Abstract

Januaria is described as a new monospecific genus of Rubiaceae, based on material from Januária, northern Minas Gerais, Brazil. The new taxon is endemic to Brazil, occurring in a vegetation type that is known locally as “carrasco”, in the southern limit of the Caatinga biome. Morphological (including palynological and SEM analyses) and molecular phylogenetic analyses based on nuclear (ETS, ITS) and plastid (atpB-rbcL, peth, rps16, trnL-trnF) sequence data were performed in the Spermacoce clade (tribe Spermacoceae). The molecular position and morphological features (a unique fruit dehiscence type, and pollen exine with simple reticulum) support Januaria as a new genus, with Mitracarpus as sister group, from which it differs principally in calyx morphology, corolla shape, and fruit dehiscence. Additionally, a further comparison with other morphologically similar genera is presented. We provide a formal description of Januaria, together with a distribution map and comments on its conservation. In addition, a discussion about the Brazilian endemics of the Spermacoce clade is given, also with a key to all the genera of this group present in the country.

Key words
Caatinga; fruit; Brazilian Atlantic dry forest; Januaria lombardii

INTRODUCTION

The Rubiaceae is a family that comprises mainly shrubs or trees, however a few lineages within the family include herbaceous species (Robbrecht & Manen 2006ROBBRECHT E & MANEN JF. 2006. The major evolutionary lineages of the coffee family (Rubiaceae, Angiosperms). Combined analysis (nDNA and cpDNA) to infer the position of Coptosapelta and Luculia, and supertree construction based on rbcL, rps16, trnL-F and atpB-rbcL data. A new classification in two subfamilies, Cinchonoideae and Rubioideae. Syst Geogr Plants 76: 85-146.). The tribe Spermacoceae (sensu Andersson & Rova 1999ANDERSSON L & ROVA JHE. 1999. The rps16 intron and the phylogeny of the Rubioideae (Rubiaceae). Plant Syst Evol 214: 161-186. https://doi.org/10.1007/BF00985737.
https://doi.org/10.1007/BF00985737... ) is the largest herbaceous lineage of the family, with over 1000 species in c. 80 genera (Groeninckx et al. 2009GROENINCKX I, DESSEIN S, OCHOTERENA H, PERSSON C, MOTLEY TJ, KÅREHED J, BREMER B, HUYSMANS S & SMETS E. 2009. Phylogeny of the herbaceous tribe Spermacoceae (Rubiaceae) based on plastid DNA data. Ann Missouri Bot Gard 96: 109-132. https://doi.org/10.3417/2006201.
https://doi.org/10.3417/2006201... , Gibbons 2020GIBBONS KL. 2020. Hedyotis, Oldenlandia and related genera (Rubiaceae: Spermacoceae) in Australia: New genera and new combinations in an Asian-Australian-Pacific lineage. Taxon 69: 515-542. https://doi.org/10.1002/tax.12236.
https://doi.org/10.1002/tax.12236... , Nuñez-Florentin et al. 2022NUÑEZ-FLORENTIN M, SALAS RM, CARMO JAM, CABRAL EL, DESSEIN S & JANSSENS SB. 2022. Paganuccia icatuensis (Rubiaceae), a new genus and species from Bahia, Brazil, with a key to all the genera of the tribe Spermacoceae in the Americas. Taxon 71(3): 630-649. https://doi.org/10.1002/tax.12651.
https://doi.org/10.1002/tax.12651... , Carmo et al. 2022CARMO JAM, REGINATO M, FLORENTÍN JE, NUÑEZ-FLORENTIN M, SALAS RM & SIMÕES AO. 2022. One more piece to the puzzle: Diadorimia, a new monotypic genus in the Spermacoceae (Rubiaceae), endemic to the campo rupestre of Minas Gerais, southeastern Brazil. Taxon 71 (2): 396-419. https://doi.org/10.1002/tax.12643.). Tribe Spermacoceae s.s. (sensu Robbrecht 1988ROBBRECHT E. 1988. Tropical woody Rubiaceae. Characteristic features and progressions. Contributions to a new subfamilial classification. Opera Botanica Belgica 272. Meise: Natl. Bot. Gard. Belgium., coinciding with the classical definition) - currently known as Spermacoce clade - is historically one of the most challenging lineages in the fourth largest family of flowering plants. From a morphological point of view, the Spermacoce clade is easily recognizable from the following combination of characters: herbaceous plants, presence of raphids, fimbriate stipules, uniovulate ovary locules, and pluri-aperturate pollen grains.

In America, the Spermacoce clade is currently represented by 23 genera, including the recently described genus Paganuccia R.M. Salas (Nuñez-Florentin et al. 2022NUÑEZ-FLORENTIN M, SALAS RM, CARMO JAM, CABRAL EL, DESSEIN S & JANSSENS SB. 2022. Paganuccia icatuensis (Rubiaceae), a new genus and species from Bahia, Brazil, with a key to all the genera of the tribe Spermacoceae in the Americas. Taxon 71(3): 630-649. https://doi.org/10.1002/tax.12651.
https://doi.org/10.1002/tax.12651... ). In 2015, Salas et al. published the first phylogenetic study focussing on the Spermacoce clade using nuclear markers (ITS and ETS), thereby describing the new genus, Carajasia R.M. Salas, E.L. Cabral & Dessein, based on morphological and molecular evidence. Despite various taxonomic and phylogenetic studies carried out in the past to further revise and elucidate generic boundaries and phylogenetic relationships within the Spermacoce clade (Miguel & Cabral 2013MIGUEL LM & CABRAL EL. 2013. Borreria krapocarmeniana, a new cryptic species recovered through taxonomic analyses of Borreria scabiosoides and Borreria linoides (Spermacoceae, Rubiaceae). Syst Bot 38: 769-781. https://doi.org/10.1600/036364413X670368.
https://doi.org/10.1600/036364413X670368... , Salas et al. 2015SALAS RM, CABRAL EL, VIANA PL, DESSEIN S & JANSSENS S. 2015. Carajasia (Rubiaceae), a new and endangered genus from Carajás mountain range, Pará, Brazil. Phytotaxa 206: 14-29. http://dx.doi.org/10.11646/phytotaxa.00.0.0.
https://doi.org/10.11646/phytotaxa.00.0.... , Florentín et al. 2017FLORENTÍN JE, CABAÑA FADER AA, SALAS RM, JANSSENS S, DESSEIN S & CABRAL EL. 2017. 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. Peer J 5: e4012. https://doi.org/10.7717/peerj.4012.
https://doi.org/10.7717/peerj.4012... , Miguel et al. 2018MIGUEL LM, SOBRADO SV, JANSSENS S, DESSEIN S & CABRAL EL. 2018. The monotypic Brazilian genus Diacrodon is a synonym of Borreria (Spermacoceae, Rubiaceae): Morphological and molecular evidences. An Acad Bras Cienc 90: 1397-1415. https://doi.org/10.1590/0001-3765201820170314.
https://doi.org/10.1590/0001-37652018201... ), there are still different opinions among specialists on the delimitation of some genera (e.g. Spermacoce-Borreria complex).

The north of Minas Gerais (Brazil) is an area characterized by predominantly xerophytic and deciduous vegetation, which constitutes a mosaic of physiognomies, or vegetational complexes. It is also considered as the southern limit of the Caatinga biome, an area of transition between the Caatinga and the Cerrado (Velloso et al. 2002VELLOSO AL, SAMPAIO EVSB & PAREYN FGC (Eds). 2002. Ecorregiões – Propostas para o bioma Caatinga. Recife, Associação Plantas do Nordeste/Instituto de Conservação Ambiental The Nature Conservancy do Brasil., Queiroz 2006QUEIROZ LP. 2006. The Brazilian caatinga: phytogeographical patterns inferred from distribution data of the Leguminosae. In: PENNINGTON RT, LEWIS GP & RATTER JA (Eds), Neotropical savannas and seasonally dry forests: plant diversity, biogeography, and conservation. Boca Raton: CRC Press, Taylor & Francis Group, p. 121-157., Queiroz et al. 2017QUEIROZ LP, CARDOSO DBOS, FERNANDES MF & MF MORO. 2017. Diversity and Evolution of Flowering Plants of the Caatinga Domain. In: SILVA JMC, LEAL IR & TABARELLI M (Eds), Caatinga: the Largest Tropical Dry Forest Region in South America. Springer, Cham. 23-63. https://doi.org/10.1007/978-3-319-68339-3.
https://doi.org/10.1007/978-3-319-68339-... , Fernandez et al. 2020). Lombardi et al. (2005)LOMBARDI JA, SALINO A & TEMONI LG. 2005. Diversidade florística de plantas vasculares no município de Januária, Minas Gerais, Brasil. Lundiana 6(1): 3-20. conducted a floristic survey in this area, specifically in the municipality of Januária. Among the arboreous/shrub Rubiaceae taxa founded, the authors identified one specimen as “Borreria sp.” During a recent herbarium study, this specimen was analysed in greater depth and although the primary traits indicated that it undoubtedly belongs to the Spermacoce clade, a more detailed analysis revealed that it could not be considered as a member of the genus Borreria G. Mey (following the concept of Miguel & Cabral 2013MIGUEL LM & CABRAL EL. 2013. Borreria krapocarmeniana, a new cryptic species recovered through taxonomic analyses of Borreria scabiosoides and Borreria linoides (Spermacoceae, Rubiaceae). Syst Bot 38: 769-781. https://doi.org/10.1600/036364413X670368.
https://doi.org/10.1600/036364413X670368... ) or another morphologically similar taxon (e.g. Spermacoce L. sensu Nuñez-Florentin et al. 2020NUÑEZ-FLORENTIN M, FLORENTÍN JE & SALAS RM. 2020. Integrative taxonomic analyses sheds light on three historically disputed american Spermacoce species, and a key to the american species of Spermacoce (Spermacoceae, Rubiaceae). Syst Bot 45(3): 585-606. https://doi.org/10.1600/036364420X15935294613464.
https://doi.org/10.1600/036364420X159352... ).

Therefore, in order to elucidate the taxonomic position of this new taxon, a molecular phylogenetic analysis of the Spermacoce clade was carried out using molecular markers from the nuclear ribosomal (ITS and ETS) and chloroplast genomes (atpB-rbcL, peth, rps16, and trnL-trnF). By applying a complementary molecular and morphological analysis (e.g., fruit dehiscence, pollen, and seed observations), the evolutionary history of the new taxon and its relatives could be inferred. In addition, a geographic overview of Brazilian Spermacoceae, focused on endemic genera of Spermacoce clade is given.

