Monadelpha (Euphorbiaceae, Plukenetieae), a new genus of Tragiinae from the Amazon rainforest of Venezuela and Brazil

Abstract Monadelpha L.J.Gillespie & Card.-McTeag., gen. nov., is described as a new member of Euphorbiaceae tribe Plukenetieae subtribe Tragiinae, to accommodate Tragiaguayanensis, a species known from western Amazonas, Venezuela and, newly reported here, from Amazonas, Brazil. The genus is unique in the subtribe for having 5-colpate pollen and staminate flowers with filaments entirely connate into an elongate, cylindrical staminal column terminated by a tight cluster of anthers. Phylogenetic analyses based on nuclear rDNA ITS and sampling 156 accessions across the diversity of Tragiinae (all 12 genera and 77 of ~195 species) also support Monadelpha as a distinct lineage that is separate from Tragia. A revised key to the genera of Tragiinae in South America and Central America is provided.

Among Tragiinae, the most species-rich genus is Tragia, which includes ~150 species, whereas the other 11 genera are much smaller with only one to 11 species each ( Table 1). The complex infrageneric classification of Tragia currently comprises two subgenera, six sections (including sect. Monadelphae L.J.Gillespie), one species group, and two unplaced species (Table 1). A single species has been segregated as T. subg. Mauroya (Leandri 1971); however, our preliminary research suggests this species is closely allied with sect. Agirta and does not warrant subgeneric status. All other species belong to T. subg. Tragia. Three other sections, sects. Leptorhachis (Klotzsch) Müll. Arg., Leucandra (Klotzsch) Müll.Arg. and Ratiga Müll.Arg., that are sometimes considered distinct are included here within sect. Tragia, a position supported by pollen (Gillespie 1994a) and molecular studies (Cardinal-McTeague and Gillespie 2016). Tragia species exhibit very diverse pollen and floral morphology that is correlated in part with its infrageneric classification (Gillespie 1994a, b). The genus was suggested to be highly paraphyletic based on this morphological diversity (Gillespie 1994a), which is confirmed by recent molecular phylogenetic studies focused on Plukenetieae (Cardinal-McTeague and Gillespie 2016). Three sections of Tragia, sects. Bia (Klotzch) Müll. Arg., Ctenomeria (Harv.) Benth., and Zuckertia (Baill.) Müll.Arg., were recently reinstated as genera (Webster 2007(Webster , 2014Medeiros et al. 2013) based on inferences from pollen morphology (Gillespie 1994a), floral morphology, and preliminary molecular results (Wurdack et al. 2005), and are supported by our more in-depth molecular study (Cardinal-McTeague and Gillespie 2016).
One of the most unusual species of Tragia is T. guayanensis L.J.Gillespie, which was considered so distinct as to merit its own monotypic section, Monadelphae L.J.Gillespie (Gillespie 1994b). The species is characterized by two features unique in Tragiinae: 5-colpate pollen and filaments entirely connate into an elongate cylindrical staminal column (Figs 1, 2). All other Tragia species have 3-aperturate pollen, which is mostly 3-colpate, sometimes 3-porate or with three poorly defined apertures (Table 1), with the exception of 4-colpate in T. rubiginosa Huft (preliminary observations in Gillespie 1994b), a species unplaced in the sectional classification. Filaments in staminate flowers of Tragia are usually distinct to sometimes basally connate. The only other species having filaments entirely connate is T. lassia Radcl.-Sm. & Govaerts of sect. Lassia, which has stamens connate into a very short disc-like structure (Baillon 1858: pl. 4, figs 24, 25;pers. obs.), and very different from that of T. guayanensis. When describing T. guayanensis, Gillespie (1994b) suggested the species was distinct and not closely related to any other Tragia species. Nevertheless, she maintained the species within Tragia in its own section pending further study and anticipating that a major reclassification along phylogenetic lines would be necessary.
Here we present molecular phylogenetic results placing T. guayanensis within subtribe Tragiinae that supports its recognition as a distinct genus. The new genus Monadelpha is described for T. guayanensis based on its unique pollen and floral morphology and isolated phylogenetic position within Tragiinae. This is the first of several contributions towards a new phylogenetic classification of subtribe Tragiinae.

