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Molecular systematics of the Amazonian genus Aldina, a phylogenetically enigmatic ectomycorrhizal lineage of papilionoid legumes
Accepted Manuscript Molecular systematics of the Amazonian genus Aldina, a phylogenetically enigmatic ectomycorrhizal lineage of papilionoid legumes Gustavo Ramos, Haroldo Cavalcante de Lima, Gerhard Prenner, Luciano Paganucci de Queiroz, Charles E. Zartman, Domingos Cardoso PII: DOI: Reference:
S1055-7903(15)00396-6 http://dx.doi.org/10.1016/j.ympev.2015.12.017 YMPEV 5389
To appear in:
Molecular Phylogenetics and Evolution
Received Date: Revised Date: Accepted Date:
19 November 2015 23 December 2015 28 December 2015
Please cite this article as: Ramos, G., Lima, H.C.d., Prenner, G., Queiroz, L.P.d., Zartman, C.E., Cardoso, D., Molecular systematics of the Amazonian genus Aldina, a phylogenetically enigmatic ectomycorrhizal lineage of papilionoid legumes, Molecular Phylogenetics and Evolution (2015), doi: http://dx.doi.org/10.1016/j.ympev. 2015.12.017
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Molecular systematics of the Amazonian genus Aldina, a phylogenetically enigmatic ectomycorrhizal lineage of papilionoid legumes
Gustavo Ramosa, Haroldo Cavalcante de Limab, Gerhard Prennerc, Luciano Paganucci de Queirozd, Charles E. Zartmane, Domingos Cardosoa,d,*
a
Departamento de Botânica, Instituto de Biologia, Universidade Federal da Bahia, Rua Barão de Jeremoabo, s.n., Ondina, 40170-115, Salvador, Bahia, Brazil
b
Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Rua Pacheco Leão, 915, 22460-030, Rio de Janeiro, Brazil c
Comparative Plant and Fungal Biology Department, Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3DS, UK d
Programa de Pós-Graduação em Botânica, Universidade Estadual de Feira de Santana, Av. Transnordestina, s.n., Novo Horizonte, 44036-900, Feira de Santana, Bahia, Brazil
e
Instituto Nacional de Pesquisas da Amazônia (INPA), Department of Biodiversity, Av. André Araújo, 2936, Petrópolis, 69060-001, Manaus, Amazonas, Brazil
*Corresponding author. Address: Departamento de Botânica, Instituto de Biologia, Universidade Federal da Bahia, Rua Barão de Jeremoabo, s.n., Ondina, 40170-115, Salvador, Bahia, Brazil. Email address: [email protected] (D. Cardoso).
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ABSTRACT Aldina (Leguminosae) is among the very few ecologically successful ectomycorrhizal lineages in a family largely marked by the evolution of nodulating symbiosis. The genus comprises 20 species exclusively distributed in Amazonia and has been traditionally classified in the tribe Swartzieae because of its radial flowers with an entire calyx and numerous free stamens. The taxonomy of Aldina is complicated due to its poor representation in herbaria and the lack of a robust phylogenetic hypothesis of relationship. Recent phylogenetic analyses of matK and trnL sequences confirmed the placement of Aldina in the 50-kb inversion clade, although the genus remained phylogenetically isolated or unresolved in the context of the evolutionary history of the main earlybranching papilionoid lineages. We performed maximum likelihood and Bayesian analyses of combined chloroplast datasets (matK, rbcL, and trnL) and explored the effect of incomplete taxa or missing data in order to shed light on the enigmatic phylogenetic position of Aldina. Unexpectedly, a sister relationship of Aldina with the Andira clade (Andira and Hymenolobium) is revealed. We suggest that a new tribal phylogenetic classification of the papilionoid legumes should place Aldina along with Andira and Hymenolobium. These results highlight yet another example of the independent evolution of radial floral symmetry within the early-branching Papilionoideae, a large collection of florally heterogeneous lineages dominated by papilionate or bilaterally symmetric flower morphology.
Keywords: Floral symmetry; Leguminosae; Papilionoideae; phylogeny; Swartzieae.
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1. Introduction The ancient evolution of root-nodulating bacteria not only played a key role during the ecological success of legumes (Leguminosae) across global biomes (Doyle et al., 1997; Sprent et al., 2013), but such a symbiosis has also greatly contributed to ecosystem services because it is tightly related to nitrogen biogeochemical cycling (Hedin et al., 2009). Recent advances in datedphylogenies have addressed the evolutionary history of such ecologically important root-nodulating legume lineages (e.g. Bontemps et al., 2010; Cannon et al., 2010; Werner et al., 2014; Li et al., 2015). However, little is still known about another important evolutionary interaction that has led to an impressive ecological dominance of legume trees in tropical forests: the ectomycorrhizal symbiosis (Torti et al., 2001; Henkel, 2003; Smith et al., 2011). Ectomycorrhizal fungi have been mainly reported in African and Asian caesalpinioid species as well as in the Australian mimosoid genus Acacia Mill. (Alexander, 1989; Duponnois and Plenchette, 2003), and recently in the neotropical caesalpinioid genus Dicymbe Spruce ex Benth. & Hook.f. (Smith et al., 2011). On the other hand, the phylogenetically enigmatic and florally disparate Amazonian genus Aldina Endl. is among the very few known examples to form ectomycorrhizal associations within the species-rich Papilionoideae (Singer and Aguiar, 1986; Henkel et al., 2002; Smith et al., 2011). Robustly resolved phylogenies are crucial for understanding the seemingly advantageous function of nodulating bacteria during legume diversification and for explaining why other traits such as ectomycorrhizal symbiosis are less so. In this context we can track when exactly the rare ectomycorrhizal root evolved in the legume phylogeny. Revealing the evolutionary history of legumes may also allow us to investigate whether traditional classifications that rely primarily on flower morphology accurately reflect phylogenetic relationships. For example, whether contrasting floral morphologies in Papilionoideae evolved in a
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conserved fashion or independently may be addressed by investigating specific cases of floral evolution in a given clade (Pennington et al., 2000; Cardoso et al., 2012b, 2013b). The genus Aldina represents an excellent opportunity to do this. It comprises ca. 20 species predominantly distributed in Amazonia (Stergios and Cowan, 1999; Pennington et al., 2005) and has been traditionally classified in tribe Swartzieae because of the uncommon radial flowers, entire calyx in bud, undifferentiated free petals, and numerous free stamens (Cowan, 1981; Ireland, 2005). The genus is one of many examples in Swartzieae that previously have appeared scattered across the phylogenetic tree of the Papilionoideae, as revealed in analyses of chloroplast DNA sequences (Doyle et al., 1997; Pennington et al., 2001; Wojciechowski et al., 2004; Cardoso et al., 2012a, 2013a). The placement of Aldina in the Papilionoideae phylogeny has been controversial, so that the genus is to date still classified in Swartzieae (Ireland, 2005). The first molecular phylogeny of legumes placed Aldina in a large clade marked by an inversion of 50 kb in the chloroplast genome (Doyle et al., 1997). However, further phylogenetic analyses of trnL intron sequences, involving a large sampling of genera from Sophoreae and Swartzieae, placed Aldina among the first diverging lineages of Papilionoideae, all of them known for lacking the 50-kb inversion (Ireland et al., 2000; Pennington et al., 2001). This controversy or incongruence in the chloroplast data has been solved only recently when a more comprehensive phylogenetic study of the early-branching papilionoids sequenced the trnL intron and the coding gene matK from entirely new collections of Aldina and confirmed the placement of the genus in the large 50-kb inversion clade (Cardoso et al., 2012a, 2015). Nevertheless, in those molecular analyses Aldina still remained phylogenetically unresolved within the main lineages of Papilionoideae. A wide polytomy across the 50-kb inversion clade includes Aldina, Amphimas Pierre ex Dalla Torre & Harms, Dermatophyllum Scheele, the small
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Andira, Lecointeoid, and Vataireoid clades, and the megadiverse lineages Dalbergioids, Genistoids, and the non-protein-amino-acid-accumulating (NPAAA) clade (Cardoso et al., 2012a, 2013a, 2015; LPWG, 2013). The enormous number of chloroplast DNA sequences that have been accumulated from phylogenetic studies of the Papilionoideae (e.g. Doyle et al., 1997; Kajita et al., 2001; Lavin et al., 2001; McMahon and Hufford, 2004; Wojciechowski et al., 2004; Delgado-Salinas et al., 2011; Cardoso et al., 2012, 2013, 2015; Queiroz et al., 2015) provide a largely untapped body of data for placing the remaining enigmatic branches in the legume phylogeny. In this study, we revisit the phylogeny of the early papilionoid radiation by exploring the phylogenetic signal of missing data in order to shed light on the obscure phylogenetic placement of Aldina, to understand the evolution of the striking floral diversity of the early-branching papilionoids, and to add data to a more consistent phylogenetic classification proposal.
