Taxonomic revision of Habenaria josephi group (sect. Diphyllae s.l.) in the Pan-Himalaya

Abstract Species of the Habenaria josephi group in the Pan-Himalaya region are revised, based on their morphological characters and results of previous molecular phylogenetics. Eight distinctive species are recognised; key to the species, taxonomic descriptions, illustrations and distribution maps are provided. Habenaria josephi is re-instated, based on morphological and molecular evidence; H. wolongensis is synonymised with H. aitchisonii, a neotype for H. tibetica and the lectotypes for H. balfouriana, H. fargesii, H. glaucifolia and H. clarkei are designated.


Introduction
Habenaria Willd. is a large genus in the Orchidaceae (Orchidoideae, Orchideae, Orchidinae), with about 891 species (Govaerts et al. 2020), most of which are terrestrial plants. The genus is distributed along the tropical, subtropical and temperate zones of the Old and New Worlds (Pridgeon et al. 2001;Batista et al. 2011) and has three main centres of diversity, i.e. eastern Asia, central and southern Africa and Brazil (Kurzweil and Weber 1992). The plants are characterised by frequently having simple or bifid petals, a tripartite lip, long rostellar arms, stalked stigmas and a well-developed nectariferous spur (Dressler 1993;Pridgeon et al. 2001).
Of about 208 species of Habenaria occurring in the south to east Asian biodiversity hotspot, roughly one fifth are represented in the Pan-Himalaya (Pearce and Cribb 2002;Chen and Cribb 2009;Rajbhandari and Rai 2017;Govaerts et al. 2020). Amongst the Asian clades of Old World Habenaria, one group with two basal leaves and with a temperate or alpine distribution is of particular taxonomic interest: the Habenaria josephi Rchb.f. complex, as the species belonging to this group formed a close alliance (clade XXIV) in a recent molecular study (Jin et al. 2017). Additionally, they share many morphological traits (e.g. pubescent scape and floral parts, mostly 2-lobed petals) and similar habitats, which often renders the species delimitation difficult; owing to this, some species were either misidentified or assigned different ranks in the past (Lang 1984;Pearce and Cribb 2002;Lucksom 2007;Chen and Cribb 2009;Choudhury et al. 2011;Maity et al. 2019). To address these taxonomic inconsistencies, here we attempt to revise the group in light of the recent molecular works (Jin et al. 2017;Raskoti and Ale 2019) and a broader examination of the herbarium specimens and literature. Our study shows eight species from the group occurring in the region, for which general morphological features, taxonomic description and illustrations are provided. Furthermore, a brief history of Habenaria section Diphyllae Kränzl. and an artificial key to the species are also given.

Study area
The study area Pan-Himalaya (also referred to as the PH hereafter) ranges from parts of Afghanistan in the west to the Yunnan Province of China in the east, forming a natural phytogeographic unit; it is further divided into 17 subregions (Hong 2015, Fig. 1).
This work is based on the review of relevant literature and examination of herbarium specimens, supplemented with observations made on living plants in natural habitats. The specimens of Habenaria collected from the PH (Fig. 1), preserved at the herbaria AMES, B, CAL, E, K, KATH, KUN, LD, LE, P, PE, S, TI, TUCH, UPS, W and WU (herbarium acronyms according to Thiers 2020) were thoroughly examined; CAL, KATH, PE and TUCH were personally visited and for others, online catalogues were utilised (e.g. 'www.cvh.ac.cn' for Chinese herbaria) and high-resolution images of putative type materials were requested.
More than 500 specimens were sorted and about 200 of them, occurring within the PH, were considered for taxonomic characterisation. Species descriptions are based on vegetative and reproductive features as observed directly and/or through stereomicroscopes (Nikon SMZ1000 and Leica S8 APO) for details. Floral parts were rehydrated in boiling water before their observation and measurements were made under the microscopes. Lip and spur morphology was particularly regarded as taxonomically-informative characters. For species delimitation, morphological species concept  (Cronquist 1978;Stuessy 2009), along with recent molecular phylogeny (Jin et al. 2017), was taken into consideration.
Data on phenology, habitat and distribution were derived from specimen labels and the distribution maps were prepared from the occurrence locations approximated to the corresponding PH county or district; the list of examined specimens is arranged in the geographical order of the PH (Hong 2015). Information on distribution outside the PH and on illustrations available from literature are also provided.

