A taxonomic revision of the myrmecophilous species of the rattan genus Korthalsia (Arecaceae)

The rattan genus Korthalsia Blume (Arecaceae: Calamoideae: Calameae) is widespread in the Malesian region. Among the 28 accepted species are 10 species that form intimate associations with ants. The ants inhabit the conspicuous ocreas that are produced by these species, using them as domatia to care for their young and aphids. As a foundation for future work, we present here a taxonomic treatment of the myrmecophilous Korthalsia species, based on extensive research pursued both in the herbarium and the field. In addition, we conduct detailed morphological characterisation of the structure and development of ocrea using light and scanning electron microscopy. Descriptions, illustrations, keys and distribution maps are presented for all 10 species, along with microscopic images of ocrea morphology and development for selected species.


Introduction
Korthalsia Blume (1843) is one of the eight genera of rattans, which are spiny climbing palms in the subfamily Calamoideae (Baker et Couvreur et al. 2015;Vorontsova et al. 2016). The genus is distributed from northern Indochina, Burma and the Andaman Islands south-eastwards to Sulawesi and New Guinea (Dransfield et al. 2008). Currently, 28 species are accepted (WCSP 2017). The highest diversity of Korthalsia is found in Borneo, Malay Peninsula and Sumatra, with 15, nine and nine species respectively. Korthalsia is an isolated group within the Calamoideae, being the sole genus of subtribe Korthalsiinae in tribe Calameae of subfamily Calamoideae (Dransfield et al. 2008).
Korthalsia is highly distinctive among rattans in its morphology. It has a unique combination of characters, including (usually) diamond-shaped leaflets with jagged distal margins, aerially branching stems, hapaxanthic stems (that die after a single flowering event) and inflorescences with catkin-like rachillae. In addition, all species bear a conspicuous extension of the leaf sheath above the point of attachment of the leaf petiole. This structure, known as an ocrea, is diverse in form in Korthalsia. Dransfield (1981) classified the ocreas of Korthalsia into four types: inflated, divergent, tightly sheathing and fibrous net-like.
The ten species of Korthalsia that possess either inflated or divergent ocreas form mutualistic relationships with ants, which exploit the ocreas as domatia (Dransfield et al. 2008). The ants nest within the ocreas, brooding their young and aphids from which they harvest honeydew. The ants defend the rattan by attacking any animal that disturbs it. In addition, ants that occupy the two species with divergent ocreas (K. hispida and K. robusta) make a pulsing, hissing sound when the rattan is disturbed by rhythmically striking their mandibles against the dry wall of the ocrea. Several ant genera have been recorded inhabiting Korthalsia ocreas: Camponotus, Crematogaster, Dolichoderus, Iridomyrmex or Philidris (Bequaert & Wheeler 1922;Dransfield 1981;Mattes et al. 1998;Moog et al. 2003;Chan et al. 2012). This association does not occur throughout the distribution of Korthalsia, the myrmecophilous species being restricted to Borneo, Malay Peninsula, Philippines (Palawan and Mindanao), Singapore and Thailand (Map 1). Although phylogenetic evidence is limited for Korthalsia, recent data indicate that the species with inflated and divergent ocreas are closely related to each other, potentially forming a monophyletic group (Shahimi 2018). Curiously, inflated ocreas occupied by ants occur in other rattan genera, specifically the African genus Laccosperma (Sunderland 2004(Sunderland , 2012 and some species of Calamus, especially on the island of New Guinea (e. g. Baker & Dransfield 2002;Dransfield & Baker 2003;Merklinger et al. 2014) The genus Korthalsia has been known to scientists for nearly two centuries, having been first described in 1843 by Carl Ludwig Blume. However, the last treatment of the genus in its entirety was published 100 years ago (Beccari 1918), building on a series of earlier contributions (Beccari 1884(Beccari , 1893(Beccari ,1909. Since that time, a synoptic treatment has been published (Dransfield 1981), as well as a series of regional accounts (Furtado 1951;Dransfield , 1984Dransfield , 1992Dransfield , 1997Henderson 2009;Lapis 2010) and some new species (Dransfield 1981;Henderson & Nguyen 2013), but no complete, modern taxonomic revision has yet been produced. Here we address this knowledge gap by presenting a taxonomic account of the myrmecophilous species of Korthalsia across their entire geographical range. We focus especially on the structural biology of the ocrea, characterising its morphology and development with light and scanning electron microscopy. This revision will serve as a valuable tool and data source for those with special interests in the ecology and evolution of ant-plant mutualism in Korthalsia (e.g. Mattes et al. 1998;Chan et al. 2012). Moreover, it represents a contribution towards the completion of a monograph of all species currently recognised in the genus.

