INTRODUCTION

Amphibians were the first group of vertebrates which were adapted to

aquatic as well as terrestrial mode of life during the course of evolution. Amphibians

have a unique place in the evolutionary history of vertebrates for being first to

establish life on land (Anderson, 2008; Frost et al., 2008). Amphibians are

intermediate between the fully aquatic fishes and the truly terrestrial amniotes. The

successful perpetuation of an amphibian species and its survival in the new

terrestrial environment depended on the development of limbs, lungs, other

anatomical modifications and more importantly on the evolution of new reproductive

strategies (Shine, 1979; Prado et al., 2005). Reproductive success for amphibians

involves spermiation, ovulation, oviposition, fertilization, embryonic development

and metamorphosis (Brown and Cai, 2007). Amphibian history suggests that the

developmental pattern evolved between the Triassic and the mid-Jurassic period

(Anderson, 2008; Boisvert, 2009). The estimate for the date of the origin of modern

amphibians (Liss amphibia: frogs, salamanders, and the limbless caecilians but not

amniota) can lie between 351 and 266 Mya (Marjanovi and Laurin, 2007) which

phylogenetically placed near the Batrachians divergence (Anderson, 2008; Frost et

al., 2008). During these years, as a consequence of interactions with the nature,

they have evolved different modes of reproduction such as oviparity, ovo-viviparity

(e.g., Pipa pipa) and viviparity (e.g., Salamandra salamandra) (Wake, 1998). It,

thus, seems that, as compared to other terrestrial vertebrates (e.g., reptiles, birds

and mammals), amphibians have radiated a wide range of diversity of reproductive

modes (Diwan, 1996). Since amphibians are poikilothermic and also that the

majority of them depend upon water for breeding, their reproductive activities are

greatly affected by the ever-changing climatic factors such as air and water

temperature, rainfall, daylength and relative humidity (Delgado et al., 1992; Sumida

et al., 2007).

In India, there are only few reports on the breeding biology of some

amphibians like Rana limnocharis (Roy and Khare, 1978), Rana alticola (Sahu and

Khare, 1983), Polypedates maculates (Mohanty and Dutta, 1986; Dutta et al.,

2000), Rhacophorus malabaricus (Sekhar, 1989), Polypedates leucomystax

(Ahmed and Lahkar, 1999; Iangrai, 2007), Rhacophorus pseudomalabaricus

(Vasudevan and Dutta, 2000), Chirixalus simus (Deuti, 2001a, b), Hyla annectans

(Ao and Bordoloi, 2001) and Rhacophorus bipunctatus (Iangrai, 2007). The

distribution and life cycle of Gegeneophis ramaswmii (Oommen et al., 2000) and

Rhacophorus pseudomalabaricus (Vasudevan and Dutta, 2000) are also well

documented in Western Ghats.

The general reproductive patterns of amphibians are (i) caecilians reproduce

biennially (Exbrayat and Delsol, 1985; Oomen et al., 2000), (ii) salamanders

reproduce annually (Cryptobranchidae, Hynobiidae and Sirenidae) or biennially

(Plethodontidae and Bolitoglossini), and (iii) anurans have seasonal reproduction

(Duellman, 1995). Reproductive cycles in amphibians are regulated by a complex

neuroendocrine mechanism involving the hypothalamus-hypophyseal-gonodal axis

(HHG) (Griffith and Wilson, 2003). The HHG axis is influenced by endogenous and

exogenous factors (Norris, 2007). Gonadotropin releasing hormone (GnRH)

secreted from the hypothalamus plays a major role in the regulation of reproductive

functions. GnRH acts on the gonadotrophic cells of the anterior pituitary to stimulate

the release of gonadotropins, namely luteinizing hormone (LH) and follicle

stimulating hormone (FSH) (Griffith and Wilson, 2003). The gonadotropins stimulate

gametogenesis and the synthesis of gonodal steroid hormones such as androgens,

estrogens and progestogens. The FSH initiates spermatogenesis in males and

follicular development in females. LH induces androgen synthesis by interstitial cell

in males and estrogen synthesis and ovulation in females (Suzuki et al., 1985).

Recent research into the relationships between testicular androgens and male

behaviors, mainly using castration/steroid treatment studies, generally supports the

conclusion that androgens are necessary but not sufficient to enhance male

behaviors. Prolactin acts synergistically with androgens and induces reproductive

development, sexual behaviors, and pheromone production (Moore et al., 2005).

This interaction between prolactin and gonodal steroids helps to explain why

androgens alone sometimes fail to stimulate amphibian behaviors. Vasotocin also

plays an important role and enhances specific types of behaviors in amphibians

(frog calling, receptivity in female frogs, amplecting and clasping, courtship

behaviors, etc.) (Moore et al., 2005).

Metamorphosis (Brown and Cai, 2007) is a post embryonic period of

profound morphological changes by which the animal alters its mode of living, gill

breathing aquatic to air-breathing terrestrial adult mode of life (Mc Diarmid and

Altig, 2000). Amphibian metamorphosis is a complex development process, and

results in reorganization of most of tissues and organs of tadpole (Galton, 1988,

Brown and Cai, 2007). Amphibian requires thyroid hormones (TH) for larval

development and metamorphosis, and secretion of TH by the thyroid gland is

greatly increased just before the onset of metamorphic climax (Dent, 1988). The

thyroid hormones help in development from the pre-metamorphic stages to the

onset of metamorphic climax. Radio Immunoassays show that levels of thyroid

hormones (TH) rise to a peak during metamorphic climax. Accompanying peaks are

reported for adrenocorticotrophic hormone (ACTH), adrenal corticoids (AC) and

prolactin (PRL) (Dent, 1988). Prolactin is widely considered to be the juvenile

hormone of anuran tadpoles and to counteract the effects of thyroid hormones (TH)

(Huang and Brown, 2000). Melatonin may also have a role in metamorphosis

(Lincoln, 1999; Wright, 2002; Udin, 2005) because it is a thyroid antagonist, whose

level falls at the metamorphic climax when the thyroid hormones peak. Melatonin

rhythms in plasma and eyes are entrained to the light/dark (LD) cycle and affected

by temperature (Wright, 2002). Consequently, melatonin could transduce

environmental information to regulate endocrine periodicity and larval circadian

organization and influence metamorphic rate.

A critical review of literature on breeding biology, breeding behaviors,

parental care, gonadal cycles, developmental biology, and metamorphosis are

given order-wise in the following sections.

Gymnophiona/Apoda:

Apodans belong to an order of amphibians with distribution in several of the

tropical and temperate zone countries (Smita et al., 2004). The Western Ghats is a

home to many Indian and regional endemic species of apodan (Oommen et al.,

2000). Fourteen out of 17 Indian species of caecilians are found in the Western

Ghats and all the species are endemic (Bhatta, 1998). Three species of

gymnophiona namely, Ichthophis garoensis, Ichthophis hussaini, and Ichthophis

sikimensis have been reported in North-Eastern region of India (Zoo Outreach

Organization, 2001; Ahmed et al., 2009). Caecilians are known to have internal

fertilization, and probably about 75% of the species are viviparous, meaning give

birth to developed young ones (Gower et al., 2008). About 25% of the species are

oviparous (egg-laying), and the eggs are guarded by the female (Wake, 1980;

1998). Caecilians, unlike other amphibians so far known, have internal insemination

and internal fertilization (Bhatta, 1998). Further, the copulatory mechanism unique

to members of the order Gymnophiona is the insertion of the male intromittent

organ (copulatory organ) in to the cloaca of the female following courtship. A major

dichotomy in caecilian reproductive modes is that of oviparity versus viviparity.

Members of aquatic family Typhlonectidae are viviparous and produce juvenile

aquatic young one (Wilkinson and Nussbaum, 1997). Viviparity has evolved from

oviparity. The transition from oviparity to viviparity requires the retention of fertilized

eggs in the female reproductive tract. Gestation requires a full year, females have

at least a biennial cycle, but males have active spermatogenesis throughout the

year (Oomen et al., 2000). Viviparity is reported for Gegeneophis seshachari

(Gymnophiona: Caeciliidae) from a gravid female containing four oviductal fetuses.

The oviducts are highly vascularized and contain patches of thickened, layered

tissue similar to fetal gut content (Gower et al., 2008). Viviparous caecilians go

through metamorphosis while inside the eggs, so they hatch with the body form of

juvenile young one. The developing young one uses the teeth to chew a nutrient

liquid made by the inner lining of the oviduct inside the mother. The fetal teeth are

shed at or near birth (Kupfer et al., 2006).

The caecilian reported so far from India are all terrestrial and oviparous

(Bhatta, 1998, Oommen et al., 2000). Oviparous caecilians lay eggs by digging a

hole close to the surface in a damped ground near water that hatch into free-living

larvae having small gills and tail fins (Bhamrah and Juneja, 1990). Many species lay

their eggs on land in burrows, crevices, under logs and debris, or at the bases of

bunch of grasses. No species of caecilians, so far known, lays the eggs in water

(Wake, 1998). Maternal care of the clutch has been mentioned in many species like

Ichthyophis glutinosus, Ichthyophis kohtaoensis, etc. Female Ichthyophis glutinosus

coils around the clutch at hatching and the larvae wriggle from the burrow to nearby

streams, where they spend approximately a year before metamorphosis (Kupfer et

al., 2006). Larval and adult caecilians are similar in morphology with the notable

exception of the presence of three pairs of external gills in the larvae. These

external gills degenerate during late embryonic or fetal life, although it is not clear

whether the gills are resorbed or broken off.

