Phylogeny of the tribe Colletieae, Rhamnaceae

Botanical Journal of the Linnean Society (1999) 131: 1–43. With 2 figures Article ID: bojl.1999.0254, available online at http://www.idealibrary.com on

Phylogeny of the tribe Colletieae, Rhamnaceae LONE AAGESEN Ca´tedra de Bota´nica, Facultad de Agronomı´a, Universidad de Buenos Aires, Av. San Martı´n 4453, RA-1417 Buenos Aires, Argentina Received November 1998; accepted for publication March 1999

A cladistic analysis based on 63 morphological characters was carried out on the tribe Colletieae including two presumed closely related genera, Ceanothus and Noltea as outgroups. In addition to a parsimony analysis of the equally weighted characters, analyses investigating the effects of character weighting, removal of a presumed hybrid species as well as the impact of uncertainly scored characters were undertaken. In all analyses Noltea was placed as sister group to a well supported monophyletic Colletieae. Nineteen different ingroup topologies were found, with the additional analyses mainly supporting two of them. Within the Colletieae a basal dichotomy divides Trevoa and Retanilla from the remainder of the tribe. While the Trevoa–Retanilla clade is fully resolved, the second lacks detailed resolution. Within this clade the Colletia species form a well supported monophyletic group, while monophyly of the disjunct genus Discaria could not be confirmed.  1999 The Linnean Society of London

ADDITIONAL KEY WORDS:—morphology – leaf anatomy – Adolphia – Colletia – Discaria – Kentrothamnus – Retanilla – Trevoa. CONTENTS

Introduction . . . . . Material and methods . Ingroup taxa . . . Outgroup taxa . . Material . . . . Phylogenetic analyses Characters . . . . . Habit . . . . . Leaves . . . . . Inflorescences . . . Perianth . . . . Androecium . . . Gynoecium . . . Disc . . . . . . Fruit . . . . . . Seed . . . . . . Results . . . . . . Phylogenetic analyses Weighting . . . .

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Excluding Discaria americana from the matrix . . Uncertainly scored characters . . . . . . Discussion . . . . . . . . . . . . . . Sister group of the Colletieae . . . . . . . The Colletieae . . . . . . . . . . . The Trevoa–Retanilla clade . . . . . . . . Retanilla . . . . . . . . . . . . . . The Adolphia–Kentrothamnus–Discaria–Colletia clade Colletia . . . . . . . . . . . . . . Adolphia and Kentrothamnus . . . . . . . . Discaria . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . References . . . . . . . . . . . . . . Appendix 1: specimens examined . . . . . . . Appendix 2: data matrix . . . . . . . . . .

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INTRODUCTION

The Colletieae belong to the family Rhamnaceae of the order Rhamnales which at least in older classifications (e.g. Dahlgren, 1975; Cronquist, 1981) also includes the Vitaceae and the Leeaceae. The Rhamnaceae differ from the two other families mainly by a combination of characters: simple leaves, valvate sepals enclosing the flower bud, fruits never being a berry, and seeds with a comparatively large embryo and scant endosperm ( Johnston, 1982). Johnston considered it likely that the Rhamnales as defined above forms a polyphyletic assemblage, an assumption supported by recent phylogenetic analyses based on sequence data from the chloroplast gene rbcL (Chase et al., 1993; Gunther, Kochert & Giannasi, 1994; Conti, Litt & Sytsma, 1996; Gustafsson, Backlund & Bremer, 1996). In all analyses the Vitaceae and the Leeaceae group far away from the Rhamnaceae, which comes out close to the Urticales, Fagaceae, Myricaceae, and Rosaceae. According to Suessenguth (1953) the Rhamnaceae consists of about 45 genera grouped in five tribes. The Colletieae are one of the smaller tribes of the Rhamnaceae, and presently the only one within the family in which all genera have been revised recently: Kentrothamnus Suess. & Overkott ( Johnston, 1973; 1 sp.), Discaria Hook. (Tortosa, 1983a; 8 spp.), Colletia Comm. ex A.Juss. (Tortosa, 1989; 5 spp.), Retanilla (DC.) Brongn. (Tortosa, 1992; 4 spp.), Trevoa Miers ex. Hook. (Tortosa, 1992; 1 sp.), and Adolphia Meisn. (Tortosa, 1993; 1 sp.). Furthermore, the Colletieae are one of the three tribes that was retained as monophyletic in the phylogenetic analysis of Richardson et al. (1997) based on plastid DNA sequence data. Most Colletieae species are distributed in South America, particularly in the area defined by Crisci et al. (1991) as southern South America, viz. the area south of 30° southern latitude as well as the Andean highlands. A few members of the tribe are found in southern North America and Mexico as well as in Australia, Tasmania, and New Zealand. Apart from the exclusion of a few misplaced species, the circumscription of the Colletieae has never been disputed. Decussate leaves, abundance of spines and the presence of serial meristems in the leaf axils have traditionally been the diagnostic characters of the tribe (e.g. Miers, 1860; Suessenguth, 1953). Although the above combination of characters characterizes the Colletieae within

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the Rhamnaceae, the individual characters are paralleled in other taxa of the family, and hence not readily support for monophyly of the tribe. Decussate leaves are found in various other taxa of the family (Suessenguth, 1953), e.g. in Ceanothus L. (subgenus Cerastes [S. Watson] McMinn) a genus considered to be closely related to the Colletieae by Johnston (1971). Spines are also found throughout the family (Suessenguth, 1953), while the occurrence of serial meristems is of more restricted distribution. Within the Colletieae serial meristems are found in all species except in Retanilla stricta Hook. & Arn. and in Discaria articulata (Phil.) Miers (Tortosa, 1983a, 1992). The serial buds emerge by asymmetric growth of a single meristem, first forming the distal bud and later the proximal one (Tourn, Medan & Tortosa, 1989). The distal bud originates a sylleptic spine, which in some species branches into higher order axes. To avoid confusion, the whole shoot-system developed from the distal bud will herein be refered to as ‘spine’. The development of the proximal bud is proleptic, giving rise to either a synflorescence or a new macroblast, or, in some species, a brachyblast (Tortosa, Aagesen & Tourn, 1996). Within the Rhamnaceae multiple buds are furthermore found in at least five species belonging to Ziziphus Mill., Paliurus Mill., and Gouania Jacq. (Urban, 1924; Cremers, 1974; Tourn et al., 1989). The multiple meristems of P. spina-christi Mill., Z. jujuba Mill., and Z. mistol Griseb. were studied by Tourn et al. (1989), who found that the ontogenetic development of the meristems in the two former species resembled the one known from Colletieae. Both Paliurus and Ziziphus belong, however, to the tribe Zizipheae characterized by the presence of drupes with only one stone not separating into mericarps, while Gouania belongs to the tribe Gouanieae diagnosed by the presence of winged fruits and/or cirri (Suessenguth, 1953). Apart from the scattered presence of serial buds in the two above-mentioned tribes the Colletieae do not share other likely synapomorphies with either Zizipheae or Gouanieae, and the presence of serial buds in the three tribes is probably due to homoplasy. The character is therefore a likely synapomorphy for the Colletieae provided that serial meristems are secondarily lost in Retanilla stricta and Discaria articulata. The Colletieae are principally shrubs but nearly all species can reach the size and shape of small trees; an exception to this is Discaria nana (Clos) Weberb., a dense, prostrate shrub from the southern Andes above 2000 m a.s.1. Adaptation to xeric conditions is common within the tribe; most xerophytic species are aphyllous or subaphyllous, with photosynthetic activity mainly taking place in younger axes and/or spines. Two species—Trevoa quinquenervia Gillies & Hook. and Retanilla trinervia (Gillies & Hook.) Hook. & Arn.—both endemic to the mattoral of central Chile, are summer deciduous as an adaptation to the Mediterranean climate of this region (Hoffmann, 1972; Montenegro, Riveros & Alcalde, 1980). The mesophytic members of the tribe are restricted to Discaria, which also includes xeromorphic species. Discaria is furthermore the only genus with a disjunct distribution, with species occurring in South America, Australia, Tasmania, and New Zealand. Mesophytes and xerophytes are found both in South America and Australia. Variation in water requirements combined with variation in number of petals has lead some authors to segregate genera from Discaria, e.g. Ochetophila Poepp. (Endlicher, 1840) and Notophaena Miers (Miers, 1860). Ochetophila and Notophaena include all mesophytic species of Discaria, but differ in the presence and absence of petals respectively. Ochetophila was furthermore segregated because of slightly different

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stipule morphology. Both genera were, however, treated as synonyms of Discaria by Hooker (1862), later retained as sections within this genus by Suessenguth (1953). Further disagreements about the delimitation of genera within the Colletieae are restricted to the genera Trevoa and Retanilla, a species-group mainly endemic to central Chile between 30° to 38° southern latitude with one species, Retanilla patagonica (Speg.) Tort. occurring in Patagonia. In disagreement with Hooker (Hooker, 1830; Hooker & Arnott, 1833) Miers (1860) separated the summer deciduous Retanilla trinervia from the remaining aphyllous or subaphyllous Retanilla species in the monotypic genus Trevoa (what is now Trevoa quinquenervia was then placed in the monotypic genus Talguenea Miers). Miers’ grouping was not maintained in the recent revision of the genera (Tortosa, 1992, who also documents the dispute between Hooker and Miers). In addition to reviewing the morphology and taxonomy of the tribe, Miers (1860) made one of the few attempts to subdivide the tribe, arranging it into three informal divisions according to presence of petals and fruit type. No later reviewer has maintained these divisions. The discovery of nitrogen-fixing root nodules containing the actinomycete endophyte Frankia Brunch. has renewed interest in the tribe. Root nodules have now been found in all but three species of the tribe (Morrison & Harris, 1958; Bond, 1976; Medan & Tortosa, 1976, 1981; Rundel & Neel, 1978; Hall & Parsons, 1978; Tortosa & Medan, 1989; Cruz Cisneros & Valdes, 1991; Tortosa, 1992). Colletia ulicina Gillies & Hook. and C. spartioides Colla have never been investigated, while in Discaria pubescens (Brongn.) Druce root nodules have not been found in natural conditions, although they do develop in the laboratory (Hall & Parsons, 1987). Nitrogen-fixing root nodules containing actinomycete endophytes are only found in a few scattered angiosperm genera (Bond, 1976) and it is striking that the same type of root nodule has been found in most species of the genus Ceanothus from southern North America and Mexico (Bond, 1976). The main aim of the present paper is to provide a reliable estimate of the phylogenetic relationships within Colletieae, essential for ongoing studies on reproductive biology and complementary studies of character evolution of the species in the tribe (Medan, 1991, 1993; D’ambrogio & Medan, 1993; Medan & D’ambrogio, 1998). Furthermore, supplementary analyses of specific topics such as the influence of a supposed hybrid (Discaria americana Gillies & Hook.) on tree structure, the impact of characters uncertainly scored in part of the taxa as well as weighting of characters have been carried out. Possible changes in generic delimitation based on the results are not considered. This topic awaits further analyses including DNA sequence data in order to draw more firmly based conclusions.

MATERIAL AND METHODS

Ingroup taxa All presently accepted Colletieae species have been included in this analysis. Presumed autapomorphies for the species are listed in Table 1 as are general notes on species distributions. All possible hybrids have been left out of the analyses. Presumed hybrids between the recognized species have been found throughout the tribe (Tortosa, 1983b, 1988,

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1992) identified by their morphological intermediacy. In some cases presumed natural hybrids are frequent where both putative parents occur in proximity. This is the case of Discaria chacaye × D. articulata where specimens with intermediate characters were found as well as individuals showing different combinations of presumed parental species characters (Tortosa, 1983b). Discaria americana is so far the only polyploid species found within the Colletieae. It is not known whether D. americana is an auto- or allopolyploid; only a single specimen has been examined to establish chromosome number (Tortosa, 1983a) and specimens with a different number could theoretically occur. Polyploidy could suggest a hybrid origin of D. americana. Similarity between this species and some of the morphologically intermediate Discaria chacaye × D. articulata specimens has been observed (Tortosa, pers. comm.); however, morphological intermediacy is a tenuous basis for recognizing hybrids (McDade, 1992). In spite of its possible hybrid origin, D. americana has been included in the data matrix, which would be correct if D americana were shown to be an autopolyploid. Furthermore, the studies by McDade (1992) suggest that hybrids included in cladistic analyses do not necessarily cause major problems. Additional searches were, however, performed excluding D. americana from the matrix as would be proper if the species is shown to be an allopolyploid, hence of hybrid origin. Outgroup taxa In the phylogenetic analyses of the Colletieae both Ceanothus and the monotypic genus Noltea Rchb. were included as outgroups, as these genera share potential synapomorphies with the tribe. Johnston (1971) considered the genus Ceanothus as closely related to the Colletieae but provided no justification for this assumption. The presence of a pedestal left on the plant after dehiscence of the fruit (see ch 59) is, however, a possible synapomophy shared by Ceanothus and part of the Colletieae. Pedestals are not found in other species of the Rhamnaceae, though an exception to this might be the South African species Colubrina nicholsonii A.E. van Wyk & Schrire, where according to Van Wyk & Schrire (1986) the basal part of the fruit is left on the plant at dehiscence. Fruits of C. nicholsonii were unfortunately not available for the present study. The presence of root nodules is not considered as a synapomorphy for Ceanothus and the Colletieae species in this study. Including the presence of root nodules as a character implies presumed monophyly of the endophyte strains of Frankia infecting Ceanothus and the Colletieae. The phylogeny of Frankia is unknown and the endophyte is known to infect presumably distantly related angiosperm taxa (see, however, Swensen [1996] for a recent analysis concerning phylogeny of actinorhizal plants including Ceanothus and three species of the Colletieae). Due to the size of Ceanothus—it comprises 55 species (Mabberley, 1990)—ten species, five from each subgenus, were included as placeholders. As the relationships among outgroup terminals may affect the ingroup topology (Nixon & Carpenter, 1993), including alternative placeholders or a different number of placeholder species of Ceanothus could potentially result in different hypotheses concerning the phylogeny of the Colletieae. The included species were chosen pseudorandomly, selecting those where the most comprehensive herbarium material was available, and on the basis that characters found both in the ingroup and in Ceanothus were represented in at

Autapomorphies – long exserted filaments flat triangular spines; rhizomes – – long, narrow, red-coloured hypanthium tube chromosome number 2n=441

– –

Species

Adolphia infesta Meisn.

