Identification of pollinators of lesser twayblade Neottia cordata with DNA barcoding reveals strong links with pine forest‐related fauna

Abstract

Many European terrestrial orchids are in decline. To curb this negative trend and preserve remaining populations, more ecological knowledge is needed. Surprisingly little is known yet about the identity and efficiency of pollinators of lesser twayblade Neottia cordata , a small terrestrial orchid species associated with pine trees through joint mycorrhizae. We identified its small and inconspicuous pollinators with DNA barcoding and assessed its fruit set with the help of observations submitted to various nature platforms. We caught pollinators on Terschelling in the Netherlands during the flowering season of 2013 and 2014. Insects were identified with 28S and COI sequences obtained from both fresh and museum‐preserved material identified by specialists. Several pollinators were detected, belonging to either parasitoid wasps (Braconidae) and spider wasps (Pompilidae), active during sunny periods, or fungus gnats (Mycetophilidae and Sciaridae), active during overcast conditions. Combined pollinator efforts resulted in a continuous average fruit set above 70% in Europe over the past 135 years. The parasitoid wasps were identified as Bracon pineti and Blacus sp., which strongly depend on pine trees for their prey. The fungus gnats were identified as Austrosciara hyalipennis, Trichosia lengersdorfi, Allodia lugens and Phronia forcipata . All four species are known to deposit their eggs in the vicinity of fruiting bodies of cone caps Strobilurus stephanocystis , mushrooms growing on pine cones, as their larvae feed on the fungi. Priocnemis pertubator and Anoplius viaticus (Pompilidae) are also important pollinators. Results obtained show that lesser twayblade is even more intricately linked to pine forest ecosystems than previously thought. Management of growth sites of N. cordata should be tailored towards preserving pine tree forests with plots that are constantly being rejuvenated to generate young pine trees and prevent succession towards birch–oak forests. Such forests not only provide mycorrhizal fungi and layers of needle litter needed for germination and development of the seeds of this orchid but also pinewood‐decaying fungi that provide egg deposition sites and food for its pollinators.

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NORDIC JOURNAL OF
BOTANY
Nordic Journal of Botany
Page 1 of 11
Subject Editor: Magne Friberg
Editor-in-Chief: Sara Cousins
Accepted 14 October 2024
doi: 10.1111/njb.04396
00
1–11
Published 3 December 2024
2024: e04396
© 2024 Nordic Society Oikos. Published by John Wiley & Sons Ltd
Many European terrestrial orchids are in decline. To curb this negative trend and
preserve remaining populations, more ecological knowledge is needed. Surprisingly
little is known yet about the identity and eciency of pollinators of lesser twayblade
Neottia cordata, a small terrestrial orchid species associated with pine trees through
joint mycorrhizae. We identied its small and inconspicuous pollinators with DNA
barcoding and assessed its fruit set with the help of observations submitted to various
nature platforms. We caught pollinators on Terschelling in the Netherlands during
the owering season of 2013 and 2014. Insects were identied with 28S and COI
sequences obtained from both fresh and museum-preserved material identied by
specialists. Several pollinators were detected, belonging to either parasitoid wasps
(Braconidae) and spider wasps (Pompilidae), active during sunny periods, or fungus
gnats (Mycetophilidae and Sciaridae), active during overcast conditions. Combined
pollinator eorts resulted in a continuous average fruit set above 70% in Europe over
the past 135 years. e parasitoid wasps were identied as Bracon pineti and Blacus sp.,
which strongly depend on pine trees for their prey. e fungus gnats were identied as
Austrosciara hyalipennis, Trichosia lengersdor, Allodia lugens and Phronia forcipata. All
four species are known to deposit their eggs in the vicinity of fruiting bodies of cone
caps Strobilurus stephanocystis, mushrooms growing on pine cones, as their larvae feed
on the fungi. Priocnemis pertubator and Anoplius viaticus (Pompilidae) are also impor-
tant pollinators. Results obtained show that lesser twayblade is even more intricately
linked to pine forest ecosystems than previously thought. Management of growth sites
of N. cordata should be tailored towards preserving pine tree forests with plots that
are constantly being rejuvenated to generate young pine trees and prevent succession
towards birch–oak forests. Such forests not only provide mycorrhizal fungi and layers
of needle litter needed for germination and development of the seeds of this orchid
but also pinewood-decaying fungi that provide egg deposition sites and food for its
pollinators.
Keywords: Braconidae, fungus gnats, Ichneumonidae, Mycetophilidae, orchids,
parasitoid wasps, Pompilidae, Sciaridae
Identification of pollinators of lesser twayblade Neottia
cordata with DNA barcoding reveals strong links with pine
forest-related fauna
JeanClaessens ✉1, Ceesvan Achterberg 1, Emmade Haas1, MarijkeClaessens-Janssen2 and
BarbaraGravendeel 1
1Naturalis Biodiversity Center-Evolutionary Ecology, Leiden, the Netherlands
2Geulle, the Netherlands
Correspondence: Jean Claessens (jean.claessens@naturalis.nl)
Research article
11

Page 2 of 11
Introduction
With an estimated 28 000 species, the orchid family
(Orchidaceae) is diverse (Cribbetal. 2003, Tsaietal. 2017,
Fay 2018), and distributed throughout the world, except in
the polar and desert regions. In their evolution, orchids have
shown a rapid diversication, due to several factors: dierent
pollination strategies, aggregation of pollen in larger units,
and the production of dust-like seeds (Tremblayetal. 2005,
Breitkopfetal. 2015, Givnishet al. 2015). Other hypoth-
eses include the evolution of epiphytism and whole genome
duplication (Gravendeeletal. 2004, Moriyama and Koshiba-
Takeuchi 2018).
