A Re-assessment of Relationships within Epacridaceae

Annals of Botany 77 : 305–315, 1996

A Re-assessment of Relationships within Epacridaceae J. M. P O W E L L*s, D. M. C R A Y N†‡‡, P. A. G A D E K†, C. J. Q U I N N†**, D. A. M O R R I S ON‡, and A. R. C H A P M AN§ * National Herbarium of New South Wales, Royal Botanic Gardens, Sydney 2000, † School of Biological Science, UniŠersity of New South Wales, Sydney 2052, ‡ Department of Applied Biology, UniŠersity of Technology, Sydney 2007 and § Western Australian Herbarium, Como, WA 6152, Australia Received : 2 February 1995

Accepted : 1 June 1995

An extensive database of predominantly morphological characters has been assembled for the family Epacridaceae. Problems of homology across the family and its outgroups were encountered for several characters. A phylogenetic analysis, using only those characters for which we were fairly confident of our assessment of homology, was undertaken to establish broad relationships within the family as a basis for a re-assessment of the supra-generic classification. The resultant phylogeny is weakly resolved and lacks robustness in the basal clades. Previous classifications of the family are assessed in the light of this analysis, and an alternative arrangement of four tribes is proposed. # 1996 Annals of Botany Company Key words : Cladistic, Epacridaceae, phylogeny, relationships, morphology.

INTRODUCTION The Epacridaceae is a medium-sized, predominantly Australian family comprising approx. 30 genera and at least 450 species. It was erected as the ‘ order ’ Epacrideae by Brown (1810), who recognized two ‘ sections ’ : I. ovules solitary in each cell of the ovary, fruit indehiscent ; II. ovules several in each cell of the ovary, fruit a capsule. This distinction was accepted by most workers and was eventually formalized as the tribes, Styphelieae and Epacreae, respectively (Bentham, 1869 ; Table 1). A third tribe, Prionoteae, defined by ‘ minute dentate leaves and the stamens free of the corolla ’, was established for Prionotes R.Br. and Lebetanthus Endl. (Drude, 1889), which were previously assigned to the Epacreae. Burtt (1948) added the montane south-east Australian endemic Wittsteinia F. Muell. to Prionoteae, and Hutchinson (1969) raised the tribe to family rank (Table 1), characterized by hypogynous free stamens and bilocular anthers. Watson (1967), however, rejected this arrangement, placing Prionotes and Lebetanthus close to Epacris, and questioning the inclusion of Wittsteinia within the family since it ‘ is not closely comparable with any other epacridaceous plant ’ ; Van Steenis (1984) has since provided convincing evidence for the placement of Wittsteinia in Alseuosmiaceae. Drude (1889) recognized two groups within his tribe Epacreae : (a) those with leaf bases expanded, more or less sheathing, and in most cases having free stamens ; (b) those with leaves attached by a narrow base, and stamens mostly epipetalous (Table 1). s Current address : Hawkesbury-Nepean Catchment Trust, PO Box 556, Windsor, NSW 2756, Australia. ** For correspondence. ‡‡ Current address : Botany Department, James Cook University, Cairns Campus, PO Box 6811, Cairns 4870, Australia.

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Watson, Williams and Lance (1966, 1967) reported on phenetic analyses of the family, on the basis of which Watson (1967) proposed a re-organization at the tribal level and above. The group comprising Dracophyllum, Richea and Sphenotoma were recognized as the sub-family Richeoideae ; all remaining genera were assigned to Epacridoideae. Within the latter he distinguised five tribes : Cosmelieae, Epacrideae, Styphelieae and two monotypic tribes, Needhamiellieae and Oligarrheneae (Table 1).

Relationships between Ericaceae and Epacridaceae The similarities between Ericaceae and Epacridaceae have long been recognized (Metcalfe and Chalk, 1950 ; Copeland, 1954 ; Stevens, 1971) and include both embryological (unitegmic tenuinucellar ovules, cellular endosperm development, and testa one cell thick and formed from the outermost layer of the integument) and floral characters (style usually hollow, the anthers introrse at maturity and lacking a fibrous layer in the wall, and the pollen grains borne in tetrads or modified tetrads or pseudomonads). The two families differ in stamen number and morphology, leaf venation and leaf hair type. Ericaceae usually bear two whorls of choripetalous (free) stamens with anthers dehiscing by basal pores (the anthers invert during development) and commonly with awns or spurs ; Epacridaceae have a single whorl of usually epipetalous stamens with unornamented anthers that dehisce by longitudinal slits. Leaf venation is predominantly reticulodromous in Ericaceae, and leaf hairs are multicellular and often complex. Epacridaceae have mainly actinodromous or parallelodromous venation, and leaf hairs are simple and mostly unicellular. Copeland (1954) considered that ‘ The palmate venation # 1996 Annals of Botany Company

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Powell et al.—Relationships within Epacridaceae T     1. Arrangement of genera according to published infra-familial classifications of Epacridaceae Bentham and Hooker, 1876 Epacreae Dracophyllum Richea Sphenotoma Andersonia Cosmelia Sprengelia Archeria Epacris Lebetanthus Lysinema Prionotes Rupicola Woollsia

Drude, 1889 Epacreae (i) Dracophyllum Richea Sphenotoma Andersonia Cosmelia Sprengelia (ii) Archeria Epacris Lysinema Rupicola Woollsia Prionoteae Lebetanthus Prionotes