2. Materials and methods 2.1. Taxon sampling and molecular data Our sampling prioritized representatives of the main Papilionoideae lineages with a particular focus on early-branching genera that appear immediately outside the NPAAA clade. This sampling strategy has been proved useful in previous densely-sampled phylogenies across the taxonomic diversity of the papilionoid tribes Sophoreae and Swartzieae (Cardoso et al., 2012a, 2013a, 2015). Because all previous phylogenetic analyses of individual molecular datasets have failed to resolve the relationship of Aldina (e.g. Doyle et al., 1997; Cardoso et al., 2012a, 2013a, 2015), here we combine analyses of three chloroplast loci: trnL intron and the protein-coding genes matK and rbcL. Robust phylogenetic analyses involving dense sampling and a higher number of molecular
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markers may improve phylogenetic accuracy because they can break or subdivide long branches (Graybeal, 1998; Soltis et al., 1998; Pollock et al., 2002; Zwickl and Hillis, 2002). Also, they potentially provide better resolution for ancient rapid radiations (Rokas et al., 2005; Rokas and Carroll, 2006), which is particularly the case in Aldina and the main lineages in the legume 50-kb inversion clade (Lavin et al., 2005; Cardoso et al., 2012a, 2013a). A large sampling of available sequences in GenBank (http://www.ncbi.nlm.nih.gov/genbank/) for the genes matK, rbcL, and trnL allowed us to cover both morphological and taxonomic diversity of the tribes Sophoreae and Swartzieae (Appendix S1). Most sequences used for this study represent significant contributions of phylogenetic studies across different legume clades (e.g. Doyle et al., 1997; Kajita et al., 2001; Lavin et al., 2001; Pennington et al., 2001; Wojciechowski et al., 2004; Boatwright et al., 2008; Bruneau et al., 2008; Queiroz et al., 2010, 2015; Cardoso et al., 2012a, 2012b, 2013b, 2015). We built the largest combined chloroplast datasets which focus on the early branches of the Papilionoideae. The eight different datasets included between 94 and 310 terminals and up to 3996 characters (Table 1). In three datasets we explored the effect of taxa with incomplete data (i.e. terminals lacking at least one complete sequence) in recovering robust phylogenetic signal for the placement of Aldina. Combined analyses of empirical data and computational simulations have demonstrated that taxa with incomplete data do not negatively affect the results and, even when the taxa included present less than 50% of data, they can break long branches and improve phylogenetic accuracy (Wiens, 2003, 2005, 2006; Wiens and Tiu, 2012). In our datasets taxa lacked 13.5 and 24.7% of sequence data (Table 1). Datasets were designed so as to result in all taxa having a matK sequence, because this gene is better represented in GenBank and is more phylogenetically informative than rbcL and trnL.
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2.2. Alignment and phylogenetic analyses Sequences retrieved from GenBank were aligned manually in SeaView version 4 (Gouy et al., 2010), using the similarity criterion of Kelchner (2000) in order to avoid inconsistencies derived from automated multiple alignment. Because the matK matrices showed high number of indels, we followed the suggestion of Wojciechowski et al. (2004) in looking for homologies among amino acid translated sequences. We ran the model-based Bayesian and maximum likelihood methods of phylogenetic reconstruction. Parsimony was not run because its performance is not robust for reconstructing phylogenies of large datasets, where homoplasies are so common and branch length varies considerably (Huelsenbeck et al., 2001; Holder and Lewis, 2003). Bayesian analyses were run in MrBayes version 3.2.1 (Ronquist et al., 2012). A best-fit nucleotide substitution model was first selected via the Akaike information criterion (AIC), as implemented in MrModelTest version 2.2 (Nylander, 2004). The best-fit model for all datasets was the most complex GTR+I+G. In two separate runs of a Metropolis-coupled Markov Chain Monte Carlo (MCMC) permutation of parameters, eight simultaneous chains were initiated with a random tree for 10 million generations through the phylogenetic tree space, sampling one tree at each 10,000th generation. Nonautocorrelated samples at the stationary phase were summarized in a Bayesian majority-rule consensus tree at 50% after a burn-in of 25%. Clade frequencies or posterior probabilities (PP) represent support measures (Huelsenbeck et al., 2002). Visualization and partial editing of the Bayesian tree for graphical presentation were done in FigTree version 1.4.0 (Rambaut, 2012). Maximum likelihood trees were inferred in RAxML v7.2.8 (Stamatakis, 2006), using the evolutionary model GTRAC and invariant sites and gamma distribution estimated during the run.
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Branch supports were estimated through 1000 bootstrap (BS) replications (Stamatakis et al., 2008). All phylogenetic analyses were run through the CIPRES Science Gateway v. 3.3 (Miller et al., 2010; http://www.phylo.org).
3. Results All phylogenetic analyses, except for the individual analysis of rbcL, confirmed with high support the position of Aldina in the large 50-kb inversion clade (Table 1). We found for the first time excellent phylogenetic resolution to define a sister group for Aldina, where all combined analyses resolved the genus as sister of a small clade comprising Andira Lam. (ca. 29 spp.) and Hymenolobium Benth. ex Mart. (ca. 15 spp.) (Fig. 1; Table 1; Appendix S2). This unexpected relationship was unequivocally resolved with high support (0.99 PP) in the Bayesian analysis of combined matK and rbcL sequence data. All other Bayesian and maximum likelihood analyses, involving taxa with either complete or incomplete data, also presented from very low (0.78 PP and 52% BS) to higher (0.98 PP and 69% BS) phylogenetic signal in resolving Aldina in the same clade with Andira and Hymenolobium (Table 1). A decrease in branch support in the combined analyses involving the trnL intron is probably related to intrinsic features of this gene. Microsatellites, nucleotide repeats of variable length, duplications, and large deletions so often found in the trnL intron sequences (Kelchner, 2000) may hamper the establishment of homologies during the alignment of relatively large matrices.
