![Localities of nine Dactylorhiza populations in northeastern Poland. D. majalis (DM), KA, SKI, and SKII; D. incarnata var. incarnata (DI), ZB, MR, and RO; D. fuchsii (DF) CM, BR, and GR (Wróblewska et al. 2024a [24]).](https://www.researchgate.net/publication/387826783/figure/fig1/AS:11431281371335123@1744367747459/Localities-of-nine-Dactylorhiza-populations-in-northeastern-Poland-D-majalis-DM-KA_Q320.jpg)
![Spatial correlograms for D. majalis, D. incarnata var. incarnata, and D. fuchsii populations with the mean pairwise kinship coefficients (Fij) of distance classes for AFLPs with respect to the hypothesis of random genetic structure obtained by permuting individual spatial locations, as implemented in SPAGeDi 1.4 [6]. The dotted lines indicate the 99% confidence intervals obtained from 10,000 permutations of genotypes. Codes of populations (KA, SKI, SKII, ZA, MR, RO, CM, BR, and GR; see Table 1); * p < 0.05.](https://www.researchgate.net/publication/387826783/figure/fig2/AS:11431281371412863@1744367749663/Spatial-correlograms-for-D-majalis-D-incarnata-var-incarnata-and-D-fuchsii_Q320.jpg)
![Locations of D. majalis (DM), D. incarnata var. incarnata (DI), and D. fuchsii (DF) populations in NE Poland and summary statistics of the genetic diversity and spatial genetic structure estimated using SPAGeDi 1.4 [6]. N-number of AFLP samples; PL%-frequency of polymorphic loci; H-Nei's gene diversity; F IS -inbreeding coefficient; CI-the upper and lower 99% confidence interval values; F ij(1) -mean pairwise kinship coefficient among individuals at the first distance class; b 1 -regression slope of pairwise kinship at the first distance; S p -the intensity of the FSGS according Veckemans and Hardy [6]. Voucher specimens were collected by Ada Wróblewska and deposited in the herbarium of the Faculty of Biology, University of Bialystok, Poland. * p < 0.05.](https://www.researchgate.net/publication/387826783/figure/tbl1/AS:11431281302231439@1736360487135/Locations-of-D-majalis-DM-D-incarnata-var-incarnata-DI-and-D-fuchsii-DF_Q320.jpg)
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Academic Editor: Hui Li
Received: 7 December 2024
Revised: 1 January 2025
Accepted: 6 January 2025
Published: 8 January 2025
Citation: Wróblewska, A.;
Ostrowiecka, B.; Jermakowicz, E.;
Tałałaj, I. Concordant Patterns of
Population Genetic Structure in
Food-Deceptive Dactylorhiza Orchids.
Genes 2025,16, 67. https://doi.org/
10.3390/genes16010067
Copyright: © 2025 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license
(https://creativecommons.org/
licenses/by/4.0/).
Article
Concordant Patterns of Population Genetic Structure in
Food-Deceptive Dactylorhiza Orchids
Ada Wróblewska * , Beata Ostrowiecka, Edyta Jermakowicz and Izabela Tałałaj
Faculty of Biology, University of Bialystok, Ciołkowskiego 1J Street, 15-245 Białystok, Poland;
b.ostrowiecka@uwb.edu.pl (B.O.); edytabot@uwb.edu.pl (E.J.); izagry@uwb.edu.pl (I.T.)
*Correspondence: adabot@uwb.edu.pl
Abstract: Background: The patterns of inbreeding coefficients (F
IS
) and fine spatial genetic
structure (FSGS) were evaluated regarding the mating system and inbreeding depression
of food-deceptive orchids, Dactylorhiza majalis,Dactylorhiza incarnata var. incarnata, and
Dactylorhiza fuchsii, from NE Poland. Methods: We used 455 individuals, representing nine
populations of three taxa and AFLPs, to estimate percent polymorphic loci and Nei’s gene
diversity, which are calculated using the Bayesian method; F
IS
;F
ST
; FSGS with the pairwise
kinship coefficient (Fij); and AMOVA in populations. Results: We detected a relatively
high proportion of polymorphic fragments (40.4–68.4%) and Nei’s gene diversity indices
(0.140–0.234)
. The overall F
IS
was relatively low to moderate
(0.071–0.312)
. The average
Fij for the populations of three Dactylorhiza showed significantly positive values, which
were observed between plants at distances of 1–10 m (20 m). F
ST
was significant in each
Dactylorhiza taxon, ranging from the lowest values in D. fuchsii and D. majalis (0.080–0.086,
p< 0.05
) to a higher value (0.163, p< 0.05) in D. incarnata var. incarnata. Molecular variance
was the highest within populations (76.5–86.6%; p< 0.001). Conclusions: We observed
concordant genetic diversity patterns in three food-deceptive, allogamous, pollinator-
dependent, and self-compatible Dactylorhiza.F
IS
is often substantially higher than Fij
with respect to the first class of FSGSs, suggesting that selfing (meaning of geitonogamy)
is at least responsible for homozygosity. A strong FSGS may have evolutionary conse-
quences in Dactylorhiza, and combined with low inbreeding depression, it may impact the
establishment of inbred lines of D. majalis and D. incarnata var. incarnata.