Styphelieae Acrotriche Astroloma Brachyloma Choristemon Coleanthera Conostephium Cyathodes Cyathopsis Decatoca Leucopogon Lissanthe Melichrus Monotoca Needhamiella Oligarrhena Pentachondra Styphelia Trochocarpa

Styphelieae Acrotriche Astroloma Brachyloma Choristemon Coleanthera Conostephium Cyathodes Cyathopsis Decatoca Leucopogon Lissanthe Melichrus Monotoca Needhamiella Oligarrhena Pentachondra Styphelia Trochocarpa

… of the Epacridaceae is the only character which distinguishes the bulk of the family from Ericaceae : the floral structure of Sprengelia and of the Epacrideae with free stamens would not be grossly out of place in Ericaceae ’ (p. 221). He considered, however, that the tribe Styphelieae is in many features markedly distinct from Ericaceae, noting inter alia, the epipetalous stamens, single anther slit, ovules reduced to one per locule, and solitary pollen grains. Recent cladistic analyses of the morphoplogical data base (Anderberg, 1993 ; Judd and Kron, 1993) have strongly supported the monophyly of the order, but have provided clear evidence of the paraphyly of Ericaceae as commonly circumscribed : Epacridaceae and Empetraceae (as well as other segregate familes sometimes recognized) are nested within Ericaceae. Anderberg (1993) also provides evidence to support the monophyly of Epacridaceae (including Lebetanthus and Prionotes), which he suggests ‘ are descendents from a clade comprising the bulk of the Vaccinioideae and the Monotropoid-Pyroloid taxa ’, and that Prionotes and Lebetanthus retain some plesiomorphs that show evidence of this ancestry.

Watson, 1967 Richeoideae Dracophyllum Richea Sphenotoma Epacridoideae Cosmelieae Andersonia Cosmelia Sprengelia Epacrideae Archeria Epacris Lebetanthus Lysinema Prionotes Rupicola Woollsia Styphelieae Acrotriche Astroloma Brachyloma Choristemon Coleanthera Conostephium Cyathodes Cyathopsis Decatoca Leucopogon Lissanthe Melichrus Monotoca Pentachondra Styphelia Trochocarpa Needhamielleae Needhamiella Oligarrheneae Oligarrhena

The results of an analysis of rbcL sequence data for representatives of the order (Kron and Chase, 1993) are in general agreement with the above, with representatives of Epacridaceae being nested within Ericaceae as sister group to a set of vaccinioid taxa. Taxon density in all these analyses has been low, and especially so for Epacridaceae. Although there is evidence to support the monophyly of the family, and to show that its closest relationships lie within Ericaceae, relationships within Epacridaceae have received little detailed taxonomic attention since Bentham (1869) and Mueller (1864, 1867, 1882, 1889). While a number of new taxa have been described, little revisionary work and no reappraisal of generic limits has been attempted. Some discussion of supra-generic classification has occurred (eg. Watson, 1967), but these are based on intuitive methods, and little reliance can be placed on the generally accepted infra-familial classification. This paper sets out to conduct a rigorous cladistic analysis of the morphological database in order to generate hypotheses on the phylogeny of the group that can be tested by a molecular database now being assembled.

Powell et al.—Relationships within Epacridaceae MATERIALS AND METHODS The terminal taxa used in the analyses were either genera, sub-genera or sections as currently circumscribed. As many species as possible were examined for each of these groups. Heuristic parsimony analyses were performed in PAUP (Version 3.1.1 ; Swofford, 1993) set for TBR branchswapping. Branch lengths for trees were calculated using the ACCTRAN optimization ; only unambiguous characterstate changes were recorded on the branches in the final figures. Support for clades was inferred by bootstrap (Felsenstein, 1985) and decay, a measure of the independence of the parsimony criterion (Bremer, 1988 ; Donaghue et al., 1992). The decay analysis was performed using the Auto Decay program (version 2.4 ; Eriksson, 1995). Output trees from PAUP were also transferred into MacClade (Maddison and Maddison, 1992) and manipulated to test other topologies and to explore character state evolution. Character polarities were determined by outgroup analysis (Maddison, Donaghue and Maddison, 1984). The outgroup for the analysis comprised representatives of the ericaceous tribes Andromedeae (Gaultheria) and Vaccinieae (Vaccinium) sensu Stevens (1971), identified in recent cladistic analyses (Judd and Kron, 1993 ; Anderberg, 1993 ; Kron and Chase, 1993) as members of the sister clade to the Epacridaceae, as well as the more distant Erica (Ericae).

Ingroup taxa The circumscription of genera within the Epacridaceae has varied somewhat amongst authors, the number recognized ranging from approx. 20 to 31. Wittsteinia, which showed no affinity with Epacridaceae in earlier analyses (Powell, Chapman and Doust, 1987 ; see also Van Steenis, 1984), has been excluded. Earlier analyses (Powell et al., 1987 ; Morrison and Powell, 1990) showed Styphelieae, including Needhamiella and Oligarrhena, constitute a well defined clade. Due to problems of homology, many characters which were informative within the tribe could not be scored with confidence across the entire family and outgroups. Relationships within this tribe, therefore, were not considered in the present study ; they will be examined in a later paper. Here, the Styphelieae is represented by five genera : Astroloma, Melichrus, Needhamiella, Oligarrhena and Styphelia. Dracophyllum currently includes approx. 48 species. Oliver (1928) defined three subgenera on the basis of inflorescence characters : Oreothamnus (28 species, all New Zealand except for one in Tasmania) with flowers solitary or in racemes ; Eudracophyllum (18 species from Australia, New Caledonia and New Zealand) with panicles ; and Cordophyllum (one species in New Caledonia) with flowers in dense whorled fascicles that form a terminal compound spike-like raceme. These subgenera are used as terminal taxa, denoted Dracophyllum O, E and C, respectively. Sphenotoma, erected for six species originally assigned to Dracophyllum (Sweet, 1828), was re-submerged in Dracophyllum by Bentham (1869) but accepted by Mueller (1889).

