Chromosome number variation in a promising oilseed woody crop, Plukenetia volubilis L. (Euphorbiaceae)

Abstract

Cytology of a promising oilseed woody crop, Plukenetia volubilis, was analyzed for the first time here. There are in total 11 different chromosome numbers, ranging from 2n = 50 to 2n = 86 in P. volubilis. Different numbers were found not only in different root tips but also in different cells of the same tip. Among the cells, 2.78% cells had the lowest chromosome number of 2n = 50; and 1.39% cells had the highest chromosome number of 2n = 86. The most common chromosome number is 2n = 58 and about 27.79% cells had this quantity of chromosomes. The chromosomes were extremely small in size, at less than 2 μm. Thus, analysis of chromosome morphology was impaired. Polyploidy, confirmed as the significant evolutionary trend in chromosome number within this species, is briefly discussed. This species is a problematic and polymorphic taxon and our results indicate that its polyploidy can be regarded as the reason.

Keywords:

• chromosome number
• karyotype
• Plukenetia volubilis
• polyploidy

Introduction

Plukenetia, a poorly defined classification genus, belongs to the tribe Plukenetieae of subfamily Acalyphoideae in the family Euphorbiaceae. Plukenetia is a pantropical genus of over 20 species of twining vines and lianas, which were distributed throughout Africa and Latin America, and as far southeast as Malesia (Airy-Shaw 1982; Gillespie 1994, 2007; Bussmann et al. 2009). It is notable for its four-carpellate ovary and the associated character of four pistillate sepals, styles that are partially to entirely fused and often massive, and scandent habit (Gillespie 1994, 2007). However, on the basis of an evolutionary study of the floral characteristics of some groups in Euphorbiaceae, Plukenetia is a paraphyletic genus lacking any uniquely derived defining characters (Webster 1994; Gillespie 1994, 2007; Suarez-Cervera et al. 2001; Bussmann et al. 2009). The most well known and commercially valuable Plukenetia species is P. volubilis L., containing high levels of omega fatty acids in its seed (Hamaker et al. 1992; Cai et al. 2011). P. volubilis, a promising oilseed crop commonly known as Sacha Inchi, was native to the Amazon rainforest and was restricted to South America and the Lesser Antilles, where it has been cultivated by indigenous people for centuries (Semino et al. 2008). It flowers five months after being planted, and bears seeds around the eighth month (Cai 2011). The male flowers are small, white, and arranged in clusters. Two female flowers are located at the base of the inflorescence and it often produces seeds nearly year-round in the tropical region.

Due to their worldwide distribution and the fact that they include many economically important plants, many genera and species of the family Euphorbiaceae have been the subject of cytological studies (Perry 1943; Miller and Webster 1966; Hans 1973; Vosa and Bassi 1991; Vanzela et al. 1997; Soontornchainaksaeng and Chaiyasut 1999; Krahenbuhl et al. 2002; Aarestrup et al. 2008). Karyotypic changes are frequent in the evolution of higher plants and are often used as the basis for deductions about phylogenetics, thus the results of chromosomal studies may be useful in plant taxonomy and phylogeny analysis (Levin 2002). In addition, information about number and structure of chromosomes of both wild resources and cultivars is an important prerequisite for effective crop genetic and breeding studies (Levin 2002). Although some cytological data on Euphorbiaceae have been generated (Perry 1943; Vosa and Bassi 1991; Vanzela et al. 1997; Soontornchainaksaeng and Chaiyasut 1999; Krahenbuhl et al. 2002; Xu et al. 2007), there exists to date no literature on the karyotype analysis of the genus Plukenetia.

This is the first cytological study of P. volubilis. The aim of this study is (1) to identify the chromosome number and morphology of this oilseed crop species for further selection and breeding of new improved cultivars; and (2) to supply cytological proof for the taxonomy and phylogeny of Plukenetia, an economically important genus.

Materials and methods

Plukenetia volubilis seeds, collected from Peru, South America, were germinated on wet filter papers in Petri dishes in suitable lab conditions. Root tips approximately 1 cm long were cut and pre-treated in a saturated dichlorobenzene solution at room temperature in darkness for 2 h, then fixed with a carnoy solution, comprised of absolute alcohol:glacial acetic acid (3:1), at approximately 4°C for 1 h, then rinsed in distilled water twice for approximately 20 minutes. After hydrolysing in 1 mol l⁻¹ HCl at 60°C for 7 minutes and staining with 1% aceto-orcein overnight, the meristematic zones of the tips was squashed for cytological observation.

Chromosome spreads were observed and photographed using a Nikon E800 microscope (Nikon, Japan). Cells with well-scattered chromosomes from five root tips were observed in detail and chromosome numbers were carefully counted.

Results

Among the 72 cells observed in P. volubilis, there are in total 11 different chromosome numbers, ranging from 2n = 50 to 2n = 86 (Figure 1). Different chromosome numbers were found not only in different root tips but also in different cells of the same tip (Table 1). The lowest chromosome number of 2n = 50 was found in 2.78% of the cells, and 1.39% cells had the highest chromosome number of 2n = 86. The most common chromosome number is 2n = 58 and about 27.79% cells had this quantity of chromosomes.

Figure 1 Metaphase chromosomes of P. volubilis.

Table 1. Chromosome numbers found in root tip cells of P. volubilis.

