NORDIC JOURNAL OF
BOTANY
Research
Pollination of Habenaria tridactylites on the Canary Islands
Jean Claessens, Juan José Bacallado, Diego Bogarin, Liliane Dedroog, Reinout Heijungs, Rob Langelaan,
Erik J. van Nieukerken, Kees van den Berg and Barbara Gravendeel
J. Claessens (https://orcid.org/0000-0002-9174-3592) ✉ (jean.claessens@naturalis.nl), R. Langelaan, E. J. van Nieukerken, K. van den Berg, B. Gravendeel
and D. Bogarin, Naturalis Biodiversity Center-Endless Forms, Darwinweg 2, NL-2300 Leiden 2300 RA, the Netherlands. – J. J. Bacallado, Puerto de la
Cruz, Spain. – L. Dedroog, Hasselt, Belgium. – R. Heijungs, Amsterdam, the Netherlands.
Nordic Journal of Botany
2019: e02401
doi: 10.1111/njb.02401
Subject Editor: Pietro Maruyama
Editor-in-Chief: Torbjörn Tyler
Accepted 2 July 2019
We investigated the pollination of Habenaria tridactylites, an endemic orchid of the
Canary Islands. The entirely green, widely open flowers have a long spur containing
nectar. We carried out fieldwork, a molecular clock analysis, herbarium surveys, identified pollinators by both morphology and DNA barcoding, and measured the length
of floral spurs and insect tongues using a combination of traditional and innovative
micro-CT scanning methods to 1) determine the pollinator of this orchid and 2)
investigate correlations between local mean spur length and age, altitude and longitude
of the island. Habenaria tridactylites was found to be pollinated on Tenerife by both
small and intermediate sized moth species with variable tongue lengths and mostly
belonging to Geometridae and to a lesser extent Crambidae, Erebidae, Noctuidae and
Tortricidae. Of the sixteen moth species identified, nine are endemic to the Canary
Islands or Macaronesia. The different local populations of H. tridactylites on the islands
of Gran Canaria, El Hierro, La Gomera, La Palma and Tenerife with different ages and
distances from mainland Africa, did not show a significant correlation of mean spur
length and altitude, but did show a significant and positive linear correlation with longitude and the geological age of the island. The latter is congruent with the evolutionary arms race theory first proposed by Darwin, suggesting that flowers gradually evolve
longer spurs and pollinators longer tongues.
Keywords: endemics, free spur space (FSS), Lepidoptera, orchids, spur, Tenerife
Introduction
Orchids display a wide variety of pollination mechanisms (Darwin 1877, Camus
1929, Godfery and Godfery 1933, Kullenberg 1961, Nilsson 1981a). In allogamous
orchids, the plants rely on animals for the transportation and deposition of pollen.
About four-fifth of all orchid species are pollinated by animals, mostly insects (Van
der Pijl and Dodson 1966, Grimaldi 1999), although there are various transitions
between complete allogamy and some degree of autogamy (Tałałaj and Brzosko 2008,
Claessens and Kleynen 2011, 2012, Jacquemyn et al. 2014). Pollinators may be
attracted by various rewards like pollen, nectar, oil or food-hairs (Kull et al. 2009),
but in European orchids, nectar is the main reward. Nectar is a key component in the
––––––––––––––––––––––––––––––––––––––––
www.nordicjbotany.org
© 2019 Naturalis Biodiversity Center. Nordic Journal of Botany published by John Wiley & Sons Ltd
on behalf of Nordic Society Oikos
This is an open access article under the terms of the Creative Commons
Attribution License, which permits use, distribution and reproduction in any
medium, provided the original work is properly cited.
1
relationship between plants and pollinators (Lenaerts et al.
2014), and in orchid flowers it can be secreted on the inflorescences (Subedi et al. 2011), sepals (Subedi et al. 2011,
Karremans et al. 2015), lip, lip base or in an elongation of the
lip, or the spur (Kocyan et al. 2008). The place where nectar
is presented greatly influences the visitor spectrum: an orchid
with easily accessible and copious nectar like Neottia ovata
(L.) Bluff & Fingerh. attracts a wide variety of pollinators
(Claessens and Kleynen 2011, Nilsson 1981b). In contrast,
a spur is an effective means of selecting a certain guild of
pollinators only. It also acts as a means of enticing the insect
to enter the stigmatic cavity, enhancing the chances that it
touches the forward projecting viscidium.
Nine European orchid genera (Epipogium J.G.Gmel.
ex Borkh., Gymnadenia R.Br., Gennaria Parl., Habenaria
Willd., Herminium Guett., Limodorum Böhm., Neottianthe
Schltr. and Pseudorchis Ség., Platanthera L.C.Rich.) from the
31 genera in total produce nectar in a spur, whereas three
genera (Anacamptis Rich., Dactylorhiza Neck. ex Nevski
and Neotinea Rchb.f.) have only a single species presenting nectar (A. coriophora (L.) R.M.Bateman, D. viridis (L.)
R.M.Bateman and N. maculata (Desf.) Stearn). Four of the
nectar presenting genera are pollinated by Hymenoptera,
four by Lepidoptera and one by Coleoptera (Claessens and
Kleynen 2011, 2016). The Hymenopteran pollinated species have a constant spur length, whereas in the Lepidopteran
pollinated species spur length can differ considerably.
Co-evolution between spur length of orchids and the length
of the proboscides of Lepidopteran pollinators is the driving
force in the evolution of Lepidopteran pollinated orchid species (Nilsson 1988, Bateman and Sexton 2008, Boberg and
Ågren 2009, Sletvold and Ågren 2010).
The pantropically distributed genus Habenaria
(Habenariinae) encompasses about 848 species (Govaerts et al.
2011, Pedron et al. 2012) and has its main centres of diversity
in Africa and Meso-America. Most of the species are terrestrial orchids that preferably grow in damp or wet habitats,
from low elevations to high montane areas. The main diagnostic features are the often bifid petals, the deeply divided
lip and the convex stigma, which can be entire or two-lobed
and which often has long, stalked stigmatic lobes (Senghas
1992, Dressler 1993, Pridgeon et al. 2001). Most Habenaria
species have greenish or pale flowers with nectar secreted in a
spur and many species have naked viscidia and long caudicles
(Szlachetko and Rutkowski 2000). Scent emission is crepuscular or nocturnal; two characteristics of moth-pollinated
flowers. Indeed, most studies refer to moths as their pollinators (Singer 2001, Singer et al. 2007, Pedron et al. 2012,
Suetsugu and Tanaka 2014, Ikeuchi et al. 2015, Xiong et al.
2015), followed by butterflies (Moreira et al. 1996) or crane
flies (Singer 2001). Pollinaria are deposited on the surface
of the eye or on the (base of the) proboscis of the pollinator.
Tao et al. (2018) demonstrate the importance of the distance
between the viscidia for the place of pollinaria attachment.
Habenaria tridactylites Lindl. is the only representative
of Habenaria in Europe and it is endemic to the Canary
Islands. The species has two long stigmatic lobes named
2
the stigmaphores (Claessens and Kleynen 2011). The
Canary Islands (Fig. 1) are situated between 27°37′ and
29°25′N and 13°20′ and 18°10′W; its easternmost island
Fuerteventura lies approximately 110 km from the African
mainland, whereas El Hierro, the westernmost island, lies at
a distance of 474 km from the mainland. The Canaries are
famous for their high diversity of plants, with an exceptionally high number of endemics. They are considered a hotspot
for plant and animal diversity (Reyes-Betancort et al. 2008)
and accommodate 2066–2091 taxa of vascular plants (species
and subspecies), of which 536–539 species are endemic, that
is 25.6% of the total botanical diversity (Arechavaleta et al.
2010, Aedo et al. 2013). All islands are of volcanic origin;
the oldest extant island, Fuerteventura, was formed about
20 million years ago (Mya). The other islands were formed
subsequently between 16 and 1.1 Mya (Del-Arco et al.
2006, Steinbauer and Beierkuhnlein 2010, Carracedo and
Perez-Torrado 2013) (Fig. 1).
The Canary Islands have never been connected to the
African continent. The oldest part of Tenerife is the Roque
del Conde volcano, formed between 11.9 and 8.9 Mya. The
Teno volcano developed between 6 and 5 Mya, and finally,
the Anaga volcano developed between 5 and 4 Mya (Fig. 2).
