Biodiv. Res. Conserv. 23: 15-27, 2011 BRC www.brc.amu.edu.pl DOI 10.2478/v10119-011-0017-7 Chelystachya, a new genus of the subtribe Polystachyinae (Orchidaceae) Joanna Mytnik-Ejsmont*, Dariusz L. Szlachetko & Marcin GÛrniak Abstract: A new genus of Orchidaceae (Polystachyinae), Chelystachya Mytnik & Szlach. is described and illustrated basing on results of molecular and morphological studies. It is related to Polystachya Hook. but is distinct in having large, singlenoded, spherical, succulent pseudobulbs flattened on the substratum, pendent and densely pubescent inflorescence, lowermost lip, and lip veins directed to the lip base. Results of phylogenetic analyses based on plastid markers validate the reason for a new genus. Keys for determination of all genera of the subtribe Polystachyinae and both species of Chelystachya are included. Two new combinations are made. A comprehensive description and full synonymy, as well as detailed ecological, phenological and distribution data and a list of 125 examined specimens are provided for each taxon treated. Because Polystachya affinis Lindl. is considered a holotype of Polystachya Hook. sect. Affines Kraenzl., this name is automatically a synonym of the newly introduced generic name Chelystachya. Other species of the former section Affines are classified within the section Humiles. Key words: Africa, molecular phylogeny, morphology, nomenclature, Polystachya, rpl16, rpl32-trnL, taxonomy, Polystachya sect. Humiles 1. Introduction The genus Polystachya Hook., described in 1824, received only one monographic treatment so far (Kraenzlin 1926). Since then, six sections (Summerhayes 1942, 1948; Cribb 1978) and a large number of new species have been described. Additionally, recently, some species placed in the traditionally conceived genus Polystachya were placed in separate genera (Mytnik-Ejsmont 2007, 2008; Mytnik-Ejsmont & Szlachetko 2007a, 2007b, 2008a, 2008b, 2011; Mytnik et al. 2011). That made Kraenzlinís (1926) classification unsatisfactory and outof-date. Therefore, there is a need to revise this large and complex genus, which consists of about 230 species. Kraenzlin (1926) established section Affines within the genus Polystachya. It includes about 40 species but it is a highly heterogeneous taxon which is difficult to circumscribe (La Croix & La Croix 1997; Davies et al. 2002). According to Kraenzlin (1926) and la Croix and Cribb (1998), it is characterized as follows: (1) small to medium-sized plants with clustered, ovoid to conical, usually erect but occasionally bilaterally compressed and appressed to the substrate, (2) 2-3-leaved pseudobulbs, (3) erect or rarely arcuate, racemose and often densely pubescent (including flowers) inflorescences, (4) conspicuous bracts, fleshy, medium-sized to large for the plant, (5) usually hairy flowers with inconspicuous to somewhat prominent mentum, (6) lateral sepals free or connate only at the base, and (7) three-lobed fleshy and strongly recurved lip. Due to the high polymorphism of the section, its characterization in floras is always full of exceptions, especially in the shape of pseudobulbs, the number of stem nodes or the type of inflorescence. Extracting two species from the group, Polystachya affinis Lindl. and P. bancoensis Van der Burg, makes the section morphologically homogenous. In this paper, it is suggested that both P. affinis and P. bancoensis, reclassified into a new genus, are two very closely related species forming a group morphologically distinct from the rest of the section and from the genus Polystachya. Both species occur in west and westcentral Africa, whereas the remaining species of the section are almost exclusively East African taxa (P. camaridioides Summerh., P. obanensis Rendle, P. reticulata StÈvart & Droissart, and P. testuana Summerh. are the only exceptions, which constitute 12% of all species of the section Affines). The results of molecular analyses of plastid markers presented in this paper and those © Adam Mickiewicz University in PoznaÒ (Poland), Department of Plant Taxonomy. All rights reserved. Unauthenticated Download Date | 6/30/16 11:08 PM VARIABILITY, TAXONOMY AND PHYLOGENY Department of Plant Taxonomy and Nature Conservation, GdaÒsk University, Al. LegionÛw 9, 80-441 GdaÒsk, Poland, *e-mail: dokjom@univ.gda.pl 16 Joanna Mytnik-Ejsmont et al. Chelystachya, a New Genus of the Subtribe Polystachyinae (Orchidaceae) recently published by Russell et al. (2010) confirm the legitimacy of such a treatment. Considering the morphological and molecular data we therefore suggest transferring P. affinis and P. bancoensis to a new taxon of the generic rank. To show an isolated position of Chelystachya affinis and C. bancoensis compared to other members of the Polystachya genus, plastid fragments were sequenced: rpl16 intron and rpl32-trnL intergenic spacer. The average length of rpl16 in Polystachyinae is 1000 bp. Data from the rpl16 region has been primarily used for a phylogenetic analysis at the infrageneric and familial levels (Kelchner & Clark 1997; Schnabel & Wendel 1998; Applequist & Wallace 2000). The average length of rpl32-trnL in Polystachyinae is 650 bp. Shaw et al. (2007) suggest that this is the best region of the 34 regions surveyed for low-level molecular studies. Until now, rpl32-trnL has not been widely used in phylogenetic studies in orchids. Barrett and Freudenstein (2009) used this marker in population study in Corallorhiza Gagnebin. Additionally, to increase the number of molecular characters in phylogenetic analyses, available data for the same set of samples (Russell et al. 2010) from plastid genome were used. The present work is a part of a larger project on the taxonomy of the subtribe Polystachyinae Schltr. (Mytnik-Ejsmont 2007, 2008; Mytnik-Ejsmont & Szlachetko 2007a, 2007b, 2008a, 2008b, 2011; Baranow & MytnikEjsmont 2009; Mytnik-Ejsmont & Baranow 2010; Mytnik-Ejsmont et al. 2011). 2. Materials and methods 2.1. Morphological characters The present study was based on the examination of 125 herbarium specimens from ten herbaria: BM, BR, C, F, K, MA, MO, P, W, WAG. The standard procedure of preparing the herbarium material to facilitate stereomicroscopic observation was applied. The vegetative and generative characters of individual plants were analyzed, the flowers were taken from the middle part of the inflorescence. Particular parts of the flower were boiled, dissected, measured and drawn under a stereomicroscope. The results were then analyzed and compared with the type material, diagnoses and original illustrations. The database of the drawings and photographs of all studied specimens is available in the first authorís archives and are available upon request. For all species and lower taxa, type material was available. The abbreviations of titles are according to those in BPH (Lawrence et al. 1968), BPH/S (Bridson 2004), TL2 (Stafleu & Cowan 1976-1982) and TL2/S (Stafleu & Mennega 1992), the abbreviations of the authorsí names are according to Brummitt and Powell (1992). 