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Ghent University, Department of Biology, Research Group Mycology, K.L. Ledeganckstraat 35, 9000 Ghent, Belgium
Steven L. Miller
University of Wyoming, Botany Department, Laramie, Wyoming 82071-3165
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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A phylogenetic analysis of Lactarius sect. Deliciosi was performed based on collections of all known species. Several samples of each species were included, originating from a wide geographic range. The two DNA regions we used (ITS and a part of the gene encoding glyceraldehyde-3-phosphate dehydrogenase) showed an incongruent phylogenetic signal. Much attention was paid to carefully observed macro-and micromorphological characters to draw taxonomic conclusions. We currently accept 38 taxa (31 species and seven varieties) in Lactarius sect. Deliciosi worldwide; four species are new to science. More sampling is needed to resolve the status of the North American varieties. Our knowledge of the Asian species in this section remains fragmentary. The monophyly of the section and its position within Lactarius subgenus Piperites, as proposed in recent morphology-based classification schemes, is confirmed. The intrasectional relationships however do not coincide with the color of the latex (as previously supposed). Intercontinental conspecificity is low in general. The name L. deliciosus is wrongfully applied in North and Central America and only two species seem to occur in both Asia and Europe.
Key words: Dapetes, L. deliciosus, L. porninsis, Russulales
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Several macroscopic character states make Lactarius sect. Deliciosi an easy group to recognize but differ only slightly between species. The latex ranges from carrot orange and dingy yellow to vinaceous red, brown and indigo blue. These colors change more or less rapidly by an enzymatic activity once the latex is exuded on the context and in most cases ends up green; the time needed for this change is species specific. Most species however start with orange latex, slowly (30 min to 1 h) changing to red and finally to green. Recent research has proven that L. porninsis Rolland, a species with unchanging white latex, surprisingly falls within Lactarius sect. Deliciosi (Nuytinck 2005
). Other macroscopical features that members of the section have in common concern the general aspect of the pileus and stipe: color, zonation, size, presence of scrobicules, etc. Weather and growing conditions unfortunately have a significant influence on these characters, thus hampering identifications.
Microscopical characters of more limited use are the size and ornamentation of the spores and the size and abundance of pleuro- and cheilomacrocystidia. The spore size and ornamentation are clearly divergent in only a few species and are similar in most other species. These spore characters are useful for identification when reference specimens are available. The size and especially the abundance of macrocystidia seem quite variable, making this character less reliable.
So far 74 names have been published in Lactarius sect. Deliciosi, 41 from Europe, 20 from America and 13 from Asia. Previous molecular and morphological research has lead to the acceptance of 10 species in Lactarius sect. Deliciosi in Europe (Nuytinck 2005, Nuytinck and Verbeken 2005). The situation in North and Central America and Asia is less well studied and remains unclear. Hesler and Smith (1960, 1979) provided an important step forward in the knowledge of Lactarius sect. Deliciosi and genus Lactarius in general in North America. However several European names are encountered in their work as well as numerous varieties, indicating uncertainty on the status of several taxa and illustrating their variability. Central America and Asia remain largely under explored and mycologists often use European or North American names here because of superficial resemblance, without evaluating intercontinental conspecificity.
About 400 Lactarius species are known worldwide (Verbeken 2001). Systematic research in tropical Africa, Australia and South America revealed until now only endemic species (except some introduced species in plantations, Verbeken 2001). In contrast many European Lactarius epithets circulate in North America and North American and European names are being applied often in Asia, although few comparative studies focusing on real conspecificity have been carried out. Kytövuori (1984) reported that all American records of L. scrobiculatus (Scop. : Fr.) Fr. he examined are erroneous. Other authors report the same species from northern Europe and Greenland or Alaska after morphological comparison of the material (Gulden et al 1988, Knudsen and Borgen 1994). Molecular studies never have been used to confirm any of these observations. Lactarius deliciosus (L. : Fr.) Gray, L. salmonicolor R. Heim & Leclair and L. deterrimus Gröger are European names commonly used for American and Asian taxa with orange latex. This is the first study critically comparing these and other European Lactarius taxa with material from outside Europe both by morphological and molecular methods.
