American Journal of Botany 87(4): 547-564. 2000. SYSTEMATIC AFFINITIES OF RHIZOPHORACEAE AND ANISOPHYLLEACEAE, AND INTERGENERIC RELATIONSHIPS WITHIN RHIZOPHORACEAE, BASED ON CHLOROPLAST DNA, NUCLEAR RIBOSOMAL DNA, AND MORPHOLOGY1 ANDREA E. SCHWARZBACH23 AND ROBERT E. RICKLEFS3-4 'University of Missouri St. Louis, Department of Biology, 8001 Natural Bridge Road, St. Louis, Missouri 63121-4499 USA; and "Smithsonian Tropical Research Institute, Unit 0948, APO AA 34002-0948 A cladistic analysis of sequences from the chloroplast gene rbcL was used to determine the systematic affinities of Rhizophoraceae and Anisophylleaceae. This analysis rejects close relationships of Rhizophoraceae with Celastraceae or Elaeocarpaceae, suggested previously, and identifies Erythroxylaceae as sister group within the Malpighiales, supported by several morphological and anatomical characters. Our molecular results also indicate that Anisophylleaceae are nested within Cucurbitales. Although this placement is novel, this affinity is also well supported by shared morphological characters. Tribal and generic relationships within Rhizophoraceae are evaluated with a combination of six molecular data sets (rbcL, atpB-rbcL intergenic spacer, trnL-trnF intergenic spacer, ITS1, ITS2, and 5.8S) and a morphological data set. These rela- tionships are compared with results from previous morphological cladistic analyses. Against the background of the molecular results, we briefly discuss the evolution of morphological characters traditionally used for tribal subdivision as well as characters presumably significant for adaptation to mangrove habitats, namely, aerial stilt roots and vivipary. Key words: Anisophylleaceae; atpB-rbcL spacer; cpDNA; Erythroxylaceae; ITS; mangroves; nrDNA; rbcL; Rhizo- phoraceae; trnL-trnF spacer; vivipary. The Rhizophoraceae comprise 15 genera and ?140 species (Table 1). Although often described as a man- grove family, only four genera, including 16 species, live exclusively in mangrove habitats (Tobe and Raven, 1988a). The family is pantropical, and all members are either trees or shrubs. Rhizophoraceae mangroves are widely distributed along tropical coastlines and the ter- restrial species grow in both primary and successional moist forests (Juncosa and Tomlinson, 1988a). A few 1 Manuscript received 14 April 1998; revision accepted 27 July 1999. The authors thank Andre Chanderbali, Joachim Kadereit, Robert Ku- zoff, Susanne Renner, and Peter Stevens for their comments on the manuscript, Peter Wilson (Royal Botanical Gardens, Sydney), Gerardo Aymard, Paul Berry, Shing-Fan Huang, Sylvain Razifimandimbison, Susanne Renner, and James Solomon (Missouri Botanical Garden), J. W. H. Yong (University of Singapore), Gudrun Clausing and Karsten Meyer (Johannes Gutenberg University, Mainz), Stans Kofman (Rijk- sherbarium, Leiden), Norman Duke (Australian Institute of Marine Sci- ences, Townsville), Gordon Guymer (Queensland Herbarium), the Sin- gapore Botanical Garden, Phil Cameron (Mt. Coo-tha Botanical Garden, Brisbane), and the Darwin Botanical Garden (Darwin) for help with collecting plant material and providing information on plant locations, Eldredge Bermingham (Smithsonian Tropical Research Institute, Pana- ma), John Benzie, and Beth Bailment (Australian Institute of Marine Sciences, Townsville) for allowing AES to use their laboratory and sequencing facilities. AES was supported by a research grant from the University of Missouri Research Board and a research fellowship of the German Research Foundation (DFG; Schw 705/1-1). Laboratory work and sequencing were supported by a research grant from the University of Missouri Research Board and a research supplement to RER's Cu- rators' Professorship. - Author for correspondence, current address: Indiana University, De- partment of Biology, Bloomington, Indiana 47405 USA (e-mail: aschwarz@bio.indiana.edu). species, mainly in the genera Cassipourea and Dactylo- petalum, inhabit drier environments. The Anisophylleaceae consist of four woody genera and ?45 species growing in both New and Old World tropics. They occur in wet lowland primary forest, except for the monotypic genus Combretocarpus, which is re- stricted to peat swamp forests on Borneo (Juncosa and Tomlinson, 1988a). The relationships of the two families and their delim- itation have long been subjects of controversy (Table 1). Several authors have regarded the pantropical Aniso- phylleaceae as either closely related to Rhizophoraceae (Melchior, 1964) or as a separate tribe (Bentham and Hooker, 1865; Baillon, 1876; Ridley, 1922) or subfamily (Schimper, 1898) within the Rhizophoraceae. Takhtajan (1997) recently established a superorder Rhizophoranae containing the two single-family orders Anisophylleales and Rhizophorales, suggesting that although immediate relatives, the two families are not very close to each oth- er. A series of thorough morphological and anatomical studies (Behnke, 1988; Dahlgren, 1988; Juncosa and Tobe, 1988; Juncosa and Tomlinson, 1988a, b; Keating and Randrianasolo, 1988; Tobe and Raven, 1988b; Raven and Tomlinson, 1988; Vezey et al., 1988) supported the exclusion of Anisophylleaceae from the Rhizophoraceae. In spite of these studies, the systematic positions of these families still have not been resolved. Several authors (Cronquist, 1981; Tobe and Raven, 1988a; Thome, 1992) proposed that Anisophylleaceae are related to Rosales based mainly on floral and embryological characters. However, studies of wood anatomy (Van Vliet, 1976; Van Vliet and Baas, 1984) and leaf architecture supported the 547 548 AMERICAN JOURNAL OF BOTANY [Vol. 87 TABLE 1. Circumscription of Rhizophoraceae and Anisophylleaceae used by different authors. Numbers in parentheses mean (number of genera/ species) or [species], respectively. Juncosa and Tomlinson, 1988b. and this paper Tobe and Raven, 1988a; Takhtajan, 1997" Melchior, 1964 Schimper, 1898 Rhizophoraceae (15/~ 143) Macarisieae (7/87) Anopyxis Pierre ex Engl. [3] Macurisia Thou. [7] Blepharistemma Wall, ex Benth. Comiphyton Floret [1] Dactylopetalum Benth. [14]b Cassipourea Aubl. [51]c Sterigmapetalum Kuhlm. [9] [2] Gynotrocheae (4/?40) Carullia Roxb. [ca. 15] Crossostylis J. R. Forst. & Gynotroches Blume [2?4] Pellacalyx Korth. [8] G. Forst. [13] Rhizophoreae (4/16) Rhizophora L. [6] Ceriops Arn. [3] Kandelia (DC.) Wight & Arn. Bruguiera Sav. [6] [1] Anisophylleaceae (4/~45) Anisophyllea R. Br. ex Sabine [ca. 40] Combretocarpus Hook.f. [1?2] Pogu Pierre [1] Polygonanthus Ducke [2] Rhizophoraceae Rhizophoraceae Rhizophoraceae Macarisieae Macarisieae Rhizophoroideae Anopyxis Macarisia Macarisieae Macurisia Blepharistemma Macarisia Blepharistemma Dactylopetalum Comiphyton Cassipourea Gynotrocheae Dactylopetalum Weihea Gynotrochineae Cassipourea Crossostylis Sterigmapetalum Gynotrocheae Ceriops Carallia Gynotroches Crossosty lideae Crossostylis Kandelia Crossostylis Gynotroches Pellacalyx Rhizophora Gynotrocheae Carallinae Carallia Rhizophoreae Carallia Gynotroches Rhizophora Pellacalyx Ceriops Anisophylloideae Kandelia Anisophyllea Rhizophoreae Bruguiera Combretocarpus Rhizophora Ceriops Anisophylleaceae Kandelia Anisophyllea Bruguiera Combretocarpus Anisophylleaceae Anisophyllea Combretocarpus Poga Polygonanthus " Takhtajan (1997) separated the two families in two orders Anisophylleales and Rhizophorales, both kept in the superorder Rhizophoranae. b Floret (1988) treated Dactylopetalum as a distinct subgenus in Cassipourea. c Including subgenera Weihea (32 species), Lusiopetalum (3), Cassipourea (13), Pumiloweihea (1), Zenkeroweihea (1), and Dinklgeoweihea (1) sensu Floret (1988), but not subgenus Dactylopetalum. close relationship of the Anisophylleaceae and Rhizo- phoraceae to each other. One goal of the present analysis was to determine the relationships of these families with- in angiosperms. With Anisophylleae removed, the Rhizophoraceae show less variation, but their position within the angio- sperms still remains unclear. Several authors have placed the family in the Myrtales (Emberger, 1960; Melchior, 1964; Soo, 1975; Takhtajan, 1980), perhaps close to the Combretaceae with which they share several floral, veg- etative, pollen (tricolporate), and embryological charac- ters. The occurrence of other mangrove genera in the Myrtales, e.g., Laguncularia (Combretaceae) and Son- neratia (Lythraceae or Sonneratiaceae), also supported this grouping. The Rhizophoraceae have also been placed in the Cor- nales (Cronquist, 1968; Thome, 1968), and Airy Shaw (1966) postulated relationships with Combretaceae, Elaeocarpaceae, and Tiliaceae, and later with Combreta- ceae, Rubiaceae, and