Phylogeny and Phylogeography of the Circumpolar GenusFraxinus(OLEACEAE) Based on Internal Transcribed Spacer Sequences of Nuclear Ribosomal DNA

MOLECULAR PHYLOGENETICS AND EVOLUTION

Vol. 7, No. 2, APRIL, pp. 241–251, 1997 ARTICLE NO. FY960393

Phylogeny and Phylogeography of the Circumpolar Genus Fraxinus (OLEACEAE) Based on Internal Transcribed Spacer Sequences of Nuclear Ribosomal DNA Sylvain Jeandroz, Alice Roy, and Jean Bousquet Centre de Recherche en Biologie Forestie`re, Faculte´ de Foresterie et de Ge´omatique, Universite´ Laval, Sainte-Foy, Que´bec, Canada G1K 7P4 Received July 15, 1996; revised November 8, 1996

The DNA sequences of the nuclear internal transcribed spacers (1 and 2) of nuclear ribosomal DNA were determined for 27 taxa of Fraxinus in an attempt to reconstruct the phylogeny of the genus and interpret its biogeographic history in a phylogenetic context. Minimal intraindividual and intraspecific polymorphisms were observed and infrageneric taxa were all recovered in phylogenetic analyses. The tree topologies estimated from parsimony and neighbor-joining analyses of one- and two-parameter substitution rates were congruent and supported by high bootstrap estimates. Indels were also found to be phylogenetically informative. The phylogenetic trees obtained were in quite good agreement with the current taxonomy of the genus but homoplasy was observed in the evolution of floral characters. Polyploidy also appeared to have evolved several times in the genus. Fraxinus nigra was more closely related to European and Asian taxa than to the other American ashes. The Asian F. platypoda had a closer relationship to Asian and European ashes in section Bumelioides than to its presumed North American allies of section Melioides. With respect to the position of F. quadrangulata as sister group to the remainder to the remaining species, the subgenus Fraxinus appeared paraphyletic. Phylogeographical analysis indicated that Fraxinus likely originated in North America, with two subsequent events of intercontinental migration from North America to Asia. r 1997 Academic Press

INTRODUCTION The genus Fraxinus (Oleaceae) is composed of about 40 woody plant species distributed across the Northern Hemisphere, with two main centers of diversity in Asia and North America (Bean, 1925). The origin of the genus is unclear. The oldest known Fraxinus fossils, samaras of Fraxinus wilcoxiana, have been recently reported from the Middle Eocene (49 to 39 Myr) in southeastern North America (Call and Dilcher, 1992), but these fossils cannot be assigned to a modern subgenus or section of the genus. Fraxinus remains do

not appear in European or Asian floras until the Oligocene or later (36 Myr) (see Call and Dilcher, 1992 for a review). Fossilized pollen of Fraxinus has been recorded from the Upper Miocene, 11 to 5 million years ago (Myr), in North America and Europe (Mu¨ller, 1981). The clarification of phylogenetic relationships among members of this genus could help to elucidate the ancestral events of diversification and dispersal underlying its biogeography. The current distribution of Fraxinus might be explained by several dispersal events from an unknown center of origin, which would be promoted by the intermittent past connections between continents in the Northern Hemisphere. During the Cenozoic (Eocene) (55 Myr) and the first half of the Pleistocene, the Bering land bridge between Alaska and Siberia provided a passage for plants and animals, likely explaining the presence of a somewhat continuous flora (Brown and Gibson, 1983). The genus Fraxinus is divided into two subgenera, Ornus and Fraxinus. Subgenus Ornus, mainly restricted to Asia, comprises two sections (Ornus and Ornaster). The subgenus Fraxinus, restricted mainly to North America and Europe, is divided into three sections (Bumelioides, Melioides, Dipetalae) with fairly strong geographic limits (Nikolaev, 1981). Within subgenus Fraxinus, sections Bumelioides and Melioides contain numerous species (12 and 13 species, respectively), whereas the third section, Dipetalae, has only two species. The current classification of Fraxinus is based on morphological characters, particularly floral characters. Polyploid taxa are found in both subgenera. The biogeography and the taxonomy of the genus are sometimes at odds. For example, F. quadrangulata and F. nigra, North American species of subgenus Fraxinus, are associated with the European ashes (section Bumelioides) rather than with the other North American ashes (section Melioides). Similarly, F. platypoda, a species restricted to China, is grouped with the American ashes (section Melioides) rather than with other EurAsian ashes (section Bumelioides). Taxonomical ambiguities also exist. Fraxinus cuspidata was classified by Miller (1955) as the only American species belonging to subgenus Ornus or was classified by

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TABLE 1 Classification of Fraxinus Taxa Used in the Phylogenetic Analyses, According to Nikolaev (1981) Fraxinus species studied Subgenus Fraxinus Section Bumelioides Subsection Paniculatae F. excelsior F. excelsior cv. Kimberly Blue F. mandshurica F. nigra F. quadrangulata (2)a Subsection Racemosae F. oxyphylla F. pallisae F. syriaca Section Melioides F. americana (2)a F. anomala F. biltmoreana F. latifolia F. pennsylvanica F. pennsylvanica var. aucubaefolia F. pennsylvanica var. subintegerrima F. platypoda F. tomentosa (2)a F. velutina Section Dipetalae F. cuspidata Subgenus Ornus Section Ornus F. ornus Section Ornaster F. chinensis F. longicuspis (2)a F. rhynchophylla Syringa vulgaris

Common name

Distribution

Source

GenBank Accession No.

Common ash — Manchurian ash Black ash Blue ash

Europe, Asia minor — China (Manchuria), Japan USA (Northeast), Canada (East) USA (Ohio, Mississippi)

MBG MBG MBG MBG MBG

U82864–U82865 U82866–U82867 U82874–U82875 U82878–U82879 U82880–U82881/U82882–U82883

Narrow-leaved ash — Syrian ash

Europe (South), Africa (North) Europe Syria, Afghanistan

MBG MBG MBG

U82868–U82869 U82870–U82871 U82872–U82873

White ash Utah ash Biltmore ash — Red ash

North America USA (Colorado, Utah) USA (East) USA North America (East of Rocky Mts)

MBG MBG MBG MBG MBG

U82906–U82907/U82908–U82909 U82914–U82915 U82910–U82911 U82912–U82913 U82902–U82903

North America

MBG

U82900–U82901

North America China USA (Illinois, Indiana, Ohio) USA (Southwest)

MBG TAAHU MBG MBG

U82894–U82895 U82876–U82877 U82896–U82897/U82898–U82899 U82904–U82905

USA (Southwest)

SABG

U82916–U82917

Manna ash

Mediterranean area, Asia (West)

MBG

U82892–U82893

— Japanese flowering ash — Common lilac

Asia, China Japan China Europe (East)

MBG MBG TAAHU MBG

U82898–U82885 U82888–U82889/U82890–U82891 U82886–U82887 U82918–U82919

— — — — Arizona ash —

Note. (—) Unknown. MBG, Montre´al Botanical Garden; TAAHU, The Arnold Arboretum of Harvard University; SABG, San Antonio Botanical Garden. a Numbers in parenthesis indicate the numbers of taxa for which ITS regions were sequenced.

