Four new species of Capsicum (Solanaceae) from the tropical Andes and an update on the phylogeny of the genus

Four new species of Capsicum (Capsiceae, Solanaceae) from Andean tropical forests in South America are described. Capsicum benoistii Hunz. ex Barboza sp. nov. (incertae sedis) is endemic to a restricted area in south-central Ecuador and is most similar to the more widespread C. geminifolium (Dammer) Hunz. (Colombia, Ecuador, and Peru). Capsicum piuranum Barboza & S. Leiva sp. nov. (Andean clade) is found in northern Peru (Department Piura) and is morphologically most similar to C. caballeroi M. Nee of the Bolivian yungas (Departments Santa Cruz and Cochabamba) but closely related to C. geminifolium and C. lycianthoides Bitter. Capsicum longifolium Barboza & S. Leiva sp. nov. (Andean clade) occurs from northern Peru (Departments Amazonas, Cajamarca, and Piura) to southern Ecuador (Province Zamora-Chinchipe), and is morphologically most similar to C. dimorphum (Miers) Kuntze (Colombia, Ecuador, and Peru). Capsicum neei Barboza & X. Reyes sp. nov. (Bolivian clade) is endemic to southeastern Bolivia (Departments Chuquisaca and Santa Cruz) in the Boliviano-Tucumano Forest, is morphologically most similar to another Bolivian endemic species C. minutiflorum Rusby (Hunz.), and is closely related to C. caballeroi. Complete descriptions, illustrations, distributions and conservation assessments of all new species are given. Chromosome numbers for C. piuranum and C. longifolium are also provided. Three of the new species were included in a new phylogenetic analysis for Capsicum; their positions were strongly resolved within clades previously recognized in the genus.


Introduction
. Measurements of dried material were made from dissections of flowers or fruits rehydrated in hot water. Information about flower, fruit, and seed color was taken mainly from our own observations in the field; we also tested pungency of immature and mature fruits by tasting them in the field. The geographic distribution for each species was plotted using QGIS 2.8 (QGIS Development Team, 2018) and was based on georeferenced data of all the herbarium 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) [15]. The extent of occurrence and area of occupancy were calculated using the Geospatial Conservation Assessment Tool GeoCAT [16,17].

Nomenclature
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Karyology
One population each of C. longifolium (Barboza and Leiva 4821) and C. piuranum (Barboza and Leiva 4841) were studied. Somatic chromosomes were observed in squashed root meristems obtained from germinated seeds. The root apices were fixed in 3:1 ethanol: acetic acid mixture for 12 hr after a pretreatment in 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. Fluorochrome-stained chromosomes of somatic metaphases were observed in pectinase-cellulasemacerated root tip squashes [18]. Fluorescent chromosome banding to reveal the type and distribution of constitutive heterochromatic regions was performed using the triple staining technique (CDD) with the fluorochromes chromomycin A3, distamycin A and 4'-6-diamidino-2-phenylindole (CMA/DA/DAPI) [19]. Metaphase chromosomes were observed and photographed with epifluorescence using an Olympus BX61 microscope equipped with the appropriate filter sets (Olympus, Shinjuku-ku, Tokyio, Japan) and a JAI CV-M4 + CL monocromatic digital camera (JAI, Barrington, N.J., USA). For the karyotype description, chromosomes were arranged in groups according to the position of the centromere and in order of decreasing size within each type. Chromosome terminology followed Levan et al. [20]. The ideograms were based on chromosome measurements of fluorochrome banded metaphase plate photomicrographs, according to Moscone et al. [21]. The number of metaphases and individuals used for the karyotype analysis of each taxon is shown in S1 Table. species, one from Lycianthes and one from Dunalia (S3 Table). Five molecular markers were sequenced: intergenic spacers psbA-trnH, rpl32-trnL, ndhF-rpl32 and trnL-trnF from the chloroplast genome, and the single-copy nuclear gene waxy (GBSSI, granule-bound starch synthase, exons 2 to 7). With the exception of a few samples (see below), psbA-trnH and waxy were amplified as previously done for Capsicum species [1], rpl32-trnL after Sang et al. [22], ndhF-rpl32 according to Miller et al. [23], and trnL-trnF following Taberlet et al. [24]. For rpl32-trnL, ndhF-rpl32, and trnL-trnF, the reaction mixtures were prepared using the Reddy-Mix PCR Master Mix (Thermo Fisher Scientific, Waltham, USA) as for psbA-trnH [1]. DNA extraction for some samples yielded degraded and/or insufficient concentrations of DNA. In those cases, amplifications were achieved following two different