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Phylogenetic relationships and ¯oral in the papilionoid legume clade

MICHELLE M. MCMAHON

McMahon, M. M. (University of , Davis, One Shields Avenue, Davis, CA, 95616, U.S.A.; e-mail: [email protected]). Phylogenetic relation- ships and ¯oral evolution in the papilionoid legume clade Amorpheae. Brittonia 57: 397±411. 2005.ÐAmorpheae (: Papilionoideae) was ®rst considered a natural group by Rupert Barneby in his illustrated monograph Daleae Imagines. Amorpheae currently comprise eight genera, ca. 250 spp., and extensive ¯oral diversity, including loss of corolla and addition of a stemonozone. The Amor- pheae and many of Barneby's subtribal groups are supported as monophyletic by previous phylogenetic analysis of nuclear ribosomal and chloroplast sequence data. However, some relationships remain unclear. A nuclear marker derived from a genomic study in Medicago, CNGC4, was sequenced in selected Amorpheae. is one of the ®rst applications of this marker for phylogenetic study. The new data con®rm some relationships inferred using trnK and ITS, but also provide evidence for new arrangements. Combined data were used to explore several aspects of Barneby's taxonomic framework. The phylogeny, in concert with data on ¯oral , implies that simpli®cation of the complex papilionoid ¯ow- er has occurred several times in the history of the Amorpheae. Key words: Amorpheae, Barneby, CNGC4, , ¯oral evolution, Papiliono- ideae.

Amorpheae Borrissova emend. Barneby was conducted, Barneby presented a hy- is a group of ca. 250 papilionoid legume pothesis of relationships among groups in with much variation in ¯oral form. Amorpheae (Fig. 2). This hypothesis was Although nested within the Papilionoideae based on several informative morphological (Lavin et al., 2001; McMahon & Hufford, characters, some of which are consistent 2004; Wojciechowski et al., 2004) the with the phylogeny based on DNA se- group contains many members with ¯owers quences, some of which are not. that depart from the standard papilionoid Previous analyses resulted in very strong form. Aspects that vary include a unique support for a clade of three genera (the dal- structure (the ``stemonozone'', Fig. 1A, B; eoids: Dalea, , ), sis- McMahon & Hufford, 2002), losses of ter to a clade of the remaining ®ve genera structures such as and , and (the amorphoids: , , the loss of attributes such as asym- , , and ) metry and strong blade/claw distinctions (McMahon & Hufford, 2004). Furthermore, (Fig. 1C, D; McMahon & Hufford, 2005). relationships among many lineages within First considered a natural group by Rupert the daleoids were strongly supported. How- Barneby (1977), Amorpheae is natively dis- ever, within the amorphoids, the previously tributed from southern Canada to northern used markers (ITS and the trnK intron) var- Chile, with areas of highest diversity in the ied little. These taxa are particularly inter- arid and semi-arid regions of Mexico, the esting because they include several appar- Andes, and the prairies of North America. ent simpli®cations from the complex papi- Although no explicit phylogenetic analysis lionoid ¯ower. Therefore, additional data

Brittonia, 57(4), 2005, pp. 397±411. ISSUED: 28 December 2005 ᭧ 2005, by The New York Botanical Garden Press, Bronx, NY 10458-5126 U.S.A. 398BRITTONIA [VOL. 57

FIG.1. Selected aspects of variation among ¯owers of Amorpheae. A. Schematic of a generalized member of Marina or Dalea showing the lateral petals attaching above the hypanthium (asterisks). The region between the petal insertion and the hypanthium is the stemonozone (McMahon & Hufford, 2002). B. Scanning electron micrograph of Gentry; proximal portion of bud near anthesis (calyx mostly removed), showing lateral petal attachment points. C. Line drawing of lateral petals, (A. Gray) Rydb., showing difference between wing and keel, asymmetry within each petal, and distinction between claw (narrow proximal region) and blade (distal laminate region). D. Line drawing of lateral petals, Errazurizia megacarpa (S. Wats.) I. M. Johnst., showing similarity of wing to keel, near symmetry within petals, and gradual tapering of distal region to proximal region. [In all, distal is up and adaxial (with respect to the in¯orescence axis) is to the left. b, banner petal; c, calyx; g, gynoecium; h, hypanthium (arrow points to rim); k: keel petal; st, stamens; w: wing petal. Scale bar (B) ϭ 0.5 mm.]

are required, ideally from a region that is independent from the ribosomal and chlo- roplast sequences used previously. Markers derived from genomic data in Medicago truncatula have been recently developed for comparative genome studies across crop le- gumes (Choi et al., 2004b). Application of these markers to phylogenetic questions is promising but not always straightforward (Scherson et al., 2005). Extending their ap- plication from Medicago to a group such as

FIG.2. Evolutionary relationships among mem- Amorpheae, diverged for perhaps tens of bers of Amorpheae as previously hypothesized, from millions of years (Choi et al., 2004b), may Barneby's Figure 1 (Barneby, 1977: 13). help provide resolution within Amorpheae. 2005] McMAHON: & FLORAL EVOLUTION OF AMORPHEAE 399

