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A COMPARISON OF MORPHOLOGICAL, CHEMICAL AND MOLECULAR CHARACTERS IN SOME PARMELIOID GENERA
Lichenologist 31(5): 451–460 (1999) Article No. lich.1999.0222 Available online at http://www.idealibrary.com on
A COMPARISON OF MORPHOLOGICAL, CHEMICAL AND MOLECULAR CHARACTERS IN SOME PARMELIOID GENERA Ana CRESPO*, Rosario GAVILÁN*, John A. ELIX† and Gabriel GUTIÉRREZ§
Abstract: Morphological and chemical data from 64 genera of Parmeliaceae, including the most common genera in Europe, were analysed by means of neighbour-joining and parsimony analysis. Two presence/absence matrices were constructed, one for the 64 genera and a smaller one for the most common European genera. Probabilities were too low to allow grouping of the genera and only a few pairs of genera had a probability of >50% for both matrices. A molecular matrix (nuclear rDNA-ITS) of the most common genera in Europe was constructed and analysed by the method of maximum likelihood. These results were much more informative than the non-molecular matrix for establishing phylogenetic 1999 The British Lichen Society relationships among the genera.
Introduction The Parmeliaceae is undoubtedly one of the largest families of lichen-forming fungi, containing approximately 2319 species in 85 genera (Hawksworth et al. 1995). The cetrarioid group of genera within this family has been studied from taxonomic and phylogenetic perspectives (Kärnefelt et al. 1992) on the basis of morphological characters. Although the genera of the parmelioid taxa (most of which are included in the broad definition of Parmelia s. lat., the largest group in the family) have been studied from morphological and chemical perspectives (Elix 1993), their phylogeny had not. In recent years molecular methodology has provided a useful means for grouping and establishing phylogenetic relationships among the parmelioid taxa by using sequences of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (rDNA) (Crespo & Cubero 1998). However, there are clear advantages for using both molecular and morphological data (Tehler 1995a, b) in the study of phylogenetic relationships since they have complementary strengths. The aim of our study was to compare the results of different phylogenetic analyses of parmelioid genera by using the extensive morphological data as summarized by Elix (1993), and the data available from ITS sequences. A *Departamento de Biología Vegetal II, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain. ‡Department of Chemistry, Australian National University, Canberra, A.C.T. 0200, Australia. §Departmento de Genética, Universidad de Sevilla, 41080 Sevilla, Spain. 0024–2829/99/050451+10 r30.00/0
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similar methodology was used to study the two datasets in order to determine whether they were congruent, whether one was more informative than the other, and to test different analytical methods. Materials and Methods Taxa sampled For the general analysis based on non-molecular characters, all 64 genera included in Elix (1993) were used. However, the genera selected for the comparative study included most of those that occur in the Iberian Peninsula and for which we have molecular information: Flavoparmelia, Flavopunctelia, Hypogymnia, Hypotrachyna, Melanelia, Neofuscelia, Parmelia, Parmelina, Parmelinopsis, Parmotrema, Platismatia, Pleurosticta, Pseudevernia, Rimelia and Xanthoparmelia. Non-molecular data Data were taken from the synoptic study of the Parmeliaceae (Elix 1993), which considered 64 genera distributed worldwide. The characters were grouped in eight major categories: upper cortex, pseudocyphellae, cell-wall polysaccharides, apothecial and spore characters, pycnidial and conidial characters, secondary thalline characters, medullary chemistry, geographical distribution and ecology (Table 1). A total of 116 character states could be recognizd for these characters and their presence or absence (1/0) was coded for each of the 64 genera. Molecular data Nuclear rDNA sequences of the ITS regions for 16 parmelioid species in 13 genera were already available (Crespo & Cubero 1998). Two new sequences, from Melanelia exasperata and Pseudevernia furfuracea, have been added. For analysis of molecular data, only a single species of each genus was considered, with the exception of the genus Melanelia. The type species was chosen when the data were available. The sequence of Hypogymnia tubulosa was included in the dataset since it may belong in the Parmeliaceae (Hawksworth et al. 1995). Usnea rigida (Grube, unpublished) was used as the outgroup because it is considered to be more closely related to the ingroup than the three species of the Physciaceae employed in Crespo & Cubero (1998). Information on the isolation of DNA, primers and PCR conditions are described in Crespo et al. (1997). Analysis of the non-molecular data Two different matrices were analysed by parsimony and neighbour-joining methods: one with all of the genera and 116 character states identified in Elix (1993) and a second, from the same source, but restricted to the 14 genera for which molecular data were also available. Hypogymnia was not included since it was not recognized as a parmelioid by Elix (1993). This latter matrix was comprized of the 88 non-molecular character states that were present