Biological Flora of Central Europe: Dactylorhiza Sambucina (L.)

Home , Anacamptis, Anacamptis coriophora, Anacamptis morio, Coeloglossum, Dactylorhiza, Dactylorhiza fuchsii

Perspectives in Plant Ecology, Evolution and Systematics 17 (2015) 318–329

Contents lists available at ScienceDirect

Perspectives in Plant Ecology, Evolution and Systematics

jo urnal homepage: www.elsevier.com/locate/ppees

Biological ﬂora of Central Europe

Biological ﬂora of Central Europe: Dactylorhiza sambucina (L.) Soó

a,∗ b a c

Jana Jersáková , Iva Traxmandlová , Zdenekˇ Ipser , Matthias Kropf ,

d e f b,g

Giuseppe Pellegrino , Bertrand Schatz , Vladan Djordjevic´ , Pavel Kindlmann ,

Susanne S. Renner

Faculty of Science, University of South Bohemia, Branisovskáˇ 1760, Ceskéˇ Budejoviceˇ 37005, Czech Republic

Department of Biodiversity Research, Global Change Research Centre AS CR, Belidlaˇ 4a, 602 00 Brno, Czech Republic

Institute for Integrative Nature Conservation Research, University of Natural Resources and Life Sciences, 1180 Vienna, Austria

Department of Biology, Ecology and Earth Science, University of Calabria, Rende, Italy

Centre d’Ecologie Fonctionnelle et Evolutive (CEFE), UMR 5175, CNRS – Université de Montpellier (EPHE), 1919 route de Mende, 34293 Montpellier, France

Institute of Botany and Botanical Garden, Faculty of Biology, University of Belgrade, Takovska 43, 11000 Belgrade, Serbia

Institute for Environmental Studies, Charles University, Benátská 2, Prague, Czech Republic

Institute of Systematic Botany and Mycology, University of Munich (LMU), Munich, Germany

a r a

t i b s

c t

l e i n f o r a c t

Article history: Dactylorhiza sambucina (L.) Soó is a polycarpic perennial herb occurring in the Central European, East-

Received 16 February 2015

ern European, and Balkan ﬂoristic provinces. At the European scale, the IUCN considers it a species of

Received in revised form 7 April 2015

“least concern”. This paper reviews the taxonomic status, morphology, distribution, habitat requirements,

Accepted 27 April 2015

mycorrhizal associations, and life cycle of D. sambucina, with special emphasis on its reproduction. We

Available online 16 May 2015

also summarize information on chromosome numbers and genetic variation. Our data from 12 years

of monitoring D. sambucina in the Czech Republic show that three to four leaves have to be produced

Keywords:

prior to flowering; plants with five and more leaves flower regularly. Juvenile plants near adult plants

Colour polymorphism

Dormancy suggest recruitment from seeds. About 20% of our 450 monitored plants underwent dormancy (failure of

mature plants to produce above-ground parts in one or more growing seasons), the maximum duration

Ecological niche

Life cycle being eight years. After reappearance, these individuals were usually sterile for the next year. Mortality

Reproductive biology was highest (24%) at the seedling stage. Regarding the purple/yellow ﬂower colour polymorphism that

Seed germination characterizes D. sambucina, we found no correlation between morph frequency and soil properties (pH,

calcium content), population density, or altitude above sea level.

Contents

Morphology and taxonomy ...... 319

Distribution and habitat requirements ...... 319

Geographical and altitudinal distribution ...... 319

Substratum...... 320

Habitats and plant communities ...... 320

Life cycle, phenology and growth ...... 322

Phenology ...... 322

Life cycle and dormancy ...... 322

Seed production and dispersal ...... 323

Seed germination in situ and seedling morphology ...... 324

Seed germination in vitro ...... 324

Mycorrhiza ...... 324

Spatial distribution of plants within populations ...... 324

∗

Corresponding author. Tel.: +420 387 775 357.

E-mail address: [email protected] (J. Jersáková).

http://dx.doi.org/10.1016/j.ppees.2015.04.002

J. Jersáková et al. / Perspectives in Plant Ecology, Evolution and Systematics 17 (2015) 318–329 319

Responses to abiotic and biotic factors ...... 324

Response to climate factors...... 324

Response to competition and management ...... 324

Herbivores and pathogens ...... 324

Floral biology ...... 325

Pollination ...... 325

Colour polymorphism ...... 325

Patterns in colour polymorphism across Europe ...... 325

Maintenance of colour polymorphism by pollinator morph discrimination ...... 325

Factors affecting fruit set ...... 326

Physiological and biochemical information...... 326

Physiological data ...... 326

Biochemical data ...... 326

Genetic data ...... 326

Chromosome number...... 326

Genetic variation...... 326

Hybrids ...... 327

Conservation ...... 327

Acknowledgements ...... 327

Appendix A. Supplementary data ...... 327

References ...... 327

Morphology and taxonomy Mediterranean from Spain to Italy, North Africa) (Pedersen, 2006).

Pedersen (2006) provides color photos and a key to these taxa.

Dactylorhiza sambucina (L.) Soó is a polycarpic perennial geo-

Two of the D. romana subspecies, namely romana and georgica,

phyte with a palmate root tuber slightly or moderately divided into

have the same ﬂower colour dimorphism as D. sambucina, while D.

several lobes (Fig. 1A). Between 3 and 6 adventitious roots that can

cantabrica, D. insularis, and D. romana subsp. guimaraesii only pro-

be up to 6 cm long develop at the base of an innovation bud (Fig. 1A).

duce yellow ﬂowers. Forms with very pale ﬂowers as are frequently

The stem is 10–20 (30) cm high, sturdy, and hollow. It bears 2 scale

observed in other sections of Dactyloriza are unknown from section

leaves and 3–7 green leaves, depending on age and nutrient sta-

“Sambucinae” (Delforge, 2005; Schatz et al., 2013).

tus, with the lower ones oblong-lanceolate 5–10 × 1–2.5 cm, and

the upper ones lanceolate; the leaves are homogenously green and

do not develop brownish lilac spots. The inﬂorescences are ovoid Distribution and habitat requirements

(egg-shaped) or shortly cylindrical dense racemes (without a ter-

minal ﬂower), (3.7) 5–7.5 (10.5) (2.7) 3.5–4.5 (5) cm in length. Geographical and altitudinal distribution

The lower bracts are longer, the upper ones equal to the ﬂowers

in length; young inﬂorescences are sheeted by 1–8 (11) cataphylls The geographical distribution of D. sambucina and D. romana

(Pedersen, 2006). The ﬂowers are zygomorphic and either yellow- (and their allies in section “Sambucinae”) ranges from Portugal in

ish or purple-red. The outer perianth segments (often called sepals) the west to northern Iran in the east, and from southern Scan-

are 0.7–0.9 cm long, oblong-ovate in shape with an obtuse tip, and dinavia in the north to northern Morocco, Algeria, and Lebanon

stand out upwards, the median perianth segment forms a helm in the south. A map of its total range may be found in Meusel

together with the two shorter, obliquely ovate lateral perianth et al. (1965: map 110d, sub nom. D. sambucina s.l.). The south-

segments. The labellum measures 0.7–1.0 0.7–0.9 cm, is rounded ern range limit of D. sambucina sensu stricto stretches from Central

with a slightly three-lobed apex and in both colour morphs is speck- Spain to the southern Peloponnese (Fig. 2, Appendix 1). Its east-

led with light reddish to dark purple spots; in the purple morph it ern border runs from eastern Bulgaria to the Dnepr River in the

has an yellowish base. The cylindrical spur is 1.0–1.5 cm long (in Ukraine (Didukh, 2009) and the Bryansk region in Russia close

Dactylorhiza insularis distinctly shorter), bent downward, and con- to the Ukrainian border (Evstigneev and Fedotov, 2004). In the

tains no nectar. The column is erect and 4.5 mm high; each of the north, it extends to southern Poland and Central Germany. Scan-

two sectile pollinia is tapering into a caudicle, attached to the viscid- dinavian populations, slightly disjoint from the main distributional

ium; the rostellum is three-lobed and forms a roof-like projection range, occur in southern Norway, eastern Denmark, in Southeast

above the stigma; one large bursicle covers the two separate sticky Sweden, and southernmost Finland. D. sambucina is absent from

viscidia; the stigma is three-lobed with large lateral lobes. The the British Isles and western Siberia. The whole of Scandinavia and

ovary is twisted, glabrous, and develops into an erect 1.1–1.3 cm much of the British Isles were covered with ice 18,000 years ago,

long capsule. The seeds are numerous, 0.5–0.6 0.15–0.25 mm and periglacial conditions persisted until 11,700 years ago. Thus,

large, spherical or slightly ellipsoid in shape and have a hyaline the northern European populations of D. sambucina all established

yellowish brown testa (Bojnanskˇ y´ and Fargasová,ˇ 2007). post-glacially, whereas southern populations may antedate the last

The species epithet apparently refers to the ﬂoral scent of glacial maximum (Pedersen, 2006; Pillon et al., 2006, 2007). The

Sambucus nigra L. (Adoxaceae) (Delforge, 2005). Taxonomically, absence of D. sambucina from the British Isles might be due to a fail-

D. sambucina belongs to sect. Sambucinae (Parl.) Smoljan, which ure to re-colonize the island over the past 10,000 years. Historically,

besides D. sambucina, includes Dactylorhiza romana (Sebast.) Soó D. sambucina also occurred in Estonia, and it was unsuccessfully

with subsp. romana (Sicily, Eastern Mediterranean, Crimea), geor- reintroduced at Saaremaa Island in 1989 (Kuusk, 1994). Reported

gica (Klinge) Soó ex Renz & Taubenheim (Northeastern Turkey occurrences in Asia Minor (including Caucasus, Crimea, Turkey), the

to Caucasus, Transcaucasia, W- and N-Iran, Turkmenistan), and Baltic countries (Latvia, Lithuania), Belarus, Sardegna, and north-

guimaraesii (D.G. Camus) H.A. Pedersen (Spain, Algeria, Morocco), ern Africa are erroneous (e.g. Stefaniak and Dabrowska,˛ 2013;

as well as Dactylorhiza cantabrica H.A. Pedersen (northern Spain), Govaerts, 2014; but see following orchid ﬂora lists: G.I.R.O.S., 2009;

and D. insularis (Sommier) O. Sanchez & Herrero (Western Gudzinskas,ˇ 2001; Gudzinskasˇ and Ryla, 2006; Khoruzhyk et al.,

320 J. Jersáková et al. / Perspectives in Plant Ecology, Evolution and Systematics 17 (2015) 318–329

Fig. 1. (A) Underground organs of Dactylorhiza sambucina. Abbreviations refer to: ot – old tuber from previous season supporting the ﬂowering stem (fs), nt – developing

new tuber, te – root-like extensions of the tuber, oar – old adventitious roots from previous season, nar – new adventitious roots. (B) Stages in the development of three-year

old seedlings, as – apical shoot, ar – adventitious root, t – tuber. (C) Cross-section of a seedling, brown coils of fungal hyphae (pelotons) are visible within the cells. (D)

Cross-section of a mycorrhizal hypha with dolipore septum that is composed of a pore cap surrounding a septal swelling and septal pore. Photo A by J. Brabec, C and D by J.

