dna barcoding alaskan willow rosette gall makers (diptera ... · volume 11, issue 1, august 2018 12...

Volume 11 Issue 1 August 2018 8

References

Andersen J C and N J Mills 2012 DNA extraction frommuseum specimens of parasitic Hymenoptera PLoSONE 7e45549 doi101371journalpone0045549

Burges H D 1959 Studies on the dermestid beetle Tro-goderma granarium Everts ecology in malt stores An-nals of Applied Biology 47445ndash462 doi101111j1744-73481959tb07278x

Gilberg M and A Brokerhof 1991 The control of insectpests in museum collections the effects of low temper-ature on Stegobium paniceum (Linneaus) the drugstorebeetle Journal of the American Institute for Conserva-tion 30197ndash201 doi101179019713691806066737

R Core Team 2017 R A Language and Environment forStatistical Computing R Foundation for Statistical Com-

puting Vienna Austria URL httpswwwR-project

org

Su N-Y and R H Scheffrahn 1990 Efficacy of sul-furyl fluoride against four beetle pests of museums(Coleoptera Dermestidae Anobiidae) Journal of Eco-nomic Entomology 83879ndash882 doi101093jee833879

Suarez A V and N D Tsutsui 2004 Thevalue of museum collections for research andsociety BioScience 5466ndash74 doi1016410006-3568(2004)054[0066TVOMCF]20CO2

Zaitseva G A 1987 Protection of museum textiles andleather against the dermestid beetle (Coleoptera Der-mestidae) by means of antifeedants Studies in Conser-vation 32176ndash180 doi101179sic1987324176

DNA barcoding Alaskan willow rosette gall makers(Diptera Cecidomyiidae Rabdophaga)

doi107299X7833SBX

by Matt Bowser1 Apphia M Bowser1 Ethan L Bowser1Miriam D Bowser1 Dominique M Collet2 and Tracy Melvin3

Introduction

Members of the Rabdophaga rosaria group form conspicu-ous rosette galls on a variety of willow (Salix spp) hosts(Collet 2002 Amendt 2003) and have a holarctic distri-bution Gall formation halts elongation of willow stemsand alters the morphology and chemical makeup of hosttissues (Gailite et al 2005 Samsone et al 2011) Eco-logically these flies are a keystone species for a commu-nity of insects associated with rose galls including mul-tiple parasitoid hyperparasitoid and commensal species(Van Hezewijk and Roland 2003 Collet 2006 Skuhravaacuteand Thuroacuteczy 2007) The larvae serve as food for chick-adees and tits which pick them out of galls in winter(Van Hezewijk and Roland 2003 Nyman et al 2011 Fig-ure 1) The galls themselves are avoided by moose (KenaiNational Wildlife Refuge staff 1981 Ford et al 1995 Rea2012) and snowshoe hares (Ford et al 1995)

Figure 1 A black-capped chickadee dismantling a gall in-duced by Rabdophaga strobiloides in central Michigan De-cember 30 2017 (httpsflickrp232e1Yn) Image copyJ D Sommer Used with permission

1US Fish amp Wildlife Service Kenai National Wildlife Refuge Soldotna Alaska Matt_Bowserfwsgov2Homer Alaska dominiquemcolletgmailcom3Michigan State University East Lansing Michigan swemtracmsuedu

AKES Newsletter httpwwwakentsocorgnewsletterphp


The taxonomy of North American members of thisgroup is problematic Although Gagneacute (1989) provided akey and illustrations to some members of this group theonly descriptions available are the original descriptions ofOsten Sacken (1862) Walsh (1864) Osten Sacken (1878)and Packard (1869) which do not enable separation of thespecies except through willow host species and gall mor-phology Also North American species have not been com-pared to Palearctic species

Through a literature search for species that formrosette or rosette-like galls on willows we found a to-tal of nine named Rabdophaga species Rabdophaga clavifex(Kieffer 1891) Rabdophaga jaapi Ruumlbsaamen 1916 Rab-dophaga rosaria Loew 1850 and Rabdophaga rosariella (Ki-effer 1897) from the Palearctic and Rabdophaga salicisbras-sicoides Packard 1869 Rabdophaga saliciscoryloides OstenSacken 1878 Rabdophaga salicisgnaphaloides Osten Sacken1878 Rabdophaga salicisrhodoides Osten Sacken 1878 andRabdophaga strobiloides Osten Sacken 1862 from the Nearc-tic