Keywords: Dactylorhiza fuchsii;Dactylorhiza incarnata var. incarnata;Dactylorhiza majalis;F
IS
;
spatial genetic structure
1. Introduction
The mating system influences the genetic structure of plant populations by altering
the drift/migration equilibrium, which is defined by the effective population size
[1,2]
.
Theoretical and experimental studies have frequently shown that pollen transfer in out-
crossing species results in lower genetic structure and higher genetic diversity than in
self-pollinating species. In the latter, it is often attributed to a significant founder effect,
which tends to increase inbreeding in selfers [
2
–
4
]. However, pollen flow may act in concert
with life-history traits, such as dispersal mechanisms (by wind or animals), and com-
bined, they exhibit high statistical power for predicting the scale of the fine spatial genetic
structure (FSGS) within populations [
5
,
6
]. When pollen and seed dispersal are restricted,
resulting in significant intra-population structure, biparental inbreeding can also influence
the inbreeding coefficient. Therefore, following an isolation-by-distance model, a strong
Genes 2025,16, 67 https://doi.org/10.3390/genes16010067
Genes 2025,16, 67 2 of 11
FSGS is frequent even within allogamous or potentially allogamous plants [
6
]. Higher
levels of FSGS have also been highlighted for selfing and clonal species in low-density
populations [
6
–
8
]. Variations in mating systems and different soil and climatic conditions
may additionally contribute to different FSGS patterns [
9
]. F
IS
also reflects inbreeding in
previous generations of perennials, resulting in the Wahlund effect on the population [
10
].
Mating systems and seed dispersal also influence F
ST
via its impact on pollen-mediated
and short and/or leptokurtic gene flow and the effective population size, especially during
selfing and mating between relatives, by increasing inbreeding, which enhances genetic
drift. A summary report by Duminil et al. [
11
], which analyzed data from 263 plant species,
indicated that the inbreeding coefficient (F
IS
) observed at the adult plant stage enables the
assessment of the effects of both biparental inbreeding and inbreeding depression on popu-
lation genetic structure. In the early stages of the plant life cycle, inbreeding depression
primarily impacts inbred progeny. As a result, the F
IS
of adult plants reflects information
about both the selfing rate and inbreeding depression.
The mating system and gene flow within and among populations in Orchidaceae can
generate common genetic diversity patterns and FSGSs [
12
]. Pollinator-mediated gene flow
among populations, e.g., was higher in deceptive than in rewarding orchids
[12,13]
. The
deceived pollinators generally visit only a limited number of flowers among plants within
populations, facilitating cross-pollination and decreasing the chances of inbreeding [
14
–
19
].
Therefore, it can be hypothesized that the FSGS of these orchids is weak. In contrast, within-
population genetic structure could be stronger in rewarding ones due to geitonogamy
and mating among close relatives. However, based on earlier experimental orchid stud-
ies, dusty-like seed dispersal was usually limited, e.g., refs. [
20
–
25
]. These results agree
with studies that have investigated the FSGSs of both deceptive orchids—e.g., Caladenia
tentaculata [
26
], Cephalanthera longibracteata [
22
], Orchis cyclochila [
23
], Orchis purpurea [
27
],
Orchis mascula
[28,29]
, and Cymbidium goeringii [
30
]—and rewarding ones—e.g., Gymnadenia
conopsea [
31
], Pogonia ophioglossoides [
32
], and Epipactis thunbergii [
33
]. Moreover, orchid
germination success has been reported to be higher in the vicinity of mother plants because
a mycorrhiza could favour the establishment of seedlings [34,35].
In this study, we focused on Dactylorhiza taxa, which are food-deceptive orchids that do
not provide any rewards for their pollinators [
36
]. This genus can be considered a model due
to its plant–pollinator interactions, natural selection, and consequent female reproductive
success and its impact on genetic structure in food-deceptive plant groups
[37–39]
. In
this context, the mating system and ID in food-deceptive D. majalis,D. incarnata var.
incarnata, and D. fuchsii populations from NE Poland were documented in detail [
40
–
43
].
