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0 CB#3280, Department of Biology, University of North Carolina, Chapel Hill, North Carolina 27599 USA
Received for publication November 3, 1998. Accepted for publication September 10, 1999.
ABSTRACT
We investigated phylogenetic and biogeographic relationships within Juglans (walnuts), a Tertiary disjunct genus, using 15 species of Juglans and related (Juglandaceae) outgroups. The relationships were analyzed using nucleotide sequences of the chloroplast gene matK and its flanking spacers and of the internal transcribed spacers (ITS) and 5.8S gene of the nuclear ribosomal DNA. The DNA sequences provided 246 informative characters for parsimony analysis. ITS data supported as monophyletic groups the four generic sections, Cardiocaryon, Dioscaryon, Rhysocaryon, and Trachycaryon. Within Rhysocaryon, the temperate black walnuts and the tropical black walnuts were supported as monophyletic groups. When the two data sets were combined, J. cinerea was nested within Cardiocaryon. Combined analysis with published nuclear DNA restriction site data placed J. cinerea in a monophyletic group with Cardiocaryon. These analyses consistently supported Juglans as a monophyletic group and as the sister group to the genus Pterocarya. The results of this work are consistent with the known geological history of Juglans. The fossil record suggests that the butternuts had evolved by the early Oligocene in North America. The presence of butternuts in Eurasia could be the result of migration from North America to Eurasia during the warming trend of the mid Oligocene.
Key Words: ITS • Juglandaceae • Juglans • matK • phylogeny • Tertiary disjunct • walnut
The study of the floristic similarity among the temperate forests of North America, Europe, and Asia dates back to the 18th century, when Halenius, a student of Linnaeus, defended a dissertation on the similarity of nine eastern North American and eastern Asian plants (Boufford and Spongberg, 1983
). An array of early botanists (including Halenius, 1750
; Kalm, 1770
; Nuttall, 1818
; Pursh, 1814
; Thunberg, 1784
) also recognized the floral similarity of the two regions. Asa Gray (1856, 1859, 1873)
, encouraged by his correspondence with Charles Darwin (Darwin, 1887, 1903
; Gray, 1893
), studied the disjunction from the 1850s on into the 1870s. Asa Gray felt that the contraction of a once widespread temperate flora had caused the similarity between the Asian and North American floras. Later scientists (Axelrod, 1959
; Chaney, 1940, 1947
) recognized that this disjunction spanned all the temperate forests of the northern hemisphere, and expanded on Gray's theory. Axelrod and Chaney believed that a temperate Arcto-Tertiary Geoflora, found throughout the high northern latitudes during the Eocene (50 million years before present), had migrated southward and been restricted in range as a result of a gradual, global cooling and drying trend. Scientists (Yurtsev, 1972
; Wolfe, 1978
; Tiffney, 1985a, b
) now know that a complex history of tectonics, climate fluctuations, repeated emergence and submergence of land bridges, multiple migrations, and other factors have led to the current distribution of northern temperate forests, which were contiguous during the Miocene (15 million years before present), when the northern continents were themselves in contact (Davis, 1981
; Tiffney, 1985a, b
; Delcourt and Delcourt, 1987
).
The purpose of this study was to examine the biogeography of the genus Juglans L. as a function of Juglans' phylogeny, as well as to address disagreement about the taxonomy of Juglans. Juglans is a Tertiary disjunct with ~21 species (Manning, 1978
) occurring from temperate northern China to the arid southwestern United States to the cloud forests of tropical South America. Its range (Fig. 1) includes eastern and western Asia, southern Europe, eastern and western North America, Central America, western South America, and the West Indies (Meusel, Jager, and Weinert, 1965
; Wilken, 1993
). Walnuts are among the most economically important nut trees in the world, and two species, J. nigra L. (eastern black walnut) and J. regia L. (Persian walnut), are widely cultivated for this reason (Jaynes, 1969
; Woodruff, 1979
). Juglans nigra is also important in commercial wood production. Most walnut species, however, are of low economic value and are used only occasionally as timber or as a source of brown dye. Consequently, certain species of Juglans have been well studied, while little is known about others. This study includes three taxa not included in previous molecular studies of the genus: J. boliviana (C. DC.) Dode, J. guatemalensis Mann., and J. cathayensis var. formosanum Hayata.
