Biochemical Systematics and Ecology 55 (2014) 296e304

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Biochemical Systematics and Ecology

journal homepage: www.elsevier .com/locate/biochemsyseco

Evolutionary and demographic history among MaghrebianMedicago species (Fabaceae) based on the nucleotidesequences of the chloroplast DNA barcode trnH-psbA

Zitouna Nadia a, Gharbi Maroua a, Ben Rhouma Hela a,Chennaoui-Kourda Houda a, Haddioui Abdelmajid b, Trifi-Farah Neila a,Marghali Sonia a,*

a Laboratory of Molecular Genetics, Immunology and Biotechnology, Biology Department, Sciences Faculty of Tunisia, Campus University,2092 El Manar, Tunis, Tunisiab Laboratory of Genetics and Vegetal Biotechnology, Biology Department, Sciences and Technics Faculty, University of Sultan MoulaySlimane, Beni-Mellal, Morocco

a r t i c l e i n f o

Article history:Received 9 October 2013Accepted 22 March 2014Available online

Keywords:MedicsMaghrebPhylogenyDemographyEvolutiontrnH-psbA

Abbreviation: CBOL, Consortium of Barcode of LifRAPD, Random Amplified Polymorphic DNA; SSR, SDevelopment Institute.* Corresponding author. Tel.: þ216 71 87 26 0; fa

E-mail addresses: soniamarghali@yahoo.fr, sonia

http://dx.doi.org/10.1016/j.bse.2014.03.0160305-1978/� 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t

Data from trnH-psbA intergenic spacer (cpDNA) were analyzed to elucidate molecularevolution within and among Maghrebian species of Medicago. The spacer highlighted ahigh interspecific variation and a low intraspecific diversity among species. Haplotype andnucleotide diversities revealed high level of variation. Parsimony and median-joiningNetwork methods revealed (1) the segregation into 17 haplotypes; (2) the ancestralbehaviour of the annual Medicago minima and (3) the clusters are independent of thegeographic origin. The neutral evolution of Wright and Fisher is rejected since the Tajima’sD values deviated from 0. Besides, the statistical analyses are in agreement with an evo-lution into stable populations’ size.

� 2014 Elsevier Ltd. All rights reserved.

1. Introduction

Medicago, a genus of Eurasian origin, has spread throughout the world. The Mediterranean Basin is the primary center ofdiversification of most of the annual species. In North Africa, these species can be found in wide-ranging habitats (Haddiouiet al., 2012). Usually called Medics, the existed 87 species and 18 intraspecific taxa, two-thirds are annuals and derived fromperennial ancestors at the end of the Tertiary era. Annual species ofMedicago are anticipated to show high levels of nucleotidesequence’s conservation and similar genetic organization (Steele et al., 2010).

Several molecular data have been used to assess the phylogenic relationships ofMedicago L. based on ITS and ETS sequencedata (Bena et al., 1998; Downie et al., 1998). Maureira-Butler et al. (2008) sampled 56 species of Medicago for the nuclear-

e; ITS, Internal Transcribed spacers; ETS, External transcribed spacers; PCR, Polymerase Chain Reaction;imple Sequences Repeats; IRLC, Inverted Repeat-Lacking Clade; SARDI, South Australian Research and

x: þ216 70 86 04 32..marghali@fst.rnu.tn (M. Sonia).

Z. Nadia et al. / Biochemical Systematics and Ecology 55 (2014) 296e304 297

encoded genes CNCG5 and b-cop and the mitochondrial gene rps14-cob. The use of chloroplastic DNA intergenic spacers toassess the phylogeny of the genus has been focused on trnK/matK (Steele et al., 2010).

Characterized by a highly conserved organization among flowering plant species, Medicago’s chloroplast DNA (cpDNA) ismarked by the loss of one copy of the large inverted repeat and belongs to the IRLC (Inverted Repeat-Lacking Clade). This clusteris dominated by temperate herbaceous genera such as Pisum, Cicer arietinum, Medicago sativa and Trifolium but also lessfamiliar genera such as Astragalus (Magee et al., 2012).

