Plagiochila punctata (Plagiochilaceae) in Tennessee, new to North America

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1 Plagiochila punctata (Plagiochilaceae) in Tennessee, new to North America PAUL G. DAVISON Department of Biology, University of North Alabama, UNA Box 5232, Florence, AL , U.S.A. DAVID K. SMITH Department of Ecology and Evolutionary Biology, University of Tennessee, Knoxville, TN , U.S.A. KATHRIN FELDBERG, MELANIE LINDNER AND JOCHEN HEINRICHS Department of Systematic Botany, Albrecht von Haller Institute of Plant Sciences, Georg August University, Untere Karspüle 2, D Göttingen, Germany s: ABSTRACT. Plagiochila (sect. Arrectae) punctata, previously known from the Neotropics, Africa and Europe, has been collected on a sandstone cliff in a rockhouse type environment in Tennessee, new to North America. Maximum likelihood analyses based on nrits1 5.8S- ITS2 sequences of several species of Plagiochila sect. Arrectae resolve the Tennessee specimen in a robust P. punctata subclade with accessions from Ecuador and the Democratic Republic of the Congo. KEYWORDS. North America, Tennessee, distribution, Plagiochila punctata, Plagiochila sect. Arrectae, internal transcribed spacer (ITS), nuclear ribosomal DNA. ^ ^ ^ Systematics and distribution of species of Plagiochila sect. Arrectae Carl is the subject of recent studies (e.g., Heinrichs et al. 2004a; Rycroft et al. 2002; Sim Sim et al. 2005). A major outcome of these studies is the finding that members of sect. Arrectae are usually not local endemics but widespread species with disjunct ranges. Heinrichs et al. (2005) extended the range of the assumed European endemic Plagiochila punctata (Taylor) Taylor to Africa and the Neotropics. This small to medium-sized Plagiochila species is characterized by lateral-intercalary branching, broad, not or shortly decurrent leaves with an ill-defined, short vitta, homogeneous to coarse-segmented oil bodies, and vegetative reproduction by caducous leaves on flagelliferous shoots. In 2001, PGD collected a tiny Plagiochila on a sandstone cliff in the Cumberland Plateau of Tennessee, U.S.A., which superficially resembled P. exigua (Taylor) Taylor. However, the most vigorous shoots had broad leaves with to 15 teeth and could thus not THE BRYOLOGIST 109(2), pp Copyright Ó2006 by the American Bryological and Lichenological Society, Inc /$0.65/0

2 Davison et al.: Plagiochila punctata in the U.S. 243 belong to P. exigua (see Schuster 1980). These shoots instead resembled small phenotypes of P. punctata that, however, had previously not been recorded from North America (Heinrichs et al. 2004b). Here we present the results of a morphological and molecular investigation of the Tennessee specimen. MATERIALS AND METHODS DNA extraction PCR amplification, and sequencing from Tennessee plants collected in 2004, Davison 6831 (GOET, UNAF), Van Buren County, Fall Creek Falls State Park, follows the protocol described in Heinrichs et al. (2004c). The new nrits sequence was compared with GenBank sequences using the BlastN program and integrated into a large alignment of Plagiochilaceae ITS sequences. In all cases sequences of Plagiochila sect. Arrectae were identified as most similar to the new sequence (data not shown). Representatives of Plagiochila sect. Arrectae were sampled. Plagiochila deflexa Mont. & Gottsche and P. rutilans Lindenb. (P. sect. Rutilantes Carl) were chosen as outgroups, based on the results of Heinrichs et al. (2005). Twenty-four ITS1 5.8S-ITS2 sequences (Table 1) from Groth et al. (2003), Heinrichs et al. (2002, 2004a, 2005), Lindner et al. (2004), Rycroft et al. (2002), Sim Sim et al. (2004) and the new sequence were aligned manually in BioEdit version (Hall 1999), resulting in an alignment including 761 putatively homologous sites (alignment available from JH). Phylogenetic trees were inferred using maximum likelihood (ML) criteria as implemented in PAUP* version 4.0b10 (Swofford 2003). To decide on the nucleotide substitution model with the smallest number of parameters that best fits the data, the program Modeltest 3.06 (Posada & Crandall 1998) was used. Based on hierarchical likelihood ratio tests, the K80 model was selected with gamma shape parameter (G) for among site variation calculated from the data set (K80þG). A ML analysis (with the Table 1. Geographic origins, voucher numbers and GenBank/EMBL accession numbers of the investigated taxa. Specimens are at GOET if not indicated otherwise. Species Origin Voucher Accession number P. bidens Gottsche Brazil Gradstein 5378 (G) AF P. bifaria (Sw.) Lindenb. Bolivia Heinrichs et al AY P. bifaria Brazil Costa & Gradstein 3805 AY P. bifaria British Isles Rycroft AY P. bifaria Costa Rica Heinrichs et al AY P. bifaria Ecuador Holz EC AJ P. bifaria Tenerife Drehwald 3922 AJ P. deflexa Costa Rica Heinrichs et al AJ P. punctata (Taylor) Taylor British Isles Rycroft AJ P. punctata Comoros Pócs et al. 9270/J AJ P. punctata D.R. Congo Pócs et al AJ P. punctata Ecuador Holz EC AJ P. punctata Tennessee Davison 6831 AM P. retrorsa Costa Rica Heinrichs et al AJ P. rubescens (Lehm. & Lindenb.) Lindenb. Chile Rycroft AJ P. rutilans Lindenb. Bolivia Groth 101 AJ P. spinulosa (Dicks.) Dumort. Belgium Dauphin et al AY P. spinulosa British Isles Rycroft AJ P. stricta Lindenb. Costa Rica Heinrichs et al.4401 AJ P. stricta Ecuador Holz EC AJ P. stricta Madagascar Pócs et al. 9868/AF AJ P. stricta Madeira Sim Sim s.n. (LISU) AY P. stricta Tenerife Drehwald 3920 AJ P. stricta Tenerife Rycroft AJ416648

