Morphological and anatomical investigation of New Caledonian graminoid Dendrobium (Orchidaceae) with the description of two new species

. Morphological characters support the description of two new species of Orchidaceae from New Caledonia: Dendrobium butinii M.Pignal & Munzinger sp. nov. and Dendrobium letocartiorum Munzinger & M.Pignal sp. nov. The lectotypes of Dendrobium camaridiorum Rchb.f. and D. crassifolium Schltr. are designated here. A leaf anatomical study was conducted and provided useful characters to distinguish the five New Caledonian graminoid Dendrobiineae species. Two identification tools are provided: an anatomical key for sterile material of grass-like Dendrobiineae and a morphological key for the New Caledonian graminoid Dendrobium . We propose preliminary conservation assessments with IUCN criteria for the new taxa. pseudo-petiole. asymmetrical, two diverging. striated in its and in a beak to the and on the 1-flowered, through a slit of the foliar sheath. Pedicel 3.5 mm long. Ovary ca 3 long, non-resupinate. Bract acuminate, 2–3 mm long. ca 12 mm long. sepal triangular, 12 2 mm. sepals triangular, 12 4 mm at the base, 7 nerves. linear or slightly lanceolate, 11 ca 1 mm. Lip approximately elliptic, margin thin and undulate, 10 mm long and 3 mm wide, obscurely tricarinated. Mentum, 3 mm long. Column, 2.5 mm high and 2 mm wide with a fimbriate margin. Fruits very few despite an abundant flowering, yellow-greenish to brown when mature, ovoid, 8 mm in diameter, with base slightly narrower. Bracts persistent. Dried seed with transparent testa, ca 267 × 86 µm, fusiform, with extremity slender and base truncate, extremity strongly spiraled. Hydrated seed ovate, sacciform. Cells cubic or with extremities rounded, elongate, ca 43–110 × 7.3–10.2 µm, with thick edges, spiral orientation and smooth cellular walls. Embryo spherical elongate, ca 6–7 µm wide, inserted in the lower ⅔ of the testa.


Introduction
The New Caledonian flora is remarkable, both for its endemism, estimated at ca 75% (Morat et al. 2012), and for the presence of numerous primitive angiosperm groups (Lowry II 1998;Pouteau et al. 2015). Morat (1993) estimated that 5-10% of the flora remained undescribed, an interpretation supported by the ongoing description of one new endemic plant species on average per month (Gâteblé et al. 2018), including some in the Orchidaceae (Pignal & Munzinger 2011;Faria 2016). Many years ago, our colleague and friend Jean-Pierre Butin (Forest Service, Northern Province) brought to our attention some potentially new species of Orchidaceae. These belong to the genus Dendrobium Sw. and are characterized by long thin stems, and narrowly elliptical leaf blades evoking the Poaceae Barnhart, that we call therefore 'graminoid' species. Two New Caledonian species were known at that time to have this strange habit: D. camaridiorum Rchb.f. and D. crassifolium Schltr. Another graminoid Dendrobium, D. unicarinatum Kores (1989: 97) is now known to occur on the island. We quickly noticed that several herbarium specimens of graminoid Dendrobium did not match these three known species, and that Hallé's annotations on herbarium sheets suggested two potential new species he provisionally named: Dendrobium crassifomentum Hallé in herb. and D. crassiformatum Hallé in herb. In order to clarify the taxonomy of New Caledonian graminoid Dendrobium and evaluate the status of the putative new species, we have undertaken a revision based on morphology of all material in P and NOU. As part of this study, we also examined leaf anatomy, which has provided very informative features for classifying members of Dendrobium s. lat. (Yukawa et al. 1992;Stern et al. 1994;Morris et al. 1996;Yukawa & Uehara 1996;Carlsward et al. 1997). The main objectives were to 1) clarify the taxonomy of graminoid Dendrobium in New Caledonia; 2) provide keys for sterile and fertile material; 3) evaluate how anatomical characters provide information about the placement of these new species in the three sections: Dendrobium Sw., Grastidium (Blume) J.J.Sm. (Smith 1905a: 348) and Biloba J.J.Sm. (Smith 1905b: 67), incl. Monanthos Schltr. (Schlechter 1912: 251).
The anatomy of leaves of five graminoid taxa belonging to sections Grastidium and Biloba was studied. Most samples were taken from P and NOU herbaria, or from cultivated material available at several institutions (see Acknowledgements and Material examined). Dried leaves were rehydrated in boiling water, transversely sectioned by hand, bleached in 10% sodium hypochlorite for 5 min, washed and mordanted with acetic acid. The sections were then stained with methylene blue in saturated aqueous solution (5 min) and with Safranin (5 min), dehydrated by 95% alcohol and mounted with Euparal.
For the observation of small pieces of leaves using the Scanning Electron Microscope (SEM) Jeol of the 'Service Commun de Microscopie Électronique du MNHN', samples were cleaned with butanol, fixed on aluminium stubs and sputter-coated with gold.
In parallel of that work, IUCN status was evaluated by the New Caledonian Red List Authority (RLA-NC) and the results are reported there. Species phenology is based on both herbarium data and Christian Laudereau's precise observations.

