| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
0 Rancho Santa Ana Botanic Garden, 1500 North College Avenue, Claremont, California 91711 USA
Received for publication April 1, 1999. Accepted for publication January 14, 2000.
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Key Words: Coronilleae • Fabaceae • internal transcribed spacer • Loteae • Lotus • phylogeny
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
). This TH group includes the Galegeae, Carmichaelieae, Cicereae, Hedysareae, Fabeae, and Trifolieae, as well as the Loteae and Coronilleae. Members of this group are distinguished by a closed vascular system, epulvinate leaves, stipules adnate to the petiole, and centers of diversity in Old World regions (Polhill, 1981b
). In addition, all eight tribes except the Loteae and Coronilleae exhibit a unique molecular feature, the loss of one copy of the inverted repeat (IR) sequence of chloroplast DNA (Lavin, Doyle, and Palmer, 1990
; Liston, 1995
). This IR anomaly has led some researchers to postulate independent origins for the Loteae and Coronilleae (Lavin, Doyle, and Palmer, 1990
) and to dissociate them from the Galegeae (Polhill, 1994
). Reinterpretation of the presumed morphological specializations of the Loteae and Coronilleae (e.g., umbellate inflorescences, determinate root nodules) has also led to the supposition that they may be derived from tropical ancestors similar to the Phaseoleae and Milleteae (Lavin, Doyle, and Palmer, 1990
). Recent, family-wide phylogenetic studies of nucleotide sequence variation in the chloroplast gene rbcL, however, do not support this hypothesis (Kass and Wink, 1996
; Doyle et al., 1997
). Rather, both rbcL studies and recent phylogenetic analyses based on matK sequence data (Hu et al., in press) suggest that the Loteae and Coronilleae are closely related to core members of the TH group (i.e., Galegeae etc.) and a New World tropical tribe, Robinieae.
In addition to the IR anomaly, several morphological features distinguish the Loteae and Coronilleae from other members of the TH group. These include a specialized pollen type (3–4 colporate with little aperturate specialization; Ferguson and Skvarla, 1981
), a base chromosome number of x = 7 (Goldblatt, 1981
), determinate root nodules (Sprent, 1981
), umbellate inflorescences (Polhill, 1981b
), and seedlings with narrowly linear cotyledons and a suppressed plumule (Duke and Polhill, 1981
). Individually, however, the Loteae and Coronilleae are less easily distinguished. Apart from the presence of jointed fruits and branched root nodules in the Coronilleae, the two tribes are morphologically very similar. Nevertheless, they have traditionally been treated as distinct entities. In general, the Loteae (sensu Polhill, 1981b
) include 
170 species distributed in four genera (Table 1). Three of these genera, Cytisopsis Jaub., & Spach, Anthyllis L., and Hymenocarpos Savi, are restricted to Old World regions in the Mediterranean, with some taxa extending into northern Europe, the Atlantic Islands, and northeast Africa. The fourth, Lotus L., has major centers of diversity in the Mediterranean region and western North America, with a minor occurrence in western South America and western Australia. Members of Loteae are usually characterized by epulvinate, generally distichous phyllotaxy, glandular, membranous or herbaceous stipules, apically dilated filaments, flowers in axillary pedunculate heads or umbels, dehiscent or indehiscent unjointed fruits, and unbranched root nodules.
|
) traditionally include six genera with 51 species (Table 1). Five of these genera, Coronilla L., Hammatolobium Fenzl, Hippocrepis L., Scorpiurus L., and Ornithopus L., are largely restricted to Old World regions (northern Mediterranean region, Europe and western Asia). One species, Ornithopus micranthus (Benth.) Arechav, however, also occurs in southern South America. A sixth, monotypic genus, Antopetitia A. Rich., is restricted to tropical Africa (Polhill, 1981b
). The Coronilleae are generally distinguished from the Loteae by their jointed or elongated fruits, branched root nodules and stamens that are not apically dilated (Polhill, 1981a, b
). Base chromosome number for both Loteae and Coronilleae is x = 7, with some aneuploid reduction and occasional polyploidy reported in a few Old World species (Larsen, 1955a, b
).
Tribal delimitation of Loteae and Coronilleae
The tribal delimitation of Loteae and Coronilleae has been the subject of numerous inquiries. Dormer (1945, 1946)
, for example, studied vegetative features and seedlings and documented the absence of a plumule in some members of both the Loteae and Coronilleae. This feature, along with similarities in shoot and leaf vascularization, suggested the establishment of a single tribe. Corby (1981)
, based on a study of root nodule morphology, however, found unbranched root nodules in the Loteae, but branched ones in the Coronilleae, lending support for the retention of a two-tribe system. Similarly, Ferguson and Skvarla (1981)
found preliminary differences in pollen morphology, which supported a two-tribe system. A more extensive palynological study, however, revealed more similarities than differences, providing evidence in favor of a single tribe (Diez and Ferguson, 1990, 1994, 1996
). Given the equivocal findings of these studies, it remains unclear whether the Loteae and Coronilleae should be treated as two separate tribes, or a single one united under Loteae.
Understanding how different genera of Loteae and Coronilleae are related to one another is pertinent to the problem of tribal classification. Two genera, Lotus and Coronilla, for example, each contain species that exhibit overlapping morphological features that suggest a close, yet morphologically complex relationship (Polhill, 1981b
). Similarly, Hippocrepis, with its distinctive horseshoe-shaped fruits, is considered closely related to Coronilla, but only insofar as having a lomentaceous (i.e., jointed) fruit morphology. The genera Anthyllis, Hymenocarpos and Scorpiurus are reasonably well defined, but their affinities to one another and to other members of Loteae and Coronilleae are unknown. Similarly, the Old World genus, Ornithopus, is believed to be closely related to Antopetitia, but their genealogical relationship remains unclear. Analyses presented here focus on elucidating the phylogenetic relationships among these genera, with a sampling emphasis on the genus Lotus.
