During this study, we only observed flowering of the V
. Miss Joaquim (Fig. E) and V. teres
(Fig. B) plants. Vanda hookeriana
, V. teres
and V. teres
did not flower. In order to identify the plants, we relied heavily on leaf morphology (Fig. ) and the plant labelling of V. hookeriana
and V. teres
at the Singapore Botanic Gardens. Identification of V. hookeriana
(Fig. A) was straightforward as it unmistakably possessed the characteristic leaf kink. Vanda hookeriana
stem and leaves were also clearly less stout than in V. teres
, a growth form that seems less vigorous. Our V. teres
(Fig. C) has leaves that are curved towards the stem, matching Holttum's description (Holttum 1953
). In addition, it possesses purple spots, which although not mentioned by Holttum to be present in V. teres
, is specifically mentioned as being absent from the variety aurorea
). Thus, our identification of V. teres
(Fig. D) relies on the absence of spots; the slight curvature of the leaves towards the stem is less pronounced than that in V. teres
; and it is the other most common variety besides V. teres
. Other V. teres
varieties are unlikely as V. teres
was mentioned by Holttum as having ‘straight purple-spotted leaves’ (Holttum 1953
Fig. 7 Pictures of fresh plant specimens used for PCR. (A) V. hookeriana; (B) V. teres var. alba; (C) V. teres var. andersonii; (D) V. teres var. aurorea; and (E) V. Miss Joaquim ‘Agnes’. Inset pictures show an enlarged view of leaves and white (more ...)
We were unable to find any sequence variation in the 600-bp region of the rbcL
gene sequenced. This finding is in accordance with many other studies that have shown that rbcL
has a slower rate of nucleotide substitution compared with other loci used for phylogenetic analyses. It is possible that sequencing a larger fragment of rbcL
may reveal some sequence variations. We found matK
to be a very useful locus for delineating the relationships between the plants studied. Sequence information obtained from matK
alone facilitated the identification of the pod parent of V.
Miss Joaquim. The matK
sequences alone were sufficient to differentiate V. teres
to the variety level. It is interesting to note that V. teres
has a sequence that is more similar to V. hookeriana
than any of the other V. teres
varieties (Fig. and Table ). Vanda teres
differs from V. hookeriana
by three bases and from the other two V. teres
varieties by seven bases. Only one base substitution distinguishes V. teres
from V. teres
(Fig. and Table ). We found that the nucleotide substitution rate throughout all three codon positions in matK
was fairly consistent, with a frequency of 0.4 for codon 1 and 0.3 for positions 2 and 3 (Table ). Despite our small data set, our finding is in agreement with other reports that the substitution rate is comparable between all codon positions (Johnson and Soltis 1994
; Hilu and Liang 1997
; Xiang et al. 1998
; Hilu et al. 2003
; Barthet and Hilu 2008
). The high rate of amino acid substitution in matK
seen in our study has also been noted in previous studies (Olmstead and Palmer 1994
; Barthet and Hilu 2008
The sample size of this study was restricted by the difficulty in finding plants in bloom or that were clearly labelled. In particular, while it was easy to find plants labelled V. teres, only two plants were found labelled to the level of variety—these were both labelled V. teres var. andersonii. Additional samples of V. hookeriana, V. teres var. alba, V. teres var. andersonii and V. Miss Joaquim ‘Agnes’ were obtained to address sampling effects. Amplification and sequencing of the extended 1.5-kb fragment of the matK gene, encompassing the standard barcode regions, from these additional plant samples and our existing specimens confirmed our earlier conclusions derived using the matK56F and matK1027R primers. We therefore conclude that the sequences obtained using the matK56F and matK1027R primers were indicative of the variation observed within the standard barcode regions defined by the 1R_KIM, 3R_KIM, matK390F and matK1326R primers.
The herbarium specimens served to confirm that V. teres
was the pod parent of the original V.
Miss Joaquim described in 1893. We have shown that the fresh sample of V. teres
is identical in sequence to the herbarium specimen. In addition, the fresh sample of V.
Miss Joaquim ‘Agnes’ is also identical in sequence to the herbarium specimen of V.
Miss Joaquim from 1893. This suggests that the contemporary and herbarium samples of V.
Miss Joaquim ‘Agnes’ share the same inferred maternal parent. The flowers of the present-day V.
Miss Joaquim remain morphologically similar to that described by Ridley (1893
) and depicted by the Royal Horticultural Society in 1897 (Royal Horticultural Society Orchid Committee 1897
; Royal Horticultural Society 2010
) (Fig. ).
Fig. 8 Pictures of flowers of specimens used in this study. (A) V. teres var. andersonii (J. B. Tay); (B) V. hookeriana (courtesy of the Singapore Botanic Gardens Library); (C) V. Miss Joaquim painted by Nelly Roberts in 1897 (Royal Horticultural Society 2010 (more ...)
Identification of plants can be challenging, if not impossible, when labels are lost or destroyed. We have shown that matK can be used to identify and distinguish terete Vanda species and varieties. In orchid hybridization, records of parentage are occasionally lost, ambiguous or erroneous. Due to the complexity of many orchid hybrids, sequencing nuclear genes could potentially be a long and difficult process. We have shown that DNA barcoding using the chloroplast gene matK is a useful technique for identification and/or confirmation of parentage, particularly when the maternal parent is disputed. We have shown that the direct PCR technique can be used to obtain amplification products from herbarium specimens. Due to the lack of high-molecular-weight DNA, the nested PCR technique may be employed to produce a higher yield, yet specific amplification product.