3 244 the bryologist 109(2): 2006 K80þG model) was implemented as a heuristic search with ten random addition sequence replicates. The confidence of branching was assessed using 1000 bootstrap resamplings in ML-analysis. RESULTS Systematic position of the Tennessee specimen based on phylogenetic analyses of the ITS dataset. Representatives of Plagiochila sect. Arrectae Carl are placed sister to P. rubescens (Lehm. & Lindenb.) Lindenb. in a robust sister relationship (Fig. 1). The robust P. bifaria (Sw.) Lindenb. clade is placed sister to a well-supported clade with accessions of P. spinulosa (Dicks.) Dumort., P. stricta Lindenb. and P. punctata. The monophyly of P. spinulosa and the Neotropical and Macaronesian subclades of P. stricta achieve good bootstrap support. Accessions of P. punctata from the Neotropics, Africa and Europe form a weakly-supported monophyletic lineage. The Tennessee specimen is nested in a robust subclade of P. punctata made up of accessions from Ecuador and the Democratic Republic of the Congo. Morphology. The phylogenetic analysis corroborates the assignment of the sterile Tennessee specimen (Fig. 2) to P. punctata. The North American collection represents a small form of P. punctata which develops only flagelliferous shoots (Fig. 2A C). Weak shoots (Fig. 2C) are morphologically inseparable from P. exigua whereas the most vigorous ones carry broad leaves with as many as 15 teeth (Fig. 2F). DISCUSSION Figure 1. Single most likely tree (ln ¼ ) recovered during 10 random taxon addition heuristic searches of the nrits1 5.8S-ITS2 DNA data set under the K80 model with estimated gamma shape (G¼0.58). Bootstrap support (.50%) is indicated at branches. The phylogenetic analysis supports identification of the Tennessee specimen as P. punctata and range extension of this species to North America. Plagiochila punctata in Tennessee occurs at the base of a tall (40 m) sandstone cliff in a rockhouse type environment. The plants here are in direct influence of subterranean air flow emitted from a large, vertical rock fissure that extends into the bedrock of the cliff. As in other rockhouse environments temperatures as compared to nearby areas are cooler in summer and warmer in winter. The cooler summer microclimate has been noted by hikers who have named the site the refrigerator. Associated plants in the same microhabitat occurring with P. punctata include the bryophytes Dicranum fulvum Hook., Frullania asagrayana Mont., Harpalejeunea ovata subsp. integra R. M. Schust. [H. molleri (Steph.) Grolle], Herbertus aduncus subsp. tenuis (A. Evans) H. A. Mill. & E. B. Bohrer, Lejeunea laetevirens Nees & Mont., Lejeunea ruthii (A. Evans) R. M. Schust., Leucobryum albidum (Brid.) Lindb., Leucolejeunea clypeata (Schwein.) A. Evans, Metzgeria sp., Microlejeunea ulicina (Taylor) Steph., Plagiochila undata Sull., Pylaisiadelpha tenuirostris (Bruch & Schimp.) W. R. Buck, Tortella humilis (Hedw.) Jenn., and the fern gametophyte Vittaria appalachiana Farrar & Mickel. Within the Cumberland Plateau, only H. aduncus subsp. tenuis and V. appalachiana are restricted to rockhouse habitats. In eastern North America rockhouse cliff for-

4 Davison et al.: Plagiochila punctata in the U.S. 245 Figure 2. Plagiochila punctata. A, C. top of shoots, dorsal view. B. top of shoot, ventral view. D. cells from leaf center with oil bodies. E. tooth from leaf apex. F. leaves [all from Davison 6831, Tennessee (GOET)]. mations are considered important refugia for tropical fern disjuncts and endemic ferns derived from tropical taxa (Farrar 1998; Walck et al. 1996). Considered to be Tertiary relicts in the United States, these ferns, many persisting as asexually reproducing gametophytes, presumably had a broader distribution in eastern North America million years ago when tropical/subtropical climates extended over the eastern United States (Farrar 1998). The extensive sequence similarities of the Tennessee plants of P. punctata and specimens from South America and Africa allow for considering rather recent longdistance dispersal (Heinrichs et al. 2005) as a feasible explanation for the single occurrence of P. punctata in eastern North America. Such an explanation has also been suggested for the present-day North American

