Ulmaceae, One Family Or Two? Evidence from Chloroplast DNA Restriction Site Mapping

Home , Ampelocera, Ampelocera hottlei, Aphananthe, Aphananthe aspera, Cannabaceae, Chaetachme

--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.:

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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. Classification follows CRONQr:IST (1988) Taxon Source Voucher

Ulmaceae (Urticales) 1. Ulmus thomasii SARg. cult.; UW-Madison, Arlington Research S. WIEGREFE74 (WIS) Station, Dane County, WI 2. Zelkova serrata (THvt,m.) MAK. cult.; Morton Arboretum, Lisle, IL. S. WmaR~ZE 195 (WIS) 3. Hemiptelea davidii PLANCH. cult.; UW-Madison campus, WI S. WmGP,EFE 82 (WIS) (W#316-1; S. Korea) 4. Planera aquatica (WALT.) J. E GMEL. wild; SC S. WIEOREFE& T. ENDO 138 (WIS) 5. HolopteIea integrifolia PLANCH. cult.; Fairchild Tropical Gardens, W. T. GmLIS 9235, 10335 (Fr) Miami, FL (PI#116039) 6. Aphananthe aspera (TmJNB.) PLANCH. cult.; UW-Madison, Forest Genetics S. WmaRE~ 176 (WIS) greenhouses, WI (W#2063; Japan) 7. Pteroceltis tatarinowii MAXIM. cult.; Morris Arboretum of the S. Wn~GP,ZFE & H. University of Pennsylvania, STONEI-nLL126 (WIS) Philadelphia, PA (MA#1366) 8. Celtis occidentalis L. cult.; UW-Madison campus, WI S. Wm~RêVZ 80 (WIS) 9. Trema micranthe BLUME wild; Naolinco, Veracruz, Mexico S. WIEGREFE& G. CASTmLOC. 133 (WIS) 10. LozaneIla enantiophylla wild; S. R Coapan, Veracruz, Mexico S. WmaP,Z~ & G. CASTILLOC. (DoNN. SM/TH) KILLIP~¢ MORTON 132 (WIS) 11. Ampelocera hottlei (STANDL.) STANDL. wild; "Los Tuxtlas" botanical garden, S. WmGREW & al. 137 (WIS) San Andreas Tuxtlas, Veracruz, Mexico Urticaceae (Urticales) 12. Boehmeria nivea (L.) GAUDICH. cult.; UW-Madison greenhouses, WI S. WIE~REW 183 (WIS) Moraceae (Urticales) 13. Morus alba L. naturalized; UW-Madison campus,WI S. WmaREFE 172 (WIS) Cannabaceae (Urticales) 14. Cannabis sativa L. namralized; Sauk County, WI S. WnaGRE~ 178 (WIS) Hamamelidaceae (Hamamelidales) 15. Hamamelis virginiana L. cult.; UW-Madison campus, WI S. WIEGREFE90 (WIS) 254 S.J. WtEOREFE & al.:

leaf tissue using a modified CTAB protocol (SMITH • al. 1992). Details of the necessary steps required to obtain high quality DNAs for restriction site analysis in Ulmaceae are provided in WlZGI~~ & al. (1994). Detailed restriction site mapping was conducted in order to improve detection and verification of homologous mutations. Nine restficfion endonucleases with six base recognition sequences were used: PstI, KpnI, SmaI, SalI, XhoI, SphI, PvulI, SstI, and BgII. Each restricfion endonuclease was used singly and doubly with at least two other restricfion endonucleases (usually PstI and Sstl) to permit the inference of restriction site locations. In this procedure, each lane containing singly digested DNA was positioned between lanes of doubly digested DNA to aid in the interpretation of resulting restricfion fragments. The DNA restriction fragments were size-separated by electrophor- esis in 0.9% horizontal agarose gels. Fragment transfers, nick translation and hybridizafion were performed as described in SYTSMA & SCHAAL(1985). For the large and small single copy regions of cpDNA, Nicotiana L. (Solanaceae) probes (OLMsTEAD & PALMER 1992) were used due to their small size and thus greater facility in interpretation of fragment patterns when smaller portions of the genome are sampled with each probing. Up to three tobacco clones were combined per probing of the large single copy region of the chloroplast genome, whereas the small single copy region was divided into three probings. The inverted repeat was divided into two probings, one using Petunia (Solanaceae) clones PstI 12 and PstI 14 (SYTSMA & GOTTLmB 1986), and the other using Lactuca (Asteraceae) clones PstI / SstI 6.2, SstI 3.5 and SstI 1.8 (JANSEN & PALMER 1988). Restriction site locations for Ulmus thomasii SARO. were mapped and given a co- ordinate location relative to the SmaI site just inside the inverted repeat from the large single copy region. This site (orte on each side of the inverted repeat) is conserved in many taxonomic groups and provides a convenient reference point on the circular molecule (cut site = nucleotide position 87503 and 155025 in Nicotiana cpDNA; SH~OZAVa& al. 1986). The restriction sites in the other taxa were also mapped and compared to this Ulmus map. Sites within 0.3 kb of a reference site were considered homologous barring other evidence to the contrary. The presence or absence of all sites were coded and entered in MacClade 3.0 (MAI)DISON & MADDISON 1992). Phylogenefie analyses. The most parsimonious cpDNA trees were identified using PAUP version 3.1.1 (SwoFFORD 1993) using a Mac Power PC. Both Wagner parsimony (FARedS 1970) and several character-state weighting schemes (ALBERT & al. 1992) were employed. Wagner parsimony was done with the branch-and-bound option. The character- state weighting schemes used values from 1.05 to 2.0 (gains : losses) to bracket the values estimated by AL~ERT & al. (1992) as appropriate for cpDNA studies at this taxonomic level. Their suggested weights are 1.3 for "low level" analyses (e.g. interspecific and/or inter- generic) and 2.0 for "high level" analyses (e.g. up to divisions). Each weighting analysis was done with a heuristic option (50 random addition sequences, TBR branch swapping, MULPARS and steepest descent on). Two sets of weighted analyses were employed: one specifying no aneestral conditions for each site (ALBERT& al. 1992) and one specifying the ancestral conditions as all site absences (HoLsTNaER & JANSEN 1993). The amount of phylogenetic information in the dataset was estimated using the consistency index (CI; KLUaE & FARR~S 1969) and retention index (RI; FARPaS 1989). This last measure was estimated from 10000 random trees using the RANDOM TREES option in PAUE Character support for monophyly of specific groups was evaluated using bootstrap analysis (FELSENSTEIN1985, SANDERSON 1989) and decay analysis (BREMER 1988, SMITH Æ SYTSMA 1990, DONOGHUE& al. 1992). Bootstrap analysis (using phylogenetically infor- mativ~ characters only) was conducted in PAUP using one thousand replicafions, each involving the same heurisfic search described above. Decay values were obtained by Circumscription of Ulmaceae s. 1., cpDNA evidence 255 relaxing parsimony by four steps (the limit at which all trees could be effectively analyzed due to computer memory limitations), filtering out trees at one-step shorter intervals from the resulting set of trees, and determining how many extra steps were required to lose monophyletic groups. The decay values for all branches maintained after relaxing parsimony by four steps were determined by using topological constraints and searching strategies described in SwoFmm~ (1993) and as implemented in BAUM & al. (1994). Rooting was determined by the outgroup method (WAT~OUS & WREEIùER 1981) using Hamamelis virginiana L. (Hamamelidaceae R. BaowN) designated as the root. At the time this study was initiated, the closest relatives of the order Urticales were still being debated vigorously (BE~a 1977, CRONQt~ST1981, DA~Lam~N 1983, T~om',rE 1983, JuDI~ & al. 1994), but included Hamamelidae (sensu CRONQUrST 1981), Malvales, and Euphorbiales. Later analyses based on rbcL sequencing (CHAsE & al. 1993, GUNTER& al. 1994, SVTSMA& al. 1996) and morphological data (Hu~voRD 1992) suggest that Rhamnaceae A. L. DE JUSSlEU, Barbeyaceae RENDI~E, and Rosaceae A. L. DE JVSSIEUare now better candidates. Alternative topologies suggested by previous studies and the number of extra steps they require were examined using MacClade 3.0 (MADDISON~; MADDISON 1992) and by invoking topological constraints in PAUP. Maximum parsimony has been shown to perform poorly in certain situations such as deep phylogenetic branching events (FEI~SENSTEIN1978) or when rates of substitution and/or levels of homoplasy are relatively high, particularly when multiple substitutions at a single nucleotide site occur (e.g. NEI & TAJn~A 1985). Although only rarely cutting restriction endonucleases (for cpDNA) were mapped in this study to reduce the chance of comparing nonhomologous sites, the great taxonomic breadth covered in this analysis makes multiple substitutions of nucleotides likely and suggested the need for an alternative evaluation of the dataset. Phylogenetic reconstructions were also made using the Neighbor-joining method (SAITOU & NEI 1987) implemented in PHYLIP 3.4 (FELsEYsTE~~ 1992). Distance matrices were calculated based on the algorithms of NEI & LI (1979).