Colletia hystrix Clos

C. paradoxa (Spreng.) Escal.

C. spartioides Bertero ex Colla C. spinosissima Gmel.

C. ulicina Gill. & Hook. Discaria americana Gill. & Hook.

D. articulata (Phil.) Miers

D. chacaye (G. Don) Tort.

In the mountain area from southwest Texas U.S.A. to Oaxaca Mexico (>18° northern latitude) and from southernmost California to northern Baja California (>28° northern latitude). In Chile between 28° 30′ to 47° southern latitude, from the Pacific coast to the Andes below 2000 m. In Argentina in the Andes between 40° to 47° southern latitude. In the mountain area of southeast Brazil (between >25° to 30° southern latitude), at the coast of Uruguay, and in the Argentinean coast at >38° southern latitude. The island Mas a Tierra of the Juan Ferna´ndez archipelago (Chile). In Ecuador, Peru, Bolivia, and northwest and central Argentina to Uruguay (>36° southern latitude). Living between 2000 m. to 4000 m. and at sea level in the southeastern extreme of it’s distribution. In Chile between 30° to 35° 30′ from sea level to 1000 m.a.s. East of the Andes from southeast Brazil and northern Argentina (>28° southern latitude) through Uruguay to the Valde´s Peninsula (>42° southern latitude) in southern Argentina, reaching the Andes at >32° southern latitude. Near the Andes in Chile between >36° to 38° southern latitude and in Argentina from >38° to 44° southern latitude. Also found on the Somuncura´ Mesa east of the main distribution area. In Chile from >33° southern latitude to Tierra del Fuego and in Argentina from >36° southern latitude to Tierra del Fuego.

Distribution

T 1. Autapomorphies and geographical distribution of the Colletieae species. Information compiled from Willis (1955), Tortosa (1983a, 1989, 1992, 1993), Hall & Parsons (1987), Medan & Mantese (1989), and Medan & Aagesen (1995)

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– – – – edge of aril deeply lobed; abscission line of flower tube sinusoid leaving the filaments on the pedestal producing several long slender unbranched macroblasts from the proximal bud of the bud complex, giving the plant an ‘ephedralike’ appearance –

campanulate flowers fruit an indehiscent drupe fruit an achene; mucilaginous thickening presents at the periclinal outer wall of some epidermis cells

D. nitida Tort.

D. toumatou Raoul

D. trinervis (Hook. & Arn.) Reiche

D. pubescens (Brong.) Druce

Kentrothamnus weddelianus (Miers) Johnst.

R. stricta Hook. & Arn. R. trinervia (Gill. & Hook.) Hook. & Arn. Trevoa quinquenervia Gill. & Hook.

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In west Argentina from >36° to 43° southern latitude and in the area of the Golf of San Jorge (from >44° to 47° southern latitude), between the two areas only known from one locality: Lago Musters (>45° 20′S 69° 25′W). Also found on the Somuncura´ Mesa isolated from the main distribution area. In Chile from >30° to 36° southern latitude between 200 m. and 1250 m. In Chile from >30° to 35° southern latitude between 25 m. and 900 m. Central Chile from 80 m. to 2000 m. Between 31° and 36° southern latitude.

In the Chilean and Argentinean Andes above 2000 m., between 31° and 46° 20′ southern latitude. In the mountains of southeast Australia (eastern New South Wales and Victoria) above 1100 m. Only four localities are known. New Zealand, most common on the South Island east of the Southern Alps; Chatham Island. In Chile from >31° to 38° southern latitude and in Argentina from >31° to 48° southern latitude. Also found east of the main distribution area at Sierra de los Chacays south of the Somuncura´ Mesa. In the mountains of southeast Australia (eastern New South Wales and Victoria earlier also present in southeast Queensland) and east Tasmania from 200 m. to 1400 m. The Altiplano of Bolivia east of 67° western longitude reaching the limit with Argentina. In Chile from >32° to 38° southern latitude between 120 m. and 1500 m.

In all other species 2n=22 (known for Colletia paradoxa, Discaria articulata, D. chacaye, D. nana, D. toumatou, and D. trinervis) (see Tortosa 1983a, 1989 and references therein).

R. patagonica (Speg.) Tort.

Retanilla ephedra (Vent.) Brong.

densely branched, prostrate shrub

D. nana (Clos) Weberb.

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least one terminal. As recommended by Nixon & Carpenter (1993), some characters were included in the analyses in order to resolve outgroup relations within Ceanothus. Including characters likely to resolve relationships within the outgroups can prevent some of the erroneous ingroup topologies found due to incorrect outgroup resolutions (Nixon & Carpenter, 1993). The monotypic genus Noltea Rchb. was furthermore included as outgroup. Possible synapomorphies for the Colletieae and Noltea include the presence of a prolonged hypanthium tube (see ch 34), and the flower disc which resembles the one found in Adolphia and Kentrothamnus (see ch 55). For the purpose of rooting, Colubrina asiatica (L.) Brongn. was included in the analysis in order to include one taxon having character states found commonly in the Rhamnaceae. The genus Colubrina Rich. ex. Brongn. was considered ‘primitive’ by Johnston (1971) who diagnosed it in negative terms by the lack of apomorphic characters found in other genera. Colubrina asiatica was also chosen as placeholder for the genus as information concerning the morphology of this species has appeared recently (Medan & Hilger, 1992). Material Authors of generic names are in accord with Brummitt (1992) while authors of subgeneric names and species names follow Suessenguth (1953) for species not belonging to the Colletieae. All authorities are cited in agreement with Brummitt & Powell (1992). The morphological characters were examined on herbarium material of all species except Colletia spartioides. Material of this latter species was unfortunately not available for the present analyses. Characters scored for C. spartioides are based on the literature indicated in the discussion of each character. Specimens held by the following herbaria were examined (Abb. according to Holmgren, Holmgren & Barnett, 1990): AK, BA, BAA, CONC, LIL, MEL, MO, NH, NSW, SGO, and SI. For a complete list of specimens studied, see Appendix 1. Additionally, living material of Colletia spinosissima J.F.Gmel., C. paradoxa (Spreng.) Escal., Discaria americana, Noltea africana (L.) Rchb., and Retanilla trinervia cultivated in the Lucien Hauman Botanical Garden, Facultad de Agronomı´a, Universidad de Buenos Aires (Argentina) was studied. Field observations were done on Colletia hystrix Clos, C. spinosissima, Discaria americana, D. articulata, D. chacaye (G. Don) Tort., D. nana, and D. trinervis (Hook. & Arn.) Reiche. Morphological characters of the inflorescence (ch 22–32) are scored in accordance with the theories of Troll (see Weberling, 1989). Characters concerning Colubrina asiatica were partly found in Medan & Hilger (1992). Anatomical characters of leaves followed Medan (1974, 1986), Medan and Mantese (1989) and Mantese and Medan, (1992, 1993) and were confirmed on the original material kindly provided by these authors. Adult leaves of herbarium specimens of all outgroup species included in the analyses were soaked overnight in weak detergent solution, hand sectioned, mounted in glycerine jelly to which 1% water-soluble safranin was added and studied under a light microscope. Phylogenetic analyses Cladistic analyses were based on maximum parsimony (Farris, 1983) using the program NONA ver. 1.8 (Goloboff, 1997a). All analyses were run with both equal

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weighted and weighted characters. Characters were weighted according to their implied weights (see Goloboff [1993] for a discussion concerning appropriate weighting functions) using Pee-Wee ver. 2.8 (Goloboff, 1997b). Due to the high number of terminals a heuristic approach was adopted. All multistate characters were treated as nonadditive. Bremer supports (Bremer, 1994) were calculated using PAUP ver. 3.1.1 (Swofford, 1991). Jackknife group frequencies (Farris et al., 1996) were calculated by NONA using jak.run (1000 iterations, randomly deleting 36% of the characters, with the search option mult∗5) and fq.exe, which calculates a majority rule consensus trees of the output (see Goloboff, 1997a, b for details). The data matrix is included in Appendix 2. Equal weighted analyses The data matrix was analysed with NONA using: (1) amb- (only unambiguous support, collapsing branches when the set of possible states of the ancestral node and the set of possible states of the descendant node have one or more character states in common); (2) mult∗50 (randomizes the order of taxa in the matrix, creates a Wagner tree and submits it to tree bisection and reconnection swapping [TBR], repeated 50 times); (3) max∗ (TBR swapping on initial trees); (4) multiple branch swapper sswap∗ (see Goloboff, 1997a, b for details). Swapping on suboptimal trees, in an attempt to find possible islands (Maddison, 1991), was done by applying options jump∗1 and jump∗2, which perform TBR swapping on trees 1 or 2 steps longer than the input trees (found in the previous analyses). Weighted analyses Weighting of the characters was carried out using implied weights (Goloboff, 1993) available in Pee-Wee. Pee-Wee weights characters according to how well they fit a specific tree. The strength by which it weights against a homoplastic character depends on an adjustable constant (conc=1, 2, 3, . . . 6) with low values weighting strongest against homoplasy. Pee-Wee was used with the options conc 3, amb-, mult∗ 20, sswap∗ and mswap∗2. Additional searches were performed on PeeWee using all possible conc values (1, 2 . . . 6) with the search options amb-, mult∗ 5 and sswap∗. Swapping on suboptimal trees was done by options jump∗1 to jump∗20, doing TBR swapping on trees having a fit 0.1 to 2.0 lower than the input trees (all fittest trees found in the initial heuristic analyses). Influence of the possible hybrid Discaria americana The data matrix excluding Discaria americana was analysed with NONA (default settings, mult∗5 and sswap) and with Pee-Wee (default settings, mult∗5 and sswap). Influence of uncertainly scored characters Characters 5, 23, and 53 all varied within the same individual among the South American Discaria species. Hence to be correctly scored, abundant herbarium

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material or field observations were required. Since only limited herbarium material was available for the Australian and the New Zealand Discaria species, the above mentioned characters were unreliably scored for these taxa. The above three characters were left out consecutively using NONA (default settings, mult∗20, sswap), and by Pee-Wee (default settings, mult∗20 and sswap∗).

CHARACTERS

Of the 63 characters investigated 52 are phylogenetically informative at the ingroup level. Some of the remaining characters are included as possible synapomorphies uniting Ceanothus and the Colletieae or Noltea and the Colletieae; others are informative for Ceanothus only. These latter characters are included to provide some information concerning structure within Ceanothus, thus reducing the number of most parsimonious trees found during analyses, due to multiple equal parsimonious resolutions within Ceanothus. Autapomorphies are not included in the analyses (concerning these characters see Table 1). When informative characters used by Johnston (1973) or Tortosa (1983a, 1989, 1992, 1993) in their generic revisions of the Colletieae are omitted or scored differently, this is discussed in the text. Habit Although at first glance quite variable, the habit of the species of the Colletieae is very uniform (Tortosa et al., 1996). All species are built of macroblasts with limited growth—developing during one growth season—and linked sympodially. In his revision of Discaria, Tortosa (1983a) found that the first node of the spines may be either proximally or distally located. While informative for Discaria, this character has shown to be difficult to apply in other species of the Colletieae, where the nodes are not clearly placed in one of the two positions found in Discaria. Consequently the character was not included in the analyses. 1. Growth form: erect (0); prostrate (1). While Discaria nana is always a prostrate shrub, intraspecific variation exists in D. chacaye, where only plants growing at high altitudes are prostrate. According to Cockayne (1905) D. toumatou Raoul, mostly a shrub, sometimes reach the size of a small tree in wet mountain regions, while in extreme xerophytic habitats it becomes prostrate. In Ceanothus, two species (C. prostratus Benth. and C. pumilus Greene) are prostrate shrubs, while in five species prostrate forms are sometimes found, apparently associated with exposure to wind (McMinn, 1942). Of the seven prostrate Ceanothus species, three are included in the outgroup (C. prostratus, C. cuneatus [Hook.] Nutt. and C. thyrsiflorus Esch.). In one, C. cuneatus, the prostrate forms are occasionally found in Sierra Nevada in localities where C. cuneatus and C. prostratus occur. According to McMinn (1942) these plants may belong to a hybrid complex formed by the two species. Considering this information, C. cuneatus is not scored as polymorphic. All other species, which show variation in growth form, are scored as polymorphic in the matrix. 2. Phyllotaxis: alternate (0); decussate (1). Although not informative at the ingroup level, the character has been included in the analyses to investigate, whether the

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decussate leaves found in part of the subtribe Cerastes have evolved in parallel with the decussate leaves found in the Colletieae. 3. Serial meristems in leaf axils: absent or only extremely seldom present (0); present at all nodes, only seldom lacking (1); lacking at various nodes, frequently leaving some branches unarmed (2). The presence of serial meristem complexes in the leaf axils is probably an autapomorphy for species of the Colletieae (though serial meristems are lacking in Discaria articulata and Retanilla stricta). In most species, spines (hence serial meristems) are found at nearly all nodes, but in D. chacaye, D. nana and D. trinervis spines are often lacking at several nodes and completely unarmed branches are frequently found. The same condition seems to be found in D. nitida Tort., described by Tortosa (1977) as a slightly or very spiny shrub, although this could not be observed on the scanty material available for this study. Cockayne (1905) noted that no spines were formed in seedlings (or branches of adult plants) of D. toumatou grown in higher moisture and more feeble light conditions than normal. Populations with few almost completely spineless plants were later recorded (Cockayne, 1922). According to Cockayne (1922) although some of these plants seemed to be unarmed due to fungus attacks (witches’ broom), others lacked the dense habit caused by this disease. These latter plants had some branches with a few spines while other branches were completely spineless. The occurrence of these partly unarmed specimens seems more restricted in D. toumatou than in other Discaria species scored as having character state (henceforth ch) 2. Thus, it is with some doubt D. toumatou is coded in this way. As above mentioned, Retanilla stricta and Discaria articulata completely or almost completely lack serial meristems, while D. chacaye and R. ephedra are polymorphic for the character, including completely unarmed plants as well as plants possessing serial meristems in some or all nodes, respectively. 4. Vegetative brachyblasts: absent (0); present (1). The vegetative brachyblasts are here distinguished from the brachyblasts formed by the reduced internodes of the synflorescence in the genus Colletia (see ch 24). The vegetative brachyblasts found in the Colletieae function as assimilating structures during one to several years, and may then produce a terminal synflorescence. After flowering, growth of the brachyblasts is continued by lateral branching. These lateral branches repeat the growth pattern of the primary axes of the brachyblast. Macroblasts are often seen sprouting from one or more apical buds of a brachyblast. Vegetative brachyblasts are found in Trevoa quinquenervia and in several species of Discaria. In D. pubescens a series of bracteose, reduced internodes are often seen at the proximal part of a synflorescence, but assimilating brachyblasts are never found. In the outgroups brachyblasts are apparently found in Ceanothus. In C. leucodermis Greene and C. cuneatus several nodes long brachyblasts are formed, while in C. papillosus Torr. & A.Gray, C. purpureus Jeps., C. thyrsiflorus, C. verrucosus Nutt. and C. greggii A. Gray lateral branches may initially produce two or three short internodes and then produce long internodes. Although it is not clear whether these short internodes are homologous to the brachyblasts seen in C. leucodermis and C. cuneatus, they are here provisionally coded as such. 5. Apex of macroblasts, developed from the proximal meristem of axillary meristem complexes: never differentiated into a spine (0); sometimes differentiated into a spine (1). In

