EuropeanJournalofTaxonomy452:1–36	ISSN2118-9773https://doi.org/10.5852/ejt.2018.452	www.europeanjournaloftaxonomy.eu2018·StecD.etal.

ThisworkislicensedunderaCreativeCommonsAttribution3.0License.

Researcharticle

urn:lsid:zoobank.org:pub:76171A51-C284-455F-9504-C4CCEFE42D9D

IntegrativedescriptionofMacrobiotuscanaricussp.nov.withnoteson

M.recens(Eutardigrada:Macrobiotidae)

DanielSTEC1,*,ŁukaszKRZYWAŃSKI2&ŁukaszMICHALCZYK3

1,2,3DepartmentofEntomology,InstituteofZoologyandBiomedicalResearch,JagiellonianUniversity,Gronostajowa9,30-387Kraków,Poland.

*Correspondingauthor:daniel_stec@interia.eu

2Email:lukasz.s.krzywanski@gmail.com

3Email:LM@tardigrada.net

1urn:lsid:zoobank.org:author:C148C15D-9F12-4312-9702-B1E1513C63CC2urn:lsid:zoobank.org:author:0B32C511-BC10-4946-8191-7623AF798C653urn:lsid:zoobank.org:author:6592787E-AAB1-4717-90C9-B967488F06F4

Abstract.InthispaperwedescribeMacrobiotuscanaricussp.nov.,anewtardigradespeciesoftheMacrobiotushufelandigroupfromtheCanaryIslands.Moreover,withtheuseofDNAsequencing,weconfirmthatMacrobiotusrecensCuénot,1932representsthehufelandigroup,eventhougheggslaidbythisspeciesdonotexhibitthetypicalhufelandigroupmorphology.Ourstudyisbasedonbothclassicaltaxonomicmethodsthatincludemorphologicalandmorphometricanalysesconductedwiththeuseoflightandscanningelectronmicroscopy,andontheanalysisofnucleotidesequencesoffourmolecularmarkers(threenuclear:18SrRNA,28SrRNA,ITS-2,andonemitochondrial:COI).OuranalysesrevealedthatM.canaricussp.nov.ismostsimilartoMacrobiotusalmadaiFontouraetal.,2008fromtheArchipelagooftheAzores,fromwhichitdiffersbytheabsenceofgranulationpatchesontheexternalandinternalsurfacesoflegsI–IIIaswellasbytheabsenceofacuticularporeinthecentreoftheexternalpatchonlegsI–III.MolecularsequencesallowedustopinpointthephylogeneticpositionsofM.canaricussp.nov.andM.recenswithintheM.hufelandigroup.

Keywords.CanaryIslands,COI,Macrobiotushufelandigroup,phylogeny,tardigrades.

StecD.,KrzywańskiŁ.&MichalczykŁ.2018.IntegrativedescriptionofMacrobiotuscanaricussp.nov.withnotesonM.recens(Eutardigrada:Macrobiotidae).EuropeanJournalofTaxonomy452:1–36.https://doi.org/10.5852/ejt.2018.452

Introduction

Tardigradesareaphylumofgloballydistributedmicroinvertebrateswhichinhabitterrestrialandmarinehabitats(Nelsonetal.2015).However,themajorityoftheover1200knownspecieswerefoundanddescribedfromterrestrialmossesandlichens(Guidetti&Bertolani2005;Degma&Guidetti2007;Degmaetal.2009–2017).Althoughtardigradefaunisticstudieshavebeenconductedformorethantwocenturies,manyregionsarestillverypoorlyknownintermsoftardigradespeciescomposition.OnesuchplaceistheCanaryIslands(Spain),whereonlytwostudiesontardigrades

EuropeanJournalofTaxonomy452:1–36(2018)

havebeenperformeduntilnow(Heinis1908;Guil&Guidetti2005).Intotal,13taxaidentifiedtospecieslevelhavebeenreportedfromtheCanaryIslands:EchiniscusarctomysEhrenberg,1853,

E.canadensisMurray,1910,E.quadrispinosusRichters,1902,E.mediantusMarcus,1930,E.trisetosus

Cuénot,1932,Isohypsibiustuberculatus(Plate,1888),MacrobiotusechinogenitusRichters,1904,

M.furcatusEhrenberg,1859,M.hufelandiC.A.S.Schultze,1834,M.occidentalisMurray,1910,MilnesiumtardigradumDoyère,1840,Minibiotusintermedius(Plate,1888)andM.gumersindoiGuil&Guidetti,2005.Moreover,bothstudiesreportedundeterminedspeciesoftheMacrobiotushufelandigroupandHeinis(1908)notedanundeterminedspeciesofEchiniscusC.A.S.Schultze,1840.However,notallofthethirteenspeciesidentificationscanbeconsideredcertainaccordingtomoderntaxonomicstandards.Forexample,recordsofM.hufelandiandM.tardigradumpredatetheredescriptionsofthesespecies(Bertolani&Rebecchi1993andMichalczyketal.2012,respectively),andthustheyneedtobeverifiedagainstmodernliterature.

WeexaminedmossandlichensamplesfromtheislandofGranCanaria.Intwoofthem,wefoundtwospeciesoftheMacrobiotushufelandigroup,onenewtoscienceandtheotherconformingtothedescriptionofMacrobiotusrecensCuénot,1932.Forboththesepopulations,weprovidetaxonomicdescriptionsbasedonmorphologicalandmorphometricdataacquiredwithphasecontrastlightmicroscopy(PCM)aswellasfromscanningelectronmicroscopy(SEM).Additionally,wesequencedfourDNAmarkers,threenuclear(18SrRNA,28SrRNA,ITS-2)andonemitochondrial(COI),thatallowedustopinpointthephylogeneticpositionsofthetwospecies.SpecimensofM.cf.recensconformtothediagnosisofthehufelandigroup,buttheylayeggsthatdepartfromthetypicalhufelandigroupeggornamentation,i.e.,insteadofinvertedgoblet-shapedprocesses,theeggsareequippedwithconicalprocesses.Thus,thespecieseitherhasnot(Biserov1990a,1990b)orhas(Bertolani&Rebecchi1993;Kaczmarek&Michalczyk2017b)beenclassifiedwithinthehufelandigroup.Therefore,thefirstDNAsequencesfor

M.recensallowedustoverifywhetherthespeciesrepresentsthehufelandigroup.

Materialandmethods

Sampleprocessingandtardigradeculturing

ThemossandlichensamplescontainingthenewspeciesandM.cf.recens,respectively,werecollectedfromtwolocalitiesinGranCanariabyMartaKapałaon16February2017(seeTable1fordetails).SampleswereexaminedforterrestrialtardigradesusingstandardmethodsdescribedbyDastych(1980)withmodificationbyStecetal.(2015).Atotalof21individualsofthenewspeciesand29individualsofM.cf.recenswereextractedfromthefirstandthesecondsample,respectively.Liveanimalswereplacedinaculturetoobtainmoreindividualsandeggsrequiredforintegrativeanalyses(seeTable1fordetails).AnimalswererearedonplasticPetridishesaccordingtotheprotocolinStecetal.(2015).

Microscopyandimaging

SpecimensforlightmicroscopyweremountedonmicroscopeslidesinasmalldropofHoyer’smediumandsecuredwithacoverslip,followingtheprotocolinMoreketal.(2016b).Slideswerethendriedforfivetosevendaysat60°C.DriedslidesweresealedwithatransparentnailpolishandexaminedunderaNikonEclipse50iphasecontrastlightmicroscope(PCM)associatedwithaNikonDigitalSightDS-L2digitalcamera.InordertoobtaincleanandextendedspecimensforSEM,tardigradeswereprocessedaccordingtotheprotocolinStecetal.(2015).Inshort,specimenswerefirstsubjectedtoa60°Cwaterbathfor30mintoobtainfullyextendedanimals,followedbyawater/ethanolandanethanol/acetone

2 series,andthenCO

criticalpointdryingandfinallysputtercoatedwithathinlayerofgold.Specimens

wereexaminedunderhighvacuuminaVersa3DDualBeamScanningElectronMicroscopeattheATOMINfacilityoftheJagiellonianUniversity,Kraków,Poland.Bothstudiedpopulationswerealsoexaminedforthepresenceofmaleswithaceto-orceinstaining(Bertolani1971)inaccordancewithStecetal.(2016a).

STECD.etal.,TardigradesfromtheCanaryIslands

Table1.SamplecollectiondetailsandnumbersofindividualsandeggsusedforintegrativeanalysesofM.canaricussp.nov.andM.cf.recensCuénot,1932.(PCM=phasecontrastmicroscopy;SEM=scanningelectronmicroscopy;DNA=DNAsequencing;ORC=orceinstaining)

*Eggsweretakenfromthecultureandincubatedindividuallyuntilhatching,thenthejuvenileswereusedforDNAsequencingwhereaseggchorions(isogenophores)weremountedonmicroscopeslidesinHoyer’smedium.

Species	Sample code	Coordinates; altitude	Sampletype,habitat	PCM	SEM	DNA	ORC
M.canaricus sp.nov.	ES.004	28°03′05″N, 15°38′21″W; 1050ma.s.l.	mossonatreetrunkinapineforest	120ind. 42eggs	20ind. 15eggs	4ind. 3eggs*	15ind. 0eggs
M.cf.recens	ES.006	28°02′38″N, 15°40′22″W; 885ma.s.l.	lichenonastonewall	87ind. 38eggs	8ind. 10eggs	0ind. 4eggs*	15ind. 0eggs
M.macrocalixPL.110	49°31′27″N, 21°02′23″E; 254ma.s.l.	mossonaroof	60ind. 72eggs	0ind. 0eggs	4ind. 0eggs	0ind. 0eggs

AllfigureswereassembledinCorelPhoto-PaintX6,v.16.4.1.1281.Forstructuresthatcouldnotbesatisfactorilyfocusedinasinglephotograph,astackof2–8imageswastakenwithanequidistanceofca

0.2μmandassembledmanuallyintoasingledeep-focusimage.

