecial (non-poplar) host specificity of the taxa and their related clades (green: angiospermshost [pale green = monocotyledonous and dark green = dicotyledonous]; orange: coniferous host; black: unknown). Species abbreviations: [M. medusae trem.] M. medusae f.sp. tremuloidae; [M. medusae delt.] M. medusae f. sp. deltoidae; [M. abietis-can.] M. abietis-canadensis; [M. larici-trem.] M. larici-tremulae; [M. larici-pop.] M. larici-populina; [M.allii-pop.] M. allii-populina. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


between clades A, C and D (CF = 0.19, CI [0–0.50]). Species treereconstruction methods are still under debate, and their accuracy

in retrieving the correct species trees has not been completelyevaluated (Allman et al., 2011; Blair and Murphy, 2011; Chung

Fig. 4. Bayesian 50% majority-rule consensus tree based on the MS208 nuclear region (mean LogLikelihood = �1124.35). Nodes with PP 6 70% and BS 6 50% are representedas unresolved. Statistical supports indicate Bayesian posterior probabilities/maximum likelihood bootstraps/maximum parsimony bootstrap values. Only statistical supportshigher than 85/65/65 are indicated. Arrow indicates conflicting node between the different gene region phylogenies. Dotted lines indicate independent evolutionary lineagesidentified and braces indicate deep clades identified. Species names in bracket represent one unique evolutionary lineage. Orange and green colors indicate the aecial (non-poplar) host specificity of the taxa and their related clades (green – angiosperms host [pale green = monocotyledonous and dark green = dicotyledonous]; orange – coniferoushost; black – unknown). Species abbreviations: [M. medusae trem] M. medusae f. sp. tremuloidae; [M. medusae delt.] M. medusae f. sp. tremuloidae; [M. abietis-can.] M. abietis-canadensis; [M. larici-trem.] M. larici-tremulae; [M. larici-pop.] M. larici-populina; [M. allii-pop.] M. allii-populina. (For interpretation of the references to color in this figurelegend, the reader is referred to the web version of this article.)


and Ané, 2011; Leaché and Rannala, 2011). Doubts especially per-sist concerning the veracity of clades retrieved with CFs smaller

than 33% (Allman et al., 2011). Due to the suspicion of misleadingbranching patterns (CF < 33%), the phylogenetic relationships of M.


larici-populina clade C (aecial host: Larix spp.) and M. nujiangensisclade D (aecial host unknown) with the other poplar rust speciesremain indeterminate (Fig. 5).

The rate of polymorphism observed between sequences withinmitochondrial loci was very low. Among the 1110 sites of the finalmitochondrial alignment, only 12.5% were variable and 1.3% wasparsimony informative (Supplementary Data 2). The low numberof parsimony informative sites resulted in poorly resolved consen-sus trees with short internal nodes and low statistical support.Bayesian PP appears to be more sensitive to the phylogenetic signalwhen fewer characters are involved to retrieve internal nodes.Bayesian and ML consensus trees shared the same topology, andall nodes retrieved from the ML consensus tree were poorlysupported (BS < 70%) (Fig. 2). Globally, the MP consensus treenodes were also poorly supported (BS < 70%), except for threenodes. Those three well-supported nodes (BS P 75%) revealed adifferent topology concerning the position of the clade composedby M. nujiangensis specimens (clade D). Bayesian and ML mito-chondrial analyses differentiated this clade independently fromthe others (PP = 100%, BS = 66%), whereas MP analysis retrievedthe M. nujiangensis clade within the M. allii-populina clade (speci-mens 1335MEAP-PON-IS, 1349MEAP-POL-CHI, 1362MEAP-PON-IS, 94IV7 and PFH3–33; BS = 97%), and related to M. microspora(1407MEMI-PON-IRQ; BS = 96) (Fig. 2, MP topology box). We thusdecided to run a MP analysis on two reduced datasets: the firstwithout M. nujiangensis specimens (1423MEN-POY-CHI and1418MEN-POY-CHI), the second without M. allii-populina and M.microspora specimens (1335MEAP-PON-IS, 1349MEAP-POL-CHI,1362MEAP-PON-IS, 94IV7, PFH3-33 and 1407MEMI-PON-IRQ).With these two reduced datasets, we obtained a topology concor-dant with ML and Bayesian consensus trees (data not shown). Such