Nikolaev (1981) in subgenus Fraxinus (section Dipetalae). Taxonomic conflicts also arise within sections, where some species have been identified by several names or have been considered subspecies. In areas of sympatry, the presence of intermediate morphological forms is likely the result of natural introgressive hybridization between closely related species (Jeandroz et al., 1995). This renders species identification precarious when based on morphological characters alone. The objectives of this study were to use sequences of the internal transcribed spacers (ITS1 and ITS2) of the nuclear ribosomal DNA unit to determine the phylogenetic relationships in the genus Fraxinus and to improve our understanding of the possible past dispersal patterns of this circumpolar, woody genus. ITS regions have been shown to evolve rapidly and be useful in inferring phylogenetic relationships among plant taxa at the generic and intrageneric levels (e.g., Baldwin, 1992; Savard et al., 1993; Baldwin et al., 1995).

MATERIALS AND METHODS Plant Material A total of 27 taxa of Fraxinus was sampled. Their distribution, source, and the GenBank accession numbers for their ITS1 and ITS2 DNA sequences are presented in Table 1. According to the classification of Nikolaev (1981), 20 species of Fraxinus were included, representing five sections and two subsections of the genus. Subsection Sciandanthus (containing two species) of section Melioides was not sampled. For F. americana, F. quadrangulata, F. longicuspis, and F. tomentosa, two individuals each were sampled to assess intraspecific variation. Syringa vulgaris, also of Oleaceae, was used as the outgroup. DNA Amplification and Sequencing Strategy Total DNA was extracted from buds following methods described previously (Bousquet et al., 1990). The conditions for ITS DNA amplification by PCR were: 50 mM KCl,

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PHYLOGENY AND PHYLOGEOGRAPHY OF Fraxinus

10 mM Tris–HCl (pH 9.0), 1.5 mM MgCl2, 0.2 mM of each dNTP, 50 pmol of each primer, 10 ng of genomic DNA, and 2 units of Taq polymerase in a final reaction volume of 50 µl. Amplifications were conducted in a DNA thermal cycler (Perkin–Elmer–Cetus 480) for 35 cycles, each consisting of a denaturing step of 1 min at 94°C, an annealing step of 1 min at 55, 57, or 62°C, depending on primer sets, and an extension step of 1 min at 72°C. The last cycle was followed by 10 min at 72°C to ensure that primer extension reactions proceeded to completion. Eight different primers were used to amplify the ITS1 and ITS2 regions (Fig. 1). Primers its-1 to its-5 have been described (White et al., 1990). Three new primers were designed from preliminary DNA sequence information: primer its-6 (reverse), 58 GGACGCCCAGGCAGACGTG 38, was usually used with primer its-1 for the amplification of the ITS1 region, and the primer pair its-7 (forward), 58 CTTGGCTCTCGCATCGATG 38, and its-8 (reverse), 58 AGGGTTGTCCGCCTGACCT 38, was used for the amplification of ITS2. Amplified fragments were visualized after agarose gel electrophoresis and then purified on Prep-A-Gene DNA purification columns (BioRad), QIAquick Spin PCR purification columns, or with QIAquick Gel extraction kit (Qiagen). Direct sequencing of DNA strands was conducted with an ABI 302 automated sequencer and performed on both strands. Phylogenetic Analyses The ITS rDNA nucleotide sequences were aligned with the PILEUP option of the computer program GCG (Genetic Computer Group, University of Wisconsin; Devereux et al., 1984). Alignments were verified and adjusted manually. Phylogenies were estimated by two methods: parsimony using PAUP 3.1.1 (Swofford, 1993) and neighbor-joining (NJ) using MEGA 1.0 (Kumar et al., 1993). For parsimony, a heuristic search was conducted with 100 random additions of sequences and the TBR branch-swapping option. Bootstrap values were calculated from 500 replicates. The number of additional steps required to force particular taxa into a monophyletic group was examinated using the CONSTRAINTS option in PAUP 3.1.1. Numbers of substitutions were also estimated using the one-parameter method of Jukes and Cantor (1969) and the two-

FIG. 1. Organization of the nuclear ribosomal DNA. The positions of primers used for DNA amplification and sequencing of the ITS1 and ITS2 regions are indicated by arrows.

parameter method of Kimura (1980). The matrices of pairwise substitution rates were then submitted to the neighbor-joining method of phylogenetic tree reconstruction (Saitou and Nei, 1987). In all cases, bootstrap values were calculated from 500 replicates. To correct for the conservative bias of the standard bootstrap procedure, a double bootstrapping method was used a posteriori to verify the monophyly of specific lineages (Zharkikh and Li, 1995). This technique estimates the effective number of competing alternative topologies and gives confidence levels of monophyly to clades or inferred tree topology. With this method, corrected bootstrap values that can be interpreted in terms of confidence were calculated from 5000 replicates as recommended (Zharkikh and Li, 1995). The significance level for these bootstrap values was preset at 1 2 a 5 95%. RESULTS Sequence Analysis The ITS1 and ITS2 regions of 27 sampled taxa of Fraxinus (Oleaceae) and of the outgroup, Syringa vulgaris (Oleaceae), were amplified, and both DNA strands were sequenced. No intraindividual ITS length variants were observed after electrophoresis of the PCRamplified products or after sequencing. However, in several taxa, two nucleotides were sometimes encountered at particular sites. These cases of polymorphism were seldom found in a given sequence, with one to two polymorphic sites encountered in 17 of the 28 sequences. The sequence from F. pallisae showed a maximum of four polymorphic sites. These ambiguous nucleotides were treated as missing data and not used in calculating substitution rates or in the parsimony analysis. The boundaries of the ITS and rDNA coding regions were identified by comparison to Daucus carota (Yokota et al., 1989) and Populus deltoides (D’Ovidio, 1992). The length of ITS1 was nearly constant within the genus Fraxinus, varying from 211 to 212 bp. It was 212 bp long for the outgroup Syringa. The length of ITS2 ranged from 215 to 222 bp among Fraxinus taxa and was 234 bp long for Syringa. The G 1 C content of the ITS1 and ITS2 regions combined ranged from 59.3% (F. quadrangulata) to 67.1% (F. mandshurica). The average G 1 C content was 63.7% (63.2%). To obtain a correct alignment of Fraxinus ITS sequences, 20 insertions/deletions were required (Fig. 2): 13 indels were of 1 bp, 5 were of 2 bp, 1 was of 3 bp, and 1 was of 7 bp. To permit alignment with the Syringa sequence, two additional gaps of 1 bp (I and L) were positioned in the Fraxinus sequences (Fig. 2). Some of the indels greater than 1 bp were associated with similar flanking sequences (data not shown). For instance, the two GC insertions in F. excelsior/F. excelsior KB (position 70) and in F. mandshurica/F. platypoda (position 95) were preceded by a (GC) 2 motif. Similarly, the 7-bp deletion at position 360, 58 (GCG)2C 38, shared by the species of section Bumelioides (except for F. nigra), resembled the

