Short title: Two new species of Lactarius

Two new species of Lactarius associated with Alnus acuminata subsp. arguta in Mexico

Leticia Montoya1

Victor M. Bandala

Edith Garay

Biodiversidad y Sistemática, Instituto de Ecología, A.C., P.O. Box 63, Xalapa, Veracruz 91000,

Mexico

Abstract: In pure stands of Alnus acuminata subsp. arguta trees from Sierra Norte de Puebla

(central Mexico) two undescribed ectomycorrhizal species of Lactarius were discovered.

Distinction of the two new species is based on morphological characters and supported with

phylogenetic analyses of the nuclear ribosomal DNA ITS region and part of the gene that encodes

for the second largest subunit of RNA polymerase II (rpb2). The phylogenies inferred recovered

the two species in different clades strongly supported by posterior probabilities and bootstrap

values. The new Lactarius species are recognized as part of the assemblage of ectomycorrhizal

fungi associated with Alnus acuminata. Information about these taxa includes the morphological

variation achieved along 16 monitories 2010–2013. Descriptions are provided. They are

accompanied by photos including SEM photomicrographs of basidiospores and information on

differences between them and other related taxa from Europe and the United States.

Key words: ectomycorrhizal fungi, ITS, Neotropical fungi, rpb2, Russulaceae, Russulales

INTRODUCTION

According to Heywood (1993) Alnus trees occur worldwide with about 30 known species,

commonly recorded growing along the banks of streams, rivers and swamps, forming dense pure

stands, in wet floodplains or on moist mountain slopes (Russo 1990) and on hillsides, anchored

by their lateral and widespread root system. In Mexico, as in other areas, alder trees are valued

In Press at Mycologia, preliminary version published on June 3, 2014 as doi:10.3852/14-006

Copyright 2014 by The Mycological Society of America.

for timber, soil improvement and, according to Carranza-Gonzalez and Madrigal-Sánchez (1995),

in some regions for medicinal purposes. Members of A. section Alnus, such as A. acuminata

Kunth (with subsp. arguta [Schlecht] Furlow and subsp. glabrata [Fernald] Furlow) and A.

jorullensis H.B.K. (with subsp. jorullensis and subsp. lutea Furlow) are known in Mexico,

occurring along streams and rivers or establishing successional stages of development after

deforestation (Rzedowski 1978, Carranza-González and Madrigal-Sánchez 1995; CONABIO

www.conabio.gob.mx). Alnus acuminaa is recorded from Mexico to South America and,

according to Becerra et al. (2005), it reaches its southernmost distribution in the Catamarca

province of Argentina.

Field observations and pure-culture syntheses of ectomycorrhizal (ECM) fungi related to

Alnus species have suggested a marked specialization of this genus regarding its ectomycorrhizal

fungus associates (Molina 1981, Brunner et al. 1992, Polme et al. 2013, Roy et al. 2013); indeed

it is recognized that Alnus trees have a species-poor assemblage of ectomycorrhizal fungi (Pritsch

et al. 1997) recorded with about 50 fungal species (Rochet et al. 2011). Our knowledge of species

diversity that includes information about the sporocaps/ectomycorrizae counterparts associated

with Alnus trees has not been thoroughly investigated, however there is taxonomic evidence

based on both fruiting and/or mycorrhiza (the latter from molecular-based studies) that certain

species belonging to approx. 17 basidiomycetous and ascomycetous genera are consistently or

occasionally present as part of the dominant mycorrhizal community of alder forests (Froidevaux

1973; Miller et al. 1991, 1992; Pritsch et al. 1997; Dilly et al. 2000; Becerra et al. 2005a, b;

Tedersoo et al. 2009; Rochet et al. 2011). Relevant information on alder ECM communities

produced at regional and global scale, scrutinizing ECM root tips, based on the amplification of

ITS region, detected 86 molecular operational taxonomic units (MOTUs) of ECM fungi sampled

in five species of Alnus in France and Corsica (Roy et al. 2013) or up to 146 MOTUs of ECM

fungi from 22 Alnus species in 96 stands across the distribution range of Alnus, except North

Africa (Polme et al. 2013). Soil calcium concentration positively affects taxonomic richness of

ECM fungi in alder communities, according to Polme et al. (2013).

