BRC Biodiv. Res. Conserv. 1-2: 18-24, 2006 www.brc.amu.edu.pl

Phylogenetic relationships within the subtribe s.l. () inferred from the nuclear ITS region

Marcin GÛrniak*, Joanna Mytnik-Ejsmont, Piotr Rutkowski, Piotr Tuka≥≥o, Julita Minasiewicz & Dariusz L. Szlachetko

Department of and Nature Conservation, University of GdaÒsk, Al. LegionÛw 9, 80-441 GdaÒsk, Poland. *e-mail: [email protected]

Abstract: Results of an analysis of ITS sequences for the subtribe Spiranthinae s.l. (Orchidaceae) are presented, and compared with results of morphological studies. To evaluate the monophyly of the subtribe, nuclear ribosomal DNA internal transcribed spacers (ITS1 and ITS2) and the 5.8S gene were sequenced for 19 taxa of the tribe Spirantheae, with Erythrodes sp., Bonatea speciosa, Cynorkis sp., curta and Chloraea flavescens designated as an outgroup. For 8 taxa, sequences were taken from the GenBank. The results confirmed the current subtribal position of Cyclopogoninae, with members of the subtribe forming a moderately supported clade. The unexpected occurrence of Odontorrhynchos (Spiranthinae) within the Cyclopogoninae is discussed with reference to the results of morphological studies. Although some of the genera representing the subtribe Stenorrhynchidinae Szlach. form a weakly supported clade, the remaining taxa are variously grouped with Spiranthinae representatives, receiving moderately weak bootstrap support. The results do not provide evidence for the strict monophyly of Spiranthinae s.l. and Stenorrhynchidinae.

Key words: Orchidaceae, Spiranthinae, Cyclopogoninae, Odontorrhynchos, molecular taxonomy, ITS

1. Introduction he included Prescottiinae, Cyclopogoninae, Stenorrhyn- chidinae and Spiranthinae. The generic and species composition of Spiranthinae, The subtribe Cyclopogoninae Szlach., as defined by due to its high polymorphism, has stirred up a lot of Szlachetko (1995), embraces 9 genera. The distinguishing controversy from the beginning. Most of its taxa were feature of this group is the characteristic viscidium initially included in the L. C. Rich., produced on the dorsal surface of the rostellum. Its but Schlechter (1920) divided it into 24 genera. His work lower, firm layer is built of sclerenchymatous cells, was harshly criticized by other scientists, mainly from while the upper, soft layer is made of partially macerated the United States (Ames 1922; Williams 1951; cells. The viscidium is enclosed by wishbone-like Schweinfurth 1958). They accused him of inconsistent pollinium apices, and caudiculae are lamellar. The usage of criteria to distinguish the taxa and, most of all, rostellum is soft, ribbon-like, entire or apically furculate, subjective choice of diagnostic features. In the 1980ís, and partly surrounds the pollinarium. However, the Garay (1982) and Burns-Balough (1982) independently generic delimitations between the largest genera, i.e. undertook attempts to revise the Spiranthinae. Their , and , are not clear results turned out to diverge to a large extent, so Szlachetko (Szlachetko et al. 2005). At first, Szlachetko (1995) made further attempts to verify the classification of distinguished 7 genera within Cyclopogoninae: Pelexia Spiranthinae (Szlachetko 1991a, 1991b, 1992a-e, Poit. ex L.C. Rich. (1818), Cyclopogon Presl (1827), 1993a-e, 1994a-c; Szlachetko & Tamayo 1996; Tamayo Sarcoglottis Presl (1827), Stigmatosema Garay (1982), & Szlachetko 1998). He also introduced a new concept Cocleorchis Szlach. (1994), Warscaea Szlach. (1994), of the tribe Spirantheae (Szlachetko 1995), into which and Szlach. (1995). A few years later he proposed VARIABILITY, AND VARIABILITY, TAXONOMY PHYLOGENY

