Phylogeny and Evolution of Orchis and Allied Genera Based on ITS DNA Variation: Morphological Gaps and Molecular Continuity

Molecular Phylogenetics and Evolution Vol. 13, No. 1, October, pp. 67–76, 1999 Article ID mpev.1999.0628, available online at http://www.idealibrary.com on

Phylogeny and Evolution of Orchis and Allied Genera Based on ITS DNA Variation: Morphological Gaps and Molecular Continuity Serena Aceto,* Paolo Caputo,† Salvatore Cozzolino,‡ Luciano Gaudio,* and Aldo Moretti† *Dipartimento di Genetica, Biologia Generale e Molecolare, Universita` Degli Studi di Napoli Federico II, Via Mezzocannone 8, I-80134 Naples, Italy; and †Dipartimento di Biologia Vegetale e ‡Orto botanico, Universita` Degli Studi di Napoli Federico II, Via Foria, 223, I-80139 Naples, Italy Received July 10, 1998; revised December 14, 1998

ers in the Northern regions of Europe and Asia (Dressler, 1993). Orchis is clearly related to other, mainly Mediterranean, genera, i.e., Aceras R. Br., Anacamptis L. C. M. Richard, Barlia Parl., Dactylorhiza Necker, Gymnadenia R. BR., Himantoglossum Koch, and Neotinea Reichenb. fil. The striking beauty of these taxa, their wide occurrence in countries with a long botanical tradition, as well as their endangered status has fostered much interest in these plants which, accordingly, have been intensively studied over the past 100 years. Despite these studies, the pervasive parallel evolution and ecological convergence which characterize European orchids (Dressler, 1981, 1993) as well as a tendency to reticulate evolution in some groups (e.g., Ehrendorfer, 1980) greatly hinder a clear understanding of the relationships both within Orchis and between it and its allied taxa. Floral features have traditionally been employed in segregating groups within orchids. However, floral traits usually show strong discontinuities, often corresponding to unique apomorphies, which are useless for inferring phylogenetic relationships. Moreover, floral characters are often inconsistent compared to other taxonomic evidence (e.g., chromosome number). As a result, the few generic-level taxonomic treatments available (e.g., Vermeulen, 1972; Cauwet-Marc and Balayer, 1984) and the scanty but novel circumscriptions for the genus (e.g., Lo¨ve and Lo¨ve, 1972) are often contradictory. In addition, studies based on isozymes (Schlegel et al., 1989; Rossi et al., 1994) have shown a pervasive lack of correspondence between floral traits and genetic relationship, indicating that some of the generic and infrageneric boundaries may be artificial. More recently, nucleic acid data have been used in order to infer relationships among members of Orchis and allied genera (Caputo et al., 1995; Cozzolino et al., 1998). These studies, based on chloroplast DNA restriction analysis, have shown that both Aceras and Dactylorhiza Necker are nested within Orchis. An exploratory

Phylogenetic relationships among members of genus Orchis and allied genera Aceras, Anacamptis, Barlia, Dactylorhiza, Gymnadenia, Himantoglossum, Neotinea, Ophrys, Platanthera, and Serapias were inferred from nucleotide sequence variation in the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA. Sequences were subjected to various alignments by changing the gap opening and extension parameters. After a preliminary parsimony analysis, the alignment with the lowest homoplasy indicators was chosen as optimal. The phylogenetic analysis, carried out on the optimal alignment by using Gennaria as an outgroup and a total of 31 taxa, showed that all the genera considered in this study are nested in Orchis despite their distinct morphological features. Genus Orchis is divided into two major clades, each of which includes one or more of the other genera in this study. The resulting phylogenetic hypothesis does not match previous conclusions based on vegetative and floral morphology of the taxa involved but is congruent with isoenzyme, karyological, and chloroplast DNA restriction data. Our results indicate that floral morphology is highly flexible and current generic and infrageneric limits are artificial. Even if some floral characters closely correspond to the molecular data, most are highly homoplastic and thus unsuitable for phylogenetic reconstruction. Various traits pertaining to floral morphology may be interpreted as a result of ecological convergence related to pollinator-mediated selection; such characters can undergo drastic modifications without correspondingly dramatic genetic changes. r 1999 Academic Press Key Words: internal transcribed spacer; floral evolution; molecular systematics; Orchidaceae; Aceras; Anacamptis; Barlia; Dactylorhiza; Gymnadenia; Himantoglossum; Neotinea; Ophrys; Orchis; Platanthera; Serapias.

INTRODUCTION Orchis L. (Orchidaceae), in its present circumscription, is a rather homogeneous genus with over 30 species centered in the Mediterranean basin and outli67

1055-7903/99 $30.00 Copyright r 1999 by Academic Press All rights of reproduction in any form reserved.

