PHYLOGENETIC EVALUATION of INFRAGENERIC GROUPS of the GENUS USNEA BASED on ITS REGIONS in Rdna I

J Hattori Bot. Lab. No. 92: 231-243 (Aug. 2002)

PHYLOGENETIC EVALUATION OF INFRAGENERIC GROUPS OF THE GENUS USNEA BASED ON ITS REGIONS IN rDNA I

2 YOSHIHITO OHMURA

ABSTRACT. A phylogenetic tree, inferred from the sequences ofITS regions (including ITSI, 5.8S rDNA and ITS2) of rDNA, has been constructed in order to confirm the infrageneric ranks of the genus Usnea in Japan. Twenty-one species of Usnea mainly collected from Japan were analyzed using the neighbor-joining (NJ) method. Samples were selected from various infrageneric groups of the genus. The data supports the segregation of the subgenera Dolichousnea and Eumitria from sub genus Usnea, and the treatment of sections Usnea and Ceratinae. Although the bootstrap values of the clades of the subgenera Dolichousnea and Eumitria and section Usnea are highly reliable, the values of the subgenus Usnea and the section Ceratinae c1ades are not enough to support the branch es. This suggests that the subgenus Usnea and the section Ceratinae need to be divided into several phylogenetic groups. Therefore, OTUs of more species belonging to these groups should be analyzed in order to elucidate the relationships within the groups.

INTRODUCTION When Ohmura (2001) revised the infrageneric groups of the genus Usnea in Japan and Taiwan, subgenus Dolichousnea Y.Ohmura was newly segregated from the subgenus Usnea. The subgenus Dolichousnea is characterized by a pendent thallus, isotomic-di chotomous branching, the presence of annular-pseudocyphellae between segments, a much thicker hypothecium [(70-)100-160(- 200) ,urn], and a positive iodine reaction in its axis. Ohmura (2001) also recognized the subgenus Eumitria (Stirt.) Zahlbr. based on its fistulose axis, and sections Usnea and Ceratinae (Motyka) Y.Ohmura in the subgenus Usnea distin guished by differences in cortical hyphae (leptodermatous type for section Usnea and pachydermatous type for section Ceratinae). However, these distinctions differ consider ably from Motyka's (1936-38) proposal. Sequences of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (rDNA) are known to have phylogenetic utility at both the infraspecific and infrageneric levels (Bruns et aI. , 1991). The purpose of this study is to test the validity of the infrageneric groupings in the genus Usnea in Japan using molecular phylogenetic analysis methods based on sequences of the ITS regions in rDNA.

MATERIALS Fresh specimens of various species collected between 1996 and 1999 were selected as

I Part of a dissertation for the degree of Doctor of Science submitted to the University of Tokyo. 2 Domestic Research Fellow, Japan Society for the Promotion of Science. Contact address: Biodiversity and Phylogenetic Study Section, Environmental Biology Division, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba-shi, Ibaraki-ken, 305-8506 Japan. 232 J. Hattori Bot. Lab. No. 92 2 0 0 2 the operational taxonomic unit (OTU). DNA was extracted within two years of the collec tion date. Collection data are shown in Table I. All voucher specimens are located in the National Science Museum, Tokyo (TNS).

METHODS DNA extraction Total DNA was extracted by CTAB methods (Murray & Thompson 1980) with small modifications. (I) Approximately 50 mg dry weight of each species was washed in flowing tap water for 24 hr. Each sample was then ground to a fine powder using liquid nitrogen in a mortar. (2) Each powdered sample was then suspended in 700,u1 of 2% CTAB extraction buffer [2% CTAB (hexadecyl trimethylammonium bromide), 100 mM Tris- HCl buffer (PH 8.0), 20 mM EDTA buffer (PH 8.0), 1.4 M NaCI] . Sample tubes were incubated for I hr at 65 °C and gently mixed at 10 min intervals. (3) 700,u1 of CIA (chloroform : isoamylalcohol = 24 : I) was then added, and the tubes were gently shaken using a microdisc rotor for 20min. (4) Each sample was centrifuged at 10,000 rpm for 20 min. at 20°e. (5) The aque ous phase of each sample was then transferred to a fresh tube. (6) 500,u1 of isopropanol was added to each tube, and gently mixed. (7) The samples were then recentrifuged at 15 ,000 rpm for 10 min. (8) The precipitate was rinsed with 70% ethanol, dried, and dis solved in 45,u1 of distilled water. (9) 5,u1 of 100 ,ug/ml RNase solution was then added to each sample, after which they were incubated for I hr at 37°e. (10) The extracted DNA was cleaned using an Ultra Clean™ Gel Spin DNA Purification Kit (MO BIO) as per the manufacturer's instruction. (11) Samples were stored at - 20°C prior to analysis (-80°C for long period storage). peR amplification PCR reactions were performed using Ready-To-Go PCR Beads (Amersham Bio sciences). PCR primers were ITSI-F (Gardes & Bruns 1993) as the 5' primer and modified LRI (LRI-I) (5'-GGTTGGTTTCTTTTCC-3') (LRI: Vilgalys & Hester 1990) as the 3' primer. 1.0,u1 of extracted DNA solution, 1.0,u1 each of 10 pmol/,ul primer and 22,u1 of sterile water were added to the sample tube. The program for amplification using a Perkin Elmer DNA thermal cycler was initial by 15 cycles for I min. 30 sec. at 95 °C, I min. 30 sec. at 57°C, and 2 min. at n oc, and 25 cycles for I min. at 95 °C, 1 min. 30 sec. at 57°C, and 2 min. at noc, followed by a final extension step of 10 min. at n°e. PCR prod ucts were analyzed electrophoretically using a 1.0% ethidium bromide stained agarose gel. Concentrations were determined using a molecular weight standard (A/StyI digest, NIP PON GENE). PCR products were prepared for sequencing with a PCR product pre-se quencing kit (Amersham Biosciences) as per the manufacturer's instruction.

Sequencing Direct sequencing of PCR products was performed using a Thermo Sequenase fluo rescent-Iabeled primer cycle sequencing kit (Amersham Biosciences). Manufacturer's in structions were followed. Cy5-labelled primers used for the sequencing reaction were ITS5 y. OHMURA: Phylogeneric evaluation of infrageneric groups of the genus Usnea 233

(White et al. 1990) as the 5' primer and modified LRl (5'-GGTTGGTTTCTTTTCCTC CG-3') as the 3' primer. Samples were exposed to the following temperature profiles using a Perkin-Elmer DNA thermal cycler: one cycle of 5 min. at 95°C, 30 cycles of 30 sec. at 95°C, 30 sec. at 55°C and 1 min. 20 sec. at n oc, and one cycle of 10 min. at n°e. The products were analyzed by electrophoresis using an ALFred DNA autosequencer (Amer sham Biosciences) after addition of 4 Jllloading buffer. ALF data were manually corrected for ambiguous sites.

Sequence alignment and phylogenetic analysis The sequences of ITS regions (including ITSl, 5.8S rDNA and ITS2) were aligned using the ClustalW (Thompson et al. 1994) alignment program. After removing gaps, alignments of 447 bp sequences were used for the phylogenetic analysis. A neighbor-join ing (NJ) tree (Saitou & Nei 1987) was constructed by MEGA ver. 2.1 (Kumar et al. 2001) using Kimura 2-parameter distance (Kimura 1980). Bootstrap analysis with 1000 replica tions was performed to estimate statistical support for each branch.