Morphometricsandmorphologicalnomenclature

Allmeasurementsaregiveninmicrometres(μm).SamplesizewasadjustedfollowingrecommendationsbyStecetal.(2016b).Structuresweremeasuredonlyiftheirorientationwassuitable.Bodylengthwasmeasuredfromtheanteriorextremitytotheendofthebody,excludingthehindlegs.TheterminologyusedtodescribeoralcavityarmatureandeggshellmorphologyfollowsMichalczyk&Kaczmarek(2003)andKaczmarek&Michalczyk(2017b).MacroplacoidlengthsequenceisgivenaccordingtoKaczmareketal.(2014).BuccaltubelengthandthelevelofthestyletsupportinsertionpointweremeasuredaccordingtoPilato(1981).Theptindexistheratioofthelengthofagivenstructuretothelengthofthebuccaltubeexpressedasapercentage(Pilato1981).AllothermeasurementsandnomenclaturearegiveninaccordancewithKaczmarek&Michalczyk(2017b).Inbrief,thebuccaltubewidthwasmeasuredastheexternalandinternaldiameteratthelevelofthestyletsupportinsertionpoint.Lengthsoftheclawbranchesweremeasuredfromthebaseoftheclaw(i.e.,excludingthelunula)tothetopofthebranch,includingaccessorypoints.Distancebetweeneggprocesseswasmeasuredastheshortestlineconnectingbaseedgesofthetwoclosestprocesses.Morphometricdatawerehandledusingthe‘Parachela’v.1.3templateavailablefromtheTardigradaRegister(Michalczyk&Kaczmarek2013).TardigradetaxonomyfollowsBertolanietal.(2014).

Comparativematerial

ThetaxonomickeyforthehufelandigroupprovidedbyKaczmarek&Michalczyk(2017b)wasusedtodeterminewhethertheisolatedspecieshadpreviouslybeendescribed.Afteronespeciescouldnotbeidentifiedwiththekey,wecompareditwiththeoriginaldescriptionofthemostsimilarspeciesofthehufelandigroup,MacrobiotusalmadaiFontouraetal.,2008.Additionally,wealsocomparedournewspecieswiththreeparatypesandoneeggofM.almadaikindlyloanedtousbyProfessorPauloFontoura.Moreover,thankstothecourtesyofMatteoVecchi,weanalysedphotomicrographsofanimalsandeggsidentifiedas‘M.recens’byMaucci(1979)fromseveralPortuguesepopulationsdepositedintheMauccicollection.AlthoughourpopulationconformedtotheoriginaldescriptionofM.recens,thedescription

EuropeanJournalofTaxonomy452:1–36(2018)

Table2.PrimersandreferencesforPCRprotocolsforamplificationofthefourDNAfragmentssequencedinthestudy.

DNAfrag-ment

Primername

Primerdirection

Primersequence(5’-3’)	Primersource	PCRprogramme

18SrRNA18S_Tar_Ff1	forward	AGGCGAAACCGCGAATGGCTC	Stecetal.

18S_Tar_Rr1	reverse	GCCGCAGGCTCCACTCCTGG

(2017a)	Zeller(2010)

28SrRNA

28SF0001	forward	ACCCVCYNAATTTAAGCATAT	Mironovetal.

28SR0990	reverse	CCTTGGTCCGTGTTTCAAGAC	(2012)

ITS2_Eutar_Ffforward	CGTAACGTGAATTGCAGGAC	Stecetal.

Mironovetal.(2012)

ITS-2

ITS2_Eutar_Rrreverse	TCCTCCGCTTATTGATATGC

(2018c)	Stecetal.(2018c)

LCO1490	forward	GGTCAACAAATCATAAAGATATTGG	Folmeretal.

Michalczyketal.

COI

HCO2198	reverse	TAAACTTCAGGGTGACCAAAAAATCA

(1994)

(2012)

isoutdatedand,inouropinion,anaccurateidentificationofthespeciesisnotpossibleuntilamodernredescriptionisavailable(seeDiscussionformoredetails).Therefore,weidentifiedourpopulationas

M.cf.recens.Nevertheless,giventhatourpopulationfitsthedescriptionofM.recens,ithastorepresentatleastacloselyrelatedspeciesand,assuch,itcanbeusedtoestimatethephylogeneticpositionof

M.recenss.str.

Genotyping

TheDNAwasextractedfromindividualanimalsfollowingtheChelex®100resin(Bio-Rad)extractionmethodofCasquetetal.(2012)withmodificationsdescribedindetailinStecetal.(2015).WesequencedfourDNAfragments,threenuclear(18SrRNA,28SrRNA,ITS-2)andonemitochondrial(COI).AllfragmentswereamplifiedandsequencedaccordingtotheprotocolsdescribedinStecetal.(2015);primersandoriginalreferencesforspecificPCRprogramsarelistedinTable2.SequencingproductswerereadwiththeABI3130xlsequencerattheMolecularEcologyLab,InstituteofEnvironmentalSciencesoftheJagiellonianUniversity,Kraków,Poland.SequenceswereprocessedinBioEditv.7.2.5(Hall1999)andsubmittedtoGenBank.

Comparativemolecularanalysis

Formolecularcomparisons,allpublishedsequencesofthefourabove-mentionedmarkersforspeciesofthehufelandicomplexweredownloadedfromGenBank(listedinTable3).Additionally,wealsosequencedthefourDNAfragmentsforanewpopulationofMacrobiotusmacrocalixBertolani&Rebecchi,1993collectedinŁękawica,southernPolandbyDSinOctober2015(seeTable1forsampledetails).Thesequenceswerealignedusingthedefaultsettings(inthecaseofCOI)andtheQ-INS-Imethod(inthecaseofribosomalmarkers:18SrRNA,28SrRNAandITS-2)ofMAFFTv.7(Katohetal.2002;Katoh&Toh2008)andmanuallycheckedagainstnon-conservativealignmentsinBioEdit.Then,thealignedsequencesweretrimmedto763(18SrRNA),712(28SrRNA),317(ITS-2)and618(COI),bp.AllCOIsequencesweretranslatedintoproteinsequencesinMEGA7v.7.0(Kumaretal.2016)tocheckagainstpseudogenes.AccordingtotherecommendationbySrivathsan&Meier(2012),uncorrectedpairwisedistanceswerecalculatedusingMEGA7insteadofgeneticdistancescorrectedbyKimura2parametermodel(K2P).

Datadeposition

RawmorphometricmeasurementsunderlyingthedescriptionofMacrobiotuscanaricussp.nov.aregiveninSupplementaryMaterials(SM.1)andaredepositedintheTardigradaRegister(Michalczyk&Kaczmarek2013)underwww.tardigrada.net/register,whereasrawmorphometricdataunderlyingthedescriptionofthepopulationofM.cf.recensaregiveninSupplementaryMaterials(SM.2).TheDNA

STECD.etal.,TardigradesfromtheCanaryIslands

Table3.SequencesofspeciesoftheMacrobiotushufelandigroupusedformolecularcomparisonsandthephylogeneticanalysesofMacrobiotuscanaricussp.nov.andM.cf.recensCuénot,1932.UnderlinedGenBankaccessionnumbersindicatetypeorneotypesequences,whereasboldednumbersindicatenewsequencesobtainedinthisstudy.

DNA marker	Species	Accessionnumber	Source

“M.hufelandi”Schultze,1834	GQ849024	Giribetetal.(1996)

M.hufelandigroupspecies	HQ604971,FJ435738–40

Bertolanietal.(2014),

Guil&Giribet(2012)

18SrRNA

28SrRNA

ITS-2

M.hannaeNowak&Stec,2018	MH063922	Nowak&Stec(2018)

“M.joannae”Pilato&Binda,1983

[=M.hannaeNowak&Stec,2018]	HQ604974–5	Bertolanietal.(2014)

M.kristenseniGuidettietal.,2013	KC193577	Guidettietal.(2013)

M.macrocalixBertolani&Rebecchi,1993	HQ604976,MH063926	Bertolanietal.(2014),thisstudy

M.papeiStecetal.,2018	MH063881	Stecetal.(2018d)

M.paulinaeStecetal.,2015	KT935502	Stecetal.(2015)

M.polypiformisRoszkowskaetal.,2017	KX810008	Roszkowskaetal.(2017)

M.polonicusPilatoetal.,2003	HM187580	Wełniczetal.(2011)

M.sapiensBinda&Pilato,1984	DQ839601	Bertolanietal.(2014)

M.scoticusStecetal.,2017	KY797265	Stecetal.(2017b)

M.shonaicusStecetal.,2018	MG757132	Stecetal.(2018a)

M.hannaeNowak&Stec,2018	MH063924	Nowak&Stec(2018)

M.hufelandigroupspecies	FJ435751,FJ435754–5	Guil&Giribet(2012)

M.macrocalixBertolani&Rebecchi,1993	MH063935	thisstudy

M.papeiStecetal.,2018	MH063880	Stecetal.(2018d)

M.paulinaeStecetal.,2015	KT935501	Stecetal.(2015)

M.polypiformisRoszkowskaetal.,2017	KX810009	Roszkowskaetal.(2017)

M.scoticusStecetal.,2017	KY797266	Stecetal.(2017b)

M.shonaicusStecetal.,2018	MG757133	Stecetal.(2018a)

M.hannaeNowak&Stec,2018	MH063923	Nowak&Stec(2018)

M.macrocalixBertolani&Rebecchi,1993	MH063931	thisstudy

M.papeiStecetal.,2018	MH063921	Stecetal.(2018d)

M.paulinaeStecetal.,2015	KT935500	Stecetal.(2015)

M.polonicusPilatoetal.,2003	HM150647	Wełniczetal.(2011)

M.polypiformisRoszkowskaetal.,2017	KX810010	Roszkowskaetal.(2017)

M.sapiensBinda&Pilato,1984	GQ403680	Schilletal.(2010)

M.scoticusStecetal.,2017	KY797268	Stecetal.(2017b)

M.shonaicusStecetal.,2018	MG757134–5	Stecetal.(2018a)

M.hannaeNowak&Stec,2018	MH057764	Nowak&Stec(2018)

