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 longifolium Barboza & S. Leiva , sp. nov. [urn:lsid:ipni.org:names: 77192558–1]. Type : Ecuador . Zamora-Chinchipe : Area of Estación Científica San Francisco , road LojaZamora , ca. 35 km from Loja , transect Q2, 03˚58’S, 79˚04’W, 1900 m , 12 Jun 2005 (fl, fr), F . A . Werner 1548 ( holotype , QCA [QCA-160608]; isotypes , LOJA , NY [NY-01130066]). Figs 3 and 4 Fig 3. Capsicum longifolium Barboza & S. Leiva. (A) Flowering and fruiting branch. (B) Flower. (C) Opened corolla. (D, E, F) Anther, ventral, lateral and dorsal view, respectively.(G) Gynoecium.(H) Ovary in cross section. (I) Fruit. (J) Seed. Drawn by S. Leiva González. https://doi.org/10.1371/journal.pone.0209792.g003 Diagnosis . Similar to Capsicum dimorphum (Miers) Kuntze but differing in the long and narrow coriaceous leaves, the number of flowers (3–9), the unequal thick calyx appendages, and the glabrous vegetative organs and calyx. Description . Scandent shrubs (0.60) 1.40–3 m tall, laxly branched, slightly plagiotropic. Young stems green, fragile, glabrous, striate with abundant ovoid and light dark lenticels; bark of older stems dark green, glabrous, striate with sparse white lenticels. Sympodial units difoliate, geminate, the leaf pair markedly anisophyllous in size and shape. Leaves simple, coriaceous, slightly discolorous, adaxial surface dark green and shiny, abaxial surface light green and opaque, glabrous on both surfaces and margins; the larger leaves with blades (7) 8.5–17 (18) cm long, (0.8) 1–2.5 cm wide, narrowly elliptic (ratio 6–10.8), major veins (11)13-17 on each side of midvein, base asymmetric and attenuate, margin entire, apex acuminate; petioles 0.2 (0.5–1.4) cm long, glabrous; the minor leaves 2.5–5.7 cm long, 1–2 cm wide (ratio 1.78–4), ovate or broadly elliptic, major veins 4–5 on each side of midvein, base short attenuate, sometimes asymmetric, margin entire, apex obtuse; petioles 0.1–0.5 cm long, glabrous. Flowers in fascicles of 3–7 (9) on a short shoot leaving evident scars when fallen, rarely solitary; flowering pedicels green, filiform, terete, pendent, slightly curved, not geniculate at anthesis, widening to the apex, 0.3–0.8 cm long, glabrous. Flower buds ovoid, yellow or purplish yellow. Calyx 2.5–3 mm long, 2.8–3 mm wide, cup-shaped, very thin, transparent, light green or greenish purple, the margin truncate, glabrous, with 2–3 thick appendages like triangular-compressed wings, 2–2.5 mm long, 1.8–2.2 mm wide, green or purple, glabrous. Corolla 6–8.5 mm long, 8–11 mm diam, stellate-campanulate, thick, entirely yellow or yellow with red-brown coloration at margin lobes or inside, without interpetalar tissue; tube (3) 4–5 mm long, glabrous inside and outside; lobes 3–3.5 (4) mm long, ca. 3 mm wide, broadly ovate, erect or patent, glabrous adaxially and abaxially, the tips papillose and cucullate. Stamens 5, equal, filaments equal, 2–2.6 mm long, white or red-brown, glabrous, inserted on the corolla ca. 2 mm from the base, with inconspicuous auricles at point of insertion; anthers 2–2.75 mm long, not connivent, elliptic, purplish white or brown. Ovary 1.6–1.8 mm long, 1.2 mm diam, subglobose, white or light green, glabrous; nectary 0.3–0.5 mm tall, white; style 5–5.8 mm long, white and lilac at the apex, widening distally, glabrous; stigma 0.3 mm long, 0.2–0.4 mm wide, light green, somewhat bilobed. Berry 0.8–1.3 cm diam, globose, slightly flattened at the apex, green when immature, orange at maturity, glabrous, not pungent, the pericarp lacking giant cells (endocarp smooth) and stone cells; fruiting pedicels 1–1.6 cm long, pendent, terete, widened distally; the fruiting calyx persistent, non-accrescent, 4–5.5 mm diam, discoid, green-purple or green, the appendages spreading or reflexed, short and wide ( 2–2.8 mm long, 2.4–2.6 mm wide at base) or long and more slender ( 4.5–5.5 mm long, ca. 1.5 mm wide at base), fleshy and subulate. Seeds ca. 24 per fruit, 1.7–2.3 mm long, 1.7–2.2 mm wide, not compressed, obconic, black, the surface reticulate, cells rectangular or polygonal in shape, lateral walls straight or slightly sinuate. Distribution and ecology . Endemic to northern Peru ( Amazonas , Cajamarca and Piura ) and southern Ecuador ( Zamora-Chinchipe ) ( Fig 2 ), growing in montane wet forests at mid elevations ( 1800–2200 m ), associated with other Solanaceae shrubs ( Capsicum geminifolium (Dammer) Hunz. , Solanum spp. , and Deprea spp. ), Cyathea Sm. ( Cyatheaceae ), Miconia Ruiz & Pav. ( Melastomataceae ), Piper L. ( Piperaceae ), Ocotea Aubl. ( Lauraceae ), Anthurium Schott ( Araceae ), amongst other shrubs and trees. It grows in the interior of primary forest in shady areas. Fig 4. Capsicum longifolium Barboza & S. Leiva. (A) Plant. (B) Internode with lenticels. (C, D) Flower buds. (E) Flower, longitudinal section. (F) Flowers showing corolla yellow with brownish center. (G) Same flower as in F, lateral view. (H, I) Flowers with completely yellow corollas, upper and lateral view, respectively. (J, K) Flowers with yellow corollas with red-brown edges, upper and lateral view, respectively. (L, M) Immature fruits. (N) Mature fruit. Photos by S. Leiva González and G. E. Barboza. https://doi.org/10.1371/journal.pone.0209792.g004 Phenology . Flowering and fruiting from December to August, and probably all year. Etymology —The species epithet refers to the shape of the leaves, which are the longest and narrowest elliptic leaves known thus far in the genus. Species Conservation Assessment . Following the IUCN Criteria (IUCN 2017), we suggest C . longifolium deserves a status of Endangered. The extent of occurrence is calculated to be 19207 km 2 (Criterion B1 < 20000 km 2, Vulnerable), the area of occupancy, 24 km 2 (Criterion B2 < 500 km 2, Endangered). Although the species has been collected many times in the last 12 years, in San Francisco Biological Reserve (SFBR, Zamora-Chinchipe , Ecuador ), it is known from only other 5 locations (Criterion B1a � 10, Vulnerable), in areas not included in a National System of Protected Areas which would indicate a risk to the quality of its habitat (Criterion B2b). Karyology . This taxon possesses a 2n = 2x = 26 karyotype with 9 m pairs (1–9) of decreasing but rather similar size, 3 sm pairs (10–12), and one st pair (13) ( Fig 5 , S 1 Table). Pair 10 (sm) is satellited. Two types of constitutive heterochromatin are found in this taxon, GC-rich heterochromatin ( CMA +/DAPI-) and moderately GC-rich heterochromatin ( CMA +/DAPIo). The fluorescent banding pattern is quite simple, with most of the chromosomes having similar-sized small terminal bands, except for pairs 8, 12 and 13, which are not banded, an intercalary band on the long arm of pair 3, all of them carrying moderately GC-rich heterochromatin, and the large heterochromatic nucleolar organizer region ( NOR )-associated block on the short arm of satellite pair 10, bearing GC-rich heterochromatin ( Fig 5 , S 4 Table). Affinities . Capsicum longifolium is strongly resolved within the Andean clade, as the first diverging branch within the clade, sister to the rest of the species included in it ( Fig 6 ). Additional specimens examined . PERU . Amazonas : Rodríguez de Mendoza , Omia , entre la Cordillera y Quebrada de Agua Sal , 06˚26’18’’-06˚25’4.4’’S, 77˚10’30.8’’-77˚10’2.3’’W, 2457– 2118 m, 22 Sept 2008 (fl, fr), V . Quipuscoa S . et al . 4374 ( HUSA , HUT, F, USM ) . Cajamarca : San Ignacio , Huarango , Quebrada El Mirador , 05˚16’12"S, 78˚40’01"W, 2200 m, 13 Mar 2000 , J . Campos et al . 6607 ( MO ) . Piura : Huancabamba , distr. Carmen de la Frontera , Río Samaniego , margen derecha, zona de amortiguamiento del Santuario Nacional Tabaconas-Namballe , 2150–2200 m, 25 Apr 2003 (fl, fr), S . M . Baldeón et al . 