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2 Botanical Institute, Göteborg University, P.O. Box 461, SE-405 30 Göteborg, Sweden; and 3 Department of Biological Sciences, The University of Alabama, Tuscaloosa, Alabama 35487 USA.
Received for publication September 9, 1999. Accepted for publication February 22, 2000.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Key Words: cpDNA • Dimetra • Myxopyrum • Nyctanthes • phylogeny • Oleaceae • rps16 • trnL-F.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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600 species in 25 genera(Table 1). The family isdistributed on all continents except the Antarctic, from northern temperateto southern subtropical regions and from low to high elevations. Somegenera are widespread and occur on more than one continent, e.g.,Chionanthus, Menodora, and Fraxinus (authors of names aregiven only if not listed in the Appendix or Table 3
, and only the first time they are mentioned). The genus Jasminum is the largest with over 200 species. Many of the genera are economically important, e.g., the olive (Olea europaea) is cultivated for its fruit and oil, species of Fraxinus are grown for timber, and Jasminum, Forsythia, Syringa, and Ligustrum are planted as ornamentals.
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The Oleaceae have by recent molecular studies been placed in Lamiales, sister group to the rest of the order (Wagstaff and Olmstead, 1997
), and APG (1998)
classified it in this order. The family has also been treated in an order of its own, Oleales, by, e.g., Takhtajan (1997)
. Most classifications of Oleaceae divide the family into two subfamilies, Jasminoideae and Oleoideae (Table 2). Knoblauch (1895)
based his division on the point of attachment of the ovules and the presence of a constriction through the apex of the fruit. Taylor (1945)
rearranged some genera on the basis of chromosomal data and fruit morphology. The most recent review of the entire family is that of Johnson (1957)
. His division of Oleaceae into subfamilies and tribes follows Taylor (1945)
, with a few exceptions. The members of the subfamily Oleoideae apparently form a monophyletic group. They all have x = 23 and are thought to be of an allopolyploid origin (Taylor, 1945
). In addition, they share a number of anatomical, morphological, and chemical apomorphies. In contrast, the Jasminoideae are a heterogeneous assemblage of those genera that do not fit in the Oleoideae. Except for the family-wide characters, the tribes of Jasminoideae share no apomorphies, but they are well distinguished from the Oleoideae. Therefore, most authors have placed them in a separate subfamily.
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and Dimetra next to Nyctanthes by Kerr (1938)
. Later, both genera were suggested to belong to the Verbenaceae (Airy Shaw, 1952
; Stant, 1952
), or in a family of their own, Nyctanthaceae (Kundu and De, 1968
). The exclusion from the Oleaceae was based mainly on the plants' "Verbenaceous appearance" (Airy Shaw, 1952
). Since then, the morphology of these genera has been investigated and compared to Oleaceae and Verbenaceae in a number of studies (cf. Kiew and Baas, 1984
), all reaching the conclusion that they should belong to Oleaceae. In an attempt to test this hypothesis with molecular data, the two genera were included in this study.
The position of the distinct genus Myxopyrum within Oleaceae has also been uncertain. It was first assigned to the Oleineae by Bentham (1876)
, kept in the Oleoideae-Oleineae by Knoblauch (1895)
, then Taylor (1945)
thought that it was "not Oleineae," and Johnson (1957)
put it in a tribe of its own in the Jasminoideae. Later, arguments for placement in the subfamily Oleoideae have come from Kiew (1983, 1984)
, but Baas et al. (1988)
and Rohwer (1996)
have doubted this. One of the aims of the present study has been to let molecular data shed new light on possible relationships for this genus incertae sedis.