One of the fossorial amphibians that are members of the order

Gymnophiona, Ichthyophis kohtaoensis (Southeast Asia) is an oviparous species in

which maternal care of the clutch is provided. In Ichthyophis kohtaoensis,

development from the end of neuralation to metamorphosis has been divided in to

20 developing stages (Dunker et al., 2000). In India, research work on caecilians

are mainly reported in distribution of the caecilians of the Western Ghats (Bhatta,

1998), where previtellogenic ovarian follicles of the caecilians Ichthyophis tricolor

and Ichthyophis ramaswami (Beyo et al., 2007), the assembly of ovarian follicles

and ultra structural feature of ovary (Beyo et al., 2007), distribution and abundance

of Gegeneophis ramaswami in southern Kerala have been reported (Oommen et

al., 2000). Further, the stages of spermatogenesis of two species of caecilians,

Ichthyophis tricolor and Uraeotyphlus cf. narayani (Amphibia: Gymnophiona)

involving light and electron microscopic studies have been conducted (Smita et al.,

2004). The spermatogenesis in these species has been divided in to three phases,

namely active spermatogenesis (July–November), early regression (December–

March) and spermatogenic quiescence (April–June) (Smita et al., 2005). There is

practically no information on the stages of development and metamorphosis of

apodans found in India.

Parental care has also been studied only in a few species of apodans. A

remarkable form of parental care and mechanism of parent-offspring nutrient

transfer has been reported in Boulengerula taitanus, which is a direct-developing,

oviparous caecilian in which the skin in brooding females is transformed to provide

a rich supply of nutrients for the developing offspring (Kuffer et al., 2006). Further,

the young individuals of this species are equipped with a specialized dentition,

which is used to peel and eat the outer layer of their mother's modified skin. This

new form of parental care provides a plausible intermediate stage in the evolution of

viviparity in caecilians (Kuffer et al., 2006; Wilkinson, 2008). So far there is no

report on any kind of parental care in caecilians in India.

Caudata/ Urodela:

Most of urodelans are four-legged and lizard-like in shape, but some are

elongate and eel-like with the degenerated limbs (e.g., Amphuima). The tail is never

lost following metamorphosis. Many salamanders have a biphasic life cycle

containing an aquatic larval form with external gills and a metamorphosed terrestrial

adult form that respires by lungs and/or through moist skin. Some species lack

metamorphosis and retain a larval appearance throughout their life (e. g., Axolotl

larva), whereas other species lack the aquatic larval stage and hatch on land as

terrestrial forms that resemble miniature adults (Buckley, 2007).

Sexual dimorphism is common with respects to breeding colors and median-

fin enlargement in the males of long-toed salamanders. Breeding males having

long-toed (Ambystoma macrodactylum columbianum), which scramble for mating

opportunities, are reportedly better in recognizing and/or locating potential breeding

female mates. All the three modes of reproduction (oviparity, ovo-viviparity and

viviparity) are displayed in Urodelans (Wake, 1998; Bhamrah, 2003). Though

salamanders display lesser diversity in reproductive modes than anurans, there is

still variation in the type of fertilization, oviposition site, seasonality, oviparity/ovo-

viviparity/viviparity and in parental care (Nussbaum, 1987). Chemosensory cues

(Pheromones) reportedly play an important role in the daily lives of salamanders in

mediating foraging, conspecific recognition and territorial advertising (Bee and

Gerhardt, 2002; Park et al., 2004). It has been established that male newts emit

pheromones that attract females of the same species (Kikuyama et al., 1997; Watts

et al., 2004). It has been found that male Ambystoma increase their general activity

when exposed to female odorants, but that activity levels in females were not

affected by conspecific odorants of males (Park et al., 2004). It has been seen that

male newts emit pheromones by the cloacal glands that attract females of the same

species (Kikuyama et al., 1997).

Two major reproductive patterns are exhibited by Urodelans. The classical

annual breeders depended upon rise of temperature, saturation of ground by

melting snow and spring rains. The majority of the salamanders have seasonal

reproductive patterns based on cyclic climatic changes, fertilization is external and

oviposition occurs within a few hours to several days after mating (Iwasaki and

Wakahara, 1999; Osikowski and Rafinski, 2001). However, in some species mating

occurs in the autumn, and spermatozoa are stored in the spermatophores for egg

fertilization until the following spring (Wake and Dickie, 1998). Sperm competition

appears to be an important aspect of any mating system in which individual female

organisms mate with multiple males and store sperm in spermatophores (Sharon et

al., 1997). Mating usually occurs in late summer or autumn and many occur again

in the spring in the same populations (Walsh, 2007). Spawning was observed in

early spring, and hatched larvae metamorphosed by August-September, but

duration of development and metamorphosis of larva varies in different species. In

some species it takes 2 years, while in others it takes 1-year or less (Iwasaki and

Wakahara, 1999). Cycles of oogenesis and oviposition may be annual or longer,

depending on the taxon and the population location (Miller and Robbins, 2005).

Post-copulatory sexual selection may be particularly important in species

that store sperm throughout long breeding seasons, because the lengthy storage

period may permit extensive interactions among rival sperm (Adams et al., 2005).

Ocoee salamander (Desmognathus ocoee) has been reported to store sperm up to

9 months prior to fertilization (Adams et al., 2005). Multiple paternities are displayed

in a natural population of a salamander with long-term sperm storage (Liebgold et

al., 2006).

Breeding activity in urodelans is initiated by rainfall and rise of temperature

in coastal population (e.g., Ambystoma tigrinum) (Hassinger et al., 1970). Some

aquatic cold stream species (e.g., Rhyacotriton olympicus) have been reported to

breed throughout the year, but other species (e.g., Dicamptodon ensatus) exhibit

seasonal reproduction (Iwasaki and Wakahara, 1999; Osikowski and Rafinski1,

2001). Most species fertilize the eggs internally, with the male depositing a sac of

sperm in the female's cloaca (Green, 1997; Wake and Dickie, 1988). However, the

most primitive salamanders grouped together as the Cryptobranchoidea exhibit

external fertilization (Selmi et al., 1997). Some species are ovo-viviparous, with the

female retaining the eggs inside her body until they hatch (Wake, 2005).

After fertilization, a larval stage follows in which the organism is fully

aquatic or land dwelling, and possesses gills. The most noticeable morphological

changes are the resorption of the three sets of external gills and the tail fin at the

final stages of metamorphosis (Brown and Cai, 2007). Depending on species, the

larval stage may or may not possess legs (Ohmura and Wakahara, 2002). The larval

stage may last from days to years (Iwasaki and Wakahara, 1999). Some species

exhibit no larval stage at all, with the young ones hatching as miniature version of

the adult (Buckley, 2007). Many urodelans exhibit direct development, in which the

most part of ontogenesis takes place in the egg and a miniature copy of the adult

adapted to the terrestrial mode of life emerges from the egg (Brown and Zippel,

2007). The transition from the larval type to the miniature adult occurs in several

species of Urodelans (Smirnov, 2008). Neoteny has been observed in all

salamander families, in which an individual may retain gills after sexual maturity

(Shi, 2000).

Metamorphosis in the urodelans, regulatory mechanisms, amplitude of

metamorphic transformations, progressive divergence of the larval and the adult

morphology and evolution are regulated by thyroid hormones (TH) (Smirnov, 1992;

Dunn, 2004). In contrast to anurans, many salamanders do not undergo

metamorphosis in nature; however, they can be induced to undergo metamorphosis

via exposure to thyroxine (T4) (Dunn, 2004). Treatment of pre-metamorphic larvae

of urodelans with TH can lead to precocious metamorphic changes even in

facultative neotenes or pedomorphic salamanders such as axolotl that do not

undergo natural metamorphosis (Dunn, 2004). However, the obligatory neotenes

such as Necturus maculosus do not metamorphose either in nature or when treated

with exogenous thyroid hormones (Brown, 2005). The Mexican axolotl, like a

number of other urodelan species, is an obligatory neoteny, which completes its full

life cycle without undergoing metamorphosis (Rosenkilde and Ussing, 2005).

The normal stages of development in urodelans are based on Ambystoma

maculatum. The anuran tadpole changes into a tail less frog or toad, whereas the

urodelan larva hardly changes in general appearance. The existing table of stages

of the normal development of the axolotl (Ambystoma mexicanum) ends just after

hatching. At this time, the forelimbs are found as small buds (Nye et al., 2003).

Anurans/Salentia:

Anurans have a biphasic life cycle, and breed in a variety of water bodies

ranging from highly ephemeral to permanent ponds (Krishna et al., 2004). Two

basic reproductive patterns are evident in anurans. Most tropical and subtropical

anurans species are capable of reproduction throughout the year, rainfall seems to

be the primary extrinsic factor controlling the timing of reproductive activity. The

breeding cycles in anurans in tropical and temperate regions are greatly influenced

by climatic factors and latitudinal distribution (Wiens, 2006; 2007).

Tropically breeding anurans that require heavy rainfall in order to reproduce

are subject to favorable breeding conditions that are sporadic. Although there is an

increased probability of rain during the rainy season, the probability of local rainfall

is unpredictable and this may influence female anurans reproductive strategies

(Lynch and Wilczynski, 2005). In most temperate species, reproductive activity is

cyclic and dependent on a combination of temperature and rainfall. Temperate zone

anurans are explosive breeders (Miwa, 2007). Based on the annual activity,

reproductive cycle of anurans is divided into four phases namely emerging and pre-

breeding phase, spawning and breeding phase, post-breeding phase, and

hibernation phase (Roy, 1990; Huang et al., 1997; Pancharatna and Saidapur,

2009).