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Despite some doubt as to its distinctiveness, the genus, which is endemic in Western Australia, is scored separately in these analyses. It differs from Dracophyllum in having a narrow corolla tube with the throat almost closed by longitudinal folds at the base of the lobes, and the filaments always adnate to the corolla tube. Richea, comprising 11 species from southeastern Australia, is separated into two sections on inflorescence characters following Bentham (1869) : Cystanthe, with simple contracted spikes and persistent bracts, and considered a distinct genus by Brown (1810) ; and Dracophylloides, with elongate spikes or compound panicles and deciduous bracts. The two sections are scored separately as Richea C and D, respectively. Richea is considered to be closely related to both Dracophyllum and Sphenotoma (Watson, 1967), but is readily distinguished from them in having the corolla lobes fused to form a calyptra that splits transversely and falls off leaving a persistent basal ring. Sprengelia comprises four species and is found in all states except Western Australia and the Northern Territory. Andersonia, which is endemic in Western Australia, was included within Sprengelia by Mueller (1867) but later accepted by him as distinct (Mueller, 1882, 1889). The two genera can be readily separated on floral features : Sprengelia has a glabrous corolla, the tube is much shorter than the lobes and often separates into petal claws, and the anthers are connivent or cohering in a ring around the style, the filaments incurved. In Andersonia (22 species) the corolla is pubescent inside, the tube is usually longer than the lobes and never splits, and the filaments are straight, with the anthers free. Watson (1962 a) recognized two sections within Andersonia based on the number of bracts subtending the flowers : Multibracteatae and Bibracteatae. These sections are scored separately as Andersonia M and B, respectively. The monotypic Western Australian genus Cosmelia was considered by Bentham (1869) to be closely allied to Epacris, but Watson (1967), on the basis of leaf morphology, placed it close to Sprengelia and Andersonia. Cosmelia differs from these genera in having filaments adnate to the corolla tube. The monotypic South American genus Lebetanthus has at times been considered a species of Prionotes (Hooker, 1837 ; Skottsberg in Arroyo, 1975) but is currently accepted as distinct. The two genera differ in flower shape, number of ovules per locule, placentation type, leaf size and venation pattern (Arroyo, 1975). Prionotes is a monotypic Tasmanian endemic. Archeria, as currently circumscribed, comprises six species from Tasmania and New Zealand. Mueller (1867, 1882, 1889) united it with Epacris, but it differs from that genus in having caducous bracts, almost basal attachment of the style and near-basal placentation. The endemic Western Australian genus Lysinema, comprising five species, is also considered to be closely related to Epacris but is distinguished by the hypocrateriform corolla, the tube often splitting at the base, the lobes contortedimbricate in the bud and the filaments free or only slightly adnate to the corolla tube. The monotypic eastern Australian endemic, Woollsia, was included in Lysinema by Bentham (1869) but was separated by Mueller (1875) on the basis of

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Powell et al.—Relationships within Epacridaceae T     2. Description of characters and their states Character no.

Description (state)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

Habit : shrub (0), climber (1) Leaf scars : present (0), absent (1) Hairs : multicellular (0), unicellular (1), unicellular­multicellular (2) Leaf insertion : non-sheathing (0), sheathing (1) Leaf venation : camptodromous}actinodromous (0), parallel (1) Leaf vein fibre caps : adaxial­abaxial (0), abaxial only (1) Leaf vein fibre caps : not reaching epidermis (0), reaching same epidermis (1), reaching opposite epidermis (2) Nodal anatomy : unilacunar (0), multilacunar (1) Pith composition : homogenous (0), heterogenous (1) Stomatal type : anomocytic (0), paracytic (1), cyclocytic (2), anomo-­para-cytic (3) Stomatal distribution : abaxial only (0), abaxial­adaxial (1) Flowers pedicellate above upper bracts : present (0), absent (1) Petal fusion type : fused (0), splitting at base (1) Aestivation : imbricate (0), valvate (1) Anther number : twice petals (0), equal to petals (1), less than petals (2) Anther coherence : free from style (0), cohering around style (1) Anther cell number : 2-celled (0), 1-celled (1) Filaments : free (0), epipetalous (1) Anther dehiscence : pores (0), longitudinal slits (1) Ovules per locule : 2-many (0), 1 (1) Placentation : axile (0), apical (1) Nectary : present (0) absent (1) Fruit type : capsule (0), drupe (1), berry (2) Inflorescence type : anthotelic (0), blastotelic (1), reduced anthotelic (2) Leaf margin : serrate (0), entire(1)

floral tube characters and differences in ovule number and placentation. Epacris as used here includes some 40 or more species from all Australian states except Western Australia and Northern Territory, and two New Zealand endemics. It is characterized by having short filaments inserted in the throat, the anthers attached at or above the mid-point and dehiscing by a longitudinal slit. The New South Wales endemic Rupicola resembles Epacris in general habit, bract structure and aestivation (Maiden and Campbell, 1898) ; it differs in having flat filaments attached to the base of the corolla tube, the anthers adnate to the filament, connivent around the style and opening by a short apical slit. Rupicola is accepted here in the sense of Telford (1992) as comprising four taxa, including R. apiculata (previously assigned to Epacris) but excluding Rupicola gnidioides, which now constitutes the monotypic Budawangia (Telford, 1992). This last genus is distinguished from Rupicola by its filiform filaments inserted in the throat, and by anthers that spread and dehisce longitudinally. Characters used in the analysis Table 2 lists the characters used in the analyses ; Table 3 is the data matrix. In the following discussion all suprageneric taxa are sensu Watson (1967) except for the Styphelieae, which is taken to include also Needhamiella and Oligarrhena. Habit (character 1). The outgroups and most Epacridaceae are shrubby, erect or occasionally decumbent [0], but two monotypic genera within the Epacridaceae, Prionotes and Lebetanthus, are climbers [1].

