Genetic Diversity and Differentiation of Eleven Medicago Species from Campania Region Revealed by Nuclear and Chloroplast Microsatellites Markers

The species belonging to the genus Medicago are considered a very important genetic resource at global level both for planet’s food security and for sustainable rangelands management. The checklist of the Italian flora (2021) includes a total number of 40 Medicago species for Italy, and 27 for Campania region, with a number of doubtful records or related to species no more found in the wild. In this study, 10 Medicago species native to Campania region, and one archaeophyte (M. sativa), identified by means of morphological diagnostic characters, were analyzed in a blind test to assay the efficacy of nine microsatellite markers (five cp-SSRs and four n-SSRs). A total number of 33 individuals from 6 locations were sampled and genotyped. All markers were polymorphic, 40 alleles were obtained with n-SSRs ranging from 8–12 alleles per locus with an average of 10 alleles per marker, PIC values ranged from 0.672 to 0.847, and the most polymorphic SSR was MTIC 564. The cp-SSRs markers were highly polymorphic too; PIC values ranged from 0.644 to 0.891 with an average of 0.776, the most polymorphic cp-SSR was CCMP10. 56 alleles were obtained with cp-SSRs ranging from 7 to 17 alleles per locus with an average of 11. AMOVA analysis with n-SSR markers highlighted a great level of genetic differentiation among the 11 species, with a statistically significant fixation index (FST). UPGMA clustering and Bayesian-based population structure analysis assigned these 11 species to two main clusters, but the distribution of species within clusters was not the same for the two analyses. In conclusion, our results demonstrated that the combination of the used SSRs well distinguished the 11 Medicago species. Moreover, our results demonstrated that the use of a limited number of SSRs might be considered for further genetic studies on other Medicago species.

Comparative Chloroplast Genomics of Litsea Lam. (Lauraceae) and Its Phylogenetic Implications

Litsea Lam. is an ecological and economic important genus of the “core Lauraceae” group in the Lauraceae. The few studies to date on the comparative chloroplast genomics and phylogenomics of Litsea have been conducted as part of other studies on the Lauraceae. Here, we sequenced the whole chloroplast genome sequence of Litsea auriculata, an endangered tree endemic to eastern China, and compared this with previously published chloroplast genome sequences of 11 other Litsea species. The chloroplast genomes of the 12 Litsea species ranged from 152,132 (L. szemaois) to 154,011 bp (L. garrettii) and exhibited a typical quadripartite structure with conserved genome arrangement and content, with length variations in the inverted repeat regions (IRs). No codon usage preferences were detected within the 30 codons used in the chloroplast genomes, indicating a conserved evolution model for the genus. Ten intergenic spacers (psbE–petL, trnH–psbA, petA–psbJ, ndhF–rpl32, ycf4–cemA, rpl32–trnL, ndhG–ndhI, psbC–trnS, trnE–trnT, and psbM–trnD) and five protein coding genes (ndhD, matK, ccsA, ycf1, and ndhF) were identified as divergence hotspot regions and DNA barcodes of Litsea species. In total, 876 chloroplast microsatellites were located within the 12 chloroplast genomes. Phylogenetic analyses conducted using the 51 additional complete chloroplast genomes of “core Lauraceae” species demonstrated that the 12 Litsea species grouped into four sub-clades within the Laurus-Neolitsea clade, and that Litsea is polyphyletic and closely related to the genera Lindera and Laurus. Our phylogeny strongly supported the monophyly of the following three clades (Laurus–Neolitsea, Cinnamomum–Ocotea, and Machilus–Persea) among the above investigated “core Lauraceae” species. Overall, our study highlighted the taxonomic utility of chloroplast genomes in Litsea, and the genetic markers identified here will facilitate future studies on the evolution, conservation, population genetics, and phylogeography of L. auriculata and other Litsea species.

