Evaluating species nonmonophyly as a trait affecting genetic diversity: a case study of three endangered species of Antirrhinum L. (Scrophulariaceae)

Molecular markers are routinely used to assess levels of diversity within and among populations, particularly with regard to species of conservation concern. However, when interpreting the level and partitioning of diversity observed, an implicit assumption is often made that the populations of the species in question form a monophyletic group. We tested this assumption in three endemics of Antirrhinum (A. charidemi, A. subbaeticum, and A. valentinum) using 79 nuclear [internal transcribed spacer (ITS)] and 85 plastid (psbA-trnH, trnT-trnL, trnK-matK, trnS-trnG) sequences representing multiple accessions of each of 24 Antirrhinum species (single accession of A. cirrhigerum). These species share six life history traits implicated in levels of genetic diversity, and have been the subject of previous population genetic studies. Populations of all three species formed monophyletic groups on ITS analysis. In contrast, none of the three species formed monophyletic groups on plastid sequence analysis: populations of A. charidemi fall in a monophyletic group including one accession of A. mollissimum, populations of A. subbaeticum form a polyphyletic group with plastid sequences shared with A. pulverulentum, and populations of A. valentinum are unresolved within a clade containing six other species. Lack of monophyly using plastid sequences is interpreted as a combination of shared ancestral polymorphism and hybridization in a reticulate evolutionary history of these species. Monophyly in the ITS tree may reflect a more recent sequence homogenization. We draw attention to the evaluation of species monophyly alongside the contribution of other life history traits in the historical interpretation of the level and partitioning of genetic diversity, and its use in recommendations for species conservation programs.     

The development of 10 novel polymorphic microsatellite markers through next generation sequencing and a preliminary population genetic analysis for the endangered Glenelg spiny crayfish, Euastacus bispinosus

The Glenelg spiny crayfish, Euastacus bispinosus, is an iconic freshwater invertebrate of south eastern Australia and listed as ‘endangered’ under the Environment Protection and Biodiversity Conservation Act 1999, and ‘vulnerable’ under the International Union for Conservation of Nature’s Red List. The species has suffered major population declines as a result of over-fishing, low environmental flows, the introduction of invasive fish species and habitat degradation. In order to develop an effective conservation strategy, patterns of gene flow, genetic structure and genetic diversity across the species distribution need to be clearly understood. In this study we develop a suite of polymorphic microsatellite markers by next generation sequencing. A total of 15 polymorphic loci were identified and 10 characterized using 22 individuals from the lower Glenelg River. We observed low to moderate genetic variation across most loci (mean number of alleles per locus = 2.80; mean expected heterozygosity = 0.36) with no evidence of individual loci deviating significantly from Hardy–Weinberg equilibrium. Marker independence was confirmed with tests for linkage disequilibrium, and analyses indicated no evidence of null alleles across loci. Individuals from two additional sites (Crawford River, Victoria; Ewens Ponds Conservation Park, South Australia) were genotyped at all 10 loci and a preliminary investigation of genetic diversity and population structure was undertaken. Analyses indicate high levels of genetic differentiation among sample locations (F _ST  = 0.49), while the Ewens Ponds population is genetically homogeneous, indicating a likely small founder group and ongoing inbreeding. Management actions will be needed to restore genetic diversity in this and possibly other at risk populations. These markers will provide a valuable resource for future population genetic assessments so that an effective framework can be developed for implementing conservation strategies for E. bispinosus.     

De novo development and characterization of polymorphic microsatellite markers in a schilbid catfish, <Emphasis Type="Italic">Silonia silondia</Emphasis> (Hamilton, 1822) and their validation for population genetic studies