5316 ( USM ) . ECUADOR . Zamora-Chinchipe : Estación Biológica San Francisco ( EBSF ) , camino hacia la antena, pasando el río San Francisco , 3˚58’22.5”S, 79˚04’40.9”W, 1830 m, 2 May 2017 (fl), G . E . Barboza & S . Leiva González 4821 ( CORD ) ; at the same place, S . Leiva González 6531 ( HAO ) ; EBSF, a unos 300 m después del cruzar el Río San Francisco , por el camino del Atajo , 3˚ 58’21.6”S, 79˚04’41.4”W, 1839 m, 17 Aug 2017 (fl, fr), G . E . Barboza & S. Leiva González 4846 & 4851 ( LOJA , duplicates to be sent to CORD & HAO ) ; EBSF, después de cruzar el Río San Francisco , 3˚58’22.5”S, 79˚04’39.3”W, 1888 m, 17 Aug 2017 (fl, fr), G . E . Barboza & S . Leiva González 4849 & 4850 ( LOJA , duplicates to be sent to CORD ) ; road Loxa-Zamora , 5 km W of Tambo , 2100 m, 14–19 Jul 1959 (fl), G . Harling 5867 (S) ; above Valladolid on road to Yanganá , 2700 m, 2 Feb 1985 (fl), G . Harling & L. Andersson 21464 ( GB ) . Capsicum longifolium is unique in the genus in having the longest and narrowest leaves and the striking calyx appendages that arise from the calyx tube as lateral compressed thick expansions or wings ( Fig 4C–4E and 4L–4N ). Apart from that, it is morphologically most similar to C . dimorphum with which it shares the shape and color of the corolla, fruit and seeds. Capsicum longifolium can be distinguished by having completely glabrous vegetative organs and calyces, long and narrow (ratio 6–10.8) coriaceous major leaves, flowers in fascicles of 3–7 (9) on a short shoot and calyces with 2–3 thick appendages like triangular-compressed wings compared to the pubescent vegetative organs and calyces, the shorter and wider (ratio 4–5.25) membranaceous major leaves, the solitary or up to 5 axillary flowers, and the toothless calyx or with 3 tiny appendages of C . dimorphum . Another species of Capsicum sympatric with C . longifolium (especially in SFBR, Ecuador ) is C . geminifolium that has a dense indumentum, long apiculate leaves, longer pedicels ( 5 cm long), thin calyx appendages, and funnel-shaped yellow corollas with many purple or maroon spots inside. Fig 5. Somatic metaphase chromosomes and ideogram of Capsicum longifolium . (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.g005 Variation in corolla color and length of the fruiting calyx appendages can be observed in the field in individuals growing under the same environmental conditions. The corolla is mainly pure yellow ( Fig 4E, 4H and 4I ), but occasional specimens have corolla lobes red-to brown-edged ( Fig 4J and 4K ), or with a red-brown ring inside the corolla limb ( Fig 4F and 4G ); in this latter case, the filaments and the style are also red-brown. In general, the fruiting calyx appendages do not enlarge considerably ( Fig 4L and 4N ) but some specimens have long appendages ( Fig 4M ). Fig 6. Bayesian majority-rule consensus tree of Capsicum . Posterior probabilities values indicated by each branch. New species are highlighted in bold-colored letters and the clade to which they belong is indicated. https://doi.org/10.1371/journal.pone.0209792.g006 The chromosome number 2n = 26 found in C . longifolium is the same as that of C . rhomboideum (Dunal) Kuntze [ 32 ], C . lanceolatum (Greenm.) C.V. Morton & Standl. [ 33 ] and C . lycianthoides Bitter [ 34 ], all belonging to the Andean clade. Their karyotype formulas are quite similar, but that of C . longifolium is closest to C . lycianthoides ( 9 m + 3 sm + 1 st) than to C . rhomboideum ( 10 m + 1 sm + 2 st). The species of this clade share small amounts of heterochromatin, a single pair of NOR, short karyotype lengths, and small chromosomes in comparison with other species of the genus [ 32 ]. The karyotype of C . longifolium is almost half the length of C . rhomboideum , the latter with the shortest karyotype length known until now for the entire genus.