© Adam Mickiewicz University in PoznaÒ (Poland), Department of Plant Taxonomy. All rights reserved. Biodiv. Res. Conserv. 1-2: 18-24, 2006 19 additional genera: Zhukowskia Szlach., R. Gonzalez T. et al. 2003), Laeliinae (Berg et al. 2000), Disinae & Rutk. (2000), Pachygenium (Schltr.) Szlach., R. Gonzalez (Douzery et al. 1999), Pogoniinae (Cameron & T. & Rutk. (2001), and (Schltr.) R. Gonzalez Chase 1999), Orchidinae (Pridgeon et al. 1997), T. & Szlach. (2003). Currently Cyclopogoninae, as Pleurothallidinae (Pridgeon et al. 2001); and on the delimited by Szlachetko, embrace 10 genera. Such generic level: Stanhopea (Whitten et al. 2000), Lycaste a group is also consistent with results of palynological and Anguloa (Ryan et al. 2000), Cypripedium, studies by Balogh (1982). Selenipedium and Paphiopedilum (Cox et al. 1997). The subtribe Stenorrhynchidinae Szlach., proposed Also Batemanís (1997) work shows that ITS is very by Szlachetko (1995), includes about 20 genera. The helpful to determine phylogenetic relations of closely distinguishing features are: the sheath-like viscidium, related genera and species within the Orchideae. To produced of the outer layer of rostellum cells, and the provide a basis for a clear distinction of Cyclopogoninae subulate rostellum remnant. Stenorrhynchidinae have sensu Szlachetko, we compared the results of our 2 distinct centres of diversity, located almost symmetrically phylogeneticanalysis with results of morphological in relation to the equator: in central , somewhat studies. below the tropic of Cancer (Jalisco, Michoacan, Veracruz, San Luis Potosi), and in south , near 2. Material and methods the tropic of Capricorn (Espirito Santo, Rio de Janeiro 2.1. Sampling of taxa and S„o Paulo). The subtribe Spiranthinae Lindl. sensu Szlachetko Nineteen taxa representing Spirantheae sensu 1995 embraces about 30 genera. The distinguishing Szlachetko were originally selected for this study, with features are: the viscidium produced on the adaxial layer Erythrodes sp., Bonatea speciosa, Cynorkis sp., Pterostylis of rostellum cells, and the deeply notched or foveolate curta and Chloraea flavescens designated as the rostellum remnant. outgroup. For 8 taxa, sequences were taken from the In contrast, Dressler (1993) proposed different GenBank: Burnsbaloghia diaphana (AJ 539484), subtribal and tribal concepts, with Spirantheae sensu hyemalis (AJ 539495), lucayanus Szlachetko in the subtribal rank within (AJ 539488), Odontorrhynchos variabilis (AJ 539498), Endl. Following his scheme, Salazar et al. (2003) llaveana (AJ 539487), Spiranthes cernua conducted a phylogenetic analysis of Cranichideae, with (AJ 539495), aurantiacus (AJ 539485), special focus on Spiranthinae sensu Dressler (1993). Stenorrhynchos speciosum (AJ 539505). Vouchers and That study involved 42 species representing all the GenBank accession number for new taxa are available subtribes of Cranichideae of Dresslerís (1993) concept, upon request from first author. including taxa recognized by Szlachetko as Cyclopogo- ninae. Nevertheless, the study conducted by Salazar et 2.2. Amplification and sequencing al. (2003) comprised only 3 taxa of Cyclopogoninae, Total genomic DNA was extracted from 100 mg of representing the genera Cyclopogon, Pelexia and fresh-frozen or 20 mg of silica-dried leaves (Chase & Sarcoglottis. In our opinion their sampling strategy was Hillis 1991) by using the DNeasy Plant Mini Kit (Qiagen not extensive enough to clearly evaluate the taxonomic GmbH, Hilden, Germany), according to manufacturerís status of the subtribe. Members of Cyclopogoninae protocol. formed a nearly monophyletic clade, disrupted by The ITS region (ITS1-5.8s-ITS2) was amplified via the occurrence of Odontorrhynchos variabilis Garay. polymerase chain reaction (PCR) in a Biometra T1 thermal The genus Odontorrhynchos Correa has been included cycler. PCR was carried out in a volume of 50 µl. The by Szlachetko in Spiranthinae. According to extensive PCR mixture contained: dd H2O, 5†µl 10 ◊ polymerase morphological data, those genera of Cyclopogoninae buffer with 15†mM MgCl2, 1†µl of 10mM mix of each and Odontorrhynchos are only distantly related. dNTP (200†µM), 10†µl 5 ◊ Qsolution, 0.5 µl of 20†mM To verify the subtribal boundaries we decided ITS4 and ITS5 primers (White et al. 1990), 2.5 units of to expand the sampling of the taxa representing Taq DNA Polymerase (Qiagen), and genomic DNA. The Cyclopogoninae and to include additional taxa thermal cycling protocol of the PCR consisted of 4 min representing genera of subtribes Spiranthinae and of initial denaturation at 94oC, followed by 30 cycles, Stenorrhynchidinae sensu Szlachetko. To evaluate the each with 45 sec of denaturation at 94oC, 45 sec of monophyly of the subtribe, we sequenced the nuclear annealing at 52oC and 45 sec of extension at 72oC, and ribosomal DNA internal transcribed spacers (ITS1 and ending with 5 min of extension at 72oC. ITS2), and the 5.8S gene, which is a part of the nrDNA Amplified products were cleaned with a High Pure multigene family. Earlier analyses of ITS sequences of PCR Product Purification Kit (Roche Diagnostic GmbH, orchids succeeded to determine the degree of Mannheim, Germany), according to manufacturerís relationship on the subtribe level: Spiranthinae (Salazar protocol. 20 Marcin GÛrniak et al. Phylogenetic relationships within the subtribe Spiranthinae...

