Chromosomes of Some West African Orchids the Stain Used
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1971 487 Chromosomes of Some West African Orchids A. H. ar-Rushdi Department of Botany, University of Ibadan, Ibadan, Nigeria Received February 10, 1970 The "Flora of West Tropical Africa" lists over 400 species of orchids. About 300 of these are estimated to occur in Nigeria and West Cameroun. In contrast to the wide range of data on American and Asian orchids, cytological information is available for only a few West African species. An opportunity for increasing such information was provided by Mr. W. W. Sanford's living collection of over 2,000 orchids, mainly from Nigeria and some from West Cameroun. His assistance in the identification of over 200 collected species and in making available the right material for cytological studies is duly acknowledged. Materials and methods The main source of material is Mr. Sanford's collection at the University of Ife. These are designated in Table 1 by the prefix WS. Additional material. collected by the staff of the Botany Department, University of Ibadan, appears in the table as herbarium number with the prefix UIH. As far as possible, more than one specimen was examined. Root-tip cytology in orchids, especially of subterranean roots, is beset with difficulties. It was therefore decided to use the sporogenous cells, since flowers were readily available. Post-meiotic metaphase in the tetrad of micro -spores was found to be a suitable and easily prepared stage for haploid counts. One pollinium was directly stained, the other being kept in case the spores were found premature for metaphase studies. Such pollinia were cultured in distilled water at room temperature for periods of 3-12 hours, depending on their stage of division. Culturing was successful only for pollinia with micro - spores in late prophase or prometaphase stages. The stain used was a preparation of 1% (w/v) synthetic orcein, dissolved by gentle heating in 40% propionic acid, cooled, filtered, and diluted with the acid as required. Spores were teased out of the pollinium into a drop of stain and gently covered. The cover was temporarily sealed to the slide, which was examined, photographed and often made permanent by the freezing method. The same staining procedure was followed in preparing smears of pollen-mother-cells but the pollinia were fixed for 15 minutes in glacial acetic alcohol (2:1) prior to staining. 1 Present Address: Biology Department, Pahlavi University, Shiraz. Iran. 33* 488 A. H. ar-Rushdi Cytologia 36 Table 1. Chromosome numbers 1971 Chromosomes of Some West African Orchids 489 Table 1. (continued) 490 A. H. ar-Rushdi Cytologia 36 1971 Chromosomes of Some West African Orchids 491 Results and discussion Sixty-three plants, derived from about 32 different species belonging to eight genera, were studied. Table 1 shows the record of their sporophytic chromosome numbers. Plate 1 illustrates some representative photomicro graphs. In the genus Polystachya, Jones (1966) reported the chromosome numbers of about 21 species. At least 14 species are reported here, confirming the constancy of the basic number (n=20) for the genus. A polyploid number, hitherto not found in West African species of Polystachya, is reported for P. odorata (WS-4450), (Figs. 7 and 8). Its chromosomal association in meiosis was not observed but lack of variability in 58 spores examined from the plant indicates its chromosomal stability. The plant was collected from the summit of Mt. Orosun. Morphologically, it could not be separated from the other diploid members of the species. In the genus Eulophia (where six species were investigated), the basic chromosome number varies, and basic numbers of n=20, 22, 23, 31, and possibly 24, have been observed. Jones (1967) reports the chromosome numbers in species belonging to ten genera of the subtribe Aerangidinae. He concludes that the predominant basic number is x=25, that most of the species are diploid but polyploid species do occur. He also records two exceptions, Ae. rhodostica (2n=42) and Cyrorchis sp. (2n=46), the latter collected from Southern Nigeria. Ancistrohyncus clandestinus (2n=48) is another West African exception. In all species studied, tetrad analysis of the chromosomes was readily possible as the spores remained