Male meiosis and behaviour of sex chromosomes in different populations of Rumex acetosa L. from the Western Himalayas, India Umer Farooq, Lovleen & M. I. S. Saggoo
Plant Systematics and Evolution ISSN 0378-2697 Plant Syst Evol DOI 10.1007/s00606-013-0881-z
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Author's personal copy Plant Syst Evol DOI 10.1007/s00606-013-0881-z
ORIGINAL ARTICLE
Male meiosis and behaviour of sex chromosomes in different populations of Rumex acetosa L. from the Western Himalayas, India Umer Farooq • Lovleen • M. I. S. Saggoo
Received: 14 November 2012 / Accepted: 14 July 2013 Ó Springer-Verlag Wien 2013
Keywords Rumex acetosa Sex trivalent and distribution Translocation North-west Himalaya Meiotic abnormalities
biologists for many years. Rumex acetosa (common garden sorrel) is a perennial dioecious herbaceous weed, which grows in a wide variety of habitats such as mountain slopes, moist valleys, meadows and along the streams. Infact, R. acetosa has a complex sex chromosome system in which females have two Xs (2n = 12 ? XX), whilst males have one X and two distinctive Ys (2n = 12 ? XY1Y2). The sex chromosomes are bigger in size and although the X is slightly larger than each one of the Ys, the two Ys still account for about 26 % of the male genome (Wilby and Parker 1988). Both the Ys are heterochromatic and differentiated from the euchromatic X chromosome (Clark et al. 1993; Ruiz Rejon et al. 1994; Lengerova and Vyskot 2001; Mosiolek et al. 2005). During meiosis I only a small teleomeric region of each Y chromosome pairs with an extreme region of the X and forms a trivalent Y1–X–Y2 (Parker and Clark 1991). Sex determination in this species is controlled by the ratio between the number of Xs and the number of autosome sets (Parker 1990). No cytogenetic study has been carried from India on this interesting species. The present paper herein discusses the male meiosis in R. acetosa on population basis to know the basic behaviour of sex chromosomes among plants of different populations of R. acetosa for the first time from India.
Introduction
Materials and methods
The subject of the factors determining sexual differentiation in dioecious organisms has been of great interest to
For meiotic studies, flower buds have been collected from plants of R. acetosa growing under natural conditions inhabiting different geographical regions of Himachal Pradesh and Kashmir Himalayas between 1,900 and 3,800 m. Young flower buds were collected from 30 randomly selected plants of each location and fixed in Carnoy’s fixative (6 ethanol:3 chloroform:1 acetic acid v:v:v)
Abstract Detailed meiotic analysis in 28 North West Himalayan populations of dioecious plant Rumex acetosa L. was carried out. The species is generally discussed as an important plant having sex chromosomes. Male meiosis in all the studied populations clearly showed the formation of six bivalents and one trivalent during diakinesis and metaphase-I. The sex chromosomes in male plants exhibit a chain of trivalent (Y1–X–Y2). In addition, among the presently investigated populations ring-shaped trivalents were also observed for the first time in the species. Varied frequency of abnormal segregation of sex trivalent was also observed leading to XY:Y segregation instead of normal X:Y1Y2 segregation. A majority of the populations exhibit normal meiosis. Plants of six populations show meiotic abnormalities like cytomixis, laggards, bridges, chromatin stickiness, etc., leading to reduced pollen fertility. Translocation between an autosome and sex chromosomes was also observed in some of the populations. 0–1B chromosomes were noticed in one population. This is the first ever meiotic analysis of the species from India.
