Romain Lebarbenchon

Ewen Camberlein

Diego Di Carlo

Clément Gaultier

Antoine Deleforge

Nancy Bertin

Diego Di Carlo

Evaluation of an Open-Source Implementation of the SRP-PHAT Algorithm within the 2018 Locata Challenge

d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Many source localization methods are based on the estimation of time-difference-of-arrival (TDOA) between microphones. In the two-microphone case, the thorough evaluation carried out in [START_REF] Blandin | Multi-source TDOA estimation in reverberant audio using angular spectra and clustering[END_REF] highlighted the good performance of the Generalized Cross-Correlation with PHAse Transform (GCC-PHAT) algorithm [START_REF] Knapp | The generalized correlation method for estimation of time delay[END_REF] among several other methods. A general principle for extending this method to the multichannel setting, named SRP-PHAT, was proposed in [START_REF] Dibiase | Robust localization in reverberant rooms[END_REF]. However, no reference implementation of this family of algorithms (including all its possible variants) was available to date, to the best of the authors' knowledge. In particular, choices of parameters (pooling in time and frequency, grid search resolution) and management of the multiple coordinate systems between pairs of microphones and the whole antenna have not been systematically documented.

We present here our participation to the 2018 LOCATA challenge [START_REF] Löllmann | The locata challenge data corpus for acoustic source localization and tracking[END_REF]. This aims at evaluating our implementation of SRP-PHAT, whose good performance was already probed in realistic environments in [START_REF] Bertin | Voicehome-2, an extended corpus for multichannel speech processing in real homes[END_REF], but which was not confronted to other methods on a common, realistic dataset so far. It is here applied to task 1 (single, static source and static array), task 3 (single moving source and static array) and task 5 (single moving source and moving array) of the challenge. Evaluation of the method on the development dataset shows encouraging results, confirming the legitimate place of SRP-PHAT among state-of-the-art methods for multichannel source localization in reverberant environments.

PRINCIPLES

TDOA and angular spectrum

The general principle of SRP-PHAT is to compute a function Φ(θ, ϕ) termed "angular spectrum", where θ and φ are azimuth and elevation variables, which is expected to exhibit local maxima in the directions of active sources. More precisely, the following steps are followed:

• build in each time-frequency (t, f ) bin a local angular spectrum function φ(t, f, θ, ϕ) that is large for directions (θ, ϕ) which are compatible with the observed signal at (t, f ) and small otherwise, • integrate (or pool) this function over the time-frequency plane, leading to a global angular spectrum, • find the peaks of this spectrum above a certain threshold and distant from a certain minimum angle.

GCC-PHAT

The GCC algorithm [START_REF] Knapp | The generalized correlation method for estimation of time delay[END_REF] generalizes cross-correlation by multiplying the cross-spectral density (CSD) of two signals by complex weights in the frequency domain. The PHAT weighting consists of normalizing the CSD to have unit amplitude at all frequencies, thus considering phase differences only and minimizing the influence of the source's power spectral density. In the case of a single broadband source, two microphones and under far-field (source-array distance much larger that the array aperture h), free-field (only the direct source-to-microphone sound paths are considered) and noiseless assumptions, the asymptotic GCC-PHAT of the microphone signals in the discrete frequency domain is e -2iπf τ cos(α) where f is the frequency index, α is the angle of arrival (AOA) of the source signal at the array and τ is the maximum observable delay in samples. We have τ = hFs/C where C denotes the speed of sound and Fs is the frequency of sampling. This leads to the following natural local angular spectrum for a microphone pair:

φ GCC-PHAT (t, f, α) = x1(t, f )x * 2 (t, f ) |x1(t, f )x * 2 (t, f )| e -2iπf τ cos(α)
(1) where x1, x2 denote the microphone signals in the short-time Fourier transform (STFT) domain and α is a local azimuth in the coordinate system defined by the microphone pair. Note that this spectrum does not depend on elevation.

Extension to multichannel

SRP-PHAT's principle is to first compute local angular spectra for each microphone pair, then to aggregate them across pairs (bringing them back in the global coordinate system) before being pooled and maximized. The computation consists of the following steps:

1. Define the search space, i.e. a grid of possible DOAs (θj, ϕ k ) for which we want to evaluate Φ in the global coordinate system;

2. For each microphone pair n: Pooling methods (such as maximum or sum) and their order over each of the f, t, n indexes must be chosen for this purpose.

4. Find the indexes j and k of the largest peak (single source case) or peaks (multiple source case) of Φ(θj, ϕ k ), yielding the estimated source azimuth(s) θj and elevation(s) ϕ k .

A Matlab toolbox allowing easy and flexible implementations of SRP-PHAT as well as 7 other angular spectrum-based was made freely available online under the name Multichannel BSS Locate 1 .

EXPERIMENTS AND RESULTS

We now evaluate our implementation of SRP-PHAT on tasks 1, 3 and 5 of the LOCATA challenge [START_REF] Löllmann | The locata challenge data corpus for acoustic source localization and tracking[END_REF] with the robot head and the Eigenmike arrays for azimuth and elevation estimation. The other two antennas of the challenge are discarded since they are nearlinear (preventing elevation estimation). Moreover, DICIT is incompatible with our far-field assumption while the dummy head is incompatible with our free-field assumption. All microphones (12) and all microphone pairs (66) are used with the robot head. All microphones are also used with the Eigenmike, but microphone pairs with a curvilinear distance lesser than 90 • are discarded in order to reduce the overall algorithm complexity, resulting in 240 microphone pairs usage instead of the 496 available pairs. A sphere sampled with 1 • resolution both in azimuth and elevation is used as the search space (see Sec. 2.3 -1). For each microphone pair, the evaluated AOAs (see Sec. 2.3 -2.(b)) are computed with 5 • resolution. SRP-PHAT is applied every 256 ms to 512 ms signals (downsampled to 16 kHz) using an overlapping sliding analysis window. The STFT is applied to each channel of the signals using 64 ms Fourier frames (1024 samples) with 50% overlap and sine windows. This results in 15 Fourier frames per analysis window. The pooling methods used are first summations over microphone pairs and frequencies and then maximum over time frames. Only the largest value 1 http://bass-db.gforge.inria.fr/bss_locate/ over the global angular search space is returned as a DOA estimate, as only single source localization is considered. DOA estimates are linearly interpolated over time in order to match the one-estimateper-time-stamp requirement of LOCATA.

Table 1. shows localization errors obtained with the proposed method. Encouragingly, mean azimuth errors (first row) are 2 to 6 times smaller than those reported for the baseline MUSIC method [START_REF] Löllmann | The locata challenge data corpus for acoustic source localization and tracking[END_REF] for the same arrays and tasks. To reduce the effect of gross errors on reported mean values, average successful localization errors for different success threshold are also reported. The proposed method localizes the target source with less than 20 • error in at least 92% of the tests for all tasks and arrays. Nearly 100% correct localization is achieved in the static scenario (task 1). While the robot head array enables better performance in all tasks, average errors never exceed 10 • regardless of the task and array.

CONCLUSION

In this paper, we reported competitive single sound source localization results using the SRP-PHAT implementation of the Multichannel BSS Locate toolbox on the 2018 LOCATA challenge dataset. The toolbox's flexibility allows one to easily adapt the method to arbitrary array geometries, to various types of emitted signals and environments, and to easily generalize any two-channel localization method to more channels. It can also be tuned to find a trade-off between computational time and accuracy, although we focused on accuracy for this challenge. While the toolbox can output multiple source direction estimates for a given input signal, it does not yet incorporate source counting or source tracking solutions, preventing its use in multiple sources scenario. Such extensions will be considered for future releases of the toolbox.

( a ) 3 .

 a3 Compute the corresponding AOAs {α (n) jk } jk with respect to the microphone pair; (b) Resample {α (n) jk } jk into a smaller set {α (n) i }i in order to reduce computational time; (c) Compute the GCC-PHAT local angular spectrum φn at angles {α (n) i }i for microphone pair n according to formula (1); (d) Linearly interpolate φn back to the original angle resolution and global coordinate system; Compute the global spectrum Φ(θj, ϕ k ) by pooling the local angular spectra φn over all time-frequency bins (t, f ) and across all microphone pairs n.

Table 1: Sound localization results obtained on the LOCATA development dataset using the proposed SRP-PHAT method. For each task and array, different success thresholds (in degrees) are considered. A localization is considered successful when both azimuth and elevation estimation errors are below these thresholds. The percentage of successful localizations (suc.) and their average azimuth (az.) and elevation (el.) errors in degrees are showed for each threshold.

Success threshold


Tomáš Figura
email: figura.tomas@gmail.com

Edita Tylová

Jan Šoch

Marc-André Selosse

Jan Ponert

In vitro axenic germination and cultivation of mixotrophic Pyroloideae (Ericaceae) and their post-germination ontogenetic development

Keywords: Pyrola, Monotropa, Ericaceae, seed germination, in vitro culture, Chimaphila, protocorm, orchid, convergent evolution, seed dormancy, mixotrophy, Moneses

Background and Aims. Pyroloids, forest subshrubs of the Ericaceae family, are an important model for their mixotrophic nutrition, which mixes carbon from photosynthesis and from their mycorrhizal fungi. They have medical uses but are difficult to cultivate ex situ, in particular their dust seeds contain undifferentiated, few-celled embryos, whose germination is normally fully supported by fungal partners. Their germination and early ontogenesis thus remain elusive.

