challenges

Article

A Method for Citizen Scientists to CatalogueWorldwide Chlorociboria spp Distribution

Sarath M Vega Gutierrez 1 ID Olivia E Kramer 2 Claudia C Andersen 1Patricia T Vega Gutierrez 1 and Seri C Robinson 1

1 Wood Science amp Engineering Oregon State University Corvallis OR 97331 USAsarathvegaoregonstateedu (SMVG) anderclaoregonstateedu (CCA)patriciavegaoregonstateedu (PTVG)

2 Department of Botany and Plant Pathology Oregon State University Corvallis OR 97331 USAkramerooregonstateedu

Correspondence serirobinsonoregonstateedu

Received 24 January 2018 Accepted 12 March 2018 Published 14 March 2018

Abstract The blue-green pigment known as xylindein that is produced by species in the Chlorociboriagenus is under heavy investigation for its potential in textile dyes wood dyes and solar cellsXylindein has not yet been synthesized and while its production can be stimulated under laboratoryconditions it is also plentiful in downed decayed wood in forested lands Unfortunately little isknown about the wood preference and forest type preference for this genus especially outside NewZealand To map the genus would be a massive undertaking and herein a method by which citizenscientists could contribute to the distribution map of Chlorociboria species is proposed The initialtrial of this method found untrained participants successfully identified Chlorociboria stained woodin each instance regardless of forest type This simple easy identification and classification systemshould be well received by citizen-scientists and is the first step towards a global understanding ofhow xylindein production might be managed for across various ecosystems

Keywords Chlorociboria spalting xylindein citizen-scientist sampling protocol

1 Introduction

Large-scale organism sampling studies that focus on the species level are often costly and difficultto achieve if done by a single scientist team [12] Relying on citizen scientists to expand and contributeto field data on different research topics such as ornithology [1] mycology [3] and entomology [4]is increasingly common Initiatives such as eBird developed by Cornell University has shown thefeasibility accuracy and reliability of data generated by citizen scientists [45]

Citizen scientist initiatives focus on citizen groups that are in contact and have a degree ofknowledge about the subjects of study eBird for example collects data for bird identification anddistribution and is targeted at bird watching hobbyists around the globe [6] Another example ofreliance on citizen-scientists for data collection is the survey of fungi in Warwickshire in the UnitedKingdom where local members of various mycological societies were trained by experts and forfifteen years collected one of the most detailed and reliable data sets of the fungi in the area [7]Another example is the ten-year chanterelle (Cantharellus cibarius) project which evaluated differentmushroom harvesting techniques in ten plots and how it affected future fruiting of the species [8ndash10]A third example specific to mycology in the North American Pacific Northwest (PNW) used amateurmycologists belonging to mushroom societies of the PNW such as the Oregon Mycological Societyfor one of the first long-term studies about the impact of different harvesting techniques on wild

Challenges 2018 9 11 doi103390challe9010011 wwwmdpicomjournalchallenges

Challenges 2018 9 11 2 of 11

mushrooms [8ndash10] Most of the members in these societies are college educated and had a high degreeof mycological knowledge [11]

Reliance on citizen scientist data can make big sampling studies more affordable and also makesscience more approachable to the general public By involving citizens in scientific studies a widernetwork is made where the scientific team and the population are more involved with the objective ofachieving a shared goal [2]

The genus Chlorociboria (phylum Ascomycota) produces wood spalting (pigmentation ofwood produced by fungi) [12ndash15] This genus is present in Europe [15] North America [16ndash18]New Zealand [19] India [20] and certain areas of Central and South America [21ndash23] Species of thisgenus are found growing on decaying wood often already colonized by white rot fungi with specieshaving been isolated from both hardwoods and softwoods [18192425] There are two notable speciesfound in North America and Europe Chlorociboria aeruginosa (Oeder) Seaver ex CS RamamurthiKorf and LR Batra and Chlorociboria aeruginascens (Nyl) Kanouse ex CS Ramamurthi Korf andLR Batra (1 3ndash4 6ndash7) [16ndash182627] Like many in this genus these two species create a blue-greencolor in wood due to the production of the pigment xylindein [2829] and have been historically usedin European woodworking as early as the 13th century [30ndash33]

Databases containing the distribution of Chlorociboria spp are also available from mycologicalsocieties such as the Danish Mycological Society and in online resources as the Global BiodiversityInformation Facility (GBIF) [34] However these databases carry information that is usually focusedin certain areas (such as the mycological societies) or they rely on the information collected by otherinstitutions that focus on other areas of interest This focus on one or two particular genera or types offungi generates geographical gaps that can benefit from citizen-scientistsrsquo data collection

Currently there is renewed interest in xylindein for use in UV-resistant wood stains textiledyes [35ndash37] and solar cells [38] Attempts to synthesize xylindein have been unsuccessful [39] howeverpigment production of Chlorociboria can be stimulated under certain laboratory conditions [4041]The pigment can also be extracted from the naturally occurring fungal substrate using non-polarsolvents with dichloromethane (DCM) the most effective [42] Experiments have also been done usingcentrifugal partition chromatography to isolate and purify xylindein [43] and the extracted pigmenthas been used directly on wood [3536] bamboo [44] and textiles [4546] to test its effectiveness atcausing coloration to mimic the effects of Chlorociboria natural growth

With the increasing research interest in xylindein and the ability to use the naturally occurringpigment in addition to generating xylindein in the lab distribution and density of Chlorociboria speciesare of interest Particularly valuable would be the management of woodland understories (especiallythe slash wood) for Chlorociboria by woodland owners This would require a better understanding ofwood species preference and habitat preference for the genus than we currently have In additionunderstanding wood preference of Chlorociboria species would help with laboratory production ofxylindein as Chlorociboria species are notoriously difficult to grow in the lab and require specializedwood-amended media [40] for maximum xylindein production

The purpose of this work was to develop an accessible and repeatable method for citizen scientiststo map occurrences of the Chlorociboria genus across the world in an effort to catalogue its distributionhabitat preference and wood species preference with a mind to future biotechnology developments

2 Materials and Methods

21 Sampling AreamdashUSA

Samples of blue-green wood were collected in the Benton County area of Oregon USA wheresightings of blue-green stained wood had been previously reported Two forests were selected basedon their accessibility and recreational value The first forest was Maryrsquos Peak which belongs to theSiuslaw National Forest On this forest Dinner Creek and Woods Creek were sampled The second

Challenges 2018 9 11 3 of 11

forest was the McDonald-Dunn forest which belongs to the College of Forestry of Oregon StateUniversity There three creeks were sampled Soap Creek Jackson Creek and Oak Creek

The collection was focused on riparian areas due to the fact that previous reports on Chlorociboriaspecies state their preference for humid areas [18] The main factors for the selection of areas to besampled were drivability accessibility and terrain The sampled creeks were close to delimited pathsUSA samples were collected from November 2015 to March 2016

22 Surveying AreamdashPeru

Peruvian surveying areas were within the Inkaterra property located in the district of Las Piedrasin Madre de Dios Peru This surveying area was selected due to the reported biodiversity of flora andfauna [4748] and its accessibility Previous scouting and collections by experts in this area discussedan abundance of spalting fungi [49]

In this area two sites were sampled The first one was coded as Reserva Amazonica (RA) ReservaAmazonica (RA) corresponds to a lower terrace tropical rainforest and has a perennial wetland withinthe area [50] The forest at RA corresponded to a primary forest with little disturbance besidesthe use of the area for ecotourism The other site was coded Hacienda Concepcion (HC) This sitecorresponded to a high terrace [50] and the forest corresponded to secondary growth mixed with cocoa(Theobroma cacao) Surveying (no samples were collected due to permitting restrictions) was done inJune of 2016

23 Training

Field crews consisting of three to six non-specialized people were trained on the rapid assessmentof Chlorociboria spp on downed wood by direct observation of sampling techniques (described below)The training consisted of recognizing the blue-green coloration of the inner downed wood which ischaracteristic for this genus and the recognition of the fruiting bodies produced by this fungus locallyknown as ldquoelfrsquos cuprdquo The field crews went into the selected areas first supervised by a graduatestudent or an instructor and then alone or accompanied by a field guide (Field Station Inkaterra only)

24 Sampling

Samples were collected during the rainy seasons of the respective countries which increasedthe likelihood of encountering Chlorociboria spp fruiting bodies Sampling was done in two differentmanners differing due to safety concerns in the Amazon For the USA all sampling was done using aline-intercept method along 400 m transects on both sides of the stream Transects were run parallel tothe stream 8 m from the edge of the creek A Spencerreg 950 loggers tape (Spencer Products CO SeattleWA USA) was used to determine distance from the stream being re-measured approximately every15 m to account for bends in the stream All downed wood (trees and branches) with a diameter of atleast 5 cm encountered along the 400 m transects were sampled using a ruler to determine whetherthe wood met this requirement The starting location of the transects was determined by choosing theclosest point of the stream from the entry site using Google Maps (version 4280 Google MountainView CA USA) for iPhone as a reference The entry site was defined as the closest area to the creeksaccessible by car

For Peruvian surveying transects were confined to known premade trails in the areaand researchers were allowed to venture only up to 15 m perpendicular to them due to safety concernsThe starting point was defined as the area closest to the field station or lodge

Regardless of country all downed dead wood within 8 m of the trailtransect (no wood smallerthan 5 cm was considered) was opened using a machete to determine if Chlorociboria spp was presentThe wood was cut 3 cm deep along a 10-cm segment on the top side of the log with repeat cutsevery 5 cm along the length of the log This method was selected because spalting fungi mostlydo not produce fruiting bodies and it is therefore necessary to look internally into the dead logs tofind colonization evidence (the blue-green color which is unique to the genus [1521304251ndash53])

Challenges 2018 9 11 4 of 11

The presence of fruiting bodies was also used when fruiting bodies occurred Only downed deadwood was sampled as Chlorociboria spp is generally a slow growing wood decomposing fungus [54]If no pigmentation was found after going approximately 3 cm deep into the wood it was considered tonot contain Chlorociboria spp If pigmentation was present the GPS location was determined using theEasy GPS application for iPhone (Apple Cupertino CA USA) made by Kraus und Karnath GbR 2kit(Duumlsseldorf Germany) for the forests located in Benton County while a Garmin GPSmap 64s (GarminSchaffhausen Switzerland) was used in the Madre de Dios Peru area as phone signal was limited

Sample collection was performed in the US site only due to permitting restrictions in PeruThe samples were from downed wood that had the characteristic blue-green pigmentation and thatwere not completely decomposed (samples which did not crumble in the collectorrsquos hand) as somestructure integrity was required for further examination The size of the samples collected was aminimum of 5 cm in width and length and at least 04 cm in thickness

The GPS coordinates were then input into an Excel spread sheet (Microsoft) and uploaded to ArcMap(ESRI version 1051 Manufacturer Redlands CA USA) to generate maps of the collection sites

25 Wood Identification

Researchers were not allowed to remove wood from Peruvian forests therefore only field woodID could be performed For US occurrences any samples that occurred on wood hard enough to becollected were taken to the Applied Mycology laboratory at Oregon State University to determine thewood species Wood slides of 16 microm were obtained utilizing a Spencer-Buffalo Company microtometo create uniform sections of each plane The obtained wood slides were placed on a glass slide(VWRreg Radnor PA USA) and covered with a 15 glass cover-slip (VWRreg) The slides were thenexamined using a Nikon Eclipse Ni-U light microscope (Nikon Instruments Inc Melville NY USA)and photographed using a Nikon DS-Ri2 microscope camera

Once the images of the wood slides were obtained different anatomical aspects were notedThe first aspect was to identify if the wood was a conifer or a hardwood species by analyzing thepresence of vessels Ray types (upright andor procumbent) length and width (unicellular andormulticellular) were also annotated The parenchyma type was described per specimen and specialcharacteristics (as presence of gums or prismatic crystals) were noted After collecting the informationper wood sample the characteristics were run through the North Carolina State University Insidewooddata base [55]

26 Verification

Microscopic verification of the wood species as well as confirmation of Chlorociboria speciescolonization was done by wood anatomy experts using a Nikon Eclipse Ni-U microscope to evaluatethe presence of hyphae and the location of the pigment in the wood

For the Peruvian samples the confirmation of the possible presence of Chlorociboria spp wasperformed by visual inspection by a professor andor postdoctoral researcher and was entirely basedupon the correct color being present in the wood (blue-green) Verification was necessary for thesampling as this was the first time the method was applied and verification was required to makesure the collections and fungal identification by wood pigmentation was performed correctly and thatthe explanations given were sufficient for a non-technical person

3 Results

31 Sampling

In the USA the trained students successfully identified eight logs that contained Chlorociboriaalong the various transectstrails All of the logs identified by the students as having Chlorociboriaspecies were confirmed as hosts of Chlorociboria spp

Challenges 2018 9 11 5 of 11

In Peru identification of Chlorociboria-stained wood was made through visual determination ofthe pigment as no specialized equipment was available for further inspection However per themethods for the USA identification a professor andor post-doctoral research did verify that thecorrect color was present to indicate Chlorociboria spp All student identifications were correct

32 Site Accessibility

The selection of the sampling sites allowed for easy accessibility by one person or a team up tothree As accessibility is a main factor for lsquocitizen-scientistsrsquo this was taken into consideration for theexperimental design Using this technique downed trees and branches carrying Chlorociboria sppwere successfully identified with minimal danger to the participants The use of an iPhone app wassuccessful (and highly accessible) and the consumer grade GPS used for the mapping in Peru alsoperformed well and all data points were saved (Tables 1 and 2)

Table 1 GPS coordinates of fallen wood containing Chlorociboria spp taken with Easy GPS in MaryrsquosPeak and McDonald-Dunn Forests

Location Sample Latitude (Decimal Degrees) Longitude (Decimal Degrees) Wood Species

Oak CreekO1 4460565 minus12333231 Acer macrophyllumO2 4460805 minus12333216 -

Soap Creek

S1 4464328 minus12332298 -S2 4464355 minus12332475 Acer macrophyllumS3 4464348 minus12332486 -S4 4464348 minus1233233 -S5 4464366 minus1233233 -

Woods Creek

W1 4454433 minus12345474 Acer macrophyllumW2 4454446 minus12345468 Acer macrophyllumW3 4454446 minus12345468 Acer macrophyllumW4 4454446 minus12345088 -W5 4454446 minus12345048 Acer macrophyllumW6 4454448 minus12344968 -

Dinner Creek D1 4447248 minus1234909 Acer macrophyllum

Jackson Creek J1 446134 minus12329206 Salix spp

Table 2 GPS coordinates of fallen wood with suspected Chlorociboria spp taken with GPSmap 64s inField Station Inkaterra Madre de Dios Peru

Location Sample Latitude (Decimal Degrees) Longitude (Decimal Degrees) Wood Species

Reserva Amazonica

331A minus125333 minus690437 -441C minus125347 minus690481 -578 minus125385 minus690505 -641 minus125373 minus690544 -

679B minus125328 minus690511 -709 minus125359 minus690518 -737 minus125293 minus690455 -

755B minus125282 minus690451 -941B minus125258 minus690456 -

Hacienda Concepcion1001B minus126068 minus690816 -1201 minus126058 minus690789 -1359 minus126078 minus690736 -

33 Wood Species Identification

A total of eight wood samples were collected Of these samples seven were found to be bigleaf maple (Acer macrophyllum Pursh) and all of these seven samples were correctly identified to thegenus (maple) by the participants without the use of a microscope The samples presented exclusiveprocumbent multiseriate ray cells Helical thickening on the vessel cell walls was also present and thischaracteristic determined the identification of the specimens as a big leaf maple as helical thickenings