Generic delimitation of Lotus
The genus Lotus is by far the most taxonomically complex of all the genera within Loteae or Coronilleae. This is largely because of its high degree of morphological and geographical diversity: in addition to being the largest genus in either tribe, it is the only one which has centers of diversity in both the New and the Old World. This diversity in morphology and biogeography has sparked contentious debate, resulting in disagreement over whether to include the New World species in Lotus, or recognize them as a distinct genus, Hosackia (Greene, 1890
; Ottley, 1923, 1944
; Callen, 1959
; Grant and Sidhu, 1967
; Isley, 1981
; Polhill, 1981b
; Kirkbride, 1994
; Bentham, 1837
; Torrey and Gray, 1838
; Gray, 1864
; Bentham and Hooker, 1865
; Watson, 1876
; Abrams, 1944
). In a revision of the New World species, Ottley (1923)
placed all the members in Lotus and designated three subgenera: Hosackia Bentham, characterized by scarious or leaf-like stipules; Syrmatium Vogel, recognized by a combination of gland-like stipules and indehiscent fruits; and Acmispon (Raf.) Ottley, distinguished by gland-like stipules and dehiscent fruits (Ottley, 1923
). Acmispon was later subdivided into two sections, Microlotus Bentham and Simpeteria Ottley, in order to distinguish those taxa (Simpeteria) that have a penicillate stigma (Ottley, 1944
). A revision of the North American species by Isley (1981)
distinguished four informal groups, which correspond to Ottley's three subgenera, plus section "Simpeteria." Simpeteria, in turn, has recently been redescribed as the genus, Ottleya D. D. Sokoloff (Sokoloff, 1999
).
In the Old World, Lotus comprises several well-marked species groups, some of which have been regarded as segregate genera (Table 1). One of these, Dorycnium Miller, is usually accepted at the generic level (Taubert, 1894
; Gillett, 1958
; Ball, 1968
; Lassen, 1986
; Greuter and Burdet, 1989
; Sokoloff, 1998
), but some species have been treated as subg. Canaria (Rikli) Gillett within Lotus (Gillett, 1958
). As it is most often recognized, Dorycnium includes ten species, which are found in the Mediterranean region and the Canary Islands. Species of Dorycnium are distinguished by an erostrate keel, flowers in a capitate umbel, and leaves with or without a petiole (Isley, 1981
). Another segregate genus, Tetragonolobus Scop., is sometimes included at the subgeneric level within Lotus (Lassen, 1986
; Greuter and Burdet, 1989
), but this view is not unanimous (Isley, 1981
). When recognized, Tetragonolobus includes five species, which are widespread throughout central and southern Europe and the Mediterranean. Species of Tetragonolobus are generally distinguished by having stipular, trifoliate leaves, a distally dilated style, and squarish, winged pods. In addition to Dorycnium and Tetragonolobus, there are approximately six other Old World species groups that have been recognized either as segregate genera, or as subgenera or sections within Lotus (Table 1).
The taxonomic problem of delimitation at both the tribal and generic levels in Loteae and Coronilleae has resisted resolution using conventional techniques. This suggests that an alternative approach is needed. Here, we use a molecular phylogenetic method to evaluate the systematics of Loteae and Coronilleae. Specifically, we employ DNA sequencing of the internal transcribed spacer regions (ITS1, 5.8S and ITS2) of nuclear ribosomal DNA to: (1) evaluate the monophyly of the two tribes; (2) investigate evolutionary relationships among their component genera; (3) assess the utility of key morphological characters traditionally used in tribal delimitation; and (4) examine the delimitation and biogeography of Lotus in light of ITS sequence evolution.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
) were included in this study (Table 2). Representatives from the Loteae include 19 species of Lotus (comprising two Old World and all three New World subgenera), two species of the segregate genus Tetragonolobus and four of Dorycnium, two species of Anthyllis, and one of Hymenocarpos. Representatives from the Coronilleae include two species each of Ornithopus, Scorpiurus, Hippocrepis, and Coronilla, and one of Securigera DC (a segregate of Coronilla). Accession numbers and country of origin for each taxon are given in Table 2.
|
, based on a combination of morphological and molecular characters, found that some Fabeae are among members of an unresolved temperate legume clade that includes the Loteae and Coronilleae, while Robinieae and then Phaseoleae are each more distantly related. Kass and Wink (1997a)
, based on DNA sequences of ITS and rbcL, place Coronilleae, Robineae, and Fabeae together with Galegeae and Trifoleae near the base of the Faboideae. Based on rbcL data alone, Kass and Wink (1996)
suggest that Coronilleae is sister to a clade containing Fabeae, which in turn, is internal to a clade containing Phaseoleae. Results based on rbcL (Doyle et al., 1997
), however, imply a more distant relationship of Phaseoleae to Loteae and Coronilleae. Hu et al. (in press), using matK sequence data, indicate a close relationship between Loteae and Coronilleae, members of the TH group and Robinieae. In an effort to be conservative in estimating the phylogenetic history of Loteae and Coronilleae, we have chosen one species each from Robineae (Robinia pseudoacacia L.) and Fabeae (Vicia faba L.) for outgroup comparison.
DNA extraction and PCR
Total genomic DNA was isolated from either field-collected leaf material (kept on ice for 1–7 d) or herbarium collections using the 2X CTAB (hexadecyltrimethyl ammonium bromide) method of Doyle and Doyle (1987)
. This method was modified by the addition of 1% sodium bisulfite. Isolations from fresh leaf tissue were performed in 15-mL corex tubes, followed by two extractions with phenol/chloroform. DNAs were purified using the EluQuick DNA Purification Kit (Schleicher & Schuell) and standardized to 10 ng/µL using fluorometry. Isolations from herbarium collections (Table 2) employed a microprep method of Cullings (1992)
and modified after Doyle and Doyle (1987)
. Herbarium specimens used for DNA isolation were verified using published keys. Vouchers for all representative taxa are deposited in the Rancho Santa Ana (RSA) Botanic Garden herbarium.
The polymerase chain reaction (PCR) was used to amplify the ITS and 5.8S and flanking regions. Single-stranded DNAs of the ITS region were directly amplified by asymmetric PCR using the primers ITS4 and ITS5 (White et al., 1990
) in a 1:1 ratio. Final concentrations or amounts of each reagent (based on 25 µL) were as follows: ITS4 and ITS5 primers (0.4 µmol/L), DNTPs (20 µmol/L in equimolar ratios), sterile water (16.1 µL), glycerol (1.25 µL), Taq polymerase buffer (2.5 µL), Amplitaq (0.1 µL at 5 units/mL), and genomic DNA (0.4 ng/µL). Reaction mixtures were sealed with a drop of mineral oil to prevent evaporation during thermal cycling.
