J\Jycologia, 92(4),2000, pp. 728-735. 728-735. 92(4), 2000, pp. Mycologia, © 2000 by The The Mycological Mycological Society of America, Lawrence. KS KS 66044-8897 66044-8897 © 2000 by America,Lawrence, Societyof The Physdaceae in in Fennoscandia: Fennoscandia: phylogeny phylogeny inferred inferred from from The family familyPhysciaceae ITS ITS sequences sequences Key Words: Ascomycetes, Lecanorales, Lecanorales, lichenized lichenized Words: Ascomycetes, Key fungi fungi Katileena Lohtander' Lohtander 1 Katileena SEBotaniska institutionen, institutionen, Stockholms universitet, SEBotaniska Stockholms universitet, 106 91 Stockholm, Stockholm, Sweden Sweden 106 91 Mari Mari Killersj6 Källersjö INTRODUCTION INTRODUCTION Naturhistoriska riksmuseet, Molekylärsystematiska Naturhistoriska riksmuseet, Molekyldrsystematiska laboratoriet, Box 50007, SE-l04 05 Box 05 Stockholm, laboratoriet, Stockholm, 50007, SE-104 Sweden Sweden Roland Moberg Roland Moberg Botaniksektionen, Evolutionsmuseet, Evolutionsmuseet, Uppsala Botaniksektionen, Uppsala universitet, Norlryvägen 16, SE-752 36 36 Uppsala, universitet, 16, SE-752 Uppsala, Norbyvdgen Sweden Sweden Anders Tehler Anders Tehler Naturhistoriska Sektionen Naturhistoriska riksmuseet, riksmuseet, Sektionen för for Box 05 Stockholm, kryptogambotanik, Box 50007, SE-104 05 50007, SE-104 Stockholm, kryptogambotanik, Sweden Sweden internal transcribed transcribedspacers Abstract: The The internai Abstract: (ITS1 spacers (ITSl and ITS2), ITS2), and and the the nuclear nuclear riboriboand the 5.8S 5.8S region of the region of DNA were for 52 somal DNA were sequenced somal rep52 specimens specimens repsequenced for of the the lichen resenting 35, mostly foliose, taxa of family lichen family foliose,taxa 35, mostly resenting The sequences Physciaceae (Lecanorales) were phyphy(Lecanorales).. The sequences were Physciaceae using parsimony parsimony jackknifing. jackknifing. logenetically analyzed analyzed using logenetically Xanthoria and X. were chosen chosen as Xanthoria parietina parietina and X. sorediata sorediata were as Because of of the the variable natureof of the outgroups. variable nature the ITS ITS outgroups.Because regions parsimony jackknifing jackknifing analyses we re perperanalyses were regions parsimony formed on on different different alignments. The analyses analyses resultresultformed alignments.The ed in trees trees with ed in with several several shared, shared, well-supported well-supported A the trees groups. A consensus tree, summarizing trees consensus all the tree, groups. summarizingall In that from alignments was also calculated. In that was also calculated. fromdifferent different alignments formed a tree the family Physciaceae formed a monophyletic tree the familyPhysciaceae monophyletic Some of of the of Physciaceae group. Some the crustose crustose species Physciaceae group. species of the genera (in the genera Amandinea, Amandinea, Buellia Buellia and and Dimelaena) Dimelaena) (in of the sistergroups thefamily. appeared as as two two sister to the the rest rest of groupsto family. appeared In this latter group Physcia, and In this latter Physcia, Heterodermia Heterodermia speciosa speciosa and constituted sistergroup Mobergia calculiformis constituted the the sister group to to Mobergia calculiformis which included the the remaining the which included the genera genera AnAnremainingfamily, family, aptychia, Hyperphyscia, Phaeorrhiza, Phaeophyscia, aptychia, Hyperphyscia, Phaeorrhiza, was Physconia, and and Rinodina. Rinodina. The The genus Phaeophyscia was genus Phaeophyscia Physconia, found to found to be be monophyletic monophyletic with with 100% An100% support. support. Anwith Phaeorrhiza and Physconia, aptychia Physconia, together Phaeorrhiza together with aptychia and formed its sister nimbosa and and Rinodina turfacea formed nimbosa Rinodina turfacea its sister was found found in triHyperphyscia adglutinata was in aa trigroup. Hyperphyscia other two twogroups. chotomy with the the other groups. chotomywith forpublication Accepted for publicationJanuary 2000. 31, 2000. Accepted January31, 1 Email: Kati.Lohtander@botan.slI.se Kati.Lohtander@botan.su.se lEmail: The The family Physciaceae (Ascomycetes, Lecanorales) familyPhysciaceae (Ascomycetes,Lecanorales) about 30 30 genera. genera. has aa worldwide has with about worldwidedistribution distributionwith Som e of fruticose and foliose lichens lichens [e.g., Some of them are fruticose and foliose them are [e.g., Anaptychia K6rb., Körb., Physcia Physcia (Schreb.) PhaeoMichx., Phaeo(Schreb.) Michx., Anaptychia Physconia Poelt], Poelt], while others physcia Moberg and Physconia while others physcia Moberg and or placoid are crustose crustose or placoid [e.g., Buellia De De Not., Not., RinoRinoare [e.g., Buellia primarily charactercharacterdina (Ach.) dina The family is primarily (Ach.) Gray]. Gray].The familyis ised ascus and and spore structure.These ised by by ascus These characters, characters, spore structure. together with pycnoconidial characters, secondary with characters, secondary together pycnoconidial chemistry and cortical structures, have been used to and cortical have been used to structures, chemistry Severalmorphoseparate genera within within Physciaceae. Physciaceae. Several morphoseparate genera studiesin logical studies in Physciaceae have been been carried carried out out Physciaceaehave logical (Kurokawa Poelt 1965, 1974, Moberg Moberg 1977, 1962, Poelt (Kurokawa 1962, 1977, 1965, 1974, and Ruef Scheidegger Ruef 1988, et al al 1996, 1988, Mayrhofer 1996, Mayrhoferet Scheidegger and Matzer and and Mayrhofer and additional studies Matzer Mayrhofer 1996), additional studies 1996), and have evaluated evaluated the some morphological have the utility utility of of some morphological for taxonomical characters for taxonomical purposes characters purposes (Mayrhofer (Mayrhofer et al 1982, Ramboldt et al 1994, Nordin 1997). According 1982, Ramboldt 1994, Nordin 1997). According to Stenroos and DePriest SSU rDNA data sugto Stenroos and DePriest (1998) (1998) SSU rDNA data sugex Humb.) that Physcia Physcia aipolia Furnr. gests that (Ehrh. ex Humb.) Fiirnr. gests aipolia (Ehrh. and Santessonia and namibensis Hale Hale & & Vobis, both bebeSantessonianamibensis Vobis, both sistergroup longing to to Physciaceae, Physciaceae, are are the the sister group to to aa spespelonging cies of of the the genus cies Leprocaulon Nyl. Ny!. within within Lecanorales. Lecanorales. genus Leprocaulon This group, This group, in in turn, forms aa sister to TelosTelossistergroup turn,forms group to chistales. and DePriest chistales. However, Stenroos and DePriest (1998) However, Stenroos (1998) used of Physciaceae. used only two species Physciaceae. Previously no only two species of Previouslyno of the comprehensive phylogenetic analysis of the family family comprehensivephylogeneticanalysis out. Physciaceae has been been carried carried out. Physciaceaehas This is the first firstattempt the phylogeny This is the attempt to to examine exarnine the phylogeny of the cladisticmethmethof the family by applying applying cladistic familyPhysciaceae Physciaceaeby ods to DNA sequence data. We our study ods to DNA We restricted restricted our sequence data. study the to Fennoscandia. Our to reconstruct to Fennoscandia. Our go al was was to reconstruct the goal of the the family to find phylogeny of and to find out out whether whether the the familyand phylogeny the Physciaceae are monophyletic. genera within the monophyletic. Our Our Physciaceaeare generawithin of determining researchhad the secondary research had the purpose of determining secondarypurpose the putative whether whether the putative species pairs suggested by Poelt Poelt species pairs suggestedby and Moberg (1970) Moberg (1977) form monophyletmonophyletwould form (1970) and (1977) would ic in the Furtherwe to find find ic units units in the analyses. analyses. Further we wanted wanted to for our appropriate outgroups for our subsequent species appropriate outgroups subsequent species studies(Lohtander et al and we we wanted pair studies al unpubl) unpubl) and wanted (Lohtander et pair to determine the placoid and and crustose crustosespeto determine whether whether the spe- 728 728 This content downloaded from 130.238.82.159 on Thu, 2 Oct 2014 09:17:08 AM All use subject to JSTOR Terms and Conditions LOHTANDER ET ET AL: AL: PHYSCIACEAE PHYSCIACEAE IN LOHTANDER IN FENNOSCANDIA FENNOSCANDIA cies cies within within Physciaceae Physciaceae would fall fall into one one or or several monophyletic groups. METHODS MATERIALS AND METHODS MATERIALS AND Voucher are deposited deposited at at Voucherspecimens.