Stelis (Orchidaceae) encompasses approximately 1100 species of epiphytic orchids distributed throughout the Neotropics, with the highest diversity in Andean South America. Sixty-two species were recorded previously in Mexico.
We formally describe here Stelis zootrophionoides as a new species from Chiapas, Mexico. To determine its systematic position, we conducted a morphological comparison with other members of Pleurothallidinae and a phylogenetic analysis of nucleotide sequences from the plastid matK/trnK and trnL/trnF regions, as well as the nuclear ribosomal ITS region for 52 species of Pleurothallidinae. Sequences of 49 species were downloaded from GenBank and those of three species, including the new taxon, were newly generated for this work. The new species is described and illustrated; notes on its ecological preferences and a comparison with closely related species are presented.
The new species, known only from one location and apparently restricted to the cloud forest in the central highlands of Chiapas, Mexico, is considered a rare species. This small epiphyte is unique among the Mexican species of Stelis by the combination of dark purple flowers with the distal third of the dorsal sepal adhered to the apices of the lateral sepals, which are partially united into a bifid synsepal, leaving two lateral window-like openings, and sagittate labellum. Stelis jalapensis, known from southern Mexico and Guatemala, also has the apices of the sepals adhered to each other, but it is distinguished by its larger flowers with lanceolate, acute dorsal sepal, completely fused lateral sepals (i.e. the synsepal is not bifid), and oblong-elliptic labellum. The phylogenetic analysis shows that S. zootrophionoides is closely related to other Mexican Stelis and corroborates previous suggestions that fused sepal apices have arisen independently in different lineages of Pleurothallidinae.
Citation: Ramos-Castro SE, Castañeda-Zárate M, Solano-Gómez R, Salazar GA (2012) Stelis zootrophionoides (Orchidaceae: Pleurothallidinae), a New Species from Mexico. PLoS ONE 7(11): e48822. doi:10.1371/journal.pone.0048822
Editor: Martin Heil, Centro de Investigación y de Estudios Avanzados, Mexico
Received: April 5, 2012; Accepted: October 3, 2012; Published: November 7, 2012
Copyright: © 2012 Ramos-Castro et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study received support from Instituto de Biología, Universidad Nacional Autónoma de México to G.A.S. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Stelis Sw. (Orchidadeae) is the most species-rich genus in subtribe Pleurothallidinae , . However, the number of species recognized varies depending on the classification. According to Luer , , the genus includes ~900 species, whereas in the circumscription of Pridgeon and Chase  there are almost 1100 species.
Stelis species are epiphytic, lithophytic, or rarely terrestrial orchids restricted to the American tropics, distributed from southwest Florida and northwestern México through the Antilles to Bolivia and Brazil, with the highest diversity concentrated in Andean South America , . The taxa occur in a wide variety of habitats, from sea level to 4000 m in elevation . Sixty-two species of Stelis were recorded previously from Mexico , , . Recently, during a field trip in the central highlands of Chiapas, we collected specimens of a small plant belonging to subtribe Pleurothallidinae in which the distal part of the dorsal sepal was adhered to the apices of the lateral sepals, but otherwise was separated from them to form two lateral window-like openings. Species with similar floral features are known to occur in several unrelated genera of Pleurothallidinae, such as Acianthera Scheidw., Phloeophila Hoehne & Schltr., Specklinia Lindl., Stelis Sw., and Zootrophion Luer. After a comparison of the newly collected plant with all the species of Pleurothallidinae known from México and Central America, it was evident that it represents an unknown species, which is described and illustrated here for the first time.
Materials and Methods
We collected material in the Reserva Ecológica Cerro Huitepec Chiapas, Mexico, which is a protected area that is privately owned by Pronatura Mexico, AC. Pronatura permits research in their reserves and no specific permits are required for field studies like the one described here.
