New subtribal and generic limits in the tribe Athroismeae (Asteraceae) and further disintegration of the subtribe Madagasterinae of the tribe Astereae

Abstract: The Astereae subtribe Madagasterinae (Asteraceae) sensu Nesom encompassing four Malagasy genera (Apodocephala, Madagaster, Rochonia and Vernoniopsis) is polyphyletic, as Apodocephala is a member of the Malagasy subtribe Lowryanthinae of the tribe Athroismeae. While Madagaster was shown to belong to Astereae, the phylogenetic positions of Rochonia and Vernoniopsis (now Jalantzia) in this tribe remained to be tested with molecular data. Lowryanthinae presently contains Apodocephala with nine species and the monospecific Lowryanthus. The monophyly of Apodocephala remained to be assessed using a comprehensive sampling. The aims were to: (1) assess the phylogenetic placements of Rochonia and Jalantzia (Vernoniopsis) and test the monophyly of Jalantzia; (2) assess phylogenetic relationships within Lowryanthinae; and (3) re-assess the subtribal and generic limits within the tribe Athroismeae. New phylogenetic analyses based on plastid sequence data confirmed the position of Rochonia in Astereae. Jalantzia (Vernoniopsis) was resolved as sister to the subtribe Lowryanthinae and therefore transferred to Athroismeae. A new subtribe, Jalantziinae, is described to accommodate Jalantzia. Apodocephala is paraphyletic with respect to Lowryanthus, which is formally merged with Apodocephala. A description of the emended subtribe Madagasterinae containing Madagaster and Rochonia is presented. Citation: Bengtson A. & Razafimandimbison S. G. 2024: New subtribal and generic limits in the tribe Athroismeae (Asteraceae) and further disintegration of the subtribe Madagasterinae of the tribe Astereae. – Willdenowia 54: 103–116. Version of record first published online on 25 April 2024 ahead of inclusion in April 2024 issue.


