Plant Systematics and Evolution (2020) 306:26 https://doi.org/10.1007/s00606-020-01630-6 ORIGINAL ARTICLE It is not a disaster: molecular and morphologically based phylogenetic analysis of Rondeletieae and the Rondeletia complex (Cinchonoideae, Rubiaceae) Alejandro Torres‑Montúfar1,2 · Thomas Borsch3 · Susy Fuentes3 · Jorge Gutierrez4 · Helga Ochoterena2 Received: 13 September 2019 / Accepted: 8 January 2020 © Springer-Verlag GmbH Austria, part of Springer Nature 2020 Abstract The circumscription of Rondeletieae has changed considerably over time. Historically, 85 genera have been included, characterized by small trees, imbricate corolla aestivation and capsules. Molecular studies led to a narrower circumscription and stressed out that Rondeletieae is mainly an Antillean clade. As historically circumscribed, Rondeletieae is both polyphyletic (involving several tribes, including Condamineae, Sipaneeae, etc.) and paraphyletic with respect to Guettardeae. Uncertainties persist on the tribal circumscription, and its generic limits and relationships. We report results of phylogenetic analyses under parsimony, maximum likelihood and Bayesian analyses including 179 taxa, using concatenated datasets of three chloroplast DNA regions (petD, trnL–F and trnK–matK), their DNA microstructural characters as well as morphology. Our objectives are to: test the monophyly of Rondeletieae and evaluate phylogenetic relationships, evaluate potential characters for tribal and generic delimitation and propose a circumscription for Rondeletieae according to phylogenetic hypothesis using morphological characters. The tribes Guettardeae and Rondeletieae are strongly supported as sister: quincuncial aestivation and spathulate corolla lobes are proposed as a unique combination of characters supporting this relationship. We did not find synapomorphies for each tribe, but there is a set of character combinations that allows distinguishing both tribes that includes the corolla and pollen ornamentation as well as fruit and seed morphology. After evaluating other genera not included in the phylogenies, our proposal is that Rondeletieae contains approximately 120 species in 20 neotropical genera: Acrosynanthus, Acunaeanthus, Blepharidium, Donnellyanthus, Mazaea, Phyllomelia, Rachicallis, Roigella, Rondeletia, Rovaeanthus, Suberanthus, Tainus, plus one tentatively included and recently described (Jamaicanthus). Keywords Guettardeae · Morphology · Phylogeny · Taxonomy · Tribal delimitation Introduction Handling Editor: Livia Wanntorp. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00606-020-01630-6) contains supplementary material, which is available to authorized users. * Helga Ochoterena helga@ib.unam.mx 1 Posgrado en Ciencias Biológicas, Universidad Nacional Autónoma de México, Ciudad Universitaria AP 70-367, 04510 Coyoacán, CdMx, Mexico 2 Departamento de Botánica, Instituto de Biología, Universidad Nacional Autónoma de México, Ciudad Universitaria AP 70-367, 04510 Coyoacán, CdMx, Mexico The tribe Rondeletieae currently entails a group of Neotropical Rubiaceae, most of which are trees or shrubs occurring on the Caribbean Islands, Mexico (south the Isthmus of Tehuantepec) throughout Mesoamerica and, in the case of the genus Rondeletia L., extending into the Caribbean basin of South America (Venezuela and Colombia). All 3 Botanischer Garten und Botanisches Museum Berlin, Freie Universität Berlin, Königin-Luise-Straße 6-8, 14195 Berlin, Germany 4 Jardín Botánico Nacional, Universidad de la Habana, Carretera del Rocío Km 3½, Calabazar, 19230 Ciudad de la Habana, Cuba 13 Vol.:(0123456789) 26 Page 2 of 25 species possess entire stipules, heterostylic flowers with imbricate corolla lobes. Stamens can be adnated at varying basal, medial or distal positions on the corolla tube. Anthers dehisce longitudinally, pollen is tricolporate and capsules are few- to many-seeded with the exception of Phyllomelia Griseb. that has two-seeded samaras (Robbrecht 1988; Delprete 1999a, b). The tribe Rondeletieae has a complex taxonomic history since its classification and generic content have remarkably changed over time varying from 15 to 44 genera, with either pantropical or neotropical geographical distribution (De Candolle 1830; Hooker 1873; Robbrecht 1988; Robbrecht and Bridson 1993; Delprete 1999a, b; Rova et al. 2002, 2009; Manns and Bremer 2010). For a comprehensive taxomic history of this tribe, see Delprete (1999a). Here, it suffices to mention that through time it not only has changed in content, but it also has been placed in different subfamilies. For instance, mainly based on fruit morphology (bilocular many-seeded capsular fruits), it was firstly described by Chamisso and Schlechtendal (1829) as a pantropical subtribe Rondeletineae of the large tribe Hedyotineae and validated in De Candolle’s Prodomus (1830); both classification schemes included 19 genera. Posteriorly, Hooker (1873) separated Rondeletieae from Hedyotideae mainly based on the entire stipules and the imbricate corolla lobes and included 16 genera of pantropical distribution. Schumann’s classification (1891) agreed in the generic content and geographical circumscription with that of Hooker’s, but he recognized two subfamilies and included Rondeletieae in Chinchonoideae including 17 genera. Many years later, Verdcourt (1958) divided the Rubiaceae into three subfamilies based on several characters such as embryo type, seed albumen and raphides (Rubioideae, Cinchonoideae and Guettardioideae). Rondeletieae was also included within Cinchonoideae by the lack of raphides, bilocular ovary with many horizontal ovules, comprising genera from tropical America and Asia without specifying the genera included. Bremekamp (1966) divided Rubiaceae into eight subfamilies; Rondeletieae was also considered as Cinchonoideae and characterized by the contorted or imbricated corolla aestivation, without specifying geographical affinity or number of genera. Posteriorly, Robbrecht (1988) divided the family into four subfamilies (Antirheoideae, Cinchonoideae, Ixoroideae and Rubioideae); Rondeletieae was included in Cinchonoideae as a pantropical tribe recognized by the entire stipules, imbricate corolla lobes and capsular fruits, with 34 genera. Within Rondeletieae, the circumscription of the genus Rondeletia has been particularly conflictive. A number of segregates have been proposed by several authors (Planchon 1849; Steyermark 1964, 1967; Borhidi et al. 1980, 2004, 2011; Borhidi and Fernandez-Zequeira 1981a, b; Borhidi and Jarai-Komlodi 1983; Fernández-Zequeira 1994), while others have argued that practically no subdivisions can be 13 A. Torres-Montúfar et al. made based on morphology and did not accept the segregation until more evidence was evaluated (Lorence 1991, 1999; Burger and Taylor 1993; Taylor 2001). In total, the “Rondeletia complex” includes nine genera directly splitted from Rondeletia (Acunaeanthus Borhidi, Járai-Koml. & Moncada, Arachnothryx Planch., Donnellyanthus Borhidi, Javorkaea Borhidi & Járai-Koml., Renistipula Borhidi, Roigella Borhidi & M.Fernández, Rogiera Planch., Rovaeanthus Borhidi, Suberanthus Borhidi & M.Fernández and Tainus Torr.-Montúfar, H.Ochot. & Borsch) or included by molecular evidence (Gonzalagunia Ruiz & Pav., Mazaea Krug & Urb., Phyllomellia, Rachicallis DC. and Stevensia Poit.). Then, the “Rondeletia complex” practically constitutes the tribe Rondeletieae. More recently, molecular biology has caused a revolution in the Rubiaceae classification, but all of them agree that Rondeletieae belongs in Cinchonoideae and that its geographical range is restricted to tropical America (as Rova et al. 2002; Delprete and Cortés 2004; Mouly et al. 2009; Manns and Bremer 2010; Razafimandimbison et al. 2011; Delprete and Jardim 2012; Kainulainen et al. 2013; Stranczinger et al. 2014). Among the molecular phylogenetic studies directly related to Rondeletieae, Rova et al. (2002) used sequences of the plastid trnL–F region representing most genera of the Rondeletieae sensu Robbrecht (1988) with one to a few species. Their phylogeny set the basis for the circumscription of a monophyletic Rondeletieae tribe within a more narrowly defined and well-supported Cinchonoideae clade. Several genera formerly included in Rondeletieae were shown to belong to Condamineeae and Sipaneeae (subfamily Ixoroideae). Although they could not provide significant statistical support for their phylogenetic hypotheses, the authors stated that Rondeletieae could be sister to Guettardeae as Robbrecht and Manen (2006) later supported by using a supertree approach. Later, Rova et al. (2009) focused more specifically on the Rondeletieae; they combined plastid rps16 and trnL–F and nuclear ITS and broadened the representation of species, in particular for Rondeletia. In the resulting combined tree, deep nodes and the clade corresponding to Rondeletieae remained unsupported and were depicted as nested in a paraphyletic assemblage of the “Guettardeae s.l.” (Rova et al. 2009). Manns and Bremer (2010) so far provided the current Rondeletieae circumscription based on the most extensive molecular phylogenetic hypothesis for the Cinchonoideae using Bayesian analysis of a combined matrix of several plastid regions (atpB–rbcL, ndhF, rbcL, rps16, trnL–F) and nuclear ITS, but including only few representative species per genus across the subfamily; their results reveal two wellsupported sister clades corresponding to Rondeletieae and Guettardeae. This study broke out previous Rondeletieae Page 3 of 25 The Rondeletieae tribe revisited circumscription schemes and confirmed ten genera to be part of it (Acrosynanthus Urb., Acunaeanthus, Blepharidium Standl., Mazaea, Phyllomelia, Roigella, Rondeletia, Rovaeanthus, Rachicallis and Suberanthus) although insights on generic limits remained scarce. The diverse genus Rondeletia (ca. 100 species) itself was just sampled with five species, and for the full sampling of Rondeletieae and Guettardeae only a Bayesian tree based on a dataset with concatenated plastid and nuclear ITS sequences was presented. Recently, Torres-Montúfar et al. (2017) analyzed a sequence dataset of three plastid genomic regions (trnK–matK, trnL–F and petD) with the main objective to solve the status of the Hispaniolan endemic Rondeletia pitreana Urb. & Ekman; their study revealed Acrosynanthus as sister to a well-supported clade including Rondeletia (15 species sampled), Mazaea, Rachicallis and Phyllomelia (therein called “Rondeletia assemblage”), albeit with largely unresolved relationships within this clade. Additionally, R. pitreana was described as a monotypic genus Tainus. Moreover, Torres-Montúfar et al. (2017) included Donnellyanthus for the first time in any phylogenetic analysis, which was resolved as sister to Rovaeanthus and as a member of the Rondeletieae clade. This result contrasts the floristic work by Lorence (2012a, b) that treats the two genera as congeneric with Arachnothryx and Rogiera, respectively, both as part of Guettardeae according to Manns and Bremer (2010). Remarkably, all these studies pointed to a close relationship between Guettardeae and Rondeletieae, the first even paraphyletic according