MATERIALS AND METHODS

Taxonomic treatment and conservation assessment

Conventional taxonomic techniques were followed for the description and analysis of the new monotypic genus and its species. Additional data was retrieved from herbarium specimens from BHCB and W [herbarium codes according to Thiers (2021THIERS B. 2021. (continuously updated). Index Herbariorum: a global directory of public herbaria and associated staff. New York Botanical Garden’s Virtual Herbarium Available at http://sweetgum.nybg.org/science/ih. (accessed on 03 March 2021).
http://sweetgum.nybg.org/science/ih... , continuously updated)]. Information concerning the habitat, flowering period, and qualitative characteristics, such as the colour of the flowers, were obtained from the herbarium labels.

An assessment of the conservation status was carried out following the IUCN Standards and Petitions Committee (2019)IUCN STANDARDS AND PETITIONS COMMITTEE. 2019. Guidelines for Using the IUCN Red List Categories and Criteria. Version 14. Prepared by the Standards and Petitions Committee. Retrieved from http://www.iucnredlist.org/documents/RedListGuidelines.pdf.
http://www.iucnredlist.org/documents/Red... recommendations.

Morphological analyses

For the morphological observations, floral and vegetative parts were rehydrated in warm soapy water and analysed under a stereomicroscope (SM) Leica MZ6 and measured using an electronic digital caliper (Schwyz). The morphological terminology follows Stearn (1986)STEARN WT. 1986. Botanical Latin. Ed. 3. London: David and Charles Publishers plc, 289 p.. For scanning electron microscopy (SEM) analyses, flowers were obtained from herbarium material and rehydrated for 12 hours in water at 60°C with a drop of detergent. After the preparation step, the material was dried to critical point with CO² and mounted on aluminium stubs. Fruits and seeds were mounted on aluminium stubs without any treatment. All material was sputter coated with 20 nm of gold-palladium. Observations were performed at 20 kV with a SEM Jeol LV 5800 at the Electron Microscopy unit of the Universidad Nacional del Nordeste (UNNE).

Palynological analyses

Pollen grains were acetolysed according to the technique described by Erdtman (1966)ERDTMAN G. 1966. An introduction to palynology, vol. 1, Pollen morphology and plant taxonomy; Angiosperms. New York & London: Hafner, 541 p. and mounted in glycerine jelly for analysis by light microscopy (LM). The shape of the pollen grains, the ratio of the polar axis (P) and the equatorial diameter (E) were studied by photographing at least 20 grains with a LM Leica DM LB2 microscope equipped with a digital camera and then measured afterwards using the program ImageJ v.1.51k (Rasband 2020RASBAND WS. 2020. Image J, version 1.51k. Bethesda, MD: U.S. National Institutes of Health. http://imagej.nih.gov/ij/. (accessed 30 Sept 2021).
http://imagej.nih.gov/ij/... ). The exine structure was analysed using SEM. The morphological terminology for pollen follows Punt et al. (2007)PUNT WS, HOEN PP, BLACKMORE S, NILSSON S & LE THOMAS A. 2007. Glossary of pollen and spore terminology. Rev Palaeobot Palynol 143: 1-81. https://doi.org/10.1016/j.revpalbo.2006.06.008.
https://doi.org/10.1016/j.revpalbo.2006.... .

Taxon and gene sampling

The sampling included 77 ingroup accessions from the Spermacoce clade (Appendix I; Fig. 1). The present sampling represents approximately 30% of the species and 80% of the genera of the Spermacoce clade. Two nuclear ribosomal (ITS, ETS) and four plastid (atpB-rbcL, peth, rps16, trnL-trnF) DNA regions were selected since they have proven to be phylogenetically informative within the Rubiaceae (Kårehed et al. 2008KÅREHED J, GROENINCKX I, DESSEIN S, MOTLEY TJ & BREMER B. 2008. The phylogenetic utility of chloroplast and nuclear DNA markers and the phylogeny of the Rubiaceae tribe Spermacoceae. Molec Phylogen Evol 49: 843-866. https://doi.org/10.1016/j.ympev.2008.09.025.
https://doi.org/10.1016/j.ympev.2008.09.... ). The new sequences were added to existing alignments used by Nuñez-Florentin et al. (2021, 2022). Bouvardia ternifolia Cav. was chosen as an outgroup taxon based on its placement in previous phylogenetic analyses (Kårehed et al. 2008KÅREHED J, GROENINCKX I, DESSEIN S, MOTLEY TJ & BREMER B. 2008. The phylogenetic utility of chloroplast and nuclear DNA markers and the phylogeny of the Rubiaceae tribe Spermacoceae. Molec Phylogen Evol 49: 843-866. https://doi.org/10.1016/j.ympev.2008.09.025.
https://doi.org/10.1016/j.ympev.2008.09.... , Salas et al. 2015SALAS RM, CABRAL EL, VIANA PL, DESSEIN S & JANSSENS S. 2015. Carajasia (Rubiaceae), a new and endangered genus from Carajás mountain range, Pará, Brazil. Phytotaxa 206: 14-29. http://dx.doi.org/10.11646/phytotaxa.00.0.0.
https://doi.org/10.11646/phytotaxa.00.0.... ). The full details of the vouchers used in the phylogenetic inference analysis are provided in Appendix I.

DNA extraction, amplification, purification, and sequencing

In order to assess the phylogenetic position of the new taxon within the Spermacoce clade, its genomic DNA was extracted from herbarium material, using a modified CTAB protocol (Doyle & Doyle 1987DOYLE JJ & DOYLE JL. 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull Bot Soc Amer 19: 11-15.) according to the protocol outlined by Janssens et al. (2006)JANSSENS S, GEUTEN K, YUAN YM, SONG Y, KÜPFER P & SMETS E. 2006. Phylogenetics of Impatiens and Hydrocera (Balsaminaceae) using chloroplast atpB-rbcL spacer sequences. Syst Bot 31: 171-180. https://doi.org/10.1600/036364406775971796.
https://doi.org/10.1600/0363644067759717... . Amplification reactions were carried out on a GeneAmp PCR system 9700 (Applied Biosystems) for six markers, two nuclear (ITS, ETS), and four plastid (atpB-rbcL, peth, rps16, trnL-trnF). Primers and thermocycler programs used for the amplification of nuclear and plastid markers were those described in Nuñez-Florentin et al. (2022)NUÑEZ-FLORENTIN M, SALAS RM, CARMO JAM, CABRAL EL, DESSEIN S & JANSSENS SB. 2022. Paganuccia icatuensis (Rubiaceae), a new genus and species from Bahia, Brazil, with a key to all the genera of the tribe Spermacoceae in the Americas. Taxon 71(3): 630-649. https://doi.org/10.1002/tax.12651.
https://doi.org/10.1002/tax.12651... . Purified amplification products were sent to Macrogen, Inc. (Seoul, South Korea) for sequencing. DNA extraction, PCR and amplification were carried out at the molecular lab of Meise Botanic Garden.

Alignment and phylogenetic analyses

The sequences were edited and assembled de novo using Geneious v11.1.4 (Biomatters, Auckland, New Zealand). Automatic multiple alignments were carried out with AliView (Larsson 2014LARSSON A. 2014. AliView: A fast and lightweight alignment viewer and editor for large datasets. Bioinformatics 30(22): 3276-3278. https://doi.org/10.1093/bioinformatics/btu531.
https://doi.org/10.1093/bioinformatics/b... ), using the Muscle algorithm, also with subsequent manual editing to improve homology for more variable regions. The combined matrix (nuclear + plastid markers) is available in Supplementary Material – Alignment. combined matrix.

Bayesian inference (BI) and Maximum likelihood (ML) were used to explore the phylogenetic relationships in the study group. The best-fit nucleotide substitution model for each nuclear and plastid dataset was selected with jModelTest v.2.1.4. using the Akaike information criterion (AIC; Posada 2008POSADA D. 2008. jModelTest: Phylogenetic model averaging. Molec Biol Evol 25: 1253-1256. https://doi.org/10.1093/molbev/msn083.
https://doi.org/10.1093/molbev/msn083... ). The chosen models are shown in Table I. Selected models which are not implemented in MrBayes (for instance, TVM and TIM1) were substituted by the closest over-parameterized model.

BI analyses were run in the Cyber infrastructure for Phylogenetic Research (CIPRES Science Gateway; Miller et al. 2010MILLER MA, PFEIFFERW & SCHWARTZ T. 2010. Creating the CIPRES Science Gateway for inference of large phylogenetic trees. In: Proceedings of the Gateway Computing Environments Workshop (GCE), New Orleans, Louisiana, 14 Nov 2010. Piscataway: IEEE, p. 45-52 https://doi.org/10.1109/GCE.2010.5676129.
https://doi.org/10.1109/GCE.2010.5676129... ) using MrBayes v3.1 (Huelsenbeck & Ronquist 2001HUELSENBECK J & RONQUIST F. 2001. MrBayes: Bayesian inference of phylogenetic trees. Bioinformatics 17: 754-755. https://doi.org/10.1093/bioinformatics/17.8.754.
https://doi.org/10.1093/bioinformatics/1... ), first on each individual data partition and then on the three combined data matrices constructed: “the nuclear matrix”, “the plastid matrix”, and the “nuclear + plastid combined matrix” (in the absence of supported conflict between the resulting gene trees). For the combined analyses, a mixed model approach was used in which the data set was partitioned, and the models of evolution were applied to the different partitions. Two independent Metropolis coupled Markov chain Monte Carlo (MCMC) runs, each consisting of one cold and three heated chains, were started simultaneously from a random tree and run for 20 million generations, with the trees being sampled every 10,000 generations. At the end of the run, chain convergence and estimated sample size (ESS) parameters were assessed with Tracer v.1.6.0 (Rambaut et al. 2014RAMBAUT A, SUCHARD MA, XIE D & DRUMMOND AJ. 2014. Tracer, version 1.6. Retrieved from https://doi.org/10.1111/j.1438-8677.1996.%0Atb00515.x.
https://doi.org/10.1111/j.1438-8677.1996... ). Burn-in was set at 25% and the remaining posterior topologies summarized as a 50% majority-rule consensus tree, with branch support expressed as posterior probabilities (PP). PP values from 0.5 to 0.95 were considered as weak to moderate support, whereas posterior probabilities > 0.95 were considered as strong to very strong support (Suzuki et al. 2002SUZUKI Y, GLAZKO GV & NEI M. 2002. Overcredibility of molecular phylogenies obtained by Bayesian phylogenetics. Proc Natl Acad Sci USA 99: 16138-16143. https://doi.org/10.1073/pnas.212646199.
https://doi.org/10.1073/pnas.212646199... , Alfaro et al. 2003ALFARO ME, ZOLLER S & LUTZONI F. 2003. Bayes or bootstrap? A simulation study comparing the performance of Bayesian Markov chain Monte Carlo sampling and bootstrapping in assessing phylogenetic confidence. Molec Biol Evol 20: 255-266. https://doi.org/10.1093/molbev/msg028.
https://doi.org/10.1093/molbev/msg028... ).

