Morphological variability of prickly pear cultivars (Opuntia spp.) established in ex-situ collection in Tunisia

Scientia Horticulturae 248 (2019) 163–175

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Morphological variability of prickly pear cultivars (Opuntia spp.) established in ex-situ collection in Tunisia

T

Elhani Amania, Louati Marwaa, Ben Salem Hichemb, Salhi-Hannachi Amela, Baraket Ghadaa,

⁎

a

University of Tunis El Manar, Faculty of Sciences of Tunis, Laboratory of Molecular Genetics, Immunology & Biotechnology LR99ES12, Campus University, 2092, El Manar, Tunis, Tunisia b National Institute for Agronomic Research of Tunis, Hedi Karray Street, 2049, Ariana, Tunisia

ARTICLE INFO

ABSTRACT

Keywords: Barbary fig Opuntia Morphologic variability Tunisia

Belonging to the succulent plants’ family, the Cactaceae, Barbary fig tree (Opuntia ficus-indica) is an excellent arid-resistant species, implicated in erosion control. In Tunisia, the Barbary fig is one of the country's main fruit species, mainly used for its edible fruits. Thus, forty-eight ecotypes belonging to four species of Opuntia genus (Opuntia ficus-indica, Opuntia engelmannii, Opuntia tomentosa and Opuntia undulata), were collected from the INRAT collection in northern Tunisia. In order to conduct a morphological study aiming to estimate the morphologic variation degree, 63 phenotypic characteristics that comprised 30 quantitative and 33 qualitative traits recorded to Opuntia trees, cladodes, flowers and fruits were detected. Based on quantitative and qualitative parameters separately, results of dendrograms were consistent with Principal Component Analysis (PCA) and Multiple Correspondence analysis (MCA) results. These analyses showed that the four quantitative variables: (the mean number of seeds per fruit, the mean number of areolas per cladode, the mean number of areolas at the cladode border and fruit mean weight) and the three qualitative variables: (cladode shape, pulp color and fruit color) are the most discriminating parameters in differentiation between the four studied species. Indeed, dendrograms showed that studied ecotypes were classified independently of their geographical origins and corresponding species. In this study, fourteen quantitative parameters were revealed using one-way analysis of variance (ANOVA). For the qualitative variables; Z test, Kruskal-Wallis test and chi-square analysis were performed and non-significant values were detected. In conclusion, our results revealed a very high level of morphological variability and a great similarity between O. ficus-indica and O. engelmannii that will be very useful for genetic breeding and conservation programs.

1. Introduction Although natural adverse climate variation is main background factor in arid and semi-arid areas, many factors are contributing massively to desertification and land degradation such as human abuses of the land and poor resource management (Alary et al., 2007). For this reason, dry lands prone to desertification have exceeded more than one third of the globe and support more than 20% of the world’s population (Wu, 2001). North Africa presents many arid and semi-arid areas becoming desert. Thus, it is important to found a solution to limit land degradation by the use of natural barriers. In this context, cactus is known to be the best plant for reforesting arid and semi-arid areas because of its resistance to limited and erratic rainfall and high temperatures (Alary et al., 2007). Indeed, it can be used in combination with cement barriers or cut palm leaves to reduce wind erosion and

⁎

sand movement maintaining the soil and improving vegetative cover (Mondragón-Jacobo and Pérez-González, 2001). Opuntia genus is an Eudicot Angiosperm and is a member of the succulent plant family Cactaceae (Pottier-Alapetite, 1979; Shetty et al., 2012). It includes 250 species according to Britton and Rose (1963), 181 according to Anderson (2001) and 195 according to Hunt and Taylor (2002). This genus belongs to one of the most popular, easily recognizable, and morphologically distinct families of plants that include approximately 130 genus with 1600 species according to Gibson and Nobel (1986) and Barthlott and Hunt (1993). According to Anderson (2002) and Peña-Valdivia et al. (2008), Opuntia genus includes approximately 181 species. It has been introduced in the Mediterranean basin in the 15th century (Donkin, 1977; Scalisi et al., 2016) and now naturalizes in other countries due to its adaptability (Chougui et al., 2016), its extraordinary capacity to stock up water (Scalisi et al.,

Corresponding author. E-mail address: [email protected] (B. Ghada).

https://doi.org/10.1016/j.scienta.2019.01.004 Received 19 October 2018; Received in revised form 31 December 2018; Accepted 7 January 2019 0304-4238/ © 2019 Elsevier B.V. All rights reserved.

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Fig. 1. Origin of Opuntia species studied.

