Four new species of Capsicum (Solanaceae) from the tropical Andes and an update on the phylogeny of the genus Author Barboza, Gloria E. Author García, Carolina Carrizo Instituto Multidisciplinario de Biología Vegetal (IMBIV), CONICET, Córdoba, Argentina, & Department of Botany and Biodiversity Research, University of Vienna, Vienna, Austria, Author González, Segundo Leiva Author Scaldaferro, Marisel Instituto Multidisciplinario de Biología Vegetal (IMBIV), CONICET, Córdoba, Argentina, & Facultad de Ciencias Exactas, Físicas y Naturales, Universidad Nacional de Córdoba, Córdoba, Argentina, Author Reyes, Ximena Centro de Investigaciones Fitoecogenéticas de Pairumani, Cochabamba, Bolivia text PLoS ONE 2019 e 0209792 2019-01-16 14 1 1 26 http://dx.doi.org/10.1371/journal.pone.0209792 journal article 301510 10.1371/journal.pone.0209792 447a44cd-2c7e-4499-b31b-6e529c586ed5 1932-6203 PMC6334993 30650102 12630921 Capsicum piuranum Barboza & S. Leiva , sp. nov. [urn:lsid:ipni.org:names: 77192559–1] Type : Peru . Piura : Prov. Huancabamba, borde de carretera y riachuelo, 5˚22’46”S, 79˚ 33’47”W, 2311–2459 m , 22 Mar 2011 (fl, fr), T . Mione 812 ( holotype , CORD [CORD-00006936]; isotype , NY [NY-03231447]) . Figs 7 and 8 Diagnosis . Like Capsicum caballeroi M. Nee but differing in the purple calyx, the 5 equal calyx appendages, the longer tubular-campanulate corolla, the globose orange non-pungent mature fruit, and the black seeds. Description . Scandent shrubs 2–2.20 (3) m tall, densely branched. Young stems green, shiny, fragile, flexuous, glabrous, striate; bark of older stems green to dark brown, glabrous, striate; lenticels absent. Sympodial units difoliate, geminate, leaf pair markedly anisophyllous in size and shape. Leaves simple, membranaceous, discolorous, adaxial surface dark green and shiny, abaxial surface light green and opaque, glabrous or with sparse simple antrorse trichomes 0.5–1.2 mm long adaxially and abaxially, occasionally trichomes more abundant on main veins and margins; the larger leaves with blades (8) 12–17.7 cm long, (2) 2.5–4.5 cm wide, elliptic, major veins 7–9 on each side of midvein, base asymmetric and attenuate, margin entire, apex long-acuminate; petioles 0.7–1.4 (1.7) cm long, slightly winged from the decurrent leaf bases, glabrous or glabrescent with trichomes like those of the leaves; the minor leaves 2.5– 4.5 cm long, 1.5–2.6 cm wide, ovate or elliptic, major veins 3–4 on each side of midvein, base short attenuate or rounded, asymmetric, margin entire, apex acute or slightly rounded; petioles 0.2–0.5 cm long, glabrescent or pubescent. Flowers solitary or in fascicles of 3; flowering pedicels green, filiform, terete, pendent, slightly curved, not geniculate at anthesis, 1.9–2.6 cm long, glabrous or glabrescent, the trichomes simple, non-glandular, multicellular, antrorse, 0.30– 0.45 mm long. Flower buds ovoid, yellow or pale yellow. Calyx 1.5–2.6 (3) mm long, 3–4 mm wide, cup-shaped, thick, purple or greenish purple, the margin truncate, glabrescent to pubescent, with 5 appendages (0.9) 2.5–3 mm long, 0.5–0.8 mm wide, thick, erect, subulate, inserted close to the margin, glabrous or glabrescent with the same trichomes as pedicels and calyx tube. Corolla 14.5–17 mm long, 12–17 mm diam, tubular-campanulate, thick, entirely yellow; tube 11–12 mm long, glabrous inside and outside; lobes 3.5–5 mm long, 4.5–5 mm wide, broadly ovate, erect, glabrous adaxially and abaxially, the tips papillose. Stamens 5, equal, filaments subequal, 3–5 mm long, greenish white, glabrous, inserted on the corolla 3–4 mm from the base, with inconspicuous auricles at point of insertion; anthers 2–2.5 (2.8) mm long, slightly connivent before anthesis, elliptic, yellowish white. Ovary 1.25–1.5 mm long, 1.5 mm diam, subglobose, white, glabrous; nectary ca. 0.5 mm tall, inconspicuous, yellowish white; style 7.5–8 mm