Revista de Biología Tropical, ISSN: 2215-2075, Vol. 74: e2026160, enero-diciembre 2026 (Publicado Feb. 25, 2026)

Genetic diversity, population structure, and conservation of Cattleya trianae

(Orchidaceae) through molecular marker analysis

María Eloísa Aldana-Jauregui1*; https://orcid.org/0009-0001-7601-9061

Héiber Cárdenas-Henao2; https://orcid.org/0000-0003-2823-8443

Nelson Toro-Perea2; https://orcid.org/0000-0002-2835-7285

1. Departamento de Biología, Facultad de Ciencias, Universidad del Tolima, Calle 42 # 1-02, Ibagué, Colombia; ealdana@

ut.edu.co (*Correspondence)

2. Departamento de Biología, Facultad de Ciencias Naturales y Exactas, Universidad del Valle, Calle 13 # 100-00, Cali,

Colombia; heiber.cardenas@correounivalle.edu.co, nelson.toro@correounivalle.edu.co

Received 12-IX-2024. Corrected 04-VII-2025. Accepted 30-I-2026.

ABSTRACT

Introduction: Cattleya trianae Linden & Rchb., celebrated for its botanical splendour, holds a revered status as

the national floral emblem of Colombia, commonly known as the “Flor de Mayo” (Flower of May). However, its

survival is threatened by a variety of environmental and anthropogenic pressures, needing urgent and tailored

conservation and management strategies to conserve the remarkable genetic diversity of C. trianae in the central

Andean range of Colombia.

Objective: To elucidate the genetic architecture of C. trianae populations using random amplified polymorphic

DNA (RAPD) and chloroplast microsatellite repeat (cpSSR) markers, providing critical insights for conservation

planning, sustainable management, and the use of this species.

Results: RAPD analysis reveals unexpectedly high levels of heterozygosity, suggesting considerable genetic

variability. Genetic distances and dendrogram topologies indicate significant relatedness between populations,

while analysis of molecular variance (AMOVA) reveals considerable population structuring, primarily due to

intrapopulation differentiation. FST and Nm values challenge the assumption of isolation by distance, reflecting

a complex genetic landscape. Notably, despite the limited population sizes, there is no substantial evidence of

genetic erosion.

Conclusions: The results reveal alarmingly low intraspecific genetic diversity within these orchid populations,

highlighting their vulnerability to environmental change and stochastic events. Furthermore, the marked genetic

divergence between populations suggests the influence of multiple evolutionary forces shaping the genetic struc-

ture and distribution of C. trianae in Colombia. These findings underscore the urgency for tailored conservation

strategies to mitigate potential extinction risks.

Key words: biodiversity; endangered species; genetic analysis; nuclear markers; cpSSR; Orchidaceae.

RESUMEN

Diversidad genética, estructura poblacional y conservación de Cattleya trianae (Orchidaceae)

mediante el análisis de marcadores moleculares

Introducción: Cattleya trianae Linden & Rchb., célebre por su esplendor botánico, ostenta un venerado estatus

como emblema floral nacional de Colombia, comúnmente conocida como la “Flor de Mayo”. Sin embargo, su

supervivencia está amenazada por una variedad de presiones ambientales y antropogénicas, lo que requiere

https://doi.org/10.15517/kdq4we73

GENÉTICA

2Revista de Biología Tropical, ISSN: 2215-2075 Vol. 74: e2026160, enero-diciembre 2026 (Publicado Feb. 25, 2026)

INTRODUCTION

Cattleya trianae Linden & Rchb., an exem-

plar of botanical beauty, occupies a promi-

nent place as the national floral emblem of

Colombia, affectionately known as the ‘Flor

de Mayo’ (Flower of May). This horticultural

tribute pays homage to the eminent Colombian

botanist José Jerónimo Triana (Ossenbach &

Jenny, 2021).

Nestled in the heart of the Central Cordil-

lera, C. trianae thrives on the Eastern slopes of

the Magdalena River basin in Colombia, within

an elevation range of 700 to 1 400 m.a.s.l. This

region, within the Colombian Andes region, is

a testament to the dynamic forces of anthro-

pogenic change. The relentless growth of the

human population, the encroachment of once

virgin forests, extensive land use practices

and the expansion of illicit crops have com-

bined to create a landscape deeply affected by

human activities. These disruptions have cast a

shadow over the once undisturbed habitats of

C. trianae and forced us to take a critical look

at the genetic diversity within the populations

of this unique endemic species (Reina-Rodrí-

guez et al., 2017).

In a world increasingly concerned with bio-

diversity conservation, the utility of molecular

markers has become paramount. These mark-

ers provide a means of objectively quantifying

genetic diversity, a prerequisite for efficient

prioritisation, cost-effective resource alloca-

tion and judicious optimisation of management

strategies within conservation programmes

(Salgotra & Chauhan, 2023). The advent of

Polymerase Chain Reaction (PCR) technology,

as pioneered by Powell et al. (1995), marked a

watershed in genetic analysis. This monumen-

tal scientific achievement paved the way for the

development of techniques such as Random

Amplified Polymorphic DNA (RAPD), which

have the invaluable advantage of not requir-

ing prior knowledge of the target organism’s

genome (Nadeem et al., 2018).

C. trianae, classified in the global category

of the International Union for Conservation

of Nature (IUCN), faces the ominous prospect

of possibly joining the ranks of endangered

species, as highlighted in the “Red Book” of

Colombian plants (Calderón-Sáenz, 2007) and

in the Plan for the study and conservation of

Orchids in Colombia (Ministerio de Ambiente

y Desarrollo Sostenible & Universidad Nacional

de Colombia, 2015). This categorisation casts a

gloomy light on the vulnerability of this species,

with the main threat coming from the ongo-

ing degradation of its habitat quality. In this

estrategias de conservación y manejo urgentes y adaptadas para conservar la notable diversidad genética de C.

trianae en el área de distribución andina central de Colombia.

Objetivo: Dilucidar la arquitectura genética de las poblaciones de C. trianae utilizando marcadores de ADN

polimórfico amplificado al azar (RAPD) y microsatélites de cloroplasto repetidos (cpSSR), proporcionando

información crítica para la planificación de la conservación, la gestión sostenible y la utilización de esta especie.

Resultados: El análisis RAPD revela niveles inesperadamente altos de heterocigosidad, lo que sugiere una variabi-

lidad genética considerable. Las distancias genéticas y las topologías de los dendrogramas indican un parentesco

significativo entre poblaciones, mientras que el análisis de la varianza molecular (AMOVA) revela una conside-

rable estructuración de las poblaciones, debida principalmente a la diferenciación intrapoblacional. Los valores

de FST y Nm desafían la hipótesis del aislamiento por distancia, reflejando un paisaje genético complejo. Cabe

destacar que, a pesar del limitado tamaño de las poblaciones, no existen pruebas sustanciales de erosión genética.