Introduction
Madagascar is known for its biodiversity and high level of endemism.The daisy family, Asteraceae, with c. 555 species on Madagascar, 86.9 % of which are endemic, is one of the most species-rich flowering plant families (Madagascar Catalogue 2022).The species represent 103 different genera, but only 20 of these are endemic to Madagascar.Five of the endemic genera are formally grouped in two subtribes: the Athroismeae subtribe Lowryanthinae (Bengtson & al. 2017(Bengtson & al. , 2021) ) and the Astereae subtribe Madagasterinae (Nesom 2020).
Athroismeae, as defined by Bengtson & al. (2017), is a small, morphologically diverse tribe, whose circumscription has gradually changed over the years (Panero & Funk 2002;Wagstaff & Breitwieser 2002;Panero 2005;Anderberg 2009;Pruski 2014;Bengtson & al. 2017Bengtson & al. , 2021)).Bengtson & al. (2017) presented the first molecular phylogenetic study of the tribe, which resulted in an amended circumscription.The tribe currently consists of ten genera classified in four morphologically diverse sub-tribes (Anisopappinae, Athroisminae, Lowryanthinae and Symphyllocarpinae; Bengtson & al. 2017Bengtson & al. , 2021)).Lowryanthinae consist of two genera: Lowryanthus Pruski and Apodocephala Baker.Lowryanthus was described by Pruski (2014), who postulated it to be closely related to the Blepharispermum group (= subtribe Athroisminae).Bengtson & al. (2017) later confirmed its position in the tribe Athroismeae, where it was resolved as sister to subtribe Athroisminae.Lowryanthus was, however, placed in its own subtribe, Lowryanthinae, mainly due to differences in synflorescences and capitula.Lowryanthus is a monospecific genus endemic to Madagascar and consists of shrubs or small trees with red synflorescences with coral-red discoid capitula and florets with reddish to pink corollas (Fig. 1B).A recent phylogenetic study by Bengtson & al. (2021) revealed the Malagasy endemic genus Apodocephala to be sister to Lowryanthus and therefore another member of subtribe Lowryanthinae.This study, however, included only two of the nine described Apodocephala species.The Apodocephala species most morphologically resembling Lowryanthus were not included and, therefore, the phylogenetic relationships within subtribe Lowryanthinae remained unclear.
Apodocephala was earlier placed in the tribe Astereae, where it has, based on morphology, been associated with the Malagasy genera Vernoniopsis Humbert, Rochonia DC. and Madagaster G. L. Nesom (Humbert 1960;Bremer 1994;Nesom 2020; Fig. 1A, C, D, E), later classified in Nesom's (2020) subtribe Madagasterinae.Apodocephala was, however, omitted from Astereae by Nesom & Robinson (2007) in their account of the tribe.Subtribe Madagasterinae are characterized by their arborescent habit, coriaceous and abaxially tomentose leaves and their caudate anthers.Bengtson & al. (2021) have shown Madagasterinae to be non-monophyletic, because Apodocephala belongs to the tribe Athroismeae.Apodocephala consists of nine species of shrubs or trees with discoid capitula and florets with white or whitish corollas (Fig. 1A).Molecular phylogenetic studies have confirmed the position of Madagaster within Astereae (Brouillet & al. 2009), but the phylogenetic placement of the Malagasy Rochonia and Vernoniopsis within Astereae and their relationships to Apodocephala remain untested by molecular analysis.Rochonia encompasses four species and has campanulate, radiate capitula with yellow ray florets (Fig. 1D).This genus is morphologically more similar to Madagaster.Vernoniopsis, however, resembles Apodocephala in having discoid 1-to few-flowered capitula with cylindric involucres and florets with whitish corollas.Vernoniopsis was originally described as monospecific with two subspecies differing in leaf and cypsela sizes (Humbert 1955), V. caudata (Drake) Humbert subsp.caudata and subsp.lokohensis Humbert.The latter subspecies was later raised to species level (Callmander & Phillipson 2011).The monophyly of Vernoniopsis has never been tested.
The aims of the study were to: (1) assess the phylogenetic placements of Rochonia and Vernoniopsis and test the monophyly of Vernoniopsis as defined by Callmander & Phillipson; (2) assess phylogenetic relationships within the subtribe Lowryanthinae; and (3) to re-assess the subtribal and generic limits within the tribe Athroismeae.

Phylogenetic analyses
Sequences were aligned using MUSCLE v.3.8.425 (Edgar 2004) as implemented in AliView v.1.24(Larsson 2014) and manually edited using BioEdit v.7.2.5 (Hall 1999).Two different datasets were compiled and analysed (Supplementary appendices S1, S2); we initially performed analyses of an ndhF matrix, including 139 accessions and a representing wide coverage of the Asteraceae tribes, in order to determine the tribal positions of Vernoniopsis and Rochonia within the family.A second dataset with a focus on the tribe Athroismeae consisting of ETS, ITS, ndhF and trnL-trnF sequence data from 60 accessions was then analysed.Boopis anthemoides Juss.(Calyceraceae) was used as outgroup in analyses of the Asteraceae ndhF matrix and Callilepis salicifolia Oliv. in analyses of the combined Athroismeae dataset, following Bengtson & al. (2017).All the analyses were conducted with Bayesian and parsimony methods.Prior to analyses of the combined dataset each region was analysed separately to check for incongruences by simply comparing the topologies.
Bayesian inference analyses were conducted using MrBayes v.3.2.2 (Ronquist & al. 2012) using the online XSEDE platform in the CIPRES Science Gateway (Miller & al. 2010).Nucleotide substitution models were set to GTR+I+G for the Asteraceae ndhF dataset, and to GTR+G for ETS and GTR+I+G for ITS and the plastid markers (ndhF, trnL-trnF) for the combined dataset, selected as best fit for the data using the Akaike information criterion (AIC) as implemented in jModeltest v. 2.1.10(Guindon & Gascuel 2003;Darriba & al. 2012).Analyses consisted of two independent runs, with eight chains each.The Markov Chain Monte Carlo (MCMC) was run for 75 million generations with a sampling frequency of 7500 for the ndhF matrix, and for 50 million generations with a sampling frequency of 5000 for the combined dataset.Convergence of Markov chains was examined using Tracer v.1.7.1 (Rambaut & al. 2018) as well as by checking the average standard deviation values of split frequencies.The first 25 % of the trees were excluded as a burn-in phase.Consensus trees were visualized using FigTree v.1.4.4 (http://tree.bio.ed.ac.uk/software /figtree/).Maximum parsimony analyses were conducted using PAUP v.4.0a (Swofford 2002), using the settings described in Bengtson & al. (2021).All analyses were run multiple times using the settings described above.