to Rova et al. (2009). Before the molecular age, Guettardeae was considered to be morphologically isolated among Rubiaceae, to the point that it was considered as monotribal subfamily Guettardioideae by Verdcourt (1958) and Bremekamp (1966), mainly characterized by the axillary inflorescences, a single ovule on each ovary locule and drupaceous fruits. With the molecular evidence, some genera segregated from Rondeletia and included in Rondeletieae (as Arachnothryx and Rogiera) were placed in Guettardeae (Rova et al. 2002, 2009; Manns and Bremer 2010; Torres-Montúfar et al. 2017, 2018) and so, the previously clear morphological circumscription of Guettardeae was broken down to include taxa with terminal inflorescences and many-seeded capsular fruits. Rova et al. (2002) suggest as a possibility to merge Rondeletieae and Guettardeae in one single large tribe; however, they considered that morphological support and a comprehensive sampling of both tribes were needed before applying such taxonomic decision. For Rondeletieae, the most complete comparative assessment of morphological characters with a phylogenetic context to date was made by Rova et al. (2009). Although their emended diagnosis lists a broad spectrum of characters belonging to genera that are currently classified within Guettardeae, such as Rogiera (Manns and Bremer 2010), it 26 lacks representatives of genera that were later segregated from Rondeletia, such as Donnellyanthus and Tainus. The main problem was that Rova et al. (2009) used a largely unsupported tree that depicted Guettardeae as paraphyletic to Rondeletieae, so that they apparently maintained Rogiera as part of the Rondeletieae based on previous pre-phylogenetic treatments. Torres-Montúfar et al. (2018) reconstructed the evolution of fruit characters within Cinchonoideae but neither found synapomorphies for Guettardeae or Rondeletieae, respectively. In both tribes, fruits are capsules except for Phyllomelia that has samaras or Guettarda L. and allies that have mesocarpic drupes. Therefore, the goal of our study was first to generate a comprehensive character matrix both for DNA sequence data from the plastid genome and for morphology including as many as possible of the other taxa that were recently classified as members of the Rondeletieae and their putative relatives, in particular from Guettardeae. We therefore extended the matK–trnK + trnL–F + petD datasets of TorresMontúfar et al. (2017, 2018) to include a broader species sampling, for both Rondeletieae (with particular emphasis on Rondeletia) and of the sister tribe Guettardeae. These rapidly evolving plastid regions were shown to provide high levels of hierarchical phylogenetic signal within Rubiaceae, both within genera and at greater distances (Tosh et al. 2009; Krüger et al. 2012; Torres-Montúfar et al. 2017) while still allowing a robust alignment. The morphological characters include features that had been selectively used to characterize in particular genera. We then aimed to reconstruct a baseline phylogeny of the Rondeletieae and allies based on combined molecular and morphological characters. Using the obtained trees, we specifically aimed at evaluating the monophyly of Rondeletieae and its genera and at analyzing in how far the morphological characters that have been used for defining taxonomic entities are synapomorphic or homoplastic. Based on these phylogenetic results, we then provide an updated diagnosis of the tribe Rondeletieae and its genera. Materials and methods Taxon sampling In order to decide which genera to include in the phylogenetic analysis, we did a literature survey to generate a list of genera historically included in Rondeletieae (Online Resource 1) and to examine whether they were already included in any molecular phylogenetic analysis. From the total of 85 genera in this list, we excluded those in our own investigation that already were unambiguously shown to be phylogenetically placed outside Rondeletieae and Guettardeae (Online Resource 1). The same applies to 13 26 A. Torres-Montúfar et al. Page 4 of 25 generic names that can now be treated as synonyms of other accepted genera based on phylogenetic insights. We sampled all the genera currently corroborated in Rondeletieae according to several studies (Rova et al. 2002, 2009; Robbrecht and Manen 2006; Manns and Bremer 2010; Torres-Montúfar et al. 2017). Although at generic level our sampling is similar to Manns and Bremer (2010), Rova et al. (2009) or Torres-Montúfar et al. (2017), our study represents by far the most complete dataset at species level for several genera in Rondeletieae. We also included all genera with dry fruits that are potentially part of Guettardeae in addition to a few representatives of the tribe with fleshy fruits. Despite our attempts, the only genera that were not included into the molecular analyses due to the lack of suitable samples are Ariadne Urb., which is from the Antilles, the monotypic genera Acrobotrys K.Schum. & K.Krause, Glionnetia Tirveng., Habroneuron Standl., Holstianthus Steyerm., Spathichlamys R.Parker, Standleya Brade, Stylosiphonia Brandegee and the recently described Jamaicanthus Borhidi (2018). Nevertheless, the pertinence to tentatively include each of these eight genera as putative Rondeletieae was evaluated based on the morphological evidence supported by the phylogeny. The morphological features of this genera were evaluated trough the examination of types (physically or with highresolution images mainly obtain from www.JStorPlants), the protologues and, in the few cases from other collections from which the species are known. Full names with authors, vouchers and GenBank accession numbers for all taxa included in the analyses are presented in the “Appendix”. Generation of the morphological matrix Morphological data were predominantly obtained from original descriptions and type specimens, complemented by personal observations, from the field and from specimens from the following herbaria: B, F, MEXU and MO. In addition, publications with descriptive data (Poiteau 1804; Standley 1918; Liogier 1962, 1995; Aiello 1979; Delprete 1999a, b; Lorence 2012a, b, c) were used as a source of information to code characters. All characters and character states used in the analyses are listed in Table 1. DNA extraction, amplification and sequencing Silica gel dried leaves were used for DNA extraction when available, complemented with herbarium material. DNA was extracted using the NucleoSpin Plant II kit (Macherey–Nagel, Düren, Germany) following the manufacturer’s protocol or a three-fraction cetyltrimethylammonium bromide (CTAB) method (Borsch et al. 2003). Three plastid markers (trnK–matK, trnL–F and petD) were amplified and sequenced in this study. The amplification of each marker was performed in reaction volumes of 50 μL, containing 2 μL of extracted DNA (with a concentration of 10–20 ng/μL), 14.7 μL of H2O, 5 μL of 10 × peqLab Taq. buffer S containing MgCl2, 3 μL of MgCl2 (25 mM), 10 μL of betaine monohydrate (5 M), 1 μL of BSA (10 μg/ µL), 2 μL of forward primer (20 pm/μL), 2 μL of reverse primer (20 pm/μl), 10 μL dNTPs (each 0.25 mM) and 0.3 μL Taq polymerase 5 units/μL (PeqLab, Erlangen Germany). For trnK–matK, the amplification was performed in two halves using the primers trnKFbryo (Wicke and Quandt 2009) and COmatK670R (Tesfaye et al. 2007) plus NYmatK480F (Hilu et al. 2003) and psbA5R (Steele and Vilgalys 1994) that also covered the spacer between the trnK 3′ exon and the psbA gene. PCR conditions were: one cycle of denaturation (90 s at 96 °C, 60 s at 50 °C, 120 s at 68 °C), 35 cycles of annealing (30 s at 95 °C, 60 s at 48 °C, 120 s at Table 1 Morphological characters evaluated Character Character states 1. Corolla aestivation 2. Merosity 3. Corolla shape 4. Corolla color 5. Corolla lobes form 6. Corolla throat ornamentation (0) Quincuncial; (1) valvated; (2) contorted; and (3) induplicated (0) 4; (1) 5; (2) 6 (0) Infundibuliform; (1) rotate (0) White; (1) reddish–pinkish; and (2) yellow (0) Spatulate; (1) triangular (0) Naked; (1) fleshy ring; (2) yellow hairy ring; (3) papillate ring; (4) white hairy ring; and (5) yellow hairy and fleshy ring (0) Basal; (1) medium; and (2) near throat (0) Dry; (1) fleshy (0) Capsule; (1) berry; (2) samara; (3) mesocarpic drupe; (4) endocarpic drupe; and (5) schizocarp (0) Loculicidal; (1) septicidal; and (2) indehiscent (0) One; (1) two to six; and (2) > 30 (0) Winged; (1) wingless (0) Echinate; (1) smooth 7. Stamen insertion 8. Fruit consistency 9. Fruit type 10. Fruit line dehiscence 11. # seeds per locule 12. Seed type: 13. Pollen exine ornamentation 13 The Rondeletieae tribe revisited 68 °C), extension (20 min at 68 °C). The trnL–F region was amplified using the primers trnTc and trnTf (Taberlet et al. 1991). PCR conditions were: 30 cycles of denaturation (60 s at 96 °C), annealing (60 s at 50 °C) and extension (120 s at 72 °C). The petD intron including the petB–petD spacer was amplified using the primers PIpetB1411F or PIpetB1365F and PIpetD738R or PIpetD346R (Löhne and Borsch 2005). PCR conditions were: 35 cycles of denaturation (60 s at 97 °C), annealing (60 s at 48 °C), extension (45 s at 72 °C) and a final extension step (7 min at 72 °C). In DNA isolated from herbarium specimens, not all amplification products were obtained for each taxon. All amplification products were purified by QIAquick PCR Purification Kit (QIAGEN GmbH, Hilden, Germany). Sequencing was performed by Macrogen Inc., South Korea (http://www.macrogen.com). Sequence assembly, alignment and microstructural character coding The matrix comprises 179 taxa with a total of 428 sequence contigs corresponding to the three molecular markers, 204 of which were newly generated for this study. Further 219 sequences were generated by Torres-Montúfar et al. (2017) and 15 downloaded from GenBank, originally generated by Rova et al. (2002, 2009). New pherograms were edited and assembled using PhyDE v.0 995 (Müller et al. 2005). Sequences were then added to the multiple sequence alignment of Torres-Montúfar et al. (2017) following the criteria proposed by Löhne and Borsch (2005). Microstructural mutations were manually coded, for gaps following the simple gap criterion of Simmons and Ochoterena (2000) and for Simple Sequence Repeats (SSR’s) and Inversions following Ochoterena (2009). Regions of uncertain homology (mutational hot spots) were removed from the matrices prior to phylogenetic analyses; final matrices and the microstructural character matrices and annotated alignments (including hot spots and the identified microstructural mutations) are available at Zenodo (https://zenodo.org/record/3518369). Phylogenetic analyses Maximum parsimony (MP) analyses were performed using the concatenated matrix with nucleotides and microstructural characters, only including potentially parsimony informative characters. A heuristic search with 10,000 replicates of Wagner trees constructed with random taxon addition followed by TBR branch swapping was performed in TNT version 1.1 (Goloboff et al. 2003); ten trees were saved on each replicate, and a further TBR was conducted to completion saving up to 10,000 