CIAT Research Online - Accepted Manuscript Morphological characterization in the genus Passiflora L.: an approach to understanding its complex variability The International Center for Tropical Agriculture (CIAT) believes that open access contributes to its mission of reducing hunger and poverty, and improving human nutrition in the tropics through research aimed at increasing the eco-efficiency of agriculture. CIAT is committed to creating and sharing knowledge and information openly and globally. We do this through collaborative research as well as through the open sharing of our data, tools, and publications. Citation: Ocampo Pérez, John Albeiro; d’Eeckenbrugge, Geo Coppens. 2017. Morphological characterization in the genus Passiflora L.: an approach to understanding its complex variability. Plant Systematics and Evolution 303(4): 531-558. Publisher’s DOI: http://dx.doi.org/10.1007/s00606-017-1390-2 Access through CIAT Research Online: http://hdl.handle.net/10568/80128 Terms: © 2017. CIAT has provided you with this accepted manuscript in line with CIAT’s open access policy and in accordance with the Publisher’s policy on self-archiving. This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License. You may re-use or share this manuscript as long as you acknowledge the authors by citing the version of the record listed above. You may not use this manuscript for commercial purposes. For more information, please contact CIAT Library at CIAT-Library@cgiar.org. 1 Morphological characterization in the genus Passiflora L.: an approach to understanding its complex variability Morphological characterization in the genus Passiflora John Ocampo Pérez1* and Geo Coppens d’Eeckenbrugge2 1Universidad Nacional de Colombia, sede Palmira. Departamento de Ciencias Biológicas / International Center for Tropical Agriculture – CIAT. Carrera 32 No 12 - 00 Chapinero, vía Candelaria Palmira - Valle del Cauca – Colombia. 2CIRAD, UMR AGAP, Avenue Agropolis, 34398 Montpellier, France. *Corresponding author: jaocampop@unal.edu.co Abstract Morphological variation was studied in 124 accessions from 61 species of genus Passiflora, using variance component and principal component analyses (PCA) on 43 quantitative traits, and neighbor joining cluster analysis on 85 qualitative traits. Coefficients of variation were generally higher for subgenus Decaloba, indicating stronger differentiation when compared with subgenera Passiflora. Twenty-four quantitative descriptors showing high variation at the subgenus level were selected for the PCA. The five principal components retained 84% of the total variation, being the first (32%) closely associated with flower length (hypanthium, nectary-chamber, androgynophore) and secondarily with floral cup constriction above the nectary chamber. The second (27%) is associated with flower width (length of bracts and length of corolla and corona-elements) and bract shape, and the third (14%) with peduncle branching, stem width and leaf length, which relates it clearly with variation between subgenera Astrophea and Deidamioides. The projection of accessions in the resulting tridimensional space consistently separates subgenera. A selection of 32 qualitative traits and four categorized quantitative variables, whose segregation follows divisions among Killip’s subgenera, allowed classifying consistently our species sample using a strictly phenetic approach. Eight of the nine subgenera established by Killip represented in our sample were supported by the morphological analysis, although subgenus Deidamioides is only supported in the quantitative analysis. Our results support seven of the eight Killip’s subgenera of our sample, but no infrasubgeneric classifications. However, Feuillet & MacDougal´s classification of subgenus Decaloba was partly supported and converged on many points with previous phylogenetic results obtained with DNA sequences. Key words Passiflora, systematics, morphological descriptors, principal component analysis, plant genetic resources. Introduction Manuscript Click here to download Manuscript Morphological descriptor_Passiflora_07_07_2016_JO.doc Click here to view linked References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 2 With more than 576 species, Passiflora L. is by far the most important genus in the family Passifloraceae, both numerically and economically. It is mostly distributed in the Neotropics, from coastal zones up to 4,300 m.a.s.l. in Andean páramos; only 23 species are native to the Old World in Southeast Asia, Australia and Oceania (Killip 1938; Ulmer and MacDougal 2004). The origin of Passifloraceae is very ancient from 65.5 million years ago (Paleocene) and seems to follow a biogeographic scenario proposed for several plant groups, originating in Africa, crossing to Europe/Asia and arriving in the New World by way of land bridges (Muschner et al., 2012). The chromosome base number varies among x = 6 and x = 9 (De Melo et al. 2001), and the average genome size is 1.03 pg (Yotoko et al. 2011). Passionflowers are herbaceous or woody vines usually climbing with tendrils, but a few are trees or shrubs. Other typical vegetative traits include alternate leaves, axillary stipules, and petiolar and/or laminar nectary glands. In addition, the genus exhibits several unique floral features such as an androgynophore, a complex corona constituted by one or several concentric rows of filaments, and a limen-operculum system limiting access to the nectary chamber, with impressive interspecific variation in size, shape and colors (Killip 1938). Their wide morphological variation seems to be the result of their habitat diversity as well as their co-evolutionary relationships with many organisms, including a wide range of pollinators as small and large insects, birds and bats (Ulmer and MacDougal 2004). Many species exhibit interesting fruits, pharmacological properties (e.g. sedative effect) and/or ornamental potential. More than 80 Passiflora species produce edible fruits, all of them belonging to subgenus Passiflora (Yockteng et al. 2011). The two botanicals forms of P. edulis Sims, flavicarpa Degener (yellow passion fruit) and edulis (purple passion fruit) are by far the most important crops in the family, with a world production estimated at ca. 640.000 tons. Other cultivated passion fruits are P. tripartita var. mollissima (Kunth) Holm-Nielsen & Jørgensen (curuba de Castilla), P. tarminiana Coppens & Barney (curuba India), P. ligularis Juss. (sweet granadilla), P. maliformis L. (granadilla de piedra or stone granadilla), P. quadrangularis L. (giant granadilla), P. popenovii Killip (granadilla de Quijos), P. alata Curtis (fragrant granadilla) and P. setacea DC (sururuca). The high potential of Passiflora for crop diversification and economic development induced research institutions of different countries to prioritize the characterization and evaluation of wild and cultivated populations (Cerqueira-Silva et al. 2016), and develop strategies for conservation and improvement of these genetic resources. The high diversity of character combinations related to nectary glands, stipules, corona, operculum and limen have been heavily used to delimit infrageneric divisions in Passiflora taxonomy, separating subgenera, sections, and series. Although most species appear well delimited, there are many cases in low level taxa as sections and series, where two or more species are very difficult to distinguish. The largest monograph and most important reference work was that of Killip (1938) who described 355 American species plus 20 species in 1960, placing them in 22 subgenera. Escobar (1988a,b, 1989, 1990, 1994) reviewed subgenera Astrophea, Distephana, Manicata, Rathea and Tacsonia, merging subgenera Tacsoniopsis and Tacsonia, subdivided into sections and series, and proposing one additional subgenus, Porphyropathanthus. MacDougal and Feuillet (2004) have proposed a new infrageneric classification of Passiflora, taking into account the Old World species and recognizing only four subgenera, Astrophea (unchanged; trees and shrubs, rarely lianas), Decaloba (vines with small flowers and fruits, the latter usually black), Deidamioides (vines, with two-flowered peduncles, not clearly defined as a morphological 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 3 group) and Passiflora (vines to lianas, with large flowers and fruits). This classification needs to be revised, and completed with the explicit inclusion of many more species. Thus, Krosnick et al. (2009) with DNA molecular data recognized a fifth subgenus, Tetrapathea (vines, inflorescences, small flowers and fruits, yellow-green to red at maturity) from Oceania. Both classifications of Killip (1938) and Feuillet and MacDougal (2003) are based on the extreme morphological richness and complexity of Passiflora, whose species present numerous particular traits, including a wide variation in leaf shape, even within species and within individuals (heterophylly and heteroblasty are very common), the presence of extrafloral nectaries in different parts (on leaf petiole, lamina or margins, on bracts, on sepals), floral traits showing a high level of coevolution with particular pollinators particularly in corolla color, the variable development of the hypanthium and that of the corona. However, no clear hierarchy emerges in the relative contributions of these traits to the taxonomy of the genus. On the other hand, the first molecular studies carried out on significant Passiflora species samples have consistently validated three of the four major subdivisions proposed by MacDougal and Feuillet (2004). The results of Muschner et al. (2003), on nuclear ribosomal internal transcribed spacers (ITS-1 and ITS-2) and plastid trnL-trnF intergenic spacer, Yockteng (2003) on chloroplast matK, Yockteng and Nadot (2004) on chloroplast-expressed glutamine synthetase (ncpGS), Hansen et al. (2006), on sequences analysis of the chloroplast (rpoC1 and trnL-trnT), and Krosnick et al. (2013), on nuclear (nrITS and ncpGS) and chloroplast (cp trnL-F and ndhF), all confirm the clear separation of three clades