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; Miller, 1976
; Manning, 1978
; Fjellstrom and Parfitt, 1995
; Whittemore and Stone, 1997
) have been largely in agreement about the classification within Juglans. In the only published revision of the genus, Dode (1906, 1909a, b)
divided Juglans into four sections based on leaf and flower morphology: Dioscaryon Dode (traditionally Juglans), Rhysocaryon Dode (black walnuts), Cardiocaryon Dode (Asian butternuts), and Trachycaryon Dode (American butternut). The four sections were also supported by Manning's (1978)
work on fruit and nut morphology. Manning did disagree with Dode about the number of species in the genus. For instance, within Rhysocaryon, Manning (1978)
recognized J. californica S. Wats. and J. major (Torr. ex Sitsgr.) Heller as distinct species, while Dode (1909b)
considered them to be synonymous. Another species of interest is J. guatemalensis, which Manning named as a new species (Manning, 1952
), but later merged with J. olanchana Stadl. et L. O. Williams (Manning, 1957
). All four of the aforementioned taxa (J. californica, J. major, J. guatemalensis, and J. olanchana) have been included in this study.
Dioscaryon contains just one species, J. regia (Persian walnut), which is native to southeastern Europe and western Asia Minor. The 16 species of black walnuts, section Rhysocaryon, are found only in America. Based on differences in wood anatomy between the black walnuts of the United States and northern Mexico and the tropical black walnuts of southern Mexico, Guatemala, and South America, Miller (1976)
suggested dividing Rhysocaryon into two groups, the north-temperate black walnuts and the tropical black walnuts. The butternuts are divided into two sections, the Asian section Cardiocaryon, containing three species native to China, Korea, and Japan and the American section Trachycaryon, consisting solely of J. cinerea L. of the eastern United States (Dode, 1906, 1909a, b
; Manning, 1978
). Miller (1976)
also found evidence from wood anatomy supporting the placement of all the butternuts into one group, but he did not suggest uniting the butternuts in a single section. The recent nuclear RFLP (restriction fragment length polymorphism) work of Fjellstrom and Parfitt (1995)
supported Dioscaryon (or Juglans), Rhysocaryon, the butternuts (Cardiocaryon plus Trachycaryon), Cardiocaryon, and Trachycaryon as monophyletic groups. The molecular study (Fjellstrom and Parfitt, 1995
) did not support the temperate black walnuts or the tropical black walnuts as monophyletic lineages.
According to various authors (Heimsch and Wetmore, 1939
; Conde and Stone, 1970
; Smith and Doyle, 1995
), Pterocarya Kunth is the genus most closely related to Juglans. A cladistic analysis (Smith and Doyle, 1995
) reported that Carya Nutt. was the sister group to the Juglans–Pterocarya clade, and Platycarya Sieb. et Zucc. the basal sister group to the Carya–(Juglans–Pterocarya) group. Three more genera of the Juglandaceae, Alfaroa Standl., Engelhardia Lesch. ex Blume, and Oreomunnea Oerst., were reported to be more distantly related to Juglans than Pterocarya, Carya, and Platycarya (Smith and Doyle, 1995
). Cyclocarya Iljinsk., considered to be another Juglandaceous genus by some authors (Iljinskaya, 1953
; Manchester, 1987
), has often been considered to be congeneric with Pterocarya (Leroy, 1955
; Conde and Stone, 1970
; Manning, 1978
).
To further explore the systematics of Juglans, our paper presents a phylogenetic analysis of nucleotide sequences from 15 walnut species and four outgroup taxa using the internal transcribed spacer (ITS) region of nuclear ribosomal DNA and the plastid gene matK with its surrounding spacers. The ITS region consists of two internal transcribed spacers, ITS1 and ITS2, and the intervening 5.8S nuclear ribosomal gene. The small and rather conserved 5.8S region does not yield many characters for phylogenetic analysis, even at the division and class levels (Jorgensen and Cluster, 1988
; Hillis and Dixon, 1992
). However, the highly variable ITS1 and ITS2 regions are commonly used for species-level comparisons—a level at which few genes have provided sufficient characters for phylogenetic analysis (Baldwin, 1992
; Baldwin, et al., 1995
; Wen and Zimmer, 1995
; Moller and Cronk, 1997
; Stanford, Harden, and Parks, 1997
; Vargas, Morton, and Jury, 1999
).