In an orchid’s life history, there are two major bottlenecks:
pollination and seed germination (Tremblay and Otero 2009).
Most orchids are dependent on pollinators for pollen transfer,
and on the access to mycorrhizal fungi for germination and
seed development (Jersákováet al. 2006, Micheneauetal.
2009, Rasmussenet al. 2015). Seed germination is limited
by the access to mycorrhizal fungi for germination and seed-
ling development. Orchids are renowned for the amount and
complexity of pollination strategies (Schiestletal. 1999, van
der Cingel 2001, Jersákováet al. 2006). Orchids can pro-
vide some kind of reward: nectar, pollen, resin, fragrances,
oil or wax (Dressler 1981); among European orchids, nectar
is the most common reward (Neiland and Wilcock 1998,
Johnsonetal. 2004, Jersákováetal. 2008). Approximately
30–40% of all European orchids are deceptive species,
relying primarily on food deception or sexual deception
(Schiestletal. 1999, Cozzolino and Widmer 2005, Schiestl
2005, Jersákováetal. 2006, Brzoskoetal. 2021).
e genetic material of orchids is aggregated in pollinia
(coherent masses of pollen grains that are transferred as a unit
during pollination), and the attraction of potential pollina-
tors and the precise placement of the pollinia on the insect’s
body is essential for successful fertilization. Precise adaptation
of the ower structure to the insect’s morphology ensures an
increased precision of pollen transfer, thereby inuencing
plant tness (Pottsetal. 2010). is close match between
ower and pollinator is a result of phenotypic selection
(Moréetal. 2012, Trunschkeetal. 2020. However, a very
strong adaptation to a specic pollinator or a class of pollina-
tors can also be a threat to the survival of the species, as the
disappearance of the insect could reduce seed- and/or fruit
set. Phenological mismatches between pollinators and orchids
have already been reported (Hutchingsetal. 2018). e early
spider orchid Ophrys sphegodes relies on male bees for pollina-
tion. e males emerge before the females and pollinate the
orchid as they search for a mate, but researchers have found
that climate change is causing the females to emerge before
the orchid's peak owering time, reducing the orchid's repro-
ductive success (Robbirtetal. 2011, Hutchingsetal. 2018).
However, not all orchid species have specialized pollination
systems (Tremblay 1992, Jersáková 2006). Orchids with a
wide pollinator spectrum can suer severe pollen losses due
to inecient pollen transfer by non-specic vectors but can
benet from a high visitation rate.
Despite their omnipresence, many orchids are highly
threatened (Fay 2018). In Europe, habitat loss is the main rea-
son for the decline of orchid populations (Kull and Hutchings
2006, Kolanowska and Jakubska-Busse 2020, Štípková and
Kindlmann 2021). One example of a threatened European
orchid species is lesser twayblade, Neottia cordata (Arbeitskreise
Heimische Orchideen 2005, Kotilíneketal. 2018, Tsiftsisetal.
2019). It has a circumpolar distribution, occurring in the
boreal-temperate zone and the foothills of the arctic zone in
Europe, Asia and large parts of North America. Its south-
ernmost boundary is formed by the mountainous regions in
the south (Pyrenees, Alps, Caucasus). It is widespread in the
northern parts of its range (Scotland, Scandinavian countries),
reaching as far as Greenland and Iceland (Hultén and Fries
1986, Tsiftsisetal. 2019). In the Netherlands, it is very rare
and now only grows on the Wadden Islands (NDFF 2021).
e rst mention of N. cordata on the Wadden Islands dates
back to 1949 (Weijer 1949), since then N. cordata spread to
all the Wadden Islands, except Texel. It is a Red List species,
protected by law (van der Meijdenetal. 2000).
In most European orchids, it takes four to six weeks after
pollination for the seed to mature. However, in N. cordata
fruit set occurs very quickly: within two weeks after pollina-
tion, the seed is ripe and the capsules open. Regularly we
found plants that had swollen capsules in the lower part,
whereas the uppermost owers were newly opened (Löjtnant
and Jacobsen 1977). Neottia cordata is often mistaken for
long-owering, because the perianth stays green after polli-
nation, whereas in most orchids the perianth dries out and
turns brown. Ziegenspeck (1936) supposes this is to support
the supply of nutrients. Pollination data for this orchid are
almost exclusively available from North American localities
(Ackerman and Mesler 1979). In North America, fungus
gnats (Sciaridae and Mycetophilidae) are the most impor-
tant and abundant pollinators; pollination eciency is high
(61–78% of all studied owers from dierent stations set
fruit (Ackerman and Mesler 1979). In Europe, the species
shows a strong decline, mainly due to changes in its biotope
or habitat loss (Arbeitskreise Heimische Orchideen 2005,
Kotilíneketal. 2018, Tsiftsisetal. 2019).
e aims of this study were to 1) identify the pollinator
spectrum of N. cordata in the Netherlands, and 2) investi-
gate if photos showing fruit set of N. cordata, obtained from
data uploaded to various publicly accessible nature observa-
tion platforms in Europe gathered by citizen scientists, could
be used for computing fruit set. By unravelling the relation-
ships between orchid, pollinators and biotope, we hope to
contribute to the conservation of this rare orchid species in
the Netherlands and beyond.