Despite containing such well-known and economically important genera, no recent classification of the entire family based on an explicit phylogeny has been published. The first author to present a "phylogeny" for the Oleaceae was Taylor (1945)
, who drew a phylogenetic chart based on cytological data. Later, Johnson (1957)
made an important contribution to the systematics of the family by reviewing its taxonomy and classification. So far, only two studies have employed cladistic methods in evaluating phylogenetic hypotheses for Oleaceae. Baas et al. (1988)
studied wood anatomy of the whole family and based cladistic and phenetic analyses on wood anatomical characters. Rohwer (1996)
based his cladistic analyses mainly on fruit and seed characters. Our molecular phylogeny is the first to be documented [see also Kim and Jansen (1993)
and Kim (1999)
] and contributes new insights towards a revised classification of the family.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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), the approximate number of species in each genus, the number of species sequenced, and the world distribution of the genera. At least two representatives from each genus in the family were sequenced, including Nyctanthes and Dimetra. Where possible we tried to use material from the type-bearing species of the genus. In the monotypic genera Abeliophyllum, Dimetra, and Picconia two different individuals of each species were sequenced. The genus Hesperelaea is also monotypic, but because it is extinct and known solely from the type collection, only a sample from this could be used. About a third of the material studied was silica-gel dried plant material, and a few fresh samples from Göteborg Botanical Garden and the New York Botanical Garden, collected by Eva Wallander. Silica-gel dried material of three Australian and New Zealand taxa were received from Wayne K. Harris (BRI), a sample of Nestegis sandwicensis from Timothy J. Motley (NY), and a recent collection of Dimetra craibiana from S. Suddee (by courtesy of the Bangkok Forestry Department, Thailand). Another third of the DNA was isolated from herbarium specimens held at BM, C, GB, MO, and NY. DNA extracts from plants cultivated at the Royal Botanic Gardens at Kew and herbarium specimens held at K were received from Mark W. Chase. Vouchers for all sequenced taxa are listed in Table 3 along with their GenBank accession numbers. As outgroup taxa we chose species of Verbenaceae and Myoporaceae (Lamiales) to test the position of Dimetra and Nyctanthes, and members of Rubiaceae, Loganiaceae, Strychnaceae, and Gelsemiaceae (Gentianales) were included to provide a root hypothesis for Oleaceae. Except for two Verbenaceae sequences, outgroup sequences were received from various authors, which are also listed in Table 3.
DNA extraction
Fresh leaf tissue was manually ground with a pestle in an Eppendorf tube immersed in liquid nitrogen, and dried tissue was homogenized using the FastPrep® instrument (BIO 101, Vista, California, USA). Total DNA was extracted using a lysis buffer consisting of 2% CTAB (cetyltrimethylammonium bromide), 1% PEG 6000 (polyethylene glycol, molecular weight 6000), 1.4 mol/L NaCl, 10 mmol/L Tris-HCl, and 20 mmol/L EDTA. Lysis was performed at 74°C with 2% mercaptoethanol added, followed by cleaning with the Geneclean® II kit (BIO 101). Cleaned DNA was transferred to 10 mmol/L Tris and kept in freezer.
cpDNA regions and primers
For our study, we chose two noncoding chloroplast regions, the trnL-F region and the intron of rps16. The trnL-F region consists of the trnL intron and the trnL-trnF intergenic spacer (Taberlet et al., 1991
). The primer pair tRNc/tRNf (Table 4) was used to amplify the entire region of 900 bp in one PCR (polymerase chain reaction). In some cases, the tRNc/tRNd and the tRNe/tRNf primer pairs were used to amplify the intron and the spacer, respectively. The intron of rps16 is a group II intron that was first used for phylogenetic studies by Oxelman, Lidén, and Berglund (1997)
. The primer pair rpsF/rpsR2 was used to amplify the entire 800–900 bp region. For DNA of low quality, internal primers were used with each of the end primers to split that region into two approximately equal halves. The position of the internal forward primer (rpsMF2) is located 50 base pairs downstream of the internal reverse primer (rpsMRP), giving sufficient overlap for determining a full sequence.
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Sequencing
Sequencing reactions, using the same primer sequences as in the PCR, were performed on a Perkin Elmer GeneAmp® PCR System 9600 version 2.01 (1 min at 95°C, followed by 32 cycles of [95°C 10 sec, 50°C 5 sec, 60°C 3 min]), using the dRhodamine Terminator Cycle Sequencing Ready Reaction DNA sequencing kit with AmpliTaq® DNA polymerase (Perkin Elmer Applied Biosystems, Foster City, California, USA) and HT1000 halfTERM Dye Terminator Reagent (GENPAK Inc., Stony Brook, New York, USA). Before gel separation, the sequence reaction products were cleaned using Sephadex® G-50 Fine DNA Grade (Amersham Pharmacia Biotech AB) in Centrisep Spin Columns (Princeton Separations, Philadelphia, Pennsylvania, USA). Separation of the fragments was done on a 5% Long RangerTM gel (FMC BioProducts, Rockland, Maine, USA) on an ABI PrismTM 377 DNA Sequencer (Perkin Elmer Applied Biosystems). The ABI PrismTM 377 Collection software version 2.1 was used to evaluate the sequences. Some sequencing reactions were also performed on an ALFexpressTM DNA Sequencer (Amersham Pharmacia Biotech AB). Reactions were then performed using the ThermoSequenase fluorescent labeled primer cycle sequencing kit with 7-deaza-dGTP (Amersham Pharmacia Biotech AB) and Cy5-labeled primers. Cycle sequencing reactions were performed on a Perkin Elmer Cetus 480 version 1.1 (2 min at 96°C, followed by 18 cycles of [95°C 30 sec, 60°C 40 sec]). The reaction products were loaded without further cleaning on a 0.5 mm 5.28% Page-Plus gel (Amresco®, Solon, Ohio, USA). Sequences were evaluated with the ALFwinTM software version 1.10.