Results
The taxonomic history of Habenaria sect. Diphyllae Habenaria sect. Diphyllae is one of the 32 sections established by Kränzlin (1892) in his first worldwide revision of Habenaria, including 17 species from Africa and Asia. The sections he assigned were based on the degree of dissection of the petals and labellum and on the structure of the gynostemium. The presence of 3-lobed labellum, simple or bilobed broad petals and thick fleshy stigmatic processes were taken as the diagnostic characters for the section Diphyllae (Kränzlin 1892;Kränzlin 1901). Later, Summerhayes (1968) designated H. diphylla as the type for the section and ascribed 24 species from East Africa to it. Several sectional treatments of Habenaria are available for Neotropics and Africa (Batista et al. 2013;Summerhayes 1968), but comprehensive accounts of Asian species are lacking, though country-level treatments were attempted previously (e.g. Pearce and Cribb 2002). The present study has uncovered the occurrence of 11 species of H. sect. Diphyllae in the PH. Eight of these species are found in high mountain habitats (i.e. collected at an altitude above 1500 m) and represent a monophyletic clade, which is here designated as the H. josephi group. The three remaining species (H. reniformis, H. diphylla and H. acianthoides), in contrast, inhabit the tropical climate and are morphologically distinct (e.g. glabrous scape and floral parts, petals always simple) and will not be considered here.

General morphology
Tuber: One or two, globose, oval or elliptic, underground, with few roots dispersed around their junction to the stem. fargesii are deflexed. A prominent needle-like appendage present at the base of the lip near the opening of the spur is characteristic of H. szechuanica. Spur, too, exhibits remarkable variation; H. diplonema bears a very short spur, in H. aitchisonii, spur is shorter than ovary, while in the rest of the species, the spur is robustly longer than the ovary. The column is well demarcated in all of the species with parallel anthers and stout caudicles; H. josephi and H. glaucifolia have parallel, closely lingulate stigmatic process; the stigma of H. aitchisonii are concave surrounding the opening of the spur.
Habitat. Moist grassy hillsides, stream banks, in Betula forest; 3000-4600 m elev.   (1890) Kränzlin (1892Kränzlin ( , 1901 proposed the section Diphyllae to accommodate the species with two basal leaves and included H. clarkei, H. glaucifolia, H. diphylla, H. reniformis and H. aitchisonii in the section along with a few other Habenaria species. He followed Hooker's view regarding the position of H. josephi as a variety of H. aitchisonii, albeit with a note "Die var. Josephi Hook. f. ist nur eine Form, aus den höchsten der oben angegebenen Standorte stammend (the variety josephi is only a form occurring in the highest of the above-mentioned locations)" suggesting that the plant is merely a higher elevation variety of H. aitchisonii. Paradoxically, Kränzlin described H. clarkei in the same publication, based on a duplicate of Hooker 42 (the type specimen of H. josephi) from Sikkim, which was kept at B (distributed from Hooker's Herbarium at K). After an extensive search, it now appears that Hooker 42 had at least six duplicates: two at K and one each at P, AMES, B and LE, of which the specimen at B was lost during the Second World War, while at LE, the specimen could not be found during a recent search (fide Petr Efimov). The remaining four duplicates are still extant.

Note. Hooker
In the past, the taxonomic identity of Habenaria josephi became doubtful, often shifting from one name to another, sometimes as Habenaria aitchisoni var. josephi (Rchb.f.) Hook.f. or as H. diphylla var. josephi (Rchb.f.) Pearce & Cribb. Even in recent literature on the orchid species of Sikkim, its type locality is not uniform in this regard. Some botanists treat it as a variety of H. diphylla (Pearce et al. 2001;Pearce and Cribb 2002;Lucksom 2007;Choudhury et al. 2011), while Maity et al. (2019 regard it merely as another synonym of H. diphylla. A closer look at this species reveals it to be not only distinct morphologically, but also well characterised in terms of habitat and distribution. Whereas H. diphylla is predominantly a tropical species of moderate size (10-40 cm tall), broadly distributed from peninsular India to the Philippines, H. josephi is a small-sized (5-20 cm tall) high-elevation temperate to alpine species occurring from Sikkim eastwards to the Hengduan Mountains, i.e. it is endemic to the Pan-Himalaya. Pearce et al. (2001) provided an elaborate discussion on the phenetic variations that delineate this taxon from the other species, yet they assigned it to a varietal rank under H. diphylla. Examination of the type and other dried specimens, as well as living individuals, clearly shows that it is distinct. Short stature, scape without sterile bracts, 2-6-flowered rachis, curved, pubescent ovary, deflexed lateral sepals and lip with stooping (reflexed) lateral lobes that ultimately coil around terminally are amongst the unique diagnostic morphological features. Furthermore, recent molecular studies (Jin et al. 2017; Raskoti and Ale 2019) have consolidated its distinction from similar-looking species. Therefore, a specific rank seems fully justified here. Description. Terrestrial herbs, 4-15 cm tall. Tubers globose-oblong. Stems densely papillate-pubescent. Leaves 2, opposite, basal; sheathing at base; leaf blade ovate to orbicular, 1-2.4 cm long, 1-2.2 cm broad, adaxially with yellowish-white venation, densely papillate, apex acute to acuminate. Inflorescence 3-12 cm long, sparsely 2-14-flowered; rachis 2-5.5 cm long, pubescent; floral bracts lanceolate, 3-6 mm long, apex acuminate. Flowers green, faintly fragrant; ovary and pedicel curved, 6-8 mm long, pubescent. Dorsal sepal broadly ovate, ca. 3.5 mm long, ca. 3 mm broad, glabrous, apex obtuse; lateral sepals oblique, ovate-elliptic, deflexed, ca. 4 mm long, ca. 2.5 mm broad, glabrous, apex obtuse. Petals obliquely falcate-ovate, ca. 3.5 mm long, 2-2.5 mm broad, glabrous, entire; lip 3-lobed, spurred; lateral lobes filiform, ca. 10 mm long; mid-lobe linear-lingulate, ca. 3 mm long; spur pendulous, clavate, 1-4 mm long. Column short; anther apex retuse; caudicles short; stigma processes clavate. (Fig. 6).
Phenology. Flowering in July to September.