One of the most variable non-coding intergenic spacers is trnH (GUG)-psbAwhich is located downstream of the trnK intron(Shaw et al., 2007). Because of its short length (often<500 bp), trnH-psbA has been proposed as a barcode for plants, eitheralone or in conjunction with other sequences such as rbcL and matK (Kress and Erickson, 2007).

In the present survey, wewill investigate the spacer trnH-psbA, of Moroccan and Tunisian species ofMedicago. The specificaims of this work were: (i) to examine global patterns of cpDNA genetic variation in Medicago and to compare the geneticpolymorphism between seeds from Tunisian and Moroccan accessions (ii) to infer phylogeographic relationships amongspecies (iii) to affiliate the evolution demographic scenario within the genus in the Maghreb region.

2. Material and methods

2.1. Taxon sampling

Nine populations of Medicago are used for screening and 5 specimens represented every species. The Tunisian biologicalmaterial consists of seeds fromMedicagominima, Medicago orbicularis andMedicago polymorpha. This collection is available inthe core-collection located in the Faculty of Sciences of Tunisia (an 8 �C room). Concerning the Moroccan germplasm, the sixanalyzed species:Medicago truncatula,Medicago scutellata,Medicago murex,M. minima,M. orbicularis andM. polymorpha, areprovided from the SARDI (South Australian Research and Development Institute) core-collection under codes noted on thesupplementary material Table 1.

2.2. Molecular methods

2.2.1. Primers design and PCR amplificationsAbout 0.1 g of harvested seedlings is used for total cellular DNA extraction using the commercial Invitrogen- purification

DNA kit and following the manufacturer protocol.As many of different non-coding regions of the chloroplast genome have been investigated, the growing number of

published sequences from several plant species provides the opportunity to design universal angiosperm PCR primers (Shawet al., 2007). The corresponding trnH-psbA primers are upstream (50-TGATCCACTTGGCTACATCCGCC-30) and downstream (50-GCTAACCTTGGTATGGAAGT-30). This pair is used for PCR screening and for sequencing reactions.

The PCR are carried-out in a total mixture of 25 ml containing 20 ng of template DNA, 1.5 ml of Taq Buffer, 3 ml MgCl2(25mM),1.5 ml dNTPmix,1.5 U of taq polymerase (Invitrogen), 0.2 mMof each reverse and forward primer and QSP of Nucleasefree Water.

After an initial denaturation step at 94 �C for 4min, 35 PCR cycles are followedwith the denaturation step at 94 �C for 1min,the annealing step at 51 �C for 1 min and the extension step at 72 �C for 2polymorphamin. A final extension step at 72 �C for10 min is also included. PCR products were first checked on 1.5% agarose gel in (0.5�) TBE at 120 V for 2 h, before beingcleaned with Pure Link PCR Purification Kit-Invitrogen. Direct sequencing followed the amplification of target sequences.

2.2.2. Sequencing trnH-psbAThe sequencing step is performed with the ABI Prism 3130 DNA automated sequencer (Applied Biosystems; HTDS,

Tunisia). To ensure the accuracy of trnH-psbA, both strands were sequenced.

2.2.3. Softwares and analysesThewhole dataset is firstly aligned usingMEGA5 (Tamura et al., 2011) and then checked by eye and refinedmanually. Gaps

were positioned to minimize the mismatches and sequences are finally verified by BLAST function.The analyzed sequences are deposited in the GenBank database (www.ncbi.nih.gov) under accessions numbers listed in

Table 1 (Supplementary material).After trimming off a 10 bp fragment at the upstream of the sequences for which no unambiguous alignment could be

achieved, Geneious Pro software (Drummond et al., 2011) is used to explore the polymorphism by estimating the followingparameters: length, nucleotides’ rates, GC content, identical sites and pairewise identity; for each species.

