--Plant Pl. Syst. Evol. 210:249-270 (1998) Systematics and Evolution © Springer-Verlag 1998 Printed in Austria The Ulmaceae, one family or two? Evidence from chloroplast DNA restriction site mapping SUSAN J. W~EGREFE, KENNETH J. SYTSMA, and RAYMOND P. GURmS Received October 22, 1996; in revised version January 22, 1997 Key words: Ulmaceae, Celtidaceae, Urticales, Cannabis.- Chloroplast DNA, cladistics, restriction site mapping. Abstraet: The Ulmaceae is usually split into two subgroups, referred to as either tribes or more commonly subfamilies (Ulmoideae and Celtidoideae). The two groups are separated, with some exceptions, on the basis of leaf venation, fruit type, seed morphology, wood anatomy, palynology, chemistry, and chromosome number. Proposifions to separate the two groups as distinct families have never gained general acceptance. Recent morphological and anatomical data have suggested, however, that not only is family status warranted but that Celtidaceae are more closely related to Moraceae and other Urticales than to Ulmaceae. In order to test these alternative sets of relationships, restriction site mapping of the enfire cpDNA was done with nine rare cutting enzymes using 11 genera of Ulmaceae s. 1., three other families of the Urticales, and an outgroup family from the Hamamelidae. Cladistic analysis of the data indicates that Ulmaceae s. 1. is not monophyletic and that distinct families (Ulmaceae and Celtidaceae) are warranted; that Ulmaceae is the sister group to Celtidaceae plus all other families in the order; and that Cannabaceae might be nested within Celtidaceae. Familial placements of vafious problemafic genera (e.g. Ampelocera, Aphananthe) are resolved and charäcter evolution of key morphological, anatomical, chemical, and chrornosomal features are discussed. The elm family, Ulmaceae MIRBEL, as originally described contained two genera, Ulmus L. and Celtis L. (MmBEL 1815). Subsequently, the number of genera included in the family has ranged as high as 18 (CRONQUIST 1981), although 15 (Table 1) are now recognized (MANCHESTER 1989, OGrNUMA & al. 1990, OMORI & TERABAYASHI 1993, TAV~HAS~ 1989, TERABAYASHI 1991). The family is distributed throughout the temperate and tropical regions of the world in the form of trees, shrubs, or lianas. Two subgroups, associated with each of the original genera, traditionally have been recognized and referred to as subfamilies (Ulmoideae and Celtidoideae: ENGLER c% PRANTL 1893, THORNE 1968, CRONQUIST 1988) or some- fimes tribes (HUTCH1NSON 1967). Typical members of the two groups (hereafter referred to, in a taxonomically neutral fashion, as the "ulmoids" and "celtoids," respectively) differ in an impressive list of characters (summarized in SWEITZER 1971, CRONQUIST 1981, JUDD & al. 1994). The ulmoids are generally separated from 250 S. J. W~EG~~ & al.: O~ ..~ ¢.,) ~.) ~ O .~ ..= ~<~ O O O © ¢..) I O tt~ r,,,~ r,,,~ v..~ v,,,~ tl') O UZ xA~ oo 7 ~ I O~ © ~3 II II i © ==r~3 eD Circumscription of Ulmaceae s. 1., cpDNA evidence 251 the celtoids based on leaves strictly pinnately veined or craspedodromous vs. leaves pinnipalmately veined or brochidodromous (GRUDZINSKAYA 1967); on generally bisexual vs. generally unisexual flowers (GRUDZINSKAYA 1967); on fruit dry and commonly a samara vs. fruit drupaceous (GRUDZINSKAYA 1967; CHERNICK 1980); on seeds flattened, with straight embryo vs. seeds globose, with a curved embryo (GRUDZ~NStC~Ya 1967); pollen 4-5 porate vs. pollen 2-3 porate (KuPRIANOVA 1962, TAKAHASHI1989); and chromosome number n = 14 vs. n = 10 or 11 (MEHRA & GILL 1974, RAVEN 1975, OGINUMA & al. 1990). As SWErTZER (1971) has pointed out, however, the placement of some genera can be debated because not all characters unambiguously place these genera into one or the other group. Early interpretations of subgroup affiliations with fewer recognized genera (PLANCHON 1873, ENCL~R & PRANTL 1893) were consistent in their assignment of Ulmus, Planera J. E GMEL., and Holoptelea PLANCH. to the Ulmoideae, and Celtis, Chaetachme PLANCH., and others to the Celtidoideae; they differed primarily on the assignment of Zelkova SPACH within the family (Table 2). More recent interpretations (e.g. GRUDZINSKAYA1967, HUTCHINSON 1967, SWEITZER 1971, GIANNASI 1978), with larger numbers of genera included in each subfamily, Table 2. Ulmaceae s. 1. subfamily compositions according to various taxonomists: PLANCnON (1873), ENCLER & PRANTL (1893), HUTCmNSON(1967), GRUDZINSKAYA(1967), SWE~TZER (1971), and GIANNASI (1978); and based on cpDNA (current study). Symbols: U Ulmoideae; C Celtidoideae; - not included in classification or study. 1GRuDZINSKAYA included Chaetoptelea within Ulmus. 2WmcREW & al. (1994) show with cpDNA restriction sites that Chaetoptelea is included within Ulmus. 3pLANCHON(1873) included Trema under the name of Sponia. 