We sought to determine whether one polyphagousspecies or multiple species of Rabdophaga caused rosettegalls on southcentral Alaskan willow species using DNAbarcodes We also wanted to relate these entities to de-scribed Rabdophaga species where possible

Methods

Initially as a homeschool science project the first four au-thors collected willow rosette galls from Salix sitchensisSanson ex Bong and Salix barclayi Andersson to deter-mine whether or not these willows were galled by distinctspecies of Rabdophaga We later expanded our sampling toa variety of willow hosts in southcentral Alaska using theillustratations and text of Collet (2002) as a guide to thesetypes of galls Alaskan willow specimens that could not beidentified with confidence by MLB were identified by DCAdditional specimens were obtained by TM from KodiakIsland and Michigan A map of collection localities is givenin Figure 2

40degN

50degN

60degN

70degN

180deg

150degW

120degW

90degW

60degW

30degW

0deg

30degE

60degE

90degE

120deg

E

150degE

R rosariaR cf rosariaR strobiloidesR cf strobiloidesGBIF records

Figure 2 Polar map of collection localities color-coded as in clades in Figure 4 See the Methods section for details on theGBIF records



Figure 3 Representative photographs of galls sampled Top left Rosette gall on Salix barclayi Alaska Kenai PeninsulaSummit Creek collected February 14 2016 Arctos GUID KNWREnto11156 Image CC0 Matt Bowser (httparctosdatabasemuseummedia10510773) Top center Rosette gall on Salix bebbiana Alaska Soldotna Ski Hill Road February20 2018 Arctos GUID KNWREnto11281 Image CC0 Matt Bowser (httpswwwinaturalistorgphotos14507792)Top right Rosette gall on Salix commutata Alaska Kenai Peninsula Turnagain Arm collected February 14 2016Arctos GUID KNWREnto11114 Image CC0 Matt Bowser (httparctosdatabasemuseummedia10522732) Middleleft Rosette gall on Salix fuscescens Alaska Kenai Peninsula Soldotna Ski Hill Road April 11 2016 Arctos GUIDKNWREnto11124 Image CC0 Matt Bowser (httparctosdatabasemuseummedia10508965) Center Rosette gall onSalix hookeriana Alaska Kenai Peninsula Turnagain Arm collected September 23 2016 Arctos GUID KNWREnto11179Image CC0 Matt Bowser (httparctosdatabasemuseummedia10518370) Middle right Rosette gall on Salix pul-chra Alaska Kenai Peninsula Sterling Moose River Drive September 26 2016 Arctos GUID KNWREnto11169 Im-age CC0 Matt Bowser (httparctosdatabasemuseummedia10508962) Bottom left Rosette gall on Salix richard-sonii Alaska Kenai Peninsula Palmer Creek August 13 2016 Arctos GUID KNWREnto11115 Image CC BY MattBowser (httpswwwinaturalistorgphotos4583073) Bottom center Rosette gall on Salix sitchensis Alaska Ke-nai Peninsula Jims Landing collected on December 7 2015 Arctos GUID KNWREnto11097 Image CC0 Matt Bowser(httparctosdatabasemuseummedia10522737) Bottom right Willow pinecone gall on Salix nigra Michigan Kala-mazoo County Al Sabo Preserve Portage Creek September 16 2017 Arctos GUID KNWREnto11280 Image CC BY-NCTracy Melvin (httpswwwinaturalistorgphotos13524200)



