A multi-gene phylogeny of Chlorophyllum (Agaricaceae, Basidiomycota): new species, new combination and infrageneric classification

Abstract Taxonomic and phylogenetic studies of Chlorophyllum were carried out on the basis of morphological differences and molecular phylogenetic analyses. Based on the phylogeny inferred from the internal transcribed spacer (ITS), the partial large subunit nuclear ribosomal DNA (nrLSU), the second largest subunit of RNA polymerase II (rpb2) and translation elongation factor 1-α (tef1) sequences, six well-supported clades and 17 phylogenetic species are recognised. Within this phylogenetic framework and considering the diagnostic morphological characters, two new species, C. africanum and C. palaeotropicum, are described. In addition, a new infrageneric classification of Chlorophyllum is proposed, in which the genus is divided into six sections. One new combination is also made. This study provides a robust basis for a more detailed investigation of diversity and biogeography of Chlorophyllum.


Introduction
The genus Chlorophyllum Massee, 1898 (Agaricaceae, Agaricales) is typified by Chlorophyllum molybdites (G.Mey.) Massee.This genus currently accommodates ca.16 species (Kirk et al. 2011) and 30 records can be found in Index Fungorum (http://www.indexfungorum.org/Names/NAMES.ASP).Traditionally, this genus was monotypic, only containing the green-spored species, C. molybdites.Based on similarities in morphology and/or molecular evidence, a few species previously placed in Macrolepiota Singer or Lepiota (Pers.)Gray, were transferred into it (Vellinga 2002).Similarly, Endoptychum agaricoides was also transferred to this genus based on molecular evidence and the proposal to conserve Chlorophyllum (hereafter abbreviated as C.) against Endoptychum was submitted to retain the genus name for the well-known toxic species C. molybdites (Vellinga and De Kok 2002) and accepted (Gams 2005).Members of this genus are characterised by the following unique combination of morphological characters: the pileus covering is hymenidermal, the stipe (if present) is smooth and basidiospores lack a germ pore or have a germ pore caused by a depression in the episporium without a hyaline covering.The basidiospores are white, green, brownish or brown in deposit and the habit varies from agaricoid, secotioid to gasteroid (Crous et al. 2015a;Ge and Yang 2006;Vellinga 2003a;2004b;Vellinga et al. 2003).Species within this genus are saprotrophic and distributed worldwide, often growing in urban and ruderal habitats, with a preference for tropical and subtropical regions (Vellinga 2004a).
Recently, three species, C. lusitanicum G. Moreno, Mohedano, Manjón, Carlavilla & Altés, C. pseudoglobosum J. Sarkar, A.K. Dutta & Acharya and C. sphaerosporum Z.W. Ge & Zhu L. Yang were described from Spain, India and China, respectively (Crous et al. 2015a;Crous et al. 2015b;Ge and Yang 2006).These studies provided a better understanding of the species diversity within the genus, but are confined to certain specific regions and samples from other poorly explored areas such as Africa have seldom been included.Such studies have been focused on new species descriptions, but an infrageneric classification for the genus is still lacking because infrageneric relationships are poorly known.
Phylogenetic studies have shown that Chlorophyllum is nested within Agaricaceae (Ge and Yang 2017;Vellinga 2004b;Vellinga et al. 2003;Vellinga et al. 2011).However, due to limited taxon sampling and /or use of the ribosomal RNA genes only (ITS and /or nrLSU), limited information on infrageneric relationships could be gleaned.Further sampling of more species and phylogenetic analyses based on protein coding genes are needed to clarify relationships within Chlorophyllum.
Based on investigations of lepiotoid fungi in China, Dominican Republic, Germany, Italy, South Africa, Thailand, United Kingdom and the United States of America, detailed morphological and molecular studies were carried out in this study.The aims were to: 1. elucidate species diversity within Chlorophyllum based on both morphological characters and phylogenetic analysis, describe novel species and provide more information on poorly known species; 2. use a combined multi-gene dataset (ITS, nrLSU, rpb2 and tef1) to provide a robust hypothesis for relationships amongst Chlorophyllum species; 3. examine diagnostic characters for recognised clades and establish an infrageneric classification that best reflects the evolutionary history of the genus.

