Biological flora of Britain and Ireland: Neottia nidus- avis

1. This account provides information on all aspects of the biology of Neottia nidus avis (L.) Rich. (Bird's- nest Orchid) that are relevant to understanding its eco logical characteristics and behaviour. The main topics are presented within the standard framework of the Biological Flora of Britain and Ireland: distribution, habitat, communities, responses to biotic factors, responses to environment, structure and physiology, phenology, reproductive characteristics, herbivory, history and conservation. 2. Neottia nidus- avis is a native mycoheterotrophic orchid; it is found most fre quently in the deep humus of densely shaded beech woodlands, on limestone or chalky soils in the British Isles. The species extends throughout temperate Eurasia. Neottia nidus- mostly Epidendroideae,


| G EOG R APHIC AL AND ALTITUDINAL DIS TRIBUTION
Neottia nidus-avis was recorded from 928 10-km squares (hectads) in the British Isles (about 23.8% of the total, with 874 hectads in Great Britain and 154 hectads in Ireland; Figure 1; Table 1). The species is widely scattered throughout much of the British Isles. It is locally common in mature woodland in parts of southern England (a belt from the northern parts of East Anglia through Lincolnshire and the north Midlands to northern and western Wales) but scarce in the rest of the British Isles. Although recorded from more than half of Scottish vice-counties, it is a very rare plant there and is very scattered in lowlands. Similarly, in the Republic of Ireland, although recorded from most counties, each has only a handful of populations. It is absent from the Isle of Man, Shetland, Orkney and the Inner and Outer Hebrides, except for the islands Skye and Mull (Foley & Clarke, 2005;Harrap & Harrap, 2005). In Northern Ireland, southern England, where it is locally abundant. The main threats are deforestation, changes in woodland management and coniferization.

K E Y W O R D S
communities, conservation, germination, mycoheterotrophic plant, mycorrhiza, Orchidaceae, reproductive biology the species is widespread in the southwest and scattered through the rest of the country.
It grows from the British Isles throughout most of central Europe (Bartha et al., 2015;Presser, 2000;Procházka & Velísek, 1983;Vlčko et al., 2003) with an eastern limit in western Siberia (Vakhrameeva et al., 2008, map. 25). It is found as far north as c. 65° in Scandinavia (Anderberg & Anderberg, 2016) and south to northern Portugal (Tyteca & Bernardos, 2003), Spain (Anthos, 2012), Corsica, the Balearic Islands, Sardinia, Sicily, the Balkans (e.g., Romania;Săvulescu & Pop, 1972), Bulgaria (Assyov et al., 2002), former Jugoslavia states (Beck-Mannagetta, 1903;Josifovic, 1976), forests of central and north continental mountains of Greece (Tsiftsis et al., 2008), Black Sea region of Turkey (Kreutz, 1998), Crimea (Kubcova, 1972) and Caucasus (Grossgejm, 1949). It is absent from Crete and Cyprus (Alibertis, 1998;Kreutz, 2004). The species is largely confined to mountains in the south of the range and is absent from the Mediterranean lowlands. It grows in the forest zones of Armenia, Azerbaijan, Belarus, the Baltic states, Georgia, Kazakhstan, Moldova, Ukraine and the European part of Russia except in its northern regions (Nevski, 1935;Vakhrameeva et al., 2008). In Siberia, the species occurs in the regions Tomsk, Altai, Omsk, Kemerovo, Tyumen and Novosibirsk (Malyschev & Peschkova, 1987, map 197). The reported occurrence in the Far East and Japan is considered incorrect (Foley & Clarke, 2005;Harrap & Harrap, 2005). In Japan, there is a plant with similar ecology (woods in mountain areas) but with a F I G U R E 1 The hectad distribution map of Neottia nidus-avis in the British Isles. Each dot represents at least one record in a 10-km square of the National Grid: open circles -localities present before 1970; grey circles -present 1970-1999; black circles -present 2000 onwards; this is mapped by K. J Walker, using Dr A. Morton's DMAP software, Biological Records Centre, Centre for Ecology and Hydrology, Monks Wood, mainly from data collected by members of the Botanical Society of Britain and Ireland.
TA B L E 1 Total number of hectads (10 km × 10 km grid squares) in which Neottia nidus-avis has been recorded in the British Isles (derived from Botanical Society of Britain and Ireland's Distribution Database (BSBI, 2021), accessed November 2021; supplied by K. J. Walker of the Botanical Society of Britain and Ireland).  (Ƭi et al., 1965), but its affiliation to N. nidus-avis is debated (Yagame et al., 2016) and requires further molecular analysis.
Isolated occurrences have been discovered on Mount Babor, part of the Tell Atlas Mountain chain in north Algeria (De Smet & Bouanza, 1984) and in the oak forests of the Kroumirie, in the northwestern Tunisia (El Mokni et al., 2010). Preston and Hill (1997) assign N. nidus-avis to the Eurosiberian temperate element of the British flora. It includes species whose main distribution reaches their easterly limit between 60° E and 120° E.

