Phytotaxa 175 (3): 121–132 www.mapress.com/phytotaxa/ Copyright © 2014 Magnolia Press ISSN 1179-3155 (print edition) Article PHYTOTAXA ISSN 1179-3163 (online edition) http://dx.doi.org/10.11646/phytotaxa.175.3.1 A new paludicolous species of Malaxis (Orchidaceae) from Argentina and Uruguay JOSÉ A. RADINS1, GERARDO A. SALAZAR2,5, LIDIA I. CABRERA2, ROLANDO JIMÉNEZ-MACHORRO3 & JOÃO A. N. BATISTA4 1 Dirección de Biodiversidad, Ministerio de Ecología y Recursos Naturales Renovables, Calle San Lorenzo 1538, Código Postal 3300, Posadas, Misiones, Argentina 2 Departamento de Botánica, Instituto de Biología, Universidad Nacional Autónoma de México, Apartado Postal 70-367, 04510 Mexico City, Distrito Federal, Mexico 3 Herbario AMO, Montañas Calizas 490, Lomas de Chapultepec, 11000 Mexico City, Distrito Federal, Mexico 4 Departamento de Botânica, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Caixa Postal 486, 31270−910 Belo Horizonte, Minas Gerais, Brazil 5 Corresponding autor; e-mail gasc@ib.unam.mx Abstract Malaxis irmae, a new orchid species from the Paraná and Uruguay river basins in northeast Argentina and Uruguay, is described and illustrated. It is similar in size and overall floral morphology to Malaxis cipoensis, a species endemic to upland rocky fields on the Espinhaço range in Southeastern Brazil, which is its closest relative according to a cladistics analysis of nuclear (ITS) and plastid (matK) DNA sequences presented here. However, M. irmae is distinguished from M. cipoensis by inhabiting lowland marshy grasslands, possessing 3−5 long-petiolate leaves per shoot (vs. 2 shortly petiolate leaves), cylindrical raceme (vs. corymbose), pale green flowers (vs. green-orange flowers) and less prominent basal labellum lobules. Malaxis irmae is morphologically also similar to the Brazilian M. warmingii, which differs in its much larger plants and prominent basal labellum lobes. Key words: ITS, Malaxis irmae, marshy grasslands, matK, phylogenetics Introduction As traditionally delimited, the genus Malaxis Solander ex Swartz (1788: 119) s.l. included about 300 species and had a worldwide distribution (Cribb 2005). However, a recent molecular phylogenetic analysis of tribe Malaxideae (Cameron 2005) showed that Malaxis s.l. is polyphyletic. Although much work on the phylogeny and taxonomy of the whole tribe Malaxideae remains to be done to clarify the generic limits, several morphology-based taxonomic studies have started to recognize less-inclusive segregated genera. Some of the current segregates of Malaxis s.l. include Old World tropical groups like Crepidium Blume (1825: 387), Dienia Lindley (1824: sub t. 825) and Orestias Ridley (1887: 197) (e.g. Szlachetko 1995, Clements & Jones 1996, Cribb 2005), whereas the New World segregated genera comprise Crossoglossa Dressler & Dodson (1993: 148), Tamayorkis Szlachetko (1995: 121) and Crossoliparis Margońska (2009: 298−299). Excluding such segregates, Malaxis s.s., including Microstylis (Nuttall 1818: 196) Eaton (1822: 115), encompasses approximately 120 species restricted to the New World and temperate regions of Eurasia (G. A. Salazar, unpubl. data). Recently, Margońska et al. (2012) published a review of “Malaxidiinae” Bentham & Hooker (1883: 463, 465), a polyphyletic assemblage of taxa allegedly distinguishable from other Malaxideae by morphological traits such as column length relative to anther length, angle of the anther relative to the column, position of anther openings, degree of concavity of the stigma and structure of the nectary, but none of these traits is consistent in any of their purported subtribes, the limits of which grossly contradict the results of both, the molecular phylogenetic analysis of Cameron (2005) and Margońska et al.’s own cladogram based on ITS sequences (Margońska et al. 2012: Fig. 5). Likewise, the cumbersome infrageneric classification proposed in that work, which recognizes sections, subsections and series often based on unreliable characters (e.g. “Raceme apically dense and conical” vs. “Raceme dense and distinctly cylindrical all along its