Incidence and frequency of desynapsis in Eremurus persicus (Jaub. & Spach) Boiss. (Asphodelaceae) – A native and important medicinal plant species of Western Himalaya

. Eremurus persicus (Jaub. & Spach) Boiss. (Asphodelaceae); a little known species of genus Eremurus grows in arid and semi-arid regions, on rocky mountains in Central Asia and Middle East. The species is native and an important medicinal plant used to treat various diseases. In the current study, we examined male meiosis, karyotypic variability, pollen viability and reproductive output of three populations of the target species. All the studied populations have diploid chromosome count of 2n = 2x =14. Meiotic course in majority of the pollen mother cells (PMCs) in all the three populations is not normal due to the presence of desynapsis at diplotene and meta-phase-I. Instead of 7IIs most of the PMCs examined reveal varying number of biva-lents and univalents. Anaphasic segregation is also affected in few PMCs. Present study also revealed that the species has asymmetric karyotype of 10 long and 4 short chromosomes. Species exhibits fruit abortion that results in extremely low fruit/seed set. The meiotic abnormalities and low fruit/seed set may be attributed to restricted distribution of this important plant species. Present study may prove helpful in devising the conservation and management strategies for this prized plant species.


INTRODUCTION
Pairing of chromosomes is the first step that leads to the organized segregation of chromosomes during meiosis and is the basis of continuity of life (Fabig et al., 2020).In addition being the first step of reduction division; this is also important as it leads to crossing over and reshuffling of alleles and contributes to the generation of genetic variations (Cai & Xu, 2007).Though it is religiously followed in stable diploid species during zygotene, instances where the deviation from norms occurs are not rare.Chromosomes in some taxa fail to pair (asynapsis) (Jakson et al., 2002;Ansari et al., 2022) or their pairing breaks precociously (desynapsis) (Golubovskaya,1979;Wani & Bhat, 2017).These anomalies arerecognized when most or all the chromosomes remain as univalents at diakinesis and metaphase-I (Visser et al.,1999).Although theoretically the phenomenon of asynapsis & desynapsis are distinct from each other, it is difficult to distinguish them in practice.This is true for those plants where chromosomes are long and pairing cannot be followed during earlier stages (Koul, 1962;Wani & Bhat, 2017).
As a characteristic of a particular variety or ecotype, desynapsis has been reported in several plant genera viz Zea (Beadle, 1933); Nicotiana (Clausen, 1931); Datura (Bergner et al., 1934); Gossypium (Beasely and Brown, 1942); Triticum (Li et al.,1945), Alopecurus (Jhonson, 1944) etc.But as an inherent feature of a taxa as a whole; it is a rare occurrence.Here we report this phenomenon in Eremurus persicus (Jaub.& Spach) Boiss.Described as a feature of meiosis in few plants of this species by Verma (2001) as well, it was found to be a regular feature of this plant species across all the plants in all populations scanned in present study.
Genus Eremurus M. Bieb. is commonly known as Foxtail lilies/ Desert candles of family Asphodelaceae comprises of more than 60 species that are native to Central Asia and Caucasia (Kamenetsky & Akhmetova, 1994).The species of the genus can be easily recognized by their long scapes bearing numerous colourful flowers along with rosette of leaves.Most of the species of this genus are important commercially as ornamental plants for landscaping and cut flower markets (Schiappacasse et al., 2013).In addition to their ornamental value, Eremurus sp. have been used in traditional medicine and are potential sources for anti-inflammatory, anti-bacterial and anti protozoal drugs (Hashemi et al., 2014 ;Mati et al., 2011).Also bio-oil and adhesives prepared from this genus play an important role in industrial applications (Vala et al., 2011).In India Genus Eremurus is represented by only two species viz Eremurus himalaicus Baker and Eremurus persicus (Jaub.& Spach) Boiss.
Eremurus persicus is a perennial herb which perennates through underground fleshy tuberous rootstocks that sprout every year in the month of February-March in the sub-tropical areas of Jammu, UT of J&K, India.The above ground portion of the plant appears in the form of rosettes of leaves which are light to dark green in colour, radical, thick and lanceolate.The inflorescence is raceme which bears flowers in an acropetal manner.Flowers are creamish to light pink in colour, pedicellate, bracteate, bisexual and glabrous.Species holds immense value for its medicinal attributes in addition to being used as a vegetable for its edible leaves.The species is widely distributed in South, East and West of Iran where it is locally known as Sarish (Karl, 1982;Safar et al., 2009;Vala et al., 2011).It is also reported from Kibri Kuch Ziarat of Pakistan (Khan et al., 2011).
In India there are scanty reports of the distribution of the species, however; the species is growing in hilly terrains of Jammu province (Hamal & Karihaloo, 1983).Extensive field surveys, perusal of relevant literature and herbarium studies revealed that the plant species has a restricted distribution in the study area.Only few populations (7 populations) of the species have been located from district Reasi of J&K.Keeping in view the rare distribution and medicinal importance of the target plant species the present study was carried out to provide detailed meiotic, mitotic account, pollen viability and fruit/seed set of E. persicus.The knowledge generated in present study may prove helpful in devising conservation strategies for this prized plant species.

