Molecular phylogeny and character-mapping support the synonymy of Cordobia and Gallardoa in Mionandra (Malpighiaceae)

Background and aims – Cordobia , Gallardoa , Mionandra , and Peixotoa (Stigmaphylloid clade, Malpighiaceae) are four small, closely related genera comprising shrubs or lianas endemic to South American savannas, dry forests, and temperate steppes. Their generic limits have significantly changed in the last century, and past molecular phylogenetic studies of Malpighiaceae have not tested the morphological characters of this group to identify synapomorphies supporting these clades/genera. Material and methods – We sampled the monospecific Cordobia and Gallardoa , one species of Mionandra (out of 2 spp.), nine species of Peixotoa (out of 29 spp.), and a single species of Camarea and Janusia as outgroups. Bayesian and Maximum Likelihood analyses were carried out for this clade based on five molecular markers (i.e. ETS, ITS, PHYC, matK , and ndhF ). A set of 16 macromorphological characters was scored and coded for identifying synapomorphies under the Maximum Likelihood criteria. Key results – Our molecular phylogeny recovered Peixotoa as monophyletic and sister to the clade comprising Cordobia + Gallardoa + Mionandra , strongly corroborating previous phylogenetic studies of Malpighiaceae. The character-mapping analyses recovered two synapomorphies supporting the Cordobia + Gallardoa + Mionandra + Peixotoa clade, six supporting Mionandra s.l. (i.e. Cordobia + Gallardoa + Mionandra ), and five supporting Peixotoa . Cordobia and Gallardoa are proposed as synonyms of Mionandra , alongside the necessary combinations, typifications, and identification keys. Conclusions – Morphological characters related to the degree of connation of the stipules, leaf indumentum type, petiole length, inflorescence architecture, number of flowers per inflorescence, presence of a peduncle in the 1-flowered cincinni, sepal connation, posture and texture, petal width and margin integrity, staminode presence, shape and size, and the shape of the apex of styles were key in circumscribing these lineages. Mionandra s.l. is proposed and characterised, including a new combination, an identification key to distinguish its species, a distribution map, and taxonomy notes.


INTRODUCTION
Malpighiaceae has undergone unparalleled changes in its traditional classification in the last two decades due to the publication of several molecular phylogenetic studies (Cameron et al. 2001;Davis et al. 2001;Davis and Anderson 2010).Several new lineages were resolved, bringing to light taxonomic problems regarding the monophyly of its subfamilies (e.g.Byrsonimoideae W.R. Anderson, Banisterioideae A.Juss., and Malpighioideae A.Juss.), tribes (e.g.only Gaudichaudieae Horan.was recovered as monophyletic), and genera (e.g.Banisteriopsis C.B.Rob., Mascagnia (Bertero ex DC.) Bertero, Stigmaphyllon A.Juss., and Tetrapterys Cav.) (Cameron et al. 2001;Davis et al. 2001;Davis and Anderson 2010;Almeida et al. 2017Almeida et al. , 2023;;Almeida andvan den Berg 2020, 2021).Subsequently, numerous authors have gradually proposed new genera and combinations to accommodate these newly identified relationships (Anderson 2006(Anderson , 2011;;Anderson et al. 2006;Almeida and van den Berg 2021).Although morphological characters were used to reconstruct the latest generic phylogeny for Malpighiaceae (Davis and Anderson 2010), no morphological characters were ever recovered and/or discussed for its newly resolved relationships (Almeida andvan den Berg 2021, 2022).
Their taxonomic history is quite convoluted, with most species of Cordobia, Gallardoa, and Mionandra having been treated under the latter.Mionandra was first described by Grisebach (1874) based on its bifid stipules (i.e.connate only at the base) and the presence of 5 fertile stamens alternating with 5 staminodes.This author only accepted two species (M.argentea Griseb.and M. camareoides Griseb.) as part of his new genus (Grisebach 1874).Kuntze (1898) described a new species of Peixotoa, P. cordobensis Kuntze, based on a specimen he collected from Córdoba, Argentina.It was readily synonymised by Niedenzu (1912) under the type specimen of his newly proposed genus Cordobia Nied., based on Grisebach's Mionandra argentea.Soon after, Chodat and Vischer (1917) added a new species to the recently monospecific Mionandra, M. paraguariensis Chodat, which was later combined as a variety of M. camareoides by Niedenzu (1928) in his revisionary study of Malpighiaceae.In the meantime, Hicken (1916) described another new genus related to Mionandra and Cordobia, the monotypic Gallardoa Hicken, comprising only G. fischeri Hicken. Finally, Niedenzu (1928) transferred G. fischeri to Cordobia, synonymising Gallardoa under the latter.Since then, contemporary taxonomists, such as William R. Anderson, have chosen to individually accept all three related genera, a decision followed by all major checklists and online databases to date (e.g.POWO 2023).
In this study, we reconstructed a molecular phylogeny of the Peixotoid clade based on three nuclear (ETS, ITS, and PHYC) and two plastid (matK and ndhF) genes to answer the questions: 1. Are Cordobia, Gallardoa, and Mionandra supported by morphological synapomorphies?2. If not, would Mionandra s.l.(including Cordobia and Gallardoa) be supported by any morphological synapomorphies?