Elaeocarpaceae (Airy Shaw, 1973). More recent taxonomic treatments have excluded the Rhizophoraceae from the Myrtales (Johnson and Briggs, 1984) and treated them as a separate order Rhizophorales, close to Linales, Malpighiales, and Geraniales (Cronquist, 1981; Thome, 1992). Other suggestions include relation- ships with Erythroxylaceae (Behnke, 1982, 1988) and with Hugoniaceae, Linaceae, Oxalidaceae, Celastraceae, and Lepidobotryaceae (Dahlgren, 1988). Dahlgren (1988) conducted a systematic search for the relatives of Rhizophoraceae. He was guided by a com- parison of characters that occur in most or all Rhizo- phoraceae, or at least in some taxa he regarded as having plesiomorphic character states. Additionally, he chose characters that are unusual in angiosperms at large, or have at least a limited distribution. Altogether six char- acters were chosen and their individual and combined occurrences within the angiosperms were evaluated: pres- ence of endothelium in combination with crassinucellate ovules, presence of an aril formed by the exostome, fi- brous exotegmic seed coat, chlorophyllous embryo, type of sieve-tube plastids, and presence of certain types of alkaloids. Some families that exhibit character similarities with the Rhizophoraceae are listed in Table 2. Dahlgren (1988) summarized his findings in a list of most likely sister taxa to Rhizophoraceae: 1, Elaeocarpaceae; 2, Ce- lastraceae; 3, Erythroxylaceae; 4, Lepidobotryaceae and Ctenolophonaceae; 5, other Geraniales; 6, Theales, Eben- ales, Sapindales, and Rutales; and 7, Myrtales. Recently, Conti, Litt, and Sytsma (1996) suggested in a molecular cladistic analysis that the Rhizophoraceae do not belong in the Myrtales. Their rbcL tree shows a sister- group relationship to Drypetes Vahl. (Euphorbiaceae), but they sampled few groups outside the Myrtales and thus could not clarify phylogenetic affinities of Rhizophora- ceae. In addition to the limitation of sparse sampling, Drypetes and Rhizophoraceae were united by few syna- April 2000] SCHWARZBACH AND RlCKLEFS SYSTEMATIC AFFINITIES OF RHIZOPHORACEAE 549 TABLE 2. Presence of characters according to Dahlgren (1988) for different angiosperm families. Families that share only one of the listed characters with Rhizophoraceae are not shown. Character Erythro- Elaeocar- Celas- Euphor- Oxali- phoraceae xylaceae paceae traceae biaceae Linaceae daceae Clusi- Zygophylla- aceae ceae Violaceae 1. Presence of endothelium in combination with crassinucellate ovules 2. Presence of aril formed from the exostome 3. Seed coat with fibrous exotegmen 4. Chlorophyllous embryo 5. Sieve-tube plastids with numerous, vari- ably large, square or polygonal protein bodies 6. Presence of certain types of alkaloids: Hygroline Tropane alkaloids Pyrrolizidine alkaloids X of shared characters pomorphies (4), particularly compared to the large num- ber of autapomorphies in both (64 and 30, respectively). Such a pattern often results from long-branch attraction (Huelsenbeck, 1997; Lyons-Weiler and Hoelzer, 1997). Despite considerable effort, the relationships of Rhi- zophoraceae and Anisophylleaceae were still unclear. Previous morphological studies were ambiguous, and sampling problems have limited the molecular studies. To resolve the relationships of the family Rhizophoraceae and Anisophylleaceae, we have sequenced the chloroplast gene rbcL for members of all tribes of the Rhizophora- ceae and two (Anisophyllea and Combretocarpus) of the four genera of Anisophylleaceae. A large number of pu- tative sister taxa were included to find the closest rela- tives of the families. Intrafamiliar relationships?After excluding Aniso- phylleae from Rhizophoraceae, the remainder of the fam- ily has traditionally been subdivided into three tribes, the Anopyxls Macarisia Sterigmapetalum Cassipourea Blepharistemma Comiphyton Dactylopetalum Crossostylis 1 1 rt 1 k 5 9 4 r^- 5 2 1 4 4 10 2 4 1 Llf" Gynotroches Pellacalyx Carallia Bruguiera Kandelia Ceriops Rhizophora 0) m (3 a. o .c cc Fig. 1. Phylogenetic relationships of Rhizophoraceae based on a cladistic analysis of morphological characters by Juncosa and Tomlin- son (1988a). Figures above branches indicate number of characters. Fig. 2. Phylogenetic relationships of Rhizophoraceae based on seed morphology and anatomy by Tobe and Raven (1988b). Figures above branches indicate number of characters. 550 AMERICAN JOURNAL OF BOTANY [Vol. 87 Based on floral characters, the genera Sterigmapetalum and Cassipourea (including Dactylopetalum) were sepa- rated as an additional tribe from the rest of the Macari- sieae (they used the illegitimate name Hypogyneae). This separation is also supported by mostly brochidodromous leaf venation in "Hypogyneae" instead of mostly eu- camptodromous (or intermediate) venation in the Macar- isieae (Keating and Randrianasolo, 1988). To examine intrafamiliar relationships as well as monophyly of proposed tribes, we combined information from three chloroplast markers (rbcL, atpB-rbcL inter- genic spacer, and trnL-trnF intergenic spacer), three nu- clear ribosomal DNA regions (ITS1, ITS2, and 5.8S), and morphological data to construct a phylogenetic hypothe- sis for the Rhizophoraceae. In addition, we assess the evolution of morphological characters, especially those traditionally used for delimitation of systematic units in the Rhizophoraceae (e.g., fruit and seed characters), and those regarded as adaptations to mangrove habitats (e.g., aerial stilt roots and vivipary). The historical biogeogra- phy of the family will be discussed elsewhere (Schwarz- bach and Ricklefs, unpublished data). In summary, the main goals of the present study were (1) to find the closest relatives of Rhizophoraceae and Anisophylleaceae, (2) to reconstruct the intergeneric re- lationships within Rhizophoraceae and compare them with previously proposed classifications, and (3) to dis- cuss the evolution of morphological attributes in Rhizo- phoraceae associated with exploitation of mangrove hab- itats. MATERIALS AND METHODS Taxon sampling?For the family-level analysis, rbcL was sequenced for 11 of the 15 genera and 17 of ~143 species of Rhizophoraceae representing all three tribes. Two species of Anisophyllea and the mono- typic Combretocarpus represented the Anisophylleaceae. RbcL sequenc- es of several putative sister families and representatives of groups within which these families may be nested were downloaded from GenBank. The following search strategies were employed to sample taxa. (1) Guided by large phylogenetic analyses of angiosperms based on differ- ent molecular markers (Chase et al., 1993; Soltis et ah, 1997a, b), we sampled rbcL sequences from representatives of all major angiosperm clades and combined these data with our Rhizophoraceae/Anisophyl- leaceae sequences. This analysis narrowed the possible relationships of Rhizophoraceae/Anisophylleaceae to the rosid clades. (2) Accordingly, we next sampled representatives of each family in the rosid clade for which rbcL sequences were available, guided by phylogenetic analyses of groups within this clade (Albert, Williams, and Chase, 1992; Price and Palmer, 1993; Swensen, Mullin, and Chase, 1994; Conti, Litt, and Sytsma, 1996; Swensen, 1996; Fay, Swensen, and Chase, 1997; Savo- lainen, Spichinger, and Manen, 1997; Alverson et al., 1998; Fay et al., 1998). When sequences for several species of one family were available, we chose one or two representative species. We preferred species of unquestionable position in the above-mentioned analyses and for which a complete sequence was available. Due to the large number of taxa, this search could be conducted only with limited search strategies (es- pecially reduced branch swapping procedures). (3) After this initial search, we added all available sequences for groups identified as close to either Rhizophoraceae or Anisophylleaceae. At the same time we removed several families that did not appear as close relatives to either one of our families in order to reduce the total number of taxa and to allow faster computing. The results of steps 1 and 2 are not shown in this paper, but can be requested from AES. The 81 taxa used for the final search are listed in Appendix 1 (sequences obtained from Gen- Bank) and Appendix 2 (sequences produced for this study). For determining intrafamiliar relationships in Rhizophoraceae we sampled 13 genera (material for the monotypic genera Comiphyton and Blepharistemmu was not available) and 28 species of this family as well as all four genera of the Erythroxylaceae and Drypetes (Euphorbiaceae) as outgroups. Representatives of Rhizophora mangle from the Atlantic coast (AO; Ricklefs 