M.cf.hufelandi,Schultze,1834	HQ876589–94,HQ876596	Bertolanietal.(2011a)

M.hufelandi,Schultze,1834	HQ876584,HQ876586–8	Bertolanietal.(2011a)

M.kristenseniGuidettietal.,2013	KC193575–6	Guidettietal.(2013)

Cesarietal.(2009),

M.macrocalixBertolani&Rebecchi,1993	FJ176203–07,FJ176208–17,

HQ876571,MH057767

Bertolanietal.(2011a)

thisstudy

COI

M.papeiStecetal.,2018	MH057763	Stecetal.(2018d)

M.paulinaeStecetal.,2015	KT951668	Stecetal.(2015)

M.polypiformisRoszkowskaetal.,2017	KX810011–2	Roszkowskaetal.(2017)

M.sandraeBertolani&Rebecchi,1993	HQ876566–67,HQ876569–70,

HQ876562–73,HQ876574–83

Bertolanietal.(2011a)

M.scoticusStecetal.,2017	KY797267	Stecetal.(2017b)

M.shonaicusStecetal.,2018	MG757136–7	Stecetal.(2018a)

M.terminalisBertolani&Rebecchi,1993	JN673960,AY598775	Cesarietal.(2011),

Guidettietal.(2005)

M.vladimiriBertolanietal.,2011	HM136931–2,HM136933–4,

Bertolanietal.(2011a,2011b)

HQ876568

EuropeanJournalofTaxonomy452:1–36(2018)

sequencesforthetypepopulationaredepositedinGenBank(https://www.ncbi.nlm.nih.gov/genbank).UncorrectedpairwisedistancesaregiveninSupplementaryMaterials(SM.3).

Phylogeneticanalysis

ToverifythephylogeneticpositionsofthenewspeciesandM.cf.recens,phylogenetictreeswereconstructedusing(1)allmacrobiotid18SrRNAsequencesavailablefromGenBank,(2)concatenated18SrRNA+28SrRNA+ITS-2+COIsequencesofmacrobiotidspeciesforwhichatleastthreeofthesemarkersweresequenced,and(3)allpublishedM.hufelandigroupCOIsequences.InadditiontothesequencesofthehufelandigrouplistedinTable3,wealsousedsequencesofotherspeciesofthefamilyMacrobiotidaepublishedsofar:Sandsetal.(2008),Guidettietal.(2009),Guil&Giribet(2012),Bertolanietal.(2014),Mapaloetal.(2016),Vecchietal.(2016),Zawieruchaetal.(2016),Mapaloetal.(2017),Stec&Kristensen(2017),Stecetal.(2018b).ThesequencesofMilnesiumvariefidumMoreketal.,2016aandMi.berladnicorumCiobanuetal.,2014fromMoreketal.(2016a)wereusedastheoutgroupinthefirsttwoanalyses,whereasfourspeciesofMesobiotusservedastheoutgroupinthethirdanalysis:Me.ethiopicusStec&Kristensen,2017,Me.hilariaeVecchietal.,2016,Me.insanisMapaloetal.,2017andMe.philippinicusMapaloetal.,2016.

Thesequenceswerealignedusingthedefaultsettings(inthecaseofCOI)andwiththeQ-INS-Imethod(inthecaseofribosomalmarkers:18SrRNA,28SrRNAandITS-2)ofMAFFTv.7(Katohetal.2002;Katoh&Toh2008)andtheneditedandcheckedmanuallyinBioEdit.Thealignmentsof18SrRNAandCOIsequencesweretrimmedto739bpand618bp,respectively,whereasthealignedsequencesthatwereusedtoconstructtheconcatenateddatamatrixweretrimmedto:728bp(18SrRNA),754bp(28SrRNA),570bp(ITS-2)and621bp(COI).ThesequenceswereconcatenatedinSequenceMatrix(Vaidyaetal.2011).Theconcatenateddatamatrixcomprisesspeciesforwhichatleastthreeoftheaforementionedmolecularmarkersareavailable.Thisresultedinonlytwogapswithinthematrix:forthe18SrRNAsequenceofMe.insanis(sequencetooshort)andtheITS-2sequenceofMe.ethiopicus(sequencenotavailable).UsingPartitionFinderv.2.1.1(Lanfearetal.2016)undertheBayesianInformationCriterion(BIC),thebestschemeofpartitioningandsubstitutionmodelswerechosenforposteriorphylogeneticanalysis.Werantheanalysistotestallpossiblemodelsimplementedintheprogram.AstheCOIisaproteincodinggene,beforepartitioning,wedividedouralignmentsofthismarkerinto3datablocksconstitutingseparatedthreecodonpositions.Asbest-fitpartitioningscheme,PartitionFinderalwayssuggestedtheretentionofallpredefinedpartitionsseparately.SeeTable4forspecificsubstitutionmodelssuggestedforalltesteddatasetsandpartitions.

Bayesianinference(BI)marginalposteriorprobabilitieswerecalculatedforbothdatasetsusingMrBayes

v.3.2(Ronquist&Huelsenbeck2003).Randomstartingtreeswereusedandtheanalysiswasrunfortenmilliongenerations,samplingtheMarkovchainevery1000generations.Anaveragestandarddeviationofsplitfrequenciesof<0.01wasusedasaguidetoensurethetwoindependentanalyseshadconverged.TheprogramTracerv.1.7(Rambautetal.2018)wasthenusedtoensureMarkovchainshadreachedstationarityandtodeterminethecorrect‘burn-in’fortheanalysis,whichwasthefirst10%ofgenerations.TheESSvaluesweregreaterthan200andaconsensustreewasobtainedaftersummarisingtheresultingtopologiesanddiscardingthe‘burn-in’.AllfinalconsensustreeswereviewedandvisualisedbyFigTree

v.1.4.3,availablefromhttp://tree.bio.ed.ac.uk/software/figtree.

Abbreviations

PCM	=phasecontrastlightmicroscopySEM	=scanningelectronmicroscopy

IZiBB=InstituteofZoologyandBiomedicalResearch,JagiellonianUniversity,Kraków,Poland

STECD.etal.,TardigradesfromtheCanaryIslands

Table4.ThebestschemeofpartitioningandsubstitutionmodelschosenforposteriorphylogeneticanalysisusingPartitionFinderv.2.1.1undertheBayesianInformationCriterion(BIC).Theanalyseswereruntotestallpossiblemodelsimplementedintheprogram.

18S	28S	ITS-2	COI-1st	COI-2nd	COI-3rd
18S	SYM+I+G	–	–	–	–	–
Dataset

Substitutionmodelforthegivenpartition

COI	–	–	–	SYM+I+G	GTR+I+G	TRN+G

18S+28S+

ITS-2+COI	SYM+G	GTR+G	SYM+G	GTR+I+G	GTR+I+G	GTR+G

Results

Taxonomicaccount

PhylumTardigradaDoyère,1840ClassEutardigradaRichters,1926OrderParachelaSchusteretal.,1980

SuperfamilyMacrobiotoideaThulin,1928(inMarleyetal.2011)FamilyMacrobiotidaeThulin,1928

GenusMacrobiotusC.A.S.Schultze,1834

Macrobiotuscanaricussp.nov.

urn:lsid:zoobank.org:act:AE3AAEA9-D20E-4917-8ECD-12B30DB514B6

Figs1–7,Tables5–6

Etymology

ThespecificepithetreferstotheCanaryIslands,theplacewherethenewspecieswasfound.

Materialexamined(162animals,57eggs)

SpecimensmountedonmicroscopeslidesinHoyer’smedium(120animals+42eggs),fixedonSEMstubs(20+15),processedforDNAsequencing(7animals),andaceto-orceinstaining(15animals).

Holotype

SPAIN:CanaryIslands,GranCanaria,Fagajesto,28°03′05″N,15°38′21″W,mossonatreetrunkinapineforest(slideIZiBBES.004.04).

Paratypes

SPAIN:127specimens,samedataasforholotype(slidesIZiBBES.004.01–24);42eggs,samedataasforholotype(slidesIZiBBES.004.25–33).

Description

Animals(measurementsandstatisticsinTable5)

Bodywhiteinadults,afterfixationinHoyer’smediumtransparent(Fig.1A).EyespresentbothinliveanimalsandinspecimensmountedinHoyer’smedium.Roundandovalpores(0.4–0.7μmindiameter),visibleunderPCMandSEM,scatteredrandomlyonentirebodycuticle(Fig.2A–F),includingexternalandinternalsurfaceofalllegs(Fig.2A–F).Extremelyfinebodygranulation(granules0.06–0.09μm

7

EuropeanJournalofTaxonomy452:1–36(2018)

Table5.Measurements(inµm)ofselectedmorphologicalstructuresofindividualsofMacrobiotuscanaricussp.nov.mountedinHoyer’smedium(N=numberofspecimens/structuresmeasured;Range=thesmallestandthelargeststructureamongallmeasuredspecimens;SD=standarddeviation)

