Delimitation of Epacridaceae: Preliminary Molecular Evidence

Annals of Botany 77 : 317–321, 1996

Delimitation of Epacridaceae: Preliminary Molecular Evidence D. M. C R A Y N*‡, K. A. K R O N†, P. A. G A D E K* and C. J. Q U I N N* * School of Biological Science, UniŠersity of New South Wales, Sydney 2052, Australia and † Department of Biology, Wake Forest UniŠersity, Winston-Salem, NC 27109, USA Received : 2 February 1995

Accepted : 1 June 1995

Comparative sequence data for the chloroplast encoded rbcL gene has been obtained for species representing the basal lineages in the proposed phylogeny of Powell et al. (Annals of Botany 77 : 305–315, 1996), with the aim of testing the delimitation of the family and the validity of the supra-generic taxa proposed, and estimating relationships within the family. Cladistic analyses indicate that Epacridaceae sensu Powell et al. (1996) is not monophyletic. Lebetanthus, a monotypic genus from South America commonly placed within the family, mostly near the Tasmanian endemic Prionotes, is shown to be closer to Gaultheria and other members of the ericaceous tribe Andromedeae. The hypothetical phylogeny of Powell et al. (1996) is evaluated in the light of this preliminary analysis of relationships within the family. The data do not support the recognition of the two subfamilies, Richeoideae and Epacridoideae, of Watson (New Phytologist 66 : 495–504, 1967). # 1996 Annals of Botany Company Key words : Epacridaceae, Ericales, molecular systematics, rbcL.

INTRODUCTION The Epacridaceae is a predominantly Australasian family with a few outliers extending the range to SE Asia, Hawaii, Tierra del Fuego and Patagonia. The limits of the family have varied little since it was described (e.g. Brown, 1810 ; Bentham, 1869 ; Drude, 1889 ; Watson, 1967). Although Wittsteinia F. Muell. has occasionally been allied to the family (e.g. Burtt, 1948), it is now accepted as a member of Alseuosmiaceae (van Steenis, 1984). Hutchinson (1969) removed Lebetanthus Endl. and Prionotes R. Br. (and Wittsteinia) to a new family, Prionotaceae, but this has not been taken up by subsequent authors. Recent cladistic studies of the Ericales based on morphology, embryology, chemistry and anatomy (Anderberg, 1993 ; Judd and Kron, 1993) suggest Epacridaceae sensu Watson (1967) is monophyletic, but its recognition renders Ericaceae sensu Stevens (1971) paraphyletic (the nomenclature of Watson and Stevens will be followed hereinafter). Nucleotide sequence data from the chloroplast gene rbcL (Kron and Chase, 1993) support this conclusion. In each case, however, sampling of tribes was restricted to five or fewer epacridaceous taxa, and only Anderberg (1993) included Prionotes and Lebetanthus. Previous classifications of Epacridaceae have been intuitive (e.g. Bentham, 1869) or based on few characters (e.g. Watson, 1967). A recent attempt to address this problem by a rigorous cladistic analysis of a primarily morphological database (Powell et al., 1996) identified several monophyletic lineages within Epacridaceae but failed to provide a clear resolution of the basal relationships between them. These authors also make the point that even those relationships ‡ For correspondence.

0305-7364}96}040317­05 $18.00}0

that were resolved are only weakly supported by the morphological database. Hence, this study was undertaken to test the limits and affinities of Epacridaceae and the validity of and relationships between the four tribal groupings of Powell et al. (1996) (Table 1) against a new database derived from nucleotide sequences of the chloroplast-encoded rbcL gene. MATERIALS AND METHODS Voucher specimens and their location, as well as GenBank accession numbers or sources of other sequences used in the analyses, are listed in the Appendix. Some positions are yet to be determined for the taxa sequenced for this study ; the sequences will be submitted to GenBank when complete (the raw data used in these analyses are available upon request from the corresponding author). DNA extraction, amplification (PCR) and sequencing techniques were identical to those we have employed in earlier studies (e.g. Fernando et al., 1993), except that double-stranded templates were sequenced using a modified single-stranded protocol (initial denaturation was allowed to proceed for 10 min). Double-stranded templates were purified with WizardTM PCR preps (Promega) prior to sequencing. A heuristic parsimony search strategy in PAUP (Version 3±1±1 ; Swofford, 1993) set for TBR branch-swapping, was employed. The choice of ingroup and outgroup taxa was based on the rbcL analysis of Kron and Chase (1993). The ingroup comprised a broad representation of epacrid taxa as well as representatives of the Ericales ; Cyrilla Garden ex L. (Cyrillaceae) was used as the outgroup. Initially, the length of the most parsimonious solution(s) was estimated by a single heuristic search with the steepest descent option invoked. In order to find multiple islands of most# 1996 Annals of Botany Company

