Fruit Morphological Descriptors As a Tool for Discrimination of Daucus L. Germplasm

Genet Resour Crop Evol (2014) 61:499–510 DOI 10.1007/s10722-013-0053-6

RESEARCH ARTICLE

Fruit morphological descriptors as a tool for discrimination of Daucus L. germplasm

Najla Mezghani • Imen Zaouali • Wided Bel Amri • Slim Rouz • Philipp W. Simon • Che´rif Hannachi • Zeineb Ghrabi • Mohamed Neffati • Be´chir Bouzbida • David M. Spooner

Received: 22 July 2013 / Accepted: 1 October 2013 / Published online: 18 October 2013 Ó Springer Science+Business Media Dordrecht (outside the USA) 2013

Abstract Fruits present major morphological char- genetic diversity within the collection. Multivariate acters used to define genera and species within the analysis of factorial correspondence and cluster ana- Apiaceae. Northern Africa represents a major center of lysis on morphological descriptors permitted the diversity of Daucus, with Tunisia containing at least 12 subdivision of the Daucus collection into five distinct species and six subspecies. We assessed 14 mature fruit groups including one single accession group, two characters from the Daucus L. germplasm collection at groups with six accessions, one group of nine acces- the National Gene Bank of Tunisia. Quantification of sions and one large group with 81 accessions corre- variability for each character was investigated using sponding each one to a species among Daucus except the standardized Shannon–Weaver diversity index the large group corresponding to D. carota and D. (H0). Diversity was established by factorial analysis capillifolius. The grouping of populations did not of correspondence and cluster analysis. The computing reflect bioclimatic and geographic patterns, suggesting H0 index ranged from 0.31 for stylopodium shape to a adaptation of populations to local environments. Of maximum of 0.81 for spine shape. A mean diversity equal importance, our study shows the effectiveness of index for all traits recorded across all populations fruit characters alone to identify species in this averaged 0.58 indicating existence of an important collection of Daucus.

N. Mezghani (&) I. Zaouali W. Bel Amri C. Hannachi Banque Nationale de Ge`nes, Boulevard du Leader Yasser Institut Supe´rieur Agronomique de Chott Mariem, Chott Arafat Z. I Charguia 1, 1080 Tunis, Tunisia Mariem, Tunisia e-mail: [email protected] Z. Ghrabi W. Bel Amri Institut National Agronomique de Tunisie, Tunis, Tunisia Faculte´ des Sciences de Tunis, Tunis, Tunisia M. Neffati B. Bouzbida S. Rouz Institut des Re´gions Arides, 4119 Medenine, Tunisia Institut Supe´rieur Agronomique de Mogran, Mogran, Tunisia

P. W. Simon D. M. Spooner (&) USDA-ARS, Vegetable Crops Research Unit, Department of Horticulture, University of Wisconsin, 1575 Linden Drive, Madison, WI 53706-1590, USA e-mail: [email protected] 123 500 Genet Resour Crop Evol (2014) 61:499–510