A mixed mating system was observed in all three studied Dactylorhiza taxa, similarly
to Hedrén and Nordström’s study [
44
]. Ostrowiecka et al. [
40
] reported that pollinator
behaviour in D. majalis likely encourages geitonogamy, which accounts for the formation
of selfed seeds in fruits at various inflorescence levels, exhibiting germination potential
comparable to that of outcrossed seeds within populations. Vallius et al. [
45
] and Hedrén
and Nordstöm [
44
] proved that different D. incarnata varieties were characterized by a high
level of inbreeding, and populations might consist of several inbred lines that were fixed
for characters, especially with respect to flower colour. Wróblewska et al.’s [
42
] results
corroborate with previous studies on Dactylorhiza concerning the low or medium level of
fruit sets ranging from 7.4% to 77.5% [
36
,
45
,
46
].
In vitro
experiments revealed that the seed
germination of three Dactylorhiza taxa from both natural pollination and hand treatments
(selfing and outcrossing) occurred at a relatively low level, up to 35% (with the exception
of D. fuchsii and outcrossing experiments) [
42
].
In vitro
asymbiotic seed germination was
similar or slightly higher in selfing than crossing experiments in D. incarnata var. incarnata
and D. majalis, while it was reversed in D. fuchsii [
42
]. Spontaneous autogamy in three
Genes 2025,16, 67 3 of 11
Dactylorhiza taxa existed in <1% of pollination in the studied populations and most likely
did not affect reproductive success [
47
,
48
]. The taxa are characterized as terrestrial, long-
lived, self-compatible, tuberous perennial orchids that reproduce primarily via seeds, with
vegetative reproduction occurring rarely [
40
,
49
]. Pollination is carried out by various
insect groups, including Hymenoptera, Diptera, and Coleoptera and predominantly bees
and bumblebees [
40
,
41
]. Molecular markers such as cpDNA (trnL, trnF, and psbC–trnK),
internal transcribed spacer (ITS) sequences, and flow cytometry data have confirmed the
taxonomic status of the three orchids studied [41].
Based on the estimates of an earlier ecological survey, e.g., natural fruit sets, a mixed
mating system, and inbreeding depression from a controlled crosses treatment, orchid
taxa from NE Poland were studied [40,42,43], in addition to the genetic reports of Hedrén
and Nordstöm [
44
] and Naczk et al. [
49
]. We tested the following hypothesis: inbreeding
coefficients are shaped at a high level in food-deceptive orchids D. majalis,D. incarnata var.
incarnata, and D. fuchsii. We also assumed that seed dispersal mainly occurs over a short
distance in orchids that are close to the mother plant, as was observed by many authors
who experimentally researched seed dispersal; therefore, fine-scale genetic structure is
stronger due to the effect of inbreeding and short-distance dispersal. Finally, the purpose
of this study is to (1) estimate the inbreeding coefficient and the intensity of the FSGS
using AFLP markers and (2) discuss how similar mating systems and different inbreeding
depression shape the genetic diversity patterns of three food-deceptive Dactylorhiza taxa.
2. Materials and Methods
2.1. Study Sites
The present study was conducted from May to July between 2014 and 2017 across three
populations of D. majalis (KA, SKI, and SKII), three populations of D. incarnata var. incarnata
(ZB, RO, and MR), and three populations of D. fuchsii (BR, CM, and GR) in northeastern
Poland (Figure 1). D. majalis grows in wet meadows filled with abundant, entomophilous,
and rewarding plants. The study sites varied in the number of D. majalis individuals, with
approximately 120–200 flowering individuals in SKI and SKII and ca. 1000 in KA. All
meadows were managed extensively and mowed annually in late July or early August,
and they were not subjected to artificial fertilization. The three populations of D. incarnata
var. incarnata were of similar size, with MA having ca. 68–100 flowering plants, ZB with
approximately 30–100, and RO with 35–200 (Figure 1). These populations were located in
the Biebrza Valley and Rospuda Valley, occupying sedge communities with a low cover of
rewarding plant species (ca. 10%). Dactylorhiza fuchsii was found in open hornbeam forests
with a limited number of rewarding plants, specifically in the Białowie˙
za Primeval Forest
and nearby areas (CM and BR, with population sizes of approximately 84–133 flowering
plants). One D. fuchsii population (GR, with ca. 140–193 flowering plants) was situated in
the Biebrza Valley [42].
The study involved samples from 455 individuals across nine populations of three
Dactylorhiza taxa, including 162 individuals of D. majalis (DM), 129 of D. incarnata var.
incarnata (DI), and 164 of D. fuchsii (DF) (Table 1; Figure 1). Although Dactylorhiza rarely
regenerates clonally, one leaf sample was collected from individual shoots at least 1 m
apart within D. majalis populations to minimize the effects of population substructure. For
D. incarnata var. incarnata and D. fuchsii, samples were collected based on the positions
of individuals within their respective populations, which were characterized by different
flowering individual densities. Each sample from all populations was mapped using a
grid coordinate system with a handheld GPS (Garmin GPSMAP 65s) to facilitate distance
calculations between samples.
