Content uploaded by Andrea Clavijo McCormick
Author content
All content in this area was uploaded by Andrea Clavijo McCormick on Aug 09, 2023
Content may be subject to copyright.
Citation: Alfaro Pinto, A.; McGill, C.;
Nadarajan, J.; Archila Morales, F.;
Clavijo McCormick, A. Seed
Morphology of Three Neotropical
Orchid Species of the Lycaste Genus.
Seeds 2023,2, 331–339. https://
doi.org/10.3390/seeds2030025
Academic Editors: JoséAntonio
Hernández Cortés and Alba
Cuena Lombraña
Received: 10 June 2023
Revised: 1 July 2023
Accepted: 24 July 2023
Published: 7 August 2023
Copyright: © 2023 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
Seed Morphology of Three Neotropical Orchid Species of the
Lycaste Genus
Alejandra Alfaro Pinto 1, * , Craig McGill 1, Jayanthi Nadarajan 2, Fredy Archila Morales 3
and Andrea Clavijo McCormick 1, *
1School of Agriculture and Environment, Massey University, Private Bag 11-222,
Palmerston North 4442, New Zealand; c.r.mcgill@massey.ac.nz
2The New Zealand Institute for Plant and Food Research Limited, Private Bag 11-600,
Palmerston North 4442, New Zealand; jayanthi.nadarajan@plantandfood.co.nz
3Experimental Orchid Station Farm of the Archila Family, Coban 16001, Alta Verapaz, Guatemala;
archilae@gmail.com
*Correspondence: malealfaro92@gmail.com (A.A.P.); a.c.mccormick@massey.ac.nz (A.C.M.)
Abstract:
Neotropical orchids are vulnerable to extinction due to overharvesting, habitat destruction
and climate change. However, a basic understanding of orchid seed biology to support conservation
efforts is still lacking for most species. Seed morphology is linked to plant adaptation and evolution,
influencing seed dispersal, dormancy, longevity, and germination, which are valuable traits for
conservation. In this study, we characterized and compared the morphological traits of seed capsules
(size, shape, and colour) and seeds (seed and embryo shape and size and internal airspace volume)
for three epiphytic Neotropical orchid species of the genus Lycaste native to Guatemala: L. cochleata,
L. lasioglossa, and L. virginalis. The three species show qualitative similarities in seed capsule colour
and appearance and in seed morphology (i.e., scobiform oval-shaped seeds and prolate-spheroid
embryos). All species have small-sized seeds (length of L. cochleata: 210
µ
m, L. lasioglossa: 230
µ
m, and
L. virginalis: 260
µ
m), with proportionally large embryos (length of L. cochleata: 140
µ
m, L. lasioglossa:
120
µ
m, and L. virginalis: 150
µ
m) and an internal air-space volume that occupies less than half
of the seed (L. cochleata: 17%, L. lasioglossa: 42%, and L. virginalis: 30%). This finding is consistent
with previous reports for other epiphytic orchid species, which typically have lower air volumes
than terrestrial orchids. These differences are likely a result of evolutionary changes associated with
different habits and may influence seed dispersal. We also found some significant differences in seed
morphology between the studied species, but their taxonomic, biological, and ecological relevance
remain to be elucidated. More comparative studies, including on other Lycaste species with different
habits, are needed to explore relationships between seed morphology, taxonomy, biology, and ecology
in this genus to support its conservation.
Keywords: adaptation; airspace; conservation; embryo; epiphytic; morphometry
1. Introduction
Worldwide, the greatest diversity of orchids can be found in the Neotropics, particu-
larly in cloud forests, which provide favourable habitats for orchid growth [
1
,
2
]. Neotropical
orchids have attractive floral displays and are extremely desired for horticulture, medicine,
and food [
3
,
4
]. However, they have complex biological and ecological features, with most
of them being epiphytic, relying on animal pollination for reproduction and fungal sym-
bionts for germination, alongside having unique physiological adaptations to tropical
climates [
5
,
6
]. Therefore, unsustainable harvesting and the destruction of their natural
habitat, combined with climate change, make these orchids particularly vulnerable to
extinction. For this reason, it is urgent to develop both in situ as well as ex situ conservation
programs to preserve them. One of the limitations in developing these programs is the lack
of information on the basic ecology and biology for most Neotropical orchid species.
Seeds 2023,2, 331–339. https://doi.org/10.3390/seeds2030025 https://www.mdpi.com/journal/seeds
Seeds 2023,2332
Guatemala is a biodiversity hotspot, home to over 1400 identified orchid species [
7
,
8
].
Among these, Lycaste species have high ethnobotanical significance since some species have
traditionally been used by indigenous peoples dating back to the Mayan civilization [
9
]. The
genus Lycaste Lindl. Belongs to the subfamily Epidendroideae and the tribe Maxillarieae and
consists of about thirty-six (36) accepted species with epiphytic, terrestrial, or lithophytic
habits [
10
,
11
]. These species are naturally distributed throughout the south of Mexico,
Central America, and the north of South America [
12
]. They are characterized by short
and thickened pseudobulbs, with flowers that are generally large, very attractive, often
with a pleasant, sweet scent, and which develop three sepals, two petals, and a third sepal
called labellum [
12
]. Many species of Lycaste are highly desirable in horticulture due to
their appearance and colour variations, which make them more susceptible to overharvest.
