New Report on Karyotypes in Three Endemic Species of Murdannia Royle (Commelinaceae) from India

Abstract

Somatic chromosome numbers and karyotypes of three Murdannia species are reported here for the first time. M. assamica and M. fadeniana are endemic to India while M. striatipetala is found only in India and Sri Lanka. M. assamica has chromosome number and karyotype formula 2n=20 and 8m+1sm+1st, M. striatipetala with 2n=20 and 8m+1sm+1st, and M. fadeniana with 2n=40 and 20m. Chromosome length in somatic metaphase varies from 1.15–3.26 µm with a total length of 20.07 µm for M. assamica, 1.63–3.14 µm with a total length of 22.36 µm for M. striatipetala and 0.96–2.04 µm, with a total length of 27.75 µm for M. fadeniana. Karyological data are found useful in assessing relationship in the family Commelinaceae.

Murdannia Royle is one of the largest genera of family Commelinaceae currently placed in subfamily Commelinoideae, tribe Commelineae and consists of about 60 species worldwide (Pellegrini et al. 2016). It is one of the only six genera of the family that has native species in both the Old World and the New World (Faden 1998) with its center of diversity in India (Ancy and Nampy 2015, Pellegrini et al. 2016) representing 52% of the global diversity. The genus is characterized by enantiostylic flowers with a 60°-degree torsion in the floral display, 2–3 antesepalous stamens, 3–4 antepetalous stminodes, 3-lobed antherodes ranging from cordate to sagittate to hastate, trilocular capsules, 1–many-seeded capsule locules, and uni- or bi-seriate seed arrangement (Ancy and Nampy 2015, Pellegrini et al. 2016).

The cytology of 42 out of 49 genera of Commelinaceae are available (Faden and Suda 1980) while data on 15 genera from India are known: Commelina L. (Sharma and Sharma 1958, Kammathy and Rao 1961, 1964, Lewis 1964, Morton 1966, Faden and Suda 1980), Cyanotis D.Don (Sharma and Sharma 1958, Kammathy and Rao 1961, 1964, Faden and Suda 1980), Floscopa Lour. (Faden and Suda 1980), Streptolirion Edgew. (Sharma and Sharma 1958, Suda and Faden 1980), Murdannia Royle (Kammathy and Rao 1961, 1964, Shetty and Subramanyam 1962, Faden and Suda 1980), Amischotolype Hassk. (Zheng et al. 1989), Belosynapsis Hassk. (Jones and Jopling 1972), Callisia Loefl. (Alam and Sharma 1984, Jones and Jopling 1972, Pitrez et al. 2001), Dictyospermum Wight (Sreenath and Rama 1984, Kammathy and Rao 1961, Morton 1966), Pollia Thunb. (Faden and Suda 1980, Jones and Jopling 1972, Morton 1967), Porandra D.Y.Hong (Yang et al. 1994, Yuan and Yang 2006), Rhopalephora Hassk. (Kammathy and Rao 1964, Rao et al. 1970, Sreenath and Rama 1984), Tinantia M.Martens and Galeotti (Heitz 1968, Jones and Jopling 1972), Tradescantia L. (Darlington 1937, Heitz 1968, Jones and Jopling. 1972, Jones et al. 1981) and Tricarpelema J.K.Morton (Morton, 1966, Rao et al. 1970). There is widespread polyploidy in the family because populations from different habitats exhibit karyotype variations (Rao et al. 1968). Aneuploidy is common in Commelinaceae because several genera have multiple numbers of basic chromosome numbers (Faden and Suda 1980). In high polyploid forms of some plants, natural meiosis and the absence of multivalent, as well as high pollen fertility were found, making allopolyploidy a significant force in evolution (Bhattacharya 1975). As evidenced from chromosome number records, allopoliploidy and aneuploidy played much more important roles than autopolyploidy in the speciation event in Commelinaceae (Bhattacharya 1975). Chromosome complements of different ecotypes differ from each other in Commelinaceae and the karyotypic studies indicated that alteration in chromosome complements is the principal factor in the speciation event (Sharma and Sharma 1958).

