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

Contents lists available at ScienceDirect

Perspectives in Plant Ecology, Evolution and Systematics

jo ur nal ho me p age: www.elsev ier .com/ locate /ppees

iological flora of Central Europe

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

ana Jersákováa,∗, Iva Traxmandlováb, Zdenek Ipsera, Matthias Kropfc,iuseppe Pellegrinod, Bertrand Schatze, Vladan Djordjevic f, Pavel Kindlmannb,g,usanne S. Rennerh

Faculty of Science, University of South Bohemia, Branisovská 1760, Ceské Budejovice 37005, Czech RepublicDepartment of Biodiversity Research, Global Change Research Centre AS CR, Belidla 4a, 602 00 Brno, Czech RepublicInstitute for Integrative Nature Conservation Research, University of Natural Resources and Life Sciences, 1180 Vienna, AustriaDepartment of Biology, Ecology and Earth Science, University of Calabria, Rende, ItalyCentre d’Ecologie Fonctionnelle et Evolutive (CEFE), UMR 5175, CNRS – Université de Montpellier (EPHE), 1919 route de Mende, 34293 Montpellier, FranceInstitute of Botany and Botanical Garden, Faculty of Biology, University of Belgrade, Takovska 43, 11000 Belgrade, SerbiaInstitute for Environmental Studies, Charles University, Benátská 2, Prague, Czech RepublicInstitute of Systematic Botany and Mycology, University of Munich (LMU), Munich, Germany

r t i c l e i n f o

rticle history:eceived 16 February 2015eceived in revised form 7 April 2015ccepted 27 April 2015vailable online 16 May 2015

eywords:olour polymorphismormancy

a b s t r a c t

Dactylorhiza sambucina (L.) Soó is a polycarpic perennial herb occurring in the Central European, East-ern European, and Balkan floristic provinces. At the European scale, the IUCN considers it a species of“least concern”. This paper reviews the taxonomic status, morphology, distribution, habitat requirements,mycorrhizal associations, and life cycle of D. sambucina, with special emphasis on its reproduction. Wealso summarize information on chromosome numbers and genetic variation. Our data from 12 yearsof monitoring D. sambucina in the Czech Republic show that three to four leaves have to be producedprior to flowering; plants with five and more leaves flower regularly. Juvenile plants near adult plantssuggest recruitment from seeds. About 20% of our 450 monitored plants underwent dormancy (failure of

cological nicheife cycleeproductive biologyeed germination

mature plants to produce above-ground parts in one or more growing seasons), the maximum durationbeing eight years. After reappearance, these individuals were usually sterile for the next year. Mortalitywas highest (24%) at the seedling stage. Regarding the purple/yellow flower colour polymorphism thatcharacterizes D. sambucina, we found no correlation between morph frequency and soil properties (pH,calcium content), population density, or altitude above sea level.

© 2015 Geobotanisches Institut ETH, Stiftung Ruebel. Published by Elsevier GmbH. All rights reserved.

ontents

Morphology and taxonomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319Distribution and habitat requirements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319

Geographical and altitudinal distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 319Substratum. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .320Habitats and plant communities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320

Life cycle, phenology and growth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322Phenology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322Life cycle and dormancy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322Seed production and dispersal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323Seed germination in situ and seedling morphology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324

Seed germination in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Mycorrhiza . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .Spatial distribution of plants within populations . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author. Tel.: +420 387 775 357.E-mail address: jersa@centrum.cz (J. Jersáková).

ttp://dx.doi.org/10.1016/j.ppees.2015.04.002433-8319/© 2015 Geobotanisches Institut ETH, Stiftung Ruebel. Published by Elsevier G

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mbH. All rights reserved.

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

Responses to abiotic and biotic factors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324Response to climate factors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .324Response to competition and management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324Herbivores and pathogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324

Floral biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325Pollination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325Colour polymorphism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325Patterns in colour polymorphism across Europe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325Maintenance of colour polymorphism by pollinator morph discrimination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325Factors affecting fruit set . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326

Physiological and biochemical information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .326Physiological data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326Biochemical data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326

Genetic data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326Chromosome number. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .326Genetic variation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .326Hybrids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327Conservation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327

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

. . . . . .

M

psbTlttd(mTi(iastswlhDttiavolly

SDbwgtgaa

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

orphology and taxonomy

Dactylorhiza sambucina (L.) Soó is a polycarpic perennial geo-hyte with a palmate root tuber slightly or moderately divided intoeveral lobes (Fig. 1A). Between 3 and 6 adventitious roots that cane up to 6 cm long develop at the base of an innovation bud (Fig. 1A).he stem is 10–20 (30) cm high, sturdy, and hollow. It bears 2 scaleeaves and 3–7 green leaves, depending on age and nutrient sta-us, with the lower ones oblong-lanceolate 5–10 × 1–2.5 cm, andhe upper ones lanceolate; the leaves are homogenously green ando not develop brownish lilac spots. The inflorescences are ovoidegg-shaped) or shortly cylindrical dense racemes (without a ter-

inal flower), (3.7) 5–7.5 (10.5) × (2.7) 3.5–4.5 (5) cm in length.he lower bracts are longer, the upper ones equal to the flowersn length; young inflorescences are sheeted by 1–8 (11) cataphyllsPedersen, 2006). The flowers are zygomorphic and either yellow-sh or purple-red. The outer perianth segments (often called sepals)re 0.7–0.9 cm long, oblong-ovate in shape with an obtuse tip, andtand out upwards, the median perianth segment forms a helmogether with the two shorter, obliquely ovate lateral perianthegments. The labellum measures 0.7–1.0 × 0.7–0.9 cm, is roundedith a slightly three-lobed apex and in both colour morphs is speck-

ed with light reddish to dark purple spots; in the purple morph itas an yellowish base. The cylindrical spur is 1.0–1.5 cm long (inactylorhiza insularis distinctly shorter), bent downward, and con-

ains no nectar. The column is erect and 4.5 mm high; each of thewo sectile pollinia is tapering into a caudicle, attached to the viscid-um; the rostellum is three-lobed and forms a roof-like projectionbove the stigma; one large bursicle covers the two separate stickyiscidia; the stigma is three-lobed with large lateral lobes. Thevary is twisted, glabrous, and develops into an erect 1.1–1.3 cmong capsule. The seeds are numerous, 0.5–0.6 × 0.15–0.25 mmarge, spherical or slightly ellipsoid in shape and have a hyalineellowish brown testa (Bojnansky and Fargasová, 2007).