Keywords Daucus Diversity Multivariate statistical analysis of several vigor descriptors related analysis Species Tunisia to fruit. It aimed to elucidate the interrelationship between the conserved accessions and to verify the suitability of morphological characterization for species identification in our collection. The generated Introduction information will be helpful to curators in the management and manipulation of the conserved germplasm. Daucus carota L. is a morphologically diverse species found in wild or feral form throughout the Mediter- ranean, southwest Asia, Africa, Australia, New Zea- Materials and methods land and the Americas (Peterson and Simon 1986; Vaughan and Geissler 2009). The gene centers for the Plant material species include Asia Minor, Transcaucasia, Iran, Turkmenistan, northwest India, Afghanistan, Tadjiki- One hundred and three accessions of Daucus con- stan, Uzbekistan and western Tian-shan mountain served at the National Gene Bank of Tunisia were used system of central Asia (Bradeen et al. 2002). Tunisia is in this study. They consisted of seven landraces and 96 considered a center of biodiversity for Daucus and wild populations of Daucus collected in August 2008 many other crops and contains a diversity of ecosys- and 2009 from road and field sites in Tunisia that tems and climatic conditions (Pottier Alapetite 1979; covered central, northern, southern, western, and Le Floc’h et al. 2010). coastal regions. Based on morphology of umbels and The latest taxonomic monograph of Daucus by fruits, taxonomic designations were made in the field Saénz Laıń(1981) recognized 20 species. Pottier during the collecting expedition. Passport data Alapetite (1979) recognized 11 Daucus species in included accession number, collection date, location, Tunisia with several subspecies, to which we add D. collector names, site name, and precise coordinates as capillifolius Gilli (described from western Libya, but obtained by a geographic positioning system device, found by us in adjacent eastern Tunisia). The basic bioclimatic zone, habit, and ecology. The collection, chromosome number for Daucus ranges from n = 9to geographic and bioclimatic registration information is n = 11. There are only four n = 9 chromosome shown in Table 1. species of Daucus: D. carota (all subspecies), D. capillifolius, D. syrticus Murb. and D. sahariensis Murb. (Grzebelus et al. 2011). The n = 9 chromo- Morphological characterization some species are clearly interrelated based on molecular data (Iorizzo et al. 2013; Spooner et al. 2013a) Daucus accessions were examined for three quantita- shared karyotypes (Iovene et al. 2008) and crossability tive and 11 qualitative traits related to fruit (Table 2). data (McCollum 1975, 1977). The selection of characters (descriptors) was made Daucus has been studied by a variety of DNA following the International Board for Plant Genetic sequence techniques (Spalik and Downie 2007; Resources guidelines for wild and cultivated carrot (D. Spooner et al. 2013a). However, knowledge of the carota) descriptors (IPGRI 1998). Some additional phenotype via morphological and agronomical characters were also taken into account according to descriptors is critically important for practical identi- description given by Pottier Alapetite (1979) and fications and traits of use for plant breeders (Sudre´ Saénz Laıń(1981) for taxonomic classification of et al. 2010). There are limited studies on morpholog- Daucus species. ical characterization of Daucus, but all of these Data were recorded under a stereoscopic micro- focused on variation within D. carota (Small 1978; scope. Fruit length (FL) and diameter were mea- Spooner et al. 2013b), and none focused on Tunisia. sured using a micrometer with an accuracy of The present study was conducted to analyze the 0.01 mm. The observations were made on thirty patterns of phenotypic diversity in the Tunisian randomly selected seeds of each accession. Weight Daucus collection, conserved at the National Gene was obtained for 100 seeds, in three replicates, using Bank of Tunisia, using uni- and multi-variate a precision balance. 123 Genet Resour Crop Evol (2014) 61:499–510 501

Table 1 Taxon name, geographic coordinates and bioclimatic zones of the studied populations of Daucus Code Taxon name Source (province, location) Latitude Longitude Bioclimatic zonea