Molecular analyses
We sampled 14 species in this study representing 12 species from the Peixotoid clade (out of 31 spp.), including the type species of all four genera and a single species of Camarea and Janusia, respectively, as outgroups, representing tribe Gaudichaudieae as their sister group (Supplementary material 1).For DNA extraction, we used leaf fragments from herbarium specimens deposited at Universidade Estadual de Feira de Santana (HUEFS).Genomic DNA was extracted using the CTAB 2× protocol, modified from Doyle and Doyle (1987).Protocols to amplify and sequence the ETS and ITS regions followed Almeida et al. (2017).PCR products were purified using PEG 11% (polyethylene glycol) and were sequenced directly with the same primers used for the PCR amplification.Sequence electropherograms were produced in an automatic sequencer (ABI 3130XL Genetic Analyzer) using Big Dye Terminator 3.1 (Applied Biosystems).Additional sequences from a single nuclear (PHYC) and two plastid (matK and ndhF) regions were retrieved from GenBank (Supplementary material 1).Sequences were edited using Geneious v.4.8.4 (Kearse et al. 2012) and aligned using Muscle v.1.0(Edgar 2004), with subsequent adjustments in the preliminary matrices made manually by eye.The complete data matrices are available on Figshare (https:// doi.org/10.6084/m9.figshare.23823105).
All trees were rooted in tribe Gaudichaudieae (Camarea + Janusia), the sister group of the Peixotoid clade, according to Davis and Anderson (2010).A combined analysis of plastid + nuclear regions was carried out using Bayesian Inference and Maximum Likelihood.We selected the model using hierarchical likelihood ratio tests (HLRT) with jModelTest2 (Darriba et al. 2012).Both model-based methods were conducted with a mixed model (GTR+G+I) and unlinked parameters, using MrBayes v.3.1.2(Ronquist and Huelsenbeck 2003) and RAxML v.8 (Stamatakis 2014) implemented in RAxMLGUI2 (Edler et al. 2021).For the Bayesian inference, the Markov Chain Monte Carlo (MCMC) was run using two simultaneous independent runs with four chains each (one cold and three heated), saving one tree every 1,000 generations for a total of ten million generations.We excluded 20% of retained trees as 'burn in' , and checked for a stationary phase of Likelihood, checking for ESS values higher than 200 for all parameters with Tracer v.1.7 (Rambaut et al. 2018).The clades' posterior probabilities (PP) were based on the majority rule consensus, using the stored trees, and calculated with MrBayes v.3.1.2.ML analyses were performed with 10 independent replicates, and default settings and support values were estimated using parametric bootstrapping with 500 replicates.Support values are presented on the branches, with bootstrap values shown above and posterior probabilities shown below the branches.

Phylogenetic analyses
The nuclear dataset represented 2,366 characters of the dataset, the plastid dataset represented 1,729 characters, and the combined plastid + nuclear dataset included 4,095 analysed characters.Topologies produced by BI and ML analyses, based on the individual nuclear and plastid datasets, did not exhibit incongruences among the topologies produced, so we performed a combined analysis of plastid + nuclear datasets (Fig. 1).The BI and ML analyses recovered a partially resolved tree with seven well-supported clades (> PP 0.95 / BS 65) at generic levels and six poorly-supported clades (< PP 0.95 / BS 65) within Peixotoa (Fig. 1).The Peixotoid clade was recovered as monophyletic and highly supported by both BI and ML analyses (1/100) comprising two major clades: the first highly supported represented by the specimens of Cordobia + Gallardoa + Mionandra (PP 1.0 / BS 100) and a second clade represented by the highly supported Peixotoa (PP 1.0 / BS 100) (Fig. 1).Combined plastid + nuclear datasets provided higher support for more clades than the results based on single plastid or nuclear datasets (Fig. 1A).

