Journal of Threatened Taxa |
www.threatenedtaxa.org | 26 April 2023 | 15(4): 22940–22954
ISSN 0974-7907
(Online) | ISSN 0974-7893 (Print)
https://doi.org/10.11609/jott.8180.15.4.22940-22954
#8180 | Received 07
September 2022 | Final received 31 March 2023 | Finally accepted 09 April 2023
Floral biology of Baccaurea courtallensis
– an endemic tree species from peninsular India
Karuppiah Nandhini
1, Vincent Joshuva David 2,
Venugopal Manimekalai 3 & Perumal Ravichandran 4
1,2,4 Department of Plant Science, Manonmaniam Sundaranar
University, Abishekapatti, Tirunelveli, Tamil Nadu
627012, India.
3 Department of Botany, Sri Parasakthi College for Women, Courtallam,
Tamil Nadu 627802, India.
1 nandhuwin19@gmail.com, 2 joshuvadavi@gmail.com,
3 sehimaravi@gmail.com, 4 grassravi@msuniv.ac.in
(corresponding author)
Editor: A.G. Pandurangan,
Thiruvananthapuram, Kerala, India. Date
of publication: 26 April 2023 (online & print)
Citation: Nandhini, K., V.J. David,
V. Manimekalai & P. Ravichandran (2023). Floral biology of Baccaurea
courtallensis – an endemic tree species from
peninsular India. Journal of Threatened Taxa 15(4): 22940–22954. https://doi.org/10.11609/jott.8180.15.4.22940-22954
Copyright: © Nandhini et al. 2023. Creative Commons Attribution 4.0
International License. JoTT allows unrestricted use, reproduction, and
distribution of this article in any medium by providing adequate credit to the
author(s) and the source of publication.
Funding: Department of Biotechnology (DBT), Ministry of Science and Technology, New Delhi, India, Project
No. BT/PR29631/FCB/125/13/2018 dt.25.02.2019, Network project for three years 2019-2022.
Competing interests: The authors declare no competing interests.
Author details: Karuppiah Nandhini is a Ph.D. research scholar & worked as Assistant Professor (Temporary) in the Department of Plant Science at Manonmaniam Sundaranar University. She is very passionate about the field of Plant Tissue culture and is also strongly interested in the fields of plant reproductive biology and molecular biology. She is keenly interested in the conservation of IUCN red-listed plants of Western Ghats. Vincent
Joshuva
David, a DBT Junior Research Fellow, is currently working on his Ph.D. in the Department of Plant Science, Manonmaniam Sundaranar University, Tirunelveli. He has a keen interest in practical
exposure to Biodiversity, Plant Reproductive Biology, Plant Molecular and Biotechnology, Plant anatomy, and Conservation of Biodiversity. Venugopal Manimekalai is currently working as an Assistant Professor in the Department of Botany, Sri Parasakthi College for Women, Courtallam. Her fields of research include Plant anatomy and histochemistry, Natural dyeing of miscellaneous plant fibres, Plant tissue culture, and Plant reproductive biology. She was a recipient of the DST young women scientist (WOSB) award. She has bagged three times best student’s research project awards from the Tamil Nadu State Council for Science and Technology. Perumal
Ravichandran is working at present as a Professor in Manonmaniam Sundaranar University. His fields of research are the Conservation of Biodiversity, Agrostology, Grassland Management, Natural Dyeing, and Plant Biotechnology. He has over 25 years of teaching and research experience. He has extensively surveyed the Western Ghats and other forest areas in India for Botanical and Ecological investigations.
He was a member of IUCN – SSC- Indian Sub-Continent Plant Specialist Group (ISPSG).
Author contributions: KN – Field & experimental study, data collection, compilation, writing, VJD- Field study, Field Photography, and Photo editing, Map preparation, VM-writing, proofreading and editing, PR- Field study, Hypothesis, conceptualization, Microphotography, writing, editing, and supervision.
Acknowledgements: The authors are thankful to the
Department of Biotechnology (DBT), Ministry of Science and Technology, New
Delhi, India for providing financial assistance through a research project and
support Joshuva David with a JRF, Project No.
BT/PR29631/FCB/125/13/2018 dt.25.02.2019. The authors extend their thanks to
Tamil Nadu Forest department for providing permission (WL5 (A)/49087/2018 dated
06/12/2018) to collect plant parts and carry out field visits. The authors thank the first two unknown
reviewers, and Dr. AGP (subject editor) for their
critical comments, suggestions, recommendations, and proofing to improve the
quality of the research article. The authors also thank Dr.
D. Narasimhan, retired professor of Botany, Madras Christian College, Tambaram,
Chennai for editing the Tamil version of the abstract within a short span of
time. The authors acknowledge the
co-coordinator of SPC-DST FIST for taking a Few fluorescent and light
microscopic images using Advanced Fluorescent Trinocular Microscope Work
Station with Digital Documentation, Imaging Facility - Nikon Eclipse Ni-U
(SPC-DST-FIST-2018-19)
Abstract: Baccaurea courtallensis,
a member of the Phyllanthaceae
family is a tree species endemic to peninsular India. Despite the fact that
this plant is naturally propagated through seeds, there is no information on
its reproductive biology. To understand the reproductive biology of this
species, its floral biology is very important. Hence, this study was conducted
to comprehend the detailed aspects of its flowering and fruiting characters.