Fig. 5. Primary concordance tree retrieved by Bayesian concordance analysis. Node annotunresolved. Species abbreviations: [M. medusae trem.] M. medusae f. sp. tremuloidae; [M.larici-trem.] M. larici-tremulae; [M. larici-pop.] M. larici-populina; [M. allii-pop.] M. allii-popuon leaves on the tree (� indicates a phylogenetic lineage not retrieved with GCPSR, ? indicadefined deep aecial host-specific clades. Annotations on edges indicate the aecial host p

methodological discordance suggests a long branch attraction phe-nomenon (Huelsenbeck, 1997; Schwarz et al., 2004; Bergsten,2005). Visual examination of the mitochondrial alignments re-vealed that all these MP-clustered specimens possessed a repeti-tion of a six-nucleotide insert (TCTAAA) within the Nad6sequence (position 941–959 in the mitochondrial gene region finalalignment). Melampsora nujiangensis (1423MEN-POY-CHI and1418MEN-POY-CHI), M. microspora (1407MEMI-PON-IRQ), and M.allii-populina (1335MEAP-PON-IS, 1349MEAP-POL-CHI, 1362MEAP-PON-IS, 94IV7, PFH3-33) respectively had three, two and onerepeats of this insert within their Nad6 sequences, whereas allother sequences included in our dataset lacked an insert at that po-sition. By removing this region, the MP analysis retrieved exactlythe same topology as the ML and Bayesian consensus trees. Asfor the ML consensus tree, MP statistical supports were very low(BS < 70%).

Due to the lack of resolution in mitochondrial phylogenies, wedecided to consider only clades with higher PP (P95%) as stronglysupported in the mitochondrial topology for the non-discordancecriteria.

3.2. Independent evolutionary lineages among poplar rust fungi

Using the criterion of congruence between individual gene re-gion phylogenies, 12 independent evolutionarily lineages wereidentified among the 15 taxa (species and f. sp.) included in thisstudy (Figs. 1–4). Three of the independent lineages retrieved werein accordance with three morphologically described species (M.larici-populina [M. larici-pop.], M. nujiangensis and M. occidentalis).Melampsora abietis-canadensis [M. abietis-can.] was also retrievedas an independent lineage apart from all M. medusae specimens.

ations indicate sample-wide CF. Nodes with sample-wide CF < 0.33 are presented asmedusae delt.] M. medusae f. sp. deltoidae; [M. abietis-can.] M. abietis-canadensis; [M.lina. Sample-wide CFs obtained for the independent lineages identified are indicatedtes an uncertainty in clade branching). Orange and green circles correspond to well-lant group corresponding to the following clade.


Melampsora medusae f. sp. deltoidae [M. medusae delt.] and M.medusae f. sp. tremuloidae [M. medusae trem.] specimens were in-cluded in two distinct lineages. Four species of the M. populnea spe-cies complex (M. magnusiana, M. pulcherrima, M. rostrupii and M.aecidioides) were split into four independent lineages. Finally,two evolutionarily independent lineages grouped more than onespecies. The first one included M. allii-populina [M. allii-pop.] andtwo hemicyclic species, M. pruinosae and M. microspora, and theother one grouped the two species of the M. populnea species com-plex that have their aecial stage on conifers, M. larici-tremulae [M.larici-trem.] and M. pinitorqua.

The primary concordance tree obtained with the BCA analysisgrouped our taxa into 13 distinct groups; 12 of them correspondedto independent phylogenetic lineages identified by GCPSR. The BCAprimary concordance tree clustered the two M. pruinosae speci-mens independently from the M. allii-populina [M. allii-pop.] /M.microspora specimens with a sample-wide CF = 0.52 (Fig. 5),whereas the GCPSR grouped M. allii-populina, M. microspora andM. pruinosae in a unique lineage. The separation inferred by BCAsuggested an evolutionary distinction of M. pruinosae from M. al-lii-populina/M. microspora, but both lineages remain closely relatedsince they appeared as sister groups in the primary concordancetree (Fig. 5).

