Resource allocation in relation to meiotic system in two species of Artemisia from North-West Himalayas

Abstract

Artemisia scoparia Waldst & Kit and Artemisia nilagirica (C. B. Clarke) Pamp. are two economically important species of Artemisia L. (F: Asteraceae) forming distinct populations in subtropical to subtemperate regions of Jammu division in Jammu and Kashmir state, India. Both these species have perennial root stocks through which they produce aerial offshoots each year and spread vegetatively. Species differ in their sex expression as well as in reproductive output through sexual reproduction. A probe into their genetic system revealed them to be matching in breeding system as both were predominantly outcrossed. However they differ markedly in their meiotic system and reproductive effort (both sexual and vegetative). The differences were both inter as well as intraspecific. Fruit set in plants of Artemisia scoparia is high and plants are perfect diploids with 2n = 16 (n = 8). Fruit set in A. nilagirica is comparatively low. It also exhibits interpopulation chromosomal variability in the form of polyploid and aneuploid races. In A. nilagirica, two different chromosome numbers which were predominant are 2n = 34 and 2n = 54. Species are also variable in their reproductive effort. The population with lower reproductive output through sexual reproduction has a higher investment in rootstock and vice versa. This paper tries to elaborate the variability in chromosome complement, reproductive output and reproductive effort of these species.

Keywords:

• Artemisia
• multivalent
• outcrossed
• polyploidy
• reproductive effort
• resource allocation

Introduction

Artemisia L., the largest genus of tribe Anthemideae of family Asteraceae, has a high number of taxa of both ecological and economic importance. Because of its diverse nature, this genus has been controversial taxonomically. Based on inflorescence architecture alone, five large sections of this genus are recognised. These include Absinthium DC, Artemisia L., Dracunculus Besser, Seriphidium Besser and Tridentatae (Rydb.) McArthur (Nazar and Mehmood 2011). In India, 27 species of Artemisia have been reported, of which 10 are common in Jammu division of Jammu and Kashmir, India (Kaul and Bakshi 1984). The present study is based on the details of reproductive effort, reproductive output and the meiotic system of two of these species, A. nilagirica (C. B. Clarke) Pamp. and A. scoparia Waldst & Kit. The choice of the species was based on the fact that they belong to different sections of genus Artemisia and were different in their sex expression and reproductive output through sexual reproduction (Sharma et al. 2014). Both these species have a perennial rootstock, which is used both for survival during harsh weather as well as for multiplication. This study was designed (a) to correlate the reproductive output with the meiotic system of the species; and (b) to check for a possible trade-off between sexual reproduction and ramification by rootstock.

Materials and methods

Different populations of A. scoparia and A. nilagirica were tagged in Jammu region of Jammu and Kashmir, India; while four different populations of A. scoparia could be localised only at lower altitudes ranges (280–327 m asl), populations (n = 5) of A. nilagirica occurred at higher elevations too (327–1152 m asl). Many observations on these plants were made at natural sites. Young plants in vegetative state were also collected randomly from all these sites and transplanted in experimental plots in the Botanical Garden at Jammu University. Plants came to bloom in the year of transplant itself and these were tagged for detailed studies. The data included in this compilation spans studies conducted over four flowering seasons of 2010–2013. Herbarium Specimens of the species are available in Jammu University herbarium as HBJU-14071, 14072 and 14073.

Plant morphology

Plants were studied for gross and floral morphology in the field. Details on morphological features e.g. number of offshoots per plant, plant height, length of inflorescence and total number of florets (ray and disc) per inflorescence were studied from the live plants in the field itself. Floral structure of the plants was studied in detail under stereomicroscope SM Z 800 (Nikon, New Delhi) in the laboratory.

Anthesis and anther dehiscence

For anthesis and anther dehiscence, tagged plants were observed in the field every day. During the period of bloom, flowers were regularly monitored throughout the day so as to record the time of peak anthesis. Time taken by a flower, single inflorescence and the full plant to bloom was recorded seperately. To check anther dehiscence and pollen shedding, flowers were monitored at different timings before and after anthesis.

Pollen viability

In order to study pollen stainability, freshly dehisced anthers were squashed in 1% acetocarmine. Filled and deep red pollen grains were considered stainable/viable and shrivelled and weakly stained grains were taken as non-stainable/nonviable.

Fruit set

Fruit set was observed in the plants growing open in the field. Number of flowers per capitulum and number of achenes set per capitulum were counted for these plants.