ML analyses were performed using RAxML-MPI v.8.2 (Stamatakis et al. 2008STAMATAKIS A, HOOVER P & ROUGEMONT J. 2008. A rapid bootstrap algorithm for the RAxML web-servers. Syst Biol 75: 758-771. https://doi.org/10.1080/10635150802429642.
https://doi.org/10.1080/1063515080242964... ), as implemented on the CIPRES Science Gateway web server (RAxML-HPC2 on XSEDE 8.1.11) (Miller et al. 2010), with the following settings: rapid bootstrap analysis with 1000 replicates and searching for the best-scoring ML tree starting with a random seed and utilizing the GTRGAMMA model. Rapid bootstrapping was performed on the ML tree using RAxML at 1000 replicates to determine branch support. Only the ML bootstrap (BS) values (≥ 0.5) are provided. Internodes with BS ≥75% were considered statistically significant.

Congruency between the different datasets was inferred using different methods. Due to the known sensitivity issues of the ILD test (Barker & Lutzoni 2002BARKER FK & LUTZONI FM. 2002. The utility of the incongruence length difference test. Syst Biol 51: 625-637. https://doi.org/10.1080/10635150290102302.
https://doi.org/10.1080/1063515029010230... ), possible conflict between the marker datasets was also assessed by visually inspecting the topologies, thereby searching for putatively conflicting relationships and the way those were supported within each topology (hard vs. soft incongruences; Johnson & Soltis 1998JOHNSON LA & SOLTIS SE. 1998. Assessing congruence: Empirical examples from molecular data. In: SOLTIS DE, SOLTIS PS & DOYLE JJ (Eds), Molecular systematics of plants, vol. 2, DNA sequencing. Boston: Kluwer Academic Publishing, p. 297-348., Wiens 1998WIENS JJ. 1998. Combining data sets with different phylogenetic histories. Syst Biol 47: 568-581. https://doi.org/10.1080/106351598260581.
https://doi.org/10.1080/106351598260581... ).

Geographic distribution

The geographic record obtained was plotted in QGis 3.4.2-Madeira (QGIS Development Team 2018QGIS Development Team. 2018. QGIS Geographic Information System. Open Source Geospatial Foundation Project. http://qgis.osgeo.org.
http://qgis.osgeo.org... ). The distribution of the species was superimposed with a layer of the Ecoregions derived from Olson’s classification (2001). To study the distribution of the species in relation to protected areas (PAs), we used spatial data from the world database on protected areas (WDPA https://protectedplanet.net/; visited in October 2021) and from Dinerstein et al. (2017)DINERSTEIN E ET AL. 2017. An ecoregion-based approach to protecting half the terrestrial realm. BioScience 67(6): 534-545. https://doi.org/10.1093/biosci/bix014.
https://doi.org/10.1093/biosci/bix014... .

RESULTS

Properties of the sequence data

Dataset characteristics and models of nucleotide substitution selected by AIC are presented in Table I. The nuclear ITS and ETS DNA regions were more phylogenetically informative than the four plastid markers. Of the 632 aligned ITS characters, 210 potentially informative sites (33.2%) were obtained, and ETS yielded 186 informative sites out of 364 characters (51.1%), for a total of 396 potentially informative nuclear characters, out of a total of 996 (39.8%). The aligned lengths of the four plastid markers ranged from 458 bp (trnL-trnF) to 1155 bp (peth), and all together they yielded 3068 characters, of which 212 (6.9%) were informative.

Separate analyses and assessment of incongruence

The individual nuclear and plastid gene datasets were first analysed separately (results available as Figures S1, S2). Most of them resulted in polytomies or only produced poorly resolved clades, except for the ITS and ETS, which provided the highest proportion of well-resolved clades. No significant incongruence or decreased resolution was observed after analysing a combined nuclear (ITS + ETS) dataset versus a combined plastid (atpB-rbcL + peth + rps16 + trnL-trnF) dataset.

Combined plastid and nuclear analyses

The concatenated ML trees (Figure S2) were very similar to the Bayesian topologies, therefore, only the 50% majority rule Bayesian consensus tree from the combined analysis (nuclear + plastid data) is shown and used for further discussion (Fig. 1).

Despite the poorly supported backbone of the Spermacoce clade, several well-supported clades have been recovered, most of these clades coinciding with genera such as Crusea Cham. & Schlthl., Micrasepalum Urb., Ernodea Sw., Psyllocarpus Mart. & Zucc., Richardia L., and Staelia Cham. & Schlthl., etc. which were recovered with high to maximum support values. Borreria, Hexasepalum, and Spermacoce however were found to be polyphyletic. Two major clades branch off in the early evolutionary history of the Spermacoce clade: clade “A” (PP = 0.95, BS = 59) including Galianthe Griseb. and the monotypic genera Schwendenera tetrapyxis K. Schum. and Carajasia cangae R.M. Salas, E.L. Cabral & Dessein, and clade “B” (PP = 0.96, BS= 58) comprising all the remaining genera, including the new taxon. The BI analysis (Fig. 1) show that the new taxon is a well-supported sister lineage of Mitracarpus (PP = 1), however in the ML analysis this relationship is not well supported (BS = 68). In addition, Mitracarpus is recovered as a strongly supported, monophyletic genus (PP = 1, BS = 100).

Taxonomic treatment

In the following paragraphs we present the formal description of the new genus and species based on morphological and phylogenetic evidence.

Januaria lombardii R.M. Salas & Nuñez Florentin, M. gen. et. sp. nov.

Type: BRAZIL. Minas Gerais, Januária, distrito de Fabião, 2 Km na estrada partindo do abrigo do Malhador [Parque Nacional Cavernas de Peruaçu], 15°07’58’’ S, 44°15’17’’ W, 23 May 1997, Lombardi J.A. & A. Salino 1674 (Holotype: BHCB37156!). Figs. 2-4.

Diagnosis

The genus Januaria (consisting of a single species J. lombardii) differs from all the other genera of the Spermacoce clade due to the particular fruit dehiscence (dehiscence longitudinal-transverse, with a longitudinal-septifragal dehiscence that starts in one of the carpels up to the middle of the fruit, and from there, it follows an transverse-loculicidal line, resulting in one indehiscent carpel remaining on the pedicel and another dehiscent carpel which falls off and releases the seed) and 8-zonoaperturate pollen grains with simple reticulate exine pattern.

Description

Shrub decumbent, height unknown. Stems simple, tetragonal to subcylindrical, glabrous in the basal branches to pubescent in the apical flowering branches; internodes 1.8–4.5 cm long. Stipular sheath 7.5–9.5 mm long, pubescent, with 5–7 fimbriae, 5.2–8.9 mm long, filiform, glabrous, colleter-tipped. Leaves opposite, pseudopetiolate to subsesille; petiole 1.2–1.7 mm long; leaf blade ovate to elliptic, 27.5–77 × 7.7–35.1 mm, concolorous, membranaceous, base attenuate, apex acute; adaxial surface hirsute, abaxial surface pubescent primarily on the middle vein and 2–3 secondary veins. Inflorescences with determinate growth; partial inflorescences compressed, axillar, 4–5 flowered. Flowers subsessile, homostylous; hypanthium 2.6–2.8 mm long, obconic, pubescent; calyx 4-lobate, calyx tube reduced; lobes triangular, 0.95–1.3 mm long, pubescent; corolla infundibuliform, white, 3.2–6.5 mm long, corolla tube 2.1–5 mm long, corolla lobes 1.1–3 mm long, 4-lobate; externally glabrous with trichomes on the apical portion of lobes, internally with a fringe of trichomes on the inferior half of corolla tube; stamens exserted, inserted at the sinuses of the corolla lobes, 3.1–3.4 mm long; anthers obovate to oblong, 0.6–0.8 mm long; orbicules absent; pollen grains (7–) 8 zonocolporate, small [P = 23.3–26 µm, E = 22.8–25.8 µm], oblate-spheroidal (P/E = 0.9–1); circular outline in polar view; ectocolpus long and narrow, 11–18.4 µm long; endoapertures laterally fused, forming an endocingulum; exine 2.3–3 µm thick, reticulate, lumina 1.1–2.1 µm long, and muri 0.4–0.7 µm long, with nanospines uniformly present; ovary 2-carpellate, 2-locular, locules 1-ovulate; style exserted, 2.6–3 mm long, bifid. Fruits dry, subsessile, turbinate, 4.8–6.5 × 1.5–2.3 mm, pubescent on the superior half; dehiscence longitudinal-transverse, with a longitudinal-septifragal dehiscence that starts in one of the carpels up to the middle of the fruit, and from there, it follows an transverse-loculicidal line, resulting in one indehiscent carpel remaining on the pedicel and another dehiscent carpel which falls off and releases the seed; calyx lobes persistent. Seeds ellipsoid, 1.9–2.1 × 0.4–0.6 mm, dark castaneous, dorsal surface convex, ventral surface ± plane, with longitudinal groove covered by the strophiole with abundant raphides; exotesta reticulate-foveate, cells polygonal, rectangular, periclinal walls concave, anticlinal walls straight.

Distribution and Habitat

Januaria is endemic from Minas Gerais (municipality of Janúaria), Brazil. Figure 4a represents the geographic distribution of this new taxon, which coincides with the ecoregion “Brazilian Dry Atlantic Forest” from Olson’s classification (2001). According to the information on the label of the collected specimen, this species occurs in a vegetation type that is known locally as “carrasco” (see further details in Discussion section).

Phenology

The sole specimen of this taxon was collected in May and contained flowers, floral buds and mature fruits, indicating that the fertile period of this species could cover the months from December to May-June approximately, coinciding with the rainy season.

Etymology

The generic name refers to the locality where the holotype was collected, Januária, Minas Gerais, Brazil. The specific epithet honours Julio Antonio Lombardi, who collected the holotype of Januaria lombardii, and has made significant contributions to the study of the Brazilian Flora, especially in the taxonomy of Celastraceae, Oleaceae, and Vitaceae.