Results
The Asteraceae ndhF dataset consisted of 2292 aligned characters, 559 of which were parsimony informative, and the combined Athroismeae dataset of 4436 aligned characters, 773 of which were parsimony informative.

Taxon sampling
Herbarium specimens from MO, P and S (herbarium codes according to Thiers 2023+), as well as newly collected specimens from Madagascar were included in the study.Thirteen specimens of Apodocephala, representing all but one species (no specimens of A. coursii Humbert suitable for DNA-extraction could be located), both species of Vernoniopsis, and Rochonia cinerarioides DC. were included in the molecular study.The systematic positions of Vernoniopsis and Rochonia were analysed in a large dataset, including 139 taxa and representing a wide coverage of Asteraceae tribes.Molecular phylogenetic analyses of the Athroismeae, with a focus on the subtribe Lowryanthinae were then conducted, including 60 taxa and representatives of all known genera of the tribe.Four different loci were sequenced for the study, two from the nuclear genome (ETS, ITS) and two from the plastid genome (ndhF, trnL-trnF).A complete list of sampled taxa and voucher information is given in Appendix 1.

Discussion
Phylogenetic positions of Vernoniopsis and Rochonia Nesom (2020) placed the Malagasy genera Apodocephala, Vernoniopsis, Rochonia and Madagaster in his new subtribe, Madagasterinae of the tribe Astereae, based on their arborescent habit with coriaceous abaxially tomentose leaves, caudate anther thecae and isolated geographic distribution.Madagaster has been confirmed to be a member of the tribe Astereae (Brouillet & al. 2009); however, Apodocephala was recently shown to belong to the tribe Athroismeae and transferred there (Bengtson & al. 2021), implying the non-monophyly of Madagasterinae as defined by Nesom (2020).The monophyly of Madagasterinae is further rejected by our analyses, because Vernoniopsis was resolved sister to the Apodocephala-Lowryanthus clade (Fig. 2, 3).Vernoniopsis is therefore another addition to the Athroismeae (Fig. 2, 3).
The phylogenetic position of Rochonia (here represented by R. cinerarioides) within Astereae is strongly supported (Fig. 2), consistent with Nesom (2020).Rochonia consists of four species of shrubs or subshrubs with radiate capitula bearing ray florets with yellow corollas (Fig. 1D).The genus resembles Madagaster in morphology, and Humbert (1932) even described the taxa currently placed under Madagaster and Rochonia (then Aster L.) as mainly differing in the colour of the ray floret corollas (Madagaster have ray florets with white to bluish corollas).Nesom (1993) described the genus Madagaster for the white-rayed species, considered to be a lineage separate from, but close to, Rochonia.We agree that Madagaster and Rochonia are likely to be closely related based on their morphological similarities; however, whether they are sisters remains to be seen.In summary, only two out of the four genera of Nesom's (2020) subtribe Madagasterinae, Madagaster and Rochonia, remain.