Murdannia is widespread in Asia and its evolutionary trend follows two different patterns, one with a basic number of x=10 and the other with x=6 (Rao et al. 1968). Other numbers such as x=9 and 11, 12 were sometimes observed (Kammathy and Rao 1961, Shetty and Subramanyam 1962, Lewis 1964, Briggs 1966, Morton 1966, Mitra and Datta 1967, Rao et al. 1968). The least chromosome number, n=6 is reported for M. semiteres (Dalzell) Santapau, while n=12 is also reported for the same species including M. juncoides (Wight) Rao & Kammathy. This is the only species in the genus with a chromosome number n=6 (Brenan 1952). Most species of Murdannia studied previously show n=10 or its multiple numbers (Rao et al. 1968). According to Lewis (1964), the basic number for Murdannia is x=5, which is present now as a dominant line of x=10 and basic numbers such as x=9 and 11 might have been secondarily derived from the dominant line, x=10. M. zeylanica shows a bimodal complement with an approximately equal number of chromosomes of two size classes (Faden and Suda 1980).

Karyomorphology provides insight into potential evolutionary characteristics of karyotypes, as well as the cytological mechanisms driving the evolution of plant diversity on a phylogenetic scale. The purpose of this study is to unravel the karyotypes of three species of Murdannia, M. assamica, M. striatipetala and M. fadeniana.

Materials and methods

Plant material and treatments

M. assamica, M. striatipetala and M. fadeniana brought from the field were grown in the Calicut University Botanical Garden. Their collection details were provided in Table 1. Voucher specimens were deposited at Calicut University Herbarium (CALI).

Table 1. Voucher information for three Murdannia species studied.

Name of species	Locality	Coordinates	Altitude (m)	Voucher
M. assamica	Goalpara, Near town railway station, Assam	26°7′27.19″N, 90°37′20.05″E	33.4	CALI 167962
M. fadeniana	Mankulam, Idukki district, Kerala	10°06′96.7″N, 076°58′80.7″E	1,023	CALI 167971
M. striatipetala	Thirumullaivoyal, near Red hill Lake, Thiruvallur district, Tamil Nadu	13°9′32.02″N, 80°9′18.43″E	14.7	CALI 167972

The stems with a nodal region (about 4–5 cm in length) were planted in clean sand for rooting. Healthy root tips were harvested at peak mitotic period; 12 : 40 pm for M. assamica and M. striatipetala, and 1:00 pm for M. fadeniana. Karyomorphological studies were done using a squash technique following Faden and Suda (1980). The root tips were washed thoroughly in distilled water and treated in a cold saturated solution of para-dichlorobenzene mixed with saponin (1 : 0.01). They were kept under refrigeration at 12–16°C for 3 h, followed by washing in distilled water and fixed in modified Carnoy’s fluid (4 chloroform : 3 ethanol : 1 glacial acetic acid) at room temperature for about 3 h. The root tips were washed in distilled water 2–3 times and hydrolyzed in 1.2 M HCl for 40 min at room temperature. They were then stained with aceto-orcein. Microslides were prepared in 45% acetic acid and photographs were taken with a DMC 4500 camera attached to a DM 2000 compound microscope (Leica, Heerbrugg).

Karyotype analysis

Image processing and karyotyping were done by using computer-aided programs such as Adobe Photoshop and Microsoft Excel. Chromosomes were identified and classified based on the length of the chromosome, arm ratio, and centromeric indices according to Levan et al. (1964). The number of chromosomes was finalized by counting more than 15 somatic cells and karyotype analyses were based on three mitotic metaphase preparations of each species. Karyograms were arranged based on decreasing order. The Karyotypic formula was based on the position of the centromere and the number of chromosomes. Disparity index (DI) (Mohanty et al. 1991), Variation coefficient (VC) (Verma 1980), Total forma percentage (TF%) (Huziwara 1962), Karyotype asymmetry index (As K%) (Arano 1963), Syi index (Greilhuber and Speta 1976), Rec index (Venora et al. 2002), Intrachromosomal asymmetry index (A1), Interchromosomal asymmetry index (A2) (Zarco 1986) and Asymmetry index (AI) (Paszko 2006) were calculated.

