Morphological and molecular data confirm the transfer of homostylous species in the typically distylous genus Galianthe (Rubiaceae), and the description of the new species Galianthe vasquezii from Peru and Colombia

Galianthe (Rubiaceae) is a neotropical genus comprising 50 species divided into two subgenera, Galianthe subgen. Galianthe, with 39 species and Galianthe subgen. Ebelia, with 11 species. The diagnostic features of the genus are: usually erect habit with xylopodium, distylous flowers arranged in lax thyrsoid inflorescences, bifid stigmas, 2-carpellate and longitudinally dehiscent fruits, with dehiscent valves or indehiscent mericarps, plump seeds or complanate with a wing-like strophiole, and pollen with double reticulum, rarely with a simple reticulum. This study focused on two species that were originally described under Diodia due to the occurrence of fruits indehiscent mericarps: Diodia palustris and D. spicata. In the present study, classical taxonomy is combined with molecular analyses. As a result, we propose that both Diodia species belong to Galianthe subgen. Ebelia. The molecular position within Galianthe, based on ITS and ETS sequences, has been supported by the following morphological characters: thyrsoid, spiciform or cymoidal inflorescences, bifid stigmas, pollen grains with a double reticulum, and indehiscent mericarps. However, both species, unlike the remainder of the genus Galianthe, have homostylous flowers, so the presence of this type of flower significantly modifies the generic concept. In this framework, a third homostylous species, Galianthe vasquezii, from the Andean region is also described. Until now, this species remained cryptic under specimens of Galianthe palustris It differs however from the latter by having longer calyx lobes, the presence of dispersed trichomes inside the corolla lobes (vs. glabrous), fruits that are acropetally dehiscent (vs. basipetally dehiscent), and its Andean geographical distribution (vs. Paranaense). Additionally, a lectotype has been chosen for Diodia palustris, Borreria pterophora has been placed under synonymy of Galianthe palustris, and Galianthe boliviana is reported for the first time from Peru. A key of all Galianthe species with indehiscent mericarps is also provided.


INTRODUCTION
Galianthe Griseb. is a neotropical genus belonging to tribe Spermacoceae (Groeninckx et al., 2009). The genus was revised by Cabral (2002) and divided into two subgenera (Cabral & Bacigalupo, 1997): Galianthe subgen. Galianthe, from South America with 39 species, and Galianthe subgen. Ebelia (Rchb.) E.L. Cabral & Bacigalupo,with 11 Mesoamerican and South American species. Historically, Galianthe was associated with Diodia L., which has been described based on only D. virginiana L. The type species has a palustrine habit, pauciflorous axillary inflorescences, filiform corolla tube, bifid style with two long filiform stigmatic lobes, and indehiscent fruits. Later, others authors (i.e., Swartz, 1788;Walter, 1788;Candolle, 1830;Small, 1913) added other species into this genus with diverse kinds of habits and inflorescences, different floral morphology (e.g., distyly or homostyly, infundibuliform or campanulate corollas, bifid or bilobate stigma), and 2-carpellate schizocarpic fruits, being currently comprised by ca. 180 names (called Diodia s. lat.). Later, Bacigalupo & Cabral (1999) revised the genus Diodia and maintained only five species that were morphologically similar to the type species D. virginiana L. (description as above, and constituting Diodia s. str.). Species that did not match with these diagnostic features, were transferred to other genera as follows: eight species to Borreria subgen. Dasycephala (DC.) Bacigalupo & E.L. Cabral (Bacigalupo & Cabral, 1996), 12 species to Hexasepalum Bartl. ex DC. (Kirkbride, 2014;Kirkbride & Delprete, 2015;Cabaña Fader et al., 2016), and four species to Galianthe subgen. Ebelia (Cabral & Bacigalupo, 1997) Fader, pers. comm., 2017). In this sense, (Bacigalupo & Cabral, 1996;Bacigalupo & Cabral, 1998) transferred these species to Borreria subgen. Dasycephala because of their homostylous flowers and indehiscent mericarps, while Delprete, Smith & Klein (2005) and Delprete (2007), alluding to a broad concept, transferred the two species to Spermacoce mainly based on fruit characters. Dessein (2003) informally proposed to consider Diodia spicata as part of Galianthe based on molecular data (ITS intron), palynological data (double reticulum), and fruit morphology. The aim of this work is to confirm the taxonomic position of D. palustris and D. spicata based on morphological and molecular data, and perform their formal combination in Galianthe. In addition, a third homostylous species (Galianthe vasquezii R.M Salas & J. Florentín) is described and illustrated based on specimens from Colombia and Peru (previously identified as D. palustris). Additionally, a lectotype has been chosen for Diodia palustris whereas Borreria pterophora has been placed under synonymy of Galianthe palustris. Moreover, Galianthe boliviana E.L. Cabral is for the first time recorded in Peru. Finally, we provided a distribution map for the species investigated in this study, as well as a dichotomous key for all taxa with indehiscent mericarps.