Blooms occur during February–May; fruits develop and mature from June to
September. Flowers are unisexual, and dioecious. The present study reports on
the rare occurrence of monoecious flowers in many inflorescences of a few
trees. Crimson red fruits are arranged in a racemose type of inflorescence and
hang in symmetric clusters. Inflorescence clusters are observed all along the
trunk from base upwards. Wind and insect pollinations were observed in this
species during field visits: honey bees and black ants
were observed as the major floral visitors. Pollen grains showed 96.24%
fertility in the acetocarmine glycerin test and 80% viability in the fluro-chromatic reaction test. Pollen germination was 63.1%
in Brewbaker and Kwack’s
medium containing 10% sucrose. The detailed aspects of flower and fruit
morphology and anatomy respectively are reported for the first time.
Keywords: Anthesis, blooms, frugivores,
phenology, pollen germination, monoecious, pollen viability, pollination, seed
biology.
INTRODUCTION
Baccaurea courtallensis
(Wight). Müll. Arg., an endemic species to peninsular India (Kumar
2012; Narasimhan & Irwin 2021) requires special conservation measures to
increase its population. The species survives the wet tropical biome and
occasionally grows along river or stream banks in the moist deciduous forests
of southern India. It is locally called Muttakaipu or
Muttathuri in Malayalam and Mootupazham,
Pulichampazham in the Tamil language. The habit is a
medium-sized tree, growing to a height of 15–25 m, and produces crimson-red
edible fruits, sour to sweet in taste when fully
ripe. The fruit is reported to be a good source of vitamin ‘C’ and antioxidants
(Nazarudhin 2010). This tree species has ornamental
value as well, at full bloom it is a treat to watch the color contrast of
inflorescences (Yogeesha et al. 2016). Tropical
evergreen forests of the southern Western Ghats, Eastern Ghats, and Odisha
(Balakrishnan & Chakrabarty 2007; Narasimhan & Irwin 2021) are the home
of this evergreen tree. It is distributed in evergreen and semi-evergreen
forest areas in the Western Ghats especially the southern Sahyadri and central
Sahyadri (up to the Coorg region). Understory trees in low and medium-elevation
evergreen forests up to 1,000 m (Abhishek et al. 2011). B. courtallensis is an underutilized fruit tree. In the
broadest sense, the term “underutilized fruit tree” refers to a group of fruit
trees that are currently growing in a dispersed and unattended manner on
roadsides, homestead land, and wasteland despite having the potential for
intensive utilization (Jisha et al. 2015). Fruits are
crimson red in colour and acidic in taste. Only the
local tribal population of the Western Ghats region consumes these fruits and
it’s not widely known to others. Fruits of B. courtallensis
are eaten for their medicinal properties too. Fruits are used to induce
fertility in men and women (Daniel et al. 2005). Fruits are also used to
prepare jam, squashes, and wine. The fruits are also consumed in greater
quantities by tortoises and Sloth Bears, which possibly reduces the likelihood
of natural regeneration (Mohan 2009). The flowering period of B. courtallensis starts in February and extends up to
April; the peak flowering month is March and the fruits develop and mature from
June to September, during the rainy season. Literature on the reproductive
biology or floral biology of this tree is sporadic. The present investigation
was carried out to study plant morphology, flower, fruit morphology, anatomy,
pollen biology, and fruit set.
MATERIALS
AND METHODS
Study area
The present
research work was carried out from 2018 to 2022, in the southern Western Ghats
of Tamil Nadu and Kerala. The Western Ghats is a magnificent mountain range,
next only to the Himalaya, and has rich biological wealth with a high degree of
endemism. Southern Western Ghats is one of the richest areas of India in the
context of floristic, diversity, composition, holding a large number of endemic
taxa. B. courtallensis is one among the
endemic species of south Western Ghats and is distributed in Tenkasi, Tirunelveli, and Kanyakumari districts of Tamil
Nadu and in Kerala, it is located in all districts but more abundantly in
Kollam, Thiruvananthapuram, Pathanamthitta, and
Idukki districts. To locate the candidate species, field expeditions were made
to the forests in the KMTR zones, especially the Mundanthurai
range of Ullaru, Kannikatti,
and Kodamaadi beats, Kadayam
range of Kadana beat, Therkumalai,
and Mylodai estate, as well as at Kollam forest
division of Achenkovil forest range. A total of about
2,500 individuals were found in the Ullaru (1700), Kodamadi (600), and Achenkovil
(200) forest areas. Detailed field investigations on the reproductive biology
and phenology of the species were conducted in these forest areas (Figure 1).
Selection
of trees
Baccaurea courtallensis is an
evergreen medium-sized tree that grows up to 15–25 m in height. It is a
dioecious tree; the morphological characters are similar in both male and
female trees, except for the stem region. The distinguishing factor for the
identification of male and female trees during the non-flowering season is
based on the scars on the trunk. The inflorescence-arising zones of the male
tree are numerous, while those of the female tree are fewer. The trunk size of
the female tree is larger than the male tree and scars of several floral
primordia regions were observed in the entire male tree. For the purpose of
observing plant morphological traits, 25 female, and 25 male trees from
fourteen population sites were chosen. The tree’s height and width, stem, bark colour, branching pattern, leaf arrangement, and the places
where inflorescences emerge in both sexes were all documented in the field.
Floral
phenology
Flowering
phenology was observed in a sequential manner from the bud to the seed maturity
stage. Twenty-five healthy trees were selected randomly from the study sites,
during the period 2018–2022. Observations were made on the reproductive phases
of plants, with respect to the ontogeny of the floral primordium, development
of flowers, maturation, anthesis, pollination, and natural withering of
flowers. Floral visitors were also recorded among these plants (Image 1 F, G
& Image 2 I, J).
