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Gowardia (Parmeliaceae)—a new alectorioid lichen genus with two species PEKKA HALONEN Botanical Museum, Department of Biology, P.O. Box 3000, FI-90014 University of Oulu, Finland e-mail:
[email protected] LEENA MYLLYS AND SAARA VELMALA Botanical Museum, Finnish Museum of Natural History, P.O. Box 7, FI-00014 University of Helsinki, Finland e-mails:
[email protected];
[email protected] HEINI HYVA¨RINEN Botanical Museum, Department of Biology, P.O. Box 3000, FI-90014 University of Oulu, Finland e-mail:
[email protected] ABSTRACT. In recent years, Alectoria in the Acharian sense has been shown to contain several discordant elements, now recognized as separate genera, including Bryoria, Nodobryoria, Pseudephebe and Sulcaria. Here we describe a new segregate genus Gowardia. At present, Gowardia contains two species: G. arctica sp. nov. and G. nigricans (Ach.) comb. nov. (syn. Alectoria nigricans). In addition to several morphological and chemical characters, our decision to recognize Gowardia as distinct from Alectoria is supported by phylogenetic analysis based on combined ITS and GAPDH data. Gowardia arctica is known from Arctic regions of Canada and Russia, while G. nigricans has a wider range. Alectoria vancouverensis is documented from Finland, which is the first report of the species outside the Pacific coast of North America. KEYWORDS. Alectoria, alpine, Arctic, Canada, Gowardia, lichen, molecular phylogeny, secondary chemistry, taxonomy
¤ Alectoria Ach. and its allies (sensu Brodo & Hawksworth 1977) are among the taxonomically most difficult of macrolichens, although they contain many conspicuous and frequently collected species. These lichens are fruticose and beard-like, and may be either pendent or less frequently shrubby and divergent. Apothecia are usually rare and many taxa are known only in a sterile condition. Most species
¤
¤ are confined to trees with acidic bark (Brodo & Hawksworth 1977). Mainly on the basis of the cortical characters and ascospores Brodo and Hawksworth (1977) separated the genus Bryoria Brodo & D. Hawksw. from Alectoria, leaving only eight species in the latter genus. Before this major revision Choisy (1930) had recognized Pseudephebe Choisy as a distinct genus,
The Bryologist 112(1), pp. 138–146 Copyright E2009 by The American Bryological and Lichenological Society, Inc.
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while Bystrek (1971) had published Sulcaria Bystr. Later, Common and Brodo (1995) separated a new genus Nodobryoria Common & Brodo for Bryoria sect. Subdivergentes (Motyka) Brodo & D. Hawksw. Three genera, Alectoria, Oropogon Th. Fr. and Sulcaria, have until recently been assumed to form a monophyletic family, the Alectoriaceae, owing to their large, brown and occasionally multicelled spores (Ka¨rnefelt & Thell 1992). Molecular studies, however, have revealed these genera to form a polyphyletic assemblage (Mattsson & Wedin 1999; Thell et al. 2002, 2004). More comprehensive taxon sampling is still needed to assess the relationships between and within the genera. The purpose of this study is to examine the delimitation of one alectorioid genus, i.e., Alectoria. We show that the genus is not monophyletic, but consists of two separate although rather closely related groups in the Parmeliaceae.