The species epithet apparently refers to the floral scent ofambucus nigra L. (Adoxaceae) (Delforge, 2005). Taxonomically,. sambucina belongs to sect. Sambucinae (Parl.) Smoljan, whichesides D. sambucina, includes Dactylorhiza romana (Sebast.) Soóith subsp. romana (Sicily, Eastern Mediterranean, Crimea), geor-

ica (Klinge) Soó ex Renz & Taubenheim (Northeastern Turkey

o Caucasus, Transcaucasia, W- and N-Iran, Turkmenistan), anduimaraesii (D.G. Camus) H.A. Pedersen (Spain, Algeria, Morocco),s well as Dactylorhiza cantabrica H.A. Pedersen (northern Spain),nd D. insularis (Sommier) O. Sanchez & Herrero (Western

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327

Mediterranean from Spain to Italy, North Africa) (Pedersen, 2006).Pedersen (2006) provides color photos and a key to these taxa.Two of the D. romana subspecies, namely romana and georgica,have the same flower colour dimorphism as D. sambucina, while D.cantabrica, D. insularis, and D. romana subsp. guimaraesii only pro-duce yellow flowers. Forms with very pale flowers as are frequentlyobserved in other sections of Dactyloriza are unknown from section“Sambucinae” (Delforge, 2005; Schatz et al., 2013).

Distribution and habitat requirements

Geographical and altitudinal distribution

The geographical distribution of D. sambucina and D. romana(and their allies in section “Sambucinae”) ranges from Portugal inthe west to northern Iran in the east, and from southern Scan-dinavia in the north to northern Morocco, Algeria, and Lebanonin the south. A map of its total range may be found in Meuselet al. (1965: map 110d, sub nom. D. sambucina s.l.). The south-ern range limit of D. sambucina sensu stricto stretches from CentralSpain to the southern Peloponnese (Fig. 2, Appendix 1). Its east-ern border runs from eastern Bulgaria to the Dnepr River in theUkraine (Didukh, 2009) and the Bryansk region in Russia closeto the Ukrainian border (Evstigneev and Fedotov, 2004). In thenorth, it extends to southern Poland and Central Germany. Scan-dinavian populations, slightly disjoint from the main distributionalrange, occur in southern Norway, eastern Denmark, in SoutheastSweden, and southernmost Finland. D. sambucina is absent fromthe British Isles and western Siberia. The whole of Scandinavia andmuch of the British Isles were covered with ice 18,000 years ago,and periglacial conditions persisted until 11,700 years ago. Thus,the northern European populations of D. sambucina all establishedpost-glacially, whereas southern populations may antedate the lastglacial maximum (Pedersen, 2006; Pillon et al., 2006, 2007). Theabsence of D. sambucina from the British Isles might be due to a fail-ure to re-colonize the island over the past 10,000 years. Historically,D. sambucina also occurred in Estonia, and it was unsuccessfullyreintroduced at Saaremaa Island in 1989 (Kuusk, 1994). Reportedoccurrences in Asia Minor (including Caucasus, Crimea, Turkey), the

Baltic countries (Latvia, Lithuania), Belarus, Sardegna, and north-ern Africa are erroneous (e.g. Stefaniak and Dabrowska, 2013;Govaerts, 2014; but see following orchid flora lists: G.I.R.O.S., 2009;Gudzinskas, 2001; Gudzinskas 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 – developingnew 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-yearo of a

C ore caJ

2w

ÖRi12225p2(1CDriR(ia

S

oT

ld seedlings, as – apical shoot, ar – adventitious root, t – tuber. (C) Cross-sectionross-section of a mycorrhizal hypha with dolipore septum that is composed of a p

ersáková.

005; Lai, 2009; Pashkov et al., 2005) and probably due to confusionith D. romana and D. insularis.

D. sambucina occurs from sea level (on the Swedish islands ofland and Gotland) up to 2400 m in the Alps (Baumann et al., 2006).egional altitudinal ranges reported in literature are: 193–2300 m

n Switzerland (AGEO, 2014), 250–2200 m in Austria (Novak, 2010),75–530 m in the Rhineland-Palatinate in Germany (Kropf, 2008,011), 1060–1200 m in Taormina Mt. in Sicily (Cristaudo and Galesi,010), 900–1800 m in Galicia in Spain (Tyteca and Bernardos,003), 600–1500 m in Mt. Giona in Greece (Aplada et al., 2012),00–2100 m in East Macedonia (Tsiftsis et al., 2008), 400–1300 m inrovince Hermannstadt in Romania (Dragulescu and Rösler, 2005),50–2500 m in France, with greatest abundance at 750–1750 mDusak and Prat, 2010), and up to 1700 m in Albania (Ziegenspeck,936). In Poland, the species occurs mainly in the Sudeten andarpathian mountains, reaching 1115 m in Pieniny (Stefaniak andabrowska, 2013). Our own observations described later in this

eview were made at 781–1300 m alt. in the National Park Cévennesn Southern France (21 populations), 467–900 m in the Czechepublic (29), 1149–1709 m in Sila National Park in Calabria in Italy17), 1240–1510 m in Dolomites in Trentino in Italy (3), 175–445 mn the Rhineland-Palatinate (19), 295–820 m in Lower Austria (13),nd 871–1795 m in western Serbia (117 populations).

ubstratum

D. sambucina occurs on nitrogen-poor soils, usually with a pHf 5.2–6.8 (Sundermann, 1970) or 5.0–6.3 (Ziegenspeck, 1936; ourable 1). However, in Southern France, Italy, Greece, and on Öland

seedling, brown coils of fungal hyphae (pelotons) are visible within the cells. (D)p surrounding a septal swelling and septal pore. Photo A by J. Brabec, C and D by J.

and Gotland it grows on calcareous soils with a pH of up to 7.7.In the Ukrainian Carpathians, it grows on brown mountain soilswith pH 4.7–4.9 (Zagulskii et al., 1998), and in western Serbiaon soils derived from limestone, serpentine, ophiolitic mélange,schists, phyllites, quartz latite, porphyrite, or Quaternary sediments(VDj. pers. obs.; Djordjevic et al., 2014). Different from other speciesof Dactylorhiza, D. sambucina does not occur in wet biotopes butinstead prefers dry soils. The maximum water-holding capacityof such soils in Central Europe ranges from 30.7 to 54.4% withmean ± SD: 43.0 ± 7.3% (JJ unpubl. data). Further mechanical andphysical soil properties (bulk density, porosity) are detailed in Mróz(1994). In France, D. sambucina sites are characterized by quarterlyprecipitations between 50–250 mm and 1120–1620 mm and tem-peratures between −8.2 ◦C to −6.2 ◦C and 21.7 ◦C to 26.2 ◦C (Dusakand Prat, 2010).

