Download chap-5 - Shodhganga

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
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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
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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
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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
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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
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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).
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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
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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).
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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
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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
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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
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(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.
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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
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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
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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
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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
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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.
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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
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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
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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.
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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
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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
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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
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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.
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