A Morphological Study of Populations of Lotus Glaber Mill

A morphological study of populations of Lotus glaber Mill. (Fabaceae) Mariana Kade, Eduardo Pagani, Rodolfo Mendoza

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Mariana Kade, Eduardo Pagani, Rodolfo Mendoza. A morphological study of populations of Lotus glaber Mill. (Fabaceae). Agronomie, EDP Sciences, 2003, 23 (3), pp.203-207. 10.1051/agro:2002083. hal-00886172

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HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Agronomie 23 (2003) 203–207 203 © INRA, EDP Sciences, 2003 DOI: 10.1051/agro:2002083 Original article

A morphological study of populations of Lotus glaber Mill. (Fabaceae)

Mariana KADEa*, Eduardo A. PAGANIb, Rodolfo E. MENDOZAb

a IBYF-CONICET, Facultad de Agronomía-UBA, Av. San Martín 4453, 1417 Buenos Aires, Argentina b CEFYBO-CONICET, Serrano 669, 1414 Buenos Aires, Argentina

(Received 6 February 2001; revised 26 November 2001; accepted 18 June 2002)

Abstract – The pasture legume Lotus glaber Mill. has colonized the Flooding Pampa (Argentina) in spite of high environmental heterogeneity. Morphological characters of plants from different populations were compared to evaluate if plastic or genetic differentiation could have contributed to such a widespread geographic distribution. Plants were collected at random from six different sites and grown in a greenhouse for five months. Clonal replicates of those plants were cultivated in pots on a native and a common soil. Populations were replicated three times and each replicate (pot) had three plants from the same clone. The number of primary shoots, internode length, leaf length and width and shoot length were recorded and the significance of population mean differences determined. All characters, except for leaf width, differed between populations on each sampling in both sets of experiments. This variability is therefore best explained by genetic differentiation of the populations. morphological variation / environmental heterogeneity / naturalized species / Lotus glaber Mill. / genetic differentiation

Résumé – Étude de la variabilité morphologique dans les populations de Lotus glaber Mill. (Fabaceae). La légumineuse fourragère Lotus glaber Mill a colonisé la Pampa inondée en Argentine malgré la très grande hétérogénéité des conditions environnementales. Dans cette étude nous comparons la morphologie de différentes populations afin de déterminer si une plasticité phénotypique ou une différenciation écotypique ont permis une aussi large dispersion géographique de l'espèce. Des plantes représentatives de 6 sites ont été transplantées et cultivées en serre durant 5 mois. Des boutures de ces différents plants ont également été cultivées dans des pots remplis avec le sol original des sites correspondants ainsi qu'avec un sol unique provenant d'un autre site. Le nombre de pousses primaires, la longueur des inter-nœuds, la longueur et la largeur des feuilles et la longueur des pousses ont été mesurées et la validité statistique des différences à la moyenne de la population a été déterminée. Toutes les caractéristiques morphologiques, sauf la largeur des feuilles ont montré des différences pour chaque échantillon et dans chaque série d'expériences. Ce type de variabilité pourrait être mieux expliqué par une différenciation d'écotypes que par une plasticité phénotypique. variabilité morphologique / hétérogénéité environnementale / adaptation des espèces / Lotus glaber Mill. / écotype

1. INTRODUCTION individuals of that species possess extensive phenotypic plasticity, thereby reducing the importance of local adaptation, or Lotus glaber Mill. is one of the most conspicuous exotic if there is a genetic variation among them [14]. legumes on lowland grasslands in the Flooding Pampa of Argentina [9]. These natural grasslands, which cover about Chrtková-Zertrová [3] recorded 14 ecotypes in Europe for 5 million ha of the region [2], are the main forage source for L. corniculatus, a species closely related to L. glaber. She cattle breeding. Because of L. glaber's high nutritive value and argued that the high variability of this tetraploid species ena- low bloat hazard, it is considered as an important pasture leg- bled it to spread into a large number of different habitats. Spe- ume by farmers of the region. cifically in L. glaber (2n = 2x = 12), genetically controlled In spite of the environmental heterogeneity that character- variation was assessed for different populations among physi- izes the Flooding Pampa, L. glaber has become naturalized ological characters related to phosphorus (P) nutrition [6, 8]. throughout the mentioned region in the last decades. Such a Also, Vignolio et al. [13] reported morphological adaptations widespread geographic distribution may be achieved either if under flooding conditions in five populations.

