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Morphological and molecular variability of wild diploid and polyploid populations of Chrysolaena flexuosa (Sims) H. Rob.: relevance for ornamental breeding
Scientia Horticulturae 260 (2020) 108875
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Morphological and molecular variability of wild diploid and polyploid populations of Chrysolaena flexuosa (Sims) H. Rob.: relevance for ornamental breeding Echeverría María Lisa, Camadro Elsa Lucilab,
T
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a
Universidad Nacional de Mar del Plata (UNMdP), Facultad de Ciencias Agrarias (FCA). Ruta Nacional 226, Km 73.5 (7620), Balcarce, Buenos Aires, Argentina Universidad Nacional de Mar del Plata (UNMdP), Facultad de Ciencias Agrarias (FCA) and Instituto Nacional de Tecnología Agropecuaria (INTA), EEA Balcarce, Laboratorio de Genética, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Ruta Nacional 226, Km 73.5 (7620), Balcarce, Buenos Aires, Argentina
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A R T I C LE I N FO
A B S T R A C T
Keywords: Lepidaploa Germplasm characterization Polyploidy Morphological phenotypes AFLP patterns Urban green spaces
Chrysolaena flexuosa is a South American species of ornamental interest, whose variability has not been explored. The objective of this work was to perform joint analyses of morphological and molecular traits in seven accessions from its geographic distribution in Argentina: diploid, tetraploid and hexaploid, along an approximately 1400 km transect. To this end, 26 morphological traits were recorded in 10–24 plants/accession, and two combination of AFLP markers were used for molecular characterization. The data was subjected to multivariate and AMOVA analyses, and the genetic diversity among accessions was evaluated by using the Fst index. The multivariate analyses for morphological traits allowed the differentiation of groups that were in accordance with their geographical origin and, thus, their respective ploidies; with the diploids exhibiting smaller-sized organs but higher numbers of leaves and heads. In the AMOVA, differences between accessions according to geographic origin were not significant (p = 0.123), and the highest percentage of the molecular variance corresponded to intra-population variation (88.1%). The Fst index indicated moderate genetic differentiation among accessions (Fst = 0.11), as observed in other allogamous species as a consequence of inter-population gene flow. In turn, the detected variability for morphological traits of interest would allow the use of these accessions in ornamental breeding for cultivar development.
1. Introduction Plant biodiversity is a very important source of goods and services for humankind. Broadly speaking, plant biodiversity satisfies human needs by providing much of the world agricultural and pharmaceutical goods, and participates in vital ecosystem services, such as nutrient cycling and oxygen production (Sala and Paruelo, 1997; Díaz et al., 2018). Most of the natural ecosystems are being disturbed by direct anthropogenic action (Valiente‐Banuet et al., 2015). With the steady fragmentation of natural environments due to agricultural intensification (Benton et al., 2003; Tscharntke et al., 2012), the importance of urban green spaces for biodiversity conservation steadily increases (Goddard et al., 2010). In this context, the utilization of novel ornamental crops in landscaping is not only a way of appreciating plant resources but also of promoting their use and care by the community. Vernonieae Cass, the major tribe of the Compositae (Asteraceae) family, could be a source of novel ornamental crops. This tribe has more
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than 1500 taxa distributed in America, Asia, and Africa (Jones, 1979; Keeley et al., 2007; Robinson and Skvarla, 2007); thus, it is considered to be one of the most complex groups in the family (Keeley et al., 2007). One of the genera in tribe Vernonieae is Chrysolaena H. Rob., which has been assigned to the Lepidaploa complex (Robinson, 1988a). The center of diversification of the actually recognized eighteen Chrysolaena taxonomic species is Southern Brazil (Robinson, 1988a; Dematteis, 2014). In particular, Chrysolaena flexuosa (Sims) H. Rob. (Vernonia flexuosa Sims) appears to be the species of the tribe with the farthest known southern geographical distribution. Natural populations of this species have been found from southern Brazil to central-eastern Argentina (Cabrera, 1963; Dematteis, 2014), in almost undisturbed environments, some of them close to farming lands (Alonso et al., 2009; Echeverría and Camadro, 2017). Ch. flexuosa is a perennial xylopodial wild herb with potential ornamental value (Echeverría and Alonso, 2012; Echeverría and Camadro, 2017). It has heads with pure white to deep purple florets
Corresponding author. E-mail addresses: [email protected] (M.L. Echeverría), [email protected] (E.L. Camadro).
https://doi.org/10.1016/j.scienta.2019.108875 Received 10 July 2019; Received in revised form 15 September 2019; Accepted 18 September 2019 Available online 27 September 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.
Scientia Horticulturae 260 (2020) 108875
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Fig. 1. Geographic distribution of the seven Chrysolaena flexuosa accessions from Argentina used in this study (modified from Echeverría and Camadro, 2017). Ref. provinces of Misiones (MI), Corrientes (CO),Entre Ríos (ER), Buenos Aires (BA). Table 1 Chrysolaena flexuosa accessions, geographical origin, ploidy level, number of sampled individuals (N) and number of individuals successfully analyzed with AFLP markers (N AFLP). Accession
Location (province, county/department)
Geographic coordinates
VdPDFV 26 VdPDFV 45 VdPDFV 14 AloEch 1 AloEch 2 AloEch 3 Nu 1
Misiones, Capital city Corrientes, Itá Ibaté, Entre Ríos, Federación Buenos Aires, Gral. Pueyrredón Buenos Aires, Balcarce Buenos Aires, Balcarce Buenos Aires, Balcarce
27° 27° 30° 37° 37° 37° 37°
26' 26' 42' 34' 53' 52' 47'
47" 47" 12" 16" 40" 13" 52"
S; S; S; S; S; S; S;
55° 57° 58° 57° 58° 58° 58°
54' 20' 00' 28' 16' 22' 08'
11" 01" 40" 04" 14" 40" 36"
W W W W W W W
Ploidy level
N
N AFLP
2x 2x 4x 6x 6x 6x 6x
15 24 20 10 21 21 14
9 14 12 7 11 11 9
Argentina, Echeverröa and Camadro (2017) reported diploid and polyploid cytotypes and an apparently positive relationship between ploidy and latitude. To complement that study -and as a first step towards the domestication of Ch. flexuosa for the floriculture market and the eventual development of commercial cultivars- the same populations were characterized for morphological traits of ornamental value and AFLP molecular marker patterns.
grouped in a larger inflorescence with a zig-zag branching pattern (Cabrera, 1963; Echeverría and Alonso, 2012; Dematteis, 2014; Echeverría and Camadro, 2017). Most of the morphological descriptions of this species have been carried out in populations of the central distribution area, and very little is known about those with the farthest southern distribution. The basic chromosome number of Ch. flexuosa is x = 10 (Dematteis, 2014). Diploid (2n = 2x = 20) and tetraploid (2n = 4x = 40) cytotypes have been reported for the central distribution area, encompassing Uruguay, Paraguay, Brazil and northeastern Argentina (Dematteis, 2014), whereas hexaploid (2n = 6x = 60) cytotypes have only been reported in the Tandilia hill range (Fig. 1), in southeastern Buenos Aires province (Hunziker et al., 1990; Echeverría and Camadro, 2017). In many flowering plants, it has been reported that polyploid cytotypes have larger organ size than their diploid counterparts (Otto and Whitton, 2000). For this reason, the use of polyploids, both natural and induced, has been a frequent practice in Floriculture (Emsweller and Ruttle, 1941; Väinölä, 2000; Van Tuyl and Lim, 2003; Zlesak et al., 2005). For accessions of Ch. flexuosa from its distribution range in
2. Material and methods 2.1. Plant material Seven accessions of Ch. flexuosa (Table 1, Fig. 1) that differed in ploidy level -2x, 4x and 6x- were used in the study, which was based on the experiment carried out by Echeverría and Camadro (2017) in pots in the open field, at Balcarce, Buenos Aires province, Argentina (37°45′48″ S; 58°17′60″ W). These accessions are samples of natural populations that cover the distribution range of the species in Argentina, over various macro- and microenvironments. The accessions are ex situ conserved at the Botany Laboratory and the reference specimens at 2
Scientia Horticulturae 260 (2020) 108875
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combinations with different specific 3 bp overhangs. The core primers were the same that were used in the pre-amplification, with the addition of two selective nucleotides at the 3′-end: EcoRI-ACA/Msel-CAG and EcoRI-ACA/Msel-CTC. The EcoRI selective primer was 5´-labeled with green fluorescent HEX dye. The PCR amplification temperature profile was performed with an initial touchdown protocol: 94 °C for 30 s, 65 °C for 30 s and 72 °C for 1 min for 12 cycles; in each cycle the annealing temperature was decreased by 0.7 °C, followed by 23 cycles at: 94 °C for 30 s, 56 °C for 30 s and 72° for 1 min. The amplified products were separated by using an automatic DNA sequencer (Applied Biosystems 3500xl/Genetic Analyzer). To determine the length of the sample fragments, an internal standard (GeneScan 500 (-250) Rox) was used. To minimize errors, 8.0% of the samples were run in duplicate.