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several species of the Colletieae the apex of the macroblast, developed from the proximal meristem of the meristem complexes, may differentiate into a spine before ceasing growth. Not all macroblasts are thorn-tipped, but in Colletia, Adolphia, Kentrothamnus as well as some species of Retanilla and Discaria, some thorn-tipped macroblasts are found, while in Trevoa, Retanilla trinervia and other Discaria species they are never found. To avoid hypotheses of homology between one of the two serial meristems of the meristem complexes, and the single bud found in at least in some nodes of all species, the character is only applied to macroblasts developed from the proximal bud of the serial bud complexes. 6. Sprouting of macroblasts or brachyblasts from prophylls of older macroblasts: absent (0); present (1). Sprouting from the prophylls of older macroblasts results in three verticillately arranged branches or brachyblasts. In several species the sprouted macroblasts may also give rise to macroblasts from their prophylls, resulting in three verticillately arranged branches more or less perpendicular to the first row. The character is seen in all species of Colletieae, though most clearly in Retanilla ephedra and Discaria articulata. A similar situation has also been observed in Ceanothus greggii (with alternate leaves), where paired branches were found in one specimen. The character has with some hesitation been scored as present in this species although it may be to overemphasize this single observation. Leaves Some characters from Medan and Mantese’s treatment of the leaf anatomy of the species in the Colletieae (Medan, 1974, 1986; Medan & Mantese, 1989; Mantese & Medan, 1992, 1993) have been included here. The scoring of characters from the above mentioned data sets is somewhat complicated, due to sample problems related to the relatively small amount of leaves cut per specimen or species. Characters have been chosen or scored in order to minimize this amount of uncertainty, discussed for each corresponding character. On revising the material, all characters regarding indumentum were excluded from the analyses. Although apparently discrete, characters such as hair shape, density of hair cover and presence or absence of hairs from the abaxial or adaxial surfaces, have all shown continuous variation. Concerning nervation patterns, the authors classified all species according to the system of Hickey (1973). The variation in the Colletieae is, however, continuous to an extent that the only character that seems discrete is the presence of one or three main nerves. 7. Foliage: leafy, leaves deciduous or long-lasting (0); aphyllous, leaves ephemeral, sparse almost lacking (1). In most species of the Colletieae assimilation takes place either mainly in the leaves or mainly in the branches. Nearly all species belong clearly to one of these categories, but the character has shown to be difficult to apply both in Retanilla patagonica and Discaria toumatou. During dry summers Retanilla patagonica—a subaphyllous species—is nearly leafless and assimilation takes place mainly in younger branches and spines, while in moister summers, leaves are quite abundant (Arce, Tortosa and Medan, pers. comm.).

PHYLOGENY OF THE TRIBE COLLETIEAE

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Judging from the herbarium specimens Discaria toumatou is subaphyllous. Cockayne (1905) noted that the leaves of D. toumatou, though most abundant in spring (principally due to developing synflorescences and young branches) are never very numerous at any time of the year. According to Cockayne, assimilation chiefly takes place in the spines and younger branches, while the leaves only play a notable part during spring. In herbarium specimens leaves are also seen at vegetative brachyblasts, hence D. toumatou is more leafy than, for example, D. americana and D. articulata. Attributing the same character state to all three species seems, therefore, inappropriate. 8. Stipules: herbaceous to scale-like (0); ‘corky’ (1). Within Ceanothus the species of the subgenera Cerastes are provided with conspicuous stipules commonly referred to as ‘corky stipules’ (according to Suessenguth [1953] the corky appearance originates from tannin accumulation in the cells). This kind of stipule is a synapomorphy for Cerastes, included in the analyses in order to minimize the number of most parsimonious trees found due to multiple equal parsimonious resolutions within the outgroups. 9. Base of stipules: caducous (0); persistent (1). In the Colletieae all species have herbaceous to scale-like stipules. These are in part caducous, but the base always persists, most markedly so in Adolphia infesta Meisn. The stipules in Cerastes (see ch 8) are persistent as well as the herbaceous stipules of Colubrina asiatica. In Noltea africana and in Ceanothus subgenus Euceanothus (Parry) McMinn, except Ceanothus papillosus, the stipules, including the base, are deciduous. 10. Base of opposite leaves: not united (0); united, forming a line (1). In some species the bases of opposite leaves are united, forming a line. Whenever the character occurs it is generally seen at all nodes; only subopposite nodes are not united. In Trevoa quinquenervia, however, the line is only found at some nodes. In Ceanothus subgenus Cerastes opposite leaves are also united by a line (the character is common in species of the Rhamnaceae having opposite leaves [Suessenguth, 1953]). 11. Leaf margin: serrate (0); dentate (1); crenate (2); entire (3). The leaf margins in the Colletieae are entire, dentate or crenate. Many species have leaves with an entire margin and leaves with a dentate margin mixed on the same plant and are coded polymorphic in the matrix. The leaf margins of Discaria chacaye are serrate or crenate and also scored as polymorphic. According to Tortosa (1992) teeth are occasionally found in Trevoa quinquenervia. This observation does, however, refer to a single specimen with glandular teeth, all other specimens had entire leaf margins (Tortosa, pers. comm.). In mixed populations of T. quinquenervia and Retanilla trinervia supposed hybrids combining characters from both presumed parents may be seen (see above concerning supposed hybrids). As mentioned above glandular teeth were seen in one specimen of T. quinquenervia, which could be due to introgression with R. trinervia (which has leaf margins with glandular teeth). Coding T. quinquenervia as polymorphic for this character seems to overemphasize a single observation, therefore in the analyses T. quinquenervia is scored as having an entire leaf margin. According to McMinn (1942) some species of Ceanothus, subgenus Euceanothus also vary with regard to the leaf margin. These species are similarly coded as polymorphic. 12. Glands at leaf margin: present (0); absent (1). In the Colletieae glands at the leaf margin are found in Retanilla trinervia and Discaria chacaye (as regards T. quinquenervia,

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see ch 11). When describing Discaria nitida, Tortosa (1977) noted the occasional presence of glands at the leaf margin. Although the occurrence of glands in D. nitida was not mentioned by Medan (1986) in his revision of leaf anatomy in Discaria, glands were found in some of the specimens revised during this study; consequently, D. nitida is scored as being polymorphic for this character. Cockayne (1900) studied the development of seedlings in D. toumatou. Both the first and second leaf pairs were found to be dentate, the teeth being glandular in the early stages of development. In the material available for this study leaves were found to be glandless. As no information concerning leaf development in either seedlings or adult plants is available for other Colletieae species, it seems best not to score D. toumatou for the presence of glands, and only apply the character to fully developed leaves. Glands are found in all species of Ceanothus subgenus Euceanothus included in the analyses. Only in C. leucodermis some variation exists. According to McMinn (1942), in this latter species leaves with an entire margin can sometimes be found mixed with leaves having a glandular-denticulate margin on the same plant. Species of subgenus Cerastes never possess glands while glands are found both in Noltea and in Colubrina asiatica. 13. Nervation: leaves with one main nerve (0); leaves with three main nerves (1). The only qualitative character related to leaf nervation is the presence of one or three main nerves. This character is, however, variable in Colletia spinosissima, C. ulicina, and Discaria nana, where leaves with one main nerve occur together with leaves having three main nerves on the same plant, as well as leaves not clearly showing to one of the two states. The same is found in Ceanothus coeruleus Lag. and in herbarium specimens of Colubrina asiatica ( Johnston [1971] only mentioned 3-nerved leaves). McMinn (1942) noted that in Ceanothus, species normally having three nerves appear almost 1-nerved when grown in dry sites, due to the reduction of the two lateral nerves. No such observations have been made for Colletieae. All above mentioned species are coded as polymorphic for the character. 14. Adaxial stomata: absent (0); present (1). In a few species of the Colletieae, scattered stomata are found on the adaxial leaf surface. In Adolphia infesta adaxial stomata were found rarely in some specimens (not originally noted by Mantese & Medan [1993]). Due to insufficient material it has not been possible to state whether the character is present only in some individuals or whether all individuals have at least some leaves with adaxial stomata. In the analyses the character is scored as being polymorphic in Adolphia. 15. Stomata type: anomocytic (0); paracytic (1). In the Colletieae only the Colletia has paracytic stomata while all other species (including the outgroup) have anomocytic stomata. 16. Position of stomata: not in pits (0); in pits (1). Stomata in pits is a synapomorphy for Cerastes, included in the analyses to minimize the number of most parsimonious trees found due to multiple equal parsimonious resolutions within the outgroups. 17. Symmetry of leaves: dorsiventral (0); subisolateral (1). Subisolateral leaves appear in few species of the Colletieae. Gemoll (1902) considered the leaves in Ceanothus coeruleus and Ceanothus subgenus Cerastes as subisolateral.

PHYLOGENY OF THE TRIBE COLLETIEAE

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The leaves of Ceanothus coeruleus sectioned during this study were all dorsiventral. The leaf anatomy in the subgenus Cerastes is somewhat complicated by the pronounced stomatal pits, the amount of mucilaginous-tanning containing cells (see ch 20) and the restricted occurrence of parenchyma. Gemoll (1902) probably referred exclusively to the areas of parenchyma when stating that the leaves were subisolateral. This interpretation is followed in this analysis. 18. Main nerves: in some parts adhered to the adaxial epidermis through bundle sheath extension (0); not adhered (1). This character is common in the Colletieae. A bundle sheath extension is normally not found in Discaria pubescens, only in one case (of 18 specimens revised) a weak bundle sheath extension was seen connecting the main nerve and the adaxial epidermis. With some hesitation D. pubescens is therefore scored as polymorphic for the character. 19. Periclinal inner wall of epidermis: cells with mucilaginous thickening present (0); absent (1). Linsbauer (1930) treated epidermis cells with a mucilaginous thickening on the periclinal inner wall. Mucilage containing-cells in the epidermis were not originally noted by Mantese & Medan (1993) in Adolphia infesta. Their occurrence is sparse, and it is not clear whether all plants have at least some leaves with mucilagecontaining cells. The character is scored as polymorphic for Adolphia. 20. Mucilaginous-tannin containing cells: present (0); absent (1). The character refers to cells, often of considerable size, in the epidermis or mesophyll. The cells contain an amber-coloured substance, presumably tannin and mucilage (Gemoll, 1902; Bo¨cher & Lyshede, 1972). These cells are often quite abundant and may frequently form an almost continuous layer below the adaxial epidermis. The mucilaginous-tannin containing cells may be present in epidermis, mesophyll or bundle sheaths. In some species the cells seem restricted to only one of the tissues. Confirmation of this demands a more thorough investigation of each species, and only the presence or absence of mucilaginous-tannin containing cells has been chosen as a character in this analyses. Mucilaginous-tannin containing cells were found only in one specimen of Discaria nitida, hence the species has been coded polymorphic for the character. 21. Abaxial hypodermis: absent (0); present (1). An abaxial hypodermis is only found in Discaria nana and Retanilla stricta. Inflorescences The complete analyses concerning morphology of the inflorescences, including illustrations, is presented elsewhere (Tortosa et al., 1996). 22. Second order synflorescences sprouting from the prophylls of old first-order synflorescences: no (0); yes (1). In all but three species of the Colletieae the synflorescences may develop from the prophyll axils of older synflorescences. These new synflorescences are here considered second-order synflorescences, as they sprout from the buds of the primary synflorescence. Second-order synflorescences were not found in Discaria nana, Retanilla ephedra, R. stricta, or in the outgroup species. 23. Sprouting of second-order synflorescences during anthesis of first-order synflorescence: no (0); yes (1). In most Colletieae the sprouting of the second-order synflorescences is

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delayed to the succeeding growth season (while the anthesis of the first-order synflorescence take place in the current one) or even later. In all examined Colletia species sprouting of second-order synflorescences frequently takes place during the anthesis of the primary synflorescence. In richly flowering specimens of Adolphia infesta, Discaria americana, D. articulata, and D. pubescens second-order synflorescences can similarly be found arising from the prophylls of the flowering first-order synflorescence. This has not been observed in other species of Discaria; however, as only limited material of D. toumatou and D. nitida was available, character state 1 cannot be ruled out. 24. Internodes of synflorescences: all visible (0); internodes of the main axis and at least first order paracladia visible, internodes of higher order paracladia condensed (1); internodes of the main axis visible, internodes of all paracladia condensed (2); synflorescence forming a condensed brachyblast, no internodes visible (3). In the Colletieae all internodes of the paracladia are reduced, hence the flowers and prophylls, when present (see ch 29), all appear to branch from the same point. The internodes of the main axis are, however, unreduced, except in Colletia where all internodes of the synflorescences are reduced, consequently the synflorescences resemble condensed flowering brachyblasts. Synflorescences with highly-branched paracladia occur in some species of Ceanothus. Here the internodes of at least the first-order paracladia are visible, while higherorder paracladia have reduced internodes. In other species of Ceanothus the synflorescences are poorly branched and all internodes of the paracladia are reduced (as in the Colletieae). In Noltea and Colubrina asiatica all internodes are visible in the synflorescences. 25. Apical meristem of synflorescences: developing a terminal flower (0); proliferating (1). In Noltea, Ceanothus, Trevoa, and Retanilla the synflorescences end in a terminal flower (they are anthotelic [Briggs & Johnson, 1979]), which is occasionally lacking in Retanilla patagonica and R. ephedra. In Colletia, Discaria, Adolphia, and Kentrothamnus the synflorescences proliferate (they are auxotely [Briggs & Johnson, 1979], forming new macroblasts). 26. Time of proliferation: proliferation occurring always during anthesis (0); proliferation in same growth season, but often delayed until after anthesis (1); proliferation at least delayed to the next growth season (2). Proliferation of the synflorescences may be delayed until after the anthesis. Some proliferating synflorescences can be found during anthesis in all species (except in Colletia), but in Discaria chacaye and Kentrothamnus weddellianus (Miers) Johnst. the proliferation is never delayed. In Colletia, the apical meristem enters dormancy after producing flowers, if growth is continued at all this is delayed until the next growth season or even later, where the apical meristem continues forming new paracladia. The synflorescences in Colletia produce second-order synflorescences (and third-order synflorescences in C. paradoxa) as well as accessory synflorescences, all with dormant apical meristems. All apical meristems are apparently capable of proliferating, and several macroblasts are often seen sprouting from the same brachyblasts. 27. Apical meristem of synflorescences: fertile during one growth season (0); fertile during more than one growth season (1). The formation of paracladia from the apical meristem of the synflorescences during more than one growth season (see ch 26) is only found in Colletia.

