Phylogenetic and distributional data on boletoid fungi (Boletaceae) in Cyprus and description of a new sampling methodology

The data presented here was obtained during a decade-long macromycete inventory on the island of Cyprus and is supplementary to the research article “Present status and future of boletoid fungi (Boletaceae) on the island of Cyprus: cryptic and threatened diversity unravelled by ten-year study” [1]. A new, rainfall-based sampling protocol for documenting fungal diversity in Mediterranean ecosystems, is described in detail.


Data
The high-profile family of Boletaceae accommodates rare as well as economically important terrestrial fungi with tubular hymenophores and dark, usually fusiform or subfusiform spores [2e4]. Although the family has been intensively studied in recent years and extensive systematic re-arrangements have been proposed, boletoid fungi in Mediterranean and inslular ecosystems remain poorly documented. The data presented here is supplementary to the research paper "Present status and future of boletoid fungi (Boletaceae) on the island of Cyprus: cryptic and threatened diversity unravelled by ten-year study" [1], and was obtained during a 10-year macromycete inventory on the Mediterranean island of Cyprus. A new, rainfall-based sampling methodology is introduced and described in detail, designed to produce maximum yields of fungal diversity in Mediterranean ecosystems, where rainfall is unpredictable and uneven in distribution, and fungal fruitings are consequently prolific but localized and brief (Table 3). Thirty representative sites dominated by ectomycorrhizal trees and shrubs, were preselected and systematically surveyed, following rainfall episodes (Tables 1 and 2, Fig. 1). In addition, the known distribution of boletoid species documented on the island is depicted (Fig. 2), and the ITS polymorphism within the /Butyriboletus fechtneri sensu lato clade is demonstrated (Table 4).

Data collection and sampling methodology
Data on fungi belonging to the Boletaceae family was gathered during a decade-long macromycete inventory on the island of Cyprus, between 2006 and 2017, following a modified protocol based on Subject area Mycology, Ecology, Biological Conservation More specific subject area Molecular phylogenetics, Systematics, Island biogeography, Climate change Type of data

Experimental features
Thirty sites representative of all major ectomycorrhizal (EcM) habitats on the island were preselected and systematically surveyed following rainfall episodes, as part of a general inventory carried out on the island (see Tables 1 and 2 and Fig. 1). All data belonging to the Boletaceae family was then extracted from the general inventory data and analyzed separately.  [1].

Value of the data
The rainfall-based sampling methodology followed in this 10-year-long inventory, allowed for the coverage of a very large area across an extended altitudinal gradient and provided excellent yields of fungal diversity, including large numbers of previously undocumented and rare species The gene flow detected between the two subclades of Butyriboletus fechtneri sensu lato helps clarifying the phylogenetic boundaries of this taxon and can be used to determine which infraspecific rank, if any, should be assigned to genetically variable populations Distributional maps of presently known diversity of Boletaceae fungi can be useful for future research and conservation efforts on a local or regional scale       [5]. Thirty loosely delimited sites were pre-selected and regularly surveyed (see Table 1), in addition to other less frequently visited localities. Fungal diversity was for the most part undocumented on the island prior to this inventory, therefore the sampling strategy was designed to cover as large an area as possible as possible and yield the maximum possible number of species. As a result, pre-selected sites were consequently large, ranging in size from~500m 2 to~2km 2 . Selection of the permanent sites included all major habitat types formed by ectomycorrhizal (EcM) trees and shrubs on the island, and preliminary observations, anecdotal reports, altitudinal range, accessibility and mean annual precipitation were also taken into consideration. Single-tree communities are rare on Cyprus, therefore the majority of sites were comprised of mixed-tree communities. Of these, mixed Pinus brutia/Quercus alnifolia habitats are the most widely distributed woodland habitats on the island and as such were better represented among the permanent sites, but mixed P. brutia/Q. coccifera subsp. calliprinos, and P. nigra subsp. pallasiana/Q. alnifolia habitats were also well-represented (see Table 1). Because fungal fruiting episodes in the Mediterranean region are typically prolific but brief, and seasonal rainfall in Cyprus is uneven in distribution and highly unpredictable, surveys within permanent sites systematically followed rainfall episodes. Precipitation data for each locality was retrieved at least three times a week from the Cyprus Department of Meteorology official website http://www.moa.gov. cy/moa/ms/ms.nsf/DMLindex_en/DMLindex_en?OpenDocument, and forays were planned accordingly. Surveying usually spanned between September and April, 18e20 days following the first substantial rainfall of the season (>20 mm) and regularly thereafter, usually 1e2 days following subsequent rainfall episodes, or 2e4 times a week. In a typical season, surveying begun from the higher elevations of the Troodos massif (1,200e1,950 m above sea level) and, as temperatures dropped and precipitation increased, surveys gradually shifted to the lower elevations, where most of the fruiting occurs during the colder winter months. Surveying for spring species followed the opposite pattern, beginning from the lowlands in late winter and gradually extending to the higher elevations, until mid-to late spring. Exceptionally, surveys were also carried out in the summer months, following substantial precipitation (>30 mm) at the higher elevations of the Troodos mountains (>1,400 m a.s.l.), where brief localized fruitings sometimes occurred. Collection of specimens within the permanent sites was mostly opportunistic and followed fructification patterns, though identified hotspots within each site and certain tree-hosts of interest were regularly checked. Surveys usually lasted 2e4 hours on each site, with 1e4 sites visited in each foray. Highly productive seasons with abundant precipitation and prolific fructifications were more intensively surveyed than seasons with low precipitation and poor fructifications. Overall, a total of 767 forays were carried out during the   Table 2). Over this period, more than 3,500 vouchered collections belonging to over 1,200 species were gathered and archived, from which all relevant data to Boletaceae fungi was extracted and analyzed separately.

