68 Cytologia 28

Chromosome Numbers and Genome Relationships

of Some Species in the Nigrohirsutae Section
of Dendrobium1

K. Shindo and H. Kamemoto

Received August 6, 1962

Among the 600 or more recognized genera in the Orchidaceae, Den

drobium is perhaps the largest and most diverse, comprising over 1,000
described species (Schlechter 1927; Holttum 1953; Hawkes 1957). This
large and unwieldy genus has been subdivided into numerous sections on
the basis of the external morphology of species. According to Holttum
(1953), the section Nigrohirsutae includes about 35 species distributed from
the Himalayas and Southern China through Malaysia to the Philippines. The
characteristic feature of this group is the black or brown hair covering the
leaf sheaths. The large, attractive, long-lasting white flowers of several
members of this group and their hybrids are considered highly desirable
horticultural characteristics.
Although information on the cytology of orchids has been accumulating
rapidly in recent years (Duncan 1959), only a small fraction of Dendrobium
species in existence has been cytologically investigated. In the Nigrohirsutae
section, the chromosome number of only 2 species, D. infundibulum and
D. formosum, has been recorded. The former was reported to be 2n=40
(Hoffmann 1930), while the latter was recorded as 2n=38 (Ito and Mutsu
ura 1957; Kosaki and Kamemoto 1961). The chromosome numbers of
species in other sections of Dendrobium also fall into either 2n=40 or 38
with the latter predominating (Ito and Mutsuura 1957; Kosaki 1958;
Vajrabhaya and Randolph 1960; Kosaki and Kamemoto 1961).
The present investigation was undertaken to expand the chromosome
number determinations of species in the section Nigrohirsutae and to possibly
elucidate species relationship through observations on meiotic behavior in
interspecific hybrids.

Materials and methods

Excepting D. draconis, all species and hybrids investigated were obtained

from the Foster Botanical Garden and orchid nurseries in Honolulu, Hawaii.

Root tips were severed, pretreated in 0.002 M aqueous solution of 8-hydroxy

- quinoline for 5-6 hours at 20-25•Ž, fixed in 1:1:2 mixture of chloroform,

1 Published with the approval of the Director of the Hawaii Agricultural Experiment

Station as Technical Paper No. 590. This study was supported by the National Science

Foundation (G-13582).
 1963 Chromosome Numbers and Genome Relationships in Dendrobium 69

Figs. 1-10. Species and hybrids in the Nigrohirsutae section of Dendrobium. 1, D. schuetzei.

2, D. formosum. 3, D. draconis. 4, 8, D. schuetzei •~D. sanderae. 5, 9, D. schuetzei •~

D, dearei. 6, D. sanderae •~D. dearei. 7, 10, D. dearei •~D. formosum. Figs. 1-7, ca,
3/10•~. Figs. 8-10, ca. 1/18•~.
 70 K. Shindo and H. Kamemoto Cytologia 28

95% ethyl alcohol, and glacial acetic acid for 30 or more minutes at room
temperature, macerated in 1:1 mixture of 95% ethyl alcohol and concentra
ted hydrochloric acid for 5 minutes at room temperature, kept in 45% acetic
acid for a minimum period of 10 minutes to promote the stainability of
chromosomes, and finally squashed and stained in 1% aceto-orcein. For
observation of meiosis, bud materials were prepared essentially as above but
omitting the pretreatment and maceration.

Results

Chromosome Number

Both D. schuetzei (Fig. 1) and D. sanderae were found to be 2n=40

(Fig. 11), while D. formosum (Figs. 2, 12) and D. draconis (Figs. 3, 13) were

2n=38. The chromosome numbers of hybrids were 2n=40 for D. schuetzei

•~ D. sanderae and D. schuetzei•~D. dearei, 2n=60 for D. sanderae•~D.

dearei and 2n=39 for D. dearei•~D. formosum (Table 1). From the above

observations, it can be concluded that the chromosome of D. dearei, which

was not examined cytologically, is 2n=40.