4. Discussion 4.1. Resolving the position of Aldina in the Papilionoideae phylogeny
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The analyses of combined chloroplast sequences allowed us to confidently define a phylogenetic position for Aldina in the Papilionoideae. Traditional concepts of generic level relations or tribal circumscriptions within the Papilionoideae have never hypothesized a relationship of Aldina to Andira or Hymenolobium, most likely because these taxa have disparate floral morphologies (Fig. 2). Andira and Hymenolobium are characterized by bilaterally-symmetrical papilionate flowers, with a strongly differentiated corolla composed of an adaxial standard or banner, two lateral wing petals, and an abaxial keel composed of two petals enveloping the stamens. This floral morphology greatly contrasts with that of the radially symmetrical flowers of Aldina composed of five undifferentiated petals and a polyandrous androecium with free stamens. Both the papilionate flowers and indehiscent fruits of Andira and Hymenolobium have traditionally been used to place these genera in the tribe Dalbergieae (Bentham, 1860; Polhill, 1981; Lima, 1990; Klitgaard and Lavin, 2005). Despite its tardily-dehiscent drupaceous fruits that are very similar to those of Andira (Fig. 2), Aldina was never grouped with this genus or any other Dalbergieae. Instead, Aldina was always classified within the most “primitive” tribe Swartzieae (Cowan, 1981) that collectively grouped many genera that deviate from the truly papilionate flowers. We show here, once again, how taxonomy influenced strongly by floral traits may result in artificial classifications that lead to evolutionary misconceptions (Pennington et al., 2001; Cameron, 2005; Martin et al., 2008; Salazar and Dressler, 2011; Cardoso et al., 2012a, 2012b, 2013b). Perhaps the most dramatic case of homoplasy in floral symmetry among the Papilionoideae was identified in the early-branching genus Acosmium s.l. in which molecular evidence has shown that this taxon, as traditionally defined, amalgamated five phylogenetically distant genera due to the superficial similarity of their radial flowers (Rodrigues and Tozzi, 2007; Cardoso et al., 2012b, 2012c).
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The recurrent theme of taxa with radially symmetric flowers interspersed in or sister to clades with monosymmetric flag blossoms presents an interesting challenge for floral morphologists and evolutionary biologists. Previous work has shown that the detailed study of floral ontogeny can help to clarify character homologies and to better understand the relationships within morphologically heterogeneous clades (cf. Tucker, 1987; Prenner, 2004; Zimmerman et al., 2013; Bruneau et al., 2014; Prenner et al., 2015). Based on the study of the enigmatic Duparquetia orchidacea Baill. Prenner and Klitgaard (2008) proposed an experimental phase in the flowers of caesalpinioid legumes that later was canalized and which has led to the highly successful papilionoid flag blossoms. The present study reinforces this hypothesis and shows that an experimental phase was inherent among early-branching Papilionoideae and that only the rise of the megadiverse NPAAA lineage in the early Eocene marks the ultimate canalization of a monosymmetric flag blossom. Future studies will have to clarify the ontogenetic basis for the morphological diversity in the newly circumscribed Andira clade (Aldina, Andira, and Hymenolobium) and in other clades with a high morphological diversity (Prenner et al., in prep.). The Andira clade is not only supported by molecular data, but also by some morphological characters and an ecological predilection for tropical rain forests. Almost all species of the clade occur in Amazonia and the Atlantic Forest, except for only a few that reach the central Brazilian cerrado (savanna-like) vegetation (Pennington, 2003; Lewis et al., 2005). In addition to the drupaceous woody fruits with fibrous endocarp of Aldina and Andira, the three genera in the Andira clade share terminal paniculate inflorescences, leaves often clustered at the branch apex, a 1–3ovulate ovary, and overgrown seeds (the seed has an undifferentiated testa and no endosperm, sensu Corner, 1951). Ferguson and Skvarla (1991) discussed how the tricolporate pollen of Aldina is distinctive among the genera of Swartzieae s.l., highlighting the unique exine stratification
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involving a lamellate endexine adjacent to the endoapertures. They also described Aldina pollen as having a granulate sometimes operculate colpus membrane, finely perforate tectum and long, dense columellae in the mesocolpium, and the colpi extending onto the poles. Most of these pollen features also have been reported in Hymenolobium and Andira (Gurgel et al., 2000; Pennington, 2003). Our main taxonomic message here is that any newly revised phylogenetic classification of the early-branching Papilionoideae should neither place Aldina in Swartzieae (Cowan, 1981; Ireland, 2005) nor in a monotypic tribe (Gontscharov et al., 2006). In fact, irrespective of how any new infra-familial level classification of legumes may be circumscribed (Cardoso et al., 2013a; Wojciechowski, 2013) our results clearly show that Aldina should be placed along with Andira and Hymenolobium. Furthermore, we recommend the need for a robustly-supported phylogeny-derived classification in legumes in order to avoid proliferation of new tribal names based upon small unresolved genera or clades (Cardoso et al., 2013a; LPWG, 2013).
4.2. Future prospects for phylogenetic and taxonomic studies of Aldina Understanding the complex taxonomic and evolutionary history of Aldina is particularly important because the genus is ecologically confined in the still poorly-known Amazon region, mainly in Brazil, a country notably recognized for its high floristic diversity and endemism (Forzza et al., 2012; BFG, 2015). Although Aldina is a small genus, some species such as A. heterophylla Spruce ex Benth. and A. latifolia Spruce ex Benth. are among the most widely distributed angiosperms in Amazonian campina (sandy) and igapó (periodically flooded) forests, respectively. A recent ecological estimate listed A. heterophylla among the dominant 227 species from an assemblage of ca. 16,000 woody species that make up the Amazon forest, whereas ca. 11,000
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species are rare or only narrowly distributed (ter Steege et al., 2013). In the Guyana Shield, Aldina is also listed among the 10 most abundant genera (ter Steege et al., 2006). Thus, Aldina can serve as a key lineage to foster our understanding on ecological and diversification processes across the extremely diverse environments of Amazonia. In the ubiquitous legume family, notably marked by a root-nodulating ability involving symbiotic association with nitrogen-fixing bacteria (Sprent et al., 2013), Aldina, in direct contrast, has achieved ecological success all over the Amazon region most likely because of its long evolutionary association with many lineages of ectomycorrhizal fungi (Henkel et al., 2002; ter Steege et al., 2006; Smith et al., 2011). Remarkably, Smith et al. (2011) found a diverse ectomycorrhizal assemblage of 17 fungal lineages associated with the roots of an Aldina and Dicymbe dominated forest in the Guyana Shield. They also showed some phylogenetic evidence that ectomycorrhizal symbiosis evolved at least four times in legumes. However, those conclusions should be interpreted with caution, since Smith et al. (2011) worked on a very limited sample that resulted in many clades having poor resolution, an issue that also has been a challenge for recent comprehensively-sampled phylogenies which address the rapid early radiation of legumes (Bruneau et al., 2008; Cardoso et al., 2012a, 2013a, 2015). Given the ecological and evolutionary importance of Aldina in the context of the Papilionoideae diversification, it is critical to establish an even more robust phylogenetic hypothesis, to include, for example, nuclear DNA markers and a complete sampling of the species diversity in the genus. We could not test here the monophyly of Aldina, because this depends on evaluating some rare or morphologically disparate species (Stergios and Aymard, 2008). A sound phylogenetic analysis of Aldina will potentially help to understand how ecology and geography interact to shape its phylogeny (Pennington et al., 2009; Oliveira-Filho et al., 2013; Pennington and Lavin, 2015). Furthermore, this will permit accurate species delimitation in a solid taxonomic
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monograph of the genus. In part the current complex taxonomy of Aldina is the result of its poor representation in herbaria, as well as a relatively high proportion of mistakenly published names. This has resulted in inconsistent identification keys and poor species circumscriptions (Cowan, 1953, 1961; Gontscharov et al., 2009). Several putative new species have been known about for some time (e.g. Stergios and Cowan, 1999), but some careless decisions have resulted in taxonomic and nomenclatural confusion. For example, some new species of Aldina have been mistakenly described based on a mixture of herbarium collections involving material from Andira (Gontscharov et al., 2006). We believe that only an integrative taxonomic approach on Aldina will allow us to better understand its evolutionary history and contribute to consistent species identification without overestimating its diversity.