2.2. Plant material, DNA extraction and PCR amplification and sequencing The taxa sampling has been guided by a previous morphology-based systematic study of Polystachyinae. DNA sequences for rpl16 intron and rpl32-trnL intergenic spacer were obtained from 42 and 45 taxa, respectively. Four species (Adrorhizon purpurascens (Thwaites) Hook.f., Sirhookera lanceolata (Wight) Kuntze, Bromheadia finlaysoniana (Lindl.) Miq., and B. srilankensis Kruiz. & de Vogel) were selected as outgroup taxa. Voucher information and GenBank accession numbers are provided in the Appendix. DNA samples were obtained from the DNA Bank of the Jodrell Laboratory, Royal Botanic Gardens, Kew (http://data.kew.org/dnabank/homepage.html). DNA extractions were performed using a modification of the CTAB protocol (Doyle & Doyle 1987), followed by purification on caesium chloride/ethidium bromide gradients. The rpl16 intron and rpl32-trnL intergenic spacer were amplified using primers developed by Shaw et al. (2005, 2007 respectively). Polymerase chain reaction (PCR) amplifications were carried out in a total volume of 25 µl containing 2.5 µl 10x buffer, 1 µl 25mM MgCl2, 1 µl 5mM dNTPs, 0.5 µl of 10µM of each primers and 1 unit of Yellow Perpetual DNA polymerase (Eurx, Poland), and H2O. The PCR cycling conditions were template denaturation at 80oC for 5 min, followed by 30 cycles of denaturation at 95oC for 1 min, primer annealing at 50oC for 1 min, followed by primer extension at 65oC for 4 min. The final extension step was of 5 min at 65oC. Amplified products were cleaned with the High Pure PCR Product Purification Kit (Roche Diagnostic GmbH, Germany), following the manufacturerís directions. Cycle sequencing was carried out using Big Dye Terminator v 3.1 Cycle Sequencing Kit (Applied Biosystems, Inc., ABI, UK) with the same primers used for PCR amplification: 2.0 µl of 5x sequencing buffer, 1 µl of Big Dye terminator with 1.5 µl of 10 µM primer (1.5 pmol), 1-2 µl of amplified product (30-90 ng/µl), and H2O in a total of 10 µl reaction volume. Cycle sequencing conditions were as follow: 25 cycles each with 15 sec denaturation (94oC), 5 sec annealing (50oC) and 4 min elongation (60oC). The sequences were generated on an ABI 3720 automated capillary DNA sequencer. Both strands were sequenced to assure accuracy in base calling. Sequence Navigator (ABI) was used to edit the sequences, and the two complementary strands were assembled using AutoAssembler (ABI). 2.3. Sequence alignment and phylogenetic analyses The rpl16 intron and rpl32-trnL intergenic spacer were aligned by eye using SeaView v. 4 (Gouy et al. 2010). Matrices are available upon request from MG. Unauthenticated Download Date | 6/30/16 11:08 PM Biodiv. Res. Conserv. 23: 15-27, 2011 An AT-rich region of rpl16 intron (161 bp) was excluded from the analysis because of ambiguous alignment. To increase molecular data, additional DNA sequences obtained from the same DNA sample as in our study (extraction available from RBG Kew) by Russell et al. 2010 were used in phylogenetic analyses. Chelystachya bancoensis (Van der Burg) Mytnik and Szlach. was not presented in Russellís data matrix. Maximum parsimony analysis was undertaken on the combined plastid matrix (rpl16 intron, rpl32-trnL + data matrix rps16-trnK from Russell et al. 2010) using heuristic searches in PAUP* version beta 10 (Swofford 2000) with tree-bisectionreconnection (TBR) branch swapping and the MULTREES (holding multiple trees) option in effect with 1000 replicates of random sequence addition. Only 10 trees were saved for each replicate to reduce the time spent in swapping large numbers of suboptimal trees. Standard tree parameters were estimated (tree length, consistency index, CI, and retention index, RI). Internal support of clades was evaluated using non-parametric bootstrapping (Felsenstein 1985) with 1000 replicates and the same settings as above, except for the simple sequence addition. All characters were treated as unordered and as equally weighted (Fitch 1971). 3. Results 3.1. Molecular data The parsimony analysis showed 54 most parsimonious trees. Statistics for one of the parsimonious is shown in Table 1. The strict consensus tree is depicted in the figure 1. The tree topology from the parsimony analysis of combined data shows the newly proposed genus, Chelystachya, to be monophyletic (Bootstrap support (BS) = 100%, Fig. 1) and is a sister clade to the rest of the Polystachyinae species (BS: 100%). A support for Chelystachya-Polystachyinae clade is below 50%. Going up the tree, there are several clades successively sister to the Polystachya s.str. clade, the grade of species-poor lineages are: Polystachya ottoniana, Polystachya longiscapa, Dendrobianthe-Neobenthamia clade and Polystachya vaginata-Polystachya goetzeana clade. 3.2. Taxonomic treatment and morphological data Chelystachya Mytnik & Szlach., gen. nov. (Fig. 2) ≡Polystachya Hook. sect. Affines Kraenzl., Rep. Sp. Nov. Regni Veget., Beih. 39: 95. 1926. Generitype: Chelystachya affinis (Lindl.) Mytnik & Szlach. (≡Polystachya affinis Lindl.). Plantae Polystachyae affines sed pseudobulbis magnis, uninodis, sphaericis, succulentis, complanatis, ad substratum appressis, inflorescentis pendulis, dense pilosis, venis labelli basim versus differunt. Epiphytic plants up to 50 cm long with a pendent habit. Pseudobulbs 1-5 cm in diameter, single-noded, dorsoventrally compressed, several, elliptic, subspherical or almost transversely elliptic, prostrate on the substratum, apically (1)2-3-leaved, glossy green to dull bluish green, sometimes tinged purple. Leaves 2-28 cm long, 0.7-6 cm wide, bluish green, greyish green to reddish blue with purplish midrib and margins, petiolate; petiole 0.5-14 cm long, narrow, subcylindrical, clasping the scape; blade oblanceolate, oblong-lanceolate, oblong-elliptic to obovate, acute to shortly acuminate. Inflorescence 1.5-40 cm long, laxly 1-28(60)-flowered, pendent, simple or branched, branches 1-5 cm long, peduncle covered with imbricating scarious acute sheaths, peduncle and rachis densely pubescent. Flowers fragrant, densely pubescent, the sepals yellowish green, pale brown to yellow or mustard yellow washed with green and yellow with pale red purplish markings, the petals and the lip yellow, the veins sometimes brownish purple. Floral bracts 3-14 mm long, 3-6 mm wide, ovatelanceolate to transversely ovate or ovate, longacuminate, papery, densely pubescent. Pedicel and ovary 1.6-10 mm long, slender, densely pubescent, more densely than the sepals. Dorsal sepal 5-9 mm long, 2.74 mm wide, elliptic-lanceolate, oblong-elliptic to lanceolate-ovate, obtuse to subacute, 5-nerved. Lateral