Jersáková.

2005; Lai, 2009; Pashkov et al., 2005) and probably due to confusion and Gotland it grows on calcareous soils with a pH of up to 7.7.

with D. romana and D. insularis. In the Ukrainian Carpathians, it grows on brown mountain soils

D. sambucina occurs from sea level (on the Swedish islands of with pH 4.7–4.9 (Zagulskii et al., 1998), and in western Serbia

Öland and Gotland) up to 2400 m in the Alps (Baumann et al., 2006). on soils derived from limestone, serpentine, ophiolitic mélange,

Regional altitudinal ranges reported in literature are: 193–2300 m schists, phyllites, quartz latite, porphyrite, or Quaternary sediments

in Switzerland (AGEO, 2014), 250–2200 m in Austria (Novak, 2010), (VDj. pers. obs.; Djordjevic´ et al., 2014). Different from other species

175–530 m in the Rhineland-Palatinate in Germany (Kropf, 2008, of Dactylorhiza, D. sambucina does not occur in wet biotopes but

2011), 1060–1200 m in Taormina Mt. in Sicily (Cristaudo and Galesi, instead prefers dry soils. The maximum water-holding capacity

2010), 900–1800 m in Galicia in Spain (Tyteca and Bernardos, of such soils in Central Europe ranges from 30.7 to 54.4% with

2003), 600–1500 m in Mt. Giona in Greece (Aplada et al., 2012), mean ± SD: 43.0 ± 7.3% (JJ unpubl. data). Further mechanical and

500–2100 m in East Macedonia (Tsiftsis et al., 2008), 400–1300 m in physical soil properties (bulk density, porosity) are detailed in Mróz

province Hermannstadt in Romania (Dragulescu˘ and Rösler, 2005), (1994). In France, D. sambucina sites are characterized by quarterly

250–2500 m in France, with greatest abundance at 750–1750 m precipitations between 50–250 mm and 1120–1620 mm and tem-

◦ ◦ ◦ ◦

(Dusak and Prat, 2010), and up to 1700 m in Albania (Ziegenspeck, peratures between −8.2 C to −6.2 C and 21.7 C to 26.2 C (Dusak

1936). In Poland, the species occurs mainly in the Sudeten and and Prat, 2010).

Carpathian mountains, reaching 1115 m in Pieniny (Stefaniak and

Dabrowska,˛ 2013). Our own observations described later in this

Habitats and plant communities

review were made at 781–1300 m alt. in the National Park Cévennes

in Southern France (21 populations), 467–900 m in the Czech

D. sambucina is a light-demanding species. It prefers nutrient-

Republic (29), 1149–1709 m in Sila National Park in Calabria in Italy

poor meadows and pastures (see section “Substratum”), including

(17), 1240–1510 m in Dolomites in Trentino in Italy (3), 175–445 m

grassy patches in stony thickets, well-drained forest meadows, for-

in the Rhineland-Palatinate (19), 295–820 m in Lower Austria (13),

est borders, clearings, and open broad-leaved or coniferous forests.

and 871–1795 m in western Serbia (117 populations).

Plant communities in which it occurs are the so-called Molinio-

Arrhenatheretea and Nardo-Callunetea classes or less frequently,

Substratum forest communities of the Querco-Fagetea class (e.g. Czech Rep. –

Tlusták and Jongepierová-Hlobilová, 1990; Polish Sudeten – Mroz,

D. sambucina occurs on nitrogen-poor soils, usually with a pH 1994; Franconian Forest – Balzer, 2000; Sicily – Cristaudo and

of 5.2–6.8 (Sundermann, 1970) or 5.0–6.3 (Ziegenspeck, 1936; our Galesi, 2010; Rivas-Martinez et al., 2002; Romania – Dragulescu˘

Table 1). However, in Southern France, Italy, Greece, and on Öland and Rösler, 2005; Rösler, 2013). In Central Europe, D. sambucina

J. Jersáková et al. / Perspectives in Plant Ecology, Evolution and Systematics 17 (2015) 318–329 321

Fig. 2. Natural range of Dactylorhiza sambucina based on the references in Appendix S1. Red circles – recent recording or recording date not speciﬁed in the literature, green

circles – between 1950 and 1980, blue circles – older than 1950, black circles – occurrence indicated in Baumann and Künkele (1982), but not veriﬁed by further sources.

(For interpretation of the references to color in this ﬁgure caption, the reader is referred to the web version of this article.)

Table 1

Properties of the soils that support Dactylorhiza sambucina populations. Mean ± SD, range and number of analysed populations.

Country pH Potassium Calcium Available Total nitrogen Organic matter (%)

(g/kg) (g/kg) phosphorus (mg/100 g)

(mg/kg)

± ±

Austria 5.4 0.5 2.7 2.5 2.3 ± 1.3 8.8 ± 2.3

(4.7–6.1) (0.6–6.7) (1.2–4.1) (6.1–11.9)

13 5 6 5

Czech 5.3 ± 0.3 2.5 ± 1.1 1.3 ± 0.6 6.3 ± 2.5

Rep. (4.7–5.9) (1.0–5.2) (0.3–2.3) (2.6–11.9)

29 18 21 15

France 6.4 ± 1.0 2.9 ± 2.9 9.6 ± 14.4 4.3 ± 2.1

(4.9–8.0) (1.0–8.9) (0.5–40.4) (2.1–7.1)

21 6 7 6

Germany 4.7 ± 0.5 1.4 ± 0.6 0.9 ± 0.3 10.1 ± 4.0

(4.2–5.9) (0.7–2.4) (0.5–1.4) (4.2–14.6)

19 6 6 6

Italy 5.5 ± 0.3 2.7 ± 1.5 2.2 ± 2.2 4.7 ± 2.0

(4.9–6.0) (1.0–4.6) (1.1–7.1) (3.1–8.4)

20 6 7 6

Poland 4.5 ± 0.5 0.09 ± 0.05 0.9 ± 0.3 5.95 ± 4.5 307.8 ± 86.1

(4.0–5.6) (0.05–0.2) (0.5–1.8) (1.8–14.9) (160.3–418.5)

11 11 11 11 11

a a a

East 6.2 2.9 12.6

Macedonia (4.3–7.7) (1.2–6.7) (0.97–35.9)

96 96 96

Southern Norway 5.5 (0.4–1.2)

Source: Jersáková et al. (unpubl. data), Poland – Mróz (1994), Macedonia – Tsiftsis et al. (2008), Southern Norway – Norderhaug et al. (1997).

a Median.

grows predominantly in mesophytic grasslands of the Arrhen- grasslands of the Violion caninae alliance, broad-leaved dry grass-

atherion elatioris alliance (Trifolio-Festucetum rubrae community), lands of the Bromion erecti alliance (Brachypodio pinnati-Molinietum

pastures of the Cynosurion cristati alliance (Anthoxantho odorati- arundinaceae community) and acidophilous dry grasslands of

Agrostietum capillaris community), submontane and montane the Koelerio-Phleion phleoidis alliance (Mróz, 1994; Kropf, 1995;

322 J. Jersáková et al. / Perspectives in Plant Ecology, Evolution and Systematics 17 (2015) 318–329

Jersáková and Kindlmann, 2004b). In Ukraine, it grows in acidic 600

Sterile

grasslands of the Cynosurion cristati alliance, in the Ukrainian

Carpathians it inhabits the Quercion robori–petraeae woodlands, as Flowering

well as grasslands (Festucetum rubrae community) developed on

former beech and spruce forest sites (Didukh, 2009). In Greece, D. 400

sambucina inhabits subalpine grasslands and open Fagus and Pinus

woodlands, often in clearings and forest margins, open Carpinus-

r of pl

Ostrya scrubs, on a variety of substrates (Tsiftsis et al., 2006,

be 200

2007; Aplada et al., 2012). In the Central and Southern Apen-

nines in Italy, it occurs in the community Nardo-Luzuletum pindicae

Num

of the Ranunculo-Nardion alliance (Tomaselli et al., 2003), the

Juniperus shrub community Daphno oleoidis-Juniperetum alpinae,

and in neutral-subacidophilous grassland community Potentillo

1 2 3 4 5 6 7

rigoanae-Brachypodietum genuensis (Phleo ambigui-Bromion erecti

alliance, Di Pietro et al., 2005). In western Serbia, D. sambucina Number of leaves

can be found in the following grassland communities: Nardetum

Fig. 3. Number of leaves in sterile and ﬂowering individuals of Dactylorhiza sam-

strictae, Danthonietum calycinae, Koelerietum montanae, Brome-

bucina. Pooled data from 2018 records over 12 years (1998–2010) of monitoring of

tum erecti, Brachypodietum pinnati, Festucetum valesiacae, Festuco

a single population in the Czech Republic.

rubrae-Agrostetum capillaris, and Poetum violaceae, and the wood-

land communities: Pinus sylvestris forests, Fagetum montanum, and

Betuletum pendulae (VDj. pers. obs.).

who monitored a population of D. sambucina for 43 years in South-

ern Sweden report consecutive ﬂowering of 49% of the individuals,

while 47% of ﬂowering plants failed to ﬂower in the next year

Life cycle, phenology and growth

(Appendix 2).

