This article was downloaded by: [Ros, R. M.] On: 10 February 2010 Access details: Access Details: [subscription number 919179156] Publisher Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37- 41 Mortimer Street, London W1T 3JH, UK Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology Publication details, including instructions for authors and subscription information: http://www.informaworld.com/smpp/title~content=t713737104 Molecular and morphological studies on the Didymodon tophaceus complex O. Werner a; H. Köckinger b; J. A. Jiménez a; R. M. Ros a Departamento de Biología Vegetal, Universidad de Murcia, Spain b Roseggergasse 12, AT-8741 Weisskirchen, Austria Online publication date: 09 February 2010 To cite this Article Werner, O., Köckinger, H., Jiménez, J. A. and Ros, R. M.(2009) 'Molecular and morphological studies on the Didymodon tophaceus complex', Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology, 143: 3, S136 — S145 To link to this Article: DOI: 10.1080/11263500903226965 URL: http://dx.doi.org/10.1080/11263500903226965 PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.informaworld.com/terms-and-conditions-of-access.pdf This article may be used for research, teaching and private study purposes. Any substantial or systematic reproduction, re-distribution, re-selling, loan or sub-licensing, systematic supply or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material. Plant Biosystems, Vol. 143, Supplement, 2009, pp. S136–145 BRYOPHYTE DIVERSITY & CONSERVATION Molecular and morphological studies on the Didymodon tophaceus complex O. WERNER1, H. KÖCKINGER2, J. A. JIMÉNEZ1, & R. M. ROS1 1Departamento de Biología Vegetal, Universidad de Murcia, Spain and 2Roseggergasse 12, AT-8741 Weisskirchen, Austria Taylor and Francis Abstract Recently two Mediterranean species closely related to Didymodon tophaceus (Brid.) Lisa have been described in the genus Didymodon Hedw.: Didymodon sicculus M. J. Cano et al. and Didymodon erosus J. A. Jiménez & J. Guerra. The former has been proved to be widespread around the Mediterranean basin in recent years, and this study points to a considerable extension of its distribution area towards north, since it has also been discovered in the Netherlands. D. erosus, with its narrow morphological characterization, was only described four years ago and has hitherto only been known from the original localities in Spain. The molecular study based on nrITS sequence data of some selected specimens of all the three species suggests that D. erosus is much more frequent and widespread in Europe than supposed and, furthermore, is morphologically much more variable than previously described. A key for the identification of the three species, an amended description of D. erosus and a map showing the known distribution area are presented. Keywords: Didymodon tophaceus, Didymodon sicculus, Didymodon erosus, nrITS sequences, taxonomy, morphological studies, European distribution et al. (2004) and later completed with new data of Introduction Cvetic[acuet] ´c and Sabovljevica[ue]t ´ (2004) and Puglisi et al. Didymodon sicculus M. J. Cano, Ros, García-Zamora (2004), extending its area through Algeria and & J. Guerra was described from south Spain by Morocco in northern Africa; France, Greece, Italy Cano et al. (1996). In the original publication, it was (mainland and Linosa Island), Montenegro, Portu- Downloaded By: [Ros, R. M.] At: 08:24 10 February 2010 indicated that it is morphologically related to Didym- gal (Azores) and Spain (mainland and Balearic and odon luridus Hornsch. and Didymodon tophaceus Canary Islands) in Europe; and Israel, Turkey and (Brid.) Lisa. Jiménez (2006) confirmed this Yemen in western Asia. similarity and concluded that the prime important Didymodon erosus J. A. Jiménez & J. Guerra has characters to distinguish D. sicculus from D. luridus more recently been described from north-western despite their ecological similarities are the papillose Spain by Jiménez, Guerra, et al. (2004). No addi- upper laminal cells and the yellowish-green colour tional specimens were detected belonging to this of the lamina with alcoholic KOH. This author species besides the detailed revision carried out by distinguished D. tophaceus from D. sicculus by the Jiménez (2006) of the genus Didymodon in Europe, elongated epidermal cells of the ventral side of the North Africa and south-west and central Asia. costa and its occurrence on vertical calcareous rock