Research Article |
Corresponding author: Jens J. Ringelberg ( jens.ringelberg@systbot.uzh.ch ) Academic editor: Patrick Herendeen
© 2022 Jens J. Ringelberg, Erik J. M. Koenen, João R. Iganci, Luciano P. de Queiroz, Daniel J. Murphy, Myriam Gaudeul, Anne Bruneau, Melissa Luckow, Gwilym P. Lewis, Colin E. Hughes.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Ringelberg JJ, Koenen EJM, Iganci JR, de Queiroz LP, Murphy DJ, Gaudeul M, Bruneau A, Luckow M, Lewis GP, Hughes CE (2022) Phylogenomic analysis of 997 nuclear genes reveals the need for extensive generic re-delimitation in Caesalpinioideae (Leguminosae). In: Hughes CE, de Queiroz LP, Lewis GP (Eds) Advances in Legume Systematics 14. Classification of Caesalpinioideae Part 1: New generic delimitations. PhytoKeys 205: 3-58. https://doi.org/10.3897/phytokeys.205.85866
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Subfamily Caesalpinioideae with ca. 4,600 species in 152 genera is the second-largest subfamily of legumes (Leguminosae) and forms an ecologically and economically important group of trees, shrubs and lianas with a pantropical distribution. Despite major advances in the last few decades towards aligning genera with clades across Caesalpinioideae, generic delimitation remains in a state of considerable flux, especially across the mimosoid clade. We test the monophyly of genera across Caesalpinioideae via phylogenomic analysis of 997 nuclear genes sequenced via targeted enrichment (Hybseq) for 420 species and 147 of the 152 genera currently recognised in the subfamily. We show that 22 genera are non-monophyletic or nested in other genera and that non-monophyly is concentrated in the mimosoid clade where ca. 25% of the 90 genera are found to be non-monophyletic. We suggest two main reasons for this pervasive generic non-monophyly: (i) extensive morphological homoplasy that we document here for a handful of important traits and, particularly, the repeated evolution of distinctive fruit types that were historically emphasised in delimiting genera and (ii) this is an artefact of the lack of pantropical taxonomic syntheses and sampling in previous phylogenies and the consequent failure to identify clades that span the Old World and New World or conversely amphi-Atlantic genera that are non-monophyletic, both of which are critical for delimiting genera across this large pantropical clade. Finally, we discuss taxon delimitation in the phylogenomic era and especially how assessing patterns of gene tree conflict can provide additional insights into generic delimitation. This new phylogenomic framework provides the foundations for a series of papers reclassifying genera that are presented here in Advances in Legume Systematics (ALS) 14 Part 1, for establishing a new higher-level phylogenetic tribal and clade-based classification of Caesalpinioideae that is the focus of ALS14 Part 2 and for downstream analyses of evolutionary diversification and biogeography of this important group of legumes which are presented elsewhere.
Fabaceae, generic delimitation, mimosoid clade, monophyly, morphological homoplasy, phylogenomics
In 2017, the Legume Phylogeny Working Group established a new subfamily classification of the Leguminosae (
Caesalpinioideae sensu
Numbers of genera across Caesalpinioideae have increased progressively through the last 270 years, but are difficult to track, because of the altered delimitation of the subfamily. However, the history of generic delimitation in mimosoids illustrates the overall trajectory of numbers of genera.
