54 (2) • May 2005: 361–368 Werner & al. • Phylogeny of Trichostomoideae Molecular phylogeny of Trichostomoideae (Pottiaceae, Bryophyta) based on nrITS sequence data Olaf Werner1, Rosa María Ros1 & Michael Grundmann2 1 Departamento de Biología Vegetal (Botánica), Facultad de Biología, Universidad de Murcia, Campus de Espinardo, 30100 Murcia, Spain. [email protected] (author for correspondence), [email protected] 2 Department of Botany, The Natural History Museum, Cromwell Road, London, SW7 5BD, U.K. m.grund- [email protected] Bayesian analysis of an alignment of 83 nrITS sequences belonging to 66 taxa of Pottiaceae revealed repre- sentatives of subfamily Trichostomoideae in a well supported monophyletic lineage. Two robust clades with- in Trichostomoideae include species of Tortella and Weissia, respectively. Eucladium verticillatum is part of Trichostomoideae, and Anoectangium, Gymnostomum, Hymenostylium, and Tuerckheimia form a robust basal clade. Affinities of Hyophila and Leptobarbula to Trichostomoideae are unclear. Within Trichostomoideae, Pleurochaete squarrosa is resolved in Tortella while Trichostomum tenuirostris, Pseudosymblepharis, and Chionoloma are placed as a separate genus or part of a broadly defined Tortella. The type species of Trichostomum, T. brachydontium, is nested in Weissia together with T. brittonianum, T. crispulum, and T. jamaicensis. Trichostomum unguiculatum and W. ayresii are resolved as closely related and separate from the rest of the Weissia clade. The genus Astomum, erected for cleistocarpous species of Weissia, is also nested with- in Weissia. Genetic divergence between selected accessions of Weissia controversa and Astomum is sometimes smaller than between accessions of Weissia controversa. KEYWORDS: bryophyta, molecular phylogeny, nrITS sequence data, Pottiaceae, Trichostomoideae. Beauv., Dialytrichia (Schimp.) Limpr., Ephemerum INTRODUCTION Hampe, Kingiobryum H. Rob., and Splachnobryum Pottiaceae are a large and taxonomically difficult Müll. Hal., which are usually treated separately, are family of mosses, whose generic delimitations and rela- members of this family. Later, Werner & al. (2004b) tionships are complicated and often controversial. Since included Kingiobryum in the genus Didymodon Hedw. the description of the family by Bruch & al. (1836– based on nrITS1 and nrITS2 sequence data. 1851), many generic classifications have been proposed At subfamily level, a subdivision into two to seven based on morphology. Spagnuolo & al. (1999), in a first different subfamilies has been proposed. One of these, molecular-phylogenetic study on Pottiaceae, sequenced subfam. Trichostomoideae, has been variously defined the nrITS 1 region in a few species and demonstrated the morphologically (e.g., Hilpert, 1933; Chen, 1941; Saito, close relationship between Pleurochaete Lindb., Tortella 1975; Zander, 1993) based on (1) stem sclerodermis (Lindb.) Limpr. and Weissia Hedw., the placement of poorly differentiated from the central cylinder, (2) hyalo- Weissia in Trichostomoideae rather than Pottioideae, and dermis commonly present, (3) leaves mostly narrow the separation of Syntrichia Brid. from Tortula Hedw. lanceolate with unistratose, plane or incurved margins, Werner & al. (2002a) studied the relationships of Tortula (4) leaf base, when differentiated, separated obliquely and allied genera using rps4 sequences and confirmed from the lamina, (5) upper laminal papillae crowded, (6) that Syntrichia and Tortula are clearly separated as upper lamina KOH reaction yellow (seldom yellowish observed by Spagnuolo & al. (1999). In addition, they orange or reddish brown), (7) costa with two bands of found: that the rhynchostegiate species of Pottia (Ehrh. stereids but lacking a differentiated dorsal epidermis, (8) ex Rchb.) Fürnr. are clearly separated from the conoste- clavate propagula absent, (9) 5-layered amphitecial giate ones and closer to Tortula, that Crossidium Jur. is derivatives of the capsule and stomata present, and (10) nested within Tortula, and that Desmatodon Brid., Ptery- peristome split into two segments from the base, without goneurum Jur., Stegonia Venturi, and some species of or with a very short basal membrane. Phascum Hedw., are closely related. Using the same Werner & al. (2004a), using rps4 sequences, accept- marker, Werner & al. (2004a) demonstrated that Hypo- ed three subfamilies in Pottiaceae: Merceyoideae, Tri- dontium Müll. Hal. and Timmiella (De Not.) Limpr. chostomoideae, and Pottioideae. Trichostomoideae were should be excluded from Pottiaceae while Cinclidotus P. delimited in a very broad sense and included many taxa 361 Werner & al. • Phylogeny of Trichostomoideae 54 (2) • May 2005: 361–368 not previously assigned to this subfamily on morpholog- µM, in the presence