Transatlantic disjunction in fleshy fungi III: Gymnopus confluens

Phylogeographic data indicate that DNA differences consistently exist between the North American and European allopatric populations of Gymnopus confluens. Conversely, pairing experiments show that collections from both populations were sexually compatible in vitro and detailed morphological examinations of numerous fresh and dried basidiomata do not produce qualitative differences. Percent ITS sequence divergence between Europe and North American collections of G. confluens was 3.25%. Species delineation metrics including Rosenberg’s PAB statistic, PID metrics, RRD (randomly distributed) and PTP (Poisson Tree Processes) gave mixed indications that North American and European populations were distinct at species rank. The North American populations are described as Gymnopus confluens subsp. campanulatus (Peck) R.H. Petersen.


Introduction
North American species numbers for fleshy fungi are certainly underestimated by the historical practice of assigning European names to North American taxa but the extent to which North American taxon numbers are underestimated is unknown and is dependent on species concepts used by investigators.A number of studies documenting cryptic geographical species argue that species may be more localized and more numerous than historical treatises might suggest.A recent study by Talbot et al (Talbot et al. 2014) demonstrated that most soil fungi are regionally endemic and limited by factors such as climate and dispersal ability.Geographical partitioning may be the rule for basidiomycete taxa, rather than the exception (Taylor 2008;Taylor et al. 2000).
In agaric systematics, discrepancy among parameters used to make taxonomic judgments at species rank is becoming more widely recognized.Three such standards, DNA sequence data, sexual compatibility and morphological characters of basidiocarps have evolved as important in taxonomic judgments, including proposal of new taxa [Lentinus (Grand et al. 2011); Megacollybia (Hughes et al. 2007); Lentinellus (Hughes and Petersen 2004;Petersen and Hughes 2004); Sparassis crispa complex (Hughes et al. 2014); Artomyces (Lickey et al. 2003)], and revision of generic or species complexes [(Panellus stypticus (Jin 2001); Marasmius scorodonius (Gordon and Petersen 1998); M. androsaceus (Gordon and Petersen 1997); Strobilomyces (Sato et al. 2007); Omphalotus (Kirchmair et al. 2004;Petersen and Hughes 1998); Pleurotus (Vilgalys et al. 1993;Vilgalys and Sun 1994); Gymnopus s. l. (Mata et al. 2007)].As phylogenetic analyses based on molecular data have increased, it has become increasingly clear that genetic differentiation of fungi may proceed in the absence of observable morphological change in basidiocarps.Taylor et al. (2006) note that eukaryotic microbes (predominantly fungi) have few discriminating morphological characters on which to base species assessments.Further, the basidiomycete fungal mating system acts to slow or prevent establishment of reproductive barriers (James et al. 1999).In the absence of morphological change and reproductive isolation, delineation of taxa may rest on evaluation of observed genetic change among populations.
Few primary literature sources are available for identification of Gymnopus s.str.Peck's proposals of new species, often under Marasmius, were included by Halling (1983) in a publication intended to summarize the taxa (under Collybia) of northeastern USA and adjacent Canada.Murrill (Kimbrough 1972) accepted Gymnopus Roussel to represent the "collybioid" taxa of Florida and described several taxa of Gymnopus (i.e.Collybia) and Marasmius s.l.from central Florida [see Kimbrough (1972); Halling (1983)].This left a geographical hiatus between the New England region and Florida.Without publishing a summary of his study, Hesler included observations on Murrill specimens in his (Hesler) notebooks (http://trace.tennessee.edu/utk_hesler/), and Smith [see (Petersen et al. 2014)] also examined Murrill's type specimens.Lennox (1979) published her dissertation on Pacific Northwest collybioid genera, but included only a single species under Collybia s. str.Desjardin (1987) published a summary of collybioid fungi from California which further expanded geographical and taxonomic coverage of Gymnopus.
A molecular discontinuity between European and eastern North American populations of Gymnopus confluens has been known for some years (Mata et al. 2007), most recently implied in a phylogeny placing G. eneficola from Newfoundland, Canada (Petersen et al. 2014).Additional data now permit a more detailed report as part of a wider project on transatlantic disjunctions in fleshy fungi.
In this study, we evaluate morphology, ability to dikaryotize in vitro and ITS-LSU sequence divergence to determine whether intercontinental allopatric populations of Gymnopus confluens represent separate taxa at some rank.In this paper, we consider the term population to comprise a group of currently interbreeding individuals.This paper is third in a series of papers exploring the nature of transatlantic disjunctions.The first in the series was Hughes et al. (2014), the second is Petersen et al. (in press).

Collections
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.

Pairing experiments
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.

Macromorphology
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.

Micromorphology
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).

Molecular studies
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).

Species-delineation metrics
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.

Data resources
The data underpinning the analyses reported in this paper are deposited in the Dryad Data Repository at doi: 10.5061/dryad.8239h.

Results
Gymnopus confluens basidiomata from North America and Europe are shown in Figs 1, 2.

Morphological parameters -macromorphology
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.

Morphological parameters -micromorphology
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.
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 cheilo-  croscopic exudate of slime which conserves water as a microscopic moist chamber in which the apical portion of cheilocystidia can proliferate.
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.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).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.

