The taxonomic identity of three varieties of Lecanorchis nigricans (Vanilleae, Vanilloideae, Orchidaceae) in Japan

Abstract To elucidate the taxonomy of the Lecanorchis nigricans Honda, 1931 species complex, the present study investigated the detailed morphology of three L. nigricans varieties in Japan. While L. nigricans var. patipetala Y.Sawa, 1980 and L. nigricans var. yakusimensis T.Hashim., 1990 have often been treated as synonyms of L. nigricans var. nigricans, the present study demonstrates that the three varieties are morphologically distinct. More specifically, L. nigricans var. nigricans only produces complete cleistogamous flowers and is distinct from the plants currently called “L. nigricans”, which are identical to the chasmogamous variety L. nigricans var. patipetala. The other chasmogamous variety L. nigricans var. yakusimensis can be easily distinguished from L. nigricans var. patipetala by its more spatulate tepals and higher cucullate lip. Therefore, the present study provides emended description of the three L. nigricans varieties based on type specimens and specimens collected from type localities. In addition, the isotype specimen of L. nigricans var. patipetala is designated as the lectotype because the holotype has been lost.


Introduction
The genus Lecanorchis Blume, 1856 (Vanilleae, Vanilloideae, Orchidaceae) is a group of mycoheterotrophic plants that includes ca. thirty species and/or varieties (Hashimoto 1990;Szlachetko and Mytnik 2000;Govaerts et al. 2016;. Members of the genus are characterised by the presence of a calyculus, a cup-like structure located between the base of the perianth and the apex of the ovary (Hashimoto 1990) and are distributed across Southeast Asia, including India, Thailand, Laos, Vietnam, Malaysia, China, Taiwan, Japan, the Philippines, Indonesia, New Guinea and the Pacific Islands (Hashimoto 1990;Szlachetko and Mytnik 2000;Averyanov 2011).
Precise identification of Lecanorchis taxa is often hindered by the similar morphology and brief flowering periods (Hashimoto 1990;Averyanov 2005;Suddee and Pedersen 2011;Tsukaya and Okada 2013;). In addition, important diagnostic characters are often lacking in herbarium specimens because the flowers of Lecanorchis members are easily dropped during preservation and the important diagnostic characters of some species have yet to be described in detail, especially for species that were first described many decades ago (reviewed by Suetsugu et al. , 2017a. Therefore, adequate taxonomic studies of the genus have yet to be conducted (reviewed by Suetsugu et al. , 2017a. The taxonomic identity of L. nigricans Honda, 1931 has remained particularly unclear. The species was first described from Wakayama Prefecture (Kinki District, Japan ;Honda 1931) and was subsequently reported from Taiwan, China, Thailand and Vietnam (Su 2000;Chen et al. 2009;Suddee et al. 2010;Hsu and Chung 2010;Vuong and Sridith 2016). Even though three L. nigricans varieties were described from Japan (Honda 1931;Sawa 1980;Hashimoto 1990), Yokota et al. (2016) defined the species in a broad sense, thereby synonymising L. nigricans var. patipetala Y. Sawa, 1980 andL. nigricans var. yakusimensis T. Hashim., 1990 as L. nigricans var. nigricans. However, it is possible that these treatments are based on the ambiguity of the original species description (Honda 1931;Sawa 1980;Hashimoto 1990) and that the species complex, in fact, comprises three entities.
To elucidate the taxonomy of the L. nigricans species complex, the present study investigated the detailed morphology of type specimens and specimens collected from type localities of three L. nigricans varieties in Japan. These findings revealed that the three varieties were morphologically distinct. More specifically, L. nigricans var. nigricans produces only complete cleistogamous flowers and is distinct from L. nigricans var. nigricans sensu Hashimoto 1990;Hashimoto et al. 1991;Nakajima and Ohba 2012;Yokota et al. 2016 (hereafter, the plants currently called "L. nigricans"), which is identical to the chasmogamous variety L. nigricans var. patipetala that was originally described from Kochi Prefecture (Shikoku District, Japan;Sawa 1980). In addition, the other chasmogamous variety L. nigricans var. yakusimensis, from Yakushima Island (Ryukyu Islands, Kagoshima Prefecture, Japan; Hashimoto 1990) can be distinguished from L. nigricans var. patipetala by its more spatulate tepals and higher cucullate lip. Therefore, the present study provides emended description of the three L. nigricans varieties based on type specimens and specimens collected from type localities.

Morphological observation
In order to compare the morphologies of the three Lecanorchis nigricans varieties with previously recorded species, the authors reviewed the literature, conducted field sampling throughout Japan and examined both digitised plant specimens from online databases such as JSTOR Global Plants (http://plants.jstor.org/) and Plants of Taiwan (http://tai2.ntu.edu.tw/specimeninfo.php) and specimens from the following herbaria: TI, TNS, KYO, KPM, OSA, MBK, KOCH and KAG. Herbarium abbreviations follow Index Herbariorum (Thiers 2017, http://sweetgum.nybg.org/science/ih/). In total, at least 30 flowers were examined from 10 flowering plants to understand the morphological variations for each variety.