Character N	Range µm pt	Mean µm pt	SD µm pt	Holotype µm pt
BodylengthBuccopharyngealtube	30	306–431	902–1206	368	1094	30	72	359	1088
Buccaltubelength	30	29.5–37.6	–	33.7	–	1.8	–	33.0	–
Styletsupportinsertionpoint	30	22.4–28.1	74.3–76.8	25.4	75.4	1.3	0.7	24.6	74.5
Buccaltubeexternalwidth	30	3.2–5.4	10.3–15.5	4.3	12.8	0.4	1.1	4.3	13.0
Buccaltubeinternalwidth	30	2.1–3.3	6.6–9.5	2.7	7.9	0.3	0.8	2.5	7.6
Ventrallaminalength	28	16.2–24.4	53.4–68.2	20.5	60.8	1.5	3.7	19.5	59.1
Placoidlengths
Macroplacoid1	30	5.5–9.5	17.7–28.4	7.3	21.5	1.0	2.4	8.0	24.2
Macroplacoid2	30	4.6–7.1	14.6–20.5	5.8	17.2	0.6	1.4	6.0	18.2
Microplacoid	30	1.6–2.7	4.7–7.8	2.0	6.0	0.3	0.8	2.1	6.4
Macroplacoidrow	30	11.5–17.5	36.8–52.0	14.1	41.8	1.4	3.1	14.4	43.6
PlacoidrowClaw1lengths	30	14.0–19.4	42.6–55.3	16.6	49.2	1.5	3.0	17.0	51.5
Externalprimarybranch	30	8.3–11.3	25.1–33.5	9.5	28.3	0.7	2.0	8.3	25.2
Externalsecondarybranch	30	6.2–9.3	19.0–26.2	7.4	22.1	0.7	1.9	6.5	19.7
Internalprimarybranch	29	7.5–10.6	23.8–32.0	9.2	27.4	0.6	1.9	8.6	26.1
Internalsecondarybranch	29	6.0–8.4	17.4–23.8	7.0	20.7	0.6	1.7	6.6	20.0
Claw2lengths
Externalprimarybranch	30	8.6–11.8	25.3–35.6	9.8	29.3	0.7	2.2	9.0	27.3
Externalsecondarybranch	30	5.8–8.7	18.4–26.0	7.4	22.1	0.7	2.2	6.3	19.1
Internalprimarybranch	29	8.2–10.8	24.2–31.2	9.4	28.0	0.6	1.8	8.2	24.8
Internalsecondarybranch	29	5.4–8.6	15.9–25.1	7.2	21.5	0.6	2.0	7.4	22.4
Claw3lengths
Externalprimarybranch	28	8.9–11.0	25.8–32.6	10.0	29.8	0.6	1.9	9.2	27.9
Externalsecondarybranch	28	6.4–9.7	18.8–28.4	7.7	22.8	0.7	2.4	7.2	21.8
Internalprimarybranch	28	5.6–11.3	16.5–31.6	9.5	28.1	1.0	3.0	8.1	24.5
Internalsecondarybranch	28	6.0–8.9	16.8–25.7	7.3	21.7	0.7	1.9	6.9	20.9
Claw4lengths
Anteriorprimarybranch	27	8.6–20.5	28.5–57.1	11.2	33.1	2.0	5.2	10.3	31.2
Anteriorsecondarybranch	25	6.7–9.7	18.5–28.8	8.1	24.1	0.8	2.3	6.9	20.9
Posteriorprimarybranch	26	9.6–13.2	28.3–37.6	11.2	33.2	0.8	2.7	10.5	31.8
Posteriorsecondarybranch	23	6.6–9.5	19.4–27.3	8.1	24.0	0.9	2.3	7.4	22.4

indiameter),visibleonlyunderSEM,presentonthedorso-posteriorcuticle(Fig.2E–F).GranulationpatchesonexternalsurfaceoflegsI–IIIclearlyvisiblebothunderPCMandSEM(Fig.3A–B).GranulationpatchesoninternalsurfaceoflegsI–IIIweaklyvisibleunderPCMbutclearlyvisibleunderSEM(Fig.3C–D,emptyindentedarrowheads).Single,large,ovalporepresentatcentreofeachexternalpatchonlegsI–III(Fig.3A–B,filledflatarrowheads).Cuticularbulge,resemblingpulvinus,presentoninternalsurfaceoflegsI–III(Fig3C–D,filledindentedarrowheads).Thisstructureisvisibleonlyiflegsarefullyextendedandcorrectlyorientedonslide.CuticulargranulationonlegsIVpresentandalwaysclearlyvisiblebothunderPCMandSEM(Fig.3E–F).

Mouthantero-ventral.Bucco-pharyngealapparatusoftheMacrobiotustype,withtheventrallaminaandtensmallperibuccallamellaefollowedbysixbuccalsensorylobes(Fig.4A–C).UnderPCM,theoralcavityarmatureisofthemaculatustype,i.e.,onlythethirdbandofteethisvisible(Fig.4A).

STECD.etal.,TardigradesfromtheCanaryIslands

UnderSEM,theoralcavityisalwayscomposedofthreebandsofteeth(Fig.4B–C).Thefirstbandofteethiscomposedofnumerousextremelysmallconesarrangedinoneortworows,situatedanteriorlyintheoralcavity,onthebasalpartoftheperibuccallamellae(Fig.4B–C,filledarrowhead).Thesecondbandofteethissituatedbetweentheringfoldandthethirdbandofteethandconsistsofcones,clearlylargerthanthoseofthefirstband(Fig.4B–C,emptyarrowhead).Theteethofthethirdbandarelocatedwithintheposteriorportionoftheoralcavity,betweenthesecondbandofteethandthebuccaltubeopening(Fig.4B–C).Thethirdbandofteethisdiscontinuousanddividedintoadorsalandaventralportion.UnderPCM,thedorsalteethformatransversalridgeweaklydividedintothreeteeth,whereastheventralteethappearastwoseparatelateraltransverseridgesbetweenwhicharoundishmediantoothisvisible(Fig.4A).UnderSEM,thedorsalteetharedividedintothreeseparateteeth:onemedianandtwolateral,themediantoothhasaslightlyserratededge(Fig.4B).Theventralteetharealsoseparatedintoonemedianandtwolateralteeth(Fig.4C).Themedio-ventraltoothismuchsmallerthanthemedio-dorsaltooth(Fig.4B–C).Pharyngealbulbspherical,withtriangularapophyses,tworod-shapedmacroplacoidsandasmallmicroplacoid(Fig.4A).Thefirstandthesecondmacroplacoidshaveafinecentralandasubterminalconstriction,respectively.Themacroplacoidlengthsequenceis2<1.

Fig.1.Macrobiotuscanaricussp.nov.,holotype,habitus.A.Dorso-ventralprojection(Hoyer’smedium,PCM).B.DorsalviewunderSEM.Scalebarsinμm.

EuropeanJournalofTaxonomy452:1–36(2018)

Fig.2.Macrobiotuscanaricussp.nov.,paratypes,bodycuticle.A–B.Poresonthedorsalandventralcuticle,respectively(PCM).C–D.Poresonthedorsalandventralcuticle,respectively(SEM).E.Poresandfinegranulationonthedorso-posteriorcuticle(SEM).F.Close-upofaporeandgranulationonthedorso-posteriorcuticle(SEM).Scalebarsinμm.

STECD.etal.,TardigradesfromtheCanaryIslands

Fig.3.Macrobiotuscanaricussp.nov.,paratypes,cuticularstructuresonlegs.A–B.ThepatchofgranulationontheexternalsurfaceoflegIIIwithalargeovalcuticularporeatthecentreofthepatch(PCMandSEM,respectively).C–D.Thepatchofgranulationandthecuticularbulge(pulvinus)ontheinternalsurfaceoflegII(PCMandSEM,respectively).E–F.GranulationonlegIV(PCMandSEM,respectively).Filledflatarrowheadsindicatethelargeporeinthecentreoftheexternalgranulationpatch,filledindentedarrowheadsindicatethecuticularfold(pulvinus)ontheinternallegsurface,andemptyindentedarrowheadsindicategranulationontheinternallegsurface.Scalebarsinμm.

EuropeanJournalofTaxonomy452:1–36(2018)

ClawsY-shaped,ofthehufelanditype(Fig.5A–D).Primarybrancheswithdistinctaccessorypointsandwithanevidentstalkconnectingtheclawtothelunula(Fig.5A–D).Lunulaeunderallclawssmooth(Fig.5A–D).CuticularbarsunderclawsabsentbutmuscleattachmentsarevisibleunderclawsI–III(Fig.5A,C,filledarrowhead).

Fig.4.Macrobiotuscanaricussp.nov.,paratypes,buccalapparatusandtheoralcavityarmatureseenunderPCMandSEM.A.Dorsalprojectionoftheentirebuccalapparatus;thelowerinsertshowsaventralviewoftheoralcavityarmature,theupperinsertshowsmacroplacoidmorphology(PCM).B–C.TheoralcavityarmatureofasingleparatypeseenunderSEMfromdifferentangles.Filledarrowheadsindicatetheteethofthefirstband,emptyarrowheadsindicatetheteethofthesecondband,medianteetharemarkedwithM,andlateralteetharemarkedwithL.Scalebarsinμm.

STECD.etal.,TardigradesfromtheCanaryIslands

Eggs(measurementsandstatisticsinTable6)

Laidfreely,white,sphericalorslightlyoval(Figs6A–B,7A).Thesurfacebetweenprocessesofthehufelanditype,i.e.,coveredbyareticulumwithverythinwalls(Figs6D–E,7A–F).Peribasalmeshesslightlylargerandwithslightlythickerwallscomparedtointerbasalmeshes(Figs6D–E,7B–F).Themeshdiameterisalwayslargerthenmeshwallsandnodes/knots(Figs6D–E,7B–F).Themeshesare0.3–1.0μmindiameter,polygonalbutwithroundededges.UnderSEM,meshesdeepandemptyinside(Fig.7C–F).Processesintheshapeofinvertedgobletswithconcaveconicaltrunksandwell-definedterminaldiscs(Figs6C–F,7A–F).Terminaldiscsstronglyserrated,withaconcavecentralarea(Figs6C–F,7B–F).SparseultragranulationontheedgesofterminaldiscsvisibleonlyunderSEM(Fig.7E–F).Threetofivemicrogranules(0.25–0.30μmindiameter),coveredwithultragranulation,presentinthecentreoftheterminaldisc(visibleonlyunderSEM;Fig.7B–F,emptyarrowheads).

Reproductivemode

Theexaminedpopulationisdioecious(gonochoristic).Maleswereidentifiedusingaceto-orceinstaining,whichrevealedtesticlesfilledwithspermatozoa.However,nomorphologicalsecondarysexualdimorphism,suchasgibbositiesonhindlegsinmales,wasidentified.

Fig.5.Macrobiotuscanaricussp.nov.,paratypes,claws.A–B.ClawsIIIandIVseenunderPCM.C–D.ClawsIandIVseenunderSEM.Arrowheadsindicatefaintmuscleattachmentsunderclaws;FigsAandBassembledfromseveralphotos.Scalebarsinμm.

EuropeanJournalofTaxonomy452:1–36(2018)

Fig.6.Macrobiotuscanaricussp.nov.,eggseenunderPCM.A.Midsectionunder400×magnification.