Phenology

Life cycle and dormancy

The appearance of leaves above ground depends on spring

humidity and temperature. The ﬁrst leaves may appear in early D. sambucina is a non-bulbous geophyte showing limited veg-

March and are fully developed during ﬂowering and fruit produc- etative spread. The wintering organs are a renewal bud with

tion. The time of ﬂowering depends on a combination of factors, a tuber and adventitious roots (Vöth, 1971; Rasmussen, 1995;

especially latitude and altitude. At lower altitudes, ﬂowering starts Vakhrameeva et al., 2008, Fig. 1A). The innovation bud produces

in mid-April (South and Central Europe, Alsace in France), peaks a new shoot that eventually terminates with an inﬂorescence

between mid-May and mid-June in Scandinavia and in subalpine (Rasmussen, 1995). The inﬂorescence is formed and remains inside

altitudes (lower altitudes in the Alps and Apennines), and extends the bud for more than a year (Vakhrameeva et al., 2008).

to early July in high alpine areas (Nilsson, 1980; Presser, 2000). D. sambucina frequently forms clumps but it is not known if they

The capsules mature for ca. 1.5 month, and seeds are shed rather are of vegetative or generative origin. During our 12 years of mon-

quickly within a few sunny days during June or July. During sum- itoring of D. sambucina, we sometimes observed new plants below

mer, the aerial parts die and the plants stay underground until the mature plants, with their small size suggesting juveniles germi-

next growing season. nated from seeds. In three of 450 monitored plants, we observed

The development of a new tuber starts in early March, when a replacement of a single adult plant by two daughter plants of a

a ﬂat, palmately divided daughter tuber and adventitious roots moderate size. Similarly, Inghe and Tamm (1988) during 43 years

develop at the base of an innovation bud, which is located in the of monitoring 74 plants observed only two vegetative clones. The

axilla of a second cataphyll at a compressed shoot base (Vöth, 1971; most widespread pattern of vegetative reproduction in orchids is

our Fig. 1A). During the ﬁrst year, the tuber elongates and becomes the formation and germination of two or more buds, including dor-

slightly or moderately divided with up to four root-like extensions mant ones, on axial organs such as rhizomes, creeping shoots, and

emerging from its distal end. The old tuber gradually shrinks and root tubers (Dactylorhiza case). The daughter shoots in orchids with

dies during capsule development. root tubers (tuberoids) detach after 0.5–1 years (Batygina et al.,

The factors determining whether a plant will ﬂower are complex 2003).

and little understood; they include both internal and environmen- Adult vegetative dormancy (Shefferson, 2009), the failure of a

tal variables. According to Wells et al. (1998), orchids have to reach a plant to produce above-ground parts in one or more growing sea-

critical size before they can ﬂower, and once that size is reached, the sons followed by reappearance of full-sized photosynthetic plants

probability of ﬂowering increases with leaf number. Our data from in subsequent seasons, has been observed in D. sambucina and typ-

a long-term monitored population of D. sambucina in the Czech ically lasts for one year (Fig. 5A). During 12 years of monitoring, we

Republic show that a plant has to reach at least three, or better four observed dormancy in 20% of the 450 monitored plants, the maxi-

leaves to start ﬂowering, while plants with ﬁve and more leaves mum length of dormancy being eight years. Inghe and Tamm (1988)

almost always ﬂower (Fig. 3). Plants with one or two leaves are recorded dormancy in 31% of their 74 plants, all but one dormant

juveniles. The probability of consecutive ﬂowering in orchids is for one year while the exceptional plant was dormant for two years.

usually highly limited by costs of ﬂowering and fruiting (Primack Most dormant plants in the Czech population recruit from ﬂower-

and Stacy, 1998; Jacquemyn and Hutchings, 2010; Jersáková et al., ing ones, and after reappearance most plants are sterile (Fig. 5B).

2011; but see Shefferson et al., 2003). In our long-term monitored The transition to dormancy thus seems to be triggered by the high

population, 77% of flowering plants develop an inflorescence also cost of flowering and fruiting.

in the following year (although some inﬂorescences aborted), 17% D. sambucina appears to be long-lived. Out of cohort of 49

became sterile (Fig. 4). Similarly, 65% of plants with aborted inﬂo- plants followed since 1942, 11 plants were still alive 43 years

rescences, which did not set fruits and thus saved energy, ﬂowered later (Inghe and Tamm, 1988). Ziegenspeck (1936) reported two

during the next year. Inghe and Tamm (1988; also Tamm, 1972) years from germination to the ﬁrst leaf appearance and 12 years

J. Jersáková et al. / Perspectives in Plant Ecology, Evolution and Systematics 17 (2015) 318–329 323

Fig. 4. Transition probabilities between life stages from year t to year t + 1 in a Dactylorhiza sambucina population in the Sumavaˇ mountains (Czech Republic). Pooled data

from 10 years (2000–2009) and 2533 transitions. The values in parentheses indicate the number of plants involved in each transition. The thickness of the lines denotes

probabilities 0–25%, 26–50%, 51–75%.

A 80 to ﬁrst ﬂowering. Our germination and seedling monitoring data

shows that ﬁrst leaf may appear above-ground after two or three

years in the soil (Appendix 3A), and the ﬁrst ﬂowering event on

average occurs ﬁve years after emergence (25 juvenile plants with

one leaf ﬂowered for the ﬁrst time 2–10 years after emergence,

± ±

plants mean SD = 5.2 1.8, median = 5; number of leaves at ﬁrst ﬂow-

f 40

± ±

o ering: mean SD = 4.2 0.7, median = 4, range 3–5). In the Czech

population, the seedlings stage had the highest mortality (24%; No 20 Fig. 4).

Seed production and dispersal 1 2 3 4 5 6 7 8

Dormancy (years)

D. sambucina is an allogamous, self-compatible species, with

84–97% fruit set after hand self-pollination, and incapable of

B 80

Juvenile agamospermy or spontaneous autogamy (Jersáková, 1998; Kropf

and Renner, 2005; Pellegrino et al., 2005; Juillet et al., 2006). The

Sterile

60 species shows high inbreeding depression after self-pollination.

Flowering

For example Nilsson (1980) reported fewer developed embryos

in self-pollinated seeds (43%) than in cross-pollinated ones (75%). plants

40 Inbreeding depression coefﬁcients for the percentage of seeds with o

a well developed embryo after self- or cross-pollination were 0.42

(Nilsson, 1980), for seed viability estimated by the tetrazolium test

0.63 (Jersáková et al., 2006), for germination rates at 65 and 130

days 0.46 and 0.60, respectively, and for survival rate 0.75 (Juillet

et al., 2006).

0 1 2

Before After Wind-dispersed seeds are numerous, with spherical or slightly

ellipsoid embryos enclosed in an elongated testa of yellowish-

Fig. 5. Dormancy in Dactylorhiza sambucina monitored over 12 years from 1998 to brown hollow cells (Bojnanskˇ y´ and Fargasová,ˇ 2007). The number

2010. (A) Frequency and length of dormancy. (B) Life stage of marked individuals

of seeds per capsule is unknown, but in D. romana it is 4736 seeds

before and after dormancy. Juvenile plants are newly emerged plants with one or

per capsule (n = 14; Nazarov, 1995, 1998). The fruit set reported

two leaves; sterile plants are non-ﬂowering individuals with three and more leaves.

from 50 populations in six countries ranged from 0.4% to 56.1%

with a mean of 22.9% ± 15.5% (reviewed in Claessens and Kleynen,

2011). In a 12-year monitored population in the Czech Republic,

324 J. Jersáková et al. / Perspectives in Plant Ecology, Evolution and Systematics 17 (2015) 318–329

the mean fruit set varied from 1.1% to 30.1% in different years single fungal strain from Tulasnellaceae or Ceratobasidiaceae fam-

(Appendix 4). ily, the adult plants frequently associated with two or three fungal

strains of the same fungal families simultaneously (Appendix 3).

Seed germination in situ and seedling morphology These results resemble ﬁndings in Orchis and Serapias in which mul-

tiple fungal associations have also been documented (Jacquemyn

According to Fuchs and Ziegenspeck (1927), D. sambucina et al., 2010; Luca et al., 2014; Pellegrino et al., 2014).

germinates in the spring (Fig. 1C). However, other Dactylorhiza In addition to rhizoctonia, some ectomycorrhizal ascomycetes

species germinate already in autumn after dissemination dur- have also been found in adult D. sambucina plants (Pellegrino and

ing the summer (Dactylorhiza majalis, Teˇsitelováˇ and Jersáková, in Bellusci, 2009). Though known mainly as mycobionts of forest-

prep.; Dactylorhiza fuchsii, Leeson et al., 1991; Dactylorhiza macu- dwelling members of Neottieae (reviewed in Dearnaley, 2007),

lata, Möller, 1990), thus the start of germination in D. sambucina here they co-occur with dominant rhizoctonia fungi. The electron

deserves further study. The mycorhizome is extensive, with the microscopy of D. sambucina roots has not revealed any ascomycetes

ﬁrst root developing during the ﬁrst winter. It is mycotrophic but forming pelotons. Dearnaley et al. (2013) proposed that the pres-

contains a certain amount of xylem. During the following spring, ence of endophytic fungi in root tissues may lead to evolution into

the leafy shoot of D. sambucina unfolds after which growth becomes true mycorrhizal partners.

sympodial and the ﬁrst root tuber with 2–4 vascular bundles devel-

ops (Fig. 1B). Spatial distribution of plants within populations

The seed bank of D. sambucina is short-lived. Our in situ germina-

tion experiments using seed packets buried in natural conditions D. sambucina grows in nutrient poor meadows and pastures,

(Rasmussen and Whigham, 1993) revealed a germinating rate of with the spatial distribution of individuals depending on popu-

0.2% and 2% intact seeds after three years in the soil. This is similar lation age, micro-habitat conditions, and grassland management

to Dactylorhiza lapponica with 0.2% of live seeds after 3 years in the practice. In Germany, dense patches had 1.4–4.4 individuals per

soil (Øien et al., 2008). In that species, the probability of a seed to m ; the highest recorded number was 12 ﬂowering plants in a

form a protocorm declined from 4.4% after one year to 0.1 and 0.2% single m (MK unpubl. data). In the Polish Sudety Mountains,

after two and three years to 0% in the fourth year (out of initially Mróz (1994) found 1.7–5.6 plants per m , with a mean of 3.4

20 thousands of seed buried). plants. In Southern Italy, 1.7–6.4 plants per m have been recorded

(Pellegrino et al., 2005). A 1 m -monitoring plot initiated by Tamm

Seed germination in vitro in 1942 contained 11–46 ﬂowering or vegetative individuals in the

course of 43 years (Inghe and Tamm, 1988).