Leaf scars (character 2). Leaf abscission in Ericaceae and most Epacridaceae leaves a distinct, often crescent-shaped scar ; an annular scar characterizes Richoideae while Cosmelieae display a curious senescence mechanism whereby the cortex from the internode below each leaf detaches with it, leaving the axis smooth and without scars (Dormer, 1945 ; Watson, 1967). Two states are recognized : scars present [0] or absent [1]. Hair type (character 3). Ericaceae mainly have multicellular hairs only [0], or occasionally both multicellular and unicellular (Stevens, 1971), while Epacridaceae mostly possess only unicellular hairs [1] (Metcalfe and Chalk, 1950). Prionotes and Lebetanthus are the exceptions within the Epacridaceae, having multicellular glandular hairs, as well as unicellular hairs [2]. Leaf insertion (character 4). In the majority of Epacridaceae and in the outgroups, leaf bases are nonsheathing [0], the leaves being sessile or petiolate ; in Richeoideae and Cosmelieae, however, the leaf bases extend down around the axis to form an encircling sheath [1]. Leaf Šenation (character 5). Two venation types were identified using Hickey (1973) as a guide. The expression of this character within the ingroup may be influenced by the differential contributions of the leaf base, petiole and lamina to the leaf of various taxa. Hence this character may not be independent of character 4. Camptodromous venation based on a prominent midrib bearing pinnate secondary veins with inter-connecting reticulations [0] is characteristic of the outgroup and three ingroup genera, Šiz. Lysinema, Prionotes and Lebetanthus (see also Arroyo, 1975). Reticulo-actinodromous venation [1], defined for Epacridaceae as usually comprising both parallel and reticulate elements (often three to five primary veins diverging from near the base of

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T     3. Data matrix used in the analyses of Epacridaceae. Taxa displaying more than one state of a character are coded ‘ V ’. Missing data are coded ‘ N ’. Character number Outgroup (Ericaceae) Erica Gaultheria Vaccinium Ingroup (Epacridaceae) Richea C Richea D Sphenotoma Dracophyllum O Dracophyllum E Dracophyllum C Cosmelia Sprengelia Andersonia M Andersonia B Prionotes Lebetanthus Archeria Epacris Woollsia Lysinema Rupicola Budawangia Needhamiella Oligarrhena Astroloma Styphelia Melichrus

5

1 0

1 5

2 0

2 5

00000 00000 V0000

00000 000V3 00001

0N000 V0000 V0000

00000 00000 00000

00001 000V0 003V0

00111 00111 00111 00111 00111 00111 01111 01111 01111 01101 10200 10200 00100 00100 00100 00100 00100 00100 00101 00101 00100 00100 00101

01111 01111 01111 01111 01111 01111 12000 12002 12002 12002 10000 10000 10000 10000 10000 10000 10000 10000 11000 11000 11000 11000 11000

01011 01011 01001 11001 00001 01001 11001 11101 11011 11011 00001 01001 00001 01001 01001 01101 01001 01001 11001 01012 01001 01001 01001

01010 01010 01110 01110 01110 01110 01110 11010 00010 00010 00010 00010 01110 01110 01010 01010 11110 01110 01111 01111 01111 01111 01111

01021 00001 00021 00021 00001 00001 00021 01021 00021 00021 00020 00020 00021 00021 00021 00021 01021 01021 10111 10121 10121 10121 001N1

the leaf which may or may not converge toward the apex, and with or without secondary reticulations between them and}or branching towards the margin), is typical of all remaining Epacridaceae. In some taxa this state is simplified as a result of leaf reduction. Leaf fibre patterns. In Ericaceae there are usually some fibres associated with the midrib vascular bundle (Stevens, 1971), often as a thin cylinder enclosing it (Lem, 1964). The midrib is typically well raised from the abaxial leaf surface. In Gaultheria, Vaccinium and Erica there is a fibre bundle on both adaxial and abaxial sides of the major veins, but there is no contact between the epidermal cells and the fibre bundles. In Epacridaceae the midrib is more often equivalent to the other veins and all are associated with conspicuous groups of fibres. Simon (1891, cited in Stevens, 1971) noted three different types of leaf anatomy in Epacridaceae and Watson (1967) added a fourth : Richea type, in which the main veins are characteristically linked to both the adaxial and abaxial epidermis by a column of fibres ; Cosmelia type, in which the veins possess an abaxial arc of fibres and are embedded in the mesophyll except toward the leaf base, where they abut the adaxial epidermis ; Epacris type, in which the veins are always embedded in the mesophyll and possess an abaxial arc of fibres ; Styphelia type, in which the veins have an abaxial arc of fibres that abuts the abaxial epidermis. This variation was scored as two characters. Character 6 : abaxial and adaxial fibre bundles both commonly associated with the major veins [0] ; abaxial bundles only [1]. Character 7 : fibre bundles not contacting