Cycle sequencing was carried out directly on the Spiranthinae representatives, receiving moderately purified product by using a Big Dye Terminator v. 3.1 Cycle weak bootstrap support (BP 75 ñ clade C). Sequencing Kit (Applied Biosystems, Warrington, Cheshire, UK): 2 µl of sequencing buffer, 4†µl of Big Cyclopogoninae (clade A) Dye Terminator with 4†µl of 0.08†mM primer (3.2 This clade consists of taxa that represent Cyclo-

pmol), 5-8 µl of amplified product, and dd H2O in a total pogoninae sensu Szlachetko 1995 (Cyclopogon, reaction volume of 20†µl. The PCR primers ITS4 and Sarcoglottis and Pelexia). Szlachetko (1995) singled ITS5 (White et al. 1990) were used to sequence both out Cyclopogoninae from the subtribe Spiranthinae strands of the ITS region. Cycle sequencing conditions sensu Dressler (1993) on the basis of the difference in for those strands were as follows: 20 sec of initial structure of the viscidium and rostellum (see above). denaturation, followed by 25 cycles, each with 15 sec Previously Greenwood (1982) noted that Cyclopogon, of denaturation at 94oC, 20 sec of annealing at 52oC, Sarcoglottis and Pelexia have the specific type of and 4 min of elongation at 60oC, by using a Biometra viscidium (wedge-type viscidium), which was also thermal cycler. Sequencing reactions were purified with confirmed by Burns-Balogh & Robinson (1983). an ExTerminator Kit (A&A Biotechnology, Gdynia, (a sister group to the Cyclopogoninae and Poland), according to manufacturerís protocol. Pelleted Odontorrhynchos clade) is similar to Odontorrhynchos samples were sequenced on an Applied Biosystems 377 and with respect to the structure of the automated sequencer. Forward and reverse strands were and perianth. Is there any explanation of the sequenced to assure accuracy in base calling. close relationship of Sauroglossum to Cyclopogoninae? Gerardo Salazar (2001) in Genera Orchidacearum 2.3. Phylogenetic analyses (2001) noted that Sauroglossum shares many features DNA sequences were aligned by íClustalXôí and with Pelexia: a long slender column, ovate cordate adjusted manually. ITS sequences were analysed by us- anther with an apical projection, and also lip structure ing the heuristic search method of PAUP* version similar to that of Pelexia weberbaueriana Schltr. On 4.0b10 (Swofford 2000). The optimality criterion was the other hand, Szlachetko (1995) on the basis of the parsimony with tree-bisection-reconnection (TBR) structure of the column, especially the rostellum and branch swapping and the MULTREES option in effect, viscidium, placed Sauroglossum in Spiranthinae. simple addition and ACCTRAN optimization. Gaps Molecular data and some morphological features indicate were coded as missing values. Internal support of clades that Sauroglossum and Cyclopogoninae could have had was evaluated by the bootstrap (Felsenstein 1985) the same ancestor. However, many differences, especially method with 1000 bootstrap replicates. All characters the structure of the column, eliminate Sauroglossum were unordered and equally weighted. from the subtribe Cyclopogoninae. The clade is well supported and constitutes a compact group, except the 3. Results and discussion unusual position of