3.1. Agronomic Traits
ANOVA showed a significant block effect for grain yield (GY) in 2013/14 and 2015/16, Harvest Index (HI) in 2014/15 and biomass in 2015/16 (
Table 1). Highly significant differences (
p < 0.001) were detected among
Lathyrus spp. (S) for all parameters in the three-years. Significant accession effect (A) was observed for all traits, except for days to flowering (DF) in the three-years and Grain Yield (GY) in 2014/15 (
Table 1). Moreover, the interaction S*A resulted significant for many traits except for biomass (2013/14), 1000 SW and GY (2014/15), whereas the interaction was not significant for DF and protein content (
Table 1).
The variability within
Lathyrus spp. (among accessions) for each parameter is reported in
Table 2. L. sativus showed a lower 1000 SW variability (CV = 3.9%) compared to
L. cicera and
L. ochrus (5.6 and 6.3%, respectively). The average 1000 SW in
L. sativus was 75.72 g, ranging from 71.32 g (accession 4, Ethiopia) to 81.54 g (accession 11, Ethiopia), whereas
L. ochrus showed an average value of 103.51 g, ranging from 92.04 g (accession 8, Greece) to 115.89 g (accession 3, Greece) (
Table S4). Finally,
L. cicera was characterized by the lowest average 1000 SW (71.58 g) in which the accessions 8 (Greece) and 14 (Greece) exhibited contrasting values (64.2 and 82.04 g, respectively) (
Table S4). A high 1000 SW heritability was observed in
L. cicera,
L. sativus and
L. ochrus with 0.92, 0.88 and 0.81, respectively (
Table 2).
The coefficient of variability for GY was different among species (5.8, 8.2 and 7.1 for
L. cicera,
L. sativus and
L. ochrus, respectively) as well as within species. The highest GY average value was detected in
L. cicera (3.49 t ha
−1) followed by
L. sativus (2.74 t ha
−1) and finally
L. ochrus (2.24 t ha
−1); further, the heritability appeared low in two species (0.55 and 0.46 for
L. cicera and
L. sativus, respectively), while
L. ochrus showed a higher value (0.69) (
Table 2). In detail, Ethiopia 2 (
L. sativus), Australia (
L. cicera) and Greece 5 (
L. ochrus) were the most productive accessions (
Table S4).
The highest biomass CV was detected among
L. ochrus accessions with an average of 6.92 t ha
−1 ranging from 6.15 (Greece 3) to 7.46 t ha
−1 (Greece 5) (
Table S4), whereas
L. sativus and
L. cicera exhibited an average of 7.57 and 8.2 t ha
−1, respectively (
Table 2). The heritability of this trait varied among species,
L. ochrus and
L. cicera showed similar values (0.68 and 0.63, respectively), whereas the lowest (0.44) was recorded in
L. sativus. The averages of days to flowering (DF) were of 98.3, 96.58 and 95.81 days for
L. cicera,
L. sativus and
L. ochrus respectively; this trait showed a high heritability (0.97, 0.97 and 0.96, respectively) and low CV values (1, 1.6 and 1.9, respectively) in all species.
Moreover, Protein content was higher in
L. sativus and
L. ochrus (30.67 and 30.20, respectively) respect to
L. cicera (27.57); the former showed a higher heritability for this trait (0.66) compared to
L. sativus (0.41) and
L. ochrus (0.48) (
Table 2). Finally,
L. cicera recorded a higher HI (0.42) compared to
L. sativus (0.36) and
L. ochrus (0.33), although the latter showed the highest heritability (0.94) and CV (9.2) for this trait, followed by
L. cicera (0.82–5.2, respectively) and
L. sativus (0.58–5.8, respectively) (
Table 2).
