AOBPreview originally published online on May 16, 2006
Annals of Botany 2006 98(1):237-244; doi:10.1093/aob/mcl094
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Exine Micromorphology of Orchidinae (Orchidoideae, Orchidaceae): Phylogenetic Constraints or Ecological Influences?
1 Orto Botanico and 2 Dipartimento delle Scienze Biologiche, Università di Napoli Federico II, Naples, Italy and 3 Insitute of Systematic Botany, Ludwig Maximilians University, Menzinger Strasse 67, 80638 Munich, Germany
* For correspondence. E-mail kocyan{at}lrz.uni-muenchen.de
Received: 31 January 2006 Returned for revision: 2 March 2006 Accepted: 13 March 2006 Published electronically: 16 May 2006
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUPPLEMENTARY INFORMATIONACKNOWLEDGEMENTSLITERATURE CITED |
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• Background and Aims Pollen characters have been widely used in defining evolutionary trends in orchids. In recent years, information on pollination biology and phylogenetic patterns within Orchidinae has become available. Hence, the aim of the presented work is to re-evaluate exine micromorphology of Orchidinae in light of recent phylogenetic studies and to test whether pollen micromorphology strictly depends on phylogenetic relationships among species or whether it is influenced by the marked differences in pollination ecology also reported among closely related species.
• Methods Pollen sculpturing of 45 species of Orchidinae and related taxa was investigated using scanning electron microscopy. To cover potential intraspecific variation, several accessions of the same species were examined.
• Key Results Orchidinae show remarkable variation in exine sculpturing, with a different level of variation within species groups. In some genera, such as Serapias (rugulate) and Ophrys (psilate to verrucate), intrageneric uniformity corresponds well to a common pollination strategy and close relationships among species. However, little exine variability (psilate–scabrate and scabrate–rugulate) was also found in the genus Anacamptis in spite of striking differences in floral architecture and pollination strategies. A larger variety of exine conditions was found in genera Dactylorhiza (psilate, psilate–scabrate and reticulate) and Orchis s.s. (psilate, reticulate, perforate–rugulate and baculate) where no unequivocal correspondence can be found to either phylogenetic patterns or pollination strategies.
• Conclusions Changes in pollen characteristics do not consistently reflect shifts in pollination strategy. A unique trend of exine evolution within Orchidinae is difficult to trace. However, the clades comprising Anacamptis, Neotinea, Ophrys and Serapias show psilate to rugulate or scabrate pollen, while that of the clade comprising Chamorchis, Dactylorhiza, Gymnadenia, Orchis s.s., Platanthera, Pseudorchis and Traunsteinera ranges from psilate to reticulate. Comparison of the data with exine micromorphology from members of the tribe Orchidieae and related tribes suggests a possible general trend from reticulate to psilate.
Key words: SEM, exine, phylogeny, pollination biology, pollen, orchids
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUPPLEMENTARY INFORMATIONACKNOWLEDGEMENTSLITERATURE CITED |
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Orchidaceae are known for their large diversity in pollen morphology (Schill and Pfeiffer, 1977
; Burns-Balogh, 1983; Freudenstein and Rasmussen, 1997). This diversity is present at different levels: variability in packaging of pollen in pollinia, in pollen wall structure, as well as in pollen surface sculpturing (Burns-balogh and Hesse, 1988). Hence, the structure and shape of pollinia have been used frequently for orchid classification (Burns-Balogh, 1983; Burns-Balogh and Funk, 1986; Rasmussen, 1999, and citations therein).
