Nordic Journal of Botany 26: 61Á65, 2008 doi: 10.1111/j.1756-1051.2008.00135.x, # The Authors. Journal compilation # Nordic Journal of Botany 2008 Subject Editor: Anne Krag Brysling. Accepted 25 September 2008 Seed micromorphology supports the splitting of Limnorchis from Platanthera (Orchidaceae) Roberto Gamarra, Pablo Gala´n, Irene Herrera and Emma Ortu´n˜ez R. Gamarra ([email protected]), I. Herrera and E. Ortu´n˜ez, Depto de Biologı´a, Univ. Auto´noma de Madrid, C/ Darwin, 2, ESÁ28049 Madrid, Spain. Á P. Gala´n, Depto de Produccio´n Vegetal, Bota´nica y Proteccio´n Vegetal, E.U.I.T. Forestal, Univ. Polite´cnica, ESÁ28040 Madrid, Spain. Seeds of several species of the genera Platanthera and Habenaria have been studied by means of scanning electron microscopy. In the genus Platanthera, two morphological patterns were found. One of these appears in the so-called ‘dilatataÁhyperborea complex’. It is proposed that this pattern is a good diagnostic character to split off this group of species into the genus Limnorchis.InHabenaria, the morphological patterns vary between the species and differs completely from those found in Platanthera. According to Pridgeon et al. (2001), the genus Platanthera Platanthera in the genus Habenaria is misapplied. All (L.) Rich. comprises about 200 species, and the genera contemporary specialists on the Orchidaceae recognize Tulotis Raf., Blephariglottis Raf., Limnorchis Rydb., Lysiella Platanthera and Habenaria as distinct genera (Luer 1975, Rydb., Gymnadeniopsis Rydb. and Piperia Rydb. are Case 1987, Smith 1993) and, in fact, the genus Habenaria included as synonyms. It belongs to the subtribe Orchidinae is referred to the subtribe Habenariinae whereas Platanthera (Dressler 1993). Platanthera is distributed throughout the belongs in subtribe Orchidinae (Dressler 1993, Pridgeon north temperate area, extending their distribution to et al. 2001). Central America, northern Africa and New Guinea. Most Other North American species of the genus Pla- of the species occur in North America and Asia. tanthera, such as P. unalascensis (Spreng.) Kurtz and P. Habenaria Willd. comprises about 600 species in elegans Lindl. were included in the genus Piperia by tropical and temperate regions, principally in America and Rydberg (1901), and Luer (1975) and Ackermann (1977) Africa (Buttler 2001). It belongs to the subtribe Habena- recognised three and four species each in this genus, riinae (Dressler 1993). respectively. The last author distinguished Piperia from Several North American taxa such as Platanthera dilatata Platanthera by several morphological characters (globose (Pursh) Lindl. and P. hyperborea (L.) Lindl. are included in tubers vs fusiform tubers, very short and inconspicuous the ‘dilatataÁhyperborea’ complex. This critical group was caudicles, senescence of leaves during anthesis). However, assigned to the genus Limnorchis by Rydberg (1900, 1901) in Europe no segregate genera have been proposed and the and this taxonomical treatment was followed by several genus Platanthera has been traditionally circumscribed authors like Britton and Brown (1943) and Lo¨ve and (Webb 1980, Delforge 1994). Simon (1968). However, Ames (1910), Mohlenbrock In the phylogenetic analysis of Hapeman and Inoue (1970) and Voss (1972) included the species of this group (1997), using the ITS regions, the genus Platanthera is in the genus Habenaria. On the other hand, Luer (1975) monophyletic and distant from Habenaria s. s. In this recognised this complex as the section Limnorchis within the analysis, five major clades could be recognized within genus Platanthera and this view was later accepted by Platanthera and one of them agrees with the section Hapeman and Inoue (1997) and Sheviak (1999a). Sheviak Limnorchis. However, according to these authors, the (1999b) proposed the new name P. aquilonis for the North relationships between the clades are weakly supported. American populations previously referred to as P. hyper- Simultaneously, Bateman et al. (1997), obtained a different borea, restricting the usage of the latter name to a species topology that indicates that the genus is polyphyletic, with that only occurs in Greenland and Iceland. Therefore, the P. hyperborea being the most divergent of the studied actualised name of this complex must be the ‘dilatataÁ species, although the studied material of this taxon is from aquilonis complex’. North America and thus rather belongs to P. aquilonis. The former inclusion by American botanists (Ames Later, Bateman et al. (2003) proposed to encompass the 1910, Correll 1950) of North American species of genera Piperia and Limnorchis within Platanthera. 