The three shield volcanoes were connected by younger volcanism from the post-erosional felsic Las Cañadas volcano,
constructed from about 3.5 Mya (Guillou et al. 2004,
Carracedo and Perez-Torrado 2013). The three oldest parts
of the island (Roque del Conde, Anaga and Teno) are termed
‘Tenerife old’ in this article. The rest of the island is termed
‘Tenerife young’.
About 730 species of Lepidoptera are known from the
Canary Islands (Karsholt and van Nieukerken 2017) of
which ca 235 species or subspecies are endemic to the islands,
ca 32% (Báez 2010). Noctuidae form the largest family, with
119 species, followed by Geometridae with 75 species.
We hypothesized that the spur length of H. tridactylites
would be different on islands with different geological ages
and that plants on geologically older islands would have longer spurs. We based our hypothesis on the evolutionary arms
race theory first proposed by Darwin (1877). According to
this theory, proboscis length of pollinators and floral spur
length are reciprocally influenced by positive feedback, leading to a constantly growing elongation of both. This feedback
loop is caused by the match between spurs and mean tongue
lengths of local pollinator communities that forage on nectar
produced in the bottom of the spurs. Too long spurs cause
pollinators to lose interest in the orchids as they cannot reach
the nectar anymore, too short spurs cause the pollinia to
remain in the anther during floral visits. Both changes would
result in a loss of reproductive success of the orchids, hence
the hypothesized growing spur elongation over time. To collect data to either support or reject this hypothesis, we investigated 1) by which insect species this orchid is pollinated
on Tenerife and 2) whether there is a correlation between
local mean spur length, age of the island, altitude and longitude on Gran Canaria, El Hierro, La Gomera, La Palma and
Tenerife.
Figure 1. The Canary Islands with their respective geological age. Illustration by Erik-Jan Bosch, modified from Carracedo and
Perez-Torrado 2013.
Material and methods
Study sites and fieldwork periods
Habenaria tridactylites is the earliest-flowering orchid of
the Canary Islands and its flowering period starts in midNovember and can extend to mid-January, depending on
the seasonal shifts. We visited Tenerife in 2013, 2014 and
2016 during different periods of the year. The study sites
were situated near El Tanque (site 1–3) and Icod de los Vinos
(site 4–5) in the north and Los Carrizales (site 6) in the
north-western part of the island (Fig. 3).
Del-Arco et al. (2006) distinguish a number of bioclimatic
belts on Tenerife. Study site 1–5 were all situated in the north
between 600 and 800 m altitude and belonged to the dry to
subhumid pluviseasonal thermomediterranean bioclimatic
zone under the influence of trade-wind clouds. Vegetation
cover was sparse, especially in sites 1, 2 and 3 (El Tanque)
Figure 2. Different parts of Tenerife. The oldest parts Anaga, Teno and Roque del Conde are referred to as Tenerife old, the rest is referred
to as Tenerife young. Illustration by Erik-Jan Bosch, modified from Dóniz-Páez et al. 2012.
3
Figure 3. Localities on Canary Islands of which spur lengths of H. tridactylites were measured indicated in blue. In addition, pollination was
studied in the field in the sites indicated in red. Illustration by Erik-Jan Bosch.
where the orchids grow in a former lava flow resulting from
the eruption of the Montaña Negra in 1706 (Solana and
Aparicio 1999). Sites 4 and 5 (Icod de los Vinos) were open
pine forest with little undergrowth. Accompanying species in
all sites were Davallia canariensis (L.) Sm., Pinus canariensis
C.Sm. ex DC., Ulex europaeus L. and Erica arborea L. The
exotic invasive Centranthus ruber (L.) DC. was present in
(close vicinity of ) all five sites. Study site 6 was situated in
the northwest, at 670 m altitude and belonged to the lower
to upper semiarid xeric thermomediterranean bioclimatic
zone, without the influence of trade-wind clouds. The vegetation lacked trees but was dominated by Euphorbia atropurpurea Brouss. Other accompanying species were e.g. various
Aeonium Webb & Berthel. species, Greenovia aurea (C.Sm. ex
Hornem.) Webb & Berthel, Kleinia neriifolia Haw., Sonchus
L. sp., Agyranthemum Webb ex Sch.Bip. sp., Monanthes Haw.
sp., Geranium molle L. and Opuntia ficus-indica (L.) Mill.;
Centranthus ruber was absent from this site.
Habenaria tridactylites is restricted to the northern part of
Tenerife, in the zone influenced by the trade winds, providing enough moisture. On the other Canary Islands, the species grows in similar conditions, a sufficient humidity level is
a critical condition for the survival of the species. It preferably grows in light shade, although it can also be found in
full sun; most localities are exposed to the north. It grows in
crevices where some humus has accumulated, on rocky slopes
or on moist crags within Laurisilva forests and also within
scrublands in less humid environments. It can regularly be
found in secondary biotopes like edges of roads or old stone
walls surrounding fields, especially in the region of Icod de
los Vinos. The plant prefers slightly acid soils. In general, it is
the most common orchid of Tenerife and the Canary Islands
and can be locally abundant.
In addition to fieldwork, we also surveyed herbarium
vouchers of H. tridactylites collected from El Hierro, Gran
Canaria, La Gomera, La Palma and Tenerife preserved
as dried plants in the herbaria of Naturalis Biodiversity
Center in Leiden, the Netherlands and the Natural
4
History Museum of the University of Oslo, Norway (see
Supplementary material Appendix 1 Table A1 for more
details and Fig. 3 and Table 1 for an overview of all localities from which data were retrieved). In order to see if we
could compare data obtained from fresh plants with dried
specimens, we measured 17 fresh flowers of H. tridactylites
in November 2014 from populations visited by us in El
Tanque, dried these and re-measured the dried flowers after
two weeks. Data from fresh and dried flowers did not differ significantly (Supplementary material Appendix 1 Table
A2), indicating that we could use both fresh and dried
plants for our measurements.
Plant measurements
Habenaria tridactylites is 10–40 cm high and has two
large ovate, basal leaves. The stem is leafless and carries a
lax, cylindrical inflorescence with 2–12 yellowish, odorous flowers. The most distinctive feature is the deeply
three-lobed lip, giving the species its name (tridactylites
means ‘with three fingers’ in Latin). The lateral sepals are
spreading; the median sepal forms a hood with the petals.
The slender spur is longer than the ovary, pendant and
downward curved, containing nectar. The column is short,
broad and sloping. The anther cells are wide apart and
extend into upward bent prolongations. The spur entrance
is placed between the anther cells. The entrance to the
spur from the front is hampered by a tongue-shaped outgrowth at the base of the lip. Under the protruding tips of
the anther cells lie two fingerlike stigmaphores (Dressler
1981, Szlachetko and Rutkowski 2000, Claessens and
Kleynen 2011).
We recorded the length of the spur of the third flower
from the base of the inflorescence (Supplementary material
Appendix 1 Fig. A1) in both fresh plants in the field and
dried plants from herbarium collections. In addition, spur
length and accumulated nectar level of all open flowers from
ten plants of a short-spurred location (Icod de los Vinos) and
Table 1. Islands and localities where populations of H. tridactylites were studied. F = fieldwork; H = herbarium survey; SL = spur length;
P = pollination.
Code + Site
G1 Telde
G2 Bandama
G3 Valsequillo
G4 Firgas
G5 San Isidro
G6 Los Berrazales
G7 Agaete
T1 Taborno (Anaga)
T2 San Diego del Monte (Anaga)
T3 La Laguna (Anaga)
T4 Los Carrizales (Teno)
T5 Huente de la Lunz
T6 Risco de Oro
T7 Orotava
T8 Fasnia
T9 Icod de los Vinos
T10 San Juan del Reparo
T11 El Tanque
G1 La Gomera1
P1 Fuencaliente
P2 San Isidro
H1 La Frontera
H2 El Hierro1
H3 Sabinar
1
Island
Gran Canaria
Gran Canaria
Gran Canaria
Gran Canaria
Gran Canaria
Gran Canaria
Gran Canaria
Tenerife old
Tenerife old
Tenerife old
Tenerife old
Tenerife young
Tenerife young
Tenerife young
Tenerife young
Tenerife young
Tenerife young
Tenerife young
La Gomera
La Palma
La Palma
El Hierro
El Hierro
El Hierro
Longitude
−15.425049
−15.459113
−15.501214
−15.5617
−15.562
−15.661963
−15.684021
−16.261830
−16.328255
−16.346097
−16.856676
−16.37948
−16.536449
−16.545014
−16.438050
−16.700935
−16.757800
−16.775856
−17.213361
−17.850417
−17.798141
−18.007956
−17.9775932
−18.131423
Latitude
Field work/herbarium survey Measurement type
27.999277
28.035493
27.990528
28.02894
28.029
28.068977
28.095430
28.555203
28.501168
28.496701
28.315473
28.388129
28.380516
28.373370
28.248553
28.341999
28.364091
28.354791
28.164907
28.487590
28.635680
27.748227
27.7432053
27.754878
H
F
F
F
F
F
F
H
H
H
F
H
H
H
F
F
F
F
H
F
F
H
H
H
SL
SL
SL
SL
SL
SL
SL
SL
SL
SL
P
SL
SL
SL
SL
P
SL
P
SL
SL
SL
SL
SL
SL
Site on island not known.
a long-spurred location (Los Carrizales) on Tenerife were
measured. This was measured to obtain an estimate of the
proboscis length needed to reach nectar in average flowers.