Pollen characters have been considered useful in defining evolutionary trends in plant families such as Araceae (Grayum, 1986), Callitrichaceae (Osborn et al., 1991, 2001; Osborn and Philbrick, 1994), Hydrocharitaceae (Tanaka et al., 2004), Fabaceae (Papilionoideae; Ferguson and Skvarla, 1982), Scrophulariaceae (Pedicularis; Wang et al., 2003) and Orchidaceae (Erdtman, 1960; Caspers and Caspers, 1976; Cronquist, 1981; Burns-Balogh, 1982, 1983; Burns-Balogh and Bernhardt, 1985; Averyanov, 1990; Dressler, 1993; Freudenstein and Rasmussen, 1999). In particular, the exine micromorphology has been frequently used as a reference character in taxonomical and phylogenetic analyses and, in orchids, this pollen character shows a remarkable diversity among closely related taxa (Schill and Pfeiffer, 1977). However, in some orchid subtribes, such as Disinae, pollen exine sculpture patterns were found to be too variable to allow a distinction even at the species level or, as in Coryciinae, too uniform for taxonomic resolution level (Chesselet and Linder, 1993). Nevertheless, at the generic and subtribal level, the same data produced phylogenetic information (Chesselet and Linder, 1993). Similar contrasting patterns were found by Schill and Pfeiffer (1977) in the genera Ophrys and Orchis (both of the subtribe Orchidinae): Ophrys had a very uniform pollen surface, but Orchis (s.l.) showed an astonishing pollen diversity.
There have been several attempts to correlate pollen surface sculpturing and pollen stratification with pollination strategies (Hesse, 2000), and certain general patterns seem to be well established: elaborate pollen sculpturing is often correlated with entomophily, and psilate pollen grains may be characteristic for anemophilous or hypohydrophilous plants (Walker, 1974). Tanaka et al. (2004) showed that there is a strong correlation between pollen morphology and pollination mechanisms in Hydrocharitaceae where entomophily seems to be the plesiomorphic state and hypohydrophily is the apomorphic state. Morphologically, the entomophilous pollen grains can be distinguished by the conspicuous spines and a two-layered exine, whereas hypohydrophilous pollen showed a reduced exine structure with a smooth surface.
Differences of pollen grains in cases of extremely different pollination mechanisms are functionally understandable and phylogenetically traceable. However, it may be more difficult to trace evolutionary tendencies within plant groups with similar pollination strategies. This was shown by Wang et al. (2003) in Pedicularis (Scrophulariaceae) with entomophilous pollen. They found a correlation between pollinators and corolla shape, but none with pollen characters. In orchids, the situation is complicated further by the fact that the pollen grains are not directly attached on the pollinator's body. The whole pollen mass (pollinium) is placed on a stalk (caudiculum or stipes) that ends on the viscidium which is responsible for the attachment on the pollinator (Dressler, 1993).
In an attempt to explain the evolution of orchid pollen surfaces, Burns-Balogh (1983) proposed that the pollen surface characteristics of Orchidaceae can be interpreted as the result of reversal processes going from primitive tectate–perforate to the derivate intectate condition with a reversion to a tectate–imperforate condition both in Epidendroideae and in Neottioideae (today in Epidendroideae). Orchidoideae show conditions with tectate imperforate, semitectate or intectate exine and the general absence of the foot layer. The evolution of exine in Orchidoideae as detailed in Burns-Balogh (1983) implicates a series of exine reductions going from a tectate–imperforate condition, with baculae maintaining lateral extensions residual from tectum demolition, to a semitecate–tectate condition with exine globules laying on the endexine. The scheme proposed by Burns-Balogh has been the subject of criticism, however, due to the small sampling within the family (Zavada, 1990; Pridgeon, 1999).
Nevertheless, independently of any evolutionary reconstruction, Orchidoideae have the widest range of pollen features in the orchid family (Hesse and Burns-Balogh, 1984). Pollen grains of orchidoids vary subtly in surface sculpture among species (Schill and Pfeiffer, 1977) but, according to Bateman et al. (2003), no clear phylogenetic patterns are evident. In contrast, Pridgeon (1999) stated that the pollen heterogeneity of Orchideae may have promising systematic utility. The main limitation in recognizing a phylogenetic signal in pollen characters of orchids depends on the influence that ecological factors, such as differences in pollination strategies, may have on the pollen morphology in spite of evolutionary affinities among taxa. In fact, often, traits pertaining to floral morphology may be interpreted as the results of pollinator-mediated selection and have more ecological than phylogenetic implications.
