Phylogenetic systematics of Hoya ( Apocynaceae )

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RESEARCH ARTICLE InTRoduCTIon
The genus Hoya of the family Apocynaceae includes 200-300 species distributed from India to the Pacific Islands (Fig. 1).The species are mostly epiphytic climbers with succulent leaves, inhabiting the tropical rainforest.The spectacular flowers are clustered in pseudoumbelliform inflorescences that are either concave and positively geotropic, convex and positively geotropic, or convex and negatively geotropic (Rintz 1978).The pentamerous corollas vary extensively in shape and size, from revolute over salver-shaped to urceolate and even tubular.Hoya flowers have staminal coronas consisting of five more or less erect lobes.The basal parts of the anther margins are mostly fused and form an 'anther skirt', a structure that has a nectariferous function (Kunze & Wanntorp in press).The dorsal margins of the pollinia are generally provided with a crest, termed 'pellucid margin'.The presence of the anther skirt and of pellucid margins on the pollinia seem to be unique to Hoya in the tribe Marsdenieae.Fruits in Hoya are follicles that enclose narrow, spindle-shaped seeds without conspicuous wings (Omlor 1998).
The number of taxa described within Hoya probably greatly exceeds the real diversity in the genus and there are over 500 names of taxa listed in The Index of Plant Names (1999).This indicates the problems in the taxonomy of the genus, which is badly in need of revision.Despite several recent attempts from the horticultural world to divide the genus into sections using mainly leaf and flower characters (e.g., Burton 1985, 1995, 1996, Kloppenburg 1993, 2001)

New classifications should be based on monophyletic groups
Today, systematists agree that classification should be based on monophyletic groups so as to reflect the true phylogenetic relationships between organisms.Taxa recognized in older classifications may, however, often conform to phylogenetic systematics, demonstrating the validity of many morphological characters used in the past.Molecular phylogenetic studies, based on chloroplast and nuclear gene sequences, were recently started in order to clarify intrageneric relationships in Hoya (Wanntorp et al. 2006a, b) as a first step towards a new phylogenetic classification of the genus.Aims of this paper are to discuss the topology of the phylogenetic tree (Fig. 2) obtained in Wanntorp et al. (2006b) in light of Schlechter's traditional classification (1913,1916), and to list possible contradictions found between the molecular and the morphological approaches.
The phylogenetic tree in Wanntorp et al. (2006b) was not fully resolved showing that the molecular markers used were not informative enough to resolve all relationships in Hoya.Three main monophyletic clades were, however, clearly identified in the phylogenetic tree, in addition to a main group, the core Hoya group, and several species whose exact phylogenetic positions are not yet identified.Below, I discuss each of these clades and compare the molecular phylogenetic results to what previously known on the species included in each clade.

The Acanthostemma clade
In 1848, Blume established the genus Acanthostemma for species of Hoya having the following flower characters: hairy corolla lobes that are completely revolute so as to make the flowers appear round or globular in shape (Fig. 3a); coronas with outer lobes ending in two lateral extensions turning inwards on each other and forming conspicuous anther skirts (Wanntorp & Forster 2007).Pollinaria of Acanthostemma species have obliquely elongate pollinia, with pellucid margins that extend along almost the entire dorsal edge, rhomboid and thick corpuscula with narrowly winged adhesive pads basally, and broadly winged caudicles (Wanntorp 2007) (Fig. 3b).Later on, Acanthostemma was lumped with Hoya and the name was subsequently used to delimitate a section in that genus (Kloppenburg 1993).The molecular analysis identified H. kentiana, H. 'tsangii', H. 'gracilis', and H. bilobata (Wanntorp et al. 2006b) as part of the Acanthostemma clade, showing a general agreement with the previously described section (Fig. 2).Another species, H. heuschkeliana, relatively newly described in the genus (Meve 2001), was nested in the Acanthostemma clade (Wanntorp et. al. 2006b) (Fig. 3c).The close affinity of H. heuschkeliana to Acanthostemma was already suggested by Kloppenburg (1993) on basis of morphology.Despite having corollas of urceolate or pseudourceolate shape, unusual in Hoya, H. heuschkeliana has other characters in common with Acanthostemma, such as pollinia with broadly winged caudicles.An unexpected result was the position of H. pseudolittoralis (= H. anulata) (Fig. 3d), as sister to the remaining species of the Acanthostemma clade.Hoya pseudolittoralis is the single species from New Guinea among those examined, that was not found clustered in the New Guinean/Australian clade (see below and Fig. 2) (Wanntorp et al. 2006b).Hoya pseudolittoralis was originally put in Schlechter's species-rich sect.Euhoya (1913).Its salver-shaped corollas and its inconspicuous anther skirts do not support a close relationship to the Acanthostemma species.Hoya pseudolittoralis has, however, pollinia with broadly winged caudicles that are very similar in morphology to the pollinia of species of Acanthostemma, thus possibly supporting the position found in the molecular phylogenetic tree (Wanntorp et al. 2006b).