Inflorescence
and floral morphology
Male and
female inflorescences and floral morphologies were noted. The inflorescence
type, position, average number of inflorescences per cluster, floral
characteristics, and flowering time were all closely monitored. A Nikon SMZ 800
stereomicroscope fit with a digital dark box and facilitated with Nikon
NIS-Elements basic research imaging software was used for standard research
applications such as analysis and photo documentation of fluorescent imaging,
four-dimensional acquisition, and advanced device control capabilities.
Male and
female flowers
Flowers are
one of the key characters to distinguish male and female trees of B. courtallensis, however, it is hard to distinguish male
and female inflorescences at the very early stages of their development and
it’s difficult to count male and female inflorescences from cluster racemes of
primordial regions due to several clusters. Twenty-five flowering trees, as
mentioned earlier, of both sexes were selected in a population for counting
inflorescences and flower buds. The mean number of clustered inflorescences and
flower buds of males and females was calculated and tabulated (Table 3).
Fruit
morphology and anatomy
The fresh
and mature fruits were harvested and brought to the laboratory immediately.
After harvest, the collection was completely randomized without seeing the
size. The collected fruits were stored in plastic bags and kept in a
refrigerator. Fruits’ fresh and dry weights were measured using a digital
balance. The maximum length (linear distance between peduncular and stylar ends) and maximum diameter (linear distances across
the width of the fruits) were measured using a digital Vernier caliper.
Free hand
cross and longitudinal sections of fruit and seed were taken and observed under
Nikon 80i fluorescent microscope fitted with a digital photography workstation
and SNZ stereo microscope. Specific stains and reagents such as Toluidine Blue
‘O’, Safranin, and oil red were used to detect specialized structures. After
staining, sections were mounted on clean micro-slides using glycerin, as a
mounting solution. The anatomical features were observed and microphotographs
were taken.
Pollen
Biology
Pollen-Ovule
ratio
Healthy, undehisced mature flower buds were taken to determine the
pollen-ovule ratio. Mature and fresh anthers from 20 flowers were randomly
collected during the early morning and squashed separately in a mixture of
ethanol (0.5%), methylene blue, and detergent (0.9ml+0.3ml+0.4ml) (Dafni et al.
2005). Thus, collected pollen grains were mixed thoroughly by the repeated
drawing of the liquid into a disposable syringe and expelling it with
force. Pollen suspensions of 100 µl were
taken on a clean hemocytometer and the total number of pollen grains were
counted in a sample under the light microscope. The procedure was repeated with
10 samples of a suspension and the average number of pollen grains was
calculated. Twenty young pistils were collected from the flowers used for ovule
count and the average number of ovules per ovary was determined by dissecting
the ovaries under the microscope. The pollen-ovule ratio was calculated by the
following formula (Cruden 1977). The percent of fruit
set was calculated by dividing the mean number of matured fruits by the mean
number of female mature flowers in an inflorescence.
The
pollen-ovule ratio is the mean number of pollen grains per flower/mean number
of ovules per flower.
Pollen
fertility
The dye
acetocarmine and glycerine reagent method was used to
determine the fertile pollen grains by the colour
change of cytoplasm alone into deep red and the pollen wall remained uncoloured (Shivanna & Rangaswamy 1992). Fresh pollen grains were collected from
ten flowers and transferred to a clean slide and two drops of the acetocarmine glycerine mixture were added in a ratio of 3:1 and mixed
thoroughly. After 15 minutes the slides were examined under a light microscope.
The number of stained and unstained pollen grains was counted. The stained
pollen grains were considered as fertile and the unstained as sterile.
Pollen
viability
The
viability of pollen grains was examined by three different reagents such as flurochromatic reaction test (FCR), 2, 3, 5-Triphenyl Tetrazolium
Chloride (TTC) and Iodine-Potassium-iodide test (I2KI). The pollen
grain was considered viable if it appeared green in FDA (Fluorescein diacetate)
under a fluorescent microscope (Heslop-Harrison, 1970), dark red in TTC test (Shivanna & Rangaswamy 1992),
and brown in the I2KI test (Sulusoglu
& Cavusoglu 2014) under a light microscope.
Viable and non-viable pollen grains were counted in each field of view for
calculating the percentage of viable and non-viable pollen grains.
In vitro
pollen germination
Fresh
pollen grains were collected on the day of anthesis for pollen germination
studies. In vitro pollen germination was conducted in Brewbaker and Kwack (BK) medium (Brewbaker & Kwack 1963) in
different levels of sucrose (5, 10, 15, and 20% solutions), in order to
determine the effect of different nutrients like boron, and calcium nitrate at
various concentrations of sucrose. The fresh pollen samples were placed into
the BK medium and kept in petri dishes lined with moist filter paper and
incubated for 12 hours. After incubation for twelve hours, pollen samples were
observed under a light microscope. The observed data was recorded and the
percentage of pollen germination was calculated using the following formula.
Percent of
pollen germination = Number of pollen grains germinated / Total number of
pollen grains observed × 100
Floral
visitors
During the
flowering period field visits were carried out to record floral visitors and
pollinators. The flowers are small and red in colour
and male flowers emit mild musky fragrance. Floral visitors and their behaviour were recorded from 0700 h to 1330 h at each study
site using Canon D-SLR Camera. Insect visitation starts around 0730 h.
Observations were made on insect floral visitors, visiting time, the purpose of
visiting, foraging activity and time spent on each flower. Floral visitors were
photographed using a Canon D-SLR camera. Some of these floral visitors were
collected and preserved for identification.