DNA barcoding
For DNA isolation, the flowers of L. nigricans var. nigricans, L. nigricans var. patipetala, L. nigricans var. yakusimensis and their closely-related species L. taiwaniana S.S.Ying 1987 emend. Suetsugu, T.C. Hsu, S. Sawa, & Fukunaga 2016 were collected and desiccated in the field using silica gel (Table 1). DNA was extracted from these silica-dried plant materials, using the CTAB method (Wu et al. 2001). The rDNA internal transcribed spacer (ITS) region was amplified from the extracted DNA samples in 10 μL PCR mixtures that contained 2 μL extracted DNA, 0.05 μL TaKaRa Ex Taq Hot Start Version (Takara Bio, Japan), 10 μM of each primer (AB101 and AB102; Douzery et al. 1993), 0.25 μM of each dNTP and 1 μL 10× buffer, using an iCycler (BioRad, Japan) and the following conditions: initial denaturation at 94 °C for 5 min; followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min; followed by a final elongation at 72 °C for 7 min. The amplified PCR products were purified using EconoSpin (Gene Design, Inc.) columns and the subsequent samples were sent for sequencing to Eurofins Genomics (Ebersberg, Germany). The primers used for amplification were also used for sequencing.

Results and discussion
Even though Honda's original description of Lecanorchis nigricans was insufficient in that most diagnostic characteristics were overlooked, he noted that neither the species' sepals nor petals were open and that they, instead, were united, forming a cylindrical perianth tube (Honda 1931). This is quite different from the characteristics of the plants currently called "L. nigricans", whose flowers are widely open (Hashimoto 1990;Hashimoto et al. 1991;Nakajima and Ohba 2012;Yokota et al. 2016). Indeed, the analysis of type specimens and specimens collected from type localities revealed that the flowers of L. nigricans var. nigricans remain completely closed throughout their flowering period (  ). Therefore, the same name should not be used for both the plants currently called "L. nigricans", whose flowers are widely open and which is much more common throughout Japan (Hashimoto 1990;Hashimoto et al. 1991;Nakajima and Ohba 2012;Yokota et al. 2016). The chasmogamous variety of L. nigricans was initially described by Sawa (1980) as L. nigricans var. patipetala. It was found that there are no clear morphological differences amongst the plants currently called "L. nigricans", L. nigricans var. patipetala lectotype specimens and L. nigricans var. patipetala type locality specimens (Table 2; Figs 4-5). Therefore, the name L. nigricans var. patipetala should be used for the common chasmogamous variety of L. nigricans that is found throughout Japan, with the exception of the Ryukyu Islands. Detailed morphological investigation revealed that L. nigricans var. patipetala could also be distinguished from L. nigricans var. nigricans by its larger perianth tube (14-17 mm vs. 11-14 mm), the shorter coloured area of its lip (ca. apical 1/3-1/5 vs. ca. apical 1/2-1/3), the shape of its lip apex in the natural situation (broadly rounded vs. acute), the status of lip hairs near apex (scarce, long and rarely branched multicellular hairs vs. dense, short and frequently branched multicellular hairs), the shape of the column (recurved vs. slightly recurved), the width of its petal base (narrow ca. 1.0-1.3 mm vs. relatively wide ca. 1.5-2.5 mm) and the shape of its anther cap (strongly bilobed v.s. slightly bilobed; Table 2; Figs 1-5).
In addition, the other chasmogamous variety L. nigricans var. yakusimensis was described from Yakushima Island (Ryukyu Islands, Japan). However, even though pubescence at the ventral side of the column was highlighted as the variety's diagnostic character (Hashimoto 1990;Hashimoto et al. 1991), the column of L. nigricans var. patipetala also varies from glabrous to slightly hairy. Nonetheless, the column of L. nigricans var. patipetala is less hairy than that of L. nigricans var. yakusimensis. In addition, L. nigricans var. yakusimensis possesses more spatulate sepals and petals, as well as more highly cucullate lips, whereas L. nigricans var. patipetala possesses more oblong sepals and less cucullate lips. Furthermore, L. nigricans var. yakusimensis can be distinguished from L. nigricans var. patipetala by its wider anther caps (ca. 2.0 mm. vs. ca. 1.5 mm) and more recurved column. Thus, L. nigricans var. yakusimensis can ata of the related species from  and  be distinguished not only by its more hairy column, but also its tepals, lip, anther cap and column shape (Table 2; Figs 4-7). As L. nigricans var. yakusimensis is more common than L. nigricans var. patipetala in both Yakushima and Taiwan (Suetsugu and Hsu, unpublished data), it is likely that the variety is also distributed on the other Ryukyu Islands. Thus, L. oligotricha Fukuy. 