Methods. Optimised in vitro cultivation system of five representatives from the subfamily Pyroloideae, to study the strength of seed dormancy and the effect of different media and conditions (incl. light, gibberellins and soluble saccharides) on germination. The obtained plants were analysed for morphological, anatomical and histochemical development.

Key Results. Thanks to this novel cultivation method, which breaks dormancy and achieved up to 100% germination, leafy shoots were obtained in vitro for representatives of all pyroloid genera (Moneses, Orthilia, Pyrola and Chimaphila). In all cases, the first post-germination stage is an undifferentiated structure, from which a root meristem later emerges, well before formation of an adventive shoot.

Conclusions. This cultivation method can be used for further research or for ex situ conservation of pyroloid species. After strong seed dormancy is broken, the tiny globular embryo of pyroloids germinates into an intermediary zone, which is anatomically and functionally convergent with the protocorm of other plants with dust seeds such as orchids.

Like the orchid protocorm, this intermediary zone produces a single meristem: however, unlike orchids, which produce a shoot meristem, pyroloids first generate a root meristem.

Introduction

Mycorrhiza is worldwide symbiosis of most plants [START_REF] Smith | Mycorrhizal symbiosis[END_REF]. Usually, plants provide photosynthates in exchange for fungal mineral nutrients [START_REF] Van Der Heijden | Mycorrhizal ecology and evolution: the past, the present, and the future[END_REF]. However, mycorrhizal exchange may be less reciprocal, and perhaps exploitative, in mycoheterotrophic plants that derive carbon resources from their mycorrhizal fungi [START_REF] Leake | Tansley Review No. 69. The biology of myco-heterotrophic ("saprophytic") plants[END_REF]. Mycoheterotrophic plants evolved independently in various plant lineages [START_REF] Merckx | Mycoheterotrophy: The biology of plants living on fungi[END_REF], and beyond full mycoheterotrophs, which are achlorophyllous, some green species display a mixed strategy, obtaining carbon from both mycorrhizal fungi and their own photosynthesis, so-called mixotrophy [START_REF] Selosse | Green plants that feed on fungi: facts and questions about mixotrophy[END_REF]. There are two non-exclusive mixotrophic conditions [START_REF] Merckx | Mycoheterotrophy: The biology of plants living on fungi[END_REF]: some species are mixotrophic at adulthood, while other species germinate in a mycoheterotrophic way, before switching to autotrophy or mixotrophy during ontogeny (such as orchids; Dearnaley et al., 2016;[START_REF] Selosse | Mixotrophy in mycorrhizal plants: extracting carbon from mycorrhizal networks[END_REF][START_REF] Těšitel | Mixotrophy in land plants : why to stay green?[END_REF].

Little is known about the biology of mycoheterotrophic and mixotrophic plants, perhaps due to the difficulty of studying them [START_REF] Merckx | Mycoheterotrophy: The biology of plants living on fungi[END_REF]. The only cultivable group of mixotrophic plants is the orchid family, although many of them have so far escaped cultivation [START_REF] Rasmussen | Terrestrial orchids: from seed to mycotrophic plant[END_REF]. For this reason, orchids are the most explored mixotrophic models, but unfortunately it is largely unknown which results obtained for them can be generalised to other mixotrophic plants. Developing cultivation protocols for other mycoheterotrophic or mixotrophic plants is therefore urgently needed to understand general mechanisms connected with mycoheterotrophy.

Within Ericaceae, the subfamily Pyroloideae (hereafter pyroloids) comprises initial mycoheterotrophs that develop, depending on the species, into auto-or mixotrophic adults [START_REF] Tedersoo | Parallel evolutionary paths to mycoheterotrophy in understorey Ericaceae and Orchidaceae: Ecological evidence for mixotrophy in Pyroleae[END_REF][START_REF] Matsuda | Seasonal and environmental changes of mycorrhizal associations and heterotrophy levels in mixotrophic Pyrola japonica (Ericaceae) growing under different light environments[END_REF]Hynson et al., 2013a), with the exception of one completely mycoheterotrophic species [START_REF] Hynson | Evidence of a myco-heterotroph in the plant family Ericaceae that lacks mycorrhizal specificity[END_REF]. This similarity with orchids is an evolutionary convergence and it is therefore interesting to compare adaptations of both groups to mycoheterotrophy. Pyroloids encompass about 40 subshrub species divided into four genera, mostly distributed in northern temperate and boreal ecosystems [START_REF] Takahashi | Seed Morphology and Its Systematic Implications in Pyroloideae (Ericaceae)[END_REF][START_REF] Liu | A molecular phylogeny and a new classification of Pyrola (Pyroleae, Ericaceae)[END_REF], and are of medicinal interest, mostly in Asia (e.g. [START_REF] Ma | Chimaphilin induces apoptosis in human breast cancer MCF-7 cells through a ROS-mediated mitochondrial pathway[END_REF][START_REF] Wang | Phytochemical Composition, Antioxidant Activity and HPLC Fingerprinting Profiles of Three Pyrola Species from Different Regions[END_REF].

The seeds of pyroloids consist of oval, central living tissue surrounded by a coat of dead cells called a testa [START_REF] Fürth | Zur Biologie und Mikrochemie einiger Pirola-Arten[END_REF][START_REF]Untersuchungen über mykotrophen Verhältnisse der "Ericales" und die Keimung von Pirolaceen[END_REF][START_REF] Lück | Zur keimung der heimischen Pirola-Arten[END_REF][START_REF] Pyykkö | Embryological and anatomical studies on Finnish species of the Pyrolaceae[END_REF][START_REF] Takahashi | Seed Morphology and Its Systematic Implications in Pyroloideae (Ericaceae)[END_REF]. Despite the fact that the oval living part seems to be anatomically homogeneous, it is composed of a one-layered endosperm and triploid nutritive tissue of 40 to 50 cells surrounding a smaller embryo inside [START_REF] Hofmeister | Neuere Beobachtungen über Embryobildung der Phanerogamen[END_REF][START_REF] Fürth | Zur Biologie und Mikrochemie einiger Pirola-Arten[END_REF][START_REF]Untersuchungen über mykotrophen Verhältnisse der "Ericales" und die Keimung von Pirolaceen[END_REF][START_REF] Pyykkö | Embryological and anatomical studies on Finnish species of the Pyrolaceae[END_REF]. Similar seed structure was observed in the related, mycoheterotrophic genus Monotropa, where the embryo consists of only two to three cells [START_REF] Olson | Patterns of embryo and endosperm formation in Monotropa hypopitys (Monotropaceae) from North America and Western Sweden[END_REF] to 5-9 cells [START_REF] Goebel | Outlines of classification and special morphology of plants[END_REF] in addition to the endosperm, although minute seeds evolved independently in this Ericaceae genus [START_REF] Lallemand | The elusive predisposition to mycoheterotrophy in Ericaceae[END_REF]. The embryo of pyroloids is larger, with 8 or 16 cells [START_REF] Goebel | Outlines of classification and special morphology of plants[END_REF] to 30 cells [START_REF]Untersuchungen über mykotrophen Verhältnisse der "Ericales" und die Keimung von Pirolaceen[END_REF]. Similarly to Monotropa, the living part of the seed contains limited reserves of lipids and proteins [START_REF] Fürth | Zur Biologie und Mikrochemie einiger Pirola-Arten[END_REF][START_REF]Untersuchungen über mykotrophen Verhältnisse der "Ericales" und die Keimung von Pirolaceen[END_REF][START_REF] Lück | Zur Biologie der heimischen Pirola-arten[END_REF]. Other mycotrophic plants exhibit a similar seed structure, but, for example, orchids lack endosperm and the only living part of the seed is undifferentiated globular embryo [START_REF] Arditti | Numerical and physical properties of orchid seeds and their biological implications[END_REF].