Distribution and ecology
Dendrobium unicarinatum grows in Fiji and in New Caledonia dense humid forest (500-750 m), as an epiphyte on trunks (at the base or at several metres high) and can be very abundant, especially on palms. C. Laudereau (pers. comm.) separates two populations by habit and light exposition: the first erect and in sunny situations, the other pendulous, in shade.

Taxonomic notes
In New Caledonia, D. unicarinatum can be easily identified by a cylindrical or slightly flattened stem, 1-flowered inflorescence and white, non-resupinate flowers. Leaf section shows D. unicarinatum to be the only graminoid New Caledonian Dendrobium having large, obvious and numerous (> 20) supernumerary fibre bundles on both sides, and having the largest vascular bundle in position 2.

Phenology
Further observations are needed to determine the flowering and fruiting periods. In the available herbarium material, flowers were observed on cultivated plants (grown in New Caledonia) in January and in the field from February to March, and fruits in May to January.

IUCN status
This species was not recognized in New Caledonia until now, and was evaluated as Data Deficient (DD) by the New Caledonian Red List Authority.     (Fig. 3A-B).

Distribution and ecology
The endemic plant is widespread in humid or gallery forests all over the main island 'Grande-Terre', from 50 to 900 m, but has not been observed in sclerophyll forest (west coast), or in the extreme northwest (Fig. 6).

Taxonomic notes
Dendrobium camaridiorum produces numerous adventive plantlets ('keikis'), which is not the case of

Phenology
Flowers are observed throughout the year. It seems that the hours of opening (in the morning) and closing of the flowers (in the afternoon) are synchronous throughout the territory.

IUCN status
Dendrobium camaridiorum is the most abundant species of the graminoid group in New Caledonia, and was evaluated as Least Concern (LC) by the New Caledonian Red List Authority.

Typification
Vieillard's numeration cannot be understood as true collection numbers (Morat 2010). Indeed, he often attributed the same number to different gatherings that he regarded as comprising a single species, however he confused many taxa, thereby generating some nomenclatural problems as many of his collections were chosen as types (see for example Pierre et al. 2014). Comparison of the material deposited at W with a specimen at P that clearly represents the same collection indicates that an error occurred in transcribing the number on the former, which is probably a fragment removed from the original specimen. Labels on the Paris specimen bear the number Vieillard 1332, whereas the sheet at W is numbered 1192, which in fact corresponds to material of Phyllanthus cornutus Baill. (Euphorbiaceae Juss.). The material in Vienna consists of a stem fragment, several leaves, a single, dissected, immature flower, and a drawing of the Paris material. The latter is a tracing of the Paris plant (Hallé, pers. comm.). This interpretation is consistent with Reichenbach's visit to P in April 1864 (Paris Herbarium Visit Catalogue). Paris's material comprises a bud and a tuft of 3 stems, and is clearly conspecific with the fragmentary collection at W. We can reasonably assume that Reichenbach studied both P and W material, which would thus represent syntypes. We have selected the material at W as the lectotype as it clearly served as the basis of the description of the flower.

Distribution and ecology
This endemic species is found in a broad ecological range, quite commonly scattered in several places on the main island 'Grande-Terre' (Fig. 7), an epiphyte in humid or gallery forest. It grows from near sea level (10 m) to 900 m.

Taxonomic notes
In New Caledonia, in the sect. Grastidium, D. crassifolium is the only species to have parts of the perianth rounded at the apex. Dendrobium crassifolium has the thickest leaves of the graminoid New Caledonian Dendrobium, it is also the only species having vascular bundles in two lines (vs one), more than 32 vascular bundles (vs < 30), and the largest vascular bundle with diameter < ½ the thickness of the leaf blade (vs > ¾ the thickness).