The stock characterization of wild populations of Silonia silondia is important for its scientific management. At present, the information on genetic parameters of S. silondia is very limited. The species-specific microsatellite markers were developed in current study. The validated markers were used to genotype individuals from four distant rivers. To develop de novo microsatellite loci, an enriched genomic library was constructed for S. silondia using affinity–capture approach. The markers were validated for utility in population genetics. A total number of 76 individuals from four natural riverine populations were used to generate data for population analysis. The screening of isolated repeat sequences yielded eleven novel polymorphic microsatellite loci. The microsatellite loci exhibited high level of polymorphism, with 6–24 alleles per locus and the PIC value ranged from 0.604 to 0.927. The observed (Ho) and expected (He) heterozygosities ranged from 0.081 to 0.84 and 0.66 to 0.938, respectively. The AMOVA analysis indicated significant genetic differentiation among riverine populations (overall F_ST = 0.075; P < 0.0001) with maximum variation (92.5 %) within populations. Cross-priming assessment revealed successful amplification (35–38 %) of heterologous loci in four related species viz. Clupisoma garua, C. taakree, Ailia coila and Eutropiichthys vacha. The results demonstrated that these de novo polymorphic microsatellite loci are promising for population genetic variation and diversity studies in S. silondia. Cross-priming results indicated that these primers can help to get polymorphic microsatellite loci in the related catfish species of family Schilbidae.     

Genetic variation and population differentiation of the endochitinase gene family in Pinus monticola

In the present study, we characterized nucleotide variations of the Pinus monticola class IV endochitinase (PmCh4) family. Using primers targeting at conserved amino acid motifs of plant class IV endochitinases, genomic DNA was amplified. Sequence data analysis identified five novel genes in the PmCh4 family with one pseudogene. Single nucleotide polymorphisms of the PmCh4 family were surveyed in seven open-pollinated seed lots representing diverse geographical distribution. Variable levels of average pairwise nucleotide diversity (π = 0.00422–0.02079) and relatively high levels of haplotype diversity (H _d = 0.85–0.96) were revealed at PmCh4 loci. Based on nucleotide variation, P. monticola populations were clustered into two main groups by phylogenetic analysis based on Nei’s genetic distance. The Mantel test revealed no correlation between geographical and genetic distances (r = ?0.11, P = 0.59). A further SNP genetic diversity study on more P. monticola populations throughout Western North America may help the design of sampling regimes for tree breeding, genetic conservation and assisted migration under climate change.     

Microsatellite Polymorphisms in Cassava Landraces from the Cerrado Biome, Mato Grosso do Sul, Brazil

Using nine microsatellite loci, we investigated genetic structure and diversity in 83 Brazilian cassava accessions, including several landraces, in the Cerrado biome in Mato Grosso do Sul, Brazil. All nine loci were polymorphic, averaging 6.00 alleles per locus. Treating each of seven municipalities as a cassava group or population, they averaged 3.5 alleles per locus, with 97% polymorphic loci, high values for observed heterozygosity (0.32) and gene diversity (0.56). Total genetic variability was high (0.668), and most of this genetic variability was concentrated within municipalities (0.577). Cluster and structure analyses divided accessions into two major clusters or populations (K = 2). Also, a significant genetic versus geographic correlation was found (r = 0.4567; P < 0.0260). Migratory routes in the Cerrado are considered main contributors to the region’s high cassava diversity and spatial genetic structure, amplifying interactions between traditional farmers and the evolutionary dynamics of this crop.     

Assessing genetic diversity for the USA endemic carnivorous plant <Emphasis Type="Italic">Pinguicula ionantha</Emphasis> R.K. Godfrey (Lentibulariaceae)

Understanding patterns of genetic diversity and population structure for rare, narrowly endemic plant species, such as Pinguicula ionantha (Godfrey’s butterwort; Lentibulariaceae), informs conservation goals and can directly affect management decisions. Pinguicula ionantha is a federally listed species endemic to the Florida Panhandle in the southeastern USA. The main goal of our study was to assess patterns of genetic diversity and structure in 17 P. ionantha populations, and to determine if diversity is associated with geographic location or population characteristics. We scored 240 individuals at a total of 899 AFLP markers (893 polymorphic markers). We found no relationship between the estimated population size with either of two measures of diversity (proportion of loci polymorphic, P = 0.37; Nei’s gene diversity, P = 0.50). We also found low levels of population genetic structure; there was no clear relationship of genetic isolation by distance (P = 0.23) and only a small (but significant) proportion of genetic variation was partitioned amongst regions (2.4 %, P = 0.02) or populations (20.8 %, P < 0.001). STRUCTURE analysis found that the model with two inferred clusters (K = 2) best described the AFLP data; the dominant cluster at each site corresponded to the results from PCoA and Nei’s genetic distance analyses. The observed patterns of genetic diversity suggest that although P. ionantha populations are isolated spatially by distance and both natural and anthropogenic barriers, some gene flow occurs among them or isolation has been too recent to leave a genetic signature. The relatively low level of genetic diversity associated with this species is a concern as it may impair fitness and evolutionary capability in a changing environment. The results of this study provide the foundation for the development of management practices that will assist in the protection of this rare carnivorous plant.     