The description of the new species was based on examination under a stereomicroscope of fresh, pickled, and pressed specimens of the new species. A line drawing was prepared from fresh material with the aid of a drawing tube adapted to the stereomicroscope. The morphological comparison with other species of Pleurothallidinae was based on study of live plants in the field and in cultivation, herbarium specimens, and information gathered from the literature.
We downloaded from GenBank sequences of 49 species belonging to 30 genera of subtribe Pleurothallidinae sensu Pridgeon et al. . Additionally, for this work we sequenced three species, Stelis nigriflora (L.O.Williams) Pridgeon & M.W.Chase, S. rubens Schltr., and the new species. We used Dilomilis montana Summerh. as outgroup, following previous phylogenetic studies . GenBank accessions for all the DNA sequences and voucher information for the new ones are given in Table S1. The aligned matrix is available on request from G.A.S. (firstname.lastname@example.org).
We analyzed the nucleotide sequences of the plastid matK/trnK and trnL/trnF regions as well as the nuclear ribosomal ITS region. All these regions have been used previously to infer phylogenetic relationships among the Pleurothallidinae  and other orchid lineages . Genomic DNA was extracted from fresh plant tissue using a 2× cetyltrimethylammonium bromide (CTAB) protocol based on Doyle and Doyle  modified by the addition of 2% polyvinyl pyrrolidone (PVP) to the extraction buffer. Amplification (PCR) and sequencing were carried out using the methods described in Pridgeon et al. . The chromatograms were assembled and edited with Sequencher v. 4.8 (Gene Codes Corp., Ann Arbor, Michigan, USA).
Sequence alignment and cladistic analysis
Alignment of the sequences was carried out using the default settings of the online version of the program MAFFT v. 6 , with minor subsequent manual adjustment. Individual gap positions were treated as missing data and all characters were treated as unordered with equal weights. Pridgeon et al.  found no evidence of conflict among the trees resulting from separate analyses of the matK/trnK, trnL/trnF and ITS regions. Accordingly, we analyzed all three data sets in combination, conducting a parsimony analysis using the program PAUP* version 4.02b . The analysis consisted of a heuristic search with 1000 replicates of random taxon addition for the starting trees and tree rearrangements using tree bisection-reconnection (“TBR”) branch swapping, saving all most-parsimonious trees (MPTs). Clade support was assessed by means of a nonparametric bootstrap analysis , consisting of 1000 bootstrap replicates, each with 20 heuristic replicates with random taxon addition and TBR branch swapping, saving up to 20 MPTs from each replicate.
Results and Discussion
Figure 1. Stelis zootrophionoides.
A) habit, B) flower, C) dissected flower, D) column and lip lateral view, E) column ventral view, F) anther cap. G, pollinia, H) fruit, I) detail of the column base. Drawn by SE Ramos-Castro from Castañeda-Zárate & SE Ramos-Castro 448.doi:10.1371/journal.pone.0048822.g001
Figure 2. Stelis zootrophionoides.
A, B) plants in situ during the season of vegetative growth, C) fruit, D) inflorescence. (Photographs A,D by SE Ramos-Castro, and B,C by M Castañeda-Zárate).doi:10.1371/journal.pone.0048822.g002
Type: MEXICO. Chiapas: Municipio San Cristóbal de Las Casas, Reserva Ecológica Huitepec, cloud forest, epiphytic on oak, rare, 5 September 2010, M. Castañeda-Zárate & S.E. Ramos-Castro 448 (HOLOTYPE: MEXU; ISOTYPES: CHIP, MO, OAX, XAL).