B.Surfaceunder400×magnification.C.Midsectionunder1000×magnification.D–E.Surfaceunder1000×magnification.F.Outlinesofterminaldiscsofeggprocessesunder1000×magnification.Scalebarsinμm.

STECD.etal.,TardigradesfromtheCanaryIslands

Fig.7.Macrobiotuscanaricussp.nov.,eggchorionmorphologyseenunderSEM.A.Entireegg.

B.Closeupoftheeggsurfaceandprocesses.C–D.Detailsoftheeggsurface.E–F.Detailsoftheterminaldiscs.Arrowheadsindicategranulesinthecentreoftheterminaldiscs.Scalebarsinμm.

EuropeanJournalofTaxonomy452:1–36(2018)

Table6.Measurements(inµm)ofselectedmorphologicalstructuresoftheeggsofMacrobiotuscanaricussp.nov.mountedinHoyer’smedium.(N=numberofeggs/structuresmeasured;Range=thesmallestandthelargeststructureamongallmeasuredspecimens;SD=standarddeviation)

Character	N	Range	Mean	SD
Eggbarediameter	30	59.9–81.6	68.7	5.0
Eggfulldiameter	30	70.2–96.2	80.8	5.4
Processheight	90	3.9–8.2	5.5	1.0
Processbasewidth	90	3.3–7.5	5.3	0.8
Processbase/heightratio	90	60%–160%	99%	19%
Terminaldiscwidth	90	3.4–7.5	4.8	0.8
Inter-processdistance	90	1.5–5.5	3.4	0.8
Numberofprocessesontheeggcircumference	30	23–31	25.5	2.0

DNAsequences

Weobtainedsequencesforallfouroftheabove-mentionedmolecularmarkers.Thetwoconservativenuclearmarkers(18SrRNA,28SrRNA)wererepresentedbysinglehaplotypes,whereasITS-2andCOIexhibitedthreeandtwohaplotypes,respectively.Thep-geneticdistancebetweentheITS-2haplotypesrangedfrom0.5to1.1%andbetweenCOIhaplotypesitwasequalto1.3%.The18SrRNAsequence(GenBank:MH063925)was1033bplong.The28SrRNAsequence(GenBank:MH063934)was721bplong.TheITS-2haplotypes1–3were413bplong(GenBank:MH063928,MH063929andMH063930,respectively).TheCOIhaplotypes1–2were658bplong(GenBank:MH057765andMH057766,respectively).

Phenotypicdifferentialdiagnosis

Bytheoralcavityarmatureofthemaculatustypeandhufelanditypeofeggshellornamentation,smoothlunulesunderclawsofalllegsandgranulationatleastonlegsIV,thenewspeciesissimilartoM.almadaiFontouraetal.,2008,M.humilisBinda&Pilato,2001,andM.rawsoniHorningetal.,1978,butcanbedifferentiatedspecificallyfrom:

Macrobiotusalmadai,knownonlyfromtheAzores(Fontouraetal.2008),bythepresenceoftheexternalandtheinternalpatchofgranulationonlegsI–III(legsI–IIIsmoothinM.almadai)andbythepresenceofasinglelargeporeinthecentreoftheexternalpatchonlegsI–III(occasionally,regularcuticularporesmaybepresentonsomelegs,butsuchporesaresmallandneverpresentonalllegsinthesameplaceinM.almadai).

Macrobiotushumilis,reportedonlyfromitstypelocalityinSriLanka(Binda&Pilato2001),bythepresenceofthreeseparateddorsalteethofthethirdband(dorsalteethfusedintoasingletransversalridgeinM.humilis),thepresenceofasubterminalconstrictioninthesecondmacroplacoid(secondmacroplacoidwithoutconstrictionsinM.humilis),moreposteriorlyinsertedstyletsupports(pt=74.3–

76.8inthenewspeciesvspt=71.1–71.3inM.humilis),slightlyhigherptofthesecondmacroplacoidlength(pt=14.6–19.6inthenewspeciesvspt=12.5–14.4inM.humilis)andbyirregularlyserratededgesoftheterminaldiscsoneggprocesses(edgesofterminaldiscsregularlyindentedinM.humilis).

Macrobiotusrawsoni,knownonlyfromitstypelocalityinNewZealand(Horningetal.1978;Kaczmarek&Michalczyk2017a),bythepresenceofgranulationonalllegs(granulationpresentonlyonlegsIVinM.rawsoni),thepresenceofasubterminalconstrictioninthesecondmacroplacoid(secondmacroplacoidwithoutconstrictionsinM.rawsoni),theabsenceofcuticularbarsundertheclawson

16

STECD.etal.,TardigradesfromtheCanaryIslands

legsI–III(thinpairedbarspresentinM.rawsoni),moreanteriorlyinsertedstyletsupports(pt=74.3–

76.8inthenewspeciesvspt=77.0–77.1inM.rawsoni),adifferentmorphologyofreticulationontheeggsurfacebetweenprocesses(severallinesofmeshbetweenneighbouringeggprocessesinthenewspeciesvstwolinesofmeshbetweenneighbouringeggprocessesinM.rawsoni)andbyasmallermeshsizeinthechorionreticulum(0.3–1.0μmindiameterinthenewspeciesvs1.8–2.5μmindiameterin

M.rawsoni).

Genotypicdifferentialdiagnosis

Therangesofuncorrectedgeneticp-distancesbetweenthenewspeciesandspeciesoftheMacrobiotushufelandicomplex,forwhichsequencesareavailablefromGenBank,areasfollows:

18SrRNA:0.5–3.7%(2.0%onaverage),beingmostsimilartotwoundeterminedspeciesofthe

M.hufelandigroupfromSpain(FJ435738–9)andtoM.macrocalixfromPoland(MH063926)andtheleastsimilartoM.polypiformisRoszkowskaetal.,2017fromEcuador(KX810008)

28SrRNA:1.9–13.2%(6.1%onaverage),beingmostsimilartothreeundeterminedspeciesoftheM.hufelandicomplexfromSpain(FJ435751andFJ435754–5)andtheleastsimilarto

M.polypiformisfromEcuador(KX810009)

ITS-2:5.3–27.8%(17.0%onaverage),withthemostsimilarbeingM.macrocalixfromPoland(MH063931)andtheleastsimilarbeingM.polypiformisfromEcuador(KX810010)

COI:17.2–24.7%(19.2%onaverage),withthemostsimilarbeingM.hannaeNowak&Stec,2018fromPoland(MH057764)andtheleastsimilarbeingM.papeiStecetal.,2018fromTanzania(MH057763)

Macrobiotuscf.recensCuénot,1932Figs8–14,Tables7–8

Materialexamined(114animals,48eggs)

SPAIN:CanaryIslands,GranCanaria,PresadeLugarejos,28°02′38″N,15°40′22″W,885ma.s.l.,lichenonastonewall.SpecimensmountedonmicroscopeslidesinHoyer’smedium(87animals+38eggs),fixedonSEMstubs(8+10),processedforDNAsequencing(4animals)andusedforaceto-orceinstaining(15animals).Slidedepositories:87animals(slides:ES.006.*,wheretheasteriskcanbesubstitutedbythefollowingnumbers1–4,10–12,14–17)and38eggs(slides:ES.006.*:5–9,13)(IZiBB).

DescriptionofthepopulationfromGranCanariaAnimals(measurementsandstatisticsinTable7)

Bodywhiteinjuvenilesandslightlyyellowishinadults,afterfixationinHoyer’smediumtransparent

(Fig.8A).EyespresentinliveanimalsandinspecimensmountedinHoyer’smedium.Ellipticalandsometimesroundishpores(1.0–1.8μmindiameter),visibleunderPCMandSEM,scatteredrandomlyonentirebodycuticle(Fig.8B–E),includingtheexternalandinternalsurfaceofalllegs(Fig.9A–I).Insideporesseveralgranules,visibleonlyunderSEM,alwayspresent(Fig.8E).GranulationpatchonexternalandinternalsurfacesoflegsI–IIIpresent(Fig.9A–E).Singleporepresentatcentreofeachexternalgranulationpatch(Fig.9A–C).Granulationpatchonexternalsurfacelargerandmoredistinctthantheoneoninternalsurface(Fig.9A–E).FaintcuticularfoldpresentonexternalsurfaceoflegsI–IIIjustaboveclaws(Fig.9A–B,emptyarrowhead),whereasoninternalsurfaceoflegsI–IIIthereisacuticularbulgeresemblingpulvinus(Fig.9D–E,filledarrowhead).Bothexternalfoldandinternalbulgevisibleonlyiflegsarefullyextendedandcorrectlyorientedonslide(particularlycuticularfoldaboveclaws).GranulationonlegsIValwaysclearlyvisibleandconsistsoftwogranulationpatches:thedistal

17

EuropeanJournalofTaxonomy452:1–36(2018)

Table7.Measurements(inµm)ofselectedmorphologicalstructuresofindividualsofMacrobiotuscf.recensCuénot,1932fromtheCanaryIslandsmountedinHoyer’smedium.(N=numberofspecimens/structuresmeasured;Range=thesmallestandthelargeststructureamongallmeasuredspecimens;SD=standarddeviation)