318

Crayn et al.—Delimitation of Epacridaceae : Preliminary Molecular EŠidence

T     1. The infra-familial classifications of Epacridaceae of Powell et al. (1996) and Watson (1967) 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

Powell et al. (1996) Richeeae Dracophyllum Richea Sphenotoma 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 Needhamiella Oligarrhena

parsimonious trees (Maddison, 1991), a second search consisting of 100 replicate analyses with random taxon addition was performed. Alternative topologies were tested by means of further heuristic searches with enforced topological constraints. 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). A modified bootstrap (1000 replicates) was performed in PAUP with the steepest descent option invoked and saving only one tree from each replicate. This allowed the search to be completed within a reasonable time period. The decay analysis was performed using the Auto Decay (version 2±4) program (Eriksson, 1995). RESULTS AND DISCUSSION Near complete rbcL sequences (up to 1398 bases) were obtained for 16 taxa representing all the major lineages in the analysis of Powell et al. (1996).

A preliminary analysis showed Wittsteinia to have no affinity with Epacridaceae nor with Ericales in any sense, but to cluster within the Asterales s.l. sensu Olmstead et al. (1993). It was omitted from subsequent analyses. A more detailed examination of the affinities of this genus will be published elsewhere. The analysis of the complete dataset, containing 440 variable nucleotide sites, found a single island of four equally parsimonious trees of 842 steps with a rescaled consistency index of 0±395. Figure 1 is the strict consensus tree. In all trees, Epacridaceae is paraphyletic : Lebetanthus is isolated from the remaining Epacridaceae, and most notably, from Prionotes, to which it has usually been linked [Hooker (1837) and Skottsberg (in Arroyo, 1975) considered them congeneric]. Lebetanthus clusters in a position basal to Gaultheria L. and LeucothoeX D. Don, within a clade of ericaceous taxa comprising representatives of the tribes Andromedeae (Vaccinioideae : Gaultheria, LeucothoeX , Vaccinium L. and Zenobia D. Don) and Daboecieae (Rhododendroideae : Daboecia D. Don). Constraining Lebetanthus to form a clade with Prionotes increased the treelength by 16 steps ; a position basal to Prionotes increased the treelength by six steps. The placement of Lebetanthus among vaccinioid genera requires parallelisms in several key morphological characters that would otherwise link it with Epacridaceae. These include the absence of anther ornamentation and disintegration tissue, the presence of a lignified leaf epidermis, unifacial fibre bundles associated with minor veins, and anther dehiscence by longitudinal slits. All of these characters, however, occur among Ericaceae : anther ornamentation and disintegration tissue is absent from many Ericaceae (Stevens, 1971), a lignified epidermis occurs in Agarista D. Don, Aguaria (DC.) Hook., Arcterica Cov., Craibiodendron W. W. Sm., some Lyonia Nutt. and some Pieris D. Don (Andromeda L. show an intermediate state ; Stevens, 1971), and unifacial leaf vein fibre bundles are known from Agarista, Agauria, and LeucothoeX (Judd, 1979). Anther dehiscence by slits also occurs in Ericaceae (Stevens, 1971). Thus, the synapomorphs uniting Lebetanthus and Epacridaceae are not unique. Indeed, Powell et al. (1996) concede their morphological ‘ database contains little to distinguish Lebetanthus from Prionotes ; neither does it provide strong evidence of a particularly close affinity between them ’. The remaining epacrid taxa form a robust (decay ­4, bootstrap 90 %) monophyletic group that occupies a derived position among Ericaceae, as sister to the Andromedeae} Daboecieae clade. This is consistent with the initial analysis of Kron and Chase (1993) and supports the view that ‘ … the evolutionary origin of the Epacridaceae [lies] among Ericaceae of the tribes Andromedeae and Gaultherieae … ’ (Copeland, 1954). Indeed, the Gondwanan distribution of capsular-fruited Epacridaceae (considered basal in the family) overlaps that of only Gaultheria and Pernettya Gaudich. (Andromedeae) among Ericaceae in New Zealand and southern Australia. In view of this, a southern origin for Epacridaceae appears highly plausible. Within Epacridaceae three major clades are recognized ; the Richea, Styphelia and Epacris groups. These correspond