Epiphytic, caespitose, herb up to 13.5 cm in height. Roots thin, terete, flexuous, white, 0.7–1.2 mm in diameter. Rhizome short, covered by tubular, scarious sheaths. Stems straight, thin, formed by 2 internodes, 2–5.5 cm long, 0.9–1.5 cm in diameter, with a thickened ring about 2.5–5 mm below the apex; covered by tubular sheaths, these obtuse, dilated towards the apex, carinate, mucronate, scarious, brownish-red when dry, lowermost sheath 0.9–2 cm long, uppermost sheath 1.6–3 cm long. Leaves oblanceolate to elliptic, rounded and minutely trilobed with mucronate apex, carinate on the abaxial surface, 3.8–8.4 cm long, 1.3–2.1 cm wide; petiole short, canaliculate, 10–15 mm long; blade fleshy, slightly arched. Inflorescence arising from the ring of the stem, racemose, shorter than the leaf, 1–3 per flowering season, 1.2–7 cm long, covered at the base by a spathaceous bract, the latter triangular, conduplicate, carinate, 3–5 mm long; peduncle straight, cylindrical, 4–13 mm long, 0.4–0.6 mm in diameter, covered by 2 tubular sheaths, these membranaceous, imbricating, obtuse, dilated and oblique towards the apex, 1.4–2.2 mm long; rachis slightly flexuous, elongating progressively towards the apex as new flowers develop, with up to 12 flowers, 1–2 open at a time. Floral bracts obliquely infundibuliform, obtuse, shortly apiculate, carinate, membranaceous, 2.7–3.8 mm long. Ovary slightly arched, thickened towards the apex, trigonous, glabrous, purple, 2.5–3.5 mm long, 0.8–1 mm diameter, articulate with the pedicel; pedicel united to the rachis above the base of floral bract, straight, cylindrical, thickened towards the apex, longer than the floral bract, 3.7–7 mm long, 0.4–0.6 mm in diameter. Flowers 4–8.1 mm long, resupinate, slightly pendent, the apex of the dorsal sepal adhered to the apex of the synsepal, forming two lateral, window-like openings; floral segments and column creamy yellow or greenish yellow, densely blotched with dark purple, anther white. Dorsal sepal united to the lateral sepals near the base, incurved, conduplicate, oblong-lanceolate, obtuse, fleshy, 3-veined, keeled on the outer surface, 6.8–7.3 mm long, 2.2–2.6 mm wide, sparsely pilose on the inner surface above the middle, hairs purple. Lateral sepals fused by two-thirds of their length to form a cymbiform, bifid synsepal, 6.5–7.1 mm long, 5.4–5.7 mm wide; the base of the synsepal forms a chin in which fits the column foot; lateral sepal apices obtuse, shortly apiculate and slightly oblique, 3-veined, keeled on the outer surface, sparsely pilose on the inner surface, hairs purple. Petals nearly parallel to the column, obovate-spatulate, fleshy, 3-veined, carinate at the abaxial surface, with rounded lateral angles, apical margins slightly erose, 2.5–3.6 mm long, 1.5–1.8 mm wide. Lip recurved, conduplicate, 3-veined, unguiculate, acute, margins erect on the proximal two-thirds, fleshy, 3.3–3.6 mm long, 0.7–1 mm wide; claw oblong, provided at each side of the base with a retrorse auricle, 1 mm long, 0.7 mm wide; blade sagittate when spread out, slightly crenulate at the margins, inner surface papillose; sides obtuse, provided with a submarginal callus, slightly thickened. Column slender, arcuate, winged, channeled ventrally, 2.8–3.2 mm long; wings erect, 0.6–0.7 mm wide; clinandrium surpassing the anther, dentate; column foot elongate, papillose at the apex. Stigma ventral, subquadrate, concave, covered by a viscous substance. Rostellum laminar, convex. Anther ventral, oblong-ovoid, two-celled, papillose-verrucose, 0.7 mm long, 0.5 mm wide. Pollinarium formed by 2 obovoid, laterally compressed, yellowish pollinia, 0.35–0.4 mm long, provided with granulose caudicles. Capsule ellipsoid, trigonous, with persistent perianth, 8.2–10 mm long, 3.7–4.9 mm in diameter.
Distribution and habitat.