long, white, widening distally, glabrous; stigma 0.5 mm long, 0.8–1 mm wide, green, somewhat bilobed. Berry 0.9–1.2 cm in diameter, globose, slightly flattened at the apex, green or white when immature, orange to red at maturity, glabrous, not pungent, the pericarp lacking giant cells (endocarp smooth), sclerotic granules 2, polyhedral, yellowish white; fruiting pedicels 2.8–3.6 cm long, pendent, slightly striate and widened distally; the fruiting calyx persistent, non-accrescent, ca. 4 mm diam, discoid, green-purple or green, the reflexed appendages 5–6.1 mm long, 0.8–1 mm wide at base, fleshy and subulate. Seeds ca. 50–80 per fruit, 2–2.2 mm long, ca. 2.5 mm wide, somewhat compressed, subreniform or obconic, dark brown, the surface reticulate, cells polygonal in shape, lateral walls straight or slightly sinuate. Distribution and ecology. Endemic to a restricted area in northern Peru ( Piura , Fig 2 ) growing in montane misty rain forests, associated with other Solanaceae shrubs ( Solanum spp. Fig 7. Capsicum piuranum Barboza & S. Leiva. (A) Flowering branch. (B) Calyx. (C) Flower. (D) Opened corolla. (E, F, G). Anther, ventral, dorsal and lateral view, respectively. (H) Gynoecium. (I) Fruit. (J) Seed. Drawn by S. Leiva González. https://doi.org/10.1371/journal.pone.0209792.g007 and Streptosolen jamesonii (Benth.) Miers ), Begonia L. ( Begoniaceae ), Otholobium C.H.Stirt. ( Fabaceae ), Aphelandra R. Br. ( Acanthaceae ), Juglans L. ( Juglandaceae ) amongst other herbs and shrubs. It grows in margins of forest and near streams, between 2300–2860 m elevation, in areas of low temperature and rich soils. Phenology. Flowering from November to May, with a peak of fruiting in March–May. Etymology. The new species is named in allusion to its very restricted habitat in Department Piura ( Peru ). Species Conservation Assessment. According to IUCN criteria (IUCN, 2017), C . piuranum is proposed as Critically Endangered ( CR ) species. The extent of occurrence is calculated to be 10.195 km 2 (Criterion B1 < 100 km 2, Critically Endangered), the area of occupancy, 8 km 2 (Criterion B2 < 10 km 2, Critically Endangered). The species is known from only three locations (Criterion B2a � 5, Endangered) and the number of mature individuals observed in each subpopulation is � 50 (Criterion C2a, Critically Endangered). Karyology. A somatic chromosome number of 2n = 2x = 26 was found in this species. The karyotype comprises 9 m pairs of rather similar length (1–9), 3 sm pairs (10–12), and one st pair (13) ( Fig 9 , S 1 Table). One pair is satellited (10 sm). As in C . longifolium , this species bears two types of constitutive heterochromatin, GC-rich heterochromatin ( CMA +/DAPI-) located in the large heterochromatic band associated to the NOR in pair 10, and moderately GC-rich heterochromatin ( CMA +/DAPIo), located in the small terminal bands and in the intercalary band on the long arm of pair 3. The fluorescent banding pattern is quite simple and very similar to C . longifolium , except for the presence of 3 small bands that are not seen in that species ( Fig 9 , S 4 Table). Affinities . Capsicum piuranum is resolved within the Andean clade, strongly supported as sister to the C . lycianthoides - C . geminifolium assemblage ( Fig 6 ). Additional specimens examined. PERU . Piura : Prov. Huancabamba, carretera Canchaque-Huancabamba , km 98–99 (ca. 20 km de Canchaque rumbo a Huancabamba ), subiendo a Cuello del Indio , 05˚22’43”-05˚22’44”S, 79˚33’34”-79˚33’37”W, 2341–2346 m, 8 May 2017 (fl, fr), G . E . Barboza & S. Leiva González 4841 & 4842 ( CORD ); carretera Huancabamba-Canchaque, a una hora del Abra Cruz Blanca, 05˚22’25"S, 79˚34’02"W, 2860 m, 13 Apr 2017 (fl, fr), M . Cueva 2912 ( USM ); distrito Canchaque, km 98–99 (ruta Canchaque-Huancabamba), 5˚ 22’44.1”-5˚ 22’42.7”S, 79˚ 33’37.2”-79˚ 33’34.1” W, 2341–2346 m, 8 May 2017 (fl, fr), S . Leiva González & G. E . Barboza 6561 & 6562 ( HAO ); Prov. Morropon: Chalaco, Bosque Mijal, 05˚ 03’51.1"S, 79˚43’25.9"W, 2800 m, 1 Nov 2015 (fl, fr), M . Cueva et al . 