Jorinde Nuytinck

Lactarius volemus sensu lato (Russulales) from northern Thailand: morphological and phylogenetic species concepts explored

Lactarius volemus (Fr.: Fr.) Fr. is a well known and morphologically easily recognizable milkcap of the Northern hemisphere, forming ectomycorrhiza with both deciduous and coniferous trees. It was originally described from Europe, but is also reported in other continents. Although it is characterized by several unique macro- and micromorphological features, substantial variation in colour, lamellae spacing and changing and staining of the latex has been recorded and it is therefore considered as a putatively unresolved species complex. This study explores the concordance between morphological and phylogenetic species concepts within L. volemus sensu lato of northern Thailand, combining a critical morphological scrutiny with a multiple gene genealogy based on LSU, ITS and rpb2 nuclear sequences. Twelve strongly supported monophyletic clades and six terminal branches are discernable in all phylogenetic trees and represent 18 phylogenetic species. Six of the monophyletic clades can be morphologically distinguished and are described as new species: L. acicularis, L. crocatus, L. distantifolius, L. longipilus, L. pinguis and L. vitellinus. Five other clades also show some morphological differences, but these are too subtle and do not allow for a clear-cut species delimitation without the corroboration of molecular data. Lactarius volemus sensu lato of northern Thailand is therefore still considered as a partially cryptic species complex. Pleurolamprocystidia, pileipellis hairs and to a lesser degree also pileus colour are important diagnostic characteristics. Spore morphology, latex discoloration and pileus surface texture are less useful as diagnostic features. Whether this rich diversity is the result of in situ Pleistocene survival or post-glacial expansion and subsequent radiation, has yet to be revealed.

Phylogenetic relationships of Puccinia horiana and other rust pathogens of Chrysanthemum × morifolium based on rDNA ITS sequence analysis.

Isolates of the most important Puccinia species that have been reported on Chrysanthemum x morifolium were collected and the sequences of their ribosomal DNA internal transcribed spacers ITS1 and ITS2 were determined and used as phylogenetic markers. The focus of this study was on Puccinia horiana, due to its quarantine status and its impact in commercial chrysanthemum production. Three technical adjustments were needed to reliably obtain the nucleotide sequences starting from fresh or dried samples. The complete rDNA ITS nucleotide sequences of P. horiana, Puccinia chrysanthemi, and Puccinia tanaceti isolates of varying age and geographic origin were determined. We also identified an as yet undescribed Puccinia species on six old herbarium samples from chrysanthemum. This new species is morphologically similar to P. chrysanthemi and near identical to recent rust samples from Artemisia tridentata. P. tanaceti could not be confirmed as a pathogen of chrysanthemum. Different rDNA ITS sequences were present in P. horiana, with intra-isolate and inter-isolate variability in the length of three nucleotide repeat regions in the different rDNA tandem copies. We also identified three ITS types within P. horiana, with the rarer types displaying up to 67 bp nucleotide sequence differences. These rarer ITS types were detected at low copy number in all isolates. In general, very little rDNA ITS sequence variation was observed between P. horiana isolates from 1903 and 2003, and among isolates from different continents. Phylogenetic analyses using distance, Maximum Likelihood and Bayesian methods confirmed P. horiana, P. chrysanthemi, and the new Puccinia sp. as well-resolved groups, with P. horiana clustering in the clade where the economically important rust species of the Poaceae are located, and P. chrysanthemi and the new Puccinia sp. clustering in the clade where the majority of the rust fungi with hosts in the Asteraceae is located.

Exposing hidden diversity by concordant genealogies and morphology—a study of the Lactifluus volemus (Russulales) species complex in Sikkim Himalaya (India)

Although Sikkim belongs to one of the Global Biodiversity Hotspots, little is known about its ectomycorrhizal fungi, and even less about the main genera of Russulales, i.e. Lactarius, Lactifluus, Multifurca and Russula. Combining a multilocus genealogical and morphological study, we aimed to document the diversity within Lactifluus volemus sensu lato of Sikkim Himalaya. We compared nuclear ITS and LSU rDNA, nuclear rpb1 and rpb2 protein-coding, and mitochondrial atp6 protein-coding genealogies to determine species boundaries. Interspecific relationships were inferred from the combined dataset. Bayesian and maximum likelihood single-locus genealogies are concordant and support recognition of six species. Three of these could be identified by unique morphological characteristics and are described as new species: L. dissitus, L. leptomerus and L. versiformis.

Lactarius sanguifluus versus Lactarius vinosus — Molecular and morphological analyses

Lactarius vinosus is often considered as a variety of L. sanguifluus. Morphological (especially macroscopical characters and spore-ornamentation) and molecular arguments (based on ITS-sequencing) are given here to treat it as a separate species. Its relationship to closely related species of Lactarius section Deliciosi is discussed.

New combinations in Lactifluus, 1 : L. subgenera Edules, Lactariopsis, and Russulopsis

In this first of a series of three papers, new combinations in the genus Lactifluus are proposed. This paper treats the subgenera Edules, Lactariopsis, and Russulopsis (all proposed here as new combinations in Lactifluus). In Lactifluus subg. Edules, eight combinations at species level are proposed. In Lactifluus subg. Lactariopsis, the following three new combinations are proposed at sectional level: Lactifluus sect. Lactariopsis with seven newly combined species, L. sect. Chamaeleontini with eight newly combined species, and L. sect. Albati with four newly combined species plus two species previously combined in Lactifluus. Finally, in L. subg. Russulopsis, eight new combinations at species level are proposed.