Interspecific genetic analysis of orchids in Brazil using molecular markers

Several species of Orchidaceae, one of the largest plant families, are considered endangered throughout South America and legal protection policies are needed so they can be preserved. Inter simple sequence repeats (ISSRs) markers are a potential tool to be used in the phylogenetic reconstruction of closely related species. In this study, we evaluate the polymorphic information content (PIC) and optimum number of ISSR markers (ONM) for five Laeliinae orchids in order to assess genetic diversity. The phylogenetic relationships between Cattleya granulosa, an endangered Brazilian orchid, and four other native Brazilian species (Brassavola tuberculata, Cattleya bicolor, Cattleya labiata and Cattleya schofieldiana) were analyzed for genetic diversity and differentiation. The 11 selected primers generated 166 unambiguous loci (PIC = 0.354; ONM = 156). Of the five studied species, C. bicolor exhibited the highest level of genetic diversity (H _E  = 0.219), while C. labiata exhibited the lowest level (H _E  = 0.132). The percentage of genetic variation among species (analysis of molecular variance) was 23.26 %. The principal component analysis (PCA) of ISSR data showed that unifoliate and bifoliolate species are genetically divergent. Additionally, PCA indicated a close relation between C. granulosa and C. schofieldiana, a species considered to be a variety of C. granulosa by many researchers. Thus, we conclude that ISSR genetic markers are effective in detecting genetic differentiation among orchid species.     

Population genetic diversity and divergence of the halobiotic herb Limonium sinense estimated by AFLP and ISSR, and implications for conservation

Limonium sinense is a halobiotic herb endemic to China that has been traditionally used for hundreds of years for its good restorative function. Genetic variation and population structure of this species were investigated by using amplified fragment length polymorphisms (AFLPs) and inter simple sequence repeats (ISSRs). A high level of genetic diversity was detected [AFLP: H _E = 0.284, percentage of polymorphic loci (PPL) = 92.68 %; ISSR: H _E = 0.257, PPL = 85.71 %] at the species level with POPGENE. Based on analysis of molecular variation (AMOVA), the among-population component accounted for 29.03 % (AFLP) and 28.81 % (ISSR) of the genetic variation, indicating that most of the genetic variation was between individuals within populations. The Shannon diversity index (I) was higher for AFLP (0.432) than for ISSR (0.395). Five main clusters were shown in the unweighted pair-group method with arithmetic mean (UPGMA) dendrogram created using TFPGA, consistent with the result of principal coordinate analysis using NTSYS. In situ conservation is advocated first. Keeping a stable environment for this halobiotic herb is necessary. For ex situ conservation, it is important to establish a germplasm bank. AFLP and ISSR markers were proved to be efficient tools in assessing the genetic variation among populations of L. sinense. The patterns of variation appeared to be consistent for these two marker systems, and they can be used for management of genetic structure, protection of the halobiotic plant, and conservation of germplasm.     

Negligence in the Atlantic forest,northern Brazil: a case study of an endangered orchid

Currently, many Brazilian orchids are threatened with extinction resulting from habitat loss and intense harvesting pressure stemming from their value as ornamental plants. Therefore, the genetic diversity in remaining populations is fundamental to the survival of these species in natural environments. In order to inform conservation strategies, this study evaluated the genetic diversity and structure of Cattleya granulosa populations. The sample consisted of 151 individuals from 12 populations in the Atlantic Forest, northeastern Brazil, evaluated using 91 ISSR markers. Genetic variability was assessed through molecular variance, diversity indexes, clusters of genotypes through Bayesian analysis, and tests for genetic bottlenecks. From all polymorphic loci, genetic diversity (H_E) varied between 0.210 and 0.321 and the Shannon index ranged from 0.323 and 0.472. Significant genetic differentiation between populations (Φ_ST = 0.391; P < 0.0001) resulted in the division of the populations into five groups based on the log-likelihood Bayesian analysis. We found significant positive correlation between geographical and genetic distances between populations (r = 0.794; P = 0.017), indicating isolation by distance. Patterns of allelic diversity within populations suggest the occurrence of bottlenecks in most C. granulosa populations (n = 8). Therefore, in order to maintain the genetic diversity of the species, the conservation of spatially distant groups is necessary.     