KNWREnto11124 S fuscescens KP Soldotna

KNWREnto11118 S pulchra KP Soldotna

KNWREnto11169 S pulchra KP Sterling

GBDP3703minus07 S purpurea Poland


KNWREnto11196 S barclayi KodiakKNWREnto11281 S bebbiana KP Soldotna

KNWREnto11115 S richardsonii KP Palmer Creek

KNWREnto11280 S nigra MI Kalamazoo CoKNWREnto11195 Salix MI Oakland Co

KNWREnto11194 Salix MI Oakland Co

KNWREnto11097 S sitchensis KP Jims Landing

KNWREnto11096 S sitchensis KP Jims LandingKNWREnto11113 S commutata KP Kasilof

KNWREnto11156 S barclayi KP Summit Creek

KNWREnto11107 S barclayi KP Soldotna

KNWREnto11106 S barclayi KP SoldotnaKNWREnto11114 S commutata KP Placer River

KNWREnto11179 S hookeriana KP Turnagain Arm

KNWREnto11178 S commutata MS Butte




012 01 008 006 004 002 0

Nucleotide substitutions per site

R rosaria

R cf strobiloides

R cf rosaria

R strobiloides

R rigidae (outgroup)

76

75

92

9882

75

98

81

92

90

Figure 4 Phylogram of Rabdophaga sequences generated by PhyML Node support values are given as percentages in blueboxes where support values were greater than 50 KP Kenai Peninsula Alaska MI Michigan MS Matanuska-SusitnaBorough Alaska

We dissected Rabdophaga larvae from galls and sub-mitted whole specimens for DNA barcoding using Life-Scanner kits (httpwwwlifescannernet) Sequencesfrom the DNA barcoding region of the COI gene were ob-tained by the Center of Biodiversity Genomics using pro-prietary methods We downloaded from BOLD (Ratnasing-ham and Hebert 2007) one sequence of Rabdophaga rosariafrom Poland obtained by Sato and Yukawa (2006) Weincluded a sequence of Rabdophaga rigidae (Osten Sacken1862) as an outgroup

Sequences were aligned using Clustal Omega (Sieverset al 2011) on EMBL-EBI (McWilliam et al 2013 Li et al2015) accepting default parameters A phylogentic treewas generated from the aligned sequences using PhyML

(Lefort et al 2017) accepting default parametersFor making a map we searched GBIF (httpswww

gbiforg) for all records of Rabdophaga on April 25 2018These data are available as a GBIF Occurrence Download(doi1015468dlluzr8v) For mapping we included onlythe nine Rabdophaga species of willow rosette gall mak-

ers listed above We also added a single observationrecord from from Russia (httpswwwinaturalistorgobservations7431914)

Results

We obtained DNA barcode sequences of rosette gall mak-ers from the following willow hosts Salix barclayi Salixbebbiana Sarg Salix commutata Bebb Salix fuscescens Ander-sson Salix hookeriana Barratt ex Hook Salix pulchra ChamSalix richardsonii Hook and Salix sitchensis from southcen-tral Alaska S barclayi from Kodiak Island and Salix nigraMarshall from Michigan (Figure 3)

Specimen data are available on Arctos (httparctosdatabasemuseum) via a saved search(doi107299X7PG1S1N) Sequence data are available onBOLD

In the phylogenetic analysis rosette gall makers wereplaced in two main clades (Figure 4) with distances be-



tween members of the clades of 005ndash009 nucleotide sub-stitutions per site

One clade included Rabdophaga rosaria from Poland andgallers from S pulchra and S fuscescens hosts with dis-tances among members of 000ndash004 nucleotide substitu-tions per site Distances between the Alaskan specimensand R rosaria were 004 nucleotide substitutions per siteWe tentatively identified Alaskan members of this clade asRabdophaga cf rosaria

The other clade included Rabdophaga strobiloides fromMichigan and Alaskan specimens from S barclayi S beb-biana S commutata S hookeriana and S richardsonii Dis-tances between members of this clade were 000ndash003 nu-cleotide substitutions per site The smallest distance be-tween R strobiloides and Alaskan members of this cladewas 001 nucleotide substitutions per site We tentativelyidentified Alaskan members of this clade as Rabdophaga cfstrobiloides

Our sequences were organized into correspond-ing clades by BOLDrsquos BIN algorithm (Ratnasinghamand Hebert 2013) Members of the clades includingR rosaria R strobiloides and R rigidae were placedin BINs BOLDADA9342 (doi105883BOLDADA9342)BOLDACZ0652 (doi105883BOLDACZ0652) andBOLDADB1445 (doi105883BOLDADB1445) respec-tively BINs BOLDADA9342 and BOLDACZ0652 wereseparated by 375 (p-dist) maximum within BIN dis-tances were 000 to 201