An MJ Research (Watertown, Massachusetts, USA) thermal cycler was programmed for 1 min denaturation (97°C), 1 min annealing (48°C), and 45 sec primer extension (72°C). Primer extension times were increased by 4 sec each subsequent cycle for a total of 38 cycles. A final 7-min incubation cycle (72°C) completed the primer-template extensions. Reactions were monitored by the inclusion of negative controls in each sample set. PCR products were electrophoresed using a 1.5% agarose gel in a 0.5x tris-borate-EDTA (TBE) buffer, stained for 15 min in ethidium bromide, destained for 15 min, and photographed on an UV transilluminator. Size of the amplification products was estimated using a 1-kb ladder (Gibco). PCR products were purified by differential filtration in Millipore Ultrafree-MCTM tubes (Millipore UFC3 THK 00). Cycle sequencing employed primers ITS2, ITS3, ITS4I, and ITS5I (see Porter, 1996
). An Applied Biosystems 373A automated DNA sequencer, and the PRISMTM Dye DeoxyTM Terminator Kit (Perkin Elmer, Norwalk, Connecticut, USA) were used to collect nucleotide sequence data. DNA of all taxa was sequenced in both directions in order to ensure the accuracy of base calls.
Sequence analysis
DNA sequences were initially aligned using the program ClustalW (Higgins, Bleasby, and Fuch, 1992
). This sequence alignment was manually adjusted by sequential pairwise comparisons. Manual alignment required the introduction of 36 (insertion/deletion) indel zones ranging in size from one to 21 bp scattered across ITS1 and ITS2, and a 1-bp indel in the 5.8S region. The boundaries of the ITS1 and ITS2 regions for all 42 taxa were identified based on comparison to published sequences of Daucus carrota L., Vicia faba (Yokota et al., 1989
), and species of Astragalus (Wojciechowski et al., 1993
). Transition to transversion ratio, GC content, number of informative characters, sequence length, and pairwise divergence values were determined using test version 4.0d63 of PAUP* (Swofford, 1998
; used with permission, D. L. Swofford, Smithsonian Institution, personal communication).
Phylogenetic analysis
A baseline maximum parsimony analysis was performed on the 42 ITS sequences using Fitch parsimony as implemented in PAUP* 4.0d63. Two initial heuristic searches were employed, one with uninformative characters included, and one without. Each search employed TBR (tree bisection-reconnection) branch swapping in conjunction with saving all minimal trees (MULPARS), accelerated transformation, (ACCTRANS) and branches of zero length collapsed. Three different regimes of stepwise addition sequences were employed, SIMPLE, CLOSEST and RANDOM (100 replicates), for a total of six initial heuristic searches. In each search, characters were given equal weight and gaps were treated as missing data.
The integrity of tree topology was explored through recoding of the indel zones as binary characters: 15 unambiguous indels were coded and appended to the end of the data matrix. Additionally, outgroup taxa were successively excluded from the data matrix in order to examine the effect on ingroup topology. A step matrix of user-defined character types employing a weight of 1:1.1 (transitions:transversions; based on estimates of Loteae and Coronilleae sequences) was also implemented. A neighbor-joining (NJ) distance analysis was performed for comparison to maximum parsimony: genetic distances were calculated using the Tamura-Nei algorithm, which corrects for any transition/transversion bias and uneven codon usage.
For each heuristic analysis, a strict consensus tree was generated. To evaluate levels of support for clades, both bootstrap (Felsenstein, 1985
) and decay (Bremer, 1994
; Donoghue et al., 1992
) analyses were performed. Bootstrap values were calculated from 10 000 fast, stepwise addition replicates as implemented by PAUP* 4.0d63 using a heuristic search with CLOSEST addition, TBR swapping, MULPARS, and ACCTRAN options in effect. Decay values were calculated using the reverse constraint option in PAUP* 4.0d63.
To explore the taxonomic utility of key morphological characters used in traditional classifications of Loteae and Coronilleae, character state evolution of two characters, fruit type (jointed vs. unjointed) and root nodule branching (branched vs. unbranched), was reconstructed using maximum parsimony assumptions and the TRACE CHARACTER function in MacClade (Maddison and Maddison, 1992
). The biogeography of Lotus was similarly examined by tracing the character "region" onto the strict consensus tree. In both sets of reconstructions, the strict consensus tree was modified to represent well-supported lineages with a single branch, and naming that branch using a representative from that clade, or in the case of monophyletic subgenera, the name of the subgenus (e.g., Syrmatium). All possible paths of character evolution given the topology and the distribution of character states were determined using the EQUIVOCAL CYCLING option in MacClade.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
The entire ITS region included a total of 681 nucleotide sites. Multiple sequence alignment of these sites required the inclusion of 168 gapped positions (24.6% of all sites). Of the 681 nucleotide sites, 304 were constant and 377 were variable, with more variable sites (229) occurring in ITS1 than in ITS2 (158). A total of 277 nucleotide sites (40.6%) were potentially parsimony informative (data available upon request).
Phylogenetic analysis
The baseline maximum parsimony analysis excluding uninformative characters produced eight minimal length trees, 1066 steps in length (CI = 0.480, RI = 0.707). When uninformative characters were included 12 most parsimonious trees (MPTs) of length 1191 were generated (CI = 0.535). The strict consensus of each set of trees is identical, and is well resolved, as indicated by the cladogram shown in Fig. 1. Weighting transitions relative to transversions (1:1.1) resulted in two trees (CI = 0.613, RI = 0.818, uninformative characters excluded), these being a subset of the trees obtained in the unweighted baseline analyses. One of these trees is shown in Fig. 1, with branch lengths, bootstrap support, and decay values indicated. Analyses that included indels (weighted equally with substitution characters) resulted in the same set of 12 trees generated under Fitch assumptions, but with a length of 1121 steps (or 1228 steps when uninformative characters are included). Using weighting (transitions to tranversions) and indels resulted in the same two trees obtained with weighting alone (uninformative characters excluded). Neighbor-joining analysis yielded a topologically identical tree to that derived from maximum parsimony, with one exception: Hippocrepis emerus and H. biflora are placed sister to a large clade containing mostly New World Lotus. Bootstrapping of the NJ tree, however, reveals no significant support (<50%) for this sister group relationship (tree not shown).
|
Assessment of relationships within these two clades yields four main conclusions. First, members of the Loteae and Coronilleae do not form individual monophyletic groups. Rather, some members of Coronilleae are strongly supported as being more closely related to members of Loteae than to other Coronilleae. For example, Coronilla scorpioides is placed in a well-supported (100%) clade with members of Lotus subg. Hosackia. In addition, C. valentina is nested within members of New World Lotus subg. Acmispon (87% support).