TABLE I GENBANK ACCESSION NUMBERS FOR NEWLY GENERATED AND DOWNLOADED SEQUENCES

GenBank Collection accession Sequences submitted: CNGC4 Amorpha apiculata Wiggins Fishbein 3745 (ARIZ) DQ023320 Amorpha californica Nutt. ex Torr. & A. Gray Grable 6434 (WS) DQ023322 Pursh McMahon 683 (WS) DQ023323 Apoplanesia paniculata C. Presl Cabrera C. 5479 (ARIZ) DQ023327 Errazurizia benthami (Brandegee) I. M. Johnst. McMahon 438 (WS) DQ023324 Errazurizia megacarpa (S. Watson) I. M. Johnst. Fishbein 3773 (WS) DQ023321 Errazurizia rotundata (Wooton) Barneby McMahon 452 (WS) DQ023325 (A. Gray) S. Watson Fishbein 3683 (ARIZ) DQ023328 Scheele Van Devender 29V79 (ARIZ) DQ023326 Parryella ®lifolia Torr. & A. Gray ex A. Gray Porter 8868 (ARIZ) DQ023329 (Torr. & A. Gray) Barneby McMahon 339 (ARIZ) DQ023330 Psorothamnus kingii (S. Watson) Barneby McMahon 688 (ARIZ) DQ023331 (Torr.) Barneby McMahon 150 (ARIZ) DQ023332 GenBank Taxon Sequence region accession Sequences downloaded ITS AFU59890 Amorpha fruticosa matK AF270861 Apoplanesia paniculata ITS AF187093 Apoplanesia paniculata matK AF270860 Dalea pulchra matK AY386860 Eysenhardtia orthocarpa matK AY386909 Eysenhardtia sp. Lavin 5052 ITS AF187096 matK AY386859 Marina sp. Lavin 5341 ITS AF187095 Medicago truncatula CNGC4 BV164997 Medicago truncatula ITS AF233339 Medicago truncatula matK AF522109 Pisum sativum CNGC4 BV165002 Pisum sativum ITS AY143481 Pisum sativum matK AY386961 For all other accession numbers, see McMahon and Hufford (2004).

It will also be of value in exploring the ben- la as CNGC4 (cyclic nucleotide-regulated e®ts of transferring data from comparative ion channel like protein) (Choi et al., genomic analyses to phylogenetic system- 2004a) was sequenced for 13 Amorpheae atics. (Table I). Although many markers will be The goals of this paper are (1) to present examined in the future, this marker was se- a preliminary set of data from one genomic lected to be ®rst based on its potential for derived nuclear marker, CNGC4, alone and variation among species (Choi et al., 2004b; in combination with previously reported Scherson et al., 2005). Additionally, the DNA sequence data, (2) to summarize cur- primers uniformly produced a single PCR rent understanding of Amorpheae relation- band and a clean sequencing chromato- ships, (3) to review patterns of ¯oral evo- gram, consistent with the possibility that the lution in Amorpheae, and (4) to compare region is single-copy in Amorpheae, al- these inferences to Barneby's hypotheses of though this is not yet con®rmed. Taxa were ¯oral evolution in the group. chosen based on their placement in previ- ous phylogenetic analyses, in which many Methods and Materials relationships were resolved with strong sup- The nuclear region identi®ed in the ge- port. However, relationships among key nome-wide analysis of Medicago truncatu- taxa that are involved in ¯oral simpli®ca- 400BRITTONIA [VOL. 57 tions were not well resolved (McMahon & the trees obtained. No strongly supported Hufford, 2004, 2005). Therefore, available relationships (Ն 90% bootstrap; Felsen- members of Eysenhardtia, Errazurizia, stein, 1985) differed among the trees. Ad- Amorpha, Parryella, and Apoplanesia (the ditionally, the partition homogeneity test (or amorphoid clade) were selected for se- incongruence length difference test, Farris quencing CNGC4. Three members of Pso- et al., 1994) as implemented in PAUP* rothamnus were chosen to span the root of (Swofford, 2003) with 1000 replicates, the daleoid clade (Graybeal, 1998; McMa- failed to reject homogeneity (P ϭ 0.8220). hon & Hufford, 2004) in an effort to resolve This method may be inappropriate as a test the position of the amorphoid root. Se- for combinability (Barker & Lutzoni, quences from Medicago truncatula and Pis- 2002), but the negative result, in concert um sativum were obtained from GenBank with the lack of strong con¯icting signal, (Table I) and included as outgroups. Addi- provides justi®cation for combining the tional ITS and trnK sequences from Amor- data. pheae have been submitted to GenBank by Parsimony and likelihood optimality cri- other labs; these were downloaded in teria were used to compare phylogenetic to use all available data (Table I). trees, and support for relationships was as- Leaf tissue was removed from dried her- sessed using the nonparametric bootstrap barium specimens or from in the ®eld (Felsenstein, 1985). For the likelihood anal- and dehydrated with silica gel. Total DNA yses, models of sequence evolution were was isolated using a standard CTAB pro- compared using hierarchical likelihood ra- tocol (Doyle & Doyle, 1987). Fragments tio tests as calculated by Modeltest 3.0 (Po- were ampli®ed using the primers 5Ј-AGA- sada & Crandall, 1998). The comparison fa- GATGAGAATCAAGAGGAGGGA- vored a model in which transitions and TGCA-3Ј and 5Ј-CATGATGAAGAGCA- transversions may have different evolution- TTTCGTCCACTGGA-3Ј (Choi et al., ary rates (the Hasegawa-Kishino-Yano 2004a) and the following pro®le: 15 min at model, HKY, Hasegawa et al., 1985) and 95Њ, 35 cycles of 1 min at 94Њ, 1 min at 52Њ, this was the model used for the CNGC4 1 (or 3) min at 72Њ, with a ®nal extension data. Model comparisons favored more of 5 min at 72Њ. Sequences were generated complicated models for the ITS data and for using the same primer pair. the trnK data, notably the inclusion of rate Phylogenetic analysis was conducted on heterogeneity across sites. A compromise the data from CNGC4 alone and in com- model was selected for the combined data bination with previously published se- set in which separate rates were estimated quences of Amorpheae (see McMahon & for transitions and for transversions and Hufford, 2004, for voucher information). variation in rates across sites was estimated The three data partitions differed in taxo- using a gamma distribution with four dis- nomic coverage: 59 sequences were from crete categories (Yang, 1994), the HKY⌫ ITS, 47 from trnK, and 13 from CNGC4. model. Likelihood analysis of the small For combined analyses, missing data were data set (CNGC4 alone; 13 taxa) began added to complete the matrix (a superma- with a random-taxon-addition starting tree, trix approach; Driskell et al., 2004). Com- and parameter values were optimized dur- bining data sets may not be appropriate ing the search. Likelihood analysis of the when processes such as sorting or larger combined data (trnK, ITS, and introgression could cause different markers CNGC4; 62 ingroup and 2 taxa) to have different histories (Maddison, was made more computationally tractable 1997). However, this is generally dif®cult by starting with a reasonably good tree (the to detect unless there is strong and con¯ict- ®rst most-parsimonious tree found in a par- ing signal that cannot be explained by ho- simony analysis limited to 100 trees), esti- moplasy. The three data sets were evaluated mating the HKY⌫ parameter values on that for evidence of strong con¯icting signal by tree, and using these values throughout the ®rst pruning the data sets to only those taxa tree search. with all three genes, and then comparing To explore hypotheses of , 2005] McMAHON: PHYLOGENETICS & FLORAL EVOLUTION OF AMORPHEAE 401