| Climatic and topographical limitation
In the British Isles, they have a similar distribution to those of the European temperate element, although there is a more marked gradient between the south-eastern England, where the greatest concentration is found, and the north-western Scotland. N. nidusavis occupies the warmer and drier regions: the mean January and mean July temperatures, and mean annual precipitation in the 10km squares occupied by N. nidus-avis in Britain are 3.5°C, 15.2°C and 938 mm, respectively ( Figure 3; Hill et al., 2004).
Neottia nidus-avis is mainly a lowland plant in the British Isles.

| Substratum
Neottia nidus-avis is found in moist soils with deep humus and leaf-litter layers. Ellenberg's indicator values for edaphic characteristics at the sites where N. nidus-avis is found (indicator values modified for UK; Hill et al., 2004) are 7 for soil reaction (the species favours sites with weakly acid to weakly basic pH and never occurs on strongly acidic soils), 4 for moisture (soil moderately moist), 5 for nutrients (moderately rich sites, rarely on nutrient-poor or nutrient-rich soils) and 0 for salinity (absent from saline habitats). In the British Isles, the species grows mostly not only on chalk and limestone soils but also on clays and sands that have a chalky or limestone component, such as boulder clay (Harrap & Harrap, 2005). Elsewhere in Europe, N. nidus-avis also prefers base-rich soils; Sundermann (1970) reported a pH of 7.2-8.1 for European sites based on 3 measurements of soil around N. nidusavis roots and 6.4-8.5 for 21 measurements in the vicinity of N. nidusavis plants. Procházka and Velísek (1983) mentioned pH values 6.1-8.5 in Czech and Slovak republics, while Tsiftsis et al. (2008) reported values from north-eastern Macedonia ranging from 4.14 to 7.74, with a F I G U R E 2 The circumpolar distribution of Neottia nidus-avis. Reproduced from Hultén and Fries (1986) modified. The main distribution area of N. nidus-avis is hatched; (•) isolated, more exactly indicated occurrences. median of 5.47. The latter study, which sampled soil at rooting depth (5-15 cm), also found organic-matter content ranging from 0.97 to 35.7% (median 7.3%) and available phosphorus concentration ranging from 0.89 to 10.2 mg/100 g (median 3.3 mg/100 g). A major requirement for the orchid is the presence of its mycorrhizal associates, fungi from the family Sebacinaceae whose ecological preferences, beyond the presence of trees that are their carbon sources (see 6.2), remain unknown (Brzosko et al., 2017;Weiß et al., 2016).
Occasionally, the species grows in spruce forests (Picea abies) with scattered beech trees on places of former beech forests.
In Northern Europe, the species typically inhabits beech woodlands of community Fagion sylvaticae (Diersen, 1996), but it can be also found in rich basiphilous pine forests (Pinus sylvestris) on very shallow calcareous soils (Bjørndalen, 2015). In northern parts of  (Bjørndalen, 2015). In the European-Mediterranean region, the species is found in shady beech woodlands and sclerophyllous oak forests, dominated by Quercus ilex or (a) Q. suber, and pinewoods with various native and planted subspecies of Pinus nigra (Pignatti, 1982;Tison et al., 2014).
In the eastern part of its distribution area, N. nidus-avis grows in different types of forest (Querco-Fagetea) on rich substrata with a sparse grass cover (Didukh, 2009). For example, the species occurs in mountain pine forest in Crimea (community Erico-Pinetea: Brachypodio-Pinion pallasianae) and oak-hornbeam woodlands of community Carpinion orientalis-Quercion pubescentis, mountain beech and valley broad-leaved forests in the Caucasus, smallleaved birch or aspen forests, as well as in mixed coniferous forest in Moscow Province (Didukh, 2009;Vakhrameeva et al., 2008). For the Russian territory, Mirkin and Naumova (2012)  All the communities described above share the presence of tree species hosting fungal species from the family Sebacinaceae that are the strictly required mycorrhizal associates of N. nidus-avis (see 6.2).