MATERIALS AND METHODS Materials. The studied Alectoria and Gowardia material is deposited in CANL, H, LE, OULU, UPS, and personal herbaria of Trevor Goward in Clearwater (HERB. GOWARD) and Mikhail Zhurbenko in St. Petersburg (HERB. ZHURBENKO). DNA techniques. We used two gene regions, i.e., the ITS (Internal Transcribed Spacers) of the nuclear ribosomal DNA and partial sequences from the protein-coding GAPDH (glyceraldehyde 3-phosphate dehydrogenase gene), which were sequenced to allow simultaneous analysis of the data sets without introducing large amounts of missing data. In the taxon sampling it was essential to take in as many representatives as possible of the Parmeliaceae with a special focus on Alectoria. Unfortunately, three alectorioid genera, i.e., Nodobryoria, Oropogon and Sulcaria were not included as GAPDH sequences could not be obtained. Consequently, altogether 59 terminals were included in the analysis (Fig. 1). All the terminals were sequenced for both gene regions with one exception: GAPDH sequence of Gowardia nigricans Ra¨ma¨ s.n. was included, although ITS sequence for this specimen was not available. Cladonia arbuscula was used as an outgroup. Total genomic DNA of the lichen samples was extracted using either QIAamp DNA Mini Kit or
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DNeasy Tissue Kit (Qiagen), following the instructions of the manufacturer except that the liquid nitrogen phase was omitted. Instead, thallus fragments of approximately 1–3 mm in diameter were ground with a mini-pestle in 40–50 mL of the lysis buffer provided with the kits. The extracted DNA was eluted in 120–140 mL of the elution buffer included in the kits. ITS regions of the nuclear ribosomal DNA were amplified and sequenced using the following primers: ITS1F (Gardes & Bruns 1993), ITS4 (White et al. 1990), ITS1LM (Myllys et al. 1999), ITS2KL (Lohtander et al. 1998) and ITS5 (White et al. 1990). Partial GAPDH gene (approximately 600 bases) was amplified and sequenced with the primer pair Gpd1LM/Gpd2-LM (Myllys et al. 2002). PCR reactions were performed using Ready To Go PCR beads (Pharmacia Biotech) following the manufacturer’s instructions. The following PCR-profile was used: initial denaturation at 95uC for 5 min, followed by 5 cycles of 30 s at 95uC, 30 s at 58uC and 1 min at 72uC, followed by 30 cycles of 30 s at 95uC, 30 s at 56uC and 1 min at 72uC. The cycle ended with a 7 min extension at 72uC. Fragments were purified with three alternative kits: Qiagen’s QIAquick PCR Purification kit (PCR products eluted in 30 ml elution buffer), Qiagen’s mini Elute PCR purification Kit (PCR products eluted 10 ml elution buffer), or with GE Healthcare’s illustra GFX2 PCR DNA and Gel Band Purification Kit (PCR products eluted in 50 ml dH2O). The sequencing reactions were prepared using the BigDye Terminator Cycle Sequencing Reaction Kit v. 2.0 (PE Applied Biosystems). The following schedule was used: denaturation for 1 min at 96uC, then 30 cycles with 30 s at 96uC, annealing for 15 s at 50uC and extension for 4 min at 60uC. The samples were run on an ABI Prism 377 automatic sequencer from Applied Biosystems. Phylogenetic analysis. Unambiguous alignment of the sequences appeared to be impossible, mainly due to the extensive length variation of the ITS and GAPDH intron sequences. This is problematic, since phylogenetic hypotheses are dependent on primary homology assumptions made during alignment, e.g., different alignments may produce different cladograms. Therefore, we decided to base our study on the concept of dynamic homology (Wheeler 1996, 2001) by using direct optimization as implemented in POY (Gladstein
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Figure 1. A strict consensus tree based on ITS and GAPDH data. Bremer support values are shown at nodes.
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& Wheeler 2001). This method requires no separate alignment step prior to analyses, but the search for optimal tree topology and character transformations are combined into one process. Sequences were initially aligned with ClustalX (Thompson et al. 1997) using default parameters. Based on these alignments we divided both ITS and GAPDH sequences into three or four separate data partitions (ITS1, 5.8S, ITS2, and intron 1, exon 1, intron 2, exon 2, respectively) to accelerate direct optimization (Giribet 2001). This partition was done within conservative regions that did not show variation between terminals. After data partitioning the gaps were removed and unaligned sequences were submitted to phylogenetic analysis. We used parsimony as an optimality criterion in our analysis. Transitions, transversions, and indels were given the same weight. Bremer support values (Bremer 1994) were calculated using POY. The command line used in the analyses is given in Appendix 1. Analyses were run in a parallel environment of eight processors of the IBMSC cluster in the CSC, the Finnish IT center for science. Chemistry. The lichen substances of Alectoria and Gowardia specimens were examined by means of thin-layer chromatography (TLC) in solvents A and B according to the methods described by Orange et al. (2001).