Measurements were all conducted in 2014 and within a few
days, in order to avoid seasonal differences.
In order to examine the variations in the volume of the
nectar, we measured the spur length and nectar volume
of all flowers of a total of 20 plants. Spur length and nectar level in the spur was measured with digital callipers. We
also measured the spur length of C. ruber on Tenerife, which
always accompanied H. tridactylites in the northern sites but
not in Los Carrizales. In order to investigate if there was a
relationship between spur length of H. tridactylites and
C. ruber, we measured spur length of C. ruber in two locations
on Tenerife, El Tanque (n = 31) and Icod de Los vinos (n = 24)
in 2016 (Supplementary material Appendix 1 Table A5). We
also noted the elevation of all sample sites.
Pollination success
On one site at Tenerife, we counted the total number of
flowers as well as the number of pollinated flowers. Female
fitness was calculated by dividing the number of capsules by
the total number of flowers.
Pollinator observations
Observations were made in the field on Tenerife at three different sites (El Tanque, Icod de los Vinos and Los Carrizales)
and took place at various times during the day and between
19:30 and 23:00 h at night. We observed for visitors and
pollinators during 11 days (32 h) and 21 evenings (73.5 h)
with a total of 105.5 observation hours. All plants observed
at night were constantly inspected using a powerful torch
and a head lamp. All visitors or pollinators were noted, photographed and caught if possible. No insect was observed
during the daytime, whereas observations during night-time
proved to be successful. If the visiting insect had pollinaria
attached to its body and visited several flowers, it was defined
as a pollinator. If the insect inspected the flower but did not
carry or remove any pollinaria, it was recorded as a visitor.
On 10 out of 21 observation nights we observed visitors or
pollinators.
Insect identifications
Samples and photographs of specimens that could not be
collected were identified morphologically partly by JJB
in Santa Cruz de Tenerife, and partly by EJvN in Leiden.
Moths brought to Leiden were also dissected to compare
the genitalia, and a leg was taken for DNA extraction and
DNA barcode assessment. There is no general handbook
for all Lepidoptera of the Canary Islands, but a field guide
(Báez 1998) allows identification of most larger moths. In
addition, genitalia and externals were checked with several
sources (Pinker 1965, Klimesch 1987, Hacker and Schmitz
1996, Goater et al. 2005, Fibiger et al. 2010, Witt et al. 2011,
Lepiforum e.V. 2017).
5
Measurement of proboscis lengths
Proboscis length of five pollinators was measured in the field
using digital calipers (Supplementary material Appendix 1
Fig. A1). All other proboscis lengths were measured by analyzing X-ray photographs of dried museum specimens with
the program ImageJ. The X-ray photographs were made by a
3D X-ray with a Sealed transmission 30–160 kV, max 10 W
X-ray sources.
Scanning was performed using the following settings:
acceleration voltage/power 80 kV/7 W; source current
78.5 μA; exposure time 4–7 s; pictures per sample 1001–1601;
camera binning 2; optical magnification 4×, with a pixel
size of 1.4–4.7 μm. The total exposure time was approximately
2.5–4 h.
Comparison of measurements of proboscis made in the
field (five measurements) to those made with ImageJ showed
that there was no significant difference between both, indicating that the program and dried specimens could also be
used as a means for measuring proboscis length.
DNA barcoding of pollinators
Morphological identifications were also checked with DNA
barcodes of 23 specimens. The DNA barcode for animals,
the partial cytochrome oxidase subunit 1 (COI) gene was
amplified with the primer mixture M13_LepFolF and
M13_LepFolR (Folmer et al. 1994, Hebert et al. 2004) using
the methodology described in Hebert et al. (2003) and in
Ratnasingham and Hebert (2007). Details of all barcoded
specimens and barcodes, barcode identification numbers
(Ratnasingham and Hebert 2013) plus NCBI Genbank
accession numbers are provided in Table 2 and in BOLD
dataset DS-HABPOL (doi: 10.5883/DS-HABPOL).
Taxon sampling for molecular clock analysis
We sampled 279 accessions of 274 orchid species belonging
to Habenaria and closely related genera. In addition, we generated new nrITS and matK sequences for Habenaria arenaria
Lindl., H. erichmichelii Christenson (= Habenaria rhodocheila
Hance), H. macrandra Lindl., H. medusa Kraenzl. and H.
tridactylites cultivated at the Hortus Botanicus Leiden, the
Netherlands. These sequences were complimented with
sequences from published studies in Orchidinae (Inda et al.
2012, Batista et al. 2013). Disa uniflora P.J.Bergius was used
as outgroup.
DNA extraction, amplification, sequencing and
alignments
Total genomic DNA was extracted from about 100 mg of
silica gel dried leaf tissue following the 2× CTAB (hexadecyltrimethylammonium bromide) protocol for isolating
DNA (Doyle and Doyle 1987). We used the 17SE and
26SE primers for amplification of nrITS and 2.1aF and
5R primers for amplification of the plastid matK region.
6
The polymerase chain reaction (PCR) mixture and amplification profiles followed Inda et al. (2012) and Kisel et al.
(2012) and sanger sequencing was conducted by BaseClear
(< www.baseclear.com >) on an ABI 3730xl genetic analyzer. Newly generated sequences were deposited in NCBI
GenBank (Table 1, Supplementary material Appendix
1 Table A8). We used Geneious R9 (Biomatters Ltd.,
Kearse et al. 2012) for the editing of chromatograms and
alignment of sequences.
Phylogenetic analyses and molecular clock analysis
We analyzed the individual and concatenated datasets of
the two molecular markers nrITS and matK with maximum likelihood (ML) in the CIPRES Science Gateway ver.
3.1 (< www.phylo.org/sub_sections/portal/ >) (Miller et al.
2010). The incongruence between plastid and nuclear datasets was assessed with the pipeline implemented by PérezEscobar et al. (2017) using the procrustean approach to
cophylogeny (PACo) application (Balbuena et al. 2013) in
R (< http://data-dryad.org/review?doi=doi:10.5061/dryad.
q6s1f >). The matK sequences from the conflicting terminals were removed in the concatenated dataset (PérezEscobar et al. 2016). The new concatenated matrix was
re-aligned and used as input to calculate divergence times
in Habenaria and close relatives. The divergence times were
estimated in BEAST ver. 1.8.2 using the CIPRES Science
Gateway (Miller et al. 2010) with GTR + G substitution
model and four gamma categories, lognormal relaxed uncorrelated clock and tree prior Yule process (Y) model. We used
the age estimates from a fossil-calibrated chronogram of
Orchidaceae by Pérez-Escobar et al. (2017) in order to perform secondary calibrations. We assigned a normal prior distribution of 43.07 (± 2.5 SD) Mya to the root node of Disa
P.J.Bergius + Habenaria and close relatives and 33.78 (± 2.0
SD) Mya to the node of divergence of Disa and Habenaria
and close relatives. Another calibration point was used to
constrain the node of the two endemic species H. tridactylites
and G. diphylla to the age of the oldest island, Fuerteventura
(20.6 Mya) (Ojeda et al. 2012). We performed two MCMC
with 60 × 106 generations and sampling every 1000 generations and burnin of 10%. We inspected the convergence of
independent runs size in Tracer ver. 1.6.