In vitro, D. sambucina readily germinates asymbiotically. Ponert

et al. (2011) described two cultivation media on which 90% of seeds Responses to abiotic and biotic factors

germinated after pretreatment of 2–5 min in 70% ethanol and 5 min

◦

in 5% Ca(OCl)2. When cultivated at 23 or 17 C, the protocorms Response to climate factors

stopped growing, started to turn brown and then died. When trans-

◦

ferred to 4 C, the protocorms also stopped growth, but remained D. sambucina ﬂowering starts in mid-April (South and Central

white in a good condition and after this cold treatment they started Europe, Alsace in France) and peaks between mid-May and mid-

to grow again and produced shoots. Van Waes and Debergh (1986) June; ﬂowers are frequently aborted due to frost (Nilsson, 1980;

achieved the highest seed germination (80.3%) with a pretreat- Balzer, 2000; pers. obs. JJ, MK). Summer drought has a negative

ment in 5% Ca(OCl)2 and 1% emulsiﬁer (Tween-80) for four hours. effect on ﬂowering in the subsequent year (Inghe and Tamm, 1988).

The percentage of coloured embryos using the tetrazolium test

after this pretreatment was 83.2%. Continuous darkness induced Response to competition and management

higher germination rates (53.3%) than illumination between 1.2

−2 −1

␮

and 30.4 mol m s (maximum germination 5.2%). Cultivation As a light-demanding species adapted to nutrient-poor soils,

medium without macroelements yielded a higher germination rate D. sambucina depends on traditional land-use, while fertilization,

(71.7%) than a medium with 0.47 mM of macroelements (55.3%) frequent mowing, and succession following abandoned land use

(Van Waes and Debergh, 1986). affect populations negatively (Kropf, 1995; Balzer, 2000). When

overgrown by stronger competitors, D. sambucina will ﬁrst react

Mycorrhiza by increasing individual plant height, later by non-ﬂowering, and

ﬁnally, by non-appearance (MK pers. obs.) in a similar way as Orchis

The symbiotic mycorrhizal partners of D. sambucina have been morio (Jersáková et al., 2002). Therefore, nature conservation man-

little studied. The ﬁnger-like extensions of the tubers are col- agement often involves maintaining traditional land-use practices,

onized with mycorrhizal fungi, as are the narrow, adventitious including no fertilization and a single mowing or grazing within a

horizontal roots (Fig. 1A). Transmission electron microscopy of growing season; ideally, after ﬂowering and fruit set of D. sambucina

roots of adult plants revealed rhizoctonia-like hyphae possessing (and other target species; Balzer, 2000). In a German study region,

septal dolipores with imperforated parenthesomes, consisting of of 16 populations with ﬂowering individuals in 2006, 12 (75%) are

two electron-dense layers separated by an electron-transparent under nature conservation management (with shrub clearance and

zone (JJ unpubl. data; Fig. 1D). Such ultrastructure of the septal sheep grazing), which protects at least 2684 ﬂowering plants or

pore and parenthesome is typical for the family Tulasnellaceae 93.6% of all ﬂowering individuals in this region (Kropf, 2008, 2011).

(Shimura et al., 2009). Molecular investigations of the fungal com-

munity conﬁrmed that both seedlings and adult plants associate Herbivores and pathogens

with various members of Tulasnellaceae, less frequently Ceratoba-

sidiaceae and Sebacinales (Pellegrino and Bellusci, 2009, JJ & GP There are no data on phytophagous insects, parasites, or diseases

unpubl. data; Appendix 3). Tulasnella is a very common symbiont of D. sambucina. In the Czech Republic, Austria and France, brows-

of terrestrial orchids (reviewed in Dearnaley, 2007), observed also ing deer frequently damage plants, and wild boar and rodents may

in other Dactylorhiza species (Kristiansen et al., 2001; Jacquemyn eat the tubers (Kropf, 2008; JJ, BS pers. obs.). Heavy grazing by cat-

et al., 2012). While each analysed seedling associated only with a tle during spring and early summer has been identiﬁed as a major

J. Jersáková et al. / Perspectives in Plant Ecology, Evolution and Systematics 17 (2015) 318–329 325

reason for the decline of D. sambucina on the Danish island Born- France Czech R. S Italy

holm (Sonne and Hauser, 2014). Germany Austria N Italy 1.00

Floral biology

0.80

Pollination

D. sambucina has conspicuous yellow and purple inﬂorescences 0.60

(Appendix 4B) that in mid-April and May attract newly emerged

and inexperienced bees, especially bumblebee queens. The pollina-

0.40

tors recorded across its distribution area (Austria, Czech Republic,

France, Germany, Italy, Sweden, Switzerland, Ukraine) are mainly

bumblebees (Bombus bohemicus, B. hortorum, B. hypnorum, B. lap- 0.20

Proportion of yellow morph

idarius, B. lucorum, B. muscorum, B. pascuorum, B. ruderarius, B.

ruderatus, B. sylvarum, B. soroeensis and B. terrestris), but also cuckoo

0.00

bumblebees Psithyrus vestalis and P. barbutellus, honeybees Apis 0 200 400 600 800 1000 1200 1400 1600 1800

mellifera, and solitary bees Andrena nigroaenea, Osmia bicolor and Altitude

Anthophora aestivalis (reviewed in Claessens and Kleynen, 2011). In

Sweden, ﬂower visitors (not pollinators because they have not been Fig. 6. Altitudinal pattern in the proportion of the yellow morph compared to the

purple morph in 79 European populations with more than 50 individuals. Purely

found to transport pollinia) include females of Halictus sp., butter-

yellow populations were found in the Rhineland-Palatinate in Germany and in Lower

ﬂies (Pyrgus malvae and Gonepteryx rhamni), and an unidentiﬁed

Austria.

ﬂy (Nilsson, 1980). In the South of France, Gonepteryx cleopatra has

been observed on the ﬂowers (BS, pers. obs.). Pollinator spectra vary

frequencies, with purely yellow morph populations in Germany

locally; for example, the bee A. nigroaenea was the unique pollinator

(n = 19), yellow morph-biased populations in Southern France (66%,

on Stora Karlsö Island (Pettersson and Nilsson, 1983).

n = 21; 69% in Gigord et al., 2001), Italy (62%, n = 20), and Austria

While probing the ﬂowers for nectar, bees will touch the bursicle

(57%, n = 13), and purple-biased populations in Sweden (Nilsson,

with its two separate viscidia. Based on the size of an insects’ head,

1980) and in the Czech Republic (37% of yellow form, n = 28). In

the viscidia become attached either to its forehead or its clypeus

addition, populations within regions and among years vary in their

(Nilsson, 1980; Appendix 4E). After pollinaria removal, the caudicle

purple/yellow frequencies. Such ﬂuctuations must be seen against

starts to bend and moves into a position suitable for contacting

the backdrop of dormancy (section “Life cycle and dormancy”), and

the stigmatic cavity of the next D. sambucina ﬂowers visited. The

switches of the dominant colour morph have been observed in both

caudicle bending time is 20–40 s, with a mean of 25 s (Nilsson, 1980;

yellow- and purple-dominated populations in single years, albeit

Peter and Johnson, 2006; Kropf and Renner, 2008; Claessens and

rarely and only in small-sized populations (Kropf and Kriechbaum,

Kleynen, 2011; BS pers. obs.). Floral visits are short (few seconds),

2009). We tried to relate the colour polymorphism in D. sambucina

typically to one or two ﬂowers of an inﬂorescence, rarely up to

to soil properties (pH and calcium content), population size, and

four (Nilsson, 1980; Kropf and Renner, 2005). As a result, individual

altitude above sea level, but found no continent-wide relationships

bees carry only one or two pollinia, the largest number found on

(Fig. 6, Appendix 6).

a single bumblebee is 15 (Nilsson, 1980; Kropf and Renner, 2005;

Claessens and Kleynen, 2011, BS pers. obs.). Pollinia color-coding

experiments have revealed that most pollen is dispersed over short Maintenance of colour polymorphism by pollinator morph

distances (median 1.23 m); the longest distance was 176 m (Kropf discrimination

and Renner, 2008).

It has been suggested that colour polymorphism in D. sambucina

Colour polymorphism is maintained by negative frequency-dependent selection induced

by food-deceived pollinators that over-visit the rare colour morph,

Colour polymorphism has been described in many food- having learned to avoid the frequent morph (Gigord et al., 2001).

deceptive orchid species (Claessens and Kleynen, 2011). D. Subsequent studies have failed to support this hypothesis (Kropf

sambucina is unusual in having populations in with highly balanced and Renner, 2005; Pellegrino et al., 2005; Jersáková et al., 2006;

purple/yellow morph frequencies as well as monochromatic popu- Smithson et al., 2007), which also does not agree with what is

lations (next section). This has led to many studies of the suspected known about learning in naïve bumblebee individuals. A suggested

underlying density-dependent selective processes maintaining the alternative explanation for the maintenance of the colour polymor-

polymorphism. As mentioned in the section “Morphology and tax- phism is differential fertility among colour morphs. This is based

onomy”, occasional salmon pink individuals have been recorded on Jersáková et al.’s (2006) ﬁnding of a lower seed viability of the

(Appendix 4C), probably reﬂecting hybridization among the pur- yellow morph in purple-biased populations in the Czech Republic.

ple and yellow morphs. Their frequency does not exceed 5%. These Another factor that may inﬂuence morph frequencies is local

pink plants have been ranked as D. sambucina subsp. zimmerman- pollinator colour-ﬁdelity due to the colours of co-ﬂowering reward-

nii A.Camus (a misapplication of the concept of subspecies, which ing plants. In greenhouse experiment, bumblebees preferred the D.

is supposed to have geographic coherence) or D. sambucina f. zim- sambucina colour morph that resembled the colour of previously

mermannii (A.Camus) P.Delforge (Souche, 2004; Delforge, 2005). visited rewarding ﬂowers (Gigord et al., 2002). Colour constancy

Rarely, somatic mutations produce individuals with unusual ﬂoral has also been observed in situ in France (BS unpubl. data) and

patterns (Appendix 4D). Sweden (Nilsson, 1980; our Table 2). Experiments in which nat-

ural D. sambucina populations were enriched with rewarding Viola

Patterns in colour polymorphism across Europe aethnensis further support these ﬁndings (Pellegrino et al., 2008):

aggregation with yellow Viola plants increased fruit set in yellow D.