Sequences from three California specimens identifiedas Rabdophaga salicisbrassicoides have not yet been publiclyreleased4 but the sequences are available for searching viaBOLDrsquos Identification Engine They are 95 and 96 sim-ilar (p-dist) to Alaskan R cf strobiloides and R cf rosariarespectively

Discussion

Both our analysis and BOLDrsquos BIN algorithm placedAlaskan Rabdophaga cf strobiloides with Rabdophaga stro-biloides from Michigan The Alaskan specimens may beconspecific with R strobiloides consistent with identifica-tions of this species on S scouleriana in British Columbia(Rea 2012) and on S bebbiana in Alberta (Van Hezewijkand Roland 2003)

We found Alaskan members of the R cf strobiloidesgroup induced galls on five species of willows in sub-genus Vetrix clade C2 of Lauron-Moreau et al (2015) Ourfinding that members of this group galled S barclayi andS commutata corroborates the rearing experiment of Col-let (2010) who found that midges reared from S barclayiformed rosette galls on S commutata Elsewhere in NorthAmerica R strobiloides has been recorded from Salix cordata

Michx Salix eriocephela Michx Salix humilis Marshall Salixnigra Marshall (Wilson 1968 Gagneacute 1989) and Salix scoule-riana Barratt ex Hook (Rea 2012) members of multiplesubgenera of Salix in clades C1 and C2 of Lauron-Moreauet al (2015)

Interestingly even though R strobiloides parasitizes Salixscouleriana in British Columbia (Rea 2012) rosette galls onS scouleriana appear to be extremely rare on the KenaiPeninsula DC is aware of a single example of a rosettegall on S scouleriana from Sterling Alaska MLB has notseen a rosette gall on S scouleriana

Alaskan specimens of Rabdophaga cf rosaria were differ-ent enough from the one Old World sequence of R rosariathat they may represent separate species The Alaskanspecimens all taken from wetlands were collected fromS fuscescens and S pulchra both facultative wetland species(Collet 2002) and both members of the C2 clade of Lauron-Moreau et al (2015) It seems more likely that the AlaskanR cf rosaria is a specialist on these willow species ratherthan a specialist on the wetland habitat because R cf stro-biloides co-occurs with R cf rosaria on separate willowspecies in the same wetlands

It remains to be resolved whether some of the geneticstructure within these two groups is correlated with spe-cialization on willow host species geographic separationor morphological differences We could not conclusivelyanswer our initial question of whether rosette makers on Ssitchensis and S barclayi were distinct because the observeddifferences could have been due to geographic patterns Inthe future we would like to obtain sequences from R cfstrobiloides gall midges on S sitchensis from other locali-ties to help resolve this question DC and MLB have ob-served that at some localities where both S barclayi and Ssitchensis are present rosette galls could only be found onS barclayi suggesting that the two willow species may begalled by distinct Rabdophaga species Alternatively it maybe that a single species of Rabdophaga prefers S barclayi overS sitchensis where both hosts are available

Morphologically all galls of Alaskan Rabdophaga thatwe collected were more open than what is typical of Rstrobiloides In the key of Gagneacute (1989) they would havebeen identified as R salicisbrassicoides R salicisrhodoides orRabdophaga saliciscoryloides which form more open rosettegalls Most Alaskan rosette galls more closely resembleillustrations and photographs of European R rosaria thanany of these North American species An exception is theunusual morphology of rosette galls on S sitchensis whichoften contain more than one midge larva and which oftenhave new stems originating from leaf axils within the galls

At present only a single sequence from this group isavailable from the Old World It would be helpful to ob-tain DNA barcode sequences from more Palearctic rosettegall makers to clarify the relationships of these entities and

4see httpwwwboldsystemsorgindexphpTaxbrowser_Taxonpagetaxid=355957 and doi107299X73J3D8D