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the interspecific divergence values were lower (0.7 to 6.5%, mean 2.8 6 3.0%) than those encountered in subgenus Fraxinus (3 to 21%, mean 12.2 6 5.7%). For many interspecific sequence comparisons, a bias in the transition/transversion ratios (Ts/Tv ratios) was observed: the average Ts/Tv ratio was 1.45 (transitions were more frequent than transversions) although a ratio of 0.5 would be expected if there was no bias (Schlo¨tterer et al., 1994). Plotting Ts/Tv ratios versus sequence divergence values showed that a high bias was encountered between closely related species: the average Ts/Tv ratio was 2.75 6 2.6 when divergence values were less than 5% (Table 2). Phylogenetic Analyses

FIG. 2. The complete ITS1 and ITS2 sequences of Syringa vulgaris (outgroup). The position of phylogenetically informative indels is indicated above the sequence, where uppercase letters in boxes indicate deletions and lowercase inserted letters indicate insertions in Fraxinus ITS relative to the S. vulgaris sequence. The mapping of indels on the phylogeny is reported in the legend of Fig. 4. Insertions are as follow: b, C; c, T, or G; d, A; g, GC; h, GC; k, G; m, T; o, C.

flanking sequence found upstream (58 GCG 38). These indels may thus result from replication slippage events (or slipped-strand mispairing), which has been shown to be an important force of evolution of simple repetitive DNA (Levinson and Gutman, 1987). After the introduction of minimal gaps, a total of 442 sites were aligned. Of these sites, 228 were polymorphic (51%) among Fraxinus and Syringa sequences when gaps were not considered as state characters: 132 sites in ITS1 and 96 sites in ITS2. Gaps were ignored in estimating phylogenetic trees even though they are shown below to be useful phylogenetic characters. For parsimony analysis, 142 polymorphic sites were found informative among the sequences of Fraxinus and the outgroup Syringa (89 in ITS1 and 53 in ITS2). Sliding windows of nucleotide divergence showed two conserved regions from bases 107 to 139 of ITS1 (13% of variable sites) and from bases 121 to 150 of ITS2 (10% of variable sites). Sequence Divergence DNA divergence values (uncorrected p-distances, Kumar et al., 1993) are shown in Table 2. The average sequence divergence between Fraxinus and Syringa was 22.1% (62%). The average sequence divergence between Fraxinus taxa was 13.5% (65.4%). The highest value (21%) occurred between F. cuspidata and F. syriaca. The lowest value (0.7%) was observed between F. chinensis and F. longicuspis. The average sequence divergence value between the two subgenera Ornus and Fraxinus was 14% (62.9%). In subgenus Ornus,

In Fraxinus, like the majority of the angiosperm families studied, the sequence length, G 1 C content, and evolutionary rate of ITS1 and ITS2 were similar enough to allow ITS1 and ITS2 to be used in combination for the phylogenetic analyses (see Baldwin et al., 1995). With parsimony analysis, three most parsimonious trees of 441 steps were found. The consistency index was 0.71 when uninformative characters were included and 0.64 when they were excluded. The retention index was 0.83. A consensus of most parsimonious trees derived from this analysis is shown in Fig. 3. The topologies obtained from the neighbor-joining analysis of substitution rates calculated with the one- or the two-parameter methods were identical, and, therefore, only the results obtained with the two-parameter method are presented (Fig. 4). The 50%-bootstrap consensus tree obtained from NJ analysis had a topology quite similar to the consensus tree obtained from parsimony analysis. It showed only one polytomy, within section Melioides, that included F. pennsylvanica taxa, F. velutina, F. tomentosa, and F. anomala (Fig. 4). Infrageneric Taxa For F. americana, F. quadrangulata, F. longicuspis, and F. tomentosa, ITS sequences were determined from each of two individuals. Minimal intraspecific nucleotide variation was observed. The two individuals of F. americana showed a maximum of 1.8% divergence (Table 2) as their sequences differed by only two nucleotides. NJ and parsimony analyses confirmed infrageneric taxa as individuals from the same species were regrouped together with variable bootstrap support (Figs. 3 and 4). Within section Ornaster, the two individuals of F. longicuspis formed a coherent group only with the NJ analysis. The consensus of three most parsimonious trees showed a polytomy between the individuals of F. longicuspis and F. rhynchophylla (Fig. 3). Subgenus Fraxinus–Section Melioides Besides the earlier divergence of F. quadrangulata (see below), the neighbor-joining tree showed a separation between, on one hand, the taxa from the sections Melioides and Dipetalae and on the other hand, the Asian F. platypoda regrouped with the other North

F. excelsior F. excelsior KB F. oxyphylla F. pallisae F. syriaca F. mandshurica F. platypoda F. nigra F. quadrangulata (1) F. quadrangulata (2) F. chinensis F. rhynchophylla F. longicuspis (1) F. longicuspis (2) F. ornus F. penn. subin. F. tomentosa (1) F. tomentosa (2) F. penn. aucub. F. pennsylvanica F. velutina F. americana (1) F. americana (2) F. biltmoreana F. latifolia F. anomala F. cuspidata Syringa vulgaris

0.3 0.7 1.2 0.6 0.8 1.0 1.4 1.5 1.5 1.3 1.3 1.2 1.3 1.2 1.0 1.0 1.0 1.2 1.0 0.8 1.0 1.2 1.0 0.9 0.8 1.0 1.9

b

Uncorrected p-distances. No transversion present. c No transition present.

a

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28.