Fruit set on unassisted selfing was checked by bagging single as well as (9–10) inflorescences with butter paper bags of appropriate sizes. These were later checked for fruit and seed set by geitonogamous pollination.

Since all the flowers in A. nilagirica bear ovules, the percentage fruit set both on open pollination and bagging was calculated as per Sharma et al. (2014).

In case of A. scoparia, the percentage fruit set was calculated by using a slightly altered formula (as only ray florets have ovules in this species). The formula used by us presently is: (1)

Resource allocation

Resource allocation studies were carried out by calculating dry biomass (Cumraswamy and Bawa 1989; Sharma et al. 1999). For this, open pollinated plants of both species i.e. A. scoparia and A. nilagirica (both populations) were randomly selected from open fields, when they were in full bloom. Each plant was uprooted and separated into various parts: rootstock, main stem, branchlets and flowers and fruits. In A. nilagirica dry biomass of leaves was also taken. Each of these parts was oven dried at 60°C for about 24 hours and weighed separately on a K-16 analytical single pan electric balance. Reproductive effort was calculated as per Sharma et al. (1999). The resources allocated to the rootstock were estimated as vegetative reproductive effort (VRE).

Cytology

Pollen mother cell meiosis was studied from young immature flower buds fixed during morning hours in a mixture of three parts of ethyl alcohol and one part of acetic acid. After fixation for 24 hours, the buds were washed in water and preserved in 70% ethyl alcohol at 4–6°C. Finally, anthers were squashed in 1% propiocarmine. Chromosome preparations were made permanent by removing the paraffin ring and inverting the slides in a Petri dish containing 1:1 mixture of n-butylalcohol and acetic acid. The slides were removed only after the coverglass was detached. Both the slide and coverslip were then transferred to a Petri dish containing n-butylalcohol. The slides were removed after 2–3 minutes and coverglass was restored using euperol. All the photomicrography was done by using a Zeiss Primostar photographic unit (Zeiss, New Delhi) fitted with Axio Cam ERc 5 s.

Results

Plant and floral morphology

Artemisia scoparia and Artemisia nilagirica are aromatic shrubs with perennial rootstocks enabling them to perennate during winter and sprout in March–May to produce aerial offshoots. They are present in the subtropical to subtemperate climes of Jammu and Kashmir, India. From each rootstock, a single aerial offshoot arises each year. The offshoot is larger in A. nilagirica, where it attains an average height of 4.1 m; in A. scoporia it has an average height of 1.78 m (n = 50 for both the species). The stem tends to be highly branched in both species and bears green pinnatisect leaves.

Flowers are borne in axillary spikes terminating in heads (Figure 1A, and 1B). Ray florets in both species are pistillate, while the disc florets are apparently hermaphrodite (Figure 1C). In disc florets of A. scoparia, the pistil is sterile. It comprises of a discoid stigma, elongated style and rudimentary empty ovary (Figure 1D). A. scoparia is thus structurally gynomonoecious but functionally monoecious. Disc florets of A. nilagirica bear a fertile pistil, this species is thus structurally and functionally gynomonoecious (Figure 1E). Hermaphrodite flowers in both these species are highly protandrous with anther dehiscence preceding stigma receptivity by 4–5 days. Also ray and disc florets open on different days with an average gap of 3–4 days between them. The sequence of anther dehiscence and stigma receptivity reveals both species to be predominantly outcrossers.

Figure 1. Flowers and seeds of A. nilagirica and A. scoparia. (A) Capitulum of A. scoparia; (B) capitulum of A. nilagirica; (C) ray florets of Artemisia; (D) sterile pistil of A. scoparia; (E) fertile pistil of A. nilagirica; (F) seeds of A. scoparia; (G) seeds of A. nilagirica.

Reproductive output

Achenes in A. scoparia are produced only by ray florets, while in A. nilagirica both disc and ray florets produce them. Two types of achene are produced by each species. These have been classified as healthy and shrivelled. Healthy achenes look plump and are capable of germination while the shrivelled ones do not show any germination. The relative proportion of these two varies in the species.