epidermal cells [0] ; fibre bundles contacting the epidermal cells on the same side of the vein [1] ; fibre bundle contacting epidermal cells on opposite side of vein [2]. Nodal anatomy (character 8). Nodes are unilacunar [0] throughout Ericaceae and Epacridaceae except for Richeoideae, where they are multilacunar [1], having three or more vascular traces to each leaf. Cosmelieae have unilacunar nodes, the vascular trace dividing immediately on entering the leaf. Pith composition (character 9). The outgroups possess mainly homogeneous pith [0], although a range of pith types occur in Ericaceae (Stevens, 1971). Epacridaceae have homogeneous pith except for Richeoideae, which are heterogeneous [1] (Watson, 1967). Cosmelia is scored as homogeneous (Dormer, 1945 ; Watson, 1967), despite it being listed as heterogeneous in Metcalfe and Chalk (1950). Stomatal types (character 10). Anomocytic stomata are widespread in Ericaceae (Stevens, 1971) and Epacridaceae ; they are lacking in Vaccinium (Ericaceae), Richeoideae and Cosmelieae. Vaccinium and Richeoideae are characterized by paracytic stomata, which also occur in Gaultheria. Cyclocytic stomata occur only in Cosmelieae. The following states are recognized : anomocytic only [0] ; paracytic only [1] ; cyclocytic only [2] ; paracytic plus anomocytic [3]. Stomatal distribution (character 11). Stomata are confined to the lower surface of the leaf [0] in most Ericaceae and Epacridaceae. Needhamiella, Cosmelia, Sprengelia and Andersonia have stomates on both surfaces [1], as do Dracophyllum O (Watson, 1962 b).

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Powell et al.—Relationships within Epacridaceae

Flowers pedicellate aboŠe upper bracts (character 12). Most Epacridaceae lack distinct pedicels above the uppermost bracts [1]. In the outgroups, and in Archeria, Prionotes and Dracophyllum E, the bracts are inserted at a distance from the calyx [0]. Petal fusion (character 13). Almost without exception, Ericaceae and Epacridaceae are sympetalous [0]. Among Epacridaceae, however, Lysinema and Sprengelia show corolla tubes which often split at the base [1]. AestiŠation (character 14). The outgroups and most Epacrideae, Cosmelieae and Richeoideae possess imbricate aestivation [0]. Valvate aestivation [1] is characteristic of Richea, Andersonia and most Styphelieae. Number of anthers (character 15). Most Ericaceae are diplostemonous [0] ; exceptions occur, however, such as Loiseleuria with only a single whorl of five stamens. Epacridaceae are haplostemonous, usually with as many stamens as petals [1], although in Oligarrhena (Styphelieae) there are fewer stamens than petals [2]. Anther coherence (character 16). Among Epacridaceae, Sprengelia and Rupicola have anthers which are connivent around the style [1]. This feature is not known to occur elsewhere in the Ericales. Anther cell number (character 17). Almost without exception, Ericales have dithecal anthers [0] (Anderberg, 1993). Epacridaceae are mostly monothecal [1], the only exceptions being Andersonia, Lebetanthus and Prionotes. Filaments (character 18). Ericaceae, with few exceptions, have stamens free from the corolla. Epacridaceae are commonly epipetalous, with the anthers usually inserted in the throat, although Richea, Sprengelia, Andersonia, Prionotes, Lebetanthus, Woollsia and Lysinema, and some species of Dracophyllum O have free stamens. Two states only are recognized : stamens free of corolla tube [0] ; stamens epipetalous [1]. Anther dehiscence (character 19). Anthers in most Ericaceae dehisce by basal pores or short slits [0]. Epacridaceae dehisce by a full-length longitudinal slit [1] ; the sole exception in our analysis is Rupicola, which has less than full-length slits, but this is still considered distinct from the outgroup condition. OŠules per locule (character 20). Cosmelieae, Epacrideae, Richeoideae and the outgroups possess numerous ovules in each locule [0]. Styphelieae have a solitary ovule per locule [1]. Placentation (character 21). Axile placentas [0] occur throughout Ericaceae and in all Epacridaceae except Styphelieae, which are characterized by apical ovule attachment [1]. Nectary (character 22). Nectary discs are lacking [1] among Epacridaceae in only Sprengelia, Richea C, Rupicola and Budawangia. The outgroups all possess nectary discs [0], as do most Ericales. Fruit type (character 23). Most Ericaceae possess capsular fruits which may dehisce septicidally or loculicidally (Stevens, 1971). Gaultheria and Erica have fruits that dehisce loculicidally [0]. The fruit of Vaccinium is a berry [2]. In Epacridaceae, only Styphelieae have fleshy (drupaceous) fruit [1] ; all other genera have loculicidal capsules. Inflorescence type (character 24). The most common types

of inflorescences found in Ericales are racemes and spikes, either terminal or in the axils of upper leaves. The inflorescence may be terminated by either a flower [0] (anthotelic : Briggs and Johnson, 1979) or a non-floral bud [1] (blastotelic). Both states occur in the outgroup. Only one member of the ingroup, Needhamiella, possesses indeterminate leafy racemes of non-bracteate flowers [2]. Inflorescences in Epacridaceae consisting of an indeterminate leafy shoot bearing a series of flowers borne singly in the axils, each terminating an axis bearing empty bracts, may be interpreted as a leafy blastotelic raceme of multibracteate flowers [i.e. state 1]. Alternatively, each flower may be interpreted as a reduced anthotelic spike or raceme (or panicle), the bracts being the pherophylls that originally subtended lateral flowers (or inflorescences) (Briggs and Johnson, pers. comm.). In that case the inflorescences should be coded as state 0. The latter interpretation was initially adopted, with the reduced anthotelic condition being treated as a third state (see Tables 2 and 3). Subsequently, the character was recoded as a two-state character according to the first interpretation to explore the effect on the analysis. Leaf margin (character 25). Serrate leaf margins [0] occur in many Ericaceae (Stevens, 1971), including Gaultheria and Vaccinium. Among Epacridaceae, only Prionotes and Lebetanthus have distinctly serrate leaf margins.