Results A total of 102 cleavage sites (excluding one copy of each site found within the inverted repeat) were detected for the nine restriction enzymes in the chloroplast genome of Ulmus thomasii (Table 4) which was found to be approximately 160 kb in length and collinear with the chloroplast genomes of Petunia and Nicotiana. The presence or absence of these 102 sites in U. thomasii and 166 additional mapped restriction sites seen in other taxa were scored, where possible, for the 15 taxa (Appendix 1). Of the 268 total sites mapped across all taxa, 29 were invariant. An additional 121 sites were variable but phylogenetically uninformative, leaving 118 sites that were phylogenetically informative. Under Wagner parsimony, three shortest trees were found at 347 steps (226 steps without autapomorphies). These trees had consistency (CI) values of 0.689 (0.522 with autapomorphies excluded) and a retention index of 0.671. The CI values are seemingly low for molecular studies with 15 taxa (SANDERSON & DONOGH~ 1989, DONOCmJE & SANDERSON 1992), but lower CI values are also correlated with broader taxonomic sampling as in this study (GIvNIsH & SYTSMA 1997b). The three trees differ in the placement of three genera of ulmoids. Depicted in Fig. 1 is the least resolved tree of the three with a polytomy involving Ulmus, Zelkova, and Planera. The alternative topologies place Zelkova as the 256 S.J. Wm~REFZ & al.:

Table 4. Restriction site positions (in kb) for the 160.1 kb chloroplast genome of Ulmus thomasii. The 102 sites (counting sites within the inverted repeat only once) are listed relative to the more interior of two generally conserved SmaI sites (0.7 kb apart) in the inverted repeat, although U. thomasii has lost the site (i.e. these two sites are at positions 0.0 and 0.7). For reference, the starting site is found in Nicotiana tabacum at positions 87501-87506 (SHrNOZArd & al. 1986). Numbering proceeds through the large single copy region starting on the side nearest rbcL. Restriction sites in bold are located within the inverted repeat; corresponding sites from each side of the inverted repeat are identified by letter for each restriction enzyme. Only one of the two corresponding sites was included in the phylogenetic analyses PstI KpnI SmaI SalI XhoI SphI Ssd Bgl PvulI 2.2 8.3 0.7 a 4.5 2.3 1.8 2.3 4:7 2.2 5.1 12.0 48.5 5.8 5.8 27.9 22.9 8.7 33.9 30.6 21.5 61.1 6.4 8.9 30.5 32.4 25.3 38.2 51.6 36.3 87.4 18.1 36.8 46.1 47.5 41.4 43.4 52.6 66.8 90.3 a 25.7 44.6 51.7 51.9 45.8 51.7 68.1 71.0 107.0 b 38.9 63.7 55.8 63.9 98.2 a 71.7 73.9 71.6 111.4 c 78.4 70.8 64.4 67.7 104.9 b 85.1 77.1 76.0 113.40 98.4 a 82.7 105.7 a 75.8 109.5 c 88.3 88.1 110.6 a 137.7 d 100.8 b 87.1 108.7 b 82.8 141.6 c 105.8 a 98.2 a 111.4 b 139.7 c 150.3 b 92.3 a 120.7 85.1 146.2 b 108,5 b 102.8 b 120.4 144.1 b 152.7 a 92.6 b 142.4 b 108.7 a 152.9 a 112.7 e 123.0 131.2 95.8 ~ 145.4 a 110.3 b 138.4 c 128.6 139.7 b 107.9 ° 112.3 c 142.6 b 148.3 b 140.5 a 108.8 e 114.40 145.3 a 152.9 a 112.3 f 115.0 e 123.1 132.6 138.8 f 136.1 e 142.3 e 136.7 d 143.2 d 138.8 c 155.3 ~ 140.8 b 158.5 b 142.4 a 158.8 ~

sister group to Ulmus and Planera, or as sister to only Planera. The tree in Fig. 1 thus also represents the strict consensus of the three most parsimonious Wagner trees. The Wagner trees separate the ulmoids as the sister group to the rest of the Urticales. The celtoids are placed within the remainder of the order. The separation of these two clades within the Urticales is one of the strongest inferences that can be made from these decay and bootstrap values (Fig. 1), and lends supports to the two family (Ulmaceae and Celtidaceae) proposition. Four extra steps are required to force the celtoids and ulmoids together as a basal paraphyletic Ulmaceae s. 1. (i.e. still containing Cannabis; see below), and 13 extra steps to form a monophyletic Ulmaceae s. 1. The problematic genera Zelkova and Ampelocera Circumscription of Ulmaceae s. 1., cpDNA evidence 257