length”, a feature that often varies in the same plant depending on the stage of development of the inflorescence), results in grouping together disparate plants and segregating in different Accepted by Cássio van den Berg: 6 Jul. 2014; published: 8 Aug. 2014 Licensed under a Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0 121 supraspecific taxa species that hardly can be told apart, e.g. M. excavata (Lindley 1838: misc. 51) Kuntze (1891: 673) and M. lepanthiflora (Schlechter 1918: 200–201) Ames (1922: 84), which are ecologically and morphologically nearly indistinguishable but are placed in different sections by Margońska et al. (2012). FIGURE 1. Phylogenetic relationships of selected Malaxideae inferred in the separate analyses of matK and ITS DNA sequences. A. The single most-parsimonious tree found by the analysis of matK. B. One of the three most-parsimonious trees recovered by the analysis of ITS. Numbers above the branches are branch lengths; numbers below the branches are bootstrap percentages. Most published work on New World Malaxis s.s. consists of descriptions of new species, both as a result of access to previously unexplored areas and of the greater attention paid to these inconspicuous, little-studied orchids by local taxonomists (e.g. de Barros 1996, Salazar 1990, 1997, Salazar & de Santiago 2007, Dressler 2003, González et al. 2008, Carnevali & Noguera 2008). Here we describe an additional new species restricted to water-logged lowland vegetation in north-eastern Argentina and Uruguay. The phylogenetic position of the new species is assessed by means of a cladistics parsimony analysis of the same two molecular markers employed in Cameron’s (2005) molecular phylogenetic analysis of Malaxideae, namely the ITS region of nuclear ribosomal DNA (Baldwin et al. 1995) and the putative pseudogene matK of the plastid genome (Hilu & Liang 1997). The morphological and ecological peculiarities of the new species are discussed against the phylogenetic framework. Materials and methods Taxon sampling for the phylogenetic analysis:—We analysed samples of 28 species of Malaxideae, of which 18 belong to Malaxis s.s. and the others represent individual species of ten other genera of the tribe. One species each of Dendrobium Swartz (1799: 82) and Bulbophyllum Petit-Thouars (1822: table 3) were used as outgroups, following earlier works that indicate that Dendrobieae is the sister clade of Malaxideae (Cameron 2005, Cameron et al. 1999, Chase & Cribb 2005). Seventy five percent of the sequences were newly generated for this work and the rest downloaded from GenBank. A list of the taxa analysed, including voucher information and accession numbers in GenBank and the European Nucleotide Archive (http://www.ebi.ac.uk/ena/data/view/HG970137-HG970159) are provided in Table 1. 122 • Phytotaxa 175 (3) © 2014 Magnolia Press RADINS ET AL. FIGURE 2. Phylogenetic relationships of selected Malaxideae inferred from combined matK and ITS DNA sequences. The main tree is the single most-parsimonious tree found; numbers above the branches are branch lengths; numbers below the branches are bootstrap percentages. The inset on the upper left hand is the same tree with branches drawn proportional to branch lengths. MALAXIS (ORCHIDACEAE) Phytotaxa 175 (3) © 2014 Magnolia Press • 123 TABLE 1. Taxa studied, voucher information or literature reference and GenBank/European Nucleotide Archive accessions. Species Voucher or reference ITS matK Bulbophyllum lobbii Lindl. van den Berg et al. (2005) AF521074 AY121740 Dendrobium aphyllum (Roxb.) C.E.C.Fisch. Ding, X., Xu, L. & Wang, Z. (unpubl.) Teng, Y.-F., Wu, X.-J., Wang, Z.-T. & Yu, G.