RESULTS AND DISCUSSION Sequencing and alignment ITS regions in rDNA were successfully sequenced for 56 samples (22 species, includ ing one species for the out group) (see Tables 1 and 2). PCR products amplified by the primers (ITS I-F and LRl-l) included the following re gions: the 3' end of the 18S rDNA (SSU), ITSl, 5.8S rDNA, ITS2, and the 5' end of28S rDNA (LSU). Each was ca. 560 or 860 bp in length. In the latter case, an insertion of ca. 300 bp was present in the SSu. A long insertion in the SSU can be a group I intron, judging from the number of bases and their position. Further research is needed to determine the nature of this long insertion. Sequences of ITS regions obtained in this study are shown in Table 2. The ITSl sec tion extends from position 1 to 196; the 5.8S rDNA gene extends from position 197 to 354; and the ITS2 section lies between positions 355 and 512.

Phylogenetic analysis for infrageneric groups Although the genus Neuropogon has been considered the closest related group to the genus Usnea (Krog 1976, 1982), molecular data have not been available. Therefore, Ever nia esorediosa (MiilI.Arg.) DuRietz was used as an out group. The genus Evernia is also thought to be closely related to the genus Usnea (Krog 1982). The phylogenetic tree based on ITS regions including 5.8S rDNA for the infrageneric analysis of the genus Usn ea is shown in Fig. I. The tree is preliminary because it still lacks some species commonly found in the study area. A monophylous relationship for the sub genera Usnea, Dolichousnea and Eumitria and the sections Usnea and Ceratinae in the subgenus Usnea are suggested by the tree, although some of them have a low bootstrap value. The OTUs belonging to the subgenus Dolichousnea, which was newly proposed by Ohmura (2001), and the subgenus Eumitria form monophyletic clades with high bootstrap values, 98% and 99% respectively. These results show that the segregation of the subgenera N Table I. Collection data for DNA analysis samples. w """ DDBJ/EMBUGenBank Species Locality and collection date Specimen number OTU name accession number

Usnea aCiculi/era Japan, Honshu, Prov. Kai, Hirano, Yamanakako-mura, Minamitsuru-gun, on Carpinus tschonoskii, V. Ohmura 4400 Ac4400 ABOSI049 ca. ISOO malt., 8.S .1998. U baileyi Japan, Honshu, Prov. Kii, en route from Ichinobashi to Okunoin, MI. Koya, on Cryptomeriajaponica, V. Ohmura 4488A Bai4488a ABOS IOSO ca. 800m all., 1.4.1999. U baileyi U.S.A., Hawaii, Maui Island, Hana District, Auwahi (200 38'N, IS6°20'W), on Dodonea sp., V. Ohmura 4S16 Bai4S16 ABOSIOSI ca. 1200 malt., 10.6. 1998. U ceratina Japan, Honshu, Prov. Kii, en route from Ichinobashi to Okunoin, MI. Koya, on Cryptomeria japonica, V. Ohmura 4486A Cer4486a ABOSIOS2 ca. 800 m all., 1.4.1999. U ceratina Japan, Honshu, Prov. Kii, en route from Ichinobashi to Okunoin, MI. Koya, on Cryptomeria japonica, V. Ohmura 4486B Cer4486b ABOSIOS3 !- ca. 800 m all., 1.4.1999. ::c U ceralina Japan, Honshu, Prov. Kii, en route from Ichinobashi to Okunoin, MI. Koya, on Cryptomeria japonica, V. Ohmura 4492A Cer4492a ABOSIOS4 '" ca. 800 m all., 1.4.1999. §. U ceratina Japan, Honshu, Prov. Kii, en route from Ichinobashi to Okunoin, MI. Koya, on Cryptomeriajaponica, V. Ohmura 4492B Cer4492b ABOSIOSS CO ~ ca .. 800 m all., 1.4.1999. r U dasaea Japan, Honshu, Prov. Mikawa, MI. Horaiji , Kadoya, Horai-cho, Minamishitara-gun V. Ohmura 2842 Das2842 ABOSIOS6 '"~ (34°S8'N, 137°3S'E), on twigs of Prunus sp., ca. 340 malt., S.12.1996. Z 0 U difJracta Japan, Honshu, Prov. Shimotsuke, W Lakeside ofYunoko, Nikko (36°48'N, 139°2S'E), V. Ohmura 1124 Difl l24 ABOSIOS7 'D N on Prunus ssiori, ca. 1480m alt., 16.7.199S. U difJracta Japan, Hokkaido, Prov. Kushiro, along Route 44, Akkeshi-cho, Akkeshi-gun, on Abies sachalinensis, V. Ohmura 2762 Dif2762 ABOSlOS8 ca.IOOm alt., 3.9.1996. U difJracta Japan, Honshu, Prov. Musashi, en route from Jyumonji Pass to MI. Ohyama, Chichibu-gun V. Ohmura 2889 Dif2889 ABOSIOS9 (3S0S6'N, 138°44'E), on TSllga diversi/olia, 1980-2160 m all., 9.12.1996. U difJracta Japan, Honshu, Prov. Shinano, SE slope of MI. Nyukasa, Fujimi-cho, Suwa-gun (3S0S3'N, 138° 11 'E), V. Ohmura 3232 Dif3232 ABOSI060 on Abies sp., 1830m all., 30.6.1997. U difJracta Japan, Hokkaido, Prov. Tokachi, Mt. Tsurugi, Memuro-cho, Kamiikawa-gun (42°S1 'N, 142°S3' E), V. Ohmura 3438 Dif3438 ABOS I061 on weathered bark of Betula ermanii, 900- 1200 m alt., 20.7.1997. U difJracta Japan, Hokkaido, Prov. Tokachi, Mt. Tsurugi, Memuro-cho, Kamiikawa-gun (42°S1 'N, 142°S3'E), V. Ohmura 3439 Dif3439 ABOSI062 on Abies sachalinensis, 900- 1200 m, 20.7. 1997. U difJracta Japan, Hokkaido, Prov. Kitami, Tokusyoppe, Furu-ume, Bihoro-cho, Abashiri-gun V. Ohmura 3S98 Dif3S98 ABOSI063 N (43°42'N, 144° IS 'E),on Tiliajaponica,ca. 330m alt., 26.7.1997. 0 0 N Table I. (Continued).

DDBJ/EMBLlGenBank Species Locality and collection date Specimen number OTU name accession number