Genes 2025,16, 67 4 of 11
Genes2025,16,xFORPEERREVIEW4of12
Figure1.LocalitiesofnineDactylorhizapopulationsinnortheasternPoland.D.majalis(DM),KA,
SKI,andSKII;D.incarnatavar.incarnata(DI),ZB,MR,andRO;D.fuchsii(DF)CM,BR,andGR
(Wróblewskaetal.2024a[24]).
Thestudyinvolvedsamplesfrom455individualsacrossninepopulationsofthree
Dactylorhizataxa,including162individualsofD.majalis(DM),129ofD.incarnatavar.
incarnata(DI),and164ofD.fuchsii(DF)(Table1;Figure1).AlthoughDactylorhizararely
regeneratesclonally,oneleafsamplewascollectedfromindividualshootsatleast1meter
apartwithinD.majalispopulationstominimizetheeffectsofpopulationsubstructure.For
D.incarnatavar.incarnataandD.fuchsii,sampleswerecollectedbasedonthepositionsof
individualswithintheirrespectivepopulations,whichwerecharacterizedbydifferent
floweringindividualdensities.Eachsamplefromallpopulationswasmappedusinga
gridcoordinatesystemwithahandheldGPS(GarminGPSMAP65s)tofacilitatedistance
calculationsbetweensamples.
Table1.LocationsofD.majalis(DM),D.incarnatavar.incarnata(DI),andD.fuchsii(DF)populations
inNEPolandandsummarystatisticsofthegeneticdiversityandspatialgeneticstructureestimated
usingSPAGeDi1.4[6].N—numberofAFLPsamples;PL%—frequencyofpolymorphicloci;H—
Nei’sgenediversity;FIS—inbreedingcoefficient;CI—theupperandlower99%confidenceinterval
values;Fij(1)—meanpairwisekinshipcoefficientamongindividualsatthefirstdistanceclass;b1—
regressionslopeofpairwisekinshipatthefirstdistance;Sp—theintensityoftheFSGSaccording
VeckemansandHardy[6].VoucherspecimenswerecollectedbyAdaWróblewskaanddeposited
intheherbariumoftheFacultyofBiology,UniversityofBialystok,Poland.*p<0.05.
TaxaPopulationGPSNPL%HFIS(CI)Fij(1)b1Sp
DMKA52°53′00″N
23°40′29″E4962.20.2050.293(0.000–1.000)0.095*−0.051*0.056
SKI52°49′50″N
23°43′10″E5959.60.2050.312(0.000–1.000)0.038*−0.009*0.001
SKII52°49′50″N
23°43′10″E5440.40.1400.192(0.000–1.000)0.071*−0.021*0.022
DIZB53°29′02″N
22°59′28″E4858.60.2170.179(0.101–0.284)0.008−0.0020.0002
Figure 1. Localities of nine Dactylorhiza populations in northeastern Poland. D. majalis (DM), KA,
SKI, and SKII; D. incarnata var. incarnata (DI), ZB, MR, and RO; D. fuchsii (DF) CM, BR, and GR
(Wróblewska et al. 2024a [24]).
Table 1. Locations of D. majalis (DM), D. incarnata var. incarnata (DI), and D. fuchsii (DF) populations
in NE Poland and summary statistics of the genetic diversity and spatial genetic structure estimated
using SPAGeDi 1.4 [
6
]. N—number of AFLP samples; PL%—frequency of polymorphic loci; H—Nei’s
gene diversity; F
IS
—inbreeding coefficient; CI—the upper and lower 99% confidence interval values;
F
ij(1)
—mean pairwise kinship coefficient among individuals at the first distance class; b
1
—regression
slope of pairwise kinship at the first distance; S
p
—the intensity of the FSGS according Veckemans and
Hardy [
6
]. Voucher specimens were collected by Ada Wróblewska and deposited in the herbarium of
the Faculty of Biology, University of Bialystok, Poland. * p< 0.05.