trees. These trees were submitted to further analysis using the “new technology” option, alternating ratchet parsimony (Nixon 1999a), sectorial, drift Page 5 of 25 26 and tree fusion (Goloboff 1999) options. One hundred initial sequences were used until the consensus was stabilized ten times using a 100% factor. All the most parsimonious trees were collected and opened in WinClada (Nixon 1999b) and summarized in a strict consensus tree. Additionally, a Jackknife analysis (JK) was executed in TNT version 1.1 (Goloboff et al. 2003), re-sampling the matrix 10,000 times with the same parameters used for the new technology searches; we considered a node to be moderately supported if the JK value is 64–85% (Farris et al. 1996), while we treated the node as highly supported only if it is above 85%. Nodes with JK below 64% are considered statistically not supported. For Bayesian inference (BI) and maximum likelihood (ML) analyses, the optimal models of sequence evolution were selected for each marker following the results of the Akaike Information Criterion in Modeltest version 3.6 (Posada and Crandall 1998) using the following parameters: 203 substitution schemes, +F base frequencies, +I and +G rate variation, nCat = 4 and SPR tree search. The GTR + I + G model of molecular evolution was selected for all three plastid markers. For the morphological and the microstructural characters, the MK model (Lewis 2001) was implemented which assumes that a character can change its state at any time with equal probability for all instantaneous time intervals along the branch. BI analyses of the concatenated matrix partitioned by molecular markers plus microstructural and morphological data were conducted using MrBayes v.3.1.2 (Huelsenbeck and Ronquist 2001). Four independent Markov Chain Monte Carlo (MCMC) runs were carried out, each with four parallel chains. Each chain was performed for 1,000,000 generations, saving one random tree every 1000 generations. The burn-in was determined using Tracer v1.5 (Rambaut and Drummond 2012), and it was set at generation 20,000, where the stability of the chains was reached as judged from log likelihood plots and standard deviation of split frequencies (ESS < 575). Phylogenetic trees were visualized using Figtree (Rambaut and Drummond 2012) and edited in TreeGraph2 (Stöver and Müller 2010). For posterior probabilities (PP), we considered a clade to be well supported if PP was equal or greater than 0.95 (according to Alfaro and Holder 2006), and moderately supported if PP was between 0.85 and 0.95. Nodes with PP below 0.85 were considered not supported. Maximum likelihood (ML) analysis was performed with RAxML (Stamatakis 2014) using RAxML GUI v. 1.3. (Silvestro and Michalak 2012) implementing the “ML + thorough bootstrap” option with 10,000 bootstrap replicates. Nodes with bootstrap (BS) values equal to or above 70% were treated as statistically supported (according to Felsenstein and Kishino (1993) and Hillis and Bull (1993)), but we considered them only to be well supported if they had BS ≥ 85%. 13 26 A. Torres-Montúfar et al. Page 6 of 25 Assessment of morphological characters Results Among the morphological characters, we define for the first time the quincuncial corolla aestivation, referring to a particular imbricate type in which “two lobes are outside, two inside and one is half in half out” (Bell 1993: 148). In this case, as the corolla only has four petals, the half in half out petal is missing. Another term not commonly used in the traditional taxonomy of the tribe is shape of the corolla lobes as spathulate, referring to a condition in which the petals have a “rounded blade above gradually tapering to the base” (Harris and Harris 2000: 110). This is to contrast the unique aestivation and corolla shape in Rondeletieae with respect to the contorted aestivatin and elliptic or obovate corolla lobes shape in other taxa. All other characters follow the terminology traditionally used for the tribe. For most analyses, the morphological matrix was concatenated with the molecular data (nucleotides plus microstructural characters). Nevertheless, the morphological data were excluded for ML due to the existence of polymorphic characters, which are not supported in the used version of RAxML GUI v. 1.3. (Silvestro and Michalak 2012). Furthermore, we optimized the entire morphological matrix onto the MP trees using the Fitch optimization criterion (Fitch 1971) as implemented in Winclada (Nixon 1999b). Figures then show different subsets of characters, useful at the corresponding taxonomic levels. The figures were created using the either CorelDraw v. 13 or Photoshop CS3. Characteristics of the datasets Table 2 Contribution of the different dataset to the total matrix The final alignment contained 5175 nucleotides. After exclusion of hot spots, the resulting nucleotide matrix comprised 5028 nucleotides, of which 904 were parsimony potentially informative. The DNA microstructural mutations were coded as a combined matrix from all three markers, which had 170 characters, of which 141 were parsimony potentially informative. The morphological matrix comprised 13 characters, all informative for parsimony. The concatenated matrix (sequence data and morphology) includes 179 terminals and 5211 characters, of which 1058 were parsimony potentially informative. A summary of the phylogenetic information for each dataset is presented in Table 2. The addition of morphological characters to the molecular matrix improved the resolution of the phylogenetic hypothesis. (Full trees based only in the molecular dataset are presented in Online Resource 2.) Overall relationships The tree topologies are congruent independently of the inference method, but there are only minor differences in resolution and/or support among the analyses even when including the few morphological characters (Online Resource 2). In Fig. 1, we present an overview of the Bayesian tree obtained with the concatenated dataset (179 taxa, nucleotides from trnK–matK, petD and trnL–F, DNA microstructural characters and morphology) in which the clades are presented that were well supported in at least one of the inference methods. (Detailed trees for each inference method including actual # Characters Total # Potentially informative for MP Excluding regions of uncertain homology Molecular: nucleotides trnK–matK 3007 2957 trnL–F 1060 1019 petD 1108 1052 Total 5175 5028 Molecular: microstructural characters trnK–matK 78 trnL–F 41 petD 51 Total 170 Morphological characters Morphology 13 Total 5211 13 Evolution model 535 179 190 904 GTR + I + G 67 33 41 141 MK 13 1058 MK The Rondeletieae tribe revisited Page 7 of 25 26 Fig. 1 Overview of phylogenetic relationships as inferred from the concatenated dataset (179 taxa, nucleotides from trnK–matK, petD and trnL–F, DNA microstructural characters and morphology) showing the well supported clades in either the BI, MP (L = 2409, Ci = 0.53, Ri = 0.87), or ML (ln = 8257.73178) topologies (trees are presented as Online Resources 1–3, respectively). Colors indicate tribes. The clade corresponding to Rondeletieae is annotated in blue with Guettardeae being sister. Light gray lines correspond to medium support, whereas unsupported nodes were collapsed branch support values are presented as Online Resources 3–5). This tree shows that tribes Rondeletieae, Guettardeae, Gardenieae, Sabiceeae, Condamineeae and Chiococceae are monophyletic. The clades for Rondeletieae and Guettardeae (together 157 terminals as a focus in our study with dense sampling) are supported as sisters. The phylogenetic position of Chione DC. with respect to Chiococceae and Guettardeae–Rondeletieae is in conflict 13 26 Page 8 of 25 A. Torres-Montúfar et al. Fig. 2 Parsimony consensus tree obtained from the concatenated analysis showing the variation in morphological characters and their states at the level of genera within Guettardeae and Rondeletieae in comparison with other tribes. Rondeletieae are morphologically uniform in these seven floral, fruit and pollen characters as circumscribed here, except Phyllomelia which differs by a different fruit type (samara with a single seed per ovule). At tribal level, circles indicate the number of DNA microstructural characters supporting the branch as synapomorphic (black) or homoplastic (white). The morphological synapomorphies were recognized using the Fitch optimization criterion. An asterisk (*) indicates taxa formerly classified as part of the Rondeletia complex. Line thickness stands for confidence into the reconstructed branches: thick lines indicate good support with all three methods (MP JK ≥ 85%, ML-BS ≥ 85% and BI-PP ≥ 0.95); medium-thick lines indicate either moderate support obtained by all methods (JK 64–85%, BS 80–85% and PP 0.85–0.95) or high support by at least one of them; thin dashed lines reflect low support by all methods (JK ≤ 64%, BS ≤ 80% and PP ≤ 0.85); gray branches include a single terminal among the trees. In the Bayesian analysis, it is sister to Chiococceae–Guettardeae–Rondeletieae (Online Resource 3); in parsimony (Online Resource 4), it is retrieved in a polytomy together with Chiococceae, Guettardeae and Rondeletieae, and in ML (Online Resource 5) it is sister to Chiococceae. For this reason, we still consider it as incerta sedis. Except for the conflicting position of Chione, there are no substantial differences among the inference methods or the inclusion versus exclusion of morphology. Figure 2 is the consensus of the parsimony tree based on the concatenated dataset in which tribes and genera within Guettardeae and Rondeletieae were condensed as single terminals. This figure shows the synapomorphies and variation of the relevant characters at this hierarchical level. In addition to individual nucleotides, all tribes are supported by DNA microstructural characters and/or by one of the seven morphological characters as synapomorphies. Exceptions are Guettardeae and Rondeletieae that are 13 supported as a sister group. The Guettardeae as annotated here refers to the now molecularly based accepted sensu lato circumscription of the tribe (e.g., Manns and Bremer 2010), in contrast to Guettardeae sensu stricto (e.g., Robbrecht 1988) in the traditional morphologically based circumscription of the tribe based on fruit morphology. The clade Rondeletieae–Guettardeae s.l. is well defined with respect to the other Cinchonoideae tribes (including those not sampled here) by the combination of quincuncial corolla aestivation and spathulate corolla lobes, and statistical support for it is high (97% JK, 98% BS, 1.0 PP). Support is also high for the Guettardeae s.l. (98% JK, 98% BS, 1.0 PP) and Rondeletieae (96% JK, 99% BS, 1.0 PP) clades as such (Online Resources 3–5 for trees of the different inference methods; BS values refer to ML without morphology). Within Cinchonoideae Chione shares the quincuncial corolla with Guettardeae s.l.