U diffracta Japan, Hokkaido, Prov. Ishikari, Ukishima Marsh, Kamikawa-cho, Kamikawa-gun Y. Ohmura 3663 Dif3663 ABOSI064 :-< (44°S6'N, 142°S8'E), on Picea glehnii, ca. 900m alt., 28.7.1997. 0 :x: U fulvoreagens Japan, Honshu, Prov. Shinano, ca. 3 km ESE of Azusayama, Kawakami-mura, Minamisaku-gun, y. Ohmura 2906 Fulv02906 ABOSI638 ;:: c:: on Larix kaempferi, 1460-ISOOm alt., 9.12.1996. ~ Japan, Honshu, Prov. Shinano, en route from Gyozya-goya to Minoto-sanso, Yatsugatake Mts., y. Ohmura 3824B Glab3824b ABOSI639 U glabrescens '"Cl Chino-city, on Salix sp., 1740-1800 malt., 29.8.1997. ~ 0- U himalayana Malaysia, Sabah, along summit trail, Kinabalu National Park, on tree in sparse forest, M. Kato S.n. (herb. Him4374 ABOSI640 ()Q e> ::l ca. 3S00m alt. , 23.12.1997. y. Ohmura 4374) e> ::I . U intumescens Japan, Honshu, Prov. Shinano, precinct in small shrine, en route from Nakakawara to Tsuetsuki Pass, y. Ohmura 3112 Intum3112 ABOSI641 n e> Chino-city (3soS9'N, 138°09 'E), on Zelkava serrata, 870 malt., 27.6.1997. .,< U /ongissima Japan, Honshu, Prov. Shinano, en route from Azusayama to Jyumonji Pass, Minamisaku-gun y. Ohmura 2877 Long2877 ABOS I 642 aC- (3soS6'N, 138°44'E), on Betula ermanii, 1860-2030 malt., 9.12.1996. 0' ::l U /ongissima Japan, Honshu, Prov. Shinano, en route from Azusayama to Jyumonji Pass, Minamisaku-gun y. Ohmura 2881 Long288I ABOSI643 ....,0 (3soS6'N, 138°44'E), on Betula ermanii, 1860-2030 malt., 9.12.1996. 5 U longissima Japan, Honshu, Prov. Shinano, around Oh-ahara Marsh, Hase-mura, Kamiina-gun y. Ohmura 32S0 Long32S0 ABOS I 644 .,!:i' ()Q e> (3soS3'N, 138° 11 'E), on twigs of Ti/iajaponica, 1820 malt., 30.6.1997. ::l e> U longissima Japan, Hokkaido, Prov. Ishikari, Ukishima Marsh, Kamikawa-cho, Kamikawa-gun y. Ohmura 3664 Long3664 ABOSI64S ::I. n (44°S6'N, 142°S8'E), on twigs of Picea glehnii, ca. 900 m all., 28.7.1997. ()Q U /ongissima Japan, Honshu, Prov. Shinano, en route from Gyozya-goya to Minoto-sanso, Yatsugatake Mts., y. Ohmura 3816A Long3816a ABOSI646 a Chino-city, on Salix sp., 1740-1800 malt., 29.8. 1997. '"'" '"0 U /ongissima Japan, Honshu, Prov. Shinano, en route from Gyozya-goya to Minoto-sanso, Yatsugatake Mts., y. Ohmura 3816B Long3816b ABOSI647 ...., 9- Chino-city, on Salix sp., 1740-1800m alt., 29.8.1997. e> ()Q y. e> U /ongissima Japan, Honshu, Prov. Shinano, MI. Tateshina, Tateshina-cho, Kitasaku-gun (36°06 'N, 138° 18'E), Ohmura 3844 Long3844 ABOSI648 ::l on Larix kaempferi, 1830 malt., 30.8.1997. '" U merrillii Japan, Honshu, Prov. Kai, Aokigahara-marubi, Karnikuishiki-mura, Nishiyashiro-gun, on dead conifer Y. Ohmura 4397 Merr4397 ABOSI649 ;:,s: tree, ca. 1000 malt., 8.S. 1998. ~ U mutabilis Japan, Honshu, Prov. Kai, Tokyo University Forest, Asahigaoka, Minamitsuru-gun, on Larix kaempferi, y. Ohmura 4407 Mut4407 ABOSI6S0 ca. ISOO m all., 8.S. 1998. U mutabilis Japan, Honshu, Prov. Kii, en route from Ichinobashi to Okunoin, MI. Koya, on Cryptomeriajaponica, y. Ohmura 4493A Mut4493a ABOSI6S1 ca. 800 m all., 1.4.1999. N w V> N Table 1. (Continued). w 0-

DDBJ/EMBLlGenBank Species Locality and collection date Specimen number OTU name accession number

U nipparensis Japan, Honshu, Prov. Shinano, en route from Gyozya-goya to Minoto-sanso, Yatsugatake Mts., Y. Ohmura 3825 Nipp3825 AB051652 Chino-city, on Salix sp., I 74D--1 800 m, 29.8.1997. Upangiana Japan, Honshu, Prov. Shinano, ca. 4.3 km SW of Lake Chiyoda, Fujisawa, Takato-cho, Kamiina-gun Y. Ohmura 3132 Pang3132 AB051653 (35°47'N, 138°08'E), on Larix kaempferi, 1220 malt., 28 .6. 1997. U pangiana Japan, Honshu, Prov. Shinano, Ikenodaira, Takato-cho, Kamiina-gun (35°55'N, 138°10'E), Y. Ohmura 3144D Pang3144d AB051654 on Larix kaempferi, 1620m alt., 29.6.1997. U pectinata Japan, Kyushu, Prav. Bungo, Kakizaka, Yabakei-cho, Shimoge-gun (33°27'N, 131 °07'E), Y. Ohmura 2989 Pect2989 AB051655 on rocks along Yamakuni River, ca. 110 malt., 3.3.1997. U pectinata Indonesia, Sumalra, Prov. Jambi, SW slope of Gunung (Mt.) Tujuh, Gunung Kerinci Seblat National M. Kato S. n. (herb. PectMK-T AB051656 ~ Park, on tree in mossy forest, ca. 2000 m alt., 27.11.1997. Y. Ohmura 4373) .,::r: Upygmoidea Japan, Hokkaido, Prav. Kushiro, along Route 44, Akkeshi-cho, Akkeshi-gun, on Abies sachalinensis, Y. Ohmura 2736 Pygm2736 AB051657 ;; 0 ca. lOOm ait., 2.9.1996. ::I. 1:0 Upygmoidea Japan, Honshu, Prov. Shinano, Ikenodaira, Takato-cho, Kamiina-gun (35°55'N, 138°10' E), Y. Ohmura 3144C Pygm3144c AB051658 P- on Larix kaempferi, 1620m all., 29.6.1997. .,r U rubicunda Japan, Honshu, Prov. Shinano, Todai, Hase-mura, Kamiina-gun , on Pin us densiflora, Y. Ohmura 3114 Rubi3 114 AB051659 ca. 1040 m ait., 27.6.1997. "'"Z !=' U rubrotincta Japan, Honshu, Pref. Nagano (Prov. Shinano), ca. 4 km ESE of Azusayama, Kawakami-mura, Y. Ohmura 2910 Rubr2910 AB051660 \0 N Minamisaku-gun (35°56'N, 138°43 'E), on Larix kaempferi, 140D--15OOm alt., 10.12.1996. U rubrotincta Japan, Kyushu, Prov. Bungo, en route from Hanamure to Oike, Shonai-cho, Ohita-gun, on Y. Ohmura 3057 Rubr3057 AB051661 Quercus acutissima, ca. 900 m all., 5.3.1997. U subfloridana Japan, Honshu, Prov. Shinano, en route fram Azusayama to Jyumonji Pass, Minamisaku-gun, Y. Ohmura 2879 Subf2879_T AB051662 on Betula ermanii, I 86D--2030 m, 9.12.1996. U subfloridana Japan, Honshu, Prov. Shinano, W slope of Mt. Tateshina, Tateshina-cho, Kitasaku-gun, Y. Ohmura 3338 Subfl338_T AB051663 on Alnus hirsuta, 167D--1760m alt., 2.7.1997. U subfloridana Japan, Honshu, Prov. Shinano, en route from Gyozya-goya to Minoto-sanso, Yatsugatake Mts., Y. Ohmura 3823 Subfl823_S AB051664 Chi no-city, on Salix sp., 1740- 1800m all., 29.8.1997. U trichodeoides Japan, Honshu, Prov. Shinano, ca. 4 km ESE of Azusa-yama, Kawakami-mura, Minamisaku-gun Y. Ohmura 2911 Tric29 I I AB051665 (35°56'N, 13 8°43 'E), on Larix kaempferi, 1400- 1500 m all., 10.12.1996. U trichodeoides Japan, Honshu, Prov. Shinano, Nyukasa Pasture Land, NW slope of MI. Nyukasa, Takato-cho, Y. Ohmura 3215 Tric3215 AB051666 N Kamiina-gun (35°54'N, 138°1O'E), on Larix kaempferi, 1780 m all., 30.6.1997. 0 0 N Table I. (Continued).