10773 D. syrticus Tataouine 33.1099 10.3658 S 10775 D. sahariensis Tataouine 32.3281 10.3539 S 10779 D. sahariensis Tataouine 32.2140 10.3539 S 10782 D. syrticus Medenine, Djerba Island 33.2578 10.3696 ACW 10784 D. syrticus Medenine, Djerba Island 33.3600 10.3487 ACW 10794 D. syrticus Medenine, Djerba Island 33.2057 10.3427 ACW 10795 D. syrticus Gabes 33.3576 9.2939 ASW 10796 D. syrticus Gabes 34.3049 9.1582 ASW 10802 D. muricatus Sousse, Enﬁdha 35.3053 10.1339 SASW 10807 D. muricatus Nabeul 36.5016 10.2249 SASW 10808 D. carota Nabeul 36.3121 10.2979 SASW 10810 D. muricatus Nabeul 36.3229 10.8076 SASW 10812 D. muricatus Nabeul 36.3268 10.2459 SASW 10814 D. muricatus Bizerte 37.8022 9.1193 SH 10815 D. carota Bizerte 37.2916 9.9774 SH 10817 D. muricatus Beja 36.2458 9.1642 SH 10820 D. capillifolius Medenine, Djerba Island, Boumellel 33.4488 10.1024 ASW 10822 D. capillifolius Medenine, Djerba Island, Houmet Souk 33.2039 10.1687 ASW 10823 D. capillifolius Medenine, Djerba Island, between Ajim and Guellala 33.4698 10.1763 ASW 10824 D. capillifolius Medenine, Djerba Island, between Ajim and Guellala 33.1687 10.2107 ASW 10826 D. capillifolius Medenine, Djerba Island Betwwen El May and Midoun 33.1299 10.3486 ASW 10827 D. capillifolius Medenine, Djerba Island betwwen El May and Ajim 33.1051 10.1010 ASW 10828 D. capillifolius Medenine, Djerba Island between El May and Ajim 33.1051 10.1010 ASW 10829 D. capillifolius Medenine, Djerba Island between El May and Ajim 33.1051 10.1010 ASW 10833 D. capillifolius Medenine, road between Zarzis and Djerba 33.4362 10.1208 ACW 10834 D. capillifolius Medenine, road between Zarzis and Djerba 33.1223 10.1160 ACW 10835 D. sahariensis Medenine, North of Bir Lahmar 33.1524 10.1039 ACW 10836 D. capillifolius Medenine, road between Ksar Haddada and Beni Khedache 33.1267 10.6348 ACW 10837 D. sahariensis Medenine, between Ksar Haddada and Beni Khedache 33.1267 10.6348 ACW 10839 D. carota landrace Tataoiune, Farm of Ghrabi Family, north of Tataouine 33.2577 10.9696 S 10840 D. carota landrace Tataouine, farm of Ghrabi Family, north of Tataouine 33.2577 10.9696 S 10842 D. sahariensis Tataouine, road between Bir Lahmar and Tatounine 33.3312 10.1477 S 10843 D. capillifolius Gabes, road from Medenine to Matmata 33.1412 10.1690 ASW 10844 D. capillifolius Gabes, road from Medenine to Matmata 33.1619 10.2100 ASW 10845 D. capillifolius Gabes, road between Matmata and Gabes 33.1982 9.3048 ASW 10846 D. capillifolius Gabes, road between Matmata and Gibbs 33.1454 9.5142 ASW 10847 D. sahariensis Gafsa, road between Matmata and Gibbs 33.1945 9.2081 ACW 10849 D. carota Gafsa, road to El Guettar 34.2159 8.8231 ACW 10850 D. capillifolius Gafsa, road from El Guettar to Bou Omrane 34.1503 9.1841 ACW 10851 D. capillifolius Gafsa, near Senad 34.1228 9.6840 ACW 10853 D. capillifolius Gafsa, road from Gafsa to Bir El Hefay 34.1472 9.1710 ACW 10854 D. capillifolius Sidi Bouzid, Mezarra 34.3300 9.1008 ACW 10862 D. capillifolius Sidi Bouzid, road from Sidi Bouzid to Regueb 35.1966 9.1334 ACW 10864 D. carota landrace Sidi Bouzid, road from Sidi Bouzid to Regueb 35.1966 9.1334 ACW

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Table 1 continued Code Taxon name Source (province, location) Latitude Longitude Bioclimatic zonea