Taxon sampling and morphological studies
Fifty-nine collections were newly sampled from China, Dominican Republic, Germany, Italy, South Africa, Thailand, the United Kingdom and United States of America and deposited in HMAS, PREM, HKAS (Herbarium of Cryptogams, Kunming Institute of Botany, Chinese Academy of Sciences) and MFLU.Twelve out of the 16 recognised species, plus two recently described species, as well as two putative new species and a new combination were represented in this study.Morphological characters were studied from field notes, colour images of the material and complemented with literature data.Colour names and codes are from Kornerup and Wanscher (1978).Microscopic character observations were conducted under a light microscope using thin handmade sections rehydrated in 5% aqueous potassium hydroxide (KOH) (w/v).Melzer's reagent was used to test the amyloidity of basidiospores and cresyl blue was used to study the metachromatic reaction (Largent et al. 1977)

DNA extraction, primers, PCR and sequencing
A small piece of dried basidiocarp was excised from a specimen and ground in an Eppendorf tube.Genomic DNA was isolated using the CTAB method (Doyle and Doyle 1987).Optimal dilutions of the DNAs were used to amplify the following regions: internal transcribed spacer (ITS), the large subunit nuclear ribosomal DNA (nrLSU), the second largest subunit of RNA polymerase II (rpb2) and the translation elongation factor 1-α gene (tef1).PCR amplifications used the previously described primers: ITS1F/ITS4 for ITS, LR0R/LR5 for nrLSU (Gardes and Bruns 1993;White et al. 1990), bRPB2-6F /bRPB2-7.1Rfor rpb2 (Matheny 2005)

Phylogenetic analyses
In this study, 144 sequences were produced using standard methods and deposited in GenBank (MG741961-MG742106), viz., 59 ITS, 29 nrLSU, 28 rpb2 and 28 tef1 (Figure 1 and Table 1) sequences.To obtain an estimate of Chlorophyllum genetic diversity, 96 ITS sequences were also retrieved from GenBank and included in the phylogenetic analyses (GenBank nos.included in Figure 1).The ITS data set included 155 Chlorophyllum sequences.From these, a subset of 29 collections was chosen to represent the full range of phylogenetic diversity sampled for a four-locus dataset comprising portions of the ITS, nrLSU, rpb2 and tef1 (Table 1).To test the monophyly of Chlorophyllum within Agaricaceae, ML analysis of the rpb2 dataset, which has much fewer ambiguous aligned sections in the matrix compared to the ITS dataset, was conducted with representative genera of the Agaricaceae (Ge and Yang 2017;Ge et al. 2015;Vellinga et al. 2011).As Chlorophyllum is confirmed as a monophyletic group close to Agaricus L. and allied genera in the present study (Suppl.material 1) and recent studies (Ge and Yang 2017;Ge et al. 2015;Vellinga et al. 2011), representative species of these genera were included as outgroups for rooting purposes for analyses; these are Agaricus campestris L., Clarkeinda trachodes (Berk.)Singer, Coniolepiota spongodes (Berk.& Broome) Vellinga, Eriocybe chionea Vellinga, Heinemannomyces splendidissimus Watling and Pseudolepiota zangmui Z.W. Ge.
Sequences were aligned using MAFFT 6.8 (Katoh et al. 2009) and further optimised visually.The best-fit evolutionary model for each dataset was determined using MRMODELTEST (Nylander 2004).Maximum Likelihood (ML) analyses were conducted in RAxML 7.2.6-WIN with default settings using a GTRGAMMA model (Stamatakis et al. 2007).Clade robustness was assessed using a bootstrap analysis with 1000 replicates (Felsenstein 1985).
The ITS-nrLSU, rpb2 and tef1 datasets were analysed separately before concatenation.As no significant (bootstrap support above 70 %) incongruence was detected, the resulting three alignments (ITS-nrLSU, rpb2 and tef1) were combined for further multigene analyses.Unavailable sequences of loci from a few species were treated as missing data in the phylogenetic analyses.Final alignments have been deposited in TREEBASE (http://www.treebase.org)with accession number (S22068).