Since anurans are poikilothermic and the majority of them depend upon

water for breeding, their reproductive activities are greatly affected by the changing

external climatic factors such as temperature (Saidapur and Hoque, 1995), rainfall

(Grafe et al., 2004; Lynch and Wilczynski, 2005), daylength (Saidapur, 1989;

Edwards and Pivorun, 1991), environmental iodine levels (Dunn, 2004) and pool

desiccation (Lind et al., 2008). Lunar cycle has also been reported to influence

breeding cycle of some species of anurans (Granta et al., 2009). The administration

of exogenous hormones and hibernation increases the breeding behavior and

gamete release by boreal toads, Bufo boreas borea (Roth et al., 2010). The three

major environmental factors temperature, rainfall and photoperiod have been

implicated in the regulation of the amphibian breeding cycles (Lofts, 1984; Dodd

and Dodd, 1976; Roy, 1994).

The environmental temperature plays a key role in regulating population

density, physical activity, metamorphosis and developmental process of anurans

(Dodd and Dodd, 1976; Reading, 2003; Brown and Cai, 2007). In ectothermic

vertebrates, environmental temperature is believed to play a key role in the control

of metabolic activity, sexual behavior and reproductive activity (Fraile et al., 1989).

Latitudinal distribution and temperature are dependent for embryonic survival,

growth and developmental rates in the common frog, Rana temporaria (Laugen et

al., 2003). Thermal acclimatization at higher temperature increases reproductive

activity such as calling and amplexus where as at lower temperature decreases

reproductive activities in Limnodynastes peronii (Rogers et al., 2007).

Growth, sexual maturation and body size dimorphism in the Indian bull frog,

Hoplobatrachus tigerinus depends upon ambient temperature (Gramapurohit et al.,

2004). Formation of growth marks in the bones of the tropical frog, Rana

cyanophlyctis takes place under natural temperature and daylength (Kumbar et al.,

2002). Effect of temperature on development time and energy expenditure was

studied in terrestrially breeding moss frog, Bryobatrachus nimbus (Mitchell and

Seymour, 2000). Environmental temperature is positively correlated with the rate of

metamorphosis and growth of anurans (Dodd and Dodd, 1976; Hayes et al., 1993).

There is paucity of information on the effects of low temperature on amphibians

metamorphosis. The thermal acclimatization ability in tropical and subtropical

amphibians is dependent on seasons (Chang and Lucy, 2005; Newman, 1998).

The effects of variation in climatic temperature were studied on breeding

activity and metamorphosis in the common toad, Bufo bufo (Reading, 2003).

Breeding activity is highly correlated with variation in climatic temperature in

common toad, Bufo bufo (Reading, 2003). Although the specific response to

temperature can vary widely between species, the most frequent observation in

amphibians with a potentially continuous cycle is that exposure to mild

temperatures (15-200C), stimulates spermatogenesis even during the period of

testicular quiescence (Paniagua et al., 1990). Effects of photoperiod and

temperature on testicular function seems to be the most important external factors

involved in the regulation of breeding cycle in many amphibian species (Lehman,

1997).

The histological evidence indicates that although proliferation of cell nests is

present throughout the year, the most important spermatogenetic activity is initiated

in summer (Delgado et al, 1989). Low temperature and short photoperiod

(daylength) in winter induced the arrest of the maturation phase of

spermatogenesis/spermiogenesis and activation of primary spermatogonia

proliferation in the frog, Rana perezi (Delgado et al., 1992). Thereby, temperature

and photoperiod regulate seasonal testicular activity. Further, sexual differentiation

of gonads has been shown to be temperature-sensitive in many species of

amphibians (Dournon et al., 1990).

Ovarian cycle is also under the control of temperature in bull frog, Rana

tigerina (Pancharatna and Saidapur, 1990). Ovary mass is larger in the temperate

than in the subtropical population (Huang and Yu, 2005). Therefore, seasonal

changes in the first growth phase oocytes (FGP) and second growth phase oocytes

(SGP) in anurans may be influenced either by a change in the gonadotrophic

hormones of the pituitary.

Rainfall seems to be another important environmental factor in regulation of

the breeding activity and reproductive cycle in both temperate and tropical anurans

(Grafe et al., 2004; Lynch and Wilczynski, 2005; Brown and Shine, 2007). The first

rain triggers the anurans to come out from their hibernation/hiding and feeding

place. Since their early life history passes in aquatic system, hence water is the

most essential for breeding cycle and metamorphosis of the anuran species.

Reproductive modes also depend upon availability of water (Touchon and

Warkentin, 2008). Water is also essential for male calling, male calls from wetter

nests are more significant for embryonic development (Mitchell, 2000). Further,

males occupying drier nests may have risked of dehydration by calling, and so were

less able to signal to females. Hydration states, therefore, have the potential to

influence the reproductive success of terrestrial male frogs (Mitchell, 2001). There

was a significant interaction between rainfall and sex, dry weather having a stronger

negative effect on males than females as in afro-tropical pig-nosed frog, Hemisus

marmoratus (Grafe et al., 2004).

As in other vertebrates (Reptiles, Birds and Mammals), daylength

(photoperiod) plays an important role in regulation of the annual breeding cycle in

tropical and subtropical anurans. Available evidence suggests that photoperiod,

temperature and rainfall, as the proximate factor determine their seasonal variation

in physiology and physical activity. Environmental temperature and photoperiod

regulate seasonal testicular activity in the frog, Rana perezi (Delgado et al., 1992).

Strong temperature vs. photoperiod significantly interacts in growth and

development of Rana temporaria tadpole southern populations (Laurila, 2001).

An experiment with blinding and exposure to red light stimulated ovarian

growth and demonstrated that melatonin counteracts blinding or red-light-induced

stimulation of ovarian activity (Joshi and Udaykumar, 2000). A deep brain

photoreceptor molecule „pinopsin‟ discovered in the toad hypothalamus is

reportedly responsible for photoreception (Yoshikawa et al., 1988). In Rana pipiens,

the incidence of both ovulation and normal embryonic development were increased

following exposure of the animals to low temperatures and short daylength

(Lehman, 1997). However, light had no positive role in regulation of

spermatogenesis in the frog, Rana cyanophlyctis (Shivakumar, 1999). Most

amphibians exhibit an annual testicular cycle characterized by a quiescent period

(late autumn-winter) and a spermatogenic period (spring and summer) (Saidapur,

1989). At the end of the period of spermatogenesis, undifferentiated interstitial cells

transform into steroid-secreting leydig cells which regress at the beginning of the

new spermatogenetic cycle (Paniagua et al., 1990). Experiments performed during

the period of germ-cell proliferation indicated that low temperatures (below 110C) as

well as short photoperiods (less than 8 hrs.) hinder germ-cell proliferation where as

moderately high temperatures (about 300 C) and long photoperiod (above 12 hrs.)

accelerate this proliferation (Paniagua et al., 1990). The spermatozoa are normally

retained in the testis in winter (low photoperiod) where as spermatozoa are

released during breeding period (high photoperiod) in bullfrog, Rana catesbeiana

(Sprando and Russel, 1988). Continuous normal light for 30 days increased

gonado-somatic index (GSI), whereas continuous injections of melatonin decreased

the GSI in the skipper frog, Rana cyanophlyctis (Udaykumar and Joshi, 1996,

1997).

The sense of olfaction (odor/pheromone/chemosensory) is another cue for

migration to breeding sites by anurans (Ishii et al., 1995; Park et al., 2004). Odor or

scent is the main factor in recognisition of sex and species (Diwan, 1995; Ishii et al.,

1995). Odor seems to be the major factor in orientation and movements to the

breeding sites for some species like Amazonian frog (Ishii et al., 1995). The sexual

selection has been reported to drive behavioral isolation and speciation among

populations of an Amazonian frog, Physalaemus petersi. Sex-pheromone secreted

by males and females also attract for mating in frogs (Kikuyama, 2002). Moreover,

each pheromone secreted by the male acts on conspecific females (Kikuyama et

al., 2005; Kikuyama, 2008).

The major factor in anuran courtship is the production of advertisement calls

(vocalization) by males (Kelly, 2004). Male advertisement vocalization in frogs is

known to be one of the energetically most expensive activities of ectothermic

vertebrates (Emerson and Hess, 2001). Vocalizations attract female anurans to

breeding sites, and there is growing experimental evidence to support auditory

orientation in anurans (Ryan et al., 2001, 2007). The vocalization of frogs has

provided means for investigating acoustic communication (Emerson and Boyd,

1999; Kelly, 2004). Vocal communication plays an important role in behavior

ranging from territorial defense to reproduction. The anuran calls are classified

according to the particular behaviors that they serve. Vocal advertisement is

generally the domain of males (Kelly, 2004; Shen, 2008). Sexual communication in

anuran amphibians has focused heavily on the advertisement call made by

reproductive males (Bowcock et al., 2007). Adult male anurans produce a species-

specific mating call which is used to attract conspecific females during their mating

season, and this call serves as a mechanism to maintain reproductive isolation from

other sympatric species (Roy, 1994; Ryan, 2007). Males produce specific calls as

an attractive courtship signal. In addition other kinds of calls are also emitted such

as territorial call, encounter call and mating call (Lode, 2001; Filho et al., 2008). The

recognition of courtship calls in a chorus may play a useful role in long-term

regulation of anuran breeding activity, especially in distantly placed partner. Male

produce three distinct vocalizations: (1) an advertisement call that attracts both

males and females, (2) an encounter call which is used in territorial interactions and

(3) a courtship call that is only produced when males perceive a female in their

immediate vicinity (Robertson, 2006). Male bullfrogs emit multicroak, quasi

harmonic advertisement calls that function in mate attraction and neighbor

recognition (Simmons, 2004).