In Mexico Kennedy et al. (2011), using ITS and LSU rDNA gene sequences, identified 23

taxa among ECM root samples in four stands of Alnus trees sampled. Particularly in A.

acuminata stands they found an ectomycorrhizal fungal assemblage containing similar lineages

of nine of the 17 aforementioned genera that includes Lactarius, as well as new associates

belonging to Clavulina and Sebacinaceae. While other Alnus species in the Americas host some

members of Lactarius that include one related to the European L. obscuratus (Lasch: Fr.) Fr.

(Miller et al. 1991, 1992; Brunner et al. 1992; Pritsch et al. 1997; Kennedy and Hill 2010), in

Mexico Alnus acuminata is recorded in ectomycorrhizal association with two unidentified

Lactarius species (Kennedy et al. 2011) and in Argentina, also with an unidentified Lactarius

species (Becerra et al. 2005a, b) and with L. omphaliformis Romagn. (Becerra 2005a, b; Pritsch

et al. 2010). From Argentina Singer (1961) described Lactarius alni Singer a putative ECM

species of Alnus jorullensis and probably contaxic with L. obscuratus.

The present work is based on the study of Lactarius basidiomes recorded in two pure

stands of Alnus acuminata subsp. arguta at Sierra Norte de Puebla (central Mexico) where

weekly explorations were performed over 3 mo during each year, 2010–2013. The patterns of

morphological variation observed in the samples together with the molecular information, as

revealed by the ITS and rpb2 sequences, support recognizing that the specimens represent two

new Lactarius species that are described here. According to macro- and micromorphological

characters and molecular data, one of the new species is closely related to the group of species

around L. obscuratus and the other to L. lilacinus (Lasch) Fr., which are ectomycorrhizal obligate

partners of Alnus trees in European forests.

MATERIALS AND METHODS

Sampling.—Basidiomes of Lactarius were gathered during 16 weekly visits conducted Aug–Oct in four consecutive

years (2010–2013). The stands of Alnus acuminata subsp. arguta are situated in Cuacuilco village hills, W of

Zacapoaxtla, appox. 5 km from Actopan, Zaragoza-Zacapoaxtla Road, Puebla, at 97°32′37″W, 19°51′04″N, at 2280

m. Collections are kept in XAL Herbarium.

Morphological and color study.—Macroscopic features of basidiomes and their colors were recorded in fresh

condition. Colors were described with the notations from Kornerup and Wanscher (1967) (e.g. 6C7) and Munsell

color charts (1994) (e.g. 7.5 YR 6/6). Micromorphological study of basidiomes was carried out on dried specimens,

rehydrated in 3% aqueous potassium hydroxide solution, except basidiospores, which were analyzed in Melzer's

reagent. We followed the protocol of Montoya and Bandala (2003) to calculate spore size ranges. Thirty

basidiospores per collection were measured (length and width), and the results are given in the descriptions as the

range of values and with the symbol Xm, the interval of mean values per collection. Q represents the basidiospore

length/width ratio and is given as an interval of mean values. Line drawings were made with the aid of a drawing

tube. Terminology is according to Hesler and Smith (1979). The specimens studied were compared with types of

Lactarius oculatus (Lasch) Fr. and L. occidentalis A.H. Sm. Acronyms for herbaria follow Holmgren and Holmgren

(1998).

DNA extraction, PCR amplification and sequencing.—Genomic DNA was extracted from fruit bodies with the

DNAeasy Plant Mini Kit (QIAGEN, Hilden, Germany) as recommended by manufacturer. PCR was performed to

amplify the internal transcribed spacer 1, 5.8S, internal transcribed spacer 2 and part of large subunit of 28S

ribosomal RNA gene, using primers ITS1F, ITS5/ITS4, LR21 (White et al. 1990, Gardes and Bruns 1993). Regions

6 and 7 of nuclear genes that encode the second largest subunit of RNA polymerase II (rpb2) were amplified with

primers bRPB2 6f/fRPB2 7CR (Liu et al. 1999, Matheny 2005). PCR conditions: (i) initial denaturation at 94 C for

3 min; (ii) 35 cycles of 1 min at 94 C, 1 min at 55 C, and 2 min at 72 C; and (iii) a 7 min final elongation at 72 C.

Amplified PCR products were purified with the DNA Clean & Concentrator Kit, (Zymo Research, USA), as

recommended by manufacturer. Cycle sequencing reactions were made with

BigDye Terminator 3.1 Cycle Sequencing kit (Applied Biosystems, USA); reactions were purified with ZR DNA

Sequencing Clean-up Kit (Zymo Research, USA), and run in a sequencer, ABIPrism 310 Genetic Analyzer (Applied

Biosystems). Once sequences were assembled and edited, they were deposited at GenBank

(http://www.ncbi.nlm.nih.gov), with accession numbers (SUPPLEMENTARY TABLE I).