Challenges 2018 9 11 6 of 11

are a common identifying feature for the Acer genus (Figure 1) This species is a hardwood that iscommonly found close to riparian forests in the Pacific Northwest

Challenges 2018 9 x 6 of 11

Wood species identification in Peru was generally not successful Those logs containing blue-green pigments were often too decayed to even attempt a genus identification The participants were not able to do any in-field wood identification at this site

The identification of the wood species is not required for citizen-scientists but if such people are capable it is a nice addition to the data

Figure 1 Radial section of a big leaf maple with red arrows pointing the helical thickenings on the vessel cell walls Magnification 200times

Figure 2 Tangential section of a willow with arrows pointing to the upright ray cells All rays are uniseriate Magnification 200times

Figure 3 Radial section of a willow showing alternate vessel wall pitting and Chlorociboria spp pigmentation Magnification 200times

Figure 1 Radial section of a big leaf maple with red arrows pointing the helical thickenings on thevessel cell walls Magnification 200times

One specimen was identified as willow (Salix spp) The sample presented both procumbent andupright rays The rays were exclusively uniseriate (Figure 2) and the vessel cell walls presented pitting(Figure 3) which are all identifying features of Salix sp L The participants incorrectly identified thissample as a maple in the field

Challenges 2018 9 x 6 of 11

Wood species identification in Peru was generally not successful Those logs containing blue-green pigments were often too decayed to even attempt a genus identification The participants were not able to do any in-field wood identification at this site

The identification of the wood species is not required for citizen-scientists but if such people are capable it is a nice addition to the data

Figure 1 Radial section of a big leaf maple with red arrows pointing the helical thickenings on the vessel cell walls Magnification 200times

Figure 2 Tangential section of a willow with arrows pointing to the upright ray cells All rays are uniseriate Magnification 200times

Figure 3 Radial section of a willow showing alternate vessel wall pitting and Chlorociboria spp pigmentation Magnification 200times

Figure 2 Tangential section of a willow with arrows pointing to the upright ray cells All rays areuniseriate Magnification 200times

Challenges 2018 9 11 7 of 11

Challenges 2018 9 x 6 of 11

Wood species identification in Peru was generally not successful Those logs containing blue-green pigments were often too decayed to even attempt a genus identification The participants were not able to do any in-field wood identification at this site

The identification of the wood species is not required for citizen-scientists but if such people are capable it is a nice addition to the data

Figure 1 Radial section of a big leaf maple with red arrows pointing the helical thickenings on the vessel cell walls Magnification 200times

Figure 2 Tangential section of a willow with arrows pointing to the upright ray cells All rays are uniseriate Magnification 200times

Figure 3 Radial section of a willow showing alternate vessel wall pitting and Chlorociboria spp pigmentation Magnification 200times

Figure 3 Radial section of a willow showing alternate vessel wall pitting and Chlorociboria spppigmentation Magnification 200times

Wood species identification in Peru was generally not successful Those logs containing blue-greenpigments were often too decayed to even attempt a genus identification The participants were notable to do any in-field wood identification at this site

The identification of the wood species is not required for citizen-scientists but if such people arecapable it is a nice addition to the data

34 Verification

Performing the sampling under supervision and further verification showed that the methodwas effective Therefore the collection method can be done by citizen-scientists without professionalsupervision as it has shown to be accurate

4 Discussion

41 Training Sampling and Sample Collection

In terms of simple mapping of Chlorociboria distribution the training was deemed successfulas eight logs containing Chlorociboria spp were identified within the McDonald-Dunn and MaryrsquosPeak Forests and twelve logs suspected to contain Chlorociboria spp were found in Madre de DiosPeru and all were successfully identified by the participants Wood identification was less successfulunfortunately especially in the tropical rainforests of Peru It seems likely that this aspect of theproposed citizen scientist survey for Chlorociboria would generally not be useful as researchers wouldhave no way to verify correct wood ID and when there is an abundance of diversity the likelihood ofmisidentification is great

A key element that allowed for the successful identification of the logs containing or suspected tocontain Chlorociboria spp was opening the logs well into the sapwood to find the blue-green coloredwood This helped students discount fungi lichens and bacteria that can colorize the surface and thefirst few centimeters of wood and thus limited them to general decay-capable fungi Another importantelement that was unique to this study was that it allowed for fungi without fruiting forms to be surveyed(when said fungi have a unique coloration system such as Chlorociboria spp) [39] This method couldeasily be applied to non-fruiting bodied pigment producing fungi such as Scytalidium cuboideumwhich develops red pigmentation [27] and Scytalidium ganodermophthorum which develops yellowpigmentation [5657] although these fungi do not have lsquouniquersquo colorants A final factor that aided inthe accurate identification was likely the focus on a single distinct genus allowing the participantsto focus their attention Surveys asking for a wider number of specimens to be located risk over orunderestimation [45859]

Challenges 2018 9 11 8 of 11

Another useful aspect of this technique was the use of common and accessible surveying toolssuch as machetes (which can be easily replaced by hatchets andor field knives) measuring tapessmart phone GPS applications andor GPS These tools require little to no training for their use and aregenerally accessible around the world This is important for future reliance on citizen-scientists forthe evaluation of Chlorociboria spp in the wild and should be effective Previous studies have showncitizen science to be very useful through works like eBird for data collection of bird diversity andmigration patterns which fully relies on bird hobbyists around the world [56] Specific to mycologythe active participation of Pacific Northwest mycological societies in the ten-year chanterelle projectresulted in the evaluation harvesting techniques of wild chanterelles in Oregon [8ndash10] Similarlythe long-term survey in Warwickshire in the UK from 1965 to 1980 gave one of the most detailedsurveys for fungi in the UK and was performed by lay mycologists of the area [7]

As with other citizen scientist surveys the technique detailed herein is similar to a rapid assessmentsurvey (RAPD) This method is commonly used for field surveys by non-specialized personnel in specificareas [60] The rapid assessment focuses on a group of genera in small diverse areas RAPD has beenwidely used for mycological surveys but if used for a wide number of genera can become unreliable [58]Focusing on a single group increases its accuracy The modified RAPD method proposed herein has beenshown to be accurate across different ecosystems (temperate rainforest in Benton County OR USA andthe tropical rainforest in Madre de Dios Peru) for the identification of the Chlorociboria spp on downedwood by non-specialized personnel In the future this method could be applied by citizen-scientists tocollect data on the worldwide distribution of the genus Chlorociboria

The method proposed can also be applied without setting a transect as it was performed in theAmazon rainforest This variation can improve the participation of citizen-scientists as some of themare mycology aficionados that can mark the location of Chlorociboria spp while on their foragingactivities or simply on general hikes

The distribution data (GPS points) which could be easily uploaded into a worldwide databasesuch as GBIF allowed the researchers on this project to identify forest types using Forest Service mapsand forest data from the Peruvian government This allowed for generation of a distribution mapand an understanding of forest types preferred by Chlorociboria species Continuation of this surveyby citizen scientists could greatly expand our understanding of the types of trees and systems thegenus of fungi prefer even if exact tree species cannot be reliably noted This data could then be madeavailable to forested landowners seeking to manage for Chlorociboria species xylindein productionand the economic opportunities therein

42 Future Research

Currently the location of the species is determined with a GPS app and field notes In thefuture the development of a smartphone application (like eBird [56]) specific to the distribution ofChlorociboria spp will be necessary to make the sampling data collection and data analysis simplerThis would also give the researchers involved one main input point for data making analysis and mapgeneration easier

As for the implementation of this initiative the unique laws and access to natural landscapesin different countries must be taken into account if the final goal is to apply it worldwide In theimmediate future the sampling of Chlorociboria spp can be done in association with mycologysocieties from the Pacific Northwest This can help to improve the location method and to develop theapplication with them as they have already participated in long-term studies (such as the Chanterelleproject) [10] In the intermediate future contacts with other mycological societies could be done on aworldwide scale (such as with the Danish Mycological Society) In the long-term the authors hopethat this method might be introduced into countries that do not have a culture of foraging (like Peru)

Finally the goal of the developed method and initiative is to gather data that can generate adetailed map of distribution of Chlorociboria spp and with it the identification of areas that present amore frequent presence of the fungus This information will be evaluated to determine the type of

Challenges 2018 9 11 9 of 11

forests and the dominant wood species in those areas and to then apply it to land management for thegrowth of these rare and useful fungi should this be of interest to landowners

5 Conclusions

The proposed method utilized simple tools and a smart phone or GPS to survey populations ofChlorociboria spp across temperate and tropical forests The method for identification of Chlorociboria sppcontaining wood was shown to be successful however the wood identification was not Howeverin general Chlorociboria identification can be easily taught to members of mycological societieseco tourists and other interested parties that wish to be involved in recording the distribution ofChlorociboria spp Over time the data collected could be used to build a worldwide picture of foresttype and wood preference by Chlorociboria species which will be of use to those who wish to managefor xylindein production on their forested lands

Acknowledgments The authors would like to thank Joseacute Koechlin Von Stein Inkaterra and the Inkaterra Asociacioacuten

Author Contributions Seri C Robinson Olivia E Kramer and Claudia C Andersen conceived and designed theexperiments Olivia E Kramer Claudia C Andersen and Patricia T Vega Gutierrez executed the experimentsand Sarath M Vega Gutierrez analyzed the data and wrote the paper

Conflicts of Interest The authors declare no conflict of interest

References

1 Stanley ES Susan RD Conservation commentary Ornithology and the national biological survey Auk1995 112 804ndash806

2 Tulloch AIT Possingham HP Joseph LN Szabo J Martin TG Realising the full potential of citizenscience monitoring programs Biol Conserv 2013 165 128ndash138 [CrossRef]

3 Cook R Encouraging the amateur Survey work in north-west England Mycologist 1992 6 162ndash164 [CrossRef]4 Gardiner MM Allee LL Brown PMJ Losey JE Roy HE Smyth RR Lessons from lady beetles

Accuracy of monitoring data from us and uk citizen-science programs Front Ecol Environ 2012 10 471ndash476[CrossRef]

5 Sullivan BL Wood CL Iliff MJ Bonney RE Fink D Kelling S Ebird A citizen-based bird observationnetwork in the biological sciences Biol Conserv 2009 142 2282ndash2292 [CrossRef]

6 Sullivan BL Aycrigg JL Barry JH Bonney RE Bruns N Cooper CB Damoulas T Dhondt AADietterich T Farnsworth A et al The ebird enterprise An integrated approach to development andapplication of citizen science Biol Conserv 2014 169 31ndash40 [CrossRef]

7 Clark MC The fungus flora of Warwickshire further records Bull Br Mycol Soc 1986 20 105ndash119[CrossRef]

8 Norvell L Studying the effects of harvesting on chanterelle productivity in Oregonrsquos Mt Hood nationalforest In Wild Mushroom Harvesting Discussion Session Minutes British Columbia Ministry of Forests PacificForestry Centre Victoria BC USA 1992 pp 9ndash15

9 Norvell L Kopecky F Lindgren J Roger J The chanterelle (cantharellus cibarius) A peek at productivityIn Proceedings of the Business and Science of Special Forest ProductsmdashA Conference and ExpositionAt Hillsboro OR USA 26ndash27 January 1994 pp 109ndash120

10 Norvell L Loving the chanterelle to death The ten-year Oregon chanterelle project McIlvainea 199512 6ndash25

11 McLain RJ Christensen HH Shannon MA When amateurs are the experts Amateur mycologists andwild mushroom politics in the Pacific Northwest USA Soc Nat Resour 1998 11 615ndash626 [CrossRef]

12 Robinson SC Richter DL Laks PE Colonization of sugar maple by spalting fungi For Prod J 200757 24ndash32

13 Persoon CH Observationes mycologicae Ann Bot 1795 15 1ndash3914 Persoon CH Synopsis Methodica Fungorum Apud Henricum Dieterich Gottingae Germany 180115 Persoon CH Mycologica Europaea Impensibus iI Palmii Erlangae Germany 1822 Volume 1 p 29116 Seaver FJ The North American Cup Fungi (Inoperculates) Hafner Publishing Co Inc New York NY

USA 1951

Challenges 2018 9 11 3 of 11

forest was the McDonald-Dunn forest which belongs to the College of Forestry of Oregon StateUniversity There three creeks were sampled Soap Creek Jackson Creek and Oak Creek

The collection was focused on riparian areas due to the fact that previous reports on Chlorociboriaspecies state their preference for humid areas [18] The main factors for the selection of areas to besampled were drivability accessibility and terrain The sampled creeks were close to delimited pathsUSA samples were collected from November 2015 to March 2016

22 Surveying AreamdashPeru

Peruvian surveying areas were within the Inkaterra property located in the district of Las Piedrasin Madre de Dios Peru This surveying area was selected due to the reported biodiversity of flora andfauna [4748] and its accessibility Previous scouting and collections by experts in this area discussedan abundance of spalting fungi [49]

In this area two sites were sampled The first one was coded as Reserva Amazonica (RA) ReservaAmazonica (RA) corresponds to a lower terrace tropical rainforest and has a perennial wetland withinthe area [50] The forest at RA corresponded to a primary forest with little disturbance besidesthe use of the area for ecotourism The other site was coded Hacienda Concepcion (HC) This sitecorresponded to a high terrace [50] and the forest corresponded to secondary growth mixed with cocoa(Theobroma cacao) Surveying (no samples were collected due to permitting restrictions) was done inJune of 2016

23 Training

Field crews consisting of three to six non-specialized people were trained on the rapid assessmentof Chlorociboria spp on downed wood by direct observation of sampling techniques (described below)The training consisted of recognizing the blue-green coloration of the inner downed wood which ischaracteristic for this genus and the recognition of the fruiting bodies produced by this fungus locallyknown as ldquoelfrsquos cuprdquo The field crews went into the selected areas first supervised by a graduatestudent or an instructor and then alone or accompanied by a field guide (Field Station Inkaterra only)

24 Sampling

Samples were collected during the rainy seasons of the respective countries which increasedthe likelihood of encountering Chlorociboria spp fruiting bodies Sampling was done in two differentmanners differing due to safety concerns in the Amazon For the USA all sampling was done using aline-intercept method along 400 m transects on both sides of the stream Transects were run parallel tothe stream 8 m from the edge of the creek A Spencerreg 950 loggers tape (Spencer Products CO SeattleWA USA) was used to determine distance from the stream being re-measured approximately every15 m to account for bends in the stream All downed wood (trees and branches) with a diameter of atleast 5 cm encountered along the 400 m transects were sampled using a ruler to determine whetherthe wood met this requirement The starting location of the transects was determined by choosing theclosest point of the stream from the entry site using Google Maps (version 4280 Google MountainView CA USA) for iPhone as a reference The entry site was defined as the closest area to the creeksaccessible by car

For Peruvian surveying transects were confined to known premade trails in the areaand researchers were allowed to venture only up to 15 m perpendicular to them due to safety concernsThe starting point was defined as the area closest to the field station or lodge

Regardless of country all downed dead wood within 8 m of the trailtransect (no wood smallerthan 5 cm was considered) was opened using a machete to determine if Chlorociboria spp was presentThe wood was cut 3 cm deep along a 10-cm segment on the top side of the log with repeat cutsevery 5 cm along the length of the log This method was selected because spalting fungi mostlydo not produce fruiting bodies and it is therefore necessary to look internally into the dead logs tofind colonization evidence (the blue-green color which is unique to the genus [1521304251ndash53])

