The dynamics of evolution on the Dactylorhiza meadow

Dactylorhiza fuchsii f. albiflora

Seven years after my first observations on a meadow in the valley of the small river Bieber in the northern region of the Spessart mountains I visit this special habitat again. The meadow with a wet trench in the middle is in full flowering. My estimation is that about 600 Dactylorhiza plants are flowering here: About 500 Dactylorhiza fuchsii, 50 already withered Dactylorhiza majalis and 50 hybrids Dactylorhiza fuchsii x majalis. Among the accompanying plants are Rhinanthus spec., Pedicularis palustris, Campanula patula and even a group of Arnica montana. Platanthera bifolia is just showing the first flowers.

Among the 500 Dactylorhiza fuchsii are about 400 plants with very bright flowers and a pattern of bright violet loops on the labellum. Furthermore, I count 22 albiflora forms with white flowers and non-spotted leafs. This is a rate of 4.4 per cent – much higher than you could expect if those were just spontaneous mutations. When I first visited this place on 20.6.2010 and 1.6.2012, there were less albiflora plants. Only about 80 of the 500 Dactylorhiza fuchsii have an intense violet colour. This meadow presumably has its own dynamics of evolution, developing increasingly bright forms of Dactylorhiza fuchsii.

Dactylorhiza fuchsii f. albiflora

Dactylorhiza fuchsii f. albiflora

There might be a relation with the learning experience of bees: With the earlier flowering Dactylorhiza majalis, many pollinators may have already made the experience that there is no nectar in the spur of violet orchid flowers. While developing brighter flowers, Dactylorhiza fuchsii might counteract this learning experience. Young honey bees still have to make this experience like this one on the flowers of the hybrid Dactylorhiza fuchsii x majalis:

Albiflora abundance on Sardinia

Anacamptis morio subsp. longicornu
Anacamptis morio subsp. longicornu

There are two forms of albiflora mutations with orchids:

  • the spontaneous growth of a white-flowered form as the consequence of a genetical defect in the process of creating anthocyanine pigments, traditionally called a freak of nature, Occurence: 1-5 among 1000 plants
  • populations of white-flowered mutations as the result of an evolutionary adaptation to environmental conditions, e.g. the concurrence of other violet-flowered food-deceptive orchids, occurrence: 10 to 500 among 1000 plants

I’ve found both forms on the Sarcidano plateau, a central region of Sardinia.

On lengthy hikes between Láconi, Ortuabis und Santa Sophia I’ve seen only one single albiflora form of Orchis mascula subsp. ichnusae, with the crimson marking of the flower labellum still preserved:

Orchis mascula subsp. ichnusae
Orchis mascula subsp. ichnusae

Before, I had already seen a single Orchis anthropophora without its typical flower colouring, in the forest of Domusnovas, on southern Sardinia:

Orchis anthropophora
Orchis anthrophora

Much more frequently are the white-flowered forms of Anacamptis morio subsp. longicornu in Sarcidano. Respectively one third of the overall several thousand plants in this region has the dark violet colouring, a bright violet (or rose) colouring or are white-flowered.

Anacamptis morio subsp. longicornu
Anacamptis morio subsp. longicornu
Anacamptis morio subsp. longicornu
Anacamptis morio subsp. longicornu

You can’t find this accumulation of albiflora forms in other regions on Sardinia which I’ve visited, neither at Domusnovas/Iglesias nor in the North or at Monte Albo. There, Anacamptis morio subsp. longicornu are consistently flowered in the regular violet. The albiflora forms of Sarcidano possibly have an evolutionary advantage. In this region there are also many Orchis mascula subsp. ichnusae giving pollinators as bees the learning experience that there is no nectar in the spur at flowers with this colour and form. In the other regions Orchis mascula subsp. ichnusae was less common or not present.

Anacamptis morio subsp. longicornu
Anacamptis morio subsp. longicornu

Orchids are not a young plant family at all

In a paper published in the latest edition of the Journal Europaeischer Orchideen (vol 44, 2/2012, p. 421-426), Wolfgang Wucherpfennig reviews the recent publications about the phylogenetic tree of the Monocotyledonae. He points out that the Orchidaeceae are between 104 and 120 million years old (A in the phylogenetic tree) and that the first orchids have been grazed by dinosaurs. So, orchids are in fact older than their relatives in the Amaryllis family (Amaryllidaceae) or the Asparagus family (Asparagaceae). Only the differentiation of the genera of orchids (B in the phylogenetic tree) has a more recent age and happened between 76 and 84 million years ago. Wucherpfennig concludes: “So, orchids are not a young plant family at all, they have a dignified age. But very old families also have small children which are enterprising and adventurous such as Ophrys and Dactylorhiza.”

