Genetic Diversity and Geographic Differentiation in the Alternative Legume Tripodion Tetraphyllum (L.) Fourr

Plant Biology ISSN 1435-8603

RESEARCH PAPER Genetic diversity and geographic differentiation in the alternative legume Tripodion tetraphyllum (L.) Fourr. in North African populations E. Bellucci, L. Nanni, E. Bitocchi, M. Rossi & R. Papa Dipartimento di Scienze Ambientali e delle Produzioni Vegetali, Universita` Politecnica delle Marche, Ancona, Italy

Keywords ABSTRACT Anthyllis tetraphylla; conservation; genetic differentiation; inter-simple sequence repeats; Wild legumes constitute an important component of widespread pastures in the Leguminoseae; Mediterranean. Mediterranean basin. This region is experiencing remarkable effects from climate change, and continuous monitoring of species and population dynamics is impor- Correspondence tant in order to plan and enact valuable conservation programmes. Tripodion tetra- R. Papa, Dipartimento di Scienze Ambientali phyllum (L.) Fourr. [=Anthyllis tetraphylla L.] (2n = 16), belongs to the tribe Loteae e delle Produzioni Vegetali, Universita` (Fabaceae), and could be very important for soil protection and sward improvement Politecnica delle Marche, Via Brecce Bianche, in abandoned or degraded Mediterranean areas. This alternative pasture legume is 60131 Ancona, Italy. very closely related to Lotus japonicus and has some important characteristics for E-mail: [email protected] survival of the species in difﬁcult and overgrazed Mediterranean areas. In this study, we have investigated the molecular diversity and population structure of Editor T. tetraphyllum from North Africa using ISSR markers and plastidial microsatel- O. Loudet lites. To date, this is the ﬁrst study concerning the genetic diversity and geographic differentiation of T. tetraphyllum. Ninety genotypes from three North African coun- Received: 7 January 2010; Accepted: 3 May tries were analysed according to ISSRs, cpSSRs and one phenotypic trait. T. tetra- 2010 phyllum shows a clear geographical structure, with differentiation associated with longitudinal differences; moreover, there is a general reduction in genetic diversity doi:10.1111/j.1438-8677.2010.00370.x from Morocco to Tunisia. With all the markers used, strong differentiation was seen among collection sites. Our data highlight a genetic diversity gradient and cline of distribution, indicating that T. tetraphyllum has extended its area of distribution from Morocco to Tunisia.

(L.) Fourr. (2n = 16) belongs to the Fabaceae, tribe Loteae; it INTRODUCTION is a predominantly selfing, annual prostrate legume with yel- Arid and semi-arid land makes up approximately one-third low flowers (Couderc 1980), that occurs on calcareous soils of the land surface of the Earth. Such areas are expanding, from sea level to 1000 m a.s.1. (Pignatti 1982), and has good which probably represents another indication of global palatability (Salsano 1996). Although little is known about weather change (Dregne 1986; Archibold 1995; Stafford this alternative pasture legume, which is usually neglected Smith 1996; Dore 2005). Consequently, much productive and regarded as unimportant, T. tetraphyllum may be very land is being degraded and livestock productivity is increas- important for soil protection and sward improvement on ingly suffering from feed shortage. slopes and in abandoned or degraded Mediterranean areas Wild legumes constitute a crucial component of wide- (such as rocky soils, steep slopes with very shallow soils, spread pastures in the Mediterranean basin. These plants are sandy salt soils). Furthermore, this species has ornamental an important source of fat, protein and minerals for sheep, importance because of its peculiar swollen calyx that persists horses, goats and cows, and can represent up to 80% of total until seed maturity and its corolla colour (Meloni et al. forage material, especially for sheep. Wild legume species in 2000). T. tetraphyllum also has important characteristics for the Mediterranean area occupy about 40% of the grazed survival of the species in difficult and over-grazed Mediterra- meadows (Viano et al. 1995). Pasture and forage legumes also nean areas: the production of numerous pods and seeds, have important roles in arable land where, especially in dry small seed size, hard seeds, early flowering and prostrate areas, they have the potential to replace fallow in cereal-pro- growth. ducing farming systems through their ability to fix nitrogen Using Internal Transcribed Spacer (ITS) and Chloroplast (Bennett & Cocks 1999). The alternative legumes deserve Simple Sequence Repeat (cpSSR) markers, T. tetraphyllum more attention, and investigations need to be carried out to [=Anthyllis tetraphylla L.] was shown to be closely related to determine their potential roles for multiple uses in Mediter- Lotus japonicus (Nanni et al. 2004), which, together with ranean environments. Medicago truncatula, was selected as a model plant species. Among the wild legumes, Loteae are widely distributed L. japonicus is the focus of large, multinational genome pro- throughout the Mediterranean basin. Tripodion tetraphyllum jects and is the second legume species to be sequenced

Plant Biology 13 (2011) 381–390 ª 2010 German Botanical Society and The Royal Botanical Society of the Netherlands 381 Genetic diversity and geographic differentiation in T. tetraphyllum Bellucci, Nanni, Bitocchi, Rossi & Papa