Crayn et al.—Delimitation of Epacridaceae : Preliminary Molecular EŠidence Epacris impressa

+3

Rupicola

+1

72

Epacris lanuginosa Woollsia

+4

Epacris group

33

80

319

Lysinema

30

Pentachondra

+1 99

Leucopogon fraseri

+1

Astroloma 70

Acrotriche

+4

Cyathodes

+1 39

Melichrus

33 <25

31

+1

+1 <25

+1

Styphelia group

+8

30

Brachyloma

+1 80 +2

35

Monotoca

+1

Leucopogon muticus Sprengelia

<25

+4

55

+1

+2

Dracophyllum Sphenotoma Richea

Richea group

90

Prionotes 37 52 <25 +2 <25 +1

+1

94

+1

+5

Leucothoë Gaultheria Lebetanthus

42

67

Zenobia

+1

+3

Daboecia Vaccinium Chamaedaphne

82

Befaria

+4

41 +1

49 +1 100

89

Calluna

96

34

+6

+1

+4

+11

Erica Rhododendron Elliotia Cassiope Arbutus Pyrola Enkianthus Cyrilla

F. 1. Strict consensus tree of the four most parsimonious trees found for the dataset. Numbers above the branches are bootstrap percentage values ; below the branches are decay values.

to the Richeoideae, Styphelieae and some of the Epacrideae, respectively. Prionotes and Sprengelia Sm. are placed in isolated positions.

The data show a tendency (decay ­2, bootstrap 80 %) to place Prionotes as the basal member of the Epacridaceae. Several authors (Smith-White, 1948 ; Stevens, 1971 ;

320

Crayn et al.—Delimitation of Epacridaceae : Preliminary Molecular EŠidence

Anderberg, 1993) have considered Prionotes and Lebetanthus may constitute an intermediate link between Epacridaceae and Ericaceae since they display characteristics of both families. The molecular data support this view, with respect to Prionotes. Powell et al. (1996) considered Epacrideae basal within the family, and probably paraphyletic ; they placed the Richeoideae­Cosmelieae as sister to the Styphelieae. According to the molecular data, the Richea group form a basal clade subtending Sprengelia (the sole representative of Cosmelieae), and two derived clades including taxa drawn from the Epacrideae and Styphelieae, respectively. None of this structure, however, is robust : the branches defining the order of association of these groups collapse at ­1. Sprengelia arises separately to the Richea group ; morphological data (Powell et al., 1996) favour a single origin for Cosmelieae ­ Richeoideae. Constraining Sprengelia to form a clade with the Richea group requires an increase in the treelength of only two steps. In view of this, the order of association of the major clades within Epacridaceae must be viewed as uncertain. The Styphelia group constitute a highly robust (decay ­8, bootstrap 99 %) clade that occupies a derived position within the Epacridaceae. This accords well with evidence from morphology (Copeland, 1954 ; Powell et al., 1996). It is probable Styphelieae arose from the capsular epacrids and subsequently diversified to occupy habitats throughout Australasia. The tribe, on the basis of morphology, DNA sequences, and cytology and pollen (Smith-White, 1948, 1955), is clearly divergent. Patterns of evolution within this group and the identities of its closest relatives among Epacridaceae are as yet unclear and may only be resolved with an expanded database and with greater taxon density. The Epacris clade, containing all sampled representatives of the Epacrideae except Prionotes and Lebetanthus, is placed as sister to the Styphelia clade. The molecular data indicate strong support for the monophyly of these taxa (decay ­4, bootstrap 72 %). This contrasts with their apparent paraphyly in the morphological analysis (Powell et al., 1996), although that arrangement was poorly supported by the data (all the branches in that part of the tree decayed at ­1). In view of this, the topology derived from the molecular data is to be preferred. Rupicola Maiden and Betche and Woollsia F. Muell. cluster within Epacris Cav. The relationship between Epacris impressa Labill. and Rupicola is particularly well supported (decay ­3, bootstrap 80 %). This topology may change with an increase in taxon density, but the present result suggests the limits of the genus Epacris may need to be reassessed. The rbcL-based trees do not support the subfamilial distinction proposed by Watson (1967, Table 1) : the recognition of Richeoideae renders his Epacridoideae paraphyletic. Further, there is no evidence to support Watson’s belief that Richeoideae are highly divergent. Like the analysis of Powell et al. (1996), the molecular data provide only limited support for some of the linkages and especially for the basal relationships in the family. It may be that the evolutionary events giving rise to these basal clades occurred over a relatively short period of time.

