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V. Discussion: During the present work, information has been generated on distribution pattern, ecological preferences, ethnobotany, biology of reproduction and distribution of genetic diversity in three overexploited medicinally important angiosperms of North – Western Himalayan region. Two of these species i.e. Picrorhiza kurrooa and Valeriana wallichii are described as endangered (Kala, 2004) and critically endangered (kumara et al., 2012) respectively where as Ferula jaeschkeana has been reported as vulnerable (Kala, 2000; Kumar et al., 2011). The information thus generated is expected to help in evolving better and robust strategies for their conservation, both in and ex situ. V.1 Threat perception to the species in nature: As per the IUCN (1994) criteria for categorizing plant species into different risk groups, population size i.e. the number of individuals of a taxon occurring in wild is an important parameter. According to this criterion a taxon is categorized as critically endangered, endangered or vulnerable when the number of mature individuals of this taxon in wild is less than 250, 2500 and 10,000 respectively. Other criteria formulated by IUCN to assess the threat status of species include, decline in area of occupancy, geographic range or extent of occurrence, habitat quality, levels of exploitation and effects of introduced taxa, hybridization, pollutants, competitors or pathogens (Keith, 1998). Judging the taxa under discussion by these parameters, the observations made during this study revealed that going by population size alone, none of these species could be categorized under either of these categories. Number of individuals scanned for all the three plant species is fairly above the threshold 94 mark decided for the categories endangered and vulnerable. Similar observations have been made by some other workers also in P. kurrooa and F. jaeschkeana (Kala, 2000, 2004, 2005). These species are however, under stress because of several other reasons which are described species wise hereunder. A common feature of all these species is their occurrence in disjunct patches like most of the rare and threatened medicinal plants (Schemske et al., 1994). V.1a Picrorhiza kurrooa: The species occurs in patches separated by varying distances and propagates mainly by stolons. Habitat range of this species has undergone a sharp reduction during the recent times. Interaction with the local people regarding the extent of distribution in past and its overexploitation as well as the personal observations made during 4 successive years in some of the habitats have brought us to this conclusion. The species although occurring in the protected areas, faces a great stress of exploitation from the locals for use in traditional medicines as well as from traders. People living in adjoining areas are well aware of all the possible sites where the species grows and inspite of strict restrictions imposed over its collection from wild, stolons of the species are being collected ruthlessly and are sold in local markets. The dried roots and stolons are used to treat a number of liver ailments and stomach problems like jaundice, fever and indigestion. It also has blood purifying properties. The risk of extinction is accentuated as plants are uprooted and destroyed by collectors or grazed by cattle (both foliage and flowering racemes) leaving no chance for them to recover and regenerate. Except for the major stress due to 95 overexploitation, habitat destruction and grazing, other factors such as pollution and pathogens or competitors do not seem to be threatening the populations in their alpine and subalpine habitats where the species apparently has adapted to grow luxuriantly through vegetative means. V.1b Ferula jaeschkeana: Individuals of this species cluster together in distinct populations like most of the rare and threatened medicinal plants (Schemske et al., 1994). All the populations surveyed in Ladakh, Sonamarg and Kishtwar were disjunct. No commercial exploitation of the species was observed at any of these locations. However, the species is exploited in Drass area of Ladakh by aboriginals as firewood and a source of medicine for joint pains and for increasing milk production in the cattle. The plant parts targeted by locals are the dried peduncles as firewood and underground rootstock for medicinal purposes. Peduncles are collected after the plants have completed reproductive phase with the seeds ripened and are undergoing senescence. But rootstock is harvested at any time during its life span. The Resin obtained from the stem is used after drying to heal the wounds. There are reports of heavy exploitation of the species in parts of Lahul and Spiti of Himachal Pradesh for its seeds and roots which are used by Amchis for medicinal purposes (Kala. 2000). This kind of anthropogenic stress to the species in the study area is not a cause of concern but the habitats of the species face acute pressure of getting destroyed due to increasing human population resulting in change in the land use patterns there. A heavy natural threat to this species is in form of widespread herbivory by beetle larvae. Despite very high reproductive output, the species 96 suffers a great loss as nearly 90% of the seeds get infested and damaged by the larvae. V.1c Valeriana wallichii: The habitat of Valeriana wallichii is prone to destruction through natural as well as anthropogenic reasons. Natural reasons include landslides and soil erosion on the slopes harbouring its populations. Anthropogenic reasons include developmental activities, direct collection of plant material for medicinal purpose and grazing in the area of occurrence. Like P. kurrooa, plants of this species are uprooted, as rhizome is the part sought after, leaving no chance for their regeneration and survival. The populations located in J&K are safer with respect to direct collection pressures as there is little if any exploitation for commercial purposes. Some tribal communities use its rhizome to treat hallucination and also some gynecological problems but that amounts to only negligible exploitation. However, personal observations as well as reports by some other workers suggest that rate of exploitation of this species is considerably higher in other states like Uttrakhand and Himachal Pradesh. Heterogeneity in sex expression occurring in natural populations of this species is beneficial for species fitness as heterozygosity is maintained and new genotypes appear after each sexual cycle through the progeny of female individuals which is a result of exclusive outcrossing. This kind of population structure