Challenges 2018 9 11 4 of 11

The presence of fruiting bodies was also used when fruiting bodies occurred Only downed deadwood was sampled as Chlorociboria spp is generally a slow growing wood decomposing fungus [54]If no pigmentation was found after going approximately 3 cm deep into the wood it was considered tonot contain Chlorociboria spp If pigmentation was present the GPS location was determined using theEasy GPS application for iPhone (Apple Cupertino CA USA) made by Kraus und Karnath GbR 2kit(Duumlsseldorf Germany) for the forests located in Benton County while a Garmin GPSmap 64s (GarminSchaffhausen Switzerland) was used in the Madre de Dios Peru area as phone signal was limited

Sample collection was performed in the US site only due to permitting restrictions in PeruThe samples were from downed wood that had the characteristic blue-green pigmentation and thatwere not completely decomposed (samples which did not crumble in the collectorrsquos hand) as somestructure integrity was required for further examination The size of the samples collected was aminimum of 5 cm in width and length and at least 04 cm in thickness

The GPS coordinates were then input into an Excel spread sheet (Microsoft) and uploaded to ArcMap(ESRI version 1051 Manufacturer Redlands CA USA) to generate maps of the collection sites

25 Wood Identification

Researchers were not allowed to remove wood from Peruvian forests therefore only field woodID could be performed For US occurrences any samples that occurred on wood hard enough to becollected were taken to the Applied Mycology laboratory at Oregon State University to determine thewood species Wood slides of 16 microm were obtained utilizing a Spencer-Buffalo Company microtometo create uniform sections of each plane The obtained wood slides were placed on a glass slide(VWRreg Radnor PA USA) and covered with a 15 glass cover-slip (VWRreg) The slides were thenexamined using a Nikon Eclipse Ni-U light microscope (Nikon Instruments Inc Melville NY USA)and photographed using a Nikon DS-Ri2 microscope camera

Once the images of the wood slides were obtained different anatomical aspects were notedThe first aspect was to identify if the wood was a conifer or a hardwood species by analyzing thepresence of vessels Ray types (upright andor procumbent) length and width (unicellular andormulticellular) were also annotated The parenchyma type was described per specimen and specialcharacteristics (as presence of gums or prismatic crystals) were noted After collecting the informationper wood sample the characteristics were run through the North Carolina State University Insidewooddata base [55]

26 Verification

Microscopic verification of the wood species as well as confirmation of Chlorociboria speciescolonization was done by wood anatomy experts using a Nikon Eclipse Ni-U microscope to evaluatethe presence of hyphae and the location of the pigment in the wood

For the Peruvian samples the confirmation of the possible presence of Chlorociboria spp wasperformed by visual inspection by a professor andor postdoctoral researcher and was entirely basedupon the correct color being present in the wood (blue-green) Verification was necessary for thesampling as this was the first time the method was applied and verification was required to makesure the collections and fungal identification by wood pigmentation was performed correctly and thatthe explanations given were sufficient for a non-technical person

3 Results

31 Sampling

In the USA the trained students successfully identified eight logs that contained Chlorociboriaalong the various transectstrails All of the logs identified by the students as having Chlorociboriaspecies were confirmed as hosts of Chlorociboria spp

Challenges 2018 9 11 5 of 11

In Peru identification of Chlorociboria-stained wood was made through visual determination ofthe pigment as no specialized equipment was available for further inspection However per themethods for the USA identification a professor andor post-doctoral research did verify that thecorrect color was present to indicate Chlorociboria spp All student identifications were correct

32 Site Accessibility

The selection of the sampling sites allowed for easy accessibility by one person or a team up tothree As accessibility is a main factor for lsquocitizen-scientistsrsquo this was taken into consideration for theexperimental design Using this technique downed trees and branches carrying Chlorociboria sppwere successfully identified with minimal danger to the participants The use of an iPhone app wassuccessful (and highly accessible) and the consumer grade GPS used for the mapping in Peru alsoperformed well and all data points were saved (Tables 1 and 2)

Table 1 GPS coordinates of fallen wood containing Chlorociboria spp taken with Easy GPS in MaryrsquosPeak and McDonald-Dunn Forests

Location Sample Latitude (Decimal Degrees) Longitude (Decimal Degrees) Wood Species

Oak CreekO1 4460565 minus12333231 Acer macrophyllumO2 4460805 minus12333216 -

Soap Creek

S1 4464328 minus12332298 -S2 4464355 minus12332475 Acer macrophyllumS3 4464348 minus12332486 -S4 4464348 minus1233233 -S5 4464366 minus1233233 -

Woods Creek

W1 4454433 minus12345474 Acer macrophyllumW2 4454446 minus12345468 Acer macrophyllumW3 4454446 minus12345468 Acer macrophyllumW4 4454446 minus12345088 -W5 4454446 minus12345048 Acer macrophyllumW6 4454448 minus12344968 -

Dinner Creek D1 4447248 minus1234909 Acer macrophyllum

Jackson Creek J1 446134 minus12329206 Salix spp

Table 2 GPS coordinates of fallen wood with suspected Chlorociboria spp taken with GPSmap 64s inField Station Inkaterra Madre de Dios Peru

Location Sample Latitude (Decimal Degrees) Longitude (Decimal Degrees) Wood Species

Reserva Amazonica

331A minus125333 minus690437 -441C minus125347 minus690481 -578 minus125385 minus690505 -641 minus125373 minus690544 -

679B minus125328 minus690511 -709 minus125359 minus690518 -737 minus125293 minus690455 -

755B minus125282 minus690451 -941B minus125258 minus690456 -

Hacienda Concepcion1001B minus126068 minus690816 -1201 minus126058 minus690789 -1359 minus126078 minus690736 -

33 Wood Species Identification

A total of eight wood samples were collected Of these samples seven were found to be bigleaf maple (Acer macrophyllum Pursh) and all of these seven samples were correctly identified to thegenus (maple) by the participants without the use of a microscope The samples presented exclusiveprocumbent multiseriate ray cells Helical thickening on the vessel cell walls was also present and thischaracteristic determined the identification of the specimens as a big leaf maple as helical thickenings

Challenges 2018 9 11 6 of 11

are a common identifying feature for the Acer genus (Figure 1) This species is a hardwood that iscommonly found close to riparian forests in the Pacific Northwest

Challenges 2018 9 x 6 of 11

Wood species identification in Peru was generally not successful Those logs containing blue-green pigments were often too decayed to even attempt a genus identification The participants were not able to do any in-field wood identification at this site

The identification of the wood species is not required for citizen-scientists but if such people are capable it is a nice addition to the data

Figure 1 Radial section of a big leaf maple with red arrows pointing the helical thickenings on the vessel cell walls Magnification 200times

Figure 2 Tangential section of a willow with arrows pointing to the upright ray cells All rays are uniseriate Magnification 200times

Figure 3 Radial section of a willow showing alternate vessel wall pitting and Chlorociboria spp pigmentation Magnification 200times

Figure 1 Radial section of a big leaf maple with red arrows pointing the helical thickenings on thevessel cell walls Magnification 200times

One specimen was identified as willow (Salix spp) The sample presented both procumbent andupright rays The rays were exclusively uniseriate (Figure 2) and the vessel cell walls presented pitting(Figure 3) which are all identifying features of Salix sp L The participants incorrectly identified thissample as a maple in the field

Challenges 2018 9 x 6 of 11

Wood species identification in Peru was generally not successful Those logs containing blue-green pigments were often too decayed to even attempt a genus identification The participants were not able to do any in-field wood identification at this site

The identification of the wood species is not required for citizen-scientists but if such people are capable it is a nice addition to the data

Figure 1 Radial section of a big leaf maple with red arrows pointing the helical thickenings on the vessel cell walls Magnification 200times

Figure 2 Tangential section of a willow with arrows pointing to the upright ray cells All rays are uniseriate Magnification 200times

Figure 3 Radial section of a willow showing alternate vessel wall pitting and Chlorociboria spp pigmentation Magnification 200times

Figure 2 Tangential section of a willow with arrows pointing to the upright ray cells All rays areuniseriate Magnification 200times

Challenges 2018 9 11 7 of 11

Challenges 2018 9 x 6 of 11

Wood species identification in Peru was generally not successful Those logs containing blue-green pigments were often too decayed to even attempt a genus identification The participants were not able to do any in-field wood identification at this site

The identification of the wood species is not required for citizen-scientists but if such people are capable it is a nice addition to the data

Figure 1 Radial section of a big leaf maple with red arrows pointing the helical thickenings on the vessel cell walls Magnification 200times

Figure 2 Tangential section of a willow with arrows pointing to the upright ray cells All rays are uniseriate Magnification 200times

Figure 3 Radial section of a willow showing alternate vessel wall pitting and Chlorociboria spp pigmentation Magnification 200times

Figure 3 Radial section of a willow showing alternate vessel wall pitting and Chlorociboria spppigmentation Magnification 200times

Wood species identification in Peru was generally not successful Those logs containing blue-greenpigments were often too decayed to even attempt a genus identification The participants were notable to do any in-field wood identification at this site

The identification of the wood species is not required for citizen-scientists but if such people arecapable it is a nice addition to the data

34 Verification

Performing the sampling under supervision and further verification showed that the methodwas effective Therefore the collection method can be done by citizen-scientists without professionalsupervision as it has shown to be accurate

4 Discussion

41 Training Sampling and Sample Collection

In terms of simple mapping of Chlorociboria distribution the training was deemed successfulas eight logs containing Chlorociboria spp were identified within the McDonald-Dunn and MaryrsquosPeak Forests and twelve logs suspected to contain Chlorociboria spp were found in Madre de DiosPeru and all were successfully identified by the participants Wood identification was less successfulunfortunately especially in the tropical rainforests of Peru It seems likely that this aspect of theproposed citizen scientist survey for Chlorociboria would generally not be useful as researchers wouldhave no way to verify correct wood ID and when there is an abundance of diversity the likelihood ofmisidentification is great

A key element that allowed for the successful identification of the logs containing or suspected tocontain Chlorociboria spp was opening the logs well into the sapwood to find the blue-green coloredwood This helped students discount fungi lichens and bacteria that can colorize the surface and thefirst few centimeters of wood and thus limited them to general decay-capable fungi Another importantelement that was unique to this study was that it allowed for fungi without fruiting forms to be surveyed(when said fungi have a unique coloration system such as Chlorociboria spp) [39] This method couldeasily be applied to non-fruiting bodied pigment producing fungi such as Scytalidium cuboideumwhich develops red pigmentation [27] and Scytalidium ganodermophthorum which develops yellowpigmentation [5657] although these fungi do not have lsquouniquersquo colorants A final factor that aided inthe accurate identification was likely the focus on a single distinct genus allowing the participantsto focus their attention Surveys asking for a wider number of specimens to be located risk over orunderestimation [45859]

Challenges 2018 9 11 8 of 11

Another useful aspect of this technique was the use of common and accessible surveying toolssuch as machetes (which can be easily replaced by hatchets andor field knives) measuring tapessmart phone GPS applications andor GPS These tools require little to no training for their use and aregenerally accessible around the world This is important for future reliance on citizen-scientists forthe evaluation of Chlorociboria spp in the wild and should be effective Previous studies have showncitizen science to be very useful through works like eBird for data collection of bird diversity andmigration patterns which fully relies on bird hobbyists around the world [56] Specific to mycologythe active participation of Pacific Northwest mycological societies in the ten-year chanterelle projectresulted in the evaluation harvesting techniques of wild chanterelles in Oregon [8ndash10] Similarlythe long-term survey in Warwickshire in the UK from 1965 to 1980 gave one of the most detailedsurveys for fungi in the UK and was performed by lay mycologists of the area [7]

As with other citizen scientist surveys the technique detailed herein is similar to a rapid assessmentsurvey (RAPD) This method is commonly used for field surveys by non-specialized personnel in specificareas [60] The rapid assessment focuses on a group of genera in small diverse areas RAPD has beenwidely used for mycological surveys but if used for a wide number of genera can become unreliable [58]Focusing on a single group increases its accuracy The modified RAPD method proposed herein has beenshown to be accurate across different ecosystems (temperate rainforest in Benton County OR USA andthe tropical rainforest in Madre de Dios Peru) for the identification of the Chlorociboria spp on downedwood by non-specialized personnel In the future this method could be applied by citizen-scientists tocollect data on the worldwide distribution of the genus Chlorociboria

The method proposed can also be applied without setting a transect as it was performed in theAmazon rainforest This variation can improve the participation of citizen-scientists as some of themare mycology aficionados that can mark the location of Chlorociboria spp while on their foragingactivities or simply on general hikes

The distribution data (GPS points) which could be easily uploaded into a worldwide databasesuch as GBIF allowed the researchers on this project to identify forest types using Forest Service mapsand forest data from the Peruvian government This allowed for generation of a distribution mapand an understanding of forest types preferred by Chlorociboria species Continuation of this surveyby citizen scientists could greatly expand our understanding of the types of trees and systems thegenus of fungi prefer even if exact tree species cannot be reliably noted This data could then be madeavailable to forested landowners seeking to manage for Chlorociboria species xylindein productionand the economic opportunities therein

42 Future Research

Currently the location of the species is determined with a GPS app and field notes In thefuture the development of a smartphone application (like eBird [56]) specific to the distribution ofChlorociboria spp will be necessary to make the sampling data collection and data analysis simplerThis would also give the researchers involved one main input point for data making analysis and mapgeneration easier

As for the implementation of this initiative the unique laws and access to natural landscapesin different countries must be taken into account if the final goal is to apply it worldwide In theimmediate future the sampling of Chlorociboria spp can be done in association with mycologysocieties from the Pacific Northwest This can help to improve the location method and to develop theapplication with them as they have already participated in long-term studies (such as the Chanterelleproject) [10] In the intermediate future contacts with other mycological societies could be done on aworldwide scale (such as with the Danish Mycological Society) In the long-term the authors hopethat this method might be introduced into countries that do not have a culture of foraging (like Peru)

Finally the goal of the developed method and initiative is to gather data that can generate adetailed map of distribution of Chlorociboria spp and with it the identification of areas that present amore frequent presence of the fungus This information will be evaluated to determine the type of

Challenges 2018 9 11 9 of 11

forests and the dominant wood species in those areas and to then apply it to land management for thegrowth of these rare and useful fungi should this be of interest to landowners

5 Conclusions

The proposed method utilized simple tools and a smart phone or GPS to survey populations ofChlorociboria spp across temperate and tropical forests The method for identification of Chlorociboria sppcontaining wood was shown to be successful however the wood identification was not Howeverin general Chlorociboria identification can be easily taught to members of mycological societieseco tourists and other interested parties that wish to be involved in recording the distribution ofChlorociboria spp Over time the data collected could be used to build a worldwide picture of foresttype and wood preference by Chlorociboria species which will be of use to those who wish to managefor xylindein production on their forested lands

Acknowledgments The authors would like to thank Joseacute Koechlin Von Stein Inkaterra and the Inkaterra Asociacioacuten

Author Contributions Seri C Robinson Olivia E Kramer and Claudia C Andersen conceived and designed theexperiments Olivia E Kramer Claudia C Andersen and Patricia T Vega Gutierrez executed the experimentsand Sarath M Vega Gutierrez analyzed the data and wrote the paper

Conflicts of Interest The authors declare no conflict of interest

References

1 Stanley ES Susan RD Conservation commentary Ornithology and the national biological survey Auk1995 112 804ndash806

2 Tulloch AIT Possingham HP Joseph LN Szabo J Martin TG Realising the full potential of citizenscience monitoring programs Biol Conserv 2013 165 128ndash138 [CrossRef]