DDBJIEMBLlGenBank ~ Species Locality and collection date Specimen number OTU name o accession number

U trichodeoides Japan, Honshu, PrOY. Shinano, SE slope ofMt. Nyukasa, Fujimi-cho, Suwa-gun (35°53'N, 138°11 'E), Y. Ohmura 3236 Tric3236 AB051667 I. on Larix kaempferi, 1830m alt. , 30.6.1997. ." ~ U trichodeoides Japan, Honshu, PrOY. Shinano, around Oh-ahara Marsh, Hase-mura, Kamiina-gun (35°53'N, 138°11 'E), Y. Ohmura 3249 Tric3249 AB051668 on Larix kaempferi, 1820m alt., 30.6.1997. c2 1'6'" U trichodeoides Japan, Hokkaido, Proy. Tokachi, along the trail trom Tomuraushi Hot Spring to the top of y. Ohmura 3495 Tric3495_S AB051669 ::1 . (l Mt. Tomuraushi, Daisetsu Mts., Shintoku-cho, Kamikawa-gun (43°28'N, 142°52 'E), on weathered twigs of Pinus pumila, 680-1000m alt., 22.7.1997. ~ y. Ohmura 3809 [ U trichodeoides Japan, Honshu, Proy. Shinano, en route from Gyoza-goya to Minoto-sanso, Yatsugatake Mts., Tric3809 AB051670 o· Chino-city, on Abies veitchii, 2100 malt., 29.8.1997. :: o U trichodeoides Japan, Honshu, PrOY. Shinano, en route trom Gyoza-goya to Minoto-sanso, Yatsugatake Mts., y. Ohmura 3822 Tric3822 AB051671 ...., S· Chino-city, on Salix sp., 1740-1800 malt., 29.8.1997. ::;> U trichodeoides Japan, Honshu, Proy. Kai, Okuniwa, NW slope of Mt. Fuji, Minamitsuru-gun, on twig of y. Ohmura 4413 Tric44ILS AB05 I 672 ~ Tsuga diversifolia, ca. 2300 malt., 9.5.1998. ::" ::1 . U wasmuthii Japan, Honshu, Proy. Shinano, ca. 3 km ESE of Azusayama, Kawakami-mura, Minamisaku-gun, Y. Ohmura 2900B Was2900b AB051673 (l'" (JQ on Larix kaempferi, 1460-1500 malt., 9.12.1996. U wasmuthii Japan, Honshu, Proy. Shinano, ca. 4 km ESE of Azusayama, Kawakami-mura, Minamisaku-gun, y. Ohmura 2915 Was29 I 5 AB051674 ~ on Betula ermanii, 1400-1500m alt., 10.12.1996. ....,'"o U wasmuthii Japan, Honshu, PrOY. Shinano, Shiozawaseki, Shirakaba-kogen, Tateshina-cho, Kitasaku-gun, y. Ohmura 3281 Was328I AB051675 So on Larix kaempferi, 1540m alt., 1.7.1997. (JQ'" ~ U wasmuthii Japan, Honshu, PrOY. Shinano, en route trom Gyozya-goya to Minoto-sanso, Yatsugatake Mts., Y. Ohmura 3821 Was382 I AB051676 !il Chino-city, on Picea sp., 1740-1800 malt., 29.8.1997. Evernia esorediosa Japan, Honshu, Proy. Shinano (pref. Nagano), along Takinoyu RiYer, Mt. Tateshina, Chino-city T. Arikawa S. n. (herb. EY-es04458 AB05 I 677 s:;:, (36°06'N, 138°17'E), on Larix kaempferi, 1960m alt., 4.8.1998. y. Ohmura 4458) ~

IV .....V.> Table 2. Aligned sequences ofITS regions from 55 samples of Usnea with Evernia esorediosa as the out group. Hyphens IV v.> are gaps required for alignment. Identical residues with Ac4400 are represented by dots. OTU names are the same as in Table I. 00 The ITS I section extends from position I to 196. The 5.8S rDNA gene extends from position 197 to 354. The ITS2 section lies between positions 355 and 512.

