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Molecular studies about two rare species of the genus Tillandsia L. (T. califanii Rauh and T. tomaselii De Luca, Sabato et Balduzzi).

Introduction

During the late 1960s, two independent botanical expeditions to Mexico were undertaken to study the genus Tillandsia. Two unusual specimens were collected. In 1966, Prof. Werner Rauh (University of Heidelberg, Germany) collected a specimen that grew on big cacti in the Tehuacan region of Puebla State, and classified it as a new species with the name T. califanii (Figure 1) (Rauh, 1971). In 1969, Prof. Paolo De Luca, Sergio Sabato, and Alberto Balduzzi (the first two of the University of Naples, and Pavia the third one, Italy) encountered a species of Tillandsia on the tree trunks along the river Rio Hondo in Oaxaca State, which was successively identified as T. tomasellii (Figure 2) (De Luca et al. 1979).

These plants have some coincidence as: (1) they have been dedicated to two botanical Italians (Prof. Luigi Califano, Pathologist Doctor, a keen collector of Tillandsia, and Ruggiero Tomaselli, Professor of Botany, very good phytosociologist); (2) morphologically similar to other Tillandsia species (Rauh, 1971, Smith & Downs 1977, De Luca et al. 1979); (3) endemic to Mexico (Smith & Downs 1977); and (4) very rare plants (Smith & Downs 1977). Relatively little has been published on these species because of the difficulty in sampling them. The available data for T. califanii are concerned with taxonomical characterisation (Rauh, 1971, Smith & Downs 1977) and ecology (Garcia-Suarez et al. 2003, 2006). There are no studies published on T. tomasellii, except one by De Luca et al. (1979) about species classification.

In the present study, we assessed the phylogenetic relationships between these two Tillandsia species. We used nucleotide sequences from six regions of the chloroplast genome, which have already been employed in an earlier phylogenetic study on Tillandsioideae (Barfuss et al. 2005), and compared them with Tillandsioideae sequences in GenBank and other Tillandsia species sequenced. The plastidial markers investigated were: rps16 intron, [trnL.sup.(UAA)] intron, [trnL.sup.(UAA)]--trnF(GAA) intergenic spacer, atpB-rbcL intergenic spacer, rbcL gene with a part of rbcL-accD intergenic spacer, and partial matK gene with a part of the flanking [trnK.sup.(UUU)] intron. The utility of these sequences for resolving phylogenetic relationships has been highlighted by several authors, as described by Barfuss et al. (2005).

Materials and Methods

Plant accessions: A total of 133 species of Tillandsioideae (121 from GenBank) were considered for the present investigation. Twelve samples (nine species) have been analysed directly by the authors, including some from herbaria and living specimens (Table 1). Parts of the fresh leaves from each specimen were frozen at - 80[degrees]C prior to use. The taxa are: Tillandsia califanii and T. tomasellii, T. achyrostachys, T. exserta, T. fasciculata, T. lepidosepala, T. matudae, T.paucifolia and T. xerographica. These taxa have not been sequenced (except for T. fasciculata). The other 121 GenBank taxa have been analysed by Barfuss et al. (2005). The complete list of taxa is available from the correspondence-author upon request.

DNA extraction from herbarium specimens: Several DNA extraction protocols have been tested because of the difficulty in obtaining a good DNA to amplify. DNA was extracted as follows: 50-100 mg leaf tissue were ground to a fine powder in liquid nitrogen and transferred to a 2-ml tube that contained 0.9 ml 2x CTAB extraction buffer [100 mM Tris-HCl, pH 8.0, 1.4 M NaCl, 20 mM EDTA, 2% (w/v) CTAB, 0.2% [beta]-mercaptoethanol], and incubated for 30 min at 60[degrees]C. The homogenate was extracted by an equal volume of chloroform--isoamyl alcohol (24:1), and then centrifuged at 7000 g for 5 min. The top aqueous phase was recovered, and two more extractions with chloroform--isoamyl alcohol (24:1) were carried out. The top aqueous phase was recovered again, and 70% cold isopropanol was added and mixed gently to precipitate the nucleic acids. After 5 min on ice, the sample was directly centrifuged at 10,000 g for 8 min. The DNA pellet was washed with 70% ethanol, dried and resuspended in 0.5 ml sterile distilled water. Another extraction with chloroform--isoamyl alcohol (24:1) was performed, and the DNA was successively precipitated with 1/10 of 3.5 M sodium acetate 3M (pH 5.2) and double volumes of 100% ethanol. The pellet was washed with 0.8 ml 70% ethanol followed by centrifugation for 3 min. The supernatant was removed, and the pellet was dried for 10 min and dissolved in 0.05 ml sterile distilled water.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