(Young et al. 2005). Thus, in the near future, the literature of T. tetraphyllum, (on average three per accession) was on the ecological genetics and molecular ecology of wild spe- obtained. Table 1 provides accession names, donors, states, cies closely related to L. japonicus is expected to grow rapidly, provinces, altitudes and geographical coordinates for each as was the case for Arabidopsis (Bevan & Walsh 2005). accession analysed, along with the number of individual There are many morphological structures used for taxo- plants. nomic classification in Papilionaceae (including Loteae) that The accessions collected from ICARDA reflect a broad can distinguish T. tetraphyllum from Anthyllis, so that the range of geographical distributions of T. tetraphyllum in accepted name for this species is T. tetraphyllum, with the North Africa, from Morocco to Tunisia. There are 16 Moroc- two synonyms, Anthyllis tetraphylla L. and Physanthyllis tetra- can accessions, from 16 collection sites distributed across four phylla (L.) Boiss. (Roskov et al. 2005). T. tetraphyllum has provinces (Beni Mellal, Khenifra, Marrakech and Tetouan), dehiscent (as does Lotus) and non-lomentaceous fruits, and with altitudes ranging from 40 m a.s.l. (Tetouan, accession the fruit structure is leguminous, two-seeded, pubescent and MTEe) to 1420 m a.s.l. (Beni Mellal, accession MdBMa). lomentaceous (Lassen 1986). When studying pollen morphol- Algeria is represented by 10 accessions, from 10 different ogy, Dıéz & Ferguson (1990) confirmed that T. tetraphyllum collection sites in six provinces (Alger, Constantine, Medea, differs significantly from all of the other species of Anthyllis Miliana, Setif and Tlemcen), with altitudes from 150 m a.s.l. in the size of its pollen grains. In addition, in a study of sev- (Constantine, accession ACOa) to 1000 m a.s.l. (Miliana, eral Loteae species, Allan & Porter (2000) showed that T. tet- accession AMId). Tunisia is represented by nine accessions raphyllum is more closely related to other Loteae than to from nine collection sites in six provinces (Beja, Bizerte, Jen- Anthyllis vulneraria, the most widespread species of the genus douba, Kasserine, Sousse and Zaghouan), with altitudes from Anthyllis. 5 m a.s.l. (Bizerte, accession TBIb) to 670 m a.s.l. (Kasserine, The inter-simple sequence repeat (ISSR) technique uses accession TKAa). The Portuguese accession (POR) obtained PCR primers that are composed of microsatellite sequences from the USDA was collected from the Elvas Plant Breeding (SSRs), and allows detection of size polymorphism between Station (PT), 300 m a.s.l. microsatellite repeats. Since Zietkiewicz et al. (1994) devel- For each accession, four seeds were sown and the plants oped the ISSR technique, it has proven to be a rapid, simple were grown and cultivated at the SAPROV Department, Uni- and inexpensive way to assess structure and genetic diversity versity Politecnica delle Marche, Italy. Table 1 also summaris- (Gonzalez et al. 2005; Culley et al. 2007), to analyse genetic es the number of individual plants for each accession used relationships among cultivars or landraces and to study evo- for the DNA isolation, and the subsequent molecular analy- lutionary processes (Sicard et al. 2005). Chloroplast microsat- ses. Total genomic DNA was isolated from young leaves of ellites (cpSSRs) are highly polymorphic sequences (Powell single individual plants using the DNeasy Plant Mini kit et al. 1995; Vendramin et al. 1996; Petit et al. 2005; Elbert & (Qiagen GmbH, Hilden, Germany), following the protocols Peakall 2009), generally maternally inherited in angiosperms. provided by the manufacturer. The final DNA quality and They represent another useful tool in the study of genetic concentrations were determined by running the samples on variation and evolution of plants (Olmstead & Palmer 1994) 0.8% agarose gels, and DNA concentrations were standar- ) and have been used for diversity studies in Glycine (Xu et al. dised to 5 ngÆll 1. 2002), Phaseolus spp. (Sicard et al. 2005; Angioi et al. 2009a,b) and Anthyllis (Nanni et al. 2004). ISSR analysis Here, we have investigated the molecular diversity and population structure of T. tetraphyllum from North Africa DNA amplifications were achieved using five different ISSR using ISSR markers and plastidial microsatellites. This study primers (listed in Table 2). These were selected after initial is the first step towards an understanding of the amount screening of a set of about 20 primers, because they and distribution of T. tetraphyllum genetic variability, and gave unambiguous and reproducible intra-population and of the direction for development of conservation guidelines inter-population patterns. The ISSR PCRs were performed in and strategies of more general interest. To the best of our 25 ll reaction mixtures containing 25 ng genomic DNA, 1· knowledge, the present study is the first concerning the PCR buffer (Epicentre), 2· Enhancer (Epicentre), 2 mm molecular diversity and population structure of T. tetraphyl- MgCl2 (Epicentre), 200 lm dNTPs (Epicentre), 15 pmol pri- lum. mer and 1 U Taq polymerase (Epicentre). Amplification was performed in a Perkin Elmer 9600 thermal cycler (Norwalk, CT, USA). The PCR reaction Touchdown programme con- MATERIAL AND METHODS sisted of: one cycle at 95 C for 3 min; one cycle at 94 C for 1 min; annealing for 1 min; and 72 C for 2 min. In the Plant material and DNA isolation followed 10 cycles, the annealing temperature was reduced We studied 36 accessions of T. tetraphyllum, of which the 35 for each cycle by 0.5 C to the optimal annealing from North Africa were obtained from the International Cen- temperature. Then, 26 cycles at 94 C for 1 min; annealing tre for Research in Dry Areas (ICARDA, Aleppo, Syria) and for 1 min at temperatures depending on the primer used; one accession from Portugal was obtained from the USDA followed by 72 C for 2 min. Details of amplifications for (USDA, ARS, National Genetic Resources Programme, Germ- each primer are given in Table 2. The reaction products were plasm Resources Information Network – GRIN). The acces- separated by electrophoresis on 1.5% agarose gels in a 0.5· sions from ICARDA consisted of samples obtained by Tris–borate–EDTA (TBE) buffer, stained for 15 min in bulking seeds from several plants (on average 11 plants) col- ethidium bromide, destained for 15 min, and visualised on a lected in each site. Overall, the DNA of 94 individual plants UV transilluminator.