Phylogenetic methods.—Sequences were aligned with MUSCLE (EMBL-EBI, 2013), including those from different

Lactarius species related to Mexican samples, downloaded from GenBank (http://www.ncbi.nlm.nih.gov/) with the

aid of the basic alignment search tool (BLAST) (Altschul et al. 1990) and sequences considered by Rochet et al.

(2011). The aligned datasets (rDNA ITS and rpb2 gene sequences) later were verified and refined manually. To

detect possible incongruence between the markers, in terms of conflicting well supported clades, we reconstructed a

phylogeny for each locus under Bayesian inference (BI) and maximum likelihood (ML) criteria. BI analyses were

performed with MrBayes 3.2.1 (Ronquist and Huelsembeck 2003); the substitution model for each matrix was

selected previously with jModel Test 0.1.1 (Posada 2008) under Akaike information criterion (AIC). The analyses

were performed with two independent runs. For each run we employed four chains for 10 000 000 generations,

sampling one tree every 1000 generations. Sample points collected before stationarity (convergence of likelihood

scores) were eliminated as burn-in (10%). Posterior probabilities for supported clades were determined by a 50%

majority-rule consensus of the trees retained after burn-in. For ML analyses of each locus, the GTR+G model

parameters were used with GARLI 2.0 (Zwickl 2006), applying default settings; nonparametric ML bootstrap

analyses were run for 1000 replicates. Bayesian posterior probabilities (BPP) 0.95 or greater and bootstrap values

(BS) of ML analyses of 70% or greater were considered significant in each. Because the phylogenies of each marker

did not present discordance among topologies and internal nodes with significant BPP or BS, a combined (ITS+rpb2)

dataset was generated in PhyDE 0.995 (Müller et al. 2006) and used to conduct final BI and ML analyses (with

settings indicated above) and deposited in TreeBASE (accession number S15024). Lactarius romagnesii Bon, L.

ruginosus Romagn. and L. pterosporus Romagn., three members of L. subg. Plinthogali, were included as outgroup

for all analyses. The phylogenies from BI and ML analyses were displayed with FigTree 1.3.1 (Rambaut 2009).

RESULTS

The combined (ITS and rpb2) dataset produced in this study included 29 sequences

(SUPPLEMENTARY TABLE I), of which 14 were newly generated (seven from ITS and seven from

rpb2) and 15 were taken from GenBank. The phylogeny inferred after Bayesian analysis from the

combined dataset is illustrated (FIG. 1), which includes the posterior probabilities and also the

bootstrap values obtained after maximum likelihood reconstruction. In the molecular phylogeny,

as well as on separated gene phylogenies (not shown), Mexican samples formed two strongly

supported clades. Morphologic distinctive features of the specimens also support the recognition

of two separate species. In the phylogeny, the former species appearing as sister group of

Lactarius lilacinus, classified provisionally in L. subgenus Piperites (Fr.) Kauffman, while the

samples of the second species grouped with the European L. obscuratus, L. cyathuliformis and L.

brunneohepaticus, which belong to L. subgenus Russularia (Fr.) Kauffman. Based on these data

we concluded that Mexican specimens represent two new species that are proposed here.

TAXONOMY

Lactarius cuspidoaurantiacus Montoya, Bandala & Garay, sp. nov. FIGS. 2–4

MycoBank MB806123

Diagnosis: Pileus 5–35 mm diam, conical to convex and plane-depressed with central sharp

papilla, brick-orange, pale orange-salmon to pale buff-orange, margin pubescent when young.

Stipe 17–65 × 2–10 mm. Lamellae distant, with veins at sides and at interlaminar spaces. Latex

milky to aqueous. Basidiospores (6–)6.5–9(–9.5) × (5–)5.5–7 µm, subreticulate. Pleuro- and

cheilomacrocystidia subcylindrical, fusoid or mucronate. Pileipellis trichodermoid.