3 Cook R Encouraging the amateur Survey work in north-west England Mycologist 1992 6 162ndash164 [CrossRef]4 Gardiner MM Allee LL Brown PMJ Losey JE Roy HE Smyth RR Lessons from lady beetles

Accuracy of monitoring data from us and uk citizen-science programs Front Ecol Environ 2012 10 471ndash476[CrossRef]

5 Sullivan BL Wood CL Iliff MJ Bonney RE Fink D Kelling S Ebird A citizen-based bird observationnetwork in the biological sciences Biol Conserv 2009 142 2282ndash2292 [CrossRef]

6 Sullivan BL Aycrigg JL Barry JH Bonney RE Bruns N Cooper CB Damoulas T Dhondt AADietterich T Farnsworth A et al The ebird enterprise An integrated approach to development andapplication of citizen science Biol Conserv 2014 169 31ndash40 [CrossRef]

7 Clark MC The fungus flora of Warwickshire further records Bull Br Mycol Soc 1986 20 105ndash119[CrossRef]

8 Norvell L Studying the effects of harvesting on chanterelle productivity in Oregonrsquos Mt Hood nationalforest In Wild Mushroom Harvesting Discussion Session Minutes British Columbia Ministry of Forests PacificForestry Centre Victoria BC USA 1992 pp 9ndash15

9 Norvell L Kopecky F Lindgren J Roger J The chanterelle (cantharellus cibarius) A peek at productivityIn Proceedings of the Business and Science of Special Forest ProductsmdashA Conference and ExpositionAt Hillsboro OR USA 26ndash27 January 1994 pp 109ndash120

10 Norvell L Loving the chanterelle to death The ten-year Oregon chanterelle project McIlvainea 199512 6ndash25

11 McLain RJ Christensen HH Shannon MA When amateurs are the experts Amateur mycologists andwild mushroom politics in the Pacific Northwest USA Soc Nat Resour 1998 11 615ndash626 [CrossRef]

12 Robinson SC Richter DL Laks PE Colonization of sugar maple by spalting fungi For Prod J 200757 24ndash32

13 Persoon CH Observationes mycologicae Ann Bot 1795 15 1ndash3914 Persoon CH Synopsis Methodica Fungorum Apud Henricum Dieterich Gottingae Germany 180115 Persoon CH Mycologica Europaea Impensibus iI Palmii Erlangae Germany 1822 Volume 1 p 29116 Seaver FJ The North American Cup Fungi (Inoperculates) Hafner Publishing Co Inc New York NY

USA 1951

Challenges 2018 9 11 4 of 11

The presence of fruiting bodies was also used when fruiting bodies occurred Only downed deadwood was sampled as Chlorociboria spp is generally a slow growing wood decomposing fungus [54]If no pigmentation was found after going approximately 3 cm deep into the wood it was considered tonot contain Chlorociboria spp If pigmentation was present the GPS location was determined using theEasy GPS application for iPhone (Apple Cupertino CA USA) made by Kraus und Karnath GbR 2kit(Duumlsseldorf Germany) for the forests located in Benton County while a Garmin GPSmap 64s (GarminSchaffhausen Switzerland) was used in the Madre de Dios Peru area as phone signal was limited

Sample collection was performed in the US site only due to permitting restrictions in PeruThe samples were from downed wood that had the characteristic blue-green pigmentation and thatwere not completely decomposed (samples which did not crumble in the collectorrsquos hand) as somestructure integrity was required for further examination The size of the samples collected was aminimum of 5 cm in width and length and at least 04 cm in thickness

The GPS coordinates were then input into an Excel spread sheet (Microsoft) and uploaded to ArcMap(ESRI version 1051 Manufacturer Redlands CA USA) to generate maps of the collection sites

25 Wood Identification

Researchers were not allowed to remove wood from Peruvian forests therefore only field woodID could be performed For US occurrences any samples that occurred on wood hard enough to becollected were taken to the Applied Mycology laboratory at Oregon State University to determine thewood species Wood slides of 16 microm were obtained utilizing a Spencer-Buffalo Company microtometo create uniform sections of each plane The obtained wood slides were placed on a glass slide(VWRreg Radnor PA USA) and covered with a 15 glass cover-slip (VWRreg) The slides were thenexamined using a Nikon Eclipse Ni-U light microscope (Nikon Instruments Inc Melville NY USA)and photographed using a Nikon DS-Ri2 microscope camera

Once the images of the wood slides were obtained different anatomical aspects were notedThe first aspect was to identify if the wood was a conifer or a hardwood species by analyzing thepresence of vessels Ray types (upright andor procumbent) length and width (unicellular andormulticellular) were also annotated The parenchyma type was described per specimen and specialcharacteristics (as presence of gums or prismatic crystals) were noted After collecting the informationper wood sample the characteristics were run through the North Carolina State University Insidewooddata base [55]

26 Verification

Microscopic verification of the wood species as well as confirmation of Chlorociboria speciescolonization was done by wood anatomy experts using a Nikon Eclipse Ni-U microscope to evaluatethe presence of hyphae and the location of the pigment in the wood

For the Peruvian samples the confirmation of the possible presence of Chlorociboria spp wasperformed by visual inspection by a professor andor postdoctoral researcher and was entirely basedupon the correct color being present in the wood (blue-green) Verification was necessary for thesampling as this was the first time the method was applied and verification was required to makesure the collections and fungal identification by wood pigmentation was performed correctly and thatthe explanations given were sufficient for a non-technical person

3 Results

31 Sampling

In the USA the trained students successfully identified eight logs that contained Chlorociboriaalong the various transectstrails All of the logs identified by the students as having Chlorociboriaspecies were confirmed as hosts of Chlorociboria spp

Challenges 2018 9 11 5 of 11

In Peru identification of Chlorociboria-stained wood was made through visual determination ofthe pigment as no specialized equipment was available for further inspection However per themethods for the USA identification a professor andor post-doctoral research did verify that thecorrect color was present to indicate Chlorociboria spp All student identifications were correct

32 Site Accessibility

The selection of the sampling sites allowed for easy accessibility by one person or a team up tothree As accessibility is a main factor for lsquocitizen-scientistsrsquo this was taken into consideration for theexperimental design Using this technique downed trees and branches carrying Chlorociboria sppwere successfully identified with minimal danger to the participants The use of an iPhone app wassuccessful (and highly accessible) and the consumer grade GPS used for the mapping in Peru alsoperformed well and all data points were saved (Tables 1 and 2)

Table 1 GPS coordinates of fallen wood containing Chlorociboria spp taken with Easy GPS in MaryrsquosPeak and McDonald-Dunn Forests

Location Sample Latitude (Decimal Degrees) Longitude (Decimal Degrees) Wood Species

Oak CreekO1 4460565 minus12333231 Acer macrophyllumO2 4460805 minus12333216 -

Soap Creek

S1 4464328 minus12332298 -S2 4464355 minus12332475 Acer macrophyllumS3 4464348 minus12332486 -S4 4464348 minus1233233 -S5 4464366 minus1233233 -

Woods Creek

W1 4454433 minus12345474 Acer macrophyllumW2 4454446 minus12345468 Acer macrophyllumW3 4454446 minus12345468 Acer macrophyllumW4 4454446 minus12345088 -W5 4454446 minus12345048 Acer macrophyllumW6 4454448 minus12344968 -

Dinner Creek D1 4447248 minus1234909 Acer macrophyllum

Jackson Creek J1 446134 minus12329206 Salix spp

Table 2 GPS coordinates of fallen wood with suspected Chlorociboria spp taken with GPSmap 64s inField Station Inkaterra Madre de Dios Peru

Location Sample Latitude (Decimal Degrees) Longitude (Decimal Degrees) Wood Species

Reserva Amazonica

331A minus125333 minus690437 -441C minus125347 minus690481 -578 minus125385 minus690505 -641 minus125373 minus690544 -

679B minus125328 minus690511 -709 minus125359 minus690518 -737 minus125293 minus690455 -

755B minus125282 minus690451 -941B minus125258 minus690456 -

Hacienda Concepcion1001B minus126068 minus690816 -1201 minus126058 minus690789 -1359 minus126078 minus690736 -

33 Wood Species Identification

A total of eight wood samples were collected Of these samples seven were found to be bigleaf maple (Acer macrophyllum Pursh) and all of these seven samples were correctly identified to thegenus (maple) by the participants without the use of a microscope The samples presented exclusiveprocumbent multiseriate ray cells Helical thickening on the vessel cell walls was also present and thischaracteristic determined the identification of the specimens as a big leaf maple as helical thickenings

Challenges 2018 9 11 6 of 11

are a common identifying feature for the Acer genus (Figure 1) This species is a hardwood that iscommonly found close to riparian forests in the Pacific Northwest

Challenges 2018 9 x 6 of 11

Wood species identification in Peru was generally not successful Those logs containing blue-green pigments were often too decayed to even attempt a genus identification The participants were not able to do any in-field wood identification at this site

The identification of the wood species is not required for citizen-scientists but if such people are capable it is a nice addition to the data

Figure 1 Radial section of a big leaf maple with red arrows pointing the helical thickenings on the vessel cell walls Magnification 200times

Figure 2 Tangential section of a willow with arrows pointing to the upright ray cells All rays are uniseriate Magnification 200times

Figure 3 Radial section of a willow showing alternate vessel wall pitting and Chlorociboria spp pigmentation Magnification 200times

Figure 1 Radial section of a big leaf maple with red arrows pointing the helical thickenings on thevessel cell walls Magnification 200times

One specimen was identified as willow (Salix spp) The sample presented both procumbent andupright rays The rays were exclusively uniseriate (Figure 2) and the vessel cell walls presented pitting(Figure 3) which are all identifying features of Salix sp L The participants incorrectly identified thissample as a maple in the field

Challenges 2018 9 x 6 of 11

Wood species identification in Peru was generally not successful Those logs containing blue-green pigments were often too decayed to even attempt a genus identification The participants were not able to do any in-field wood identification at this site

The identification of the wood species is not required for citizen-scientists but if such people are capable it is a nice addition to the data

Figure 1 Radial section of a big leaf maple with red arrows pointing the helical thickenings on the vessel cell walls Magnification 200times

Figure 2 Tangential section of a willow with arrows pointing to the upright ray cells All rays are uniseriate Magnification 200times

Figure 3 Radial section of a willow showing alternate vessel wall pitting and Chlorociboria spp pigmentation Magnification 200times

Figure 2 Tangential section of a willow with arrows pointing to the upright ray cells All rays areuniseriate Magnification 200times

Challenges 2018 9 11 7 of 11

Challenges 2018 9 x 6 of 11

Wood species identification in Peru was generally not successful Those logs containing blue-green pigments were often too decayed to even attempt a genus identification The participants were not able to do any in-field wood identification at this site

The identification of the wood species is not required for citizen-scientists but if such people are capable it is a nice addition to the data

Figure 1 Radial section of a big leaf maple with red arrows pointing the helical thickenings on the vessel cell walls Magnification 200times

Figure 2 Tangential section of a willow with arrows pointing to the upright ray cells All rays are uniseriate Magnification 200times

Figure 3 Radial section of a willow showing alternate vessel wall pitting and Chlorociboria spp pigmentation Magnification 200times

Figure 3 Radial section of a willow showing alternate vessel wall pitting and Chlorociboria spppigmentation Magnification 200times

Wood species identification in Peru was generally not successful Those logs containing blue-greenpigments were often too decayed to even attempt a genus identification The participants were notable to do any in-field wood identification at this site

The identification of the wood species is not required for citizen-scientists but if such people arecapable it is a nice addition to the data

34 Verification

Performing the sampling under supervision and further verification showed that the methodwas effective Therefore the collection method can be done by citizen-scientists without professionalsupervision as it has shown to be accurate

4 Discussion

41 Training Sampling and Sample Collection

In terms of simple mapping of Chlorociboria distribution the training was deemed successfulas eight logs containing Chlorociboria spp were identified within the McDonald-Dunn and MaryrsquosPeak Forests and twelve logs suspected to contain Chlorociboria spp were found in Madre de DiosPeru and all were successfully identified by the participants Wood identification was less successfulunfortunately especially in the tropical rainforests of Peru It seems likely that this aspect of theproposed citizen scientist survey for Chlorociboria would generally not be useful as researchers wouldhave no way to verify correct wood ID and when there is an abundance of diversity the likelihood ofmisidentification is great

A key element that allowed for the successful identification of the logs containing or suspected tocontain Chlorociboria spp was opening the logs well into the sapwood to find the blue-green coloredwood This helped students discount fungi lichens and bacteria that can colorize the surface and thefirst few centimeters of wood and thus limited them to general decay-capable fungi Another importantelement that was unique to this study was that it allowed for fungi without fruiting forms to be surveyed(when said fungi have a unique coloration system such as Chlorociboria spp) [39] This method couldeasily be applied to non-fruiting bodied pigment producing fungi such as Scytalidium cuboideumwhich develops red pigmentation [27] and Scytalidium ganodermophthorum which develops yellowpigmentation [5657] although these fungi do not have lsquouniquersquo colorants A final factor that aided inthe accurate identification was likely the focus on a single distinct genus allowing the participantsto focus their attention Surveys asking for a wider number of specimens to be located risk over orunderestimation [45859]

Challenges 2018 9 11 8 of 11

Another useful aspect of this technique was the use of common and accessible surveying toolssuch as machetes (which can be easily replaced by hatchets andor field knives) measuring tapessmart phone GPS applications andor GPS These tools require little to no training for their use and aregenerally accessible around the world This is important for future reliance on citizen-scientists forthe evaluation of Chlorociboria spp in the wild and should be effective Previous studies have showncitizen science to be very useful through works like eBird for data collection of bird diversity andmigration patterns which fully relies on bird hobbyists around the world [56] Specific to mycologythe active participation of Pacific Northwest mycological societies in the ten-year chanterelle projectresulted in the evaluation harvesting techniques of wild chanterelles in Oregon [8ndash10] Similarlythe long-term survey in Warwickshire in the UK from 1965 to 1980 gave one of the most detailedsurveys for fungi in the UK and was performed by lay mycologists of the area [7]

As with other citizen scientist surveys the technique detailed herein is similar to a rapid assessmentsurvey (RAPD) This method is commonly used for field surveys by non-specialized personnel in specificareas [60] The rapid assessment focuses on a group of genera in small diverse areas RAPD has beenwidely used for mycological surveys but if used for a wide number of genera can become unreliable [58]Focusing on a single group increases its accuracy The modified RAPD method proposed herein has beenshown to be accurate across different ecosystems (temperate rainforest in Benton County OR USA andthe tropical rainforest in Madre de Dios Peru) for the identification of the Chlorociboria spp on downedwood by non-specialized personnel In the future this method could be applied by citizen-scientists tocollect data on the worldwide distribution of the genus Chlorociboria

The method proposed can also be applied without setting a transect as it was performed in theAmazon rainforest This variation can improve the participation of citizen-scientists as some of themare mycology aficionados that can mark the location of Chlorociboria spp while on their foragingactivities or simply on general hikes

The distribution data (GPS points) which could be easily uploaded into a worldwide databasesuch as GBIF allowed the researchers on this project to identify forest types using Forest Service mapsand forest data from the Peruvian government This allowed for generation of a distribution mapand an understanding of forest types preferred by Chlorociboria species Continuation of this surveyby citizen scientists could greatly expand our understanding of the types of trees and systems thegenus of fungi prefer even if exact tree species cannot be reliably noted This data could then be madeavailable to forested landowners seeking to manage for Chlorociboria species xylindein productionand the economic opportunities therein