Females always select advertisement calls of a heavier male (Robertson,

2006). Males are always combating for sexual partners, but some turgid female

toads give males the slip: a new mechanism of female mate choice in the anurans

(Bruning et al., 2010). Males occupying drier nests may have risked dehydration by

calling and so were less able to signal to the females toad lets (Mitchell, 2001).

Females responded faster to high call rates, and female vocal activity was greater

in response to low-frequency male calls in Iberian midwife toad, Alytes cisternasii

(Bee and Gerhardt, 2002). Females usually exhibit strong and unequivocal

recognition of conspecific mating signals and reject those of other sympatric hetero-

specifics (Bee and Gerhardt, 2002; Ryan et al., 2007). Receptive females and

males of Bufo terrestris responded positively at a distance up to 40 m to a recording

of a conspecific chorus (Duellman and Trueb, 1994). Female poison frogs prefer to

mate with good caller because calling performance is an honest indicator of

paternal genetic quality of the male (Frosman and Hagman, 2006). Females are

typically silent, but in a few anuran species they can produce a feeble reciprocal call

or rapping sounds or rapid trills during courtship (Watson and Kelly, 1992; Elliott

and Kelly, 2007; Shen et al., 2008). Androgen levels in females at this time are

significantly higher than even those levels in males (Emerson and Boyd, 1999;

Burmeister and Wilczynski, 2001). Arginine-8 vasotocin inhibits the call by causing

an accumulation of water and internal pressure (Diakow, 1978).

In India, advertisement call (vocalization) has been studied in Polypedates

maculatus (Kanamadi et al., 1993), Ramanella montana (Kadadevaru et al., 1998),

Kaloula pulchara (Kanamadi et al., 2002), and Polypedates leucomystax (Roy,

2002). Morphological and acoustic comparisons of Microhyla ornata, Microhyla

fissipes, and Microhyla okinavensis (Anura: Microhylidae) are well described for

species identification in Western Ghats (Kuramoto and Joshy, 2006). The

advertisement calls of three Indian frogs, Ramanella triangularis (Microhylidae),

Indirana gundia (Ranixalidae) and Fejervarya rufescens (Dicroglossidae) have been

analyzed and species are characterized in Western Ghats (Kuramoto and Dubois,

2009).

Mating calls of three frog species abundant in North East India Rana

tigerina, Rana cyanophlyctis, and Rana limnocharis were recorded and analyzed in

the fields of Assam and Meghalaya during their breeding season (Roy, 1994; Roy

and Elepfand, 2007; Roy, 2008). A comparison of the mating calls of Rana

cyanophlyctis with those of the sibling Rana ehrenbergi showed differences in their

temporal and spectral characters, supporting the suggestion that these two species

are distinct species, rather than subspecies of the same species (Roy and

Elepfand, 2007). Rana limnocharis in Northeast India is composed of several sub

species. Vocalizations of Rana limnonectes/ Fejervarya limnonectes were studied

in Eastern Himalayas and accordingly species were characterized on the basis of

ossilogram (Borthakur et al., 2007). In Meghalaya, mating calls of Polypedates

leucomystax and Rhacophorus bipunctatus were studied (Iangrai, 2007). The

ossilogram of Polypedates leucomystax showed that each call was composed of

three notes, while that of Rhacophorus bipunctatus each call was composed of six

notes. The difference in the number of notes per call indicates that the call is

species specific (Roy et al., 1998; Iangrai, 2007).

In Orissa, Microhyla ornata and Ramanella variegata usually call from grass

stems or leaves and small branches (Dash and Mahanta, 1993). Polypedates

maculatus commonly calls from branches of trees from low vegetation and from

ground near the water during breeding season (Das and Dutta, 2006). In some

species (e.g., B. melanostictus, B. stomaticus, P. maculatus, Τ. Breviceps, R.

tigerina) the croaking sounds were observed only during the monsoon period (rainy

season). But other two species, (e.g., R. limnocharis and R. Cyanophlyctis) the

croaking was observed throughout the year (Dash and Mahanta, 1993).

The role of gonadotropins in the vitellogenic process and in ovarian

steroidogenesis has been investigated through in vitro experiments in Rana

esculenta (Polzonetti-Magni et al., 1998). The anuran testis is considered as a

model to study germ cell progression during spermatogenesis (Pierantoni et al.,

2002). In the bullfrog, Rana catesbeiana, testicular weight is constant throughout

the year, but the volume and densities of germinal and interstitial compartments

undergo inverse changes from winter (non-breeding) to summer (breeding) (Sasso

et al., 2004). A study in Scandinavian Peninsula found that relative testicular weight

varies and testis weight declines towards the subarctic in the frog, Rana temporaria

(Hettyey et al., 2005). In the reproductive cycle of hylid frog, Dendropsophus

minutus, testicular morphometry is characterized by a continuous gametogenesis

(Santos and Oliveira, 2007). In Rana temporaria, testicular steroid metabolism of

winter and spring frogs showed marked seasonal differences (Antila and Saure,

1979). The spermatogenic activity of Rana ridibunda living in the East Marmara

region was found to be potentially continuous type. Further, the components of

thumb pads exhibited structural changes with respect to the activities of Leydig cells

(Kaptan and Murathanoglu, 2008).

In Bufo melanostictus, plasma androgen, plasma testosterone and changes

in the weights of testes, liver, and fat bodies activity were higher during breeding

period (Huang et al., 1997). Histological evidence indicated that the spermatogenic

cycle of Bufo melanostictus is of a fluctuating continuous type. Although cell nests

of all spermatogenic cell types were present throughout the year, however, the

highest intensity of spermatogenic activity occurred in the month of March (Huang

et al., 1997). Studies have been undertaken on changes in the cytomorphology of

gonadotrophs during the breeding cycle of the male bull frog, Rana tigerina

(Pancharatna and Saidapur, 1990). The gonado-somatic index (GSI) of bullfrogs,

Rana catesbeiana showed no significant variations during different months of the

year (Sasso-Cerri et al., 2004), whereas in Rana cyanophlyctis exposure to

continuous light for 30 days stimulated the GSI and melatonin treatment for 30 days

decreased the GSI (Udaykumar and Joshi, 1997).

Temperate zone female anurans typically have annual ovarian cycles that

are seasonally correlated. In contrast, tropical anurans have diverse patterns of

ovarian cycles (Jorgensen et al., 1979; Rastogi et al., 1983). Another remarkable

characteristic of anurans is the change of ovarian cyclicity in correlation with the

variation in environmental conditions (Kanamadi and Saidapur, 1982; Pancharatna

and Saidapur, 1992). Among anuran amphibians, cyclic ovarian changes have

been reported in Xenopus laevis (Dumont, 1972), Bufo bufo (Jorgensen et al.,

1979), Rana esculenta (Rastogi et al., 1983), Rana cyanophlyctis (Pancharatna and

Saidapur, 1985), Bufo melanostictus (Kanamadi et al., 1989), Rana perezi (Delgado

et al., 1990), and Polypedates maculatus (Kanamadi and Jirankali, 1991).

The classification of developing oocytes of anurans had been carried out by

many workers (Saidapur and Hoque, 1995). Role of temperature in regulation of

ovarian cycle in bull frog, Rana tigrina was studied by exposing them to different

temperature (Pancharatna and Saidapur, 1990). Depending upon the phase of the

oogonial proliferation and the reproductive cycle and/or the pattern of oogenetic

activity, the ovary contains oogonia, first growth phase oocytes (FGP), second

growth phase oocytes (SGP) and matured ovum (Saidapur and Hoque, 1995;

Khanna and Yadav, 2005). The rate of somatic development of the ovary in

anurans is correlated with the rate of gonad differentiation and varies from species

to species (Khanna and Yadav, 2005). FGP and SGP or vitellogenic oocytes were

produced in both the captive and wild caught frogs (Rana cyanophlyctis) throughout

the year (Pancharatna and Saidapur, 1992). Ovarian follicular kinetics and

gravimetric changes in the ovary were studied in the skipper frog, Rana

cyanophlyctis (Pancharatna and Saidapur, 1992; Udaykumar and Joshi, 1996). A

quantitative study of follicular kinetics in relation to body mass, oviduct, and fat body

cycles were studied in Rana cyanophlyctis (Pancharatna and Saidapur, 2009). The

progression of ovarian cycles has been studied in, Rana tigrina (Girish and

Saidapur, 2000).

The temperate anurans exhibit breeding activity throughout the year while

subtropical anurans breed only from March to September (Huang and Yu, 2005).

The annual reproductive pattern of anurans has been studied from the temperate

and subtropical regions. Studies on the tropical anuran species are comparatively

less. Mating success of individual male frogs within explosive breeding species can

depend on their ability to compete for a mate and to hold onto that mate during

amplexus. Such importance of amplexus has resulted in the evolution of sexual

dimorphism in the morphology and anuran forelimb muscles used during amplexus

(Navas and James, 2007).