DNA extraction from fresh tissue: Total DNA was isolated according to the method of Doyle & Doyle (1990), using 100 mg leaf tissue, with the addition of a chloroform--isoamyl alcohol (24:1) purification step. DNA was resuspended in 0.08 ml sterile distilled water.

PCR amplification and sequence analyses: Chloroplast markers were amplified using the primers described by Barfuss et al. (2005). For herbarium specimens, additional internal primers were designed for Tillandsia sequences. All primers used are listed in the Table 2. According to matK, only the first part was amplified, because it yields the most variable portion through a preventive alignment of all GenBank sequences. PCR was carried out under conditions described by Barfuss et al. (2005). 7 [??]l of herbaria PCR reactions were reamplified in a 50 [??]l reaction volumes with internal primers and the PCR conditions were the same as for the fresh tissue samples. The amplified fragments were sequenced using a modification of the method of Sanger et al. (1977), using a fluorescent dye (Big Dye[TM] Terminator Cycle Sequencing Kit; Applied Biosystems) in an ABI PRISM[R] 3100-Avant Genetic Analyzer (Applied Biosystems). Complete sequences of both strands of each PCR product were processed, aligned, and visually checked by using Sequence Analysis ver. 1.8 and Sequence Navigator ver. 1.0.1 software (Applied Biosystems).

Phylogenetic analyses: Sequences were aligned using ClustalW ver. 1.4 software (Thompson et al. 1994) with default values. Alignments were carried out as daughter processes of BioEdit ver. 7.0.9 software (Hall, 1999).

Outgroups were the same as those used by Barfuss et al. (2005) and corresponded to two species of Bromelioideae (Aechmea nudicaulis var. nudicaulis, and Bromelia plumieri), eight species of Pitcairnioideae (Brocchinia micrantha, B. reducta, B. steyermarkii, B. tatei, Hecthia carlsoniae, Lindmaniaguianensis var. guianensis, Pitcarniapunicea, and Puya laxa) and two taxa of Rapateaceae (Stegolepsis ligulata, and S. parvipetala).

Aligned sequences were then visually inspected to correct gap distributions devoid of biological meaning, and to reduce the number of gaps. After alignment, all the sequenced markers were used for cladistic analysis (gaps scored as missing data), carried out using a combined matrix.

Merging of matrices and tree editing were carried out by using the cladistic software Winclada ver. 1.00.08 (Nixon, 1999-2002). Maximum parsimony (MP) analysis was carried out using Nona ver. 20.0 (Goloboff, 1999) and TNT ver. 1.0 software (Goloboff et al. 2000); the latter was also used for the bootstrap analysis (Felsenstein, 1985). One hundred replications were carried out, saving 100 trees per replication, in a tree-space of 100,000; the resulting trees underwent a further cycle of TBR swapping. A distance tree was also obtained by using a Neighbour-Joining algorithm (Saitou & Nei 1987), which was estimated with the Jukes-Cantor distances method (Jukes & Cantor 1969) available in PAUP * ver. 4.0b10 software (Swofford, 1998).

Results

For a major resolution of the phylogenetic trees, the author have modified the trees, proposing in detail the single species of Tillandsia. The complete trees are available from the correspondence-author upon request.

The obtained results from the parsimony analysis of the single sequences are not described, being equivalent to the results of Barfuss et al. (2005). In fact the matK gene is the marker with the greatest number of variable characters (19.5%) unlike the rbcL gene (7%).

The total alignment of the 121 Tillandsioideae and 12 sequences outgroups (2 Bromelioideae, 8 Pitcairnioideae and 2 Rapateaceae) has produced a matrix of 5910 characters of which 5098 not informative. The matrix has yielded 46000 MP trees, length of the cladograms was 2280, C.I. = 0.66, R.I. = 0.81 (by removing uninformative characters, L = 1754, C.I. = 0.56, R.I. = 0.81). A majority-rule consensus tree is shown in the Figure 3. Examining the bootstrap value, the position of the taxa analysed are well supported in cladistic analyses (Figure 3).