corresponding to the new contours of subgenera Decaloba, Astrophea, and Passiflora. These three major clades correspond to cytogenetic groups as they appear characterized by chromosome numbers of 2n = 12, 24, and 18, respectively. The results of Hansen et al. (2006) also support the small subgenus Deidamioides, whereas the two species that represent it in the study of Yockteng and Nadot (2004) are split in two widely divergent branches. In addition, the latter study indicated that four other small subgenera, Dysosmia (DC.) Killip, Tryphostemmatoides (Harms) Killip, Polyanthea (DC.) Killip, and Tetrapathea (DC.) Rchb., should also be recognized. Another study carried out by Muschner et al. (2012) on the phylogeny, biogeography and divergence times in 106 species and four subgenera of Passiflora with plastidial, mitochondrial and nuclear genomes, showed that the genus Passiflora is monophyletic. In addition, subgenus Deidamioides as described by MacDougal and Feuillet (2004) emerged as paraphyletic. They also situated the divergence among the four subgenera in Passiflora from 33 to 38 million years ago, during the Andes uplifting process. Recently, Krosnick et al. (2013) studied the phylogenetic relationships of subgenus Decaloba using 148 taxa and four molecular markers (nrITS, ncpGS, cp trnL-F and ndhF). The results showed that subgenus Decaloba is monophyletic and contains seven major lineages that generally correspond to currently recognized supersections. These studies have provided many new insights into the evolution of Passiflora but reveal as many new challenges that need to be addressed. Furthermore, such phylogenetic molecular studies cannot give us a definitive answer on the relationship between morphologically ill- defined species and species groups. Despite the impressive morphological diversity described among Passiflora species, few studies have compared intra- and intersubgeneric, and intra- and interspecific variation with statistical tools. A first 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 4 study was conducted by Villacís et al. (1998) on the most common species of subgenera Tacsonia and Manicata on Colombian and Ecuadorian accessions. Floral traits were mostly represented in their set of 33 qualitative descriptors, and vegetative traits in their set of 28 quantitative descriptors. The former showed limited intraspecific variation and a consistent picture of interspecific relations, while the latter provided more information on intraspecific variation but a less consistent picture on the differences between species. The descriptor list was corrected to take into account traits specific to subgenus Tacsonia, and augmented to 62 qualitative and 67 quantitative descriptors, giving a better balance between floral and vegetative traits. The same descriptor list was used to study morphological variation in the three most common cultivated (P. tarminiana and P. tripartita var. mollissima) and their hybrids, showing maternal effects in the hybrid phenotypes and confirmed the spontaneous introgression occurring between the wild P. mixta L. and the cultivated P. tripartita var. mollissima (Primot et al. 2005). A very detailed descriptor list was also used by Porter-Utley (2014) to study supersection Cieca Feuillet and MacDougal of subgenus Decaloba, and particularly the species complex around P. suberosa L. and P. coriacea Juss. Seventy quantitative traits were measured finding 33 to 39 descriptors that could be categorized and gathered with qualitative traits for neighbor joining cluster analyses of the different subsamples. On the other hand, there was considerable incongruence between molecular (ITS sequences) and morphological phylogenies which was mostly attributed to a smaller sample size and intraspecific variation in the molecular data. A Brazilian Passiflora collection including ten species was characterized by Crochemore et al. (2003) with 22 qualitative and quantitative descriptors. The results showed clear differentiation between the two botanical forms of P. edulis. More recently, De Oliveira et al. (2005) tested a new morphometric method based on leaf structures in a sample of ten Passiflora species. The method was very accurate in correctly differentiating species, with the exception of two species not consistently classified, P. foetida L., a problematic species in all classifications, and P. miersii Mast. In conclusion, despite the interest to complete an objective classification of a plant family with a huge morphological richness and complexity, only few research teams have developed and applied the necessary methodology. However, this methodology tends to be much more laborious than in plain molecular characterizations, due to the need of field germplasm collections from species with variable climatic adaptations in one or very few places where they can develop until flowering. In the case of wild Passiflora accessions, a possible solution is carrying out in situ characterization which must then be taken into account to avoid or reduce environmental bias in the analysis. The present study benefited from projects on diversity of Colombian Passifloraceae, including a component of collecting and establishing germplasm in field collections. As Colombia is the country with the highest Passiflora species diversity both for wild and cultivated material (Ocampo et al. 2007, 2010), a wide species sample could be gathered. However, practical limitations allowed describing accessions of only 51 of the 171 reported species from eight subgenera for the genus in the country. A few non-native species were added extending the sample to 61 species. Our goal was twofold, to test the utility of the revised set of descriptors over a wide range of Passiflora species, and to study morphological divergence among subgenera, species and populations. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 5 Materials and methods Study area The morphological study was carried out in three germplasm collections located in different ecological regions in Colombia according to environmental adaptations of species: El Cerrito (Tenerife) (2,700 m.a.s.l., 3°43'51.49"N; -76° 4'36.56"W) and El Cerrito (El Moral) in the department of Valle del Cauca (2,400 m.a.s.l., 3°42'53.80"N; -76° 4'37.67"W), and Buenavista (Paraguacito) in the department of Quindío (1,200 m.a.s.l., 4°23'47.57"N; -75°44'3.54"W). Plant materials The total sample was composed of 261 individuals representing 124 populations and 61 species of the Passiflora genus corresponding to nine of the 22 Killip subgenera (electronic supplementary material), of the four McDougal & Feuillet subgenera (Table 1). Geographic distribution was taken into account in the selection of accessions of a species. Narrow endemics as P. trinervia (Juss.) Poir.) are represented only by one population, and widespread species as P. edulis f. flavicarpa by one population per region. Three plants per were grown from seeds for each populations, at a distance of 3 m between rows and 3 to 5 m within rows, according to adult plant size. In this study we followed the two taxonomic treatment of the genus Passiflora proposed by Killip (1938, with emends by Escobar 1988a,b; 1989; 1994 and MacDougal 1994) and Feuillet and MacDougal (2013), as a point of comparison and discussion of our data. Data collection The descriptor list was developed in various stages by experts of Bioversity International (Regional Office for the Americas) and Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD), with adaptations to include the wide diversity of our collections. It included 43 quantitative and 84 qualitative descriptors that are presented synthetically in Table 2. These were assessed on three individuals per populations and five measures were taken for quantitative characters (in millimeters) for each individual plant. Color characters were recorded with the Royal Colour Chart (Royal Horticultural Society 2002). Quantitative fruit traits were not taken into account as they are often submitted to convergent selection processes both in wild and in cultivated species. Analyses of quantitative variation Shape descriptors were computed as ratios of crude ones. Quantitative data were submitted to an analysis of variance to compare variation among and within subgenera, species, populations, and individuals. To identify characteristics that contributed highly to differentiation among subgenera, we selected traits for which more than half of the variance was caused by variation at this level. When shape descriptors showed similar discriminating power, they were preferred over crude descriptors to avoid giving too much importance to variation in size. The selected descriptor set was submitted to a principal component analysis (PCA) applying the varimax normalized rotation option using the STATISTICA 10.0 software (Hill and Lewicki 2006), retaining those factors with an eigenvalue superior to one, and the individuals were projected onto the first three PCA axes. Cluster analyses on qualitative data 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 6 According to the PCA results, certain quantitative variables were selected on the basis of their contribution to the axes, categorized and added to the qualitative dataset, provided that the corresponding information was not yet included in a purely qualitative descriptor. The resulting dataset was treated in two steps. A first set of qualitative variables was selected on the basis of their contribution to differentiation among subgenera, discarding those that show frequent variation at lower levels. A second set included all qualitative descriptors. Both sets were submitted to a neighbor joining cluster analysis (Saitou and Nei 1987) using the coefficient of dissimilarity of Sokal & Michener and calculating bootstrap values from 1,000 replicates with the DARwin 6.0 software (Perrier et al. 2003). This phenetic approach was preferred because of the relatively poor information on morphological evolution in Passiflora. Results Quantitative variation As expected, a very high variability was observed among