Pileus 5–35 mm diam, conical with sharp papilla at very young stages, becoming convex, convex

to plano-depressed, at times irregular or eccentric, bearing a central papilla or at times in mature

specimens faintly umbonate, hygrophanous when wet but in general dry and dull; central area

dark amber-orange or ochraceous orange (6C7; 7.5YR 6/6); the remaining area orange (5AB5),

orange-brown (6C7-8) with faint brick orange in humid conditions or in immature specimens

(6C6, 7.5 YR 6/6, 5/8, 8/4) but not reddish or violaceous, fading when drying to pale orange-

salmon (6A6), pale buff orange (5A2, 5A4-5, 7.5 YR 7/6) or buff with pale orange tinges (7.5

YR8/4) and keeping dark brick orange (7.5 YR 6/8, 6C8) tinges in the center and orange-brown

(7.5 YR 6/8) in the papilla; azonate, finely rugulose when young, velutinous to finely tomentose,

becoming faintly areolate as it expands but in more mature specimens with the surface layer

breaking and acquiring an irregular areolate appearance especially in the center or in all the

surface; such areoles conserving the orange tinges even when drying and at times appearing as

squamules; margin involute when young, becoming decurved or straight with age, variably wavy

or irregular, at times faintly crenate, tomentose when young, with pale orange hairs when

young. Lamellae distant, thick, broad (0.5–3.5 mm wide), subdecurrent, arcuate, with irregular

margin, frequently forked toward the stipe attachment, at sides of lamellae or at interlaminar

spaces with evident anastomosed veins at times, giving a venose-alveolate appearance in mature

specimens, then surface below the pileus venose or rugose, 1–3 or up to 4 lamellullae between

two lamellae, pale flesh orange or orange (7.5YR 7/6; 5A3-4) or grayish ochraceous orange

(7.5YR 7-8/4, 7/6). Stipe 17–65 × 2–10 mm, subcylindrical or at times broadened toward the

base, other sinuous or curved, central or eccentric, smooth, single or fasciculate, dry, matted

fibrillose to innately fibrillose, finely pubescent to tomentose, the tomentum more conspicuous

when immature and present at apex and base where it is more dense and conspicuous, buff-

orange (6A4-B5, 5A5-6, 7.5 y 8/4-6, 7.5 YR 7/8-6), at times darker (6C7-8, 6B5-6), fistulous in

mature specimens, in young stages filled with mycelium or more or less compact, base at times

with strigose hairs. Context buff (5A2-3, 5B2, 5B5, 6B3), at times with orange-flesh tinges; odor

Pelargonium-like, agreeable. Latex milky white to serous, at times staining white paper yellow,

sticky, mild or slightly acrid. KOH grayish on context and pileus and stipe surfaces.

Basidiospores (6–)6.5–9(–9.5) × (5–)5.5–7 µm, Xm = 7.7–8.1 × 6.3–6.4 µm, Q = 1.22–

1.23, broadly ellipsoid, subreticulate with short warts in the angles of the reticulum and isolated

verrucae or ridges; ornamentation up to 0.8 µm high, plage partly amyloid. Basidia 43–52 × 8–10

µm, clavate, sometimes sinuous, tetrasporic. Pleuromacrocystidia 66–90 × 7–11 µm,

subcylindrical, sinuous; apex fusoid, rounded or at times mucronate. Cheilomacrocystidia 48–73

× 6-8.5 µm, subcylindrical, attenuate toward the apex. Pileipellis a layer 360–420 µm thick,

composed of short hyphae that are 5–12 µm broad, intermixed; trichoderm-like patches frequent,

formed by projected chains of elements, ending in subcylindrical, subclavate or ventricose

terminal elements of 16–48 × 3.5–8.5 µm; intercalar elements in the chains subcylindrical 5–12

µm broad or at times versiform, even furcate, and other somewhat inflated, 10–42 × 9.5–21.5 µm;

in some mounds it is possible to observe (in periclinal arrangement or covering the mounds) thin

cylindrical terminal elements, which are 10–38 × 3.5–6 µm; elements pale yellowish in KOH or

yellowish brown in distilled water. Pileus trama with hyphae 1.5–5 µm diam, laticifers 3.5–8.0

µm diam, sphaerocytes 10.5–17.5 µm diam. Hymenophoral trama composed of hypha of 1.5–3

µm diam and laticifers 1–1.5 µm diam.

Holotype: MEXICO. PUEBLA, MPIO. ZACAPOAXTLA, Cuacuilco, growing in

fascicles, on soil, in ectomycorrhizal association with Alnus acuminata subsp. arguta trees,

11 Oct 2010, Montoya 4809 (XAL).

Habit, habitat and distribution: Gregarious, commonly growing in fascicles, in

ectomycorrhizal association with Alnus acuminata subsp. arguta trees.