In India, based on observations on the annual breeding cycle of

Rhacophorus maculatus (Mishra and Das, 1984), Rana limnocharis (Roy, 1990),

Bufo melanostictus (Huang et al., 1997), Polypedates maculatus (Das et al., 2001),

Hyla annectans (Ao and Bordoloi, 2001), Chirixalus simus (Deuti, 2001), Paa

annandalii (Bordoloi et al., 2001), Polypedates leucomystax (Iangrai 2007), and

Rhacophorus bipunctatus (Iangrai, 2007) have been established. It has been found

that the annual testicular cycle of these species consists of four phases, though the

breeding timings differ from species to species. Based on observations on annual

activity cycle and gonadal histology, the annual breeding cycle of male anurans has

been divided into four phases, namely emerging and pre-breeding period, spawning

and breeding period, post-breeding period and hibernation period (Huang et al.,

1997; Roy, 2003; Iangrai, 2007; Pancharatna and Saidapur, 2009).

Generally the female frogs select oviposition sites based on factors such as

water depth, water temperature, water pH, presence or absence of predators

(Khanna and Jadav, 2005). According to Duellman and Trueb (1994), there are 29

ways of egg deposition. But according to Haddad and Prado (2008), there are more

than 29 reproductive modes in anurans. Based on daily monitoring of data on

anuran oviposition, it has been reported that there can be 69 types of natural

oviposition sites during a complete reproductive season (Rudolf and Rodel, 2005).

The most common and phylogenetically widespread site of oviposition is in standing

water (Mode 1), or flowing water (Mode 2), eggs arboreal and tadpoles aquatic

(Mode 4, 6, 18 19 & 20), and eggs terrestrial and tadpoles aquatic (Mode 12, 13, 14

& 24) (Duellman and Trueb, 1994). Many anurans evolved reproductive modes to

meet the special conditions. Such modes include breeding terrestrially and

arboreally, making foam nests, parental transport of eggs and/or tadpoles, direct

development. Other modes are ovo-viviparity and viviparity (Wake and Dickie,

1998, Buckley et al., 2007).

In Western Ghats, diversity of egg laying had been reported. In

Rhacophorus pseudomalabaricus, foam nest construction is done by females, and

eggs are fertilized by sperm secreted by male malabaricus (Vasudevan and Dutta,

2000). In case of a rare microhylid frog, Ramanella montana while in axillary

amplexus, the male clasped the female and pressed her abdomen against the tree

trunk, which apparently facilitated egg deposition. Eggs were deposited and

attached to the surface of the tree trunk just above the water and on the floating,

dried leaves (Krishna et al., 2004). In case of Nyctibatrachus humayani, the female

determines oviposition sites, and lays eggs exactly at the spot from where the male

had been calling. There was no amplexus or any physical contact between the

sexes (Kunte, 2004). In case of shrub frog (Philautus glandulosus), eggs

development is direct and takes place in egg membrane, and there are no free

swimming tadpole stages. Further, the eggs undergo direct development and

hatching of frog lets occurs after 28 days (Biju, 2003).

In North eastern region there is less information on egg laying habits of

anurans. All the Ranid frogs like Rana cyanophlyctis (Mahanta-Hejmadi and Dutta,

1979), Rana limnocharis (Roy, 1990; Borthakur et al., 2007), Bufo melanostictus

(Huang et al., 1997), and Paa annandalii (Bordoloi et al., 2001) laid eggs upon the

water surface attached to a substratum, especially to aquatic plants. After courtship,

the amplecting pair lays the eggs in batches covered with jelly capsules.

In contrast to apodans and urodelans, practically all anurans exhibit external

fertilization (Sever, 2002; Beck, 2002; Marjanovi and Laurin, 2007; Buckley et al.,

2007). Internal fertilization is known only in Ascaphus truei (Sever, 2002) and two

species of Eleutherodactylus (Townsend et al., 1981). In India, so far no anuran

species with internal fertilization has been reported.

In anurans, maternal care is restricted in species with internal fertilization,

and male parental care is limited in species with external fertilization (Beck, 2002).

Male parental care is prevalent in neotropical frog, Eleutherodactylus coqui, where

males brood clutches of direct-developing eggs in non-aquatic nest sites and

defend eggs against cannibalistic nest intruders (Townsend, 1986). The male

Australian pouched frog (Assa darlingtoni) has pouches along its side in which the

tadpoles reside until metamorphosis. Male parental care has also been reported in

the genus Alytes (Rafel, 1993). The care of the strings of eggs is carried by male

partner in Alytes cisternasii (Iberian midwife toad), A. dickhilleni (Bentic midwifw

toad), A. obstetricans (Common midwife toad), and A. mulelensis (Mallorcan

midwife toad) (Rafel, 1993). A unique example of parental care is found in the

female gastric brooding frogs, Rheobatrachus silus from Australia (Tyler et al.,

1983). The female carries embryos and young-ones in the stomach and gives births

to the juveniles orally without any injury to young ones (Tyler et al., 1983). There is

no report regarding the male parental care in Indian species.

Metamorphosis (Gr. meta- "change" + morphe "form") is a biological process

generally attributed to amphibians (Bishop et al., 2006). Metamorphosis in anurans

involves spectacular changes such as resorption of tail, development of fore and

hind limbs, changes in organ system such as gill breather to lung breather, etc.

(Dodd and Dodd, 1976). The transformation of tadpole into frog is one of the most

spectacular processes in nature and, consequently, one of the most thoroughly

investigated event (Shi, 2000; Mc Diarmid and Altig, 2000). Anuran metamorphosis

is divided into three specific periods: pre-metamorphosis, pro-metamorphosis and

metamorphosis climax (Mc Diarmid and Altig, 2000; Brown and Cai, 2007).

In Anurans, the metamorphosis and developmental stages reach a higher

degree of modifications and specialization in comparison to apodans and urodelans

(Mc Diarmid and Altig, 2000). Metamorphosis is a model system to study anuran

organogenesis (Brown and Cai, 2007). Metamorphosis has been studied as a

series of transcriptional programs controlled by thyroid hormones (TH). During

metamorphosis, distinct remodeling has been reported in tail resorption (Huang

and Brown, 2000; Yaoita and Nakajina,1997), muscles (Nicolas et al., 1998;

Gaillord et al., 1999; Cai et al., 2007), intestine (Shi and Brown, 1993), pancreas

(Shi and Brown, 1990; Maake et al., 1998), kidney (pronephros to metanephros),

respiratory organs (gills to lungs) (Dodd and Dodd,1976), liver (Atkinson et al.,

1998), immune system (Rollins-Smith, 1998), brain and spinal cord (Kollros, 1981),

eyes (Hoskins, 1986; Mann and Holt, 2001), nose (Higgs and Burd, 2001), pituitary

gland (Kikuyama et al., 1993; Huang et al., 2001), hematopoetic system (Weber,

1996) and most of the skeleton (Trueb and Hanken, 1992).

In nature, anuran metamorphosis is accelerated by a number of ecological

factors (extrinsic factors) such as increasing temperature (Saidapur and Hoque,

1995), rainfall (Lynch and Wilczynski, 2005), photoperiod (Saidapur, 1989), pool

desiccation (Lind et al., 2008), diet quality (Nicieza et al., 2006), environmental

iodine levels (Dodd and Dodd, 1976) and pond hydrology (Ryan and Winne, 2001).

These factors play an important role in determining the rate and fate of

metamorphosis (Hayes, 1997). The iodine is essential for the synthesis of thyroid

hormones. Hence, sufficient amounts of iodine must be present in the diet and/or

water. Another factor that is likely to function through a neuroendocrine pathway is

light, which regulates melatonin synthesis, and hence thyroid physiology and

metamorphosis (Wright et al., 1990).

Metamorphosis is mainly controlled by thyroid hormones (TH) secreted by

the thyroid gland (Hanken and Hall, 1988; Huang and Brown, 2000). The anuran

metamorphosis is controlled by the hypothalamus-pituitary-thyroid axis involving

actions of several hormones (Hanken and Hall, 1988; Page et al., 2008; Huang and

Brown, 2000). Environmental factors stimulate release of thyroid releasing hormone

(TRH) by the hypothalamus, which stimulates secretion of thyroid stimulating

hormone (TSH) from the pituitary. TSH stimulates secretion of thyroid hormones

(TH) namely 3, 5, 3‟-triiodothyronine (T3) and 3, 5, 3‟5‟-tetraiodothyronine (T4) from

the thyroid gland. An increased concentration of T4 has been reported to accelerate

metamorphosis of anuran tadpoles (Page et al., 2008).

Thyroid hormones play a critical role in the morphological transformations

during metamorphosis in larval bullfrogs, Rana catesbeiana (Galton, 1988;

Fernandez-Mongil et al., 2009). Its effects are mainly mediated through

transcriptional regulation by T3 receptor (TR) (Das et al., 2008). Anuran

metamorphosis serves as an excellent model to study T3 function during post-

embryonic development in vertebrate due to its total dependence on TH (Wang et

al., 2006; Page et al., 2009). The thyroid hormone receptor functions as a master

control factor that can both activate and repress genes in controlling the

transformation of the larval tadpole to the adult frog. Transcription studies have

shown that TR activates or represses TH-inducible genes by recruiting co-activators

or co-repressors in the presence or absence of TH, respectively. However, the

developmental roles of the co-activators or co-repressors of TR remain largely

unexplored (Lorenz et al., 2009).