Ecological, morphological, phenological and chorological analyses
Over 200 Boletaceae collections were gathered and analyzed during this ten-year-inventory. All specimens were photographed in situ, the altitude and soil characteristics were annotated, and the host plant was assigned based on analysis of plant community composition. For collections found in mixed stands, the putative host-plant was assigned based on analysis of the fruiting pattern, spatial distribution of ECM plants, and known host preferences for each species following original descriptions and monographic works [3,6e11]. When the precise ectomycorrhizal symbiont was uncertain, no hostplant was assigned. For the purpose of evaluating estimated abundance, collections found >25 m apart from one-another were considered as fruiting from different mycelia following Dahlberg & Stenlid 1994 [12], and Hirose et al. 2004 [13]. Detailed macromorphological observations were made on fresh fruit bodies, when possible from various developmental stages. Oxidation of the context was observed after bruising the hymenium and longitudinally slicing one or more fresh fruit bodies from each collection. Microscopic studies were performed on both fresh and dried material under a Leica BM E binocular, an AmScope T360B trinocular plan achromatic, and a Zeiss axioskop microscopes at Â100, Â400 and Â1000 magnifications. For spore study, normal tap water was used as a mounting medium. A minimum of 30 naturally discharged, normally developed spores were measured from each basidiocarp, after placing fragments of the pileus on a glass slide overnight. When fresh material was not available, naturally discharged spores deposited on the stipe apex were measured. The Me (average length and width), Q (minimum and maximum length/width ratio) and Qm (average length/width ratio) were calculated for each collection, based on methods described by Peintner et al. (2003) [14], and Assyov (2012) [15]. Melzer's solution was used to observe possible amyloidity of the hyphae at the stipe base, following Singer (1965) [6], and Ladurner & Simonini (2003) [10]. Congo red in 10% ammonia (NH 4 OH), lactophenol cotton blue (LPCB), and 5% potassium hydroxide (KOH) were used to highlight the basidia, cystidia and pileipellis. All climatological data cited in this study (including normal, actual and cumulative actual/normal monthly precipitation), was retrieved from Cyprus Department of Meteorology. Correlation between climatological variables (monthly, seasonal and annual precipitation levels), and fruiting abundance of boletoid fungi, was performed using Pearson's product-moment tests in R 3.

DNA extraction, amplification and sequencing
Following morphological studies, representative specimens identified to belong to distinct species were selected for molecular analysis. A number of collections from atypical habitats or displaying unusual features were also molecularly analyzed, along with comparative collections from Bulgaria, Croatia, France, Greece and Switzerland. DNA extraction and PCR amplification were conducted with the REDExtract-N-Amp tm Plant PCR Kit (Sigma-Aldrich, St. Louis, MO, USA), following the manufacturer's instructions. The internal transcribed spacers and 5.8S rDNA (ITS) were amplified from each collection, with the ITS-1F/ITS-4b primer pair, as described in   [16]. When no band was detected by agarose-gel electrophoresis analysis, 1 mL of the PCR product was used as template in a second PCR using the ITS1F/ITS4 primer pair [17]. Amplicons were purified and sequenced by Eurofins Genomics, Ebersberg, Germany. Raw sequence data were edited and assembled with Codon Code Aligner 4.1.1 (CodonCode Corp., Centerville, MA, USA), and deposited in Genbank under the accession numbers indicated in Table 1 [1].

Phylogenetic analyses
Phylogenetic analyses were performed online at www.phylogeny.lirmm.fr. Multiple sequence alignment was carried out with MUSCLE 3.7 [18], using full processing mode and 16 iterations.  56 76 79 81 89 118 184 207 259 443 461 471 478 550 619 623 693 704 768   FR2015676  A  A  A  e  G  T  C  T  T  e  e  e  e  Y  G  T  e  T  T  FR2015677  A  A  A  e  G  T  C  T  T  e  e  e  e  Y  G  T  e  T  T  FR2016688 A T  T  Y  T  FR2017043  A  A  A  e  G  T  C  T  T  e  e  e  e  C  G  T  e  T  T  FR2017060  A  A  A  e  G  T  C  T  T  e  e  e  e  C  G  T  e  T  T  FR2017041 A Italic represents the sequences resolved in the "pan-European" subclade in Fig. 1D; Bold represents the gene flow-revealing heterozygocities.