The plastid gene matK, previously known as orfK, is a maturase-encoding gene located in the intron of trnK (the transfer RNA gene for lysine), found in the large, single-copy region of the chloroplast genome. The matK gene, which is more variable than the widely sequenced rbcL gene, has been used in molecular systematics at the genus and family levels (Steele and Vilgays, 1994
; Johnson and Soltis, 1995
; Liang and Hilu, 1996
; Kron, 1997
; Manos and Steele, 1997
).
MATERIALS AND METHODS
We were able to obtain 16 Juglans taxa, representing all four sections of the genus, for this analysis. Included were 15 species widely accepted in the literature plus two varieties of J. cathayensis Dode, J. cathayensis var. cathayensis and J. cathayensis var. formosanum. Four outgroups, Carya, Cyclocarya, Platycarya, and Pterocarya, were also sequenced for this study. These outgroups were chosen based on previous phylogenetic analyses (Heimsch and Wetmore, 1939
; Conde and Stone, 1970
; Smith and Doyle, 1995
). The matK analyses were also performed using as outgroups sequences taken from GenBank for Alfaroa (GenBank accession number GBAN-U92849), Betula (GBAN-U92853), Casuarina (GBAN-U92858), Comptonia (GBAN-U92857), and Myrica (GBAN-U92857). (The prefix GBAN-has been added for linking the online version of American Journal of Botany to GenBank, but is not part of the actual GenBank accession number). All taxa were vouchered as herbarium specimens and deposited at the University of North Carolina Herbarium in Chapel Hill (NCU) (Table 1).
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) leaf tissue using a modified version of the hot cetyltrimethylammonium bromide (CTAB) method outlined by Doyle and Doyle (1987)
. Amplifications of the ITS region of the nrDNA were obtained using the ITS5A/ITS4 primer pair (Table 2). Amplifications of the matK region were primarily performed using primers trnK1 and trnK2r (located within the flanking trnK gene), and sometimes using primers matK161f (located within the matK gene) and trnK2r (Table 2). Double-stranded DNA amplifications were performed in a 25-µL volume containing 12 µL of sterile distilled water, 4 µL of 200 µmol/L dNTPs in equimolar ratio, 2.5 µL of 10X Taq DNA Polymerase buffer (Promega, Madison, Wisconsin, USA), 1.5 µL 25 mmol/L MgCl2, 2.5 µL dimethyl sulfoxide (DMSO), 0.5 µL each of 10 mmol/L primer, 0.125 µL 10% BSA, 0.5 units of Taq DNA Polymerase (Promega), and 1 µL of genomic DNA (1–10 ng). For DNA amplifications, the first cycle was 1 min at 94°C for denaturation, 1 min at 50°C for annealing, and 1.5 min at 72°C for primer extension. Primer extension time was increased by 3 s in each cycle; an additional 10 min of extension time followed the final cycle. Prior to manual sequencing, 40 cycles were used, and prior to automated sequencing, 30 cycles were used. Positive and negative controls were included in each set of amplifications. Except for Cyclocarya, all of the taxa listed in Table 1 were sequenced for both the ITS and the matK regions. Cyclocarya was sequenced for the ITS region only.
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Initially, sequences were generated according to manual dideoxy nucleotide sequencing methods outlined by Chase et al. (1993)
, using SequenaseTM (USB, Cleveland, Ohio, USA) and alpha 32P labeled dATP. However, most data were generated by cycle sequencing, and the sequences were produced through automated sequencing on a Perkin-Elmer (Norwalk, Connecticut, USA) Applied Biosystems, Inc. model 377 according to the manufacturer's protocols. For sequencing of the ITS region, primers ITS2, ITS3, ITS4, and ITS5A were used (Table 2). For sequencing of the matK region, primers trnK1, matK161f, matK720f, matK4, matK5, matK7, matK77, trnK2r, matK7r, matK820r, matK1290r, and matK1300r, were used (Table 2). Autoradiograms generated through manual sequencing were read by sight, rechecked, and recorded by hand. Sequences generated through automated methods were manually edited and assembled into a consensus sequence. Sequences were aligned manually. All sequences can be obtained through GenBank (Table 1).