Results

M. assamica is an annual ascending herb with a terminal single pedunculate cincinnus, easily identified by its large caducous bracteoles, having two fertile stamens and four staminodes, hastate or knob like antherodes and widely obovoid capsules with one-seeded locules. The species is so far known only from the type locality Goalpara in Assam, India (Fig. 1A).

Fig. 1. Habits of Murdannia species studied. A. M. assamica; B. M. fadeniana; C. M. striatipetala (photographed by Sreekutty, T. K.).

The karyomorphological data of M. assamica is given in Table 2. The chromosome number is 2n=20, with ten pairs of homologous chromosomes composed of eight pairs of the median, one pair of submedian, and one pair of subterminal. The karyotype is formulated as 8m+1sm+1st. The chromosome length varied from 1.15 to 3.26 µm, and the total length and average length are 20.07 µm and 1.00 µm, respectively (Fig. 2A, B).

Table 2. Karyomorphology of M. assamica.

No. of chromosomes	Length of long arm (l) (µm)	Length of short arm (s) (µm)	Chromosome length (c) (µm)	Arm ratio (s/l)	Centromeric index (s/TCL×100)	Chromosome shape
1	2.63	0.98	3.25	0.37	27.38	sm
2	2.13	0.82	2.95	0.38	27.79	m
3	1.58	0.98	2.56	0.62	38.28	m
4	1.30	0.80	2.10	0.61	38.09	m
5	1.50	0.33	1.83	0.22	18.03	st
6	0.92	0.74	1.66	0.80	44.57	m
7	1.01	0.61	1.62	0.60	37.65	m
8	0.99	0.63	1.62	0.63	38.88	m
9	0.84	0.40	1.24	0.47	32.25	m
10	0.67	0.48	1.15	0.71	41.73	m

　

Fig. 2. Mitotic metaphase (A) and karyogram (B) of M assamica. Mitotic metaphase (C) and karyogram (D) of M. fadeniana. Mitotic metaphase (E), and karyogram (F) of M. striatipetala.

M. fadeniana is a perennial rosette herb with scapose inflorescence, consisting of terminal or axillary thyrses with erect to ascending cincinni, having three fertile stamens and three staminodes, and 10–16 seeds arranged biseriately in the capsule locules (Fig. 1B).

The karyomorphological data of M. fadeniana is detailed in Table 3. The chromosome number is 2n=40, with 20 pairs of homologous chromosomes composed of 20 pairs of median and the karyotype formulated as 20m. The size of the chromosomes varied from 0.96 to 2.04 µm, and the total length and average length are 27.75 µm and 0.69 µm, respectively (Fig. 2C, D).

Table 3. Karyomorphology of M. fadeniana.

No. of chromosomes	Length of long arm (l) (µm)	Length of short arm (s) (µm)	Chromosome length (c) (µm)	Arm ratio (s/l)	Centromeric index (s/TCL×100)	Chromosome shape
1	1.24	0.79	2.04	0.63	0.38	m
2	1.12	0.71	1.83	0.63	0.38	m
3	1.03	0.74	1.77	0.71	0.41	m
4	1.06	0.65	1.71	0.61	0.38	m
5	1.02	0.59	1.61	0.58	0.36	m
6	1.06	0.49	1.56	0.46	0.31	m
7	0.80	0.68	1.49	0.84	0.45	m
8	0.81	0.63	1.45	0.77	0.43	m
9	0.79	0.53	1.32	0.66	0.40	m
10	0.81	0.48	1.30	0.59	0.37	m
11	0.70	0.57	1.28	0.81	0.44	m
12	0.78	0.46	1.25	0.58	0.37	m
13	0.67	0.57	1.24	0.84	0.45	m
14	0.63	0.56	1.19	0.88	0.46	m
15	0.66	0.52	1.18	0.78	0.43	m
16	0.64	0.51	1.16	0.79	0.44	m
17	0.60	0.53	1.14	0.89	0.47	m
18	0.58	0.51	1.09	0.88	0.46	m
19	0.59	0.49	1.09	0.84	0.45	m
20	0.59	0.37	0.96	0.61	0.38	m

M. striatipetala is an annual herb with spirally arranged leaves, terminal and axillary thyrses consisting of 1–3 pseudo umbellate cincinni. It is easily distinguished by its orbicular petals with dark contrasting veins (Fig. 1C).