Table 2 Morphological traits evaluated in seven accessions of Chrysolaena flexuosa, including phenological stage at the time of the measurement (stage 1: full flowering; stage 2: end of fruiting), trait abbreviation, cycle (1: 2012/13; 2: 2013/ 14) and unit/scale of measurement. Stage
Morphological trait
Abbreviation Cycle 1
Cycle 2
NL1 LL1 LW1 IH1 NB1 NH1 HW1 HL1 NI1 CL1
NL2 LL2 LW2 IH2 NB2 NH2 HW2 HL2 NI2 CL2 – –
Unit/scale
1 1 1 1 1 1 1 1 1 1 1 1
Leaves Largest leaf length Largest leaf width First inflorescence height Branches per inflorescence Heads per inflorescence Central head width Central head length Inflorescences per plant Mean length of three corollas Phyllaries color Corolla color
1
Leaf pubescence
–
2
Number of leaves at the end of fruiting Largest leaf length at the end of fruiting Largest leaf width at the end of fruiting
NLA
number cm cm cm number number cm cm number cm green; violet purple; lilac; pink; white pubescent; slightly pubescent number
LLA
cm
LWA
cm
2 2
2.4. AFLP scoring and genetic analysis Electropherograms were read with Peak Scanner 1.0 (Applied Biosystems; http://products.invitrogen.com/ivgn/ product/4381867). Marker scoring was performed with RawGeno v2.0–1 (Arrigo et al., 2009; http://sourceforge.net/projects/rawgeno/) in R software interface (http://www.R-project.org/), with the following parameters: scoring range, 50–500 bp; minimum intensity, 100 rfu; minimum bin width, 1.0 bp; maximum bin width, 1.5 bp. With this software, every peak per sample was scored in a binary matrix for presence/absence of the fragment. Following Smith et al. (2015), fragments that were observed in only one sample were removed from the analysis.
the BAL Herbarium, both belonging to the Instituto Nacional de Tecnología Agropecuaria (INTA) and Universidad Nacional de Mar del Plata (UNMdP), Balcarce, Buenos Aires, Argentina.
2.5. Data analyses Mean values and coefficients of variation were estimated for the 23 quantitative morphological traits recorded in each of the seven accessions, and correlations analyses between the 23 morphological traits was carried out by using R software (http://www.R-project.org/). Moreover, in plants that were successfully processed with the selected molecular markers (Table 1), Principal Coordinate Analyses, genetic diversity estimations and an analysis of molecular variance were performed.
2.2. Morphological characterization In 10–24 individual plants per accession, vegetative and reproductive morphological traits of ornamental interest were registered in two growth cycles: 2012/13 (cycle 1) and 2013/14 (cycle 2) (Table 2). Measurements were performed at two phenological stages: full flowering (stage 1) and end of fruiting (stage 2). A total of 26 morphological traits were recorded: 23 quantitative and three qualitative (Fig. 2). The quantitative traits were measured in both cycles, whereas the qualitative traits were only measured in cycle 2.
2.5.1. Relationships between accessions Various Principal Coordinate Analyses based on similarity matrices were carried out to observe the relationships between the studied accessions, by using R software. These analyses were of three types: (1) with ploidy levels and morphological data, using Gower similarity coefficient; (2) with the AFLP data, using Jaccard similarity coefficient; (3) with ploidy level, morphological and AFLP data, using Gower similarity coefficient (Gower, 1971; Cuadras, 2014). On the other hand, with the qualitative morphological traits and ploidy levels, a factorial correspondence analysis was carried out with data from all accessions.
2.3. Molecular characterization Total DNA was extracted from leaves of 15 to 24 individual plants per accession according to Haymes (1996). For each DNA sample, 2 μl of RNAse were added, and samples were incubated at room temperature for 2 h. After spectrophotometric measurement of its concentration (BIO-RAD SmartSpect TM 3000), DNA was diluted in TE buffer to 1000 ng μl−1. Amplified fragment length polymorphism (AFLP) analysis was performed as described by Vos et al. (1995). For each sample, 1 μg of DNA was digested at 37 °C for 3 h in a thermocycler (Applied Biosystems, Veriti) with 2 U of EcoRI (Promega) and 1 U of Msel (New England Biolabs). The specific adaptors EcoRI (5′-CTC GTA GAC TGC GTA CC-3′ and 5′-AAT TGG TAC GCA GTC TAC-3′) and Msel (5′-GAC GAT GAG TCC TGA G- 3′ and 5′-TAC TCA GGA CTC AT-3′) were ligated to the digested DNA at 20 °C for 3 h, with 3 U/μl of T4 DNA ligase (Promega). Preamplification reactions were performed by using the adaptorligated template DNA and a set of pre-selective primers with one selective nucleotide (EcoRI + 1: 5′-AGA CTG CGT ACC AAT TC A-3′ and Msel+1: 5′-GAC GAT GAG TCC TGA GTA AC-3′). The polymerase chain reaction (PCR) programme consisted of 20 cycles at: 94 °C for 30 s, 56 °C for 1 m and 72 °C for 1 min. The selective amplification was performed by using two primer
2.5.2. Genetic diversity and analysis of molecular variance The percentage of polymorphism (P), Shannon diversity index (I) and Nei's genetic diversity (h) were estimated by using the POPGENE software version 1.32 (Yeh et al., 1999). The average genetic diversity was calculated at the level of both individual accessions and all accessions considered together. Nei’s genetic distance among accessions was also estimated and a dendrogram was constructed with UPGMA (Unweighted Pair Group Method using Arithmetic averages) by using the POPGENE software (Yeh et al., 1999). A correlation analysis was performed between Nei´s genetic distances among accessions and the geographical distances among the sampled populations from which the accessions were derived using R software. An analysis of molecular variance (AMOVA) was carried out to partition the observed variation in three components: within populations, among populations, and among geographic regions (Excoffier et al., 1992). Variance components were tested for significance with a 3
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Fig. 2. Adult specimen of Chrysolaena flexuosa in pre-anthesis with indication of morphological traits recorded in seven wild accessions. Corolla color (a) white, (b) pink, (c) lilac, (d) purple; leaf pubescence (e) slightly pubescent leaves, (f) pubescent; phyllary color (g) green, (h) violet. Ref.: IH: inflorescence height. Arrows: inflorescence branches. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
hexaploid accessions from Buenos Aires -AloEch 2, AloEch 3, Nu1, AloEch 1-, and those belonging to the tetraploid accession from Entre Ríos -VdPDFV 14- had lower number of leaves, inflorescences, and heads per inflorescence than those belonging to the diploid accessions from Corrientes and Misiones -VdPDFV 45 and VdPDFV 26-. On the other hand, polyploid plants had larger leaves, heads and corollas than the diploid ones (Table 3, Figs. 4 and 5).
non-parametric re-sampling approach with 1023 permuted data sets. Genetic differentiation among populations was estimated by using Wright´s fixation index (Fst). These analyses were performed with Arlequin software version 3.1 (Excoffier et al., 2005).
3. Results 3.1. Morphological characterization
3.2. Factorial correspondence analysis of qualitative morphological traits and ploidy level per accession
In the 125 studied individual plants belonging to the seven accessions, the most variable quantitative morphological traits were number of leaves and number of inflorescences per plant in cycle 2 (CV = 44.0% and 40.0%, respectively) (data not shown). Regardless the growth cycle, the traits number of leaves, leaf length and number of heads were highly and positively correlated among them, and highly but negatively correlated with leaf width, corolla length, and diameter and height of the central chapter (Fig. 3). Plants belonging to the four
The factorial correspondence analysis of qualitative morphological traits and ploidy level per accession allowed the distinction of three groups. These groups were respectively composed of the hexaploid accessions from Buenos Aires, the tetraploid accession from Entre Ríos, and the diploid accessions from Corrientes and Misiones (Fig. 6). The first two components explained > 70.0% of the total variability. The 4
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Fig. 3. Correlation between 23 morphological traits recorded in seven accessions of Chrysolaena flexuosa from Argentina. Cold colors = positive correlations, warm colors = negative correlations, no color = lack of correlation. Correlations increase with increasing circle diameter and color intensity. Ref.: NL = number of leaves, LL = largest leaf length, LW = largest leaf width, HW = central head width, HL = central head length, IH = first inflorescence height, NB = number of branches per inflorescence, NH = number or heads, NI = number of inflorescences per plant, CL = mean length of three corollas, LLA = largest leaf length at the end of fruiting, LWA = largest leaf width at the end of fruiting, NLA = number of leaves at the end of fruiting. Numbers 1 and 2 indicate growth cycle: 1 = 2012/13, 2 = 2013/14. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
Table 3 Mean values and coefficients of variation (CV) of 26 morphological traits recorded in plants of seven accessions of Chrysolaena flexuosa from Argentina. Ref.: Buenos Aires Province: AloEch 1 (6x), AloEch 2 (6x), AloEch 3 (6x), Nu 1 (6x); Entre Ríos Province: VdPDFV 14 (4x); Corrientes province: VdPDFV 45 (2x); Misiones province: VdPDFV 26 (2x). MORPHOLOGICAL TRAIT
QUANTITATIVES NL1 NL2 LL1 LL2 LW1 LW2 HW1 HW2 HL1 HL2 IH1 IH2 NB1 NB2 NH1 NH2 NI1 NI2 CL1 CL2 LLA LWA NLA QUALITATIVES Leaf pubescence Phyllaries color Corolla color
ACCESSION AloEch 1 (6x)
Nu 1 (6x)
Mean
CV (%)
Mean
12.3* 42.1* 9.5** 9.4** 3.1** 2.2** 2.7** 2.5** 2.5** 2.5** 20.2** 31.0** 2.8* 3.0* 5.3* 8.3* 1.0* 1.7* 0.119** 0.149** 6.7** 2.4** 11.1*
16.7 50.0 13.7 20.9 17.7 17.7 10.2 14.7 4.1 3.1 20.9 23.8 26.6 54.4 71.8 35.3 0.0 28.5 15.8 13.3 32.5 36.8 50.1
15.8* 45.5* 8.0** 9.6** 2.2** 1.9** 2.4** 2.6** 2.0** 2.2** 14.6** 27.0** 1.8* 2.9* 4.0* 7.6* 1.1* 2.0* 10.32** 0.130** 6.2** 2.2** 15.8*
pubescent violet purple