can be deleterious as it can disrupt the balanced genetic structure and population fitness in case any one kind of individuals are collected from a population. In case of selective elimination of hermaphrodite individuals from a population, the female plants are likely to fail in 97 reproducing sexually in event of pollen scarcity or non availability at all. If on the other hand female individuals get eliminated from any population, the proportion of heterozygous genotypes generated through outcrossing will be reduced in that population and homozygous genotypes will outnumber them which is likely to effect the species fitness. Plants from Chakrata and Almora also show a tendency to multiply vegetatively through stolons and thus have increased the possibility of geitonogamy in patches derived from vegetative multiplication. Increased selfing in outcrossing populations leads to inbreeding depression consequently increasing genetic load of populations (Ralls et al., 1979; Kirkpatrick and Jarne, 2000; Fox et al., 2008; Charlesworth and Willis, 2009; Hedrick and Fredrickson, 2010). Loss of genetic variability resulting from increased homozygosity is also found to be associated with reduced fitness and have low evolutionary potential (Fisher, 1958; Charlesworth and Charlesworth, 1987; Da Silva et al., 2006; Hanski and Saccheri, 2006; Grueber et al., 2008). Since we have observed that V. wallichii is self as well as cross compatible and seed set under both selfing and outcrossing is also comparable, effects of inbreeding depression may appear at later stages of life cycle or under different environmental conditions. Moreover, it has been reported that in some cases, genetic degradation may show its effects on fitness and lead to extinction only after level of inbreeding depression has reached a threshold in small populations (Frankham, 1995a). On the whole, the loss of genetic variability as a result of shrinking population size in all species and particularly disruption of the normal ratios of different sex types in V. wallichii may trigger the loss of fitness and adaptability of 98 these species to changing environmental conditions and drive them towards extinction. Besides population structure and stress faced by these taxa through extrinsic factors, other attributes of reproductive biology studied during present investigation have highlighted some features of breeding system, meiotic system, pollination biology and seed germination and dispersal in these species, some of which have been discussed in terms of their adaptive significance to the species whereas some are apparently a constraint in the success of sexual reproduction and hence pose an intrinsic threat to the species in nature. Impact of various features of genetic system on the threat status is discussed as under for each of the three taxa studied: V.2 Genetic System: Genetic system refers to the state of generation and distribution of genetic variability in a species (Darlington, 1940). Genetic system has two components: a) meiotic system which creates genetic variation through the process of recombination in male and female sex tracks and b) breeding system which distributes the variation thus generated among the progenies. 2a Meiotic system: Meiosis is a complex and genetically controlled process of cell division occurring in the germ line cells which is responsible for production of haploid and genetically variable gametes. It forms the basis for sexual reproduction in both plants and animals (Golubovskaya, 1979; Falistocco et al., 1994; Pankratz and Forsburg, 2005). 99 Components of meiosis responsible for generation of genetic variation include chromosome number, chromosome size, ploidy level, pairing between homologous chromosomes and degree of crossing over as chiasmata frequency at diplotene, diakinesis and metaphase - I. Irregularities during the course of meiosis are known in a number of plant species and invariably result in the formation of non - functional gametophytes (Lattoo et al., 2006; Kumar et al., 2010; Larrosa et al., 2012). Many of the reported irregularities arise due to disruption in the pairing process leading to asynapsis (Beadle, 1930; Rieger et al., 1991), due to failure in maintaining chiasmata leading to desynapsis (Li et al., 1945) or formation of multivalent associations, chromosome bridges and laggards due to structural hybridity (Darlington, 1929; Pagliarni, 2000; De Souza et al., 2003; Chibber et al., 2007). Since irregularities in meiosis directly affect the viability of gametes and are thus important in determining the success of sexual reproduction, studies on meiosis have been very useful in understanding the quantum of genetic variability, and also the causes of poor fertility and low seed set in several plant species. 2a (i) Unusual Pollen Mother Cell meiosis in V. wallichii: Two modes of PMC meiosis were observed in V. wallichii i) desynaptic, a predominant mode in the species which without affecting the meiotic course, results in the formation of viable and functional pollen grains and ii) normal chiasmata formation up to metaphase - I, but beyond that several anomalies are manifested which lead to a significant level of pollen sterility. Desynapsis is described as a condition of having univalents instead of bivalents at later prophase and metaphase - 1 which may arise either due to failure of 100 homologous chromosomes to form chiasmata (Li et al., 1945) or failure in maintaining chiasmata (Maguire, 1978, 1990; Maguire, et al., 1991). Most of the studies focusing desynapsis have regarded it as a meiotic anomaly which further leads to other irregularities and affects the fertility of the resulting gametophytes (Jauhar and Singh, 1969; Krishnan et al., 1970; Jackson et al., 2001; Vergilio et al., 2008; Sharma et al., 2010; Sharma et al., 2011; Pagliarini et al., 2011). . Genetic basis of desynapsis has been intensively studied and desynaptic genes in maize, tomato and Haplopappus gracilis are known (Jackson, 1985; Maguire et al., 1991). These mutations may be hologenomic i.e. affecting all the bivalents as in case of Tomato (Jackson, 1985) or may be chromosome specific which affect only a specific bivalent as in Haplopappus gracilis (Jackson et al., 2002). Occurrence of univalents instead of bivalents at metaphase - I triggers irregularities in the events that follow. These include irregularities in spindle organization like appearance of extra minispindles, presence of tripolar, quadripolar and multiple spindles in the same cell (Koduru and Rao, 1981; Dawe, 1998), random distribution of univalents at anaphase - I, precocious chromatid separation and cytokinesis (Dawe, 1998; Krishnaswamy and