Introduction

Argyreia Lour. is taxonomically complex genus of the family Convolvulaceae. It is native to continental Asia, with a majority of the species distributed in Southeast Asia, the Indian subcontinent and China. India comprises 40 taxa which includes 37 species and 3 varieties [15] among which 17 species are endemic to India (Table 1). Hooker [2] recognized two sections in Argyreia based on sepal morphology as Pomifera C.B. Clarke and Euargyreia C.B. Clarke. The three species investigated in this report belong to sect. Euargyreia (Argyreia). Ooststroom [7] in Flora Malesiana divided Argyreia into two sections, viz. Ptyxanthus G. Don and Schizanthus G. Don on the basis of lobed or entire corolla and included or exserted reproductive whorls. A. cuneata and A. setosa belongs to sect. Ptyxanthus while A. osyrensis belong to sect. Schizanthus. But the above said sectional names are not correct according to the ICN. As in case of sect. Euargyreia, infrageneric names starting with prefix ‘Eu’ are not validly published and should be named as sect. Argyreia. Another case of sect. Schizanthus, it includes the type of Argyreia hence must be called as sect. Argyreia [15, 17]. Argyreia cuneata (Willd.) Ker Gawl. is unique in being a medium-sized erect shrub; purple–carmine red colored flowers with included stamens (Fig. 1a). A. osyrensis (Roth) Choisy and A. setosa (Roxb.) Sweet are climbing, twinning shrubs with exserted stamens or stamens up to corolla mouth (Fig. 1c, e). The studied species are distributed in the Indian subcontinent and Southeast Asia.

Table 1 Argyreia species in India, their distribution and somatic counts (2n)
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Sampathkumar and Ayyangar [10], who have intensively studied the karyophyletic affinities among the members of Convolvulaceae and suggest that the similarity in overall chromosome morphology, somatic chromosome counts and almost similar chromosome sizes in the genera Argyreia, Rivea and Stictocardia may represent a common line of evolution. Argyreia is the second most species-rich genus of Indian Convolvulaceae with immense ornamental potential and considerably longer chromosome size. Even though, cytotaxonomic records for the genus are very few [1, 3, 6, 9,10,11,12,13, 18] and are mainly confined to somatic counts. The only meiotically screened species A. bella exhibits basic number, x = 14 [12]. Among the studied taxa, seven species have been recorded with somatic chromosome count 2n = 2x = 30 chromosomes while two species, A. argentea and A. bella, had 2n = 2x = 28 chromosomes (Table 1). There are no chromosome reports for the Southeast Asian endemic species of Argyreia considered to be species rich centre for the genus.

In the genus Argyreia almost 80% of the species are yet to be cytologically investigated. Hence, the paper deals with the detailed karyomorphological studies of the three species of Argyreia, viz. A. cuneata, A. osyrensis. A. setosa. Of these three, cytotaxonomic details of A. osyrensis and A. setosa are reported for the first time.

Materials and methods

Three species of Argyreia were collected from their natural habitats from the Indian states of Maharashtra and Karnataka. Herbarium specimens of the species have been deposited in Shivaji University Herbarium (SUK). Seeds were nicked near the hilum, soaked in distilled water for 12 h and sown in soil for germination. Root tips ca. 1 cm in length were excised, pretreated for 3–4 h in p-Dichlorobenzene at 12–14o C, hydrolyzed in 1 N. HCl for 45 s and stained with 2% propionic-orcein. Suitable somatic plates were photographed with LEICA DM 2000 microscope fitted with camera at × 1000 magnification. The karyotypic analysis was done with 8–10 well separated mitotic plates. The chromosome nomenclature followed was that proposed by Levan et al. [4]. The relative variation in chromosome length was evaluated using CVCL [8] parameter while the relative variation in centromeric position of chromosomes in complement was evaluated using CVCI [8] parameter. Stebbins classification [16] and asymmetry index (AI) by Paszko [8] were employed to estimate overall asymmetries of the karyotypes.

Results

The three species studied had somatic chromosome count, 2n = 30 chromosomes (Fig. 1b, d, f). The shortest chromosome pair observed was 2.43 μm from A. cuneata, while the longest chromosome pair of 4.81 μm incurred in A. osyrensis. Argyreia osyrensis had the highest Mean Chromosome Length (MCL) and highest Total Chromosome Length (TCL), 3.62 μm and 54.25 μm, respectively. Argyreia setosa had the lowest Mean Chromosome Length, 3.07 μm and lowest Total Chromosome Length (TCL), 46.04 μm. Argyreia cuneata had MCL and TCL values intermediate between the above two (Table 2).

