402 Cytologia 27 Genome Relationships of Neofinetia Hu and Some Allied Genera of Orchidaceae1 K. Shindo and H. Kamemoto Hawaii Agricuttural Experiment Station, University of Hawaii, Honolulu, Hawaii, U. S. A. Received April 11, 1962 Neofinetia falcata (Thumb) Hu, an endemic species in Japan (Ohwi 1953), belongs to a monotypic genus in the sub-tribe Sarcanthinae of the Or chidaceae. Its phylogenetic relationship has not been clearly defined. Hawkes (1961) recently placed it in the group Saccolabiae, while Lindley included it in the genus Angraecum, and Schlechter in the genus Angraecopsis (Schlechter 1927). Angraecoid orchids are native to the African region and are geo graphically isolated from Vandaceous orchids. If N. falcata is an Angraecoid, the evolution of this species is indeed perplexing in view of its wide disjunction in geographical distribution. Recent attempts by orchid breeders of Hawaii in hybridizing N. falcata with some Vandaceous species have met with rewarding success. The flower ing of some of these unique, miniature intergeneric hybrids has enabled the analysis of the genomes of parental species and the elucidation of their phylogenetic relationships. The significance of cytology in clarifying the taxonomy and evolution of orchids has been established in earlier reports (Tanaka and Kamemoto 1961, Kamemoto and Sindo 1962). This paper presents cytological evidences that N. falcata is very closely related to Van daceous orchids. Materials and methods Plants for this study were obtained from M. Yamada and E. Iwanaga2, orchid breeders of Hawaii. N. falcata•~Vanda lamellata and N. falcata •~ Ascocentrum miniatum were hybridized and flowered by M. Yamada, while N. falcata•~Asc. ampullaceum and N. falcata•~Aerides jarckianum were hybridized and flowered by E. Iwanaga. After critical cytological examinations, careful description of plant and flower characteristics and photographing in color and black and white, plants were returned to the respective owners. The value of orchid plants prohibited the preparation of herbarium specimens. As a substitute, kodachrome slides 1 Published with the approval of the Director of the Hawaii Agricultural Experiment Station as Technical Paper No. 568. This study was supported by the National Science Foundation (G-13582). 2 The authors express their gratitude to both breeders for generously providing plant materials. 1962 Genome Relationships of Neofinetia Hu and Some Allied Genera 403 were made and field for validating purposes. Pollinia were fixed in a 1:1:2 mixture of chloroform, 95% ethanol and Figs. 1-9. Species and hybrids of Neofinetia and allied genera. 1, Neofinetia falcata. G, Vanda lamellata. 3, flowers of V. lamellata. 4, N. falcata•~V. lamellata. 5, flower of N. falcata•~Ascocentrum miniatum. 6, Aerides jarckianum. 7, flower of A. jarckianum. 8 , N. falcata•~A. jarckianum. 9, flowers of N. falcata•~A. jarckianum, 404 K. Shindo and H. Kamemoto Cytologia 27 glacial acetic acid for 10 to 30 minutes at room, temperature. They were transferred to 45% acetic acid for 5 to 10 minutes and stained with 1% aceto-orcein. Observation N, falcata•~Vanda lamellata. N. falcata is a relatively small plait with fleshy leaves of about 1.2cm wide and 10cm long (Fig. 1). The small white flowers, measuring about 1.5cm. across, are characterized by a long falcate spur of about 5cm in length. V. lamellata Ldl. (Figs. 2, 3), one of the diminutive members of strap-leaved Vanda, is native to the Philip pines. The plant habit is similar to N. falcata except for its slightly larger size. Its recurved, channelled leaves are about 2.5cm wide and 20cm long. Flowers are 3-4cm across, are yellow with brown spots and have short spurs of about 5mm. The hybrid between these two species is somewhat intermediate in general plant habit (Fig. 4). The pale orange flowers have slightly curved spurs of about 1.3cm in length. The chromosome number of the hybrid was 2n=38. At metaphase I, 19-17 bivalent chromosomes were observed (Table 1, Fig. 10). The arrange - Table 1. Metaphase I configurations in PMCs of intergeneric hybrids of Neofinetia, 2n=38 ment of the bivalents at the equatorial plane was regular. Each bivalent consisted of 2 chromosomes of equal size, and was of the rod or ring form with terminalized or nearly terminalized chiasmata. At anaphase I, the bivalents separated regularly to both poles, while the univalents often lagged between the two anaphase groups. The products of meiosis were observed as tetrads and tetrads with 1-2 microcytes (Table 3). As a consequence of the slight irregularity in the distribution of chromosomes during meiosis, the chromosome, number of microspores varied from 17 to 21, with the highest frequency at 19. Microcytes contained one or two chromosomes. N. falcata•~Ascocentrum miniatum. Asc. miniatum (Ldl.) Schltr. is similar to V lamellata except for the slight overall reduction in plant parts. It is distributed from the Himalayas to Java. The flower color is bright yellow or orange-yellow, and the length of spur is about 8mm. The hybrid has fleshy, linear leaves and dark orange-yellow flowers with spurs of about 1.3cm long (Fig. 5). The chromosome number of the hybrid was 2n=38. Chromosome pair 1962 Genome Relationships of Neofinetia Hu and Some Allied Genera 405 ing at meiosis was nearly regular, forming 19 bivalents in a majority of PMCs (Table 1, Fig. 11). Only a few PMCs had 18 bivalents and 2 univalents. The bivalents were of rod or, ring form with terminalized chiasmata and con Figs. 10-15. Chromosomes of intergeneric hybrids of Neofinetia falcata. •~1400. 