Stelis zootrophionoides is known from a single location in the central highlands of Chiapas, Mexico. The only population recorded was found growing epiphytically in humus accumulations on trunks of Quercus laurina in a cloud forest at 2,554 m elevation. Other epiphytes sharing the same habitat are Peperomia quadrifolia (L.) Kunth, Epidendrum propinquum A.Rich. & Galeotti, and various bryophytes and ferns such as Campyloneurum angustifolium (Sw.) Fée, Dryopteris parallelogramma (Kunze) Alston, and Pleopeltis macrocarpa (Bory ex Willd.) Kaulf. var. interjecta (Weath) A.R.Sm.
Flowering occurs from July to September; developing fruits were observed in August, and seed dispersal occurred between October and December.
So far, this species is known from only one population and we consider it as rare, although additional field studies are required to ascertain its conservation status more objectively. It was located within a private protected area, the Reserva Ecológica Cerro Huitepec; however, outside the reserve natural vegetation is being strongly affected by logging and collecting of epiphytes . It is interesting to note that the new species was discovered in a relatively accessible area and it was immediately obvious that it represented a novelty for the Mexican orchid flora, which highlights the fact that there are still species waiting to be discovered, while the remnant habitats are being lost rapidly.
The specific epithet refers to the resemblance of the flowers to those of the genus Zootrophion; the latter was derived from Greek zootrophion, “a menagerie”, in allusion to the flowers that resemble the heads of animals .
Stelis zootrophionoides is unique among Mexican species of this genus by its raceme shorter than the leaf, with successive flowers whose sepals united at their apices leave lateral openings. Vegetatively, S. zootrophionoides is similar to Stelis nigriflora, S. retusa (Lex.) Pridgeon & M.W.Chase, and S. sotoarenasii Solano. Stelis nigriflora is distinguished from S. zootrophionoides by its larger plants, leaves spotted with purple on the abaxial surface, and abbreviated raceme; moreover, it has a restricted distribution around Tepoztlán, Morelos . Stelis retusa and S. sotoarenasii are similar to one another and both differ from S. zootrophionoides in their narrower leaves with very short petiole and free sepal apices; S. retusa is found in the Transverse Volcanic Belt and the Sierra Madre del Sur , whereas S. sotoarenasii is known only from the mountains surrounding the central valleys of Oaxaca . The only other Mexican or Central American species of Stelis in which the sepals adhere to one another at their apices is Stelis jalapensis (Kraenzl) Pridgeon & M.W.Chase, but this is a more robust plant with a many-flowered inflorescence much longer than the leaf, larger, pendulous flowers with sepals ≥10 mm long, sepals prominently ciliate and adhered only at their apex, and lateral sepals completely connate forming an entire, acute synsepal , .
In our phylogenetic analysis, the aligned matrix consisted of 4392 characters, of which 1683 (38%) were variable and 868 (20%) were parsimony-informative. The analysis found four MPTs with a length of 4258 steps, consistency index (excluding uninformative characters) of 0.41, and retention index of 0.52. One of the four MPTs, on which bootstrap percentages are indicated, is shown in Figure 3. Overall, relationships agree with those recovered in the phylogenetic analyses of Pridgeon et al. . Stelis zootrophionoides is embedded in strongly supported Stelis sensu Pridgeon and Chase , being strongly supported as sister to S. nigriflora. This species-pair is in turn sister to S. pilosa Pridgeon & M.W.Chase/S. segoviensis (Rchb.f.) Pridgeon & M.W.Chase, although this group collapsed in the strict consensus. This whole clade was recovered as sister to a weakly supported group including successively S. rodrigoi (Luer) Pridgeon & M.W.Chase, S. emarginata (Lindl.) Soto Arenas & Solano, S. neoharlingii (Garay) Pridgeon & M.W.Chase, S. velaticaulis (Rchb.f.) Pridgeon & M.W.Chase, and S. rubens/S.argentata Lindl.