How ancient forest fragmentation and riparian connectivity generate high levels of genetic diversity in a micro-endemic Malagasy tree

Understanding landscape changes is central to predicting evolutionary trajectories and defining conservation practices. While human-driven deforestation is intense throughout Madagascar, exception in areas like the Loky-Manambato region (North) raises questions. This region also harbors a rich and endemic flora, whose evolutionary origin remains poorly understood.We assessed the genetic diversity of an endangered micro-endemic Malagasy olive species (Noronhia spinifolia) to better understand the vegetation dynamic in the Loky-Manambato region and its influence on past evolutionary processes. We characterized 72 individuals sampled across eight forests through nuclear and mitochondrial restriction associated sequencing data (RADseq) and chloroplast microsatellites (cpSSR).Extremely high genetic diversity was revealed in the three genomic compartments (chloroplast h = 0.99, mitochondrial h = 0.85, and nuclear HO = 0.07-0.20). Combined population and landscape genetics analyses indicate that N. spinifolia diversity is best explained by the current forest cover (R2 = 0.90), highlighting a long-standing forest fragmentation in the region. Our results further suggest a predominant role of forestdwelling organisms in mediating pollen and seed dispersals.This sustains a major and long-term role of riparian corridors in maintaining connectivity across those antique mosaic-habitats, calling for the study of organismal interactions that promote gene flow.

Population genetic structure and variability in Lindera glauca (Lauraceae) indicates low levels of genetic diversity and skewed sex ratios in natural populations in mainland China

Lindera glauca (Lauraceae) is a tree of economic and ecological significance that reproduces sexually and asexually via apomictic seeds. It is widely distributed in the low-altitude montane forests of East Asia. Despite the potential implications of a mixed reproductive system in terms of genetic diversity, few studies have focused on this aspect. In this study, the genetic structure of wild populations of L. glauca was investigated via genetic analyses. Overall, 13 nuclear microsatellites (nSSRs) and five chloroplast microsatellites (cpSSRs) were used to genotype 300 individual plants, taken from 20 wild populations (a small sample size in some wild populations is due to the limitation of its specific reproduction, leading to certain limitations in the results of this study) and two cultivated populations ranging across nearly the entire natural distribution of mainland China. The populations exhibited low levels of genetic diversity (nSSR: AR = 1.75, Ho = 0.32, He = 0.36; cpSSR: Nb = 2.01, Hrs = 0.40), and no significant effect of isolation by distance between populations existed, regardless of marker type (nSSR: R2 = 0.0401, P = 0.068; cpSSR: R2 = 0.033, P = 0.091). Haplotype networks showed complex relationships among populations, and the H12 haplotype was predominant in most populations. Analyses of molecular variance obtained with nuclear markers (Fsc = 0.293, FST = 0.362) and chloroplast markers (Fsc = 0.299, FST = 0.312) were similar. The migration ratio of pollen flow versus seed flow in this study was negative (r = −1.149). Results suggest that weak barriers of dispersal between populations and/or the similarity of founders shared between neighbors and distant populations are indicative of the gene flow between populations more likely involving seeds. Wild L. glauca in mainland China was inferred to have highly skewed sex ratios with predominant females. In addition, some populations experienced a recent bottleneck effect, especially in Gujianshan, Chongqing, and southwest China (population GJS). It is suggested that few wild male individuals should be conserved in order to maintain overall genetic diversity in the wild populations of this species. These findings provide important information for the sustainable utilization and preservation of the overall genetic diversity of L. glauca.

Chloroplast microsatellites reveal genetic diversity and population structure in natural populations of Himalayan Cedar (Cedrus deodara (Roxb.) G. Don) in India

Himalayan cedar (Cedrus deodara) is one of the most important temperate timber species of Western Himalayas and is considered to be among the endangered conifer species in the region. Knowledge of genetic diversity and population structure will help guide gene conservation strategies for this species. Ten polymorphic chloroplast microsatellites (cpSSR) were used to study genetic diversity and population structure in twenty one natural populations of C. deodara throughout its entire distribution range in Western Himalayas. When alleles at each of the 10 loci were jointly analysed, 254 different haplotypes were identified among 1050 individuals. The cpSSRs indicate that C. deodara forests maintain a moderately high level of genetic diversity (mean h = 0.79 ). AMOVA analysis showed that most of the diversity in C. deodara occurs within populations. Bayesian analysis for population structure (BAPS) revealed spatial structuration of the variation (22 % of the total variation) and substructuring captured nineteen genetic clusters in the entire divisions of the populations. Most of the populations were clustered independently with minor admixtures. The distribution of genetic diversity and sub-structuring of C. deodara may be due to restricted gene flow due to geographic isolation, genetic drift, and natural selection. These findings indicated existence of genetically distinct and different high diversity and low diversity clusters, which are potential groups of populations that require attention for their conservation and management. The results are interpreted in context of future conservation plans for C. deodara.