Habitats and plant communities

D. sambucina is a light-demanding species. It prefers nutrient-poor meadows and pastures (see section “Substratum”), includinggrassy patches in stony thickets, well-drained forest meadows, for-est borders, clearings, and open broad-leaved or coniferous forests.Plant communities in which it occurs are the so-called Molinio-Arrhenatheretea and Nardo-Callunetea classes or less frequently,forest communities of the Querco-Fagetea class (e.g. Czech Rep. –

Tlusták and Jongepierová-Hlobilová, 1990; Polish Sudeten – Mroz,1994; Franconian Forest – Balzer, 2000; Sicily – Cristaudo andGalesi, 2010; Rivas-Martinez et al., 2002; Romania – Dragulescuand 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, greencircles – 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 1Properties of the soils that support Dactylorhiza sambucina populations. Mean ± SD, range and number of analysed populations.

Country pH Potassium(g/kg)

Calcium(g/kg)

Availablephosphorus(mg/kg)

Total nitrogen(mg/100 g)

Organic matter (%)

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

CzechRep.

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

East 6.2a 2.9a 12.6a

Macedonia (4.3–7.7) (1.2–6.7) (0.97–35.9)96 96 96

S t al. (2

gapA

Southern Norway 5.5 (0.4–1.2)

ource: Jersáková et al. (unpubl. data), Poland – Mróz (1994), Macedonia – Tsiftsis ea Median.

rows predominantly in mesophytic grasslands of the Arrhen-therion elatioris alliance (Trifolio-Festucetum rubrae community),astures of the Cynosurion cristati alliance (Anthoxantho odorati-grostietum capillaris community), submontane and montane

008), Southern Norway – Norderhaug et al. (1997).

grasslands of the Violion caninae alliance, broad-leaved dry grass-lands of the Bromion erecti alliance (Brachypodio pinnati-Molinietumarundinaceae community) and acidophilous dry grasslands ofthe Koelerio-Phleion phleoidis alliance (Mróz, 1994; Kropf, 1995;

3 gy, Evolution and Systematics 17 (2015) 318–329

JgCwfswO2noJaracstrlB

L

P

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atcpaRlajua2pibrd

Numbe r of lea ves

Num

ber o

f pla

nts

1 2 3 4 5 6 70

200

400

600SterileFlowering

Fig. 3. Number of leaves in sterile and flowering individuals of Dactylorhiza sam-bucina. Pooled data from 2018 records over 12 years (1998–2010) of monitoring of

22 J. Jersáková et al. / Perspectives in Plant Ecolo

ersáková and Kindlmann, 2004b). In Ukraine, it grows in acidicrasslands of the Cynosurion cristati alliance, in the Ukrainianarpathians it inhabits the Quercion robori–petraeae woodlands, asell as grasslands (Festucetum rubrae community) developed on

ormer beech and spruce forest sites (Didukh, 2009). In Greece, D.ambucina inhabits subalpine grasslands and open Fagus and Pinusoodlands, often in clearings and forest margins, open Carpinus-strya scrubs, on a variety of substrates (Tsiftsis et al., 2006,007; Aplada et al., 2012). In the Central and Southern Apen-ines in Italy, it occurs in the community Nardo-Luzuletum pindicaef the Ranunculo-Nardion alliance (Tomaselli et al., 2003), the

uniperus shrub community Daphno oleoidis-Juniperetum alpinae,nd in neutral-subacidophilous grassland community Potentilloigoanae-Brachypodietum genuensis (Phleo ambigui-Bromion erectilliance, Di Pietro et al., 2005). In western Serbia, D. sambucinaan be found in the following grassland communities: Nardetumtrictae, Danthonietum calycinae, Koelerietum montanae, Brome-um erecti, Brachypodietum pinnati, Festucetum valesiacae, Festucoubrae-Agrostetum capillaris, and Poetum violaceae, and the wood-and communities: Pinus sylvestris forests, Fagetum montanum, andetuletum pendulae (VDj. pers. obs.).

ife cycle, phenology and growth

henology

The appearance of leaves above ground depends on springumidity and temperature. The first leaves may appear in earlyarch and are fully developed during flowering and fruit produc-

ion. The time of flowering depends on a combination of factors,specially latitude and altitude. At lower altitudes, flowering startsn mid-April (South and Central Europe, Alsace in France), peaksetween mid-May and mid-June in Scandinavia and in subalpineltitudes (lower altitudes in the Alps and Apennines), and extendso early July in high alpine areas (Nilsson, 1980; Presser, 2000).he capsules mature for ca. 1.5 month, and seeds are shed ratheruickly within a few sunny days during June or July. During sum-er, the aerial parts die and the plants stay underground until the

ext growing season.The development of a new tuber starts in early March, when

flat, palmately divided daughter tuber and adventitious rootsevelop at the base of an innovation bud, which is located in thexilla of a second cataphyll at a compressed shoot base (Vöth, 1971;ur Fig. 1A). During the first year, the tuber elongates and becomeslightly or moderately divided with up to four root-like extensionsmerging from its distal end. The old tuber gradually shrinks andies during capsule development.