Besides thyroid hormones, prolactin also plays a critical role in regulation of

anuran larval development and metamorphosis (Dodd and Dodd, 1976; Takada and

Kasai, 2003). Prolactin mainly helps in metamorphosis in early part of the life

history. It has not been detected after 34 day of developing tadpole in gray tree

frog, Hyla versicolor (Beachy et al., 1999). The growth of post-metamorphic

anurans is stimulated by somatotropin but not by prolactin (Frye et al., 2004).

Corticoids (e.g., corticosterone) and the sex steroids (especially 17ß-

estradiol) potentially regulate thyroid hormone activity both by affecting

hypothalamic and pituitary control of thyroid hormone secretion and also by

interacting with thyroid hormones peripherally (Hayes, 1997). Corticosteroids disrupt

amphibian metamorphosis by complex modes of action including increased

prolactin expression (Lorenz et al., 2009).

In India, out of 303 species of amphibians, the developmental stages of only

few species have been studied and documented (Das and Dutta, 2007, Ahmed et

al., 2009). The total duration of metamorphosis of anurans varies from species to

species as reported in Bufo melanostictus (35-50 days, Khan, 1965), Polypedates

maculates (55 days, Mohanty and Dutta, 1986), Rhacophorus malabaricus (68

days, Sekar, 1989), Rana pipens (90 days, Taylor and Shumway, 1990), Hyla

annectans (64 days, Ao and Bordoiloi, 2001), Philautus glandulosus (28 days, Biju,

2003), Polypedates leucomystax (60-61 days, Iangrai, 2007), and Rhacophorous

bipunctatus (59-60 days, Iangrai, 2007).

Appropriate staging of the larval period is fundamental to life history of

anurans. Gosner (1960) gave a simplified table for staging anuran embryos and

larvae with notes of identification. Mc Diarmid and Altig (2000) suggested the

complete tables of development for accurate comparison of development stages in

different anurans with 46 Gosner stages. There is paucity of information on the

development and metamorphosis in Indian anuran species.

According to the IUCN Red Data Book (Version 2009.1), there are 787

species of amphibians worldwide listed in the endangered category (Frost et al.,

2008). These species need serious attention for their in situ conservation. As a first

step in this direction, the breeding biology of the listed endangered species needs

to be investigated in their natural habitat as well as under captivity (Daniels, 1990;

Hoffman, 2009).

The global amphibian crisis has resulted in renewed interest in captive

breeding as a conservation tool for amphibians (Gupta, 1998; Griffiths and

Pavajeau, 2008). Captive breeding programme and reintroduction of anurans are

employed for species which are locally extinct and might help in sustaining

populations (Daniels, 1990). Tropical frogs and toads are disappearing worldwide

due to habitat damage or destruction. The tropical forests of India are also under

human pressure, and many species of anurans are believed to be locally extinct or

at the verge of extinction. The bronzed frog (Rana temporalis) and Malabar torrent

toad (Ansonia ornate) in south India are extinct, but were present 50 years ago

(Daniels, 1992). Most captive breeding and reintroduction programme for

amphibians have focused on threatened species in industrialized countries with

relatively low amphibian diversity (Griffith and Pavajeau, 2008). The conservation

status of all the amphibians in China is analyzed, and the country has shown

priority for conservation in comparison to many other countries of the world (Xie et

al., 2007).

Significant advances have been made during the last decade for amphibian

assisted reproduction including the use of exogenous hormones for induction of

spermiation and ovulation, in vitro fertilization, short-term cold storage of gametes

and long-term cryopreservation of spermatozoa (Kouba and Vance, 2009). The

endangered Wyoming toad (Bufo baxteri) is the subject of an extensive captive

breeding and reintroduction programme. Because Wyoming toads in captivity rarely

ovulate spontaneously, and therefore, hormonal induction is used to ovulate

females or to stimulate spermiation in males (Browne et al., 2006). The Mullorcan

mid wife toad (Alytes muletensis) are conserved by captive breeding programmed

by Jersy Wildlife Preservation Trust, Jersy (Morgan et al., 2008).

In India, captive breeding of common Indian frogs and toads has been

undertaken in Bufo melanostictus, Euphylyctis cyanophlyctis, Euphylyctis

hexadactylus, Hoplobatrachus crassus, Limnonectes keralensis, Limnonectes

limnocharis, Rana temporalis and Tomopterna breviceps (Gupta, 1998). The

captive breeding programme is being introduced to conserve the near-threatened

species Ramanella montana endemic to the Western Ghats (Krishna et al., 2004)

and an endangered tree frog, Rhacophorus lateralis located in coffee plantation in

Kerala (Dinesh et al., 2010).

A critical review of the literature clearly indicates that most of the studies on

breeding biology, reproductive behavior, development and metamorphosis of

amphibians have been conducted on temperate zone species. Limited information

is available on the breeding cycle, reproductive behavior, development and

metamorphosis of amphibian species found in India. Further, the available

information on breeding biology of amphibians in India is fragmentary in nature. So

far no attempt has been made to study the breeding biology of any endangered

and/or threatened amphibian species in any part of the country. Rana leptoglossa is

one of the rare and endangered anuran amphibian species in India (Biodiversity

Conservation Prioritization Project, India, CAMP Workshops REPORT, 2001).

There is paucity of information on population density, breeding biology, gonadal

cycle, developmental stages and metamorphosis of the frog, Rana leptoglossa

under its natural habitat as well as under captive condition in India. Therefore,

keeping in view the endangered and data deficient status of the frog, it was thought

worthwhile to investigate reproductive biology, gonadal cycle, developmental stages

and metamorphosis of the frog, Rana leptoglossa at the Kakoijana Reserve Forest

(KRF), Bongaigaon, Assam. The present dissertation will provide basic information

on reproductive biology, development and metamorphosis of the endangered frog,

Rana leptoglossa (Cope, 1868).

DECLARATION

NORTH- EASTERN HILL UNIVERSITY

SHILLONG -793 022

I, Mr. Biplab Kumar Saha, hereby declare that the subject

matter of this thesis is the record of the work done by me, that the

contents of this thesis did not form the basis of the award of any

previous degree to me or to the best of my knowledge to any body

else, and that the thesis has not been submitted by me for any

research degree in any other University /Institute.

This is being submitted to the North-Eastern Hill University for

award of the degree of Doctor of Philosophy in Zoology.

Prof. R. N. K. Hooroo Prof. B. B. P. Gupta Mr. B. K. SAHA (Head) (Supervisor) (Candidate)

Fig.1.1: Male Rana leptoglossa (Cope, 1868)

Fig.1.2: Female Rana leptoglossa (Cope, 1868)

Fig.1.3: Dorsal view of Rana leptoglossa (Cope, 1868)

B

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To Guwahati

Rivers

International Boundary

State Boundary

Roads

Railway

INDEX

AIE VALLEY DIVISION

AN

T

BRAHMAPUTRA RIVER

HU

B

D

H

U

B

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K

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A

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H

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MANAS RESERVED FOREST

KAKAIJANA RESERVE

FOREST

NH 31

BONGAIGAON

NH31

MANAS RIVER

MANAS RIVER

Map1.1: Map of Kakoijana Reserve Forest in Aie Valley Division, Assam, India.

Map1.2: Map of Kakoijana Reserve Forest (KRF) showing selected breeding sites.

Table 1. 1: Latitudes, longitudes and altitudes of selected breeding sites of

Rana leptoglossa at Kakoijana Reserve Forest (K. R. F)

Site/ Plot No. Latitudes Longitudes Altitude (ASL)

Site 1 260 28' 6.7″ N 900 38' 33.5″ E 59 m

Site 2 260 27' 50″ N 900 38 ' 35.9″ E 46 m

Site 3 260 27' 46.1″ N 900 38' 35.6″ E 49 m

Site 4 260 27 ' 2″ N 900 38' 35.3″ E 57 m

Site 5 260 27 ' 20″ N 900 38' 36″ E 67 m

Site 6 260 28' 8″ N 900 38' 36.4″ E 48 m

Site 7 260 28 ' 16″ N 900 37' 6.9″ E 45 m

Site 8 260 27' 50″ N 900 37 ' 9″ E 71 m

Site 9 260 27 ' 57.9″ N 900 36' 50.9″ E 70 m

Site 10 260 28' 5.4″ N 900 37' 10.9″ E 76 m

Table 1.2: Criteria for morphometric measurements (Chanda, 1994)

Table 1.3: Morphometric measurements of Rana leptoglossa

Sl. No.