-Rondeletieae as The Rondeletieae tribe revisited Page 9 of 25 26 Fig. 3 Relationships within Guettardeae–Rondeletieae based on the concatenated dataset (complete tree also depicting the other clades in online Appendix). Gray boxes indicate the taxa formerly placed in Rondeletieae. Support values are shown on each branch (PP and ML-BS above; MP-JK below). Branches were collapsed if PP, BS and JK values were equal or less than 0.85, 80% and 64%, respectively 13 26 Page 10 of 25 the only other genus outside this clade, but Chione differs by the shape of the corolla lobes (Fig. 2). The tribe Guettardeae (Fig. 3) The genera Arachnothryx, Javorkaea, Renistipula and Rogiera that were formerly placed in the Rondeletia complex do not share a most recent common ancestor with Rondeletieae, and they are also not part of the Guettardeae s.s. These genera, together with Gonzalagunia and Machaonia Bonpl. are paraphyletic with respect to Guettardeae s.s., so that Guettardeae s.l. comprises three well-supported Fig. 4 Parsimony consensus tree obtained from the concatenated analyses showing morphological characters and their states that are variable among the genera within Rondeletieae. The synapomorphies were recognized using the Fitch optimization criterion. Monophyletic genera are depicted as single terminals, and gray branches indicate that a terminal includes only a single species. Line thickness is related to support: thick branches are well supported by the three methods (MP-JK ≥ 85%, ML-BS ≥ 85% and BI-PP ≥ 0.95); medium branches have either moderate support in all methods (JK 64–85%, BS 80–85% and PP 0.85–0.95) or high support in at least one of them; thin dashed branches reflect low support throughout (JK ≤ 64%, BS ≤ 80% and PP ≤ 0.85) 13 A. Torres-Montúfar et al. clades: Machaonia pauciflora-Rogiera (98% JK, 72% BS), Guettardeae s.s. (99% JK, 100% BS, 1.0 PP) and Arachnothryx–Gonzalagunia–Javorkaea–Renistipula (Arachnothryx complex; 98% JK, 100% BS, 1.0 PP). Within this tribe, only Rogiera and Gonzalagunia were retrieved as monophyletic. (We only included one sample of Machaonia; therefore, its monophyly is not properly tested). Arachnothryx is paraphyletic with respect to Gonzalagunia and forms a polytomy with Javorkaea and Renistipula. Also Guettarda forms a polytomy with the morphologically related Antirhea Comm. ex Juss., Chomelia L. and Stenostomum C.F. Gaertn. The Rondeletieae tribe revisited Page 11 of 25 26 Fig. 5 Parsimony consensus tree obtained from the concatenated analysis depicting the numbers of synapomorphic versus homoplastic DNA microstructural and morphological characters that are variable among the genera within Rondeletieae. Characters were optimized using the Fitch criterion. Monophyletic genera are depicted as single terminals, and gray branches indicate that a terminal includes only a single species. Line thickness is related to support: thick branches are well supported by the three methods (JK ≥ 85%, BS ≥ 85% and PP ≥ 0.95); medium branches have either moderate support in all methods (JK 64–85%, BS 80–85% and PP 0.85–0.95) or high support in at least one of them; thin dashed branches reflect low support throughout (JK ≤ 64%, BS ≤ 80% and PP ≤ 0.85) The tribe Rondeletieae (Figs. 4, 5) lis–Rondeletia–Roigella. Within Clade I (Figs. 3, 4), the Mesoamerican monotypic genus Blepharidium is the earlier divergent taxon, and it is sister to two subclades (66% JK, 81% BS, 0.92 PP). One subclade includes the Mexican–Mesoamerican genera Donnellyanthus (monotypic) and Rovaeanthus (bitypic) (97% JK, 97% BS, 1.0 PP). Both genera can be distinguished by the number of floral parts (four in Donnellyanthus vs five in Rovaeanthus) and the corolla throat ornamentation (naked in Donnellyanthus vs with both a fleshy and hairy ring in Rovaeanthus). The other subclade includes the Antillean endemic genera Acunaeanthus (monotypic) and Suberanthus (nine species) (66% JK, 45% BS, 0.74 PP). Both genera can be distinguished by the corolla throat ornamentation (naked in Acunaeanthus and with a fleshy ring in Suberanthus) and the capsule dehiscence (loculicidal in Acunaeanthus vs septicidal in Suberanthus). All genera in Clade II (Fig. 4) are well represented in or even restricted to the Antilles, and, as in the case of the genera in Clade I, they do not have morphological synapomorphies or a unique combination of Within Rondeletieae, all genera represented with more than one species (or individuals in the case of monotypic genera) are supported as monophyletic: Acrosynanthus (100% JK, 100% BS, 1.0 PP), Donnellyanthus (95% JK, 99% BS, 1.0 PP), Rachicallis (99% JK, 100% BS, 1.0 PP), Rovaeanthus (99% JK, 100% BS, 1.0 PP), Suberanthus (93% JK, 92% BS, 1.0 PP) and Tainus (99% JK, 100% BS, 1.0 PP), except for Rondeletia, which is paraphyletic to Stevensia (85% JK, 88% BS, 0.98 PP). The genus Stevensia, therefore, will be treated hereafter as Rondeletia. In general, there is little if any structure within genera, but the relations among genera are overall fairly well supported both in MP and probabilistic inference methods. Within Rondeletieae, two clades are recovered: Clade I (67% JK, 86% BS, 0.92 PP), including Blepharidium–Rovaeanthus–Donnellyanthus–Acunaeanthus–Suberanthus and Clade II (98% JK, 97% BS, 1.0 PP) including Tainus–Acrosynanthus–Phyllomelia–Mazaea–Rachical- 13 26 A. Torres-Montúfar et al. Page 12 of 25 morphological features in common. There is a grade at the earlier divergent point of Clade II formed by Tainus (99% JK, 100% BS, 1.0 PP), followed by Acrosynanthus (100% JK, 100%, 1.0 PP), and further followed by the Rondeletia assemblage (88% JK, 91% BS, 0.99 PP): Phyllomelia–Mazaea–Rachicallis–Rondeletia–Roigella. Tainus is a monotypic genus endemic to Hispaniola Island, characterized by the papillate corolla throat ornamentation and also two SSRs in the trnL–F region (TGAT in the aligned positions 396–399 and TTTTC in the aligned positions 435–439, as in Torres-Montúfar et al. 2017 and Fig. 4). The genus Acrosynanthus, with seven species, is endemic to the Antilles and easily recognizable within the tribe by the stamens inserted at the base of the corolla tube (a feature not common among Rubiaceae), a white hairy ring at the corolla throat and a synapomorphic inversion in the trnK–matK region (TTATGAAA inverted to TTTCATAA in the aligned positions 331–338) (Fig. 4). The Rondeletia assemblage includes mostly Antillean species (particularly in Cuba), but Rachicallis and Rondeletia have some representatives in Mesoamerica and Rondeletia even in the Caribbean basin in South America (Fig. 4). Within this clade, the monotypic genus Roigella, which does not have a unique feature distinguishing it among other genera in the clade, is the earliest diverging lineage. The next is Rondeletia, which is the most specious and diverse genus in the entire tribe, encompassing around 100 species. The genus Rondeletia does not have a unique morphological feature distinguishing it within the tribe or even within Clade II, but it is supported by a synapomorphic SSR in the trnL–F region (ACTATATCAAA in aligned positions 261–271, Fig. 4); it is sister to Rachicallis, Mazaea and Phyllomelia (Fig. 4). The genus Rachicallis (monotypic) is unique within the tribe by the yellow corolla, and within Clade II by the stamens inserted near the middle of the corolla tube; in addition, it has a SSR in the petD region (GTAAG in the aligned positions 832–836) (Fig. 4). The genera Mazaea (bitypic) and Phyllomelia (monotypic) are endemic to Cuba. The genus Mazaea could be confused with Rondeletia by the fleshy ring at the corolla throat; however, it has wingless seeds (vs. winged in Rondeletia) and its septicidal capsules only carry a few seeds (2–6), a unique feature within the tribe. The genus Phyllomelia is easy to recognize by the one-seeded samaras, also unique within Rondeletieae (Fig. 4). The contribution of each data partition to the concatenated tree (parsimony tree inferred from nucleotide, microstructural and morphological characters) is summarized in Fig. 5. Morphological characters used in this analysis do not provide support for generic relationships as synapomorphies but are useful for the recognition of most genera. Exceptions are Blepharidium, Donnellyanthus, Acunaeanthus and Roigella, which do not have even homoplastic conditions. Only Rovaeanthus, Tainus, Phyllomelia and Rachicallis have unique conditions that distinguish them from 13 the rest (Fig. 5). Regarding molecular markers, trnK–matK and petD only contribute to clade support for and resolution within Clade II. In contrast, trnL–F is more or less evenly distributed among the Rondeletieae clades. Within Clade I, it provides the only synapomorphy for a sister group between Donellyanthus and Rovaeanthus (Fig. 5). Most DNA microstructural characters just allow to distinguish genera with their species, as only Phyllomelia–Mazaea share a synapomorphic four-nucleotide deletion in trnL–F (aligned positions 226–229). In contrast, most clades and genera are supported by synapomorphic point mutations of nucleotides; this is clearly correlated with clade support (Fig. 5). Discussion The sister tribes Guettardeae and Rondeletieae To date, our analysis includes the largest representation of Guettardeae and Rondeletieae together. Our results agree with previous studies (Rova et al. 2002; Robbrecht and Manen 2006; Manns and Bremer 2010) in that the Arachnothryx complex and Rogiera, both segregated from Rondeletia, do not share a most recent common ancestor with Rondeletieae. Previous authors (Robbrecht 1988) considered the corolla aestivation in the Arachnothryx complex and Rogiera to be imbricate, which is a common feature within Cinchonoideae. We found that imbricate corolla buds, better described as quincuncial corolla aestivation of Rondeletieae and Guettardeae s.l. and Chione, are unique among Cinchonoideae. Also, we describe the corolla lobes in Rondeletieae–Guettardeae s.l. as spathulate, a synapomorphy that contrasts with the common triangular or linear shape in other genera of Cinchonoideae (Fig. 2). Since the use of molecular phylogenetics, it has become clearer that Rondeletieae is more restricted than previously thought. Despite the agreement in the general tribal composition, it has become difficult to characterize Guettardeae s.l. versus Rondeletieae, even referred to as a complex of tribes by Robbrecht and Manen (2006). Previous studies retrieved the close relationship among these tribes having moderately to highly support values. Rova et al. (2002) using sequences of the plastid trnL–F region (including the trnL intron and the trnL–trnF spacer) found the clade with a parsimony JK value of 72%. Manns and Bremer (2010) combined matrix of several plastid regions (atpB–rbcL, ndhF, rbcL, rps16, trnL–F) and nuclear (ITS) reported the clade with a Bayesian PP 1.00. Torres-Montúfar et al. (2017) using the plastid regions (petD, trnL–F, trnK–matK) retrieved the clade highly supported with a Bayesian PP 1.0, parsimony JK 99% and maximum likelihood BS of 100%. Only the study by Rova et al. (2009) using the plastid marker trnL–F, the ribosomal rps16 and Page 13 of 25 The Rondeletieae tribe revisited nuclear ITS found an unsupported Rondeletieae clade and nested in a “paraphyletic assemblage with Guettardeae” (Rova et al. 2009). In our study, using the plastid regions (petD, trnL–F, trnK–matK) plus morphological characters, we found the clade