RESULTS
Distribution
and morphology of the plant
The
selected trees were found at altitudes between 180 to 1,000 m in forest areas
of Kadana, Kannikatti, Ulllaru, Ingikuzhi, Kodamadi, Ueipattrai and Valaiyaru, of Tirunelveli District and Courtallam
of Tenkasi District (as per state government orders)
of Tamil Nadu. In Kerala, it is located from Achenkovil
and Thenmala of Kollam District and Kallaru, and Bonacaud Estate on
the way to Ponmudi, at an elevation of 400 to 700 m
in the Thiruvananthapuram District (Figure 1). It is a shade-loving understory
tree that can reach a height of 15 to 25 m, like other evergreen trees found in
the Western Ghats. It is a slow-growing, dioecious tree and rarely monecious.
Few monoecious trees were identified in Ullaru and Kodamadi forest regions. B. courtallensis
trees are closely associated with Antidesma
menasu, Elaeocarpus
venustus, Eugenia singampattiana,
Polyalthia korintii,
Celosia polygonoides, Calamus sp., Hydnocarpus pentandra, and
H. alpina.
Fourteen
population sites were selected for observing the stand height, GBH, and density
of the selected candidate species. Among the 14 populations, only a few forest
areas had a large number of trees specifically Ullaru,
Kodamadi, and Achenkovil.
The distribution of the population was observed continuously to a stretch of 10
km in Ullaru, while in Kodamadi
the distribution was discontinuous and broken into smaller groups. The
population size in Ullaru and Kodamadi
forest areas was about 250 individuals per 5 km radius whereas in Kadana, Achenkovil, and Kallaru to Ponmudi the population
size was 50–100 (Table 1). The number of adult individuals, stand height and
GBH was quantified using population structural data. In every population, there
were more male trees (77.56%) than female (22.43%) trees. The height of male
trees ranges from 10–25 m, whereas the female trees were 10–20 m. Male trees’
mean GBH was 54 cm, whereas female trees’ mean GBH was 58 cm.
The main
threats to candidate species are natural landslides, habitat disturbance,
formation of roads and dams, and other non-forestry activities, since mud roads
have been extended and anthropogenic activities have made the species more
susceptible. The majority of populations were found along roadsides and in
neighboring regions. If no specific precautions are taken for this plant across
the sites, the species will be more vulnerable to catastrophic events resulting
in a reduction of populations.
Baccaurea courtallensis grows up to
15–25 m tall (Image 1A & Image 2A). The colour of
the bark is grey usually smooth or scaly, blaze light orange covered by
lichens. Branchlets terete, glabrous. Leaves simple, alternate, clustered at
twig ends, stipules ovate, acute, hairy, and caducous. 1.2–3.8 cm long, swollen
at both ends, terete, puberulous when young. Leaf
lamina 7.5–17.8 × 3–7.6 cm, oblanceolate, apex bluntly caudate acuminate, base
cuneate, glabrous, midrib slightly raised above, secondary nerves 4–8,
ascending, tertiary nerves slender, distantly per count. As per earlier
reports, the trees are unisexual and dioecious. The present study reports for
the first time that the trees are rarely monoecious (Image 4). At two different
forest locations namely Kodamadi and Ullaru a few trees were observed with monoecious flowering
conditions.
Floral phenology
The term ‘phenology’ describes
how living things react to climatic and seasonal changes in a cyclic manner.
Seasonal patterns are observed in a variety of reproductive processes known as pheno-events in flowering plants, including the
commencement of flowering, fruiting, and seed distribution (Shivanna
& Tandon 2014). The phenological data of B. courtallensis
was observed and recorded based on repeated field observations. When the
tree attains maturity (nearly 10–12 years), floral primordia appear from the
base to the middle portion of the main trunk, from which crimson-red flowers
emerge (Yogeesha et al. 2016). Baccaurea
courtallensis was confirmed as unisexual,
dioecious, and infrequently monoecious trees. The seasonal and climatic changes
influence the flowering phenology of evergreen trees as witnessed over a period
of four years. Baccaurea courtallensis begins flowering in the month of February
and extends up to May, however, reaches mass flowering during March. Both male
and female floral buds primordial are eventually developed into brown buds and
then mature into dark red coloured buds (Image 1B,D).
The flower buds take 16–24 days from initiation to full bloom. Flowers open in
the morning from 6.30–10.00 h and anther dehiscence was noticed around 1330 h
after anthesis. However, seasonal and climatic changes have made a strong
impact on the flowering of B. courtallensis (Image
4). Continuous observations helped us to determine the flowering period,
occasionally flowering occurred in early February and in some other times cauliflorous initiates from March and extended up to May
(Image 4). These changes occur due to rainfall and other environmental factors
and such factors significantly influence the flowering phenomenon of these
trees.
Floral morphology
The inflorescence is cauliflory,
a large population of trees that produce dark crimson colour
flowers, in densely clustered slender racemes on the old stem. Rarely trees
were observed in the monoecious status where male and female flowers are
produced in the same inflorescence stalk but such cases have yielded fewer
fruits (Image 5). The flower size of candidate species was observed in female
flowers with 3–4 mm (Image 2C,D) and in male flowers 1 mm to 1.5 mm (Image 1E). Female flowers are
relatively larger than male flowers and the tepals of the female are also
larger than the male flower (Image 5). Both male and female flowers emit a mild
musky odour. Floral traits of B. courtallensis are listed in (Table 2).
Male flowers
The male inflorescence appears in
clustered racemes on short tubercles all over the trunk, red in colour. Bracts, linear lance-shaped or triangular, free,
conduplicate, encircling the base of lateral branches; tepals 4–5, 1.5–2 x 1
mm, linear, oblong, elliptic, nearly round or inverted lance-shaped, glabrous
or sparsely puberulous; stamens 6–7, free and
fertile; anthers basifixed; pistillode club-shaped
(Image 1H).