Genetic relationship and genetic diversity among Typha taxa from East Asia based on AFLP markers

The genetic relationship and diversity among four Typha taxa in East Asia were evaluated using amplified fragment length polymorphism (AFLP) markers. Three AFLP selective primer combinations generated a total of 707 amplification products, of which 704 (99.6%) were polymorphic. The unweighted pair-group method with arithmetic mean (UPGMA) dendrogram and principal component analysis (PCA) plot confirmed the taxonomic status of four separate species. East Asian Typha taxa separated into two groups: the first with Typha angustifolia and the second with T. orientalis, T. laxmanni, and T. latifolia with a high bootstrap value for UPGMA (93%) and a low first score for PCA (25%). The two clusters corresponded with two sections based on the bracteoles in the female flower: section Bracteolatae and section Ebracteolatae. T. angustifolia showed the highest genetic diversity among the four Typha taxa (percentage of polymorphic loci [PPL] = 71%, H_o = 0.157), whereas T. latifolia had the lowest genetic diversity (PPL = 40%, H_o = 0.117). Genetic diversity was related to the presence of the gap between male and female inflorescences. A positive correlation between genetic distance and geographic distance was clearly found in the two species with continuous inflorescences (T. latifolia and T. orientalis). This positive correlation was not observed in the other species with discontinuous spikes (T. angustifolia and T. laxmanni).     

Planchonella, first record of gynomonoecy for the family Sapotaceae

Inter-simple sequence repeat (ISSR) markers were used to investigate the levels and pattern of genetic variation within and among populations of Pteroceltis tatarinowii Maxim., an endangered plant endemic to China. Of the 76 primers screened, 11 produced highly reproducible ISSR bands. A total of 118 bands were presented from the 11 selected primers across all individuals of five natural populations, corresponding to an average of 10.73 bands per primer. The size of the ISSR bands ranged from 200 to 2,000 bp. The percentage of polymorphic loci at the population level ranged from 77.97 to 86.44%, with an average value of 82.54%. Genetic differentiation among populations was revealed based on Nei’s genetic diversity analysis (19.41%) and the nonparametric analysis of molecular variance (20.62%). The Mantel test showed a significant positive correlation between geographic distance and genetic distance (r = 0.7758, P < 0.05), indicating a role of geographic isolation in shaping the present population genetic structure of P. tatarinowii. The size of the natural populations of P. tatarinowii was noted in field observations to be very small, chiefly owing to habitat destruction and overexploitation in the past decades. Therefore, effective measures for preserving genetic diversity of this species at the population level are needed and should include protecting its natural habitats and increasing the numbers of individuals. To meet the commercial demand for this species, P. tatarinowii plantations and cultivation facilities should be established as an alternative source of raw materials.     

Utilization of in silico EST–SSR markers for diversity studies in castor (<Emphasis Type="Italic">Ricinus communis</Emphasis> L.)

Castor (Ricinus communis L.) a chief non-edible oilseed crop has numerous industrial applications. Systematic genetic diversity analysis utilizing DNA based markers has been quick and reliable method that ensures selection of diverse parents for exploitation of higher levels of heterosis in breeding programs. From NCBI database, 63,852 EST sequences of castor were mined. One thousand one hundred and five (1105) EST–SSRs and 1652 repeat motifs sequences were identified from 20,495 non-redundant unigene sequences. Repeat motifs consisted of 29.7 % mono nucleotide repeats, 24.8 % di nucleotide repeats, 27.27 % tri nucleotide repeats and 3.94 % tetra nucleotide repeats. Twenty eight primer pairs were chosen from SSR-containing ESTs to determine genetic diversity among 27 castor accessions. Twelve EST–SSRs showed polymorphism. Number of alleles detected were 2–3 with an average of 2.33 per locus. 150–400 bp was the size of an allele. Dendrogram analysis grouped the 27 accessions into two separate clusters. Genetic similarity coefficient of dendrogram ranged from 0.24 to 0.83. The polymorphic information content value of 0.28–0.49 revealed medium level of diversity in castor. Results of present study indicated that EST–SSRs to be efficient markers for genetic diversity studies. Knowledge on level of diversity existing in castor genotypes would be useful for breeders to plan efficient hybrid breeding programme.     