Meenakshi, 1957; Vergilio et al., 2008). Most of these anomalies affect the fertility of resulting gametophytes. In this backdrop, the present observations on the pmc meiosis in V. wallichii are exceptional to the usual meiotic pathway. In the species, desynapsis does not occur as an anomaly but is a predominant mode of meiosis without affecting the pollen viability. Pollen mother cells in such plants have univalents at stages as early as diplotene and metaphase - I, the subsequent events like anaphasic segregation (both at AI and AII) proceeding normally, and no 101 irregularities as mentioned in literature (Dawe, 1998; Krishnaswamy and Meenakshi, 1957; Vergilio et al., 2008) were however, observed in this study. In the species desynaptic mode seems useful since the species has adopted it as the most successful mechanism of meiosis in the male tract. Though the homologous chromosomes separate at diplotene, the migration of univalents to two poles is regular and so is the segregation of chromatids at anaphase - II. Thus, the four daughter cells are genetically balanced which is also reflected by high viability of pollen grains. Interestingly, the plants that deviate from the desynaptic mode with pmcs following the normal course of meiosis up to metaphase - I, show highly unusual anaphasic segregation that is not known so far in any sexual organism. This meiotic pathway has a deleterious impact on the fertility of pollen, and a large proportion of pollen produced by such plants is sterile. Interestingly, in these plants, chiasmata once formed fail to resolve in all or most of the chromosome pairs and bivalents persist even at stages beyond metaphase - I, which is an unusual event. The non - disjointed bivalents move together towards opposite poles during anaphase - I creating great genetic imbalance at both poles, as each pole receives a particular chromosome in duplicate where as the other end lacking any member of this altogether. The bivalents at the poles in such cells remain as paired units and the anaphasic segregation of homologues is postponed till anaphase – II. After reaching the four poles, chromatid separation takes place. The final chromosomal complement at four poles depends upon the manner of segregation at anaphase - I and anaphase - II. Lagging chromosomes in the form of either bivalents or univalents are also a common feature of this meiotic 102 pathway. Most of the gametes thus produced are genetically imbalanced and sterile. Prevalence of meiotic irregularities and resulting pollen sterility in this species is also analyzed with reference to the variation in sex expression presently observed in this species. The species exibits gynodioecy all over but some individuals in the populations presently scanned are gynomonoecious. As per the earlier held view on the origin of gynodioecy, it is believed to be controlled by cytoplasmic and nuclear genes acting antagonistically, former to cause male sterility and the later to restore this function (Ross, 1978; Richards, 1986; Couvet et al., 1990). Gynodioecy is also considered as an evolutionary intermediate stage in transition of sexual system from hermaphroditism to dioecy (Darwin, 1877; Lloyd, 1975; Charlesworth and Charlesworth, 1978; Ross, 1978; Bawa, 1980). Gynomonoecious plants represent a state of partial male sterility and can be considered an intermediate stage in transition from hermaphrodite to completely male sterile state in pistillate flowers. Thus, V. wallichii presents an interesting example of a species undergoing evolution of sexual system with all the possible intermediate stages well represented in nature. The present study suggests that, disruption in the normal functioning of genes operating during pmc meiosis may be one of the mechanisms underlying the transition from male fertile to male sterile condition in Valeriana wallichii and thus producing different sexual phenotypes. In addition to the unusual meiosis observed in this species, polymorphism was observed in pollen mother cells of an anther with respect to their size and chromosome number. Among the three classes of pmcs observed in this species 103 (viz. small, medium and large), large pmcs exhibited mixoploidy i.e. occurrence of diploid and tetraploid numbers. These pmcs however, represent the smallest fraction of total pmcs in any anther with their frequency ranging from 0 - 8.7%. Diploid cells had 16 bivalents which show desynapsis characteristic of this species. Tetraploid pmcs were noticed at late metaphase – I with initial stages of segregation of 32IIs arranged on equatorial plate, and also at anaphase – I with 32 Is at each pole of the cell. Occurrence of pmcs with Different chromosome number in different or same anthers of a plant have been reported in Allium tuberosum (Sharma and Gohil, 2004) and in Capsicum annum L. (Lakshmi et al., 1991). There is however, no report of size variation associated with mixoploidy. Origin of mixoploidy has been attributed to a number of cellular phenomenon. Polyploid cells have been explained to arise from mixing of chromosomes from different cells (Smith, 1942), due to fusion of neighbouring cells during early stages of meiosis (Levan, 1941) or due to defective cell wall formation (Lin, 1977). Cytomixis has also been described as a mode of origin of aneu- and polyploids (Sarvella, 1958; Falistoco et al., 1995). In the present case a thorough cytological examination of anthers from different plants did not reveal even a tendency towards cytomixis but fusion of pmcs at early prophase was observed. Commensurate with the size difference in the pmcs, difference in pollen size within the same anther confirms that the apparent coalescing of pmcs at early stage is not an artifact. The low pollen viability of small pollen can be explained on the basis of some genic imbalance. This assertion is supported by the fact that the viability is upto 100% in large sized pollen in which because of increased number of chromosomes, the imbalance perhaps gets neutralized. 