10865 D. capillifolius Sidi Bouzid, road from Sidi Bouzid to Regueb 35.1966 9.1334 ACW 10866 D. capillifolius Sidi Bouzid, road from Sidi Bouzid to Regueb 34.1166 9.1918 ACW 10867 D. capillifolius Sidi Bouzid, road from Sbitla to Sfax 34.4266 9.1061 ACW 10868 D. capillifolius Sfax, road from Markez El Meawey to Sfax 34.1128 10.1210 ASW 10869 D. capillifolius Sfax, Sakiat Eddayer 34.1693 10.1019 ASW 10870 D. capillifolius Sfax, Sakiat Eddayer 34.1693 10.1019 ASW 10871 D. carota landrace Sfax, Ellouza Village 35.1851 11.6480 ASW 10872 D. capillifolius Sfax, Ellouza Village 35.1851 11.6480 ASW 10873 D. carota landrace Sfax, Ellouza Village 35.1315 10.1673 ASW 10874 D. capillifolius Sfax, Ellouza Village 35.1315 10.1673 ASW 10876 D. carota landrace Sfax, seed market in village of Hagez 35.1336 10.1672 ASW 10882 D. carota landrace Mahdia, road between Chebba and Ksour Essaf 35.1778 11.5808 SASW 10886 D. capillifolius Monastir, road from Monastir to M’Saken 35.1017 10.1533 SASW 10887 D. capillifolius Sousse, near M’Saken 35.8466 10.1411 SASW 10890 D. capillifolius Sousse, City of Chott Mariem 35.2178 10.1600 SASW 10891 D. capillifolius Sousse, road from Sousse to Tunis 27 km north of Sousse 0.0000 10.2150 SASW 10895 D. capillifolius Sousse, road between Enﬁdha and Kairouan 36.1839 10.1215 SASW 10896 D. muricatus Sousse, road between Enﬁdha and Kairouan 36.1839 10.1215 SASW 10898 D. capillifolius Sousse, road between Sousse and Hammamet 36.4152 10.1213 SASW 10900 D. capillifolius Sousse, Hergla 36.7746 10.1974 SASW 10901 D. carota Ben Arous, El Mourouj 36.2175 10.1673 SASW 10902 D. aureus Ben Arous, north of Zaghouan 36.1544 10.2016 SASW 10903 D. capillifolius Ben Arous, north of Zaghouan 36.1544 10.2016 SASW 10904 D. carota Zaghouan, highway between Bir Mecherga and Fahs 36.2754 9.1495 SASW 10905 D. capillifolius Zaghouan, road between Fahs and El Magren 36.8706 9.1855 SASW 10906 D. carota Zaghouan, Fahs 36.2556 9.1104 SASW 10907 D. capillifolius Zaghouan, Fahs 36.2556 9.1104 SASW 10910 D. carota Zaghouan, Fahs 36.1267 10.9936 SASW 10911 D. carota Nabeul, road between Saliman and Sidi Aissa 36.1456 10.1946 SASW 10912 D. carota Nabeul, road to Takelsa 36.4524 10.8460 SASW 10913 D. carota Nabeul, road between Takelsa and El Haouaria 36.5406 10.3228 SASW 10914 D.carota Nabeul, Sidi Daoud 36.1576 10.1321 SASW 10916 D.carota Nabeul, road between El Haouaria and Dar Allouche 37.2316 11.2958 SASW 10918 D. carota Ariana 36.9534 10.5940 SASW 10922 D. carota Ariana, road to Bouhnach 36.1186 10.2005 SASW 10923 D. carota Bizerte, south of Bizerte 37.1926 10.1106 SH 10927 D. carota Bizerte, south side of Ichkeul 37.2202 9.1452 SH 10928 D. carota Bizerte, west of Ghezab 37.1436 9.2262 SH 10929 D. carota Bizerte, Sajnane 37.1269 9.6702 SH 10932 D. carota Bizerte, road between Sajnane to Cap Negro 37.2964 9.1486 SH 10933 D. carota Bizerte, Sajnane 37.1970 9.1664 SH 10934 D. muricatus Bizerte, Sajane 37.1970 9.1664 SH 10935 D. carota Beja, road from Sajnan to Tabarka 36.9750 9.1230 SH 10936 D. carota Jendouba, Tabarka 36.9732 8.9558 H

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Table 1 continued Code Taxon name Source (province, location) Latitude Longitude Bioclimatic zonea

10937 D. carota Jendouba, Ain Draham 36.7176 8.1651 H 10943 D. carota Jendouba, road from Aı¨n Draham to Beja 36.4602 8.5988 SH 10944 D. carota Jendouba, road from Ain Draham to Beja 36.1839 9.1217 SH 10947 D. carota Beja, road between Beja and Tunis 36.2163 9.8868 SH 10949 D. aureus Beja, near Village El Munchar 36.8904 9.7062 SH 10950 D. carota Beja, near Village El Munchar 36.8904 9.7062 SH 10951 D. carota Beja, road between Beja and Nefza 36.1666 9.5340 SH 10955 D. carota Jendouba, Tabarka 36.3618 8.1234 SH 10956 D. carota Sousse, road between Sousse and Kairouan 35.7584 10.1879 SASW 10957 D. capillifolius Kairouan, road between Kairouan and Fahs 35.1382 10.5736 ACW 10958 D. carota Kairouan, road between Kairouan and Nassr Allah 35.1376 10.9120 ACW 10959 D. carota Kairouan, road between Kairouan and Skhira 35.1930 10.1723 ACW 10960 D. carota Kairouan, road between Bouhajla and Menzel Hached 35.1736 10.1443 ACW 10961 D. capillifolius Kairouan, road between Bouhajla and Menzel Hached 35.5718 10.2101 ACW 10962 D. capillifolius Sfax, direction Skhira before Bir Ali Ben Khelifa 34.6702 10.8778 ASW a Bioclimatic zones are deﬁned according to Emberger’s (1966) coefﬁcient: ASW arid at soft winter, SAWS semi-arid at soft winter, ACW arid at cool winter, SACW semi-arid at cool winter, S Saharian