2016) and to grow in dry and even desert conditions (Chalak et al., 2014). In Tunisia, this species is considered as an important fodder crop mainly for sheep during seasons of low feed availability in Central and Southern regions of the country (Zoghlami et al., 2007). In fact, after harvesting, chopping pads into small pieces it given directly to animals as an energy supplement of low-quality stubble (Alary et al., 2007). Appreciate to its considerable number of uses; cactus has immense potential to be the food of future (Shetty et al., 2012). Over the world, it is mainly used for fruit production (Nobel et al., 1992) added to the young tender spring vegetative growth of wild cactus (nopalitos) that has been extensively consumed by Hispanics during Lent (Russell and Felker, 1987; Shetty et al., 2012). In addition, it has a growing interest in nonfood uses especially in medical applications (Malainine et al., 2003) and in cosmetic industry (Sáenz and Berger, 2006; Hadj Sadok et al., 2009) for several industrial purposes such as jam and spirit production (Vigueras and Portillo, 2001). The taxonomic classification of the Opuntia genus (Cactaceae, Opuntioidae) has been reported as complex, which explains the existence of many reports of misclassification of Opuntia species (Samah et al., 2016). In fact, continuous morphological variation, and limited morphological descriptors for cultivar discrimination are the most difficult obstacles to achieve a stable classification (Labra et al., 2003; Caruso et al., 2010; Valadez-Moctezuma et al., 2014; Samah et al., 2016). Thus, the difficulties in morphological interpretation have led to publication of a large number of binomials, many of which are synonyms, homonyms, or false attributions (Gibson and Nobel, 1986). In Tunisia, the superficies, the impact of cactus on natural resource preservation and on economy has not been well documented since some years. In 2000, Nefzaoui and Ben Salem (2000) have mentioned that an area of 700.000–1 million ha have been planted cactus in the last 50 years in North Africa. Inglese et al., 2001 have mentioned also that

recently, large plantations have been established near the region of Kasserine. Bayar et al., 2018 mentioned that actually Opuntia ficus-indica is largely distributed and it occupies extended areas throughout Tunisia estimated at 600 000 ha according to (DGACTA, 2015). Despite its important role, Barbary fig is menaced by drastic erosion due to biotic and abiotic stresses particularly and intensive urbanization (Bendhifi et al., 2013). Hence, it is imperative to elaborate a research program aiming the identification of the Tunisian germplasm in order to preserve it especially by setting up rational decisions concerning the establishment of collections of Barbary fig to conserve the biodiversity existing. The INRAT collection is the first Barbary fig’ collection established in Tunisia, so it is recommended to study genetic diversity among collected ecotypes. The aim of this research was to study the morphology of cladodes, flowers and fruits from four cactus Opuntia species, in order to start a program of establishing a catalogue collecting all varieties and to conserve our genetic resources. For this object, in the herein study, we analyzed the diversity observed among 48 spineless Opuntia ecotypes originated from 10 countries over the world based on 63 morphological traits. 2. Material and methods 2.1. Sampling A set of forty-eight spineless Opuntia ecotypes belonging to four species of Opuntia genus: Opuntia ficus-indica, Opuntia engelmannii, Opuntia tomentosa and Opuntia undulate were collected from the collection established by ‘the National Institute of Agronomic Research of Tunisia (INRAT)’ and installed in Bourbia from Zaghouan governorate in Northern Tunisia (Fig. 1). These different ecotypes have originated from ten countries over the word such as Tunisia, Italy (Sicily), 164

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Table 1 Opuntia ecotypes used for morphological characterization. Code

ecotype

species

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

B1C1 B1C2 B1C3 B1C4 B1C5 B1C6 B1C7 B1C8 B1C9 B1C10 B2C1 B2C2 B2C3 B2C4 B2C5 B2C6 B2C7 B2C8 B2C9 B2C10 B3C1 B3C2 B3C3 B3C4 B3C5 B3C6 B3C7 B3C8 B3C9 B3C10 B4C1 B4C2 B4C3 B4C4 B4C5 B4C6 B4C7 B4C8 B4C9 B4C10 B5C1 B5C2 B5C3 B5C4 B5C5 B5C6 B5C7 B5C8

Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia Opuntia

ficus-indica ficus-indica ficus-indica ficus-indica tomentosa ficus-indica ficus-indica ficus-indica ficus-indica ficus-indica ficus-indica ficus-indica ficus-indica ficus-indica ficus-indica ficus-indica ficus-indica ficus-indica ficus-indica ficus-indica ficus-indica ficus-indica ficus-indica ficus-indica ficus-indica ficus-indica ficus-indica engelmannii ficus-indica ficus-indica ficus-indica ficus-indica ficus-indica ficus-indica ficus-indica ficus-indica ficus-indica ficus-indica ficus-indica ficus-indica ficus-indica ficus-indica ficus-indica undulata ficus-indica ficus-indica ficus-indica ficus-indica