-Specimens examined are specimens.-Specimensexamined the Swedish Museum of Natural History History (S) or in in Uppsala Uppsala the of Natural SwedishMuseum (S) or = Nor(UPS). D == Denmark, F F == Finland, lI == Iceland, Iceland, N N = Nor(UPS). D S == Sweden. numbersare are given in parentheway, given in parentheGenBank numbers Sweden. GenBank way,S ses. Amandinea Amandineapunctata ses. punctata (Hoffm.) (Hoffm.) Coppins & Scheidegger: Coppins & Scheidegger: 5346 (UPS) S, Upl, Uppsala, Uppsala, Nordin Ncrrdin 5346 S, Upl, (UPS) (AF224353). (AF224353). AnaptyAnaptyK6rb.: S: S: Upl, 498 (S) chia ciliaris(L.) chia ciliaris Upl, Tullgarn, Tullgarn, Lohtander Lohtander 498 (S) (L.) Körb.: 301 (S) Upl, Velamsund, (AF224366); Lohtander 301 Velamsund, Lohtander (S) (AF224366); Upl, runcinata (With.) R. Laundon: Laundon: (AF224353). Anaptychia Anaptychia runcinata (AF224353). J. R. (With.)J. D, Brnh, Brnh, Erteholmerne, Wedin 557 557 (UPS) Erteholmerne,Wedin D, (UPS) (AF224364). (AF224364). Th. Fr.: S: Gtl, Tehler Buellia alboatra Fr.: S: Buellia alboatra (Hoffm.) Sundre, Tehler Gtl, Sundre, (Hoffm.) Th. 5345 (UPS) 8043 Upl, Uppsala, Uppsala, Nordin Nordin 5345 (UPS) 8043 (S) (S) (AF224350); (AF224350); Upl, S6derBuellia disciformis (Fr.) Mudd: S, His, Söder(AF224351). Buellia S, Hls, (Fr.) Mudd: (AF224351). disciformis hamn, Agren Ågren 436 436 (UPS) (AF224349). (AF224349). Dimelaena Dimelaena crreina oreina hamn, Norman: S, Nordin 4800 (UPS) 4800 (UPS) S, Jmt, Jmt, Berg, (Ach.) Norman: Berg, Nordin (AF224352). Heterodermia Trevis: Heterodermia speciosa (WulfeninJacq.) inJacq.) Trevis: speciosa(Wulfen (AF224352). 8950 (UPS) S, Gstr, Hille, Hille, Moberg (AF224360). Hyperphyscia Hyperphyscia S, Gstr, (UPS) (AF224360). Moberg8950 & Poelt: Poelt: S, adglutinata (Flörke) H. H. Mayrhofer Mayrhofer & Gtl, Visby, Visby, S, Gtl, adglutinata (Florke) Moberg 12045 (UPS) (AF224361). Mobergia calculiformis calculifcrrmis 12045 (UPS) (AF224361). Mobergia Moberg (W. Weber) H. H. Mayrhofer & Sheard: Baja CaliforCaliforSheard: Mexico, Mexico, Baja (W. Weber) Mayrhofer& Manuela, Moberg Moberg 10422 (AF224359). nia, Laguna 10422 (UPS) nia, (UPS) (AF224359). Laguna Manuela, Tehler7904 7904 Phaeophyscia ciliata (Hoffm.) Moberg: ciliata (Hoffm.) S, Visby, Moberg: S, Visby,Tehler Phaeophyscia 7892a (S) Tehler 7892a Uppland, Väddö, (S) Vaddd, Tehler (AF224354); Uppland, (S) (AF224354); Mob& Nyl.) (Norr!. & Ny!.) Mob(AF224448). Phaeophyscia (AF224448). constipata(Norrl. Phaeophysciaconstipata s.n. (UPS) 1998 Lö/gren Vsm, Arboga Arboga 1998 (AF224374). erg: S: Vsm, (UPS) (AF224374). erg: S: Lofgrens.n. I: Sudur-Mulasysendococcina(Körb.) Phaeophyscia endococcina Moberg: l: (Korb.) Moberg: Sudur-MulasysPhaeophyscia 4824 (UPS) Ncrrdin 4824 N: Fnm, Fnm, la, Egilsstadir, Egilsstadir, Nordin la, (AF224444); N: (UPS) (AF224444); 408 (S) Alta, Lohtander Lohtander & Jalonen 408 (AF224358). PhaeophysPhaeophysAlta, (S) (AF224358). &Jalonen S: Halland, cia cia endophoenica (Harm.) Moberg: Halland, Abild, Abild, HjuHjuMoberg: S: endophoenica(Harm.) Nordin 4408 4408 (UPS) (AF224445). Phaeophyscia nigrinigrileberg, Nordin (UPS) (AF224445). Phaeophyscia leberg, 12046 S: Upl, cans (Flörke) Moberg: Uppsala, Moberg Moberg 12046 cans (Flo6rke) Moberg: S: Upl, Uppsala, (UPS) (AF224375). Phaeophyscia crrbicularis (Neck.) (Neck.) MobMob(UPS) (AF224375). Phaeophysciaorbicularis & Jalonen 325 (S) F: Ta, Karjalohja, Lohtander Lohtander & erg: F: Ta, Karjalohja, (S) Jalonen 325 erg: 7893a (S) Tehler 7893a Väddö, Tehler (AF224355); S: Uppland, (S) (AF224355); S: Uppland, Vadd6, sciastra (Ach.) F: Sa, (AF224356). Phaeophyscia Phaeophyscia sciastra Moberg: F: Sa, (Ach.) Moberg: (AF224356). S: Vsm, Ars.n. (S) 1996 Myllys Myllys s.n. Savitaipale, Vsm, Ar(AF224357); S: (S) (AF224357); Savitaipale, 1996 1998 Lofgren s.n. (UPS) Lö/gren s.n. (AF224446). Phaeorrhiza Phaeorrhiza boga, 1998 (UPS) (AF224446). boga, Sudur& Poelt: (Fr.) H. H. Mayrhofer Mayrhofer & Poelt: I, l, Nordur-I, Nordur-l, Sudurnimbosa (Fr.) nimbosa 4916a (UPS) (UPS) (AF224363). (AF224363). Physcia Physcia Thingeyrarsysla, Ncrrdin Nordin4916a Thingeyrarsysla, 1995 Myllys adscendens F: Nyl, Nyl, Siuntio, s.n. (Fr.) H. H. Olivier: Olivier: F: adscendens(Fr.) Siuntio,1995 Myllyss.n. Lohtander 381 (S) (AF224422); Lk, Sodankylä, Lohtander & Jalonen 381 Lk, (S) (AF224422); Sodankyla, &Jalonen ex Humb.) (S) (AF224423). (Enrh. ex Humb.) Ffirnr.: Fiirnr.: (S) (AF224423). Physcia Physciaaipolia aipolia (Enrh. 314 (S) Lohtander&Jalonen F: F: Nyl, Nyl, Emasalo, Emäsalo, Lohtander & Jalonen 314 (S) (AF224390); (AF224390); Tehler7894 S: Upl, S: Upl, Vadd6, Väddö, Tehler 7894 cc (S) Physcia aipolia (AF224398). Physcia (S) (AF224398). aipolia F: Ks, var. var. alnophila Lynge: F: Ks, Ruka, Lohtander & JaRuka, Lohtander &Jaalnophila (Vain.) (Vain.) Lynge: Lohtander& lonen380 380 (S) (S) (AF224382); Jalonen lonen Savukoski,Lohtander (AF224382); Savukoski, &Jalonen Tehler7894 399 (S) S: Upl, 7894 aa (S) 399 (S) (AF224381); Upl, Vadd6, Väddö, Tehler (S) (AF224381); S: F: Nyl, Ffirnr.:F: caesia (Hoffm.) (Hoffm.) Fiirnr.: Nyl, HelsinHelsin(AF224383). Physcia (AF224383). Physciacaesia 346 (S) S: Upl, Lohtander346 (S) (AF224389); (AF224389); S: Upl, Uppsala, Uppsala, Moberg ki, ki, Lohtander Moberg ornata dimidiata var. 12019 (UPS) 12019 var. crrnata (UPS) (AF224384). (AF224384). Physcia Physcia dimidiata (Nädv.) Moberg: N: Sogn Fjordarna, Laerdal, Moberg Laerdal, Moberg (Nadv.) Sogn og Fjordarna, Moberg: N: dubia Lettau:N: 6663 (UPS) 6663 (UPS) (AF224414). bia (Hoffm.) (Hoffm.) Lettau: N: (AF224414). Physcia Physciadu 401 Lohtander& Fnm, & Jalonen 401 (S) (AF224401); Fnm, Skibottn, Skibottn,Lohtander (S) (AF224401); Jalonen 12016 (UPS) S: Upl, S: Upl, Uppsala, Uppsala, Moberg (UPS) (AF224412). (AF224412). Physcia Physcia Moberg12016 729 Gtl, Östergarn, Moberg 12031 (UPS) leptalea (Ach.) DC.: DC.: S: S: Gtl, 12031 (UPS) Ostergarn,Moberg leptalea(Ach.) \F224428); 12034 (UPS) (AF224429). Physcia magnussonii 12034 (UPS) kF224428); (AF224429). Physciamagnussonii Jmt, Undersåker, Lundstedt s. n. (UPS) Frey: S: S: Jmt, 1998 Lundstedt s.n. Undersaker, 1998 (UPS) Frey: Ny!.: S: Äng, Ullanger, Ullånger, (AF224376). Physcia stellaris stellaris(L.) S: Ang, (L.) Nyl.: (AF224376). Physcia & Moberg 12009 Mo, Lohtander 12009 (UPS) Lohtander& (UPS) (AF22441O); (AF224410); Äng, Ang, Mo, Moberg 453 (S) (AF224406). Physcia Physcia tenella tenella (Scop.) (Scop.) DC.: DC.: F: Jalonen 453 F: (S) (AF224406). Jalonen 311 (S) Nyl, Sipoo, Lohtander & Jalonen 311 (S) (AF224425); Siun(AF224425); SiunSipoo, Lohtander Nyl, &Jalonen Myllys s.n. s.n. (S) (S) (AF224427). (AF224427). Physcia Physcia tenellavar. tenellavar. mamatio, 1995 1995 Myllys tio, rina Ny!.) Lynge: Lynge: S: Öregrund, Tehler 8050 8050 rina (E. Tehler S: Uppland, (E. Nyl.) Uppland, Oregrund, (S) (AF224426); Uppland, Väddö, Tehler Tehler 8057 8057 (S) (S) (AF224426); (S) Uppland, Vadd6, F: Nyl, (AF224424). Nyl, Sipoo, Physconia detersa detersa (Nyl.) (Ny!.) Poelt: Poelt: F: (AF224424). Physconia Sipoo, Lohtander 306 (S) Lohtander & Jalonen 306 (S) (AF224372). (AF224372). Physconia distorPhysconiadistor&Jalonen ta Laundon: F: ta (With.) J. R. R. Laundon: F: Nyl, Nyl, Sipoo, Lohtander & Jalo(With.) J. Sipoo, Lohtander &Jalo(AF224373); S: Moberg 12036 nen 313 (S) S: Gtl, 12036 nen 313 Gtl, Östergarn, (S) (AF224373); Ostergarn,Moberg (UPS) (Ny!.) Poelt: F: enteroxantha F: (UPS) (AF224371).Physconia (Nyl.) Poelt: (AF224371).Physconiaenteroxantha 302 Nyl, Sipoo Lohtander & Jalonen 302 (S) (AF224370). PhysLohtander (S) (AF224370). Nyl,Sipoo Phys&Jalonen conia grisea S: Gtl, 12031 Moberg 12031 (Lam.) Poelt: Poelt: S: conia grisea (Lam.) Gtl,Östergarn, Ostergarn,Moberg (UPS) (AF224368). Physconia Physconia muscigena (Ach.) Poelt: S: Poelt: S: (UPS) (AF224368). muscigena(Ach.) Gtl, (UPS) (AF224369). (AF224369). Physconia Physconia perper12044 (UPS) Gtl,Tofta, Tofta,Moberg Moberg12044 & isidiosa (Erichsen) Moberg: Moberg: F: Ta, Tammela, Lohtander & F: Ta, isidiosa (Erichsen) Tammela, Lohtander 600 (S) Jalonen 600 (S) (AF224367). Rinodina turfacea turfacea (Wahlenb.) (Wahlenb.) (AF224367). Rinodina Jalonen 10422 (UPS) Nordingrå, Moberg Moberg 10422 Körb.: K6rb.: S, S, Ång, (UPS) Ang, Nordingra, Th. Fr.: Th. Fr.: S, Velam(AF224362). Xanthoria parietina (L.) Xanthoria S, Upl, (L.) (AF224362). parietina Upl, Velams.n. (S) soresund, Lohtander s.n. (S) (AF224347). Xanthoria sore1999 Lohtander sund, 1999 (AF224347). Xanthoria diata (Vain.) (Vain.) Poelt: Poelt: S, Uppland, Vadd6, Väddö, Tehler 7883 (S) (S) Tehler7883 diata S, Uppland, (AF224348) . (AF224348). of the Fennoscandian For this study we sequenced most of the Fennoscandian For this studywe sequenced most with som some species treated treated by Moberg (1977) e exceptions: (1977) with by Moberg exceptions: species Moberg and phaea Phaeophyscia kairamoi kairamoi (Vain.) and Physcia (Vain.) Moberg Physciaphaea Phaeophyscia because of of fresh Thoms. (Tuck. ) Thoms. are missing be cause of lack of fresh matelack mateare (Tuck.) missing some rial. Other species studiedwere species studied were Heterodermia Heterodermia speciosa, speciosa, some ria!. Other crustose crustose lichens lichens belonging belonging to to Physciaceae Physciaceae (from (from the the genera genera Amandinea, Buellia, Phaeorrhiza and and Rinodina), Rinodina) , Amandinea, Buellia,Dimelaena, Dimelaena,Phaeorrhiza All Mexico.All and and Mobergia Mobergia calculiformis calculifcrrmis from from Baja California, California,Mexico. fromDenmark, the other specimens have been been collected collected from the other Denmark, specimens have and Sweden. Finland, Norway and Xanthoria parietina parietina Sweden. Xanthoria Finland, lceland, Iceland, Norway as outgroup and X. scrrediata sorediatawere chosen as and X. were chosen outgroup specimens. specimens. DNA internal transcribed DNA techniques.-The transcribed spacers ITSl spacers ITS1 techniques.-The internai rDNA of DNA and and the 5.8S rDNA nuclear ribosomal and ITS2 ITS2 and the 5.8S of the the nuclear ribosomal DNA forthe the analyses. The ITS ITS region been were region has has been were sequenced analyses.The sequenced for of lichens, studies of successfully used in in several lichens, at severalprevious at successfullyused previousstudies and Wei level (Niu Goffinetand species and genus genus level Wei 1993, and 1993, Goffinet (Niu and species and et al al 1999a, al 1998a, Lohtander et et al Bayer 1997, b, Myllys Myllys et 1999a, 1997, Lohtander 1998a, b, Bayer Thell 1998), weil as as at at higher higher taxonomic taxonomic level Thell as well level (Berbee (Berbee 1998), as et al al 1999b), in the al 1995, latter et Luzoni 1997, et al Myllys et the latter 1995, Luzoni 1997, Myllys 1999b), in case in combination combinationwith small subunit subunit (SSU) withthe case usually usually in the small (SSU) rDNA. rDNA. DNA extractedfrom collectionsand and herDNA was was extracted from both both fresh fresh collections herThe QlAamp Tissue Kit barium barium material material using using The Kit from from QiaQiaQIAamp Tissue The oldest fromwhich oldestspecimen whichDNA DNA was was successfully gen. The successfully gen. specimenfrom extractedwas 1980. For For extraction extracted was from from 1980. extraction we we used used small small fragfragor apothecia. The procedure ments ments of of thalli, thalli, or apothecia. The procedure was was performed performed the manufacturers' instructionswith folaccording to the manufacturers' instructions with the the folaccording to The tissue tissuewas was macerated withaa minimacerated with minilowing modifications. modifications. The lowing in 50 the liquid pL of of buffer pestle in 50 fLL buffer AT, AT, omitting nitrogen omittingthe liquid nitrogen pestle 130 fLL of buffer AT and and 20 pL bufferAT 20 fLL phase. After Mter maceration, maceration, 130 iLL of phase. K were and the incubatedin in Proteinase was incubated Proteinase K were added, the sample added, and sample was C overnight. DNA was 55-60 C extractedDNA was eluted eluted in 55-60 The extracted in 40 40 fLL ILL overnight.The This content downloaded from 130.238.82.159 on Thu, 2 Oct 2014 09:17:08 AM All use subject to JSTOR Terms and Conditions 730 730 MVCOLOGIA MYCOLOGIA of elution buffer AE. AE. Before Before PCR PCR the DNA was was further further didithe DNA of elution buffer luted times with with distilled 10 times distilledH luted 10 2 0. H20. Pharmacia Biotech Biotech PCR-reactions were performed using using Pharmacia PCR-reactions were performed Ine. Ready Ready To To Go Go PCR beads, 1l.5 H 0, 1l.5 [LL DNA 11.5 DNA PCR beads, 11.5 [LL Inc. F4L HO2, 2 p^L dilution and 1 l [LL primers ITS1-F ITS1-F (Gardes (Gardes and and 10 [LM of 10 dilution and F&Mprimers FLLof Bruns ITS4 (White (White et al 1990), in aa 30-cycle et al reBruns 1993) and ITS4 1990), in 1993) and 30-cyclereof 60 60 ss at at 95 C (denaturation), (denaturation ), action with with aa PCR PCR profile profile of action 95 C C (annealing), (annealing), and 60 ss at All 60 ss at at 72 C (extension). 60 at 60 60 C and 60 72 C (extension).All PCR products were were purified purified with with the the QIAquick PCR PuriPuriPCR products QIAquick PCR and eluted eluted with 40 [LL dHzO. fication Kit Kit of of Qiagen, pLLdH2O. fication with40 Qiagen, and Both strands re sequenced. labelled (Cy5) Both strandswe Fluorescentlabelled were sequenced. Fluorescent (Cy5) primers used used for for sequencing (White et et al al 1990), were ITS5 ITS5 (White 1990), primers sequencingwere et al b) and and ITS1-LM et ITS2-KL (Lohtander al 1998a, ITS1-LM (Myllys ITS2-KL 1998a, b) (Lohtanderet (Myllyset al 1999a). The sequencing al re actions were produced using reactions were 1999a). The sequencing produced using Amersham's in aa reacAmersham's ThermoSequenase ThermoSequenase Sequencing Kit, in reacSequencing Kit, min at C (l tion with 95 C tion with aa profile profile of of 22 min at 95 by followedby (1 cycle), cycle),followed 30 ss at 30 ss at 50 C, min at at 95 95 C, at 50 and 1 l min at 72 C (30 30 72 C C, 30 C, and (30 cycles). cycles). The sample run on The s were we re run on aa 6% Long Ranger in aa PharPhar6% Long samples gel in Rangergel macia automatic macia Biotech Biotech Inc. Ine. ALFExpress automatic sequencer. ALFExpress sequencer. Sequences we re aligned aligned using using the the ClustalW (Thompson ClustalW (Thompson Sequences were et al Since different et al 1994) program. Since different alignment 1994) alignment alignmentprogram. alignment parameter settings may produce produce different different alignments alignments and and parameter settingsmay affect the tree tree topology, topology, we we chose affectthe chose an an approach described approach described by Farris Farris et al (1999) (1999) also used by by Myllys et al al (I 999b ). et al also used (1999b). by Myllyset Several alternative we re analyzed with parsimoparsimoSeveral alternative alignments alignmentswere analyzedwith order to that are are shared shared jackknifing in in order to identity ny jackknifing identifygroups ny groups that in every tree. tree. The following combinations re performed performed in every The following combinationswe were extensionpenalty): (gap opening penalty/gap penalty/gap extension penalty): 1/1,2/4,1/ 1/1, 2/4, 1/ (gap opening 5,3/5,6/3,5/5,8/6,5/7,7/5,10/1,10/5,9/7,5/10,13/ 5, 3/5, 6/3, 5/5, 8/6, 5/7, 7/5, 10/1, 10/5, 9/7, 5/10, 13/ Each align5, and 30/10. 