Preliminary conservation status

Januaria is known from a single collection from the Cavernas do Peruaçu National Park, a conservation unity of 564.48 km², located in the municipalities of Itacarambi, Januária, and São João das Missões. Even though the vegetation cover within the limits of the park is relatively well preserved, its adjacent areas, as well as most of the north of Minas Gerais are in constant transformation due to increased anthropogenic land use (Fig. 4b). Although extensive field work was carried out in April-May 2012, no additional populations of the species were found. Accordingly, based on the available information, and following the criteria and recommendations of the IUCN Red List Status (2019) and Callmander et al. (2005)CALLMANDER MW, SCHATZ GE & LOWRY II PP. 2005. IUCN Red List assessment and the Global Strategy for Plant Conservation: taxonomists must act now. Taxon 54(4): 1047-1050. https://doi.org/10.2307/25065491.
https://doi.org/10.2307/25065491... , we consider this new taxon as Vulnerable (VU), under criterion D2, for the time being.

Notes

Due to its morphological characteristics, especially compressed, axillary, pauciflorous, inflorescences with homostylous flowers the new taxon resembles some species of Borreria, Hexasepalum, and Spermacoce. However, it differs from each of them due to the unique dehiscence of its fruits, by the 8-zonoaperturate pollen grains with simple reticulum (vs. primarily perforate to perforate-microreticulate, and eutectate in Borreria, Hexasepalum, and Spermacoce) and the presence of a shortly branched, bifid stigma (vs. capitate-bilobate to bilobate in Borreria, Hexasepalum, and Spermacoce). For a further comparison between these taxa see Table II. In the vegetative state, they are very similar, and maybe this is the reason for the scarce collections and/or misidentification.

Additional specimens analysed (paratype)

BRAZIL: sine loco, sine data, Gardner 2191 (W, proparte).

DISCUSSION

The phylogenetic position of Januaria in the Spermacoce clade, and its distinctive morphology

The topologies recovered in the current study agree with previous studies of the Spermacoce clade that were obtained from nuclear data only (Salas et al. 2015SALAS RM, CABRAL EL, VIANA PL, DESSEIN S & JANSSENS S. 2015. Carajasia (Rubiaceae), a new and endangered genus from Carajás mountain range, Pará, Brazil. Phytotaxa 206: 14-29. http://dx.doi.org/10.11646/phytotaxa.00.0.0.
https://doi.org/10.11646/phytotaxa.00.0.... , Florentín et al. 2017FLORENTÍN JE, CABAÑA FADER AA, SALAS RM, JANSSENS S, DESSEIN S & CABRAL EL. 2017. 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. Peer J 5: e4012. https://doi.org/10.7717/peerj.4012.
https://doi.org/10.7717/peerj.4012... , Miguel et al. 2018MIGUEL LM, SOBRADO SV, JANSSENS S, DESSEIN S & CABRAL EL. 2018. The monotypic Brazilian genus Diacrodon is a synonym of Borreria (Spermacoceae, Rubiaceae): Morphological and molecular evidences. An Acad Bras Cienc 90: 1397-1415. https://doi.org/10.1590/0001-3765201820170314.
https://doi.org/10.1590/0001-37652018201... ) or from a combined nuclear and plastid data matrix (Nuñez-Florentin et al. 2021NUÑEZ-FLORENTIN M, SALAS RM, JANSSENS SB, DESSEIN S & CARDOSO D. 2021. Molecular-based phylogenetic placement and revision of Micrasepalum (Spermacoceae-Rubiaceae). Taxon 70(6): 1300-1316. https://doi.org/10.1002/tax.12593.
https://doi.org/10.1002/tax.12593... , 2022).

Using molecular sequence data, we demonstrated that new described taxon, Januaria lombardii, belongs to the Spermacoce clade, one of the most taxonomically complex groups within the tribe Spermacoceae (for discussion see Groeninckx et al. 2009GROENINCKX I, DESSEIN S, OCHOTERENA H, PERSSON C, MOTLEY TJ, KÅREHED J, BREMER B, HUYSMANS S & SMETS E. 2009. Phylogeny of the herbaceous tribe Spermacoceae (Rubiaceae) based on plastid DNA data. Ann Missouri Bot Gard 96: 109-132. https://doi.org/10.3417/2006201.
https://doi.org/10.3417/2006201... , Salas et al. 2015SALAS RM, CABRAL EL, VIANA PL, DESSEIN S & JANSSENS S. 2015. Carajasia (Rubiaceae), a new and endangered genus from Carajás mountain range, Pará, Brazil. Phytotaxa 206: 14-29. http://dx.doi.org/10.11646/phytotaxa.00.0.0.
https://doi.org/10.11646/phytotaxa.00.0.... ). Specifically, the new taxon is recovered within clade “B”, in closed relationship with the genus Mitracarpus (Fig. 1).

Mitracarpus is a morphologically and phylogenetically well circumscribed Neotropical genus (Souza et al. 2010SOUZA EB, CABRAL EL & ZAPPI DC. 2010. Revisão de Mitracarpus (Rubiaceae–Spermacoceae) para o Brasil. Rodriguésia 61(2): 319-352. https://doi.org/10.1590/2175-7860201061213.
https://doi.org/10.1590/2175-78602010612... ), strongly supported, but largely unresolved in relationship with the other genera of the Spermacoce clade (Salas et al. 2015SALAS RM, CABRAL EL, VIANA PL, DESSEIN S & JANSSENS S. 2015. Carajasia (Rubiaceae), a new and endangered genus from Carajás mountain range, Pará, Brazil. Phytotaxa 206: 14-29. http://dx.doi.org/10.11646/phytotaxa.00.0.0.
https://doi.org/10.11646/phytotaxa.00.0.... , Nuñez-Florentin et al. 2021NUÑEZ-FLORENTIN M, SALAS RM, JANSSENS SB, DESSEIN S & CARDOSO D. 2021. Molecular-based phylogenetic placement and revision of Micrasepalum (Spermacoceae-Rubiaceae). Taxon 70(6): 1300-1316. https://doi.org/10.1002/tax.12593.
https://doi.org/10.1002/tax.12593... , 2022). In the present analyses, Januaria, results a monospecific genus sister to Mitracarpus, from which it differs by its calyx morphology (4 lobes of the same length vs. 2 smaller lobes and 2 larger lobes), corolla shape (infundibuliform vs. hypocrateriform), seed morphology (ellipsoid seed with a longitudinal ventral groove vs. ovoid or ellipsoid seed with a quadrangular or rectangular, “X-shaped” or “inverted Y-shaped” ventral groove), and fruit dehiscence (longitudinal-transverse dehiscence with one dehiscent carpel and one indehiscent carpel vs. circumscissile dehiscence, with both dehiscent carpels). For a further comparison with Mitracarpus and other morphologically similar genera see Table II.

One of the most striking features of Januaria is the fruit dehiscence, which is unique within the Spermacoce clade. The taxonomic value of the fruit morphology in the Spermacoce clade has already been shown in the description of the tribe Spermacoceae by Berchtold & Presl (1820)BERCHTOLD F & PRESL KB. 1820. Spermacoceae. Prir. Rostlin. Krala Wiljma Endersa, Praga, 256 p.. Subsequently, classic taxonomists, such as Candolle (1830)CANDOLLE AP. 1830. Prodromus Systematis Naturalis Regni Vegetabilis 4: 561. Paris: Treuttel & Würts. or Schumann (1888SCHUMANN K. 1888. Rubiaceae-Spermacoceae. Flora Brasiliensis 6(6): 6-99., 1891SCHUMANN K. 1891. Rubiaceae. In: ENGLER A & PRANTL K (Eds), Die naturlichen Pflanzenfamilien. Leipzig: Engelmann. 1-156.), considered the type of dehiscence as the only criterion to support or conserve genera. There are some genera in which the fruit morphology is so unique that their circumscription is based on only that particular character (e.g. Mitracarpus, Ernodea, Crusea). The main role attributed to the type of dehiscence has resulted in several taxa that have been considered artificial groups, not supported by morphological characters other than such features (e.g. Diodia L., Borreria, Spermacoce, Staelia, etc. Cabaña Fader 2013CABAÑA FADER AA. 2013. Estudios biosistemáticos en especies americanas de Diodia s. lat. (Rubiaceae). Ph.D. Dissertation. Universidad Nacional del Nordeste, Corrientes, Argentina., Miguel 2016MIGUEL ML. 2016. Estudios biosistemáticos en especies sudamericanas de Borreria subsecc. Borreria y filogenia de las especies del complejo Spermacoce-Borreria (Rubiaceae). Ph.D. Dissertation. Universidad Nacional del Nordeste, Corrientes, Argentina., Salas 2011SALAS RM. 2011. Revisión de Staelia s.l. (Rubiaceae). Ph. D. Dissertation. Universidad Nacional del Nordeste. Corrientes, Argentina.). Therefore, in the case of the genus Januaria, the longitudinal-transverse dehiscence in combination with the specific morphological pollen characters and the phylogenetic position support its recognition as a new genus.