The factors determining whether a plant will flower are complexnd little understood; they include both internal and environmen-al variables. According to Wells et al. (1998), orchids have to reach aritical size before they can flower, and once that size is reached, therobability of flowering increases with leaf number. Our data from

long-term monitored population of D. sambucina in the Czechepublic show that a plant has to reach at least three, or better four

eaves to start flowering, while plants with five and more leaveslmost always flower (Fig. 3). Plants with one or two leaves areuveniles. The probability of consecutive flowering in orchids issually highly limited by costs of flowering and fruiting (Primacknd Stacy, 1998; Jacquemyn and Hutchings, 2010; Jersáková et al.,011; but see Shefferson et al., 2003). In our long-term monitoredopulation, 77% of flowering plants develop an inflorescence also

n the following year (although some inflorescences aborted), 17%ecame sterile (Fig. 4). Similarly, 65% of plants with aborted inflo-escences, which did not set fruits and thus saved energy, flowereduring the next year. Inghe and Tamm (1988; also Tamm, 1972)

a single population in the Czech Republic.

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(Appendix 2).

Life cycle and dormancy

D. sambucina is a non-bulbous geophyte showing limited veg-etative spread. The wintering organs are a renewal bud witha tuber and adventitious roots (Vöth, 1971; Rasmussen, 1995;Vakhrameeva et al., 2008, Fig. 1A). The innovation bud producesa new shoot that eventually terminates with an inflorescence(Rasmussen, 1995). The inflorescence is formed and remains insidethe bud for more than a year (Vakhrameeva et al., 2008).

D. sambucina frequently forms clumps but it is not known if theyare of vegetative or generative origin. During our 12 years of mon-itoring of D. sambucina, we sometimes observed new plants belowmature plants, with their small size suggesting juveniles germi-nated from seeds. In three of 450 monitored plants, we observeda replacement of a single adult plant by two daughter plants of amoderate size. Similarly, Inghe and Tamm (1988) during 43 yearsof monitoring 74 plants observed only two vegetative clones. Themost widespread pattern of vegetative reproduction in orchids isthe formation and germination of two or more buds, including dor-mant ones, on axial organs such as rhizomes, creeping shoots, androot tubers (Dactylorhiza case). The daughter shoots in orchids withroot tubers (tuberoids) detach after 0.5–1 years (Batygina et al.,2003).

Adult vegetative dormancy (Shefferson, 2009), the failure of aplant to produce above-ground parts in one or more growing sea-sons followed by reappearance of full-sized photosynthetic plantsin subsequent seasons, has been observed in D. sambucina and typ-ically lasts for one year (Fig. 5A). During 12 years of monitoring, weobserved dormancy in 20% of the 450 monitored plants, the maxi-mum length of dormancy being eight years. Inghe and Tamm (1988)recorded dormancy in 31% of their 74 plants, all but one dormantfor one year while the exceptional plant was dormant for two years.Most dormant plants in the Czech population recruit from flower-ing ones, and after reappearance most plants are sterile (Fig. 5B).The transition to dormancy thus seems to be triggered by the highcost of flowering and fruiting.

D. sambucina appears to be long-lived. Out of cohort of 49plants followed since 1942, 11 plants were still alive 43 yearslater (Inghe and Tamm, 1988). Ziegenspeck (1936) reported twoyears 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 Sumava mountains (Czech Republic). Pooled datafrom 10 years (2000–2009) and 2533 transitions. The values in parentheses indicate theprobabilities 0–25%, 26–50%, 51–75%.

A

B

1 2 3 4 5 6 7 80

20

40

60

80

Dormancy (years)

No.

of p

lant

s

1 20

20

40

60

80JuvenileSterileFlowering

Before After

No.

of p

lant

s

Fig. 5. Dormancy in Dactylorhiza sambucina monitored over 12 years from 1998 to2010. (A) Frequency and length of dormancy. (B) Life stage of marked individualsbefore and after dormancy. Juvenile plants are newly emerged plants with one ortwo leaves; sterile plants are non-flowering individuals with three and more leaves.

number of plants involved in each transition. The thickness of the lines denotes

to first flowering. Our germination and seedling monitoring datashows that first leaf may appear above-ground after two or threeyears in the soil (Appendix 3A), and the first flowering event onaverage occurs five years after emergence (25 juvenile plants withone leaf flowered for the first time 2–10 years after emergence,mean ± SD = 5.2 ± 1.8, median = 5; number of leaves at first flow-ering: mean ± SD = 4.2 ± 0.7, median = 4, range 3–5). In the Czechpopulation, the seedlings stage had the highest mortality (24%;Fig. 4).

Seed production and dispersal

D. sambucina is an allogamous, self-compatible species, with84–97% fruit set after hand self-pollination, and incapable ofagamospermy or spontaneous autogamy (Jersáková, 1998; Kropfand Renner, 2005; Pellegrino et al., 2005; Juillet et al., 2006). Thespecies shows high inbreeding depression after self-pollination.For example Nilsson (1980) reported fewer developed embryosin self-pollinated seeds (43%) than in cross-pollinated ones (75%).Inbreeding depression coefficients for the percentage of seeds witha well developed embryo after self- or cross-pollination were 0.42(Nilsson, 1980), for seed viability estimated by the tetrazolium test0.63 (Jersáková et al., 2006), for germination rates at 65 and 130days 0.46 and 0.60, respectively, and for survival rate 0.75 (Juilletet al., 2006).

Wind-dispersed seeds are numerous, with spherical or slightlyellipsoid embryos enclosed in an elongated testa of yellowish-brown hollow cells (Bojnansky and Fargasová, 2007). The numberof seeds per capsule is unknown, but in D. romana it is 4736 seeds

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

3 gy, Ev

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24 J. Jersáková et al. / Perspectives in Plant Ecolo

he mean fruit set varied from 1.1% to 30.1% in different yearsAppendix 4).

eed germination in situ and seedling morphology

According to Fuchs and Ziegenspeck (1927), D. sambucinaerminates in the spring (Fig. 1C). However, other Dactylorhizapecies germinate already in autumn after dissemination dur-ng the summer (Dactylorhiza majalis, Tesitelová and Jersáková, inrep.; Dactylorhiza fuchsii, Leeson et al., 1991; Dactylorhiza macu-

ata, Möller, 1990), thus the start of germination in D. sambucinaeserves further study. The mycorhizome is extensive, with therst root developing during the first winter. It is mycotrophic butontains a certain amount of xylem. During the following spring,he leafy shoot of D. sambucina unfolds after which growth becomesympodial and the first root tuber with 2–4 vascular bundles devel-ps (Fig. 1B).

