Population Biology and Ecology in the Rare and Endangered Hapalopilus Croceus

Population biology and ecology in the rare and endangered Hapalopilus croceus

Deanne Redr Greaves

Degree project in biology, Master of science (2 years), 2017 Examensarbete i biologi 45 hp till masterexamen, 2017 Biology Education Centre, Uppsala University, and SLU: Department of Forest Mycology and Plant Pathology, Almas allé 5, 756 51, Uppsala, Sweden Supervisor: Audrius Menkis External opponent: Anushree Sanyal Abstract Landscape management has altered the population dynamics of many species residing in old growth ecosystems. As a result, fragmentations in residual old oak habitats in Sweden have led to the decline of endangered and long-lived polypores including the bright orange Hapalopilus croceus. Since little is known about this species, the aim was to investigate its growth, mating, and population biology using molecular and ecological methods. Specifically, what its growth patterns are, what its mating type is, and whether there is genetic variation among sampled populations in Sweden and the Baltic area. Fruiting body samples were collected from 34 localities in Sweden and 6 localities in neighboring Baltic States. Heterokaryotic mycelia were then cultivated from these samples and the genomes sequenced. 42 single-spore isolates from SLU’s culture bank were also revived and cultured. These homokaryons were then crossed to determine mating type. Mating type was examined using morphological observations and calculated via mating matrix. To examine growth rate and establishment, H. croceus mycelia were grown in comparison to Fomitopsis pinicola mycelia. To explore the population biology, the potential for gene flow was analyzed among Swedish and Baltic isolates. As a result, H. croceus was found to be slow growing, exhibit a tetra polar mating type, and with limited genetic variation in the Baltic Sea area. Its slow growth could provide a possible insight into colonization strategy, whereas its multiallelic mating system could possibly increase outcrossing in the future. Minimal genetic variation could result from previous connectivity between oaks, where the fungus may have outbred readily and colonized more hosts, maintaining a more uniform genetic structure. However, recent habitat alteration has not been analyzed for this species, nor has community interaction among other fungi and fruiting dynamics. Thus, further examination of these factors would help expand this study and possibly provide conservation and management guidelines for future projects.

Background Introduction Producing brightly colored orange fruiting bodies, Hapalopilus croceus is a rare and endangered polypore. Its nature and ecology however, remain largely unexplored. Often indicative of thermophilous and deciduous forests in Central-European and Sarmatic regions (IUCN, 2013), its fruiting bodies are generally found on old coarse oak trees. It is a white-rot species, whose hyphae (strands of fungal cells) are capable of producing combustive peroxidases that break down plant cells containing the complex polymer lignin (Aust, 1995). This fungus is thought to enter the host through hollowed-out areas below branches or damaged sections of the trunk as a weak parasite before gradually developing into a lignicolous saprotroph (ArtDatabanken, 2015), but this still remains uncertain. Its mycelia (groups of hyphae, essentially the “body” of the fungus) consists of a long-lived single genetic individual who may perhaps be several hundreds years old, with an annual sporocarp that may appear yearly if environmental

1 conditions permit (Environmental Protection Agency, 2017). Little is known about this fungus, with few publications on general ecology and distribution (Fraiture and Otto, 2015; Ryvarden and Melo, 2014). The genome of H. croceus has also never been fully sequenced, with only a small portion of ribosomal DNA currently available (Dvorak et al., 2014).

Aims The overall objective was to study ecology and population genetics of the basidiomycete polypore H. croceus. Specific aims: a. Examine ecology by recording data such as fruiting body location on the host tree, surrounding habitat, and sporocarp occurrence. b. Determine mycelial growth rate of cultures, to provide a larger picture of factors possibly influencing the polypore’s establishment patterns. c. Discover mating type system, since it has never been determined for H. croceus and might play a role in outcrossing. d. Examine sequenced genomes of H. croceus in Sweden and other Baltic states to determine whether there is gene flow in the Baltic Sea area, which will provide insight into the population genetics of the species.

Hypotheses 1: H. croceus is slow growing, a trait that influences its establishment in substrate. 2: H. croceus is tetrapolar, resulting in a 25% mating compatibility between sibling spores. 3: Swedish populations of H. croceus are likely to have low genetic diversity and exhibit minor to absent gene flow from neighboring European populations.

Any information uncovered in this study can assist conservation efforts and increase general knowledge of threatened communities, for other critically red listed wood-inhabiting oak fungi as well.

Background Ecology and population genetics Of the 170 species of oak-inhabiting red-listed fungi, a large quantity of the European population exists in Swedish oak (Quercus) habitats (Dahlberg, 2006). This makes Sweden internationally important in fungal conservation, particularly for H. croceus, which has been listed as critically endangered (CR) in a large portion of European forests (Dahlberg and Croneborg, 2003). Sweden is expected to contain ⅓ of the estimated European population of this polypore. The number of individuals with unique genotypes is expected to be at most 250; possibly scattered throughout localities that are expected to not exceed 120 sites (only 40 sites

2 are known thus far (ArtDatabanken, 2015)). Furthermore, due to disappearing old growth forest habitats, H. croceus is in decline and is predicted to decrease in population size by 50% over the next 100 years (ArtDatabanken, 2015).

However, landscape history implies this species may have previously occurred in greater REMAINING ICE numbers. Pollen data suggest oak was not present in Sweden until 8500 BP, where it possibly Finland migrated across a land bridge connecting Sweden and Denmark (Jensen Svejgaard et al., 2002), but still was unable to colonize ANCYLUS LAKE more northerly regions due to Estonia Ancylus lake (fig. 1). Then, Sweden between 6000-2500 BP, oaks thrived and were in much larger Latvia numbers than they are today Denmark (Jensen Svejgaard et al., 2002). If oaks thrived, it is likely that H. Lithuania croceus did as well, if it followed the host tree up through Sweden. Fig. 1: A land bridge existed between Denmark 8.9BP where oaks Thus when combined, these could have migrated across (Modified from Björck,1995). Part of Sweden was also covered under Ancylus lake. factors could all show interesting variation in the H. croceus genome, since the species currently has a fragmented distribution and limited gene flow between remaining populations is likely.

Molecular methods in exploring genetic variation For species identification in fungi, ITS rDNA primers are chosen, which anneal to the ITS (internal transcribed spacer) region of the genome. This region acts as a barcode for fungal species and can be used to examine phylogenies as well as species differentiation in community studies (Schoch et al., 2012). Thus, the ITS region of the DNA can be examined closely to provide a more detailed insight into the varied or similar population structure within a fragmented species. This is due to the evolutionary nature of the ITS region, where there is less evolutionary pressure on non-functional sequences. So in part to its high degree of variation, differences between even the closest related species can be observed. In addition, when

3 combined with other data, this can potentially be used to provide a sort of indicator for geographic differentiation between species (Grantham et al., 2015). However, published research regarding geographic differentiation between individual ITS regions in fungi is currently lacking. Nonetheless, examining the ITS region may be a helpful tool when no previous population structure information has been obtained.

For examining genetic distance and gene flow between populations, it is important to identify dissimilarity among different individuals. Among various lab tools, Random Amplification of Polymorphic DNA (RAPD) can be used as a fast and inexpensive method. RAPD is a DNA fingerprinting method based on standard PCR techniques, that can be used to identify genetic variation between samples (Catană et al., 2013). Following DNA extraction, genomic DNA is amplified using a general JO-1 primer that targets random strands of polymorphic DNA sequences during the PCR process (Soll, 2000); thus producing more favorable results for deciphering population genetic variation.

Basidiomycota life cycle and mating To better understand population structure, how a species propagates can be a useful tool, particularly for polypores belonging to Basidiomycota. Basidiomycota exhibit a very unique life cycle and mating system (fig.2; Casselton and Olesnicky, 1998). A single spore germinates in substrate as a haploid homokaryon; hyphae containing a single karyotype (chromosome pattern). When an individual of an incompatible mating type is present in substrate, pheromone-signaling in fungi will often lead to morphological traits such as barriers or toxins to ward off offending mycelia (Jones and Bennett, 2011). However, if compatible mycelia are present, they fuse together to undergo plasmogamy where fusion of nuclear cells occur and heterokaryon mycelia form. In fungi, compatibility is regulated by different mating systems and mating will only proceed if the meeting hyphal strains are of different mating types. The hyphae resulting from mated strains are often recognized in many Basidiomycota for showing clamp- connections; small linkages crossing cell walls (septa), depositing divided nuclei into adjacent cells following mitosis (fig.2). Fruiting bodies can only develop from heterokaryon hyphae and together the mycelia build a sporocarp containing diploid nuclei containing cells which will divide following meiosis and provide countless homokaryotic spores that can deposit into additional substrate to repeat the cycle (fig. 2).

