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 ()

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 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, MAF 67090 Spain, 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 stored in small envelopes. Others were obtained from the SD herbaria MA (Real Jardı´n Bota´nico, Madrid) and MAF 9 12.57 13.60 19.43 12.57 18.37 15.92 9.22 10.55 12.63 10.31 14.82 12.51 15.69 17.76 (Facultad de Farmacia, Madrid), and the private collec- 17.19 9.69 17.83 9 9 9 9 9 9 9 9 9 9 9 9 9 9 tions of Ilona Blinova (BLINOVA, Polar-Alpine Bot. 9 9 9 m) mean Gard. Inst., Murmansk, Russia), Pablo Gala´n Cela (PG, m Univ. Polite´cnica de Madrid, Spain) and Roberto Gama- rra (ORCH, Univ. Auto´noma de Madrid, Spain). A list of the sources of the specimens with localities is given in

Table 1. SD Width ( The samples were mounted on SEM stubs, sputter- 9 26.20 102.33 25.05 125.33 23.11 164.00 20.4121.81 104.33 104.00 30.5221.59 100.00 16.48 85.00 16.9219.3121.52 84.33 13.84 99.33 16.0727.99 102.67 52.67 103.67 103.00 206.67 coated with gold (SEM coating system, Bio-Rad SC 502), 26.95 119.00 20.7521.37 81.67 172.33 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9

and viewed with a Philips XL-30 or a Hitachi S-3000N m) mean SEM at an acceleration voltage of 20 kV. Morphological m observations were undertaken, particularly of the seed coat and the ornamentation of the periclinal and anticlinal walls. Size and colour of seeds and embryos, mounted using

polyvinyl alcohol, were recorded from at least 30 seeds per SD Length ( species under the light microscope. Seed and embryo colour 9 0.62 149.33 0.57 177.00 0.28 238.33 0.72 166.67 0.76 163.67 0.380.56 154.67 0.520.48 137.33 0.560.270.70 131.33 1.00 140.67 0.23 157.33 149.67 155.33 181.00 303.67 are described in subjective terms. 1.060.56 167.33 0.46 146.00 229.67 9 9 9 9 9 9 9 9 9 9 9 9 9 9 The terminology and micromorphological methods were 9 9 9 adopted from Arditti et al. (1979), Barthlott and Ziegler (1981), Chase and Pippen (1988) and Molvray and Kores (1995). SD L/W ratio mean 9 Results 11.59 4.08 32.09 2.30 17.16 3.94 22.53 4.11 19.15 5.18 17.229.43 3.24 16.8817.11 4.03 23.0013.00 4.14 21.21 3.29 18.4324.64 3.97 2.24 4.05 7.28 2.07 18.71 4.94 9.4836.03 4.07 3.04 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9

In the studied species of Platanthera, two different patterns m) mean on the seed coat were found. All the European species (i.e. m Platanthera algeriensis Batt. & Trab., P. bifolia (L.) Rich., P. chlorantha (Custer) Rchb. and P. micrantha Schltr.) have fusiform seeds (Fig. 1), with cells of the basal zone isodiametric and polygonal (2), the medial cells elongate SD Width ( and rectangular (3), a truncate apical zone with an ending 9 52.39 117.00 46.09 266.33 46.36 147.00 57.77 143.00 74.25 130.00 49.0839.47 149.67 42.1923.96 99.00 48.5117.78 134.67 38.00 127.33 64.3629.66 176.67 121.00 139.67 139.00 261.67 cell (4), periclinal walls with transversal ridges (5) and 73.37 143.67 33.4750.00 99.67 245.00 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9 9

anticlinal walls without lamella. This type of seed coat also m) mean appears in the North American species Platanthera orbicu- m lata (Pursh) Lindl. and P. transversa (Suksd.) R. M. Bateman (6). However, Platanthera dilatata and P. aquilonis show clavate seeds (7), generally globose in the apical region (8), with cell size similar along the longitudinal axis (7, 8, 9), smooth periclinal walls (7, 8, 9, 10) and a distinct lamella in the anticlinal walls (10). In both types of seeds, 7 574.00 87 693.67 10 401.67 730.00 Á testa and embryo are brown in colour, and the spherical Á Á Á 6 (7) 472.33 6 (7) 606.00 9 572.00 8 662.67 7 (8)7 481.00 395.33 77134 (5)556 550.00 411.67 690.00 268.00 552.00 999.00 537.33 Á Á Á Á Á Á embryo fills the central area. Á Á Á Á Á Á Á There are no numerical differences in seed and embryo 87 4

