Convergent Evolution in Ophrys Kotschyi (Orchidaceae) Revisited: a Study Using Nrits and Cpigs Sequences
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Ann. Bot. Fennici 48: 97–106 ISSN 0003-3847 (print) ISSN 1797-2442 (online) Helsinki 29 April 2011 © Finnish Zoological and Botanical Publishing Board 2011 Convergent evolution in Ophrys kotschyi (Orchidaceae) revisited: a study using nrITS and cpIGS sequences Gábor Sramkó1*, Gergely Gulyás2 & Attila Molnár V.1 1) Department of Botany, University of Debrecen, Egyetem tér 1, H-4010 Debrecen, Hungary (*corresponding author’s e-mail: [email protected]) 2) BioAqua Pro Ltd., Soó Rezső út 21, H-4032 Debrecen, Hungary Received 19 Jan. 2009, revised version received 23 Mar. 2010, accepted 23 Apr. 2010 Sramkó, G., Gulyás, G. & Molnár V., A. 2011: Convergent evolution in Ophrys kotschyi (Orchidaceae) revisited: a study using nrITS and cpIGS sequences. — Ann. Bot. Fennici 48: 97–106. Convergence in the endangered European bee-orchid species Ophrys kotschyi was studied using a molecular phylogenetic approach. We sequenced the nuclear ribosomal internal transcribed spacer (nrITS) and the Rrn5–TrnR intron of the chloroplast DNA (cpIGS) to resolve conflicting interpretations of its relationships. Some authors include all morphologically similar Greek taxa in the study species, others believe that similar- ity results from convergent evolution driven by a shared pollinator. Parsimony-based network building and three approaches of phylogenetic tree reconstruction provided a basic insight into the phylogeny of the studied taxa, revealing that the inclusion of the various Greek taxa in O. kotschyi results in a polyphyletic species. This implies the consideration of the species as a narrow endemic to Cyprus, and corroborates the view that convergent evolution is responsible for apparent morphological similarity. Addi- tionally, nrITS sequencing revealed additive polymorphic sites in the nrITS, which implies significant inter-specific gene flow. Introduction flowers. Most Ophrys species supposedly have a unique pollinator species, and sympatric popu- For evolutionary plant biologists, one of the lations often differ in the preferred pollinators most exciting and fascinating genera of Europe (Paulus & Gack 1990a). This pollination system is probably the orchid genus Ophrys, which induces rapid species diversification (Cozzolino has undergone a rapid and presumably adaptive & Widmer 2005): the plants are strongly iso- radiation that has produced remarkable floral lated through prezygotic reproductive barriers by variability. This radiation is commonly thought having a specific pollinator, but following isola- to reflect its striking pollination system, which tion new taxa can emerge from shift to another occurs by sexual deceit (Schiestl et al. 1999), specific pollinator (Schiestl & Ayasse 2002). making these plants intriguing for both botanists The above scenario, although leaving open and entomologists. In this system, the flow- the question of how the descendant is perfectly ers are pollinated by naïve hymenoptera males, adapted to the new pollinator, is to our knowl- who were sexually stimulated, and thus deceived edge the best explanation of why the sexually by the female-mimicking odour bouquet of the deceptive genus Ophrys has radiated into more 98 Sramkó et al. • ANN. BOT. FeNNICI Vol. 48 than 260 supposed species (Delforge 2006). kotschyi (Soó 1926). The taxonomic treatment Although that number is undoubtedly inflated and systematic position of this species remain by inappropriate recognition of variants at spe- controversial. Though many monographers clas- cies level (Pridgeon et al. 2001), it highlights the sified the taxon differently, they all considered spectacular biodiversity of the genus. it to be distinct from others. This view was The above interpretation of the genus’ diver- changed by Sundermann (1975), who combined sity, e.g. that the diversity of described species O. cretica in O. kotschyi as subsp. cretica, thus is connected to highly specific but unstable pol- defining the taxon in a broader sense with a lination (Paulus 2006), has been repeatedly chal- wider distribution (Fig. 1). Later, Pedersen and lenged by recent works (Schiestl 2005, Pedersen Faurholdt (2002) also included another taxon, & Faurholdt 2007, Devey et al. 2008, Bateman et O. ariadnae, in O. kotschyi as subsp. ariadnae al. 2011) because of the lack of genetic isolation (Fig. 1). Although these attempts to define the among the supposed species in the genus. In fact, species O. kotschyi with subspecies were not several works (Soliva & Widmer 2003, Gulyás adopted by later, more comprehensive works, the et al. 2005, Devey et al. 2008, Pellegrino et al. latest Ophrys monograph (Pedersen & Faurholdt 2008) showed significant gene flow between 2007) presented O. kotschyi