Evolutionary classification of European orchids

J. Eur. Orch.

41 (2): 243 – 318. 2009.

Richard M. Bateman

Evolutionary classification of European orchids: the crucial importance of maximising explicit evidence and minimising authoritarian speculation

Keywords Cladistics; classification; DNA; evolutionary mechanisms; evolutionary tree; genus; monophyly; morphology; nuclear ribosomal ITS; Orchidinae; phylogeny; plastid; reticulation; species delimitation.

Summary Bateman, R.M. (2009): Evolutionary classification of European orchids: the crucial importance of maximising explicit evidence and minimising authoritarian speculation. – J. Eur. Orch. 41 (2): 243–318.

The controversial Bateman–Pridgeon–Chase recircumscription of genera in the dominantly European subtribe Orchidinae (formulated in 1997 and updated in 2003) was generated by applying an explicit set of self-imposed rules to a phylogeny of 186 samples of Orchidinae and Habenariinae analysed for the nuclear ribosomal ITS region. Here, the five prioritised rules that were used to generate that classification are elucidated for the first time, and their implications for circumscribing several genera within Orchidinae are reviewed. During the last decade, many criticisms have been levelled at both molecular phylogenies and the resulting monophyletic classifications.

Although some criticisms have some validity, none represents a serious threat to the increasing dominance of statistically assessed monophyly. DNA-based phylogenies clearly provide the strongest basis for orchid classification, particularly at the genus level. The molecular phylogenies provide the best framework for comparing and interpreting additional data-sets that describe morphology, cytology and/or various of aspects of reproductive isolation; it is important that orchidologists continue to collect such intrinsic data. Extrinsic data such as geographical distribution, ecological preference, and pollinator

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and mycorrhizal specificity are also valuable but they alone are not sufficient to delimit species and higher taxa, nor are speculations regarding which of the many evolutionary mechanisms might have allowed particular taxa to originate.

Authors who reject monophyly as the key criterion for classification have not yet proposed a credible alternative approach that links explicit concepts and methodologies and shares the ability of monophyly to allow a wide range of quantitative data to be converted logically and directly into a natural classification. Despite the phylogenetic revolution of the last 30 years, many formal classifications are still being generated that reflect the ‘instincts’ of experienced taxonomists. Such authoritarian classifications are typically hybrids, compiled from portions of various previous classifications; these ad hoc constructs are far more difficult to justify than classifications that result from a single logical and explicit analysis of a particular carefully compiled, robust data matrix. Authoritarian classifications also tend to be geographically restricted (e.g. to Europe), whereas effective classification requires global analysis (i.e. a monographic rather than a floristic approach to taxonomy is necessary). Monophyletic classifications best serve conservationists and the many other users of taxonomic data, who are justly losing patience with a taxonomic community that apparently cannot agree its primary goals or the best means of achieving them.

Zusammenfassung Bateman, R.M. (2009): Über die entscheidende Bedeutung einer Maximierung von Befunden und Minimierung autoritärer Spekulation zur abstammungsgeschichtlichen Klassifizierung der europäischen Orchideen. – J. Eur. Orch. 41 (2): 243–318.

Die Revision der Gattungen der vorwiegend europäischen Subtribus Orchidinae nach Bateman–Pridgeon–Chase, veröffentlicht im Jahre 1997 und überarbeitet im Jahre 2003 war in der letzten Zeit vielfach Gegenstand kontoverser Bewertungen. Die Ermittlung der Phylogenie von 186 untersuchten Taxa aus den Subtriben Orchidinae und Habenariinae anhand der Analyse ihrer nuklearen ribosomalen ITS-Abschnitte gründete auf klar festgelegten, selbstdefinierten Regeln. Hier werden diese fünf prioritären Regeln zur ihrer Klassifizierung erstmal erläutert, ihre Auswirkung auf die Umschreibung verschiedener Gattungen der Orchidinae wird untersucht. In den letzten zehn Jahren wurde vielfach Kritik an molekularen Phylogenien und an den davon abgeleiteten monophyletischen Stammbäumen geäußert.

Auch wenn manche Kritik eine gewisse Berechtigung zu haben scheint, kann keine die gegenwärtig hervorragende Bedeutung statistisch abgesicherter

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Monophylie ernsthaft in Frage stellen. DNA-basierte Phylogenien liefern eindeutig die aussagekräftigste Grundlage für eine Klassifizierung der Orchideen, insbesondere auf Gattungsebene. Darüberhinaus sind molekulare Phylogenien bestens geeignet, zusätzliche Daten über Morphologie, Zytologie und/oder verschiedener Aspekte der Reproduktionsisolierung zu interpretieren. Deshalb ist die Erhebung solcher Daten durch Orchideenspezialisten weiterhin von großer Bedeutung. Ebenfalls wertvoll sind Daten über äußere Faktoren wie geographische Verbreitung, ökologische Präferenz und Bestäuber- und Mykorrhiza-Spezifität, allerdings sind sie alleine zur Abgrenzung von Arten und höheren Taxa nicht ausreichend, ebensowenig wie Spekulationen, welche der vielen möglichen Evolutionsmechanismen zur Entstehung bestimmter Taxa beigetragen haben.

Autoren, die Monophylie als Schlüsselkriterium zurt Klassifizierung ablehnen, haben bislang keine glaubwürdige Alternative angeboten, die explizit Konzepte und Methoden verbindet und wie die Monophylie die Eigenschaft besitzt, ein weites Spektrum quantitativer Daten logisch und direkt in ein natürliches Klassifizierungssystem zu überführen. Obwohl die letzten 30 Jahre eine phylogenetische Revolution erlebt haben, werden nach wie vor formale Klassifizierungen getroffen, die mehr den „Instinkt“ erfahrener Taxonomen widerspiegeln. Solche autoritären Klassifizierungen sind typischerweise hybridogener Natur, zusammengestellt aus Anteilen verschiedenen früherer Klassifizierungen; dabei sind diese ad hoc-Konstrukte weitaus schwieriger zu gerechtfertigen als Klassifizierungen auf Basis einer einzelnen, logisch durchgeführten Analyse definierter, sorgfältig zusammengestellter, robuster molekulargenetischer Daten. Autoritäre Klassifizierungen neigen auch stark zu geographischen Einschränkungen (z.B. auf Europe), während effektive Klassifizierungen weltweite Analysen erfordern (so erscheinen zur Klärung der Taxonomie bestimmter Gruppen eher monographische statt floristische Bearbeitungen erforderlich). Monophyletische Klassifizierungen sind bestens geeignet für die Bedürfnisse von Naturschützern und vieler anderer Anwender taxonomischer Daten, die allmählich die Geduld mit einer Gemeinschaft von Taxonomen verlieren, die sich nicht auf ihre primären Ziele und über den besten Weg, diese zu erreichen, einigen kann.

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1. Introduction: a brief history of biological classification I was encouraged to write this paper primarily by the recent reclassification of European members of the orchid subtribe Orchidinae by TYTECA & KLEIN (2008), which sought to critique, and ultimately to replace, the phylogenetic and systematic synthesis of the subtribe developed by PRIDGEON et al. (1997) and BATEMAN et al. (1997, 2003) – hereafter termed the ‘BPC’ (BATEMAN– PRIDGEON–CHASE) classification. The present review is deliberately broad in topics covered and personalised in style of presentation. It builds on previously published accounts of orchid systematics (BATEMAN 2001) and of the ITS phylogeny of tribes Orchideae (BATEMAN et al. 2003) and Neottieae (BATEMAN et al. 2005). My aim is to review the current status of phylogenetic classifications, to explore their relationship with evolutionary process (acknowledging that this year marks the 150th anniversary of the publication of The Origin by Charles DARWIN), and to explain how monophyletic classifications are more objective than, and thus preferable to, more traditional ‘authoritarian’ classifications.

Authoritarianism is defined by Chambers Dictionary as “setting authority above freedom; relating to, governed by, or stressing the importance and power of authority, or of a small group representing it; domineering, disciplinarian”. It is important to distinguish between something that is authoritarian and something that is authoritative: “having the sanction or weight of authority; accepted, approved, definitive, reliable”. Both terms are derived from the same Latin root and are frequently employed to describe the formal classifications produced by taxonomists – classifications that are intended to organise biological diversity into a framework that is both maximally informative and maximally utilitarian.

The contention underlying this paper is my belief that classifications should be authoritative rather than authoritarian. In other words, the relative merits of competing classifications should be assessed not according to the academic status and enthusiasm of their advocates but rather according to (1) the quality and quantity of the evidence (data) on which they are based, and (2) the explicitness and biological relevance of the rules by which those data have been converted into a specific formal classification. To help illustrate this point I will briefly review key events in the history of biological classification, beginning with an author who was, within his own lifetime, both authoritarian and authoritative: Carolus Linnaeus.

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1.1. Carolus Linnaeus – the organiser The father of formal classification, CAROLUS LINNAEUS (LINNÉ 1759), grouped organisms according to explicit rules that he himself formulated. In the case of flowering plants, his systematis sexualis (sexual system) used only the architectural features of the flower in a strict sequence of decreasing priority: (1) the number of stamens per flower, (2) the number of stamen whorls, (3) whether stamens are fused with each other, or with the pistil, (4) whether stamens occur in the same flower as the pistils, and (5) whether the flowers are visible to the naked eye (Figure 1). Thus, Linnaeus reflected the social politics of his day in prioritising the male parts of the flowers over the female and the sterile parts (SCHIEBINGER 1996; BATEMAN & SIMPSON 1998).

The resulting classification (and the associated diagnostic key) functioned well for its intended purposes. It provided a hierarchical framework that could be translated directly into a classification of ascending taxonomic ranks (subsequently formalised as species, genus, tribe, family, order, class). This in turn allowed organisation of binomial names (genus plus species, as in Orchis militaris L.) and provided both himself and other authors with a checklist of morphological characters of the flower that should be included in the formal description of any flower. And the combination of the name plus the morphological description (preferably supported by one or more illustrations) provided the basis for other interested persons to assign ‘unknown’ individual plants to that species – a routine, yet crucial, process that we now term plant identification.

This entire approach to organismal classification is rooted in a simple logic, clearly explained rules, and works well in practice. It is not surprising that it rapidly achieved dominance in the developed world, as it was both pragmatic and effective. Anyone seeking a deeper philosophical meaning in the pattern of morphological variation simply aimed to understand the detailed thought processes of God when He designed these organisms over the space of a mere three working days, generating all of the natural world in a near-instantaneous burst of supernatural diversification.

1.2. Charles Darwin – the heretic This perception of a beautifully simple, ordered and precisely and instantaneously crafted fixed universe was, of course, wrecked by CHARLES DARWIN’s (1859) infuriating success in identifying an equally simple

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mechanism – natural selection – that could credibly explain organic evolution through natural rather than supernatural means. Darwin replaced the instantaneous diversification engineered by a conscious creator with a genealogy of life that required an almost unimaginable period of time to elapse between the origin of life and the diversity of species observed by nineteenth century natural historians. In the intervening period, species periodically diverged to generate the diversity of species observed today.

Not surprisingly, DARWIN, together with contemporary advocates of Darwinism such as T.H. HUXLEY and E. HAECKEL, immediately selected the tree motif as being the most appropriate way to represent the diversification (and occasional extinction) of species through time. The obvious comparison with human genealogy also encouraged natural historians to think in terms of ancestors and descendants, and relative closeness of relationship (e.g. sisters versus cousins). Most importantly from a taxonomic perspective, the trees could be divided by taxonomists at specific branches in order to generate a natural hierarchical classification – one that was intended to reflect the relative closeness of relationship of different branches in the tree of life but retained the formal hierarchical levels of its Linnean predecessors. Evolutionary relationships offered the appealing theoretical prospect of using the shared evolutionary history of species to develop a more objective means of classifying them.

However, DARWIN’s evolutionary insights presented taxonomists with a serious dilemma. Natural historians were forced, often unwillingly, to reexamine the patterns of morphological variation that they were studying.

Instead of the perfection predicted by the biblical myth of creation, they now perceived an appallingly chaotic reality. Instead of a carefully organised universe of fixed invariant species, each carefully crafted by God and thus readily distinguished from all other species, they began to appreciate that DARWIN’s deceptively simple evolutionary mechanism led to complex and dynamic variations in morphology – variations that appeared so anarchic that it was difficult to discern any clear patterns. Also, even rational observers who accepted both DARWIN’s emphasis on the genealogy of species and his explanation of how it operated in nature remained perplexed by the failure of Victorian science to discern a credible mechanism by which morphological features (traits) could be inherited across successive generations.

Moreover, no explicit methodology was developed for reconstructing the tree (phylogeny) of life, or for independently estimating the accuracy of any particular inferred version of that tree, or for converting such a tree into a

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formal classification. Consequently, instead of rapidly realising its potential to unify phylogenetics and taxonomy (thereby helpfully reducing the range of opinions regarding how best to classify organisms), placing evolution at the centre of taxonomy further increased the diversity of opinions on how best to classify organisms. Thus, ironically, the widespread acceptance of evolution increased rather than decreased the role of authoritarianism in taxonomy, as contrasting inferences of evolutionary mechanisms were superimposed onto observed patterns of character variation. Classification consequently remained more of an art than a science.

1.3. Francis Crick – the mechanist We have already seen that even Linnaeus, a committed creationist, awarded a key classificatory role to reproductive characteristics. DARWIN similarly emphasised the importance of the relative reproductive success of individual organisms. Most notably, he reported, and sometimes even conducted, artificial crosses to demonstrate the importance of reproduction in underpinning his theory of evolution by natural selection. Reproduction was necessary to allow morphological change. However, a reliable process ensuring heritability between generations was also necessary, not only to perpetuate any morphological novelties but also limit their potential for further change, so that advantageous features that increased fitness would more likely be retained by a lineage. This truth was evident to the influential proponents of the ‘modern synthesis’ (e.g. MAYR, SIMPSON, STEBBINS: cf. HUXLEY 1940) in the 1930s and 1940s, even though they, like DARWIN, were unaware of the nature of the molecular mechanisms that permitted inheritance.

Most biologists would cite the discovery of the biochemical structure of DNA

by WATSON & CRICK (1953a, b) as the critical moment in understanding how traits are inherited, and thus how evolutionary changes can be preserved within lineages. However, I would identify as even more critical the subsequent discovery by FRANCIS CRICK (e.g. 1958) of how the sequence of triplets of bases in DNA is translated (via RNA) to dictate the sequence of amino acids when assembling proteins – the fundamental building blocks of life. If the nucleic acids DNA and RNA are the designs for life, proteins are the builders and also a significant portion of the construction materials.

As well as revealing the primary mechanism of inheritance, these scientific breakthroughs identified the sequence of bases in nucleic acids as being the best means of quantifying differences between genomes. This in turn

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permitted the development of a marvellous range of techniques for comparing the genomes of different species, in order to infer both their evolutionary relationships and whether they have become reproductively isolated (and, if so, for how long). Subsequent technical advances have served primarily to increasingly automate the production of such sequence data and thus to exponentially increase the volume of data available to taxonomists and other comparative biologists (e.g. PAGE & HOLMES 1998).