The Australasian species of Lactarius subgenus Gerardii (Russulales)

This paper provides an overview and identification key of the Australasian species of Lactarius subg. Gerardii. A molecular phylogeny based on the nuc DNA markers ITS, LSU and rpb2, combined with detailed morphological observations resulted in the delimitation of 12 strongly supported and morphologically recognizable species. Five new agaricoid species are described here: Lactarius hora, L. coniculus, L. limbatus, L. leae and L. leonardii. Other agaricoid species belonging to subg. Gerardii are L. atrovelutinus, L. bicolor, L. ochrogalactus and L. reticulatovenosus. Lactarius venosus is also considered as a member but could not be included in the molecular analysis. Two new pleurotoid species are discovered: L. genevievae and L. conchatulus, both close relatives of L. uyedae, but each with distinct characters. Their position within subg. Gerardii is supported in the phylogeny and by their microscopic characters. This study also confirms the existence of multiple cryptic species and species complexes for which species recognition or species delimitation remains problematic as is the case for the Australian species L. wirrabara. In general, detailed macroscopic and microscopic observations are needed to identify species of L. subg. Gerardii.

A multi-gene phylogeny of Lactifluus (Basidiomycota, Russulales) translated into a new infrageneric classification of the genus

Infrageneric relations of the genetically diverse milkcap genus Lactifluus (Russulales, Basidiomycota) are poorly known. Currently used classification systems still largely reflect the traditional, mainly morphological, characters used for infrageneric delimitations of milkcaps. Increased sampling, combined with small-scale molecular studies, show that this genus is underexplored and in need of revision. For this study, we assembled an extensive dataset of the genus Lactifluus, comprising 80 % of all known species and 30 % of the type collections. To unravel the infrageneric relationships within this genus, we combined a multi-gene molecular phylogeny, based on nuclear ITS, LSU, RPB2 and RPB1, with a morphological study, focussing on five important characteristics (fruit body type, presence of a secondary velum, colour reaction of the latex/context, pileipellis type and presence of true cystidia). Lactifluus comprises four supported subgenera, each containing several supported clades. With extensive sampling, ten new clades and at least 17 new species were discovered, which highlight the high diversity in this genus. The traditional infrageneric classification is only partly maintained and nomenclatural changes are proposed. Our morphological study shows that the five featured characteristics are important at different evolutionary levels, but further characteristics need to be studied to find morphological support for each clade. This study paves the way for a more detailed investigation of biogeographical history and character evolution within Lactifluus.

Lactifluus volemus in Europe: Three species in one--Revealed by a multilocus genealogical approach, Bayesian species delimitation and morphology.

This study provides morphological and molecular evidence (from nuclear ITS, LSU, and rpb2 DNA sequences) for three previously unrecognized species within the morphospecies Lactifluus volemus from Europe. Phylogenetic species are supported by both a multi-locus tree-based method and Bayesian species delimitation. Lactifluus volemus and Lactifluus oedematopus are provided with a new description, and a third species, Lactifluus subvolemus, is described as new to science. Lactifluus oedematopus can be easily recognized by its short pileipellis hairs. Both L. volemus and L. subvolemus have longer pileipellis hairs and can only be distinguished from each other based on cap colour. Intermediary colour forms, however, occur as well, and cannot be identified as either L. volemus or L. subvolemus without molecular data. Revealing that L. volemus--already considered extinct in the Netherlands and the Belgian Flemish region, and declining in other European countries--is actually a complex of three species that are even more vulnerable to extinction, this study emphasizes the fundamental role of taxonomy in species conservation.

Morphology and phylogeny of four new Lactarius species from Himalayan India

Four new species of Lactarius are described from Himalayan India. Lactarius olivaceoglutinus, L. pyriodorus, and L. yumthangensis belong to L. subg. Piperites and L. indochrysorrheus is closely related to some representatives of L. subg. Russularia. An ITS based phylogeny confirms the phylogenetic placement of the four new species, although the monophyly of neither Lactarius subg. Piperites nor L. subg. Russularia can be confirmed. The ITS data also suggest that the Indian species are closely related to some European and American species.

Lactarius subgenus Russularia (Basidiomycota, Russulales): novel Asian species, worldwide phylogeny and evolutionary relationships

Lactarius subg. Russularia is a large group of milkcaps occurring almost worldwide and dominant in many ecosystems. In this study we focus on new diversity, evolutionary relationships, divergence time, and origin of the subgenus. Six conifer symbionts are described as new to science: Lactarius atrii, L. aurantionitidus, L. dombangensis, L. flavigalactus, L. lachungensis, and L. sikkimensis. Species delimitation is assessed based on the concordance between morphological characteristics and an ITS phylogeny. Infrageneric relationships were studied using a phylogeny constructed from concatenated ITS-rpb2 data using Maximum Likelihood and Bayesian inference. Results show that species in this subgenus do not cluster together according to their geographic origin. Intercontinental sister relationships between Europe/Asia/North America are common but actual conspecificity is rare. This result suggests that allopatric speciation has played an important role within this subgenus. Only few morphological characteristics tend to be phylogenetically informative, with the most important being presence or absence of true cystidia and the pileipellis structure. Two datasets were generated in order to estimate the age of L. subg. Russularia. The results suggest the origin of L. subg. Russularia to be in the Mid Miocene period.