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28. Foliage of synflorescences: frondo-frondulose to frondo-bracteose, with leaves proximal and bracts (or smaller leaves) distal (0); frondo-frondulose to frondo-bracteose, with bracts proximal and leaves distal (1) frondose (2); bracteose (3). The foliage of the synflorescences is quite variable. In the anthotelic synflorescences with cymose paracladia (see ch 31) the foliage is frondo-frondulose to frondo-bracteose, with the size of the leaves decreasing towards the apex of the synflorescence. The synflorescences in Retanilla ephedra and R. stricta with uniflorous paracladia always have bracteose foliage. In R. patagonica bracteose foliage is occasionally found in sparsely flowering synflorescences. The foliage is frondose in Discaria chacaye, D. trinervis, D. nana, and Kentrothamnus weddellianus. In Adolphia infesta and all other Discaria species the foliage is frondobracteose (occasionally frondo-frondulose) with the size of the leaves increasing towards the apex of the synflorescence. Frondose foliage is occasionally found in A. infesta. In all species of Colletia as well as in most of the outgroup the foliage is bracteose. 29. Pherophylls of primary flower is paracladia: partially covering paracladium before anthesis (0); covering whole paracladium before anthesis, this resembling a cone (1); not covering paracladium before anthesis (2). In Ceanothus the pherophylls (sensu Briggs & Johnson [1979]) of the primary flower in the paracladia cover the young synflorescences, thus resembling a cone. The condition, which was present in all species examined, is a possible autapomorphy for Ceanothus and apparently not found in other Rhamnaceae. Curiously the character was found in one specimen of Colletia hystrix (D. Medan 597 [BAA]), where aberrant growth of the pherophylls in several flowering brachyblasts caused them to resemble small cones. In Noltea and Colubrina asiatica the pherophylls only partly cover the young synflorescences, while in the Colletieae the developing synflorescences are uncovered. 30. Persistence of primary flower pherophylls in paracladia: caducous (0); persistent (1). In Noltea and all Ceanothus species the bracts covering the young synflorescences are shed at the initiation of anthesis, while in the Colletieae and in Colubrina asiatica the pherophylls are persistent. 31. Paracladia: cymose (0); uniflorous (1). The paracladia found in the Colletieae are 3–7-flowered cymes or solitary flowers. Adolphia, Kentrothamnus and most Discaria species have three-flowered cymes while seven-flowered cymes are found in Trevoa and Retanilla trinervia. Variation in the number of flowers does, however, exist, while the only distinction that has been made here is between cymes and uniflorous paracladia. In some species with cymose paracladia (Discaria americana, D. articulata, and Retanilla patagonica) specimens with uniflorous paracladia can also be found. A similar reduction was never seen in other species. It may be justified to treat this potential reduction as a separate character state that could be informative especially for Discaria. It seems probable that the character state may vary according to ecological conditions; correct scoring of this would then require abundant material for each species, a condition not met in the Australian and New Zealand species. Uniflorous paracladia have therefore been scored only when cymes were never been observed in the species concerned. 32. Prophylls of primary flower in paracladia: present (0); absent (1). Both first- and (when present) second-order flowers, are provided with pherophylls and prophylls in most

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species of the Colletieae. In Retanilla ephedra, R. patagonica, and R. stricta only the pherophylls of the primary flower develop, while all flowers lack prophylls. Perianth Most micro-characters concerning flower and fruit have been derived from Medan and Aagesen (1995). The flowers in the Colletieae are normally 4- or 5-merous, but 3- and 6-merous flowers occasionally appear. The amount of intraspecific variation found in all species makes the character unsuitable for this analysis. Tortosa (1982) studied the vasculature of the flowers of Colletia paradoxa, C. spinosissima, Discaria americana, D. articulata, and D. trinervis. Most differences occurred in degree of branching of vascular bundles in sepals and petals; nervation of the disc was the only qualitative difference found among the species. Tortosa noted that veinlets composed of poorly differentiated elements were found in all species studied except D. trinervis. No attempt has been made to use this character in the analyses; although serial sections of all species belonging to the Colletieae were available, these were not properly stained to identify phloem. Unfortunately flowers of Colletia spartioides were not available, so information concerning these was derived from Miers (1860) and Skottsberg (1928). 33. Flower colour: white to white-cream (0); white-rose (1); red (2); bluish to purple (3); yellowish (4). Most species of the Colletieae have white to white-cream flowers, the only exceptions being Colletia ulicina that has red flowers and Kentrothamnus weddellianus which has flowers that are white-cream with rose base and rose lines leading to the filaments according to Johnston (1973). Adolphia and some species of Colletia, Discaria, and Retanilla are, however, polymorphic for the character, having some specimens with completely white to whitecream flowers as well as specimens with the basal part of some flowers rose to reddish while other flowers are white. These species are coded as polymorphic for flower colour. The polymorphism has been observed on living material of Colletia spinosissima, Discaria americana, and D. nana, while Arce (pers. comm.) provided information concerning Retanilla patagonica. All other species in the Colletieae are scored according to Tortosa (1983a, 1989, 1992, 1993). Data on flower colour have been derived from McMinn (1942) Ceanothus, Suessenguth (1953) Noltea, and Johnston (1971) Colubrina asiatica. 34. Hypanthium: disc formed (0); forming a tube (1). The long hypanthium common in the Colletieae is often mentioned as an autapomorphy for the tribe. There is, however, considerable variation in the length of the hypanthium, which varies from 1–1.6 mm in Adolphia infesta up to 11.5 mm in Colletia ulicina. A short flower tube is sometimes mentioned in descriptions of Ceanothus (e.g. Brizicky, 1964) making the probable distinctiveness of the hypanthium of, for example A. infesta, uncertain. In this analysis, a distinction has been made between a tube formed hypanthium as always seen in the Colletieae, and the disc-like hypanthium, only uniting the sepals at the level of the insertion of the petals, found in many Rhamnaceae, including Ceanothus. A short prolonged hypanthium forming a tube is also present in Noltea.

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35. Form of hypanthial tube: widening towards the mouth (0); centrally constricted (1). The cylinder formed by the hypanthium can either widen throughout its length towards the mouth or be centrally constricted. In most species where the tube is centrally constricted, it widens out towards the mouth without further constrictions. In Colletia ulicina an indistinct second constriction is, however, present at the tube mouth (an autapomorphy). 36. Post-anthetic hypanthium behaviour: hypanthium attached to the growing fruit (0); hypanthium shed by an abscission layer (1). In the Colletieae the hypanthium is normally shed early by an abscission layer at the basal part of the flower. This is not the case in Retanilla trinervia where the hypanthium remains attached to the growing fruit until this finally breaks the hypanthium. When that happens, the hypanthium is generally shed in irregular pieces, leaving only the basal part attached to the fruit. As this basal part of the hypanthium is delimited by a well-defined layer, and as it furthermore seems to correspond to what is left attached to the growing fruits in species of the Colletieae where the hypanthium is shed early, an abscission layer is considered present in R. trinervia although not of primary importance in the abscission of the hypanthium tube. In Trevoa quinquenervia no abscission layer exists and the flower tube remains attached to the mature fruit. The very short cup-formed hypanthial tube of Adolphia infesta covers the base of the growing fruit, as well as the mature one, resembling the situation in Ceanothus. No observations are available for Noltea, but inspection of mature fruits suggests that also here an abscission layer sheds the hypanthium. 37. Outer surface of hypanthial tube and sepals: completely glabrous (0); tube and sepals hairy (1); only apex of sepals hairy (2). The hypanthium and the sepals may be covered with hairs on the outside. The density of this indumentum varies, being very dense in, e.g. Kentrothamnus weddellianus and sparse in, e.g. Discaria chacaye. The variation is continuous, and no attempt has been made to divide this state any further. In some species of Discaria the indumentum is, however, restricted to the apex of the sepals, and is then scored as a separate character state. In various species of the Colletieae, as well as in all outgroup taxa except Colubrina asiatica, the hypanthial tube and the sepals are completely glabrous. 38. Style and upper part of flower tube hairy inside: no (0); yes (1). In Retanilla and Trevoa hairs cover the style, ovary and inside of hypanthium. The indumentum may be part of a secondary pollen presentation mechanism, where the pollen grains fall from the anthers and are captured by the hairs in the flower tube and on the style (see also ch 44). The pollen may adhere to the mouth parts of visiting insects that must traverse the hairy barrier to reach the nectar in the bottom of the flower tube (Medan & Aagesen, 1995). As the lowermost part of the flower tube, at the level of the nectar secreting tissue (see introduction to disc, ch 54–56), is nearly glabrous in R. trinervia, this character has been scored separately, as well as the character ‘ovary hairy’, as the ovary in Discaria chacaye is covered with hairs while the flower tube and the style are glabrous. 39. Lower part of flower tube hairy inside: no (0); yes (1). See ch 38. 40. Ovary hairy: no (0); yes (1). See ch 38.

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41. Behaviour of sepals after anthesis: curved inwards (0); curved outwards (1); reflexed (2). After anthesis the sepals can be slightly curved outwards as in Adolphia and Retanilla. In Discaria the sepals are perpendicular to the hypanthium tube, or slightly reflexed, while totally reflexed sepals that touch the hypanthium tube with their apices are seen in Colletia, Kentrothamnus and Trevoa. The degree of sepal reflexion in Discaria, Colletia, Kentrothamnus, and Trevoa varies continuously and is scored as a single character state. In Ceanothus the sepals are curved inward after anthesis, while in Noltea the sepals maintain a position similar to the one seen in Adolphia. In Colubrina asiatica the sepals are totally reflexed after anthesis. 42. Petals: present (0); absent (1). Petals are present in most of the Colletieae, but in Colletia and some Discaria species they are lacking. In C. spinosissima petals are sometimes present, mainly in cultivated individuals, but petals have also been observed in one natural population. These petals are lanceolate and only found in some flowers of an individual; furthermore, the petals very often carry more or less rudimentary pollen sacs. The presence of these petals is here considered an abnormality, and in the analyses C. spinosissima is coded as lacking petals. Ceanothus, Noltea, and Colubrina asiatica all have petals. 43. Form of petal: cucullate (0); lanceolate (1). When petals are present these may be cucullate, totally covering the young anthers, or lanceolate as in D. americana, articulata, and D. pubescens. Although in D. americana and D. pubescens the petals are slightly more concave than in D. articulata all are coded as lanceolate, while the pronounced cucullate petals found in other species of the tribe and in the outgroup, are scored as a separate character state. 44. Petal/anther forming a pollen dosing unit: no (0); yes (1). The cucullate petals cover the young anthers. During pollen presentation the petals in many species reflex permitting access to the pollen. In Trevoa and Retanilla the cucullate petals continue covering the anthers while the thecae are open presenting the pollen. This prevents direct access to the pollen that falls down in the flower tube where it is captured by hairs on the style and in the hypanthium tube (see ch 38). The petal/anther complex is here provisionally called a pollen dosing unit, and is only found in Retanilla and Trevoa. If a secondary pollen presentation does exist ch 38 and 44 are redundant, and it may be more correct to consider both as a single character. Androecium Pollen characters have not been used in the present study due to lack of information. Some data can be found in Schirarend & Kohler (1993) but this work is not complete for the tribe. In general, discrete characters seem difficult to find, and characters differentiating Colletieae from the outgroup or uniting the tribe with one or more of the outgroups seem to be lacking. 45. Filament: erect (0); curved inward (1); reflexed (2). During anthesis the filaments are erect (to slightly leaned inwards) in all species of the Colletieae and in Noltea. The long filaments in Ceanothus curve inwards, while in Colubrina asiatica the filaments are totally reflexed during anthesis.

PHYLOGENY OF THE TRIBE COLLETIEAE

21

46. The angle between proximal and distal part of the filament: a curve (0); a ‘knee’ (1). The filament consists of a proximal part (its position is discussed in ch 45), and a distal part that carries the anther. This distal part is horizontal to downward pointing. The proximal and distal parts may meet in a soft curve as seen in various Colletieae species or in a sharp angle (a ‘knee’), as seen in other species of the Colletieae and in all Ceanothus-species examined. In Noltea the angle is not pronounced and Noltea is consequently scored as ‘?’ in the matrix. 47. Outer pair of pollen sacs: separate (0); apically united (1). The anthers consist of two thecae each with two pollen sacs. Often all four pollen sacs are separated before opening, but opposite pollen sacs may be apically united (Tortosa [1989]) mentioned them as resembling a horseshoe). In this case, the outer pair of pollen sacs is often clearly larger than the inner pair. In some species both pairs of pollen sacs are united, while in other species the outer (this character) or inner pollen sacs (ch 48) are united. In Colletia and some Discaria species variation exists between anthers belonging to the same flower. In Noltea the pollen sacs are sometimes united, while in Ceanothus and Colubrina asiatica all pollen sacs are separated (apically united pollen sacs do occasionally occur in other Rhamnaceae, e.g. Phylica L. [see Suessenguth, 1953]). 48. Inner pollen sacs: separated (0); apically united (1). See ch 47. 49. Dehiscence of the pollen sacs: as two vertical separate units (0); 4 separate sacs opening as one inverse u-shaped unit (1); apically united sacs opening as one inverse u-shaped unit (2); as one disc-shaped horizontal unit (3). When all pollen sacs are separated as in Ceanothus, Colubrina asiatica, and part of the Colletieae, the anthers are normally latrorse, dehiscing as two separate vertical units. In Colletia where one or both pollen sacs are apically united with the corresponding pollen sac of the opposite theca, the anther dehisces as one latrorse/introrse, inverse u-shaped unit. The same type of dehiscence occurs in Adolphia, Kentrothamnus and Trevoa, even though all four pollen sacs are separated in these genera. Transverse sections of the apical part of the anthers show that the connective in this region is very thin, often only consisting of a single cell layer. When the anther opens the connective breaks, leaving the two formerly separated pollen-presenting surfaces as one single surface. Dehiscence of the anthers as an inverse u-shaped unit in Adolphia, Kentrothamnus, Trevoa and Colletia seems then non-homologous, as it is reached in what appears to be two different ways. Transitions between shapes of the anthers are, however, seen in Colletia and Noltea; in the latter all possible combinations were found in one single flower. Variation in anther dehiscence was also recorded in Discaria americana, D. nana and D. pubescens. Here an inverse u-shaped pollen-presenting surface is formed if one pair of opposite pollen sacs is apically united. Dehiscence of the anthers in these species is consequently scored as a polymorphy. In Retanilla, where both pairs of pollen sacs are always perfectly united, the anther initially opens as one u-shaped unit but then becomes completely disc-shaped and horizontal. 50. Anther position relative to the receptive stigma: anthers and stigma at same level (0); anthers below the stigma (1); stigma below the anthers (2). During anthesis the anthers and the stigma may be at the same level, or one may be shorter than the other. It should be noted that the character may be based on a underlying continuum

22

L. AAGESEN

(see Stevens, 1991) and should as such be avoided in the analyses (but see Chappill [1989] for a different point of view). The same observation is valid for ch 51.