The topology of the tree shows a subdivision in clades in accordance with the molecular results of Barfuss et al. 2005. The arrangement of the taxa under examination is in agreement with the available morphological data in literature (Rauh, 1971, Smith & Downs 1977, De Luca et al. 1979). In fact T. califanii is sister-groupto T. achyrostachys and T. tomaselii is in basal polytomy together with T. paucifolia and T. klausii. This polytomy is a sister group to another collapsed clade formed by T. xerographica and T. fasciculata, T. caput-medusae, T. juncea, T. ionantha var. ionantha (Fig. 3). The analysis of the JukesCantor distances has produced a phenogram (not present) where the same groups are recognizable in the cladistic analysis (Figure 3). The distances and sequences alignment matrix is available from the correspondence-author upon request.

Discussion

Tillandsia califanii--The plant lives in dry and semiarid region of Tehuacan- Zapotitlan de las Salinas Valley System (Puebla State, Mexico). It is epiphyte on columnar cacti or succulent Liliales [e.g., Cephalocereus columna-trajani (Karwinsky ex Pfeiff.) K. Schum., C. hoppenstedtii (F.A.C. Weber) K. Schum., Neobuxbaumia tetetzp (J.M. Coult.) Backeb., or Beaucarnea gracilis Lem.]. According to Garcia-Suarez et al. (2006), most T. califanii individuals (90%) use B. gracilis as phorophyte, and only 2% live on the columnar cactus C. columna-trajani.

Considering the molecular data, two accessions of T. califanii have been examined: the first corresponds to the sample of herbarium collected by the author of the species (Prof. W Rauh) (Figure 1); the second is a sample cultivated in the greenhouse. Both samples have produced the same results (the sequences have resulted identical) and according to the parsimony analysis they are sister to the two accessions of T. achyrostachys with a good bootstrap support (Figure 3). The analysis of the genetic distances has confirmed these results; in fact the T. califanii sequences have more similarity with T. achyrostachys sequences. These results confirm the hypothesis of Rauh (1971), according to which T. califanii is very close to T. achyrostachys, but differs from it by habitat, size, and morphology. T. achyrostachys prefers tropical dry forest (e.g., Acacia Mill., Bursera Jacq. ex L.), but some varieties are also epiphytes on cacti and trees in dry habitat. The main differences to T. achyrostachys are the pale carmine-red, nearly whitish, densely white lepidote flower bracts, the dark blue-violet flowers, while these are pale yellow green in T. achyrostachys and the flower bracts of a bright carmine red with prominent nerves (as reported in Rauh, 1971).

According to the parsimony analysis, the clade organization is well structured and supported. The T. califanii clade is sister group to a clade where most Tillandsia spp. sequenced in this research (except for T. lepidosepala) are present. These two clades have in common an ancestor which very probably was an epiphyte in dry environments. In fact in basal position there is T. disticha and successively T. paniculata. Both species do not live in Mexico but are epiphytes in xeric regions of South America (Colombia, Peru, Ecuador for T. disticha, and Haiti, Dominican Republic for the second one). This ecological feature (i.e., epiphytism in xeric regions) lost in other species of T. califanii clade (i.e., T. punctulata, T. andrieuxii and T. carlos-hankii) has reappeared in T. califanii and T. achyrostachys.

Tillandsia tomasellii--It is epiphytic on trees (e.g., Amphipterigium adstringens (Schltdl.) Standl., Andira inermis (Sw.) Kunth ex DC, AstroniumgraveolensJacq., Lysiloma microphyllum Benth., and Stenocereus chacalapensis (Bravo et Macdoug.) Buxb.) in semiarid regions (low deciduous tropical forest) of the Municipality of San Carlos Yautepec (Oaxaca State, Mexico). Nowadays the only well-known station is that of the type collection (De Luca et al. 1979).

Only one specimen has been analysed which corresponds to the Isotype (PAV) (Figure 2), in fact other Botanical Gardens, and Tillandsia lovers or sellers have been contacted unsuccessfully to find other samples. In August of 2007, a botanical expedition of ours was undertaken to sample T. tomaselhi specimens. Unfortunately, no samples have been found because of a strong anthropogenic impact which has compromised the habitat of T. tomaselhi.