Table 2 Morphological traits studied. Origin

Code

Tunisia Tunisia Tunisia Beja Tunisia Sbeitla Algeria Tunisia INRAT Tunisia Tunis INRAT Tunisia Tunisia Tunisia Tunisia Tunisia Tunisia Tunisia Tunisia Tunisia Tunisia Morocco Morocco Algeria Algeria Algeria Algeria Algeria Algeria Algeria Algeria New Mexico New Mexico South Africa South Africa Mexico Mexico Morocco Morocco Morocco Morocco Argentina Argentina France (Sarson) France Italy (Sicily) Italy (Sicily) Italy (Sicily) Ethiopia

Tree Ht Fl Frct Fqb/a Nf/r Psf Fqf/a Fqfr/a Cladode Rt/Rpt PFR Lgr lgr Epbr Dibr Eppr Nma/r Nma/b Dsma Dima Lgr/lgr Fr Cr Epn Tr Dg Flowers Np/f Lgpf lgpf Lgpf/lgpf Ns/f Dicf Lgrf Lgpif Ff Tf Cpf Fpf Caf Cpif Cfif Cstf Fruits Nfr/r Pfr Lgfr Difr Dicvfr Na/cvfr Ngrfr P100gr Ffr Tfr Cmfr Cfr Cpufr Cchfr Fcvfr Cgfr Fqgr/fr Tgrfr Fgrfr Mucifr Gfr Chtfr

Argentina, Algeria, Morocco, Ethiopia, France, Mexico, USA (New Mexico) and South Africa (Fig. 1). Plant material used for morphological characterization is considered of five mature cladodes, five flowers and five mature fruits sampled from different orientations of each ecotype. The main characteristics (code, ecotype, species and origin) for the 48 considered ecotypes are summarized in Table 1. 2.2. Morphological descriptors-measurements Forty-eight representative old ecotypes (planted since 2004 by INRAT) were chosen to measure sixty-three morphological descriptors related to trees, cladodes, flowers and fruits. For each ecotype, 30 quantitative and 33 qualitative traits were examined to characterize morphologically the four studied species of Opuntia (Table 2). Data were obtained from March and August 2017.

descriptor Plant height Flowering Fructification Burgeon frequency/tree flowers mean number/cladode Flowers position on cladodes Flowers frequency/tree Fruits frequency/tree Length cladode(2015)/length cladode(2016) cladode fresh weight Length cladode Width cladode Thickness at cladode base Diameter cladode Thickness at cladode tip areoles mean number/cladode areoles mean number at the cladode border Mean distance between areoles Areole mean diameter Length cladode/width cladode Cladode shape Cladode color Spines (present/absent) Cladode size Glochides density Petal mean number Petal mean length Petal mean width Petal mean length/ Petal mean width Sepal mean number Mean diameter flower Floral receptacle mean length Pistil mean length Flower shape Flower size Petal color Petal shape Anther color Pistil color Filaments color Stigma color Fruits mean number/cladode Fruit mean weight Fruit mean length Fruit mean diameter Recepticular scar mean diameter Areoles mean number/recepticular scar Seed mean number 100 seeds mean weight Fruit shape Fruit size Fruit edibility Fruit color Pulp color Peel color Recepticular scar position Glochides color Seed frequency/fruit Seed size Seed shape Mucilage in fruits (present/absent) Fruit taste Fruit fall

transported to the laboratory and treated within 24 h. The samples were brushed cautiously to remove the maximum of glochides. They were then weighed before being peeled. The edible portion of each fruit was crushed and was filtered through a sieve to separate the seeds from the

2.3. Determination of seeds number Each sample was made up of five fruits, which were collected from several plants in the same sampling ecotype. The fruits harvested were 165

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Fig. 2. Morphological variabilityof different Organ of Opuntia speciesstudied. a: Opuntiacladodes diversity; a1: (B5C2) OFI, a2: (B2C8) OFI, a3: (B5C4) OUD, a4:(B5C8) OFI, a5:(B1C5) OTM, a6:(B3C6) OFI, a7: (B3C7) OFI. b: Opuntia flowers diversity; b1: (B1C6) OFI,b2: (B1C10) OFI,b3: (B5C4)OUD,b4: (B1C5) OTM,b5: (B5C2) OFI. c: Opuntia fruits diversity; c1: (B3C5) OFI,c2: (B5C4) OUD,c3: (B3C9) OFI,c4: (B5C8) OFI. (OFI: Opuntia ficus-indica, OTM: Opuntia tomentosa, OUD: Opuntia undulata).

3.1. Quantitative morphological traits variability

juicy pulp. Once the seeds have been recovered, they were placed on an absorbent paper and exposed to full sunlight to completely dry, this step may take a few hours. Then, the dry seeds are weighed to determine 100 seeds mean weight of each ecotype (P100gr).

