Field collections made by the authors were dried overnight on the day they were collected. Prior to drying, a fragment was stored in silica gel for later DNA extraction and spores were deposited on malt extract agar (MEA) plates to obtain cultures. Collections were accessioned into TENN, cultures into CULTENN.

Establishment of single basidiospore isolates (SBIs) and pairing experiments were performed as described in Gordon and Petersen (1992). All SBIs were examined microscopically to determine monokaryon status before mating experiments.

Macromorphological characters observed were stipe length, stipe vesture, color of living and dried material, and lamella structure (distance between lamella, number, narrow vs. broad, attachment). “Complete” lamellae (those which extended from pileus margin to attachment juxtaposed to stipe) were interspersed with numerous lamellulae, usually of at least two and occasionally three ranks. The most accurate assay counted all lamellae (and lamellulae) which reached the pileus margin. This tally was performed by counting lamellae for approximately ¼ pileus circumference and multiplying by four.

Basidiospore statistics were gathered for “European” vs. “American” collections. Two spore metrics were especially examined: Q^m (median ratio of spore length to width) and L^m (median spore length). Pileipellis hyphae were examined for presence of short side branches reported by Antonín and Noordeloos (2010). Terminal cell shape of these side branches was noted. Cheilocystidia vary within subg. Vestipedes, and were evaluated for G. confluens in both size and shape (i.e.non-strangulate to strangulate).

DNA was extracted from dried specimens and/or cultures, and ITS and LSU sequences were amplified and sequenced as described in Hughes et al. (2013). Sequences were deposited in GenBank (Table 1). Sequences were manually aligned using GCG (2000). PhyML with 100 bootstrap replicates was performed in Geneious V9. Gymnopus eneficola (Petersen et al. 2014) was selected as the outgroup because it is the most closely related species and is sister to G. confluens. G. eneficola is often mistaken for G. confluens in the field. Trees were visualized in TreeView (Page 1996) and deposited in Dryad (Petersen and Hughes 2015).

Several species-delineation metrics including Rosenberg’s P_AB statistic (Rosenberg 2007), P_ID (strict) and P_ID(liberal) (Ross et al. 2008), P_RD (Rodrigo et al. 2008) and PTP (Zhang et al. 2013). P_AB, P_ID (strict), P_ID (liberal) and P_RD were implemented in Geneious V9 (Geneious 2005; Masters et al. 2011). PTP was implemented at http://species.h-its.org/ using web default settings for generations (100,000) burn-in (0.1, MCMC convergence was reached) and thinning (100). Rosenberg’s P_AB statistic is the probability that a putative species will be monophyletic with respect to a sister clade under the model of random coalescence. The null hypothesis is that monophyly is a chance outcome of random branching. The P_ID statistics provide the frequency with which a member of a putative species can be correctly identified given a specific alignment of sequences. P_ID(strict) requires that an unknown specimen falls within but not sister to the species clade. P_ID(liberal) requires that an unknown specimen falls either sister to or within the species clade. P_RD (Probability Randomly Distributed) is the probability that a clade has the observed degree of distinctiveness due to random coalescent processes. A probability value less than 0.05 rejects the null hypothesis of random coalescence and suggests that the clade is a cryptic species. PTP (Poisson Tree Processes; Zhang et al. 2013) estimates the number of species using both maximum likelihood and Bayesian approaches.