Snap-shot of the evolution: preliminary report of albiflora studies

Peter Zschunke: Albiflora-Formen der Orchidaceae - mehr als eine Laune der Natur
Thanks to all the contributions to this project website I’ve compiled a first paper about the white-coloured forms of orchids, published in Berichte aus den Arbeitskreisen Heimische Orchideen (1/2012, p. 141-170). Following a short overview about taxonomic aspects the relationship of flower colour and fertilizers are discussed. The main part considers the differences of albiflora forms with particular genera of orchids. The paper finishes with a discussion of high frequencies of albiflora forms with Dactylorhiza fuchsii in Western Ireland and certain regions in Germany. Where does random mutation ends and where begins an evolutionary process? One possible scenario might be that nectar deceptive orchids flowering earlier than Dactylorhiza fuchsii – as there are Orchis mascula or Dactylorhiza majalis – impart fertilizing insects the experience that flowers with a certain form and a purple colour don’t grant them any nectar. Thus, a colour change to white might be an advantage. The German language paper can be downloaded here.

Three albino plants of Cephalanthera damasonium

Cephalanthera damasonium It is well studied that Cephalanthera damasonium belongs to those orchids which can live without chlorophyllum – together with other species of the tribe of Neottieae or the genus Epipactis. While exploring a mixed forest near Lahnstein (Rhineland-Palatinate) together with Ingo Beller of the Arbeitskreis Heimische Orchideen (AHO) Rheinland-Pfalz, we found a group of three albino plants in addition to three green-leafed Cephalanthera damasonium. Of the apochromic plants one has two flowers, one only one flower and one has no flower. Those albino plants receive their organic carbon with the help of fungi. A study of V. Tranchida-Lombardo, M. Roy, E. Bugot, G. Santoro, Ü.Püttsepp, M. Selosse and S. Cozzolino, published in 2010 in Plant Biology suggests that the albino Cephalanthera damasonium may be viewed as “an intermediate step in the evolutionary emergence of mycoheterotrophy”, or of the ability to be nourished both by fungi and photosynthesis. By means of genetic analyses the authors declare: “Albinos could be either permanent mutants, as suggested by phenotype stability over the years, or transitory phenotypic stages, in which genes involved in the photosynthetic pathway can switch off depending on micro-environmental conditions (e.g., the amount of C resources provided by the nearby fungal mycelia or tree roots) that prevent greening.”

Lecturing about albiflora orchids in Koblenz

Invited by the Arbeitskreis Heimische Orchideen (AHO) Rhineland-Palatinate I’ve presented some thoughts about albiflora orchids at a meeting in Koblenz. After introducing the basics of the bio-chemistry and genetics of flower colours I pointed to the striking differences in the frequency of albiflora forms with certain orchid species. With frequencies of more than 0.1 per cent one might assume that albiflora forms are not just the result of spontaneous mutations but may indicate a certain evolutionary process. With regard to Dactylorhiza fuchsii there is reason to believe that there are ecological pressures favouring albiflora forms – a hypothesis, which will be elaborated in an upcoming article in AHO’s journal “Berichte aus den Arbeitskreisen Heimische Orchideen”.

Richard Bateman on stability and change

Richard Bateman
Still looking for any hints to shed some light on the albiflora phenomenon, I visited Richard Bateman in London. The white-flowered morphs “are of greatest interest to me because of their relative frequencies in the diploid and the tetraploid groups of Dactylorhiza”, he told me. “If your mind works its ways through the different diploids – incarnata, fuchsii, sambucina – they all show colour polymorphism. And they all have a certain number of very pale or white individuals.” Quite different are the observations with tetraploid Dactylorhiza species like praetermissa, majalis or alpestris. Bateman noted: “I’ve only found one albino praetermissa and one albino traunsteinerioides in 30 years of going in the field.” Quite younger is the project – but up to now only some scattered findings of white-flowered Dactylorhiza majalis have been reported – and none of praetermissa or traunsteineri. As a possible explanation Bateman noted: “Presumably, in the tetraploids there has to be a minimum of four copies of a gene that is malfunctioning to cause the albinism. So I think the tetraploids are buffered against albinism by having additional copies of the genes that generate the anthocyanin pigments.”

