The Bryologist 107(2), pp. 215 221 Copyright ᭧ 2004 by the American Bryological and Lichenological Society, Inc.

The Systematic Position of the Kingiobryum paramicola () Based on Molecular and Morphological Data

OLAF WERNER,JUAN A. JIMEÂ NEZ, AND ROSA M. ROS Departamento de BiologõÂa Vegetal, Facultad de BiologõÂa, Universidad de Murcia, Campus de Espinardo, 30100- Murcia, Spain; e-mail: [email protected]

Abstract. ITS1 and ITS2 sequences and a morphological analysis con®rm the position of the Andean species Kingiobryum paramicola H. Rob. within the Pottiaceae and the genus Didymodon. It is closely related to Didymodon australasiae and D. umbrosus. A new combination is proposed: Didymodon paramicola (H. Rob.), comb. nov.

Keywords. Didymodon paramicola, ITS, Kingiobryum, phylogenetic analysis, Pottiaceae.

Robinson (1967) in his studies on the Bryophytes lanceolate leaves with sheathing leaf bases e.g., Er- of Colombia, described from the Colombia Eastern ythrophyllopsis, Leptodontium (MuÈll. Hal.) Lindb., Cordillera, Dept. Cundinamarca, at 3,490 m ele- and Rhexophyllum Herzog in the Pottiaceae, and vation, the genus Kingiobryum that included the Holomitrium, Oncophorus, and Symblepharis in the new species K. paramicola H. Rob. He placed it in Dicranaceae. The authors concluded that, in spite the Dicranaceae. He considered his new genus to of the above mentioned similarities, the genus Kin- be most closely related to the group of genera in- giobryum should be retained in the Dicranaceae be- cluding Oncophorus (Brid.) Brid., Symblepharis cause of the following: ``stem hyalodermis of en- Mont., and Holomitrium Brid., and distinguishable larged rectangular cells; perichaetial leaves smaller from these genera and all other Dicranaceae by the than the cauline leaves, oval, prosenchymatous presence of what may be called cancelline areas of throughout; cauline leaves with upper leaf cells enlarged, hyaline, porose cells in the leaf base that moderately and evenly thickened, heterogeneous in had been previously noted only in the Calympera- shape; upper laminal papillae bi®d and centered ceae and Leucobryaceae. FlorschuÈtz-de-Waard and over the transverse walls; and costa in cross section FlorschuÈtz (1979) compiled a list of the moss spe- with eight guide cells in one layer''. Churchill and cies known at that time in Colombia and included Linares (1995) summarized the information con- a new locality for Kingiobryum paramicola (Dept. cerning the species and pointed out that this An- of Meta). New collections bearing capsules from dean species grows on soil or thin layers of soil on the State of MeÂrida in Venezuela allowed a more rocks of the ``paÂramos'' from the Eastern Cordillera complete study of the and ecology of the of Colombia and the Cordillera of MeÂrida of Ve- species (Zander & Cleef 1982). These authors made nezuela, at 3,490±4,200 m elevation. a detailed morphological description of the game- Zander (1993), in a re-investigation of the family tophyte and the sporophyte, because the original placement, stated that slits formed by transverse re- description of Robinson (1967) was brief and lim- sorption across the longitudinal walls of the medial ited to the gametophyte. Placement in the Dicran- basal cells, such as those of Kingiobryum parami- aceae was questioned because of some morpholog- cola, are also present in Didymodon herzogii R. H. ical characteristics typical of the Pottiaceae, such as Zander and Gertrudiella validinervis (Herzog) the papillose upper cells and the acid-base color Broth. He emphasized that the latter two species reaction, which is similar to that of the genus Er- are not closely related, and that further studies were ythrophyllopsis Broth. The aperistomate nature of needed to clarify whether Kingiobryum belongs to the capsules of the species prevented more accurate the Pottiaceae or the