Statistical analyses
Data were analysed with one-way ANOVA tests and various
posthoc procedures as well as with linear regression analysis
using SPSS Statistics 24 to investigate possible correlations
between spur length, elevation, longitude and age of the
islands, respectively.
Data deposition
Data are available from the BoLD Digital Repository:
<http://doi.org/10.5883/ds-habpol> (Claessens et al. 2019).
Table 2. Pollinators and visitors of Habenaria tridactylites recorded in the field on Tenerife, all belonging to the order Lepidoptera. See also dataset <https://dx.doi.org/10.5883/
DS-HABPOL>.
Family
Species
Crambidae
Eudonia angustea
(Curtis, 1827)
Crambidae
Eudonia lineola
(Curtis, 1827)
Evergestis isatidalis
(Duponchel, 1833)
Crambidae
Erebidae
Erebidae
Geometridae
Geometridae
Geometridae
Geometridae
Geometridae
Geometridae
Geometridae
Eilema albicosta witti
Kobes, 1993
Schrankia
costaestrigalis
(Stephens, 1834)
Ascotis fortunata
(Blachier, 1887)
Charissa canariensis
canariensis (Rebel,
1911)
Costaconvexa
centrostrigaria
(Wollaston, 1858)
Cyclophora
maderensis trilineata
(Prout, 1934)
Episauris kiliani (Rebel,
1898)
Gymnoscelis
rufifasciata
(Haworth, 1809)
Nebula ibericata
(Staudinger, 1871)
NCBI
GenBank
accession no.
No.
individuals
Pollinator?
4+
yes
S, SW Europe
Los Carrizales;
El Tanque
1
yes
S, SW Europe
El Tanque
3
yes
S Europe
Los Carrizales
2
yes
2
no
1
yes
Macaronesia
Icod de los Vinos
Bacallado s.n.
1
yes
Endemic Canary
Islands
Los Carrizales
RMNH.5011609
1
yes
Macaronesia, N
America
Icod de los Vinos
Bacallado s.n.
–
34
yes
Endemic Canary
Islands
Icod de los Vinos
Bacallado s.n.
–
2
yes
Icod de los Vinos
Bacallado s.n.
–
1
no
Endemic Canary
Islands
Europe
El Tanque
RMNH.5011616
BOLD:ADL3671
3
yes
SW Europe
El Tanque
BOLD:ACF0717
BOLD:ADL3062
MK566739
MK566755
MK566749
MK566742
MK566758
Distribution
Sample sites
RMNH registry number
RMNH.5009039
RMNH.5011624
RMNH.5011625
RMNH.5011626
Bacallado s.n.
20131204_DSC9368
RMNH.5011610;
RMNH.5011611;
RMNH.5011627
Endemic Tenerife Los Carrizales; Icod RMNH.5011612;
de los Vinos
RMNH.5011618
Europe
El Tanque
RMNH.5011617;
RMNH.5011622
BIN
–
MK566751
–
BOLD:ADL3576
BOLD:ACD0672
BOLD:AAD1543
MK566741
MK566743
MK566744
MK566750
MK566746
MK566756
MK566754
–
BOLD:ADL4101
MK566753
MK566745
Noctuidae
Cucullia calendulae
(Treitschke, 1835)
6
yes
S Europe
El Tanque, Los
Carrizales
Noctuidae
Paranataelia whitei
(Rebel, 1906)
Clepsis coriacanus
(Rebel, 1894)
Acroclita sonchana
Walsingham, 1908
1
no
Los Carrizales
1
no
Los Carrizales
RMNH.5011613
BOLD:ADL2652
MK566740
3
yes
Endemic Canary
Islands
Endemic Canary
Islands
Endemic Canary
Islands
RMNH.5009041
RMNH.5009042
RMNH.5011615
RMNH.5009040
RMNH.5011605;
RMNH.5011606;
RMNH.5011607
RMNH.5011608
Los Carrizales; El
Tanque
RMNH.5011614;
RMNH.5011621;
RMNH.5011623
BOLD:ADL4602
MK566747
MK566757
MK566748
Tortricidae
Tortricidae
MK566759
BOLD:ABX5041
7
Figure 4D. Biotope of H. tridactylites with Pinus canariensis and
Davallia canariensis. Photograph by Jean Claessens at El Tanque on
19 Nov 2014. Scale bar = 10 mm.
Figure 4A–B. (A) Habitus of H. tridactylites. (B) Close-up of part of
the inflorescence of H. tridactylites. Photographs by Jean Claessens
on Tenerife, El Amparo, on 01.01.2008. Scale bar = 10 mm.
Results
Plant measurements
Mean nectar level was 3.2 ± 2.1 mm (n = 49) at Icod de los
Vinos, Los Carrizales and El Tanque on Tenerife. Figure 5D
shows that mean spur length was the longest in the easternmost, oldest island (Gran Canaria, n = 86) and the shortest
in the two westernmost, youngest islands (La Palma, n = 71
and El Hierro, n = 2). The spur length in Tenerife showed
the highest variation for a single island. The longest spurs,
comparable to those of Gran Canaria, were found in the old
parts of the island, Anaga and Teno, indicated as Tenerife old
(n = 120). The shortest spurs were found in Tenerife young
(n = 51).
Nectar levels in the spurs of H. tridactylites plants differed
considerably, both between flowers of a single flower spike as
well as between plants. Supplementary material Appendix 1
Figure A1, Table A4 give an indication of the minimal length
of the proboscis (in mm) needed to reach the nectar. We
found a considerable difference in nectar level, resulting in
Figure 4C. Longitudinal section of a flower of H. tridactylites.
A = anther, RA-rostellar arm, T = tongue-shaped elevation on the
lip base, V = viscidium, SL = stigmatic lobe, S = spur, O = ovary.
Photograph by Jean Claessens. Scale bar = 1 mm.
8
a variable distance between spur entrance and nectar level,
further indicated as free spur space (FSS). This ranged from
15.1% to 153.5%, indicating that in each population there
was nectar within reach of even the shortest-tongued pollinators. In the most extreme case, the proboscis length needed
for reaching nectar ranged from 4.3 to 10.9 mm. In the shortspurred site (Icod de Los Vinos), pollinators with a proboscis
of 4.3 mm long could already reach the nectar. In flowers of
the long-spurred site (Los Carrizales), FSS was much higher
and nectar could only be reached by pollinators with a proboscis of at least 9.9 mm long. Spur length of Centranthus
ruber differed between the sites of Tenerife: at El Tanque the
length was significantly higher than that of H. tridactylites
(p = 0.00) whereas in Icod de los Vinos the length of the spurs
of both plant species did not differ significantly (p = 0.67)
(see also Supplementary material Appendix 1 Table A5).
Fruit set of H. tridactylites – in a total of 40 flowers, no
signs of autogamy could be detected. In six flowers, one
or both pollinaria were still present whereas the flower was
already pollinated. Out of 27 plants with 174 open flowers, a
Figure 4E–F. (E) Eudonia lineola pollinating H. tridactylites with a
viscidium sticking to its eyes at El Tanque on 8 Dec 2013. (F)
Nebula ibericata numidata pollinating H. tridactylites with a bunch
of pollinaria sticking to the base of the proboscis at El Tanque on
4 Dec 2013. Photographs by Jean Claessens. Scale bar = 1 mm.
Figure 4G. Cyclophora maderensis trilineata carrying pollinaria of
H. tridactylites at Tenerife, El Tanque on 15 Jan 2016. The viscidia
stick to the eyes. Photograph by Jean Claessens. Scale bar = 1 mm.
total of 130 flowers (74.1%) were pollinated. On Tenerife the
average fruit set was 59.4% (n = 21) (see also Claessens and
Kleynen 2011, 2016).
Pollinators of H. tridactylites on Tenerife
We never observed a pollinator or a visiting insect during the
daytime on any study site. In total, we caught 61 insects of
which 53, belonging to 16 different species, were actual pollinators of H. tridactylites. All pollinators were Lepidoptera
(see Fig. 4, Table 2 for more details). Only on two occasions,
we saw a visiting snout moth (Crambidae). Most pollinators belonged to the Geometridae: five different species were
noted as pollinators. On one site, we collected 34 specimens
of Cyclophora maderensis trilineata Prout.
The pollinator spectrum varied between the three sites,
due to the different biotopes. Two of the four Geometridae
species only seen at Icod de los Vinos feed as caterpillars on
Erica arborea, only common at this site.
