POLLINATION BIOLOGY OF HUMBOLDTIA VAHLIANA WIGHT. (FABACEAE) – AN ENDEMIC TREE OF SOUTHERN WESTERN GHATS

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POLLINATION BIOLOGY OF HUMBOLDTIA
VAHLIANA WIGHT. (FABACEAE) – AN ENDEMIC
TREE OF SOUTHERN WESTERN GHATS
K.P. Deepthy Kumary1, A.K. Sreekala2 and Anoosh Varghese2
1Plant Systematics and Evolutionary Science, 2Conservation Biology
Division, Jawaharlal Nehru Tropical Botanic Garden and Research Institute,
Palode, Trivandrum 695 562
ABSTRACT
A study on pollination biology of Humboldtia vahliana Wight., an
endemic plant of Southern Western Ghats was carried out in wild conditions.
The plant starts flowering in the month of February and extends up to May and
reaches a peak during March. The flowers are white coloured and open in the
evening between 1800-2100 h. Stigma remains receptive at the time of anthesis
and extends up to the next day evening. Different pollen viability tests indicated
that 80-85% pollen grains are viable on the day of anthesis and its viability
gradually decreased on successive days after anthesis. Honey bees, butterflies,
moths and ants are the major floral visitors. Habitat destruction, environmental
factors, over exploitation of the species, recalcitrant nature of the seeds, seed
infestation and poor seedling recruitment in the natural habitat could be the reason
for its narrow distribution.
Keywords: Anthesis, Humboldtia vahlian, Phenology, Pollination biology,
Western Ghats.
INTRODUCTION
Conservation of biodiversity of the world is the need of the hour. Plants
are the basis of life on earth, and their survival is key to human existence.
Throughout the world, this rich part of our natural heritage is being threatened.
Wild habitats are fast disappeared and thousands of species are lost due to several
biotic and abiotic factors. However, plants are vital natural resources and are
irreplaceable part of world’s natural heritage. Reproductive constrains may also
lead to rarity of species in certain cases. The reproductive biology of flowering
Journal of Palynology Vol. 53 (2017) : 13-23
Editor: A.J. Solomon Raju
Today and Tomorrow’s Printers & Publishers, New Delhi-110 002 (India)

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plants is important for determining barriers to seed and fruit set for conservation,
pollination and breeding systems that regulate the genetic structure of populations
(Tandon et al. 2003). Adequate knowledge on reproductive biology is essential
for conservation, management and recovery of endemic and endangered plants.
Therefore, detailed information on the reproductive biology of rare, endangered
and threatened plants is essential for developing effective strategies for their
conservation and sustainable utilization.
Humboldtia Ruiz. & Pav. is a genus of legume in the family Fabaceae
which includes 9 species in Kerala. H. vahliana Wight. belong to the subfamily,
Caesalpinioideae is a large tree growing up to 20 m tall, usually found adjacent
to rivers/streams in semi-evergreen and evergreen forests up to an altitude of 750
m .The tree is endemic to Southern Western Ghats of Tamilnadu and Kerala
(Sasidharan 2004; Nair et al. 2006). The hardwood of the tree is used for making
match boxes and also for fuel. Bark powder or decoction is used to cure
biliousness, impure blood, ulcers and epilepsy (Sanjappa 1984). A comprehensive
study on the reproductive biology of this species has not so far been made. In
this background, a study has been conducted on reproductive biology of
Humboldtia vahliana, an endemic tree of Southern Western Ghats with an intension
of understanding the causal factors that lead to population reduction.
MATERIALS AND METHODS
Study area
Humboldtia vahliana was located along river banks of evergreen forest
areas of Aryankavu of Kollam and Chittar river of Trivandrum districts up to an
altitude of 750 m asl (Figure 1A). Each population consists of more than 150
individual species. It is a tree of up to 20 m height having bark 5-6 mm thick,
dark brown mottled with white; blaze is pink in colour (Figure 1C) . The plant
grows in association with other wet evergreen forest species such as Aporosa
acuminata, Baccaurea courtallensis, Myristica malabarica, Vateria indica, Hopea
parviflora, Acacia caesia, Lagerstroemia microcarpa, Vitex altissima, Ochlandra
wightii, Xanthophyllum arnottianum, Entada rheedei, Myxopyrum smilacifolium,
Syzygium occidentale, Dracaena sp. etc. in a given community. The study was
conducted during 2013-2016. Two populations were selected for the present
investigation in the natural condition.
Morphology of the flower and flower parts were studied by using a hand
lens and dissecting microscope. The measurements of the floral parts were recorded
on 10 flowers selected randomly at each population with the help of scale and
vernier calipers. Twenty healthy plants were selected in the community and
observations were made on day to day basis in natural habitat on flowering