Dicranaceae. Another species taxonomic placement at the time. They also pointed that presents porose basal cells is Didymodon chal- out that the porose basal cells observed by Robin- laense R. H. Zander that was previously considered son, besides appearing in the Calymperaceae and a member of the genus Erythrophyllopsis Broth. the Leucobryaceae, may also be present in the Pot- (Zander 1993). tiaceae and the Encalyptaceae, and mentioned that It was La Farge et al. (2002) who ®rst supported the intermediate position between Dicranaceae and the hypothesis of Zander and Cleef (1982) and Zan- Pottiaceae is also evident in other genera showing der (1993), and concluded that Kingiobryum be-

0007-2745/04/$0.85/0 216 THE BRYOLOGIST [VOL. 107 longs to the Pottiaceae in a study of the circum- For the morphological study, additional specimens were scription of the Dicranaceae based on the chloro- examined: Kingiobryum paramicola. COLOMBIA. CUNDINA- plast regions trnL-trnF and rps4. These authors se- MARCA. Eastern Cordillera, Municipality of Calera, Haci- quenced a total of 84 species, among them, 50 enda La Siberia, PaÂramo de Palacio, 3,490 m, King C- species belonging to 29 genera selected as exem- 1015, Jaramillo-Mejia & Guevara (holotype, US; isotype, plars from the Dicranaceae sensu Vitt (1984). Ex- NY). META.PaÂramo de Sumapaz, Cerro Nevado del Su- mapaz, superpaÂramo del lado W. Lajas, 4,200 m, Cleef emplar taxa from the Bartramiaceae, Bryaceae, En- 8073 (NY); lado SW, subida al Alto del Buque, 3,700 m, calyptaceae, Funariaceae, and Timmiaceae were se- Cleef 7691 (NY). lected as remote outgroup taxa in this analysis. The Didymodon herzogii. BOLIVIA. COCHABAMBA. Cho- study clearly showed that Kingiobryum is not close- cayatal, 3,300 m, Herzog 3606 (holotype of Trichostomum ly related to Holomitrium, Oncophorus, Symble- ferrugineum Herzog, JE). Gertrudiella validinervis. BOLIVIA. LA PAZ.Inden pharis, or any other Dicranaceae. Kingiobryum was Dornbuschsteppe des Palo, 1,600 m, Herzog 4344 (holo- placed in a clade with Tortula and Stegonia with type of Gertrudia validinervis Herzog, JE). Cordillera de 100% bootstrap support. Santa Cruz, in schattigen Felsen bei Tres Cruces, 1,400 Another study using chloroplast rps4 sequences m, Herzog 3473 (JE). of more than 50 species belonging to the family DNA extraction. Total DNA was extracted by the NaOH extraction method described by Werner et al. Pottiaceae con®rmed that Kingiobryum is a member (2002). Five ␮l of the crude NaOH extract were diluted of this family (Werner et al. 2004). These authors by the addition of 45 ␮l of 100 mM Tris±1 mM EDTA recognized only three of the seven subfamilies pro- (pH 8.3) and stored frozen at Ϫ18ЊC until the PCR reac- posed by Zander (1993): Merceyoideae, Trichos- tion was carried out. tomoideae, and Pottioideae. The subfamily Tim- DNA sequencing. PCR reactions were performed in an Eppendorf Mastercycler using 4 ␮l of the DNA solu- mielloideae was excluded from the Pottiaceae, tion in 50 ␮l ®nal volume. The reaction mix contained the while the subfamily Gertrudielloideae could not be primers 18S (5Ј-GGAGAAGTCGTAACAAGGTTTCCG- studied because no material was available. 3Ј), designed by Spagnuolo et al. (1999) and ITS4 (5Ј- The Merceyoideae (in the sense of Zander) are TCCTCCGCTTATTGATATGC-3Ј; White et al. 1990), at a ®nal concentration of 400 M, in the presence of 200 clearly polyphyletic. The genera Barbula Hedw. ␮ ␮M each of dNTP, 2 mM MgCl2, 2 units Taq polymerase (partly), Bryoerythrophyllum P. C. Chen, Dialytri- (Oncor Appligene), one ␮l BLOTTO (10% skimmed milk chia (Schimp.) Limpr., Didymodon Hedw., Kin- powder, 0.2% NaN3 in water), and the buffer provided by giobryum, Leptodontium, Pseudocrossidium R. S. the supplier of the enzyme. BLOTTO attenuates the in- Williams, and Triquetrella MuÈll. Hal., all included hibition of PCR by plant compounds (De Boer et al. 