The different pollinator families behaved very differently.
Crambidae and Tortricidae sat generally immobile on a
flower spike for a long time, from several minutes to more
than half an hour. They sometimes moved from flower to
flower, may be incited by the light of the torch. The pollinaria were attached to their eyes. In contrast, Geometridae
regularly moved from flower to flower and probed the flowers from a few seconds up to several minutes. Pollinaria were
generally attached to the proboscis base or sometimes to the
Figure 4H. Cucullia calendulae pollinating H. tridactylites at Tenerife
on Los Carrizales on 2 Dec 2014. Photograph by Jean Claessens.
Scale bar = 10 mm.
Figure 4I. CT scan of the proboscis of RMNH5011606. Photograph
by Rob Langelaan. Scale bar = 0.44 mm.
eyes. Noctuidae were the most active pollinators, constantly
moving from flower to flower. They moved between flower
spikes in less than ten seconds. The number of pollinators is
underestimated because we were not able to catch all insects
during the fieldwork due to the rocky terrain.
Proboscis lengths of pollinators of H. tridactylites from
Tenerife differed between 4 mm and 15.7 mm and are summarized in Supplementary material Appendix 1 Table A3 and
Fig. 5A. Mean spur lengths of H. tridactylites from various sites
in Gran Canaria, Tenerife, La Palma, La Gomera and El Hierro
are summarized in Supplementary material Appendix 1 Table
A6 and Fig. 5A. The ANOVA tests indicate that the mean spur
length differs significantly for some of the islands (p < 0.000).
A non-parametric Kruskal–Wallis test, which is less sensitive for deviations of the requirements of ANOVA, confirms
this (p < 0.000). Significant differences were found between
El Hierro/La Palma and Tenerife/Gran Canaria (excluding la
Gomera) (Supplementary material Appendix 1 Table A7).
Identification of Lepidoptera
All identifications are given in Table 2. Unfortunately,
since there has not yet been a systematic barcode campaign
for Canarian Lepidoptera, for several species no matching barcodes could be found. This resulted in new barcode
Figure 4J. CT scan of RMNH5011610. Photograph by Rob
Langelaan. Scale bar = 0.44 mm.
9
Spur length of third flower
B
Figure 4K. CT scan of RMNH5011611. Photograph by Rob
Langelaan. Scale bar = 0.44 mm.
identification numbers (BIN’s) for the endemic Paranataelia
whitei Rebel, Charissa canariensis canariensis Rebel, Acroclita
sonchana Walsingham and Clepsis coriacanus Rebel plus new
BIN’s for the Tenerife populations of Evergestis isatidalis
Duponchel and Gymnoscelis rufifasciata Haworth. In contrast,
the barcodes for Nebula ibericata Staudinger, Cucullia calendulae Treitschke and Schrankia costaestrigalis Stephens are not
or hardly different from continental European populations.
One specimen that was destructively extracted delivered a
barcode with 100% similarity to Lamoria adaptella Walker.
We consider this an unlikely outcome, as this species is
mainly known from Australia and East Asia, with as closest
only a single record from Gambia (De Prins and De Prins
2017). Since we cannot confirm the identity, the record is not
listed in Table 2, nor on BOLD.
Molecular clock results
The most recent common ancestor of H. tridactylites and
G. diphylla was dated to ca 18.5 ± 2.5 Mya (Supplementary
Longitude
Figure 5B. Scatterplot of spur length (mm) of H. tridactylites against
distance to mainland Africa (measured by longitude).
material Appendix 1 Fig. A2). This clade was strongly supported by the Bayesian Inference with a posterior probability
of 1.0.
Discussion
Very little was previously known about the pollination of
Habenaria tridactylites. There was only one observation of
a pollinator recorded by Paulus (1999) on Gran Canaria,
identified as possibly Mamestra brassicae L. (Noctuidae), with
one pollinarium attached to the proboscis base. However,
this is an unlikely identification as this species has not been
recorded from the Canary Islands (Báez 2010, Vives Moreno
C
Spur length of third flower
Spur length of third flower
A
Age (Mya)
Island
Figure 5A. Boxplot of spur length (mm) of H. tridactylites per
island.
10
Figure 5C. Scatterplot of spur length (mm) of H. tridactylites against
mean geological age of the locality of the plant in the field (measured in million years ago).
Spur length of third flower
D
Altitude (m)
Figure 5D. Scatterplot of spur length against altitude.
2014) and close inspection of the photograph shows that
this is most likely Mniotype usurpatrix Rebel (Noctuidae), a
Canarian endemic, common in winter on Gran Canaria (B.
Skule unpubl.). During our fieldwork, we discovered that
H. tridactylites is well suited for pollination by insects. The
species is well adapted to night-flying moths: scent emission
augments in the afternoon and the pale green coloured flowers are visible when there is little light. The flowers are well
accessible to insects: due to the downward arching lip the
entrance to the column is wide open. The lateral spreading
sepals provide a holdfast for visiting Lepidoptera: they can
rest their forelegs on the sepals while searching for nectar
(Fig. 4). The long, downward curved spur forces visiting
moths to enter the entire proboscis into the spur. The tonguelike ridge on the lip base forces the moths to enter the flower
from above. In this position the chances of touching the viscidia are high. If the pollinarium is attached to a pollinator,
the caudicle slowly bends forward after being dislodged. This
process takes 5–10 min (Claessens and Kleynen 2011) and
is a means of preventing self-pollination (Nunes et al 2016).
The pollinaria bend downward and inward and are placed
in the ideal position for touching the stigmaphores, lying
quite close to another. We regularly saw stigmaphores abutting to each other. Autogamy is not likely, because the anther
cells lean backwards and the pollinaria cannot be dislodged
without external help.
Pollination efficiency is high, but the coverage of the stigmaphores with pollen varied considerably: some had only
a few massulae (pollen packages) adhering, whereas others
were covered with many massulae. Pollinators of various
Lepidopteran families were observed. Paulus (1999) already
found that in Gran Canaria the spur length of H. tridactylites
flowers differed considerably. Based on the distribution of
the spur lengths found, these could be divided in four classes
(12–14.4, 14.5–46.9, 17–19.4 and 19.5–21 mm); all spur
classes were equally represented, with the exception of the
longest spurs. According to Paulus (1999) this is an indication
that H. tridactylites attracts potential pollinators with different proboscis lengths. Our findings confirm this hypothesis.
Our observations show that H. tridactylites is a
Lepidopteran pollinated species. In one site Cyclophora
maderensis trilineata was an abundant and efficient pollinator.
This moth is endemic to the Canary Islands and Madeira. It
inhabits the laurisilva and adjacent bushes with its hostplant
Figure 5E. Mean spur length of Habenaria tridactylites on the Canary Islands.
11
tree heath, Erica arborea (Fayal-brezal vegetation). It can be
found all year round in many annual generations. In one
case, we observed six moths, feeding on a group of nine
H. tridactylites plants. Most pollinators recorded in the North
of Tenerife were Lepidoptera commonly associated with the
Fayal-brezal (Myrico fayae–Ericion arboreae), and the majority
are also endemic (sub)species.
According to Inda et al. (2012), the divergence between
H. tridactylites and G. diphylla took place in the Miocene,
13–23 Mya. This estimate is in accordance with our own
molecular clock analyses inferred from an expanded sampling of Habenaria and close relatives (Supplementary
material Appendix 1 Fig. A2). Tenerife was formed only
11.9 Mya, so H. tridactylites might first have colonised the
geological older island Lanzarote and then spread westwards.
Alternatively, an ancestral lineage occurring elsewhere might
have spread to the Canary Islands after these were formed.
Population genetic analyses of H. tridactylites and G. diphylla
are needed to answer this question but that was beyond the
scope of this study.
Spur length is an important feature determining which
insect can act as a pollinator (Darwin 1877, Nilsson 1988,
Maad and Nilsson 2004, Boberg et al. 2014). Yet, spur length
is not fixed but depends on various factors. Bateman and
Sexton (2008) and Bateman et al. (2012) showed that spur
length in European species of the orchid genus Platanthera
has a latitudinal cline, decreasing northward. Also, spurs of
plants in shaded habitats are on average longer than those of
plants growing in more open habitats. In our study we found
no correlation between altitude (data not shown) and spur
length, but a significant linear correlation between island
longitude and spur length (Fig. 5B) and between island age
and spur length (Fig. 5C). A simple regression of spur length
with one explanatory variable (either age or longitude) gives
a significant model (R2 = 36–38%, p < 0.000). In both cases,
the regression coefficient is significantly positive. A multiple
regression with both explanatory variables (age and longitude) also gives a significant model (R2 = 38%, p < 0.000)
(Supplementary material Appendix 1 Table A7). However,
there is a multicollinearity problem, as the explanatory
variables age and longitude are correlated. This discredits
the significance calculations of the coefficients in a multiple regression; and for that reason, we preferred to address
separate simple regressions.