Acknowledgments The authors immensely thank Matt Lavin, Gwil Lewis, Toby Pennington, Marty Wojciechowski, Colin Hughes, Alfonso Delgado-Salinas, Brigitte Marazzi, Anne Bruneau, Brian Schrire, and Charles Stirton for the many insightful and rich discussions on legume systematics and evolution; Matt Lavin and an anonymous reviewer for detailed critical comments and suggestions that enhanced the final version of the manuscript; Dirce Komura for helping DC and CZ during fieldwork in Amazonia, which resulted in new collections of Aldina and other early-branching Papilionoideae; André Cruz for helping with fieldwork in Bahia that resulted in interesting flowering collection of Hymenolobium janeirense; André Gil for providing valuable bibliography; Cecília Azevedo for kindly permitting us to use the photo of Andira fraxinifolia; Nádia Roque, Maria Lenise Guedes, and the staff at ALCB and HUEFS herbaria for providing appropriate facilities to work on the legume collections. The first author acknowledges the undergraduate grant
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from the PIBIEX/UFBA program and colleagues at FLORA lab for providing a stimulating scientific environment in which to work. Fieldwork, the study of herbarium collections, and DNA sequencing were partially sponsored by the projects Sistema Nacional de Pesquisa em Biodiversidade (SISBIOTA CNPq 563084/2010-3 / FAPESB PES0053/2011), Programa de Pesquisa em Biodiversidade do Semi-árido (PPBIO), and REFLORA (CNPq 563546/2010-7 / FAPESB PES0054/2011). We are also thankful to FAPESB (PET0039/2012) for the financial support to our research on molecular phylogenetics.
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Fig. 1. Bayesian majority-rule consensus tree of combined matK, rbcL, and trnL intron sequence data showing the placement of Aldina in the Andira clade, one of the early-branching Papilionoideae lineages within the large 50-kb chloroplast inversion clade. Posterior probabilities in the close-up of the Andira clade are only presented in each branch supported by 0.98–1. Branches in bold in the summary Papilionoideae phylogeny are supported by posterior probability of 0.98–1. Floral morphology of the genera Aldina, Andira, and Hymenolobium is illustrated by a representative species: Aldina kunhardtiana (Photo: Domingos Cardoso), Andira fraxinifolia (Photo: Cecília Azevedo), and Hymenolobium janeirense (Photo: Domingos Cardoso). A complete version of this Bayesian tree including all tip names is available in the Appendix S2.
Fig. 2. Flower and fruit diversity in the Andira clade (Aldina, Andira, and Hymenolobium). A–B. Bilaterally-symmetrical papilionate flowers of Andira fraxinifolia (A) and Andira anthelmia. (B). C. Drupaceous fruits of Andira legalis. D–E. Papilionate flowers of Hymenolobium janeirense (D) and Hymenolobium alagoanum (E). F. Indehiscent samaroid fruits of Hymenolobium petraeum. G–I. Radial flowers of Aldina latifolia (G), Aldina kunhardtiana (H), and Aldina heterophylla (I). J. Drupaceous fruits of Aldina latifolia. Photos: Cecília Azevedo (A), Domingos Cardoso (B–D, F–J), and Luciano Queiroz (E).
Appendix S1. Molecular sequences from the chloroplast loci matK, rbcL, and trnL intron and associated GenBank accession number that were used for the individual and combined phylogenetic analyses of the Amazonian genus Aldina and the early-branching papilionoids.
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Appendix S2. Majority-rule consensus tree derived from a Bayesian combined analysis of the chloroplast loci matK, rbcL, and trnL intron from a comprehensive sampling of legumes with focus set on the early-branching papilionoid clades. Representative clades of the caesalpinioids and mimosoids were sampled as outgroups. Posterior probabilities are omitted for the resolved branches weakly supported by 0.50–0.70. Branches in bold are those supported by a posterior probability of 0.98–1.0. The early-branching papilionoid clades, those that fall outside the large non-proteinamino-acid-accumulating (NPAAA) clade are named according to Cardoso et al. (2012a, 2013a).
27
Figure 1
a
Sw
ae
ie
rtz
1
0.98
Andira clade
50-kb inve rsion clade
Aldina heterophylla Aldina heterophylla Aldina insignis Aldina kunhardtiana Aldina discolor 0.99 Aldina latifolia 0.98 Aldina latifolia Hymenolobium janeirense Hymenolobium mesoamericanum Hymenolobium petraeum Hymenolobium heringerianum 1 Hymenolobium grazielanum Hymenolobium alagoanum Hymenolobium heterocarpum 0.98 Hymenolobium sericeum 1 Andira humilis Andira marauensis Andira anthelmia 1 Andira legalis Andira carvalhoi Andira inermis Andira galeottiana 1 Andira ormosioides
Da
ds
oi
t is
en
G
AA NPA
lbe
0.01 changes
rgi
oid
s
Figure 2
Table 1 Summary of the phylogenetic analyses for the placement of Aldina in the Papilionoideae phylogeny. Characteristics of different molecular datasets are shown, where “N” is the number of terminals (taxa) and incomplete taxa show the percentage of missing sequence of rbcL and/or trnL in the analyses. Support values (posterior probability of Bayesian inference/maximum likelihood bootstrap) are only shown for the large 50-kb inversion clade, where Aldina is recovered as sister to Andira+Hymenolobium, the newly circumscribed Andira clade (Ald (And, Hym)). Unresolved nodes or supported by less than 50% are represented by a trace (–).
Datasets
N
Alignment length Incomplete (base pairs) taxa 1731 –
Aldina in the (Ald (And, Hym)) 50-kb clade 0.92/87% –/–
matK
310
rbcL
226
1428
–
trnL
151
852
–
0.97/73%
matK+rbcL
226
3141
–
1/93%
0.99/69%
matK+rbcL incomplete
310
3159
13.5%
0.98/92%
0.78/52%
matK+rbcL+trnL
94
3905
–
1/99%
0.92/60%
matK+rbcL+trnL incomplete
260
3996
24.7%
1/100%
0.97/54%
matK+rbcL+trnL incomplete
156
3966
14.5%
1/100%
0.98/50%
–/–
–/– –/–
28
Graphical Abstract
Andira clade
Aldina heterophylla Aldina heterophylla Aldina insignis Aldina kunhardtiana Aldina discolor Aldina latifolia Aldina latifolia Hymenolobium janeirense Hymenolobium mesoamericanum Hymenolobium petraeum Hymenolobium heringerianum Hymenolobium grazielanum Hymenolobium alagoanum Hymenolobium heterocarpum Hymenolobium sericeum Andira humilis Andira marauensis Andira anthelmia Andira legalis Andira carvalhoi Andira inermis Andira galeottiana Andira ormosioides
Highlights
Combining analyses with missing data improve resolution in the legume phylogeny.