the 124 populations. Table 3 gives mean values and coefficients of variation for the whole sample per subgenus. Coefficients of variation are generally higher for subgenus Decaloba compared with Passiflora that have comparable representation in number of species. This higher relative variation can be seen much clearer in inflorescences (18 out of 20 traits) and shape ratios (10 out of 13 ratios), than for vegetative parts (9 of 21 descriptors), suggesting a higher interspecific differentiation in subgenus Decaloba as compared to the other two. The relative variance components for 57 quantitative descriptors shows that all present a residual variance under 25% and then a high repeatability (Fig. 1). Many descriptors appear to be efficient in discriminating among subgenera. Thus, the proportion of variance at this level exceeds 50% for 27 descriptors, including stem diameter, leaf margin serration, leaf length, numbers of nectar glands on leaf margins and petiole, diameter of peduncle, length of first and second order peduncle segments, dimensions and shape of bracts, length of flower, hypanthium, sepals and petals, nectary chamber, crown longest series, androgynophore, stamens and ovary, relative constriction above nectary chamber, and bract/hypanthium length ratio. At the species level, 28 characters are more important and they are related to dimensions of stipule, lobation (angle between lateral nerves, shape of central lobe, length of lateral lobe, distance between leaf sinus and petiole insertion), number of laminar nectary glands, position of petiolar nectary glands, length of peduncle, diameter of hypanthium, length of gynophore, shape of petals and sepals, androgynophore/hypanthium length ratio (defining protrusion of gynoecium and androecium) and pedicel/peduncle ratio. At lower levels, variance among and within populations rarely contributes more than 20% of the total. Correlations and principal components analysis (PCA) A Pearson correlation (r) matrix shows high coefficients ranging between 0.71 and 0.97 among dimensions of certain floral components as petals and sepals, hypanthium, nectary chamber, operculum, androgynophore, stamens and ovary. The length of the corona is correlated with the upper hypanthium diameter (r = 0.75). Bract dimensions are moderately correlated with these traits (0.33 to 0.65, but 0.75 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 7 with ovary length). Stem diameter is correlated with peduncle branching (r = 0.73) which is due to the association of these traits in representatives of subgenus Astrophea. From the 27 quantitative descriptors showing high variation at the subgenus level, 24 were selected for the PCA discarding three of them to avoid redundancy between shape ratios and the original traits. Five principal components were retained, representing 84% of total variation (Table 4). The first component (32%) is primarily associated with flower length (hypanthium, nectary chamber, androgynophore) and secondarily with the constriction of the floral cup above the nectary chamber. The second one (27%) is associated with flower width (length of bracts and length of corolla and corona elements) and bract shape. The third one (14%) is associated with peduncle branching, stem width and leaf length, which relates it clearly with variation between subgenus Astrophea, and secondarily with subgenus Deidamioides, on one hand, and all other subgenera. The fourth one (5%) is associated with the number of nectaries on leaf margins, which essentially relates it in supersection Distephana. The last one (5%) is only correlated with leaf serration. Fig. 2 shows the accessions in the three first axes showing a clear grouping by subgenus and supersection. The representatives of supersection Tacsonia are placed on the right along the first axis, in relation to their long and wide flowers. A few accessions, with shorter flowers as P. luzmarina Jørgensen and P. pinnatistipula Cav., appear closer to the origin of this axis, together with P. manicata individuals. P. trinervia, of subgenus Decaloba (supersection Decaloba) is placed even further on the right thanks to its very long floral tube; additionally, it is clearly separated on the second axis by its much narrower flowers and minute setaceous bracts. On the left side, subgenera Passiflora and Decaloba are not differentiated by the flower length-related axis 1, but by the second, flower width-related, axis 2. On this second axis, we find the large-flowered P. alata Curtis and P. quadrangularis (section Quadrangulares of subgenus Passiflora) at one extreme, and those of the small-flowered P. arbelaezii and P. gracillima Killip of subgenus Deidamioides at the other one. As expected, the third axis clearly differentiates subgenera Astrophea and Deidamioides from the five others. In general, subgenera Passiflora, Decaloba, Astrophea and Deidamioides, as likewise the supersections Tacsonia (subg. Passiflora) and Decaloba (subg. Decaloba) are clearly separated in the main tridimensional space. As expected, P. manicata (section Manicata) takes an intermediate position between the supersection Tacsonia and the other sections of subgenus Passiflora. This species not only combines morphological traits typical of both subgenera, but also intermediate eco-climatic requirements, as it may be found at lower elevations than tacsos (Tacsonia), but higher elevations than representatives of subgenus Passiflora. The representatives of the supersection Tacsonia that come closest to P. manicata are P. pinnatistipula and P. luzmarina, two tacsos with relatively shorter floral tubes. The former is also differentiated by a filamentous corona, instead of the typical reduced tacso coronas. Other species taking a particular position are P. foetida and P. vesicaria of subgenus Passiflora (supersection Stipulata / section Dysosmia), placed near both subgenera Passiflora (other supersections) and Decaloba, but closer to the former. Qualitative variation among and within subgenera Our first attempt to reduce the number of qualitative descriptors led us to retain 32 of them on the basis of their potential to discriminate among subgenera. The criterion was that the descriptor appears 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 8 monomorphic or shows a highly dominant condition in at least one subgenus, while polymorphic among other subgenera. Three quantitative descriptors were categorized and added because of their high correlations with the principal components of quantitative variation. Thus, the first component was represented by androgynophore length, the second one by sepal length, and the third one by stem diameter and leaf length. The fourth and fifth ones were not included to avoid redundancy with very similar qualitative descriptors. Table 5 synthesizes the observations for these descriptors. Our Colombian species of subgenus Astrophea exhibit the highest number of unique/rare traits including tree habit, wide stems of irregular section, very long leaves, absence of tendrils, short triangular stipules, dorsal scar-like nectaries (appressed against or near petiole), branched peduncles, bright-yellow sickle-sword-shaped corona filaments, and tricostate ovaries. Unique and rare traits of the two species of subgenus Deidamioides include peduncle branching (shared with species of subgenus Astrophea and P. sexflora Juss. of subgenus Decaloba), the presence of tendrils at the axil of the peduncles, and the retuse leaf apex (unique in our sample, although this trait can be observed in individuals of P. emarginata Humb. & Bonpl. subg. Astrophea). P. vitifolia Kunth (subgenus Passiflora / supersection Distephana) is differentiated by its tubular corona formed by the partial fusion of its elements and conspicuous nectary glands on leaf sinus and bracts, P. foetida and P. vesicaria (subgenus Passiflora / section Dysosmia) only by their pinnatisect bracts, and P. trinervia (subgenus Decaloba / supersection Decaloba) by the absence of a limen. Most of these traits are typical for each of these subgenera ensuring that they will not bias the cluster analysis in terms of subgeneric classification. Subgenera Decaloba (except P. trinervia) and Passiflora do not show unique traits, however they are clearly segregated by combinations of non- exclusive traits. Thus, in subgenus Decaloba, the presence of a flat hypanthium is only shared with subgenus Tryphostemmatoides, and the relatively small flower size with subgenera Astrophea, Deidamioides and the section Dysosmia of subgenus Passiflora. On the other side, subgenus Passiflora share wide flowers and the general presence of petiolar nectaries. The last three and subgenus Decaloba (P. trinervia, section Decaloba) also share large red or pink corollas and long tubular flowers (long androgynophores), typical of hummingbird-pollinated species. Floral tube length reaches extreme values in P. trinervia and Tacsonia species, with the exceptions of P. pinnatistipula, P. luzmarina and P. manicata related to their pollinators (hummingbirds). In addition, they present reduced coronas of short filaments or tubercles generally in one row only, while two-row coronas are most common in subgenera Decaloba, Deidamioides, Astrophea, and Passiflora (supersection Distephana), and highly complex coronas (more than three rows) are typical in subgenera Passiflora (except supersec. Tacsonia). Bracts are foliaceous in subgenera Passiflora and Tacsonia. Fruit shape is generally globose to short ovate/obovate in subgenera Astrophea, Decaloba (except P. trinervia), Deidamioides and Passiflora (supersections Passiflora, Distephana and Stipulata), and oval to fusiform in the supersections Tacsonia (tacsos with the exception of P. pinnatistipula) and Decaloba (P. trinervia). Fruit color seems also an interesting trait, with a particular frequency of blackish fruits in subgenus Decaloba, however this descriptor was not observed in all species. Certain species show unusual trait combinations in their subgenus. This is particularly clear in subgenus Decaloba, where P. adenopoda Moc. & Sessé ex DC. shows foliaceous bracts, serrate leaf margins with conspicuous nectary glands, orbicular petiolar glands, and an uniseriate corona. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 9 P. guatemalensis S. Watson also shows foliaceous bracts and glandless leaves as well as peltate leaves and a yellow uniseriate corona. P. sexflora shows multiple peduncles. P. coriacea, P. suberosa, P. capsularis L. and P. rubra L. lack bracts; in addition, the first two species show petiolar nectaries while the last two lack such glands in all their organs and produce an elongated fruit with a bright red color. Cluster analysis on the reduced descriptor list Fig. 3 presents a Neighbor Joining dendrogram obtained from the observations on the first set of descriptors. The three best-represented subgenera, i.e. Passiflora, Astrophea and Decaloba, and the supersection Tacsonia (except section Manicata) are supported by the analysis. Their placement on the dendrogram shows a polarization of the latter according to several traits. On one side, we find the supersections Distephana, Tacsonia and Manicata (subgenus Passiflora), i.e., species presenting large flowers and fruits as well as petiolar nectaries and foliaceous bracts, with a base chromosome number of n = 9. They are further divided between the carpenter bee-pollinated species (subgenus Passiflora) and the hummingbird-pollinated species of the supersections Tacsonia and Distephana. The consistency of this sub-classification compensates for the low associated bootstrap values. On the opposite side of the tree we find subgenera whose species produce small to medium sized flowers and fruits, with relatively simple coronas of generally two rows of filaments (rarely one or three), where petiolar nectaries are rare, with a base chromosome number of 12 for tree species and 6 for the others. As expected from the number of their rare traits, the tree species of subgenus Astrophea appear very uniform and clearly separated in a very distant cluster. P. trinervia (subgenus Decaloba) is placed on another long branch inserted at the same position. A third much larger cluster is constituted by all the species of subgenera Deidamioides and Decaloba, except for P. trinervia and P. adenopoda (subgenus Decaloba). The latter species is placed on a well-separated branch inserted in an intermediate position between the Passiflora-Distephana-Tacsonia-Manicata clusters and the Astrophea-Decaloba- Deidamioides clusters. Indeed, this species shows several unusual features and trait combinations when compared to Decaloba as a group. P. foetida and P. vesicaria (subgenus Passiflora) take a very similar intermediate position and appear well differentiated from one another, supporting the recent revision of section Dysosmia (Vanderplank 2013). Although the branch bearing the representatives of subgenus Deidamioides (P. gracillima and P. arbelaezii) is relatively long, it is clearly inserted within subgenus Decaloba, suggesting that the qualitative morphological differentiation of subgenus Deidamioides is fragile. This is consistent with the very low number of traits supporting it, but contrasting with the PCA results for quantitative traits. The first set of qualitative data also allows distinguishing some structures within clusters corresponding to subgenera. Thus, within the Passiflora cluster, one main branch corresponds to P. edulis f. edulis and medium-flowered species as P. bahiensis Kl. and P. guazumaefolia Juss., another one to large-flowered species of supersection Passiflora i.e. P. edulis f. flavicarpa, P. incarnata, and P. cincinnata Mast., a different one to series Quadrangulares (P. quadrangularis and P. alata) and typical representatives of series Tiliifoliae (P. tiliifolia and P. ligularis). Another main branch relates to typical representatives of series Laurifoliae (P. popenovii and P. nitida), one to accessions of P. maliformis, and 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 10 another one to species of section Kermesinae (P. lehmannii and P. smithii). Killip’s series Lobatae is shared between the Incarnatae subcluster (including P. caerulea L. and P. gibertii Brown) and the Kermesinae subcluster (P. subpeltata Ortega), which shows the fragility of these subclusters, as P. gibertii and P. subpeltata are morphologically very similar. Within the Tacsonia-Manicata cluster there are three main branches. A first one includes the common and widely dispersed P. mixta, P. tripartita, P. tarminiana, P. cumbalensis and endemic species related to them as P. mathewsii (Mast.) Killip, and P. luzmarina. A second one includes species of Escobar’s section Colombiana (eg. P. tenerifensis and P. linearistipula), and the last one includes the relatively short-tubed species P. pinnatistipula and P. manicata. Within the Decaloba-Deidamioides cluster, one branch corresponds to section Cieca (P. coriacea and P. suberosa), whose species are not clearly distinguished, and another one to section Xerogona (P. capsularis and P. rubra). P. guatemalensis, the only representative of supersection Hahniopathantus, is placed apart. Section Decaloba is split between three distinct branches, one for the closely related P. alnifolia Kunth and P. bogotensis Benth., one for P. auriculata Kunth, and another one for all its other representatives. Discussion Morphological and molecular diversity To appreciate the organization of Passiflora morphological diversity, we can compared some of the interspecific associations or divergences with results obtained in phenetic studies based on biochemical and molecular markers, on samples including some of the species we studied. A first series of genetic studies were carried out on smaller samples, mostly from Colombia. In the trees obtained with RAPD and cpDNA RFLP markers by Fajardo et al. (1998) and Sánchez et al. (1999), the species of subgenus Tacsonia constitute one subcluster within a large cluster, gathering them with species of subgenus Passiflora. Subgenus Decaloba is represented by P. coriacea and P. adenopoda, both species strongly diverging from this Passiflora-Tacsonia cluster, but also between themselves, which is consistent with our results. In the RAPD study, subgenera Distephana and Astrophea are represented respectively by P. vitifolia and P. spinosa (Poepp. & Endl.) Mast., and both species are placed at a considerable distance from the Passiflora-Tacsonia cluster, their divergence being intermediate between that of P. adenopoda and that of P. coriacea. Within the Tacsonia subcluster the distances between the species P. tripartita var. mollissima, P. cumbalensis, P. pinnatistipula, and P. antioquiensis follow the same order as in the our morphological cluster. This result is still the same when the comparison is extended to subgenus Manicata and comparing them to the ones obtained by Segura et al. (2002, 2003) with AFLP markers (P. tenerifensis and P. parritae also included), and with isozymes, although P. antioquiensis is placed closer to the most common tacsos than P. pinnatistipula in the isozyme study. Another convergence between morphological and AFLP markers is the clear separation of P. maliformis from the typical species of series Tiliifolia of subgenus Passiflora (Ocampo et al. 2004) and in the same way it does not support the inclusion of this species in series Laurifoliae according to Feuillet & MacDougal (2013). 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 11 Genetic relationships between subgenera and between particular species can also be deduced from subsequent phylogenetic studies carried out on wider species samples by Muschner et al. (2003) with ITS, trnL-trnF and rps4 sequences, by Yockteng (2003) with chloroplastic matK sequences, Yockteng and Nadot (2004) with sequences of the nuclear chloroplast-expressed glutamine synthetase gene (ncpGS), and Hansen et al. (2006) with trnL/trnT sequences. All these studies support the existence of three major clades, one corresponding to subgenus Astrophea, a second one formed around subgenus Decaloba, and a third one formed around subgenus Passiflora. Species of the supersection Tacsonia form a subclade within the Passiflora clade (i.e. all studies except that of Muschner et al. 2003). P. vitifolia and other representatives the supersection Distephana in Yockteng’s studies are also included in this large Passiflora clade, although they are not grouped consistently in the matK tree (Yockteng 2003). The relative positions of the three major clades differ among studies. Subgenus Astrophea takes an intermediate position in the ITS tree; it is closer to the Passiflora clade in the other trees of Muschner et al. (2003), but closer to the Decaloba clade in the trnL-trnT tree of Hansen et al. (2006) while the Decaloba clade appears closer to the Passiflora clade in the sample of Yockteng (ncpGS and matK trees). P. foetida (section Dysosmia) is clearly placed within the Passiflora clade according to rps4 and trnL- trnF sequences, but more distant although basal to this clade, in the trees obtained with ITS, ncpGS, and matK sequences. P. adenopoda is basal to the Decaloba clade in the ncpGS tree as well as in the ITS study of Krosnick and Freudenstein (2005). P. morifolia Mast., another species of section Bryonioides is also basal to the Decaloba clade, on a branch between Decaloba and Astrophea in the ITS and trnL-trnF trees. In the ncpGS tree of Yockteng and Nadot (2004) and the ITS study of Krosnick and Freudenstein (2005), subgenus Deidamioides, represented by P. tryphostemmatoides Harms and by P. arbelaezii L. Uribe respectively, is placed close to subgenus Astrophea. Strangely, in the study of Yockteng and Nadot (2004) (ncpGS tree), P. sanguinolenta Mast. is placed in a Passiflora subclade dominated by representatives of series Tiliifolia. According to Killip (1938), the placement of this species in subgenus Psilanthus is “anomalous”; indeed, his suggestion to consider its relation with P. rubra was validated by the phylogeny of Krosnick et al. (2009), Within the Decaloba clade we can recognize the higher level of divergence between subclusters, i.e. the