Second, Lotus is not monophyletic. This is indicated by the fact that New and Old World Lotus form separate, robustly supported (100%) clades. Moreover, these clades reveal a deep phylogenetic split between Old and New World Lotus (>40 nucleotide changes separate the two clades; phylogram not shown). This, however, does not account for a group of three New World Lotus species (L. crassifolius, L. formosissimus, and L. oblongifolius), which comprise a well-supported (100%) group, but remain ambiguously placed (<50% support) relative to both the New and Old World Lotus clades (Fig. 1). Nevertheless, even if the affinities of these three taxa could be resolved, Lotus would not be monophyletic.
Third, New World Lotus is not monophyletic. Rather, Coronilla valentina and C. scorpioides appear more closely related to different New World Lotus (L. oroboides, 97%, and L. crassifolius, 70%, respectively) than to one another or to other Coronilleae. The New World Lotus s.l. clade, however, contains a well-supported (96%) group comprising members of subg. Syrmatium. In contrast, neither subgenus Acmispon nor Hosackia appears monophyletic (Fig. 1).
Fourth, the representatives of Old World Lotus are monophyletic (75% support), but only if Tetragonolobus is included. Lotus and Tetragonolobus, in turn, appear closely allied (97% support) to a moderately supported (66%) clade containing members of the segregate genus Dorycnium. The well-supported (100%) pairing of Hippocrepis emerus and H. biflora is also noteworthy in that it supports the recent transfer of the formerly recognized taxon Coronilla emerus from Coronilla to Hippocrepis (Lassen, 1989
). This transfer was based largely on similarity in base chromosome number (Coronilla is x = 6; H. emerus and Hippocrepis are x = 7) and is also supported by studies of pollen exine structure (Diez and Ferguson, 1996
).
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
), there are reasons to express caution when using a single gene to infer phylogeny. In particular, three biological processes, introgression, lineage sorting, and gene duplication, have been noted as problems that can potentially impact phylogenetic history (Doyle, 1992
). Further, systematic error, such as long-branch attraction (Hendy and Penny, 1989
), and differential sampling can directly impact the outcome of phylogenetic analyses. However, if the ITS phylogeny presented here is taken as an accurate representation of the relationships of Loteae and Coronilleae, then we can ask what its implications are for (a) the tribal classification of Loteae and Coronilleae, (b) the utility of key morphological features used in tribal delimitation, and (c) the taxonomic delimitation and biogeography of Lotus.
Implications for tribal classification
The earliest description of the Loteae and Coronilleae is that of DeCandolle (1825)
who recognized two distinct tribes containing three and five genera, respectively. Bentham and Hooker (1865)
recognized only a single tribe, an expanded Loteae, and transferred all members of the Coronilleae to tribe Hedysareae. Taubert (1894)
accepted only Loteae, and created subtribe Coronillinae under Hedysareae to contain members of the formerly recognized Coronilleae. Hutchinson (1964)
restored the two-tribe system, while recognizing several new genera in both Loteae and Coronilleae. These genera have either been reduced in number while retaining a two tribe system (Polhill, 1981b
) or combined under a single tribe, Loteae (Polhill, 1994
; Sokoloff, 1998
).
The traditional two-tribe classification presented by Polhill (1981b)
and previous workers (DeCandolle, 1825
; Bentham and Hooker, 1865
; Taubert, 1894
; Hutchinson, 1964
) is not consistent with the evolutionary history inferred from the phylogenetic analysis based on ITS sequences (Fig. 1). Rather, these data are in agreement with Polhill (1994)
and Sokoloff (1998)
, who have each arranged genera of Loteae and Coronilleae under Loteae. It is important to note, however, that the results presented here do not formally rule out the possibility of a monophyletic "Coronilleae" minus Coronilla. This is due in part to the lack of significant support for the resolution of branch order among Hippocrepis, Scorpiurus, Ornithopus and Securigera, and the absence of some Loteae (sensu Polhill, 1994
) genera (e.g., Dorycnopsis, Tripodion, Hammatolobium). Additional sampling of these genera may help to resolve relationships at the base of the phylogeny and ultimately decide the question of whether members of the Coronilleae, except Coronilla, are monophyletic.
However, if we assume that a goal of classification is to recognize monophyletic genera and higher taxa, then the hypothesis of a two-tribe system is falsified. By contrast, recognition of an expanded Loteae is readily accommodated. Data supporting the synthesis of the Loteae and Coronilleae include robust relationships between species of Coronilla and both Lotus subg. Acmispon and Lotus subg. Hosackia, as well as an association between Anthyllis, Hymenocarpos (Loteae) and Securigera (Coronilleae). Two representatives of Ornithopus also occur sister to a major clade containing North American species of Lotus. This alliance, however, is not well supported and should be viewed cautiously.
Utility of key morphological characters
Key morphological features that have traditionally been used to support the recognition of a two-tribe system include jointed fruits (lomented pods) and branched root nodules of Coronilleae vs. unjointed fruits and unbranched root nodules of Loteae. Mapping of these characters onto one of the MPTs (Fig. 2) clearly shows that these features are homoplastic. This finding is perhaps not surprising given that lomented pods in general have evolved several times in other temperate and subtropical groups of Fabaceae (Dudik, 1981
; Polhill and Raven, 1981
). In Loteae s.l., the taxonomic utility of jointed fruits has also been called into question. Lassen (1989)
, for example, reassessed morphological relationships in Coronilla, Securigera, and Hymenocarpos and suggested that lomented pods have evolved more than once in these genera. Polhill (1981b)
, upon considering whether to combine the Loteae and Coronilleae, also referred to the problem of jointed fruits, calling it a "technical distinction." This distinction is further accentuated by observations made on two morphologically complex species, Old World Lotus ornithopodioides and the monotypic segregate genus Podolotus: L. ornithopodioides, for example, has lomented pods, whereas Podolotus has the unjointed, dehiscent pod characteristic of Lotus, but a calyx similar to that of Coronilla (Polhill, 1981b
). These examples, coupled with ITS evidence, underscore the fact that fruit type characters may be unreliable for constructing higher-level (e.g., generic) classifications in Loteae. By contrast, morphological features such as a suppressed plumule, a base chromosome number of x = 7, pollen that is 3–4 (6–7) colporate, staminal filaments that are basally dilated, and compound leaves with at least three terminal leaflets, appear to be well-defined synapomorphies for the more broad circumscription of Loteae.
