Capsicum regale Barboza & Bohs, sp. nov., a new species from the tropical wet forests of the eastern Andean slopes (Colombia, Ecuador, and Peru) is described and illustrated. This new species belongs to the Andean clade (all species 2n = 26) of Capsicum and is similar to C. longifolium Barboza & S.Leiva in its glabrescence, calyx morphology, and corolla and seed color but differs in its membranous and elliptic leaves, fleshy calyces, deeper stellate corollas, longer filaments, longer and purple fruiting pedicels, purple berries, and larger seeds. Its chromosome number was counted (2n = 26), a preliminary assessment of conservation status is given and discussed, and an updated identification key to the species of the Andean clade is provided.

Andean clade, Capsicum, chromosomes, phylogeny, South America, taxonomy

Capsicum L. (Capsiceae, Solanaceae), the chili pepper genus, consists of approximately 42 species distributed in temperate and tropical Central and South America, Mexico and the West Indies (Barboza et al. 2020). It includes five species cultivated worldwide as vegetables, spices, and medicines (C. annuum L., C. frutescens L., C. chinense Jacq., C. baccatum L. and C. pubescens Ruiz & Pav.). Capsicum peppers are major crops worldwide, and along with potato, tomato, and eggplant in the genus Solanum L., are amongst the most economically important members of the Solanaceae (Samuels 2015).

The Andes are one of the main centers of diversity for Capsicum, where new species continue to be discovered (Nee et al. 2006; Barboza et al. 2019). Approximately 50% of the species (ca. 20 species) occur in tropical Andean forests or in dry inter-Andean valleys (Barboza et al. in prep.). The tropical montane forest ecoregion is located on the slopes of the Andes extending north to south from southern Colombia, through Ecuador, and into northern Peru (WWF 2020). This region is characterized by a lush vegetation with evergreen seasonal broad-leaved forests and a rich fauna (Stewart et al. 2020; WWF 2020). It is one of the most biologically diverse ecosystems in the world (Gentry 1992; Bruijnzeel et al. 2010; Tapia-Armijos et al. 2015) with a high level of species endemism (Myers et al. 2000). Khoury et al. (2020) have demonstrated that the highlands of Colombia, Ecuador, Peru, and Venezuela represent one of the hotspots for Capsicum that need further investigation in terms of collecting taxa for ex situ conservation of the wild species.

During recent field explorations in the Colombian Cordillera Oriental (Dept. Caquetá), an atypical species of Solanaceae was collected. Despite the presence of several Solanaceae experts in the group, no one was sure what genus it belonged to. Its deeply stellate yellowish corollas, long-exserted stamens, and purple fruits and fruiting pedicels were striking and called to mind some characters of the poorly known genus Cuatresia Hunz., whereas its thick, triangular-compressed, and reflexed calyx appendages resembled those of some Lycianthes (Dunal) Hassler taxa, whose species are not well understood in Colombia. Puzzled, we provisionally named it “Cuatresianthes” and placed some bets on its eventual generic identity. DNA was extracted and sequenced in the Bohs lab from leaf material collected on these field trips. BLAST results indicated that the species belonged not to Cuatresia or Lycianthes, but to Capsicum. A preliminary molecular study placed the collection unequivocally in the Andean clade of Capsicum, but it did not belong to any known species. Through an exhaustive search amongst unidentified Cuatresia collections in herbaria, we found other specimens from Ecuador and Peru that matched our Colombian gatherings. Here, we describe this species as new to science and provide information on its morphology, distribution, karyology and phylogenetic position in the genus Capsicum.

Two field trips were made in Colombia (Dept. Caquetá) during 2016 and 2019. Fresh material was preserved in 70% alcohol to perform measurements of reproductive organs using a Zeiss Stemi 2000-C stereomicroscope at 6.5–50× magnification. Descriptions were based on living plants observed during field work and examination of digital images of herbarium specimens housed at the following seven herbaria: BM, COAH, COL, F, MO, QCNE, US. Seeds were also examined using scanning electron microscopy (SEM); they were prepared using enzyme etching (Lester and Durrands 1984) to dissolve outer cell walls, affixed to aluminum stubs with double-sided adhesive tape, coated with gold, and examined using a FE-SEM Sigma (LAMARX, National University of Córdoba, Argentina) microscope.

Information about flower, fruit, and seed color was taken mainly from our own observations in the field and photographs sent by some collectors; we tested pungency in the field on immature and mature fruits.