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analysis of the Internal Transcribed Spacer 1 (ITS1) of the nuclear ribosomal DNA in five Orchis species was recently conducted (Cozzolino et al., 1996) to test the feasibility of ITS1 sequencing as a tool to infer relationships in the genus. The evidence obtained from this preliminary investigation showed good correlation with the isozyme and chloroplast DNA data. Among the various sequences employed in phylogenetic inference, ITS1 and 2 appear to have a mutation rate which makes them suitable (Baldwin et al., 1995 and references therein) for studies of intergeneric relationships (e.g., Suh et al., 1993; Susanna et al., 1995; Bogler and Simpson, 1996) or of infrageneric relationships in rapidly evolving taxa (e.g., Kim and Jansen, 1994; Bayer et al., 1996). In this paper we present evidence gathered by sequencing ITS1 and 2 for 31 orchid taxa. Data will be discussed in the light of the selective pressures which have probably acted on floral morphology, consequently hindering the recognition of the phylogenetic pattern based upon floral morphology. MATERIALS AND METHODS Plant Material The following taxa were employed in this study: Aceras anthropophorum (L.) R. Br. ex Aiton fil., Anacamptis pyramidalis (L.) L. C. M. Richard, Barlia robertiana (Loisel.) Greuter, Dactylorhiza romana (Sebastiani) Soo´, Gennaria diphylla (Link) Parl., Gymnadenia conopsea (L.) R. Br. ex Aiton fil., Himantoglossum hircinum (L.) Spreng., Neotinea maculata (Desf.) Stearn, Ophrys tenthredinifera Willd., O. collina Banks et Solander ex A. Russel, O. coriophora L. subsp. fragrans (Pollini) Sudre, O. italica Poir., O. lactea Poir., O. laxiflora Lam., O. longicornu Poir., O. mascula (L.) L. subsp. mascula, O. militaris L., O. morio L. subsp. morio, O. palustris Jacq., O. papilionacea L. subsp. papilionacea, O. patens Desf., O. pauciflora Ten., O. provincialis Balbis ex Lam. et DC., O. purpurea Huds., O. quadripunctata Cyr. ex Ten., O. simia Lam., O. spitzelii Sauter ex Koch, O. tridentata Scop., O. ustulata L., Platanthera chlorantha (Custer) Reichenb., and Serapias lingua L. All specimens were field collected by the authors and cultivated at the Botanical Garden of Naples, Italy. Leaves were collected at flowering time. Voucher specimens of the examined plants are deposited at NAP. DNA Extraction and Sequencing Leaves (1 g per sample) were ground in liquid nitrogen and total DNA was extracted following the procedure described in Caputo et al. (1991). ITS1 and 2 were amplified by using two pairs of primers which anneal in the 38 region of the 18S (58-GGAGAAGTCGTAACAAGGTTTCCG-38) and in the 58 region of the 5.8S (58-ATCCTGCAATTCACACCAAG-

TATCG-38), or in the 38 region of the 5.8S (58TTGCAGAATCCCGTGAACCATCG-38) and in the 58 region of the 26S (58-CCAAACAACCCGACTCGTAGACAGC-38), respectively. PCRs were carried out for 30 cycles in a Perkin–Elmer–Cetus 9600 thermocycler. Initial conditions were as follows: 1 min denaturation at 94°C, 1 min annealing at 55°C, and 45 s extension at 72°C. Samples were denatured for 5 min at 94°C before the beginning of the first cycle; extension time was increased by 3 s/cycle; and extension was further prolonged for 7 min at the end of the last cycle. PCR fragments were then purified by using Microcon 100 microconcentrators (Amicon, Danvers, MA) and double-strand sequenced in both directions by using a modification of the Sanger dideoxy method (Sanger et al., 1977) as implemented in a double-strand DNA cycle sequencing system with fluorescent dyes. Sequence reactions were then loaded into a 373A Applied Biosystems Automated DNA sequencer (Applied Biosystems, Foster City, CA). Various sequencing experiments were repeated to solve all uncertainties. Data Analysis A raw alignment was accomplished by using Clustal W ver. 1.6 (Thompson et al., 1994) with default settings. Sequences were then reduced to only ITS1 and ITS2 by aligning them with the 38 termini of 18S and 5.8S and with the 58 termini of 5.8S and 26S of various monocot sequences available in the literature. When differences of starting and ending points occurred, the sequence of Vanilla planifolia Andrews (GenBank Accession no. U66819, Mai and Coleman, unpublish.) was used. Six fictitious unknowns (N’s) were added at the 38 terminus of both ITS1 and ITS2 in all taxa to prevent terminal misalignments. The optimal alignment was searched separately for ITS1 and ITS2 by employing Clustal W. An alignment software was preferred to sequential pairwise comparison (Swofford and Olsen, 1990) used by other workers (e.g., Oxelman et al., 1995; Susanna et al., 1995; Bayer et al., 1996; Sang et al., 1996) because of the difficulty in interpreting a fairly large amount of small (1–5 bp) insertions/deletions (indels) in various ingroup taxa. The approach used in Clustal W is two-stepped: a first step in which pairwise alignments are carried out to determine which are the closest sequences in each and every possible pair and a second step in which multiple alignments are calculated according to the results of the first step. In each step, gaps are opened to maximize sequence similarities. Gap opening and extension are controlled by four parameters: PWGAPOPEN (which controls the gap opening cost in the first step), PWGAPEXT (which controls the gap extension cost in the first step), and GAPOPEN and GAPEXT (which are the corresponding parameters for the second step). The optimal alignment was determined using a strategy similar to that described in Bogler and Simp-