30/10. Each align5, 15/5, 15/5, 10/8, 10/8, 10/10,20/10,25/10, 10/10, 20/10, 25/10, and ment estimatedby was estimated ment was by eye eye and and those those that that appeared as clearclearappeared as to initiate ly unrealistic unrealistic (the alignment program program failed failed to initiate the the (the alignment ly same site the beginning of the the ITS1 alignment from from the the same site at at the beginning of ITSl alignment A consensus were omitted omittedin region) were in the the analyses. analyses. A consensus of of all all the the region) trees as Farriset resulting trees was constituted, as suggested by Farris et al was al constituted, resulting suggestedby withgap (1999). alignment with gap opening penalty 9.0 9.0 (1999). Sequence Sequence alignment opening penalty and gap 7.0 is is deposited TreeBASE and gap extension extension penalty penalty 7.0 in TreeBASE deposited in (S478, M700). M700). (S478, Phylogenetic analysis.-The analysis.-The data data were were analyzed by parsimoparsimoPhylogenetic analyzedby et al jackknifing (Farris (Farris et al 1996) the computer prony jackknifing 1996) using ny using the computerproS. Farris and discussed discussedin gram Xac (designed by J. S. Farris and in Killersjo Källersjö gramXac (designedbyJ. et al 1000 replicates et al 1998), with the the following following settings: replicates were were 1998), with settings:1000 and 55 random performed with with branch-swapping branch-swapping and random addition addition performed each. In sequences In parsimony parsimony jackknifing jackknifing (Farris et al al 1996) (Farriset sequences each. 1996) so that the that approx the original original data data set set is is resampled approx 63% 63% of of resampled so the and without are chosen the characters chara.cters are chosen randomly randomly and without replacereplacement for are calculated ment for parsimony parsimony analysis. analysis. Trees Trees are calculated for for each each replicate and combined combined into into jackknife jackknife trees trees showing only replicate and showingonly in at groups that that are are present present in at least least 50% of the the replicates. replicates. 50% of groups One of of the the benefits of using is its its speed One benefits of Xac is to using Xac speed compared compared to other uninformative do other programs. programs. Furthermore, uninformative characters characters do Furthermore, in parsimony not affect values in not affect support parsimony jackknifing jackknifing as as they supportvalues they do were treated do in in bootstrapping bootstrapping (Carpenter treated 1996). Gaps (Carpenter 1996). Gaps were fifthcharacter as aa fifth characterstate stateor or coded coded as as missing as missing data. data. RESULTS RESULTS The ITS sequences The ITS obtained were we re approx approx 500 500 bp, sequences obtained some exceptions. Heterodermia speciosa with with some exceptions. Heterodermia speciosa had had aa to the slightly ITS region region (477 bp) bp) compared compared to the shorterITS slightlyshorter other The sequences of Anaptychia Anaptychia ciliaris ciliaris otherspecimens. specimens.The sequences of varied 532-563 bp. The The aligned aligned data data matrix matrixranged varied 532-563 bp. ranged from 584 penalty 10; 584 (alignment with gap from 10; (alignmentwith gap opening opening penalty 679 gap extension penalty 10) to 679 sites (alignment extension to sites penalty gap (alignment with gap opening opening penalty penal ty 1.0; gap extension penalty extensionpenalty withgap 1.0; gap 1.0). The number of informative characters varied The number of informative characters varied 1.0). from' 274 274 to to 302 gaps were were treated treated as as character character from when gaps 302 when states, being approx 45% of of all The numnumall characters. characters.The states,being approx 45% ber in alignments 33 (alignment ber of of gaps gaps in alignments varied varied from from 33 (alignment with gap gap opening opening penalty penalty 15.0; gap extension penalty with extensionpenalty 15.0; gap 79 (alignment 5.0) to 79 with gap gap opening penalty 1.0; 1.0; 5.0) to (alignmentwith opening penalty extensionpenalty gap extension penalty 1.0). gap 1.0). above 15.0 15.0 resulted resultedin Gap penalties above in clearclearGap opening opening penalties unrealisticalignments and such such alignments ly unrealistic alignments and alignments were were ly abandoned. Seventeen Seventeen alignments abandoned. alignments were were left left to to be be in the used in In addition used the analyses. analyses. In addition to the consensus to the consensus tree we also present tree we also present the the tree best resotree that thathad had the the best resolution (the in the lution number of of nodes nodes in the tree) tree) as well as as as well (the number the best best support (the number of nodes with support the number of nodes with support (the support of or more of 95% 95% or more in in the the tree). alignment with with gap The alignment tree). The gap 9.0 and opening penalty 9.0 and gap extension penalty penalty 7.0 7.0 opening penalty gap extension resulted in the the best best resolved resolved tree (with 37 groups) resulted in tree (with 37 groups) and and received received the the best best support values (16 nodes nodes with with supportvalues aa support or more) in the of 95% 95% or more) in the Xac Xac analysis (FIG. supportof analysis(FI(G. The consensus consensus tree in FIG. FIG. 2. l). The tree is is presented presented in 2. 1). The of different influence of The influence different alignment alignment parameter parameter low. Conflict Conflictbetween settings in the the trees trees was was rather rather low. between settingsin the on the the different the trees trees based based on different alignments was seen seen alignmentswas in the the Buellia Otherwisethe the only in Buellia group group (FIGS. (FIGS. 1, l, 2). 2). Otherwise only 17 trees uniform.The topologies of all all 17 rather uniform. The treeswere were rather topologies of main difference differencewas of resolution. resolution.In main was in in the the degree degree of In all trees the the family formed a all trees Physciaceae formed a monophyfamilyPhysciaceae monophyletic group with letic with 100% l, 2). 2). 100% support (FIGS. (FIGS. 1, The influenceof the gaps the phylogenetic The influence of the gaps on on the phylogenetic rereconstruction not drastic drasticeither. construction was was not either. Alignment with with Alignment and gap extensionpenalty gap opening penalty 9.0 9.0 and gap extension penalty gap opening penalty 7.0 an almost almostidentical identicaltree 7.0 gave gave an tree regardless of whethregardlessofwhether or not not the characterstates. er or the gaps gaps where where used used as as character states.The The resolution was somewhat somewhatlower when the the resolution was lower (29 groups) when (29 groups) The other information information provided provided by by gaps gaps was was omitted. omitted. The other sixteentrees sixteen trees based based on on different alignments had more different more alignmentshad in degree deviationin of resolution deviation degree of between the trees resolutionbetween the trees where were treated treatedas statesand and the the where gaps gaps were as character character states were coded coded as trees trees where where gaps as missing data. No No concongaps were missingdata. to be flict these trees, the flict was was to be observed observed between between these trees, but but the latter much latter ones ones (with gaps as as missing we re much (with gaps data) were missingdata) more the trees more poorly poorly resolved resolved compared compared to to the trees with with as character states. gaps character states. gaps as Buellia all trees the base bas e of of all trees two two Buellia Buellia group. At At the Buellia group. alboatra formed aa sister sisterpair with 100% alboatra specimens pair with 100% specimens formed the parameter support l, 2). 