Fig. 2: Life cycle of Basidiomycota, showing spore germination, homokaryon, mating and resulting heterokaryon with clamp connections. Fruiting body formation with n+n basidiospores follows (Casselton and Olesnicky, 1998. Modified).

Under Basidiomycota, the class Agaricomycetes are known to typically express either a bi-polar or tetra-polar mating type (Esser et al., 1994). However, 77% of polypore species exhibit tetrapolar mating types (James et al., 2013). In a bi-polar mating system, genomic regions consisting of two types of MAT loci exist with two different alleles at each locus that determine sexual identity similar to how sex chromosomes do in animals (Coelho et al., 2010). It is possible however, for four types of MAT loci to emerge during each meiosis cycle, as a result of sexual selection restricting two different molecular recognition sites that encode each respective MAT region (Coelho et al., 2010). Here, A and B mating type genes occur in four different combinations; the resulting haploid hyphae must contain two of these genes that are also different from one another (fig. 2). So, in tetrapolar mating systems, these four mating types mean only 25% of spores released from the fruiting body are compatible, potentially resulting in more recombination at the MAT locus (James, 2015). When the mating system for a fungal species is unknown, a lab setup where strains of homokaryotic mycelia are crossed with one another is helpful in determining mating type (Nieuwenhuis et al., 2011). To study this, morphological traits such as the wall-building between unmated mycelia (Coates et al., 1985) and clamp connections of mated mycelia could be used to evaluate compatible and incompatible strains.

Methods Sample collection of sporocarp segments Localities were visited between 08/2015-10/2015, using known GPS coordinates from vaxvakt.ekoo.se and fruiting body data from artportalen.se: 4 sites in Uppsala county, 10 sites in Stockholm county, 5 sites in Södermanland county, and 9 sites in Östergötland county (see fig. 3) were personally visited by me. 30 additional cultures were provided by Stellan Sunhede, who collected sporocarp tissues in localities ranging from Öland, Västergötland, Småland, and others counties. While initially all localities in the database since Fig. 3: Map of southern Sweden, displaying localities of H. croceus for 1990 were to be sampled, time 2015. restraints resulted in a narrowing down of sites according to the highest probability of H. croceus fruiting in the last ten years. Thus, in total, 45 sporocarps were sampled (one sporocarp segment per location), representing 45 genotypes throughout Sweden (37 sampled sites), Latvia (4 sampled sites), and Lithuania (4 sampled sites) (appendix 2). Permissions from all appropriate county administrations were obtained prior to sampling. The database was also updated with recent coordinates if fruiting bodies were or were not discovered. I documented the localities via observational notes consisting of location of sporocarp on oak tree, distance of fruiting body from ground, surrounding vegetation and habitat, and also photographs for future use. Each site was recorded if a fruiting body was found, to later calculate rate of occurrence (# of sites fruiting bodies found/ # lacking fruiting bodies). Fruiting bodies, when found, were sampled via a 5 x 5 cm extraction that left the remaining sporocarp attached to the host tree. Extractions were placed in a 1 liter ziplock bag, labeled with locality and sample name, then brought back to the SLU department lab.

Heterokaryon and homokaryon mycelial cultivation Sporocarp segments collected in the field from Sweden, Latvia, and Lithuania were cultured to develop heterokaryon mycelia used for the population genetics portion of this study. These samples were also used for genome-wide Illumina sequencing. Three of these heterokaryons were also randomly chosen to compare growth with the commonly occurring species Fomitopsis pinicola. H. croceus homokaryons were cultured by me from 42 single-spore isolates previously collected from Lithuania, Fig. 4: Mycelial growth study displaying how replicates obtained from Rimvys Vasaitis at the SLU were ordered, 18 total. Each replicate was measured daily at north, east, west, and south coordinates on each culture bank with permission. These respective petri dish. Orange figures represent H. croceus, homokaryons were then used to establish blue F. pinicola. Red square denotes mycelial sample. the mating type for H. croceus by crossing to determine sibling spore compatibility. One homokaryon sample was also used for PacBio sequencing as a reference library to the Illumina sequenced heterokaryons.

Following field sample collection, sporocarp extractions were immediately brushed clean of debris and transferred to a fume hood, where I used sterile forceps to transfer three 1 x 1 cm pieces from each fruiting body onto previously prepared 9 cm Hagem agar petri dishes (Stenlid, 1985). Two replicates were generated per sporocarp and were each labeled (lab ID, appendix 2.), wrapped in parafilm, then sealed and cultured for up to 6 days at 23 C° until at least 2 cm of radial heterokaryon mycelial growth was observed. Next, to ensure purity of the samples, a 5 x 5 mm square was cut from each cultured dish, containing existing agar and fresh mycelia, and transferred to new Hagem agar dishes of the same dimensions and allowed to culture again under the exact procedure as before. This process was repeated for a third time, except mycelia were allowed to grow to the end of the plate, after which a sterilized spatula was used in a fume hood to carefully scrape the surface and transfer the mycelia into labeled Eppendorf tubes, where they were stored at 4 C° until further use. Fruiting body segment samples of H. croceus collected by Stellan Sunhede in southern Sweden were also transferred to Hagem agar dishes by him, then packed and shipped to the SLU department of Forest Mycology and Plant Pathology, where I cultured them on new dishes following the same protocol mentioned above.

To compare mycelial growth rates, I sampled five fruiting bodies of the commonly occurring

7 polypore F. pinicola in Stadskogen, Uppsala in October 2015 and cultured them in the same manner as H. croceus. Then, three random cultures from each species were selected, transferred to 14 cm diameter Hagem agar petri dishes and divided into three replicates each, labeled respectively, totaling 18 cultures (fig. 4). Each dish was measured daily along a coordinate plane (north, east, south, west), and the furthest-reaching mycelial growth along each axis was recorded. After ten days, averages of coordinate measurements were taken per replicate, then condensed into one overall average per species, and analyzed in excel to display mycelial growth and rate of growth. I then ran a t-test to obtain a p-value for the growth rate and growth area measurements and also a Pearson’s R value for positive or negative correlation on growth rates.

To determine mating type, the 42 homokaryon single-spore isolates were each removed from the respective tubes in 5mm sample sizes via sterilized forceps and transferred to the same Hagem agar medium, labeled HC6.1 through HC6.42. I followed the exact protocol as field- collected samples listed above. 22 of the 42 samples regenerated and were used for the study while the remaining dead were discarded.

Fig. 5: LEFT: Example of homokaryon crossings, showing variety of structures between mated and unmated strains. RIGHT: Unmated strain displaying mycelial barrier between incompatible partners.

Mating type determination 5 x 5 mm agar samples with established fungal mycelia were cut from each of the 22homokaryon cultures and transferred to separate 9 cm diameter agar plates containing a 5 x 5 mm complimentary homokaryon samples, with a 2 cm gap dividing them, until I crossed all pairs once with an opposing strain in a non-repetitive manner (fig. 5). This resulted in 231 dishes, which were labeled, sealed, and allowed to culture for 50-55 days at 23 C° until I observed visible changes in growth patterns and mycelia covered at least 90% of the plate.

Plates were then scored according to morphological differences. Dishes showing visible incompatibility traits (mycelial barriers, discoloration along walls built, or lack of mycelia between the two samples) were marked as incompatible, while those displaying a uniform merging of mycelia were examined for clamp connections under a Leica microscope (Leica, Wetzlar, Germany) (fig. 6). I then generated a matrix in an Excel sheet where each sample was marked with an “x” for mated (clamp connections observed) and “u” for unmated (plates with visible signs of rejection). The ratio of “x” to “u” was used to generate a percentage for mating type using the following formula: (x/total plates)*100 = sibling compatibility %. 25% of “x” meant the species is tetrapolar and 50% of “x” meant the species is bipolar.