MATERIAL AND METHODS
A total of 7 populations of E. persicus were located during present investigation, which include: Slal, Kharjala, Slal Kotli, Sarmega I, Sarmega II, Ponsli I and Ponsli II (Figure 1 and 2).Of these three populations viz.Slal (Population-I), Sarmega I (Population-II) and Ponsli I (Population-III) were selected for detailed meiotic, mitotic investigation, pollen viability and seed set in the present study.Materials for male meiotic studies were collected from wild plants growing on rocky slopes of hilly areas of Trikuta hills of Reasi.Young unopened flower buds of suitable sizes were collected randomly from different plants of each population during the peak flowering period, i.e., March-April, when minimum and maximum temperature of this area averages 13-25°C.The floral buds were fixed in Carnoy's fixative for 24 h.Subsequently, the materials were transferred in 70% ethanol and stored under refrigerator at about 4°C until analysis.For meiotic preparations, smears of the fixed anthers were made in 1% propiocarmine.Photomicrographs of Pollen Mother Cells (PMCs) for chromosomal counts at different stages and meiotic irregularities were made from the freshly prepared slides using EVOS XL microscope.Chiasmata number was counted for cells at diplotene.Data was used to calculate Recombination Index using the following formulae RI= Chiasmata frequency per cell + haploid chromosome number of the species For Karyological studies, healthy root tips from seedlings were collected and washed with distilled water.These were pre-treated with 0.3% colchicines solution for 4 hours and fixed in acetic acid: ethanol (1:3) for 24 hours.Thereafter, the root tips were transferred to 70% ethanol for further use.For mitotic chromosomal preparations, preserved root tips were washed with water and hydrolyzed in 1 drop of 1N HCL and 9 drops of 1% aceto-orcein at 60°C for 12-15 min.Thereafter these root tips were squashed in 1% acetocarmine.Karyotype formula was determined according to Levan et al., 1964.Karyotypes were compared using Stebbins classification, karyotype asymmetry index (As.K %) (Arano, 1963), total form percent (TF %) (Huziwara,1962), Rec and Syi indices (Venora et al., 2002), intrachromosomal symmetry index (A1) and interchromosomal asymmetry index (A2) (Romeo- Zarco, 1986), dispersion index (DI) (Lavania & Srivastava, 1999), degree of asymmetry of karyotype ( A index) (Watanabe et al.,1999), asymmetry index (AI) (Paszko, 2006), the coefficient of variation of chromosome length (CV CL ) and the coefficient of variation of centromeric index (CV CI ) (Paszko, 2006) and mean centromeric asymmetry (M CA ) (Peruzzi et al., 2009).The strength of the association between karyotype asymmetry indices was tested using Pearson correlation analysis using PAST software.
To calculate number of pollen grains per anther and/ flower, 30 flowers ready to open with intact anthers were selected.Pollen quantity was estimated by squashing one anther (several times) in 10 drops of distilled water in a cavity block and shaken with a glass rod.
The following equations were used to calculate the number of pollen per flower: where p is the mean pollen count per drop of water; q is the number of water drops taken initially in which one anther was squashed; r is the mean number of pollen per  anther; s is the mean number of anthers per flower; t is the total count per flower (Ganie et al., 2021).
For calculation of pollen count per flower the average pollen number in one anther was multiplied by total number of anther per flower Average ovule number per pistil was counted using dissection microscope (Ganie et al., 2021).
Pollen viability was determined by subjecting the pollen grains to 1% acetocarmine, TTC and FDA.In first method, mature anthers were squashed in 1% acetocarime and kept for 10 minutes and then slides were scanned under microscope, well stained and plump pollen grains were considered viable while as lightly stained and shriveled pollen grains were considered as non-viable.In second method, the ready-to-dehisce mature anthers were placed in 1% tetrazolium chloride for one hour and squashed to check for viability (Rashid et al. 2023) and in third method pollen grains were mounted in Fluorescein diacetate (FDA) solution and incubated for 3-5 min.The pollens with fluorescent and non-fluorescent cytoplasm were treated as viable and nonviable, respectively (Rashid et al. 2023).In all the three methods, percentage of pollen viability was determined by following formula:

Pollen viability = Number of pollengrains stained X 100 Total number of observed pollen grains
To check the reproductive efficiency on open pollination, 20 different inflorescences were tagged and kept for pollination to take place as it does in nature.These were monitored regularly from the time of flower opening to that of fruit maturation.These flowers were observed after some days to check the percentage fruit set.Percentage fruit set on unassisted selfing was performed by bagging large number of buds from 10 different inflorescences in transparent butter paper bags.Total number of fruits as an estimate of female success was calculated.Mature fruits were harvested shortly before dehiscence and scored for the presence of seeds.Percentage fruit set and seed set was then calculated by using Bharti et al., 2021 method.RESULT AND DISCUSSION

PMC meiosis
All the three populations of E. persicus matched in having 14 chromosomes in their PMCs revealing 2n= 2x = 14.PMCs were analysed at diplotene, diakinesis, metaphase-I and anaphase-I.Desynapsis was observed as a regular feature in majority of the cells at diplotene and all the cells at Metaphase-I.Meiotic behavior of three populations differed slightly and the meiotic behavior of the studied populations is given as under:

Population-I (P-I)
In P-1, a total of 97 cells were scanned at different stages of meiosis with 26 cells (26.80%) at diplotene, 15 cells (15.46%) at diakinesis, 39 cells (40.20%) at metaphase-I and 17 cells (17.52%) at anaphase-I (Figure 3 a-e).Out of 26 cells at diplotene only 2 cells were having perfect 7IIs (Figure 3 a).Of observed bivalents, 5 bivalents/chromosomes were large and 2 were small.Interestingly in these bivalents, chromosomes were mostly held by terminal chiasmata only.At metaphase-I, cells were observed with different configurations and with varying number of univalent and bivalents in all PMCs.It has also been observed that 38.46% of cells showed all the 14 chromosomes as univalents (Figure 3 b) while 25.64% of cells showed the presence of 2IIs along with 10Is.Other configurations like 1,3,4 & 5 IIs were also observed.The univalents at this stage are arranged close to each other.At anaphase-I, PMCs usually showed normal segregation of 7:7 chromosomes at each pole (Figure 3 c-e).Chiasmata frequency per PMC calculated at diplotene in this population is 9.15 while RI calculated is 16.5.

Population-II (P-II)
In P-II, 91cells were observed, out of which 33 cells (36.26%) were at diplotene, 37 cells (40.65%) were at metaphase-I and 21cells (23.07%) were at anaphase-I (Figure 3 f-i).In this population, formation of 7IIs was lacking even at the diplotene stage.At this stage only few II's were held by terminal chiasmata.At metaphase-I varied number of IIs and their configurations were observed, in addition varied number of Is were also observed in all the scanned PMCs.At this stage also chromosomes in IIs were associated terminally only.In 51.35% of cells all the 14 chromosomes as Is were observed (Figure 3 g-h) followed by 3,2&1 IIs along with Is.However, at anaphase-I usually normal segregation of 7:7 chromosomes was observed (Figure 3 i).Chiasmata frequency per PMC calculated at diplotene in this population is 6.9 and RI thus comes out to be 13.9.