2). When When the parameter settings in (FIGS. 1, support (FIGS. settingsin the exthe alignments were low low (gap (gap opening opening and and gap alignmentswere gap extension alboatraformed tension penalties penalties under under 3.0), B. alboatra formed aa 3.0), B. the rest sister group the family the analysis. family in analysis. sister group to to the rest of of the in the This content downloaded from 130.238.82.159 on Thu, 2 Oct 2014 09:17:08 AM All use subject to JSTOR Terms and Conditions 731 731 LOHTANDER LOHTANDER ET ET AL: AL: PHYSCIACEAE PHYSCIACEAE IN IN FENNOSCANDlA FENNOSCANDIA . - - - - - - - - - - - - - - - - - - - - - - - - - Xanthoria Xanthoria parietina parietina t - - - - - - - - - - - - - - - - - - - - - - Xanthoria Xanthoria sorediata sorediata ra II g' Buellia alboat alboatra 100 - Buellia Buellia alboatra Buellia alboat ra l ｾ＠ 95 Amandinea Amandineapunctata a I tO punctata Buellia Buellia disciformis disciformis l 3 991c Dimelaena 77Dimelaena oreina oreina I-I-§ .--------------------He t erodermia speciosa Heterodermia speciosa I I . - - - - - - - - - - - - - - - - - - Mobergia 77 calculiformis 77 Mobergia calculiformis I . - - - - - - - - - - - - - - - - - Physcia magnussonii Physcia magnussonii I .-----------------Physcia var. ornatal dimidiata var. ornata Physcia dimidiata I - - - - - - - - - - - - - - P h y sPhyscia c i a dubia 67 dubia 67 II II t - - - - - - - - - - - - - - P h y sPhyscia c i a dubia dubia I 100 100 t - - - - - - - - - - - - - - P h y sPhyscia c i a stellaris stellaris 98 98 I - - - - - - - - - - - - - - P h y sPhyscia c i a stellaris stellaris Physcia aipolia var. alnophila Physcia aipolia var. alnophilaI ""O 100 I Physcia aipolia var. alnophila var. alnophila ｾ＠ IL.1 Physcia aipolia Physcia 100 var. alnophila 95 100 aipolia var. alnophilaI. ｾ＠ Physcia aipolia iii' .------- Physcia caesia caesia 62 62 Physcia tO .., L - _ 1 - - - - Physcia caesia I oc:: Physcia caesia 96 61 Physcia "O var. aipolia -§ aipolia var. aipolia Physcia aipolia Physcia var. aipolia Physcia aipolia aipolia var. aipolia ...--------Physcia tenella tenella var. var. tenella Physcia tenella I-------Physcia t enella var. tenella var. tenella tenella Physcia 83 83 t-------Physcia tenella var. var. marina marina Physcia tenella 9::!,3:L_ _- t - - - - - - - Physcia t enella var. marina L _ _93 tenella var. marina Physcia Physcia adscendens adscendens I 559 1Physcia Physcia adscendens Physcia adscendens Physcia alea 100 100 I leptalea Physcialept Physcia lept alea alea Physcia lept ...---------Phaeophyscia nigricans Phaeophyscia nigricans .-------Phaeophyscia constipata l Phaeophyscia constipata 100 58 100 .----'52..!8L-t---Phaeophyscia endophoenica I Phaeophysciaendophoenica Phaeophyscia endococcina endococcina l ｾ＠ 57 |-Phaeophyscia Phaeophyscia endococcina endococcina II ｾ＠ Phaeophyscia 7'8 78 .---------Phaeophyscia Phaeophyscia sciastOra {l -g. sciastra I '< 78 t------Phaeophyscia sciastra I ｾ＠ Phaeophyscia sciastra 90 Phaeophyscia ciliata l iii' 85 Phaeophyscia ciliata ｾＹｪＭｐｨ｡･ｯｰｹｳ｣ｩ＠ 85 ciliata I Phaeophyscia ciliata 99 90 1 Phaeophyscia Phaeophyscia orbicularis orbicularis l Phaeophyscia orbicularis orbicularis I Phaeophyscia . . . - - - - - - - - - - - - - Hyperphyscia Hyperphyscia adglutinata adglut inata Rnodina turfacea t urfacea I 8- 78 . . . - - - - - - - - - - - - - RFinodina II Phaeorrhiza nimbosa .--------------Phaeorrhiza nimbosa I 96 Anapt hycia ciliaris ciliaris I 100 1-Anapthycia 60 60 ' ciliaris o Anapthycia ciliaris 1::3l Anapthycia ...---------Anapthycia runeinata I ＠ ｾ runcinata I< 62 Anapthycia 62 ...--------Physconia grisea l o Physconia grisea perisidiosa II iii' Physconia perisidiosa .74 .---------Physconia Q .--------Physconia muscigena I tO Physconia muscigena 77 co -_ _ Physconia enteroxantha ent eroxant ha I -§ Physconia 97 97 a I Physconia distort distorta l Physconia Physconia distorta distorta I { 99 Physconia ! Physconia detersa detersa 741 Physconia o § o FIG. FIG. 1. L Jackknife jackknife tree tree based based on on ITS ITS sequence sequence data data (alignment (alignment with with gap gap opening opening penalty penalty 9.0 9.0 and and gap gap extension extension penalty penalty 7.0). The The numbers numbers given given at at each each node node indicate indicate the the jackknife jackknife support. support. 7.0). This content downloaded from 130.238.82.159 on Thu, 2 Oct 2014 09:17:08 AM All use subject to JSTOR Terms and Conditions MVCOLOGIA MYCOLOGIA 732 732 Xant horia pari et ina Xanthoria parietina Xant horia sorediata Xanthoria sorediata Buellia alboat ra ra Buellia alboat Buellia alboat ra Buellia alboatra Amandinea punctata Amandinea punctata Buellia discifo rmis Buellia disciformis Dimelaena oreina Dimelaenaoreina Mobergia rmis calculiformis Mobergia calculifo Het erodermia speciosa Heterodermia speciosa Physcia magnussonii Physcia magnussonii Physcia dimidiat a var. var. ornata ornata Physciadimidiata Physcia dubia Physciadubia Physcia dubia Physciadubia Physcia stellaris Physcia stellaris stellaris Physcia Physcia stellaris var. alnophila Physcia aipolia var. alnophila Physciaaipolia Physcia aipolia var. alnophila aipolia var. alnophila 'Physcia Physcia aipolia var. alnophila alnophila aipolia var. *Physcia caesia Physcia Physcia caesia caesia Physcia Physcia caesia var. aipolia Physcia aipolia aipolia var. aipolia *Physcia Physcia var. aipolia aipolia var. aipolia Physciaaipolia tenella var. tenella Physcia var. tenella Physciatenella Physcia enella var. tenella var. tenella Physciattenella Physcia tenella var. var. marina marina Physciatenella Physcia t eneiia var. marina tenella var. marina Physcia adscendens Physcia Physciaadscendens adscendens Physcia Physciaadscendens alea Physcia lept leptalea 'Physcia alea Physcia leptalea Physcia lept Phaeophyscia Phaeophyscianigricans nigricans Phaeophyscia ipata constipata Phaeophysciaconst Phaeophyscia endophoenica Phaeophysciaendophoenica endococcina Phaeophyscia Phaeophysciaendococcina endococcina Phaeophyscia Phaeophysciaendococcina ra sciastra Phaeophyscia Phaeophysciasciast ra Phaeophyscia sciast ra Phaeophysciasciast ciliata Phaeophyscia a Phaeophysciaciliat Phaeophyscia ciliata Phaeophysciaciliata orbicularis Phaeophyscia orbicularis -Phaeophyscia Phaeophyscia orbicularis orbicularis -Phaeophyscia inataa Hyperphyscia adglut adglutinat Hyperphyscia Rnodinatturfacea Rnodina urfacea nimbosa Phaeorrhizanimbosa Phaeorrhiza ciliaris Anapt hycia -Anapt hycia ciliaris ciliaris Anapt hycia ciliaris *Anapthycia runcinata Anapthycia Anapthyciaruncinata Physconia Physconiagrisea grisea Physconia perisidiosa Physconia perisidiosa Physconia muscigena Physconiamuscigena Physconia ent eroxant ha Physconiaenteroxantha distorta Physconia Physconiadistorta -Physconia distorta Physconiadistorta -Physconia detersa det Physconia ersa r- L- '- -----cc '- r--I..