association between representatives of series Punctatae and Miserae (Killip´s classification) in the widest subclade (trnL-trnF tree of Muschner et al. 2003), the relative separation of P. sexflora from this group, and the even more distant position of P. coriacea and of the couple formed by P. capsularis and P. rubra (ITS, trnL-trnF, and ncpGS sequences). However, the last two species are closely associated with P. sexflora in the ncpGS tree. Additionally, these results are consistent with the ones found by Krosnick et al. (2013) and Milward-de-Azevedo et al. (2014) regarding their position in the series and they also support subgenus Decaloba as monophyletic. As in our morphological study, the Passiflora clade identified in phylogenetic studies generally shows loose relations among species. The interpretation of the poorly supported subclades is very uneasy, with the partial exception of the ncpGS tree presented by Yockteng and Nadot (2004), where branches are better defined although not easier to interpret, given for example the dispersion of species belonging to series Incarnatae and Laurifoliae (Killip´s classification). Among the close associations documented by 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 12 our morphological study, we can only recognize those of P. quadrangularis with P. alata and P. incarnata with P. edulis (ITS and ncpGS trees). As a first major point of conclusion on this comparison between morphological and molecular diversity, we can underline that the major morphological divisions observed in our study find support in the genetic studies. The cytological groups are always validated with a clear separation of subgenera Astrophea (n = 12) and Deidamioides and Decaloba (n = 6), both among themselves and from subgenera Passiflora, Tacsonia, and Distephana (n = 9). Concerning subgenus Deidamioides, the consistency between morphological and genetic studies is clear only when considering our quantitative analysis, where it is associated with subgenus Astrophea mostly on peduncle traits (third principal component). This trait is also represented in the qualitative descriptors but its effect is blurred by the high number of traits shared with subgenus Decaloba. While the comparison is difficult for subgenus Deidamioides, it is impossible for P. trinervia, which is not duly represented in molecular studies. The species P. adenopoda and P. foetida and their close relatives that take an intermediate position in the general morpho- cytological pattern are consistently placed in intermediate positions. In most phylogenetic studies P. adenopoda or P. morifolia (supersection Bryonioides of subgenus Decaloba) appear basal to a general Decaloba clade, and P. foetida and P. vesicaria (section Dysosmia) basal to the general Passiflora clade (Krosnick et al. 2013). The comparison becomes more difficult at lower infrasubgeneric levels. Subgenus Decaloba appears better structured than the other subgenera, and its morphological and molecular diversity patterns appear consistent, with the grouping of Killip’s sections Punctatae and Miserae, and the differentiation of species of sections Xerogona, Cieca, and series Auriculata, and less clearly Sexflorae. The position of P. adenopoda may look surprising, as it is not consistent with either classification (Decaloba section Pseudosysosmia for Killip, supersection Bryonioides for Feuillet & MacDougal), however, this species showed several unusual features as compared to Decaloba as a group. P. foetida (section Dysosmia), takes a very similar position, that is clearly more consistent with its classification in subgenus Dysosmia by Killip (1938) than with its inclusion in subgenus Passiflora by Feuillet & MacDougal (2003). Interestingly, these two problematic species materialize the separation between the two cytogenetic groups in our tree. Indeed, chromosome counts for P. adenopoda give 2n = 12 (MacDougal 1994) as in most species of subgenus Decaloba, while those for P. foetida are 2n = 20 (De Melo et al. 2001; Yockteng and Nadot 2004). According to De Melo et al. (2001) and De Melo and Guerra (2003), P. foetida appears cytologically quite isolated but closer to the n = 9 group, its smaller chromosomes and articulate interphase nuclei being similar to species with n = 6, while its chromosome number, higher karyotype symmetry, CMA staining properties, and the number of 45S rDNA sites make it similar to species of subgenus Passiflora. In any case, more species should be gathered in a same morphological study before revising objectively the morphological classification. Within the n = 9 group, molecular data with DNA sequence studies partially allow the distinction of a Tacsonia-Manicata group and fail to separate clearly the supersection Distephana (or subgenus according to Killip), placing both of them within a Passiflora clade, while morphological analysis supports these three subgenera at the same level of differentiation. The fact that species of subgenera Distephana, Tacsonia and Manicata according to Killip´s classification have developed ornithophyly is obviously 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 13 related to their strong morphological differentiation, which does not minor the importance of their separation from subgenus Passiflora. Whether their probable evolution from a “Passiflora-like” common ancestor justifies their inclusion in the bee-pollinated Passiflora subgenus as proposed in the new classification, it is just the same classical question about considering birds as dinosaurs. In the end, it seems an issue of giving more emphasis to the adaptative forces commanding evolution or to the genetic structure that subtends them. Concerning subgenus Passiflora sensu Killip, no clear structure appears at the interspecific level that could result in clear subdivisions into the series. The study of sequence variation for the ncpGS gene provides the only tree with reasonably well supported structure at this level. However, several obvious abnormalities question the robustness of the information. Our morphological observations only confirm closer associations between the most typical representatives of some series, although the number of contradictions with the classification and the lack of a clear hierarchy in the branch structure point to the difficulty of the work and the risk of under or over classification, which will lead to chose between a limited number of poorly supported series or a great number of poorly represented series. Similarly, the structure of the Tacsonia-Manicata branch does not support clearly sections and series in supersection Tacsonia, but it allows differentiation between two groups of tacsos; one corresponding to common species that probably have their center of diversity in Ecuador as is obvious for P. cumbalensis, P. luzmarina and P. matthewsii, and very likely for P. mixta, P. tripartita and P. tarminiana (Segura et al. 2005), and another cluster including only species endemic to Colombia with a slight but clear differentiation related to extreme variation for peduncle length. On the other hand, P. vitifolia remained as a good representative of the uniform supersection Distephana, so its position does not support its downgrading to a supersection within subgenus Passiflora proposed by Feuillet and MacDougal (2003). Conclusions A shorter list of 32 qualitative traits selected after analyzing variation among Killip’s and Feuillet & MacDougal´s subgenera allowed to classify our 61 species sample consistently, using a strictly phenetic approach. Most discriminant characters include size of stems and leaves, presence of tendrils, number and distribution of extrafloral nectaries, dimensions and general shape of bracts, width and length of flowers, corona complexity, and, although they were not systematically analyzed, fruit size and color. Furthermore, the smaller number of descriptors providing valuable information used in this approach will also allow a reduction in the labor, time and resources spent in the characterization of genetic resources of genus Passiflora. Eight of the nine of Killip’s subgenera represented in our sample are supported by the morphological analysis, although subgenus Tryphostemmatoides (Deidamioides) is only supported in the quantitative analysis. On the other hand, the simplification proposed by Feuillet and MacDougal is not clearly supported in our analyses, except for the possible inclusion of P. manicata in subgenus Tacsonia as this species is intermediate with subgenus Passiflora for quantitative traits but very similar to tacsos for most qualitative traits. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 14 Comparing molecular results with our phenetic approach provides a better resolved vision of relations among passifloras. Beyond the differentiation between cytological groups (n = 6, 12 or n = 9; De Melo et al. 2001) underlined in most analyses, other features of considerable importance for their evolution were found. The division between the two cytological groups is particularly paralleled by a division on the presence and position of extrafloral nectaries and the complexity of the corona, showing the importance of coevolution. In the same line, Yockteng (2003) underlined the differentiation appearing in the spectrum of cyanogenic components developed against herbivores in the two cytological groups. Even clearer appears the coevolution with pollinators causing the main line of floral divergence between subgenera Passiflora pollinated by large bees, Tacsonia pollinated only by the sword-billed hummingbird, and Distephana, pollinated by other hummingbirds can be seen even clearer. In the n = 6/12 group where small to medium size insects dominate as pollinators, the morphological divergence of P. trinervia (subgenus Psilanthus) showing exactly the same adaptation to Andean highlands and pollination by the same bird species as subgenus Tacsonia is not less significant. This convergence is logically expressed in the evolution of the corona. However, the minute corona of P. trinervia and other typical representatives of subgenus Psilanthus (Killip´s classification) probably results from the transformation of the two- ranked corona common in subgenera Astrophea and Decaloba, while the reduced corona of most tacsos has evolved from the complex corona observed in all species of subgenus Passiflora at the same time as their specialized hypanthium. Remnants of these complex coronas can still be observed in species with a less developed hypanthium as P. manicata and P. antioquiensis, with one or two external whorls plus a few other series of thinner filaments under the floral tube throat, and even in typical long-tubed tacso flowers. Thus, a more or less developed second whorl is not rare in P. mixta, while loose whorls of very thin white filaments can be observed far in the hypanthium of P. tripartita var. mollissima. The evolution has probably been different for subgenus Distephana (Killip´s classification) and its differentiation within the n = 9 group earlier in the history of Passiflora, as its species show no indication of an ancestral complex corona. Instead, they are constituted by two or three rows, which can be compared with numbers observed in subgenera Decaloba and Astrophea. The fusion of the elements into a prolongation of the floral tube is paralleled in certain representatives of subgenus Decaloba such as P. tulae Urban (Murucuja, Killip´s classification), forming another striking case of convergence between species from very different evolutionary backgrounds. Acknowledgements This research has been funded by Colciencias and the Colombian Ministry for Environment, with support from the Research Center of the Colombian Coffee Grower Federation (Cenicafé) through the projects: ‘Conservación y utilización de los recursos genéticos de pasifloras’ and ‘Estudio de la diversidad de las Passifloraceae y Caricaceae de la zona cafetera de Colombia’. The first author gratefully acknowledges the financial support from the Gines-Mera Fellowship Foundation (CIAT - CBN). 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 15 Compliance with Ethical Standards  Funding: This study was funded by Colombian Ministry for Environment (grant number CEN-303-2003)  Conflict of Interest: The authors declare that they have no conflict of interest References Cerqueira-Silva CBM, Faleiro FG, Jesus ON, Santos ESL, Souza AP (2016) The genetic diversity, conservation, and use of passion fruit (Passiflora spp.). In: Ahuja MR, Jain SM (eds.). Genetic Diversity and Erosion in Plants, Sustainable Development and Biodiversity 8. Springer International Publishing Switzerland. doi: 10.1007/978-3-319-25954-3_5 Crochemore M, Molinare H, and Colauto N (2003) Caracterizao agromorfologica do Maracujazeiro (Passiflora spp.). 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Springer Verlag, Berlin, Heidelberg. doi: 10.1007/978-3-642-20447-0 Table 1 List of accessions of the genus Passiflora L. used in this study. Taxonomy according to infrageneric classification proposed by Feuillet and MacDougal (2003) Table 2 List of 127 descriptors used in the morphological characterization study Table 3 Mean values and coefficients of variation for all the descriptors per subgenera Table 4 Factor loadings from the principal component analysis (varimax normalized rotation) carried out on 24 quantitative descriptors Table 5 Variation for 32 qualitative and four categorized quantitative descriptors in the different subgenera sampled Fig. 1 Relative variance components for 57 quantitative descriptors. Bold characters are used for traits displaying more than 50% of variance among subgenera Fig. 2 Tridimensional plot of the scores of Passiflora accessions for the first three quantitative variation components. Colors refer to subgeneric classification Fig. 3 Dendrogram obtained with a first set of qualitative data. Distances of Sokal & Michener. Images courtesy of PhyloPic (phylopic.org) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 Figure_1 Click here to download Figure Figure 1_Variance components.jpg Figure_2 Click here to download Figure Figure_2_tridimensional plane_PCA.jpg Figure_3 Click here to download Figure Figure_3_dendogram.jpg Table 1 List of accessions of the genus Passiflora L. used in this study. Taxonomy according to infrageneric classification proposed by Feuillet and MacDougal (2003). Subgenus / Supersection / Section / Series / Species Number of individuals Country Locality Subgenus Astrophea (DC.) Masters, 1871 Section Astrophea Passiflora arborea Spreng. 2 Colombia Manizales (Caldas) Passiflora emarginata Humb. & Bonpl. 2 Colombia Manizales (Caldas) Passiflora sphaerocarpa Triana & Planch. 2 Colombia Cali (Valle del Cauca) Subgenus Deidamioides (Harms) Killip, 1938 Passiflora arbelaezii L. Uribe 1 Colombia Quibdó (Chocó) Passiflora gracillima Killip 1 Colombia Salento (Quindío) Subgenus Decaloba (DC.) Rchb., 1828 Supersection Auriculata MacDougal & Feuillet Passiflora auriculata Kunth 2 Colombia Victoria (Caldas) Supersection Bryonioides (Harms.) MacDougal & Feuillet Passiflora adenopoda Moc. & Sessé ex D.C 2 Colombia Buenavista (Quindío) 2 Colombia Ibagué (Tolima) Supersection Cieca (Medic.) Feuillet & MacDougal Passiflora coriacea Juss. 2 Colombia Palestina (Caldas) Table_1 2 Colombia Cartago (Valle del Cauca) 2 Colombia Palmira (Valle del Cauca) 2 Colombia Ibagué (Tolima) Passiflora suberosa L. 2 Colombia Jericó (Antioquia) 2 Colombia Manizales (Caldas) 2 Colombia Manizales (Caldas) 2 Colombia Barichara (Santander) 3 Colombia Palmira (Valle del Cauca) Supersection Decaloba (DC.) MacDougal & Feuillet Section Decaloba (DC.) Mast Passiflora misera Kunth 2 Colombia Jamundí (Valle del Cauca) Passiflora trifasciata Lemaire 2 Ecuador Misahuallí (Napo) Passiflora alnifolia Kunth 2 Colombia Manizales (Caldas) 2 Colombia Chachagüí (Nariño) 2 Colombia Tona (Santander) Passiflora bogotensis Benth. 2 Colombia Bogotá (Cundinamarca) Passiflora biflora Lam. 2 Colombia Mariquita (Tolima) 2 Colombia La Mesa (Cundinamarca) Passiflora cuspidifolia Harms 1 Colombia Duitama (Boyacá) Passiflora erythrophylla Mast. 2 Colombia Duitama (Boyacá) Passiflora magdalenae Triana & Planch. 2 Colombia Victoria (Caldas) Passiflora sexflora Juss. 2 Colombia Calarcá (Quindío) Passiflora trinervia (Juss.) Poir. 2 Colombia Salento (Quindío) P. sp. nov 2 Colombia Pereira (Risaralda) Section Xerogona (Raf.) Killip Passiflora capsularis L. 2 Colombia Jericó (Antioquia) 2 Colombia Barichara (Santander) 2 Colombia Cartago (Valle del Cauca) Passiflora rubra L. 2 Colombia Manizales (Caldas) 2 Colombia Buenavista (Quindío) Supersection Hahniopathanthus (Harms.) Killip Passiflora guatemalensis S. Wats. 2 Colombia Filadelfia (Caldas) Subgenus Passiflora Supersection Passiflora Series Passiflora Passiflora cincinnata Mast. 3 Brazil 3 Brazil Passiflora edulis f. edulis Sims 3 Colombia Salamina (Caldas) 2 Colombia Manizales (Caldas) 2 Colombia Puracé (Cauca) 1 Peru 1 Colombia Génova (Quindío) Passiflora edulis f. flavicarapa Degener 4 Brazil 3 Colombia La Unión (Valle del Cauca) 3 Colombia Rivera (Huila) 3 Peru 3 Peru 3 Colombia Anserma (Caldas) Passiflora incarnata L. 1 U.S.A Miami (Florida) Passiflora bahiensis Klotzsch 3 Brazil Salvador (Bahia) Supersection Stipulata Feuillet & MacDougal Section Kermesinae (Cervi) Feuillet & MacDougal Passiflora lehmanni Mast. 2 Colombia Buenavista (Quindío) Passiflora smithii Killip 2 Colombia Ibagué (Tolima) Section Granadillastrum Triana & Planch. Passiflora caerulea L. 3 France Montpellier (Herault) Passiflora subpeltata Ortega 3 Colombia Santa Marta (Magdalena) Passiflora gibertii N.E. Brown 2 Brazil Section Dysosmia DC. Passiflora foetida L. var. foetida 2 Colombia Quibdó (Chocó) Passiflora vesicaria L. var. vesicaria 3 Colombia Armero (Tolima) Supersection Laurifolia (Cervi) Feuillet & MacDougal Series Laurifoliae Killip ex Cervi Passiflora guazumaefolia Juss. 2 Colombia Plato (Magdalena) Passiflora nitida Kunth 1 Colombia Quibdó (Chocó) Passiflora popenovii Killip 2 Colombia Timbío (Cauca) Passiflora maliformis L. 2 Colombia Fredonia (Antioquia) 3 Colombia Palestina (Caldas) 2 Colombia Buga (Valle del Cauca) 2 Colombia Calarcá (Quindio) 2 Colombia Rivera (Huila) 3 Colombia Tuluá (Valle del Cauca) 1 Colombia La Unión (Valle del Cauca) 2 Colombia Ibagué (Tolima) Series Quadrangulares Feuillet & MacDougal 2 Colombia Chachagüí (Nariño) Passiflora alata Curtis 3 Brazil 3 Brazil Passiflora quadrangularis L. 3 Colombia Paicol (Huila) 2 Colombia Palmira (Valle del Cauca) Series Tiliiefolia Feuillet & MacDougal Passiflora ligularis Juss. 3 Colombia Anserma (Caldas) 1 Colombia Cuenca (Azuay) 3 Colombia Salento (Quindio) 1 Colombia Santa Rosa (Risaralda) 2 Colombia Génova (Quindio) Passiflora serrulata Jacq. 2 Colombia Plato (Magdalena) Passiflora tiliifolia L. 2 Colombia El Cerrito (Valle del Cauca) Passiflora serratodigitata L. 2 Brazil Supersection Distephana (DC.) Feuillet & MacDougal Passiflora vitifolia Kunth 2 Colombia Victoria (Caldas) 2 Colombia Ibagué (Tolima) 1 Colombia El Cerrito (Valle del Cauca) Supersetion Tacsonia (Juss.) Feuillet & MacDougal Section Colombiana Escobar Series Leptomischae Escobar Passiflora antioquiensis Karst. 2 Colombia Sta. Rosa Osos (Antioquia) 2 Colombia Manizales (Caldas) Passiflora flexipes Triana & Planch. 