Morphological study
This study is based on classical taxonomy techniques . Collections deposited at the  BA, BHCB, CEPEC, CTES, ESA, FUEL, FPS, FURB, HAS, HOXA, HUT, IAC, IAN,  IFFSC, IPA, K, LIL, MBM, MO, NY, P, PR, SI, SP, UB, UFRN, USB, US, USM and UEC herbaria were analysed. Furthermore, the databases of the 'Catálogo de plantas e fungos do Brazil' and 'Missouri Botanical Garden' were examined. In order to carry out scanning electron microscope (SEM) analyses, flowers were dehydrated using a graded series of ethanol solutions and afterwards critically point dried and sputter-coated with gold-palladium. SEM images were obtained with a JEOL 5800 LV scanning electron microscope. Pollen grains were acetolyzed according to Erdtman (1966) and mounted in glycerine jelly for analysis by light microscopy (LM). Conventional parameters (P = polar axis, E = equatorial axis) of at least 20 grains were measured under LM, and the exine was analyzed using SEM. Pollen terminology follows Punt et al. (2007). Species distribution maps were generated from distribution data that was present on the herbarium labels for each specimen and subsequently georeferenced using Google Earth (www.google.earth.com.ar) and Hijmans (2013).
The electronic version of this article in Portable Document Format (PDF) will represent a published work according to the International Code of Nomenclature for algae, fungi, and plants (ICN), and hence the new names contained in the electronic version are effectively published under that Code from the electronic edition alone. In addition, new names contained in this work which have been issued with identifiers by IPNI will eventually be made available to the Global Names Index. The IPNI LSIDs can be resolved and the associated information viewed through any standard web browser by appending the LSID contained in this publication to the prefix ''http://ipni.org/''. The online version of this work is archived and available from the following digital repositories: PeerJ, PubMed Central, and CLOCKSS.

Molecular study
In total, 45 species (47 accessions genera, and Bouvardia ternifolia (Cav.) Schltdl. as the outgroup. Leaf samples of these studies were obtained from silica gel-dried material or herbarium materials. Forty-three species (44 accessions) were previously used by Salas et al. (2015). Four accessions belonging to D. palustris has been added. All studied species with geographical information, collector, herbarium and GenBank accession numbers are provided in the Appendix.

Molecular protocols
Total genomic DNA was isolated from silica-dried leaf material using a modified CTAB protocol (Doyle & Doyle, 1987). Nuclear ribosomal ETS and ITS fragments were amplified following Baldwin & Markos (1998) and Negrón-Ortiz & Watson (2002), and White et al. (1990, respectively. PCR reactions for both gene markers investigated in this study consisted of 2 min initial denaturation at 94 • C and 30 cycles of 30 s denaturation at 94 • C, 30 s primer annealing at primer specific temperature and 1 min extension at 72 • C. Primer annealing for ETS and ITS were at 47 • C and 48 • C respectively. Amplification reactions were carried out on a GeneAmp PCR system 9700 (Applied Biosystems, Foster City, CA, USA). Purified amplification products were sent to Macrogen, Inc. (Seoul, South Korea) for sequencing. Sequences obtained in this study were deposited at GenBank [Diodia palustris, Verdi et al. 1905