Population-III (P-III)
In P-III, a total of 318 cells were observed at different stages, 45 cells (14.15%) at diplotene (Figure 4  Erratic distribution of 7:6, 6:6 and 6:5 chromosomes at each pole was observed in 7 cells (2.20%), 8 cells (2.51%) and 13 cells (4.08%) respectively.Also 27 cells (8.49%) showed presence of laggards and chromosomal bridges along with clumping of chromosomes (Figure 4 f-m).At anaphase-I and anaphase-II the formation of laggards and bridges was observed as a common feature.Chromosomal bridges were observed at metaphase-II also.All the cells scanned at metaphase-II and anaphase-II showed the presence of laggards and chromosomal bridges (Figure 5 a-d).Therefore the second meiotic division exhibited drastic irregularities in this population.Chiasmata frequency per PMC calculated at diplotene in this population is 5.3 and RI is 12.3.
Detailed studies on meiotic system revealed that in the species, majority of the PMCs at diplotene, diakinesis and metaphase consist of univalents ranging from 2-14 in all the three studied populations.At diplotene loosely bound IIs are seen mostly with terminal and rarely with intercalary chiasmata.As meiosis proceeds, intercalary chiasmata are not seen in any of the bivalent and the frequency of univalents goes on increasing.At later stage few bivalents and more frequency of univalents are observed.Subsequently the chromosomes fall apart and remain as univalents during rest of the cell cycle till metaphase-I.In most of the PMCs univalents were found to lie in pairs (distant pairing) were also observed in the present study.The frequency of univalent and bivalents observed in three populations are depicted in Figure 6.

SOMATIC CHROMOSOME COMPLIMENT
All the three populations studied show chromosomes in their somatic compliment confirming 2n= 2x = 14 as the diploid chromosome number of the species.Th ese fall into two size groups.Group I includes 10 long chromosomes, all of them being subterminal while group II includes 4 small chromosomes which are submedian and subterminal.Th e total chromatin length of the somatic compliment for population I, II and III is 160.2, 158.4 and 168.3 µm.Th e kayotype formula for the three populations is 10ST+2ST+2SM, 10ST+3SM+1ST and 10ST+1ST+3SM respectively (Figure 7).Th ese populations thus varied slightly in their karyotypic formulae too, although the chromosomes were of subtelocentric and submetacentric type only.Th ese fallin 3C category of the Stebbin's (1971) chart of asymmetry.Among all the populations studied, P-III possessed the highest value (17.6 µm) of longest chromosome while P-I (15.54 µm) possessed the lowest value for the same.Th e highest and lowest values for the smallest chromosome occurred in P-III (4.4µm) and P-II (4.95µm) respectively (Table 1).Th e most asymmetrical karyotype was observed in P-III (Table 2) P-III possessed the highest value for CV showing the highest variation among its chromosomes compared to the other populations.Pearson coeffi cient of correlation determined for karyotypic parameters among the populations studied revealed a high correlation signifi cant at p≤0.05 (Table 3).Th e AI value and scatter diagram based on the CV CL and CV CI seem best suited to assess overall classifi cation strength and display relationships among E.persicus populations with respect to karyotype asymmetry (Table 4; Figure 8 and 9).
Cluster analysis of the populations of E. persicus based on relative karyotypic data revealed that P-I and P-II are placed close to each other while P-III joined them with a distance (Figure 10).However, there is morphological similarity between them but due to karyotypic diff erence it stands separate from the rest of the two populations.Th is is also in accordance with their meiotic studies where P-III showed high variation in comparison to P-I and P-II.According to the As.K %, TF% and Syi and Rec indices P-III is more asymmetric than P-I and P-II.

POLLEN COUNT AND VIABILITY
Pollen grains of E. persicus are ovate, smooth walled.These are shed at two celled stage.Highest pollen production per anther is observed for POP III ; 6619.4±381.46.Different anthers in a flower display some difference in pollen content, so that the average pollen output per flower comes out to be 38506.6±2298.49(Table 5).In POP II, pollen production per anther is averaging 6252.9±361.03and total pollen production per flower comes out to be 37517.4±2166.22.Pollen production per anther averages 5428.36±351.13 in POP I and total pollen production per flower comes out to be 32570.2±2106.79.
Highest pollen stainability by 1% acetocarmine was observed in population II followed by population I and III.However pollen viability by TTC and FDA averages 89.61±0.26 and 90.78±0.53 in population I, 90.53±0.17and 92.61±0.82 in population II and 90.40±0.33 and 91.74±0.61 in population III by TTC and FDA respectively(Table 5 and Figure 11).