RESULTS AND DISCUSSION We obtained altogether 31 new sequences from two loci. These include 15 complete ITS sequences and 16 partial GAPDH sequences (Table 1). The remaining 87 sequences were obtained from GenBank. The combined analysis of ITS and GAPDH data resulted in four equally parsimonious optimizations with a length of 2422 steps. The CPU time used for the analysis was 22091 seconds and during this time 23.5 million trees were evaluated. In the highly resolved strict consensus tree (Fig. 1), Alectoria as currently delimited (Brodo & Hawksworth 1977) is polyphyletic, since Pseudevernia furfuracea (L.) Zopf is nested inside the genus and forms a sister taxon to the new genus Gowardia described in this paper. Within Gowardia, both G. arctica and G. nigricans are monophyletic. The remaining Alectoria forms a well-supported group with A. ochroleuca (Hoffm.) A. Massal. being
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the basal taxon. The relationships of A. imshaugii V. Marcano & A. Morales, A. sarmentosa (Ach.) Ach. and A. vancouverensis (Gyeln.) Brodo & D. Hawksw. remain unresolved. The analysis suggests that the three previously mentioned species are conspecific, but at this point we refrain from making any taxonomic conclusions because many morphological characters separate the species such as isidia or isidia-like spinules of A. imshaugii, and the number of specimens used in our analysis was relatively low; only one A. imshaugii specimen was included, for instance. The analysis also contains a specimen of A. vancouverensis from Finland (Oulun Pohjanmaa, Kiiminki, 2006, Halonen [OULU]), and this is the first record of the species outside the Pacific coast of North America. Furthermore, our results suggest that the other alectorioid genera included in this study, i.e., Bryoria and Pseudephebe are closely related to Alectoria and Gowardia, and together form a monophyletic group where Pseudevernia is included. This is in contrast with the results of Thell et al. (2004), where these genera were widely distributed in the Parmeliaceae. It should be noted, however, that that study also suffered from insufficient sampling. In our future studies, we will continue to add taxa and characters to assess the relationships between and within the alectorioid genera.
Gowardia P. Halonen, L. Myllys, S. Velmala & H. Hyva¨rinen, gen. nov. Thallus erectus aut decumbens, terrestris vel saxatilis, raro corticola. Basi cinerascens vel pallide brunnea et apici cinereus vel nigrescens aut rami omnino nigrescens. Pseudocyphellae albae, fusiformes vel fissural. Apothecia rara. Soralia rarissima. Isidia et pycnidia non visa. TYPE SPECIES: Gowardia nigricans (Ach.) P. Halonen, L. Myllys, S. Velmala & H. Hyva¨rinen Etymology. This lichen genus is dedicated to Trevor Goward, B.C., Canada, for his remarkable and ongoing work on North American lichens published in his lichen guides and numerous articles. Discussion. Gowardia is easily distinguished from Alectoria on the basis of cortical pigments. Alectoria contains usnic acid and has a yellowish or greenish-yellow hue, while this substance is lacking in Gowardia, which instead has melanic pigments yielding a grayish to blackish color. Other
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Table 1. Specimens used in the analyses and GenBank accession numbers for ITS and GAPDH sequences. Collection data are provided for samples sequenced in this study (accession numbers are marked in bold).
Species Ahtiana pallidula Ahtiana sphaerosporella Alectoria imshaugii Alectoria ochroleuca Alectoria sarmentosa Alectoria sarmentosa Alectoria cf. sarmentosa Alectoria sarmentosa ssp. vexillifera Alectoria sarmentosa ssp. vexillifera Alectoria vancouverensis Alectoria vancouverensis Allantoparmelia alpicola Bryoria fuscescens Cetraria commixta Cetraria islandica Cetraria sepincola Cetrariella delisei Cetrelia olivetorum Cladonia arbuscula Coelopogon epiphorellus Cornicularia normoerica Evernia prunastri Everniastrum americanum Everniastrum sp. Everniopsis trulla Flavocetraria cucullata Flavopunctelia flaventior Gowardia arctica Gowardia arctica Gowardia arctica Gowardia arctica Gowardia nigricans Gowardia nigricans Gowardia nigricans Gowardia nigricans Hypogymnia physodes Karoowia sp. Melanelia stygia Menegazzia cincinnata Menegazzia terebrata Namakwa exornata Nephromopsis pseudocomplicata Parmeliopsis ambigua Parmotrema cetratum Parmotrema fistulatum Parmotrema perlatum Parmotrema reticulatum
Locality
Canada, B.C.