5 246 the bryologist 109(2): 2006 distribution of the tropical fern Grammitis nimbata (Jenman) Proctor, in the U.S. known only from one location in western North Carolina (Farrar 1967, 1985) where it is restricted to a rockhouse environment. Farrar (1967: 1267) pointed out that Possibly, this entire colony in North Carolina may have originated from the chance landing of a single spore from the south. The North Carolina population of G. nimbata is maintained by asexually reproducing gametophytes as the sporophytes fail to mature and produce spores. Both P. punctata and G. nimbata occur in mountainous regions in the tropics where both are known to produce spores. Like G. nimbata in North Carolina, P. punctata in Tennessee relies on asexual reproduction for continued existence. ACKNOWLEDGMENTS This study was supported by the Tennessee Department of Environment and Conservation, Division of Natural Heritage (Contract # FA , for the field survey of the rare fern Hymenophyllum tayloriae Farrar and Raine) and the German Research Foundation (DFG grant HE 3584 / 1). We would like to thank Keith Bowman, Stuart Carroll and David Lincicome for field assistance. LITERATURE CITED Farrar, D. R Gametophytes of four tropical fern genera reproducing independently of their sporophytes in the southern Appalachians. Science 155: Independent fern gametophytes in the wild. Proceedings of the Royal Society of Edinburgh 86B: The tropical flora of rockhouse cliff formations in the eastern United States. Journal of the Torrey Botanical Society 125: Groth, H., M. Lindner, R. Wilson, F. A. Hartmann, M. Schmull, S. R. Gradstein & J. Heinrichs Biogeography of Plagiochila (Hepaticae): natural species groups span several floristic kingdoms. Journal of Biogeography 30: Hall, T. A BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/ NT. Nucleic Acids Symposia Series 41: Heinrichs, J., H. Groth, Holz I., D. S. Rycroft, C. Renker & T. Pröschold The systematic position of Plagiochila moritziana, P. trichostoma and P. deflexa, based on ITS sequence variation of nuclear ribosomal DNA, morphology and lipophilic secondary metabolites. The Bryologist 105: ,, M. Lindner, K. Feldberg & D. S. Rycroft. 2004a. Molecular, morphological and phytochemical evidence for a broad species concept of Plagiochila bifaria (Sw.) Lindenb. (Hepaticae). The Bryologist 107: , M. Lindner & H. Groth. 2004b. Sectional classification of North American Plagiochila (Hepaticae, Plagiochilaceae). The Bryologist 107: ,, & C. Renker Distribution and synonymy of Plagiochila punctata (Taylor) Taylor, with hypotheses on the evolutionary history of Plagiochila sect. Arrectae (Plagiochilaceae, Hepaticae). Plant Systematics and Evolution 250: , & T. Pócs. 2004c. nrdna internal transcribed spacer data reveal that Rhodoplagiochila R. M. Schust. (Jungermanniales, Marchantiophyta) is a member of Plagiochila sect. Arrectae Carl. Organisms, Diversity and Evolution 4: Lindner, M., T. Pócs & J. Heinrichs On the occurrence of Plagiochila stricta on Madagascar, new to Africa. Journal of the Hattori Botanical Laboratory 96: Posada, D. & K. A. Crandall Modeltest: testing the model of DNA substitution. Bioinformatics 14: Rycroft, D. S., W. J. Cole, J. Heinrichs, H. Groth, C. Renker & T. Pröschold Phytochemical, morphological and molecular evidence for the occurrence of the neotropical liverwort Plagiochila stricta in the Canary Islands, new to Macaronesia. The Bryologist 105: Schuster, R. M The Hepaticae and Anthocerotae of North America, vol. 4. Columbia University Press, NY. Sim-Sim, M., M. G. Esquível, S. Fontinha & S. Carvalho Plagiochila stricta Lindenb. new to Madeira. Morphological and molecular evidence. Nova Hedwigia 79: , M. Stech, M. G. Esquível, A. C. Figueiredo, M. M Costa, J. G. Barroso, L. G. Pedro, S. Fontinha & C. Lobo Plagiochila spinulosa (Dicks.) Dumort. (Plagiochilaceae, Hepaticophytina) in Madeira Island - morphological, phytochemical, and molecular evidence. Journal of the Hattori Botanical Laboratory 98: Swofford, D. L PAUP*, phylogenetic analysis using parsimony (*and other methods). Sinauer Associates, Sunderland, MA. Walck, J. L., J. M. Baskin & C. C. Baskin Sandstone rockhouses of the eastern United States, with particular reference to the ecology and evolution of the endemic plant taxa. The Botanical Review 62: ms. received October 31, 2005; accepted February 21, 2006.