Character N	Range µm pt	Mean µm pt	SD µm pt
BodylengthBuccopharyngealtube	30	353–731	684–1174	576	1042	66	90
Buccaltubelength	30	48.5–70.3	–	55.4	–	5.8	–
Styletsupportinsertionpoint	30	39.3–57.2	79.1–83.5	44.7	80.8	4.9	1.0
Buccaltubeexternalwidth	30	8.0–15.6	15.4–22.8	9.8	17.6	1.6	1.4
Buccaltubeinternalwidth	30	6.1–12.2	11.6–17.8	7.5	13.4	1.3	1.3
Ventrallaminalength	30	24.9–42.3	47.3–63.8	32.9	59.6	3.2	4.2
Placoidlengths
Macroplacoid1	30	9.4–19.4	18.1–30.5	13.3	24.0	2.3	2.6
Macroplacoid2	30	8.2–17.0	15.9–24.8	10.6	19.0	2.0	1.8
Microplacoid	30	3.1–8.4	5.8–12.3	4.7	8.5	1.1	1.3
Macroplacoidrow	30	12.5–39.1	24.1–57.1	25.4	45.7	5.1	6.2
PlacoidrowClaw1lengths	30	17.3–52.6	33.4–76.8	32.7	58.7	6.1	6.3
Externalprimarybranch	30	12.3–18.0	23.1–32.7	14.6	26.6	1.5	2.4
Externalsecondarybranch	29	9.6–14.6	17.9–23.8	11.7	21.1	1.2	1.6
Internalprimarybranch	30	12.0–17.0	22.7–29.1	14.1	25.6	1.2	1.9
Internalsecondarybranch	30	8.2–13.7	15.7–24.5	11.1	20.1	1.3	2.4
Claw2lengths
Externalprimarybranch	30	12.8–18.3	23.2–31.6	15.0	27.2	1.3	2.3
Externalsecondarybranch	30	8.0–14.6	16.5–26.4	11.7	21.2	1.5	2.5
Internalprimarybranch	30	12.0–20.5	22.2–39.7	14.7	26.6	1.8	3.3
Internalsecondarybranch	30	8.5–14.9	15.5–25.6	11.2	20.4	1.6	2.7
Claw3lengths
Externalprimarybranch	30	13.3–18.2	23.9–33.7	15.2	27.6	1.2	2.4
Externalsecondarybranch	30	8.9–16.2	17.2–26.1	11.7	21.2	1.5	2.0
Internalprimarybranch	30	11.1–17.5	20.4–32.7	14.5	26.3	1.4	2.5
Internalsecondarybranch	30	9.1–15.1	16.7–29.2	11.5	20.8	1.3	2.5
Claw4lengths
Anteriorprimarybranch	29	11.8–22.2	22.6–36.7	17.3	31.3	1.9	3.2
Anteriorsecondarybranch	28	11.1–17.2	20.6–29.0	13.1	23.7	1.5	2.0
Posteriorprimarybranch	27	14.5–23.4	28.0–38.9	18.1	32.7	1.6	2.5
Posteriorsecondarybranch	26	11.1–17.4	20.2–32.0	13.7	24.6	1.6	3.1

patchwithdenselydistributedgranulessituatedjustaboveclawsandtheproximalpatchbeingwiderwithmoresparselydistributedgranuleslocatedimmediatelyabovedistalpatch(Fig.9G–I).

Mouthantero-ventral.Bucco-pharyngealapparatusoftheMacrobiotustype(Fig.10A–C),withventrallaminaandtensmallperibuccallamellaefollowedbysixbuccalsensorylobes.UnderPCM,oralcavityarmatureofthehufelanditype,i.e.,withallthreebandsofteethalwaysvisible(Fig.10B–C).Firstbandofteethcomposedofnumerousverysmallconesarrangedinfourtosixrowssituatedanteriorlyinoralcavity,justbehindbasesofperibuccallamellae(Figs10B–C,11A–B,filledarrowhead).Secondbandofteethsituatedbetweenringfoldandthirdbandofteethandcomprises4–5rowsofsmallcones,slightlylargerthanthoseoffirstband(Figs10B–C,11A–B,emptyarrowhead).Teethofthethirdbandlocatedwithinposteriorportionoforalcavity,betweenthesecondbandofteethandbuccaltube

STECD.etal.,TardigradesfromtheCanaryIslands

opening(Figs10B–C,11A–B).Thirdbandofteethdiscontinuousanddividedintodorsalandventralportions.UnderPCM,dorsalteethseenasthreedistincttransversalridges,whereasventralteethappearastwoseparatelateraltransversalridgesandaroundishmediantooth(Fig.10B–C).UnderSEM,bothdorsalandventralteethclearlydistinct(Fig.11A–B).Medio-ventraltoothrarelydividedintotwoorthreesmallerteeth(Fig.11B).UnderSEM,marginsofdorsalandlatero-ventralteethslightlyserrated(Fig.11A–B).Pharyngealbulbspherical,withtriangularapophyses,tworod-shapedmacroplacoidsand

Fig.8.Macrobiotuscf.recensCuénot,1932,GranCanaria,habitusandcuticularpores.A.Dorso-ventralprojection(PCM).B–C.Poresonthedorsalandventralcuticle,respectively(PCM).D.Poresonthedorsalcuticle(SEM).E.Granulationinsideasinglepore(SEM).Scalebarsinμm.

EuropeanJournalofTaxonomy452:1–36(2018)

onebigrod-shapedmicroplacoid(Fig.10D–E).Macroplacoidlengthsequence2<1.Firstandsecondmacroplacoidwiththecentralandthesubterminalconstriction,respectively(Fig.10D–E).

ClawsYshaped,ofthehufelanditype(Fig.12A–D).Primarybrancheswithdistinctaccessorypointsandwithanevidentstalkconnectingclawtolunula(Fig.12A–D).LunulaeI–IIIsmooth(Fig.12A,C),whereaslunulaeIVslightlycrenulated(Fig.12B,D).FaintcuticularbarsunderclawsI–IIIpresent,morevisibleinlargerspecimens(Fig.12A,C).Horseshoe-shapedstructureconnectsanteriorandposteriorlunules(Fig.12B).

Fig.9.Macrobiotuscf.recensCuénot,1932,GranCanaria,structuresonforelegsseenunderPCMandunderSEM.A–C.ThepatchofgranulationontheexternalsurfaceoflegII,withthecuticularporeatthecentreofthepatchandthecuticularfoldabovetheclaws.D–F.Thepatchofgranulationandthecuticularbulge(pulvinus)ontheinternalsurfaceoflegIII.G–I.GranulationonlegIV.Filledflatarrowheadsindicatethelargeporeatthecentreoftheexternalgranulationpatch,emptyflatarrowheadsindicatethecuticularfoldabovetheclawsontheexternallegsurface,filledindentedarrowheadsindicatethecuticularfold(pulvinus)ontheinternallegsurface,andemptyindentedarrowheadsindicategranulationontheinternallegsurface.Scalebarsinμm.

STECD.etal.,TardigradesfromtheCanaryIslands

Eggs(measurementsandstatisticsinTable8)

Laidfreely,white/lightyellow,sphericalorslightlyoval(Figs13A–B,14A).Surfacebetweenprocessesofthehufelanditype,i.e.,chorionsurfacebetweenprocessescoveredbyreticulumwithverysmallmeshes(Figs13C–D,14B–E).UnderPCM,surfacebetweenprocessesseemstobecoveredbydarkdots(Fig.13C)andonlysometimesaclearreticulationisvisible(Fig.13D).Severalrowsofmeshesbetweeneggprocesses(usually7–8).Meshbordersandnodes/knotsthickandsometimeswiderthanmeshdiameter(Fig.14B–E).Meshescircularorslightlyoval(0.4–0.9μmindiameter)andunderSEM

Fig.10.Macrobiotuscf.recensCuénot,1932,GranCanaria,buccalapparatusandtheoralcavityarmatureseenunderPCM.A.Dorsalprojectionoftheentirebuccalapparatus.B–C.Dorsalandventralviewoftheoralcavityarmature,respectively.D–E.Placoidmorphology.Scalebarsinμm.

EuropeanJournalofTaxonomy452:1–36(2018)

Table8.Measurements(inµm)ofselectedmorphologicalstructuresoftheeggsofMacrobiotuscf.recensCuénot,1932fromtheCanaryIslandsmountedinHoyer’smedium.(N=numberofeggs/structuresmeasured;Range=thesmallestandthelargeststructureamongallmeasuredspecimens;SD=standarddeviation)

Character	N	Range	Mean	SD
Eggbarediameter	30	86.1–113.7	102.1	8.0
Eggfulldiameter	30	113.6–151.4	137.6	9.3
Processheight	90	13.4–26.3	20.0	2.7
Processbasewidth	90	3.2–10.7	6.3	1.4
Processbase/heightratio	90	16%–45%	32%	6%
Inter-processdistance	90	3.7–74.0	6.6	7.3
Numberofprocessesontheeggcircumference	30	21–27	24.6	1.5

allporesemptyinside(Fig.14C–D).Meshdiameterdecreasesgraduallyfromperibasaltointerbasalmeshes(Fig.14C–D).ShortthickeningsradiatingfromprocessbasesareoftenvisibleunderPCMandalwaysvisibleunderSEM;thickeningsmaycreateacrownaroundprocessbases(Figs13C,14C–D,filledarrowheads).Processesintheshapeofhighandthinconeswithstraightconicaltrunks,devoidofterminaldiscsandsometimesbifurcated(Figs13A–F,14A–F).Trunksofprocessesslightlyundulated.Undulationscoveredbynumerousgranulescomposedofmicrogranuleaggregations.UndulationsandgranulespoorlyvisibleunderPCM(Fig.13C–F).Undulations,granulesandmicrogranulesalwaysclearlyvisibleunderSEM(Fig.14B,E–F).

Reproductivemode

Theexaminedpopulationisdioecious(gonochoristic).Maleswereidentifiedusingaceto-orceinstaining,whichrevealedtesticlesfilledwithspermatozoa.However,nomorphologicalsecondarysexualdimorphism,suchasgibbositiesonhindlegsinmales,wasidentified.

Fig.11.Macrobiotuscf.recensCuénot,1932,GranCanaria,oralcavityarmatureseenunderSEMfromdifferentangles.A.Viewofthedorsalportion.B.Viewoftheventralportion.Filledarrowheadsindicatetheteethofthefirstband,emptyarrowheadsindicatetheteethofthesecondband,medianteetharemarkedwithM,andlateralteetharemarkedwithL.Scalebarsinμm.