Our screening of 101 D. sambucina populations (Appendix 4C) sambucina and vice versa (“magnet species effect”, cf. Johnson et al.,

and published records reveal a strong regional bias in morph 2003).

326 J. Jersáková et al. / Perspectives in Plant Ecology, Evolution and Systematics 17 (2015) 318–329

Table 2

Physiological and biochemical information

Colour constancy (>2 consecutive visits to the same colour morph) of individual bees

visiting Dactylorhiza sambucina ﬂowers on Öland in Sweden (from Nilsson, 1980) or

◦ ◦ Physiological data

in the south of France (Site A, Camprieu: 44 06 N, 3 31 E, 1147 m; Site B, Licide:

◦ ◦ ◦ ◦

44 06 N, 3 21 E, 904 m; Site C, Labastide: 43 30 N, 3 13 E, 832 m; orig. data of B.

Schatz collected in April 2006). The density of stomata on the upper leaf surface is about

2 2

1680 per cm , on the lower leaf surface 5044 per cm (Ziegenspeck,

Site Öland Site A Site B Site C

1936).

Frequency of yellow morph 0.13 0.46 0.56 0.9

Total pollinator visits 25 11 8 13

Biochemical data

Constant visits to yellow ﬂowers 3 0 1 9

Constant visits to purple ﬂowers 18 6 3 0

Inconstant visits 3 5 4 4

Infection of the tubers of D. sambucina (cited as O. latifolia)

Ratio of constant to inconstant pollinators 0.84 0.55 0.5 0.69

with a strain of Rhizoctonia repens from Orchis militaris resulted

in the synthesis of the phytoalexin orchidinol (2,4-dimethoxy-7-

hydroxy-9,10-dihydrophenanthrene) and p-hydroxybenzylalcohol

The most plausible current explanation for what maintains

(Nüesch, 1963; Gäumann et al., 1960). Quercetin 3-O-ˇ-d-glucoside

colour polymorphism in D. sambucina thus is a combination of post-

(isoquercetin) has been isolated from air-dried ﬂowers of D.

pollination barriers among the morphs (affecting seed viability),

sambucina (Tira, 1971). Further work by Pagani (1976) revealed

presence, density and colours of co-ﬂowering rewarding plants, and

the presence of quercetin 3-O-ˇ-d-glucoside, quercetin 7-O-

innate vs. learned colour preferences of bees. ˇ d

- -glucoside and quercetin 3,7-di-O,O-ˇ-d-glucoside, and the

phenylpropanoid esters caffeoyl-1-glucoside and p-coumaroyl-1-

Factors affecting fruit set glucoside. The species contains cyanidin 3,5-diglucoside (cyanin)

and the pigments orchicyanin I and II (Strack et al., 1989), these

Fruit set ﬂuctuates between years and among populations three compounds being also present in related species D. majalis

(Nilsson, 1980; Pellegrino et al., 2005, Appendix 5). D. sambucina and D. maculata (Arditti, 1992).

is pollinator-limited because it offers neither nectar nor pollen as The ﬂoral fragrance on both morphs is a blend of at least 3

a reward for bee pollinators, and the addition of nectar to D. sam- mono- and 7 sesquiterpene hydrocarbons (dominated by limonene

bucina ﬂowers increased pollinia removal and deposition in both and trans-caryophyllene, Nilsson, 1980). Similarly, Salzmann and

colour morphs (Jersáková et al., 2008). There is a positive corre- Schiestl (2007) found the scent proﬁle of D. romana mainly com-

lation between the number of ﬂowers and fruit set, illustrating posed of monoterpenes making up at least 60% of all the ﬂoral

the effect of ﬂoral display (Nilsson, 1980). However, a negative compounds, with the mean relative amounts of compounds not

parabolic relationship between reproductive success and the num- differing between morphs, with the exception of linalool (high in

ber of ﬂowers has also been demonstrated, which means that the purple morph) and benzaldehyde (high in the yellow morph).

plants with unusually small or large inﬂorescences are less success- The absolute amounts of the macroelements N, P, K, Ca, and Mg

ful than those with medium-sized inﬂorescences (Jersáková and in blooming plants of D. sambucina were reported by Mróz (1994).

Kindlmann, 2004a, 1998). Since the ﬂowers open gradually from

the bottom towards the top, and bumblebees visit inﬂorescences

Genetic data

from the bottom upwards, the lower parts of inﬂorescences typ-

ically set more fruits than the upper parts (Nilsson, 1980; Vogel,

Chromosome number

1993, JJ unpubl. data). Pellegrino et al. (2005), however, found no

effect of ﬂower position on fruit set in Italy, and Kropf and Renner

D. sambucina has 2n = 40 (exceptionally 2n = 42) chromosomes

(2005), who analysed the proportional pollination success of each

(Hagerup, 1938; Heusser, 1938; Del Prete et al., 1980; Gathoye and

ﬂower position argued for the opposite pattern, with mid-position

Tyteca, 1989). The same number is reported from D. romana s.str.

ﬂowers having a higher fruit set rate than low-position ﬂowers.

(Del Prete et al., 1980; Bianco et al., 1987; Alba et al., 2003), while

Another factor that may affect fruit set is density of conspeciﬁc

D. insularis is a triploid, with 2n = 60 (Scrugli, 1977; Bernardos et al.,

plants (cf. Maintenance of colour polymorphism). In a pollen-tracking

2002, 2005) or 2n = 60 + 1B chromosomes (Bernardos et al., 2004),

experiment Kropf and Renner (2008) found most pollen being

and D. cantabrica a tetraploid (2n = 80; Pedersen, 2006). The role of

deposited to plants at the edge of higher density patches after

polyploidy in the evolution of this group of species has attracted

bee ﬂight distances from a few tens to hundreds meters, possible

much interest (Bullini et al., 2001; Pedersen, 2006; Nordström and

due to the visual attractiveness of dense patches from a distance.

Hedrén, 2007; Pillon et al., 2007), it has been suggested that there

In yellow-dominated populations in Italy, the relative male and

are two allopatric basal diploid species, D. sambucina (western)

female reproductive success was independent of total D. sam-

and D. romana (eastern and southern), with D. insularis likely an

bucina density, but positively correlated with the yellow morph

allotriploid, and D. cantabrica an allotetraploid (Pedersen, 2006).

frequency, again pointing to the visual attractiveness to bees of

dense patches of yellow ﬂowers (Pellegrino et al., 2005). Contrary

Genetic variation

to this, Internicola et al. (2006) in Southern France found that aggre-

gation of yellow and purple D. sambucina with a blue rewarding

Modern genetic studies on D. sambucina and its close allies are

species (Muscari neglectum) diminished the fruit set of the orchid.

scarce (Pedersen, 2006; Nordström and Hedrén, 2007; Pillon et al.,

In this case, bumblebees probably learned to avoid the high-density

2006, 2007, older allozyme studies are cited therein). Allozyme

patches of the non-rewarding D. sambucina and instead to visit the

diversity suggests that D. romana s.str. is the least derived mem-

near-by rewarding patch of blue ﬂowers (Internicola et al., 2006).

ber of the D. sambucina complex and that local populations may be

Experimental defoliation (simulation of herbivory) of mature

subdivided into demes determined by the distinct colour morphs

individuals had no effect on capsule production but signiﬁcantly

and partial morph constancy of individual bumblebees (Pedersen,

decreased weight of fruits (Pellegrino and Musacchio, 2006). Since

2006). Using 19 allozyme loci in one Spanish and six Italian popula-

the dry weight of a capsule and the number of seeds it contains are

tions of D. insularis, nine Italian populations of D. sambucina, and 11

strongly positively correlated (Vallius, 2001), one can assume that

Italian populations of D. romana, Bullini et al. (2001) suggested that

defoliated plants produced fewer seeds than control ones.

D. insularis may be allotriploid and originate from D. sambucina and

J. Jersáková et al. / Perspectives in Plant Ecology, Evolution and Systematics 17 (2015) 318–329 327

D. romana. However, this species and the apparently allotetraploid locally (Kropf, 2008), as is impropriate timing of cattle grazing

D. cantabrica probably originated multiple times (Pedersen, 2006). during spring and early summer (Sonne and Hauser, 2014; cf. Herbi-

vores and pathogens). An attempted re-introduction on the Estonian

Hybrids island Saaremaa, using plants from the Åland islands, was unsuc-

cessful (Kuusk, 1994).

Intergeneric hybrids involving D. sambucina have been

described with Gymnadenia conopsea (×Dactylodenia zollikoferi Acknowledgements

(Stoj.) Peitz) and Platanthera bifolia (×Dactylanthera fournieri (E.

Royer) J.M.H. Shaw) (for original publication details and distri- We would like to thank Andreas Braun for sampling and soil

bution maps see e-monocot.org). Souche (2004) also observed analyses in our Austrian study region. We thank Eckehart Jäger and

natural hybrids with Coeloglossum viride, Gymnadenia conopsea, Karl Peter Buttler for their comments on an earlier version, and

Orchis mascula, Orchis pallens, Platanthera bifolia, Pseudorchis albida Henrik Pedersen for checking the section on taxonomic issues. This

and Anacamptis morio. The Collectif SFO-RA (2012) conﬁrmed the research was supported by the grants GAJU 04-145/2013/P (to ZI),

report of natural hybrids with O. pallens. Occurrence of hybrids No. 14-36098G of the GACR and No. LO1415 of the MSMT (to PK

× ×

with the genera Serapias ( Serapirhiza), Orchis ( Orchidactyla), and IS), No. 173030 of the Ministry of Education, Science and Tech-

and Pseudorchis ( Pseudorhiza) remains doubtful, while Souche nological Development of the Republic of Serbia (to VDj), and the

(2004) states that viable hybrids from experimental crossings with Hochschuljubiläumsstiftung der Stadt Wien (to MK).