Odontorrhynchos. That genus represents the subtribe Spiranthinae sensu Szlachetko (1995) or The ITS region included a total of 760 aligned ìBrachystele allianceî sensu Balogh (1982). The positions, of which 348 were constant, and 98 variable differences between Odontorrhynchos and Pelexia are characters were parsimony-uninformative. There were very significant, as those genera differ in many 314 parsimony-informative characters. Figure 1 depicts morphological characters regarded as evolutionarily a single, 50% majority rule bootstrap consensus tree, conservative. In Odontorrhynchos the gynostemium is with a length of 1143 steps, CI (consistency index) of short and rather massive, its column part shorter than 0.55 and RI (retention index) of 0.66. Four groups can the anther, and the column foot is rudimentary, whereas be identified, with Prescottiinae as a monophyletic sister Pelexia is characterized by an elongate, slender group with high bootstrap (BP) support in relation to gynostemium with the column part being longer than the rest of spiranthoids (BP 100). Cyclopogoninae the anther, and a prominent column foot. The genera differ (Cyclopogon, Pelexia and Sarcoglottis) with Odontorrhyn- in the presence (Pelexia) or absence (Odontorrhynchos) chos form a well-supported clade (BP 97). Sauroglossum of caudiculae, spur and claw of the lip. Those orchid Lindl., with moderately weak support (BP 72) are a taxa also exhibit different kinds of viscidium, which is sister group to the latter (clade A). The position of small, elongate, produced on the dorsal surface of the Schltr. remains unresolved. The results rostellum apex, and glued apically (acrotonically) with do not provide evidence for the strict monophyly of pollinia in Pelexia, and produced on the central part of Spiranthinae sensu Szlachetko (1995) and Stenorrhynchi- the rostellum base and glued to the basal part of pollinia dinae Szlach. Although some of the genera representing (basitonically) in Odontorrhynchos. The rostellum the latter subtribe form a weakly supported clade B (BP remnant is tridentate and fleshy in the latter genus, and 76), the remaining taxa are variously grouped with linear with foveolate apex, slender and soft in the former. Biodiv. Res. Conserv. 1-2: 18-24, 2006 21

Pelexia has oblique and lateral , which are the hypochile in Pelexia is slightly concave near the decurrent on the column foot, whereas tepals in top, and the epichile is strongly bent forward. Auricles Odontorrhynchos are free and straight. Besides, the are produced on the outer margin of the lip in the former genera differ in lip morphology, especially in the genus, but they are produced on the inner part of the lip hypochile structure. In Odontorrhynchos it is sigmoid, margin in the latter one. Also leaves differ in shape and its margins are folded, and the basal part is saccate, but proportions: those of Odontorrhynchos are oblong-lanceolate

Fig. 1. Bootstrap 50% majority rule consensus tree from the parsimony analysis of ITS sequences from taxa of the tribe Spirantheae. Bootstrap percentages >50 are indicated above the branches; A, B, C, D ñ clades 22 Marcin GÛrniak et al. Phylogenetic relationships within the subtribe Spiranthinae...