Character mapping
We recovered three synapomorphies (stipules connate at the base or up to the middle, 1-flowered cincinni lacking peduncles [i.e.sessile], and sepals revolute at apex) for the Peixotoid clade alongside the outgroups representing tribe Gaudichaudieae (Fig. 1B).The three synapomorphies recovered by us for tribe Gaudichaudieae (thyrsi, cincinnus peduncle present, and petals widely elliptic) are interpreted as sampling artefacts caused by the limited outgroup sampling of our study.These characters most likely represent plesiomorphic states in the family, but a study focusing on Malpighiaceae as a whole is necessary to address this issue.
The first clade recovered within the Peixotoid clade included the genera Cordobia + Gallardoa + Mionandra supported by six synapomorphies (sepals free at base, chartaceous, and entirely revolute, antherodes filiform and minute, apex of styles truncate to expanded) and a single homoplasy (1-flowered cincinni) (Fig. 1B).The second clade recovered within the Peixotoid clade comprised only species of Peixotoa, being supported by five synapomorphies (stipules completely connate, secondary arrangement of inflorescences [i.e.synflorescences] of umbels arranged in thyrsi, petals orbicular, petal margins dentate, and filaments of staminodes as long as fertile stamens) and two homoplasies (long petioles and leaves glabrescent) (Fig. 1B).

DISCUSSION
The Peixotoid clade was recovered as highly supported (PP 1.0 / BS 100) in our tree (Fig. 1A), corroborating previous phylogenetic studies of Malpighiaceae (Davis and Anderson 2010;Davis et al. 2014;Willis et al. 2014;Cai et al. 2016).This clade was also recovered with three morphological synapomorphies: partially to completely connate stipules, sessile 1-flowered cincinni, and sepals completely revolute.Partially connate stipules are not exclusive to the Peixotoid clade, with several genera from the distantly related Byrsonimoid clade also showing this character (Anderson 1981).In contrast, sessile 1-flowered cincinni are quite rare in Malpighiaceae, additionally found only in Diplopterys and Coleostachys A.Juss.(Almeida et al. 2020).However, the flowers in Coleostachys are completely sessile, lacking both peduncle and pedicel (Almeida and Hall 2016).Finally, sepals revolute at the apex are very common in Malpighiaceae (Almeida et al. (including Cordobia and Gallardoa) is well-circumscribed with six synapomorphies and a single homoplasy (Fig. 1B), being easily differentiated from Peixotoa by the sepals free at base, chartaceous and entirely revolute, antherodes filiform and minute, and apex of styles truncate to expanded.Since several morphological synapomorphies supporting Mionandra s.l. were recovered in our analysis, we have chosen to follow Grisebach's (1874) broader concept of Mionandra (including Cordobia) but also synonymising Gallardoa under it (see taxonomic treatment).For more information, see the identification key in the taxonomy section, differentiating the genera of the Peixotoid clade accepted in this study.
Even though Anderson (1982) did not accept any infrageneric ranks in her monograph of Peixotoa, Niedenzu (1928) accepted two sections for this genus in his taxonomic revision for Malpighiaceae: P. sect.Balantiopsis Nied.and P. sect.Perinopsis Nied.The first was characterised by its leaves and stems pubescent and anthers with connectives 1-lobed (Niedenzu 1928).In contrast, the latter was characterised by glabrous leaves and stems, and anthers with connectives 2-lobed (Niedenzu 1928).Our analysis evidenced that the sections of Peixotoa proposed by Niedenzu (1928) are not monophyletic (Fig. 1B, P. glabra, P. hatschbachii, and P. hispidula in red represent P. sect.Perinopsis, while the remaining species in black represent P. sect.Balantiopsis) and further morphological studies must be carried out within this genus to shed light on any infrageneric classification to be proposed.

Peixotoid clade
Diagnosis.Distinguished from the remaining genera of the Stigmaphylloid clade by its stipules connate, cincinni sessile and 1-flowered, and sepals completely distally revolute or involute along margins.Notes.The Peixotoid clade currently comprises two monophyletic and morphologically well-circumscribed genera divided into a total of 32 species.Peixotoa is the largest genus of the two, currently with 29 species (Anderson 1982(Anderson , 2001)), with Mionandra comprising only four species (this study).As aforementioned, both genera share some peculiar morphological characters but can be easily differentiated based on both vegetative and reproductive characters (see Table 1 and the key).

1.
Stipules connate at base or up to the middle (i.e.bifid); umbels 1-flowered, bract and bracteoles absent; sepals free, completely revolute or involute along margins; antherodes filiform, minute; styles apex truncate to slightly expanded....  from Argentina, southern Bolivia, and western Paraguay in South America (Fig. 2).Notes.A comprehensive treatment for the genera comprising Mionandra s.l.(including Cordobia and Gallardoa) and three of their four species are presented by Aliscioni and Torretta (2017) within the Flora of Argentina project, with M. paraguariensis (which does not occur in Argentina) not included in the treatment.Thus, we provide an updated key to all species of Mionandra s.l., plus comments on the recognition of M. paraguariensis.