STECD.etal.,TardigradesfromtheCanaryIslands

DNAsequences

Weobtainedsequencesforallfouroftheabove-mentionedmolecularmarkers.Thetwoconservativenuclearmarkers(18SrRNA,28SrRNA)wererepresentedbysinglehaplotypes,whereasbothITS-2andCOIexhibitedtwohaplotypes.Thep-geneticdistancebetweentheITS-2aswellasbetweentheCOIhaplotypeswas1.1%.The18SrRNAsequence(GenBank:MH063927)was1033bplong,the28SrRNAsequence(GenBank:MH063936)was725bplong,theITS-2haplotype1and2sequences(GenBank:MH063932andMH063933,respectively)were420bplong;theCOIhaplotype1and2sequences(GenBank:MH057768andMH057769,respectively)were658bplong.

Genotypicdifferentialdiagnosis

Therangesofuncorrectedgeneticp-distancesbetweenthenewspeciesandspeciesoftheMacrobiotushufelandicomplex,forwhichsequencesareavailablefromGenBank,areasfollows:

Fig.12.Macrobiotuscf.recensCuénot,1932,GranCanaria,claws.A–B.ClawsIIIandIVseenunderPCM,withsmoothandweaklycrenulatedlunules,respectively.C–D.ClawsIIandIVseenunderSEM,withsmoothandweaklycrenulatedlunules,respectively.AandBassembledfromseveralphotos.Filledarrowheadsindicatefaintcuticularbarsunderclawswhereasemptyarrowheadindicatesahorseshoe-shapedstructure.Scalebarsinμm.

EuropeanJournalofTaxonomy452:1–36(2018)

Fig.13.Macrobiotuscf.recensCuénot,1932,GranCanaria,eggseenunderPCM.A.Midsectionunder400×magnification.B.Surfaceunder400×magnification.C–D.Surfaceunder1000×magnification.E–F.Midsectionunder1000×magnification.Filledarrowheadsindicateshortthickeningsradiatingfromtheprocessesbases.Scalebarsinμm.

STECD.etal.,TardigradesfromtheCanaryIslands

Fig.14.Macrobiotuscf.recensCuénot,1932,GranCanaria,eggchorionmorphologyseenunderSEM.A.Entireegg.B.Closeupoftheeggsurfaceandprocesses.C–D.Detailsoftheeggsurface.E–F.Detailsofprocessmorphology.Filledarrowheadsindicateshortthickeningsradiatingfromtheprocessbases.Scalebarsinμm.

EuropeanJournalofTaxonomy452:1–36(2018)

18SrRNA:0.4–4.0%(2.0%onaverage),withthemostsimilarbeingM.macrocalixfromPoland(MH063926)andtheleastsimilarbeingM.polypiformisfromEcuador(KX810008)

28SrRNA:1.3–13.6%(6.5%onaverage),withthemostsimilarbeingM.macrocalixfromPoland(MH063935)andtheleastsimilarbeingM.polypiformisfromEcuador(KX810009)

ITS-2:3.7–26.7%(16.9%onaverage),withthemostsimilarbeingM.macrocalixfromPoland(MH063931)andtheleastsimilarbeingM.polypiformisfromEcuador(KX810010)

COI:16.4–24.7%(19.1%onaverage),withthemostsimilarbeingM.macrocalixfromItaly(FJ176203–7andFJ176213–7)andtheleastsimilarbeingM.papeifromTanzania(MH057763)

Molecularphylogeny

Phylogeneticanalysesconductedonmacrobiotid18SrRNAsequencesaswellasontheconcatenatedmacrobiotiddatasetunambiguouslyconfirmedthatthetwostudiedspeciesrepresenttheM.hufelandigroup(Figs15–16).ThephylogenybasedontheCOIsequencesofthehufelandigroupalsocorroboratedtheseresults,sincenoneofthetwospecieswererecoveredexternaltothespeciesofthehufelandigroup(Fig.17).Inallanalyses,twocladeswithinthehufelandigroupwerepresent,althoughthespeciescompositionvariedslightlybetweenphylogeniesbasedondifferentmarkers.Onecladegroupedexclusivelyspeciesthatexhibitmodifiedeggprocesses(M.paulinaeStecetal.,2015,M.polypiformis,

M.papei,M.shonaicusStecetal.,2018a,M.scoticusStecetal.,2017bandM.kristenseniGuidetti,Peluffo,Rocha,Cesari&MolydePeluffo,2013);‘thekristenseniclade’henceforth.Theothercladecomprisedmostlyspecieswithtypicalinvertedgoblet-shapedeggprocesses(‘thehufelandiclade’hereafter).Incontrasttoourpredictions,M.cf.recens,withitsatypicaleggprocesses,wasalwaysembeddedwithinthehufelandiclade.ThetwocladeswerewellsupportedinphylogeniesbasedontheconcatenateddatasetandonCOIsequences,butweaklysupportedinthe18SrRNAtree(Figs15–17).Moreover,inthe18SrRNAanalysisthekristenseniclade,inadditiontothemajorityofspecieswithmodifiedeggprocesses,comprisedM.sapiensBinda&Pilato,1984(DQ839601)andundeterminedspeciesoftheM.hufelandigroup(HQ604971),ofwhichatleastthefirstspeciesexhibitsthetypicaleggmorphology.Incontrasttootheranalyses,the18SrRNAphylogenyrecoveredaclade,with

X.pseudohufelandi(Iharos,1966)andM.polonicusPilato,Kaczmarek,Michalczyk&Lisi,2003,thatwasinasisterrelationshiptoallotherspeciesofthehufelandigroup,suggestingthatthehufelandigroupispolyphyleticorthatXerobiotusbelongstothehufelandigroup(Fig.15).

Discussion

Thankstothedetailedmorphologicalandmolecularanalyses,wewereabletodescribeanewspecies

M.canaricussp.nov.andcharacteriseapopulationofM.cf.recens,bothcollectedontheCanaryIslands.Moreover,bytheuseofphylogeneticinference,weconfirmedtheaffinityofbothstudiedspecieswiththeM.hufelandigroup.

Macrobiotusrecenswasdescribedin1932byLucienCuénot,butsoonafter,someresearchersstartedquestioningthestatusofthisspeciesandclassifieditas‘M.hufelandiformarecens’(Marcus1936;Ramazzotti1945,1962,1972;Rudescu1946;Grigaricketal.1973).However,Maucci(1979)analysedseveralPortuguesepopulationsheidentifiedasM.recensmorphologicallyandconfirmedthatthetaxonisindeeda‘bonaspecies’.However,itshouldbenotedthatthelocustypicusforM.recensisLaTardière,acommuneintheVendéedepartment,inthePaysdelaLoireregioninwesternFrance.Thus,similarlytoourstudy,thecomparisonpresentedbyMaucci(1979)hastobetreatedwithcaution,asitmaybebasedonarelatedspeciesratherthanM.recenss.str.Sincetheoriginaldescriptionisoutdatedandincomplete,aconfidentidentificationofM.recensiscurrentlynotpossible.AlthoughPilato&Bertolani(2004)supportedtheidentificationofthePortuguesepopulationsstudiedbyMaucci(1979)asM.recens,wethinkthatwithoutanintegrativeredescriptionofthespeciesitisnotpossibletotestwhetherM.recensisasinglespeciesoracomplexofmorphologicallysimilartaxa.Thus,insteadofdesignatingtheCanary

26

STECD.etal.,TardigradesfromtheCanaryIslands

IslandspopulationasM.recens,wetreatitasM.cf.recens,atthesametimehighlightingtheneedforaredescriptionbasedonnewmaterialfromthelocustypicus.ItcouldbehypothesisedthatourCanarianpopulation,aswellaspreviousrecordsofM.recensoutsidethetypelocality,i.e.,fromRomania(Rudescu1946),Italy(Ramazzotti1945),Greece(Marcus1936),Portugal(Maucci1979)andChina(Rahm1937;Bartoš1963),mayrepresentdistincttaxathatcouldbecomeidentifiableonlyafteramodernredescriptionofM.recens.Thiswouldnotbesurprising,assuchapatternhasbeenobservedbefore,forexample,whennominaltaxainothergroupsweredescribed:Macrobiotushufelandi(redescribedbyBertolani&Rebecchi1993andBertolanietal.2011a),Milnesiumtardigradum(redescribedbyMichalczyketal.2012),Mesocristaspitsbergensis(redescribedbyGąsioreketal.2016),Hypsibiusdujardini(redescribedbyGąsioreketal.2018)orRamazzottiusoberhaeuseri(redescribedbyStecetal.2018c).Thus,inour

0.92

M.cf.recens(MH063927)

1.00

0.86	M.macrocalix(HQ604976)

M.canaricus(MH063925)

1.00	M.aff.hufelandi(FJ435740)

Macrobiotushufelandigroup M.aff.hufelandi(FJ435739)

M.aff.hufelandi(FJ435738)

M.hannae(MH063922)

M.hannae[”M.joannae”](HQ604975)

M.hannae[”M.joannae”](HQ604974)

0.86

0.82

M.scoticus(KY797265)

Macrobiotidae M.paulinae(KT935502)

1.00

0.65

M.papei(MH063881)

M.shonaicus(MG757132)

M.kristenseni(KC193577)

1.00

1.00	M.aff.hufelandi(HQ604971)

M.sapiens(DQ839601)

M.polypiformis(KX810008)

M.polonicus(HM187580)

1.00	X.pseudohufelandi(HQ604989)

X.pseudohufelandi(HQ604990)

1.00

1.00

0.89

0.66

0.97

1.00	1.00

1.00

Paramacrobiotusspp.

T.voronkovi(KX810045)

M.furcatus(HQ604977–8,FJ435746–7)

Minibiotusspp.

Mesobiotusspp.

1.00

Milnesiumvariefidum(KT951664)

Milnesiumberladnicorum(KT951660)

0.02

Fig.15.TheBayesianInference(BI)phylogenyconstructedfrom18SrRNAsequencesofthefamilyMacrobiotidae.NumbersatnodesindicatetheBayesianposteriorprobability.Speciesofthehufelandigroupwithtypicalandatypicaleggprocessesareindicatedbyblueandredfonts,respectively.The

X.pseudohufelandi+M.polonicusclade,whichisinasisterrelationshiptoallotherspeciesofthehufelandigroup,isindicatedbygreenbranches.SeeTable2andthePhylogeneticanalysissubsectioninMaterialandmethodsfordetailsonspeciessequencesusedintheanalysis.Theremainingmacrobiotidsaremarkedwithblackandtheoutgroupismarkedwithgrey.Scalebarrepresentssubstitutionsperposition.