Alignment and indel coding
The forward and reverse sequences were checked and edited using the SequencherTM software version 3.1 (Gene Codes Corporation, Ann Arbor, Michigan, USA). Consensus sequences from each of the two chloroplast loci were aligned separately. The two sequences of each of Abeliophyllum and Picconia were found to be identical so only one of them was included. Alignment was done using the assembly feature in Sequencher, and then manually adjusted using criteria described in Andersson and Rova (1999)
. Adding new sequences to the alignment was relatively easy because of conserved regions and shared indels. Twenty-two indels in the rps16 matrix and 20 in the trnL-F were considered informative, and indel characters were added to the combined matrix (using A/T for present/absent). A few insertions, which did not contain informative characters, were then deleted. Autapomorphic insertions were also removed. The alignment is available from the corresponding author upon request.
Cladistic analyses
The combined matrix consisted of 78 ingroup taxa and ten outgroup taxa. Two taxa in this matrix, Hesperelaea palmeri and Priogymnanthus apertus, were represented by the rps16 sequence only, and in the case of Hesperelaea, only the first 423 bp of the sequence were included (because of sequencing problems with the second part). The two data sets were subjected to parsimony analyses separately, and in combination, and in the latter case with and without indel characters, using PAUP* version 4.0b4a (Swofford, 2000
) on a Power Macintosh. All characters were analyzed using equal weights (=1), and gaps were treated as missing data.
Initial rounds of PAUP analyses yielded tree overflow with maximum memory settings so the following search strategy was adopted: first a search for multiple tree islands was conducted by doing 100 random addition sequence replicates, limited to only ten saved trees from each. The resulting most parsimonious trees were then used as starting trees for TBR (tree bisection-reconnection) branch swapping in an additional heuristic search for shorter trees. Up to 5000 additional trees of equal or shorter length were allowed to be saved and were then compared to the starting trees as consensus trees.
Another strategy was also adopted, namely excluding some taxa thought to cause most of the problems with thousands of equally parsimonious trees. By starting out with only two of the closest outgroup taxa (Verbenaceae) and 16 ingroup "backbone taxa," and then restoring a few taxa in successive runs, we were able to determine which were causing the problem. It was also evident from the first runs, and from inspecting the alignment, that the taxa of Johnson's tribe Oleeae are very closely related and not many characters are available to support any particular interrelationship. By only representing each genus in this tribe with one sequence, but excluding taxa with an incomplete sequence (e.g., Hesperelaea palmeri), the number of trees obtained drastically decreased. In an analysis that made the best compromise between computational time and fewest excluded taxa, 64 of the 78 ingroup taxa were included, and 4312 trees were found with complete TBR branch-swapping.
Parsimony jackknifing (Farris et al., 1996
) was performed on the combined matrix, with and without indel characters added, using XAC (J. S. Farris, Swedish Museum of Natural History, Stockholm, Sweden). One thousand jackknife replicates, each with ten random addition sequences and nonrotational branch-swapping (J. S. Farris, Swedish Museum of Natural History, Stockholm, Sweden, personal communication), were conducted.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Previously, Gielly and Taberlet (1994)
have shown that the variation in the trnL-F region is too low to resolve relationships within Fraxinus. For the Oleaceae family, the rps16 intron is more informative than the trnL-F region, but still, the combined data set does not contain enough informative characters to resolve the inter-tribal relationships outside Jasmineae and Oleeae (i.e., branch lengths between the basal tribes are almost zero).
Congruence between molecular data and other characters
The result of the molecular analyses agrees well with nonmolecular data, e.g., chromosomal data (Taylor, 1945
), wood anatomy (Baas et al., 1988
), ovule number and position (Taylor, 1945
; Rohwer, 1996
), fruit anatomy (Rohwer, 1996
), flavonoid glycosides (Harborne and Green, 1980
), and iridoids (Jensen, 1992
). The monophyly of the Oleoideae is not only supported by the present molecular analysis, but also by numerous morphological, anatomical, chemical, and chromosomal synapomorphies. The subfamily Jasminoideae, on the other hand, is not supported by this study, nor by any nonmolecular synapomorphies. Some of the many characters supporting the results from this study are shown on a summary tree (Fig. 3) and are discussed under the tribal and subtribal sections further below. Fruit type, which varies considerably in the Oleaceae (Rohwer, 1996
), is also discussed.