RESULTS AND DISCUSSION A heuristic analysis with 500 replicates of random taxon addition (using the initial coding of character 24) gave 78 equally parsimonious trees of 59 steps with a rescaled consistency index of 0±53 and a retention index of 0±83. Despite the large number of trees found, there is a good deal of structure in the strict consensus (Fig. 1). Three major clades, labelled the Richea, Cosmelia and Styphelia clades, can be recognized. The first two clades are clustered as sister-group to the third, while the taxa in Watson’s Epacrideae are attached basally. The ingroup is shown to be monophyletic, with Prionotes and Lebetanthus basal ; these two genera form a clade in 77 % of trees. Apart from the clustering of Budawangia and Rupicola, there is little agreement on the relationships of the remaining Epacrideae apart from a tendency to attach Woollsia and Lysinema below the rest (in 54 % of trees). Re-analysis of the data after recoding character 24 (inflorescence type) in two states produced 108 equally parsimonious trees of 58 steps, but there was no change to the topology of the strict consensus (Fig.1). There was, however, a slightly stronger tendency to group Prionotes and Lebetanthus (in 83 % of trees). Hence, the interpretation of this character is not critical to the main structure of the cladogram. An examination of all 78 trees from the first analysis revealed that 12 of them had a distinctly lower F ratio (0±173 ; Farris, 1972). The most highly resolved of these trees was loaded into MacClade to investigate patterns of character evolution (Fig. 3). Monophyly of the ingroup is supported by unique synapomorphs in characters 6, 15 and

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Gaultheria Vaccinium Richea C Richea D

Dracophyllum E Dracophyllum O

Richea clade

Sphenotoma

Dracophyllum C Cosmelia

Andersonia M Andersonia B

Cosmelia clade

Sprengelia

Asrtoloma Styphelia

Styphelia clade

Oligarrhena

Ingroup clade

Needhamiella

Melichrus Archeria Epacris Woollsia Lysinema Rupicola Budawangia Prionotes Lebetanthus Erica F. 1. Strict consensus tree for the 78 most parsimonious trees found in a heuristic search with 500 replicates of random taxon addition.

19 (fibre caps, anther number and anther dehiscence), as well as an unambiguous change in character 24 (inflorescence type). The Richea clade is also well supported, possessing unique synapomorphs in characters 8 (nodal anatomy) and 9 (pith composition), and a reversal in 6 (fibre caps), as well as sharing the paracytic state [10–1]. There are two unique synapomorphs defining the Cosmelia clade [2–1 and 7–2] and the Styphelia clade [20–1 and 23–1]. The sister relationship between the Richea and Cosmelia clades is supported by only a single synapomorph [4–1], while the clustering of the combined clade with the Styphelia clade is supported by two unique synapomorphs [5–1 and 7–1]), the former showing a reversal in Astroloma and Styphelia.

When parsimony was relaxed by one step, the ingroup clade and its three major sub-clades remained erect in the strict consensus of the 8352 trees found, but only the Cosmelia clade retained any internal structure (Fig. 2). The order of association of the three sub-clades also decayed at ­1. All other clades decayed at ­2 except for the ingroup clade, the Richea clade, and Andersonia B­M, which decayed at ­3. A bootstrap analysis exhausted the memory in its early stages (on replicate 24). A repeat analysis was then conducted, with the steepest descent option selected, saving only one tree from each replicate. The results obtained after 500 such replicates are shown in Fig. 2. The Richea and ingroup clades were present in the highest frequency (both

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Powell et al.—Relationships within Epacridaceae Erica Gaultheria Vaccinium Richea C

57%

Richea D Dracophyllum E

Richea clade

Dracophyllum C 88% Sphenotoma Dracophyllum O

54%

Cosmelia 72%

Sprengelia 63% 78%

52%

Andersonia M

Cosmelia clade

Andersonia B Needhamiella 50%

Oligarrhena Astroloma

74%

Styphelia clade

Styphelia Melichrus Archeria

66%

Epacris Rupicola 60% Budawangia Woollsia Lysinema

88% Lebetanthus Prionotes

F. 2. Cladogram with branch lengths proportional to the number of unambiguous changes for one of the 78 most parsimonious trees chosen as having the lowest F ratio and showing greatest resolution. Bootstrap frequencies are shown for branches present in more than 50 % of replicates. Dotted branches decayed at 0 (i.e. not present in strict consensus) ; dashed branches decayed at ­1 step ; thin solid branches decayed at ­2 ; thick branches decayed at ­3.

in 88 % of replicates). The Styphelia and Cosmelia clades were found less frequently (74 and 72 %, respectively). The order of association of these three clades was only weakly supported, being present in just over half the replicates (Fig. 2). Hence, decay and bootstrap analyses are in broad agreement in showing the only major clades that can be considered robust are the Richea and ingroup clades ; the remainder of the topology is weak.