Etymology: Referring to both pileus shape and color.

Specimens examined: MEXICO. PUEBLA, MPIO. ZACAPOAXTLA, Cuacuilco, 11 Oct 2010, Montoya

4806; 11 Oct 2010, Montoya 4808; 11 Oct 2010, Montoya 4809 (Holotype); 11 Aug 2011, Montoya 4823; 19 Sep

2011, Montoya 4828; 30 Sep 2011, Ramos 471; 21 Aug 2012, Montoya 4893; 29 Aug 2012, Montoya 4900; 20 Sep

2012; Montoya 4908; 1 Oct 2012, Montoya 4918, 1 Oct 2012, Montoya 4925; 8 Aug 2013, Montoya 5059; 21 Aug

2013, Montoya 5070; 28 Aug 2013, Montoya 5059; Oct 15 2013, Montoya 5100 (all at XAL). Other specimens

examined: USA. New York, North Elba, Peck s.n., September (Holotype of L. subdulcis var. oculatus, NYS).

Comments: The species is characterized by the basidiome colors, the pileus shape and the

thick and distant lamellae (frequently anastomosed with veins); microscopically by basidiospore

size and ornamentation. The molecular analysis suggests that Lactarius cuspidoaurantiacus is

most closely related to L. lilacinus, a European species strictly associated with Alnus trees

(Heilmann-Clausen et al. 1998, Basso 1999). Lactarius lilacinus differs by more robust and

pinkish lilaceous color. The pileus in L. lilacinus can be pale brownish rose or light vinaceous

gray (Heilmann-Clausen et al. 1998) or pink vinaceous, pinkish purple, pinkish lilaceous with

squamules dark purple violaceous (Basso 1999). The pileus is 15–50(–120) mm or 25–80(–140)

mm and stipe 15–50(–70) × 3–12(–25) mm or 35–55 × 8–20 mm. Furthermore the pileus is not

sharply conical or papillate when young as in Mexican collections and the lamellae are medium

crowded to distant, these and pileus are more pinkish lilaceous. The macroscopic characters are

very different between these two taxa, while the basidiospores, according to the authors, present a

more or less similar variation, being 6.4–9.2 × 5.3–6.9 µm, Xm = 7.7–8.2 × 6–6.4 µm, (Q = 1.15–

1.45) (Heilmann-Clausen et al. 1998) or 6.7–8.5(–9) × 6–7 µm (Basso 1999) but distinguished by

having a more or less complete reticulum. The pileipellis in Mexican species is frequently present

in hyphal mounds and the elements lack brown violaceous vacuolar pigments as those mentioned

in L. lilacinus by Basso (1999).

Lactarius oculatus, an American species described by Hesler and Smith (1979), could be

related to the Mexican taxon but is distinguished by its pileus “… tacky (subviscid) and shiny,

soon dry but not areolate … dark reddish brown to dark or medium reddish cinnamon over and

around papilla or over disc (if papilla absent) and marginal area a dingy vinaceous pink or (faded)

dingy vinaceous buff …”, and as observed in the type specimen, it possesses larger

basidiospores (7.5–)8–10.5 × 6–7(–7.5) µm, having a more disarticulated ornamentation pattern,

consisting mostly of isolated warts and some ridges (FIG. 4e–f), scarce and shorter

pleuromacrocystidia (35.5–75 × 8–10 µm), the pileipellis slightly gelatinous and lacks veins

among lamellae and naked stipe. Moreover, according to Hesler and Smith (1979), L. oculatus

grows in association with conifers.

Lactarius herrerae Montoya, Bandala & Garay, sp. nov. FIGS. 5–7

MycoBank MB806124

Diagnosis: Pileus 6–27 mm diam, convex, papillate, dark brown with reddish orange tones,

developing olivaceous tinges, margin striate. Stipe 3–26 × 1–2.5 mm. Lamellae close to

subdistant, adnate, ventricose, pinkish salmon, pale flesh orange. Latex aqueous to whitish.

Basidiospores 7–10 × 6.5–8 µm, almost completely reticulate. Pleuro- and cheilomacrocystidia

fusoid. Pileipellis an epithelium with terminal elements clavate, sphaeropendiculate or somewhat

narrowly utriform.