NORTH AMERICA, CANADA, New Brunswick, Fundy National Park, vic. Alma, Caribou Plains Trail, 45°38.59'N, 65°06.94'W, 25.IX.2013, coll Stephen Clayden, TFB14409 (TENN-F-69073); Fundy National Park, vic. Alma, Maple Grove Backroad, 45°35.34'N, 64°59.014'W (stop 1), 24.IX.2013, coll. Unknown, TFB 14389 (TENN-F-69053); Newfoundland, Moccasin Lake, Abitibi Trail, 10.ix.2008, coll. Maria Voitk, mixed woods, MS4-009 (TENN-F-69133). UNITED STATES, New York, Tompkins Co., Ringwood Preserve, 42°27.03'N, 76°21.80'W, 4.IX.2013, coll TJ Baroni & RHP, TFB 14357 (TENN-F-69006); Tompkins Co., vic. Dryden, Ringwood Preserve, 42°28.11"N, 76°19.06"W, 13.IX.1984, leg. & det. R.E. Halling, REH no. 3851 (dupl. NY), TENN-F-47030; North Carolina, Macon Co., vic. Highlands, Shortoff Mt. area, 35°05.47'N, 083°11.25'W, 18.VII.1994, coll. J. Johnson, TFB7219 (TENN-F- 53522)[annot. R.E.Halling as C. confluens]; Tennessee, Sevier Co., GSMNP, Rainbow Falls Parking Area, 28.VII.1989, coll RHP (as Collybia ?acervata), TFB 2033 (TENN-F-48376); Sevier Co., vic. Gatlinburg, GSMNP, “Mt. LeConte,” 8.VIII.1941, coll L.R. Hesler & S.L. Meyer, LRH 13883 (TENN-F-13833); Virginia, Smyth Co., vic, Sugar Grove, Mt. Rogers National Recreation Area, Appalachian Trail, 8.VIII.2008, coll. P.B. Matheny, PBM 2991 (TENN-F-63806).

EUROPE, BELGIUM, Domain Massembre vic. Hevre, 50°09.62’ N, 4°51.48'E, 7.IX.2010, coll RHP, TFB13754 (TENN-F-65131). FINLAND, Hämeenlinna, Torronguo National Park, 60°44.32'N, 23°38.63'E, 8.VIII.2002, coll. J.L. Mata, TFB 11055 (TENN-F-59469). FRANCE, Rhône-Alpes, Dpt. Savoie (73), commune St. Germain laChambotte, 45°46.62'N, 5°53.07'E, 10.IX.2001, RHP & Pierre-Arthur Moreau, TFB11335 (TENN-F-59219). GERMANY, Thuringia, vic. Menteroda, 51°18.04'N, 10°31.44'E, 28.VIII.12, coll RHP 2012, TFB14132 (TENN-F-67882); Thuringia, vic. Schlotheim by Pöthen, 51°16.90'N, 10°33.434’ E, 27.VIII.2012, coll. RHP, TFB14115 (TENN-F-67865). RUSSIA, Leningrad Reg., vic. Lodeynoe Pole, 60°41.70'N, 33°17.98'E, 30.VIII.1999, coll RHP, TFB10650 (TENN-F-58239); Novgorod Region, Valdai District, National Park Valdaiski, vic. Road to National Park, 57°57.88'N, 33°19.32'E, 19.VIII.2003, coll. RHP, TFB 11615 (TENN 59578); Valdai National Park at resort, 58°00.511’ N, 33°21.543’ E, 22.VIII.2003, coll. RHP, TFB11641 (TENN-F-59603); Samara Region, Bakhilovo district, vic. Bakhilovo, Shiryayeva Valley, 53°24.30'N, 49°55.03'E, 17.VIII.2004, coll. RHP, TFB12171 (TENN-F-60109). SWEDEN, Uppland, vic. Uppsala, Gottsundaborgen, 59°48.8'N, 17°37.40'E, 7.IX.1994, coll Svenngunnar Ryman, TFB7262 (TENN-F-53546). SWITZERLAND, Graubunden, Chur, Lenzerheide, 46°40.17'N, 9°38.97'E, 19.IX.2001, coll. E. Horak, TFB11400 (TENN-F-59285).

North America — AK=USA, Alaska, GSMNP=USA, Great Smoky Mountains National Park (TN or NC); NB=Canada, New Brunswick; NC=USA, North Carolina; NL=Canada, Newfoundland; TN=USA, Tennessee; VA=USA, Virginia; WA=Washington. Europe — BE=Belgium; FI=Finland; FR=France; GE=Germany; RU=Russia; SZ=Switzerland; SW=Sweden. SBI=single-basidiospore isolates; h=haplotype; c=clone.

Macromorphological characters readily distinguish G. confluens (at least in Europe and North America) from other Gymnopus taxa. In nature and in herbarium specimens, basidiomata generally exhibit long stipes compared to pileus diameter.