Conclusiones: Los resultados revelan una diversidad genética intraespecífica alarmantemente baja dentro de estas

poblaciones de orquídeas, destacando su vulnerabilidad al cambio ambiental y a los eventos estocásticos. Además,

la marcada divergencia genética entre poblaciones sugiere la influencia de múltiples fuerzas evolutivas que mol-

dean la estructura genética y la distribución de C. trianae en Colombia. Estos hallazgos subrayan la urgencia de

estrategias de conservación a medida para mitigar los riesgos potenciales de extinción.

Palabras clave: biodiversidad; especies amenazadas; análisis genético; marcadores nucleares; cpSSR; Orchidaceae.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 74: e2026160, enero-diciembre 2026 (Publicado Feb. 25, 2026)

context, the economic importance of C. trianae

in the fields of floriculture and orchidology is

significant. Paradoxically, however, many spe-

cies within the genus Cattleya remain shrouded

in commercial obscurity, lacking the benefit of

efficient propagation protocols and, regrettably,

often overshadowed by conservation efforts.

This unfortunate situation has paved the way

for a worrying trend: the illegal collection of

plants from wild populations. This clandestine

practice, which has been going on for more

than a century and a half, has inflicted severe

damage on the species. It has not only taken its

toll in terms of population decline, but also in

the gradual erosion of the natural habitats that

C. trianae calls home (Salazar-Mercado & Vega-

Contreras, 2017).

In this context, the primary aim of our

study was to elucidate the current status of

C. trianae populations. We sought to provide

essential information not only to safeguard the

existing genetic diversity within this endan-

gered orchid species, but also to provide the

basis for the development of sustainable man-

agement strategies. In this respect, our research

plays a pivotal role in the conservation and

rejuvenation of the ecological environment of

these orchids.

MATERIAL AND METHODS

Study areas and collection of C. trianae:

Sampling was carried out in the upper Mag-

dalena Basin, located in the central Colom-

bian Cordillera, and covering the geographical

coordinates 4-4°48’0’’ N & 75-75°18’0’’ W. This

study covered an elevation range of 800 to

1 700 m.a.s.l. focusing on montane forest veg-

etation. A total of ten to 30 samples were

meticulously collected, ensuring plant viability

by taking a single leaf from each plant. These

samples were then carefully preserved in silica

gel with a cobalt indicator, following the proto-

col outlined by Chase & Hills (1991). The har-

vested tissues were then dehydrated, macerated

in liquid nitrogen and securely stored at -70 °C.

Geospatial processing and analysis: The

georeferencing coordinates were meticulously

documented and this data was systematically

organised using ArcView version 3.2 ® software.

Subsequent analysis of the datasets was carried

out using ArcView 3.2 ®, supplemented by the

Spatial Analyst and 3D Analyst add-ons. Dis-

tance analysis was carried out by considering

population distribution and road coverage with

the ArcView Find Distance function. To ensure

consistency and compatibility, all information

was reprojected into the Universal Transverse

Mercator (UTM) coordinate system, specifi-

cally within Zone 18 North using the WGS-84

datum. Distance calculations between locations

were skilfully performed using the Distance

Matrix extension within the ArcView platform.

Taxonomic identification: The informa-

tion about orchid species documented in the

upper Magdalena Basin, located in the central

Colombian Cordillera, their distribution in

the study area, their geographical and alti-

tudinal range, and the habitat requirements

were obtained during the fieldwork, and the

revision of herbarium material. Herbarium

specimens were examined according to the

standard procedures. Every studied sheet was

photographed, and the data were taken from

the labels. Both vegetative and reproductive

characters of each plant were studied. All infor-

mation was complemented by data obtained

from the literature, mostly protologues and

Neotropical orchid floras (Bateman et al., 2003;

Cuatrecasas, 1958; Dodson & Luer, 2010; Espi-

nal & Montenegro, 1977).

DNA extraction: For DNA extraction, 70

mg of finely powdered leaf tissue was processed

according to the method originally described

by Kobayashi et al. (1998), adapted for recal-

citrant plant tissues. Notable modifications to

the protocol were the inclusion of Proteinase K

at a concentration of 60 μg/ml in buffer 2 and

the addition of RNAse at 20 μg/ml in the final

step. The extracted DNA was then stored at

-20 °C. DNA concentration was quantified by

spectrophotometric analysis using a Pharmacia

4Revista de Biología Tropical, ISSN: 2215-2075 Vol. 74: e2026160, enero-diciembre 2026 (Publicado Feb. 25, 2026)

Biotech Gene Quant kit. Simultaneously, spec-

trophotometric measurements were used to

assess protein absorbance, which provided an

indication of the purity of the DNA samples.

DNA integrity was check through an elec-

trophoresis in 0.8 % agarose gel stained with

ethidium bromure.

Random Amplified Polymorphic DNA

(RAPD): The RAPD-PCR reaction protocol

for Cattleya was standardised according to the

method described by Weeden et al. (1992).

Amplification was performed in a GeneAmp

PCR System 9700 thermal cycler from Applied

Biosynthesis. PCR conditions included an ini-

tial denaturation at 94 °C for 4 minutes, fol-

lowed by 40 cycles of 1 minute at 94 °C, 1

minute at 35 °C and 2 minutes at 72 °C, culmi-

nating in a final extension at 72 °C for 7 min-

utes. PCR amplification was performed using a

mixture containing 1X buffer, 2.5 mM MgCl2,

0.2 mM dNTPs, 0.8 μM oligonucleotide, 0.6 μg/

μl BSA, 0.8 U Taq polymerase and 2.5 ng DNA,

adjusted with ultrapure water to a final volume

of 12 μl. The reproducibility of the markers was

ensured by repeating all PCRs.

Amplification of cpSSR: The PCR condi-

tions were as follows: 4 minutes of denaturation

at 94 °C, followed by 30 cycles of denaturation

at 94 °C for 1 minute, annealing at 54 °C for 1

minute, and extension at 72 °C for 2 minutes.

This was followed by a final extension at 72

°C for 7 minutes. PCR amplification was per-

formed using 1X PCR buffer, 3 mM MgCl₂,

0.25 mM dNTPs, 0.2 µM forward and reverse

oligonucleotides, 0.6 µg/µl BSA, 0.75 U Taq

polymerase, and 0.4 ng/µl DNA. The total vol-

ume was 20 µl, adjusted with ultrapure water.

Reproducibility was confirmed for all ampli-

fications. The PCR primers for cpSSR were

developed by another study of the author that

is currently being published.

