Karyotype analysis in Machaerium lunatum (Linn. f.) Ducke

The paper gives an account of the results of karyological investigation on Machaerium lunatum (Linn. f.) Ducke using root squash technique. Root tips (approximately 1 cm) were excised and pretreated in 0.002M 8-hydroxyquinoline for 3–3.5 hrs, fixed in 3:1 ethanol-acetic acid for 24 hrs, hydrolised with 5% HCl and squashed in drops of FLP orcein on clean glass slides. Mitotic chromosome slides were observed under research microscope and photomicrographs of 10 good quality metaphase plates were taken and recorded. A chromosome number of 2n = 30 was recorded for the taxon and the chromosomes varied from 0.83 – 2.71 μm in length with karyotype of 7m+4sm+4st. The length of the long and short arms ranged from 0.64 – 1.66 μm and 0.19 – 1.05 μm respectively. The chromosomes varied from metacentric to subtelometacentric. This is the first report of the karyotype and chromosome of this species from Nigeria. DOI: https://dx.doi.org/10.4314/jasem.v21i7.24 Copyright @ 2017 Ekeke and Ugiomoh. This is an open access article distributed under the Creative Commons Attribution NonCommercial License (CC-BY-NC), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Received 15 September 2017, received in revised form 10 October 2017, accepted 11 December 2017

Machaerium lunatum (Linn.f.) Ducke (syn. Drepanocarpus lunatus (Linn. f.) G.F.W. Mey) belongs to the family Fabaceae-Papilionaceae (Hutchinson and Dalziel 1954). It has about 130 species distributed from Mexico to Argentina which are difficult to delimit taxonomically (Rudd 1987) and 150 species distributed from Mexico to South America, and the West Indies (Keay 1985;Airy-Shaw 1985) and coastal West Africa (Hutchinson and Dalziel 1954). In West Africa, it has monotypic species M. lunatum (Hutchinson and Dalziel 1954) which grows along the tropical belt from the Democratic Republic of Congo through Angola, Cameroun, and Nigeria to the Gambia all along the West African coast (Nyananyo 2006). The plant is perennial and is found along fresh and brackish river banks especially salt marshes and tidal portions. The leaves are compound 5-7 foliate. Leaflets are oblong, elliptic and about 5cm long, 2cm wide and rounded at each end. It is well endowed with recurved stipular thorns (Nyananyo 2006). The inflorescence of M. lunatum is a panicle consisting of more than one flower, and usually comprises distinct individual flowers. The flowers are purple and are found at the axial or terminal portions of the stem. The fruit is in a pod and is flat and sickle shaped green when unripe and brown when ripe and about 3cm in diameter. It is said to flower between the months of December and March (Husaini and Gill 1986). Traditionally, Ugiomoh and Anyanwu (2016) reported that the root of the plant is used in the treatment of diarrhea, dysentery, edema, gout and stomach ulcers.
Existing literature on the chromosome number and cytological studies on the members of this genus is scanty (Carlos et al. 2002), however a basic chromosome number (n) equals 10 and 9 has been reported among the genus (Husaini and Gill 1986;Carlos et al. 2002). Coleman and De Menezes (1980) reported n = 10 in M. aculeatum Raddi; Husaini and Gill (1986) reported n = 9 in M. lunatum (L.f.) Ducke; Bandel (1974)  In some other members of Fabaceae family the chromosome number, length and size including karyotypes have been described (Tabur et al. 2009; Adesoye and Nnadi 2011) but this information is lacking in the genus Machaerium. Also, the chromosome number of the West African species of M. lunatum is not yet determined. Therefore this work presents the first report on the chromosome number and karyotype of M. lunatum from Nigeria, West Africa.