Morphologically, it was characterized by Jiménez, faces with seeping water or similarly humid places, Guerra, et al. (2004)as follows: (1) rhizoidal tubers whereas D. sicculus shows quadrate or shortly underground or on rhizoids in the lower part of the rectangular ventral epidermal cells on the costa and stem, (2) lanceolate to narrowly ovate-lanceolate grows in dry habitats. leaves, (3) generally unistratose lamina, sometimes The distribution area of D. sicculus has been bistratose in patches, (4) margins that are markedly progressively widened around the Mediterranean erose and papillose-crenulate in the upper middle basin. It has been summarized by Jiménez, Ros, part of the leaf, plane or lightly recurved from the Correspondence: R. M. Ros, Departamento de Biología Vegetal, Universidad de Murcia, Campus de Espinardo, Murcia 30100, Spain. Tel: +34- 968364989. Fax: +34-968363963. Email: [email protected] ISSN 1126-3504 print/ISSN 1724-5575 online © 2009 Società Botanica Italiana DOI: 10.1080/11263500903226965 Studies on Didymodon tophaceus S137 base to 1/2 or 3/4 of the leaf, and (5) perichaetia that confirmed or revised according to the molecular are terminal or on short lateral branches, generally in results. Most specimens were obtained from the the bifurcation of two vegetative branches. They also herbaria of the authors and also from the Jan Kuocar[n]ˇera considered D. erosus to be morphologically very Herbarium in CBFS, which holds a large collection closely related to Didymodon sinuosus (Mitt.) of the genus Didymodon. Based on the results of the Delogne because of the similar stance of the leaves earlier publication on the phylogenetic relationships when moist and the erose and papillose-crenulate within the genus Didymodon using nrITS data, margins, and to Didymodon tomaculosus (Blockeel) D. luridus, D. maximus (Syed & Crundw.) M. O. Hill M. F. V. Corley because of the rhizoidal tubers. and D. spadiceus (Mitt.) Limpr. were chosen as Furthermore, it was observed that despite the outgroup species. Details of the geographical origin morphological differences with D. tophaceus, both of the plant material, vouchers and GenBank acces- species share the same habitat. sion number of the obtained sequences are given in D. tophaceus is a well-known species, very variable Table I. morphologically, for which many synonyms have been given and which continues to be found when DNA isolation and amplification of ITS some profound studies are carried out (Zander 1994; Jiménez et al. 2005). It is distributed DNA extraction. Total DNA was extracted by the worldwide (Jiménez 2006; Zander 2007). NaOH extraction method described by Werner et al. The systematic studies based on nrITS sequences (2002), in which 5 µl of crude NaOH extract was carried out in the genus Didymodon by Werner et al. diluted by the addition of 45 µl of 100 mM Tris – 1 (2005) withdraw support from most of the species mM EDTA (pH 8.3) and stored frozen at –18°C relationships based on the morphology proposed for until the PCR was carried out. both D. erosus and D. sicculus. They observed that D. sicculus is not phylogenetically related to D. luri- DNA sequencing. PCR was performed in an Eppen- dus and that D. erosus is neither closely related to D. dorf Mastercycler using 4 µl of the DNA solution in sinuosus nor to D. tomaculosus. On the contrary, a 50 µl final volume. The reaction mix contained the highly supported monophyletic clade was obtained primers 18S (5′-GGAGAAGTCGTAACAAG- including D. tophaceus, D. erosus and D. sicculus. GTTTCCG-3′), designed by Spagnuolo et al. Recently, some complex European specimens of (1999), and ITS4 (5′−TCCTCCGCTTAT- the genus Didymodon have been found that could be TGATATGC-3′; White et al. 1990), at a final related to this group of species. An example of this concentration of 400 µM, in the presence of 200 µM complexity can be observed in the note published by of each dNTP, 2 mM MgCl2, 2 units of Taq poly- Frahm (2001), who made a call for help to identify merase (Oncor Appligene), 1 µl BLOTTO (10% a specimen found on an artificial slope. It was consid- skimmed milk powder, 0.2% NaN3 in water) and the ered to be morphologically closely related to D. topha- buffer provided by the supplier of the enzyme. Downloaded By: [Ros, R. M.] At: 08:24 10 February 2010 ceus, but showed some differences, such as the more BLOTTO attenuates PCR inhibition by plant acute apex, the shortly rectangular ventral cells on the compounds