The legacy of Bentham’s generic system has been long-lasting. At the heart of Bentham’s system were a set of large, geographically widespread genera, including Acacia, Calliandra Benth., Pithecellobium Mart. and Prosopis L., all of which, with the advent of molecular phylogenetics, have been shown to be non-monophyletic. The disintegration of Acacia into (currently) seven segregate genera (Acacia, Acaciella Britton & Rose, Mariosousa Seigler & Ebinger, Parasenegalia Seigler & Ebinger, Pseudosenegalia Seigler & Ebinger, Senegalia and Vachellia), based on 20 years of molecular phylogenetic studies (
By 1981, the number of mimosoid genera had risen to 62 in Advances in Legume Systematics Part 1 (
Across the non-mimosoid Caesalpinioideae generic delimitation has also seen many changes. The most complex problems have been, without doubt, in the Caesalpinia Group and, especially, the genus Caesalpinia L. s.l. (
Since
Despite this rapid on-going progress to align genera with clades in recent years, generic delimitation across Caesalpinioideae and, especially, the mimosoid clade, remains in a state of considerable flux and there is evidence to suggest that several more genera are non-monophyletic: Prosopis (
Another issue has been delimitation of the mimosoid clade with on-going uncertainties surrounding the inclusion or not of certain genera (
Several other issues have hindered a more complete understanding of the phylogeny and tribal / generic classification of subfamily Caesalpinioideae. First, the legacy of the traditional subfamily classification meant that taxon sampling in previous phylogenetic studies focused primarily on either old sense Caesalpinioideae (i.e. the grade subtending mimosoids (the ‘Caesalpinieae grade’ of
More robust foundations to overcome these difficulties were established by
This new phylogeny provides the basis for testing the monophyly of genera (the main focus of this paper and of this Special Issue Advances in Legume Systematics (ALS) 14, Part 1), establishing a new higher-level classification of the subfamily (the focus of ALS 14, Part 2) and for downstream analyses of biogeography, trait evolution and diversification (
To test generic monophyly as thoroughly as possible, we sampled taxa to encompass known or suspected cases of generic non-monophyly, as well as sets of representative species spanning the root nodes of larger genera in Caesalpinioideae (Suppl. material
We sequenced a set of 997 nuclear genes specifically selected for phylogenomic analyses of the mimosoid clade (
Phylogeny of Caesalpinioideae, part 1 (continued in Figs
To explore evolution of morphological traits that have been important for generic delimitation, we scored variation in armature, aspects of floral heteromorphy and mode of fruit dehiscence and mapped their distribution across the Caesalpinioideae phylogeny. Our goal was to highlight how an over-reliance on broadly-defined character complexes or functional traits may have misled classification in the past, rather than to perform detailed reconstructions of character evolution through time or to thoroughly assess the homology of various character states.
The three character complexes and their states were defined as follows:
Data were assembled from taxonomic monographs, revisions and floras. Character evolution was simulated across the phylogeny using the ‘make.simmap’ function in the phytools (
A tree file of the ASTRAL phylogeny based on the single-copy genes (depicted in Figs
For full results of the sequencing, orthology assembly and phylogenetic inference, see
Hybrid capture and sequencing yielded a large phylogenomic dataset with little missing data: the concatenated nucleotide alignment of the 821 single-copy nuclear genes (a subset of all 997 genes, see below) contains 944,871 sites, 824,713 alignment patterns (i.e. an indication of the phylogenetic informativeness of the alignment, determined by RAxML) and only 11.88% gaps. The ten nuclear species trees that were inferred using different phylogenetic methods are well-supported in terms of gene tree congruence measures (Figs
The plastid phylogeny (Suppl. material
Hereafter the ASTRAL phylogeny based on the subset of 821 single-copy nuclear gene trees is used as the ‘reference’ Caesalpinioideae backbone phylogeny (Figs
The resultant ASTRAL phylogeny is, in general, robustly supported across the majority of nodes using measures of gene tree support and conflict (Figs
All the informally named clades of
Twenty-two genera were recovered as non-monophyletic or were nested within another genus and, therefore, likely require generic re-delimitation (Figs
Appendix
Armature, types of inflorescence heteromorphy and pod dehiscence type each show high levels of homoplasy (Figs
The new Caesalpinioideae phylogeny (Figs
Given the extensive re-arrangements of genera in Caesalpinioideae over the last two decades, the question arises why such a significant fraction of genera is still non-monophyletic in these new phylogenomic analyses. We identify two main reasons for this. First, extensive morphological homoplasy has misled generic delimitation and second, lack of pantropical taxonomic synthesis and phylogenetic sampling have resulted in failure to identify clades that span the Old World and New World or, conversely, amphi-Atlantic genera that are non-monophyletic, i.e. potential trans-continental connections and disconnects.