pubescent violet purple
AloEch 2 (6x)
AloEch 3 (6x)
VdPDFV 14 (4x)
VdPDFV 45 (2x)
VdPDFV 26 (2x)
CV (%)
Mean
CV (%)
Mean
CV (%)
Mean
CV (%)
Mean
CV (%)
Mean
CV (%)
32.7 33.6 24.1 10.8 16.1 36.6 16.3 22.4 3.5 17.4 27.8 60.6 59.1 65.6 55.1 74.2 31.4 46.3 18.2 12.4 32.6 19.2 31.8
21.8* 56.6* 11.0** 10.7** 2.6** 1.9** 2.5** 2.5** 2.5** 2.4** 17.7** 26.7** 2.3* 3.0* 6.1* 8.4* 1.0* 1.9* 0.135** 0.138** 7.9** 2.6** 11.4*
45.8 13.4 13.4 11.2 21.1 46.0 19.9 24.4 2.8 7.6 36.4 27.7 41.2 47.1 49.8 36.1 0.0 44.5 19.1 17.1 32.3 36.3 54.4
20.8* 67.9* 8.5** 8.6** 2.4** 1.6** 2.4** 2.7** 2.2** 2.4** 17.3** 18.9** 2.2* 2.0* 6.0* 4.5* 1.0* 1.8* 0.117** 0.125** 8.0** 2.3** 15.3*
54.9 26.5 21.9 25.3 23.0 49.2 32.7 20.1 15.4 9.7 36.5 34.2 35.9 59.8 50.9 47.5 0.0 79.4 16.1 10.7 14.1 25.4 54.1
24.7* 49.9* 9.0** 10.6** 2.3** 1.7** 2.2** 2.6** 2.2** 2.0** 17.2** 24.6** 2.3* 2.7* 5.4* 8.4* 1.1* 2.0* 0.107** 0.119** 8.2** 2.6** 22.8*
41.3 32.0 18.0 23.1 22.7 41.4 11.3 17.7 10.1 14.6 28.4 31.4 21.0 17.1 25.0 37.9 28.7 44.7 19.1 22.1 22.0 30.8 50.8
31.2* 88.2* 13.5** 11.5** 1.3** 0.9** 1.7** 2.2** 1.6** 1.9** 25.3** 23.8** 2.8* 2.9* 12.9* 13.6* 1.4* 3.7* 0.81** 0.115** 11.7** 1.4** 25.1*
38.1 24.1 19.0 13.2 32.1 24.7 16.5 14.6 14.5 19.8 14.1 23.2 35.0 33.2 41.5 55.3 62.0 32.4 11.4 17.3 27.6 38.5 52.7
26.3* 74.1* 12.0** 10.2** 1.5** 0.9** 1.5** 2.0** 1.5** 1.6** 22.9** 28.1** 3.0* 3.3* 9.5* 12.7* 1.0* 4.0* 0.77** 0.88** 9.4** 1.3** 22.4*
22.4 27.5 18.2 27.5 28.4 30.2 18.2 31.4 12.5 25.9 22.2 35.3 35.6 45.5 56.5 40.3 0.0 47.9 12.3 11.5 27.3 29.0 44.1
pubescent violet purple
pubescent violet purple
pubescent green white
slightly pubescent green lilac; pink; white
slightly pubescent green white; pink
Note.: * scale: number; ** scale: length in cm; NL = number of leaves, LL = largest leaf length, LW = largest leaf width, HW = central head width, HL = central head length, IH = first inflorescence height, NB = number of branches per inflorescence, NH = number or heads, NI = number of inflorescences per plant, CL = mean length of three corollas, LLA = largest leaf length at the end of fruiting, LWA = largest leaf width at the end of fruiting, NLA = number of leaves at the end of fruiting. Numbers 1 and 2 indicate growth cycle: 1 = 2012/13, 2 = 2013/14. 5
Scientia Horticulturae 260 (2020) 108875
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Fig. 4. Characteristic flower heads of seven accessions of Chrysolaena flexuosa from Argentina.
polymorphic loci ranged from 60.55% in the hexaploid accession AloEch 1 to 73.05% in the hexaploid accession AloEch 3 (Table 4).
Buenos Aires accessions were associated with pubescent leaves, violet phyllaries and purple corollas; the Entre Ríos accession, with pubescent leaves, green phyllaries, and white corollas; and the Corrientes and Misiones accessions, with slightly pubescent leaves, green phyllaries and lilac and pink corollas.
3.4.2. Genetic diversity and AMOVA Genetic diversity in the seven accession was revealed by the two selected AFLP marker combinations. Nei´s genetic diversity ranged from 0.1850 in the tetraploid accession VdPDFV 14 to 0.2204 in the hexaploid accession Nu 1, with an average of 0.2324 over all accessions. Similarly, Shannon diversity index ranged from 0.29 (VdPDFV 14) to 0.3374 (Nu 1), with an average 0.3707 over all accessions (Table 4). Furthermore, the average diversity of all the accessions did not vary significantly in relation to the individual values of each accession; thus, no association was observed between the estimated values of genetic diversity and ploidy level of the accessions or the geographical origin of the populations from which the accessions were derived. The smaller genetic distance (0.0373) was observed between accessions AloEch 2 and AloEch 3, and the largest (0.0930) between accessions VdPDFV 45 and AloEch 1 (Table 5). In the dendrogram based on Nei’s genetic distance with UPGMA (Fig. 8), it can be seen that clustering reflects geographical relationships. Moreover, the correlation between genetic distances among accessions and the geographical distances among the sampled populations was positive (r = 0.83; p < 0.0001). The results of the AMOVA performed to analyze the partition of the genetic variance according to the accession grouping obtained with the
3.3. Affinity among accessions based on joint information of ploidy and morphological traits The results of Principal Coordinates Analysis performed with the combined data of ploidy levels and the 26 morphological traits from at least 10 individual plants per accession, in each of the seven wild populations under study, can be observed in Fig. 7. In this analysis, the accessions were grouped as in the factorial correspondence analysis, with the first three eigenvalues explaining, respectively, 50.0%, 16.0% and 9.6% of the total variability. 3.4. Molecular characterization 3.4.1. AFLP amplification products The selected AFLP primers generated information to describe 73 individuals out of 125 that were morphologically analyzed (Table 1). A total of 256 polymorphic bands were detected, with fragment length ranging from 54 bp to 470 bp. EcoRI/ACA Msel-CAG primer combination allowed the detection of a higher number of polymorphic bands than EcoRI-ACA/Msel-CTC (151 vs. 105). The percentage of
Fig. 5. Characteristic morphological phenotypes at the vegetative stage of seven accessions of Chrysolaena flexuosa from Argentina. a: AloEch 2 (6x); b: AloEch 3 (6x); c: Nu 1 (6x); d: AloEch 1 (6x); e: VdPDFV 14 (4x); f: VdPDFV 45 (2x); g: VdPDFV 26 (2x). 6
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Fig. 6. Factorial correspondence analysis of seven accessions of Chrysolaena flexuosa from Argentina (dots) based on four qualitative morphological traits: leaf pubescence, phyllaries color, corolla color, and ploidy level.
Principal Coordinates Analysis with combined data from ploidy and morphological traits are presented in Table 6. This analysis revealed that the differences in molecular variance among geographic regions (Buenos Aires, Entre Ríos, Corrientes and Misiones provinces) were not significant. The AMOVA revealed that the highest percentage of the total molecular variance (88.1%) was due to variation among plants within accessions (p = 0.00001), with a smaller percentage corresponding to variation among accessions within geographic regions (1.5%), which was not statistically significant (p = 0.126). Wright´s Fixation Index (Fst) was 0.11, indicating moderate differentiation among accessions.
Table 4 Genetic diversity and percentage of polymorphic loci of seven accession of Chrysolaena flexuosa from Argentina estimated with AFLP markers. Ref.: h: Nei´s genetic diversity; I: Shannon genetic diversity index; P%: percentage of polymorphic loci.
3.4.3. Affinity among accessions based on molecular markers The first two coordinates of the Principal Coordinates Analysis using the AFLP markers contributed to explain 19.0% of the total variability. Individual plants exhibited a large dispersion around the first coordinate, revealing the presence of intra-accession or intra-population variability. In this analysis, a tendency of the individuals to be ordered according to the second coordinate is observed. The hexaploid plants from the Buenos Aires accessions were mostly located in the lower and central area of the graphic, the diploids from Misiones and Corrientes were located in the upper area, and the tetraploids from Entre Ríos were located in the central area (Fig. 9).
Accession
h
I
P%
VdPDFV 26 (2x) VdPDFV 45 (2x) VdPDFV 14 (4x) AloEch 1 (6x) AloEch 2 (6x) AloEch 3 (6x) Nu 1 (6x) All
0.2068 0.1850 0.1864 0.1946 0.1897 0.1918 0.2204 0.2324
0.3171 0.2900 0.2902 0.2917 0.2918 0.3028 0.3374 0.3707
66.02 71.09 67.58 60.55 64.45 73.05 71.09 100.00
3.4.4. Affinity among accessions based on joint information of ploidy level and morphological and molecular traits The first two coordinates of the Principal Coordinates Analysis using combined data of ploidy, morphological traits and AFLP markers contributed to explain more than 70% of the total variability. In the twodimensional graphic, some scattered points were observed. Notwithstanding, individual plants exhibited intra-accession o intrapopulation variability around the first coordinate. They also tended to group according to the second coordinate, forming strata that were
Fig. 7. Principal Coordinates Analysis using combined data of ploidy level and 26 morphological traits in 73 plants belonging to seven accessions of Chrysolaena flexuosa from Argentina on the two first principal coordinates. The first three coordinates contributed, respectively, 50.0%, 16.0% and 9.6% of the total variability. 7
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Table 5 Nei´s genetic distance (below diagonal) obtained with AFLP markers data and geographic distance in kilometers (above diagonal) between seven populations of Chrysolaena flexuosa from Argentina. Accession
VdPDFV 26
VdPDFV 45
VdPDFV 14
AloEch 1
AloEch 2
AloEch 3
Nu 1
VdPDFV 26 VdPDFV 45 VdPDFV 14 AloEch 1 AloEch 2 AloEch 3 Nu 1
– 0.0307 0.0704 0.0807 0.0858 0.0682 0.0878
147 – 0.0472 0.0930 0.0618 0.0591 0.0765
411 366 – 0.0837 0.0459 0.0536 0.0506
1.182 1.170 810 – 0.0493 0.0373 0.0493
1.178 1.163 797 43 – 0.0202 0.0254
1.185 1.161 796 53 10 – 0.0269
1.165 1.147 787 36 16 23 –
Fig. 8. UPGMA dendrogram based on pairwise genetic distance among seven accessions of Chrysolaena flexuosa from Argentina, and their geographical localization. Table 6 Analysis of molecular variance (AMOVA) based on AFLPs of 73 plants of seven accessions of Chrysolaena flexuosa from Argentina. Source of variation
df
Sum of squares
Variance components
% of variation
P
Among geographic regions Among accessions within geographic regions Among plants within accessions
2 4 66
232.548 143.232 2026.11
3.61740 0.52179 30.8378
10.4 1.5 88.1
0.123 0.126 0.0000
Fig. 9. Principal Coordinates Analysis using molecular markers based on individual values of 256 polymorphic AFLP bands in 73 plants belonging to seven accessions of Chrysolaena flexuosa from Argentina. Total contribution of the first three main coordinates: 20.7%.