Clearly, an independently acquired knowledge of species relationships may help in elucidating correlations between pollen morphology and pollination strategies. In orchids, in particular in Orchidinae, such an attempt is still lacking, but in recent years several independent and largely congruent studies (Bateman et al., 1997, 2003; Pridgeon et al., 1997; Aceto et al., 1999; Cozzolino et al., 2001) defined the patterns of phylogenetic relationships of the subtribe Orchidinae (Orchidoideae) based on nuclear internal transcribed spacer (ITS) sequences. In particular, most members of the old genus Orchis have been split into three related genera: Anacamptis, Neotinea and Orchis (s.s.) (Bateman et al., 1997). These clades found support from karyological data and root tuber characteristics, though, in general, additional morphological synapomorphies defining these clades are still wanted (Bateman et al., 2003).
Often, information on pollination biology of Orchidaceae is relatively scarce as most orchid species are epiphytes living high up on trees, thus making it difficult to observe pollination. Information on pollination is thus more easily accessible in terrestrial orchids. In particular, Orchidinae have a substantial pollination observation record (e.g. van der Cingel, 1995; Pridgeon et al., 2001, and references therein). This makes Orchidinae an ideal candidate for a case study on the correlation of pollen characteristics and pollination biology when phylogenetic patterns are known.
The aim of this study is a re-evaluation of the exine micromorphology, based on newly produced and literature data, of allied members of the subtribe Orchidinae in the light of the recent molecular phylogenetic reconstructions of the group (Bateman et al., 2003). In particular, we are interested in ascertaining whether the variation in pollen micromorphology reflects phylogenetic relationships among species or whether it may be significantly influenced by the striking difference in pollination ecology also found among closely related species (Neiland and Wilcock, 1994; Aceto et al., 1999; Cozzolino et al., 2001).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUPPLEMENTARY INFORMATIONACKNOWLEDGEMENTSLITERATURE CITED |
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Plant material
Forty-five species of Orchidinae, of which three species were formerly included in Habenariinae (Table 1), have been investigated. Pollinaria were collected from plants cultivated at the Botanical Garden of Naples, Italy or in the wild (see Table 1). To cover intraspecific variation, pollinaria of 2–6 individuals per species were sampled. Sampling was conducted over 3 years. Pollinaria were removed by sticking the viscidium on a small piece of Parafilm pellicle.
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Taxa classification, pollination biology and pollen nomenclature
Classification of Orchidinae (including the former Habenariinae) follows those of Bateman et al. (1997
, 2003), Dressler (1993) and Pridgeon et al. (2001). Data regarding orchid pollination are reported in van der Cingel (1995) and reference therein. Pollen nomenclature follows that of the ‘Glossary of pollen and spore terminology’ (available on-line at http://www.bio.uu.nl/~palaeo/glossary/glos-int.htm).
Scanning electron microscopy (SEM)
Pollinia were fixed in FAA (formalin–acetic acid–alcohol 10 : 5: 50), dehydrated in an ethanol series, critical-point dried in liquid CO2 and sputter-coated with approx. 30 nm of gold. Alternatively, air-dried pollinia were coated with gold. We observed no differences of the investigated structures between critical-point- and air-dried material. To preserve the exine and intine, no acetolysis was carried out (Hesse and Waha, 1989). Specimens were observed under a Cambridge 250Mark3 and under a FEI-Quantas 200 ESEM, at the CISME centre, Università degli Studi di Napoli ‘Federico II’. As pollen characters may vary depending on the position on the massulae (Caspers and Caspers, 1976), we decided to compare only exine structures at the distal massulae ending to achieve maximum comparability (Fig. 1). In addition, we focused on exine structures at the outer surface of the pollen tetrad or massula, respectively, as the exine structure between the pollen grains can be largely reduced (Averyanov, 1990).
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUPPLEMENTARY INFORMATIONACKNOWLEDGEMENTSLITERATURE CITED |
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Neither intra- nor interindividual variation in exine micromorphology was observed for the species for which different accessions were examined.
