Updated taxonomy of LactifluussectionLuteoli: L.russulisporus from Australia and L.caliendrifer from Thailand

Abstract Lactifluusrussulisporus Dierickx & De Crop and Lactifluuscaliendrifer Froyen & De Crop are described from eucalypt forests in Queensland, Australia and different forest types in Thailand, respectively. Both species have recently been published on Index Fungorum and fit morphologically and molecularly in L.sect.Luteoli, a section within L.subg.Gymnocarpi that encompasses species with alboochraceous basidiomes, white latex that stains brown and typical capitate elements in the pileipellis and/or marginal cells.


Introduction
Since the division of Lactarius into Lactarius sensu novo and Lactifluus (Buyck et al. 2008), our understanding of both genera has increased significantly. Although Lactifluus is the smaller of the two genera, it is characterised by a higher genetic diversity with subgroups in very different and genetically distant clades . Recently, efforts in Lactifluus culminated in a new infrageneric classification based on checked for in Cotton blue in lactic acid and Cresyl blue (Clémençon 1997;2009) Spore ornamentation is described and illustrated as observed in Melzer's reagent. A total of 40 spores (20 per collection) were measured for each of the two new species. For details on terminology we refer to Verbeken (1998) and Verbeken and Walleyn (2010). Line-drawings were made with the aid of a drawing tube (Zeiss camera lucida on a Zeiss Axioskop 2 microscope equipped with a magnification changer of 2.5× for spores and an Olympus U-DA on an Olympus CX21 microscope for individual elements and pileipellis structures) at original magnifications: 6000× for spores, 1500× for individual elements and sections. Basidia length excludes sterigmata length. Spores were measured in side view, excluding the ornamentation, and measurements are given as (MINa) [AVa-2*SD]-AVa-AVb-[AVb+2*SD] (MAXb), with AVa = lowest mean value for the measured collections and AVb = greatest mean value for the measured collections, SD = standard deviation, MINa = lowest extreme value of collection "a" and MAXb = greatest extreme value of collection "b". The Q-value (quotient length/ width) is given as (MIN Qa) Qa-Qb (MAX Qb), with Qa = lowest mean ratio for the measured collections and Qb = greatest mean ratio for the measured collections, MIN Qa = lowest extreme ratio of collection "a" and MAX Qb = greatest extreme ratio of collection "b". Other measurements are given as MIN-MAX values. Colour codes refer to Kornerup and Wanscher (1978). Microscopic photographs were taken using a Nikon eclipse NI-U-microscope equipped with a DX-Fi1c camera and Nikon NIS-Elements software including EDF module.

Molecular work
DNA from dried collections was extracted using the protocol described by Nuytinck and Verbeken (2003) with modifications described in Van de Putte et al. (2010), and from fresh material using the CTAB extraction method described in Nuytinck and Verbeken (2003). Protocols for PCR amplification follow Le et al. (2007). The internal transcribed spacer (ITS) was sequenced for a second collection for each new species using the primers ITS1-F and ITS4 (Gardes and Bruns 1993;White et al. 1990). PCR products were sequenced using an automated ABI 3730 XL capillary sequencer (Life Technology) at Macrogen. Forward and reverse sequences were assembled into contigs and edited where needed with SequencherTM v5.0 software (Gene Codes Corporation, Ann Arbor, MI, USA).

Results
In congruence with De Crop et al. (2017), our molecular results show that the collections from Australia as well as those from Thailand belong to Lactifluus. subg. Gymnocarpi sect. Luteoli (Fig. 2). The newly generated sequences for Halling 9674 and Wisitrassameewong 392 belong to the same species as Halling 9398 and Wisitrassameewong 378 respectively. These two species are supported by morphological and geographical differences (see discussion) and are fully described below as L. russulisporus and L. caliendrifer. Original diagnosis. Basidiocarps small (up to 4 cm cap diam.). Cap and stipe dry, matt, yellowish white to pale brown. Context with unpleasant, fishy smell. Latex copious, watery white, staining tissues brown. Basidiospores broadly ellipsoid 7.0-7.8-7.9-8.7 × 5.7-6.4-6.5-7 μm (n=40, Q = 1.14-1.23-1.40); ornamented with irregular and isolated warts which are up to 1.3 μm high. True pleurocystidia absent, but with few to abundant sterile elements in the hymenium. Pileipellis a lampropalisade. L. russulisporus differs from its sister species, L. caliendrifer, by its longer basidia, slightly bigger spores with a somewhat heavier and more irregular ornamentation and the absence of abundant thick-walled marginal cells.