The karyomorphological data is given in Table 4. The chromosome number is 2n=20, with 10 pairs of homologous chromosomes composed of eight pairs of the median, one pair of submedian and one pair of subterminal, and the karyotype formulated as 8m+1sm+1st. The chromosome length varied from 0.63–3.14 µm, and the total length and average length are 22.36 and 1.11 µm, respectively (Fig. 2E, F).

Table 4. Karyomorphology of M. striatipetala.

No. of chromosomes	Length of long arm (l) (µm)	Length of short arm (s) (µm)	Chromosome length (c) (µm)	Arm ratio (s/l)	Centromeric index (s/TCL×100)	Chromosome shape
1	1.91	1.23	3.14	0.64	39.28	m
2	2.25	0.64	2.90	0.28	22.34	st
3	1.55	1.03	2.58	0.66	40.00	m
4	1.39	0.84	2.24	0.60	37.88	m
5	1.31	0.81	2.12	0.61	38.06	m
6	1.32	0.73	2.05	0.55	35.61	sm
7	1.14	0.80	1.94	0.70	41.36	m
8	1.08	0.71	1.79	0.65	39.68	m
9	1.13	0.77	1.91	0.68	40.77	m
10	0.94	0.69	1.63	0.73	42.38	m

Discussion

Chromosome numbers of 24 species of Murdannia have been reported so far (Rice et al. 2015). The size of chromosomes in this genus is particularly small and the numbers vary from n=6 to 30 (Faden and Suda 1980). Most of the species have the chromosome number n=10 or its multiples.

In this study, we found that M. assamica and M. striatipetala have the same chromosome number n=10 and M. fadeniana has n=20. Thus, the chromosome numbers reported here are multiples of the most common basic number x=10 reported for Murdannia.

For M. assamica, the karyotype is formulated as 8m+1sm+1st. This species is morphologically close to M. nudiflora (L.) Brenan in its general appearance, in their caducous bracts, terminal single pedunculate cincinnus inflorescence, and presence of two stamens and four staminodes, but can be easily distinguished by its larger bracteoles and sepals, single seed in each locule, and the absence of basal rosette arrangement of leaves. M. assamica which is included in a complex of M. nudiflora, M. simplex (Vahl) Brenan, M. loriformis (Hassk.) R.S.Rao & Kammathy and M. gigantea (Vahl) G.Brückn., in which except for M. gigantea, all the others exhibit n=10 or its multiples, while M. gigantea exhibits n=11 (Kammathy and Rao 1961, Panigrahi and Kammathy 1963). Here we compare M. nudiflora with M. assamica to check whether the cytological data supports the placement of this species in M. nudiflora complex. There are different reports available for the chromosome number of M. nudiflora, from that, the report of 2n=22 by Saravanakumar (2013) correlates with the previous reports of Rao et al. (1960) and Panigrahi and Kammathy (1963) having a karyotype formula 4M+2m+10sm+6st. Recently, Kaur et al. (2017) and Kaur (2018) reported n=12, making an additional record for the species on a worldwide basis as the species is previously known to exhibit 2n=20 (Bhattacharya 1975, Renugadevi and Sampathkumar 1986) at a diploid level based on x=10 from India and outside, respectively. The cytological evidence from the studies also delimit the two species from each other by comparing the chromosome morphology. The two species also differ in their karyotype formula.

M. striatipetala is close to M. spirata (L.) G.Brückn., the common and most variable species of Murdannia in India. M. striatipetala possesses a definite base, narrow leaves, dark veined petals, short appressed hairs on the filament, and having more seeds per locule. It is included in the group of species with terminal or axillary thyrse inflorescence. Different chromosome numbers were reported for M. spirata: 2n=18 (Raghavan and Rao 1961), 2n=20, 40 (Rao et al. 1970) and 2n=40 (Kammathy and Rao 1961). Recently, Kaur (2018) reported 2n=24. The chromosome number for this group is concluded as n=10, the only exception n=12 reported above. M. striatipetala also has the same chromosome number n=10 with karyotype formula 8m+1sm+1st.