Data analysis total), showing a wide range of variability for all the traits. Data analyses were performed using the statistical procedures in SAS 9.1 software (SAS 1990). Estimate of Estimate of variation using the Shannon–Weaver variability for each character was computed using the diversity index (H0) standardized Shannon and Weaver (1949, as referred by 0 0 Al Khanjari et al. 2008) DiversityP Index, designed as H Diversity indices (H ) for quantitative and qualitative 0 and has the formula: H =- pi (log2 pi)/log2 n, characters (Table 2) ranged from 0.31 for stylopodium where pi = frequency proportion of each descriptor shape to 0.81 for spine shape at the top with a mean state, n = number of states for each descriptor. Fre- diversity value of 0.58. The diversity values showed quency distributions for all the morphological traits low variation in stylopodium shape (0.31) and size were computed using the Proc Freq procedure in SAS (0.32), fruit shattering, and spine rigidity (0.45). High 9.1 software. The Shannon–Weaver diversity index has phenotypic variability was observed and was largest a value ranging from 0 to 1 where 0 indicates absence of for spine shape (0.81), followed by fruit color at diversity and 1 indicates maximum diversity. maturity (0.79), fruit shape (0.78) and spine shape at Frequencies determined for each descriptor state were the base (0.68). All other characters exhibited inter- subjected to factorial analysis of correspondence (FAC) mediate variation (0.5–0.59). Low variation indicated using the Proc Corresp procedure. Principal factors of FAC the dominance of one character state over the others served as input variables for the cluster analysis using the while high variation indicated equitable distribution of unweighted pair-group method of averages (UPGMA) to the different states as shown by frequency distribution. generate a dendrogram using the Proc Cluster. Population structure

Results The general structure of sample variability was established by factorial analysis of correspondence We measured 30 replications of 14 characters for all (FAC) and cluster analysis. FAC is a multivariate 103 Daucus accessions (43,260 measurements in technique that detects associations and oppositions 123 504 Genet Resour Crop Evol (2014) 61:499–510

Table 2 Morphological descriptors, descriptor states, their codes for numerical analysis, frequency distribution, and diversity index of Daucus accessions listed in Table 1 Trait/descriptor Descriptor acronym Descriptor Class Frequency (%) Diversity state index (H0)

Quantitativea Fruit length FL Short (\3 mm) 1 16.7 0.56 Intermediate (3–4 mm) 2 73.7 Elongated (5–6 mm) 3 8.7 Very elongated ([6 mm) 4 0.9 Fruit diameter FD Narrow (1.25–1.5 mm) 1 12.1 0.59 Intermediate (1.6–2) 2 79.1 Wide ([2 mm) 3 8.8 100 Fruit weight HFW Low (0.10–0.2 g) 1 12.1 0.5 Intermediate (0.21–0.6 g) 2 78.2 High (0.61–2 g) 3 8.8 Very high (2.1–3.0 g) 4 0.9 Qualitative Fruit shape FSh Ovoid 1 23.3 0.78 Ellipsoid 2 76.7 Fruit color at maturity FCM Yellowish 1 12.6 0.79 Yellowish to brownish 2 19.4 Brownish 3 59.3 Purplish 4 8.7 Fruit shattering FSha Low 3 9.5 0.45 High 7 90.5 Spine shape at base SShB Not enlarged at base 1 74.2 0.68 Widened 2 13.3 Dilated 3 12.5 Spine shape at top SShT Simple 1 75 0.81 Stellate 2 25 Spine rigidity SR Soft 1 90.2 0.45 Rigid 2 9.8 Spine confluency SC Separate 1 86.4 0.57 Confluent 2 13.6 Spine size on secondary rib SSi Shorter than fruit diameter 1 5.5 0.75 The length of fruit diameter 2 33.5 Longer than fruit diameter 3 61 Spine number on secondary rib SNSR Few (\15) 3 86.4 0.57 Many ([20) 7 13.6 Stylopodium shape StSh Conical 1 94.4 0.31 Globular 2 5.6 Style size StSi 2 9 stylopodium length 1 94.2 0.32 4–5 9 stylopodium length 2 5.8 a Quantitative characters were converted to phenotypic classes with the class boundaries as defined by Pottier Alapetite (1979) and IPGRI descriptors (1998)