Lactifluus russulisporus Dierickx & De Crop
Basidiomes rather small. Pileus 20-40 mm diam., convex to plano-convex and depressed on disc to uplifted and slightly depressed, yellowish white (4A2) to pale brown, dry, matted, subtomentose to finely subvelutinous and somewhat subrugulose to subcorrugate; margin inrolled. Stipe 10-30 × 5-10 mm cylindrical, dry, matt, yellowish white, sometimes paler brownish towards the base, with white mycelium at the base. Lamellae adnexed to subdecurrent, rather close, pale greyish white to yellowish white, turning darker to near pale brown with age. Context white, solid to somewhat pithy in the stipe; smell unpleasant, fishy; taste mild. Latex copious, watery white, staining tissues brown.

Distribution. Known from Eastern Australia.
Ecology. East-Australian wet sclerophyll and subtropical rainforest, scattered to gregarious on soil under Leptospermum, Syncarpia, and Eucalyptus spp.
Etymology. Named after the spores which are reminiscent of the spore ornamentation and shape of many Russula species.
Distribution Remarks. Lactifluus caliendrifer differs from its sister species, L. russulisporus, by the abundant thick-walled marginal cells, very long pileipellis hairs and slightly smaller basidia and spores with more regular and lower warts.

Discussion
The morphological distinction between Lactarius and Lactifluus is not always straightforward in the field and can only be based on some general trends. For example, the genus Lactifluus is generally characterised by the complete absence of zonate and viscose to glutinose caps, and it contains many species with veiled and velvety caps (Buyck et al. 2008;Verbeken and Nuytinck 2013). A cellular hymenophoral trama and a lampropalisade as pileipellis structure are both characters which are more often observed in Lactifluus than in Lactarius.
The newly described species can macroscopically be recognised as members of genus Lactifluus by the tomentose to velvety appearance of their caps and the exuded milk that changes to brownish (which is more common in Lactifluus and very rare in Lactarius). Microscopically the presence of a lampropalisade and a cellular trama indicate the affinity with Lactifluus.
Lactifluus russulisporus and L. caliendrifer belong to L. subg. Gymnocarpi, which is supported by molecular (Fig. 2)  ) and morphological data (e.g. brown discolouration of the latex and the absence of true pleurolamprocystidia). Both new species are placed in L. sect. Luteoli, which consists of seven species from all continents except South America and Antarctica, and are characterised by capitate elements in the pileipellis and/or the presence of differentiated marginal cells.
The sister species Lactifluus russulisporus and L. caliendrifer are clearly delimited molecularly, which is reflected in both geographical and morphological characters. Geographically, L. russulisporus is only known from Eastern Australia (Queensland), while L. caliendrifer is only known from Southeast Asia (Thailand). In the field, both species can be recognised by their cream to yellowish white basidiomes, dry and finely velvety to pruinose pilei, rather crowded white to concolorous lamellae and copious watery latex that stains brown. These features are common to most species in L. sect. Luteoli.
Lactifluus caliendrifer can be distinguished macroscopically by its velvety pileus, whiter basidiomes and its strong and fruity smell. Lactifluus russulisporus differs from its sister species by having a more yellowish-brown shade and an unpleasant, fishy smell.
Lactifluus rubrobrunnescens is known to occur in Java (Indonesia) and can easily be recognised by a hollow stipe, latex that stains reddish brown, more globose spores (average Q = 1.16) and distinctly capitate elements in the pilei-and stipitipellis, and marginal cells (Verbeken et al. 2001).