Evolution of fruit dehiscence types across the mimosoid clade. Character states were defined as: indehiscent; inertly dehiscent along one or both sutures; explosively dehiscent, whereby the woody valves twist and split along both sutures along whole length of pod simultaneously; elastically dehiscent from the apex, the valves recurving, but not laterally twisting; craspedium, i.e. fruits breaking up into free-falling one-seeded articles leaving a persistent replum or whole valve breaking away intact from replum (valvately dehiscent); lomentiform fruit, i.e. the valves readily cracking between the seeds into one-seeded articles, taken here to include crypto-lomentiform fruits. Branch lengths are not informative in this figure. Photos a–e elastically dehiscent a Acacia argyraea Tindale b Calliandra prostrata Benth. c Calliandropsis nervosa (Britton & Rose) H.M. Hern. & P. Guinet d Alantsilodendron mahafalense (R. Vig.) Villiers e Zapoteca portoricensis (Jacq.) H.M. Hern f–h craspedium f Entada polystachya (L.) DC. g Lysiloma tergeminum Benth. h Mimosa montana var. sandemanii Barneby i–l lomentiform i Albizia moniliformis (DC.) F. Muell. j Albizia subdimidiata (Splitg.) Barneby & J.W. Grimes k Albizia pistaciifolia (Willd.) Barneby & J.W. Grimes l Prosopidastrum globosum (Gillies ex Hook. & Arn.) Burkart. Photos a Bruce Maslin b, c, e–h Colin Hughes d http://clubbotatoliara.e-monsite.com/pages/posters-films-rapports/photos.html i Garry Sankowsky http://www.rainforestmagic.com.au j Marcelo Simon k Xavier Cornejo l https://www.floramendocina.com.ar.
First, and most importantly, the likely extent of homoplasy of morphology and functional traits across Caesalpinioideae is only now starting to be revealed using this new phylogeny (Figs
Evolution of types of floral heteromorphy across the mimosoid clade. Character states were defined as: homomorphic, i.e. with no conspicuous modification or variation amongst flowers within an inflorescence (here we include inflorescences that can comprise proportions of male and bisexual flowers, but no other more conspicuous variation); heteromorphic 1 = basal flowers of the inflorescence with showy staminodia; heteromorphic 2 = flowers dimorphic within an inflorescence, the central flower (or flowers) enlarged/sessile cf. the peripheral (sometimes pedicellate) flowers. Branch lengths are not informative in this figure. Photos a–h heteromorphic 1 a Neptunia plena (L.) Benth. b Dichrostachys cinerea (L.) Wight & Arn. c Dichrostachys myriophylla Baker d Gagnebina pterocarpa (Lam.) Baill. e Dichrostachys bernieriana Baill. f Dichrostachys akataensis Villiers g Parkia bahiae H.C. Hopkins h Parkia nitida Miq. i–l heteromorphic 2 i Pseudosamanea guachapele (Kunth) Harms j Albizia obliquifoliolata De Wild. k Hydrochorea corymbosa (Rich.) Barneby & J.W. Grimes l Albizia grandibracteata Taub. Photos a, b, g, i Colin Hughes c, k, l Erik Koenen d Melissa Luckow e, f Dave Du Puy h Giacomo Sellan https://identify.plantnet.org/the-plant-list/observations/1012799991 j Jan Wieringa.
Fruits are highly diverse across Caesalpinioideae reflecting adaptations for hydrochory, anemochory, endozoochory, ornithochory, and myrmecochory, as well as several forms of mechanical seed dispersal via explosively, elastically and inertly dehiscent fruits. Here, we show that fruit dehiscence type shows extensive homoplasy across the mimosoid clade, with repeated evolution of, for example, pods elastically dehiscent from the apex, craspedia and lomentiform fruits (Fig.
Evolution of different types of armature across Caesalpinioideae. Character states were defined as: unarmed; nodal or internodal prickles on stem; stipular spines; nodal axillary thorns including modified inflorescence axes of Chloroleucon; spinescent shoots. Branch lengths are not informative in this figure. Photos a and b axillary thorns a Parkinsonia andicola (Griseb.) Varjão & Mansano b Prosopis juliflora (Sw.) DC. c, d, h internodal prickles c Senegalia tamarindifolia (L.) Britton & Rose d Mimosa ophthalmocentra Mart. ex Benth. e spinescent shoots, Prosopis kuntzei Harms f and g stipular spines f Prosopis ferox Griseb. g Vachellia cornigera (L.) Seigler & Ebinger h Cylicodiscus gabunensis Harms. All photos Colin Hughes, except h William Hawthorne.