Several clustered racemes are
found all over the trunk. The stem shows several dark brown coloured
scars, where the floral primordium abscised. The male flower buds take 5 to 24
days from initiation to maturation (Image 1B,C,D). At the time of blooming each
cluster produces numerous inflorescences. Each cluster has a maximum of 96 male
inflorescences, a minimum of 13, and an average was 58.32. The single
inflorescence consists of many of floral buds, maximum numbers of floral buds
were 85, the minimum was 39 and the average number of buds was 64.32. The
length of male inflorescence was also measured; the maximum length was 19 cm;
the minimum was 7.5 cm and the average was 12.10 cm (Table 3).
Female flowers
Female inflorescences are born on
clustered racemes mostly confined to the base of the trunk (Image 2B). Small
projections were observed on such trunks where the female inflorescence arises.
Each cluster had a maximum of 19 female inflorescences, a minimum of 6, and an
average of 10.56. The single inflorescence contained many flower buds with a
maximum number of 45 buds, a minimum of 23, and an average of 34. The length of
female inflorescence was also measured by using a scale; the maximum length was
25 cm, the minimum was 13 cm, and the average was 18.22 cm (Table 3).
Bracts 1–1.5 mm long,
lance-shaped; tepals 4–5, 2.5–3 x 0.6–1.5 mm, linear, oblong or
oblong-elliptic, sparsely puberulous to hairless,
fringed with hairs; the ovary is superior, woolly, 3-angled, 2.5 x 2–3 mm,
ovoid or sub-spherical, trilocular; ovules 2 in each locule (Image 2F); as the flower is trimerous stigma is
with three flabellate sticky surfaces to be successful by wind pollination
(Image 2G,H). Style and stigma are reduced close to the gynoecium. The ovule
structure of B. courtallensis is anatropous
with amphistomal making up the micropyle, crassinucellate, origin from axile
placentation, bitegmic integuments, and obturator forming a nucellar beak. The
position of the ovule is ventral epitropous. The
ovary normally has two ovules in a locule. The embryo
is chlorophyllous and the endosperm is cellular in
nature. Seeds are arillate with white mucus fibers on the surface. The cross-section
of the flower showed three distinct locules (Image
2E), in each fruit two–three seeds are produced at the time of maturation and
the remaining ovules are either aborted or unfertilised.
Fruit morphology and anatomy
The fruit is a capsule and Bacca (fleshy fruit) thus the name Baccaurea
(Image 3A). Fruits are crimson to brown after maturity, globose, beaked,
1.5–2.5 cm across; ribbed, pubescent when young, seeds broad composed with
fleshy aril and have exocarp, mesocarp, and stony endocarp (Image 3B). The
fruit shows tricarpellary ovary with well-developed
three locules (Image 3D). The average fruit size in
length is 25.36 mm and its width is 25.30 mm. The thickness of the fruit varies
from place to place with a range of 23.91 to 35.38 mm. Similarly, the fresh
weight of the fruit varies from 19.5 to 26.52 g (average 22.77 g), and the dry
weight has an average of 5.22 g. The fruit has about 77.06 % of moisture
content at the time of harvest.
Exocarp is made of a single layer
of epidermis with thin wall cells (Image 3F), rectangular in shape, it contains
more amount of anthocyanin pigment, and chloroplast is deposited within the
epidermis cells. The size of the epidermis has a maximum length of 229.79 µm
and a minimum of 103.08 µm (average 155.93 µm) and width maximum of 134.28 µm
and a minimum of 60.04 µm (average 106.45 µm). Fruit cover has stomata of cyclocytic type. Stomata are surrounded by one or two
narrow rings of subsidiary cells and two guard cells. The pubescent nature of
fruits is due to the presence of trichomes and hooked hair-like structures with
thick-walled cells (Image 3E). Mesocarp is 2 to 3 mm thick and has
approximately 40–50 layers of parenchymatous cells. Mesocarp consists of two types of cells,
thick-walled cells, and thin-walled cells. The size of thin-walled cells has a
maximum of 267.62 x 239.82 µm and a minimum of 118.94 x 124.42 µm.
Consequently, the thick-walled cells have a maximum of 195.13 x 179.48 and a
minimum of 63.63 x 76.49 µm.
Seeds are covered with an extra,
fleshy layer outside the seed coat. This fleshy layer more or less envelops the
seed coat and it is known as aril (Image 3C). In transverse sections, the
mucilage layer is present below the testa, and
mucilage cells are thin-walled in nature, with fiber-like structures containing
polyphenol compounds (Image 3G,H). Endocarp or testa
is made of stone cells, it covers the endosperm, and two to five layers of
cells are tightly packed; the length of a stone cell varies 73.04–37.85 µm
(average 52.89 µm), and its thickness is between 95.61–216 µm (average 140.82
µm). The testa is followed by a palisade layer of
thin-walled cells, 2 to 6 layers occur below the endocarp; a group of
large-size druse crystals is located in the palisade layer. The length of the
palisade cell varies from 86.54–343.21 µm (average 205.29 µm) and its width is
49.35–133.58 µm. Almost the entire region of the endosperm is occupied by the
developing embryo with large etiolated cotyledons which are fleshy in texture
and circular in shape (Image 3I). The length of endosperm cells varies from
94.04–167.95 µm (average 128.39 µm) and its width is between 48.75–198.9 µm
(average 108.2 µm). The longitudinal section of the embryo axis shows
developing cotyledons, hypocotyl region, and radicle with root primordium (root
meristematic region) and well-developed root cap region (Image 3J,K,L).