Evaluation of gene flow patterns via pol-

len and seed dispersal: We performed a com-

parative analysis of gene distances derived from

RAPD and cpSSR data to identify potential

correlations. Our aim was to determine the

interaction between pollen and seed gene

flow. To achieve this, we implemented the

formula introduced by Ennos (1994), denoted

in equation 1.

Where: mp and ms, represent the migration

rates of pollen and seeds, respectively. These

values were calculated by extrapolation from

the FST estimates obtained in AMOVA for both

the RAPD and cpSSR datasets.

Association between genetic distances

and geographical distances: We carried out

a comprehensive analysis of Jaccard distances

among populations of each species studied.

These distances were calculated from RAPD

and cpSSR data. The aim was to investigate

potential relationships between genetic dis-

tances and geographical distances separating

localities, thus evaluating the isolation-by-dis-

tance model. To facilitate this investigation,

we used the Mantel test as explained by Sokal

& Rohlf (1995).

Correlation between anthropogenic

influence and the genetic diversity of the Cat-

tleya populations: We conducted an analysis

to detect correlations between genetic diver-

sity, as measured by expected heterozygosity

values derived from RAPD and cpSSR data,

and the proximity of sampling sites to the near-

est road and human population centres. This

study served as an indirect assessment of the

potential anthropogenic impact on the plant

populations studied.

Data analysis: We calculated FST and φST

for all locations and location pairs, assuming

RAPDs followed Hardy-Weinberg equilibrium

and random mating (FIS = 0). For cpSSR data,

which considers maternally inherited alleles,

we estimated gene flow and migration between

populations using Wright´s (1931) island

model. Notably, we adjusted the Nm calcula-

tion for chloroplast data, using a multiplication

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 74: e2026160, enero-diciembre 2026 (Publicado Feb. 25, 2026)

factor of 1 / 2 instead of 1 / 4 due to the haploid

nature of the data. Molecular analysis of vari-

ance (AMOVA) was then used with parameters

from Excoffier et al. (1992) using GENALEX

6.0® software, following the instructions of

Peakall & Smouse (2006). Population structure

was assessed at two levels: between sites and

within sites. We evaluated variance components

and fixation indices through 999 permutations

and calculated ΦST for a measure analogous

to FST, using the same software and interpreta-

tion of population differentiation according to

Wright (1978). Finally, a DAPC was made with

the RAPD matrix using the package Adegenet

(Jombart, 2008) from R software.

RESULTS

Study areas and population collection:

Populations inhabited patches of secondary

forest ranging from five to ten hectares in size,

with canopies over 25 metres high. These areas

were interspersed with other soil uses such as

mixed crops, coffee plantations and mining

areas. The region’s altitudes ranged from 854 to

1 550 m.a.s.l. and temperatures ranged from 17

to 25 °C, with an average annual temperature

of 25.3 °C. Annual rainfall averaged between

1 700 and 2 400 mm. According to Holdridge’s

ecological formations (Holdridge, 1982), these

zones straddle the tropical dry forest (bs-T)

and the very humid premontane forest (bmh-

PM). The Magdalena Basin comprises agroeco-

systems of mechanised rice (Oryza sativa L.),

coffee (Coffea arabica L.), diversified peasant

systems and undifferentiated rural areas.

Plant habitat and lyophytic occurrence:

Individuals of C. trianae were found on vari-

ous tree species including Anacardium excel-

sum Skeels., Guazuma ulmifolia Lam., Samanea

saman (Jacq.) Merr., Cordia alliodora (Ruiz &

Pav.) Oken, Jacaranda caucana Pittier., Ficus

spp., Pseudolmedia rigida (Klotzsch & H.Karst.)

Cuatrec., Trichilia pallida Sw., Guarea gigantea

Triana & Planch. and Guarea spp. at heights

between 15 and 35 metres. We also confirmed

the presence of lyophytic habitats, as plants

were also identified on rocks.

Chloroplast microsatellites (cpSSR): Of

the 14 chloroplast genome sequences analysed,

four showed repetitive variable regions in the

different Cattleya species analysed, from which

specific oligonucleotide pairs were designed for

the amplification of chloroplast microsatellites

for this plant genus.

Estimation of cpSSR frequencies in C.

trianae populations: The rps16 and matK 5

loci were found to be monomorphic. Three

alleles were found for matK 3, with allele 100

exhibiting the highest frequency (0.97948). The

rbcL-atpB system was the most polymorphic

with three alleles (124, 125, 126), with allele

(126) being the highest frequency (0.9793).

Although the absence of heteroplasmy is gener-

ally reported in cpSSRs (Provan et al., 2001), in

C. trianae heteroplasmy of MatK 3 was identi-

fied in the La Chapa and Boquerón populations

and another in an individual from La Chapa for

rbcL-atpB.

Analysis of RAPD and cpSSR molecular

data for C. trianae populations: The RAPD

results (Table 1) showed that these populations,

despite occupying disturbed and relatively

small areas, had remarkable genetic diversity

values (He = 0.31).

The genetic diversity from RAPD in Table

1, shows the expected heterozygosity for each

of the populations of C. trianae, showing simi-

lar values and an average value for the total

population of 0.31, highlighting those obtained

for the populations: El Pital, Congoja, Gaviota

and Boquerón. Furthermore, Genetic diver-

sity from cpSSR, shows the heterozygosity val-

ues obtained, which were very low (He =

0.013), while the percentage of polymorphic

loci observed per population was less than 40 %

and for five populations of C. trianae (Table 1).

The RAPD-derived FST values (Table 2)

correspond to moderate to very high genetic

differentiation, indicating different levels of

gene flow between the pairs of C. trianae

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populations studied. The RAPD dendrogram of

Jaccard distances (Fig. 1) shows the differentia-

tion of the populations.

Expected heterozygosity values calculated

with cpSSR and range from 0 to 0.036, indicat-

ing that cpSSR detects little polymorphism and

very little genetic variation at the cytoplasmic

level (Table 1). The results derived from cpSSR

in C. trianae (Table 1, Fig. 2) indicate that there

is no significant differentiation between popu-

lations. The dendrograms obtained showed

high similarity of the chloroplast genome. The

FST (Table 3) indicated low to moderate dif-

ferentiation between populations, which would

be due to frequent chloroplast alleles in the

populations, possibly derived from a common

maternal ancestor.

Similarity, genetic distance and DAPC:

Fig. 1 shows the Jaccard distance-based den-

drogram for RAPD markers in C. trianae.

Three groups can be identified: one for El

Agrado, El Boquerón, La Chapa, and La Gavi-

ota; one for El Pital, Guayabal, La Argentina,

Table 1

Genetic diversity values in C. trianae based on RAPD and cpSSR.