1

3

2.4 6.2 7.4 0.7 1.0 1.3 0.6 0.7 0.9 0.7 1.4 0.8 1.1 1.3 1.3 1.2 1.2 1.2 1.1 1.0 1.1 1.0 1.1 0.9 1.2 1.1 1.1 1.1 0.9 0.8 0.8 0.8 0.9 0.8 0.9 0.9 0.9 0.8 0.6 0.8 0.9 0.9 1.0 1.0 0.9 0.8 0.9 0.7 0.7 0.7 0.8 0.7 1.6 1.5

2

5

6

5.3 8.1 9.3 5.3 7.8 8.4 2.2 5.9 11.2 3.9 9.7 0.8 11.4 1.0 0.5 1.1 0.6 0.5 1.7 0.7 0.8 1.4 1.0 1.1 1.4 1.0 1.1 1.2 0.9 1.1 1.2 0.9 1.1 1.2 0.9 1.1 1.3 0.9 1.1 1.2 0.9 1.0 1.0 0.7 0.9 0.9 0.7 0.9 1.0 0.8 0.9 1.1 0.8 0.9 1.0 0.8 0.8 0.9 0.6 0.7 1.0 0.8 0.8 1.1 0.8 0.9 1.0 0.7 0.9 0.9 0.8 0.8 0.8 0.6 0.8 0.9 0.7 0.8 1.6 1.3 1.6

4

8

9

10

11

5.2 7.8 18.9 19.3 15.8 5.2 7.9 19.4 19.9 16.8 8.3 10.0 19.8 20.2 17.2 6.5 8.4 18.9 19.4 16.3 9.0 10.2 22.1 22.6 19.5 5.4 8.8 19.7 20.1 17.8 6.6 19.2 19.6 16.1 0.8 18.0 18.5 15.0 1.3 1.2 0.7 9.6 1.2 1.2 2.0 10.3 1.0 1.0 1.6 1.6 1.0 1.1 1.6 1.6 b 0.9 1.0 1.4 1.4 2.0 1.0 1.1 1.6 1.6 4.0 1.0 1.1 1.4 1.4 0.9 0.9 1.1 2.5 2.4 2.0 0.8 1.1 2.5 2.4 1.9 0.9 1.1 2.5 2.5 1.9 1.0 1.2 2.8 2.8 2.3 0.9 1.1 2.0 2.0 1.7 0.7 0.8 1.6 1.6 1.4 0.8 1.1 2.4 2.4 1.8 0.9 1.1 2.4 2.4 1.8 0.9 1.2 3.0 2.9 2.1 0.8 1.2 1.8 1.8 1.5 0.7 0.8 1.7 1.7 1.2 0.8 0.9 1.5 1.5 1.7 1.7 1.5 1.7 1.7 1.8

7

0.7 1.1 1.7 1.6 1.6 1.8 1.4 1.2 1.5 1.7 1.9 1.3 1.1 1.6 1.7

2.0 1.0 1.9 1.8 1.8 2.1 1.6 1.3 1.8 1.9 2.0 1.5 1.2 1.7 1.8

c

16.2 17.2 17.6 16.7 19.9 18.4 16.8 15.9 10.0 10.7 0.7 0.5

13

15.7 16.7 17.1 16.2 19.4 18.0 16.3 15.4 9.5 10.2 0.5

12

1.2 1.8 1.7 1.7 2.1 1.5 1.3 1.8 2.1 2.1 1.6 1.2 1.7 1.8

16.3 17.3 17.2 16.8 19.5 18.1 16.8 15.5 9.8 10.5 1.2 0.7 1.2

14

1.8 1.7 1.7 2.0 1.5 1.3 1.7 1.7 1.9 1.4 1.2 1.5 1.5

18.1 19.1 18.6 18.5 21.7 19.2 18.3 17.1 10.5 11.2 5.4 6.0 6.3 6.5

15

2.0 b

1.6 0.4 4.0 3.2 6.0 2.0 0.5 1.8 2.2

6.0 b

1.8 0.4 4.7 4.0 7.0 1.9 0.7 1.9 2.3

b

14.3 14.8 15.5 14.0 17.8 15.4 14.0 15.5 12.1 12.8 10.7 10.9 11.3 11.4 13.3 0.7

17

14.6 15.1 16.0 14.3 18.1 15.7 14.2 15.7 12.4 12.8 11.2 11.4 11.8 11.9 13.7

16

11 1.8 0.7 3.5 3.2 4.7 1.8 0.6 1.8 2.2

15.2 15.3 16.4 14.5 18.3 15.8 14.9 16.4 13.0 13.7 11.4 11.6 12.0 12.1 13.9 1.6 0.7

18

1.4 1.0 5.0 3.8 7.5 2.0 1.2 2.3 2.4

16.0 16.2 17.3 15.5 19.2 16.3 15.6 16.6 13.3 14.0 12.1 12.3 12.5 12.8 14.7 1.9 2.1 2.8

19

0.8 2.1 2.1 2.7 1.5 0.8 1.7 2.1

16.2 17.0 17.3 16.2 19.9 16.6 16.0 17.3 13.9 14.4 13.0 13.2 13.4 13.5 15.3 2.6 3.0 3.9 2.8

20

1.1 1.2 1.7 0.9 0.4 1.3 1.7

15.9 16.2 16.8 15.2 18.5 17.0 15.5 16.6 14.1 14.8 12.9 13.1 13.6 13.6 15.0 3.1 3.0 4.0 4.2 4.9

21

3.0 2.3 1.3 1.3 2.4 2.3

15.0 15.8 16.9 15.3 18.8 16.1 14.7 15.9 11.2 11.9 10.3 10.2 10.4 11.0 12.4 4.0 3.5 4.2 4.2 5.8 5.9

22

3.3 1.4 1.3 2.4 2.3

16.5 17.2 18.3 17.0 20.0 17.8 16.3 16.9 12.6 13.3 11.7 12.1 12.3 12.1 13.1 4.7 4.9 5.8 5.6 7.2 7.3 1.9

23

0.4 1.4 2.2 2.3

15.1 15.9 16.1 14.9 18.5 16.5 15.0 16.6 12.2 12.9 11.0 11.2 11.5 11.7 13.4 3.8 3.3 4.0 4.0 6.1 5.7 2.4 3.1

24

1.1 1.7 1.9

14.8 15.6 15.8 14.6 18.1 16.4 14.9 15.7 12.3 13.0 12.1 12.1 12.5 12.6 14.2 5.4 4.9 5.8 5.6 7.7 7.5 3.7 5.1 2.6

25

1.3 1.8

18.1 18.6 19.5 17.9 21.3 18.7 17.7 18.5 15.6 16.2 13.5 13.9 14.3 14.4 15.6 4.6 4.2 4.9 6.0 6.9 7.0 7.0 7.9 6.8 8.6