Fig. 6: LEFT: Unmated homokaryon hyphae, no clamp connections and possible pheromones/lipids involved in wall construction detected, as indicated by red arrows. RIGHT: Red arrows point to clamp connections that confirm existence of mated heterokaryon hyphae.

DNA extraction from fungal mycelia for RAPD DNA extraction for RAPD was performed on all 42 heterokaryon samples. I followed SLU guidelines for DNA extraction, purification, amplification, and electrophoresis (Clemmensen et al., 2011). For extraction, a 1 cm mycelial ball was placed in respectively labeled 2 ml screw-cap tubes containing 6 small glass beads. 1 μl of 3% CTAB was pipetted into each tube and agitated by hand after sealing to ensure chemical saturation of contents. The samples were then placed in a Precellys 24 homogenizer (Bertin Corp, Rockville, MD, USA) and centrifuged for two cycles at 5000 RPM for 30 seconds before being vortexed briefly via VortexGenie (Scientific Industries, Bohemia, NY, USA) and placed in a ceramic heating block at 65 C° for one hour to facilitate DNA denaturing. Following denaturing, 900 μl of each sample was pipetted into new and labeled 1.5 ml Eppendorf tubes (old tubes containing suspension remnants were discarded), topped with

600 μl of chloroform, and centrifuged for 15 minutes at 9.6 x g. Following the centrifuge cycle, the upper phase containing separated DNA of each sample was pipetted into new and respectively labeled 1.5 ml Eppendorf tubes containing 600 μl of isopropyl alcohol. These samples then precipitated for 30 minutes at 22 C°. To facilitate DNA pellet formation, samples were centrifuged at 16.0 x g for 10 minutes. Pellets were then washed with 200 μl of 70% ethanol after the Fig. 7: Standard PCR protocol for RAPD method, displaying supernatant was pipetted out, and temperature and duration for each cycle. centrifuged at 16.0 x g for 5 minutes. The resulting supernatant was pipetted from each respective tube and pellets were left to dry under strict supervision in a 65 C° heating block just until all traces of ethanol evaporated. Each pellet was then suspended in 30 μl milliQ water and stored in -20 C°.

To evaluate DNA content, an ND-1000 program was used in conjunction with a NanoDrop 3300 fluorospectrometer (Thermo Fisher Scientific, Waltham, MA, USA) where 2 μl of each respective sample was pipetted onto the sensor head and recorded for concentration. A 2 μl sample was removed from each tube and diluted in new and labeled Eppendorf tubes to a DNA concentration range of 10-10.8 ng / μl using 10mM TRIS, with a backup stock reserve of original extracted DNA saved in -20 C°.

PCR amplification, RAPD analysis and electrophoresis 28 μl of a master mix consisting of DreamTAQ polymerase buffer, DreamTAQ Polymerase, dNTP, MgCl, TAQpol, and 2.4 μl of JO-1 primer was added to each PCR tube containing 2 μl of template DNA. PCR reactions ran with the DNA mixtures but also a control consisting of 30 μl MilliQ water and 30 μl pure master mix. Samples were centrifuged briefly using a table top mini centrifuge (Denville Scientific, Holliston, MA, USA) and inserted into an Applied Biosystems 2700 thermal cycler (Applied Biosystems, Foster City, CA, USA), programmed for 40 cycles using a custom program (fig. 7). Post PCR samples were stored at 4 C° until needed.

DNA purity was tested via NanoDrop prior to electrophoresis. Gels were prepared with a 1% agar to 99% SB buffer ratio and 1.5 μl stain (Nancy-520). Each gel contained a GR DNA ladder marker on each end of the tray, PCR product, and sample master mix to measure control and contamination of foreign DNA in samples.

Electrophoresis chambers were surrounded by ice packs (which were rotated out every four hours) then ran for 16 hours at 60 volts using randomly selected samples 8 times and 8 hours at 140 volts with the same run number. Each gel was then scanned and saved using QuantityOne software (Piovanelli, 2006).

External genomic sequencing preparations Sequenced samples were from varying localities throughout Sweden, Latvia, and Lithuania (fig. 9), with several replicates in some localities (“36” represents replicates in Öland, appendix 2).

Fig. 9: Map of the southern and central Baltic Sea area representing location of genome sequenced heterokaryon individuals of H. croceus.

Pacific Biosciences platform (PacBio) Of the homokaryotic cultures grown, I chose one (HC27) for genome DNA sequencing using Pacific Biosciences platform, as its mycelia grew the most rapidly during initial culturing. HC27 underwent the same DNA extraction and purification protocol mentioned above. These homokaryons provided a genomic library to be used as a template for genome assembly for the heterokaryon reads later provided by NGI SciLifeLab (NGI) labs. The sequenced reads were then assembled by Uwe Menzel at the SLU Forest Mycology and Plant Pathology bioinformatics department.

DNA extraction and purification for NGI Illumina sequencing: NGI provided a preparation protocol I used for all heterokaryon samples (Gillner, 2016), under which new cultures grown for DNA extraction were also measured for quality control via electrophoresis and NanoDrop. This protocol included RNAse treatment for purification of samples, which was followed according to SLU’s specifications (Personal communication, Ihrmark, 2013). New cultures were grown using the same methods as previously described, in three replicates for each of the 45 heterokaryons sampled, for three weeks until mycelia covered the agar plate. Resulting mycelia was then carefully removed from each dish and transferred to an appropriately labeled tube to be stored for 12 hours at -4 C° (45x3, 115 tubes total). Each sample then underwent DNA extraction identical in procedure as previously extracted samples, with the addition of DNA purification as a final step.

For DNA purification, tubes from the previous DNA extraction process containing heterokaryotic samples were filled with 10mM TRIS (pH8) until they were all 200 μl in volume. 1 μl of RNAse was added to each tube and allowed to rest for 60 minutes. Then, 200 μl of chloroform was added and samples were centrifuged for 5 minutes at 9.6 x g, after which the suspension was pipetted into new and appropriately labeled 1.5 μl Eppendorf tubes. 20 μl of NaAC and 500 μl EtOH was added and samples were allowed to rest for 10 minutes prior to centrifuging for 7 minutes at 17.0 x g. The remaining suspension was removed and purified DNA pellets were washed with 70% EtOH, centrifuged again for 5 minutes at 4.1 x g, and allowed to dry after the suspension was again removed. Dry pellets were suspended in 56 μl 10 mM TRIS solution and stored at 4 C° overnight.

Fig. 10: Sample gel results example with sample names and Concentration for all purified samples markers at top, sent along with barcode plate as requested by via NanoDrop were recorded NGI. (acceptable concentration levels ranged from 50-300 ng/μl) and diluted if necessary with the same 10 mM TRIS solution. Concentration and quality of samples were documented prior to shipping and measured via Nanodrop and quality via electrophoresis on 0.8% agar gels (fig. 10) in TAE buffer solution (80V for 90 minutes), as specified by NGI protocol. The resulting gel images, concentration values, sample number and volume were added to an excel spreadsheet provided by the company. NGI

12 also provided a 96-well barcoded colored plate; following approval (records of gels and nanodrop images were sent prior to sample-loading, samples were loaded in columns from column 1 row A onward and recorded in the spreadsheet as well. Genomes were then sequenced via NGI SciLifeLab in Stockholm using the Illumina HiSeq2500 platform (Illumina, San Diego, CA, USA).

Bioinformatics analysis of resulting sequenced data All Illumina and PacBio sequenced data was processed and analyzed with help from Uwe Menzel at the bioinformatics division in the Department of Forest Mycology and Plant Pathology at SLU. PacBio’s reference sequence was used as a library for all 41 heterokaryon sequence reads, which were aligned to the long homokaryon reference sequence using an index created via bowtie2 (Langmead et al., 2009). Trimming was completed using trimmomatic (Bolger et al., 2014), where adapters and low quality nitrogenous bases were removed. Following trimming, sequences were run through fastQC, a quality control tool primarily used to screen high-throughput sequence data (Andrews, 2010). Next, since nearly 97% of the sequences were paired, unpaired sequences were not used for the study and overrepresented ones were removed. Mapping was completed using hisat2 (Kim et al., 2015), where paired and trimmed sequences were aligned concordantly one time with an average of 94.7% overall alignment rate. These sequences were then run through SAMtools for variant calling and mapping quality check (Li et al., 2009). Next, to confirm that the samples sequenced were all of the same species and to identify foreign fungal contaminants all genomes were BLASTed using a previously sequenced H. croceus ribosomal DNA (JQ821320 ribosomal sequence in GENBANK; Dvorak et al., 2014). Samples were also BLASTed against a portion of F. pinicola genome containing highly conserved ITS regions.