size between the species studied (Table 2). Only, the Á 417 5 407 (3) 4 440415 4 310 (4) 5 5 Á Á number of cells along the longitudinal axis is higher in Á Á Á the two species of the ‘dilatataÁaquilonis complex’ than the 4461 4 Á PG ORCH ORCH MAF 76096 4 BLINOVA rest of the species of Platanthera. ORCH ORCH ORCH MA 522351MAF 158447MAF (3) 155474 4 4 MA 202539 4 MA 202535MA (6) 192276 7 MA 8 29385MAF 3 419357MAF 67090 3 3 4 Four species of the genus Habenaria (i.e. H. edwallii Cogn., H. ligulata C. Schweinf., H. repens Nutt., H. tridactylites Lindl.) were analysed. They show different types of seed coat, not related to Platanthera. Within Habenaria, the micromorphological patterns are distinct among themselves. For example, H. repens have filiform seeds with smooth periclinal walls (11) and H. tridactylites show reticulation in the periclinal walls in the fusiform

seeds (12). Table 2. Average sizes of seeds andSpecies embryos in the studied species. Platanthera algeriensis Platanthera bifolia Platanthera chlorantha Platanthera micrantha VoucherPlatanthera orbiculata NumberPlatanthera of transversa cellsPlatanhera aquilonis Platanthera dilatata LengthHabenaria ( edwallii Habenaria ligulata Habenaria repens Habenaria tridactylites

64 Discussion Buttler, K. P. 2001. of Orchidaceae tribus Orchideae, a traditional approach. Á J. Eur. Orch. 33: 7Á32. Healey et al. (1980) observed two distinct seed morpholo- Case, F. W. 1987. Orchids of the western Great Lakes region. gical patterns for the studied species of Platanthera and Á Cranbook Inst. Sci. Chase, M. W. and Pippen, J. S. 1988. Seed morphology in the Piperia. The seeds of the genus Piperia agree with the Oncidiinae and related subtribes (Orchidaceae). Á Syst. Bot. samples studied by us in the European species of 13: 313Á323. Platanthera. However, the three species of Platanthera (P. Clifford, H. T. and Smith, W. K. 1969. Seed morphology and dilatata, P. hyperborea, P. saccata) studied by these authors, classification of Orchidaceae. Á Phytomorphology 19: 133Á all of which belong in the ‘dilatataÁaquilonis complex’, have 139. smooth seeds, as also shown in the present study. Correll, D. S. 1950. Native orchids of North America north of During our research about seed micromorphology of the Mexico. Á Stanford Univ. Press. European genera in the subtribe Orchidinae, we have Delforge, P. 1994. Guide des Orchide´es d’Europe, d’Afrique du observed a strong agreement between the qualitative Nord et du Proche-Orient. Á Delachaux et Niestle´. characters and the genera (Gamarra et al. 2007). According Dressler, R. L. 1993. Phylogeny and classification of the orchid to this criterion, Platanthera has reticulate seeds with a family. Á Timber Press. truncate apical zone but without lamella. This pattern is Gamarra, R. et al. 2007. Seed micromorphology in the genus also found within the species previously referred to Piperia. Neotinea Rchb. f. (Orchidaceae, Orchidinae). Á Bot. J. Linn. Our results largely support the result of the molecular Soc. 153: 133Á140. Á phylogeny published by Bateman et al. (2003), who nested Hapeman, J. R. and Inoue, K. 1997. pollinator interactions and floral radiation in Platanthera (Orchidaceae). Á In: the genus Piperia within Platanthera. On the contrary, the Givnish, T. J. and Sytsma, K. J. (eds), Molecular evolution studied species of the ‘dilatataÁaquilonis complex’ have and adaptive radiation. Á Cambridge Univ. Press, pp. 433Á smooth seeds with a rounded apical zone and presence of 454. lamella. This pattern supports splitting off this group from Healey, P. L. et al. 1980. Morphometry of orchid seeds. III. Platanthera to include it in the segregate genus Limnorchis, Native California and related species of Goodyera, Piperia, and these results are consistent with the paraphyletic Platanthera and Spiranthes. Á Am. J. Bot. 67: 508Á518. Platanthera obtained by Bateman et al. (1997) and with Kurzweil, H. 1993. Seed morphology in southern African the separate clade formed by Limnorchis in the analysis by (Orchidaceae). Á Plant Syst. Evol. 185: 229Á Hapeman and Inoue (1997). 247. 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