42 Future Research

Currently the location of the species is determined with a GPS app and field notes In thefuture the development of a smartphone application (like eBird [56]) specific to the distribution ofChlorociboria spp will be necessary to make the sampling data collection and data analysis simplerThis would also give the researchers involved one main input point for data making analysis and mapgeneration easier

As for the implementation of this initiative the unique laws and access to natural landscapesin different countries must be taken into account if the final goal is to apply it worldwide In theimmediate future the sampling of Chlorociboria spp can be done in association with mycologysocieties from the Pacific Northwest This can help to improve the location method and to develop theapplication with them as they have already participated in long-term studies (such as the Chanterelleproject) [10] In the intermediate future contacts with other mycological societies could be done on aworldwide scale (such as with the Danish Mycological Society) In the long-term the authors hopethat this method might be introduced into countries that do not have a culture of foraging (like Peru)

Finally the goal of the developed method and initiative is to gather data that can generate adetailed map of distribution of Chlorociboria spp and with it the identification of areas that present amore frequent presence of the fungus This information will be evaluated to determine the type of

Challenges 2018 9 11 9 of 11

forests and the dominant wood species in those areas and to then apply it to land management for thegrowth of these rare and useful fungi should this be of interest to landowners

5 Conclusions

The proposed method utilized simple tools and a smart phone or GPS to survey populations ofChlorociboria spp across temperate and tropical forests The method for identification of Chlorociboria sppcontaining wood was shown to be successful however the wood identification was not Howeverin general Chlorociboria identification can be easily taught to members of mycological societieseco tourists and other interested parties that wish to be involved in recording the distribution ofChlorociboria spp Over time the data collected could be used to build a worldwide picture of foresttype and wood preference by Chlorociboria species which will be of use to those who wish to managefor xylindein production on their forested lands

Acknowledgments The authors would like to thank Joseacute Koechlin Von Stein Inkaterra and the Inkaterra Asociacioacuten

Author Contributions Seri C Robinson Olivia E Kramer and Claudia C Andersen conceived and designed theexperiments Olivia E Kramer Claudia C Andersen and Patricia T Vega Gutierrez executed the experimentsand Sarath M Vega Gutierrez analyzed the data and wrote the paper

Conflicts of Interest The authors declare no conflict of interest

References

1 Stanley ES Susan RD Conservation commentary Ornithology and the national biological survey Auk1995 112 804ndash806

2 Tulloch AIT Possingham HP Joseph LN Szabo J Martin TG Realising the full potential of citizenscience monitoring programs Biol Conserv 2013 165 128ndash138 [CrossRef]

3 Cook R Encouraging the amateur Survey work in north-west England Mycologist 1992 6 162ndash164 [CrossRef]4 Gardiner MM Allee LL Brown PMJ Losey JE Roy HE Smyth RR Lessons from lady beetles

Accuracy of monitoring data from us and uk citizen-science programs Front Ecol Environ 2012 10 471ndash476[CrossRef]

5 Sullivan BL Wood CL Iliff MJ Bonney RE Fink D Kelling S Ebird A citizen-based bird observationnetwork in the biological sciences Biol Conserv 2009 142 2282ndash2292 [CrossRef]

6 Sullivan BL Aycrigg JL Barry JH Bonney RE Bruns N Cooper CB Damoulas T Dhondt AADietterich T Farnsworth A et al The ebird enterprise An integrated approach to development andapplication of citizen science Biol Conserv 2014 169 31ndash40 [CrossRef]

7 Clark MC The fungus flora of Warwickshire further records Bull Br Mycol Soc 1986 20 105ndash119[CrossRef]

8 Norvell L Studying the effects of harvesting on chanterelle productivity in Oregonrsquos Mt Hood nationalforest In Wild Mushroom Harvesting Discussion Session Minutes British Columbia Ministry of Forests PacificForestry Centre Victoria BC USA 1992 pp 9ndash15

9 Norvell L Kopecky F Lindgren J Roger J The chanterelle (cantharellus cibarius) A peek at productivityIn Proceedings of the Business and Science of Special Forest ProductsmdashA Conference and ExpositionAt Hillsboro OR USA 26ndash27 January 1994 pp 109ndash120

10 Norvell L Loving the chanterelle to death The ten-year Oregon chanterelle project McIlvainea 199512 6ndash25

11 McLain RJ Christensen HH Shannon MA When amateurs are the experts Amateur mycologists andwild mushroom politics in the Pacific Northwest USA Soc Nat Resour 1998 11 615ndash626 [CrossRef]

12 Robinson SC Richter DL Laks PE Colonization of sugar maple by spalting fungi For Prod J 200757 24ndash32

13 Persoon CH Observationes mycologicae Ann Bot 1795 15 1ndash3914 Persoon CH Synopsis Methodica Fungorum Apud Henricum Dieterich Gottingae Germany 180115 Persoon CH Mycologica Europaea Impensibus iI Palmii Erlangae Germany 1822 Volume 1 p 29116 Seaver FJ The North American Cup Fungi (Inoperculates) Hafner Publishing Co Inc New York NY

USA 1951

Challenges 2018 9 11 5 of 11

In Peru identification of Chlorociboria-stained wood was made through visual determination ofthe pigment as no specialized equipment was available for further inspection However per themethods for the USA identification a professor andor post-doctoral research did verify that thecorrect color was present to indicate Chlorociboria spp All student identifications were correct

32 Site Accessibility

The selection of the sampling sites allowed for easy accessibility by one person or a team up tothree As accessibility is a main factor for lsquocitizen-scientistsrsquo this was taken into consideration for theexperimental design Using this technique downed trees and branches carrying Chlorociboria sppwere successfully identified with minimal danger to the participants The use of an iPhone app wassuccessful (and highly accessible) and the consumer grade GPS used for the mapping in Peru alsoperformed well and all data points were saved (Tables 1 and 2)

Table 1 GPS coordinates of fallen wood containing Chlorociboria spp taken with Easy GPS in MaryrsquosPeak and McDonald-Dunn Forests

Location Sample Latitude (Decimal Degrees) Longitude (Decimal Degrees) Wood Species

Oak CreekO1 4460565 minus12333231 Acer macrophyllumO2 4460805 minus12333216 -

Soap Creek

S1 4464328 minus12332298 -S2 4464355 minus12332475 Acer macrophyllumS3 4464348 minus12332486 -S4 4464348 minus1233233 -S5 4464366 minus1233233 -

Woods Creek

W1 4454433 minus12345474 Acer macrophyllumW2 4454446 minus12345468 Acer macrophyllumW3 4454446 minus12345468 Acer macrophyllumW4 4454446 minus12345088 -W5 4454446 minus12345048 Acer macrophyllumW6 4454448 minus12344968 -

Dinner Creek D1 4447248 minus1234909 Acer macrophyllum

Jackson Creek J1 446134 minus12329206 Salix spp

Table 2 GPS coordinates of fallen wood with suspected Chlorociboria spp taken with GPSmap 64s inField Station Inkaterra Madre de Dios Peru

Location Sample Latitude (Decimal Degrees) Longitude (Decimal Degrees) Wood Species

Reserva Amazonica

331A minus125333 minus690437 -441C minus125347 minus690481 -578 minus125385 minus690505 -641 minus125373 minus690544 -

679B minus125328 minus690511 -709 minus125359 minus690518 -737 minus125293 minus690455 -

755B minus125282 minus690451 -941B minus125258 minus690456 -

Hacienda Concepcion1001B minus126068 minus690816 -1201 minus126058 minus690789 -1359 minus126078 minus690736 -

33 Wood Species Identification

A total of eight wood samples were collected Of these samples seven were found to be bigleaf maple (Acer macrophyllum Pursh) and all of these seven samples were correctly identified to thegenus (maple) by the participants without the use of a microscope The samples presented exclusiveprocumbent multiseriate ray cells Helical thickening on the vessel cell walls was also present and thischaracteristic determined the identification of the specimens as a big leaf maple as helical thickenings

Challenges 2018 9 11 6 of 11

are a common identifying feature for the Acer genus (Figure 1) This species is a hardwood that iscommonly found close to riparian forests in the Pacific Northwest

Challenges 2018 9 x 6 of 11

Wood species identification in Peru was generally not successful Those logs containing blue-green pigments were often too decayed to even attempt a genus identification The participants were not able to do any in-field wood identification at this site

The identification of the wood species is not required for citizen-scientists but if such people are capable it is a nice addition to the data

Figure 1 Radial section of a big leaf maple with red arrows pointing the helical thickenings on the vessel cell walls Magnification 200times

Figure 2 Tangential section of a willow with arrows pointing to the upright ray cells All rays are uniseriate Magnification 200times

Figure 3 Radial section of a willow showing alternate vessel wall pitting and Chlorociboria spp pigmentation Magnification 200times

Figure 1 Radial section of a big leaf maple with red arrows pointing the helical thickenings on thevessel cell walls Magnification 200times

One specimen was identified as willow (Salix spp) The sample presented both procumbent andupright rays The rays were exclusively uniseriate (Figure 2) and the vessel cell walls presented pitting(Figure 3) which are all identifying features of Salix sp L The participants incorrectly identified thissample as a maple in the field

Challenges 2018 9 x 6 of 11

Wood species identification in Peru was generally not successful Those logs containing blue-green pigments were often too decayed to even attempt a genus identification The participants were not able to do any in-field wood identification at this site

The identification of the wood species is not required for citizen-scientists but if such people are capable it is a nice addition to the data

Figure 1 Radial section of a big leaf maple with red arrows pointing the helical thickenings on the vessel cell walls Magnification 200times

Figure 2 Tangential section of a willow with arrows pointing to the upright ray cells All rays are uniseriate Magnification 200times

Figure 3 Radial section of a willow showing alternate vessel wall pitting and Chlorociboria spp pigmentation Magnification 200times

Figure 2 Tangential section of a willow with arrows pointing to the upright ray cells All rays areuniseriate Magnification 200times

Challenges 2018 9 11 7 of 11

Challenges 2018 9 x 6 of 11

Wood species identification in Peru was generally not successful Those logs containing blue-green pigments were often too decayed to even attempt a genus identification The participants were not able to do any in-field wood identification at this site

The identification of the wood species is not required for citizen-scientists but if such people are capable it is a nice addition to the data

Figure 1 Radial section of a big leaf maple with red arrows pointing the helical thickenings on the vessel cell walls Magnification 200times

Figure 2 Tangential section of a willow with arrows pointing to the upright ray cells All rays are uniseriate Magnification 200times

Figure 3 Radial section of a willow showing alternate vessel wall pitting and Chlorociboria spp pigmentation Magnification 200times

Figure 3 Radial section of a willow showing alternate vessel wall pitting and Chlorociboria spppigmentation Magnification 200times

Wood species identification in Peru was generally not successful Those logs containing blue-greenpigments were often too decayed to even attempt a genus identification The participants were notable to do any in-field wood identification at this site

The identification of the wood species is not required for citizen-scientists but if such people arecapable it is a nice addition to the data

34 Verification

Performing the sampling under supervision and further verification showed that the methodwas effective Therefore the collection method can be done by citizen-scientists without professionalsupervision as it has shown to be accurate

4 Discussion

41 Training Sampling and Sample Collection

In terms of simple mapping of Chlorociboria distribution the training was deemed successfulas eight logs containing Chlorociboria spp were identified within the McDonald-Dunn and MaryrsquosPeak Forests and twelve logs suspected to contain Chlorociboria spp were found in Madre de DiosPeru and all were successfully identified by the participants Wood identification was less successfulunfortunately especially in the tropical rainforests of Peru It seems likely that this aspect of theproposed citizen scientist survey for Chlorociboria would generally not be useful as researchers wouldhave no way to verify correct wood ID and when there is an abundance of diversity the likelihood ofmisidentification is great

A key element that allowed for the successful identification of the logs containing or suspected tocontain Chlorociboria spp was opening the logs well into the sapwood to find the blue-green coloredwood This helped students discount fungi lichens and bacteria that can colorize the surface and thefirst few centimeters of wood and thus limited them to general decay-capable fungi Another importantelement that was unique to this study was that it allowed for fungi without fruiting forms to be surveyed(when said fungi have a unique coloration system such as Chlorociboria spp) [39] This method couldeasily be applied to non-fruiting bodied pigment producing fungi such as Scytalidium cuboideumwhich develops red pigmentation [27] and Scytalidium ganodermophthorum which develops yellowpigmentation [5657] although these fungi do not have lsquouniquersquo colorants A final factor that aided inthe accurate identification was likely the focus on a single distinct genus allowing the participantsto focus their attention Surveys asking for a wider number of specimens to be located risk over orunderestimation [45859]

Challenges 2018 9 11 8 of 11

Another useful aspect of this technique was the use of common and accessible surveying toolssuch as machetes (which can be easily replaced by hatchets andor field knives) measuring tapessmart phone GPS applications andor GPS These tools require little to no training for their use and aregenerally accessible around the world This is important for future reliance on citizen-scientists forthe evaluation of Chlorociboria spp in the wild and should be effective Previous studies have showncitizen science to be very useful through works like eBird for data collection of bird diversity andmigration patterns which fully relies on bird hobbyists around the world [56] Specific to mycologythe active participation of Pacific Northwest mycological societies in the ten-year chanterelle projectresulted in the evaluation harvesting techniques of wild chanterelles in Oregon [8ndash10] Similarlythe long-term survey in Warwickshire in the UK from 1965 to 1980 gave one of the most detailedsurveys for fungi in the UK and was performed by lay mycologists of the area [7]

As with other citizen scientist surveys the technique detailed herein is similar to a rapid assessmentsurvey (RAPD) This method is commonly used for field surveys by non-specialized personnel in specificareas [60] The rapid assessment focuses on a group of genera in small diverse areas RAPD has beenwidely used for mycological surveys but if used for a wide number of genera can become unreliable [58]Focusing on a single group increases its accuracy The modified RAPD method proposed herein has beenshown to be accurate across different ecosystems (temperate rainforest in Benton County OR USA andthe tropical rainforest in Madre de Dios Peru) for the identification of the Chlorociboria spp on downedwood by non-specialized personnel In the future this method could be applied by citizen-scientists tocollect data on the worldwide distribution of the genus Chlorociboria

The method proposed can also be applied without setting a transect as it was performed in theAmazon rainforest This variation can improve the participation of citizen-scientists as some of themare mycology aficionados that can mark the location of Chlorociboria spp while on their foragingactivities or simply on general hikes

The distribution data (GPS points) which could be easily uploaded into a worldwide databasesuch as GBIF allowed the researchers on this project to identify forest types using Forest Service mapsand forest data from the Peruvian government This allowed for generation of a distribution mapand an understanding of forest types preferred by Chlorociboria species Continuation of this surveyby citizen scientists could greatly expand our understanding of the types of trees and systems thegenus of fungi prefer even if exact tree species cannot be reliably noted This data could then be madeavailable to forested landowners seeking to manage for Chlorociboria species xylindein productionand the economic opportunities therein

42 Future Research

Currently the location of the species is determined with a GPS app and field notes In thefuture the development of a smartphone application (like eBird [56]) specific to the distribution ofChlorociboria spp will be necessary to make the sampling data collection and data analysis simplerThis would also give the researchers involved one main input point for data making analysis and mapgeneration easier

As for the implementation of this initiative the unique laws and access to natural landscapesin different countries must be taken into account if the final goal is to apply it worldwide In theimmediate future the sampling of Chlorociboria spp can be done in association with mycologysocieties from the Pacific Northwest This can help to improve the location method and to develop theapplication with them as they have already participated in long-term studies (such as the Chanterelleproject) [10] In the intermediate future contacts with other mycological societies could be done on aworldwide scale (such as with the Danish Mycological Society) In the long-term the authors hopethat this method might be introduced into countries that do not have a culture of foraging (like Peru)