Densely-sampled analyses resolve the placement of the enigmatic papilionoid Aldina.
The early-branching Andira clade is newly circumscribed to include Aldina.
A remarkable example of independent evolution of radial flowers is revealed.
29
S1055-7903(15)00396-6 http://dx.doi.org/10.1016/j.ympev.2015.12.017 YMPEV 5389
To appear in:
Molecular Phylogenetics and Evolution
Received Date: Revised Date: Accepted Date:
19 November 2015 23 December 2015 28 December 2015
Please cite this article as: Ramos, G., Lima, H.C.d., Prenner, G., Queiroz, L.P.d., Zartman, C.E., Cardoso, D., Molecular systematics of the Amazonian genus Aldina, a phylogenetically enigmatic ectomycorrhizal lineage of papilionoid legumes, Molecular Phylogenetics and Evolution (2015), doi: http://dx.doi.org/10.1016/j.ympev. 2015.12.017
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Molecular systematics of the Amazonian genus Aldina, a phylogenetically enigmatic ectomycorrhizal lineage of papilionoid legumes
Gustavo Ramosa, Haroldo Cavalcante de Limab, Gerhard Prennerc, Luciano Paganucci de Queirozd, Charles E. Zartmane, Domingos Cardosoa,d,*
a
Departamento de Botânica, Instituto de Biologia, Universidade Federal da Bahia, Rua Barão de Jeremoabo, s.n., Ondina, 40170-115, Salvador, Bahia, Brazil
b
Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Rua Pacheco Leão, 915, 22460-030, Rio de Janeiro, Brazil c
Comparative Plant and Fungal Biology Department, Royal Botanic Gardens, Kew, Richmond, Surrey, TW9 3DS, UK d
Programa de Pós-Graduação em Botânica, Universidade Estadual de Feira de Santana, Av. Transnordestina, s.n., Novo Horizonte, 44036-900, Feira de Santana, Bahia, Brazil
e
Instituto Nacional de Pesquisas da Amazônia (INPA), Department of Biodiversity, Av. André Araújo, 2936, Petrópolis, 69060-001, Manaus, Amazonas, Brazil
*Corresponding author. Address: Departamento de Botânica, Instituto de Biologia, Universidade Federal da Bahia, Rua Barão de Jeremoabo, s.n., Ondina, 40170-115, Salvador, Bahia, Brazil. Email address: [email protected] (D. Cardoso).
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ABSTRACT Aldina (Leguminosae) is among the very few ecologically successful ectomycorrhizal lineages in a family largely marked by the evolution of nodulating symbiosis. The genus comprises 20 species exclusively distributed in Amazonia and has been traditionally classified in the tribe Swartzieae because of its radial flowers with an entire calyx and numerous free stamens. The taxonomy of Aldina is complicated due to its poor representation in herbaria and the lack of a robust phylogenetic hypothesis of relationship. Recent phylogenetic analyses of matK and trnL sequences confirmed the placement of Aldina in the 50-kb inversion clade, although the genus remained phylogenetically isolated or unresolved in the context of the evolutionary history of the main earlybranching papilionoid lineages. We performed maximum likelihood and Bayesian analyses of combined chloroplast datasets (matK, rbcL, and trnL) and explored the effect of incomplete taxa or missing data in order to shed light on the enigmatic phylogenetic position of Aldina. Unexpectedly, a sister relationship of Aldina with the Andira clade (Andira and Hymenolobium) is revealed. We suggest that a new tribal phylogenetic classification of the papilionoid legumes should place Aldina along with Andira and Hymenolobium. These results highlight yet another example of the independent evolution of radial floral symmetry within the early-branching Papilionoideae, a large collection of florally heterogeneous lineages dominated by papilionate or bilaterally symmetric flower morphology.
Keywords: Floral symmetry; Leguminosae; Papilionoideae; phylogeny; Swartzieae.
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1. Introduction The ancient evolution of root-nodulating bacteria not only played a key role during the ecological success of legumes (Leguminosae) across global biomes (Doyle et al., 1997; Sprent et al., 2013), but such a symbiosis has also greatly contributed to ecosystem services because it is tightly related to nitrogen biogeochemical cycling (Hedin et al., 2009). Recent advances in datedphylogenies have addressed the evolutionary history of such ecologically important root-nodulating legume lineages (e.g. Bontemps et al., 2010; Cannon et al., 2010; Werner et al., 2014; Li et al., 2015). However, little is still known about another important evolutionary interaction that has led to an impressive ecological dominance of legume trees in tropical forests: the ectomycorrhizal symbiosis (Torti et al., 2001; Henkel, 2003; Smith et al., 2011). Ectomycorrhizal fungi have been mainly reported in African and Asian caesalpinioid species as well as in the Australian mimosoid genus Acacia Mill. (Alexander, 1989; Duponnois and Plenchette, 2003), and recently in the neotropical caesalpinioid genus Dicymbe Spruce ex Benth. & Hook.f. (Smith et al., 2011). On the other hand, the phylogenetically enigmatic and florally disparate Amazonian genus Aldina Endl. is among the very few known examples to form ectomycorrhizal associations within the species-rich Papilionoideae (Singer and Aguiar, 1986; Henkel et al., 2002; Smith et al., 2011). Robustly resolved phylogenies are crucial for understanding the seemingly advantageous function of nodulating bacteria during legume diversification and for explaining why other traits such as ectomycorrhizal symbiosis are less so. In this context we can track when exactly the rare ectomycorrhizal root evolved in the legume phylogeny. Revealing the evolutionary history of legumes may also allow us to investigate whether traditional classifications that rely primarily on flower morphology accurately reflect phylogenetic relationships. For example, whether contrasting floral morphologies in Papilionoideae evolved in a
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conserved fashion or independently may be addressed by investigating specific cases of floral evolution in a given clade (Pennington et al., 2000; Cardoso et al., 2012b, 2013b). The genus Aldina represents an excellent opportunity to do this. It comprises ca. 20 species predominantly distributed in Amazonia (Stergios and Cowan, 1999; Pennington et al., 2005) and has been traditionally classified in tribe Swartzieae because of the uncommon radial flowers, entire calyx in bud, undifferentiated free petals, and numerous free stamens (Cowan, 1981; Ireland, 2005). The genus is one of many examples in Swartzieae that previously have appeared scattered across the phylogenetic tree of the Papilionoideae, as revealed in analyses of chloroplast DNA sequences (Doyle et al., 1997; Pennington et al., 2001; Wojciechowski et al., 2004; Cardoso et al., 2012a, 2013a). The placement of Aldina in the Papilionoideae phylogeny has been controversial, so that the genus is to date still classified in Swartzieae (Ireland, 2005). The first molecular phylogeny of legumes placed Aldina in a large clade marked by an inversion of 50 kb in the chloroplast genome (Doyle et al., 1997). However, further phylogenetic analyses of trnL intron sequences, involving a large sampling of genera from Sophoreae and Swartzieae, placed Aldina among the first diverging lineages of Papilionoideae, all of them known for lacking the 50-kb inversion (Ireland et al., 2000; Pennington et al., 2001). This controversy or incongruence in the chloroplast data has been solved only recently when a more comprehensive phylogenetic study of the early-branching papilionoids sequenced the trnL intron and the coding gene matK from entirely new collections of Aldina and confirmed the placement of the genus in the large 50-kb inversion clade (Cardoso et al., 2012a, 2015). Nevertheless, in those molecular analyses Aldina still remained phylogenetically unresolved within the main lineages of Papilionoideae. A wide polytomy across the 50-kb inversion clade includes Aldina, Amphimas Pierre ex Dalla Torre & Harms, Dermatophyllum Scheele, the small
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Andira, Lecointeoid, and Vataireoid clades, and the megadiverse lineages Dalbergioids, Genistoids, and the non-protein-amino-acid-accumulating (NPAAA) clade (Cardoso et al., 2012a, 2013a, 2015; LPWG, 2013). The enormous number of chloroplast DNA sequences that have been accumulated from phylogenetic studies of the Papilionoideae (e.g. Doyle et al., 1997; Kajita et al., 2001; Lavin et al., 2001; McMahon and Hufford, 2004; Wojciechowski et al., 2004; Delgado-Salinas et al., 2011; Cardoso et al., 2012, 2013, 2015; Queiroz et al., 2015) provide a largely untapped body of data for placing the remaining enigmatic branches in the legume phylogeny. In this study, we revisit the phylogeny of the early papilionoid radiation by exploring the phylogenetic signal of missing data in order to shed light on the obscure phylogenetic placement of Aldina, to understand the evolution of the striking floral diversity of the early-branching papilionoids, and to add data to a more consistent phylogenetic classification proposal.