Pollen Biology
Pollen morphology and production
The male flowers have a reduced pistillode and well-developed anthers (Image 1H). Pollen
grains are spherical, trilobed, or four-lobed, circular in polar view, tricolporate to tetracolporate, colpus slender, and pore elongate with an unclear outline.
The estimation of pollen production is important to understand many aspects of
pollen biology. The amount of pollen produced in each anther/flower varies
greatly. In B. courtallensis each anther
produces a minimum of 1,648 to a maximum of 2,144 pollen grains; the mean
number of pollen grains in a single anther was 1,446. The sizes of pollen
grains vary from 10–12 µm in diameter.
Pollen ovule ratio
The study on pollen ovule ratios
was used to predict the efficiency of pollination in a particular species (Oskay 2017). The cross-section of a pistil shows three locules and each locule has two
ovules. The total number of ovules in an ovary is six. A single anther
consists of around 1,446 pollen grains and thus a flower has around 10,122
pollen grains. The pollen ovule ratio was counted as 1687:1. This study
indicates that external agents are needed for pollination and successful fruit
set. The percent of fruit set for the candidate species is 74.
Pollen fertility and viability
Pollen fertility and viability is
a critical factor, which is considered important parameter of pollen quality
(Dafni & Firmage 2000). Pollen grains treated
with reagents such as acetocarmine, TTC, and I2KI essentially impart
colouration to the cytoplasmic contents of the pollen
to indicate whether the pollen grains are fertile or not. The stained pollen
grains were considered fertile and the unstained as sterile (Image 1I). This technique
revealed that 96.24% of pollen grains are fertile on the day of anthesis and
this is used to know the pollen longevity. Pollen viability was determined by
using the Flurochromatic reaction test (FCR), 2, 3,
5-Triphenyl Tetrazolium chloride (TTC) test, and the Iodine Potassium iodide (I2KI)
test. The percentage of pollen viability on the day of anthesis in the FCR test
showed 80.00% (Image 1J) TTC test was 86.72% and the I2KI test was
91.03% (Image 1K). Among the three staining tests I2KI expressed the
highest pollen viability (91.03%).
In vitro pollen germination
Pollen germination was carried
out in Brewbaker and Kwack
medium at different concentrations of sucrose such as 5%, 10%, 15%, and 20%.
The maximum percentage of pollen germination was observed in 10% sucrose after
12 hours of incubation. Pollen germination was low in 5, 15, and 20% levels of
sucrose-supplemented BK medium. BK medium augmented with 10% of sucrose, showed
63.15% of pollen germination. (Image 1L). Increasing the concentration of
sucrose beyond 10% inhibits pollen germination in B. courtallensis.
A similar kind of response was reported in Tectona
grandis tree (Hine et al. 2019) and this study
corroborates such a finding.
Floral visitors
The candidate species is a
unisexual-dioecious tree; male and female trees produce flowers separately, so
obviously, cross-pollination is predetermined. Dioecious plants have a great
advantage over other plants wherein their cross-fertilisation
is guaranteed (Renner 2014). Further, the cluster of inflorescences and the
miniature size of flowers also designate that they may be cross-fertilised
by wind and biotic means. During the flowering period, many floral visitors
were observed. In female flowers; black ants and
spiders were found however in male flowers, honeybees were also observed.
During many such field visits, black ants and
honeybees were detected in male flowers abundantly (Image 1F,G Image 2 I,J).
Though, floral visitors were observed in large numbers, the presence of nectar
glands or nectar organs in either male or female flowers could not be located.
Floral visitation happens from 0730 h to 1000 h. The flowers are small, dark
crimson colour with a mild musky fragrance. The
floral visitors are attracted by the mass flowering with contrasting colour combinations.
DISCUSSION
The knowledge of phenology, and
flower biology is essential to understand the persistence, dispersion,
pollination, and breeding systems of plants (Munguia-Rosa et al. 2011).
Phenological studies are essential to increase knowledge of the specific
functions of plants in natural populations and they must be taken into account
in conservation and rational management schemes (Oskay
2020). The development of successful conservation and sustainable use
strategies for endangered, indigenous plants depends on comprehensive knowledge
of their reproductive biology. The current findings revealed the species floral
phenology, male and female flower morphology, and pollen biology. The selected
species is an understory semi-evergreen to evergreen tree, endemic to Southern
Western Ghats and Eastern Ghats. Fruits of this tree are edible and used by Kani, Kadar, Muthuvan tribes for
medicinal purposes specifically to treat infertility problems (direct
conversation with them).
An altitudinal gradient, which
supports the significance of elevation in the distribution of a tree species,
is the foundation upon which a forest community depends for its survival (Liu
et al. 2007; Barni et al. 2012). Based on our random
and repeated field observations this tree occurs from 200 m to 1000 m altitude
in Tamil Nadu and Kerala forest areas. This tree prefers a canopy region to
grow and reach up to 15 m in height. The current investigation revealed that
the vegetative phase of both sexual plants displayed similar phenological
characteristics throughout the season. Both male and female trees share a
similar kind of morphological pattern except for the stem portion. Only the
flowering phenomenon helps to identify both sexes.