4. Discussion

4.1. Identification of host-specific lineages

Four deep clades (A–D) were identified in single gene regionphylogenies and were both retrieved and clearly distinct in the pri-mary concordant tree (Figs. 1–5). However, even if they are glob-ally concordant, the four gene region phylogenies presented inthis study do not support exactly the same relationships amongthe phylogenetic lineages identified. A specific incongruence be-tween the ribosomal and MS208 phylogenies for the relative posi-tion of M. nujiangensis (clade D) was confirmed by topological tests.Numerous processes (e.g., saturation of the phylogenetic signal,horizontal transfer, incomplete lineage sorting) can lead to discor-dance between gene trees. In such cases, combined data ap-proaches (concatenation of the loci, consensus or supertree), ifused to estimate the species tree and to infer relationships be-tween the phylogenetic species identified, could result in strongsupport for an incorrect tree (Kolaczkowski and Thornton, 2004;Mossel and Vigoda, 2005; Kubatko and Degnan, 2007; Knowles,2009).

The BCA offers an alternative approach by integrating uncer-tainty among single gene region phylogenies (Ané et al., 2007;Baum, 2007). Primary concordance and population trees retrievedwith BCA analyses shared the same topology, indicating that ouranalysis fell outside of the ‘too greedy zone’ defined by Degnanand Rosenberg (2009). This zone corresponds to the failure of themethod to return the true topology of the species tree when thecoalescent process causes incongruence between gene trees only.In such cases, the estimation of the population tree implementedin BUCKy provides a consistent alternative (Ané et al., 2010). Inour analysis, since the population tree obtained was concordantwith the primary concordance tree, the latter provided a consistentestimate of species trees (Chung and Ané, 2011) (Fig. 5). The BCAprimary concordance tree revealed a clear separation between allevolutionary independent lineages with alternation on floweringplants (Angiosperms, clade B) and the majority of evolutionaryindependent lineages with alternation on conifers (clade A). Thistopology confirmed the phylogenetic relationships already re-ported and supported by individual gene region phylogenies (deepclades A and B). Clade A included the majority of phylogenetic lin-

eages corresponding to species that alternate exclusively on coni-fers (M. occidentalis, M. medusae f. sp. deltoidae, M. medusae f. sp.tremuloidae, M. abietis-canadensis and M. larici-tremulae/M. pinitor-qua), whereas clade B included all phylogenetic lineages corre-sponding to species that alternate on flowering plants (M.pulcherrima, M. magnusiana, M. rostrupii, M. allii-populina) togetherwith phylogenetic lineages including species with unknown aecialhosts (M. aecidioides, M. pruinosae and M. microspora). Such evolu-tionary separation among these species suggests that the aecialhost specialization may act in ancestral species divergence duringspeciation events. Aecial host specificity seems to be responsiblefor deeper phylogenetic lineages within the Melampsora genus.Nevertheless, the separation of M. medusae f. sp. deltoidae and M.medusae f. sp. tremuloidae into two distinct sister groups (Figs. 1and 5) indicates that telial host specificity can also be an importantmechanism in the speciation process. In this case, telial host spec-ificity is likely to have evolved relatively recently from an aecialhost-specific ancestor occurring on Larix spp. Such cryptic diversityassociated with telial host specificity has already been revealed bymolecular studies within Melampsora species retrieved in willowtrees (Bennett et al., 2011; Milne et al., 2012). Moreover, molecularstudies based solely on ribosomal DNA revealed cryptic geneticstructures in rust fungi at the species level correlated with life cy-cle and host specificity (Pei et al., 2005; Szabo, 2006; Chatasiri andOno, 2008; Alaei et al., 2009). Finally, at higher taxonomic levels(class, order and genus), other molecular studies have alreadynoted a strong correlation between rust clusters and host specific-ities (Maier et al., 2003; Wingfield et al., 2004; Aime, 2006; Aimeet al., 2006).

Host specialization as the only barrier to gene flow resulting indistinct species has already been suggested in closely related fun-gal pathogen species (Guérin and Le Cam, 2004; Peever et al., 2007;Giraud et al., 2010). However, in our study, more than one distinctspecies infect the same host in the same geographical area. Forexample, in western North America, M. medusae f. sp deltoidaeand M. occidentalis both occur on Larix spp., and M. larici-tremulaeand M. larici-populina also occur on Larix spp. in Europe. Moreover,despite differences in their telial host range, M. medusae f. sp. del-toidae and M. occidentalis are able to hybridize, giving rise toM. � columbiana, which is capable of infecting both parents’ telialhost range (Newcombe et al., 2001). Finally, M. allii-populina occurson more than one distinct aecial plant host species (Muscari spp.,Allium spp., and Arum spp.) without gene flow barrier. Thus, thespeciation process within the genus Melampsora may have beendriven by host specialization coupled with additional evolutionaryforces that have led to species splitting.