61 Table 1. List of studied species, localities and voucher specimens. Species Locality Voucher Platanthera algeriensis Batt. & Trab. Spain, Teruel: Fortanete ORCHÁ440 Spain, Cuenca: Buenache de la Sierra ORCHÁ417 Platanthera bifolia (L.) Rich. Spain, Le´rida: Borreda` ORCHÁ415 Russia: Murmansk peninsula BLINOVAÁ87 Platanthera chlorantha (Custer) Rchb. Spain, Huesca: Plan-Chı´aPGÁ4461 Spain, Barcelona: Figols ORCHÁ407 Spain, Le´rida: Serra del Cadı´ ORCHÁ310 Platanthera micrantha Schltr. Portugal, Azores: Corvo MA 522351 Platanthera orbiculata (Pursh) Lindl. Canada, British Columbia MAF 158447 Platanthera transversa (Suksd.) R. M. Bateman USA, California: Sierra Nevada MAF 155474 Platanthera (Limnorchis) aquilonis Sheviak Canada: Pere Louis-Marie MA 202539 Platanthera (Limnorchis) dilatata (Pursh) Lindl. Canada, British Columbia MA 202535 Habenaria edwallii Cogn. Brazil, Nova-Friburgo MA 192276 Habenaria ligulata C. Schweinf. Peru, Cuzco: Moubamba MA 293985 Habenaria repens Nutt. Mexico, Guadalajara: Tesistan MA 419357 Habenaria tridactylites Lindl. Spain, Gran Canaria MAF 67090 Spain, Tenerife MAF 76096 Figure 1Á6. Morphology of the seeds in the Platanthera species. 1) P. bifolia: fusiform seed (scale bar300 mm), 2) P. bifolia: cells of the chalazal pole (scale bar100 mm), 3) P. bifolia: medial cells (scale bar100 mm), 4) P. algeriensis: apical cells (scale bar20 mm), 5) P. bifolia: periclinal walls (scale bar20 mm), 6) P. transversa: apical pole (scale bar50 mm). 62 Figure 7Á10. Morphology of the seeds in the Platanthera (Limnorchis) species. 7) P. aquilonis: clavate seed (scale bar200 mm), 8) P. aquilonis: apical and medial cells (scale bar100 mm), 9) P. dilatata: medial cells (scale bar50 mm), 10) P. aquilonis: anticlinal walls (scale bar10 mm). Previous studies on orchid seeds have demonstrated the Piperia and Platanthera, previously proposed by Luer diagnostic and phylogenetic value of certain quantitative (1975). and qualitative characters (Clifford and Smith 1969, Arditti During our research about the seed micromorphology of et al. 1979, Barthlott and Ziegler 1981, Chase and Pippen the European taxa of the subtribe Orchidinae (Orchidoi- 1988, Kurzweil 1993). Besides, recent publications (Gama- deae, Orchidaceae), we have analysed the testa cells of rra et al. 2007) emphasize the congruence between seed several species of the genus Platanthera. The aim of this micromorphology and the molecular phylogeny of the paper is to describe the qualitative and quantitative subtribe Orchidinae (Bateman et al. 2003). In yet earlier characters of the seeds, to compare them between species publications, Healey et al. (1980) studied seeds of several of the same genus from distinct geographical areas and with species of the genera Platanthera (P. dilatata, P. hyperborea, species of the genus Habenaria. Finally, seed morphology is P. saccata (Greene) Hulte´n) and Piperia (P. elegans, P. contrasted with the phylogenetic studies of Hapeman and maritima Rydb. and P. unalascensis). Their results show two Inoue (1997) and Bateman et al. (1997, 2003). morphological patterns. In Platanthera, the seeds have smooth periclinal walls, whereas in Piperia the testa cells show transversal ridges in the periclinal walls. These authors Material and methods consider that the presence or absence of reticulation is a good character to distinguish closely related genera, and Seeds were carefully taken from mature capsules in the their study support the taxonomic division of the genera field, and were later dried for at least one month and Figure 11Á12. Morphology of the seeds in Habenaria species. 11) H. repens: fusiform seed (scale bar200 mm), 12) H. tridactylites: apical cells (scale bar50 mm). 63 seeds (12). show reticulation inseeds with the smooth periclinal periclinalamong walls walls themselves. (11) in and ForHabenaria the example, fusiform types ofH. seed tridactylites coat,Cogn. not related to rest of the species of P. chlorantha Platanthera algeriensis on the seed coat were found. All the European species (i.e. 64 the two species ofnumber the ‘ of cellssize along between the the longitudinal axis speciesembryo is studied fills higher the (Table central in testa area. 2). and embryo Only,lamella are in the brown the in anticlinal9), walls colour, smooth (10). and In periclinal(8), the both with walls spherical types cell of (7, sizeshow seeds, similar clavate 8, seeds along (7), 9, the generallyBateman globose 10) longitudinal (6). in However, axis the and (7, apicallata region a 8, distinct appears in the Northanticlinal American walls without species lamella.cell This type (4), of seed periclinaland coat rectangular walls also (3), a withisodiametric truncate and transversal apical polygonal zone ridgesfusiform with (2), (5) an the seeds ending and medial (Fig. cells 1), elongate with cells ofIn the the studied basal species of zone Results (1995).