Table 1. Chromosome numbers of species and hybrids in the Nigrohirsutae section of

Dendrobium

Conspicuous differences in chromosome size were apparent in root-tip

cells of species. The chromosomes of D. formosum were twice as large as

those of D. sanderae, while those of D. draconis were of intermediate size

(Figs. 11-13).

Meiotic Behavior

D. schuetzei-As to be expected, meiosis in D. schuetzei was regular,

showing 20 bivalents in metaphase I (Fig. 14, Table 2). Two exceptionally

large bivalents were evident.

D. schuetzei•~D. sanderae (Figs. 4, 8)-Meiosis was regular in this

hybrid with the formation of 20 bivalents (Fig. 15, Table 2), the orderly

separation of sister chromosomes at anaphase I, and the distribution of 20

 1963 Chromosome Numbers and Ge
nome Relationships in Dendrobium 71

Table 2. Mean metaphase I configurations of chromosomes i

n PMCs of species and hybrids
in the Nigrohirsutae section of Dendrobium

Figs. 11-13. Somatic metaphase chromosomes of species. 2100•~. 11, D. sanderae. 12,

D. formosum. 13, D. draconis.

chromosomes to each microspore. Two conspicuously heteromorphic bival

ents were visible at metaphase I. These represented the pairing of the 2

large chromosomes contributed by the D. schuetzei parent with their homo

logues from D. sanderae which are much smaller in size. These large

chromosomes which were again visible at metaphase of microspore division

were dumbbell shaped and approximately twice as large as the rest of the

chromosomes (Fig. 19).

D. schuetzei•~D. dearei (Figs. 5, 9)-Meiotic behavior of this hybrid

was similar to that of the above, exhibiting 20 bivalents at metaphase I and

72 K. Shindo and H. Kamemoto Cytologia 28

20 chromosomes in each of the microspores (Fig. 16, Table 2). The large
chromosomes of D. schuetzei formed heteromorphic pairs at metaphase I and
were easily distinguishable at microspore division.
D. sanderae•~D. dearei
Table 3. Metaphase I configurations of chromo
(Fig. 6)-Metaphase I of this somes in PMCs of the triploid hybrid D. sanderae
triploid plant exhibited configu •~ D. dearei

rations with various proportions

of trivalents, bibalents, and uni

valents (Fig. 17, Tables 2, 3).

The number of trivalents varied

from 19 to 13 and bivalents and

univalents from 7 to 1, but

within a single PMC, the num

ber of bivalents usually equaled

that of univalents. Chromosome

distribution at anaphase I was

unequal and ultimately gave rise to tetrads or tetrads with microcytes (Fig.
20).
D. dearei•~D. formosum (Figs. 7, 10)-Unlike the above diploid hybrids,

this hybrid exhibited considerable irregularity of meiosis (Fig. 18, Table 2).

Bivalents varied from 19 to 11 with the mean of 15.9 and univalents from

14 to 1 with the mean of 6.9. Trivalents were also observed, but these

were considered to be pseudo-associations. The majority of bivalents were

heteromorphic, reflecting the chromosome size difference of the parental

genomes. As indicated in the photomicrographs of mitotic chromosomes

(Figs. 11, 12), the larger chromosomes of this hybrid must have been con

tributed by D. formosum.

The bivalents separated normally to both poles, while the univalents

often lagged but eventually got included in the daughter nuclei or remained

at the plate to become incorporated into micronuclei. The products of meiosis

were predominantly tetrads, with smaller proportions of tetrads plus micro

cytes, triads, triads plus microcytes and dyads (Fig. 21).