attached to one another and their stages of division were synchronised. Furthermore, in the majority of the species, a high degree of synchronisation was also observed between tetrads within a pollinium. Tetrad analysis was useful in detecting trisomics, triploidy and occasional distributional errors of meiosis in diploids. The trisomic condition in Eulophia euglosa (UIH 10288), suspected from tetrad analysis (Figs. 3 and 4) and from the occurrence of micronuclei in some tetrads was verified by meiotic observations. In Polystachya rhodoptera (WS-203-66), the trisomic plant also showed complementary counts of 20 and 21 in the tetrad, but its meiotic associations were not examined. Plant UIH 10285 was recognised as a triploid by the highly variable counts in the spores and by the occur rence of micronuclei of variable sizes and chromosome content. It was further verified as an autotriploid by examining the first meiotic metaphase where 22 Figs. 1 and 3-11. Metaphase chromosomes, ca. •~2,000 in the microspores of: 1, Eulophia gracilis (n=22). 3, Eulophia euglossa (n=20). 4, Eulophia euglossa (n+1=21). 5, Eulophia cristata (n=23). 6, Eulophia guineensis (n=23). 7, diploid Polystachya odorata (n=20). 8, tetraploid Polystachya odorata (n=40). 9, Ancistrorhynchus clandestinus (n=24). 10, Euclophidium saundersianum (n=29). 11, Eulophia quartiniana (n=48). Fig. 2. Chromo some configurations at metaphase I of meiosis in triploid Eulophia gracilis (22III). •~1,600. 492 A. H. ar-Rushdi Cytologia 36 trivalents invariably occurred (Fig. 2). The fertility of this plant was much impaired, yet three capsules of seeds were set in two flowering seasons. Observed errors of meiosis in diploids consisted of occasional non - disjunction of individual chromosomes, recognised as exceptional counts of n-1 and n+1 in the tetrad, and of non-reduction of the chromosome number. Pooling the data from all the diploid species investigated, non-disjunctional counts were recorded for 0.22% of the spores. Data from individual plants were considered insufficient to associate such error with specific plants or species. Restitutional dyads were observed in individual plants belonging to different species and genera. Their incidence was rare, but their occurrence in each of the three plants of Polystachya rhodoptera investigated, including the trisomic plant, is perhaps noteworthy. Restitution was also observed to have occurred in one of the products of the first meiotic division, giving rise to a "Triad" with one diploid and two haploid spores . Barber (1942) drew attention to the physiological implications of the spores of orchids remaining attached in tetrads. He suggested that the possibility of nuclear interaction between adherent spores made the group of four cells, rather than the individual cell, act as a unit of development. He concluded that the synchronisation of cell division was a climax of the unitary control of cell processes within the tetrad. He also pointed out the significance of the condition in permitting the division of chromosomally deficient spores, even of micronuclei with only a few chromosomes, in harmony with the balanced members of the tetrad. These explanations appear to be corroborated by the present observations. Furthermore, Barber's data as well as the observation made by Miduno (1940) on a haploid plant of Bletilla striata, indicated no pollen abortion of chromosomally unbalanced spores. It would therefore appear that the natural occurrence of chromosomal variants in spores and the lack of stringent selection against such spores, coupled with prevalent vegetative reproduction, provide a combination of features permitting variation in chromosome number in orchids. This probably explains the diversity of basic chromosome numbers reported for numerous genera of orchids, and may be a means for exploiting the profusion of habitats available in the tropics. References Barber, H. N. 1942. The pollen-grain division in the Orchidaceae. J. Genet. 43: 97-103. Jones, K. 1966. The chromosomes of orchids I. Polystachya Hook. Kew Bull. 20: 357 - 359.- 1967. The chromosomes of orchids II. Vandeae Lindl. Kew Bull. 21: 151-156. Miduno, T. 1940. Chromosomenstudien an Orchidazeen III. Uber das Vorkommen von haploiden Pflanzen bei Bletilla striata Reichb. f. var. gebina Reichb. f. Cytologia 11: 156..