Finally the goal of the developed method and initiative is to gather data that can generate adetailed map of distribution of Chlorociboria spp and with it the identification of areas that present amore frequent presence of the fungus This information will be evaluated to determine the type of

Challenges 2018 9 11 9 of 11

forests and the dominant wood species in those areas and to then apply it to land management for thegrowth of these rare and useful fungi should this be of interest to landowners

5 Conclusions

The proposed method utilized simple tools and a smart phone or GPS to survey populations ofChlorociboria spp across temperate and tropical forests The method for identification of Chlorociboria sppcontaining wood was shown to be successful however the wood identification was not Howeverin general Chlorociboria identification can be easily taught to members of mycological societieseco tourists and other interested parties that wish to be involved in recording the distribution ofChlorociboria spp Over time the data collected could be used to build a worldwide picture of foresttype and wood preference by Chlorociboria species which will be of use to those who wish to managefor xylindein production on their forested lands

Acknowledgments The authors would like to thank Joseacute Koechlin Von Stein Inkaterra and the Inkaterra Asociacioacuten

Author Contributions Seri C Robinson Olivia E Kramer and Claudia C Andersen conceived and designed theexperiments Olivia E Kramer Claudia C Andersen and Patricia T Vega Gutierrez executed the experimentsand Sarath M Vega Gutierrez analyzed the data and wrote the paper

Conflicts of Interest The authors declare no conflict of interest

References

1 Stanley ES Susan RD Conservation commentary Ornithology and the national biological survey Auk1995 112 804ndash806

2 Tulloch AIT Possingham HP Joseph LN Szabo J Martin TG Realising the full potential of citizenscience monitoring programs Biol Conserv 2013 165 128ndash138 [CrossRef]

3 Cook R Encouraging the amateur Survey work in north-west England Mycologist 1992 6 162ndash164 [CrossRef]4 Gardiner MM Allee LL Brown PMJ Losey JE Roy HE Smyth RR Lessons from lady beetles

Accuracy of monitoring data from us and uk citizen-science programs Front Ecol Environ 2012 10 471ndash476[CrossRef]

5 Sullivan BL Wood CL Iliff MJ Bonney RE Fink D Kelling S Ebird A citizen-based bird observationnetwork in the biological sciences Biol Conserv 2009 142 2282ndash2292 [CrossRef]

6 Sullivan BL Aycrigg JL Barry JH Bonney RE Bruns N Cooper CB Damoulas T Dhondt AADietterich T Farnsworth A et al The ebird enterprise An integrated approach to development andapplication of citizen science Biol Conserv 2014 169 31ndash40 [CrossRef]

7 Clark MC The fungus flora of Warwickshire further records Bull Br Mycol Soc 1986 20 105ndash119[CrossRef]

8 Norvell L Studying the effects of harvesting on chanterelle productivity in Oregonrsquos Mt Hood nationalforest In Wild Mushroom Harvesting Discussion Session Minutes British Columbia Ministry of Forests PacificForestry Centre Victoria BC USA 1992 pp 9ndash15

9 Norvell L Kopecky F Lindgren J Roger J The chanterelle (cantharellus cibarius) A peek at productivityIn Proceedings of the Business and Science of Special Forest ProductsmdashA Conference and ExpositionAt Hillsboro OR USA 26ndash27 January 1994 pp 109ndash120

10 Norvell L Loving the chanterelle to death The ten-year Oregon chanterelle project McIlvainea 199512 6ndash25

11 McLain RJ Christensen HH Shannon MA When amateurs are the experts Amateur mycologists andwild mushroom politics in the Pacific Northwest USA Soc Nat Resour 1998 11 615ndash626 [CrossRef]

12 Robinson SC Richter DL Laks PE Colonization of sugar maple by spalting fungi For Prod J 200757 24ndash32

13 Persoon CH Observationes mycologicae Ann Bot 1795 15 1ndash3914 Persoon CH Synopsis Methodica Fungorum Apud Henricum Dieterich Gottingae Germany 180115 Persoon CH Mycologica Europaea Impensibus iI Palmii Erlangae Germany 1822 Volume 1 p 29116 Seaver FJ The North American Cup Fungi (Inoperculates) Hafner Publishing Co Inc New York NY

USA 1951

Challenges 2018 9 11 6 of 11

are a common identifying feature for the Acer genus (Figure 1) This species is a hardwood that iscommonly found close to riparian forests in the Pacific Northwest

Challenges 2018 9 x 6 of 11

Wood species identification in Peru was generally not successful Those logs containing blue-green pigments were often too decayed to even attempt a genus identification The participants were not able to do any in-field wood identification at this site

The identification of the wood species is not required for citizen-scientists but if such people are capable it is a nice addition to the data

Figure 1 Radial section of a big leaf maple with red arrows pointing the helical thickenings on the vessel cell walls Magnification 200times

Figure 2 Tangential section of a willow with arrows pointing to the upright ray cells All rays are uniseriate Magnification 200times

Figure 3 Radial section of a willow showing alternate vessel wall pitting and Chlorociboria spp pigmentation Magnification 200times

Figure 1 Radial section of a big leaf maple with red arrows pointing the helical thickenings on thevessel cell walls Magnification 200times

One specimen was identified as willow (Salix spp) The sample presented both procumbent andupright rays The rays were exclusively uniseriate (Figure 2) and the vessel cell walls presented pitting(Figure 3) which are all identifying features of Salix sp L The participants incorrectly identified thissample as a maple in the field

Challenges 2018 9 x 6 of 11

Wood species identification in Peru was generally not successful Those logs containing blue-green pigments were often too decayed to even attempt a genus identification The participants were not able to do any in-field wood identification at this site

The identification of the wood species is not required for citizen-scientists but if such people are capable it is a nice addition to the data

Figure 1 Radial section of a big leaf maple with red arrows pointing the helical thickenings on the vessel cell walls Magnification 200times

Figure 2 Tangential section of a willow with arrows pointing to the upright ray cells All rays are uniseriate Magnification 200times

Figure 3 Radial section of a willow showing alternate vessel wall pitting and Chlorociboria spp pigmentation Magnification 200times

Figure 2 Tangential section of a willow with arrows pointing to the upright ray cells All rays areuniseriate Magnification 200times

Challenges 2018 9 11 7 of 11

Challenges 2018 9 x 6 of 11

Wood species identification in Peru was generally not successful Those logs containing blue-green pigments were often too decayed to even attempt a genus identification The participants were not able to do any in-field wood identification at this site

The identification of the wood species is not required for citizen-scientists but if such people are capable it is a nice addition to the data

Figure 1 Radial section of a big leaf maple with red arrows pointing the helical thickenings on the vessel cell walls Magnification 200times

Figure 2 Tangential section of a willow with arrows pointing to the upright ray cells All rays are uniseriate Magnification 200times

Figure 3 Radial section of a willow showing alternate vessel wall pitting and Chlorociboria spp pigmentation Magnification 200times

Figure 3 Radial section of a willow showing alternate vessel wall pitting and Chlorociboria spppigmentation Magnification 200times

Wood species identification in Peru was generally not successful Those logs containing blue-greenpigments were often too decayed to even attempt a genus identification The participants were notable to do any in-field wood identification at this site

The identification of the wood species is not required for citizen-scientists but if such people arecapable it is a nice addition to the data

34 Verification

Performing the sampling under supervision and further verification showed that the methodwas effective Therefore the collection method can be done by citizen-scientists without professionalsupervision as it has shown to be accurate

4 Discussion

41 Training Sampling and Sample Collection

In terms of simple mapping of Chlorociboria distribution the training was deemed successfulas eight logs containing Chlorociboria spp were identified within the McDonald-Dunn and MaryrsquosPeak Forests and twelve logs suspected to contain Chlorociboria spp were found in Madre de DiosPeru and all were successfully identified by the participants Wood identification was less successfulunfortunately especially in the tropical rainforests of Peru It seems likely that this aspect of theproposed citizen scientist survey for Chlorociboria would generally not be useful as researchers wouldhave no way to verify correct wood ID and when there is an abundance of diversity the likelihood ofmisidentification is great

A key element that allowed for the successful identification of the logs containing or suspected tocontain Chlorociboria spp was opening the logs well into the sapwood to find the blue-green coloredwood This helped students discount fungi lichens and bacteria that can colorize the surface and thefirst few centimeters of wood and thus limited them to general decay-capable fungi Another importantelement that was unique to this study was that it allowed for fungi without fruiting forms to be surveyed(when said fungi have a unique coloration system such as Chlorociboria spp) [39] This method couldeasily be applied to non-fruiting bodied pigment producing fungi such as Scytalidium cuboideumwhich develops red pigmentation [27] and Scytalidium ganodermophthorum which develops yellowpigmentation [5657] although these fungi do not have lsquouniquersquo colorants A final factor that aided inthe accurate identification was likely the focus on a single distinct genus allowing the participantsto focus their attention Surveys asking for a wider number of specimens to be located risk over orunderestimation [45859]

Challenges 2018 9 11 8 of 11

Another useful aspect of this technique was the use of common and accessible surveying toolssuch as machetes (which can be easily replaced by hatchets andor field knives) measuring tapessmart phone GPS applications andor GPS These tools require little to no training for their use and aregenerally accessible around the world This is important for future reliance on citizen-scientists forthe evaluation of Chlorociboria spp in the wild and should be effective Previous studies have showncitizen science to be very useful through works like eBird for data collection of bird diversity andmigration patterns which fully relies on bird hobbyists around the world [56] Specific to mycologythe active participation of Pacific Northwest mycological societies in the ten-year chanterelle projectresulted in the evaluation harvesting techniques of wild chanterelles in Oregon [8ndash10] Similarlythe long-term survey in Warwickshire in the UK from 1965 to 1980 gave one of the most detailedsurveys for fungi in the UK and was performed by lay mycologists of the area [7]

As with other citizen scientist surveys the technique detailed herein is similar to a rapid assessmentsurvey (RAPD) This method is commonly used for field surveys by non-specialized personnel in specificareas [60] The rapid assessment focuses on a group of genera in small diverse areas RAPD has beenwidely used for mycological surveys but if used for a wide number of genera can become unreliable [58]Focusing on a single group increases its accuracy The modified RAPD method proposed herein has beenshown to be accurate across different ecosystems (temperate rainforest in Benton County OR USA andthe tropical rainforest in Madre de Dios Peru) for the identification of the Chlorociboria spp on downedwood by non-specialized personnel In the future this method could be applied by citizen-scientists tocollect data on the worldwide distribution of the genus Chlorociboria

The method proposed can also be applied without setting a transect as it was performed in theAmazon rainforest This variation can improve the participation of citizen-scientists as some of themare mycology aficionados that can mark the location of Chlorociboria spp while on their foragingactivities or simply on general hikes

The distribution data (GPS points) which could be easily uploaded into a worldwide databasesuch as GBIF allowed the researchers on this project to identify forest types using Forest Service mapsand forest data from the Peruvian government This allowed for generation of a distribution mapand an understanding of forest types preferred by Chlorociboria species Continuation of this surveyby citizen scientists could greatly expand our understanding of the types of trees and systems thegenus of fungi prefer even if exact tree species cannot be reliably noted This data could then be madeavailable to forested landowners seeking to manage for Chlorociboria species xylindein productionand the economic opportunities therein

42 Future Research

Currently the location of the species is determined with a GPS app and field notes In thefuture the development of a smartphone application (like eBird [56]) specific to the distribution ofChlorociboria spp will be necessary to make the sampling data collection and data analysis simplerThis would also give the researchers involved one main input point for data making analysis and mapgeneration easier

As for the implementation of this initiative the unique laws and access to natural landscapesin different countries must be taken into account if the final goal is to apply it worldwide In theimmediate future the sampling of Chlorociboria spp can be done in association with mycologysocieties from the Pacific Northwest This can help to improve the location method and to develop theapplication with them as they have already participated in long-term studies (such as the Chanterelleproject) [10] In the intermediate future contacts with other mycological societies could be done on aworldwide scale (such as with the Danish Mycological Society) In the long-term the authors hopethat this method might be introduced into countries that do not have a culture of foraging (like Peru)

Finally the goal of the developed method and initiative is to gather data that can generate adetailed map of distribution of Chlorociboria spp and with it the identification of areas that present amore frequent presence of the fungus This information will be evaluated to determine the type of

Challenges 2018 9 11 9 of 11

forests and the dominant wood species in those areas and to then apply it to land management for thegrowth of these rare and useful fungi should this be of interest to landowners

5 Conclusions

The proposed method utilized simple tools and a smart phone or GPS to survey populations ofChlorociboria spp across temperate and tropical forests The method for identification of Chlorociboria sppcontaining wood was shown to be successful however the wood identification was not Howeverin general Chlorociboria identification can be easily taught to members of mycological societieseco tourists and other interested parties that wish to be involved in recording the distribution ofChlorociboria spp Over time the data collected could be used to build a worldwide picture of foresttype and wood preference by Chlorociboria species which will be of use to those who wish to managefor xylindein production on their forested lands

Acknowledgments The authors would like to thank Joseacute Koechlin Von Stein Inkaterra and the Inkaterra Asociacioacuten

Author Contributions Seri C Robinson Olivia E Kramer and Claudia C Andersen conceived and designed theexperiments Olivia E Kramer Claudia C Andersen and Patricia T Vega Gutierrez executed the experimentsand Sarath M Vega Gutierrez analyzed the data and wrote the paper

Conflicts of Interest The authors declare no conflict of interest

References

1 Stanley ES Susan RD Conservation commentary Ornithology and the national biological survey Auk1995 112 804ndash806

2 Tulloch AIT Possingham HP Joseph LN Szabo J Martin TG Realising the full potential of citizenscience monitoring programs Biol Conserv 2013 165 128ndash138 [CrossRef]

3 Cook R Encouraging the amateur Survey work in north-west England Mycologist 1992 6 162ndash164 [CrossRef]4 Gardiner MM Allee LL Brown PMJ Losey JE Roy HE Smyth RR Lessons from lady beetles

Accuracy of monitoring data from us and uk citizen-science programs Front Ecol Environ 2012 10 471ndash476[CrossRef]

5 Sullivan BL Wood CL Iliff MJ Bonney RE Fink D Kelling S Ebird A citizen-based bird observationnetwork in the biological sciences Biol Conserv 2009 142 2282ndash2292 [CrossRef]

6 Sullivan BL Aycrigg JL Barry JH Bonney RE Bruns N Cooper CB Damoulas T Dhondt AADietterich T Farnsworth A et al The ebird enterprise An integrated approach to development andapplication of citizen science Biol Conserv 2014 169 31ndash40 [CrossRef]

7 Clark MC The fungus flora of Warwickshire further records Bull Br Mycol Soc 1986 20 105ndash119[CrossRef]

8 Norvell L Studying the effects of harvesting on chanterelle productivity in Oregonrsquos Mt Hood nationalforest In Wild Mushroom Harvesting Discussion Session Minutes British Columbia Ministry of Forests PacificForestry Centre Victoria BC USA 1992 pp 9ndash15

9 Norvell L Kopecky F Lindgren J Roger J The chanterelle (cantharellus cibarius) A peek at productivityIn Proceedings of the Business and Science of Special Forest ProductsmdashA Conference and ExpositionAt Hillsboro OR USA 26ndash27 January 1994 pp 109ndash120