Three Lycaste species were selected to explore their seed capsules and morphology:
L. virginalis (Scheidw.) Linden, L. lasioglossa Rchb.f., and L.cochleata Lindl. (synonym
of Selbyana cochleata (Lindl.) Archila). Lycaste virginalis f. alba (B.S. Williams) Archila &
Chiron, commonly known as “Monja Blanca” (white nun), has been the National Flower of
Guatemala since 1934. It holds high cultural and patriotic values, and it is often displayed at
national celebrations. However, L. virginalis is at a high and increased risk of extinction [
13
].
Lycaste cochleata is very rare and has a limited distribution from southeastern Mexico to
Nicaragua [
12
,
13
]. Lycaste lasioglossa also is very rare, and is distinguished from all other
Lycaste species found in Guatemala by its densely pubescent labellum; this species is not at
risk of extinction yet, but its trade is regulated [12–14].
Seeds are essential for the species’ adaptation, regeneration, distribution, and persis-
tence, playing a significant role in orchid conservation strategies [
15
,
16
]. Seed morphology
is associated with important processes such as seed dispersal, dormancy, germination, and
establishment, which are all relevant to seed conservation [
15
]. Seed traits are thought to
be more conservative than floral and vegetative characteristics, and consequently they are
also valuable indicators of the taxonomy, phylogenetics, and phytogeographic distribution
of orchid species [16,17].
Most orchid seeds are small (dust-like), with a low weight. A single capsule (the
reproductive unit) can produce thousands of seeds [
17
]. Due to their minuscule size, seed
characteristics remain undescribed for many orchid species. Orchid seeds typically consist
of a seed coat that incorporates an embryo and an internal air space volume (between the
seed coat and embryo), lacking an endosperm [17].
The relationship between seed and embryo volumes determines the proportion of
air space within the seeds. The internal air space of the seeds varies depending on the
species [
15
]. Previous studies suggest that epiphytic orchids typically have lower air-
space volumes (~30% on average), while terrestrial species have larger air volumes (~60%
on average) [
16
,
18
]. These differences are likely to impact seed dispersal, germination,
dormancy, and establishment [15–19].
Understanding seed traits is essential to inform conservation efforts. Therefore, species-
specific studies to characterize seed morphological traits (including the airspace volume) are
useful to identify the most suitable conservation approaches for each taxon [
15
]. Therefore,
this study aimed to characterize and compare the seed capsule and seed morphological
traits of three tropical rare and threatened epiphytic species from Guatemala: L. cochleata,
L. lasioglossa, and L. virginalis. To this end, we measured qualitative traits (seed capsule
colour and shape, seed and embryo shape) and quantitative traits (capsule size, seed size,
embryo size, airspace volume), and calculated the relationships between quantitative traits
to assess the relative occupancy of the embryo and air space within the seeds.
2. Materials and Methods
2.1. Seed Material
Lycaste cochleata,L. lasioglossa, and L. virginalis (Figure 1) were grown at the Experimen-
tal Orchid Station Farm of the Archila family located in the tropical forest of Guatemala
(temperatures ranging from 12.5
◦
C to 27.8
◦
C, relative humidity from 76% to 91%, latitude
Seeds 2023,2333
15
◦
29
0
0
00
N, longitude 90
◦
22
0
0
00
W, and an altitude of 1316 m a.s.l.) [
20
]. The flowers were
self-pollinated by hand and the seed capsules harvested when mature (i.e., capsules that
completed development and were fully grown) and about to naturally dehisce (i.e., gape or
burst open). After harvesting, the seed capsules were placed in different plastic containers
with silica gel to absorb humidity and tissue paper on the top and bottom to minimize
movement, surrounded by a cold shipping package that consisted of a standard duration
cooling system of 96 h at a constant 2 to 5
◦
C (provided by a Guatemalan supplier, SM
Soluciones S.A.) and sent by courier to Massey University Turitea Campus in New Zealand
(1–2 weeks). Upon arrival, the seed capsules were placed in plastic containers to dry in a
controlled environment room operating at 20
◦
C and 55% relative humidity (RH). The seed
capsules were allowed to open naturally under those conditions, and exposed seeds were
then collected and stored in glass vessels under the same conditions (20
◦
C and 55% RH)
before being assessed.