Notes on terminology
When it comes to terminology used in the genera Lactarius and Lactifluus, most authors tend to follow Verbeken and Walleyn (2010) and Verbeken (1998). Unfortunately, some confusion seems to exist concerning hymenophoral cells that can be termed either leptocystidia or sterile elements. Even though this type of cell is frequently present in Lactifluus (pers. observations), these cells are only rarely reported in species descriptions Delgat et al. 2017), probably often being dismissed as basidioles and/or of limited taxonomic value. This problem presented itself during the description of the two new species and a consensus between the authors of this paper was pursued.
The term leptocystidium is composed of the Greek leptós, meaning "smooth, thin-walled" and cystidium, meaning "a sterile body, frequently of distinctive shape, occurring at any surface of a basidiome, particularly the hymenium from which it frequently projects" (Ainsworth 2008). In Clémençon (1997), leptocystidia are described in a similar manner, with the addition that they often have an excretory function. For the latter, we could not find evidence in our collections. According to Verbeken and Walleyn (2010), leptocystidia can be regarded as "thin-walled cystidia without remarkable content and thus only deviating by their shape. They are tapering at the top and often have a rostrate apex, which makes them easy to confuse with monosterigmatic basidia. One can consider them to be cystidia if they are regularly observed and if they never bear a spore or spore primordium". In the two new species, and by extension in most Lactifluus species, thin-walled sterile cells with no remarkable content occur in the hymenium. Furthermore, they do not exhibit a deviating shape, being cylindrical and usually ending blunt. If shape deviation is seen as a vital component for being a cystidium, these cells cannot be named as such. In addition, we dismiss the idea that these cells represent basidioles. Firstly, no intermediate forms between these cells and basidioles were observed. Secondly, in L. russulisporus these cells display a different morphology in both collections. In RH 9674, and by extension in general, they do not protrude from the hymenium and do not exhibit a deviant form, leaving open the possibility that they constitute basidioles or protobasidia (Fig. 7C). However, in RH 9398, they grow out strikingly, protruding clearly from the hymenium (Fig. 7A, B). The same behaviour is seen in the pseudocystidia and marginal cells in this collection. According to Moore (2005), principle nine of fungal developmental biology states that "meiocytes appear to be the only hyphal cells that become committed to their developmental fate. Other highly differentiated cells retain totipotency-the ability to generate vegetative hyphal tips that grow out of the differentiated cell to re-establish a vegetative mycelium." A possible hypothesis is that some stimulus, perhaps environmental, caused the totipotent cells in the hymenium to grow out, giving rise to the protruding sterile elements, pseudocystidia and marginal cells in RH 9398. This explanation adds to the idea that these cells are not precursor cells of meiocytes (basidia).
As these sterile elements are argued not to be cystidia or basidioles, the question remains as to what they are. Several terms might have been used to indicate the same kind of cells. For example, haplohyphidia refers to unmodified, unbranched or little branched terminal hyphae in the hymenium of (mostly) Aphyllophorales. An intriguing term, paraphyses, is used in the works on the developmental biology of the hymenium done in Coprinopsis cinerea (Horner and Moore 1987;Rosin and Moore 1985a). These cells originate as branches of sub-basidial cells and insert into the basidial layer, later inflating so that they become the main structural component as a pavement from which basidia and cystidia protrude (Horner and Moore 1987;Moore 1985;Rosin and Moore 1985a;. This description fits well with the sterile elements observed in Lactifluus (Figs 7, 8F). Nevertheless, paraphyses is a term strongly associated with Ascomycota, used for more hair-like (filiform) cells. It cannot be stated with certainty that Ascomycete paraphyses are homologous to the cells we find in Lactifluus.
Given the lack of a distinctive deviating shape in most cases, the improbability of being basidioles and the neutrality of the term, we recommend the use of the term 'sterile elements' over the terms 'leptocystidia' and 'paraphyses' to refer to these cells.
Thereto can be added that marginal cells often bear a striking resemblance to sterile elements (Fig. 8). Furthermore, in Inocybe, little differentiated cystidia are referred to as paracystidia, which also show similar morphology to marginal cells and might constitute the same type of cell (Jacobsson and Larsson 2012;Kuyper 1986). Presently it is difficult to argue whether this is due to homology or homoplasy. Marginal cells are sterile elements on a sterile edge that differ from pleurocystidia and are, in fact, 'hairs' sensu Romagnesi (Verbeken and Walleyn 2010). In species where the edge is fertile, sterile elements are also present on the edge. It is possible that, when no differentiated marginal cells are present on an infertile edge, sterile elements are present and consequently reported as being marginal cells. We suggest paying more attention to these sterile elements which occur predominantly in Lactifluus. Given the variation that we observe within L. russulisporus, it is likely that the taxonomic value of this character is rather low, but this needs more observations. of the 'Bijzonder Onderzoeksfonds Gent University' (BOF) Gent University and the Thailand Research Fund (BRG5580009) under the research grant entitled 'Taxonomy, Phylogeny, and Biochemistry of Thai Basidiomycetes". Roy Halling was partially supported by National Science Foundation (USA) funds from grant DEB 1020421. The National Geographic Society Committee for Research and Exploration provided funding via grant 8457-08. The Queensland Herbarium (BRI) collaborated generously with assistance and support for herbarium and field studies in Australia. We would like to thank Viki Vandomme for conducting lab work.