Ecology and abundant occurrence of Genea (Pyronemataceae) in western Oregon, U.S.A.

11 Genea (Pyronemataceae, Ascomycota) develops hypogeous ascomata (truffles) that are 12 often small and soil-colored. This group is considered common but challenging to find. We used 13 a truffle diversity dog to locate Genea truffles in the southern Willamette Valley and associated 14 mountains in western Oregon, U.S.A, recording when the sporocarps were present at a wide 15 range of elevations (113 to 1879 m). We found Genea was present in half of our surveys, and 16 noted that it rarely fruited in areas that had experienced wildfire. This study demonstrates the


Introduction
The truffle genus Genea (Pyronemataceae, Ascomycota) is characterized by lobed and often inconspicuous hypogeous ascomata and was originally circumscribed in Italy in the 1800s (Vittadini 1831).It is considered common in both Europe and North America (Alvarado et al. 2016), but because it fruits underground and is often difficult to differentiate from soil aggregates, we have limited knowledge of its ecology and distribution.One of the most comprehensive studies in the U.S. Pacific Northwest focused on oak grasslands (Smith et al. 2006) and described two new species.However, Genea forms ectomycorrhizal associations with a wide variety of plants (Smith et al. 2006), suggesting that it may be widely distributed throughout the Douglas-fir (Pseudotsuga menziesii) forests of the Cascades and Coast Range.
We tested this possibility using a novel tool: a truffle dog with an uncanny ability to find Genea truffles.
Truffle dogs are most commonly trained to find mature culinary truffles that command a high commercial price.However, some dogs have a natural inclination towards generalizing on any hypogeous odorous sporocarps.If the trainer rewards all finds (rather than just culinarily valuable species), they can train "diversity dogs" who can locate a broad range of truffles that may be of interest to science.Most truffle surveys are currently performed by raking, where a researcher uses a small garden hand rake to peel back the upper layer of the soil.This method has a visual bias towards what the researcher spots under the duff, and often misses rare and inconspicuous taxa.In contrast, truffle dogs have a scent bias driven by mature sporocarps of any size or color, can find truffles deeper than most people rake, and do not have preconceived ideas of where truffles 'should' be.By documenting which truffle species are found throughout a range of sites and seasons, we can use truffle diversity dogs to broaden our understanding of hypogeous fungal ecology.

Methods
All truffles were located by trained truffle diversity dog Rye, who had found 23 genera of truffles in two years of truffling as of February 2023.Rye demonstrated particular persistence in locating Genea truffles as of the time of this study.He received a reward for each Genea truffle located, reinforcing his pursuit of truffles with this type of aroma.
We performed 87 general truffle surveys and forays in all seasons between October 2021 and November 2022.We visited 66 sites, 12 of which we visited more than once throughout the year (Figure 1; Table S1).Most surveys were conducted along existing public trails that allowed us easy access to varied landscapes.We performed these surveys at a range of elevations from 113 m to 1879 m (elevation data extracted from WorldClim (Fick and Hijmans 2017)).On each survey, we primed Rye to find truffles of any species and rewarded his finds with praise and a brief game of fetch with a tennis ball (his favorite reward).He was given freedom to roam offleash with voice recall during these surveys so that he could cover more ground and follow his nose.We conducted our surveys primarily in conifer-dominated forests of varying age classes and densities, with the exception of the site at Mt. Pisgah which was oak savanna (Table S1).
Our sites had a variety of fire histories, from no recent fire to recently burned (1-11 years).Most low-mid elevation conifer forest sites were primarily composed of Douglas-fir (Pseudotsuga menziesii) and western hemlock (Tsuga heterophylla), and high elevation sites often included mountain hemlock (Tsuga mertensiana), true fir (Abies spp.), and pine (Pinus spp.).All sites were located in Benton, Deschutes, Lane, and Linn Counties in southern Willamette Valley, Oregon, USA.These counties encompass the western Cascades, southern Coast Range, and Willamette Valley.We excluded observations from coastal Lane County because we performed only spatiotemporally limited surveys on the coast.
We only recorded truffle presence/absence at each site.Where multiple truffles were found, this counted as a single 'presence' for a site.If we surveyed multiple habitat types at a single location (for example, inside and outside a burn area), this was counted as multiple sites.