Seeds 2023, 2 333
2. Materials and Methods
2.1. Seed Material
Lycaste cochleata, L. lasioglossa, and L. virginalis (Figure 1) were grown at the Experi-
mental Orchid Station Farm of the Archila family located in the tropical forest of Guate-
mala (temperatures ranging from 12.5 °C to 27.8 °C, relative humidity from 76% to 91%,
latitude 15°29′0” N, longitude 90°22′0” W, and an altitude of 1316 m a.s.l.) [20]. The flowers
were self-pollinated by hand and the seed capsules harvested when mature (i.e., capsules
that completed development and were fully grown) and about to naturally dehisce (i.e.,
gape or burst open). After harvesting, the seed capsules were placed in different plastic
containers with silica gel to absorb humidity and tissue paper on the top and boom to
minimize movement, surrounded by a cold shipping package that consisted of a standard
duration cooling system of 96 h at a constant 2 to 5 °C (provided by a Guatemalan sup-
plier, SM Soluciones S.A.) and sent by courier to Massey University Turitea Campus in
New Zealand (1–2 weeks). Upon arrival, the seed capsules were placed in plastic contain-
ers to dry in a controlled environment room operating at 20 °C and 55% relative humidity
(RH). The seed capsules were allowed to open naturally under those conditions, and ex-
posed seeds were then collected and stored in glass vessels under the same conditions (20
°C and 55% RH) before being assessed.
Figure 1. Flowers of (a) Lycaste cochleata, (b) L. lasioglossa, and (c) L. virginalis (images by Fredy Ar-
chila Morales).
2.2. Seed Capsule Assessment
To observe the morphological variation of the seed capsules, we assessed ten capsules
from different individual plants per species (upon arrival in NZ).Each capsule was treated
as a replicate. Each capsule’s length, top diameter, central diameter, and boom diameter
were measured using a vernier calliper. The measurement consisted of placing the seed
capsules longitudinally (for length) and transversely (for diameter) between the two ver-
nier scales. In addition, we visually evaluated and compared the capsule shape, particu-
larly the number of ribs per capsule and the colour of the seed capsules, which was exam-
ined using the Pantone Colour Matching System [21].
2.3. Seed and Embryo Morphology
Prior to undertaking the measurements, seed samples of five different capsules per
species were stained with tetrazolium chloride [22]. We used this staining method for ease
of observation under the microscope, and recorded seed viability data (indicated by red
staining the embryo), which will be reported as part of a separate study. The tetrazolium
test was performed following the method employed by Diantina [23]. Firstly, seed sam-
ples were placed in plastic vials with a sucrose solution at 10% (w/v) in a room at 20 °C for
twenty-four hours. Afterwards, the sucrose solution was replaced with 2,3,5-triphenyl te-
trazolium chloride at 1% (w/v), and then the seeds were incubated in a dark incubator at
40 °C for another twenty-four hours. Lastly, ten red-stained seeds per capsule (50 seeds
per species) were measured for the length and width of both the seed and the embryo
under a binocular microscope Olympus SZX7 at magnification 5.6×.
Figure 1.
Flowers of (
a
)Lycaste cochleata, (
b
)L. lasioglossa, and (
c
)L. virginalis (images by Fredy
Archila Morales).
2.2. Seed Capsule Assessment
To observe the morphological variation of the seed capsules, we assessed ten capsules
from different individual plants per species (upon arrival in NZ).Each capsule was treated
as a replicate. Each capsule’s length, top diameter, central diameter, and bottom diameter
were measured using a vernier calliper. The measurement consisted of placing the seed
capsules longitudinally (for length) and transversely (for diameter) between the two vernier
scales. In addition, we visually evaluated and compared the capsule shape, particularly the
number of ribs per capsule and the colour of the seed capsules, which was examined using
the Pantone Colour Matching System [21].
2.3. Seed and Embryo Morphology
Prior to undertaking the measurements, seed samples of five different capsules per
species were stained with tetrazolium chloride [
22
]. We used this staining method for ease
of observation under the microscope, and recorded seed viability data (indicated by red
staining the embryo), which will be reported as part of a separate study. The tetrazolium
test was performed following the method employed by Diantina [
23
]. Firstly, seed samples
were placed in plastic vials with a sucrose solution at 10% (w/v) in a room at 20
◦
C for
twenty-four hours. Afterwards, the sucrose solution was replaced with 2,3,5-triphenyl
tetrazolium chloride at 1% (w/v), and then the seeds were incubated in a dark incubator at
40
◦
C for another twenty-four hours. Lastly, ten red-stained seeds per capsule (50 seeds per
species) were measured for the length and width of both the seed and the embryo under a
binocular microscope Olympus SZX7 at magnification 5.6×.
The seed volume (SV), embryo volume (EV), and percentage of air space within the
seed (ASV) were estimated using the formulas established by Arditti and Ghani [
24
] for
seeds with prolate spheroid embryos:
SV =2(( SW/2)2∗(SL/2)∗1.047)
Seeds 2023,2334
EV =4/3π∗(EL/2)∗(EW/2)2
ASV (%)=SV −EV
SV ∗100
where SW = seed width, SL = seed length, EW = embryo width, and EL = embryo length.
The seed length and seed width (SL/SW), embryo length and embryo width (EL/EW),
and seed volume with embryo volume (SV/EV) relationship were assessed to confirm the
relative occupancy of the embryo and air space within the seeds.