1.4. Willi Hennig – the formaliser At the same time as the ‘code of life’ was being broken by biochemists and geneticists, a few unusually visionary systematists, epitomised by the statistically-inclined pheneticists SOKAL & SNEATH (1963) and the pioneering cladist WILLI HENNIG (1950, 1966), were attempting to formalise how morphological data (but not yet DNA sequence data) could be scored in a matrix of species × characters that could then used to infer the nature of the underlying evolutionary tree. Both the pheneticists and the cladists also realised that describing the trees, and the character states that constituted the trees, would require the development of a new set of carefully and explicitly defined scientific terms. Their goals were to develop a conceptually rigorous analytical approach that sought to delimit natural (evolutionary cohesive) groups that were delimited by specific, identifiable character states. This required a tree-building method that was simple, logical, explicit and minimised circularity of logical argument.

Each species to be compared is scored for a set of characters that ideally should describe all of the organs of the plant, dividing each character into two or more contrasting character states. One of several methods (the simplest being parsimony, which constitutes the cornerstone of cladistics) is then used to build an evolutionary tree, termed a cladogram, from that basic data matrix. The shortest tree that can be constructed from the original matrix is considered to be the most likely approximation of the true relationships of the species included in the analysis. If the direction of character evolution is also to be inferred the tree must be rooted, such that the base of the tree can be viewed as coinciding with the origin of the study group and the tips of the ultimate branches represent the species analysed in that group. Today, rooting is almost always achieved by identifying and analysing one or more taxa that are closely related to the study group (which is termed the ingroup) but that are not considered to be part of the study group; these related taxa are termed the outgroups (cf. BATEMAN 2001).

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The resulting tree, whether phenetic or cladistic (Figure 2), has a shape (topology) that is considered to reflect the relationships among the species analysed. Points in the tree from which two or more lineages diverge are termed nodes, and can if desired be viewed as hypothetical ancestral species.

Two groups that link directly to the same node are said to be sister groups, and are considered to be of equal status. Three kinds of groups of species can be identified in any particular tree: monophyletic, paraphyletic and polyphyletic.

The main aim of cladistics is to identify monophyletic groups (clades); each clade consists of a hypothetical ancestor and all of its descendants. Only monophyletic groups are self-defining products of evolution and thus truly natural. Paraphyletic groups are also descended from a single hypothetical ancestor (i.e. have a single evolutionary origin) but one or more species is deliberately excluded from the group by the systematist, making the residual group artificial – it is no longer self-delimiting. The earlier diverging members of the paraphyletic group are said to be primitive relative to the monophyletic group nested within them, which is said to be derived (Figure 2). Both monophyletic and paraphyletic groups have only a single putative ancestor, but only a monophyletic group includes all of its descendents. In contrast, polyphyletic groups have two or more hypothetical ancestors (i.e. are evolutionarily separated by other species that are not considered to be part of the group), and so are clearly artificial; they are therefore the product of taxonomy rather than the product of evolution.

This set of explicit terminology extends to character states once these have been used to construct the tree. Transitions between contrasting states of the same character (e.g. long spur > short spur) can be related to particular branches of the tree. The older state (i.e. the state occurring closer to the root of the tree) is said to be plesiomorphic and the state that it changes into is said to be apomorphic. Character states uniting two or more sister species are said to be synapomorphic, whereas those that delimit just one analysed species are termed autapomorphic. Where a character undergoes the same transition in two or more different places on the tree, or becomes apomorphic but does so later (i.e. higher in the tree) by reverting to the plesiomorphic condition, that character is said to be homoplastic.

In theory, these statistical methods and terms can be applied to any evolutionary tree based on any kind of quantifiable data. In the 1970s and 1980s, it was easier and cheaper to score species for morphological data than for molecular (typically DNA-based) data, but the ease of generating DNA data greatly increased during the 1990s and 2000s; it is now much cheaper and less time consuming to generate DNA data than to generate morphological

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data. Also, there is a limit to the number of morphological characters that can readily be scored from an orchid (typically ca 50), whereas the number of molecular characters that can be scored has become astronomical (analyses of >10,000 DNA base-pairs are increasingly common). And, most importantly, molecular data simultaneously allow both evolutionary relationships among species and reproductive isolation to be inferred, whereas morphological data provide no direct evidence of isolation.

Given these facts, it should be no surprise that DNA data are today preferred by the great majority of professional systematists. Moreover, the cost of DNA analyses is now sufficiently low that such analyses need no longer be confined to professional researchers; soon, orchidologists will be able to carry into the field hand-held devices capable of instantaneously identifying orchids using DNA-based analyses (BATEMAN 2009a), an approach to identification termed DNA barcoding (e.g. SAVOLAINEN et al. 2005). However, there is a danger in these developments that morphological characters will be completely forgotten. Such arbitrary rejection of traditional data would be disastrous, as accurate morphological data are essential in order to understand the biology and the ecological role of each species (e.g. BATEMAN 2001).

1.5. Should monophyly supersede authoritarianism?

The inevitable consequence of these phylogenetic advances has been that, certainly above the species level, classification of orchids (and that of all other kind of organisms) has become dominated by the recognition of monophyletic groups that have been identified in trees built largely, or more often completely, from DNA data (e.g. ANGIOSPERM PHYLOGENY GROUP 1998, 2003, 2009; STEVENS 2009). Despite sporadic ongoing resistance, the phylogenetic approach now dominates comparative biology and classification of all groups of organisms, and guides the research of most professional systematists in the developed world.

Orchidologists remain leaders in the field of reconstructing phylogenies and developing monophyletic classifications (e.g. the Genera Orchidacearum project: PRIDGEON et al. 1999 et seq.), but the level of scepticism expressed by some parts of the orchidological community for the resulting classifications has also been unusually strong compared with the reactions of specialists in other plant families. As noted by many recent observers, including TYTECA & KLEIN (2008), some authors still refuse to accept the principles and/or the products of monophyletic classifications. And even if monophyly is accepted

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as the pre-eminent criterion for classification, some difficult decisions must still be made by the analyst when generating formal classifications from phylogenies. My motivations in writing this paper are therefore to: (1) make explicit the rules on which the BPC genus-level classification of European orchids (summarised by BATEMAN et al. 2003) was based; (2) explain and alleviate specific concerns raised about the construction and interpretation of phylogenetic trees, including those recently expressed by TYTECA & KLEIN (2008); (3) review the many evolutionary mechanisms that can cause speciation in orchids and explain why they are not directly relevant to supraspecific orchid classification, and, most importantly; (4) explain why the uniform application of monophyly is greatly preferable to maintaining the unscientific authoritarianism that has hitherto dominated, and consistently weakened, orchid taxonomy.

2. How to Derive a Monophyletic Classification From a Phylogeny The approach to converting any particular evolutionary tree into a classification that is recommended here is designed to provide a clear prioritisation of five explicit criteria and to minimise the number of decisions that must be made by the systematist. I have chosen to explain those criteria in terms of first the properties that I most wish my classification to possess and then the classificatory rules that are needed to adequately describe those properties: Property 1: Classification should consist only of natural (evolutionarily inclusive, self-circumscribing) groups; Rule 1: Recognise only monophyletic groups (clades) evident in the tree.

Property 2: Classification shows considerable stability when further data of the same or other kinds are gathered; Rule 2: Preferentially divide the tree at branches that are relatively robust; the best evidence of robustness is provided by various measures of statistical support, though relative branch length is usually an adequate proxy (but see discussion of long branches in Section 7.3).

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Property 3: Classification generates taxa at the same rank that show similar levels of divergence in the characters that were used to construct the tree; Rule 3: Preferentially divide the tree at branches that receive similar levels of statistical support (typically these are of approximately equal length).

Property 4: Each rank of the classification provides grouping information; Rule 4: Minimise the proportion of branches in the tree that simultaneously represent more than one taxonomic rank (most notably, terminal branches that represent supposed monotypic genera, such as Chamorchis alpina: cf.

BACKLUND & BREMER 1998).

Property 5: Classification minimises alterations necessary to existing Linnean names; Rule 5: Preferentially divide the tree in a way that minimises the need to (a) create new names and (b) create new combinations of existing names.

These five rules are sufficient to generate an explicit, logical, robust, and biologically justifiable classification from any substantial data matrix; they underlie the BPC classification of Orchidinae as summarised by BATEMAN et al. (2003; see also BATEMAN et al. 1997; PRIDGEON et al. 1997), though they have not been formally presented until now. Explicitly stating these rules helps to highlight the differences between our views and those of TYTECA & KLEIN (2008), who challenged Rule 1, supported Rule 2 (but did not discuss how robustness could be assessed), did not directly address Rule 3, and ignored the issues underlying Rules 4 and 5. Let us consider the many taxonomic consequences of adopting these contrasting viewpoints.

3. Optimally Dissecting the Orchidinae Tree I will now review eight of the most controversial groups in Orchidinae, together with brief reappraisal of the next-most diverse group of European orchids, the Neottieae. My aim is to explain how the protocol and rules described in the previous Section have been applied to generate the BPC classification (Figure 3). I will then consider two relevant properties that were emphasised by TYTECA & KLEIN (2008) but do not figure directly in the above protocol: (1) other evidence of reproductive isolation (most notably, frequency of natural hybridisation and chromosome numbers), and (2) the estimated amount of morphological divergence that separates taxa. Both of these issues are discussed at greater length in subsequent sections.

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3.1. Dactylorhiza group Controversy within this group focuses on the incorporation of the former monotypic genus Coeloglossum viride into an expanded Dactylorhiza by BPC. BATEMAN et al. (2003) and PILLON et al. (2007) used nuclear ITS sequences to place D. viridis only one node above the base of the Dactylorhiza clade. Using plastid trnL sequences, BATEMAN & DENHOLM (2003) placed D. viridis on the basal node, alongside three other major lineages within the genus (the D. incarnata group, the D. romana–sambucina group, and the D. fuchsii– maculata group). DEVOS et al. (2006a, b) gathered sequence data from the ITS region and the similar nuclear ribosomal region ETS. Combining the data placed D. viridis as sister to the remaining species of Dactylorhiza. However, the outgroup used by DEVOS et al. contained only Gymnadenia, and three of the four species of Gymnadenia were not sequenced for ETS, raising the possibility that the tree was not optimally rooted.

Applying the five rules listed above, Rule 1 (monophyly) precludes recognition of Coeloglossum as a separate genus in the trees published by BATEMAN et al. (2003), BATEMAN & DENHOLM (2003) and PILLON et al.

(2007), but genus-level recognition is in principle permitted by the tree published by DEVOS et al. (2006b). However, the tree of DEVOS et al. (2006b) fails Rule 2; the branch separating D. viridis from the other Dactylorhiza species is no longer than the branch subtending the D. incarnata group, whereas the branch leading to the sister genus, Gymnadenia, is significantly longer than any branch within Dactylorhiza s.l. Thus, the tree also fails Rule 3; even in the topology of DEVOS et al., the branch that should be cut to circumscribe genera is the longest one – that separating Dactylorhiza s.l. from Gymnadenia s.l. Also, Coeloglossum fails Rule 4, as the supposed genus is monotypic and so provides no information on grouping together multiple species. And when expanding genus Dactylorhiza, we deliberately avoided major nomenclatural changes by conserving the genus Dactylorhiza against Coeloglossum, so that only one new nomenclatural combination was required (D. viridis); thus, Rule 5 was satisfied.

Hybridisation and chromosomal evidence also favour inclusion of ‘Coeloglossum’ in Dactylorhiza, as it has the 2x = 40 chromosome number that characterises the Dactylorhiza s.l.–Gymnadenia s.l. group and hybridises infrequently but widely with other co-occurring species of Dactylorhiza.

Thus, any case for recognition of Coeloglossum must rely on (a) preferring the tree of DEVOS et al. (2006b) over the other competing trees so that

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Dactylorhiza minus D. viridis can be tentatively viewed as monophyletic, and (b) emphasising the importance of several morphological differences enumerated by DEVOS et al. (2006b) and TYTECA & KLEIN (2008) between the flowers of D. viridis and those of other Dactylorhiza species.

This conflict illustrates well the difference between overall phenetic distance (given three taxa, which two are most similar?) and phylogeny (given three taxa, which two are most closely related – in other words, which two have the most recent shared ancestor?). Often these two distinct principles agree but sometimes they disagree. For most of the history of taxonomy, some form of similarity (typically assessed crudely and without quantification or explicit rules) has dominated, whereas today it is more commonly (and justifiably) subordinated to closeness of relationship (Section 9). Similarity has been constrained to a more appropriate role, specifically circumscribing species from individuals via populations (BATEMAN 2001).

3.2. Nigritella group The ITS tree of BATEMAN et al. (2003) placed the former genus Nigritella well within the genus Gymnadenia, but all species in the group are separated by relatively short branches. In contrast, a plastid trnL analysis that was reported in summary by BATEMAN & DENHOLM (2003) showed Gymnadenia and Nigritella as sister taxa, again separated by short branches – a result similar to that obtained via phenetic analyses of allozyme (protein) data by HEDRÉN et al.

(2000).

Thus, it remains unclear whether Nigritella is monophyletic or paraphyletic under Rule 1). However, it is clear that there are only small molecular differences between species of Nigritella and species of Gymnadenia s.s., whereas the branch that subtends the group combining the two subgenera is considerably longer. Thus, like Coeloglossum, Nigritella fails Rules 2 and 3; the obvious branch to cut is the long, robust branch that subtends both Gymnadenia and Nigritella. Because Nigritella contains at least a dozen species (e.g. TEPPNER & KLEIN 1985, 1998) rather than being monotypic, it does not contradict Rule 4. Nonetheless, there are arguably more species of Gymnadenia s.s. than of Nigritella, thereby justifying the synonymisation of Nigritella into Gymnadenia.

If we now consider reproductive isolation, hybrids are occasional but widespread between ‘Nigritella’ and Gymnadenia s.s., and have led to at least

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one stabilised hybrid combination that has been described as an apomictic species (HEDRÉN 1999). Moreover, both groups have a base chromosome number of 2x = 40 and show a tendency to generate tetraploids and occasional hexaploids (TEPPNER & KLEIN 1985). Overall, the situation regarding Nigritella is very similar to that regarding Coeloglossum. Any attempt to justify continued recognition of Nigritella must accept trees showing Gymnadenia s.s. as monophyletic and reject trees showing Gymnadenia s.s. as paraphyletic. It must also justify the generic distinction on the basis of the many indisputable differences between the flowers of Gymnadenia s.s. and ‘Nigritella’. Again, the modern concept of relationships must be rejected in favour of the traditional concept of overall similarity if Nigritella is to be recognised as a genus.