In the studied collection, seventeen cladode characteristics were evaluated (Table 2). Two ecotypes were typified by very large cladodes with an oblong curved shape (B5C2 and B5C4) belonging to Opuntia ficus-indica and Opuntia undulata respectively with a mean width of 24.8 cm and the lowest mean number of areoles per cladodes equal to 67 and 92 areoles respectively. These two ecotypes had areoles with mean diameter of 2 mm. The smaller characterized one was the only representative ecotype of Opuntia tomentosa (B1C5) not exceeding 0.29 kg with 23.4 cm of length and 10.4 cm of width and having a velvety texture. The biggest flowers were observed among (B5C4) with the longest floral receptacle (6.73 cm) compared to B5C1 having the smallest flowers with a floral receptacle of 2.96 cm. Concerning the weight of harvested fruits varied considerably; an interval of 9.4–99.6 g was recorded. The longest fruit was (B5C4) (Opuntia undulata) followed by (B5C3) belonging to Opuntia ficus-indica, while, the smallest ones were represented by B5C1 of Opuntia tomentosa.

2.4. Data analysis Morphological diversity of Opuntia ecotypes in INRAT collection was estimated using several statistical procedures in order to elucidate relationships between ecotypes. Data were subdivided into two categories (quantitative and qualitative variables). Based on quantitative data, a Hierarchical Cluster Analysis (HCA) using Euclidean distance of Bray & Curtis following the Ward’s method and Principal Component Analysis (PCA) were performed using the XLSTAT software version 2014.5.03. In addition, analysis of the variance (ANOVA) was carried using the IBM SPSS Statistics software version 20 (Nie et al., 2011). For the qualitative variables; a HCA using chi-square distance and Multiple Correspondence analysis (MCA) were constructed using the XLSTAT software version 2014.5.03. In addition, Z test, Kruskal-Wallis test and chi-square analysis were performed using STATISTICA (data analysis software system) version 8.0 (StatSoft, Inc., 2007), being the 5% the adopted significance level.

3.2. Qualitative morphological traits variability Results indicate that most of the accessions had a semi upright habit and a round shape only three accessions had a flat shape and three other accessions were found with upright habit and elongated shape. Variability was noticed for the cladodes characteristics among the 48 studied cultivars. The prickly pear can be distinguished according to the presence or absence of spines and glochids, color and shape. All the Opuntia ecotypes studied were spineless. Glochids were present in low density on the cladodes of all ecotypes while they were present in high

3. Results The morphological study shows very important and significant differences between samples. We were interested in 63 parameters relative to trees, cladodes, flowers and fruits (Table 2).

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Fig. 3. Dendrogram constructed by Euclidean distance using Ward's method, to study the relationships among the 48 Opuntia cultivars based on 30 quantitative traits.

density in B5C8 ecotype belonging to Opuntia ficus-indica and native to Ethiopia. Cladodes color was almost similar among ecotypes (Fig. 2). All cultivars had green cladodes, only B5C2, B5C8 (Opuntia ficus-indica) and B5C4 (Opuntia undulata) had light green and the only representative cultivar of Opuntia tomentosa (B1C5) having dark green cladodes. Six shades of flower color was detected in the collection; yellow, light yellow, orange, light orange, red-pink and red-orange (Fig. 2). B1C5 ecotype belonging to Opuntia tomentosa has the smaller flowers comparing to the others ecotypes and was characterized by the red-pink color very attractive. In addition, we have detected a special flowers having light-yellow color corresponding to B5C4 ecotype belonging to Opuntia undulata. All others cultivars of Opuntia ficus-indica and the only cultivar of Opuntia engelmannii have similar flowers very difficult to differentiate between them. The color of ecotypes fruits varied from green to purple (Fig. 2). We have distinguished six shades

of color: green, yellow, greenish-yellow, orange, red and purple. (B5C4), belonging to Opuntia undulata, is the only ecotype having purple fruits. Fruits shape observed were ovoid, elongated, round or Funnel shaped and the recepticular scar position in fruits may be either flat or sunken. 3.3. Quantitative morphological traits analysis In order to study the morphological diversity between the 48 ecotypes of Opuntia basing of 30 quantitative parameters, the HCA, PCA and ANOVA test were performed. 3.3.1. Hierarchical clustering analysis (HCA) The dendrogram constructed via the Hierarchical Ascending Classification allowed as identifying three major groups (Fig. 3). Group

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Table 3 ANOVA analysis (Species). Code PFR