10, N. falcata•~Vanda lamellata, 19II. 11-12, N. falcata•~Aseocentrum miniatum. 11, 1911. 12, tetrad of microspores with 19 chromosomes each. 13, N. falcata•~Asc ampullaceum, 1911. 14-15, N. falcata•~Aerides jarckianum. 14, 2IV+1III+6II+15I. 15, microspores and microcytes with 24. 24, 13, 13 and 2 chromosomes. sisted of two chromosomes of the same size. Most of the PMCs formed normal tetrads at the end of meiosis, but a few formed tetrads with 1-2 microcytes (Table 3, Fig. 12). The chromosome number of microspores ob 406 K. Shindo and H. Kamemoto Cytologia 27 served at pollen mitosis was 19 in the majority of sporad groups. N. falcata•~Ascocentrum ampullaceum. Asc. ampullaceum Schltr. is similar to Asc. miniatum in general plant characteristics. It is distributed from the Himalayas to Burma. The small violet-purple flowers measuring 1.7cm across have spurs of about 1.2cm long. The general plant habit of the hybrid is similar to those of N. falcata•~Asc. miniatum. It was not possible to obtain details on flower structure, size and coloration due to the complete use of the few buds that were available for meiotic studies. The chromosome number of this hybrid was 2n=38. The number of bivalents observed at meta phase I was 19-17 with a mean of 18.5 (Table 1, Table 2, Fig. 13). The bivalents regularly arranged themselves at the equatorial plane and at anaphase I separated to both poles, while univalents often remained suspended between the two anaphase groups to form micronuclei at the end of the first division. N. falcata•~Aerides jarckianum. A. jarckianum Schltr. has large, recurved strap leaves of 15-25cm in length and light burgundy-colored flowers that are slightly smaller than those of N. falcata (Figs. 6, 7). The slightly curved spurs are about 5mm long. It is indigenous to the Philippines. The hybrid has medium fleshy leaves of about 10cm long and 1.8cm wide, and purplish flowers of about 1.7cm across with falcate spurs of about 1.2cm long (Figs. 8, 9). The chromosome number of the hybrid was 2n= 38. Meiosis was highly irregular. Chromosome con figurations were comprised of various combinations of univalents, bivalents, and multivalents (Fig. 14). In cluding pseudobivalents, the number of bivalents varied from 10 to 2 with a mean of 6.0, and that of univalents from 22 to 6 with a mean of 14.8 (Table 2). Trivalents were most common among multivalents, but the majority of trivalents and almost all of the higher multivalents appeared to he associations of chromosomes by matrix connection. The resulting sporads contained microspores and microcytes of variable number and size, but dyads and tetrads with or without microcytes were most common (Table 3, Fig. 15). The following Table 2. Mean metaphase I configurations of PMCs in intergeneric hybrids of Neofinetia 1962 Genome Relationships of Neofinetia Hu and Some Allied G enera 407 chromosome numbers were observed in nuclei of sporads at microspore di vi sion: 1-4, 6, 8, 10-13, 16-22, 24-28 , 30, 32, 34, 36-38, and 40 . Table 3. Sporad formation in intergeneric hybrids of Neofi netia Discussion N. falcata•~V. lamellata , N. falcata•~Asc. nuniatum, and N. falcata •~ Asc. ampullaceum showed 19-17 bivalents with means of about 18 .6 at metaphase I, indicating a strong homology of the parental genomes , while N. falcata•~A. jarckianum showed only 10-2 bivalents with a mean of 6 .1. A conclusion immediately drawn from these observations is that N . falcata is phylogenetically very closely related to strap-leaved Vanda1 and Ascocentrum , and more distantly related to Aerides. These results confirm the conclusion of Tanaka and Kamemoto (1961) that Vanda and Ascocentrum are phylo genetically very closely related to each other, whereas Aerides is somewhat more distantly related to both. Deta on chromosome pairing did not provide any positive evidence on the evolutionary sequence among Vanda , Ascocentrum, and Neofinetia, but on the basis of the evolutionary trends of morphological characteristics of Orchidaceae (Holttum 1958, Dressler and Dodson 1960) , Vanda might be considered ancestral to both Ascocentrum and Neofinetia . Vanda and Ascocentrum have their evolutionary center in Southeast Asia.