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son (1996), which employs homoplasy indicators (C.I. and R.I.) as optimality criteria, and gap opening and extension parameters were varied across every alignment with unit increments from 4 to 15 and from 4 to 10, respectively. We also imposed the following constraint: PWGAPOPEN ⫽ GAPOPEN ⱖ PWGAPEXT ⫽ GAPEXT within any single alignment. In all the tests, transitions and transversions were equally weighted, and the parameter MAXDIV (which controls the delay of the alignment for the most divergent sequences) was set to 85%. Each alignment was then converted to numeric format by means of a word processor macro, fictitious unknowns were removed, and the resulting matrix was subjected to a heuristic parsimony analysis (mh*; bb*;) employing the Hennig86 software (Farris, 1988). Gaps were coded as missing data and all characters were treated as unordered (nonadditive). For each analysis, consistency and retention indices (C.I. and R.I.) were recorded. The optimal alignments were chosen as those with highest C.I. ⫻ R.I. values. In case of ties, all the alignments with identical C.I. ⫻ R.I. values were analyzed further. The optimal alignments for ITS1 and ITS2 were then united (for a total of eight matrices because of ties within ITS1) and subjected again to a parsimony analysis and to the same optimality criterion. Ties in this case were resolved by choosing the most parsimonious solution in terms of the number of steps. This yielded a single optimal alignment. Because number of steps is positively correlated with the gap opening and extension costs (see also Bogler and Simpson, 1996), we checked that all equally homoplasious alignments yielding the minimal number of cladograms indicated the same topology. Because the gap parameters of the optimal alignments, although yielding the least homoplasious solutions, are arbitrary per se, in order to verify to what extent ambiguous gap positions may influence topology and to avoid the problem that a single set of parameters may influence the final result, we used the elision approach, as described by Wheeler et al. (1995). This approach consists in ‘‘eliding’’ various individual alignments into a single combined alignment on which a phylogenetic analysis is carried out. The elision approach, although afflicted by theoretical difficulties related to homology assessment (Wheeler et al., 1995), tends to give more resolved solutions compared to other methods of the same family (e.g., Gatesy et al., 1993). Because merging all the matrices obtained by varying the gap parameters would have been computationally infeasible, we sampled our set of matrices at wide intervals according to the variation of gap parameters. Eight matrices were sampled and merged in such a way that a single combined matrix of 4260 characters was obtained. This matrix was then analyzed by using a recent version of Nona (Goloboff, 1993), with the following parameters: hold 1000; hold/25; mult*25; max*.

Once the optimal alignment was found as indicated above, the calculation of pairwise distances was carried out independently for ITS1 and ITS2 using the ‘‘distance matrix’’ option of Clustal W and correcting for gaps but not for multiple substitutions. A heuristic Hennig86 analysis was run (mh*; bb*;) on the optimal alignment by using Gennaria diphylla as an outgroup. The resulting cladograms were examined and manipulated with the Clados software package (Nixon, 1992). Bootstrap percentages (Felsenstein, 1985) and presence of phylogenetic signal (Permutation Tail Probability test as implemented by Faith and Cranston, 1991) were calculated by using the SEQBOOT (1000 and 500 replicates, respectively) and DNAPARS programs of the Phylip 3.57 package (Felsenstein, 1993). Bremer support (Bremer, 1988, 1994; Ka¨llersjo¨ et al., 1992) was calculated by using a recent version of Nona (Goloboff, 1993) that explored the trees up to six steps longer (BS 6) than the most parsimonious solutions. Finally, although we preferred to treat indels as missing data and not as a fifth state in order to avoid overweighting, we also carried out a heuristic phylogenetic analysis on the optimal data matrix by coding indels as a fifth state. RESULTS Basic ITS Information The lengths and GC contents of both the ITS’s for all taxa in the study are reported in Table 1 (EMBL Accession nos.: Z94059 to Z94120; the increasing numbers correspond to the alphabetical order of the genus/ species initial, Z94059 being the ITS1 sequence of A. anthropophorum and Z94120 being the ITS2 sequence of S. lingua). ITS1 length ranged from 226 to 250 bp and GC content from 39.6 to 50.4%. ITS2 length ranged from 225 to 247 bp and GC content from 41.3 to 51.3%. ITS1 sequence divergence in the ingroup ranged from 0.4% (pairwise distance between O. mascula and O. provincialis and between O. purpurea and O. simia) to 27.5% (pairwise distance between O. lactea and S. lingua); the minimum intergeneric distance was 2.8% (pairwise distance between A. anthropophorum and O. italica) and the maximum infrageneric distance within Orchis was 25.5% (pairwise distances between O. collina and O. patens). ITS2 sequence divergence in the ingroup ranged from complete identity (O. mascula and O. provincialis) to 25.8% (pairwise distance between O. morio and P. chlorantha). The minimum intergeneric distance was 2.1% (pairwise distance between A. anthropophorum and O. italica) and the maximum infrageneric distance within Orchis was 23.6% (pairwise distances between O. papilionacea and O. patens).