3.3. Orchis s.s. group The ITS phylogeny shows that Orchis s.s. is undoubtedly monophyletic and contains three well-supported species groups: Group 1 contains the O. quadripunctata plus O. anatolica plus O. spitzelii subgroups, Group 2 is built around O. mascula and O. pauciflora, and Group 3 contains most of the anthropomorphic species such as O. simia and O. punctulata (Figure 3). The phylogenetic (and thus taxonomic) problems are caused by the uncertain placements of the O. provincialis–O. pallens lineage (which morphology predicts should place in the O. mascula group) and of O. italica and O. (formerly Aceras) anthropophora, whose basal placements within Orchis s.s. render the anthropomorphic Orchis group apparently paraphyletic. However, neither of these inconvenient phylogenetic placements gains statistical support from bootstrap analysis; it is therefore possible that the anthropomorphic Orchis species and the O. mascula group s.l. (i.e. including O. provincialis) are each monophyletic, but such relationships are not presently demonstrable.

Thus, any comprehensive genus-level division within the Orchis s.s. clade, such as that advocated by TYTECA & KLEIN (2008), will contravene Rule 1.

In the absence of robust supporting branches, and given the risk of future reassessment of the group based on additional gene regions, Rule 2 prevents reliable classification within the group, and certainly does not encourage reassignment of the non-anthropomorphic Orchis species to the separate genus, Androrchis, proposed by TYTECA & KLEIN (2008). Separation of any new genera is also discouraged by Rule 3: the length of the branch subtending the non-anthropomorphic Orchis species is sufficiently short that it is exceeded by the branch subtending the O. quadripunctata–anatolica–spitzelii

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group (Figure 3). TYTECA & KLEIN’s ‘Androrchis’ passes Rule 4, as it avoids establishing monotypic genera, but it fails Rule 5, as it requires many new nomenclatural combinations.

The case for separating the anthropomorphic Orchis species from the remainder relies on their apparent reproductive isolation. Although they show rampant hybridisation among themselves (PEITZ 1970; BOURNÉRIAS & PRAT 2005; BATEMAN et al. 2008), credible reports of hybrids between anthropomorphic Orchis and species of other groups are rare (KRETZSCHMAR et al. 2007). It would be helpful if our knowledge of their chromosomes (e.g.

BIANCO et al. 1991) could be raised to the same level as that of the former species of Orchis now placed in Anacamptis (D’EMERICO et al. 1996). This is a valid argument for separating the anthropomorphic species from the remainder, but it is not a compelling one.

3.4. Anacamptis group Anacamptis pyramidalis is undoubtedly deeply embedded within the remainder of the expanded genus. Thus, there is no question that recognising the remaining species of Anacamptis s.l. as a new genus, Herorchis (TYTECA & KLEIN 2008), formalises a paraphyletic genus, thereby clearly failing Rule 1. The substantial and approximately equal molecular divergence evident among the several recognisable species-groups that constitute Anacamptis s.l. would mean that, to satisfy Rule 3, Anacamptis s.l. would need to be divided not into two genera but into at least five, based respectively on A. laxiflora, A. collina, A. pyramidalis, A. coriophora, A. papilionacea and A. morio–boryi.

Branches within the group are (just) sufficiently long to give the level of robustness required by Rule 2. Most of these lineages would be monotypic or near-monotypic, thereby breaking Rule 4, and recognition of ‘Herorchis’ requires numerous new combinations, thereby contravening Rule 5.

Considerable karyotypic diversity has been documented within Anacamptis s.l., including reduction from 2n = 36 to 2n = 32 in A. papilionacea (D’EMERICO et al. 1996), but there is nothing chromosomally distinctive about A. pyramidalis. Moreover, A. pyramidalis generates sporadic natural hybrids with several other species of Anacamptis, most commonly members of the A. morio–boryi group. Thus, the case for separating A. pyramidalis at genus level relies entirely on the happenstance adaptation of its flowers for pollination primarily by lepidoptera rather than by bees; this is a wholly inadequate justification, given the diversity of pollinators documented within other genera of Orchidinae (e.g. CINGEL 1995).

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Overall, the case for splitting Anacamptis s.l. into five genera is stronger than the case for dividing it in just two. Nonetheless, continued recognition of a single, morphologically heterogeneous Anacamptis s.l. receives greatest support from the available data.

3.5. Neotinea group TYTECA & KLEIN (2008) chose to return to a monotypic concept of the genus Neotinea, recognising only N. maculata (syn. N. intacta), and so argued that the remaining species in the genus should be transferred to a new genus, Odontorchis. This would be permitted by monophyly (Rule 1), as N. maculata is undoubtedly sister to the remaining species (Figure 3). However, the branch subtending the remaining species is relatively short (despite TYTECA & KLEIN’s argument that the genetic distance is “large”); thus, it contravenes Rules 2 and 3. Also, cutting this branch recognises a monotypic genus, contravening Rule 4, and requires several new combinations, contravening Rule 5.

TYTECA & KLEIN (2008) rightly state that there is no convincing evidence of hybridisation between N. maculata and other species assigned by BPC to Neotinea, but as N. maculata is autogamous, the lack of reported hybrids is hardly surprising. In addition, TYTECA & KLEIN’s argument that N. maculata has a chromosome number of 2n = 40, whereas other species of Neotinea have 2n = 42, masks a far more interesting pattern. The overall topology of the ITS tree clearly indicates an ancestral chromosome number of 2n = 42 for Neotinea s.l., but there is no doubt that there are reliable counts of both 2n = 42 and 2n = 40 within the genus. However, these values are not species-specific. In Italy, N. tridentata s.l. maintains both diploid (2n = 42) and tetraploid (2n = 84) cytotypes. Significantly, all three species of Neotinea occurring within the Iberian Peninsula (N. maculata, N. conica and N. ustulata) have yielded reliable counts on both 2n = 42 and 2n = 40. Moreover, there is some evidence of geographic concentration of contrasting cytotypes (BERNARDOS & AMICH 2002; BERNARDOS et al. 2004; AMICH et al. 2007). Thus, instead of chromosome number distinguishing N. maculata from the remainder of the genus, as argued by TYTECA & KLEIN, the ability to maintain contrasting karyotypes within several species of the genus actually constitutes a polymorphic character that unifies the expanded genus Neotinea. There is no substantive case for recognising a monotypic genus based on N. maculata.

I will now consider four clades of European orchids that were not discussed by

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TYTECA & KLEIN (2008) but that also present taxonomic conundra at the genus level. Decisions made for the Himantoglossum clade and the Traunsteinera– Chamorchis clade in particular are more open to criticism than the more widely debated examples, discussed above, that have attracted the attention of numerous commentators.

3.6. Platanthera group The Platanthera s.l. clade is relatively poorly represented in Europe; in order to understand its phylogeny, Asian and North American species should also be considered. Platanthera is in some ways analogous to Anacamptis; it contains several lineages that are sufficiently divergent in both DNA and morphology that they could be (and in the past have been) treated as several genera, each relatively species-poor (cf. HAPEMAN & INOUE 1997; SHEVIAK 2002; BATEMAN et al. 2003, 2009a).

Contrary to figure 7 of TYTECA & KLEIN (2008), the ITS tree of BATEMAN et al. (2003) tentatively placed Pseudorchis as sister to the unequivocally monophyletic Galearis–Neolindleya–Platanthera clade rather than the Gymnadenia s.l.–Dactylorhiza s.l. clade (Figure 3). Interestingly, the molecular branch subtending the genus is unusually short relative to most other lineages in Orchidinae, suggesting a comparatively low mutation rate. The Asiatic–North American genus Galearis is clearly monophyletic, and has as its unequivocal sister the monotypic genus Amerorchis (BATEMAN et al. 2003).

However, the greatest instability in this region of the Orchidinae tree is caused by another monotypic genus, Neolindleya, which could equally be sister to either Galearis or Platanthera (BATEMAN et al. 2003, 2009a). Indeed, a recent study used morphology to arbitrate between these two molecularly defined options, suggesting that Neolindleya is an autogamous lineage that is more closely related to Galearis (EFIMOV et al. 2009). The ITS branches subtending Galearis and Amerorchis are arguably sufficiently long to warrant genus status, though they are shorter than some branches within the genus Platanthera (Figure 3).

Thus, there are several contrasting ways that this clade could be divided into genera. Within the constraint of monophyly (Rule 1), a case could be made for incorporating the two monotypic genera, Amerorchis and Neolindleya, into an expanded Galearis. This solution would usefully eliminate two genera that are monotypic and so currently contravene Rule 4 (and only two new combinations would be required under Rule 5). This taxonomic change has

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recently been suggested for Amerorchis (BATEMAN et al. 2009a). In contrast, including Neolindleya in Galearis is discouraged by Rule 2; the branch supporting this inferred relationship is insufficiently robust.

3.7. Himantoglossum group The challenges posed by the Himantoglossum group are very similar to those presented by the Platanthera group. The crown of the clade consists of several closely similar species of Himantoglossum s.s. Molecular branches of approximately equal and considerable length separate this species group from (in succession, moving down the tree) ‘Barlia’, ‘Comperia’ and Steveniella.

‘Barlia’ contains only two species, and both ‘Comperia’ and Steveniella are monotypic. The branches supporting these relationships are long and robust.

However, it could be argued that, in compiling the BPC classification, we did not follow our own prioritisation of the rules in the case of these taxa. The considerable length of these branches could permit recognition of these species as separate genera; we thereby prioritised Rule 4 (the desire to eliminate monotypic genera) over Rule 3 (recognising genera subtended by molecular branches of similar length). It may also appear inconsistent to have failed to incorporate Steveniella into Himantoglossum, but in this case the putative sister-group relationship of Steveniella to Himantoglossum s.l. is insufficiently strongly supported; Rule 2 therefore prevents their amalgamation.

There is also considerable morphological divergence between Steveniella and the rest of the Himantoglossum s.l. clade (cf. DELFORGE 1999), while hybridisation records show occasional gene flow between Himantoglossum s.s. and the former genera Barlia and Comperia (KARATZAS 2004). In contrast, literature survey suggests that no gene flow has thus far been recorded with Steveniella. Sadly, we have not yet obtained an ITS sequence from Himantoglossum formosum, which appears morphologically intermediate between Himantoglossum s.s. and the former genus Barlia (BATEMAN et al.

2003).

3.8. Chamorchis/Traunsteinera group To the best of my knowledge, no comment has ever been made regarding the tentative decision of BATEMAN et al. (2003) to maintain the Alpine specialists Chamorchis and Traunsteinera as separate, arguably monotypic genera. The

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ITS phylogeny clearly shows that they are sister species, but the molecular branches subtending each species are short (even shorter than those subtending Dactylorhiza s.l. and Gymnadenia s.l.), whereas the branch subtending the two species together is longer (though still short; these species appear to share with Pseudorchis, another alpine specialist, a characteristically slow mutation rate). Thus, the two species constitute a monophyletic group, and the most appropriate branch to cut would be that subtending both genera (thereby satisfying Rules 1–3). In addition, if both genera are viewed as monotypic, they do not provide any useful grouping information, whereas a single genus combining the two genera (Chamorchis has nomenclatural priority) would convey more phylogenetic information, thus satisfying Rule 4 and requiring only at most two new combinations under Rule 5.

If we seek relevant information beyond the ITS phylogeny, the two putative genera also have congruent geographical distributions and similarly alpine habitat preferences. The chromosomes of Chamorchis (D’EMERICO & GRÜNANGER 2001) have been studied in greater detail than those of Traunsteinera (e.g. CAUWET-MARC & BALAYER 1984), proving to be comparatively distinct. Thus, at present, the only valid arguments against their unification into a single genus are their relatively low mutation rate (reflected in the short subtending branches) and their strong morphological divergence in both floral and vegetative features. Following our own rules, the junior genus, Traunsteinera REICHENBACH (1841), should perhaps have been incorporated into Chamorchis RICHARD (1817). It is interesting to note that our decision to maintain the taxonomic status quo for these genera, which arguably was inconsistent with our principles, did not attract comment from other orchidologists. This observation well illustrates the general principle that too much attention is paid to taxonomic change relative to taxonomic stasis; when new sources of data come to light, classification of the entire group should be reconsidered, paying equal attention to each putative species under scrutiny.

3.9. Neottia group (Neottieae) I am surprised that our subsequent decision to amalgamate Listera and Neottia (BATEMAN et al. 2005; BATEMAN 2006) has sparked little discussion in subsequent literature (one exception was BOURNÉRIAS & PRAT 2005). This decision was based on molecular comparison of only three European and one North American species of tribe Neottieae: Neottia (Listera) ovata, Neottia (Listera) cordata, Neottia (Listera) smallii and Neottia nidus-avis. Far

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stronger species sampling of the clade is desirable. Nonetheless, within the context of this comparison, N. nidus-avis and N. ovata are clearly sister species, relatively distantly related to N. cordata; this topology was obtained not only from nuclear ITS sequences but also from a combination of four plastid genic regions. Thus, Listera as traditionally circumscribed is undoubtedly paraphyletic, and both monophyly (Rule 1) and robustness of the branch subtending the combined group (Rule 2) requires amalgamation of Neottia and Listera, even though the branch subtending Neottia is also sufficiently long to justify genus status (Rule 3). Eliminating Neottia, the far less species-rich of the two former genera, would best satisfy Rule 4.

Unfortunately, recognising nomenclatural priority for Neottia contravenes Rule 5, as far more new combinations are required than if we had synonymised Neottia into Listera. We therefore considered recommending nomenclatural conservation of Listera over Neottia. However, rather than introduce novel recombinations of Neottia species into Listera, we took a pragmatic decision to use the combinations of Listera into Neottia that had been made 12 years earlier by SZLACHETKO (1995; also SZLACHETKO & RUTKOWSKI 2000) on the basis of the strongly similar gynostemium morphology of the two former genera.

Examining morphology more broadly confirms the strikingly similar floral morphologies of Neottia nidus-avis and Listera ovata. Although there are several obvious differences in vegetative morphology between the two species, they can all be attributed to the one-way evolutionary transition from autotrophic to obligate mycoheterotrophic (‘saprophytic’) nutrition that accompanied the origin of Neottia s.s. (BATEMAN et al. 2005). Acquisition of mycoheterotrophy is not in itself sufficient justification for recognition as a genus; several other orchid genera combine photosynthetic and mycoheterotrophic species (not least Cephalanthera, which includes the Asian-American mycoheterotroph C. austiniae: LUER 1975; BATEMAN et al.

2005).

Having briefly considered several specific case-studies, I will now address some of the relevant conceptual and practical issues in modern classification, including all of those raised by TYTECA & KLEIN (2008).

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4. Rising Above Reticulation: Reassessing Hybridisation Although the classification of TYTECA & KLEIN (2008) primarily concerns the circumscription of genera, their many criticisms of cladistics fail to distinguish between applying the cladistic method at or below the species level and above the species level (Figure 4). They rightly draw particular attention to the complications caused by what are known as reticulation events, when genes ‘disobey’ the requirement that, once they have diverged, evolutionary lineages should become and remain reproductively isolated (Figure 5). Under these circumstances, some of the genetic interactions contradict the tree motif, and instead form a network that can, in theory, introduce errors into a molecular tree. Orchids are, of course, especially prone to hybridisation, partly due to the frequent weakness or even absence of post-zygotic (i.e. internal chemical or chromosomal) barriers to gene exchange. Instead, many orchids rely heavily on pre-zygotic barriers, notably pollinator preference (e.g. TREMBLAY et al.