2. Materials and methods 2.1. Taxon sampling and molecular data Our sampling prioritized representatives of the main Papilionoideae lineages with a particular focus on early-branching genera that appear immediately outside the NPAAA clade. This sampling strategy has been proved useful in previous densely-sampled phylogenies across the taxonomic diversity of the papilionoid tribes Sophoreae and Swartzieae (Cardoso et al., 2012a, 2013a, 2015). Because all previous phylogenetic analyses of individual molecular datasets have failed to resolve the relationship of Aldina (e.g. Doyle et al., 1997; Cardoso et al., 2012a, 2013a, 2015), here we combine analyses of three chloroplast loci: trnL intron and the protein-coding genes matK and rbcL. Robust phylogenetic analyses involving dense sampling and a higher number of molecular
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markers may improve phylogenetic accuracy because they can break or subdivide long branches (Graybeal, 1998; Soltis et al., 1998; Pollock et al., 2002; Zwickl and Hillis, 2002). Also, they potentially provide better resolution for ancient rapid radiations (Rokas et al., 2005; Rokas and Carroll, 2006), which is particularly the case in Aldina and the main lineages in the legume 50-kb inversion clade (Lavin et al., 2005; Cardoso et al., 2012a, 2013a). A large sampling of available sequences in GenBank (http://www.ncbi.nlm.nih.gov/genbank/) for the genes matK, rbcL, and trnL allowed us to cover both morphological and taxonomic diversity of the tribes Sophoreae and Swartzieae (Appendix S1). Most sequences used for this study represent significant contributions of phylogenetic studies across different legume clades (e.g. Doyle et al., 1997; Kajita et al., 2001; Lavin et al., 2001; Pennington et al., 2001; Wojciechowski et al., 2004; Boatwright et al., 2008; Bruneau et al., 2008; Queiroz et al., 2010, 2015; Cardoso et al., 2012a, 2012b, 2013b, 2015). We built the largest combined chloroplast datasets which focus on the early branches of the Papilionoideae. The eight different datasets included between 94 and 310 terminals and up to 3996 characters (Table 1). In three datasets we explored the effect of taxa with incomplete data (i.e. terminals lacking at least one complete sequence) in recovering robust phylogenetic signal for the placement of Aldina. Combined analyses of empirical data and computational simulations have demonstrated that taxa with incomplete data do not negatively affect the results and, even when the taxa included present less than 50% of data, they can break long branches and improve phylogenetic accuracy (Wiens, 2003, 2005, 2006; Wiens and Tiu, 2012). In our datasets taxa lacked 13.5 and 24.7% of sequence data (Table 1). Datasets were designed so as to result in all taxa having a matK sequence, because this gene is better represented in GenBank and is more phylogenetically informative than rbcL and trnL.
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2.2. Alignment and phylogenetic analyses Sequences retrieved from GenBank were aligned manually in SeaView version 4 (Gouy et al., 2010), using the similarity criterion of Kelchner (2000) in order to avoid inconsistencies derived from automated multiple alignment. Because the matK matrices showed high number of indels, we followed the suggestion of Wojciechowski et al. (2004) in looking for homologies among amino acid translated sequences. We ran the model-based Bayesian and maximum likelihood methods of phylogenetic reconstruction. Parsimony was not run because its performance is not robust for reconstructing phylogenies of large datasets, where homoplasies are so common and branch length varies considerably (Huelsenbeck et al., 2001; Holder and Lewis, 2003). Bayesian analyses were run in MrBayes version 3.2.1 (Ronquist et al., 2012). A best-fit nucleotide substitution model was first selected via the Akaike information criterion (AIC), as implemented in MrModelTest version 2.2 (Nylander, 2004). The best-fit model for all datasets was the most complex GTR+I+G. In two separate runs of a Metropolis-coupled Markov Chain Monte Carlo (MCMC) permutation of parameters, eight simultaneous chains were initiated with a random tree for 10 million generations through the phylogenetic tree space, sampling one tree at each 10,000th generation. Nonautocorrelated samples at the stationary phase were summarized in a Bayesian majority-rule consensus tree at 50% after a burn-in of 25%. Clade frequencies or posterior probabilities (PP) represent support measures (Huelsenbeck et al., 2002). Visualization and partial editing of the Bayesian tree for graphical presentation were done in FigTree version 1.4.0 (Rambaut, 2012). Maximum likelihood trees were inferred in RAxML v7.2.8 (Stamatakis, 2006), using the evolutionary model GTRAC and invariant sites and gamma distribution estimated during the run.
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Branch supports were estimated through 1000 bootstrap (BS) replications (Stamatakis et al., 2008). All phylogenetic analyses were run through the CIPRES Science Gateway v. 3.3 (Miller et al., 2010; http://www.phylo.org).
3. Results All phylogenetic analyses, except for the individual analysis of rbcL, confirmed with high support the position of Aldina in the large 50-kb inversion clade (Table 1). We found for the first time excellent phylogenetic resolution to define a sister group for Aldina, where all combined analyses resolved the genus as sister of a small clade comprising Andira Lam. (ca. 29 spp.) and Hymenolobium Benth. ex Mart. (ca. 15 spp.) (Fig. 1; Table 1; Appendix S2). This unexpected relationship was unequivocally resolved with high support (0.99 PP) in the Bayesian analysis of combined matK and rbcL sequence data. All other Bayesian and maximum likelihood analyses, involving taxa with either complete or incomplete data, also presented from very low (0.78 PP and 52% BS) to higher (0.98 PP and 69% BS) phylogenetic signal in resolving Aldina in the same clade with Andira and Hymenolobium (Table 1). A decrease in branch support in the combined analyses involving the trnL intron is probably related to intrinsic features of this gene. Microsatellites, nucleotide repeats of variable length, duplications, and large deletions so often found in the trnL intron sequences (Kelchner, 2000) may hamper the establishment of homologies during the alignment of relatively large matrices.