The seed bank of D. sambucina is short-lived. Our in situ germina-ion experiments using seed packets buried in natural conditionsRasmussen and Whigham, 1993) revealed a germinating rate of.2% and 2% intact seeds after three years in the soil. This is similaro Dactylorhiza lapponica with 0.2% of live seeds after 3 years in theoil (Øien et al., 2008). In that species, the probability of a seed toorm a protocorm declined from 4.4% after one year to 0.1 and 0.2%fter two and three years to 0% in the fourth year (out of initially0 thousands of seed buried).

eed germination in vitro

In vitro, D. sambucina readily germinates asymbiotically. Ponertt al. (2011) described two cultivation media on which 90% of seedserminated after pretreatment of 2–5 min in 70% ethanol and 5 minn 5% Ca(OCl)2. When cultivated at 23 or 17 ◦C, the protocormstopped growing, started to turn brown and then died. When trans-erred to 4 ◦C, the protocorms also stopped growth, but remainedhite in a good condition and after this cold treatment they started

o grow again and produced shoots. Van Waes and Debergh (1986)chieved the highest seed germination (80.3%) with a pretreat-ent in 5% Ca(OCl)2 and 1% emulsifier (Tween-80) for four hours.

he percentage of coloured embryos using the tetrazolium testfter this pretreatment was 83.2%. Continuous darkness inducedigher germination rates (53.3%) than illumination between 1.2nd 30.4 �mol m−2 s−1 (maximum germination 5.2%). Cultivationedium without macroelements yielded a higher germination rate

71.7%) than a medium with 0.47 mM of macroelements (55.3%)Van Waes and Debergh, 1986).

ycorrhiza

The symbiotic mycorrhizal partners of D. sambucina have beenittle studied. The finger-like extensions of the tubers are col-nized with mycorrhizal fungi, as are the narrow, adventitiousorizontal roots (Fig. 1A). Transmission electron microscopy ofoots of adult plants revealed rhizoctonia-like hyphae possessingeptal dolipores with imperforated parenthesomes, consisting ofwo electron-dense layers separated by an electron-transparentone (JJ unpubl. data; Fig. 1D). Such ultrastructure of the septalore and parenthesome is typical for the family TulasnellaceaeShimura et al., 2009). Molecular investigations of the fungal com-

unity confirmed that both seedlings and adult plants associateith various members of Tulasnellaceae, less frequently Ceratoba-

idiaceae and Sebacinales (Pellegrino and Bellusci, 2009, JJ & GP

npubl. data; Appendix 3). Tulasnella is a very common symbiontf terrestrial orchids (reviewed in Dearnaley, 2007), observed alson other Dactylorhiza species (Kristiansen et al., 2001; Jacquemynt al., 2012). While each analysed seedling associated only with a

olution and Systematics 17 (2015) 318–329

single fungal strain from Tulasnellaceae or Ceratobasidiaceae fam-ily, the adult plants frequently associated with two or three fungalstrains of the same fungal families simultaneously (Appendix 3).These results resemble findings in Orchis and Serapias in which mul-tiple fungal associations have also been documented (Jacquemynet al., 2010; Luca et al., 2014; Pellegrino et al., 2014).

In addition to rhizoctonia, some ectomycorrhizal ascomyceteshave also been found in adult D. sambucina plants (Pellegrino andBellusci, 2009). Though known mainly as mycobionts of forest-dwelling members of Neottieae (reviewed in Dearnaley, 2007),here they co-occur with dominant rhizoctonia fungi. The electronmicroscopy of D. sambucina roots has not revealed any ascomycetesforming pelotons. Dearnaley et al. (2013) proposed that the pres-ence of endophytic fungi in root tissues may lead to evolution intotrue mycorrhizal partners.

Spatial distribution of plants within populations

D. sambucina grows in nutrient poor meadows and pastures,with the spatial distribution of individuals depending on popu-lation age, micro-habitat conditions, and grassland managementpractice. In Germany, dense patches had 1.4–4.4 individuals perm2; the highest recorded number was 12 flowering plants in asingle m2 (MK unpubl. data). In the Polish Sudety Mountains,Mróz (1994) found 1.7–5.6 plants per m2, with a mean of 3.4plants. In Southern Italy, 1.7–6.4 plants per m2 have been recorded(Pellegrino et al., 2005). A 1 m2-monitoring plot initiated by Tammin 1942 contained 11–46 flowering or vegetative individuals in thecourse of 43 years (Inghe and Tamm, 1988).

Responses to abiotic and biotic factors

Response to climate factors

D. sambucina flowering starts in mid-April (South and CentralEurope, Alsace in France) and peaks between mid-May and mid-June; flowers are frequently aborted due to frost (Nilsson, 1980;Balzer, 2000; pers. obs. JJ, MK). Summer drought has a negativeeffect on flowering in the subsequent year (Inghe and Tamm, 1988).

Response to competition and management

As a light-demanding species adapted to nutrient-poor soils,D. sambucina depends on traditional land-use, while fertilization,frequent mowing, and succession following abandoned land useaffect populations negatively (Kropf, 1995; Balzer, 2000). Whenovergrown by stronger competitors, D. sambucina will first reactby increasing individual plant height, later by non-flowering, andfinally, by non-appearance (MK pers. obs.) in a similar way as Orchismorio (Jersáková et al., 2002). Therefore, nature conservation man-agement often involves maintaining traditional land-use practices,including no fertilization and a single mowing or grazing within agrowing season; ideally, after flowering and fruit set of D. sambucina(and other target species; Balzer, 2000). In a German study region,of 16 populations with flowering individuals in 2006, 12 (75%) areunder nature conservation management (with shrub clearance andsheep grazing), which protects at least 2684 flowering plants or93.6% of all flowering individuals in this region (Kropf, 2008, 2011).