Evolutionary relationships among species in Lactarius sect. Deliciosi are unclear. Morphological hypotheses on these relationships invariably group those species with similarly colored latex. Several European authors for example have divided the section into subsections (and stirps) using the color of the latex as a main character (Basso 1999, Bon 1980, Schaefer 1970). To understand the relationships between these species and to elucidate morphological and ecological characters supporting these relationships we included all known species in the section in our phylogenetic analyses.
In this study we addressed these questions: (i) Does Lactarius sect. Deliciosi form a monophyletic clade within Lactarius subgenus Piperites (Fr. ex J. Kickx f.) Kauffman, as proposed by most current authors? (ii) Which morphologically defined species are confirmed by our molecular analyses? (iii) Are morphologically similar taxa that occur on different continents conspecific? (iv) What are the relationships among species; (v) Is the color of the latex useful for a further division of the section? We used nrDNA ITS sequences and an 800-bp fragment of the gene encoding glyceraldehyde-3-phosphate dehydrogenase (gpd) and combined the results with morphological data to address these questions.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Other ITS sequences used are L. acerrimus (AJ278139
[GenBank]
), L. blennius (AY331015
[GenBank]
), L. chrysorrheus (AF096983
[GenBank]
), L. controversus (AJ272246
[GenBank]
), L. fluens, (AY331014
[GenBank]
), L. fulvissimus (AF204679
[GenBank]
), L. hepaticus (AF096989
[GenBank]
), L. intermedius (AF140256
[GenBank]
), L. mitissimus (AF157412
[GenBank]
), L. olympianus, USA, Wyoming, JN 2003-032 (GENT) (EF685079
[GenBank]
), L. quietus (AF096982
[GenBank]
), L. repraesentaneus (AY331011
[GenBank]
), L. rufus, Norway, JN 2002-008 (GENT) (EF685089
[GenBank]
), L. scrobiculatus (AF140263
[GenBank]
), L. serifluus s.l. (AY332558
[GenBank]
), L. tabidus (AF349716
[GenBank]
), L. tesquorum (AF096986
[GenBank]
), L. trivialis (AJ534935
[GenBank]
), L. uvidus (AJ534936
[GenBank]
), L. subsericatus (AF140254
[GenBank]
), L. pterosporus (AY331013
[GenBank]
), L. fuliginosus (AY606947
[GenBank]
) and L. lignyotus (AY606949
[GenBank]
). Other gpd sequences used are L. croceus, USA, Virginia, SLM 8-17-1997 (RMS) (EF685107
[GenBank]
), L. olympianus, USA, Wyoming, JN 2003-032 (GENT) (EF685128
[GenBank]
), L. rufus (DQ890419
[GenBank]
) and L. tesquorum (DQ890418
[GenBank]
).
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, Nuytinck (2005), Nuytinck and Verbeken (2003) and purified with GENECLEAN III (Qbiogene, Carlsbad, California). Aqueous genomic preparations were frozen at –20 C until use.
PCR amplification of the ITS region was performed with tailed primer pair M13-ITS5/M13-ITS4 (White et al 1990) or primers ITS1-F and ITS4-B (Gardes and Bruns 1993). A touchdown PCR profile was used as in Nuytinck and Verbeken (2003). The gpd gene was amplified with primers CTK-107, CTK-132 and CTK-108rev and the PCR program described for the Lactarius-specific primers in Kreuzinger et al (1996). PCR products were purified with ExoSAP (USB, USA) or Wizard PCR Preps (Promega Corp., Madison, Wisconsin). DNA sequencing reactions were performed with the ABI PRISM® BigDyeTM Terminators v3.0 Cycle Sequencing Kit using the same primers on an ABI PRISM® 377 DNA Sequencer or using primers M13-Forward (–29) and M13-Reverse labeled respectively with IRD-700 and IRD-800 in preparation for simultaneous bidirectional sequencing (LI-COR Biotechnology Division, Lincoln, Nebraska). Amplified PCR products were sequenced with the Se-quiTherm EXCEL II DNA Sequencing Kit (Epicentre Technologies, Madison, Wisconsin) and analyzed on a LI-COR Gene ReadIR 4200-2 automated sequencer. Phred and Phrap software (Ewing et al 1998, Ewing and Green 1998) or Base ImagIR (v4.0, LI-COR) was used to process raw data. Sequences were deposited in GenBank (accession numbers see TABLE I
).