For example, as pointed out by
There are several other examples of classifications and especially genera being misled by parallel evolution of fruit types. For example, the polyphyly of the genus Enterolobium Mart. (
Of course, homoplasy per se in no way negates the value and importance of morphology for classification, but instead prompts re-evaluation of homology and the utility of specific morphological characters via reciprocal illumination with new molecular phylogenetic evidence. For example, armature is also homoplasious across Caesalpinioideae with repeated evolution of stipular spines, nodal and internodal prickles, axillary thorns and spinescent shoots (Fig.
Detailed phylogenetic reconstructions for other characters, based on more rigorous and detailed anatomical assessment of homology, will undoubtedly be worthwhile, but it is already clear that the three traits mapped here (Figs
Pre-eminence of certain morphological characters over others in classification of a group and the prevalence of ‘organogenera’ (sensu
A second important reason for the extensive generic non-monophyly is the lack of pantropical synthesis and integration that has been the hallmark of much taxonomic work on Caesalpinioideae up to now and the lack of adequate pantropical sampling of taxa in previous phylogenies. In this light, it is notable that two of the most productive and influential mimosoid taxonomists of the twentieth century, both of whom significantly reshaped the generic classification – Rupert Barneby and Ivan Nielsen – worked largely independently in different geographical areas, especially on genera of the former tribe Ingeae. While both were very much aware of the wider pantropical dimensions and elements of their groups, Barneby focused primarily on New World mimosoids (e.g.
Our new phylogeny with its near-complete generic sampling reveals several instances of Old World – New World connections and disconnects that have important implications for generic delimitation and which were not fully apparent before. First, the amphi-Atlantic genus Prosopis is shown to be non-monophyletic (Figs
We recover both Chidlowia and Sympetalandra as firmly nested in the mimosoid clade (Fig.
The mimosoid clade, i.e. the subfamily formerly known as the mimosoideae, was traditionally diagnosed by petals valvate, as opposed to imbricate, in bud. Valvate petal aestivation is mostly a reflection of whether or not the flowers are actinomorphic vs. zygomorphic, i.e. as the flowers become radially symmetrical the petals become valvate in bud. Across the non-mimosoid grade of Caesalpinioideae subtending the mimosoid clade, taxa with imbricate and valvate aestivation are phylogenetically intermingled. Although the vast majority of mimosoids do, indeed, have valvate petal aestivation, three exceptions: Chidlowia (as indicated above), alongside Mimozyganthus Burkart and Parkia R.Br., both of which are deeply nested within the mimosoid clade, show imbricate petal aestivation, providing further evidence of the homoplasy of this character. Further work to characterise petal aestivation across all relevant genera of Caesalpinioideae is needed, but it is clear that valvate aestivation does not provide a unique diagnostic synapomorphy for the mimosoid clade.
All other aspects of higher-level relationships are discussed in ALS14 Part 2.
Once purely the domain of morphological analyses (e.g.
The phylogeny of Caesalpinioideae presented here (Figs
Finally, despite providing the main (usually sole) source of information for classification for centuries, morphology was rapidly eclipsed as a source of data for phylogeny reconstruction with the advent of molecular data (e.g.
Here, we present a series of phylogenomic analyses including detailed assessment of gene tree conflict and support that suggest that about one quarter of mimosoid genera are non-monophyletic (Figs
However, despite including 420 taxa in the current analyses, it is clear that additional taxon sampling will be needed to fully resolve all the possible non-monophyly issues within Caesalpinioideae. Several priorities for future research are apparent. First, denser taxon sampling across Senegalia and allies is needed to address the unusual dilemmas posed by extreme lack of resolution and cytonuclear discordance surrounding delimitation of the genera across the paraphyletic grade comprising Senegalia, Pseudosenegalia, Parasenegalia and Mariosousa (Fig.