North American Rabdophaga cf rosaria and Rabdophaga cfrosaria It would also be desirable to obtain access to se-quences of North American R salicisbrassicoides R salicis-rhodoides and Rabdophaga saliciscoryloides for comparison

Finally we hope that our exploratory work using DNAbarcoding is followed up by a more integrated approachincluding morphology of the gall midges and galls

Acknowledgments

We are grateful to Willem N Ellis and Junichi Yukawafor reviewing and providing helpful comments on thismanuscript Esther L Bowser enthusiastically participatedin collecting and dissecting rosette galls We also thankMariah Mcinnis for her help collecting willow rosette gallsI am especially grateful to my wonderful wife for her pa-tience when I would stop the car on family trips to collectwillow galls The contents of this article are the work ofthe authors and do not necessarily represent the views ofany government entity

References

Amendt J 2003 Taxonomie Biologie und Fein-dartenkomplex ausgewaumlhlter Gallmuumlckenarten(Diptera Cecidomyiidae) auf Weiden (Salix spp)Phd thesis Goethe University Frankfurt GermanyURL httppublikationenubuni-frankfurtde

frontdoorindexindexdocId684

Collet D M 2002 Willows of Southcentral AlaskaKenai Watershed Forum Soldotna Alaska URLhttpwebacsalaskanet~kenaiwatershedforum

willowguidehtml

Collet D M 2006 Genus Rabdophaga and associated in-sects in Alaska URL httpwwwakentsocorgdoc

Collet_DM_2006pdf

Collet D M 2010 Rearing experiment to deter-mine the willow host range of Rabdophaga spp inAlaska Newsletter of the Alaska Entomological So-ciety 39ndash11 URL httpwwwakentsocorgdoc

AKES_newsletter_2010_Ipdf

Ford E E Berg and R McAvinchey 1995 Brows-ing avoidance of galled willow stems by winteringmoose on the Kenai Peninsula Alaska Technical reportUS Fish amp Wildlife Service Kenai National WildlifeRefuge Soldotna Alaska URL httpwwwfwsgov

uploadedFilesFord_E_et_al_1995pdf

Gagneacute R J 1989 The Plant-Feeding Gall Midges of NorthAmerica Cornell University Press Ithaca New York

Gailite A U Andersone and G Ievinsh 2005Arthropod-induced neoplastic formations on treeschange photosynthetic pigment levels and oxidative en-zyme activities Journal of Plant Interactions 161ndash67doi10108017429140500254728

Kenai National Wildlife Refuge staff 1981 Kenai NationalWildlife Refuge Annual Narrative Report CalendarYear 1981 US Department of the Interior Fish andWildlife Service National Wildlife Refuge SystemSoldotna Alaska URL httpscatalogdatagov

datasetkenai-national-wildlife-refuge-annual-

narrative-report-calendar-year-1981

Lauron-Moreau A F E Pitre G W Argus M Labrecqueand L Brouillet 2015 Phylogenetic Relationships ofAmerican Willows (Salix L Salicaceae) PLoS ONE10e0121965 doi101371journalpone0121965

Lefort V J-E Longueville and O Gascuel 2017 SMSSmart Model Selection in PhyML Molecular Biology andEvolution 342422ndash2424 doi101093molbevmsx149

Li W A Cowley M Uludag T Gur H McWilliamS Squizzato Y M Park N Buso and R Lopez 2015The EMBL-EBI bioinformatics web and programmatictools framework Nucleic Acids Research 43W580ndashW584 doi101093nargkv279

McWilliam H W Li M Uludag S Squizzato Y M ParkN Buso A P Cowley and R Lopez 2013 AnalysisTool Web Services from the EMBL-EBI Nucleic AcidsResearch 41W597ndashW600 doi101093nargkt376

Nyman T R Paajanen S Heiska and R Julkunen-Titto 2011 Preference-performance relationship inthe gall midge Rabdophaga rosaria insights from acommon-garden experiment with nine willow clonesEcological Entomology 36200ndash211 doi101111j1365-2311201101260x