Rondeletieae–Guettardeae highly supported: with a parsimony JK 97% and Bayesian PP 1.00, while the maximum likelihood bootstrap (excluding morphological characters) was also high, with 98% (Online Resources 3–5). Additionally, the monophyly of each tribe is highly supported in most analyses. We retrieved a monophyletic and highly supported Rondeletieae (1.0 PP, 99% ML-BS, and 96% MP-JK) and Guettardeae s.l. (1.0 PP, 98% ML-BS, and 98% MP-JK), while the tree provided by Manns and Bremer (2010) has Bayesian 1.0 PP, in contrast to Rova et al. (2002) where the support was lower for Rondeletieae (MP 87% JK) and Guettardeae (MP 91% JK). The current Rondeletieae and Guettardeae circumscription is provided by Manns and Bremer (2010), which does not provide a morphological framework to distinguish both tribes and the genera within Rondeletieae. Our study does provide a morphological diagnosis for the Rondeletieae tribe and explore potential synapomorphic characters (both molecular and morphological) to diagnose it and to distinguish from morphologically closely related genera within Guettardeae s.l. Regarding the close relationship among Rondeletieae–Guettardeae, the earlier phylogenetic analysis by Rova et al. (2002) is suggested as a possibility to accept one large Rondeletieae tribe including Guettardeae. However, their analysis resulted in poorly resolved and statistically unsupported trees. Despite the highly supported monophyly for the tribes and also for their sister relationship, in our study we also did not find morphological or DNA microstructural synapomorphies for each individual tribe; however, in the wait for further evidence including analyses with more species, particularly within the Guettardeae s.l. clade, and markers, we prefer to maintain both tribes. We believe that there is increased evidence for the existence of two sister clades that can be circumscribed with Rondeletieae on the one hand and Guettardeae on the other (Fig. 2). Furthermore, it is still possible to provide a diagnosis for Rondeletieae based on unique combination of characters: among genera conforming the tribal complex Guettardeae s.l. and Rondeletieae, when fruits are capsular multiseeded and bilocular, the pollen lacks endofissures or endocracks in Rondeletieae (Torres-Montúfar et al. in rev). Because the new diagnosis of Rondeletieae is based on pollen nexine continuity, a feature not usually reported, the following combinations of nonsynapomorphic characters serve to place genera in Rondeletieae with respect to Guettardeae s.l.: if the capsular fruits bear winged seeds, the corolla throat has a thick ring or the capsule has few seeds; 26 if the capsular fruits bear wingless seeds, the corolla throat is ornamented or the stamens are inserted in the middle portion of the corolla tube. For instance, those taxa that were formerly placed within the Rondeletia complex can be excluded from Rondeletieae by the lack of some morphological features in Rondeletieae. For example, Rogiera, which has capsular fruits with winged seeds, does not have a fleshy ring at the corolla throat and has pollen with endofissures (Torres-Montúfar et al. in rev.); the Arachnothryx complex does not have a fleshy ring or winged seeds and has pollen with endocracks (Torres-Montúfar et al. in rev.); Guettardeae s.s. has mesocarpic drupes (Torres-Montúfar et al. 2018). The Rondeletieae tribe All phylogenetic studies (Rova et al. 2002; Robbrecht and Manen 2006; Manns and Bremer 2010) suggest that there is a set of taxa that conform a natural group. In all analyses, there is a clade that includes: Blepharidium, Mazaea, Phyllomelia, Rachicallis, Roigella, Rondeletia, Rovaeanthus (sometimes as Rogiera suffrutescens) and Suberanthus. The genera Acunaeanthus, Donnellyanthus (Borhidi et al. 2011; treated as Rondeletia deamii), and Tainus (TorresMontúfar et al. 2017; treated as Rondeletia pitreana) were only included in some analyses (Rova et al. 2002; Manns and Bremer 2010). The genera Ariadne and Stevensia were treated as such in some studies, but they are now considered as synonyms of Mazaea and Rondeletia, respectively (Delprete 1999b; Borhidi 2010; Manns and Bremer 2010). Both, Acrosynanthus and Tainus are sister to Guettardeae–Rondeletieae in Rova et al. (2009), but they still included them in their synthesis of Rondeletieae. In summary, there is then either consensus or at least not disagreement in that there is a lineage of 12 genera (Acrosynanthus, Acunaeanthus, Blepharidium, Donnellyanthus, Mazaea, Phyllomelia, Rachicallis, Roigella, Rondeletia, Rovaeanthus, Suberanthus and Tainus) that we are treating as the Rondeletieae tribe, comprising around 120 species which are entirely neotropical with a diversity center in the Caribbean basin primarily at the Antilles island system, and only few genera and species in Mesoamerica. The excluded genera are discussed below. Phylogenetic relationships within the Rondeletieae tribe—Our trees include the largest set of genera (12 vs 9–12) and species (47 vs 17–34) within Rondeletieae compared to previous phylogenetic studies. The topology presented here most strikingly contrasts with the tree by Robbrecht and Manen (2006) that, however, depicted not only a differing overall branching order but several genera as nonmonophyletic. We believe that this could be an artifact of the phylogenetic reconstruction method, as they used a supertree approach, later shown to have several pitfalls (Von Haeseler 2012). Our results also 13 26 Page 14 of 25 differ considerably with respect to the trees by Rova et al. (2002, 2009), who provided the first trees of Rondeletieae inferred by parsimony methods from consistent sequence matrices. Rova et al. (2002) et al. analyzed a trnL–F data set, but their phylogeny has very little resolution inside Rondeletieae (annotated as clade “C5b” in their study). They basically recovered a clade with Acrosynanthus sister to Rondeletia, Mazaea, Phyllomelia, Rachicallis and Roigella within Rondeletieae. Their later paper (Rova et al. 2009) rather added confusion because the authors presented an ITS tree that was better sampled taxonomically but nodes were even less supported. Essentially, they found Rondeletia to be monophyletic when including Stevensia minutifolia (94% and 96% JK; R. pitreana was not sampled). Another tree that was published Rova et al. (2009) was based on ITS combined with trnL–F and rps16 sequences but much less taxon coverage, lacking most genera of Rondeletieae and lacking any node support values. In our analysis (Fig. 3, Online Resources 3–5), as well as in Manns and Bremer (2010), two main clades are recovered within Rondeletieae. The main difference is regarding the Blepharidium position, which in Manns and Bremer (2010) is found as the first divergent genus inside our Clade II, while we found it inside our Clade I. It is difficult to explain these differences without the precise alignment used by Manns and Bremer (2010) and without trees based on individual loci, i.e., the plastid genomic compartment and ITS since reticulate evolution cannot be excluded. On the other hand, the plastid dataset analyzed by TorresMontúfar et al. (2017) did not provide support for Clade I at all, by the three inference methods, depicting its genera as a polytomy except a Donellyanthus–Rovaeanthus lineage; however, individual analyses retrieved a supported Clade I in the Bayesian (PP 0.94) and MP studies (JK 80%), while in ML the Clade I is unsupported (BS 42%); in all these trees, the position of Blepharidium is uncertain regarding the two main Rondeletieae clades (see Supplementary material of Torres-Montúfar et al. 2017). Our plastid dataset also shows evidence for the Clade I (85% BS) in which Blepharidium branches first (81% BS, Online Resource 5). Including the morphological dataset, the clade is confirmed as weakly supported by MP (67% JK) to highly supported by Bayesian study (0.96 PP). There seems to be one morphological character state transformation supporting Clade I (Fig. 5). The position of Blepharidium was uncertain until the study by Rova et al. (2002); before that, Blepharidium was classified as Cinchoneae (Robbrecht 1988) or Hillieae (Robbrecht and Bridson 1993 and Andersson 1995). In the ITS tree of Rova et al. (2009), it is placed as sister to Suberanthus, but without any statistical support. Our results are in line with Manns and Bremer (2010) and Torres-Montúfar et al. (2017) in recovering the genus Donnellyanthus as sister to Rovaeanthus, in contrast to the 13 A. Torres-Montúfar et al. proposal by Lorence (2012a, b) to synonymize them with Arachnothryx and Rogiera (Guettardeae s.l.), respectively. Both genera are easily distinguishable by the flower merosity and the corolla throat ornamentation. In our analysis as well as in Manns and Bremer (2010), Acunaeanthus is sister to Suberanthus. Both genera have a homoplastic SSR (GT) in the aligned positions 765–766 of trnL–F also shared with Gardenieae, Sabiceeae and Calycophylleae (Fig. 5). Although the Acunaeanthus–Suberanthus clade is only weakly supported in our analyses (Fig. 3, Online Resource 5), this clade gained 1.0 PP in Manns and Bremer (2010) including ITS. Further increased character and taxon sampling is needed to test whether Suberanthus is monophyletic. At this point, we accept both genera because Acunaeanthus lacks ornamentation in the corolla throat and has loculicidal fruits, whereas Suberanthus has a fleshy ring on the corolla throat and the capsules are septicidal. For Clade II, our study (Figs. 3, 4, 5, Online Resources 3–5) confirms the finding by Manns and Bremer (2010) with increased posterior probability and now also high support from parsimony and likelihood reconstructions that Tainus (as R. pitreana in Manns and Bremer 2010) is the sister to all remaining genera. In our analysis, Roigella is the earliest divergent genus within the Rondeletia assemblage (Fig. 3), whereas Manns and Bremer (2010) recovered it sister to Rondeletia (1.0 PP). Torres-Montúfar et al. (2017) could not resolve relationships within the Rondeletia assemblage and depicted Roigella in a broad polytomy, whereas our ML analysis of the same plastid regions (trnK–matK, petD, and trnL–F) but an improved species sampling of Rondeletia recovered 90% BS for a sister group relationship of Roigella and Rondeletia (Online Resource 5). In our combined tree with morphology, the node for a core of the Rondeletia assemblage excluding Roigella received 0.95 PP and 86% JK (Fig. 3). However, there is only one morphological character supporting this node (Fig. 5) which is also homoplastic: flower merosity. The genus Roigella can have either five or six floral parts; therefore, the optimization is ambiguous and the rest of the clade have four floral parts, also shared with many other lineages in Rondeletieae, Guettardeae, Chiococceae or Calycophylleae, but with many shifts to five floral parts or even six, within Rondeletia species. Considering the presence of very short branches, it will be crucial to add further characters (e.g., phylogenomics) to further test the phylogenetic relationships of Roigella, which differs from all Rondeletia species by lacking a fleshy