Post-germination development of such seeds in pyroloids with undifferentiated embryo has attracted attention for a long time. First, researchers searched for seedlings in nature [START_REF] Irmish | Bemerkungen über einige Pflanzen der deutschen Flora[END_REF][START_REF] Velenovský | O biologii a morfologii rodu " Monesis[END_REF][START_REF] Velenovský | Über die Keimpflanzen der Pirolaceen[END_REF][START_REF] Fürth | Zur Biologie und Mikrochemie einiger Pirola-Arten[END_REF], albeit with limited success. They found only older seedlings, which revealed that a small root-like structure grows into an extensively branched structure which is formed before the first shoot emerges [START_REF] Irmish | Bemerkungen über einige Pflanzen der deutschen Flora[END_REF][START_REF] Velenovský | O biologii a morfologii rodu " Monesis[END_REF][START_REF] Fürth | Zur Biologie und Mikrochemie einiger Pirola-Arten[END_REF]. By contrast, orchids, which also start mycoheterotrophic development from an undifferentiated embryo, first form a specific structure called a protocorm, from which shoots and roots later develop [START_REF] Rasmussen | Terrestrial orchids: from seed to mycotrophic plant[END_REF]Dearnaley et al., 2016). This raised three questions: Are the structures observed in germinating pyroloids true roots? How does this root-like structure develop from a tiny undifferentiated embryo? What is its relationship to a protocorm? Many attempts have been made to answer these questions, but with only ambiguous results to the date. [START_REF] Velenovský | O biologii a morfologii rodu " Monesis[END_REF][START_REF] Velenovský | Über die Keimpflanzen der Pirolaceen[END_REF] believed this belowground structure is "neither a root nor a stem" and called it "prokaulom," while [START_REF]Untersuchungen über mykotrophen Verhältnisse der "Ericales" und die Keimung von Pirolaceen[END_REF] suggested that this structure is a root. Later, [START_REF] Lihnell | Keimungsversuche mit Pyrolasamen[END_REF] and [START_REF] Copeland | Observations on the structure and classification of the Pyroleae[END_REF] did the same anatomical analyses and found a typical root anatomical structure, where the radial vascular bundle is diarch, rarely triarch (i.e. has two or rarely three xylem strands). The middle layer of the root called the cortex is made of three to four (or rarely more) cell layers from which the innermost forms the endodermis [START_REF] Lihnell | Keimungsversuche mit Pyrolasamen[END_REF][START_REF] Copeland | Observations on the structure and classification of the Pyroleae[END_REF]. The outermost root layer, the epidermis, is one-layered and consists of isodiametric cells. Lateral branches grow from the outer layer of the stele [START_REF] Lihnell | Keimungsversuche mit Pyrolasamen[END_REF].

The method of burying seed packets at natural sites yields pyroloid seedlings easily, but the anatomical structure of such seedlings has not been studied in detail. These root-like structures, called "root-like structures" [START_REF] Hashimoto | Mycoheterotrophic germination of Pyrola asarifolia dust seeds reveals convergences with germination in orchids[END_REF][START_REF] Johansson | Recruitment limitation, germination of dust seeds, and early development of underground seedlings in six Pyroleae species[END_REF], live heterotrophically below ground for months, if not years [START_REF] Lihnell | Keimungsversuche mit Pyrolasamen[END_REF]Hynson et al., 2013a;[START_REF] Johansson | Specificity of fungal associations of Pyroleae and Monotropa hypopitys during germination and seedling development[END_REF].

Other researchers tried to germinate pyroloid seeds in vitro axenically [START_REF]Untersuchungen über mykotrophen Verhältnisse der "Ericales" und die Keimung von Pirolaceen[END_REF][START_REF] Lück | Zur Biologie der heimischen Pirola-arten[END_REF][START_REF] Lihnell | Keimungsversuche mit Pyrolasamen[END_REF] or symbiotically [START_REF] Fürth | Zur Biologie und Mikrochemie einiger Pirola-Arten[END_REF][START_REF] Lück | Zur keimung der heimischen Pirola-Arten[END_REF][START_REF] Lihnell | Keimungsversuche mit Pyrolasamen[END_REF].

Germination was achieved, although the results were "mostly uneven, not very consistent" [START_REF] Lihnell | Keimungsversuche mit Pyrolasamen[END_REF] and the seedlings usually stopped growing very soon after germination [START_REF]Untersuchungen über mykotrophen Verhältnisse der "Ericales" und die Keimung von Pirolaceen[END_REF][START_REF] Francke | Beiträge zur Kenntnis der Mykorrhiza von Monotropa hypopitys L[END_REF][START_REF] Lück | Zur Biologie der heimischen Pirola-arten[END_REF][START_REF] Lück | Zur keimung der heimischen Pirola-Arten[END_REF], sometimes being only a "few tenths of a mm long" [START_REF]Untersuchungen über mykotrophen Verhältnisse der "Ericales" und die Keimung von Pirolaceen[END_REF]. Similar tiny seedlings, which ceased growth, were obtained for Monotropa [START_REF] Francke | Beiträge zur Kenntnis der Mykorrhiza von Monotropa hypopitys L[END_REF]. The best results were achieved by [START_REF] Lihnell | Keimungsversuche mit Pyrolasamen[END_REF] and yielded a few branched root-like seedlings of Pyrola rotundifolia.

In these studies, observing different pyroloids, a polarised cone-shaped structure resembling a root with big epidermal cells grows from the tiny globular embryo [START_REF]Untersuchungen über mykotrophen Verhältnisse der "Ericales" und die Keimung von Pirolaceen[END_REF][START_REF] Lück | Zur Biologie der heimischen Pirola-arten[END_REF][START_REF] Lück | Zur keimung der heimischen Pirola-Arten[END_REF][START_REF] Lihnell | Keimungsversuche mit Pyrolasamen[END_REF]. At this stage, the seedling breaks the testa and forms an approximately 1-mm long roll-shaped structure [START_REF] Lihnell | Keimungsversuche mit Pyrolasamen[END_REF], whose central cells undergo elongation [START_REF] Lück | Zur keimung der heimischen Pirola-Arten[END_REF]. At this point growth usually ceases [START_REF] Lück | Zur Biologie der heimischen Pirola-arten[END_REF][START_REF] Lihnell | Keimungsversuche mit Pyrolasamen[END_REF]. Seedlings rarely grow further, producing a rod-shaped stage, where vessels start differentiation [START_REF] Lihnell | Keimungsversuche mit Pyrolasamen[END_REF], and, soon after, a root cap. Then, the seedling becomes darker, possibly because of the accumulation of tannins (e.g. [START_REF] Holm | Pyrola aphylla: A Morphological Study[END_REF][START_REF]Untersuchungen über mykotrophen Verhältnisse der "Ericales" und die Keimung von Pirolaceen[END_REF][START_REF] Lück | Zur Biologie der heimischen Pirola-arten[END_REF][START_REF] Lihnell | Keimungsversuche mit Pyrolasamen[END_REF]. [START_REF] Lihnell | Keimungsversuche mit Pyrolasamen[END_REF] showed that such older seedlings have the structure of a root and their illumination does not lead to the formation of chlorophyll. Shoots were never produced in vitro.

Growth suddenly stopped in all in vitro experiments [START_REF]Untersuchungen über mykotrophen Verhältnisse der "Ericales" und die Keimung von Pirolaceen[END_REF][START_REF] Lück | Zur Biologie der heimischen Pirola-arten[END_REF][START_REF] Lück | Zur keimung der heimischen Pirola-Arten[END_REF][START_REF] Lihnell | Keimungsversuche mit Pyrolasamen[END_REF], which raised the question of the ideal conditions for in vitro growth.

Germination usually started four to eight months after sowing [START_REF] Lück | Zur Biologie der heimischen Pirola-arten[END_REF][START_REF] Lück | Zur keimung der heimischen Pirola-Arten[END_REF][START_REF] Lihnell | Keimungsversuche mit Pyrolasamen[END_REF] and it was difficult to keep the cultures moist for such a long period [START_REF] Lihnell | Keimungsversuche mit Pyrolasamen[END_REF]. It is also hard to say if a specific experiment was really axenic, as the authors admitted themselves (for example, [START_REF]Untersuchungen über mykotrophen Verhältnisse der "Ericales" und die Keimung von Pirolaceen[END_REF], discussed whether worms were the reason for the failure of cultivation), and in symbiotic cultures it was not clear whether the fungus really formed symbiosis or not [START_REF] Lihnell | Keimungsversuche mit Pyrolasamen[END_REF]indeed, sometimes "symbiotic" seedlings grew away from the fungus). Moreover, in vitro cultivation media often contained substances of variable composition, such as potato extract, yeast extract, malt, peptone, or even humus, soil extracts [START_REF]Untersuchungen über mykotrophen Verhältnisse der "Ericales" und die Keimung von Pirolaceen[END_REF][START_REF] Lück | Zur keimung der heimischen Pirola-Arten[END_REF][START_REF] Lihnell | Keimungsversuche mit Pyrolasamen[END_REF]. Previous results from in vitro cultures are therefore based on a few plants only and do not indicate cultivation conditions that are ideal for more detailed observation.

Possible seed dormancy was also discussed. [START_REF]Untersuchungen über mykotrophen Verhältnisse der "Ericales" und die Keimung von Pirolaceen[END_REF] noticed that it is difficult to soak pyroloid seeds in water, indicating impermeability of the seed coat. Seeds disinfected with calcium hypochlorite solution showed the best germination after the longest bleaching time (15 and 30 minutes; [START_REF] Lihnell | Keimungsversuche mit Pyrolasamen[END_REF]. [START_REF] Lihnell | Keimungsversuche mit Pyrolasamen[END_REF] and [START_REF] Lück | Zur keimung der heimischen Pirola-Arten[END_REF] suggested that some "water-soluble substances" have an inhibitory effect on germination, and [START_REF] Harley | The biology of mycorrhiza. The biology of mycorrhiza[END_REF] hypothesised that some substances could be removed by soaking in solution. Although dormancy in minute seeds may seem unexpected, long bleaching of seeds enhances germination in many orchids (e.g. [START_REF] Burgeff | Samenkeimung der Orchideen und Entwicklung ihrer Keimpflanzen[END_REF][START_REF] Rasmussen | Seed dormancy patterns in Epipactis palustris (Orchidaceae): Requirements for germination and establishment of mycorrhiza[END_REF][START_REF] Rasmussen | Terrestrial orchids: from seed to mycotrophic plant[END_REF] probably because hypochlorite solutions have high pH and strong oxidative effects on a wide range of compounds, which could break impermeable seed coats (Arditti, 1967;[START_REF] Rasmussen | Terrestrial orchids: from seed to mycotrophic plant[END_REF][START_REF] Zeng | Seed biology and in vitro seed germination of Cypripedium[END_REF]. In some hardly germinating orchid species, pre-treatment with a weak H 2 SO 4 solution enhances germination, probably ensuring stronger degradation of seed coats (e.g. [START_REF] Malmgren | Asymbiotisk fröförökning i stor skala av Anacamptis, Ophrys, Orchis och andra orkidéer med runda rotknölar[END_REF][START_REF] Ponert | Asymbiotic germination of mature seeds and protocorm development of Pseudorchis albida (Orchidaceae) are inhibited by Nitrates even at extremely low concentrations[END_REF][START_REF] Malmgren | Orchid Propagation[END_REF]. However, the mechanisms are unclear in orchids and strong differences exist between species. Dormancy clearly requires further study in pyroloid seeds.