in some of the 15 genera, including the outgroup. All analyses were performed using the PHYLIP 3·5 c package (Felsenstein 1993). Trees were derived from 100 and 500 bootstrap replications and were performed for the large and small matrices, respectively. As the main aim of this paper was to compare the relationships among the parmelioid genera, the cetrarioid genus Coelocaulon [according to the definition of Elix (1993)] was chosen as the internal outgroup in both matrices. Analysis of the molecular data As a similar dataset had already been analysed by neighbour-joining and parsimony methods (Crespo & Cubero 1998), the maximum likelihood method was selected here for tree reconstruction. This method uses the HKY mathematical model (Hasegawa et al. 1985). Since the maximum likelihood method required extensive computation time, an exhaustive search could not be carried out with more than ten sequences. To overcome this limitation the MOLPHY software package was employed (Adachi & Hasegawa 1996). Initially, a maximum likelihood distance matrix between all sequences was calculated using the HKY model. The NUCML
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Parmelioid genera—Crespo et al. T 1. List of non-molecular characters taken from Elix (1993) Upper cortex (18) Nature of epicortex 1. Pored epicortex 2. Non-pored epicortex 3. Minutely-pored epicortex Anatomy of the upper cortex 4. Bilayered 5. Palisade plectenchymatous 6. Paraplectenchymatous 7. Prosoplectenchymatous 8. Vaulted Maculae 9. Reticulatae 10. Effigurate 11. Simple Colour and chemistry of upper cortex 12. Yellow-green or yellow-brown, usnic acid dominant 13. Grey, atranorin dominant 14. Grey, lichexanthone dominant 15. Brown (HNO3 +blue-green) 16. Brown (HNO3 +violet or red) 17. Brown (HNO3 ) 18. Dull grey yellow Pseudocyphellae (6) Position 19. On upper surface 20. On lower surface 21. Absent Nature of pseudocyphellae 22. Linear or effigurate 23. Punctiform 24. Tuberculate Cell-wall chemistry (4) 25. Xanthoparmelia-type lichenan 26. Cetraria-type lichenan 27. Intermediate-type lichenan 28. Isolichenan and/or other polysaccharides Apothecial and spore characters (18) Position of apothecia 29. Laminal (aspicilioid) 30. Laminal (stipitate) 31. Laminal (sessile or substipitate) 32. Marginal 33. Nephromoid 34. Terminal Disc 35. Perforate Number of spores 36. Asci multi-spored 37. Asci 8-spored Length of ellipsoid spores 38. More than 20 m 39. 15–20 m 40. 10–15 m 41. 6–10 m
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THE LICHENOLOGIST T 1. Continued. Diameter of spores 42. Less than 6 m 43. More than 6 m Length of curved spores 44. Less than 7 m 45. More than 10 m Spores bicornute 46. 10–20 m long Pycnidial and conidial characters (16) Pycnidial position 47. Apothecial 48. Laminal 49. Marginal 50. Terminal Pycnidial emergence (thalline) 51. Emergent 52. Immersed 53. Verruciform Type of conidiophore 54. Psora-type 55. Parmelia-type Shape of conidia 56. Bifusiform 57. Cylindrical 58. Falcate 59. Filiform 60. Fusiform 61. Sublageniform 62. Unciform Secondary thalline characters (25) Growth form 63. Subcrustose 64. Subfruticose or fruticose 65. Umbilicate 66. Vagrant (unattached) 67. Foliose Lower surface. Type of rhizines 68. Agglutinate 69. Dichotomous 70. Dimorphous 71. Fasciculate 72. Furcate 73. Simple 74. Squarrose Other lower surface features 75. Pseudocyphellae 76. Velvety 77. Thick hypothallus present 78. Canaliculate 79. With basal holdfast or umbilicus 80. With hapters or rhizoids 81. Ecorticate (at least in part) 82. Convex (upper surface canaliculate) 83. Convex (lobs terete)
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T 1. Continued. Cilia 84. Simple 85. Branched 86. Bulbate 87. Tapered Medullary chemistry (13) 88. Orcinol depsides 89. Orcinol depsidones 90. -Orcinol depsides 91. -Orcinol depsidones 92. Aliphatic acids 93. Triterpenes 94. Benzyl esters 95. Anthraquinones or other quinones 96. Secalonic acids 97. Amino acid derivatives 98. Pulvinic acid derivatives 99. Diphenyl ethers 100. Xanthones Geography and ecology (16) Phytogeography 101. Cosmopolitan 102. Africa 103. Asia 104. Australasia 105. Europe 106. North America 107. South America Ecology 108. Arctic (or antarctic)-alpine 109. Boreal (or austral-cool temperate) 110. Arid-subarid 111. Temperate 112. Subtropical-tropical 113. Montane tropical Substrate 114. Saxicolous 115. Terricolous 116. Corticolous **The number of states included in the eight major categories of characters is indicated in brackets. All 116 character states were used in the analysis of 64 genera matrix. The 88 character states used in the matrix of 15 genera are indicated in bold type.
program of MOLPHY was then used with option D. Next, a neighbour-joining method was employed to reconstruct a tree from the matrix using the NJDIST programme of MOLPHY. This seed tree was then improved using the maximum likelihood method of option R (local rearrangement search) of the NUCML programme. In this limited search, branches are changed until the tree reaches maximum likelihood under the HKY substitution model. The tree obtained in this way may not be the maximum likelihood tree but may be considered to be a good approximation to it. This programme also calculates a local bootstrap probability (LBP, Hasegawa & Kishino 1994) to indicate the reliability of the tree nodes.