Voucher
Goward 05-32 (HERB. GOWARD)
Finland, Oulun Pohjanmaa Halonen s.n. (OULU) Canada, B.C. Goward 05-38 (HERB. GOWARD) U.S.A., Alaska Wright 2005-4-9 (HERB. GOWARD) Finland, Oulun Pohjanmaa Halonen s.n. (OULU) Canada, B.C. Goward 01-542 (HERB. GOWARD) Canada, B.C. Goward 01-810 (HERB. GOWARD) Finland, Oulun Pohjanmaa Halonen s.n. (OULU)
Canada, Nunavut Canada, Nunavut Canada, N.W.T. Russia, Yamalia Russia, Nenetsia Finland, Inarin Lappi Finland, Enontekio¨n Lappi Finland, Enontekio¨n Lappi
Mattsson 5142 (UPS) Mattsson 5115 (UPS) Mattsson 5255 (UPS) Pajunen s.n. (OULU) Kumpula, Strengell & Moilanen s.n. (OULU) Hyva¨rinen s.n. (OULU) Virtanen s.n. (OULU) Ra¨ma¨ s.n. (OULU)
GenBank ITS
GenBank GAPDH
AY353709 AF141859 EU282496 AF451735 EU282494 EU282495 EU282491 EU282493 EU282492 EU282497 EU282498 AY251410 AF451736 AF451796 AF228296 AF152468 AF228305 AF451763 AY170787 AF254633 AY251416 AF451740 AY251418 AY251417 AY251419 AF451793 AY251420 EU282505 EU282504 EU282502 EU282503 EU282500
AY249602 AY249604 EU282511 AY249638 EU282509 EU282510 EU282506 EU282508 EU282507 EU282512 EU282513 AY249626 AY249636 AY249596 AY249594 AY249597 AY249595 AY249611 AY170750 AY249634 AY249623 AY249639 AY249614 AY249613 AY249620 AY249601 AY249606 EU282521 EU282520 EU282518 EU282519 EU282515
EU282499 EU282501 — AF141368 AY251425 AF451775 AF451741 AY251430 AY251432 AF404131 AF451764 AY251449 AY251415 AY586566 AY251450
EU282514 EU282517 EU282516 AY249610 AY249629 AY249607 AY249609 AY249608 AY249630 AY249603 AY249625 AY249616 AY249621 AY249618 AY249617
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Table 1. Continued. Species
Locality
Voucher
Parmotrema sp. Parmotrema tinctorum Platismatia glauca Pseudephebe minuscula Pseudevernia furfuracea Psiloparmelia distincta Tuckermanopsis chlorophylla Usnea florida Usnea (‘‘Neuropogon’’) sp. Xanthomaculina hottentotta Xanthoparmelia conspersa Xanthoparmelia semiviridis
distinctions between the genera appear in Table 2, and include differences in the pseudocyphellae and chemistry. Gowardia is a circumpolar genus essentially restricted to Arctic-alpine localities. By contrast, Alectoria is much more widely distributed, with a center of diversity in temperate regions of western North America (Brodo & Hawksworth 1977). Alectoria ochroleuca and A. sarmentosa ssp. vexillifera (Nyl.) D. Hawksw. occur in Arctic and alpine regions, where they could be found in many areas growing together with Gowardia species.