3.2. Correlations Among Agronomic Traits
The relationships between agronomic traits within
Lathyrus spp. were also calculated by Pearson’s correlation (
Table S5). Correlation coefficient between pairs of agronomic traits highlighted specific significant correlations; in detail, negative correlation between 1000 SW and DF was detected in
L. sativus (−0.67) and
L. cicera (−0.58) and positive correlation between GY and HI was observed in
L. sativus (0.70) and
L. ochrus (0.59), while 1000 SW was significantly correlated with biomass (0.87) in
L. cicera. Finally, negative correlation was also observed in
L. ochrus between biomass and HI (−0.58), DF and HI (−0.74) and protein content and HI (−0.71). By contrast, positive significant correlations were detected between DF and protein content (0.72) as well as between DF and biomass (0.65) in
L. ochrus (
Table S5).
3.3. Multivariate Analysis of the Agronomic Traits
Cluster analysis was performed based on the three-years mean for each parameter (
Figure 1). The dendrogram generated using Euclidean distance and Ward’s method was able to distinguish three major groups clustered by species, and this result was also confirmed by the gap statistics method (data not shown). Cluster I (blue) included all genotypes belonging to
L. cicera, which was the latest ripening species, with the best performance for GY and biomass as well as the highest HI. Cluster II (yellow) included all the
L. sativus accessions, characterized by a high protein content, as well as one accession from Cyprus (O6_C) belonging to
L ochrus; finally, Cluster III (orange) was represented by the remaining
L. ochrus accessions characterized by high 1000 SW (
Figure 1). The distances among clusters was higher between
L. cicera and
L. ochrus (32.67) followed by
L. sativus vs.
L. ochrus (26.67) and
L. sativus vs.
L. cicera (6.84). In addition, a higher intra-group variance was detected in
L. sativus and
L. ochrus (47.9 and 46.9, respectively) than in
L. cicera (18.1) (data not shown). Moreover, similarities between some accessions from same geographical origin were also highlighted as
L. cicera accessions from Australia and
L. ochrus accessions from Cyprus (
Figure 1).
Principal component analysis based on agronomic traits displayed two components contributing for 84.2% of the variability among species (
Figure S1). PC1 explained 69.3% of total variance, where 1000 SW and protein content were mainly involved, whereas PC2 explained 14.9% of the variance and the traits involved were DF, HI, GY and biomass (
Figure S1).
3.4. Genetic Diversity Among Species
Despite the high PIC values previously reported for the 10 selected SSR markers, only 50% of them showed polymorphism among
Lathyrus accessions. SSR summary statistics and genetic parameters among 40
Lathyrus accessions are reported in
Table 3.
Five polymorphic SSR markers amplified a total of 48 polymorphic alleles (Na) with an average of 9.6 alleles per locus. Allele number for each locus ranged from 6 to 13 for the SSR G157 and G67, respectively. Number of effective alleles (Ne) ranged from 2.485 (G26) to 4.733 (G67) with a mean of 3.392 alleles per locus. Gene diversity (H) varied from 0.662 to 0.881 for G157 and G67, respectively with an average of 0.784 in the whole
Lathyrus collection. Meanwhile, polymorphic information content (PIC), which provide an estimate of the discriminatory power of each SSR locus, ranged from 0.607 (G157) to 0.856 (G67) with an average of 0.745 (
Table 3).
Shannon’s information index (I), commonly used to assess population genetic diversity, was also estimated and ranged from 1.078 (G157) to 1.571 (G67) with a mean of 1.226. A relatively important genetic differentiation was observed among 40
Lathyrus accessions with an average Fst value of 0.227 (
Table 3).
3.5. Genetic Diversity Within Lathyrus Species
The genetic diversity parameters for each species are listed in
Table 4. According to SSR markers analysis,
L. sativus had the highest PIC (0.641) whereas
L. cicera harbor the lowest one (0.528). The study also revealed that
L. sativus showed the highest genetic diversity according to Shannon’s information index and gene diversity values (I = 1.492 and H = 0.694, respectively). Furthermore, 58.33% (28 alleles) of total allele number were private, of which 64.3% (18 alleles) were exclusively detected in
L. sativus, and this result indicated that grasspea shared less than 50% of total alleles (34) with the other two studied species (
Table 4).
Finally, the genetic differentiation within the species was the highest in L. ochrus (Fst = 0.256) and the lowest in L. cicera (Fst = 0.137). These results suggested that the gene flow within L. ochrus accessions is highly limited being a strictly autogamous species by contrast with the two other species which are primarily autogamous, with some out-crossing likely.