4. Discussion 4.1. Resolving the position of Aldina in the Papilionoideae phylogeny
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The analyses of combined chloroplast sequences allowed us to confidently define a phylogenetic position for Aldina in the Papilionoideae. Traditional concepts of generic level relations or tribal circumscriptions within the Papilionoideae have never hypothesized a relationship of Aldina to Andira or Hymenolobium, most likely because these taxa have disparate floral morphologies (Fig. 2). Andira and Hymenolobium are characterized by bilaterally-symmetrical papilionate flowers, with a strongly differentiated corolla composed of an adaxial standard or banner, two lateral wing petals, and an abaxial keel composed of two petals enveloping the stamens. This floral morphology greatly contrasts with that of the radially symmetrical flowers of Aldina composed of five undifferentiated petals and a polyandrous androecium with free stamens. Both the papilionate flowers and indehiscent fruits of Andira and Hymenolobium have traditionally been used to place these genera in the tribe Dalbergieae (Bentham, 1860; Polhill, 1981; Lima, 1990; Klitgaard and Lavin, 2005). Despite its tardily-dehiscent drupaceous fruits that are very similar to those of Andira (Fig. 2), Aldina was never grouped with this genus or any other Dalbergieae. Instead, Aldina was always classified within the most “primitive” tribe Swartzieae (Cowan, 1981) that collectively grouped many genera that deviate from the truly papilionate flowers. We show here, once again, how taxonomy influenced strongly by floral traits may result in artificial classifications that lead to evolutionary misconceptions (Pennington et al., 2001; Cameron, 2005; Martin et al., 2008; Salazar and Dressler, 2011; Cardoso et al., 2012a, 2012b, 2013b). Perhaps the most dramatic case of homoplasy in floral symmetry among the Papilionoideae was identified in the early-branching genus Acosmium s.l. in which molecular evidence has shown that this taxon, as traditionally defined, amalgamated five phylogenetically distant genera due to the superficial similarity of their radial flowers (Rodrigues and Tozzi, 2007; Cardoso et al., 2012b, 2012c).
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The recurrent theme of taxa with radially symmetric flowers interspersed in or sister to clades with monosymmetric flag blossoms presents an interesting challenge for floral morphologists and evolutionary biologists. Previous work has shown that the detailed study of floral ontogeny can help to clarify character homologies and to better understand the relationships within morphologically heterogeneous clades (cf. Tucker, 1987; Prenner, 2004; Zimmerman et al., 2013; Bruneau et al., 2014; Prenner et al., 2015). Based on the study of the enigmatic Duparquetia orchidacea Baill. Prenner and Klitgaard (2008) proposed an experimental phase in the flowers of caesalpinioid legumes that later was canalized and which has led to the highly successful papilionoid flag blossoms. The present study reinforces this hypothesis and shows that an experimental phase was inherent among early-branching Papilionoideae and that only the rise of the megadiverse NPAAA lineage in the early Eocene marks the ultimate canalization of a monosymmetric flag blossom. Future studies will have to clarify the ontogenetic basis for the morphological diversity in the newly circumscribed Andira clade (Aldina, Andira, and Hymenolobium) and in other clades with a high morphological diversity (Prenner et al., in prep.). The Andira clade is not only supported by molecular data, but also by some morphological characters and an ecological predilection for tropical rain forests. Almost all species of the clade occur in Amazonia and the Atlantic Forest, except for only a few that reach the central Brazilian cerrado (savanna-like) vegetation (Pennington, 2003; Lewis et al., 2005). In addition to the drupaceous woody fruits with fibrous endocarp of Aldina and Andira, the three genera in the Andira clade share terminal paniculate inflorescences, leaves often clustered at the branch apex, a 1–3ovulate ovary, and overgrown seeds (the seed has an undifferentiated testa and no endosperm, sensu Corner, 1951). Ferguson and Skvarla (1991) discussed how the tricolporate pollen of Aldina is distinctive among the genera of Swartzieae s.l., highlighting the unique exine stratification
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involving a lamellate endexine adjacent to the endoapertures. They also described Aldina pollen as having a granulate sometimes operculate colpus membrane, finely perforate tectum and long, dense columellae in the mesocolpium, and the colpi extending onto the poles. Most of these pollen features also have been reported in Hymenolobium and Andira (Gurgel et al., 2000; Pennington, 2003). Our main taxonomic message here is that any newly revised phylogenetic classification of the early-branching Papilionoideae should neither place Aldina in Swartzieae (Cowan, 1981; Ireland, 2005) nor in a monotypic tribe (Gontscharov et al., 2006). In fact, irrespective of how any new infra-familial level classification of legumes may be circumscribed (Cardoso et al., 2013a; Wojciechowski, 2013) our results clearly show that Aldina should be placed along with Andira and Hymenolobium. Furthermore, we recommend the need for a robustly-supported phylogeny-derived classification in legumes in order to avoid proliferation of new tribal names based upon small unresolved genera or clades (Cardoso et al., 2013a; LPWG, 2013).
4.2. Future prospects for phylogenetic and taxonomic studies of Aldina Understanding the complex taxonomic and evolutionary history of Aldina is particularly important because the genus is ecologically confined in the still poorly-known Amazon region, mainly in Brazil, a country notably recognized for its high floristic diversity and endemism (Forzza et al., 2012; BFG, 2015). Although Aldina is a small genus, some species such as A. heterophylla Spruce ex Benth. and A. latifolia Spruce ex Benth. are among the most widely distributed angiosperms in Amazonian campina (sandy) and igapó (periodically flooded) forests, respectively. A recent ecological estimate listed A. heterophylla among the dominant 227 species from an assemblage of ca. 16,000 woody species that make up the Amazon forest, whereas ca. 11,000
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species are rare or only narrowly distributed (ter Steege et al., 2013). In the Guyana Shield, Aldina is also listed among the 10 most abundant genera (ter Steege et al., 2006). Thus, Aldina can serve as a key lineage to foster our understanding on ecological and diversification processes across the extremely diverse environments of Amazonia. In the ubiquitous legume family, notably marked by a root-nodulating ability involving symbiotic association with nitrogen-fixing bacteria (Sprent et al., 2013), Aldina, in direct contrast, has achieved ecological success all over the Amazon region most likely because of its long evolutionary association with many lineages of ectomycorrhizal fungi (Henkel et al., 2002; ter Steege et al., 2006; Smith et al., 2011). Remarkably, Smith et al. (2011) found a diverse ectomycorrhizal assemblage of 17 fungal lineages associated with the roots of an Aldina and Dicymbe dominated forest in the Guyana Shield. They also showed some phylogenetic evidence that ectomycorrhizal symbiosis evolved at least four times in legumes. However, those conclusions should be interpreted with caution, since Smith et al. (2011) worked on a very limited sample that resulted in many clades having poor resolution, an issue that also has been a challenge for recent comprehensively-sampled phylogenies which address the rapid early radiation of legumes (Bruneau et al., 2008; Cardoso et al., 2012a, 2013a, 2015). Given the ecological and evolutionary importance of Aldina in the context of the Papilionoideae diversification, it is critical to establish an even more robust phylogenetic hypothesis, to include, for example, nuclear DNA markers and a complete sampling of the species diversity in the genus. We could not test here the monophyly of Aldina, because this depends on evaluating some rare or morphologically disparate species (Stergios and Aymard, 2008). A sound phylogenetic analysis of Aldina will potentially help to understand how ecology and geography interact to shape its phylogeny (Pennington et al., 2009; Oliveira-Filho et al., 2013; Pennington and Lavin, 2015). Furthermore, this will permit accurate species delimitation in a solid taxonomic
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monograph of the genus. In part the current complex taxonomy of Aldina is the result of its poor representation in herbaria, as well as a relatively high proportion of mistakenly published names. This has resulted in inconsistent identification keys and poor species circumscriptions (Cowan, 1953, 1961; Gontscharov et al., 2009). Several putative new species have been known about for some time (e.g. Stergios and Cowan, 1999), but some careless decisions have resulted in taxonomic and nomenclatural confusion. For example, some new species of Aldina have been mistakenly described based on a mixture of herbarium collections involving material from Andira (Gontscharov et al., 2006). We believe that only an integrative taxonomic approach on Aldina will allow us to better understand its evolutionary history and contribute to consistent species identification without overestimating its diversity.