The data were analyzed using PAUP 3.1 (Phylogenetic Analysis Using Parsimony; Swofford, 1993
) software. Pairwise distances were calculated for the data using the PAUP 3.1 software. All informative base-pair differences were used in the analyses, and indels were coded plus(1)/minus(0) or treated as missing data. Uninformative characters were excluded from the analysis. In analyses of matK, all indels were treated as missing data. In analyses of ITS, data were analyzed twice: in one analysis, five indels, coded for presence or absence were included. The ITS data were then reanalyzed with indels treated as missing data. Based on previous analyses of the Juglandaceae (Heimsch and Wetmore, 1939
; Conde and Stone, 1970
; Smith and Doyle, 1995
), outgroups were specified as paraphyletic to the ingroup. Branch-and-bound searches were executed to identify the most parsimonious trees. Several statistical measures were calculated, including bootstrap (Felsenstein, 1985
) with 100 replicates, consistency indices (Kluge and Farris, 1969
), decay indices (Bremer, 1994
), retention indices (Farris, 1989
), homoplasy indices (Kluge and Farris, 1969
), and Hillis and Huelsenbeck's g1 statistic (1992)
, obtained by generating 1 000 000 random trees. Separate phylogenetic analyses of the matK spacers, the matK coding region, the entire matK region, the ITS region alone, and the combined matK and ITS data sets were conducted. Because transitions were 1.8 times more common than transversions in the ITS data set, an analysis of ITS data was also performed with transversions weighted 1.8 times more than transitions. A combined analysis of the molecular data (ITS and matK) generated in this study and the RFLP data generated by Fjellstrom and Parfitt (1995)
was also conducted.
To study the biogeography of the genus, species distributions were mapped onto the phylogeny to create an area cladogram for Juglans.
RESULTS
The aligned matK data matrix consisted of 2427 nucleotide sites. Of these sites, only 173 were parsimony informative. The matK region yielded a minimum of 50 pairwise differences at the species level in this study. The ratio of transitions to transversions in the matK data set was 1 to 1.1. The aligned ITS data matrix contained 658 sites, of which 73 were parsimony-informative. The ratio of transitions to transversions in the ITS data set was 1.8 to 1. Sequences for either region proved to be identical within one species and nonidentical between species, including controversial species J. californica, J. major, J. guatemalensis, and J. olanchana.
All analyses supported Cyclocarya as a separate genus from Pterocarya, and Juglans as the monophyletic sister group to Pterocarya. Within Juglans, analyses of all but the matK data supported section Rhysocaryon as the sister group to the other three Juglans sections. All combined analyses supported a temperate clade and a tropical clade of black walnuts; the tropical clade included J. olanchana.
Separate analysis of the matK spacer regions (5' and 3') and coding region yielded trees that were largely unresolved due to the small number of informative characters contained in any one of the three regions.
When the entire matK region, including both spacers and the coding region, was analyzed, pairwise distances ranged from 0.7% within Rhysocaryon to 9.3% between J. boliviana and J. cathayensis. The same results were obtained both when Alfaroa, Betula, Casuarina, Comptonia, and Myrica were used as outgroups and when they were not. (These genera have therefore been excluded from our cladograms.) The search yielded four most parsimonious trees (Fig. 2), which differed only in the placement of J. major. The consensus tree contained a Cardiocaryon clade with J. ailantifolia Carr. and J. cathayensis as the sister group to J. mandshurica Maxim. Juglans regia was the sister group to Cardiocaryon, and the Cardiocaryon–Dioscaryon clade was the sister group to a Rhysocaryon–Trachycaryon clade. Bootstrap values were <50% for most branches, and decay values were between one and two steps.