-D. (unpubl.) AF355573 -- -AF447068 Crepidium acuminatum (D.Don) Szlach. Ohi-Toma et al. (2007) AB290884 AB290892 Crossoglossa fratrum (Schltr.) Dressler ex Dodson Costa Rica, Dressler s.n. (USJ) HG970119 HG970141 Crossoliparis wendlandii (Rchb.f.) Marg. Mexico, Salazar et al. 6425 (MEXU) HG970118 HG970140 Dienia ophrydis (J.Koenig) Seidenf. Cameron (2005) AY907114 AY907181 Diteilis nervosa (Thunb.) M.A.Clem. & D.L.Jones Cameron (2005) AY907092 AY907158 Hippeophyllum micranthum Schltr. Philippines, Salazar 7637 (MEXU) HG970115 HG970137 Liparis loeselii (L.) Rich. U.K., Chase 7238 (K) HG970117 HG970139 Malaxis brachyrrhynchos (Rchb.f.) Ames Mexico, Salazar et al. 7484 (MEXU) HG970121 HG970143 Malaxis carpinterae (Schltr.) Ames Costa Rica, Salazar s.n. (MEXU, spirit) HG970125 HG970147 Malaxis cipoensis F.Barros Brazil, Batista 2328 (BHCB) HG970126 HG970148 Malaxis hagsateri Salazar Mexico, Salazar 6773 (MEXU) HG970123 HG970145 Malaxis hastilabia (Rchb.f.) Kuntze Costa Rica, Salazar s.n. (MEXU, spirit) HG970128 HG970150 Malaxis histionantha (Link, Klotzsch & Otto) Garay & Dunst. Mexico, Soto 8958 (AMO) HG970124 HG970146 Malaxis irmae Radins & Salazar Argentina, Radins 105 (CTES) HG970127 HG970149 Malaxis lepanthiflora (Schltr.) Ames Mexico, Reyes 5469 (MEXU) HG970129 HG970151 Tribe Dendrobieae Tribe Malaxideae Malaxis lepidota (Finet) Ames Mexico, Soto 9733 (AMO) HG970122 HG970144 Malaxis maxonii Ames El Salvador, Salazar & Linares 7519 (MEXU) HG970130 HG970152 Malaxis molotensis Salazar & de Santiago Mexico, de Santiago 1320 (MEXU) HG970131 HG970153 Malaxis moritzii (Ridl.) Kuntze Venezuela, Jardín Botánico Universidad de Mérida 27-07 (MEXU, spirit) HG970132 HG970154 Malaxis pandurata (Schltr.) Ames Mexico, Rojas 54 (MEXU) HG970135 HG970158 Malaxis parthonii C.Morren Argentina, Radins s.n. (MEXU, photograph) HG970133 HG970155 Malaxis rosilloi R.González & E.W.Greenw. Mexico, Salazar & Carnevali 6078 (MEXU) HG970134 HG970156 Malaxis soulei L.O.Williams Mexico, Soto 9741 (AMO) HG970136 HG970159 Malaxis spicata Sw. Without locality, Chase 377 (K) AF521068 HG970157 Malaxis zempoalensis López-Ferr. & Espejo Mexico, Espejo et al. 5714 (AMO) HG970120 HG970142 Oberonia wappeana J.J.Sm. Cameron (2005) AY907138 AY907206 Stichorkis gibbosa (Finet) J.J.Wood South East Asia, Heildelberg Botanical Garden s.n. (HEID) HG970116 HG970138 Tamayorkis porphyrea (Ridl.) Salazar & Soto Arenas Cameron (2005) AY907115 AY907182 DNA extraction, amplification and sequencing:—Genomic DNA was extracted from fresh, silica gel-dried or herbarium material using a 2× cetyltrimethylammonium bromide (CTAB) protocol based on Doyle & Doyle (1987), modified by the addition of 2% of polyvinylpyrrolidone (PVP) to the extraction buffer. PCR was carried out in 25 μL reactions using a commercial kit (Taq PCR Core Kit, Qiagen, Hilden, Germany), adding to the reaction mix 0.25 μL of each primer at a concentration of 100 ng/μL and 0.5 μL of a 0.4% aqueous solution of bovine serum albumin (BSA) to neutralize potential inhibitors (Kreader 1996). In the case of the ITS region, 0.5 μL of dimethylsulfoxide (DMSO) were 124 • Phytotaxa 175 (3) © 2014 Magnolia Press RADINS ET AL. added to the reaction tube to reduce problems associated with DNA secondary structure. The PCR profile for matK consisted of a 2 min 30 s initial premelt at 94°C, 28−30 cycles with 1 min denaturation at 94°C, 1 min annealing at 52°C, a first 2 min 30 s extension at 72°C, increased by 8 s on each consecutive cycle, and final extension of 7 min at 72°C. The PCR profile for the ITS region consisted of an initial 2 min premelt at 94°C, 30 cycles of 1 min denaturation at 94°C, 1 min annealing at 50°C, and 2 min extension at 72°C, with final extension of 7 min at 72°C. The primers used for PCR and sequencing are listed in Table 2. TABLE 2. Primers used for PCR and sequencing. Primer name DNA region Primer sequence Reference 17SE ITS 5’-ACG AAT TCA TGG TCC GGT GAA GTG TTC-3’ Sun et al. (1994) 26SE ITS 5’-TAG AAT TCC CCG GTT CGC TCG CCG TTA-3’ Sun et al. (1994) matK -19F matK 5’-CGT TCT GAC CAT ATT GCA CTA TG-3’ Molvray et al. (2000) matK 458F matK 5’-CTA CTA ATA CCC YAT CCC ATC-3’ Molvray et al. (2000) matK 556R matK 5’-GAA GRA ACA TCT TTK ATC CA-3’ Molvray et al. (2000) matK 731F matK 5’-TCT GGA GTC TTT CTT GAG CGA-3’ Molvray et al. (2000) matK 1326R matK 5’-TCT AGC ACA CGA AAG TCG AAG T-3’ Cuénoud et al. (2002) trnK 2R matK 5’-AAC TAG TCG GAT GGA GTA G-3’ Steele & Vilgalys (1994) PCR products were purified using QIAquick silica