4.2. Phylogenetic species recognition concordant with ecologicalspecies recognition

The GCPSR approach applied to poplar rust species providedevidence of the occurrence of 12 independent evolutionary lin-eages evolving without gene flow during a sufficient time periodto allow fixation of ancestral polymorphism in descendant popula-tions. Several groups were strongly supported as monophyletic bythe four independent gene phylogenies (Figs. 1–4). The primaryconcordance tree inferred by BCA confirmed these 12 phylogeneticspecies (Fig. 5) and suggested a 13th independent lineage byretrieving M. pruinosae independently from M. allii-populina/M.microspora, although these three taxa are considered as single phy-logenetic species by GCPSR. In general, we observed a concordancebetween ecological species recognition (i.e., morphological speciesdelineation complemented with host specificity) and phylogeneticspecies recognition. Seven of the phylogenetic species corre-sponded to species previously described based on their hostspecificity (aecial or telial host) and sometimes considered to be


formae speciales based on their morphology (M. abietis-canadensis,M. aecidioides, M. magnusiana, M. medusae f. sp. deltoidae, M. medu-sae f. sp. tremuloidae, M. pulcherrima, and M. rostrupii), while onlythree phylogenetic species corresponded to species recognizedstrictly based on their morphology (M. larici-populina, M. nujiang-ensis, and M. occidentalis).

4.2.1. M. abietis-canadensis recognized as a phylogenetically distinctspecies

Melampsora larici-populina, M. occidentalis, M. nujiangensis andM. abietis-canadensis were unequivocally identified as five inde-pendent lineages and thus recognized as phylogenetic species. Syn-onymy between M. abietis-canadensis and M. medusae is oftenencountered in the literature (Vialle et al., 2011). These two speciesshare a telial host, but were originally differentiated based on theirability to infect Tsuga canadensis and Larix spp., respectively(Thümen, 1878; Ludwig, 1915). However, the similar uredinio-spore morphology between both species, and especially the abilityof M. abietis-canadensis to infect Larix spp. under laboratory condi-tions, led to synonymy between the two species (Arthur andCummins, 1962; Tian and Kakishima, 2005). Thus, for a few dec-ades, M. abietis-canadensis was reduced to synonymy with M.medusae. Our results confirmed those from a recent DNA barcodestudy that highlighted the distinction between the two speciesbased on their ITS sequences (Feau et al., 2009). Despite theconvergence in morphological traits between the two species,M. abietis-canadensis remains phylogenetically distinct from M.medusae as is reflected by their predominant aecial host specificityto Tsuga spp. versus Larix spp. (Ludwig, 1915).

4.2.2. The Melampsora medusae formae speciales recognized as twodistinct phylogenetic species

Melampsora medusae was split into two f. sp. differing in telial(poplar) host range (Shain, 1988). Melampsora medusae f. sp. deltoi-dae is pathogenic primarily to P. deltoides (section Aigeiros),whereas M. medusae f. sp. tremuloidae is pathogenic primarily toP. tremuloides (section Populus) (Shain, 1988; Newcombe, 1997).

The two formae speciales of M. medusae were identified as twoindependent evolutionary lineages based on their telial host spec-ificities, underlying the influence of telial host specificity in theevolution process within the Melampsora genus. This result sup-ports the concluding remarks of Shain (1988) who wondered ifM. medusae f. sp. deltoidae and M. medusae f. sp. tremuloidae werein fact two distinct species since their telial hosts belonged totwo distinct, non-interfertile Populus sections.