sepals 5-9 mm long, 4.5-7 mm wide, obliquely ovate-triangular, acute or obtuse, slightly cochleate, 4-nerved, reflexed at the apex. Mentum 3-6 mm high, saccate. Petals 4.3-7.5 mm long, 1.6-2.2 mm wide, oblanceolate, oblong-obovate to spathulate, rounded at apex, 3-nerved. Lip 5.2-8 mm long, 3-6 mm wide, strongly Table 1. Statistics for one of the most parsimonious trees for combined plastid matrix Matrix No. of taxa Included positions in matrix Variable site Parsimony-informative sites Trees (MPT) Fitch tree length CI RI ts:tv Combined plastid 46 rpl32-trnL (1119); rpl16 (1592) rps16-trnK* 1707 777 54 2620 0.76 0.75 0.55 Explanations: CI ñ consistency index, RI ñ retention index, ts:tv ñ transition/transversion ratio, * - from Russell et al. 2010 (5340) Unauthenticated Download Date | 6/30/16 11:08 PM 17 18 Joanna Mytnik-Ejsmont et al. Chelystachya, a New Genus of the Subtribe Polystachyinae (Orchidaceae) Fig. 1. The strict consensus tree of 54 most parsimonious trees from the analysis of combined plastid data for Polystachyinae. Bootstrap percentages >50 are given for supported clades above branches Unauthenticated Download Date | 6/30/16 11:08 PM Biodiv. Res. Conserv. 23: 15-27, 2011 to slightly recurved, three-lobed just above the middle, sometimes very obscurely pubescent at the base, lowermost due to apical drooping of the peduncle, the disc furnished with an inconspicuous central, fleshy, oblong, and pubescent callus along the mid-nerve between the lateral lobes; the lip nerves directed to its base; the middle lobe 2-3 mm long, 2-3.2 mm wide, oblong-ovate to semi-elliptic, subacute to rounded or retuse, slightly fleshy; the lateral lobes erect, narrowly oblong. Column (a free part of gynostemium) 1-2.7 mm high, semiterete, stout; column foot 2.5-8.5 mm long. Viscidium transversely narrowly-elliptic. E t y m o l o g y : The name derives from Chelys, the Greek word for turtle and is an allusion to the pseudobulbs looking like turtles grouped closely. D i s t r i b u t i o n : Republic of Guinea, Sierra Leone, Liberia, Ivory Coast, Ghana, Nigeria, Chad, Cameroon, Central African Republic, Equatorial Guinea, S„o TomÈ, Gabon, Republic of the Congo, Democratic Republic of the Congo, Angola, Uganda. Key for determination of the species. The genus comprises two species, which can be distinguishable in the following key: 1. Plant 15-50 cm long; pseudobulbs glossy green, spherical to transversely elliptic, 2.5-5 cm in diameter; lip distinctly three-lobed..............Chelystachya affinis 1*. Plant up to 8.5 cm long; pseudobulbs dull bluish green; elliptic to subspherical, 0.7-1.5 cm in diameter; lip obscurely three-lobed..........Chelystachya bancoensis Chelystachya affinis (Lindl.) Mytnik & Szlach., comb. nov. Basionym: Polystachya affinis Lindl., Gen. Sp. Orchid. Pl.: 73. 1830. ñ TYPE: SIERRA LEONE. G. Don s.n. (lectotype: K!; isolectotype: W!). ≡ Polystachya bracteosa Lindl., Edwardsís Bot. Reg. 26 (Misc.): 48. 1840. ≡ Dendrorkis bracteosa (Lindl.) Kuntze, Revis. Gen. Pl. 2: 658. 1891. ñ TYPE: S√O TOM…. Masson s.n. (holotype: K ñ not seen; W! ñ drawing in Reichenbachís collection). ≡ Polystachya villosa Cogn., J. OrchidÈes 7: 139. 1896, hom. illeg. (non Polystachya villosa Rolfe, Bull. Misc. Inform. Kew 1894: 393. 1894). ≡ Polystachya villosula Schltr., Beih. Bot. Centralbl. 33(2): 414. 1915. Madagascar (?), sphalm.ñ TYPE: MADAGASCAR (?). cult. Pauillac (Medoc), Çreceived from Linden in 1896í, M. Adde s.n. (holotype: BR! Herb. Cogniaux). Fig. 2. Chelystachya Mytnik & Szlach. Explanations: A ñ habit, B ñ flower, C ñ lip (in natural position), D ñ lip (flattened), E ñ dorsal sepal, F ñ petal, G ñ lateral sepal (drawn by J. Mytnik-Ejsmont from the lectotype of Chelystachya affinis (Lindl.) Mytnik & Szlach.) Unauthenticated Download Date | 6/30/16 11:08 PM 19 20 Joanna Mytnik-Ejsmont et al. Chelystachya, a New Genus of the Subtribe Polystachyinae (Orchidaceae) Epiphytic plants 15-50 cm long. Pseudobulbs 2.5-5 cm in diameter, 1-noded, dorsoventrally compressed, several, subspherical, prostrate on the substratum, 2-3-leaved at apex (weak individuals 1-leaved), glossy green. Leaves 8-28 cm long, 2.6-6 cm wide, bluish green with purplish midrib and margins, petiolate; petiole 3-14 cm long, narrow, subcylindrical, clasping the scape; blade oblanceolate, oblong-lanceolate, oblong-elliptic to obovate, acute to shortly acuminate, coriaceous. Inflorescence 6-40 cm long, laxly 8-28(60)flowered, most often pendulous, simple or branched, branches 4-5 cm long, peduncle apically drooping, covered with imbricating scarious acute sheaths, peduncle and rachis densely pubescent. Flowers medium-sized, fragrant, densely pubescent, the sepals pale brown to yellow or mustard yellow washed with green and yellow with pale red purplish markings, the petals and the lip yellow, the veins purple. Floral bracts 6-14 mm long, 5-6 mm wide, ovate-lanceolate, long-acuminate, papery, densely pubescent. Pedicel and ovary 3.5-10 mm long, slender, densely pubescent, more densely than the sepals. Dorsal sepal 5.3-9 mm long, 2.7-4 mm wide, elliptic-lanceolate, oblong-elliptic, lanceolate-ovate, obtuse to subacute, 5-nerved. Lateral sepals 6.5-9 mm long, 4.5-7 mm wide, obliquely ovate-triangular, acute or obtuse, slightly cochleate, 4-nerved, reflexed at the apex. Mentum 3.5-6 mm high, saccate. Petals 5.7-7.5 mm long, 1.6-2.2 mm wide, oblanceolate to oblong-obovate, rounded at the apex, 3-nerved. Lip 6.2-8 mm long, 4.7-6 mm wide, recurved, lowermost, obscurely three-lobed just above the middle, pubescent at the base, furnished with a not prominent central fleshy oblong and pubescent callus along the mid-nerve between the lateral lobes, merged with the disc gradually, 1.8-2 mm long; the nerves prominent, directed to the base of the lip; the middle lobe 2-3 mm long, 2.5-3.2 mm wide, oblong-ovate to semi-elliptic, subacute to rounded, slightly fleshy; the lateral lobes erect, narrowly oblong. Column 1 mm high, semiterete, stout; column foot 4-8.5 mm long. (Fig. 2) D i s t r i b u t i o n : Republic of Guinea, Sierra Leone, Liberia, Ivory Coast, Ghana, Nigeria, Chad, Cameroon, Central African Republic, Equatorial Guinea, S„o TomÈ, Gabon, Republic of the Congo, Democratic Republic of the Congo, Angola, Uganda. H a b i t a t : A shade epiphyte in rain forest, riverine forest, savanna. F l o w e r i n g t i m e : December to February. A l t i t u d e : 50-1350 m. S e l e c t e d s p e c i m e n s e x a m i n e d : REPUBLIC OF GUINEA. Faranah: Dabola, Kouroufig, Pobeguin 1523 (P!); Chevalier 20258 (BR!, P!); Sambadougou Chevalier 20550 (K!, P!); Cerde de Faranah, Sambadougou, 26 Jan 1909, Chevalier 20990 (K!). LabÈ: Pita, Pobeguin 2195 (P!). SIERRA LEONE. Bombali: Karina, 10 Jan 1929, Glanville 150 (K!); Makali, 13 Jan 1947, Deighton 4567 (K!). Kailahun: Bumbuma, alt. 200 m, 14 Oct 1914, Thomas 3239 (K!); Thomas 9018 (1915), (K!); Makuta, alt. 120 m, Thomas 483, 7 Jun 1914, (K!); Thomas 9018 (1915), (K!); Bunambu, in riverine forest, 28 Feb 1982, Segerb‰ck 1501a (K!); 24 May 1981, Segerb‰ck 1501 (K!). Kankan: Wallia, Scott-Elliot 4586 (P!). Kanema: near Kenema Town bridge, May 1914, Poole 226 (K!). Port Loko: near Wallia, Jan 1930, Scott Elliot 4627 b (K!). Senehun: along Tibai river, Senehun (Kamjei), 16 Apr 1960, Bakshi 128 (K!).Western Area Urban: Hastings, Feb 1956, Gledhill 48 (K!). LIBERIA. Nimba: Ganta, 14 Feb 1951, Harley 1772 (K!); Sanninquelle, Nimba Mts., alt. 800 m, 16 Jan 1969, Johansson 450 (C!, K!). Grand Bassa: Herbel, behind Botanical research garden Firestone, 18 Feb 1969, Jansen 1568 (K!). Sine loc.: alt. 500 m, 26 Jan 1965, Adam 20761 (K!). IVORY COAST. Dix-Huit Montagnes: Tiapleu, 15 Feb 1967, AkÈ Assi 9418 (K!, ABI); Mt. Tonkoui, Jan 1950, Schnell 4082 (P!); Mt. Tonkoui, Dec 1971, Perez-Vera 175 (ABI, P!). Bas-Sassandra: Cavally, seashore between Tabou and BÈrÈby, 16-17 Aug 1907, Chevalier 19967 bis (P!); Cavally bank, Liepleu, 24 Feb 1974, Perez-Vera 583 (K!). Worodougou: SÈguÈla-Mankono, 11 Mar 1974, Perez-Vera 599 (K!). GHANA. Ashanti: Efiduase, May 1936, Cox 31 (K!); Brafu Edru, 26 Jan 1896, Cummins 15139 (K!). Eastern: Kwahu Nteso to Ankoma, bank of River Asuboni, 9 Jan 1968, Bowling 36638 (K!); Tafo, cocoa farm, 5 Sep 1967, Bowling 36628 (K!). Volta: Amedzofe, Jan 1953, Westwood 32 (K!). NIGERIA. Ebo: Benin Prov., Sapoba Distr., 13 Nov 1948, Keay 24666 (K!); Sapoba Forest Reserve, fresh water swamp forest on banks of Jamieson River, 15 Jan 1959, Keay 37735 (K!); Benin Prov., Iyekuselu, 6 Dec 1961, Daramola 45653 (K!). Ogun: near Ogere and Abeokuta, 28 Jan 1945, Burtt 27 (K!). Olibo: S Nigeria, Dagema Distr., 1916, Talbot 3776 (K!). Ondo: Owo Distr., Owo, Sep 1948, Keay 22714 (K!); Keay 22714 (K!); Akure Distr., Akure Ofosu, 4 Dec 1961, Adebuauyi 4531 (45324?) (K!). Plateau Province: Kwarra, Proiunca (?), King 538 (K!). CHAD. Haut Oubangui: Krebedje (Fort Sibut), Chevalier 10780 (P!); Bangui Chevalier 10837 (P!); Chevalier 10914 (P!); Chevalier s.n. (P!). CAMEROON. South: Bitye pres riv. Dja, 50 km ENE Sangmelima, 28 Dec 1921, Bates 1886 (K!); Bitye, near the River Dja Bates s.n. (P!); Bella, 45 km NE Kribi, 24 Jan 1962, Letouzey 4122 (K!, P!); 10 km N of Kribi Raynal 13497 (P!); Embouchure de líestuaire de la Lobe, S Kribi, 10 Feb 1969, Sanford 5701 (K!, P!); Kribi, 1912, Zenker 4382 (BM!, K!); Nkomo pres Ngoase, S riv. Lobe, 14 Feb 1962, Letouzey 4235 (K!, P!); Lokundje, Feb 1911, Zenker 4 (F!). East: Entre Alat Makay et riv. Djable, 20 km NNE Mintom II, 4 Jan. 1973, Letouzey 11783 (K!, P!). Littoral: Douala-Edea Unauthenticated Download Date | 6/30/16 11:08 PM Biodiv. Res. Conserv. 23: 15-27, 2011 Forest, 1978, Thomas 206 (K!). Southwest: alt. 50 m, 30 Oct 1985 Thomas 4911 (MO!). Centre: Bord de la riv. Soo, 78 km S de Yaounde Villiers 1107 (P!). Sine loc.: Bates 1254 (BM!); Bates 1265 (BM!). CENTRAL AFRICAN REPUBLIC. Haute-Kotto: Yalinga, 16 Oct 1922, Le Testu 4221 (BM!); Yalinga, 16 Oct 1922, Le Testu 4228 (BM!, BR!, K!). Ouaka: Rocher chutes Gumburu, Wamire, 60 km N Bambari, 27 Nov 1928, Tisserant 2063 (BM!). EQUATORIAL GUINEA. Rio Muni: Nkolendangen, alt. 450 m, 8 Jan 1908, Tessmann 99 (K!); Bata-Sendje (Senye), estrada km 40, a jussante da ponte sobre Rio Benito, bosque primario, 30 May 1993, Carvalho 5320 (MA!); Bata-Bicomo, en las margenas del Rio Ecucu, al lado de la central hidroelectrica, bosque primario, 30 Jun 1997, Carvalho 6377 (MA!). Pagalu: Mt. Cacinca, S of San Pedro, alt. 250 m, 21 Jul 1959, Melville 176 (K!). S√O TOM…. Sine loc.: Masson s.n (W!). GABON. Ngounie: 2 km on road from Lebamba to Mbigou, 2o13íS, 11o29íE De Wilde & Sosef 10418 (WAG!). Ogooue-Ivindo: Station díIpassa, 10 km S de Makokou, Nyame Pende, Florence 1122, (P!-p.p.); Ayem, 10 km SW de Ndjole HallÈ 1965 (P!); Francesville, Thollon 816 (P!). Haute-Ngounye: bord de la Ngounye, Litsila, Moupounde, 20 Jun 1927, Le Testu 6511 (BM!, K!, P!); Ngwa Le Testu 9174 (BM!, P!). Estuaire: ca. 30 km E of Cocobeach, Ekorodo-village, 0 o56íN, 9o57íE, Reitsma 2312 (WAG!). Sine loc.: 12 Apr 1920, Le Testu 8017 (BM!). REPUBLIC OF THE CONGO. Likouala: Impfondo, Chevalier 10961 (P!); Impfondo, Chevalier 11011 bis (P); Komi, Lecomte s.n. (P!). Bouenza: Mouyondzi Farron 4599 (P!). DEMOCRATIC REPUBLIC OF THE CONGO. Bas-Congo: Kinshasa, Bequaert 8039 (BR!); Zongo, Breyne 149 (BR!); Zundu, Compere 1684 (BR!); Kisantu, Gillet s.n. (BR). Kasai: Tshimboa, Callewaert s.n. (BR!). Forestier Central: Dundusana, Mortehan 987 (BR!); Ngazi Riv., Aruwimi, Louis 12180 (BR!); Yangambi, Louis 6318 (BR); Irebu, Laurent 1 (BR!); Likako, Evrard 3040 (BR!); lac Tumba, Laurent s.n. (BR!). Ubangi-Uele: Suronga, Seret 155 bis (BR!); route Bima-Bambili, Seret 155 (BR!). ANGOLA. Lunda Sul: Lunda, Dundo, Rio Lunachi, 18 Apr 1937, Carrisso & Sousa s.n. (BM!); River Luembe near Cossa, alt. 700 m, 12 Jun 1948, Gossweiler 14077 (BM!, K!). UGANDA. Mengo Distr.: Mpanga Forest, Feb 1965, Karnya in Leakey s.n. (K!). N o m e n c l a t u r a l N o t e s . Polystachya villosa was described from a plant cultivated by M.†Adde from Pauillac, who Ñreceived it from Madagascarî (according to Perrier 1936). It has not been rediscovered again and the provenance of the material remains doubtful. Moreover, Polystachya villosula was merely a sphalm for P. villosa Cogn. (Kraenzlin 1926). Chelystachya bancoensis (Van der Burg) Mytnik & Szlach., comb. nov. Basionym: Polystachya bancoensis Van der Burg, Misc. Papers Agric. Univ. Wag. 19: 23. 1980. ≡ Polystachya affinis var. nana J.B.Hall, Kew Bull. 29(2): 428. 1974. ñ TYPE: GHANA. Western region: Tanoso, Bowling in GC38171 (Holotype: K!; Isotype: GC ñ not seen, Photo: WAG!). Dwarf epiphytic plants up to 8.5 cm long. Pseudobulbs 1-2.5 cm long, 0.7-1.5 cm in diameter, dorsoventrally compressed, subspherical to elliptic, prostrate on the substratum, 1-2-leaved at apex, dull bluish green, sometimes tinged purple. Leaves 2-6 cm long, 0.7-1.5 cm wide, bluish green, greyish green to reddish blue with purplish midrib and margins, thin-textured, petiolate; petiole up to 1 cm long, narrow, subcylindrical; blade oblanceolate, lanceolate-elliptic to lanceolateovate, acute to shortly acuminate. Inflorescence 1.5-7 cm long, laxly 1-6(13)-flowered, pendent, simple, sometimes branching, branches 1-2, short; peduncle with 1-2 bracts, apically drooping, rachis flexuous, peduncle and rachis pubescent. Flowers rather small, minutely and densely pubescent outside, yellowish green with pale red purplish markings, the pedicel and ovary purplish red, the base of the anther and the base of the column reddish, and sometimes the lip nerves brownish. Floral bracts 3-5 mm long, 3 mm wide, suborbicular, ovate to transversely ovate, acuminate to tridentate, papery, densely