Criterion Males (N=10) Females (N=10) Results of one-way ANOVA

Range (mm)

Mean ± S. E

Range (mm)

Mean ± S. E

F-ratio Level of significance

1 Snout-vent 41-59.1 49.83 32.5-69 57±3.4* 3.158 0.092

Criterion Abbreviations

Details of the morphometric parameters

Snout-vent length SVL From tip of snout to vent

Head length HL From the angle of the jaw to tip of snout

Head width HW At angle of jaw

Eye Diameter ED Distance from posterior corner to anterior corner of eye

Inter-orbital space IOS Maximum gap between two eyes

Snout length SL From tip of snout to anterior corner of eye

Tympanic Diameter TD Greatest tympanum diameter along horizontal plane

Length of Forelimb LF From the proximal end of forelimbs to tip of longest finger

Length of Hand LH From the base of the palm to tip of longest finger

1st Finger length F1 From the base of palm to tip of 1st finger

2nd Finger length F2 From the base of palm to tip of 2nd finger

3rd Finger length F3 From the base of palm to tip of 3rd finger

4th Finger length F4 From the base of palm to tip of 4th finger

Hind limb length HLL From mid-ventral line of leg with body to tip of longest toe

Length of Tibia TBL Distance between surface of knee to surface of heel

Foot length FL From the base of foot to tip of longest toe

1st toe length T1 From the base of phalange to tip of 1st toe

2nd toe length T2 From the base of phalange to tip of 2nd toe

3rd toe length T3 From the base of phalange to tip of 3rd toe

4th toe length T4 From the base of phalange to tip of 4th toe

5th toe length T5 From the base of phalange to tip of 5th toe

length (SVL) ±1.9*

2 Head length (HL)

14.5-17.2 15.75±0.35 15.6-19.5

17.52±0.69c 14.02 0.001

3 Head width (HW)

13.5-15.7 14.5±0.25 14-18 16.0±0.70a

7.505 0.013

4 Eye diameter (ED)

5.5-6.3 5.95±0.09 6.1-6.5 6.3±0.07c 17.64 0.001

5 Inter-orbital space (IOS)

3.1-3.9 3.49±0.08 3.5-6 5.2±0.53c 43.22 0.001

6 Snout length (SL)

6.5-7.5 7.04±0.10 7.6-9 8.83±0.27c 260.967 0.001

7 Tympanic diameter (TD)

3.1-4.1 3.6±0.10 3.8-5 4.6±0.21c 32.609 0.001

8 Length of forelimb (LF)

25-26.1 25.55±0.10 26.4-30.1

28.18±0.65c 39.348 0.001

9 Length of Hand (LH)

11-14.75 13.17±0.44 13.5-15.3

14.4±0.31b

10.608 0.004

10 1st Finger length

(F1)

8-8.9 8.5±0.1 12.5-13.8

13.24±0.25c 807.542 0.001

11 2nd

Finger length (F2)

6.9-7.8 7.35±0.09 10.8-11.3

11.04±0.09c 1410.183 0.001

12 3rd

Finger length (F3)

10.5-11.5 10.99±0.11 15.5-15.9

15.7±0.07c 2018.776 0.001

13 4th Finger length

(F4)

8.3-9.2 8.75±0.09 13.8-14.9

14.28±0.23c 1261.935 0.001

14 Hind limb length (HLL)

78.1-87.2 82.58±0.97 88-92 90.0±0.70c 52.095 0.001

15 Length of Tibia (TBL)

25.4-26.3 25.85±0.09 28-31.5 29.9±0.64c 102.196 0.001

16 Foot length (FL) 20.8-21.8 21.32±0.10 27-31 29.0±0.70c 275.963 0.001

17 1st Toe length

(T1)

4.6-5.5 5.05±0.09 10-12 10.8±0.37c 663.462 0.001

18 2nd

Toe length (T2)

9.6-10.6 10.1±0.11 13-15 14.2±0.37c 268.245 0.001

19 3rd

Toe length (T3)

14.8-15.9 15.37±0.11 18-22 20.0±0.70c 101.006 0.001

20 4th Toe length

(T4)

20.5-21.8 21.08±0.14 27-31 29.0±0.70c 296.183 0.001

21 5th Toe length

(T5)

16-16.9 16.45±0.09 20-23 21.7±0.53c 221.781 0.001

*All values are expressed as mean ± Standard error (S.E.); N = 21.

a, b, c

Differ significantly from the respective parameter of the male: p < 0.05, 0.01 and 0.001,

respectively.

Table 1.4: Environmental parameters of Kakoijana Reserve Forest during 2005

Year/ Month

Minimum Temperature

Maximum Temperature

Average Temperature

Rain fall

Daylength (hour)

Relative Humidity

(oC) (oC) (oC) (mm) (%)

2005 JAN

12.54 ± 0.25*

21.08 ± 0.47*

16.81 ± 0.30*

0

10.70 ± 0.02*

81.35 ± 1.47*

FEB

17.06± 0.44

25.83 ± 0.25

21.45± 0.25

0.6

11.26±

0.03

73.92 ±

1.09

MAR

21.38 ± 0.33

28.76 ± 0.17

25.07 ± 0.12

22.8

11.96 ±

0.03

63.27±

1.01

APR

23.19 ± 0.13

27.53 ± 0.07

25.36 ± 0.06

30.5

12.65 ±

0.05

77.78 ±

0.50

MAY

22.28 ± 0.24

28.85 ± 0.38

25.46 ± 0.28

75.6

13.40±

0.02

78.67±

0.83

JUN

24.57 ± 0.25

30.77 ± 0.36

27.73 ± 0.23

16.1

13.73

±0.006

76.90 ±

1.22

JUL

25.74 ± 0.26

30.64 ± 0.50

28.19 ± 0.35

52.3

13.6 ± 0.02

87.0 ± 0.86

AUG

26.82 ± 0.15

31.26 ± 0.33

29.04 ± 0.21

263.2

13.05 ±

0.03

83.1 ± 1.00

SEP

26.12 ± 0.31

32.01 ± 0.47

29.06 ± 0.35

99.0

12.31 ±

0.04

79.22 ±

2.73

OCT

22.36 ± 0.21

25.30 ± 0.26

23.83 ± 0.13

66.4

11.55 ±

0.03

84.85 ±

1.15

NOV

18.99 ± 0.28

25.00 ± 0.20

21.99 ± 0.20

1.4

10.88 ±

0.03

77.4 ± 1.89

DEC

14.44 ± 0.25

23.46 ± 0.14

18.92 ± 0.16

0

10.53

±0.006

73.7.9 ±

0.60

* All values are expressed as mean ± standard error (S. E.), N=12.

Table 1.5: Environmental parameters of Kakoijana Reserve Forest during 2006

Year/ Month

Minimum Temperature

(0C)

Maximum Temperature

(0C)

Average Temperature

(0C)

Rain fall

(mm)

Day Length (hour)

Relative Humidity

(%)

2006

JAN

12.54 ± 0.25*

21.08 ± 0.47*

16.81 ± 0.30*

51.2

10.70

± 0.02*

81.35 ± 1.47*

FEB

16.85 ± 0.41

25.72 ± 0.24

21.28 ± 0.22

9.1

11.26 ± 0.03

74.19 ±

1.29

MAR

19.88 ± 0.27

30.63 ± 0.37

25.25 ± 0.21

16.8

11.96 ± 0.04

56.56 ±

1.69

APR

21.45 ± 0.35

29.04 ± 0.64

25.24 ± 0.44

37.8

12.73 ± 0.03

73.15 ±

1.98

MAY

24.14 ± 0.30

32.03 ± 0.42

28.09 ± 0.32

119.1

13.40 ± 0.02

70.25 ±

1.73

JUN

25.52 0.18

30.5 0.24

28.01 0.16

136.1

13.72

0.006

80.88 1.00

JUL

26.97 ± 0.13

32.31 ± 0.18

29.64 ± 0.12

53.6

13.58 ± 0.02

75.58 ±

2.6

AUG

27.24 ± 0.17

33.20 ± 0.30

30.22 ± 0.21

18.3

13.02 ± 0.03

75.95 ±

1.07

SEP

25.41± 0.18

30.25 ± 0.47

27.83 ± 0.30

7.8

12.29 ± 0.03

80.06 ±

1.09

OCT

23.28 ± 0.33

29.57 0.18

26.43 ± 0.22

0

11.52 ± 0.03

77.53 ±

0.73

NOV

18.83 ± 0.39

25.96 ± 0.34

22.39 ± 0.35

3.1

10.87 ± 0.28

79.26 ±

0.97

DEC

14.70 ± 0.17

22.93 ± 0.17

18.81 ± 0.14

0

10.53 ± 0.007

81.90 ±

1.04

* All values are expressed as mean ± standard error (S. E.), N=12.

Table 1.6: Environmental parameters of Kakoijana Reserve Forest during 2007

Year/ Month

Minimum Temperature

(0C)

Maximum Temperature

(0C)

Average Temperature

(0C)

Rain fall

(mm)

Day Length (hour)

Relative Humidity

(%)

2007 JAN

11.6 ± 0.30*

21.32 ± 0.29*

16.46 ± 0.25*

0

10.38 ± 0.31*

81.74 ± 0.73*

FEB

15.04 ± 0.23

22.63 ± 0.53

18.84 ± 0.31

2.6

11.25 ±

0.03

77.5 ±

1.9

MAR

18.69 ± 0.57

28.12 ± 0.38

23.40 ± 0.38

8

11.98 ±

0.04

62.53 ±

2.36

APR

22.13 ± 0.38

30.15 ± 0.52

26.14 ± 0.41

25.2

12.76 ±

0.03

76.56 ±

1.09

MAY

24.97 ± 0.23

33.30 ± 0.43

29.14 ± 0.30

38.6

13.40 ±

0.02

69.56 ±

1.24

JUN

26.27 0.31

30.56 0.50

28.41 0.36

70.3

13.69 0.008

76.55 0.59

JUL

26.90 ± 0.19

30.64 ± 0.58

28.77 ± 0.38

55.5

13.55 ± 0.024

81.51 ±

0.80

AUG

26.48 ± 0.20

31.66 ± 0.40

29.07 ± 0.28

15.8

13.43 ±

0.06

82.98 ±

0.85

SEP

26.50± 0.07

28.15 ± 0.20

27.32 ± 0.12

6

12.30 ±

0.04

72.42 ±

1.4

OCT

25.15 ± 0.30

30.38 0.38

27.76 ± 0.33

15.4

11.56 ±

0.04

80.59 ±

0.69

NOV

19.46 ± 0.38

26.46 ± 0.31

22.96 ± 0.32

0

10.88 ±

0.02

77.4 ± 0.80

DEC

13.84 ± 0.28

22.36 ± 0.30

18.10 ± 0.27

0

10.54 ±

0.04

83.8 ± 0.56

* All values are expressed as mean ± standard error (S. E.), N=12.