Phylogenetic analyses.— –
We aligned the sequences with Clustal x 1.83 (Thompson et al 1997) and manually corrected and refined the alignments. Both ITS and gpd sequences were aligned easily across all taxa studied. The alignments are available through TreeBase (S1838).
To verify nonrandom structuring of the data, a "left-skewness" (g1) test was performed (Hillis and Huelsenbeck 1992) with 10 000 randomly generated trees under the parsimony criterion. Maximum parsimony (MP) analyses were performed with PAUP* 4b10 (Swofford 2002) using 100 or 1000 heuristic searches, employing TBR branch swapping and random sequence addition with a limit of 1000 trees saved per replicate. Bootstrap supports were evaluated with 1000 bootstrap replicates with 10 heuristic searches per replicate, random sequence addition and TBR branch swapping.
Maximum likelihood (ML) analyses were performed with PAUP*. The model of sequence evolution was optimized with likelihood ratio tests as implemented in Modeltest version 3.06 (Posada and Crandall 1998). Gaps were treated as missing data and phylogenies were obtained with the heuristic search option and TBR branch swapping. One MP tree was used as a start. Bootstrap support for branches was calculated with 10 000 replicates of the fast bootstrap option in PAUP*.
MrBayes 3.0b4 (Huelsenbeck and Ronquist 2001) was used to perform Bayesian analyses. Parameters of the likelihood model were set to correspond with the results of the hierarchical likelihood ratio tests. Three independent analyses of 2 x 106 generations were run starting with a random tree and keeping one tree every 100 generations. The burn-in value was set to 20%. The remaining trees were used to calculate a 50% majority rule consensus tree and to determine the posterior probabilities for the individual branches.
Compatibility of the datasets was determined with the partition homogeneity test (Farris et al 1995); we used PAUP* to perform 1000 replicate searches.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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) and 21 were obtained from GenBank. All 78 gpd sequences, with the exception of L. deterrimus GenBank U30876
[GenBank]
, were generated (TABLE I).
Data quality and hierarchical structure.— –
The "skewness" value g1 equalled –0.62 for the ITS dataset and –0.31 for the gpd dataset. This indicates that significant (p > 0.01) nonrandom structure is present, reflecting phylogenetic signal (Hillis and Huelsenbeck 1992).
ITS phylogeny.— – The alignment of the 109 ITS sequences resulted in a 930-bp dataset, of which 397 bp were variable and 247 bp were parsimony informative. A total of 24 species not belonging to Lactarius sect. Deliciosi were included in the analyses to test the monophyly of the section and its position within Lactarius. Lactarius pterosporus Romagn., L. lignyotus Fr. and L. fuliginosus (Fr. : Fr.) Fr. (representatives of Lactarius subgenus Plinthogali [Burl.] Hesler & A.H. Sm.) were assigned to the outgroup.
The TrN+I+G model (Tamura and Nei 1993) was chosen as the best fitting with Modeltest. Variable sites were assumed to follow a gamma distribution (shape = 0.6712), nucleotide frequencies were set to A 0.2471, C 0.2463, G 0.2345 and T 0.2721 and substitution rates to 3.4613 (AG), 5.6180 (CT) and 1 for all transversions. The proportion of invariable sites was set to 0.2337. The ML phylogeny (-ln Likelihood = 7050.37) is depicted (FIG. 1). The overall topology of the ML tree corresponds with the strict consensus tree of the MP analysis and the 50% majority rule consensus tree resulting from the Bayesian analysis. The Bayesian analysis shows that Lactarius sect. Deliciosi, including L. porninsis, forms a monophyletic group (supported by a posterior probability of 100%) within Lactarius subgenus Piperites. The MP analysis (100 replicates, saving maximum 1000 trees per replicate) produced 57 000 shortest trees divided over 57 islands with a length of 1008 steps (CI = 0.5188, RC = 0.3904, RI = 0.7524).