104 V.2a (ii) PMC meiosis in Ferula jaeschkeana: The normal course of meiosis followed in the male sex tract of this species is a source of generation of genetic variability as reflected through its recombination index. An efficient meiotic system operating in the species is responsible for the production of fertile pollen in large numbers which provide the basic requisite for the successful reproduction. Cytomixis and other meiotic anomalies observed in the species although of rare occurrence directly affect the pollen viability. Cytomixis has been described as a meiotic irregularity regulated by genetic and environmental factors and is generally associated with low pollen fertility (Latoo et al., 2006; Song et al., 2009). V.3 Breeding system: Information on the breeding system provides a framework to understand the pattern of diversity found in plant species. The type and success of a particular breeding system is a decisive force in establishing co-sexuality (hermaphroditism) or separation of sexes (dioecy) in a species in nature (Castillo and Argueta, 2009). Understanding the mating patterns especially in rare plant taxa can provide valuable information for their effective management including the conservation of natural genetic diversity (Neel et al., 2001). V.3a Breeding system of V. wallichii seems to be evolving from hemaphroditism to dioecy: Valeriana wallichii displays a mixed mating system due to cross as well as self compatibility in bisexual flowers and entomophilous pollination. While the bisexual flowers present opportunities for both self (geitonogamous) and cross pollination, female flowers offer for exclusive outcrossing. In manual crossing experiments, females outperformed the hermaphrodites in terms of both 105 reproductive output and percentage seed germination. Hermaphrodites although showed nearly comparable reproductive output under manual crossing and manual selfing, but the seeds derived from manual cross pollination showed a better germinability than those derived from manual self pollination. Increased reproductive performance and better offspring quality in females relative to hermaphrodites is mostly observed in gynodioecious plant populations and has been explained as a compensation for the loss of male function that allows for increased allocation of resources towards female function (Williams et al., 2000). It is also a means to balance the contribution to the gene pool with that of hermaphrodites which use both male (pollen) and female (ovule) function to contribute their genes and are thus likely to be favoured by natural selection (Charnov, 1982). It has also been postulated that females in any population perform better than hermaphrodites because they are never susceptible to inbreeding depression and hence loss of performance and fitness arising due to this reason is not a chance for them (Darwin, 1877; Lewis, 1941; Llyod, 1975, 1976; Ross, 1978; Charlesworth and Charlesworth, 1978; Charlesworth and Ganders, 1979; Charnov, 1982). Breeding system in this species is interesting and unique as it represents gynodioecy i.e. co-existing of female as well as hermaphrodite individuals in a population but at the same time gynomonoecious individuals also were found to exist in the species, though they represent the smallest fraction of any population. Breeding systems in plants have been characterized as hermophroditic, monoecious and trioecious having male, female and hermaphrodite individuals in a species (Castillo et al., 2009). According to the existing views about evolution of breeding system in plants, hermaphroditism was 106 the original condition represented in about 75% species (Williams et al., 2000) from which evolved dioecy or unisexuality (Darwin, 1877; Weiblen et al., 2000). Two evolutionary pathways involved in the change from hermaphroditism to dioecy have been described; one operates through the invasion of male sterile mutants in a hermaphrodite population and their subsequent selection over the later due to better performance and fitness. In this pathway, gynodioecy represents the intermediate stage. (Darwin, 1877; Llyod, 1975; Charlesworth and Charlesworth, 1978; Ross, 1978; Bawa, 1980; Barrett, 2002). The other mechanism is explained to work via monoecy under the influence of disruptive selection (Barrett, 2002). The gynomonoecious individuals although represent the smallest fraction of any population, their presence in all the populations indicates the evolutionary significance of this sexual morph. Such individuals represent the effect of male sterility mutations as some of the male sterile flowers in these plants have rudimentary anthers with sterile pollen. The role of meiotic aberrations in this respect has already been discussed. In such a case, these individuals represents intermediaries in transition from hermaphrodite to female state. On the other hand, if the natural selection favours male function in the bisexual flowers of such plants, they may evolve into monoecious individuals during in course of time. These plants, therefore, may also represent a transition to monoecy from hermaphroditism which may eventually lead to gynodioecy or dioecy as described by Barrett (2002). Breeding system of V. wallichii on one hand is biased towards outcrossing as is evident by evolution of exclusively outcrossing female plants and also contrivances for promoting outcrossing in hermaphrodite flowers i.e. dichogamy and herkogamy, but on the other hand have 107 retained the possibilities of selfing by being self compatible. Inspite of mixed mating strategy, it was observed that cross pollen perform better by having a better rate of pollen tube growth the self pollen. Crossing is therefore, preferred over selfing in these plants. In female flowers, pollen tubes reach the ovary considerably quicker than in bisexual flowers either crossed or selfed. This can be due to the shorter style length in the former. In the species, the female flowers are much smaller than their hermaphrodite counterparts and also lack pollen as a reward for pollinators. As a result they receive comparatively poor pollinator services than the hermaphrodites. During the present study, this was experimentally demonstrated that at the end of the season percentage of stigma with pollen load was much lower in females as compared to hermaphrodites. This could prove highly detrimental to the reproductive success of the female plants as they become deprived of pollen. But an additional strategy adapted by females is to increase the duration as well as level of stigma receptivity much above that of hermaphrodites so as to make best utilization of pollinator visits. It was observed that in female flowers, stigma remains fully receptive at all the times for which the flowers remain on plants. Adopting this strategy, flowers of all the stages present in an inflorescence get effectively pollinated even in a single visit by pollinators and this assures seed set. Hermaphrodites prefer to be fertilized by cross pollen under the conditions of ample pollen supply of both kinds, but also assure reproductive success by selfing under the conditions of pollen limitation that may arise due to pollinator scarcity under adverse climatic conditions. Besides retaining self compatibility, mechanical movement of style as detailed below is a strategy to ensure some seed set. Such