Results
We found Genea fruiting bodies in slightly over half (51.7%) of the site surveys.We reported occurrences of Genea nearly year-round, except the months of August and September (Figure 2) which are the driest months of the year due to western Oregon's Mediterranean climate.Low and mid elevation sites had the most consistent Genea presence.The highest site we found Genea at was at 1716 m elevation.Genea truffles were not present in any burned conifer forests, but were found during two oak savanna surveys that had burned three years prior.Odor and morphology of the truffles suggested that there are likely multiple species present.We noted that some visually similar truffles collected from shared locations had a mellow cheese odor, while others had a sharp garlic-truffle odor.Microscopic examination of these particular variants showed ellipsoid spores in the former and subglobose spores in the latter.Subsequent examinations of additional collections suggest extensive hidden diversity in the dark-colored Genea found in this region.

Discussion
Using a dog trained to find truffle diversity, we found Genea grew abundantly nearly year-round.In the literature, Genea is known to be common (Alvarado et al. 2016); however, there can be an impression of local scarcity due to the difficulty of finding small and soil-like sporocarps.The commonness of Genea suggests that this genus may be a key ectomycorrhizal partner for trees in the area, especially the dominant Douglas-fir timber trees.Although the decrease in abundance with elevation may be partially due to a sampling bias, our findings suggest that Genea is most prevalent at lower elevations.Douglas-fir trees have limited distribution at higher elevations (Case and Peterson 2005), where the landscape is more likely to be dominated by mountain hemlock (Tsuga mertensiana) or true firs (Abies), enforcing the likelihood of a strong mycorrhizal association between Douglas-fir and Genea.
Our surveys demonstrate that human truffle surveys have a visual bias that can be overcome by complementing with scent surveys.The dog strongly indicates, requiring the accompanying human to look more closely and find the "hidden" Genea.However, our surveys were limited to the abilities of the truffle dog.He cannot find truffles in hot weather, so our summer surveys were focused at higher elevations where the temperatures were cooler.
Similarly, we were unable to access higher elevation sites until after snowmelt and the roads had opened, but that also would have been true of the more traditional truffle-finding method of raking.
Genea truffles are difficult to identify from morphological features alone, and even spores can be indistinct.Most Genea collections from conifer forests in the Pacific Northwest have typically been identified as G. harknessii (species complex) or G. gardneri, as the only two species of dark Genea currently known from this habitat (Smith et al. 2006;Trappe et al. 2007).
We have observed that there are likely several additional species that are morphologically similar and difficult to distinguish without microscopic examination and sequence data.In some instances we noted the change in odor and texture of the Genea we found, but we did not consistently track these traits given that these characteristics do not reliably separate different species in many hypogeous fungi.This group would benefit from further taxonomic study using modern molecular methods to determine which species are present in the area in the different habitats and elevations.
Eleven of our sites were burned, including eight conifer sites that had experienced wildfire in the past 1 to 11 years.In light of the increasing frequency and severity of wildfire in western Oregon (Halofsky et al. 2020;Dye et al. 2024), the lack of Genea at these sites is notable and suggests that some species of Genea may be sensitive to wildfire, at least in the earlier years of recovery.On the other hand, we did find Genea during both surveys at the burned oak savanna site, suggesting that not all Genea species may be equally affected by fire.This genus is relatively abundant in the Willamette Valley; however, it may decline in abundance as Douglas-fir forests are affected by climate change and high severity fire events.
Our observations of Genea as ubiquitous in modified and disturbed habitat suggests it is resilient to anthropogenic effects, but extreme fire may be an exception for some species.We must prioritize document fungal species distributions, particularly ones that are difficult to locate like Genea, to understand what can be lost through climate change.

Figure 1 :
Figure 1: Sites and methods used to document Genea ecology.A) Map of all sites surveyed as part of this study.Blue points are sites where we found at least one Genea truffle.Symbols indicate elevation category (circle = high elevation, triangle = mid elevation, square = low elevation).B) Example of truffle dog Rye finding a truffle.C-E) Photographs of Genea truffles demonstrating lobing, dark peridium and light gleba, and sometimes very small size and resemblance to soil clusters.

Figure 2 :
Figure 2: Genea presence in the southern Willamette Valley and associated mountains throughout a single year.Numbers within the bars indicate the number of surveys where Genea was found (blue) or not found (gray).Parenthetical numbers indicate how many of those sites were burned.