110 110 I 100 Ac44()() COCICCCCCCCGTTTCCCCCICClICICfTCICCClllCccllCCl1ccmm illccrcccmcccCfcClCIrnccmmrncm·crmrcmcmF"flClCICClCCCIF-lli1l-mr:11 I ICI Ai 11111 Bai4488a .•••.••.•••. C••••••• C•..•...••••••..••. IC .•• I...... ••..• .••••••••• ••.• CC •• cc...... CCO. I •• C.. C...... C,. ·CI. IC. IC.,. C. .. 11 -1...... I ...... -, ..•. • ... 01 ..... C.' Bai4616 ...... ••••••. C..•.•.•.•••• . ...•.••••• C•. .••••••••.. •..•. IC ••. I •••....•.•.••.. ••..••....•.•• CC .. CC ...... CCO. 1.. C•. C...... C.• ·CI. IC. IC ••• C... 11-1...... I ...... • ..... • ... 01 ..... C.· Cer4486a ••• •••••• •••• C...... 1•••••• C•• C...... C...... C•• ·I...... C...... - ...... Cer4486b I ...... C...... I ...... l ..... C•• C...... I ...... C•• -I...... • .. •...... , •• -...... -. , ...... Cer4492a •••• ••. •• •••• C••••••• •••.• ••• •• •••. •• •• ••• ••• •••••• ••••••.••.••.•••• •• ••••••.•••. •••• 1• ••••• C•• C•• ...... C...... C.. ·I...... C...... -...... Cer4492b ...... C...... 1•• •• •• C•• C...... C...... C•. -I...... C...... -...... D6;2842 ...... I ...... C.• C...... 1...... ·1 ...... -...... • .. C.· ...... Oifll24 ...... C...... C...... O. •• • C.• I.O ...... CO ...... O.. ... -.. C•• C•. ... II...... C.. C...... O..... C.... OO . .. I ...... CUI·O. , C.. I ...... O.. C-... -.. CO· ...... O· Oi12762 . 1...... C...... c...... O.... C•• I.O ...... CO ...... O..... -.. C•• C.. ••• TT .. , •. •••••. C. • C• ..•••..... 0.•.•• C.. ..00 .•. I ...•••. OUl·O .. C.• I .• .. ••. O.. C· .• . • .. CO· ...... O· Oi12889 ...... C...... C...... 0.... C.. 1.0 ...... CO ...... O..... -•• C• • C.. ... TT ...... C.. C, ...... 0.... . t. . ..00 . .. I ...... CIII·O .. C• • !...... C--C· . •• • .. CC· ...... C· Oif3232 ...... C.... C...... C...... O... . C•. I.O ...... CO ...... O...•• -•• C• • C.. · .. 11...... C.. C...... C..... C.... cc ... 1. •••.•• OUl·O .. C•. I ...... C--C· ••. ' .• cc· ...... C· Oif3438 ...... C...... c...... O• •.• C•• I.O ...... CO ...... O..... -.. C• . C.. • .• TT ...... C.. C...... 0..... C.... cc ••• I. , .... . CIII·O •• C.. I ...... C--C-••• ' .. cc· ...... C· Oif3439 ····-.... •• .. C...... C...... O•.•• C.• I. O...... CO ...... O..... -.. C•. C.. • .. TT ...... C.. C...... C... .. C. . ..cc ... I •• •• ••. CIII·C •• C• . I ...... C--C· •. . ' .. cc· ...... O· Oif3698 ., C...... c...... 0..•• C. . I. 0...... CO ...... 0•••• • • •• C•. C.. .. • TT ...... C. . C...... C..... C.. ..co ... I ...... 0111 -0.. C• . I ...... O--C· ... ' •• co· ...... O· Oi13863 .. C...... C...... 0.... C.. 1.0 ...... CO ...... 0••••• • •. C•. C.. .. • TT ...... C. • C...... C..... c.. .. 00 ... I ...... 0111-0 .. C•. I ...... O--C· ••• ' •• CC· ...... O· Fulvo2906 ...... C...... I .. .. . • .. C•• CC. • ...... C.• c.... . C...... C.... C . . .. I •••••• C...... I ... • •. C...... 1. ~ Gl.b3824b ..•...•...... • •• .. .. C.••..•.... .•• ••.••.•••••••••••••.•••.•••••• I ••••• • •• C•• Cc...... C.. C..••• C...... C.... C .... I...... ---.1. .. • .. C.· ...... ::r: Him4374 ...... C...... C...... OC .... C...... 1••••. Cl. C•. CC! ...... I. CTIC. IC ...... C. . -1... •. I .• C.• C.... -- ...... 1...... ---. ....••. C.· .... . Intum3112 ...... C...... C...... C...... I. .. . I ...... I ... I ..... • .. C. . CCT ...... I ...... I ...... c.. -1 ...... 1----...... I ...... ---.. 1. . ·IOC.· .. . C. g 1.ong2877 ...... c...... C...... OC ••• C• • I ...... • ...... O..... • .. C, .. C. C... I . .. . CC ... .. C.. t...... C. . -!-C ... I ...... OIlI·CC ... . U ...... ---C ... . • ••. C· ... I. ::l. Lon1288 1 ...... C...... C...... OC ••• C•• I ...... • ...... O..... • .. C... C. C... I .... CC .. ... C.. C...... C.. ·I·C . •• I ...... CIII·CC . .. . I ...... ---C ••.. • ... 0· ... I .. 1.ong3260 ...... C...... C...... OC ••• C•• I ...... • ...... O••• •• • .. C... C. C. .. I. ... CC ..... C.. C...... t..-!-O . •• I ...... OIII·CC .... I ...... --·C . .. . • ... O· ... I. 0:1 1.ong3664 ...... C...... OC ... C.. I ...... • ...... O..... • .. C•• C.. C.• TT .... C...... C.. C...... C.• · I·C ••• 1...... CIII·CC .... I .. ..•.• ---C •. . . • ... 0- ... I. ::a. 1.ong38lS. .. .• C...... C...... OC ••. C•• TT ...... · ...... 0... •. • .. C•• C.. C.. TT ••• • C...... C•• C...... C•• ·H... I ••••••• OIll·CC .... I ...... ---C •... • .. • 0· ... I .. t"" 1.onB38IGb .... C...... C...... OC ... C.. I...... •...... O••••• • .• C.. CC . C... I .... c...... C.. C...... C.. -!-O ... 1...... • OIIl·CC .... I ...... ---c .... ' ... 0-... I .. 1.ong3844 ...... C...... C...... OC .. . C.. I...... •...... C. ••• • • .• C... c. C... I .... CC ..... C.. C...... C•. '!-G... I ...... OIlI·CC .... I ...... --·C •. . • • •.. 0-... I ... • ?''" Merr4397 ... C...... C...... C•• •.. ·C.C .. C...... I ...... C.. -I...... 1--·· ...... --•.....•.. C.·...... • Z Mut4407 .C ...... C...... I ••• ••• • C•• • ... .. •...... ••.•..•..•. C...... •. C...... • C•. -I. C•... C.II •... ----..... C••...... --· CO ... · .. CO · ...... • !=' Mut4493a .C ••..••. .•...... C..• .• •.•.. ...•...... ••. .••.•...... •...•...... ••• I ....•.• C• • • ••••. . •. .••••••.••..••.. C..••••... C..•.••• C•• -1. C. •.• C. II •. .. --...... C••..•.•. --·CO .•. • .. CO· ..•• 'D Nipp3826 ••.•. 0 ...... •.. 1..•...... •...••••...... ••..••.•.• •.••.. •.. ..• •••..••. .. •...... •• I •. .. • • •• C•• C•• 0...... 0..... C.. -I...... C.•••• ---...... ---C .... O. C. C· ...... • IV Pang3132 C... I .• C...... C...... C...... C...... I ••.. I ...... I ..... CC . ... CC. .. .• •••.... I . ... •. .•..••...... C•. ·I. .• ••...... I .. --...... --·. •. I.·I.C. · ... C.. I· PangSl44d C...... C...... C...... I ... . I ...... I ..... CC .... CC...... I ...... t. •• I...... C.. --...... --...... ' 1. C.· .. . C.. I· Poct2989 ...... C...... C...... IC ...... C...... • .. C•• CC. .. .. I .. C... CC ... C.. C...... C.. ·CI. 11. IC ... C.I. 11-1 ...... I ...... --... . I.· .. C.I .... . C.· Poc'4373 ...... C...... C...... C... C...... IC ...... C...... • .. C•• CC. .. .. I .. 0... OC ... C.. C...... C.. ·CI. TT. IC ... C.I. 11-1...... I ...... ---... I.· .. C.I .... . C.' Pygm2736 •..•.••••• •• . C.....•••••...... •.•...... •• C•...... • O•. •••••• I •••• I .•....••••••• ••••• I•••• • • •. C•• CC...... Cl •• • ...... C•• -I...... O. I----C ...... --••. •.• -1 •.• • ... C... • Pygm3144c ...... C...... C...... I •••. I ...... I •.•.. • •. C... C. . .• •..• .... I ...... •...... •• C•• -1 •.•.•... C.... 1----....•..•.....• --•. .... -1 ... • ... C... • Rubi3114 ...... 1...... C. C...... IT ...... l .... · .. C.. C...... CH·IC ...... 1. ----...... 111 . . ... • .. t...... Rubr2910 ...... I ...... C...... l •••• • .. C•• C...... I .. . . . C...... O..... C.. ·I...... I.----...... • ..... • .. C.· Rubr3067 ...... C...... I ...... I ••.•• C.. • •• CC • ...... O..... C. . ·I ...... I.----...... --· . .... • .. C.· ...... • Sub12879_T ...••••• I ••• • •.•.••••••••••...••...•..•• C•• ... ••..•••.•••••. ...• •.•••• •.• •.. •• .••••• I ••••• -•• C• • CCI •••••••••• C• • C•.••. C•••.• •. .••.. ••••• C• • --C •••• I .•••.••• ----•••••••.• ••••• --'. I . •. • •• C.· ••.... 1. Subf3338_T I ...... C...... I ..... • .. C•. CCI •• . 1...... C•. C.... • C....••...... •• C•• --C .... I .•....•. ----..• C•.•...... --'. I ... • .. C.· •..... I. Sub1"3823_S ...... C...... I ..... • .. C•• CC! ...... C•. C..... C...... C.. --C .••. I ...... ----...... --'. I ... • .. C. · ...... I. Tric29 11 ...... C...... C...... O.... C.• I...... O..... • .. C.. C.. C... 1...... 1. ... C.• C...... C•• -!-O ... 1...... CUI·C ...... --·C •.•• • ... 0- ..... C.' TrieS216 ...... C...... I ...... C...... O. ... C. • I ...... O••.•• • .• C• • C.. C••• I. .•••• I ...• C.• C•• •...... , •.••.•. C.. -!-O ... I •. .•... OIII-C ..•. .•• ...... --·C•••. • ... O· .•..... • Tric32S6 ...... C...... C...... O.... C•. I ...... O•. . •• • .• C.. C.. C.•• I ..•••• I •... C•• C•••• ••..•••••••• . C.. '!-G... I .•• •..• OUI ·C•. ..•....•. ,. --·C •... • ... 0- •.... C.' Tric3249 ...... C...... C...... O.... C.. I ...... O• . ••• • .. C.. C•. C... I .•• ••. I .... C•• C.•••• ... . , ..••••• C•• -!-O .• • I •••.••• OIIl·C ..••..• . ...•• --·C •.•• • ... 0- ••.. . C. ' Tric3495_S .C •..•••. I •..• . .. C...... • C• •. .•.••••..•• O•••• C•. I. .••.• O•• •..•••. • , ••••• • O••••• • .• C•• CC. C..• I. , •.•• 1. •• . C.. C.• ••• .... ••..••.• C•• -!-O ••• I •• C•• •• OCII·C •••.. I .•••.•• --·C .. . • • .•• 0- ...... · Tric3809 .. C...... C..•...... •....•. .••.• C•••. C•• I. •••••.•• .•.•• ••• , •.. ••• O• •••• • .. C.. C•. C. .. I ...... I . ••. C.. C...... C. . -!-O .•• I ...... OIII·C ...... ---C ... . • ... O· .. ... C.· Tric3822 C...... C...... O.... C.. I ...... O.•••• • •• C.• C.. t. .. 1. ••••. I .... C.. C...... C••. !-G .. . I ...... OU I·C...... --·C.... • ... 0-..... C.' Tric4413_S C...... C...... C.·.C ...... O.••• C.. I .•. I ...... O..... • •• C•• C.. C.• 11 ...... I .•• . C.• C...... C••• !-G ... I •• C... . OUI·C .... . I ...... --·CO . . . • ... O· Wai2900b ...•.• ••.•• ...... •. ...••• • C....•....•. ••• •••••. ••...... ••••. ••••.•••. I .•••• • .. C•. CC...... 01.. C...... C.. --C .... I ...... ----...... --'. I .. . • .. C.· ..... 1. Wa.2915 •••••... . •.• ...•.•...... C.... . •.... .•.•.•...... • I •••.• .. •. , ..• •••••• I •••• • • •• C•. CC. • •••.•••• • C. • C••.••• •• •••..•••••••••. C.• --C •••• I ••••..•• --...... 1--. I ..• • •. C.· ...... I. IV W.. 3281 ...... C...... I.I.... . • .. C•• CC ...... C.. C••••••• •••••••••••••••• C. . --C.... I ...... ----...... ---. I .•. • .. C. · ...... I. o Wu3821 ..... C...... I ...•. • .. C•• CC...... C...... C. . --C •••• IC...... ----...... " -. I ... • .. C. · ...... 1. EV"ei04458 .C ...... •...... • .• IC •... . •...... •...•. ... I •• IC. I •• O.. C.. CC. O. .. I ... I ...... C...... I ...... m ·l .. 1-. 1...... I"IC.IC •• I ...... • T·I .. ... 1.. C...... • o IV Table 2. (Continued).