adaptations are acquired by plant species to 108 avoid complete failure of reproduction and contribute genes to next generation even through inbred progeny. V.3b Breeding system in F. jaeschkeana: Self as well as cross compatibility, pollination mediated by insects and occurrence of plants with characteristic pattern of sex expression favour a mixed mating strategy in the species. Since the hermaphrodite flowers are strongly dichogamous, outcrossing is the preferred strategy. However, most of the pollinators visit staminate and bisexual flowers in their female phase on a plant and therefore, geitonogamous pollen is likely to be more abundantly deposited over the stigma. However, type B plants i.e. exclusively hermaphrodites have very remote chances of getting selfed because when umbels on a peduncle are passing through female phase, their own anthers are already shed and umbels of successive orders are just emerging from the enclosing sheaths and as such cannot offer their pollen for pollination. On the other hand andromonoecious plants present a self sustained strategy of reproductive assurance even if no other plant of the species is present in the vicinity which was the case in isolated plants in some populations. In such plants, synchronization of male phase in staminate umbels on a peduncle with the female phase on the central umbel of the same peduncle ensures supply of self pollen to them. Reproductive output under self and cross pollination is nearly same, so no effect of inbreeding depression upto fruit set was manifest in the species. 109 V.3c Breeding system in P. kurrooa: Understanding of breeding system of this species through floral architecture and floral biology parameters points to its outcrossing nature. However, present observations on compatibility status show that self as well as cross compatibility exists in the species and moreover the three morphoforms (Table IV.35) are mutually compatible. Since, results were not followed upto seed set and their germination, fluorescence microscopy has confirmed that no prezygotic barriers exist in case of inter morphoform crosses also. In all the three morphoforms, the flowers are protogynous and there is a gap of about 15 h between stigma receptivity and anther dehiscence. Moreover, in Morphoforms - 1 and - 3, style and stamens protrude out more than double the length of corolla tube and are directed away from each other leaving no chance for unassisted selfing. In Morphoform - 2 corolla is bilipped and forms a hood and the stamens are arranged in two pairs, of which the upper pair is placed just at the margins of hood petal. Stigma after the maximum elongation of the style is placed just under the hood but as soon as stamens elongate and reach at that level, style takes a sharp bend and moves downwards and inclines to one side in the groove between the lip and lateral petal of that side. Thus, in this case too autonomous selfing is denied. However, some basal flowers in some inflorescences which had their stigmata confined within corolla after anthers had shed pollen present some chance to receive self pollen sticking to corolla. Since populations of these morphoforms are spatially separated due to specific habitat requirement of each morphoform and also insect activity is negligible, the possibility of inter - morphoforms is very low. These 110 morphoforms although inter - fertile, seem to be reproductively isolated and provide suitable conditions for speciation to occur due to fixation of variation generated as well as reproductive isolation. V. 4 Stylar movement in V. wallichii – a contrivance for reproductive assurance and species survival: Typically the flowers of this species have straight style standing erect in the middle of the corolla tube. Also dichogamy prevalent in the species, prevents intra floral autogamy. Stylar bending has evolved as a strategy to ensure pollination and reproductive success under stressful conditions which restrict the pollinator visitation to the flowers. Comparison of pollen load on stigma of female and hermaphrodite flowers towards end of the season when pollinator frequency is reduced, reveals that a significantly higher percentage of stigmata in hermaphrodite flowers bear pollen load than those of female plants. This can be explained on the basis of the phenomenon of stylar bending operating in hermaphrodite flowers which enables them to receive self pollen from anthers of same or neighboring flowers on the same inflorescence. Stylar movement is reported to operate in other plant species too where it promotes outcrossing (Li et al., 2001; Verma et al., 2004), or helps achieve delayed selfing (Sun et al., 2007; Ruan et al., 2009). Reproductive assurance is the final outcome of all these strategies. V.5 Pollination Biology: Pollination in F. jaeshkeana and V. wallichii is insect dependent and as revealed during the present investigation, both species receive abundant pollinator services in natural populations. This is confirmed by the high frequency of stigmata carrying heavy pollen load (Table IV. 26) in natural 111 populations and also by the large number of pollen deposited per stigma in these two species. Since the ovary contains a single ovule in V. wallichii and one ovule in each locule of F. jaeschkeana, even a single viable pollen is sufficient to fertilize the ovule and initiate fruit and seed set in these species. However, the average pollen load per stigma is much more than the minimum requirement. Moreover, mass flowering effect, attractive inflorescences and pollen and nectar as rewards attract pollinators in plenty. Since, most of the populations are separated widely in space, possibility of inter – population pollen flow seems to be remote. The chances of pollen exchange between closely situated populations however, cannot be ruled out. Pollination by a variety of pollinators in these species also reduces the risk of pollen limitation which is the case in many threatened plant species dependent on a single vector for pollination. In any case, pollination in these two species is exclusively insect mediated and may fail altogether if pollinators get eliminated from their habitat range due to some change in climatic conditions, habitat alteration, introduction of alien pollinators or pesticide poisoning (Bond, 1994). The effect of absence or reduction of pollinators was evident in case of V. wallichii through an examination of the percentage of stigmata carrying pollen towards the end of flowering season. Percentage of such stigmata was significantly low as compared to those recorded during peak flowering in case of female flowers. The difference was comparatively less pronounced in hermaphrodite flowers because at least some of the stigmata in these flowers were able to acquire pollen through stylar bending. 