To summarize, no reliable protocol for in vitro germination of pyroloids exists and their early ontogenetic development remains elusive. We therefore sought to develop an efficient protocol for in vitro culture. We successfully report early steps of the postgermination development from undifferentiated embryo to leafy plant, in terms of storage compounds, and morphological and anatomical development, which enabled us to clearly answer the long-standing abovementioned questions about the nature of root-like structures of pyroloid seedlings, their development from a tiny undifferentiated embryo and their relationship to a protocorm.

Materials and methods

Plant material

We selected five European species as representatives of all four genera of the subfamily Pyroloideae (Pyrola media, Pyrola minor, Orthilia secunda, Moneses uniflora, Chimaphilla umbellata; Table S1). To compare with another subfamily of Ericaceae where minute seeds and mycoheterotrophic germination independently evolved [START_REF] Freudenstein | Phylogenetic relationships at the base of ericaceae: Implications for vegetative and mycorrhizal evolution[END_REF][START_REF] Lallemand | The elusive predisposition to mycoheterotrophy in Ericaceae[END_REF], we used Monotropa uniflora from the Monotropoideae (Table S1). Ripe capsules were collected, dried at room temperature, and extracted seeds were stored in the dark and dry conditions at +4°C (Table S1).

Cultivation media

To find a suitable cultivation medium for germination and growth, we tested nine media originally designed for orchid in vitro culture (Table S2). All media 0.7% agar (w/v, Sigma-Aldrich) and 1 -3 % sucrose (Table S2). After pH was adjusted to 5.8 using NaOH, media were autoclaved at 144 kPa, 121°C (Tuttnauer 2540 EK-N) for 20 min and poured into 5 cm plastic Petri dishes, unless otherwise indicated. Medium Knudson C with activated charcoal (Sigma-Aldrich) was used for all subsequent experiments, unless otherwise indicated. Activated charcoal is used to improve germination and growth of orchids and slowgrowing tissue cultures generally, perhaps due to its ability to adsorb toxic products of plant metabolism [START_REF] Van Waes | Effect of activated charcoal on in vitro propagation of Western European orchids[END_REF][START_REF] Thomas | The role of activated charcoal in plant tissue culture[END_REF]. To test the effect of different soluble saccharides that could mimic the carbon provided by the fungi in natural situations, sucrose was excluded or replaced with the monosaccharide 100 mM glucose or the disaccharide 50 mM sucrose or trehalose. To test the effect of gibberellins, 0.01, 0.1 or 1 mg/L of GA 3 (Sigma-Aldrich) was added to the medium before autoclaving.

Seed disinfection and sowing

Seeds were disinfected in 5 mL syringes and sown as a suspension in sterile deionised water as described previously [START_REF] Ponert | European orchid cultivationfrom seed to mature plant[END_REF][START_REF] Ponert | Asymbiotic germination of mature seeds and protocorm development of Pseudorchis albida (Orchidaceae) are inhibited by Nitrates even at extremely low concentrations[END_REF], but the application times of disinfection solutions of H 2 SO 4 and Ca(OCl) 2 were modified in a fully factorial design to find proper seed treatment. All treatments of disinfection were pre-incubated in 70% ethanol for 5 minutes, washed three times with deionised water (< 0.2 µm.cm -1 ), treated with 2% H 2 SO 4 for 10 minutes or not, treated with Ca(OCl) 2 solution for 5, 10 or 15 minutes, and finally washed three times with sterile deionised water. Ca(OCl) 2 solution was prepared by dissolving 20 g of chlorinated lime (Kittfort, Czech Republic) in 100 mL of deionised water, filtering through filter paper and adding a drop of Tween 20. For all subsequent experiments, we selected the most efficient treatment: 70% ethanol for 5 minutes, washed three times with deionised water, 2% H 2 SO 4 for 10 minutes, Ca(OCl) 2 solution for 10 minutes, washed three times with sterile deionised water. Seven Petri dishes sealed with air permeable foil (Parafilm M) were prepared for each experimental treatment.

Cultivation conditions

Because seeds ripen late in the season, all cultures were incubated in the dark at 4°C for three months after sowing to simulate winter, and then transferred to the dark at 20°C, except for experiments where the effect of light or cold stratification period was tested (see below). Cultures were observed every two weeks using a Krüss, MSZ 5400 Stereo Zoom Microscope (magnification 40x) and an Olympus Provis AX70 microscope for higher magnification. Germination rate was counted three times after end of cold stratification, at 30, 60 and 90 days. The last count (3 rd ; after 90 days at 20°C) was used to count total germination rate, because there no further germination was observed. To count the germination rate, seeds without embryo or with obviously undeveloped embryo were excluded and well-developed seeds with broken testa were regarded as germinated seeds (Fig. S1). To estimate seedling size in selected experiments, the total length of all branches of each seedling was measured from pictures taken with a Nikon D7000 + Micro Nikkor 55/2.8 using ImageJ 1.6.0_24 software.

To study the establishment of shoots in detail, we used approximately 3-5 mm long seedlings of P. minor pre-cultivated on Knudson C medium (Table S2) for 60 days after transfer to temperature 20°C. Each seedling (n=22) was transplanted to an individual nine cm Petri dish with BM-1 medium (Table S2). Pictures of plants were taken every 10 days. Plants with established shoots were continuously collected for anatomical analyses.

Plants with growing etiolated shoots were transferred to light (16 h light / 8 h dark) and cultivated for the next month to produce green leafy shoots. Plants with green shoots were deflasked, washed with water and potted in a mixture of coarse expanded perlite, fine pumice gravel, fine pine bark and loamy soil (1:1:1:2) in clay pots. Pots were sealed in polyethylene bags to keep air humidity high and were kept on a windowsill at 25°C in moderate light.

Anatomical analysis

Plant material (seeds, seedlings of P. minor) was fixed in 4% formaldehyde in phosphate buffer (0.1 M, pH 7.1). Selected samples were embedded in paraplast after dehydration using an ethanol-butanol series (for details, see [START_REF] Soukup | Essential Methods of Plant Sample Preparation for Light Microscopy[END_REF].

Sections (10 µm) were prepared using a Leica 2155 microtome and collected on microscopic slides coated with alum gelatine adhesive. Cryosections were prepared on a Shandon cryomicrotome. Hand sections were prepared on a Leica hand-microtome. For whole-mount preparations, samples were gradually equilibrated in 65% glycerol and mounted in NaI-based clearing solution of high refractive index [START_REF] Soukup | Essential Methods of Plant Sample Preparation for Light Microscopy[END_REF]. Histochemical tests involved staining with safranin O (2-h incubation) and Fast Green FCF (2 min). Lipids were detected with Sudan Red 7B (1 h) according to [START_REF] Brundrett | Efficient Lipid Staining in Plant Material with Sudan Red 7B or Fluoral Yellow 088 in Polyethylene Glycol-Glycerol[END_REF]. Proteins were stained with Ponceau 2R in 2% acetic acid (10 min) and Azur II (10 s) according to Gutmann et al. (1996). Detection of starch involved staining with Lugol solution. Observations were made with an Olympus BX51 microscope (Olympus Corp., Tokyo, Japan) equipped with an Apogee U4000 digital camera (Apogee Imaging Systems, Inc., Roseville, CA, USA) or with a Zeiss LSM 880 confocal microscope.

Endogenous starch HPLC analysis

To confirm the presence of starch in seedlings, we characterised the endogenous saccharide spectrum of selected pyroloids. Six-month-old (including period of cold stratification) seedlings of Mone. uniflora, O. secunda and P. minor (n=3 for each species) cultivated on BM-1 medium were collected in liquid nitrogen. Soluble carbohydrates were extracted following the protocol of [START_REF] Kubeš | Robust carbohydrate dynamics based on sucrose resynthesis in developing Norway spruce somatic embryos at variable sugar supply[END_REF].

The pellets left after soluble saccharide extraction were carefully washed with Milli-Q ultrapure water (sonicated in 1 mL of water for 15 min, centrifuged at 14000 rpm for 15 min, supernatant removed) and used for starch analysis. Starch was enzymatically degraded by αamylase (Sigma-Aldrich) and amyloglucosidase (Sigma-Aldrich) following the protocol of system described above, except for the use of an IEX Ca form 8 μm column in this case.