Phenology
Flowering probably throughout the year (not observed in April).

IUCN status
The species is common in New Caledonia and was evaluated as Least Concern (LC) by the New Caledonian Red List Authority.

Etymology
The species is dedicated to our good friend, Jean-Pierre Butin, an enthusiastic lover of botany, who first recognized and collected this taxon.

Distribution and ecology
This endemic species has been located so far on one mountain (Mont Colnett) and in one valley (Tchamba) and appears restricted to humid forest of the North-East of the main island. Dendrobium butinii sp. nov. appears to occur in areas with the same ecological conditions as those of D. unicarinatum.
It can also occur in windy areas, as a lithophytic cushion plant on mossy micashists, in a dense population of cushion-like plants (Butin, pers. comm.). The species is known from 280 to 700 m elevation. The species has been observed also in the forest of Sailles (Thio) and Pénari (C. Laudereau, pers. comm.).

Taxonomic notes
The material studied that we attribute to D. butinii sp. nov. clearly does not correspond to D. minutiflorum Kraenzl. (Kränzlin 1914: 84), which was considered an insufficiently known species by Hallé (1977) and was based on a single collection (Sarasin 579) that was probably destroyed in Berlin. According to the original description (Kränzlin 1914), D. minutiflorum has bilobed leaves that are 23 mm long and 6-7 mm wide, 2-3-flowered inflorescences, suggesting that this taxon would be closest to D. isochiloides Dendrobium butinii sp. nov. matches the characters of the "fourth group" so called by Hallé (1977), characterized by having leaves along the stem, internodes hidden by leaf sheaths that are not imbricate and 2-flowered inflorescences on the leafy stems, opposite the lamina and developing from a small, sessile sheath. In New Caledonia this morphological group is represented by three species: D. butinii sp. nov., D. crassifolium and D. camaridiorum. This group is also remarkable by the presence of a 'V-like' central imprint on the leaves, always clearly present in D. camaridiorum and D. crassifolium, sometimes less apparent in D. butinii sp. nov. (Figs 1R, 8).
Dendrobium camaridiorum is the closest species to D. butinii sp. nov. Based on habit, D. butinii sp. nov. can be distinguished by its more spindly aspect, its smaller leaves, the presence of numerous adventive roots without adventive plantlets ('keikis') and its abruptly narrowed sepals.
Floral features, including the presence of papillose hairs on the lip and its fimbriate aspect, suggest a close affinity between D. butinii sp. nov. and D. crassifolium, although the hairs and fibres are much more developed in the latter species. Dendrobium butinii sp. nov. has thin leaves and vascular bundles in one line as D. camaridiorum, but differ by having vascular bundles < 15 (vs > 22), midrib not prominent (vs prominent), largest bundles in position 3(4-5) (vs 5(-6)) and presence of supernumerary fibre bundles (vs absence). Dendrobium butinii sp. nov., D. camaridiorum and D. crassifolium were all observed by the authors growing together on a single tree trunk in the Tchamba valley in NE New Caledonia.

Phenology
Flowering and fructification periods need further observations. The fruits were observed from the field and in cultivation (in New Caledonia) in January, April to May, October to November. Flowering specimens were observed in January, April, May and October to November.

IUCN status
Even if uncommon, D. butinii sp. nov. was evaluated as Least Concern (LC) by the New Caledonian Red List Authority, because no specific threat to the species or its habitat is known.