Mean spur length was the highest in the oldest island,
Gran Canaria and showed a decrease from east to west, that
is from the oldest to the youngest islands, La Palma and El
Hierro (Fig. 5D). Tenerife showed a large variation in spur
length, but the longest spurs were found on the oldest parts
of the island, Anaga and Teno, two of the shield volcanoes
that were eventually merged by the eruption and subsequent
emergence of the column of the Teide volcano. A shorter spur
length was found in younger parts, which were colonised
only after the formation of the Teide. The pollinator shift
theory (Wasserthal 1997, 1998, Whittall and Hodges 2007,
Hodges and Whittall 2008) assumes that saltatory mutations
12
driving the length of the spur evolve as an adaption to the
tongue length of a new pollinator guild. This theory seems
not applicable to H. tridactylites on the Canary Islands, since
there is only one pollinator guild (Lepidoptera) and the flowers are attractive to Lepidoptera with various tongue lengths.
The spur length does not show discrete length differences
between the different islands or elevation either, but a gradual
elongation from the youngest to the oldest islands instead.
The increasing mean spur length on older islands might be
in accordance with the evolutionary arms race theory as first
pointed out by Darwin (1877). According to this theory, proboscis length and spur length are reciprocally influenced by
positive feedback, leading to a constantly growing elongation
of both. This idea was later on elaborated by various other
authors (Nilsson 1988, 1998, Whittall and Hodges 2007,
Anderson and Johnson 2008, Pauw et al. 2009). According
to this theory, spur length is age-dependent and could thus
explain the distribution of mean spur length of H. tridactylites on the various Canary Islands as confirmed in this study.
We only collected data on pollinator tongue length from
three regions of a single island, Tenerife (El Tanque, Icod de
los Vinos and Los Carrizales) so more data on local pollinator
tongue lengths are needed from the other Canary Islands with
contrasting geological ages. The shortest spurs of H. tridactylites on El Hierro and La Palma are probably related to the
fact that these are geologically the youngest Canary Islands
and that these islands are also separated from the mainland
by the greatest distance. When comparing island specimens
of butterflies to mainland specimens of the same species, the
individuals of island populations tend to be smaller, especially
for small, weak fliers that cannot easily make the crossing
(Garth and Tilden 1986). It might be that the mean tongue
length of the pollinators of H. tridactylites on El Hierro and
La Palma is shorter due to this so-called island effect. More
experimental data are needed from common garden studies
to further investigate this.
Dispersal of H. tridactylites by human activities seems
highly unlikely as the plants, seeds and roots do not have any
agricultural value and preferably grow in sites that are unsuitable for cultivation. The fact that these orchids are nowadays
found in secondary biotopes is caused by increasing human
presence on the Canary Islands, reducing the cover of the
original biotopes of this orchid species. The seeds are dust-like
and can easily travel long distances through the air, enabling
colonisation of new biotopes on adjacent islands. Arditti and
Ghani (2000) for instance describe that orchids were among
the first plants to grow on newly emerging volcanic islands.
Therefore, we postulate that H. tridactylites spread westwards
over the Canary Islands via seed dispersal by wind.
Analysing the free spur space (FSS) seems to be more
realistic than measuring spur length, because this determines
whether a potential pollinator can reach nectar accumulated in the spur. Our analyses showed that this measure is
quite flexible, enabling even insects with a short proboscis
to consume at least some nectar. Combined with the general
poverty of flowering plants during the flowering period of
H. tridactylites this mechanism promotes visits of insects with
various proboscis length. It also incites insects with a short
proboscis to bend over deeply in order to reach the nectar. In
doing so they will almost certainly touch the viscid discs and
remove the pollinaria. If they already had pollinaria attached,
they will press them firmly onto the stigmatic lobes while trying to reach the nectar.
Whereas there seems to be a mismatch in some Habenaria
species between pollinator and spur length (Moré et al.
2012), on Tenerife the female success, as expressed in fruit
set, is high in both the old and the young parts of the island.
Crambidae and Tortricidae had only few pollinaria attached,
and seem to be less important pollinators judging from their
behaviour. Geometridae were the most abundant pollinators, and five different species acted as pollinators. The largest, fastest-moving pollinators were the Noctuidae, which
were exclusively observed in north-western Tenerife (Los
Carrizales, geologically belonging to the Teno massif ). This
is also where the flowers with the longest spurs were found.
We do not know if flowers of H. tridactylites in the Anaga
mountains are also pollinated by long tongued moths. We
did record a long spur length here, but the local pollinator
spectrum has not yet been investigated.
When the flowering period of H. tridactylites is ending, another orchid, Gennaria diphylla, starts flowering.
Interestingly, we observed that there is an overlap in pollinator spectrum between both orchid species. Several pollinators
of H. tridactylites were also observed pollinating the rewarding orchid G. diphylla (Claessens et al. unpubl.). The paucity
of co-flowering nectar plants might incite these insects to
probe all flowering plants in the area.
During our fieldwork on Tenerife, we noticed that
C. ruber was frequently visited by butterflies and moths.
This species therefore probably also plays a role in the nectar
supply of orchid pollinators. Mean spur length of C. ruber and
H. tridactylites on Icod de los Vinos was similar in both species, whereas there was a considerable difference on El Tanque
(Supplementary material Appendix 1 Table A5). Given
the shortage of flowering plants in the flowering period of
H. tridactylites, it might be that the same pollinators visit
both species. However, C. ruber is not an indigenous species
on the Canary Islands. It is strongly associated with human
settlements (Stierstorfer and von Gaisberg 2006). It can have
a positive influence on the pollinator spectrum, but it cannot
be associated with the evolution of longer spurs over millions
of years as assumed for H. tridactylites. It would, therefore, be
interesting to investigate whether populations of other plant
endemics such as for instance Viola cheiranthifolia Humb.
& Bonpl., which is pollinated by bees (Seguí et al. 2017)
also have longer spurs on older parts of Tenerife as compared
with populations occurring in more recent parts. To the best
of our knowledge, correlations in spur length of other plant
species and geographical ages of islands have not yet been
published. If such data would become available, preferably
backed up by real-time divergent evolution experiments, this
would provide further support for the theory that either a
pollinator shift or evolutionary arms race might be at play
between plants and pollinators on the Canary Islands.
Acknowledgements – We thank Helmut Läpple, Sheila Edwards,
Rogier van Vugt, Petra Sonius, Felix Baeten, Gustavo Peña and
Jürgen Vetter for providing locality data and other information
of H. tridactylites, Kevin Beentjes for DNA barcoding of the
pollinators and Erik-Jan Bosch for help with the illustrations.
We thank Marijke Claessens for critically reading earlier versions
of the manuscript and Hugo de Boer for providing a constructive
review of the manuscript. Gyulá László is acknowledged for the
preparation of genitalia slides, and Rob de Vos for assistance with
collections. We thank Bjarne Skule, Hermann Hacker, Per Falck,
Leif Aarvik and Berend Aukema for help with several Lepidoptera
and Hemiptera identifications. DNA barcoding of the insects was
co-financed by Fonds Economische Structuurversterking (FES).
References
Aedo, C. et al. 2013. Species richness and endemicity in the Spanish
vascular flora. – Nord. J. Bot. 31: 478–488.
Anderson, B. and Johnson, S. D. 2008. The geographical mosaic
of coevolution in a plant–pollinator mutualism. – Evolution 62:
220–225.
Arditti, J. and Ghani, K. A. 2000. Numerical and physical
properties of orchid seeds and their biological implications.
– New Phytol. 145: 367–421.
Arechavaleta, M. et al. 2010. Lista de especies silvestres de Canarias.
Hongos, plantas y animales terrestres. 2009. – Gobierno de
Canarias, p. 577.
Báez, M. 1998. Mariposas de Canarias. – Editorial Rueda, S.L.