This assertion needs to be tested against additional data from a less conservative locus. The molecular phylogeny presented here is based on a single gene, rbcL. Whilst there may be factors in the evolutionary history of this gene that may confound the relationships inferred in our phylogenetic analysis, we consider their likely impact to be minimal (see Doyle, 1992, and Chase et al., 1993, for a discussion of these factors). Nevertheless, we regard it to be essential to corroborate relationships inferred from single gene phylogenies with other data sources before strong reliance is placed on them. Apart from incorporating morphological and chemical characters into molecular analyses, we are presently working to accumulate sequence data from the more rapidly evolving matK gene in the hope of being able to resolve the basal relationships in the family as well as to test the pattern of relationships defined above. A C K N O W L E D G E M E N TS The authors wish to thank Dr G. P. Findlay, Professor A. E. Ashford and the directors of the National Botanic Gardens, Canberra, and the Royal Botanic Gardens, Sydney, for living material, and Dr J. M. Powell for useful discussion. This work was supported by Australian Research Council grant no. A19330712 to C.J.Q. and NSF grant nos. BSR-8821264 and DEB-94073502 to K.A.K. 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(2/4) : 312–330. Bentham G. 1869. Flora Australiensis. Vol. 4. London : Reeve & Co. Bremer B. 1988. The limits of amino acid sequence data in angiosperm phylogeny reconstruction. EŠolution 42 : 795–803. 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. Chase MW, Soltis DE, Olmstead RG, Morgan D, Les DH, Mishler BD, Duvall MR, Price RA, Hills HG, Qiu Y-L, Kron KA, Rettig JH, Conti E, Palmer JD, Manhart JR, Sytsma KJ, Michaels HJ, Kress WJ, Karol KG, Clark WD, Hedre! n M, Gaut BS, Jansen RK, Kim K-J, Wimpee CF, Smith JF, Furnier GR, Strauss SH, Xiang Q-Y, Plunkett GM, Soltis PS, Swensen S, Williams SE, Gadek PA, Quinn CJ, Eguiarte LE, Golenberg E, Learn Jr GH, Graham SW, Barrett SCH, Dayanandan S, Albert VA. 1993. Phylogenetics of seed plants : an analysis of nucleotide sequences from the plastid gene rbcL. Annals of the Missouri Botanical Garden 80 : 528–580. 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 Botanical Garden 79 : 333–345. Doyle JJ. 1992. Gene trees and species trees : molecular systematics as one-character taxonomy. Systematic Botany 17(1) : 144–163. Drude O. 1889. Epacridaceae. In : Engler A, Prantl K. Die natuX rlichen Pflanzenfamilien. Edn. 1. 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). Felsenstein J. 1985. Confidence limits on phylogenies : an approach using the bootstrap. EŠolution 39 : 783–791.

Crayn et al.—Delimitation of Epacridaceae : Preliminary Molecular EŠidence Fernando ES, Gadek PA, Crayn DM, Quinn CJ. 1993. Rosid affinities of Surianaceae : molecular evidence. Molecular Phylogenetics and EŠolution 2 : 344–350. 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. 1979. Generic relationships in the Andromedeae (Ericaceae). Journal of the Arnold Arboretum 60 : 477–503. Judd WS, Kron KA. 1993. Circumscription of Ericaceae (Ericales) as determined by preliminary cladistic analyses based on morphological, anatomical, and embryological features. Brittonia 45(2) : 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. Maddison DR. 1991. The discovery and importance of multiple islands of most-parsimonious trees. Systematic Zoology 40 : 315–328. Olmstead RG, Bremer B, Scott KM, Palmer JD. 1993. A parsimony analysis of the Asteridae sensu lato based on rbcL sequences. Annals of the Missouri Botanical Garden 80 : 700–722. Powell JM, Crayn DM, Gadek PA, Quinn CJ, Morrison DA, Chapman AR. 1996. A re-assessment of relationships within Epacridaceae. Annals of Botany 77 : 305–315. Smith-White S. 1948. A survey of chromosome numbers in the Epacridaceae. Proceedings of the Linnean Society of New South Wales 73 : 37–56. Smith-White S. 1955. Chromosome numbers and pollen types in the Epacridaceae. Australian Journal of Botany 3 : 48–67. Stevens PF. 1971. A classification of the Ericaceae : subfamilies and tribes. Botanical Journal of the Linnean Society 64 : 1–53. Swofford DL. 1993. PAUP—Phylogenetic Analysis Using Parsimony, Version 3±1±1. Champaign : Illinois Natural History Survey. van Steenis CGGF. 1984. A synopsis of Alseuosmiaceae in New Zealand, New Caledonia, Australia and New Guinea. Blumea 29 : 387–394. Watson L. 1967. Taxonomic implications of a comparative anatomical study of Epacridaceae. New Phytologist 66 : 495–504.