Lamellae: Lamellae in G. confluens appear to be quite consistent; crowded, narrow and significantly seceding upon drying. In an attempt to statistically measure the first two items, lamellae in numerous collections were carefully examined for breadth (rarely exceeding two mm) and number. The number of lamellae reaching pileus margin ranged from 116–147, consistently more than 120, and with no discernible intercontinental difference. In similar morphological taxa (i.e. G. subnudus, G. eneficola, etc.) this number ranged from 65–87, significantly fewer than in G. confluens.

Stipe: Pileus Ratio: Overall, stipe length:pileus diameter usually exceeded 3:1 (with range from 1.5:1 to 6:1). This ratio varied little between North American and European populations, but both clades exhibited some ratios downward, usually explainable due to dry weather or poor nutrition.

Stipe vesture: In most cases, stipe vesture is most sparse upward on the stipe and there (at 10X) exhibiting densely distributed spikes. Downward, vesture becomes denser, and toward the stipe base, a felty subiculum subsumes individual spikes and often is strigose. In dried material, stipe vesture takes on a gray coloration, sometimes with a very slight olive tint. Stipe vesture varied considerably in both populations/clades. In fact, vesture variation within the major populations exceeded that between clades. No suitable metric was devised to summarize this situation, but macroscopic vesture characters did not prove distinctive.

Variation in spore shape is shown in Figs 3 and 4. Spores of at least three European collections were shorter (and somewhat narrower) than those of eastern North American material, and also tapering more obviously proximally (TENN-F-67865 GE, TENN-F-67882 GE, TENN-F-59212 FR). Spores of most European collections approach the metrics of eastern North American material (Table 2). A few collections of otherwise mature basidiomata were devoid of spores. Whether this is a function of in vivo drying followed by mechanical but not biological resuscitation is not known. Counter to this hypothesis is the presence on such basidiomata of mature (sterigmate), turgid basidia which appear to be fecund.

Figure 3. 

Gymnopus confluens Europe. Outlines of basidiospores showing variation in sizes and shapes. A TENN-F-59219 FR B TENN-F-67865 GE C TENN-F-65131 BE D TENN-F-59578 RU E TENN-F-53546 SW F TENN-F-59285 SZ. Standard bar = 5 μm.

Figure 4. 

Gymnopus confluens subsp. campanulate. North America. Outlines of basidiospores showing variation in sizes and shapes: A TENN-F-47030 NY B TENN-F-48376 TN C TENN-F-63806 VA D TENN-F-48376 TN E WTU-F-021394 AK F WTU-F-024005 AK. Standard bar = 5 μm.

When spore statistics from numerous collections were compared, little difference was apparent, and spore statistics were concluded to be inconclusive for morphological separation of the phylogenetic clades of G. confluens. Median spore length for North American collections was 7.72 μm (n=10 collections); for European collections it was 7.52 μm (n=10 collections) (Table 2). This was not significantly different based on a 2-tailed T-test (P=0.40, df=19). Q^m for North American collections was 2.34 (n=10 collections); for European collections it was 2.30μ (n=14 collections). Q^m values were not significantly different based on a two-tailed T-test (P=0.71). The two collections from Alaska (MICH139598 and MICH139602) which clearly fell within the European clade by ITS sequence, were included in calculations of spore statistics for Europe.

Cheilocystidia: Cheilocystidum size and shape vary within Gymnopus subg. Vestipedes, and this variation was closely examined for numerous collections of G. confluens from both continents. Variation in both size and shape (i.e.non-strangulate to strangulate) was expected based on previous reports and illustrations. As expected, cheilocystidia varied in abundance, size and shape, but without correlation to geographic origin. Although cheilocystidia of European collections generally appear to be longer and longer-stalked than those from eastern North American specimens, the range of sizes and complexities seems parallel across the two populations. Overall variation of cheilocystidia is shown in Figs 5 and 6, where cheilocystidia are arranged according to increasing complexity. Tentacularly branched cheilocystidia (TENN-F-53546 SW) and the formation of a thatch out of the withered cheilocystidial apices (TENN-F-60109 RU) seem unique in Gymnopus sect. Vestipedes. Cheilocystidia may be involved in microscopic exudate of slime which conserves water as a microscopic moist chamber in which the apical portion of cheilocystidia can proliferate.