112 Pollination mechanism in P. kurrooa, however, could not be understood with certainty. High pollen - ovule ratio as well as floral architecture (long and deep purple corolla tube, densely hairy from inside) indicate the possibility of cross pollination through entomophily in case of Morphoform - 2. On the other hand, in Morphoforms - 1 and - 3, long protruding stamens and style, small, green and unattractive corolla and pollen grains with smooth exine presented some of the features suited for anemophily. Nectar is absent in all the morphoforms. Pollinators are hardly observed in these habitats except for the rare visits by a species of Bombus that too avoids to visit the less attractive, non- scented and sparse inflorescences of P. kurrooa and prefers other larger, fragrant and luxuriantly flowering species of herbaceous annuals or perennials growing in the vicinity. Sparse flowering in this species and absence of rewards suited to the needs of the pollinator may explain why the species is altogether neglected by the floral visitors. Climatic conditions too have a role in keeping the pollinators away. Flowering period of the species is characterized by continuous rain for several days. Low pollinator availability is a common feature of alpine habitats which present harsh climatic conditions (Arroyo et al., 1982; Bingham and Orthner, 1998; Medan et al., 2002; Sieber et al., 2011) and it has been hypothesized that to combat the effects of decreased pollinator abundance, alpine plants have resolved to selfing as a method to assure seed set and in principle selfing rate should increase with altitude (Schroter, 1926; Bliss, 1962; Garcia et al., 2009; Korner and Paulsen, 2009). Some later studies have however, contradicted this view (Wirth et al., 2010). This hypothesis can be analysed in present case. In the species under 113 discussion, all the three morphoforms are self compatible but autonomous selfing is denied due to incompatible floral structure and dichogamy in Morphoforms - 1 and - 3. Both temporal as well as spatial separation of the two sexual phases leaves no chance for self pollen to arrive on the stigmatic surface. In Morphoform - 2 however, flower architecture might assist in selfing but in this case too, dichogamy and downward bending of the styles prior to anther dehiscence deny the stigma to access the self pollen. Here, the stigma is capitate and wet, remains just beneath the outer margin of the hood petal but as soon as the stamens elongate and reach at the same level and dehiscence of anthers begins, styles take a sharp bend and move to one side thus escaping any chance to receive self pollen sticking to hood petals and anthers. This seems a strategy to avoid selfing. However, some flowers on a few inflorescences have their stigmata confined within the corolla tube even after anther dehiscence. These may present a possibility of autonomous selfing. On the other hand, stigmata collected from these habitats showed considerable pollen load which justifies good fruit and seed set inspite of the pollinator limiting conditions. This is a pointer towards some other mechanism of pollination operating in this species and anemophily seems to be the most probable mode under the alpine conditions where strong winds are a characteristic climatic feature. V.6 Pattern of nectar secretion in F. jaeschkeana assures pollinator visits to the flowers in female phase: Presence of andromonoecious plants in natural populations of this species assures adequate supply of pollen from staminate umbels to the flowers of 114 lower umbels going through female phase. But this strategy alone is not enough to ensure pollination until the flowers in female phase having shed their anthers are capable of attracting pollinators. To achieve this, the species has evolved a unique mechanism to ensure pollination by secreting nectar in two phases during the lifetime of a flower. In hermaphrodite flowers nectar secretion begins with and continues till the end of male phase which spans for 50 - 52 h from onset of anthesis. During this phase, both pollen and nectar are presented as reward to the pollinators which visit these flowers in great abundance. As soon as the male phase is over, nectar secretion also stops. Staminate flowers start drying soon after shedding of all the stamens, whereas hermaphrodite flowers undergo stylar movement till they reach a characteristic position when the two stigmata become receptive and this marks beginning of the female phase. During this phase, flowers do not possess any contrivances to attract pollinators. To overcome this constraint, nectar secretion starts again after a gap of about 36 – 40 h as a result of which pollinators visit the flowers with receptive stigmata and effect pollination. During the intermittent period, nectar is reabsorbed as is evident from the fact that even in bagged flowers, stylopodial discs become devoid of nectar during this period. Thus, nectar secretion in two phases has evolved as an important contrivance to ensure the success of pollination consequently adding to the efficiency of reproduction in this species. Nectar resorption is a well known phenomenon in plants having being described in several species (Burquez and Coebet, 1991; Nepi et al., 1996a, b; Davis, 1997; Langenberger and Davis, 2002) and its significance for the plant has been explained variously. Plants utilize a considerable portion of 115 energy in production and secretion of nectar (Pyke, 1991) which may account for as much as 37% of plant’s total photosynthetic energy (Southwick, 1984). Thus the resorption of nectar is mainly considered as a strategy of plants to prevent energy expenditure and also to utilize the materials like carbon for the synthesis of other important plant products like stigmatic exudates (Shuel, 1961) or in seed development (Stpiczynska, 2003). Thus, in the present case too nectar resorption during the intermittent period saves energy expenditure of plant and resumption of its secretion during female phase assures pollination. V.7 Three morphoforms of P. kurrooa in the light of taxonomy of the genus: Taxonomy of the genus Picrorhiza has been confusing since Hooker (1885) first described the genus as monotypic having one species kurrooa with floral dimorphism in respect of corolla and stamens, one form having long stamens and a short corolla tube with 5 equal lobes and the other having short stamens and a long bilipped corolla. Earlier, Royle (1835) depicted the long stamened form as P. kurrooa in an illustration. Pennell (1943), based upon the