2.4. Data Analysis
The Shapiro–Wilk test was used to confirm that data met the premises of normality
(p> 0.05). A general linear model (GLM) was performed to explore differences in the
quantitative seed coat and seed traits among species. Afterwards, means were compared
with a post hoc Tukey’s test (significance 5%), using the software ‘IBM SPSS Statistics’
version 28.0.1.1. [
25
]. In addition, the relationships of the different seed morphological
parameters studied, SL/SW, EL/EW, and SV/EV, were analysed as described above.
3. Results
3.1. Seed Capsule Morphology
The seed capsules of the three species had six longitudinal ribs, as is common for
other orchid species [
26
,
27
]. The seed capsules were considered ripe when their colour was
yellowish green (Pantone 3604-382) (Figure 2). On drying, the capsules turned brown with
some small green–yellow spots (Pantone 1255-1265-582-457) (Figure 2).
Seeds 2023, 2 334
The seed volume (SV), embryo volume (EV), and percentage of air space within the
seed (ASV) were estimated using the formulas established by Ardii and Ghani [24] for
seeds with prolate spheroid embryos:
SV 2 SW 2
∗SL 2
∗1.047
EV 43
𝜋 ∗ EL 2
∗EW 2
ASV %
SVEV
SV ∗100
where SW = seed width, SL = seed length, EW = embryo width, and EL = embryo length.
The seed length and seed width (SL/SW), embryo length and embryo width (EL/EW),
and seed volume with embryo volume (SV/EV) relationship were assessed to confirm the
relative occupancy of the embryo and air space within the seeds.
2.4. Data Analysis
The Shapiro–Wilk test was used to confirm that data met the premises of normality
(p > 0.05). A general linear model (GLM) was performed to explore differences in the quan-
titative seed coat and seed traits among species. Afterwards, means were compared with
a post hoc Tukey’s test (significance 5%), using the software IBM SPSS Statistics’ version
28.0.1.1. [25]. In addition, the relationships of the different seed morphological parameters
studied, SL/SW, EL/EW, and SV/EV, were analysed as described above.
3. Results
3.1. Seed Capsule Morphology
The seed capsules of the three species had six longitudinal ribs, as is common for
other orchid species [26,27]. The seed capsules were considered ripe when their colour
was yellowish green (Pantone 3604-382) (Figure 2). On drying, the capsules turned brown
with some small green–yellow spots (Pantone 1255-1265-582-457) (Figure 2).
Figure 2.
Variability of ripe and dry seed capsules of Lycaste virginalis,L. lasioglossa, and L. cochleata.
The scale in the left pictures is a 30 cm ruler, and in the right ones each square is 1 cm2.
Table 1shows the results of a general linear model (GLM), which revealed that
L. cochleata capsules were significantly different in mean length (F (2, 27) = 13.28,
p= 0.0
)
and in top diameter (F (2, 27) = 47.88, p= 0.0) compared with L. lasioglossa and L. virginalis.
For the central diameter, there were no significant differences in means across the three
Seeds 2023,2335
species (F (2, 27) = 1.5, p= 0.24). For the bottom diameter, there was a statistically significant
difference in the mean between all three species (F (2, 27) = 28.5, p= 0.0), with L. virginalis
having the largest diameter and L. cochleata the smallest.
Table 1.
Variability in seed capsule length and diameter (Ø) of the three different Lycaste or-
chid species.
Species Capsule Measurement (cm)
Length Ø Top Ø Central Ø Bottom
L. cochleata 7.90 ±0.74 b1.0 ±0.00 b2.1 ±2.13 n.s. 1.0 ±0.00 c
L. lasioglossa 9.80 ±1.03 a2.1 ±0.32 a2.9 ±0.32 n.s. 1.6 ±0.52 b
L. virginalis 10.3 ±1.42 a2.2 ±0.42 a3.0 ±0.47 n.s. 2.0 ±0.00 a
Each individual value (mean
±
SD) is followed by letters that indicate significant differences when present (Tukey
test, p< 0.05). n.s. indicates that no significant differences were found for this trait.
3.2. Seed Morphology
Lycaste has fine, yellowish, sand-like seeds. The colour intensity varies slightly be-
tween each species (L. cochleata: Pantone 393, L. lasioglossa: Pantone 615, and L. virginalis:
Pantone 459) (Figure 3) [
21
]. Under the microscope, it was possible to distinguish the seed
coat and embryo. The three species have scobiform oval-shaped seeds and prolate spheroid
embryos, as defined by [15,16] (Figure 3).
Seeds 2023, 2 335
Figure 2. Variability of ripe and dry seed capsules of Lycaste virginalis, L. lasioglossa, and L. cochleata.
The scale in the left pictures is a 30 cm ruler, and in the right ones each square is 1 cm
2
.