columns (Qiagen) and used in cycle sequencing reactions with the ABI Prism Big Dye® Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq® DNA polymerase version 3.1 (Applied Biosystems Inc., Foster City, California, USA). Cycle sequencing reactions included 2 μL terminator mix, 0.25 μL primer at the same concentrations as for PCR and 3 μL PCR product. Cycle sequencing products were purified with Centri-Sep sephadex columns (Princeton Separations, Inc., Adelphia, New Jersey, USA) and analysed in a 3100 Genetic Analyzer (Applied Biosystems Inc.). Phylogenetic analysis:—The chromatograms were edited and assembled with Sequencher (GeneCodes Corp., Ann Arbor, Michigan, USA), and the resulting sequences were aligned by eye, trying to maximize sequence similarity (Simmons 2004). Individual gap positions were treated as missing data. The aligned matrix is available from TreeBase (http://purl.org/phylo/treebase/phylows/study/TB2:S15688). We analysed the matK and ITS data separately and in combination under the parsimony optimality criterion using the software PAUP* v. 4.02b (Swofford 2002). Each analysis consisted of a heuristic search with 1,000 replicates of random order of taxa for calculating the starting trees, tree-bisection-reconnection (TBR) branch-swapping and the “Multrees” option activated, saving all the mostparsimonious trees (MPTs) found. Clade support was assessed by means of 1,000 bootstrap replicates (Felsenstein 1985), each consisting of 20 heuristic searches conducted as above but saving up to 20 MPTs per heuristic replicate. Morphological observations:—The description of the new species was based mainly on study and measurements from living, pressed and ethanol-preserved plants from Argentina under a stereomicroscope. Complementary information on the Uruguayan record was obtained from literature (Izaguirre 2010). Results of the phylogenetic analysis The matK matrix included 1,411 characters, of which 160 were parsimony-informative. The heuristic search found a single MPT (Fig. 1A) with a length of 509 steps, Consistency Index (CI, excluding uninformative characters) = 0.69 and Retention Index (RI) = 0.87. The ITS matrix consisted of 705 characters, of which 299 were parsimonyinformative, and the search recovered 3 MPTs with a length of 1,034 steps, CI = 0.56 and RI = 0.71. One of the 3 MPTs is shown in Fig. 1B. As in the study of Cameron (2005), matK and ITS recovered similar relationships. The combined matrix consisted of 2,116 characters, of which 459 were parsimony-informative, and the analysis found a single MPT with a length of 1,453 steps, CI = 0.59 and RI = 0.75. The single MPT, with clade support from the bootstrap analysis (Bootstrap Percentages, BP), is shown in Fig. 2. In the following, we will refer only to this tree, which we consider as our best estimate of the phylogenetic relationships among the taxa analysed. The earliest groups to diverge in Malaxideae consist of a grade of two strongly supported, successively diverging clades containing Old World taxa (Fig. 2). The first such clade encompasses Stichorchis Petit-Thouars (1809: 318) as sister to [Hippeophyllum Schlechter (1905: 107)-Oberonia Lindley (1830–1840: 15)], whereas the second clade includes Diteilis Rafinesque (1833: 73–74) and [Crepidium-Dienia] and is in turn sister to a strongly supported group (BP 100) in which Northern Temperate Liparis loeselii (Linnaeus 1753: 947) Richard (1817: 38) is the sister of a New World clade encompassing the remaining taxa. Within the latter, Crossoliparis, Crossoglossa and Tamayorkis MALAXIS (ORCHIDACEAE) Phytotaxa 175 (3) © 2014 Magnolia Press • 125 are successive sisters to weakly supported (BP 67) Malaxis s.s. The latter consists, in ascending branching order, of a group formed by M. soulei Williams (1934: 343) and M. pandurata (Schlechter 1906: 77–78) Ames (1922: 84) (BP 91), the clade M. brachyrrhynchos (Reichenbach filius 1888: 152–153) Ames (1922: 84)-[M. lepidota (Finet 1907: 531-532) Ames (1922: 84)-M. zempoalensis López-Ferrari & Espejo (2009: 45)] (BP 100) and a further clade (BP 