pubescent. Pedicel and ovary 1.6-4 mm long, slender, densely hairy, hairs up to 0.4 mm long. Dorsal sepal 5-6.7 mm long, 3-4 mm wide, elliptic-ovate or oblong-elliptic, obtuse. Lateral sepals 5-6 mm long, obliquely elliptic- to triangular-ovate, subacute or subobtuse, slightly cochleate. Mentum 3-4 mm long, saccate. Petals 4.3-5.7 mm long, 2 mm wide, obovate to spathulate, rounded at apex, glabrous. Lip 5.2-7.3 mm long, 3-3.8 mm wide, obscurely three-lobed, lowermost, fleshy along the mid-nerve, slightly recurved, elliptic to oblong-elliptic in general outline, furnished with an oblong inconspicuous callus in the basal half; the middle lobe 2-3 mm long, 2-2.4 mm wide, semielliptic, retuse to round at the apex, slightly fleshy; the lateral lobes 1-2.2 mm long, porrect, transversely semielliptic. Column 2.7 mm high, column foot 3-3.5 mm long, viscidium transversely narrowly-elliptic. D i s t r i b u t i o n : Liberia, Ivory Coast, Ghana. H a b i t a t : A fully exposed trunk epiphyte on high branches, in humid evergreen forest. Flowering time. November. A l t i t u d e : 0-400 m. Representative Specimens Examined: LIBERIA. Sine loc.: cult., De Wit s.n. (WAG!). IVORY COAST. AgnÈby: Yapo, 3 Nov 1974, Perez-Vera 722 (K!, P!). Bas-Sassandra: San Pedro, km 14 road GrandBereby-Tabou, 4o43íN, 7o00íW, Kruif 709 (WAG!). Dix-Huit Montagnes: Tiapleu, Nov 1966, Ake Assi 9228 (K!); TÈkÈ, 22 Nov 1966, Ake Assi 9308 (K!). Haut- Unauthenticated Download Date | 6/30/16 11:08 PM 21 22 Joanna Mytnik-Ejsmont et al. Chelystachya, a New Genus of the Subtribe Polystachyinae (Orchidaceae) Sassandra: Mt. Mafa, 28 Oct 1965, Bonardi s.n. (P!). Lagunes: Banco Forest Reserve, 22 Oct 1975, van der Burg 1245 (WAG!); Banco Forest Reserve, near entrance, 10 Dec 1974, de Koning 5005 (WAG!); AdiopodoumÈ, 4o08íW, 5o20íN, Dekker 328 (WAG!); AdiopodoumÈ Perez-Vera 152 (K!, P!); Anguededou, 7 Jan 1975, de Koning 5120 A (WAG!). Sine loc.: van der Burg 1269 (WAG); de Koning 6102 (WAG!); de Koning 6107 (WAG!); de Koning 6115 (WAG!); de Koning 6116 (WAG!). GHANA. Western region: Tanoso, Bowling in GC 38171 (K!). 4. Discussion 4.1. Phylogenetic relationships The tree obtained in this study (Fig. 1) provides a valuable resolution at the subtribal level, permitting the comparison of the molecular phylogenetic framework with the traditional classification of Polystachya. The results of recent molecular studies conducted by Russell et al. (2010) and our morphological and molecular analyses presented here confirm isolated positions of some taxa being classified within Polystachya till now. The first clade comprises the complex of two species: Chelystachya affinis (classified as Polystachya affinis) and Chelystachya bancoensis (classified as Polystachya bancoensis). The tree indicates the need to distinguish the P. affinis-group at the generic rank and morphological characters also confirm the status change (see below for detailed discussion). Thus, the newly proposed genus is shown to be monophyletic. The monophyly of the clade formed by the two species of the new genus is well supported. Another taxon that deserved to be placed in a separate genus is Polystachya longiscapa Summerh., a member of ígrade of species-poor lineageë as it was defined by Russell et al. (2010). The results obtained by these authors and our phylogenetic tree show the taxon occupies an isolated position within Polystachyinae. The problem with the classification of P. longiscapa was presented by many authors, e.g. Summerhayes (1934), Cribb (1984) and Cribb and King (2006). Summerhayes (1934) observed that P. longiscapa approaches the remarkable monotypic genus Neobenthamia Rolfe, Cribb (1984) classified the species within the section Dendrobianthe (Schltr.), Mytnik, Cribb and King (2006) noticed the species occupies rather an isolated position within Polystachya. In our opinion, P. longiscapa occupies an intermediate position between Dendrobianthe and Neobenthamia and detailed morphological studies and the results of Russell et al. (2010) confirm the taxon deserves a generic rank. In 2011, Polystachya longiscapa was placed in a separate monotypic genus Neoburttia (Mytnik et al. 2011). Neobenthamia, representing the next lineage, is a well defined monotypic genus closely related to Dendrobianthe and ÑP. longiscapa cladeî (Neoburttia) and we disagree with Russell et al. (2010) that it should be included within Polystachya. Neobenthamia is sister to Dendrobianthe dendrobiiflora (Rchb.f.) Mytnik but the former genus differs from Polystachya in many aspects (e.g. lack of pseudobulbs, lowermost lip, obscure column foot and mentum, pollinia subsessile on the viscidium) and in our opinion they are two distinct genera. Including Neobenthamia within Polystachya makes the latter genus heterogenic and difficult to define. Dendrobianthe dendrobiiflora is a species representing one of recently described genera and in the present analysis forms another well supported clade. Dendrobianthe, excluding Dendrobianthe longiscapa, comprises three species [D. dendrobiiflora, D. bequaertii (Summerh.) Mytnik, and D. zuluensis (L. Bolus) Mytnik] clearly differing from Polystachya s.str. by e.g. slender stems thickened into pseudobulbs at the base only, narrow, grass-like leaves predominantly absent at the flowering time, widely open flowers, unlobed or rarely very obscurely trilobed lip with a central pubescent thickening. Polystachya goetzeana (Kraenzl.) Mytnik & Szlach. and P. vaginata (Summerh.) Mytnik & Szlach. form the last lineage of the base of the tree and the clade is sister to Polystachya s. s. The results of both molecular analyses (Russell et al. 2010 and that presented here) show the species, being representative of the section Isochiloides Summerh., occupy an isolated position in the tree and it was distinguished as another taxon of generic rank within Polystachyinae Isochilostachya Mytnik & Szlachetko (2011). From closely related Polystachya, the species of Polystachya goetzeana-vaginata clade differ in the structure of foliage, shape and size of leaves and floral bracts, form and length of sepals, lip pubescence and the gynostemium length. Molecular analyses revealed a striking position of P. ottoniana Rchb.f., the clade being sister to Neobenthamia, Dendrobianthe, Polystachya section Isochiloides and Polystachya s.str. clades. The position of P. ottoniana, a representative of the section Affines Kraenzl., is difficult to explain from a morphological point of view. We examined over forty specimens of the species in details and we cannot find any explanation for such position of P. ottoniana in the tree. In our opinion further detailed studies are needed to resolve the problem. To sum up, we found that Polystachya s.l., including Neobenthamia, newly proposed Chelystachya, Neoburttia (P. longiscapa), Dendrobianthe, Isochilostachya (P. vaginata-P.goetzeana clade) and other five recently described genera, not present in our analysis because of lack of DNA material [Disperanthoceros Mytnik & Szlach., Epiphorella Mytnik & Szlach., Geerinckia Mytnik & Szlach., Szlachetkoella Mytnik, and Unguiculabia Mytnik & Szlach.], is a heterogeneous taxon, difficult to define. Thus we recognize Polystachya in a narrow Unauthenticated Download Date | 6/30/16 11:08 PM Biodiv. Res. Conserv. 