Data analysis

Statistical analyses were performed using statistical software R 3.2.3 (R Core Team 2015). The normality of data was tested using the Shapiro-Wilk test [START_REF] Shapiro | An analysis of variance test for normality (complete samples)[END_REF] and homogeneity of variances was tested using the Bartlett test [START_REF] Bartlett | Properties of sufficiency and statistical tests[END_REF]. Differences between the measurements were statistically tested with ANOVA, followed by the Tukey-Kramer test [START_REF] Kramer | Extension of multiple range tests to group means with unequal numbers of replications[END_REF] for data with a normal distribution and the Kruskal-Wallis test [START_REF] Kruskal | Use of ranks in one-criterion variance analysis[END_REF], followed by pairwise comparisons using Wilcoxon's rank-sum test for data that did not have normal distribution. To compare the effects of different disinfectants on seed germination, we used two-way ANOVA, followed by a Tukey-Kramer test.

Results

Effect of seed disinfection

It turned out to be impossible to sow non-disinfected seeds in vitro because of overwhelming contamination (data not shown). The highest germination rate of all tested species was reached after H 2 SO 4 treatment and the effect of H 2 SO 4 was significant in all tested species (P. minor: F [1,24] =193.96; p=5.4x10 -13 ; M. uniflora: F [1,40] =8.23; p=0.0065; O. secunda: F [1,23] =24.32; p=5.5x10 -5 ) except for C. umbellata; F [1,28] =0.06; p=0.8; Fig 1). The effect of Ca(OCl) 2 ; was significant in P. minor (F [2,24] =57.42; p=7.1x10 -10 ) and O. secunda (F [1,23] =3.79; p=0.038), but the optimal length of disinfection differed between these taxa.

Longer incubation in Ca(OCl) 2 strongly promoted germination of P. minor, but slightly inhibited germination of O. secunda (Fig. 1). Mone. uniflora germination was also higher after longer incubation in Ca(OCl) 2 , especially after the pre-treatment with H 2 SO 4 (Fig. 1B), but the effect was not significant (F [2,40] =2.13; p=0.13 ). The interaction between the effects of H 2 SO 4 and Ca(OCl) 2 was significant for P. minor (F [2,24] =35.97; p=6.0x10 -8 ) and for O. secunda (F [1,23] =4.05; p=0.031) only.

Effect of different media

Optimal media differed between species (Fig. 2A-C). Generally, the highest germination rates were observed on the media BM-1 (with or without activated charcoal), DS and MoN (Fig 2; (7) = 8.45; p= 0.29; Fig. S3B). The best medium for germination of P. minor was BM-1 (with or without activated charcoal), with a germination rate of almost 100% (χ 2 (8) = 32.59; p= 7.29x10 -5 ; Fig. 2A). For Mone. uniflora, the highest germination rates were achieved on media BM-1 and DS (χ 2 (5) = 7.34; p= 0.20; Fig. 2B).

Orthilia secunda germinated on all media tested, however, after longer incubation the highest germination rate was achieved on BM-1 medium without activated charcoal (F [7,41] =3.21; p=0.0083; Fig. 2C).

Media also differed in their suitability for seedling growth in a more or less similar way (Fig. 2D-F). We were unable to compare the effects of the different media on C.

umbellata growth because few seedlings developed on medium Knudson C. The biggest seedlings of P. minor developed on medium BM-1 (with or without activated charcoal) and on DS medium, although the differences between treatments were not significant (F [7,16] =0.984; p=0.48; Fig. 2D). The largest seedlings of Mone. uniflora developed on media DS and BM-1 without activated charcoal (F [6,15] =21.76; p =1.32x10 -6 ; Fig. 2E). The largest seedlings of O. secunda developed on MoN medium followed by BM-1 medium with activated charcoal (F [7,17] =14.54; p=1.19x10 -5 ; Fig. 2F).

Effect of light

The germination of both tested species (P. minor and O. secunda) was inhibited by light (16/8 h photoperiod; Fig. S4A,B). For O. secunda, this difference was significant (Fig. S4B;χ 2 (1) = 5.4; p= 0.02 in 2 nd month; only one plate in the 3 rd month because light cultures became contaminated). For P. minor, a similarly small proportion of seeds germinated after the 1 st month in both treatments and further germination was only slightly lower on light (differences not significant; F [1,8] =0.058; p=0.82; Fig. S4A). However, after another 6 months of cultivation, seedlings on light did not grow, while the seedlings in the dark grew, produced many branches and even some buds and first shoots (data not shown). Interestingly, seedlings cultivated on light displayed a reddish colour (Fig. 3B).

Effect of gibberellic acid and selected soluble saccharides

We found little effect of GA 3 on germination of the tested species, namely P. minor (Fig. S4C,D) and O. secunda (Fig. S4E,F). For O. secunda, there was no difference in germination rate between GA 3 concentrations on both media tested (Fig. S4E,F), except after the 1 st month on Knudson C (higher germination on the highest GA 3 concentration;

F [3,15] =7.94; p=0.002; Fig. S4F). For P. minor, there was a weak stimulatory effect after the 1 st month on BM-1 medium at the highest (1 mg/L) GA 3 concentration (χ 2 (3) = 4.719; p= 0.002; Fig. S4C).

Tested saccharides stimulated germination of seeds of both tested species. Pyrola minor was stimulated by all saccharides, with sucrose being the best (χ 2

(3) = 20.57, p= 0.0001, Fig. 4A), and the seeds cultivated without soluble saccharides germinated very rarely.

O. secunda reached higher germination rates only on sucrose (χ 2

(3) = 16.34, p= 0.001, Fig. 4B), while glucose and trehalose hardly enhanced germination. Seedling size was similarly affected by saccharides, with all saccharides acting on P. minor (Fig. S5A, Fig. S6A, F [3,14] =27.11; p=4.32x10 -6 ) and sucrose only on O. secunda (Fig. S5B, Fig. S6B; F [3,16] =9.57; p =0.00074). After three additional months of cultivation, seedlings from all saccharidesupplemented treatments produced at least a few long roots, while those from saccharide-free cultures did not, indicating that trehalose and glucose were also partially utilised. Moreover, after cultivation for one year, the individual seedlings on media with glucose and trehalose showed significant growth (Fig. S7).

Ontogenesis of seedlings

All species tested produced elongated seedlings of similar morphology (Fig. 3), irrespective of the medium. We used P. minor as a model species to analyse seedling development in detail. Like other pyroloids, P. minor has a minute seed with few-celled embryo surrounded by a cellular endosperm enclosed in single-layered transparent testa with pitted cell walls (Fig. 5A). Histochemical test indicates storage of lipids (Fig. S8C,E) and proteins (Fig. S8B,D) but not starch (Fig. S8A) in mature seeds. When germinated aseptically on media supplemented with soluble carbohydrate, germination starts with enlargement of the embryo at the micropylar pole (Fig. 5B) and with the formation of an apical meristematic area (in seedlings < 0.5 mm long; Fig. 5F) that later passes into root meristem (Fig. 5G). The transition from embryo into growing root is fast, but gradual. Less organised tissue is produced during a short initial phase to form an "intermediary zone" (Fig. 5C-H) that exhibits a heterogeneous surface cell pattern (Fig. 5D) and differentiates vascular tissues in its central part (Fig. 5G,H). In seedlings above 0.5 mm long, typical root organisation is achieved within the growing apex (Fig. 5G). The root produced includes radial vascular bundle devoid of pith (mostly diarch), exarch xylem with protoxylem close to the pericycle, suberised endodermis, and a root cap (Fig. 5I,J, Fig. S8I,L).

Seedling establishment on sucrose-amended media is accompanied by gradual disappearance of lipids (Fig. S8C,F,J) and accumulation of starch (Fig. S8F,H,K). Starch grains are located in cortical cells (Fig. S8H-I,K-L). High starch content was confirmed by HPLC analyses (Table S3.). Conversely, germination on media lacking carbohydrates terminated in the very initial phase, with very limited enlargement of embryo even after 11 months of cultivation (Fig. S8G).

The emerging root started to branch very early and produced an extensive root system (Fig. 6A), which initiates the first shoot bud much later (Fig. 6B). To observe the timing and positioning of the emergence of the first bud, we transferred seedlings individually (n=16) to new Petri dishes soon after germination on sucrose-amended BM-1 media. Seedlings produced their first buds at different times, starting 35 days after transplantation, and 7 out of 16 plants did not sprout after six months. The first shoot always sprouted a few mm away from the original position of the embryo, often at the nearest root branching (8 out of 9 plants; Fig. 6C-E). Very often additional buds appeared approximately one to two months after the first one, emerging along the root axis at sites of branching (Fig. 6D). One individual displayed 7 sprouts after six months. The buds grew quickly into leafy shoots which became green when transferred to the light (Fig. 6F,G). Seedlings left in a small Petri dish with other seedlings grew much slower and did not form buds till the end of the experiment. We also successfully germinated branching Mono. uniflora (n=3; Fig. S9A on BM-1 with activated charcoal; Fig. S3A) from which one plant produced multiple buds within a dense nest-like root cluster after one year of cultivation (Fig. S9B).