SEM observations of all investigated Serapias species showed a substantial uniformity in the genus with a characteristic rugulate exine (Fig. 2; Supplementory Information, avalable online). This uniformity corresponds to the close phylogenetic relationship among all species and also to the common pollination strategy based on the mimicry of a sleeping hole for solitary insects. The autogamous S. parviflora is notable as it shows the same exine pattern as all other allogamous species.
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In the sexually deceptive genus Ophrys, SEM observations show only small differences in the exine micromorphology of investigated taxa ranging from psilate to scabrate–verrucate exine (Fig. 2; Supplementay Information). In detail: O. apifera and O. bertolonii show a psilate–scabrate to verrucate exine; in the O. fusca–lutea complex, O. fusca shows a psilate–scabrate to verrucate exine and O. lutea shows a psilate–scabrate exine; O. sphegodes shows a psilate exine; O. tenthredinifera shows a psilate–scabrate to verrucate exine; and O. lacaitae shows a scabrate–verrucate exine. Our results are in accordance with the findings of the work of Caspers and Caspers (1976)
where a general psilate condition of Ophrys pollinia surface was reported. These authors, in their study, also reported the presence of pores. However, if pores were visible, they were mainly from acetolysed pollinia. Caspers and Caspers (1976) even stressed that the observed differences can be detectable on the same massula, or at least in different massulae of the same pollinia. Therefore, they doubted the real usefulness of such sculptural characters for differential diagnosis. Schill and Pfeiffer (1977) pointed out two different types of pollinia surface structures, namely the ±verrucose–hamulate type of Ophrys attica, O. bertolonii, O. biscutella, O. reinholdii, O. speculum and O. tenthredinifera, and the psilate–scabrose type of Ophrys atrata, O. fuciflora, O. fusca, O. kurdica, O. parviflora and O. scolopax. However, they also mentioned that some taxa (Ophrys apifera, O. garganica, O. lutea and O. sphegodes) represent transitional stages between the states described above. Our current study comprises species of both groups of Schill and Pfeiffer (our Ophrys lacaitae was formerly described as O. fuciflora ssp. lacaitae), but we have not found any significant differences that could confirm their proposed division into two groups. Thus, we are more inclined to consider these ambiguous exine structures, also observed within the same Ophrys species, as intraindividual variations rather than real distinct categories. Similarly, little variability has been found in the exine of the genus Anacamptis, where the exine reduction leads to a psilate–scabrate to scabrate–rugulate condition (Fig. 2; Supplementary Information). In fact, A. caspia and A. papilionacea show a scabrate–rugulate exine; A. coriophora and A. morio show a psilate–scabrate exine; A. laxiflora and A. longicornu show a psilate exine; and A. pyramidalis shows a perforate to rugulate exine.
The low exine variability detected contrasts sharply with the substantial differences in floral architecture and pollination strategies observed within this group: most members of Anacamptis have short to long spurs and are nectar cheaters, including the aberrant long-spurred Anacamptis pyramidalis that is pollinated by long-tongued day- and night-flying Lepidoptera. Two species, however, are nectar rewarding (represented by Anacamptis coriophora in our study). Neither the moth-pollinated (A. pyramidalis) nor the nectar-rewarding species (A. coriophora) show remarkable differences in their pollen sculpturing. This corresponds well to the phylogenetic position of A. pyramidalis and A. coriophora, both deeply nested in the Anacamptis clade (Bateman et al., 2003).
Interestingly, in the related clade Himantoglossum (Fig. 2), H. hircinum shows a rugulate exine while H. (Barlia) robertianum shows a psilate exine. The two species, in spite of their phylogenetic affinity, are clearly distinct in pollination strategies (van der Cingel, 1995).
In the Neotinea clade (Fig. 2), Neotinea maculata, that is sister to the rest of Neotinea in Bateman et al. (2003), is different in exhibiting a rugulate exine surface whereas the remaining investigated species have psilate exine. Our results are similar to those reported by Schill and Pfeiffer (1977). The somewhat isolated position of N. maculata can be interpreted in accordance with its pollination strategy: N. maculata is self-pollinating (cleistogamous) whereas other Neotinea species have allogamous, food deceptive flowers.