Cladode freshweight

lgr

Width cladode

Dibr

Diameter

Nma/r

Areolesmeannumber/cladode

Dsma

Mean distance between areoles

Lgrf

Floral receptacle mean length

Lgpif

Pistil mean length

Pfr

Fruit mean weight

Lgfr

Fruit mean length

Difr

Fruits mean diameter

Dicvfr

Recepticular scar mean diameter

Na/cvfr

Areoles mean number/recepticular scar

Ngrfr

Seed mean number

P100gr

100 seeds mean weigth

Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total

Sum of squares

ddl

Square average

Test F

p < 0.05

0.462 1.608 2.070 105.533 254.105 359.638 1.181 5.868 7.049 4666.238 22600.572 27266.810 2.739 9.592 12.331 7.886 7.080 14.966 0.749 1.069 1.818 4242.213 12954.872 17197.085 16.006 21.828 37.834 5.033 13.209 18.242 0.350 1.666 2.016 62.022 236.978 299.000 54234.139 162907.778 217141.917 6.336 4.867 11.203

3 44 47 3 44 47 3 44 47 3 44 47 3 44 47 3 44 47 3 44 47 3 44 47 3 44 47 3 44 47 3 44 47 3 44 47 3 44 47 3 44 47

0.154 0.037

4.211

0.011*

35.178 5.775

6.091

0.001*

0.394 0.133

2.952

0.043*

1555.413 513.649

3.028

0.039*

0.913 0.218

4.188

0.011*

2.629 0.161

16.337

0.000*

0.250 0.024

10.270

0.000*

1414.071 294.429

4.803

0.006*

5.335 0.496

10.754

0.000*

1.678 0.300

5.589

0.002*

0.117 0.038

3.081

0.037*

20.674 5.386

3.839

0.016*

18078.046 3702.449

4.883

0.005*

2.112 0.111

19.094

0.000*

(I) included ecotypes with consists of the Algerian ecotype (B1C5) belonging to Opuntia tomentosa, the French ecotype (B5C4) of Opuntia undulata and the two ecotypes of Opuntia ficus-indica (B5C2) (B5C8) originating to Argentina and Ethiopia respectively. The second group noted II consists only of Opuntia ficus-indica ecotypes native of seven countries, namely; Tunisia (B1C7, B1C10, B2C4, B2C9 and B2C10), Algeria (B3C4, B3C5, B3C6 and B3C10), Morocco (B4C10), Mexico (B4C6), South Africa (B4C4), Italy (Sicily) (B5C7) and USA (New Mexico) (B4C1 and B4C2). The third group (III) contains a large number of ecotypes belonging to eight countries. It consists of the majority of Tunisian (B1C1, B1C2, B1C3, B1C4, B1C6, B1C8, B1C9, B2C1, B2C2, B2C3, B2C5, B2C6, B2C7 and B2C8) and Moroccan (B3C1, B3C2, B4C7, B4C8 and B4C9) Opuntia ficus-indica ecotypes, in addition to 3 Algerian ecotypes (B3C7, B3C8, and B3C9), 2 Sicilian ecotypes (B5C5 and B5C6), 1 French ecotype (B5C3), 1 Mexican ecotype (B4C5), 1 South African ecotype (B4C3) and 1 cultivar native to Argentina (B5C1).

3.3.2. Analysis of the variance (ANOVA) The analysis of the variance among the 30 quantitative variables for all the data was carried out. Mean values obtained for the variables studied in the different ecotypes were compared by one-way analysis of variance ANOVA, assuming that there were significant differences among them when the statistical comparison gives p < 0.05. Depending on species (Table 3), 14 discriminator parameters were observed (PFR, lgr, Dibr, Nma/r, Dsma, Lgrf, Lgpif, Pfr, Lgfr, Difr, Dicvfr, Na/cvfr, Ngrfr and P100gr). Furthermore, 22 discriminator traits were notated through ANOVA depending on ecotypes origins (Ht, PFR, Lgr, lgr, Lgr/lgr, Epbr, Dibr, Eppr, Nma/r, Nma/b, Dsma, Dima, Nf/r, Np/f, Lgpf, lgpf, Lgpf/lgpf, Dicf, Lgrf, Lgpif, Nfr/r and Na/cvfr) (Table 4). Results suggested by ANOVA analysis reflects a very substantial number of significant quantitative parameters used in this study. 3.3.3. Principal component analysis (PCA) We have performed a PCA analysis taking into account the 30 quantitative traits (PCA1). Result of PCA analysis shows that 98.79% of

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Table 4 ANOVA analysis (Origins). Code Ht

Plant height

PFR

Cladode freshweight

Lgr

Length cladode

lgr

Width cladode

Lgr/lgr

Length cladode/ Width cladode

Epbr

Thickness 1

Dibr

Diameter

Eppr

Thickness 2

Nma/r

Areolesmeannumber/cladode

Nma/b

Areoles mean number at the cladode border

Dsma

Mean distance between areoles

Dima

Areole mean diameter

Nf/r

Flowers mean number/cladode

Np/f

Petal mean number

Lgpf

Petal mean length

lgpf

Petal mean width

Lgpf/lgpf

Petal mean length/petal mean width

Dicf

Mean diameter flower

Lgrf

Floral receptacle mean length

Lgpif

Pistil mean length

Nfr/r

Fruits mean number/cladode

Na/cvfr

Areoles mean number/recepticular scar

Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total Inter-groups Intra-groups Total