Table 1 shows the results of a general linear model (GLM), which revealed that L.
cochleata capsules were significantly different in mean length (F (2, 27) = 13.28, p = 0.0) and
in top diameter (F (2, 27) = 47.88, p = 0.0) compared with L. lasioglossa and L. virginalis. For
the central diameter, there were no significant differences in means across the three spe-
cies (F (2, 27) = 1.5, p = 0.24). For the boom diameter, there was a statistically significant
difference in the mean between all three species (F (2, 27) = 28.5, p = 0.0), with L. virginalis
having the largest diameter and L. cochleata the smallest.
Tab l e 1. Variability in seed capsule length and diameter (Ø) of the three different Lycaste orchid
species.
Species Capsule Measurement (cm)
Length Ø Top Ø Central Ø Boom
L. cochleata 7.90 ± 0.74
b
1.0 ± 0.00
b
2.1 ± 2.13
n.s.
1.0 ± 0.00
c
L. lasioglossa 9.80 ± 1.03
a
2.1 ± 0.32
a
2.9 ± 0.32
n.s.
1.6 ± 0.52
b
L. virginalis 10.3 ± 1.42
a
2.2 ± 0.42
a
3.0 ± 0.47
n.s.
2.0 ± 0.00
a
Each individual value (mean ± SD) is followed by leers that indicate significant differences when
present (Tukey test, p < 0.05). n.s. indicates that no significant differences were found for this trait.
3.2. Seed Morphology
Lycaste has fine, yellowish, sand-like seeds. The colour intensity varies slightly be-
tween each species (L. cochleata: Pantone 393, L. lasioglossa: Pantone 615, and L. virginalis:
Pantone 459) (Figure 3) [21]. Under the microscope, it was possible to distinguish the seed
coat and embryo. The three species have scobiform oval-shaped seeds and prolate sphe-
roid embryos, as defined by [15,16] (Figure 3).
Figure 3. Lycaste seeds without (left photographs) and with 5.6× magnification (right photographs).
The seeds enlarged at 5.6× are stained with 2,3,5-Tetrazolium Chloride (TTC) for ease of visualiza-
tion under the microscope. Each square on the left side is 1 cm
2
.
Figure 3.
Lycaste seeds without (left photographs) and with 5.6
×
magnification (right photographs).
The seeds enlarged at 5.6
×
are stained with 2,3,5-Tetrazolium Chloride (TTC) for ease of visualization
under the microscope. Each square on the left side is 1 cm2.
GLM analysis revealed that there was a statistically significant difference in the seed
dimensions (SL = seed length, SW = seed width, and SV = seed volume), and significant
differences within the embryo dimensions (EL = embryo length, EW = embryo width, and
EV = embryo volume) between the three species (Table 2). The data showed that the mean
air-space percentage differed significantly between the three species (
F (2, 147) = 105.62
,
p= 0.0
), with L. lasioglossa having the highest seed air space volume and L. cochleata the low-
est, but all were less than 50% of the total seed volume. Likewise, the post hoc comparison
Seeds 2023,2336
using Tukey’s test after GLM found that the mean value of the seed morphological traits
was significantly different between the three species, but this differed for individual traits,
with L. virginalis having the longest seed but an embryo equal in length to L. cochleata. The
largest embryo volume was found in L. cochleata.
Table 2. Seed and embryo dimensions for three Lycaste species.
Seed Dimensions
Species Length (mm) Width (mm) Volume (mm3)
L. cochleata 0.21 ±0.02 c0.11 ±0.01 a0.00063 ±0.0002 a
L. lasioglossa 0.23 ±0.03 b0.09 ±0.01 b0.00051 ±0.0001 b
L. virginalis 0.26 ±0.02 a0.09 ±0.01 b0.00060 ±0.0002 a
Embryo dimensions Air-space % in the seed
Species Length (mm) Width (mm) Volume (mm3)
L. cochleata 0.14 ±0.01 a0.082 ±0.01 a0.00051 ±0.00011 a17.4 ±8.0 c
L. lasioglossa 0.12 ±0.01 b0.066 ±0.01 c0.00029 ±0.0007 c42.3 ±8.1 a
L. virginalis 0.15 ±0.01 a0.073 ±0.01 b0.00041 ±0.00009 b29.7 ±9.5 b
Each individual value (mean
±
SD) is followed by letters that indicate significant differences when different
(Tukey test, p< 0.05).
Table 3shows comparison of the relationship between some of the morphological
parameters evaluated, i.e., seed length (SL) with seed width (SW); the ratio of embryo
length (EL) with embryo width (EW); and seed volume (SV) with embryo volume (EV).
The SL/SW and SV/EV differed across all three species.
Table 3.
Relationship between seed length and seed width (SL/SW), embryo length and embryo
width (EL/EW), and seed volume with embryo volume (SV/EV) of three Lycaste species.