DnaSP5 (Librado and Rozas, 2009) allowed to calculate the nucleotide diversity (p), the number of haplotypes and thehaplotype diversity (Hd), the gene flow (Nm) and the interspecific variation (Fst). This software estimated also the totalnumber of: indels, sites (variable, conserved and parsimony information), mutations and singletons. The same softwarerepresented the substitutions’ patterns.

MEGA5 software conducted the following statistics 1) Tajima’s D (1993) for the neutral evolution test and 2) the transition/transversion rates (R) under Kimura (1980) model.

Z. Nadia et al. / Biochemical Systematics and Ecology 55 (2014) 296e304298

The evolutionary study concerned also two additional genebank sequences corresponding to the perennial M. sativa andthe annual, biannual or occasionally short-lived perennial Medicago lupulina. The pairewise genetic distances matrix, isestablished using the algorithm of Tajima and Nei (1984). Mesquite version 2.75 (Maddison and Maddison, 2011) alloweddrawing the heuristic evolution of Medicago, in a phylogenetic context using a Parsimony analysis.

A median-joining analysis is applied to display the haplotypic representation (relationships and ancestor). The graphicalNetwork is constructed using Network software version 4.5.0.0.

In order to test the evidence of demographic range expansions, the curves Mismatch distributions are reported.

3. Results

3.1. Sequences characteristics and gene content

Even if the PCR were easily conducted using the universal primers, the analysis of trnH-psbA in this work required thedouble-sensed sequencing (forward and reverse). Herein, the rates of amplificationwere ranged from 60% (M. polymorpha) to100% (M. murex).

The spacer varies in length from 268 bp to 492 bp for individuals respectively belonging to theMorrocanM.minima andM.truncatula, while the consensus sequence is 568 bp (Table 1). The alignment has obvious a 40 bp-gap between both M.polymorpha’s populations (Morrocan, 422 bp; Tunisian, 464 bp). In addition, a high variation is recorded at the intrapopu-lation level mainly for the Tunisian population of M. polymorpha (392e464 bp).

The pairwise identities exposed themaximum of similarities among TunisianM. orbicularis (98.8%) unless the minimum isamong M. truncatula (76.2%). This model legume is considered by this analysis as the most polymorphic species (Table 1).

The rate of Adenine (A) is higher than Guanine (G), with respectively 34.8% and 11.7%. The pyrimidine bases’ ratio ofThymine (T) and Cytosine (C), are also not uniformly distributed (T¼ 37.9%; C¼ 15.6%). These results echoed consequently thelower rate in GC content.

The spacer trnH-psbA undergo several ponctual mutational’ events (transversion and transition), represented in table 2.The total transition’s rate among overall North AfricanMedics is equal to 57.42% (53.38% for Moroccan material and 27.02% forTunisian one). The puric transition G0A is more relevant in the Moroccan species (21.42%), while for Tunisian species thepyrimidic motif C0T was the most abundant (15.16%). The transversion’ rate was higher for Tunisian Medics (72.98%).

Besides, several long insertions and deletion events are shown among the 45 analyzed sequences (Table 3). Actually, M.minima lost a 176 bp frame comparing with the other species. In addition, five insertions were detected, among them fourwere considered as long (between 4 bp and 100 bp) and a specific frame-shift insertion of (T) characterized MorrocanMedicago orbicualris in the 277th position.

3.2. Genetic divergence among Medics

Length consensus sequences among regions were 552 bp for Morrocan Medicago species, 508 bp for Tunisian and 568 bpfor the overall Maghrebian ones (Table 4). The total number of indel sites is twice time higher among Morrocan species(80.98%). For the whole analyzed sequences, 463 indel sites are counted, with a rate of 82.51%.

Among excluding-indel sequences, the number of conserved sites is higher in Tunisian species (275 sites) while 76conserved sites are obtained through all combined sequences.

Sequences of the core-collected Morrocan species carried more variable and parsimony informative sites with 27 and 24respectively.