Habenaria aitchisonii
Phenology. Flowering from July to September.
More illustrations. Wu et al. (2010, fig. 192, 4-6).  Note. This species is distributed along the whole range of the Himalaya up to the Hengduan Mountains at elevations between 2000 and 4500 m (temperate to alpine) and thus is the most widespread Habenaria species in the Pan-Himalaya. The type material was collected from the Darban Valley along the Pakistan-Afghanistan border in the western end of the distribution (Aitchison 1888). Similar plants from the Hengduan Region were described as new species by Schlechter (1912Schlechter ( , 1924 and Kränzlin (1921), respectively, but were later reduced to synonyms (Chen and Cribb 2009). There is considerable variation in the size of the plant, in the colouring of the leaves, with yellowish-white markings that occasionally give a false impression of this being a new species; the peduncle is also quite variable in length as is the density of the inflorescence, from subdensely few-flowered to densely many-flowered. Habenaria wolongensis from Sichuan also falls within the range of variation of H. aitchisonii and is here reduced to synonymy. Description. Terrestrial herbs, 10-24 cm tall. Tubers oblong. Stems densely pubescent. Leaves 2, opposite, basal; leaf blade ovate or ovate-orbicular, 2-4.5 cm long, 2-4 cm broad, fleshy, apex acuminate or acute. Inflorescence 8-20 cm long, subdensely 3-12-flowered; rachis 5-10 cm long; floral bracts lanceolate, apex acuminate. Flowers yellowish-green; ovary and pedicel arcuate, fusiform, 0.8-1 cm long, finely papillatehairy. Dorsal sepal forming a hood with petals, erect, ovate, concave, 5-6 mm long, 3.5-4 mm broad, margin ciliate-denticulate, apex obtuse; lateral sepals oblique, ovate-oblong, reflexed, 6-7 mm long, 3.5-4 mm broad, apex subacute. Petals 2-lobed, glabrous; upper lobe obliquely ovate-lanceolate, 5-6 mm long, 2-2.2 mm broad; lower lobe a tooth at base of upper lobe, ca. 0.5 mm long; lip deeply 3-lobed above base, spurred; lateral lobes linear, retrorse, almost embracing ovary, linear, 1-1.2 cm long, apex bent; mid-lobe linear, reflexed, ca. 1 cm long; spur pendulous, slightly curved, clavate, 1.2-2 cm long. Column stout, anthers parallel, connective wide; stigmatic processes sub-oblong. (Fig. 14).

Habenaria balfouriana
Phenology. Flowering in July and August.
Phenology. Flowering from June to August. Habitat. Thickets, alpine grasslands; 3200-4900 m elev. Distribution. N and S Hengduan, also in NE Qinghai of China. (Fig. 20). More illustrations. Wu et al. (2010, fig. 195, 5-7). Note. According to the protologue, Habenaria tibetica was described by Schlechter, based on two specimens from China: East Tibet, Ta tsien lu, 3600 m elev., Limpricht 2303 and Batang-Litang, 4800-4900 m elev., Limpricht 2277 (Limpricht 1922); consequently, both of these are syntypes as per Art. 9.6 of the ICN (Turland et al. 2018). Many of the type specimens, described by Schlechter together with H. tibetica (e.g. Platanthera minax  Schltr. & P. winkleriana Schltr.), were believed to be kept at B and later, their duplicate specimens were recovered at other European herbaria WU and WRSL; however, despite an extensive search, none of the type materials of H. tibetica could be located in any of the world's major herbaria and could have been destroyed during the Second World War at B. Furthermore, we were unable to find any other original material related to the species. Thus, assuming that all the original material of H. tibetica is lost, it warrants designating a neotype, which is here accomplished. For that purpose, X.L. Jiang 36260 (PE) is designated the neotype according to Art. 9.8 of the ICN (Turland et al. 2018); this specimen was also collected from the original type locality (Kangding, Sichuan). The designated neotype specimen conforms to the description in the protologue and is consistent with the current application of the taxon name (e.g. Chen and Cribb 2009).