description by Hooker (1885) and examination of samples collected by Smith and Cave in 1911, from Zemu glacier in Sikkim, bifurcated the genus into two species, P. kurrooa for the form having long stamens found in Western Himalaya and P. scrophulariiflora for the form having short stamens and bilipped corolla found in Eastern Himalaya. Later, the two species were studied in some more detail by Hong (1984), who on the basis of some differences in pollen of the two species suggested to raise two species to the 116 level of two genera naming them as Picrorhiza and Neopicrorhiza, each having a single species kurrooa and scrophulariiflora respectively. Many subsequent explorations made during recent times in Himalayan region by different workers have led to a consensus that Neopicrorhiza scrophulariiflora is confined to Eastern Himalayan region and Picrorhiza kurrooa to the Western Himalayan region (Hara et al., 1982; Chandra et al., 2006; Purohit et al., 2008; Bantawa et al., 2009a, 2010). Based on this history of identification and taxonomy of the genus, the recovery of three distinct morphoforms during the present investigation can be seen as a case of coexistence of the two genera in the same habitat which has not been reported before. Morphoforms - 1 and 2 correspond to P. kurrooa and N. scrophulariiflora respectively as analyzed through a comparison of different morphological features of these with those described for the two genera in literature (Flora of China. Vol. 18). But occurrence of these morphoforms along the altitudinal gradient and their specificity to particular habitat conditions in all the study sites indicates that these morphoforms represent the variants of a single species that have evolved in response to varying habitat conditions encountered by a vegetatively propagating species along an altitudinal gradient. Presence of Morphoform - 3 in the transition zone and having nearly all the features intermediate between the two forms, presents another strong evidence that variation exhibited by P. kurrooa is not abrupt but gradual and has a correlation with change in the habitat conditions while moving along the altitudinal gradient. No difference in pollen of these forms was observed during present study as mentioned by Hong (1984). Also, all the three morphs are inter - compatible and inter 117 fertile without any pre - fertilization barrier although spatially separated. Keeping all these factors in mind, taxonomic status of this taxon needs reviewing and may present an interesting case of species evolution in alpine Himalayas. V. 8 Analysis of genetic diversity in the three species: The amount of genetic variability and its distribution within a species is an important factor to determine its persistence, fitness and evolutionary potential in nature (Maki et al., 1999). Parameters like the total genetic diversity in a species, its apportionment between and within the populations, genetic differentiation of populations and degree of gene flow between them are among the important parameters that are utilized to understand the genetic structure of threatened taxa. Quantification of genetic diversity available in a threatened plant species helps to assess the degree of genetic erosion that the species has undergone and also in prioritizing sites (Godt et al., 1996; Petit et al., 1998) and design strategies for its conservation (Brown, 1989; Ceska et al., 1997; Neel et al., 2003). In the present study, magnitude and distribution pattern of genetic diversity in three species was determined using the parameters mentioned above. In case of V. wallichii and F. jaeschkeana, the analysis was made to calculate interand intra – population diversity and to derive genetic relatedness of different populations of these species. In case of P. kurrooa, the purpose of genetic analysis was to establish the nature of variation observed among the morphology of three morphoforms. To achieve the target these morphoforms were screened for inter - and intra - morphoform variation at molecular level 118 using ISSR markers. Also, RAPD markers were used in V. wallichii and ISSR in F. jaeschkeana. The value of different parameters for all the three species is given in table V. 41. Table V. 41 Average values of different parameters to calculate genetic Diversity in taxa under study. Name of the H taxon. I Total genetic diversity (HT) Proportion of diversity within morphoforms Degree of gene flow (Nm) 0.7421 Proportion of diversity among populations (GST) 0.2579 V. wallichii 0.4027 0.5858 0.4060 F. jaeschkeana 0.2826 0.4320 0.2906 0.7836 0.2164 1.15 P. kurrooa 0.0786 0.1222 0.1908 0.4943 0.5057 0.488 1.35 H – Nei’s genetic diversity index; I – Shannon’s information index The values of Nei’s genetic diversity index (H) and Shannon’s information index (I) for V. wallichii and F. jaeschkeana are very high indicating high genetic polymorphism existing in these two species. Also, the value of gene flow was greater than 1 in both cases suggesting the possibility of interpopulation flow of genetic material through pollen or seeds in nature. This goes in positive correlation with the mating system of the two species, which favours outcrossing, accomplished through the intervention of insect pollinators. Thus, insect mediated migration of genetic material in the form of pollen may be responsible for the pattern of polymorphism and gene flow observed in this case. Moreover, occurrence of species in the form of clusters with a number of plants growing close to each other within a small area is also a feature that promotes successful outcrossing besides the occurrence of dichogamy as the main contrivance. The occurrence of inter - population gene 119 flow leads to little differentiation among populations, consequently, in such species the proportion of genetic variation existing within population is more than that occurring between the populations (McDermott and McDonald, 1993). This is clearly evident in these two species, where respectively 74.21% and 78.36% of total genetic diversity is present within a population. The values of apportionment of genetic diversity as well as gene flow suggest that two taxa (V. wallichii and F. jaeschkeana) go in line with other outcrossing species (Hamrick and Loveless, 1989; White and Powell, 1997; Hamrick and Godt, 1996; Verma et al., 2007). Total genetic diversity of the three species presently worked out when compared with some other rare and endangered plant species (Table V. 42) falls at the lower end of that range. Should the genetic diversity between V. wallichii and one of its sister species V. ciliata (Faivre et al., 2002) be compared, it becomes evident that the extent of genetic