Results
The ITS alignment contained 787 sites, all of which were included in the analyses.The ML tree is shown in Figure 1 with the final ML optimisation likelihood at -5625.939348.According to the ML tree, 17 phylogenetic species were recovered.The new species were nested within Ellipsoidospororum clade (C.africanum) and Chlorophyllum clade (C.palaeotropicum).
The combined data set included subsamples of the 17 species recovered in the ITS tree.This alignment contained 2896 nucleotide sites (including gaps), consisting of 785, 851, 699 and 561 sites (including gaps) for ITS, nrLSU, rpb2 and tef1, respectively.The analyses identified six distinct well-supported clades within Chlorophyllum, each representing one to four species (Figure 2).These clades are: Chlorophyllum clade, Ellipsoidospororum clade,  Each of these clades received 100 percent maximum boot strap (ML-BP) support in the combined data set and strong support (≥87 % boot strap support) in the individual data sets (ITS-nrLSU, rpb2 and tef1 respectively).Species relationships within these six clades are largely resolved, but relationships amongst all clades were not resolved with confidence.
Emphasising both molecular data and morphological characters, a new infrageneric classification for Chlorophyllum and two new distinct species, C. africanum and C. palaeotropicum are proposed.

Infrageneric classification of Chlorophyllum
The genus Chlorophyllum is divided into six sections: sect.Diagnosis.This species is distinguished from other Chlorophyllum species by relatively small basidiocarps with yellow grey to grey orange (5B4) furfuraceous squamules, the Description.Pileus 30-50 mm broad, hemispherical to convex when young, expanding to broadly convex or applanate with age, sometimes with a prominent umbo; margin sulcate striate; surface covered with thin, yellow grey (4B2-4B3), orange grey (6B2) to grey orange (5B4) furfuraceous squamules, these remaining intact on the disc but elsewhere diffracted-scaly with expansion and receding from pileus margin.Lamellae free and remote from stipe, white to off white, crowded, narrow, up to 6 mm deep, with 1-2 series of lamellulae; edges entire, white.Stipe 35-60 × 3-6 mm, subcylindric, slightly enlarged at base, glabrous, white to light brown, hollow, nearly stuffed, with a simple annulus about 10-15 mm from top of the stipe.Context white, 1-2 mm thick in pileus, discolouring brown to red where bruised or handled, with strong mushroom odour, taste mild.Spore print white to cream.
Chlorophyllum africanum is also similar to C. hortense on account of the smallsized basidiocarps, ellipsoid basidiospores and subcylindrical cheilocystidia.However, C. hortense differs from C. africanum by 2-spored basidia and the whitish context of the stipe becoming reddish where bruised (Akers and Sundberg 1997;Vellinga 2003b).
Leucocoprinus zeylanicus (Berk.)Boedijn, described from Sri Lanka, is also similar to C. africanum due to the small-sized pileus with a distinct umbo, the subcylindric cheilocystidia and the short ellipsoid basidiospores (Pegler 1986).However, the finely radially silky-striate pileus of Lc. zeylanicus beset with sparse, minute, blackish-brown repent squamules and the basidiospores with a small germ pore (Pegler 1986), differentiate this species from C. africanum.
Lepiota zeyheri (Berk.)Sacc., a species also found in South Africa, is somewhat similar to C. africanum on account of the whitish pileus with a clay brown umbo that is elsewhere covered with cream or brown squamules and the ellipsoid basidiospores.However, L. zeyheri has much larger basidiocarps measuring 10-22 cm or larger, a pale pink spore deposit, larger broadly ellipsoid basidiospores (15.0-17.0× 10.0-12.0μm) with a germ pore and clavate cheilocystidia (Pearson 1950).