The distribution map was produced using QGIS 3.8 (QGIS Development Team 2019) and was based on georeferenced data of all the collections analyzed. Conservation status was assessed using IUCN criteria B, geographic range in the form of B1 (EOO: extent of occurrence) and B2 (AOO; area of occupancy) (IUCN 2019). The extent of occurrence and area of occupancy were calculated using the Geospatial Conservation Assessment Tool GeoCAT (Bachman et al. 2011; GeoCAT 2020).

Somatic metaphases were examined in root tip squashes obtained from germinated seeds. The root apices were fixed in 3:1 ethanol: acetic acid mixture for 12 hr after a pretreatment in 2 mM 8-hydroxyquinoline solution for two hr at room temperature and two hr at 4 °C. The material was kept at –20 °C until examination. The root tips were macerated in pectinase-cellulase solution (Moscone et al. 1993), and chromosomes were stained with 4’–6-diamidino-2-phenylindole (DAPI) (Schweizer and Ambros 1994). Metaphase chromosomes were observed and photographed with epifluorescence using an Olympus BX61 microscope equipped with the appropriate filter sets (Olympus, Shinjuku-ku, Tokyo, Japan) and a JAI CV-M4 + CL monochromatic digital camera (JAI, Barrington, N.J., USA). Three individual seeds from the collection Orejuela et al. 3034 were germinated and grown until root tips were produced, and 10 cells from each seedling were studied in metaphases.

Phylogenetic affinities were explored using DNA sequences from four markers, namely: the intergenic spacers psbA-trnH, ndhF-rpl32 and trnL-trnF from the plastid genome, and the single-copy nuclear gene waxy (GBSSI, granule-bound starch synthase, exons 2 to 10). Representatives of different clades recognized within Capsicum and several outgroup species were included. Genomic DNA of C. regale was extracted from silica-gel dried leaves using the Qiagen DNeasy Plant mini kit (Qiagen Inc., Valencia, California, EUA) and a modified CTAB protocol. Most sequences included in this study were used in previously published analyses and therefore were retrieved from GenBank, except for a few sequences from outgroup species (see Suppl. material 1: Table S1), for which DNA extracts were already available. Amplification and sequencing protocols for the markers used were as in Carrizo García et al. (2016, 2020) and Barboza et al. (2019). PCR amplicons were sequenced on an automated capillary sequencer [University of Vienna (Vienna, Austria), and the University of Utah HSC Core Research Facility (Salt Lake City, Utah, USA)]. A single concatenated dataset was assembled in MEGA 7 (Kumar et al. 2016). Phylogenetic reconstructions were done using maximum parsimony [MP, in PAUP* 4.0b10 (Swofford 2003)], maximum likelihood [ML, in RAxML v8.2.10 (Stamatakis 2014)] and Bayesian inference [BI, in MrBayes 3.2.2 (Ronquist et al. 2012)] approaches as in Carrizo García et al. (2016, 2020). The GTR+R nucleotide substitution model was selected a priori following the Akaike Information Criteria in jModelTest 2.1.3 (Darriba et al. 2012) for ML and BI analyses.

Capsicum regale belongs to the Andean clade of Capsicum (Carrizo García et al. 2016; see below). It is a very striking species due to its unbranched (Figs 1B, 2E, J) or forked inflorescence (Fig. 2D) with 5–13 deciduous flowers on an elongate rachis (Fig. 2D), fleshy and laterally compressed calyx appendages (Fig. 2D, E), deeply stellate corollas (Fig. 2F, G), strongly thickened and brilliant purple fruiting pedicels (Fig. 2H–K), dark blue to purple fruits (Fig. 2J, K), and flattened black seeds with spine-like projections at the margins (Fig. 3). This species is morphologically most similar to C. longifolium (Barboza et al. 2019) with which it shares lack of pubescence, multi-flowered inflorescences, yellow corollas, laterally compressed calyx appendages, and black seeds (see contrasting characters in the key below).