Gowardia arctica P. Halonen, L. Myllys, S. Velmala & H. Hyva¨rinen, sp. nov. Figs. 2, 3
GenBank ITS
GenBank GAPDH
AY251448 AY251443 AF451758 AY251446 AF451768 AY251447 AF451789 AF451739 AY251434 AY251452 AF451748 AF451746
AY249615 AY249619 AY249593 AY249635 AY249624 AY249622 AY249600 AY249640 AY249637 AY249631 AY249633 AY249628
Thallus decumbens, terrestris, ad 13 cm longus. Rami leviter nitidi et in parte complanati, diametro inaequales, 0.3–0.5(–1) mm crassus, omnino nigrescens vel ex parte fuscus. Pseudocyphellae albae, plerumque planae. Soralia, isidia, pycnidia et apothecia non visa. Acidum alectorialicum et duas substantias ignotas continens. HOLOTYPE: CANADA. N.W.T.: Inuvaluit, Banks Island, Swan Lake, mainly mesic mountain heath, 100 m, 1999, Mattsson 5255 (UPS, isotypes H, OULU). Description. Thallus erect to decumbent, to 13 cm in diam., richly branched, generally shiny, matte in parts, black to brown-black, occasionally lighter brown or even pale gray in basal portions appressed to the substrate; branching pattern anisotomic-
Table 2. Characters separating Alectoria and Gowardia. * 5 not seen in some Alectoria species and G. arctica. ** 5 spore sizes according to Brodo and Hawksworth (1977). Alectoria Habit Color of surface Pseudocyphellae Pycnidia Apothecia* Spores** Usnic acid Alectoronic acid Alectorialic acid Barbatolic acid Substrate Center of diversity
Erect to pendent Yellow, greenish-yellow, or greenish-gray Usually convex, yellowish white, mainly 0.5–1 mm long Present in some taxa Usually plane or concave 28–43.5 3 16–27 mm Present 6 present Absent Absent Mainly corticolous Temperate
Gowardia Erect or decumbent Pale/dark bicolorous to entirely black Usually plane, white, mainly 0.2–0.5 mm long Unknown Usually plane or convex 30–39 3 20–23 mm Absent Absent Present 6 present Mainly on ground Arctic
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Figure 2. Gowardia arctica. Shiny branches of Gowardia arctica. Note flattened parts (Mattsson 5115).
dichotomous becoming isotomic near apices; main branches 0.3–0.5(–1) mm in diam., irregularly shaped, often flattened especially at the axes (Fig. 2), sometimes foveolate; terminal branches 0.1–0.3 mm in diam., tapering or more often varying in diameter; pseudocyphellae (Fig. 3) sparse to rather abundant, white, plane, sometimes slightly raised or depressed, fusiform to fissural, short, mainly 0.2–0.5 mm long. Soralia, isidia, pycnidia and apothecia not seen. Discussion. Together with Gowardia nigricans (see the discussion under G. nigricans), G. arctica could be confused with Bryoria nitidula (Th. Fr.) Brodo & D. Hawksw. That species, however, has dark-colored pseudocyphellae and produces fumarprotocetraric acid (K2, PD+ red). Bryocaulon divergens (Ach.) Ka¨rnefelt, which is a common Arctic-alpine lichen with shiny thallus and white pseudocyphellae, is readily separated by its red-brown color. Chemistry. Lichen substances were investigated from all specimens except the collection from Yamalia (small thallus). The chemistry is uniform and consists of alectorialic acid and two unknown substances A: 3, 2–3/B: 5, 3 (K+ red or orange, C+ green-black, KC+ red, PD+ yellowish). Distribution and ecology. Gowardia arctica has been found from northern regions of Canada and
Figure 3. Gowardia arctica. Shiny branches of Gowardia arctica. Note white pseudocyphellae (arrow) (Mattsson 5255, holotype).
Russia along the Arctic Ocean coast and islands (Fig. 5), where it grows on xeric to moist tundra soil. Brodo and Hawksworth (1977: 64) noted that the specimen from Southampton Island differs in some aspects from the typical Gowardia (Alectoria) nigricans, but they did not want to make a taxonomic assessment having seen but a single specimen. However, this specimen has a somewhat paler color and longer pseudocyphellae than the other G. arctica material. The collections have contained the following associate species: Bryoria nitidula, Caloplaca sp., Cetraria spp., Flavocetraria cucullata, Gowardia nigricans, Hypogymnia cf. subobscura, Leptogium sp., Parmelia omphalodes, Polytrichum piliferum, Pseudephebe pubescens, Stereocaulon alpinum, Thamnolia vermicularis (frequently present) and some unidentified species. Additional specimens examined. CANADA. NUNAVUT: Baffin, Bathurst Island, Dyke Acland Bay, 100–150 m, 1999, Mattsson 5115, 5142 (UPS); Southampton Island, Salmon Pond, 1970, Parker SP70-14 (CANL). RUSSIA. FRANZ-JOSEF LAND: Scott-Keltie Island, 1930, Savicz 985 (LE); WESTERN SIBERIA, YAMALIA: central part of Yamal Peninsula, by Khaleovta Lake, Jul 2005, Pajunen (OULU); CENTRAL SIBERIA, KRASNOYARSK TERRITORY: N part of Bolshevik Island, 20–40 m, 1996, Zhurbenko 96218 (HERB. ZHURBENKO, OULU), 96847
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Figure 4. Bicolor thallus of Gowardia nigricans with slender and dark terminal branches (VIII 1994, Walker). Scale bars 5 5 mm.