In summary, germination was successful to the stage of green leaves in P. minor (Fig. 6F-G) and also in Ch. umbellata, O. secunda, Mone. uniflora and P. media (not shown). The seedlings of P.minor were successfully transferred to ex-vitro conditions, and cultivated for the next six months (not shown).

Discussion

We developed an efficient novel protocol for in vitro axenic sowing of pyroloids, which enable us, for the first time, to successfully germinate representatives of all pyroloid genera and a related mycoheterotroph Monotropa uniflora. This protocol allowed us to overcome problems with seed germination and seedling grow cessation that were previously reported. Germination is probably driven mostly by physical dormancy because gibberellins had little effect on germination, while intensive pre-treatment (chemical scarification) of seeds greatly improved germination. This new, reliable protocol allowed us to produce seedlings in great number and to describe ontogenesis clearly, from early germination to leafy plantlet. In all cases, a tiny undifferentiated embryo produces an intermediary zone, which subsequently establishes a first root. Adventitious shoots grow later from this and secondary roots (Fig. 7, on the right). Our results suggest convergence in mixotrophic plants with dustlike seeds in regressive evolution leading to a single meristem that builds mycorrhizal tissue at germination.

Germination conditions

Gibberellic acid (GA 3 ), which greatly stimulates germination of many plant species [START_REF] Shu | Two Faces of One Seed: Hormonal Regulation of Dormancy and Germination[END_REF], had little effect on pyroloid germination. A strikingly similar situation occurs in orchid minute seeds, where gibberellins usually have little effect on seed germination (Arditti, 1967;[START_REF] Rasmussen | Terrestrial orchids: from seed to mycotrophic plant[END_REF], despite occasional reports of inhibition [START_REF] Van Waes | In vitro germination of some Western European orchids[END_REF] or stimulation [START_REF] Pedroza-Manrique | Evaluation of the Effect of three growth regulators in the germination of Comparettia falcata seeds under In vitro conditions[END_REF][START_REF] Pierce | Asymbiotic germination of the White Mountain Orchid (Pseudorchis albida) from immature seed on media enriched with complex organics or phytohormones[END_REF]. Low sensitivity to gibberellins may be a general feature of dust-like seeds.

The situation in orchids is sometimes explained by the absence of endosperm [START_REF] Arditti | Numerical and physical properties of orchid seeds and their biological implications[END_REF][START_REF] Yeung | A perspective on orchid seed and protocorm development[END_REF], because the stimulation of germination by gibberellins is mostly connected to the induction of the expression of genes encoding enzymes hydrolysing the endosperm (Groot and Karssen, 1987;[START_REF] Groot | Gibberellin-induced hydrolysis of endosperm cell walls in gibberellin-deficient tomato seeds prior to radicle protrusion[END_REF][START_REF] Schuurink | Dormancy of the barley grain is correlated with gibberellic acid responsiveness of the isolated aleurone layer[END_REF][START_REF] Leubner-Metzger | Effects of gibberellins, darkness and germination[END_REF]. Yet, since pyroloid seeds have a one-layered endosperm [START_REF] Hofmeister | Neuere Beobachtungen über Embryobildung der Phanerogamen[END_REF][START_REF] Fürth | Zur Biologie und Mikrochemie einiger Pirola-Arten[END_REF][START_REF]Untersuchungen über mykotrophen Verhältnisse der "Ericales" und die Keimung von Pirolaceen[END_REF][START_REF] Pyykkö | Embryological and anatomical studies on Finnish species of the Pyrolaceae[END_REF], the low sensitivity to gibberellins of dustlike seeds may not be associated with the absence of endosperm but with other characteristics yet to be clarified.

The strong inhibitory effect of light on germination could serve as a protection against germination on the soil surface, where mycorrhizal fungi may not be present. A similar mechanism of light inhibition is well known in many terrestrial species of orchids (Arditti, 1967[START_REF] Arditti | Micropropagation of orchids[END_REF][START_REF] Rasmussen | Terrestrial orchids: from seed to mycotrophic plant[END_REF]. We also found a strong effect of disinfection on seed germination. More intensive seed disinfection stimulated germination and longer incubation in calcium hypochlorite solution after sulphuric acid treatment was often most effective.

Similarly, [START_REF] Lihnell | Keimungsversuche mit Pyrolasamen[END_REF] achieved the highest germination rate after the longest incubation in calcium hypochlorite solution, which he was unable to explain. Since these solutions also act corrosively to destroy the impermeable coats of dust-like seeds (e.g. Arditti, 1967;[START_REF] Rasmussen | Terrestrial orchids: from seed to mycotrophic plant[END_REF][START_REF] Zeng | Seed biology and in vitro seed germination of Cypripedium[END_REF], we propose that strong physical dormancy due to the testa explains the long pre-germination period and the low germination rates observed in situ [START_REF] Hashimoto | Mycoheterotrophic germination of Pyrola asarifolia dust seeds reveals convergences with germination in orchids[END_REF][START_REF] Johansson | Recruitment limitation, germination of dust seeds, and early development of underground seedlings in six Pyroleae species[END_REF]Hynson et al., 2013b;[START_REF] Johansson | Specificity of fungal associations of Pyroleae and Monotropa hypopitys during germination and seedling development[END_REF]. After proper chemical scarification, we reached 100% and 99% germination for P.

minor and O. secunda seeds, respectively, which is much higher than observed in situ [START_REF] Hashimoto | Mycoheterotrophic germination of Pyrola asarifolia dust seeds reveals convergences with germination in orchids[END_REF]Hynson et al., 2013b) or in vitro [START_REF] Lück | Zur Biologie der heimischen Pirola-arten[END_REF][START_REF] Lihnell | Keimungsversuche mit Pyrolasamen[END_REF] in different species of the same genera. It is hard to discuss possible actions of disinfection agent's on seeds in detail, because chemical composition of pyroloid seeds is unknown. In orchids, seed coats seems to consist of lignin, lipids and other uncharacterized compounds [START_REF] Barsberg | Composition of Cypripedium calceolus (Orchidaceae) seeds analyzed by attenuated total reflectance IR spectroscopy: in search of understanding longevity in the ground[END_REF]2018). Ethanol is a good solvent for many lipids and wax compounds and it usually makes seeds less hydrophobic and more accessible for subsequent treatment with aqueous solutions. Lignin is highly sensitive to oxidative effect of hypochlorite solutions, so we could expect disintegrating of seed coat during hypochlorite treatment. The effect of H 2 SO 4 is hard to guess, but we could expect existence of some other compound sensitive to acid hydrolysis, but not the alkaline one.

Chimaphila umbellata and Mone. uniflora germinated at significantly lower rates comparing with P. minor and O. secunda in our experiments. Since these two genera form a clade separate from that encompassing Pyrola and Orthilia [START_REF] Freudenstein | Phylogenetic relationships at the base of ericaceae: Implications for vegetative and mycorrhizal evolution[END_REF][START_REF] Lallemand | The elusive predisposition to mycoheterotrophy in Ericaceae[END_REF], some additional dormancy mechanism may occur in this clade, which remains to be identified. In soils, dormancy, added to photoinhibition, may increase the ability of the plant to wait for suitable fungi required for germination.

Our in vitro methods provided seedlings in great number and shed light on the early development of pyroloids, with replicates for dissection to an extent that was never reached before, allowing a clear discussion of early ontogenesis of pyroloidsassuming, of course, that our artificial, non-symbiotic conditions do not alter ontogeny, as supported below.

Cultivation protocol

Based on our results, we propose a cultivation protocol that would allow to germinate all pyroloids generally. This procedure uniquely combines different techniques used in orchid culture previously to satisfy needs of pyroloids. Seed disinfection implies: 70% ethanol for 5 minutes, washed out three-times with a deionised water, then 2% H 2 SO 4 for 10 minutes, followed by Ca(OCl) 2 solution for 10 minutes, washed three times with sterile deionised water. The best cultivation medium is BM-1, but DS and MoN work also well. For cultivation conditions, we recommend 4°C for 3 months after sowing, and subsequently 20°C, in dark. Seedlings can be transferred to the new medium when they are approximately 5 mm long. When shoots will start to grow, seedlings should be transferred to the light and, at that time, it is better to put them separately into 100 ml cultivation jars.