HO III 110 lOO lIO l-IO---- 400 Ac4400 TTCCICCIICCIICIICUCCCICCCUIICCCITUCIUICICII"CCICuTTCICICllICIfe01llcm IICICCCCICIICCCIICClfCCIITlCCCI-crCCCCcc-cccCcC 8ai4_ ...... c...... T...... CI : .. .. 8ai4516 ...... C...... I ...... Cl .... . Cer4486a ...... c...... -...... -I .... . Cer4486b ...... c...... -...... -TT ... . ~ Cer4492a ...... c...... -...... -I .... . o Cer4492b ...... c...... -...... -I .... . ::c 0 ..2842 ...... -...... -..... 1 ;:: Oilll24 :: :::: :::::: ::::: :::: C:::::: :: : ::: ::::::: :C::: :::::::: ...... 1. c...... •. CCI ...... -...... Oif27S2 ...... C...... C...... I.C ...... CCI ...... -...... Oil'l889 ...... C...... C...... I.C ...... CCI •...... ~ Difll2S2 ...... C...... C...... I.C ...... CCI ...... Oi!ll438 •••••• C••••••• ••••• •••••••• C.•••••••••...... •. c...... CCI ...... ~'" Oi!ll439 ...... C...... C...... ••. c...... CCI ...... Oifll588 ...... C...... C...... •..... I.C ...... CCI ...•... Oi!ll66S ...... C...... C...... •. c...... CCI •..•••. ~ Fulv02506 .•. ...•..•.. ICC ...... -...... ~ G1.b3824b ...... c...... -...... -...... ::J. Him4374 .••••.••.••••••••.•...•..•••. -.•••.... -TT .. () Intum3112 ...... c...... -...... -. 1.1 .. Long2877 :::::::::: :::::::::::c:::::::: .. ····· .. ··· ··· .. .. ····· ...... •.... c.c ...... •...... CCI .• ... .. -...... ~ Long2881 ...... c...... c.c ...... •• CCI ...... -...... ;:: LongS250 ., ...... •.. c.c ...... • CCI ...•... -...... a Long3664 :::::: :::::::: :::::::1:::::::: .. ········ ·· ········ .. ·· .•.•.•...... c.c ...... CCI ...•... -...... Long38ISa ...... c...... c.c ...... CCT .•••••. -I.... . g' Long38ISb ...... c.c ...... CCI . ••• .. . -.. ...• o Lonl3844 :: :::::::::::: ::: :: :: ~::: ::: :: :: :: ...... ccc ...... CCI . •. . ••• -...•...... , Morr4S97 ...... c...... -IC ...... -I .... . s· Mut4407 ...... c..... c-...... -...... ;::r> Mut4493a ...... •...... c..... c-.. . Nipp3826 ...... c..... c-...... -...... ~::; Pang3132 ...... , .. . , , .. . , ...... , ...... ,.., .. , Pang3144d ...... -...... -1 .. .. . "::J . Peet298'J ..... I...... c...... I ...... -TT .. .. () Pect4S7S ...... c...... I ...... -TT .. .. (JQ Pygm2736 •..•••••.•.•..••...•.•.••.••• -•••••... -TT •.•. a Pygm3144c ...... c...... -...... -.I... . RubiSII4 .... c-...... -I .... . ~'" Rubr2910 ..... -...... -1 .... . o RubrS067 •.. .• ..•• •. •...... •• ...•.• •• -•. • ..••. -1 •••...... , Sub1'l879_T .• ....•..... IC ...... •..• -...... -I .... . 5' SubfllS38_T ...••...•...•C ...... ••• .•.. .. -...... • -I .... . Sub!ll82S_S ...... •....•C •••...... •.. . • -.•...•.• -I .... . (JQ" Tric.2911 •...... •. IC ...... C•..•.... -...... ::; . a...... IC ...... Cl ...... -...... " TricS216 ~ TricS2S6 :::::::::::::::::::::j:':::.::;:::: .. ::::::.::.::':::: ...... IC ...... C...... -...... TricS249 •••••••••••• IC .•••••••••••••• C•• •.•••• -••• •. • TricS496_S c...... •.•.•. .... IC ...... •.•• CI ••..... -•.. ... s: TricS800 ...... •..•. IC ..•....•...... C•.. •.• . . -...... Tric3822 ••...••.•••. IC ...... C...... -...... '"~ Tric44IS_S ...... :::::::::::::: !:::::::: ...... •.•...... ICC .....•.....•.. Cl ...... -...... Wa.29OOb •....•..••.. IC ...... C....•. -•..••... W.. 2916 ..•...... •• IC •..•....•...... -.. •..•.• W•• 3281 ...... IC ...... -.C ...... Wa.S821 ••..•..•••.. IC •••..... •••.... -...... EV'e004468 .. ~ ...... ~K ...... , ""'0 N Table 2. (Continued) "'"o