Capsicum regale possesses unusual characters of the genus. Normally, Capsicum species have unbranched inflorescences lacking peduncles, with the flowers solitary or congested on a very short axis. Flowers can be arranged on a short or relatively elongated rachis in a few species, e.g., C. rhomboideum (Dunal) Kuntze, C. coccineum (Rusby) Hunz., C. lycianthoides Bitter (Barboza pers. obs.), C. longifolium (Barboza et al. 2019), and C. regale, but none of them have short peduncles or forked inflorescences as occurs occasionally in C. regale. In most Capsicum species the calyx appendages, when present, are usually cylindrical or subulate, and green-colored. It is very rare to find laterally compressed calyx appendages that appear as wing-like structures, as occur in C. longifolium (Barboza et al. 2019), in some plants of C. dimorphum (Miers) Kuntze (Barboza, pers. obs.), and in C. regale. Stellate corollas lobed about halfway to the base are common in the genus; exceptions to this are found in C. benoistii Barboza (Barboza et al. 2019) and C. regale, both of which have deeply stellate corollas lobed more than halfway to the base. In most Capsicum species, the fruiting pedicels and fruiting calyx are generally green or green with purple tones or lines; only C. caatingae Barboza & Agra (Carrizo García et al. 2016) and sometimes C. dimorphum and C. geminifolium (Dammer) Hunz. (Jarret et al. 2019) have pedicels and calyces uniformly violet-colored, while those of C. regale are uniformly purple-colored. An unusual constriction at the junction of the thickened fruiting pedicels with the fruiting calyx is clearly evident in C. regale (Fig. 2K), a character also present in some other species, i.e., C. chinense Jacq. (Baral and Bosland 2004), C. caatingae (Carrizo García et al. 2013), C. minutiflorum (Rusby) Hunz. (Carrizo García et al. 2016), and C. lanceolatum (Greenm.) C.V. Morton & Standl. (Barboza pers. obs.). The dark blue to purple fruits are unique to C. regale among the wild Capsicum species, which have red, orange-red, or greenish-golden yellow fruits at maturity (Hunziker 2001; Carrizo García et al. 2016).

Carrizo García et al. (2016) were the first to provide an extensive phylogenetic analysis of Capsicum using broad sampling of 34 of the approximately 35 species of the genus known at the time. They identified and named 11 well supported clades within Capsicum. One of these is the Andean clade, which includes species native to Central America and the Andes in northwestern South America. Morphological characters of the Andean clade species include leaves borne in anisophyllous pairs, flowering pedicels straight (not geniculate), corollas mainly yellow, fruits red to orange-red and non-pungent with the pericarp lacking giant cells, seeds black or blackish-brown, and chromosome base numbers of x = 13 (Jarret et al. 2019; Scaldaferro and Moscone 2019). Capsicum regale exhibits all of these characters except for its dark blue or purple fruits and the occasional forked inflorescences, which are not known in any other wild Capsicum species. Molecular data from both chloroplast and nuclear regions place Capsicum regale within the Andean clade (Fig. 6). Although C. regale is morphologically most similar to C. longifolium, the combined molecular data places it in a clade with C. rhomboideum and C. hookerianum with moderate support. Nevertheless, its closest specific affinities need to be further studied using additional data.

Capsicum regale inhabits the Andean-Amazonian Piedmont, encompassing the eastern slopes of the Cordillera Oriental from southern Colombia to the Cerros de Kampanquis, the easternmost branch of the Andes in northern Peru. This area is home to a transitional ecosystem with a distinctive vegetation and biodiversity due, in part, to the juxtaposition between the Amazon basin and the Andean forests (Gentry 1992; Pitman et al. 2002); this unique biodiversity is rapidly disappearing due to intense deforestation, clearing, and fragmentation (Pitman et al. 2002; Mulligan 2010; Tapia-Armijos et al. 2015; Alvarez-B et al. 2019). Some localities where C. regale has been collected are protected areas (Parque Nacional Natural Alto Fragua Indi Wasi, Colombia; Reserva Ecológica Antisana, Ecuador; Estación Biológica Jatun Sacha, Ecuador), and it is expected that in these reserves it is not under serious threat. Other sites in which it occurs are susceptible to human disturbance such as crop planting and high levels of deforestation; these locations include Correg. Caraño (Caquetá, Colombia, Alvarez-B et al. 2019, our observations), Río Bermejo to Cerro Sur Pax (Sucumbíos, Ecuador, Pitman et al. 2002), and Cuenca del Río Morona, Pongo Chinim (Loreto, Peru, Pitman et al. 2012). In these areas, C. regale is considerably threatened, and a conservation strategy is urgently needed to protect these species-rich ecoregions.

We are indebted to the curators and assistants of the herbaria cited who provided digital images of their collections, to E. Trujillo, A. Orejuela and C. I. Orozco for their assistance in field explorations, to S. Montecchiesi and G. Aburrá for preparing the illustrations, to V. Palchetti for her assistance with the map, to L. Coca, D. Hoyos, D. Sanín, P. Gonzáles, and A. Orejuela for providing photographs or specimen vouchers, to the reviewers for their helpful suggestions and critical reading of the manuscript, and to the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, PIP 0100147 and joint project 2014-0401 PCB-FWF AI2119), the Secretaría de Ciencia y Técnica (SECyT-UNC), and the U.S. National Science Foundation ARTS program (DEB 1457366) for funding.