2655 ( USM ); same locality and date, M . Cueva et al . 2656 , 2657 & 2658 ( USM , duplicates to be sent to CORD, HUSA ) . Capsicum piuranum is morphologically most similar to C . caballeroi M. Nee of the Bolivian yungas ( Santa Cruz and Cochabamba ) based on their campanulate yellow corollas. However, these species can be distinguished in the calyx color, the calyx appendages (number, size, and shape), the position of the corolla lobes at anthesis, the fruit size, shape, color and pungency, the presence of stone cells, and the seed color. Capsicum piuranum has a purple or greenish purple calyx with 5 equal subulate appendages ( Fig 8D and 8E ), while C . caballeroi has green calyx with 10 unequal linear appendages. Corolla lobes are erect compared to those of C . caballeroi which are recurved. Mature fruits are smaller (up to 1.2 mm diam), globose, orange and not pungent in C . piuranum but are larger (up to 1.6 mm diam), globose-depressed to globose, bright red and pungent in C . caballeroi . Capsicum piuranum has two stone cells ( Fig 8H ) and dark brown smaller seeds ( 2–2.2 mm long, ca. 2.5 mm wide) while C . caballeroi lacks of stone cells and the seeds are pale yellow or light brown and larger ( 3.2–4 mm long, 3.8–5 mm wide). Fig 8. Capsicum piuranum Barboza & S. Leiva. (A) Plant. (B) Leaves, abaxial surface. (C) Fruiting branch. (D) Flower bud. (E) Flower and immature fruit. (F) Mature fruit. (G) Fruit, transverse section, showing placenta and seeds. (H) Fruit transverse section, showing a stone cell at the apex (arrow). Photos by S. Leiva González and G. E. Barboza. https://doi.org/10.1371/journal.pone.0209792.g008 Fig 9. Somatic metaphase chromosomes and ideogram of Capsicum piuranum . (A) Methaphase chromosomes. (B) Ideogram. Solid black blocks or dots denote CMA+/DAPI- (NOR) or CMA+/DAPIo (terminal and intercalary) heterochromatic bands. The NOR is indicated as a separate block. https://doi.org/10.1371/journal.pone.0209792.g009 Capsicum piuranum is sympatric with other two Andean species, C . geminifolium (Dam- mer) Hunz. and C . rhomboideum , both of which have also yellow corollas and non-pungent fruits, but a moderate to dense pubescence on stems and leaves. Capsicum geminifolium differs in having longer calyx appendages ( 3–6.5 mm long) compared to C . piuranum ( 2.5–3 mm long) and funnel-shaped generally purple spotted yellow corollas (tubular-campanulate and pure yellow in C . piuranum , Fig 8E ). Capsicum rhomboideum has ovate or rhomboid-ovate leaves, up to 12 axillary flowers, campanulate-rotate smaller corollas ( 0.6–0.95 cm long), and smaller (up to 0.9 cm diam) bright red to blackish red fruits in contrast to C . piuranum where leaves are elliptic or narrowly elliptic (sometimes the minor leaves are ovate, Fig 8B and 8C ), the flowers are solitary or in fascicles of 3 ( Fig 8E ), the corolla is tubular-campanulate and longer ( 14.5–17 mm long), and the fruits are larger ( 0.9–1.2 cm diam) and orange colored. This species exhibits the same number of chromosomes as C . longifolium and the species that belong to the Andean clade [ 32 – 34 ]. In addition, C . piuranum and C . longifolium share the same karyotype formula, little heterochromatin, one only pair of NOR, and the smallest chromosomes in the genus [ 32 ]. The markedly anisophyllous leaves, the deflexed non-geniculate pedicels, the yellow corollas, the globose orange to red non-pungent fruits, the absence of giant cells and the presence of stone cells in the pericarp, the black seeds, and the chromosome number 2n = 26 place C . piuranum in the Andean clade proposed by Carrizo García et al. [ 1 ], as it has been determined in this work based on DNA data.