Species SL/SW EL/EW SV/EV
L. cochleata 1.98 ±0.26 c1.78 ±0.24 b1.22 ±0.13 c
L. lasioglossa 0.57 ±0.37 b1.86 ±0.19 b1.77 ±0.25 a
L. virginalis 2.75 ±0.38 a2.04 ±0.26 a1.45 ±0.21 b
Different letters indicate significant differences (p< 0.05) between species for each trait (mean
±
SD) after a Tukey
post hoc test.
4. Discussion
4.1. Seed Capsule Morphology
In this study, we characterized the seed capsule morphology of three tropical epiphytic
species of the genus Lycaste. The seed capsule morphological assessment was based on the
capsule’s length, diameter (i.e., top, middle, bottom), and colour. Our results show that
the seed capsules of the three species are very similar in appearance and colour (Figure 2).
Other studies have reported similarities in seed capsule shape and colour for other Lycaste
species [12,17,27].
Despite qualitative similarities, the capsules’ quantitative traits varied between species,
with L. cochleata having comparatively smaller capsules than the other species (Table 1).
We also observed some intraspecific variation in capsule size (Figure 2). These differences
could reflect species-specific adaptations to their natural environment [
23
,
28
] related to
capsule position, or could be a result of differences in resource availability to plants when
developing the capsules (the seed capsules were produced in the same location at the same
time, and the collected capsules were positioned in the same location on different plants;
however, the plants grew outdoors, where many factors were not controlled). Other factors,
such as intra-plant competition, may also have contributed to the observed differences
(i.e., competition between individuals of the same species for resources, edge effects). More
research over several production seasons and under controlled conditions is required to
elucidate the reasons behind inter- and intraspecific variability.
Seeds 2023,2337
4.2. Seed Morphology
Like for the seed capsules, multiple traits are shared by the seeds of the three Lycaste
species, e.g., they seeds are small, oval-shaped, scobiform, and have comparatively large
embryos and lower air volumes. Similar traits (small ~300
µ
m, scobiform seeds) have been
previously reported for Lycaste skinneri by [
17
], who also described testa seed characteristics
(not explored here).
Orchid seed sizes can be categorized based on their length, from very small (
100–200 µm
),
small (200–500
µ
m), medium (500–900
µ
m), and large (900–2000
µ
m), to very large
(2000–6000
µ
m) [
17
]. Based on this classification, L. cochleata,L. lasioglossa, and
L. virginalis
are all considered species with small seeds (210, 230, and 260
µ
m, respectively). Dur-
ing the evolutionary process, morphological differences occurred in orchid seeds as an
adaptation to different habitats (e.g., epiphytic vs. terrestrial), climates (e.g., tropical vs.
temperate), and modes of dispersal (e.g., anemochory, zoochory, hydrochory) [
16
]. The
three Lycaste species studied are tropical epiphytes, and more information is needed about
their dispersal mechanisms.
Our results show that the three Lycaste species have low air volumes, which is con-
sistent with other epiphytic orchids [
15
,
16
,
18
]. Epiphytic orchids (e.g., many Dendro-
bium spp.) typically have smaller air spaces than their terrestrial counterparts (e.g., Paphio-
pedilum) [
15
]. It has been suggested that a larger airspace was an advantage for ancestral
terrestrial orchids; it causes higher buoyancy and floatability, and thus wider distribution
areas [
15
,
16
,
18
]. The shift in seed aerodynamic traits is likely to be related to their evolution-
ary changes in growth habit, from terrestrial to epiphytic, suggesting lower dispersibility
in epiphytes [18].
We found the embryo dimensions to be similar to those reported for other tropical
epiphytic species, e.g., Dendrobium spp. [
29
,
30
]. A comparative study between closely
related terrestrial and epiphytic species of the Liparis genus in Japan found that epiphytic
species had larger embryos and suggested that seeds with larger embryos are likely to be
developmentally advanced and have the potential to germinate earlier, facilitating seed
establishment in epiphytic species which lack a soil substrate [
18
]. However, embryo size
is highly variable among orchids [
17
], and other studies found no relationship between
embryo size and habit for other genera [
23
,
31
]. More studies on Lycaste species with
different habits are needed to explore whether there is a relationship between embryo size,
habit, and germination in this genus.
Seeds with large embryos have also been reported as non-dormant, an adaptation that
may allow them to germinate faster in tropical environments, as opposed to in temperate
environments where seeds may require dormancy until certain environmental conditions
are met to support germination [
15
,
30
]. In this study, seed dormancy was not evaluated
but we hypothesize that seeds are non-dormant, given their natural distribution (tropical
habitats). Further research is needed to validate whether Lycaste’s seed morphological traits
are associated with a lack of dormancy, as predicted.
While many similarities exist, we also recorded some variability in the seed morpho-
logical traits across Lycaste species. Of particular interest are the differences in embryo
volume and air space percentage, which differ significantly between species and could
influence important processes such as seed dispersal, dormancy, germination, and estab-
lishment. L. lasioglossa has smaller seeds, smaller embryos, and a larger air volume than the
other two species, but the biological and ecological implications of these differences remain
to be elucidated.