Locations Individuals

RAPD cpSSR

Average expected

heterozygosity % of

polymorphic loci

Average estimated

heterozygosity % of

polymorphic loci

unbiased Nei (1978) unbiased Nei (1978)

Porvenir 14 0.27 83.7 0.028 20

La Argentina 16 0.24 76.0 0.000 0

Congoja 20 0.28 90.4 0.020 20

San Jacinto 13 0.25 82.7 0.000 0

Guayabal 15 0.25 80.8 0.028 20

El Pital 20 0.30 93.3 0.000 0

El Agrado 13 0.29 76.9 0.000 0

Gaviota 22 0.29 89.4 0.036 40

Chapa 20 0.25 75.0 0.020 20

Boquerón 20 0.29 89.4 0.010 10

Totumo 19 0.25 78.8 0.000 0

Total/Average 192 0.31 83.3 0.013 60

Table 2

FST and Nm values between pair of populations of C. trianae using RAPD markers. Below the diagonal, FST values, above the

diagonal, Nm values.

Populations Porv. Argen. Congoja San Jacinto Guay. El Pital El Agrado Gaviota Chapa Boquer. Totumo

Porvenir - 1.4 2.6 2.2 1.9 2.0 0.9 0.7 0.9 1.1 2.1

Argentina 0.1511 - 3.8 2.1 1.8 1.4 0.8 0.7 0.7 0.8 1.0

Congoja 0.0879 0.0611 - 6.1 3.2 2.0 1.0 0.7 0.8 0.9 1.4

San Jacinto 0.1036 0.1068 0.0393 - 3.6 2.4 1.0 0.6 0.8 0.8 1.2

Guayabal 0.1177 0.1204 0.0728 0.0642 - 3.3 0.8 0.6 0.7 0.8 1.1

El Pital 0.1106 0.1515 0.1094 0.0959 0.0698 - 2.8 1.4 1.2 1.5 1.2

El Agrado 0.2193 0.2435 0.1931 0.1993 0.2279 0.0826 - 0.9 1.1 1.0 0.7

Gaviota 0.2649 0.2665 0.2610 0.2801 0.2811 0.1528 0.2227 - 1.1 1.1 0.6

Chapa 0.2246 0.2608 0.2323 0.2371 0.2580 0.1678 0.1907 0.1838 - 1.4 0.7

Boquerón 0.1821 0.2451 0.2131 0.2374 0.2284 0.1431 0.1949 0.1803 0.1493 - 1.0

Totumo 0.1064 0.2065 0.1488 0.1700 0.1910 0.1766 0.2505 0.2915 0.2649 0.1962 -

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La Congoja, and San Jacinto; and one for Por-

venir and Totumo. DAPC analysis suggests a

structure of three populations (k = 3), and the

membership probability plot (Fig. 3) reflects

the same population configurations for the

three groups as the Jaccard distances. The

populations of Porvenir and El Pital showed the

highest intrapopulation variance.

Fig. 2 shows the UPGMA dendrogram of

Nei (1978), specifically in terms of the genetic

distance between populations from the cpSSR

in C. trianae. The values above the nodes

indicate the percentage of agreement for each

grouping, according to bootstrap analysis with

1 000 replicates. From cpSSR, estimated FST

values between pairs of C. trianae populations

ranged from 0 to 0.027. Except for nine out of

55 population pairs where there is little differ-

entiation, the values hover around 0, indicating

no genetic differentiation. This is supported by

the fact that for the populations evaluated, all

four cpSSR systems, matK 5 and rps16 were

monomorphic, matK 3 and rbcL-atp each had

3 different alleles, but one of the three had the

highest frequency (0.98). As all FST values are

less than 0.33, the estimated values are greater

than one migrant per generation, again indicat-

ing very little differentiation of the chloroplast

genome in C. trianae (Table 3, Fig. 2).

The population structure, analysed by

means of an AMOVA analysis of variance with

two hierarchical levels, showed that there were

significant differences between the populations

of C. trianae (Table 4), but that the variance

within populations was greater than the vari-

ance between populations. At this level, the La

Congoja, El Pital, Gaviota and Boquerón popu-

lations are the ones that contribute the most

to the within-population variance (Table 4). It

should be noted that the populations with a

Fig. 1. Dendrogram according to Jaccard distances, using RAPD markers in C. trianae.

Fig. 2. UPGMA dendrogram according to Nei (1978),

genetic distance among populations from cpSSR in C.

trianae. Values above the nodes indicate the percentage

of consistency of each cluster, based on bootstrap analysis

with 1 000 replicates.

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lower contribution to the variance have a lower

number of individuals. The ΦST estimator of

population structure indicates high genetic dif-

ferentiation between populations (Fig. 1, Fig. 2).

The AMOVA of the cpSSR data with two

levels of hierarchy showed that, according to

the ΦST value, there is no significant variation

between populations, while the P value within

populations is significant (between individuals),

indicating that the variation between indi-

viduals is high, suggesting variation at the local

level. La Gaviota population contributes the

most to the variation, followed by El Porvenir,

Guayabal and La Chapa. The contribution to

variation is zero for La Argentina, San Jacinto,

El Pital, El Agrado and Totumo (Fig. 1, Fig. 2).

Using RAPD data, the FST estimator

showed that genetic differentiation among pairs

Fig. 3. Membership probability plot from the DAPC (k=3) for populations of C. trianae.

Table 3

FST and Nm values between pair of populations of C. trianae using cpSSR markers.

Populations Porv. Argen. Congoja San Jacinto Guay. El Pital El Agrado Gaviota Chapa Boquer. Totumo

Porvenir – 50 – – – 18.2 – – 124.5 42.6 18,2

Argentina 0.010 – – – 110.6 – – – – – –

Congoja -0.059 -0.012 – – 199.5 – – – – – –

San Jacinto -0.006 0.000 -0.023 – – – – – – – –

Guayabal 0.000 0.005 0.003 -0.010 – 24.5 – – 199.5 59.7 24,5

El Pital 0.027 0.000 0.000 0.000 0.020 – – – – – –

El Agrado -0.006 0.000 -0.023 0.000 -0.010 0.000 – – – – –

Gaviota -0.043 -0.015 -0.034 -0.026 -0.041 -0.004 -0.026 – – – –

Chapa 0.004 -0.012 0.00 -0.023 0.003 0.000 -0.023 -0.001 – – –

Boquerón 0.012 -0.012 0.000 -0.023 0.008 0.000 -0.023 -0.003 -0.034 – –

Totumo 0.027 0.000 0.000 0.000 0.020 0.000 0.000 -0.004 0.000 0.000 –

Below the diagonal, FST values, above the diagonal, Nm values.