Among the Maghrebian variable sites 4 singletons are observed, 5 are shown in the Tunisian material and 3 in theMorrocan one.

Table 1Sizes ranging, Pairwise intraspecific identity and Nucleotides’ content of the trnH-psbA amplicons.

Species Origin Length ranging(bp)

% Pairwiseidentity

%T %C %A %G %GC

M. orbicularis Morrocco 385 (375e400) 97.8 38.3 16.3 34.8 10.6 13.4M. orbicularis Tunisia 394 (389e405) 98.8 38.9 16.1 34.5 10.4 13.3M. polymorpha Morrocco 464 (458e468) 98 36.2 14.2 37.9 11.8 13M. polymorpha Tunisia 422 (392e464) 84.8 37.9 15.1 35.9 11.1 13.1M. minima Morrocco 298 (268e325) 87.4 37.0 16.6 32.7 13.7 15.2M. minima Tunisia 309 (304e320) 98.5 36.9 17.0 34.1 12.1 14.5M. truncatula Morrocco 478 (455e492) 76.2 35.7 16.9 33.3 14.1 15.5M. scutellata Morrocco 369 (345e384) 81.7 40.4 14.1 35.0 10.5 12.3M. murex Morrocco 443 (418e467) 88 39.3 14.1 35.3 11.2 12.7Overall 568 90.09 37.9 15.6 34.8 11.7 13.6

bp: Base pairs.In bold: sizes ranging; Surligned: Consensus size ranging; Italic: pairwise identity ranging.

Table 2Estimation of the trnH-psbA substitution patterns among Morrocan, Tunisian and Maghrebian species under the Tamura-Nei (1993) (a, b and c respectively).

A T C G

(a)A e 8.89 3.47 6.92T 8.28 e 7.03 2.68C 8.28 18.01 e 2.68G 21.42 8.89 3.47 e

Mean Transition rate53.38%

Transversion rate46.64%

(b)A e 13.78 5.96 1.28T 12.71 e 6.57 4.04C 12.71 15.16 e 4.04G 4.01 13.78 5.96 e

Mean Tansition rate27.02%

Tranversion rate72.98%

(c)A e 6.73 1.70 6.52T 10.55 e 4.28 2.32C 10.55 16.99 e 2.32G 29.63 6.73 1.70 e

Mean Transition rate57.42% Transversion rate

42.60%

Transitional substitutions are in bold; Transversional substitutions are in italics.Only substitutions within rows should be compared.

Table 3Specific indel mutations characterizing Medicago species and populations (Long and frame-shift).

Event Species Sites (bp)

Deletion Medicago minima 90e266Insertion Medicago polymorpha (100% of Morrocan and

40% of Tunisian seeds)166e258

Medicago murex 166e266Insertion Medicago truncatula 170e266Insertion Medicago orbicularis Morrocco 277Insertion Medicago scutellata 297e309Insertion Medicago scutellata

Medicago truncatula351e356

Table 4Characteristics and divergence of Medicago species in correlation with their geographic origins.

Morrocan species Tunisian species Maghrebian species

Number of analyzed sequences 30 15 45Consensus length 552 508 568Length excluding indel sites 447 (80.98%) 214 (42.12%) 463 (82.51%)Variable sites 27 18 28Conserved sites 77 275 76Parsimony information 24 13 24Singleton variable sites 3 5 4R 1.63 0.35 1.64Number of haplotypes 15 13 17Hd 0.899 0.981 0.823p 0.057 0.021 0.042D �1.045

NS p > 0.10�0. 195NS p > 0.10

�1.498NS p > 0.10

Hd: Haplotypic Diversity; p: Nucleotidic Diversity; D: Tajima’s neutrality Test; NS: Non Significant, R: transition/Transversion ratio.

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The transition/transversion ratio (R) is higher than 1 in Morrocan and overall analyzed species, against a (0.35) value forthe random Tunisian seeds. This result confirms the highest level of transversion among the Tunisian Medicago species. Apositive correlation between R and the genetic divergence in trnH-psbA, is showed. Actually, the transversion’s rate is abovethe transition’s rate until a 31.6% of genetic divergence yielding an apparent ratio equal to 0.14 (Data not shown).