Báez, M. 2010. Orden Lepidoptera. – In: Arechavaleta, M. et al.
(eds), Lista de especies silvestres de Canarias. Hongos, plantas
y animals terrestres. Gobierno de Canarias, pp. 302–318.
<http://www.gobiernodecanarias.org/medioambiente/piac/
descargas/Biodiversidad/Listas-Especies/Lista_Especies_
Silvestres>.
Balbuena, J. A. et al. 2013. PACo: a novel procrustes application
to cophylogenetic analysis. – PLoS One 8: e61048.
Bateman, R. et al. 2012. Contrast in levels of morphological versus
molecular divergence between closely related Eurasian species
of Platanthera (Orchidaceae) suggests recent evolution with a
strong allometric component. – New J. Bot. 2: 110–148.
Bateman, R. and Sexton, R. 2008. Is spur length of Platanthera
species in the British Isles adaptively optimized or an evolutionary red herring? – Watsonia 27: 1–22.
Batista, J. A. N. et al. 2013. Molecular phylogenetics of the speciesrich genus Habenaria (Orchidaceae) in the New World based
on nuclear and plastid DNA sequences. – Mol. Phylogen. Evol.
67: 95–109.
Boberg, E. and Ågren, J. 2009. Despite their apparent integration,
spur length but not perianth size affects reproductive success in
the moth-pollinated orchid Platanthera bifolia. – Funct. Ecol.
23: 1022–1028.
Boberg, E. et al. 2014. Pollinator shifts and the evolution of spur
length in the moth-pollinated orchid Platanthera bifolia. – Ann.
Bot. 113: 267–275.
Camus, E. G. 1929. Iconographie des Orchidees d’Europe et du
Bassin mediterraneen. vol. 2. – P. Lechevalier, Paris.
13
Carracedo, J. C. and Perez-Torrado, F. J. 2013. Geological and
Geodynamic context of the Teide Volcanic complex. Teide
Volcano. – Springer, pp. 23–36.
Claessens, J. and Kleynen, J. 2011. The flower of the European
orchid – form and function. – Jean Claessens & Jacques
Kleynen.
Claessens, J. and Kleynen, J. 2011. Bestäubung bei Europäischen
Orchideen zwischen Allogamie und Autogamie – einige
Beispiele. – Ber. Arbeitskrs. Heim. Orchid. Beiheft 8:
14–31.
Claessens, J. and Kleynen, J. 2016. Orchidées d’Europe, fleur et
pollinisation. – Biotope Éditions.
Claessens, J. et al. 2019. Data from: pollination of Habenaria
tridactylites on the Canary Islands. – BoLD Digital Repository,
<http://doi.org/10.5883/ds-habpol>.
Darwin, C. 1877. The various contrivances by which British and
foreign orchids are fertilised by insects. – John Murray.
De Prins, J. and De Prins, W. 2017. Afromoths, an online database
of Afrotropical moth species (Lepidoptera). – Belgian
Biodiversity Platform. <http://www.afromoths.net/>, accessed
20 February 2018.
Del-Arco, M. et al. 2006. Bioclimatology and climatophilous
vegetation of Tenerife (Canary Islands). – Ann. Bot. Fenn. 43:
167–192.
Dóniz-Páez, J. et al 2012. Quantitative size classification of scoria
cones: the case of Tenerife (Canary Islands, Spain). – Phys.
Geogr. 33: 514–535.
Doyle, J. and Doyle, J. 1987. A rapid DNA isolation procedure for
small quantities of fresh leaf tissue. – Phytochem. Bull. 19:
11–15.
Dressler, R. L. 1981. The orchids. – Harvard Univ. Press.
Dressler, R. L. 1993. Phylogeny and classification of the orchid
family. – Cambridge Univ. Press.
Fibiger, M. et al. 2010. Noctuidae Europaeae, volume 12: Rivulinae,
Boletobiinae, Hypenodinae, Araeopteroninae, Eublemminae,
Hermiinae, Hypeninae, Phytometrinae, Euteliinae and
Micronoctuidae. – Entomological Press.
Folmer, O. et al. 1994. DNA primers for amplification of
mitochondrial cytochrome c oxidase subunit 1 from diverse
metazoan invertebrates. – Mol. Mar. Biol. Biotechnol. 3:
294–299.
Garth, J. S. and Tilden, J. W. 1986. California butterflies. – Univ.
of California Press.
Goater, B. et al. 2005. Pyraloidea I (Сrambidae: Acentropinae,
Evergestinae, Heliothelinae, Schoenobiinae, Scopariinae).
Microlepidoptera of Europe. – Apollo Books.
Godfery, M. and Godfery, H. 1933. Monograph and iconograph
of native British Orchidaceae. – Univ. Press.
Govaerts, R. et al. 2011. World checklist of Orchidaceae. – The
Board of Trustees of the Royal Botanic Gardens, Kew. <http://
apps.kew.org/wcsp/>.
Grimaldi, D. 1999. The co-radiations of pollinating insects and
angiosperms in the Cretaceous. – Ann. Miss. Bot. Gard. 86:
373–406.
Guillou, H. et al. 2004. Implications for the early shield-stage
evolution of Tenerife from K/Ar ages and magnetic stratigraphy.
– Earth Planet. Sci. Lett. 222: 599–614.
Hacker, H. and Schmitz, W. 1996. Fauna und Biogeographie der
Noctuidae des makaronesischen Archipels. – Esperiana 4:
167–221.
Hebert, P. D. et al. 2004. Ten species in one: DNA barcoding
reveals cryptic species in the neotropical skipper butterfly
14
Astraptes fulgerator. – Proc. Natl Acad. Sci. USA 101:
14812–14817.
Hebert, P. D. et al. 2003. Barcoding animal life: cytochrome c
oxidase subunit 1 divergences among closely related species.
– Proc. R. Soc. B 270: S96–S99.
Hodges, S. and Whittall, J. 2008. One-sided evolution or two? A
reply to Ennos. – Heredity 100: 541–542.
Ikeuchi, Y. et al. 2015. Diurnal skipper Pelopidas mathias
(Lepidoptera: Hesperiidae) Pollinates Habenaria radiata
(Orchidaceae). – Entomol. News 125: 7–12.
Inda, L. A. et al. 2012. Phylogenetics of tribe Orchideae
(Orchidaceae: Orchidoideae) based on combined DNA
matrices: inferences regarding timing of diversification and
evolution of pollination syndromes. – Ann. Bot. 110: 71–90.
Jacquemyn, H. et al. 2014. Biological flora of the British Isles:
Epipactis palustris. – J. Ecol. 102: 1341–1355.
Karremans, A. P. et al. 2015. Pollination of Specklinia by nectarfeeding Drosophila: the first reported case of a deceptive
syndrome employing aggregation pheromones in Orchidaceae.
– Ann. Bot. 116: 437–455.
Karsholt, O. and van Nieukerken, E. 2017. Fauna Europaea:
Lepidoptera. – Fauna Europaea, ver. 2017.06. <https://faunaeu.org/>.
Kearse, M. et al. 2012. Geneious basic: an integrated and extendable
desktop software platform for the organization and analysis of
sequence data. – Bioinformatics 28: 1647–1649.
Kisel, Y. et al. 2012. Testing the link between population genetic
differentiation and clade diversification in Costa Rican orchids.
– Evolution 66: 3035–3052.
Klimesch, J. 1987. Beitrage zur Kenntnis der Microlepidopterenfauna des Kanarischen Archipels. 9. Beitrag: Tortricidae,
Cochylidae. – Vieraea 17: 297–322.
Kocyan, A. et al. 2008. Molecular phylogeny of Aerides (Orchidaceae)
based on one nuclear and two plastid markers: a step forward
in understanding the evolution of the Aeridinae. – Mol.
Phylogen. Evol. 48: 422–443.
Kull, T. et al. 2009. Orchid biology: reviews and perspectives X.
– Springer.
Kullenberg, B. 1961. Studies in Ophrys pollination. – Almqvist and
Wiksells Boktryckeri AB.
Lenaerts, M. et al. 2014. Rosenbergiella australoborealis sp. nov.,
Rosenbergiella collisarenosi sp. nov. and Rosenbergiella epipactidis
sp. nov., three novel bacterial species isolated from floral nectar.
– Syst. Appl. Microbiol. 37: 402–411.