Key to the species of Mionandra
1.
Leaves 2-glandular near base; sepals involute along margins; stamens dimorphic (the latero-posterior ones with shorter, stout and curved filaments, the posterior ones and the anterior filaments thin and straight but the anterior shorter in length); mericarps rugose, dorsal wing well-developed .Notes.After revisiting the original description of Chodat and Vischer (1917) and analysing the type specimen, we disagree with Niedenzu's (1928) treatment of M. paraguariensis as a variety of M. camareoides.Aside from the difference in leaf indumentum, M. paraguariensis differs from M. camareoides due to its larger flowers, differently shaped petals, stamens with pubescent filaments, and 5 staminodes the same length as the filaments.Thus, we unambiguously recognise it as a distinct species.
Mionandra fischeri (Hicken)  Type species.Peixotoa glabra A.Juss.Diagnosis.Distinguished from Mionandra s.l. by its stipules completely connate, leaves long-petiolate, never hirsute-sericeous; umbels 4-flowered; sepals connate at base, coriaceous, revolute only at apex; petals orbicular, margin dentate; fertile stamens 5, staminodes 5, antherodes globose, conspicuous; style apex capitate; mericarps with wings well-developed, dorsal wing dominant.Distribution, habitat, and ecology.Peixotoa comprises 29 species occurring in dry forests, rainforests, and savannas in Brazil, eastern Bolivia, and eastern Paraguay in South America (Fig. 2).Notes.Peixotoa has a contemporary taxonomic revision available for 28 of its species (Anderson 1982) and the taxonomic treatment for a new species endemic to Paraguay (Anderson 2001).Nonetheless, misguided morphological interpretations drawn by this author from the inflorescence architecture have made the identification keys provided in these studies challenging to use by the general public and even for Malpighiaceae specialists (pers.obs.).Since C. Anderson published more than twothirds of the species diversity of Peixotoa, only species with conspicuous morphological traits (e.g.glabrous leaves -Peixotoa glabra A.Juss.) or specific geographical distributions (e.g.Peixotoa catarinensis C.E. Anderson and P. hispidula A.Juss.) are correctly identified in Brazilian herbaria (pers.obs.).For a preliminary revisionary study of Peixotoa in Brazil, see Almeida et al. (2020).A revised monograph for this genus is urgently needed to enable the correct application of names in Peixotoa (pers.obs.).

CONCLUSIONS
Studies mapping the evolution of macro-morphological characters in molecular phylogenies are the steppingstone to challenge traditional classifications and propose new predictive systems in Malpighiaceae, reflecting the evolutionary history of their taxa (Almeida and van den Berg 2021).As a continuation of the studies of Cameron et al. (2001), Davis et al. (2001), and Almeida and van den Berg (2021), we revised the circumscription within the genera of the Peixotoid clade based on molecular and morphological data to finally ensure the taxonomic stability of generic circumscriptions within this clade.Cordobia and Gallardoa were synonymised under Mionandra, and the necessary combination was made alongside typifications and identification keys.

Figure 1 .
Figure 1.Phylogeny and character-mapping of the Peixotoid clade.A. Phylogram recovered from the Maximum Likelihood analysis showing bootstrap values above the branches and posterior probability values below the branches.B. Consensus tree showing the character-mapping summarisation resulting from the Mesquite Maximum Likelihood analysis and WinClada visualisation.Red circles represent synapomorphies, and white circles represent homologies.The numbers above the circles represent the character numbers, and those below the circles represent the character states provided in Supplementary materials 2 and 3. Terminals of Peixotoa in red represent P. sect.Perinopsis Nied.and those in black represent P. sect.Balantiopsis Nied.

Figure 3 .
Figure 3. Mionandra argentea.A. Shrubby habit.B. Detail of a floral shoot showing the partially connate stipules in the node and the sessile 1-flowered cincinnus with a long-pedicellate floral bud.C. Flower in frontal view.D. Flower in lateral view showing the sepals involute along margins.Photographs A-B by Étienne Lacroix-Carignan; C-D by Andrea Cocucci.

Figure 4 .
Figure 4. Mionandra camareoides.A. Shrubby habit.B. Detail of floral branches.C. Detail of flowers.D. Flower in frontal view.Photographs A-C by Eduardo Alfredo; D by William Anderson.

Figure 5 .
Figure 5. Mionandra fischeri.A. Shrubby habit.B. Detail of a flower in frontal view.C. Detail of a flower in lateral view.D. Detail of floral branches.Photographs A-B by Anival Prina; C by Michelle Delaloye; D by Ivan Federico Ebrecht.

Figure 6 .
Figure 6.Peixotoa catarinensis.A. Detail of a sterile branch showing the connate stipules.B. Detail of the base of a leaf in abaxial view.C. Detail of a flowering branch.D. Floral bud in lateral view.E. Flower in frontal view.F. Winged mericarps in lateral view.Photographs by Marco Pellegrini.