.... L,_ Cannabaceae

Celtidaceae

I Celtidaceae i'" Cannabaceae

: ...... Morus Urticaceae

; ...... Boehmeria Moraceae

Ulmaceae

...... Hamamelidaceae

Fig. 1. Consensus phylogram of three most parsimonious Wagner trees of the Ulmaceae and other Urticales using mapped restriction sites in the chloroplast genome. One of the three trees is identical to this consensus tree and the alternative topologies place Zelkova as the sister group to Ulmus plus Planera or as sister to only Planera. Numbers above the branches refer to the range of site mutations using both accelerated and delayed transformation optimality criteria. Numbers below the branches refer to bootstrap percentages and decay values. The bootstrap majority rule tree differs from the strict consensus tree in placing Morus, rather than Boehmeria, as sister to the Celtidaceae; this arrangement and its bootstrap support are indicated by dashed lines. The circumscription of the Ulmaceae (= Utmoideae) and Celtidaceae (= Cehidoideae) are indicated by grey overlays. The Celtidaceae is paraphyletic with Cannabis derived from within the family 258 S.J. WIEaREFE & al.:

KLOTZSCH are solidly within the ulmoid clade; likewise, Aphananthe PLANCH. is firmly within the celtoid clade. The Cannabaceae, represented here only by Cannabis L., is strongly nested within the celtoid clade, a surprising and never before suggested relationship. A bootstrap support of 94% is seen for the clade containing Cannabis and two genera of celtoids. Six extra steps are required to move Cannabis out of the entire celtoid clade. Character-state weighting (ancestral conditions not specified) using gain to loss ratios up to 1.05 provides a single most parsimonious tree equivalent to the stfict Wagner tree (Fig. 1). At weights up to 1.4 a single tree (not shown) is generated that is basically similar to the strict Wagner tree. Morus and Boehmeria switch places and Lozanella shifts from its association with Aphananthe to Trema; this tree is found in the set of trees one step longer than the three shortest trees under Wagner parsimony. Character-state weighting using ancestral condifions specified as absent, generated one tree at all weights up to 1.4, this tree being equivalent to that found between 1.05 and 1.4 weighting when no ancestral condifions are specified. At extreme weights of 1.5 to 2.0 (using either ancestral condition), major differences are seen relative to the Wagner trees. In these trees (not shown), Moraceae and Urticaceae individually are the sister groups to portions of the celtoid group, or the Urticaceae shifts to a basal position in the Urticales. Neighbor-joining based on pairwise distance values produced a tree similar in part to the trees based on Wagner or weighted parsimony (tree not shown). This tree is found among trees six steps longer than the shortest trees using Wagner parsimony. The placement and relationships of genera within the ulmoids are congruent with that in the Wagner trees. However, relationships among the celtoid genera and other families in Urticales are incongruent with trees based on either Wagner or weighted parsimony. Most importantly, Aphananthe is separated from the celtoid clade and placed between the Urticaceae and Moraceae.