|
Second, New World Lotus does not share common ancestry with Old World Lotus to the exclusion of several other genera. This result is pertinent to the debate on whether to include the New World species in Lotus or exclude a portion of them as the New World endemic genus Hosackia. Resolving this controversy, however, is complicated by the placement of Ornithopus and Coronilla within the "New World" Lotus clade. While there is only weak support for the placement of Ornithopus as sister to this clade, both C. valentina and C. scorpioides are each robustly supported as being closely related to different New World species of Lotus.
An alternative explanation for the association of at least one Coronilla species (C. valentina) with New World Lotus is hybridization: leaf tissue of C. valentina was obtained from a waif collection of C. valentina from California (Table 2). Examination of the herbarium specimen from which DNA was obtained, however, revealed no evidence of morphological introgression with Lotus. Nevertheless, hybridization appears to occasionally occur between Lotus species (Isley, 1981
; Grant, 1965
; Allan, 1998
), suggesting that intergeneric hybridization cannot be discounted. The fact that Old World C. scorpioides also appears closely allied to New World Lotus, however, strongly suggests that the association between New World Lotus and Coronilla may be accurate. Additional sampling of other Old World Coronilla species will be an important priority for future analyses; these should help verify the validity of the novel relationships suggested here.
Third, the non-monophyly of Lotus is underscored by the fact that even with the sparse sampling of New World Lotus included in this study, two of the three subgenera are not monophyletic. This is the case, for example, with both Acmispon and Hosackia. By contrast, subg. Syrmatium appears monophyletic (Fig. 1). Finally, it is important to note that only a few Old World species of Lotus were included in this study. Consequently, it may be that some Old World species, which have not been sampled, are more closely related to species in the New World, than to other Old World Lotus. This question is the subject of ongoing phylogenetic analyses (Allan, Zimmer, and Wagner, unpublished data).
Agreement with morphology
In general, data from ITS are in agreement with relationships based on morphology. For example, the observation that Old and New World Lotus form separate clades is consistent with the fact that Old World and New World species are palynologically distinct: Old World Lotus are uniformly three-aperturate, while those of the New World with the exception of subg. Hosackia are generally four-aperturate (Crompton and Grant, 1993
; Diez and Ferguson, 1994
). In addition, a number of well-supported, morphologically recognizable groups are found within these two clades. New World subg. Syrmatium, for example, forms a well-supported (96% bootstrap) clade and is easily recognized by a combination of gland-like stipules and indehiscent fruits. Subgenus Acmispon (with the exception of C. valentina), distinguished by gland-like stipules and dehiscent fruits, is also reasonably well supported (76% bootstrap). Note, however, that this clade does not contain L. salsuginosus, a species generally included in Acmispon. Among the members of Old World Loteae, three morphologically cohesive groups receive moderate to high bootstrap support. These include Lotus with its distinctive pollen (75% bootstrap support), Dorycnium recognized by its erostrate keels and capitate inflorescences (66% support), and Tetragonolobus with its winged and squarish fruits (90% support).
In cases where ITS and morphology do not strictly agree there is a posteriori evidence which suggests that a reexamination of morphology may prove insightful. Consider, for example, the pairing of Coronilla valentina and New World Lotus oroboides. These taxa, in general, are phenotypically dissimilar: L. oroboides is a low-growing, perennial herb with linear-lanceolate leaves, whereas C. valentina is a small, woody shrub with obovate to slightly obcordate leaves. Despite these dissimilarities, it is noteworthy that both C. valentina and L. oroboides are nested within a well-supported (85% bootstrap) clade containing both prostrate herbaceous and upright, semiwoody taxa. Further, the leaves of C. valentina are morphologically reminiscent of those of L. cedrosensis, also found in this clade. Thus, barring the possibility of hybridization as an alternative explanation for the inclusion of C. valentina in the New World clade, there may be reason to reevaluate the morphological affinities of taxa within this New World clade.
In a similar case of apparent morphological incongruence, C. scorpioides is placed among members of subg. Hosackia. Here, the annual habit, suborbicular leaves, and absence of stipules in C. scorpioides are contrasted with the elliptic to obovate leaves and leaf-like stipules characteristic of Hosackia. Stipule morphology in Lotus, however, has been the subject of considerable inquiry: the anatomical distinction between stipules and true leaves is not clear (Heyn, 1976
). Taking this into consideration, it may be that the axillary and slightly clasping leaves of C. scorpioides are homologous with the leaf-like stipules of Hosackia. It is also important to point out that the low-growing, herbaceous habit of C. scorpioides is morphologically more similar to members of Hosackia than to Coronilla. This observation is made all the more pertinent by the fact that L. formosissimus, also a member of this clade, is distinguished from other species of subg. Hosackia by having petal claws that are exserted from the calyx tube (Isley, 1981
). Moreover, this feature is one of several diagnostic characters used to describe the genus Coronilla (Polhill, 1981b
). Thus, both in the case of C. scorpioides and C. valentina, there is a posteriori evidence of morphological resemblance to Lotus that is consistent with relationships based on ITS.
Implications for the biogeography of Lotus
Nuclear ITS data display varying degrees of support for different clades in the phylogeny. However, if the common structure observed in the eight MPTs is taken to be consistent with the true phylogeny of this group, then these data have a direct bearing on our understanding of the geographic origin and subsequent dispersal of Lotus.