Single nucleotide polymorphisms (SNPs) were used from the Illumina data of heterokaryons to construct a map for population differences in fastStructure (Raj et al., 2014), where maps were generated according to K=2 through K=6 and arranged by country, county, and locality site. HHOAK17 from the site Halltorpshage on Öland was set as a control, since cultures from the site were collected from the same tree and fungal genetic individual during 1992, 1994, and 2015. Several phylogenetic trees using Illumina data were created using PhyloPhlan (Segata et al., 2013), which mapped out population structure using FastTree 2.1 Maximum Likelihood trees.

Results

Ecology

FIG. 11: Sample name, Localities, position of fruiting bodies, and images of respective H. croceus sporocarps and their environments upon collection. In general, I found H. croceus fruited on oaks that were 300+ years of age and around old wounds from previously broken branches, where nearly all sporocarps apart from HC5 were at least 3 meters above the ground (fig. 11, appendix 1). HC5 was fruiting between 1 and 1.5 m above the ground in a stem crack. During field observations, old oaks that grew in suffocating environments consisting of young and mature trees (predominately mixed hardwood species including oak, hazel, and linden) produced the most fruiting bodies. There was also no predictable pattern to sporocarp production: some sites where H. croceus was found showed no fruiting structures, even though previous site records on artdatabanken indicated regular annual fruiting several years before. In some areas, there were no documented observations of sporocarps in the last ten years (appendix 1). Of the 28 sites visited, I found 7 had fruiting bodies, resulting in a 25% rate of occurrence.

Mycelial growth comparison The 18 cultures of F. pinicola and H. croceus I used for estimation of mycelial growth rate showed significant differences, where p≤0.0001 (t-value: 7.42, df: 10, appendix 4) in growth rate (millimeters per day) (fig. 12a). F. pinicola grew 42.6% faster than H. croceus in the first

Mycelial growth rate in F. pinicola and H. croceus 6

4 R² = 0.67186

3 F. pinicola 2 Growth (mm) R² = 0.55651 1

Date Fig. 12a: Top: Growth rate trends comparing the two species, showing increase for H. croceus and decrease for F. pinicola for the last six days. 12b: Bottom: Areal mycelial growth measured during 11-day trial, F. pinicola grew more than H. croceus.

Measured growth area comparison between F. pinicola and H. croceus

45 40 y = 3.8029x - 161164 R² = 0.99677 35 ) 2 30 F. pinicola 25 H. croceus 20 15 y = 1.0803x - 45781 Growth (mm R² = 0.95661 10 5 0 1/9/2016 1/11/2016 1/13/2016 1/15/2016 1/17/2016 1/19/2016 1/21/2016 1/23/2016 Date

five days, but then slowed for the remainder of the trial by an average of 24% . After five days, H. croceus increased in growth by 43.8%. H. croceus experienced an initial delay in growth on the second and ninth days but then increased again on the third and 10th days.

Areal growth was significantly (p≤0.00002, t-value: 6.22, df: 10, fig. 12b, appendix 4) more for F. pinicola and resulted in 271.2 mm and 60.7 mm of total growth area for F. pinicola and H. croceus respectively over 11 days. Pearson’s R showed a negative correlation (-0.23, appendix 4) in growth rates between both species.

Mating type determination All 231 homokaryon single spore isolate plates showed either compatible or incompatible interactions (fig. 13). After scoring these plates according to the methods stated earlier, 57 of the 231 tested plates had mated. Thus, this resulted in a 24.7% (57/231 = 0,2468*100 = 24.7%) sibling compatibility, placing H. croceus under a tetrapolar mating type. Fig. 13: Example of some plates of homokaryotic H. croceus strains H6 showing mated mycelia on the left and unmated with barriers on right.

RAPD electrophoresis I originally used a Randomly Amplified Polymorphic DNA (RAPD) method to study the genetic population structure of H. croceus. However, resulting gels from the electrophoresis did not produce readable results of sufficient resolution. Bands were undifferentiated or smeared and thus could not provide insight into population structure or genetic distance among different individuals (Fig. 14, right). F. pinicola was also tested using the same methods, but produced more readable results on gels (fig. 14, left), showing some band differentiation between

Fig. 14: Left: RAPD gel of F. pinicola, showing some band distribution and differences between samples. Right: RAPD gel of H. croceus, displaying bands of nearly uniform resolution between samples. individuals. On several occasions, the buffer in which gels ran overheated, even when icepacks were rotated every four hours. Nearly all populations resembled one another closely or DNA within gels degraded during electrophoresis runs to where band differentiation was unreadable. Changing run time and voltage seemed to have no effect on readable results.

PacBio and Illumina genome sequencing 32.6 GB of data was generated in total for the species, PacBio and Illumina data showed H. croceus to have a genome length of 32.837.733 bp. 40 out of 44 samples were sequenced on Illumina’s HiSeq2500 (HiSeq Control Software 2.2.58/RTA 1.18.64) platform by NGISciLife labs in Stockholm, 4 samples failed the sequencing process. A project report was generated, consisting of sample names, (appendix 2).

Population structure PhyloPhAn’s maximum likelihood tree generated a population structure with sample sites

17 arranged randomly, except for a small subset of individuals from Halltorpshage oak17 (HHoak17), but still produced outliers within this population (fig. 15). Latvian and Lithuanian populations were not grouped together and were also distributed around the tree randomly.

SNP analysis via fastStructure showed two distinct population groups; regardless of whether population differentiation was selected to sort according to K=2 or K=6 variants. The k variants were selected to group each population according to the number of possible variants in a population subset (2 variants, 3 variants …6, etc.), but the results were always that the groups diverged into the same two categories, regardless of what k variant was selected. The separation is shown in the site map for HHoak17 (fig. 16a, 16b), where some samples belonged to the same North-European group as all samples, while another subset belonged to its own distinct population. The mean Q value (probability that the specimen belongs to a population) for this statistical test also supported these populations diverging into their own groups since HHoak17 under K=2 showed nearly 100% probability that it belonged in a group separate from the other North European samples (appendix 3).

Fig. 15: Maximum likelihood tree from PhyloPhlAn, displaying all strains from Swedish, Latvian, and Lithuanian localities. Names of H. croceus strains at the end of branches are as in Appendix 2.

Fig 16a: Population structure where K=2 using single nucleotide polymorphisms (SNPs), arranged according to country (TOP), county (CENTER), and site (BOTTOM). Swedish population showing two distinct populations, blue representing a separate outgroup from the Northern European population (red). References in appendix 3.

Fig 16b: Population structure where K=6 using single nucleotide polymorphisms (SNPs), arranged according to country (TOP), county (CENTER), and site (BOTTOM). Swedish population showing two distinct populations, red representing a separate outgroup from the Northern European population (blue). References in appendix 3.

Discussion Ecology The surrounding habitats of the sampled sporocarps on old oak trees varied, although most from successful fruiting cultures were in areas that were becoming increasingly colonized by younger, aggressively growing hardwood trees (appendix1). Sporocarps were rarely observed in landscapes where oaks thrived in open pastures without threat from competition. It is possible that several factors vary between open pasture and forest oak trees. Light, substrate moisture, and temperature have all shown to impact fruiting cycles in fungi (Boddy et al., 2014), and habitats in open areas may be drier. Additionally, occurrence of H. croceus was at 25% (appendix 1) of visited sites, meaning that fruiting bodies on the majority of trees do not occur on an annual basis. Although the sporocarps of this fungus are annual (ArtDatabanken, 2015), whether a fruiting body is produced annually can rely on other factors; perhaps environmental conditions and resource availability for buildup of fungal fruiting bodies. Climate change, for example, may also influence fruiting schedule as moisture levels and temperatures vary annually (Ágreda et al., 2016). However, upon discussing ecology of this species with Stellan Sundhede, who provided the samples for southern Sweden, it was found that several old oaks growing in open pastures fruited annually for decades (Sunhede, 1997). Thus, it is likely that ecological notes from 28 localities is simply not enough to make an informed decision regarding the fruiting patterns in H. croceus and further sampling or input from other sources is needed.