4 Based on rbcL sequence evidence (SYTSMA& al. 1996) Genus PLANCHON E. & E HUTCHIN GRUDZ SWEITZER GIANNASI cpDNA Son inskaya Ampelocera -- C C C C U U Aphananthe C C C C C U C Celtis C C C C C C C Chaetachme C C C C C C -- Chaetoptelea -- __ __ 1 U U U 2 U Gironniera C C C C C U/C 4 C Hemiptelea U -- C U C U U Holoptelea U U U U U U U Lozanella -- -- C C C C C Mirandaceltis -- -- C -- C U -- Parasponia C C C C C C 4 C Phyllostylon -- U U U U U -- Plagioceltis .... C C -- Planera U U U U U U U Pteroceltis -- C C C C C C Trema 3 C C C C C C C Ulmus U U U U U U U Zelkova U C C U C U U 252 S.J. Wm~~FE &al.: are offen discordant (Table 2). The problem of seemingly parallel evolution in the key morphological characters used to separate the two subfamilies is exemplified by Zelkova. The genus was originally placed in the celtoid group based on its drupaceous fruits, but is more similar to the ulmoids based on other characters, especially its strictly pinnate veined leaves. The division of the Ulmaceae into separate families (Ulmaceae and Celtidaceae LIyK) was first proposed by LINS: (1831) over a century and a half ago, but never gained general acceptance. Opponents of the two family classification (e.g. SWEITZZR 1971, CROYQUISr 1981, 1988) cited the existence of "intermediate" genera and the small size of each of the resultant families as deterrents to the split, because it was assumed that the two families would be sister groups to each other (CRONQUIST 1988). However, GRUDZINSKAYA(1967) proposed that not only are the two subgroups sufficienfly distinct to warrant elevation to the rank of separate families, but more importantly that each of the two groups had been imprecisely characterized and that the Celtidaceae were actually more closely related to the Moraceae LINK than to the Ulmaceae. A number of the similarities (pollen shape and pore number, carpel number, gynoecial vasculature, and curved embryos) shared between the Celtidaceae and the other families of Urticales (excluding Ulmaceae) are likely synapomorphic (GRUDZINSKAYA1967, JuDo & al. 1994, OMORI & TERABAYASHI1993), thus placing Ulmaceae basal in the order and the sister group to the rest of the order. Her two family proposition has been supported by two recent preliminary cladistic analyses. The morphological analysis of the order Urticales (JUDO & al. 1994) and the rbcL analysis of Juglandaceae A. RICHARD EX KUNTH and relatives in Hamamelidae (GUNTERH al. 1994) both place Ulmus basal in the Urticales and Celtis as the sister group to the remainder of the order. However, only Ulmus and Celtis were sampled from the Ulmaceae s. 1. in both studies. An independent data set analyzed cladistically is needed to re-evaluate these morphological, anatomical, and chemical data and to determine whether individual characters are synapomorphies, symplesiomorphies, or possibly parallelisms. We employed detailed restfiction site mapping tecbniques of chloroplast DNA (cpDNA) to determine phylogenetic relationships within and among the ulmoids, celtoids, and other families of Urticales. The conservative rate of cpDNA evolution, the precision provided by restriction endonuclease site mapping (PALMER & al. 1988, OLMSTEADH PALMER 1994), and the lower levels of homoplasy seen in cpDNA restriction site studies (GIVNISH & SYTSMA 1997a, b) make this approach useful for producing an independent data source for assessing phylogenetic relationships, cpDNA analysis has proven useful in examining relationships among species and genera (see SYTSMA H HAHN 1994, 1996 for recent reviews), but few cpDNA studies have been done at the familial and ordinal levels (e.g. JANSENH al. 1992, OLMSTEAD H SWEERE 1994). Specific questions addressed were: (1) What is the genefic composifion of the ulmoid and celtoid groups? Does the molecular evidence support the various morphological, anatomical, palynological, and chemical results in terms of group composition? (2) What is the nature of the relafionsbip between the two groups? Are they one monophyletic lineage, is one paraphylefic relative to the other (both suggesting retention of Ulmaceae in the broader sense), or are they distinct and unrelated lineages (suggesting the Circumscription of Ulmaceae s. 1., cpDNA evidence 253 recognition of Ulmaceae and Celtidaceae)? (3) What are the relationships of the ulmoids and celtoids to the other families in Urticales? Are the ulmoids basal in the order and the celtoids more closely related to the Moraceae or other families (e.g., Cannabaceae, Urticaceae)? This restriction site mapping study was designed primarily to address questions 1 and 2, but with enough sampling in the order to gain insight into the relationships of the ulmoids and celtoids to each other and to other families. A subsequent analysis based on rbcL sequencing throughout the Urticales addresses the latter question in more detail (SVTSMA& al. 1996). Materials and methods DNA extraction and restriction site mapping. Eleven of the 15 genera of Ulmaceae s. 1., representatives of three other families in the Urticales (Moraceae, Urticaceae A. L. DE Jusslzu, and Cannabaceae E~LICI-mR), and a representative from the order Hamamelidales as a remote outgroup were collected (Table 3). Total cellular DNAs were extracted from Table 3. Ulmaceae s. 1. and outgroup genera examined, source and voucher information.