Material and methods
Study site
Within the Terschelling dune system there are some culti-
vated coniferous woodlands with Pinus nigra J.F.Arnold,
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Page 3 of 11
planted at the beginning of the 20th century to reduce the
eects of strong sand drifts on the island. In such pine forests,
we can distinguish several stages of succession. At rst, there
is no vegetation at all, the vegetation is dominated by terres-
trial growing lichens of the genera Cladonia and Cladina; this
type of forest is known as lichen forest or Cladonio–Pinetum
(Westho 1959, Zumkehr 2011, 2012). As the forest ages,
it enters a next phase called a moss forest or Leucobryo–
Pinetum, characterised by e.g. Leucobryum glaucum Hedwig,
Rhytidiadelphus loreus (Hedw.) Warnst. and Lepidozia reptans
Dumort. (van Toorenetal. 2002). Gradually phanerogams
appear, and after about 25 years the biotope is suitable for
orchids, including N. cordata and Goodyera repens R.Br.
(Westho 1959, Weeda etal. 1994). Neottia cordata pref-
erably grows in a thick layer of needle litter, providing the
needed moist environment; it is not found in a dry pine
forest (Vermeulen 1958). As the moss layer thickens, the
forest reaches its nal stage, the birch–oak forest, Betulo–
Quercetum roboris. Herbs and shrubs develop, the canopy
is less dense, the forest gets drier, the humidity decreases and
the growing conditions are no longer suitable for N. cordata
(Zumkehr 2011).
Study species
Neottia cordata is a boreal–montane species; in Europe it
occurs in wet heathlands, bogs, mires and in coniferous for-
ests. It prefers very acidic to acidic substrates, nutrient-poor
humus or peat (Harrap and Harrap 2005, Kotilínek etal.
2018). Neottia cordata reproduces by means of long roots
that do not penetrate deep into the substrate (Kotilíneketal.
2018). Neottia cordata is mainly associated with nonecto-
mycorrhizal Sebacinales Clade B (Těšitelová et al. 2015).
Less frequent are rhizoctonias from Ceratobasidiaceae and
Tulasnellaceae, ectomycorrhizal fungi from Russulaceae,
Atheliaceae (Tylospora), elephoraceae, and numerous
presumably endophytic ascomycetes and basidiomycetes
(Yagameetal. 2016, Schieboldetal. 2018).
Neottia cordata has two opposite, heart-shaped leaves; the
stem is hairy, green to reddish purple, carrying generally 6–12,
up to 20 yellowish-green to reddish-purple owers (Fig. 1A).
Sepals and petals form a loose hood. e lip splits and forms
two elongated lobes; in its centre is a median nectar-secreting
groove (Fig. 1B). At the lip base is another nectar-secreting
zone, similar to its sister species Neottia ovata Blu & Fingerh.
(Claessens and Kleynen 2011). e rostellum has sensitive
hairs at its apex, acting as a lever that enables the extrusion
of viscid uid when touched, thus gluing the pollinia to a
visiting insect. Neottia cordata has foetid-smelling nectar
(Brackley 1985), which is often an indication that ies ovi-
posit on the plant, but such behaviour has never been observed
(Ackerman and Mesler 1979, Hoy 2002). Observations of
pollinators of N. cordata in North America included fungus
gnats of the genera Mycetophila (Mycetophilidae), Sciara and
Corynoptera (Sciaridae), other occasional pollinators were
crane ies (Tipulidae) and parasitoid wasps (Braconidae and
Ichneumonidae) (Ackerman and Mesler 1979). In Europe,
fungus gnats and occasionally beetles have been observed
(Summerhayes 1968, Claessens and Kleynen 2011, 2016).
e nectar is freely accessible, and there is no adaptation to a
specic group of pollinators. Visiting insects can land in dif-
ferent positions on the ower and eventually move toward the
column, guided by the nectar trail on the lip. Darwin (1877)
described the pollination mechanism of Neottia as highly spe-
cialized, but with unspecialized pollinators, which was later
conrmed by (Nilsson 1981).
e species is self-compatible, usually not self-pollinating
(Kirchner 1922); the coherence of the tetrads in the pollinia
is much higher than in other Neottia species (Ackerman and
Williams 1980).
Study site
e study site was located on Terschelling in Hoornse bos,
53°24′19″N, 5°22′7″E. It belongs to the cushion moss-pine
forest community (Leucobryo–Pinetum) (Schaminée et al.