Arguing with him about the negative connotations of the term malfunction, Bateman answered: “Most organisms are ‘designed’ to remain the way they are and not to change [substantially]. So, from a genetic viewpoint, any change that is expressed is a malfunction. I agree the malfunction could be beneficial rather than negative but most times it’s negative.”

Bateman’s main interests in research are the questions of speciation or at least the question of what might lead to evolutionary divergence between populations: “The genus level – for me at least – is solved. The species level interests me the most now. That’s were the most challenging topics still have to be addressed – how orchids speciate.” Despite a huge literature on the topic, Bateman noted, those questions are still not adequately solved. “Each time I address a particular set of orchids, the answer [to this question] is different.” So up to now, no generalization is possible. But, Bateman said, “I strongly believe that the importance of pollinator [specificity] is being exaggerated by a lot of workers.”

At least one general observation can be stated according to Bateman: “New [evolutionary] strategies are tried out constantly – more than most people believe – but I think they succeed less often than most people believe.”

In addition to such reflections on stability and change in the genetics of orchids, we wondered why hypochrome forms of Ophrys are rather green than white – obviously the Ophrys flowers still contain chlorophyll even if they lack the anthocyanins – and with good reason: “The rosette leaves of Ophrys (and Himantoglossum as well) tend to be dead before the flowers properly open”, Bateman said. “So I don’t think there is much supply of nutrient coming from [root to flower]. The flower has become autonomous … whereas in Orchis or Anacamptis the flowers are far less independent.” So, Ophrys flowers are quite special. “One of the most striking things when you start working on the flowers, cutting them up looking at them under microscope, you realize how much energy is invested in an Ophrys flower. There is a lot of tissue.”

After the visit, the Royal Botanical Gardens in Kew have been the very right place to further contemplate on the miracles of nature. (With thanks to Richard Bateman for reviewing his quotes, additional notes are marked with brackets)
Kew Gardens

Colour influences pollinator behaviour

The Flower of the European Orchid

Form and function of the flower organs are the main focus of the new fascinating book The Flower of the European Orchid by Jean Claessens and Jacques Kleynen. Illustrated by great macro as well as microscopic photos this important opus presents a comprehensive description of the structure of orchid flowers with the different European genera. In a foreword, Richard Bateman writes: „No other family of plants can match the orchids for their sheer charisma“. But the excitement goes along with a certain scientific pain – Bateman stresses that there still remain major scientific uncertainties which „further torment us“ – among them questions of evolutionary adaptation.

The orchids’ strategies of fertilization are manifold and the book explains how the specific construction of the column (gynostemium) supports allogamy by pollinators or autogamy (self-fertilization). Especially intriguing are the strategies of Dactylorhiza, Orchis and other genera without any nectar in the spur. Claessens and Kleynen explain that the pollinators of Orchis mascula are „recently emerged, naïve bees or exploratory insects that have not yet learned that the flowers offer no reward” (p. 220). The authors also cite the study of L. Dormont, R. Delle-Vedove, J.-M. Bessière, M. Hossaert-Mc Key und B. Schatz about the presence of white-flowered Orchis mascula which underlines „the importance of visual cues for attracting pollinators“ (p. 220).

In the Dactylorhiza chapter the authors write: „Colour can also influence pollinator behaviour“ (p. 240). With regard to the red and the yellow forms of Dactylorhiza sambucina they refer to experiments showing that experienced bumblebees „preferred by far the morph that most resembled the rewarding plant on which they have fed previously“. Vice versa it may be presumed that there may be a form of evolutionary adaptation directed to develop visual cues which are different from non rewarding plants being abundant in a certain region – as it could be the case in Western Ireland with the many white-flowered forms of Dactylorhiza fuchsii on meadows with earlier flowering Orchis mascula.

Albiflora plants influence naïve pollinators

White-flowered orchid varieties are not just a “freak of nature” – they have quite obviously some biological function. A group of scientists in Montpellier in Southern France has found that the existence of albiflora plants in a population of Orchis mascula is connected with a much higher fruit set of the purple-flowered plants than in populations where there are no white-flowered Orchis mascula:

“Surprisingly, our study showed that the presence of co-occurring white-flowered individuals led to significantly higher reproductive success of nearby purple-flowered individuals (mean fruit set 27%), while white-flowered plants themselves had the same low fruit set (6%)”, the authors of the study – L. Dormont, R. Delle-Vedove, J.-M. Bessière, M. Hossaert-Mc Key and B. Schatz – wrote in their article in New Phytologist (2010) 185: 300–310. The flowers studied – overall 11 709 at 805 plants – showed almost the same increased fruit set when the researchers planted some ping-pong balls which mimic the white Orchis mascula inflorescences: “The effect was virtually identical in magnitude (fruit set increased from 6 to 27%), whether the nearby white-coloured object was an O. mascula inflorescence or a ping-pong ball.” The nearer a purple-flowered plant to the white colour, the higher was the fruit set developed due to a successful pollination.