Discussion One family, or two? Based on the strong support for the basal division of the order into two ¢lades and the non-monophyleti¢ nature of Ulmaceae s. 1., the ulmoids and ¢eltoids hereafter will be referred to as the Ulmaceae and Celtidaceae. Thirteen additional steps beyond the length of the most parsimonious Wagner tree are required to place the two families into one monophyletic group. These ¢pDNA results, placing the Celtidaceae in the major clade containing Moraceae, Urticaceae, and Cannabaceae, support the ¢areful analyses (although not framed in expli¢it ¢ladistic fashion) and re¢ent conclusions of a number of researchers using floral morphology (GRuDZ~S~YA 1967), gynoecial vasculature (O~ORI & TE~BAYASHI 1993), embryology and seed coat morphology (Ch-~m,a~: 1975, TA~SO 1987, TA~SO & TOBE 1990), fruit morphology (GRUDZ~S~YA 1967, C~n~~,rIK 1980), pollen (ERDTMAN 1971, KUPRIANOVA1962, ZAVADA 1983, TAKAHASHI 1989), chromosome number (MEHRA 8z GILL 1974, OGINLrMA & al. 1990), leaf venation (GRUDZINSKAYA 1967), leaf/stipule vernation (TERABAYASHI1991), wood anatomy (Twpo 1938, ZHONG • äl. 1992), and secondary chemistry (GIANNASI 1978). These results also are consistent with those obtained in the preliminary cladistic analysis Circumscription of Ulmaceae s. 1., cpDNA evidence 259 of the order Urticales using a limited set (17) of morphological characters and only Ulmus and Celtis as representatives of the two families (JUOD & al. 1994). The cpDNA restriction site data also are consistent with the results of the rbcL analysis of Juglandaceae and relatives, a study in which Ulmus, Celtis, and one genus each of Moraceae and Urticaceae were included (Gt:N~R & al. 1994), and of the more comprehensive rbcL analysis of the Urticales (SYTSMA & al. 1996). A morphological cladistic analysis conducted alongside the rbcL analysis in Gt:N~R & al. (1994) indicates that Ulmaceae s. 1. is polyphyletic, but with Ulmus placed within but near the base of the "higher hamamelids" and Celtis placed at the base of Urticales. Identification of the morphological synapomorphies that link Celtidaceae with all other Urticales (minus Ulmaceae) depends critically on the outgroups chosen. The Juglandales, Fagales, and Malvales have been suggested most orten (e.g. BERG 1989) and the latter have been used cladistically as an outgroup (Tiliaceae A. L. DE Jussmu in JUDD & al. 1994). Some workers have ruled out any of these groups as closely related to Urticales (e.g. BEHN~ 1989 based on sieve element plastids). Recent rbcL analyses point to Rhamnaceae, Elaeagnaceae, Barbeyaceae, and Rosaceae as the closest families to Urticales (CHASE & al. 1993, SYTSMA & al. 1996). Of all the major classification systems these relationships are evident only in the works of LrNDt.EY (1853), placing the Ulmaceae in his expanded Rhamnales, and THORNE (1992), placing the Urticales between Malvales and Rhamnales in his Malvanae (see also summary in SWEITZER 1971). The Rhamnales share an impressive list of floral and vegetative character states with the Urticales, but SWErrZER(1971) ruled out a close relationship of Urticales with Rhamnales (instead opting for a relationship with Juglandales) based on the specialized wood characters exhibited by Rhamnales. Without the detailed morphological, anatomical, and pollen data available for Rhamnaceae and Barbeyaceae that is presently available for Ulmaceae and many other Urticales, it is premature to decide polarity for most of the characters in the impressive list differentiating the Ulmaceae and Celtidaceae. Most of the detailed studies on Ulmaceae and Celtidaceae listed above do not and cannot provide information on whether each character is a synapomorphy for Celtidaceae + other Urticales, an apomorphy for Celtidaceae, or an apomorphy for Ulmaceae. Attempts to polarize character states generally have utilized taxa now considered to be quite distinct from the Urticales (e.g. Tiliaceae in JUDD & al. 1994, Eucommiaceae ENGLEg in OMORI & TERABAYASHI 1993), and MANCHESTER(1989) claims that polarity of many characters in Urticales varies depending on whether Malvales or Hamamelidales are used as outgroups. In any event, discussions on whether Ulmaceae or Celtidaceae is more "primitive" (i.e. contains more plesiomorphic states) (BERG 1989, CRONQUIST1981, MANCHESTER 1989, TAKAI-IASI-n1989, TAKASO & TOBE 1990, ZAVADA~ CREPET 1981, Z~IONG & al. 1992) are premature, because the polarity of many characters is still equivocal. Likewise, arguments against the two family proposition on the grounds that some genera (e.g. Ampelocera, Aphananthe, Gironniera GAUDICH.) can appear either ulmoid or celtoid depending on the character (CRONQUIST 1981, BERG 1989, MANCHESTER 1989, SWEITZER 1971, ZI~ONa & al. 1992) often are confusing symplesiomorphies and synapomorphies (see GIANNASI 1978 for further discus- 260 S.J. WIEGREFE¢~ al.: sion). For example, Ampelocera often has been placed in Celtidaceae (or the Celtidoideae, Table 2) based in part on its drupaceous fruits. The drupaceous fruit is plesiomorphic if one uses Rhamnaceae as the outgroup and if the fossil record is examined (MANCHESTER 1989). Thus the placement of Ampelocera in Ulmaceae based on cpDNA evidence (Fig. 2) is not necessarily contradictory, because Ampelocera could be retaining the plesiomorphic state (see more detailed discussion on Ampelocera below). Similar examples in Ampelocera, Zelkova and other genera are seen with pollen (TAKAHASHI 1989), vemation (TERABAYASHI1991), flavonoids (GIANNASI 1978), karyology (O6INUMA & al. 1990), and seed coat morphology (TAKASO & TOBE 1990). The placement of Cannabaceae (only Cannabis in this study) within Celtidaceae and sister to a clade comprising Celtis and Pteroceltis MAXlM. is surprising because Cannabaceae is orten placed within Moraceae (see CRONQUIST 1981 and HUMPHRIES & BLACKMORE1989 for discussion). Difficulties, however, in obtaining high quality Cannabis DNA, in reliably mapping some of the sites in Cannabis, and the resulting number of missing data for Cannabis (see Appendix 1), suggest some caution in evaluating this result. However, based on scorable sites, this relationship has high bootstrap and decay support (Fig. 1). Considering that portions of Celtidaceae and Cannabaceae alone in the Urticales share the basic chromosome number x = 10 (MEHRA& GILL 1974), this relationship merits further investigation, especially by increasing the taxon coverage of Moraceae, Urticaceae, and Cecropiaceae. Indeed, rbcL sequence data involving both genera of Cannabaceae (Cannabis and Humulus) and considerably more representatives of the Urticales consistently place Cannabaceae within Celtida- ceae (SYTSMA & al. 1996). If this relationship holds up to further analyses, either Cannabaceae must be merged into Celtidaceae or the latter family must be divided into separate families. The relationships among Celtidaceae (plus Cannabaceae), Moraceae, and Urticaceae are not well resolved by these restriction site data (note low bootstrap values in Fig. 1) and such resolution will require greater numbers of molecular characters and greater coverage of taxa. One step longer trees place Moraceae rather than Urticaceae as sister to Celtidaceae, and two step longer trees place Celtidaceae as sister to both Moraceae and Urticaceae. Relationships within this clade of families (minus Ulmaceae) based on rbcL (SYTSMA• al. 1996) place Celtidaceae (plus Cannabaceae) as sister to the remaining Urticales, results more in accord with that based on morphology (JUDD & al. 1994). The relationships among families of Urticales supported by both the cpDNA restriction site data and rbcL data (SYTSMA& al. 1996) need to be evaluated by a more comprehensive (taxa and characters) morphological cladistic analysis including at least Rhamnaceae and Barbeyaceae as outgroups. Generic composition of Ulmaceae and Celtidaceae. The cpDNA restriction site data suggest that the Ulmaceae comprises Ulmus (including Chaetoptelea LIEBM.; WIEGREFE& al. 1994), Planera, Zelkova, Hemiptelea PLANCH., Holoptelea, and Ampelocera (Phyllostylon CAPPAN. ex BEYrH., not sampled here, would presumably belong as well; see Table 2). The Celtidaceae comprises Aphananthe (including Mirandaceltis), Celtis (including Plagioceltis MmOBR. ex BAEHNI), Lozanella, Pteroceltis, and Trema. Parasponia MIQ., Gironniera, and Chaetachme Circumscription of Ulmaceae s. 1., cpDNA evidence 261