2010). e dominant tree species is Pinus nigra with little
undergrowth of Sorbus aucuparia, Prunus serotina Ehrh.,
Dryopteris dilatata (Hom.) A.Gray, Polypodium vulgare,
Betula pendula Roth., Ilex aquifolium, Lonicera periclyme-
num, Goodyera repens, Hypnum jutlandicum Holmen &
E.Warncke, Hypnum cupressiforme Hedw. and Lophocolea
bidentata Dumort. Neottia cordata is the most widespread
species with several thousands of individuals. e study site
is part of a Natura 2000 reserve (Natura 2000 2021) and
is specially protected because of the presence of this orchid
species. On Terschelling, 95% of the total occurrence of N.
cordata in the Netherlands is found, of which 40% occurs on
the study site.
Floral visitors
Observations of visitors were made on Terschelling in 2013
and 2014. In 2013 observations were made between 11:30
Figure 1. (A) Neottia cordata, owering plant. Hoorn (the
Netherlands), 4 May 2013, (B) Neottia cordata, ower. Hoorn (the
Netherlands), 7 May 2013. Photographs by Jean Claessens.
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Page 4 of 11
and 16:30 h for ve days and between 19:30 and 22:00 h
for two days. Several large plant clusters were checked for
the presence of insect visitors by patrolling along a transect;
visitors and pollinators were collected and preserved in alco-
hol. In 2014 we observed for 7 h, totalling 42 h of observa-
tion. Fresh insect material was collected on 5–10 May 2013
and on 24 April 2014 and afterwards identied using both
morphology and DNA barcoding. For morphological com-
parison, we used the specimens deposited in the entomology
collection of Naturalis Biodiversity Center.
DNA barcoding of pollinators
For extraction of total genomic DNA from the insects stud-
ied, the Dneasy Blood & Tissue Kit (Qiagen, Valencia,
California, USA) was used. Two modications in the proto-
col were made. First of all, instead of cutting the insect into
small parts previous to extraction, as indicated in the origi-
nal protocol, the entire insect was submerged in 180 µl ATL
buer + 20 µl Proteinase K, followed by 3 h of incubation at
57°C. e Diptera collected in 2013 were incubated over-
night at 57°C. is modication was done so that the speci-
mens would be available for resampling. e insects remained
intact and could therefore be added to the entomology col-
lection of Naturalis Biodiversity Center after drying. DNA
elution was done twice with 50 µl AE buer. Between each
pipetting step, the column was centrifuged for 1 min at 8000
rpm. Dierent DNA barcoding regions were chosen based
on earlier studies: 16S, 28S and COI (Kambhampatietal.
2000, Shietal. 2005, Zaldivar-Riverónetal. 2006). As both
fresh and museum-preserved material were analysed, we used
both long (600–660 bp) regions as well as minimalistic bar-
code regions (100–130 bp). Primers for the latter region were
designed during this study. Amplications were done in a Bio-
Rad S1000 ermal Cycler under the following conditions:
COI: denaturation for 3 min at 94° followed by 40 cycles
of 15 s at 94°, 30 s at 45° and 40 s at 72°, and a nal exten-
sion of 5 min at 72°. 16S: denaturation for 3 min at 94°
followed by 35 cycles of 15 s at 94°, 30 s at 48° and 1 min at
72°, and a nal extension of 7 min at 72°. 28S: denaturation
for 3 min at 94° followed by 45 cycles of 15 s at 94°, 30 s at
55° and 1 min at 72°, and a nal extension of 8 min at 72°.
MiniCOI: denaturation for 3 min at 94° followed by 40
cycles of 15 s at 94°, 30 s at 45° and 40 s at 72°, and a
nal extension of 5 min at 72°. Amplication was performed
in a 25 µl reaction volume consisting of 18.8 µl Ultrapure
mQ, 2.5 µl Qiagen PCR buer CL 10x concentration, 1 µl
Forward primer, 1 µl 10 pmol µl–1 reverse primer, 0.5 µl 2.5
mM dNTPs, 0.25 µl 5 U µl–1 Taq and 1 µl DNA template.
For a second COI amplication and COI mini bar-
codes the following 25 µl reaction volume was used: 17.8
µl Ultrapure mQ, 2.5 µl Qiagen PCR buer CL 10x con-
centration, 0.5 µl 25 mM MgCl2, 0.5 100 mM BSA, 1 µl
Forward primer, 1 µl 10 pmol µl–1 reverse primer, 0.5 µl 2.5
mM dNTP, 0.25 µl 5 U µl–1 Taq and 1 µl DNA template.
e resulting amplicons were Sanger sequenced on an ABI
3730 at Macrogen, Amsterdam. Electropherograms were
analyzed with Sequencher ver. 4.10.1 and DNA sequences
were blasted against NCBI Genbank and the BOLD database
(Supporting information). Identications were considered
correct only for similarity hits of 99% or higher.
Pollen export and import
Pollinia removal and pollen deposition (the male and female
tness component) of 33 plants with 330 owers was
recorded in Schluderbach (Italy) in June 2006 and Hoornse
bos, Terschelling (the Netherlands) in May 2013. Pollinia
removal or deposition was observed with a 10x-magnifying
lens and a powerful torch (Table 1, Supporting information).
Each ower was inspected and recorded as pollinated if pol-
len was deposited on the stigma. Pollinia removal and deposi-
tion did not always occur simultaneously, in some owers the
pollinia were still in the anther while the pollen was already
deposited on the stigma.