2005; SCHIESTL & COZZOLINO 2008). It is also possible for minute portions of genomes to be transferred between even very distantly related lineages via, for example, viral vectors (WON & RENNER 2003; BERGTHORSSON et al. 2004).

This process is termed lateral gene transfer and was raised as a possible cause of erroneous tree topologies by TYTECA & KLEIN (2008). However, to the best of my knowledge, lateral gene transfer has not yet been recorded in orchids, despite the routine infection of their roots by endomycorrhizae which offers one obvious potential source of ‘jumping genes’. Hybridisation, rather than lateral gene transfer, therefore remains the main concern with regard to possible reticulation events.

The most crucial factor in assessing, and if necessary mitigating, the effects of natural hybridisation is being aware of the risk that it could have occurred. We can then deploy a range of analytical techniques to explore hybridisation, including producing our own artificial hybrids of known parentage (e.g.

MALMGREN 1992, 2008; SCOPECE et al. 2007; COZZOLINO & SCOPECE 2008).

Suspected natural hybridisation is most effectively explored by gathering DNA sequence data for the same individuals from two different genomes within the plant: the chromosomes in the nucleus, and the plastids in the cytoplasm. Chromosomal genes are inherited equally from both parents, whereas the plastids are inherited only from the ‘mother’ (more correctly termed the seed parent) (BATEMAN 2001; BATEMAN et al. 2008). Thus, it is highly desirable that we generate separate evolutionary trees from at least one chromosomal gene region (e.g. ITS or ETS) and at least one plastid region (e.g. rbcL, trnL, matK), and compare the resulting topologies (tree shapes).

The topologies are always broadly similar, but the placement of one species in

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radically different locations in the two trees might well indicate recent hybridisation. In addition, sequencing the ITS or ETS region of a hybrid would reveal at least two contrasting copies, one from the mother and one from the father, for a minimum of several generations after the hybridisation event.

Admittedly, a process termed concerted evolution eventually eliminates all but one copy; current evidence suggests that this process occurs relatively rapidly in orchids (e.g. PILLON et al. 2007).

Almost all bona fide orchid species native to Europe have now been sequenced for nuclear genomes and many for plastid genomes, though publication of the plastid data by various authors (including myself) has been undesirably slow, largely because the plastid genomes routinely generate the same topologies as ITS but produce fewer informative characters and consequently yield branches that are less robust (cf. SOLIVA & WIDMER 2003; BATEMAN & DENHOLM 2003; BATEMAN et al. 2008; DEVEY et al. 2008).

Deriving similar results from the near-independent nuclear and plastid genomes within each individual orchid helpfully demonstrates the reliability of the molecular phylogenetic method. Difficulties were encountered exactly where experience told us that we should expect them – specifically, between infraspecific taxa of the same species or between supposed species whose species status was already considered questionable.

For example, TYTECA & KLEIN (2008) cite my own studies comparing Platanthera bifolia with P. chlorantha (e.g. BATEMAN et al. 2009b) as showing that DNA sequencing cannot discriminate between all bona fide European orchid species. Certainly, until gene exchange between potentially divergent lineages has reached a very low level, the ‘embryonic’ species in question will not be able to develop their own unique genetic signatures through fixation of novel mutations (Figure 6). But until they have reached that low level of gene exchange, should the embryonic lineages actually be regarded as separate species? If one follows TYTECA & KLEIN in choosing to emphasise the importance of reproductive isolation, the answer is likely to be ‘No’. The lag phase between the achievement of a high degree of reproductive isolation and fixation of unique mutations in gene regions such as ITS is short (Figure 6). One could perhaps argue that P. chlorantha has very recently separated from P. bifolia, though this possibility seems unlikely, given the extensive and coincident geographical ranges of the two putative species. In the case of the genus Ophrys, only at most ten molecularly circumscribable species have been detected within the genus, irrespective of whether ITS sequences, plastid sequences or AFLPs are under consideration (DEVEY et al.

2008, 2009). Can we realistically argue that 241 of DELFORGE’s (2006) 251

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putative species of Ophrys are, by some remarkable coincidence, currently existing in that brief period of ‘phylogenetic limbo’ between achieving reproductive isolation and acquiring their own distinct ITS sequences? This coincidence is too improbable to consider seriously (BATEMAN et al. (2009C).

Moreover, similarly weak divergence characterises another archetypal taxonomic nightmare, Serapias (BATEMAN et al. 2003; PELLEGRINO et al.

2005).

Another problematic example that could legitimately have been raised by TYTECA & KLEIN (2008) concerns the anthropomorphic Orchis taxa (their ‘genus Orchis’ sensu strictissimo). The recent study by BATEMAN et al. (2008) showed that in western and central Europe there is extensive hybridisation among O. purpurea, O. militaris and O. simia, demonstrating the closeness of relationship of these imperfectly separated putative species. In contrast, the more molecularly divergent anthropomorphic Orchis species, O. italica and O. anthropophora, do not show blurred genetic boundaries. Although they also are occasionally observed to hybridise with each other and with the O. purpurea–militaris–simia group (e.g. BOURNÉRIAS & PRAT 2005), levels of gene flow are clearly insufficient to render these species genetically inseparable.

One important issue that has not received sufficient discussion in the literature is the question of which genetic markers should be prioritised when seeking to delimit species and supraspecific taxa. On the one hand, there is no doubt that fully expressed genic regions such as the popular plastid regions rbcL and matK are too conservative (i.e. mutate too slowly) to discriminate phylogenetically among all bona fide orchid species. On the other hand, increasingly popular genetic ‘fingerprinting’ techniques such as AFLP (cf.

HEDRÉN et al. 2001; BATEMAN et al. 2008; DEVEY et al. 2008, 2009; STÖKL et al. 2008) and nuclear microsatellites (e.g. MANT et al. 2005) are guaranteed to yield some discrimination among cross-pollinating individuals even if they are all conspecific. With the possible exception of the very similar ETS (DEVOS et al. 2006), no other genic region has proven able to reliably differentiate between all but the most controversial species circumscribed using morphology but show high levels of uniformity within morphospecies to the degree achieved by ITS. A particularly interesting recent study was the exploration of sequence variation in the low-copy nuclear gene LFY (Leafy) within the Ophrys fusca s.l. group by SCHLÜTER et al. (2007). This gene family ostensibly shows considerable promise, as it appears able to discriminate among microspecies that shared the same sequence in the ITS studies of BATEMAN et al. (2003) and DEVEY et al. (2008). However, three microspecies

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within the fusca group were analysed for either two or three individuals yielding LFY sequences, and despite the very limited sampling, two of these three supposed species failed to resolve as monophyletic. This implies either that the morphologically circumscribed microspecies are not bona fide species or that LFY mutates too readily to be conserved at the species level – an observation consistent with the exceptional variation in the length of this genic region reported by SCHLÜTER et al. (2007).

One of the features that I have chosen to emphasise in the past (e.g. BATEMAN et al. 1997, 2003; BATEMAN 2008) when advocating the BPC generic recircumscriptions within Orchidinae is that they almost eliminated supposed natural hybrids between genera (a strength of the BPC classification that was acknowledged by TYTECA & KLEIN 2008). Intergeneric hybrids had previously been considered commonplace as a result of inadequate generic circumscription. The most notable exception is hybridisation between the two re-circumscribed genera that were separated by the shortest branches in the ITS tree of BATEMAN et al. (2003): Dactylorhiza (including the former Coeloglossum) and Gymnadenia (including the former Nigritella). Any large mixed population of, for example, Dactylorhiza fuchsii and Gymnadenia conopsea found in the British Isles generally yields a few hybrid individuals upon close inspection. But here again, as one would expect, levels of gene flow are far too low to blur the reliable genetic distinctions between the species involved.

Evidently, the relationship between species circumscription and genetic divergence is of considerable importance. For example, Salvatore COZZOLINO and colleagues have repeatedly used the BPC ITS tree as a key element in their exceptional series of papers inferring different speciation mechanisms, reflecting contrasting approaches to achieving a high degree of reproductive isolation, in rewarding, food-deceptive and sexually-deceptive groups of orchids (e.g. COZZOLINO & WIDMER 2005a, b; SCOPECE et al. 2007; COZZOLINO & SCOPECE 2008). The average degree of genetic divergence within these three reproductively defined groups of orchids is critical to the process-based interpretation, which in turn means that it is essential that species showing little if any sequence divergence are indeed bona fide species.

Evolutionary interpretations are inextricably linked to taxonomy.

Can we draw any broader conclusions regarding gene flow? Most importantly, TYTECA & KLEIN (and some other influential European orchidologists) contrast molecular phylogenetics with reproductive isolation, apparently arguing that isolation is a more important criterion for classification than

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molecular divergence. Yet molecular phylogenetic data directly reflect the levels of gene flow that in turn allows us to assess degrees of reproductive isolation. Admittedly, we are inferring levels of gene flow that have been generalised across both the geographic distribution and recent history of the species in question. More ecologically-oriented students of reproductive isolation, who operate through direct observation of pollination events (e.g.

DAFNI 1987; PAULUS & GACK 1990; CINGEL 1995; PAULUS 2006), would no doubt argue that such generalisation is a crude, post hoc echo of actual reproductive behaviour. In contrast, I would argue that the time-averaged nature of sequence data is a strength rather than a weakness, given that direct ecological observations of pollination are too limited in space and time to greatly help us delimit species – pollination observations are usually restricted to small geographical areas and are inevitably confined to periods of observation far shorter than evolutionary timescales. In contrast, DNA sequences summarise the entire evolutionary history of the lineage.

5. “Almighty Monophyly” is Most Effective Above the Species Level But Still Requires Statistical Tests Although monophyly can be applied successfully at or below the species level, we have already seen that reticulation through hybridisation complicates its application and weakens its general utility. I suspect that most embryonic orchid lineages (‘prospecies’) never achieve the biological independence that accompanies a high degree of reproductive isolation. They are eventually reunited with their ancestral lineage and so fail to attain a status most appropriately recognised as full species (this statement especially applies to the great majority of the many supposed species listed by some authorities within Ophrys: [BATEMAN et al. (2009C]). However, once a lineage has achieved the ‘escape velocity’ needed to emerge from its genetically complex, ‘embryonic’ genetic divergence lag phase, it rapidly acquires fixed mutations that allow the DNA-based recognition and identification of all members of that lineage (Figure 6). At this point, most of the criticisms of monophyly as the pre-eminent criterion for classification (cf. SOSEF 1997; WUCHERPFENNIG 1999, 2002; BRUMMITT 2002; HÖRANDL 2006; ZANDER 2007, 2008; TYTECA & KLEIN 2008) cease to apply.

When we develop cladistic classifications, we are not just seeking groups that appear monophyletic in trees based on the particular dataset that we have accumulated – we are actually seeking groups that are so strongly statistically supported that they will be perceived as being monophyletic in most or all of

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the datasets likely to be gathered by researchers in the future. In other words, the primary aim of phylogeny-based taxonomy is to generate a formal classification that is reliably predictive. Thus, the BPC classification divided the ITS tree for Orchidinae at the most robust – and hence most predictive – branches. In general, the most robust branches are the longest. In order to compare branch lengths, cladistic trees should routinely be presented as phylograms, showing branch lengths proportional to the number of characterstate transitions on that branch (Figure 3), rather than as cladograms, with all branches shown artificially as being of equal length (contra figure 7 of TYTECA & KLEIN 2008).

Fortunately, there are better measures of the robustness of branches than simply their relative lengths. This is why most cladists include on their trees statistical estimates of the robustness of the branches on their trees (surprisingly, these measures were not discussed by TYTECA & KLEIN 2008).

For parsimony trees these statistical tests take two forms. Resampling techniques such as the bootstrap and jackknife, together with posterior probability, are expressed as percentages, whereas the decay index (= Bremer support) records the number of additional steps that the evolutionary tree can withstand while still circumscribing that particular branch. All of these measures are relative rather than absolute – branches can only be compared within the context of that particular dataset, rather than between trees for the same species generated from different datasets. The decay index is arguably the better measure, as it has a theoretically infinite maximum value. In contrast, bootstrap, jackknife and especially posterior probability assessments reach the maximum value of 100% rather easily, so they cannot be used to distinguish a strongly supported branch from a very strongly supported branch (e.g. FROHLICH 2006). I have previously argued that many of my colleagues rely too heavily on these measures of statistical support, and noted that a bootstrap value of 100% is no guarantee of a genuinely monophyletic group (e.g. BATEMAN et al. 2006). Nonetheless, all of these measures of robustness within a particular dataset provide valuable guidance to taxonomists when they convert a tree into a formal classification.

TYTECA & KLEIN (2008) caution against making “hasty decisions” in classification. The approach used in generating the BPC classification was indeed duly cautious, but it focused on generating a genus-level classification most likely to survive both (a) the accumulation of further phylogenetic data (for example, gathering plastid sequence data or scoring morphological characters to generate phylogenies for comparison with the nuclear ribosomal ITS phylogeny) and (b) using different mathematical techniques to generate

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trees from the same data matrix (another concern understandably raised by TYTECA & KLEIN 2008). Thus, TYTECA & KLEIN are right to identify the phylogenetic position of Pseudorchis as uncertain. Pseudorchis was placed as sister to Platanthera + Galearis (+ Neolindleya) in the parsimony trees of PRIDGEON et al. (1997) and BATEMAN et al. (2003) (Figure 3), but as sister to Gymnadenia s.l. + Dactylorhiza s.l. in the ITS tree of BATEMAN (2001), which was generated using not parsimony but a different (and arguably inferior) mathematical technique, neighbour joining. However, this uncertain placement is irrelevant to the generic circumscription, as all trees agreed consistently and strongly that Pseudorchis albida and P. straminea formed a monophyletic group subtended by a branch of at least moderate length. It is this robust branch that confers on Pseudorchis its rightful status as a genus, not whether it is more closely related to the Platanthera group or the Gymnadenia group.

The key (and reliable) fact is that Pseudorchis is closely related to neither group, and therefore deserves to be a genus in its own right. Its relationship to other genera would become relevant only if we were attempting to circumscribe taxa at a level above the genus and below the subtribe.

We should perhaps pause at this point to consider which biological factors could theoretically generate a robust (and most likely relatively long) branch in a phylogeny. It could mean that the lineage mutated for a long period without undergoing speciation (i.e. without dividing). Alternatively, it could mean that the lineage did in fact divide during that period in its evolution, but that only one of the species that evolved during that period survived to reach the present so that we could sequence its DNA. Or it could simply mean that we have not adequately sampled the taxonomic group in question, and that if we had sampled more extant species within that group, we would find one or more species that would be inserted into the evolutionary tree on that particular long branch, thereby dividing it into two or more shorter branches (BATEMAN & DIMICHELE 2002). This is why, when developing the BPC classification, we worked hard to acquire representatives of all morphologically-delimited species groups, and why we sampled and analysed the entire subtribe Orchidinae rather than restricting our studies to European species. We have continued to add species to the ITS matrix of BATEMAN et al.

(2003) (cf. BATEMAN et al. 2009a), and the very few desirable species still missing from our ITS matrix (e.g. Himantoglossum formosum, Aceratorchis tschiliensis) occur in remote areas outside Europe. Species sampling has proven critically important in developing the most robust phylogenies (see Section 7).