Baccaurea courtallensis is found to be unisexual and
dioecious tree in abundance however; monoecious trees were also recorded
sporadically based on our findings. Though, Monoeceae
status was reported by Yogeesha et al. (2016) where
the male and female flowers were observed in different inflorescence stalks,
however, the present study reports that both male and female flowers are
present in the same inflorescence stalks. A similar kind of morpho-variation
was observed in Jatropha curcas flowers (Singh
et al. 2010). This tree begins to flower in February and continues till April,
with a peak flowering period being recorded in March. In comparison to female
flowers, male trees begin flowering earlier. These changes might be influenced
by local climate and edaphic conditions. In dioecious species, the timing of
flowering varies between the sexes, with the male typically starting to flower
before the female tree (Lloyd & Webb 1977; Beach 1981). In dioecious species, male plants often
produce a greater number of flowers than female plants (Vaughtton
& Ramsey 1998) to provide sufficient pollen grains for successful
pollination. Fruit growth and development were observed from April and need 4–5
months to attain full maturation. Fruits that have matured fall off from June
to September. Fruits are eaten by frugivores especially elephants, sloth bears,
monkeys, squirrels, bats, and tortoises. The tortoises possibly consume a
greater number of fruits than any other due to the nature of cauliflory and
close abundance to the floor. As a consequence of this behavior, seeds are also
dispersed by these frugivores. The plant and animal interactions signify a
mutualistic network of seed dispersal systems in B. courtallensis.
Studying anthesis and anther
dehiscence is essential for understanding how pollen grains disperse into the
atmosphere (Bhattacharya & Datta 1992). Flowers
of B. courtallensis open in the morning
6.30–10.00 hours, male flowers are short-lived compared to females. Dehiscence
of anthers was observed after 30–45 minutes of a flower opening. The mean
number of pollen grains in a single anther was 1,446. In the acetocarmine
glycerin and the flurochromatic tests, pollen grains
displayed a 96.24% fertility rate and an 80% of viability rate, respectively.
However, I2KI test showed 91.03% of pollen viability, it may be due
to more amount of starch content present in the pollen grains. This method is
commonly used to stain the starch content in pollen grains (Bolat
et al. 1999). Iodine broke up in a watery arrangement of potassium iodide the
tri-iodide-anion edifices with starch, and it distinguishes the viable and
non-viable pollen grains. The same kind of result was observed in hybrid banana
pollen grains (Ssali et al. 2012). It has been known
that pollen viability is an important factor in assessing the good quality of
pollen (Sulusoglu et al. 2014). Brewbaker
and Kwack’s medium supplemented with 10% sucrose had
a pollen germination rate of 63.1%. Pollen germination rate was higher in 10%
sucrose-containing medium in many Euphorbiaceae
species. 71% of pollen germination rate was observed in Jatropha curcas in a medium containing sucrose (Abdelgadir et al. 2012). Sucrose serves as a nutritive
resource for pollen germination and assists in the maintenance of the osmotic
balance between pollen cytoplasm and germinated medium (Johri & Vasil
1961). However, a higher concentration of sucrose inhibits the growth of pollen
tubes (Hine et al. 2019). Pollination efficiency and pollinator availability
are influenced by the timing of flowering (Bawa
1983). Female flowers have a lifespan of 5–7 days, while male flowers have a
lifespan of 2–3 days after anthesis. Female flowers have pistils and reduced
staminodes, and male flowers have well-developed anthers and diminished pistillode. Cross-pollination is facilitated in B. courtallensis by the flower architecture.. In the
present study honey bees, black ants, and spiders were observed in male and
female flowers. Black ants and spiders were
considered floral visitors because they were found on both male and female
flowers. A male flower offers nectar and pollen grains as food resources to
floral visitors. There is a possibility for pollination by these floral
visitors. A comparable type of insect visitation and pollination that occurred
in Phyllanthus emblica was reported by Halder
et al. (2019). Amla tree flowers are mostly
cross-pollinated by wind and honey bees. The pollen ovule ratio is an important
factor that determines the fruit set rate (Cruden
1977). The mean number of pollen/ovule ratio was 1687:1 and the percentage of
fruit set was 74. Successful pollination and fruit set are greatly influenced
by pollinators, as well as, by the abundance of male trees (Dafni et al. 2005).
The fruit is a bacca (berry) which has a thick and juicy pericarp with a coloured exocarp, fleshy mesocarp, and a membranous
endocarp. The contrasting inference is offered by a few authors that fruits of phyllanthaeceae are dehiscent schizocarps (Fahn & Zohary 1955; Roth
1977; Gagliardi et al. 2013). However, the fruit of Baccaurea
courtallensis is a bacca
which is dehiscent naturally. The mesocarp encloses a fragile aril that
develops from one of the structures like funiculus, raphe, or integuments (Fahn 1967). Arils
are very common in tropical and subtropical plants. These arillate seeds are
well adapted to dispersal by animals (Corner 1976). The fruit is a dehiscent
type and develops from three carpels. Species of Phyllanthaceae
have tricarpellary ovary with two ovules in each locule (Gagliardi et al. 2013). Though, fertilization takes
place uniformly in a flower the number of seeds set at maturity is either one
or two per fruit. Thus, out of six ovules, only one or two become fertile seeds
and the rest either abort or are underdeveloped. As the flower and fruit set
ratio is unbalanced a large number of flowers are produced on the tree trunks
in order to increase the availability of fertile seeds. The fruits are fleshy
and edible and hence, they attract animals which subsequently help in seed
dispersal. Fleshy fruits are opened by dispersing animals or naturally by the
rotting of the outer tissues (Mauseth 1988).
The seed is covered by a
protective layer of cells called testa which is made
of an outer thick layer with macrosclerides and thin
inner layers (Gagliardi et al. 2013). The scleride
nature of these cells is easily distinguished while observing under polarized
light facilitated with first order red plate. The thickness of the testa varies from region to region within a seed. Below the
testa, two degrading layers of cells are found during
the developmental stage which may be the perisperm
and endosperm (Image 3G). Just beneath the digesting endosperm layer, the
storage cells of cotyledon are found abundantly. These storage cells house
starch and oil bodies enormously. Oil bodies in cotyledons are distinguished by
rhodamine stain and Sudan III (Image 3H).