According to the ecological sampling data of fruiting body location, it is possible that H. croceus favors broken branches above hollows or other areas (fig. 11, appendix 1), as an entry point in damaged tree sections. If this is indeed evidence for a favorable colonization point for the fungus, then conservation efforts could concentrate on management methods such as oak veteranisation to create future suitable habitats for the species (Bengtsson et al., n.d.). This would mean that conservationists could manually break branches on oak trees to increase chances of H. croceus colonization. However, more research is needed before a conclusion can be made, since it is also possible that these areas are simply an exit point for the fruit bodies. In addition, many external factors including competition from other wood-inhabiting fungi, substrate availability and/or condition of the oak trees (Schilling et al., 2015), could influence establishment of this species.

Growth comparisons in Petri dishes between H. croceus and F. pinicola demonstrated that F. pinicola mycelia grew at a significantly faster rate and also expressed more areal mycelial growth (fig. 12). H. croceus’s growth rate increased 43.8% in the last five days of the trial however, while F. pinicola’s decreased 42.6% over the same time interval, suggesting a possible colonization strategy difference between the two species. I chose F. pinicola, specifically because it is commonly occurring which provided a contrast to the rare H. croceus. Examining

22 growth patterns between the two species could help shed light on what makes one fungus abundant while another limited in wood-decay communities. When competing with other species that are faster growing, H. croceus could be out-competed. Hence, F. pinicola’s aggressive growth could mean it is more suited as a colonizer who rapidly establishes and consumes its substrate to mate and sporulate as quickly as possible, much like ruderal species do following primary succession (Dighton and White, 2005). If the fungal colony present while H. croceus grows is highly concentrated, providing competition for available substrate, then this species may have adapted to more slow growing methods; maybe correlating to its mycelial longevity. Or, similar to Phellinus nigrolimitatus, it could exhibit a dominant decay strategy where the species establishes in material that is at the final stage of decomposition (Jönsson et al., 2008), where only highly decomposed material is left for digestion. H. croceus’s decomposition potential may play a role in this, since this fungus is a white-rot species which has evolved to break down hemicellulose and lignin, the last remaining compounds in plant cell walls left after brown-rot fungi (such as F. pinicola) have broken down the initial cellulose (Cowling, 1961). Thus, the results of my experiment support my first hypothesis; H. croceus is slow growing and when considering the factors mentioned above, this trait likely impacts its establishment.

However, this growth comparison was made in a lab under controlled conditions and the species were not permitted to interact on the same substrate. In nature, it is probable that these species may both grow differently. Either way, this helps to understand how H. croceus grows, especially when compared to other more abundant fungi.

Mating type determination Since Basidiomycetes can exist for years before fruiting occurs, paired with long distances between host trees and habitats containing compatible partners creates a selection process that would favor higher outcrossing, largely in part because most homokaryotic genotypes encountered at random are compatible with one another (James, 2015). Thus, tetrapolarity would prove beneficial for H. croceus, whose occurrence is fragmented and rare. Although, inbreeding depression could still result from offspring mating with one another in a fragmented population, tetrapolar fungi are less likely to experience it since sexual compatibility between sibling homokaryons (mycelia containing only one mating type (Ainsworth, 2008)) is 50% less probable than in bipolar mating types (James, 2015). Thus, even though this species is highly fragmented in population size, its chances of inbreeding depression are still lower since this mating type is thought to be far less vulnerable to inbreeding than bipolar fungi (James, 2015). These factors all support hypothesis 2, where I predicted H. croceus to exhibit a tetrapolar mating type which results in a 25% mating compatibility between sibling spores. For many polypore species, spores will fall within the vicinity of the sporocarp (Stenlid and Gustafsson,

2001), meaning a 25% compatibility between these spores could act as protection against inbreeding. Mating type alone however, does not provide the entire picture of how this species propagates. Further researching components involving sporocarp formation including environmental factors, spore release and deposition, and substrate colonization could help expand our understanding of H. croceus sexuality and perhaps other polypore species as well.

RAPD and electrophoresis RAPD electrophoresis was initially chosen because it allows us to process and compare large amounts of strains quickly and at a low cost. In addition, in order to run the method, no previous sequence knowledge of the studied species is needed (Feng et al., 2009). This process was repeated approximately 15 times. As can be seen from fig. 14, repeated attempts failed to produce gels with a high enough resolution to decipher differences among the genotypes. This method is used readily in research labs for fungi, although it was not successful for H. croceus, likely in part due to its homogenous genetic structure. RAPD can also be sensitive in that it can fluctuate from gel to gel; there have been previous observations regarding reproducible results with some specimens (Tommerup et al., 1995). Since it can take up to 16 hours to run, many variables are present that can skew results. For example, the buffer can overheat and degrade the DNA samples, which are sensitive to higher temperatures (Westermeier, 2016).

Even on gels that were somewhat readable, bands were too clustered together to show any clear reading of smaller bands. However for F. pinicola, gels contained more differentiated bands at a readable resolution when compared to H. croceus (fig. 14). This proves a difficult situation for when genetic population diversity is to be examined and readable band width is required to evaluate differences. H. croceus has never been used for this method and it is possible the species’ genetic material is too sensitive. Even with the adjustment of gel density, varying voltages and run times, there were no readable results. Thus, after these failed attempts, an alternative method was devised where full sequencing of sample genomes was to be completed via PacBio and Illumina sequencing at NGI SciLifelab.

Population structure

Fig. 17: PhyloPhlAn maximum likelihood tree displaying sites from Sweden, Latvia, and Lithuania. HHoak17 does not form separate clade as expected, nor does it form an out-group with other Halltorpshage oaks on Öland. Lithuania and Latvia also do not form out groups and are instead distributed throughout the tree. Names of H. croceus strains at the end of branches are as in Appendix 2. Using FastTree (which infers maximum likelihood relatedness) constructed by PhyloPhlAn, individuals were highlighted according to region to possibly shed light on any correlations between the population groups. As can be seen from fig. 17, there is not enough of a pattern detected between individuals to be considered significant. There are outliers in every group presented. Halltorpshage oak17 (HHoak17) for example, comes from a control group where each sporocarp sample stems from the same tree and genetic individual sampled in 1992, 1994, and 2015, yet HHoak17 shows up in other clades when it should be clustered together. Upon further investigation, it was discovered that PhyloPhlAn uses 400 universal bacterial proteins as

25 a comparison to construct its trees, thus this could present a problem when strictly fungal genomes are to be analyzed. A different method is currently under way, but the results will not be finished in time for this paper.

However, when analyzing SNP data, HHoak17 showed up as two different populations (fig. 16a, 16b), one of which was considered to be outside of the North European group with 99.9% probability (meanQ, appendix 3), suggesting it may be a different species. However, when BLASTed against ribosomal DNA previously deposited on the NCBI website, there was no indication that this strain belonged to any other species except for H. croceus. Later attempts were made to use other programs, such as progressiveMAUVE (Darling et al., 2010), but the results took weeks to process and are currently ongoing. One possibility is that there was a labeling error with HHoak17, and during collection it was sampled from another genetic individual and mislabeled. Thus, when analyzed it appears as two distinct populations. However, HHoak17 also appears as a separate population from the pan-European subset, which is puzzling. It is possible that depending on seasonality, many spores can travel vast distances (Savage et al., 2012), thus HHoak17 could be an individual from another continent that falls outside of the pan European population group.

There is also a possibility that simply not enough time has passed to properly illustrate a genetic differentiation between individuals in population groups. If H. croceus is indeed a long-lived individual, it may be possible that its mycelia can exist in substrate for centuries before mating even takes place. Mated heterokaryons can continue to grow and digest substrate indefinitely before environmental conditions are met and fruiting can proceed (Casselton and Olesnicky, 1998). Thus, centuries could pass from the time it takes for the slow-growing fungus to colonize substrate, establish, mate, and produce fruiting structures.