4. Discussion
slightly defined by the geographical region. The diploid plants from the Misiones and Corrientes accessions were located in the upper area, the hexaploids from Buenos Aires in the lower area, and the tetraploids from Entre Ríos in the central area (Fig. 10). These results are in agreement with those obtained with the correlation analysis between genetic distances among accessions and the geographical distances among the natural populations from which the accessions were derived, and to the dendrogram based on Nei’s genetic distances (Fig. 8).
4.1. Molecular and morphological characterization The large number of species belonging to the Lepidaploa complex that thrive in southern Brazil (Robinson, 1987a, 1987b, 1987c, 1988a, 1988b) had led Keeley et al. (2007) to propose that this area is their center of diversification. Thus, the evolution of the species of the 8
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Fig. 10. Principal Coordinates Analysis using combined data of ploidy, 26 morphological traits and 256 polymorphic AFLP bands in 73 plants belonging to seven accessions of Chrysolaena flexuosa from Argentina. Total contribution of the first two main coordinates: 73.7%.
Lepidaploa complex should have occurred eccentrically in the rest of South America; extending their distribution towards South America, with Ch. flexuosa reaching the southeastern area of Buenos Aires province, in Argentina. In this area, populations of the species have only been found in the Tandilia hill range. In the molecular characterization with AFLP markers, the Fst index reflected a moderate genetic differentiation among accessions, regardless of their geographic origin. Nei´s genetic diversity and Shannon diversity index of the individual accessions were similar and not related to geographic origin or ploidy level. Moreover, the AMOVA and the Principal Coordinates Analysis revealed high intra-population variability (among plants within accessions). Although the diploid accessions were obtained from natural populations that grew closer to the center of diversification of the Lepidaploa complex than the polyploid ones, the estimated genetic diversity of the latter could be explained by the greater capacity of polyploids vs. diploids to store alleles in their repeated genomes (Carputo et al., 2006). These results are in line with those expected in allogamous species, and in contrast to the expected in autogamous ones (see Camadro, 2012). In turn, the Principal Coordinates Analysis based on the AFLP markers revealed a slight association between accessions from populations of nearby regions and same ploidy level. In the morphological characterization, however, individual plants from geographically closer populations shared some or even all of the qualitative attributes, in contrast with plants from more distant populations, which did not share any of the qualitative traits considered in this study. Furthermore, the correlation analysis between genetic and geographical distances as well as the dendrogram based on Nei’s genetic distance among accessions revealed an important association between those that came from populations of nearby regions and had the same ploidy level. Being a species that presents both sexual and vegetative reproduction via the xylopodia and exhibits the typical allogamous behavior, it could be speculated that, depending on the distance and under sexual compatibility, proximity between nearby populations could promote inter-population gene flow via biotic and/or abiotic vectors of either pollen grains (i.e., wind, insects) and/or seeds or xylopodia (i.e., water currents, soil movements). Gene flow will result in inter-population exchange of alleles, genetically homogenizing geographically separated populations. This aspect should be considered by plant breeders in genotype selection according with the objective(s) of their programs.
organ size was observed between tetraploid and hexaploid plants. Notwithstanding, the correlation between vegetative and reproductive organ size and ploidy level was high and positive, in coincidence with the reported in many flowering plants (see Stebbins, 1947; Levin 1983; Otto and Whitton, 2000; Sugiyama, 2005; Greilhuber and Leitch, 2013), although this correlation does not hold for all plant groups (see Otto and Whitton, 2000; Ibañez et al., 2014). Moreover, both morphological and molecular variability was detected among and within these accessions. Thus, the studied accessions are amenable to selection and manipulations for ornamental breeding purposes. However, in order to obtain commercial cultivars with desirable attributes, the parental genotypes have to be sexually compatible. Therefore, pre- and post-zygotic sexual compatibility relationships within and between populations should be investigated, regardless of their geographic origin and ploidy. To advance in this line of inquiry, it must be tested if plants can be directly hybridized or if, for that end, they are amenable to manipulations. In crop breeding in general, pre-zygotic barriers can be circumvented by choice of either the appropriate parental genotypes or the crossing direction. But in otherwise pollen-pistil compatible genotypic combinations, post-zygotic barriers could be acting (see Hadley and Openshaw, 1980), being endosperm abortion the most frequently encountered barrier in interploid crosses (Camadro et al., 2004). In a breeding programme, the endosperm barrier can be eventually overcome either asexually, by induced chromosome duplication of the lower ploidy parent, or sexually, by interploid crossing if the lower ploidy progenitor produces 2n gametes (see examples in Pfeiffert and Bingham, 1983; Orjeda et al., 1990; Carputo et al., 1997; Camadro et al., 2004). In this regard, Echeverría and Camadro (2017) reported the production of 2n pollen in the accessions characterized in the present study and, based on the underlying cytogenetic mechanisms, suggested that this phenomenon could be inherited. The use of both morphological and AFLP molecular markers in this exploratory study provided valuable information for the eventual incorporation of the Argentinian accessions of Ch. flexuosa in a breeding program. For more advanced studies, however, the use of omic-based technologies could be of interest, for example, to evaluate metabolic differences between diploids and polyploids and their consequence at the phenotypic level, as it has been done in plants of agronomic importance (see Tan et al., 2015, 2017, as examples).
4.2. Ploidy level and ornamental breeding
5. Conclusions
In the Ch. flexuosa plants of the present study, no differences in
The high morphological variability detected in qualitative and 9
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quantitative traits of ornamental interest and the molecular variability revealed by the AFLP markers in the studied accessions would allow their use in breeding programs for commercial purposes. Moreover, advantage can be taken from ploidy level differences -reflected as variations in the size of organs of ornamental interest-, for obtaining commercial cultivars with particular quantitative attributes. The conservation of biodiversity is an important global issue. In populated areas, urban environments can play a role in the conservation of local/regional species (Hostetler et al., 2011). Therefore, successful ornamental cultivar production of Ch. flexuosa would not only become a way to appreciate this genetic resource but also to care for it and to generate conscience in the society on the importance of protecting plant resources and the ecosystems where they thrive.