Gynoecium 51. Position of receptive stigma relative to hypanthium mouth: stigma exserted (0); stigma and hypanthium mouth at same level (1); stigma included (2).The receptive stigma may be exserted through the hypanthium mouth, the stigma and the hypanthium opening may be at the same level or the stigma may be included. 52. Stylar branches: the upper part of the style clearly separated into branches (0); only the most distal part separated, not forming real stylar branches (1); the distal parts of style completely fused (2). In Colubrina asiatica and in Ceanothus the upper part of the style is clearly divided into three stylar branches. In Noltea and several species of the Colletieae only the uppermost part of the style is separated into two or three branches, while in other Colletieae species the stylar branches are completely fused laterally. In some Retanilla and Discaria species variation exists, occasionally even in the same flower, where some stylar branches may be laterally fused while others are separated. 53. Abscission of style: style often persistent on fruit at least for some time, then shed (0); shed together with hypanthium tube (1); persistent on fruit (2). In Ceanothus, Noltea (according to Suessenguth [1953]) and most of the Colletieae, the style persists on the growing fruit for some time and is then shed. In nearly all Colletia species, the style is always shed with the hypanthium, and an abscission layer is clearly seen at the base of the style. The only exception is C. spartioides where, according to Miers (1860), the style persists on the growing fruit. In Trevoa quinquenervia and Retanilla trinervia the style is still present on the mature fruit.

Disc In most Rhamnaceae a nectar-secreting disc is found surrounding the gynoecium (see Suessenguth, 1953). Secreting tissue can be recognized anatomically by the simultaneous occurrence of modified stomata with permanently open ostioles and 3–6 subepidermal, continuous layers of small, strongly staining cells (see Medan & Aagesen, 1995). 54. Disc: present (0); absent (1). In most species of the Colletieae a morphologically distinguishable disc is seen between the androecium and the gynoecium. In Trevoa quinquenervia, a small elevated rim was recorded in the lower part of the hypanthium tube. Below this line, the hairs seemed more scattered than above, but not conspicuously so as in Retanilla trinervia (ch 39). Although inconspicuous, this structure is here considered homologous to the free elevated part of the disc found in other species. Only in Retanilla a disc is completely lacking, and the nectar-secreting tissue is included in the wall of the lower part of the hypanthium tube.

PHYLOGENY OF THE TRIBE COLLETIEAE

23

55. Disc type: annular, flat (0); annular, elevated (1); adpressed, lining the lower part of hypanthium (2); a free, revolute lamina (3); a small elevated rim delimiting nectarsecreting tissue (4). When a disc is present, it may be voluminous, annular and flat as in Colubrina asiatica. This kind of disc occupies the entire disc-formed flower cup, and nectar is secreted all over the surface (see Medan & Hilger, 1992). In Ceanothus, also with disc-formed flower cups, the nectar-secreting disc is annular with a free elevated part, only covering the area next to the gynoecium. Modified stomata are scattered all over the surface of the free elevated part. This kind of disc is also found in Discaria, although in this genus the disc seems slightly more elevated. Some variation concerning size and degree of pleating of the free elevated part, seems to exist in Discaria. There may also be a difference in the size of the hypanthial area covered by the disc. These differences are, however, subtle and the only variation that may be safely scored is whether the free part of the disc is plicate or nearly totally smooth (ch 56). In Noltea, Kentrothamnus, and Adolphia the disc is less conspicuous, adpressed and lining the hypanthium up to the insertion of the sepals. In Kentrothamnus the rim of the disc is somewhat sinusoid also lining the lowermost part of the filaments (an autapomorphy). Nectar secreting tissue is found all over the disc surface. The disc type described for Trevoa is here scored as a separate character state. Below the rim, i.e. in the lower part of the hypanthium tube, nectar-secreting tissue is included in the hypanthial wall as in Retanilla. In Colletia the disc is a free and revolute lamina, located in the lower part of the hypanthium tube. Nectar-secreting tissue is restricted to the most distal part of this lamina. 56. Free part of disc: plicate (0); smooth (1). The free part of the disc may be plicate or nearly smooth. A smooth disc is found only in Colletia and Discaria americana.

Fruit 57. Fruit: explosive capsule (0); drupe (1); achene (2). In Colubrina asiatica, Ceanothus, and most Colletieae species the fruit is an explosive capsule, as commonly found in the Rhamnaceae (see Suessenguth, 1953). Dehiscence is due to the endocarp, which mainly consists of fibres with different form and orientation. When the mature fruit dries, the amount of tension in the endocarp increases and the fruit finally opens along predefined weaker sutures (Medan, 1985). In Retanilla the fruit is a drupe with exocarp, endocarp, and septa varying in thickness: it is thin in R. trinervia, more extended in R. patagonica, and reaching the maximum extension in R. ephedra and R. stricta. The fruit of Trevoa quinquenervia is an achene. 58. Fruit dehiscence: instantaneous (0); late (1); fruit indehiscent (2). In the species with explosive fruits the opening of the mature fruit is instantaneous. In Retanilla the drupe generally drops from the plant and opens on the ground, although in R. patagonica endocarp splitting might commence while still attached to the plant. This opening process is slow, with the endocarp breaking gradually along weak sutures, leaving two or tree indehiscent endocarpids (Medan & Aagesen, 1995). In Trevoa quinquenervia and Retanilla trinervia the fruits are indehiscent.

24

L. AAGESEN

59. Pedestal: absent (0); present (1). At fruit dehiscence a pedestal may separate from the rest of the fruit. Johnston (1973) first applied the term pedestal for the accresced lower part of the floral cup lined by the disk. The pedestal was further defined as “. . . the fruiting pedicel, and all carpellary tissues belonging to the inferior part of the ovary—endocarp excepted—as well as those floral tissues not eliminated by flower tube abscission . . .” by Medan & Aagesen (1995). Such a pedestal is found in Ceanothus and in all Colletieae species with explosive capsules. In Colubrina asiatica and Noltea, the lower part of the fruit breaks irregularly when the fruit opens. In Trevoa and Retanilla trinervia no pedestal is formed, while in all other species of Retanilla a small pedestal adheres to the fruit when this is released from the plant, and separates from the fruit at dehiscence. Although the pedestal in Retanilla is less conspicuous than the one found in other Colletieae (due to little or no accrescence in the lower part of the hypanthium) it otherwise corresponds to the definition given above. Variation in the degree of accrescence in the basal part of the hypanthium, occurs in species with explosive capsules, particularly in Discaria and no characters relating to the form of the pedestal have been used. 60. Pedestal remains on the plant: yes (0); no (1). normally remains on the plant. The pedestal above, shed with the fruit. Only in Retanilla remain on the plant. This coincides with early

When the fruit opens the pedestal found in Retanilla is, as mentioned patagonica do pedestals occasionally dehiscence.

Seed The number and form of the seeds have been omitted from the analyses. The number of seeds shows a considerable intraspecific variation. In most species of the Colletieae three (occasionally four) seeds are formed. In most Retanilla species two seeds are commonly formed, but the number may vary between two and four. In Trevoa one seed is formed; two or three seeds are found only rarely (Tortosa, 1992). The shape of the seed depends on the number of seeds in the fruit. 61. Aril: attached to seed after opening of fruit (0); released from seed after opening of fruit (1). In all species included in the analyses a small aril may be found on the developing seeds. When the fruit opens, the aril often separates from the seed. It remains attached in Ceanothus only. 62. Aril type: disc-shaped (0); long obpyramidal (1); short obpyramidal (2). In Retanilla and Trevoa a long obpyramidal aril is found, while all other species of the Colletieae have disc-shaped arils, also found in Noltea and Colubrina asiatica. The persistent aril in Ceanothus is short and obpyramidal. 63. Aril rim: entire or only slightly lobed (0); deeply lobed (1). The border of the arils is entire to slightly lobed in most species. In Noltea and Kentrothamnus the rim is deeply lobed which is scored as a separate character state. RESULTS

Phylogenetic analyses Analysing the data matrix NONA found 57 equally parsimonious trees each 178 steps long (CI=0.49, RI=0.79). Among the 57 cladograms 19 distinct ingroup

PHYLOGENY OF THE TRIBE COLLETIEAE

25

topologies were found, seven of these being caused by multiple equally parsimonious placements of Colletia spartioides within the Colletia. The remaining cladograms were caused by rearrangements within Ceanothus, where three different groupings of the taxa appeared. Two jumps were performed—jump∗1 and jump∗2—neither of which found additional trees. The following discussion limits itself to rearrangements among the ingroup species. In the strict consensus tree (Fig. 1A) the Colletieae form a monophyletic group having Noltea africana as sister taxon. The tribe is divided into two major clades, one containing Trevoa and all Retanilla species, the second consisting of Adolphia, Kentrothamnus, Discaria and Colletia. While the Trevoa–Retanilla clade is fully resolved, the second major clade lacks detailed resolution, only including a Colletia clade where C. paradoxa and C. spinosissima are sister species. Weighting Heuristic searches using implied weights and default conc value in Pee-Wee resulted in three trees of equal fit (fit=470.9, length=179, rescaled fit=0.61). All trees included the same ingroup topology—similar to one of the trees found during the equal weighted analyses (see Fig. 1B)—but differ from this in placing Colletia hystrix rather than C. ulicina basal within the Colletia clade. None of the 20 jumps performed found additional trees. Analysing the matrix with lower conc values (weighting stronger against homoplastic characters) altered the result only when using the lowest conc value (conc=1). This yielded a single tree which only differed from the one previously found by the placement of Adolphia infesta and Discaria chacaye (tree not shown). When weighting less strongly against homoplastic characters, using conc=4, in addition to the tree previously found yet another tree found in the equal weighted analyses appeared (tree Fig. 1C). Analysing the matrix using conc=5 only this latter tree (Fig. 1C) was found. Using the highest conc value (conc=6) a single tree, also found during the equal weighted analyses, appeared. This tree differs from the tree shown in Figure 1C by placing the ((D. nana D. trinervis)(D. chacaye (D. nitida D. toumatou))) clade as sister group to the Adolphia–Kentrothamnus–Discaria p. p.–Colletia clade (tree not shown). Excluding Discaria americana from the matrix When analysing the data matrix without Discaria americana, NONA found 39 trees (175 steps long, CI=0.50, RI=0.79), a subset of the initial 19 ingroup trees found by NONA including those topologies where D. americana does not appear as sister group to the Colletia clade. A strict consensus of the 39 trees includes a D. pubescens–D. articulata clade in addition to the clades appearing in the consensus tree shown in Figure 1A. Analysing the same matrix with Pee-Wee resulted in a single ingroup topology (175 steps long, rescaled fit=0.62) also found in the initial equal weighted analyses. This tree differs from the one shown in Figure 1C by the placement of Adolphia and Kentrothamnus as basal branches to the Colletia clade while D. nana and D. trinervis are placed as basal branches to the D. pubescens–D. articulata clade (tree not shown).

26

L. AAGESEN

A 86 4

1

1

91 5

1

1

1 98 4

50 1

89 3

92 93 4 3

91 5

54 2

98 7

B

77 1

Colb. asiatica Cea. cuneatus Cea. greggii Cea prostratus Cea. purpureus Cea. verrucosus Cea. papillosus Cea. coeruleus Cea. americana Cea. thyrsiflorus Cea. leucodermis Nol. africana Tre. quinquenervia Ret. trinervia Ret. patagonica Ret. ephedra Ret. stricta Ado. infesta Ken. weddellianus Dis. americana Dis. articulata Dis. chacaye Dis. nana Dis. nitida Dis. pubescens Dis. toumatou Dis. trinervis Col. paradoxa Col. spinosissima Col. hystrix Col. spartioides Col. ulicina

Colb. asiatica Cea. cuneatus Cea. greggii Cea prostratus Cea. purpureus Cea. verrucosus Cea. papillosus Cea. coeruleus Cea. americana Cea. thyrsiflorus Cea. leucodermis Nol. africana Tre. quinquenervia Ret. trinervia Ret. patagonica Ret. ephedra Ret. stricta Ado. infesta Ken. weddellianus Dis. chacaye Dis. nitida Dis. toumatou Dis. trinervis Dis. nana Dis. pubescens Dis. articulata Dis. americana Col. ulicina Col. spartioides Col. hystrix Col. paradoxa Col. spinosissima

PHYLOGENY OF THE TRIBE COLLETIEAE

27

Colb. asiatica Cea. cuneatus Cea. greggii Cea prostratus Cea. purpureus Cea. verrucosus Cea. papillosus Cea. coeruleus Cea. americana Cea. thyrsiflorus Cea. leucodermis Nol. africana Tre. quinquenervia Ret. trinervia Ret. patagonica Ret. ephedra Ret. stricta Ken. weddellianus Ado. infesta Col. hystrix Col. ulicina Col. spartioides Col. paradoxa Col. spinosissima Dis. pubescens Dis. articulata Dis. americana Dis. trinervis Dis. nana Dis. chacaye Dis. nitida Dis. toumatou

C

Figure 1. Trees produced during the phylogenetic analysis. Within Ceanothus three different topologies were found, shown as a polytomy within the genus in tree B and C. A, strict consensus of all most parsimonious trees found during the equally weighted analysis. Numbers above branches indicate jackknife group frequencies. Numbers below branches refer to the Bremer support (the high support for the Colletia clade is calculated excluding C. spartioides from the matrix—see text for explanation). B, one of the most parsimonious trees found by NONA. Pee-Wee (conc. 3 or 4) found a nearly identical tree placing Colletia hystrix and not C. ulicina basal within the Colletia clade. C, one of the most parsimonious trees found by NONA. Pee-Wee found the same tree when using conc 4 or 5.