Fig. 18: Differences in deciduous forest cover from 2000BP to present day (Björse and Bradshaw, 1998), showing decrease in deciduous cover.

Other endangered fungal species have been found to have similar homogenous genetic structure, which has possibly been attributed to ecological history following glacial retreat (Kauserud and Schumacher, 2002). 10,000 years ago, an ice sheet covered northern Europe and oaks had not colonized the area until much later, rendering this plausible. Landscape history thus should be considered especially for slowly growing and long-lived species, as they are unable to quickly reproduce and generate progeny; so their slow generational times serves as a disadvantage in unstable environments (Verin et al., 2015). 8000 BP, water covered the area where many H. croceus samples were recently taken in Sweden (fig. 1; Björck, 1995. Modified), meaning oaks had not colonized there yet while they did begin to colonize more southern regions (Jensen Svejgaard et al., 2002). Further, agriculture has been present in Sweden for the last 6,000 years (Berglund, 1991), suggesting landscape alteration could have also affected host oaks. Additionally, further analysis of 2,000 years of forest dynamics in Sweden shows human intervention in landscape alteration to result in significant decline of deciduous habitat (Fig. 18; Björse and Bradshaw, 1998). Thus, when considering all of these variables and the slow

27 generational times of this species, it is possible that if H. croceus migrated with its host tree into Sweden ca. 8,000 years ago, thrived for 6,000 years, and then experienced habitat fragmentation in the last 2000 years, changes in the genome would be little to non-existent. This could explain why population variation between individuals in our sequenced heterokaryons appears to be minimal. Hence, even though genetic variation between populations in the Baltic Sea area appears to be minimal, the source of this variation cannot be concretely determined at this time. Thus hypothesis 3, which predicted that Swedish populations of H. croceus would exhibit low genetic diversity and minor to absent gene flow from neighboring European populations, can neither be confirmed nor rejected.

Since the genetic homogeneity of H. croceus was not directly pinpointed, further research into this study is needed. A tremendous amount of data has been collected and it is only a matter of sorting through it more thoroughly than could be possible under the relatively short duration of this project. Results from this study when analyzed more extensively could be expanded into other projects; including habitat management for endangered wood-inhabiting species and conservation projects involving rehabilitation of various threatened fungi. These efforts would all help expand our understanding of Northern Europe’s beloved ancient oak forests.

Acknowledgements I thank Audrius Menkis and Anders Dahlberg for their support and guidance as supervisors during this study, Uwe Menzel for his contribution to the bioinformatics section, Rimvys Vasaitis for allowing me to use his 42 homokaryon single-spore Lithuanian isolates, and Stellan Sunhede for providing samples from southern Sweden and ecological insight from his decades of H. croceus observations. Your assistance throughout this project has been most appreciated.

References Ágreda, T., Águeda, B., Fernández-Toirán, M., Vicente-Serrano, S.M., Òlano, J.M., 2016. Long-term monitoring reveals a highly structured interspecific variability in climatic control of sporocarp production. Agric. For. Meteorol. 223, 39–47. doi:10.1016/j.agrformet.2016.03.015 Ainsworth, G.C., 2008. Ainsworth & Bisby’s dictionary of the fungi. Cabi. Andrews, S., 2010. . FastQC Qual. Control Tool High Throughput Seq. Data. URL http://www.bioinformatics.babraham.ac.uk/projects/fastqc ArtDatabanken, 2015. Hapalopilus croceus - Saffronsticka [WWW Document]. URL http://artfakta.artdatabanken.se/taxon/121 (accessed 1.26.16). Aust, S., 1995. Aust SD. Mechanisms of degradation by white rot fungi. Environ. Health Perspect. 103, 59–61. Bengtsson, V., Hedin, J., Niklasson, M., n.d. Veteranisation of oak–managing trees to speed up habitat production. Trees Wood Colour 61. Berglund, B.E., 1991. The cultural landscape during 6000 years in southern Sweden: the Ystad project. Ecol. Bull., Munksgaard, Copenhagen 41. Björck, S., 1995. A review of the history of the Baltic Sea, 13.0-8.0 ka BP. Quat. Int. 27, 19–40. Björse, G., Bradshaw, R., 1998. 2000 years of forest dynamics in southern Sweden: suggestions for forest management. For. Ecol. Manag. 104, 15–26. Boddy, L., Büntgen, U., Egli, S., Gange, A.C., Heegaard, E., Kirk, P.M., Mohammad, A., Kauserud, H., 2014. Climate variation effects on fungal fruiting. Fungal Ecol. 10, 20–33. doi:10.1016/j.funeco.2013.10.006 Bolger, A.M., Lohse, M., Usadel, B., 2014. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120. doi:10.1093/bioinformatics/btu170 Casselton, L.A., Olesnicky, N.S., 1998. Molecular genetics of mating recognition in basidiomycete fungi. Microbiol. Mol. Biol. Rev. 62, 55–70. Catană, R., Mitoi, M., Ion, R., 2013. The RAPD techniques used to assess the genetic diversity in <i>Draba dorneri</i>, a critically endangered plant species. Adv. Biosci. Biotechnol. 04, 164–169. doi:10.4236/abb.2013.42024 Coates, D., Rayner, A.D.M., Boddy, L., 1985. Interactions Between Mating and Somatic Incompatibility in the Basidiomycete Stereum hirsutum. New Phytol. 99, 473–483. Coelho, M.A., Sampaio, J.P., Gonçalves, P., 2010. A Deviation from the Bipolar-Tetrapolar Mating Paradigm in an Early Diverged Basidiomycete. PLoS Genet. 6, e1001052. doi:10.1371/journal.pgen.1001052 Cowling, E.B., 1961. Comparative biochemistry of the decay of sweetgum sapwood by white-rot and brown-rot fungi. US Dept. of Agriculture. Dahlberg, A., 2006. Dahlberg, A., 2006. The fauna and flora on oaks - how important are the Swedish oak habitats in a Europaen perspective? . The oak - history, ecology, management and planning, Linköping, Sweden, pp 18-19. Dahlberg, A., Croneborg, H., 2003. 33 threatened fungi in Europe. Complementary and revised information on candidates for listing in Appendix I of the Bern Convention T-PVS (2001). Darling, A.E., Mau, B., Perna, N.T., 2010. progressiveMauve: Multiple Genome Alignment with Gene Gain, Loss and Rearrangement. PLoS ONE 5, e11147. doi:10.1371/journal.pone.0011147 Dighton, J., White, J., 2005. The Fungal Community: Its Organization and Role in the Ecosystem, in: Mycology. CRC, pp. 383–397. Dvorak, D., Betak, J., Tomsovsky, M., 2014. Aurantiporus alborubescens (Basidiomycota, Polyporales)– first record in the Carpathians and notes on its systematic position. CZECH Mycol. 66, 71–84. Environmental Protection Agency, 2017. Artportalen [WWW Document]. Rapp. För Växter Djur Och Svampar. URL https://www.artportalen.se/ Esser, K., Lemke, P.A., Bennett, J.W. (Eds.), 1994. The Mycota: a comprehensive treatise on fungi as experimental systems for basic and applied research. Springer-Verlag, Berlin ; New York. Feng, K., Wang, S., Hu, D.J., Yang, F.Q., Wang, H.X., Li, S.P., 2009. Random amplified polymorphic DNA (RAPD) analysis and the nucleosides assessment of fungal strains isolated from natural Cordyceps sinensis. J. Pharm. Biomed. Anal. 50, 522–526. doi:10.1016/j.jpba.2009.04.029 Fraiture, A., Otto, P., 2015. Distribution, Ecology and Status of 51 Macromycetes in Europe: Results of the ECCF Mapping Programme. Scripta Botanica Belgica, Meise.