169

Sum of squares

ddl

Square average

Test F

p < 0.05

2.660 3.813 6.473 0.816 1.254 2.070 401.668 307.132 708.800 215.002 144.635 359.638 1.389 2.401 3.790 5.926 4.394 10.321 3.083 3.966 7.049 3.010 3.688 6.698 18559.874 8706.936 27266.810 5638.715 2183.391 7822.106 7.902 4.430 12.331 14.793 8.092 22.886 221.485 316.827 538.313 73.681 97.194 170.875 3.573 1.629 5.202 1.131 0.703 1.834 1.024 0.953 1.978 0.423 0.443 0.867 5.843 9.123 14.966 0.837 0.981 1.818 221.485 316.827 538.313 154.839 144.161 299.000

10 37 47 10 37 47 10 37 47 10 37 47 10 37 47 10 37 47 10 37 47 10 37 47 10 37 47 10 37 47 10 37 47 10 37 47 10 37 47 10 37 47 10 37 47 10 37 47 10 37 47 10 37 47 10 37 47 10 37 47 10 37 47 10 37 47

0.266 0.103

2.581

0.018*

0.082 0.034

2.405

0.026*

40.167 8.301

4.839

0.000*

21.500 3.909

5.500

0.000*

0.139 0.065

2.141

0.046*

0.593 0.119

4.990

0.000*

0.308 0.107

2.877

0.009*

0.301 0.100

3.020

0.007*

1855.987 235.323

7.887

0.000*

563.872 59.011

9.555

0.000*

0.790 0.120

6.600

0.000*

1.479 0.219

6.764

0.000*

22.149 8.563

2.587

0.017*

7.368 2.627

2.805

0.011*

0.357 0.044

8.115

0.000*

0.113 0.019

5.953

0.000*

0.102 0.026

3.976

0.001*

0.042 0.012

3.533

0.002*

0.584 0.247

2.370

0.028*

0.084 0.027

3.156

0.005*

22.149 8.563

2.587

0.017*

15.484 3.896

3.974

0.001*

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Fig. 4. (A): PCA1 plot of 48 Opuntia cultivars based on the first two principal axes accounting for 96.66% of the total genetic variation (first axis = 87.11% and second = 9.89% of total variation) based on 30 quantitative variables.(B): PCA2 projection of the discriminant fourteen variables according to ANOVA analysis on the factor plan (1–2) (97.97% of the total genetic variation).

the total variation was explained by the first three principal components, which accounted for 87.11%, 9.89% and 1.79% respectively of the observed variation. Four traits (Ngrfr), (Nma/r), (Nma/b) and (Pfr) found to explain the variation in the PCA (Fig. 4A). The fourteen morphological parameters found to be discriminant according to ANOVA analysis depending on species was used to conduct a second PCA in order to improve ecotypes distribution (Fig. 4B). This second PCA shows that 99.80% of the total variation was explained by the first three principal components. The axes 1 and 2 represent 97.97% of the total variations with 89.59% and 8.38% respectively. The most important traits found to explain the variation, in the PCA2: Ngrfr and Pfr were associated with PC1. On the other hand, Nma/r was associated with PC2. Projection of ecotypes in the PCA 1–2 plan shows the presence of two major groups. The first group noted (I) consists of two ecotypes of Opuntia ficus-indica (B5C2) and (B5C8) originating from Argentina and Ethiopia respectively, the Algerian ecotype (B1C5) of Opuntia tomentosa and the French ecotype (B5C4) belonging to the species Opuntia undulata. Group (I) bring together all ecotypes characterized by the lowest numbers of seeds per fruit (Ngrfr) and the lowest number of areoles per cladodes (Nma/r). The second group (II) gathers all Tunisian, Algerian and Moroccan ecotypes as well as other ecotypes native to Argentina, Italy, France, Mexico, Italy (Sicily), USA (New Mexico) and South Africa belonging to the species Opuntia ficusindica as well as the ecotype of the species Opuntia engelmannii (B3C8). In this analysis, all cultivars appeared dispersed in the PCA plot without clear aggregations.