The New Guinean/Australian clade
Only eight species of Hoya are endemic to Australia (Forster & Liddle 1996) while New Guinea seems to be one of the main centres of diversity for the genus, and Forster (1996) lists not less than 74 species from that area.The New Guinean species H. hypolasia, H. inflata (Fig. 3e), H. patella, H. venusta, and an undetermined taxon (H.spec.Chase 17132), as well as the Australian H. albiflora, H. australis and H. macgillivrayi were found together in a well-supported monophyletic clade in the tree (Wanntorp et al. 2006b) (Fig. 2).A sister relationship between taxa from New Guinea and taxa from Australia is concordant with the results of other phylogenetic studies and is consistent with the fact that New Guinea and Australia lie on the same continental plate (McLoughlin 2001), and taxa from these two areas are often found to be closely related (e.g., Linder & Crisp 1995, Wanntorp & Wanntorp 2003).The species of Hoya examined have large flowers with bell-shaped corollas (Fig. 3e), pollinaria with obovate pollinia, that are protruding outwards basally and thick rhomboid corpuscula (Fig. 3f).Hoya inflata (Fig. 3e) was originally described as the single species in the genus Madangia but phylogenetic analyses based on molecular as well as morphological characters have shown that the genus has to be included in Hoya (Wanntorp et al. 2006a, b, Wanntorp & Forster 2007).The other species examined were previously ascribed to three different sections by Schlechter (1913).Hoya macgillivrayi, H. patella and H. venusta were placed in sect.Physostelma, H. hypolasia in sect.Plocostemma, while H. albiflora and H. australis were included in sect.Pterostelma.According to the molecular phylogenetic studies, these three sections have to be merged, although molecular and morphological phylogenetic studies of several species from New Guinea and Australia are necessary in order to confirm this result.

The Eriostemma clade
Hoya affinis, H. ariadna and H. ciliata are three of the eleven species traditionally grouped in sect.Eriostemma (Schlechter 1913).These three species formed a distinct clade in the molecular phylogenetic tree (Wanntorp et al. 2006b) (Fig. 2).Also in this case, there seems to be agreement between traditional and modern systematics.Eriostemma species are characterised by a terrestrial (not epiphytic) habit and flowers with small coronas lying above large salver-shaped pubescent corollas (Fig. 4a).
The club-shaped pollinia lack pellucid margins and have twisted and winged caudicles (Fig. 4b).Altogether this morphology well defines sect.Eriostemma and supports the molecular results.Kloppenburg & Gilding (2001) even suggested that Eriostemma should be excluded from Hoya, based on the morphological characters listed above.Although it cannot be ruled out, this suggestion does not yet find enough support in the molecular analyses (Fig. 2) (Wanntorp et al. 2006b).

The core Hoya group
Many of the species of Hoya examined were found as part of a large core Hoya group in the phylogenetic tree of Wanntorp et al. (2006b).This group includes several of the species originally listed in Schlechter's sect.Euhoya.The exact relationships of several of these species were, however, not retrieved and further molecular studies are needed to resolve the present polytomies (Wanntorp in prep. ).An example of species in the core Hoya clade whose position in the tree is especially intriguing is the commonly cultivated H. lacunosa (Fig. 4c, d), which is one of the species of Hoya appreciated for the sweet smell of their flowers.Hoya lacunosa has flowers very similar to those of the Acanthostemma species, with hairy corolla and revolute lobes (Fig. 4c), and pollinia with broadly winged caudicles (Fig. 4d).This species has, however, conspicuous anther skirts, which are not typical for the Acanthostemma species, thus supporting a position for H. lacunosa outside the Acanthostemma clade proper.In fact, morphological characters of the flowers of Hoya seem to have been switched on and off during the evolution of the different species, and it is difficult to point out specific morphological characters as phylogenetically informative throughout the genus.This should, however, not hinder the study of morphological characters and their use in combination with molecular data.

ConCLudIng REmARKS And FuTuRE STudIES
The molecular phylogenetic results have shown that several traditional sections, such as Acanthostemma and Eriostemma (Schlechter 1913, 1916, Hooker 1885) are in fact reflecting true relationship between the species that they include.Other sections, such as Physostelma, Plocostemma and Pterostelma will probably have to be revised in order to mirror monophyletic groups in Hoya.We are only at the beginning of the examination of the phylogenetic relationships within Hoya.We have not yet analysed the positions of all species of this genus and there are still sections such as Peltostemma (Schlechter 1916) and Ancistrostemma (Hooker 1885) that have not yet been examined in light of the molecular phylogenetic tree.It is also indispensable to go on with α-taxonomic studies of Hoya in parallel with the phylogenetic studies.Taxonomic revisions of groups of Hoya, are, at the moment, in progress in many Southeast Asian areas where Hoya occurs and will be very important to interpret the phylogenetic results.Traditional taxonomy, molecular data as well as the use of a phylogenetic approach will together form the base of a new classification of a dominant but poorly understood genus of Southeast Asia.