382 Plant Biology 13 (2011) 381–390 ª 2010 German Botanical Society and The Royal Botanical Society of the Netherlands Bellucci, Nanni, Bitocchi, Rossi & Papa Genetic diversity and geographic differentiation in T. tetraphyllum

Table 1. Details of the T. tetraphyllum accessions analysed in the present study.

no. of original altitude genotypes no. of no. of accession accession m geographical analysed genotypes genotypes name donor code state province a.s.l. coordinates per accession per province per state

MdBMa ICARDA 14357 Morocco Beni Mellal 1420 W06 30 N32 03 2 12 36 MBMb ICARDA 14360 Morocco Beni Mellal 1070 W06 27 N32 10 2 MBMc ICARDA 14364 Morocco Beni Mellal 820 W06 44 N32 02 2 MBMd ICARDA 14369 Morocco Beni Mellal 1280 W06 32 N31 55 2 MBMe ICARDA 14370 Morocco Beni Mellal 530 W06 20 N32 35 2 MBMf ICARDA 14371 Morocco Beni Mellal 770 NA 2 MKHa ICARDA 14372 Morocco Khenifra 1300 W05 12 N33 31 4 4 MMAa ICARDA 14359 Morocco Marrakech 350 W07 49 N33 00 2 8 MMAb ICARDA 14362 Morocco Marrakech 300 W07 34 N32 58 2 MMAc ICARDA 14363 Morocco Marrakech 390 W07 34 N32 44 2 MMAd ICARDA 14368 Morocco Marrakech 350 W07 36 N33 00 2 MTEa ICARDA 14358 Morocco Tetouan 220 W05 19 N35 10 4 12 MTEb ICARDA 14361 Morocco Tetouan 140 W05 24 N35 35 2 MTEc ICARDA 14365 Morocco Tetouan 60 W06 00 N35 34 2 MTEd ICARDA 14366 Morocco Tetouan 100 W05 36 N35 04 2 MTEe ICARDA 14367 Morocco Tetouan 40 W05 27 N35 41 2 AALa ICARDA 14348 Algeria Alger 350 E02 35 N36 27 4 4 28 ACOa ICARDA 14356 Algeria Constantine 150 E06 42 N36 47 4 4 AMEa ICARDA 14346 Algeria Medea 720 E03 40 N36 17 2 4 AMEb ICARDA 14347 Algeria Medea 825 E02 53 N36 07 2 AMIa ICARDA 14349 Algeria Miliana 650 E02 20 N36 19 2 8 AMIb ICARDA 14350 Algeria Miliana 550 E02 15 N36 12 2 AMIc ICARDA 14351 Algeria Miliana 700 E02 12 N36 06 2 AMId ICARDA 14352 Algeria Miliana 1000 E02 10 N36 00 2 ASEa ICARDA 14354 Algeria Setif 950 E04 23 N36 06 4 4 ATLa ICARDA 14353 Algeria Tlemcen 395 W01 40 N34 46 4 4 TBEa ICARDA 111290 Tunisia Beja 300 E09 04 01 N36 45 53 3 5 26 TBEb ICARDA 111262 Tunisia Beja 150 E09 05 43 N36 34 50 2 TBIa ICARDA 111634 Tunisia Bizerte 30 E09 40 03 N37 12 33 2 6 TBIb ICARDA 111713 Tunisia Bizerte 5 E09 41 59 N37 06 35 2 TBIc ICARDA 111900 Tunisia Bizerte 10 E09 56 59 N37 08 48 2 TJEa ICARDA 111351 Tunisia Jendouba 10 E08 50 45 N36 57 14 4 4 TKAa ICARDA 109019 Tunisia Kasserine 670 E09 02 N35 23 4 4 TSOa ICARDA 109033 Tunisia Sousse 100 E10 26 N35 51 3 3 TZAa ICARDA 111065 Tunisia Zaghouan 150 E10 13 29 N36 21 08 4 4 POR USDA PI239858 Portugal Alentejo 300 NA 4 4 4 Average 2.6 5.5 30a Total 94

NA = Not Available. aCalculated excluding the Portuguese genotypes.