Development of pyroloid seedlings

The dust seed structure with an undifferentiated globular embryo is typical for many mycoheterotrophic plants [START_REF] Leake | Tansley Review No. 69. The biology of myco-heterotrophic ("saprophytic") plants[END_REF] and plants with mycoheterotrophic early development (Dearnaley et al., 2016). Such dust seeds have limited reserves, the storage compounds of which are mostly unknown. In pyroloids, previous studies found storage lipids and proteins in mature seeds [START_REF] Fürth | Zur Biologie und Mikrochemie einiger Pirola-Arten[END_REF][START_REF]Untersuchungen über mykotrophen Verhältnisse der "Ericales" und die Keimung von Pirolaceen[END_REF][START_REF] Lück | Zur Biologie der heimischen Pirola-arten[END_REF]. Similarly, ripe seeds of orchids usually contain lipids and proteins [START_REF] Harrison | Ultrastructural and Histochemical changes during the Germination of Cattleya aurantiaca (Orchidaceae)[END_REF][START_REF] Manning | The development and mobilisation of seed reserves in some African orchids[END_REF][START_REF] Rasmussen | Cell differentiation and mycorrhizal infection in Dactylorhiza majalis (Rchb. f.) Hunt & Summerh. (Orchidaceae) during germination in vitro[END_REF][START_REF] Richardson | Seed reserves and early symbiotic protocorm development of Platanthera hyperborea (Orchidaceae)[END_REF][START_REF] Yam | Orchid Embryos In[END_REF][START_REF] Li | Embryology of two mycoheterotrophic orchid species, Gastrodia elata and Gastrodia nantoensis: ovule and embryo development[END_REF], and few orchid species display starch in mature embryos [START_REF] Tian | In vitro seed germination and developmental morphology of seedling in Cymbidium ensifolium[END_REF][START_REF] Guo | Studies on the changes of cell ultrastructure in the course of seed germination of Bletilla striata under fungus infection conditions[END_REF][START_REF] Yeung | Embryology of Calypso bulbosa. II. Embryo development[END_REF]. Our histochemical tests confirmed storage lipids and proteins, but absence of starch in P. minor seeds, thus further amplifying the convergence with orchids.

During germination, lipids and storage proteins were utilised and seedlings shifted to use starch as the main storage compound, as confirmed by histochemistry and HPLC. This switch to starch was already observed in several pyroloids [START_REF]Untersuchungen über mykotrophen Verhältnisse der "Ericales" und die Keimung von Pirolaceen[END_REF][START_REF] Lück | Zur Biologie der heimischen Pirola-arten[END_REF][START_REF] Lihnell | Keimungsversuche mit Pyrolasamen[END_REF] and is common among orchids [START_REF] Manning | The development and mobilisation of seed reserves in some African orchids[END_REF][START_REF] Rasmussen | Cell differentiation and mycorrhizal infection in Dactylorhiza majalis (Rchb. f.) Hunt & Summerh. (Orchidaceae) during germination in vitro[END_REF][START_REF] Richardson | Seed reserves and early symbiotic protocorm development of Platanthera hyperborea (Orchidaceae)[END_REF]. The transition from lipid and protein reserves in seeds to starch in seedlings therefore also convergently characterizes mycoheterotrophic germination.

Mycoheterotrophic pyroloid seedlings grow in vitro as branching roots for a long time before the first green shoot emerges. Such non-green seedlings grow below ground in nature very slowly, for an even longer time [START_REF] Hashimoto | Mycoheterotrophic germination of Pyrola asarifolia dust seeds reveals convergences with germination in orchids[END_REF][START_REF] Johansson | Recruitment limitation, germination of dust seeds, and early development of underground seedlings in six Pyroleae species[END_REF]Hynson et al., 2013b;[START_REF] Johansson | Specificity of fungal associations of Pyroleae and Monotropa hypopitys during germination and seedling development[END_REF]. Seedling growth was also promoted by more complex cultivation media (e.g. BM-1, compared to Knudson C or MS), which could indicate dependency on some other organic compounds provided in nature by fungus. Complex media are beneficial also for many mixotrophic orchids [START_REF] Rasmussen | Terrestrial orchids: from seed to mycotrophic plant[END_REF][START_REF] Arditti | Micropropagation of orchids[END_REF].

Post-germination development differs from orchids despite some parallels. Orchid germination forms a protocorm whose enlargement establishes a shoot meristem on its anterior pole [START_REF] Burgeff | Samenkeimung der Orchideen und Entwicklung ihrer Keimpflanzen[END_REF][START_REF] Leroux | Morphogenesis of the protocorm of Cypripedium acaule (Orchidaceae)[END_REF][START_REF] Yeung | A perspective on orchid seed and protocorm development[END_REF]. In pyroloids, germination starts with enlargement of the embryo at the suspensor pole, which develops into a small elongated structure growing apically-the 'cone stadium' of [START_REF] Lück | Zur keimung der heimischen Pirola-Arten[END_REF] and [START_REF] Lihnell | Keimungsversuche mit Pyrolasamen[END_REF].

Soon after, the apical region establishes a root meristem and grows into a typical root.

Although the transition is pretty fast, a short region (app. 0.5 mm) close to the former embryo exhibits a slightly different internal structure (Fig. 7), which is why we call this part the "intermediary zone" (see discussion below).

The first root grows and establishes lateral roots to develop an extensively branched root system as also observed in situ [START_REF] Irmish | Bemerkungen über einige Pflanzen der deutschen Flora[END_REF]Velenovsky, 1892Velenovsky, , 1905;;[START_REF] Bobrov | Grushankovye Rossii[END_REF][START_REF] Hashimoto | Mycoheterotrophic germination of Pyrola asarifolia dust seeds reveals convergences with germination in orchids[END_REF]. For a long time, it was unclear whether these structures are true roots (see [START_REF] Goebel | Gibberellins regulate seed germination in tomato by endosperm weakening: a study with gibberellin-deficient mutants[END_REF][START_REF] Lihnell | Keimungsversuche mit Pyrolasamen[END_REF]. [START_REF] Velenovský | O biologii a morfologii rodu " Monesis[END_REF][START_REF] Velenovský | Über die Keimpflanzen der Pirolaceen[END_REF] suggested these are "neither root nor stem". We clearly show a typical root organisation (incl. root cap, endogenous origin of lateral roots and radial vascular bundle; Fig. 5 H,I,J), as concluded in earlier anatomical studies [START_REF] Lihnell | Keimungsversuche mit Pyrolasamen[END_REF][START_REF] Copeland | Observations on the structure and classification of the Pyroleae[END_REF]. The abovementioned discussions were probably caused by insufficient anatomical examination and confusion between the intermediary zone and the true roots. As in the observations of [START_REF] Velenovský | Über die Keimpflanzen der Pirolaceen[END_REF] and [START_REF] Lihnell | Keimungsversuche mit Pyrolasamen[END_REF] and adult roots [START_REF] Hynson | Evidence of a myco-heterotroph in the plant family Ericaceae that lacks mycorrhizal specificity[END_REF], the roots observed had no root hairs. Our data clearly show that the first shoot sprouts from a small section between the original position of embryo and the first root branching site. Shortly after, other adventitious shoots usually emerge from other places of the root system. Such root sprouting also exists in adult pyroloids in natural conditions [START_REF] Copeland | Observations on the structure and classification of the Pyroleae[END_REF][START_REF] Klimešová | Root-sprouting in myco-heterotrophic plants: prepackaged symbioses or overcoming meristem limitation?[END_REF].

Is the intermediary zone a protocorm?

Typical plant embryos develop from both radicle (basal pole establishing the primary root) and plumule (apical pole establishing the primary shoot) poles, while in pyroloids or orchids one embryo pole does not grow: respectively, the primary shoot or the primary root is completely missing (Fig. 7). Other mycoheterotrophic species with dust seeds that have been investigated to the date, Afrothismia hydra (Burmanniaceae; [START_REF] Imhof | Ontogeny of the mycoheterotrophic species Afrothismia hydra (Burmanniaceae)[END_REF] and the genus Voyria (Gentianaceae; [START_REF] Imhof | Are Monocots Particularly Suited to Develop Mycoheterotrophy?[END_REF][START_REF] Imhof | Subterranean morphology and mycorrhizal structures In[END_REF], also germinate by a single, basal (radicle) embryo pole. This independent loss of one meristematic embryo pole in plants with dust-like seeds indicates some general selection pressure in mycoheterotrophic germinations perhaps for several non-exclusive reasons: (i) the limited resources available cannot support two growth sites [START_REF] Imhof | Ontogeny of the mycoheterotrophic species Afrothismia hydra (Burmanniaceae)[END_REF] and /or (2) the absence of requirement for shoots and roots in mycoheterotrophy, since a single organ for interaction with the fungus is sufficient, and/or (3) the later mycorrhizal organ makes one embryo pole unable to differentiate into a meristem. We therefore propose that transformation of one embryo pole into mycorrhizal tissue is a necessary evolutionary step enabling reduction of seed reserves to a minimum of the production of a true dust-like seed structure.

It could be argued that plants cultivated in vitro exhibit different development from plants in nature. However, the seedlings observed by us fully fit these observed in situ [START_REF] Irmish | Bemerkungen über einige Pflanzen der deutschen Flora[END_REF]Velenovsky, 1892Velenovsky, , 1905;;[START_REF] Hashimoto | Mycoheterotrophic germination of Pyrola asarifolia dust seeds reveals convergences with germination in orchids[END_REF][START_REF] Johansson | Recruitment limitation, germination of dust seeds, and early development of underground seedlings in six Pyroleae species[END_REF]Hynson et al., 2013a). In particular, the drawings and photographs in vitro by [START_REF]Untersuchungen über mykotrophen Verhältnisse der "Ericales" und die Keimung von Pirolaceen[END_REF], [START_REF] Lück | Zur Biologie der heimischen Pirola-arten[END_REF][START_REF] Lück | Zur keimung der heimischen Pirola-Arten[END_REF] and [START_REF] Lihnell | Keimungsversuche mit Pyrolasamen[END_REF] reveal similar seedlings.