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TABLE 1 ITS Length and GC Content for the Taxa in Study ITS1

ITS2

Taxon

Length (bp)

GC%

Length (bp)

GC%

Aceras anthropophorum Anacamptis pyramidalis Barlia robertiana Dactylorhiza romana Gennaria diphylla Gymnadenia conopsea Himantoglossum hircinum Neotinea maculata Ophrys tentrediniphera Orchis collina O. coriophora O. italica O. lactea O. laxiflora O. longicornu O. mascula O. militaris O. morio O. palustris O. papilionacea O. patens O. pauciflora O. provincialis O. purpurea O. quadripunctata O. simia O. spitzelii O. tridentata O. ustulata Platanthera chlorantha Serapias lingua

249 240 243 246 248 247 244 240 237 241 241 249 248 241 243 250 243 245 246 226 244 249 250 249 244 249 241 248 247 250 240

45.78 39.58 46.91 48.78 43.95 46.56 45.08 45.42 45.15 40.66 42.74 46.59 41.13 41.67 46.91 47.60 43.21 46.94 41.06 42.92 50.41 46.59 48.00 42.97 48.36 43.37 49.38 42.34 41.30 42.40 46.25

240 234 243 243 247 240 241 230 239 238 237 240 225 240 236 240 238 234 240 226 239 240 240 240 239 240 239 230 229 240 237

45.00 47.86 50.62 46.09 42.51 48.33 47.72 44.78 46.03 46.22 48.95 49.16 44.00 46.67 50.42 49.58 46.64 51.28 46.67 47.35 49.79 47.08 50.00 47.08 49.37 46.67 50.20 44.35 45.85 41.25 48.10

Phylogenetic Analysis The cladistic analysis carried out on the optimal alignment (ITS1: PWGAPOPEN ⫽ GAPOPEN ⫽ PWGAPEXT ⫽ GAPEXT ⫽ 4, consensus length 261 bp, informative characters 44.8%; ITS2: PWGAPOPEN ⫽ GAPOPEN ⫽ PWGAPEXT ⫽ GAPEXT ⫽ 4, consensus length 262 bp, informative characters 40.8%; total consensus length 523, informative characters 42.8%) yielded eight equally parsimonious cladograms (839 steps, C.I. ⫽ 0.60, R.I. ⫽ 0.69). The consistency index of our cladograms is quite low but altogether comparable with those obtained when dealing with large genera (e.g., Yuan et al., 1996). Moreover, as has been demonstrated by Goloboff (1991), the C.I. is negatively correlated with both the number of terminal taxa and the number of characters. The consensus tree of two of the eight topologies obtained as well as the consensus among all topologies are shown in Fig. 1. The cladogram(s) of Fig. 1 show that all the genera considered in this study are nested in Orchis (except Gennaria, which was used as an