Figure 5. 

Gymnopus confluens. Europe. Outlines of cheilocystidia, showing variation in sizes and complexity. Basidia furnished for size comparison. A TENN-F-59285 SZ B TENN-F-59469 FI C TENN-F-53546 SW D TENN-F-59219 FR E TENN-F-67882 GE F TENN-F-60109 RU. Standard bar = 20 μm.

Figure 6. 

Gymnopus confluens subsp. campanulatus North America. Outlines of cheilocystidia, showing variation in sizes and complexity. Basidia furnished for size comparison. A MS4-009 NF B TENN-F-47030 NY C TENN-F-69053 NB D TENN-F-63806 VA E TENN-F-48376 TN F TENN-F-69074 NB.Standard bar = 20 µm.

Caulocystidia: In observing caulocystidial hyphae composing stipe vesture, especially those gathered to form the characteristic vesture “spikes,” some variation was perceived in the shape of the terminal cells, whether equal (parallel-sided and bluntly rounded at apex), tapering distally (and therefore narrowly rounded at apex), or some variation in shape (i.e. subsagitate, lobed, etc.). Once noted, special care was taken to observe this character. Caulocystidial hyphae are invariably clamped within their emergent length. Caulocystidia, correctly depicted by Antonín and Noordeloos (2010)often include small side lobes with narrow attachments to parent hyphae. It is easy to observe the erect hyphae which compose the spikes, but more difficult is observation of the subicular hyphae which produce the hyphal complexes which elongate into the spikes. The complexity and depth of the subiculum vary from arachnoid (and then revealing the color and glassy surface of the dried stipe cortex) (TENN-F-59282 SZ; TENN-F-59219 FR) to opaque-felty with subsumed spikes (TENN-F-67865 GE) to shaggy (TENN-F-67882 GE). The felty subiculum increases downward and appears as a sheath toward the stipe base, somewhat reminiscent of Connopus acervatus.

Pleurocystidia: Whether pleurocystidia are commonly present has not been thoroughly investigated. Structures resembling cheilocystidia were observed in three collections (TENN-F-59578 RU, TENN-F-67865 GE, TENN-F-59212 FR) among basidia rather than being clustered at the temini of lamellar tramal hyphae.

Basidia: Basidia varied little across both continents. Rarely, an individual two-spored basidium was detected, and four-spored basidia accounted for almost all mature basidia observed (Figs 5, 6).

A basidium is produced as a terminal cell of a subhymenial hypha. The hyphae then proliferates through the subtending clamp connection and another basidium is produced in the same fashion. After several such proliferations and basidial discharge (usually leaving little or no residue), the subhymenial hypha appears asymmetrically notched, and superficially resembles some cheilocystidia (TENN-F-67865 GE), for which they are easily mistaken, especially as seen in considerable numbers in older hymenia.

Side branches from pileipellis: The side branches from pileipellis hyphae reported and illustrated by Halling (Halling 1983) and Antonín and Noordeloos (2010) were consistently observed. Shapes ranged from short, simple and digitate (i.e. TENN-F-13883, TENN-F-48376, TENN-F-69133, TENN-F-53522 from North America; TENN-F-65131, TENN-F-60109, TENN-F-67882, TENN-F-59469 from Europe) to over 100 μm long and branched in a tentacular fashion (i.e. TENN-F-63806 from North America; TENN-F-67865 from Europe). Quantitatively, however, these side branches seem longer (TENN-F-67882 GE, TENN-F-60109 RU) and usually more complex (i.e. branched in a coralloid fashion (TENN-F-67865 GE, TENN-F-59469 FI) in European specimens than in American. It may be that this proliferation is aided by microscopic local moisture idiosyncratic to individual conditions (including some mucoid matrix if present). The variation was wide both within and between clades, and no clade separation was possible based on this character (Figs 7, 8).

Figure 7. 

Gymnopus confluens. Europe. Outlines of side branches of pileipellis hyphae showing variation in size and complexity. A TENN-F-65131 BE B TENN-F-60109. RU C TENN-F-59469 FI D TENN-F-67865 GE. Standard bar = 20 μm.

