﻿Re-evaluating monotypic Eleutherostylis from New Guinea and the Moluccas and its inclusion in Grewia (Malvaceae, Grewioideae)

﻿Abstract Morphological and molecular phylogenetic evidence indicate that Eleutherostylis Burret (Malvaceae, Grewioideae), a monotypic genus described from New Guinea, is best considered a synonym of Grewia L., a species-rich genus widespread across the Paleotropics and found in Africa, Arabia, Asia, Australia and the western Pacific. A new combination, based on E.renistipulata Burret, G.renistipulata (Burret) Dorr, comb. nov., is proposed. Original material of the basionym could not be located and a neotype is designated. A lectotype is designated for G.morotaiensis Kosterm., a synonym of G.renistipulata.


Introduction
Generic circumscriptions in Malvaceae s.l.remain surprisingly unsettled and many genera of clearly uncertain circumscription have not had their monophyly tested in well-sampled phylogenies.Sufficient taxon sampling in such phylogenies, even with limited genetic loci, can often identify problems and suggest alternative taxonomies (e.g.Eriolaena DC., see Dorr and Wurdack (2021)).Grewia L. (Malvaceae, Grewioideae), one of the largest genera in the family, with 280-300 species distributed across Africa, Arabia, Asia, Australia and the western Pacific has a history of generic circumscription problems with multiple segregates.One of those segregates, the monotypic genus Eleutherostylis Burret, endemic to New Guinea and the Moluccas, is the focus of this study.When Burret (1926) created Eleutherostylis, he stated how it differed from Grewia while tacitly admitting the two genera were closely allied.He observed that his new genus differed by having several styles that are free to the base and a gynoecium with more locules.Eleutherostylis has 4(5) locules while Grewia sensu Burret has 1-2(3) locules.These two genera along with Vincentia Bojer (non Gaud.) and Microcos L. were the only genera Burret (1926) included in his circumscription of Grewieae Endl.(Grewioideae).Hutchinson (1967) retained Eleutherostylis in an expanded circumscription of Grewieae in which he included seven genera.His concept of the genus Grewia included Vincentia Bojer and Microcos as synonyms rather than distinct genera.In Hutchinson's (1967) key to the tribe, Eleutherostylis was distinguished from Grewia by a single character, viz.flowers dioecious versus flowers bisexual.Earlier, Burret (1926) had rather pointedly dismissed floral sexuality as a useful systematic character ("Systematischer Wert ist [die Geschlechtsverh] nicht beizumessen").Hutchinson also noted that he did not follow Burret (1926) who retained Vincentia Bojer and Microcos as genera separate from Grewia because the "stigmatic characters used for distinguishing [these two genera] are scarcely of generic importance".The former is considered now a synonym of Grewia (Capuron and Mabberley 1999) while there is strong evidence the latter is distinct (see Brunken and Muellner (2012)).
Few other authors have discussed the relationships of Eleutherostylis.Kostermans (1972) considered the genus to be close to both Grewia and Trichospermum Blume and he placed G. morotaiensis Kosterm., described from the Moluccas, in synonymy under E. renistipulata.Bayer and Kubitzki (2003) maintained Eleutherostylis as a distinct genus, separating it from Grewia by the lack of a style in female flowers (versus styles cylindrical or dilated apically) and its heteromorphic (versus usually monomorphic) stipules.The latter is scarcely a generic character and positionally varies within a plant (heteromorphic pairs of reniform plus filiform stipules on lateral plagiotropic branches, but only reniform stipules on orthotropic branches).Brunken and Muellner (2012) included Eleutherostylis in their circumscription of the Grewieae and within that tribe in their "Grewia subclade".They based their argument on a morphological phylogenetic analysis, but did not elaborate on either characters or relationships within the subclade they recognised, which also included Colona Cav., Desplatsia Bocq., Mollia Mart., Tetralix Griseb.and Trichospermum, based on molecular data and Duboscia Bocq.and Vasivaea Baill., based solely on morphological data.Their phylogenetic analysis did not support the inclusion of Eleutherostylis within Grewia, although there was not strong support for any of the relationships that they found.Jennings (2021) treated Eleutherostylis in a floristic account of the trees of New Guinea, but the key couplet in that work used to separate Eleutherostylis from Grewia is based only on leaf and stipule characters.
Pollen data presented by Perveen et al. (2004) do not permit one to distinguish Eleutherostylis from Grewia.The pollen grains of both are described as coarsely reticulate and 3-colporate.The pollen shape ratio (P/E) of the former (1.66) falls within the range (1.27-2.05) of the limited number of species sampled of the latter.Pollen grains of both genera, prolate with long colpi, are described as "Corchorus-type", which can be applied to most Grewioideae, except for Apeiba Aubl., which Brunken and Muellner (2012) placed in Apeibeae Benth., a tribe distinct from Grewioideae.Hoorn et al. (2019), relying on the information and images provided by Perveen et al. (2004), noted that, within Malvaceae, the occurrence of prolate-subprolate bireticulate pollen with long colpi and distinct margos is restricted to Grewioideae.
Eleutherostylis was recently sampled with genomic-scale data for the Plant and Fungal Trees of Life Project (PAFTOL) and it was resolved as the strongly-supported sister-group to the single Grewia sampled (G.flavescens Juss.) (see Tree of Life Explorer, https://treeoflife.kew.org/tree-of-life;Baker et al. (2022)).The PAFTOL generic-exemplar approach to taxon sampling, however, did not test the monophyly of Grewia, which requires a more synoptic representation for that genus.The goals of our study, as part of broader phylogenetic work within Grewioideae, were to test the relationships of Eleutherostylis more adequately with respect to Grewia.