27

EuropeanJournalofTaxonomy452:1–36(2018)

opinion,allrecordsofM.recensoutsideitstypelocalityshouldbeconsideredasuncertainidentificationsuntilaredescriptionofM.recensisavailableandatleastsomeofthereportscanbeverified.Followingthislogic,iftherecordofM.cf.recensfromtheCanaryIslandsturnsouttorepresentM.recenss.str.,thenthisstudywillbeanadditiontoourpoorknowledgeoftheintraspecificvariabilityintardigrades.If,however,thecurrentrecordrepresentsanewspecies,then,withthedataalreadypresentedinourstudy,onlyanewnamewillhavetobeproposedtoerectanewspecies.

Recently,Stecetal.(2018a)showedtwowellsupportedCOIlineageswithintheM.hufelandigroup:onecladegroupingspecieswithhufelandi-typeeggprocessesintheshapeofinvertedgoblets,andtheotherwithmodifiedeggprocess(conicalprocessesorprocesseswithfilamentsgrowingoutofterminaldiscs).Thus,M.cf.recens,withitsconicaleggprocesses,shouldbeexpectedtoclusterwithspecieswithmodifiedeggprocesses.However,incontrasttothisprediction,M.cf.recensisembeddedwithinthecladewithspeciesexhibitingtypicaleggmorphologyinourCOIanalysis(Fig.17).Moreover,the

1.00

1.00

MacrobiotushannaeMacrobiotusmacrocalix

1.00

1.00

Macrobiotuscf.recens

1.00

1.00

Macrobiotuscanaricus

Macrobiotuspapei

0.611.00

Macrobiotidae Macrobiotusshonaicus

1.00

1.00

1.00

Macrobiotuspolypiformis

1.00

Macrobiotushufelandigroup Macrobiotuspaulinae

1.00

0.96

1.00

Macrobiotusscoticus

ParamacrobiotuslachowskaeMesobiotusinsanisMesobiotusethiopicus

Mesobiotusphilippinicus

1.00

MilnesiumvariefidumMilnesiumberladnicorum

0.5

Fig.16.TheBayesianInference(BI)phylogenyconstructedfromconcatenatedsequences(18SrRNA

+28SrRNA+ITS-2+COI)ofthefamilyMacrobiotidae.NumbersatnodesindicatetheBayesianposteriorprobability.Speciesofthehufelandigroupwithtypicalandatypicaleggprocessesareindicatedbyblueandredfonts,respectively.SeeTable2andthePhylogeneticanalysissubsectioninMaterialandmethodsfordetailsonthespeciessequencesusedintheanalysis.Theremainingmacrobiotidsaremarkedwithblackandtheoutgroupismarkedwithgrey.Thescalebarrepresentssubstitutionsperposition.

STECD.etal.,TardigradesfromtheCanaryIslands

samepositionofthespecieswasrecoveredinthe18SrRNAphylogeny(Fig.15)andintheanalysisbasedonfourconcatenatedmarkers(Fig.16).Also,M.sapiens,withtypicaleggprocesses,shouldbepresentinthehufelandiclade,butinouranalysesitclusteredwithspeciesexhibitingmostlymodifiedprocesses(Fig.15).Importantly,however,the18SrRNAsequenceidentifiedasM.sapiens(DQ839601;Schill&Steinbrück2007)comesfromCroatia,whereasthetypelocalityofthespeciesisinSicily(Binda&Pilato1984).Thus,giventhattherearenotype/neotypesequencesforM.sapiensandSchill&Steinbrück(2007)didnotprovideSEMphotomicrographsofCroatianeggs,itisnotpossibletoverifytheidentityoftheDQ839601sequence.Inotherwords,thesequencemayrepresentasimilarspeciesofthehufelandigroupthatmayexhibitterminaldiscfilaments.Nevertheless,regardlessofthephylogeneticpositionofM.sapiens,theaffinityofM.cf.recenswithspeciesexhibitingtypicaleggprocessesshowsthatthemorphologicalcriterionproposedbyStecetal.(2018a)todistinguishthetwocladesisnot

0.67

1.00

0.98

0.93

Macrobiotuscf.hufelandi

Macrobiotushufelandis.s.

0.60

0.61

1.00

1.00

Macrobiotushannae

Macrobiotuscanaricus

Macrobiotuscf.recens

Macrobiotusmacrocalix

0.99

0.87

0.50

1.00

Macrobiotusvladimiri

0.50

Macrobiotussandrae

Macrobiotusterminalis(JN673960)

Macrobiotusterminalis(AJ598775)

1.00

0.67

1.00

1.00

1.00

MacrobiotusshonaicusMacrobiotuspolypiformis

0.59

1.00

0.94

Macrobiotuspaulinae

MacrobiotuspapeiMacrobiotusscoticus

1.00

1.00

0.55

0.99

Macrobiotuskristenseni

MesobiotushilariaeMesobiotusphilippinicus

Mesobiotusinsanis

Mesobiotusethiopicus

0.2

Fig.17.TheBayesianInference(BI)phylogenyconstructedfromCOIsequencesofthespeciesoftheMacrobiotushufelandigroup.NumbersatnodesindicatetheBayesianposteriorprobability.Speciesofthehufelandigroupwithtypicalandatypicaleggprocessesareindicatedbyblueandredfonts,respectively.SeeTable2fordetailsonthespeciessequencesusedintheanalysis.Theoutgroupismarkedwithgrey.Thescalebarrepresentssubstitutionsperposition.

EuropeanJournalofTaxonomy452:1–36(2018)

universal.Therefore,morespeciesrepresentingthetwocladesneedtobesequencedtoelucidatethetaxonomicstatusofthetwoclades.

AnotherspecieswhosephylogeneticpositiondivergesfromthepredictedisM.polonicus.Thisspecies,withtypicaleggprocesses,shouldbeembeddedwithinthehufelandiclade.However,inthe18SrRNAphylogeny,notonlydoesitnotclusterwithinthehufelandiclade,butitclusterswithXerobiotuspseudohufelandiinacladethatisinasisterrelationshiptotheentirehufelandigroup.TheM.polonicus+

X.pseudohufelandicladewasalsofoundtobeasistergrouptoallotherhufelandigroupspeciesbyBertolanietal.(2014).ThegenusXerobiotusBertolani&Biserov,1996sharesasimilarmorphologyofthebuccalapparatus,eggshellandspermatozoawithspeciesoftheM.hufelandigroup,butitdiffersfromthembyhavingstronglyreducedclaws.GiventhattheM.polonicus+X.pseudohufelandicladecontainsonlyasinglehufelandigroupsequence,thecladecouldbeastatisticalartefactorpossiblythe18SrRNAmarkeristooconservativetosolvetherelationshipsbetweenthehufelandigroupandthegenusXerobiotus.Therefore,itiscrucialtoanalysemorepopulationsofM.polonicus,otherspeciesofthepersimilissubgroup(sensuKaczmarek&Michalczyk2017b)andspeciesofthegenusXerobiotus,bothintermsofthe18SrRNAmarkerandadditional,morevariableDNAfragments.If,however,morespeciesofthehufelandigroupturnouttoclusterwiththegenusXerobiotus,thenthetaxonomicstatusofthegenusandofthehufelandigroupshouldbereconsidered.

Althoughourmultilocusphylogenysupportsthepresenceoftwodistinctevolutionarylineageswithinthehufelandigroup,thetaxonsamplesizeisstillverylowandmorepreciseconclusionsconcerningthephylogenyofthegroupcannotcurrentlybemade.Thus,toprovidemorereliableconclusions,muchmoreeffortshouldbemadetoobtainmultilocusmoleculardatalinkedtomorphologyforalargernumberofspeciesandpopulations.Particularly,itwouldbebeneficialtoincreasethesamplesize,bothintermsofmarkersaswellasspecies,fortheM.polonicus+X.pseudohufelandicladetotestwhetherthegenusXerobiotusandthehufelandigrouparemonophyletic.Anothercladewhichwouldbenefitfromanincreasedsamplesizeisthecladethatcomprisesspecieswithmodifiedeggprocesses.Currently,thecladeconsistsofspecieswithtwoverydifferentprocessmorphotypes,i.e.,specieshavingprocesseswithflexiblefilamentsontheterminaldiscsandspecieshavingconicalprocessesdevoidofterminaldiscs.Thus,itmightbepossiblethatincreasedsamplingcouldrevealthepresenceoftwofurthercladesthatdifferbytheeggprocessmorphology.Nevertheless,ourfindingssupportthepreviousresultsthateggmorphologyseemstobeevolvingfasterthananimalmorphology,whichunderlinestheusefulnessofchorionornamentationinthedelineationofcloselyrelatedspecies(Guidettietal.2013;Stecetal.2016a,2017a,2018a).

Acknowledgments

WearegratefultoMartaKapałaforcollectingthesamplesontheCanaryIslands.WewouldalsoliketothankProfessorPauloFontoura(UniversityofPorto,Portugal)forlendingusslideswithM.almadaiandtoMatteoVecchi(UniversityinModena,Italy),whokindlysentusthephotosofspecimensandeggsfromPortugalidentifiedbyMaucci(1979)asM.recensanddepositedintheMauccicollection.WearealsogratefultoMatteoVecchiandananonymousreviewer,whosecommentshelpedtoimproveourwork.WewouldliketothanktheAquaticEcosystemsTeamfromtheInstituteofEnvironmentalSciences(JagiellonianUniversity,Kraków)forprovidinguswiththerotifers.ThestudywassupportedbytheLinneanSocietyofLondonandtheSystematicsAssociation(agrantfromtheSystematicsResearchFundawardedtoDS)andbytheJagiellonianUniversity(subsidyno.K/ZDS/007357toŁM).

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EuropeanJournalofTaxonomy452:1–36(2018)

Manuscriptreceived:10April2018

Manuscriptaccepted:25June2018

Publishedon:24July2018Topiceditor:RudyJocqué

Deskeditor:KristiaanHoedemakers

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