outgroup). In particular, the ingroup appears divided into two major clades. One clade is composed of Anacamptis, Barlia, Himantoglossum, Ophrys, Orchis collina, O. coriophora, O. laxiflora, O. longicornu, O. morio, O. palustris, O. papilionacea, and Serapias. In this clade, Barlia and Himantoglossum are a sister group and, together with Ophrys, are in a sister group relationship with the rest of the listed species. This latter group consists of Serapias plus a not fully resolved clade in which sister group relationships between O. laxiflora and O. palustris, between O. longicornu and O. morio, and between the latter two and O. papilionacea can be observed. The other major clade is composed in turn of two monophyletic groups. The first group has Dactylorhiza and Gymnadenia in a sister group relationship, with a pectinate sequence of Platanthera, Neotinea, O. tridentata, O. ustulata, and O. lactea. The second group has O. italica in a basal position to a clade composed of Aceras as sister group to a unit in which a trichotomy including O. militaris, O. purpurea, and O. simia is basal to a poorly resolved monophylum. In this clade, sister group relationships between O. mascula and O. provincialis, between O. quadripunctata and O. spitzelii, and between the latter two and O. patens can be observed. Repeated successive weighting (Farris, 1969) on the eight cladograms yielded two cladograms (3336 steps, C.I. ⫽ 0.83, R.I. ⫽ 0.86) with a greater resolution. These two topologies are a subset of the eight obtained for the original matrix. For this reason, we choose the consensus tree between them, topologically identical to the cladogram in Fig. 1, as a working hypothesis of the phylogenetic relationships in our ingroup. ITS1 and ITS2 data, when analyzed independently (mh*; bb*;), yielded 180 cladograms (437 steps, C.I. ⫽ 0.58, R.I. ⫽ 0.70) and 77 cladograms (393 steps, C.I. ⫽ 0.64, R.I. ⫽ 0.70), respectively. Neither ITS1 nor ITS2 topologies are fully congruent with those expressed by the total data set. In particular, the consensus tree for ITS1 shows Serapias as sister group to the rest of the ingroup and Ophrys as sister group to O. laxiflora and O. palustris. The consensus tree for ITS2 shows Barlia and Himantoglossum at the base of the tree in a sister group relationship to the rest of the ingroup. Bootstrap analysis on the complete data set (bootstrap values ⬎50% are shown in Fig. 1) generated a majority-rule consensus tree topologically similar to the cladogram in Fig. 1. The only difference is in the position of Dactylorhiza and Gymnadenia compared to the rest of their clade. Despite the fact that the majority of the clades have bootstrap values above 50%, the relationships among the three major clades into which the ingroup is split as well as those between Ophrys, Platanthera, and their respective sister groups, are weakly supported.

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FIG. 1. Consensus tree between two of the eight equally parsimonious topologies obtained (839 steps, C.I. ⫽ 0.60, R.I. ⫽ 0.69). This tree is identical to the consensus topology obtained after successive weighting of the original matrix. Numbers above the branches indicate Bremer’s support; bootstrap support above 50% is indicated below each branch. Boxes: general consensus topology.

The evaluation of phylogenetic signal as carried out by the PTP test (Faith and Cranston, 1991) showed that no tree obtained from any of the 500 fictitious matrices was comparable in length to the most parsimonious cladograms mentioned above. The length of the resulting 3342 cladograms ranged from 1331 to 1369 steps (mean value 1350.0, SD 6.65), that is 37 to 39% longer than the most parsimonious trees found for the original data set, and at least 30% longer than the longest topology obtained in the preliminary analyses. In such cases, PTP equals 0.01 (Faith and Cranston 1991) and therefore the null hypothesis was rejected. Bremer support (Fig. 1) is high for the majority of the clades. Notable exceptions are the clade with Dactylorhiza and Gymnadenia at the base, the clade with A. anthropophorum at the base, the clade immediately above the latter, the clade with O. papilionacea at the base, and the clade above O. patens. The consensus among the four cladograms obtained through the elision approach described above (7002 steps long) is extremely similar to the consensus tree obtained by the original data set (Fig. 1), the only difference being the pectinate position of O. papiliona-

cea, Anacamptis, and O. collina, which is not present in any of our original cladograms. The consensus tree between the four most parsimonious cladograms obtained by coding indels as a fifth state (1032 steps, C.I. ⫽ 0.63, R.I. ⫽ 0.71) is congruent with that obtained in the original matrix (Fig. 1), with the only difference being a greater resolution in the relative positions of Anacamptis, O. collina, and O. coriophora (this resolution is present also in the consensus tree shown in the boxes of Fig. 1). This is further empirical proof of the fact that different indel treatments affect ITS topologies only minimally (Baldwin et al., 1995). DISCUSSION Phylogenetic Relationships The analyses presented here address various longdebated issues in the study of European orchid phylogeny. Perhaps the most relevant result of this study is the indication that all the genera considered here are nested in Orchis. Indications of the inclusion of Aceras