[FIGURE 3 OMITTED]

Molecular Studies about two Rare Species of Tillandsia.

According to the morphology, this species presents some likenesses with two Tillandsia spp. (De Luca et al. 1979). On the basis of its carinate posterior sepals it is related to T. xerographica and to T. fasciculata; but it is different from the second because its leaves are more cretaceous-coated, its spikes are few-flowered and smaller, its inflorescence is denser and much longer; and from T. xerographica because it is larger, its inflorescence is denser and much longer, its spikes are more numerous, its flowers and its floral bracts are smaller, its primary bracts are very pale pink and not yellow-red.

The molecular results confirm the above, even though the clade of T. tomasellii is not completely resolved due to a collapse with T. paucifolia and T. klausii (Figure 3). It is possible to observe a closeness of T. tomasellii with T. xerographica and T. fasciculata. This last species is part of another collapse with T. caput-medusae, T. juncea and T. ionantha var. ionantha. Worthy of note is the genetic distances matrix, where T. tomaselli has an elevated similarity of sequence with T. xerographica. Overall the clade of T. tomasellii is well structured as that of T. califanii (Figure 3). Both belong to a greater clade.

The species analyzed additionally place among other members of the subgenus Tillandsia, except for T. lepidosepala, confirming the new taxonomical collocation performed by Espejo-Serna (2002), in fact according to the author this species belongs to Viridantha genus. This could be possible due to the morphological complexity of these plants, in fact until now an exhaustive taxonomical treatment is not present in literature determining some inaccuracies in the taxonomical collocations in this genus.

Acknowledgements

Mr Luca Paino and Dr Chiara Marzano are thanked for the technical help. We are also grateful to the Dr Jose Armando Lozada Garcia for the field activity and Dr Walter Till for help with T. paucifolia.

Literature Cited

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Authors

De Castro, O. Universita degli Studi di Napoli Federico II, Dip delle Scienze Biologiche, Sez. Biologia Vegetale, Via Foria 223, I-80139 Napoli, Italy (olga.decastro@ unina.it)*corresponding author

Cennamo, P. Universita degli Studi Suor Orsola Benincasa, Facolta di Lettere, Conservazione dei Beni culturali, Via Santa Caterina da Siena, I-80135 Napoli, Italy (pcennamo@unina.it)

Vazquez-Torres, M. Institute) de Investigaciones Biologicas, Universidad Veracruzana. Av. Dr. Luis Castelazo s/n Col. Industrial Animas, Xalapa-91190, Veracruz, Mexico (savazquez@uv.mx)

De Luca, P. Universita degli Studi di Napoli Federico II, Dip delle Scienze Biologiche, Sez. Biologia Vegetale, Via Foria 223, I-80139 Napoli, Italy (pdeluca@unina.it)
Table 1. Specimens analysed directly by authors and their GenBank
accessions (rps16 intron, trnL intron and trnL-trnF intergenic spacer,
atpB-rbcL intergenic spacer, rbcL with a part of rbcL-accD intergenic
spacer, and partial trnK intron and matK).

Taxa                     Locality and Voucher     GenBank No.s

Tillandsia               Mexico (cultivated       FM211650, FM211661,
achyrostachys-1          in NAP), Batterer TA1    FM210798, FM211061,
E. Morren ex Baker       10.2007                  FM210787

T. achyrostachys-2       Mexico, Larson TA2       FM211653, FM211662,
                         11.2007 (NAP)            FM210799, FM211062,
                                                  FM210788

T. califanii-1 Rauh      Mexico, Rauh 36219       FM211651, FM211663,
                         17.07.1974 (heid)        FM210800, FM211063,
                                                  FM210789

T. califanii-2           Mexico (cultivated       FM211652, FM211664,
                         in NAP), Wrinkk TC5      FM210801, FM211064,
                         10.2007                  FM210790

T. exserta Fernald       Mexico, Larson TE2       FM211654, FM211665,
                         11.2007 (NAP)            FM210802, FM211065,
                                                  FM210791