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saw the need to "ultimately abandon the subfamilies and to treat the allotetraploid Oleoideae as equivalent to the other tribes." Rohwer (1996)
stated that "the Jasminoideae is so heterogeneous in its present circumscription that it seems advisable to dismember it as a taxonomic unit," and Qin (1996)
, who based his conclusions on leaf peroxidases and morphology of a few genera of the family, revoked "the subfamily rank because the tribes in subfamily Jasminoideae have no points in common." Kiew and Baas (1984)
proposed to abandon the use of subfamilies and revert to the old tribes sensu Bentham (1876)
. In this case, the Oleoideae would fall apart to Fraxineae, Syringeae, and Oleeae, and the jasminoids would stay in their assigned tribes (sensu Johnson). However, our present findings suggest that only dropping the subfamily rank and keeping the tribes unaltered is unsatisfactory. Because chromosomal data and a number of morphological characters support the monophyly of Oleoideae, we think it is important to recognize this. We suggest that changing rank of the subfamily Oleoideae to tribe Oleeae, and changing all previous tribes of Oleoideae to subtribes, is a better solution. In this way the monophyly of this group is shown, equal in status to the jasminoid tribes. Therefore, we present a revised classification of Oleaceae (Table 2; Appendix), shown on a summary tree in Fig. 3. We recognize five tribes: Myxopyreae (Myxopyrum, Nyctanthes, and Dimetra), Fontanesieae (Fontanesia), Forsythieae (Forsythia and Abeliophyllum), Jasmineae (Jasminum and Menodora) and Oleeae. The Oleeae now contain four subtribes: Ligustrinae (Syringa and Ligustrum), Schreberinae (Schrebera and Comoranthus), Fraxininae (Fraxinus), and Oleinae (the remaining 12 genera). The subtribes Schreberinae and Fraxininae are new (Appendix).
From this point forward, when we discuss and compare our results with those of other studies, there are cases where it is simpler to refer to the old taxonomy, i.e., subfamilial groupings. In order not to cause confusion when using the tribal name Oleeae, we will state whether it is our new tribe Oleeae (former subfamily Oleoideae) or subtribe Oleinae (former tribe Oleeae). The term "jasminoids" is used to refer to the tribes Myxopyreae, Fontanesieae, Forsythieae, and Jasmineae, i.e., the former subfamily Jasminoideae.
As can be seen by comparing our classification with that of Johnson (1957)
, apart from alterations in ranks, the changes are: (1) transfer of the tribe Schrebereae (as subtribe Schreberinae) back to our tribe Oleeae, (2) reinstatement of the subtribe Ligustrinae (with Syringa and Ligustrum) in tribe Oleeae, and (3) inclusion of the formerly incertae sedis Nyctanthes and Dimetra with Myxopyrum in Myxopyreae.
The new tribe Oleeae
The Oleeae are clearly a monophyletic group, supported by numerous data (Fig. 3). The haploid chromosome number n = 23 is basic in all genera of the new tribe Oleeae. In contrast, the basic number of the jasminoids is either x = 11, (12), 13, or 14. Chromosome numbers served as one of the fundamental characters upon which Taylor (1945)
based his division of Oleaceae into subfamilies and tribes. It was suggested by Taylor that the x = 23 group has an allopolyploid origin (from two unknown and now probably extinct jasminoids with x = 11 and 12).
The ovaries of all Oleaceae are bilocular and the number of ovules in each locule vary from one to many. All genera of Oleaceae have pendulous ovules, except Myxopyreae, which has ascending ovules (see below). The synapomorphy for the new tribe Oleeae is two pendulous ovules per locule (except Schrebera, which has four; Taylor, 1945
). In contrast, the tribes Fontanesieae and Forsythieae have varying numbers of ovules per locule, but never two. Jasminum and Menodora in the Jasmineae have 1–2 and 2–4 ovules per locule, respectively, but their position is more horizontal.
Harborne and Green (1980)
carried out an investigation of flavonoid glycosides in leaves of all genera of Oleaceae. The pattern they found was clear: all the jasminoid genera have only the plesiomorphic flavonols present, but more complex flavonoids, including flavones and flavanones, were found to be a synapomorphy for taxa with x = 23, i.e., the new tribe Oleeae. Unfortunately, they did not recognize Nyctanthes and Dimetra in Oleaceae, which were excluded from the study despite containing two common flavonols. Harborne and Green, also investigating flavonoid patterns in other closely related families came to the conclusion that keeping Oleaceae in an order of its own (Oleales) was justified based on the fact that the flavonoid pattern in this family differed from other sympetalous families.