strategies: a. using the Phusion Green Hot Start II High-Fidelity PCR Master Mix (Thermo Fisher Scientific, Waltham, USA) for waxy, and b. after a two-steps nested reaction, the first one using the ReddyMix PCR Master Mix and the second one with the Phusion Green Hot Start II High-Fidelity PCR Master Mix, for the plastid markers. The following reaction mixture was used with the Phusion Green Hot Start II High-Fidelity PCR Master Mix in all cases: 1 μL DNA, 5 μL mix, 1 μl each primer (5 μM), 0.3 μL dimethyl sulphoxide, 0.1 μL bovine albumin, and 2.6 μL trehalose. For amplification with the Phusion Green Hot Start II High-Fidelity PCR Master Mix, the conditions were adjusted for each marker (S2 Table), following the manufacturer's recommendations. In all cases, PCR products were cleaned using a combination of the enzymes exonuclease I (Exo I, Thermo Scientific) and thermosensitive alkaline phosphatase (FastAP, Thermo Scientific), following Werle et al. [25], and sequenced on an automated capillary sequencer [Macrogen Inc. (Seoul, South Korea) and University of Vienna (Vienna, Austria)].
Phylogenetic analysis. A bayesian inference (BI) analysis was conducted to explore the affinities of three of the new species proposed. A preliminary test (see next paragraph for details) was made to have a first insight on the placement of the new species within the framework of the 11 clades recognized in Capsicum [1]. A single sample of representative species of most clades (exception made for the Longidentatum clade) was included and only the sequences of the plastid markers were used for this scope. Based on the result (not shown), the number of samples was increased for the clades were the new species were resolved to perform the definitive analyses; cultivated species/varieties were avoided in all steps, except for C. pubescens. The materials studied and their collection data are listed in S3 Table. New sequences were obtained for most samples for the five markers, which were deposited in GenBank, and a few were downloaded from GenBank (mostly used in Carrizo García et al. [1]; S3 Table).

Taxonomic treatment
Distribution and ecology. Endemic to a restricted area in central-southern Ecuador (Tungurahua, Loja, Fig 2) growing in thickets in montane forests, between 1500-2600 m elevation.
Phenology. Flowering from March to May. Fruiting time unknown. Etymology-The new species is named in honor to Raymond Benoist (1881-1970), a French botanist, who collected in French Guyana, Morocco and Ecuador; the holotype is a nice specimen collected by her in 1931.
Species Conservation Assessment. Following the IUCN Red List Criteria (IUCN 2017), this species is proposed as Endangered (EN). The extent of occurrence is calculated to be 2050 km2 (Criterion B1 < 5000 km2, Endangered), the area of occupancy, 12 km2 (Criterion B2 < 500 km2, Endangered) and the species is known from only three localities (Criterion B1a � 5, Endangered). It is possible that its geographic range has declined (EOO and AOO, Criterion B2b i & ii) because the species has not been collected since 1978 despite recent intensive searches in the same locations. Capsicum benoistii was identified as a new species by the late Solanaceae specialist Armando T. Hunziker (CORD) who annotated the epithet name benoistii on the specimen housed at P (Benoist 4204), but this name was never published. It is a poorly known species collected only three times in Ecuador; none of these collections have fruits. Extensive recent field explorations in Tungurahua were unsuccessful in finding this species. It is distinctive in its deeply lobed stellate corolla (lobes three times longer than the tube, Fig 1B) and in the presence of heterostylous flowers (Fig 1G and 1H). These features plus the short flowering pedicels (1.3-2 cm long) distinguish C. benoistii from C. geminifolium, which has funnel-shaped corollas lobed about halfway, homostylous flowers, and longer pedicels (5 cm long).
As some data are still unknown (e.g. corolla color, fruit and seed characters, and chromosome number) for this species and freshly collected leaf material is not available for DNA extraction, we cannot suggest in which of the different clades of the current phylogeny of Capsicum [1] it could be placed.
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  New species of Capsicum from the Andes 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.
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 New species of Capsicum from the Andes 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).
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 New species of Capsicum from the Andes 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.
Capsicum piuranum Barboza  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.