Acknowledgments The authors immensely thank Matt Lavin, Gwil Lewis, Toby Pennington, Marty Wojciechowski, Colin Hughes, Alfonso Delgado-Salinas, Brigitte Marazzi, Anne Bruneau, Brian Schrire, and Charles Stirton for the many insightful and rich discussions on legume systematics and evolution; Matt Lavin and an anonymous reviewer for detailed critical comments and suggestions that enhanced the final version of the manuscript; Dirce Komura for helping DC and CZ during fieldwork in Amazonia, which resulted in new collections of Aldina and other early-branching Papilionoideae; André Cruz for helping with fieldwork in Bahia that resulted in interesting flowering collection of Hymenolobium janeirense; André Gil for providing valuable bibliography; Cecília Azevedo for kindly permitting us to use the photo of Andira fraxinifolia; Nádia Roque, Maria Lenise Guedes, and the staff at ALCB and HUEFS herbaria for providing appropriate facilities to work on the legume collections. The first author acknowledges the undergraduate grant
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from the PIBIEX/UFBA program and colleagues at FLORA lab for providing a stimulating scientific environment in which to work. Fieldwork, the study of herbarium collections, and DNA sequencing were partially sponsored by the projects Sistema Nacional de Pesquisa em Biodiversidade (SISBIOTA CNPq 563084/2010-3 / FAPESB PES0053/2011), Programa de Pesquisa em Biodiversidade do Semi-árido (PPBIO), and REFLORA (CNPq 563546/2010-7 / FAPESB PES0054/2011). We are also thankful to FAPESB (PET0039/2012) for the financial support to our research on molecular phylogenetics.
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Fig. 1. Bayesian majority-rule consensus tree of combined matK, rbcL, and trnL intron sequence data showing the placement of Aldina in the Andira clade, one of the early-branching Papilionoideae lineages within the large 50-kb chloroplast inversion clade. Posterior probabilities in the close-up of the Andira clade are only presented in each branch supported by 0.98–1. Branches in bold in the summary Papilionoideae phylogeny are supported by posterior probability of 0.98–1. Floral morphology of the genera Aldina, Andira, and Hymenolobium is illustrated by a representative species: Aldina kunhardtiana (Photo: Domingos Cardoso), Andira fraxinifolia (Photo: Cecília Azevedo), and Hymenolobium janeirense (Photo: Domingos Cardoso). A complete version of this Bayesian tree including all tip names is available in the Appendix S2.
Fig. 2. Flower and fruit diversity in the Andira clade (Aldina, Andira, and Hymenolobium). A–B. Bilaterally-symmetrical papilionate flowers of Andira fraxinifolia (A) and Andira anthelmia. (B). C. Drupaceous fruits of Andira legalis. D–E. Papilionate flowers of Hymenolobium janeirense (D) and Hymenolobium alagoanum (E). F. Indehiscent samaroid fruits of Hymenolobium petraeum. G–I. Radial flowers of Aldina latifolia (G), Aldina kunhardtiana (H), and Aldina heterophylla (I). J. Drupaceous fruits of Aldina latifolia. Photos: Cecília Azevedo (A), Domingos Cardoso (B–D, F–J), and Luciano Queiroz (E).
Appendix S1. Molecular sequences from the chloroplast loci matK, rbcL, and trnL intron and associated GenBank accession number that were used for the individual and combined phylogenetic analyses of the Amazonian genus Aldina and the early-branching papilionoids.
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Appendix S2. Majority-rule consensus tree derived from a Bayesian combined analysis of the chloroplast loci matK, rbcL, and trnL intron from a comprehensive sampling of legumes with focus set on the early-branching papilionoid clades. Representative clades of the caesalpinioids and mimosoids were sampled as outgroups. Posterior probabilities are omitted for the resolved branches weakly supported by 0.50–0.70. Branches in bold are those supported by a posterior probability of 0.98–1.0. The early-branching papilionoid clades, those that fall outside the large non-proteinamino-acid-accumulating (NPAAA) clade are named according to Cardoso et al. (2012a, 2013a).
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Figure 1
a
Sw
ae
ie
rtz
1
0.98
Andira clade
50-kb inve rsion clade
Aldina heterophylla Aldina heterophylla Aldina insignis Aldina kunhardtiana Aldina discolor 0.99 Aldina latifolia 0.98 Aldina latifolia Hymenolobium janeirense Hymenolobium mesoamericanum Hymenolobium petraeum Hymenolobium heringerianum 1 Hymenolobium grazielanum Hymenolobium alagoanum Hymenolobium heterocarpum 0.98 Hymenolobium sericeum 1 Andira humilis Andira marauensis Andira anthelmia 1 Andira legalis Andira carvalhoi Andira inermis Andira galeottiana 1 Andira ormosioides
Da
ds
oi
t is
en
G
AA NPA
lbe
0.01 changes
rgi
oid
s
Figure 2
Table 1 Summary of the phylogenetic analyses for the placement of Aldina in the Papilionoideae phylogeny. Characteristics of different molecular datasets are shown, where “N” is the number of terminals (taxa) and incomplete taxa show the percentage of missing sequence of rbcL and/or trnL in the analyses. Support values (posterior probability of Bayesian inference/maximum likelihood bootstrap) are only shown for the large 50-kb inversion clade, where Aldina is recovered as sister to Andira+Hymenolobium, the newly circumscribed Andira clade (Ald (And, Hym)). Unresolved nodes or supported by less than 50% are represented by a trace (–).
Datasets
N
Alignment length Incomplete (base pairs) taxa 1731 –
Aldina in the (Ald (And, Hym)) 50-kb clade 0.92/87% –/–
matK
310
rbcL
226
1428
–
trnL
151
852
–
0.97/73%
matK+rbcL
226
3141
–
1/93%
0.99/69%
matK+rbcL incomplete
310
3159
13.5%
0.98/92%
0.78/52%
matK+rbcL+trnL
94
3905
–
1/99%
0.92/60%
matK+rbcL+trnL incomplete
260
3996
24.7%
1/100%
0.97/54%
matK+rbcL+trnL incomplete
156
3966
14.5%
1/100%
0.98/50%
–/–
–/– –/–
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Graphical Abstract
Andira clade
Aldina heterophylla Aldina heterophylla Aldina insignis Aldina kunhardtiana Aldina discolor Aldina latifolia Aldina latifolia Hymenolobium janeirense Hymenolobium mesoamericanum Hymenolobium petraeum Hymenolobium heringerianum Hymenolobium grazielanum Hymenolobium alagoanum Hymenolobium heterocarpum Hymenolobium sericeum Andira humilis Andira marauensis Andira anthelmia Andira legalis Andira carvalhoi Andira inermis Andira galeottiana Andira ormosioides
Highlights
Combining analyses with missing data improve resolution in the legume phylogeny.
Densely-sampled analyses resolve the placement of the enigmatic papilionoid Aldina.
The early-branching Andira clade is newly circumscribed to include Aldina.
A remarkable example of independent evolution of radial flowers is revealed.
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