Herbivores and pathogens

There are no data on phytophagous insects, parasites, or diseases

of D. sambucina. In the Czech Republic, Austria and France, brows-ing deer frequently damage plants, and wild boar and rodents mayeat the tubers (Kropf, 2008; JJ, BS pers. obs.). Heavy grazing by cat-tle during spring and early summer has been identified as a major

gy, Evolution and Systematics 17 (2015) 318–329 325

rh

F

P

(atFbirbmASfflflblo

wt(stcPKtfbaCeda

C

dsplupo(ppnimRp

P

a

0.00

0.20

0.40

0.60

0.80

1.00

0 200 400 600 800 10 00 12 00 14 00 16 00 18 00

Altitude

Pro

porti

on o

f yel

low

mor

ph

France Czech R. S ItalyGermany Austria N Italy

Fig. 6. Altitudinal pattern in the proportion of the yellow morph compared to the

J. Jersáková et al. / Perspectives in Plant Ecolo

eason for the decline of D. sambucina on the Danish island Born-olm (Sonne and Hauser, 2014).

loral biology

ollination

D. sambucina has conspicuous yellow and purple inflorescencesAppendix 4B) that in mid-April and May attract newly emergednd inexperienced bees, especially bumblebee queens. The pollina-ors recorded across its distribution area (Austria, Czech Republic,rance, Germany, Italy, Sweden, Switzerland, Ukraine) are mainlyumblebees (Bombus bohemicus, B. hortorum, B. hypnorum, B. lap-

darius, B. lucorum, B. muscorum, B. pascuorum, B. ruderarius, B.uderatus, B. sylvarum, B. soroeensis and B. terrestris), but also cuckooumblebees Psithyrus vestalis and P. barbutellus, honeybees Apisellifera, and solitary bees Andrena nigroaenea, Osmia bicolor andnthophora aestivalis (reviewed in Claessens and Kleynen, 2011). Inweden, flower visitors (not pollinators because they have not beenound to transport pollinia) include females of Halictus sp., butter-ies (Pyrgus malvae and Gonepteryx rhamni), and an unidentifiedy (Nilsson, 1980). In the South of France, Gonepteryx cleopatra haseen observed on the flowers (BS, pers. obs.). Pollinator spectra vary

ocally; for example, the bee A. nigroaenea was the unique pollinatorn Stora Karlsö Island (Pettersson and Nilsson, 1983).

While probing the flowers for nectar, bees will touch the bursicleith its two separate viscidia. Based on the size of an insects’ head,

he viscidia become attached either to its forehead or its clypeusNilsson, 1980; Appendix 4E). After pollinaria removal, the caudicletarts to bend and moves into a position suitable for contactinghe stigmatic cavity of the next D. sambucina flowers visited. Theaudicle bending time is 20–40 s, with a mean of 25 s (Nilsson, 1980;eter and Johnson, 2006; Kropf and Renner, 2008; Claessens andleynen, 2011; BS pers. obs.). Floral visits are short (few seconds),

ypically to one or two flowers of an inflorescence, rarely up toour (Nilsson, 1980; Kropf and Renner, 2005). As a result, individualees carry only one or two pollinia, the largest number found on

single bumblebee is 15 (Nilsson, 1980; Kropf and Renner, 2005;laessens and Kleynen, 2011, BS pers. obs.). Pollinia color-codingxperiments have revealed that most pollen is dispersed over shortistances (median 1.23 m); the longest distance was 176 m (Kropfnd Renner, 2008).

olour polymorphism

Colour polymorphism has been described in many food-eceptive orchid species (Claessens and Kleynen, 2011). D.ambucina is unusual in having populations in with highly balancedurple/yellow morph frequencies as well as monochromatic popu-

ations (next section). This has led to many studies of the suspectednderlying density-dependent selective processes maintaining theolymorphism. As mentioned in the section “Morphology and tax-nomy”, occasional salmon pink individuals have been recordedAppendix 4C), probably reflecting hybridization among the pur-le and yellow morphs. Their frequency does not exceed 5%. Theseink plants have been ranked as D. sambucina subsp. zimmerman-ii A.Camus (a misapplication of the concept of subspecies, which

s supposed to have geographic coherence) or D. sambucina f. zim-ermannii (A.Camus) P.Delforge (Souche, 2004; Delforge, 2005).arely, somatic mutations produce individuals with unusual floralatterns (Appendix 4D).

atterns in colour polymorphism across Europe

Our screening of 101 D. sambucina populations (Appendix 4C)nd published records reveal a strong regional bias in morph

purple morph in 79 European populations with more than 50 individuals. Purelyyellow populations were found in the Rhineland-Palatinate in Germany and in LowerAustria.

frequencies, with purely yellow morph populations in Germany(n = 19), yellow morph-biased populations in Southern France (66%,n = 21; 69% in Gigord et al., 2001), Italy (62%, n = 20), and Austria(57%, n = 13), and purple-biased populations in Sweden (Nilsson,1980) and in the Czech Republic (37% of yellow form, n = 28). Inaddition, populations within regions and among years vary in theirpurple/yellow frequencies. Such fluctuations must be seen againstthe backdrop of dormancy (section “Life cycle and dormancy”), andswitches of the dominant colour morph have been observed in bothyellow- and purple-dominated populations in single years, albeitrarely and only in small-sized populations (Kropf and Kriechbaum,2009). We tried to relate the colour polymorphism in D. sambucinato soil properties (pH and calcium content), population size, andaltitude above sea level, but found no continent-wide relationships(Fig. 6, Appendix 6).

Maintenance of colour polymorphism by pollinator morphdiscrimination

It has been suggested that colour polymorphism in D. sambucinais maintained by negative frequency-dependent selection inducedby food-deceived pollinators that over-visit the rare colour morph,having learned to avoid the frequent morph (Gigord et al., 2001).Subsequent studies have failed to support this hypothesis (Kropfand Renner, 2005; Pellegrino et al., 2005; Jersáková et al., 2006;Smithson et al., 2007), which also does not agree with what isknown about learning in naïve bumblebee individuals. A suggestedalternative explanation for the maintenance of the colour polymor-phism is differential fertility among colour morphs. This is basedon Jersáková et al.’s (2006) finding of a lower seed viability of theyellow morph in purple-biased populations in the Czech Republic.