Osten Sacken C R 1862 V On the North American Ce-cidomyidae Pp 173ndash205 pl i in H Loew editor Mono-graphs of the Diptera of North America Part I Smith-sonian Miscellaneous Collections 6 Smithsonian Institu-tion Washington URL httpsdoiorg105962bhl

title1718

Osten Sacken C R 1878 Catalogue of the describedDiptera of North America Number 270 in Smithso-nian Miscellaneous Collections Smithsonian InstitutionWashington doi105962bhltitle1728

Packard A S 1869 Guide to the Study of Insectsand a Treatise on those Injurious and Beneficial toCrops Naturalistrsquos Book Agency Salem Massachusettsdoi105962bhltitle49880



Ratnasingham S and P D N Hebert 2007 BOLD TheBarcode of Life Data System (wwwbarcodinglifeorg)Molecular Ecology Notes 7355ndash364 doi101111j1471-8286200701678x

Ratnasingham S and P D N Hebert 2013 A DNA-Based Registry for All Animal Species The BarcodeIndex Number (BIN) System PLoS ONE 8e66213doi101371journalpone0066213

Rea R V 2012 The influence of willow Salixsp rose insect galls on moose Alces alces winterbrowsing The Canadian Field-Naturalist 126189ndash193doi1022621cfnv126i31350

Samsone I U Andersone and G Ievinsh 2011 Gallmidge Rhabdophaga rosaria-induced rosette galls on Salixmorphology photochemistry of photosynthesis and de-fense enzyme activity Environmental and ExperimentalBiology 929ndash36 URL httpeeblulvEEB201108

EEB_9_Samsonepdf

Sato S and J Yukawa 2006 Resurrection of Rabdophagasalicivora Shinji (Diptera Cecidomyiidae) a Japanesegall midge formerly misidentified as a North Americanspecies Rabdophaga rigidae (Osten Sacken) with observa-tions on the phylogenetic relationships of its populationsin Japan and the Russian Far East Entomological Science9423ndash434 doi101111j1479-8298200600188x

Sievers F A Wilm D Dineen T J Gibson K KarplusW Li R Lopez H McWilliam M Remmert J Soumld-ing J D Thompson and D G Higgins 2011 Fastscalable generation of high-quality protein multiple se-quence alignments using Clustal Omega Molecular Sys-tems Biology 7539 doi101038msb201175

Skuhravaacute M and C Thuroacuteczy 2007 Parasitic Hy-menoptera reared from galls of Cecidomyiidae (Diptera)in Europe Acta Zoologica Universitatis Comenianae47203ndash221

Van Hezewijk B H and J Roland 2003 Gall size de-termines the structure of the Rabdophaga strobiloides host-parasitoid community Ecological Entomology 28593ndash603 doi101046j1365-2311200300553x

Walsh B D 1864 On the insects coleopterous hy-menopterous and dipterous inhabiting the galls of cer-tain species of willow Proceedings of the Entomo-logical Society of Philadelphia 3543ndash641 URL https

wwwbiodiversitylibraryorgpart15908

Wilson L F 1968 Life history and habits of the pinewillow gall midge Rhabdophaga strobiloides (Diptera Ce-cidomyiidae) in Michigan The Canadian Entomologist100430ndash433 doi104039Ent100430-4

How heating affects growth rate of Dubia roachesby Fionn D Pietsch

Introduction

I have always loved entomology My favorite insectsare cockroaches I currently am raising two colonies ofcockroaches one colony of Madagascar Hissing cock-roaches (Gromphadorhina portentosa) and the other of DubiaRoaches (Blaptica dubia) Dubia roaches live in central andsouth America Madagascar hissing cockroaches live inMadagascar Both of these areas are in the tropics and havewarm climates but the Madagascar Hissing cockroachesprefer cooler habitats relative to the Dubia roaches Theonly cockroaches that live in Alaska are non-native speciesthat live in warm buildings

You may wonder why you usually only see insects inthe warmer months of the year You may think it is be-cause there is less food and water in the winter which maybe partially true However the primary reason is becauseof heat Heat is one of the most important things for insectgrowth I formed a hypothesis that supplementary heating

would increase the growth rate of Dubia roaches comparedto their growth rate at room temperature