Fruit set
Fruiting success was calculated as the number of fruits
divided by the number of owers (Table 2, Supporting infor-
mation). Fruiting plants of N. cordata are easy to identify
because the fruits are spherical (Fig. 3). During seed ripen-
ing the ovary almost doubles in size. A spherical fruit is a
characteristic found in only two other European species,
Neottia ovata and Goodyera repens. Neottia ovata has much
larger leaves and is a much larger plant. In G. repens, the
fruits are angled upwards, close together and on one side of
the inorescence, whereas in N. cordata the fruits are hori-
zontal and arranged around the stem in a very loose ino-
rescence. Also, the fruits of G. repens do not appear until
much later in the season, when the plants of N. cordata have
already disappeared. is makes confusion with other species
in the eld very unlikely. e fruits are delicate and easily
compressed, so that they remain attached to the stem when
dried. Due to the large dierence in shape between fertilized
and unfertilized fruiting ovaries, it was possible to determine
fruit set even in herbarium specimens, with only intact and
undamaged specimens included in the count. Data on fruit
Table 1. Pollen export and import of Neottia cordata in Hoornse bos (the Netherlands) and Schluderbach (Italy).
Pollen export and import of N. cordata (n = 33)
A B C D C + D B + D
Pollinia present, not
pollinated
Pollinia present,
pollinated
Pollinia absent, not
pollinated
Pollinia absent,
pollinated
Total pollinia
removal
Total pollinia
receipt
Hoornse bos 69 64 18 146 164 210
Schluderbach 0 3 0 30 30 33
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Page 5 of 11
set were obtained with three dierent methods. Firstly, dur-
ing visits to dierent sites in Germany, France, Switzerland
and the Netherlands in 2004, 2012, 2014, 2015 and 2021,
the total number of owers and swollen, pollinated fruits
was counted (Table 2). e second method was the use of
herbarium specimens. To assess whether a swollen ovary was
equivalent to pollination, the inorescence of one dried her-
barium specimen of N. cordata (deposited at the herbarium
of Naturalis Biodiversity Center) was boiled up and two ow-
ers were examined. In both cases, remains of pollinia could
be observed on the stigma. We therefore concluded that a
swollen ovary was equivalent to pollination. is allowed us
to use available herbarium vouchers (94 vouchers of plants
collected in the period 1887–1978 throughout Europe and
deposited at Naturalis Biodiversity Center) to calculate fruit
set. An herbarium search of European vouchers from 1887
until 1930 (Herbarium Renz) yielded an additional 51 usable
vouchers. In a third method, we used photos of 141 fruit-
ing N. cordata plants from the Wadden Islands Terschelling,
Schiermonnikoog and Vlieland, made in the period 2006–
2022 and posted on the publicly available internet reposi-
tories Waarneming.nl (2003), Observation.org (2003) and
GBIF (2003). ese websites are widely used for uploading
nature observations. To investigate if these photos could be
used for counting fruit set, we examined a population of
12 fruiting N. cordata in the Bavarian Alps, near Garmisch-
Partenkirchen (Germany). We numbered each plant, noted
the fruit set of each plant and also took photographs of all
plants. Afterwards, we counted the fruit set of the numbered
and photographed plants and compared the results with our
counts in situ. Our counts in the eld matched the counts
from the photos. Having established that photographs of
pollinated orchids could be used to determine pollinator
eciency, we examined 1416 photographs in the databases
of Waarneming.nl, Observation.org and GBIF, which are all
Table 2. Fruit set of Neottia cordata in the Netherlands and in Europe.
Fruit set Neottia cordata
Location Date Plants Flowers Pollinated Not pollinated
Garmisch-Partenkirchen (D) August 2004 47 382 347 (90.8%) 35 (9.2%)
Scuol (CH) July 2012 22 167 143 (85.6%) 24 (14.4%)
Terschelling (NL) May 2014 40 435 414 (95.2%) 21 (4.8%)
Chichiliane (F) June 2015 21 191 162 (84.8%) 29 (15.2%)
Gresse-en-Vercors (F) June 2015 24 208 153 (73.6%) 55 (26.4%)
Garmisch-Partenkirchen (D) July 2021 12 100 91 (91%) 9 (9%)
Figure3. Neottia cordata, fruiting plant, inorescence with swollen
capsules. Photograph by Jean Claessens.
Figure2. (A) Mycetophilidae pollinating Neottia cordata. Pramaran
(CH), 16 June 2012, (B) Braconidae pollinating Neottia cordata.
Hoorn (NL), 8 May 2013. A bunch of pollinia, collected by the
insect, sticks to the stigma and prevents the parasitoid wasp from
getting away. Photographs by Jean Claessens.
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Page 6 of 11
checked for correct species identications by validators. A
search of the photos in Waarneming.nl retrieved 141 utiliz-
able records, searches of GBIF and Observation.org revealed
another 158 and 90 records, respectively. Only photos show-
ing the entire inorescence with sucient detail were used.