Baas et al. (1988)
studied wood characters for the whole family (including Nyctanthes), and made both phenetic and cladistic analyses of the data. Trees from both analyses agree in principal with our results. The distribution of fiber and vessel characters especially agrees with the molecular phylogeny presented here, and libriform fibers and multiple vessels form synapomorphies for the new tribe Oleeae (exceptions in Ligustrinae, see below).
The jasminoids
The tribes of the former subfamily Jasminoideae, viz. Fontanesieae, Forsythieae, Myxopyreae, and Jasmineae, share no apparent morphological apomorphies with each other, nor with the new tribe Oleeae (except Jasmineae, see under this subheading below). Taylor (1945)
, in his cytological study of the family, found varying low basic chromosome numbers (x = 11, 12, 13, or 14) in the jasminoids. George, Geethamma, and Ninan (1989)
have proposed that x = 11 (found in Menodora and Myxopyrum) is the basic chromosome number of the family, and that all other low numbers have originated by aneuploidy. In the study of flavonoid glycosides by Harborne and Green (1980)
, the jasminoids were shown to contain only the plesiomorphic flavonols (except Myxopyrum that also contained advanced flavones, but not of the same type as in Oleoideae). In the wood anatomical study by Baas et al. (1988)
, fiber-tracheids and solitary vessels were shown to be plesiomorphic characters in common for the jasminoids.
Tribe Jasmineae
In the strict consensus tree (Fig. 1), Jasmineae are resolved as sister group to Oleeae, supported by eight steps and a jackknife value of 76%. Nonmolecular support has also come from Jensen (1992)
, who investigated iridoids in a number of species of Oleaceae, and the results fit very well with the molecular phylogeny. The tribes Jasmineae and Oleeae both contain oleoside, whereas in Fontanesieae (Damtoft, Franzyk, and Jensen, 1995
), Forsythieae (Damtoft, Franzyk, and Jensen, 1994
), and Myxopyreae (S. R. Jensen, Technical University of Denmark, unpublished data) this compound is absent. Our results also indicate that Jasminum is paraphyletic, as Kim and Jansen (1993)
and Rohwer (1996)
have suggested, because Menodora is nested within it. There is, however, no doubt that the tribe is monophyletic. The phylogram (Fig. 2) shows that the clade is supported by 61 steps, and based on fruit anatomy the Jasmineae are unique in the family in having bilobed fruits. Jasminum has a bilobed berry (each lobe one-to-two-seeded, one lobe frequently aborted) and Menodora, the New World counterpart of Jasminum, has a bilobed circumscissile capsule. The development of these two seemingly different fruit types is in fact very similar except for the final stages (Rohwer, 1995, 1997
).
The position of Nyctanthes and Dimetra
The molecular results presented here clearly show that both Nyctanthes and Dimetra belong to Oleaceae. Their inclusion in the family is supported by a jackknife value of 100%. Nyctanthes arbor-tristis L. was placed in Oleaceae by Bentham (1876)
, Knoblauch (1895)
, and Taylor (1945)
(Table 2). Takhtajan (1997)
placed Nyctanthes in its own subfamily in Oleaceae (Nyctanthoideae). The second species of Nyctanthes, N. aculeata Craib, was described in 1916 and placed by the author in Oleaceae-Jasmineae. When Kerr (1938)
described the new monotypic genus Dimetra, he assigned it to Oleaceae without hesitation. He stated that its closest alliance clearly was with Nyctanthes. Later, Airy Shaw (1952)
transferred both of them to Verbenaceae (in subfam. Nyctanthoideae) because "the Verbenaceous facies of Nyctanthes almost hits one in the eye." Stant (1952)
supported this view with a study of some anatomical characters, and Johnson (1957)
agreed. This transfer generated a number of papers investigating various morphological aspects of Nyctanthes and Dimetra. Kundu and De (1968)
investigated cytology, palynology, and leaf, wood, and floral anatomy of Nyctanthes and compared it with members of Oleaceae, Verbenaceae, and Loganiaceae. They came to the conclusion that it should be placed in a family of its own, Nyctanthaceae, because of differences with both Oleaceae and Verbenaceae. They described Nyctanthaceae as a new family, not knowing that it had already been described by Agardh in 1858 (as Nyctantheae). Support for placing Nyctanthes in Oleaceae has come from studies of embryology (Kapil and Vani, 1966
), structure and vascular anatomy of the gynoecium (Kshetrapal and Tiagi, 1970
), vessel anatomy (Murthy et al., 1978
), leaf morphology (Mohan and Inamdar, 1983
), wood anatomy (Baas et al., 1988
), ultrastructure and morphology of intranuclear proteinic inclusions in the mesophyll parenchymatic cells (Bigazzi, 1989
), and fruit anatomy (Kuriachen and Dave, 1989
; Rohwer, 1994, 1996
). These and other studies are reviewed in detail by Kiew and Baas (1984)
, who summarized the overwhelming evidence that Nyctanthes belongs to Oleaceae. Because Nyctanthes shares a number of characters with Jasminum and Menodora (Kiew and Baas, 1984
), and because they did not want to erect a monogeneric tribe, they proposed that Nyctanthes should be kept in Jasmineae sensu Bentham. Although no one has disputed a close relationship between Dimetra and Nyctanthes, Dimetra was not included in most of the studies and was not mentioned in the review by Kiew and Baas (1984)
. Since our results clearly point to the close relationship between Nyctanthes and Dimetra, grouped with Myxopyrum rather than with Jasmineae, we argue for placing them in the tribe Myxopyreae. The pertinent node is supported by a jackknife value of 100%.