Material and methods
The taxon sampling here of 107 tips was a subset of data from an ongoing broader phylogenetic study and included Eleutherostylis renistipulata (Schodde & Craven 4438, US [01210662], the neotype designated below), along with a wide geographic sampling of Grewia (61 species) and 23/25 genera of Grewioideae (missing Erinocarpus Nimmo ex J. Graham and Goethalsia Pittier, which have been sampled elsewhere) recognised by Bayer and Kubitzki (2003).Outgroups included three Byttnerioideae (Malvaceae).Appendix 1 provides details of data sources, including vouchers and GenBank accession numbers.The nuclear ribosomal Internal Transcribed Spacer (ITS) region was selected for its ability to resolve the relevant taxa and recoverability from herbarium-specimen sourced DNAs.While single/few locus phylogenetic studies may appear dated in this genomics era, they remain appropriate for the scale of questions addressed here.Molecular methods for the 97 newly-generated sequences and phylogenetic analyses largely followed Dorr and Wurdack (2021) from DNeasy Plant Mini Kit (Qiagen Inc., Valencia, California, USA) extractions through to fluorescent Sanger sequencing of ITS amplification products on an ABI 3730xl DNA Analyzer (Thermo Fisher Scientific, Waltham, Massachusetts).The multiple sequence alignment (MSA) used MAFFT v.7.272 (Katoh and Standley 2013) under the G-INS-i refinement method, followed by minor manual refinements, based on a similarity criterion.Sensitivity analyses with different MAFFT optimality criteria, alignment without the divergent Byttnerioideae outgroups and exclusion sets to reduce missing data (i.e.removal of 119 MSA columns with > 50% missing data that reduced overall missing data from 16.2 to 3.85%) had little impact on the phylogenetic resolution, except in a few poorly-supported nodes.While there is sequence length variation within Grewia, there are few ambiguously aligned regions within our sampling for the genus.The MSA with the tested exclusion set is archived in the Dryad data repository (https://doi.org/10.5061/dryad.cnp5hqcbx).Final Maximum Likelihood (ML) analyses using all data were with IQ-TREE v.1.6.12 (Trifinopoulos et al. 2016) under GTR+F+I+G4 (selected by ModelFinder; Kalyaanamoorthy et al. (2017)) and RAxML-NG (Stamatakis 2014) as implemented on CIPRES XSEDE under GTR+I+Γ and clade support estimated by 1000 rapid bootstrap replicates.Bayesian Inference (BI) under GTR+I+Γ was performed using MrBayes v.3.2.7a (Ronquist et al. 2012) with two concurrent runs, each with four Markov chains (three cold and one heated), a 0.2 temperature coefficient and sampling every 1000 generations over 50 million generations and a conservative 25% burn-in.Topology and support value differences between the ML programmes were slight (IQ-TREE values are presented in Fig. 1); however, more pronounced were ML versus BI differences with shifts in some deeper nodes (albeit mostly poorly supported).