In total, 389 photos (27.5%) out of 1416 were used for
counting fruit set (Supporting information)
Results
Visitors and pollinators
All visiting insects, with one exception, belonged to the order
Diptera or Hymenoptera, spread over seven families. Insects
of ve families were pollinators; in most cases we observed
one or several pollinators per family, but in three families
we observed larger numbers of pollinators (Table 3). Insects
were dened as pollinator if they had pollinaria attached
to their body and visited several owers, leaving and/or
depositing pollinaria. Species identications were obtained
by matching DNA sequences with reference data in NCBI
GenBank and BOLD. Species from eight families were pol-
linators; Sciaridae and Mycetophilidae were most abun-
dant, followed by Braconidae, Tipulidae, Anthomyiidae,
Pompilidae, and one observation of Bolitophilidae and
Ichneumonidae (Table 3, Supporting information). Sciaridae
and Mycetophilidae (Fig. 2A) alighted on the inorescence
and actively searched for nectar, visiting and revisiting most
owers, thereby removing and depositing pollinia. ey were
active during overcast conditions and showed an alternation
between inspecting behaviour and immobile periods, which
lasted from 25 s to two minutes.
Braconidae (Fig. 2B) were found predominantly when it
was not too windy. ey were alternating between moving on
the plant for a short time, then checking the ower and licking
the lip. en they stayed immobile for one to 18 minutes, after
which they repeated the movement and inspection behaviour.
Of the individuals observed, 94% (79 out of 84 individuals)
carried and deposited pollinia. In total, they stayed on one
plant for a long time, up to almost 1 h. In some cases, we
observed that an insect was glued to the stigmatic surface.
e spider wasp Priocnemis perturbator Harris was an
important pollinator, constantly patrolling, mostly active in
spots that were in full sun. After landing on a plant, it crawled
up and down the plant several times, inspected several ow-
ers and then ew to the next plant. Meanwhile, it deposited
already attached pollinia on the stigma and got new pollinia
glued to its clypeus. It was also observed in windy conditions,
but only if the plant was in direct sunlight.
Ants (Formica fusca and Myrmica rubra) inspected the
owers; in one case an ant removed pollinia but was able to
rub them o. Spiders (Metellina sp.) were regularly seen wait-
ing on the ower spike or in their web for prey. Some carried
pollinia, but they are not considered pollinators because we
did not observe them depositing any pollinia on a stigma.
Table 3. Pollinators and visitors of Neottia cordata recorded in this study.
Pollinators and visitors of Neottia cordata
Order Family Species Sex
No. of
individuals
Pollinator/
visitor Locality Date
Hymenoptera Braconidae ?unknown 2 pollinator Pramaran (CH) 18 June 2012
Hymenoptera Braconidae ?male abundant pollinator Terschelling (NL) 9 May 2013
Hymenoptera Braconidae Blacus sp. unknown 1 pollinator Terschelling (NL) 24 Apr. 2014
Hymenoptera Braconidae Bracon pineti Thomson unknown 1 pollinator Terschelling (NL) 8 May 2013
Hymenoptera Formicidae Formica fusca L. unknown 3 visitor Terschelling (NL) 4 May 2013
Hymenoptera Formicidae Myrmica rubra L. unknown 8 visitor Terschelling (NL) 5 May 2013
Hymenoptera Pompilidae Anoplius viaticus L. unknown 1 pollinator Terschelling (NL) 24 Apr. 2014
Hymenoptera Pompilidae Priocnemis perturbator
Harris
female 26 pollinator Terschelling (NL) 5 June 2013,
24 Apr. 2014
Diptera Bolitophilidae Bolithophila (Cliopisa)
aperta Lundström
male 2 pollinator Pramaran (CH) 15 June 2012
Diptera Empididae ?unknown 1 visitor Pramaran (CH) 15 June 2012
Diptera Mycetophilidae ?unknown 9 pollinator Pramaran (CH) 16 June 2012,
26 June 2014
Diptera Mycetophilidae Mycetophila trinotata
Staeger
female 1 pollinator Pramaran (CH) 15 June 2012
Diptera Mycetophilidae Phronia sp. female 1 pollinator Pramaran (CH) 15 June 2012
Diptera Lonchopteridae Lonchoptera lutea Panzer unknown 1 visitor Terschelling (NL) 24 Apr. 2014
Diptera Mycetophilidae Allodia lugens
Wiedemann
unknown various pollinator Terschelling (NL) 24 Apr. 2014
Diptera Mycetophilidae Phronia forcipata
Winnertz
male/female 4 pollinator Terschelling (NL) 24 Apr. 2014
Diptera Sciaridae Austrosciara hyalipennis
Meigen
male 1 pollinator Terschelling (NL) 9 May 2013
Diptera Sciaridae Trichosia lengersdorfi
Heller, Köhler & Menzel
male 2 pollinator Terschelling (NL) 24 Apr. 2014
Araneae Tetragnathidae Metellina sp. unknown 8 visitor Terschelling (NL) 7 May 2013
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Page 7 of 11
Pollen export/import and fruit set
In Hoornse bos (the Netherlands) (n = 28) pollen export was
63.2% and pollen import 68.4%; in Schluderbach (Italy)
(n = 5) pollen export was 90.9% and pollen import 100%.
In Hoornse bos, in 20.8% of all owers pollen was already
deposited onto the stigma while the pollinia were still in the
anther (Table 1, Supporting information). No autogamous
pollination was observed.
e mean fruit set in European populations (without the
Netherlands) was 83.8 ± 15.3%. e mean fruit set of the
Wadden Islands was 73.5 ± 28.1% (Table 2, Supporting
information).