as a species with Ophrys “species”, albeit within morphologically subspecies cretica and ariadnae, thus recognis- definable groups, i.e. between closely related ing a large distributional area (Fig. 1) and total taxa. The lack of evidence for genetic isolation population size for this species. of the > 250 currently described species led It seems that the above authors disregarded Pedersen and Faurholdt (2007) to define species the study of Gölz and Reinhard (1985) which, much more widely than Delforge (2006). On the based on floral morphometrics, proved the sta- other hand, those authors neglected molecular tistical distinctness of O. kotschyi from species phylogenetic information, and so developed their of the O. reinholdii group, to where the other system without referring to the phylogenetic relevant taxa (O. cretica and O. ariadnae) belong background provided by Bateman et al. (2003). (Delforge 2006, Devey et al. 2008). Gölz and More recently, Devey et al. (2008, 2009) pro- Reinhard (1985) invoked convergence driven vided the deepest insight so far into the phylog- by the same pollinator to interpret the striking eny of these plants by sequencing the nrITS and morphological similarity of O. kotschyi and the cpDNA trnD–trnT IGS, and by generating AFLP other taxa. Indeed, all taxa currently included in data from 85 putative species. They found nrITS O. kotschyi are pollinated by bees of the genus to be the most valuable source of information on Melecta (Paulus & Gack 1990b), whereas the phylogenetic tree reconstruction in Ophrys. The other presumed relatives of O. kotschyi from the phylogenetic tree presented had reliable support O. umbilicata group are pollinated by bees of the on the “spine” of the tree, but the “tips” (i.e. genus Eucera (Paulus & Gack 1990a). relationship between the currently defined spe- A clear picture on the taxonomic state of O. cies) remained unresolved; also, many morpho- kotschyi is especially important, because it is one logically similar species were segregated among of the four Ophrys species currently listed in the clades. This finding was interpreted as evidence annexes of European Union’s Habitats Directive of a high level of hybridisation, and of the limi- (92/43/EEC). In fact, the experts on the spe- tations of a morphological species-concept that cies (Baumann & Künkele 1994, Kreutz 2004) is presently widely applied in the genus — a reported that fragmented, very small populations concept that, in effect, neglects the problem of occur in Cyprus. Although the species can be morphological convergence, i.e. convergence in found on the island frequently (R. M. Bateman flower morphology toward similar pollinators. in litt. and our pers. obs.), only a few individuals The recognition of convergence could be occur at each site, so the perceived threat to the crucial for distinguishing between morphologi- species can be justified. cally similar, but genetically isolated species A better understanding of the taxonomic (Avise 2004). One species likely to be influenced status and species delimitation can be crucial by the phenomenon of convergence is Ophrys to plan an adequate conservation strategy of ANN. BOT. FeNNICI Vol. 48 • Convergent evolution in Ophrys kotschyi revisited 99 Ophrys kotschyi Ophrys kotschyi Ophrys kotschyi subsp. cretica subsp. ariadnae subsp. kotschyi Fig. 1. Appearance and distribution range of Ophrys kotschyi and its subspecies as defined by Pedersen and Faurholdt (2007). Original photo- graphs and drawing by the authors. endangered species (Mace 2004), and is espe- Material and methods cially important in groups such as European orchids, where “taxonomic inflation” (Isaac et Plant material al. 2004) is caused by geopolitical bias in sys- tematics (Pillon & Chase 2007). Our present Field-collected leaf-pieces from populations of paper focuses on one outcome of our molecular O. kotschyi and all its presumed relatives, plus survey of the genus Ophrys, namely the impli- an additional population of O. apifera as out- cations for taxonomy and conservation conse- group (Table 1) were sampled. Although the quences concerning the endangered Ophrys kot- work of Delforge (2006) is more comprehensive, schyi. nrITS is one of the most widely applied its taxonomic treatment of the genus was repeat- markers in plant molecular systematics (Álvarez edly criticised (Pridgeon et al. 2001, Pedersen & Wendel 2003), and also seems to be the most & Faurholdt 2002, Devey et al. 2008, Devey et powerful tool in the molecular systematics of al. 2009), therefore the nomenclature and thus Ophrys (Devey et al. 2008). Here, we apply the the taxonomic treatment of the latest Ophrys sequencing of the ribosomal ITS of the nucleus monograph by Pedersen and Faurholdt (2007) is and the Rrn5–TrnR intron of the chloroplast applied here. DNA (hereafter also referred to as