We encourage further comparative studies including other Lycaste species to estab-
lish relationships between seed morphology and biological and ecological traits. The
exploration of other morphological traits not investigated here (e.g., additional seed coat
characters, ovary size, testa cell shape and dimensions, etc.) could also add valuable
information for species comparisons. This is only a small contribution to the knowledge
of Guatemalan (and Neotropical) orchid species, but more studies on seed morphology,
Seeds 2023,2338
ecology, chemistry, and storage such as those conducted for other species, e.g., [
23
,
30
,
32
,
33
],
are vital to ensure the conservation of Neotropical orchids for future generations.
5. Conclusions
Overall, our results show similarities in the qualitative traits of the seed capsules
and the seeds of Lycaste. While similarities exist, we also report significant differences
in the quantitative characteristics of the seed capsules and seed micromorphology of the
three studied species. The implication of these differences for taxonomy, seed biology, and
ecology remain to be elucidated.
Like other epiphytic orchids, Lycaste species have relatively small air volumes com-
pared to terrestrial species, which is thought to be an evolutionary adaptation related to seed
dispersal. Smaller air volumes are associated with less floatability and buoyancy, restricting
seed dispersal, and probably contribute to the limited distribution of these species.
This characterization is of value to systematic botany because it contributes to our
understanding of orchid seed micromorphology in three Neotropical species of the Lycaste
genus. However, we only contributed to the study of a small proportion of the total number
of species in the Lycaste genus (3 species out of 36); more comparative studies including
other Lycaste species are needed to explore the relationships between seed morphology,
taxonomy, biology, and ecology to inform conservation efforts.
Author Contributions:
Conceptualization and methodology A.A.P., A.C.M., C.M. and J.N.; data
collection A.A.P.; data analysis A.A.P. and A.C.M.; revision and discussion A.A.P., A.C.M., C.M., J.N.
and F.A.M.; manuscript written by A.A.P., A.C.M., C.M. and J.N. All authors have read and agreed to
the published version of the manuscript.
Funding:
This research was funded by a Manaaki Scholarship granted by the Ministry of Foreign
Affairs and Trade of New Zealand and by Massey University Research Fund (MURF).
Informed Consent Statement: Not applicable.
Data Availability Statement:
The data presented in this study are available on request from the
corresponding authors.
Acknowledgments:
We are grateful to the Ministry of Foreign Affairs and Trade of New Zealand
(MFAT) for providing financial assistance to carry out this research and to Massey University for
admitting us to use their research laboratories and equipment to collect the data. We also want to
thank the Experimental Orchid Station Farm of the Archila Family for donating the seed material.
We are grateful to the Guatemalan authorities, especially CONAP (National Council of Protected
Areas of Guatemala) and MAGA (Ministry of Agriculture, Livestock and Food of Guatemala), for
issuing the certificates needed for the seed material exportation, and to the MIP (Ministry of Primary
Industries of New Zealand) for allowing the importation of the seeds into New Zealand.
Conflicts of Interest: The authors declare no conflict of interest.
References
1. Didier, L.; Tamay, C. Orquídeas: Importancia y uso en México. Bioagrociencias 2013,6, 7.
2.
Meisel, J.E.; Kaufmann, R.S.; Pupulin, F. Orchids of Tropical America: An Introduction and Guide; Cornell University Press:
Ithaca, NY, USA; Comstock Publishing Associates: New York, NY, USA, 2014.
3. Cavero, M.; Collantes, B.; Patroni, C. Orquídeas del Perú; Centro de datos para la Conservación del Perú: Lima, Perú, 1991.
4.
Lawson, C.; Wraith, J.; Pickering, C. Regulating wild collected orchids? The CBD, Nagoya Protocol and CITES overlaps. Environ.
Plan. Law J. 2019,36, 339–361.
5. Martija-Ochoa, M. El Gran Libro de las Orquídeas; Parkstone International: New York, NY, USA, 2019.
6. Sailo, N.; Rai, D.; De, L. Physiology of temperate and tropical orchids-an overview. Int. J. Sci. 2014,3, 3–8.
7. Archila Morales, F. Listado actualizado de orquídeas de Guatemala. Guatemalensis 2022,25, 129–419.
8.
Gaskett, A.C.; Gallagher, R.V. Orchid diversity: Spatial and climatic patterns from herbarium records. Ecol. Evoution
2018
,22,
11235–11245. [CrossRef] [PubMed]
9. Archila Morales, F.; Chiron, G.R. El género Lycaste en Lindley en Guatemala. Guatemalensis 2011,14, 1–55.
10.
World Flora Online, Lycaste Lindl. Available online: http://www.worldfloraonline.org/taxon/wfo-4000022472;jsessionid=9948
75A46326DB4656AA9765C0EF5F5C (accessed on 16 July 2021).
Seeds 2023,2339
11.
KEW, Plants of the World Online: Lycaste Lindl. Available online: https://powo.science.kew.org/taxon/urn:lsid:ipni.org:names:
30000072-2 (accessed on 29 June 2023).