FIG.3. Phylogenetic relationships inferred using the nuclear marker CNGC4. A. Maximum likelihood es- timate of topology and branch lengths. Numbers above branches indicate likelihood bootstrap values. B., C. Parsimony analysis resulted in three equally parsimonious trees (EPTs), one of with matches the topology shown in A. The other two are depicted here, with some taxa removed. Note that the three EPTs include all possible relationships between Eysenhardtia, Errazurizia, and the rest of the sampled amorphoid taxa. Parsimony boot- strap analysis resulted in values nearly identical to the ML analysis except for the branch leading to Psorotham- nus (90%) and the branch leading to Eysenhardtia ϩ Errazurizia (43%). analyses were conducted in which the fol- quence, and this differed little from the se- lowing four clades were constrained to be quences found in the third Amorpha,inE. monophyletic: Dalea, Amorpha, Errazuri- rotundata, and in Parryella ®lifolia. All zia, and Psorothamnus. Optimal trees with three species of Psorothamnus have a ϳ95 and without each constraint were compared bp insertion relative to the outgroup and the using parsimony and likelihood. To assess remaining ingroup. Other than this large in- signi®cance of difference in the likelihood sertion, few gaps were necessary (c. 1%) to values with and without the constraint, the align the sequences. The sequences have set of trees that included the best trees with been submitted to GenBank (Table I) and and without all of the constraints was sub- the aligned data set is available on the au- jected to an SH test (Shimodaira & Hase- thor's website (ginger.ucdavis.edu/mmcma- gawa, 1999) as implemented in PAUP* hon/data). (Swofford, 2003) using the RELL approx- CNGC4 provides strong evidence for imation to estimate log likelihoods and several clades among the small set of taxa 1000 bootstrap replicates to estimate the sampled (Fig. 3). An amorphoid clade con- distribution of the test statistic. sisting of Amorpha, Errazurizia, Eysen- hardtia, Parryella, and Apoplanesia is re- Results covered with strong support [99% bootstrap The nuclear marker CNGC4, sequenced (BS) in likelihood and parsimony analyses]. for 13 Amorpheae, resulted in an aligned The branch leading to the daleoids, here data set of 535 nucleotides. Aligning the represented by only three members of Pso- ingroup to two outgroup sequences from rothamnus, is weakly supported by likeli- GenBank (Pisum sativum and Medicago hood BS but is supported with 90% by par- truncatula) required the addition of four simony bootstrap. The pair of species from gapped characters to the ingroup for a ®nal Eysenhardtia are supported as sisters with matrix of 539 sites. The maximum pairwise 100% BS, as are two of the three Errazur- divergence (uncorrected distance) between izia species, E. benthami ϩ E. megacarpa. ingroup taxa was ca. 10% between Psoro- Parryella ®lifolia groups together with the thamnus kingii and Errazurizia benthami. Amorpha species and the third Errazurizia, Two species of Amorpha had the same se- E. rotundata, but the relationship among 402BRITTONIA [VOL. 57 them is not clear. This clade's sister is Apo- sulted in the largest addition to the number planesia, with moderate support. Poorly of parsimony steps required (50) and the supported are the relationships among greatest decrease in likelihood value (106 Eysenhardtia, Errazurizia, and the Apopla- lnL units) and was highly signi®cant (p Ͻ nesiaÐAmorpha clade. The ML analysis 0.0001). Forcing Amorpha to be a clade had inferred a sister relationship between little effect; tree length remained the same Eysenhardtia and Errazurizia, but with low (although fewer trees were recovered), and support (68% BS). Parsimony analysis re- the log-likelihood changed insigni®cantly sulted in three equally parsimonious topol- (Table II). ogies re¯ecting the three possible resolu- tions of this node (Fig. 3A±C). Discussion Combining these data with ITS and trnK Continuing research into the for a larger sample of taxa resulted in 1080 of Papilionoideae con®rms multiple inde- MP trees and one ML tree (Fig. 4). The pendent simpli®cations of the papilionoid strict consensus MP tree has nearly the corolla. Although some lineages have re- same topology as the ML tree except that tained an ancestral non-papilionoid ¯ower several nodes with less than 60% BS (Fig. form, others have had it and lost it (Pen- 4) are collapsed in the strict consensus tree nington et al., 2000), contradicting infer- (not shown). Additionally, in the MP strict ences that non-papilionoid forms should be consensus tree, Eysenhardtia is sister to the considered plesiomorphic (Mansano et al., remaining amorphoid taxa (as depicted in 2004). In Amorpheae, the loss has occurred Fig. 3C) whereas the ML tree places Eysen- several times (McMahon & Hufford, 2004, hardtia sister to Errazurizia s. str., together 2005). The addition of data from a nuclear sister to Apoplanesia (Fig. 4). Species in marker helps re®ne inferences of relation- Psorothamnus are strongly supported to be ships within the amorphoid clade, in which in two clades by substantial branch lengths corolla loss and dedifferentiation has oc- and 100% BS (Fig. 4). All sampled mem- curred. The nuclear marker strongly sup- bers of Dalea form a clade except one: Dal- ports the result that all species with zero or ea ®liciformis is placed in or sister to Ma- one petal are a clade. Data from ITS also rina. Excluding D. ®liciformis, Marina and support this, but weakly, and data from Dalea are sisters, and the data support some trnK disagree, although weakly (McMahon relationships within each of these genera. & Hufford, 2005). Therefore, the new anal- Errazurizia rotundata is placed with Par- yses do not overturn previous conclusions ryella ®lifolia with strong support (97% regarding ¯oral evolution and simpli®cation BS), and these are nested within or sister to in Amorpheae, but instead add evidence for the Amorpha clade (100% BS, Fig. 4). more precise inferences. Three of the four constrained analyses re- To summarize recent phylogenetic re- sulted in trees that were considerably longer search in Amorpheae, it is important to (and signi®cantly less likely) than the un- place it in the context of its taxonomic his- constrained trees (Table II). Forcing Dalea tory, primarily the work of Rupert Barneby, to be monophyletic (i.e., forcing D. ®lici- to whom this symposium is dedicated. formis to group with the other daleas in- stead of with the marinas) resulted in an RELATIONSHIPS AMONG AMORPHEAE increase of 19 steps required to explain the tree using parsimony, and a likelihood val- Rupert Barneby's classi®cation of the ue decrease of 57 ln likelihood units. The Amorpheae revolutionized our understand- SH test assessed this difference as signi®- ing of this part of the legume tree of . cant (P ϭ 0.0350). Likewise, forcing the Prior to his treatment, workers had consid- three Errazurizia species together was ered all glandular legumes with indehiscent found to be signi®cantly less likely (P ϭ single-seeded pods and simple basi®xed 0.0010) than the best tree in which E. ro- to belong together in Psoraleeae tundata is placed with Parryella. Constrain- Benth. (with variations in rank and spelling) ing Psorothamnus to be monophyletic re- (Bentham, 1865; Taubert, 1894; Rydberg, 2005] McMAHON: PHYLOGENETICS & FLORAL EVOLUTION OF AMORPHEAE 403