Gillner, K., 2016. Sample requirements for genomics projects. Grantham, N.S., Reich, B.J., Pacifici, K., Laber, E.B., Menninger, H.L., Henley, J.B., Barberán, A., Leff, J.W., Fierer, N., Dunn, R.R., 2015. Fungi identify the geographic origin of dust samples. PloS One 10, e0122605. Ihrmark, K., 2013. DNA-prep mycel för genomsekvensering. IUCN, 2013. Hapalopilus croceus [WWW Document]. URL http://iucn.ekoo.se/iucn/species_view/445931/ (accessed 1.26.16). James, T.Y., 2015. Why mushrooms have evolved to be so promiscuous: Insights from evolutionary and ecological patterns. Fungal Biol. Rev. 29, 167–178. doi:10.1016/j.fbr.2015.10.002 James, T.Y., Sun, S., Li, W., Heitman, J., Kuo, H.-C., Lee, Y.-H., Asiegbu, F.O., Olson, A., 2013. Polyporales genomes reveal the genetic architecture underlying tetrapolar and bipolar mating systems. Mycologia 105, 1374– 1390. doi:10.3852/13-162 Jensen Svejgaard, J., Gillies, A., Munro, R., Flemming Madsen, S., Roulund, H., Lowe, A., Csaikl, U., 2002. Chloroplast DNA variation within the Nordic countries. For. Ecol. Manag. Jones, S.K., Bennett, R.J., 2011. Fungal mating pheromones: Choreographing the dating game. Fungal Genet. Biol. 48, 668–676. doi:10.1016/j.fgb.2011.04.001 Jönsson, M.T., Edman, M., Jonsson, B.G., 2008. Colonization and extinction patterns of wood-decaying fungi in a boreal old-growth Picea abies forest: Colonization-extinction dynamics of wood-decaying fungi. J. Ecol. 96, 1065–1075. doi:10.1111/j.1365-2745.2008.01411.x Kauserud, H., Schumacher, T., 2002. Population structure of the endangered wood decay fungus Phellinus nigrolimitatus (Basidiomycota). Can. J. Bot. 80, 597–606. doi:10.1139/b02-040 Kim, D., Langmead, B., Salzberg, S.L., 2015. HISAT: a fast spliced aligner with low memory requirements. Nat Meth 12, 357–360. Langmead, B., Trapnell, C., Pop, M., Salzberg, S.L., 2009. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biol. 10, R25. Li, H., Handsaker, B., Wysoker, A., Fennell, T., Ruan, J., Homer, N., Marth, G., Abecasis, G., Durbin, R., 1000 Genome Project Data Processing Subgroup, 2009. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079. doi:10.1093/bioinformatics/btp352 NIETO FELINER, G., GUTIÉRREZ LARENA, B., FUERTES AGUILAR, J., 2004. Fine-scale Geographical Structure, Intra- individual Polymorphism and Recombination in Nuclear Ribosomal Internal Transcribed Spacers in Armeria (Plumbaginaceae). Ann. Bot. 93, 189–200. doi:10.1093/aob/mch027 Nieuwenhuis, B.P.S., Debets, A.J.M., Aanen, D.K., 2011. Sexual selection in mushroom-forming basidiomycetes. Proc. R. Soc. B Biol. Sci. 278, 152–157. doi:10.1098/rspb.2010.1110 Piovanelli, M., 2006. Quantity One User Guide for Version 4.6.3 Windows and Macintosh. Raj, A., Stephens, M., Pritchard, J.K., 2014. fastSTRUCTURE: Variational Inference of Population Structure in Large SNP Data Sets. Genetics 197, 573–589. doi:10.1534/genetics.114.164350 Ryvarden, L., Melo, I., 2014. Poropoid fungi of Europe. Fungiflora. Savage, D., Barbetti, M.J., MacLeod, W.J., Salam, M.U., Renton, M., 2012. Seasonal and Diurnal Patterns of Spore Release Can Significantly Affect the Proportion of Spores Expected to Undergo Long-Distance Dispersal. Microb. Ecol. 63, 578–585. doi:10.1007/s00248-011-9949-x Schilling, J.S., Kaffenberger, J.T., Liew, F.J., Song, Z., 2015. Signature Wood Modifications Reveal Decomposer Community History. PLOS ONE 10, e0120679. doi:10.1371/journal.pone.0120679 Schoch, C.L., Seifert, K.A., Huhndorf, S., Robert, V., Spouge, J.L., Levesque, C.A., Chen, W., Fungal Barcoding Consortium, Fungal Barcoding Consortium Author List, Bolchacova, E., Voigt, K., Crous, P.W., Miller, A.N., Wingfield, M.J., Aime, M.C., An, K.-D., Bai, F.-Y., Barreto, R.W., Begerow, D., Bergeron, M.-J., Blackwell, M., Boekhout, T., Bogale, M., Boonyuen, N., Burgaz, A.R., Buyck, B., Cai, L., Cai, Q., Cardinali, G., Chaverri, P., Coppins, B.J., Crespo, A., Cubas, P., Cummings, C., Damm, U., de Beer, Z.W., de Hoog, G.S., Del-Prado, R., Dentinger, B., Dieguez-Uribeondo, J., Divakar, P.K., Douglas, B., Duenas, M., Duong, T.A., Eberhardt, U., Edwards, J.E., Elshahed, M.S., Fliegerova, K., Furtado, M., Garcia, M.A., Ge, Z.-W., Griffith, G.W., Griffiths, K., Groenewald, J.Z., Groenewald, M., Grube, M., Gryzenhout, M., Guo, L.-D., Hagen, F., Hambleton, S., Hamelin, R.C., Hansen, K., Harrold, P., Heller, G., Herrera, C., Hirayama, K., Hirooka, Y., Ho, H.-M., Hoffmann, K., Hofstetter, V., Hognabba, F., Hollingsworth, P.M., Hong, S.-B., Hosaka, K., Houbraken, J., Hughes, K., Huhtinen, S., Hyde, K.D., James, T., Johnson, E.M., Johnson, J.E., Johnston, P.R., Jones, E.B.G., Kelly, L.J., Kirk, P.M., Knapp, D.G., Koljalg, U., Kovacs, G.M., Kurtzman, C.P., Landvik, S., Leavitt, S.D.,

Liggenstoffer, A.S., Liimatainen, K., Lombard, L., Luangsa-ard, J.J., Lumbsch, H.T., Maganti, H., Maharachchikumbura, S.S.N., Martin, M.P., May, T.W., McTaggart, A.R., Methven, A.S., Meyer, W., Moncalvo, J.-M., Mongkolsamrit, S., Nagy, L.G., Nilsson, R.H., Niskanen, T., Nyilasi, I., Okada, G., Okane, I., Olariaga, I., Otte, J., Papp, T., Park, D., Petkovits, T., Pino-Bodas, R., Quaedvlieg, W., Raja, H.A., Redecker, D., Rintoul, T.L., Ruibal, C., Sarmiento-Ramirez, J.M., Schmitt, I., Schussler, A., Shearer, C., Sotome, K., Stefani, F.O.P., Stenroos, S., Stielow, B., Stockinger, H., Suetrong, S., Suh, S.-O., Sung, G.-H., Suzuki, M., Tanaka, K., Tedersoo, L., Telleria, M.T., Tretter, E., Untereiner, W.A., Urbina, H., Vagvolgyi, C., Vialle, A., Vu, T.D., Walther, G., Wang, Q.-M., Wang, Y., Weir, B.S., Weiss, M., White, M.M., Xu, J., Yahr, R., Yang, Z.L., Yurkov, A., Zamora, J.-C., Zhang, N., Zhuang, W.-Y., Schindel, D., 2012. Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. Proc. Natl. Acad. Sci. 109, 6241–6246. doi:10.1073/pnas.1117018109 Segata, N., Börnigen, D., Morgan, X.C., Huttenhower, C., 2013. PhyloPhlAn is a new method for improved phylogenetic and taxonomic placement of microbes. Nat. Commun. 4. doi:10.1038/ncomms3304 Soll, D.R., 2000. The ins and outs of DNA fingerprinting the infectious fungi. Clin. Microbiol. Rev. 13, 332–370. Stenlid, J., 1985. Population structure of Heterobasidion annosum as determined by somatic incompatibility, sexual incompatibility, and isozyme patterns. Can. J. Bot. 2268–2273. Stenlid, J., Gustafsson, M. arten, 2001. Are rare wood decay fungi threatened by inability to spread? Ecol. Bull. 85– 91. Sunhede, S., 1997. Vedsvampar på ek-Saffransticka. Ekbladet 12. Tommerup, I.C., Barton, J.E., O’brien, P.A., 1995. Reliability of RAPD fingerprinting of three basidiomycete fungi, Laccaria, Hydnangium and Rhizoctonia. Mycol. Res. 99, 179–186. Verin, M., Bourg, S., Menu, F., Rajon, E., 2015. The biased evolution of generation time. ArXiv Prepr. ArXiv150205508. Westermeier, R., 2016. Electrophoresis in Practice: A Guide to Methods and Applications of DNA and Protein Separations, 5th ed. Wiley-VCH.