dextran blue, 0.25 mm EDTA), the PCR products were Chloroplast microsatellite (cpSSR) analysis resolved on 6% polyacrylamide gels run at 100 W constant A screening with three pairs of chloroplast SSR universal power for 1.5 h, and then visualised using GENOMIX primers, previously used by Nanni et al. (2004) in Anthyllis (Beckman, CA, USA). spp. and by Angioi et al. (2009a,b) in Phaseolus vulgaris, was conducted for all of the accessions of T. tetraphyllum exam- Phenotypic characterisation ined with ISSR markers. The primers used (Table S1) were designed based on chloroplast sequences of Nicotiana taba- Each individual of each accession was analysed for colour of cum from Weising & Gardner (1999) to be ampliﬁed across the epicotyls. One month from sowing, the T. tetraphyllum a broad range of species. The cpSSR PCRs were performed plants showed the absence or presence of anthocyanic coloura- in total volumes of 25 ll containing 25 ng genomic DNA, tion at the epicotyl level. On the basis of epicotyl colour, with the reaction mixture and PCR conditions as described three discrete phenotypic classes were identiﬁed: absence of in Nanni et al. (2004). After addition to each sample colour (class 0), presence of light-red coloration (class 1) and (2.5 ll) of 2.5 ll loading buffer (98% formamide, 2% intense red colouration (class 2).

Plant Biology 13 (2011) 381–390 ª 2010 German Botanical Society and The Royal Botanical Society of the Netherlands 383 Genetic diversity and geographic differentiation in T. tetraphyllum Bellucci, Nanni, Bitocchi, Rossi & Papa

Table 2. Details of each ISSR primer screened. geographic distances was carried out to evaluate the correlation between the two data matrices, using the GenALEx, bands version 6 (Peakall & Smouse 2006). total polymorphic annealing Population structure primer base sequence (5¢–3¢) temperature (C) NN% With the aim to further investigate population structure, we used the assignment method implemented in structure, ISSR 8081 (GA)9 C 54 9 7 77.8 version 2.1 (Pritchard et al. 2000). This infers the number of ISSR 8082 (CT)9 G 58 3 3 100.0 a ISSR TE GT (GGT)3 GRC 58 7 4 57.1 clusters (populations), K, present in a sample by comparing b ISSR 10 (AG)7 YC 54 5 4 80.0 the posterior probability for different numbers of putative

ISSR 15 GGTC (AC)7 56 8 8 100.0 populations specified by the user, and it assigns individuals Total 32 26 81.3 to these clusters, giving the percentage of membership (q). Mean 6 5 83.3 Twenty independent runs for each K (from 1 to 12) were performed using 30,000 MCMC (Markov chain Monte Carlo) aY=CorT. b repetitions and 30,000 burn-in periods, using no prior infor- R=GorA. mation and assuming correlated allele frequencies and admixture. The number of clusters (K) was estimated by computing the ad-hoc statistic, DK, based on the rate of change in the log probability of the data between successive Data analysis K values (Evanno et al. 2005). K was set to 2, as this number From the ISSR amplification profiles, only those bands that maximised the DK parameter (Evanno et al. 2005). In this showed consistent amplification were scored (smeared and case, the run length was set to 100,000 burn-ins and 100,000 weak bands were excluded). The ISSR bands were scored as iterations. The output of the software gives, for each individ- present (1) or absent (0) for each sample. A total of 32 bands ual, the percentage membership for the K clusters. We then (26 of which were polymorphic) from 250 to 1700 bp were computed the average percentage membership (q) of the scored. Electrophoretic ISSR patterns were translated into a three populations and of each collection site. To display the binary data matrix of 32 bands · 94 individuals. The matrix membership coefficients on the North Africa map and to was then analysed using different computer packages. As obtain an interpolated map, we used the r software (version T. tetraphyllum is a predominantly selfing species (Couderc 2.8.1, ‘maps’ package), using the Kriging method (R Develop- 1980), all of the individuals studied were considered as ment Core Team 2008). homozygous, and the data were therefore analysed assuming Correlation between the membership coefficient q a haploid genome. (obtained by structure analysis based on ISSR markers) and epicotyl colour classes or cpSSR markers was estimated using a non-parametric test (Spearman’s q, multivariate Genetic diversity, population analysis and differentiation method) implemented in jmp 7.0 software (SAS Institute Inc. Genetic diversity analyses for the ISSR molecular marker data 2007, Cary, NC, USA). were carried out by estimating allele frequencies using the aflp-surv 1.0 computer program (Vekemans 2002); the fre- Geographic structure quency of the marker allele at each locus (1 or +) is set equal to the frequency of the ISSR fragment in the sample. This Spatial autocorrelation between spatial (geographical and alti- method assumes that individuals are haploids or that there is tude) and genetic distances were computed separately using fixed homozygosity at each locus due to complete self-fertili- Spatial Genetic Software (sgs), version 1.0d (Degen et al. sation. Estimates of allele frequencies were used, following 2001). Moran’s I (Moran 1950; Sokal & Oden 1978) was used the approach of Lynch & Milligan (1994), to calculate the for spatial distance classes (in metres), the dimensions of effective number of alleles (ne), the unbiased expected hetero- which were 136 km for geographic distances (10 classes) and zygosity, He, (Nei 1987), genetic distances (Nei 1978) and 94 m for altitude differences (10 classes). The significances of FST values (Wright 1969) between populations. To test the the observed average Moran’s I values were assessed by com- significance of differences in estimates of gene diversity, ne paring them with the corresponding values derived by ran- and He, we used the non-parametric Wilcoxon signed-ranks domly permuting (500 bootstrap) genotypes over the spatial test for two groups arranged as paired observations (Wilco- coordinates of samplings. Ninety-nine per cent confidence xon 1945). To determine the number of haplotypes and envelopes were estimated. The mean overall pairs, corre- for analysis of molecular variance (amova), we applied the sponding to a random distribution, constituted a reference population genetics package arlequin, version 3.1 (Excoffier value against which spatial genetic structure was measured. et al. 2005). Using the tfpga software (Miller 1997), we cal- Values higher than the 99% confidence limit indicate positive culated the Nei’s unbiased heterozygosity, He (Nei 1987) for spatial genetic structure (i.e. proximal individuals are geneti- each locus and at different hierarchical levels, from state to cally more similar than expected if the distribution of geno- collection site. tfpga was also used to compute frequency types was spatially random). Values below the lower distributions and to calculate genetic diversity for the pheno- confidence limit indicate negative spatial structure. Values typic trait (epicotyl colour) and for the chloroplast microsat- within the 99% confidence limits do not deviate significantly ellites used. The Mantel test (Mantel 1967) of genetic and from a random distribution.