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THE LICHENOLOGIST Tuckermannopsis Allantoparmelia Masonhalea Cornicularia Arctoparmelia
56 52
80
88
56
Cetrariastrum Everniastrum Canomaculina Rimeliella Flavoparmelia Parmelina Myelochroa Parmotrema Melanelia Parmelia Relicina Bulbothrix Parmelinella Psiloparmelia Xanthoparmelia Karoowia Xanthomaculina Almbornia Omphalophora Omphalodium Rhizoplaca Parmotrema Punctelia Cetrelia Flavopunctelia Platismatia Everniopsis Placoparmelia Omphalodiella Pleurosticta Pannoparmelia Parmeliopsis Imshaugia Parmelaria Ahtiana Esslingeria
Vol. 31 Vulpicida Brodoa Asahinea Pseudevernia Concamerella
Canoparmelia Parmelinopsis Rimelia Hypotrachyna Anzia
Relicinopsis Neofuscelia Paraparmelia Chondropsis Namakwa
Pseudoparmelia
Coelopogon
Cetreliopsis Nephromopsis Cetrariopsis Allocetraria Coelocaulon Cetraria
F. 1. Neighbour-joining analysis of 64 genera of the Parmeliaceae based on 116 character states (including chemical, biogeographical and ecological data). Bootstrap values over 50 are indicated.
Results Non-molecular data The two methods yielded similar results when applied to the complete dataset (Fig. 1: only the neighbour-joining tree is shown; the parsimony tree is available upon request). Although Coelocaulon was chosen as the internal outgroup, Cetraria also remained independent of the rest of the genera. Most of the genera remained independent from one another, but several pairs of genera exhibited over 50% probability in the bootstrap analysis: Cetrariastrum and Everniastrum, Canomaculina and Rimeliella, Relicina and Bulbothrix, Omphalophora and Omphalodium, Cetrelia and Flavopunctelia (both of which were
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Parmelia
Pleurosticta Pseudevernia Platismatia Flavopunctelia Parmelina
Flavoparmelia Parmelinopsis Rimelia 74 Parmotrema
Hypotrachyna Xanthoparmelia 83 Neofuscelia
Melanelia
Coelocaulon F. 2. Neighbour-joining analysis of 15 genera of the Parmeliaceae occurring in the Iberian Peninsula. The study is based on 88 characters (including chemical, biogeographical and ecological data). Bootstrap values over 50 are indicated.
weakly related to Punctelia), Placoparmelia and Omphalodiella, and Parmeliopsis and Imshaugia. Results obtained from the two analytical methods from the 14 species selected for the comparative study were quite similar (Fig. 2), as they were with those extracted from the complete generic dataset. However, in this case, the pairs of genera that exhibited the highest bootstrap probabilities were Rimelia and Parmotrema and Xanthoparmelia and Neofuscelia.
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Pleurosticta acetabulum Pseudevernia furfuracea 58 85
Hypogymnia tubulosa Hypotrachyna revoluta
96
Parmelinopsis horrescens Parmelina quercina 89
75
Platismatia glauca Melanelia glabra Parmelia saxatilis Melanelia exasperata
83 94
Xanthoparmelia tinctina
73
Neofuscelia delisei Flavopunctelia flaventior 97
Flavoparmelia caperata 76
Parmotrema crinitum 100
Rimelia reticulata Usnea rigida
F. 3. Maximum likelihood analysis of 15 genera of the Parmeliaceae occurring in the Iberian Peninsula (Hypogymnia tubulosa was also included). The study is based on nuclear rDNA ITS sequences. Bootstrap values over 50 are indicated.
Molecular data Analysis of the sequence data gave a single maximum likelihood tree (Fig. 3). In this figure the numbers indicate nodes with bootstrap values over 50%. Pleurosticta acetabulum was placed as the initial sister group in the first node but this node had a low probability (58% LBS). The next branch had a well-supported second node (89% LBS) that grouped three branches of all the remaining taxa. In one of these branches there was a node grouping a further three branches each supporting a monophyletic group—namely Pseudevernia, Hypogymnia tubulosa and another well-supported group (96% LBS) of Hypotrachyna revoluta and Parmelinopsis horrescens. The second branch of the second node bore a further node (75% LBS) with three branches, representing Parmelina, Platismatia and Melanelia glabra. The third branch of the second
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node presented a new node (83% LBS) with two clades, where Parmelia was the sister group to the remaining taxa. The remaining taxa arose from a node (94% LBS) with three branches; with Melanelia exasperata as one terminal branch, Xanthoparmelia tinctina and Neofuscelia delisei forming a monophyletic group and, finally, the monophyletic branch with a node (97% LBS) in which Flavopunctelia was the sister group to Flavoparmelia and to Parmotrema crinitum and Rimelia reticulata. The latter pair also form a monophyletic group. Discussion The analysis of the non-molecular results for the 64 genera of the family yielded some relevant information. A previous phylogenetic study of the cetrarioid genera (Kärnefelt et al. 1992) revealed Coelocaulon and Cetraria to be more closely related to one another than to the remaining taxa. The tree provided less information about the remaining 62 genera. In the neighbourjoining analysis, eight pairs of genera were found to be closely related morphologically with a bootstrap probability greater than 50%. These included Cetrariastrum and Everniastrum, Relicina and Bulbothrix, Omphalophora and Omphalodium and Parmeliopsis and Imshaugia. The relationships of these taxa were discussed previously by Elix (1993). The close relationship between Canomaculina and Rimeliella was first described very recently (Elix 1997) and was confirmed by the current analysis, with a support of 64%. Canomaculina was formerly considered to be close to Parmotrema (Elix 1993; Krog & Swinscow 1981), but this relationship was not confirmed in the present study. In the neighbour-joining analysis Cetrelia and Flavopunctelia formed a