Figure 3. Phylogenetic tree.
One of the four most parsimonious trees found by the combined analysis of matK/trnK, trnL/trnF, and nrITS DNA sequences. Numbers above branches are number of changes, numbers below branches are bootstrap percentages. The arrowheads point to clades that collapse in the strict consensus.doi:10.1371/journal.pone.0048822.g003
The most salient feature distinguishing S. zootrophionoides from its close relatives is the adhesion of the distal portions of the sepals. As mentioned earlier, adhesion of the sepal apices is found in other distantly related groups of Pleurothallidinae . Such similarity might be the result of use of the same or similar pollinators, as several studies have shown that homoplasy in floral attributes involved in pollination among unrelated clades is a widespread phenomenon in the Orchidaceae , . According to the pollination syndromes concept, flowers can converge on a suite of floral traits (i.e. flower size, color, odor, rewards) associated with the attraction and utilization of a specific group of animals as pollinators . Pleurothallidinae is mainly a group pollinated by small flies (myophily) of families Drosophilidae, Chlorophidae and Phoridae , , and the myophillous syndrome is characterized generally by bowl-shaped or flat flowers with windows or filiform appendages .
Taxa analyzed, voucher information or literature reference, and GenBank accessions for the DNA sequences.
We are grateful with Luis Sanchez-Saldaña and Rolando Jiménez-Machorro (AMO) for their assistance; Pablo A. Merino-García, César F. Ramos-Castro and Carlos F. Ramos-Castro for their help during field work; PRONATURA-Chiapas for granting us permission to work at the Reserva Ecológica Cerro Huitepec; Laura Márquez-Valdelamar (Laboratorio de Biología Molecular, Instituto de Biología, Universidad Nacional Autónoma de México) for assistance for DNA sequencing and Rubén Pérez-Ishiwara (Laboratorio de Interacción Planta-Animal, Instituto de Ecología, Universidad Nacional Autónoma de México) for assistance with microscopy. David Gernandt (Departamento de Botánica, Instituto de Biología, Universidad Nacional Autónoma de México) and two anonymous reviewers made useful suggestions to a previous versions of the manuscript.
Conceived and designed the experiments: SERC MCZ GAS. Performed the experiments: SERC MCZ RSG GAS. Analyzed the data: SERC MCZ GAS RSG. Contributed reagents/materials/analysis tools: SERC GAS. Wrote the paper: SERC MCZ RSG GAS.
- 1. Luer CA (2007) Icones Pleurothallidinarum XXIX: A third century of Stelis of Ecuador. Monogr Syst Bot Mo Bot Gard 112: 1–2.
- 2. Luer CA (2009) Icones Pleurothallidinarum XXX: Systematics of Stelis of Ecuador, part four. Monogr Syst Bot Mo Bot Gard 115: 31–36.
- 3. Pridgeon AM, Cribb PJ, Chase MW, Rasmussen FN (2005) Genera Orchidacearum Volume 4: Epidendroideae (Part One). New York USA: Oxford University Press. 672 p.
- 4. Solano R, Salazar GA (2007) A new species of Stelis (Orchidaceae, Pleurothallidinae) from Guerrero México. Rev Mex Biodivers 78: 253–256. doi: 10.1371/journal.pone.0048822
- 5. Solano R (2010) Dos especies nuevas de Pleurothallidinae (Orchidaceae) para México. Lankesteriana 9: 447–454.
- 6. Solano R (2011) Three New species of Stelis (Orchidaceae) for México. Brittonia 63: 325–333. doi: 10.1007/s12228-010-9171-z
- 7. Pridgeon AM, Solano R, Chase MW (2001) Phylogenetic relationships in Pleurothallidinae (Orchidaceae): combined evidence from nuclear and plastid DNA sequences. Am J Bot 88: 2286–2308. doi: 10.2307/3558390
- 8. Cameron KM (2007) Molecular phylogenetics of Orchidaceae: the first decade of DNA sequencing. In: Cameron KM, Arditti J, Kull T, editors. Orchid Biology: Reviews and Perspectives IX. New York: New York Botanical Garden Press. pp. 163–200.