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6. The Seductiveness of Evolutionary Process – And How Best to Avoid it 6.1. Non-directionality is a greater challenge than non-linearity I share the interest of TYTECA & KLEIN in non-linear processes. TYTECA & KLEIN (2008, figure 4) chose to plot molecular change against morphological/ behavioural (?= ecological) change as a stepwise graph, and argued that this showed “nonlinearity” between these two properties of evolutionary lineages.

In fact, the fundamental truth encapsulated by their graph (a truth that I have long recognised) is better presented as two separate graphs that contrast first morphological change and then molecular change against evolutionary time (BATEMAN 1999, 2002) (Figure 6). This shows that non-linearity is indeed present but it actually occurs between morphological change and time, rather than between morphological and molecular change. Although the accuracy of ‘molecular clocks’ has been exaggerated by some advocates, there is no doubt that mutation in lineages follows a broadly clock-like pattern (admittedly, the mutational universe is more Einsteinian than Newtonian; it is subject to local distortions, such as those noted above in the alpine orchid genera Chamorchis, Traunsteinera and Pseudorchis). Thus, molecular change is undoubtedly nearlinear while, in my opinion, morphological change is highly sporadic and follows a punctuationist pattern (sensu ELDREDGE & GOULD 1972; ELDREDGE 1989; GOULD & ELDREDGE 1993) (Figure 6).

6.2. Many evolutionary mechanisms cause speciation in orchids 6.2.1. Saltation—I also admire the bravery of TYTECA & KLEIN in advocating a saltationist view of morphological evolution, though I am not certain that their definition of saltation (they do not offer one) would coincide with my own: “a genetic modification that is expressed as a profound phenotypic change across a single generation and results in a potentially independent evolutionary lineage termed a prospecies” (BATEMAN & DIMICHELE 2002: 112). Hypotheses of saltational evolution have long been unpopular with the neoDarwinian (perhaps better described as the neoDawkinsian) orthodoxy.

Consequently, saltation as a mode of macroevolution has gained only a small number of advocates (BATEMAN & DIMICHELE 1994, 2002; RUTISHAUSER 1995; ERWIN 2000; TUCKER 2000; VERGARA-SILVA 2003; THEISSEN 2006), most commonly in the emerging field of evolutionary-developmental genetics (e.g. HINTZ et al. 2006; NUTT et al. 2006; MONDRÁGON-PALOMINO & THEISSEN 2009). In my opinion, saltation has much to offer students of orchid evolution, and naturally occurring mutants provide excellent illustrations of how, if

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fortunate enough to encounter a suitable vacant niche, radically altered morphotypes could on rare occasions immediately establish new evolutionary lineages (RUDALL & BATEMAN 2002; BATEMAN & RUDALL 2006).

6.2.2. Epigenesis.—I also agree with TYTECA & KLEIN that epigenetics (modification of the expression of a gene by means other than the addition, substitution or deletion of bases) could play a significant role in the evolution of such lineages; indeed, I am currently progressing a collaborative project designed to explore epigenetic floral mutants of Platanthera chlorantha (R. BATEMAN, C. KIDNER, K. JAMES & P. RUDALL unpublished data). The most probable role for epigenesis is cytosine methylation, which alters the expression of key developmental genes without altering the identities of any of the bases in that gene (i.e. without conventional base mutation occurring).

Differences between alleles in their relative degrees of methylation have been shown to strongly influence the expression of key developmental genes that dictate the floral architecture of several different families of flowering plant (JABLONSKA & LAMB 1995; CUBAS et al. 1999; KALISZ & PURUGGANAN 2004; GRANT-DOWNTON & DICKINSON 2006), and it seems highly likely that this statement also applies to orchid flowers.

6.2.3. Heterotopy and heterochrony.—We should also consider another category of major morphological shifts (one that was not addressed by TYTECA & KLEIN 2008), specifically heterotopy and heterochrony (cf. GOULD 1977; ALBERCH et al. 1979; MCKINNEY & MCNAMARA 1991; ZELDITCH & FINK 1996; BATEMAN & DIMICHELE 2002). Heterotopy is the replacement of one kind of organ by another kind of organ between putative ancestor and descendant. Heterochrony is a change in the initiation, cessation or rate of growth of a particular organ between putative ancestor and descendant. Both kinds of developmental change, especially heterochrony, have played significant roles in the evolution of European orchids (e.g. BATEMAN & RUDALL 2006). Heterochrony has helped to explain the origin of humans from a chimpanzee-like ancestor. Adult humans resemble juvenile chimpanzees, a category of heterochrony known as paedomorphosis; this shows that the development of our bodies (though, fortunately, not that of our brains) has become truncated (e.g. GOULD 1977).

6.2.4. Organ suppression.—In addition, suppression of organs has played an important role in orchid evolution. There is little doubt that the ancestor of the orchids possessed six stamens, yet only two of these stamens are expressed in Cypripedium and only one stamen (positionally different from either of the stamens expressed in Cypripedium) is expressed in Orchidinae (e.g. RUDALL

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& BATEMAN 2002). Similarly, in the case of spurs, the ITS phylogeny of Orchidinae (Figure 7) demonstrates that spurs have been greatly reduced on several occasions (e.g. in Dactylorhiza viridis, Orchis anthropophora and Gymnadenia subgenus Nigritella: BELL et al. 2009). Moreover, they have been lost completely from the genera Serapias and Ophrys; indeed, I suspect that, because of developmental constraints, the strongly convex Ophrys labellum could not have evolved without first losing its ancestral spur.

It is the availability of an explicit, data-based phylogeny that allows us to recognise these evolutionary events as reductions or losses; if we did not possess the phylogeny, we could equally easily perceive these species as having evolved from ancestors that also lacked spurs – in other words, as being primitively spur-less. It is our ability to reconstruct the evolution of characters from the base of the tree to its outermost branches that permits us to infer evolutionary patterns, and to identify the directionality (termed the polarity) of particular transitions between contrasting states of the same character.

So, it would appear that both TYTECA & KLEIN (2008) and my own research group believe that epigenesis and saltation are significant causes of evolution, while I also perceive heterotopy and heterochrony as probable causes of many of the putatively saltational evolutionary shifts in morphology. This belief in extremely rapid evolutionary shifts presently distinguishes TYTECA & KLEIN and myself from most evolutionary biologists, who consider relatively slow and gradualistic natural selection to be the dominant (for many authorities, the only) evolutionary mechanism leading to speciation.

6.2.5. Adaptation through selection.—Adaptation to pollinators is perhaps the single most famous biological feature of orchid flowers (e.g. DARWIN 1877). The many elegant adaptations encourage us to believe that all orchid speciation is adaptive and driven by pollination biology (though the orchid’s other partners, the mycorrhizal fungi, might justly feel neglected, since their roles in initiating seed germination and providing life-long nutrition to the orchid arguably contribute even more to its ultimate ecological success and fitness). It is very easy to become over-enthusiastic about the power of coevolution and to credit each orchid species with having acquired its own special pollinator – a trend in evolutionary thinking that has reached its logical extreme in the recent ‘hyper-split’ taxonomy of the genus Ophrys (cf.

DEVILLERS & DEVILLERS-TERSCHUREN 1997; DELFORGE 2006 versus DEVEY et al. 2008; PEDERSEN & FAURHOLDT 2008).

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Perhaps we should pause for a moment to ask whether this extraordinary level of pollinator fidelity could function in practice. It is easy to see how an orchid species with several pollinators, some more important than others, could gradually and adaptively shift its floral morphology to better fit one or more of the previously subdominant pollinators (admittedly, it will thereby become less well adapted to its previously dominant pollinator(s)). But if the ancestral orchid species has not formed a liaison with a spectrum of pollinators but has instead acquired just a single pollinator, how can one orchid species ever evolve into another? There would inevitably be a transitional period when the gradually changing flower would attract no pollinators at all.

The only possible alternative explanation would be a saltational shift such that the morphology of the flower was radically altered but nonetheless was fortuitously and instantaneously sufficiently functional to allow visitation by another pollinator. Although I have argued above that saltational evolutionary events have occurred in orchids, I do not believe that they immediately render the lineage better adapted to acquire novel pollinators; this scenario is too improbable to allow even my acceptance. I suspect that saltation most commonly generates self-pollinating or apomictic orchid species, where pollinators are no longer required so that the decreased functionality of the flower resulting from the saltation event is not in practice a handicap.

Admittedly, polyploidy (a commonly occurring mode of saltation) can result is a phenological shift in the daughter lineage that could give access to new pollinators, but I would argue that the polyploid orchid would most likely continue to sample at least a portion of the pollinator spectra employed by the parental species. Lastly, in my opinion, demonstrating the ability to switch pollinators would, by definition, also demonstrate that pollinators are not the key factor limiting the fitness of the orchids in question, since the relationship with the new pollinator(s) will not initially be even close to optimally adaptive. Instead, pollinator switching would demonstrate the ability of the orchid lineage to perpetuate itself despite possessing a suboptimal phenotype.

6.2.6. Profound niche shifts.—If radical evolutionary transitions do indeed occur, some are likely to radically alter the niche occupied by the lineage.

Certainly, major niche transitions (e.g. terrestrial to aquatic, terrestrial to epiphytic, autotrophic–photosynthetic to mycoheterotrophic) are inevitably accompanied by substantial morphological changes in the lineage.

Among European orchids, perhaps the most interesting such transition is the origin of mycoheterotrophic (‘saprophytic’) lineages. All four obligately mycoheterotrophic orchid lineages present in Europe belong to subfamily

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Epidendroideae rather than subfamily Orchidoideae. Two are the evolutionary apices of tribes that are more common in Asia and/or North America: Epipogium (Gastrodieae) and Corallorhiza (Vandeae). Limodorum and Neottia both occur in tribe Neottieae, but they nonetheless represent independent origins of mycoheterotrophy (BATEMAN et al. 2005). The small number of Limodorum species are all obligate mycoheterotrophs. However, it is clear from ITS and plastid sequencing that the mycohetrotrophic Neottia nidus-avis is nested phylogenetically within the former genus Listera, which consequently is rendered paraphyletic. Thus, we are confident that Neottia and Listera should be amalgamated. Their flowers are very similar, despite their radically different vegetative organs.

6.2.7. Chromosomal and ploidy changes.—Chromosome counts of European orchids have gradually accumulated through the last century (e.g. BRANDHAM 1999), accompanied by more detailed explorations of karyotypes (e.g.

D’EMERICO et al. 1996,1999; LEVIN 2002). Two beneficial trends of recent years have been to obtain counts from multiple accessions of each species, preferably representing different geographical areas (e.g. AMICH et al. 2007), and to examine the structure of the chromosomes as well as simply counting their numbers (e.g. D’EMERICO et al. 1996; D’EMERICO 2000), which can allow the evolutionary history of the karyotype to be inferred. Mapping chromosome counts across the ITS phylogeny allowed PRIDGEON et al. (1997) and BATEMAN et al. (2003) to infer an ancestral chromosome number of 2n = 42 and two critical chromosomal fusions that define major clades: 2n = 40 in Dactylorhiza s.l. plus Gymnadenia s.l., and 2n = 36 in the clade containing Ophrys, Serapias, Anacamptis s.l. and the Himantoglossum group.

However, surveying the subtribe reveals a large number of ploidy increases, most commonly leading to tetraploids, though triploids, pentaploids and hexaploids have also evolved. Polyploid species are uncommon in most clades of Orchidinae – for example, they constitute less than 5% of species in the species-rich genera Ophrys, Serapias, Anacamptis s.l., Neotinea s.l. and Platanthera s.l., while in Orchis s.s. they have been reported only from O. patens and O. canariensis (e.g. BIANCO et al. 1991; KRETZSCHMAR et al.

2006). The notable exception is the clade defined by 2n = 40, consisting of Dactylorhiza s.l. plus Gymnadenia s.l. Both allotetraploids and, to a lesser degree, autotetraploids are common in Dactylorhiza, many representing repeated allopolyploidisation events between the D. incarnata and D. fuchsii aggregates (e.g. DEVOS et al. 2006a; HEDRÉN et al. 2007; PILLON et al. 2007).

Occasional triploids (e.g. Dactylorhiza insularis) and hexaploids (e.g. some populations attributed to D. traunsteineri) also occur. Similarly diverse ploidy

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levels have been documented in Gymnadenia s.l., both within Gymnadenia s.s. (e.g. MRKVICKA 1993) and, especially, within the former genus Nigritella (e.g.

TEPPNER & KLEIN 1985). In addition, some species of both Dactylorhiza s.l. and Gymnadenia s.l. have been shown to be at least facultatively autogamous or, more commonly, apomictic.

6.2.8. Drift.—The mathematical theories of population genetics that underpin inferences of natural selection are based primarily on large, freely interbreeding populations. Although some orchid species are autogamous, the great majority of orchid species are indeed freely interbreeding, fulfilling one of the two theoretical requirements. However, several authors, most notably TREMBLAY et al. (2005), have noted that orchid populations are often very small and that individual plants flower infrequently (typically once every three years in the case of Orchidinae). TREMBLAY et al. convincingly argued that, under such circumstances, orchid populations are especially vulnerable to genetic drift – random fixation of mutations that are neither strongly selected for not strongly selected against (e.g. WRIGHT 1968; KIMURA 1991; OHTA 1992). Such mutations are equally likely to increase or decrease in frequency and so, by chance, some will reach fixation (i.e. characterise every plant in the population).

By this method, a population can gradually acquire a series of novel characteristics that distinguish it from all other populations, even though none of those novel features will have increased the fitness of the individual members of that population. Nonetheless, the novelties may prove sufficient to permit its reproductive isolation and so underpin the emergence of a new species, albeit one that is initially highly geographically localised. Drift and natural selection are often viewed as antagonistic (e.g. PATTERSON 1999), but they are perhaps better seen as processes that are most likely to by active under contrasting ecological conditions.

6.2.9. Anagenesis.—I used to argue that anagenetic speciation (speciation involving progressive, directional change in a single lineage) does not occur in nature. However, phylogenetic consideration of island floras in general (e.g.

STEUSSY & ONO 1998; BATEMAN 1999), and of the Macaronesian orchid flora in particular, forced me to reconsider my earlier position. The non-tropical Macaronesian islands (the Canary Isles, Madeira and the Azores, omitting the more southerly Cape Verde islands) have yielded 13 orchid species (BATEMAN 2001b; BATEMAN & DEVEY 2006). Remarkably, these 13 species belong to no fewer than 12 genera: only Platanthera appears to have undergone a possible speciation event that involved a single immigration event followed by

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divergence of two putative species, on the Azores (e.g. BATEMAN et al. 2009a, b; CARINE & SCHAEFER 2009). Once, the majority of these geographically isolated populations were treated taxonomically as being conspecific with orchids found on the areas of land closest to these islands: northwest Africa and the Iberian peninsula. However, in recent years, most of the Macaronesian species have gradually been segregated off as Macaronesian island endemics, often with inadequate evidence (e.g. BATEMAN & HOLLINGSWORTH 2006).