384 Plant Biology 13 (2011) 381–390 ª 2010 German Botanical Society and The Royal Botanical Society of the Netherlands Bellucci, Nanni, Bitocchi, Rossi & Papa Genetic diversity and geographic differentiation in T. tetraphyllum

Table 3. ISSR marker analysis for each of the North African states. and 1.19-fold higher, respectively, than the Tunisian population. The differences between the Algerian and markers Tunisian populations for both He and ne were significant N N (P < 0.05, Wilcoxon signed-ranks test); the difference state total polymorphycs % private rarea between the Moroccan and the Algerian populations was significant only for ne (P < 0.05), but marginally not Morocco 30 26 86.7 1 0 significant for He (P = 0.056). Algeria 29 25 86.2 0 0 In Table S2, for the ISSR markers, the number of haplo- Tunisia 27 22 81.5 0 3 types, ISSR marker distribution and genetic diversity were Overall 32 26 81.3 NA NA calculated for each North African state at different hierarchi- Mean 29 24 84.9 NA NA cal levels (state, province and accession). The province with NA = not applicable. the highest genetic diversity identified was Tetouan, Morocco a Rare marker, occurring in <5% of all individuals, frequency <0.05. (He = 0.34), near the Straits of Gibraltar. In this case, 10 haplotypes out of 12 genotypes were found, and 85.7% of the ISSR markers were polymorphic. Miliana province showed RESULTS the higher genetic diversity in an Algerian state (He = 0.32), and Beja province showed the highest genetic diversity within Genetic diversity, population analysis and differentiation Tunisia (He = 0.20). A lower genetic diversity was detected ISSRs for Constantine province (Algeria) with He = 0.01 and two All of the five ISSR primers used gave reproducible banding haplotypes out of four genotypes analysed, even though only profiles; the total number of bands from 250 to 1700 bp was one collection site was available. Considering the accession 32, 26 of which (81.3%) were polymorphic, with an average level, the collection site showing the highest diversity was of six total bands and five polymorphic bands per primer AMIb (in Miliana Province, Algeria, He = 0.27), followed by (Table 2). The percentages of polymorphic band (PPB) values AALa (in Alger Province, Algeria, He = 0.20) and by MBMb ranged from 57.1% for ISSR-TE to 100.0% for ISSR-15 and (in Beni Mellal Province, Morocco, He = 0.18). Two acces- ISSR-8082. For the three states analysed (Table 3), the aver- sion from Morocco (MBMd and MMAd), two from Tunisia age PPB was 84.9%, and varied little among the states, with (TBIa and TBIb) and one from Algeria (AMEb) were com- the Morocco accessions having the highest number of poly- pletely monomorphic (He = 0.00, and single haplotype). morphic bands (30; 86.7%). The Moroccan accessions also had a private marker (with a frequency of 0.40) and for the CpSSR and epicotyl colour Tunisian population we identified three rare markers (occur- Two (ccmp2 and ccmp10) out of three cpSSRs used were ring in no more than 5% of all individuals; frequency <0.05). monomorphic in all of the T. tetraphyllum genotypes analy- The diversity for ISSR markers was analysed in the three sed. Among the 94 genotypes analysed, amplification with North African states for a total of 90 genotypes (Table 4). ccmp7 gave four alleles, with a size ranging from 127 to The total number of haplotypes detected was 73 (81.1%): 31 130 bp. Even with ccmp7, Morocco showed the highest (86.1%) for the Moroccan population, 21 (75.0%) for Algeria genetic diversity (He = 0.58), with three alleles out of four and 21 (80.8%) for Tunisia. The highest genetic diversity (chlorotypes A, B and C); Algeria also had three out of four (He) was found in Morocco (He = 0.33, standard deviation, alleles (chlorotypes A, B and D), but with an overall SD = 0.19), followed by the Algerian population (He = 0.28, He = 0.42 and the frequency of allele cp-129 (chlorotype B) SD = 0.18) and Tunisia (He = 0.20, SD = 0.19). The same equal to 0.70; Tunisia, instead, showed the presence of only pattern was observed for the average effective number of two alleles (chlorotypes A and B) (Tables 4 and S2). More- alleles (ne), with the Moroccan population showing the high- over, Morocco and Algeria each had a private chlorotype: C est value (ne = 1.56, SD = 0.37), the Tunisian population a (with a frequency of 0.11) for the Moroccan accessions and lower value (ne = 1.31, SD = 0.33), and the Algerian popula- D (with a frequency of 0.04) for the Algerian accessions. tion at the halfway point (ne = 1.44, SD = 0.34). Using the With respect to the phenotypic marker evaluated (epicotyl non-parametric Wilcoxon signed-ranks test, the values of He colour), we found that the Moroccan accessions presented and ne for the Moroccan population were significantly the three phenotypic classes with similar frequencies and an higher than for the Tunisian population (P < 0.001). Indeed, overall phenotypic diversity of 0.67, while for the Algerian the Moroccan population had values of He and ne 1.65-fold accessions the class ‘absence of colour’ was the most frequent

Table 4. Diversity analysis for each of the North African states.