Another factor that may influence morph frequencies is localpollinator colour-fidelity due to the colours of co-flowering reward-ing plants. In greenhouse experiment, bumblebees preferred the D.sambucina colour morph that resembled the colour of previouslyvisited rewarding flowers (Gigord et al., 2002). Colour constancyhas also been observed in situ in France (BS unpubl. data) andSweden (Nilsson, 1980; our Table 2). Experiments in which nat-ural D. sambucina populations were enriched with rewarding Viola

aethnensis further support these findings (Pellegrino et al., 2008):aggregation with yellow Viola plants increased fruit set in yellow D.sambucina and vice versa (“magnet species effect”, cf. Johnson et al.,2003).

326 J. Jersáková et al. / Perspectives in Plant Ecology, Ev

Table 2Colour constancy (>2 consecutive visits to the same colour morph) of individual beesvisiting Dactylorhiza sambucina flowers on Öland in Sweden (from Nilsson, 1980) orin 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).

Site Öland Site A Site B Site C

Frequency of yellow morph 0.13 0.46 0.56 0.9Total pollinator visits 25 11 8 13Constant visits to yellow flowers 3 0 1 9Constant visits to purple flowers 18 6 3 0

cppi

F

(iabcltpbpfKtfi1e(flflApedbdIfbfdtgsIpn

idtsd

Inconstant visits 3 5 4 4Ratio of constant to inconstant pollinators 0.84 0.55 0.5 0.69

The most plausible current explanation for what maintainsolour polymorphism in D. sambucina thus is a combination of post-ollination barriers among the morphs (affecting seed viability),resence, density and colours of co-flowering rewarding plants, and

nnate vs. learned colour preferences of bees.

actors affecting fruit set

Fruit set fluctuates between years and among populationsNilsson, 1980; Pellegrino et al., 2005, Appendix 5). D. sambucinas pollinator-limited because it offers neither nectar nor pollen as

reward for bee pollinators, and the addition of nectar to D. sam-ucina flowers increased pollinia removal and deposition in botholour morphs (Jersáková et al., 2008). There is a positive corre-ation between the number of flowers and fruit set, illustratinghe effect of floral display (Nilsson, 1980). However, a negativearabolic relationship between reproductive success and the num-er of flowers has also been demonstrated, which means thatlants with unusually small or large inflorescences are less success-ul than those with medium-sized inflorescences (Jersáková andindlmann, 2004a, 1998). Since the flowers open gradually from

he bottom towards the top, and bumblebees visit inflorescencesrom the bottom upwards, the lower parts of inflorescences typ-cally set more fruits than the upper parts (Nilsson, 1980; Vogel,993, JJ unpubl. data). Pellegrino et al. (2005), however, found noffect of flower position on fruit set in Italy, and Kropf and Renner2005), who analysed the proportional pollination success of eachower position argued for the opposite pattern, with mid-positionowers having a higher fruit set rate than low-position flowers.nother factor that may affect fruit set is density of conspecificlants (cf. Maintenance of colour polymorphism). In a pollen-trackingxperiment Kropf and Renner (2008) found most pollen beingeposited to plants at the edge of higher density patches afteree flight distances from a few tens to hundreds meters, possibleue to the visual attractiveness of dense patches from a distance.

n yellow-dominated populations in Italy, the relative male andemale reproductive success was independent of total D. sam-ucina density, but positively correlated with the yellow morphrequency, again pointing to the visual attractiveness to bees ofense patches of yellow flowers (Pellegrino et al., 2005). Contraryo this, Internicola et al. (2006) in Southern France found that aggre-ation of yellow and purple D. sambucina with a blue rewardingpecies (Muscari neglectum) diminished the fruit set of the orchid.n this case, bumblebees probably learned to avoid the high-densityatches of the non-rewarding D. sambucina and instead to visit theear-by rewarding patch of blue flowers (Internicola et al., 2006).

Experimental defoliation (simulation of herbivory) of maturendividuals had no effect on capsule production but significantly

ecreased weight of fruits (Pellegrino and Musacchio, 2006). Sincehe dry weight of a capsule and the number of seeds it contains aretrongly positively correlated (Vallius, 2001), one can assume thatefoliated plants produced fewer seeds than control ones.

olution and Systematics 17 (2015) 318–329

Physiological and biochemical information

Physiological data

The density of stomata on the upper leaf surface is about1680 per cm2, on the lower leaf surface 5044 per cm2 (Ziegenspeck,1936).

Biochemical data

Infection of the tubers of D. sambucina (cited as O. latifolia)with a strain of Rhizoctonia repens from Orchis militaris resultedin the synthesis of the phytoalexin orchidinol (2,4-dimethoxy-7-hydroxy-9,10-dihydrophenanthrene) and p-hydroxybenzylalcohol(Nüesch, 1963; Gäumann et al., 1960). Quercetin 3-O-ˇ-d-glucoside(isoquercetin) has been isolated from air-dried flowers of D.sambucina (Tira, 1971). Further work by Pagani (1976) revealedthe presence of quercetin 3-O-ˇ-d-glucoside, quercetin 7-O-ˇ-d-glucoside and quercetin 3,7-di-O,O-ˇ-d-glucoside, and thephenylpropanoid esters caffeoyl-1-glucoside and p-coumaroyl-1-glucoside. The species contains cyanidin 3,5-diglucoside (cyanin)and the pigments orchicyanin I and II (Strack et al., 1989), thesethree compounds being also present in related species D. majalisand D. maculata (Arditti, 1992).

The floral fragrance on both morphs is a blend of at least 3mono- and 7 sesquiterpene hydrocarbons (dominated by limoneneand trans-caryophyllene, Nilsson, 1980). Similarly, Salzmann andSchiestl (2007) found the scent profile of D. romana mainly com-posed of monoterpenes making up at least 60% of all the floralcompounds, with the mean relative amounts of compounds notdiffering between morphs, with the exception of linalool (high inthe purple morph) and benzaldehyde (high in the yellow morph).

The absolute amounts of the macroelements N, P, K, Ca, and Mgin blooming plants of D. sambucina were reported by Mróz (1994).