Furthermore, although there is no evidence that any large clades in Caesalpinioideae are subtended by whole genome duplication (WGD) events (
Finally, our preliminary assessments of homoplasy (Figs
The authors thank B. Adhikari, D. Lorence, B. Marazzi, É. de Souza, the G, K, JRAU, L, MEL, NY, FHO, P, RB, WAG and Z Herbaria, the Millennium Seed Bank, Kew, the South African National Botanical Institute, the Direction de Environment, New Caledonia and the National Tropical Botanical Garden, Hawaii, U.S.A. for provision of leaf or DNA samples; P. Ribeiro, É. de Souza, M. Morim, M. Simon and F. Bonadeu in Brazil and staff of the Kew Madagascar Conservation Centre and Botanical and Zoological Garden of Tsimbazaza, Madagascar for fieldwork support; the national regulatory authorities of Brazil for access to DNA of Brazilian plants (authorised through SISGEN n° A464716), and Madagascar (research permit 006/14/MEF/SG/DGF/DCB.SAP/SCB); U. Grossniklaus, V. Gagliardini, Dept Plant & Microbial Biology, Univ. Zurich for use of their TapeStation; the S3IT, Univ. Zurich for use of the ScienceCloud computational infrastructure; and the Club Botanique de Toliara, Madagascar, the Flora Mendocina project, Argentina, B. Maslin, G. Sankowsky, M. Simon, and X. Cornejo for permission to use photos included in Figs
Generic non-monophyly in Caesalpinioideae – towards a new generic system for the subfamily
Caesalpinia
Divergent circumscriptions of the genus Caesalpinia L. were largely resolved by
Dimorphandra
In line with previous studies (
Xylia and Calpocalyx
The non-monophyly of Xylia with Calpocalyx nested within it was documented using matK sequences (
Entada and Elephantorrhiza
A close relationship between Entada Adans. and Elephantorrhiza Benth. has long been suggested in all molecular phylogenies that sampled these genera (e.g.
Prosopis
One of the most striking and robustly supported examples of generic non-monophyly in our analyses is Prosopis s.l. whose species are placed in four separate lineages (Figs
Desmanthus
The non-monophyly of Desmanthus with the monospecific Hawaiian endemic Kanaloa Lorence & K.R. Wood nested within it (Fig.
Dichrostachys , Gagnebina and Alantsilodendron
Dichrostachys (DC.) Wight & Arn. and Alantsilodendron Villiers are both recovered as non-monophyletic in our sparsely sampled analysis (Fig.
Stryphnodendron and Pseudopiptadenia
Our analyses support the monophyly of the Stryphnodendron clade sensu
The remaining genera in the Stryphnodendron and Mimosa clades are all monophyletic (Fig.
Senegalia and allied genera
The striking cytonuclear discordance whereby Senegalia Raf. appears as non-monophyletic in the analyses of nuclear gene sequences, but as monophyletic in the analyses of plastomes, was first revealed by
Calliandra
Following reduction of
Pithecellobium and allies
While the Pithecellobium alliance is the only one of the informal alliances of
The Archidendron clade
The genera and lineages of the large Archidendron clade comprising Acacia Mill., Archidendron F. Muell. and six smaller genera (Fig.
Our results weakly support Paraserianthes lophantha as sister to Acacia (Fig.
Albizia
At the start of this study, the genus Albizia was dubbed the last pantropical so-called ‘dustbin’ genus pending resolution (
Abarema , Hydrochorea and Balizia
The recent re-circumscription of Abarema Pittier to include just two species and transfer of the remaining species to the re-instated Punjuba Britton & Rose and Jupunba Britton & Rose (
Leucochloron
Zygia , Macrosamanea and Inga
Alongside Archidendron, the large Neotropical, mainly rainforest genus Zygia remains one of the least well-documented genera of mimosoids, with many species known from incomplete material (
Table S1
Data type: excel file.
Explanation note: Samples included in this study.
Table S2
Data type: excel file.
Explanation note: Trait data used for character evolution analyses.
Figure S1
Data type: Pdf file.
Explanation note: Chloroplast phylogeny of Caesalpinioideae. Only bootstrap support values lower than 100% are shown.
Supplementary tree file
Data type: Tree file (Newick format).
Explanation note: Tree file of the ASTRAL phylogeny based on the single-copy genes (depicted in Figs