10 Norvell L Loving the chanterelle to death The ten-year Oregon chanterelle project McIlvainea 199512 6ndash25

11 McLain RJ Christensen HH Shannon MA When amateurs are the experts Amateur mycologists andwild mushroom politics in the Pacific Northwest USA Soc Nat Resour 1998 11 615ndash626 [CrossRef]

12 Robinson SC Richter DL Laks PE Colonization of sugar maple by spalting fungi For Prod J 200757 24ndash32

13 Persoon CH Observationes mycologicae Ann Bot 1795 15 1ndash3914 Persoon CH Synopsis Methodica Fungorum Apud Henricum Dieterich Gottingae Germany 180115 Persoon CH Mycologica Europaea Impensibus iI Palmii Erlangae Germany 1822 Volume 1 p 29116 Seaver FJ The North American Cup Fungi (Inoperculates) Hafner Publishing Co Inc New York NY

USA 1951

Challenges 2018 9 11 7 of 11

Challenges 2018 9 x 6 of 11

Wood species identification in Peru was generally not successful Those logs containing blue-green pigments were often too decayed to even attempt a genus identification The participants were not able to do any in-field wood identification at this site

The identification of the wood species is not required for citizen-scientists but if such people are capable it is a nice addition to the data

Figure 1 Radial section of a big leaf maple with red arrows pointing the helical thickenings on the vessel cell walls Magnification 200times

Figure 2 Tangential section of a willow with arrows pointing to the upright ray cells All rays are uniseriate Magnification 200times

Figure 3 Radial section of a willow showing alternate vessel wall pitting and Chlorociboria spp pigmentation Magnification 200times

Figure 3 Radial section of a willow showing alternate vessel wall pitting and Chlorociboria spppigmentation Magnification 200times

Wood species identification in Peru was generally not successful Those logs containing blue-greenpigments were often too decayed to even attempt a genus identification The participants were notable to do any in-field wood identification at this site

The identification of the wood species is not required for citizen-scientists but if such people arecapable it is a nice addition to the data

34 Verification

Performing the sampling under supervision and further verification showed that the methodwas effective Therefore the collection method can be done by citizen-scientists without professionalsupervision as it has shown to be accurate

4 Discussion

41 Training Sampling and Sample Collection

In terms of simple mapping of Chlorociboria distribution the training was deemed successfulas eight logs containing Chlorociboria spp were identified within the McDonald-Dunn and MaryrsquosPeak Forests and twelve logs suspected to contain Chlorociboria spp were found in Madre de DiosPeru and all were successfully identified by the participants Wood identification was less successfulunfortunately especially in the tropical rainforests of Peru It seems likely that this aspect of theproposed citizen scientist survey for Chlorociboria would generally not be useful as researchers wouldhave no way to verify correct wood ID and when there is an abundance of diversity the likelihood ofmisidentification is great

A key element that allowed for the successful identification of the logs containing or suspected tocontain Chlorociboria spp was opening the logs well into the sapwood to find the blue-green coloredwood This helped students discount fungi lichens and bacteria that can colorize the surface and thefirst few centimeters of wood and thus limited them to general decay-capable fungi Another importantelement that was unique to this study was that it allowed for fungi without fruiting forms to be surveyed(when said fungi have a unique coloration system such as Chlorociboria spp) [39] This method couldeasily be applied to non-fruiting bodied pigment producing fungi such as Scytalidium cuboideumwhich develops red pigmentation [27] and Scytalidium ganodermophthorum which develops yellowpigmentation [5657] although these fungi do not have lsquouniquersquo colorants A final factor that aided inthe accurate identification was likely the focus on a single distinct genus allowing the participantsto focus their attention Surveys asking for a wider number of specimens to be located risk over orunderestimation [45859]

Challenges 2018 9 11 8 of 11

Another useful aspect of this technique was the use of common and accessible surveying toolssuch as machetes (which can be easily replaced by hatchets andor field knives) measuring tapessmart phone GPS applications andor GPS These tools require little to no training for their use and aregenerally accessible around the world This is important for future reliance on citizen-scientists forthe evaluation of Chlorociboria spp in the wild and should be effective Previous studies have showncitizen science to be very useful through works like eBird for data collection of bird diversity andmigration patterns which fully relies on bird hobbyists around the world [56] Specific to mycologythe active participation of Pacific Northwest mycological societies in the ten-year chanterelle projectresulted in the evaluation harvesting techniques of wild chanterelles in Oregon [8ndash10] Similarlythe long-term survey in Warwickshire in the UK from 1965 to 1980 gave one of the most detailedsurveys for fungi in the UK and was performed by lay mycologists of the area [7]

As with other citizen scientist surveys the technique detailed herein is similar to a rapid assessmentsurvey (RAPD) This method is commonly used for field surveys by non-specialized personnel in specificareas [60] The rapid assessment focuses on a group of genera in small diverse areas RAPD has beenwidely used for mycological surveys but if used for a wide number of genera can become unreliable [58]Focusing on a single group increases its accuracy The modified RAPD method proposed herein has beenshown to be accurate across different ecosystems (temperate rainforest in Benton County OR USA andthe tropical rainforest in Madre de Dios Peru) for the identification of the Chlorociboria spp on downedwood by non-specialized personnel In the future this method could be applied by citizen-scientists tocollect data on the worldwide distribution of the genus Chlorociboria

The method proposed can also be applied without setting a transect as it was performed in theAmazon rainforest This variation can improve the participation of citizen-scientists as some of themare mycology aficionados that can mark the location of Chlorociboria spp while on their foragingactivities or simply on general hikes

The distribution data (GPS points) which could be easily uploaded into a worldwide databasesuch as GBIF allowed the researchers on this project to identify forest types using Forest Service mapsand forest data from the Peruvian government This allowed for generation of a distribution mapand an understanding of forest types preferred by Chlorociboria species Continuation of this surveyby citizen scientists could greatly expand our understanding of the types of trees and systems thegenus of fungi prefer even if exact tree species cannot be reliably noted This data could then be madeavailable to forested landowners seeking to manage for Chlorociboria species xylindein productionand the economic opportunities therein

42 Future Research

Currently the location of the species is determined with a GPS app and field notes In thefuture the development of a smartphone application (like eBird [56]) specific to the distribution ofChlorociboria spp will be necessary to make the sampling data collection and data analysis simplerThis would also give the researchers involved one main input point for data making analysis and mapgeneration easier

As for the implementation of this initiative the unique laws and access to natural landscapesin different countries must be taken into account if the final goal is to apply it worldwide In theimmediate future the sampling of Chlorociboria spp can be done in association with mycologysocieties from the Pacific Northwest This can help to improve the location method and to develop theapplication with them as they have already participated in long-term studies (such as the Chanterelleproject) [10] In the intermediate future contacts with other mycological societies could be done on aworldwide scale (such as with the Danish Mycological Society) In the long-term the authors hopethat this method might be introduced into countries that do not have a culture of foraging (like Peru)

Finally the goal of the developed method and initiative is to gather data that can generate adetailed map of distribution of Chlorociboria spp and with it the identification of areas that present amore frequent presence of the fungus This information will be evaluated to determine the type of

Challenges 2018 9 11 9 of 11

forests and the dominant wood species in those areas and to then apply it to land management for thegrowth of these rare and useful fungi should this be of interest to landowners

5 Conclusions

The proposed method utilized simple tools and a smart phone or GPS to survey populations ofChlorociboria spp across temperate and tropical forests The method for identification of Chlorociboria sppcontaining wood was shown to be successful however the wood identification was not Howeverin general Chlorociboria identification can be easily taught to members of mycological societieseco tourists and other interested parties that wish to be involved in recording the distribution ofChlorociboria spp Over time the data collected could be used to build a worldwide picture of foresttype and wood preference by Chlorociboria species which will be of use to those who wish to managefor xylindein production on their forested lands

Acknowledgments The authors would like to thank Joseacute Koechlin Von Stein Inkaterra and the Inkaterra Asociacioacuten

Author Contributions Seri C Robinson Olivia E Kramer and Claudia C Andersen conceived and designed theexperiments Olivia E Kramer Claudia C Andersen and Patricia T Vega Gutierrez executed the experimentsand Sarath M Vega Gutierrez analyzed the data and wrote the paper

Conflicts of Interest The authors declare no conflict of interest

References

1 Stanley ES Susan RD Conservation commentary Ornithology and the national biological survey Auk1995 112 804ndash806

2 Tulloch AIT Possingham HP Joseph LN Szabo J Martin TG Realising the full potential of citizenscience monitoring programs Biol Conserv 2013 165 128ndash138 [CrossRef]

3 Cook R Encouraging the amateur Survey work in north-west England Mycologist 1992 6 162ndash164 [CrossRef]4 Gardiner MM Allee LL Brown PMJ Losey JE Roy HE Smyth RR Lessons from lady beetles

Accuracy of monitoring data from us and uk citizen-science programs Front Ecol Environ 2012 10 471ndash476[CrossRef]

5 Sullivan BL Wood CL Iliff MJ Bonney RE Fink D Kelling S Ebird A citizen-based bird observationnetwork in the biological sciences Biol Conserv 2009 142 2282ndash2292 [CrossRef]

6 Sullivan BL Aycrigg JL Barry JH Bonney RE Bruns N Cooper CB Damoulas T Dhondt AADietterich T Farnsworth A et al The ebird enterprise An integrated approach to development andapplication of citizen science Biol Conserv 2014 169 31ndash40 [CrossRef]

7 Clark MC The fungus flora of Warwickshire further records Bull Br Mycol Soc 1986 20 105ndash119[CrossRef]

8 Norvell L Studying the effects of harvesting on chanterelle productivity in Oregonrsquos Mt Hood nationalforest In Wild Mushroom Harvesting Discussion Session Minutes British Columbia Ministry of Forests PacificForestry Centre Victoria BC USA 1992 pp 9ndash15

9 Norvell L Kopecky F Lindgren J Roger J The chanterelle (cantharellus cibarius) A peek at productivityIn Proceedings of the Business and Science of Special Forest ProductsmdashA Conference and ExpositionAt Hillsboro OR USA 26ndash27 January 1994 pp 109ndash120

10 Norvell L Loving the chanterelle to death The ten-year Oregon chanterelle project McIlvainea 199512 6ndash25

11 McLain RJ Christensen HH Shannon MA When amateurs are the experts Amateur mycologists andwild mushroom politics in the Pacific Northwest USA Soc Nat Resour 1998 11 615ndash626 [CrossRef]

12 Robinson SC Richter DL Laks PE Colonization of sugar maple by spalting fungi For Prod J 200757 24ndash32

13 Persoon CH Observationes mycologicae Ann Bot 1795 15 1ndash3914 Persoon CH Synopsis Methodica Fungorum Apud Henricum Dieterich Gottingae Germany 180115 Persoon CH Mycologica Europaea Impensibus iI Palmii Erlangae Germany 1822 Volume 1 p 29116 Seaver FJ The North American Cup Fungi (Inoperculates) Hafner Publishing Co Inc New York NY

USA 1951

Challenges 2018 9 11 8 of 11

Another useful aspect of this technique was the use of common and accessible surveying toolssuch as machetes (which can be easily replaced by hatchets andor field knives) measuring tapessmart phone GPS applications andor GPS These tools require little to no training for their use and aregenerally accessible around the world This is important for future reliance on citizen-scientists forthe evaluation of Chlorociboria spp in the wild and should be effective Previous studies have showncitizen science to be very useful through works like eBird for data collection of bird diversity andmigration patterns which fully relies on bird hobbyists around the world [56] Specific to mycologythe active participation of Pacific Northwest mycological societies in the ten-year chanterelle projectresulted in the evaluation harvesting techniques of wild chanterelles in Oregon [8ndash10] Similarlythe long-term survey in Warwickshire in the UK from 1965 to 1980 gave one of the most detailedsurveys for fungi in the UK and was performed by lay mycologists of the area [7]

As with other citizen scientist surveys the technique detailed herein is similar to a rapid assessmentsurvey (RAPD) This method is commonly used for field surveys by non-specialized personnel in specificareas [60] The rapid assessment focuses on a group of genera in small diverse areas RAPD has beenwidely used for mycological surveys but if used for a wide number of genera can become unreliable [58]Focusing on a single group increases its accuracy The modified RAPD method proposed herein has beenshown to be accurate across different ecosystems (temperate rainforest in Benton County OR USA andthe tropical rainforest in Madre de Dios Peru) for the identification of the Chlorociboria spp on downedwood by non-specialized personnel In the future this method could be applied by citizen-scientists tocollect data on the worldwide distribution of the genus Chlorociboria

The method proposed can also be applied without setting a transect as it was performed in theAmazon rainforest This variation can improve the participation of citizen-scientists as some of themare mycology aficionados that can mark the location of Chlorociboria spp while on their foragingactivities or simply on general hikes

The distribution data (GPS points) which could be easily uploaded into a worldwide databasesuch as GBIF allowed the researchers on this project to identify forest types using Forest Service mapsand forest data from the Peruvian government This allowed for generation of a distribution mapand an understanding of forest types preferred by Chlorociboria species Continuation of this surveyby citizen scientists could greatly expand our understanding of the types of trees and systems thegenus of fungi prefer even if exact tree species cannot be reliably noted This data could then be madeavailable to forested landowners seeking to manage for Chlorociboria species xylindein productionand the economic opportunities therein

42 Future Research

Currently the location of the species is determined with a GPS app and field notes In thefuture the development of a smartphone application (like eBird [56]) specific to the distribution ofChlorociboria spp will be necessary to make the sampling data collection and data analysis simplerThis would also give the researchers involved one main input point for data making analysis and mapgeneration easier

As for the implementation of this initiative the unique laws and access to natural landscapesin different countries must be taken into account if the final goal is to apply it worldwide In theimmediate future the sampling of Chlorociboria spp can be done in association with mycologysocieties from the Pacific Northwest This can help to improve the location method and to develop theapplication with them as they have already participated in long-term studies (such as the Chanterelleproject) [10] In the intermediate future contacts with other mycological societies could be done on aworldwide scale (such as with the Danish Mycological Society) In the long-term the authors hopethat this method might be introduced into countries that do not have a culture of foraging (like Peru)

Finally the goal of the developed method and initiative is to gather data that can generate adetailed map of distribution of Chlorociboria spp and with it the identification of areas that present amore frequent presence of the fungus This information will be evaluated to determine the type of

Challenges 2018 9 11 9 of 11

forests and the dominant wood species in those areas and to then apply it to land management for thegrowth of these rare and useful fungi should this be of interest to landowners

5 Conclusions

The proposed method utilized simple tools and a smart phone or GPS to survey populations ofChlorociboria spp across temperate and tropical forests The method for identification of Chlorociboria sppcontaining wood was shown to be successful however the wood identification was not Howeverin general Chlorociboria identification can be easily taught to members of mycological societieseco tourists and other interested parties that wish to be involved in recording the distribution ofChlorociboria spp Over time the data collected could be used to build a worldwide picture of foresttype and wood preference by Chlorociboria species which will be of use to those who wish to managefor xylindein production on their forested lands

Acknowledgments The authors would like to thank Joseacute Koechlin Von Stein Inkaterra and the Inkaterra Asociacioacuten

Author Contributions Seri C Robinson Olivia E Kramer and Claudia C Andersen conceived and designed theexperiments Olivia E Kramer Claudia C Andersen and Patricia T Vega Gutierrez executed the experimentsand Sarath M Vega Gutierrez analyzed the data and wrote the paper