-. ｾ＠ L---c L-c '- '--- L FIG. 2. FIG. differentalignments. of seventeen tree of A strict consensus tree seventeen jackknife jackknife trees trees based based on on different aiignments. A strict consensus This content downloaded from 130.238.82.159 on Thu, 2 Oct 2014 09:17:08 AM All use subject to JSTOR Terms and Conditions LOHTANDER ET AL: PHYSCIACEAE PHYSCIACEAE IN IN FENNOSCANDIA FENNOSCANDIA ET AL: LOHTANDER containOtherwise B. B. alboatra alboatra grouped group ed with with aa clade clade containOtherwise and ing Amandinea Amandinea punctata, punctata, B. B. disciformis and Dimelaena Dimelaena disciformis ing oreina Together they they formed group sistergroup formedaa sister oreina (FIc. (FIG. 1). 1). Together in all to the of the the family family with with 83-99% all the rest rest of to 83-99% support supportin The resolution within the Amandinea-Buelliatrees. The the Amandinea-Buelliaresolutionwithin trees. fromone one tree to another. varied from tree to another. Dimelaena-clade varied Dimelaena-clade Physcia group. The The next next clade clade (FIGS. (Frcs. 1, 2) concon1, 2) Physcia group. tained the genus Physcia, and and the the species Heterodertained the species Heterodergenus Physcia, Mobergia calculiformis (the Physcia mia speciosa speciosa and and Mobergia mia (the Physcia calculiformis group) forming the sister Phaeophyscia (the to Phaeophyscia sistergroup (the group to formingthe group) Phaeophyscia group) the genera Anaptychia, HyHyand the genera Anaptychia, group) and Phaeophyscia and Rinodina (the perphyscia, Phaeorrhiza, Physconia and Rinodina Phaeorrhiza, (the Physconia perphyscia, Physconia group) Physconia group).. The Physcia either either formed formedaa monophyletic The genus monophyletic genus Physcia group together with Heterodermia speciosa and and MobMobwith Heterodermia group together speciosa or formed a polytomy with ergia calculiformis (FIc. 1) or a with formed (FIG. 1) polytomy ergiacalculiformis the those and the rest of the family (FIc. and the rest of those two two species 2). species family(FIG. 2). The support for a monophyletic Physcia always The support for a monophyleticPhysciawas was always In the the best best tree Physcia magnusmagnusabove 90%. above tree (FIc. 90%. In (FIG. 1) 1) Physcia to the taxon to the rest rest of of Physcia. Physcia. sonii soniiappeared as aa sister sistertaxon appeared as There were were two two large clades within within Physcia Physcia in in most most There large clades of the the trees. first clade, which was was present present in in all all of trees.The The first clade, which trees (with contained all the spectrees 95-100% support) all the (with95-100% support) contained specimens in two and Physcia imens of of Physcia Physcia aipolia aipolia and Physcia caesia two discaesia in distinctgroups. tinct One with aipolia var. var. alnophila alnophila withPhyscia groups. One Physciaaipolia had other with withPhyscia had aa 100% The other Physcia aipolia 100% support. support.The aipolia unresolved var. var. aipolia aipolia grouping together with with two two unresolved grouping together in all caesia specimens Physcia caesia had aa 91-96% 91-96% support all Physcia supportin specimenshad trees (FIcs. 1, trees (FIGS. 1, 2). 2). The second clade the ciliate ciliate species The second clade contained contained the species tenella Physcia adscendens, Physcia leptalea, Physcia Physcia adscendens,Physcia leptalea,Physcia tenella var. tenellaand and Physcia tenellavar. var. tenella Physcia tenella var. marina. marina. Physcia Physcia lepleptalea formed a talea formed a monophyletic monophyletic group 2), as as 1, 2), (FIGS. 1, group (FIcs. did adscendens(FIG. did Physcia Physcia adscendens and Physcia Physcia tenella tenella (FIG. 1) 1) and some cases. (trees not not shown) in some cases. The The clade clade containcontain(trees shown) in and Physcia ing Physcia Physcia adscendens, Physcia leptalea leptalea and Physcia ing adscendens,Physcia tenellacollapsed when gaps tenella gaps were were coded coded as as missing collapsed when missing data (trees not shown). data (trees not shown). var. ornata, Physcia dimidiata dimidiata var. ornata, Physcia Physcia dubia, and dubia, and Physcia stellarisremained in many unresolved in Physcia stellaris remained unresolved many cases cases Physcia did Physcia Physcia magnussonii magnussonii (FIc. 2), but but (FIcs. 2), as as did 1, 2), (FIGS. 1, (FIG. 2), in some some of treesthey in of the the trees they appeared appeared as as unresolved unresolved taxa taxa in in aa clade clade containing the group group with with Physcia Physcia adscenadscencontainingthe and tenella not dens, Physcia Physcia leptalea and Physcia tenella (trees not dens, (trees leptalea Physcia shown). shown). The species of Phaeophyscia Phaeophyscia group. The Phaeophyscia Phaeophyscia group. species of formed a with formed a monophyletic monophyletic group in 100% support group with 100% supportin all trees all trees (FIcs. 2). Phaeophyscia Phaeophyscia nigricans nigricans appeared 1, 2). (FIGS. 1, appeared as aa sister sistertaxon the rest as taxon to to the rest of of the the genus. Phaeophyscia genus. Phaeophyscia Pha. endophoenica and Pha. eiconstipata, and Pha. endococcina endococcina eiendophoenica constipata,Pha. therformed or were ther formed aa monophyletic (FIG. 1) were 1) or monophyleticgroup group (FIG. in restof of in aa polytomy polytomy with with the the group group containing containing the the rest In the tree Pha. Phaeophyscia (FIG. (FIG. 2). the best best tree Pha. constipata 2). In Phaeophyscia constipata and Pha. and Pha. endophoenica grouped together with two two endophoenicagrouped togetherwith Pha. Pha. endococcina endococcina specimens Phaeophyscia cilcil(FIG. 1). 1). Phaeophyscia specimens (FrG. 733 733 iata, Pha. orbicularis and Pha. Pha. sciastra formed aa orbicularisand sciastra formed iata, Pha. monophyletic group in every analysis (FIcs. in 1, 2). every analysis (FIGS. 1, monophyleticgroup specimens appeared as a Two Pha. sciastra Two Pha. sciastra specimens appeared as a sister sister group to to the the other other two two species in some some alignments alignments group species in (trees they formed at the the or they formedaa trichotomy at not shown) (treesnot shown) or trichotomy next group base of group (Frcs. 2). Phaeophyscia Phaeophyscia cilbase of the the next cil1, 2). (FIGS. 1, iata Pha. orbicularis iata and and Pha. orbicularisformed formed aa well well supported supported monophyletic each tree. Pha. orin each ortree. The The two two Pha. monophyleticgroup group in bicularis specimens ed aa monophyletic group bicularis formed specimens form monophyleticgroup in all within group with with 90-91 % support all trees. trees. withinthat thatgroup 90-91% supportin sistergroup Physconia group. The The sister group to to the the PhaeoPhaeoPhysconia group. contained the physcia group the genera genera Anaptychia, Anaptychia, physcia group contained and Rinodina Phaeorrhiza, Physconia Physconia and Rinodina (the Physconia Phaeorrhiza, (the Physconia FIGS. group; FIcs. 1, 2). Hyperphyscia adglutinata was was either 1, 2). group; Hyperphyscia adglutinata either in the found as basal sister taxon in the Physconia Physconia group group found as aa basal sistertaxon with (FIc. or in in trichotomy with the groups (FIG. the two two groups 1) or (FIG. 1) (FIG. trichotomy 2), depending on the alignment used in the analysis. in the analysis. on the used depending alignment Rinodina Rinodina turfacea and Phaeorrhiza nimbosa formed formed aa Phaeorrhizanimbosa turfaceaand the genera grade basal basal to to the genera Anaptychia Anaptychia and and Physconia. Physconia. grade Anaptychia ciliaris either formed formed aa sister the ciliariseither sistergroup to the Anaptychia group to the specimens rest of the (FIG. 1) or remained remained unreunrerest of 1) or specimens (FIG. solved with runcinataand and Physconia solved with A. A. runcinata grisea (FIG. (FIG. Physconiagrisea Anaptychia ciliaris ciliaris always formed aa monophyletic 2). Anaptychia alwaysformed monophyletic the best group with with 100% In the best tree tree Anaptychia Anaptychia 100% support. group support.In and Physconia Physconia perisidiosa perisidiosa runcinata, Physconia Physconia grisea grisea and runcinata, the group formedaa grade formed grade basal basal to to the containing the the rest rest group containing of the the Physconia of Physconia specimens Physconia musmus(FIG. 1). 