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) Bataille with L. sanguifluus (Paulet) Fr. (clade B) and L. miniatosporus Montoya & Band.-Muñoz with L. paradoxus Beardslee & Burl. (clade F); all clades received high support. The Bayesian inference tree also shows strong support for the clade containing L. porninsis, L. rubrilacteus, L. paradoxus, L. miniatosporus, L. deliciosus and the varieties of L. deliciosus described from North America. Lactarius salmonicolor, L. thyinos and L. laeticolor (S. Imai) Imazeki ex Hongo show a basal position, as well as L. sp.2, an undescribed species from Hunan, China. The ITS data seem to support the morphologically identified entities and almost all species receive high bootstrap support. Lactarius thakalorum Bills & Cotter (sequence obtained from the Nepalese type specimen) invariably is placed together with L. sanguifluus and is the only species not confirmed here. Most identification problems have emerged in the clade that unites the varieties of L. deliciosus described from North America.
gpd phylogeny.— – Alignment of the gpd sequences resulted in a 835-bp dataset, of which 337 bp were variable and 203 bp were parsimony informative. Four species not belonging to Lactarius sect. Deliciosi were included: L. tesquorum Malençon, L. olympianus Hesler & A.H. Sm., L. rufus (Scop. : Fr.) Fr. and L. croceus Burl.
The TrN+I+G model (Tamura and Nei 1993) again was chosen as the best fitting model. Variable sites were assumed to follow a gamma distribution (shape = 0.8657), nucleotide frequencies were set to A 0.2391, C 0.2773, G 0.2357 and T 0.2479 and substitution rates to 4.2907 (AG), 5.4938 (CT) and 1 for all transversions. The proportion of invariable sites was set to 0.2953. One of the > 100 000 shortest trees obtained by the MP analysis (100 replicates, saving max. 1000 trees per replicate) is shown (FIG. 2). MP, Bayesian and ML topologies are the same overall. Lactarius sect. Deliciosi received bootstrap support of 75% in the MP analysis and a 100% posterior probability in the Bayesian analysis. The gpd data support most of the morphologically recognized species. Exceptions are the collections identified as L. deliciosus or a variety of that species from North America that fall into two clades and the collection identified as L. indigo var. diminutivus Hesler & A.H. Sm. that does not group with L. indigo. Lactarius sp.1 and L. horakii are not separated from L. hatsudake. Again the basal relationships in Lactarius sect. Deliciosi did not receive significant support. Lactarius barrowsii and L. rubriviridis; L. laeticolor, L. thyinos and L. salmonicolor and L. porninsis and L. rubrilacteus do group with high support. The Australian collection included was growing under Pinus radiata, an American pine species, but groups with the European and Asian collections of L. deliciosus.
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). Both phylogenies are not well resolved in the basal nodes of Lactarius sect. Deliciosi but show resolution for the morphologically recognized taxa and seem to support mostly the same groups of species. The general topology differs strongly however; in trees based on the gpd gene L. subindigo Verbeken & E. Horak, L. sp.2 and L. salmoneus Peck show a basal position in the section, while ITS trees show L. salmonicolor and its Asian and American counterparts at the base. The MP strict consensus trees from the individual datasets were compared and examined for conflicts involving nodes with bootstrap values > 70% (Mason-Gamer and Kellogg 1996). Most striking is the different placement of specimens L. deliciosus JN 2001-046, L. deliciosus var. areolatus A.H. Sm. JW 381, L. indigo var. diminutivus MCA 811, L. sp.1 K112 and L. hatsudake HKAS 38541. Moreover the ITS data provide high support for species L. akahatsu Tanaka, L. horakii and L. fennoscandicus Verbeken & Vesterh. while the gpd data do not. Vice versa gpd data strongly support L. porninsis, L. sanguifluus and L. aurantiosordidus Nuytinck & S.L. Miller while ITS data do not.