The position of Myxopyrum
The genus Myxopyrum consists of four species distributed in subtropical and tropical east Asia (Kiew, 1984
). They are scandent shrubs with quadrangular stems and conspicuously triplinerved leaves. They share the common basic characters with other Oleaceae, but some divergent features have made the genus difficult to place, and there has therefore been different opinions on where it belongs (Table 2). Bentham (1876)
and Knoblauch (1895)
put it in the Oleineae (sensu Bentham), but according to Taylor (1945)
it differed in so many characters that it should probably be separated from the Oleineae. Johnson (1957)
erected a new tribe for it, Myxopyreae, and placed it in the heterogeneous Jasminoideae. The results from this study strongly support the placement of Myxopyrum as sister to Nyctanthes and Dimetra, as discussed above, even though there are no apparent outer morphological similarities between them. However, the three genera share the apomorphic character of ascending ovules (Fig. 3), and Nyctanthes and Myxopyrum both have quadrangular stems with cortical bundles in the corners (Kiew, 1984
; Kiew and Baas, 1984
). Rohwer's (1996)
investigation of fruit and seed characters of the Oleaceae showed that Myxopyrum and Nyctanthes, apart from ascending ovules, also share a deep stylar canal and the presence of a distinctive tissue in the center of the ovary septum. In contrast to the ovary, the fruit of Myxopyrum (a one-to-four-seeded berry) is not similar to that of Nyctanthes (a dry schizocarp that splits into two one-seeded mericarps), and Myxopyrum has varying one to three ovules per locule, whereas Dimetra and Nyctanthes have only one. Apart from the above synapomorphies, it is difficult to find morphological characters that unite these quite distinct genera. Most characters are either plesiomorphic and found in other jasminoid genera as well, or autapomorphic. For example, the wood anatomical study by Baas et al. (1988)
showed that Myxopyrum only shared plesiomorphies with the other jasminoids, and the phytochemical study by Harborne and Green (1980)
showed that Myxopyrum contains three apigenin glycosides that are not found in any of the other genera of Oleaceae. This finding of advanced glycosides in Myxopyrum led Kiew (1984)
, together with her own investigation of the morphology, to conclude that Myxopyrum should be retained within the Oleoideae. The chromosome number of Myxopyrum was unknown at that time, but now there are two reports: 2n = 22 for M. hainanense Chia (synonym to M. pierrei Gagnep.) (Weng and Zhang, 1992
) and 2n = 24 for M. smilacifolium Blume (George and Geethamma, 1983
). At least the former fit well with 2n = 44 reported for Nyctanthes arbor-tristis (George and Geethamma, 1984
), which would suggest that the ancestor of Nyctanthes (and Dimetra) arose by polyploidy from the ancestor in common with Myxopyrum. The chromosome number of Dimetra is not known. Chromosome counts in Nyctanthes are notoriously variable (Rohwer, 1996
), however, so one should not draw any conclusions based on chromosome number alone.
New chemical evidence (S. R. Jensen, Technical University of Denmark, unpublished data) on two new carbocyclic iridoid glucosides in Myxopyrum smilacifolium shows that these are very similar to the compounds found in Nyctanthes and structurally represent the same biosynthetic pathway (myxopyroside). Also, the three genera do not contain oleoside, a compound that only occurs in the two tribes Jasmineae and Oleeae (Jensen, 1992
). These findings, together with chromosome numbers, further strengthens the conclusion that Myxopyrum does not belong in the former Oleoideae. To conclude, a number of nonmolecular synapomorphies do support the Myxopyreae clade, despite no obvious outer morphological similarities.
