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. The ancestral species of N. falcata presumably suffered dispersal from this center to Japan. Being isolated, it developed specialized morphological characteristics that warranted the taxonomist's separation into the new genus Angraecum, Angraecopsis or Neofinetia. Undoubtedly the long spur of N. falcata was an important criterion for placing this species in the Angraecoid groups, Angraecum by Lindley, and Angraecopsis by Schlechter. However, in view of the fact that to date no hybrids have been realized between Angraecoids and Vandaceous orchids despite several attempts at hybridizations by orchid breeders, that hybrids between N. falcata and Vandaceous orchids

1 Vanda includes two distinct sub-groups , the strap-leaved and terete-leaved. Because genomes of these two groups are weakly homologous, Tanaka and Kamemoto (1961) sug gested that these groups "might possibly be classified into subgenera or even separated into two separate genera on the basis of cytological studies". Also, according to Holttum (1953), Schlechter proposed a new genus Papilionanthe for the terete-leaved group because of the specialized form. Since V. lamellata involved in this study belongs to the strap eaved group, Vanda will hereinafter refer to the strap-leaved group.

Cytologia 27, 1962 27 408 K. Shindo and H. Kamemoto Cytologia 27

have been obtained with facility, and that these hybrids exhibit strong homo logy of parental genomes, it can be concluded that N. falcata is not an Angraecoid, but a close relative of Vanda and Ascocentrum. On the basis of the strong homology of chromosomes of parental species in hybrids involving species of strap-leaved Vanda, Ascocentrum and Neo finetia, it might be logical to merge the above three genera. Tanaka and Kamemoto (1961) have suggested that Ascocentrum be included with the strap-leaved Vanda. The close relationship of Vanda and Ascocentrum is further indicated in the statement by Holttum (1953): "If the flowers of Vandas were reduced, without change of shape to a very small size, they would most nearly resemble Ascocentrum; and the species here called As cocentrum micranthum is perhaps no more than a very much reduced Vanda". If Ascocentrum is to be merged with Vanda, then it is logical to include Neofinetia falcata which shows strong chromosome affinity to both Vanda and Ascocentrum. The degree of phylogenetic relationship among the above three genera based on chromosome homology is comparable to that of species within the genus Renanthera (Kamemoto and Shindo 1962). If the separate genera are to be retained, then any phylogenetic scheme should exhibit the close relationship of these groups. Accordingly N. falcata should be removed from the group Saccolabiae in Hawkes' (1960) recent classification and placed in the Vandeae. Also, the Angraecoid synonyms of N. falcata should not be construed to represent a close phylogenetic relationship between N. falcata and the Angraecoid orchids. The close homology of the genomes of Neofinetia and those of Vanda and Ascocentrum suggests that N. falcata can be crossed to numerous species and hybrids of the two groups. Because miniturization is a recent trend in breeding of orchids, N. falcata may assume an important role in the breeding of orchids.

Summary

Meiotic chromosome behavior in four intergeneric hybrids of Neofinetia

falcata Hu was investigated. N. falcata•~Vanda lamellata Ldl., N. falcata •~ Ascocentrum miniatum Schltr., and N. falcata•~Ascocentrum ampullaceum

Schltr. showed 19-17 bivalent chromosomes with means of about 18.6 at metaphase I, indicating a strong homology of the parental genomes, whereas

N. falcata•~Aerides jarckianum Schltr. exhibited 10-2 bivalents with a mean

of 6.1, indicating a relatively poor homology of the parental genomes. On

the basis of chromosomal affinity, Neofinetia is very closely related to As

cocentrum and strap-leaved Vanda and more distantly related to Aerides.

Literature cited

Dressler, R. L. and Dodson, C. H. 1960. Classification and phylogeny in the Orchidaceae. Annals of The Missouri Botanical Garden 47: 25-68. 1962 Genome Relationships of Neofinetia Hu and Some Allied Genera 409

Hawkes, A. D. 1961. Orchids, Their Botany and Culture. Harper and Brothers, New York. 297 pp. Holttum, R. E. 1953. Flora of Malaya, Vol. 1. Orchids. Government Printing Office, Singa pore. 753 pp.- 1958. Evolutionary trends in the Sarcanthine orchids. Proc. Second World Orchid Conference. Harvard University Printing Office, Cambridge, Mass. pp. 40-48. Kamemoto, H. and Shindo, K. 1962. Genome relationships in interspecific and intergeneric hybrids of Renanthera. Amer. Jour. Bot. 49: 737-748. Ohwi, J. 1953. Flora of Japan. (In Japanese). Shibundo, Tokyo. 1383 pp. Schlechter, R. 1927. Die Orchideen. Paul Parey, Berlin. 959 pp. Tanaka, R. and Kamemoto, H. 1961. Meiotic chromosome behavior in some intergeneric hybrids of the Vanda alliance. Amer. Jour. Bot. 48: 573-582.

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