1942 that has been described from Iriomote Island (Ryukyu Islands), may actually be identical to L. nigricans var. yakusimensis, while L. oligotricha has been considered as a synonym of L. nigricans var. nigricans. Even so, the name L. nigricans var. yakusimensis is preferred because the taxon should be recognised as an intraspecific variety, instead of an independent species. In addition, it should be noted that some L. nigricans specimens from the Ryukyu Islands were misidentifications of L. taiwaniana. However, it is unlikely that L. oligotricha is synonymous with L. taiwaniana, owing to differences in sepal and petal shape, according to the protologue (Fukuyama 1942). Unfortunately, the type materials of L. oligotricha in KPM are poorly preserved and no mature flowers are available for dissection (Inoue et al. 1998). Therefore, further investigation of L. oligotricha specimens from the species' type locality will be critical to clarifying the species' taxonomic status.
Based on the findings of the present study, it is suggested that the two varieties L. nigricans var. yakusimensis and L. nigricans var. patipetala should be revived since the distinct morphological characteristics of the three varieties are clear and stable. It is also considered that the aforementioned differences amongst the three varieties are relatively minor and represent interspecific variation. The identical DNA barcode sequences of L. nigricans, L. nigricans var. patipetala and L. nigricans var. yakusimensis also support this conclusion, whereas the sequence divergence of the three L. nigricans varieties and L. taiwaniana (i.e. 5 substitutions) support the independent specific status of both L. nigricans and L. taiwaniana, even though the two are sometimes considered synonymous (e.g. Su 2000). Actually, L. taiwaniana and its closely-related species L. tabugawaensis Suetsugu & Fukunaga 2016 can easily be distinguished from the three varieties of L. nigricans by having taller inflorescences, longer and lighter coloured rachis, yellowish-white, narrower sepals and petals and brighter brown suberect capsules ).
Note. When describing L. nigricans var. patipetala, Sawa (1980) cited the specimens that he had collected from Ikku (Kochi Prefecture) on 5 August 1978. However, even though Sawa reported that the holotype specimen had been deposited in MBK and that the isotype specimens had been deposited in KYO and KOCH, no specimens fitting Sawa's description could be located, despite intensive surveys of MBK, KYO and KOCH. The only putative original specimen that was found was a specimen in MBK that was collected by Sawa from Ikku (Kochi Prefecture) on 5 August 1979. This specimen has already been treated as an isotype by the MBK curator. The status of the specimen is somewhat controversial since both the collection date (5 August 1979vs. 5 August 1978 and collection number (O-101 vs. O-135) differ from those of the L. nigricans var. patipetala protologue. However, the MBK specimen should still be recognised as an L. nigricans var. patipetala isotype, because the collection dates are similar enough that the difference could be regarded as a typing error. Actually, Hashimoto (1990), who investigated the L. nigricans var. patipetala holotype when the specimen was still preserved in MBK, cited the collection date as 5 August 1979 and collection number O-101. Actually, the collection number O-101 was cited as a collection number for the Gastrodia pubilabiata holotype in the paper by Sawa (1980) that described both L. nigricans var. patipetala and G. pubilabiata. It is highly possible that the collection number for L. nigricans var. patipetala and G. pubilabiata was somehow reversed in the protologue. Therefore, in order to stabilise the taxonomic status of L. nigricans var. patipetala, the MBK isotype was designated as the lectotype, according to Articles 9.11 and 9.12 of the ICN (McNeill et al. 2012   J-shaped or complex, ligneous. Roots simple, radiate numerous, horizontally or downward elongate to 20-30 cm long, yellowish brown. Rachis 2-8 cm, 3-15 flowered, internode length of upper-half of rachis, 1-6(-10) mm. Floral bracts triangular, acute, 0.7-2.0 mm long. Pedicellate ovary ascending, 14-30 mm long. Flowers widely opening, ca. 2.5 cm in diameter. Sepals purplish white, linear, oblanceolate-spatulate, ca. 13-17 mm long, 3.3-4.0 mm wide, apex obtuse. Petals purplish white, linear, oblanceolate-spatulate, 13-17 mm long, 3.3-4.0 mm wide, apex obtuse. Lip spatulate to cucullate, strongly concave, 12-15 mm long, ca. 4.5 mm wide in natural situation, 7.5-8.0 mm wide when flattened, disc with rather scarce, long multicellular hairs which are rarely branched, near apex. Column 10-13 mm long, recurved, fused with lip about 1/2 its length, ventrally densely puberulent; anther purplish white, ca. 2.0 mm wide. Capsule 20-30 mm long, cylindrical-fusiform, black, ascending at 70-90 degree angle from axis. Flowering in mid-July to mid-September.