ring on the corolla throat and by wingless seeds. The latter are present in several other genera of the Rondeletia assemblage (Fig. 4), and as a result of convergent evolution the wings may rather support the monophyly of Rondeletia (including Stevensia) than being useful for inferring the relationships of Roigella. Rova et al. (2002, 2009) did not include this species in their analyses. Page 15 of 25 The Rondeletieae tribe revisited Rondeletia was retrieved as monophyletic by some studies (Rova et al. 2009; Manns and Bremer 2010), whereas others (Rova et al. 2002; Torres-Montúfar et al. 2017, 2018) could not resolve relationships with Mazaea, Phyllomelia, Rachicallis and Roigella. Our results show a well-supported Rondeletia clade (Fig. 3, as well as plastid sequence data alone, see Online Resource 2) and corroborate Rova et al. (2009) and Manns and Bremer (2010) in that Rondeletia is paraphyletic to Stevensia. The presence of an incipient fleshy ring and the winged seeds in Stevensia provides morphological support for the monophyly of Rondeletia including Stevensia as a SSR in trnL–F (Fig. 4) for the taxonomic decision to reduce Stevensia as a synonym of Rondeletia, also proposed by Borhidi (2010). There is agreement between our results and those of Manns and Bremer (2010) in that Mazaea, Phyllomelia, and Rachicallis share a most recent common ancestor within the Rondeletia assemblage. Mazaea and Phyllomelia were already found as sisters in Rova et al. (2002) based on ITS and in Torres-Montúfar et al. (2017) based on plastid sequences. Rova et al. (2002) and Manns and Bremer (2010) sampled both species of Mazaea, while we could only include M. shaferi, which was at some point transferred to a separate genus, Ariadne (Urban 1922). In Rova et al. (2002) these three species form a polytomy, while in Manns and Bremer (2010) Mazaea is paraphyletic with respect to Phyllomelia. Even though the paraphyly of Mazaea has perfect support in Manns and Bremer (2010), we prefer to maintain both as distinct genera because they are morphologically very different: Mazaea has a fleshy ring on the corolla throat and capsular dehiscent fruits with two to six seeds per locule, while Phyllomelia has a naked corolla throat and samaras with one-seeded locules. To be consistent with the phylogeny, Ariadne (M. shaferi) should then also be recognized as a different genus, but the morphological characters used to distinguish M. shaferi from M. phialanthoides are quantitative and there is no justification to use them at generic level (for a more thorough discussion, see Delprete 1999b). It remains to be tested whether plastid data support the paraphyly of Mazaea. At this moment, considering the lack of resolution between the three species in Rova et al. (2002) that opens the possibility of the Mazaea monophyly and the lack of morphological/molecular characters to distinguish Mazaea and Ariadne, we prefer to tentatively maintain Ariadne as a synonym. The genus Rachicallis is also morphologically very different and, in fact, it has the longest branch within the clade, including a SSR: GTAAG in the aligned positions 832–836 of petD (Fig. 4). It was first classified within Hedyotideae (De Candolle 1830; now Rubioideae) probably at least in part because it is small shrub (instead of small trees), but overall it is the most distinctive genus inside Rondeletieae: the yellow corollas with the stamens inserted in the middle 26 portion of the tube are unique; in addition, its coastline habitat is reflected in very succulent small leaves with short internodes. The genus Jamaicanthus The recently described monotypic genus Jamaicanthus (Borhidi 2018) was segregated from Rondeletia (R. laurifolia). The species was collected by Olof Swartz in Jamaica between 1784 and 1786 (Stearn 1980) and later described by Swartz in 1797. The type specimens are not in a perfect stage, but it is still possible to see a few flower buds and a couple of open fruits with winged seeds. Both the original description and the type images (JStor Global Plants) are consistent with Rondeletia, except that the fairly detailed species original description does not mention the presence of a ring on the corolla throat. Borhidi (2018) highlights in the description of the genus several morphological features, the most relevant of which are the small corolla tube (1.5–2.5 mm long) and the naked corolla throat. This species has not been included in any phylogenetic analysis, but the lack of a ring on the corolla throat would be determinant to not consider its inclusion in Rondeletia. A definitive test of the phylogenetic position of this species is needed to firmly accept the status of this genus, but with the available information we decide to tentatively include the genus as part of the tribe. Genera excluded from Rondeletieae Despite the fact that our study includes the most comprehensive taxon sampling for Rondeletieae, it is still necessary to test the position of a few genera never included in a phylogenetic context due to the difficulty to access material. Manns and Bremer (2010) suggested to tentatively include six genera in Rondeletieae that they could not sample: Acrobotrys, Glionnetia, Habroneuron, Holstianthus, Spathichlamys and Standleya. Thus, efforts to rediscover populations of Acrobotrys, Habroneuron and Stylosiphonia, which are only known from the type collections, will be important. In the case of R. pitreana that was known only from the type collection made in 1929, it was rediscovered in 2003 (Peguero et al. 2007) through fieldwork in Hispaniola Island, which after its rediscovery could be included in a phylogenetic analysis and proved to represent a new genus (TorresMontúfar et al. 2017). In the absence of robust phylogenetic results, we revised the original descriptions, the type specimens (JStor Global Plants) and further relevant literature in order to assess the morphological characters that were analyzed here in a phylogenetic context and to suggest the inclusion or exclusion of the respective from Rondeletieae using the emended diagnosis of the tribe of this study (see 13 26 Page 16 of 25 below). For a number of genera, we are confident that they do not belong to Rondeletieae as circumscribed here. The Colombian monotypic genus Acrobotrys is only known from the type collection from 1908. Rova (1999) tentatively included it within Condamineeae. The genus Acrobotrys has a four-locular ovary typical of Condamineeae (Kainulainen et al. 2010) but no spathulate corolla lobes. So we agree with the exclusion of this genus from Rondeletieae, contrary to Manns and Bremer (2010). After Manns and Bremer (2010), the monotypic African genus Glionnetia was found with an unresolved position relative to Vanguerieae and the Aleisanthieae–Greeneeae–Ixoreae clade within Ixoroideae (Razafimandimbison et al. 2011). Furthermore, in the original description (Tirvengadum 1984) the corolla aestivation was referred to as contorted. Therefore, its tentative inclusion in Rondeletieae is rejected, which is also in line with the geographical distribution outside the range of Rondeletieae. The Mexican monotypic genus Habroneuron was described as closely related to Plocaniophyllon Brandegee, Sommera Schltdl. and Sabicea, which all have lineolate venation in common (Standley 1927). Nevertheless, Habroneuron differs from the previous by the contorted corolla aestivation, which was used as an argument to place it within Rondeletieae (Robbrecht 1988) when the tribe had a broader circumscription. This genus is known only from two collections from the 1800 hundreds and its fruits are unknown. Darwin (1980) remarks the close morphological affinity between Habroneuron and Lindenia Benth., both classified within Rondeletieae by Hooker (1873), by the creeping habit, membranaceous and striate leaves, rounded corolla lobes and included anthers. The phylogenetic analysis by Kainulainen et al. (2013) suggested the treatment of Lindenia as a synonym of Augusta Pohl and placed it within the Coffeae alliance (Ixoroideae). Despite the fact that the fruits of Habroneuron are still unknown and that it has never been included in phylogenetic studies, based on the creeping habit, and the contorted corolla aestivation, we support its exclusion from Rondeletieae. The monotypic genus Holstianthus from the Amazonian Guyana was accommodated in tribe Rondeletieae by Robbrecht (1988). However, it differs from the current Rondeletieae genera by its herbaceous habit and the indehiscent dry fruits (aside from Phyllomelia). In the protologue, Steyermark (1986) compared it with Sipaneopsis Steyerm. also included in Rondeletieae sensu Robbrecht (1988). The genus Sipaneopsis nevertheless has a densely barbate corolla throat with elongated yellow hairs (as in Rogiera, now in Guettardeae). Molecular phylogenetic analysis (Delprete and Cortés 2004) recovered Sipaneopsis within the Sipaneeae tribe as part of Ixoroideae. Because Holstianthus is herbaceous and the corolla aestivation is undoubtedly contorted, we propose to exclude it from Rondeletieae. 13 A. Torres-Montúfar et al. The monotypic Asian genus Spathichlamys was included within Rondeletieae by Robbrecht (1988). The morphology suggests a close relation to Greenea Wight & Arn., also included within Rondeletieae by Hooker (1873), placed within Ixoreae by Rova (1999) and Greeneae by Mouly et al. (2009). The morphological affinity between both genera led Tange (2013) to treat them as synonyms. Following these studies, and taking into consideration the geographical distribution, we agree that Spathichlamys should be tentatively included within Ixoroideae. The monotypic Brazilian genus Standleya was proposed to be part of Rondeletieae by Robbrecht (1988); however, it is herbaceous. In the protologue, Brade (1932) suggested that it is related to Bradea Standl. ex Brade, traditionally placed at Hedyotideae (Robbrecht 1988). Based on morphological similarities and on unpublished DNA sequences, Delprete and Jardim (2012) suggested including both genera in Coussareeae, result recently corroborated by Löfstrand et al. (2019). In addition to the previous genera, three other genera deserve discussion: Blandibractea Wernham, Renistipula and Stylosiphonia. The monotypic Brazilian genus Blandibractea was originally placed in Rondeletieae (Werham 1917) based on three original collections, which lack fruits. Posteriorly, Delprete (1998) considered this species as a synonym of Simira glaziovii (K.Schum.) Steyerm., a proposition that has been generally accepted. The genus Simira Aubl. was at some point considered part of Rondeletieae, but phylogenic analyses place them now in Condamineeae (Rova et al. 2002; Kainulainen et al. 2010). We support that Blandibractea should be excluded from Rondeletieae. The Mesoamerican genus Renistipula Borhidi comprises three species; it was described to encompass Rondeletia species with reniform stipules, and hence it was included within Rondeletieae (Borhidi et al. 2004). Nevertheless, it was not included by Rova et al. (2002, 2009) or Manns and Bremer (2010). By its morphology, we consider it as a synonym of Arachnothryx (Guettardeae), which was also proposed