Calanthe xhsinchuensis (Orchidaceae), a new natural hybrid from Taiwan

Background Natural hybridization in plants is a vital mechanism of speciation. Calanthe arisanensis and C. sieboldii occur in the same habitat in northern Taiwan, where there are a number of plants whose morphologically characters are similar in between of these two species. In this report, a new natural hybrid, Calanthe species – C. xhsinchuensis Y.I. Lee putatively derived from the natural hybridization of C. arisanensis and C. sieboldii was described and illustrated. Besides, somatic chromosome number was counted. Results The morphological and histological data of flowers, capsules, roots and leaves of C. xhsinchuensis show an intermediate condition between its putative parents. The chromosome number, 2n = 40, is also congruent with its putative parents. Color plates and line drawings are provided to aid in identification. Conclusion Base on the morphological characteristics of reproductive and vegetative organs, and the distributions of putative parents, it is proposed that C. xhsinchuensis is a natural hybrid between C. arisanensis Hayata and C. sieboldii Decaisne ex Regel. Electronic supplementary material The online version of this article (doi:10.1186/1999-3110-54-25) contains supplementary material, which is available to authorized users.


Background
Natural hybridization is an important mechanism of plant evolution (Arnold, 1997). Fertile hybrids may intercross or backcross with parental species, producing abundant genetic and phenotypic variation for natural selection. The genus Calanthe comprises more than 170 species widely distributed from Africa, Asia, Pacific Islands and Australia (Jin and Li, 2007). This genus is characterized by pubescent roots, often clustered leaves, petals and sepals similar, spurred lip and eight pollinia. In Taiwan, there are 19 species of Calanthe (Su, 2000;Flora of Taiwan). Calanthe arisanensis is an endemic species with pinkish white flowers that could be found throughout the island at the altitudes about 1000 meter (Lin, 1988;Su, 2000). Calanthe sieboldii is closely related to Calanthe arisanensis with bright yellow flowers that could be found in the northern areas of Taiwan (Lin, 1988;Su, 2000). C. sieboldii is also distributed in Ryukyu Islands, southern Japan and Korea. In northern Taiwan, C. arisanensis and C. sieboldii occur in the same habitat, and they both bloom during March and April. In some natural habitats, besides these two Calanthe species, there are a number of plants whose morphologically characters are similar in between of C. arisanensis and C. sieboldii that are considered to be their natural hybrid. In this report, a new Calanthe species putatively derived from the natural hybridization of C. arisanensis and C. sieboldii was documented.

Material
Specimens of Calanthe xhsinchuensis, C. arisanensis and C. sieboldii were collected from Jiashih Township of Hsinchu County, Taiwan and cultivated in the greenhouse at National Museum of Natural Science, Taiwan for further morphological comparison, SEM, histological and cytological studies. The vouchers are deposited at TNM.

Chromosome preparation
The actively growing root tips were cut and pretreated in 2 mM 8-hydroxyquinoline at 25°C for 5 h to accumulate prometaphase cells, rinsed with distilled water, then fixed in fresh prepared Farmer's fluid (three parts of ethanol to one part of glacial acetic acid). Root tips were macerated with 6% cellulose (Onoauka R-10, Yakukt Honsha, Japan) and 6% pectinase (Sigma Chemical Co., St. Louis, Mo.) in 75 mM KCl, pH = 4.0 at 37°C for 30 min. After a brief wash with 45% acetic acid solution, root tips were squashed as described by Aoyama (1989), then the chromosomes were stained with DAPI in an antifade solution (Vector Laboratories, CA, USA). The images were captured digitally by a CCD camera attached to a Zeiss Axioskop 2 microscope (Carl Zeiss AG, Germany).

Scanning electron microscopy
The dry seeds were mounted on aluminum stubs with adhesive or silver paint, coated with 30 nm gold in a sputter coater and observed using a scanning electron microscope (S-3000 N; Hitachi, Japan).

Histological observations
Root sections were cut and fixed in 2.5% glutaraldehyde and 1.6% paraformaldehyde buffered with 0.05 M phosphate buffer, pH 6.8, for 24 hours at 4°C. After fixation, the samples were rinsed in three 15-min changes of buffer. The materials were dehydrated in an acetone series and embedded in the Spurr's resin (Electron Microscope Sciences, Washington, PA). Sections of 1 μm were obtained by a glass knife on an ultramicrotome (Leica, Wetzlar, Germany), and were stained with 0.1% alkaline TBO for 1 min at 60°C on a hot plate. The sections were viewed and the images were captured digitally using a CCD camera attached to a light microscope (Carl Zeiss AG).

Distribution
It is known mainly from the mountain area in Jiashih Township (1500 m) of Hsinchu County in northern Taiwan.

Etymology
The name of this species is derived from the Hsinchu County, the place of its discovery in northern Taiwan.

Notes
C. xhsinchuensis only occurs at the sites where both C. arisanensis and C. sieboldii could be found together. Base on the comparison of morphological characteristics of reproductive and vegetative organs of C. xhsinchuensis and its putative parental species (Table 1, Figures 2, 3 and 6), it is proposed that this unusual plants occur in Jiashih Township of Hsinchu County is a natural hybrid between C. arisanensis and C. sieboldii.
The root diameter of C. xhsinchuensis shows an intermediate condition between its putative parents (Table 1;    Figure 6). The root diameter of C. arisanensis ( Figure 6B) is the thickest among the three species, whereas the root diameter of C. sieboldii ( Figure 6C) is the thinnest. The velamen of C. xhsinchuensis is 4 to 5 celled-wide, and the cortex is 8 to 9 celled-wide ( Figure 6A). The hyphae, and pelotons could be found in the cells of the cortex. The other features, including leaf shape ( Figure 3) and flower color (Figures 2A,D and G) of C. xhsinchuensis are intermediate between those of C. arisanensis and C. sieboldii. The spur shape of C. xhsinchuensis is curved that is similar to the spur shape of C. arisanensis; while the ovary color of C. xhsinchuensis is light green that is similar to the ovary color of C. sieboldii ( Figures 2B,E and H). The capsule shape of C. arisanensis is characterized by having 6 longitudinal wing-like ridges, while C. sieboldii has no conspicuous wing-like ridges on its capsule. C. xhsinchuensis has 6 lightly longitudinal ridges on the capsule, showing an intermediate condition ( Figures 2C,F and I).

Conclusion
All the available data and the distributions of putative parents support the recognition of the new species C. xhsinchuensis is a natural hybrid between C. arisanensis Hayata and C. sieboldii Decaisne ex Regel. Width (cm) 1.6 -2.0 2.0 -2.3 1.5 -1.7 Figure 6 The cross sections of Calanthe roots. (A) Calanthe xhsinchuensis; (B) C. arisanensis; (C) C. sieboldii. Scale bar = 3 mm.