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 km2 (Criterion B1 < 100 km2, Critically Endangered), the area of occupancy, 8 km2 (Criterion B2 < 10 km2, 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, S1 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, S4 Table).
Affinities. Capsicum piuranum is resolved within the Andean clade, strongly supported as sister to the C. lycianthoides-C. geminifolium assemblage (Fig 6). 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).  Capsicum piuranum is sympatric with other two Andean species, C. geminifolium (Dammer) 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][33][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. Description. Small shrubs 0.70-2 (3) m tall, thin, erect, laxly branched above. Young stems green, slim, fragile, glabrescent, and slightly striate, without lenticels; bark of older stems light brown, glabrous, with a few oblong lenticels. Sympodial units difoliate, geminate, leaf pair not markedly anisophyllous in size and shape. Leaves simple, membranaceous, glabrescent on both surfaces and margins with 4-7-celled non glandular trichomes 0.2-0.5 mm long; the larger leaves with blades (5.5) 6.7-11 cm long, 2.1-4 (4.5) cm wide, elliptic or ovate, major veins 3-4 on each side of midvein, base attenuate, margin entire, apex acute; petioles 0.3-0.8 (1.5) cm long; the minor leaves 2.7-4.6 (6) cm long, 1.2-1.8 (2.3) cm wide, elliptic or ovate, major veins 2-3 on each side of midvein, base attenuate, margin entire, apex obtuse or acute; petioles 0.2-0.5 (0.8) cm long, with similar pubescence as in larger leaves. Flowers 2-4 per axil, rarely solitary; flowering pedicels green, filiform, striate, pendent, slightly curved, not geniculate at anthesis, (0.65) 0.8-1.5 cm long, with sparse 5-6-celled non-glandular trichomes and tiny dark glandular trichomes (stalk unicellular, head multicellular). Flower buds ovoid, greenish pale yellow. Calyx 1.7-2.5 mm long, 2-3 mm wide, cup-shaped, green, with 10 nerves clearly evident, the margin truncate, pubescent, with non-glandular trichomes 0.3-0.6 mm long outside and dense glandular pubescence inside (head multicellular, stalk unicellular), 10 unequal linear appendages, green, the five longer appendages (0.7) 0.9-1.75 (2) mm long, emerging almost from the margin, the five shorter 0.2-0.8 (1.2) mm long, emerging 0.8-1 mm below the margin, with the same non glandular trichomes of the calyx tube. Corolla (6) 8-10 mm long, 5-6 mm diam, stellate, delicate, entirely yellow or with small brownish green spots in the base of the lobes and tube inside, with a thin interpetalar tissue; tube 3-4.5 mm long, with tiny glandular trichomes (head and stalk one celled each) inside and glabrescent outside; lobes 3.5-5.5 mm long, ca. 2 mm wide, ovate, erect, glabrous adaxially and with sparse nonglandular trichomes abaxially, the tips papillose and cucullate. Stamens   Distribution and ecology. Endemic to southeastern Bolivia (Fig 12), mainly in the Serranías Iñao, Yahuañanca and Khaskha Orkho (Dpt. Chuquisaca). A few collections have been recorded from the Yungas (Dpt. Santa Cruz). Capsicum neei is most commonly collected in the Boliviano-Tucumano Forest in both Departments [35] from understories at the foot of Phenology. Flowering and fruiting from October to May. Etymology. The epithet is in honor to Dr. Mike Nee (NY), a solanaceous specialist who carried out extensive explorations in the Bolivian territory and separated specimens of this species as a rare or probable new species in various herbaria.
Species Conservation Assessment. According to IUCN criteria [15], C. neei is proposed as Near Threatened species. The species meets the area requirements under criterion B for threatened (EOO: 16912 km2, B1 < 20000 km2, Vulnerable; AOO: 44 km2, B2 < 500 km2, Endangered) and is declining, but the population is not severely fragmented and occurs in more than 10 locations. Capsicum neei has been collected many times in the last 23 years in a recently Protected Area: National Park and Integrated Management Natural Area "Serranía Iñao" [36], and in nearby areas which suggests that both the decline in its geographic range (EOO and AOO) and the population size will not be significantly affected in the forthcoming years.