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In line with the relatively high level of variation found in the Maghrebian species, trnH-psbA sequences exhibited 17haplotypes. The 15 Tunisian individuals involved into 13 haplotypes while the 30 Morrocan Medics were divided into 15haplotypes. The mean haplotype diversity Hd was 0.823 (Hd for Tunisian species ¼ 0.981; Hd for Morrocan species ¼ 0.899).The high haplotype variation is contrasted with low nucleotide diversity (p ¼ 0.057 and 0.021).

Tajima’s D was used to test the neutral evolution hypothesis under the Wright and Fisher Equilibrium. In this survey,values were non significant and generally skewed towards negative values (Morocco, �1.045; Tunisia, �0.195 and Maghreb,�1.498). Consequently, the estimated neutrality tests rejected the neutrality assumption in the total analyzed sequences.

3.3. Species evolution based on clustering trees and haplotypic networks

In order to investigate the evolutionary history of the annual analyzed Medicago species, two additional sequences cor-responding to Medicago sativa and M. lupulina; were added to our dataset.

The genetic distances GD (Tajima and Nei, 1984) were balancing between 0.003 and 0.282. The low genetic distancesconfirm the recent evolution of this forage genus and specifically in the Maghrebian regions. The GD shows also the closenessof M. sativa and M. lupulina with the annual analyzed Medics.

An UPGMA tree was drawn using Maximum Parsimony method (Fig. 1). The clusters showed close profiles of Tunisian andMorrocan materials. Sequences representing M. murex were straddling with the other clusters regardless to the geographicorigin. Although, specimens voucher ofM. orbicularis, M. minima, M. scutellata andM. truncatulawere grouped together in thedendrogram. Indeed,M. sativa, the unique perennial species, was related to the cluster gathering the speciesM. murex andM.truncatula. M. lupulina behaved close to M. minima.

To confirm the genealogical hierarchies amongmaghrebianMedicago species, an unrooted phylogenetic network based onvariation of the inferred haplotypes resulting from non-coding cpDNA trnH-psbA was illustrated in Fig. 2. The Network isrepresented by 17 haplotypes with 5 median vectors corresponding to intermediate evolutionary step. These median vectorsshematized by dark black nodes were not collected in our survey. The central haplotype H1 is considered as the majorhaplotype classes composed of 18 accessions of M. orbicularis and polymorpha. This haplotype was inferred as the ancestralone based on both frequency and position. From H1, six haplotypes were diverged. The H13 regroupedM. minima sequences,

Fig. 1. UPGMA tree revealing the evolutionary relationships of the Medicago genus and species.

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and constituted the second main haplotype; whereas H3 (Medicago truncatula) occurred the highest level of mutations (30).The perennial Medicago sativa is represented by the H11 and occurred 2 mutations in the 335th and 339th sites.

3.4. Demographic expansion

To test the hypothesis of population expansion in Moroccan, Tunisian and Maghrebian species, we calculated thefrequency distribution of pairwised nucleotide differences among individual haplotypes (Mismatches). The mismatchdistributions (Fig. 3) were clearly inconsistent with the bell-shaped curve expected for an expanding population. Actually,the observed mismatch distributions for cpDNA haplotypes are calculated separately for the Morrocan (a), Tunisian (b)and Maghrebian (c). The Morrocan mismatch revealed a bimodal representation, which can be explained by a slowlygrowth in the populations’ size. For Tunisian species the obtained profile was unimodal and thus congruates with pre-dicted under a model of sudden range expansion. Gene Flow (Nm) and inter-specific variations (Fst) were highlysignificative (Nm ¼ 3.50 and Fst ¼ 0.06). For overall Maghrebian species, the resulting profile exhibited a multimodaldomain suggesting contractional populations. These deviations were contrasted by negative and non-significant Tajima’sD values (Table 3).