26

Pairwise Uncorrected DNA Divergence Values a (%) (above Diagonal) and Transition/Transversion Ratios (below Diagonal) between Taxa for ITS1 and ITS2 Combined Data

TABLE 2

2.3

17.4 18.4 19.1 17.9 21.4 18.5 18.0 17.8 12.3 13.0 13.1 13.2 13.7 14.0 15.6 11.0 10.5 11.1 12.1 13.0 13.4 11.8 13.5 12.0 12.6 13.0

27

25.2 26.2 27.5 25.8 28.6 27.1 25.9 25.0 18.7 19.4 19.5 19.7 20.1 19.3 21.2 22.6 22.1 22.7 23.0 24.4 24.2 21.4 21.8 21.6 21.4 25.5 24.7

28

PHYLOGENY AND PHYLOGEOGRAPHY OF Fraxinus

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JEANDROZ, ROY, AND BOUSQUET

FIG. 3. Strict consensus of the three minimal trees (441 steps) obtained from parsimony analysis of ITS1 and ITS2 sequences combined. Numbers on branches indicate bootstrap values from 500 replicates (in %). NA, North America; AS, Asia; EU, Europe. A posteriori rooting of the tree with Jasminum attenuatum did not change the position of the root (see results).

American taxa of the section Bumelioides (Fig. 4). These three sections appeared essentially as a trichotomy in the parsimony tree (Fig. 3). The section Melioides was divided into two groups: one composed of F. pennsylvanica, F. velutina, F. tomentosa (three taxa known as the red-ash complex), and F. anomala, and the second of F. americana, F. biltmoreana (two taxa known as the white-ash complex), and F. latifolia. Each of these two groups was strongly supported by high bootstrap values with both parsimony and neighbor-joining analyses (Figs. 3 and 4). Fraxinus platypoda, an Asian species of section Melioides, was placed within the Bumelioides group in both trees. Subgenus Fraxinus–Section Bumelioides The separation between the two subsections Paniculatae (F. excelsior, F. mandshurica, and F. nigra) and Racemosae (F. oxyphylla, F. pallisae, and F. syriaca) was not well defined (Figs. 3 and 4). DNA divergence values between F. mandshurica or F. nigra and F. excelsior (9.2 or 7.8%, respectively), all of subsection Paniculatae, were higher than the value of 6.1% observed between F. excelsior and F. oxyphylla (subsection Racemosae) (Table 2). Fraxinus excelsior (subsection

Paniculatae) was more closely related to the taxa of the subsection Racemosae than to the other species of the subsection Paniculatae (Figs. 3 and 4). F. excelsior and F. oxyphylla are sympatric and some cases of natural interspecific hybridization have been reported between these species (Jeandroz et al., 1995). The phylogenetic relationships of species in these subsections, as determined by analysis of ITS sequences, seemed to correspond better to the geographic distribution of the taxa rather than to the current taxonomy (Figs. 3 and 4). The North American species F. nigra grouped with the Asian and European ashes rather than with other North American species of its section, F. quadrangulata. F. quadrangulata was sister group to the remaining species of Fraxinus sampled and was distantly related to the other members of section Bumelioides. This position was supported by parsimony and neighborjoining analyses as well as by a significant doublebootstrap estimate (P . 0.95) (Fig. 4). An a posteriori analysis using a second Oleaceae as outgroup, a sequence from Jasminum attenuatum (GenBank No. U82920 for ITS1 and U82921 for ITS2, average divergence value of 33% with Fraxinus species) showed the

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FIG. 4. 50% bootstrap consensus tree obtained from the neighbor-joining analysis of numbers of substitutions estimated from the two-parameter method of Kimura (1980) for ITS1 and ITS2 regions combined. Numbers on branches indicate bootstrap estimates from 500 replicates (in %). Parametric bootstrap values obtained a posteriori from 5000 replicates by double-bootstrapping (Zharkikh and Li, 1995) are indicated in parentheses. Indels (except for autapomorphies) are indicated above each branch, where uppercase letters indicate deletions and lowercase indicate insertions relative to the Syringa vulgaris sequence (see Fig. 2 for definition). NA, North America; AS, Asia; EU, Europe. A posteriori rooting of the tree with Jasminum attenuatum did not change the position of the root (see results).

same placement of the root of the trees either with parsimony or neighbor-joining method, and therefore confirmed F. quadrangulata as sister group to all other Fraxinus species. Because of this early divergence of F. quadrangulata, section Bumelioides appeared polyphyletic. Constraining the parsimony analysis to force section Bumelioides into a monophyletic group produced a minimal-tree length of 12 additional steps. When F. quadrangulata was constrained to be part of a monophyletic group with the other members of subgenus Fraxinus, the most parsimonious tree was six steps longer. Subgenus Ornus The taxonomic division of subgenus Ornus into sections Ornus and Ornaster was supported by both parsimony and neighbor-joining trees. Other references to the taxonomy of this subgenus are limited by the minimal taxon sampling for this mainly Asian group. Indels Although excluded from the phylogenetic analyses, 15 of 22 indels (Fig. 2) appeared phylogenetically informative when mapped on the NJ tree (Fig. 4). These indels are reported with reference to the less divergent outgroup sequence, from Syringa (Fig. 2). Five indels of

1 bp and two of 2 bp were autapomorphies and were not reported on the tree. I and L are deletions common to all the species of the genus Fraxinus. d is an insertion shared by the two individuals of F. quadrangulata. Insertion m was shared by all species of the genus Fraxinus except F. quadrangulata. J and k (deletion and insertion, respectively) were found in all members of section Bumelioides (excepting F. quadrangulata). N was a deletion found in species of section Bumelioides occurring in Asia and Europe, and which formed a clade distinct from the North American F. nigra. The two closely related species F. platypoda and F. mandshurica shared insertion h. A, E, F, b, and c (three deletions and two insertions, respectively) were shared only by members of subsection Racemosae. Fraxinus excelsior and the cultivar F. excelsior KB shared insertions g and o. Floral Characters The states of floral characters used in the current taxonomy of the genus were also mapped on the NJ tree to study character transformation (Fig. 5). Following the current taxonomy, the species belonging to subgenus Ornus have complete flowers with calyx and corolla and have been given the vernacular name of flowering

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FIG. 5. NJ phylogenetic tree showing transformation of floral characters in Fraxinus. The number of state changes was the same whether ancestral flowers were complete (with calyx and corolla) or completely reduced (without calyx and corolla). Asterisks indicate polyploid taxa.