Molecular phylogeny
To determine the phylogenetic relationships of Monadelpha, we sequenced and analyzed the Internal Transcribed Spacer (ITS) region (including complete ITS1, 5.8S, and ITS2, and flanking portions of 18S and 26S) of nuclear rDNA. ITS has been  Gillespie (2016) with pollen characters from Gillespie (1994aGillespie ( , 1994b and taxonomic updates from this paper. ). The paratype of T. guayanensis (Williams 14990, US) was sampled and sequenced at the Smithsonian separately from all other new data, under more stringent conditions for degraded museum samples following protocols in Dorr et al. (2018). That specimen is well preserved and the data appear authentic based on appropriate negative controls and unique phylogenetic placement. The sequences were aligned using the auto-select algorithm of MAFFT ver. 7.450 (Katoh and Standley 2013) in Geneious ver. 11.1.5 (BioMatters, Auckland, New Zealand), and the optimal model of nucleotide evolution was ranked by AIC (Akaike Information Criterion) using default search parameters across three substitution schemes in jModeltest2 ver. 2.1.6 on XSEDE (Darriba et al. 2012;Miller et al. 2010). Subsequent analyses were conducted on all data in the alignment and potentially ambiguous regions were few.
We estimated a phylogenetic tree using Bayesian inference with MrBayes ver. 3.2.6 on XSEDE (Ronquist et al. 2012), executing an (MC) 3 analysis with two runs of 3 million generations and sampling every 1000 generations, using the optimal model of nucleotide evolution on an unpartitioned alignment (remaining parameters as default). Runs were considered converged if ESS (effective sample size) of each parameter were >500 in Tracer ver. 1.7 (Rambaut et al. 2018), and if PSRF (potential scale reduction factor) and the standard deviation of split frequencies were close to 1.0 and <0.005, respectively, as determined by the MrBayes output. A 50% majority rule consensus tree was calculated following a 25% burn-in, resulting in Bayesian posterior probability (PP) values based on posterior distribution of 4500 trees from the combined runs. For an additional estimate of branch support, we inferred maximum likelihood bootstrap percentages (MLBP) using 1000 rapid bootstrap replicates under default parameters with RAxML-HPC ver. 8 on XSEDE (Stamatakis 2014). In the Results, we interpret strong branch support as PP >0.95 and MLBP >85. Discussion of the subclades (T1-T10) follows the naming convention of Cardinal-McTeague and Gillespie (2016) with minor adjustments.

Data resources
The data underpinning the analyses reported in this paper (DNA alignment and resulting Bayesian tree) are deposited in the Dryad Data Repository at https://doi. org/10.5061/dryad.5hqbzkh4d.

Morphology
Specimens were examined at CAN and US, on loan from MO, NY, and P (herbarium acronyms following Index Herbariorum (http://sweetgum.nybg.org/science/ih/), and from other herbaria via online images in the Global Biodiversity Information Facility (GBIF.org 01 Oct 2020 Occurrence Download https://doi.org/10.15468/dl.upmgky).
The key was adapted from Gillespie (1994b), modified and updated based on examination of specimens and the following references: Mulgura de Romero and Gutierrez de Sanguinetti (1989); Radcliffe-Smith (2001)

Phylogenetic results
Our 159-terminal (80 taxa) ITS dataset, including 55 new sequences, had an aligned length of 795 characters (410 variable, 353 parsimony informative [44%], 0.7% missing data), and GTR+I+G was identified as its optimal model of nucleotide evolution. Bayesian and ML analyses revealed very similar results. The 50% majority rule Bayesian topology was well resolved with most clades strongly supported by PP and MLBP (Fig. 3).