Fruit set is highest in the populations of A. scoparia where it averages 74.6% on open pollination with 40.5% of these fruits being healthy (Figure 1F). In Rajouri populations of A. nilagirica the fruit set on open pollination averages 51.2%, with the majority of these, 46.7%, being healthy, whereas in the Jammu population fruit set averages only 21.38% with only a small fraction (2.6%) being healthy (Figure 1G). No fruit set occurred in all these species, when a single capitulum was bagged, revealing lack of intra capitulum fertilization. Bagging of 9–10 inflorescences together on different individuals resulted in an average of 65% fruit set in plants of A. scoparia and 39.4% and 20% in plants of Rajouri and Jammu populations of A. nilagirica. In both the species, all the achenes set in all the bagged inflorescences were shrivelled and did not show any germination on moist filter paper in laboratory conditions. Germination of healthy achenes averaged 63% and 35.4% in A. scoparia and Rajouri population of A. nilagirica (n = 500). In Jammu population nil germination was recorded as even the healthy achenes failed to germinate (n = 500).

Estimation of the sexual reproductive effort (SRE) by dry biomass allocation method reveals highest values (32.3%) in A. scoparia (Table 1). Of the two populations of A. nilagirica, the Rajouri population had higher value (21.6%) than that of Jammu population (14.29%). Dry weight of rootstock taken as vegetative reproductive effort (VRE) revealed the highest value in Jammu population of A. nilagirica (29.35%). Rajouri population of this species had lowest VRE, i.e. 8.87%, while in A. scoparia the values were intermediate (16.71%) (Table 1).

Table 1. Data on pollen viability, seed set and reproductive effort.

	Treatment	Species
		A. nilagirica		A. scoparia
		Jammu population	Rajouri population
1	Percentage pollen viability (n = 50)	47.77 ± 1.36	90.60 ± 1.17	94.2 ± 0.59
2	Seed set (%)
2a	Open pollination (n = 80)	21.38 (2.6% healthy)	51.2 (46.7% healthy)	74.6 (40.5% healthy)
2b	Bagging
2b(i)	Single capitulum (n = 50)	—	—	—
2b(ii)	9–10 capitula (n = 50)	20	39.4	65
3	SRE% (n = 25)	14.29	21.6	32.3
4	VRE %(n = 25)	29.35	8.87	16.71

Meiotic studies

Plants of A. scoparia investigated cytologically during the present work were perfect diploids with 2n = 16. During meiosis the 16 chromosomes pairs regularly form eight bivalents (Figure 2A) with chiasmata frequency per pollen mother cell at diplotene averaging 14.9. Cells at anaphase I revealed normal segregation of chromosomes with 8:8 distribution at the two opposite poles (Figure 2B). The recombination index of this species averaged 22.9 (Table 2). No anomalies were noted during meiosis. Plants of Rajouri and Jammu populations of A. nilagirica differed drastically at the cytological level. In plants of the Rajouri population the number of bivalents per cell was much higher. Most of the plants scanned for pollen mother cell meiosis showed haploid number as n = 27 (Figure 2C). Chromosomes paired during meiosis to form bivalents of different shapes. However, in a number of cells, univalents, quadrivalents as well as hexavalents were observed. Segregation was normal in most of the cells i.e. 27:27 at anaphase I of meiosis (Figure 2D). The chiasmata frequency per pollen mother cell at diplotene averaged 43.4. The recombination index of this population averaged 70.4 (Table 2). When plants of the Jammu population were analyzed cytologically, the number of bivalents was shown to be 17 in 73.49% of pollen mother cells scanned. This reveals a chromosome number of 2n = 34 (Figure. 2E). In many of the pollen mother cells chromosomes were observed to be involved in different types of multivalent associations (Figure 2F). The segregation of chromosomes was found to be normal in few cells at anaphase I (Figure. 2G). In many cells however, bridges and laggards were observed (Figure 2H). The chiasmata frequency per pollen mother cells at diplotene averaged 29.8 and the recombination index was 46.8 (Table 2). Percentage pollen viability of A. scoparia averages 94.2 ± 0.59, whereas in the case of A. nilagirica it averages 47.77 ± 1.36 and 90.60 ± 1.17 in Jammu and Rajouri populations respectively.

Figure 2. Pollen mother cells at different stages of meiosis. (A, B) A. scoparia at diplotene and anaphase I. (C, D) A. nilagirica (Rajouri population) at diakinesis and anaphase I; (E) A. nilagirica (Jammu population) at metaphase I; (F) associations at diakinesis (2IVs + 13IIs); (G) anaphase I; (H) laggard at anaphase I.

Table 2. Recombination index in species of Artemisia.