- 9. Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure from small quantities of fresh leaf tissue. Phytochem Bull 19: 11–15.
- 10. Katoh K, Asimenos G, Toh H (2009) Multiple alignment of DNA sequences with MAFFT. In: Posada D, editor. Bioinformatics for DNA sequence analysis; Methods in Molecular Biology 537. New York: Humana Press. pp. 39–64.
- 11. Swofford DL (2002) PAUP*: Phylogenetic analysis using parsimony (and other methods) 4.0 beta. Sunderland: Sinauer.
- 12. Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783–791. doi: 10.2307/2408678
- 13. Enríquez-Rocha OL, Rangel-Salazar JL (2009) La Reserva Ecológica Huitepec. Biodiversitas 85: 6–10.
- 14. Luer CA (2004) Icones Pleurothallidinarum XXVI: Systematics of Epibator, Ophidion, and Zootrophion. Monogr Syst Bot Mo Bot Gard 95: 211–228.
- 15. Solano R, Soto Arenas MA (2003) Stelis jalapensis (Kraenzl.) Pridgeon & MW Chase. In: Hágsater E, Soto Arenas MA, editors. Icones Orchidacearum 5–6: Orchids of México 2–3. México DF: Herbario AMO. pp. 683.
- 16. Solano R, Soto Arenas MA (2008) Stelis retusa (Llave & Lex.) Pridgeon & MW Chase. In: Hágsater E, Soto Arenas MA, editors. Icones Orchidacearum 10: Orchids of México 4. México DF: Herbario AMO. pp. 1098.
- 17. Soto Arenas MA, Hágsater E, Salazar GA, Jiménez-Machorro R, Solano R et al.. (2007) Orchids of México; Digital Catalogue. México: Instituto Chinoin.
- 18. Pridgeon AM, Chase MW (2001) A phylogenetic reclassification of Pleurothallidinae (Orchidaceae). Lindleyana 16: 235–271.
- 19. Luer CA (1986) Icones Pleurothallidinarum III: Systematics of Stelis. Monogr Syst Bot Mo Bot Gard 20: 1–50. doi: 10.2307/4110977
- 20. Chase MW, Williams NH, Donisete de Faria A, Neubig KM, Amaral MCE, et al. (2009) Floral convergence in Oncidiinae (Cymbidieae; Orchidaceae): an expanded concept of Gomesa and a new genus Nohawilliamsia. Ann Bot 104: 387–402.
- 21. Salazar GA, Cabrera LI, Figueroa C (2011) Molecular phylogenetics, floral convergence and systematics of Dichromanthus and Stenorrhynchos (Orchidaceae, Spiranthinae). Bot J Linn Soc 167: 1–18. doi: 10.1111/j.1095-8339.2011.01161.x
- 22. Fenster CB, Armbruster WS, Wilson P, Dudash MR, Thomson JD (2004) Pollination syndromes and floral specialization. Annu Rev Ecol Evol S 35: 375–403. doi: 10.1146/annurev.ecolsys.34.011802.132347
- 23. Christensen DE (1992) Notes on the reproductive biology of Stelis argentata Lindl. (Orchidaceae: Pleurothallidinae) in Eastern Ecuador. Lindleyana 7: 28–33.
- 24. Albores-Ortiz O, Sosa V (2006) Polinización de dos especies simpátricas de Stelis (Pleurothallidinae: Orchidaceae). Acta Bot Mex 74: 155–168.
- 25. Pellmyr O (2002) Pollination by animals. In: Herrera CM, Pellmyr O, editors. Plant-Animal Interactions, an Evolutionary Approach. United Kingdom: Blackwell Science Ltd. 313 p.