U. Farooq (&) Lovleen M. I. S. Saggoo Department of Botany, Punjabi University, Patiala 147002, Punjab, India e-mail:
[email protected]
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for 24 h. Flower buds were washed and preserved in 70 % ethanol at 4 °C until used. Smears of appropriate sized flower buds were made, using standard acetocarmine technique. To confirm the chromosome number in case of normal meiosis, around 200 PMCs were observed at different stages of meiosis, preferably at diakinesis/metaphase-I/anaphase-I, II. To authenticate the orientation pattern of the sex trivalent almost 2,000 PMCs were analysed. In case of abnormal meiosis, however, more than 500 PMCs were considered to ascertain the type and frequency of various abnormalities per plant. Pollen fertility was estimated by glycero-acetocarmine method (1:1) mixture (Marks 1954). Nearly 500–700 pollen grains were analysed in each case for evaluating pollen fertility and pollen size. Photomicrographs of pollen mother cells and pollen grains were made from freshly prepared slides using Nikon 80i eclipse digital imaging system. Voucher specimens are deposited in the Herbarium, Department of Botany, Punjabi University, Patiala (PUN).
Results Detailed meiotic studies have been carried out on as many as 28 populations of R. acetosa from North West Himalayas. The data regarding specific locality with altitude, voucher number, pollen fertility and pollen size of these populations are presented in Table 1. It was observed that at diakinesis and metaphase-I, in all the pollen mother cells, 15 chromosomes paired to form six bivalents and a single trivalent (Fig. 1a). The chain type trivalent comprising the Y1–X–Y2 chromosome assumes a different ‘‘V’’ shape at later stage (Fig. 1b). Both at diakinesis and anaphase-I, the X chromosome could be easily distinguished from two Ys because of its large size and central position in the trivalent (Fig. 1c). However, the two Y chromosomes could not be identified individually as there is little size difference between Y1 and Y2 and it was not possible to differentiate these two individually during meiosis. In addition to the chain shaped sex trivalent, we noticed ringshaped trivalent that is not reported in the literature so far (Fig. 1d). Out of a total 1,897 PMCs analysed at diakinesis and metaphase-I stages the ring-shaped sex trivalent was present in 12.85 % of PMCs. At late metaphase-I the trivalent comprising three sex chromosomes usually oriented in such a way (convergent type) that the central large X chromosome faced one of the poles, while the two Ys faced the opposite pole (Fig. 1e). This characteristic orientation of the trivalent always resulted in 8:7 segregation of the chromosome at anaphaseI and metaphase-II (Fig. 1f, g) with one of the poles receiving 6A ? Y1Y2 chromosomes and the other pole receiving 6A ? X chromosomes, i.e. it follows the X:YY
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Fig. 1 a A PMC at metaphase-I showing six bivalents and one c chain-shaped sex trivalent (arrowed). b A PMC at metaphase-I showing sex trivalent in convergent shape (arrowed). c A PMC at metaphase-I showing the central position of the X chromosome. d A PMC showing ring sex trivalent (arrowed). e A PMC showing both the Y chromosome facing the opposite poles. f A PMC at anaphase-I showing unequal distribution of chromosomes (see X and both Y chromosomes to opposite poles) (arrowed). g A PMC at metaphase-II showing 8:7 distribution. h A PMC showing XY:Y distribution. i– j PMC’s showing translocation between the sex chromosome and autosome. k–l PMCs showing late and early disjunction of the chromosomes (arrowed). n A PMC showing stickiness. o A PMC showing 1B chromosome (arrowed)
