An amazing new Capsicum (Solanaceae) species from the Andean-Amazonian Piedmont

Abstract 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.

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.

Materials and methods
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. (2016García et al. ( , 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) García et al. (2016García et al. ( , 2020. The GTR+R nucleotide substitution model was selected a priori following the Akaike Information Criteria in jModelTest 2. Diagnosis. Capsicum regale is morphologically most similar to C. longifolium Barboza & S.Leiva, but the former differs in having membranous and elliptic leaves, fleshy calyces, more deeply stellate corollas, longer filaments, longer and purple fruiting pedicels, dark blue to purple berries, larger seeds, smooth seed coats, and spine-like projections along the seed margins. Type Description. Slender shrubs (1-) 1.8-2.5 (-3) m tall, with the main stem somewhat thick, ca. 0.8 cm in diameter at base, sparsely branched toward apex, the branches dichotomous, weak, spreading horizontally. Stems solid and terete at base, the young stems pale green, glossy, striate, glabrous, the nodes green; bark of older stems dark brown, glabrous; lenticels present. Sympodial units difoliate, geminate, the leaf pairs markedly differing in size. Leaves simple, membranous, slightly discolorous, green adaxially, pale green with the midvein prominent and purple and the secondary veins lilac or green abaxially; adaxial and abaxial surfaces glabrous; major leaves with blades 17-20 (-24) cm long, 4.7-8 (-9.2) cm wide, elliptic, the major veins 6-8 on each side of midvein, the base unequal and attenuate, the margins entire and glabrous, the apex apiculate to long-apiculate; petioles (0.8-) 1.5-2.3 cm long, green adaxially and purple abaxially, glabrous; the minor leaves 2-5 cm long, 1-3 cm wide, ovate, the major veins 3-5 on each side of midvein, the base unequal, the margins entire, glabrous, the apex obtuse; petioles 0-0.4 cm long, green, glabrous. Inflorescence ca. 10 mm long, unbranched or rarely shortly forked, with 5-13 flowers, the axes glabrous; peduncle 0-5.5 mm; rachis 4.5-6 mm long; pedicels 1.2-1.4 cm long, thin, 2-3-edged, erect to spreading, straight, purple to green, glabrous, nearly contiguous, articulated at the base, leaving conspicuous scars. Buds ellipsoid, green. Flowers 5-merous, all perfect. Calyx 2-3 mm long, ca. 2 mm wide, cup-shaped, fleshy, green or greenish purple, the margin truncate, circular in outline, glabrous, the appendages (0-) 4-5, 1-1.8 mm long, 0.8-1.1 mm wide, purple, thick, triangular-compressed, reflexed, inserted very close to the margin. Corolla 7-8 mm long, ca. 10 mm in diameter, deeply stellate, thick, with narrow interpetalar tissue, pure yellow or yellow with maroon pigmentation abaxially and greenish yellow with lobes marginally maroon adaxially, glabrous, the tube 2-2.5 mm long, the lobes 5-5.5 mm long, ca. 2 mm wide, triangular, the tips papillose,   the margins with short eglandular trichomes. Stamens subequal, one filament longer than the others; long filament 3.5-4.3 mm long, shorter filaments (2) 3-3.2 mm long, white, glabrous, inserted on the corolla ca. 1 mm from the base, with inconspicuous auricles; anthers ca. 2 mm long, elliptic, not connivent, the thecae lilac or pale bluish, opening into longitudinal slits. Ovary ca. 1.3 mm long, ca. 1 mm in diameter, light green, ovoid, glabrous; nectary ca. 0.4 mm high, paler than the ovary, conspicuous; style 4.3-4.5 mm long, white, clavate, glabrous; stigma ca. 0.1 mm long, ca. 0.8 mm wide, light green, globose or somewhat discoid. Fruit a berry, globose, 6-9 mm in diameter, green when immature, turning nearly white and translucent during transition to maturity, then becoming dark blue to purple when mature, glabrous, non-pungent, the pericarp opaque, without giant cells, the endocarp smooth; stone cells absent; fruiting pedicels ca. 1.8 cm long, 1.8-2 mm in diameter proximally, 2.5-2.6 mm in diameter distally, brilliant dark purple, erect, fleshy, slightly angled and strongly thickened distally; fruiting calyx 3.75-4.25 mm in diameter, persistent, not accrescent, discoid, brilliant purple, with a conspicuous annular constriction at the junction with the swollen pedicel, the appendages reflexed, brilliant purple, fleshy and laterally compressed. Seeds 7-17 per fruit, 2.7-3.4 mm long, 2.2-2.7 mm wide, flattened, C-shaped, black, the seed coat smooth except for small spine-like projections on the seed margin, the cells irregular in shape to polygonal at seed margins, the lateral walls sinuate to straight.
Distribution. Capsicum regale occurs in southern Colombia, eastern Ecuador, and northern Peru, known mainly on the eastern slopes of the Andes (the Andean-Amazonian Piedmont), between 700-1900 m elevation (Fig. 4).
Ecology. The small populations inhabit the understory of the premontane or montane humid tropical forests of the Amazonian slopes of the Andes.
Phenology. The species has been collected in flower and fruit in April and from August to December.
Etymology. The specific epithet comes from the Latin regalis, royal or regal, referring to the regal, princely, or magnificent appearance of this special plant and also making reference to the royal purple color that suffuses the leaves, fruits, and fruiting pedicels.
Preliminary assessment of conservation status. Assessment using the IUCN Red List Criteria (IUCN 2019) suggests a status of Endangered (EN) B2ab(iii) for C. regale. Although this species has an extent of occurrence (EOO) of 47,806.378 km 2 , its area of occupancy (AOO) is calculated to be 32 km 2 (criterion B2 < 500 km 2 ), and the habitat quality has experienced a continuing decline, especially associated with fragmentation and deforestation.
Chromosome number. The somatic chromosome number found in C. regale is 2n = 2x = 26 (Fig. 5), as for all of the species of the Andean clade (Scaldaferro and Moscone 2019; Barboza et al. 2019).
Phylogenetic affinities. Capsicum regale is strongly resolved within the Andean clade of Capsicum in all analyses. Within the Andean clade, C. regale is moderately supported in a clade with C. rhomboideum and C. hookerianum. Within this clade, it is weakly supported as sister to C. rhomboideum (Fig. 6).

Discussion
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  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 , 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 , 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) (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), andCuenca 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.