ISSR cpSSR7 epicotyl colour

genotypes haplotypes state N N (%) He ne chlorotypes (N) He phenotypic diversity

Morocco 36 31 (86.1) 0.33 1.56 A ⁄ B ⁄ C (3) 0.58 0.67 Algeria 28 21 (75.0) 0.28 1.44 A ⁄ B ⁄ D (3) 0.42 0.61 Tunisia 26 21 (80.8) 0.20 1.31 A ⁄ B (2) 0.48 0.27

Overall 90 73 (81.1) 0.27 1.44 A ⁄ B ⁄ C ⁄ D (4) 0.55 0.65

Plant Biology 13 (2011) 381–390 ª 2010 German Botanical Society and The Royal Botanical Society of the Netherlands 385 Genetic diversity and geographic differentiation in T. tetraphyllum Bellucci, Nanni, Bitocchi, Rossi & Papa

Table 5. Analysis of molecular variance. membership (q1 = 0.87), Tunisia was assigned to cluster 2 (q = 0.79), while Algeria showed a high level of admixture, sum of variance %of 2 with q = 0.47 and q = 0.53. In Fig. 1a and b, we also com- sources of variation df squares component variation F 1 2 ST puted the percentages membership for each collection site, Among States 2 54.22 0.76 14.79 0.15** where each vertical line represents an accession (collection Within States 87 383.38 4.41 85.21 site) and the percentage membership is the mean of individual Total 89 437.60 5.17 100 qs. All of the Moroccan collection sites showed a high percentage membership for cluster 1, with the highest q1 for the **P < 0.0001. collection sites MTEb and MTEe (0.96), and the lowest for collection sites MKHa (q1 = 0.70). The Tunisian collection Table 6. Nei’s genetic distances (above diagonal) and FST values (below sites showed medium-high percentages of membership to diagonal). cluster 2, from q2 = 0.96 for TKAa and TSOa, to q2 = 0.66 Morocco Algeria Tunisia for TBEa and TBIb, with the exception of only one collection site, TBIc, which showed a high percentage membership to Morocco – 0.05 0.08 cluster 1 (q1 = 0.85), and collection site TBEb showing Algeria 0.11 – 0.06 admixture (q1 = 0.44). The Algerian collection sites had very Tunisia 0.19 0.17 – variable percentages of membership: three sites (AMIa, AMIb and AMId) were assigned to cluster 1 (average q1 = 0.92), three (ACOa, ATLa and ASEa) to cluster 2 (average (0.52), with an overall phenotypic diversity of 0.61; for the q2 = 0.89) and the remaining four collection sites had high Tunisian accessions, the class ‘absence of colour’ was clearly levels of admixture with average q1 = 0.58 and q2 = 0.42. dominant (frequency of 0.85) and He = 0.27 (Tables 4 and At the genotype level, the same trend as for the collection S2). sites was observed, with individuals belonging to the same collection site showing the same percentages of membership FST to the same cluster, with few exceptions. All of the Portu- The overall amova analysis indicated that there is higher guese genotypes were assigned to cluster 1, with a very high genetic variation within states (85.21%) than among states percentage of membership (q1 = 0.95). If we set a threshold (14.79%), with an FST of 0.15 (Table 5). Hierarchical analysis value for identification of the admixed genotype of 0.70 (the of molecular variance indicates that genetic variation is lowest in Moroccan collection sites), we observe that the greater within each state (85.21%) and province (64.80%) Moroccan genotypes were 94% assigned to cluster 1 (with than among similar hierarchical levels; at the level of collec- 6% admixed genotypes), the Tunisian genotypes were 77% tion site, the highest genetic differentiation is between sites assigned to cluster 2 and 12% to cluster 1 (with 11% (70.73%), rather that within. Tunisia had the highest FST admixed genotypes), while the Algerian genotypes showed value (0.16), followed by Algeria (0.15) and Morocco (0.14). 36% membership to cluster 1, 39% to cluster 2 and 25% Nei’s (1978) unbiased genetic distance, and FST matrices were admixed genotypes. estimated over all states, using the 32 ISSR markers (Table 6): The correlation between epicotyl colour classes and the lowest value of genetic distance among pair values was STRUCTURE membership coefficient (q) was significant that between Morocco and Algeria (Nei’s unbiased genetic (Spearman’s correlation q = )0.35, P = 0.0004, pair-wise cor- distance = 0.05, FST = 0.11), while the highest was between relation = 0.33, P = 0.001), while for the marker cpSSR7 the Morocco and Tunisia (Nei’s unbiased genetic distance = 0.08, correlation was slightly significant (Spearman’s correlation FST = 0.19). All the pair-wise comparisons between the three q = 0.28, P = 0.01, pair-wise correlation 0.23, P = 0.05). states were significant (110 permutations, P < 0.05). Mantel test Population structure The Mantel test was applied to the matrix of Nei’s unbiased Population structure analysis was conducted using all 94 genetic distances and the matrix of geographic distances, genotypes and 32 ISSR markers. The first step was estimation which showed a weak, but significant, correlation between of the number of identifiable populations (K) that character- the two matrices (R = 0.22 and P < 0.01). Moreover, we ised the sample without considering any prior information; tested the correlation between molecular and altitudinal dis- the plot of the average log_{e} likelihood values over 20 runs tances and observed an R = 0.08 with P < 0.01. These signifi- for K values ranging from 1 to 12, showed that the log_{e} cant relationships between genetic structure and both the likelihood estimates increased progressively as K increased geographical and altitudinal variations were further investi- (data not shown). Therefore, we used an ad-hoc statistic, DK gated and validated using spatial autocorrelation analysis. (Evanno et al. 2005) to infer the real number of populations While no significant trends were found for altitude levels, the and found that the genetic structure of our sample was char- spatial autocorrelation analysis confirmed the significant acterised by two groups, K = 2, named cluster 1 and cluster 2. effect for geographic distances obtained by the Mantel test. The second step of the analysis was assignment of the 36 collection sites and 94 individual genotypes to inferred clusters 1 Geographical structure and 2. We calculated the proportions of membership (q1 for Within the three distance classes (below about 400 km), posi- cluster 1, and q2 for cluster 2, with q1 +q2 = 1) of each pre- tive Moran’s I values were detected, indicating that ‘proximal’ defined population in each of the two clusters. The state of individuals (including those of the same collection site) Morocco was assigned to cluster 1, with a high percentage were more genetically similar than expected from a random

