Phylogenetic systematics of the tribe Millettieae (Leguminosae) and its relationship to temperate herbaceous legumes

Jer-Ming Hu

NSF grant proposal 1997.10.09

PROJECT SUMMARY

Phylogenetic studies show that several temperate angiosperm groups appear to be derived from their tropical woody relatives. The trend from woody tropical to herbaceous temperate goups can also be found in the large family Leguminosae, which contains tremendous radiations in several temperate tribes. The goal of this study is to reconstruct the phylogeny of the tropical tribe Millettieae and its temperate counterparts in order to gain insights into evolutionary history of these groups. Preliminary data from nuclear ribosomal DNA and chloroplast DNA have provided considerable of new phylogenetic information on the poorly understood tribe Millettieae. The specific objectives of this study are to: (1) Collect a more complete sample of taxa for Millettieae and related temperate herbaceous taxa. (2) Reconstruct the phylogeny of Millettieae and related groups based on DNA sequences using several analytic approaches. (3) Reconstruct the evolutionary history of key morphological characters associated with the origin of the temperate taxa.

PROJECT DESCRIPTION

Tropical flowering plants have long attracted biologists' attention because of their great diversity in morphology. However, some of the predominantly temperate families like Brassicaceae and Apiaceae have more species than their tropical relatives, Capparaceae and Araliaceae (Judd, et al., 1994). The tropical members in these family pairs are generally paraphyletic, some members being sister groups to their temperate counterparts. The transition from woody tropical to herbaceous temperate also occurs in several other angiosperms including the large family Leguminosae, especially in the subgroup Papilionoideae. The temperate herbaceous tribes, consisting of the major radiation in Papilionoideae, all share several vegetative features, such as the loss of stipels and pulvini, and the lack of secondary thickening and secretory reservoirs (Dormer, 1945, 1946; Polhill, 1981). However, recent studies from molecular data suggest that these temperate herbaceous tribes might not form a monophyletic group (Lavin et al., 1990). Most of these tribes lack one copy of the 25 kb inverted repeat (IR) in their chloroplast genome (Lavin et al., 1990). The presumably monophyletic group with the absence of IR also includes the temperate woody Wisteria and Millettia japonica, and the tropical genus Callerya, all of which are members of the tribe Millettieae. On the other hand, the two temperate herbaceous tribes, Loteae and Coronilleae, which contain the IR, are considered to be very close to the IR-lacking clade as suggested by a phylogenetic study of the rbcL gene (Doyle et al., 1997).

The new phylogeny from molecular data (Doyle et al., 1997; Lavin et al., in press) indicates that temperate herbaceous tribes or epulvinate series might evolve independently more than once, or alternatively the loss of IR occurred more than once if we consider epulvinate series is monophyletic. To understand the evolutionary trends in these groups, a more complete investigation of basal temperate herbaceous groups and their closest tribe, Millettieae, is proposed in this study. Millettieae, despite its tropical habitat and some unspecialized features, is generally thought to be very close to the temperate tribe Galegeae because of some shared similarities in flower structure and chemical compunds in their seeds (Evans et al., 1985). The Millettieae species lacking the IR, including Wisteria, Callerya and Millettia japonica, however, do not have any morphological features distinguishing them from other Millettieae members (Geesink, 1984). For example, the genus Callerya can only be distinguished from Millettia by its paniculate inflorescence (Schot, 1994), but this character can be found in several other Millettieae genera as well. The phylogenetic analyses (Zandee & Geesink, 1987; Chappill, 1995) based on morphological data had just too many homoplasies and low support to give resolution for Callerya, Wisteria, and other members in the tribe Millettieae. It is necessary to survey more Millettieae species in molecular analyses from taxa related to Callerya and Wisteria to have a closer look at the evolutionary history and at other morphological trends.

The tribe Millettieae, comprising over 40 genera and nearly 1,000 species, is itself one of the most problematic groups in legume systematics. The circumscription of this tribe is vague and can only be defined as those tropical woody papilionoids with derived flower features (fused keel petals) and seeds containing non-protein amino acids (Polhill, 1981; Lavin et al., in press). Nevertheless, many studies have suggested that Millettieae is at a critical position in the radiation of a large portion of the subfamily Papilionoideae (Geesink, 1981; Polhill, 1981, 1995), including the THC, and other tribes such as Phaseoleae, Indigofereae, etc. (Fig. 1).