3.6. Genetic Differentiation and Relationship Among the Three Species
Nei and Li [
34] genetic distance varied from 0.635 to 1.326 among
Lathyrus species (
Table 5). The greatest genetic distance was observed between
L. ochrus and
L. cicera, contrary to
L. sativus vs.
L. cicera. Pairwise Fst estimates followed the trend of genetic diversity and revealed a high degree of differentiation between
L. ochrus and the other
Lathyrus species (
Table 5).
Furthermore, the gene flow (Nm) showed that the major gene flow occurred between
L. sativus and
L. cicera with Nm = 1.139 (data not shown). Moreover, AMOVA revealed that most of the total genetic diversity was explained by within species variation (77%) (
Table 6).
The clustering analysis by Unweighted Pair Group Method using the Arithmetic Averaging dendrogram (UPGMA) divided the forty accessions into two distinct groups which correlated with the sections Lathyrus and Clymenum (
Figure 2A).
The first group in red and cyclamen represented the Lathyrus section, including the accessions of the two most cultivated species aggregated in two distinct subgroups: L. cicera and L. sativus. The second group, in light blue, represented the Clymenum section, comprising all L. ochrus accessions. Interestingly, one accession of L. cicera appeared as an out-group being the most distant accession.
To further highlight the genetic relationships among accessions of different origin within each species, a second clustering analysis was based on the genetic distances among each geographic group or population (
Figure 2B). The genetic distance matrix between each population pairs was computed based on Nei and Li [
34] algorithm (
Table S6). The UPGMA dendrogram grouped the
Lathyrus accessions by species. In particular,
L. cicera and
L. sativus accessions clustered in the same group, even if the Italian accession appeared the most distant. The second cluster grouped all the
L. ochrus accessions, in which the German accession was the most diverse compared to the others.
3.7. Genetic Structure
The genetic structure of the 13
L. sativus, 15
L. cicera and 12
L. ochrus accessions was revealed by using Structure software (
Figure 3). The optimal number of genetic groups (K) was identified based on posterior probability (∆K) values. K was tested from one to ten based on the distribution of the 48 alleles at the 5 SSR loci among the 40 individuals. Structure simulation demonstrated that the highest ∆K values was at K = 3 (
Figure S2). Estimated membership coefficients inferred for K = 3 for each individual is presented in
Figure 3. Every accession corresponded to a vertical line which is divided into K segments. The length of each segment is proportional to the K assigned to each cluster. The results revealed three clear distinct gene pools with few admixture rates among the species. However, the accession C5_S showed more than 80% admixture indicating a frequent gene flow between this accession and the others from different clusters. However, the representation of the three clusters (
Figure 3) revealed a clustering by species.
Additional evidence for
Lathyrus spp. genetic structure was furnished by Principal Coordinate Analysis (PCoA). This was performed to provide a spatial representation of the pair-wise estimated genetic differentiation of forty accessions and the different populations of diverse origins within
Lathyrus species (
Figure 4A, B). The first two principal coordinates explained 27.17% and 15.05% of total variation, respectively, and the 50.22% of the variation was explained considering the three coordinates. In agreement with cluster analysis, accessions of each
Lathyrus species formed clear separated groups; however, overlap among the three species was still detected, highlighting the level of genetic admixture among the individuals, as revealed by structure analysis. Moreover, most of the accessions of the same species clustered regardless the geographic origin:
L. sativus (Syria, Italy and Ethiopia);
L. cicera (Australia and Syria, Greece) and
L. ochrus (Germany, Greece and Cyprus). However, two Greece accessions of
L. cicera and most
L. ochrus accessions were defined positively by the first coordinate and negatively by the second, suggesting a genetic relatedness probably correlated to the geographic origin. In addition, most of the accessions of both species
L. sativus and
L. cicera were defined negatively by the first coordinate contrary to
L. ochrus accessions, indicating that
L. sativus and
L. cicera are closely related.
Finally, significant correlation existed between agronomic and genetic distance tested by Mantel test (r = 0.504) (
Figure S3).