Late Miocene origin and recent population collapse of the Malagasy savanna olive tree (Noronhia lowryi)

Debates regarding the origin of tropical savannas have attempted to disentangle the role of human, biotic and abiotic factors. Understanding the origins of savanna remains essential to identifying processes that gave rise to habitat mosaics, particularly those found in the Central Plateau of Madagascar. Documenting the evolutionary history and demography of native trees occurring in open habitats may reveal footprints left by past and recent environmental changes. We conducted a population genetic analysis of an endangered Malagasy shrub (Noronhia lowryi, Oleaceae) of the Central Plateau. Seventy-seven individuals were sampled from three sites and genotyped at 14 nuclear and 24 chloroplast microsatellites. We found a highly contrasting nuclear and plastid genetic structure, suggesting that pollen-mediated gene flow allows panmixia, while seed-based dispersal may rarely exceed tens of metres. From a phylogeny based on full plastomes, we dated the surprisingly old crown age of maternal lineages back to ~6.2 Mya, perhaps co-occurring with the global expansion of savanna. In contrast, recent demographic history inferred from nuclear data shows a bottleneck signature ~350 generations ago, probably reflecting an environmental shift during the Late Pleistocene or the Holocene. Ancient in situ adaptation and recent demographic collapse of an endangered woody plant highlight the unique value and vulnerability of the Malagasy savannas.

Identification, distribution and comparative analysis of microsatellites in the chloroplast genome of Oryza species

Chloroplast genome is important because of its maternally inherited, conserved within species and stable in structure. This allows elucidation of inter-specific comparison, identification and establishment of evolutionary relationship among different species. Different classes of DNA elements were present in chloroplast genome of Oryza species, among them chloroplast microsatellites were supposed to be highly variable. In this study, a total of 102 sequences of chloroplast genome of 23 species of Oryza genus belonging to ten different genome types were downloaded from NCBI nucleotide database. The chloroplast DNA length varied from 134,401bp to 136,133bp. Microsatellite analysis revealed that total SSR number varied from 268 in O. coarctata to 281 in O. granulata. Comparative analysis of repeat type revealed that mononucleotide and trinucleotide were least present, while penta and hexa nucleotide motif were the most common type among all the Oryza species. Frequency analysis of the repeat revealed that T repeat among mono, AT among di, AAT among tri, AATA among tetra, AATTC among penta and AAAGAA among hexa nucleotide repeat was found to be common among most of the Oryza species. Total of five set of SSR markers flanking to penta and hexa nucleotide repeat types were developed which uniquely present among species, namely, O. barthi, O. sativa Japonica, O. cocarctata, O. rhizomatis and O. ridley. Phylogeny based on SSR markers indicate separate evolution of different SSR markers among individual species.

The complete chloroplast genome of Fagus crenata (subgenus Fagus) and comparison with F. engleriana (subgenus Engleriana)

This study reports the whole chloroplast genome of Fagus crenata (subgenus Fagus), a foundation tree species of Japanese temperate forests. The genome has a total of 158,227 bp containing 111 genes, including 76 protein-coding genes, 31 tRNA genes and 4 ribosomal RNA genes. Comparison with the only other published Fagus chloroplast genome, F. engeleriana (subgenus Engleriana) shows that the genomes are relatively conserved with no inversions or rearrangements observed while the proportion of nucleotide sites differing between the two species was equal to 0.0018. The six most variable regions were, in increasing order of variability, psbK-psbI, trnG-psbfM, rpl32, trnV, ndhI-ndh and ndhD-psaC. These highly variable chloroplast regions in addition to 160 chloroplast microsatellites identified (of which 46 were variable between the two species) will provide useful genetic resources for studies of the inter- and intra-specific genetic structure and diversity of this important northern hemisphere tree genus.