Lepiforum e.V. 2017. Bestimmungshilfe fur die in Europa
nachgewiesenen Schmetterlingsarten. – <http://www.
lepiforum.de/lepiwiki.pl?Bestimmungshilfe>, accessed 28
September 2017.
Maad, J. and Nilsson, L. A. 2004. On the mechanism of floral shifts
in speciation: gained pollination efficiency from tongue-to
eye-attachment of pollinia in Platanthera (Orchidaceae). – Biol.
J. Linn. Soc. 83: 481–495.
Miller, M. A. et al. 2010. Creating the CIPRES Science Gateway
for inference of large phylogenetic trees. 2010 gateway
computing environments workshop (GCE). – Ieee, pp. 1–8.
Moré, M. et al. 2012. Armament imbalances: match and mismatch
in plant–pollinator traits of highly specialized long-spurred
orchids. – PLoS One 7: e41878.
Moreira, G. R. et al. 1996. Pollination of Habenaria pleiophylla
Hoehne & Schlechter (Orchidaceae) by Heliconius erato phyllis
Fabricius (Lepidoptera, Nymphalidae). – Rev. Bras. Zool. 13:
791–798.
Nilsson, L. A. 1981a. Pollination ecology and evolutionary processes
in six species of orchids. – Almqvist and Wiksell Int.
Nilsson, L. A. 1981b. The pollination ecology of Listera ovata
(Orchidaceae). – Nord. J. Bot. 1: 461–480.
Nilsson, L. A. 1988. The evolution of flowers with deep corolla
tubes. – Nature 334: 147–149.
Nilsson, L. A. 1998. Deep flowers for long tongues. – Trends Ecol.
Evol. 13: 259–260.
Nunes, C. E. et al. 2016. The dilemma of being a fragrant flower:
the major floral volatile attracts pollinators and florivores in the
euglossine-pollinated orchid Dichaea pendula. – Oecologia 182:
933–946.
Ojeda, I. et al. 2012. The origin of bird pollination in Macaronesian
lotus (Loteae, Leguminosae). – Mol. Phylogen. Evol. 62:
306–318.
Paulus, H. F. 1999. Bestäubungsbiologische Untersuchungen an
Ophrys bombyliflora, Orchis canariensis und Habenaria
tridactylites (Orchidaceae) in Gran Canaria (Spanien). – Ber.
Arbeitskrs. Heim. Orchid. 16: 4–22.
Pauw, A. et al. 2009. Flies and flowers in Darwin’s race. – Evolution
63: 268–279.
Pedron, M. et al. 2012. Pollination biology of four sympatric
species of Habenaria (Orchidaceae: Orchidinae) from southern
Brazil. – Bot. J. Linn. Soc. 170: 141–156.
Pérez-Escobar, O. A. et al. 2016. Rumbling orchids: how to assess
divergent evolution between chloroplast endosymbionts and
the nuclear host. – Syst. Biol. 65: 51–65.
Pérez-Escobar, O. A. et al. 2017. Recent origin and rapid speciation
of Neotropical orchids in the world’s richest plant biodiversity
hotspot. – New Phytol. 215: 891–905.
Pinker, R. 1965. Interessante und neue Funde und Erkenntnisse
fur die Lepidopterenfauna der Kanaren III. – Zeitschrift der
Wiener Entomologischen Gesellschaft 50: 153–167, pls
119–123.
Pridgeon, A. et al. 2001. Genera Orchidacearum, Vol. 2:
Orchidoideae (Part 1). – Oxford Univ. Press.
Ratnasingham, S. and Hebert, P. D. 2007. BOLD: the barcode of
life data system (<www.barcodinglife.org>). – Mol. Ecol.
Notes 7: 355–364.
Ratnasingham, S. and Hebert, P. D. 2013. A DNA-based registry
for all animal species: the barcode index number (BIN) system.
– PLoS One 8: e66213.
Reyes-Betancort, J. A. et al. 2008. Diversity, rarity and the evolution
and conservation of the Canary Islands endemic flora. – Anales
Jard. Bot. Madrid, pp. 25–45.
Seguí, J. et al. 2017. Species–environment interactions changed by
introduced herbivores in an oceanic high-mountain ecosystem.
– AoB Plants 9, doi: 10.1093/aobpla/plw084.
Senghas, K. 1992. Habenaria. – In: Schlechter, R. et al. (eds), Die
Orchideen: ihre Beschreibung, Kultur und Züchtung. Erster
Band. Teil A, Botanische Grundlagen der Orchideenforschung,
Taxonomischer Teil. Parey, p. 1128.
Singer, R. et al. 2007. The pollination mechanism of Habenaria
pleiophylla Hoehne & Schlechter (Orchidaceae: Orchidinae).
– Funct. Ecosyst. Commun. 1: 10–14.
Singer, R. B. 2001. Pollination biology of Habenaria parviflora
(Orchidaceae: Habenariinae) in southeastern Brazil.
– Darwiniana 39: 201–207.
Sletvold, N. and Ågren, J. 2010. Pollinator-mediated selection on
floral display and spur length in the orchid Gymnadenia
conopsea. – Int. J. Plant Sci. 171: 999–1009.
Solana, M. and Aparicio, A. 1999. Reconstruction of the 1706
Montaña Negra eruption. Emergency procedures for Garachico
and El Tanque, Tenerife, Canary Islands. – Geol. Soc. Lond.
Spec. Publ. 161: 209–216.
Steinbauer, M. J. and Beierkuhnlein, C. 2010. Characteristic pattern of species diversity on the Canary Islands. – Erdkunde 64:
57–71.
Stierstorfer, C. and von Gaisberg, M. 2006. Annotated checklist
and distribution of the vascular plants of El Hierro, Canary
Islands, Spain. – Englera 27: 3–221.
Subedi, A. et al. 2011. Pollination and protection against herbivory
of Nepalese Coelogyninae (Orchidaceae). – Am. J. Bot. 98:
1095–1103.
Suetsugu, K. and Tanaka, K. 2014. Diurnal butterfly pollination
in the orchid H. abenaria radiata. – Entomol. Sci. 17:
443–445.
Szlachetko, D. L. and Rutkowski, P. 2000. Gynostemia orchidalium
I. – Acta Bot. Fenn. 169: 1–380.
Tałałaj, I. and Brzosko, E. 2008. Selfing potential in Epipactis
palustris, E. helleborine and E. atrorubens (Orchidaceae). – Plant
Syst. Evol. 276: 21–29.
Tao, Z.-B. et al. 2018. Nocturnal hawkmoth and noctuid moth
pollination of Habenaria limprichtii (Orchidaceae) in
sub-alpine meadows of the Yulong Snow Mountain (Yunnan,
China). – Bot. J. Linn. Soc. 187: 483–498.
Van der Pijl, L. and Dodson, C. H. 1966. Orchid flowers: their
pollination and evolution. – Fairchild Tropical Garden and the
Univ. of Miami Press.
Vives Moreno, A. 2014. Catálogo sistemático y sinonímico de los
Lepidoptera de la Península Ibérica, de Ceuta, de Melilla y de
las Islas Azores, Baleares, Madeira y Salvajes (Insecta:
Lepidoptera).
Wasserthal, L. 1997. The pollinators of the Malagasy star orchids
Angraecum sesquipedale, A. sororium and A. compactum and the
evolution of extremely long spurs by pollinator shift. – Bot.
Acta 110: 343–359.
Wasserthal, L. T. 1998. Deep flowers for long tongues. – Trends
Ecol. Evol. 13: 459–460.
Whittall, J. B. and Hodges, S. A. 2007. Pollinator shifts drive
increasingly long nectar spurs in columbine flowers. – Nature
447: 706–709.
Witt, T. J. et al. 2011. Subfamilia Arctiinae. – In: Witt, T. and
Ronkay, L. (eds), Noctuidae Europaeae, volume 13. Lymantriinae
and Arctiinae including phylogeny and check list of the
Quadrifid Noctuoidea of Europe. Entomological Press,
pp. 81–217, pls 214–220, 271–312, 323–361.
Xiong, Y.-Z. et al. 2015. Mast fruiting in a hawkmoth-pollinated
orchid Habenaria glaucifolia: an 8-year survey. – J. Plant Ecol.
8: 136–141.
Supplementary material (available online as Appendix njb02401 at < www.nordicjbotany.org/appendix/njb-02401 >).
Appendix 1.
15