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(Rossi et al., 1994; Caputo et al., 1995; Cozzolino et al., 1998) and Dactylorhiza (Caputo et al., 1995; Cozzolino et al., 1998) in Orchis were already available, but in this case nesting is hypothesized for Anacamptis, Barlia, Gymnadenia, Himantoglossum, Neotinea, Ophrys, Platanthera, and Serapias. However, the inclusion of several of these genera in Orchis is only weakly supported (i.e., bootstrap percentages are below 50% for the basal clades of our analysis). In order to evaluate the strength of our hypothesis more accurately, we estimated the increase in step number by forcing the above-mentioned genera outside Orchis. The results of these attempts show that the above mentioned genera fall into two groups. The first group is composed of Barlia–Himantoglossum, Dactylorhiza–Gymnadenia, Ophrys, Platanthera, and Serapias. The separate exclusion of each of them increases the tree length by 6–8 steps. The second group is composed of Aceras, Anacamptis, and Neotinea, which when forced outside Orchis increases tree length by 25–35 steps. Making Orchis strictly monophyletic costs over 50 steps, and excluding only the genera of the first group causes an increase of 23 steps. This substantial increase in the step number may depend upon how far removed from the ingroup is the chosen outgroup. More closely related possible outgroups were discarded after preliminary results showed that the selected taxa, all belonging to subtribe Orchidinae (namely, Anacamptis, Barlia, Himantoglossum, Ophrys, Platanthera, and Serapias), were included in our ingroup. We therefore chose a member of subtribe Habenariinae Bentham (the only other subtribe of tribe Orchideae according to Dressler, 1993) as an outgroup. Also a previous analysis based on chloroplast DNA RFLPs (Cozzolino et al., 1998) demonstrated that chloroplast DNA in our orchids was less conserved than expected and thus led us to avoid selecting far removed outgroups (e.g., Cephalanthera L. C. M. Richard, which was the outgroup in that analysis, or any other taxa not belonging to Orchideae) because ITS sequences are normally less conserved than chloroplast DNA as a whole. Correlation with Previous Evidence Morphology. The phylogenetic hypothesis shown here does not match in entirety previous knowledge on the gross morphology of the taxa involved. Floral traits, in particular the shape of the labellum, the presence of a spur, and the convergence of outer tepals into a galea, which have been traditionally used to discriminate among species and related genera within orchids, do not appear to be completely congruent with the pattern of relationships depicted by ITS’s (Fig. 2). Therefore, our proposal of phylogenetic relationships does not match any previous treatment based on morphology. Vermeulen (1972), in the last taxonomic revision avail-

FIG. 2. Plotting of the chromosome numbers and of the key morphological characters traditionally used for diagnostic purposes onto the cladogram of Fig. 1. Characters are as follows: 1, haploid chromosome number (1.0 ⫽ 17; 1.1 ⫽ 20; 1.2 ⫽ 21; 1.3 ⫽ 18; 1.4 ⫽ 16); 2, shape of the tuberoids (2.0 ⫽ entire; 2.1 ⫽ palmately lobed); 3, shape of the labellum (3.0 ⫽ three equal lobes; 3.1 ⫽ entire; 3.2 ⫽ three lobes, central one with lobules; 3.3 ⫽ five tapering lobes, in the shape of a standing man); 4, spur (4.0 ⫽ present; 4.1 ⫽ absent); and 5, galea formed by the outer tepals (5.0 ⫽ absent; 5.1 ⫽ present). Character 3 has been scored following the traditional views of orchidologists; states 3.2 and 3.3 may be also scored as an independent binary character. Terminal polymorphic states are not shown.

able for Orchis, divided the genus into four sections which are at least paraphyletic if not altogether polyphyletic according to our data (Table 2 and Fig. 1). Also, the groups recognized by Cauwet-Marc and Balayer (1984) on chromosomal and morphological grounds (Table 2), as well as the segregated genera Vermeulenia Lo¨ve and Lo¨ve (Lo¨ve and Lo¨ve, 1972) and Anteriorchis E. Klein and D. Strack (Strack et al., 1989), are not supported by our analysis. In contrast, some small groups already recognized in previous works (e.g., Vermeulen’s subsections) are congruent with our data. Notable in this respect are subsect. Moriones (Rchb. f.) Parl. and subsect. Laxiflorae Nevskij. Despite the incongruencies with previous work based on morphology, some of the clades in our analysis show evident morphological

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TABLE 2 Synopsis of the Groups Recognized by Previous Authors Vermeulen’s (1972) sections Labellointegrae O. caspia O. collina O. papilionacea

Labellotrilobatae O. laxiflora O. mascula O. morio O. pallens O. palustris O. patens O. pauciflora O. provincialis O. quadripunctata

Informal groups by Cauwet-Marc and Balayer (1984) O. coriophora O. morio O. collina O. papilionacea

Informal groups by Rossi et al. (1994) O. morio O. papilionacea O. coriophora O. sancta