Taxa Population GPS N PL%H FIS (CI) Fij (1) b1Sp
DM KA 52◦53′00′′ N
23◦40′29′′ E49 62.2 0.205 0.293 (0.000–1.000) 0.095 * −0.051 * 0.056
SKI 52◦49′50′′ N
23◦43′10′′ E59 59.6 0.205 0.312 (0.000–1.000) 0.038 * −0.009 * 0.001
SKII 52◦49′50′′ N
23◦43′10′′ E54 40.4 0.140 0.192 (0.000–1.000) 0.071 * −0.021 * 0.022
DI ZB 53◦29′02′′ N
22◦59′28′′ E48 58.6 0.217 0.179 (0.101–0.284) 0.008 −0.002 0.0002
RO 53◦54′39′′ N
22◦56′32′′ E48 58.6 0.197 0.071 (0.022–0.149) 0.224 * −0.055 * 0.063
MR 53◦47′25′′ N
22◦57′22′′ E33 58.1 0.206 0.098 (0.032–0.218) 0.092 * −0.037 * 0.041
DF CM 52◦41′03′′ N
23◦39′07′′ E58 68.4 0.234 0.113 (0.034–0.244) 0.078 * −0.021 * 0.019
BR 52◦50′59′′ N
23◦53′40′′ E57 63.9 0.211 0.134 (0.068–0.226) 0.084 * −0.026 * 0.028
GR 53◦60′68′′ E
22◦84′68′′ N49 56.7 0.197 0.079 (0.024–0.169) −0.008 0.002 −0.0002
2.2. AFLP Analysis
Genomic DNA was extracted from dried leaf tissues using the Genomic Mini AX
Plant kit (A & A Biotechnology, Gdansk, Poland), and the samples were genotyped for
AFLP markers. The AFLP procedure, as outlined by Vos et al. [
50
], was adapted following
the Applied Biosystems protocol (AFLPTM Plant Mapping). Initially, 12 primer pair
combinations were tested on four selected samples from each Dactylorhiza taxon. The
Genes 2025,16, 67 5 of 11
GeneScan 500 Liz-labelled size standard (Applied Biosystems, Waltham, MA, USA) was
employed for DNA analysis on an ABI 3130. Subsequently, seven primer combinations
were selected that yielded polymorphic, clear, and reproducible fragments of consistent
intensities across the three Dactylorhiza taxa (D. majalis EcoR1-ACC/MseI-CAG, EcoR1-
AGG/MseI-CAC; D. incarnata var. incarnata EcoR1-ACA/MseI-CAG, EcoR1-ACA/MseI-
CTA; D. fuchsii EcoR1-AGG/MseI-CAG, EcoR1-ACC/MseI-CAT, EcoR1-ACC/MseI-CTA).
Variable fragments in the 70–500 bp size range were recorded as present (1) or absent
(0) using GeneMapper 4.0 (Applied Biosystems). To assess the repeatability of the AFLP
results, three individuals from each population were fully replicated, starting from the
restriction/ligation step of the AFLP process. The potential resampling of clones was
evaluated using the AFLPdat R-script but was determined to be insignificant and therefore
not corrected for.
To evaluate genetic diversity, the proportion of polymorphic fragments (PL%) and
Nei’s gene diversity (H) were calculated using the Bayesian method with a nonuniform
prior distribution of allele frequencies as proposed by Zhivotovsky [
51
] and implemented
in AFLP-Surv version 1.0 [
52
]. The Fstatistic was determined through analysis of molecular
variance (AMOVA) using Arlequin 3.11 [
53
], with the significance of variance components
assessed using 1000 independent permutation runs.
The fine-scale genetic structure (FSGS) was analyzed through spatial autocorrela-
tion using the pairwise kinship coefficient F
ij
for dominant markers [
54
]. Mean F
ij
esti-
mates for pairs of individuals across specified distance classes were calculated and plotted
against distance on a logarithmic scale with SPAGeDi 1.4 [
6
,
54
]. Distinct distance classes
were created for each population of D. majalis,D. incarnata var. incarnata, and D. fuchsii
due to varying spatial distribution/density patterns. To evaluate the significance of the
FSGS, the regression slopes (b) of kinship coefficients against the natural logarithm of
distance were compared to slopes obtained from the permutations of individual genotypes
(
10,000 random permutations
). The extent of the FSGS was quantified using the S
p
statistic
as proposed by Vekemans and Hardy [
6
] and calculated as S
p
=
−
b/(1
−
F
1
), where bis the
regression slope and F
1
represents the average F
ij
between individuals. For each spatial
distance class, the 99% confidence interval was determined using 10,000 permutations
(with SPAGeDi) [
55
]. The probability value (p) was computed for each spatial distance class
and coefficient.
To investigate the F
IS
, the Metropolis–Gibbs algorithm was applied in the I4A software
based on dominant markers [
56
]. The data were run using prior values of beat distri-
bution equal to
α
=
β
= 1.0 (corresponding to an “uninformative” flat distribution) and
60,000 repetitions, including a 10,000-step burn-in.
3. Results
Overall, 193, 215, and 263 polymorphic bands were scored in D. majalis,D. incarnata
var. incarnata, and D. fuchsii, respectively. Considering the error rates (2%, 1.3%, and 1.5%,
respectively), none of the samples may have represented clones.