186) and from the Pacific coast (PO; Ricklefs 171) of Panama were sequenced. For the combined analysis it was necessary to use sequences from different species in two instances: (1) Erythrox- ylum confusum (rbcL) combined with Erythroxylum argentinum (trnL- trnF, atpB-rbcL, and nrDNA) and (2) Drypetes roxburghii (rbcL) com- bined with Drypetes deplanchei (trnL-trnF). Protocols for the molecular study?Total DNA from frozen, silica gel-dried, and herbarium material was extracted using the DNA easy extraction kit (QIAGEN Inc., Chatsworth, California, USA) mainly fol- lowing the protocol provided by QIAGEN. The plant material was ground in Eppendorf tubes using sterilized sea-sand instead of liquid nitrogen. Herbarium and silica gel-dried material was ground in a dry state before adding extraction buffer, while the frozen material was ground after adding extraction buffer. DNA amplifications were per- formed in 50 u,L reactions containing 1.25U Taq polymerase (Promega, Madison, Wisconsin, USA), reaction buffer A supplied by Promega, 1.5 mmol/L MgCl2, 25 pmol of each primer and 0.2 mmol/L of each dNTR The flanking primer sequences for rbcL (IF and 1460R) were used as in Fay et al. (1998); additional internal primers were designed for max- imum fit in the Rhizophoraceae (674F: 5 -TTTATAAAGCACA- GGCGGAA-3'; 795R: 5 -CTGTTAAGTAGTCATGCATT-3'). The trnL-trnF spacer region was amplified using primers E and F from Ta- berlet et al. (1991). The atpB-rbcL intergenic spacer region was ampli- fied using the forward primer "oligo 2" described by Manen, Natali, and Ehrendorfer (1994) and a reverse primer complement to the forward primer (IF) that was used for amplification of rbcL. For amplification of the nuclear ribosomal DNA, primers designed by White et al. (1990) were used and 10% DMSO was added to the PCR mix. The PCR profile included 32 cycles of 30 s at 94?C, 30 s at 55?C, and 1 min at 72?C, and a final cycle of 30 s at 95?C, 1 min at 55?C, and 8 min at 72?C. The extension time per cycle was increased to 2 min for rbcL. The product was separated from residual primers, dNTPs, and Taq poly- merase by running the entire product on a low melting agarose gel. Amplified DNA was recovered by using the QIAquick Gel Extraction kit (QIAGEN Inc.). Part of the purified DNA (60-120 ng) was used for the cycle sequencing reaction following the protocol of the DyeDeoxy Terminator Cycle Sequencing kit (Applied Biosystems Inc., Foster City, California). The samples were run on an acrylamide gel using an ABI 373 or ABI 377 DNA Sequencing System (Applied Biosystems Inc.). In most cases, both strands were sequenced. Morphological data?We expanded the genus-level morphological data sets of Juncosa and Tomlinson (1988a), Keating and Randrianasolo (1988), and Tobe and Raven (1988b) to all species in the molecular study (Appendix 3) and added the outgroups Erythroxylaceae (Schulz, 1907; Badre, 1973a, b; Boesewinkel and Geenen, 1980; Verdcourt, 1980; Weberling, Lorcher, and Bohnke, 1980; Rury, 1981; Behnke, 1988) and Drypetes (Levin, 1986; Kapil and Bhatnagar, 1994; Dunlop, Leach, and Cowie, 1995; Tokuoka and Tobe, 1999). Additionally, some characters from published studies were recoded. For example, Juncosa and Tomlinson (1988a) coded fruit type (dehiscent or baccate) and seed appendages (no appendages, wings, or arils) as two separate characters. We treated these as nonindependent characters because certain combi- nations are not possible (e.g., berries with winged seeds or arils). There- fore, "fruit type" and "seed appendages" were united into a single character (see character 23 for coding, Appendix 3). Data analysis?The DNA sequences were assembled and used to April 2000] SCHWARZBACH AND RlCKLEFS SYSTEMATIC AFFINITIES OF RHIZOPHORACEAE 551 construct consensus sequences. All sequences were aligned manually. For the ITS regions a Clustal W alignment (Thompson, Higgins, and Gibson, 1994) was performed in addition to the manual alignment, but this resulted in only minor differences that had no effect on the tree topologies. Gaps were coded as missing. Missing sequences were coded as missing data in the combined data set. All sequences have been deposited in GenBank (Appendix 2). The aligned sequences were exported to PAUP* versions 4.0d64 (kindly provided by D. Swofford). The heuristic search algorithm, with the MULPARS and TBR branch swapping options, and 100 RANDOM additions to search for multiple islands of trees (Maddison, 1991), was used for all final tree searches. The morphological data were analyzed with the same PAUP settings. Larger preliminary analyses for the fam- ily relationships were performed with NNI branch swapping and CLOS- EST taxon addition. For the intergeneric relationships the different data sets were analyzed both separately and combined. Strict consensus trees were constructed from all most parsimonious trees obtained from the individual and combined searches. Consistency (CI) and retention in- dices (RI) were calculated. Bootstrap analyses (Felsenstein, 1985) were used to assess support for monophyletic groups. The same PAUP set- tings were used for the bootstrap analysis (100 replicates) as for the original search, but SIMPLE addition was used instead of RANDOM. For the trnL-trnF data set a "fast bootstrap" with 10 000 replicates was performed, because a regular bootstrap could not be finished due to computational constraints. MacClade 3.05 (Maddison and Maddison, 1992) was used for map- ping characters and computation of tree lengths for alternative topolo- gies. Both ACCTRAN and DELTRAN optimization were used and compared. The Templeton test (Templeton, 1983; Mason-Gamer and Kellogg, 1996) was used to test whether the alternative topologies ob- tained by moving branches in the family analysis were significantly longer than the most parsimonious trees. The same test was used to test for significant differences between the morphological and combined molecular trees. The molecular trees re- sulting from the different molecular data sets do have slightly different taxon composition and were therefore only compared visually by com- paring topology and branch supports, respectively. RESULTS Family-level search?During the third step of the fam- ily-level search 81 taxa were included (Appendices 1 and 2). The aligned sequences included 1402 bp, of which 411 characters were phylogenetically informative. The search based on rbcL sequences revealed three islands of trees, each containing 16 equally most parsimonious trees, 2365 steps long (excluding autapomorphies). One of the trees, which is identical to the majority rule con- sensus tree containing all compatible groupings, is shown in Fig. 3, but branches collapsing in the strict consensus tree are also indicated. The consistency index is 0.282 (excluding autapomorphies); the retention index is 0.558. Our results show that the family Rhizophoraceae is sister group to Erythroxylaceae, supported by a bootstrap value of 90%. Both families are part of the order Malpighiales within eurosids I (sensu APG, 1998). Additionally, our tree suggests a close relationship of Anisophylleaceae and Cucurbitaceae within the order Cucurbitales. Although this relationship is not well supported, Anisophylleaceae and Rhizophoraceae clearly are not immediate relatives. Forcing the Rhizophoraceae into a sister group relation- ship with Anisophylleaceae requires 37 extra steps on the tree and this new topology is significantly different (P < 0.0001). Conversely, moving Anisophylleaceae to the base of Rhizophoraceae creates a tree 32 steps longer than the most parsimonious trees. Similarly, placing Rhi- zophoraceae in close relationship to families that were previously suggested as close relatives results in trees be- tween 19 and 42 steps longer than the shortest trees (Fig. 3). All these alternative topologies require significantly more steps (P < 0.0001). Intrafamiliar search?rbcL?Altogether, 27 rbcL se- quences, each 1402 bp long, were analyzed. The aligned data set contained 235 variable characters, of which 91 were phylogenetically informative. The tree search re- sulted in 144 equally most parsimonious trees (Table 3). All most parsimonious trees show the monophyly of the three tribes Macarisieae, Gynotrocheae, and Rhizopho- reae (Fig. 4). The rbcL sequence data resulted in mainly well-resolved and well-supported deeper branches, but they fail to resolve more recent relationships. AtpB-rbcL?Twenty-eight taxa were sequenced for this cpDNA spacer, which is adjacent to the rbcL gene. The sequences were between 647 and 736 bp long, and the aligned sequences contained 969 positions, of which 137 were variable