Pileus 6–27 mm diam, convex to plane-convex, to slightly centrally depressed, frequently

papillate when immature to broadly umbonate or faintly papillate or without papilla when mature,

with decurved margin, finely rugulose, at times wrinkled in the center when young, brown (5F8,

6B6, 6F6, 6F8; 5YR3/3, 2.5YR 2.5/3, 7.5YR 2.5/2-3/4), brown with orange tinges (6D7, 6E8),

dark brown toward the center (7.5YR 2.5/2-3/3, 5YR 2.5/2-2.5/3; 6F7-8) and orange-brown

(6C6-7) in the remaining area or even paler toward the margin, hygrophanous, with desiccation

developing olivaceous tinges, greenish gray (1C3, 3C3-4) and finally fading to grayish buff-

brown (5CD5; 10YR 6/3) or whitish gray, surface faintly pruinose especially when young;

margin pruinose to tomentose and decurved when young to straight when mature, at times

lobulate, faintly striate or sulcate to translucently striate when expanded. Lamellae 1–4 mm

broad, ventricose, thick, close to subdistant, adnate but not decurrent, frequently becoming

detached from the stipe and leaving a remaining tooth, with regular and straight edge, pinkish

salmon to pinkish gray (5YR 6-7/4, 7/6, 8/3-4; 5YR 6/4, 7/6, 7/4, 8/3-4; 7.5 YR 8/6, 7/8, 7/6, 6/8;

6B3-4; 5A3-4, 6A4) (no yellowish tinges observed), with pale pinkish tinges (6A3), pale flesh

orange (5A3-6A3) from side view, with lamellulae of 5–7 sizes, with 2–3 lamellulae between two

lamellae, some nearly reaching the stipe attachment. Stipe 3–26 × 1–2.5 mm, subcylindrical,

central or eccentric, smooth, dry, dull, subpruinose or finely pubescent especially at apex and all

over when young, solid or hollow in parts, orange-brown (5YR 4-5/6, 7.5YR 6/6-5, 8-6/8; 6C7)

to brick or deep brown-orange (5YR 5/6) or flesh-orange (6B4, 6C5, 6B6), paler than pileus; base

with white tomentum. Latex serous to whitish, mild or slightly peppery. Context of the stipe

compact, dark brown (6E8) below the pileus cuticle, orange-brown (6D8) in the rest, pale flesh

orange (5A2-A3) in the stipe, tending to fistulose in the stipe with age; odor sweet, somewhat

Pelargonium-like; flavor mild. Spore deposit whitish. KOH negative.

Basidiospores 7–10 × 6.5–8 µm, Xm = 8.3–8.6 × 7–7.2 µm, Q = 1.18–1.22, broadly

ellipsoid, subreticulate to almost completely reticulate, with some isolated verrucae and ridges;

ornamentation 1–1.2 µm high. Basidia 39–60 × 9–14 µm, clavate, bi- and tetrasporic.

Pleuromacrocystidia 30–106 × 4–13 µm, fusoid, ventricose, with apex enlarged and acute, arising

from the trama or from the hymenial layer, thin-walled, abundant. Cheilomacrocystidia 35–52 ×

5–7 µm, fusoid, some subventricose, with apex enlarged and acute, thin-walled, scarce. Pileipellis

an epithelium composed of a cellular layer that is 70–90 µm thick; terminal elements clavate,

sphaeropendiculate or somewhat narrowly utriform, 16–23 × 8–11 µm, originating from

subisodiametric, ovoid or subellipsoid cells, which are 14–25 × 7–16 µm, thin-walled, pale

yellowish brown in KOH. Context with sphaerocytes of 30–40 µm diam, laticifers 2–8 µm diam

and hyphae 2.5–13 µm diam. Hymenophoral trama composed of hyphae 4–6 µm diam and

laticifers 3–7 µm diam.

Holotype: MEXICO. PUEBLA, MPIO. ZACAPOAXTLA, Cuacuilco, growing in groups,

on soil, in ectomycorrhizal association with Alnus acuminata subsp. arguta trees, 11 Oct 2010

Montoya 4811 (XAL).

Habit, habitat and distribution: Gregarious, on soil in ectomycorrhizal association with

Alnus acuminata subsp. arguta trees.

Etymology: dedicated to Dr Teófilo Herrera a leader and beloved friend of Mexican

mycologists, on occasion of his 90th birthday.