Chlorophyllum palaeotropicum Z.W. Ge & A. Jacobs, sp. nov. MycoBank MB 823862 Figs 3D, 5
Diagnosis.This species is distinguished from other Chlorophyllum species by mediumsized basidiocarps with distinct brownish squamules composed of a trichodermal layer of subcylindrical brownish hyphae and slightly enlarged terminal the greenish subglobose basidiospores without a germ pore and the clavate to narrowly clavate cheilocystidia with brownish to fuscous brown vacuolar pigments.
Chlorophyllum palaeotropicum is also similar to C. molybdites, C. globosum and C. pseudoglobosum in general appearance due to the brownish to reddish discolourations where bruised, but C. palaeotropicum differs from these three species in having subglobose to globose basidiospores without a germ pore (apex rounded), while C. molybdites, C. globosum and C. pseudoglobosum have amygdaliform basidiospores with large germ pores (apex truncate).
Chlorophyllum palaeotropicum also resembles C. sphaerosporum on account of the basidiocarps bearing similar subglobose basidiospores without a germ pore.However, the squamules of C. sphaerosporum are made up of a hymenidermal layer composed of clavate to broadly clavate terminal elements.Furthermore, the context of C. sphaerosporum does not change colour when bruised.So far, C. sphaerosporum has only been recorded from temperate regions in northern China.These two species also belong to two different sections (Figure 1).
Distribution.Known from China and Vietnam in Asia.
Discussion.The distinctive characters of Chlorophyllum demangei are the discolouration of basidiocarps when bruised, the ellipsoid basidiospores without a germ pore and the pileal squamules composed of clavate to narrowly clavate elements.From the examination of specimens newly collected from southern Yunnan in China, not far away from the locality where the type of Lepiota demangei was collected, the distinctive characters were found that fit the description of Lepiota demangei (Yang 2000) very well.Thus, Lepiota demangei is transferred from Lepiota to Chlorophyllum.

Monophyly and infrageneric classification of Chlorophyllum
In the present study, based on the extensive dataset comprising 75 % of all known species, four gene regions were used to clarify the evolutionary relationships of Chlorophyllum, in separate and multi-locus analyses.Based on molecular data, the genus Chlorophyllum is monophyletic and the genus Clarkeinda appeared to be the likely sister clade to Chlorophyllum (Figure 2).A six-section infrageneric classification of Chlorophyllum was proposed based on the demarked morphological characters of wellsupported clades.This phylogeny also elucidated the systematic positions of previously not included taxa, such as C. demangei, C. sphaerosporum and C. subrhacodes (Figure 2).In addition, two new species, C. africanum and C. palaeotropicum have been added.