I approached this taxonomic assertion of high endemism in Macaronesia with considerable scepticism. However, where molecular phylogenetic data have been obtained, they have indeed shown significant divergence between Macaronesian orchid populations and their closest morphological analogues in Africa and/or Iberia: examples include Himantoglossum metlesicsianum versus H. robertianum and Orchis canariensis versus O. patens (e.g.

BATEMAN et al. 2003; BATEMAN & HOLLINGSWORTH 2006). It is evident that the ancestors of these Macaronesian endemics migrated from the mainland to the islands a significant period of time ago, despite having subsequently achieved only subtle morphological divergence from their probable ancestral populations and despite the relatively young ages of the more ecologically suitable islands (e.g. CARRACEDO 2001).

Anagenetic speciation on these islands is most likely to reflect a combination of founder effect and drift. On rare occasions, orchid seeds carried in the wind from North Africa or Iberia reached the Macaronesian islands and, having encountered suitable mycorrhizae, successfully germinated. These successful seeds (perhaps as few as a single seed) could carry only a small proportion of the total genetic diversity in the source population, and so would be unlikely to be representative of that population. This narrowing of and randomness to the sampled genetic diversity is termed the founder effect. Once on the island, the new colonisers would be likely to experience less severe selection pressure than that constraining their forbears on the mainland. In addition, the initially small population size would encourage genetic drift (see Section 6.2.8), allowing further random deviation in genetics (and thus potentially in morphology) from the ancestral species, and ultimately permitting legitimate recognition of the majority of the island endemics as new species. The difficult question is not how anagenetic speciation could have occurred on these islands but rather why they appear to have witnessed only a single incidence of divergent speciation (BATEMAN & HOLLINGSWORTH 2005; BATEMAN & DEVEY 2006).

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6.2.10. Synposis.—In summary, we have successfully identified eight major categories of evolutionary mechanism. The list includes, but extends well beyond, DARWIN’s (1859) key concept of imperceptibly gradual changes via natural selection. The main message that I have drawn from these studies is that formal species descriptions should obligatorily contain both morphological and molecular characters (BATEMAN 2009b, c). Were he alive today, I believe that DARWIN would have embraced this diversity of evolutionary mechanisms with much greater enthusiasm than hard-core neodarwinians who have achieved prominence more recently (e.g. DAWKINS 1986; MAYNARD SMITH 1989). Certainly, his ally T.H. HUXLEY and influential cousin Francis GALTON were more broad-minded. But of course we will never know for certain. We can only applaud DARWIN’s extraordinary legacy to natural history and move on.

6.3. Morphology can mislead attempts to reconstruct phylogeny I am one of a diminishing number of phylogeneticists who still believes that it can be cost-effective to code morphological features in order to generate morphological phylogenies (cf. CHASE 1999; CHASE et al. 2000; BATEMAN et al. 2006). Nonetheless, this does not mean that I am blind to the many hazards of using morphology to reconstruct phylogeny.

When briefly considering human origins, TYTECA & KLEIN (2008) expressed sympathy for MORGAN’s (1997) controversial theory that a significant proportion of the morphological characters that distinguish humans from their closest relatives could reflect a hypothesised period in the evolution of our lineage when humans largely occupied an aquatic niche. However, TYTECA & KLEIN did not develop this argument to its logical conclusion, which is that, in consequence, mankind came to resemble in some characteristics other aquatic mammals – a pattern known as evolutionary convergence. Convergence toward similar function is also responsible for the superficial similarity of the wings of pterosaurs, birds and bats – lineages that acquired the ability to fly independently. More pertinently, convergence is (along with plesiomorphy) probably the greatest source of phylogenetic error when attempting to use morphology to infer the relationships among European orchids.

Comparison of molecular and morphological data shows clearly how adaptation in particular frequently leads similar floral characters to arise in species that are not sisters (i.e. are not each others closest relatives). Examples of floral convergence include Anacamptis pyramidalis and Gymnadenia

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conopsea, Anacamptis boryi and Orchis quadripunctata, Ophrys scolopax and Ophrys oestrifera. Molecular phylogenies show clearly that these taxa are not each other’s closest relatives (i.e. sisters); instead of indicating shared ancestry, such similarities probably originated independently in separate lineages as a result of adaptation mediated by natural selection, most likely to attract similar pollinators. Convergence can also occur through a similar genetic or epigenetic change occurring independently in different lineages, such as the origination of white-flowered individuals in many orchid lineages; this can be achieved by suppressing any one stage in the biosynthetic pathway that generates anthocyanin pigments.

Nor is convergence confined to flowers. The most spectacular cases of vegetative convergence occur in lineages that become obligate mycoheterotrophs; all such orchid species lose most or all of their chlorophyll, have leaves that are reduced to scales, and develop highly branched rootstocks with an unusually large surface-to-volume ratio that maximises mycorrhizal infection. In this case, we must rely primarily on floral morphology to indicate the most likely photosynthetic relatives of mycoheterotrophic groups. But it is molecular phylogenies that have allowed precise estimation of the number of times that obligate mycoheterotrophy has originated within orchids.

Even more usefully, molecular phylogenies also demonstrate that the evolutionary transition to mycohetrotrophy cannot be reversed (e.g. BATEMAN et al. 2005). Once a lineage has become obligately mycoheterotrophic, at least some of its photosynthetic pathways are no longer required, so there is no evolutionary penalty when they subsequently mutate and become dysfunctional. After a few generations the photosynthetic apparatus is sufficiently disabled that it can no longer be resurrected. This makes especially interesting the occurrence in some European Epipactis species of facultative mycoheterotrophy; individual plants within an otherwise photosynthetic population become non-photosynthetic and thus wholly reliant for nutrition on their symbiotic mycorrhizae. It is likely that the obligate mycoheterotrophs originated via facultative mycoheterotrophy (BATEMAN et al. 2005; JULOU et al. 2005; WATERMAN & BIDARTONDO 2008). There are also indications that other major evolutionary transitions, such as the transition from allogamy (cross-pollination) to autogamy (self-pollination), may also be irreversible (e.g. BATEMAN et al. 2005; HOLLINGSWORTH et al. 2006).

Similarly, the same two parental species have repeatedly given rise via allopolyploidy to subtly distinct lineages of tetraploid dactylorhizas, each lineage having the D. fuchsii aggregate as its mother and the D. incarnata

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aggregate as its father (e.g. PILLON et al. 2007). And like mycoheterotrophy, polyploidy is rarely reversible; the duplicated complement of chromosomes is retained by all of the descendants of the original evolutionary event that combined hybridisation with genome duplication (though there is a gradual conversion to a functionally diploid condition: e.g. PILLON et al. 2007).

However, many (perhaps the majority) of evolutionary transitions are readily reversible. For example, the length of the labellar spur (measured either on an absolute scale or, preferably, relative to the length of the entire labellum: Figure 7) undergoes evolutionary expansions and contractions with relative ease, while its ability to generate nectar (an energy-consuming luxury) also changes frequently and apparently easily during the evolution of particular groups of orchids (BOX et al. 2008; BELL et al. 2009). The composition of the dominant pigments in orchid flowers is similarly evolutionary malleable (cf.

STRACK et al. 1989; BATEMAN 1999; GIGORD et al. 2001). Reversibility means that the concept of primitive and derived features is, for most characters, dependent on the phylogenetic context of the species under comparison. For example, current evidence suggests that the ancestor of the orchid family was nectiferous. However, if one considers only the genus Anacamptis s.l., phylogenetic reconstruction of the ancestor of the genus infers that it lacked nectar, even though at least one group within Anacamptis (the coriophora group) produces some nectar. Thus, nectar production is most likely primitive relative to the orchid family as a whole but is derived within the genus Anacamptis. It is in part the realisation that morphological evolution can be so complex and lacking in overall directional trends that undermines repeated attempts to describe species as relatively ‘primitive’ or ‘advanced’ when comparing them.

This principle is illustrated well by reconsidering TYTECA & KLEIN’s (2008, tables 3–7) morphological tables that compare ‘Coeloglossum’ with Dactylorhiza s.s., ‘Nigritella’ with Gymnadenia s.s., Anacamptis pyramidalis with other Anacamptis species, Neotinea maculata with other Neotinea species, and the anthropomorphic versus non-anthropomorphic species groups within Orchis s.s. By definition, such two-way comparisons cannot identify primitive versus derived groups (and thus ancestral versus derived features). A minimum of three groups must be compared, as any two of the three groups will inevitably be sisters. By definition, sisters are groups of equal rank, status and evolutionary ‘advancement’ (e.g. CRISP & COOK 2005); consequently, they can only be considered derived relative to a third group (Figure 7). Thus, even if the tree of DEVOS et al. (2006b) were correct, and ‘Coeloglossum’ had diverged from the lineage slightly earlier than the earliest divergence within

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Dactylorhiza s.s., the nectar-producing ‘Coeloglossum’ would have an evolutionary status equal to the entire species group in Dactylorhiza s.s.

DEVOS et al. rightly observe that ‘Coeloglossum’ viride has a short spur and secretes modest amounts of nectar, whereas species of Dactylorhiza s.s. have longer spurs and do not secrete nectar. But we can decide whether these character states of D. viridis are primitive or derived only by looking further down the tree, to see which groups are located beneath D. viridis and which comparable features they possess. It is this broader comparison that can, on occasion, determine whether groups higher in the tree are primitive or derived for particular characters such as spur length or nectar secretion.

So far, this discussion has concentrated on the randomness of evolutionary transitions. But it is also worth noting that several of the evolutionary mechanisms listed above – saltation, epigenesis, organ suppression, heterotopy, radical niche shifts – all have the potential to permit sudden evolutionary jumps that will not involve intermediate morphologies. Any observer seeking a progressive chain of intermediate morphologies that could then potentially be reconstructed entirely using morphological characters may be severely disappointed. The gradualism sought by the observer will be evident only in the underlying genomes.

Rather, our understanding of orchid evolution progresses most rapidly when we compare morphological characters within the framework provided by molecular phylogenies. The molecular phylogenies are invaluable for identifying relationships between species that differ radically in morphology but are actually closely related or, conversely, have come to closely resemble each other through convergence to similar habitats or modes of reproduction but are actually only distantly related. Such superficial similarities, perhaps reflecting similar evolutionary mechanisms but certainly not reflecting shared ancestry, are most effectively revealed by molecular phylogenetics. They can then be identified as an interesting evolutionary interpretation but discounted from classification as ‘noise’ that obscures the true primary goal of modern classification – reflecting evolutionary relationships.

6.4. How do we relate evolutionary mechanisms to classification?

Even this superficial review has identified nine arguably distinct categories of evolutionary mechanism, each of which is implicated in orchid evolution (Section 6.2), and which collectively at least partly undermine phylogenies based solely on morphological data (Section 6.3). One question that appears to

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be of great concern to TYTECA & KLEIN (2008) is how we can incorporate all of this complexity of evolutionary process when we generate a formal supraspecfic classification. I have repeatedly argued that it is of considerable importance at the species level (e.g. BATEMAN 2001), because inferring a credible mode of origin for a particular species helps to justify its morphological and molecular circumscription. However, crucially in my opinion, we should not even attempt to link classification to suspected evolutionary mechanism above the species level. A supraspecific taxon containing more than a very small number of species is highly likely to represent more than one speciation mechanism.

Please don’t misunderstand me. The primary objective of my orchidological research programme is to better understand the processes by which orchids evolve and speciate. In order to achieve this goal I gather any information that I can that appears relevant to comparative biology – morphological phylogenetics, morphometrics, developmental morphology, DNA sequences, DNA fragments (e.g. AFLP), proteins (e.g. allozymes), gene expression patterns, epigenetic expression patterns, and ecological observations. I should probably also be attempting to gather sequence data from small RNAs (typically 20–24 nucleotides), as they have recently been identified as playing crucial roles in plant development and their numbers appear to be positively correlated with the relative complexity of organisms (e.g. GRIMSON et al.

2008). But the studies of speciation in which I have been involved have resulted in interpretations of speciation mechanisms that range from very tentative at worst to strongly supported inferences at best; no study has resulted in unequivocal proof. Many other assertions of evolutionary mechanisms have no basis in relevant scientific data, and were eloquently described as ‘Just So’ stories by S.J. GOULD (GOULD & LEWONTIN 1979; GOULD 1980, 1997) – scenarios that are potentially interesting but have not yet acquired a genuine scientific basis (echoing the decidedly Lamarckian children’s stories of British author Rudyard Kipling).

For example, one of the reviewers of this manuscript argued in their review that whereas Ophrys species are undoubtedly subject to selective pressures via the adaptation of their flowers to specific pollinators, the pollinators themselves are not subject to selective pressures caused by their interactions with the flowers. Thus, the reviewer considered that the Ophrys flowers effectively parasitise their pollinators. I find this argument appealing.

However, I could counter by arguing that the naïve males of the pollinating species could in fact benefit from their interaction with the flower. The practice gained from attempting to mate with the flower could increase the

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likelihood of successful reproduction of the duped naïve male when he finally encounters a female of his own kind (we could call this the ‘inflatable doll hypothesis’). Alternatively, the naïve male’s chances of reproducing might decrease, as he wastes crucial energy and focus in his fruitless interaction with the flower (the ‘unrequieted love hypothesis’). Thus, I can simultaneously argue that the insect’s interaction with the flower could either increase or decrease his fitness. Given that gathering sufficient data to adequately test either hypothesis would be nightmare, both hypotheses will remain ‘Just So’ stories.

Evolutionary process clearly remains a matter of opinion – often a matter of unusually strong opinion. My views on the likely processes of orchid evolution, summarised in Section 6.2, actually appear broadly similarly to those of TYTECA & KLEIN (2008). Nonetheless, it is unlikely that any two taxonomists will agree on precisely which evolutionary processes have generated the particular species they are studying, or how best to incorporate those hypotheses of speciation mechanisms into a logical classification.

Attempting to build a classification on the basis of assumed evolutionary mechanisms is probably the best way to guarantee that the perennial controversies regarding the classification of European orchids continue for the foreseeable future, as the resulting classifications will inevitably be highly subjective and highly individualistic. Instead, we need criteria for classification that are derived directly and explicitly from available scientific data – in other words, criteria such as those presented in Section 2.

In addition, attempting to inject information on evolutionary mechanisms into classifications is to place the cart before the horse. In order to understand evolutionary process we should first carefully describe evolutionary pattern.

This is the primary goal of a cladogram. Having generated an evolutionary tree through an explicit and repeatable analytical protocol, we can then use that evolutionary tree to both provide a classification and to help interpret evolutionary mechanisms. But it is important to delay that interpretational phase of the analysis until as late in the analytical protocol as is feasible. Our classification should emerge from the data, rather than be pre-programmed into the data analysis in a way that will inevitably reinforce our existing authoritarian prejudices.