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Table 3 Eigenvalues, percentage variation, and cumulative variance and was positively correlated with style size variation explained by the first four principal factors of facto- (StSi) and stylopodium shape (StSh) (Table 4). rial analysis of correspondence The scatter plot of factorial analysis of correspon- Principal Eigenvalue Variation of each Cumulative dence in the plan defined by the two first axes (Fig. 1) factor factor (%) variation (%) separated Daucus populations on the basis of their 1 6.77 33.48 33.48 fruit characteristics into 5 distinct groups. The first 2 6.40 29.94 63.42 group (G1, D. syrticus) included accessions 10773, 3 3.30 7.99 71.41 10782, 10784, 10794, 10795 and 10796 characterized 4 2.81 5.76 77.17 as having the smallest fruits (2–3 mm 9 1.25–1.5 mm) and the lowest 100 fruit weight (0.1–0.2 g) with yellow color at maturity, an ovoid Table 4 Correlation between the characters and the two first shape, long and widened spines at the base but not principal factors of factorial analysis of correspondence confluent with a soft structure, a conical shape for the Character Factor 1 Factor 2 stylopodium and a style of twice stylopodium length (Fig. 2a). Fruit length 2.4597 1.7855 The second group (G2, D. sahariensis) comprised Fruit diameter 2.4438 1.7900 accessions 10775, 10779, 10835, 10837, 10842 and 100 fruit weight 0.8577 0.5964 10847 distinguished from the first group by a yellow- Fruit shape -0.4692 0.0715 ish to brownish color, a globular shape for the Fruit color at maturity 2.4438 1.7900 stylopodium and a style of 4–5 times stylopodium Fruit shattering -1.5635 1.5356 length (Fig. 2b). Spine shape at the base 2.1901 1.6321 The third group (G3, D. muricatus L.) formed by Spine shape at the top 0.7357 0.4995 accessions 10802, 10807, 10812, 10814, 10817, Spine rigidity -1.6155 1.5320 10896, 10934 and 10949 exhibited relatively large Spine confluency 1.8751 1.3758 (5–6 m 9 2–3 mm) and heavy fruits (0.61 g \100 Spine size on secondary rib 0.4767 0.1968 fruit weight \2.0 g) with an ellipsoid form and a Spine number on secondary rib 1.8956 1.3974 distinctive purplish color. The spines are dilated and Stylopodium shape -2.2180 2.3423 confluent at the base, longer than FD, stellate at the top Style size -2.2163 2.3402 and present a rigid structure (Fig. 2c). The style showed a conical shape and was twice the stylopodium Values in bold represent the coordinates of the most important morphological characters supporting the formation of the two length. Accession 10810 belonging also to this group first principal factors of FAC was marked by its biggest size (FL 8 mm) and highest weight (100 fruit weight 2.9 g). existing between subjects (the populations in our case) The fourth group (G4, D. aureus Desf.) with a and variables (morphological descriptors), measuring single accession 10902 differed from group (G3) by its their contribution to the total inertia for each factor. fruit color, spine shape and confluency. This accession This enables two dimensional graphs to be drawn presented fruit with yellowish golden color, widened which offer aid in the interpretation of the results (Teil but non confluent spines (Fig. 2d). 1975). In the end, this analysis results in the identi- The fifth group (G5, D. capillifolius, D. carota) fication of major attributes that are responsible for the composed of 81 accessions characterized by fruits observed variation within a given collection. with intermediate size (3–4 mm 9 1.6–2 mm) and Factorial analysis of correspondence showed that weight (0.21–0.6 g), ovoid or ellipsoid shape, brown- the first four principal factors accounted for 77.1 % of ish color, separate spines with a soft structure and the total variance (Table 3). Factor 1 explained different spine sizes. Group G5 was divided into two 33.4 % of the total variance and was positively subgroups: G5-1 contained accessions 10929, 10933, correlated with FL, fruit diameter (FD), fruit color at 10944 and 10951 with spines shorter than FD, maturity (FCM), spine shape at the base (SShB), spine widened at the base and stellate at the top (Fig. 2e) number on secondary rib (SNSR) and spine conflu- related to D. carota subsp. maximus (Desf.) Ball and ency (SC). Factor 2 accounted for 29.9 % of total G5-2 composed of remaining accessions with the 123 506 Genet Resour Crop Evol (2014) 61:499–510

Fig. 1 Scatter plot grouping of Daucus accessions based on the ﬁrst two principal factors of factorial analysis of correspondence