Relatively high proportions of polymorphic fragments (PL% = 40.4–68.4%) and Nei’s
gene diversity indices (H= 0.140–0.234) were detected among the three orchid species
(Table 1). The overall F
IS
was relatively low to moderate, and it equaled 0.071–0.224 in
D. incarnata var. incarnata and 0.079–0.134 in D. fuchsii; it reached the highest values of
0.192–0.312 in D. majalis.
The correlograms of the average F
ij
values for the populations of three Dactylorhiza taxa
exhibited significantly positive values, which were observed in the short-distance classes.
In D. majalis, the relatives were noted at a distance from 1 m to 10 m (Figure 2), and the
values were significantly negative with respect to the longer distance classes (52–72 m) in
Genes 2025,16, 67 6 of 11
the two other populations. Similar observations were made in two of the three populations
of D. incarnata var. incarnata and D. fuchsii. Significant positive values were observed at a
distance from 2 m to 20 m in D. incarnata var. incarnata (Figure 2) and from 2 m to 10 m in
D. fuchsii (Figure 2). The b
F
values for D. majalis (
−
0.051–0.009), D. incarnata var. incarnata
(
−
0.055–0.002), and D. fuchsii (
−
0.026–0.002) were almost all significant (permutation test,
p< 0.05
) (Table 1). The highest Sp values were observed for D. incarnata var. incarnata
(0.063) and D. majalis (0.056) (Table 1).
Genes2025,16,xFORPEERREVIEW6of12
3.Results
Overall,193,215,and263polymorphicbandswerescoredinD.majalis,D.incarnata
var.incarnata,andD.fuchsii,respectively.Consideringtheerrorrates(2%,1.3%,and1.5%,
respectively),noneofthesamplesmayhaverepresentedclones.
Relativelyhighproportionsofpolymorphicfragments(PL%=40.4.5–68.4%)and
Nei’sgenediversityindices(H=0.140–0.234)weredetectedamongthethreeorchidspe-
cies(Table1).TheoverallFISwasrelativelylowtomoderate,anditequaled0.071–0.224
inD.incarnatavar.incarnataand0.079–0.134inD.fuchsii;itreachedthehighestvaluesof
0.192–0.312inD.majalis.
ThecorrelogramsoftheaverageFijvaluesforthepopulationsofthreeDactylorhiza
taxaexhibitedsignificantlypositivevalues,whichwereobservedintheshort-distance
classes.InD.majalis,therelativeswerenotedatadistancefrom1mto10m(Figure2),
andthevaluesweresignificantlynegativewithrespecttothelongerdistanceclasses(52–
72m)inthetwootherpopulations.Similarobservationsweremadeintwoofthethree
populationsofD.incarnatavar.incarnataandD.fuchsii.Significantpositivevalueswere
observedatadistancefrom2mto20minD.incarnatavar.incarnata(Figure2)andfrom
2mto10minD.fuchsii(Figure2).ThebFvaluesforD.majalis(−0.051–0.009),D.incarnata
var.incarnata(−0.055–0.002),andD.fuchsii(−0.026–0.002)werealmostallsignificant(per-
mutationtest,p<0.05)(Table1).ThehighestSpvalueswereobservedforD.incarnatavar.
incarnata(0.063)andD.majalis(0.056)(Table1).
Figure2.SpatialcorrelogramsforD.majalis,D.incarnatavar.incarnata,andD.fuchsiipopulations
withthemeanpairwisekinshipcoefficients(Fij)ofdistanceclassesforAFLPswithrespecttothe
hypothesisofrandomgeneticstructureobtainedbypermutingindividualspatiallocations,asim-
plementedinSPAGeDi1.4[6].Thedoedlinesindicatethe99%confidenceintervalsobtainedfrom
10,000permutationsofgenotypes.Codesofpopulations(KA,SKI,SKII,ZA,MR,RO,CM,BR,and
GR;seeTable1);*p<0.05.
Figure 2. Spatial correlograms for D. majalis, D. incarnata var. incarnata, and D. fuchsii populations
with the mean pairwise kinship coefficients (F
ij
) of distance classes for AFLPs with respect to the
hypothesis of random genetic structure obtained by permuting individual spatial locations, as
implemented in SPAGeDi 1.4 [
6
]. The dotted lines indicate the 99% confidence intervals obtained
from
10,000 permutations
of genotypes. Codes of populations (KA, SKI, SKII, ZA, MR, RO, CM, BR,
and GR; see Table 1); * p< 0.05.