12. Ames, O.; Correll, D.S. Orchids of Guatemala; Chicago Natural History Museum: Chicago, IL, USA, 1953; Volume 26, Number 2.
13.
CONAP. Lista de Especies Amenazadas de Guatemala (LEA); Departamento de Vida Silvestre del Consejo Nacional de Áreas
Protegidas de Guatemala: Guatemala City, Guatemala, 2009.
14. CITES Species Lists. 2021. Available online: https://www.speciesplus.net/species (accessed on 14 July 2021).
15.
Diantina, S.; Clavijo McCormick, A.; Pritchard, H.W.; Millner, J.; Nadarajan, J.; Mastur, M.; McGill, C. Orchid seed micro-
morphometry: Importance to species biology, ecology, and conservation. II Int. Symp. Trop. Subtrop. Ornam.
2021
,1334, 153–162.
[CrossRef]
16.
Verma, J.; Sharma, K.; Thakur, K.; Sembi, J.K.; Vij, S.P. Study on seed morphometry of some threatened Western Himalayan
orchids. Turk. J. Bot. 2014,38, 234–251. [CrossRef]
17. Barthlott, W.; Große-Veldmann, B.; Korotkova, N. Orchid seed diversity. Englera 2014,32, 1–245.
18.
Tsutsumi, C.; Yukawa, T.; Lee, N.S.; Lee, C.S.; Kato, M. Phylogeny and comparative seed morphology of epiphytic and terrestrial
species of Liparis (Orchidaceae) in Japan. J. Plant Res. 2007,120, 405–412. [CrossRef] [PubMed]
19. Leck, M.A.; Parker, V.T.; Simpson, R. Seedling Ecology and Evolution; Cambridge University Press: Cambridge, UK, 2008.
20.
Weather Atlas, Monthly Weather Forecast and Climate Cobán, Guatemala. 2021. Available online: https://www.weather-atlas.
com/en/guatemala/coban-climate (accessed on 28 December 2021).
21. Pantone, I. PANTONE®Colours. 2017. Available online: http://www.pantone-colours.com/ (accessed on 28 December 2021).
22. ISTA. International Rules for Seed Testing; The International Seed Testing Association: Bassersdorf, Switzerland, 2022.
23.
Diantina, S.; McGill, C.; Millner, J.; Nadarajan, J.; WPritchard, H.; Clavijo McCormick, A. Comparative seed morphology of
tropical and temperate orchid species with different growth habits. Plants 2020,9, 161. [CrossRef] [PubMed]
24.
Arditti, J.; Ghani, A. Numerical and physical properties of orchid seeds and their biological implications. New Phytologist.
2000
,
146, 569.
25. IBM Corp. IBM SPSS Statistics for Windows; IBM Corp: Armonk, NY, USA, 2022.
26. Rasmussen, F.N.; Johansen, B. Carpology of orchids. Selbyana 2006, 44–53.
27. Ames, O.; Correll, D.S. Orchids of Guatemala; Chicago Natural History Museum: Chicago, IL, USA, 1952; Volume 26, Number 1.
28.
Vafaee, Y.; Mohammadi, G.; Nazari, F.; Fatahi, M.; Kaki, A.; Gholami, S.; Ghorbani, A.; Khadivi, A. Phenotypic characterization
and seed-micromorphology diversity of the threatened terrestrial orchids: Implications for conservation. S. Afr. J. Bot.
2021
,137,
386–398. [CrossRef]
29.
Hariyanto, S.; Pratiwi, I.A.; Utami, E.S.W. Seed morphometry of native indonesian orchids in the genus Dendrobium.Scientifica
2020,2020, 3986369. [CrossRef]
30.
Prasongsom, S.; Thammasiri, K.; Pritchard, H.W. Seed micromorphology and ex vitro germination of Dendrobium orchids. I Int.
Symp. Trop. Subtrop. Ornam. 2016,1167, 339–344. [CrossRef]
31.
Ortúñez, E.; Gamarra, R. Seed morphology, life form and distribution in three Bromheadia species (Epidendroideae, Orchidaceae).
Diversity 2023,15, 195. [CrossRef]
32.
Diantina, S.; Kartikaningrum, S.; McCormick, A.C.; Millner, J.; McGill, C.; Pritchard, H.W.; Nadarajan, J. Comparative
in vitro
seed germination and seedling development in tropical and temperate epiphytic and temperate terrestrial orchids. Plant Cell
Tissue Organ Cult. (PCTOC) 2020,143, 619–633. [CrossRef]
33.
Diantina, S.; McGill, C.; Millner, J.; Nadarajan, J.; Pritchard, H.W.; Colville, L.; Clavijo McCormick, A. Seed viability and fatty acid
profiles of five orchid species before and after ageing. Plant Biol. 2022,24, 168–175. [CrossRef]
Disclaimer/Publisher’s Note:
The statements, opinions and data contained in all publications are solely those of the individual
author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to
people or property resulting from any ideas, methods, instructions or products referred to in the content.