Fig. 2 Representative fruit of each group identified in the e subgroup G5-1, D. carota subsp. maximus; f subgroup 5-2, D. Daucus collection. a Group G 1, D. syrticus; b group G2, D. carota subsp. carota and D. capillifolius sahariensis; c group G3, D. muricatus; d group G4, D. aureus; spines not enlarged at the base, the length of FD or 1.7. At the average distance of 1.1, the dendrogram longer and a simple structure at the top (Fig. 2f). It is identified four main clusters Cl1, Cl2, Cl3 and Cl4 D. carota subsp. sativus (Hoffm.) Arcangeli, D. carota including the same groups (G1–G5) identified by subsp. carota and D. capillifolius. (FAC). Accessions of G1 (D. syrticus) and G2 (D. The hierarchical cluster dendrogram (Fig. 3) sep- sahariensis) fell together in cluster Cl1. Cluster Cl2 arated the populations into several clusters with an grouped accessions of G5 (D. carota) with the two average distance between clusters ranging from 0 to subgroups G5-1 (D. carota subsp. maximus) and G5-2

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Fig. 3 Dendrogram obtained from cluster analysis of local Daucus accessions using the UPGMA

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(D. carota subsp. sativus, D. carota subsp. carota, and concordance with the criteria that supported the D. capillifolius) whereas Cl3 included accessions of taxonomic classification of Daucus species as given G3 group (D. muricatus) and Cl4 included the single by Heywood (1968), Pottier Alapetite (1979) and accession of group G4 (D. aureus). Saénz Laıń(1981), except D. capillifolius, distinguished from D. carota by yellow corollas and linear Population distribution according to bioclimatic leaflets fell throughout the D. carota group. This and geographic origins morphological result is fully in agreement with crossability and molecular data as mentioned above, Distribution of populations according to their biocli- and supports D. capillifolius as a locally-evolved matic and geographic origin (Table 1; Fig. 3) showed species from D. carota. Thus, the first identified group that the morphological distinction of the different included populations with fruit characteristics of groups was not paralleled by a congruent bioclimatic D. syrticus; the second one corresponded to D. saha- distribution. For example, two or more populations riensis; whereas the third and the fourth groups from the Saharian zone grouped with populations from showed typical characteristics of D. muricatus and arid zone at cool or/and soft winter (populations of G1 D. aureus, respectively. The final group, the largest and G2). Similarly, species in Group G3, D. muric- one, was made up of populations of D. carota. It was atus, grouped accessions from two bioclimatic zones, itself divided into two subgroups representing three the semi-arid at soft winter and the subhumid. subspecies. The first one contained D. carota subsp. Populations of group G5 were spread across all maximus, whereas the second one grouped accessions bioclimatic zones of Tunisia ranging from arid to of D. carota subsp. sativus, the cultivated form of humid through the semi-arid and the subhumid in carrot, D. carota subsp. carota, its closest wild relative different geographic area. However, in some cases, and D. capillifolius. accessions from the same bioclimatic and geographic Fruit, commonly referred to as seed in Apiaceae, origin had a tendency to stay together. For example, has been found to be a useful taxonomic feature for a this is the case for accessions 10903, 10904 from large number of species or genera in this family Zaghouan; 10957, 10958, 10959 from Kairouan, and (Hartman and Nesom 2012; Nesom 2012; Akalin 10914, 10916 from Nabeul. Urus¸ak and Kizilarslan 2013) and is a critical taxonomic character distinguishing taxa within Daucus (Pottier Alapetite 1979;Saénz Laıń 1981; Pujadas Discussion Salva` 2003). In many cases, morphological characteristics, such as seed shape and testa ornamentation, can For Daucus, apart the studies of Pottier Alapetite be used to distinguish species and varieties. (1979) and Le Floc’h et al. (2010) which focused on Fruits and seeds tend to show less phenotypic the identification of Daucus species in Tunisia, no plasticity, in comparison with other organs. Seed investigation has been carried out for morphological characters are less affected by environmental condi- characterization of a wide diversity of species using tions, and often reflect genetic differences (Bonilla- international standards. The fruit characters used in Barbosa et al. 2000). D. carota is strongly outcrossing our study revealed considerable diversity within a and populations are genetically heterogeneous (Simon local Daucus collection maintained at the National 1984). Accessions of all subspecies of D. carota, D. Gene Bank of Tunisia as shown by a mean diversity capillifolius, and D. sahariensis (all sharing n = 9 index of 0.58, confirming that Tunisia is one of the chromosomes, and all examined in this study) appar- principal centers of diversification for Daucus in the ently have no barriers to crossing (McCollum 1975, Mediterranean regions. 