ashes. Within subgenus Fraxinus, the members of section Melioides are characterized by flowers with a calyx but no corolla. The members of section Bumelioides have neither calyx nor corolla. The taxa of section Dipetalae are characterized by flowers with calyx and a corolla of only two petals. The mapping of the floral characters on the phylogenetic tree indicated the homoplasious evolution of floral characters at least twice, whether ancestral flowers were considered completely reduced (no calyx and no corolla) or complete (calyx and corolla present) (Fig. 5). Furthermore, the number of character state changes was the same whether completely reduced flowers or complete flowers were assumed ancestral, and whether the character transformation series involved one or two steps to account for the change between completely reduced and complete flowers. Polyploidy Polyploidy also appeared to have evolved several times in the genus, with polyploid species F. chinensis, F. americana, and F. velutina widely dispersed in the phylogenetic tree (Fig. 5). DISCUSSION ITS Sequences The length of the ITS regions of the 27 taxa of Fraxinus and their high G 1 C content (50 to 75%) were quite similar to those reported for other angiosperm

species (Baldwin et al., 1995). The highly conserved ITS1 sequence motif of flowering plants, GGCRY-(4 to 7n)-GYGYCAAGGAA (Liu and Schardl, 1994) was also found in Fraxinus ITS1 between positions 122 and 142. Liu and Schardl (1994) suggested that this sequence plays a role in the processing of rRNA gene transcripts. A second conserved region was observed in Fraxinus within ITS2 between bases 121 and 150 (10% of variable sites), but this region did not correspond to the conserved motif reported by Liu and Schardl (1994) and Buckler and Holtsford (1996). This region in ITS2 was not as highly conserved as the one found in ITS1. DNA divergence values between Fraxinus species (2 to 22%) were also in the range observed for other angiosperm families and genera (Nickrent et al., 1994) and allowed to reconstruct with quite high levels of confidence the phylogeny of the genus, especially with respect to ingroups. Notably, DNA divergence values at the intraspecific level were much lower than DNA divergence values between species belonging to the same section. Polymorphisms were detected at particular nucleotide sites within individuals in several species of Fraxinus. In some cases, these polymorphisms might have been caused by sequencing artifacts, as they were present on only one strand. When these intraindividual nucleotide polymorphisms were encountered at the same site on both strands, and were found again in the repeated sequencing of these strands, they could be indicative of the presence of multiple rDNA unit types, and therefore, of the presence of ITS variants that differ by a few nucleotides. While the mechanism of concerted evolution is known to homogenize the rDNA arrays, this process might be slowed down by long generation time (Sang et al., 1995) and large effective population size such as encountered in these long-lived mixed-mating Fraxinus species. The polymorphism of rDNA unit types could also result from recent mutational events, or from horizontal transfer by interspecific hybridization among closely related taxa. Fraxinus excelsior and F. oxyphylla are two closely related species known to hybridize in sympatric areas (Jeandroz et al., 1995). Two putative nucleotide polymorphisms in the ITS regions were observed between these two species but the comparison of these polymorphisms did not result in evidence for horizontal gene transfer. A strategy incorporating more taxa and the cloning and sequencing of numerous ITS for each species would need to be implemented to confirm the presence of these polymorphisms and clarify their possible origins. The low level of intraindividual and intraspecific polymorphism observed in this study contrasts with recent studies involving diploid and polyploid species (Kim and Jensen, 1994; Wendel et al., 1995a,b; Sang et al., 1995). These studies have shown that ITS sequences displayed numerous nucleotide polymorphisms, some of which show patterns of additivity, indicating that ITS regions could be useful for detecting hybridization phenomena and cases of reticulate evolution. Of 27 species and subspecies of Paeo-

PHYLOGENY AND PHYLOGEOGRAPHY OF Fraxinus

nia sampled (Sang et al., 1995), 15 showed nucleotide additivity at polymorphic sites, and up to 14 ambiguous sites could be observed per sequence. The validity of phylogenetic analyses could be affected when such large numbers of nucleotide polymorphisms are encountered, particularly in species of allopolyploid origin (Wendel et al., 1995a,b). No extensive intraindividual sequence polymorphisms or patterns of nucleotide additivity were observed in the polyploid Fraxinus species analyzed here (only one or two sites were polymorphic in two of the three polyploid species F. chinensis, F. americana, and F. velutina) or among the other Fraxinus taxa studied. However, the presence of minor DNA types within a given species or individual might not have been detected in this study because the direct sequencing of PCR-amplified products would only sample the most abundant rDNA unit types. The phylogeny of the genus Fraxinus was estimated more accurately with sequences from the ITS regions than with chloroplast DNA sequences (Gielly and Taberlet, 1994). Whereas no interspecific variation was detected between F. oxyphylla, F. excelsior, F. americana, and F. pennsylvanica by sequencing of the chloroplastic trnL intron, or the chloroplast intergenic spacer between the trnL 38 exon and the trnF gene (Gielly and Taberlet, 1994), the ITS regions provided the variability needed to infer phylogeny. Due to their rapid evolution, ITS regions have been shown to be useful in the phylogenetic studies where no or little variation is found within genera for chloroplastic genes such as rbcL (Savard et al., 1993). Nuclear rDNA ITS regions showed an overall substitution rate 1.9 times that of the matK gene in Saxifragaceae (Johnson and Soltis, 1995) and 20 times that of rbcL in Betulaceae (Savard et al., 1993). Considering the sizeable DNA divergence values for ITS between pairs of Fraxinus taxa and the small number of putative nucleotide polymorphisms within species, it appears that ITS sequences are a valuable resource for inferring phylogeny in Fraxinus. Phylogeny of the Genus Fraxinus The trees obtained from the different methods of phylogenetic tree reconstruction (parsimony and NJ) were generally congruent with the current taxonomic classification of the genus. As shown in Figs. 3 and 4, subgenus Ornus formed a coherent group composed of sections Ornaster and Ornus. Subgenus Fraxinus appeared paraphyletic, with F. quadrangulata (section Bumelioides) the sister group to the remainder of the genus. A posteriori rooting of the ingroup network with of a second outgroup (Jasminum attenuatum) did not change this topology although the divergence values between the ingroup sequences and Jasminum attenuatum were higher than with Syringa vulgaris. The remainder of subgenus Fraxinus was monophyletic. Fraxinus cuspidata (section Dipetalae), which had been classified by Miller (1955) in the subgenus Ornus, was grouped with subgenus Fraxinus, close to section Melioides (Figs. 3 and 4). This grouping