| RE S P ON S E TO B I OTI C FAC TOR S
Neottia nidus-avis typically grows in habitats with a sparse herb layer (AHO Sachsen-Anhalt, 2011), probably because shaded conditions limit photosynthesis, and thus it does not suffer from the competition of surrounding vegetation, unless the site is disturbed. Seed germination is dependent on the presence of suitable mycorrhizal fungi (see 6.2).

| Gregariousness
Neottia nidus-avis grows singly or in sparse groups, and rarely in pop- Observations on a population growing in Moscow Province showed annual dynamics from 69 to 88 plants over 4 years (Vakhrameeva et al., 2008). A 6-year observation study in a permanent 1 × 1 m 2 plot in Estonia showed that of the initial 11 plants only one was present in years 2, 3 and 5 and their distribution in the plot may suggest that they were not the same individuals (Kull & Tuulik, 1994). Similar observation of a fluctuating number of flowering shoots (35, 41, 6, 2 in successive years) was made during four-year observations in a 20 × 10 m 2 permanent plot in Poland. However, previously unobserved shoots appeared outside the permanent plot in the final year of the study and outnumbered those observed in the first 2 years on the permanent plot, indicating high spatio-temporal population dynamics (J. Minasiewicz, unpubl. data).

| Performance in various habitats
Neotia nidus-avis occupies a variety of habitats on a vast geographic area which may result in some regional habitat variability in plant performance. However, the present data are too sparse and anecdotal to allow any pattern recognition.

| Effect of frost, drought, etc.
Populations in the British Isles appear intolerant of minimum average January temperatures below 0°C ( Figure 2). However, both the Siberian and montane localities indicate that it is most likely frost tolerant.
To our best knowledge there are no data on drought response of N. nidus-avis, but reduction in the number of flowering shoots may be one response, as seen in other temperate mycoheterotrophic orchids. This may be a result of reduced fungal host availability due to drought-induced reduction in fungal biomass (McCormick et al., 2009;Verrier, 2017).
As a non-photosynthetic species Neottia nidus-avis can grow in shady places, tolerating the heaviest of shade. It is one of only four species in the British and Irish flora with an Ellenberg indicator value of 2 for light (i.e., it grows in shade to deep shade with less than 5% of relative illumination; Hill et al., 2004).

| Morphology and anatomy
Neottia nidus-avis is a brownish, heterotrophic plant ( Figure 4a) with short rhizomes bearing numerous, tightly packed, thick, glabrous and short adventitious roots (Figure 5a). The lower part of the shoot is wrapped in 3-5 brownish scales. Leaf scales have very few vascular bundles, which are located at the leaf margin and centre, but water storage and mechanical support of adjacent tissues can be assisted by tracheoid cells (Aybeke, 2012). Its mesophyll is composed of eight or nine layers of thin-walled cells with small intercellular spaces, rarely with oxalate raphides (Stern, 2014). The stomata on scale leaves are sparsely distributed on the abaxial surface, having stomatal pores (Figure 5b) that are smaller than those of photosynthesizing orchid taxa (Aybeke, 2012;Ziegenspeck, 1936). This is contrary to Stern (2014)

| Mycorrhizal diversity
Identity of fungal associates remained elusive over the 20th century, despite intensive attempts to cultivate them (see Rasmussen, 1995 for historical review), until their detection by molecular methods was possible. Then, the symbionts were shown to be highly specific, from the Sebacinaceae (McKendrick et al., 2002;Selosse et al., 2002), a family within the Sebacinales (Basidiomycota). Unlike its sister family Serendipitaceae, from which many mycorrhizal fungi of green orchids are recruited, Sebacinaceae are as-yet unculturable (Weiß et al., 2016). This identification is congruent with ultrastructural hyphal traits revealed by electron microscopy (Barmicheva, 1990).
These Sebacinaceae also form mycorrhizas of the ectomycorrhizal type on surrounding tree roots (Selosse et al., 2002), through which they are inferred to extract the carbon resources supporting the whole system, as in other mycoheterotrophic plants from the temperate zone (Merckx, 2013). Indeed, whenever Sebacinacean DNA polymorphism was observed from one orchid root to another, similar DNA variants colonised the very nearby tree roots (Selosse et al., 2002), providing evidence of a mycelial link with surrounding trees.