100) that, except for M. cipoensis Barros (1996: 31), consists of species having above-ground, ovoid pseudobulbs separated by conspicuous rhizomes (cf. Salazar 1990), in contrast with the subterranean, globose corms without rhizomes found in other species of the genus. That last major clade consists in turn of two subclades, i.e. M. moritzii (Ridley 1888: 330) Kuntze (1891: 673) through M. lepanthiflora, and M. rosilloi González & Greenwood (1984: 387) through M. cipoensis. The latter includes the lectotype species of Malaxis (M. spicata Solander ex Swartz 1788: 119). The new species, hereafter referred to as Malaxis irmae Radins & Salazar (see Taxonomy, later) occupies a derived position in this clade as the sister of M. cipoensis (BP 95). Taxonomy Malaxis irmae Radins & Salazar, sp. nov. (Figs. 3, 4). Similar to Malaxis cipoensis F.Barros, differing in inhabiting marshy lowland vegetation, 3−5 petiolate leaves per shoot, pale green flowers with somewhat darker green labellum and smaller basal lobules of the labellum. Holotype:—ARGENTINA. Misiones: Garupá, 11 July 2008, J. A. Radins 105 (CTES!). Paludicolous herb 6−14 cm in height including the inflorescence. Roots cylindrical, sparsely pilose, up to 3 cm long, 0.5−1.0 mm in diameter. Rhizome whitish, up to several cm long, 3−5 mm in diameter. Pseudobulbs inconspicuous, ovoid, up to 10 mm long and 6 mm in diameter, when young concealed by the sheathing bases of the petioles. Leaves 3−5 per shoot, ascending, petiolate; petiole white at base but becoming green towards the apex, channelled, distinctly elongate, 12−55 mm long, 3−4 mm wide; blade deep green, broadly ovate to elliptic, base widely cuneate to rounded, apex obtuse-rounded, 14−50 mm long, 10−35 mm wide. Inflorescence provided with a laterally compressed peduncle 50−80 mm long, which is concealed at base by the petiole of the upper leaf; raceme 16−60 mm long, at first condensed and thus appearing somewhat umbellate but the rachis elongates as the flowers open successively and the raceme is cylindrical; rachis slightly angled. Floral bracts slightly concave, incurved, triangular, acute, 1−2 mm long. Flowers non-resupinate; sepals and petals pale green, labellum deep green. Sepals convex, with revolute lateral margins, 3veined; dorsal sepal adpressed to the ovary, ovate, subacute, 2−2.5 mm long, ca. 1.5 mm wide; lateral sepals diverging, obliquely ovate-elliptic, obtuse, 1.8−2.3 mm long, ca. 1.8 mm wide. Petals strongly recurved, linear, subacute, 1veined, 2.1−2.2 mm long, ca. 0.2 mm wide. Labellum fleshy, broadly cordate-sagittate, 1.4−1.7 mm long, 1.9−2.8 mm wide; proximal half provided with two rounded excavations; base provided at each side with a slightly retrorse, rounded lobule 0.5−0.6 mm long; apex apiculate, the apicule somewhat incurved in natural position. Column slightly compressed dorsiventrally, ca. 7 mm long and wide; anther dorsal, emarginate; rostellum broadly obtuse; stigma apical, concave, distinctly wider than long. Pollinaria 2, each formed by 2 fused pollinia, ca. 0.5 mm long (fide Izaguirre 2010; not seen). Ovary erect, straight, slightly twisted and somewhat thinner on the proximal one-third, above the middle slightly 6-angled, 6−8 mm long, ca. 1 mm in diameter near the apex. Capsule obovoid-ellipsoid, ca. 6 mm long (plus the pedicel of about the same length), to 5 mm in diameter. Distribution and ecology:—Malaxis irmae is known only from the Río Paraná and Río Uruguay basins in Uruguay and north-eastern Argentina (Fig. 5), but it is expected also from southern Paraguay and the state of Rio Grande do Sul, Brazil. It inhabits in water-logged terrain dominated by grasses, and in neighbouring forest edges, between 60 and 100 m above sea level. Conservation status:—Only three populations of this species have been recorded, but the Argentinian and Uruguayan populations are over 700 km apart in a straight line (Fig. 5) and it is likely that other populations exist, since suitable habitats (lowland wet grasslands) are widespread over the extensive Río de la Plata basin (which, among others, encompasses the