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When the combined ITS and matK data sets were analyzed, three most parsimonious trees, differing only in the arrangement within the J. major–J. microcarpa–J. nigra clade, resulted (Fig. 4). The consensus tree was similar to the tree obtained from analysis of the ITS data set alone. The topology within Rhysocaryon was both more resolved and better supported than within the ITS-based tree. Within the tropical black walnut clade, J. olanchana was the sister group to the four species found farther south, J. guatemalensis was the sister group to the species found south of it, and J. neotropica Diels was the sister group to J. australis Griseb. and J. boliviana. The temperate black walnut clade contained two groups, a "western" clade containing J. californica and J. hindsii (Jeps.) Rehder and an "eastern" clade containing J. major, J. microcarpa, and J. nigra.
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was conducted. Analysis of 289 potentially informative characters yielded just two most parsimonious trees (Fig. 5) that differed only in the placement of J. cinerea and J. mandshurica. In these trees, Rhysocaryon was better resolved than in the ITS/matK trees. The trees had essentially the same topology as the trees resulting from the combined analysis of the ITS and matK data. The Mexican species J. mollis Engelm. ex Hemsl. (which was not included in the ITS and matK studies) fell within the temperate black walnut clade. The United States' black walnut species formed a monophyletic sister group to J. mollis.
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Phylogeny
Section Rhysocaryon was well supported as a monophyletic group in these analyses. Analyses of ITS, total sequence data, and combined sequence and RFLP data yielded a monophyletic Rhysocaryon, with a decay index greater than ten and a bootstrap value of 100% in analysis of total sequence data (Fig. 4). Although matK data did not present any well-supported hypothesis for relationships within Rhysocaryon, ITS data and total data analyses suggested that Rhysocaryon contains two monophyletic groups. The analysis supported dividing Rhysocaryon into two subclades, a temperate black walnut clade and a tropical black walnut clade. This same division was suggested by Miller (1976)
. Within the temperate black walnut clade, two monophyletic groups were supported: a clade of J. californica and J. hindsii (the two California species) and a clade of J. microcarpa, J. major, and J. nigra. Within the tropical black walnuts, the northernmost species, the Mexican J. olanchana, is the basal sister group to all the more southerly taxa, J. guatemalensis, J. neotropica, J. australis, and J. boliviana. Of those four species, the northernmost, J. guatemalensis, is the basal sister group to the remaining three. It seems likely that southward migration has been closely followed by speciation in the tropical species of the genus. All analyses supported J. californica, J. major, J. guatemalensis, and J. olanchana as distinct species.
Cardiocaryon was supported as a monophyletic group in these analyses, but it was supported less strongly than Rhysocaryon. Analyses of the matK data and of weighted ITS data indicated that J. ailantifolia was more closely related to J. cathayensis than to J. mandshurica, while analysis of unweighted ITS data indicated that J. ailantifolia was more closely related to J. mandshurica than to J. cathayensis. The closer relationship of J. ailantifolia and J. cathayensis resulting from analysis of matK data had much stronger support (with a bootstrap value of 98% and a decay index of 5) than the closer relationship of J. ailantifolia and J. mandshurica (with a bootstrap value of 67% and a decay index of 2) resulting from analysis of unweighted ITS data. However, Fjellstrom and Parfitt's (1995)
analyses consistently supported a closer relationship of J. ailantifolia and J. mandshurica.
The butternuts as a whole were strongly supported as a monophyletic group in analyses of ITS data and of total data, but analysis of matK data placed J. cinerea in a clade with Rhysocaryon. With a decay index of 10 and a bootstrap support of 99%, the monophyly of the butternuts suggested by ITS data was better supported than the relationship (with decay indices of 2 or less and bootstrap values of 50 or less) suggested by matK data.
Both matK and ITS data, when analyzed separately or together, supported section Dioscaryon as the sister group to the butternuts. Analysis of total data indicated that 16 characters supported the monophyly of the butternuts and the Persian walnut (J. regia). The butternut–Dioscaryon clade was consistently supported as the monophyletic sister group to the Rhysocaryon clade.
In general, the phylogenetic trees generated by these analyses became more resolved and better supported as additional data sets were added. This increased resolution seems to be a common result of combining data sets in the literature (Schnabel and Wendel, 1998
; Yasui and Osnishi, 1998
; Les et al., 1999
). The inclusion of multiple data sets provides additional characters, and may help cancel out "noise" present in individual data sets. Although it is not always possible, it may be advisable to include two or more data sets in all molecular phylogenetic studies.