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Funding sources This work was financially supported by Universidad Nacional de Mar del Plata [research projects AGR 448/14, AGR 503/16 and AGR 557/18] and Instituto Nacional de Tecnología Agropecuaria. Acknowledgements We would like to thank Dr. G. Via do Pico and Dr. M. Dematteis, from the Instituto de Botánica del Nordeste (Corrientes, Argentina), for providing Ch. flexuosa seeds from northeastern Argentina populations. References Alonso, S.I., Guma, I.R., Nuciari, M.C., Van Olphen, A., 2009. Flora de un área de la Sierra La Barrosa (Balcarce) y fenología de especies nativas con potencial ornamental. Rev. FCA. UNCuyo. 41 (2), 23–44. Available from [accessed 4 March 2018]. http://www. bdigital.uncu.edu.ar/objetos_digitales/3166/t41-2-03-alonso.pdf. Arrigo, N., Tuszynski, J.W., Ehrich, D., Gerdes, T., Alvarez, N., 2009. Evaluating the impact of scoring parameters on the structure of intra-specific genetic variation using RawGeno, an R package for automating AFLP scoring. BMC Bioinformatics 10 (1), 33. https://doi.org/10.1186/1471-2105-10-33. Benton, T.G., Vickery, J.A., Wilson, J.D., 2003. Farmland biodiversity: is habitat heterogeneity the key? Trends Ecol. Evol. (Amst.) 18 (4), 182–188. https://doi.org/10. 1016/S0169-5347(03)00011-9. Cabrera, A.L., 1963. Flora de la Provincia de Buenos Aires. Compuestas. Colección Científica del INTA, Buenos Aires. Camadro, E.L., Carputo, D., Peloquin, S.J., 2004. Substitutes for genome differentiation in tuberbearing Solanum: interspecific pollen–pistil incompatibility, nuclear–cytoplasmic male sterility, and endosperm. Theor. Appl. Genet. 109 (7), 1369–1376. https://doi.org/10.1007/s00122-004-1753-2. Camadro, E.L., 2012. Relevance of the genetic structure of natural populations, and sampling and classification approaches for conservation and use of wild crop relatives: potato as an example. Botany. 90 (11), 1065–1072. https://doi.org/10.1139/ b2012-090. Carputo, D., Barone, A., Cardi, T., Sebastiano, A., Frusciante, L., Peloquin, S.J., 1997. Endosperm balance number manipulation for direct in vivo germplasm introgression to potato from a sexually isolated relative (Solanum commersonii Dun.). Proc. Natl. Acad. Sci. U. S. A. 94 (22), 12013–12017. https://doi.org/10.1073/pnas.94.22. 12013. 1997. Carputo, D., Camadro, E.L., Peloquin, S.J., 2006. Terminology for polyploids based on their cytogenetic behavior: consequences in genetics and breeding. In: In: Janick, J. (Ed.), Plant Breeding Reviews Vol. 26. John Wiley and Son, London, pp. 105–124. Cuadras, C.W., 2014. Nuevos métodos de análisis multivariante. CMC Editions, Barcelona. Dematteis, M., 2014. Tribu Vernonieae Cass. In: Zuloaga, F.O., Belgrano, M.J., Anton, A.M. (Eds.), Flora Argentina. Flora vascular de la República Argentina. IBODA CONICET, Argentina, pp. 229–287. Díaz, A., Pascual, U., Stenseke, M., Martín-López, B., Watson, R.T., Molnár, Z., Hill, R., Chan, K.M.A., Baste, I.A., Brauman, K.A., Polasky, S., Church, A., Lonsdale, M., Larigauderie, A., Leadley, P.W., van Oudenhoven, A.P.E., van der Plaat, F., Schröter, M., Lavorel, S., Aumeeruddy-Thomas, Y., Bukvareva, E., Davies, K., Demissew, S., Erpul, G., Failler, P., Guerra, C.A., Hewitt, C.L., Keune, H., Lindley, S., Shirayama, Y., 2018. Assessing nature’s contributions to people. Science 359 (6373), 270–272. https://doi.org/10.1126/science.aap8826. Echeverría, M.L., Alonso, S.I., 2012. Crecimiento inicial bajo cultivo de Chrysolaena flexuosa (Sims) H. Rob., Asteraceae nativa de valor ornamental potencial. Rev. FCA UNCuyo. 44 (2), 89–98. Available from [accessed 10 March 2018]. http://www. scielo.org.ar/scielo.php?pid=S1853-86652012000200007&script=sci_arttext. Echeverría, M.L., Camadro, E.L., 2017. Chromosome numbers, meiotic abnormalities, and 2n pollen formation in accessions of the wild species Chrysolaena flexuosa (Sims) H. Rob. (Vernonieae, Compositae) from its distribution range in Argentina. Bol. Soc. Argent. Bot. 52 (4), 737–752. https://doi.org/10.31055/1851.2372.v52.n4.18860. Emsweller, S.L., Ruttle, M.L., 1941. Induced polyploidy in floriculture. Am. Naturalist. 75
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Morphological and molecular variability of wild diploid and polyploid populations of Chrysolaena flexuosa (Sims) H. Rob.: relevance for ornamental breeding Echeverría María Lisa, Camadro Elsa Lucilab,
T
⁎
a
Universidad Nacional de Mar del Plata (UNMdP), Facultad de Ciencias Agrarias (FCA). Ruta Nacional 226, Km 73.5 (7620), Balcarce, Buenos Aires, Argentina Universidad Nacional de Mar del Plata (UNMdP), Facultad de Ciencias Agrarias (FCA) and Instituto Nacional de Tecnología Agropecuaria (INTA), EEA Balcarce, Laboratorio de Genética, Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Ruta Nacional 226, Km 73.5 (7620), Balcarce, Buenos Aires, Argentina
b
A R T I C LE I N FO
A B S T R A C T
Keywords: Lepidaploa Germplasm characterization Polyploidy Morphological phenotypes AFLP patterns Urban green spaces
Chrysolaena flexuosa is a South American species of ornamental interest, whose variability has not been explored. The objective of this work was to perform joint analyses of morphological and molecular traits in seven accessions from its geographic distribution in Argentina: diploid, tetraploid and hexaploid, along an approximately 1400 km transect. To this end, 26 morphological traits were recorded in 10–24 plants/accession, and two combination of AFLP markers were used for molecular characterization. The data was subjected to multivariate and AMOVA analyses, and the genetic diversity among accessions was evaluated by using the Fst index. The multivariate analyses for morphological traits allowed the differentiation of groups that were in accordance with their geographical origin and, thus, their respective ploidies; with the diploids exhibiting smaller-sized organs but higher numbers of leaves and heads. In the AMOVA, differences between accessions according to geographic origin were not significant (p = 0.123), and the highest percentage of the molecular variance corresponded to intra-population variation (88.1%). The Fst index indicated moderate genetic differentiation among accessions (Fst = 0.11), as observed in other allogamous species as a consequence of inter-population gene flow. In turn, the detected variability for morphological traits of interest would allow the use of these accessions in ornamental breeding for cultivar development.
1. Introduction Plant biodiversity is a very important source of goods and services for humankind. Broadly speaking, plant biodiversity satisfies human needs by providing much of the world agricultural and pharmaceutical goods, and participates in vital ecosystem services, such as nutrient cycling and oxygen production (Sala and Paruelo, 1997; Díaz et al., 2018). Most of the natural ecosystems are being disturbed by direct anthropogenic action (Valiente‐Banuet et al., 2015). With the steady fragmentation of natural environments due to agricultural intensification (Benton et al., 2003; Tscharntke et al., 2012), the importance of urban green spaces for biodiversity conservation steadily increases (Goddard et al., 2010). In this context, the utilization of novel ornamental crops in landscaping is not only a way of appreciating plant resources but also of promoting their use and care by the community. Vernonieae Cass, the major tribe of the Compositae (Asteraceae) family, could be a source of novel ornamental crops. This tribe has more
⁎
than 1500 taxa distributed in America, Asia, and Africa (Jones, 1979; Keeley et al., 2007; Robinson and Skvarla, 2007); thus, it is considered to be one of the most complex groups in the family (Keeley et al., 2007). One of the genera in tribe Vernonieae is Chrysolaena H. Rob., which has been assigned to the Lepidaploa complex (Robinson, 1988a). The center of diversification of the actually recognized eighteen Chrysolaena taxonomic species is Southern Brazil (Robinson, 1988a; Dematteis, 2014). In particular, Chrysolaena flexuosa (Sims) H. Rob. (Vernonia flexuosa Sims) appears to be the species of the tribe with the farthest known southern geographical distribution. Natural populations of this species have been found from southern Brazil to central-eastern Argentina (Cabrera, 1963; Dematteis, 2014), in almost undisturbed environments, some of them close to farming lands (Alonso et al., 2009; Echeverría and Camadro, 2017). Ch. flexuosa is a perennial xylopodial wild herb with potential ornamental value (Echeverría and Alonso, 2012; Echeverría and Camadro, 2017). It has heads with pure white to deep purple florets
Corresponding author. E-mail addresses: [email protected] (M.L. Echeverría), [email protected] (E.L. Camadro).
https://doi.org/10.1016/j.scienta.2019.108875 Received 10 July 2019; Received in revised form 15 September 2019; Accepted 18 September 2019 Available online 27 September 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.
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Fig. 1. Geographic distribution of the seven Chrysolaena flexuosa accessions from Argentina used in this study (modified from Echeverría and Camadro, 2017). Ref. provinces of Misiones (MI), Corrientes (CO),Entre Ríos (ER), Buenos Aires (BA). Table 1 Chrysolaena flexuosa accessions, geographical origin, ploidy level, number of sampled individuals (N) and number of individuals successfully analyzed with AFLP markers (N AFLP). Accession
Location (province, county/department)
Geographic coordinates
VdPDFV 26 VdPDFV 45 VdPDFV 14 AloEch 1 AloEch 2 AloEch 3 Nu 1
Misiones, Capital city Corrientes, Itá Ibaté, Entre Ríos, Federación Buenos Aires, Gral. Pueyrredón Buenos Aires, Balcarce Buenos Aires, Balcarce Buenos Aires, Balcarce
27° 27° 30° 37° 37° 37° 37°
26' 26' 42' 34' 53' 52' 47'
47" 47" 12" 16" 40" 13" 52"
S; S; S; S; S; S; S;
55° 57° 58° 57° 58° 58° 58°
54' 20' 00' 28' 16' 22' 08'
11" 01" 40" 04" 14" 40" 36"
W W W W W W W
Ploidy level
N
N AFLP
2x 2x 4x 6x 6x 6x 6x
15 24 20 10 21 21 14
9 14 12 7 11 11 9
Argentina, Echeverröa and Camadro (2017) reported diploid and polyploid cytotypes and an apparently positive relationship between ploidy and latitude. To complement that study -and as a first step towards the domestication of Ch. flexuosa for the floriculture market and the eventual development of commercial cultivars- the same populations were characterized for morphological traits of ornamental value and AFLP molecular marker patterns.