Useful morphological characters in delimitation sections and species within Chlorophyllum
The morphological diversity within Chlorophyllum is mainly reflected in the general appearance of basidiocarp, colour reaction of context when bruised, the structure of pileus squamules, colour, shape and size of basidiospores and presence / absence of germ pore, shape of cheilocystidia and presence /absence of clamp connections.Based on the morphological characters chiefly used for species delimitation, morphological features in Chlorophyllum appear to be fast evolving and prone to shifts and no synapomorphic characteristics have been found to consistently separate the sections.This is probably due to the fact that major evolutionary radiations might have occurred in a relatively short time as it can be seen that most of the deep branches in the phylogenies are short.Nevertheless, several character combinations are phylogenetically informative thus are useful for delineating sections and species.
1. General habitus of basidiocarps.The sequestrate (secotioid / gasteroid) form of basidiocarps is considered to be the result of selective pressures (Bruns et al. 1989) and the loss of forcible spore discharge has been found in several predominantly agaricoid genera within the family Agaricaceae (Gube and Dörfelt 2012), e.g.Agaricus (Vellinga 2004), Macrolepiota (Lebel and Syme 2012) and Lepiota (Ge and Smith 2013;Vidal et al. 2015).The transition from agaricoid to secotioid or gasteroid is thought to be irreversible (Hibbett 2004).The majority of Chlorophyllum species is agaricoid and the secotioid / gasteroid habit is an apomorphy for the genus Chlorophyllum and consequently can be used together with other characteristics to distinguish clades.These phylogenetic analyses demonstrate that the secotioid C. agaricoides forms an independent clade, while the gasteroid C. arizonicum and C. lusitanicum jointly form a clade that is sister to the agaricoid C. sphaerosporum (Figure 2).These results suggest that the gasteroid C. arizonicum and C. lusitanicum derived from the same ancestor as C. sphaerosporum, while the secotioid C. agaricoides may have evolved independently from a different agaricoid ancestor in the genus.2. Colour reaction of the context when bruised.The context of Chlorophyllum species shows reddening changes when exposed to the air, from light sienna, pinkish, pinkish cinnamon, reddish, dull brownish-orange to orange red (Crous et al. 2015a;Crous et al. 2015b;Ge and Yang 2006;Vellinga 2003aVellinga , 2003b)).These changes are difficult to quantify and have not been uniformly recorded according to the same criteria and thus, cannot be used to distinguish sections.Nevertheless, this character can be used in combination with other characters in delimitation of species as this character varies amongst species: some species have a strong reddening reaction, some only weakly change pinkish, in others the context becomes reddish first, then changes to brown.3. Structure of pileus squamules.The squamules in Chlorophyllum are considered to be a hymeniform layer in general, but can be further divided into three different types: i. a palisade of hyphae with terminal clavate to subfusiform elements; ii. a tightly packed hymeniderm of cylindrical and flexuous, narrowly clavate or narrowly lageniform elements; and iii. a hymeniderm of loosely arranged clavate to subfusiform hyphae.These different types of structure of squamules can be used to delimit sections in combination with other characters.For instance, the squamules of species in section Chlorophyllum are of type i, those of section Rhacodium are of type ii, while those of section Ellipsoidosporum and section Sphaerosporum are of type iii. 4. Colour, shape and size of basidiospores and presence/absence of germ pore.The basidiospores of Chlorophyllum vary from subglobose to globose without germ pore, ellipsoid without germ pore and amygdaliform to ellipsoid with large germ pore that causes the spore apex to be obviously truncated.The "ellipsoid without germ pore" shape is a conspicuous synapomorphy for the Ellipsoidospororum clade.Similarly, "subglobose to globose basidiospores without a germ pore" is characteristic of the Sphaerospororum clade, while all species in section Parvispororum have relatively small (less than 10 μm long) basidiospores with a truncate apex.Basidiospores can be hyaline or olive to greenish and this character can be used to separate certain clades: species within the Chlorophyllum clade and Endoptychorum clade may have olive to greenish basidiospores, while the remaining clades have hyaline basidiospores.

Conclusions and future directions
This study constitutes the first multigene molecular phylogenetic analysis of the genus Chlorophyllum.Previous analyses included only a limited number of ITS/nrLSU sequences.This study significantly increased the molecular sampling for this group and included a wider array of taxa from a broader geographic range.Several previously unsampled species were included (i.e. C. africanum, C. demangei, C. palaeotropicum, C. sphaerosporum and C. subrhacodes).Based on these results, the genus Chlorophyllum is monophyletic and composed of six well-supported monophyletic groups that were classified as sections (Figure 2).Each section is also characterised by several morphological features.Although the relationships amongst all sections are not yet fully resolved, relationships amongst species within sections are.The majority of Chlorophyllum species occurs in disturbed or arid habitats in subtropical to tropical regions and many species have a wide distribution over more than one continent.The role of humans in some of these distribution patterns should be investigated.
. In the descriptions of basidiospores, the abbreviation [n/m/p] indicates n basidiospores measured from m basidiocarps of p collections; (a)b-c(d) stands for the dimensions of the basidiospores, with b-c containing a minimum of 90 % of the measured values and (a) and (d) extreme values.Q is used to mean "length/width ratio" of a basidiospore and Qav represents average of Q of all basidiospores studied.