and 73 phylogenetically informative (Table 3). The tree search resulted in 20 equally most parsimo- nious trees. All deep branches are well resolved (Fig. 4), but often less well supported than in the rbcL tree. This might be the result of fewer informative characters over- all or, what seems to be more plausible to us, increasing difficulty in recovering multiple substitutions further back in time and therefore underestimation of deeper branch lengths. TrnL-trnF?This cpDNA spacer region was sequenced for all 34 taxa included in this study. The sequences were between 331 and 437 bp long in the Rhizophoraceae and up to 467 bp in the outgroups. The aligned sequences were 567 bp long, of which 158 positions were variable and 87 phylogenetically informative (Table 3). The tree search could not be completed due to computational lim- itations and was stopped after obtaining 73 346 trees of 156 steps length. The strict consensus tree is shown in Fig. 4. This spacer region was the most variable, but also the shortest cpDNA piece sequenced in this study (Table 3). NrDNA?ITS1 (206-251 bp), 5.8S (162 bp), ITS2 (196- 222 bp), and a small part of the 26S nrDNA (50 bp) were sequenced for 20 taxa. We were not able to obtain a com- plete data set for two reasons. (1) Fungal and algal con- taminations of the DNA that was extracted from some of the herbarium material leaves did not allow us in a few cases to obtain the Rhizophoraceae sequences. Even the use of primers that were designed to better fit angiosperm sequences (F. R. Blattner, personal communication, IPK Gatersleben, Germany) could not discriminate between fungal and Rhizophoraceae targets. All sequences ob- tained were tested with a GenBank BLAST search for possible contaminations, a caution suggested by Liston et al. (1996). (2) For some of the taxa we obtained two different copies of this repeat region, and for other taxa only "copy one" or "copy two." For this analysis we chose the copy that we obtained for a larger number of 552 AMERICAN JOURNAL OF BOTANY [Vol. 87 Heracleum Apiaceae Libanotis Apiaceae Aceratium Elaeocarpaceae Elaeocarpus Elaeocarpaceae Bauera Cunoniaceae Sloanea Elaeocarpaceae Oxalis Oxalidaceae Euonymus Celastraceae Pamassia Parnass\aceae___^ Rhizophora mangle -p Rhizophora stylosa N o T3 3" O 3 o CD 0) CD Rhizophora apiculata Kandelia candel Ceriops tagal Bruguiera sexangulare Bruguiera exaristata Bruguiera parviflora Pellacalyx axitlaris Pellacalyx saccardianus Gynotroches axillaris Crossostylis biflora Carallla brachiata Cassipourea elliptica Cassipourea guianensis Macarisia spec. Eryihroxyium Erythroxylaceae Byrsonima Malpighlaceae Passiflora Passifloraceae Drypetes Euphorbiaceae OchnaOchnaceae Humiria Humiriaceae Licania Chrysobalanaceae Chrysobalanus Chrysobalanaceae Hymenanthera Violaceae Leonia Violaceae Euphorbia Euphorbiaceae Acaiypha Euphorbiaceae Clusia Clusiaceae Tetrameles Tetramelaceae Octomeles Tetramelaceae Coriaria Coriariaceae Corynocarpus Corynocarpaceae Anisophyliea fallax Anisophyileaceae j Anisophyllea disticha Anisophyileaceae' Combrelocarpus Anisophyileaceae Neoalsomitra Cucurbitaceae Cucurbita Cucurbitaceae Datisca cannabina Datiscaceae Datisca glomerata Datiscaceae Begonia Begoniaceae Hillebrandia Begoniaceae Nothofagus Nothofagaceae Fagus Fagaceae Rhamnus Rhamnaceae P/'/eallrticaceae Spiraea Rosaceae Baptisia Fabaceae Polygala Polygalaceae Erodiurn Geraniaceae Hypseocharis Hypseocharitaceae Terminalia Combretaceae Bucida Combretaceae Quisqualis Combretaceae Circaea Onagraceae Fuchsia Onagraceae Lythrum Lythraceae Myrcianthes Myrtaceae Rhexia Melastomataceae Vochysia Vochysiaceae Tropaeotum Tropaeolaceae Cleome Capparaceae Schinus Anacardiaceae Dirca Thymelaeaceae 77/iaTiliaceae Dillenia Oilleniaceae Vitis Vitaceae CeraV*pft)'//u/nCercidiphyllaceae Hamamelis Hamamelidaceae Sabia Sabiaceae Platanus Platanaceae asterids Oxalidales Celastrales Malpighiales Cucurbitales I Fagales Rosales Fabales Geraniales Myrtales Brassicales Sapindales Malvales outgroups Fig. 3. One of 48 equally most parsimonious trees identical to the majority-rule consensus tree showing all compatible groupings based on the rbcL-based family search. Branches collapsing in the strict consensus tree are indicated by dotted lines. Figures above branches are number of nucleotide changes; figures below branches represent bootstrap values expressed as percentage of 100 bootstrap replications. Bootstrap values <50 % are not shown. The numbers shown in the boxes indicate the number of extra steps required forcing Rhizophoraceae into the indicated position. Tree length is 2365 steps (excluding autapomorphies), consistency index is 0.282 (excluding autapomorphies), retention index is 0.558. Boxed taxa show the delimitation of Rhizophoraceae and Anisophyileaceae, respectively. Eurosids I are indicated by black bars, eurosids II by white bars. Geraniales have not yet been assigned to either group (APG, 1998). April 2000] SCHWARZBACH AND RlCKLEFS SYSTEMATIC AFFINITIES OF RHIZOPHORACEAE 553 TABLE 3. Tree statistics for the different data sets and their combinations. Number of Number of included Number of informative Number Data set taxa- characters1. characters of trees Tree length0 CI RI Family relationships rbcL 81 1402 411 (29.2%) 48 2365 0.282 0.558 Intrafamiliar relationships rbcL 27 1402 91 (6.5%) 144 153 0.686 0.887 trnL-trnL 34 567 87 (15.2%) >73,346d 156 0.673 0.877 atpB-rbcL 28 969 73 (7.6%) 20 113 0.761 0.902 ITS 1, ITS 2 and 5.8S, partial 26S 20 711 236 (33.2%) 2 614 0.658 0.734 Morphology 34 30 30 (100%) 55 77 0.649 0.899 Molecular combined 34 3649 487 (13.4%) 7 1047 0.669 0.818 All combined 34 3679 517 (14.1%) 1 1132 0.664 0.825 a List of taxa in appendices 1 and 2, respectively. b Length of aligned sequences. c Excluding autapomorphies. d Result of an incomplete tree search (see text for details). taxa, simply to avoid a large number of missing data in the combined analyses. The aligned data set was 711 bp long and contained 410 variable characters, of which 236 were phylogenet- ically informative (Table 3). The tree search resulted in two equally most parsimonious trees and the strict con- sensus tree was calculated. The nrDNA phylogeny (Fig. 4) is largely congruent with the cpDNA trees. The main difference is the sister-group relationship of Gynotroches and Crossostylis (bootstrap value 79%) instead of Gyn- otroches being sister to Pellacalyx as found for cpDNA. This may well be a long-branch attraction problem be- cause a small number of synapomorphies are opposed by a large number of autapomorphies (Fig. 4). Morphology?The tree reconstruction based on 30 morphological characters (Appendix 3) resulted in 55 equally most parsimonious trees. The strict consensus tree is shown in Fig. 4. The phylogeny is not well re- solved, most likely due to the small number of characters. ACCTRAN and DELTRAN optimizations did not result in any differences that would lead to an important dif- ference in character interpretation. Trees resulting from separate searches were highly congruent, although the separate analyses were some- times less resolved, which would tend to obscure any incongruencies. Tree searches based on the trnL-trnF spacer sequences could not be completed, even though this spacer produced more informative characters than atpB-rbcL and the morphological data. TrnL-trnF may be too variable (Table 3) to unambiguously reconstruct deep- er branches of the phylogeny. Multiple nucleotide sub- stitutions over time seem to be the cause of the blurred deep relationships. The nrDNA was even more variable (Table 3), but the variability appeared to be distributed differently. Parts of ITS1 and ITS2 and especially the 5.8S DNA are relatively conservative (see also Hersh- kovitz and Lewis, 1996; Hershkovitz and Zimmer, 1996), allowing resolution of deep branches. Combined analyses?We were not able to sequence the different regions for all taxa, mainly due to difficulties with amplification of DNA from herbarium material. For this reason the amount of data available for the different taxa differs (Appendix 2). Because some sequences were incomplete, branch lengths generally do not reflect the actual number of transformations in the combined anal- ysis. Missing data may also reduce bootstrap values in some cases. The combined molecular analysis based on an aligned data set composed of 3649 positions and 487 phylogenetically informative positions resulted in seven equally most parsimonious trees of 1047 steps length. The strict consensus tree is shown in Fig. 4. This tree is well resolved except within the Macarisieae, which might be due to the fact that most missing data are within this tribe. When comparing molecular and morphological trees the Templeton test (Templeton, 1983) revealed some in- teresting insights. By plotting the combined molecular data set onto the 55 most parsimonious morphological tree topologies, as well as onto the 50% majority rule and strict consensus trees of these 55 trees, we found that only two of the 55 single-tree topologies did not differ signif- icantly. Interestingly, even the consensus trees of the morphological analysis (and only morphological data were included in this case) differed significantly from the individual trees of this same analysis. This might indicate considerable internal conflict within the morphological trees, in our opinion most likely the result of the small number of informative characters. Conversely, plotting the morphological data onto the seven combined molec- ular trees resulted in no significant differences (P = 0.15? 