type of distribution. Thus, cytologically two types of pollen grains were produced in equal numbers, half of them having 6A ? Y1Y2 and half with 6A ? X chromosomes. The population collected from Lohararhi (P27) showed exclusively convergent type of segregation, while in the rest of the populations, the frequency of PMCs showing convergent type segregation varies between 38.71 and 90.79 % (Table 2). The remaining PMCs show linear form of sex trivalent segregation which is an abnormal type of segregation leading to XY:Y separation at anaphase-I (Table 2; Fig. 1h). The meiotic course was normal except in plants collected from Aharbal (P7), Kongwattan (P13), Lidderwat Hilltop (P21), Deot (P26), Lohararhi (P27) and Bara Gram (P28). Among these populations highest incidence of meiotic abnormalities was recorded in Aharbal population (P7, 32.14 %) followed by plants of Kongwattan (P13, 23.76 %) and Lidderwat Hill top (P21, 22.01 %) populations, while it was lowest in the plants of Lohararhi (P27) population, where the meiotic anomaly was recorded to be 3.17 % (Table 2). Phenomenon of cytomixis was observed in three populations resulting in empty, hyper and hypoploid cells (Table 3; Fig. 2a–d). Some cells with extra chromatin mass were also observed (Fig. 2e–f). Interestingly, translocation involving an autosome and a sex chromosome was noticed in plants of four populations (Fig. 1i–j). Careful observation revealed that up to 4.5 % of PMC’s showed pentavalent formation among these populations (Fig. 1i–j). Late disjunction of autosomes was observed between 0.66 and 3.06 % of PMC’s (Fig. 1k); similarly, early disjunction at metaphase-I and diakinesis was visualised in 6.38 % of the PMC’s (Table 3; Fig. 1). A large number of PMC’s were screened at meiosis-I and meiosis-II stages and it was visualised that laggards and bridges were common anomalies encountered at anaphases and telophases (Fig. 2i–l). Chromosomal stickiness was seen to occur at only metaphase-II stage of meiosis (Fig. 1n). In plants collected from Kongwattan population, few PMCs were seen to undergo pycnosis (Fig. 2g–h). Microsporogenesis was also erratic as expected with the formation of monads, diads, triads, tetrads and polyads
Author's personal copy Sex chromosomes in different populations of R. acetosa L.
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Author's personal copy Sex chromosomes in different populations of R. acetosa L. bFig. 2 a–b PMCs involved in cytomixis (arrowed). c–d PMCs showing empty, hypo and hyperploid cells (arrowed). e–f PMCs showing extra chromatin mass (arrowed). g–h PMCs showing pycnosis (arrowed). i–j PMCs showing mono and di-centric bridges with laggards (arrowed). k–l PMCs at telophase-I and telophase-II showing laggards (arrowed). m A PMC with five poles and one micronuclei (arrowed). n Monad with three micronuclei (arrowed). o Dyad with small micronuclei (arrowed). p–q Triads with micronuclei (arrowed). r–s Heterogenous sized fertile and sterile pollen grains. Scale bar 10 lm
Table 1 Showing district and specific locality with altitude, voucher number (PUN), pollen fertility and pollen size of the presently investigated populations of Rumex acetosa
Population
with or without micronuclei (Table 3; Fig. 2m–q). The results obtained from the cytological analysis of these populations revealed that the meiotic behaviour varies among genotypes. Pollen fertility was reduced in some populations and it varied in the order P21 [ P7 [ P13 (76.47–79.95 %) while in spite of the meiotic abnormalities present the populations P26, P27 and P28 pollen fertility was not