1995). Ampli®cation started with 3 min denaturation at by Zander in the Merceyoideae, were transferred to 94ЊC, followed by 35 cycles of 15 s at 94ЊC, 30 s at 50ЊC, the Pottioideae. Anoectangium, SchwaÈgr., Barbula and one min at 72ЊC. A ®nal extension step of 7 min at (partly), Gymnostomiella M. Fleisch., Gymnosto- 72ЊC completed the PCR. The primers were designed to mum Nees & Hornsch., and Hymenostylium were amplify the last bases of the 18s ribosomal RNA gene, included in the Trichostomoideae. Of these genera, ITS-1, 5.8s rRNA gene, ITS-2, and the ®rst few bases of the 26 rRNA gene. Five ␮l of the ampli®cation products only Scopelophila (Mitt.) Lindb. remained in the were visualized on a 6% polyacrylamide gel and success- Merceyoideae. The rps4 data also suggested that ful ampli®cations were cleaned with the QIAquick puri- the genus most closely related with Kingiobryum is ®cation kit (Qiagen). The ampli®cation primers were used Didymodon (Werner et al. 2004). But the insuf®- in the sequencing reactions with the Big Dye sequencing cient resolution of the rps4 sequences and the low kit and separated on a ABI-Prism 3700 sequencing ma- chine using standard protocols. number of Didymodon species included did not al- The sequences were aligned using CLUSTALX low the authors to draw ®nal conclusions. There- (Thompson et al. 1997) with the gap open penalty set to fore, we decided to use the more variable nuclear 10 and the gap extension penalty set to 0.1. BioEdit (Hall ITS region to address this problem. Additionally, 1999) was used for minor manual adjustment of the align- Kingiobryum paramicola is morphologically com- ment. An aligned matrix is available on request from the ®rst author. pared with other members of the genus Didymodon. MEGA 2.1 (Kumar et al. 2001) was used for the Min- imum Evolution (ME) analysis and PAUP* (Swofford MATERIAL AND METHODS 1998) with maximum parsimony (MP) as the optimality criterion. Gaps were excluded from the phylogenetic anal- Plant material. A total of 22 accessions were used in ysis. In the case of ME, the Kimura 2-parameter distance the molecular part of this study, 12 belonging to the genus was used. The maximum number of trees to be retained Didymodon, one to Kingiobryum, and nine to possible out- at each step was set to 100 and the CNI search level to group species. The outgroup species were selected ac- two. cording to previous results obtained during the study of a In the case of MP, all characters were given equal broader selection of Didymodon species. Trichostomum weight and the heuristic search used the following set- unguiculatum (Mitt.) R. H. Zander (subfamily Trichosto- tings: steepest descent off, TBR branch swapping, MUL- moideae) was used to root the trees. Details on geographic TREES on, and 100 random-sequence additions saving an origin, voucher data, and GenBank accession numbers are unlimited number of trees per replicate. given in Table 1. Bootstrap analysis (Felsenstein 1985) was carried out 2004] WERNER ET AL.: KINGIOBRYUM PARAMICOLA 217 Genbank AY437129 AY437130 AY437111 AY437121 AY437124 AY437115 AY437099 AY437098 AY437106 AY437090 AY437093 AY437117 AY437103 AY437122 AY437134 AY437128 AY437133 AY437132 AY437127 AY437126 AY437125 Accession No. 13.06.02) Blockeel 10325 15351 12954 13217 15415 8443 13428 12783 13654 13438 10427 9053 14049 13827 12254 6465 12218 635286 MUB MUB MUB MUB MUB MUB MUB MUB MUB MUB NY MUB MUB MUB MUB MUB MUB MUB Herb. Blockeel ( Herb. Blockeel 29/097 Herb. R. Skrzypczak 95600 Ã Èrttemberg Ânnisos Ârida Âa Âa Ellas Âlaga Ârida Spain, Almerõ Greece, Stere Yugoslavia, Serbia South Africa, Western CapeSpain, Province Ciudad Real South Africa, Western Cape province Spain, Le Spain, Balear Islands Greece, Central Greece andUnited Euboea Kingdom, England Italy, Sicily Germany, Baden-Wu Italy, Alto Adige Greece, Pelopo Morocco, Souss Massa-Draa Spain, Ma