More pronounced discontinuity in exine micromorphology is detected in the Gymnadenia–Dactylorhiza clade (Fig. 2). The ornamentation of the small genus Gymnadenia is psilate–perforate to ornate (Schill and Pfeiffer, 1977; Xi et al., 2000; Fig. 2, this study). Dactylorhiza (formerly Coeloglossum) viridis shows a reticulate exine with fragmented muri. The other Dactylorhiza species are clearly different, showing a psilate to psilate–scabrate exine. Only D. saccifera shows perforate rugulate surfaces. In general, these findings agree with those of Schill and Pfeiffer (1977). Nevertheless, they reported for some northern European taxa (D. elata, D. maculata, D. majalis and D. traunsteineri) a verrucose–hamulate sculpturing. Similar to our sculpturing types and those of Schill and Pfeiffer (1977), Averyanow (1990) describes three types of sculpturing in Dactylorhiza: psilate–scabrose, verrucose–hamulate and reticulate–fragmentimurate. This latter type was reported only for D. iberica. It would be interesting to check whether this species, not examined yet in available phylogenetic analyses, groups together with D. viridis, which shows a similar exine micromorphology.
Current phylogenetic reconstruction of Dactylorhiza (Bateman et al., 2003) indicates Dactylorhiza viridis as a sister species of a larger clade containing, among others, D. romana, D. sambucina and D. saccifera. This basal position may indicate that pollen of D. viridis possesses some basal traits for this Dactylorhiza group. However, this scenario is very unlikely because members of the other Dactylorhiza clades also have psilate exines (Schill and Pfeiffer, 1977) such as the related Platanthera chlorantha that shows a psilate–scabrate exine (Fig. 2). Rather, the reticulate exine condition found in D. viridis is more likely to be an autoapomorphy for this species and may reflect the strong ecological shift in its pollination strategy. Dactylorhiza viridis offers nectar in a short spur as a reward to a broad range of pollinators (van der Pijl and Dodson, 1966; van der Cingel, 1995) in contrast to the rest of related Dactylorhiza species which are food deceptive (Nilsson, 1980; van der Cingel, 1995).
Pollen of rewarding species is expected to be delivered to specific stigmas in a shorter time than pollen of deceptive species because of insect constancy in visiting a rewarding species (Cozzolino and Widmer, 2005). However, it is unknown if a longer permanence of pollinia on the insect body, expected in deceptive orchids, may promote a difference in exine micromorphology in order to prevent excessive pollen dehydration.
In the newly circumscribed genus Orchis (s.s.; Fig. 2; Supplementary Information), the formerly monospecific Orchis (= Aceras) anthropophora shows a reticulate–fragmentimurate exine. The closely allied species O. galilaea, O. purpurea, O. militaris and O. simia are reticulate, while O. italica has a verrucate exine. All these species are short spurred.
Among long-spurred Orchis s.s., O. mascula, O. provincialis and O. pauciflora show a psilate exine, the thin-long-spurred O. quadripunctata shows a perforate–rugulate exine, while its vicariant taxon, O. anatolica, shows a psilate–scabrate exine. These latter two species are expected to have a similar pollination biology, even if detailed studies on their pollination are still lacking (van der Cingel, 1995).