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phenology which includes season, habit, development, time of anthesis and anther
dehiscence. Fifty flower buds were selected at random and observations were made
between 1800-2200 h to study the time of anthesis and anther dehiscence. Peak
flowering time was noted when maximum number of flowers opened. Pollen
morphology was studied by the acetolysis method proposed by Erdtman (1952)
and Nair (1960). The SEM photographs were taken from the acetolysed samples.
The number of pollen grains per flower was calculated as per the method suggested
by Dafni et al. (2005). The total number of pollen grains in the suspension was
calculated by the given formula.
No. of pollens per suspension × No. of pollen suspension
Mean no. of pollen grains =
Total No. of suspension used for counting.
Figure 1. Reproductive dynamics of Humboldtia vahliana: A. Habitat, B. Flowering Twig, C. Pink
coloured blaze, D. Bud development, E. Inflorescence, F. Fruit.

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The average number of ovules per ovary was determined by dissecting the ovaries
under the microscopes. The pollen-ovule ratio was calculated as suggested by
Cruden (1977).
Mean number of pollen grains/flower
Pollen-Ovule ratio = Mean number of ovules/flower
Pollen fertility was assessed by acetocarmine-glycerin staining technique
(Radford et al. 1974). The number of stained and unstained pollen grains were
counted. The stained pollen grains were considered as fertile and the unstained
pollens as sterile. Pollen viability was assessed by FCR (fluorochromatic reaction),
IKI (Iodine Potassium Iodine) and TTC (2,3,5-Triphenyl Tetrazolium Chloride)
tests as per procedure proposed by Shivanna and Rangaswamy (1992). In FCR
test, the observations were made under (Leica DME, Germany) fluorescent
microscope using blue filter, the bright green fluorescent pollen grains were scored
as viable. The brownish red pollen grains were counted as viable in TTC test
whereas in IKI test, red and light red stained pollen grains were scored as viable.
To study pollen germination in-vitro, pollen grains were collected from the fresh
flowers was incubated in Brewbaker’s medium containing various concentrations
of sucrose.
Stigma receptivity was studied visually with the help of hand lens and
hydrogen peroxide test according to the method of Scribailo and Posluzky (1984).
Floral visitors were observed in the two sites. During the peak flowering period,
the pollinators were observed at the time of anthesis to the next day evening itself.
The number of floral visitors, foraging behavior, foraging hour, time spent on each
flower and the frequency of visit are recorded. All the visitors were collected
and identified with the help of experts and insect taxonomy manual. The percentage
of fruit set in the natural condition was noticed.
RESULT AND DISCUSSION
Floral morphology
Floral morphological studies were carried out by visual observation. The
flowers are bisexual, in racemes; corymbiform, axillary and cauline; bracts
foliaceous and persistent. Flowers are white, calyx tube cylindric, lobes 4, broadly
elliptic, unequal; Petals 4, white, clawed, elliptic-ovate; stamens 5, alternating
with 5 staminodes, filaments red coloured; style filiform, stigma capitate and ovary
stipitate. Pods oblanceolate, brownish velvety, stipitate and apex beaked. Seeds
are discoid and 3-4 in number.

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Table 1. Floral characters of Humboldtia decurrens
Floral characters Humboldtia decurrens
Flowering period February-May
Flower colour white
Odour Sweet scented
Anthesis time 1800-2100 hr
Anther dehiscence time 2000–2100 hr
Mean No. of anthers/flower 5 and 5 staminodes
Mean No. of pollen grains/flower ±5280
Pollen size 32.25 µm
Mean No. of ovules/flower 4
Pollen-ovule ratio 1320:1
Stigma type Wet and papillate
Fruit type Pod
Floral phenology
Flowering and fruiting patterns ultimately determine the reproductive
success in plants. These phenological events are strongly controlled by climatic
factors and evolutionary processes. The timing of flowering and fruiting has major
influences on biological process from the organism to ecosystem scale (Rathcke
and Lacy 1985). In south India, a majority of tropical wet evergreen and dry
deciduous forest species exhibit annual flowering patterns due to regular
reproductive cycles. The seasonal changes in reproductive phenology resulting from
environmental variability are crucial in shaping biotic interactions such as
pollination and dispersal syndromes. Humboldtia vahliana starts flowering in the
month of February and extends up to May and reaches a peak during March
(Figure 1B). The flower buds take 15-20 days from initiation to full bloom (Figure
1D-E). The flowering period extends up to 120 days in a year and average life
span of each flower is 1-2 days (Table 1). The flower open in the evening between
1800-2100 h and anther dehiscence was noticed 2 hr after anthesis (2000-2100
h). Fruit initiation, maturation and dehiscence were noticed from June to July
(Figure 1F). Seasonal variations affect the flower size, anthesis, anther dehiscence,
pollinator behavior and also in fruit setting too (Aswani and Sabu 2015). In our
present study, from the two study sites, Humboldtia vahliana starts blooming in
February and extends up to May and fruit maturation was noticed during June
- July.