The reticulate simple exine, which is relatively uncommon within the Spermacoce clade, is characteristic for Januaria lombardii. Apart from the new taxon, a micro-reticulate to reticulate exine is observed in Staelia catolensis R. M. Salas & E.L. Cabral (Salas & Cabral 2014SALAS RM & CABRAL EL. 2014. Morfología polínica de Staelia s.l. (Rubiaceae). Bol Soc Argent Bot 49(1): 51-65. https://doi.org/10.31055/1851.2372.v49.n1.7821.
https://doi.org/10.31055/1851.2372.v49.n... ), Richardia brasiliensis Gomes (Pire 1997PIRE SM. 1997. El polen de especies brasileñas de Richardia L. (Rubiaceae-Spermacoceae). Geociencias II (n° especial): 184-191.), three species of Mitracarpus [M. brasiliensis M. Porto & Waechter and M. diversifolius Souza & E.L. Cabral (Souza 2008SOUZA EB. 2008. Estudos sistemáticos em Mitracarpus (Rubiaceae-Spermacoceae) com ênfase em espécies brasileiras. Ph. D. Dissertation. Universidade Estadual de Feira de Santana, Bahia, Brazil.), M. robustus Souza & E.L. Cabral (Nuñez-Florentin, unpublished data)], four Australian Spermacoce species [e.g. S. congestanthera Harwood, S. graniticola Harwood, etc. (Dessein et al. 2005DESSEIN S, HARWOOD B, SMETS E & ROBBRECHT E. 2005. Pollen of the Spermacoce (Rubiaceae) species from the Northern Territory of Australia: morphology and taxonomic significance. Aust Syst Bot 18(4): 367-382. https://doi.org/10.1071/SB03025.
https://doi.org/10.1071/SB03025... )] and three Borreria species from Borreria subsect. Latifolia, also known as the “Borreria latifolia group” [e.g., B. bradei Standl., B. dimorpha J.H. Kirkbr, and B. shumannii (Pire 1996PIRE SM. 1996. Palynological study of American species of Borreria (Rubiaceae-Spermacoceae). Opera Bot 77: 413-423., Sobrado 2015SOBRADO SV. 2015. Estudios biosistemáticos en especies de Borreria (Spermacoceae-Rubiaceae) con énfasis en Borreria subsecc. Latifoliae. Ph.D. Dissertation. Universidad Nacional del Nordeste, Corrientes, Argentina.)]. According to the pollen size, shape, number of apertures, and colpi length, the pollen grains of Mitracarpus robustus, Spermacoce congestanthera, and Staelia catolensis are those that most closely resemble J. lombardii, yet they differ from the latter in a few slightly different features. Mitracarpus robustus has 9–10 aperturate pollen grains, whereas Januaria is (7–) 8 aperturate. Furthermore, while Staelia catolensis has a micro-reticulate to reticulate ornamentation pattern with a lumina size of 0.1–1.2 µm, Januaria has a reticulate exine with larger lumina (1.1–2.1 µm). In addition, Spermacoce congestanthera has colpi that are middle sized to short in length, whereas the ectocolpi of Januaria pollen are long. As a result, J. lombardii pollen has a morphological affinity with pollen type 20, as proposed by Dessein et al. (2005)DESSEIN S, HARWOOD B, SMETS E & ROBBRECHT E. 2005. Pollen of the Spermacoce (Rubiaceae) species from the Northern Territory of Australia: morphology and taxonomic significance. Aust Syst Bot 18(4): 367-382. https://doi.org/10.1071/SB03025.
https://doi.org/10.1071/SB03025... and then expanded by Salas & Cabral (2014)SALAS RM & CABRAL EL. 2014. Morfología polínica de Staelia s.l. (Rubiaceae). Bol Soc Argent Bot 49(1): 51-65. https://doi.org/10.31055/1851.2372.v49.n1.7821.
https://doi.org/10.31055/1851.2372.v49.n... . Table III presents a detailed comparison of the palynological characteristics between J. lombardii and other taxa with a reticulate exine.

The endemism and habitat of Januaria

Following the regionalization by Olson et al. (2001)OLSON DM ET AL. 2001. Terrestrial ecoregions of the world: a new map of life on earth. Bioscience 51: 933-938., the only georeferenced location known for J. lombardii shows that inhabits the Brazilian Atlantic Dry Forest, in an ecotonal region between Caatinga and Cerrado ecoregions (Fig. 4a). The Carrasco (a local name for sedimentary Caatinga) is sometimes referred to as being composed of a mix of seasonally dry tropical forests and woodlands (SDTFW sensu Queiroz et al. 2017QUEIROZ LP, CARDOSO DBOS, FERNANDES MF & MF MORO. 2017. Diversity and Evolution of Flowering Plants of the Caatinga Domain. In: SILVA JMC, LEAL IR & TABARELLI M (Eds), Caatinga: the Largest Tropical Dry Forest Region in South America. Springer, Cham. 23-63. https://doi.org/10.1007/978-3-319-68339-3.
https://doi.org/10.1007/978-3-319-68339-... ) and savanna elements (Araújo et al. 1998ARAÚJO FS, SAMPAIO EVSB, FIGUEIREDO MA, RODAL MN & FERNANDES AG. 1998. Composição florística da vegetação de carrasco, Novo Oriente, CE. Braz J Bot 21: 105-116. https://doi.org/10.1590/S0100-84041998000200001.
https://doi.org/10.1590/S0100-8404199800... , Fernandez et al. 2020). According to the information on the label, J. lombardii was collected in this vegetation type called “Carrasco”, characterized by a mainly deciduous shrub-tree vegetation composed of plants that are mostly no higher than 5m tall, with only a few species reaching 10m. Herbs and climbers are common in the forest margins or along roads but are rare in the interior of the vegetation (Lombardi et al. 2005LOMBARDI JA, SALINO A & TEMONI LG. 2005. Diversidade florística de plantas vasculares no município de Januária, Minas Gerais, Brasil. Lundiana 6(1): 3-20.).

The floristic study of Lombardi et al. (2005)LOMBARDI JA, SALINO A & TEMONI LG. 2005. Diversidade florística de plantas vasculares no município de Januária, Minas Gerais, Brasil. Lundiana 6(1): 3-20. highlights the floristic and physiognomic diversity of Januária, northern of Minas Gerais, and emphasizes the importance and urgency of inventories for the region as it is of high priority for conservation due to the enormous anthropogenic pressure caused by the continuously advancing agricultural frontier and the new mining concessions. In this sense, Fernandez et al. (2020) noted the importance of the border areas of the Caatinga and stated that “the adjacency and connectivity of different biomes within such a small area provide geographic opportunities for ecologically labile species to expand their ranges and be recorded in different biomes even within a small geographic area”.

Even though the new genus Januaria occurs within the limits of a protected area (National Park Carvernas do Peraçu, Fig. 4b), the national park and adjacent areas are currently considered as “Nature imperiled” areas by Dinerstein et al. (2017)DINERSTEIN E ET AL. 2017. An ecoregion-based approach to protecting half the terrestrial realm. BioScience 67(6): 534-545. https://doi.org/10.1093/biosci/bix014.
https://doi.org/10.1093/biosci/bix014... . Dinerstein and coauthors organized the 846 ecoregions recognized worldwide into four distinct categories, defined by the extent of the remaining natural habitat and protected land. The category “Nature imperiled” refers to regions in which the protected area is less or equal to 20% where the remaining habitat exists as a mosaic of isolated fragments insufficient in size and orientation to adequately conserve biodiversity. In addition, more recently, Peixoto Teixeira et al. (2021)PEIXOTO TEIXEIRA L, NIC LUGHADHA E, VINICIUS CHAGAS DA SILVA, M & FREIRE MORO M. 2021. How much of the Caatinga is legally protected? An analysis of temporal and geographical coverage of protected areas in the Brazilian semiarid region. Acta Bot Brasilica 35(3): 473-485. https://doi.org/10.1590/0102-33062020abb0492.
https://doi.org/10.1590/0102-33062020abb... using GIS, quantified the total area of Caatinga encompassed by fully protected and sustainable use reserves. The authors found that less than 8% of the Caatinga is legally protected under Brazil’s national nature reserve legislation (SNUC law), and only 1.3% is in reserves with full legal protection. Therefore, Dinerstein et al. (2017)DINERSTEIN E ET AL. 2017. An ecoregion-based approach to protecting half the terrestrial realm. BioScience 67(6): 534-545. https://doi.org/10.1093/biosci/bix014.
https://doi.org/10.1093/biosci/bix014... and Peixoto Teixeira et al. (2021)PEIXOTO TEIXEIRA L, NIC LUGHADHA E, VINICIUS CHAGAS DA SILVA, M & FREIRE MORO M. 2021. How much of the Caatinga is legally protected? An analysis of temporal and geographical coverage of protected areas in the Brazilian semiarid region. Acta Bot Brasilica 35(3): 473-485. https://doi.org/10.1590/0102-33062020abb0492.
https://doi.org/10.1590/0102-33062020abb... agreed with previous authors (e.g. Miles et al. 2006MILES L, NEWTON AC, DEFRIES RS, RAVILIOUS C, MAY I, BLYTH S, KAPOS V & GORDON JE. 2006. A global overview of the conservation status of tropical dry forests. J Biogeogr 33: 491-505. https://doi.org/10.1111/j.1365-2699.2005.01424.x.
https://doi.org/10.1111/j.1365-2699.2005... , Queiroz et al. 2006), that the tropical dry forest is one of the most endangered biomes in the world, and despite a recent expansion of the protected area network, only small portions of the Brazilian semiarid region are effectively safeguarded.

Endemism of Spermacoce clade in Brazil

As stated by Lombardi et al. (2005)LOMBARDI JA, SALINO A & TEMONI LG. 2005. Diversidade florística de plantas vasculares no município de Januária, Minas Gerais, Brasil. Lundiana 6(1): 3-20. the wide variety of vegetation types in a relatively small sample area, such as in the north of Minas Gerais, is probably conditioned by edaphic factors, including the capacity of the soil to retain water. The whole Brazilian territory shows similar conditions, thereby exhibiting a large expanse of several biomes, with different types of vegetation and remarkable plant diversity.

Studies on the ancestral area reconstruction and diversification are still lacking in the Spermacoce clade, but it has been hypothesized that the Neotropics is the centre of origin for the Spermacoce clade (Dessein 2003DESSEIN S. 2003. Systematic studies in the Spermacoceae (Rubiaceae). Ph.D. Dissertation. Katholieke Universiteit Leuven, Belgium., Janssens et al. 2016JANSSENS SB, GROENINCKX I, DE BLOCK P, VERSTRAETE B, SMETS E & STEVEN D. 2016. Dispersing towards Madagascar: biogeography and evolution of the Madagascan endemics of the Spermacoceae tribe (Rubiaceae). Mol Phylogenetics Evol 95: 58-66. https://doi.org/10.1016/j.ympev.2015.10.024.
https://doi.org/10.1016/j.ympev.2015.10.... ). According to our knowledge, Brazil comprises 16 of the 24 currently recognized genera of the Spermacoce clade. Of these, seven genera are endemic to the country: Carajasia, Denscantia E.L. Cabral & Bacigalupo, Paganuccia, Planaltina R.M. Salas & E.L. Cabral, Psyllocarpus, Schwendenera K. Schum., and the new genus Januaria. Denscantia is a climbing subshrub endemic to Alagoas, Bahia, Espírito Santo, and Rio de Janeiro, growing in different biomes: only four species inhabit the Atlantic Forest biome of Brazil, in areas of Restinga, while D. calcicola R.M. Salas & E.L. Cabral grows in a seasonally dry region in the Caatinga, north-eastern Brazil (Salas & Cabral 2012SALAS RM & CABRAL EL. 2012. Denscantia calcicola (Rubiaceae), a new species from limestone outcrops in the Brazilian Caatinga. Syst Bot 37(3): 807-810. https://doi.org/10.1600/036364412X648742.
https://doi.org/10.1600/036364412X648742... ). Planaltina is a genus with four species endemic from the central Brazilian highlands in Goiás, Minas Gerais, and Federal district states, growing between 800–1200 m (Salas & Cabral 2010SALAS RM & CABRAL EL. 2010. Planaltina nuevo género de la tribu Spermacoceae (Rubiaceae), endémico del Planalto central de Brasil y una nueva especie del estado de Goiás, Brasil. J Bot Res Inst 4(1): 193-206.). Psyllocarpus is divided in two sections (Kirkbride (1979)KIRKBRIDE JH. 1979. Revision of the Genus Psyllocarpus (Rubiaceae). Smithsonian Contributions to Botany 41: 1-32.: P. sect. Psyllocarpus, inhabits the Cerrado in the states of Bahia, Goiás, Minas Gerais, and the Distrito Federal; whereas P. sect. Amazonica J.H.Kirkbr. is restricted to white-sand Amazonian campinas in the states of Amazonas, Pará, and Rondônia. Schwendenera is endemic to the Atlantic Forest, ocurring in the interior or along the margin of the humid forest biome in the south of Brazil (São Paulo and Paraná states) (Salas et al. 2020SALAS RM ET AL. 2020. Schwendenera in Flora do Brasil 2020. Jardim Botânico do Rio de Janeiro. Disponível em: http://floradobrasil.jbrj.gov.br/reflora/floradobrasil/FB14285>. Access: 13 Oct 2021.
http://floradobrasil.jbrj.gov.br/reflora... ). Paganuccia is a recently described monospecific genus (Nuñez-Florentin et al. 2022NUÑEZ-FLORENTIN M, SALAS RM, CARMO JAM, CABRAL EL, DESSEIN S & JANSSENS SB. 2022. Paganuccia icatuensis (Rubiaceae), a new genus and species from Bahia, Brazil, with a key to all the genera of the tribe Spermacoceae in the Americas. Taxon 71(3): 630-649. https://doi.org/10.1002/tax.12651.
https://doi.org/10.1002/tax.12651... ), endemic to the dune areas of the mid São Francisco River valley (Bahia), occurring in the Caatinga. Carajasia is also a monotypic genus with a restrictive distribution, being endemic to Pará in the north of Brazil, where it only grows in ferric soil (or Canga) on the top of the Carajás mountain range (Salas et al. 2015SALAS RM, CABRAL EL, VIANA PL, DESSEIN S & JANSSENS S. 2015. Carajasia (Rubiaceae), a new and endangered genus from Carajás mountain range, Pará, Brazil. Phytotaxa 206: 14-29. http://dx.doi.org/10.11646/phytotaxa.00.0.0.
https://doi.org/10.11646/phytotaxa.00.0.... ).