0.32). In summary, this means that the morphological data are compatible with the molecular tree topologies, but the molecular data are not compatible with the gen- erally badly resolved morphological topologies. We think that the incongruence in only one direction is the result of an internal problem of the morphological data set and we therefore decided to combine the data sets. Finally, the combination of all data sets, morphological and molecular, resulted in a single most parsimonious tree 1132 steps long (Fig. 5). Altogether, 517 informative characters were available. We do not believe that the larg- er molecular data set swamps the smaller morphological one resulting in a tree that more closely resembles the molecular tree than the morphological. A small number of added characters can alter tree topologies dramatically, and Chippindale and Wiens (1994) have shown that com- 554 AMERICAN JOURNAL OF BOTANY [Vol. 87 80 32 10 93 13 100 13 96 94 83 64 61 rbcL 97 49 98 10 98 100 48 84 28 16 99 59 82 "& 12 94 88 70 63 82 13 52 8 93V 99 trnL-trnF -DRYrho -ERYcon -STEgui -MACspe -DACver -CASell -CASgui -CAScey -CARbra -GYNaxi -PELsac -PELaxi -CROsty -% BRUpar -5?BRUexa ?z?BRUgym -BRUcyl -BRUsex -KANcan -CERaus -CERtag -RHIapi -RHIsty -RHImuc -RHIman 186 -RHIman 171 -RHIrac DRYdep ANEafr PINcon NECkae ERYarg -STEgui -ANOkla -MACspe -DACver -DACbar -CASell -CASgui -CAScey -CARbra -CAReug -GYNaxi -PELsac -PELlob -PELaxi -CROsty -BRUpar -BRUexa -BRUcyl -BRUgym BRUsex KANcan ?CERaus CERtag RHIapi RHIsty RHImuc RHIman 186 RHIman 171 RHIrac 152 29 20 100 37 |23r Too1 34 15 15 56 100 100 _54_ 13 87 79 10 20 96 14 19 81 nrDNA 25 100 12 901 JI_ 10 52 25 34 100 98 L 4 74 77 -[83"L 75 61 77 10 96 59 41 97 90 62 combined molecular morphology - ERYarg -STEgui -CAScey -CASgui -CARbra -CAReug -GYNaxi -CROsty -PELsac -PELlob -PELaxi -BRUsex -BRUcyl -BRUexa -KANcan -CERaus -CERtag -RHIapi -RHImuc -RHIman 186 -DRYdep -ANEafr -PINcon -NECkae -ERYarg -STEgui -ANOkla -MACspe -DACver -DACbar -CASell -CASgui -CAScey -GYNaxi - PELsac - PELlob -PELaxi -CARbra -CAReug -CROsty -BRUpar -BRUcyl -BRUexa -BRUgym -BRUsex -KANcan -CERaus -CERtag -RHIapi -RHIsty -RHImuc -RHIman 186 -RHIman 171 -RHIrac Fig. 4. Phylogenetic analyses of Rhizophoraceae based on rbcL, utpB-rbcL spacer, trnL-trnF spacer, and nuclear ribosomal DNA sequences (ITS 1, ITS2, 5.8S, and partial 26S), all molecular data combined, and morphology. Strict consensus trees are shown; tree statistics are listed in Table 3. Due to computational constraints, the trnL-trnF tree represents the result of an uncompleted tree search (see text for details). Figures above branches are number of character changes (ACCTRAN optimization); figures below branches represent bootstrap values. Differences among trees are entirely based on badly supported or unresolved branches. binations of smaller and larger data sets do not necessar- ily produce a tree that is identical to the tree obtained for the larger data set alone. In our study the single tree ob- tained with the total evidence approach is identical to one of the seven trees that resulted from the combined mo- phological characters helped to discriminate between the alternative trees that resulted from molecular analyses. DISCUSSION Relationships of Anisophylleaceae- -Our results indi- lecular analysis, indicating that the relatively few mor- cate that the Anisophylleaceae belong in the Cucurbitales April 2000] SCHWARZBACH AND RlCKLEFS SYSTEMATIC AFFINITIES OF RHIZOPHORACEAE 555 115 23 161 100 11 14 97 31 57 103 100 99 28 72 71 74 19 22 39 89 13 55 28 94 100 100 100 15 34 29 100 61 14 86 23 100 10 73 11 12 45 32 100 fruit type: C = capsule D = drupe B = berry I = indehiscent fruit 100 17 10 90 19 33 30 39 23 56 18 91 seed appendages: W = wing A = aril N = non-appendaged 94 82 30 100 32 20 61 100 56 12 11 66 ? Dry petes* ? Aneulophus africanus ? Erythroxylum* ? Nectaropetalum kaessneri ? Pinacopodium congolense ?Sterigmapetalum guianense ? Anopyxis klaineana ? Macarisia spec. I Dactylopetalum verticillatum I Dactylopetalum barteri -Cassipourea ceylanlca -Cassipourea elliptica -Cassipourea guianensis - Carallia eugenioidea - Carallia brachiata -Crossostylis biflora -Gynotroches axillaris - Pellacalyx axillaris - Pellacalyx lobbii - Pellacalyx saccardianus - Bruguiera parviflora -Bruguiera exaristata -Bruguiera gymnorrhiza -Bruguiera cylindrica -Bruguiera sexangulare - Kandelia candel -Ceriops australis -Ceriops tagal ? Rhizophora apiculata - Rhizophora mucronata - Rhizophora stylosa - Rhizophora mangle 171 PO -Rhizophora mangle 186 AO - Rhizophora racemosa & CO 0) c] [AI CO D > o N ? C fr w 3 a / W o / V o C c s o 2 A a. '/ > > / o A ? B N ? ? CD C A o ?g I?| O to o s O B o o 2 o Q. 0 a ? c ? N S 10 o o CO 2 | a > > I I Fig. 5. Single most parsimonious tree based on the combined analysis of rbcL, trnL-trnF spacer, atpB-rbcL spacer, FTS1, ITS2, 5.8S, partial 26S sequences, and morphological characters. Figures above branches are number of character changes; figures below branches represent bootstrap values expressed as percentage of 100 bootstrap replications. Asterisks mark taxa where sequences originated from different species of the same genus (see text for details). Tree length is 1132 steps (excluding autapomorphies), consistency index is 0.664 (excluding autapomorphies), retention index is 0.825. Boxes to the right indicate distribution of morphological characters. (sensu Bremer, Bremer, and Thulin, 1997; APG, 1998), which include the Cucurbitaceae, Begoniaceae, Datisca- ceae, Tetramelaceae, Coriariaceae, and Corynocarpaceae (see also Swensen, Mullin, and Chase, 1994; Swensen, Luthi, and Rieseberg, 1988), but are not closely related to Rhizophoraceae. Our analysis results in a sister group relationship of the Anisophylleaceae and Cucurbitaceae, although this relationship is not well supported. The An- isophylleaceae and, to a large extent, the rest of Cucur- bitales share separate styles, an inferior ovary, and uni- 556 AMERICAN JOURNAL OF BOTANY [Vol. 87 sexual flowers (apart from Combretocarpus). In contrast to most other Cucurbitales (but see Octomeles, a tree up to 60 m height), the Anisophylleaceae are often large tropical trees. Similarities between the Rhizophoraceae and the Anisophylleaceae, such as incised petals and sim- ilar vascular anatomy (Tobe and Raven, 1988a), probably should be regarded as the results of parallelism or con- vergent evolution. Tobe and Raven (1988a) argued that the Anisophylleaceae should be treated as distinct from Rhizophoraceae, but they postulated an intermediate po- sition between the Rhizophoraceae and Myrtales. How- ever, because several families are more closely related to one or another of these three groups (Fig. 3), character similarities among the Anisophylleaceae, Rhizophora- ceae, and Myrtales should be regarded as plesiomorphies or parallelisms rather than synapomorphies. Cronquist (1981) and Thome (1992) treated the Anisophylleaceae as a separate family in the order Rosales, but the close relationship to Rosaceae or other Rosales (Cronquist, 1981; Dahlgren, 1988) suggested by pollen and flower characters can also be rejected on the basis of our mo- lecular results. A better understanding of the relationships of the An- isophylleaceae will require a broader study including all four genera of the family as well as more members of the Cucurbitales, and perhaps a more variable marker. The genus Polygonanthus differs especially in many characters from the rest of the Anisophylleaceae, sug- gesting that it might be not part of this family, but un- fortunately we were not able to obtain any material. Relationships of the Rhizophoraceae?The hypothe- ses proposed by several authors of a close relationship