| Mycorrhizal colonisation
Mycorrhizal colonisation intensity (the percentage of root length with mycorrhizal fungi) is high in the roots and the rhizome (80%-90%, Vakhrameeva et al., 2008)

| Perennation: Reproduction
Sexual reproduction by seed prevail in the species and is described in detail in Section 8.3. Neottia nidus-avis has a rather slow development from seeds. Because it is believed that during the early years the previous rhizome segment disappears and is replaced by a new but similar rhizome segment, the initial stages are virtually indistinguishable from each other in the soil, making any estimate of duration of the developmental stages unreliable. It is estimated that it takes 9-10 years from the protocorm stage (see 8.5) to the generative phase (Bernard, 1909;Rasmussen, 1995;Vakhrameeva et al., 2008;Ziegenspeck, 1936). However, Tatarenko (c) reported much shorter ontogenesis from Moscow Province, where it lasted for 3-5 years. When a flowering shoot develops, it remains alive until the fruits ripen, and then often dies completely together with the rhizome, suggesting a monocarpic life history (Rasmussen, 1995). However, this view of the rhizome as monopodial (i.e. growing from its tip bud exclusively) is challenged by the observations (Champagnat, 1963;Prillieux, 1856;Ziegenspeck, 1936; M.-A Selosse pers. obs.) that lateral buds, although most often dormant, allow sympodial growth so that the individual can persist. Champagnat, (1963) estimates that only 10%-60% of rhizomes die after flowering. Indeed, sympodial growth explains why some rhizomes are branched and even bear several flowering shoots (Prillieux, 1856).  Figure 6c). This unusual transition is favoured when roots become separated from the rhizome accidentally or by decay of their proximal part (Champagnat, 1963); this transition may result from a rhizome-to-root hormonal signalling, since Vanillia planifolia roots can be induced to form shoots upon auxin treatment in vitro (Philip & Nainar, 1988). The tip of the root thins and an outgrowth starts to proliferate from the root cap, with continuity of vascular bundle that soon shifts from the typical root organisation (xylem alternating with phloem) to that of a shoot (xylem internal to the phloem, with a central medulla; Champagnat, 1971). The resulting whitish bud (Figure 6c) is soon colonised by the symbiotic fungus and proliferates, first forming irregular outgrowths and then producing scaly leaf sheaths, which assemble around a vegetative meristem (Champagnat, 1971;Ziegenspeck, 1936). Later, the root bearing the bud gradually decays while new adventitious roots are formed (Figure 6c), generating the form of a juvenile plant. The initial outgrowth at the root tip is sometimes viewed as a developmental reiteration of a protocorm (Champagnat, 1963(Champagnat, , 1971), yet the presence of vascular tissues, which are absent in a protocorm, makes it different. Vakhrameeva et al. (2008) noted such asexual propagation in juvenile and flowering individuals, although they were less frequent in non-flowering individuals. Such a propagation is variable in frequency: in the Urals, it was found in 25%-30% of individuals (Knyasev & Knyaseva, 1988), while in observations from Caucasus, the Crimea and Moscow Province, it did not exceed 3% (Tatarenko, 2002). Reasons for this variability among plants and among populations remain unclear. Asexual propagation through underground organs is also known from other mycoheterotrophic plants, exemplifying convergent evolution (Klimešová, 2007;Roy et al., 2009). It may be favoured by the local presence of the vital mycorrhizal fungus, which often displays patchy distribution, making vegetative propagation an essential adaptive trait for these strictly fungal-dependent plants (Roy et al., 2009). The fact that seeds germinate better around adults (see 8.4) supports this patchiness.
Whether asexual propagation adapts somehow to fungal availability or physiological status remains unclear.
A detailed karyological study by Bartolo et al. (2010) found the karyotype of N. nidus-avis to be composed of 11 metacentric, 1 submetacentric, 2 subtelocentric and 4 telocentric pairs. Chromosome pair 1 is long; pairs 2 to 18 are progressively shorter; pair 11 possesses a small satellite on the short arm. All chromosomes have centromeric C-bands. Pair 2 shows a terminal C-band in the long arm. Pairs 3, 5 and 18 have heterochromatic short arms (Bartolo et al., 2010).
The authors draw attention to a large number of large telocentric F I G U R E 6 (a) Germination of Neottia nidus-avis in the earliest published drawings by (Bernard, 1902;from Selosse et al. (2011), with permission); (1) ungerminated seed and (2) seed at an early germination stage, with fungal penetration, with testa (t) and a region of attachment to the maternal placenta (m; side of the virtual embryo suspensor) and vegetative pole (v); (3) seedling (protocorm) with initiation of the meristem of the first roots (r), and colonised zone with living pelotons (lp) and degenerated pelotons (dp); (4) older seedling with an apical bud (ab) and cracked seed envelope (= testa, t) at its base; (b) protocorms (p) and ungerminated seeds (  chromosome pairs in comparison with the genus Epipactis (also tribe Neottieae) and frequent associations among telomeric chromosomes in metaphase.