23: 15-27, 2011 sense. Differences between the genera are presented in the key. 4.2. Morphological differences between Polystachya and Chelystachya The representatives of the newly proposed genus Chelystachya are clearly distinct from all the other representatives of Polystachya. They are small to mediumsize plants (up to 50 cm long) with a unique hanging habit (pendent inflorescence) and very characteristic flattened, spherical, and 1-noded pseudobulbs with an apical pendent and densely pubescent inflorescence. The lip is lowermost due to the drooping inflorescence. Such exposure emphasizes colours and patterns to attract pollinators and facilitate pollination (Ernst & Arditti 1994). The other distinctive feature of Chelystachya is the venation pattern on the lip. The veins are directed toward the lip basal half, not apical part, as in Polystachya. The species of the Chelystachya are exclusively trunk epiphytes in riverine or rain forests in Africa. They grow on high, mossy or naked branches. They can be found in relatively cool climates where there is high humidity throughout the year. The Chelystachya species grow at sea level up to 1,350 meters a. s. l. and flower from November till February (Hall 1974). Unlike Chelystachya, the Polystachya plants are erect plants of various sizes (from 1 cm to more than 1 m high). Polystachya are mainly epiphytic but sometimes also lithophytic or terrestrial plants. Their stems are often thickened at the basal part or the stems are almost entirely thickened. Pseudobulbs are most often narrowly to widely elliptic to cylindrical or conical, laterally compressed, not flattened on the substratum as in Chelystachya. The other characters setting Polystachya apart from Chelystachya are erect to semi-erect inflorescences, flowers with an uppermost lip and the lip venation directed to the apical part of the lip. The main differences between Chelystachya and Polystachya are presented in Table 2. 4.3. Key for determination of Polystachyinae The subtribe Polystachyinae presently includes ten genera and they are keyed as follows: Key for determination of the genera of Polystachyinae Schltr.: 1. Leaves absent at the anthesis...................................2 1*. Leaves present at the anthesis................................3 2. Lip variously lobed, furnished with a prominent cushionlike crest in the middle, deflexed just above the base, with elevated keel present at lip base........Epiphorella Mytnik & Szlach. 2*. Lip entire, with a single callus, if any, not pulvinate ...................................................Dendrobianthe Mytnik 3. Robust plants 50-120 cm tall with pseudobulbs, sepals 16-20 mm long, mentum 5-6.5 mm long...... 12.3. ........................Neoburttia Mytnik, Szlach. & Baranow 3*. Not as above..........................................................4 4. Inflorescence pendent or apically drooped, lip of lowermost position.......................................................5 4*. Inflorescence erect to semi-erect, lip of uppermost position.........................................................................6 5. Pseudobulbs large, spherical, succulent, flattened on the substratum, inflorescence pendent.......................... .................................................Chelystachya, gen. nov. 5*. Pseudobulbs absent, stem reed-like, inflorescence erect with a rachis apically drooped...Neobenthamia Rolfe 6. Spur very long and narrow, three times longer than the dorsal sepal.......Disperanthoceros Mytnik & Szlach. 6*. Spur (mentum) up to two times longer than the dorsal sepal.............................................................................7 7. Whole plant pendent or creeping, leaves fleshy, imbricate, bilaterally flattened.............Szlachetkoella Mytnik 7*. Plant neither pendent nor creeping, leaves not imbricate..........................................................................8 8. Lip with a long claw, at least third of the lip length, lip divided into 3 parts with the mesochile variously lobed, mentum elongate, saccate, swollen at the apex ....................................Unguiculabia Mytnik & Szlach. 8*. Lip shortly clawed or sessile, usually 3-lobed, then divided into the lateral and middle lobes, mentum conical-saccate, obtuse.......................................................9 9. Leaves elliptic, ovate or obovate, lip often furnished with pseudopollen, sepals up to 8 mm long, obtuse, column part short and massive, 1-2.5 mm long............. .........................................................Polystachya Hook. 9*. Leaves narrow, grass-like, lip without pseudopollen, covered with numerous clavate or capitate prominent, long hairs, sepals more than 8.5 mm long, acuminate, column part slender, 2-4 mm long................................. .................................Isochilostachya Mytnik & Szlach. Table 2. Morphological differences between Polystachya Hook. and Chelystachya Mytnik & Szlach. Pseudobulbs Inflorescence Lip Lip venation Polystachya narrowly to widely elliptic or conical or even inconspicuous, laterally compressed, not flattened on the substratum erect to semi-erect uppermost directed to the apical part of the lip Chelystachya almost spherical, dorsoventrally compressed, flattened on the substratum pendent lowermost directed to the lip base Unauthenticated Download Date | 6/30/16 11:08 PM 23 24 Joanna Mytnik-Ejsmont et al. Chelystachya, a New Genus of the Subtribe Polystachyinae (Orchidaceae) 4.4. The species of Chelystachya Chelystachya bancoensis and C. affinis are two closely related species. The differences between them were presented by Hall (1974), who described a Çdepauperate formí of Polystachya affinis (todayís Chelystachya affinis) in a varietal rank as P. affinis Lindl. var. nana. Six years later, Van der Burg (in Arends et al. 1980) raised the variety to the species rank describing it as P. bancoensis. Until 1974, Chelystachya bancoensis was considered to be a smaller form of C. affinis. But the observations by Hall (1974) and Arends et al. (1980), during a several year cultivation period, proved the species has retained its distinctive features and no intermediate forms have been found. Both species differ in many ways, especially, in the size of plants and floral bracts and the shape and pubescence of the lip. The main morphological differences are presented in Table 3. According to Hall (1974) and Arends et al. (1980), there are also ecological and phenological differences: C. affinis is a shade epiphyte found in very damp areas. It grows on mossy branches, where the climate is relatively cool with high humidity throughout the year and reduced solar radiation because of mist and clouds. The species flowers one or two months after C. bancoensis (Arends et al. 1980), from late December to February, though, this difference can be caused by ecological differences. C. bancoensis grows fully exposed on the bare bark of high branches in the evergreen forest. It grows at lower altitudes, in a climate with a dry season. C bancoensis can be deciduous; during the dry season it may drop its rather fragile leaves. It flowers in November (Hall 1974). 