Disregarding the incongruence test and combining both datasets in a total evidence approach results in improved resolution and higher bootstrap support for several nodes in the trees. The MP strict consensus tree is shown (FIG. 3). This phylogeny reflects the gpd topology in the basal nodes but shows much of the species and species groupings from the ITS topology. ML and Bayesian topologies did not show a different general topology than this MP tree. Lactarius subindigo, L. sp.2 and L. salmoneus are basal to the rest of Lactarius sect. Deliciosi. Well supported clades (bootstrap value > 70%) are: (A) a clade formed by L. barrowsii, L. rubriviridis and L. subpurpureus Peck; (B) a clade comprising L. laeticolor, L. thyinos and L. salmonicolor; and (C) a clade uniting L. deliciosus, L. hatsudake, L. quieticolor, L. sp.1 and L. horakii. Lactarius hatsudake becomes paraphyletic when excluding the latter three taxa. Furthermore the data strongly support the grouping of L. sanguifluus and L. vinosus, L. deterrimus and L. fennoscandicus, and L. paradoxus and L. miniatosporus. The specimens identified as L. deliciosus or one of its varieties collected in North America are not maintained as a monophyletic group.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Heilmann-Clausen et al 1998), is confirmed in our analyses while the classification of Hesler and Smith (1979), placing these milk caps in a separate subgenus (Lactarius subgenus Lactarius), can be rejected. That Lactarius sect. Deliciosi forms a monophyletic group is not surprising because many morphological characters are unique to this section and similar in the sometimes barely discernible species. However L. porninsis, a species with white latex, has to be included in this section and thus the definition of Lactarius sect Deliciosi will have to be broadened.
Species in Lactarius sect. Deliciosi.— –
The total of 89 samples included in this phylogenetic study represents 30 species in Lactarius sect. Deliciosi. This means all known species are included, except L. cyanopus Basso, which was included and discussed in detail in previous studies, together with the other European species (Nuytinck 2005, Nuytinck and Verbeken 2005). The current state of knowledge of the American and Asian species is discussed here. The inclusion of several collections per species for most species lets us draw conclusions on the delimitation of these species, on their intra- and interspecific variability and on the frequency of misidentifications.
Twenty names have been published from Central and North America. Hesler and Smith (1979) accept 10 species in their important monograph. Since then only L. miniatosporus and the hypogeous L. rubriviridis have been described from the American continent (Montoya and Bandala 2004, Desjardin 2003). It is important to note that many varieties of North American species have been described. Hesler and Smith (1979) mention eight varieties belonging to five species: L. chelidonium var. chelidonioides (A.H. Sm.) Hesler & A.H. Sm., L. deliciosus var. areolatus, var. deterrimus (Gröger) Hesler & A.H. Sm., var. olivaceosordidus Hesler & A.H. Sm. and var. piceus Smotl. (nom. inval.), L. indigo var. diminutivus, L. pseudodeliciosus var. paradoxiformis (Murrill) Hesler & A.H. Sm. and L. salmoneus var. curtisii (Coker) Hesler & A.H. Sm. Some are not well known and recent collections are scarce (e.g. L. pseudodeliciosus var. paradoxiformis). As a consequence we were not able to include all of them in our analyses. Lactarius indigo var. diminutivus, several North American varieties of "L. deliciosus" and L. salmoneus var. curtisii are included. We strongly doubt however that the distinction between L. salmoneus var. salmoneus and var. curtisii can be maintained. Hesler and Smith (1979) already expressed their doubts on the value of the only reported difference, presence or absence of a greenish discolouration. Observations in well known European species show that the greenish discoloration is a variable character, sometimes absent, sometimes strikingly present, and this convinced us to label the concerned specimens L. salmoneus.