Tribes Fontanesieae and Forsythieae
The molecular result shows a closer relationship between Forsythia and Abeliophyllum than between Fontanesia and Abeliophyllum, as might have been expected on the basis of fruit morphology (Taylor, 1945
; Rohwer, 1996
). Fontanesia and Abeliophyllum both have the same type of samara (differing from the one in Fraxinus, see below), but Forsythia has loculicidal capsules. Other characters, e.g., karyology (Fontanesia has x = 13 and Forsythieae x = 14; Taylor, 1945
) and chemical data (only Forsythieae contains cornoside; Damtoft, Franzyk, and Jensen, 1994
), also support the close relationship between Forsythia and Abeliophyllum. But as can be seen in Fig. 2, Fontanesia is resolved as sister group to the Forsythieae clade. This is the fact in most of the equally parsimonious trees and, although the branch length is extremely short (one step!), this relationship can be expected to be phylogenetically most probable, because Fontanesia and Abeliophyllum share fruit characters that are much easier interpreted as synapomorphies than parallelisms (J. G. Rohwer, University of Hamburg, Germany, personal communication). Because the strict consensus tree does not resolve the position of Fontanesia and because of the conflict between characters, we continue to leave Fontanesia alone in its own tribe.
Subtribe Ligustrinae
Syringa and Ligustrum form a well-supported basal clade within the new tribe Oleeae. They have dry bilocular capsules and one-to-four-seeded berries (except Ligustrum sempervirens that has dehiscent drupes), respectively. Their fruits are quite similar in development, the only differences being in the development of the mesocarp and fruit dehiscence (Taylor, 1945
). Johnson (1957)
also stated that Ligustrum is undoubtedly more closely related to Syringa than to the rest of Oleeae, but instead of including Ligustrum in the Syringeae (sensu Taylor), he placed both of them in the Oleeae. Because they form a distinct and well-supported clade in our tribe Oleeae, we have reinstated subtribe Ligustrinae Koehne to accommodate them.
In Fig. 3, libriform fibers and multiple vessels are plotted as synapomorphies for the tribe Oleeae (with the plesiomorphic states fiber-tracheids and solitary vessels). This is true in the sense that no taxa outside this clade have this type of wood anatomy, but these features are poorly developed in some taxa of both Ligustrum and Syringa. They are unique in having both fiber types and have most of their vessels solitary rather than in multiples (Baas et al., 1988
), i.e., the plesiomorphic states are retained alongside with the apomorphic. All taxa in the Oleeae clade, excluding Ligustrum and Syringa, always have vessel multiples and exclusively libriform fibers. This condition supports the position of Ligustrinae as basal in the Oleeae. The molecular results also indicate that Syringa might be paraphyletic (Fig. 2).
Subtribe Schreberinae
The genus Schrebera has a disjunct distribution in Africa and India, but Comoranthus occurs only on Madagascar and the Comores. There is also a report of Schrebera americana Gilg. from Peru. Both genera have bivalved woody capsules (Rohwer, 1996
), and, based on overall morphology, it is obvious that they are closely related, if not congeneric (P. S. Green, Royal Botanic Gardens, Kew, personal communication). Johnson (1957)
grouped Schrebera and Comoranthus in the new tribe Schrebereae and, pending chromosomal data, provisionally referred it to his subfamily Jasminoideae. Briggs (1970)
determined the haploid chromosome number of Schrebera to be 23, but the chromosome number of Comoranthus is still unknown. The present study clearly shows that these genera form a distinct clade that belongs to the same group as the other genera with x = 23, and we have therefore placed them in the subtribe Schreberinae status novus in the new tribe Oleeae. The chemotaxonomic survey by Harborne and Green (1980)
and the wood anatomical study by Baas et al. (1988)
also give support to this placement.
Subtribe Fraxininae
This new subtribe contains only the genus Fraxinus. It is a circumpolar genus of the northern hemisphere, comprising 50 species of mainly trees. The genus is characterized by large pinnate leaves and samaras, and there is no doubt that it represents a monophyletic group. Because of the fruit type, Fontanesia was included in the Fraxineae by Bentham (1876)
and Knoblauch (1895)
. However, the samaras in Fontanesia and Abeliophyllum, compared with those of Fraxinus, are neither morphologically nor developmentally similar. Instead, the samara of Fraxinus shows an internal structure very similar to that of the loculicidal capsule of Syringa (Rohwer, 1996
). The fruit of Fraxinus has two ovules per locule but usually only one ovule develops, making the samara one-seeded. In contrast, Fontanesia and Abeliophyllum have only one ovule per locule, and although both ovules start to develop, the mature fruit is usually one-seeded (Rohwer, 1996
). There are also differences in the morphology of the wing. In the long terminal wing of Fraxinus the fibers run longitudinally, and in Fontanesia's short lateral wings, they run obliquely perpendicular (Rohwer, 1993
).