Uncertainly scored characters Characters uncertainly scored in Discaria include ch 5, 23 and 53. When ch 5 (apex of macroblasts differentiated into a spine) is excluded only two ingroup trees are found, the one shown in Figure 1C and the other placing Discaria nana as sister group to the clade D. trinervis–D. chacaye–D. nitida–D. toumatou (both trees were also found during initial heuristic search). When excluding ch 23 (sprouting of secondorder synflorescences during anthesis of first-order synflorescence) only the tree in Figure 1C appeared with either Colletia hystrix or C. spartioides basally in the Colletia clade. Exclusion of ch 53 (abscission of style) resulted in six different ingroup topologies (all among the initial 19 ingroup trees). A strict consensus of these trees adds the clades (D. chacaye (D. nitida, D. toumatou)) and (D. pubescens, D. articulata, D. americana) to the strict consensus of all 19 initial trees, and places Colletia hystrix basally within the Colletia clade. When analysing the matrix with Pee-Wee omitting ch 23, the preference for the tree initially found (see Fig. 1B) was not altered. Absence of ch 5 from the matrix yielded the two trees found by NONA when excluding the same character, in

28

L. AAGESEN

addition to the tree previously found by Pee-Wee. When excluding ch 53 this tree still appears in addition to the tree in Figure 1C.

DISCUSSION

The present study confirms the frequently assumed monophyly of the tribe Colletieae. However, Noltea rather than Ceanothus appeared as sister group to the tribe. Forcing Ceanothus and the Colletieae to be monophyletic requires three extra steps. The result obtained when analysing the equally weighted matrix reveals a basal dichotomy within the Colletieae, one clade containing Trevoa and the Retanilla species, the other consisting of all remaining genera. Within the latter clade only two monophyletic groups persist in the strict consensus tree: the genus Colletia in which C. spinosissima and C. paradoxa appear as sister groups (see Fig. 1A). The result conflicts with the one obtained by Swensen (1996). In her analyses based on DNA sequences of the chloroplast gene rbcL (including Colletia ulicina, Discaria chacaye, and Retanilla trinervia) Discaria was placed basally within the Colletieae, with Retanilla and Colletia being sister groups; this topology being based on a single character state change. However, it was not the aim of Swensen to analyse the phylogeny of the Colletieae, and considering the low support for the Colletia–Retanilla clade further sampling within the tribe may change the results. To see whether a detailed phylogenetic pattern could be obtained the matrix was weighted using implied weights (Goloboff, 1993). When weighting the characters Pee-Wee found a single ingroup tree when conc=2–3. A similar tree (Fig. 1B) was among the trees found during the equal-weighted analyses, differing only in the placement of Colletia ulicina and C. hystrix. Weighting less strongly against homoplasy (conc=4–6) yielded two additional trees, which were also among the trees found when applying equal weights to the characters. One of these trees is shown in Figure 1C. Considering that Discaria americana could be of hybrid origin the effect of omitting this taxon from the matrix was examined. Discaria americana and the Colletia species share a unique character state—free part of disc smooth. Not unexpected, when examining the modified matrix NONA found only a subset of the original trees, those where D. americana did not appear as the sister taxon of Colletia. When using implied weights Pee-Wee found a single ingroup tree grouping Discaria as polyphyletic. Three characters (5, 23 and 53) were only safely scored in South American Discaria species when abundant herbarium material or field studies were available. As material of Australian and New Zealand Discaria species was limited these characters were uncertainly scored in D. nitida, D. pubescens and D. toumatou. The impact of these three characters was examined by successively eliminating each character and re-analysing the matrix. The results of the equal weighted analyses were either combinable with tree 1C, or consistently supporting tree 1C, or a slightly different tree placing D. nana and D. trinervis as basal branches to the D. chacaye–D. nitida–D. toumatou clade. When the matrix was analysed with implied weights, either tree 1B or both tree 1B and trees similar to the one in Figure 1C appeared. The tree in Figure 1B with the two basal branches in Colletia collapsed, and tree 1C (or trees differing from this not treating D. nana and D. trinervis as a clade) thus

PHYLOGENY OF THE TRIBE COLLETIEAE

29

appear to be the most strongly supported cladograms found during the heuristic search. The following discussion is mainly based on these trees with only occasional references to other results. The number of character state changes supporting a clade refers to unambiguous changes (using the amb- option of NONA) present in all equally parsimonious trees. These character changes are marked with an asterisk in Figure 2. Sister group of the Colletieae All searches place Noltea africana as sister group to the Colletieae tribe, though with a low Bremer support of 1 (this support is not altered even if the character root nodules is included in the analyses) and a group frequency of 50% (note this is the lowest possible value). The close relationship between Noltea and the Colletieae should be taken with caution because of the limited number of outgroup species included. However, the analysis suggests that the assumed close relationship between Ceanothus and the Colletieae is erroneous. In this analysis Noltea and the Colletieae share one unique character, the presence of a tube-shaped hypanthium. A more or less prolonged tubular hypanthium is present in other genera of the Rhamnaceae and a more thorough analysis of the family is needed to reveal whether this character is a synapomorphy for Noltea and the Colletieae or merely a parallelism. Furthermore, three homoplastic characters support the Noltea–Colletieae clade (the existence of an abscission layer separating the hypanthium from the growing fruit, a style having only the uppermost part separated into branches, and frondo-frondulose to frondo-bracteose foliage of the synflorescences), all being reversed or modified within the Colletieae. The Colletieae Monophyly of the Colletieae tribe is strongly supported by seven character state changes, a Bremer support of 5 and a group frequency of 91%. Only one unique character, pherophylls of primary flowers not covering the paracladium before anthesis, supports the clade. Pherophylls of primary flowers that do not cover the paracladium before anthesis may be more widely distributed at least within the Rhamneae (the same character is found in Frangula alnus Mill. and Scutia buxifolia Reissek). Two characters (decussate leaf arrangement, and sprouting of macroblasts or brachyblasts from prophylls of older macroblasts) are paralleled within Ceanothus, and two characters (presence of serial meristems, and sprouting of second-order synflorescences from the prophylls of old first- order synflorescences) are reversed within Colletieae. The last two characters (leaf margin, and presence of glands at the leaf margin) are characters with reversals both within Ceanothus and the Colletieae. The Trevoa–Retanilla clade Within the Colletieae the Trevoa–Retanilla clade is well supported by seven character state changes, a Bremer support of 4, and a high group frequency of 98%. This clade corresponds to the informal division Clithrocarpae Miers (excl. Scypharia Miers)

10:

1->0

11:

3->1

9:

1->0 36:

1->0

20:

0->1

63:

2->1

32: 53: 58: 59:

1->0

13:

0->1

38:

0->1

40:

0->1

44:

0->1

53:

0->2

58:

0->2

62:

0->1

R. ephedra 0->1

51: 1->2

41: 47: 48: 49: 51: 54:

41:

21:

17: 0->1

4: 0->1 12:

R. stricta

R. trinervia

N. africana

R. patagonica

L. AAGESEN

T. quinquenervia

30

22:

1->0

31:

0->1

35:

1->0

0->1 2->1 2->1 0->1

2->1 0->1 0->1 1->3 0->1 0->1

see Fig. 2B

0->1

2: 0->1 3: 0->1 6: 0->1 11: 0->3 12: 0->1 22: 0->1 29: 0->2 28: 34: 36: 49: 52:

3->0 0->1 0->1 0->1 0->1

A

Figure 2. Character state changes within the Noltea–Colletieae clade of the tree in Figure 1C. Characters are optimized using MacClade ver. 3 (Maddison & Maddison, 1992). Only unambiguous character state changes are shown (equal to the amb- option of NONA Pee-Wee). Characters state changes marked with an asterisk occur in all equally parsimonious trees found when analysing the complete matrix (excl. Colletia spartioides—see text for explanation), and are further discussed in the text. A, character state changes of Noltea and the Trevoa–Retanilla clade. B, character stage changes of Kentrothamnus to Colletia. C, character stage changes within the Discaria clade.

33:

0->2

50:

0->2

51:

0->2

50:

35: 1->0

36: 1->0

17: 1->0

20: 0->1

13: 15: 19: 24: 26: 27: 28: 31: 42: 56:

41: 2->1

50: 0->1

see Fig. 2c

0->1 0->1 0->1 2->3 1->2 0->1 2->3 0->1 0->1 0->1

63: 0->1

7: 10: 17: 25: 28: 59:

B

0->1

14: 0->1

33: 0->1

52: 1->2

31

Col. spinosissima

Col. paradoxa

Col. ulicina

Col. hystrix

A. infesta

K. weddellianus

PHYLOGENY OF THE TRIBE COLLETIEAE

18:

0->1

23:

0->1

0->1 1->0 0->1 0->1 0->2 0->1

Continued from Fig. 2A

Figure 2. continued.

46:

1->0

53:

0->1

3:

35:

52:

1->2

56:

0->1

1->0

11:

D. nana

D. trinervis

D. nitida

D. toumatou

D. chacaye

D. articulata

D. americana

L. AAGESEN

D. pubescens

32

3->0/2 37:

12:

1->0

26:

1->0

1->2

35:

1->0

1: 0->1 14: 0->1 17: 0->1 21: 0->1 22: 1->0 31: 0->1 50: 0->2 51: 1->2

1->0 50:

0->2

51:

1->2

40:

0->1

28:

2->1

35:

1->0

53:

28:

2->1

43:

0->1

18:

1->0

42:

0->1

3:

1->2

4:

0->1

7:

1->0

23:

10:

0->1

17:

1->0

49:

1->0

51:

0->1

1->0

1->0

C continued from Fig. 2B

Figure 2. continued.

established by Miers (1860), and furthermore confirms the assumption of a close relationship between these two genera, reflected by the earlier problems of segregating the involved species into genera (see Tortosa, [1992] for details). Three unique character states provide the most reliable support for the Trevoa–Retanilla clade: long obpyramidal aril, style and upper part of flower tube hairy inside, and sepal/anther forming a pollen-dosing unit. The long obpyramidal arils found in Trevoa and Retanilla separate from the seed at maturity. The arils remain within the indehiscent endocarpids and are probably pushed out by the embryonal root at germination (Medan & Aagesen, 1995). Within the Rhamnaceae only very limited information is available concerning arils, particularly of those separating from the seed at maturity. Consequently, although arils similar to those found in Trevoa and Retanilla

PHYLOGENY OF THE TRIBE COLLETIEAE

33

have not been reported in other genera of the Rhamnaceae, it has to be confirmed whether this aril type is a synapomorphy for Trevoa and Retanilla, not paralleled in other parts of the family. The characters, style and upper part of the flower tube hairy inside, and sepal/anther forming a pollen dosing unit, might be part of a secondary pollen presentation device and therefore not independent characters. Secondary pollen presentation has not been reported in other species of the Rhamnaceae, but flowers provided with a prolonged hypanthium and with hairs covering the lower part of the style and inner side of the flower tube are also found at least in Cryptandra Sm. (Suessenguth, 1953). All remaining characters provide less reliable support for the clade. One character, ovary hairy, is developed in parallel in Discaria chacaye. Variation of this latter character within a single genus is seen elsewhere in the Rhamnaceae, e.g. in Cryptandra, Phylica, and Pomaderris Labill. (Suessenguth, 1953). Two characters, style persistent on fruit and indehiscent fruits, are shared only by Trevoa quinquenervia and Retanilla trinervia, being reversed or modified within the remaining species of Retanilla. The last character shared by Trevoa and Retanilla (leaves with three main nerves) only provides weak support for the clade. Nervation of leaves is very variable both within the Colletieae and in Ceanothus. Variation in number of main nerves within a single genus is recorded, e.g. in Colubrina ( Johnston, 1971), while McMinn (1942) attributed intraspecific variation in Ceanothus due to ecological conditions.

Retanilla Within the above mentioned clade Retanilla forms a well supported monophyletic group in the strict consensus tree supported by four character state changes, a Bremer support of 3, and a group frequency of 89%. There seems to be no reason for segregating R. trinervia in a separate genus as proposed by Miers (1860). One unique character, absence of disc, defines the genus. Lack of a disc is not common within the Rhamnaceae, but it has been reported in the two monotypic genera Doerpfeldia Urb. and Maesopsis Engl. as well as in some Phylica species (Suessenguth, 1953). Two character states (outer pollen sacs apically united, and inner pollen sacs apically united) are similarly optimized as non-homoplastic synapomorphies supporting the Retanilla clade, as only the Retanilla species are monomorphic for these states. In fact, united pollen sacs are paralleled in Colletia, Discaria and Noltea but only in species polymorphic for the character. The last character state supporting the clade (sepals curved outwards after anthesis) is paralleled in Adolphia and Noltea. Two synapomorphies providing support for the Retanilla clade are the occurrence of drupes, and the dehiscence of the pollen sacs as one disc-shaped horizontal unit. Drupes do occur in other groups of the Rhamnaceae, e.g. in the Zizipheae (Suessenguth, 1953), but neither among other Colletieae species nor among the taxa presumed to be closely related to this tribe. However, occurrence of drupes is optimized as ambiguous support to the Retanilla clade as the fruit type of Trevoa quinquenervia (achene) is not shared by other taxa included in the analyses. Hence when optimized on a cladogram, drupes could alternatively be a synapomorphy for the more inclusive Trevoa–Retanilla clade and subsequently modified within Trevoa. As for the dehiscence of the pollen sacs, the state found in Retanilla does not occur in other taxa included in the analyses, and the state is optimized as a synapomorphy