Of all the investigated groups, the Orchis s.s. clade is the most divergent and variable in exine condition, ranging from psilate, psilate–scabrate, verrucate, perforate–rugulate to reticulate. A similar diversity was also described by Schill and Pfeiffer (1977) but when considering the old circumscription of the genus Orchis s.l. A phylogenetic reconstruction of the exine evolution in the group is difficult as the different exine types are scattered over the ITS cladogram of Bateman et al. (2003) (Fig. 2) with even closely allied species such as O. anatolica and O. quadripunctata showing different exine characters. However, within the genus, some main trends can be recognized. For instance, the long-spurred species group, mainly pollinated by social and solitary long-tongued bees (i.e. O. mascula, O. pauciflora and O. provincialis), are all characterized by psilate exine. On the contrary, the short-spurred Orchis species display a reticulate exine with the notable exception of verrucate exine of O. italica. In Bateman et al. (2003), O. anthropophora is sister to the rest of Orchis s.s., and O. militaris and O. simia are part of a large ‘core’ Orchis clade whereas O. italica is sister to all Orchis (but not to O. anthropophora). However, in the phylogenetic reconstruction of Aceto et al. (1999) and Cozzolino et al. (2001), O. italica is sister to all other Orchis species (including O. anthropophora), and in a strict consensus tree of chloroplast sequence data (A. Widmer and A. Kocyan, unpublished data), O. anthropophora is sister to O. purpurea, O. militaris, O. simia, O. galilaea and O. punctulata. According to these alternative reconstructions, the reticulate condition is a clearly defined morphological character and agrees to a certain extent with pollination. In general, short-spurred Orchis s.s. are nectar cheaters pollinated by short-tongued solitary bees, beetles and flies. The only exception is O. galilaea that is probably sexually deceptive and attracts males of solitary bees by scent emission (Dafni, 1987).
Due to the large variation in exine morphology, a phylogenetic trend of exine evolution in the subtribe Orchidinae is difficult to trace (Fig. 2). However, one main difference clearly is elucidated: the clade comprising Anacamptis, Himantoglossum, Neotinea, Ophrys and Serapias shows, in general, psilate to rugulate or scabrate pollen, whereas the clade comprising Dactylorhiza, Gymnadenia, Orchis s.s., Platanthera, Pseudorchis and Traunsteinera (unknown pollen state)/Chamorchis (reticulate–fragmentimurate; Schill and Pfeiffer, 1977) has a much wider range of pollen sculpturing, from psilate to reticulate with several intermediate stages.
The former clade, with the notable exception of the Neotinea species group, is characterized by a chromosomal number of 2n = 36 while the latter has 2n = 40 and 42 as typical chromosomal numbers (D'Emerico, 2001). Hence, this main dichotomy in pollen micromorphology finds strong correspondence in the phylogenetic reconstruction of Orchidinae proposed by Bateman et al. (2003) who, in contrast to the phylogenetic reconstructions of Aceto et al. (1999) and Cozzolino et al. (2001), suggested Neotinea (2n = 42) as sister clade of the 2n = 36 orchids.
Unambiguous reconstruction of evolutionary trends in the tribe Orchidieae cannot be firmly supported, and our data do not help in disclosing the basal relationship in Orchidinae that are still lacking (see Bateman et al., 2003). Pollen sculpturing data of sister clades comprising Amitostigma, Hemipilia, Neottianthe and Ponerorchis give an ambiguous signal. Hemipilia has reticulate pollen (Luo, 1999) and Neottianthe has a psilate–perforate to reticulate pollen surface (Schill and Pfeiffer, 1977; Xi et al., 1998). Stenoglottis, Herminium, Habenaria or Disa pollen micromorphology does not indicate a linear trend: Stenoglottis is reticulate–heterobrochate (Schill and Pfeiffer, 1977), Cynorkis purpurascens is rugulate (Fig. 2), Herminium monorchis is baculate (Fig. 2), some Habenaria species are baculate (Fig. 2), hamulate or ornate (Schill and Pfeiffer, 1977), or reticulate or spinulate (Hesse and Burns-Balogh, 1984), and Disa species are baculate–pilate, ornate or reticulate (Schill and Pfeiffer, 1977; Chesselet and Linder, 1993).