There is inadequate information
in the literature on most of the aspects of the reproductive biology of B. courtallensis like phenology, floral description,
pollen biology, pollination, and embryo development. The present study has
carried out floral biology, phenology, pollen biology, and some aspects of
fruit development for the first time in B. courtallensis,
which may be useful for efforts on the conservation of this endemic,
underutilized wild edible fruiting tree.
CONCLUSION
Baccaurea courtallensis
is one of the
few interesting and important members of the family Phyllanthaceae,
which is endemic and occurs from 180 m to 1,000 m elevations in the Western
Ghats of peninsular India. This taxon attracts attention due to its cauliflory
and the bright red flowers and fleshy fruits that are attributed with curative
properties. Apart from the ornamental purpose, the fruits of this tree are rich
in nutritional value and possess medicinal properties. Due to anthropogenic
activities and habitat destruction, this species has reduced in population.
Being devoured by animals and exploited by local trade, the species is
represented sparsely by fewer individuals. Successful management of forest
resources requires knowledge of blooming phenology, the time, duration, and
frequency of distinct phenoevents, particularly for
species of forest trees. The conservation of this species depends heavily on
the understanding of its reproductive biology, floral biology, pollen biology,
and pollination. Thus, the flowering phenology, floral morphology, fruit
anatomy, and pollen biology are reported in the current study in detail so as
to better understand them for future applications. Future efforts to protect
this unique tree might benefit a lot from this report.
Table 1. Baccaurea
courtallensis populations in selected forest
areas of Western Ghats.
|
District |
Forest location |
GPS coordinates |
No. of Individuals |
|||
Latitude |
Longitude |
Altitude (in m) |
Male |
Female |
|||
1 |
Tirunelveli |
Kannikatti |
8.63083 |
77.27417 |
795 |
26 |
12 |
Ullaru |
8.62875 |
77.29455 |
615 |
160 |
50 |
||
Ingikuzhi |
8.61955 |
77.27647 |
644 |
24 |
7 |
||
Kodamadi - Ueipattrai |
8.70730 |
77.26643 |
496 |
60 |
12 |
||
Vethalakan odai |
8.71018 |
77.26237 |
457 |
40 |
19 |
||
Kalkatodai |
8.71197 |
77.25870 |
482 |
44 |
23 |
||
Kadana |
8.966466 |
77.299725 |
181 |
13 |
4 |
||
2 |
Tenkasi |
Courtallam-Therkumalai estate |
8.90412 |
77.25780 |
569 |
9 |
2 |
Courtallam- Mayilodai estate |
8.91028 |
77.25760 |
734 |
36 |
9 |
||
3 |
Kollam |
Achenkovil |
9.07085 |
77.20057 |
382 |
65 |
7 |
Thenmala |
9.02872 |
77.09707 |
672 |
27 |
5 |
||
4 |
Thiruvananthapuram |
Kallaru |
8.74087 |
77.12062 |
394 |
14 |
2 |
Ponmudi - on the way |
8.74045 |
77.12377 |
482 |
11 |
2 |
||
Bonacaud estate |
8.67570 |
77.15845 |
514 |
7 |
1 |
||
|
Percentage distribution of male
and female trees |
77.56% |
22.43% |
Table 2. Floral characteristics
of Baccaurea courtallensis.
|
Criteria |
Result |
1. |
Flowering period |
February to March/ May |
2. |
Flower type |
Unisexual, dioecious, and
rarely monoecious |
3. |
Flower colour |
Crimson red |
4. |
Odour |
Musky fragrance |
5. |
Presence of nectar |
Present, at the base of stamens |
6. |
Anthesis time |
0630–1000 h |
7. |
Anther dehiscence time |
0900–1145 h |
8. |
No. of anthers per flower |
7 to 8 |
9. |
Total no. of pollen grains in a
male flower |
Approximately 1,500 |
10. |
No. of ovule per flower |
6 (3 locules,
2 ovules in each locule) |
11. |
Pollen size |
10 to 12 µM |
12. |
Stigma type |
3-flabellate |
13. |
Pollen fertility |
Fertile (tested by I2KI,
TTC, Acetocarmine and FDA) |
14. |
Fruit type |
Capsule/Fleshy berry |
15. |
Pollination |
Wind assisted insect
pollination |
16. |
Floral visitors |
Honey bees, flies, black ants,
spiders |
Table 3. Number of
inflorescences, buds, and length of female and male flowers.
Traits |
Sex of the trees |
Sample size (n) |
Mean±SD |
Std. Error |
Minimum |
Maximum |
No. of Inflorescences in a
cluster |
Female |
25 |
10.56±3.428 |
.68576 |
6.00 |
19.00 |
Male |
25 |
58.32±22.24 |
4.4496 |
13.00 |
96.00 |
|
|
One-way ANOVA |
F = 112.535, P <0.01% |
||||
Length of Inflorescences in cm |
Female |
25 |
18.22±2.665 |
.53314 |
13.00 |
25.00 |
Male |
25 |
12.10±3.034 |
.60690 |
7.50 |
19.00 |
|
|
One-way ANOVA |
F = 57.396, P <0.01% |
||||
No. of flower buds |
Female |
25 |
34.00±5.751 |
1.1503 |
23.00 |
45.00 |
Male |
25 |
64.32±13.06 |
2.6132 |
39.00 |
85.00 |
|
|
One-way ANOVA |
F = 112.765, P <0.01%
|
For
figure & images - - click here for complete PDF
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