of 200 µM of each dNTP, 2 mM ical grounds, e.g., Ephemerum (previously Ephemera- MgCl2, 2 units Taq polymerase (Oncor Appligene), 1µl ceae), Anoectangium Schwägr., Aschisma Lindb., Gym- BLOTTO (10% skimmed milk powder and 0.2% NaN3 nostomiella M. Fleisch., Gymnostomum Nees & in water) and the buffer provided by the supplier of the Hornsch., Hymenostylium Brid., Hyophila Brid., Lepto- enzyme. BLOTTO attenuates PCR inhibition caused by barbula Schimp., Pottiopsis Blockeel & A. J. E. Smith, plant compounds (De Boer & al., 1995, unpubl.). Pseudosymblepharis Broth., Splachnobryum, Weissia Amplification started with 3 min denaturation at 94°C, Hedw. and some species of Barbula Hedw. (other followed by 35 cycles of 15 s at 94°C, 30 s at 50°C, and sequenced species of Barbula belonged to Pottioideae). 1 min at 72°C. Concluding with a final extension step of However, Trichostomoideae were not resolved with 7 min at 72°C., 5 µl of amplification products were visu- strong support and the new classification, therefore, alized on a 6% polyacrylamide gel. Successful amplifi- remained a provisional one. cations were cleaned with the QIAquick purification kit In the present paper the delimitation of Tricho- (Qiagen). The amplification primers were used in the stomoideae and the relationships among selected genera sequencing reactions with the Big Dye sequencing kit of this subfamily are explored using new evidence from and separated on an ABI-Prism 3700 sequencing nrITS sequence data. machine using standard protocols. Sequences were aligned using CLUSTALX (Thompson & al., 1997) with the gap open penalty set to 10 and the gap extension penalty set to 1. BioEdit (Hall, MATERIALS AND METHODS 1999) was used for minor manual adjustments of the Plant material. — Selected species were sampled alignment. Sequences with high frequency of indels dif- from all major lineages of Trichostomoideae. Eighty- ficult to align were excluded from the analysis [e.g., three specimens were sequenced, representing 66 taxa. In Barbula indica (Hook.) Spreng., Ephemerum sessile many cases, more than one sample was sequenced to (Bruch) Müll. Hal., Splachnobryum obtusum (Brid.) demonstrate possible sequence variation at species or Müll. Hal.]. The aligned matrix is available on request variety level. The following species of Pottioideae sensu from the first author. Gaps outside hypervariable regions Werner & al. (2004a) were included: Barbula unguicula- were coded as present (1) or absent (0). Pairwise dis- ta Hedw., Bryoerythrophyllum recurvirostrum (Hedw.) P. tances between sequences were calculated by MEGA 2 C. Chen, Chenia leptophylla (Müll. Hal.) R. H. Zander, (Kumar & al., 2001). Didymodon rigidulus Hedw., D. sinuosus (Mitt.) Bayesian analysis was carried out using MrBayes Delogne, Pseudocrossidium hornschuchianum (Schultz) 3.0 (Huelsenbeck & Ronquist, 2001), with 500,000 gen- R. H. Zander, Tortula inermis (Brid.) Mont., T. muralis erations run, sampling every 100th generation and using Hedw., Triquetrella arapilensis Luisier, and Tr. tristicha the following settings for the sequence data: Nst=6, (Müll. Hal.) Müll. Hal. Bryoerythrophyllum recurvi- rates=invgamma (a proportion of the sites are invariable rostrum was assigned as outgroup species in the molecu- while the rate for the remaining sites corresponds to the lar analysis and used to root the tree. Voucher data and general model of DNA substitution with gamma distrib- GenBank accession numbers are given in the Appendix. uted rate variation across sites). Recoded gaps were treat- Nomenclature follows Zander (1993) except for ed as standard data. Based on empirical evaluation, burn Chionoloma bombayensis (Müll. Hal.) P. Sollman in was set at 100,000 generations. A 50% majority rule (Sollman, 2001) and Trichostomum sweetii (E. B. tree was constructed using the “sumt” command of Bartram) L. R. Stark (Stark, 1996). MrBayes. The tree was edited using TreeView version DNA extraction. — Total DNA was extracted 1.6.6 (Page, 1996). Clade credibility values (cv) below using the NaOH method of Werner & al. (2002b), in 0.75 were treated as “poor” support, between 0.75 and which 5 µl of a crude NaOH extract was diluted by the 0.95 as “moderate” and above 0.95 as “good”. addition of 45 µl of 100 mM Tris - 1 mM EDTA (pH 8.3), stored frozen at -18°C and used as template for subse- quent PCR analysis. DNA sequencing. — PCR reactions were per- RESULTS formed in an Eppendorf Mastercycler using 4 µl of the The combined length of the 18S ITS1 - 5.8S rRNA - DNA solution in a 50 µl final volume. The reaction mix ITS2 region was 662 [Trichostomum unguiculatum contained the primers 18S (5´-GGAGAAGTCGTAA- (Mitt.) R. H. Zander] to 836 [Chionoloma bombayensis] CAAGGTTTCCG-3´), designed by Spagnuolo & al. base pairs (bp). The ITS1