Locality/altitude (m)
PUN
Pollen fertility (%)
Pollen size (lm)
P1
JK: Bandipora (Mansbal) 1,900
56,898
98.05
18.70 9 17.61
P2
JK: Shopian (Sedew) 2,100
56,886
98.10
17.58 9 19.01
P3
JK: Anantnag (Pahalgam) 2,200
56,892
95.70
17.65 9 16.01
P4
JK: Baramulla (Babareshi) 2,200
56,907
99.05
19.10 9 18.02
P5
JK: Budgam (Yosmarg) 2,300
56,891
99.97
17.92 9 16.57
P6
JK: Baramulla (Gulmarg) 2,400
56,893
99.98
18.87 9 17.02
P7
JK: Kulgam (Aharbal) 2,400
56,887
78.46
21.57 9 19.34 15.03 9 14.76
P8
JK: Anantnag (Baitab Valley) 2,500
56,904
96.09
17.29 9 16.91
P9
JK: Kulgam (Kongwatan on way) 2,500
56,889
93.35
18.95 9 17.60
P10 P11
JK: Baramulla (Drang) 2,600 JK: Anantnag (Duksum) 2,600
56,906 56,905
99.01 98.07
17.58 9 16.51 18.20 9 17.31
P12
JK: Budgam (Doodganga) 2,600
56,909
98.03
19.23 9 18.61
P13
JK: Kulgam (Kongwatan) 2,600
56,890
79.95
23.48 9 22.16 13.65 9 12.67
P14
JK: Kulgam (Sangam) 2,600
56,888
97.86
17.09 9 15.96
P15
JK: Anantnag (Aru) 2,900
56,903
95.41
18.95 9 17.60 18.31 9 17.80
P16
JK: Budgam (Doodthpathri) 2,900
56,908
97.75
P17
JK: Anantnag (Aru higher reaches) 3,000
56,910
98.01
19.10 9 18.58
P18
JK: Baramulla (Gulmarg Fir forest) 3,200
56,899
96.01
17.10 9 16.58
P19
JK: Srinagar (Dagwan on slopes in meadow) 3,500
56,897
97.53
17.56 9 16.39
P20
JK: Srinagar (Mahadev) 3,500
56,902
89.07
19.10 9 18.58
P21
JK: Anantnag (Lidderwat Hilltop) 3,600
56,901
76.47
26.24 9 25.32 13.65 9 12.67 10.89 9 9.97
P22
JK: Budgam (Tosamaidan) 3,600
56,894
95.60
P23
JK: Anantnag (Amarnath on way) 3,700
56,895
97.89
17.32 9 16.20 17.22 9 16.44
P24
JK: Anantnag (Tulin lake) 3,800
56,900
97.09
18.61 9 19.23
P25
JK: Anantnag (Amarnath) 3,800
56,896
96.75
17.29 9 16.91
P26
HP: Kangra (Deot) 2,100
26,622
95.58
19.09 9 18.27 22.86 9 21.52 26.40 9 24.11
P27
HP: Kangra (Lohararhi) 2,300
26,623
98.81
19.01 9 17.58
P28
HP: Kangra (Bara Gram) 2,900
26,624
92.07
20.40 9 17.82
23.15 9 21.11 Populations in bold letters indicate abnormal populations JK Jammu and Kashmir, HP Himachal Pradesh
24.01 9 23.61 28.49 9 26.24
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Author's personal copy U. Farooq et al. Table 2 Incidence of sex trivalent in different populations of Rumex acetosa Population
PMCs observed
PMCs with linear trivalent (%)
PMCs with ring trivalent (%)
PMCs at A-I with trivalent-segregation (%)
Dia.
Dia.
Convergent
Linear
58.70
41.30
48.89
51.11
M-I
Total
M-I
Total
P1
66
27.27
63.62
90.89
3.03
6.06
P2
65
24.61
63.07
87.68
4.61
7.69
P3
65
33.84
56.92
90.76
4.61
4.61
9.22
55.00
45.00
P4
70
41.42
48.56
89.98
4.28
5.71
9.99
55.56
44.44
P5
70
42.85
49.99
92.84
2.85
4.28
7.13
47.37
52.63
P6
63
36.5
50.78
87.28
4.76
7.93
12.69
43.24
56.76
P7 P8
55 54
27.26 31.48
54.53 49.99
81.79 81.47
7.27 11.1
18.17 18.5
63.89 58.06
36.11 41.94
P9
57
26.31
57.88
84.19
10.52
5.26
15.78
61.11
38.89
P10
47
31.91
55.31
87.22
6.38
6.38
12.76
68.97
31.03
P11
63
39.68
49.2
88.88
4.76
6.34
11.1
42.86
57.14
P12
55
34.54
49.08
83.62
9.09
7.27
16.36
38.71
61.29
P13
54
35.18
48.15
83.33
9.25
7.4
16.65
60.00
40.00
P14
48
22.91
58.33
81.24
10.41
8.33
18.74
53.13
46.88
P15
83
36.14
54.21
90.35
7.22
2.4
9.62
44.68
55.32
P16
88
32.95
54.53
87.48
10.27
2.27
12.54
60.00
40.00
P17
81
40.74
44.44
85.18
9.87
4.93
14.8
50.00
50.00
P18
58
36.2
46.54
82.74
10.34
6.89
17.23
48.39
51.61
P19
50
24.00
56.00
80.00
16.00
4.00
20.00
63.33
36.67
P20
64
45.31
42.18
87.49
4.68
7.81
12.49
46.88
53.13
P21
51
25.49
56.85
82.34
11.76
5.88
17.64
68.75
31.25