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7. Expanding the Matrix: Maximising Taxa is More Beneficial Than Maximising Characters The entire enterprise of classifying global biodiversity can be viewed as an exceptionally long-term, research-driven journey from complete ignorance to complete knowledge. There are three main parameters that we can alter in order in order to gain knowledge of a particular taxonomic group such as subtribe Orchidinae; we can increase the number of taxa analysed, increase the number of individuals of each taxon analysed, and/or increase the number of characters used to describe each taxon (and each individual) analysed (Figure 8). Obviously, it is desirable to increase all three of these parameters, thereby greatly expanding the body of data available to the analyst. However, if we accept that resources available for taxonomic research are severely limited, it is necessary to prioritise these decisions. Predictably, the large number of issues that should be considered during prioritisation means that these decisions are highly controversial (cf. RYDIN & KÄLLERSJÖ 2002; PALMER et al. 2004; SOLTIS et al. 2004, 2005; MARTIN et al. 2005; PHILIPPE et al. 2005; ROKAS & CARROLL 2005; BATEMAN et al. 2006; JEFFROY et al. 2006).

Most importantly, current evidence suggests that, provided that the identities of the taxa selected for analysis and the categories of data to be recorded have both been chosen wisely, the accuracy of a phylogeny of a particular taxonomic group is increased most strongly by increasing the number of taxa included in an analysis.

7.1. Increasing the number of individuals analysed Increasing the number of individuals of each taxon sampled is most important when attempting to delimit species and infraspecific taxa. When using standard genic regions widely used for reconstructing species-level phylogenies such as ITS, one can occasionally encounter significant variation within previously circumscribed species that can simply indicate geographically related clines in genotype within species (e.g. those observed within Orchis simia and O. purpurea by BATEMAN et al. 2008) but may occasionally indicate the presence of species that are morphologically cryptic but reliably molecularly distinct (e.g. those briefly reported in the Gymnadenia conopsea aggregate by BATEMAN et al. 2003; BATEMAN 2006). In other cases, such as European species of Platanthera (BATEMAN et al. 2009b) and Dactylorhiza (HEDRÉN and colleagues), putative species that are morphologically distinct may appear indistinguishable using molecular data.

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In addition, analysing multiple accessions of each putative species occasionally reveals that a previously generated DNA sequence has been assigned by the authors to the wrong species, due either to original misidentification of the sampled plant (typically rejecting inadequate knowledge of the morphology of the species) or to cross-contamination of DNA samples in the molecular laboratory. For example, I have recently detected ITS sequences deposited in GenBank by other authors that had been mis-assigned to Platanthera chlorantha and to Cypripedium reginae, while one of my own previous phylogenies (BATEMAN et al. 1997; PRIDGEON et al.

1997) contained an ITS sequence (fortunately not deposited in GenBank) that was mis-assigned to Dactylorhiza iberica. More broadly, I gain considerable confidence from knowing that the BPC study was paralleled by another phylogenetic study that independently generated ITS sequences from representatives of Orchidinae and generated topologies similar to our own (ACETO et al. 1999; COZZOLINO et al. 2001).

7.2. Increasing the number of characters scored In my opinion, the average morphological character is more valuable than the average DNA character. Morphological information can be linked directly to the biological function of the feature being described, and so provides valuable information on evolution. However, it is difficult to find more than 50 (at most 80) characters in a group of orchids that can readily be coded for morphological comparison, either through traditional taxonomic descriptions or through morphological cladistic analyses. Thus, morphology cannot generate sufficient characters to produce a well-resolved or stable tree for more than say 25 orchid species.

The ITS region in Orchidinae commonly consists of about 650 base-pairs, though in a typical analysis only 30–50% of these characters will provide phylogenetic information; the remainder will be identical in all of the orchid species examined. The resulting 200–300 characters is the maximum number whose evolution can realistically be studied individually by the analyst. When larger (often much larger) numbers of molecular characters are scored, the procedures for aligning sequences, building and interpreting trees all become increasingly automated, and determining the relationships essentially becomes a mathematical/ statistical challenge. However, recent advances in DNA sequencing technology have made it much easier and cheaper to simultaneously analyse very large regions of each of the three genomes found in all land-plants (nucleus, plastid, mitochondrion). One popular approach is

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to sequence the entire genome of the chloroplast of each study species, which typically contains about 160,000 base-pairs per species. If say half of these bases are informative, and a group of say 100 orchid species is being studied, each branch in the resulting phylogeny will most likely be supported by an average of approximately 1,000 characters. This gives very well-resolved trees composed of branches that attract exceptionally strong statistical support. In other words, when viewed statistically, trees with such a large ratio of characters to species might be expected to be highly accurate.

7.3. Increasing the number of taxa analysed However, there are several mathematical properties that can prevent this happy outcome. Of these, the most damaging is long-branch attraction, where two branches of a tree that are supported by many characters can become linked by molecular similarities that are coincidental rather than accurately reflecting evolutionary history (PAGE & HOLMES 1998; FELSENSTEIN 2004; STEFANOVIC et al. 2004). An ideal tree contains branches of broadly equal length.

The simplest way of shortening branches in the tree is to add more relevant species to the analysis. When the analysis contains an unusually broad phylogenetic sampling (e.g. comparing flowering plants with gymnosperms, ferns and bryophytes), or groups that contain few species, this cannot be done and it becomes even more desirable to conduct a corresponding morphological cladistic analysis (e.g. BATEMAN et al. 2006). Fortunately, the orchid family diversified relatively recently (BREMER & JANSSEN 2006; RAMIREZ et al. 2007; CONRAN et al. 2009) and is rich in species (e.g. DRESSLER 1993), so that phylogenetically isolated ‘long-branch’ taxa are relatively uncommon.

However, long-branch taxa can easily be created artificially in a phylogenetic analysis if only a modest percentage of the available species in the study group is actually sampled for analysis.

7.4. Classifying European orchids and interpreting their evolution requires global comparison The easiest way to artificially weaken a molecular phylogenetic analysis is to exclude taxa from an analysis simply because they do not occur in the geographic region of greatest interest. The ITS analysis of BPC (e.g.

BATEMAN et al. 2003) sampled tribe Orchideae strongly from across the northern Hemisphere. For relatively recently originating genera that are confined to Europe and Asia Minor, notably Ophrys and Serapias, a Europe-

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only analysis would be viable. However, for genera that occur in Europe but have a broader distribution, such as Dactylorhiza and Platanthera, and genera absent from Europe, such as the Asian-American Galearis and wholly Asian Neottianthe–Ponerorchis– Hemipilia clade, the North American and especially the Asian species play crucial roles in generating a tree that is likely to give the most accurate representation of relationships among the European taxa. In other words, floristic treatments should routinely be based on monographic treatments.

Removing the non-European species from the ITS tree and reanalysing the data to generate a tree based only on European species (cf. TYTECA & KLEIN 2008) generates a different topology, and one that is less likely to yield the correct inter-species relationships. A classification of European species alone should be derived not from an analysis of European species alone but from an analysis of all known species within the group, subtracting the non-European species after the phylogenetic analysis but maintaining the previously inferred relationships among the European species.

Maximising taxon sampling is not only important for generating the most credible set of relationships; it also means that any attempt to infer the pattern of morphological evolution in the group is more likely to be accurate. For example, one of the arguments put forward by DEVOS et al. (2006b) and TYTECA & KLEIN (2008) in favour of phylogenetic placement of Dactylorhiza (Coeloglossum) viridis below the other species of Dactylorhiza is that, unlike other species of Dactylorhiza, D. viridis generates nectar (albeit in modest quantities). These authors considered this feature to be primitive on the theoretical basis that methods of food deceit and sexual deceit of pollinators are derived within Orchidaceae as a whole – in other words, they inferred that the first species to evolve that we would all have recognised as an orchid would have offered pollinators a nectar reward. This may well be true, but within the orchid family, there are clearly frequent transitions between rewarding and non-rewarding modes of pollination, and vice versa. This particular aspect of orchid biology is evolutionarily labile.

We can use the ITS phylogeny to compare the spur size and nectar production of ‘Coeloglossum’, not just with those of other species of Dactylorhiza but also with its sister genus (Gymnadenia s.l.) and with other closely related clades (Pseudorchis, Galearis plus Neolindleya, Platanthera) (Figure 7). It soon becomes evident that the Gymnadenia clade reliably provides various quantities of nectar, Pseudorchis generates modest amount of nectar, the Galearis–Neolindleya clade operates by nectar-less deceit, and Platanthera

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species are reliably nectiferous. Given these relationships and this high level of variability, it is not possible to determine whether the earliest Dactylorhiza would have been nectiferous or deceitful. Consequently, it is not possible to determine whether ‘Coeloglossum’ shows the primitive or derived condition within the group.

This example nicely illustrates the potential of phylogenies for discerning between primitive and derived states but it also highlights the difficulties of doing so, demonstrates the relative rather than absolute nature of primitive (or ‘ancestral’) states, and reveals the dangers of attempting to infer ‘primitiveness’ outside the context of an explicit phylogeny. Once again, the act of classifying organisms needs to be protected from unwarranted processbased speculation.

8. Phylogenies Facilitate Comparison of Different Categories of Systematic Data During the last decade I have discussed the BPC recircumscription of genera in Orchidinae with many orchidologists – some have been supporters and others critics. These conversations have left me with a strong impression that most of the opposition is not actually driven by explicit criticisms of molecular phylogenetic concepts or approaches. It is not the ‘tyranny of monophyly’ or the potential sources of errors in molecular phylogenies that cause greatest offence, but rather three more intuitive concerns: (1) A belief that morphological evidence is being unjustly subordinated to DNA evidence.

(2) An unwillingness to abandon intuitively (rather than quantitatively) assessed overall morphological similarity as the primary criterion for supraspecific classification, even though it has been shown repeatedly to lead to serious errors when inferring evolutionary relationships. In other words, we are reluctant to reconsider the evidence that is literally before our eyes.

(3) An understandable desire to bring into the discussion (and ultimately into the classification) all other relevant categories of systematic data available to us: intrinsic categories such as chromosome number and karyotype, and extrinsic categories such as habitat preference and presumed co-evolutionary relationships, which are often used collectively to infer likely degrees of reproductive isolation.

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I have some sympathy with each of these three concerns, but my answer to each is the same; explicit phylogenies based on substantial bodies of data provide the best remedy.

8.1. Why we still badly need morphological data Firstly, I am surprised that no-one has yet published a morphological cladistic phylogeny of subtribe Orchidinae (for an outline morphological phylogenetic analysis of the entire orchid family see FREUDENSTEIN & RASMUSSEN 1999). I am gradually collecting data toward this objective, but because previously published descriptions of species are often unreliable, many of my preferred morphological characters cannot be recorded from herbarium specimens and living plants of many species are hard to find; building this morphological phylogenetic matrix has consequently become a long-term project. In addition, I know that the investment of time and resources per useful character scored will be much greater than for morphology than for the more easily produced molecular phylogenies, and that the number of scorable characters will be severely constrained (these weaknesses are evident in the study of FREUDENSTEIN & RASMUSSEN 1999). Lastly, because mapping of morphological characters across the ITS tree has already demonstrated a high frequency of morphological convergence (see Section 6.3), we can predict with confidence that morphological phylogenies will give comparatively unreliable estimates of evolutionary relationships.

The majority of my phylogenetic colleagues no longer gather non-molecular data, and those that do gather such data generally subordinate it to molecular data. In addition, many believe that the best approach to reconstructing and interpreting phylogenies is to combine all of the data that they have accrued in a single aggregate matrix, and then use that matrix to generate trees (this is termed the ‘total evidence’ or ‘concatenation’ approach). However, in my opinion, more can be learned by analysing data sets separately than together.

Different categories of phylogenetic evidence (e.g. nuclear genes, plastid genes, morphology, chromosome numbers) are under strongly contrasting kinds of biological constraint, and so cannot be expected to behave in identical ways during evolution. Thus, my preferred approach is to code each category of data in a separate matrix, to generate trees from each matrix, and then to compare the topologies (shapes) of the trees generated from each category of data. If all the trees are similar in topology we can be confident that we have obtained an accurate picture of evolutionary relationships within the group analysed. At that point, the different data sources can indeed be combined in a

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single larger-scale analysis. However, if strongly contrasting topologies result, we are likely to be less confident in our conclusions, and we should seek process-based biological explanations for the disagreements between the trees (e.g. hybridisation). Ideally, the ITS topology for Orchidinae should be compared with trees generated from plastid genes (this has been done by us but not yet published) and from morphology.

Despite the recent focus on molecular data, the self-discipline necessary to quantify morphological characters across the subtribe, and to analyse them using phylogenetic software, would significantly improve our understanding of morphological variation in the group and, when compared with the ITS tree, would also yield insights into evolutionary patterns. The existence of the ITS tree should not suppress the production or analysis of other categories of systematic data. Instead, the tree provides an invaluable framework for comparison with, and the analysis of, all other available categories of data. A rooted tree is an exceptionally powerful tool, because it simultaneously provides us with statements of the relationships of species, the relative times at which they originated, and which character states evolved in order to generate a particular species or inclusive group of species (clade).

8.2. Why we no longer need ‘blobograms’ During the last three decades the tree motif of the cladogram has gradually replaced the ‘blobogram’, which represented higher taxa as curvaceous ‘blobs’ scattered across the page. Admittedly, blobograms were used to summarise classifications by some of the most experienced and respected plant taxonomists of the 20th Century (cf. CRONQUIST 1968; TAKHTAZHAN 1969; SPORNE 1974; THORNE 1976), but in almost all cases they were used to depict relationships in an undesirably enigmatic way; any information that underlay the relative positions of the blobs on the page was confined to the brain of the researcher who drew them. The most notable exception was DAHLGREN (1975, 1977, 1980, 1985; also DAHLGREN et al. 1985), but interestingly, he depicted the blobs in three dimensions, linked in the third dimension by dichotomising branches that represented the hypothesised evolutionary history of the group.

But even in DAHLGREN’s diagrams, there are no explicit characters present to support the branches or explain why blob A is shown as being closer to blob B than to blob C. Interestingly, DAHLGREN adopted cladistic methods just before his untimely death.

Blobograms can now be seen to have been a distraction that tended to weaken rather than strengthen plant classification. They viewed evolution from above

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when (at least, at supraspecific levels) it is actually better viewed from the side, so that we can represent time and relationships instead of (or preferably in addition to) overall similarity (Figure 4). In most such presentations (including those of TYTECA & KLEIN 2008), the blobs have no explicit meaning – in other words, no quantitative analysis has generated the spatial relationships of the blobs. Instead, they constitute an abstract impression of overall (usually dominantly morphological) similarity somehow gained by the taxonomist who drew them. In the case of TYTECA & KLEIN (2008, figures 1– 3, 6), presumably the blobograms are intended to represent “the advice and experience of well renowned orchidologists of our time” (p. 531).

In many diagrams, including those of TYTECA & KLEIN (2008), the blobs on the blobogram vary in size. Often, that size variation approximately reflects the relative numbers of species perceived to characterise each genus that is represented by a blob (though Nigritella, containing approximately 15 species, is shown as being smaller than the monotypic Neotinea s.s. by TYTECA & KLEIN). Smaller blobs are said to be ‘satellites’ of larger blobs, but again, this concept has no biological meaning; any two lineages that diverge from a single point in time are, by definition, of equal weight and rank; one cannot reasonably be considered a subordinate ‘satellite’ of another taxon simply because it subsequently speciated less often (or generated a larger proportion of species that subsequently became extinct).