34

L. AAGESEN

for the Retanilla species in all but a single cladogram where the optimization is equivocal for the Trevoa–Retanilla clade. Within the Retanilla clade, R. patagonica is the sister group to R. stricta and R. ephedra. This relationship is also well supported by four character state changes, a Bremer support of 4, and a group frequency of 92%. Two unique character states (absence of prophylls of primary flower in the paracladia, and late dehiscence of the fruit) support this clade, while presence of pedestal is paralleled in the second major clade containing all remaining genera of the tribe as well as in Ceanothus. The last character, style shed with flower tube, is homoplastic within Discaria. Retanilla ephedra and R. stricta are sister groups within the last mentioned clade. Three homoplastic character state changes, a Bremer support of 3, and a group frequency of 93% support the sister group relationship. Two characters (second-order synflorescences not sprouting from the prophylls of old first-order synflorescences, and uniflorous paracladia) are paralleled in Discaria nana, while the third character (hypanthium tube widening towards the mouth) is paralleled in various species of the tribe. The Adolphia–Kentrothamnus–Discaria–Colletia clade The second major clade within the Colletieae consists of Adolphia, Kentrothamnus, Discaria and Colletia. This clade is supported by only three character state changes and has a Bremer support of 2 and a very low group frequency of 54%. One unique character (proliferating synflorescences) defines the clade. Proliferating synflorescences have not been mentioned in other taxa of the Rhamnaceae (except occasionally in Noltea and Pomaderris apetala Labill. [Troll, 1959; 1960]) but few detailed studies concerning the inflorescences in the Rhamnaceae exist, hence proliferating synflorescences might be found in other groups of the family if searched for. The two remaining characters supporting the clade, presence of a pedestal and frondose foliation of synflorescences, are respectively paralleled in Ceanothus and parts of Retanilla or homoplastic within the clade. Colletia Resolution within this second major clade is poor and only two clades persist in the strict consensus. One of these, comprising all Colletia species, is particularly well supported by a Bremer support of 7 and a group frequency of 98%. Six character state changes define this clade, only two (paracytic stomata, and apical meristem of synflorescences fertile more than one growth season) being free of homoplasy (both Bremer support and number of character state changes have been calculated excluding the partly unknown species C. spartioides from the matrix, the Bremer support for Colletia including C. spartioides is 4. When C. spartioides is included in the matrix only two characters [15 and 42] appear as unambiguous support in all trees, the remaining 5 characters are scored as unknown in C. spartioides, hence optimized as ambiguous support in cladograms where C. spartioides is placed basally within Colletia). While information concerning the general occurrence of the latter character is lacking, the presence of paracytic stomata provides strong support for the monophyly of Colletia. Among other Rhamnaceae species anomocytic stomata are

PHYLOGENY OF THE TRIBE COLLETIEAE

35

prevailing while paracytic stomata have been found only in some Rhamnus L. and Ziziphus species (Metcalfe & Chalk, 1957). Two unique character states (synflorescence forming a condensed brachyblast, and proliferation of synflorescences delayed at least to next growth season) belonging to otherwise homoplastic characters furthermore define the Colletia clade. The two remaining character states (absence of petals, and uniflorous paracladia) are paralleled within Discaria and in Discaria and Retanilla respectively. One character state (disc with free revolute lamina) present in all Colletia species, does not appear as support in the strict consensus tree due to equivocal optimization in some trees. A disc with free revolute lamina is only seldom seen in other Rhamnaceae species, e.g. in some Spyridium Fenzl species (Suessenguth, 1953), hence this character state similarly seems to be among those providing strong support for the monophyly of Colletia. Within the Colletia clade a sister group relationship between Colletia paradoxa and C. spinosissima is retained in the strict consensus tree, though only weakly supported by a Bremer support of 1 and a moderate group frequency of 77%. The clade is defined by the presence of adaxial stomata, paralleled in Discaria nana and in some specimens of Adolphia infesta. A second character state (dorsiventral leaves) supports the clade in cladograms where Discaria americana does not appear as sister group to the Colletieae species. The position of C. hystrix, C. spartioides and C. ulicina relative to the C. paradoxa–C. spinosissima clade is not resolved in the strict consensus. In most trees C. hystrix is the basal taxon within the Colletia clade, with the resulting C. spartioides–C. ulicina–C. paradoxa–C. spinosissima clade being supported by one character (loss of mucilaginoustannin containing cells) paralleled in Retanilla trinervia and in some Adolphia infesta specimens. When D. americana appears as sister taxon to the Colletia clade, C. ulicina is placed basally within the Colletia clade (except in the single ingroup tree found by Pee-Wee when analysing the entire data matrix—one step longer but 0.7 higher in fit than the tree in Fig. 1B). The resulting C. hystrix–C. spartioides–C. paradoxa–C. spinosissima clade is supported by two character state changes (anthers and receptive stigma at same level, and stigma exserted) both highly homoplastic with a minimum of six and four extra steps, respectively. When excluding Discaria americana from the data matrix (presuming D. americana to be an allopolyploid) NONA found a subset of the initially 19 ingroup trees including only the topologies where D. americana does not appear as sister taxon to the Colletia clade, hence placing C. hystrix as basal branch within the Colletia clade. Finally, in some trees C. spartioides was placed basal within Colletia. This latter topology is caused by the persistence of the style on the growing fruit in C. spartioides, while in all other Colletia species the style is shed early. The difference found in C. spartioides is based on a drawing of Miers (1860) and lacks confirmation.

Adolphia and Kentrothamnus The position of Adolphia and Kentrothamnus within the second major clade of the Colletieae was not resolved in the present study. The two genera appear in some trees as a monophyletic sister group to a clade consisting of Colletia and Discaria or to a clade including only the Colletia species. In other trees the two genera form a grade placed in either of three different positions: with Adolphia being the sister taxon

36

L. AAGESEN

to a clade either consisting of Discaria and Colletia, or of D. americana, D. articulata, D. pubescence, and the Colletia species, or of the Colletia species. In the tree in Figure 1B, Adolphia and Kentrothamnus appear as a monophyletic sister group to a Discaria–Colletia clade. The Adolphia–Kentrothamnus clade is supported by four homoplastic character states (apex of macroblasts sometimes differentiated into a spine, leaves ephemeral almost lacking, base of opposite leaves not united, and leaves subisolateral) all paralleled within the Discaria–Colletia clade. An arrangement of Kentrothamnus and Adolphia as basal branches in a more inclusive Kentrothamnus– Adolphia–Discaria–Colletia clade appears in tree 1c. In this tree the Adolphia– Discaria–Colletia clade is defined by two character state changes (loss of bundle sheath extensions adhering the main nerve to the adaxial epidermis, and sprouting of second-order synflorescences during anthesis of the first-order synflorescences) both reversed within Discaria. Placing Adolphia and Kentrothamnus as sister groups in this tree requires two extra steps, while when analysing with Pee-Wee the same change is less optimal than placing Adolphia as sister group to the Discaria or Colletia clade. The last arrangement of Adolphia and Kentrothamnus obtaining some support when manipulating the data set, includes the two genera within a clade furthermore containing Discaria p.p. and Colletia. Depending of the placement of Adolphia and Kentrothamnus within this group the clade is supported by two to six homoplastic characters, only one (leaves ephemeral almost lacking) not being reversed within the same clade. Discaria Colletia and Discaria form a monophyletic group in trees 1B and 1C. Different characters support the clade in the alternative trees with only one character appearing in both (angle between proximal and distal part of the filament a curve). Discaria is the only genus within the Colletieae whose monophyly is not confirmed in the present analysis. Discaria appeared as monophyletic, paraphyletic or polyphyletic. These discordant results are caused by the ambiguous placement of the D. pubescens–D. articulata–D. americana group. When excluding D. americana from the matrix Discaria still appears as mono-, para-, or polyphyletic if weights are not applied to the characters. Resolution within Discaria is furthermore ambiguous, though three species groups, D. pubescens–D. articulata–D. americana, D. chacaye–D. toumatou–D. nitida, and D. trinervis–D. nana, consistent with the sections retained by Suessenguth (1953), reappear in all cladograms either as monophyletic groups or paraphyletic assemblages. In tree 1B Discaria pubescens, D. articulata, and D. americana are basal branches to the Colletia clade. The clade is defined by six homoplastic character states of which three do not occur in other Discaria species: sprouting of secondary synflorescences during anthesis of first-order synflorescences, absence of vegetative brachyblasts, and leaves almost lacking (these three characters are furthermore shared with Adolphia and occasionally Kentrothamnus, causing these genera to be associated with the above mentioned clade in some cladograms). In tree 1B D. americana is the sister taxon of the Colletia clade, based on a single unique character: free part of disc smooth. In tree 1C Discaria pubescens–D. articulata–D. americana alternatively form a clade within the monophyletic Discaria. Two homoplastic characters support the clade (presence of lanceolate petals, and foliage of the synflorescences frondo-frondulose

PHYLOGENY OF THE TRIBE COLLETIEAE

37

to frondo-bracteose with bracts proximal and leaves distal) both paralleled in D. nitida and D. toumatou. The sister group relationship between D. articulata and D. americana is mainly supported by the two highly homoplastic character states: receptive stigma below the anthers, and receptive stigma included. The Discaria clade itself is defined by four homoplastic characters (base of opposite leaves united, dorsiventral leaves—reversed in D. nana—dehiscence of the pollen sacs as two vertical separate units, and equivocal support by the states receptive stigma and hypanthium opening at same level or stigma included) all being paralleled in other parts of the tribe. In the same tree D. chacaye, D. toumatou, and D. nitida form a clade that reappears in all other trees supported by the manipulated data. This clade corresponds to the genus Notophaena established by Miers (1860) and retained by Suessenguth (1953) as a section within Discaria, but furthermore includes D. nitida segregated from D. pubescens by Tortosa in 1977. Two homoplastic characters support the clade (main nerves adhered to adaxial epidermis through bundle sheath extension, and absence of petals) the former character state being paralleled in some D. pubescens specimens, while both character states are paralleled in other parts of the tribe. Discaria toumatou and D. nitida are sister groups within this clade, supported by frondo-frondulose to frondo-bracteose foliage of the synflorescence (with bracts proximal and leaves distal) paralleled in the D. pubescence–D. articulata–D. americana clade. The placement of Discaria trinervis and D. nana varies between being sister groups to one of these two major Discaria clades respectively. The two species always appear as sister groups or as grades consistent with the genus Ochetophila, the genus segregated from Discaria but retained as a section within Discaria by Suessenguth (1953). In tree 1B Discaria trinervis and D. nana are most closely related to the Discaria pp–Colletia clade. This relationship is based on a single character (main nerves not adhered to adaxial epidermis through bundle sheath extension) only paralleled in Adolphia. Discaria nana is the sister group to the Discaria pp–Colletia clade based on the presence of a completely glabrous hypanthium tube, only modified in Discaria pubescens. In the tree in Figure 2 Discaria trinervis and D. nana are monophyletic and sister group to the D. chacaye–D. nitida–D. toumatou clade, though in two similar trees among the initially 19 ingroup trees, D. nana and D. trinervis are grades and basal branches to the D. chacaye–D. nitida–D. toumatou clade, one of either species being the sister taxon to this clade. The entire clade is supported by four character state changes, only one being free of homoplasy (serial meristems in leaf axils lacking at various nodes), while a second state (presence of vegetative brachyblasts) is paralleled in Trevoa. The monophyly of Discaria nana and D. trinervis is defined by a single character state (main nerves adhered to adaxial epidermis through bundle sheath extension) paralleled in other Discaria species. The difference in stipule morphology between D. nana–D. trinervis and the remaining Discaria species mentioned by Miers (1860) as support for the D. nana–D. trinervis clade (genus Ochetophila) could not be confirmed in the present study. Additional support for the D. nana–D. trinervis clade can, however, be found in a character not included in this analysis, concerning the location of the first nodes of the spines. In D. nana and D. trinervis this node is placed proximally, as opposed to a clear distal location of this node in all other Discaria species. As this character was not readily scored in the remaining species of the tribe, it was left out of the analyses.

38

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Summary The tribe Colletieae forms a monophyletic group with a basal dichotomy resulting in a Trevoa–Retanilla clade with the Retanilla species forming the groups (R. trinervia (R. patagonica (R. ephedra, R. stricta))). The remaining species form the second major clade in which Kentrothamnus and Adolphia probably are basal branches rather than sister groups, or less likely Adolphia and Kentrothamnus belong to a clade also including Colletia and parts of Discaria. Colletia and Discaria are likely to form a monophyletic group with Colletia being monophyletic, having C. hystrix as the most likely basal taxon and with C. spinosissima and C. paradoxa placed as sister taxa. Within Discaria, D. chacaye, D. toumatou and D. nitida presumably form a clade probably having D. nana and D. trinervis as a monophyletic sister group. Discaria pubescens, D. articulata, and D. americana either form a monophyletic sister group to this latter clade, hence Discaria is monophyletic, or D. pubescens, D. articulata, and D. americana belong to a clade including Colletia and less likely Adolphia and Kentrothamnus, leaving Discaria paraphyletic.

ACKNOWLEDGEMENTS

I am grateful to Dr Diego Medan and Ing. Agr. Roberto Tortosa (Ca´tedra de Bota´nica, Facultad de Agronomı´a, Universidad de Buenos Aires) for suggesting the subject of this study, for providing working space in the institute, and for their helpfulness during the whole project. I am also grateful to rest of the staff, especially Gabriel Rua for discussion on inflorescence morphology. I am especially grateful to Ole Seberg (Botanical Institute, University of Copenhagen) for supervising the project and helpful suggestions that improved earlier drafts of this manuscript. I also thank the curators of the various herbaria for providing material for the analyses. Part of this study has been supported by the ‘Fiedler og hustru Legat’.