(HERB. ZHURBENKO); EASTERN SIBERIA, YAKUTIA: New Siberian Islands, Zhokhov Island, 10–100 m, 1989, Samarskii (HERB. ZHURBENKO).
Gowardia nigricans (Ach.) P. Halonen, L. Myllys, S. Velmala & H. Hyva¨rinen, comb. nov. Cornicularia ochroleuca var. nigricans Ach., Lich. Univ. 615. 1810; Alectoria nigricans (Ach.) Nyl., Lich. Scand. 71. 1861. TYPE: Lapponia (H-ACH!, lectotype). Fig. 4 For further synonyms see Hawksworth (1972: 224) and Brodo and Hawksworth (1977: 63–64). Gowardia nigricans is separated from G. arctica by its matte thallus, which is usually distinctly bicolor, i.e., with dark apical parts and pale branches towards the base. Furthermore, it normally has more conspicuous main branches, while G. arctica has a more decumbent and divergent habit. For a detailed description of the morphology of G. nigricans see Brodo and Hawksworth (1977). In addition to alectorialic acid, G. nigricans also contains barbatolic acid as well as an unknown K+ red or orange compound solely in the dying basal parts (Brodo & Hawksworth 1977); in G. arctica, the latter substance occurs in the entire thallus.
ACKNOWLEDGMENTS We are grateful to Trevor Goward for his coo¨peration and kind help during over a decade with these tangled, fruticose lichen
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Figure 5. Known distribution of Gowardia arctica (the outline map produced by Natural Resources Canada). genera, including Bryoria and Usnea. We warmly thank JanEric Mattsson (Uppsala), Mikhail Zhurbenko (St. Petersburg) and Irwin M. Brodo (Ottawa) for the valuable specimens. The study was financially supported by the Finnish Ministry of Environment as a part of the research program on insufficiently known and threatened forest species (‘‘PUTTE’’).
LITERATURE CITED Bremer, K. 1994. Branch support and tree stability. Cladistics 10: 295–304. Brodo, I. M. & D. L. Hawksworth. 1977. Alectoria and allied genera in North America. Opera Botanica 42: 1–164. Bystrek, J. 1971. Taxonomic studies on the genus Alectoria. Annales Universitatis Mariae Curie-Skłodowska, Sectio C, Biologia 26: 265–279. Choisy, M. 1930. Icones lichenum universalis, Ser. II, Fasc. 2. Oullins. Common, R. S. & I. M. Brodo. 1995. Bryoria sect. Subdivergentes recognized as the new genus Nodobryoria (lichenized Ascomycotina). The Bryologist 98: 189–206. Gardes, M. & T. D. Bruns. 1993. ITS primers with enhanced specificity for basidiomycetes—application to the identification of mycorrhizae and rusts. Molecular Ecology 2: 113–118. Giribet, G. 2001. Exploring the behavior of POY, a program for direct optimization of molecular data. Cladistics 17: 60–70. Gladstein, D. & W. C. Wheeler. 2001. POY documentation and command summary. Available at ftp://ftp.amnh.org/pub/ molecular/poy. Goloboff, P. A. 1999. Analyzing large data sets in reasonable times: solutions for composite optima. Cladistics 15: 415–428.