| Physiological data
The species is mycoheterotrophic from germination to adulthood and thus obtains all mineral and carbon resources from its mycorrhizal fungi (see 6.2) that are shared with surrounding trees.
Although gene loss is less pronounced than in some other mycohet-   (Stöckel et al., 2014;Suetsugu et al., 2020), a feature that reflects the 15 N and 13 C abundance in fungi ectomycorrhizal on trees but differs from that in saprotrophic fungi (Hynson et al., 2013).
Thus, the classical view of N. nidus-avis as a 'saprotrophic' plant (Hudák et al., 1997) is incorrect, even considering only the biology of its fungal associates. The second piece of evidence for a link to surrounding trees as a carbon source comes from radiocarbon ( 14 C) measurements that evaluate the time elapsed since photosynthetic carbon fixation: the short transfer time observed in N. nidus-avis, with a radiocarbon age similar to that of autotrophic plants (Hatté et al., 2020;Suetsugu et al., 2020), strongly differs from the older radiocarbon age of mycoheterotrophic plants associated with saprotrophic fungi and can be explained by rapid carbon transfer through mycelial links. Finally, N. nidus-avis is the only mycoheterotrophic species linked to trees for which there is evidence for sharing not only the same fungal species but also the same individual fungal mycelia (Selosse et al., 2002).

| Biochemical data
The plant is often viewed as achlorophyllous, which is not entirely true (see 6.5). One anecdotal trait demonstrates this: when put into solvent, heated water, or close to a flame, the inflorescence colour shifts from brownish to greenish (Figure 5g; Mangenot & Mangenot, 1966;Reznik, 1958). Chlorophyll is most likely associated with the brown coloration of N. nidus-avis, as chlorophyll is absent in white var. nivea (Figure 4f; Reznik, 1958). The explanation for the brown colour and the experimental greening is either from a second pigment masking chlorophyll (Reznik, 1958) or, our preferred explanation, a link to thermosensitive chlorophylls and/or carotenoids-protein complexes that modify the light absorbance spectrum (Menke & Schmid, 1976), as found in brown algae (Menke, 1940). Indeed, recent transcriptome analyses confirmed that N. nidus-avis, unlike other mycoheterotrophic plants -Epipogium aphyllum and Gastrodia elata -expresses the full range of genes required for the synthesis of chlorophyll as well as some chlorophyll a/b binding proteins that are mostly activated in flowers (Jąkalski et al., 2021). These pigment-protein complexes are stored in plastids, whose altered structure reflects a loss of photosynthetic function. These are spindle-like with numerous, isolated (not grana-forming) thylakoids, arranged in parallel or coiled as globules (or droplets), but are devoid of starch in accordance with the absence of photosynthesis (Hudák et al., 1997;Mangenot & Mangenot, 1966;Menke & Schmid, 1976). These plastids are found in shoots, even in the phloem (Danilova & Barmicheva, 1990).  (Klooster et al., 2009).
The pigments may also have some other role directly related to light. Menke and Schmid (1976) offered evidence for cyclic photophosphorylation, for instance, with ATP production by cyclic functioning of Photosystem I. The possibility of this pathway generating energy with neither O 2 evolution nor CO 2 fixation deserves closer inspection; however, absence of most plastid and nuclear genes coding for Photosystem I and cytochrome b6/f does not support this hypothesis (Jąkalski et al., 2021).
The unusually high zeaxanthin content in shoots (Haspelova Horvatovicova & Holubkova, 1980;Pfeifhofer, 1989) is suggestive of a xanthophyll cycle, which plays an important role in the protection against oxidative stress. On the other hand, the absence of conversion of violaxanthin to zeaxanthin on illumination (Haspelova Horvatovicova & Holubkova, 1980), confirmed by the lack of the crucial enzyme of the cycle, points to alternative reasons for zeaxanthin accumulation (Jąkalski et al., 2021). Finally, the reason for high concentrations of tocopherol in N. nidus-avis (Reznik et al., 1969), another metabolite connected to photoprotection, deserves further investigation. A clear understanding of the role of all these metabolites in N. nidus-avis remains pending.