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McDade LA. 1992. Hybrids and phylogenetic systematics II. The impact of hybrids on cladistic analyses. Evolution 46: 1329–1346. McMinn HE. 1942. A systematic study of the genus Ceanothus. In: Van Rensselaer M, McMinn HE. Ceanothus. Santa Barbara: Santa Barbara Garden, 131–279. Maddison D. 1991. The discovery and importance of multiple islands of most parsimonious trees. Systematic Zoology 40: 315–328. Maddison W, Maddison D. 1992. MacClade. ver. 3. Sunderland, Massachusetts: Sinauer Associates Inc. Mantese A, Medan D. 1992. Anatomı´a y arquitectura foliares de Retanilla (Rhamnaceae). Darwiniana 31: 253–259. Mantese A, Medan D. 1993. Anatomı´a y arquitectura foliares de Colletia y Adolphia (Rhamnaceae). Darwiniana 32: 91–97. Medan D. 1974. Anatomı´a foliar de Kentrothamnus (Rhamnaceae). Boletin de la Sociedad Argentina de Bota´nica 16: 83–88. Medan D. 1985. Fruit morphogenesis and seed dispersal in the Colletieae (Rhamnaceae) I. The genus Discaria. Botanische Jahrbu¨cher fu¨r Systematik, Pflanzengeschichte und Pflanzengeographie 105: 205–262. Medan D. 1986. Anatomı´a y arquitectura foliares de Discaria (Rhamnaceae). Kurtziana 18: 133–151. Medan D. 1991. Reproductive phenology, pollination biology, and gynoecium development in Discaria americana (Rhamnaceae). New Zealand Journal of Botany 29: 31–42. Medan D. 1993. Breeding system and maternal success of a perennial hermaphrodite, Discaria americana (Rhamnaceae). New Zealand Journal of Botany 31: 175–184. Medan D, Aagesen L. 1995. Comparative flower and fruit structure in the Colletieae (Rhamnaceae). Botanische Jahrbu¨cher fu¨r Systematik, Pflanzengeschichte und Pflanzengeographie 117: 531–564. Medan D, D’ambrogio A. 1998. Reproductive biology of the andromonoecious shrub Trevoa quinquenervia (Rhamnaceae). Botanical Journal of the Linnean Society 126: 191–206. Medan D, Hilger HH. 1992. Comparative flower and fruit morphogenesis in Colubrina (Rhamnaceae) with special reference to C. asiatica. American Journal of Botany 79: 809–819. Medan D, Mantese A. 1989. Contribucio´n a la anatomı´a y arquitectura foliares de Talguenea (Rhamnaceae). Kurtziana 20: 95–100. Medan D, Tortosa RD. 1976. No´dulos radicales en Discaria y Colletia (Rhamna´ceas). Boletı´n de la Sociedad Argentina de Bota´nica 17: 323–336. Medan D, Tortosa RD. 1981. No´dulos actinomicorrı´cicos en especies argentinas de los ge´neros Kentrothamnus; Trevoa (Rhamnaceae) y Coriaria (Coriariaceae). Boletı´n de la Sociedad Argentina de Bota´nica 20: 71–81. Metcalfe CR, Chalk L. 1957. Anatomy of the Dicotyledons. Oxford: Clarendon Press. Miers J. 1860. On the tribe Colletieae, with some observations of the structure on the seed in the family of the Rhamnaceae. Annals and magazine of natural history, including zoology, botany, and geology ser. 3, 5: 76–96, 200–216, 267–273, 370–381, 482–492; 6: 5–14. Montenegro G, Riveros F, Alcalde C. 1980. Morphological structure and water balance of four Chilean shrub species. Flora 170: 554–564. Morrison TM, Harris GP. 1958. Root nodules in Discaria toumatou Raoul. Nature 182: 1746–1747. Nixon KC, Carpenter JM. 1993. On outgroups. Cladistics 9: 413–426. Richardson JE, Fay MF, Cronk QCB, Bowman D, Chase MW. 1997. A molecular analysis of Rhamnaceae using rbcL and trnL-F plastid DNA sequences. Systematics, first biennial international conference of the Systematic Association. Book of abstracts, 40. Rundel PW, Neel JW. 1978. Nitrogen fixation by Trevoa trinervis (Rhamnaceae) in the Chilean Matorral. Flora 167: 127–132. Schirarend C, Kohler E. 1993. Rhamnaceae Juss. World Pollen and Spore Flora, Vols 17, 18, 1–53. Skottsberg C. 1928. Pollinationsbiologie und Samenverbreiting auf den Juan-Ferna´ndez-Insel. In: ipse, The natural history of Juan Ferna´ndez and Easter Islands, vol. 2. Uppsala: Almquist and Wiksell, 503–547. Stevens PF. 1991. Character states, morphological variation, and phylogenetic analyses: a review. Systematic Botany 16: 553–583. Suessenguth K. 1953. Rhamnaceae. In: Engler A, Prantl K, eds. Die natu¨rlichen Pflanzenfamilien, 2. Aufl. 20d: 7–173. Swensen SM. 1996. The evolution of actinorhizal symbioses: evidence for multiple origins of the symbiotic association. American Journal of Botany 83: 1503–1512. Swofford DL. 1991. PAUP: Phylogenetic Analyses Using Parsimony ver. 3.1.1. Illinois State Natural History Survey, Champaign, IL.

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Tortosa RD. 1977. Una nueva especie australiana de Discaria (Rhamnaceae). Hickenia 1: 109–111. Tortosa RD. 1982. Organografia y vascularizacio´n de flores de Discaria, Colletia y Condalia (Rhamnaceae). Kurtziana 15: 19–39. Tortosa RD. 1983a. El ge´nero Discaria (Rhamnaceae) Boletı´n de la Sociedad Argentina de Bota´nica 22: 301–335. Tortosa RD. 1983b. Una especie polimorfa de Discaria: D. chacaye (G.Don) comb. nov. (Rhamnaceae) y sus hı´bridos presuntivos. Parodiana 2: 79–98. Tortosa RD. 1988. Natural hybridization in the genus Colletia (Rhamnaceae). Botanical Journal of the Linnean Society 97: 405–412. Tortosa RD. 1989. El ge´nero Colletia (Rhamnaceae). Parodiana 5: 279–332. Tortosa RD. 1992. El complejo Retanilla-Talguenea-Trevoa (Rhamnaceae). Darwiniana 31: 223–252. Tortosa RD. 1993. Revisio´n del ge´nero Adolphia (Rhamnaceae-Colletieae). Darwiniana 32: 185–189. Tortosa RD, Medan D. 1989. Novedades sobre no´dulos actinomicorrı´cicos en Angiospermas Sudamericanas. Revista de la Facultad de Agronomı´a Universidad de Buenos Aires 10: 79–86. Tortosa RD, Aagesen L, Tourn GM. 1996. Morphological studies in the tribe Colletieae (Rhamnaceae): analyses of architecture and inflorescences. Botanical Journal of the Linnean Society 122: 353–367. Tourn GM, Medan D, Tortosa RD. 1989. Yemas multiples en Rhamnaceas: organizacio´n del complejo axilar con especial referencia a Ziziphus y Paliurus. Kurtziana 20: 101–111. Troll W. 1959. Bericht d. Kommission fu¨r biologische Forschung. Akademie der Wissenschaften und der Literatur, Jahrbuch, Mainz: 112–131. Troll W. 1960. Bericht d. Kommission fu¨r biologische Forschung. Akademie der Wissenschaften und der Literatur, Jahrbuch, Mainz: 81–96. Urban I. 1924. Sertum antillanum. Repertorium specierum novarum regni vegatabilis 19: 298–308. Van Wyk AE, Schrire BD. 1986. A remarkable new species of Colubrina (Rhamnaceae) from Pondoland. South African Journal of Botany 52: 379–382. Weberling F. 1989. Morphology of flowers and inflorescences. Cambridge: Cambridge University Press. Willis JH. 1955. The Australian anchor plant. Victoria Naturalist 72: 51–55.

APPENDIX 1: SPECIMENS EXAMINED

Adolphia infesta Meisn.: Cleverland s.n. (MO 1922442), Dillon 889 (MO), Gonza´lez 30 (MO), Johnston 7456 (SI), Liston s.n. (fixed material, BAA), Lo´pez 6833 (MO), Paris 4432 (MO), Pringle 11945 (SI), Ventura & Pringle 7593 (BA). Ceanothus americanus L.: Bennett 537 (LIL), Hutchinson 626 (BAA), Meilleur & Baril s.n. (SI 28177), Smeltzer 366 (LIL). Ceanothus coeruleus Lag.: unknown collector s.n. (SI 25701), Muller 2895 (LIL), Pringle 11395 (SI), Ventura 625 (LIL). Ceanothus cuneatus (Hook.) Nutt.: Epling & Anderson s.n. (BA 14138), Solbrig 2602 (SI), Steward 7067 (LIL). Ceanothus greggii A. Gray: Harrison 1900 (MO), Venrick 577 (MO). Ceanothus leucodermis Greene: Carter 1160 (LIL). Ceanothus prostratus Benth.: Cunan s.n. (MO 1922248), Parks & Parks 24233 (SI), Pullman 45 (LIL). Ceanothus papillosus Torr. & A.Gray: Hilger & Hofmann s.n. (BAA 22421), Vanderwal 218 (LIL). Ceanothus purpureus Jeps.: Baker 10190 (LIL), Howell s.n. (LIL 262700), Howell 13062 368827), Howell 13062 (SI), Reed s.n. (LIL 368827). Ceanothus thyrsiflorus Esch.: Bracelin 1992, 2741 (LIL), Randall 321 (LIL), Solbrig 1989 (SI), Steward 7075 (LIL). Ceanothus verrucosus Nutt.: Gander 102 (LIL). Colletic hystrix Clos: Mallo et al. s.n. (BAA 18163), Marticorena et al. 1007 (BAA), Medan 579 (BAA), Tortosa s.n. (BAA 14970), Tortosa & Medan 66, 82, 952, 1209, 1232, 1237 (BAA). Colletia paradoxa (Spreng.) Escal.: D’ambrogio s.n. (BAA 18805), Frioni s.n. (fixed material, BAA), Medan 358, 672, 674 (BAA), Tortosa & Medan 222 (BAA). Colletia spinosissima J.F. Gmel.: Aagesen s.n. (fixed material, BAA), Boelcke et al. 5562 (BAA), Ca´mara Herna´ndez et al. s.n. (BAA 17419), Castagnino s.n. (BAA 14703), Guarnaschelli 33 (BAA), Kiesling & Saenz 4149 (BAA), Tortosa s.n. (BAA 18164), Tortosa & Medan s.n. (BAA 14689, 14691).

42

L. AAGESEN

Colletia ulicina Gillies & Hook.: Barrientos 1642 (CONC), Barros 872 (CONC), Bricker & Landrum 194 (CONC), Bustillos s.n. (SGO 51831), Medan 590 (BAA), Werdermann 619 (SI). Discaria americana Gillies & Hook.: Ancibor s.n. (BAA 9889), Berardo s.n. (BAA 18482), Boelcke 11937 (BAA), Boelcke et al. s.n. (BAA 2404), Leal 22327 (BAA), Legaspi s.n. (BAA 2157), Leon 3758 (BAA), Parodi 13062 (BAA), Rua 236 (BAA), Tortosa s.n. (BAA 16999), Troncoso et al. 2101 (BAA), Valla s.n. (BAA 20321), Villamil et al. s.n. (BAA 4331). Discaria articulata (Phil.) Miers: Boelcke 11417 (BAA), Correa 4182 (BAA), Dawson 1322 (BAA), Leal 18274, 26373 (BAA), Tortosa & Medan 130, 160 (BAA), Valla et al. s.n. (BAA 21112). Discaria chacaye (G.Don) Tort.: Bartoli et al. s.n. (BAA 21871), Ca´mara Herna´ndez s.n. (BAA 18730), Castelino s.n. (BAA 18731), Correa 8051 (BAA), Cusato 2551 (BAA), Gallego s.n. (BAA 19024), Tortosa & Medan 10, 68, 78, 91, 110, 114, 117, 122, 127, 130, 140, 141, 142, 172, 173, 190, 196, 955 (BAA) Discaria nana (Clos) Weberb.: Boelcke 10498 (BAA), Boelcke et al. 10300, 10316, 10398 (BAA), Boelcke et al. 16118 (BAA), Leal 23544 (BAA), Medan 676, 677, 678, 758, 760, 763 (BAA), Kiesling et al. 7615 (BAA), Rossow 846, 910, 1034, 1334 (BAA). Discaria nitida Tort.: Rood 5359 (MEL), Hall s.n. (BAA 20283, 20284), Scarlett 80–47 (BAA). Discaria pubescens (Brong.) Druce: unknown collector s.n. (MEL 56197, 56219), Barley 004 (MEL), Beauglehole 34884 (MEL), Boorman s.n. (NSW 133913), Briggs s.n. (NSW 133867), Constable s.n. (NSW 55985), Gray 444 (BAA), McGillivray & Bartlett 3191 (MEL, BAA), Parker s.n. (MEL 1543486), Pullen 2450 (MEL), Robbins s.n. (MEL 544193), Walsh 2669 (MEL), Williamson s.n. (MEL 56210), Willis s.n. (MEL 225426 p.p.). Discaria toumatou Raoul: Adams s.n. (AK 15003, 15004), Cockayne s.n. (AK 104098), Cooper s.n. (AK 24256), de Lange 1409 (AK), Harding s.n. (AK 5150), Hynes s.n. (AK 104630), Michell & MacMillam s.n. (AK 135153, SI ex. D.S.I.R. 204737), Petri s.n. (AK 5148), Willis s.n. (MEL 56254). Discaria trinervis (Hook. & Arn.) Reiche: Andrada et al. s.n. (BAA 29379), Boelcke 4507, 4511 (BAA), Caldero´n & Ru´golo 4 (BAA), Correa 8030 (BAA), Leal 26787 (BAA), Mendez & Wuilloud s.n. (BAA 37338), Roig 6995 (BAA), Rossow 1275 (BAA), Tortosa & Medan 43, 48, 146, 149, 154, 157, 176, 957, 977 (BAA), Valla et al. s.n. (BAA 21104), Vallerini s.n. (BAA 2024). Kentrothamnus weddellianus (Miers) Johnst.: Beck 9024 (BAA), Beck, Gomez & Ru´golo s.n. (SI 18041), Cabrera 17649 (BAA), Ceballos et al. 285, 306 (SI), Garcı´a, Beck & Michel 575, 594 (BAA), Mu¨rch 139 (SI), Mu¨rch 139 (SI), Ruthsatz 252, 265 (BAA), Ruthsatz s.n. (BAA 11417), Schreiter 11230 (SI), Tortosa & Medan 472, 483, 1199 (BAA). Noltea africana (L.) Reichenb.: Cooper 39 (NH), Medan 604 (BAA), Nicholson 2803 (NH). Retanilla ephedra (Vent.) Brong.: Boelcke 3848, 3903 (BAA), Medan 587 (BAA), Tortosa & Medan 1213, 1234, 1236 (BAA). Retanilla patagonica (Speg.) Tort.: Bartoli et al. s.n. (BAA 21850), Boelcke 4306, 16606 (BAA), Boelcke et al. 10432 (BAA), Boelcke et al. 13546 (BAA), Correa et al. 2524 (BAA), Correa et al. 7932 (BAA), Gomez et al. 236 (BAA), Leon 3188 (BAA), Medan 681 (BAA), Nicora 7466 (BAA), Roig 6285 (BAA), Rossow 947 (BAA), Schajovskoy 24/vii (BAA), Tortosa & Medan 305 (BAA). Retanilla stricta Hook. & Arn.: Germain s.n. (SGO 40920), Pairoa s.n. (SGO 78718), Volkmann s.n. (SGO 051901). Retanilla trinervia (Gillies & Hook.) Hook. & Arn.: Boelcke 3813 (BAA), Kausel 2480 (BAA), Mantese s.n. (BAA 21316), Medan 580, 588, 649 (BAA), Tortosa & Medan 1208, 1215, 1220, 1233, 1239 (BAA). Trevoa quinquenervia Gillies & Hook.: Flores s.n. (BAA 19574), Medan 581 (BAA), Tortosa & Medan 1210, 1221, 1241, 1243, 1244, 1247, 1248, 1249, 1262, 1286, 1288, 1289, 1297 (BAA).

APPENDIX 2: DATA MATRIX

PHYLOGENY OF THE TRIBE COLLETIEAE

43