Almost all F
ST
values were significant in each Dactylorhiza taxon, ranging from the
lowest values in D. fuchsii and D. majalis (0.080 and 0.086, p< 0.05, permutation test) to the
highest value (0.163, p< 0.05, permutation test) in D. incarnata var. incarnata. The amount
of molecular variance was highest within populations, and it was maintained at relatively
higher and similar levels in D. majalis,D. incarnata var. incarnata, and D. fuchsii (AMOVA:
76.5%, 85.5%, and 86.6%; p< 0.001, respectively).
4. Discussion
The relationships among mating systems, inbreeding depression, biparental inbreed-
ing, and their effects on F
IS
and F
ST
have been infrequently documented in plant sur-
veys [
11
]. Baskin and Baskin [
57
] summarized the impact of inbreeding depression on seed
germination across 743 instances involving 233 species from 64 families. They found that
in 50.1% of the cases, inbred and outcrossed seeds germinated at comparable frequencies,
while 8.1% of inbred ones exhibited better germination rates than outcrossed seeds. In-
terestingly, the authors observed no strong correlation between decreased germination
Genes 2025,16, 67 7 of 11
rates and increased F
IS
nor between increased germination rates and heightened levels of
population genetic diversity. However, we observed concordant genetic diversity patterns
in food-deceptive, allogamous, and pollinator-dependent populations, although these were
also self-compatible with the mixed mating systems of D. majalis,D. incarnata var. incarnata,
and D. fuchsii. Genetic diversity within studied Dactylorhiza populations was shaped at
a relatively high level comparable to the data reported by Naczk et al. [
49
] and Hedrén
and Nordström [
44
,
58
], suggesting that studied Dactylorhiza populations can be found via
genetically different individuals and/or gene flow via leptokurtic dispersal. The genetic
differentiation among them was low and significant (0.080–0.163), showing that gene flow
(historical) in northeastern Poland was relatively high or populations were established
from one source. However, the isolation processes of these Dactylorhiza populations were
observed, resulting in the formation of a substructure.
Furthermore, the inbred population was shaped from moderate to high levels in three
Dactylorhiza taxa, similarly to the studies of Hedrén and Nordström [
44
], Filippov et al. [
59
],
and Naczk et al. [
49
]. Meanwhile, in D. majalis as the allotetraploid, F
IS
exhibited a wide
range of values in the populations reported by Balao et al. [
60
], Hedrén and Nordström [
44
],
and Naczk and Zi˛etara [
61
]. Let us assume the prediction that inbreeding is solely the result
of mating among neighbouring plants. In this case, we expect F
IS
to be approximately equal
to F
ij
at the smallest distance interval in the studied Dactylorhiza populations [
6
]. In our
survey, F
IS
was substantially higher than F
ij
with respect to the first class of spatial distances
in most Dactylorhiza populations, suggesting that selfing is at least partially responsible for
homozygosity [
56
,
62
]. However, spontaneous autogamy in three Dactylorhiza taxa existed
until 1% of pollination in the studied populations [
47
,
48
]. Hence, the only explanation
of selfing in three Dactylorhiza taxa is the pollinator behaviour of bumblebees and other
pollinators, which are known to promote geitonogamy and/or autogamy, explaining the
development of selfed seeds [
40
,
41
]. The important factor shaping F
IS
was the slightly
higher selfing frequency compared to outcross seeds germinated in
in vitro
treatments
in D. majalis and D. incarnata var. incarnata, while in D. fuchsii, the germination pattern
was reversed [
42
]. This phenomenon suggested that inbred and outbred D. majalis and D.
incarnata var. incarnata seeds germinated at a similar or even slightly higher frequency. We
stress the careful interpretation of the relationship between seed germination and F
IS
. This
needs to be confirmed in further studies, including the growth and mortality observations
of plants germinated from selfed and outcrossed seeds in the following stages. However,
our data are related to a single studied Dactylorhiza species, and we can suppose that in D.
fuchsii, a similar pattern exists in two out of three populations, such as D. majalis and D.