Communicated by Gérard Guyot (Avignon, France)

* Correspondence and reprints [email protected] 204 M. Kade et al.

Table I. Soil chemical properties and textural classification from different sites where Lotus glaber plants were collected.

pH: 1:2.5 (soil/0.01 N CaCl2, stirred); EC: electrical conductivity; P: Bray1; CEC: cation exchange capacity; exchangeable cations: calcium (Ca); sodium (Na); magnesium (Mg); potassium (K); ESP: exchangeable sodium percentage; HN: hydrolyzable nitrogen Site Locality pH CEC EC P Ca Na Mg K ESP HN

CaCl2 (cmol·kg–1)(ds·m–1)(mg·kg–1)(cmol·kg–1)(cmol·kg–1)(cmol·kg–1)(cmol·kg–1) (%) (mg·kg–1) 1 Alvarez Jonte 5.3 17.6 1.49 4.7 6.5 6.3 3.3 1.0 36 429 2 Verónica 4.4 19.8 0.28 1.45 8.9 1.4 4.5 0.7 7 244 5 Monte 5.9 11.2 0.31 2.3 6.6 0.7 2.2 0.2 6 188 9 San Vicente 6.7 15.4 1.27 8.1 6.7 6.9 2.7 1.4 45 357 16 Samborombón 5.5 20.5 0.5 9.1 7.3 7.4 5.4 1.5 36 722 17 Laprida 7.8 11.3* 1.22 5.5 5.6 6.0 1.2 1.2 53 72 *Presence of soluble salts.

Site Soil classification* Geographical location Sand (%) Silt (%) Clay (%) Texture 1 Chromic Hapludert Alvarez Jonte 15.7 45.7 38.6 Silty clay 2 Aquic Hapludert Verónica 8.9 52.4 38.7 Silty clay 5 Entic Hapludoll Monte 86.7 9.6 3.7 Loamy sand 9 Typic Natraqualf San Vicente 22.5 61.6 15.9 Coarse silty 16 Typic Natraqualf Samborombón 13.1 60.2 26.7 Coarse silty 17 Typic Natraqualf Laprida 51.2 37.2 11.4 Clayey *According to Soil Taxonomy, (USDA) [10].

In this study we have quantified the morphological differ- each site, six L. glaber plants were collected at random with ences between plants of six L. glaber populations growing on roots and the undisturbed surrounding soil. Those plants were native soils from the Flooding Pampa and when they were transplanted to pots with their native soil and kept in a green- transplanted to a common soil. This should allow us to deter- house for five months (mean temperature 27 °C ± 3 without mine whether those populations showed genetic or plastic dif- humidity control and natural light). The pots were arranged in ferentiation for the mentioned characters. If the morphological groups corresponding to the six populations sampled. Seed variation has a plastic origin it is supposed to disappear when samples from the 36 plants collected were submitted to the individuals are evaluated in a uniform environment; if popula- flavonol testing procedure [5] in order to verify that they tions remain different under these conditions they have proba- belonged to the species L. glaber (2n = 2x =12). After the bly accumulated genetic differences [7, 12]. preconditioning period, three healthy donor plants from each population were selected at random and cloned by stem cuttings [6]. The cuttings were planted in undrained plastic 2. MATERIALS AND METHODS pots, which had been filled either with 200 g of soil (dry weight) from the origin sites (Tab. I) or with a local Natraquoll (common soil named 18) [pH (soil:0.01 M CaCl 1:2.5) 5.0; 2.1. Collection and propagation of plant material 2 EC 0.46 ds·m–1; P 6.1 mg·kg–1; CEC 20.1 cmol·kg–1 and ESP A survey of eighteen different L. glaber populations was 6.5%] classified as silty clay loam. Each pot contained five performed along the Flooding Pampa in Buenos Aires prov- cuttings from the same donor plant and was replicated at least ince. Finally, six populations were selected, considering dif- three times. Cuttings were grown for one month (roots ferences mainly in pH, P and the ESP (Tab. I). Sites were appeared 20–25 days after planting) in a growth cabinet as named after the nearest locality; in the text we will refer to described in Kade et al. [6]. them with the corresponding number (Tab. I). Sites 2 and 16 had an intermediate topographic position and their summer 2.2. Plant culture dominant species were Piptochaetium montevidense-Ambro- sia tenuifolia-Eclipta bellidioides and Mentha pulegium [2]. At the beginning of the experiment, the pots were trans- Sites 1 and 5 could be categorized as lowlands and contained ferred to greenhouse conditions and thinned to three plants per a community defined by M. pulegium-Leontodon taraxoides pot. The experiment consisted of three replicates for each and Paspalidium paludivagum [2]. Populations on sites 9 and donor plant from the same population cultivated on the native 17 also grew on a low topographic position but under alkaline soil. Each replicate (pot) had three plants (27 plants/popula- soil conditions with Distichlis spicata as dominant species. At tion were measured). Simultaneously, one donor plant chosen Morphological variation in Lotus glaber populations 205