Morphology
The phylogenies generated by these analyses are largely in agreement with previous, morphology-based, analyses. The four sections delineated by Dode (1906, 1909a, b)
and Manning (1978)
coincide with four monophyletic groups generated by parsimony analysis of gene sequence data performed in this study and by parsimony analysis of RFLP data (Fjellstrom and Parfitt, 1995
). Section Dioscaryon typically has leaves with five to nine entire leaflets, dehiscent husks, and smooth nuts with two winged sutures. Rhysocaryon is typified by pedicillate staminate flowers and the presence of crystal chains and heterocellular rays in the wood. Notched leaf scars and five rows of scales on embryos and seedlings distinguish Cardiocaryon from Trachycaryon, which has unnotched leaf scars and scaleless embryos and seedlings. Synapomorphies between Cardiocaryon and Trachycaryon include downy leaflets, a hairy fringe on leaf scars, nuts two-celled at the base, and flattened ray cells. The butternuts and section Dioscaryon share a few characters, including sessile staminate flowers and homocellular rays, but there is no strong morphological evidence for monophyly of these groups.
In agreement with the results of this molecular systematic study is Miller's (1976)
division of the Rhysocaryon into two groups, the tropical black walnuts and the temperate black walnuts. Miller (1976)
found that the tropical walnuts, unlike the temperate walnuts, have wood with diffuse-porous vessel distribution. The tropical walnuts also have longer crystal chains and exclusively heterocellular rays. Temperate black walnuts have semiporous ring vessel distribution, and, unlike any other Juglans species, they have reticulate thickenings in the axial parenchyma.
Not in agreement with the results of this molecular study is Dode's (1906, 1909a, b)
division of Rhysocaryon into three groups and of Cardiocaryon into three groups. Dode's (1906, 1909a, b)
subsectional divisions were based largely upon nut morphology. Cardiocaryon was divided according to the shape of the nuts and the presence of ventral furrows on the nuts. His division of Rhysocaryon was based on whether or not the nuts were deeply crested and whether the crests were pointed or dull.
Biogeography
Many events have shaped the distributions of Arcto-Tertiary disjuncts. Asia and America appear to have shared a land connection via the Bering land bridge (and possibly a periodic Aleutian bridge as well) from the Mesozoic (over 70 million years before present) until the late Miocene or early Pliocene, ~10 million years before present (Fujita, 1978
; Barron et al., 1981
; McKenna, 1983
; Briggs, 1987
). A north Atlantic European–American land bridge is speculated to have existed from the early Eocene (55 million years before present) until the late Miocene, although it was possibly interrupted during the Oligocene, 30–40 million years before present (Raven and Axelrod, 1974
; Tiffney, 1985a, b
). However, disagreement about this North Atlantic land bridge abounds. Some geologists and biologists (Kurtén, 1973
; Thiede, 1980
; McKenna, 1983
; Briggs, 1987
) have contended that it existed (from the Mesozoic) only until the mid-Eocene (50 million years before present), in which case it would not have been a viable migration route during most of the Tertiary. Hallam (1981)
has argued that the Atlantic land bridge lasted until the Oligocene, 38 million years ago. In any case, migration across the north Atlantic via island hopping may still have been possible after the land bridge was submerged. The ability of temperate species in Asia, Europe, and America to exchange genetic information during this time appears to have depended largely upon climate: only during warm periods would the land bridges have been forested with temperate species (Wolfe, 1978
; Frakes, 1979
; McKenna, 1983
). Warm periods with mean temperatures in northern latitudes ~20°C occurred at ~63 million years before present, 55 million years before present, and 30 million years before present. Less extreme warming periods, with mean temperatures around 10°C, occurred more recently, ~25, 18, and 4 million years before present (Savin, 1977
; Vail, Mitchum, and Thompson, 1977
; Wolfe, 1978
; Frakes, 1979
). The current distributions of Arcto-Tertiary disjunct plants have probably resulted from periodic contact (among the taxa of northern temperate forests) during warmer climate periods when northern land bridges were present.