Affinities. Capsicum neei is nested within the Bolivian clade, strongly resolved as sister to C. caballeroi (Fig 6). Capsicum neei is morphologically most similar to the Bolivian C. minutiflorum in having stellate yellow corolla and red fruit at maturity. It can be distinguished by the non-geniculate pendent flowering pedicels and the strongly nerved calyx with 10 unequal appendages (Figs 10C, 10E and 11B) versus the geniculate and erect flowering pedicels and the calyx weakly nerved and with 5 equal short appendages in C. minutiflorum (Rusby) Hunz. The flowers in C. neei often appear to be solitary but the remains of 2-3 early deciduous bud or flower scars can be seen in the axils. Fruit features as pungency, presence of giant cells and sclerotic granules in the pericarp and mature seeds are unknown at present but it is probable that the fruits are pungent and have giant cells in the innermost layer of the pericarp as occur in the remaining species of the Bolivian clade where C. neei is positioned.
This new species is sympatric with C. baccatum L. var. baccatum, a taxon with a much wider distribution in South America, that has geniculate pedicels, calyx with 5 equal appendages, white corollas with greenish yellow spots inside and ovoid or globose red fruits.
Capsicum neei has been resolved as a new member of the Bolivian clade, which is coherent with its geographic range and the main common feature recognized for the clade, the yellow corollas [1]. However, the Bolivian clade has a weak support, most likely due to the apparent divergence of C. coccineum from the rest of the species; indeed, C. coccineum would deserve more attention considering some morphological variability observed in the species (GEB, pers. obs.).

Final comments on Capsicum phylogenetics
The BI tree resolves the same major clades obtained in the previous study of Carrizo García et al. [1]. The position of the three new species included in the current analysis was strongly resolved, therefore just increasing the number of species within the clades where they are placed. However, the affinities of the Bolivian clade, where C. neei is resolved, need to be further analysed. Indeed, the Flexuosum clade was resolved as its sister group in a previous study [1], in both the BI and MP consensus trees (with strong and weak support, respectively), but that result was not repeated this time. That previous result was already revealing certain weakness on this matter. As regards the Andean clade, where C. piuranum and C. longifolium are nested, the latter forms the first branch to diverge within the clade, instead of C. dimorphum, as previously found [1]. Within this clade, excluding C. longifolium and C. dimorphum, two groups of species can be distinguished: C. rhomboideum and C. hookerianum, on the one side, and C. lanceolatum, C. piuranum, C. lycianthoides and C. geminifolium, on the other side ( Fig  6). The short length of the branches within the latter group (not counting the C. lanceolatum branch) would be a signal of the closeness between these species. In fact, the delimitation of/ between C. lycianthoides and C. geminifolium has required extensive and meticulous herbarium and field observations (GB, pers. obs.). Besides, it is worth mentioning that C. hookerianum has been included for the first time in a phylogenetic analysis of the genus and, in agreement with earlier hypothesis [1], the species is resolved as a member of the Andean clade. As a final comment, the present results add more evidence about the marked divergence between the Andean clade and the rest of Capsicum (Fig 6), already discussed and sustained on chemical, anatomical and cytological features [1], such us non-pungent fruits, without giant cells in the pericarp, and the 2n = 2x = 26 karyotype.
Supporting information S1 Table. Karyotype features of the Capsicum taxa studied (2n = 26). HKL haploid karyotype length; χ mean value; sd standard deviation (no. of metaphases included in the measurements indicated in S4 Table). Heterochromatin amount expressed as percentage of HKL; NOR-assoc. NOR-associated heterochromatin; Interc. Intercalary heterochromatin. (DOC) S2 Table. PCR protocols followed for DNA amplification using the Phusion Green Hot Start II High-Fidelity PCR Master Mix. Markers are specified between brackets when conditions differ. (DOC) S3 Table. Materials and taxa studied in the phylogenetic analysis: Position within Capsicum (clade) or as outgroup, provenance, voucher specimens, ID in the BI tree, and Gen-Bank accession numbers for each marker analyzed. Sequences retrieved from GenBank are marked with an asterisk (materials not specified here). (XLS) S4 Table. Karyotype measurements in C. longifolium and C. piuranum. Number of seedlings and somatic metaphases analysed per sample, respectively, given in brackets after the voucher number. χ mean value; sd standard deviation; HKL haploid karyotype length; m metacentric chromosome; sm submetacentric chromosome; st subtelocentric chromosome; m-sat metacentric chromosome with secondary constriction and satellite; fhcb fluorochrome heterochromatic band. p and q upper and lower arms, respectively, in the ideograms (Fig 1). Bands are terminal except those marked with^, which are intercalary. � Band related to NOR.