3.4.1. Hierarchical clustering analysis (HCA) and multiple correspondence analysis (MCA) The dendrogram constructed using only qualitative paramaters based on Chi-square distance show a similar topology with dendrogram constructed by quantitative traits (Fig. 5). The MCA (Fig. 6) show the presence of two distinct groups, comprising respectively 44 (Group I) and 4 (Group II) ecotypes. The first group is the same group compared to PCA 1 (Fig. 4) and Ward’s dendrogram (Figs. 3 and 5), that still regrouping the four most morphologically distinctive ecotypes of the collection; the Algerian ecotype (B1C5) with velvety texture and belonging to Opuntia tomentosa species, the two ecotypes (B5C2) (Argentina) and (B5C4) (France) belonging respectively to Opuntia ficusindica and Opuntia undulata. Both of these cultivars are characterized by curved oblong cladodes. The Ethiopian ecotype (B5C8) is the fourth ecotype constituting group I belonging to Opuntia ficus-indica. The second group brings together all the rest of ecotypes. 3.4.2. Statistical analysis based on qualitative parameters The estimation of phenotypic variation among ecotypes was obtained after sorting variables into ordinal and binomial data. Results were given as the mean ± SD of the five replicate. Data was examined by normality test (Kolmogorov Smirnov test). Non parametric analysis followed by Kruskal-Wallis test (5%) were done to define the significance between the qualitative parameters (ordinal data) (Table 5). In addition to this, statistical comparison were performed by means of the chi-square (χ2) test (Table 5). Binomial variables were compared using Z test, simple correlation was performed using Spearman’s coefficient (Table 5). For all tests, the significance level was set at 0.05. Reliability tests were performed between observers and shows the nonsignificant values obtained by means of these tests based on ordinal and binomial data separately. Qualitative parameters only in this study may not be sufficient to characterize the Opuntia ecotypes in INRAT collection. Therefore, it could be necessary to only refer to quantitative parameters that were more informative.

3.4. Qualitative morphological traits analysis In order to study the morphological variability between the 48 ecotypes of Opuntia, the HCA, MCA and Kruskal-Wallis test, Z test and chi-square analysis were carried out on 33 ordinal and binomial variables.

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Fig. 5. Dendrogram constructed by Chi-square distance using Ward's method, to study the relationships among the 48 Opuntia cultivars based on 33 qualitative traits.

3.5. Taxonomic key based on morphological traits

the species “Opuntia tomentosa”. Furthermore, Opuntia undulata seems to be one of the species having a lot of apomorphic characters; the light green color of cladodes, light yellow flowers and the purple fruits observed only in this species. All species are caracterized by synapomorphic characters that are a lot of traits very specific related to the Opuntia genus; presence of cladodes instead of stems, hermaphrodite flowers and fruits having crown covered by a great number of areoles and glochids. B5C2 and B5C8 are two ecotypes belonging to Opuntia ficus-indica that seems to be excluded from Opuntia ficus-indica species

Results revealed in the present study allowed us to build a taxonomic key (Fig. 7) using the principal disciminant traits in PCA and ANOVA. This key based on apomorphic and synapomorphic characters illustres the specific morphological characters of each species and shows the most discriminant traits allowing separating between the four studied species. Dark green cladodes with velvety texture and small red-pink flowers are the important apomorphic traits found for

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Fig. 6. Multiple Correspondence Analysis (MCA) based on qualitative parameters (60.83% of total variability).

because of their difference from the other ecotypes belonging to the same species. All apormorphic and synapomorphic characters illustrated in this taxonomic key can be very useful for genetic breeding and species characterization (Fig. 7).

According to the results obtained on morphological characteristics (quantitative and qualitative traits), significant differences were noticed between the samples. Clearly, from dendrograms (Figs. 3 and 5) PCA, MCA (Figs. 4 and 6) results, we suggest that Group (I) is distinctly different of both Group (II) and (III). It included three different Opuntia species presented by the four ecotypes most differentiated morphologically in the collection: (Opuntia undulate (B5C4), Opuntia tomentosa (B5C1) and Opuntia ficus-indica (B5C2) (B5C8)). The weight of harvested fruits in this group (Group (I)) varied (considerably). An interval of 9.4 to 99.6 g was recorded; the biggest fruit was detected in (B5C4) (Opuntia undulata) and the smallest ones were represented by (B5C1) (Opuntia tomentosa) and (B5C8) (Opuntia ficus-indica). These last two ecotypes presented the lowest number of seeds (Ngrfr) with the presence of mucilage accompanied by a very low number of areoles (Nma/ r and Nma/b). Ecotype (B5C2) belonging to Opuntia ficus-indica have very similar cladodes to Opuntia undulata; this may pose some ambiguities regarding classification because it cannot be caused by environmental modifications related to origins. B5C1 and B5C8 are both belonging to Opuntia ficus-indica and native to Argentina and Ethiopia

4. Discussion In our study, separation of all ecotypes was morphologically possible with some qualitative variables related to cladodes, flowers and fruits. Cladode dimensions as well as cladode texture added to fruit size and flowers colors were found to be significant to differentiate between species studied (Fig. 2). A similar study conducted by Gallegos-Vázquez et al., (2012) found that qualitative variables such as flesh and peel color proved useful to distinguish among potentially the twenty-one redundant accessions collected from the Hidalgo and Zacatecas states of Mexico. Gutierrez-Acosta et al., (2002) and Valdes-Zepeda et al., (2003) mentioned also that some other qualitative traits such as fruit dimensions are useful in the separation of cactus pear accessions (GallegosVázquezet al., 2012).