Discussion
D. schuetzei, D. sanderae and D. dearei are endemic to the Philippines,
while D. formosum is distributed from the Himalayas to Peninsular Siam
and D. draconis from Tenasserim to Cochinchina. The chromosome numbers
of all three Philippine species are 2n=40, while the extra-Philippine species
are 2n=38.
The complete chromosome pairing in interspecific diploid hybrids involv
ing Philippine species provides conclusive evidence of the close phylogenetic
relationship of these species. The formation of numerous trivalent associations
in the triploid hybrid between D. sanderae and D. dearei also confirms the
 1963 Chromosome Numbers and Genome Relationships i
n Dendrobium 73

Figs. 14-21. Meiosis and microspore division of species and hybrids in Nigrohirsutae
section of Dendrobium. 2100•~. 14, D. schuetzei, 20II. The arrows point to the two large
bivalents. 15, D. schuetzei•~D. sanderae, 20II. The arrows point to the two heteromor

phic bivalents. 16, D. schuetzei•~D. dearei, 20II. The arrows point to the two hetero
morphic bivalents. 17, D. sanderae•~D. dearei, 14III+6II+6I. 18, D. dearei•~D. for

mosum, 17II+5I. Note the heteromorphic bivalents. 19, D. schuetzei•~D. sanderae,

microspore division showing 20, 20, 20, 20 chromosomal distribution. Note a single large
dumbell-shaped chromosome in each microspore. 20, D. sanderae•~D. dearei, anaphase I.
21, D. dearei•~D. formosum, dyad with unreduced chromosome number.
74 K. Snindo and H. Kamemoto Cytologia 28

close homology of parental genomes. Since no tetraploid species has been

encountered, it is presumed that this triploid hybrid arose through the func
tioning of an unreduced gamete, which is not an uncommon event in orchids.
A slight morphological divergence of chromosome sets has occurred,
particularly in D. schuetzei as evidenced by the 2 extremely large chromo
somes which are absent in either D. dearei or D. sanderae.
D. formosum shows considerable chromosomal variation from the
Philippine species. The chromosome number is 38 instead of 40, chromo
somes are much larger, and furthermore, the hybrids between it and D. dearei
exhibited about 16 bivalents, the majority of which were heteromorphic owing
to the difference in size of the parental chromosomes. The retention of the
close homology of chromosomes, despite the change in chromosome number
and size, may indicate their relatively recent evolution from a common an
cestry.
The occurrence of species with chromosome numbers of 2n=38 or 2n=
40 within the section Nigrohirsutae poses the question of which is the most
likely evolved form. Earlier studies on chromosome numbers on Dendrobium
have indicated 2n=40 as the common number (Hoffman 1930; Eftimiu-Heim
1941; Ito and Mutsuura 1957), but according to the more recent reports,
2n=38 seems to predominate (Kosaki 1958; Vajrabhaya and Randolph 1960;
Kosaki and Kamemoto 1961). It might, therefore, be presumed that 2n=38 is
the more primitive form in Dendrobium. However, there is the possibility
that evolution has proceeded in either direction. Further studies on other
members of the Nigrohirsutae section as well as the entire genus are necessary
to establish the basic number for Dendrobium.

Summary

Chromosome numbers and meiotic behavior of species and hybrids in

the Nigrohirsutae section Dendrobium were investigated. The chromosome

number of the Philippine species, D. schuetzei and D. sanderae was 2n=40,

while that of the extra-Philippine species, D. formosum and D. draconis,

was 2n=38.

The chromosomes of D. formosum were about twice as large as those

of D. sanderae, and those of D. draconis were intermediate in size.

Meiosis in D. schuetzei and its hybrids with D. sanderae and D. dearei

was regular with the formation of 20 bivalent chromosome at metaphase I .

The triploid hybrid, D. sanderae•~D. dearei, formed 19-13 trivalents and

7-1 bivalents and univalents. D. dearei•~D. formosum, having 39 chromo

somes, exhibited 19-11 bivalents with a mean of 15.9, most of which were

heteromorphic.

It can be concluded that the Philippine species are very closely related

to each other while D. formosum is more distantly related.

1963 Chromosome Numbers and Genome Relationships in Dendrobium 75

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