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is in agreement with the most recent classification of the genus used in this study (Nikolaev, 1981). The topologies obtained also showed that section Melioides appeared polyphyletic. Based on ITS sequences, F. platypoda (section Melioides) appeared in a group with the ashes of section Bumelioides (particularly F. mandshurica, subsection Paniculatae), rather than with other ashes of section Melioides (Table 2). If the current taxonomy is phylogenetically correct, the ITS sequences of F. platypoda would have converged to the Bumelioides type, coincidentally (it is a noncoding region) or by contact (horizontal gene transfer by interspecific hybridization). However, no precedent has been reported for such a coincidental mode of evolution for ITS (Wendel et al., 1995b), and there are no reported cases of hybridization between F. platypoda and F. mandshurica, its closest relative based on the ITS trees. Phylogenetic trees indicated that floral characters have evolved repeatedly in the genus. Although the topologies estimated could not favor the hypothesis of ancestral flowers being complete over that of being completely reduced, it is likely that ancestral flowers were complete, as other genera in the family Oleaceae show mainly complete flowers (e.g., Fontanesia, Forsythia, Jasminum, Olea, Osmanthus, Syringa, Siphonosmantus; Bean, 1925; Rehder, 1951). Also, it is now generally accepted that the apetalous conditions and the absence of calyx in Angiosperms reflects the loss of parts rather than an original absence (Stebbins, 1974; Cronquist, 1988). If these reasonings are correct, then there would have been parallel loss of corolla in F. platypoda and in taxa from section Melioides. A complete reduction of flowers would have also occurred twice in F. quadrangulata and in the remaining taxa from section Bumelioides. Thus, in Fraxinus, floral characters seems to have evolved repeatedly and therefore may not always be reliable indicators for taxonomic purposes. The same conclusions apply for polyploidy, which appeared to have evolved repeatedly in the genus. Phylogenetically informative indels had been noted in other plant studies analyzing ITS sequences (Baldwin et al., 1995). The informative indels found in the Fraxinus sequences, although excluded from the phylogenetic analyses, strongly supported the topologies obtained (Fig. 4). A 7-bp deletion (N) was encountered exclusively in the Asian and the European members of section Bumelioides, underlying a pattern of geographical differentiation. Similarly, a 1-bp insertion (h) was only shared by F. platypoda and F. mandshurica, supporting the grouping of F. platypoda with the taxa of section Bumelioides. Moreover, deletion d was only found in F. quadrangulata, further supporting the polyphyly of section Bumelioides. Phylogeography Given these results, we can recast the biogeography of the genus in a phylogenetic framework. Two scenarios are presented in Fig. 6, with their underlying assumptions differing only according to the geographic origin of the ancestor of the genus, Asia or North

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FIG. 6. Reconstructions of the biogeographic history of Fraxinus based on the phylogenetic analysis of ITS sequences. (A) Hypothesis assuming a North American origin or (B) an Asian origin for the genus. Both North America and Asia are main centers of diversity. Arrows indicate intercontinental dispersal events.

America, that are the two main centers of diversity. Our scenarios involve several intercontinental dispersal events which would have occurred during the intermittent connections between the North American and Asian land masses during the Middle and Late Tertiary. In establishing these scenarios, we assumed that the presence of some taxa from section Bumelioides in the Middle East and in Europe (F. excelsior, F. oxyphylla, F. pallisae, and F. syriaca) is more likely due to progressive dispersal from easternAsia to Europe than to a quite recent intercontinental migration from North America to Europe. Scenario A. The position of F. quadrangulata in the phylogenetic tree as sister group to the remainder of the genus (Figs. 3 and 4) would indicate a North American origin of the genus with an early divergence of the lineage leading to F. quadrangulata. The North American origin is also supported by the location of the oldest known fossils of the genus found in southeastern North America (Call and Dilcher, 1992). Later, migration to Asia could have led to subgenus Ornus, which is more represented in Asia than the subgenus Fraxinus. The diversification of subgenus Fraxinus into sections Melioides and Dipetalae would have followed in North America. A second intercontinental migration to Asia from a North American ancestor close to the F. nigra type, followed by subsequent westward dispersion, could account for the presence of the remaining taxa mainly of the Bumelioides type in Asia and in Europe. This scenario implies a total of two events of migration from North America to Asia (Fig. 6). Scenario B. This scenario assumes an Asian origin of the genus, which is against all available fossil evidence. The first event would have been an interconti-

nental migration to North America of the lineage leading to F. quadrangulata followed by the emergence of subgenus Ornus in Asia. A second intercontinental migration event to North America would have taken place and then led to the diversification of subgenus Fraxinus into its sections Melioides and Dipetalae, which are mainly represented in North America. A third intercontinental migration would account for the presence of section Bumelioides in North America (F. nigra). Members of section Bumelioides in Asia would have dispersed westward to account for its presence in Europe. The major weakness of this scenario is that section Bumelioides would have migrated twice from Asia to account for the presence of F. quadrangulata and F. nigra in North America. This scenario implies a total of three events of migration (Fig. 6). When we consider the total number of intercontinental migrations, the origin of the genus in North America (scenario A) is more parsimonious, because only two events are necessary to explain the observed geographic distribution of taxa instead of three such events for scenario B. Scenario A is also supported by the oldest known fossils of the genus, found in North America (Call and Dilcher, 1992). Hence, the phylogenetic history of Fraxinus appears to be closely related to the natural distributions of extant taxa. Climatic changes and environmental barriers have likely led to the modification of past natural distributions and to geographic isolation of populations, which is known to play a key role in speciation (Eldredge, 1980). The strong phylogeographical structure observed in this study indicates that these factors were likely a driving force in shaping the diversity of the circumpolar genus Fraxinus.

PHYLOGENY AND PHYLOGEOGRAPHY OF Fraxinus

ACKNOWLEDGMENTS ´ . Morin (Montre´al Botanical Garden) for the opportuWe thank E nity to sample Fraxinus taxa, S. Kelley and T. Forrest (Arnold Arboretum of the Harvard University), and P. Cox (San Antonio Botanical Garden) for kindly providing Fraxinus samples. We also thank K. Dewar (University of Pennsylvania) and two anonymous reviewers for providing helpful comments on previous manuscript drafts. This research was supported by grants to J.B. from the Natural Sciences and Engineering Research Council of Canada (NSERC), the Que´bec Research Fund (FCAR). S.J. was supported by a Lavoisier fellowship from the Ministe`re des affaires e´trange`res of France.