incarnata var. incarnata. In the CM and BR populations, high inbreeding and slightly lower
kinship coefficients supported the possibility of selfing (geitonogamy). The interesting
question is whether biparental inbreeding can exist in food-deceptive Dactylorhiza taxa,
even though pollinators spend a short time period on flowers and inflorescence and learn
to avoid deceptive flowers. They typically only visit fewer flowers per plant and/or a
few flowers between inflorescences within populations, promoting cross-pollination and
skipping more plants between plant visits. In light of this outcrossing hypothesis [
15
],
biparental inbreeding is rather unlikely. Using videotaping, Ostrowiecka et al. [
40
] observed
that Apis mellifera visited three to five flowers on the same inflorescence within 11 to 40 s,
contributing to geitonogamy. Our observations noted that A. mellifera pollinators did not
return to the same flowers and avoided visiting all flowers on the inflorescences. Conversely,
the bending of pollinaria serves as a mechanism to prevent geitonogamy and biparental
inbreeding among closely related individuals. In Dactylorhiza, the average bending time
is 39–54 s, which is considered relatively long for deceptive plants and similar to other
deceptive Dactylorhiza taxa [
63
]. The bending time observed in each studied D. majalis
Genes 2025,16, 67 8 of 11
population ranged from 8 s to 2 min and 5 s [
40
]. This relatively short bending time may be
an opportunity for geitonogamy. This observation and the bending times in the studied
Dactylorhiza populations support our hypothesis that geitonogamy cannot be completely
ruled out in deceptive orchids compared to biparental inbreeding. Dactylorhiza seems
to possess a more generalized pollination system, and numerous pollinators have been
described and studied in detail. These pollinators can spend different amounts of time on
the flowers, promoting geitonogamy.
Hand pollination using emasculated flowers was employed to assess the extent of
apparent geitonogamy occurring via pollinators [
64
,
65
]. A previous fruit set observation
from controlled pollination in three Dactylorhiza documented a moderate level of fruit set
(35.4–40.5%). Simultaneously, emasculation experiments in their populations showed a
significant decrease in fruiting between these treatments (D. majalis, 28.2% fruit set from
emasculated flower, paired t = 2.68, df = 8, p< 0.002; D. incarnata var. incarnata, 14.6%
fruit set from emasculated flower, paired t = 3.46, df = 10, p< 0.006; D. fuchsii, 28.2%
fruit set from emasculated flower, paired t = 4.83, df = 10, p< 0.0007; Wróblewska et al.
unpublished data [
66
]). This study concludes that selfing in three Dactylorhiza occurs
mainly through geitonogamy. Kropf and Renner [
17
] have also pointed out the high levels
of geitonogamous pollination in Dactylorhiza; measuring biparental inbreeding can be
challenging in deceptive plants.
The F
IS
observed at the adult stage enabled the assessment of inbreeding depression
impacts on the population’s genetic structure. In long-lived plants, F
IS
reflects inbreeding
not only in the current generation but also in previous overlapping generations. However,
other factors, such as the long lifespan of plants, can affect inbreeding depression [
11
].
In D. majalis, selfing (e.g., geitonogamy) and/or progeny and likely seed dispersal in
the vicinity of the mother plant can manifest most strongly in spatial genetic structures.
In the case of D. majalis, the results of the present study are inconsistent with those of
Husband and Schemske [
67
], who concluded that purging is a significant evolutionary
force in natural populations. Without reducing the genetic load, such fixation could
reduce inbreeding depression [
67
,
68
]. However, inbreeding depression may be lower in
long-standing populations with inbreeding than in populations with outcrossing, where
selection may have purged the genome of its genetic load [
67
–
70
]. These two alternative
approaches in a laboratory should be tested at a later stage of the life cycle of D. majalis,
such as in seedlings and adult reproductive individuals.
5. Conclusions
Selfing (meaning of geitonogamy) and a strong fine-scale genetic structure may have
additional and unexplored evolutionary consequences in Dactylorhiza, and combined with
low inbreeding depression, they may strongly influence the establishment of inbred lines
in the cases of D. majalis and D. incarnata var. incarnata. Currently, we cannot state that
inbreeding depression may be widely viewed as the primary selective factor allowing
transitions to complete selfing in Dactylorhiza. On the other hand, there are still limited
studies on breeding systems and pollinator behaviour in deceptive multi-flowered orchids,
which could shed light on geitonogamy and biparental inbreeding. Our study stressed
that different Dactylorhiza food-deceptive taxa with varying levels of inbreeding depression
can characterize similar FSGSs and inbreeding coefficients. Despite these distinct patterns
of inbreeding depression, FSGSs comprise the formation of local family structures in
Dactylorhiza taxa due to limited gene dispersal (e.g., seeds) and geitonogamy.
Author Contributions: Conceptualization, A.W.; methodology, A.W. and B.O.; software, A.W.;
validation, A.W.; formal analysis, A.W.; investigation, A.W., B.O., E.J. and I.T.; writing—original draft
preparation, A.W.; writing—review and editing, A.W.; visualization, A.W.; supervision, A.W.; project
Genes 2025,16, 67 9 of 11
administration, A.W.; funding acquisition, A.W. and I.T. All authors have read and agreed to the
published version of the manuscript.
Funding: This research was funded by the National Science Center in Poland (no. 2013/09/B/NZ8/03350).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: All data cited in the study are publicly available.
Acknowledgments: We thank Emilia Brzosko and Paweł Mirski for their help and support during fieldwork.
Conflicts of Interest: The authors declare no conflicts of interest.
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