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 74: e2026160, enero-diciembre 2026 (Publicado Feb. 25, 2026)

of C. trianae populations ranges from moderate

to very high, with only the Congoja-San Jacinto

pair showing low differentiation. The pairs with

very high genetic differentiation are Gaviota

with Porvenir, Argentina, Congoja, San Jacinto

and Guayabal and La Chapa with Argentina

and Totumo.

Gene flow Nm is less than three individu-

als per generation for most population pairs.

Population pairs exchanging ≤ 10 individuals

per ten generations generally included El Agra-

do, Gaviota, La Chapa, Boquerón and Totumo

(Fig. 2). In the remaining pairs, the number of

migrants per generation varied up to a maxi-

mum of 6 individuals per generation, as can be

observed between La Congoja and San Jacinto.

When correlating genetic diversity values

derived from RAPD or cpSSR with distance

to the nearest road or distance to the nearest

town or urban centre, in both cases the cor-

relation coefficient is not significant, i.e. there

is no significant association between the pairs

of variables considered. The p-values indicate

that there is no correlation between the genetic

diversity data obtained from RAPD and cpSSR

and the distance of each plant population stud-

ied to the nearest road or highway and to the

nearest urban centre, so it can be postulated

that the distance to roads does not influence

the genetic composition of the populations and

that the proximity to human settlements does

not influence the genetic composition of the

populations of C. trianae.

Genetic distances derived from RAPD data

and cpSSR data showed no correlation (R2 = 9.3

x 10 -4 and p = 0.8253). This may indicate that

RAPD genetic distances derived from nuclear

genomes of both maternal and paternal origin,

and those derived from chloroplasts of mater-

nal origin, follow different patterns, which may

be explained by the mechanisms of transmis-

sion of this genetic information from one gen-

eration to the next.

The Mantel test to correlate RAPD and

geographical distances showed no significant

correlation values (R = 0.0497). Similarly, when

contrasting cpSSR genetic distances and geo-

graphical distances, there were no significant

correlation values (R = -0.1916, p = 0.0850).

DISCUSSION

Pollination of Cattleya species: Studies

of Cattleya species have recognised Euglos-

sina bees as their pollinators (Braga, 1977;

van der Pijl & Dodson, 1966). In the subgenus

Cattleya, male Euglossina bees facilitate cross-

pollination, thereby promoting variation within

populations, a pattern that may also apply to

C. trianae. The fact that C. trianae populations

show less genetic differentiation than other

endangered orchid species may be due to the

particular mechanisms of gene flow through

anemophilous seed dispersal and pollen flow

mediated by euglossine bees (Wu et al., 2023;

Zhang et al., 2019).

On the other hand, Euglossines, which pol-

linate Neotropical orchids, not only travel long

distances, but also adapt to scattered micro-

habitats in the landscape and colonise forested

areas during regeneration processes, so they

would be able to move through the matrices

within which forest patches are found (Cândido

et al., 2021; Ulyshen et al., 2023).

Table 4

AMOVA for C. trianae using RAPDs and SSRs markers.

Source of variation g.l Sum of Square Mean square Variance component % total P-value

RAPDs

Between populations 10 761.4 76.3 3.5 18.6 < 0.001

Between individuals 181 2 764.2 15.3 15.3 81.4 < 0.001

cpSSR

Between populations 10 0.55 0.06 -0.0007 -1.10 0.87

Between individuals 182 12.29 0.07 0.0675 101.10 < 0.001

10 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 74: e2026160, enero-diciembre 2026 (Publicado Feb. 25, 2026)

The Ennos test (Ennos, 1994) concluded

that seed dispersal predominates, a result simi-

lar to that found by Hedrén & Lorenz (2019) for

the orchid Epipactis helleborine, which seems

to be a peculiarity of the orchid family, which

produces a large number of very light seeds that

favour their anemophilous dispersal over long

distances (Brzosko et al., 2017). Fruiting within

and between populations can be influenced

by pollinator diversity and activity, can vary

between 13 and 70 % positively related to visita-

tion rate, and in C. trianae, fruit size increases

as the number of pollinia applied increases

(Zhang & Gao, 2021).

This could be explained by the fact that for

many of the pollinating bees, movement along a

route is preferred because more plant resources

are available to collect substances from flowers,

and regardless of the distance between popula-

tions, there would be higher or lower levels of

flow, resulting in different degrees of differen-

tiation found in the populations.

Analysis of RAPD and cpSSR molecular

data for C. trianae populations: The genetic

diversity obtained by RAPD exceeded those

reported for other Cattleya species in disturbed

habitats, such as Cattleya lobata Lindl. (He =

0.262) (Lemos-Gomes et al., 2018) and Cattleya

elongata Barb. Rodr. (He = 0.175) (Ueno et al.,

2015) but similar to those found by Pinheiro

et al. (2012) for Cattleya labiate (He = 0.30)

in Brazil They were significantly higher than

the values reported for other orchid species

in disturbed habitats, including Paphiopedi-

lum micranthum Tang & F. T. Wang (Li et al.,

2020), Platanthera leucophaea Lindl. (Bell et

al., 2021) and Changnienia amoena S. S. Chien

(Qian et al., 2014).

The levels of genetic diversity found in this

research for Cattleya, compared to the work

described above, could be due to the fact that

in this genus the combination of sexual and

asexual reproduction and the longevity of the

plants can ensure that genotypes are main-

tained over generations, reducing the impact of

dispersal, even in affected populations (Lemos-

Gomes et al., 2018) and that cross-pollination

is favoured, associated with long distances trav-

elled by pollinators and successive visits to

flowers of the same species. In fact, Wong &

Sun (1999) and Sun & Wong (2001) reported

for the orchids Goodyera procera Hook., Zeux-

ine gracilis (Breda) Blume, Eulophia sinensis

Miq. and Zeuxine strateumatica (L.) Schltr.,

with different mating mechanisms, that genetic

diversity differs greatly both at the species level

(He = 0.144 ~ 0.293) and at the population level

(He = 0.011 ~ 0.181) (Table 1).

The genetic diversity values obtained from

cpSSR (average He = 0.013), indicated a little

genetic variation at the chloroplast DNA level

(Table 1). To contrast these data with others

for orchids, only the data reported by Squirrell

et al. (2001), where core diversity values from

allozymes and (cpDNA RFLPs) from the orchid

E. helleborine (L.) Crantz. showed equivalent

levels of genetic diversity.

The chloroplast genome is haploid, does

not recombine and has a low mutation rate

compared to the nucleus, as noted by Vu et

al. (2020), chloroplast markers have different

resolutions for different orchid genus. van den

Berg et al. (2009) showed that the combination

of different chloroplast microsatellites is very

useful for phylogenetic studies of Laleliinae.