with short and wide petioles, whereas those of Pelexia affinity between and have are oblong-ovate with long and narrow petioles. In our varied. Dressler (1981), and Salazar et al. (2003) also opinion it is not possible for both taxa to have evolved. suggested a close relation between those taxa and As an explanation we suggest that plant material for consequently they postulated joining them. the genus Odontorrhynchos, used in the study by Salazar Both genera assemble small epiphytic with at al. (2003), could have been determined incorrectly, fasciculate, fleshy, puberulent roots. Their leaves are leading to a continuous sampling error. It could have basal, rosulate, with narrow petioles. Blades are been a plant of the genus Pelexia or Cyclopogon, or an- ovatelanceolate to oblanceolate, acute, with ciliolate other representative of Cyclopogoninae closely related margins. The scape is erect to arcuate, delicate, and to Pelexia. densely villose. The inflorescence of Eurystyles is very dense, all-sided, supported at the base by few sterile Spiranthinae and Stenorrhynchidinae (clades B and C) bracts, so it resembles the inflorescence of Asteraceae. As mentioned earlier, our results do not support The raceme of Lankesterella is few-flowered and lax. a strict monophyly of Spiranthinae (with a notched Flowers of Eurystyles are non-resupinate, which makes rostellum remnant) and Stenorrhynchidinae (with this genus unusual not only in comparison to a subulate rostellum) sensu Szlachetko, which is Lankesterella, but to all Spirantheae. Beside Eurystyles, congruent with the report by Salazar et al. (2003). This non-resupinate flowers are found only in author suggest that the structure of the rostellum and trilobata Schltr. Pseudoeurystyles Hoehne and viscidium has been determined by pressures from Aracamunia Carnevali & I.Ramirez. pollinators. According to Szlachetko et al. (2005) this In Lankesterella lateral sepals are connate and form explanation is impossible because it implies that Çthe a chin-like or conical-cylindrical spur, while the lip is notched rostellum remnant and viscidium, formed of sessile and in Eurystyles the lip is clawed. The genera the apical part of the rostellum, evolved independently differ in lip structure. The claw in Eurystyles is often 5 timesí. The resulting consensus tree (Fig. 1) shows wide and concave, with basal auricles, often terminated some weak evidence that the subtribe Spiranthinae sensu by thick appendages. There are also many differences Szlachetko tends to be more polyphyletic than Stenorr- between the two genera in gynostemium structure. It is hynchidinae. However, we were unable to extend the elongate and slender in Eurystyles and relatively short sampling of taxa and verify this. in Lankesterella. The column foot is prominent and It is worth noting that many genera from both rather massive in the latter genus, and short, incurved subtribes seem to be more closely related than indicated and adnate to the ovary apex in the former. Pollinia in by results of morphological studies. An example is the close Eurystyles are unequal in size and shape, and free from relationship between Eurystyles cotyledon Wawra and caudiculae, whereas in Lankesterella, caudiculae are Lankesterella longicollis (Cogn.) Hoehne. Both taxa were present in most species. The stigma in Eurystyles is classified by Szlachetko (1995) within different subtribes, 3-lobed, and the rostellum is elongate and ribbon-like, Spiranthinae and Stenorrhynchidinae, respectively. while in Lankesterella the stigma is 2-lobed and the Eurystyles was described by an Austrian, Heinrich rostellum is subulate and elastic. Wawra, in 1863, and considering the unusual Autogamy is observed in these groups of orchids. inflorescence for the order Orchidales, it was initially Autogamous species are characterized by a reduction classified into the order Zingiberales by mistake. or lack of rostellum and viscidium. Good examples are Schlechter (1920) included 4 species into Eurytyles, Eurystyles actinosophila (Barb. Rodr.) Schltr., E. which was described by him earlier as