Anacamptis coriophora and Orchis provincialis can be obtained.

Formally named within Dactylorhiza hybrids includes D. ver-

Appendix A. Supplementary data

meuleniana (D. ×gabretana (A.Fuchs) Soó), D. fuchsii (D. ×inﬂuenza

(Sennholz) Soó), D. incarnata (D. ×guillaumeae C. Bernard), D.

Supplementary data associated with this article can be found,

kalopissii (D. ×metsowonensis B. Baumann & H.Baumann), D. macu-

in the online version, at http://dx.doi.org/10.1016/j.ppees.2015.04.

× ×

lata (D. altobracensis (Coste & Soulié) Soó), D. majalis (D. ruppertii 002

(M.Schulze) Borsos & Soó), D. romana (D. ×rombucina (Cif. & Gia-

com.) Soó), and D. viridis (syn. Coeloglossum viride) (D. ×erdingeri

References

(A.Kern.) B.Bock). Collectif SFO-RA (2012) also observed intra-

generic hybrids with Dactylorhiza savogiensis in the French Alps, AGEO, 2014. www.ageo.ch – ofﬁcial website of the Association Working Group on

and Souche (2004) observed intrageneric hybrids with Dactylorhiza Native Orchids of Aargau. Switzerland (accessed 01.12.14).

sphagnicola. Alba, V., Alba, E., Polignano, G.B., Perrino, P., 2003. Biodiversity of wild orchids in

Basilicata region (South-Italy). J. Eur. Orch. 35, 407–418.

Aplada, E., Tiniakou, A., Georgiadis, T., 2012. The orchid ﬂora of Mount Giona (Sterea

Conservation Ellas Greece). J. Eur. Orch. 44, 765–792.

Arbeitskreis Heimische Orchideen, 2005. Die Orchideen Deutschlands. Uhlstädt-

Kirchhasel, Germany.

D. sambucina is treated by the IUCN as “least concern” (Appendix

Arditti, J., 1992. Phytochemistry Fundamentals in Orchids. Wiley, Sons Ed., pp.

7). As an orchid, D. sambucina is included in the Appendix II of the 243–258.

Balzer, S., 2000. Populationsökologische Untersuchungen an Dactylorhiza sambucina

Convention on International Trade in Endangered Species of wild

(L.) Soó (Orchidaceae) im Frankenwald Nordost-Bayern. Bayreuther Forum

fauna and ﬂora (CITES). Although not listed within the Annexes

Ökologie 81, Altendorf b. Bamberg, Germany.

of the European Habitat Directive, D. sambucina is at least region- Batygina, T.B., Bragina, E.A., Vasilyeva, V.E., 2003. The reproductive system and ger-

mination in orchids. Acta Biol. Cracov. Bot. 45, 21–34.

ally rare and a threatened species in many European countries

Baumann, H., Künkele, S., 1982. Die wildwachsenden Orchideen Europas. Kosmos,

or regions (Appendix 7). National law also sometimes protects Stuttgart.

all orchids (e.g. “Bundesartenschutz-Verordnung” in Germany or Baumann, H., Künkele, S., Lorenz, R., 2006. Die Orchideen Europas mit angrenzenden

Gebieten. Eugen Ulmer KG. Stuttgart.

strictly protected species in Poland). This all-inclusive protection

Bernardos, S., Amich, F., Gallego, F., Crespí, A., 2002. Contributions to the knowledge

covers all stages in the life cycle.

on the orchid ﬂora of Northern Portugal. J. Eur. Orch. 34, 35–50.

A decline in the number of populations and ﬂowering individ- Bernardos, S., Tyteca, D., Amich, F., 2004. Cytotaxonomic study of some taxa of the

uals is especially well documented in Germany, where the most subtribe Orchidinae (Orchidoideae Orchidaceae) from the Iberian Peninsula. Isr.

J. Plant Sci. 52, 161–170.

recent national orchid ﬂora has quantiﬁed the loss of populations

Bernardos, S., García-Barriuso, M., Amich, F., 2005. New chromosome counts in

based on country-wide grid cells (i.e. quarters of the topographical

Iberian orchids. J. Eur. Orch. 37, 639–650.

maps 1:25,000). By comparing the presence of the species in grid Bianco, P., Medagli, P., D’Emerico, S., Ruggiero, L., 1987. Numeri cromosomici per la

Flora Italiana: 1139–1155. Inf. Bot. Ital. 19, 322–332.

cells before 1950 and 2004, Kretzschmar and Blatt (2005) showed

Bojnanskˇ y,´ V., Fargasová,ˇ A., 2007. Atlas of Seeds and Fruits of Central and East-

the loss of occurrences in 83.1% of the cells, placing D. sambucina

European Flora: The Carpathian Mountains Region. Springer, Dordrecht.

among orchids with the most severe population losses in Germany. Bournérias, M., Prat, D., 2005. Les Orchidées de France, Belgique et Luxembourg,

deuxième édition. Biotope, collection Parthénope, Mèze.

However, with fewer than 100 total cells, D. sambucina has long

Bullini, L., Cianchi, R., Arduino, P., de Bonis, L., Mosco, M.C., Verardi, A., Porretta,

been rare in Germany, perhaps because of a too humid or cold

D., Corrias, B., Rossi, W., 2001. Molecular evidence for allopolyploid speciation

climate (Kretzschmar and Blatt, 2005). In France, D. sambucina is and a single origin of the western Mediterranean orchid Dactylorhiza insularis

(Orchidaceae). Biol. J. Linn. Soc. 72, 193–201.

regionally protected in the French regions of Alsace and Bourgogne.

Claessens, J., Kleynen, J. (Eds.), 2011. The Flower of the European Orchid: Form and

It has disappeared from the departments Aisne, Moselle, and Seine-

Function. Jean Claessens & Jacques Kleynen, Geuelle.

et-Marne, which are under high urbanization pressure (Vogt-Schilb Collectif SFO – RA (Société Franc¸ aise d’Orchidophile – Rhone-Alpes), 2012. A la

rencontre des orchidées sauvages de Rhone-Alpes. Biotope, Mèze (Collection

et al., 2015). D. sambucina has disappeared mainly from lowlands,

Parthénope).

such as the Parisian basin and the East of France (Bournérias and

Cristaudo, A., Galesi, R., 2010. La ﬂora orchidologica della valle dell’Alcantara e dei

Prat, 2005). Monti di Taormina (Sicilia nord-orientale). J. Eur. Orch. 42, 231–326.

There are a number of reasons for the decline of D. sambucina Dearnaley, J.D.W., 2007. Further advances in orchid mycorrhizal research. Mycor-

rhiza 17, 475–486.

(cf. Response to competition and management). Beside loss of habitats

Dearnaley, J.D.W., Martos, F., Selosse, M.-A., 2013. Orchid mycorrhizas: molecular

through land-use change, intensiﬁcation or abandonment (Kropf,

ecology, physiology evolution and conservation aspects. In: Hock, B. (Ed.), The

1995; Balzer, 2000; Arbeitskreis Heimische Orchideen, 2005), there Mycota 9 (Fungal associations). Springer, Berlin, Heidelberg, pp. 207–230.

Del Prete, C., Garbari, F., Giordani, A., 1980. Numeri cromosomici per la Flora Italiana:

are also orchid-speciﬁc hazards, such as picking and even digging-

690–695. Inf. Bot. Ital. 12, 117–120.

out of ﬂowering plants (Kropf, 1995, 2008). Naturally, increasing

Delforge, P., 2005. Orchids of Europe. In: North Africa and the Middle East. Timber

numbers of wild boar can also be a problem (see above), at least Press, Portland, Oregon.

328 J. Jersáková et al. / Perspectives in Plant Ecology, Evolution and Systematics 17 (2015) 318–329

Di Pietro, R., De Santis, A., Fortini, P., 2005. A geobotanical survey on acidophilous Kristiansen, K.A., Taylor, D.L., Kjøller, R., Rasmussen, H.N., Rosendahl, S., 2001.

grasslands in the Abruzzo. Lazio and Molise National Park (Central Italy). Lazaroa Identiﬁcation of mycorrhizal fungi from single pelotons of Dactylorhiza majalis

26, 115–137. (Orchidaceae. using single-strand conformation polymorphism and mitochon-

Didukh, Y.P. (Ed.), 2009. Red Data Book of Ukraine. Vegetable Kingdom. Izd. Glob- drial ribosomal large subunit DNA sequences). Mol. Ecol. 10, 2089–2093.

alkonsalting, Kiev. Kropf, M., 1995. Vegetationskundlicher Vergleich von Standorten des Holunder-

Djordjevic,´ V., Tsiftsis, S., Lakusiˇ c,´ D., Stevanovic,´ V., 2014. Niche analysis of orchids Knabenkrautes (Dactylorhiza sambucina) im Nahegebiet und der Rhein-

of serpentine and non-serpentine areas: Implications for conservation. Plant hessischen Schweiz unter Berücksichtigung von Pﬂege sowie Verbre-

Biosyst., http://dx.doi.org/10.1080/11263504.2014.990534 itung und Gefährdung dieser Orchideenart. Flora Fauna Rheinland-Pfalz 8,

Dusak F. and Prat D. (eds.), Atlas des Orchidées de France. Biotope, Mèze (Collection 133–145.

Parthénope), Muséum National d’Histoire Naturelle, 2010, Paris. Kropf, M., 2008. Das Holunder-Knabenkraut (Dactylorhiza sambucina [L.] Soó)

Dragulescu,˘ C., Rösler, R., 2005. Zur Chorologie der Orchideen der Regierungs- in Rheinland-Pfalz: Landesweite Bestandsaufnahme einer stark gefährdeten

bezirkes Sibiu (Hermannstadt) in Siebenbürgen (Rumänien). J. Eur. Orch. 37, Orchideenart. J. Eur. Orch. 40, 423–440.