P22 P23
50 52
32.00 28.84
54.00 51.91
86.00 80.75
10.00 9.61
4.00 9.61
14.00 19.22
68.97 72.73
31.03 27.27
P24
80
38.75
45.05
83.8
8.75
7.05
15.8
52.38
47.62
P25
61
31.14
83.59
11.47
4.91
16.38
P26
76
–
P27
129
–
P28
142
–
Average
1,897
52.45 100 98.45 100
100 98.45 100
65.71
34.29
–
–
–
90.79
9.21
–
1.55
1.55
98.45
1.55
–
–
–
85.21
14.79
59.70
40.29
87.12
affected much and it ranged from 92.07 to 98.81 % (Table 3). Besides difference in the number of sex chromosomes among pollen grains, meiotic abnormalities also play their part in forming the heterogenous sized pollen grains, the size of which varied from 10.89 9 9.97 to 26.24 9 28.49 lm. The diameter of the normal pollen grains came out to be 17.58 9 19.01 lm with the relative frequency of 99.94 % (Fig. 2r–s).
Discussion Rumex acetosa, one of the best studied dioecious plant species, has aneuploid sex chromosomes XX in females and Y1XY2 in males (Kihara and Ono 1923). Sex determination in R. acetosa is independent of the presence or absence of Y-chromosome and is simply controlled by
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10.9 7.4
9.09 12.3
12.85
whether the X: autosome ratio is 1.0 or more (resulting in females) or is 0.5 or less (resulting in males). The two Y chromosomes, however, are required for the successful progress of meiosis in pollen mother cells (Parker and Clark 1991). The species which has been chromosomally counted for the first time from India was studied in detail on the population basis covering different altitudinal areas of Western Himalayas. Meiosis in the male plants of this species was quite interesting because of the presence of a trivalent, formed by the three sex chromosomes. The origin of dioecy and the appearance of sex chromosomes in Rumex have been estimated to be about 15–16 million years old (Navajas-Pe´rez et al. 2005). It has been argued that degeneration of Y chromosomes in Rumex has been accelerated by the accumulation of RAYS1 sequences, among other repeats. It is believed that regular disjunction of the Y1XY2 trivalent in R. acetosa is
Author's personal copy Sex chromosomes in different populations of R. acetosa L. Table 3 Analysis of meiotic aberrations in various cytological abnormal populations of Rumex acetosa
Abnormalities/phases
Incidence (%) in abnormal populations P7
P13
P21
P26
P27
P28
Cytomixis at diakinesis/M-I
8.33
8.08
–
4.62
–
–
Early disjunction at diakinesis\metaphase-I
2.85
4.16
–
–
–
–
Pentavalent formation
–
4.50
2.91
3.03
0.18
–
Laggards
7.50
6.06
11.83
1.97
4.16
2.55
Bridges
5.83
5.05
7.95
1.31
2.77
–
Late disjunction of autosomes
2.66
3.06
–
0.66
–
–
–
–
–
Anaphase-I
Telophase-I Laggards Bridges Stickiness at M-II Anaphase-II
2.30
2.32
–
1.68
0.33
– –
7.27
6.07
7.50
–
–
Laggards
8.18
7.92
11.25
–
1.54
0.62
Bridges
5.45
4.95
4.54
–
–
– –
Telophase-II Laggards
3.63
2.97
6.25
–
–
Bridges
–
–
–
–
1.04
–
Micronuclei small
4.54
1.98
13.75
–
–
–
Micronuclei large
1.81
–
2.50
–
–
–
Multipolarity
2.72
–
–
–
2.08
–
Microsporogenesis Monads
–
1.20
–
–
0.05
–
Monads ? micronuclei
–
–
–
–
–
–
Diads
1.70
2.40
4.70
–
–
–
Diads ? micronuclei
3.53
–
3.52
–
–
–
Triads Triads ? micronuclei
2.65 4.42
– –
2.35 –
0.05 0.1
0.15 0.05
– –
Tetrads
78.76
79.95
76.47
95.58
98.81
92.07
Tetrads ? micronuclei
8.84
16.86
12.34
0.26
0.25
–