Ten American species are indisputably confirmed in our analyses (L. barrowsii, L. indigo, L. miniatosporus, L. paradoxus, L. pseudodeliciosus Beardslee & Burl., L. rubrilacteus, L. rubriviridis, L. salmoneus, L. subpurpureus and L. thyinos). We are not sure about L. chelidonium Peck because we only have one representative in our analyses (S.L. Miller 9649). This specimen was compared with two specimens of L. chelidonium identified by Peck (collections Peck, 21 Aug, Bolton landing [NYS] and Sep, Bethlehem [NYS]; the type is lost). The spores are similar but differences were found in the abundance of cheilo-and pleuromacrocystidia, which perhaps is not a stable feature (Nuytinck 2005). More well documented specimens are needed here. One new species was found (L. aurantiosordidus, collections SLM 213-03 and 216-03), described in Nuytinck et al (2006a). Lactarius indigo var. diminutivus formed a well supported group with L. indigo var. indigo in the ITS and the combined ITS-gpd analyses but not in the analysis of the gpd data alone, indicating a potentially strong difference between both taxa. More collections must be examined to decide the status of these varieties.
None of the North American specimens identified as L. deliciosus are conspecific with the European L. deliciosus. Detailed observations on the morphology (color and general [surface] aspect of the pileus and stipe, and color [change] of the latex) and microscopy are needed to elucidate the delimitations of taxa in the American "L. deliciosus" clade. Lactarius deliciosus var. areolatus is characterized by its distinctly larger spores and the lack of pleuromacrocystidia and is reported to be the most common variety of "L. deliciosus" in western North America (Hesler and Smith 1979, Methven 1997). Other varieties are mainly distinguished by the presence and abundance of pleuromacrocystidia, the color of the pileus and the staining reaction of the context, three characters that show intraspecific variability in this section.
From Asia only the recently described L. deliciosus var. indicus Atri, Saini & D.K. Mann and L. sanguifluus var. asiaticus Dörfelt, Kiet & A. Berg are not included in the analyses. These taxa unfortunately are accompanied by incomplete descriptions, making it difficult to draw any conclusions on their status. Lactarius akahatsu, L. laeticolor and L. subindigo form well supported clades. Lactarius hatsudake on the contrary seems to be a heterogeneous group. Based on morphological evidence we distinguish three species, L. hatsudake, L. horakii and L. sp.1.
It is obvious that our species concept in Asia is inevitably wider than in a well studied area such as Europe. The lack of detailed macroscopical descriptions from Asia, forcing us to rely on the less informative microscopy, contributes to this difference. The specimen collected in Yunnan, China, and identified as L. deliciosus indeed falls in the European L. deliciosus clade. Lactarius thakalorum, described from Nepal, is possibly conspecific with the European L. sanguifluus but more material is needed to confirm this. This study revealed three new species in Asia (L. horakii, L. sp.1 and L. sp.2). The number of Asian species in Lactarius sect. Deliciosi now adds up to nine. More species certainly remain to be discovered because our knowledge from this under explored continent is poor and fragmentary. Wrongly identified collections frequently were encountered during our research. This was most striking for the Asian material but also in the American "L. deliciosus" complex.
In conclusion we accept 38 taxa (31 species and seven varieties) in Lactarius sect. Deliciosi worldwide but admit that the status of the varieties needs further study. Moreover a few collections could not be reconciled with any of these taxa; one of those collections is included here as L. sp. MTS 3445 (originally identified as L. deliciosus var. olivaceosordidus). Because macroscopical descriptions are lacking for these collections we did not draw any further conclusions, keeping in mind the importance of macroscopical characters in this section. Microscopical descriptions of these collections can be found in Nuytinck et al (2006a, b). The fact that the majority of the species, described with the aid of morphological data alone, is confirmed by our molecular approach is striking when taking into account the strong macro-and microscopical similarity of many taxa.