Specimens examined: MEXICO. PUEBLA, MPIO. ZACAPOAXTLA, Cuacuilco, 7 Sep 2010, Ramos 365;

11 Oct 2010, Montoya 4810; 11 Oct 2010, Montoya 4811 (Holotype); 11 Aug 2011, Montoya 4820; 19 Sep 2011,

Montoya 4825; 5 Oct 2011, Ramos 475; 21 Aug 2012, Montoya 4892; 29 Aug 2012, Montoya 4899; 12 Sep 2012,

Bandala 4533; 20 Sep 2012, Montoya 4910; 1 Oct 2012, Montoya 4917; 9 Oct 2012, Bandala 4582; 21 Aug 2013,

Montoya 5071 (all at XAL). Other specimens examined: USA. Washington: Smith 2559, Lactarius occidentalis

(Holotype, MICH).

Comments: Mexican collections are characterized by pileus color variation (dark brown

with reddish orange tones and developing olivaceous tinges after desiccation), broad and adnate

lamellae, which commonly become detached from the stipe, subreticulate basidiospores, fusoid

macrocystidia and the pileipellis structure, which is epithelioid with terminal elements clavate,

sphaeropendiculate or somewhat narrowly utriform. Lactarius herrerae represents a new taxon

related to L. cyathuliformis Bon, L. brunneohepaticus Moser and L. obscuratus, which represent

a group of European species associated with Alnus trees and in general terms phenotypically

similar. The Mexican species can be distinguished from L. brunneohepaticus because in the latter

species the latex stains lemon yellow (Moser 1978, Basso 1999). In L. cyathuliformis the milk

stains yellow on white tissue (Heilmann-Clausen et al. 1998). While the basidiomes of L.

herrerae are brown with orange tinges and develop olivaceous tinges slowly after collection as a

result of lost humidity, the European taxa have the olivaceous tinges already present in the fresh

condition, often disappearing later (Moser 1978, 1983; Heilmann-Clausen et al. 1998; Basso

1999). Additionally in L. brunneohepaticus the macrocystidia are shorter, macrocystidia 55–70(–

80) × 10–11 µm (Moser 1978), pleuromacrocystidia 60–85 × 7–11 µm (Basso 1999). Regarding

L. cyathuliformis, this presents bigger basidiomes (12–45 mm broad), becoming increasingly

funnel-shaped with age (Heilmann-Clausen et al. 1998), lamellae slightly decurrent and with a

bigger basidiospore, 9–12 × 7–9 µm (Bon 1978, Basso 1999). In contrast to L. herrerae, in L.

obscuratus the basidiospores are shorter, Xm = 7.1–8.1 × 5.7–6.3 µm or (6–)6.5–8(–8.5) × (5–

)5.5–6 µm, the pileipellis consists of broader, more subisodiametric cells, 30(–35) µm broad, the

pileus becomes infundibuliform and the lamellae are slightly decurrent to decurrent (Basso 1999)

and even narrower (2–2.5 mm broad) (Heilmann-Clausen et al. 1998). Lactarius obscuratus var.

radiatus (Lange) Romagn. and L. obscuratus var. subalpinus Basso were discussed by Basso

(1999); unlike L. herrerae, these differ by lacking olivaceous tinges, the variety radiatus having a

pure orange pileus and the variety subalpinus a pinkish brown pileus, cyathiform habit, bigger

pileus (23–50 mm diam) and shorter pleuromacrocystidia (61.5–93.5 × 5–10 µm) than the

Mexican taxon.

Among the American taxa, Lactarius occidentalis could be considered as being related to

the Mexican taxon, but Hesler and Smith (1979) reported it with bigger basidiomes (pileus 10–35

mm broad, stipe 30–60 × 5–6 mm), flesh slowly becoming reddish brown where cut and latex

usually changing to yellow on exposure. Based on type observations, L. occidentalis is

distinguished also by its narrower basidiospores, 7–9(–9.6) × 5.4–6(–6.6) µm, Xm = 8.3 × 5.8

µm, Q = 1.41, with somewhat higher (0.8–2 µm) and more disarticulated ornamentation pattern,

with oblique hilar appendage and shorter pleuromacrocystidia (40–60 × 6–12 µm).

DISCUSSION

The set of morpho-anatomical features of the two newly discovered species in ectomycorrhizal

association with Alnus acuminata subsp. arguta at the study site relate them to the group of

species within the lineages of Lactarius lilacinus and L. obscuratus, which have been recognized

as part of the ectomycorrhizal fungal community of Alnus in different areas, especially Europe.