In any case, perceptions of species numbers within particular higher taxa differ radically. One might instinctively consider Anacamptis to be a satellite genus relative to its sister genus, Ophrys. However, my view that Ophrys and Serapias have been greatly over-split and that neither genus actually contains more than 10 species (DEVEY et al. 2008; BATEMAN et al. (2009C) would require me to depict both genera as ‘satellites’ of the expanded genus Anacamptis, which is said to contain between 11 (KRETZSCHMAR et al. 2007) and 20 (DELFORGE 2006) species. Surely, the diversity of characters, both morphological and molecular, within each group is a more satisfactory basis for comparing groups of equivalent taxonomic rank than traditional views of the number of species that they encompass?

Lastly, the blobograms of TYTECA & KLEIN (2008) are incompletely sampled at the genus level. Not only are all related non-European genera absent from the Figures, but also some European genera (e.g. Ophrys, Himantoglossum s.l.) that are nested phylogenetically within those genera that are depicted; the resulting diagrams resemble chess-boards that have already lost half of their pieces. Overall, blobograms are no substitute for an explicitly and mathematically generated evolutionary tree.

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9. Conclusions: Real Science Transcends Authoritarianism TYTECA & KLEIN (2008: 521) argue that classification remains “a ‘subjective’ vision … influenced by our needs to categorize living beings according to our perceptions and experiences.” My argument is that this natural instinct to apply our own “perceptions and experiences” that needs to be determinedly resisted by anyone who is seeking to generate a genuinely scientific, databased classification. Cladistic methods in general, and molecular phylogenetic methods in particular, seek to minimise that subjectivity by restricting as far as possible the number of decisions that must be made by the taxonomist rather than by the data. The protocol recommended in this paper for dividing a tree into a Linnean classification shares the goal of minimising subjectivity.

Phylogenetic methods are not restricted to DNA; they can accommodate any category of quantitative or semi-quantitative data. They allow inferences of evolutionary mechanisms to emerge from the analysis in a scientific manner, rather than being programmed into the analysis in a fundamentally unscientific, authoritarian manner.

It will then be data, rather than prior belief, that informs us whether pollinator specificity or mycorrhizal switching or mycoheterotrophy or chromosomal fusions or polyploidy or heterotopy or heterochrony or organ suppression or saltation or epigenesis … or many other evolutionary mechanisms revealed by modern science … have been most important in generating today’s remarkable diversity of orchids. We may even be able to infer whether we humans did indeed have aquatic ancestors (and how closely those ancestors resembled chimpanzees). But these insights should emerge from classification, rather than being programmed into classification. This is one crucial layer of damaging subjectivity that can easily be removed from orchid taxonomy by adopting modern concepts and methods. Without the separation of morphological and molecular patterns from the various evolutionary processes, there can be no reciprocal illumination between pattern and process and our research again becomes fundamentally unscientific. TYTECA & KLEIN (2008), and other noteworthy authorities on European orchids such as BUTTLER (2001), KREUTZ (2005: 11) and DELFORGE (2006: 17), offer us alternative classifications of European orchids, but they do not offer an alternative set of rules to explain how one can synthesise the great diversity of available scientific evidence in order to generate those classifications in a logical, repeatable and biologically defensible way.

If the taxonomy and classification of organisms is finally to become a true science, it must focus on the analysis of data (preferably intrinsic data that

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directly describe properties of the organisms) within an explicit conceptual context. As currently pursued by many of its practitioners, classification resembles a visit to the supermarket. Authors decide which names they will accept and which they will reject from the many classifications already available, as though they were selecting products from the supermarket shelves. In doing so, each author creates yet another ‘hybridised’ classification, usually without explaining the rules or principles that he or she has followed to reach those individualistic taxonomic conclusions. Such classifications are the result of personal opinion rather than any explicit analysis, and in the absence of the monophyly criterion, the resulting taxa are artificial human constructs rather than the self-defining products of evolution.

Effectively, 21st Century data are being constrained by voluntarily retaining an 18th Century approach to biological classification. Indeed, if artificial rather than natural classifications of European orchids are desired, LINNAEUS’ (1759) pioneering classification appears preferable to many of the more recent efforts. LINNAEUS sensibly followed simple, explicit, clearly stated rules, and his formal system allows reliable (if crude) classification of any flowering plant and reliable identification of a large proportion of those species (Figure 1).

Indeed, the simplicity of LINNAEUS’ work reveals to us a great truth – it is, in fact, attempts to accommodate evolutionary relationships that have complicated, and make more challenging, modern attempts to classify all of life. Yet some of us feel that the great predictivity and biological cohesion conferred on classifications by inferring closeness of evolutionary relationships justifies the lifetime of work that it requires to achieve. My own prejudices initially rebelled against the relationships inferred as a result of our early molecular phylogenetic research (BATEMAN et al. 1997; PRIDGEON et al.

1997). Some of the relationships did not fit my morphologically-based expectations; worse still, they undermined some of my most precious hypotheses about which evolutionary mechanisms might have allowed particular orchid species to originate. And I agree with TYTECA & KLEIN (2008) regarding “the necessity of avoiding hasty conclusions” (p. 506; see also their request that we “refrain from [drawing overly] quick taxonomic conclusions”: p. 525). But the generic re-circumscriptions of BPC have since been supported by many subsequent data-based studies (e.g. ACETO et al.

1999; COZZOLINO et al. 2001; BARONE LUMAGA et al. 2006; GAMARRA et al.

2007; also, though presumably they would deny the charge, DEVOS et al.

2006a, b), all actually reinforcing the main elements of our 1997 classification. If I were now to rebel, and decide to ignore more than a decade of multi-faceted research in order to maintain my own prejudices, I would

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have left the realm of scientific authority and entered the less admirable realm of unscientific authoritarianism. Orchid classification will not benefit from “multiple, complementary viewpoints” (TYTECA & KLEIN 2008: 506) or “using several hypotheses to arrive at provisional conclusions” (DELFORGE 2006: 17), but rather from multiple, complementary datasets, each analysed using the same cladistic protocol and interpreted in the same cladistic framework.

Many of my professional colleagues view debates such as the circumscription of genera of European orchids as irrelevant to both mainstream science and everyday life. They are wrong on both counts. In my opinion, the main reason that phylogenetic classifications have been more heavily debated in European orchid circles than in most others is simply because more people are deeply interested in European orchids than in most other groups of plants – we orchid enthusiasts are fortunate to have gained a constituency that is sufficiently large to maintain several specialist societies. More enthusiasts inevitably leads to more opinions. Thus, I broadly agree with TYTECA & KLEIN’s (2008) assessment of where the BPC classification currently stands within the community of European orchid enthusiasts (see also Table 1). It has been adopted in its entirety by the two main UK orchid floras (FOLEY & CLARKE 2005; HARRAP & HARRAP 2005), but this success is not entirely surprising as I was the taxonomic advisor to both publications. It has also been largely accepted by the most recent orchid flora of France and Belgium (BOURNÉRIAS & PRAT 2005), and it provided the framework for the thorough monograph of the former (polyphyletic) genus Orchis by KRETZSCHMAR et al. (2007).

Other, less enthusiastic authors have simply ‘cherry picked’ selected portions of the BPC classification that they found convenient, some choosing to use a majority of the recommended generic transfers (e.g. BOURNÉRIAS & PRAT 2005), others only a minority (e.g. BUTTLER 2001; DELFORGE 2006; KREUTZ 2007; TYTECA & KLEIN 2008), and yet others none at all (BAUMANN et al.

2006). I agree with the statement made by TYTECA & KLEIN (2008: 512) that “nomenclatural and systematic confusion followed the revised classification proposed by BATEMAN, PRIDGEON and co-workers. It can be said that almost every author has his/her own conception about what can be retained and what should be discarded.” However, I would argue that plenty of confusion also preceded the BPC classification. Clearly, the fundamental problem is not ‘confusion’ per se but rather disagreement reflecting subjective opinion. I would prefer the BPC classification to be wholly rejected (preferably on logical scientific grounds, such as rejecting the principles of monophyly and natural classification) than partly accepted on the grounds that some parts of the classification ‘feel right’ to the author and other parts do not.

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Such ongoing subjectivity has contributed to the long-term decline of taxonomy, causing the discipline to become viewed by other branches of science (with some justification) as fundamentally unscientific.

If we consider the classification of the orchid family as a whole, rather than simply its European representatives, we see that the internationally acclaimed Genera Orchidacearum project (PRIDGEON et al. 1999 et seq.) is based on molecularly-defined monophyletic groups, even though the taxonomic descriptions therein are wholly morphological and the taxonomic presentation is traditional. The genus-level classification established in this six-volume monograph has been adopted as their benchmark by the Royal Horticultural Society, the American Orchid Society, and the online World Monocot Checklist (www.kew.org/wcsp/monocots), which ensures that it will increasingly dominate the international horticultural community.

If we further expand our horizons to consider the classification of all flowering plant families, we soon realise that the professional world of plant systematics has become dominated by the extraordinarily highly cited and pervasive Angiosperm Phylogeny Group (APG) classification (ANGIOSPERM PHYLOGENY GROUP 1998, 2003, 2009; STEVENS 2009), which is again based on molecularly defined monophyletic groups. Beginning with the Royal Botanic Garden Edinburgh (HASTON et al. 2007), many of the major herbaria in the world are now being reorganised according to the APG classification; these include the Royal Botanic Gardens Kew, Natural History Museum (London), National Herbaria of the Netherlands, Natural History Museum (Paris) and the Geneva Herbarium. Thus, APG is replacing the previously dominant, authoritarian (and non-cladistic) classifications of CRONQUIST (e.g.

1988), THORNE (e.g. 1992, 2000) and TAKHTAJAN (e.g. 1997). And monophyly provides the framework for the most popular of the recent summaries of the vascular plants (e.g. MABBERLEY 2008, which also follows Genera Orchidacearum at genus level) and textbooks of systematic botany (e.g.

SOLTIS et al. 2005; JUDD et al. 2008) (see also the overview of how evolutionary relationships are depicted in popular literature, by CATLEY & NOVICK 2008).

When viewed in this wider context, the reality facing the European orchid community becomes clear: adopt modern classifications that have monophyly at their core, or become increasingly detached from the rest of the botanical community – and also from the conservation community. But here I am making a pragmatic argument that borders on authoritarianism! I would instead encourage readers to approach these issues more dispassionately,

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selecting among the available classifications of European orchids on the basis of the relative strength of the (admittedly complex) scientific arguments that underlie them.

10. Acknowledgements I thank DAVID ROBERTS, PAULA RUDALL and two anonymous referees for constructive criticisms of the manuscript, and the editors for processing this manuscript with laudable speed.

11. References ACETO, S., P. CAPUTO, S. COZZOLINO, L. GAUDIO & A. MORETTI (1999): Phylogeny and evolution of Orchis and allied genera based on ITS DNA variation: morphological gaps and molecular continuity. – Molec. Phyl.

Evol. 13: 67–76.

ALBERCH, P., S.J. GOULD, G.F. OSTER & D.B. WAKE (1979): Size and shape in ontogeny and phylogeny. – Paleobiology 5: 296–317.

AMICH, F., M. GARCIA-BARRIUSO & S. BERNARDOS (2007): Polyploidy and speciation in the orchid flora of the Iberian Peninsula. – Bot. Helv. 117: 143–157.

ANGIOSPERM PHYLOGENY GROUP (M.W. CHASE, K. BREMER, P. STEVENS & 26 CO-AUTHORS) (1998): An ordinal classification of the flowering plants. – Ann. Mo. Bot. Gard. 85: 531–553.

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Author's address: Prof. Richard M. Bateman Jodrell Laboratory -Royal Botanic Gardens Kew Richmond (Surrey) TW9 3DS U.K. e-mail: r.bateman@kew.org

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Fig. 1. – (a) The exclusively reproductive high-level plant classification of LINNAEUS (1759: 837), and (b) a relatively free translation into English by TURTON (1802) (after SCHIEBINGER 1996: 101; BATEMAN & SIMPSON 1998, figures 1, 2). Although this classification is both highly typological and highly artificial, it is also attractively simple.

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Fig. 2. – Hypothetical tree of a single outgroup (O) and eight ingroup members (A–H), illustrating the contrast between monophyletic (a), paraphyletic (b) and polyphyletic (c) groups.

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Fig. 3. – ITS phylogeny of tribe Orchideae, presented as a phylogram under Acctran optimisation, for 186 species of Orchidinae and selected Habenariinae plus a Diseae outgroup (after BATEMAN et al. 2003, figure 2A, B).

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Fig. 4. – Comparison of perspectives of evolution as viewed laterally through time, dominantly represented by the tree motif, and viewed perpendicular to the present time-plane, when taxa are best represented as clusters of individuals separated by morphological and/or genetic discontinuities (after BATEMAN 2006, figure 1).

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Fig. 5. – Hypothetical tree illustrating the role of reticulation during speciation at four different spatial and temporal scales (after MADDISON & MADDISON 1992, figure 3.2, p. 26).

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Fig. 6. – A simple hypothetical phylogeny of eight species, four extant and four extinct, all ultimately derived from a single ancestor. Evolutionary patterns are contrasted for morphological data (a), showing geologically instantaneous (punctuational) morphological divergence, and sequences from non-morphogenetic regions of the genome (b), showing constant, clock-like sequence divergence. In this example both speciation events and extinction events are roughly evenly spaced and the magnitude of morphological divergence is random through time – features that maximise the likelihood of correctly reconstructing the phylogeny of the group (modified after BATEMAN 1999, figure 19.2; BATEMAN & DIMICHELE 2002, figure 7.2).

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Fig. 7. – Phylogenetic context of the five species that are the focus of the present study (highlighted and arrowed). The topology was abstracted by BATEMAN et al.

(2006) from the broader molecular phylogenetic analysis of nuclear ribosomal ITS region in tribe Orchideae in BATEMAN et al. (2003). It shows the nonglobose tubered clade within subtribe Orchidinae (cross-bar 1), contrasting the fusiform-tubered clade (cross-bar 2) with the digitate tubered clade (cross-bar 3, a group also delimited by a chromosomal fusion from 2n = 42 to 2n = 40). A more recent analysis (EFIMOV et al. 2009) tentatively suggests that, within the fusiform-tubered clade, Neolindleya is sister to Galearis plus Amerorchis, together forming a clade characterised by highly reduced filiform tubers that is sister either to Pseudorchis plus Platanthera s.l. or to these genera plus

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Gymnadenia s.l. and Dactylorhiza s.l. Mapping across the phylogeny of nectar production and relative spur length shows substantial degrees of homoplasy in both characters. The ancestral states inferred for the basal node (cross-bar 1) are intermediate for spur length but equivocal for nectar production. Polyploid species are asterisked (after BOX et al. 2008, figure 1C).

Fig. 8. – Three different ways of increasing the data available to progress a taxonomic study from a point of near-complete ignorance (the original description, represented by most species (bottom left), to a point of complete knowledge, best represented by Arabidopsis thaliana (top right).

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