Raven (1971)
first suggested that Old and New World Lotus are only distantly related. Raven and Polhill (1981)
further postulated that Lotus, along with other temperate legume genera (e.g., Astragalus, Lupinus, and Trifolium), originated in Eurasia and reached North America via repeated long-distance dispersal. It has also been postulated, based on evidence from pollen morphology (Diez and Ferguson, 1996
) and phytogeographic patterns (Sousa and Delgado, 1993
), that the North American species of Lotus represent a secondary diversification of the genus from an Old World origin centered in the Mediterranean. It is evident, based on data presented here, that the suggested model of diversification of New World Lotus, via dispersal from Old World regions exclusive of other genera (e.g., Coronilla), may have oversimplified the patterns of intercontinental dispersal.
In evaluating different phylogeographic scenarios, however, it is important to note that the sister group to the Loteae–Coronilleae clade is not definitively identified here. Moreover, deciding the geographic origin of the ancestor of the Loteae–Coronilleae clade is not possible given the geographic distribution of the outgroup taxa (Robinia is New World, while Vicia is Old World). However, in keeping with traditional hypotheses and in an attempt to be conservative relative to assessments by Raven (1971)
, Raven and Polhill (1981)
, and Sousa and Delgado (1993)
, an assumption will be made that the ancestor of the Loteae-Coronilleae clade was Old World in distribution. Given this scenario, there are four possible reconstructions of biogeography that are equally parsimonious (Fig. 3A–D).
|
; Raven and Polhill, 1981
) or implied (Sousa and Delgado, 1993
) multiple, independent dispersal events to the New World from Old World Lotus. In one sense, however, the ITS data provide evidence refuting this idea in that there is no indication that Old World Lotus are in any way involved with dispersal to the New World. Nevertheless, multiple dispersal events to the New World are a possible explanation of the data. There are three most parsimonious reconstructions for examining multiple dispersal events to the New World. In each case, an Old World origin for New World Lotus is assumed (Fig. 3B–D). In the first, at least three independent dispersals are required (one each for members of subg. Hosackia, Acmispon, and Syrmatium), coupled with a single back migration to account for Coronilla scorpioides (Fig. 3B). The second reconstruction (Fig. 3C) requires three independent dispersals (two for subg. Hosackia and one for the remaining New World Lotus), and one back migration (C. valentina). The final reconstruction (Fig. 3D) requires two independent dispersals to account for subg. Hosackia and the remaining New World Lotus. In addition, two back migrations are required, one each for C. scorpioides and C. valentina.
Regardless of the geographic origin of the ancestor of New World Lotus, if there were at least two independent dispersal events, then at least one back migration must also have occurred. Clearly, phylogeographic explanations of the ITS phylogeny, requiring the fewest dispersal events, are still considerably more complicated than previously envisioned. If this pattern is upheld with additional sampling, it would suggest a complex series of multiple, independent continental dispersals that involve not only Lotus, but Coronilla as well. Elucidating these patterns will be important for comparison with the biogeography of other TH legumes, especially those that show a disjunct distribution between western North America and the Mediterranean (Kass and Wink, 1997b
; Ainouche and Bayer, 1999).
Summary and conclusions
Phylogenetic estimation of ITS sequences from the Loteae–Coronilleae provides independent evidence for assessing evolutionary relationships and classification within this diverse group of legumes. Analyses of bootstrap sampling and decay index show that these data provide support for recognizing a single tribe, Loteae, as envisioned by Polhill (1994)
and reaffirmed by Sokoloff (1998)
. Within this expanded Loteae two major clades are identified: one containing the majority of New World Lotus species plus one species of Coronilla; and the other comprising Old World Lotus plus Tetragonolobus, and Dorycnium. The fact that New and Old World Lotus are in separate, well-supported clades strongly suggests that Lotus is not monophyletic. New World Lotus is also not monophyletic in that it includes members of Old World Coronilleae, Coronilla and Ornithopus. At the level of subgenera, only Syrmatium appears monophyletic, with both Acmispon and Hosackia supported as paraphyletic or polyphyletic groups, respectively. Of genera in the Old World, Lotus appears to be monophyletic, but only if Tetragonolobus is included. Further, the segregate genus Dorycnium is weakly supported as a monophyletic sister group to Old World Lotus, suggesting that recognition at the generic or at least the subgeneric level is justified. Phylogeographically, ITS data present a complex picture of independent dispersal and back migration for Lotus and Coronilla, respectively. Although robust, these data suggest that future investigations of biogeography should include both additional sampling of Old World Lotus and Coronilla and an independent data source in order to confirm the alternative patterns of biogeography observed here. These data suggest that molecular methods are useful for evaluating alternative classifications within Loteae s.l. and can offer additional insight into elucidating both its morphological evolution and biogeographic history.