Sequencing of the chloroplast genome of Cat-

tleya crispata showed that it is similar to that

of the genus Cymbidium and that the differ-

ences are in the order of some genes, which is

useful for population studies (da Rocha-Perini

et al., 2016). However, the presence of rare

and private alleles (Jin et al., 1996) in two of

the four cpSSR loci in C. trianae and hetero-

plasmy (Fig. 1, Fig. 2) may indicate an origin

by recent mutations in the tested regions of

the chloroplast genome and may be related to

the fact that mutation rates of microsatellite

length variation have been found to be higher

(10–2–10–6) than point mutation rates (Amos,

2016; Wheeler et al., 2014).

The levels of genetic structure generated

by RAPD (ΦST = 0.186) correspond to high

genetic differentiation and are highly signifi-

cant; the greatest contribution to genetic differ-

entiation is within populations, suggesting that

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 74: e2026160, enero-diciembre 2026 (Publicado Feb. 25, 2026)

there is under-structuring within populations,

requiring microgeographic evaluation of locali-

ties. These values are lower than those reported

for threatened orchids such as P. leucophaea (Φ

ST = 0.21) and Platanthera integrilabia (Φ ST

= 0.27) (Wooten et al., 2020) and the orchid C.

amoena (Φ ST = 0.43) (Tikendra et al., 2021).

The Mantel test showed that for the two

species there was no correlation between the

genetic distances obtained with the two mark-

ers and the geographical distances, so there

is no isolation by distance, a result consistent

with that reported for endangered orchids such

as C. amoena and P. leucophaea, where a lack

of gene flow between populations or genetic

drift within populations has been suggested

(Qian et al., 2014).

Our research, with the primary aim of

elucidating the current status of C. trianae

populations, has provided essential information

that not only safeguards the existing genetic

diversity within this endangered orchid species,

but also serves as a cornerstone for the devel-

opment of sustainable management strategies.

This study plays a pivotal role in the conserva-

tion and rejuvenation of the ecological environ-

ment in which these orchids thrive.

The genetic diversity and structure analy-

ses using RAPD and cpSSR molecular mark-

ers revealing that the genetic diversity in the

populations of C. trianae is not as low as is

expected for species affected by habitat frag-

mentation. Additionally, the pronounced diver-

gence among these populations is staggering,

implying that various factors have significantly

shaped the genetic composition and distribu-

tion of Colombian C. trianae.

This study is only the first step in under-

standing this native orchid species. To further

our understanding, it is imperative to expand

our investigation to include a larger and more

geographically diverse sample representing the

entire country. Using co-dominant markers

and delving into sequencing data from cpDNA

or nrDNA (ITS) is emerging as a promising

avenue, with the potential to unravel the intri-

cate mechanisms governing seed and pollen

gene flow. This approach not only advances our

understanding of population biology but also

provides critical insights into the context of

invasive orchids, with far-reaching implications

for conservation and ecological management.

Implications for conservation: The com-

prehensive analyses presented in this study

clearly demonstrate the profound influence of

dominant habitats on the striking genetic dif-

ferentiation within C. trianae populations. This

research serves as a clarion call, highlighting

the transformative effects of disturbance on

the genetic fabric of these populations, and

underscoring the urgent need for conservation

action.

The implications for the conservation

of this species are clear. Urgent and concerted

efforts are imperative to mitigate the genetic

differentiation between C. trianae populations,

with a primary focus on the rapid restoration

of ecological integrity. In situ conservation is

of paramount importance, not only to pre-

vent further habitat fragmentation, but also to

protect the complex ecological relationships

involving fungi and pollinators.

Considering these findings, a strong and

effective ban on the unsustainable collection

of C. trianae resources, particularly during the

flowering season, is imperative. This measure

is a linchpin in facilitating increased gene flow

and ensuring the continued survival of these

invaluable orchids.

Taken together, these findings highlight

the urgent need for proactive and strategic

conservation approaches. This study lays the

groundwork for the formulation of effective

management and conservation protocols,

reflecting our unwavering commitment to the

preservation of these threatened orchid popula-

tions. In its guiding role, this research not only

secures the future of C. trianae but also revital-

ises the ecological sanctuaries they inhabit.

Ethical statement: The authors declare

that they all agree with this publication and

made significant contributions; that there is no

conflict of interest of any kind; and that we fol-

lowed all pertinent ethical and legal procedures

12 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 74: e2026160, enero-diciembre 2026 (Publicado Feb. 25, 2026)

and requirements. All financial sources are fully

and clearly stated in the acknowledgments sec-

tion. A signed document has been filed in the

journal archives.

ACKNOWLEDGMENTS

This study was supported by the Univer-

sidad del Tolima and FPIT Fundación para la

Promoción de la Investigación y la Tecnología

del Banco de la República de Colombia.

REFERENCES

Amos, W. (2016). Heterozygosity increases microsatelli-

te mutation rate. Biology Letters, 12(1), 20150929.

https://doi.org/10.1098/rsbl.2015.0929

Bateman, R. M., Hollingsworth, P. M., Preston, J., YI-BO,

L., Pridgeon, A. M., & Chase, M. W. (2003). Mole-

cular phylogenetics and evolution of Orchidinae

and selected Habenariinae (Orchidaceae). Botanical

Journal of the Linnean Society, 142(1), 1–40. https://

doi.org/10.1046/j.1095-8339.2003.00157.x

Bell, T. J., Bowles, M. L., Zettler, L. W., Pollack, C. A., &

Ibberson, J. E. (2021). Environmental and mana-

gement effects on demographic processes in the

U.S. Threatened Platanthera leucophaea (Nutt.)

Lindl. (Orchidaceae). Plants, 10(7), 1308. https://doi.

org/10.3390/plants10071308

Braga, P. I. S. (1977). Aspectos biológicos das Orchida-

ceae de uma campina da Amazônia Central. Acta

Amazonica, 7(2, Supplement 1), 5–89. https://doi.

org/10.1590/1809-43921977072s005

Brzosko, E., Ostrowiecka, B., Kotowicz, J., Bolesta, M., Gro-

motowicz, A., Gromotowicz, M., Orzechowska, A.,

Orzołek, J., & Wojdalska, M. (2017). Seed dispersal in

six species of terrestrial orchids in Biebrza National

Park (NE Poland). Acta Societatis Botanicorum Polo-

niae, 86(3), 3557. https://doi.org/10.5586/asbp.3557

Calderón-Sáenz, E. (Ed.). (2007). Libro rojo de plantas de

Colombia: Orquídeas, primera parte (Vol. 6). Instituto

de Investigación de Recursos Biológicos Alexander

von Humboldt

Cândido, M. E. M. B., Miranda, P. N., & Morato, E. F.