Paganuccia and Carajasia are similar to Januaria in that they are all recently described genera based on new or hitherto unstudied, or unidentified, herbarium material from areas with limited access. Their limited access could explain why these areas are poorly explored botanically resulting in large collecting deficits. As Fernandez et al. (2020) argues: “further botanical exploration with increased collecting efforts and taxonomic revisions of plant diversity, especially understudied families, may potentially reveal larger numbers of flowering plants in the dry forests, especially in the Caatinga; and that the discovery of new taxa is inevitable in light of the large number of unidentified specimens in Brazilian herbaria”. In this sense, Bebber et al. (2010)BEBBER DP ET AL. 2010. Herbaria are a major frontier for species discovery. Proc Natl Acad Sci USA 107(51): 22169-22171. https://doi.org/10.1073/pnas.1011841108.
https://doi.org/10.1073/pnas.1011841108... mentioned that only 16% of the flowering plants are described within five years of being collected for the first time. The description of the remaining 84% involves much older specimens, with nearly 25% of new species descriptions involving specimens that are over 50 years old. Extrapolation of these results suggests that, of the estimated 70,000 species still to be described, more than half have already been collected and are stored in herbaria. Januaria lombardii is indeed a good example of this interesting scenario. This taxon was collected twice, one collection by Julio Lombardi in 1997, and the oldest specimen probably collected in the middle of the nineteenth century (Gardner’s paratype), i.e., the genus remained indeterminate in a herbarium for more than 100 years. Supporting Bebber et al. (2010)BEBBER DP ET AL. 2010. Herbaria are a major frontier for species discovery. Proc Natl Acad Sci USA 107(51): 22169-22171. https://doi.org/10.1073/pnas.1011841108.
https://doi.org/10.1073/pnas.1011841108... , a study also performed in the Spermacoce clade, resulted in the recently described new species of Staelia overlooked for more than a century (Staelia schumannii R.M. Salas & E.L. Cabral, Carmo et al. 2021CARMO JAM, SOBRADO SV, CABRAL EL & SALAS RM. 2021. Staelia schumannii (Rubiaceae, Spermacoceae): an old new species from Bahia, Brazil, overlooked for more than a century. Syst Bot 46(3): 844-851. https://doi.org/10.1600/036364421X16312067913525.
https://doi.org/10.1600/036364421X163120... ).

In view of the above, we provide an updated key to all the genera of the Spermacoce clade in Brazil (adapted from Nuñez-Florentin et al. 2022NUÑEZ-FLORENTIN M, SALAS RM, CARMO JAM, CABRAL EL, DESSEIN S & JANSSENS SB. 2022. Paganuccia icatuensis (Rubiaceae), a new genus and species from Bahia, Brazil, with a key to all the genera of the tribe Spermacoceae in the Americas. Taxon 71(3): 630-649. https://doi.org/10.1002/tax.12651.
https://doi.org/10.1002/tax.12651... ).

Key to the genera of Spermacoce clade occurring in Brazil

Genera distributions (in Brazil) are presented within brackets.

1. Inflorescences lax, thyrsoid or pleiothyrsoid, with partial inflorescences glomeriform or subglomeriform. 2

1. Inflorescences mostly congested; flowers in glomerules, axillary or pseudoaxillary, pauciflorous to multiflorous, or with 1-flowered axillary inflorescences. 5

2. Plants erect, rarely climbing. Flowers heterostylous, or rarely homostylous. Seeds with a persistent or deciduous strophiole [widespread]. Galianthe

2. Plants climbing. Flowers always homostylous. Seeds always with a persistent strophiole. 3

3. Fruits indehiscent or fruits tardily separated at the apex into two indehiscent carpels. Seeds wingless or shortly winged [Bahia]. Paganuccia

3. Fruits septicidally dehiscent, dividing into two valves. Seeds winged, wing derived from a strophiole that exceeds the base and apex of the seed or from an extension of the exotesta 4

4. Flowers pedicellate, pedicel 1.7–5.5 mm long. Fruit with an apical portion of the carpels exceeding the hypanthium and forming a “beak” or “rostrum”. Strophiole developed and exceeding the length of the seed apically and basally [widespread] Emmeorhiza Pohl ex Endl.

4. Flowers subpedicellate or shortly pedicellate, pedicel 0.5–1.2 mm long. Fruit without an apical portion that exceeds the hypanthium. Strophiole as long as, or shorter than, the length of the seed [Alagoas, Bahia, Espírito Santo, Paraíba, Pernambuco, Rio de Janeiro, Sergipe]. Denscantia

5. Fruits dry or fleshy, indehiscent, or dry schizocarpic . 6

5. Fruits with longitudinal, circumscissile, longitudinal-oblique or longitudinal-transverse dehiscence. 12

6. Flowers heterostylous. Stigma 4-fid [Paraná, São Paulo]. Schwendenera

6. Flowers homostylous. Stigma bifid, 2-lobed, 3-fid or 4-lobed, never 4-fid 7

7. Ovary 3- or 4-carpellate. Stigma 3-fid, 4-lobed, rarely 2-lobed [widespread]. Richardia

7. Ovary 2-carpellate. Stigma bifid, 2-lobed, or obscurely 2–4-lobed. 8

8. Pauciflorous inflorescences (1–5 flowers per node) 9

8. Multiflowered inflorescences (40–100 flowers per node). 11

9. Herbs rupicolous, with stems and leaves reddish. Calyx lobes 0.15–0.3 mm long. Schizocarp fruits with septicidal dehiscence into two mericarps leaving a basal carpophore [Pará]. Carajasia

9. Herbs or subshrubs, generally psammophilous or growing in wet soils, rarely rupicolous, with green stems and leaves. Calyx lobes 0.6–8 mm long. Schizocarp fruits with septicidal dehiscence into two mericarps without leaving a basal carpophore, or indehiscent fruits (tardily separated at the apex into two indehiscent carpels). 10

10. Growing in marshy soils. Corolla hypocrateriform, corolla tube internally glabrous, lobes internally pubescent. Stigma and style bifid [widespread] Diodia s.s.

10. Mostly on sandy soils, not marshy. Corolla infundibuliform, tube with a ring of moniliform trichomes internally in the inferior part of the tube. Stigma 2-lobed or obscurely 2–4-lobed [widespread]. Hexasepalum

11. Inflorescences in axillary (bilateral) glomeruli. Corolla with a ring of trichomes internally on the middle of the corolla tube, lobes glabrous. Stamens and style exserted. Seeds 1(2) per capsule [widespread]. Borreria p.p.

11. Inflorescences in pseudoaxillary (unilateral) glomeruli, rarely terminal and with 1–2 axillary glomeruli [e.g. Spermacoce decipiens (K. Schum.) Kuntze]. Corolla with a fringe of trichomes internally on the inferior half of the corolla lobes or in the tube. Stamens and style included. Seeds 2 per capsule [widespread]. Spermacoce p.p.

12. Calyx 2- or 4-lobed, if 4-lobed then with 2 lobes smaller than the other 2. Fruit with circumscissile or longitudinal-oblique dehiscence. 13

12. Calyx 2- or 4-lobed, lobes equal to subequal in size. Fruit with longitudinal dehiscence, or longitudinal-transverse dehiscence. 14

13. Calyx 4-lobed, with 2 larger and 2 smaller lobes, rarely exceptions. Corolla mostly hypocrateriform. Fruit with circumscissile dehiscence; the fruit separates into two parts after dehiscence, the superior part in the shape of a “mitre” formed by the upper portion of the carpels and persistent calyx lobes, and the inferior part formed of the basal portion of the carpels and basal part of the septum [widespread]. Mitracarpus

13. Calyx 2-lobed (rarely 3–4), with lobes equal or subequal. Corolla infundibuliform. Fruit with longitudinal-oblique dehiscence, fruit separated into three parts after dehiscence, two apical caducous valves, and a basal portion formed of the basal portion of the carpels, and an intercarpellar septum that remains intact and persists on the plant [widespread]. Staelia p.p

14. Fruit divides into three parts, two caducous valves and a persistent intercarpellar septum. 15

14. Fruit divides into two dehiscent valves, or one dehiscent valve and the other indehiscent. 17

15. Flowers homostylous with included stamens, or distylous with included stamens in long-styled flowers and exserted in short-styled flowers. Fruit strongly compressed laterally [Amazonas, Bahia, Distrito Federal, Goiás, Minas Gerais, Pará, Rondônia]. Psyllocarpus p.p.