pair (52% support), and the parsimony analysis indicated a relationship with Punctelia in a node with very low support. The relationship between Omphalodiella and Placoparmelia (68% support from neighbour-joining and 56% in parsimony analysis) had not been previously recognized. Both are monotypic genera from Patagonia (Argentina) that have aspicilioid ascomata and similar ecological affinities (Elix 1993), but unfortunately their cell wall chemistry is unknown. In the case of the 14 European genera, parsimony and neighbour-joining analysis of the non-molecular data revealed a close relationship only between Rimelia and Parmotrema and between Xanthoparmelia and Neofuscelia, while the remaining taxa occupied unresolved branches. However, the results from the molecular study revealed considerably more about the relationships between these genera. Maximum likelihood analysis showed a congruent topology and a much higher resolution by which to elucidate relationships between the genera, for example, the early isolation of Pleurosticta from all other taxa. It also revealed similar relationships between Parmotrema and Rimelia, as the non-molecular neighbour joining tree, but forming a monophyletic group with Flavopunctelia and Flavoparmelia. These relationships had not been recognized previously. These results are consistent with others from a previous neighbour-joining analysis (Crespo & Cubero 1998) that identified the relationships between Flavoparmelia and Parmotrema.
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Other established relationships have been confirmed. Thus, Hypotrachyna revoluta and Parmelinopsis horrescens appear to form a monophyletic group, as do the species of Xanthoparmelia and Neofuscelia. Pseudevernia furfuracea, a hitherto unstudied species, has been placed close to Hypogymnia and to the sister pair Hypotrachyna revoluta and Parmelinopsis horrescens. The other new sequence, that of Melanelia exasperata, appears to be quite distant from M. glabra. Melanelia exasperata was introduced into this analysis because of its peculiar morphological features (verrucose pseudocyphellae). This result confirms that these two Melanelia species are not monophyletic for this gene. In contrast, M. glabra and M. subargentifera, which are morphologically closely related on the basis of their hairy upper cortex, have been found to be monophyletic (Crespo & Cubero 1998) for this gene. We thank Martin Grube (Graz), Mats Wedin (London) and Paul Blanz (Graz) for the organization of the Graz Workshop and for kindly inviting us to present this work. We are also indebted to Martin Grube for permitting us the use of the unpublished sequence of Usnea rigida. Thanks also to Oscar F. Cubero for his help with computation and for discussion. This work was supported by the Ministerio de Educación y Cultura (PB 93–1129-CO2–01; APC960082). R Adachi, J. & Hasegawa, M. (1996) Molphy Version 2·3. Programs for molecular phylogenetics based on maximum likelihood. Tokyo: Institute of Statistical Mathematics. Crespo, A. & Cubero, O. F. (1998) A molecular approach to the circumscription and evaluation of some genera segregated from Parmelia s. lat. Lichenologist 30: 369–380. Crespo, A., Bridge, P. D. & Hawksworth, D. L. (1997) Amplification of fungal rDNA-ITS regions from non-fertile specimens of the lichen-forming genus Parmelia. Lichenologist 29: 275–282. Elix, J. A. (1993) Progress in the generic delimitation of Parmelia s. lat. lichens (Ascomycotina: Parmeliaceae) and a synoptic key to the Parmeliaceae. Bryologist 96: 359–383. Elix, J. A. (1997) The lichen genera Canomaculina and Rimeliella (Ascomycotina, Parmeliaceae). Mycotaxon 65: 475–479. Felsenstein, J. (1993) PHYLIP (Phylogeny Inference Package) version 3·5c. Seattle: Department of Genetics, University of Washington. Hasegawa, M. and Kishino, H. (1994) Accuracies of the simple methods for estimating the bootstrap probability of a maximum likelihood tree. Molecular Biology and Evolution 11: 142–145. Hasegawa, M., Kishino, H. & Yano, T. (1985) Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. Journal of Molecular Evolution 22: 160–174. Hawksworth, D. L., Kirk, P. M., Sutton, B. C. & Pegler, D. J. (1995) Ainsworth & Bisby’s Dictionary of the Fungi. 8th Edn. Wallingford: CAB International. Kärnefelt, I., Mattsson, J-E & Thell, A. (1992) Evolution and phylogeny of cetrarioid lichens. Plant Systematics and Evolution 183: 113–160. Krog, H. & Swinscow, T. D. V. (1981) Parmelia subgenus Amphigymnia (lichens) in East Africa. Bulletin of the British Museum (Natural History), Botany Series 9: 143–231. Tehler, A. (1995a) Morphological data, molecular data and total evidence in phylogenetic analysis. Canadian Journal of Botany 73: 667–676. Tehler, A. (1995b) Arthoniales, phylogeny as indicated by morphological and rDNA sequence data. Cryptogamic Botany 5: 82–97. Accepted for publication 3 April 1999
A COMPARISON OF MORPHOLOGICAL, CHEMICAL AND MOLECULAR CHARACTERS IN SOME PARMELIOID GENERA Ana CRESPO*, Rosario GAVILÁN*, John A. ELIX† and Gabriel GUTIÉRREZ§
Abstract: Morphological and chemical data from 64 genera of Parmeliaceae, including the most common genera in Europe, were analysed by means of neighbour-joining and parsimony analysis. Two presence/absence matrices were constructed, one for the 64 genera and a smaller one for the most common European genera. Probabilities were too low to allow grouping of the genera and only a few pairs of genera had a probability of >50% for both matrices. A molecular matrix (nuclear rDNA-ITS) of the most common genera in Europe was constructed and analysed by the method of maximum likelihood. These results were much more informative than the non-molecular matrix for establishing phylogenetic 1999 The British Lichen Society relationships among the genera.