Number of cells	2	5	8	5	8	7	20	5	9	2	1
Number of chromosomes	50	53	54	55	56	57	58	59	60	62	86
Percentage (%)	2.78	6.94	11.11	6.94	11.11	9.72	27.79	6.94	12.5	2.78	1.39

The absolute length of the chromosomes of P. volubilis is less than 2 μm (Figure 1), thus the chromosomes were small-sized ones. Because of that, analysis of chromosome morphology was impaired by the extremely small size of the chromosomes, restricting the comparison of the species in terms of chromosome type.

Discussion

Karyotype analysis has been useful for clarifying species by measuring number, size, arm ratio, and unique shape of chromosomes at the somatic metaphase (Levin 2002). Chromosomal information is an important key to the classification, phylogenetic analysis and evolutionary biology of plant species, because chromosomal information in plants, unlike other characteristics, retains its consistency (Levin 2002; Adams and Wendel 2005). The chromosome number of different cells in the metaphase of P. volubilis was so non-uniform that the single basic chromosome number of this promising oilseed crop could not been easily and correctly postulated. Perry (1943) analyzed a large number of species in the Euphorbiaceae and observed that the basic chromosome numbers of unrelated species formed a primary series of x = 7, 8 and 9 and a secondary series of x = 6, 10 and 11. Vanzela et al. (1997) thought the chromosome numbers detected in the subfamily Acalyphoideae were quite fragmented and did not suggest a single basic number for the subfamily and an appropriate cytotaxonomic interpretation. In addition, chromosome counts performed in the subfamily Acalyphoideae have revealed that the tribe Plukenetieae consists exclusively of polyploid species (Vanzela et al. 1997). Thus, P. volubilis is a polyploid plant, like 41.38% of other species in Acalyphoideae (Vanzela et al. 1997) and 50% of other species in the Euphorbiaceae family (Perry 1943; Aarestrup et al. 2008).

Currently available chromosome data show that polyploidy is the most significant evolutionary trend in chromosome number within Euphorbiaceae (Perry 1943; Hans 1973; Vosa and Bassi 1991; Vanzela et al. 1997; Krahenbuhl et al. 2002; Aarestrup et al. 2008). Polyploid plants usually have larger cells and plants are often larger, with thicker shoots, larger leaves and fruits, and a higher nutritional content, etc. (Levin 2002). Polyploidy is also widely acknowledged as a major mechanism of adaptation and speciation in plants (Levin 2002; Adams and Wendel 2005; Jian et al. 2012). Polyploids may have superior levels of adaptability and higher probabilities of survival than their diploid relatives (Soltis and Soltis 2000). Moreover, polyploidy is thought to imbue plants with novel features that allow them to occupy new environments, expand their geographic range, or achieve new species interactions (Levin 2002). Its strong and broad ecological valence would permit rapid spreading out and coexistence of different chromosome forms, which in some cases have been noted to show diverse behavior of P. volubilis plants growing under a wide range of environmental gradients in the tropical regions (Semino et al. 2008; Cai 2011; Cai et al. 2012), This adaptive significance of polyploidy was tested recently in wild yarrow (Achillea borealis) (Ramsey 2011).

Cytogenetic variation in plants has long attracted biologists’ interest (Stebbins 1950). What is particularly interesting about the P. volubilis described here is its surprisingly high and diverse cytological variation. The presence of a total of 11 different chromosome numbers in P. volubilis is clearly an example of a chromosomally highly variable species. It seems that polyploidy is confirmed as the most significant evolutionary trend in chromosome number within this species. The same phenomenon (numerous chromosome alterations with dysploidy resulted in high and diverse chromosome numbers) was also found in Dalechampia species in the same subfamily, Acalyphoideae (Vanzela et al. 1997). On the other hand, there are many reports of the widespread occurrence of chromosome races or variants within species (Murray and Young 2001; Severns and Liston 2008; Abdolkarim et al. 2011), especially for rare plants (Severns and Liston 2008). In some cases there is a clear geographic separation of the races; in others the different chromosome numbers are due to the variable presence of chromosomes, or supernumerary chromosomes, and in a third group differences in chromosome number may indicate the need for taxonomic revision (Severns and Liston 2008). The reason for this variation and instability is the variation in chromosome number, resulting in taxonomical problems in defining its species and genus (Vanzela et al. 1997; Gillespie 2007). Moreover, the diverse chromosome number of P. volubilis might imply that this crop has rich genetic diversity. There could be many wild resources with different cytological characters in the natural habitat, which could be further used to improve the resistance, productivity or the quality of seed oil of the widely cultivated varieties. Biparental inbreeding in normally outbreeding species may be responsible for the elevated level of chromosome variation (Parker and Wilby 1989). Following enforced inbreeding in a variety of plants, increased chromosome instability was observed, resulting in high levels of spontaneous breakage and reunion of chromosomes and a reduction in chiasma frequency, with an increase in univalent formation (Murray and Young 2001; Levin 2002; Severns and Liston 2008). There is no evidence for significant levels of inbreeding in the P. volubilis plants we examined. In addition, in many other groups in the Euphorbiaceae family, such as Euphorbia L., structural rearrangements play a marked evolutionary role in karyotype differentiation (Vosa and Bassi 1991). However, because of the extremely small size of the chromosomes of P. volubilis, the rearrangements responsible for karyotype differentiation cannot be clearly pointed out.

Acknowledgments

This work was financially supported by grants (KSCX2EWQ17, KSCX2EWZ15) from the Chinese Academy of Sciences, and by a grant (31170641) from the National Natural Science Foundation of China.