| PHENOLOGY
Seeds of N. nidus-avis germinate in spring, in humid substrates after contact with a suitable mycorrhizal fungus (Rasmussen, 1995). The experiment conducted by McKendrick et al. (2002) involving successive sampling of previously buried seed samples showed a slow progression of underground seedling growth, measured as the volume of each germinated seedling. After 9 months, the volume of protocorms was 0.05-0.13 mm 3 . It successively increased to 0.14-0.37 mm 3 after 11 months; 0.38-0.99 mm 3 after 18 months; 1.00-2.72 mm 3 after 23 months; and 2.73-54.59 mm 3 after 30 months in soil. Between 18 and 23 months of growth, pear-shaped protocorms begin to branch. All seedlings with a volume greater than 1.64 mm 3 are branched. At this stage, rootlets develop from the protocorm and subsequently shoot-bud develops from the apical meristem (see 8.5).
Mature rhizome formation may take several years (see 6.3). Rhizome growth in vegetative individuals starts in April-June in areas with dry and hot summers and in May-August in milder climates. During this period, an individual sprouts a new annual segment of monopodial rhizome consisting of 6-7 growth units (Champagnat, 1963;Ziegenspeck, 1936). In unfavourable seasons, rhizome growth does not occur and the terminal bud remains dormant. The flowering shoot meristem is formed in the terminal bud of the underground rhizome one season before sprouting (Vakhrameeva et al., 2008). Flowering is from May to June. The shoot persists until fruit maturation in August-September, and then dies and dries completely, often but not always together with rhizome (see 6.3). Sometimes only a few drops are produced, depending on the air humidity (Müller, 1883). In younger flowers, nectar can be found in large quantities. The flowers produce a sweet scent, perceived as honey-like or mouldy (Summerhayes, 1951;Ziegenspeck, 1936). The anther opens before anthesis, then quickly dries out and folds up.

| Floral biology
At the same time, the rostellum margins curl up exposing the mealy pollinia in the groove of the rostellum (Figure 4b). Stigmatic lobes run either side of the rostellum. On the crest of the rostellum are minute rough points, which are particularly sensitive to touch, causing the expulsion of the viscid matter that glues pollinia to a pollinator; this so-called touch-sensitive rostellum is a unique, shared feature within the genus Neottia (Claessens & Kleynen, 2011;Darwin, 1862).
After that, the rostellum bends towards the stigma, preventing freshly removed pollinia from being deposited in the same flower.
After 2 days, the rostellum recovers its original position (Claessens & Kleynen, 2011). The rostellum then shrinks and the stigma appears swollen. At this time, pollinia hanging over the rostellar edge can touch the stigma, causing self-pollination regardless of whether the mechanism was triggered or not.

| Hybrids
No inter-or intrageneric hybrids are known.
The thousand seed weight is 0.0031 g (Török et al., 2013). The embryo (147-205 × 100-130 μ; Mrkvicka, 1994) is an undifferentiated mass of meristematic cells without a suspensor (Veyret, 1956) and is surrounded by an envelope of dead cells (i.e. testa; Figure 6a, various studies show that dust-like orchid seeds can cover distances as short as a few metres but can also travel as far as thousands of kilometres (Arditti & Ghani, 2000).

| Viability of seeds: germination
Asymbiotic in vitro germination experiments were unsuccessful, as were symbiotic germination trials with fungi normally mycorrhizal with green orchids, so-called 'rhizoctonia' (Rasmussen, 1995 and references therein). Smreciu and Currah (1989) (Stöckel et al., 2014); germination was recorded in 2.7% of seed packets, resulting in only about 0.1% of germinating seeds within a year of seed burial.

| Animal feeders or parasites
Because the underground organs of N. nidus-avis contain starch, the shoot buds are often damaged or destroyed by soil invertebrates and insect larvae (Vakhrameeva et al., 2008). Unlike other orchid species, the flowering shoots are not damaged by deer or wild boar (Jersáková, pers. obs.).

| Plant parasites and diseases
Dark necrotic spots are often noticeable on the roots (Selosse, pers. obs.), but the causative pathogen remains unknown.