Results
Eleutherostylis is resolved as well-nested with multiple strongly-supported nodes (Posterior probability = 1.0,ML bootstrap > 85%) within a paraphyletic Grewia (Fig. 1).Its sequence divergence is relatively high amongst Grewia species, although it does not present additional alignment problems.Relationships within Grewia suggest a complex biogeography, although the small taxon and gene  sampling presented here limit our conclusions.The Grewia subclade containing Eleutherostylis contains only African species, while all the other Indo-Asian species (11 sampled) are far removed and mostly in a separate well-supported subclade.Relationships amongst the genera of Grewioideae and resolution as two major clades (circumscribed as tribes Apeibeae and Grewieae) largely mirror other studies (Bayer et al. 1999;Brunken and Muellner 2012;PAFTOL), except in the weakly-supported placement here of Desplatsia further removed from Grewia.Microcos is clearly distinct from Grewia and groups with Colona and Duboscia, which are Asian and African genera, respectively.Desplatsia and Duboscia, both African, are distinct from each other despite prior uncertainty (see Bayer and Kubitzki (2003)).The Neotropical and Palaeotropical taxa of Trichospermum group as separate sister subclades.

Discussion
Grewia is a morphologically diverse and biogeographically interesting genus, with species radiations in the Paleotropics, especially in sub-arid and woodland regions of Africa with 60% of the nearly 300 species.This diversification of arid-adapted shrubby species, often with animal dispersed fleshy drupaceous fruit, is especially notable in Madagascar (ca.65 species, mostly endemic) and Sub-Saharan Africa (124 species) (Gautier et al. 2012).The Indo-Asian (including China) diversity of Grewia contains ca.73 species, with a subset of only 28 species in the Flora Malesiana Region (Peninsula Malaysia, Indonesia, New Guinea, Philippines and Borneo).The relatively isolated nature of Eleutherostylis is suggested by sequence divergence and its placement amongst African taxa away from the Indo-Asian clade.However, our limited taxon sampling (ca.20% of Grewia species) and limited phylogenetic resolution prevent us from reliably identifying finer patterns in biogeography, except to note that the Malagasy endemics do not form a monophyletic group.While Eleutherostylis fits well within the broad morphological diversity of Grewia, it is ecologically unusual in the genus inasmuch as it is a large dioecious tree with dry fruit in lowland tropical forests.Most Malvaceae are hermaphroditic and dioecy is rare.Within Grewioideae, dioecy characterises Hydrogaster Kuhlm., Tetralix, Vasivaea and some Grewia (including Eleutherostylis).In addition, Erinocarpus, Grewia, Heliocarpus L. and Triumfetta L. have some species with alternative breeding systems (e.g.polygamous, gynodioecious) and sometimes unisexual (staminate, pistillate) flowers.While the stipules of Eleutherostylis are conspicuous because of their relatively largely size and positional dimorphism, similar large stipules occur in other species of Grewia (e.g.G. falcistipula K. Schum., an African species).

Figure 1 .
Figure1.Phylogenetic relationships of Eleutherostylis and its Grewioideae relatives.Bayesian 50% majority-rule consensus tree, based on ITS sequences with posterior probability and IQ-TREE ML bootstrap values indicated, above and below branches, respectively.NP = an edge not present with ML.