of Rhizophoraceae with either Celastraceae or Elaeocarpa- ceae (Dahlgren, 1988; Juncosa and Tomlinson, 1988a; Tobe and Raven, 1988b) are not supported by our mo- lecular analysis. Character similarities with the Rhizo- phoraceae, namely stipules, similar leaf venation, unicel- lular hairs, stomatal type, and presence of oxalate crystals in Elaeocarpaceae, and laticifers, oxalate crystals, stip- ules, inflorescence structure, and identical embryological features in Celastraceae (Dahlgren, 1988), should be re- garded as plesiomorphies or parallelisms. Our analyses show, instead, that the sister family to the Rhizophora- ceae is Erythroxylaceae, which was the third of the list of putative sister families suggested by Dahlgren (1988). The relationship between Rhizophoraceae and Erythrox- ylaceae is well supported by a high bootstrap value (90%) in the molecular analysis (rbcL) and by both families sharing morphological characters with other taxa in the order Malpighiales (sensu Bremer, Bremer, and Thulin, 1997; APG, 1998). When the Rhizophoraceae are forced to be the sister group to the alternative families suggested by Dahlgren (1988), at least 19 more steps (Celastrales) are required than in the most parsimonious tree (Fig. 3). Moreover, several nonmolecular characters listed by Dahlgren (1988) also support the sister group relationship of Erythroxylaceae and Rhizophoraceae, which share a woody habit, many embryological characters, the pres- ence of the alkaloid hygroline, and, especially, a unique sieve-tube plastid type (Behnke, 1988). Indeed, the Ery- throxylaceae shared with the Rhizophoraceae the highest number of the characters listed by Dahlgren (1988), namely seven (Table 2). Compelling as this may be, Dahlgren (1988) preferred the Celastraceae and Elaeo- carpaceae over the Erythroxylaceae as potential sister groups to the Rhizophoraceae. In particular, he found no reason to remove Erythroxylaceae from the Geraniales (or from Finales, if this order is separated from Gerani- ales, as by Cronquist, 1981) and to move them to the vicinity of Rhizophoraceae. Dahlgren (1988) thought that the similarities between Erythroxylaceae and Rhizophor- aceae were "not as far-reaching" as those between Rhi- zophoraceae and Elaeocarpaceae. Consequently, accord- ing to his view, "the very particular kind of sieve-tube plastids that the two families [Erythroxylaceae and Rhi- zophoraceae] have in common has evolved by convergent evolution in the ancestors of each of the two families." Characters that vary in the Erythroxylaceae and Rhi- zophoraceae but also support a close relationship of the two families are discussed below with the results of the generic analysis. Relationships within the Rhizophoraceae?System- atic inferences from the combined analysis (Fig. 5) agree with some traditional subdivisions of the family into the three tribes Macarisieae, Gynotrocheae and Rhizophoreae (Table 1). The monophyly of all three clades is well sup- ported by bootstrap values of at least 99% (Fig. 5). The Gynotrocheae are the sister group to the mangrove tribe Rhizophoreae (bootstrap 100%) and both together are sis- ter group to Macarisieae (bootstrap 100%; Fig. 5). Al- most all separate analyses of molecular data show iden- tical relationships of the tribes (except for trnL-trnF, for which this part of the tree is unresolved), and these con- tradict in several respects the arrangement indicated by the morphological analyses of Juncosa and Tomlinson (1988a; see Fig. 1) and Tobe and Raven (1988b; Fig. 2). Our morphological analysis did not resolve the tree on a tribal level. The three analyses differ in taxon and char- acter sampling as well as in character coding, which may have been responsible for the different topologies. The main discrepancy can be found in the relationships of the Gynotrocheae and their position as a separate tribe. Based on our unambiguous molecular results and our interpre- tation of morphological character changes that will be presented below, we prefer a subdivision of the family into three tribes (Table 1). With one exception, the three tribes can be circum- scribed by fruit characters. Macarisieae share capsular fruits, Gynotrocheae (except Crossostylis) have berries, and Rhizophoreae have indehiscent fruits with seeds ger- minating on the mother plant (viviparous). In the analyses of Juncosa and Tomlinson (1988a) and Tobe and Raven (1988b), the capsular fruits with arillate seeds of Cros- sostylis resulted in a sister group relationship of Cros- sostylis to the rest of the Gynotrocheae and Rhizopho- reae, leaving the Gynotrocheae paraphyletic. According to Juncosa and Tomlinson (1988a), the most parsimoni- ous interpretation is that the capsular fruit constitutes the plesiomorphic character state in the family; a common ancestor of Carallia, Gynotroches, Pellacalyx, and the mangrove clade evolved berry-like fruits, which was fol- lowed by a shift to viviparous, indehiscent fruits in the mangrove group. Our most parsimonious interpretation April 2000] SCHWARZBACH AND RlCKLEFS SYSTEMATIC AFFINITIES OF RHIZOPHORACEAE 557 based on the combined analysis also suggests a plesiom- orphic capsular fruit. Juncosa and Tomlinson's (1988a) tree topology sug- gests that Crossostylis occupies a more basal position and retains the plesiomorphic fruit character state of the Rhi- zophoraceae. In contrast, our hypothesis suggests a more derived position for Crossostylis combined with a derived fruit type different from the plesiomorphic character state in the family. Several other apomorphic features also sup- port a more derived position of Crossostylis. Species of this genus share a polyandrous androecium, which is clearly derived and elsewhere in the family is found only in Kandelia, a member of the Rhizophoreae, the most derived mangrove clade. The absence of terminal flowers in Crossostylis is a clearly derived feature that evolved in parallel in Rhizophora and Kandelia. Species with cap- sular fruits in the Macarisieae share superior ovaries, which can be regarded as plesiomorphic, but Crossostylis has a half-inferior or inferior ovary (Tobe and Raven, 1988b; Setoguchi, Ohba, and Tobe, 1998) and the capsule opens with small slits. As far as we know, the capsular fruits of Macarisieae open with longitudinal slits or fall from the tree unopened, dehiscing on the ground upon drying out (Juncosa and Tomlinson, 1988a). The hypoth- esis of a derived position of Crossostylis also agrees with the biogeography of this genus, which is endemic to South Pacific Islands. Based on morphological and cpDNA RFLP (restriction fragment length polymorphism) data, Setoguchi et al. (1998) suggested a successive dis- tribution from more western islands (Solomon Islands, Vanuatu, and New Caledonia) to more eastern islands (Fiji, Samoa, Society Islands, and Marquesas Islands). Therefore, the arillate seeds in Crossostylis are most like- ly distributed by birds from island to island in contrast to the ant-dispersed arillate seeds of the Macarisieae. Not only fruit characters but also seed characters are important in the delimitation of clades within Rhizo- phoraceae. We have already suggested that the capsular fruit can be regarded as a plesiomorphic character state within the family and that the arillate seeds of Crossos- tylis are derived. For the entire family it is still question- able what seed characters are apomorphic or plesio- morphic. Regarding the evolution of seed appendages there have been two hypotheses. Based on their morpho- logical cladistic analysis (Fig. 1), Juncosa and Tomlinson (1988a) considered that arillate-seeded genera were de- rived from winged-seeded ancestors. In contrast, other authors (Dahlgren, 1988; Tobe and Raven, 1988b) have suggested that the presence of an aril is the plesiomorphic character state. Their argument was based on the fact that all arillate seeds occur in taxa with superior ovaries, which they regarded as the plesiomorphic state. However, this is contradicted by Crossostylis, which has a half- inferior or inferior ovary, but arillate seeds (Juncosa and Tomlinson, 1988a), and by the fact that, without excep- tion in the Rhizophoraceae, winged seeds also co-occur with superior ovaries (Anopyxis, Macarisia, and Sterig- mapetalum). Within the Erythroxylaceae, only Aneulophus has ar- illate seeds and a capsular fruit; the rest of the family has nonappendaged seeds, in either drupes or capsules. Con- cerning the entire family, it is equally parsimonious to assume nonappendaged or appendaged seeds as the ple- siomorphic state. If we look closer at Macariseae, the common ancestor of this tribe very likely had appenda- ged