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Pollen biology
Pollen morphology was studied by using SEM photography. Pollen grains
from flower buds just before anthesis were prepared for scanning electronic
microscopy (SEM) by standard acetolysis method proposed by Erdtman (1953).
Pollen grains are tricolporate and 32.25µm in diameter. Pores are ellipsoidal with
rounded end (Figure 2D). Exine thickness is 2.25 µm. Floral analysis indicated
that, each flower has five anthers and four ovules. A single anther consists of
1056 pollen grains and thus a flower has around 5280 pollen grains hence the
pollen ovule ratio had been worked out as 1320:1. This clearly indicates that,
an external agency is required for effective pollination, therefore the species
favours cross pollination. The acetocarmine staining technique revealed that
84.66% pollen grains are fertile (Figure 2A). Pollen viability by FCR , TTC and
IKI tests confirmed that 80-85% pollen grains were viable on the day of anthesis
and its viability gradually decreased on successive days after anthesis (Figure 2B,C,
Table 2). This observation was similar to that of Humboldtia decurrens and H.
sanjappae (Jayalakshmi et al. 2015, 2016). In-vitro pollen germination indicates
that the best pollen germination at 73% with 1242 µm tube elongation was noticed
in Brew baker’s medium containing 0.25% sucrose after 24 hrs of incubation.
Germination percentages were significantly low in higher concentration of sucrose.
Table 2. Pollen viability under different treatments
Treatments Percentage of Pollen viability
One day before On the day of Second day of
anthesis anthesis anthesis
Acetocarmine 62.92 84.66 53.83
FCR 58.07 83.27 44.64
TTC 59.82 85.76 42.39
IKI 54.41 80.12 42.06
Pollen viability is a critical factor, which is considered as an important
parameter of pollen quality (Dafni and Firmage 2000). Treating pollen grains with
stains such as acetocarmine and aniline blue in lactophenol essentially imparts
colouration to the contents of the pollen as well as fixed/ dead pollen. It may
be useful to determine the degree of pollen stability in plants of hybrid origin
or those grows under favourable conditions (Alexander 1969). From time to time,
the pollen viability test has been constantly upgraded. In the present investigation,
flurochromatic reaction test (FCR) and 2,3,5-Triphenyl Tetrazolium chloride (TTC)
test were used for assessing the pollen viability as suggested by Shivanna and
Rangaswamy (1993) and Dafni and Firmage (2000). In vitro pollen germination

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test was incorperated in the present study as this test reveals correlation with Fruit
and seed test. In H. vahliana, the best in vitro pollen germination and tube
elongation was noticed in Brew baker’s medium . It contains sucrose which acts
as a nutritive material for pollen germination (Johri and Vasil 1961) and helps
in maintaining osmotic balance between the germination media and pollen
cytoplasm (Mukerjee and Das 1964). Germination percentages were significantly
low in higher concentration of sucrose medium. Pollen germination and subsequent
post pollination events depends upon the receptivity of the stigma, its nature and
compatibility.
Figure 2. Pollen Biology of Humboldtia vahliana: A. Pollen fertility assessed by acetocarmine
test, B & C. Pollen viability assessed by FCR and IKI test, D. SEM Photographs of pollen.
Stigma receptivity
Stigma is wet and papillate. Stigma receptivity test indicated that stigma
remains receptive at the time of anthesis and extends up to 22 hrs. The maximum
receptivity was observed between 1800-1300 h. Stigma receptivity is a critical
factor for successful completion of post pollination events. Usually, receptivity
reaches a maximum soon after anthesis (Shivanna and Johri 1985) but the
receptivity may vary from species to species (Jashirao and Saoji 1989) depending
upon temperature and humidity. After pollination, pollen grains well adhired on
stigmatic surface and showed 56.2% in vivo pollen germination. Pollen tubes grow
towards the stylar region and reach up to the ovary and fertilize the ovules. The
fertilized ovules developed into seeds.