2 Colombia Salento (Quindío) Passiflora tenerifensis L.K. Escobar 2 Colombia El Cerrito (Valle del Cauca) 1 Colombia El Cerrito (Valle del Cauca) Series Colombianae Escobar Passiflora adulterina L.f. 2 Colombia Duitama (Boyacá) Passiflora lanata (Juss.) Poir. 2 Colombia Duitama (Boyacá) Series Quindiensae Escobar Passiflora linearistipula L.K. Escobar 2 Colombia Manizales (Caldas) Section Elkea Feuillet & MacDougal Passiflora cumbalensis var. cumbalensis (H. Karst.) Harms 2 Ecuador Tulcán (Carchi) 1 Colombia Pasto (Nariño) Passiflora luzmarina Jorgensen 1 Ecuador Loja (Loja) 1 Ecuador Loja (Loja) Passiflora tarminiana Coppens & Barney 3 Argentina Castellar 2 Colombia Boyacá 2 Colombia Silvia (Cauca) 4 Ecuador Baños (Tungurahua) 1 Peru 3 Colombia El Cerrito (Valle del Cauca) 3 Venezuela Táchira Passiflora tripartita var. mollissima Holm-Nielsen & Jørgensen 4 Colombia Cundinamarca 1 Ecuador Baños (Tungurahua) 2 Colombia Pasto (Nariño) 1 Peru 3 Colombia El Cerrito (Valle del Cauca) 1 Venezuela Táchira Passiflora tripartita var. tripartita Holm-Nielsen & Jørgensen 2 Ecuador Ambato (Tungurahua) Section Parritana Escobar Passiflora jardinensis L.K. Escobar 2 Colombia Jardín (Antioquia) Passiflora parirtae (Mast.) L.H Bailey 2 Colombia Herveo (Tolima) Section Insignes Feuillet & MacDougal Passiflora pinnatistipula Cav. 1 Colombia Boyacá Passiflora x rosea (H.Karst.) Killip 1 Ecuador Tuta (Boyacá) Section Tacsonia Passiflora mathewsii (Mast.) Killip 1 Ecuador Cuenca (Azuay) Passiflora mixta L.f. 2 Colombia El Cerrito (Valle del Cauca) 3 Colombia El Cerrito (Valle del Cauca) 3 Colombia El Cerrito (Valle del Cauca) 1 Colombia El Cerrito (Valle del Cauca) 1 Colombia El Cerrito (Valle del Cauca) Section Manicata (Harms) Feuillet & MacDougal Passiflora manicata (Juss.) Pers. 1 Ecuador Baños (Tungurahua) 2 Colombia Santander 2 Colombia Santander 2 Colombia Santander Table 2 List of 127 descriptors used in the morphological characterization study Organ Qualitative characters (84) Code Quantitative characters (43) Code Stem Habit STHA Diameter STDI Shape STSH Internode length STIN Pubescence STPU Anthocyanin STAN Tendril Presence TEPR Shape TESH Pubescence TEPU Anthocyanin TEAN Stipule Presence SPPR Length SPLE Permanence SPPE Width SPWI Color SPCO Terminal arista length SPTA Pubescence SPPU Shape SPSH Margin SPMA Anthocyanin SPAN Petiole Anthocyanin PEAN Length PELE Pubescence PEPU Distance from base to first gland PEDG Color PECO Nectary number PENM Nectary shape PENS Nectary stipe PESN Leaf Heterophylly LEPO Margin serration density LEMS Lobe number LELN Angle between lateral lobes LEAB Margin LEMA Central lobe length LELC Base shape LEBS Right lobe length LERL Apex shape LEAS Central lobe width LECL Presence of acumen LEPA Distance between leaf sinus and petiole insertion LESS Pubescence – adaxial LEAX Nectary number on lamina LELA Pubescence – abaxial LEPB Nectary number on leaf margin LENN Anthocyanin – lamina LEAL Anthocyanine – nerves LEAN Color – adaxial LECA Heteroblasty LEPH Presence of laminar nectaries LENL Distribution of laminar nectaries LEDN Presence of marginal nectaries LEPN Distribution of margin nectaries LELM Nectary shape LENS Peduncle Pubescence PDPU Length PDLE Anthocyanin PDAN Diameter PDDI Bifurcation PDBN Pedicel length PDPL Length to first bifurcation PDLF Length to second bifurcation PDBS Bract Union BRUN Length BRLE Presence BRPR Width BRWI Permanence BRPE Pubescence BRPU Position of pubescence BRPP Color BRCO Anthocyanin BRAN Shape BRSH Margin BRMA Table_2 Apex shape BRAS Marginal nectaries BRNM Flower Corona type FLCY Orientation (in degrees to vertical) FLOG Corolla type FLCT Petal length FLPL Orientation FLOR Petal width FLPW Pubescence on corolla FLPU Sepal length FLSL Hypanthium pubescence FLHP Sepal width FLSW Dominant petal color FLCP Diameter of nectary chamber FLNC Chlorophylla on exterior of sepal FLCS Hypanthium diameter above nectary chamber FLHD Keel-shaped sepals FLKS Hypanthium diameter - distal FLHS Sepal awn FLSA Flower length FLLE Presence of petals FLPP Hypanthium length FLHL Union of sepals FLUS Length of nectary chamber FLCN Color of filaments at base FLCB Number of corona series FLNS Color of filaments at apex FLCA Filament length FLFL Distribution of anthers FLDA Staminal filaments length FLSF Ovary pubescence FLOP Ovary length FLOL Color of ovary FLCO Style length FLSL Color of style FLCS Gynophore length FLGL Color distribution on styles FLDS Color of stigmas FLCG Androgynophore length FLAL Color of androgynophore FLCN Operculum length FLOP Color distribution on androgynophore FLDN Limen length FLLL Pubescence of androgynophore FLPN Limen margin FLML Nectary chamber ring FLNR Hypanthium type FLHY Internal color of hypanthium FLCI Chlorophylla on exterior of hypanthium FLCE Anthocyanin on exterior of hypanthium FLAE Nectaries on sepals FLNS Dominant sepal color FLCP Anthocyanin on exterior of sepals FLAP Fruit Type FRTY Shape FRSH Organ Factor Level Passiflora Distephana Dysosmia Tacsonia Manicata Descriptors n 504 20 25 340 35 STEDI Mean 44.01 35.00 15.00 30.00 30.00 CV 0.15 0.00 0.00 0.00 0.00 STIN Mean 321.40 61.30 40.04 285.54 321.63 CV 0.55 0.14 0.14 0.52 0.31 SPLE Mean 14.45 6.20 2.70 8.69 11.65 CV 0.73 0.17 0.26 0.55 0.12 SPWI Mean 5.98 1.00 5.35 7.48 18.64 CV 1.24 0.00 0.10 0.87 0.12 SPTA Mean 0.13 0.00 6.22 2.72 3.87 CV 5.77 - 0.04 0.94 0.19 LEMS Mean 4.26 6.10 5.00 6.81 8.63 CV 0.81 0.05 0.00 0.29 0.06 LEAB Mean 58.09 77.90 92.24 105.42 116.74 CV 1.17 0.10 0.03 0.40 0.07 LELC Mean 120.92 115.10 73.22 96.95 112.82 CV 0.26 0.09 0.04 0.21 0.24 LERL Mean 51.21 96.30 45.47 83.91 117.99 CV 1.06 0.13 0.04 0.41 0.27 LECL Mean 75.49 49.30 42.16 37.59 52.39 CV 0.56 0.11 0.05 0.39 0.23 LESS Mean 15.37 44.00 41.05 21.72 41.35 CV 1.16 0.02 0.08 0.55 0.22 LELA Mean 0.00 0.00 0.00 0.00 0.00 CV - - - - - LENN Mean 0.00 7.00 0.00 0.00 0.00 CV - 0.15 - - - PELE Mean 44.24 30.85 21.92 22.57 28.63 CV 0.58 0.18 0.16 0.34 0.21 PEDG Mean 23.91 0.50 0.00 9.25 7.89 CV 0.75 0.00 - 0.57 0.36 PENM Mean 2.90 2.00 0.00 6.09 9.20 CV 0.58 0.00 - 0.37 0.29 BRLE Mean 33.91 26.70 20.92 36.30 42.97 CV 0.50 0.19 0.11 0.29 0.10 BRWI Mean 21.50 8.70 16.20 15.70 26.04 CV 0.47 0.18 0.27 0.36 0.09 PDLE Mean 44.50 46.85 49.39 86.71 65.70 CV 0.45 0.13 0.16 1.43 0.25 PDDI Mean 2.66 2.50 1.50 2.50 2.50 CV 0.12 0.00 0.00 0.00 0.00 PDPL Mean 8.63 4.92 2.60 8.93 13.70 Killip´s classification (1938) Table 3 Mean values and coefficients of variation for all the descriptors per subgenera according to Killip´s and Feuillet & MacDougal´s classification Passiflora Feuillet & MacDougal´s classification (2003) F lo w er S te m L ea f B ra ct Table_3 CV 0.41 0.07 0.00 0.40 0.12 PDLF Mean 0.00 0.00 0.00 0.00 0.00 CV - - - - - PDBS Mean 0.00 0.00 0.00 0.00 0.00 CV - - - - - FLNS Mean 5.00 2.00 4.20 1.40 5.00 CV 0.00 0.00 0.84 0.80 0.00 FLOG Mean 114.94 110.00 142.00 10.99 158.00 CV 0.45 0.42 0.23 2.02 0.14 FLPL Mean 34.58 56.25 18.90 44.28 42.27 CV 0.28 0.06 0.14 0.25 0.08 FLPW Mean 10.36 13.58 7.92 19.39 14.65 CV 0.43 0.20 0.13 0.23 0.09 FLSL Mean 36.15 59.60 19.47 46.91 44.36 CV 0.26 0.05 0.11 0.24 0.08 FLSW Mean 14.71 16.60 7.22 18.65 14.17 CV 0.34 0.07 0.12 0.28 0.13 FLNC Mean 13.32 13.90 8.32 15.07 15.85 CV 0.26 0.13 0.05 0.17 0.09 FLHD Mean 13.04 11.90 8.40 10.21 10.93 CV 0.30 0.15 0.05 0.23 0.07 DHS Mean 17.39 12.75 8.40 12.00 14.64 CV 0.29 0.06 0.05 0.21 0.11 FLHS Mean 27.00 55.75 12.88 103.36 42.44 CV 0.23 0.07 0.08 0.17 0.07 FLLE Mean 11.97 12.07 8.35 71.01 13.03 CV 0.31 0.09 0.10 0.28 0.24 FLCN Mean 5.56 6.10 2.48 10.17 8.58 CV 0.32 0.26 0.06 0.25 0.08 FLFL Mean 35.39 24.10 11.20 2.10 5.82 CV 0.44 0.05 0.33 0.80 0.23 FLSF Mean 11.08 14.20 4.60 14.86 12.19 CV 0.25 0.12 0.13 0.32 0.12 FLOL Mean 8.30 10.76 3.71 11.60 8.20 CV 0.30 0.06 0.20 0.17 0.10 FLSL Mean 11.11 13.20 6.44 13.61 12.14 CV 0.25 0.06 0.09 0.24 0.12 FLGL Mean 1.91 2.72 1.00 4.37 2.70 CV 0.58 0.09 0.02 0.67 0.19 FLAL Mean 11.36 42.08 8.46 82.69 29.11 CV 0.29 0.08 0.11 0.21 0.09 FLOP Mean 2.75 10.00 2.32 6.61 10.37 CV 0.46 0.06 0.07 0.24 0.08 FLLL Mean 1.65 3.55 1.40 1.53 1.25 CV 0.64 0.14 0.07 0.36 0.16 PEDG/PELE Mean 0.56 0.02 0.00 0.41 0.28 CV 0.53 0.22 - 0.45 0.32 LECL/LELC Mean 0.59 0.43 0.58 0.39 0.47 CV 0.37 0.11 0.04 0.30 0.14 BRWI/BRLE Mean 0.64 0.34 0.78 0.43 0.61 CV 0.22 0.32 0.28 0.25 0.07 FLPW/FLPL Mean 0.30 0.24 0.42 0.44 0.35 S h a p e re la ti o n F lo w er CV 0.24 0.14 0.08 0.16 0.10 FLSW/FLSL Mean 0.41 0.28 0.37 0.41 0.32 CV 0.24 0.11 0.09 0.23 0.16 FLNC/FLHD Mean 1.04 1.17 0.99 1.53 1.46 CV 0.13 0.05 0.03 0.23 0.10 FLLE/FLHL Mean 2.33 4.64 1.56 1.54 3.42 CV 0.21 0.08 0.17 0.26 0.21 FLCN/FLHL Mean 0.47 0.50 0.30 0.15 0.69 CV 0.22 0.22 0.10 0.34 0.22 FLAL/FLHL Mean 1.01 3.50 1.03 1.23 2.35 CV 0.35 0.08 0.19 0.32 0.24 PDDI/PDPL Mean 0.08 0.05 0.03 0.08 0.04 CV 0.77 0.13 0.16 0.70 0.27 PDPL/PDLE Mean 0.23 0.11 0.05 0.29 0.22 CV 0.63 0.15 0.16 0.77 0.27 BRLE/FLHL Mean 2.89 2.25 2.55 0.54 3.52 CV 0.47 0.27 0.22 0.39 0.28 FLGL/FLAL Mean 0.18 0.06 0.12 0.06 0.09 CV 0.68 0.06 0.12 1.49 0.23 LECL/LECL Mean 0.58 0.47 0.58 0.39 0.50 CV 0.44 0.14 0.04 0.30 0.12 S h a p e re la ti o n Astrophea Deidamioides Decaloba Psilanthus Astrophea Thryphostematoides Total sample 310 10 50 10 1304 10.81 9.60 230.00 5.00 37.96 0.24 0.05 0.25 0.00 1.11 98.50 50.80 54.67 22.91 235.09 1.05 0.04 0.32 0.10 0.76 5.34 8.70 4.45 0.46 9.82 0.47 0.08 0.16 0.61 0.83 2.46 1.00 2.86 0.25 5.59 1.88 0.00 0.29 0.21 1.22 0.18 0.00 0.00 0.00 1.03 3.40 - - - 1.96 0.21 0.00 0.00 0.00 3.89 3.50 - - - 0.92 80.38 33.10 0.00 0.00 75.40 0.39 0.08 - - 0.75 59.80 116.50 247.27 32.11 103.05 0.38 0.01 0.26 0.38 0.46 65.31 92.70 0.00 0.00 63.42 0.35 0.03 - - 0.72 33.50 18.70 123.49 22.38 54.96 0.53 0.08 0.31 0.15 0.69 45.76 89.40 0.00 0.00 25.74 0.38 0.01 - - 0.83 4.09 6.00 0.00 0.00 1.02 1.10 0.00 - - 2.79 0.00 0.00 0.00 0.00 0.11 - - - - 8.10 25.26 24.70 26.20 19.09 31.99 0.69 0.05 0.36 0.14 0.66 15.25 0.00 26.38 19.09 16.65 1.18 - 0.37 0.14 0.98 0.81 0.00 2.00 2.00 3.27 1.22 - 0.00 0.00 0.85 2.63 8.60 2.10 1.00 25.31 1.68 0.10 0.10 0.00 0.75 1.28 2.70 1.30 0.50 13.93 2.73 0.18 0.00 0.00 0.81 19.74 99.30 17.88 23.86 49.56 0.63 0.01 0.35 0.10 1.41 1.51 1.11 2.92 0.80 2.30 0.02 0.05 0.11 0.00 0.24 4.42 3.30 13.44 8.24 7.81 Killip´s classification (1938) Table 3 Mean values and coefficients of variation for all the descriptors per subgenera according to Killip´s and Feuillet & MacDougal´s classification Decaloba Feuillet & MacDougal´s classification (2003) 0.71 0.15 0.42 0.05 0.54 0.00 0.00 23.30 26.50 1.10 - - 0.21 0.06 4.64 0.00 0.00 23.30 16.50 1.02 - - 0.21 0.10 4.68 2.00 1.00 1.00 2.00 3.10 1.10 0.00 0.00 0.00 0.91 72.73 172.50 135.00 0.00 73.71 0.96 0.03 0.20 - 0.94 7.98 11.20 24.08 13.54 30.28 0.76 0.08 0.19 0.19 0.55 2.76 4.90 8.44 3.56 10.86 0.93 0.06 0.35 0.27 0.66 14.62 39.10 24.47 14.51 33.51 0.38 0.02 0.16 0.29 0.46 5.62 7.00 9.36 5.51 13.11 0.39 0.13 0.27 0.34 0.50 8.55 10.00 7.59 7.10 12.33 0.34 0.08 0.17 0.14 0.32 8.73 8.70 7.55 6.81 10.82 0.36 0.06 0.17 0.14 0.34 9.59 7.50 8.28 6.81 13.30 