(2021). Orchid bees in riparian and terra-firme forest

fragments in an urban matrix in southwestern Bra-

zilian Amazonia. Acta Amazonica, 51(3), 214–223.

https://doi.org/10.1590/1809-4392202003781

Chase, M. W., & Hills, H. H. (1991). Silica gel: An ideal

material for field preservation of leaf samples for

DNA studies. TAXON: The Journal of the International

Association for Plant Taxonomy, 40(2), 215–220.

https://doi.org/10.2307/1222975

Cuatrecasas, J. (1958). Aspectos de la vegetación natural

de Colombia. Revista de la Academia Colombiana de

Ciencias Exactas Físicas y Naturales, 10(40), 221–264.

da Rocha-Perini, V., Leles, B., Furtado, C., & Prosdocimi,

F. (2016). Complete chloroplast genome of the orchid

Cattleya crispata (Orchidaceae:Laeliinae), a Neotro-

pical rupiculous species. Mitochondrial DNA Part A,

27(6), 4075–4077. https://doi.org/10.3109/19401736

.2014.1003850

Dodson, C. H., & Luer, C. A. (2010). Orchidaceae, Genera

Cyrtochiloides-Epibator. In G. Harling, & C. Persson

(Eds.), Flora of Ecuador (Vol. 87, pp. 225–227). Göte-

borg University.

Ennos, R. A. (1994). Estimating the relative rates of

pollen and seed migration among plant populations.

Heredity, 72(3), 250–259. https://doi.org/10.1038/

hdy.1994.35

Espinal, S., & Montenegro, M. (1977). Zonas de vida o for-

maciones vegetales de Colombia: Memoria explicativa

sobre el mapa ecológico de Colombia (1a ed.). Instituto

Geográfico Agustín Codazzi.

Excoffier, L., Smouse, P. E., & Quattro, J. M. (1992). Analy-

sis of molecular variance inferred from metric distan-

ces among DNA haplotypes: Application to human

mitochondrial DNA restriction data. Genetics, 131(2),

479–491. https://doi.org/10.1093/genetics/131.2.479

Hedrén, M., & Lorenz, R. (2019). Seed dispersal and fine-

scale genetic structuring in the asexual Nigritella

miniata (Orchidaceae) in the Alps. Botanical Journal

of the Linnean Society, 190(1), 83–100. https://doi.

org/10.1093/botlinnean/boz005

Holdridge, L. (1982). Life zone ecology. Tropical Science

Center.

Jin, L., Macaubas, C., Hallmayer, J., Kimura, A., & Mig-

not, E. (1996). Mutation rate varies among alle-

les at a microsatellite locus: Phylogenetic evidence.

PNAS: Proceedings of the National Academy of Scien-

ces, 93(26), 15285–15288. https://doi.org/10.1073/

pnas.93.26.15285

Jombart, T. (2008). adegenet: A R package for the mul-

tivariate analysis of genetic markers. Bioinforma-

tics, 24(11), 1403–1405. https://doi.org/10.1093/

bioinformatics/btn129

Kobayashi, N., Horikoshi, T., Katsuyama, H., Handa, T., &

Takayanagi, K. (1998). A simple and efficient DNA

extraction method for plants, especially woody plants.

Plant Tissue Culture and Biotechnology, 4(2), 76–81.

Lemos-Gomes, P. C., de Camargo-Smidt, E., de Fraga, C.

N., & Silva-Pereira, V. (2018). High genetic variability

is preserved in relict populations of Cattleya lobata

Revista de Biología Tropical, ISSN: 2215-2075, Vol. 74: e2026160, enero-diciembre 2026 (Publicado Feb. 25, 2026)

(Orchidaceae) in the Atlantic Rainforests inselbergs.

Brazilian Journal of Botany, 41(1), 185–195. https://

doi.org/10.1007/s40415-017-0422-z

Li, Z., Li, J., & Li, M. (2020). Effect of human disturbance

on genetic structure of rare and endangered Paphio-

pedilum micranthum implied the habitat status. Tro-

pical Conservation Science, 13(1), 1940082920942012.

https://doi.org/10.1177/1940082920942012

Ministerio de Ambiente y Desarrollo Sostenible, & Uni-

versidad Nacional de Colombia. (2015). Plan para el

estudio y la conservación de las orquídeas en Colombia.

Ministerio de Ambiente y Desarrollo Sostenible, Uni-

versidad Nacional de Colombia.

Nadeem, M. A., Nawaz, M. A., Shahid, M. Q., Doğan, Y.,

Comertpay, G., Yıldız, M., Hatipoğlu, R., Ahmad, F.,

Alsaleh, A., Labhane, N., Özkan, H., Chung, G., &

Baloch, F. S. (2018). DNA molecular markers in plant

breeding: Current status and recent advancements in

genomic selection and genome editing. Biotechno-

logy & Biotechnological Equipment, 32(2), 261–285.

https://doi.org/10.1080/13102818.2017.1400401

Nei, M. (1978). Estimation of average heterozygosity and

genetic distance from a small number of individuals.

Genetics, 89(3), 583–590. https://doi.org/10.1093/

genetics/89.3.583

Ossenbach, C., & Jenny, R. (2021). Rudolf Schlechter’s

South-American orchids IV. Schlechter’s “network”:

Venezuela and Colombia. Lankesteriana: Internatio-

nal Journal on Orchidology, 21(2), 157–232. https://

doi.org/10.15517/lank.v21i2.47581

Peakall, R., & Smouse, P. E. (2006). GENALEX 6:

Genetic analysis in Excel. Population gene-

tic software for teaching and research. Mole-

cular Ecology Notes, 6(1), 288–295. https://doi.

org/10.1111/j.1471-8286.2005.01155.x

Pinheiro, L. R., Carregosa-Rabbani, A. R., Cruz da Silva,

A. V., da Silva Lédo, A., García-Pereira, K. L., &

Cardamone-Diniz, L. E. (2012). Genetic diversity

and population structure in the Brazilian Cattleya

labiata (Orchidaceae) using RAPD and ISSR markers.

Plant Systematics and Evolution, 298(10), 1815–1825.

https://doi.org/10.1007/s00606-012-0682-9

Powell, W., Orozco-Castillo, C., Chalmers, K. J., Provan,

J., & Waugh, R. (1995). Polymerase chain reaction-

based assays for the characterisation of plant genetic

resources. Electrophoresis, 16(1), 1726–1730. https://

doi.org/10.1002/elps.11501601285

Provan, J., Powell, W., & Hollingsworth, P. M. (2001).