FIG.4. Phylogenetic relationships among Amorpheae based on trnK, ITS, and CNGC4 DNA sequence data. Topology and branch lengths represent one of the two maximum likelihood trees obtained using an HKY⌫ model of sequence evolution. Numbers above branches indicate parsimony bootstrap values greater than 50%. The dashed line leading to the daleoid clade is inserted for graphical convenience; the inferred branch length is given by the solid portion only. 404BRITTONIA [VOL. 57

TABLE II RESULTS FROM UNCONSTRAINED AND CONSTRAINED PARSIMONY AND LIKELIHOOD ANALYSES

Constraint MP trees Steps Dif. (steps) ML trees ln L Dif. (lnL) P None 1080 2893 3 Ϫ22,082.43954 Amorpha 90 2893 0 2 Ϫ22,084.56305 Ϫ2.12351 0.8490 Dalea 1080 2912 19 2 Ϫ22,139.70861 Ϫ57.26907 0.0350 Errazurizia 360 2921 28 3 Ϫ22,181.34296 Ϫ98.90342 0.0010 Psorothamnus 1620 2943 50 2 Ϫ22,189.02309 Ϫ106.58355 Ͻ0.0001 Signi®cance value for difference in likelihood score was assessed using the SH test. Boldface text indicates signi®cance at the 5% level.