REPRODUCTIVE OUTPUT
All the three populations have very low reproductive output in terms of % fruit set.The plants in their natural populations (open pollination) showed 19.00±3.19,20.21±2.45 and 18.62±3.46% of fruit set in P-I, P-II and P-III respectively.Bagged inflorescences do not produce seeds in all the selected populations.Also  The present study revealed that Eremurus persicus is diploid with chromosome number of 2n = 2x = 14.The karyotype analysis revealed that various species of the genus Eremurus have 2n chromosome counts of 14 and 28 (Hadizadeh et al., 2020).Somatic chromosome analysis of eight species of Aloe (Asphodelaceae) showed diploid chromosome number of 2n = 14 (Sánchez-Get al. 2018).The other species of the genus growing in India, i.e., E. himalaicus is also diploid with chromosome num-ber of 2n = 14 (Kumari et al., 2016).In present study meiotic abnormalities were found in the target species, similarly meiotic abnormalities were also found in E, himalaicus growing in Indian Himalayan region (Kumari et al., 2016).The abnormalities are attributed to pervading environmental conditions in the Himalaya (Wani et al. 2023).The phenomenon of desynapsis was also found in the PMCs of presently studied plant species, both extrinsic conditions such as temperature and fertilizer quality (Dhesi et al., 1975;Rao, 1975) and intrinsic conditions like gene action, loss of chromosome pair, apomixis and structural or numeral changes of chromosomes (Praaken, 1943) are responsible for the desynaptic behavior of plants.Over the years desynapsis has been established as a gene mediated phenomenon in a large number of plant species (Gottschalk & Baquar, 1971), however; the precise mode of action of these genes is not fully understood.Of the three types viz weak, medium and strong or complete desynapsis (Prakken, 1943); in present study medium desynaptic type was found in the target species because of the presence of many univalents and few loose bivalents.In spite of the lack of pairing and the formation of univalents as a consequence of desynapsis, anaphase segregation is normal in majority of cells which needs further investigation.The P-III displays irregular distribution of chromosomes with maximum frequency.Other meiotic irregularities include formation of laggards and bridges at anaphase-I, metaphase-II and anaphase-II.An interesting observation made in the present study revealed the presence of univalents of a pair close to each other, as also the regular anaphasic segregation point towards the species showing "distance pairing" in which the homologs lie  side by side but apparently do not touch.Same has been observed in Human oocytes also (Thermann & Sarto, 1977).Low recombination index of the species due to lack of adequate crossing over can limit genetic variation which may ultimately compromise with the survival of the species (Szczecińska et al., 2016).The species has asymmetric karyotype of 10 long and 4 short chromosomes.The TF% and Syi and Rec values decreases with increasing asymmetry, while values of others i.e A1 and A2, A and CV CL increase with increasing asymmetry (Zuo& Yuan, 2011) as observed in the present study.
The meiotic abnormalities observed in E. persicus may be attributed to high fruit and seed abortion rates.Most conservation biologists believe that before setting up a conservation program for plant species it is better to know about its intrinsic reproductive constraints (Friedman & Ryerson, 2009;Wani et al,. 2023).The presently investigated plant species are already facing threats such as habitat fragmentation, overexploitation, and grazing, therefore, the presence of meiotic bottlenecks can add to the factors that lead to a decrease in its population size.Therefore, keeping in view the meiotic abnormalities and anthropogenic threats sustainable conservation strategies are needed to devise for this medicinally important plant species.

Figure 7 .
Figure 7. Somatic metaphasic spread of P-I, P-II and P-III showing 14 chromosomes and the karyogram of the same.( Scale = 10µm).

Figure 10 .
Figure 10.Dendrogram for P-I, P-II and P-III.

Table 1 .
Karyomorphometric analysis of somatic metaphasic chromosomes complement of three populations of E. persicus.

Table 2 .
Levan et al. 1964 according toLevan et al. 1964and characteristics of the studied species SC -the shortest chromosome length; LC -the longest chromosome length; p -mean length of long arm; q -mean of short arm; CL -mean of chromosome; CI -mean centromeric index; ST-subtelocentric; SM-submetacentric; SD-standard deviation.

Table 3 .
Pearson correlations for asymmetry indices.

Table 4 .
Karyotypes of E. persicus using different methods of evaluating karyotype asymmetry.

Table 5 .
Data on Pollen count and pollen viability.