Introduction The Parmeliaceae is undoubtedly one of the largest families of lichen-forming fungi, containing approximately 2319 species in 85 genera (Hawksworth et al. 1995). The cetrarioid group of genera within this family has been studied from taxonomic and phylogenetic perspectives (Kärnefelt et al. 1992) on the basis of morphological characters. Although the genera of the parmelioid taxa (most of which are included in the broad definition of Parmelia s. lat., the largest group in the family) have been studied from morphological and chemical perspectives (Elix 1993), their phylogeny had not. In recent years molecular methodology has provided a useful means for grouping and establishing phylogenetic relationships among the parmelioid taxa by using sequences of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA (rDNA) (Crespo & Cubero 1998). However, there are clear advantages for using both molecular and morphological data (Tehler 1995a, b) in the study of phylogenetic relationships since they have complementary strengths. The aim of our study was to compare the results of different phylogenetic analyses of parmelioid genera by using the extensive morphological data as summarized by Elix (1993), and the data available from ITS sequences. A *Departamento de Biología Vegetal II, Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain. ‡Department of Chemistry, Australian National University, Canberra, A.C.T. 0200, Australia. §Departmento de Genética, Universidad de Sevilla, 41080 Sevilla, Spain. 0024–2829/99/050451+10 r30.00/0
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similar methodology was used to study the two datasets in order to determine whether they were congruent, whether one was more informative than the other, and to test different analytical methods. Materials and Methods Taxa sampled For the general analysis based on non-molecular characters, all 64 genera included in Elix (1993) were used. However, the genera selected for the comparative study included most of those that occur in the Iberian Peninsula and for which we have molecular information: Flavoparmelia, Flavopunctelia, Hypogymnia, Hypotrachyna, Melanelia, Neofuscelia, Parmelia, Parmelina, Parmelinopsis, Parmotrema, Platismatia, Pleurosticta, Pseudevernia, Rimelia and Xanthoparmelia. Non-molecular data Data were taken from the synoptic study of the Parmeliaceae (Elix 1993), which considered 64 genera distributed worldwide. The characters were grouped in eight major categories: upper cortex, pseudocyphellae, cell-wall polysaccharides, apothecial and spore characters, pycnidial and conidial characters, secondary thalline characters, medullary chemistry, geographical distribution and ecology (Table 1). A total of 116 character states could be recognizd for these characters and their presence or absence (1/0) was coded for each of the 64 genera. Molecular data Nuclear rDNA sequences of the ITS regions for 16 parmelioid species in 13 genera were already available (Crespo & Cubero 1998). Two new sequences, from Melanelia exasperata and Pseudevernia furfuracea, have been added. For analysis of molecular data, only a single species of each genus was considered, with the exception of the genus Melanelia. The type species was chosen when the data were available. The sequence of Hypogymnia tubulosa was included in the dataset since it may belong in the Parmeliaceae (Hawksworth et al. 1995). Usnea rigida (Grube, unpublished) was used as the outgroup because it is considered to be more closely related to the ingroup than the three species of the Physciaceae employed in Crespo & Cubero (1998). Information on the isolation of DNA, primers and PCR conditions are described in Crespo et al. (1997). Analysis of the non-molecular data Two different matrices were analysed by parsimony and neighbour-joining methods: one with all of the genera and 116 character states identified in Elix (1993) and a second, from the same source, but restricted to the 14 genera for which molecular data were also available. Hypogymnia was not included since it was not recognized as a parmelioid by Elix (1993). This latter matrix was comprized of the 88 non-molecular character states that were present in some of the 15 genera, including the outgroup. All analyses were performed using the PHYLIP 3·5 c package (Felsenstein 1993). Trees were derived from 100 and 500 bootstrap replications and were performed for the large and small matrices, respectively. As the main aim of this paper was to compare the relationships among the parmelioid genera, the cetrarioid genus Coelocaulon [according to the definition of Elix (1993)] was chosen as the internal outgroup in both matrices. Analysis of the molecular data As a similar dataset had already been analysed by neighbour-joining and parsimony methods (Crespo & Cubero 1998), the maximum likelihood method was selected here for tree reconstruction. This method uses the HKY mathematical model (Hasegawa et al. 1985). Since the maximum likelihood method required extensive computation time, an exhaustive search could not be carried out with more than ten sequences. To overcome this limitation the MOLPHY software package was employed (Adachi & Hasegawa 1996). Initially, a maximum likelihood distance matrix between all sequences was calculated using the HKY model. The NUCML