PLANCH., not sampled here, would presumably belong as well; indeed, rbcL sequence data indicate that the first two fall within the Celtidaceae (SYTSMAÆ al. 1996; see Table 2). The generic composition of the Ulmaceae and Celtidaceae based on these cpDNA restriction site data closely matches the classification system of GRUDZINSKAYA (1967) and the division based on flavonoid content detected by G~A~,~~sI (1978) (Table 2). These two systems differ only on the placements of Ampelocera and Aphananthe. The inclusion of Ampelocera in the Ulmaceae with Ulmus, Zelkova, Planera, Hemiptelea, and Holoptelea is consistent with the findings of GIANNASI (1978), while the placement of Aphananthe in the Celtidaceae with Celtis, Pteroceltis, Trema, and Lozanella concurs with the system of GRUDZINSKAYA(1967). GIANNASI'S (1978) study of flavonoid content is one of the few studies on the Urticales where outgroup information has been presented. His inclusion of Barbeyaceae, a family of debatable position but placed in the Urticales by CRONQUIST (1981) and now placed near the Urticales and Rhamnaceae by rbcL data (CHASE & al. 1993, SYTSMA • al. 1996), was fortuitous because it provides polarization of the two classes of flavonoids found in Ulmaceae and Celtidaceae. The apomorphic state of possessing glycoflavones is found in all Celtidaceae (sensu GRUDZINSKAYA1967) except Ampelocera, Aphananthe, and Gironniera subg. Galumpita. The placement of Ampelocera in the Ulmaceae using cpDNA is thus consistent with its possession of flavonols, and would suggest that GRUDZINSKAYA (1967) misinterpreted the affinities of Ampelocera. As discussed in more detail below, this does seem to be the case on the basis of more recent morphological and anatomical data as well. The presence of flavonols in Aphananthe and one of the two subgenera of Gironniera is more difficult to explain. Aphananthe groups with Celtidaceae with both non-molecular characters (see below) and these cpDNA data. Gironniera is morphologically isolated in Celtidaceae (see below), a placement supported by rbcL evidence (SYTSMA & al. 1996). The presence of plesiomorphic flavonols in these two genera of Celtidaceae is thus consistent with their basal position within Celtidaceae. Determining the exact number of times that glycoflavones have evolved in Celtidaceae and Urticales is dependent on verification of the placements for Gironniera (both subgenera) and Chaetachme within Celtidaceae, as well as wider sampling of flavonoids in the other families of Urticales. Relationships within the Ulmaceae. One of the strongest supported clades in this analysis is Ulmaceae, represented here by five of its six usually recognized genera (only Phyllostylon not sampled) and the problematic Ampelocera (Fig. 1). The considerable number of mutations supporting the monophyly of the subclade formed by Ulmus, Planera, Zelkova, and Hemiptelea unequivocally places Zelkova and Hemiptelea in the Ulmaceae rather than Celtidaceae, decisions which have been controversiat in some earlier treatments (Table 2). The Ulmaceae (with the exception of Ampelocera) are quite uniform in other characteristics including basic chromosome number (x = 14; TaKAHASN 1989), pollen morphology and pore number (4-6; OGI~JMA & al. 1990), seed coat morphology (TAKASO & TOBE 1990), and vernation (TERABAYASHI1991). The sister genus to Ulmus could not be determined, because cpDNA restriction site data could not resolve the polytomy of Ulmus, Planera, and Zelkova. 262 S.J. WIEGREFE & al.:

Neither of these latter two genera share the samaroid fruit type with Ulmus, a fruit type that probably is apomorphic based on the fossil record (MA~CHESTER 1989) and on comparison with likely related families Rhamnaceae and Rosaceae (CHASE & al. 1993, SYTSMA & al. 1996). Samaras thus probably arose at least three times independently in the Urticales: twice in Ulmaceae (Ulmus and Holoptelea) and once in Celtidaceae (Pteroceltis). The close relationship of Ulmus, Planera, and Zelkova is evident by their shared possession of the distinctive and apomorphic Pc type of sieve-element plastids, not found elsewhere in Ulmaceae (some genera not examined), Celtidaceae, other Urticales, or likely outgroups (BEHNKE 1989). The cpDNA restriction site data places Ampelocera solidly within the Ulmaceae and sister to Holoptelea (Fig. 1). Nine additional steps are required to place Ampelocera at the base of Celtidaceae. This strong placement of Ampelocera within Ulmaceae is contrary to all previous classification systems except that suggested by GIANNASI(1978) based on flavonoid data (Table 2). Ampelocera is a poorly known genus of nine tree species occurring in low- to mid- elevation rainforests from Mexico to Brazil (TODZIA 1989). It was first described by I~OTZSCH (1847) who placed it near the genus Celtis, presumably based in part on its drupaceous fruits. According to TODZIA (1989), PLANCHON (1873) considered its placement even within the Ulmaceae s. 1. doubtful due to its unusually large number of stamens (up to 16). SWZITZER (1971), in bis study of wood anatomy, found Ampelocera to have the homocellular biseriate and multiseriate rays condition shared by the Ulmaceae, but argued for a placement within Celtidaceae (Celtidoideae) to reflect the classification of GRUDZINSKAYA(1967). Most of the subsequent studies have noticed an affinity of Ampelocera with Ulmaceae. CHEm~IK (1975) found that Ampelocera was similar to the Ulmaceae in the vasculature of its floral parts. A study of pollen morphology by ZAVADA& CRZPET (1981) reported that Ampelocera with its 4 or 5 porate pollen is better placed in the Ulmaceae than with the Celtidaceae which generally possesses 2 or 3-porate pollen. A later study of pollen types by TAKAI-IASI-II(1989), however, isolated Ampelocera from all other Ulmaceae and Celtidaceae. TAKASO & TOBE (1990) described the seed coat of AmpeIocera as colliculate like other Ulmaceae (Ulmus, Zelkova, Hemiptelea, Planera, and Holoptelea), however they placed Ampelocera in a separate pollen type with no suggestion of its phylogenetic relationships. Vernation type (TERABAYASHI1991) strongly links Ampelocera with Holoptelea and Phyllostylon. Chromosome number and karyomorphology have not been examined for Ampelocera (Oa~:MA & al. 1990), but is predicted to be n = 14 with features similar to Holoptelea and Phyllostylon based on the molecular analysis and the cited morphological studies above. Ampelocera and its sister genus Holoptelea (along with likely related Phyllostylon, see below) are the only exclusively tropical members of the Ulmaceae. Both are components of moist forests, Holoptelea in moist deciduous forests in the Old World (MEHRa & GmL 1974), and Ampelocera in evergreen forests in the New World (TODZIA 1989). Both genera contain species with large leaves having entire margins and arcuate venation, in contrast to the smaller leaves with serrate margins and simply craspedodromous venation which are character- Circumscription of Ulmaceae s. 1., cpDNA evidence 263 istic of the temperate genera of Ulmaceae. Because leaf size and entire margins have been found to be correlated with abundant moisture regimes and tropical distributions, respectively (GivN~sn 1987), these similarities could be synapomorphies derived in their common ancestor as adaptations to the wet, tropical forest habitat. However, entire margins and arcuate venation in these two genera also may be plesiomorphic considering the occurrence of both character states in Rhamnaceae. The placement of neotropical Phyllostylon, not examined in this study, within the Ulmaceae is supported by a wealth of morphological data. Although its exact relationships are still ambiguous, Phyllostylon is predicted to lie near the other tropical genera, Holoptelea and Ampelocera. Pollen evidence indicates that it exhibits the general pollen type of all other Ulmaceae (TAKAHASHI1989). Chromo- some number places Phyllostylon (n = 14) within the Ulmaceae (all n-- 14) and karyomorphology further suggests an affinity with Holoptelea (O~~~A & al. 1990). Phyllostylon also shares the vernation type seen in Holoptelea and Ampelocera (TERABAYASHI1991). Seed coat morphology indicates an affinity to Ulmus and Zelkova and not to either Planera or Ampelocera; Holoptelea and Hemiptelea, however, were not examined (TAKASO & TOBE 1990). Relafionships within Celtidaceae. The Celtidaceae is not as clearly defined by these molecular results as is the Ulmaceae and appears to be composed of several quite isolated lineages. Monophyly of Celtidaceae, relationships among its various lineages, and relationships with Moraceae and Urticaceae are weakly supported and vary under Wagner or weighted parsimony conditions (Fig. 1). Sequence analysis of this clade of Urticales is needed but will require greater coverage of the Urticaceae, Moraceae, and Cecropiaceae. The close relationship shown hefe between Celtis and Pteroceltis is supported by numerous other types of data, including pollen morphology (TA~HAS~ 1989), karyomorphology (OcINU~A & al. 1990), and seed coat morphology (TAKASO & TOBE 1990). Pteroceltis appears in the fossil record during the Oligocene (ca. 30 Mya), rauch later than does Celtis which is first detected in the Paleocene (ca. 64 Mya) (MANe~STER 1989). The fossil record may reflect the more limited distri- bution and fewer number of species in Pteroceltis compared to Celtis, or it may be an indication that Celtis is paraphyletic relative to Pteroceltis rather than sister to Pteroceltis. A larger clade of Celtis, Pteroceltis, Trema, Lozanella, and Parasponia (not sampled here) is supported to varying degrees by pollen morphology (TAKAHASHI 1989), seed coat morphology (TAKASO & TOBE 1990), vernation (TERABAYASHI 1991), and n = 10 chromosome number (OG~UMA & al. 1990). The placement of Aphananthe somewhere within this larger clade is supported by a number of morphological characters. Aphananthe shares the pollen structure of Celtis (KUPRIANOVA1962) and a stratified pollen wall structure with Celtis and Pteroceltis (TAKAHASHI 1989). Vernation (TERABAYASHI1991) and gynoecial vasculature (OMo~ Æ TERABAYASHI1993) also position Aphananthe with most of the genera in this clade. An early divergence of Aphananthe within Celtidaceae, however, is suggested by seed coat morphology (TAKASO & TOBE 1990), ovule anatomy (TAKASO 1987), 264 S.J. WIEaR~~ & al.:

presence of flavonols rather than glycoflavones (GIANNASI1978), and chromosome number (O~INUMA & al. 1990). Aphananthe exhibits the medial or submedial, simple centromeres of CeItidaceae versus the predominantly terminal and subterminal diffuse-complex centromeres of Ulmaceae, but it has a reduced chromosome number (n= 13), intermediate between Ulmaceae, Urticaceae, Moraceae (n = 14) and all other sampled genera of Celtidaceae (n = 10), excluding Gironniera (n = 14) (MEHRA & GILL 1974, OGINUMA • al. 1990). The polarity of most of these characters needs to be determined before the uncertainty regarding the phylogenetic relationships of Aphananthe can be fully resolved. It should be noted that using Neighbor-joining on these restriction site data, Aphananthe is the only genus of either Ulmaceae or Celtidaceae that radically changes position by being placed between Moraceae and Urtieaceae. Two genera not sampled hefe, Gironniera and Chaetachme, are critical to fully resolving the relationships within Celtidaceae and between Celtidaceae and the remainder of the Urticales. Chaetachme is quite isolated from other Celtidaceae based on pollen (TAKAHASHI 1989), vernation (TERABAYASHI1991), and possibly seed coat morphology (TAKASO & TOBE 1990). Moreover, its chromosome number has not been determined (M~HRA & GILL 1974, O~INt;MA & al. 1990). Gironniera is perhaps even of more phylogenetic interest. Gironniera subg. Gironniera possesses glycoflavones typical of Celtidaceae, but subg. Galumpita has flavonols as in Ulmaceae and Aphananthe (GIANNASI 1978). The possibility that Gironniera is situated near the base of Celtidaceae (as argued by GIANNASI 1978), or even the Urticales, is increased by its unique type of pollen (TAKAHASHI 1989), vemation (TERABAYASHI 1991), seed coat morphology (TAKASO & TOBE 1990), and n = 14 chromosome number (OGINUMA & al. 1990). Along with Aphananthe and Chaetachme, the two subgenera of Gironniera need to be included in more comprehensive molecular and morphological cladistic analyses to better understand not only their relationships within Celtidaceae and to other Urticales, but also character evolution and biogeography within the Urticales as a whole.