4.2.3. The Melampsora populnea species complexMelampsora populnea represents a species complex, and opinions

regarding the distinction and exact number of species found in thiscomplex vary greatly. By definition, the M. populnea species com-plex includes morphologically similar Melampsora species that de-velop uredinial-telial stages on all poplars from the section Populus(Wilson and Henderson, 1966; Boerema and Verhoeven, 1972). TheM. populnea species complex is most often found to comprise fourMelampsora species recognizable by their aecial host specificity: M.larici-tremulae alternates on Larix spp.; M. magnusiana alternateson Papaveraceae species such as Chelidonium spp. and Corydalisspp.; M. pinitorqua alternates on Pinus spp.; and M. rostrupii alter-nates strictly on Mercurialis perennis. Two other species are some-times added to this complex: M. aecidioides, which occurs onPopulus alba, and the hybrid P. canescens (P. alba � P. tremula) withno aecial host recorded; and Melampsora pulcherrima, called thewhite poplar Mediterranean rust. The latter alternates exclusivelyon Mercurialis annua, but its synonymy with M. rostrupii is still un-der debate (Moriondo et al., 1989; Bagyanarayana, 1998; Pei andShang, 2005). Thus, depending on the authors, the species included

in the M. populnea complex differ in number and in classificationand could be considered as formae speciales (Viennot-Bourgin,1956; Peace, 1962; Wilson and Henderson, 1966; Boerema andVerhoeven, 1972; Bagyanarayana, 1998) or as distinct species(Klebahn, 1899; Pinon, 1973; Tian et al., 2004; Frey et al., 2005;Pei and Shang, 2005; Tian and Kakishima, 2005; Feau et al.,2009). Recent molecular studies have endorsed the genetic distinc-tion between the species/formae speciales included in this complex(Tian et al., 2004; Tian and Kakishima, 2005; Feau et al., 2009).

Our phylogenies indicate that the M. populnea complex is not amonophyletic group and is clearly divided into two distinct deeperlineages. The first one, identified as clade B (Figs. 1 and 3–5) wasretrieved in three out of four individual gene region phylogeniesand in the primary concordance tree. This clade grouped allMelampsora taxa without known aecial hosts, i.e., M. aecidioides,M. pruinosae and M. microspora, and those alternating on floweringplants, i.e., M. allii-populina, with alternation on monocotyledonousspecies, and M. rostrupii, M. pulcherrima and M. magnusiana, withalternation on dicotyledonous plants, i.e., Mercurialis perennis, M.annua and Papaveraceae, respectively. The second deeper clade,identified as clade A, grouped the two remaining species, i.e., M.larici-tremulae with alternation on Larix spp. and M. pinitorqua withalternation Pinus spp., with other species that alternate on Pina-ceae, such as M. medusae and M. occidentalis, which both alternateon Larix spp., and M. abietis-canadensis, which alternates on Tsugaspp. This result demonstrates the inadequacy of the morphologicalboundaries initially defined for this complex of species(Viennot-Bourgin, 1956; Peace, 1962; Wilson and Henderson,1966; Boerema and Verhoeven, 1972; Bagyanarayana, 1998).

Melampsora rostrupii, M. pulcherrima, M. magnusiana and M.aecidioides were retrieved as four independent evolutionary lin-eages. Thus, these four species should be considered as distincttaxa (i.e., not as formae speciales), named as distinct species andnot related to the M. populnea species name, i.e., M. magnusianaG.H. Wagner, M. rostrupii G.H. Wagner, M. pulcherrima Maire andM. aecidioides (DC.) J. Schröt., as described in Vialle et al. (2011).However, we failed to differentiate M. larici-tremulae and M. pini-torqua as two distinct phylogenetic lineages. Those two speciescan infect either Larix spp. or Pinus spp. under laboratory condi-tions (Naldini Longo et al., 1985, 1988; Desprez-Loustau, 1986),but their distinction was suggested due to their differences in ae-cial host pathogenicity, with the epicotyl infection of Pinus sylves-tris remaining exclusive to M. pinitorqua (Desprez-Loustau, 1986).Moreover, according to telial infection studies, M. larici-tremulaecould also weakly infect Populus balsamifera whereas M. pinitorquacould not (Gäumann, 1959). To avoid misidentification, our studyincluded specimens collected on Pinus sylvestris, which can beunambiguously identified as M. pinitorqua based on their aecialhost specificity. Moreover, our dataset included two specimens,1461MLT-PTA-FR and 1462MPI-PTA-FR, categorically identifiedas M. larici-tremulae and M. pinitorqua, respectively, based on mor-phological characters and pathogenicity tests (Desprez-Loustau,1986). Only one fixed SNP difference (over the six loci) was foundbetween the Nad6 sequences from the five M. larici-tremulae spec-imens and Nad6 sequences from the eight M. pinitorqua specimens.The low molecular divergence observed in our dataset suggestseither that interspecific gene flow occurs or that M. larici-tremulaeand M. pinitorqua represent a case of recent speciation. On the onehand, morphological character and aecial host range overlaps mayindicate hybridization and introgression between the two speciesdue to weak reproductive barriers, Moreover, a taxonomic studycombining morphological criteria and infection studies has alreadysuggested a lack of discrimination between M. larici-tremulae andM. pinitorqua (Gäumann, 1959). On the other hand, the exclusivepathogenicity of M. pinitorqua on Pinus sylvestris and the allochronyobserved in teliospore maturation (Desprez-Loustau, 1986)