I_JO_-___, _ 110 III uti 101 Ac4400 GIGCCCGIUIGCiGTGGCGGI·CCGGlCCGICTTTCIGCGIIGIIIITTTICITCCCCmIGIIIGIICGCCfTTGGGCTGGCClGI'lImCI-" "'TlTIC'111 Bai4488a ...... •.. •. ... I. .. G.... 1...... • .•••••...... I ...•.. IC .• I. eecC ..• Cl ..••.• -.•.•• -.H Ill. Cl .. ·GC. Bai4616 ...... •.•... .. •...... •.... I ... G... . 1.....•.. .. •... _• ••...•• . I ••...• IC .. !.CCGC •• . Cl .•• . •• -•.. .• -.H Ill. Cl .. -GC. Cn4486a ...... · .. 1...... I ...... I. . CO ... I ...... • ..... · . I-I m ..... · .. . Cer4486b ...... • .. 1...... I...... I.. CO ..•....•.. .. -.... I.. -·I m ..... -.. . Cor4492a · ...... ' .. I ...... , ...... I ••..•.. .. . I .. ca ..• I •...•.•. -.. •.• ·.H Ill... .. ' .. . Cer4492b ...... • .. 1...... •...... •...... •...•..... I ...... I..CG .•. I ..•.•••. -•..•. ·.I·I m.. ... ·.. . D.. 2642 .. . •...... •...... •...... I...... C...... • ...... -'1 Ill.... . • . . . Din 124 ... •.... I ...... CI .. .. .•..•.. I.. -. IC ...... •..•.. I ...... CGC .....•. ..•. • ..•.•.. OIC Ill.. ·C. · . . . Di12762 ...... I ...... CI...... 1.. -. IC ...... •. I ...... COC ••..•.•.•.. -...... ClC m .. ·C.·.. . Dif2669 ...... I ...... Cl ...... 1.. -. IC ...... •. I .... 1. CGC ...... ••. -•...... lAC -Il.. -c. -•.• Difl!232 ...... I ....• .. Cl ...... 1.. -. IC ...... I .... 1. COC •...... •. • . .•.... IIC Ill.. ·C.·.. . Dill!438 ...... I .•. .... Cl ...... 1.. •. IC ...... •..... I ...... CGC •...... • ...... OIC m .. · C. -•.. Dill!439 •.. -.... I ...... Cl ...... 1.. -. IC .•.••. . ...• .. I ... .. • Coc. •.....• ... -...... GIC Ill.• -C.· ••• Difl!698 ...... I ...... Cl .• ....•...• 1.. •. IC ...... •...... I ...... CGc ...... •••.. .•. OJ( TTI .• ·C.·•.. Dil1!663 ...... I ...... Cl ....•...... 1•. •. IC ...... I ...... m...... -...... m Ill.. ·C.·.. . FulY02906 . . . I ...... • ...... 1.. .. c...... I ...... I .. cc ...... c. . ·...... 11' ..I ..... • .. . G1ab3824b .. . · ...... I .... C...... •... . I ...... I .. CO ....• .• ..... • ...... u· ··I•• C•• • .. . Him4374 ... -...... 1...... I ...... I .. CG .. . . CI •.•. 1. -...... 1" Ill.. " . I . •. IntumSI12 ...... • ...... 1...... 1...... 1...... • .... •• .. •• Lonl2877 ...... I ...... C..•...... 1. C' •. C...•.. I ...... I ..... cm... C• .•.... • ... 1... 111 m .. · .. · .. . ""' Lon12881 ..... ' ...• I .•. . ... C...... 1. C' .. C...... I ...... I .. ... cm... C..•..•. -.•. 1... 111 m ...... :r: Lonl326O ..... -.... I ...... C....•...... 1. C' .. C.•.... I ...... I ....• CCCC ... C... •..• -... I .. . UI m .. · .. · .. . g'" Lonl3664 ..... -... . I. 1...... 1. C- .. C...... I ....•. I .. ... cm..• C...... -.•. 1. . . 111 Ill..••.•••• :l. Lonl38l6& .. . ..•.... I. I ...... •.. ... 1. c' .. C...... I ...... I .•.•. CCOC .•. C....••. -.•• 1... 111 m...... Lonl38ISb · ...... I ...... C...... I.C-.. c...... I ...... I. .. . . cm ... c...... ·... 1... 111 Ill.•••.•..• 00 Lonl3844 ...... I ...... C...... 1. C- .. C...... I ...... I .. ... CCCC •.. C.•.... . • ... 1... 111 TTI. . •. ••. .. ~ Merr4397 •.. IC ...... I ...... I .. C...... I .. .•.. -...... I-· Ill. . ". I... r' Mut4407 ... I ... I ...... I...... •... c...... I ...... · ...... CII· ,11.. ". 11 .. Mut4493a ...... 1. . • 1...... I...... c...... I ...... • ...... W' -11 .. -'.11.. !T'" Nipp3826 .c ...... , ..... • .. , .. , ...... IC ...... I ...... IC ..... ccC... C.. •.. I.·...... ··I OIO ... C.· .. . Z Panl3132 ... · ...... I.·...... I ...... I ... I .. CC· .... I ...... •...... 1-· Panl3144d ... • •...... •...... I. · ...... • I ...... ICI. I.CCG· . .•. I. ••... • ...... I·· Ill.. ". I .. . Pect2989 \0 ..•. , ....•..•... . I •.. G...... , ...... ••...... I ...... IC .. I. CCCC . .. Cl ...... •..•..•. ··C Ill. C... ·GC. "N Pect4373 ...... I .. . G...... I ...... IC .. I.mc... Cl ...... ·.··C Ill. C. .. ·GC. Pnm2736 ...... I·...... I ...... I...... I.CCO· .... I .... I ...... + 1-1..... 1.. . PYim3144c ...... I ...... 1...... I ...... I .. ca, .... I ...... • ...... Rubi3114 ...... I •. • ...... 1...... I ...... I .. C...... C...... • ...... 11, .11...... Rubr2910 ...... • ...... I ...... I ...... I .. C...... I ...... Rubr3067 ·· .... ·· ...... • ...... I ...... I .. C.I .... 1...... U· . 11. C.. I·.. . Subf2879.T ...... 1...... • ...... 1...... • ...... 1...... 1... I .. CO ...... U· -11. .... • .. . Subfl!338.T ...... I ...... · ...... I ...... I ...... I ... I .. CG ...... 11' -10 .. ... • .. . Subt:l823.S ...... 1...... • ...... 1...... • ...... 1...... 1... I .. CG ...... U· -11. .... • .. . Tric2911 ...... •.... I.I ...... I ...... I. C· .. C...... I ...... I ..... CCGI ... C...... IGI Ill.. ·C .... . Tric3216 · .1 ...... I· ... I. I ...... I ...... iCe' .. C...... I ...... I ..... CCOI. I. C...... Ill Ill.. • ...... Tric3236 ...... I. I ...... I ...... 1. C' .. C...... I ...... I .. ... CCOI ... C...... iCT Ill.. · C.· . .. Tric3249 ...... •...... •.... I. I .•. .. • 1...... 1. C' .. C..•.•• I •..... I ..... CCOI •.• C•..•...... iCT TTI.. · C.... . Tric3495_S ...... I ...... I ...... O.iCC· .. C...... I ...... I ...... COl ... C...... Ill CII •• • .. • . . . Tric3809 ...... I. I ...... I ...... 1. C' .. C...... I ...... I ..... CCCI, .. C...... iCT Ill.. ·C .... . Tric3822 ...... I. I ...... 1...... 1. c' .. C...... I ...... I ..... CCOI ... C...... ICT Ill. . ·C.·.. . Tric44I S.S ...... , ...... • ... . I ...... I ...... ICC· .. C...... I ...... 1..... CCcl . .. C...... Ill IGI .. • .. · . G. W.. 29OOb · ...... · ...... I ...... I ...... I .. Ca ...... · . U· -11 ... .. • .. . W.. 2916 ··· .. · ...... • ...... 1...... I ...... I .. CG ... •...... ·.CI· ,11 .. . C.· .. . Wai3281 ...... • ...... 1...... I ...... I .. CO •..•...•.•...... ·. CI· -11. .. .. • .. . W.. 3821 ...... •...... • ...... ••..... 1...... • ...... • I ...... C... I .. CO •...... ·.11- -11 .. . •••.. . EV"8i04468 ...... 1...... •.• . •.. . . C. I ...... 1...... •...... I ...... I ... .• CCOT . .. C.. . •...... TlCIC ··I.... . I .. G N ITS2 o o N y. OHMURA: Phylogeneric evaluation of infra generic groups of the genus Usnea 241