grouped in a larger inflorescence with a zig-zag branching pattern (Cabrera, 1963; Echeverría and Alonso, 2012; Dematteis, 2014; Echeverría and Camadro, 2017). Most of the morphological descriptions of this species have been carried out in populations of the central distribution area, and very little is known about those with the farthest southern distribution. The basic chromosome number of Ch. flexuosa is x = 10 (Dematteis, 2014). Diploid (2n = 2x = 20) and tetraploid (2n = 4x = 40) cytotypes have been reported for the central distribution area, encompassing Uruguay, Paraguay, Brazil and northeastern Argentina (Dematteis, 2014), whereas hexaploid (2n = 6x = 60) cytotypes have only been reported in the Tandilia hill range (Fig. 1), in southeastern Buenos Aires province (Hunziker et al., 1990; Echeverría and Camadro, 2017). In many flowering plants, it has been reported that polyploid cytotypes have larger organ size than their diploid counterparts (Otto and Whitton, 2000). For this reason, the use of polyploids, both natural and induced, has been a frequent practice in Floriculture (Emsweller and Ruttle, 1941; Väinölä, 2000; Van Tuyl and Lim, 2003; Zlesak et al., 2005). For accessions of Ch. flexuosa from its distribution range in
2. Material and methods 2.1. Plant material Seven accessions of Ch. flexuosa (Table 1, Fig. 1) that differed in ploidy level -2x, 4x and 6x- were used in the study, which was based on the experiment carried out by Echeverría and Camadro (2017) in pots in the open field, at Balcarce, Buenos Aires province, Argentina (37°45′48″ S; 58°17′60″ W). These accessions are samples of natural populations that cover the distribution range of the species in Argentina, over various macro- and microenvironments. The accessions are ex situ conserved at the Botany Laboratory and the reference specimens at 2
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combinations with different specific 3 bp overhangs. The core primers were the same that were used in the pre-amplification, with the addition of two selective nucleotides at the 3′-end: EcoRI-ACA/Msel-CAG and EcoRI-ACA/Msel-CTC. The EcoRI selective primer was 5´-labeled with green fluorescent HEX dye. The PCR amplification temperature profile was performed with an initial touchdown protocol: 94 °C for 30 s, 65 °C for 30 s and 72 °C for 1 min for 12 cycles; in each cycle the annealing temperature was decreased by 0.7 °C, followed by 23 cycles at: 94 °C for 30 s, 56 °C for 30 s and 72° for 1 min. The amplified products were separated by using an automatic DNA sequencer (Applied Biosystems 3500xl/Genetic Analyzer). To determine the length of the sample fragments, an internal standard (GeneScan 500 (-250) Rox) was used. To minimize errors, 8.0% of the samples were run in duplicate.
Table 2 Morphological traits evaluated in seven accessions of Chrysolaena flexuosa, including phenological stage at the time of the measurement (stage 1: full flowering; stage 2: end of fruiting), trait abbreviation, cycle (1: 2012/13; 2: 2013/ 14) and unit/scale of measurement. Stage
Morphological trait
Abbreviation Cycle 1
Cycle 2
NL1 LL1 LW1 IH1 NB1 NH1 HW1 HL1 NI1 CL1
NL2 LL2 LW2 IH2 NB2 NH2 HW2 HL2 NI2 CL2 – –
Unit/scale
1 1 1 1 1 1 1 1 1 1 1 1
Leaves Largest leaf length Largest leaf width First inflorescence height Branches per inflorescence Heads per inflorescence Central head width Central head length Inflorescences per plant Mean length of three corollas Phyllaries color Corolla color
1
Leaf pubescence
–
2
Number of leaves at the end of fruiting Largest leaf length at the end of fruiting Largest leaf width at the end of fruiting
NLA
number cm cm cm number number cm cm number cm green; violet purple; lilac; pink; white pubescent; slightly pubescent number
LLA
cm
LWA
cm
2 2
2.4. AFLP scoring and genetic analysis Electropherograms were read with Peak Scanner 1.0 (Applied Biosystems; http://products.invitrogen.com/ivgn/ product/4381867). Marker scoring was performed with RawGeno v2.0–1 (Arrigo et al., 2009; http://sourceforge.net/projects/rawgeno/) in R software interface (http://www.R-project.org/), with the following parameters: scoring range, 50–500 bp; minimum intensity, 100 rfu; minimum bin width, 1.0 bp; maximum bin width, 1.5 bp. With this software, every peak per sample was scored in a binary matrix for presence/absence of the fragment. Following Smith et al. (2015), fragments that were observed in only one sample were removed from the analysis.
the BAL Herbarium, both belonging to the Instituto Nacional de Tecnología Agropecuaria (INTA) and Universidad Nacional de Mar del Plata (UNMdP), Balcarce, Buenos Aires, Argentina.
2.5. Data analyses Mean values and coefficients of variation were estimated for the 23 quantitative morphological traits recorded in each of the seven accessions, and correlations analyses between the 23 morphological traits was carried out by using R software (http://www.R-project.org/). Moreover, in plants that were successfully processed with the selected molecular markers (Table 1), Principal Coordinate Analyses, genetic diversity estimations and an analysis of molecular variance were performed.
2.2. Morphological characterization In 10–24 individual plants per accession, vegetative and reproductive morphological traits of ornamental interest were registered in two growth cycles: 2012/13 (cycle 1) and 2013/14 (cycle 2) (Table 2). Measurements were performed at two phenological stages: full flowering (stage 1) and end of fruiting (stage 2). A total of 26 morphological traits were recorded: 23 quantitative and three qualitative (Fig. 2). The quantitative traits were measured in both cycles, whereas the qualitative traits were only measured in cycle 2.
2.5.1. Relationships between accessions Various Principal Coordinate Analyses based on similarity matrices were carried out to observe the relationships between the studied accessions, by using R software. These analyses were of three types: (1) with ploidy levels and morphological data, using Gower similarity coefficient; (2) with the AFLP data, using Jaccard similarity coefficient; (3) with ploidy level, morphological and AFLP data, using Gower similarity coefficient (Gower, 1971; Cuadras, 2014). On the other hand, with the qualitative morphological traits and ploidy levels, a factorial correspondence analysis was carried out with data from all accessions.
2.3. Molecular characterization Total DNA was extracted from leaves of 15 to 24 individual plants per accession according to Haymes (1996). For each DNA sample, 2 μl of RNAse were added, and samples were incubated at room temperature for 2 h. After spectrophotometric measurement of its concentration (BIO-RAD SmartSpect TM 3000), DNA was diluted in TE buffer to 1000 ng μl−1. Amplified fragment length polymorphism (AFLP) analysis was performed as described by Vos et al. (1995). For each sample, 1 μg of DNA was digested at 37 °C for 3 h in a thermocycler (Applied Biosystems, Veriti) with 2 U of EcoRI (Promega) and 1 U of Msel (New England Biolabs). The specific adaptors EcoRI (5′-CTC GTA GAC TGC GTA CC-3′ and 5′-AAT TGG TAC GCA GTC TAC-3′) and Msel (5′-GAC GAT GAG TCC TGA G- 3′ and 5′-TAC TCA GGA CTC AT-3′) were ligated to the digested DNA at 20 °C for 3 h, with 3 U/μl of T4 DNA ligase (Promega). Preamplification reactions were performed by using the adaptorligated template DNA and a set of pre-selective primers with one selective nucleotide (EcoRI + 1: 5′-AGA CTG CGT ACC AAT TC A-3′ and Msel+1: 5′-GAC GAT GAG TCC TGA GTA AC-3′). The polymerase chain reaction (PCR) programme consisted of 20 cycles at: 94 °C for 30 s, 56 °C for 1 m and 72 °C for 1 min. The selective amplification was performed by using two primer
2.5.2. Genetic diversity and analysis of molecular variance The percentage of polymorphism (P), Shannon diversity index (I) and Nei's genetic diversity (h) were estimated by using the POPGENE software version 1.32 (Yeh et al., 1999). The average genetic diversity was calculated at the level of both individual accessions and all accessions considered together. Nei’s genetic distance among accessions was also estimated and a dendrogram was constructed with UPGMA (Unweighted Pair Group Method using Arithmetic averages) by using the POPGENE software (Yeh et al., 1999). A correlation analysis was performed between Nei´s genetic distances among accessions and the geographical distances among the sampled populations from which the accessions were derived using R software. An analysis of molecular variance (AMOVA) was carried out to partition the observed variation in three components: within populations, among populations, and among geographic regions (Excoffier et al., 1992). Variance components were tested for significance with a 3
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Fig. 2. Adult specimen of Chrysolaena flexuosa in pre-anthesis with indication of morphological traits recorded in seven wild accessions. Corolla color (a) white, (b) pink, (c) lilac, (d) purple; leaf pubescence (e) slightly pubescent leaves, (f) pubescent; phyllary color (g) green, (h) violet. Ref.: IH: inflorescence height. Arrows: inflorescence branches. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
hexaploid accessions from Buenos Aires -AloEch 2, AloEch 3, Nu1, AloEch 1-, and those belonging to the tetraploid accession from Entre Ríos -VdPDFV 14- had lower number of leaves, inflorescences, and heads per inflorescence than those belonging to the diploid accessions from Corrientes and Misiones -VdPDFV 45 and VdPDFV 26-. On the other hand, polyploid plants had larger leaves, heads and corollas than the diploid ones (Table 3, Figs. 4 and 5).
non-parametric re-sampling approach with 1023 permuted data sets. Genetic differentiation among populations was estimated by using Wright´s fixation index (Fst). These analyses were performed with Arlequin software version 3.1 (Excoffier et al., 2005).
3. Results 3.1. Morphological characterization
3.2. Factorial correspondence analysis of qualitative morphological traits and ploidy level per accession
In the 125 studied individual plants belonging to the seven accessions, the most variable quantitative morphological traits were number of leaves and number of inflorescences per plant in cycle 2 (CV = 44.0% and 40.0%, respectively) (data not shown). Regardless the growth cycle, the traits number of leaves, leaf length and number of heads were highly and positively correlated among them, and highly but negatively correlated with leaf width, corolla length, and diameter and height of the central chapter (Fig. 3). Plants belonging to the four
The factorial correspondence analysis of qualitative morphological traits and ploidy level per accession allowed the distinction of three groups. These groups were respectively composed of the hexaploid accessions from Buenos Aires, the tetraploid accession from Entre Ríos, and the diploid accessions from Corrientes and Misiones (Fig. 6). The first two components explained > 70.0% of the total variability. The 4
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Fig. 3. Correlation between 23 morphological traits recorded in seven accessions of Chrysolaena flexuosa from Argentina. Cold colors = positive correlations, warm colors = negative correlations, no color = lack of correlation. Correlations increase with increasing circle diameter and color intensity. Ref.: NL = number of leaves, LL = largest leaf length, LW = largest leaf width, HW = central head width, HL = central head length, IH = first inflorescence height, NB = number of branches per inflorescence, NH = number or heads, NI = number of inflorescences per plant, CL = mean length of three corollas, LLA = largest leaf length at the end of fruiting, LWA = largest leaf width at the end of fruiting, NLA = number of leaves at the end of fruiting. Numbers 1 and 2 indicate growth cycle: 1 = 2012/13, 2 = 2013/14. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).