Discussion
The phylogenetic relationships of Tragiinae recovered here largely agree with previous phylogenetic analyses of Plukenetieae based on ITS and plastid psbA-trnH data (Cardinal-McTeague and Gillespie 2016). Our increased Tragiinae taxon sampling (77 here compared to 50 previously) improved both resolution and support, despite including only ITS data. A noteworthy difference is the revised placement of Gitara (subclade T9, formerly weakly supported as sister to Tragia sect. Tragia, T10; Cardinal-McTeague and Gillespie 2016), which is here strongly supported as sister to Zuckertia (T6), with Monadelpha sister to both of them. Monadelpha is an isolated lineage, clearly distinct from New World and Old World clades of Tragia. Support for its relationship with Gitara and Zuckertia (subclade T6/9) is not strong, which suggests that its position on the phylogeny may not be stable and could vary with additional sequence data. Inclusion of the Monadelpha ITS sequence in a broader analysis of Plukenetieae that sampled six loci (nuclear ribosomal ETS, ITS; low copy KEA1, TEB; plastid matK, ndhF; results not shown) recovered similar results with strong support for subclade T6/9, the inclusion of Monadelpha in subclade T6/9, and with weak support for generic relationships therein (Cardinal-McTeague et al., unpublished).
Monadelpha is also morphologically distinct, especially its staminate flowers with filaments completely connate into a long cylindrical staminal column bearing a tight terminal cluster of ±5 anthers. Acidoton, Bia, Gitara, Platygyna, and Zuckertia all have numerous free stamens, the large Tragia sects. Tragia and Tagira have 3 stamens (sometimes more, to 22) that are distinct or connate only at the base, and T. sect. Leptobotrys has two stamens (rarely 3) connate basally. Only the distantly related Tragia lassia (T. sect. Lassia) of Madagascar has stamens or filaments entirely connate, but this feature has obviously evolved independently. Its androecium, consisting of a small 3-anthered disc-shaped structure on a very short narrow column, is very different from that of Monadelpha.
Other characters of Monadelpha that are unusual for Tragiinae include unisexual inflorescences and long, mostly distinct styles. Whereas most Tragiinae have bisexual inflorescences with pistillate flowers proximal, Monadelpha shares unisexual inflorescences with Gitara and the Caribbean genera Acidoton and Platygyna (plus a few species of Old World Tragiinae). Styles of Monadelpha are slender, cylindrical, mostly smooth (papillose only at the apex), up to 10 mm long, and connate basally (up to ¼ their length). Most New World Tragiinae and Old World Tragia have styles that are much shorter, relatively thicker, and basally to mostly connate into a thick stylar column. Perhaps most similar is Zuckertia with somewhat longer (to 5 mm), slender cylindrical styles that are connate into a slender column, but differ in the degree of connation (1/2-3/4 their length) and the free portion papillose adaxially.
Tragia rubiginosa from Amazonian Peru is another morphologically unusual Tragia species (Huft 1989) that is unplaced in the present sectional classification. The species has 4-colpate pollen (preliminary observations in Gillespie 1994b) and is the only species in Tragiinae other than Monadelpha with a pollen aperture number greater than three. Its broad, thick, subsessile stigmas are unique in Tragiinae and its staminate flowers with five sessile anthers are unusual. The species is morphologically distinct from Monadelpha, and the two taxa are unlikely to be closely related. Molecular data is not yet available to determine its phylogenetic position within Tragiinae.
Etymology. The specific epithet is derived from Guayana, and refers both to the Guayana Shield region of northern South America and to the Guayana Region of Venezuela where the species is native. Guayana is of Amerindian derivation by European colonists, and may come from the tribe Guayanos or the Indigenous word uayana, meaning pale (see Berry et al. 1995). Distribution and preliminary conservation status. Known from only three collections. The two from Venezuela are ~220 km apart in lowland rainforests of the upper Orinoco Basin and Río Casiquiare of western Amazonas. The Brazilian collection occurs at least 500 km to the south along the Rio Juruá (locality imprecise) in Amazonas. They occur in remote, pristine rainforest and their IUCN Red List Category presently should be Data Deficient given limited information of distribution and threats.  Notes. Monadelpha guayanensis is newly reported here from Brazil based on one 1900 collection by E.H.G. Ule. Staminate inflorescences on this collection (L-0160690 sheet) are considerably longer than previously described, and are closer in length to the pistillate inflorescence. It appears that the staminate inflorescence on the holotype may be damaged and partly missing or possibly less mature. Leaf blades are more variable in size and shape than on the two Venezuelan collections, some blades being very similar, others larger and relatively broader.
Monadelpha guayanensis has unisexual inflorescences and is likely monoecious (rather than dioecious). Although unisexual inflorescences are not found on the same branch, the type collection, Stergios & Aymard 9182, has inflorescences of both sexes, and is thus monoecious if one assumes branches originate from a single individual. Further collections are needed to confirm this character.

Appendix 1
Sources for ITS data used in phylogenetic analyses. Voucher data (country, collector, number, herbarium code) are provided, with new GenBank numbers in bold and beginning with MK and MW.