The Juglans area cladogram (Fig. 6) does not support any particularly theory of the origin of the genus. However, it certainly does not conflict with the North American origin of the genus suggested by the fossil record. The earliest Juglans fossils, dating from the Eocene (50 million years before present) were found in several North American sites. It is conceivable that this early North American lineage diversified into the two main lineages, the North American lineage Rhysocaryon Dode and the primarily Eurasian butternut-Dioscaryon Dode lineage, during the Eocene (40-50 million years before present). Certainly Rhysocaryon had evolved by the mid-Eocene (45 million years before present)–fossil black walnut species J. clarensis Scott was found in the Middle Eocene Clarno Formation in Oregon (Manchester, 1987
). However, the earliest butternut fossil, extinct species J. lacunosa Manchester, came from the early Oligocene (~35 million years before present) Blakely Formation of Washington. While the phylogenies generated by this study and by Fjellstrom and Parfitt (1995)
imply that Dioscaryon Dode evolved from a common ancestor with Cardiocaryon, the absence of Dioscaryon from the fossil record makes it difficult to even speculate as to the group's origins. The history of the butternuts and Rhysocaryon seems clearer.
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; Briggs, 1987
), when the migration would have taken place. Another possibility is that Juglans arrived in Europe during the Eocene warming trend (45 million years before present), when the north Atlantic land bridge was almost certainly extant, although no European Juglans fossils are known from the Eocene time period (Manchester, 1987
). Thereafter, Juglans would have migrated to Asia, where the fossil record dates from the Miocene, ~25 million years before present (Manchester, 1987
). This migration could have been from Europe, as Yurtsev (1972)
asserts that almost all migrations over the Beringian land bridge were from Asia to America. Based on the cladogram, however, it seems more likely the migration to Asia was from North America via the Beringian land bridge during the Oligocene warming trend (30 million years before present). CONCLUSIONS
Based on matK, ITS, and nuclear RFLP data, Juglans is a monophyletic group, and Pterocarya is the sister group to Juglans. Within the Juglans clade are two subclades, one consisting of section Rhysocaryon and one consisting of sections Cardiocaryon, Dioscaryon, and Trachycaryon. Within Rhysocaryon (the black walnuts) are two groups, also suggested by Miller's (1976)
study of wood anatomy in walnuts, the temperate black walnuts and the tropical black walnuts. Within the second subclade, Dioscaryon is the sister group to Cardiocaryon and Trachycaryon (the butternuts), but phylogenetic relationships within the butternuts remain unclear. Three of the previously proposed sections (Dode, 1908, 1909; Manning, 1978
), Dioscaryon, Rhysocaryon, and Trachycaryon, are monophyletic groups. It seems likely that Cardiocaryon is also monophyletic. Although combined matK and ITS sequence data generated for this study do not clearly support monophyly, both the individual ITS and matK data and Fjellstrom and Parfitt's (1995)
RFLP data indicate that Cardiocaryon is monophyletic.
A complex history of climate changes and continental movement has shaped the current biogeography of the group. The evolution of the butternut clade may have occurred as a result of the cooling trend ~40 million years ago, since the first known fossils of the group date from 35 million years ago. This group could have migrated to Europe, where the oldest fossils date from 30 million years ago, during the warming trend which occurred ~30 million years ago. Overall, the well-resolved phylogenies generated for Juglans, combined with the well-studied fossil record of the group, present a clear picture of the history of Juglans.
FOOTNOTES
1 The authors thank Stephan Beck, Juan Fernando Hernandez, Percy Nuqez, Robert Parks, Barry Prigge, Linda Prince, Alan Stanford, Clay Weeks, George White, and Kenneth Wurdack for assistance in obtaining germplasm, Paul Manos and Kelly Steele for providing us with primers, and Hal Hill and Jonathan Wendel for other assistance. This work was supported in part by the Biology Department of the University of North Carolina and NSF Grant INT-9710505. 
2 Author for correspondence, current address: Division of Science and Mathematics, University of the Virgin Islands, 2 John Brewers Bay, Saint Thomas, United States Virgin Islands 00802.
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