Greece, Central Greece andFrance, Euboea Alps Maritimes Spain, Balear Islands Venezuela, Me Spain, Murcia (Schultz) R. H. Zander Èll. Hall. (Hedw.) P. C. Chen Species Geographic Origin Voucher Specimen Èll. Hal.) J. Guerra & M. J. Cano (Mitt.) R. H. Zander (Mu H. Rob. Èll. Hal.) R. H. Zander Âbr. & R. B. Pierrot (Hook. & Grev.) R. H. Zander Èll. Hal.) Mu Luisier He (Brid.) Lisa (Mu Hedw. Jur. (Mu (Mitt.) Delogne Hedw. (Brid.) R. H. Zander Hornsch. (Brid.) K. Saito (Hedw.) R. H. Zander Hedw. (Brid.) Mont. 1. Collection identi®cation and GenBank accession numbers for the taxa included in the molecular analysis. ABLE T Didymodon rigidulus Didymodon sinuosus Didymodon cordatus Didymodon fallax Didymodon luridus Didymodon tophaceus Didymodon umbrosus Pseudocrossidium hornschuchianum Tortula inermis Tortula muralis Triquetrella tristicha Trichostomum unguiculatum Triquetrella arapilensis Barbula unguiculata Bryoerythrophyllum recurvirostrum Didymodon acutus Didymodon australasiae Didymodon bistratosus Leptophascum leptophyllum Kingiobryum paramicola Didymodon vinealis 218 THE BRYOLOGIST [VOL. 107

FIGURE 1. One of two most parsimonious MP cladograms (221 steps; CI ϭ 0.507; RI ϭ 0.693). The genus Didy- modon forms a monophyletic clade and Kingiobryum paramicola occupies a position near Didymodon australasiae and Didymodon umbrosus. Bootstrap support values (1,000 replicates) are given below the clades. with 1,000 replicates and identical settings for both ME had the longest ITS2 region within this genus with and MP. 292 bp and Didymodon acutus (Brid.) K. Saito the shortest (273 bp). The alignment had a total length RESULTS of 1,412 bases. DNA sequencing. The complete region of the In all, 893 positions of the aligned sequences 18S rRNA gene (partial sequence)-ITS1 - 5.8S were gapped sites and were excluded from further rRNA - ITS2 26S rRNA gene (partial sequence) analysis. Of the remaining sites, 397 were constant, could be sequenced for all accessions. The length 35 variable but parsimony-uninformative and 87 of the region ranged from 735 to 1044 base pairs parsimony-informative. (bp). The shortest sequence corresponded to Didy- The MP search revealed two most parsimonious modon sinuosus (Mitt.) Delogne and the longest se- trees of 221 steps, CI ϭ 0.507, RI ϭ 0.693 (Fig. quence to D. bistratosus HeÂbr. & R. B. Pierrot. The 1). The ME criterion resulted in a similar tree with partial sequence of the 18S rRNA gene had a uni- almost identical topology (Fig. 2). The Didymodon form length of 26 bp. The ITS1 region was highly clade, in both cases, is well supported with boot- variable and contained multiple indels and ranged strap values of 97% (MP) and 98% (ME). Within from 233 bp in Trichostomum unguiculatum to 551 Didymodon, there are two clades that are probably bp in Didymodon bistratosus. Within the genus Di- monophyletic, one of them formed by D. acutus, dymodon, D. sinuosus showed the shortest ITS1 re- D. cordatus Jur., D. rigidulus Hedw., and D. vali- gion with 244 bp. The 5.8S rRNA gene had the dus Limpr. (88% bootstrap support with MP and same length in all accessions (159 bp). The ITS2 97% with ME), and the other by D. australasiae, region was less variable in length than the ITS1 D. bistratosus, D. umbrosus (MuÈll. Hal.) R. H. Zan- region, measuring between 264 bp (Trichostomum der, and Kingiobryum paramicola (99% bootstrap unguiculatum) and 305 bp (Pseudocrossidium support with MP and 100% with ME). The position hornschuchianum (Schultz) R. H. Zander). Didy- of the remaining species of Didymodon is not well modon australasiae (Hook. & Grev.) R. H. Zander resolved and at present it is not clear whether this 2004] WERNER ET AL.: KINGIOBRYUM PARAMICOLA 219