Monophyly of Vernoniopsis (now Jalantzia)
Our analyses strongly support the monophyly of Vernoniopsis (here represented by its two species, V. caudata (Drake) Humbert and V. lokohensis (Humbert) Callm.& Phillipson; Callmander & Phillipson 2011) and its phylogenetic position in the tribe Athroismeae as delimited by Bengtson & al. (2021), where it is resolved as sister to the Apodocephala-Lowryanthus clade (Fig. 2, 3).This finding is inconsistent with Bremer (1994) and Nesom (2020), who classified the genus in the tribe Astereae.Members of Vernoniopsis are shrubs or small trees with coriaceous leaves and discoid homogamous capitula containing 1-4 florets with white corollas (Fig. 1C; Humbert 1960;Callmander & Phillipson 2011).Vernoniopsis caudata, the generitype, was originally described by Drake (1899) as Vernonia caudata Drake because of its Vernonia-like habit and inflorescences, inconsistent with our results.Humbert (1955) totally rejected this taxonomic decision and described his new genus Vernoniopsis to accommodate Vernonia caudata in the tribe Astereae based on differences in the stigmatic surfaces of the style branches.The name Vernoniopsis was recently noted to be an illegitimate later homonym of Vernoniopsis Dusén and has therefore been replaced by Jalantzia D. J. N. Hind (Hind & Langhorne 2024).The recognition of Vernoniopsis (now Jalantzia) at generic level is consistent with our findings.Jalantzia (Vernoniopsis) has been suggested to be closely related to the Malagasy genus Apodocephala based on their discoid capitula with white corollas and the presence of shortly caudate anthers (Humbert 1960;Bremer 1994;Nesom 2020).The molecular phylogenetic analyses here con- firm the close relationship of Apodocephala and Jalantzia (Fig. 2, 3).Jalantzia differs from Apodocephala in having epaleate receptacles and a pappus of scabrid bristles, whereas Apodocephala has paleate receptacles and lacks a true pappus.Therefore, we maintain the current generic status of Jalantzia.Further, our analyses support the recognition of Vernoniopsis caudata subsp.lokohensis Humbert at species level (Callmander & Phillipson 2011).Jalantzia (Vernoniopsis) caudata (Drake) D. J. N. Hind and J. lokohensis (Humbert) D. J. N. Hind do not grow sympatrically.The former commonly grows along the littoral forests of the eastern coast of Madagascar, while the latter is restricted to the highland rainforests of the Marojejy massif in northeastern Madagascar between 1300 and 2000 m in elevation.In other words, Jalantzia (Vernoniopsis) contains two geographically distinct lineages, the northeastern group (J.lokohensis) and the eastern-southeastern group (J.caudata).A similar pattern has recently been reported by Razafimandimbison & al. (2022) in the Malagasy Schismatoclada farahimpensis Homolle (Rubiaceae), which was resolved in two geographically and morphologically distinct lineages: northern and southern groups, that could be recognized at subspecies level ( Razafimandimbison, unpublished data).