How to classify the structure that develops between the undifferentiated globular embryo and the typical root? It starts with polarised growth at suspensor pole of the embryo and continues with cell divisions in an emerging meristematic area at its apical part. Later, it becomes thick, with extremely heterogeneous large epidermal cells and a broad central area of vascular tissues, but without a typical root structure. We therefore called it here the intermediary zone. In previous studies, it was named a "root-like structure" [START_REF] Hashimoto | Mycoheterotrophic germination of Pyrola asarifolia dust seeds reveals convergences with germination in orchids[END_REF], a "procaulom" (Velenovsky, 1892(Velenovsky, , 1905) ) or a "protosoma" [START_REF] Bobrov | On the early stages of ontogenesis of Europaean Pyrolaceae species [In Russian][END_REF][START_REF] Bobrov | Grushankovye Rossii[END_REF][START_REF] Bobrov | Zhiznennaya forma Moneses uniflora (Pyroloideae, Ericaceae) [In Russian][END_REF]. We believe that previous studies did not explore the anatomical structure in detail (an analysis now allowed by the number of seedlings provided by our cultivation methods) and therefore did not distinguish between the intermediary zone and the true roots. Moreover, previous studies analysed seedlings that were already much larger.

Orchids also display an intermediary transition stage between the globular embryo and the first shoot meristem that is larger than the pyroloid intermediary zone and is called a protocorm [START_REF] Rasmussen | Terrestrial orchids: from seed to mycotrophic plant[END_REF][START_REF] Yeung | A perspective on orchid seed and protocorm development[END_REF], because it precedes formation of the typical plant cormus (i.e. the first shoot or root). Since the intermediary zone of pyroloids also precedes the cormus (here, the first root), we also suggest use of the term protocorm for the intermediary zone of pyroloids. The term protocorm was first used for post-embryonic stages of clubmosses [START_REF] Treub | Études sur les Lycopodiaceès[END_REF], which also form a mycoheterotrophic structure appearing before formation of the first shoot and root. This term was later transferred to orchids [START_REF] Bernard | L'évolution dans la symbiose. Les Orchideés et leurs champignons commensaux[END_REF] and it is thus historically not orchid-limited, but really naming a convergently evolved structure. Recently, protocorm was even used in the obligately parasitic Rafflesiaceae, for an endophytic structure developing from proembryonic endophytic tissue before the formation of the flower-bearing shoot [START_REF] Nikolov | Floral structure and development in Rafflesiaceae with emphasis on their exceptional gynoecia[END_REF][START_REF] Nikolov | The big, the bad, and the beautiful: Biology of the world's largest flowers[END_REF]. Interestingly, [START_REF] Harley | The biology of mycorrhiza. The biology of mycorrhiza[END_REF] [START_REF] Imhof | Are Monocots Particularly Suited to Develop Mycoheterotrophy?[END_REF][START_REF] Imhof | Subterranean morphology and mycorrhizal structures In[END_REF] produce a root as the first organ, but nothing is known about initiation of the root from the globular embryo in these plants.

Conclusion

We used unique combination of cultivation techniques which, for the first time allowed us to develop an efficient cultivation protocol to germinate and grow leafy plants of representatives from all pyroloid genera. Our unique methods for pyroloid cultivation may be used for conservation purposes and also for physiological investigations, since pyroloid mixotrophic nutrition at adulthood is currently of considerable interest (e.g., Lallemand et al., 2017). The cultures obtained allow fine analyses of germination, with the transition from lipid and protein storage in seeds to starch accumulation, based on saccharides from the environment. Seedlings form first roots, before adventitious sprouting. The first root emerges

Figure captions

  Fig. S2). Media 1/4-2, Knudson C and MS allowed slightly lower germination generally (Fig 2; Fig. S2). Surprisingly, few seedlings of C. umbellata germinated on Knudson C medium (χ 2

Fig. 1 .

 1 Fig. 1. Effect of different disinfection treatments on seed germination of (A) Pyrola minor, (B) Moneses uniflora, (C) Chimaphila umbellata, (D) Orthilia secunda. In vitro cultivation on medium Knudson C was evaluated after three months after the cold stratification. Median values are given (n=7). Different letters indicate significant difference according to the ANOVA followed by Tukey HSD post-hoc test for multiple comparison.

Fig. 2 .

 2 Fig. 2. Effect of different media on germination of (A) Pyrola minor, (B) Moneses uniflora, (C) Orthilia secunda and growth of (D) Pyrola minor, (E) Moneses uniflora, (F) Orthilia secunda. Both germination and total length of seedlings were measured 3 months after cold stratification. Different letters indicate significant difference. Results of Tukey HSD post hoc test for multiple comparisons are indicated by capital letters and results of Pairwise Wilcoxon Rank Sum test by lowercase letters.

Fig. 3 .

 3 Fig. 3. Representatives of in vitro grown seedlings of species used in this study. A) Pyrola minor cultivated in dark, B) Pyrola minor cultivated on light and C) Moneses uniflora, D) Orthilia secunda, E) Chimaphila umbellata and F) Monotropa uniflora cultivated in dark. Scale bars: 0,5 mm.

Fig. 4 .

 4 Fig. 4. Effect of selected soluble saccharides on germination of Pyrola minor (A) and Orthilia secunda (B). In vitro cultivation on Knudson C medium where sucrose was replaced by saccharide indicated. Different letters indicate significant difference. Results of Tukey HSD post hoc test for multiple comparisons indicated by capital letters and results of Pairwise Wilcoxon Rank Sum test by lowercase letters; bars indicate 1 st and 3 rd quantiles. Pairwise comparisons were calculated separately for each month.

Fig. 5 .

 5 Fig. 5. Germination and early development of Pyrola minor. (a) mature seed with globular embryo. (b) germination starts with enlargement of embryo (arrows indicate the former embryo). (c) seedling develops further into root through less organized intermediary zone with very heterogenic surface cell pattern. (d) detail of the surface cell pattern of intermediary zone. (e) seedling of 1-1.5 mm size with already established root meristem. (f) meristematic zone is established very early after germination. (g) seedling of less than 1 mm size with established meristem and differentiated vascular tissues in central part of intermediary zone close to the former embryo. (h) seedling of ca. 3 mm size with already established lateral root primordium (white arrow) and differentiated vascular tissues (in detail). (i) root structure is obvious at 1.5 cm from the position of former embryo in 3 cm long seedling. Abbreviations: en, endodermis; x, protoxylem vessels of radial vascular bundle; c, cortex; rh, rhizodermis. (j) root tip of 3 cm long seedling. Abbreviations: lrc, lateral root cap; p, protoderm; qc, quiescent center. a, b, e, j, whole-mounts, DIC; c, whole-mount, confocal image; d, whole-mount, UV; f-h, paraplast sections, Safranin O + Fast Green FCF; I, hand section, UV; j, paraplast section, Lugol + Orange G. Black arrows indicate position of former embryo; scale bars 50 µm.

Fig. 6 .

 6 Fig. 6. Transition to shoot formation in P. minor seedlings. (a) Nine-months old seedling with established root system prior the onset of shoot growth. (b) Nine-months old seedling with emerging shoot (arrow). (c) shoot bud emerges at first root branching site close to the position of former embryo (r, root; sh, shoot bud; iz, intermediary zone connecting the former embryo; x, xylem), section stained with Safranin O and Fast Green FCF. (d) later, additional shoot buds emerge along root axis (arrows). (e) detail of first shoot bud, whole-mount preparation. (f) fully-established in vitro plant. (g) adventitious roots emerging at stem node (sh, shoot). Scale bars 0.5 cm, except c, e: 200 µm.

  also suggested the term protocorm for seedlings of Monotropa, which are closely related to pyroloids. The pyroloid protocorm is significantly smaller and grows on the opposite embryo pole compared to orchids, but this transitory structure precedes the typical cormus in both mycoheterotrophic developments. Investigations of the early ontogenetic development of other unrelated mycoheterotrophic plants with dust seeds (e.g Burmaniaceae,

Gentianaceae, Triuridaceae;

[START_REF] Eriksson | The evolutionary ecology of dust seeds[END_REF]

are pending. Scarce studies have revealed that Afrothismia hydra (Burmanniaceae;

[START_REF] Imhof | Ontogeny of the mycoheterotrophic species Afrothismia hydra (Burmanniaceae)[END_REF]

and Voyria spp. (Gentianaceae;

  ). Embryo is highlighted in red, protocorm in blue, shoots in green and roots in brown. Abbreviations: a, adventive root; br, branching roots; e, embryo; end, endosperm; h, hilum; iz, intermediary zone; p, protocorm; ram, root meristem; sh, shoot; sam, shoot meristem; t, testa; x, xylem. Thin bar is 100µm, dotted bar 1mm and thick bar 0,5 cm.

Acknowledgements

We thank to Montreal Botanical Garden for sending us seeds of C. umbellata and Mono. uniflora, Yvetta Šefrnová and Marek Širl for help with anatomical study, Radim Hédl and Jitka Klimešová for help with the old literature and Karel Fajmon and Michal Štefánek for help with sampling localities. We also thank editor Margreth Sauter and two anonymous reviewers for useful comments.

Funding

This work was supported by grant of the Czech Ministry of Education, Youth and Sports [grant no. LO1417] and by Grant Agency of Charles University [grant no. 468318].

functional and developmental similarity with protocorms in other plants. Our