|
FOOTNOTES |
|---|
2 Author for correspondence, current address: Laboratory of Molecular Systematics, National Museum of Natural History, Smithsonian Institution, Washington, D.C. 20560, USA (e-mail: gallan{at}lms.si.edu)
|
LITERATURE CITED |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
AÏnouche, A.-K., and R. J. Bayer. 1999 Phylogenetic relationships in Lupinus (Fabaceae: Papilionoideae) based on internal transcribed spacer sequences (ITS) of nuclear ribosomal DNA. American Journal of Botany 86: 590–607
Allan, G. J. 1998 Molecular systematic and biogeographic studies of the temperate herbaceous papilionoid tribes Loteae and Coronilleae (Fabaceae). Ph.D. dissertation, Claremont Graduate School, Claremont, California, USA
Baldwin, B. G., M. J. Sanderson, J. M. Porter, M. F. Wojciechowski, S. S. Campbell, and M. J. Donoghue. 1995 The ITS region of nuclear ribosomal DNA: a valuable source of evidence on angiosperm phylogeny. Annals of the Missouri Botanical Garden 82: 247–277[CrossRef][Web of Science]
Ball, P. W. 1968 Dorycnium, Lotus, Tetragonolobus. In T. G. Tutin [ed.], Flora Europaea, vol. 2, 182–183. Cambridge University Press, Cambridge, UK
Bentham, G. 1837 Observations on the genus Hosackia and the American Loti. Transactions of the Linnean Society 17: 363–368
———, and J. D. Hooker. 1865 Leguminosae. Genera Plantarum: 434–600
Bremer, K. 1994 Branch support and tree stability. Cladistics 10: 295–304[CrossRef][Web of Science]
Callen, E. O. 1959 Studies in the genus Lotus (Leguminosae) I. Limits and subdivisions of the genus. Canadian Journal of Botany 37: 157–165
Chapill, J. A. 1995 Cladistic analysis of the Leguminosae: the development of an explicit phylogenetic hypothesis. In R. M. Polhill and P. H. Raven [eds.], Advances in legume systematics, part 7, Phylogeny, 1–9. Royal Botanic Gardens, Kew, UK
Corby, H. D. L. 1981 The systematic value of leguminous root nodules. In R. M. Polhill and P. H. Raven [eds.], Advances in legume systematics, part 1, 657–669. Royal Botanic Gardens, Kew, UK
Crompton, C. W., and W. F. Grant. 1993 Pollen morphology in Loteae (Leguminosae) with particular reference to the genus Lotus L. Grana 32: 129–153[Web of Science]
Cullings, K. W. 1992 Design and testing of a plant-specific PCR primer for ecological and evolutionary studies. Molecular Ecology 1: 233–240
DeCandolle, A. P. 1825 Leguminosae. Prodromus systematis naturalis regni vegetabilis, 93–170, 216–353, 381–524
Diez, M. J., and I. K. Ferguson. 1990 Studies of the pollen morphology and taxonomy of the tribes Loteae and Coronilleae (Leguminosae: Faboideae) 1, Anthyllis L. and related genera. Lagascalia 16: 77–94
———, and ———. 1994 The pollen morphology of the tribes Loteae and Coronilleae (Papilionoideae: Leguminosae) 2, Lotus L. and related genera. Review of Palaeobotany and Palynology 81: 233–255[CrossRef]
———, and ———. 1996 Studies of the pollen morphology and taxonomy of the tribes Loteae and Coronilleae (Papilionoideae; Leguminosae) 3, Coronilla L. and related genera and systematic conclusions. Review of Palaeobotany and Palynology 94: 239–257[CrossRef]
Donoghue, M., R. G. Olmstead, J. F. Smith, and J. D. Palmer. 1992 Phylogenetic relationships of Dipsicales based on rbcL sequences. Annals of the Missouri Botanical Garden 79: 333–345
Dormer, K. J. 1945 On the absence of a plumule in some Leguminous seedlings. New Phytologist 44: 25–28[CrossRef]
———. 1946 Vegetative morphology as a guide to the classification of the Papilionatae. New Phytologist 45: 145–161[CrossRef][Web of Science]
Doyle, J. J. 1992 Gene trees and species trees: molecular systematics as one-character taxonomy. Systematic Botany 17: 144–163[CrossRef][Web of Science]
———, and J. L. Doyle. 1987 A rapid DNA isolation method for small quantities of fresh leaf tissues. Phytochemical Bulletin 19: 11–15
———, ———, J. A. Ballenger, E. E. Dickson, T. Kajita, and H. Ohashi. 1997 A phylogeny of the chloroplast gene rbcL in the Leguminosae: taxonomic correlations and insights into the evolution of nodulation. American Journal of Botany 84: 541–554[Abstract]
Dudik, N. M. 1981 Morphology of the pods of Leguminales (Fabales). In R. M. Polhill and P. H. Raven [eds.], Advances in legume systematics, part 2, 897–901. Royal Botanic Gardens, Kew, UK
Duke, J. A., and R. M. Polhill. 1981 Seedlings of the Leguminosae. In R. M. Polhill and P. H. Raven [eds.], Advances in legume systematics, part 2, 941–950. Royal Botanic Gardens, Kew, UK
Felsenstein, J. 1985 Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783–791[CrossRef][Web of Science]
Ferguson, I. K., and J. J. Skvarla. 1981 The pollen morphology of the subfamily Papilionoideae (Leguminosae). In R. M. Polhill and P. H. Raven [eds.], Advances in legume systematics, 859–896. Royal Botanic Gardens, Kew, UK
Gillett, J. B. 1958 Lotus in Africa south of the Sahara (excluding the Cape Verde Islands and Socotra) and its distinction from Dorycnium. Kew Bulletin 13: 361–381
Goldblatt, P. 1981 Cytology and the phylogeny of Leguminosae. In R. M. Polhill and P. H. Raven [eds.], Advances in legume systematics, part 2, 427–463. Royal Botanic Gardens, Kew, UK
Grant, W. F. 1965 A chromosome atlas and interspecific hybridization index for the genus Lotus (Leguminosae). Canadian Journal of Genetics and Cytology 7: 457–471[Web of Science]
———, and B. S. Sidhu. 1967 Basic chromosome number, cyanogenetic glucoside variation and geographic distribution of Lotus species. Canadian Journal of Botany 45: 639–647
Gray, A. 1864 Synopsis of the species of Hosackia. Proceedings of the National Academy of Sciences, Philadelphia 1863: 346–352
Greene, E. L. 1890 Enumeration of the North American Loti. Pittonia 2: 133–150
Greuter, W. H., and M. Burdet 1989. Med-Checklist, 4. Leguminosae. Conservatoire et Jardin Botaniques, Geneve: 2–214
Hendy, M. D., and D. Penny. 1989 A framework for the quantitative study of evolutionary trees. Systematic Zoology 38: 297–309[CrossRef][Web of Science]
Heyn, C. C. 1976 An old question revived: "What are the stipules of Lotus?" Lotus Newsletter 7(8): 3–5
Higgins, D. G., A. J. Bleasby, and R. Fuchs. 1992 Clustal V: improved software for multiple sequence alignment. Computational Applications in Applied Biosciences 8: 189–191