REFERENCES Baldwin, B. G. (1992). Phylogenetic utility of the internal transcribed spacers of ribosomal DNA in plants: An example from the Compositae. Mol. Phylogenet. Evol. 1: 3–16. Baldwin, B. G., Sanderson, M. J., Porter, J. M., Wojciechowski, M. F., Campbell, C. S., and Donoghue, M. J. (1995). The ITS region of nuclear ribosomal DNA: A valuable source of evidence on angiosperm phylogeny. Ann. Missouri Bot. Gard. 82: 247–277. Bean, W. J. (1925). ‘‘Trees and Shrubs Hardy in the British Isles,’’ J. Murray, London. Bousquet, J., Simon, L., and Lalonde, M. (1990). DNA amplification from vegetative and sexual tissues of trees using polymerase chain reaction. Can. J. For. Res. 20: 254–257. Brown, J. H., and Gibson, A. C. (1983). ‘‘Biogeography,’’ Mosby, St. Louis. Buckler, E. S., and Holtsford, T. P. (1996). Zea ribosomal repeat evolution and substitution patterns. Mol. Biol. Evol. 13: 623–632. Call, V. B., and Dilcher, D. L. (1992). Investigations of angiosperms from the Eocene of southeastern North America: Samaras of Fraxinus wilcoxiana Berry. Rev. Paleobot. Palynol. 74: 249–266. Cronquist, A. (1988). ‘‘The Evolution and Classification of Flowering Plants,’’ The New York Botanical Garden, New York. Devereux, J., Haeberli, P., and Smithies, O. (1984). A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 12: 387–395. D’Ovidio, R. (1992). Nucleotide sequence of a 5.8S gene and of the internal transcribed spacers from Populus deltoides. Plant Mol. Biol. 19: 1069–1072. Eldredge, N. (1980) ‘‘Phylogenetic Patterns and the Evolutionary Process,’’ Columbia Univ. Press, New York. Gielly, L., and Taberlet, P. (1994). Chloroplast DNA polymorphism at the intrageneric level and plant phylogenies. C. R. Acad. Sci. Paris/Sciences de la Vie 317: 685–692. Jeandroz, S., Faivre-Rampant, P., Pugin, A., Bousquet, J., and Berville´, A. (1995). Organization of nuclear ribosomal DNA and species-specific polymorphism in closely related Fraxinus excelsior and F. oxyphylla. Theor. Appl. Genet. 91: 885–892. Johnson, L. A., and Soltis, D. E. (1995). Phylogenetic inference in Saxifragaceae sensu stricto and Gilia (Polemoniaceae) using matK sequences. Ann. Missouri Bot. Gard. 82: 149–175. Jukes, T. H., and Cantor, C. R. (1969). Evolution of protein molecules. In ‘‘Mammalian Protein Metabolism’’ (H. N. Munro, Ed.), pp. 21–132, Academic Press, New York. Kim, K.-J., and Jansen, R. K. (1994). Comparisons of phylogenetic hypotheses among different data sets in dwarf dandelions (Krigia, Asteraceae): Additional information from internal transcribed spacer sequences of nuclear ribosomal DNA. Plant. Syst. Evol. 190: 157–185.

251

Kimura, M. (1980). A simple method for estimating evolutionary rate of base substitutions through comparative studies of nucleotide sequences. J. Mol. Evol. 16: 11–120. Kumar, S., Tamura, K., and Nei, M. (1993). MEGA—Molecular Evolutionary Genetics Analysis, V. 1.0, Institute of Molecular Evolutionary Genetics, Pennsylvania State Univ. Levinson, G., and Gutman, G. A. (1987). Slipped-strand mispairing: a major mechanism for DNA sequence evolution. Mol. Biol. Evol. 4: 203–221. Liu, J. S., and Schardl, C. L. (1994). A conserved sequence in internal transcribed spacer 1 of nuclear rRNA genes. Plant Mol. Biol. 26: 775–778. Miller, G. N. (1955). The genus Fraxinus, the ashes in North America, North of Mexico. Memoire Cornell Univ. Agric. Exp. Stn. 335: 1–64. Mu¨ller, J. (1981). Fossil pollen records of extant angiosperms. Bot. Rev. 47: 1–142. Nickrent, D. L., Schuette, K. P., and Starr, E. M. (1994). A molecular phylogeny of Arceuthobium (Viscaceae) based on nuclear ribosomal DNA internal transcribed spacer sequences. Am. J. Bot. 81: 1149–1160. Nikolaev, E. V. (1981). The genus Fraxinus (Oleaceae) in the flora of the USSR. Botanicheskii Zhurnal 66: 1419–1432. Rehder, A. (1951). ‘‘Manual of Cultivated Trees and Shrubs,’’ MacMillan, New York. Saitou, N., and Nei, M. (1987). The Neighbor-Joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4: 406–425. Sang, T., Crawford, D. J., and Stuessy, T. F. (1995). Documentation of reticulate evolution in peonies (Paeonia) using internal transcribed spacer sequences of nuclear ribosomal DNA: Implications for biogeography and concerted evolution. Proc. Natl. Acad. Sci. USA 92: 6813–6817. Savard, L., Michaud, M., and Bousquet, J. (1993). Genetic diversity and phylogenetic relationships between birches and alders using ITS, 18S rRNA, and rbcL gene sequences. Mol. Phylogenet. Evol. 2: 112–118. Schlo¨tterer, C., Hauser, M. T., von Haeseler, A., and Tautz, D. (1994). Comparative evolutionary analysis of rDNA ITS regions in Drosophila. Mol. Biol. Evol. 11: 513–522. Stebbins, G. L. (1974) ‘‘Flowering Plants. Evolution above the Species Level,’’ The Belknap Press of Harvard Univ. Press, Cambridge. Swofford, D. L. (1993). PAUP: Phylogenetic Analysis Using Parsimony, Version 3.1.1. Illinois Natural History Survey, Champaign, Illinois. Wendel, J. F., Schnabel, A., and Seelanan, T. (1995a). Bidirectional interlocus concerted evolution following allopolyploid speciation in cotton (Gossypium). Proc. Natl. Acad. Sci. USA 92: 280–284. Wendel, J. F., Schnabel, A., and Seelanan, T. (1995b). An unusual ribosomal DNA sequence from Gossypium gossypioides reveals ancient, cryptic, intergenomic introgression. Mol. Phylogenet. Evol. 4: 298–313. White, T. J., Bruns, T., Lee, S., and Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In ‘‘PCR Protocols, a Guide to Methods and Applications’’ (M. A. Innis, D. H. Gelfang, J. J. Sninsky, and T. J. White, Eds.), pp. 315–322, Academic Press, San Diego. Yokota, Y., Kawata, T., Iida, Y., Kato, A., and Tanifuji, S. (1989). Nucleotide sequences of the 5.8S rRNA gene and transcribed spacer regions in carrot and broad bean ribosomal DNA. J. Mol. Evol. 29: 294–301. Zharkikh, A., and Li, W. H. (1995). Estimation of confidence in phylogeny: Complete- and partial-bootstrap technique. Mol. Phylogenet. Evol. 4: 49–63.