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Pollinator behaviour
Pollination is a fundamental aspect of plant reproduction and largely
considered as a co-adaptive process in which plants evolve traits to attract certain
pollinators, whereby pollinators then evolve traits to better exploit floral resources
of particular plants (Frankel and Galun 1977). Plants and pollinators may have
very close links and examples show that the loss of pollinators lead to the decline
of their associated plants. Pollination mechanisms are greatly diverse among
angiosperms and one of the first steps to assure the reproductive success of plants
and the reproduction of tropical plants depends largely on the interaction with
animals that carry pollen grains, thus performing pollination. Pollination is an
important link of successful plant reproduction and is often dependent on
mutualistic interactions with animals. A reduction in pollinator service can directly
influence reproductive output, decreasing the quantity and quality of fruit and seed
set and promoting selfing in self-compatible species (Rodriguez-Perez 2005). In
H. vahliana, observations were made on floral visitors between 1800-1300 h
showed that several visitors were attracted by mass blooming of white flowers
with fragrance. Pollen, nectar, fragrance, visual signals and brooding sites
constitute primary floral rewards for the visitors. There are some generalizes
correlations between pollinators and floral odour (Faegri and Pijl 1980) Generally
bees and butterflies pollinated flowers having mild fragrance whereas beetles
pollinated flowers have strong aminoid smells and thus beetles and flies are drawn
to the flowers which mimic that smell (Simpson 2006). In H. vahliana, the average
volume of nectar available per flower was 4µl. The concentration of nectar sugar
in samples collected from two populations ranged 20-26%. In H. vahliana, the
floral visitors are honey bees, stingless bees, ants, moths, butterflies and wasps,
but only few of them effected pollination. Apis cerana, Apis dorsata and Trigona
iridipennis are the most frequent visitors visiting more flowers than any other
pollinators for pollen and nectar (Table 3). Their inter-plant movement facilitates
pollination.
Table 3. Pollinator behaviour of H. vahliana
Visitors Visiting time Foraging nature Foraging hours
Apis cerana Day Pollen 0600-1030
Apis dorsata Day Pollen 0900-1130
Trigona iridipennis Day Pollen 0600-1200
Euploea core Day Nectar 0830-1130
Pachliopta hector Day Nectar 0730-1200
Pachliopta aristolochiae Day Nectar 0800-1230

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Danaus genutia Day Nectar 0900-1300
Papilio demoleus Day Nectar 0830-1130
Oecophylla smaragdina Day and night Nectar Throughout
Macroglossum corythus Night Nectar 2200-0200
All the floral visitors except moths foraging in the day time between 0730-
1300 h. Butterflies are also visited the flowers for nectar spending 2-3s in each
flower during day time (0800-1300 h). In H. vahliana, butterflies were active
during day time and in fine weather, they actively visit the flowers. However,
during the rainy season the butterflies are completely inactive. This observation
was similar to that of Humboldtia decurrens and some Impatiens species
(Jayalakshmi et al. 2015; Sreekala et al. 2008). Plant pollinator interactions are
one of the most common mutualisms in nature through which plants offer rewards
to floral visitors and they in advertently transfer pollen among nonspecific plants
to effect fertilization. The endemic species of particular geography is largely
depends up on animals for their pollination and seed dispersal. In the present
investigation, the visits of all the insects are not sufficient to pollinate all the
flowers in the population. This may lead to poor fruit set in natural condition.
Fruit initiation and maturation was noticed during June -July. About 38-46% fruit
set was noticed in the two study sites. The dehisced seeds germinate quickly in
the ground. The seeds are infested by borer beetle and subsequent poor seedling
recruitment in the wild.
CONCLUSION
H. vahliana is an economically important endemic tree of Southern Western
Ghats. The species is poorly distributed in the wild mainly because of the habitat
degradation/modification of habitats, environmental factors, over exploitation due
to its medicinal importance, recalcitrant nature of the seeds, seed infestation and
subsequent poor natural regeneration of the seedlings etc. Therefore, this ethno-
medicinal tree species needs special attention for its long term conservation and
sustainable utilization by adopting suitable strategies.
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