Methods

This spring for the Interior Alaska Science Fair I per-formed an experiment where I used Dubia Roaches I hadfour groups which were in plastic container with air holesin them Three of the groups sat on a 17 watt seedling heat-ing mat (Hydrofarm Seedling Heat Mat) and the controlgroup did not The heated enclosures went from 239 degC to261 degC The unheated enclosure was 161 degC to 211 degC Allgroups had the same amount of food (Nature Zones bitesfor Roaches) and water (Flukerrsquos cricket quenchers calciumfortified) In each group there were about 16 juvenile cock-roaches weighing between 15ndash2 grams per group All thegroups had similar enclosures (newspaper bedding withegg crate hides)

At the beginning I weighed all of the groups I weighedthem weekly for three more weeks At the end I calculatedhow much the groups grew in percentages



The taxonomy of North American members of thisgroup is problematic Although Gagneacute (1989) provided akey and illustrations to some members of this group theonly descriptions available are the original descriptions ofOsten Sacken (1862) Walsh (1864) Osten Sacken (1878)and Packard (1869) which do not enable separation of thespecies except through willow host species and gall mor-phology Also North American species have not been com-pared to Palearctic species

Through a literature search for species that formrosette or rosette-like galls on willows we found a to-tal of nine named Rabdophaga species Rabdophaga clavifex(Kieffer 1891) Rabdophaga jaapi Ruumlbsaamen 1916 Rab-dophaga rosaria Loew 1850 and Rabdophaga rosariella (Ki-effer 1897) from the Palearctic and Rabdophaga salicisbras-sicoides Packard 1869 Rabdophaga saliciscoryloides OstenSacken 1878 Rabdophaga salicisgnaphaloides Osten Sacken1878 Rabdophaga salicisrhodoides Osten Sacken 1878 andRabdophaga strobiloides Osten Sacken 1862 from the Nearc-tic

We sought to determine whether one polyphagousspecies or multiple species of Rabdophaga caused rosettegalls on southcentral Alaskan willow species using DNAbarcodes We also wanted to relate these entities to de-scribed Rabdophaga species where possible

Methods

Initially as a homeschool science project the first four au-thors collected willow rosette galls from Salix sitchensisSanson ex Bong and Salix barclayi Andersson to deter-mine whether or not these willows were galled by distinctspecies of Rabdophaga We later expanded our sampling toa variety of willow hosts in southcentral Alaska using theillustratations and text of Collet (2002) as a guide to thesetypes of galls Alaskan willow specimens that could not beidentified with confidence by MLB were identified by DCAdditional specimens were obtained by TM from KodiakIsland and Michigan A map of collection localities is givenin Figure 2

40degN

50degN

60degN

70degN

180deg

150degW

120degW

90degW

60degW

30degW

0deg

30degE

60degE

90degE

120deg

E

150degE

R rosariaR cf rosariaR strobiloidesR cf strobiloidesGBIF records

Figure 2 Polar map of collection localities color-coded as in clades in Figure 4 See the Methods section for details on theGBIF records

























012 01 008 006 004 002 0


R rosaria

R cf strobiloides

R cf rosaria

R strobiloides


76

75

92

9882

75

98

81

92

90







Results











Discussion














Acknowledgments


References




willowguidehtml


Collet_DM_2006pdf
















title1718









EEB_9_Samsonepdf









Introduction




Methods



























012 01 008 006 004 002 0


R rosaria

R cf strobiloides

R cf rosaria

R strobiloides


76

75

92

9882

75

98

81

92

90







Results











Discussion














Acknowledgments


References




willowguidehtml


Collet_DM_2006pdf
















title1718









EEB_9_Samsonepdf









Introduction




Methods










Discussion














Acknowledgments


References




willowguidehtml


Collet_DM_2006pdf
















title1718









EEB_9_Samsonepdf









Introduction




Methods







Acknowledgments


References




willowguidehtml


Collet_DM_2006pdf
















title1718









EEB_9_Samsonepdf









Introduction




Methods









EEB_9_Samsonepdf









Introduction




Methods




dna barcoding alaskan willow rosette gall makers (diptera ... · volume 11, issue 1, august 2018 12...

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