Table 3 Mean values and coefficients of variation (CV) of 26 morphological traits recorded in plants of seven accessions of Chrysolaena flexuosa from Argentina. Ref.: Buenos Aires Province: AloEch 1 (6x), AloEch 2 (6x), AloEch 3 (6x), Nu 1 (6x); Entre Ríos Province: VdPDFV 14 (4x); Corrientes province: VdPDFV 45 (2x); Misiones province: VdPDFV 26 (2x). MORPHOLOGICAL TRAIT
QUANTITATIVES NL1 NL2 LL1 LL2 LW1 LW2 HW1 HW2 HL1 HL2 IH1 IH2 NB1 NB2 NH1 NH2 NI1 NI2 CL1 CL2 LLA LWA NLA QUALITATIVES Leaf pubescence Phyllaries color Corolla color
ACCESSION AloEch 1 (6x)
Nu 1 (6x)
Mean
CV (%)
Mean
12.3* 42.1* 9.5** 9.4** 3.1** 2.2** 2.7** 2.5** 2.5** 2.5** 20.2** 31.0** 2.8* 3.0* 5.3* 8.3* 1.0* 1.7* 0.119** 0.149** 6.7** 2.4** 11.1*
16.7 50.0 13.7 20.9 17.7 17.7 10.2 14.7 4.1 3.1 20.9 23.8 26.6 54.4 71.8 35.3 0.0 28.5 15.8 13.3 32.5 36.8 50.1
15.8* 45.5* 8.0** 9.6** 2.2** 1.9** 2.4** 2.6** 2.0** 2.2** 14.6** 27.0** 1.8* 2.9* 4.0* 7.6* 1.1* 2.0* 10.32** 0.130** 6.2** 2.2** 15.8*
pubescent violet purple
pubescent violet purple
AloEch 2 (6x)
AloEch 3 (6x)
VdPDFV 14 (4x)
VdPDFV 45 (2x)
VdPDFV 26 (2x)
CV (%)
Mean
CV (%)
Mean
CV (%)
Mean
CV (%)
Mean
CV (%)
Mean
CV (%)
32.7 33.6 24.1 10.8 16.1 36.6 16.3 22.4 3.5 17.4 27.8 60.6 59.1 65.6 55.1 74.2 31.4 46.3 18.2 12.4 32.6 19.2 31.8
21.8* 56.6* 11.0** 10.7** 2.6** 1.9** 2.5** 2.5** 2.5** 2.4** 17.7** 26.7** 2.3* 3.0* 6.1* 8.4* 1.0* 1.9* 0.135** 0.138** 7.9** 2.6** 11.4*
45.8 13.4 13.4 11.2 21.1 46.0 19.9 24.4 2.8 7.6 36.4 27.7 41.2 47.1 49.8 36.1 0.0 44.5 19.1 17.1 32.3 36.3 54.4
20.8* 67.9* 8.5** 8.6** 2.4** 1.6** 2.4** 2.7** 2.2** 2.4** 17.3** 18.9** 2.2* 2.0* 6.0* 4.5* 1.0* 1.8* 0.117** 0.125** 8.0** 2.3** 15.3*
54.9 26.5 21.9 25.3 23.0 49.2 32.7 20.1 15.4 9.7 36.5 34.2 35.9 59.8 50.9 47.5 0.0 79.4 16.1 10.7 14.1 25.4 54.1
24.7* 49.9* 9.0** 10.6** 2.3** 1.7** 2.2** 2.6** 2.2** 2.0** 17.2** 24.6** 2.3* 2.7* 5.4* 8.4* 1.1* 2.0* 0.107** 0.119** 8.2** 2.6** 22.8*
41.3 32.0 18.0 23.1 22.7 41.4 11.3 17.7 10.1 14.6 28.4 31.4 21.0 17.1 25.0 37.9 28.7 44.7 19.1 22.1 22.0 30.8 50.8
31.2* 88.2* 13.5** 11.5** 1.3** 0.9** 1.7** 2.2** 1.6** 1.9** 25.3** 23.8** 2.8* 2.9* 12.9* 13.6* 1.4* 3.7* 0.81** 0.115** 11.7** 1.4** 25.1*
38.1 24.1 19.0 13.2 32.1 24.7 16.5 14.6 14.5 19.8 14.1 23.2 35.0 33.2 41.5 55.3 62.0 32.4 11.4 17.3 27.6 38.5 52.7
26.3* 74.1* 12.0** 10.2** 1.5** 0.9** 1.5** 2.0** 1.5** 1.6** 22.9** 28.1** 3.0* 3.3* 9.5* 12.7* 1.0* 4.0* 0.77** 0.88** 9.4** 1.3** 22.4*
22.4 27.5 18.2 27.5 28.4 30.2 18.2 31.4 12.5 25.9 22.2 35.3 35.6 45.5 56.5 40.3 0.0 47.9 12.3 11.5 27.3 29.0 44.1
pubescent violet purple
pubescent violet purple
pubescent green white
slightly pubescent green lilac; pink; white
slightly pubescent green white; pink
Note.: * scale: number; ** scale: length in cm; NL = number of leaves, LL = largest leaf length, LW = largest leaf width, HW = central head width, HL = central head length, IH = first inflorescence height, NB = number of branches per inflorescence, NH = number or heads, NI = number of inflorescences per plant, CL = mean length of three corollas, LLA = largest leaf length at the end of fruiting, LWA = largest leaf width at the end of fruiting, NLA = number of leaves at the end of fruiting. Numbers 1 and 2 indicate growth cycle: 1 = 2012/13, 2 = 2013/14. 5
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Fig. 4. Characteristic flower heads of seven accessions of Chrysolaena flexuosa from Argentina.
polymorphic loci ranged from 60.55% in the hexaploid accession AloEch 1 to 73.05% in the hexaploid accession AloEch 3 (Table 4).
Buenos Aires accessions were associated with pubescent leaves, violet phyllaries and purple corollas; the Entre Ríos accession, with pubescent leaves, green phyllaries, and white corollas; and the Corrientes and Misiones accessions, with slightly pubescent leaves, green phyllaries and lilac and pink corollas.
3.4.2. Genetic diversity and AMOVA Genetic diversity in the seven accession was revealed by the two selected AFLP marker combinations. Nei´s genetic diversity ranged from 0.1850 in the tetraploid accession VdPDFV 14 to 0.2204 in the hexaploid accession Nu 1, with an average of 0.2324 over all accessions. Similarly, Shannon diversity index ranged from 0.29 (VdPDFV 14) to 0.3374 (Nu 1), with an average 0.3707 over all accessions (Table 4). Furthermore, the average diversity of all the accessions did not vary significantly in relation to the individual values of each accession; thus, no association was observed between the estimated values of genetic diversity and ploidy level of the accessions or the geographical origin of the populations from which the accessions were derived. The smaller genetic distance (0.0373) was observed between accessions AloEch 2 and AloEch 3, and the largest (0.0930) between accessions VdPDFV 45 and AloEch 1 (Table 5). In the dendrogram based on Nei’s genetic distance with UPGMA (Fig. 8), it can be seen that clustering reflects geographical relationships. Moreover, the correlation between genetic distances among accessions and the geographical distances among the sampled populations was positive (r = 0.83; p < 0.0001). The results of the AMOVA performed to analyze the partition of the genetic variance according to the accession grouping obtained with the
3.3. Affinity among accessions based on joint information of ploidy and morphological traits The results of Principal Coordinates Analysis performed with the combined data of ploidy levels and the 26 morphological traits from at least 10 individual plants per accession, in each of the seven wild populations under study, can be observed in Fig. 7. In this analysis, the accessions were grouped as in the factorial correspondence analysis, with the first three eigenvalues explaining, respectively, 50.0%, 16.0% and 9.6% of the total variability. 3.4. Molecular characterization 3.4.1. AFLP amplification products The selected AFLP primers generated information to describe 73 individuals out of 125 that were morphologically analyzed (Table 1). A total of 256 polymorphic bands were detected, with fragment length ranging from 54 bp to 470 bp. EcoRI/ACA Msel-CAG primer combination allowed the detection of a higher number of polymorphic bands than EcoRI-ACA/Msel-CTC (151 vs. 105). The percentage of
Fig. 5. Characteristic morphological phenotypes at the vegetative stage of seven accessions of Chrysolaena flexuosa from Argentina. a: AloEch 2 (6x); b: AloEch 3 (6x); c: Nu 1 (6x); d: AloEch 1 (6x); e: VdPDFV 14 (4x); f: VdPDFV 45 (2x); g: VdPDFV 26 (2x). 6
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Fig. 6. Factorial correspondence analysis of seven accessions of Chrysolaena flexuosa from Argentina (dots) based on four qualitative morphological traits: leaf pubescence, phyllaries color, corolla color, and ploidy level.
Principal Coordinates Analysis with combined data from ploidy and morphological traits are presented in Table 6. This analysis revealed that the differences in molecular variance among geographic regions (Buenos Aires, Entre Ríos, Corrientes and Misiones provinces) were not significant. The AMOVA revealed that the highest percentage of the total molecular variance (88.1%) was due to variation among plants within accessions (p = 0.00001), with a smaller percentage corresponding to variation among accessions within geographic regions (1.5%), which was not statistically significant (p = 0.126). Wright´s Fixation Index (Fst) was 0.11, indicating moderate differentiation among accessions.