MATERIALS AND METHODS
The materials used for this work were sourced from brackish and fresh river bodies south-south Nigeria where M. lunatum grow namely Ogoni and UGIOMOH, IFEOMA GLADYS; EKEKE, CHIMEZIE

Rumuchem in Rivers State and Ogbia in Bayelsa
State. The seeds were first sterilized for 10 min in 10% ethanol solution, rinsed properly in deionised water, plated under sterile conditions in Petri dishes and incubated till germination occurred. Young primary roots (about 1cm long) were excised and pretreated in 0.002 M 8-hydroxyquinoline for 3-3.5 hr, Fixed in 3:1 ethanol-acetic acid for 24 h and the roots were stored in 70% ethanol solution before squashing. When required the roots were removed from the 70% ethanol, hydrolyzed in 5% HCl and squashed in a drop of FLP orcein (Osuji 2003). Mitotic chromosomes were observed under research microscope and photomicrographs of 10 good quality metaphase plates were taken and recorded with Leica photomicroscope equipment fitted with a digital camera. The long arm (l), short arm (s) and the total chromosome length (c) of each chromosome were measured on enlarged microphotographs. The relative lengths, arm ratios (r = l/s) and centromeric index (I = s/c 100) were calculated and used to classify and determine homologous chromosomes (Gomurgen et al. 2010). For karyotype description the chromosomes were arranged in groups according to the position of the centromere (median, m; submedian, sm; subtelocentric, st) and in order of decreasing size in each class. Chromosome nomenclature followed Levan et al. (1964). The variation in chromosome length and chromosome arm ratio within the karyotype was estimated by calculating mean and standard deviation (SD) of these parameters using Microsoft Excel 2010.

RESULTS AND DISCUSSION
A mitotic chromosome count of 2n = 30 was recorded in M. lunatum (Figure 1). This suggests that this species is triploid with basic chromosome number x = 10. The basic chromosome number observed in this species supports existing information of n = 10 reported in the genus (Carlos et al. 2002) but in contrast with Husaini and Gill (1986) who reported n = 9 in M. lunatum. The average sizes of the chromosomes in this species varied from 0.83 µm to 2.71 µm (Table 1). This is within the values previously reported among some members of Fabaceae family. Adesoya and Nnadi (2011) Osuji and Edeoga (2005) suggesting that M. lunatum is advanced phylogenically (Stebbins, 1971). The length of the short arms ranged from 0.19 -1.05 µm while the long arms varied from 0.64 -1.66 µm. These arm lengths are relatively smaller than 1.04 -1.96 µm for short arm and 1.84 -3.84 µm for long arm recorded in Phaseolus vulgaris L. cultivars (Mirela et al., 2005). From our study, the karyotype formula of M. lunatum is 7m+4sm+4st (seven of the chromosomes were metacentric, four submetacentric and four subtelometacentric) as shown in Table 1 and Figure   2. There is slight variation in the chromosome morphology however the chromosomes are predominantly mentacentric types. Osuji and Edeoga (2005), Sinha and Sinha (1980) have inferred that this UGIOMOH, IFEOMA GLADYS; EKEKE, CHIMEZIE is an indication of advanced phylogeny. Also the presence of metacentric and submetacentric chromosomes in the karyotype of M. lunatum is a common feature of Fabaceae family (Osuji and Edeoga 2005;Mirela et al. 2005;Adesoya and Nnadi 2011). Decrease in total chromatin length can be used to infer the evolutionary trend of plant species (Stebbins 1950). In M. lunatum the total chromatin length is 49.94µm and the metacentric frequency is 46.67%. The high percentage of metacentric chromosomes in plant is an indication of symmetric karyotype which is primitive in nature (Stebbins 1950). Furthermore, we observed subtelometacentric chromosomes in M. lunatum karyotype. This corresponds with the works of Mercado-Ruaro and Delgado-Salinas (1998) who reported subtelometacentric chromosomes in Phaseolus chiapasanus, Zheng et al. (1991) in Phaseolus vulgaris and Tabur et al. (2009) in Vicia, Coronilla, Trifolium and Spatium. The presence of subtelometacentric chromosomes suggests that M. lunatum is highly advanced (Oziegbe and Eludini 2013). Finally, it is also important to note that a symmetric karyotype does not necessarily implies primitivity as assumed by earlier students (Lorenzo and Halil 2013) and is evident in M. lunatum due to the presence of metacentric and submetacentric chromosomes. The findings of this work represent the first report on the chromosome number of 2n = 30, karyotype description and formula of in 7m+4sm+4st (seven of the chromosomes were metacentric, four submetacentric and four subtelometacentric) M. lunatum from Nigeria.