4. Discussion

4.1. trnH-psbA: PCR, sequencing efficiency and variation

In our survey, trnH-psbA intergenic spacer was chosen as a DNA barcode to study the polymorphism among MaghrebianMedics.

This frame presents a short length (around 300-400 bp); however the sequencing step was difficult to conduct. Starr et al.(2009) reported that sequences were unreadable for approximately 9% of trnH-psbA, which required the use of the

Fig. 2. Evolutionary haplotypic Network on the basis of the 45 analyzed sequences additionating to the two genebank-imported sequences Branch length isproportional to the number of occurring mutations Haplotype 1: 19 [Medicago orbicularis Morroco 1e5; Medicago polymorpha Morroco 24; Medicago murexMorroco 5; Medicago lupulina; Medicago orbicularis Tunisia 2e5; Medicago polymorpha Tunisia 1e5] Haplotype 2: 2 [Medicago polymorpha Morroco 1,5]Haplotype 3: 1 [Medicago truncatula Morroco 1] Haplotype 4: 2 [Medicago truncatula Morroco 2; Medicago murexMorroco 1] Haplotype 5: 1 [Medicago truncatulaMorroco 3] Haplotype 6: 1 [Medicago truncatula Morroco 4] Haplotype 7: 1 [Medicago truncatula Morroco 5] Haplotype 8: 2 [Medicago murexMorroco 2, 3]Haplotype 9: 1 [Medicago murexMorroco 4] Haplotype 10: 4 [Medicago scutellata Morroco 1e4] Haplotype 11: 1 [Medicago sativa] Haplotype 12: 1 [Medicagoscutellata Morroco 5] Haplotype 13: 6 [Medicago minima Morroco 1,5; Medicago minima Tunisia 1e4] Haplotype 14: 2 [Medicago minima Morroco 2,4] Haplotype15: 1 [Medicago minima Morroco 3] Haplotype 16: 1 [Medicago orbicularis Tunisia 1] Haplotype 17: 1 [Medicago minima Tunisia 5].

Fig. 3. Mismatch Distributions of the pairwise difference among (a) Tunisian (b) Moroccan and (c) Maghrebian species.

Z. Nadia et al. / Biochemical Systematics and Ecology 55 (2014) 296e304302

bidirectional sequences. Part of the difficulty associated with the sequencing can be explained by the presence of numeroushomopolymeric regions or by the host of pseudogene (rps19) (Starr et al., 2009).

The 45 deposited sequences’ sizes ranged from 268 to 492 bp for individuals of respectively M. minima Tunisia and M.truncatula. This size overlapping is explained by the host of rps19, and/or by the proximity of trnH-psbAwith the Large SingleCopy of the chloroplast DNA (which contracts and retracts).

The rate of the identical sites is correlated with the pairwise identities, and shows high conservation rates. This resultconfirms the efficiency of this integenic spacer (trnH-psbA) as a barcode. It supports also the high level of polymorphismcharacterizing Maghrebian Medicago crops (Maureira-Butler et al., 2008).

Analysis of the nucleotides content revealed an advantage of the AT content. This confirms the richness of the non-codingregions of the chloroplast DNA of AT (around 70%); which facilitate the occurrence of transversion substitutions (Baraket et al.,2009). This is confirmed in this survey with the upper rate of transversion occurred.

Aldrich et al. (1988) reported that the major source of size variation in intergenic spacers was the occurrence of insertions/deletions. Five long insertions/deletions and several punctual mutations were reported. Long deletions were observed onAlfalfa with Aldrich et al. (1988). An eventual common progenitor of Medicago could have a longer intergenic region. Theseobservations are in agreement with the evolutionary scheme of the Medics; which derive from a perennial ancestor.

4.2. Genetic divergence of the core collected Morrocan species versus the randomly selected Tunisian species

The rate of conservation sites is higher in Tunisian species than theMorrocan, which carriedmore variables and parsimonyinformative sites with 27 and 24 respectively. This is explained by the high variations of the SARDI seeds (Elwood et al., 2006).