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Parmelioid genera—Crespo et al. T 1. List of non-molecular characters taken from Elix (1993) Upper cortex (18) Nature of epicortex 1. Pored epicortex 2. Non-pored epicortex 3. Minutely-pored epicortex Anatomy of the upper cortex 4. Bilayered 5. Palisade plectenchymatous 6. Paraplectenchymatous 7. Prosoplectenchymatous 8. Vaulted Maculae 9. Reticulatae 10. Effigurate 11. Simple Colour and chemistry of upper cortex 12. Yellow-green or yellow-brown, usnic acid dominant 13. Grey, atranorin dominant 14. Grey, lichexanthone dominant 15. Brown (HNO3 +blue-green) 16. Brown (HNO3 +violet or red) 17. Brown (HNO3 ) 18. Dull grey yellow Pseudocyphellae (6) Position 19. On upper surface 20. On lower surface 21. Absent Nature of pseudocyphellae 22. Linear or effigurate 23. Punctiform 24. Tuberculate Cell-wall chemistry (4) 25. Xanthoparmelia-type lichenan 26. Cetraria-type lichenan 27. Intermediate-type lichenan 28. Isolichenan and/or other polysaccharides Apothecial and spore characters (18) Position of apothecia 29. Laminal (aspicilioid) 30. Laminal (stipitate) 31. Laminal (sessile or substipitate) 32. Marginal 33. Nephromoid 34. Terminal Disc 35. Perforate Number of spores 36. Asci multi-spored 37. Asci 8-spored Length of ellipsoid spores 38. More than 20 m 39. 15–20 m 40. 10–15 m 41. 6–10 m
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THE LICHENOLOGIST T 1. Continued. Diameter of spores 42. Less than 6 m 43. More than 6 m Length of curved spores 44. Less than 7 m 45. More than 10 m Spores bicornute 46. 10–20 m long Pycnidial and conidial characters (16) Pycnidial position 47. Apothecial 48. Laminal 49. Marginal 50. Terminal Pycnidial emergence (thalline) 51. Emergent 52. Immersed 53. Verruciform Type of conidiophore 54. Psora-type 55. Parmelia-type Shape of conidia 56. Bifusiform 57. Cylindrical 58. Falcate 59. Filiform 60. Fusiform 61. Sublageniform 62. Unciform Secondary thalline characters (25) Growth form 63. Subcrustose 64. Subfruticose or fruticose 65. Umbilicate 66. Vagrant (unattached) 67. Foliose Lower surface. Type of rhizines 68. Agglutinate 69. Dichotomous 70. Dimorphous 71. Fasciculate 72. Furcate 73. Simple 74. Squarrose Other lower surface features 75. Pseudocyphellae 76. Velvety 77. Thick hypothallus present 78. Canaliculate 79. With basal holdfast or umbilicus 80. With hapters or rhizoids 81. Ecorticate (at least in part) 82. Convex (upper surface canaliculate) 83. Convex (lobs terete)
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T 1. Continued. Cilia 84. Simple 85. Branched 86. Bulbate 87. Tapered Medullary chemistry (13) 88. Orcinol depsides 89. Orcinol depsidones 90. -Orcinol depsides 91. -Orcinol depsidones 92. Aliphatic acids 93. Triterpenes 94. Benzyl esters 95. Anthraquinones or other quinones 96. Secalonic acids 97. Amino acid derivatives 98. Pulvinic acid derivatives 99. Diphenyl ethers 100. Xanthones Geography and ecology (16) Phytogeography 101. Cosmopolitan 102. Africa 103. Asia 104. Australasia 105. Europe 106. North America 107. South America Ecology 108. Arctic (or antarctic)-alpine 109. Boreal (or austral-cool temperate) 110. Arid-subarid 111. Temperate 112. Subtropical-tropical 113. Montane tropical Substrate 114. Saxicolous 115. Terricolous 116. Corticolous **The number of states included in the eight major categories of characters is indicated in brackets. All 116 character states were used in the analysis of 64 genera matrix. The 88 character states used in the matrix of 15 genera are indicated in bold type.
program of MOLPHY was then used with option D. Next, a neighbour-joining method was employed to reconstruct a tree from the matrix using the NJDIST programme of MOLPHY. This seed tree was then improved using the maximum likelihood method of option R (local rearrangement search) of the NUCML programme. In this limited search, branches are changed until the tree reaches maximum likelihood under the HKY substitution model. The tree obtained in this way may not be the maximum likelihood tree but may be considered to be a good approximation to it. This programme also calculates a local bootstrap probability (LBP, Hasegawa & Kishino 1994) to indicate the reliability of the tree nodes.