Lactarius cuspidoaurantiacus and L. herrerae increase the number of the few known mycorrhizal

partners of Alnus. These findings indicate that also in the eastern extreme of the Mexican

neovolcanic axis, the close ectomycorrhizal relationship between alders and Lactarius of the

mentioned lineages is retained in congruence with the observation by Tedersoo et al. (2009) that

alders associate with a relatively restricted set of ectomycorrhizal fungi, which are highly host

specific.

The phylogenetic reconstruction performed with ITS and rpb2 sequences strongly

supports the separate positions of the two Mexican Lactarius species within the two lineages

(FIG. 1). The results, incorporating Lactarius cuspidoaurantiacus and L. herrerae to the

phylogenetic analysis of alnicolous species of Lactarius, are consistent with the relationships

found by Rochet et al. (2011), who said Alnus species are associated with specific fungal

lineages, in the case of Lactarius, to specific taxa belonging to subgenus Piperites and

Russularia. In the present study the same arrangement was displayed, with Lactarius

cuspidoaurantiacus clustered with species of subgenus Piperites and L. herrerae with species of

Russularia. Within the phylogenetic arrangement, Lactarius herrerae represents another member

of the obscuratus lineage recognized by Rochet et al. (2011), which includes the European L.

obscuratus, L. cyathuliformis and L. brunneohepaticus. These two new species represent good

examples of the specialist strategy mentioned by Roy et al. (2013), in which an ECM fungus may

associate with hosts from a single lineage and that this Alnus–ECM fungi mutualism is highly

conserved at a large geographical scale, which also is congruent with the information recovered

by Polme et al. (2013) in their biogeographic study of Alnus ECM community. They found that

intrageneric phylogenetic relations among host plants and regional processes largely account for

the global biogeographic distribution of Alnus-associated ECM fungi. After a comparison of

phylogenies of European alnicolous species of Lactarius and Alnus trees of different sections,

Rochet et al. (2011) suggested that L. lilacinus underwent phylogenetic speciation with A.

glutinosa and A. incana, two members of section Alnus. The ectomycorrhizal association now

recognized in Mexico between L. cuspidoaurantiacus and A. acuminata subsp. arguta, this latter

being grouped in the mentioned section, could reveal additional data of possible speciation of this

Lactarius lineage and alders of section Alnus.

ACKNOWLEDGMENTS

We thank the curators of NYS and MICH herbaria for loan of specimens. We appreciate the collaboration in the

monitoring of Lactarius in the field to Bióls. M. Soto, J.M. Álvarez and G. Valerio and assistance in the laboratory

by Bióls. D. Ramos and J.C. Corona. We are especially indebted to Dr Eduardo Ruíz (Instituto de Ecología A.C.) for

the revision and important suggestions during the development of this phylogenetic treatment.

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LEGENDS

FIG. 1. Phylogenetic relationships within Lactarius species inferred from the combined ITS region and part of RPB2

nuclear gene sequence data. Bayesian tree, including the posterior probability (only values ≥ 0.95 are indicated) and

bootstrap values (only ≥ 80% are indicated).

FIG. 2. Lactarius cuspidoaurantiacus basidiomes. a. Montoya 4821. b. Montoya 4823. c, d. Montoya 4808. Bars =

10 mm.

FIG. 3. Lactarius cuspidoaurantiacus (Montoya 4809, holotype). a. basidiospores. b. pleuromacrocystidia. c.

basidia. d. cheilomacrocystidia. Bars = 10 µm.

FIG. 4a–d. Lactarius cuspidoaurantiacus (Montoya 4809 holotype). a–b. pileipellis. c, d. basidiospores under SEM.

e. f = L. oculatus (Peck s.n. holotype) basidiospores under SEM. Bars: a = 50 µm, b = 10 µm, c–f = 2 µm.

FIG. 5. Lactarius herrerae (Montoya 4820) basidiomes. Bar = 10 mm.

FIG. 6. Lactarius herrerae (Montoya 4811, holotype). a. basidiospores. b. pleuromacrocystidia. c. basidia. d.

cheilomacrocystidia. Bars = 10 µm.

FIG. 7. a–c. Lactarius herrerae (Montoya 4811, holotype). a. pileipellis. b–e. basidiospores under SEM. Bars: a =

50 µm, b–e = 2 µm.

FOOTNOTES

Submitted 13 Jan 2014; accepted for publication 29 Apr 2014.

1Corresponding author. E-mail: leticia.montoya@inecol.mx