	Characteristics	Species
		A. nilagirica		A. scoparia
		Jammu population	Rajouri population
1	Haploid chromosome number (n)	17	27	8
2	Chiasmata frequency per cell (CF)	29.8	43.4	14.9
3	Recombination index(n + CF)	46.8	70.4	22.9

Discussion

Data on fruit set indicates that both the species are unable to set any achenes by intra capitulum pollination (Table 1). When inter capitulum pollination is allowed, achenes are set, but all them are shrivelled, revealing a high degree of inbreeding depression. Fruit set on open pollination is appreciable in A. scoparia. Both the populations of A. nilagirica lag behind A. scoparia, but display interpopulation variability. Of the two populations of A. nilagirica, the Rajouri population has a higher achene set and a greater percentage of healthy achenes. The Jammu population is sexually most inefficient displaying only 21.38% achene set, of which just 2.6% is healthy (Table 1). Observations on dry biomass allocation reveal almost the same trend, displaying highest value of SRE (32.3%) in A. scoparia. Of the two populations of A. nilagirica, the Rajouri population has a higher value (21.6%) than the Jammu population (14.29%). The Jammu population of A. nilagirica has the lowest fruit set and the lowest SRE, but displays the highest value of VRE (29.35%). The Rajouri population of this species had the lowest VRE (8.87%), while in A. scoparia the values were intermediate (16.71%) (Table 1).

A. scoparia populations from India have been reported to be diploid with 2n = 16 (Khoshoo and Sobti 1958; Kaul and Bakshi 1984). Elsewhere the species is reported to have 2n = 16 and 18 (Abdolkarim et al. 2010). Our report of 2n = 16 in A. scoparia thus is in line with previous reports. The A. nilagirica populations scanned presently are however interesting. These reveal chromosome numbers of 2n = 34 and 54; seemingly belonging to different base numbers. Previous work on this species from the country has reported it to be diploid with 2n = 18, based on n = 9 (Bala et al. 2012; Mehra and Remanandan 1974). B chromosomes are also reported in this species (Bala et al. 2012). No data on chromosome number of this species are available from other parts of the world. Our numbers, i.e. 2n = 34 and 54, are thus new reports for the species. In both the populations, in addition to bivalents, chromosomes seem to be involved in various types of multivalent associations, revealing their polyploid origin.

Genus Artemisia is known in the literature as being highly variable cytologically and many base numbers have been reported for this genus. These are x = 7, 8, 9, 10, 13 and 17 (Wiens and Richter 1966; Vallès et al. 2005; Chehragani and Hajisadeghian 2009; Matoba and Uchiyama 2009; Park 2009). Of these x = 8 and x = 9 are the most common. Going by these base numbers A. scoparia is a diploid based on x = 8 (2n = 16). The Rajouri population of A. nilagirica has 2n = 54 according to previous reports (2n = 18), and it can be assigned to x = 9 as a hexaploid. The Jammu population on the other hand has 2n = 34. It can be treated as diploid if based on n = 17. Due to the presence of multivalents and the fact that two base numbers are rare in a single species, this population also seems to be polyploid. With base number 9, the new number, i.e. 2n = 34, can arise from a tetraploid by deletion of two chromosomes or by fusion of two chromosome pairs. Aneuploid reductions are not uncommon in genus Artemisia and have been reported previously in some species (Mehra and Remanandan 1974). Anomalous anaphasic segregation as well as low fruit set also points towards this. Occurrence of this chromosome number in A. nilagirica is a new record for the species. More detailed studies are however required to establish the origin of this number.

The flexible meiotic system of A. nilagirica is reflected in the sexual reproductive output as well as SRE, both of which are very low for the Jammu population of the species. Highly reduced fruit set on open pollination in this population is indicative of this unstable nature, resulting from irregularities in meiosis, and also from specific gene combinations leading to a series of disharmonies produced at various stages of sexual cycle. Higher chromosome number and relatively stable pairing (Figure 2C) and recombination index (Table 2) gives the Rajouri population of A. nilagirica an advantage over the Jammu population in its sexual reproduction.

The Jammu population of A. nilagirica has the highest VRE of all the populations scanned presently. It appears that as it is unable to utilize its sexual reproductive pathway at present, the plants of this population are diverting their resources to VRE for successful perennation as well as multiplication.

Acknowledgements

The authors are grateful to the Head, Department of Botany, University of Jammu, Jammu, for providing necessary laboratory facilities. Eshan Sharma would also like to thank the Ministry of Environment & Forests, Government of India for financial support Sanction No. 22/8/2010-RE.

Disclosure statement

No potential conflict of interest was reported by the authors.