According to Chase et al. (2003), Orchidinae and Disinae are sister to Brownleeinae, all together representing the tribe Orchidieae, which is sister to the monogeneric tribe of Codonorchideae. Plotting pollen characters on their cladogram, it seems possible that there is an evolutionary trend from foveolate to reticulate (Codonorchideae), reticulate (Brownleeinae) to psilate (including intermediate stages to verrucose, scabrate, rugulate and perforate) in Disinae and culminating in the Orchidinae Anacamptis–Himantoglossum–Neotinea–Ophrys–Serapias–Steveniella clade with the largely psilate (but not reticulate) stage. The reticulate pollen types of the other Orchidinae Dactylorhiza, Gymnadenia, Orchis s.s., Platanthera, Pseudorchis and Traunsteinera/Chamorchis clade (i.e. the 2n = 40, 42 clade) should then be reversals from a psilate stage, thus implying a derivate position for these orchids compared with the 2n = 36 (namely Ophrys, Serapias, Himantoglossum and Anacamptis) plus Neotinea clade.
Orchids are considered a paramount example of evolution through floral diversification (Cozzolino and Widmer, 2005). Before the molecular era, orchid systematics were mainly based on floral morphological traits. However, these traits have turned out to be very homoplastic and thus unsuitable for phylogenetic reconstruction because, as the result of pollinator-mediated selection, they revealed more ecological than phylogenetic implications (Bateman et al., 1997; Aceto et al., 1999; Cozzolino et al., 2001). If this was true also for the exine structures investigated in the present study, we would expect a larger variation in this trait according to the frequently observed changes in pollination strategies. For instance, for A. pyramidalis and A. coriophora, species with unique floral characters within the genus Anacamptis, different pollen structures can be expected, which was not found in this study. Thus it can be assumed that, at least in some clades, such as in the Anacamptis–Himantoglossum–Neotinea–Ophrys–Serapias–Steveniella clade, with mostly psilate stage, the pollen sculpturing is more likely to reflect their evolutionary history. In contrast, in some genera such as Dactylorhiza and Orchis s.s., species groups characterized by similar pollination biology revealed marked differences in pollen sculpturing. The absence of a univocal relationship between pollen micromorphology and pollination strategies has been confirmed by recent evidence that showed that several species characterized by different pollen sculpturing show a large overlap in pollinator set. Different and unrelated orchid species such as D. romana, A. morio and O. mascula, when growing in sympatry, have been found to adopt a largely overlapping set of pollinator species (Cozzolino et al., 2005) irrespective of the marked differences in their pollen sculpturing. At the same time, closely related species with presumably identical pollination biology, such as O. quadripunctata and O. anatolica, have different exine morphology.
These pieces of evidence suggest that a convergent pollination syndrome is not always reflected in a preferential pollen sculpturing and that, at the same time, a shift to a different pollination strategy does not necessarily imply a significant change in pollen micromorphology. In light of this, the finding of species with marked difference in pollen micromorphology when compared with their close relatives (e.g. N. maculata, D. viridis and O. italica) may also reflect the effects of relaxed selection on this trait or the consequence of different evolutionary constraints of flower topology (such as pleiotropic effects induced, for instance, by the evolution of cleistogamy or by modification of floral parts) rather than the ecological adaptation per se to a different pollinator functional group.
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SUPPLEMENTARY INFORMATION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUPPLEMENTARY INFORMATIONACKNOWLEDGEMENTSLITERATURE CITED |
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SEM pictures of the following taxa are mentioned in the text but not printed in this article: Anacamptis caspia, Anacamptis morio, Anacamptis longicornu, Ophrys apifera, Ophrys fusca, Ophrys tenthredinifera, Orchis militaris, Orchis mascula, Serapias vomeracea. They are available as Supplementary Information online at http://www.aob.oxfordjournals.org/.
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ACKNOWLEDGEMENTS |
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The authors thank Jasmin Joshi and Alex Widmer for valuable discussions on orchid pollination, and Paolo Grunanger for kindly providing some orchid pollinaria. We would like to thank V. Avolio, S. Giorgio and R. Salatiello of the Naples Botanical Garden for help with plant collection and cultivation, and the PRIN Research Programme for supporting research on orchid biology.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONSUPPLEMENTARY INFORMATIONACKNOWLEDGEMENTSLITERATURE CITED |
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