High level of polymorphism for cpDNA may be associated with the evolutionary history of Medicago L. genus (60 muta-tions: punctual þ and indel mutations), yielding the mutations to be accumulated within lineage.

The high haplotype polymorphism (0.823) shown is contrasted by lower nucleotide diversity value (p¼ 0.021). This couldbe explained the specificity and discrimination of the occurred mutations.

Tajima’s test (D) was non significant but the neutral evolution under Wright and Fisher model is rejected. In fact, values ofTajima’s D test are all negative �1.045, �0.195 and �1.498, for respectively Morrocan, Tunisian and the overall Maghrebianspecies. This result predicts an excess of rare alleles (Schmidt and Pool, 2002) and pretends that Medics evolve into stablepopulation’s size. This may be explained by the well adaptation of Maghrebian Medics to their endemic areas.

Z. Nadia et al. / Biochemical Systematics and Ecology 55 (2014) 296e304 303

4.3. Evolutionary history among the Medicago genus

UPGMA dendrogram and a Median-Joining Network estimated, in the current study, the efficiency of trnH-psbA on theevolution history of the nine populations of Medics.

The infraspecific UPGMA tree is represented under a non-structured repartition. In fact, this tree branching is puzzled(independent from the geographic origin and from the nomenclature). This confirms the polymorphism characterizing theMedics species revealed previously by molecular studies Bena et al. (1998). Moreover, these ascertainments highlight theexistence of an important gene exchange and a commune gene pool, in the Maghreb territories. The related proximity of allthe analyzed species confirms the conservation of the cpDNA and the high polymorphism of trnH-psbA.

Since chloroplasts are uniparentally inherited, the Network offers more resolution on the evolutionary relationships ofcertain species with different base chromosome numbers (Fu et al., 2010). The intraspecific network based on the haplotypes’distribution, reveal the distinction of 17 haplotypes with a major H1 node representing by 18 sequences all belonging to M.orbicularis and M. polymorpha (and M. lupulina).

Our overall findings mainly the close genealogies between Morrocan and Tunisian species, heightens to enlarge theanalysis and to explore the germplasm from other geographic regions especially from Algeria (Haddioui et al., 2012). Thestudy of the gene flow movements in the Maghreb region will be more efficient.

4.4. Demographic scenario of the Maghrebian Medics’ populations

Evidence for population dynamics can be obtained from an examination of the distributions of pairwise differences amongmismatch distributions. The sudden demographic expansion and lead to an unimodal distributions of the Mismatch (Baraketet al., 2009). However, the shapes of the Mismatches, in this survey, are multimodal for Morrocan and overallMedics (Fig. 3).

Spontaneous Tunisian species (unimodal shape) seem to undergo or are undergoing periods of growth. Conversely,Morrocan populations provided from a core collection are genetically stable and tend to show a more structured population.The multimodal distribution of the North African populations suggests that the populations are influenced by gene flows butthey involove in constant sizes (Haddioui et al., 2012). This is in agreement with the high values of Nm and Fst founded amongthe two populations (Nm ¼ 3.50 and Fst ¼ 0.06). The correlation of the mismatch distributions with the statistical Tajima’s Dtest confirmed the demographic scenario (the values are negative and non-significant).

Anyway, the primary implicit goal of this study was to test the trnH-psbA barcode for the discrimination of Medicago L.genus. This study represents the first attempt to demonstrate the demographic structure among Medicago species. It is alsothe first report focusing on Medics on the Maghreb zone. Nevertheless, further analyses using other DNA barcodes such asmatK and rbcL markers are required to affiniate the evolutive scenario and to schematise the demographic scenario.

Acknowledgments

This work was partially supported by Tunisian-Moroccan inter-university Cooperation Program through project 25/MT/08and by the Ministery of high education and scientific research (Tunisia) Project Lab B02.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.bse.2014.03.016.

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