Cytological information has provided hints about the relationship between the temperate herbaceous clade (THC, not including Loteae and Coronilleae, Lavin et al., 1990) and the tribe Millettieae. Most of the THC have a basic chromosome number = 8 (Goldblatt, 1981). In comparison, either n=11 or 12 is common within Millettieae (Goldblatt, 1981 and the author's previous survey), but with exceptions that n = 8 in Wisteria and Callerya, n = 9 in Cyclolobium, n = 10 in Leptoderris and Aganope, and is extraordinarily complicated in Millettia (n = 8, 10, 11, 12, 16, 18, or 24). The result agrees with recent molecular phylogenies for the two Millettieae genera, Callerya and Wisteria, are close to the THC, these include the molecular systematic studies of rbcL (Doyle et al., 1997) and the phytochrome gene family (Lavin et al., in press).

Relationships in Millettieae-THC are poorly resolved; the tribes Robineae, Loteae, Indigofereae, and the genus Glycyrrhiza in Galegeae are all potentially related, but all support is low in this region of the rbcL phylogeny (Doyle et al, 1997). In addition, the sampling of Millettieae for the rbcL study was sparse (5 out of 43 genera). Since Millettieae is very polymorphic and is the key group to understand the diversification pattern from tropical to THC, we propose to solve this problem by two approaches: (1) to incorporate more taxa in Millettieae for our analyses, especially those putative members close to Callerya. (2) to obtain new molecular data from DNA sequences known to be phylogenetically informative regarding this group, especially for nuclear sequences that can be regarded as having an independent history relative to the chloroplast genome.

Knowledge of the phylogenetic relationships of the THC and Millettieae will provide further information regarding the origin of THC, and will serve as an example of a transition pattern from tropical to temperate zone as suggested by Judd et al. (1994). Detailed examination of character evolution in Millettieae, THC, and the related groups will allow a test of the hypothesis that Dormer (1946)'s epulvinate series is a natural monophyletic group. The results of these analyses will also provide information about relationships within Millettieae and help the design of further sampling for systematic study in this tribe. The long-term goal of this work is to commence the systematic revision of the polymorphic tribe Millettieae, but much work will have to wait until more samples are obtained from several remote areas, including tropical America and tropical Africa.

This proposal is to reconstruct the phylogeny of Millettieae and the related temperate herbaceous groups, especially for those species related to the THC, by using molecular data. This work will be able to resolve several problematic issues in the systematics of the tribe Millettieae and also the tribal relationships in Papilionoideae. There are three major objectives for this proposal:

(1) To determine which Millettieae belong in the Callerya clade, and examine the relationships among the selected species in this clade.

(2) To reconstruct the phylogeny of Millettieae and related groups in Papilionoideae. A phylogeny at the generic level will be reconstructed by using DNA sequence data.

(3) To reconstruct character evolution in key morphological features from Millettieae to the THC using the phylogenetic trees generated from above, and to test the hypothesis of the monophyly of the epulvinate series in legumes.

Considerable information regarding the phylogeny of the THC has already been obtained by the PI's for several taxa for all the gene regions being used in this proposal (Sanderson & Wojciechowski, 1996). Analysis of the DNA sequences of the nuclear ribosomal DNA internal transcribed spacer (ITS) region showed that all examined Callerya and Wisteria species formed a monophyletic group, while Wisteria was nested within the Callerya clade. This suggested that the genus Callerya is paraphyletic and the classification of Callerya and Wisteria should be reconsidered. The Callerya clade is sister to temperate herbaceous legumes (THC) in all the phylogenetic analyses of the ITS and two other chloroplast DNA sequences (see below for details). Furthermore, the whole clade containing Callerya and THC showed a closer relationship to Robineae and Loteae rather than to other Millettieae members, which agrees with rbcL data (Doyle et al., 1997), but with a much higher support. Several paniculate Millettieae species which are close to Callerya have been included in this study and preliminary results showed none of the tested species fell into Callerya clade.