1919, 1920, 1928a, 1928b; Isely, 1962). been further supported by additional char- Hutchinson (1964) ®rst suggested splitting acters, for example, gland anatomy and de- Bentham's Psoraleeae into two tribes, Dal- velopment (Turner, 1986), root nodules eae and Psoralieae, based on a single char- (Corby, 1981), pollen morphology (Fergu- acter, the position of the petals. In Hutch- son & Skvarla, 1981; Ferguson, 1990), and inson's Daleae, petals are perched above the aspects of ¯ower development (Tucker & rim of the hypanthium, appearing to attach Stirton, 1991; McMahon & Hufford, 2002; to the fused staminal column (thereby the Prenner, 2004). Molecular studies have also term ``epistemonous,'' Barneby, 1977), supported the monophyly of the two tribes whereas all petals of Psoralieae insert on and their separation on the legume tree. In the hypanthial rim. Several inconsistencies studies that include more than one species arose from this arrangement, including of Amorpheae, they form a clade with high falsely placing in Psoralieae the (then) support and are often placed as sister to the monotypic Marina, whose petals are dalbergioid clade by trnK/matK (Lavin et perched just as in Dalea. Furthermore, Ryd- al., 2001; McMahon & Hufford, 2004; Wo- berg's Psorothamnus and Psorodendron jciechowski et al., 2004), and trnL (Lavin (which have sessile petals) and the et al., 2001; but note polytomy in Penning- Dalea (which have epistemonous petals), ton et al., 2001). However, support levels had always been associated based on char- for a dalbergioid ϩ Amorpheae clade ap- acteristics of the pod, glands, habit, and fo- pear to depend on sampling: bootstrap val- liage (Munz, 1959; Shreve & Wiggins, ues were either 92% (Lavin et al., 2001), 1964, Wiggins, 1980). Indeed, most species 73%±78% (Wojciechowski et al., 2004), or of Psorothamnus and Psorodendron were 68% BS (McMahon & Hufford, 2004) us- originally described as members of Dalea. ing the same sequencing region but differ- The separation of taxa based solely on petal ent sets of taxa. On the other hand, ITS position, therefore, proved unsatisfying. places Amorpheae with the Brongiartieae Barneby's (1977) re-evaluation of the (Hu et al, 2002) or in a polytomy group provided new characters on which to (Lavin et al., 2001). Although the precise base tribal classi®cation. In¯orescence ar- placement of Amorpheae is not yet clearly chitecture became primary in delimiting established, molecular data place Psoraleae two newly circumscribed tribes: Amor- phylogenetically quite distant, not close to pheae Borrissova emend. Barneby, in which Trifolium as Rydberg (1928a) and Barneby in¯orescences are terminal, and Psoraleae s. (1977) had suggested, but nested within the str., in which in¯orescences are axillary. millettioid-phaseoloid clade (Hu et al., Furthermore, in Psoraleae s. str., leaves are 2000; Kajita et al., 2001; Wojciechowski et usually trifoliolate whereas leaves in Amor- al., 2004). pheae are mostly pinnate. This led Barneby Barneby's entirely new concept for the to suggest a relationship between Psoraleae membership of Amorpheae, therefore, has and Trifolieae, far separated from Amor- been supported by new data and new ana- pheae. Delimitation of these two tribes and lytical techniques. The monophyly of the phylogenetic distance between them has Amorpheae sensu Barneby is consistently 2005] McMAHON: PHYLOGENETICS & FLORAL EVOLUTION OF AMORPHEAE 405 supported regardless of molecular marker, Marina section Marina. The optimality criterion, or choice of outgroup count for D. ®liciformis is 2n ϭϮ8 which (Lavin et al., 2001; McMahon & Hufford, is elsewhere found only in a few daleas. 2004; Wojciechowski et al., 2004). Also, Considering the lea¯et, pod, and DNA within the tribe, many of Barneby's group- characters, a formal transfer of D. ®licifor- ings are strongly supported by sequence mis to Marina is supported, pending further data, but some re®nements to his classi®- investigation into the chromosome number. cation are required. The current best under- Errazurizia, as circumscribed by Barne- standing of relationships within Amorpheae by (1962), has four species, three of which is presented in Figure 5, in which the clad- have been sequenced, and two of these ogram was restricted to include only those form a clade (``Errazurizia s. str.''). These hypothesized clades that received strong two species are from around the support from parsimony bootstrap and were Gulf of California and have ¯owers that, present in the maximum likelihood tree. like Eysenhardtia, lack the wing/keel dis- Clearly, much concords with Barneby's tinction, and are consequently only mildly ideas, but do the few areas of discordance zygomorphic. Morphologically very similar make sense? to one of these is the only amorphoid taxon According to data from nuclear ribosom- in South America, the highly disjunct Er- al DNA and from the chloroplast trnK in- razurizia multifoliolata (Clos) I. M. Johnst. tron (including the matK gene), the nine from northern Chile, which has not yet been species in Psorothamnus fall into two included in the molecular analyses. The clades. These clades correspond neatly with third sampled taxon, E. rotundata (Wooton) Rydberg's original ideas of these taxa: (1) Barneby, has unusual morphology and the Psorothamnus Rydb., comprising generity- molecular data strongly support its place- pus P. emoryi (A. Gray) Rydb., along with ment away from the other two (the likeli- P. scoparius (A. Gray) Rydb., P. polyden- hood of it being placed with the others is ius (Torr.) Rydberg, and their (now) syno- nearly 100 lnL units worse). This Colorado nyms, to which should be added P. thomp- Plateau was ®rst assigned to the ge- sonae (Vail) S. B. Welsh & Atwood; and nus Parryella as P. rotundata Wooton be- (2) Psorodendron Rydb., comprising ge- cause it shares with P. ®lifolia Torr. & A. neritypus P. johnsoni (Torrey) Rydb. (ϭP. Gray ex A. Gray, the only other member of fremontii (S. Wats.) Rydb., ®de Barneby, Parryella, a complete lack of corolla. Pod 1977), P. kingii (S. Wats.) Rydb., P. arbo- characteristics and habit caused Barneby to rescens (Torr.) Rydb., P. schottii (Torr.) align P. rotundata with Errazurizia, but this Rydb, and P. spinosum (A. Gray) Rydb., was presented with uncertainty (Barneby, along with their (now) synonyms. Fruit and 1977), and its reassignment to the Parryella in¯orescence characters support the para- ϩ Amorpha group is not a surprise. Pollen phyly of Psorothamnus and the recognition morphology agrees with these conclusions of Psorodendron. Following additional (Guinet & Ferguson, 1989). The evidence morphological and taxonomic research, for- is mounting that Parryella and E. rotundata mal resurrection of Psorodendron will be belong together, but whether they are nested published. within or are sister to Amorpha is not yet Barneby's expansion of Marina and the clear and awaits better sampling and more transfer thereto of dalean species with quickly evolving markers. straight calyx trichomes, 10 , The largest genus, Dalea, contains at and lea¯ets with epidermal lineoles is well least 171 spp. (Barneby, 1977, 1980, 1981, supported by the molecular data, to the ex- 1988, 1990; Estrada-C. et al., 2004), ca. 2/ tent that it has been sampled. The inclusion 3 of the entire tribe. Molecular data have of Dalea ®liciformis is not outrageous, but been sampled the poorest in this genus requires further scrutiny. The lea¯ets of this (only 24 species, ϳ14%), but a few conclu- species resemble the ``typical'' marina leaf- sions can be drawn. The members of subg. let, and the pod has glands arranged in two Theodora that were included in the study concentric crescents, just like the pods of are the only sequenced species that have 406BRITTONIA [VOL. 57