651–703. Kropf, M., 2011. Das Holunder-Knabenkraut (Dactylorhiza sambucina [L.] Soó) in

Evstigneev, O.I., Fedotov, Y.P., 7 others, 2004. Red Book of Bryansk Region. Plants. Rheinland-Pfalz – Ein Nachtrag zur landesweiten Bestandsaufnahme 2006. J.

Izdatelstvo Citaj-gorod,ˇ Bryansk. Eur. Orch. 43, 581–589.

Fuchs, A., Ziegenspeck, H., 1927. Entwicklung, Axen und Blätter einheimischer Kropf, M., Renner, S.S., 2005. Pollination success in monochromic yellow popula-

Orchideen. IV. Botanisches Archiv 20, 275–422. tions of the rewardless orchid Dactylorhiza sambucina. Plant Syst. Evol. 254,

G.I.R.O.S., 2009. Orchidee d’Italia – Guida alle Orchidee spontanee. Cornaredo (MI), 185–197.

II Castello. Kropf, M., Renner, S.S., 2008. Pollinator-mediated selﬁng in two deceptive orchids

Gathoye, J.-L., Tyteca, D., 1989. Contribution à l’étude cytotaxonomique des Dacty- and a review of pollinium tracking studies addressing geitonogamy. Oecologia

lorhiza d’Europe occidentale. Mémoires de la Societé Royale de Botanique de 155, 497–508.

Belgique 11, 30–42. Kropf, M., Kriechbaum, M., 2009. Variation des Blühverhaltens und der

Gäumann, E., Nüesch, J., Rimpau, R.H., 1960. Weitere Untersuchungen über die Blütenfarben-Häuﬁgkeiten als Voraussetzung für negativ dichte-abhängige

chemischen Abwehrreaktionen der Orchideen. Phytopathol. Z. 38, 274–308. Selektion durch Bestäuber bei der dimorphen Dactylorhiza sambucina. Sauteria

Gigord, L.D.B., Macnair, M.R., Smithson, A., 2001. Negative frequency-dependent 18, 209–222.

selection maintains a dramatic ﬂower color polymorphism in the rewardless Kuusk, V., 1994. Native orchids in Estonia with some respect to Latvia and Lithuania.

orchid Dactylorhiza sambucina (L.) Soó. Proc. Natl. Acad. Sci. 98, 6253–6255. In: Kull, T. (Ed.), Orchid Ecology and Protection in Estonia. Estimaa Looduse Fond,

Gigord, L.D.B., Macnair, M.R., Stritesky, M., Smithson, A., 2002. The potential for ﬂoral Tartu, pp. 5–9.

mimicry in rewardless orchids: an experimental study. Proc. Roy. Soc. B – Biol. Lai, R., 2009. Le Orchidee della Sardegna. Tiemme Ofﬁcine Graﬁche srl, Cagliari.

Sci. 269, 1389–1395. Leeson, E., Haynes, C., Wells, T.C.E., 1991. Studies of the phenology and dry matter

Govaerts, R., 2014. World Checklist of Orchidaceae. Facilitated by the Royal Botanic allocation of Dactylorhiza fuchsii. In: Wells, T.C.E., Willems, J.H. (Eds.), Popula-

Gardens, Kew. Published on the Internet (http://apps.kew.org/wcsp) (accessed tion Ecology of Terrestrial Orchids. SPB Academic Publishing, The Hague, pp.

01.12.14). 125–138.

Gudzinskas,ˇ Z., 2001. Diversity, state, and protection of Orchidaceae species in Luca, A., Bellusci, F., Pellegrino, G., 2014. Interactions with symbionts in two closely

Lithuania. J. Eur. Orch. 33, 415–441. related hybridizing orchid species. Acta Bot. Croat. 73, 209–220.

Gudzinskas,ˇ Z., Ryla, M., 2006. Lietuvos Geguzraibiniaiˇ (Orchidaceae). Botanikos Meusel, H., Jäger, E., Weinert, E., 1965. Vergleichende Chorologie der zentraleu-

Institutas, Vilnius. ropäischen Flora. VEB Gustav Fischer Verlag, Jena.

Hagerup, O., 1938. Studies on the signiﬁcance of polyploidy. II. Orchis. Hereditas 24, Möller, O., 1990. Beobachtungen und Bemerkungen über den Wuchs der Dacty-

258–264. lorhiza maculata. Die Orchidee 41, 22–26.

Heusser, C., 1938. Chromosomenverhältnisse bei schweizerischen basitonen Mróz, L., 1994. Ekologia Dactylorhiza sambucina (L.) Soó w Sudetach. Acta Univ.

Orchideen. Ber. Schweiz. Bot. Ges. 48, 562–605. Wratisl. Prace Botaniczne LXXVI, 103–157.

Inghe, O., Tamm, C.O., 1988. Survival and ﬂowering of perennial herbs. V. Patterns Nazarov, V.V., (PhD thesis) 1995. Reproductive Biology of Crimean orchids, St. Peters-

of ﬂowering. Oikos 51, 203–219. burgh (in Russian).

Internicola, A.I., Juillet, N., Smithson, A., Gigord, L.D.B., 2006. Experimental investiga- Nazarov, V.V., 1998. Samenproduktivität europäischer Orchideen. I. Methoden zur

tion of the effect of spatial aggregation on reproductive success in a rewardless Bestimmung der Samenzahl. J. Eur. Orch. 30, 591–602 (in German).

orchid. Oecologia 150, 435–441. Nilsson, L.A., 1980. The pollination ecology of Dactylorhiza sambucina (Orchidaceae).

Jacquemyn, H., Hutchings, M.J., 2010. Biological ﬂora of the British Isles: Spiranthes Bot. Not. 133, 367–385.

spiralis (L.) Chevall. J. Ecol. 98, 1253–1267. Norderhaug, A., Bakkevik, B., Skogen, A., 1997. Søstermarihand. Dactylorhiza sam-

Jacquemyn, H., Honnay, O., Cammue, B.P.A., Brys, R., Lievens, B., 2010. Low speciﬁcity bucina, en truet art i Norge? Blyttia 55, 73–86 (in Norwegian).

and nested subset structure characterize mycorrhizal associations in ﬁve closely Nordström, S., Hedrén, M., 2007. Development of polymorphic nuclear microsatel-

related species of the genus Orchis. Mol. Ecol. 19, 4086–4095. lite markers for polyploid and diploid members of the orchid genus Dactylorhiza.

Jacquemyn, H., Deja, A., De Hert, K., Cachapa Bailarote, B., Lievens, B., 2012. Variation Mol. Ecol. Notes 7, 644–647.

in mycorrhizal associations with Tulasnelloid fungi among populations of ﬁve Novak, N., 2010. Heimische Orchideen in Wort und Bild. Leopold-Stocker Verlag.

Dactylorhiza species. PLoS ONE 7, e42212. Graz – Stuttgart.

Jersáková, J., 1998. A comparative study in fruit set of nectar-bearing and nectarless Nüesch, J., 1963. Defence reactions in orchid bulbs. Symp. Soc. Gen. Microbiol. 13,

orchids. In: Vlcko,ˇ J., Hrivnák, R. (Eds.), Európske vstavacovitéˇ (Orchidaceae) – 335–343.

vyskum´ a ochrana II. Technická univerzita vo Zvolenu. Zvolen, 43-50 (in Czech). Øien, D.-I., O’Neill, J.P., Whigham, D.F., McCormick, M.K., 2008. Germination ecology

Jersáková, J., Kindlmann, P., 1998. Patterns of pollinator-generated fruit set in Orchis of the boreal-alpine terrestrial orchid Dactylorhiza lapponica (Orchidaceae). Ann.

morio (Orchidaceae). Folia Geobot. 33, 377–390. Bot. Fenn. 45, 161–172.

Jersáková, J., Kindlmann, P., 2004a. Reproductive success and sex variation in nec- Pagani, F., 1976. Plant constituents of Orchidaceae. I. Components of Orchis sam-

tarless and rewarding orchids. Int. J. Plant Sci. 165, 779–785. bucina L., Orchis morio L. Boll. Chim. Farm. 115, 407–412.

Jersáková, J., Kindlmann, P., 2004b. Zásady péceˇ o orchidejová stanovistˇ eˇ (Manage- Pashkov, G.P., Kalenda, L.V., Logvin, V.N., Petrikov, A.M., 2005. Krasnaya Kniga

ment guidelines for orchid habitats). Kopp, Ceskéˇ Budejoviceˇ (in Czech). Respubliki Belarus. Rasteniya, Belaruskaya Entsyklapedyya Imeni Petrusya

Jersáková, J., Kindlmann, P., Stríteskˇ y,´ M., 2002. Population dynamics of Orchis morio Brovki. Minsk, 35–36.

in the Czech Republic under human inﬂuence. In: Kindlmann, P., Willems, J.H., Pedersen, H.Æ., 2006. Systematics and evolution of the Dactylorhiza

Whigham, D.F. (Eds.), Trends and Fluctuations and Underlying Mechanisms in romana/sambucina polyploid complex (Orchidaceae). Bot. J. Linn. Soc.

Terrestrial Orchid Populations. Backhuys Publishers, Leiden, pp. 209–224. 152, 405–434.

Jersáková, J., Kindlmann, P., Renner, S.S., 2006. Is the colour dimorphism in Pellegrino, G., Musacchio, A., 2006. Effects of defoliation on reproductive success in

Dactylorhiza sambucina maintained by differential seed viability instead of two orchids. Serapias vomeracea and Dactylorhiza sambucina. Ann. Bot. Fenn. 43,

frequency-dependent selection? Folia Geobot. 41, 61–76. 123–128.

Jersáková, J., Johnson, S.D., Kindlmann, P., Pupin, A.C., 2008. Effect of nectar supple- Pellegrino, G., Bellusci, F., 2009. Molecular identiﬁcation of mycorrhizal fungi in

mentation on male and female components of pollination success in the Dactylorhiza sambucina (Orchidaceae). Biologia 64, 893–897.

deceptive orchid Dactylorhiza sambucina. Acta Oecol. 33, 300–306. Pellegrino, G., Bellusci, F., Musacchio, A., 2008. Double ﬂoral mimicry and the magnet

Jersáková, J., Malinová, T., Jerábkova,ˇ K., Dötterl, S., 2011. Biological ﬂora of the British species effect in dimorphic co-ﬂowering species, the deceptive orchid Dacty-

Isles: Pseudorchis albida (L.) A. and D. Löve. J. Ecol. 99, 1282–1298. lorhiza sambucina and rewarding Viola aethnensis. Preslia 80, 411–422.