Discussion
Pollinators of lesser twayblade in the Netherlands are
all part of a pine tree community
By applying a combination of DNA barcoding and matching
specimens with reference collections in Naturalis Biodiversity
Center, the pollinators of N. cordata collected in Hoornse
bos, Terschelling could all be identied to species level. is
adds more details to previous ndings by Ackerman and
Mesler (1979) for North America, who also found that the
pollinators were parasitoid wasps and fungus gnats. Neottia
cordata is not specically adapted to pollination by one par-
ticular insect group. e easily accessible nectar, secreted in
minute quantities on the lip and the lip base is accessible
to a wide range of insects with small mouthparts and does
not require adaptation to a specic body shape. e oral
architecture facilitates pollination by fungus gnats. e sis-
ter species, Neottia ovata Blu & Fingerh. is also pollinated
by a wide range of insects from dierent families (Claessens
and Kleynen 2011, Kotilíneketal. 2015). e small amount
of nectar produced may serve to lter out certain potential
pollinators: insects with high nectar requirements are more
likely to avoid the plant because their needs are not met. is
makes the nectar supply especially suitable for insects for
whom a small amount of nectar is enough to meet their nec-
tar needs. ere is a strong relationship between fungus gnats
and plant habitat: fungus gnat pollination is mainly found in
habitats where other pollinators are absent (Mochizuki and
Kawakita 2018). In continuously moist environments fungus
gnats are present throughout the year and can act as reliable
pollinators, while other pollinators such as bees are rare in
these conditions. In fact, in Hoornse Bos on Terschelling, in
addition to fungus gnats, we only observed parasitoid wasps,
ants and spiders during our eldwork.
e foul-smelling nectar may be an adaptation to sap-
romyophily, but ovipositing behaviour was never observed
by Ackerman and Mesler (1979) and us. Lemoine (2018)
described the oral visiting behaviour of Mycetophilidae,
observed in Norway. e insects started at the top of the
inorescence, descended upside down and probed the nectar
of all owers, from time to time remaining motionless. is is
in line with our observations on Terschelling, where only nec-
tar-feeding behaviour was observed. Nectar and scent seem
to act as attractants for insect visitors (Ackerman and Mesler
1979, Claessens and Kleynen 2011, 2016). Irrespective of
the position in which the insects land, they eventually turn
towards the stigma, guided by the nectar trail on the lip and
attracted by the second nectar secretion zone at the lip base.
We inspected 33 plants with 243 owers, but never found
any signs of autogamous pollination (Table 1), nor did we
nd any mention of autogamy in literature. Neottia cordata
apparently relies on insect vectors for pollination.
e main fungus gnat pollinators found on Terschelling
belong to Mycetophilidae and Sciaridae. Trichosia lengersdor
Heller, Köhler & Menzel, Phronia forcipate Winnertz, Allodia
lugens Wiedemann and Austrosciara hyalipennis Meigen are
all fungus gnats. eir larvae feed on fungi, fungal mycelia
in rotten wood and decaying plant material (Mochizuki and
Kawakita 2018). ey are generally considered to be inef-
cient pollen vectors due to their small body size and poor
ight ability (Proctoret al. 1996). However, their ubiquity
compensates for this (Mesler et al. 1980, Mochizuki and
Kawakita 2018). Sciaridae have a worldwide distribution
and can survive even in extreme habitats such as subantarctic
islands or high altitudes. In Europe, more than 600 species
are known (Mohrig 2003).
Next to fungus gnats, fruiting bodies of the fungus
Strobilurus stephanocystis (Kühner & Romagn. ex Hora)
Singer were found by us in large numbers on Terschelling
at the same locality as the orchids during their main ow-
ering season. is fungus grows on decaying cones of pine
and spruce trees and has been found on the cones of Pinus
nigra. e larvae of Mycetophilidae and Sciaridae, regular
pollinators of N. cordata, primarily feed on fungi and decay-
ing organic matter (Mead and Fasulo 2001, Jakovlev 2011,
Põldmaaetal. 2015), providing an additional link between
this orchid and pine forest ecosystems.
Parasitoid wasps observed were Bracon pineti omson,
Blacus sp. and unidentied Braconidae. ey are known
for laying their eggs in caterpillars of microlepidoptera or
Coleoptera larvae, living in decaying wood, pine cones or
under the bark of the trees (van Achterberg and Altenhofer
1997) or on aphids feeding on pine needles (Žikić et al.
2012). ey use the nectar of N. cordata as a food source,
because no other owering plants are present at the time of
the orchid’s owering.
In addition to parasitoid wasps, Priocnemis perturbator
Harris and Anoplius viaticus, both spider wasps (Pompilidae),
were very active in sunny conditions, searching for spider prey.
Spiders were observed using the owering N. cordata plants
to capture prey by spinning webs or ambushing on the plant.
Priocnemis perturbator and Anoplius viaticus are species of
sandy soils and open forests (Peetersetal. 2004). Above all P.
perturbator was an important pollinator, which was observed
in various weather conditions. Its fast-searching behaviour
allowed it to visit and pollinate many plants in a short time.