Chloroplast microsatellites: New tools for studies

in plant ecology and evolution. Trends in Ecology &

Evolution, 16(3), 142–147. https://doi.org/10.1016/

S0169-5347(00)02097-8

Qian, X., Li, Q. J., Liu, F., Gong, M. J., Wang, C. X.,

& Tian, M. (2014). Conservation genetics of an

endangered Lady’s Slipper orchid: Cypripedium japo-

nicum in China. International Journal of Molecular

Sciences, 15(7), 11578–11596. https://doi.org/10.3390/

ijms150711578

Reina-Rodríguez, G. A., Rubiano-Mejía, J. E., Castro-Lla-

nos, F. A., & Soriano, I. (2017). Orchids distribution

and bioclimatic niches as a strategy to climate change

in areas of tropical dry forest in Colombia. Lankes-

teriana: International Journal on Orchidology, 17(1),

17–47. https://doi.org/10.15517/lank.v17i1.27999

Salazar-Mercado, S. A., & Vega-Contreras, N. A. (2017).

Asymbiotic seed germination and in vitro propaga-

tion of Cattleya trianae Linden & Reichb.f. (Orchida-

ceae). Acta Agronómica, 66(4), 544–548. https://doi.

org/10.15446/acag.v66n4.63597

Salgotra, R. K., & Chauhan, B. S. (2023). Genetic diver-

sity, conservation, and utilization of plant genetic

resources. Genes, 14(1), 174. https://doi.org/10.3390/

genes14010174

Sokal, R., & Rohlf, F. (1995). Biometry: The Principles and

Practice of Statistics in Biological Research (3rd ed.).

W.H. Freeman and Company.

Squirrell, J., Hollingsworth, P. M., Bateman, R. M., Dickson,

J. H., Light, M. H. S., MacConaill, M., & Tebbitt, M.

C. (2001). Partitioning and diversity of nuclear and

organelle markers in native and introduced popula-

tions of Epipactis helleborine (Orchidaceae). Ameri-

can Journal of Botany, 88(8), 1409–1418. https://doi.

org/10.2307/3558447

Sun, M., & Wong, K. C. (2001). Genetic structure of

three orchid species with contrasting breeding sys-

tems using RAPD and allozyme markers. American

Journal of Botany, 88(12), 2180–2188. https://doi.

org/10.2307/3558379

Tikendra, L., Potshangbam, A. M., Amom, T., Dey, A.,

& Nongdam, P. (2021). Understanding the genetic

diversity and population structure of Dendrobium

chrysotoxum Lindl. An endangered medicinal orchid

and implication for its conservation. South Afri-

can Journal of Botany, 138, 364–376. https://doi.

org/10.1016/j.sajb.2021.01.002

Ueno, S., Fernandes-Rodrigues, J., Alves-Pereira, A., Pansa-

rin, E. R., & Veasey, E. A. (2015). Genetic variability

within and among populations of an invasive, exotic

orchid. AoB Plants, 7, plv077. https://doi.org/10.1093/

aobpla/plv077

Ulyshen, M., Urban-Mead, K. R., Dorey, J. B., & Rivers, J. W.

(2023). Forests are critically important to global polli-

nator diversity and enhance pollination in adjacent

crops. Biological Reviews, 98(4), 1118–1141. https://

doi.org/10.1111/brv.12947

van den Berg, C., Higgins, W. E., Dressler, R. L., Whitten,

W. M., Soto-Arenas, M. A., & Chase, M. W. (2009).

A phylogenetic study of Laeliinae (Orchidaceae)

14 Revista de Biología Tropical, ISSN: 2215-2075 Vol. 74: e2026160, enero-diciembre 2026 (Publicado Feb. 25, 2026)

based on combined nuclear and plastid DNA sequen-

ces. Annals of Botany, 104(3), 417–430. https://doi.

org/10.1093/aob/mcp101

van der Pijl, L. & Dodson, C. H. (1966). Orchid flowers:

Their pollination and evolution. University of Miami

Press.

Vu, H. T., Tran, N., Nguyen, T. D., Vu, Q. L., Bui, M. H., Le,

M. T., & Le, L. (2020). Complete chloroplast genome

of Paphiopedilum delenatii and phylogenetic relation-

ships among Orchidaceae. Plants, 9(1), 61. https://doi.

org/10.3390/plants9010061

Weeden, N. F., Timmerman, G., Hemmat, M., Kneen, B.,

& Lodhi, M. (1992). Inheritance and reliability of

RAPD markers. In D. Hoisington, & A. McNab (Eds.),

Application of RAPD technology to plant breeding (pp

12–17). Joint Plant Breeding Symposia Series, Crop

Science Society of America.

Wheeler, G. L., Dorman, H. E., Buchanan, A., Challa-

gundla, L., & Wallace, L. E. (2014). A review of the

prevalence, utility, and caveats of using chloroplast

simple sequence repeats for studies of plant biology.

Applications in Plant Sciences, 2(12), 1400059. https://

doi.org/10.3732/apps.1400059

Wong, K. C., & Sun, M. (1999). Reproductive biology and

conservation genetics of Goodyera procera (Orchida-

ceae). American Journal of Botany, 86(10), 1406–1413.

https://doi.org/10.2307/2656923

Wooten, S., Call, G., Dattilo, A., Cruse-Sanders, J., & Boyd,

J. N. (2020). Impacts of forest thinning and white-

tailed deer herbivory on translocation of the rare

terrestrial Orchid Platanthera integrilabia. Diversity,

12(11), 412. https://doi.org/10.3390/d12110412

Wright, S. (1931). Evolution in Mendelian populations.

Genetics, 16(2), 97.

Wright, S. (1978). Evolution and the genetics of populations:

Variability within and among natural populations

(Vol. 4). The University of Chicago Press.

Wu, Q., Dong, S., Zhao, Y., Yang, L., Qi, X., Ren, Z., Dong,

S., & Cheng, J. (2023). Genetic diversity, population

genetic structure and gene flow in the rare and endan-

gered wild plant Cypripedium macranthos revealed by

genotyping-by-sequencing. BMC Plant Biology, 23(1),

254. https://doi.org/10.1186/s12870-023-04212-z

Zhang, W., & Gao, J. (2021). A comparative study on the

reproductive success of two rewarding Habenaria

species (Orchidaceae) occurring in roadside verge

habitats. BMC Plant Biology, 21(1), 187. https://doi.

org/10.1186/s12870-021-02968-w

Zhang, Z., Gale, S. W., Li, J. H., Fischer, G. A., Ren, M.

X., & Song, X. Q. (2019). Pollen-mediated gene flow

ensures connectivity among spatially discrete sub-

populations of Phalaenopsis pulcherrima, a tropical

food-deceptive orchid. BMC Plant Biology, 19(1), 597.

https://doi.org/10.1186/s12870-019-2179-y