Genetic data

Chromosome number

D. sambucina has 2n = 40 (exceptionally 2n = 42) chromosomes(Hagerup, 1938; Heusser, 1938; Del Prete et al., 1980; Gathoye andTyteca, 1989). The same number is reported from D. romana s.str.(Del Prete et al., 1980; Bianco et al., 1987; Alba et al., 2003), whileD. insularis is a triploid, with 2n = 60 (Scrugli, 1977; Bernardos et al.,2002, 2005) or 2n = 60 + 1B chromosomes (Bernardos et al., 2004),and D. cantabrica a tetraploid (2n = 80; Pedersen, 2006). The role ofpolyploidy in the evolution of this group of species has attractedmuch interest (Bullini et al., 2001; Pedersen, 2006; Nordström andHedrén, 2007; Pillon et al., 2007), it has been suggested that thereare two allopatric basal diploid species, D. sambucina (western)and D. romana (eastern and southern), with D. insularis likely anallotriploid, and D. cantabrica an allotetraploid (Pedersen, 2006).

Genetic variation

Modern genetic studies on D. sambucina and its close allies arescarce (Pedersen, 2006; Nordström and Hedrén, 2007; Pillon et al.,2006, 2007, older allozyme studies are cited therein). Allozymediversity suggests that D. romana s.str. is the least derived mem-ber of the D. sambucina complex and that local populations may besubdivided into demes determined by the distinct colour morphsand partial morph constancy of individual bumblebees (Pedersen,

2006). Using 19 allozyme loci in one Spanish and six Italian popula-tions of D. insularis, nine Italian populations of D. sambucina, and 11Italian populations of D. romana, Bullini et al. (2001) suggested thatD. insularis may be allotriploid and originate from D. sambucina and

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. romana. However, this species and the apparently allotetraploid

. cantabrica probably originated multiple times (Pedersen, 2006).

ybrids

Intergeneric hybrids involving D. sambucina have beenescribed with Gymnadenia conopsea (×Dactylodenia zollikoferiStoj.) Peitz) and Platanthera bifolia (×Dactylanthera fournieri (E.oyer) J.M.H. Shaw) (for original publication details and distri-ution maps see e-monocot.org). Souche (2004) also observedatural hybrids with Coeloglossum viride, Gymnadenia conopsea,rchis mascula, Orchis pallens, Platanthera bifolia, Pseudorchis albidand Anacamptis morio. The Collectif SFO-RA (2012) confirmed theeport of natural hybrids with O. pallens. Occurrence of hybridsith the genera Serapias (×Serapirhiza), Orchis (×Orchidactyla),

nd Pseudorchis (×Pseudorhiza) remains doubtful, while Souche2004) states that viable hybrids from experimental crossings withnacamptis coriophora and Orchis provincialis can be obtained.

Formally named within Dactylorhiza hybrids includes D. ver-euleniana (D. ×gabretana (A.Fuchs) Soó), D. fuchsii (D. ×influenza

Sennholz) Soó), D. incarnata (D. ×guillaumeae C. Bernard), D.alopissii (D. ×metsowonensis B. Baumann & H.Baumann), D. macu-ata (D. ×altobracensis (Coste & Soulié) Soó), D. majalis (D. ×ruppertiiM.Schulze) Borsos & Soó), D. romana (D. ×rombucina (Cif. & Gia-om.) Soó), and D. viridis (syn. Coeloglossum viride) (D. ×erdingeriA.Kern.) B.Bock). Collectif SFO-RA (2012) also observed intra-eneric hybrids with Dactylorhiza savogiensis in the French Alps,nd Souche (2004) observed intrageneric hybrids with Dactylorhizaphagnicola.

onservation

D. sambucina is treated by the IUCN as “least concern” (Appendix). As an orchid, D. sambucina is included in the Appendix II of theonvention on International Trade in Endangered Species of wild

auna and flora (CITES). Although not listed within the Annexesf the European Habitat Directive, D. sambucina is at least region-lly rare and a threatened species in many European countriesr regions (Appendix 7). National law also sometimes protectsll orchids (e.g. “Bundesartenschutz-Verordnung” in Germany ortrictly protected species in Poland). This all-inclusive protectionovers all stages in the life cycle.

A decline in the number of populations and flowering individ-als is especially well documented in Germany, where the mostecent national orchid flora has quantified the loss of populationsased on country-wide grid cells (i.e. quarters of the topographicalaps 1:25,000). By comparing the presence of the species in grid

ells before 1950 and 2004, Kretzschmar and Blatt (2005) showedhe loss of occurrences in 83.1% of the cells, placing D. sambucinamong orchids with the most severe population losses in Germany.owever, with fewer than 100 total cells, D. sambucina has longeen rare in Germany, perhaps because of a too humid or coldlimate (Kretzschmar and Blatt, 2005). In France, D. sambucina isegionally protected in the French regions of Alsace and Bourgogne.t has disappeared from the departments Aisne, Moselle, and Seine-t-Marne, which are under high urbanization pressure (Vogt-Schilbt al., 2015). D. sambucina has disappeared mainly from lowlands,uch as the Parisian basin and the East of France (Bournérias andrat, 2005).

There are a number of reasons for the decline of D. sambucinacf. Response to competition and management). Beside loss of habitatshrough land-use change, intensification or abandonment (Kropf,

995; Balzer, 2000; Arbeitskreis Heimische Orchideen, 2005), therere also orchid-specific hazards, such as picking and even digging-ut of flowering plants (Kropf, 1995, 2008). Naturally, increasingumbers of wild boar can also be a problem (see above), at least

olution and Systematics 17 (2015) 318–329 327

locally (Kropf, 2008), as is impropriate timing of cattle grazingduring spring and early summer (Sonne and Hauser, 2014; cf. Herbi-vores and pathogens). An attempted re-introduction on the Estonianisland Saaremaa, using plants from the Åland islands, was unsuc-cessful (Kuusk, 1994).

Acknowledgements

We would like to thank Andreas Braun for sampling and soilanalyses in our Austrian study region. We thank Eckehart Jäger andKarl Peter Buttler for their comments on an earlier version, andHenrik Pedersen for checking the section on taxonomic issues. Thisresearch was supported by the grants GAJU 04-145/2013/P (to ZI),No. 14-36098G of the GACR and No. LO1415 of the MSMT (to PKand IS), No. 173030 of the Ministry of Education, Science and Tech-nological Development of the Republic of Serbia (to VDj), and theHochschuljubiläumsstiftung der Stadt Wien (to MK).

Appendix A. Supplementary data

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.ppees.2015.04.002

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