Phylogenetic relationships within Apodocephala
Apodocephala currently consists of nine species of trees or shrubs with discoid paleate capitula in terminal corymbiform-paniculate inflorescences, all endemic to Madagascar: A. angustifolia, A. begueana, A. coursii, A. minor, A. multiflora, A. oliganthoides, A. pauciflora, A. radula and A. urschiana.Our analyses demonstrate that Apodocephala is paraphyletic with respect to the monospecific Malagasy genus Lowryanthus (here represented by two individuals of L. rubens).This is inconsistent with Bengtson & al. (2021), who resolved Lowryanthus as sister to two species of Apodocephala (Fig. 3).Apodocephala is resolved into two major clades: one containing A. minor, A. radula and L. rubens and the other formed by A. angustifolia, A. begueana, A. multiflora, A. oliganthoides, A. pauciflora and A. urschiana.Apodocephala and Lowryanthus mainly differ in the colour of the peduncles, capitula and florets (Fig. 1A, B).The latter has a striking appearance with bright red inflorescences and florets, whereas Apodocephala has florets with white or whitish corollas.Aside from that, L. rubens resembles Apodocephala, both consist of shrubs or trees with discoid mainly few-flowered paleate capitula in terminal corymbiform-paniculate inflorescences and have cypselas that lack a true pappus.Bengtson & al. (2021) noted additional similarities in the cypselas of Lowryanthus and Apodocephala (Fig. 4).Lowryanthus has geniculaterostrate cypselas with a tightly curved rostrum (Pruski 2014), a character also found in several species of Apodocephala.The flattened rostrate cypselas of A. coursii and A. radula (see Humbert 1962: 86) especially resemble those of L. rubens, but A. minor and A. urschiana also have cypselas with a narrowed curved rostrum (Fig. 4; Humbert 1960Humbert , 1962)).The character therefore occurs in both clades (Fig. 3, 4).
The two major clades of Apodocephala are geographically separated.Apodocephala minor and A. radula are both found in close geographical proximity of the known localities of Lowryanthus rubens in southeastern Madagascar.However, the three species do not grow sympatrically and occupy different habitats.Lowryanthus rubens is found in low-elevation humid evergreen forests from 100-300 m in elevation, whereas A. radula is found in siliceous rocky habitats at higher elevations and A. minor grows in rocky habitats at low elevations (Humbert 1960;Pruski 2014).The remaining sampled Apodocephala species form a well-supported clade (Fig. 3) and have a much wider geographic distribution along eastern Madagascar, ranging from Ivohibe in the southeast to Daraina in northern Madagascar.It can be concluded that geographic proximity is sometimes a better predictor of evolutionary history than morphology; a similar pattern has recently been reported from the Malagasy endemic Rubiaceae genera Payera Baill.and Schismatoclada Baker (Razafimandimbison & al. 2022).Despite the geographical separation of the two clades, we argue that Lowryanthus should be formally merged in Apodocephala (which has the priority over the former), because there is no morphological difference between the two clades that would justify dividing Apodocephala in two.Humbert (1955) divided Apodocephala pauciflora into two varieties, A. pauciflora var.pauciflora and A. pauciflora var.cacuminum, differing in leaf size and shape (see Table 1) as well as in shape of involucral bracts and capitulum size, where A. pauciflora var.cacuminum appears to have somewhat larger capitula.Morphological comparisons also show differences in the shape of the cypselas, which are shorter and more subprismatic in var.cacuminum (Fig. 4G, H).The two varieties differ in distribution and habitat.Apodocephala pauciflora var.pauciflora grows in rainforests at 800-1900 m in elevation and has a wider distribution ranging from Marojejy in the northeast to Fianarantsoa in central Madagascar.In contrast, A. pauciflora var.cacuminum is restricted to the Marojejy massif in the northeast, where it is found in ericoid vegetation in rocky places from 1300 to over 2000 m in elevation (Humbert 1955(Humbert , 1960)).Further, A. pauciflora   Apodocephala coursii could not be included in the molecular study, but morphological comparison shows similarities to A. radula and A. rubens, and it is likely to be closely related to those species.Apodocephala coursii, A. radula and A. rubens all have similar flattened cypselas with a curved rostrum, and A. coursii and A. rubens also have cypselas with long marginal and apical trichomes (Humbert 1960(Humbert , 1962;;Pruski 2014).Apodocephala coursii, A. radula and A. rubens all have distributions in southeastern Madagascar.Apodocephala coursii is only known from the Atsimo-Atsinanana region in eastern Madagascar where it grows in rainforest vegetation (Humbert 1962).