a new genus borealis A. H. Heller or Lankesterella orthantha Trachelosiphon: T. actinosophilum (Barb. Rodr.) (Kraenzl.) Garay. Autogamous taxa occur on the peripheries Schltr., T. ananassocomos Schltr., T. cogniauxii of ranges of particular genera, in Mesoamerica and in (Kraenzl.) Schltr., and T. lorenzii (Cogn.) Schltr. In the the north of South America in this instance. Synanthes same work he also described the genus Cladobium was described by Burns-Balogh & Bernhardt in 1985 (todayís Lankesterella Ames) with 5 Brazilian species, as a genus closely related to Eurystyles; those genera which were formerly included into Stenorrhynchos differ in gynostemium structure. The new genus was Rich. ex Spreng.: C. ceracifolium (Barb.Rodr.) Schltr., distinguished as a new taxon on the basis of studies of C. epiphytum (Barb.Rodr.) Schltr., C. gnomus (Kraenzl.) autogamous species only: Synanthes borealis (Heller) Schltr., C. longicolle (Cogn.) Schltr. and C. pilosum Burns-Bal., H.Rob. & Mercedes S. Foster, and S. (Cogn.) Schltr. In that work and in the next one, bertonii Burns-Bal., H.Rob. & Mercedes S. Foster. Schlechter (1920, 1926) placed Trachelosiphon and The subtribe Spiranthinae includes taxa with Cladobium in different groups of the tribe Spirantheae a relatively primitive type of gynostemium. The because of differences in gynostemium and viscidium rostellum is soft, short to elongate; the viscidium is structures. Since then, opinions about the degree of produced from the adaxial layer cells of the rostellum, Biodiv. Res. Conserv. 1-2: 18-24, 2006 23 while the rostellum remnant is deeply notched requires further studies and extensive sampling strategy or foveolate. Members of Stenorrhynchidinae are in future phylogenetic examination. characterized by a sheath-like rigid viscidium, which is produced of the outer layer of rostellum cells, and 4. General conclusions the rostellum remnant is subulate. Stenorrhynchidinae are characterized by filiform caudiculae, distinctly thin The results of our phylogenetic analysis, based on at the end, and they are absent or very poorly developed examination of the ITS region, confirm the monophyly in Spiranthinae. Both subtribes are among the most and separation of the subtribe Cyclopogoninae, as controversial groups of Orchidales. They were delimited by Szlachetko (1995). The unusual occurrence examined in detail by Balogh (1979), Garay (1982), of Odontorrhynchos variabilis within Cyclopogoninae Greenwood (1982), Balogh (1982), and Szlachetko is not supported by morphological data, and, in our (1991a, 1991b; 1992a-e; 1993a-e; 1994a-c). Thus both opinion, is probably a result of sampling error, due to subtribes as delimited by Szlachetko require further an incorrect determination of source material. The detailed morphological studies and more extensive Stenorrhynchidinae and Spiranthinae subtribes, as sampling of taxa for phylogenetic analysis. delimited by Szlachetko (1995), remain polyphyletic and thus further evaluation of subtribal concepts is Prescottiinae (clade D) required (see also Salazar et al. 2003). The clade Our results show that Prescottiinae are a monophyletic Prescottiinae is strongly supported as monophyletic, group with high bootstrap support (BP 100). Because although a lack of extensive sampling of taxa limits the we were not able to extend the sampling of taxa for this reliability of this finding. subtribe, only representatives of the genus Prescottia Acknowledgments. The first author would like to thank were included in the analysis. Thus the observed Aneta Wojewodzka and Krzysztof Spalik for a short-term monophyly seems to be of low reliability. Moreover, fellowship at the Department of Plant Systematics and Ge- the results reported by Salazar et al. (2003) indicated ography, Warsaw University. The project was supported by that Prescottiinae are paraphyletic within Cranichidinae the State Committee for Scientific Research, Warsaw and Spiranthinae sensu Dressler. Thus the subtribe (Projects no. 3 P04 C 036 24 and no. 3 P04 C 075 24).

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