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Hawksworth, D. L. 1972. Regional studies in Alectoria (Lichenes) II. The British species. Lichenologist 5: 181–261. Janies, D. & W. C. Wheeler. 2002. POY documentation and command summary. Update 6. Nov 2006. American Museum of Natural History, New York, U.S.A. Ka¨rnefelt, I. & A. Thell. 1992. The evaluation of characters in lichenized families, exemplified with the alectorioid and some parmelioid genera. Plant Systematics and Evolution 180: 181–204. Lohtander, K., L. Myllys, R. Sundin, M. Ka¨llersjo¨ & A. Tehler. 1998. The species pair concept in the lichen Dendrographa leucophaea (Arthoniales): analyses based on ITS sequences. The Bryologist 101: 404–411. Mattsson, J.-E. & M. Wedin. 1999. A re-assessment of the family Alectoriaceae. Lichenologist 31: 431–440. Myllys, L., K. Lohtander, M. Ka¨llersjo¨ & A. Tehler. 1999. Sequence insertion and ITS data provide congruent information in Roccella canariensis and R. tuberculata (Arthoniales, Euascomycetes) phylogeny. Molecular Phylogenetics and Evolution 12: 295–309. ———, S. Stenroos & A. Thell. 2002. New genes for phylogenetic studies of lichenized fungi: glyceraldehyde-3phosphate dehydrogenase and beta-tubulin genes. Lichenologist 34: 237–246. Orange, A., P. W. James & F. J. White. 2001. Microchemical Methods for the Identification of Lichens. British Lichen Society. Thell, A., S. Stenroos, T. Feuerer, I. Ka¨rnefelt, L. Myllys & J. Hyvo¨nen. 2002. Phylogeny of cetrarioid lichens (Parmeliaceae) inferred form ITS and b-tubulin sequences, morphology, anatomy and secondary chemistry. Mycological Progress 1: 335–354. ———, T. Feuerer, I. Ka¨rnefelt, L. Myllys & S. Stenroos. 2004. Monophyletic groups within the Parmeliaceae identified by ITS rDNA, b-tubulin and GAPDH sequences. Mycological Progress 3: 297–314. Thompson, J. D., T. J. Gibson, F. Plewniak, F. Jeanmougin & D. G. Higgins. 1997. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 24: 4876–4882. Wheeler, W. 1996. Optimization alignment: the end of multiple sequence alignment in phylogenetics? Cladistics 12: 1–9. ———. 2001. Homology and the optimization of DNA sequence data. Cladistics 17: 3–11. White, T. J., T. D. Bruns, S. Lee & J. W. Taylor. 1990. Amplification and direct sequencing of fungal ribosomal genes for phylogenetics. Pages 315–322. In M. A. Innis, D. H. Gelfand, J. J. Sninsky & T. J. White (eds.), PCR Protocols. Academic Press, San Diego, CA. ms. received April 23, 2007; accepted February 22, 2008.
Appendix 1. The commands and a brief explanation of the commands used in POY analysis (Janies & Wheeler 2002). Parallel: executes jobs in parallel using PVM. Solospawn: sets the number of slave jobs to be spawned in a multiprocessor computer. Molecularmatrix: reads in a cost matrix-specifying transition, transversion, and gap costs. In this case each of these transformations is weighted equally. Maxtrees 5: set maximum number of trees held in buffers to five. Holdmaxtrees 30: number of trees to hold over all random replicates set to 30. Random 25: 25 random addition sequence searches performed. Multibuild 5: specifies number of random addition builds performed on slave nodes. Ratchettbr 3: number of iterative rounds of ratcheting with TBR branch swapping. Ratchettrees 2: number of trees saved during ratchet iterations. Treefuse: performs treefusing (Goloboff 1999). Fuselimit 25: limits the number of tree fusing pairs to 25. Fusingrounds 1: performs treefusing and fuses the resulting trees once. Slop 2: check all cladogram lengths, which are within 0.2% of the minimum tree length. Checkslop 5: by adding an extra round of TBR branch-swapping checks all cladogram lengths within 0.5% of the minimum length. Seed -1: sets pseudorandom number generation to pick the system time, in seconds, to be used as a seed. Fitchtrees: ensures that trees kept in buffer are a random subset of those that would have been kept if the buffer had been larger. Noleading: leading and trailing gaps are initially accounted for to prevent a trivial nonoverlapping alignment but not counted when determining tree length. Norandomizeoutgroup: the first terminal of the first data set used as the starting point for adding the other terminals. Indices: prints out tree statistics. Impliedalignment: generates a topology specific multiple alignment.