Table II. ANOVA probability values for intrapopulation variation in different morphological characters for six Lotus glaber populations (95 DABE).

Populations Primary shoot Internode length Leaf length Leaf width Length/Width Shoot length number (mm) (mm) (mm) (mm) 1 ns ns 0.0168 ns ns ns 2 ns ns 0.0185 ns ns ns 5 ns ns 0.0098 ns ns ns 9 nsnsnsnsnsns 16 0.0007 ns ns ns ns ns 17 ns ns 0.0004 0.0085 0.0188 ns P values are significant at the 95.0% confidence level; ns: not significant.

Table III. (a) Comparison of morphological characters between 2.3. Morphological variation Lotus glaber populations growing on their native soil (50 DABE); (b) comparison of morphological characters between Lotus glaber The number of primary shoots, internode length, leaf length populations growing on a common soil (50 DABE). and width and, at the end of the experiment, the largest secondary shoot, were recorded directly on the plants. The (a) length of the sixth internode from the basal part of the plant was measured on a labeled shoot. The leaf length and width Site Primary shoots Leaf length Leaf width Length/Width measurements were taken on the middle leaflet of the fourth number (mm) (mm) leaf [1]. The leaf length/width ratio was also calculated. The 1 1.61 b 5.82 c 3.33 b 1.93 c measurements were made 50, 70 and 95 days after the beginning of the experiment (DABE). 2 2.29 a 7.63 b 4.21 a 1.86 c 9 1.64 b 8.77 a 3.27 bc 2.84 a 2.4. Statistical analysis 17 1.07 c 7.05 b 3.54 b 2.14 b Data were analyzed by separate ANOVAs to test for Different letters within a column indicate significant differences morphological differences between and within populations. (P < 0.05) between populations comparing their mean ranks. The data were checked for normality and homogeneity of variances by the Bartlett test. As in the analyses of variation (b) between populations these assumptions were violated, a nonparametric Kruskal-Wallis test [4] was performed. Site Primary shoots Leaf length Leaf width Length/Width number (mm) (mm) 1 on 18 2.00 ab nd nd 2.08 abc 3. RESULTS 2 on 18 1.22 c nd nd 1.71 d At the first sampling (50 DABE), plants from populations 5 5 on 18 1.63 abc 6.67 b 3.22 a 2.15 ab and 16 had not grown enough to be evaluated, and this was also the case during the second evaluation (70 DABE) for pop- 9 on 18 1.50 bc 8.22 a 3.44 a 2.32 a ulation 5. Because of the plants' size, it was impossible to 16 on 18 2.11 a 4.89 c 2.78 a 1.82 cd determine the internode length at the first sampling for all populations. 17 on 18 1.44 bc 6.00 bc 3.11 a 1.97 bcd Analyses of intrapopulation variation indicated some dif- Different letters within a column indicate significant differences (P < ferences between their individuals, particularly for the leaf 0.05) between populations comparing their mean ranks. nd: no data. length (Tab. II). Population 2 exhibited the highest shoot number on the original soil; on the contrary, under uniform soil conditions, at random from each population to avoid possible differences population 2 was amongst the ones with the lowest number of due to intrapopulation variation was replicated three times on shoots (Tabs. IIIb, IVb and Vb). Plants from populations 2 and the common soil (9 plants/population). During the experiment 9 showed longer internodes than the other ones on their (mean temperature 20 °C ± 3 without humidity control and original soils (Tabs. IVa and Va). Population 9 also had larger natural light), the pots were held near field capacity (25% leaves (Tabs. IIIa, IVa and Va). In contrast, populations 5 and w/w) by weighing and watering daily. The pots were randomly 16 exhibited not only a significantly shorter leaf length, but spaced and periodically rotated. also width, for the last sampling (Tab. Va). Plants from 206 M. Kade et al.