suggest that the lack of genetic differentiation could reflect relictu-al genes present in the gene pool before divergence. In fact, hostspecialization, leading to reproductive isolation and ecological spe-ciation, could be due to a change in a single locus or in a cluster oftightly linked loci (Giraud et al., 2010). In any case, such phyloge-netic species recognition among the M. populnea species complex,i.e., M. magnusiana, M. aecidioides, M. pulcherrima, and M. rostrupii,recognized as distinct phylogenetic species and clearly retrieveddistantly from M. pinitorqua and M. larici-tremulae, supports previ-ous molecular studies and puts emphasis on species delineationbased on species host range rather than urediniospore morphology(Tian et al., 2004; Tian and Kakishima, 2005; Feau et al., 2009).

4.2.4. Relationships between Melampsora allii-populina, M. pruinosaeand M. microspora

Surprisingly, M. allii-populina was not recognized as a distinctphylogenetic lineage by GCPSR or in the BCA primary concordancetree. This species is recognizable by a smooth spot at the apex of itsurediniospores and alternation on diverse monocotyledonousplants. However, our results suggest a close relationship betweenM. allii-populina and the two hemicyclic rusts, M. pruinosae andM. microspora.

The urediniospore morphology of M. pruinosae is close to that ofM. magnusiana, but it shows smaller echinulations and thus clearlydiffers from the urediniospore morphology of M. allii-populina.Melampsora pruinosae is described as being specific to poplars fromthe section Turanga, whereas M. allii-populina is recorded on pop-lars from sections Aigeiros and Tacamahaca (Klebahn, 1902; Arthur,1903; Pinon, 1973; Pei and Shang, 2005; Tian and Kakishima,2005). Melampsora pruinosae, which has no aecial host recorded,was recently described as a distinct taxon based on morphologicaland molecular phylogenetic analyses (ITS + 28S) (Tian et al., 2004).Only the BCA analysis confirmed the phylogenetic distinction of M.pruinosae, indicating the genetic isolation of this species (Baum,2007). The distinction of this species is supported by biologicalobservations (telial host specificity, Populus yunnanensis; and mor-phological criteria, such as urediniospore globoid, rarely ovoidwith thin walls). Nevertheless, M. pruinosae remains closely relatedto M. allii-populina and M. microspora in the primary concordanttree. Thus, the inability of GCSPR to establish a distinction amongthese lineages illustrates the conservative requirement of theGCPSR approach when ancestral polymorphism persists, especiallyin recently divergent species (Taylor et al., 2000; Nichols, 2001).

Melampsora microspora differs from the other poplar rustspecies by having small and faintly verrucose urediniospores(Tranzschel, 1939; Kuprevich and Tranzschel, 1957). This rare spe-cies, described as hemicyclic, presents a telial host range overlap-ping with that of M. allii-populina. In fact, M. microspora has onlybeen recorded a few times on Populus species from the sectionAigeiros, and its aecial host remains unknown (Tranzschel, 1939;Bagyanarayana, 1998; Pei and Shang, 2005). Urediniosporemorphology seems to have no phylogenetic value as this speciesappears to be conspecific with M. allii-populina. As M. microsporashares telial host range with M. allii-populina, we suggest that M.microspora could be a rare morphological variant of M. allii-populi-na retrieved in the former USSR, Iran, Iraq and Tajikistan(Bagyanarayana, 1998; Tian and Kakishima, 2005). However, sinceonly one specimen from Iraq was identified as M. microspora in thisstudy, further analyses including other specimens of M. microsporawould be needed to confirm this hypothesis.