Character eŠolution The placement of Needhamiella within a large group of taxa possessing the reduced anthotelic state [24–2] suggests that its apparently blastotelic condition is not strictly homologous with the blastotelic condition in the outgroups, but was acheived by loss of the bracts beneath the flowers in 24–2.

Powell et al.—Relationships within Epacridaceae

313

18/0

14/1

17/0

14/1

6/0

8/0 9/1 10/1

4/1 2/1 7/2 18/0

10/0

5/1 7/1

5/0

20/1

23/1

22/1

18/1

3/1

17/1

6/1 15/1 19/1 24/2 1/1

3/2

Erica Gaultheria Vaccinium Richea C Richea D Dracophyllum E Dracophyllum C Sphenotoma Dracophyllum O Cosmelia Sprengelia Andersonia M Andersonia B Needhamiella Oligarrhena Astroloma Styphelia Melichrus Epacrris Rupicola Budawangia Archeria Woollsia Lysinema Prionotes Lebetanthus

F. 3. One of the 78 most parsimonious cladograms found in PAUP, chosen on the basis of lowest F value and highest resolution of relationships, showing unambiguous character-state changes on the internal branches within the ingroup. Key : unique synapomorphs without reversals within the clade, broad bar ; unique synapomorphs with reversals, narrow bar ; synapomorphs showing homoplasy, double bar ; reversals, cross. Characters and their states numbered as in Table 1.

It should be noted that the most parsimonious solutions require a reversal in character 6 (Fig. 3). The plesiomorph of both abaxial and adaxial fibre bundles being associated with the major veins gives rise to the synapomorph for Epacridaceae by the loss of the adaxial bundles, only to be regained within the Richea clade, although in this case they take the form of a flange of fibres extending right across the mesophyll. This suggests that perhaps the Richea clade may be better placed lower on the cladogram. Analysis applying a constraint revealed that moving the Richea clade below Epacrideae increases tree length by three steps (two steps if the sister relationship with the Cosmelia clade was maintained) ; moving both clades outside Prionotes and Lebetanthus increases tree length by a further step. Hence this data base gives reasonable support to the derived position of the Richea and Cosmelia clades. Several characters show marked homoplasy on the most parsimonious trees. Character 18 was initially scored as three states to distinguish those taxa in which the filaments are inserted near the base of the corolla tube from those with filaments inserted in the throat. The second apomorph was shown to arise only once and then give rise to the first on three separate internodes leading to Rupicola, Needhamiella and Sphenotoma. Since the second apomorph was not informative about relationships above the generic level, the two apomorphs were combined. Combining the states reduced the number of equally parsimonious trees from 104 to 78, but the strict consensus tree remained

unchanged. Even in this simplified form epipetaly shows two separate reversals on the internodes leading to Richea C­D, and to Sprengelia­Andersonia M­B. Valvate aestivation [14–1] arises in three different places : Oligarrhena, Richea C­D, and Andersonia M­B. The nectary disc has been lost [22–1] on three separate occasions : in Rupicola­Budawangia, Sprengelia, Richea C. The amphistomatic state [11–1] is a synapomorph for the Cosmelia clade, but also occurs in Needhamiella and Dracophyllum O. Taxonomic implications Smith-White (1948, 1955) had some doubts about a single origin of the family on cytological evidence. While this analysis using only a limited range of outgroups was not designed to rigorously test this point, it does provide some support for the monophyly of Epacridaceae, including Prionotes and Lebetanthus (see also Anderberg, 1993). The basal position of these two genera within the family, however, decays at ­1, occurred in only 66 % of bootstrap replicates and is supported by only two unambiguous character-state changes, each of which shows at least one reversal or homoplasy (Figs 2 and 3). Our data base contains little to distinguish Lebetanthus from Prionotes ; neither does it provide strong evidence of a particularly close affinity between them. The analysis certainly does not support their separation as a distinct family (Hutchinson,

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1969). Rather, they appear to occupy an intermediate position between the Epacridaceae and Ericaceae. Because of the limited support and the restricted outgroup representation in this analysis, the above conclusion needs to be tested against new data sources and with greater taxon density. Oligarrhena and Needhamiella are both embedded within the Styphelia clade. Clearly neither warrants the individual tribal status promoted by Watson (1967) ; rather, both should be assigned to the Styphelieae. Epacrideae in any sense (Bentham and Hooker, 1876 ; Drude, 1889 ; Watson, 1967) appear to be paraphyletic, the group having been defined on the basis of retention of plesiomorphs (e.g. capsular fruit ; absence of sheathing leaf bases). The Richea, Cosmelia and Styphelia clades are all recognized as distinct monophyletic groups. Each corresponds to a previously recognized infrafamilial taxon : the Richeoideae and Cosmelieae of Watson (1967), and the Styphelieae of Bentham and Hooker (1876) and Drude (1889), respectively. While the Richea clade is more robust, the most parsimonious arrangement does not support its recognition as a subfamily as proposed by Watson (1967). In view of the poor resolution of relationships between the three clades it is preferable that they be accorded equivalent status as tribes : Richeeae—defined by the possession of both adaxial and abaxial fibre caps [6–0] that extend as a flange to contact each epidermis [7–1], multilacunar nodes [8–1], heterogeneous pith [9–1] and paracytic stomata [10–1] ; Genera : Dracophyllum, Richea, Sphenotoma. Cosmelieae—defined by the absence of adaxial fibre caps [6–1], abaxial fibre caps extending around the veins to contact the adaxial epidermis [7–2], and lack of leaf scars [8–1] ; Genera : Andersonia, Cosmelia, Sprengelia. Styphelieae—defined by uniovulate locules [20–1], apical placentation [21–1] (except in Melichrus) and drupaceous fruit [23–1]. Genera : Acrotriche, Astroloma, Brachyloma, Coleanthera, Conostephium, Cyathodes, Cyathopsis, Decatoca, Leucopogon, Lissanthe, Melichrus, Monotoca, Needhamiella, Oligarrhena, Pentachondra, ‘ Pseudactinia ’, Styphelia, Trochocarpa. The determination of the appropriate treatment for the remaining taxa must await analyses of an expanded data base ; for the present they are left in a fourth tribe : Epacrideae—Genera : Archeria, Budawangia, Epacris, Lebetanthus, Lysinema, Prionotes, Rupicola, Woollsia. Our data base provides support for the generic concepts of both Richea and Andersonia, but suggests that the generic limits of Dracophyllum need to be re-assessed. The three sub-genera do not form a monophyletic group in any of the most parsimonious trees ; in many, Sphenotoma clusters within Dracophyllum (Fig. 3). Sphenotoma, which was originally included within Dracophyllum (Brown, 1810 ; Bentham, 1869), was separated largely on the presence in the former of epipetalous stamens and pronounced longitudinal folds at the base of the corolla lobes (Sweet, 1828). Both these conditions are known to occur in Dracophyllum. Further, leaf wax morphology suggests a relationship between Sphenotoma and some species of Dracophyllum subgenus Oreothamnus (Weiller, Crowden and Powell, 1994).