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THE LICHENOLOGIST Tuckermannopsis Allantoparmelia Masonhalea Cornicularia Arctoparmelia
56 52
80
88
56
Cetrariastrum Everniastrum Canomaculina Rimeliella Flavoparmelia Parmelina Myelochroa Parmotrema Melanelia Parmelia Relicina Bulbothrix Parmelinella Psiloparmelia Xanthoparmelia Karoowia Xanthomaculina Almbornia Omphalophora Omphalodium Rhizoplaca Parmotrema Punctelia Cetrelia Flavopunctelia Platismatia Everniopsis Placoparmelia Omphalodiella Pleurosticta Pannoparmelia Parmeliopsis Imshaugia Parmelaria Ahtiana Esslingeria
Vol. 31 Vulpicida Brodoa Asahinea Pseudevernia Concamerella
Canoparmelia Parmelinopsis Rimelia Hypotrachyna Anzia
Relicinopsis Neofuscelia Paraparmelia Chondropsis Namakwa
Pseudoparmelia
Coelopogon
Cetreliopsis Nephromopsis Cetrariopsis Allocetraria Coelocaulon Cetraria
F. 1. Neighbour-joining analysis of 64 genera of the Parmeliaceae based on 116 character states (including chemical, biogeographical and ecological data). Bootstrap values over 50 are indicated.
Results Non-molecular data The two methods yielded similar results when applied to the complete dataset (Fig. 1: only the neighbour-joining tree is shown; the parsimony tree is available upon request). Although Coelocaulon was chosen as the internal outgroup, Cetraria also remained independent of the rest of the genera. Most of the genera remained independent from one another, but several pairs of genera exhibited over 50% probability in the bootstrap analysis: Cetrariastrum and Everniastrum, Canomaculina and Rimeliella, Relicina and Bulbothrix, Omphalophora and Omphalodium, Cetrelia and Flavopunctelia (both of which were
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Parmelia
Pleurosticta Pseudevernia Platismatia Flavopunctelia Parmelina
Flavoparmelia Parmelinopsis Rimelia 74 Parmotrema
Hypotrachyna Xanthoparmelia 83 Neofuscelia
Melanelia
Coelocaulon F. 2. Neighbour-joining analysis of 15 genera of the Parmeliaceae occurring in the Iberian Peninsula. The study is based on 88 characters (including chemical, biogeographical and ecological data). Bootstrap values over 50 are indicated.
weakly related to Punctelia), Placoparmelia and Omphalodiella, and Parmeliopsis and Imshaugia. Results obtained from the two analytical methods from the 14 species selected for the comparative study were quite similar (Fig. 2), as they were with those extracted from the complete generic dataset. However, in this case, the pairs of genera that exhibited the highest bootstrap probabilities were Rimelia and Parmotrema and Xanthoparmelia and Neofuscelia.
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Pleurosticta acetabulum Pseudevernia furfuracea 58 85
Hypogymnia tubulosa Hypotrachyna revoluta
96
Parmelinopsis horrescens Parmelina quercina 89
75
Platismatia glauca Melanelia glabra Parmelia saxatilis Melanelia exasperata
83 94
Xanthoparmelia tinctina
73
Neofuscelia delisei Flavopunctelia flaventior 97
Flavoparmelia caperata 76
Parmotrema crinitum 100
Rimelia reticulata Usnea rigida
F. 3. Maximum likelihood analysis of 15 genera of the Parmeliaceae occurring in the Iberian Peninsula (Hypogymnia tubulosa was also included). The study is based on nuclear rDNA ITS sequences. Bootstrap values over 50 are indicated.
Molecular data Analysis of the sequence data gave a single maximum likelihood tree (Fig. 3). In this figure the numbers indicate nodes with bootstrap values over 50%. Pleurosticta acetabulum was placed as the initial sister group in the first node but this node had a low probability (58% LBS). The next branch had a well-supported second node (89% LBS) that grouped three branches of all the remaining taxa. In one of these branches there was a node grouping a further three branches each supporting a monophyletic group—namely Pseudevernia, Hypogymnia tubulosa and another well-supported group (96% LBS) of Hypotrachyna revoluta and Parmelinopsis horrescens. The second branch of the second node bore a further node (75% LBS) with three branches, representing Parmelina, Platismatia and Melanelia glabra. The third branch of the second
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node presented a new node (83% LBS) with two clades, where Parmelia was the sister group to the remaining taxa. The remaining taxa arose from a node (94% LBS) with three branches; with Melanelia exasperata as one terminal branch, Xanthoparmelia tinctina and Neofuscelia delisei forming a monophyletic group and, finally, the monophyletic branch with a node (97% LBS) in which Flavopunctelia was the sister group to Flavoparmelia and to Parmotrema crinitum and Rimelia reticulata. The latter pair also form a monophyletic group. Discussion The analysis of the non-molecular results for the 64 genera of the family yielded some relevant information. A previous phylogenetic study of the cetrarioid genera (Kärnefelt et al. 1992) revealed Coelocaulon and Cetraria to be more closely related to one another than to the remaining taxa. The tree provided less information about the remaining 62 genera. In the neighbourjoining analysis, eight pairs of genera were found to be closely related morphologically with a bootstrap probability greater than 50%. These included Cetrariastrum and Everniastrum, Relicina and Bulbothrix, Omphalophora and Omphalodium and Parmeliopsis and Imshaugia. The relationships of these taxa were discussed previously by Elix (1993). The close relationship between Canomaculina and Rimeliella was first described very recently (Elix 1997) and was confirmed by the current analysis, with a support of 64%. Canomaculina was formerly considered to be close to Parmotrema (Elix 1993; Krog & Swinscow 1981), but this relationship was not confirmed in the present study. In the neighbour-joining analysis Cetrelia and Flavopunctelia formed a pair (52% support), and the parsimony analysis indicated a relationship with Punctelia in a node with very low support. The relationship between Omphalodiella and Placoparmelia (68% support from neighbour-joining and 56% in parsimony analysis) had not been previously recognized. Both are monotypic genera from Patagonia (Argentina) that have aspicilioid ascomata and similar ecological affinities (Elix 1993), but unfortunately their cell wall chemistry is unknown. In the case of the 14 European genera, parsimony and neighbour-joining analysis of the non-molecular data revealed a close relationship only between Rimelia and Parmotrema and between Xanthoparmelia and Neofuscelia, while the remaining taxa occupied unresolved branches. However, the results from the molecular study revealed considerably more about the relationships between these genera. Maximum likelihood analysis showed a congruent topology and a much higher resolution by which to elucidate relationships between the genera, for example, the early isolation of Pleurosticta from all other taxa. It also revealed similar relationships between Parmotrema and Rimelia, as the non-molecular neighbour joining tree, but forming a monophyletic group with Flavopunctelia and Flavoparmelia. These relationships had not been recognized previously. These results are consistent with others from a previous neighbour-joining analysis (Crespo & Cubero 1998) that identified the relationships between Flavoparmelia and Parmotrema.