Phylogenetic analysis of matK region agreed with the result that Callerya and Wisteria are distantly related to other Millettieae. The data also showed that Loteae and Robineae formed a monophyletic group and are sistered to the big clade containing THC, Callerya, and Wisteria.

ITS data suggest that the core-Millettieae as a monophyletic group is supported by high confidence (bootstrap) values, similar to the phylogeny according to the phytochrome gene family analyses (Lavin et al., in press). The phylogeny based on ITS data showed that the largest genus of Millettieae, Tephrosia, is well supported as a monophyletic group. Two other large genera in Millettieae, Lonchocarpus and Millettia, are both not monophyletic groups. A broader sampling for these two genera will provide some details about relationships at the generic and subgeneric level, but this is not the main focus of this study. The preliminary tree constructed by ITS and matK sequences is shown in Fig. 2.

Taxonomic Sampling.

Sampling will focus on likely members and allies of the Callerya clade, including 19 Callerya species recognized by Schot (1994) and 8 Wisteria species. The genera in Millettieae with paniculate inflorescences will also be collected, e.g. Endosamara, Sarcodum, Dewevrea, Behaimia, Afgekia, Craibia, Kunstleria, Aganope (=Ostryocarpus*), Philenoptera*, Platycyamus*, and Cyclolobium* (* represent genera included in the preliminary studies). Other available members in Millettieae will also be included in this study, and several species have already been used in the preliminary ITS sequence analysis, i.e. species from Lonchocarpus, Millettia, Tephrosia, Austrosteenisia, Dalbergiella, Dalhstedia, Fordia, Chadsia, Mundulea, Derris, Paraderris, Pongamiopsis, Piscidia, and Pongamia. Samples from Loteae and Coronilleae will be selected to complete the sampling in epluvinate series. Several species from Robineae, Dalbergieae, Loteae, Phaseoleae, and Hologalegeae (defined as the group which lacks IR in Papilionoideae) will serve as possible outgroups. Samples will be collected from the field or herbarium specimen. Funding is requested for two collecting trip, one in southwestern China as well as the herbarium in Kunming (KUN, Kunming Institute of Botany), where most of the problematic Callerya and Millettia are distributed (Wei, 1985a,b). Dr. Hang Sun, the curator of KUN, has agreed to collaborate and assist us in field work (see appended letter). Another collecting trip is arranged to Netherlands for examining as well as collecting samples from the herbarium in Rijksherbarium (L), one of the most complete collections of African species. This trip is necessary for collecting several African genera in Millettieae, especially for several Millettia allies. Dr. Frits Adema from Rijksherbarium has also expressed a desire for collaboration (see appended letter).

Molecular Data Analyses.

Total genomic DNA will be isolated from field-collected and herbarium specimen material using a protocol described by Scott & Playford (1995). By employing this method, genomic DNA from both fresh leaf and herbarium specimen material of interest has been successfully isolated and sequenced for nuclear and plastid DNAs used for this proposal. Three regions will be sequenced:

1). The nuclear ribosomal ITS region (Baldwin et al., 1995), the sequence length is about 700 bp;
2). The intron region in chloroplast tRNA gene trnL (Taberlet et al., 1991), the sequence length is about 600 bp;
3). The gene encoding a maturase (matK) located within the trnK intron in chloroplast (Sugita et al., 1985), the sequence length is about 2.5 kb.

Gene specific primers will be used to amplify specific target DNA sequences by Polymerase Chain Reaction (PCR) using VentR DNA Polymerase (Biolab, New England) with modifications according to Palumbi (1996). Typical conditions for PCR reaction are 4 min at 94 °C for initial denaturation, followed by 35 cycles of 30 secs at 94 °C, 30 secs at 48-50 °C for annealing, 1 min at 72 °C for primer extension, and after the cycles, a final 7 min incubation at 72 °C will be employed to complete the reaction. For matK sequence, which needs longer extension time, the reaction time in each cycle will be doubled. PCR products are then analyzed by gel electrophoresis, purified by differential filtration through Ultrafree-MC columns (Millipore, Bedford, MA). Sequence data are obtained from double stranded sequence analysis of the PCR product by using automated DNA sequencing (377 DNA Sequencer, Perkin-Elmer).