FIGURE 2. ME phylogram. This analysis con®rms the monophyly of the genus Didymodon. Here, Kingiobryum paramicola seems to be more closely related to Didymodon umbrosus. Bootstrap support values (1,000 replicates) are given below the clades. group of species is monophyletic, as suggested by The most closely related species to Kingiobryum ME, or not. paramicola from a morphological point of view are Also, many morphological similarities were ob- Didymodon australasiae and D. umbrosus. These served between Kingiobryum paramicola and some three species share most of the characteristics de- species of the genus Didymodon. According to our scribed by Zander (1993) for Didymodon sect. As- own observations and the precise description of terisciumÐoccasional stem hyalodermis; leaves Zander and Cleef (1982), this species possesses the spreading to squarrose when moist from a sheath- uniseriate axillary hairs of the leaves, composed of ing base, broadly channeled on the adaxial surface; 1±2 brownish basal cells with the remaining 5±7 leaf margins not decurrent, plane to broadly re- cells hyaline; this is the most important morpho- curved above the sheathing base, bistratose above logical feature to characterize Didymodon. Other midleaf; costa ending below the apex to shortly ex- morphological characteristics of this genus include current, adaxial surface convex, with quadrate to laminal cells of leaves well de®ned in surface view, elongate cells; upper laminal cells unistratose to mostly quadrate to short-oblong cells of the abaxial surface of the costa (Saito 1975), mostly lanceolate bistratose, papillae absent to large, simple to bi®d, to longly lanceolate leaves, and the usually green, 1±4 per lumen; transverse section of costa often shortly rectangular, poorly differentiated basal lam- with hydroids; and KOH color reaction yellow to inal cells (Zander 1993). The genus Erythrophyl- yellowish orange. The only discrepancy with the set lopsis can be distinguished from Kingiobryum par- of characteristics proposed by Zander (1993) is the amicola by bistratose leaf laminae with plane mar- development of the adaxial stereid band in the gins; absence of pores in the basal cells; elongate transverse section of the costa, which, according to cells on the abaxial face of the costa and on the Zander (1993), is absent or very small in Didymo- adaxial face only in the upper ¼ of the leaf; and don sect. Asteriscium, but strongly developed in rudimentary or short peristome. Kingiobryum paramicola. 220 THE BRYOLOGIST [VOL. 107

DISCUSSION idinervis as the only species belonging to this ge- nus. After a study of the type specimens of both The genus Didymodon, as de®ned by Zander species, we have observed that they are morpho- (1978, 1981, 1993), KucÏera (2000, 2002) and logically similar to Kingiobryum paramicola, al- JimeÂnez (2003) using morphological characters, has though some differences are observed: the adaxial been con®rmed as monophyletic within the Pot- stereid band of the costa section is present in K. tioideae based on molecular markers (Werner, pers. paramicola but absent from the other species; the comm.). According to the nuclear rDNA sequence number of guide cell layers in the costa section is data presented here, Kingiobryum paramicola is one in K. paramicola, two in D. herzogii and four placed, with good support, within the genus Didy- to ®ve in G. validinervis; the papillosity in the up- modon by both methods used in this paper for phy- per laminal cells is absent or slight in K. parami- logenetic interference. Its closest relatives are a cola and Gertrudiella validinervis, but abundant, group of species formed by D. australasiae, D. bis- simple or bi®d in D. herzogii; and the margins of tratosus, and D. umbrosus. Chloroplast rps4 data the leaf are narrowly recurved and bistratose in K. (Werner et al. 2004) also suggest these relation- paramicola, plane and bistratose in D. herzogii and ships, but the slower evolution of this region, to- revolute and unistratose in G. validinervis. We con- gether with