Taxonomy
An extensive study of specimens at the Kew herbarium (K) and three international herbaria, namely E, KEP and SING (herbarium acronyms follow Holmgren et al. 1990) underpins this study. Specimen images available online in other institutions were also consulted. Exclamation marks in the taxonomic treatment (!) indicate specimens that have been studied by the authors (including online specimen images). In addition, the first author has conducted two field expeditions in 2014 and 2015, visiting Peninsular Malaysia and Singapore, then Borneo and Singapore. Herbarium specimens were made in the field using standard preparation guidelines for palms (Dransfield 1986;Baker & Dransfield 2006).

Characterisation of ocrea morphology and development
Korthalsia stem apices measuring c. 1 m in length were collected in the field and fixed in 70% ethanol. Each shoot was carefully dissected by removing leaf sheaths until the shoot was approximately the diameter of a pencil. Images of later stages of development were captured with a photomacroscope. For examination of early leaf development close to the stem apex, we used Scanning Electron Microscopy (SEM). Dissected samples were dehydrated through an alcohol series to 100% ethanol. The samples were transferred to an Autosamdri 815B CPD for critical-point drying, mounted onto SEM pin stubs using double-sided tape, and coated with platinum using an Emitech K550 sputter coater. Samples were examined using a Hitachi S-4700 cold-field emission SEM at RBG Kew. Ocrea morphology was also studied using a Leica photomicroscope M400 (Light Microscopy).

Habit
The myrmecophilous species of Korthalsia are moderate to robust, clustering, high-climbing rattans up to 60 m or more. Some species can reach the forest canopy. They are found only in lowland and hill tropical forest, being absent in montane forest. Most of the species have a wide ecological range and are abundant in primary forest.

Stem
The stem size of myrmecophilous Korthalsia varies from slender (0.2 -0.8 cm in diameter without sheath) to moderately large (1.0 -4.0 cm in diameter without sheath). The internodes are elongate and variable in length. Nodal scars of Korthalsia species are often uneven. Aerial branching sometimes occurs due to parallel forking of stems.

Leaves
The leaf sheath is tubular, green and usually with caducous indumentum. The sheath is sometimes unarmed or variously armed with spines. The petiole ranges from 1.5 cm to 40 cm. The leaflets are regularly arranged and usually rhomboid with distal margins praemorse, but in few species (e.g. Korthalsia echinometra), they are lanceolate, although still with a praemorse distal margin. The adaxial surface of the leaflets is usually bright or dark green, and the abaxial surface is typically covered in white or grey indumentum, or sometimes with caducous, orange or brown to dark brown indumentum. The number of leaflets on each side of the rachis varies from one to 25, with the smallest number belonging to K. furcata and the highest in K. echinometra. The main veins diverge from the leaflet base. All species of Korthalsia have leaves armed with a cirrus, a leaf whip extending beyond the terminal leaflets.

Ocrea
We examined ocrea development in four species of myrmecophilous Korthalsia for which fixed material was available, representing both inflated and divergent ocrea types (sensu Dransfield 1981): inflated ocrea (K. echinometra, K. scortechinii, and K. rostrata), divergent ocrea (K. robusta). For comparison, we also examined three species with the tightly sheathing ocrea type (K. debilis, K. rigida and K. tenuissima). The fibrous net-like ocrea type is found only in K. jala, which was not encountered during our fieldwork and fixed material was not available (Fig. 1).
The three species examined with inflated ocreas ( Fig. 2A -C) differ in the origin of the inflation. Korthalsia echinometra ( Fig. 2A) shows inflation from the point of attachment of the ocrea to the leaf sheath, whereas in the other two species (Fig. 2B, C) the ocrea is tightly clasping at the base and inflated above. At the stage recorded in Fig. 2, the tightly clasping part is almost as long as the inflated part, although in the mature ocrea the clasping part is relatively shorter. Korthalsia echinometra also differs from K. scortechinii and K. rostrata in that it lacks the notch that forms in the ocrea apex of K. scortechinii and K. rostrata. In contrast, the apex is truncate in the species in which the ocrea is tightly sheathing for its entire length (K. debilis and K. rigida; Fig. 2E -F). In some species, spines cover the ocrea at an early stage ( Fig. 2A -D), whereas in others they are absent ( Fig. 2E -F).
At an earlier stage, the degree of ocrea inflation differs between Korthalsia echinometra, K. scortechinii and K. rostrata ( Fig. 3A -C). At this stage, there is slight inflation of the leaf base in K. echinometra (Fig. 3A), no inflation in K. scortechinii (Fig. 3B) and clear inflation in K. rostrata (Fig. 3C). The bifid ocrea apex is clearly visible at this stage in K. rostrata (Fig. 3C). Small spines are also visible at this stage on the ocrea of K. robusta (Fig. 3D) and a few small rounded spines were visible in K. debilis (Fig. 3E), although none was present in K. tenuissima (Fig. 3F).
Young leaf primordia at successive stages were examined for Korthalsia echinometra (Fig. 4) and K. debilis (Fig. 5), representing the inflated and tightly sheathing ocrea type respectively. In both species, early stages of leaf plication are evident (Fig. 4B, 5A), although the ocrea lacks plications in either species at any stage. In both species, the ocrea becomes visible at a relatively late stage (Fig. 4D, 5D).