erosion in the former species is considerably lesser than the later and conservation efforts if undertaken timely can save it from further loss. Results of AMOVA in V. wallichii also show that within population diversity was high but among population diversity was also statistically significant. This is quite in agreement with other reports on the genetic diversity analysis of this species undertaken by different workers on some other populations using AFLP and ISSR markers (Rajkumar et al., 2011; Jugran et al., 2013). FST distances between the present populations of V. wallichii ranged from 0.1957 (between Kund and Cha) to 0.4711 (between Thajwas and Sappanwali). Neighbour Joining tree showed Thajwas population to be genetically most distant of all the other populations. Its distinctly different genetic architecture 120 can be ascribed to the unique habitat that this population inhabits. Small size of this population may also be responsible for this pattern of genetic diversity. The dendrogram revealed two major clusters, one comprising Chakrata and Almora populations and the other comprising the rest. Chakrata and Almora are most distantly located of all the collection sites. This distinctness is also manifested through a difference in Table V. 42 Hsp values obtained in rare and threatened taxa using RAPD, AFLP and Allozyme markers S. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Species Swietenia macrophylla King. Fitzroya Cupressoides (Molina) Johnston Rosmarinum tomentosus Huber- Morath et Maire Araucaria aracana (Molina) K. Koch Valeriana ciliata T.&G. (Prairie Valerian Antirrhinum subbaeticum Guemes, Mateu et sánchez Gómez Terminalia amazonica (J.F. Gmel.) Exell. Eremostachys superba Royle ex Benth Citrus hongheensis Y. M. Ye et al. Panax ginseng C. A. Meyer Dactylorhiza hatagirea D. Doon Rhodiola imbricata Edgew. Hippophae rhamnoides ssp turkestanica Hippophae salicifolia D. Don Hippophae tibetana Schlecht HT value 0.45 0.54 3.588 0.689 0.245 1.008 Reference Gillies et al.,1999 Alnutt et al., 1999 Martin and Bermejo, 2000 Bekessy et al., 2002 Faivre et al., 2002 Jiminez et al. 2002 0.379 0.478 0.3535 0.2886 0.253 0.359 0.303 0.106 0.097 Pither et al., 2003 Verma et al., 2007 Yang et al., 2010 Li et al., 2011 Warghat et al., 2012 Gupta et al., 2012 Raina et al., 2012 Raina et al., 2012 Raina et al., 2012 morphological features observed in the plants of these two accessions which produce stolons during flowering season unlike any other population investigated. Interestingly however, Mantel test revealed non significant correlation between genetic and physical distances (r = -0.01376). Obviously, genetic distances between populations other than Chakrata, Almora and Thajwas were not in correlation with their physical distance, which led to non significant results in the mantel results. 121 In case of F. jaeschkeana, the dendrogram revealed that populations, Drass proper and Bhimbhat are genetically distant from other populations of Drass and Kashmir which cluster together. The Sonamarg population from Kashmir appears to be related closely to three populations of Drass region generating the possibility that this population established in Kashmir by means of the propagules dispersed from Drass population. Some nomadic tribes with their livestock migrating between these regions might be responsible for this dispersion. The period of their migration also coincides with the flowering season of the species. The pattern of genetic variation observed in different morphoforms of P. kurrooa was unlike the one expected for outcrossing species. The values of Shannon’s index (I) and Nei’s genetic diversity index (H) were respectively 0.0786 and 0.1222 which indicate low level of genetic polymorphism in this species. Also the low value of gene flow (NM= 0.488) indicates the remote possibility of exchange of genetic material among three morphoforms of this species. This pattern of genetic diversity can be aptly explained on the basis of observations made regarding the population structure and mating strategies of the species. The three morphoforms have strict habitat preferences and are spatially separated at each site. Non availability of pollinators is also a limiting factor in the exchange of genetic material among the populations of these morphoforms. Moreover, temporal separation in the flowering time of these morphoforms located at different altitudes further eliminates any chance of exchange of gene flow between them. Genetic exchange through seeds is also not a common phenomenon in natural populations as the fruit and seed dispersal is very poor and even if dispersal takes place seeds are not capable of spreading to distances far away from the parent populations. Lack of inter - morphoform gene flow is also reflected 122 through high value of population differentiation and relatively low genetic diversity apportioned within the populations (49.43%) than that between the populations (50.57%). The low values of H and I also point to inbreeding in this species. It prolifically multiplies through vegetative means and spreads luxuriantly, forming large patches in nature. Frequency of flowering is also very low and chances of pollination between spikes originating from the same plant are more, making geitonogamy the most prevalent breeding strategy. Variation observed in the vegetative and floral features of the three morphoforms also seems to be genetically fixed which is evident from a certain degree of polymorphism observed between these morphoforms. Thus, morphoforms appear to have originated as a result of adaptive changes in response to diverse range of habitat conditions which the species has encountered during the course of time while spreading to different parts of Himalayan mountain slopes and acclimatizing to microhabitat conditions there. Such broad scale ecological adaptations can contribute to changes in nuclear DNA, and thus become genetically fixed, producing a number of diverse populations varying in morphological features, growth habit, physiological polymorphism in flowering period and adaptability to grow in various soil conditions (Rongsen, 1992; Singh et al., 1995; Dwivedi et al., 2001; Singh, 2003). The dendrogram depicting the relatedness of three morphoforms of P. kurrooa shows that Morphoform - 2 is diverged from Morphoforms - 1 and - 3 which show a closer association. This pattern is also comparable to the level of morphological variation among these. Based upon this analysis, the three morphoforms more probably appear as three ecotypes of P. kurrooa rather than distinct species. The analysis is thus an important initiative to understand the complex taxonomy of this taxon. 123