1). Physconia specimens (FIc. enteroxantha in polytomy and Physconia were in polytomy cigena were cigenaand Physconiaenteroxantha withaa group with containing Physconia dis torta and Physdistorta and group containingPhysconia Physconia detersa detersaas as aa sister sistergroup group to to another Physconia conia another Physconia distorta distorta specimen. specimen. DISCUSSION DISCUSSION Various Various approaches approaches have have been been employed employed to to analyze analyze for phylogenetic sequence alignments for phylogenetic purposes. purposes. sequence alignments eitherused in analyses Alignments are either used in they are, or as they are, or Alignmentsare analysesas they may be manipulated manipulated in in various For examexamvariousways. they maybe ways.For in order avoid noisy ple, in order to to avoid noisy characters, characters, Berbee Berbee et al et al ple, and Luzoni Luzoni (1997) their alignments, (1995) (1995) and (1997) adjusted adjusted their alignments, offregions or regions othpruning off with long pruning regions with long gaps, gaps, or regions otherwise erwise considered considered ambiguous. The problem with The with ambiguous. problem such an an approach the information informationis such approach is is that that aa part part of of the is since such such regions contain phylogenetic lost, regions may may contain phylogenetic lost, since structurein in addition addition to to noisy structure noisy characters al characters(Myllys et al (Myllyset in noise a data set not 1999b). Furthermore, noise in a data set may may not be be 1999b). Furthermore, severe problem, since Wenzel Wenzel and Siddall (1999) aa severe problem, since and Siddall (1999) have shown shown that fractionof that aa large large fraction of noisy characters have noisycharacters are loss of of phylogenetic are needed needed to to produce produce loss phylogenetic signal. signal. We left since we We left our our alignments alignments intact, we wanted wanted to to use use intact,since all data. all available available data. are of often coded as data. It It is Gaps are ten coded as missing is aa simple Gaps missingdata. simple eliminateartifacts artifactsbased on ambiguous way to to eliminate based on alignway ambiguous alignthe disadvantage in such an approach such an ments, but the disadvantage in approach is is ments,but that information lost. Another that information may be lost. Another method method is is to to may be This content downloaded from 130.238.82.159 on Thu, 2 Oct 2014 09:17:08 AM All use subject to JSTOR Terms and Conditions 734 734 MVCOLOGIA MYCOLOGIA treat gaps gaps as as aa fifth fifth character character state. state. The The information information treat provided by by indels indels remains re mains intact, intact, but but then then long long gaps gaps provided may seem seem to to present present aa problem. problem. Should Should one one code code aa may long gap gap consisting consisting of of several several gaps gaps (nucleotide (nucleotide losslosslong es) as as one one character character (presence/absence), (presencejabsence), or or should should es) each single single position position in in aa gap gap equal equal one one character? character? each The difficulty difficulty in in the the first first alternative alternative is is how how to to treat treat The overlapping gaps. In the second alternative the gaps In the alternative the second gaps overlappinggaps. may gain too great significance in the analysis. Since in the Since too greatsignificance analysis. maygain our alignment alignment contained contained many many overlapping overlapping gaps gaps of of our which homology was difficult to assess, we coded each each difficult to we coded whichhomologywas assess, single gap gap as as aa character character state. state. The The different different alignalignsingle ments yielded yielded trees trees that that were were not not in in conflict conflict with with ments each other other whether whether or or not not gaps gaps were we re coded coded as as fifth fifth each character state, state, but but the the trees trees where where gaps gaps were were coded coded character as fifth fifth character character state state were were better better resolved resolved than than the the as other trees. trees. The The better better resolution resolution seemed seemed not not to to be be other an artifact artifact only, only, since since different different alignments alignments yielded yielded an similar groups groups in in each each analysis. analysis. This This indicates indicates that that the the similar ambiguous parts contained phylogenetic signal. FurFurcontained signal. phylogenetic parts ambiguous thermore, the the well-supported well-supported group group containing containing PhysPhysthermore, and Physcia tenella cia adscendens, adscendens, Physcia Physcia leptalea leptalea and tenella cia Physcia (FIGs. 1, l, 2), 2), species with shared morphological charcharshared morphological species with (FIGS. acter (cilia), (cilia), collapsed collapsed when when gaps gaps were were coded coded as missas missacter ing data. data. ing The parameter settings had some, alThe alignment some, alsettingshad alignmentparameter though not not aa drastic drastic influence influence on on the the resulting resulting trees. trees. though The Buellia group was was affected affected by by different different alignalignThe Buellia group ment parameter parameter settings. Perhaps more more comprehencomprehenment settings.Perhaps stable. sive would make make this this group more stable. sive sampling group more sampling would in each reOtherwise tree reeach tree overall structure structurein Otherwise the the overall in mained similar. also observed observedin et al al (1999b) also similar.Myllys mained Myllyset diftheir trees based based on on difthat the the trees Arthonialesstudy theirArthoniales studythat and were were similar topologies ferent topologies and had similar ferentalignments alignmentshad ITS senot their highly variable ITS setheir variable in conflict, not in conflict,despite highly despite inwere of the characters quence data (up to 85% of the characters were into data 85% (up quence formative, compared to 45% in our present data set). data in our to 45% set). formative, present compared Only variin the treeswas was varithe trees the degree of resolution resolutionin degree of Only the able. able. one of of the the been one Growth has traditionally form has Growthform traditionallybeen to grounds forgrouping lichenizedfungi. fungi.According Accordingto groupinglichenized groundsfor has arisarisour formshas crustose(or our study foliose) growth (or foliose) growthforms studycrustose The en withinPhysciaceae twicewithin en at at least least twice 1, 2). 