4.5. Taxonomic implications Because Polystachya affinis, the type of Polystachya sect. Affines, is now transferred to the newly established genus Chelystachya, this makes automatically this sectional name a nomenclatural synonym of this new generic name. Consequently, species that are still residual in the genus Polystachya constitute a separate section. Thus, we propose to include them in the formerly existing section Humiles, described by Summerhayes (1942). Humiles includes the vast majority of the species formerly classified within the section Affines sensu Kraenzlin (1926). Polystachya Hook. sect. Humiles Summerh. Bot. Mus. Leafl. [Harv. Univ.] 10: 284. 1942. ñ TYPE SPECIES: Polystachya ottoniana Rchb.f. Summing up, the genus Polystachya section Humiles comprises 39 species: Polystachya acridolens Summerh., P. acuminata Summerh., P. bella Summerh., P. bruechertiae Eb.Fisch., Killmann & J.-P.Lebel, P. camaridioides Summerh., P. campyloglossa Rolfe, P. confusa Rolfe, P. disiformis P.J.Cribb, P. epiphytica De Wild., P. fischeri Rchb.f. ex Kraenzl., P. haroldiana Rolfe, P. heckmanniana Kraenzl., P. holmesiana P.J. Cribb, P. holstii Kraenzl., P. johnstonii Rolfe, Polystachya kaluluensis P.J. Cribb & la Croix, P. laurentii De Wild., P. lawrenceana Kraenzl., P. leucosepala P.J. Cribb, P. mafingensis P.J.Cribb, P. magnibracteata P.J.Cribb, P. malilaensis Schltr., P. melanantha Schltr., P. mzuzuensis P.J.Cribb & la Croix, P. ngomensis McDonald & McMurtry, P. obanensis Rendle, P. ottoniana Rchb.f., P. piersii P.J.Cribb, P. praecipitis Summerh., P. proterantha P.J.Cribb, P. pubescens (Lindl.) Rchb.f., P. purpureobracteata P.J.Cribb & la Croix, P. reticulata StÈvart & Droissart, P. sandersonii Harv., P. teitensis P.J.Cribb, P. testuana Summerh., P. valentina la Croix & P.J.Cribb, P. villosa Rolfe and P. zambesiaca Rolfe. Table 3. Morphological differences between Chelystachya affinis (Lindl.) Mytnik & Szlach. and C. bancoensis (Van der Burg) Mytnik & Szlach. Plant size Pseudobulbs Flowers Flower colour Floral bracts Sepals and lip Lip Callus Hairs on the lip C. affinis up to 50 cm mostly circular to almost transversely elliptic glossy green opened brownish-yellow 6-14 mm long reach at least half-way up the dorsal sepal markedly curved backwards in the upper part with conspicuous deep red veins finishes abruptly confined to the median zone C. bancoensis up to 8.5 cm usually elliptic dull bluish green, sometimes tinged purple do not open as much as in C. affinis yellowish-green 3-5 mm long short, hardly longer than the ovary almost straight occasionally with some faint reddish lines merges with the lip gradually near the margins on the upper side of lip Unauthenticated Download Date | 6/30/16 11:08 PM Biodiv. Res. Conserv. 23: 15-27, 2011 Acknowledgments. We wish to thank the curators and staff of the cited herbaria for their kind hospitality and assistance during our visits (BM, BR, C, K, MA, P, W, WAG) and for providing the material available on loan (MO, F). We would also like to express our gratitude to Dr. Guy Chiron, the chief editor of Richardiana, for his priceless help in translating the diagnosis of the new genus. Our special thanks go to Dr. Przemyslaw Baranow for critical review of the manuscript. We are also obliged to Anton Russell for an access to his DNA sequences and stimulating discussions. This study was supported by a grant from the Ministry of Science and Higher Education (N N303 343735). References APPLEQUIST W. L. & WALLACE R. S. 2000. Phylogeny of the Madagascan endemic family Didiereaceae. Pl. Syst. Evol. 221: 157-166. ARENDS J. C., VAN DER BURG W. J. & VAN DER LAAN F. M. 1980. Notes on African orchids. In: J. C. ARENDS & H. C. D. DE WIT (eds.). Liber gratulatorius in honorem H.C.D. de Wit, pp. 23-25. 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Flora Zambesiaca, 11(2): 1-320. London, Flora Zambesiaca Management Committee. LA CROIX I. F. & LA CROIX E. A. S. 1997. African Orchids in the Wild and Cultivation. 379 pp. Portland, Or, Timber Press. LAWRENCE G. H. M., BUCHHEIM A. F. G., DANIELS G. S. & DOLEZAL H. 1968. Botanico-Periodicum-Huntianum. Pittsburgh, Hunt Botanical Library. MADDISON D. & MADDISON W. 2000. MacClade. Sinauer Associates, Sunderland, Massachusetts, USA. MYTNIK-EJSMONT J. 2007. Contribution ‡ la rÈvision de Polystachyinae Schlechter (Orchidaceae). 1. Szlachetkoella Mytnik. Richardiana 7(2): 55-60. MYTNIK-EJSMONT J. 2008. Contribution ‡ la rÈvision de Polystachyinae Schlechter (Orchidaceae). 6. Dendrobianthe (Schltr.) Mytnik. Richardiana 8(1): 23-27. MYTNIK-EJSMONT J. & BARANOW P. 2010. Taxonomic studies of Polystachya Hook. (Orchidaceae) from Asia. Pl. Syst. Evol. 290: 57-63. MYTNIK-EJSMONT J. & SZLACHETKO D. L. 2007a. Contribution ‡ la rÈvision de Polystachyinae Schlechter (Orchidaceae). 2. Geerinckia Mytnik and Szlach. Richardiana 7(2): 61-63. MYTNIK-EJSMONT J. & SZLACHETKO D. L. 2007b. Contribution ‡ la rÈvision de Polystachyinae Schlechter (Orchidaceae). 3. Disperanthoceros Mytnik and Szlach. Richardiana 7(2): 64-66. Unauthenticated Download Date | 6/30/16 11:08 PM 25 26 Joanna Mytnik-Ejsmont et al. Chelystachya, a New Genus of the Subtribe Polystachyinae (Orchidaceae) MYTNIK-EJSMONT J. & SZLACHETKO D. L. 2008a. Contribution ‡ la rÈvision de Polystachyinae Schlechter (Orchidaceae). 4. Epiphorella Mytnik and Szlach. Richardiana 8(1): 12-17. MYTNIK-EJSMONT J. & SZLACHETKO D. L. 2008b. Contribution ‡ la rÈvision de Polystachyinae Schlechter (Orchidaceae). 5. Unguiculabia Mytnik and Szlach. Richardiana 8(1): 18-22. M YTNIK -E JSMONT J. & S ZLACHETKO D. L. 2011. Isochilostachya Mytnik&Szlach., gen. nov., a new genus of Polystachyinae Schltr. (Orchidaceae). Acta Soc. Bot. Pol. 80(1): 79-85. MYTNIK-EJSMONT J., SZLACHETKO D. L. & BARANOW P. 2011. Neoburttia, a new genus for Polystachyalongiscapa (Polystachyinae, Orchidaceae). Pol. Bot. J. 56(1): 4549. PERRIER DE LA BATHIE. 