1977). Furthermore, it is noteworthy that in Tunisia The use of multivariate analysis of factorial corre- carrot is widespread both as a wild and cultivated spondence and cluster analysis on fruit descriptors plant, and that these forms grow in close proximity to permitted the subdivision of the studied populations each other; such as population 10872 which was into five morphological groups which differed mainly 3–5 km away accession 10871; moreover, cultivated by their fruit size, FCM, spine shape at the base and forms, frequently moved throughout the country (e.g., stylopodium shape. Classification results showed via seed exchange among farmers and among external 123 Genet Resour Crop Evol (2014) 61:499–510 509 markets), may be accidentally introduced into natural Botanique et Zoologie. Facult Sci Montpellier France and semi-natural ecosystems. Therefore, under some 7:1–43 Grzebelus D, Baranski R, Spalik K, Allender C, Simon PW conditions, gene flow may occur, considering the (2011) Daucus. In: Kole C (ed) Wild crop relatives: allogamous mating system of the species. genomic and breeding resources, vol 5., Vegetables, Multivariate analysis provided evidence that popu- Springer, Berlin, pp 91–113 lations are clustered independently from their biocli- Guetet A, Zammouri J, Boussaid M, Neffati M (2009) The use of reproductive vigor descriptors to study genetic variability matic origin. Thus, we noted the presence of two or more in wild populations of Allium roseum L. (Alliaceae) in bioclimatic and geographic zones in the same group. Tunisia. Sci Hortic 120:282–287 Similar results were obtained by Guetet et al. (2009)and Hartman RL, Nesom GL (2012) Taxonomy of the genus Vesper Rhimi et al. (2013) who reported that the aggregation of (Apiaceae). Phytoneuron 94:1–9 Heywood VH (1968) Daucus L. In: Tutin TG, Heywood VH, 28 populations of Allium roseum L. and 17 populations Burges NA, Moore DM, Valentine DH, Walters SM, Webb of Capparis spinosa L. using morphological data DA (eds) Flora Europaea. University Press, Cambridge, according to their geographical and bioclimatic origins pp 373–375 was not respected. This could be justified by the Iorizzo M, Senalik DA, Ellison SL, Grzebelus D, Cavagnaro PF, Allender C, Brunet J, Spooner DM, Van Deynze A, Simon reproduction mode of these species, being considered PW (2013) Genetic structure and domestication of carrot as allogamous species which therefore require external (Daucus carota subsp. sativus L.) (Apiaceae). Am J Bot pollen donors (Aradhya et al. 2003). 100:930–938 The present study demonstrated the use of fruit Iovene M, Grzebelus E, Carputo D, Jiang J, Simon PW (2008) Major cytogenetic landmarks and karyotype analysis in characters to evaluate the genetic diversity of local Daucus carota and other Apiaceae. Am J Bot 95:793–804 Daucus populations. Our morphological data provide IPGRI (1998) Descriptors for wild and cultivated carrot (Daucus considerable information that is useful to distinguish carota L.). International Plant Genetic Resources Institute, species and subspecies in this difficult genus. Further Rome Le Floc’h E, Boulos L, Vela E (2010) Catalogue synonymique collection missions are necessary to acquire additional commente´ de la flore de Tunisie. Banque Nationale des accessions to enrich the collection and have all species Ge`nes de la Tunisie, Tunis identified in Tunisia. This research will also be contin- McCollum GD (1975) Interspecific hybrid Daucus carota 9 D. ued by combining both morphological and molecular capillifolius. Bot Gaz 136:201–206 McCollum GD (1977) Hybrids of Daucus gingidium with cul- data in order to confirm the diversity observed and tivated carrots (D. carota subsp. sativus) and D. capillifo- improve Daucus germplasm management. lius. Bot Gaz 138:56–63 Nesom GL (2012) Taxonomy of Polytaenia (Apiaceae): P. nuttallii and P. texana. Phytoneuron 66:1–12 Peterson CE, Simon PW (1986) Carrot breeding. In: Basset MJ (ed) Breeding vegetable crops. AVI, Westport, pp 321–356 References Pottier Alapetite G (1979) Daucus. In: Flore de la Tunisie, Angiospermes-Dicotyledones, Apetales, Dialypetales. Akalin Urus¸ak E, Kizilarslan C (2013) Fruit anatomy of some Imprimerie Officielle de la Re´publique Tunisienne, Tunis, Ferulago (Apiaceae) species in Turkey. Turk J Bot pp 615–621 37:434–445 Pujadas Salva` A (2003) Daucus. 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