Evidence for spontaneous polyploidization in cassava Manihot esculenta Crantz

Since high levels of genetic diversity may ensure long-term survival of a plant species, it is essential to preserve the genetic diversity of the species. Tipularia japonica and Epipactis papillosa are rare terrestrial orchids in southern Korea with fewer than 50 mature individuals in a population and southern Japan and considered to be threatened (endangered or vulunerable). To obtain knowledge of how the genetic variation of these species is partitioned within and among populations in Korea, I used enzyme electrophoresis to examine the genetic diversity of each eight known populations of the two species from South Korea. Twenty-three (E. papillosa) and 24 putative loci (T. japonica) resolved from 15 enzyme systems revealed no variation either within or among populations of each species (0.0% of the percentage of polymorphic loci, %P). Previous studies, in contrast, showed that their more widely distributed disjunct congeners T. discolor and E. helleborine harbored high allozyme-based genetic diversity within populations in eastern United States (%P = 75%) and in Denmark (%P = 73.6%), respectively. In theory, small population size leads to allelic fixation at many loci over generations within a population, resulting in population genetic divergence or differentiation. In this regard, the complete lack of genetic differences between conspecific populations of T. japonica and E. papillosa cannot be explained by genetic drift. Instead, the present allozyme data suggest that recent origin from the same genetically depauperate ancestral or source population could result in this observation. The current status of T. japonica and E. papillosa (rarity and lack of genetic variation) significantly threatens the long-term survival of the species in Korea.     

Comparative Analysis of Genetic Diversity and Population Structure of Sipunculus nudus as Revealed by Mitochondrial COI Sequences

Genetic diversity and population structure of Sipunculus nudus were evaluated using a 652 base pair fragment of the mitochondrial cytochrome oxidase I gene. The populations were collected from Beihai, Sanya, and Xiamen. A total of 71 polymorphic sites defined 16 distinct haplotypes. The mean haplotype diversity and nucleotide diversity of the three populations were 0.9354 ± 0.0168 and 0.0035 ± 0.0018, respectively. Analysis at the intrapopulation level showed that the Beihai population had the greatest haplotype and nucleotide diversity, followed by the Xiamen and Sanya populations. Analysis of molecular variance showed significant genetic differentiation among the three populations (Fst = 0.0796, P < 0.05). The present results revealed that S. nudus populations had a high level of genetic diversity and distinct population structures.     

Genome-wide discovery of DNA polymorphism in Brassica rapa

Single nucleotide polymorphisms (SNPs) and/or insertion/deletions (InDels) are frequent sequence variations in the plant genome, which can be developed as molecular markers for genetic studies on crop improvement. The ongoing Brassica rapa genome sequencing project has generated vast amounts of sequence data useful in genetic research. Here, we report a genome-wide survey of DNA polymorphisms in the B. rapa genome based on the 557 bacterial artificial clone sequences of B. rapa ssp. pekinensis cv. Chiifu. We identified and characterized 21,311 SNPs and 6,753 InDels in the gene space of the B. rapa genome by re-sequencing 1,398 sequence-tagged sites (STSs) in eight genotypes. Comparison of our findings with a B. rapa genetic linkage map confirmed that STS loci were distributed randomly over the B. rapa whole genome. In the 1.4 Mb of aligned sequences, mean nucleotide polymorphism and diversity were θ = 0.00890 and π = 0.00917, respectively. Additionally, the nucleotide diversity in introns was almost three times greater than that in exons, and the frequency of observed InDel was almost 17 times higher in introns than in exons. Information regarding SNPs/InDels obtained here will provide an important resource for genetic studies and breeding programs of B. rapa.     