FIG.5. Estimate of the phylogeny of Amorpheae, showing major events in ¯ower evolution. Only those branches found in all most parsimonious trees and supported by Ͼ90% in the bootstrap analysis have been included. Clades that correspond to genera are indicated on branches and evolutionary changes in ¯oral form are indicated by ¯oral drawings. The ancestral ¯ower, based on outgroup analysis (McMahon & Hufford, 2004), was papilionoid (A). Flowers with exposed androecia and non-differentiated petals evolved in the amorphoid clade (B). Loss of four petals (C) and loss of the banner petal (D) occurred in Amorpha and E. rotundata ϩ 2005] McMAHON: PHYLOGENETICS & FLORAL EVOLUTION OF AMORPHEAE 407 chromosome counts of 2n ϭ 8, and their suggested that the order of evolution was monophyly is supported by sequence data from ®ve to smaller numbers, which is con- (100% BS, Fig. 4). Further, they are strong- sistent with the phylogeny (Fig. 5). Current ly supported to be sister to the rest of Dal- sequence data indicate that Amorpha (1 pet- ea, in which 2n ϭ 7 (all other Amorpheae al), Parryella (0 petals), and Errazurizia ro- have 2n ϭ 10, with the possible exception tundata (0 petals) form a clade (Figs. 4, 5), of D. ®liciformis), in full agreement with implying that their loss of petals is evolu- Barneby's hypothesis. The rest of the Dalea tionarily related. The ancestral condition species in the study fall in two subgenera was probably ®ve petals (McMahon & Huf- in Barneby's , subg. Dalea and ford, 2004, 2005), but it is not clear from subg. Parosela (Cav.) Barneby (treated as the phylogeny if the evolutionary events a separate genus by Rydberg, 1919, 1920). happened in a simple progression: ®ve pet- Most Dalea species are relatively similar in als changed to one petal in the ancestor of having standard papilionaceous corollas, the ``amorpha'' clade, changing again to except for the lateral petals inserting above zero petals in a lineage leading to Parryella the hypanthium. These species occupy the and E. rotundata. If the phylogeny in Fig- largest subgenus, Parosela. The second ure 4 is correct, and these two species are largest is subg. Dalea, which includes the nested within Amorpha, then this seems the American prairie clovers and other daleas most plausible scenario. However, each of with ¯owers that show little differentiation the three markers used in the molecular between wing and keel petals and in which phylogenetic analyses (CNGC4, ITS, and the androecium is exposed at anthesis. Ac- trnK) root this clade differently, with dif- cording to the phylogenetic analyses, the ferent consequences for the inference of sampled members of subg. Dalea, indicated petal number evolution (Fig. 3A; McMahon in Figure 5, are scattered within subg. Pa- & Hufford, 2005). Two species of Dalea rosela, and there is no support for mutually have also lost petals (D. urceolata Greene exclusive subgenera Dalea and Parosela. and D. confusa (Rydb.) Barneby var. hex- andra Barneby). Although they have not MORPHOLOGICAL EVOLUTION IN AMORPHEAE been sequenced, there is little doubt that Rupert Barneby reorganized the mem- they belong in Dalea, and therefore repre- bers of Amorpheae based on insightful and sent at least one other evolutionary loss of extensive observation of morphology. What petals. do the new, slightly modi®ed hypotheses of Another characteristic of several mem- relationship have to say about these char- bers of Amorpheae is the placement of the acters? And what else have we learned four lateral petals: sessile, i.e., on the rim about morphology of Amorpheae in the in- of a hypanthium, as is common in legumes, terim? or epistemonous, i.e., above the hypanthial Amorpheae includes trees, shrubs, and rim on tissue that resembles staminal col- herbs, with compound, unifoliolate, or sim- umn. Barneby suggested that this condition ple leaves, and members live in mesic is derived in Dalea and Marina, either in woodlands, grasslands, tropical deciduous concert or independently if Marina is more forests, and alpine habitats. Yet, the most closely related to Psorothamnus (Fig. 2). remarkable morphological evolution in The phylogeny supports the former hypoth- Amorpheae has occurred in the ¯ower. Pet- esis because Marina and Dalea form a al number varies from ®ve to zero; Barneby strongly supported clade, and all members