Subtribe Oleinae
The subtribe Oleinae, former tribe Oleeae, is characterized by drupes. Although this group does not receive strong jackknife support, there is nevertheless no doubt that this is a monophyletic group. Relationships between the genera in this subtribe are difficult to elucidate, with neither the cpDNA data nor morphology giving a clear answer. Several studies have found that some genera in this group may be polyphyletic as presently circumscribed, e.g., Olea (Altamura, Altamura, and Mazzolani, 1985, 1987
; Kiew, 1979
) and Osmanthus (Johnson, 1957
), and our study can only confirm this suspicion. For example, Olea brachiata (formerly placed in the separate genus Tetrapilus Lour.; Johnson, 1957
) seems to be more related to Chionanthus, and so does Osmanthus americanus, the only New World species of Osmanthus (once treated separately in Amarolea Small; Johnson, 1957
). These results are also supported by wood anatomy (Baas et al., 1988
).
Within this subtribe lies a complex of five supposedly more closely related Old World genera, distributed mainly in the subtropics: Osmanthus (except O. americanus), Phillyrea, Picconia, Nestegis, and Notelaea. There is no jackknife support for this grouping, but it is shown in the strict consensus tree (Fig. 1). Green (1958)
mentioned this generic complex and Baas et al. (1988)
found some support for its monophyly in wood anatomical characters. The synapomorphies are dendritic vessel distribution and vascular tracheids. The similarity in fruit morphology between Phillyrea and Picconia has been pointed out by Taylor (1945)
, and other characters by Johnson (1957)
. W. K. Harris (University of Queensland, personal communication) has found that, based on nuclear ITS sequences, the Australian, New Zealand, and New Caledonian taxa of Osmanthus and Nestegis should be included in Notelaea. Generic delimitations in this complex are admittedly difficult (P. S. Green, Royal Botanic Gardens, Kew, personal communication) and further studies, using loci with more variation than in the present study (e.g., ITS), are needed to clarify relationships within the entire subtribe Oleinae.
Hesperelaea
The genus Hesperelaea is now extinct (Moran, 1996
), but we were successful in obtaining an rps16 intron sequence from the type specimen, the one and only collection from 1875. It is only known from its type locality on Guadalupe, a Mexican island off Baja California. Hesperelaea was collected by Edward Palmer and described by Asa Gray (Watson, 1876
) as H. palmeri, a new monotypic genus of Oleaceae. When collected, Palmer found only three old trees alive, no young trees, but several dead ones. The area was heavily grazed by goats, which presumably led to Hesperelaea's extinction (Moran, 1996
). Not much is known about the genus; Gray's description was rather short, but noteworthy was that its flowers had four stamens. In other genera of Oleaceae, the most common condition is two stamens, but four stamens occasionally occur, e.g., in Chionanthus, Osmanthus, Noronhia, Schrebera, and Forestiera. The fruit was a drupe and so it was placed by Johnson (1957)
among the other genera with drupes in his tribe Oleeae (the new Oleinae). Despite having only part of the rps16 intron sequence to confirm this placement, we feel sure that this is correct.
Conclusions
This study has presented molecular evidence, congruent with other data, that requires a revised classification of the Oleaceae. (1) The subfamily level is abandoned because Jasminoideae is paraphyletic. (2) The monophyly of the former Oleoideae—here recognized as tribe Oleeae—is strongly supported and treated equal in status to the former jasminoid tribes Fontanesieae, Forsythieae, Myxopyreae, and Jasmineae. (3) The tribe Jasmineae is sister to Oleeae. This relationship is supported by chemical data. (4) The long-debated genera Nyctanthes and Dimetra clearly belong to the Oleaceae. (5) The position of the hitherto genus incertae sedis Myxopyrum is supported as sister to Nyctanthes and Dimetra. All three genera are placed in Myxopyreae. (6) The monophyly of the subtribe Oleinae, characterized by drupes, is supported. (7) The rps16 sequence of Hesperelaea palmeri, known only from the type specimen collected in 1875, confirms the placement of this extinct taxon in the subtribe Oleinae. (8) A closer relationship between a group of five genera in the Oleinae, viz. Osmanthus, Picconia, Phillyrea, Nestegis, and Notelaea, is suggested by molecular data and has morphological and wood anatomical support. (9) The two noncoding chloroplast loci, the rps16 intron and the trnL-F region, have proven useful for this infrafamilial study, in combination giving over 500 informative sites. In contrast, the variation at infra- and intergeneric level in the Oleeae, especially in the genus Fraxinus and in the subtribe Oleinae, is too low to be useful.
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FOOTNOTES |
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4 Author for reprint requests (e-mail: eva.wallander{at}systbot.gu.se
).
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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