Based on DI value, the heterogeneous assemblage of chromosomes was more prominent in M. assamica (47.82) followed by M. fadeniana (35.71) and M. striatipetala (31.56). Based on the VC value more heterogenous chromosomes were seen in M. assamica (67.50) while more homologous chromosomes were found in M. fadeniana (41.60). Lower TF% and higher As K% indicate asymmetry of chromosome. A1–A2 also indicates the asymmetry and heterogeneity of chromosomes. M. assamica has a TF% and As K% (33.57; 66.42) followed by M. striatipetala (37.14; 62.85) and M. fadeniana (41.34; 58.65), respectively (Table 5).

Table 5. A comparison of karyotype and asymmetry indices of the species studied.

Chromosome characteristics	M. assamica	M. fadeniana	M. striatipetala
Chromosome number (2n)	20	40	20
Karyotype formula	8m+1sm+1st	20m	8m+1sm+1st
RCL	1.15–3.26 µm	0.96–2.04 µm	1.63–3.14 µm
TCL	20.07 µm	27.75 µm	22.36 µm
ACL	1.00 µm	0.69 µm	1.11 µm
CVCI	23.22	10.30	15.00
CVCL	33.75	20.80	22.00
DI	47.82	35.71	31.56
VC	67.50	41.60	44.07
TF value (%)	33.57	41.34	37.14
As K%	66.42	58.65	62.85
Syi index	51.29	70.37	59.08
Rec index	31.25	33.95	35.54
A1	0.43	0.28	0.38
A2	0.33	0.20	0.22
AI	7.83	2.14	3.30

Karyotype asymmetry for the relations between the chromosome arms has been estimated using the A1 index and their values ranging from zero to one. The A1 index does not depend on chromosome number or chromosome size. The A1 index obtained for M. assamica is 0.43, M. fadeniana is 0.28, and for M. striatipetala is 0.38. The A2 index is also represented as 0.33, 0.20 and 0.22 respectively. The scatter diagram of A1 against A2 asymmetry indices (Fig. 3) revealed that M. fadeniana had the lowest A1 and A2 values, indicating a small difference in the length of chromosome arms and the least variation in chromosome lengths. However, M. assamica with the highest A1 value presented the greatest difference in the length of chromosome arms and the highest A2 value indicating the large variation in length among chromosomes. When compared to M. assamica, M. striatipetala had the lowest A1 and A2 values, but it is higher than M. fadeniana and it had a small difference in the length of chromosome arms and the smallest variation in length among chromosomes. The A1 and A2 values were higher for M. assamica than the other two species studied. Stebbins (1971) has stated that one of the basic features which bring about karyotype asymmetry is shifting of the centromere from median to submedian and subterminal position and it has been considered as a progressive step in karyotype evolution. Taxa with asymmetric karyotype tend to have low TF% (Huziwara 1962).

Fig. 3. Scatter diagram of A1 and A2 asymmetry of M. assamica, M. fadeniana, and M. striatipetala.

The scatter diagram of CV_CI against CV_CL asymmetry indices (Fig. 4) also showed that M. assamica possesses a higher asymmetric karyotype as compared to M. fadeniana and M. striatipetala. These results are also strongly supported by higher AI=7.83. The AI index gives a measure of the heterogeneity of chromosome length and centromere position in a given karyotype. This value is proportional to karyotype asymmetry. Based on the karyotype asymmetry indices M. assamica possess the most asymmetric karyotype and more heterogenous karyotype when compared with the other two species. M. fadeniana possesses a symmetric karyotype than the two.

Fig. 4. Scatter diagram of CV_CL and CV_CI of M. assamica, M. fadeniana, and M. striatipetala.

Acknowledgments

The authors are grateful to the Head, Department of Botany, the University of Calicut for facilities, and Sreekutty T.K to CSIR-HRDG for fellowship.

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