Intercontinental conspecificity.— – Intercontinental conspecificity in this section seems much lower than assumed so far. No overlap could be shown between America and Eurasia. Further research is needed, including more samples from boreal North America and Asia, to exclude the existence of circumboreal species. Only L. deliciosus and L. sanguifluus seem to occur in both Asia and Europe. This misconception in large has originated from insufficient attention to morphological characters. Lactarius deliciosus and its varieties recognized in North America differ strongly macroscopically from the Eurasian L. deliciosus (pers obs on fresh collections). A new name for the American L. deliciosus is not proposed yet. Full understanding of the status of the varieties is needed first, and that requires more and better macro- and microscopical observations from a wide geographic range.
L. indigo often has been reported from Asia (Hongo and Yokoyama 1978, Imazeki et al 1988, Wu and Mueller 1997) but all collections examined by us are L. subindigo. The two species show distinct morphological differences in spore size and ornamentation (Verbeken and Horak 2000). The divergent placement of L. indigo and L. subindigo in the phylogenetic trees (FIGS. 1, 2 and 3) also strengthens the argument that they are distinct species. There are records of L. salmonicolor from North and Central America, but we had no material available to check conspecificity. Describing Asian taxa under European or American names (recent examples are L. deliciosus var. indicus and L. sanguifluus var. asiaticus) is unacceptable without a thorough comparison.
Relationships between the species and evolutionary trends.— –
Due to the low resolution and support we obtained for the basal relationships and the differences resulting from the analysis of the ITS and gpd regions, it is impossible to propose a further division of Lactarius sect. Deliciosi in subsections. Several morphology-based classifications group those species with similarly colored latex (Basso 1999, Bon 1980, Schaefer 1970). The initial color of the latex is plotted (FIG. 3) and seems to be of limited value to determine relationships in Lactarius sect. Deliciosi. This color, caused by the presence of azulene and hydroazulene sesquiterpenoids with a guaiane skeleton (Schmitt 1974, Sterner and Anke 1995), apparently changed frequently during evolution of the lineage. The two species with indigo blue latex never clustered in any of our analyses; red latex must have evolved at least five times independently and L. porninsis has lost the striking pigmentation of the latex. Nevertheless some well supported clades in our phylogenetic analyses also are supported by morphological and ecological evidence. Lactarius salmonicolor, L. thyinos and L. laeticolor share the lack of a green discoloration, the large spores with a thin ornamentation and striking macrocystidia. They all are associated with Abies (the association of L. thyinos with Abies was personally communicated by Yves Lamoureux). Furthermore L. barrowsii and L. rubriviridis have similar densely ornamented spores and share the red latex and large spores with L. subpurpureus. Spore characters (in this case the heavy ornamentation) also support the L. hatsudake, L. quieticolor, L. horakii and L. sp.1 clade.
When considering the geographic origin of the samples it is striking that many clades are composed of species from distant areas. This suggests that several ancestors must have existed when migration between the continents was still possible. Recent migration between North America and Eurasia seems improbable, given the fact that until now no single conspecific taxon was found. The ancestors must have been similar in morphology to the extant species because some species in this section (e.g. the ones with orange latex that are found in nearly every clade of the tree) are strikingly similar and often difficult to distinguish. The phylogenetic trees showed generally short branch lengths within Lactarius sect. Deliciosi, indicating a low divergence between the taxa. However several mainly North American species are placed on longer branches (e.g. L. salmoneus and L. pseudodeliciosus).
Host trees were plotted on the ITS and gpd tree (FIG. 3). The majority of species form ectomycorrhiza with Pinus, but other coniferous hosts are Picea, Abies, Larix, Pseudotsuga and Tsuga. Lactarius indigo and L. subindigo are reported to be associated also with Fagaceae (Quercus and Castanopsis respectively). Host associations remain unclear for several species (e.g. L. thyinos and L. salmoneus) and deserve more attention. The well documented host specificity of the European species has yet to be confirmed for North American and Asian species. The mostly mixed woods in North America can complicate the host designation. Some species presumably are associated with more than one host (e.g. L. indigo is reported with Pinus and Quercus). The host switch from Pinaceae to Fagaceae or the other way around must have occurred at least twice. Careful comparative host-specificity and host-preference studies are necessary to verify these suggestions and draw more conclusions.
