O. Lutea × O. Tarentina
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Ann. Bot. Fennici 45: 61–67 ISSN 0003-3847 (print) ISSN 1797-2442 (online) Helsinki 29 February 2008 © Finnish Zoological and Botanical Publishing Board 2008 Morphological and molecular investigation of the parentage of Ophrys ¥ circlarium (O. lutea ¥ O. tarentina), a new hybrid orchid from Italy Giuseppe Pellegrino*, Francesca Bellusci & Aldo Musacchio Dipartimento di Ecologia, Università della Calabria, I-87036 Arcavacata di Rende, CS, Italy (*corresponding author’s e-mail: [email protected]) Received 30 Nov. 2006, revised version received 19 Feb. 2007, accepted 21 Feb. 2007 Pellegrino, G., Bellusci, F. & Musacchio, A. 2008: Morphological and molecular investigation of the parentage of Ophrys ¥ circlarium (O. lutea ¥ O. tarentina), a new hybrid orchid from Italy. — Ann. Bot. Fennici 45: 61–67. This study reports a new hybrid combination Ophrys ¥ circlarium Pellegrino, hybr. nov., which derives from two highly divergent species, O. lutea (O. fusca-lutea com- plex) and O. tarentina (O. sphegodes complex). These two species grow sympatrically in the north of Calabria region (southern Italy), in a stand where two potential hybrid individuals were found during a floristic investigation. Two of the 16 morphometric characters analyzed were intermediate relative to those of the potential parental spe- cies. PCR-RFLP analysis of nuclear ribosomal DNA Internal Transcribed Spacer sequences (rDNA ITS) confirmed that the two specimens are hybrids of the two co- occurring Ophrys species. Key words: ITS, new hybrids, Ophrys lutea, Ophrys tarentina, rDNA Introduction (Van der Cingel 1995). Noticeably, even within the sexually decep- Natural hybridization among vascular plants is a tive genus Ophrys, known for its peculiar pollina- relatively common phenomenon and has played tor specificity, interbreeding is frequent (Nelson a significant role in their evolution (Grant 1981). 1962, Danesch & Danesch 1972, Baumann & Although it is not clear whether spontaneous Künkele 1982, Delforge 2001) and likely contrib- hybrid formation is a typical feature of some utes to the difficulties in the taxonomy. Indeed, plant groups, it is likely that interbreeding might the number of Ophrys species is on the increase be frequent among still divergent taxa and, thus, (21, 68, 148, 215 or 250 species according to within the more recent and advanced families Nelson 1962, Baumann & Künkele 1988, Del- of the angiosperms. In this respect, a suitable forge 1994, 2001, 2005, respectively) and, in example is represented by the Mediterranean the course of time, several classical binomial food deceptive orchids, like Orchis, Anacamptis combinations, such as O. bertolonii, O. fuci- and Dactylorhiza, in which frequent hybridiza- flora, O. sphegodes and O. fusca, have become tion has been documented (Delforge 2001) and aggregates of numerous taxa, weakly differing attributed to their unspecific pollination system in their morphological traits, flowering period 62 Pellegrino et al. • ANN. BOT. FENNICI Vol. 45 and pollinators. In addition, hybridization events (SS92), a few kilometres north of Cerchiara di between Ophrys aggregates may be facilitated by Calabria (northern Calabria region, Italy). The their shared ploidy level (2n = 36) (Greilhuber & two hybrid flower spikes were likely emerg- Ehrendorfer 1975, Bianco et al. 1989). ing from a single rootstock suggesting that the In the course of a floristic investigation in smaller one was a recent clonal derivate of the the northern Calabria region, the authors vis- other (Fig. 1). No further similar specimens were ited a site where Ophrys tarentina was found detected in the vicinity and thus we did not col- sympatric with O. lutea. At this site, two orchid lect any of the two plants for herbarium. specimens were noticed for their strikingly unu- To minimise disturbance of the orchids, veg- sual flowers, which appeared to combine traits etative and floral measurements were made in the of the two co-occuring species and thus were field only on the more phenologically advanced suspected to be their hybrid progeny. This find- individual of the two hybrids and on ten average- ing was unexpected because the two presumed sized individuals of each putative parent. For parental orchid species possess different pollina- each specimen, eight qualitative and eight quan- tion strategies and belong to different sections of titative diagnostic floral traits (Table 1) were Ophrys (Godfery 1928, Delforge 1994). Ophrys evaluated on the second flower from the bottom tarentina belongs to the section Euophrys and its of the inflorescence. Quantitative measures were pollinators are bees of the genus Osmia, which made to the nearest 1 mm using a ruler. may carry pollinaria on their head. Ophrys lutea For molecular analysis, one fresh cauline leaf on the other hand belongs to the section Pseu- of both presumed hybrids and three specimens dophrys and is pollinated by several species of of each sympatric Ophrys species were sampled the bee genus Andrena, which remove pollinaria and stored in silica gel. For total DNA extraction with their abdomen (Paulus & Gack 1990a, approx. 