______--'1-"00"-! MJI4407JU b·l· r MJI4493 snea mUla I IS '------Nipp3825 U. nipparensis Ac4400 U. aciculiCera Das2842 U. dasaea ,.------Merr4397 U. merrillii .. III Rubr3057 ] U. rubrotincta Rubr2910 :.:= L-_____ Rubi3114 U. rubicunda f .---_.:;.::c ______Him4374 U. himalayana u.. ,.------intum3112 U. intumescens Pang3144d ] . '--___ Pang3132 U. pangJana ,----- Pygm2736] U . 71 Pygm3144c . pygmOldea cer4486bJ Cer4492a U f 8 Cer4486a . cera ma Cer4492b Subf2879.T ~ _--"'<.r-- Subf333a.T U. subnoridana Subf3823.S Fulvo2906 U. Culvoreagens 65 99 Glab3824b U. glabrescens waS2900bJ Was3821 U. wasmuthii Was2915 Was3281 ,.--__--'-'100 9 Bai4488iil U baileyi '------1 Bai4516.J . 99 '------i P9Ct298;J U. peclinala 100

diffracla ,...--______--'-'100 p~~lu.Di1'3598 9 Di1'3438 Di1'3663 a'j Dif2762 = ='"o 99 83 83 Long3250Lo~~~~77] -=-~ 98 Long3844 U. longissima Q 98 Long3816b Q Long3664 c::: o .go 76 69 60 ri-;~-~-2-!-:'_3~~~~~8l': __~. '"

Tric3236 87 Tric3809 Tric3822 '------Ev-eso4458 Evemia esorediosa t------; 0.01 Fig. J. NJ tree based on sequences ofITS regions including 5.8S rDNA (447 bp after removing gaps) for the infrageneric analysis of the genus Usnea. Kimura 2-parameter dis tance is used. Numbers at branches indicate bootstrap values (~50%) based on 1000 replica tions. OTU names are the same as in Table 1. 242 1. Hattori Bot. Lab. No. 92 2 0 0 2

Dolichousnea and Eumitria from subgenus Usnea as treated in Ohmura (2001) is highly re liable. The subgenus Usnea also forms a monophyletic clade with 81 % bootstrap value. This value is insufficient support in comparison with those of subgenera Dolichousnea and Eumitria. In the subgenus Usnea clade, the monophylous nature of sections Usnea and Ceratinae is inferred. The section Usnea is strongly supported with a high boots trap value (99%), but the section Ceratinae shows a low bootstrap value (less than 50%). As a general rule, if the bootstrap value for a given interior branch is 95% or higher, then the topology at that branch is considered "correct" (Nei & Kumar 2000). Therefore, the lower bootstrap values of the subgenus Usnea and the section Ceratinae clades are not enough to support the branches. These results suggest that the subgenus Usnea and the section Ceratinae can still be subdivided into several viable phylogenetic groups, especially for the section Cer atinae. Therefore, more OTUs of additional species belonging to these groups should be analyzed in order to elucidate the relationships within the groups. The result also suggests a more distant phylogenetic relationship between subgenus Dolichousnea and sub genera Usnea and Eumitria, and a closer relationship between the latter two subgenera, but the bootstrap value (65%) for the branch of subgenera Usnea and Eumitria is insufficient to support. Further data need to be analyzed to confirm these rela tionships. Although subgenus Eumitria is strongly supported by the molecular phylogenetic re sult, this sub genus includes morphologically ambiguous species. Subgenus Eumitria is dis tinguished from other subgenera by having a fistulose axis to the thallus. Usnea baileyi has a typical fistulose axis in the thallus, but U pectinata has a solid axis and the general habit looks like U longissima. Ohrnura (2001) considered that U pectinata belongs to the sub genus Eumitria because this species is closely related to U himantodes which has a fistu lose axis in the mature thallus. The taxonomic position of U pectinata is strongly support ed by the molecular phylogenetic tree as shown in Fig. 1 with high bootstrap value (99%). The taxonomic positions of other groups related to the genus Usnea, also need to be examined using the molecular phylogenetic methods. To date, only four related genera, Letharia, Letharielia, Protousnea and Neuropogon, have been studied (Krog 1976, Walker 1985). However, fresh samples of these groups were unavailable for this study.

ACKNOWLEDGEMENTS I wish to express my sincere gratitude to Prof. H. Kashiwadani of the University of Tokyo, Japan, for his constant and valuable guidance and encouragement throughout this study. I would like to thank Dr L. St. Clair of Brigham Young University, U.S.A. for criti cally reading and linguistically revising the manuscript. I would also like to thank Drs M. Kato, N. Kondo, H. Nozaki and M. Higuchi of the University of Tokyo, Dr M. Inoue of the University of Akita, Japan and Dr K. H. Moon of Sookmyung Women's University, Korea for their valuable advice and critical opinions regarding this study. I am also grateful to Mr H. Sakamoto and Dr H. Koyama for allowing me the opportunity to perform molecular phylogenetic analysis at the National Science Museum, Tokyo. Further thanks are due to Mr K. Miyazaki, Forestry and Forest Products Research Institute, Japan, Mr T. Arikawa, the University of Tokyo and Dr H. Doring, University of Bayreuth, Germany for their tech- y. OHMURA: Phylogeneric evaluation of infrageneric groups of the genus Usnea 243 nical advice and valuable insights concerning molecular phylogenetic analysis.

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