dependent on more or less on convergent type segregation leading to X:Y1Y2 distribution. The ratio of males to females at sexual maturity may be biased due to differences between the sexes in germination, mortality, vegetative vigour, flowering frequency, environmental responses, or due to genetic mechanism distorting the sex ratio (Korpelainen 2002). The mechanisms underlying female bias in Rumex seeds are only poorly understood. It is thought that probably the grains with lower DNA amount possess seven chromosomes, and grains with the higher DNA amount eight (Blocka-Wandas et al. 2007). Furthermore, the bias appears to increase with successive life stages (Rychlewski and Zarzycki 1973; Korpelainen 2002; Stehlik and Barrett 2005). Sex determination is probably the most typical case where evolution can produce a variety of solutions to the same basic problems in development (Hodgkin 1992) and plants are the key players in the study of the evolution of
sex determination because they offer a unique opportunity in giving access to the very early stages of X and Y history. One of the most important outstanding issues within evolutionary biology concerns the study of the origin and the evolution of sex-determining mechanisms and of sex chromosomes. Recently evolved sex chromosomes systems constitute excellent study models for the advancement of knowledge in this respect. On these grounds, dioecious plant species with heterochromatic sex chromosomes represent a unique opportunity to investigate the very early stages of sex chromosomes evolution. The discovery of new species harbouring differential differentiated sex chromosomes can open new promising opportunities to shed light on sex chromosome evolution. Meiotic course was normal in plants belonging to majority of populations of R. acetosa. As many as six populations were observed to have anomalies in the meiosis leading to heterogenous sized pollen grains. Cytomixis
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may lead to the migration of partial or whole chromatin among neighbouring meiocytes and lead to the formation of un-reduced pollen and gametes as reported in several plant species (Falistoco et al. 1995; Lattoo et al. 2006; Shabrangi et al. 2010). In the present case, hyperploid nature and presence of extra chromatin mass were seen yielding variable sized fertile and sterile pollen grains. Cytomixis is also considered an additional mechanism for the origin of aneuploidy and polyploidy. Stickiness of chromosomes is another abnormality observed in the few populations of R. acetosa. It is believed that the defects in the functioning of one or two types of nonhistone proteins (DNA topoisomerase II and peripheral proteins) which are necessary for chromatid segregation (Gaulden 1987) are responsible for the phenomenon. Some researchers consider genetic and environmental factors as responsible for chromosome stickiness (Nirmala and Rao 1996; Baptista-Giacomelli et al. 2000; Saggoo et al. 2011; Jeelani et al. 2012). Non-synchronisation in the disjunction of bivalents resulting into early or late disjunction of some bivalents is a common abnormality. This might be due to different rates of terminalisation of various chromosomes of a complement (Darlington 1939), changed homology of chromosomes (Koul 1971), or absence of coordination between chromosome and spindle (Sharma 1976). Sometimes due to late disjunction, bivalents lag behind forming micronuclei which ultimately lead to abnormal microsporogenesis. As a consequence of late disjunction of bivalents bridges are often noticed at anaphases and telophases due to interlocking of chiasmata. This phenomenon can be considered of immense cytological significance as it can lead to the formation of gametes with n ? 1 or n - 1 number of chromosomes causing numerical variation in chromosome number.
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