Table IV. (a) Comparison of morphological characters between Lotus glaber populations growing on their native soil (70 DABE); (b) comparison of morphological characters between Lotus glaber populations growing on a common soil (70 DABE).

(a)

Site Primary shoots Internode length Leaf length Leaf width Length/Width number (mm) (mm) (mm) 11.90 b 8.65 c 5.86 c 3.33 b 1.77 c 22.70 a 13.92 b 7.00 d 3.92 a 1.81 c 91.93 b 16.64 a 8.53 a 3.43 b 2.54 a 16 1.56 b nd 4.56 d 2.31 c 2.02 b 17 1.17 c 8.73 c 7.21 b 3.42 b 2.12 b Different letters within a column indicate significant differences (P < 0.05) between populations comparing their mean ranks. nd: no data.

(b)

Site Primary shoots Internode length Leaf length Leaf width Length/Width number (mm) (mm) (mm) 1 on 18 3.22 a 12.44 bc 8.78 a 3.89 a 2.27 ab 2 on 18 1.89 c 18.33 a 6.67 c 3.72 ab 1.8 cd 5 on 18 2.22 bc 14.89 ab 7.11 c 3.5 ab 2.09 bc 9 on 18 2.44 abc 16.22 ab 8.44 ab 3.27 bc 2.51 a 16 on 18 2.89 ab 9.63 c 5.11 d 2.94 c 1.79 d 17 on 18 2.67 abc 15.55 ab 7.44 bc 3.72 ab 2.00 bcd Different letters within a column indicate significant differences (P < 0.05) between populations comparing their mean ranks.

Table V. (a) Comparison of morphological characters between Lotus glaber populations growing on their native soil (95 DABE); (b) comparison of morphological characters between Lotus glaber populations growing on a common soil (95 DABE).

(a) Site Primary shoots Internode length Leaf length Leaf width Length/Width Shoot length number (mm) (mm) (mm) (cm) 1 13.43 b 9.63 c 6.19 c 3.21 c 1.93 c 11.00 c 2 24.62 a 14.21 b 7.71 b 4.21 a 1.86 c 20.42 a 5 52.11 c 6.10 d 5.05 d 2.68 d 1.92 c 3.14 e 9 92.47 c 17.33 a 9.13 a 3.27 bc 2.84 a 14.36 b 16 162.13 c 5.25 d 4.67 d 2.46 d 1.94 c 3.94 e 17 171.54 d 9.50 c 7.58 b 3.54 b 2.14 b 9.17 d Different letters within a column indicate significant differences (P < 0.05) between populations comparing their mean ranks.

(b)

Site Primary shoots Internode length Leaf length Leaf width Length/Width Shoot length number (mm) (mm) (mm) (cm) 1 on 18 6.11 ab 14.67 b 8.78 ab 3.94 a 2.24 bc 10.78 c 2 on 18 4.00 c 19.11 a 8.33 b 4.33 a 1.93 d 15.44 a 5on 18 4.89 bc 5.11 b 8.00 b 3.61 a 2.26 b 12.83 abc 9 on 18 3.44 c 17.22 ab 9.67 a 3.72 a 2.62 a 14.06 ab 16 on 18 7.00 a 10.55 c 6.67 c 3.72 a 1.80 d 10.44 c 17 on 18 6.55 ab 15.89 ab 7.89 b 3.94 a 2.01 cd 12.50 bc Different letters within a column indicate significant differences (P < 0.05) between populations comparing their mean ranks. Morphological variation in Lotus glaber populations 207 population 2 exhibited wider leaves (Tabs. IIIa, IVa and Va), REFERENCES but this trait was similar between populations under uniform conditions (Tabs. IIIb and Vb). Plants from population 2 [1] Beuselinck P.R., Ben Younes M., Mc Graw R.L., Taxonomic showed the largest secondary shoots (Tab. Va). 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