APPENDIX List of taxa used in the analyses. The voucher details (followed by the GenBank accession numbers where applicable) for each taxon are given in parentheses. Taxa marked with an asterisk were sequenced for this study. Cyrillaceae Cyrilla racemiflora L. (NCU, Kron s.n. ; L01900) Epacridaceae *Acrotriche diŠaricata R. Br. (NSW 242116) *Astroloma humifusum (Cav.) R. Br. (UNSW 22502) *Brachyloma daphnoides (Sm.) Benth. (UNSW 22543) *Cyathodes glauca Labill. (UNSW 22521) Dracophyllum longifolium R. Br. (RBG Edinburgh acc. 761673 ; L12614) Epacris impressa Labill. (RBG Edinburgh acc. 680556 ; L01915)

321

*Epacris lanuginosa Labill. (UNSW 22531) *Lebetanthus myrsinites (Lam.) Duse! n (MW Chase 891) Leucopogon fraseri A. Cunn. (RBG Edinburgh acc. 341059 ; L12620) *Leucopogon muticus R. Br. (NSW, Powell 4866) *Lysinema ciliatum R. Br. (UNSW 22193) *Melichrus procumbens (Cav.) Druce (NSW, Powell 4863) *Monotoca scoparia (Sm.) R. Br. (NSW, Powell 4865) Pentachondra pumila (J. R. and G. Forst.) R. Br. (RBG Edinburgh acc. 6810338 ; L12621) *Prionotes cerinthoides (Labill.) R. Br. (MW Chase 890) *Richea pandanifolia J. D. Hook. (UNSW, GP Findlay s.n.) *Rupicola sprengelioides Maiden & Betche (UNSW 22545) *Sphenotoma dracophylloides Sonder (CBG acc. 7900865) *Sprengelia incarnata Sm. (UNSW 22539) *Woollsia pungens (Cav.) F. Muell. (UNSW 22510) Ericaceae Arbutus canariensis Duhamel (RBG Edinburgh acc. 752562 ; L12597) Arctostaphylos uŠa-ursi (L.) Spreng. (Arnold Arb. acc. 361–68 ; L12598) Befaria racemosa Vent. (NCU, Kron 2070 ; L12600) Calluna Šulgaris (L.) Hull (RBG Edinburgh acc. 721433 ; L12601) Cassiope mertensiana (Bong.) G. Don (Arnold Arb. acc. 75–83 ; L12603) Chamaedaphne calyculata (L.) Moench. (RBG Edinburgh acc. 752181 ; L12606) Daboecia cantabrica (Hudson) K. Koch (RBG Edinburgh acc. 751770 ; L12611) Elliotia racemosa Muhl. ex Elliot (RBG Edinburgh acc. 672632 ; L12615) Enkianthus campanulatus G. Nicholson (Arnold Arb. acc. 14528-C ; L12616) Erica australis L. (RBG Edinburgh acc. 781912 ; L12617) Gaultheria eriophylla (Pers.) Sleumer (RBG Edinburgh acc. 763043 ; L12618) Leiophyllum buxifolium (Berg) Elliot (NCU, Kron 2067 ; L12619) LeucothoeX racemosa (L.) Gray (NCU, Kron s.n.) Rhododendron hippophaeoides F. Balfour and Forrest (RBG Edinburgh acc. 321022 ; L01949) Vaccinium macrocarpon Aiton (NCU, Hills 89019 ; L12625) Zenobia pulŠerulenta (Bartram) Pollard (Arnold Arb. acc. 21087 ; L12626) Pyrolaceae Pyrola rotundifolia L. (NCU, Kron 1906 ; L12622)