Paraná and Uruguay rivers). At the type locality, urban expansion of the town of Garupá, on the outskirts of the city of Posadas, represents a short-term threat to that population; besides, the wet grasslands and associated water-logged forests that constitute the habitat of this species, are one of the most endangered vegetation types within the Atlantic rain forest biome in south-eastern South America (e.g. Bitetti et al. 2003, Krauczuk 2005, Overbeck et al. 2007). According to the IUCN Red List Categories and Criteria (IUCN 2012), M. irmae would qualify as Critically Endangered CR (criteria B2a, B2b and C2a(i)). However, both plants and flowers of this species are inconspicuous and can easily go unnoticed, even to trained botanical collectors. Therefore, further field studies are required to attain an objective assessment of its conservation status. 126 • Phytotaxa 175 (3) © 2014 Magnolia Press RADINS ET AL. FIGURE 3. A–E, Malaxis irmae (from the type locality in Garupá, Misiones, Argentina). A. Flowering plant in situ. B. Fruiting plant in situ. C. Plant removed from substrate. D. Fruiting plant compared to a scale (in cm). E. Close-up of a flower. F–G. M. cipoensis (Brazil, Batista 2328, BHCB). F. Plants in situ. G. Inflorescence. H–I. M. warmingii (Brazil, Hoehne & Gehrt 35287, SP). H. Overview of the herbarium sheet with a 15 cm ruler for scale. I. Close-up of dissected flower. Photographers: A–E, J.A. Radins; F–G, J.A.N. Batista; H–I, F. de Barros. MALAXIS (ORCHIDACEAE) Phytotaxa 175 (3) © 2014 Magnolia Press • 127 FIGURE 4. Malaxis irmae. A. Habit. B. Flower frontal view. C. Flower side view. D. Dorsal sepal. E. Petal. F. Lateral sepal. G. Labellum. H. Column from above. I. Column side view. J. Column from below. Drawn with camera lucida by Rolando Jiménez-Machorro from Radins 105. 128 • Phytotaxa 175 (3) © 2014 Magnolia Press RADINS ET AL. Phenology:—Flowering from March to June. Capsules in advanced stage of development, including some already dehiscing, have been observed from May to August. Etymology:—The specific epithet honours Ms. Irma Stella Insaurralde, long-term student of the orchids, and the flora in general, of the province of Misiones, Argentina. Additional specimen examined:—ARGENTINA. Corrientes: Colonia Liebig, D. Boicho s.n. (CTES!). Other records:—URUGUAY. Florida: precise locality not indicated, E. Marchesi s.n. (see Izaguirre 2010). Discussion:—Our phylogenetic analysis strongly supports M. irmae as the sister of M. cipoensis (Fig. 1, 2), in agreement with their similar plant and flower size and overall floral morphology. However, they differ strikingly in habitat preferences, since M. cipoensis inhabits in well-drained soil on rocky field (campo rupestre) areas at 1,000 to 1,340 m elevation, having been found so far only on the Serra do Cipó and Serra da Moeda, both of which form part of the Espinhaço Range in the state of Minas Gerais, Brazil (Barros 1996, J.A.N. Batista, pers. obs.; Fig. 3F–G). Malaxis irmae thus differs from M. cipoensis in its lowland, water-logged habitat (Fig. 4 A–B), and morphologically it can also be distinguished from the latter by possessing 3−5, distinctly petioled leaves per shoot (vs. 2 shortly petiolate leaves), pale green flowers with a darker green labellum (vs. orangish-green flowers) and smaller basal labellum lobes (Fig. 4C–E; a good illustration of M. cipoensis for comparison is found in Barros 1996). Malaxis warmingii (Reichenbach 1881: 64) Kuntze (1891: 673), a relatively widespread, south-eastern Brazilian species that was not sampled for our molecular analysis, shares with M. irmae a preference for water-logged habitats, the 5−6-leaved shoots and a similar overall floral morphology, but it has much larger plants (40−60 cm in height) and prominent basal labellum lobes (Fig. 4H–I, Cogniaux 1893−1896). Malaxis hieronymi (Cogniaux 1893−1896: 279−280) Williams (1939: 363) inhabits marshes at high elevations (2,000−3,500 m) in Bolivia and the Argentinian provinces of Salta, Jujuy and Tucumán, being further distinguished from M. irmae by having pseudobulbs