Implications on the subtribal classification of the tribe Athroismeae
Athroismeae sensu Bengtson & al. (2021) is currently divided into four morphologically distinct subtribes: Anisopappinae, Athroisminae, Lowryanthinae and Symphyllocarpinae; in addition to this, the tribe also contains the unplaced monospecific Anisochaeta (Bengtson & al. 2017).The subtribe Lowryanthinae is endemic to Madagascar and now encompasses the broadly defined Apodocephala (including Lowryanthus).The subtribe has earlier been placed as sister to subtribe Athroisminae (Bengtson & al. 2017(Bengtson & al. , 2021)).Similar obcompressed carbonized asymmetrically rostrate cypselas, and cypselas with long marginal and apical twin hairs, occur in members of both subtribes (Humbert 1960;Eriksson 1990Eriksson , 1992Eriksson , 1995;;Pruski 2014), and this is also what first led Pruski (2014) to place Lowryanthus in Athroismeae.These characters are, however, not found in Jalantzia (Vernoniopsis).Phylogenetic analyses place Jalantzia here as sister to subtribe Lowryanthinae as defined by Bengtson & al. (2021), and the morphological similarities to Apodocephala support the position of Jalantzia as close to Lowryanthinae.Jalantzia, however, differs from the members of Lowryanthinae in having epaleate receptacles and a true pappus of scabrid bristles, and we have therefore chosen not to include this Malagasy genus in subtribe Lowryanthinae but instead to describe a new subtribe, Jalantziinae, to accommodate Jalantzia.

Conclusions
The Malagasy Rochonia is confirmed to be a member of the tribe Astereae, whereas Vernoniopsis (now Jalantzia) is resolved as sister to the Apodocephala-Lowryanthus clade and therefore another addition to the growing tribe Athroismeae.The non-monophyly of the Astereae subtribe Madagasterinae is further supported.This group now only contains the Malagasy genera Madagaster and Rochonia, and a description of the emended subtribe Madagasterinae is presented.Phylogenetic analyses demonstrate the paraphyly of Apodocephala with respect to the Malagasy monospecific Lowryanthus, and a broad delimitation of Apodocephala (including Lowryanthus) was adopted to render this genus monophyletic.The newly re-circumscribed Apodocephala was resolved in two geographically segregated clades.The Athroismeae now consist of ten genera that are currently classified in five morphologically different subtribes (although Anisochaeta is still unplaced).Much remains unclear regarding how the different subtribes and the Athroismeae as a whole have evolved.Also, the backbone of the tribe is still largely unresolved.It is not unlikely that more additions to the tribe are in the Malagasy flora.

Fig. 2 .
Fig. 2. Bayesian fifty-percent majority-rule consensus tree from an analysis of the Asteraceae ndhF dataset, showing position of Rochonia within Astereae and Jalantzia (Vernoniopsis) within Athroismeae.Numbers above branches indicate posterior probability (PP) and bootstrap values (BS), bootstrap values <50 are indicated by a dash.Scale bar shows number of substitutions per site.

Fig. 3 .
Fig. 3. Bayesian fifty-percent majority-rule consensus tree from an analysis of the combined Athroismeae dataset (including ETS, ITS, ndhF and trnL-trnF).Numbers above branches indicate posterior probability (PP) and bootstrap values (BS), bootstrap values <50 are indicated by a dash.Scale bar shows number of substitutions per site.
var. pauciflora and var.cacuminum also appear to differ in flowering time (Table1).Analyses place the two varieties in different clades, a specimen of A. pauciflora var.pauciflora is placed sister to A. begueana, in a clade also consisting of A. urschiana and A. angustifolia, whereas two specimens of A. pauciflora var.cacuminum form a monophyletic group sister to A. oliganthoides in a clade comprising also A. multiflora (Fig.3).Apodocephala pauciflora var.cacuminum resembles A. oliganthoides in having oblanceolate leaves and capitula with rounded Note -The type specimen in P indicated in the protologue is actually a gathering consisting of two duplicate specimens, with no indication in the protologue, or on the specimens, as to which is the holotype and which is the isotype.A holotype cannot consist of two specimens (Art.8.1).As a result, a lectotypification is needed.bracts.Our results support that A. pauciflora var.pauciflora and var.cacuminum should be considered separate species.