Appendixes

Research sites visited: LAB ID* COLLECTION COORD. FRUIT POSITION LOCATION NOTES Site 1 OLD broken branch 4 meters Checked four oaks, nothing. 2001 last seen fruiting. Coordinates off, one oak downed and decomposed. Old site? Site 2 NO Linden, hazel, oak ecosystem. h3 Site 3 YES broken branch Crowded understory, newer treeas, oaks dying. h4 Site 4 YES broken branch 7 meters CWD in surrounding area, crowded understory. h5 SD9 YES broken branch 6 meters Located on hill, rocky with lack of understory. SD2 NO 3 old oaks in area on south facing slope, showed scarring and previous fruits, nothing new. SD3 NO No fresh fruits, no evidence of older fruits and tree mostly broken up in state of severe decomposition. SD5/SD6 NO Oak hollow 3 meters May have old fruiting body SD8 YES broken branch 4 meters Also had older sporocarp, 5 meters up on west facing side. SD1&4 NO No suitable fruiting habitats, no existing fruits. Unable to access all sites due to private landowners. SNV NO Close to water, no observed fruits or previous fruits. h7 HDG YES branch scar 2 meters Located in neighborhood near wetland, dense understory brush. Old sporocarp 5 meters above new one. SLOT NO Cattle grazed area, 300-500 year old oaks presnt, none had signs of previously fruiting. S.SOL NO Two oaks, no new/old fruits. One tree matched description with branch scaring. Open canopy. S.DJUR NO Unaccessible area, gate locked. S.HERR NO No new/old fruits. Open area, well maintained with many potential oak habitats. S.EKH NO Oak logged recently, only stump left behind. CWD cleaned up, surrounding oaks younger and fruitless. Grazed area. S.SPA NO No new/old fruits, potenial oak habitats but no signs of fruiting. Hilly, grazed area. Some dried F. hepatica. ÖS.SKAP NO Habitat unlike oak, coordinates incorrect. ÖS.STJA NO One oak, no other oaks in area, along edge of ravine. Dead elms everywhere. ÖS.SÖDR YES tree base ground One old oak, dried sporocarp ÖS.TINNE NO No oaks with sporocarps, oaks were 200+ years old. Woody grazed grassland. ÖS.STUR NO Many old oaks, none with new/old fruting bodies. Close to lake. H. punicea found. ÖS.SABYB NO SKIPPED. Inaccurate coordinates. ÖS.SLATT NO SKIPPED ÖS.ADEL NO 1980-1979 was last seen fruiting, no new sporocarps found. ÖS.TROSS NO tree base ground Open, hollowed oak outside Natural History Museum. STOCKHO YES

*lab ID was given if mycelia successfully cultured in lab for sequencing Appendix 1: Ecological notes taken from each site. Lab ID indicates which sporocarps that were sampled survived culturing and sequencing. Of all 28 sites visited, only 7 had fruited, indicating a 25% chance of occurrence.

NGI ID Lab ID Site County Country Collection date P4959_201 116 Kaunas Kaunas Lithuania 1994 P4959_202 823 Dukstos Vilnius Lithuania 1994 P4959_203 lv1 Nurmizi Siguldas Latvia 2015-07-21 P4959_204 lv2 Cirgali Zvartavas Latvia 2015-07-22 P4959_205 lv5 Amatnieki Rigas pilseta Latvia 2015-09-10 P4959_206 lv6 Zalvite Nereta Latvia 2015-09-21 P4959_207 h1 Wik Uppland Sweden 2015-09-18 P4959_208 h3 Sättra Stockholm Sweden 2015-09-18 P4959_209 h4 Sättra Stockholm Sweden 2015-09-18 P4959_210 h5 Strömsholm Västmanland Sweden 2015-09-29 P4959_211 h6 Djurgården SD9 Stockholm Sweden 2015-10-08 P4959_212 h7 Huddinge Stockholm Sweden 2015-10-08 P4959_213 h8 Linköping SödraLÖstergötaland Sweden 2015-10-14 P4959_214 33 Vanås slott Skåne Sweden 2015-10-23 P4959_215 36 Halltorps hage, oak 7 Öland Sweden 2015-11-03 P4959_216 37 Halltorps hage, oak 17 Öland Sweden 2015-11-03 P4959_217 39 Toftaholm Småland Sweden 2015-10-11 P4959_218 42 Ekbacken Blekinge Sweden 2015-10-11 P4959_220 46 syd Uddebo Västergötland Sweden 2015-10-14 P4959_221 47 Halltorps hage, oak Öland Sweden 1993-06-25 P4959_222 48 Halltorps hage, oak 7 Öland Sweden 1994-09-23 P4959_223 49 Halltorps hage, oak 16 Öland Sweden 1994-09-23 P4959_224 50 Halltorps hage, oak 16 Öland Sweden 1994-09-24 P4959_225 51 Halltorps hage, oak 17 Öland Sweden 1994-09-05 P4959_226 52 Halltorps hage, oak 17 Öland Sweden 1994-09-05 P4959_227 53 Halltorps hage, oak 17 Öland Sweden 1994-09-05 P4959_228 54 Halltorps hage, oak 17 Öland Sweden 1994-09-05 P4959_229 55 Halltorps hage, oak 17 Öland Sweden 1994-09-05 P4959_230 56 Halltorps hage, oak 17 Öland Sweden 1994-09-05 P4959_232 58 Toftaholm Småland Sweden 1994-09-08 P4959_233 59 Toftaholm Småland Sweden 1994-09-09 P4959_235 61 Toftaholm Småland Sweden 1994-09-10 P4959_236 62 Råbäck Västergötland Sweden 1980-09-22 P4959_237 78 Råbäck Västergötland Sweden 1992-06-29 P4959_238 79 Råbäck Västergötland Sweden 1992-07-27 P4959_239 82 Halltorps hage, oak 17 Öland Sweden 1992-07-28 P4959_240 83 Halltorps hage, oak 18 Öland Sweden 1992-07-29 P4959_241 84 Halltorps hage, oak 19 Öland Sweden 1992-07-30 P4959_242 85 Halltorps hage, oak 20 Öland Sweden 1992-07-31 P4959_243 86 Råbäck Västergötland Sweden 1997-08-?? Appendix 2: Sequenced heterokaryons: with post-sequencing NGI -assigned lab names, our given lab names, location of sample taken, county, country, and date.

Appendix 3: Table showing meanQ (probability that specimen belongs to that population) values for K=2 (chosen number of possible variants in a population subset). HHoak17 is clearly shown as a separate population, except for P229 and P230.

Compare Means for growth (mm) Descriptive Statistics VAR Sample size Mean Standard Deviation Variance F. pinicola 11 22,4702 12,69624 161,1946 H. croceus 11 5,51894 3,82783 14,65227 Paired two-sample t-test Degrees of Freedom 10 Hypothesized Mean Difference 0, Pooled Variance 87,92344 Test Statistics 6,21958 Pearson R 0,96851 Two-tailed distribution p-level 0,0001 Critical Value (5%) 2,22814 One-tailed distribution p-level 0,00005 Critical Value (5%) 1,81246 Compare Means for growth rate (mm/day) Descriptive Statistics VAR Sample size Mean Standard Deviation Variance F. pinicola 11 3,65202 0,69779 0,48692 H. croceus 11 1,15758 0,72499 0,52561 Paired two-sample t-test Degrees of Freedom 10 Hypothesized Mean Difference 0, Pooled Variance 0,50626 Test Statistics 7,42347 Pearson R -0,22681 Two-tailed distribution p-level 0,00002 Critical Value (5%) 2,22814 One-tailed distribution p-level 0,00001 Critical Value (5%) 1,81246 Appendix 4: Paired t-test results from growth rate (mm/day) and total radial growth (mm) for F. pinicola and H. croceus, a p-values and Peason R for both comparisons.