1936. BiogÈographie des Plantes de Madagascar. Paris, SociÈtÈ díEditions, GÈographiques, Maritimes et Coloniales. RUSSELL A., SAMUEL R., RUPP B., BARFUSS M. H. J., äAFRAN M., BESENDORFER V. & CHASE M. W. 2010. Phylogenetics and cytology of a pantropical orchid genus Polystachya (Polystachyinae; Vandeae; Orchidaceae); evidence from plastid DNA sequence data. Taxon 59: 389-404. SCHNABEL A. & WENDEL J. F. 1998. Cladistic biogeography of Gleditsia (Leguminosae) based on ndhF and rpl16 chloroplast gene sequences. Am. J. Bot. 85: 17531765. SHAW J., LICKEY E. B., BECK J. T., FARMER S. B., LIU W., MILLER J., SIRIPUN K. C., WINDER C. T., SCHILLING E. E. & SMALL R. L. 2005. The tortoise and the hare II: relative utility of 21 noncoding chloroplast DNA sequences for phylogenetic analysis. Am. J. Bot. 92: 142-166. SHAW J., LICKEY E. B., SCHILLING E. E. & SMALL R. L. 2007. Comparison of whole chloroplast genome sequences to choose noncoding regions for phylogenetic studies in angiosperms: The tortoise and the hare III. Am. J. Bot. 94: 275-288. SHIMODAIRA H. & HASEGAWA M. 1999. Multiple Comparisons of Log-Likelihoods with Applications to Phylogenetic Inference. Mol. Biol. Evol. 16: 1114-1116. STAFLEU F. A. & COWAN R. S. 1976-1988. Taxonomic Literature: A Selective Guide to Botanical Publications and Collections with Dates, Commentaries and Types. 2nd ed., 7 Vols. Utrecht, The Netherlands, Bohn, Scheltema and Holkema. STAFLEU F. A. & MENNEGA E. A. 1992. Taxonomic Literature: A Selective Guide to Botanical Publications and Collections with Dates, Commentaries and Types. Supplement 6. Kˆnigstein, Germany, Koeltz Scientific Books. SUMMERHAYES V. S. S. 1934. African Orchids: VI. Bull. Misc. Inform. Kew 1934: 205-214. SUMMERHAYES V. S. S. 1942. African Orchids: XII. Bot. Mus. Leafl. 10(9): 257. SUMMERHAYES V. S. S. 1948. African Orchids: XVII. Kew Bull. 2(2): 128. SWOFFORD D. L. 2000. PAUP* version 4.0b10: Phylogenetic analysis using parsimony (*and other methods). Sinauer, Sunderland, Massachusetts, USA. Unauthenticated Download Date | 6/30/16 11:08 PM Biodiv. Res. Conserv. 23: 15-27, 2011 Appendix. Voucher information and GenBank accession numbers for samples used in this study Taxon Adrorhizon purpurascens (Thwaites) Hook.f. Bromheadia finlaysoniana (Lindl.) Miq. Bromheadia srilankensis Kruiz. & de Vogel Chelystachya affinis (Lindl.) Mytnik and Szlach. (2) comb. nov. Chelystachya bancoensis (Van der Burg) Mytnik and Szlach. comb. nov. Dendrobianthe dendrobiiflora (Rchb.f.) Mytnik (1) Dendrobianthe dendrobiiflora (Rchb.f.) Mytnik (2) Neobenthamia gracilis Rolfe (1) Neobenthamia gracilis Rolfe (1) Polystachya adansoniae Rchb.f. Polystachya bella Summerh. Polystachya bennettiana Rchb.f. (1) Polystachya bennettiana Rchb.f. (2) Polystachya bicolor Rolfe (2) Polystachya confusa Rolfe Polystachya cultriformis (Thouars) Sprengel (1) Polystachya cultriformis (Thouars) Sprengel (2) Polystachya dolichophylla Schltr. Polystachya fischeri Rchb.f. ex Kraenzl. (1) Polystachya fischeri Rchb.f. ex Kraenzl. (2) Polystachya foliosa (Hook.) Rchb.f. Polystachya fulvilabia Schltr. Polystachya galeata (Sw.) Rchb.f. (1) Polystachya galeata (Sw.) Rchb.f. (2) Polystachya goetzeana Kraenzl. Polystachya henrici Schltr. Polystachya lindblomii Schltr. Polystachya longiscapa Summerh. Polystachya melanantha Schltr. Polystachya melliodora P. J. Cribb Polystachya odorata Lindl. Polystachya ottoniana Rchb.f Polystachya paniculata (Sw.) Rolfe Polystachya piersii P. J. Cribb Polystachya polychaete Kraenzl. Polystachya pubescens (Lindl.) Rchb.f. Polystachya seticaulis Rendle Polystachya setifera Lindl. Polystachya spatella Kraenzl. (1) Polystachya spatella Kraenzl. (2) Polystachya steudneri Rchb.f. Polystachya transvaalensis Schltr. Polystachya tsaratanananae H. Perrier Polystachya undulata P. J. Cribb & A. Podzorski Polystachya vaginata Summerh. Polystachya vulcanica Kraenzl. var. acontiflora (Summerh.) P. J. Cribb & A. C. Podzorski (1) Sirhookera lanceolata (Wight) Kuntze Kew DNA 15745, Chase 15745 (K) Kew DNA 21766, Duangjai 039 (BRUN, K) Kew DNA 15745, Chase 15745 (K) Kew DNA 21165, Chase 21165 (K) HQ222153 HQ222154 HQ222155 HQ222149 GenBank No. rpl32trnL HQ222257 HQ222254 HQ222253 HQ222250 Heidelberg BG 120907 HQ222150 HQ222251 - Kew DNA 17962, PCP 063/98/1621 (EA) Kew DNA 19184, Mugambi & Odhiambo 064/98/1622 (EA) Kew DNA O-340, Carlsward 311 HBV ORCH07214 Kew DNA 17957, B 429/94/469 (EA) Kew DNA 17950, B & N 783 (EA) Kew DNA 19186, Mugambi & Odhiambo 81/01 (EA) Kew DNA 17958, B 338/94/418 (EA) Kew DNA 25884, Kew living collection 2003-406 (WU) Kew DNA 17947, B et al. 122 (EA) Kew DNA 19183, Mugambi & Odhiambo 054/98/1612 Kew DNA 19182, Mugambi & Odhiambo 054/98/1607 (EA) Kew DNA 25886, Chase 25886 (K) Kew DNA 17964, Pearce 616/94/607 (EA) Kew DNA 17965, B 674/95/1280 (EA) Kew DNA 25887, Kew living collection 2001-3986 Kew DNA 17855, Chase 17855 (K) Kew DNA O-1496, Chase O-1496 (K) Kew DNA 14650, Chase 14650 (K) Kew DNA 17955, B & L 1772 (EA) Kew DNA 17856, Chase 17856 (K) Kew DNA 17967, B 1142/98/1695 (EA) HBV ORCH06411 Kew DNA 17954, B & L 1783 (EA) Kew DNA 17923, Chase 17923 (K) Kew DNA 17857, Chase 17857 (K) Kew DNA 25888, Kew living collection 2005-964 Kew DNA 25889, Kew living collection 1984-4977 Kew DNA 17948, B et al. 101/95/1186 (EA) Kew DNA 25890, Kew living collection 2001-3987 Kew DNA O-700, Kurzweil 1849 Kew DNA 17924, Chase 17924 (K) Kew DNA O-1493, Chase O-1493 (K) Kew DNA 17951, B 949 (EA) Kew DNA 19263, Khayota 381 (EA) Kew DNA bank 17956, B 712/95/1305 (EA) Kew DNA 19264, Bytebier & Luke 1774 (EA) Kew DNA 17861, Chase 17861 (K) Kew DNA 17862, Chase 17862 (K) Kew DNA 17949, B 566/95/1140 (EA) Kew DNA 17863, Chase 17863 (K) HQ222146 HQ222147 HQ222243 HQ222242 GQ145126 GQ145127 HQ222148 HQ222138 HQ222141 HQ222240 HQ222160 HQ222233 HQ222194 GQ145087 GQ145088 GQ145095 GQ145097 HQ222140 HQ222121 HQ222193 HQ222212 GQ145096 GQ145120 HQ222101 HQ222109 HQ222173 HQ222190 GQ145121 - HQ222133 HQ222189 GQ145124 GU045350ª HQ222116 HQ222115 GU045351ª HQ222105 HQ222108 HQ222117 HQ222128 HQ222134 HQ222135 HQ222124 GU045360ª HQ222137 HQ222125 HQ222118 HQ222103 HQ222099 HQ222111 HQ222104 HQ222102 HQ222119 HQ222126 HQ222100 HQ222123 HQ222113 HQ222139 HQ222170 HQ222180 HQ222177 HQ222205 HQ222161 HQ222216 HQ222220 HQ222252 HQ222211 HQ222174 HQ222179 HQ222197 HQ222215 HQ222248 HQ222207 HQ222178 HQ222224 HQ222183 HQ222168 HQ222237 HQ222184 HQ222185 HQ222169 HQ222191 HQ222196 HQ222208 HQ222245 HQ222171 GQ145128 GQ145133 GQ145134 GQ145135 GQ145137 GQ145139 GQ145140 GQ145141 GQ145145 GQ145154 GQ145155 GQ145157 GQ145158 GQ145164 GQ145168 GQ145170 GQ145172/ GQ145178 GQ145179 GQ145186 GQ145187 GQ145188 GQ145189 GQ145190 GQ145197 GQ145199 GQ145203 GQ145204 GQ145208 HQ222152 HQ222258 GQ145211 Voucher Kew DNA 15748, Chase 15748 (K) GenBank No. rpl16 Explanations: ñ the sequence from Przemys≥aw Baranow (Ph.D. thesis 2010, unpbl.); * ñ sequences from Russell et al. (2010) a Unauthenticated Download Date | 6/30/16 11:08 PM GenBank No. rps16trnK* GQ145084 GQ145085 GQ145086 GQ145090 27