2). The (FIGS.l, Physciaceae (FIGs. the as are are the are crustose, species Buellia group in the the Buellia crustose,as group are species in in the the Physconbasal and Phaeorrhiza) basal taxa taxa (Rinodina Phaeorrhiza)in (Rinodinaand Physconet and Myllys and DePriest DePriest (1998) ia et ia group. Stenroosand (1998) and Myllys group. Stenroos difof difal also observed observedmultiple have also al (1999b) (1999b) have originsof multipleorigins Our result resulttotoin their theirstudies. studies.Our formsin ferent ferentgrowth growthforms DePriest and DePriest of Stenroos Stenroos and gether the results results of with the gether with that the the (1998) et al al (1999b) and Myllys (1999b) suggest (1998) and suggest that Myllyset usefulcharacter character not be be aa useful growth of lichens lichensmay formof maynot growthform taxonomicleveis, for studiesat at higher forphylogenetic levels, highertaxonomic phylogeneticstudies or ascolocular ascolocular either in ascohymenial eitherin (Lecanorales) or ascohymenial(Lecanorales) (Arthoniales ) lichens. lichens. (Arthoniales) the ascus have studied studied the Ramboldt et et al al (1994) ascus types types (1994) have of of several several species species in in Physciaceae Physciaceae and and found found that that two two kinds of asci asci (with (with some some intermediate intermediate types) types) are are preprekinds of sent sent in in the the family: family: Bacidia-type Bacidia-type asci asci and and Lecanora-type Lecanora-type asci. In our our analysis analysis the the Buellia Buellia group group corresponds corresponds to to asci. In the the Bacidia-type Bacidia-type of of asci, asci, while while the the rest rest of of the the species species have have asci asci of of the the Lecanora-type. Lecanora-type. Furthermore Furthermore RamRamboldt boldt et et al al (1994) (1994) suggested suggested that that the the genus genus Dimelaena Dimelaena is is more more closely closely related related to to Buellia Buellia than than to to Rinodina. Rinodina. Our Our result result is is in in agreement agreement with with that. that. The The taxon taxon dedelimitation is still still somewhat somewhat unclear. unclear. limitation in in Physciaceae Physciaceae is For For example, example, the the genus genus Buellia Buellia appeared appeared not not to to be be monophyletic. Also Also the the relationship relationship of of Anaptychia Anaptychia monophyletic. and and Physconia Physconia remained remained unresolved. unresolved. Anaptychia Anaptychia did did not not form form aa monophyletic monophyletic group group in in any any of of the the analyses analyses and and Physconia Physconia formed formed aa monophyletic monophyletic group group in in some some cases only only (FIG. (FIG. 1). l). cases The The species species pair pair concept concept was was originally originally described described by Poelt Poelt (1970). (1970). It It is is aa well weil known known concept concept in in lichenlichenby ology based based on on the the observation observation that that morphologically morphologically ology as pairs-one be grouped pairs-one indistinguishable taxa taxa may may be indistinguishable grouped as asexumember member reproducing reproducing sexually sexually and and the the other other asexuthat Our results the ally. Our results suggest that the putative species pair species pair suggest putative ally. detersa distorta and Physcia distorta (sexual) and Physcia detersa (asexual, (asexual, (sexual) Physcia Physcia Moberg 1977) might represent represent aa single but 1977) might single species, species, but Moberg Our result is needed to confirm this. more more sampling is needed to confirm this. Our result sampling also also indicates indicates that that Physcia Physcia aipolia and PhysPhys(sexual) and aipolia (sexual) as aa single cia should be be treated treated as (asexual) should cia caesia caesia (asexual) spesinglespeis nested cies since since Physcia cies nested within within Physcia aiPhyscia caesia caesia is Physciaaiis not and varietal varietal placement placement is not consistent consistent (FIGS. (FIGS. potia and polia tenella(asexual) and Physcia l, Physcia tenella Physcia leptalea 1, 2). (asexual) and 2). Physcia leptalea and Physas well as Physcia dubia (asexual) (sexual), as weil as Physcia dubia (asexual) and (sexual), Physto be be closely are supposed cia stellaris stellaris(sexual), relatcia (sexual), are closelyrelatsupposed to ed to to each otherand and forming each other ed pairs (Moberg (Moberg species pairs formingspecies either confirm confirm ITS data data set set failed failed to but the the ITS 1977) to either 1977) but The genus and or contradict contradictthat that(FIGs. or Physcia and 1, 2). (FIGS.l, 2). The genus Physcia studied it are within some species some pairs within it are being studied sepasepabeing species pairs et al al unpubl). rate ly (Lohtander unpubl). (Lohtander et rately as aa well-defined well-definedgroup, group, Phaeophyscia appeared Phaeophysciaappeared as of the having 100% support regardless of the alignment 100% alignment support regardless having was described described by Until this this genus used. Until used. by Moberg Moberg genus was in of Phaeophyscia were included in the species (1977) we re included (1977) the Phaeophyscia species of to our rethe genus the (Poelt 1974). 1974). According Accordingto our regenus Physcia Physcia(Poelt relatedto to the the Physis more more closely sultsPhaeophyscia sults is Physcloselyrelated Phaeophyscia The putative thanit it is is to to Physcia. coniagroup conia species putativespecies Physcia.The group than orbicularis ciliata (sexual)-Pha. pair (sexual)-Pha. orbicularis pair Phaeophyscia Phaeophysciaciliata monoformedaa mono(asexual, formed 1977) consistently (asexual, Moberg consistently Moberg 1977) be studied These species will be studied sepaphyletic sepaspecies will group. These phyleticgroup. In Phaeophyscia al unpubl). et al rately (Lohtander et Phaeophyscia unpubl). In rately (Lohtander the same same speof the the specimens some of some sperepresentingthe specimens representing and Pha. Pha. Pha. ciliata ciliataand Pha. endococcina, such as as Pha. cies, endococcina,Pha. cies, such not always did not sciastra, 1, 2). 2). sciastra,did together(FIGS. l, alwaysgroup group together of informative informative the low low number number of This may be due due to to the This may be that but it also possible it is is also in this thisgroup, charactersin group, but characters possible that furtherstudy. delimitationsrequire the species the study. requirefurther species delimitations corof the the trees 2) cor(FIGS.l, trees (FIGS. structureof The overall overallstructure 1, 2) The This content downloaded from 130.238.82.159 on Thu, 2 Oct 2014 09:17:08 AM All use subject to JSTOR Terms and Conditions LOHTANDER ET ET AL: AL: PHYSCIACEAE PHYSCIACEAE IN IN FENNOSCANDIA F'ENNOSCANDIA LOHTANDER respond quite quite well weil to to previous previous genus genus concepts concepts in in the the respond family (Poelt (Poelt 1965, 1974, Moberg Moberg 1977). 1977). Many Many infrainfrafamily generic relationships relationships remain to to be be resolved, especialespecialgeneric ly those in the genus genus Physcia. Physcia. However, the the ITS ITS rerely gion contained contained enough enough information to to resolve resolve many many gion relationships, such as those between the the of the other relationships, different genera genera in Physciaceae. Physciaceae. The The structure structure of of the the different appeared to be be stable despite despite the variation variation in in trees appeared ITS sequences. sequences. We We will will continue continue our our study study by by adding adding ITS data to the ITS ITS data data set. set. more data ACKNOWLEDGMENTS ACKNOWLEDGMENTS We thank thank Gullevi Gullevi Bergqvist Bergqvist for for help help with with laboratory laboratory work work We and Jyrki Jyrki Jalonen Jalonen and and Leena Leena Myllys Myllys for for collecting collecting specispeciand mens. 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Amplification Lee S, BrunsTD, WhiteTJ, White TD, Lee S, Taylor Amplification TaylorJw. TJ,Bruns for of fungal ribosomalgenes directsequencing and direct and genes for fungalribosomal sequencing of Gelfand In: Innis phylogenetics. DH, Sninsky]J, MA, Gelfand DH, SninskyJJ, phylogenetics.In: lnnis MA, Academic San PCR protocols. eds. PCR White TJ, White San Diego: Academic TJ,eds. Diego: protocols. Press.pp 315-322. 315-322. Press. This content downloaded from 130.238.82.159 on Thu, 2 Oct 2014 09:17:08 AM All use subject to JSTOR Terms and Conditions