4.3. Perspectives and impact on poplar rust taxonomy and speciesrecognition

The evolutionary scenarios proposed in our study suppose thatwithin the Melampsora genus, species sharing common aecial host

ranges should be closely related as they share common ancestors.This observed co-speciation could provide important clues in oursearch for rusts with unknown aecial hosts. This could have impli-cations for tree disease management. From such a perspective,M. aecidioides, which is actually suspected to overwinter in poplarbuds (Arthur and Cummins, 1962; Savile, 1973; Feau et al., 2009),M. pruinosae and M. microspora, which have no aecial host reportedso far, could have (or could have had in the past) the ability toalternate on angiosperms, as inferred from their inclusion in cladeB. We hypothesize that M. aecidioides could have a preference fordicotyledonous plants due to its close position to M. magnusiana,M. rostrupii and M. pulcherrima, which alternate on Papaveraceae,Mercurialis perennis and M. annua, respectively (Fig. 5). In the sameway, M. pruinosae and M. microspora could have a preference formonocotyledonous plants due to their close relationship with M.allii-populina, which alternates on Allium, Arum and Muscari spp.(Fig. 5).

To date, there is still no consensus on the number of Melamps-ora species that should be recognized on poplar and which criteria(i.e., morphology or ecology, such as host ranges) should be usedfor the recognition and delineation of species. The most usefulcriteria for species recognition will depend on the history of speci-ation of the targeted organisms (Cai et al., 2011). Host specializa-tion and host-pathogen co-evolution are probably some of themost important evolutionary forces of ecological divergence lead-ing to speciation (Giraud et al., 2008, 2010). This is particularly trueof fungal biotrophs, such as the rust fungi, which depend com-pletely on their host for nutrition and reproduction. Among theMelampsora species, increasing host specificity (telial and aecial)might have constituted a reproductive barrier leading to specia-tion. Therefore, this criterion is crucial to species delineation de-spite morphological similarities. According to our results, theaecial stage, and especially aecial host identity, should be consid-ered as important criteria in Melampsora taxonomy and for speciesdefinition. However, finding the aecial host for a rust species onlyknown from its uredinial and telial stages can be an arduous task(e.g., Jin et al., 2010). Also, reproducing complete Melampsora lifecycles under laboratory conditions is not an easy undertakingand is not always representative of natural host preferences. Incor-rect synonymies may arise from aecial pathogenicity inducedunder controlled conditions (Arthur and Cummins, 1962;Desprez-Loustau, 1986).

Phylogenetic relationships obtained for a representative num-ber of described poplar rusts significantly clarify the currently usedconflicting taxonomic concepts by corroborating the importance ofthe host range as central criterion in species delineation. The phy-logenetic species concept that we propose here for Melampsorarusts should be useful for taxonomic, epidemiological, and man-agement purposes.

Acknowledgments

The authors thank Nick Harby, herbarium assistant at theArthur Herbarium of Purdue University (PUR), Dr. Amy Rossmanfrom the U.S. National Fungus Collection (BPI), Pierre DesRochersfrom the René-Pomerleau Herbarium of Natural Resources Canada(QFB), Professor Makoto Kakishima from the Mycological Herbar-ium of the Institute of Agriculture and Forestry of the Universityof Tsukuba (TSH), Herbert Boyle from the Museum of Natural His-tory of Görlitz, Germany (GLM), Dr. George Newcombe from theUniversity of Idaho, Dr. Ming H. Pei from Rothamsted Research,UK, Dr. Marie-Laure Desprez-Loustau from INRA Bordeaux, France,and Pr. Salvatore Moricca from the University of Firenze, Italy, forkindly providing important specimens; Josyanne Lamarche fromNatural Resources Canada provided constructive comments onthe manuscript. This research was supported through funding to


the Canadian Barcode of Life Research Network from Genome Can-ada through the Ontario Genomics Institute, the Natural Sciencesand Engineering Research Council of Canada, and other sponsorslisted at www.BOLNET.org, and by Natural Resources Canada’sCanadian Biotechnology Regulatory Strategic fund.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.ympev.2012.10.021.

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Phylogenetic species recognition reveals host-specific lineages among poplar rust fungi