Seed micromorphology in Dactylorhiza Necker ex Nevski (Orchidaceae) and allied genera

Turkish Journal of Botany Turk J Bot (2015) 39: 298-309 © TÜBİTAK doi:10.3906/bot-1401-66 http://journals.tubitak.gov.tr/botany/ Research Article Seed micromorphology in Dactylorhiza Necker ex Nevski (Orchidaceae) and allied genera 1, 2 3 1 1 Roberto GAMARRA *, Pablo GALÁN , Henrik Ærenlund PEDERSEN , Emma ORTÚÑEZ , Ernesto SANZ 1 Department of Biology, Faculty of Sciences, Autonomous University of Madrid, Madrid, Spain 2 Department of Vegetal Production: Botany and Vegetal Conservation, E.U.I.T. Forestal, Polytechnic University of Madrid, Madrid, Spain 3 Botanical Garden, Natural History Museum of Denmark, University of Copenhagen, Copenhagen K, Denmark Received: 23.01.2014 Accepted: 06.08.2014 Published Online: 16.03.2015 Printed: 10.04.2015 Abstract: Seeds of 21 taxa of the genera Dactylorhiza (incl. Coeloglossum), Gymnadenia (incl. Nigritella) and Pseudorchis were examined by light microscope and SEM. Qualitative and quantitative characters were analyzed. In Dactylorhiza, the seeds are fusiform, but some populations of D. viridis show clavate seeds. According to the ornamentation of the periclinal walls, 3 types of seeds are recognized in this genus. The considerable variation in the seed coat ornamentation pattern in the taxa belonging to majalis, maculata, and praetermissa groups of the genus Dactylorhiza is congruent with the genetic processes that occurred during the history of this genus. Gymnadenia shows clavate seeds with stout and straight to slightly curved anticlinal walls, although these are straight to wavy in some taxa considered previously as Nigritella. These taxa also show low values of seed length. Pseudorchis has fusiform seeds without ornamentation in the periclinal walls and fine anticlinal walls. Our study supports the inclusion of the former genus Coeloglossum in Dactylorhiza. Key words: Dactylorhiza, Coeloglossum, Gymnadenia, Nigritella, Pseudorchis, SEM, testa, periclinal walls 1. Introduction Within the tribe Orchideae (Orchidaceae), one of the most controversial groups belongs to the so-called “digitate tuber” clade (Pridgeon et al., 1997), which includes the genera Dactylorhiza Necker ex Nevski, Gymnadenia R.Br., and Pseudorchis Ség. In the molecular phylogenetic analyses published by Pridgeon et al. (1997) and corroborated by Bateman et al. (2003), Coeloglossum Hartm. and Nigritella Rich. were nested in Dactylorhiza and Gymnadenia, respectively. Accordingly, Coeloglossum and Nigritella were synonymized in Genera Orchidacearum (Pridgeon et al., 2001). However, based on either morphological or molecular data, other authors (Aedo and Herrero, 2005; Delforge, 2006; Devos et al., 2006) have maintained them as independent genera. Dactylorhiza is distributed from Europe and the Mediterranean across temperate Asia to the Himalayas, Japan, and Alaska (Pridgeon et al., 2001; Sheviak et al., 2002). Generally, Dactylorhiza is considered a complex genus, and there is still no consensus about the delimitation of species and their classification into aggregates or groups (Averyanov, 1990; Delforge, 2006; Pillon et al., 2007). In his extensive review of the genus, Averyanov (1990) recognized 75 species within 4 sections, subdivided * Correspondence: : 298 into subsections and aggregates. Pridgeon et al. (2001) considered about 50 species in the total range of the genus, including the only species of Coeloglossum in it. Delforge (2006) reported 60 species for Europe, North Africa, and the Middle East, within 7 groups (iberica, sambucina, incarnata, majalis, traunsteineri, praetermissa, maculata). However, according to the species concept advocated by Pedersen (1998), far fewer species should be recognized (Pillon et al., 2006; Pedersen 2010). Even the overall structure in some of the currently prevailing classifications is in conflict with the patterns that have emerged from recent molecular studies. For example, the members of an allotetraploid complex in Greece that Hedrén et al. (2007), based on allozymes, AFLPs, and plastid DNA data, found to be extremely closely related (partly even synonymous) were assigned to 3 different species groups in Delforge (2006). Nowadays, the inclusion of Coeloglossum viride (L.) Hartm. in the genus Dactylorhiza is generally accepted. This species has a holarctic-boreal distribution, extending from North America to Japan (Delforge, 2006). Morphologically, it is distinguished from the rest of the genus Dactylorhiza by the spur, which is short and nectariferous, and the petals and sepals, which are joined GAMARRA et al. / Turk J Bot in a connivent helmet, while the labellum is bifid and the rostellum bears 2 bursicles rather than 1 (Szlachetko and Rutkowski, 2000; Aedo and Herrero, 2005). Gymnadenia comprises about 30 species distributed in Eurasia and along the western coast of North America (Pridgeon et al., 2001). While members of Gymnadenia s.s. have the labellum oriented downwards, taxa with the labellum oriented upwards have frequently been treated as a distinct genus, Nigritella (Teppner and Klein, 1998; Hedrén et al., 2000). Pseudorchis has a circumboreal distribution, from Canada to the Kamchatka Peninsula (Pridgeon et al., 2001). Only P. albida (L.) A. & D.Löve is recognized, but its 2 subspecies (Reinhammar, 1998) are sometimes treated at species level. Based on molecular phylogenetic analyses, Pridgeon et al. (1997, 2001) and Bateman et al. (2003) recognized Pseudorchis as an independent genus, more closely related to Platanthera Rich. than Gymnadenia, where it was assigned by Delforge (2006). From a cytogenetic point of view, all the genera mentioned above have the same chromosome base number (x = 20), and different mechanisms of aneuploidy and speciation processes by hybridization and polyploidy have been reported (Cauwet-Marc and Balayer, 1984; Amich et al., 2007). Apart from the yet unknown Coeloglossum × Nigritella and Coeloglossum × Pseudorchis, all possible hybrid combinations between these putative genera have been recorded in the field (Aedo and Herrero, 2005; Delforge, 2006; Bateman, 2009). A particularly remarkable case is the so-called Gymnigritella runei Teppner & E. Klein - tetraploid Gymadenia conopsea × Nigritella nigra hybrids that form self-reproducing populations in northern Sweden (Hedrén et al., 2000). Based on data from diverse molecular markers, patterns of allopolyploidization have also been well documented within Nigritella (Hedrén et al., 2000) and Dactylorhiza (Hedrén, 1996; Pedersen, 2006; Hedrén et al., 2007; Pillon et al., 2007). Previous studies on orchid seeds have demonstrated the taxonomic value of quantitative and qualitative characters (Clifford and Smith, 1969; Barthlott, 1976; Arditti et al., 1979; Chase and Pippen, 1988). In addition, recent publications (Tsutsumi et al., 2007; Gamarra et al., 2007, 2008, 2010, 2012) have emphasized the strong correlation between seed micromorphology and molecular analyses, as reconstructed using DNA sequence data. Toh (...truncated)