in the recent floristic treatment for Mesoamerica (Lorence 2012a). In contrast, molecular analysis suggested its inclusion within Hamelieae (Stranczinger et al. 2014). We question this last result because Renistipula does not have raphides, its inflorescences have bracts and its flowers are not yellow reddish. When we tried to replicate these results by downloading and re-analyzing the sequences used by Stranczinger and collaborators (results not shown), we corroborate the placement of Renistipula within Hamelieae, but we also found one species of Deppea (Hamelieae) inside Rondeletieae. Perhaps sequences were mixed up at some point, so they appear with wrong annotations in GenBank. However, Torres-Montúfar (2018) suggests that Renistipula Page 17 of 25 The Rondeletieae tribe revisited should indeed be considered as a synonym of Arachnothryx and hence, excluded from Rondeletieae. The type species from the Mesoamerican genus Stylosiphonia is only known from the type collection made by Purpus in 1913. The genus Stylosiphonia was placed within Rondeletieae by Robbrecht (1988). This proposal was accepted by Borhidi (2006) but has never been tested in phylogenetic analyses. In the original description, Brandegee (1914: 70) mentioned that the genus has “Corollae… limbi 5 lobi, lanceolati, elongati…” and capsular fruits with “semina angulate, rugosa.” The original material deposited at F and GH and UC (accessed as image through JStor Global Plants) shows flower characteristics which are not present among other Rondeletieae such as long-tubular flowers with long linear lobes. Standley (1921) mentioned the corolla of the genus to be contorted. Despite that Stylosiphonia glabra Brandegee needs to be included in molecular phylogenetic analysis, we propose to exclude it from Rondeletieae based on the contorted corolla aestivation, the narrow elongated lanceolate corolla lobes. Later on, Standley (1924) added a second species to the genus Stylosiphonia (Stylosiphonia salvadorensis Standl.), based on fruiting material. Even in the original description, Standley doubted the generic placement of the species due to the lack of flower material. This species is currently considered a synonym of Arachnothryx jurgensenii (Hemsl.) Borhidi, to what we agree considering the available morphological information. Considering the wingless seeds, if this species were to be Rondeletieae, among the current taxa, it is only similar to Rachicallis, but they are very different in habitat, lifeform, leave texture, shape and size. For these reasons and pending collections in the type locality S. salvadorensis, we suggest to tentatively excluding it from Rondeletieae. Biogeography Our phylogeny implies for sure more than one colonization event, at least four colonization events between the Antilles and the continental land (Fig. 4). Our methods do not allow determining the migration directionality for these colonization events. Despite the fact that our sampling of Rondeletia is incomplete, it is still possible to say that inside the Rondeletia assemblage, there must have been more than one independent migration from the Antilles to continental land because Rachicallis does not share a most recent common ancestor with the sampled Mesoamerican species (Rondeletia thiemei Donn. Sm.), which is well nested within Antillean species. It remains to be tested whether by adding more continental species of Rondeletia, their group reveal a single continental radiation or 26 they reveal several independent migrations between both areas. It is remarkable that Rondeletieae can be considered a Caribbean lineage, except for the genera Blepharidium, Donnellyanthus and Rovaeanthus, the rest of the genera and species are restricted to the Antilles (Acevedo-Rodriguez and Strong 2012), and even the South American Rondeletia species are restricted to the Caribbean basin. Sampling density of population of Rachicallis americana (Jacq.) Hitchc, which is widespread with coastline populations in the Antilles and continental land, is desirable to produce phylogeographical studies that might shade light upon specific patterns of migration among continental land and the Antilles. This opens the possibility that morphological variations formerly used to describe species could in fact correspond to genetic divergence, e.g., in ring speciation (Martins et al. 2013). Conclusions The present study improves our understanding of the phylogenetic relationships and circumscription of Rondeletieae and also of the generic limits. We provided morphological characters that are phylogenetically informative at tribal and generic ranks. Concerning the Rondeletia complex, it is confirmed that Arachnothryx, Javorkaea, Renistipula and Rogiera should be excluded from Rondeletieae and that they are paraphyletic with respect to Guettardeae s.s. The clades Guettardeae s.l. and Rondeletieae are sister: both share quincuncial aestivation and spathulate corolla lobes. Among genera conforming the tribal complex Guettardeae s.l.-Rondeletieae, when fruits are capsular multiseeded and bilocular, the pollen lacks endofissures or endocracks in Rondeletieae. These character combinations allowed us to confidently exclude genera that were previously tentatively included in Rondeletieae; the tribe should include for sure 12 genera: Acrosynanthus, Acunaeanthus, Blepharidium, Donnellyanthus, Mazaea, Phyllomelia, Rachicallis, Roigella, Rondeletia, Rovaeanthus, Suberanthus and Tainus plus one (Jamaicanthus) tentatively included. With the current circumscription, the tribe comprises approximately 120 species mainly restricted to the Caribbean region. A follow-up phylogeny focusing more on the Guettardeae tribe is still needed to determine whether the genera formerly placed in Rondeletieae should be considered part of it and to address the systematic position of other genera currently placed in that tribe. 13 26 Page 18 of 25 A. Torres-Montúfar et al. Based on the results from the present study, we propose the following taxonomic synthesis for Rondeletieae, based on our morphological observations. Tribe Rondeletieae (DC.) Miq., Flora van Nederlandsch Indie 2: 130, 156. 1856. ≡ Rondeletiinae DC., Prodr. 4: 342, 401. 1830, as tribe Hedyotideae, subtribe “Rondeletieae”. ≡ Rondeletieae DC. ex Rchb., Deut. Bot. Herb. 1: 77. 1841.—TYPE: Rondeletia L. Fig. 6 Rondeletieae. a–c Examples of genera with white flowers and lacking a fleshy corolla throat ring. a Blepharidium guatemalense (photograph by N. Hellmuth); b Roigella correifolia (photograph by R. Rankin); c Tainus pitreanus (photograph by A. Torres-Montúfar). d–g Examples of genera with reddish flowers; d Donnellyanthus deamii. e Rondeletia buxifolia (photograph by A. Torres-Montúfar). f Rondeletia merilloana (photograph by A. Torres-Montúfar). g Rovaeanthus strigosus showing the fleshy ring and the hairy ring on the corolla throat (photograph by H. Ochoterena). h Rachicallis americana with yellow flowers and succulent leaves (photograph by R. B. Foster); i Phyllomelia coronata samara fruits (photograph by J. Salazar) Taxonomic treatment 13 The Rondeletieae tribe revisited Page 19 of 25 26 Fig. 7 Schemes summarizing the main morphological character combination to distinguish the genera within Rondeletieae and heatmaps showing their geographic distribution 13 26 Page 20 of 25 A. Torres-Montúfar et al. Fig. 7 (continued) Shrubs or trees; raphides absent; thorns absent. Stipules free or connate at base, mostly entire, interpetiolar, persistent to readily caducous; leaves opposite or verticillate, petiolate to sessile; domatia variably present or absent. Inflorescences terminal or axillar, cymose, paniculate or thyrsoid, bracteate. Flowers hermaphroditic, 4- to 6(7)-merous; calyx persistent or caducous; incipient calycophylls rarely present, all calyx lobes acrescent into rotate pterophylls (green to greenish white) after anthesis rare (in Phyllomelia); aestivation quincuncial; corolla rotate, corolla lobes spathulate, corolla throat naked, with a fleshy ring (in Mazaea, Rondeletia, Suberanthus) and/ 13 or with a hairy ring (in respectively Rovaeanthus and Acrosynanthus); stamens mostly as many as the corolla lobes, inserted near the base (in Acrosynanthus), at the medial zone (in Rachicallis), or more often near the corolla throat; anthers included or exserted, dorsifixed near the base or around the middle; ovary inferior or secondary half-inferior (in Rachicallis), bilocular, with few to many ovules per locule, rarely one (in Phyllomelia). Fruits woody capsules, loculicidal or septicidal (Mazaea, Rachicallis, Suberanthus), or rarely indehiscent samaras (in Phyllomelia); seeds angulate, winged or not. The Rondeletieae tribe revisited Genera included: Acrosynanthus, Acunaeanthus, Blepharidium, Donnellyanthus, Mazaea, Phyllomelia, Rachicallis, Roigella, Rondeletia, Rovaeanthus, Suberanthus, Tainus (Figs. 6, 7). Genera tentatively included: Jamaicanthus. Acknowledgements The first author (A.T.M.) thanks the Programa de Posgrado en Ciencias Biológicas, UNAM for a graduate scholarship (CONACyT grant No. 239869), to carry out a doctoral project under the supervision of H. Ochoterena. We further thank the Verein der Freunde des Botanischen Gartens und Botanischen Museums Berlin-Dahlem for funding fieldwork in Cuba and laboratory work in the context of the program on plant diversity of Cuba and the Caribbean. We are grateful to K. Windeler and Marítima Dominicana for providing financial resources supporting additional fieldwork in the Dominican Republic; to Ricardo García and the JBSD herbarium staff and to Nora Hernández and Rosa Rankin (HAJB). Special thanks are due to the authorities in Cuba, the Dominican Republic and México for granting permits. We appreciate the technical support of Bettina Giesicke (Freie Universität Berlin). A.T.M. further thanks the BGBM for additional support during a research internship in Berlin in the course of the doctoral studies, and the programa posgrado UNAM for funding the travel cost. We also would like to acknowledge the Center for Conservation and Sustainable Development at the Missouri Botanical Garden for providing a Shirley A. Graham Fellowship, which allowed the first author to visit MO. Compliance with ethical standards Conflict of interest The authors declare that they have no conflict of interest. Appendix Taxa sampled, voucher data, geographical origin and GenBank accession numbers. Data format: Subfamilial rank: Tribal rank: Species names, followed by the country of origin, collector(s) and collection number of the specimen (herbarium and/or silica gel dried sample), the herbarium acronym in parentheses, and the DNA project code (RUB000). GenBank accession numbers refer to sequences of genomic regions in the following order: trnK/matK, trnL–F, petD. Except for new sequences from each of the three regions of eight samples that are highlighted with an asterisk (*), sequences are from Torres-Montúfar et al. (2017), Additional trnK–matK and trnL–F sequences were taken from Rova et al. (2002, 2009). Subfamily Cinchonoideae: Tribe Calycophylleae: Calycophyllum candidissimum (Vahl) DC., Cuba, T. Borsch et al. 5125 (B; HAJB; PAL), RUB336, KY785298, KY614094, -. Tribe Chiococceae: Ceuthocarpus involucratus (Wernham) Aiello, Cuba, T. Borsch 4995 (B; HAJB; PAL), RUB233, -, KY785213, KY614097; Chiococca cubensis Urb., Cuba, S. Fuentes et al. 535 (B; HAJB; PAL), RUB264, -, KY785214, KY614098; Erithalis fruticosa L., Page 21 of 25 26 Dominican Republic, S. Fuentes et al. 221 (B; JBS), RUB183, -, KY785218, KY614101; Erithalis vacciniifolia (Griseb.) C.Wright, Dominican Republic, S. Fuentes et al. 1044 (B; JBS), RUB303, KY785272, KY785217, KY614100; Exostema caribaeum (Jacq.) Schult., Dominican Republic, S. Fuentes et al. 1177 (B; JBS), RUB294, -, KY785220, KY614103; Exostema longiflorum (Lamb.) Roem. & Schult., Cuba, N. Köster 2666 (B; HAJB; PAL), RUB251, -, KY785221, KY614104; Exostema spinosum (Le Vavass.) Krug & Urb., Dominican Republic, S. Fuentes et al. 207 (B; JBS), RUB243, KY785273, KY785219, KY614102; Isidorea leonardii Urb., Dominican Republic, S. Fuentes et al. 1210 (B; JBS), RUB302, KY785280, KY785232, KY614115; Isidorea veris Ekman ex Aiello & Borhidi, Dominican Republic, S. Fuentes et al. 977 (B; JBS), RUB284, -, KY785233, KY614116; Phialanthus sp., Cuba, S. Fuentes et al. 545 (B; HAJB; PAL), RUB265, -, KY785236, KY614119; Portlandia sp., Cuba, M. Ackermann 847 (B; HAJB; PAL), RUB226, -, KY785239, KY614122. Tribe Guettardeae: Antirhea lucida (Sw.) Benth. & Hook.f., Cuba, T. Borsch et al. 5288 (B; HAJB; PAL), RUB166, KY785299, KY785196, KY614077; Arachnothryx affinis (Hemsl.) Borhidi, Mexico, H. Ochoterena 644 (MEXU), KY785257, KY785197, KY614078; Arachnothryx aspera (Standl.) Borhidi, Costa Rica, E. Lepiz 174 (MEXU), RUB094, KY785258, -, KY614079; Arachnothryx buddleioides (Benth.) Planch., Mexico, H. Ochoterena 924 (MEXU), RUB130, KY785259, KY785198, KY614080; Arachnothryx capitellata (Hemsl.) Borhidi, Mexico, H. Ochoterena 850 (MEXU), RUB076, KY785260, KY785199, KY614081; Arachnothryx guerrerensis (Lorence) Borhidi, Mexico, D. Breedlove 61973 (MEXU), RUB343, KY785300, KY785200, KY614082; Arachnothryx heteranthera (Brandegee) Borhidi, Mexico, H. Ochoterena 823 (MEXU), RUB046, KY785261, KY785201, KY614083; Arachnothryx jurgensenii (Hemsl.) Borhidi, Mexico, H. Ochoterena 803 (MEXU), RUB042, KY785262, -, KY614084; Arachnothryx leucophylla (Kunth) Planch., Mexico, H. Ochoterena 752 (MEXU), RUB043, KY785301, KY785202, KY614085; Arachnothryx manantlanensis (Lorence) Borhidi, Mexico, R. Cuevas 4978 (MEXU), RUB104, KY785263, KY785203, KY614086; Arachnothryx pumae Torr.-Montúfar & H.Ochot., Mexico, H. Ochoterena 741 (MEXU), RUB069, KY785264, KY785204, KY614087; Arachnothryx pyramidalis (Lundell) Borhidi, Mexico, M. Heath 414 (MEXU), RUB088, KY785302, KY785205, KY614088; Arachnothryx secundiflora (B.L.Rob.) Borhidi, Mexico, H. Ochoterena 743 (MEXU), RUB118, KY785265, KY785206, KY614089; Arachnothryx stachyoidea (Donn. Sm.) Borhidi, Mexico, H. Ochoterena 842 (MEXU), RUB067, KY785266, KY785207, KY614090; Arachnothryx tabascensis Borhidi, Mexico, H. Ochoterena 891 (MEXU), KY785267, KY785208, KY614091; Arachnothryx villosa 13 26 Page 22 of 25 (Hemsl.) Borhidi, Mexico, H. Ochoterena 846 (MEXU), RUB124, KY785268, KY785209, KY614092; Chomelia brachypoda Donn. Sm., Mexico, H. Ochoterena 746 (MEXU), RUB119, KY785270, KY785215, KY614099; Gonzalagunia killipii Standl., Ecuador, M. Zak 3566 (MEXU), RUB049, KY785304,, KY614105; Gonzalagunia panamensis (Cav.) K.Schum., Cuba, N. Köster 2506 (B; HAJB; PAL), RUB418, KY785274, KY785222, -; Gonzalagunia rudis (Standl.) Standl., Costa Rica, R. Forero 7419 (MEXU), RUB095, KY785305, KY785223, KY614106; Gonzalagunia thyrsoidea (Donn.Sm.) B.L. Rob., Guatemala, M. Gonzalez 1455 (MEXU), RUB047, KY785275, KY785224, KY614107; Guettarda camagueyensis Britton, Cuba; T. Borsch et al. 4028 (B; HAJB; PAL), RUB017; Cuba, -, KY785231, KY614114; Guettarda elliptica Sw., Mexico, H. Ochoterena 894 (MEXU), RUB125, KY785306, KY785225, KY614108; Guettarda ferruginea C.Wright ex Griseb., Cuba, T. Borsch et al. 5007 (B; HAJB; PAL), RUB152, KY785278, KY785229, KY614112; Guettarda lamprophylla Urb., Dominican Republic, S. Fuentes et al. 1202 (B; JBS), RUB382, KY785276, KY785226, KY614109; Guettarda monocarpa Urb., Cuba, T. Borsch et al. 4429 (B; HAJB; PAL), RUB153, KY785307, KY785227, KY614110; Guettarda prenleloupii Urb., Dominican Republic, S. Fuentes et al. 1205 (B; JBS), RUB292, KY785279, KY785230, KY614113; Guettarda pungens Urb., Dominican Republic, S. Fuentes et al. 238 (B; JBSD), RUB184, KY785277, KY785228, KY614111; Javorkaea hondurensis (Donn.Sm.) Borhidi & Jarai-Koml., Honduras, J. Linares 6241 (MEXU), KY785281, KY785234, KY614117; Machaonia pauciflora Urb., Cuba, T. Borsch et al. 5218 (B; HAJB; PAL), RUB164, KY785282, KY785235, KY614118; Rogiera amoena Planch., Mexico, H. Ochoterena 805 (MEXU), RUB131, KY785284, KY785240, KY614123; Rogiera cordata (Benth.) Planch., Mexico, H. Ochoterena 732 (MEXU), RUB072, KY785285, KY785241, KY614124; Rogiera ligustroides (Hemsl.) Borhidi, Mexico, H. Ochoterena 781 (MEXU), RUB068, KY785286, KY785242, KY614125; Rogiera macdougalli Lorence, Mexico, H. Ochoterena 841 (MEXU), RUB073, KY785288, KY785244, KY614127; Rogiera nicaraguensis (Oerst.) Borhidi, Honduras, J. Linares 3520 (MEXU), RUB064, KY785287, KY785243, KY614126; Rogiera stenosiphon (Hemsl.) Borhidi, Mexico, H. Ochoterena 749 (MEXU), RUB075, KY785289, KY785245, KY614128. Tribe Rondeletieae: Acrosynanthus revolutus Urb., Cuba, T. Borsch et al. 4156 (B; HAJB; PAL), RUB224, MF460511, MF460689, MF460590); Acrosynanthus trachyphyllus Standl., Cuba, T. Borsch et al. 4444 (B; HAJB; PAL), RUB154, KY785256, KY785195, KY614076; Acrosynanthus trachyphyllus Standl., Cuba, S. Fuentes et al. 602 (B; HAJB; PAL), RUB203,(MF460512, MF460690, MF460591); Acunaeanthus tinifolius (Griseb.) Borhidi, 13 A. Torres-Montúfar et al. Cuba, Stahl et al. s.n. (S), -, GQ852451, -; Blepharidium guatemalense Standl., Guatemala, Gustafsson et al. 211 (GB), -, AF152735, -; Donnellyanthus deamii (Donn.Sm.) Borhidi, Honduras, S. Duery 172 (MEXU), RUB056, KY785271, KY785216, -; Mazaea shaferi (Standl.) Delprete, Cuba, T. Borsch et al. 4075 (B; HAJB; PAL), RUB144, (MF460567, MF460749, MF460657); Phyllomelia coronata Griseb., Cuba, T. Borsch et al. 4620 (B; HAJB; PAL), RUB158, KY785303, KY785210, KY614093; Rachicallis americana (Jacq.) Hitchc., Cuba, N. Köster 2465 (B; HAJB; PAL), RUB170, KY785283, KY785238, KY614121; Rachicallis americana (Jacq.) Hitchc., Cuba, T. Borsch et al. 5625 (B; HAJB; PAL), RUB217, (MF460568, MF460750, MF460658); Roigella correifolia (Griseb.) Borhidi & M. Fernández, Cuba, Rova et al. 2262 (GB), -, AF152746, -; Rondeletia alaternoides A. Rich, Cuba, Rova et al. 2228 (GB), -, AF152740, -; Rondeletia baraconensis Urb., Dominican Republic, S. Fuentes et al. 315 (B; JBS), RUB187, KY785290, KY785246, KY614129; Rondeletia berteroana DC., Dominican Republic, S. Fuentes et al. 239 (B;), RUB185, (MF460575, MF460758, MF460666); Rondeletia buxifolia Vahl, Montserrat islands, Veloz et al. 1868 (MO), -, GQ852555, -; Rondeletia camarioca C.Wright, Cuba, T. Borsch et al. 5601 (B; JBN; PAL), RUB215; KY785294, KY785251, KY614134; Rondeletia camarioca C.Wright, Cuba, T. Borsch et al. 5096 (B; HAJB; PAL), RUB215, KY785294, KY785251, KY614134; Rondeletia cristalensis Urb., Cuba, N. Köster 2828 (B; HAJB; PAL), RUB178, (MF460578, MF460761, MF460669); Rondeletia fuertesii Urb., Dominican Republic, S. Fuentes et al. 1170 (B; JBS), RUB298, KY785291, KY785247, KY614130; Rondeletia hameliifolia Dwyer & M.V.Hayden, Panama, Kirkbride & Hayden 164 (NY), -, GQ852546, -; Rondeletia hypoleuca Griseb., Cuba, T. Borsch et al. 4204 (B; HAJB; PAL), RUB020, KY785292, KY785248, KY614131; Rondeletia inermis (Spreng.) Krug & Urb., Puerto Rico, Acevedo-Rodriguez et al. 7691 (NY), -, AF152745, -; Rondeletia intermixta Britton, Cuba, Rova et al. 2245 (GB), -, AF152742, -; Rondeletia merilloana Urb., Dominican Republic, S. Fuentes et al. 1136 (B; JBS), RUB353, KY785293, KY785249, KY614132; Rondeletia microphylla Griseb., Cuba, T. Borsch et al. 4169 (B; HAJB; PAL), RUB018, KY785308, KY785250, KY614133; Rondeletia nipensis Urb., Dominican Republic, Delprete et al. 8651 (UPS), -, GQ852547, -; Rondeletia portoricensis Krug & Urb., Puerto Rico, C. Taylor 11687 (MO), -, AF152743, -; Rovaeanthus strigosus (Benth.) Borhidi, Guatemala, M. Veliz 6539 (MEXU), RUB063, KY785295, KY785252, KY614135; Rovaeanthus strigosus (Benth.) Borhidi, Guatemala, D. Lorence 8920 (PTGB), -, GQ852550, -; Rovaeanthus suffrutescens (Brandegee) Borhidi, Mexico, B. Bremer 2712 (S), -, AF152738, -; Suberanthus brachycarpus (Griseb.) Borhidi & M.Fernández, Cuba, McDowell 4824 Page 23 of 25 The Rondeletieae tribe revisited (DUKE), -, HM045004, -; Suberanthus stellatus (Griseb.) Borhidi & M.Fernández, Cuba, Rova et al. 2219 (GB), -, AF152736, -; Tainus pitreanus (Urb. & Ekman) J.A.TorresMontúfar, H.Ochoterena & T. Borsch et al., Dominican Republic, S. Fuentes et al. 1110 (B; JBS), RUB311, (MF460587, MF460773, MF460686); Tainus pitreanus (Urb. & Ekman) J.A.Torres-Montúfar, H.Ochoterena & T.Borsch et al., Dominican Republic, T. Clase 4228 (JBSD, MO), RUB354, (MF460588, MF460774, MF460687). Subfamily Ixoroideae: Tribe Condamineae: Picardaea haitiensis Urb., Dominican Republic, S. Fuentes et al. 1017 (B; JBS), RUB409, KY785297, KY785237, KY614120. Tribe Gardenieae: Casasia clusiifolia (Jacq.) Urb., Cuba, S. Fuentes et al. 761 (B; HAJB; PAL), RUB271, KY785269, KY785211, KY614095; Casasia jacquinioides (Griseb.) Standl., Cuba, T. Borsch et al. 4990 (B; HAJB; PAL), RUB232, -, KY785212, KY614096; Randia aculeata L., Cuba, T. Borsch et al. 5316 (B; HAJB; PAL), RUB245, KY785255, KY785194, KY614075. Tribe Sabiceeae: Sabicea mexicana Wernham, Mexico, H. Ochoterena 876 (MEXU), RUB111, KY785309, KY785253, KY614136; Sabicea villosa Schult., Mexico, H. Ochoterena 858 (MEXU), RUB122, KY785296, KY785254, KY614137. Information on Electronic Supplementary Material All datasets generated and analyzed during the present investigation are available on Zenodo (https://zenodo.org/recor d/3518369). Online Resource 1. Summary of tribal classification (abbreviated using the first three letters) of the genera historically placed in tribe Rondeletieae. Online Resource 2. Phylogenetic hypotheses obtained from the nucleotidic dataset (179 taxa, nucleotides from trnK–matK, petD and trnL– F): parsimony consensus tree showing JK values (MPTs L = 2314, Ci = 0.54, Ri = 0.87; consensus L = 2545, Ci = 0.49, Ri = 0.84), Bayesian tree showing PP values and the maximum likelihood tree showing BS values (ln = 8657.73178). Online Resource 3. Bayesian tree obtained from the concatenated dataset (179 taxa, nucleotides from trnK–matK, petD and trnL–F, DNA microstructural characters and morphology) comparing the branch support values obtained with the three inference methods: BI-PP and ML-BS above (without morphology); MP-JK below. Online Resource 4. Parsimony consensus tree (MPTs L = 2409, Ci = 0.53, Ri = 0.87; consensus L = 2550, Ci = 0.50, Ri = 0.85) obtained from the concatenated dataset (179 taxa, nucleotides from trnK–matK, petD and trnL–F, DNA microstructural characters and morphology) showing JK values. Online Resource 5. 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