the reduced number of Didymodon spe- clude that the three species share an important set cies included in this study, did not permit a de®n- of features and that the differences are within the itive conclusion to be reached about the taxonomic usual range of variation for related species; fur- status of Kingiobryum. However, of the four species thermore, the segregation of G. validinervis into a of Didymodon included (D. giganteus (Funck) Jur., different genus considered to form an independent D. luridus, D. sinuosus, and D. australasiae), it was subfamily, Gertrudielloideae R. H. Zander, should D. australasiae that was placed with bootstrap val- be more carefully studied. Unfortunately, we have ues of 99% (Neighbor-Joining) and 100% (MP) in not been able to ®nd appropiate material to be in- a monophyletic clade together with Kingiobryum cluded in this molecular study. paramicola. Based on the rps4 data, Kingiobryum Didymodon challaense, also known from the An- paramicola is not very closely related to Erythro- des cordillera, is closely related to Kingiobryum phyllopsis. Instead, E. fuscula (MuÈll. Hal.) Hilp. is paramicola, not only in the porose basal cells, but placed in a clade with Erythrophyllastrum andinum also the hyaline sheathing leaf base and the bistra- (Sull.) R. H. Zander and Barbula unguiculata tose leaf marginsÐthis probably led Zander (1993) Hedw. to include it within the section Asteriscium of Di- The morphological characteristics that led Rob- dymodon. inson (1967) to consider Kingiobryum paramicola The following nomenclatural change is pro- as a new species were the basal laminal cells dif- posed: ferentiated medially, with porose walls. Although uncommon, at least within the family Pottiaceae, DIDYMODON PARAMICOLA (H. Rob.), comb. nov. these features are present, as mentioned previously, Kingiobryum paramicola H. Rob., THE BRYOLOGIST 70: in another two Didymodon species. Didymodon 9. 1967. TYPE: ``Colombia. Eastern Cordillera, Depart- herzogii was included by Hilpert (1933) in the ge- ment of Cundinamarca, Municipality of Calera, Hacienda nus Gertrudiella Herzog as G. ferruginea Hilp. La Siberia, PaÂramo de Palacio, el. 3490 m'' King C-1015, (basionym: Trichostomum ferrugineum, nom. il- Jaramillo-Mejia & Guevara (holotype, US!; isotypes, COL, NY!, U). leg.), as well as G. validinervis, both of them only known from the Andes. In this work, Hilpert con- ACKNOWLEDGMENTS sidered that the genus Gertrudiella is closely relat- ed to the genus Asteriscium (MuÈll. Hal.) Hilp. nom. We would like to thank the curators of JE, NY, and US, illegitimate [ϭ Didymodon sect. Asteriscium (MuÈll. for sending material and two anonymous reviewers for their helpful comments on an earlier version of the man- Hal.) R. H. Zander] because of strongly papillose uscript. This work has been carried out with ®nancial sup- upper laminal cells, anatomy of the leaf costa, leaf port from MCyT of Spain (Project BOS2001±0276) and shape, upper laminal cells, thin-walled, long rect- ``FundacioÂn SeÂneca'' of Murcia. angular basal laminal cells, and pluristratose leaf margins (this last characteristic absent from G. val- LITERATURE CITED idinervis). These af®nities between the genera Ger- CHURCHILL, S. P. & E. L. LINARES C. 1995. Prodromus trudiella and Asteriscium described by Hilpert bryologiae Novo-Granatensis. IntroduccioÂn a la ¯ora (1933), were later partly corroborated by Zander de musgos de Colombia. Part 1. Adelotheciaceae a Funariaceae. Bilbioteca Jose JeroÂnimo Triana, Santaf- (1993), who placed G. ferruginea in the genus Di- e de BogotaÂ. dymodon section Vinealis (Steere) R. H. Zander DE BOER, S. H., L. J. WARD,X.LI &S.CHITTARANJAN. with a new name: D. herzogii, but keeping G. val- 1995. Attenuation of PCR inhibition in the presence 2004] WERNER ET AL.: KINGIOBRYUM PARAMICOLA 221

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