Inflorescence
The flowering behaviour in all species of Korthalsia is hapaxanthic (individual stems flower only once in their lifetime and die subsequently). The inflorescences are borne at the apex of the stem and are lax to congested, with one to two orders of branching. The peduncle is adnate to the internode above the subtending leaf (Dransfield et al. 2008). The prophyll is 2-keeled and tightly sheathing. Rachis bracts are similar to the prophyll, but lack keels and are somewhat inflated in some species.    A -D developmental stages for Korthalsia echinometra, a species with an inflated ocrea. A leaf primordium differentiated into a distal lamina but lacking plications at this stage; B successive stages of leaf elongation, with leaf plication becoming more pronounced on both sides of the lamina and small lobes present at the top of the leaf sheath (arrow) indicating the first stage of ocrea development; C at this stage, the petiole has begun to elongate, the spines have begun to develop and the ocrea is more clearly visible; D the ocrea is a well-delimited structure that will persist into the adult organ. Bars = 100 μm. la lamina, s sheath, sp spines, pe petiole, pl leaf plications, oc ocrea. A plication inception is visible on the leaf; B the cirrus is visible above the petiole (arrow); C at this stage, the petiole has begun to elongate, the spines have begun to develop and the ocrea has become more visible; D the ocrea is a well-delimited structure that will persist into the adult organ. Bars = 100 μm. s sheath, sp spines, cr cirrus, pe petiole, pl leaf plications, oc ocrea.
The bracts can be unarmed or sparsely armed and densely covered with caducous indumentum. The rachillae are cylindrical and catkin-like, with densely arranged rachilla bracts, sometimes with hairs in between. The rachillae can be slender or congested.

Flowers
In all Korthalsia species, the flower is hermaphroditic and borne in pits in the catkin-like rachillae. Unlike many other calamoid palms, the flower is solitary, rather than borne in pairs. The calyx is tubular at the base, with three sepals and usually shorter than corolla. The corolla consists of three valvate petals. The flower contains 6 -9 stamens.

Fruits and Seeds
The fruit of Korthalsia species examined here is globose to ovoid, with one seed. The epicarp is thin and covered with vertical rows of imbricate scales. The scales are usually brown to strawcoloured. The mesocarp develops as a thin sweetfleshy layer surrounding the seed, and the endocarp is not differentiated. Unlike most species of tribe Calameae, the seed lacks a sarcotesta. The seed is attached basally. In most of the species, the endosperm is ruminate, but it in a few species (e.g. K. hispida) the endosperm is homogeneous. The fruits of most species appear to be attractive to animals (Dransfield 1981).

Korthalsia robusta
Malaysia, Perak, Kamuning. Type not found (Furtado 1951  K. furtadoana also have conspicuous, fine and closely spaced transverse veinlets. In Brunei, a form of Korthalsia rostrata occurs that has lanceolate rather than rhomboid leaflets. Although the shape of leaflets is distinctive, the form appears otherwise to be identical to remaining forms of K. rostrata.
Dransfield for sharing his encyclopaedic knowledge of rattans. Many other individuals assisted in a wide variety of ways; Abu Husin, Felix Merklinger, Marie-Hélène Weech, Mohamad Aidil, Mohd Nazri, Mohd Sukor, Peter Petoe, Serena Lee, Siti Nur Bazilah, Syed Mohd Ezham, Ummul Nazrah and Wan Nur Fasihah Zarifah. We thank two reviewers and Richard Bateman for helpful comments that have significantly improved the manuscript. This paper is an output of SS's doctoral studies, which were funded by Minister of Higher Education Malaysia and Universiti Malaysia Terengganu.
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