T. fasciculata Swartz    Mexico (cultivated       FM211655, FM211666,
                         in NAP), Wrinkle TF2     FM210803, FM211066,
                         10.2007                  FM210792

T. lepidosepala          Mexico (cultivated       FM211656, FM211667,
L. B. Smith              in NAP), Kak. Haa.       FM210804, FM211067,
                         TL001 07.2007            FM210793

T. matudae L.B. Smith    Mexico (cultivated       FM211657, FM211668,
                         in NAP), Kak. Haa.       FM210805, FM211068,
                         TM001 07.2007            FM210794

T. paucifolia Baker      Mexico, De Luca &        FN550873, FN550872,
                         Vazquez-Torres           FN550870, FN550874,
                         01.2009 (NAP, HEID)      FN550871

T. tomasellii            Mexico, De Luca &        FM211658, FM211669,
De Luca, Sabato &        al. 3777 Isoype          FM210806, FM211069,
Balduzzi                 12.07.1969 (PAV)         FM21079

T. xerographica-1        Mexico, (cultivated      FM211659, FM211670,
Rohweder                 in NAP), De Luca s.n.    FM210807, FM211070,
                                                  FM210796

T. xerographica-2        Mexico,  Lo%ada          FM211660, FM211671,
                         s.n. 08.2007 (NAP)       FM210808, FM211071,
                                                  FM210797

Table 2. Primers used for PCR amplification, reamplification and cycle
sequencing of plastidial regions of fresh and herbarium specimens of
Tillandsia sequenced in the present study.

Primer                 Sequence (5'-3')           Reference

rps16 intron

Forward--rpsF          GTG GTA GAA AGC AAC        Oxelman et al. 1997
                       GTG CGA CTT
Reverse--rpsR2         TCG GGA TCG AAC ATC        Oxelman et al. 1997
                       AAT TGC AAC
Internal-rps-For       AGA TGC TCT TGG CTC        De Castro *
                       GAC AT)
Internal-rps-Rev       TTC CTC ATA CGG CTC        De Castro *
                       GAG AA

trnL intron and trnL-trnF intergenic spacer

Forward--c             CGA AAT CGG TAG ACG        Taberlet et al. 1991
                       CTA CG)
Reverse--d             GGG GAT AGA GGG ACT        Taberlet et al. 1991
                       TGA AC
Forward--e             GGT TCA AGT CCC TCT        Taberlet et al. 1991
                       ATC CC
Reverse--f             ATT TGA ACT GGT GAC        Taberlet et al. 1991
                       ACG AG

atpB-rbcL intergenic spacer

Forward--Oligo 2       GAA GTA GTA GGA TTG        Manen et al. 1994
                       ATT CTC
Reverse--Oligo 5       TAC AGT TGT CCA TGT        Manen et al. 1994
                       ACC AG
Internal-spacer-For    TTA GTT GGT ACC GCC        De Castro *
                       CAA
Internal-spacer-Re     TTG AGG AGT TAC TCG        De Castro *
                       GAA TGC

rbcL gene with a part of rbcL-accD intergenic spacer

Forward--1F            ATG TCA CCA CAA ACA        Fay et al. 1998
                       GAA AC
Reverse--1460R         TCC TTT TAG TAA AAG ATT    Fay et al. 1998
                       GGG CCG AG
Internal-rbc-For       TTT GGT TTC AAA GCC        De Castro *
                       CTA CG
Internal-rbc-Rev       CTA GCT CAG GGC TCC        De Castro *
                       ATTT G

partial matK gene with a part of the flanking trnK intron

Forward---19F          CGT TCT GAC CAT ATT        Molvray et al. 2000
                       GCA CTA TG
Reverse--1326R         TCT AGC ACA CGA AAG        Cuenoud et aL 2002
                       TCG AAG T
Internal-mat-For       CCT GCC TCT GGC TCA        De Castro *
                       AGT AG
Internal-mat-Rev       AAT CGG TCC AGA TTG        De Castro *
                       GCT TA

* primers designed by the first author for herbarium specimens.
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Title Annotation:Scientific
Author:De Castro, Olga; Cennamo, Paola; Vazquez-Torres, Mario; De Luca, Paolo
Publication:Journal of the Bromeliad Society
Article Type:Report
Geographic Code:4EUIT
Date:Sep 1, 2009
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