0.36 0.07 0.21 0.14 0.39 11.52 162.00 23.24 8.30 44.56 0.31 0.02 0.18 0.11 0.88 6.32 112.90 5.82 4.96 26.47 2.29 0.02 0.25 0.16 1.16 3.64 29.30 4.92 3.80 6.48 0.34 0.07 0.11 0.18 0.57 7.68 2.90 13.39 5.83 17.37 0.40 0.25 0.19 0.11 1.02 4.26 8.30 6.36 2.46 10.13 0.34 0.06 0.14 0.15 0.50 3.45 11.90 4.98 2.85 7.81 0.40 0.06 0.14 0.07 0.47 4.62 5.10 6.76 3.63 9.92 0.31 0.14 0.33 0.07 0.43 0.93 9.20 1.00 0.51 2.34 0.26 0.11 0.00 0.06 0.94 6.25 150.50 12.40 4.86 30.69 0.36 0.01 0.30 0.04 1.14 2.12 4.20 1.84 0.84 3.88 0.49 0.10 0.17 0.06 0.67 0.87 0.00 0.60 0.65 1.38 0.70 - 0.34 0.08 0.65 0.60 0.00 1.01 1.00 0.52 0.67 - 0.03 0.00 0.64 0.58 0.16 0.50 0.76 0.52 0.44 0.08 0.12 0.26 0.42 0.18 0.32 0.62 0.50 0.47 1.41 0.24 0.07 0.00 0.53 0.24 0.44 0.34 0.26 0.33 0.82 0.11 0.23 0.16 0.42 0.42 0.18 0.38 0.38 0.40 0.38 0.13 0.25 0.17 0.28 0.99 1.16 1.01 1.05 1.17 0.08 0.14 0.03 0.13 0.26 5.85 1.44 4.25 1.70 3.07 0.71 0.03 0.30 0.16 0.87 1.89 0.26 0.89 0.77 0.74 0.78 0.08 0.24 0.12 1.32 3.36 1.33 2.21 1.00 1.75 0.79 0.02 0.31 0.12 0.95 0.11 0.01 0.18 0.03 0.09 0.65 0.05 0.25 0.11 0.75 0.40 0.03 0.76 0.35 0.30 1.36 0.15 0.43 0.10 1.10 0.92 0.08 0.38 0.21 1.67 1.28 0.10 0.21 0.15 0.92 0.17 0.06 0.09 0.11 0.14 0.41 0.12 0.39 0.08 0.80 0.59 0.43 0.76 0.16 0.52 0.37 0.11 0.26 0.08 0.42 Descriptors 1 2 3 4 5 STDI -0.088 0.215 -0.892 -0.023 0.041 LEAB 0.353 0.365 0.152 0.041 0.759 LELC 0.019 0.571 -0.686 0.009 -0.195 LENN -0.017 0.040 0.011 0.962 0.014 PENN 0.648 0.407 0.013 -0.055 0.158 PDDI 0.135 0.847 -0.287 0.010 0.145 PDLF -0.054 -0.136 -0.943 -0.007 -0.044 PDBS -0.054 -0.113 -0.973 -0.006 -0.046 BRLR 0.296 0.723 0.186 -0.091 0.240 FLPL 0.501 0.774 -0.021 0.235 0.042 FLPW 0.713 0.595 -0.024 0.095 0.028 FLSL 0.577 0.716 0.036 0.243 -0.028 FLSW 0.520 0.754 0.034 0.074 -0.108 FLLE 0.960 0.179 0.045 0.017 0.090 FLHL 0.904 0.063 0.086 -0.100 0.119 FLHL 0.824 0.208 0.026 -0.080 -0.137 FLCN -0.521 0.723 0.034 -0.007 -0.329 FLFL 0.529 0.651 0.082 0.097 0.171 FLSF 0.677 0.658 0.076 0.061 -0.049 FLOL 0.454 0.693 0.067 0.073 0.263 FLSL 0.964 0.064 0.047 0.033 0.099 FLOP 0.711 0.236 0.099 0.363 0.214 BRWI/BRLE -0.160 0.718 -0.221 -0.131 0.241 FLNC/FLHD 0.700 0.056 0.042 0.043 0.368 Expl.Var 7.610 6.496 3.336 1.256 1.238 Prp.Totl 0.317 0.271 0.139 0.052 0.052 % total variance 46.028 16.999 11.138 4.913 3.986 Principal components Table 4 Factor loadings from the principal component analysis (varimax normalized rotation) carried out on 24 quantitative descriptors. Table_4 Table 4 Factor loadings from the principal component analysis (varimax normalized rotation) carried out on 24 quantitative descriptors. Feuillet & MacDougal (2003) Astrophea Deidamioides Killip (1938), Escobar (1988), MacDougal (1994) Astrophea Decaloba Psilanthus Passiflora Distephana Dysosmia Tacsonia Manicata Tryphostemmatoides Species/accession number n= 3/4 n= 17/31 n= 1/1 n= 19/44 n= 1/2 n= 1/2 n= 16/36 n= 1/1 n= 2/2 Main pollinators medium (honey) bees small to large bees - wasps sword-billed large (carpenter) bees hummingbirds bees sword-billed hummingbirds bees hummingbird hummingbird Chromosome number 2n = 24 2n = 12, 22, 24, 36 2n = 12 2n = 18 2n = 18 2n = 18, 20, 22 2n = 18 2n = 18 2n = 12 Altitudinal range 1.000 - 2.200 m 60 - 2.700 m 2.600 - 3.200 20 - 2.400 m 50 - 1.200 m 30 - 1.200 m 2.100 - 3.700 m 1.900 - 2.500 m 50 - 2.000 m Descriptors Habit tree vine vine vine vine vine vine vine vine Stem section irregular terete/angular angular terete/angular/winged terete terete terete/angular angular terete Secondery xylema present absent absent absent absent absent absent absent absent Tendril position absent axillary axillary axillary axillary axillary axillary axillary axillary and peduncle Stipule short triangular setaceous/linear/foliaceous-aristate setaceous setaceous/linear/foliaceous-aristate linear foliaceous-aristate setaceous/linear/foliaceous-aristate foliaceous-aristate setaceous Stipule nectaries (conspicuous) absent absent absent absent/present (P. maliformis ) present present absent absent absent Leaf lobation one three three one/three/more three three one/three three one Leaf base cuneate-rounded cuneate-rounded/cordate/ cordate cuneate-rounded/cordate cordate cordate cuneate-rounded/cordate cordate rounded peltate (P. coriacea and P. guatemalensis ) Leaf apex obtuse/acute rounded/obtuse/acute acute/very acute rounded to very acute acute acute acute/very acute obtuse to very acute retuse Leaf margin entire entire/serrate (P. adenopoda ) entire entire/serrate serrate serrate serrate serrate entire Laminar nectaries absent present/absent (five species)b present absent absent absent absent absent absent Leaf margin nectaries (conspicuous) absent absent/sinus/ absent absent/sinus sinus absent absent/sinus (P. jardinensis ) absent leaf base all margin (P. adenopoda ) lateral lobe (P. edulis f. edulis ) Petiolar nectaries absent absent/orbicular (P. adenopoda )/ absent all except auriculate orbicular absent linear/orbicular linear absent cylindrical (P. coriaceae and P. suberosa ) /auriculate (P. auriculata ) Dorsal nectaries present absent absent absent absent absent absent absent absent Peduncle branching present absent/present (P. sexflora ) absent absent absent absent absent absent present Bract shape linear setaceous/linear/ linear foliaceous linear pinnatisect foliaceous foliaceous linear foliaceous (P. adenopoda , P. guatemalensis ) /absent (four species)* Nectary on bract absent absent absent absent/present present absent absent absent absent Flower orientation erect erect/intermediate/pendular pendular erect/intermediate/pendular erect erect erect/intermediate/pendular erect erect Corolla shape reflex intermediate/campanulate campanulate intermediate/reflex reflex intermediate/reflex campanulate/intermediate/reflex reflex reflex Dominant corolla color white white red (pink) white/red red white red (including pink or orange) red white Corona type filamentous filamentous filamentous filamentous filamentous filamentous tuberculous/ filamentous filamentous Corona filaments sickle-sword-shaped linear linear linear linear filamentous, linear linear linear free free free free fused at base free free free free Corona filaments number uniseriate biseriate/ uniseriate pentaseriate biseriate 3- to 5-seriate uni- to 5-seriate pentaseriate biseriate uniseriate (P. guatemalensis and P. adenopoda )/ triseriate (P. magdalenae and P. sp. nv) Corona color-clear (longest row) white white white red white white/purple purple white Corona color-darkest (longest row) yellow white/purple/ white purple red purple purple purple white yellow (P. guatemalensis ) white (P. guazumaefolia ) white (P. jardinensis ) Petals present present/ present present present present present present present absent (P. coriaceae and P. suberosa ) Sepal awn absent absent/present absent present present present present present absent Nectar chamber ring present absent absent present present present present present absent Hypanthium campanulate flat tubular campalunate tubular campanulate tubular tubular flat Limen present present absent present present present present present present Ovary shape tricostate globose globose globose globose globose globose globose globose Fruit shape globose globose/ elongate globose/ globose globose elongate/ elongate globose elongate (P. rubra and P. capsularis ) elongate (P. quadrangularis ) globose (P. pinnatistipula ) Categorized (from quantitatives traits) Stem diameter ≥ 120 mm ≤ 20 mm ≤ 20 mm ≥ 30 mm ≥ 30 mm ≤ 20 mm ≥ 30 mm ≥ 30 mm ≤ 20 mm < 60 mm < 60 mm < 60 mm < 60 mm Leaf length > 215 mm / < 190 mm < 190 mm < 190 mm < 190 mm < 190 mm < 190 mm < 190 mm < 190 mm < 190 mm (P. sphaerocarpa ) Sepal length ≤ 30 mm ≤ 30 mm > 30 mm > 30 mm > 30 mm ≤ 30 mm > 30 mm / > 30 mm ≤ 30 mm ≤ 30 mm (six species)ª ≤ 30 mm (P. luzmarina and P. mathewsii ) Androgynophore length < 23 mm < 23 mm > 25 mm < 23 mm > 25 mm < 23 mm > 25 mm > 25 mm < 23 mm Decaloba Passiflora P. capsularis b* , P. coriacea*, P. rubra b* , P. suberosa b* , P. sexflora b , P. guatemalensis b , P. bahiensis a , P. edulis f. edulis a , P. guazumaefolia a , P. smithii a , P. subpeltata a , P. serrulata a Table 5 Variation for 32 qualitative and four categorized quantitative descriptors in the different subgenera sampled Table_5 Electronic Supplementary Material Click here to access/download Electronic Supplementary Material Suplementary material_List accessions_Killip classification.docx Cali, July 06, 2016 Plant Systematic and Evolution Scientific Committee Dear Scientific Committee, We hereby submit the paper with the title Morphological characterization in the genus Passiflora L.: an approach to understanding its complex variability to Plant Systematic and Evolution. Our research brings new information on the genus Passiflora as there are no currently published works on its morphological variability. The present study benefited from projects on diversity of Colombian Passifloraceae, including a component of collecting and establishing germplasm in field collections. As Colombia is the country with the highest Passiflora species diversity both for wild and cultivated material (Ocampo et al. 2007; 2010), a wide species sample could be gathered. However, practical limitations allowed describing accessions of only 51 of the 171 reported species from eight subgenera for the genus in the country. A few non-native species were added extending the sample to 61 species. Our goal was twofold, to test the utility of the revised set of descriptors over a wide range of Passiflora species, and to study morphological divergence among subgenera, species and populations. The manuscript’s main text has 7059 words. Best Regards, John Ocampo Geo Coppens d'Eeckenbrugge • Ocampo, J.A., Coppens d’Eeckenbrugge, G., Restrepo, M., Salazar, M., and Jarvis, A. 2007. Diversity of Colombian Passifloraceae: biogeography and an updated list for conservation. Biota Colombiana 8(1): 1-45. • Ocampo, J., Coppens d’Eeckenbrugge, G. and Jarvis, A. 2010. Distribution of Colombian Passifloraceae Indicates Highest Conservation Priority to Andean Hillsides. Diversity 2: 1158 – 1180. Cover Letter