←

FIG.5.continued P. ®lifolia, respectively. Gain of a stemonozone occurred in the ancestor of Dalea ϩ Marina (E). Losses of lateral petal differentiation in the paraphyletic subg. Dalea are indicated by circles and dashed lines (F). All species in the Dalea clade that are neither subg. Theodora nor subg. Dalea are in subg. Parosela. Ingroup taxa downloaded from GenBank that were redundant or unidenti®ed (e.g., ``Marina sp.'') were eliminated. 408BRITTONIA [VOL. 57 of these genera have easily observed epis- evolution of ¯owers in Amorpheae? He was temonous petal placement. However, sev- rather circumspect, but he suggested some- eral species of Psorothamnus also have a thing startling: a secondary derivation of very short region below the insertion of the the papilionoid ¯ower. He speculated that lateral petals, dif®cult to see with a hand this was the case because of two things: (1) lens but apparent under scanning electron the presence of the epistemonous petals, be- microscopy (McMahon & Hufford, 2002). ing a somewhat subtle but fundamental de- Further analysis has found this ``minute ste- parture from standard papilionoid forms monozone'' in Errazurizia megacarpa and and indeed rare among angiosperms, and yet absent in E. benthami (McMahon & (2) the hypothetical phylogeny derived in Hufford, 2005). This condition is unknown part from polarizing characters such as in Eysenhardtia and Apoplanesia, inappli- wood, in¯orescence structure, chromosome cable in the ``amorpha'' clade, and awaits number, and androecial exposure. However, further investigation within the probable additional data and explicit phylogenetic to Amorpheae, the dalbergioid analysis, as well as a perhaps better under- clade (Lavin et al., 2001). Therefore, all standing of the relationship of Amorpheae that can be concluded currently is that ep- to the rest of Papilionoideae, lead to the istemonous petals serve as a synapomorphy conclusion that the ¯oral simpli®cations for the Dalea ϩ Marina clade, and the mi- seen in the amorphoids and in several dal- nute stemonozone is possibly homoplasious eas as well as the epistemonous petals are (McMahon & Hufford, 2004, 2005). all derived conditions. Indeed, it is not the Differentiation of the corolla into three case that the complex papilionoid ¯ower types of petals (a banner, a pair each of has evolved more than once in Amorpheae; wing and keel petals) is a relatively stan- rather, simpli®cations have occurred. dard condition in Papilionoideae (Polhill, Several species that were originally 1981; Endress, 1994). However, in Amor- placed in the tribes Sophoreae and Swart- pheae, several taxa have non- or weakly- zieae (Polhill, 1981) have atypical ¯owers differentiated wing and keel petals. Erra- (Polhill, 1981; Mansano et al., 2004). How- zurizia s. str., Eysenhardtia, and Apopla- ever, it is becoming clear with each addi- nesia all show poor lateral petal differenti- tional phylogenetic study that many of ation, and this is consistent with their these are not closely related, for example, phylogenetic placement together in the the several that are scattered now in the dal- amorphoid clade (Fig. 5). Phylogenetically bergioid clade (Pennington et al., 2000, independent from these cases are those in 2001; Lavin et al., 2001; Wojciechowski et Dalea. In Barneby's treatment of the genus, al., 2004). Therefore, an important question the two largest subgenera are distinguish- in legume diversi®cation is the source of able, in part, because of petal differentia- independent origins of simpli®ed ¯owers, tion: Dalea subg. Dalea has wings and of which several examples are seen in keels that are quite similar whereas subg. Amorpheae. Parosela has the standard three types of petals. Molecular phylogenetic analysis, NUCLEAR CNGC4 however, does not concord with the sub- genera. Poor resolution and insuf®cient tax- phylogenetic systematics relies on sampling within Dalea limits precise heavily on molecular data, particularly from claims, but there is evidence for at least chloroplast and nuclear ribosomal regions, three separate cases of dedifferentiation in but these regions are often insuf®ciently Dalea: (1) D. lanata Spreng., (2) D. clif- variable to resolve relationships among spe- fortiana Willd., D. purpurea Vent., D. can- cies. Therefore, discovering and testing dida Michx. ex Willd. (but note that the re- low-copy markers from the nuclear genome lated D. scandens (Mill.) R. T. Clausen has (e.g., Bailey et al., 2004) is an appealing 3 types), and (3) D. lumholtzii Robins. & approach to add data and corroborate pre- . (Fig. 5). viously used markers, none of which are What did Barneby conclude about the without problems (AÂ lvarez & Wendel, 2005] McMAHON: PHYLOGENETICS & FLORAL EVOLUTION OF AMORPHEAE 409