Table 4 Genetic diversity and percentage of polymorphic loci of seven accession of Chrysolaena flexuosa from Argentina estimated with AFLP markers. Ref.: h: Nei´s genetic diversity; I: Shannon genetic diversity index; P%: percentage of polymorphic loci.
3.4.3. Affinity among accessions based on molecular markers The first two coordinates of the Principal Coordinates Analysis using the AFLP markers contributed to explain 19.0% of the total variability. Individual plants exhibited a large dispersion around the first coordinate, revealing the presence of intra-accession or intra-population variability. In this analysis, a tendency of the individuals to be ordered according to the second coordinate is observed. The hexaploid plants from the Buenos Aires accessions were mostly located in the lower and central area of the graphic, the diploids from Misiones and Corrientes were located in the upper area, and the tetraploids from Entre Ríos were located in the central area (Fig. 9).
Accession
h
I
P%
VdPDFV 26 (2x) VdPDFV 45 (2x) VdPDFV 14 (4x) AloEch 1 (6x) AloEch 2 (6x) AloEch 3 (6x) Nu 1 (6x) All
0.2068 0.1850 0.1864 0.1946 0.1897 0.1918 0.2204 0.2324
0.3171 0.2900 0.2902 0.2917 0.2918 0.3028 0.3374 0.3707
66.02 71.09 67.58 60.55 64.45 73.05 71.09 100.00
3.4.4. Affinity among accessions based on joint information of ploidy level and morphological and molecular traits The first two coordinates of the Principal Coordinates Analysis using combined data of ploidy, morphological traits and AFLP markers contributed to explain more than 70% of the total variability. In the twodimensional graphic, some scattered points were observed. Notwithstanding, individual plants exhibited intra-accession o intrapopulation variability around the first coordinate. They also tended to group according to the second coordinate, forming strata that were
Fig. 7. Principal Coordinates Analysis using combined data of ploidy level and 26 morphological traits in 73 plants belonging to seven accessions of Chrysolaena flexuosa from Argentina on the two first principal coordinates. The first three coordinates contributed, respectively, 50.0%, 16.0% and 9.6% of the total variability. 7
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Table 5 Nei´s genetic distance (below diagonal) obtained with AFLP markers data and geographic distance in kilometers (above diagonal) between seven populations of Chrysolaena flexuosa from Argentina. Accession
VdPDFV 26
VdPDFV 45
VdPDFV 14
AloEch 1
AloEch 2
AloEch 3
Nu 1
VdPDFV 26 VdPDFV 45 VdPDFV 14 AloEch 1 AloEch 2 AloEch 3 Nu 1
– 0.0307 0.0704 0.0807 0.0858 0.0682 0.0878
147 – 0.0472 0.0930 0.0618 0.0591 0.0765
411 366 – 0.0837 0.0459 0.0536 0.0506
1.182 1.170 810 – 0.0493 0.0373 0.0493
1.178 1.163 797 43 – 0.0202 0.0254
1.185 1.161 796 53 10 – 0.0269
1.165 1.147 787 36 16 23 –
Fig. 8. UPGMA dendrogram based on pairwise genetic distance among seven accessions of Chrysolaena flexuosa from Argentina, and their geographical localization. Table 6 Analysis of molecular variance (AMOVA) based on AFLPs of 73 plants of seven accessions of Chrysolaena flexuosa from Argentina. Source of variation
df
Sum of squares
Variance components
% of variation
P
Among geographic regions Among accessions within geographic regions Among plants within accessions
2 4 66
232.548 143.232 2026.11
3.61740 0.52179 30.8378
10.4 1.5 88.1
0.123 0.126 0.0000
Fig. 9. Principal Coordinates Analysis using molecular markers based on individual values of 256 polymorphic AFLP bands in 73 plants belonging to seven accessions of Chrysolaena flexuosa from Argentina. Total contribution of the first three main coordinates: 20.7%.
4. Discussion
slightly defined by the geographical region. The diploid plants from the Misiones and Corrientes accessions were located in the upper area, the hexaploids from Buenos Aires in the lower area, and the tetraploids from Entre Ríos in the central area (Fig. 10). These results are in agreement with those obtained with the correlation analysis between genetic distances among accessions and the geographical distances among the natural populations from which the accessions were derived, and to the dendrogram based on Nei’s genetic distances (Fig. 8).
4.1. Molecular and morphological characterization The large number of species belonging to the Lepidaploa complex that thrive in southern Brazil (Robinson, 1987a, 1987b, 1987c, 1988a, 1988b) had led Keeley et al. (2007) to propose that this area is their center of diversification. Thus, the evolution of the species of the 8
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Fig. 10. Principal Coordinates Analysis using combined data of ploidy, 26 morphological traits and 256 polymorphic AFLP bands in 73 plants belonging to seven accessions of Chrysolaena flexuosa from Argentina. Total contribution of the first two main coordinates: 73.7%.
Lepidaploa complex should have occurred eccentrically in the rest of South America; extending their distribution towards South America, with Ch. flexuosa reaching the southeastern area of Buenos Aires province, in Argentina. In this area, populations of the species have only been found in the Tandilia hill range. In the molecular characterization with AFLP markers, the Fst index reflected a moderate genetic differentiation among accessions, regardless of their geographic origin. Nei´s genetic diversity and Shannon diversity index of the individual accessions were similar and not related to geographic origin or ploidy level. Moreover, the AMOVA and the Principal Coordinates Analysis revealed high intra-population variability (among plants within accessions). Although the diploid accessions were obtained from natural populations that grew closer to the center of diversification of the Lepidaploa complex than the polyploid ones, the estimated genetic diversity of the latter could be explained by the greater capacity of polyploids vs. diploids to store alleles in their repeated genomes (Carputo et al., 2006). These results are in line with those expected in allogamous species, and in contrast to the expected in autogamous ones (see Camadro, 2012). In turn, the Principal Coordinates Analysis based on the AFLP markers revealed a slight association between accessions from populations of nearby regions and same ploidy level. In the morphological characterization, however, individual plants from geographically closer populations shared some or even all of the qualitative attributes, in contrast with plants from more distant populations, which did not share any of the qualitative traits considered in this study. Furthermore, the correlation analysis between genetic and geographical distances as well as the dendrogram based on Nei’s genetic distance among accessions revealed an important association between those that came from populations of nearby regions and had the same ploidy level. Being a species that presents both sexual and vegetative reproduction via the xylopodia and exhibits the typical allogamous behavior, it could be speculated that, depending on the distance and under sexual compatibility, proximity between nearby populations could promote inter-population gene flow via biotic and/or abiotic vectors of either pollen grains (i.e., wind, insects) and/or seeds or xylopodia (i.e., water currents, soil movements). Gene flow will result in inter-population exchange of alleles, genetically homogenizing geographically separated populations. This aspect should be considered by plant breeders in genotype selection according with the objective(s) of their programs.
organ size was observed between tetraploid and hexaploid plants. Notwithstanding, the correlation between vegetative and reproductive organ size and ploidy level was high and positive, in coincidence with the reported in many flowering plants (see Stebbins, 1947; Levin 1983; Otto and Whitton, 2000; Sugiyama, 2005; Greilhuber and Leitch, 2013), although this correlation does not hold for all plant groups (see Otto and Whitton, 2000; Ibañez et al., 2014). Moreover, both morphological and molecular variability was detected among and within these accessions. Thus, the studied accessions are amenable to selection and manipulations for ornamental breeding purposes. However, in order to obtain commercial cultivars with desirable attributes, the parental genotypes have to be sexually compatible. Therefore, pre- and post-zygotic sexual compatibility relationships within and between populations should be investigated, regardless of their geographic origin and ploidy. To advance in this line of inquiry, it must be tested if plants can be directly hybridized or if, for that end, they are amenable to manipulations. In crop breeding in general, pre-zygotic barriers can be circumvented by choice of either the appropriate parental genotypes or the crossing direction. But in otherwise pollen-pistil compatible genotypic combinations, post-zygotic barriers could be acting (see Hadley and Openshaw, 1980), being endosperm abortion the most frequently encountered barrier in interploid crosses (Camadro et al., 2004). In a breeding programme, the endosperm barrier can be eventually overcome either asexually, by induced chromosome duplication of the lower ploidy parent, or sexually, by interploid crossing if the lower ploidy progenitor produces 2n gametes (see examples in Pfeiffert and Bingham, 1983; Orjeda et al., 1990; Carputo et al., 1997; Camadro et al., 2004). In this regard, Echeverría and Camadro (2017) reported the production of 2n pollen in the accessions characterized in the present study and, based on the underlying cytogenetic mechanisms, suggested that this phenomenon could be inherited. The use of both morphological and AFLP molecular markers in this exploratory study provided valuable information for the eventual incorporation of the Argentinian accessions of Ch. flexuosa in a breeding program. For more advanced studies, however, the use of omic-based technologies could be of interest, for example, to evaluate metabolic differences between diploids and polyploids and their consequence at the phenotypic level, as it has been done in plants of agronomic importance (see Tan et al., 2015, 2017, as examples).
4.2. Ploidy level and ornamental breeding
5. Conclusions
In the Ch. flexuosa plants of the present study, no differences in
The high morphological variability detected in qualitative and 9
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quantitative traits of ornamental interest and the molecular variability revealed by the AFLP markers in the studied accessions would allow their use in breeding programs for commercial purposes. Moreover, advantage can be taken from ploidy level differences -reflected as variations in the size of organs of ornamental interest-, for obtaining commercial cultivars with particular quantitative attributes. The conservation of biodiversity is an important global issue. In populated areas, urban environments can play a role in the conservation of local/regional species (Hostetler et al., 2011). Therefore, successful ornamental cultivar production of Ch. flexuosa would not only become a way to appreciate this genetic resource but also to care for it and to generate conscience in the society on the importance of protecting plant resources and the ecosystems where they thrive.
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