completely covered by fibrous leaf sheaths, two subsessile leaves and labellum lacking basal lobules. The Uruguayan material assigned here to M. irmae was identified by Izaguirre (2010) as M. spicata, a 2-leaved species with prominently lobed, brownish to orange-vermillion labellum provided with a deep triangular cavity, which is limited by a V-shaped, thickened rim. Malaxis spicata is restricted to the Antilles and the southeastern U.S.A. (Luer 1972). FIGURE 5. Known distribution of Malaxis irmae (dots). The overall phylogenetic relationships in Malaxideae are beyond the focus of this paper and will be discussed in detail elsewhere (G. A. Salazar et al., unpublished manuscript). The phylogenetic analysis conducted here was aimed mainly at setting up a context to determine the relationships of the new species, since a morphological comparison with other species of the genus indicated similarities to both M. cipoensis and M. warmingii (see earlier), and its preference MALAXIS (ORCHIDACEAE) Phytotaxa 175 (3) © 2014 Magnolia Press • 129 for water-logged habitats is shared, besides M. warmingii, by several other species, including South American M. hieronymi (as noted above) and Mexican M. zempoalensis. From our analysis, it is clear that the preference for wet places has evolved more than once in this genus. Although our sample of species of the genus is too sparse to draw conclusions at this time, the relationships recovered suggest that habitat divergence/specialisation may have played a role in promoting speciation in Malaxis s.s., which is exemplified by the contrasting habitat preferences of M. irmae and M. warmingii with respect to M. cipoensis (the closest relative of M. irmae among the taxa we sampled). This and other interesting evolutionary questions, however, will have to be revisited when a more thorough sample of the genus is available for molecular phylogenetic study. Barros (1996) placed M. cipoensis in Malaxis section Umbellulatae (Ridley 1888: 315) Barros (1996: 33) and M. warmingii in M. section Spicatae Ridley (1888: 315). The latter is obviously superfluous as it includes the (lecto-) type species of Malaxis (M. spicata), whereas our phylogenetic analysis shows that M. cipoensis belongs in the same clade as M. spicata, thus demonstrating that these infrageneric taxa are of little use. These and other infrageneric groups (e.g. those in Margońska et al. 2012), will have to be thoroughly assessed when the phylogenetic relationships in the genus are better understood. Acknowledgements The authors thank Fabio de Barros for information and photographs of M. warmingii, Luis Vivero and Daniel Boicho for photographs and material of the Corrientes population of the new species, Robert L. Dressler, José L. Linares, the Royal Botanic Gardens, Kew, UK and the Jardín Botánico de Mérida, Venezuela, for providing plant material and information, Laura Márquez-Valdelamar for assistance with DNA sequencing, Héctor Huerta for help in preparing the map and two anonymous reviewers for useful suggestions to the manuscript. Financial support and courtesies to G.A.S. from Biofábrica Misiones and Sociedad Argentina de Botánica to participate in the XXIII Jornadas Argentinas de Botánica and conduct field work required for this study are gratefully acknowledged. JANB acknowledges a grant (PQ-2) from Conselho Nacional de Desenvolvimento Científico e Tecnológico – CNPq. References Ames, O. (1922) Descriptions of new orchids from Tropical America with nomenclatorial changes. Proceedings of the Biological Society of Washington 35: 81–88. Baldwin, B.G., Sanderson, M.J., Porter, J.M., Wojciechowski, M.F., Campbell, C.S. & Donohue, M.J. (1995) The ITS region of nuclear ribosomal DNA: a valuable source of evidence on angiosperm phylogeny. Annals of the Missouri Botanical Garden 82: 247–277. http://dx.doi.org/10.2307/2399880 Barros, F. de (1996) Uma nova espécie de Malaxis Sol. ex Sw. (Orchidaceae) da Serra do Cipó (Minas Gerais, Brasil) e considerações sobre as seções brasileiras do gênero. 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