15. Flowers always homostylous. Stamens and style exserted. Fruit obovoid or subglobose. 16

16. Calyx 2-lobed. Stigma bifid. Pollen grains 7–10-aperturate, small (P = 25.7, E = 25.3 µm), with long colpi. Ventral surface of seeds without ruminations [widespread]. Staelia p.p.

16. Calyx 4–7 lobed. Stigma bilobate or obscurely 2–4-lobed. Pollen grains 10–11(13)-aperturate, large (P = 61,5–65, E = 60–64,5 µm), with short colpi. Ventral surface of seeds ruminate [Distrito Federal, Goiás, Minas Gerais] Planaltina

17. Flowers heterostylous or homostylous. Fruit strongly compressed, with a persistent membranous septum parallel to the valves [Amazonas, Bahia, Distrito Federal, Goiás, Minas Gerais, Pará, Rondônia]. Psyllocarpus p.p.

17. Flowers always homostylous. Fruit obconic, turbinate, obovoid, not compressed, without a persistent membranous septum parallel to the valves. 18

18. Pollen with a reticulate simple exine. Longitudinal-transverse dehiscence, with one indehiscent carpel and one dehiscent valve that separates from the pedicel [Minas Gerais]. Januaria

18. Pollen with an eutectate, perforate, rarely microreticulate exine. Longitudinal dehiscence, with both carpels dehiscent or both indehiscent, or one carpel indehiscent and one valve dehiscent, in all cases both valves remain together and persist on the pedicel. 19

19. Inflorescences in axillary (bilateral) glomeruli. Stamens and style exserted, stamens attached at the sinuses of the corolla lobes [widespread]. Borreria p.p

19. Inflorescences in pseudoaxillary (unilateral) glomeruli, rarely terminal and 1–2 axillary. Stamens and style included, stamens attached in the middle or at the base of the corolla tube, sometimes near to interlobular sinuses but inside the tube [widespread]. Spermacoce p.p

CONCLUSION

This study further unravels the complex phylogenetic relationships within the Spermacoce clade as part of an ongoing global revision of the group. It also further improves the taxonomic classification of the Spermacoce clade. In this opportunity, we present the description of an endemic, and probably endangered, new genus, with a combination of phylogenetic and morphological evidence. Nevertheless, many issues remain unsolved in the classification of the Spermacoce clade (tribe Spermacoceae) and further sampling will be required (especially of those poorly known taxa) in order to provide further improvements. To prevent an irreversible loss of biodiversity, more attention and funding must be devoted to the protection of the Brazilian dry forest and the many endemic species that are characteristic of these notable ecosystems.

Furthermore, we believe that the description of this new taxon, despite being a unicate, is extremely important considering that the area it inhabits is clearly threatened. This is a contribution to the recent checklists of floristic information about the Caatinga, considered as a first large step in the increase in scientific knowledge, fundamental for establishing conservation priorities, information on land use management, or even conducting further biogeographic studies.

SUPPLEMENTARY MATERIAL

Figures S1, S2.

Alignment. Combined matrix (ITS + ETS + atpB-rbcL + peth + rps16 + trnL-trnF).

ACKNOWLEDGMENTS

MNF, JEF, RMS thank the Consejo Nacional de Investigaciones Científicas y Tecnológicas (CONICET) for the grants awarded that supported this work. This study was also partially funded by the Agencia Nacional de Promoción Científica y Técnica (FONCYT PICT-2016-3517) and the Universidad Nacional del Nordeste (PI A009-17 and PI 16P001 grants). We are grateful to Elsa Leonor Cabral for her valuable comments and suggestions, and to Laura Simón for her help with the illustration of the species. We also thank the staff of the molecular laboratory of Meise Botanic Garden, Belgium, especially Pieter Asselman and Wim Baert.

REFERENCES

APPENDIX I

Voucher information and GenBank accession number for taxa used in the present study. Species, geographic origin, voucher information (collector and number, and herbarium of deposition), and GenBank accession numbers for ITS, ETS, atpB-rbcL, peth, rps16, trnL-trnF. Newly generated sequences are indicated by an asterisk (*). – : information not applicable.