386 Plant Biology 13 (2011) 381–390 ª 2010 German Botanical Society and The Royal Botanical Society of the Netherlands Bellucci, Nanni, Bitocchi, Rossi & Papa Genetic diversity and geographic differentiation in T. tetraphyllum

Fig. 1. a: Population structure as inferred by STRUCTURE, v2.1 software. Each vertical line represents an accession (collection site), and the percentage of membership is the mean of genotypes qs (q1 blue and q2 yellow). Collection sites are ordered by longitude. b: Two-dimensional representation of admixture levels: interpolated membership coefficients in the two apparent subpopulations (using the statistical software R). The colour scale on the right indicates the membership coefficient to cluster 1 (q1). POR, Portuguese accession; M_TE, Moroccan accessions from Tetouan Province; M_BM, Moroccan accessions from BeniMellal Province; M_KH, Moroccan accessions from Khenifra Province; M_MA, Moroccan accessions from Marrakech Province; A_TL, Algerian accessions from Tlemcen Province; A_AL, Algerian accession from Alger Province; A_MI, Algerian accessions from Miliana Province; A_ME, Algerian accessions from Medina Province; A_SE, Algerian accession from Setif Province; A_CO, Algerian accession from Constantine; T_BE, Tunisian accessions from Beja Province; T_BI, Tunisian accessions from Bizerte Province; T_ZA, Tunisian accession from Zaghouan Province; T_SO, Tunisian accession from Sousse Province; T_KA, Tunisian accession from Kasserine Province; T_JE, Tunisian accession from Jendouba Province. distribution (see Fig. 2). The degrees of correlation, however, rapidly decreased, and negative I values were found starting from the fourth class (above 450 km), suggesting a negative spatial structure in this range of distances (from 450 to 800 km). From the sixth class (beyond 800 km), the I values measured tended to not fall out of the 99% confidence enve- lope, indicating that above that distance I values did not deviate significantly from a random distribution.

DISCUSSION This is the ﬁrst study that has analysed molecular diversity and population structure within T. tetraphyllum. We analysed North African populations obtained from the ICARDA (International Centre for Research in the Dry Areas, Aleppo – Syria) germplasm collection centre. Our study has here pro- Fig. 2. Spatial autocorrelation analysis: Genetic distances versus geo- vided an optimised method for evaluating genetic diversity of graphic distances.

Plant Biology 13 (2011) 381–390 ª 2010 German Botanical Society and The Royal Botanical Society of the Netherlands 387 Genetic diversity and geographic differentiation in T. tetraphyllum Bellucci, Nanni, Bitocchi, Rossi & Papa