seeds but it is not clear whether wings or arils were present. We have to infer parallel or reversal evolution for either one of the seed appendages, but unfortunately all of the most parsimonious scenarios require the same number of transformations and to date we can only pro- vide ecological data that might favor the hypothesis of arillate seeds being apomorphic. Regarding arillate seeds as apomorphic within this tribe is supported by the fact that some species of the widespread and species-rich ar- illate-seeded genera Cassipourea and Dactylopetalum typically occur in drier habitats. This is clearly a derived feature within Rhizophoraceae, and the arillate seeds of these genera may be adapted to the characteristic seed dispersers of drier environments. More detailed anatom- ical studies might be able to tease apart homoplasious and homologous character states and solve this problem in the future. Evolution of presumably adaptive morphological characters related to mangrove habitats?Tomlinson (1986) listed several apparently adaptive characters, in- cluding aerial (stilt) roots and vivipary, that are associ- ated with living in mangrove habitats. We have examined the distribution of these characters and their order of oc- currence within Rhizophoraceae (Fig. 5). Vivipary?True vivipary is defined by the development of seedlings that remain attached to the parent plant. Thus, seedlings, rather than seeds or fruits, disperse (Elmqvist and Cox, 1996). Members of the tribe Rhizo- phoreae and a seagrass (Amphibolis Schott & Kotschy) are probably the only truly viviparous seed plants (Jun- cosa, 1982). Pseudovivipary sometimes occurs in the form of adventitious plantlets in inflorescences or facul- tative vivipary is found as premature germination in many cultivated fruits, e.g., Citrus L. In the case of cryp- tovivipary, the embryo emerges from the seed coat but not from the fruit before the entire entity is distributed (Carey, 1934; Juncosa, 1982). Cryptovivipary and vivi- pary have apparently evolved in parallel in many unre- lated mangrove genera, such as Pelliciera Planch. & Tri- ana (Pellicieraceae), Avicennia L. (Avicenniaceae), Ae- giceras Gaertn. (Myrsinaceae), Nypa Steck (Arecaceae), and Aegialitis Trin. (Plumbaginaceae). As Tomlinson (1986) pointed out, embryological development of seeds is a continuous process, which for a longer or shorter time period is interrupted by seed dormancy. In vivipa- rous plants this seed dormancy is either absent or very brief. Thus, vivipary represents the extreme of a contin- uum of seed dormancy relative to fruit abscission or de- hiscence. All members of the tribe Rhizophoreae are viviparous (Fig. 5). In Bruguiera, the seedling disperses initially with the fruit (Tomlinson, 1986), whereas in the more derived genera Rhizophora, Kandelia, and Ceriops, only the seedling disperses (Juncosa, 1982, 1984). The isolated position of Bruguiera from other Rhizophoreae is also evident in the phylogenetic tree (Fig. 5). Aerial stilt roots?According to Tomlinson (1986), stilt roots are branched, looping aerial roots that arise from 558 AMERICAN JOURNAL OF BOTANY [Vol. 87 the trunk and lower branches and often stabilize the tree trunk in older individuals. Aerial stilt roots can be found in many tropical plants growing in poorly aerated soils, but especially in swampy and mangrove habitats. All members of the mangrove tribe Rhizophoreae have aerial roots, although Kandelia develops them only in some limiting environments (Tomlinson, 1986). Rhizophora develops the largest and most elaborate stilt roots where- as Ceriops and Bruguiera form smaller stilt roots at the base of the trunk. In older plants of Ceriops and Bru- guiera these separate roots coalesce and form a conical trunk base (Juncosa and Tomlinson, 1988a). Interestingly, some nonmangrove members of the family also have ae- rial stilt roots, suggesting that this character preceded the entry of the Rhizophoreae into coastal habitats. Among the inland genera, at least Carallia brachiata occurring in peat swamps (DingHou, 1958) as well as Crossostylis and Gynotroches of the Gynotrocheae develop aerial stilt roots (Juncosa and Tomlinson, 1988a). Based on the to- pologies found here (Fig. 5), the common ancestor of the Gynotrocheae and Rhizophoreae evolved the capacity to develop aerial stilt roots and stilt roots were lost in par- allel in Pellacalyx and some populations of Kandelia. Conclusions?Our molecular phylogenetic analyses show how DNA sequence data can resolve systematic relationships that are difficult to infer from analysis of morphological data alone due to extensive homoplasy. Despite several excellent morphological and anatomical studies (Dahlgren, 1988; Juncosa and Tobe, 1988; Jun- cosa and Tomlinson, 1988a, b; Keating and Randriana- solo, 1988; Tobe and Raven, 1988a, b; Raven and Tom- linson, 1988; Vezey et al., 1988), it has not been possible to clearly identify the sister groups of Rhizophoraceae and Anisophylleaceae. Our molecular data allow us to reject previously suggested close relationships of the Rhi- zophoraceae with Celastraceae or Elaeocarpaceae and identify the Erythroxylaceae as sister group to the Rhi- zophoraceae and possibly the Cucurbitaceae as sister to the Anisophylleaceae. Although we were not able to ob- tain good support for the immediate relationships of the Anisophylleaceae, their position within Cucurbitales (sensu Bremer, Bremer, and Thulin, 1997; APG, 1998) can be regarded as both stable and surprising. LITERATURE CITED AIRY SHAW, H. K. 1966. J. C. Willis: a dictionary of the flowering plants and ferns, 7th ed. Cambridge University Press, Cambridge, UK. . 1973. J. C. Willis: a dictionary of the flowering plants and ferns, 8th ed. Cambridge University Press, Cambridge, UK. ALBERT, V. A., S. E. WILLIAMS, AND M. W. CHASE. 1992. Carnivorous plants: phylogeny and structural evolution. Science 257: 1491? 1495. ALVERSON, W. S., K. G. KAROL, D. A. BAUM, M. W. CHASE, S. M. 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Amplification and direct sequencing of fungal ribosomal RNA genes for phylo- genetics. In M. Innis, D. Gelfand, J. Sninsky, and T White [eds.], PCR protocols: a guide to methods and applications. Academic Press, San Diego, California, USA. April 2000] SCHWARZBACH AND RlCKLEFS SYSTEMATIC AFFINITIES OF RHIZOPHORACEAE 561 APPENDIX 1. List of species ordered by family for rbcL sequences obtained from GenBank. Species Authors of sequences or literature citation GenBank ace. noa Schinus molle L. Heracleum duluce Fisch. Libanotis coreana (Wolff.) Kitag. Hillebrandia sandwicensis Oliver Begonia herbacea Veil Cleome hasslerianu Chod. Euonymus alatus (Thunberg) Siebold Cercidiphyllum japonicum Siebold & Zucc. Chrysobalanus icaco L. Licania tomentosa Fritsch. Clusia gundlachi Stahl Bucida macrostachiu Standl. Quisqualis indica L. Terminaliu catappa L. Coriariu myrtifolia L. Corynocarpus laevigata Forst. Cucurbita pepo L. Neoalsomitra sarcophylla (Wall.) Hutchinson Bauera rubioides Andr. Datisca cannabina L. Datisca glomerata Baill. Dillenia indica L. Aceratium ferrugineum C. T. White Elaeocarpus grandis F. Muell. Sloanea latifoliu Schum. Erythroxylum confusum Britton Acalypha rhomboidea Rafin. Drypetes roxburghii (Wall.) Hurusawa Euphorbia polychroma A. Kerner Baptisia tinctoria R. Br. Fagus sylvatica L. Erodium texanum A. Gray Hamamelis mollis Oliv. Humiria balsaminifera Aubl. Hypseochuris spec. Remy Lythrum hyssopifolia L. Brysonima crassifolia (L.) Kunth. Rhexia virginica L. Myricianthes frugrans (Sw.) McVaugh Nothofagus alpinu (Poepp. & Endl.) Oerst Ochna multiflora DC. Circaeu alpina L. Fuchsia cyrtandroides J. W Moore Oxalis dillenii Jaqu. Parnassia fimbriata Banks Passiflora quadrangularis L. Platanus occidentalis L. Polygala cruciata L. Rhamnus cathartica L. Spiraea vanhouttei (Briot) Zabel Sabia spec. Colebr. Octomeles sumatrana Miq. Tetrameles nudiflora R. Br. Tilia americana L. Dirca palustris L. Tropaeolum majus L. Pilea pumila (L.) A. Gray Hymenanthera alpina (T. Kirk) W R. B. Oliv. Leonia glycycarpa Ruiz & Par. Vitis aestivulis Torr. Vochysia hondurensis Sprague Anacardiaceae Apiaceae Apiaceae Begoniaceae Begoniaceae Capparaceae Celastraceae Cercidiphyllaceae Chrysobalanceae Chrysobalanaceae Clusiaceae Combretaceae Combretaceae Combretaceae Coriariaceae Corynocarpaceae Cucurbitaceae Cucurbitaceae Cunoniaceae Datiscaceae Datiscaceae Dilleniaceae Elaeocarpaceae Elaeocarpaceae Elaeocarpaceae Erythroxylaceae Euphorbiaceae Euphorbiaceae Euphorbiaceae Fabaceae Fagaceae Geraniaceae Hamamelidaceae Humiriaceae Hypseocharitaceae Lythraceae Malpighiaceae Melastomataceae Myrtaceae Nothofagaceae