A C K N O W L E D G E M E N TS We are particularly indebted to Drs B. G. Briggs and L. A. S. Johnson for their detailed comments on drafts of this work, and Dr G. J. Jordan for discussions on venation patterns. We also thank the many colleagues with whom we have discussed this project : Drs B. Conn, M. Crisp, W. Judd, K. Kron, P. Stevens and P. Weston, and Mr A. Doust. We acknowledge funding assistance for parts of this work from the Australian Biological Resources Study and Australian Research Council grant no. A19330712. LITERATURE CITED Anderberg AA. 1993. Cladistic interrelationships and major clades of the Ericales. Plant Systematics and EŠolution 184 : 207–231. Arroyo S. 1975. Lebetanthus, genero austroamericano de Epacridaceae y sus diferencias con el genero Prionotes de Tasmania. Darwinia 19 : 312–330. Bentham G. 1869. Flora Australiensis. Vol. 4. London : Reeve & Co. Bentham G, Hooker JD. 1876. Genera Plantarum. Vol. 2. London : Reeve & Co. Bremer B. 1988. The limits of amino acid sequence data in angiosperm phylogeny reconstruction. EŠolution 42 : 795–803. Briggs BG, Johnson LAS. 1979. Evolution in the Myrtaceae—evidence from inflorescence structure. Proceedings of the Linnean Society of New South Wales 102 : 157–266. Brown R. 1810. Prodromus Florae NoŠae Hollandiae et Insulae Van Diemen. London : Johnson. Burtt BL. 1948. Studies in Ericales VIII. The taxonomic position of Wittsteinia. Kew Bulletin 3 : 493–495. Copeland HF. 1954. Observations on certain Epacridaceae. American Journal of Botany 1 : 215–221. Donaghue MD, Olmstead RG, Smith JF, Palmer JD. 1992. Phylogenetic relationships of Dipsacales based on rbcL sequences. Annals of the Missouri Botanic Garden 79 : 333–345. Dormer KJ. 1945. Morphology of the vegetative shoot in Epacridaceae. New Phytologist 44 : 149–152. Drude O. 1889. Epacridaceae. In : Engler A, Prantl K, eds. Die naturlichen Pflanzenfamilien. Edn.1. Leipzig : Engelmann, 66–79. Eriksson T. 1995. Auto Decay Šersion 2.4. Harvard University Herbaria. (copies of this program are freely available via electronic mail from torsten.eriksson!botan.su.se). Farris JS. 1972. Estimating phylogenetic trees from distance matrices. American Naturalist 106 : 645–668. Felsenstein J. 1985. Confidence limits on phylogenies : an approach using the bootstrap. EŠolution 39 : 783–791. Hickey LJ. 1973. Classification of the architecture of dicotyledonous leaves. American Journal of Botany 60 : 17–33. Hooker WJ. 1837. Icones Plantarum. Oxford : Blackwell. Hutchinson J. 1969. EŠolution and phylogeny of flowering plants. Dicotyledons : facts and theory. London : Academic Press. Judd WS, Kron KA. 1993. Circumscription of Ericaceae (Ericales) as determined by preliminary cladistic analyses based on morphological, anatomical, and embryological features. Brittonia 45 : 99–114. Kron KA, Chase MW. 1993. Systematics of the Ericaceae, Empetraceae, Epacridaceae and related taxa based upon rbcL sequence data. Annals of the Missouri Botanical Garden 80 : 735–741. Lem K. 1964. Evolutionary studies in the Ericaceae II. Leaf anatomy as a phylogenetic index in the Andromedeae. Botantical Gazette 125 : 178–186. Maddison WP, Donaghue MJ, Maddison DR. 1984. Outgroup analysis and parsimony. Systematic Zoolology 33 : 83–103. Maddison WP, Maddison DR. 1992. MaC Clade : An analysis of phylogeny and character eŠolution. Versions 3.01. Sunderland : Sinauer Associates. Maiden JH, Campbell WS. 1898. The flowering plants and ferns of New South Wales. Sydney : Government. Printer.

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