L. rubriviridis is a hypogeous sequestrate species with forcibly discharged spores (Desjardin 2003). It was the first hypogeous species described in the genus Lactarius. Based on morphological arguments, unambiguous designation to Zelleromyces or Arcangeliella turned out to be impossible. Moreover it has been demonstrated that the latter two genera are polyphyletic (Miller et al 2001, Peter et al 2001) and since then other hypogeous species have been assigned to the genus Lactarius as well (Eberhardt and Verbeken 2004, Nuytinck et al 2003). Desjardin (2003) states that the red latex, green stains, forcibly discharged basidiospores and pine association of L. rubriviridis suggest that the species is derived relatively recently from an epigeous agaricoid ancestor, allied with L. rubrilacteus. Lactarius rubriviridis in our analyses is related closely to another species with red latex, namely L. barrowsii. But as indicated above red latex originated several times in the section. The species with red latex from North and Central America seem to fall into three distinct clades: (i) a clade formed by L. rubriviridis, L. barrowsii and L. subpurpureus; (ii) a clade comprising L. paradoxus and L. miniatosporus; and (iii) a clade uniting L. rubrilacteus with L. porninsis and several North American collections identified as "L. deliciosus".
Future perspectives and open questions.— –
The phylogenetic signal in the ITS and gpd datasets is incongruent. The ITS phylogeny agrees better with our morphological observations (e.g. separating L. deterrimus and L. fennoscandicus, grouping all specimens identified as L. hatsudake and "L. deliciosus" in North America). A possible explanation for the different signal in both datasets is that we are dealing with paralogous copies of either gene. Such copies have been reported for both genes in plants (Figge et al 1999, Álvarez and Wendel 2003). But both analytical factors (limited data availability, specific assumptions in the modeling of sequence evolution) and biological factors (the action of natural selection or genetic drift) might cause the history of the genes to obscure the history of the taxa (Rokas et al 2003). Differences between datasets also can result from inclusion of reticulate taxa (Mason-Gamer and Kellogg 1996), but more research is needed to understand the evolutionary history and eventual hybridization between taxa such as L. deterrimus and L. fennoscandicus. Sequencing more genes might be a solution for obtaining a robust phylogenetic hypothesis for Lactarius sect. Deliciosi (Rokas et al 2003).
The low resolution of the phylogenies we obtained, especially in the basal clades, indicates that the genes used do not contain sufficient congruent information to solve these relationships. On the other hand both DNA regions were applied successfully at the same or even a lower taxonomic level (Berbee et al 1999, Chapela and Garbelotto 2004, Hibbett et al 1998, Shen et al 2002). A hypothesis is that rapid speciation caused this low resolution. This also would make incomplete lineage sorting a possible explanation for the incongruent phylogenetic signals we observe in Lactarius sect. Deliciosi.
The remaining taxonomic problems, such as the delimitation of taxa in the American "L. deliciosus" complex and the proposal of new names for these taxa but also for the undescribed species (named L. sp. 1 and 2 here), awaits additional sampling accompanied by detailed morphological descriptions.
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ACKNOWLEDGMENTS |
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) for providing herbarium collections. We acknowledge Dr Saisamorn Lumyong (Chiang Mai University) for providing us with a material transfer agreement for the Thai specimens. The department of Plant Systems Biology, Prof Dr G. Borgonie, the Centre for Molecular Phylogeny and Evolution (Ghent University) and Terry McClean and the Nucleic Acid Exploration Facility (University of Wyoming) are thanked for making available their infrastructure for molecular work and for their help in sequencing and analysis of the data. The research of the first author is financed by the Institute for the Promotion of Innovation by Science and Technology in Flanders (IWT), Belgium. Contributions by Miller were financed by NSF Biotic Surveys DEB-0315607 and USDA CREES 2003-01542. |
FOOTNOTES |
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1 Corresponding author. E-mail: jorinde.nuytinck{at}ugent.be
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