4 cm2 of each leaf were separately pes- 1990b, 1990c). tled in a 2-ml eppendorf using 500 µl of isola- We considered this finding worthy of report- tion buffer (2% hexadecyltrimethyl ammonium ing and sampled the presumed hybrid and both bromide). Successive procedures were in accord- parent species to facilitate subsequent morpho- ance with Doyle and Doyle (1987) protocol. To metric and molecular investigations. Molecular determine whether the intermediate plants were approaches have successfully been applied to actually of hybrid origin, rDNA Internal Tran- study taxonomic position and parental lineage scribed Spacer sequences were obtained from of hybrid specimens as well as to characterize the putative parental taxa. The internal ribos- gene flow in hybrid zones (Bateman & Holl- omal spacers (ITS 1 and ITS 2) were ampli- ingsworth 2004, Pellegrino et al. 2005). For fied with polymerase chain reaction (PCR). The Ophrys, a molecular study of presumed hybrids ITS1 was amplified using a pair of primers, was undertaken only once (Gulyás et al. 2005) which anneal in the 3´ region of the 18S (5´- to the best of our knowledge. Therefore, the GAGAAGTCGTAACAAGGTTTCCG-3´) and main goals of this study were (1) to furnish a in the 5´ region of the 5.8S (5´-ATCCTGCAAT- morphological description of the relevant fea- TCACACCAAGTATCG-3´). The ITS2 region tures of the hybrid plants in comparison with was amplified using a pair of primers annealing the co-occurring Ophrys species, (2) to ascertain in the 3´ region of the 5.8S (5´-TTGCAGAATC- with molecular analysis the hybrid origin of the CCGTGAACCATCG-3´) and in the 5´ region two specimens, and (3) to correctly identify their of the 25S (5´-CCAAACAACCCGACTCGTA- parental species. GACAGC-3´). All PCR reactions of 100 µl final volume contained 2 µl DNA template, 100 µM of each dNTP, 0.3 µM of each primer, 2 units Material and methods Taq polymerase, 2 µM MgCl2 and 10 µl reaction buffer. The site hosting O. tarentina, O. lutea and their PCR reactions were conducted in a thermal presumed hybrid was visited on 20 April 2005. cycle (Perkin Elmer 2600) for 30 cycles. Initial It is located along the right-hand side of the road conditions were as follows: 30 sec denaturation ANN. BOT. FENNICI Vol. 45 • Ophrys ¥ circlarium: a new orchid hybrid 63 Fig. 1. Ophrys ¥ circlarium and its parents. — A: The two inflorescences of O. ¥ circlarium. — B: Flowers of O. lutea. — C: Flower of O. ¥ circlarium. — D: Flower of O. tarentina. All taken at the Cerchiara di Calabria, Italy. at 94 °C, 1 min annealing at 55 °C, 45 sec exten- DNA sequencer (Applied Biosystems, Foster sion at 72 °C; extension time was increased by City, CA, USA). 3 sec/cycle; extension was further prolonged The sequences were examined using Gene- for 7 min at the end of the last cycle. Amplified Jockey to find a restriction site that would dis- ITSs were electrophoretically separated on a 2% tinguish them using Polymerase Chain Reac- agarose gel. A 100 base pair (bp) ladder (Phar- tion-Restriction Fragment Length Polymorphism macia Biotech) was used as a molecular weight (PCR-RFLP). This approach allows the exam- marker. ination of a heterozygous individual (e.g., a Amplified fragments were sequenced in both hybrid) without the necessity of cloning and sub- directions using a modification of the Sanger sequently sequencing several ITS clones (hetero- dideoxy method as implemented in a double zygous individuals give overlapping traces from stranded DNA cycle sequencing system with direct sequencing that are often difficult to inter- fluorescent dyes. Sequence reactions were then pret). Restriction enzyme MaeIII, which cuts at run on a 373A Applied Biosystems Automated GTNAC, differentiated the putative parental taxa 64 Pellegrino et al. • ANN. BOT. FENNICI Vol. 45 due to the presence of a G/A substitution about plants exhibited six features typical of one or 77 base pairs into the ITS 1 sequence (GTTGC the other co-occurring Ophrys species, two char- in O. lutea, GTTAC in O. tarentina); while AluI, acters are more or less intermediate, while two which cuts at AGCT, showed two nucleotide other characters lie outside the values of both substitution C/T, about 154 base pairs (AGCT in parents (Table 1). O. lutea, AGTT in O. tarentina), and G/T, about As regards a structure of diagnostic value, 203 base pairs (AGCT in O. lutea, ATCT in O. the labellum of the hybrid plants is 3-lobed and tarentina) into the ITS 2 sequences. marginally yellow as that of O. lutea, while its Fragments of hybrids and parental species H-shaped speculum looks like that of O. taren- (100 ng) were then digested in a final volume tina. The qualitative features of the sepals and of 20 µl with selected restriction endonucleases lateral petals are essentially those observed in O. (1U/µg DNA), according to the manufacturer’s tarentina.