Phylogenetic analyses
Contiguous sequences were assembled using Geneious v7.0.6 (Biomatters, Auckland, New Zealand). Automatic alignments were carried out with MAFFT (Katoh et al., 2002) Subsequent manual finetuning of the aligned dataset was done in Geneious v7.0.6. Congruency between the different datasets was inferred using different methods. First, a series of incongruence length difference tests (ILD; Farris et al., 1995) were carried out with PAUP* v.4. 0b10 (Swofford, 2003) using the following parameters: simple taxon addition, TBR branch swapping and heuristic searches of 1,000 repartitions of the data. Despite the well-known sensitivity of the ILD test (Barker & Lutzoni, 2002), the results of this test were compared in light of the resolution and support values of the obtained nuclear and nuclear ribosomal topologies. As a result, possible conflict between data matrices was visually inspected, searching for conflicting relationships within each topology that are strongly supported (hard vs. soft incongruence; Johnson & Soltis, 1998. Model selection for the Bayesian inference analysis was conducted with ModelTest 3.06 (Posada & Crandall, 1998) under the Akaike Information Criterium (AIC). The GTR+G model was selected for both ITS and ETS. Bayesian analyses of the concatenated dataset were carried out with MrBayes 3.1 (Huelsenbeck & Ronquist, 2001;Ronquist & Huelsenbeck, 2003). Four chains (one cold, three heated), initiated from a random starting tree were run simultaneously for 10 million generations. Every 1,000 generations, a tree was sampled from the chain for a total of 10,000 trees. Due to the burn-in, 50% of the sample points were discarded. Convergence of the chains was examined with TRACER 1.4 (Rambaut & Drummond, 2007). This resulted in an effective sampling size (ESS) parameter exceeding 100, which assumes a sufficient sampling and acceptable mixing.

Phylogenetic results
The ingroup contains 14 genera represented by 45 species of the Spermacoce clade. Of these, Diodia spicata and D. palustris are analysed for the first time in this context. ITS and ETS datasets were analysed both separately and combined. Because topology of each gene marker is very similar, we only present the results of the combined analysis ( Fig. 1). Current results indicate that most clades coincide with most currently accepted genera (e.g., Crusea, Emmeorhiza, Ernodea, Diodia s.s. (sensu Bacigalupo & Cabral, 1999), Mitracarpus, Psyllocarpus, Richardia and Staelia). Spermacoce, Borreria and Hexasepalum  100)). The species assigned to Borreria (sensu Bacigalupo & Cabral, 1996)

Ecology-Galianthe vasquezii grows in Montane
Forest of Peru and Colombia, which represents a severely fragmented type of vegetation. It grows between 1,800 and 2,500 m altitude.

DISCUSSION
Galianthe palustris and G. spicata share the same taxonomic and nomenclatural history. First, they were described under Diodia, later they were added to genus Borreria (Bacigalupo & Cabral, 1996;Bacigalupo & Cabral, 1998) due to the presence of homostylous flowers and type of fruit. Later they were, transferred to the genus Spermacoce (Delprete, Smith & Klein, 2005;Delprete, 2007). In 1998, Bacigalupo & Cabral (1998) observed that G. palustris (then still Borreria palustris) is characterized by a thyrsoid inflorescence that is similar to that of Galianthe. Despite this remarkable observation, the authors decided to transfer the species to genus Borreria. Nearly a decade later, Delprete, Smith & Klein (2005) and Delprete (2007) transferred both species to Spermacoce in an attempt to create a broad genus concept for Spermacoce. Despite overall molecular evidence, Galianthe spicata and G. palustris also share similar morphological characteristics with the other Galianthe species (e.g., spiciform and thyrsoid inflorescences, a bifid stigma and pollen grains with a double reticulum). This last character appears in most species of Galianthe, except for G. bogotensis  (1997) hypothesized that in a genus mainly represented by species with double reticulum pollen grains, the simple reticulum is the result of the absence of an infrareticulum persisting only a suprareticulum.
Current molecular data indicates that the phylogenetic position of Diodia palustris (Galianthe palustris) and D. spicata (G. spicata) make Galianthe paraphyletic. The Galianthe clade, including both former Diodia species, is strongly supported and has two molecularly well-defined clades. The (Diodia palustris + D. spicata) + G. brasiliensis clade is composed only by species with capsules separating into two indehiscent mericarps and which is a diagnostic character of Galianthe subgen. Ebelia. The sister clade, [G. eupatorioides + G. grandifolia ] + G. peruviana, includes species of Galianthe subgen. Galianthe, and is characterized by fruits with dehiscent valves. Both morphological and molecular data support the transfer of two former Diodia species to Galianthe, and more specifically in subgen. Ebelia. Additionally, and according to present sampling, the two subgenera described by Cabral & Bacigalupo (1997) seem to be monophyletic. The transfer of Diodia spicata to Galianthe was originally proposed by Dessein (2003), based on fruit, polynological and molecular features.
Even though morphological and molecular data show that three species share several characteristics with Galianthe subgen. Ebelia, there is a significant difference with the other species of the subgenus. The three species, unlike the remainder, have homostylous flowers. As a result, these results demonstrate the presence of a new floral trait in Galianthe and therefore strongly modify the generic concept of the genus.