T. tetraphyllum using ISSR markers, which is an economic lum extended its area of distribution from Morocco to Tuni- and rapid approach used on an alternative and neglected sia. Indeed, for all the markers analysed (ISSR, cpSSR, and legume species, and can be useful for further investigation. colour of the epicotyl; Table 4), a general reduction in Unlike co-dominant markers, estimation of allele frequencies genetic diversity from Morocco to Tunisia can be seen. Fur- from dominant markers, such as ISSR, presents some statisti- thermore, in Morocco we can identify a private ISSR frag- cal difficulties (Lynch & Milligan 1994). These difficulties ment and a private cpSSR allele, both with a frequency should be taken into account and can be resolved using higher than 10%. These alleles could be lost during the appropriate methods, such as Bayesian methods (Zhivotovsky migration of populations of T. tetraphyllum from Morocco to 1999), and better estimators for analysis of dominant markers Algeria and Tunisia. Similarly in Tunisia, we can find three (Lynch & Milligan 1994). rare alleles that, on the contrary, showed a frequency higher Ninety genotypes of T. tetraphyllum from three North Afri- than 5% in Morocco and Algeria. can states were analysed using five ISSR primers, yielding 32 Based on results of the genetic diversity gradient observed scorable bands; this number is lower than in similar studies and of the cline of distribution highlighted by structure based on ISSRs in other plant species, but given the some- analysis, we can state that T. tetraphyllum extended its area what preliminary flavour of this study, it proved large of distribution from Morocco to Tunisia, and we can also enough to provide some useful insights. The three popula- suggest a possible hypothesis in relation to the origin of tions show percentages of polymorphism ranging from 81.5% T. tetraphyllum. This hypothesis is also supported by the dis- (Tunisia) to 86.7% (Morocco), while the overall polymor- tribution of two species still classified in the genus Anthyllis, phism was 81.3%. Other studies in plant species with a large but very close to T. tetraphyllum, and belonging to the ‘tetra- distribution, based on a greater number of individuals and phylla clade’ (Nanni et al. 2004): A. hamosa, the distribution ISSR bands, yielded similar ISSRs values of polymorphism of which is limited to the Iberian peninsula, and A. cornicina, (in Penstemon, Wolfe et al. 1998 and in Primula obconica, which is distributed both in the Iberian peninsula and in Nan et al. 2003). The ISSR polymorphism detected revealed a Morocco (Greuter et al. 1989). The hypothesis is that T. tet- high level of variability, suggesting that ISSR technology is a raphyllum arose in Morocco, or that it originated from the powerful and efficient approach to assess population struc- Iberian Peninsula. To resolve the question of the origin of ture in T. tetraphyllum. T. tetraphyllum, a more detail analysis should be conducted, The populations of T. tetraphyllum showed clear geograph- including populations from other Mediterranean countries ical structures, with the differentiation associated mainly to and in particular from the Iberian Peninsula. It is also inter- longitudinal differences, which extended from Marrakech esting that in the province of Tetouan, the nearest to the (collection site MMAa, 0749¢ W) in Morocco to Sousse (col- Gibraltar Strait, we identified the highest value of He (0.34), lection site TSOa, 1026¢ E) in Tunisia. We were thus able to and we can find almost all of the ISSR markers (28 out of observe a cline in distribution of this species from west to 32), three out of four cp-SSR alleles, all the classes of epicotyl east in the Mediterranean region considered, and the pattern colour and the Morocco private allele, with a frequency of was highlighted using R software, with the plotted struc- 0.43. Moreover, the ITS sequences and cpSSR analysis (Nanni ture membership coefficients (see Fig. 1a and b). et al. 2004) showed that the closest species to T. tetraphyllum Both the spatial autocorrelation and amova results indi- is A. hamosa, as previously mentioned, a species endemic in cated a high population structure for nuclear and chloroplast the Iberian Peninsula. markers. This is in agreement with a largely autogamous The Mediterranean region is experiencing a remarkable breeding system, where a low frequency of out-crossing effect of climate change and human activities, and it is thus reduces migration between populations by reducing the pol- important that there is continuous monitoring of species and len flow (Hamrick & Godt 1990). Moreover, the Mantel test population dynamics, to plan and enact valuable conservation and spatial autocorrelation analysis suggested that population programmes for species such as T. tetraphyllum. This study structure was coherent with the Isolation-by-Distance model has implications for the use and conservation of this alterna- (IBD) of migration proposed by Wright (1946), with a positive pasture legume through its potential role for multiple tive and significant Moran I value between plants at distances uses in the changing environments of the Mediterranean <400 km. This indicates again that geographic distance was basin. Analysis of distribution of genetic variability suggests an important factor for genetic divergence, and genetic drift that the higher percentage of total variation is still harboured might be the primary evolutionary force during the process. within state and provinces and among collection sites, indi- Any genetic differentiation, however, was not simply a func- cating that in the North African environment, this species is tion of spatial distance, as indicated by the moderate correla- not yet endangered. tion between genetic and geographic distance matrices. Other factors known to be important include the life-history char- ACKNOWLEDGEMENT acteristics, population arrangements (patchy versus continuous), physical barriers to dispersal and habitat factors The authors would like to thank the Genetic Resources affecting geographical distribution, such as soil, temperature Unit of the International Centre for Research in Dry Areas and salinity. The high levels of variability observed within (ICARDA, Aleppo, Syria) for supplying the seeds. countries may be required to maintain plasticity in a highly patchy and diverse environment like the Mediterranean Basin SUPPORTING INFORMATION (Ortiz-Dorda et al. 2005). Based on the markers used, the most important outcome of Additional Supporting Information may be found in the this study is that the results strongly suggest that T. tetraphyl- online version of this article:

388 Plant Biology 13 (2011) 381–390 ª 2010 German Botanical Society and The Royal Botanical Society of the Netherlands Bellucci, Nanni, Bitocchi, Rossi & Papa Genetic diversity and geographic differentiation in T. tetraphyllum

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390 Plant Biology 13 (2011) 381–390 ª 2010 German Botanical Society and The Royal Botanical Society of the Netherlands