2003; Bailey et al., 2003; Wolfe & Randle, ments; and the following herbaria for their 2004). The nuclear marker CNGC4 holds generous loans: ARIZ, MO, NY, RSA/ promise for Amorpheae. CNGC4, when an- POM, UC/JEPS. alyzed alone, shows clear signal for several clades that were also inferred using ITS and Literature Cited trnK/matK, providing corroboration. How- ever, all three markers show weak but dis- AÂ lvarez, I. & J. F. Wendel. 2003. Ribosomal ITS parate signal regarding the root of the amor- sequences and plant phylogenetic inference. Mo- phoids. Additional taxon sampling for lecular Phylogenetics and Evolution 29: 417±434. Bailey, C. D., T. G. Carr, S. A. Harris & C. E. CNGC4, as well as the application of new Hughes. 2003. Characterization of angiosperm nuclear markers, will likely be needed to nrDNA , paralogy, and pseudogenes. resolve this issue. and Evolution 29: 435± 455. ÐÐÐ, C. E. Hughes & S. A. Harris. 2004. Using CONCLUSIONS RAPDs to identify DNA sequence loci for species Phylogenetic analysis is largely in agree- level phylogeny reconstruction: an example from Leucaena (Fabaceae). Systematic Botany 29: 4±14. ment with Barneby's thorough taxonomic Barker, F. K. & F. M. Lutzoni. 2002. The utility of revision of Amorpheae. Amorpheae is the incongruence length difference test. Systematic monophyletic and only distantly related to 51: 625±637. the taxa with which they were previously Barneby, R. C. 1962. A synopsis of Errazurizia. Leaf- grouped (Psoraleae s. str.). Psorothamnus lets of Western Botany 9: 209±214. ÐÐÐ. 1977. Daleae imagines: an illustrated revision should be split into the original Rydberg of Errazurizia Philippi, Psorothamnus Rydberg, genera, Psorothamnus and Psorodendron. Marina Liebmann, and Dalea Lucanus emend. Monotypic Parryella and E. rotundata may Barneby, including all species of Leguminosae be better considered as members of Amor- tribe Amorpheae Borissova ever referred to Dalea. Memoirs of The New York Botanical Garden 27: pha, and D. ®liciformis is more closely re- 1±891. lated to Marina. Plants in Amorpheae either ÐÐÐ. 1980. Three new species of Dalea sect. Pa- have typical papilionoid ¯owers (Psoro- rosela (Leguminosae: Amorpheae) from western thamnus only) or have ¯owers that are atyp- and southern Mexico. Brittonia 32: 392±396. ical in several different ways, some of ÐÐÐ. 1981. New species of Dalea section Parosela (Leguminosae: Amorpheae) from Peru and Mexico. which have apparently evolved more than Brittonia 33: 508±511. once. ÐÐÐ. 1988. The genus Dalea (Fabaceae tribe Amor- Barneby's insights into Amorpheae were pheae) in Departamento de Cajamarca, Peru, with borne of years of ®eld observations, begin- description of three new species. Brittonia 40: 1±6. ÐÐÐ. 1990. Two new taxa in Dalea (Fabaceae: ning with his mid-1960's trips to Mexico Amorpheae) from southern Mexico and northern (Crase, 2004). Having North American As- Chile. Brittonia 42: 89±91. tragalus well in hand, he turned to Dalea, Bentham, G. 1865. Leguminosae. Pages 434±600. In: about which the biographer D. Crase writes G. Bentham & J. D. Hooker, editors. Genera plan- ``the way in which Dalea most seductively tarum. Lovell Reeve & Co., London, UK. Choi, H. K., D. Kim, T. Uhm, E. Limpens, H. Lim, resembled Astragalus was not as a legume J.-H. Mun, P. Kalo, R. V. Penmetsa, A. Seres, but as a taxonomic knot to be untied.'' O. Kulikova, B. A. Roe, T. Bisseling, G. B. Kiss (Crase, 2004: 71). It is fortunate for stu- & D. R. Cook. 2004a. A sequence-based genetic dents of legumes, New World ¯oristics, and map of Medicago truncatula and comparison of marker colinearity with M. sativa. Genetics 166: ¯oral diversi®cation that Rupert Barneby 1463±1502. was intrigued in this manner. ÐÐÐ, J.-H. Mun, D.-J. Kim, H. Zhu, J.-M. Baek, J. Mudge, B. Roe, N. Ellis, J. Doyle, G. B. Kiss, Acknowledgments N. D. Young & D. R. Cook. 2004b. Estimating genome conservation between crop and model le- I thank Martin Wojciechowski and Aaron gume species. Proceedings of the National Acade- Liston for the invitation to participate in the my of Science 43: 15289±15294. symposium honoring the life work of Ru- Corby, H. D. L. 1981. The systematic value of legu- minous root nodules. Pages 657±670. In: R. M. pert Barneby; Larry Hufford for collabora- Polhill & P. H. Raven, editors. Advances in legume tion on previous work in this group; two systematics. Part 2. Royal Botanic Gardens, Kew, anonymous reviewers for helpful com- UK. 410BRITTONIA [VOL. 57

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