Figure 8. 

Gymnopus confluens subsp. campanulate. North American. Outlines of side branches of pileipellis hyphae, showing variation in size and complexity. A TENN-F-53522 NC B TENN-F-69133 NL C TENN-F-63806 VA. Standard bar = 20 μm.

Morphological summary: Morphological characteristics based on lamellae, side branches from pileipellis hyphae, cheilocystidia, caulocystidia, and pleurocystidia, varied considerably but observed differences were not continent-specific. Basidia and basidiospores showed little variability and did not differ between continents.

Results of three self-crosses of G. confluens (collections TFB 7219, NC; 9048, CA; 11400, SZ) were reported (Mata et al. 2007) as showing tetrapolarity, not unusual in Omphalotaceae. This study added TENN-F-69053 (TFB14389), New Brunswick, Canada, which we also determined to be tetrapolar. In all cases, distribution of mating types was unbalanced and in most of these self-crosses, some SBIs were found which exhibited unexplained mating results, mostly ability to dikaryotize two opposing mating types, most easily explained by either harvesting of two hemicompatible basidiospore germlings together, or occurrence of two hemicompatible nuclei within a single basidiospore (Petersen 1995). Such results have also been found in other members of Gymnopus subg. Vestipedes, such as G. subnudus (Murphy 1992; Murphy and Miller 1993; Petersen 1995) which has been reported as bipolar and tetrapolar (Petersen, ined.)

In the sexual compatibility study using G. confluens [(Mata et al. 2007);Fig. 14], a total of 11 collections were used, seven from Europe and four from North America. Five intercontinental intercollection pairings were performed, all universally sexually compatible. If clamp connection production is accepted as a proxy for sexual reproduction, no apparent prezygotic reproductive barrier exists between the continents. It was concluded, based on limited evidence, that only a single sexual recognition species was involved. No attempt to segregate morphological entities was offered in that study.

A PhyML tree based on ribosomal ITS sequences is given in Fig. 9. North American and European collections segregated largely into two distinct clades which share an average sequence identity of 96.75%. Collections from Alaska were placed in both clades. A collection from California was also affiliated with the European clade. For a smaller data set, both ribosomal ITS and LSU sequences were available and were concatenated. Results of the PhyML analysis are given in Fig. 10.

Figure 9. 

PhyML tree based on ribosomal ITS sequences. Bootstrap values based on 100 bootstrap replicates are at the left of the supported node. Analysis assumed the GTR model of evolution with the transition/transversion ratio, number of invariable sites and shape of the gamma distribution estimated. The log likelihood of the tree was -1776.7. Bold type = holotype of Gymnopus confluens subsp. campanulatus. Percent identity was based on the entire ITS1-5.8S-ITS2 sequence.

Figure 10. 

PhyML tree based on concatenated ribosomal ITS + LSU sequences. Bootstrap values based on 100 bootstrap replicates are at the left of the supported node. Analysis assumed the GTR model of evolution with the transition/transversion ratio, number of invariable sites and shape of the gamma distribution estimated. The log likelihood of the tree was -3135.8. Bold type = holotype of Gymnopus confluens subsp. campanulatus.

For the ITS data set, Rosenberg’s P_AB statistic for both European and North American clades was P=1.6 × 10^-8. Thus, a null hypothesis of reciprocal monophyly under a random coalescence model can be rejected. The probability of correctly identifying an unknown member of a putative species is given by P_ID statistics. P_ID (strict) European clade = 0.95 (σ=0.89,1.00) and P_ID (strict) North American clade = 0.98 (σ=0.93, 1.00). P_ID (strict) is the more stringent of the P_ID statistics. The probability P_RD that a clade has the observed distinctiveness under a null hypothesis of random coalescence for North America is 0.05 and for Europe is 0.21. Neither of these probabilities reject the null hypothesis. PTP species-delimitation results produce both maximum likelihood and Bayesian estimates of the number of species. PTP for both analyses partitions G. confluens European and North American populations into two species groups but without significant support (bootstrap support for both analyses was 0.23 for North America and 0.55 for Europe. Outgroup taxa are also partitioned into two species groups.