Johnson, S.D., Peter, C.I., Nilsson, L.A., 2003. Pollination success in a deceptive orchid Pellegrino, G., Luca, A., Bellusci, F., 2014. Relationships between orchid and fungal

is enhanced by co-occurring rewarding magnet plants. Ecology 84, 2919–2927. biodiversity: mycorrhizal preferences in Mediterranean orchids. Plant Biosyst.,

Juillet, N., Dunand-Martin, S., Gigord, L.D.B., 2006. Evidence for inbreeding depres- http://dx.doi.org/10.1080/11263504.2014.940071

sion in the food-deceptive colour-dimorphic orchid Dactylorhiza sambucina (L.) Pellegrino, G., Caimi, D., Noce, M.E., Musacchio, A., 2005. Effects of local density and

Soó. Plant Biol. 9, 147–151. ﬂower colour polymorphism on pollination and reproduction in the rewardless

Khoruzhyk, L.I., Sushchenia, L.M., Parﬁonov, V.I., 2005. Red Data Book of the Republik orchid Dactylorhiza sambucina (L.) Soó. Plant Syst. Evol. 251, 119–129.

of Belarus: Rare and Threatened Plant of Native Species. Pietras Brovka Belarus- Peter, C.I., Johnson, S.D., 2006. Doing the twist: a test of Darwin’s cross-pollination

sian Encyclopedia. Minsk. hypothesis for pollinium reconﬁguration. Biol. Lett. 2, 65–68.

Kretzschmar, H., Blatt, H., 2005. Statistische Auswertung der Orchideenkartierung. Pettersson, G., Nilsson, L.A., 1983. Pollinationsekologin hos Adam och Eva pa Stora

In: Arbeitskreis Heimische Orchideen (Ed.), Die Orchideen Deutschlands. , pp. Karlsö (Pollination ecology of the Elder-ﬂowered orchid on the island Stora

686–692. Karlsö). Svensk. Bot. Tidskr. 77, 123–132 (in Swedish).

J. Jersáková et al. / Perspectives in Plant Ecology, Evolution and Systematics 17 (2015) 318–329 329

Pillon, Y., Fay, M.F., Shipunov, A.B., Chase, M.W., 2006. Species diversity versus Stefaniak, A., Dabrowska,˛ M., 2013. Dactylorzhiza sambucina (L.) Soó (Orchidaceae) –

phylogenetic diversity: a practical study in the taxonomically difﬁcult genus a new site outside the continuous range in Poland. Arch. Biol. Sci. 65, 697–701.

Dactylorhiza (Orchidaceae). Biol. Conserv. 126, 4–13. Strack, D., Busch, E., Klein, E., 1989. Anthocyanin patterns in European orchids and

Pillon, Y., Fay, M.F., Hedrén, M., Bateman, R.M., Devey, D., van der Bank, A., their taxonomic and phylogenetic relevance. Phytochemistry 28, 2127–2140.

Chase, M.M.W., 2007. Insights into the evolution and biogeography of west- Sundermann, H., 1970. Europäische und Mediterrane Orchideen. Eine Bestim-

ern European species complexes in Dactylorhiza (Orchidaceae). Taxon 56, mungsﬂora mit Berücksichtigung der Ökologie. Brücke-Verlag Kurt Schmersow,

1185–1208. Hannover.

Ponert, J., Vosolsobe,ˇ S., Kmecová, K., Lipavská, H., 2011. European orchid cultivation Tamm, C.O., 1972. Survival and ﬂowering of some perennial herbs. II. The behaviour

– from seed to mature plant. Eur. J. Environ. Sci. 1, 95–107. of some orchids on permanent plots. Oikos 23, 23–28.

Presser, H., 2000. Die Orchideen Mitteleuropas und der Alpen: Variabilität, Biotope Tira, S., 1971. Isoquercitrin from Orchis sambucina. Phytochemistry 10, 1975–1976.

und Gefährdung. Ecomed, Landsberg/Lech. Tlusták, V., Jongepierová-Hlobilová, I., 1990. Orchids of the White Carpathians.

Primack, R., Stacy, E., 1998. Cost of reproduction in the pink lady’s slipper orchid Krajské vlastivednéˇ muzeum. Olomouc (in Czech with English and German sum-

(Cypripedium acaule Orchidaceae): an eleven-year experimental study of three mary).

populations. Am. J. Bot. 85, 1672–1679. Tomaselli, M., Bernardo, L., Passalacqua, N., 2003. The vegetation of the Ranunculo-

Rasmussen, H.N., 1995. Terrestrial Orchids from Seed to Mycotrophic Plant. Nardion in the Southern Apennines S. Italy. Phyton. Horn. 43, 39–57.

Cambridge University Press, Cambridge. Tsiftsis, S., Tsiripidis, I., Karagiannakidou, V., 2006. Study of the orchid ﬂora of Mount

Rasmussen, H.N., Whigham, D., 1993. Seed ecology of dust seeds in situ: a new Falakro (NE Greece). J. Eur. Orch. 38, 57–78.

study technique and its application in terrestrial orchids. Am. J. Bot. 80, Tsiftsis, S., Karagiannakidou, V., Tsiripidis, I., 2007. The orchid ﬂora of East Macedonia

1374–1378. (NE Greece). J. Eur. Orch. 39, 489–526.

Rivas-Martinez, S., Diaz, T.E., Fernandez-Gonzalez, F., Izco, J., Loidi, J., Lousa, M., Tsiftsis, S., Tsiripidis, I., Karagiannakidou, V., Alifragis, D., 2008. Niche analysis and

Penas, A., 2002. Vascular plant communities of Spain and Portugal. Addenda conservation of the orchids of east Macedonia (NE Greece). Acta Oecol. 33, 27–35.

to the Syntaxonomical checklist of 2001. Itinera Geobot. 15, 5–922. Tyteca, D., Bernardos, S., 2003. The Orchid Flora of Portugal. Addendum N. 5. Sta-

Rösler, R., 2013. Zur Chorologie der Orchideen des Regierungsbezirkes Arad in tus of some rare species in Portugal and neighbouring regions. J. Eur. Orch. 35,

Rumänien. J. Eur. Orch. 45, 31–57. 559–633.

Salzmann, C.C., Schiestl, F.P., 2007. Odour and colour polymorphism in the food- Vallius, E., 2001. Factors affecting fruit and seed production in Dactylorhiza maculata.

deceptive orchid Dactylorhiza romana. Plant Syst. Evol. 267, 37–45. Bot. J. Linn. Soc. 135, 89–95.

Schatz, B., Delle-Vedove, R., Dormont, L., 2013. Presence, distribution and pollination Vakhrameeva, M.G., Tatarenko, I.V., Varlygina, T.I., Torosyan, G.K., Zagulskii, M.N.,

effects of white, pink and purple morphs in the orchid Orchis mascula. Eur. J. 2008. Orchids of Russia and Adjacent Countries (within the Borders of the Former

Environ. Sci. 3, 119–128. USSR). A. R. G. Gantner Verlag, Ruggell, Liechtenstein.

Scrugli, A., 1977. Numeri cromosomici per la Flora Italiana: 331–347. Inf. Bot. Itali. Van Waes, J.V., Debergh, P.C., 1986. In vitro germination of some Western European

9, 116–125. orchids. Physiol. Plant. 67, 253–261.

Shefferson, R.P., 2009. The evolutionary ecology of vegetative dormancy in mature Vöth, W., 1971. Knollenentwicklung und Vegetationsrhythmus von Dactylorhiza

herbaceous perennial plants. J. Ecol. 97, 1000–1009. romana und D. sambucina. Orchidee 22, 254–256.

Shefferson, R.P., Proper, J., Beissinger, S.R., Simms, E.L., 2003. Life history trade-offs Vogel, S., 1993. Betrug bei Pﬂanzen: Die Täuschblumen. Abhandlungen

in a rare orchid: the costs of ﬂowering, dormancy, and sprouting. Ecology 84, der Akademie der Wissenschaften und der Literatur, Mathematisch-

1199–1206. Naturwissenschaftliche Klasse. Mainz.

Shimura, H., Sadamoto, M., Matsuura, M., Kawahara, T., Naito, S., Koda, Y., 2009. Vogt-Schilb, H., Munoz, F., Richard, F., Schatz, B., 2015. Decline and range shift of

Characterization of mycorrhizal fungi isolated from the threatened Cypripedium orchids in Western Europe over 20 years: a study based on a large-scale partic-

macranthos in a northern island of Japan: two phylogenetically distinct fungi ipative survey. Biol. Conserv. (in press).

associated with the orchids. Mycorrhiza 19, 525–534. Wells, T.C.E., Rothery, P., Cox, R., Bamford, S., 1998. Flowering dynamics of Orchis

Smithson, A., Juillet, N., MacNair, M.R., Gigord, L.D., 2007. Do rewardless orchids morio L. and Herminium monorchis (L.) R Br. at two sites in eastern England. Bot.

show a positive relationship between phenotypic diversity and reproductive J. Linn. Soc. 126, 39–48.

success? Ecology 88, 434–442. Zagulskii, M.N., Basamak, G.M., Buchko, G.M., Buchko, R.M., 1998. On ecology and

Sonne, M.T., Hauser, T.P., 2014. Population ﬂuctuations, losses to grazing, and repro- biology of Dactylorhiza sambucina (L.) Soó (Orchidaceae) in the Ukraine Carpathi-

ductive success of Dactylorhiza sambucina on Bornholm, Denmark. Ann. Bot. ans. In: Plant Ontogenesis in Natural and Disturbed Habitats. Proceedings of

Fenn. 51, 375–386. International Conference, Lviv, pp. 25–27 (in Ukranian).

Souche, R., 2004. Les Orchidées Sauvages de France. Les éditions du Pélican (collec- Ziegenspeck, H., 1936. Orchidaceae. Lebensgeschichte der Blütenpﬂanzen Mitteleu-

tion Grandeur Naure). ropas. Band 1. Abteilung 4. Eugen Ulmers, Stuttgart.