Ochnaceae Onagraceae Onagraceae Oxalidaceae Parnassiaceae Passifloraceae Platanaceae Polygalaceae Rhamnaceae Rosaceae Sabiaceae Tetramelaceae Tetramelaceae Tiliaceae Thymelaeaceae Tropaeolaceae Urticaceae Violaceae Violaceae Vitaceae Vochysiaceae Gadek et al., 1996 GBAN-U39270 Kondo et al., 1996 GBAN-D44569 Kondo et al., 1996 GBAN-D44573 Swensen, 1996 GBAN-U59822 Swensen, 1996 GBAN-U59816 Rodman et al., 1993 GBAN-M95755 Chase et al., 1993 GBAN-L13184 Olmstead et al., 1992 GBAN-L 11673 Morgan and Soltis, 1993 GBAN-L11178 Morgan and Soltis, 1993 GBAN-L11193 Fay, Swensen, and Chase, 1997 GBAN-Z75673 Conti, Litt, and Sytsma, 1996 GBAN-U26321 Albert, Williams, and Chase, 1992 GBAN-L01948 Conti, Litt, and Sytsma, 1996 GBAN-U26338 Albert, Williams, and Chase, 1992 GBAN-L01897 Martin and Dowd, 1993 GBAN-L28949 Swensen, Mullin, and Chase, 1994 GBAN-L21938 Swensen, 1996 GBAN-U59823 Morgan and Soltis, 1993 GBAN-L11174 Swensen, Mullin, and Chase, 1994 GBAN-L21939 Swensen, Mullin, and Chase, 1994 GBAN-L21940 Albert, Williams, and Chase, 1992 GBAN-L01903 Martin and Dowd, 1993 GBAN-U06838 Martin and Dowd, 1993 GBAN-U06842 Alverson et al., 1998 GBAN-AF022131 Chase et al., 1993 GBAN-L13183 Gunter, Kochert, and Giannasi, 1994 GBAN-U00435 Rodman et al., 1993 GBAN-M95757 Chase et al., 1993 GBAN-L13183 Kass and Wink, 1995 GBAN-Z70120 Martin and Dowd, 1993 GBAN-L13340 Price and Palmer, 1993 GBAN-L 14693 Albert, Williams, and Chase, 1992 GBAN-L01922 Albert, Williams, and Chase, 1992 GBAN-L01926 Price and Palmer, 1993 GBAN-L 14699 Conti, Fischbach, and Sytsma, 1993 GBAN-L10218 Albert, Williams, and Chase, 1992 GBAN-L01892 Conti, Litt, and Sytsma, 1996 GBAN-U26334 Conti, Litt, and Sytsma, 1996 GBAN-U26328 Martin and Dowd, 1993 GBAN-L13342 Fay, Swensen, and Chase, 1997 GBAN-Z75273 Conti, Fischbach, and Sytsma, 1993 GBAN-L 10216 Conti, Fischbach, and Sytsma, 1993 GBAN-L 10220 Albert, Williams, and Chase, 1992 GBAN-L01938 Albert, Williams, and Chase, 1992 GBAN-L01939 Albert, Williams, and Chase, 1992 GBAN-L01940 Albert, Williams, and Chase, 1992 GBAN-L01943 Albert, Williams, and Chase, 1992 GBAN-L01945 Chase et al., 1993 GBAN-L13189 Morgan and Soltis, 1993 GBAN-L 11206 Qiu et al., 1993 GBAN-L 12662 Swensen, Mullin, and Chase, 1994 GBAN-L21942 Swensen, Mullin, and Chase, 1994 GBAN-L21943 Chase et al., 1993 GBAN-AF022127 Conti, Litt, and Sytsma, 1996 GBAN-U26322 Price and Palmer, 1993 GBAN-L 14706 Gunter, Kochert, and Giannasi, 1994 GBAN-U00438 Fay, Swensen, and Chase, 1997 GBAN-Z75692 Fay, Swensen, and Chase, 1997 GBAN-Z75693 Albert, Williams, and Chase, 1992 GBAN-L01960 Conti, Litt, and Sytsma, 1996 GBAN-U26340 a The prefix GBAN- has been added to link the online version of the American Journal of Botany to GenBank but is not part of the actual accession number. 562 AMERICAN JOURNAL OF BOTANY [Vol. 87 0. 00 < S ?c s til s 0 o o o o OH PL, ^ < < z z ZZ < < < < m pq pq co 0 0 0 0 OHNn^tmn! t- O r- s ? CN ^ 2 A i oo pfl < 13 PL] > P pq - j to a OB'S ? to c 00 (M n "* ?-? .S,6llS ?rt ?a 00 00 =0 .3 ^ -H 3 ??? 2 >H o o J? < 5 a ? 00 . 3 00 PLl r-H ?3? J3 U 00 ^ s X c o 3 ? 3 X a PL, I TJ g 6 e a . _ c e 2-.2 O ? 6 5 S 1 a, u 52 s go a -3 a. S p 5- 3^^ s y y &- ^ JH ^ cd 3" i su u S " *! *! ^ ^- ^ 13 w ^j fen. ? S 2 2 -? '3 0 0 o .2 ?S- is 8 -o 00 G 3 ^ 3 a Pi E ^ % * N &^<>. o o o PL PL PL ZZ z << < PQ PQ PQ 0 0 0 -^ (N o o PL PL ZZ PQ PQ o o LL < z < pa O ^miopcooiO^Nn'tviio HHHrHH^^MlSrJOllNM P~P~P~P-P-P-P-P-P-P-P-P-P- P-P-P-P-P-P-P-P-P-P-P-P-P- (NfN(NfN(NfN(NfN(NfN(NfN(N PLpL]pL]pL]pL]pL]pL]pL]pL]pL]pL]pLpL] zzzzzzzzzzzzz PQPQPQPQPQPQPQPQPQPQPQPQPQ 0000000000000 MOio^(N^Ttvi^p?a,o ^?^inininviin^inininvi^ p~p~p-p-r-r-r-r-r-r-r-r-r- P-P-P-P-P-P-P-P-P-P-P-P-P- (NfN(NfN(NfN(NfN(NfN(NfN(N PLpL]pL]pL]pL]pL]pL]pL]pL]pL]pL]pLpL] ZZZZZZZZZZZZZ PQPQPQPQPQPQPQPQPQPQPQPQPQ 0000000000000 m^i/l\OPOOO\O^Mm^i/l ooooooooooooooo\a\0\a\0\a\ p~p~p~p-p-p-p-p-p-p~p~p~p- (NfN(NfN(NfN(NfN(NfN(NfN(N PLpL]pL]pL]pL]pL]pL]pL]pL]pL]pL]pLpL] ZZZZZZZZZZZZZ PQPQPQPQPQPQPQPQPQPQPQPQPQ 0000000000000 So' -t -.00 05 5 < " o ^p< 0.? OS s LC < SO G 4= =352^ 00 Pi 2 ' O .a .3 u (H Pi P< Pi ^ 3 E 01)5 3 ^ o (S 00 ^ oo % 3 M >-i 00 R 0% o Pi pa u 1 o S w 00 PQ H s o pq O JJ2 o ZZ z 3 << < 0 PQ PQ PQ cs 0 0 0 ?& rt rt 3 O pq 5 J O O PQ o o S 00 pq -a . e oas . a 3 t S jo a a a a a a ?\ ?% > a 5 a a O O S3 o b 3 r3 O a o 3 j ^ j a ,^ ^ "S "3 "S O O O a, ft, a. o p-a o c N ,3 ? O 0 O 0 O 0 SeC I as a c 3 a :"Q< %i ^i %i ^i %i S '3 '3 '3 '3 cq cq cq cq cq o .a .% e e o H April 2000] SCHWARZBACH AND RlCKLEFS SYSTEMATIC AFFINITIES OF RHIZOPHORACEAE 563 APPENDIX 3. Morphological data matrix. Characters Taxon- 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 DRYdep 0 0 1 0 ? 0 0 ? 0 1 3 3 2 0 ANEafr 0 0 0 0 ? 1 1 ? 0 2 3 2 0 0 NECkae 0 0 1 0 ? 0 1 ? 0 2 3 2 0 0 PINcon 0 0 1 0 ? 0 1 ? 0 2 3 2 0 0 ERYarg 0 0 1 0 ? 0 1 ? 0 2 3 2 0 0 STEgui 0 0 0 0 0 1 0 0 0 1 0 0 0 0 ANOkla 0 0 0 2 0 0 0 0 0 2 0 1 0 0 CASell 0 0 0 0 0 0 0 0 0 2 0 0 0 0 CASgui 0 0 0 0 0 0 0 0 0 2 0 0 0 0 CAScey 0 0 0 0 0 0 0 0 0 2 0 0 0 0 MACspe 0 0 0 1 & 2 0 0 & 1 0 0 0 2 0 0 0 0 DACver 0 0 0 0 0 0 0 0 1 2 0 0 0 0 DACbar 0 0 0 0 0 0 0 0 1 2 0 0 0 0 CARbra 1 1 0 0 1 0 0 0 2 1 0 1 0 CAReug 0 1 0 0 1 1 0 0 2 1 0 1 0 GYNaxi 1 1 0 1 1 1 0 1 0& 2 2 0 0 0 CRObif 1 1 0 0 1 1 0 0 2 1 1 0 1 PELsac 0 1 0 2 0 0 & 1 0 1 2 2 0 0 0 PELaxi 0 1 0 2 0 0 & 1 0 1 2 2 0 0 0 PELlob 0 1 0 2 0 0 & 1 0 1 2 2 0 0 0 KANcan 1 1 0 0 1 1 0 2 0 0 1 1 CERaus 1 1 0 0 1 1 1 2 0 0 1 0 CERtag 1 1 0 0 1 1 1 2 0 0 1 0 RHIapi 1 1 0 0 1 1 0 2 0 2 0 0 RHIsty 1 1 0 0 1 1 0 2 0 2 0 0 RHImuc 1 1 0 0 1 1 0 2 0 2 0 0 RHImanl86 1 1 0 0 1 1 0 2 0 2 0 0 RHImanl71 1 1 0 0 1 1 0 2 0 2 0 0 RHIrac 1 1 0 0 1 1 0 2 0 2 0 0 BRUsex 1 1 0 0 1 1 2 2 0 0 1 0 BRUpar 1 1 0 0 1 1 0 2 0 0 1 0 BRUgym 1 1 0 0 1 1 2 2 0 0 1 0 BRUcyl 1 1 0 0 1 1 0 2 0 0 1 0 BRUexa 1 1 0 0 1 1 2 2 0 0 1 0 a Names abbreviated, first three capital letters stand for genus, last three small letters for species. Character legend: (1) stilt roots: 0 absent, 1 present; (2) root hairs: 0 present, 1: absent; (3) phyllotaxy: 0 opposite, 1 alternate; (4) leaf venation: 0 brachididromous, 1 eucamptodromous, 2 intermediate; (5) stipule vernation: 0 valvate, 1 imbricate; (6) stipule morphology: 0: intrapetiolar, 1 interpetiolar; (7) leaf margin: 0 serrate, 1 entire; (8) hypodermis: 0 absent, 1 present; (9) salt tolerant: 0 no, 1 yes; (10) inflorescence: 0 open-branched, 1 fasciculate, 2 solitary; (11) breeding system: 0 monoecious, 1 dioecious, 2 hermaphroditic; (12) floral laticifers: 0 one layer, 1 radially expanded, 2 idioblastic, 3 none; (13) lateral fringed petal appendages: 0 present, 1 abort, 2 absent, 3 no petals; (14) petal orientation: 0 reflexed, 1 erect, 2 no petals; (15) androecium: 0 diplostemonous, 1 polyandrous; (16) hypanthium: 0 absent, 1 present; (17) ovary position: 0 superior, 1 half-inferior, 2 inferior; (18) carpels: 0 five carpels, 1 three-many carpels, 2 two carpels, 3 three carpels; (19) ovules per locule: 0 two ovules, 1 five-eight, 2 one ovule; (20) nucellus: 0 crassinucellate, 1 tenuinucellate; (21) integument: 0 not vascularized, 1 vascularized; (22) seeds per fruit: 0 several to many, 1 one seed, 2 two seeds, 3 three seeds; (23) fruit-seed appendage type: 0 capsule-aril, 1 baccate-none, 2 capsule-wing, 3 capsule-none, 4 drupe-none, 5 indehiscent hard-walled-none; (24) cotyledons: 0 separate, 1 connate; (25) viviparous: 0 no, 1 yes; (26) germination process: 0 by hypocotyl, 1 by endosperm expansion; (27) seedling establishment: 0 by radicle, 1 by lateral roots; (28) sepals: 0 ten, 1 eleven, 2 twelve, 3 thirteen, 4 four, 5 five, 6 six, 7 seven, 8 eight, 9 fourteen; (29) trichomes: 0 stellate, 1 others; (30) stamen tube: 0 absent, 1 present. 564 APPENDIX 3. Extended. AMERICAN JOURNAL OF BOTANY [Vol. 87 Characters 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 0 0 1 0 0 ? 1 4 0 0 0 0 4 & 5 0 0 0 3 0 0 ? 3 0 0 0 0 0 5 1 0 0 2 2 0 ? 2 3 0 0 0 0 5 1 0 0 2 2 0 ? 2 3 0 0 0 0 5 1 0 0 3 2 0 ? 1 4 0 0 0 0 5 1 0 0 0 0 0 0 0 2 0 0 0 0 5 0 0 0 0 0 0 0 0 2 0 0 0 0 5 0 0 0 1 0 0 0 0 0 0 0 0 0 4 & 5 0 0 0 1 0 0 0 0 0 0 0 0 0 4 & 5 0 0 0 1 0 0 0 0 0 0 0 0 0 5 0 0 0 0 0 0 0 0 2 0 0 0 0 5 0 0 0 2 0 0 0 0 0 0 0 0 0 6 & 7 0 0 0 2 0 0 0 0 0 0 0 0 0 5 & 6 0 2 0 0 0 0 0 1 0 0 0 0 4&S&6&7&8 0 2 0 0 0 0 0 1 0 0 0 0 5 0 0 0 1 1 0 0 1 0 0 0 0 4 & 5 0 1 0 0 0 0 0 0 0 0 0 4 0 1 1 1 0 0 1 0 0 0 0 4 0 0 1 1 1 0 0 1 0 0 0 0 5 0 0 1 1 1 0 0 1 0 0 0 0 4 0 0 2 0 0 1 5 1 5 & 6 0 2 0 0 1 5 1 5 0 2 0 0 1 5 1 5 0 2 2 0 0 1 5 1 4 0 2 2 0 0 1 5 1 4 0 2 2 0 0 1 5 1 4 0 2 2 0 0 1 5 1 4 0 2 2 0 0 1 5 1 4 0 2 2 0 0 1 5 1 4 0 2 0 0 1 5 0 0 0 0& 1 & 2 0 2 0 0 1 5 0 0 0 8 0 2 0 0 1 5 0 0 0 0&1&2&3&9 0 2 0 0 1 5 0 0 0 8 0 2 0 0 1 5 0 0 0 8 0