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Other established relationships have been confirmed. Thus, Hypotrachyna revoluta and Parmelinopsis horrescens appear to form a monophyletic group, as do the species of Xanthoparmelia and Neofuscelia. Pseudevernia furfuracea, a hitherto unstudied species, has been placed close to Hypogymnia and to the sister pair Hypotrachyna revoluta and Parmelinopsis horrescens. The other new sequence, that of Melanelia exasperata, appears to be quite distant from M. glabra. Melanelia exasperata was introduced into this analysis because of its peculiar morphological features (verrucose pseudocyphellae). This result confirms that these two Melanelia species are not monophyletic for this gene. In contrast, M. glabra and M. subargentifera, which are morphologically closely related on the basis of their hairy upper cortex, have been found to be monophyletic (Crespo & Cubero 1998) for this gene. We thank Martin Grube (Graz), Mats Wedin (London) and Paul Blanz (Graz) for the organization of the Graz Workshop and for kindly inviting us to present this work. We are also indebted to Martin Grube for permitting us the use of the unpublished sequence of Usnea rigida. Thanks also to Oscar F. Cubero for his help with computation and for discussion. This work was supported by the Ministerio de Educación y Cultura (PB 93–1129-CO2–01; APC960082). R Adachi, J. & Hasegawa, M. (1996) Molphy Version 2·3. Programs for molecular phylogenetics based on maximum likelihood. Tokyo: Institute of Statistical Mathematics. Crespo, A. & Cubero, O. F. (1998) A molecular approach to the circumscription and evaluation of some genera segregated from Parmelia s. lat. Lichenologist 30: 369–380. Crespo, A., Bridge, P. D. & Hawksworth, D. L. (1997) Amplification of fungal rDNA-ITS regions from non-fertile specimens of the lichen-forming genus Parmelia. Lichenologist 29: 275–282. Elix, J. A. (1993) Progress in the generic delimitation of Parmelia s. lat. lichens (Ascomycotina: Parmeliaceae) and a synoptic key to the Parmeliaceae. Bryologist 96: 359–383. Elix, J. A. (1997) The lichen genera Canomaculina and Rimeliella (Ascomycotina, Parmeliaceae). Mycotaxon 65: 475–479. Felsenstein, J. (1993) PHYLIP (Phylogeny Inference Package) version 3·5c. Seattle: Department of Genetics, University of Washington. Hasegawa, M. and Kishino, H. (1994) Accuracies of the simple methods for estimating the bootstrap probability of a maximum likelihood tree. Molecular Biology and Evolution 11: 142–145. Hasegawa, M., Kishino, H. & Yano, T. (1985) Dating of the human-ape splitting by a molecular clock of mitochondrial DNA. Journal of Molecular Evolution 22: 160–174. Hawksworth, D. L., Kirk, P. M., Sutton, B. C. & Pegler, D. J. (1995) Ainsworth & Bisby’s Dictionary of the Fungi. 8th Edn. Wallingford: CAB International. Kärnefelt, I., Mattsson, J-E & Thell, A. (1992) Evolution and phylogeny of cetrarioid lichens. Plant Systematics and Evolution 183: 113–160. Krog, H. & Swinscow, T. D. V. (1981) Parmelia subgenus Amphigymnia (lichens) in East Africa. Bulletin of the British Museum (Natural History), Botany Series 9: 143–231. Tehler, A. (1995a) Morphological data, molecular data and total evidence in phylogenetic analysis. Canadian Journal of Botany 73: 667–676. Tehler, A. (1995b) Arthoniales, phylogeny as indicated by morphological and rDNA sequence data. Cryptogamic Botany 5: 82–97. Accepted for publication 3 April 1999