Figure 1 .
Figure 1.ML tree inferred from ITS data.Bootstrap values >50 % are indicated at internodes.Names of new taxa are in bold.

Figure 2 .
Figure 2. ML tree inferred from the combined alignment based on ITS, nrLSU, rpb2 and tef1.Bootstrap values >50 % are indicated at internodes.Names of new taxa are shown in bold.

Table 1 .
Taxa, vouchers, geographic origin, andGenBank accession numbers of DNA sequences of Chlorophyllum and outgroups used in this study.New sequences generated by this work are in bold.

sect. Parvispororum Z.W. Ge, sect. nov.
(Wilson 2017 small to medium-sized, agaricoid, covered with large squamules contrasting in colour with the background.Basidiospores relatively small (less than 10 μm long), with germ pore, forming a truncated apex.Cheilocystidia clavate to mucronate clavate.Pileipellis a palisade of hyphae with cylindrical terminal elements.Hyphae without clamp connections.Discussion.This section contains the species from south-eastern North America (C.subrhacodes) and east Asia (C.neomastoideum) displaying an America-Asia disjunct distribution.Differs from other sections by the stout basidiocarps with plate-like squamules on the pileus and white lamellae, basidiospores with wide germ pore and pileipellis composed of a tightly packed hymeniderm of cylindrical and flexuous, or narrowly clavate or narrowly lageniform elements.Chlorophyllum rhacodes (Vittad.)Vellinga,Mycotaxon83:416.2002.≡AgaricusrhacodesVittad.[as'rachodes'],Descr.fung.mang.Italia: 158.1835.Description.Basidiocarps medium to large sized, stout, agaricoid, with plate like squamules, basidiospores with wide germ pore, forming a truncated apex.Cheilocystidia clavate to sphaeropedunculate.Pileipellis a tightly packed hymeniderm of cylindrical and flexuous, or narrowly clavate or narrowly lageniform elements.Discussion.This section contains C. nothorhacodes, C. brunneum, C. rhacodes, C. olivieri and C. venenatum (Bon) C. Lange & Vellinga.There was controversy over the spelling of the species epithet 'rhacodes' (originally published as 'rachodes').The Nomenclature Committee for Fungi debated the issue for years and the General Committee made the final decision that the epithet of Agaricus rhacodes Vittad.(Descr.Fung.Mang.: 158.1833) is to be so spelled, even though it was originally spelled 'rachodes', which was approved by the International Botanical Congress in Shenzhen, China(Wilson 2017).

sect. Sphaerospororum Z.W. Ge, sect. nov.
Differs from other sections by the nonporous, globose to subglobose basidiospores and gasteroid basidiocarps or globose to subglobose basidiospores and a hymenodermal pileipellis made up of loosely arranged clavate to broadly clavate elements when the basidiocarps are agaricoid.Basidiocarps agaricoid or gasteroid, covered with inconspicuous squamules.Basidiospores subglobose to globose without germ pore (with rounded apex).Cheilocystidia (if present) clavate to broadly clavate.Pileipellis a hymeniderm made up of loosely arranged clavate to broadly clavate elements.Discussion.This section contains the agaricoid C. sphaerosporum and two hypogeous taxa, C. arizonicum and C. lusitanicum.It is so far the only clade containing gasteroid species.Recognition of two new species and the transfer of Lepiota demangei from Lepiota to Chlorophyllum Type.Chlorophyllum sphaerosporum Z.W. Ge & Zhu L. Yang, Mycotaxon 96: 187.2006.Description.