Table 2 Karyotype data for A. cuneata, A. osyrensis and A. setosa
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The karyotypic formulae were deduced to be 8m + 7sm for A. cuneata, 5m + 10sm for A. osyrensis and 15m chromosome pairs for A. setosa. The karyotypes of A. cuneata and A. osyrensis belonged to the 3A category while that of A. setosa fitted with the 4A category of Stebbins [16] (Table 3). The karyotype asymmetry values of CVCL, CVCI and AI were highest for A. osyrensis and lowest for A. setosa. The values of CVCL, CVCI and AI for A. cuneata were occurred between the above two (Table 3).

Table 3 Karyotype evaluation data using different methods to evaluate karyotype asymmetry
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Fig. 1
figure 1

Argyreia cuneata: a Flower, b Mitotic metaphase plate; A. osyrensis: c Flower with exserted stamens, d Mitotic metaphase plate; A. setosa: e Flower, f Mitotic metaphase plate. Scale bar = 10 μm

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Discussion

Somatic chromosome count, base number and ploidy level of species form the primary basis of cytotaxonomy [14]. In Convolvulaceae, x = 5 is believed to be a primary basic number. The x = 15, x = 14 is prevalent among the studied taxa. This may represent the secondarily derived basic numbers [6, 10]. The chromosomes in Convolvulaceae showed great variability in length. Sampathkumar [12] has recognized two categories of chromosomes: Ipomoea type (short) and Argyreia type (large). As compared to the other Convolvulaceae members, the genera Argyreia, Rivea and Stictocardia possess large chromosomes (Argyreia type) with centromere in the median and submedian region.

Two series of somatic chromosome counts, 2n = 2x = 30 and 2n = 2x = 28 chromosomes, exists in Argyreia and fall under two basic number series viz. x = 15 and x = 14 chromosomes respectively, with no reported polyploids. There is preponderance of base chromosome number x = 15 although either of them could have derived from the other through aneuploidy [10].

The somatic chromosome counts of A. cuneata, 2n = 30 chromosomes found in this study is in congruence with the studies of Sampathkumar [12]. However, unlike Sampathkumar [12], who reported 7m + 8sm homologous pairs, we report 8 + 7sm homologous chromosome pairs in A. cuneata. Previous chromosome studies have shown the occurrence of satellite chromosomes but in our studies we have not found such satellite chromosomes.

According to Lorenzo and Halil [5] CVCI index is a measure of chromosomal heterogeneity and not of intrachromosomal asymmetry although our results suggests it is useful to show intrachromosomal asymmetry. The CVCI parameter measures relative variation in the centromeric position among chromosomes in complement. Arygyeia osyrensis exhibits the highest CVCI percentage, reflected in the karyogram as an increase in the number of submetacentric chromosomes. The lowest CVCI is in A. setosa, as is evident from its all-metacentric chromosomes (Table 3 and Fig. 2). The relative variation in chromosome length in a complement is evaluated by CVCL parameter. The CVCL value was high in A. osyrensis and was slightly variable in A. cuneata which is possibly due to their high total chromosomal length (Table 2, Table 3). The Asymmetry Index (AI) by Paszko [8], which combines two kinds of asymmetries, is proved beneficial in analyzing karyotype asymmetry in our study. The highest AI value in A. osyrensis represents a comparatively asymmetric karyotype while the lowest in A. setosa represents a symmetric karyotype (Table 3), which is also clear from their karyotypic formulae (Fig. 2).

Fig. 2
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Karyogram of A. cuneata; b Karyogram of A. osyrensis; c Karyogram of A. setosa. Scale bar = 10 μm

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According to previous studies, most of the karyotypes in the genus Argyreia have a preponderance of chromosomes with centromere in the median and sub median regions, suggesting moderately symmetrical nature of karyotypes. The karyotypes of A. osyrensis and A. cuneata fall in the 3A while that of A. setosa belong to the 4A category, which also implies a moderately symmetrical to symmetrical nature of karyotypes.

Argyreia, though cytologically poorly studied genus, is a good candidate for cytogenetic studies for establishment of infrageneric classification and better understanding of its taxonomy.