Coriophorae O. coriophora O. sancta

O. palustris O. laxiflora

O. italica O. simia A. anthropophorum O. purpurea

synapomorphies. For example, Dactylorhiza and Gymnadenia share palmate tuberoids (Fig. 2). Karyology. Our hypothesis (Fig. 1), albeit not compatible with evidence from gross morphology, is, however, broadly congruent with the cytogenetic reports for the species in this study (Fig. 2). For all taxa included in the Ophrys and Serapias clades, reports indicate n ⫽ 18, with the exception of Serapias lingua (n ⫽ 36, one of the few tetraploid species of the genus) and O. papilionacea (n ⫽ 16). All taxa included in the O. italica and Platanthera clades are n ⫽ 21 and Dactylorhiza and Gymnadenia are n ⫽ 20 (Cauwet-Marc and Balayer, 1984; Bianco et al., 1989; Del Prete et al., 1991; D’Emerico et al., 1992, 1996; D’Emerico, pers. comm.). The only karyological ambiguities in this framework are related to O. patens and O. spitzelii for which n ⫽ 21, 40 and n ⫽ 20, 21, respectively, have been reported. However, we chose to report only the apparently plesiomorphic number 2n ⫽ 42 in Fig. 2. For Gennaria, reports indicate n ⫽ 17 for Sardinia and n ⫽ 20 for Canary Islands (Dolcher and Dolcher, 1961; Scrugli, 1978; Sundermann and Von Der Bank, 1977). If n ⫽ 20 is accepted as the plesiomorphic number for the species (the most probable case for Habenariinae, according to Dressler, 1993) n ⫽ 18 and n ⫽ 21 would be apomorphic for the involved clades. In this case, Dactylorhiza and Gymnadenia would have retained the plesiomorphic feature. On the other hand, if n ⫽ 17 is regarded as the plesiomorphic state, reconstruction of chromosome number onto the cladogram would be equivocal. Previous molecular evidence. Data from isoenzymes (Schlegel et al., 1989; Rossi et al., 1994) give only

O. pallens O. mascula O. provincialis

O. laxiflora O. palustris

Militares O. italica O. lactea O. militaris O. punctulata O. purpurea O. simia O. tridentata O. ustulata

O. tridentata O. ustulata O. simia O. militaris O. purpurea O. quadripunctata

partial support to the ITS-based phylogeny. These authors recognize a group composed of O. morio and O. ustulata which is not supported by the present evidence. The same authors, however, implicitly define the positions of Dactylorhiza and Gymnadenia in a manner similar to ours. Also the grouping by Rossi et al. (1994) is not entirely congruent with ours (Table 2). However, it is worth mentioning that the latter authors placed A. anthropophorum within Orchis. It is regrettable that no previous cladistic analysis is available for morphological, karyological, and enzymatic characters. Consequently this has prevented us from carrying out a combined analysis, which could have provided further insights into the phylogeny of these orchids in a total evidence framework. We therefore resorted to simply plotting the relevant karyological and morphological characters on our consensus tree (Fig. 2) under a delayed transformation model. Finally, a chloroplast DNA restriction fragment length polymorphism analysis carried out in our laboratory on representatives species of Orchis, Aceras, Dactylorhiza, Anacamptis, and Serapias (Cozzolino et al., 1998) showed that Orchis is paraphyletic because it also contains Aceras and Dactylorhiza. The genus Orchis appeared divided into two major clades which, to the extent of the species common to both studies, are almost completely congruent with the results shown here. The only relevant difference between the two analyses was in the positions of Anacamptis and Serapias, which were external to Orchis and sister group to each other in the former study. As far as Serapias is concerned, we interpret this discrepancy as due to sampling paucity in the chloroplast analysis that in-

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volved only 14 taxa. In fact, the position of this genus is rather basal even in the present analysis, and forcing the genus out of Orchis causes only a slight deterioration of our hypothesis in terms of step number. Anacamptis, in contrast, is highly nested in our ingroup in the present analysis. The discordance between the pattern of descent depicted by ITS’s and chloroplast DNA in this genus may be interpreted as a possible chloroplast capture event (at least, for the populations of Central and Southern Italy used in the chloroplast DNA study). Plastid capture has been demonstrated to be a rather common occurrence in some groups of plants (Soltis and Kuzoff, 1995; Schilling and Panero, 1996), and in this case the event may be hypothesized as following hybridization either with a member of the stem lineage of Serapias or with some extant relative of Serapias not tested in that analysis. Besides plastid capture, some other kind of event can be hypothesized as a factor acting in the production of the floral organization typical of Anacamptis. A natural hybrid between O. collina (the sister group of Anacamptis according to the cladogram in Fig. 1) and a species of Serapias, recently found in Apulia (Italy) (Bianco et al., 1990), does not resemble Anacamptis. This monotypic genus, whose morphology is strikingly dissimilar from that of its closest relatives (it has a very long and thin spur), is pollinated mainly by Zygaenidae moths (Nazarov and Efetov, 1993) and this condition does not occur in any member of its clade. Unfortunately, the chloroplast DNA data (Cozzolino et al., 1998) are representative of less than 50% of the species employed in the present study, do not include several of the genera studied here, and are rooted by a different outgroup. This prevented us from properly exploring incongruence issues a posteriori and because chloroplast DNA is strictly matrilinear in orchids a combined analysis would perhaps not have been appropriate. CONCLUDING REMARKS Heterogeneity in pollination syndromes or in pollination-related floral characters is widespread in the study group (e.g., Aceras, Neotinea, Ophrys, and Platanthera compared to their close relatives). Orchidaceae are considered a paramount example of evolution through floral diversification which includes changes of pollinator, variations in flower color, and pollinia specialization representing only some of the mechanisms potentially involved in orchid speciation. Our results indicate that while some floral characters closely correspond to the molecular data, most are highly homoplastic and thus unsuitable for reconstructing phylogeny. Various traits pertaining to floral morphology may be interpreted as a result of pollinator-