Hu, J.-M., M. Lavin, M. F. Wojciechowski, and M. J. Sanderson. In press. Phylogenetic systematics of the tribe Millettieae (Leguminosae) based on chloroplast matK sequences, and implications for evolutionary patterns in Papilionoideae. American Journal of Botany,
Hutchinson, J. 1964 The genera of flowering plants. Clarendon Press, Oxford, UK
Isley, D. 1981 Leguminosae of the United States III. Memoirs of the New York Botanic Garden 25: 128–213
Kass, E., and M. Wink. 1996 Molecular evolution of the Leguminosae: phylogeny of the three subfamilies based on rbcL sequences. Biochemical Systematics and Ecology 24: 365–378[CrossRef]
———, and ———. 1997a Phylogenetic relationships in the Papilionoideae (Family Leguminosae) based on nucleotide sequences of cpDNA and ncDNA (ITS1 and 2). Molecular Phylogenetics and Evolution 8: 65–88[CrossRef][Web of Science][Medline]
———, and ———. 1997b Molecular phylogeny and phylogeography of Lupinus (Leguminosae) inferred from nucleotide sequences of the rbcL gene and ITS 1 + 2 of rDNA. Plant Systematics and Evolution 208: 139–167[CrossRef][Web of Science]
Kirkbride, J. H. 1994 Taxonomic circumscription of the genus Lotus Linnaeus (Fabaceae, Loteae), its tribal position, and its species. The First International Lotus Symposium, Missouri Botanical Gardens, St. Louis Missouri, University Extension, University of Missouri-Columbia, Missouri, USA
Larsen, K. 1955a Cyto-taxonomical Studies in Lotus II. Botanisk Tidsskrift 52: 8–17
———. 1955b Cyto-taxonomical studies on the Mediterranean flora. Botaniska Notiser 108: 263–275
Lassen, P. 1986 Med-Checklist Notulae 13
———. 1989 A new delimitation of the genera Coronilla, Hippocrepis, and Securigera (Fabaceae). Willdenowia 19: 49–62
Lavin, M., J. J. Doyle, and J. D. Palmer. 1990 Evolutionary significance of the loss of the chloroplast DNA inverted repeat in the Leguminosae subfamily Papilionoideae. Evolution 44: 390–402[CrossRef][Web of Science]
Liston, A. 1995 Use of the polymerase chain reaction to survey for the loss of the inverted repeat in the legume chloroplast genome. In R. M. Polhill and P. H. Raven [eds.], Advances in legume systematics, part 7, Phylogeny, 31–40. Royal Botanic Gardens, Kew, UK
Maddison, W. P., and D. R. Maddison. 1992 MacClade version 3.04. Analysis of phylogeny and character evolution. Sinauer, Sunderland, Massachusetts, USA
Ottley, A. M. 1923 A revision of the California species of Lotus. University of California Publications in Botany 10: 189–305
———. 1944 The American Loti with special consideration of a proposed new section, Simpeteria. Brittonia 5: 81–123
Polhill, R. M. 1981a Papilionoideae. In R. M. Polhill and P. H. Raven [eds.], Advances in legume systematics, part 1, 191–208. Royal Botanic Gardens, Kew, UK
———. 1981b Loteae and Coronilleae. In R. M. Polhill and P. H. Raven [eds.], Advances in legume systematics, part 1, 371–374. Royal Botanic Gardens, Kew, UK
———. 1994 Classification of the Leguminosae. In F. A. Bisby, J. Buckingham, and J. B. Harborne [eds.], Phytochemical dictionary of the Leguminosae, xxxv–lvii. Chapman and Hall, New York, New York, USA
———, and P. H. Raven. 1981 Evolution and Systematics of the Leguminosae. In R. M. Polhill and P. H. Raven [eds.], Advances in legume systematics, part 1, 1–26, and references therein. Royal Botanic Gardens, Kew, UK
Porter, J. M. 1996 Phylogeny of Polemoniaceae based on nuclear ribosomal transcribed spacer DNA sequences. Aliso 15: 57–77
Raven, P. H. 1971 The relationships between ‘Mediterranean’ floras. Plant life of southwest Asia, 119–134. Botanical Society of Edinburgh, Aberdeen, UK
———, and R. M. Polhill. 1981 Biogeography of the Leguminosae. In R. M. Polhill and P. H. Raven [eds.], Advances in legume systematics, part 1, 27–34. Royal Botanic Gardens, Kew, UK
Sokoloff, D. D. 1998 Morphological and taxonomical study of the genus Anthyllis and principles of revision of Loteae tribe (Papilionaceae). Ph.D. dissertation, Moscow University, Moscow, Russia (in Russian)
———. 1999 Ottleya, a new genus of Papilionaceae–Loteae from North America. Feddes Repertorium 110: 89–97
Sousa, M. S., and A. S. Delgado. 1993 Mexican Leguminosae: phytogeography, endemism and origins. In T. P. Ramamoorthy, R. Bye, A. Lot, and J. Fa [eds.], Biological diversity of México: origins and distribution, 459–511. Oxford University Press, Oxford, UK
Sprent, J. K. 1981 Functional evolution in some papilionoid root nodules. In R. M. Polhill and P. H. Raven [eds.], Advances in legume systematics, part 2, 671–676. Royal Botanic Gardens, Kew, UK
Swofford, D. L. 1998 PAUP*. Phylogenetic Analysis Using Parsimony (*and other methods), version 4. Sinauer, Sunderland, Massachusetts, USA
Taubert, P. H. W. 1894 Leguminosae III. Die naturlichen Pflanzenfamilien, 70–385. W. Englemann, Leipzig, Germany
Torrey, G. S., and A. Gray. 1838 Flora of North America
Watson, S. 1876 Hosackia. Botany of California, 133–139. University of California Press, California, USA
White, T. J., T. Bruns, S. Lee, and J. Taylor. 1990 Amplification and direct sequencing of fungal ribosomal genes for phylogenetics. In M. Innis, D. Gelfand, J. Sninsky, and T. White [eds.], PCR protocols: a guide to methods and applications, 315–322. Academic Press, San Diego, California, USA
Wojciechowski, M. F., M. J. Sanderson, B. G. Baldwin, and M. J. Donoghue. 1993 Monophyly of aneuploid Astragalus (Fabaceae): evidence from nuclear ribosomal DNA internal transcribed spacer sequences. American Journal of Botany 80: 711–722[CrossRef][Web of Science]
Yokota, Y., T. Kawata, Y. Iida, A. Kato, and S. Tanifuji. 1989 Nucleotide sequences of the 5.8S rRNA gene and internal transcribed spacer regions in carrot and broad bean ribosomal DNA. Journal of Molecular Evolution 29: 294–301[CrossRef][Web of Science][Medline]
This article has been cited by other articles:
|
|
 |
|
  G. J. Kenicer, T. Kajita, R. T. Pennington, and J. Murata Systematics and biogeography of Lathyrus (Leguminosae) based on internal transcribed spacer and cpDNA sequence data Am. J. Botany, July 1, 2005; 92(7): 1199 - 1209. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
) denoting members of Loteae and open triangles (
) denoting Coronilleae. The most recent taxonomic arrangements (Polhill, 1994