Conflicts of Interest The authors declare no conflict of interest

References

1 Stanley ES Susan RD Conservation commentary Ornithology and the national biological survey Auk1995 112 804ndash806

2 Tulloch AIT Possingham HP Joseph LN Szabo J Martin TG Realising the full potential of citizenscience monitoring programs Biol Conserv 2013 165 128ndash138 [CrossRef]

3 Cook R Encouraging the amateur Survey work in north-west England Mycologist 1992 6 162ndash164 [CrossRef]4 Gardiner MM Allee LL Brown PMJ Losey JE Roy HE Smyth RR Lessons from lady beetles

Accuracy of monitoring data from us and uk citizen-science programs Front Ecol Environ 2012 10 471ndash476[CrossRef]

5 Sullivan BL Wood CL Iliff MJ Bonney RE Fink D Kelling S Ebird A citizen-based bird observationnetwork in the biological sciences Biol Conserv 2009 142 2282ndash2292 [CrossRef]

6 Sullivan BL Aycrigg JL Barry JH Bonney RE Bruns N Cooper CB Damoulas T Dhondt AADietterich T Farnsworth A et al The ebird enterprise An integrated approach to development andapplication of citizen science Biol Conserv 2014 169 31ndash40 [CrossRef]

7 Clark MC The fungus flora of Warwickshire further records Bull Br Mycol Soc 1986 20 105ndash119[CrossRef]

8 Norvell L Studying the effects of harvesting on chanterelle productivity in Oregonrsquos Mt Hood nationalforest In Wild Mushroom Harvesting Discussion Session Minutes British Columbia Ministry of Forests PacificForestry Centre Victoria BC USA 1992 pp 9ndash15

9 Norvell L Kopecky F Lindgren J Roger J The chanterelle (cantharellus cibarius) A peek at productivityIn Proceedings of the Business and Science of Special Forest ProductsmdashA Conference and ExpositionAt Hillsboro OR USA 26ndash27 January 1994 pp 109ndash120

10 Norvell L Loving the chanterelle to death The ten-year Oregon chanterelle project McIlvainea 199512 6ndash25

11 McLain RJ Christensen HH Shannon MA When amateurs are the experts Amateur mycologists andwild mushroom politics in the Pacific Northwest USA Soc Nat Resour 1998 11 615ndash626 [CrossRef]

12 Robinson SC Richter DL Laks PE Colonization of sugar maple by spalting fungi For Prod J 200757 24ndash32

13 Persoon CH Observationes mycologicae Ann Bot 1795 15 1ndash3914 Persoon CH Synopsis Methodica Fungorum Apud Henricum Dieterich Gottingae Germany 180115 Persoon CH Mycologica Europaea Impensibus iI Palmii Erlangae Germany 1822 Volume 1 p 29116 Seaver FJ The North American Cup Fungi (Inoperculates) Hafner Publishing Co Inc New York NY

USA 1951

Challenges 2018 9 11 9 of 11

forests and the dominant wood species in those areas and to then apply it to land management for thegrowth of these rare and useful fungi should this be of interest to landowners

5 Conclusions

The proposed method utilized simple tools and a smart phone or GPS to survey populations ofChlorociboria spp across temperate and tropical forests The method for identification of Chlorociboria sppcontaining wood was shown to be successful however the wood identification was not Howeverin general Chlorociboria identification can be easily taught to members of mycological societieseco tourists and other interested parties that wish to be involved in recording the distribution ofChlorociboria spp Over time the data collected could be used to build a worldwide picture of foresttype and wood preference by Chlorociboria species which will be of use to those who wish to managefor xylindein production on their forested lands

Acknowledgments The authors would like to thank Joseacute Koechlin Von Stein Inkaterra and the Inkaterra Asociacioacuten

Author Contributions Seri C Robinson Olivia E Kramer and Claudia C Andersen conceived and designed theexperiments Olivia E Kramer Claudia C Andersen and Patricia T Vega Gutierrez executed the experimentsand Sarath M Vega Gutierrez analyzed the data and wrote the paper

Conflicts of Interest The authors declare no conflict of interest

References

1 Stanley ES Susan RD Conservation commentary Ornithology and the national biological survey Auk1995 112 804ndash806

2 Tulloch AIT Possingham HP Joseph LN Szabo J Martin TG Realising the full potential of citizenscience monitoring programs Biol Conserv 2013 165 128ndash138 [CrossRef]

3 Cook R Encouraging the amateur Survey work in north-west England Mycologist 1992 6 162ndash164 [CrossRef]4 Gardiner MM Allee LL Brown PMJ Losey JE Roy HE Smyth RR Lessons from lady beetles

Accuracy of monitoring data from us and uk citizen-science programs Front Ecol Environ 2012 10 471ndash476[CrossRef]

5 Sullivan BL Wood CL Iliff MJ Bonney RE Fink D Kelling S Ebird A citizen-based bird observationnetwork in the biological sciences Biol Conserv 2009 142 2282ndash2292 [CrossRef]

6 Sullivan BL Aycrigg JL Barry JH Bonney RE Bruns N Cooper CB Damoulas T Dhondt AADietterich T Farnsworth A et al The ebird enterprise An integrated approach to development andapplication of citizen science Biol Conserv 2014 169 31ndash40 [CrossRef]

7 Clark MC The fungus flora of Warwickshire further records Bull Br Mycol Soc 1986 20 105ndash119[CrossRef]

8 Norvell L Studying the effects of harvesting on chanterelle productivity in Oregonrsquos Mt Hood nationalforest In Wild Mushroom Harvesting Discussion Session Minutes British Columbia Ministry of Forests PacificForestry Centre Victoria BC USA 1992 pp 9ndash15

9 Norvell L Kopecky F Lindgren J Roger J The chanterelle (cantharellus cibarius) A peek at productivityIn Proceedings of the Business and Science of Special Forest ProductsmdashA Conference and ExpositionAt Hillsboro OR USA 26ndash27 January 1994 pp 109ndash120

10 Norvell L Loving the chanterelle to death The ten-year Oregon chanterelle project McIlvainea 199512 6ndash25

11 McLain RJ Christensen HH Shannon MA When amateurs are the experts Amateur mycologists andwild mushroom politics in the Pacific Northwest USA Soc Nat Resour 1998 11 615ndash626 [CrossRef]

12 Robinson SC Richter DL Laks PE Colonization of sugar maple by spalting fungi For Prod J 200757 24ndash32

13 Persoon CH Observationes mycologicae Ann Bot 1795 15 1ndash3914 Persoon CH Synopsis Methodica Fungorum Apud Henricum Dieterich Gottingae Germany 180115 Persoon CH Mycologica Europaea Impensibus iI Palmii Erlangae Germany 1822 Volume 1 p 29116 Seaver FJ The North American Cup Fungi (Inoperculates) Hafner Publishing Co Inc New York NY

USA 1951

Challenges 2018 9 11 10 of 11

17 Ramamurthi CS Korf RP Batra LR A revision of the North American species of Chlorociboria(sclerotiniaceae) Mycologia 1958 49 854ndash863 [CrossRef]

18 Tudor D Margaritescu S Saacutenchez-Ramiacuterez S Robinson SC Cooper PA Moncalvo JM Morphologicaland molecular characterization of the two known North American Chlorociboria species and their anamorphsFungal Biol 2014 118 732ndash742 [CrossRef] [PubMed]

19 Johnston PR Park D Chlorociboria (fungi helotiales) in New Zealand N Z J Bot 2005 43 679ndash719[CrossRef]

20 Singh H Chlorosplenium in India Trans Br Mycol Soc 1974 63 289ndash294 [CrossRef]21 Seaver FG Photographs and descriptions of cup fungi ndashxxiv Chlorociboria Mycologia 1936 28 309ndash39422 Baroni TJ Lodge DJ Cantrell SA Tropical connections Sister species and species in common between

the Caribbean and the Eastern United States McIlvainea 1997 13 4ndash1923 Medel R Morales O del Moral RC Caacuteceres R New ascomycete records from Guatemala Mycotaxon

2013 124 73ndash85 [CrossRef]24 Robinson SC Laks PE Wood species affects laboratory colonization rates of Chlorociboria sp Int Biodeterior

Biodegrad 2010 64 305ndash308 [CrossRef]25 Dixon JR Chlorosplenium and its segregates The genera Chlorociboria and Chlorencoelia Mycotaxon 1975

1 193ndash23726 Robinson SC Tudor D Cooper PA Utilizing pigment-producing fungi to add commercial value to

American beech (Fagus grandifolia) Appl Microbiol Biotechnol 2012 93 1041ndash1048 [CrossRef] [PubMed]27 Robinson SC Developing fungal pigments for ldquopaintingrdquo vascular plants Appl Microbiol Biotechnol 2012

93 1389ndash1394 [CrossRef] [PubMed]28 Blackburn GM Ekong DE Nielson AH Todd L Xylindein Chimia 1965 19 208ndash21229 Maeda M Yamauchi T Oshima K Shimomura M Miyauchi S Mukae K Sakaki T Shibata M

Wakamatsu K Extraction of xylindein from Chlorociboria aeruginosa complex and its biological characteristicsBull Nagaoka Univ Technol 2003 25 105ndash111

30 Blanchette RA Wilmering AM Baumeister M The use of green-stained wood caused by the fungusChlorociboria in intarsia masterpieces from the 15th century Holzforschung 1992 46 225ndash232 [CrossRef]

31 Raggio O Wilmering AM The Gubbio Studiolo and Its Conservation Italian Renaissance Intarsia and theConservation of the Gubbio Studiolo Metropolitan Museum of Art New York NY USA 1999 Volume 2

32 Otterstedt A Investigating green marquetry on bowed-string instruments The leaves be Greene Galpin Soc J2001 330ndash338 [CrossRef]

33 Michaelsen H Unger A Fischer C-H Blaugruumlne faumlrbung an intarsienhoumllzern des 16 Bis 18 JahrhundertsRestauro 1992 98 17ndash25

34 Facility GBI Chlorociboria Seaver ex cS Ramamurthi korf amp lR Batra Available online httpswwwgbiforgspecies8034517 (accessed on 22 February 2018)

35 Robinson SC Hinsch E Weber G Leipus K Cerney D Wood colorization through pressure treatingThe potential of extracted colorants from spalting fungi as a replacement for woodworkersrsquo aniline dyesMaterials 2014 7 5427ndash5437 [CrossRef] [PubMed]

36 Robinson SC Weber G Hinsch E Vega Gutierrez SM Pittis L Freitas S Utilizing extracted fungalpigments for wood spalting A comparison of induced fungal pigmentation to fungal dyeing J Coat 20142014 1ndash8 [CrossRef]

37 Robinson SC Vega Gutierrez SM Cespedes RA Irounme N Vorland NR McClelland A Huber MStanton S Potential for carrying pigments derived from spalting fungi in natural oils J Coat Technol Res2017 14 1107ndash1113 [CrossRef]

38 Rath JJ Organic Semiconductors Incorporating Xylindein Into (opto) Electronic Devices Bachelorrsquos ThesisOregon State University Corvallis OR USA 2016

39 Donner CD Cuzzupe AN Falzon CL Gill M Investigations towards the synthesis of xylindeina blue-green pigment from the fungus Chlorociboria aeruginosa Tetrahedron 2012 68 2799ndash2805 [CrossRef]

40 Robinson SC Tudor D Snider H Cooper PA Stimulating growth and xylindein production ofChlorociboria aeruginascens in agar-based systems AMB Express 2012 2 1ndash7 [CrossRef] [PubMed]

41 Robinson SC Tudor D Cooper PA Promoting fungal pigment formation in wood by utilizing a modifieddecay jar method Wood Sci Technol 2011 46 841ndash849 [CrossRef]

Challenges 2018 9 11 11 of 11

42 Robinson SC Hinsch E Weber G Freitas S Method of extraction and resolubilisation of pigments fromChlorociboria aeruginosa and Scytalidium cuboideum two prolific spalting fungi Color Technol 2014 130221ndash225 [CrossRef]

43 Boonloed A Weber GL Ramzy KM Dias VR Remcho VT Centrifugal partition chromatographyA preparative tool for isolation and purification of xylindein from Chlorociboria aeruginosa J Chromatogr A2016 1478 19ndash25 [CrossRef] [PubMed]

44 Vega Gutierrez S Vega Gutierrez P Godinez A Pittis L Huber M Stanton S Robinson S Feasibility ofcoloring bamboo with the application of natural and extracted fungal pigments Coatings 2016 6 37 [CrossRef]

45 Weber G Chen H-L Hinsch E Freitas S Robinson S Pigments extracted from the wood-staining fungiChlorociboria aeruginosa Scytalidium cuboideum and S ganodermophthorum show potential for use as textiledyes Color Technol 2014 130 445ndash452 [CrossRef]

46 Hinsch EM Weber G Chen H-L Robinson SC Colorfastness of extracted wood-staining fungalpigments on fabrics A new potential for textile dyes J Text Appar Technol Manag 2015 9 3

47 Puhakka L Salo M Saaksjarvi I Bird diversity birdwatching tourism and conservation in PeruA geographic analysis PLoS ONE 2011 6 [CrossRef] [PubMed]

48 Duellman WE The Reserva Cuzco Amazonico Peru Biological Investigations Conservation and EcotourismMuseum of Natural History University of Kansas Lawrence KY USA 1991

49 Vega Gutierrez SM Spalting Fungi Genetic Identification Material Interactions and Microscopical Characteristicsof Extracted Pigments Oregon State University Corvallis OR USA 2017

50 Malleux OJ Inventarios Forestales en Bosques Tropicales Universidad Nacional Agraria Managua Nicaragua 198251 Von Houmlhnel F Uumlber dothiorella tulasnei sacc Fragmente zur Mykologie (XIII Mitteilung Nr 642 bis 718)

Sitzungsber K Akad Wiss Math-Naturwiss Kl 1915 120 463ndash46452 Berthet P Formes conidiennes de divers discomycetes Bull Trimest Soc Mycol Fr 1964 80 125ndash14953 Fenwick GA Chlorociboria aeruginascens in laboratory culture Mycologist 1993 7 172ndash175 [CrossRef]54 Richter DL Glaeser JA Wood decay by Chlorociboria aeruginascens (Nyl) Kanouse (Helotiales Leotiaceae)

and associated Basidiomycete fungi Int Biodeterior Biodegrad 2015 105 239ndash244 [CrossRef]55 Wheeler EA Inside woodmdashA web resource for hardwood anatomy IAWA J 2011 32 199ndash21156 Kang H Sigler L Lee J Gibas C Yun S Lee Y Xylogone ganodermophthora sp Nov an ascomycetous

pathogen causing yellow rot on cultivated mushroom Ganoderma lucidum in Korea Mycologia 2010 1021167ndash1184 [CrossRef] [PubMed]

57 Robinson SC Tudor D Zhang WR Ng S Cooper PA Ability of three yellow pigment producing fungito colour wood under controlled conditions Int Wood Prod J 2014 5 103ndash107 [CrossRef]

58 Dickinson JL Zuckerberg B Bonter DN Citizen science as an ecological research tool Challenges andbenefits Annu Rev Ecol Evol Syst 2010 41 149ndash172 [CrossRef]

59 Galloway AWE Tudor MT Haegen WMV The reliability of citizen science A case study of Oregonwhite oak stand surveys Wildl Soc Bull 2006 34 1425ndash1429 [CrossRef]

60 Cannon PF Strategies for rapid assessment of fungal diversity Biodivers Conserv 1997 6 669ndash680 [CrossRef]

copy 2018 by the authors Licensee MDPI Basel Switzerland This article is an open accessarticle distributed under the terms and conditions of the Creative Commons Attribution(CC BY) license (httpcreativecommonsorglicensesby40)