| CONS ERVATION
Neottia nidus-avis has suffered a considerable decline throughout the 20th century, particularly between 1930 and 1970, and especially in south-eastern England (Preston et al., 2002). Although this decline appears to have slowed, localised losses have continued to occur throughout its British and Irish range over the past 20 years (K.J. Walker, pers. comm. 2022). Kull and Hutchings (2006)  declining (see below the ranking of the species in national red lists).
The species is very vulnerable to habitat disruption, and most losses are probably due to changes in woodland management, woodland clearance, use of heavy machinery in forestry operations, destruction of the habitat by fire and conversion of deciduous woodland into conifer plantations. In addition, this orchid is affected by urbanisation, tourism and related infrastructure expansion, as well as plant collection (Rankou et al., 2014). In recent years, the loss of old broadleaved woodland has slowed down as the conservation and cultural values of this habitat have become generally recognised. However, its especially rapid decline from apparently suitable habitats in south-eastern England and East Anglia suggests additional causes, possibly decreased spring and summer rainfall coupled with increased atmospheric deposition of nitrogen and other pollutants. Additionally in woodlands, tree seedlings continue to be damaged by increasing numbers of deer (particularly in southern England) and sheep (in the uplands) that hinder spontaneous regeneration of host species important for Sebacinaceae fungi.
While habitat conservation remains the only way of protecting N. nidus-avis, along with the vast majority of other mycoheterotrophic plants, attempts are also being made to develop their ex situ propagation, potentially enabling their reintroduction into suitable habitats. The most promising method for germination of mycoheterotrophic plants appears to be one that initiates an ex situ tripartite symbiosis with tree seedlings, fungi and orchids (Warcup, 1985) and although initial application of this method has failed to initiate seed germination of N. nidus-avis, there is still room for refinements (Hughes, 2018). Micro-propagation of N. nidus-avis has also been attempted, but this experiment did not develop beyond callus formation (Sheyko & Musatenko, 2011).

The species is classified as Near Threatened in the Red
List for Great Britain (Cheffings & Farrell, 2005) and of Least Concern for Wales (Dines, 2008) but was recently categorised as Vulnerable in England, due to marked decline in area of occupancy (Stroh et al., 2014). On the Irish Red List, the species ranks of Least Concern category (Wyse-Jackson et al., 2016). Globally, the species is of Least Concern on the European Red List of Vascular Plants (Bilz et al., 2011). In the continental Atlantic zone, the species is rare, as shown by its protection in Bretagne and Limousin regions of France, as well as in Belgium and Luxembourg (Bournérias & Prat, 2005). Regionally, the species is listed as Critically Endangered in Netherlands (Soortenbank, 2017), Near Threatened in the Czech Republic (Grulich, 2012) and Norway (Kålås et al., 2010), Least Concern in Upper Austria (Hohla et al., 2009) and Switzerland (BAFU, 2016), but it is not listed for the whole of Austria (Niklfeld, 1999), Estonia (Red Data Book of Estonia, 2008), Germany (Korneck et al., 1996), Sweden (Aronsson et al., 2010) or Ukraine (Didukh, 2009). It is not listed in the red lists and books of Slovak Republic (Feráková et al., 2001), Bulgaria (Petrova & Vladimirov, 2009), Greece (Phitos et al., 2009), Poland (Kaźmierczakowa et al., 2016), Spain (Banares et al., 2008), Finland (Rassi et al., 2001), Italy (Rossi et al., 2013), Slovenia (Skoberne, 2007) and Lithuania (Rašomavičius, 2007).

AUTH O R S ' CO NTR I B UTI O N
All authors contributed equally to writing this account. All authors contributed critically to the drafts and gave the final approval for publication.

ACK N OWLED G EM ENTS
We thank J. Claessens, M. Kotilínek, R. Maret, R. Bauer, C. Noel, P. Labrot and J. Rojek for permission to publish their photographs; PaperTrue™ -Editing and Proofreading Service for linguistic correction. We are grateful to K. J. Walker of the Botanical Society of Britain and Ireland for his suggestions and supportive materials. We are also grateful to the members of the editorial board for their suggestions and corrections.

CO N FLI C T O F I NTE R E S T
The authors have no conflicts of interest to declare.

PE E R R E V I E W
The peer review history for this article is available at https://publo ns.com/publo n/10.1111/1365-2745.13953.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing not applicable -no new data generated.