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Opuntia ficus-indica. HCA analyses (Figs. 3 and 5) show that the 48 cultivars distribution occurs undependably of their geographic origin. These results are similar to those obtained by Bendhifi et al. (2013) on 28 accessions of Opuntia ficus-indica collected from 19 sites in south western and central Tunisia. This is also proved by the same author (Zarroug et al., 2015)) by analyse 25 Cactus cultivars belonging to 7 different species of Opuntia (Opuntia ficus-indica, Opuntia engelmannii, Opuntia tomentosa, Opuntia undulata, Opuntia ellisiana, Opuntia streptacantha and Opuntia robusta) from 11 countries, namely; Tunisia, Algeria, Morocco, Italy (Sicily), Mexico, USA (New Mexico), France, Texas, South Africa, Argentina and Ethiopia.

Table 5 Chi-square, Kruskal-Wallis using ordinal variables and Z-test using binomial variables analysis of the qualitative parameters of Opuntia ecotypes.

Tr Fr Cr Dg Fl Ff Tf Cpf Fpf Caf Cpif Cstf Cfif Fqb/a Frct Fqf/a Fqfr/a Ffr Tfr Cfr Gfr Cpufr Cchfr Cgfr Fqgr/fr Tgrfr Fgrfr Psf Fcvfr Epn Cmfr Mucifr Chtfr

ML-Chi-square

df

p

39.031 77.184 32.034 105.408 84.780 9.721 45.621 78.543 16.627 – 108.275 61.603 124.827 96.693 39.879 96.693 96.692 48.306 38.807 136.899 92.596 172.608 180.089 9.721 86.344 67.480 58.634 22.444 46.327 Z-test

94 235 94 141 94 47 94 235 47 – 235 141 188 94 47 94 94 141 94 235 141 376 423 41 94 94 141 47 47

0.423 0.397 0.423 0.414 0.423 0.432 0.423 0.397 0.432 – 0.397 0.414 0.405 0.423 0.432 0.404 0.423 0.414 0.413 0.397 0.414 0.377 0.370 0.413 0.423 0.400 0.414 0.423 0.430 p 0.250 0.750 0.750 0.250

5. Conclusion Morphological characters are important in the botanical description of Cactus pear. According to the analysis performed, many descriptors have shown an effective discriminating capacity. Four quantitative descriptors (the mean number of seeds per fruit, the mean number of areolas per cladode, the mean number of areolas at the cladode border and the fruit mean weight) and three qualitative descriptors (cladode shape, pulp color and fruit color) have been identified as differential parameters. Those parameters allowed us to distinguish easily between the four studied species (Opuntia ficus-indica, O.engelmannii, O.undulata and O.tomentosa). Low genetic variability associated with relatively low morphological divergence has been found between Opuntia ficus indica and Opuntia engelmannii suggesting this may be typical domestication resulting in low levels of genetic differentiation. The morphological characterization of Opuntia species is indispensable and helpes to highlight a fairly high level of diversity and estimate phylogenetic relationships. Finally, based on the morphological characterization of prickly pear ecotypes, this study is a first step in a more complete conservation and improvement plan for Opuntia species in Tunisia. These results suggest that both molecular and morphological data should be used in conservation planning.

respectively, but they are so different especially regarding cladode and fruit shape. From our result, it is also noted that Opuntia engelmannii seems to be very close to the ecotypes of Opuntia ficus-indica due to the very high similarity especially regarding cladode shape and fruits color. Indeed, the work of Zarroug et al., (2015) proved the existence of a genetic approximation between the Tunisian accessions of these two species (Opuntia ficus-indica and Opuntia engelmannii). In addition, the work of Snoussi Trifa et al. (2007) suggested that Opuntia engelmannii can no longer be considered a valid species but must be a domesticated form of

Acknowledgements This work was supported by grants from the Tunisian “Ministère de l’Enseignement Supérieuret de la Recherche Scientifique’’; ED STVST of El Manar University from Tunis, Tunisia. We are also thankful to Mr. Hichem Ben Salem, responsible of international Opuntia collection established in INRAT institute.

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Fig. 7. Taxonomic Key of fourOpuntia speciesstudied using the most discriminant morphological traits in PCA and ANOVA.OFI (Opuntia ficus-indica); OUD (Opuntia undulata); OEG (Opuntia engelmannii); OTM (Opuntia tomentosa).

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