The authors explain their surprising results with pollinator behaviour after visiting Orchis mascula who belongs to the food-deceptive orchids: “It seems plausible to suppose that after unrewarding visits to purple flowers, naïve pollinators probably avoid homogeneous populations of purple flowers, and may then preferentially orient to a different colour or to a colour contrast such as a mix of white and purple flowers.” Pollinators of Orchis mascula are bumblebees (Bombus, Psithyrus), solitary bees (Eucera, Nomada, Andrena, Apis) and the beetle Cetonia aurata.

The albiflora varieties are quite rare in the populations studied in Southern France: The authors counted 0.9 to 1.4 percent in different populations. But this is much higher than the percentage which could be assumed in the case of spontaneous mutations affecting floral pigmentation genes with an average of just 0.1 percent. Regarding the higher percentage of albiflora varieties with Orchis mascula the authors state that “it is unlikely that such high frequencies could be the result of repeated spontaneous mutations alone” – and this should also apply to the case of other orchid species with a higher percentage of white-flowered plants like Anacamptis morio or Dactylorhiza fuchsii in Western Ireland.

The white-flowered Orchis mascula themselves have only a low fruit set, but they “help” the purple-flowered plants of their species to be pollinated. “In O. mascula, the presence of whiteflowered variants might be regarded as an adaptation that benefits the purple-flowered relatives of white-flowered morphs, rather than providing a direct benefit to whiteflowered individuals”, the authors wrote and assumed that there is some “mechanism of kin selection” responsible to grant a higher percentage of albiflora plants.

The scientists in Montpellier are pursuing their research with other species as well. Laurent Dormont wrote me that they have also studied white-flowered plants of Calanthe sylvatica on the Caribbean island of La Réunion (the results to be published in Plant Systematics and Evolution and also the floral volatiles of white-flowered orchis species.

Colour change more common among diploid Dactylorhiza

In an e-mail exchange following his recent article in the Journal Europaeischer Orchideen (JEO), Richard Bateman, orchid specialist at Kew Gardens, wrote me that albiflora plants “are far more common among diploid Dactylorhiza species than tetraploid species”. A possible reason might be the “buffering of mutations by having four comparable genes in the tetraploid chromosomes”. Diploid species (with 40 chromosomes) are Dactylorhiza fuchsii, D. incarnata and D. sambucina. Tetraploid species (with 80 chromosomes) are D. majalis, D. praetermissa, D. maculata, D. elata, D. sphagnicola and D. traunsteineri.

Albiflora plants of Dactylorhiza fuchsii are quite often observed, and in Ireland there is also the intriguing D. fuchsii ssp. okellyi which is diploid as well. D. incarnata and D. sambucina are known for their colour dimorphism: red and yellow with D. sambucina, purple and yellowish-white with D. incarnata and its var. ochroleuca. In a recent article in the Annals of Botany (2009), Mikael Hedrén and Sofie Nordstroem presented the results of their reasearch about the colour dimorphism with D. incarnata. They observed that there was “no clear pattern of habitat differentiation … among the colour morphs”. With D. incarnata var. ochroleuca “the lack of anthocyanins is probably due to a particular recessive allele in homozygous form” – the diploid chromosome set has both alleles determining the lack of purple in the flowers.

Besides genetics, colour also affects the pollination function of orchid flowers. Bateman wrote me that “in at least a few cases, instantaneous loss of anthocyanins (or even just radical decrease in anthocyanin production) must affect pollinator preference, and lead to lineage divergence”. A potential example of such an evolutionary process could be Gymnadenia frivaldii as a relative of Gymnadenia conopsea.

But in general the question of a certain functionality of colour change is still unanswered. Following his mentioning of white flowers in the above mentioned JEO article, Bateman wrote me it would be “more correct to use the term ‘parallelism’ rather than ‘convergence’, since in most cases no-one has demonstrated a change of function or ‘behaviour’ in the abnormal white flowers”. He further noted “the probability that many different mutations and epimutations generate white flowers”. Recognising that there are quite many open questions, Bateman also asked “whether white is actually a colour at all”, pointing to the “very simple shifts between ‘white’ flowers and ‘green’ flowers in Platanthera”.