mediated selection and thus have more ecological than phylogenetic implications. As already shown on molecular grounds for Platanthera pro parte (Haperman et al., 1996a,b) and for Oncidium and related taxa (Chase and Palmer, 1992), pollinator-mediated selection may undermine the understanding of phylogenetic relationships through morphological analysis of floral characters, because of widespread convergence. However, floral traits may be relevant in understanding isolation mechanisms within Orchidaceae. An issue which emerges in this study is that isolations appear to have occurred mainly in connection with pollinator specificity. The captivation of new pollinators (or the onset of elaborate mechanisms of deception) may have resulted in divergent floral morphologies that quickly segregated new species and species groups. We are therefore inferring that pollinator-mediated morphological selection was somehow punctuated and in some instances abruptly so. A number of markedly distinct lineages (i.e., Dactylorhiza, Ophrys, Platanthera, and Serapias types) diverged more or less simultaneously from an ancestral group and little evidence (i.e., few apomorphies) was left in the ribosomal DNA of these plants from this first dynamic but short evolutionary period. Successive similar events led to the divergence of more derived groups (i.e., Aceras, Anacamptis, Barlia, Himantoglossum, and Neotinea). It is interesting to note that taxa in the former group (i.e., those placed basally in the cladogram of Fig. 1) are multispecific, whereas those in the latter group tend to be pauci- or monotypic. In addition to pollinator-mediated processes, another frequently suggested mechanism of production of new models of floral organization, i.e., interspecific hybridization, albeit commonly observed, does not seem to be the key to the origin of new bodyplans in the taxa in this study (with the possible exception of Anacamptis, discussed above). In fact, interspecific hybrids are found regularly but rarely. Most of the hybridization in the genus Orchis is usually restricted to a few hybrid plants, and hybrid swarms have rarely been observed (Dafni and Ivri, 1981; Dafni and Baumann, 1982; field observations). Moreover, among all the sequences obtained by us, we only occasionally found paralogous sites, very few of which were of apparent hybrid origin. This is in contrast with similar findings in other groups of angiosperms, for which paralogy seems to be a consequence of past hybridization (e.g., Sang et al., 1996). We are not, of course, suggesting that hybridization or the other kinds of mechanisms usually called upon as involved in speciation do not play any role in orchid evolution. For example, Hedre´n (1996) clearly indicates that allopolyploidy, and therefore hybridization, is a key factor in the genetic differentiation within Dactylorhiza. We are only implying that they are not responsible for the production of new floral organizations.

ITS DNA VARIATION IN Orchis

In conclusion, we think that the paraphyletic status of genus Orchis has been demonstrated beyond any reasonable doubt. Orchis may be regarded as representing a very successful, probably plesiomorphic body plan which has been rather frequently modified by selective pressures. Classical grouping of species within the genus has often been based on convergence or parallelism in floral traits, which has been misconstrued as synapomorphy. Given the specialization in reproductive morphology within the family, the tendency to emphasize morphological discrepancy has possibly been the main cause of the taxonomic inflation besetting the group. The findings reported here, merged with previous investigations on different orchid groups (Chase and Palmer, 1992; Haperman et al., 1996a,b), indicate that conspicuous morphological changes may be a consequence of minimal DNA rearrangements. Most probably, sequences in which minimal modifications cause drastic morphological changes are regulatory ones. In particular, mutations in floral homeotic genes have been repeatedly demonstrated to generate major floral restructuring (Weigel and Meyerovitz, 1994; Theissen and Saedler, 1995) and may well be involved in the origin of the strongly divergent floral shapes present in many European orchids. ACKNOWLEDGMENT The authors acknowledge a MURST 1997 grant by which the present research was partly funded. Note added in proof: The authors would like to communicate that, after sending the manuscript, they became aware of the publication of the following paper, whose authors independently reached results similar to theirs: Pridgeon A. M., Bateman R. M., Cox A. V., Hapeman J. R., and Chase M. W., 1997. Phylogenetics of subtribe Orchidine (Orchidoideae, Orchidaceae) based on nuclear ITS sequences. 1. Intergeneric relationships and polyphyly of Orchis sensu lato. Lindleyana 12: 89–109.

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