Table 1 shows the taxa and sequences in Millettieae and allies which have already been assayed in PI's preliminary work.

Phylogenetic Analyses.

Sequences will be aligned manually and examined by the PILEUP program in the Sequence Analysis Software Package (GCG, Inc., 1991), and sequences will be analyzed separately and in combined data matrices when warranted by tests of combinability (Mason-Gamer & Kellogg, 1996). Phylogenies will be reconstructed using parsimony as implemented in PAUP 3.1.1 (Swofford 1993) and maximum likelihood on PAUP 4.0 (Swofford, 1997, unpublished), fastDNAml (Olsen et al. 1994), or PHYLIP v. 3.5 (Felsenstein 1993) depending on the availability. Bootstrapping will be used to assess the robustness of the trees (Felsenstein, 1985).

Character evolution studies.

MacClade 3.1 (Maddison & Maddison, 1992) will be used to examine the distribution of molecular and morphological attributes on the cladograms generated from the above studies. The molecular characters include nucleotide substitution and insertion/deletion in the sequences. Hypothesis of monophyly on epulvinate series will be tested on the generated tree. Patterns of several key morphological characters either for distinguishing Millettieae or the characters common in epulvinate series, i.e. the presence of stipels, ridge bundles in the petioles, secondary thickening, and secretory reservoirs, will be examined on the most parsimonious tree generated from molecular studies. To find all most-parsimonious reconstructions, MacClade (Maddison & Maddison, 1992)'s Equivocal Cycling calculation will be performed to examine the all possible histories in conjunction with DELTRAN and ACCTRAN optimizations (Swofford & Maddison, 1987) using options in MacClade 3.1. The characters used to distinguish Millettieae taxa include inflorescence types, pod morphologies, and several floral features, such as appendages and morphologies in petals. Other characters, such as chromosome numbers and non-protein amino acids, will also be used to examine the distribution in Papilionoideae in molecular phylogenetic trees.

Fig. 1. Schematic phylogeny showing the relationships of Callerya, Wisteria, Millettieae, Phaseoleae, Robineae, Dalbergieae, Sophoreae, and THC. THC consists of tribes Galegeae, Carmichaelieae, Hedysareae, Viceae, Cicereae, and Trifolieae. Hologalegeae is defined as the group with the loss of IR (inverted repeat) in Papilionoideae. The shaded line above Callerya and Wisteria implies these two genera are still under Millettieae in current taxonomic treatment. Satellite genera implies some Millettieae members shown to be distantly related to the core Millettieae. This diagram is modified from Doyle (1995) and phylogenetic studies of ITS, matK, and trnL intron sequences based on author's preliminary data (Sanderson & Wojciechowski, 1996).

Fig. 2. Consensus bootstrap tree (100 replicates) for selected taxa. Bootstrap values above the branches were obtained based on ITS, and those below the branches were obtained based on matK sequences using parsimony criteria. Current Millettieae species (Geesink, 1984) are shown in bold.

Table 1. List of Millettieae species and DNA sequences which have been obtained in PI's preliminary work. The numbers in the parentheses are the total species in the genus, and the numbers in each column show the number of species which have been sequenced. The taxa from most of the THC are all available from the PI's and are not listed here.

Taxa	ITS	trnL intron	matK
Austrosteenisia (2)	1	1	1
Callerya (19)	6	3	3
Chadsia (18)	1
Cyclolobium (5)	1
Dalbergiella (3)	1		1
Dalhstedia (1)	1
Derris (41)	2		1
Fordia (10)	1
Lonchocarpus (150)	13	2	1
Millettia (90)	5	4	1
Mundulea (16)	1
Ostryocarpus (6)	1
Paraderris (6)	1		1
Philenoptera (15)	1
Piscidia (7)	1	1
Platycyamus (2)	1	1	1
Pongamiopsis (2)	1
Tephrosia (400)	4	2	1
Wisteria (6)	2	1	1
Taxa in preparation: Endosamara, Sarcodum, Afgekia, Craibia, Kunstleria, Aganope, Dewevrea, Behaimia, Antheroporum, Imbrylax, Pongamia

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