Sexual recognition experiments
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.

Phylogenetic analyses
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.
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.

Discussion
Allopatric speciation may be the most common mode of speciation in fungi and other organisms, and separation of populations on different continents would, in the absence of significant gene flow, lead gradually to accumulation of genetic differences and ultimately speciation.The mode and tempo of speciation, however, must vary with reproductive strategies and selection pressures.The point at which two allopatric populations become new species is often a matter of judgment but important in terms of evaluating conservation status and estimating species diversity for a given region.Numerous studies suggest that in basidiomycete fungi, ability of allopatric populations to intercross in vitro is conserved as a function of the unique multiple allelic mating systems even while genetic divergence (usually indicated by differences in nuclear ribosomal ITS sequences) proceeds (Gordon and Petersen 1997;1998;Grubisha et al. 2012;James et al. 1999;Lickey et al. 2002;Lickey et al. 1999;Taylor et al. 2006;Vilgalys and Sun 1994).It should be noted, however, that in vitro compatibility examines only one aspect of reproductive intercompatibility and that failure to produce F 1 fruitbodies, reduced fertility of F 1 hybrids, lack of or inviability of F 2 progeny and competition failure at any level may also be involved in reproductive isolation.These factors have not been evaluated as isolated mechanisms between basidiomycete populations to date.
In Gymnopus confluens, two of three criteria used to evaluate delineation of species (morphology and ability to intercross in vitro) show no significant intercontinental separation.ITS sequences, however, are divergent (3.25% base pair difference), a level often used to suggest different species (Hughes et al. 2009), and European and North American clades are well-supported.The combination of a barcode gap, Rosenberg's P AB statistic results and P ID (strict) suggest that North American and European clades are monophyletic, that the observed differences are not due to coalescence in gene trees and that they are distinct phylogenetic species.In contrast, P RD and PVP do not reject the possibility that the observed result (Fig. 9) is due to random coalescence.The question then becomes whether to assign these populations nomenclatural rank on the basis of ribosomal ITS and LSU sequences alone.To identify these populations as comprising a single species ignores significant ITS + LSU sequence divergence indicative of speciation processes, and underestimates diversity.To identify these populations as separate species may overstate the degree of genetic divergence.A middle-ground solution seems to be to assign these populations subspecies rank, thus recognizing genetic differentiation.We do so below adhering to, insofar as possible, procedures suggested by Tripp and Lendemer (2014) for naming taxa when molecular evidence is the only evidence available.
The finding that ITS sequences for collections from Alaska represented two distinct ITS entities, one of which falls within the European clade (Fig. 9) suggests a dual origin for collections from this region.An ITS sequence of a collection from California also falls within the European clade.Possibly, these collections represent humanmediated transfer of material from Europe to North America.Alternately, movement from Eurasia to Alaska thence to California may have been feasible via the Bering land bridge during periods of glaciation, but without an understanding of G. confluens from Asia, neither hypothesis be substantiated.Intra-continental geographical partitioning is not clearly evident for either European or North American populations of Gymnopus confluens.This contrasts with findings in some other basidiomycete taxa (Geml et al. 2008;Hughes et al. 2014;Zhao et al. 2013) but intracontinental biogeographical distributions have not been extensively examined and are likely to be species-specific.Taxon diagnosis.1) ITS nrDNA sequence significantly different from sequence of Gymnopus confluens subsp.confluens; 2) basidiomata densely gregarious to subcespitose; 3) basidiomata apparently persistent beyond spore production and discharge; 4) stipe:pileus diameter ration from 2-5:1 (stipe significantly longer than pileus diameter); 5) pileus hygrophanous, brown where moist, pallid tan to pinkish buff where dry, drying to more uniform pallid color; 6) lamellae very crowded (total lamellae at pileus margin 110-140), shallow, seceding upon drying; 7) lamellar edge entire (smooth) to delicately fimbriate; 8) stipe grooved or compressed, stiff, with brown cortex (rind); 9) stipe vesture concolorous with pileus when moist and fresh, easily bleaching on drying to pallid gray shades; 10) basidiospores generally elongate-ellipsoid to sublacrymiform; 11) cheilocystidia stalked, usually lobed or strangulate, sometimes branched; 12) pileipellis hyphae smooth, firm-walled, with occasional to common side branches appearing digitate to long and branched.13) Distribution in North America.
Habitat and phenology.on duff under Quercus and other hardwoods including Acer (TENN 63806); gregarious on leaf litter under Fagus (TENN 47030) and occasionally Pinus; hardwood duff (TENN 48376).

Conclusions
Gymnopus confluens in Europe and North America shows intercontinental but not intracontinental divergence in ITS and LSU sequences but European and North American populations do not differ morphologically and retain the ability to dikaryotize in vitro.Intercontinental ITS/LSU sequence divergence is sufficient to recognize differences taxonomically.The North American population is described as G. confluens subsp.campanulatus.

Figure 9 .
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-ITS2sequence.

Figure 10 .
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.

Table 2 .
Basidiospore metrics for European and North American collections of Gymnopus confluens.