Neottia cordata is a relatively recently arrived species in o-
ristic observations of the Netherlands, having been recorded
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Page 8 of 11
from 1949 onwards on Ameland, one of the Wadden islands
(Westho 1959). In Hoornse bos it was found in 1955,
about 25 years after the planting of non-native Pinus nigra.
By that time the mycorrhiza associated with Pinus were well
established in the soil, enabling the germination of seeds of
N. cordata. Orchid seeds lack a food reserve in the form of
endosperm and rely on a mycorrhizal relationship with a fun-
gus for development (Lietal. 2021). When the Pinus nigra
forest enters the Leucobryo–Pinetum phase, it is suitable for
orchids. e pines form a dense canopy that provides su-
cient shade for the drought-sensitive orchid and maintains a
suciently high moisture level. Another advantage of the rel-
atively dense canopy is that few other plants can develop. As
more herbs and shrubs appear, the forest enters the Betulo–
Quercetum roboris phase and conditions are no longer suit-
able for the non-competitive orchid (Zumkehr 2011, 2012).
ose familiar with N. cordata in the Alps will be surprised
to nd the orchid in such a seemingly dry habitat. However,
humidity is high due to the proximity of the North Sea. e
groundwater level is high enough to provide the moisture
conditions required by the orchid.
e fungus gnats are dependent on the fungi associated
with P. nigra, as are Braconidae and Pompilidae for their prey
living on or around the pine trees or associated fungi. e
existence of such an interdependent community stresses the
importance of conserving not just a single species, but pro-
tecting all members of the community, all of which depend
on the continued existence of a Pinus nigra habitat.
Continuous high fruit set of lesser twayblade due to
its dual pollination strategy
In orchids, fruit set is generally more pollinator than resource-
limited (Nilsson 1992). High fruit and seed set result in high
recruitment rates (Jacquemyn and Brys 2010) and is there-
fore important for the ability of a population to survive. In
the Netherlands, N. ovata shows a continuous high fruit set
(Fig. 4). Apparently, in this interdependent system where
few other owering plants grow, unceasing visits from insect
visitors are guaranteed. Although fungus gnats are considered
inecient pollen vectors, their numbers and their attach-
ment to this particular biotope, which provides food and
prey, ensure a continuous high pollination rate. Nectar is
produced in minute quantities, forcing the insects to return
again and again to satisfy their energy needs. e fungi that
grow on decaying P. nigra cones are important for oviposition
and food for larvae. Pollination of N. cordata involves a dual
strategy: on the one hand, the orchid is pollinated by insects
attracted by smell, colour and nectar present. On the other
hand, additional pollination takes place because insects in the
biotope of N. cordata are looking for prey, and in the absence
of other nectar sources rely on the orchid to meet their nectar
needs. It seems that this dual strategy ensures a continuous
high fruit set.
Conclusions
We discovered a mutual dependence of both Neottia cordata
and its pollinators on a specic biotope: pine forests at the
Leucobryo–Pinetum stage of development. e pine forests of
the Dutch Wadden Islands were all planted at the same time
and all trees are therefore equally old. If natural succession
is allowed to continue, these forests will become more open
due to the death of mature pine trees and gradual transition
towards the Betulo–Quercetum roboris stage with birch and
oak trees. As a result, the needle litter and pine cones on the
forest oor will disappear, and cover of the underlying herb
layer will increase, ultimately causing the disappearance of
these orchids and their pollinators. We therefore recommend
to apply a cyclic management, where plots of pine forest are
constantly being rejuvenated (Zumkehr 2011, 2012). e
result of such a cyclic management is that there will always be
pine forest plots that are in a developmental phase favourable
for these orchids to germinate, develop into seedlings and ulti-
mately plants with owers and fruits that release new seeds.
Figure4. Fruiting percentage of Neottia cordata in the Netherlands from 1949 onwards and in Europe from 1887 onwards.
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Page 9 of 11
Acknowledgements – We would like to thank Bertie Joan van Heuven,
Mark Smeets and Frank Stokvis for their help in the laboratory.
Piet Zumkehr gave us permission to use his reports, for which we
are grateful. Jan Bunnik (Staatsbosbeheer) allowed us to carry out
eldwork on Terschelling and Frederique Bakker provided access to
the entomology collection of Naturalis Biodiversity Center.
Funding – e author(s) received no nancial support for the
research and authorship of this article.
Author contributions
Jean Claessens: Conceptualization (supporting); Data
curation (lead); Formal analysis (lead); Investigation
(lead); Methodology (lead); Project administration (lead);
Visualization (lead); Writing – original draft (lead); Writing –
review and editing (lead). Cees van Achterberg: Investigation
(supporting); Resources (supporting). Emma de Haas:
Investigation (supporting); Resources (supporting). Marijke
Claessens-Janssen: Investigation (supporting); Writing
– original draft (supporting); Writing – review and edit-
ing (supporting). Barbara Gravendeel: Conceptualization
(lead); Formal analysis (supporting); Investigation (support-
ing); Methodology (supporting); Supervision (lead); Writing
– original draft (supporting); Writing – review and editing
(supporting);
Data availability statement
Data are available from the Dryad Digital Repository: https://
doi.org/10.5061/dryad.rxwdbrvk7 (Claessensetal. 2024).
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