Table 1.7: Average temperature at KRF, Bongaigaon, Assam during three consecutive years (2005, 2006 and 2007)

Months Temperature (0C)

2005 Temperature (

0C)

2006 Temperature (

0C)

2007 Average

Temperature (0C)

Jan

16.81 ± 0.30

16.81 ± 0.30

16.46 ± 0.25

16.69 ± 0.28*

Feb

21.28 ± 0.22

21.28 ± 0.22

18.84 ± 0.31

20.44 ± 0.25

Mar

25.25 ± 0.21

25.25 ± 0.21

23.40 ± 0.38

24.63 ± 0.26

Apr

25.24 ± 0.44

25.24 ± 0.44

26.14 ± 0.41

25.52 ± 0.43

May

28.09 ± 0.32

28.09 ± 0.32

29.14 ± 0.30

28.19 ± 0.31

Jun

28.01 0.16

28.01 0.16

28.41 0.36

28.14 ± 0.68

Jul

29.64 ± 0.12

29.64 ± 0.12

28.77 ± 0.38

29.35 ± 0.20

Aug

30.22 ± 0.21

30.22 ± 0.21

29.07 ± 0.28

29.83 ± 0.23

Sept

27.83 ± 0.30

27.83 ± 0.30

27.32 ± 0.12

27.66 ± 0.24

Oct

26.43 ± 0.22

26..43 ± 0.22

27.76 ± 0.33

26.87 ± 0.25

Nov

22.39 ± 0.35

22..39 ± 0.35

22.96 ± 0.32

22.58 ± 0.34

Dec

18.81± 0.14

18.81± 0.14

18.10± 0.27

18.57 ± 0.18

* All values are expressed as mean ± standard error (S. E.), N=12.

Table 1.8: Average daylength at KRF, Bongaigaon, Assam during three consecutive years (2005, 2006 and 2007)

Months Daylength (hrs) 2005

Daylength (hrs) 2006

Daylength (hrs) 2007

Average Daylength (hrs)

Jan

10.70 ± 0.02

10.70 ± 0.02

10.38 ± 0.31

10.59 ± 0.11*

Feb

11.26 ± 0.03

11.26 ± 0.03

11.25 ± 0.03

11.25 ± 0.03

Mar

11.96 ± 0.03

11.96 ± 0.04

11.98 ± 0.04

11.96 ± 0.03

Apr

12.65 ± 0.05

12.73 ± 0.03

12.76 ± 0.03

12.71 ± 0.03

May

13.40 ± 0.02

13.40 ± 0.02

13.40 ± 0.02

13.40 ± 0.02

Jun

13.73 ± 0.006

13.72 0.006

13.69 0.008

13.71 ± 0.006

Jul

13.6 ± 0.02

13.58 ± 0.02

13.55 ± 0.024

13.57 ± 0.02

Aug

13.05 ± 0.03

13.02 ± 0.03 13.43 ± 0.06 13.16 ± 0.04

Sept

12.31 ± 0.04

12.29 ± 0.03

12.30 ± 0.04

12.30 ± 0.03

Oct

11.55 ± 0.03

11.52 ± 0.03

11.56 ± 0.04

11.54 ± 0.03

Nov

10.88 ± 0.03

10.87 ± 0.28

10.88 ± 0.02

10.87 ± 0.11

Dec

10.53 ± 0.006

10.53 ± 0.007

10.54 ± 0.04

10.53 ± 0.017

* All values are expressed as mean ± standard error (S. E.), N=12.

Table 1.9: Average rainfall at KRF, Bongaigaon, Assam during three consecutive years (2005, 2006 and 2007)

Months Rainfall 2005

Rainfall 2006

Rainfall 2007

Average Rainfall (mm)

Jan 0

51.2

0

17.06

Feb

0.6

9.1

2.6

4.10

Mar

22.8

16.8 8

15.86

Apr

30.5

37.8

25.2

31.16

May

75.6

119.1

38.6

77.60

Jun

16.1

136.1

70.3

74.14

Jul

52.3

53.6

55.5

53.80

Aug

263.2

18.3

15.8

99.10

Sept

99.0

7.8 6

37.60

Oct

66.4 0

15.4

27.26

Nov

1.4

3.1 0

1.50

Dec 0 0 0 0.0

Table 1.10: Average relative humidity at KRF, Bongaigaon, Assam during three consecutive years (2005, 2006 and 2007)

Months Relative Humidity (%)

2005

Relative Humidity (%)

2006

Relative Humidity (%)

2007

Average R. H. (%)

Jan

81.35 ± 1.47

81.35 ± 1.47

81.74 ± 0.73

81.48 ± 0.1.2*

Feb

73.92 ± 1.09

74.19 ± 1.29

77.5 ± 1.9

75.20 ± 1.42

Mar

63.27 ± 1.01

56.56 ± 1.69

62.53 ± 2.36

60.78 ± 1.68

Apr

77.78 ± 0.50

73.15 ± 1.98

76.56 ± 1.09

75.83 ± 1.19

May

78.67 ± 0.83

70.25 ± 1.73

69.56 ± 1.24

72.82 ± 1.26

Jun

76.90 ± 1.22

80.88 1.00

76.55 0.59

78.11 ± 0.93

Jul

87.0 ± 0.86

75.58 ± 2.6

81.51 ± 0.80

81.36 ± 1.42

Aug

83.1 ± 1.00

75.95 ± 1.07

82.98 ± 0.85

80.67 ± 0.97

Sept

79.22 ± 2.73

80.06 ± 1.09

72.42 ± 1.4

77.23 ± 1.74

Oct

84.85 ± 1.15

77.53 ± 0.73

80.59 ± 0.69

80.99 ± 0.85

Nov

77.4 ± 1.89

79.26 ± 0.97

77.4 ± 0.80

78.02 ± 1.22

Dec

73.79 ± 0.60

81.90 ± 1.04

83.8 ± 0.56

79.83 ± 0.73

*All values are expressed as mean ± standard error (S. E.), N=12.

Table 1.11: Correlation of average population density with different climatic factors such as temperature, daylength, rainfall and relative humidity

Months Average Coefficient of correlation (r)

population density ± S.E

(2005 - 2007)

Temperature vs.

Population density

Daylength vs.

Population density

Rainfall vs.

Population density

Relative Humidity

vs. Population

density

March 0.49 ± 0.029*

0.639d

0.863d

0.676d

-0.336

April 0.53 ± 0.033

May 0.59 ± 0.029b

June 0.62 ± 0.029c

July 0.55 ± 0.029a

August 0.40 ± 0.020

*All values are expressed as mean ± standard error (S. E.).

d

Significant positive correlation: p < 0.05 level (N=6).

a, b, c

Differ significantly from the values of August (Minimum): P < 0.05, 0.01 and 0.001,

respectively.

Fig.1.4: Average population density of Rana leptoglossa during 2005, 2006 and

2007 at KRF, Bongaigaon.

ccccccccccccc

a ba

Fig.1.5: Monthly variations between average temperature and population density.

All values are expressed as mean ± standard error (S. E.).

a, b, c

Differ significantly from the values of August: P < 0.05, 0.01 and 0.001, respectively.

Fig.1.6: Monthly variations between average daylength and population density.

All values are expressed as mean ± standard error (S. E.). a, b, c

Differ significantly from the values of August: P< 0.05, 0.01 and 0.001,

respectively.

ccccccccccccc

a ba

ccccccccccccc

a ba

Fig.1.7: Monthly variations between average rainfall and population density.

All values are expressed as mean ± standard error (S. E.). a, b, c

Differ significantly from the values of August: P < 0.05, 0.01 and 0.001,

respectively.

Fig.1.8: Monthly variations between average relative humidity and population density.

All values are expressed as mean ± standard error (S. E.).

a, b, c

Differ significantly from the values of August: P < 0.05, 0.01 and 0.001, respectively.

0

5

10

15

20

25

30

35

0 J F A M A J M J S O N D J F A M A J M J S O N D J F A M A J M J S O N D

Te

mp

era

ture

(0C

)

Months

2005

2006

2007

ccccccccccccc

a ba

Fig. 1.9: Monthly variations of air temperature at KRF, Bongaigaon during 2005, 2006 and 2007.

Fig. 1.10: Monthly variations of daylength at KRF, Bongaigaon during 2005, 2006

and 2007.

Fig. 1.11: Monthly variations of rainfall at KRF, Bongaigaon during 2005, 2006 and

2007.

0

50

100

150

200

250

300

0 J F A M A J M J S O N D

Months

J F A M A J M J S O N D J F A M A J M J S O N D

Ra

infa

ll

(mm

) (m

m)

((m

min

mm

mm

mm

m

2005

2006

2007

0 1

2

3 4

5

6 7

8

9 10

11

12 13

14

Da

yle

ng

th

(hrs

)

0 J F A M A J M J S O N D

Months

J F A M A J M J S O N D J F A M A J M J S O N D

2005

2006

2007

Fig. 1.12: Monthly variations of relative humidity at KRF, Bongaigaon during 2005, 2006 and 2007.

2005

2006

2007

0

10

20

30

40

50

60

70

80

90

0 J F A

M A J M J S O N D J F A M A J M J S O N D J F A M A J M J S O N D

Re

lati

ve h

um

idit

y (

%)

Months