OUTGROUP: Bouvardia ternifolia (Cav.) Schlthl., Oaxaca, Mexico, Ochoterena et al. 454 (BR), KF736987, KF737029, –, –, –, –; National Botanical Garden, Meise, origin unknown S2928 (BR), –, –, –, –, AF002758, –; Mexico, Spencer et al. 363 (NY), –, –, –, –, –, EU642537. — INGROUP: Borreria alata (Aubl.) DC., Goiás, Brazil, Queiroz et al. 14105 (CTES), KF736995, KF737036, –, –, –, –; Misiones, Argentina, Miguel 64 (CTES), –, –, MZ064144, MZ064115, MZ064094, MZ064059; Borreria brachystemonoides Cham. & Schlthl., Corrientes, Argentina, Miguel et al. 26 (CTES), MF166821, MF166810, MZ064145, MZ064116¸ MZ064095¸ MZ064060; Borreria capitata (Ruiz & Pav.) DC., Minas Gerais, Brazil, Sobrado et al. 135 (CTES), MF166822, MF166811, –, –, –, –; French Guiana, Andersson 1908 (GB), –, –, EU543007, EU557764, EU543069, EU543158; Borreria dasycephala (Cham. & Schlthl.) Bacigalupo & E.L.Cabral, Misiones, Argentina, Salas et Cabaña 388 (CTES), KF736991, –, –, –, –, –; Misiones, Argentina, Miguel et al. 18 (CTES), –, MF166807, MZ064146, –, –, –; Borreria diacrodonta L.M.Miguel & E.L.Cabral, Ceará, Brazil, Bolland s.n. (K), MF166816, MF166805, –, –, –, –; Brazil, Salas 432 (CTES), –, –, MZ064147¸–, –, –; Borreria latifolia (Aubl.) K.Schum., Goiás, Brazil, Queiroz et al. 14110 (CTES), KF736994, KF737035, –, –, –, –; Minas Gerais, Brazil, Sobrado 143 (CTES), –, –, MZ064148, MZ064117, MZ064096, MZ064061; Borreria loretiana E.L.Cabral, Misiones, Argentina, Keller et Paredes 9918 (CTES), MF166820, MF166809, MZ064149, MZ064118, –, MZ064062; Borreria multibracteata E.L.Cabral & Bacigalupo, Goiás, Brazil, Queiroz et al. 14261 (CTES), KF736990, KF737032, –, –, –, –; Borreria ocymifolia (Willd. ex Roem. & Schult.) Bacigalupo & E.L.Cabral, French Guiana, Andersson et al. 2040 (GB), AM939463, –, EU542952, EU557712, –, EU543108; Ecuador, Bremer 3340 (UPS), –, AM932951, –, –, –, –; Borreria orientalis E.L.Cabral, R.M.Salas et L.M.Miguel, Misiones, Argentina, Sobrado et Salas 158 (CTES), MF166823, MF166812, MZ064150, MZ064119, –, MZ064063; Borreria schumannii (Standl. ex Bacigalupo) E.L.Cabral & Sobrado, Misiones, Argentina, Cabral et al. 760 (CTES), KF736997⁴, KF737038, –, –, –, –; Corrientes, Argentina, Medina 398 (CTES), –, –, MZ064151, MZ064120, MZ064097, MZ064064; Borreria spinosa Cham. & Schlthl. ex DC., Minas Gerais, Brazil, Viana et al. 5917 (BHCB), MF166817, MF166806, MZ064152, MZ064121, –, MZ064065; Borreria tenella (Kunth) Cham. & Schlthl., Misiones, Argentina, Miguel et al. 15 (CTES), MF166819, MF166808, –, MZ064122, –, MZ064066; Tocantins, Brazil, Fonseca 6547 (IBGE), –, –, MZ064153, –, –, –; Borreria verticillata (L.) G.Mey., Corrientes, Argentina, Salas 402 (CTES), KF736998, KF737039, –, –, –, –; Minas Gerais, Brazil, Oliveira 43 (CESJ), –, –, –, MZ064123, –, MZ064067; Carajasia cangae R.M.Salas, E.L.Cabral & Dessein, Pará, Brazil, Costa et al. 588 (BHCB), KF737015, KF737057, –, –, –, –; Brazil, Mota 1972 (BHCB), –, –, MZ064154, MZ064124, MZ064098, MZ064068; Crusea calocephala DC., Oaxaca, Mexico, Ochoterena et al. 456 (BR), KF737009, KF737051, –, –, –, –; Guatemala, Gustafsson et al. 215 (GB), –, –, EU542930, EU557690, –, EU543088; Crusea coccinea DC., Oaxaca, Mexico, Ochoterena et al. 461 (BR), KF737010, KF737052, –, –, –, –; Crusea hispida (Mill.) B.L.Rob., Tabasco, Mexico, Chase 2913 (K), –, –, –, –, AF002759, –; Crusea megalocarpa (A.Gray) S.Watson, Mexico, Pringle 3852 (S), AM939439, AM932929, EU542931, EU557691, EU543025, EU543089; Diodia aulacosperma K.Schum., Kenya, Luke 9029 (UPS), AM939444, AM932934, EU542934, EU557695, EU543026, EU543092; Diodia saponariifolia Cham. & Schlthl., Misiones, Argentina, Cabaña & Salas 22 (CTES), KF737007, KF737049, –, –, –, –; Misiones, Argentina, Miguel et al. 20 (CTES), –, –, –, MZ064125, MZ064099, MZ064069; Diodia virginiana L., Missouri, U.S.A., Taylor 12758 (MO), KF737008, KF737050, –, –, –, –; U.S.A., Vincent 4296 (GB), –, –, –, –, AY764288, –; Emmeorhiza umbellata (Spreng.) K.Schum., Bahia, Brazil, Queiroz et al. 13746 (CTES), KF737000, KF737042, MZ064143, MZ064126, MZ064100, MZ064070; Ernodea littoralis Sw., Cuba, Rova et al. 2286 (GB), KF737001, KF737043, EU542937, EU557698, AF002763, EU543095; Ernodea taylori Britton, North Bimini, Correll 44186 (NY), KF737002, KF737044, –, –, –, –; Galianthe brasiliensis (Spreng.) E.L.Cabral & Bacigalupo, Misiones, Argentina, Cabral et al. 758 (CTES), KF737011, KF737053, MZ064156, –, –, –; Misiones, Argentina, Miguel 32 (CTES), –, –, –, MZ064128, MZ064102, MZ064072; Galianthe eupatorioides (Cham. & Schlthl.) E.L.Cabral, Goiás, Brazil, Queiroz et al. 14190 (CTES), KF737012, KF737054, –, –, –, –; Argentina, Schinini et Cristóbal 9811 (GB), –, –, EU542939, EU557700, EU543028, EU543097; Galianthe grandifolia E.L.Cabral, Distrito Federal, Brazil, Queiroz et al. 14015 (CTES), KF737013, KF737055, –, –, –, –; Brazil, Viana et al. 5860 (BHCB), –, –, MZ064157, MZ064129, MZ064103, MZ064073; Galianthe palustris (Cham. & Schlthl.) Cabaña Fader & E.L.Cabral, Misiones, Argentina, Miguel et al. 19 (CTES), MF166825, MF166827, MZ064158, MZ064130, MZ064104, –; Galianthe peruviana (Pers.) E.L.Cabral, Minas Gerais, Brazil, Salas et al. 408 (BHCB, CTES), KF737014, KF737056, –, –, –, –; Brazil, Salas et al. 413 (CTES, HUEFS), –, –, MZ064159, MZ064131, MZ064105, MZ064074; Galianthe spicata (Miq.) Cabaña Fader & Dessein, French Guiana, Anderson et al. 1961 (GB), AM939535, AM933008, EU542935, EU557696, EU543027, EU543093; Hexasepalum angustifolium Bartl. ex DC., Mexico, Rzedowski et al. 17792 (MEXU), KF737004, KF737046, –, –, –, –; Hexasepalum apiculatum (Willd.) Delprete & J.H.Kirkbr., Bahia, Brazil, Queiroz et al. 13727 (CTES), KF737003, KF737045, –, –, –, –; Brazil, Salas et al. 457 (CTES), –, –, MZ064160, –, –, MZ064075; Bahia, Brazil, Queiroz et al. 14601 (HUEFS), –, –, –, MZ064132, –, –; Hexasepalum sarmentosum (Sw.) Delprete & J.H.Kirkbr., Cameroon, Dessein et al. 1521 (BR), KF737005⁴, KF737047, –, –, –, –; French Guiana, Andersson et al. 2071 (GB), –, –, –, –, AF002762¹, –; Hexasepalum teres (Walter) J.H.Kirkbr., Goiás, Brazil, Queiroz et al. 14089 (CTES), KF737006, KF737048, –, –, –, –; Hydrophylax maritima L., Sri Lanka, Lundqvist 8945 (UPS), –, –, EU567457, –, –, –; Januaria lombardii R.M. Salas & Nuñez-Florentin, M., Brazil, Minas Gerais, Januaria, Lombardi & Salino 1674 (BHCB), OP921300*, OP902588*, OP902589*, –, –, OP902590*; Micrasepalum eritrichoides (C. Wright ex Griseb.) Urb., Cuba, León 12997 (US), MZ064088, –, MZ064161, MZ064133, MZ064106, MZ064076; Micrasepalum haitiense Urb. & Ekman, Dominican Republic, Liogier 14859 (US), MZ064089, –, –, –, –, –; Mitracarpus capitatus Lozada-Pérez & Borhidi, Ochoterena 543 (BR), KM215366, KM215328, –, –, KM215470, –; Mitracarpus hirtus (L.) DC., Brazil, Souza 1228 (HUEFS), KM215374, –, –, –, –, –; Argentina, Keller 11863 (CTES), –, MZ064084, –, –, MZ064107–; Argentina, Miguel 67 (CTES), –, –, MZ064162, MZ064134, –, MZ064077; Mitracarpus megapotamicus (Spreng.) Kuntze, Beck 26027 (LPB), KM215361, –, –, –, –, –; Corrientes, Argentina, Salas et Cabaña 399 (CTES), –, KF737041, –, –, –, –; Bueno 2617 (HAS), –, –, –, –, KM215465, –; Mitracarpus microspermus K.Schum., Brazil, Queiroz 14122 (HUEFS), KM215351, KM215313, –, –, –, –; Guyana, Jansen-Jacobs et al. 4785 (GB), –, –, EU542975, EU557732, EU543044, –; Brazil, Viana et al. 5888 (BHCB), –, –, –, –, –, MZ064078; Mitracarpus rigidifolius Standl., Brazil, Souza 911 (HUEFS), KM215352, KM215314, –, –, –, –; Brazil, Salas et al. 452 (CTES, HUEFS), –, –, MZ064163, MZ064135, MZ064108, –; Mitracarpus robustus E.B.Souza & E.L.Cabral, Brazil, Salas 410 (CTES, HUEFS), –, –, MZ064164, MZ064136, MZ064109, MZ064079; Paganuccia icatuensis R. M. Salas, Bahia, Brazil, Salas et al. 434 (BR, CTES, K, MO, NY, SI), MZ703642, MZ703643, MZ703645, MZ703646, –, MZ703644; Psyllocarpus asparagoides Mart. ex Mart. & Zucc., Brazil, Salas et al. 411 (CTES, HUEFS), KF737018, KF737060, MZ064165, MZ064137, –, –; Bahia, Brazil, Harley et al. 20077 (AAU), –, –, –, –, AF003611, –; Psyllocarpus laricoides Mart. ex Mart. & Zucc., Brazil, Andersson et al. 35750 (UPS), AM939531, –, –, –, –, –; Minas Gerais, Brazil, Mota 2662 (BHCB), –, MZ064085, –, –, MZ064110, –; Minas Gerais, Brazil, Monteiro 245 (SPF), –, –, MZ064166, MZ064138, –, –; Psyllocarpus phyllocephalus K.Schum., Brazil, Queiroz et al. 14016 (CTES), –, KF737061, –, –, –, –; Minas Gerais, Brazil, Viana 5885 (BHCB), MZ064090, –, MZ064167, MZ064139, MZ064111, MZ064080; Richardia brasiliensis Gomes, Brazil, Souza 966 (HUEFS), KM215369, KM215334, –, –, KM215474 –; Richardia grandiflora (Cham. & Schlthl.) Steud., Goiás, Brazil, Queiroz et al. 14055 (CTES, HUEFS), KF737027, KF737066, MZ064168, MZ064140, –, –; Brazil, Souza 967 (HUEFS), –, –, –, –, KM215475, –; Richardia humistrata (Cham. & Schlthl.) Steud., Misiones, Argentina, Cabaña et Salas 17 (CTES), KF737028, KF737067, –, –, –, –; Argentina, Keller 5268 (CTES), –, –, MZ064169, MZ064141, –, MZ064081; Richardia scabra L., Minas Gerais, Brazil, Lombardi et al. 3771 (CTES), MZ064091, –, MZ064170, MZ064142, –, –; Minas Gerais, Brazil, Viana et al. 5912 (CTES), –, MZ064086, –, –, –, –; Goiás, Brazil, Fonseca et al. 4078 (CTES), –, –, –, –, MZ064112, –; Richardia stellaris (Cham. & Schlthl.) Steud., Australia, Egeröd 85343 (GB), AM939534, –, EU543006, EU557763, EU543068, EU543157; Schwendenera tetrapyxis K.Schum., Paraná, Brazil, Marques et al. 83 (CTES), KF737017, KF737059, –, –, –, –; Spermacoce breviflora F.Muell ex Benth., Northern Territory, Australia, Harwood 1070 (BR), KF737019, KF737062, –, –, –, –; Spermacoce confusa Rendle, Oaxaca, Mexico, Ochoterena et al. 552 (CTES), KF737020, KF737063, –, –, –, –; Colombia, Andersson et al. 2074 (GB), –, –, –, –, AF003619, –; Spermacoce dibrachiata Oliv. Zambia, Dessein et al. 626 (BR), KF737021, –, –, –, –, –; Spermacoce erosa Harwood, Australia, Harwood 1148 (BR), AM939537, AM933009, EU543008, EU557765, EU543070, EU543159; Spermacoce eryngioides (Cham. & Schlthl.) Kuntze, Corrientes, Argentina, Salas et al. 378 (CTES), KF736992, KF737033, –, –, –, –; Spermacoce filituba (K.Schum.) Verdc., Kenya, Luke 9022 (UPS), AM939539, AM933011, EU543009, –, EU543071, EU543160; Spermacoce glabra Michx., Missouri, U.S.A., Taylor 12757 (MO), KF737022, KF737064, –, –, –, –; Spermacoce hispida L., Sri Lanka, Wanntorp et al. 2667 (S), AM939540, AM933017, EU543011, EU557768, EU543073, EU543162; Spermacoce incognita (E.L.Cabral) Delprete, Goiás, Brazil, Queiroz et al. 14049 (CTES), KF736993, KF737034, –, –, –, –; Spermacoce marginata Benth., Dessein s.n. (BR), –, –, KT252890, KT252886, KT252880, KT252883; Spermacoce paganucci E.L.Cabral & Bacigalupo, Brazil, Queiroz 14609 (HUEFS), –, KM215324, –, –, –, –; Spermacoce princeae (K.Schum.) Verdc., Kenya, Luke & Luke 8371 (UPS?), HM042452, HM042507, –, –, HM042566, HM042585; Spermacoce prostrata Aubl., Goiás, Brazil, Queiroz et al. 14083 (CTES), KF736996, KF737037, –, –, –, –; Colombia, Andersson et al. 2078 (GB), –, –, EU543012, EU557769, –, EU543163; Spermacoce ruelliae DC., Gabon, Andersson & Nilsson 2296 (GB), AM939543, AM933014, EU543014, EU557771, EU543074, EU543165; Spermacoce sphaerostigma (A.Rich.) Oliv., Zambia, Dessein et al. 555 (BR), MF166813, MF166801, –, –, –, –; Spermacoce stipularis Dessein, Zambia, Dessein et al. 368 (BR), MF166814, MF166802, –, –, –, –; Spermacoce subvulgata (K.Schum.) J.G.García, Zambia, Dessein et al. 216 (BR), MF166815, MF166803, –, –, –, –; Spermacoce tenuior L., Tabasco, Mexico, Novelo et al. 4160 (MO), KF737023, KF737065, –, –, –, –; Staelia culcita R.M.Salas & E.L.Cabral, Minas Gerais, Brazil, Viana et al. 5891 (BHCB), MZ064092, –, MZ064171, –, MZ064113, MZ064082; Staelia herzogii (S.Moore) R.M.Salas & E.L.Cabral, Santa Cruz, Bolivia, Soto et al. 1053 (CTES, USZ), KF737024, –, –, –, –, –; Staelia thymoides Cham. & Schlthl., Misiones, Argentina, Cabral et al. 754 (CTES), MZ064093, –, –, –, –, –; Staelia virgata (Link ex Roem. & Schult.) K.Schum., Bahia, Brazil, Salas et al. 423 (CTES), KF737025⁴, MZ064087, MZ064172, –, MZ064114, MZ064083.

Publication Dates

History