The authors thank JEFFREY PALMER and ROBERT JANSEN for the provision of cpDNA clones and the following individuals for their assistance in the collection of the plant materials used: GENE SMALLEY,GEORGE WARE, DAVID MICHENER,Mfs R. ZINMANN,MfS E. BENNETT, KATHY ZUZEK, KRISTINEMEDIC THOMAS, PAUL MEYER, HEIDI STONEHILL, WILLIAM HAWKINSON, SUZANNE GRANGER, and GONZALO CASTILLO-CAMPOS.We also thank KAREN ROSNECK,JACEK OLEKSYN, and DmK VANDERKLEINfor translation of the Russian and Dutch references, and GEOR~EWARE for making available a translafion of a Chinese reference and underwrifing a portion of the Russian translafion work. The graphic portrayal of our findings by KAND~S ELLIOT is appreciated. SUSAN SHERMAN-BROYLES,REGIS MILLER, and STEVENMANCHESTER contributed valuable information. We greatly appreciate the assistance of IRINAGRUDZINSKAYA in supplying repfints of a number of her publicafions and providing clarifications where necessary. Critical reading of an earlier manuscript by MICHAELHAVEY and DAVIDSPOONER are appreciated. The research represents a portion of the Ph.D. research of SJW. The research was supported by the College of Agficulture and Life Sciences, University of Wisconsin, Madison and by McInfire-Stennis funds (project 142-C968) to SJW and RPG; and by NSF grants (BSR-8806520, DEB-9020055) to KJS. Circumscription of Ulmaceae s. 1., cpDNA evidence 265

Appendix 1. Character state data for all 268 restriction sites scored for Ulmaceae s. 1. and outgroup genera. Mutations are numbered by restriction enzyme with the numbers directly tbllowing restriction enzyme name. The second number indicates location (in kilobases) of site mutation in chloroplast genome relative to a conserved SmaI site just inside inverted repeat near large single copy region (see Table 4). Distances are calculated from this site in the direction of the large single copy region on the side closest to the rbcL gene and progressing around molecule. Taxa are listed in order as in Table 3.9 in the data set indicates missing data due to inability to score site

PstI O1 2.2 111110110011101 KpnI 21 41.0 000000000001091 Safi 05 25.7 111111111110190 PstI 02 5.1 llllllO00000iO1 KpnI 22 21.0 000000000000190 SalI 06 38.9 111111111190191 PstI 03 30.6 111111111111111 KpnI 23 55.6 000000000000190 SalI 07 78.4 111111111119191 PstI 04 51.6 111111111111191 KpnI 24 72.9 000000000000190 SaII 08 98.4 111111111111110 PstI 05 52.6 111111111111191 KpnI 25 75.8 000000000000190 SalI 09 100.8 111111110111111 PstI Oó 68.1 111111110110191 KpnI 26 18.8 000000000001090 SalI 10 2.1 001000000000090 PstI 07 73.9 111111000000090 KpnI 27 26.2 000000000001090 SaH 11 21.7 000001111101190 PstI 08 77.1 11110900009009¤ KpnI 28 116.6 000000000000010 SalI 12 59.1 000001111100010 PstI 09 88.1 111111111110111 KpnI 29 25.8 00000000000009¤ SalI 13 120.5 000001101009000 PstI 10 98.2 111111111111111 KpnI 30 29.1 000000000000091 SalI 14 29.0 000000110000090 PstI 11 102.8 111111111111111 KpnI 31 74.3 000000000000091 SalI 15 61.6 000001111101111 PstI 12 123.0 111111001191101 SmaI O1 0.0 000011001111101 SaII 16 86.4 000000111100190 PstI 13 128.6 111100000099000 SmaI 02 0.7 110111001110110 SalI 17 124.3 000000110000000 PstI 14 131.9 011100000090000 SmaI 03 48.5 111110990010990 SalI 18 135.2 000000110000000 PstI 15 8.3 001000000001001 SmaI 04 61.1 100010000010090 SalI 19 19.8 000000000010090 PstI 16 29.4 001001009100100 SmaI 05 87.4 110111001911111 Saß 20 89.8 000000000010090 PstI 17 27.8 001000000000000 SmaI 06 107.0 11111111111111l SalI 21 94.0 000000000010000 PstI 18 6.8 000010000011100 SmaI 07 111.4 111111111111111 SaH 22 25.2 000000001001191 PstI 19 75.9 000001000000190 SmaI 08 113.4 111111111111111 SalI 23 37.3 000000000001090 PstI 20 117.4 00000¤110190900 SmaI 09 6.8 001001110101190 SalI 24 118.4 000000000001000 PstI 21 25.5 000000110001000 SmaI 10 68.7 001010000000090 SalI 25 72.8 000000000000190 PstI 22 88.9 000000110000090 SmaI 11 91.2 000011001119191 XhoI Ol 2.3 110111110110191 PstI 23 21.7 000000001001900 SmaI 12 110.2 000011111111190 XhoI 02 5.8 111111110000091 PstI 24 44.1 000000000001090 SmaI 13 33.6 000001000000090 XhoI 03 8.9 111111110111191 PstI 25 126.9 000000000001000 SmaI 14 1.0 000001009999090 XhoI 04 36.8 110110110111091 PstI 26 38.8 000000000000190 SmaI 15 56.l 000001111101199 XhoI 05 44.6 111110000000190 PstI 27 20.9 000000001101110 SmaI 16 56.5 000001000000099 XhoI 06 63.7 111100000090990 PstI 28 68.6 000000000000190 SmaI 17 75.5 000001000190011 XhoI 07 70.8 111111000091991 PstI 29 121.4 000000000000010 SmaI 18 5.2 000000111101110 XhoI 08 82.7 111111111111191 KpnI Ol 8.3 110111000011101 SmaI 19 9.9 000000110000000 XhoI 09 87.1 110100000000090 KpnI 02 12.0 110110000010000 SmaI 20 32.3 000000110000011 XhoI 10 92.3 111190000911191 KpnI 03 21.5 111100000000000 SmaI 2l 48.0 000000110090999 XhoI 11 92.6 111111111191111 KpnI 04 36.3 111111111110111 Smai2286.6 000000110000011 Xhoi1295.8 111111111101110 KpnI 05 66.8 111111111111111 SmaI 23 12.9 000000001000090 XhoI 13 107.9 111111111199111 KpnI 06 71.0 111111111111111 SmaI 24 42.0 000000001000099 XhoI 14 108.8 111110111119111 KpnI 07 71.6 111111111011011 SmaI 25 64.8 000000001000090 XhoI 15 112.3 111110111110111 KpnI 08 76.0 111111000000091 SmaI 26 24.5 000000000109090 XhoI 16 123.1 110111991991091 KpnI 09 110.6 111111111111111 SmaI 27 84.2 000000000100190 XhoI 17 47.7 010000000000090 KpnI 10 111.4 111111111110111 SmaI 28 76.5 000000000010090 XhoI 18 116.3 000010000000000 KpnI 1l 120.4 111100000000001 SmaI 29 123.0 000000000010000 XhoI 19 111.9 000001000000000 KpnI 12 131.2 111110001001011 SmaI 30 24.2 000000000001090 XhoI 20 120.3 000001000000090 KpnI 13 33.5 01000000000000 SmaI 31 38.3 000000000001099 XhoI 21 14.9 000000110000090 KpnI 14 26.9 000011991190090 SmaI 32 92.8 000000000001090 XhoI 22 40.7 000000110909090 KpnI 15 47.6 000010000010191 SmaI 33 1.7 000000000000001 XhoI 23 66.1 000000100000090 KpnI 16 3.2 000000119000010 SmaI 34 51.6 000000000000091 XhoI 24 122.8 00000119991090 KpnI 17 76.5 000000111011190 SalI O1 4.5 111110000090090 XhoI 25 7.4 000000001000090 KpnI 18 79.2 000000110000010 SalI 02 5.8 111190110090091 XhoI 26 41.6 000000000109090 KpnI 19 24.2 000000110000010 SalI 03 6.4 110111¤11111190 XhoI 27 44.0 000000000100090 KpnI 20 27.2 000000119910090 SalI 04 18.1 111111119111191 XhoI 28 123.4 000000000199090 266 S. J. WIEGREFE & al.:

Appendix 1 (continued) XhoI 29 19.2 000000000019090 Ssd 11 108.7 111111111111111 BgH 05 45.8 111101111100191 XhoI 30 37.4 000000000000190 Ss~ 12 110.3 111100000000000 BgH 06 98.2 111110111111191 XhoI 31 48.3 000000000000191 Ss8 13 112.3 111111111111111 Bg~07 104.9 111111111191101 XhoI 32 59.5 000000000000190 Ss~ 14 114.4 111111111111111 BgH 08 109.5 111111111199111 XhoI 33 86.2 000000000000190 Ss~ 15 115.0 111110111119191 BgH 09 69.1 000001111191191 XhoI 34 121.7 000000000000010 Ssd 16 132.6 101100000000000 Bgfl 10 107.6 000001000000090 XhoI 35 89.0 000000010000090 Ss~ 17 30.5 010001000000090 BgH 11 113.9 000001111190110 XhoI 36 19.0 000000000001099 Ss8 18 17.4 000010000010090 Bg~ 12 8.5 000000110000190 XhoI 37 5.2 000000000000091 Ss~ 19 16.0 000010000010090 BgH 13 51,8 000000110001090 XhoI 38 10.4 000000000000091 Ssfl 20 25.8 000001000000099 BgH 14 76,7 000000110000091 XhoI 39 27.5 000000000000091 Ssff 21 39.0 000001000000099 BgH 15 64,4 000000000100090 XhoI 40 126.7 000000000000091 Ssfl 22 62.8 000001000000099 Bg~ 16 53,4 000000000001090 SphI 01 1.8 111110000010090 Ss~ 23 72.9 000001000000090 Bg~ 17 84.7 000000000001090 SphI 02 27.9 111111111111191 Ssfl 24 92.2 000001000000090 BgH 18 122.0 000000000000010 SphI 03 30.5 111111111111191 Ssfl 25 114.9 000001000001099 BgH 19 9.2 000000000000091 Sphi0446.1 111111111111191 Ss~ 26 124.3 000001991100000 BgH 20 33.8 000000000000091 SphI 05 51.7 111110000010090 Ssfl 27 22.0 000000110100919 PvulI 01 2.2 111110000091090 SphI06 55.8 111100000000091 Ss8 28 34.4 000000110000099 PvulI 02 33.9 110101000191099 SphI 07 64.4 111111111011191 Ssfl 29 68.7 000000110000099 PvuII 03 38.2 111111111191199 SphI 08 105.7 111110111110111 Ss8 30 88.6 000000110000090 PvulI 04 43.4 111119111191199 SphI 09 108.7 111110111111111 Ss~ 31 96.2 000000111000010 PvuH0551.7 111111111110191 SphI 10 120.7 111110000090000 Ssfl 32 123.9 000000110000090 PvuII0671.7 111111111111111 Sphlll 88.1 001000000000090 Ssfl 33 1.9 000000001100099 PvuH 07 85.1 110110000010090 SphI 12 21.6 000001000000090 Ss~ 34 76.5 000000001100090 PvuII 08 88.3 111111111099191 SphI 13 107.4 000001000000000 Ss~ 35 76.5 000000001990099 PvuH 09 105.8 111111111199111 SphI 14 59.4 000000000001090 Ss~ 36 82.6 000000001000099 PvuII 10 108.5 111111111110111 SphI 15 14.2 000000000000190 Ss~ 37 83.6 000000001100190 Pvug 11 112.7 111111111110111 SphI 16 22.5 000000000000190 Ss~ 38 61.6 000000000100099 PvuII 12 47.6 001010000001099 SphI 17 16.0 000000000000091 Ssfl 39 76.9 000000000100099 PvuII 13 121.9 001000000000000 SphI18 19.4 000000000000091 Ss~ 40 70.0 000000000010090 PvuII 14 76.2 000011111111191 SphI 19 31.8 000000000000091 Ssfl 41 97.6 000000000010000 PvulI 15 29.9 000001000000090 Ssfi 01 2.3 111110110010191 Ss~ 42 18.8 000000000001090 PvuII 16 61.6 000001111001111 Ssfl 02 22.9 111111001911990 Ss~ 43 6,9 000000000000190 PvuR 17 116.8 000000100000100 Ssg 03 32.4 111111111111111 Ss~ 44 117.1 000000000000100 PvuII 18 17.3 000000001000090 Ss~ 04 47.5 111111111010119 Ssd 45 124.6 000000000000010 Pvug 19 63.8 000000000100090 Ss~ 05 51.9 111111991111199 Ssfl 46 65.6 000000000000091 PvulI 20 31.9 000000000001090 Ss~ 06 63.9 111111991011191 Ss~ 47 21.6 000000000000091 Pvu~ 21 33.3 000000000000091 Ssfl 07 67.7 111110110000090 Bgfl 01 4.7 111111111111091 PvuII 22 65.3 000000000000091 Ssfl 08 75.8 111111119901191 BgH 02 8.7 111111991111990 PvuH 23 82.6 000000000000091 Ssfl 09 82.8 111111009111991 BgH 03 25.2 100000000000090 Ssd 10 85.1 111111000111190 BGH0441.4 110100000000990

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Addresses of the authors: Dr SUSANJ. WIEGREFE,The Morton Arboretum, 4100 Illinois Route 53, Lisle, IL, 60532, USA. - Prof. Dr KBNNETrI J. SrrSMA, Department of Botany, University of Wisconsin, Madison, WI, 53706, USA. - Prof. Dr RAYMOND P. GURIES, Department of Forestry, University of Wisconsin, Madison, WI, 53706, USA.