Description
Erect epiphyte. Roots whitish, ca 1-2 mm wide, at the base of the plant (axillary roots lacking). Stems reaching 60 cm long, slightly flattened, completely covered by the sheaths of the old leaves, lamina remaining only in the upper half, yellow-orange on dry material. Internodes ca 25 mm long at the base of the stem. Leaves spirally arranged, dark shiny green above, light green below (fide collectors' notes), ca 75 × 7 mm. Midrib impressed on adaxial surface, very prominent on abaxial surface. Two lateral nerves very conspicuous, < 1-2 mm from the margin (see discussion of anatomy below), numerous prominent smaller nerves visible, all convergent at the apex. Base narrow, forming a flattened pseudopetiole. Extremity emarginate, asymmetrical, the two lobes often diverging. Sheath striated in its upper half and ending in a beak opposite to the lamina and applied on the stem. Inflorescence 1-flowered, emerging through a slit of the foliar sheath. Pedicel 3.5 mm long. Ovary ca 3 mm long, non-resupinate. Bract acuminate, 2-3 mm long. Flower pale green to yellow, ca 12 mm long. Dorsal sepal triangular, 12 × 2 mm. Lateral sepals triangular, 12 × 4 mm at the base, with 7 nerves. Petals linear or slightly lanceolate, 11 × ca 1 mm. Lip entire, red, approximately elliptic, margin thin and undulate, 10 mm long and 3 mm wide, obscurely tricarinated. Mentum, 3 mm long. Column, 2.5 mm high and 2 mm wide with a fimbriate margin. Fruits very few despite an abundant flowering, yellow-greenish to brown when mature, ovoid, 8 mm in diameter, with base slightly narrower. Bracts persistent. Dried seed with transparent testa, ca 267 × 86 µm, fusiform, with extremity slender and base truncate, extremity strongly spiraled. Hydrated seed ovate, sacciform. Cells cubic or with extremities rounded, elongate, ca 43-110 × 7.3-10.2 µm, with thick edges, spiral orientation and smooth cellular walls. Embryo spherical elongate, ca 6-7 µm wide, inserted in the lower ⅔ of the testa.

Taxonomic notes
Dendrobium letocartiorum sp. nov. has always slightly flattened stems. The species has affinities with D. bilobum Lindl., but the flower is bigger and the lip does not have a dark red spot on the adaxial surface. Leaf anatomy shows that D. letocartiorum sp. nov. is very similar to D. camaridiorum, having thin leaves, 24-28 vascular bundles in one line and largest bundle in position 5(-6). They only differ by the margins in transverse section, which are obtuse in D. letocartiorum sp. nov. and truncate in D. camaridiorum (Fig. 4B).

Phenology
Flowering and fructification periods need further observations. The fruits were observed in the field and in cultivation (in New Caledonia) during the months of January, May, July, October, November and December and flowering specimens are from May and November.

Distribution and ecology
This endemic species occurs on the main island, in the central chain and on the Panié range in North-East coast. It grows on edges of humid forest, and also on rocks as a lithophyte and is known from 700-800 m a.s.l. The species has been observed also in Plateau de Dogny (Sarraméa) (C. Laudereau, pers. comm.)

IUCN status
This species is currently known from only four locations in New Caledonia, based on herbarium specimens. Dendrobium letocartiorum sp. nov. was evaluated as Near Threatened (NT) by the New Caledonian Red List Authority, considering the species apparent rarity and the low threat to its habitat.

Anatomical characters
Analysis of the leaf sections of the five species discussed in the present paper shows that they present strong differences between samples, especially regarding the number of vascular bundles (VB). We assigned a number to each VB, starting from the margin of the lamina (Fig. 4), and observed that the position of the larger VBs is stable and has a diagnostic value for the species, as has the ratio of the size of the larger VBs compared to the thickness of the lamina. Vascular bundles exhibit little variation within a species but can be very different between species. Differences were also seen in the alignment of the VBs, which are in two lines in D. crassifolium, but in only one line in all other species studied. Differences were also observed in the size of VBs number 2 of D. crassifolium which are symmetrically situated on each side of the lamina and are larger than the others. The presence of fibre bundles was also a remarkable character, especially in D. unicarinatum. Table 1 summarizes these anatomical characters for each species, which were also used to prepare an identification key to distinguish sterile material of graminoid Grastidineae species from New Caledonia.  Yukawa et al. (1992).

Key based on leaf anatomy (transverse section) to the graminoid species of Dendrobium in New Caledonia
to have one or the other type of stomata, except Grastidium, which exhibited both types. Members of sect. Biloba were reported to have Type II stomates, and our observations of D. unicarinatum and D. letocartiorum sp. nov. agree with these results (Fig. 5G-H). Our study also confirms the presence of both types within Grastidium, with Type I stomata in D. camaridiorum (Fig. 5A-B), and the surprising presence of both types of stomata in D. crassifolium and D. butinii sp. nov. (see Fig. 5C-F).
These proposals were rejected in Pridgeon et al. (2014) and would have brought the total number of genera in New Caledonia to 13, an excessive number considering the relatively small differences between the taxa. In line with the international consensus view of a broad concept of Dendrobium (Adams 2011;Schuiteman 2011;Chase et al. 2015;Pridgeon et al. 2014) we have classified all taxa in this paper under Dendrobium.