DNA typing and genetic relations among populations of Kelussia odoratissima using ISSR and SRAP markers

Kelussia odoratissima is well known for its medicinal importance. It has been announced as an endangered species. Thus, examining the genetic variation and conservation of this plant is necessary. In the present study, inter simple sequence repeat (ISSR) and sequence-related amplified polymorphism (SRAP) molecular markers were employed for the first time to access the genetic diversity and relationships of 77 wild individual plants of K. odoratissima collected from seven populations in Central Zagros region of Iran. A total of 146 bands were amplified by 12 ISSR primers, of which 129 (87.80 %) were polymorphic, while 69 polymorphic bands (83.30 %) were observed among 86 bands amplified by 11 SRAP primers. Polymorphic information content (PIC = 0.32), resolving power (Rp = 7.80), and marker informativeness (MI = 3.48) generated by ISSR primers were higher than that of SRAP analysis (PIC = 0.30, Rp = 5.61, and MI = 1.88). The study indicated that ISSR were more effective than SRAP markers for assessing the degree of genetic variation of K. odoratissima. In both UPGMA dendrograms of ISSR and SRAP, in most cases, individuals from each population were clustered in various groups without clear separation, which demonstrates the high variability of this germplasm in Iran. UPGMA cluster analysis revealed inconsistencies in the clustering patterns, as the Mantel’s test between the dendrograms for ISSR and SRAP data indicated a poor fit for the ISSR and SRAP data types (r = 0.10). Besides, principal coordinate analysis results showed that the first three principal coordinates account for 65.57 % of the total variation and studied seven populations were separated from each other and placed into five groups. These results have an important implication for K. odoratissima germplasm characterization, improvement, and conservation.     

Assessment of molecular diversity and evolutionary relationship of kenaf (Hibiscus cannabinus L.), roselle (H. sabdariffa L.) and their wild relatives

Kenaf (Hibiscus cannabinus L.) and roselle (H. sabdariffa L.) are valuable fibre crop species with diverse end use. Phylogenetic relationship of 73 accessions of kenaf, roselle and their wild relatives from 15 countries was assessed using 44 inter-simple sequence repeat (ISSR) and jute (Corchorus olitorius L.) specific simple sequence repeats (SSR) markers. A total of 113 alleles were identified of which 61.95 % were polymorphic. Jute specific SSR markers exhibited high polymorphism and resolving power in kenaf, although ISSR markers exhibited higher resolving power than SSR markers. Number of polymorphic alleles varied from 1 to 5 for ISSR and 1 to 6 for SSR markers. Cultivated species exhibited higher allele polymorphism (57 %) than the wild species (35 %), but the improved cultivars exhibited lower genetic diversity compared to germplasm accessions. Accessions with common genetic lineage and geographical distribution clustered together. Indian kenaf varieties were distinct from cultivars bred in other countries and shared more genetic homology with African accessions. High genetic diversity was observed in the Indian (J = 0.35–0.74) and exotic kenaf germplasm collections (J = 0.38–0.79), suggesting kenaf might have been introduced in India from Africa through Central Asia during early domestication. Genetic similarity-based cluster analysis was in close accordance with taxonomic classification of Hibiscus.     

Estimation of genetic variability and population structure in Sapindus trifoliatus L., using DNA fingerprinting methods

Genetic variability and population structure of Sapindus trifoliatus L. (Sapindaceae), collected from Gujarat, Karnataka and Uttar Pradesh states were estimated using three DNA fingerprinting methods viz., random amplified polymorphic DNA (RAPD), directed amplification of minisatellite DNA (DAMD) and inter-simple sequence repeats (ISSR). The cumulative data analysis carried out for all three markers showed 69.42 % polymorphism. The intra-population genetic diversity analysis revealed the highest values of Nei’s genetic diversity (0.16), Shannon information index (0.24) and polymorphic loci (43.99 %) among Bhavnagar (BH) population, whereas lowest values were found in Junagarh (JU) population. The maximum inter-population average genetic distance (0.20) was between Allahabad (AL) and JU populations. Analysis of molecular variance (AMOVA) showed highest percentage of variation among individuals of populations (56 %) followed by 25 % among populations and 19 % among regions. Principal coordinate analysis and UPGMA dendrogram revealed that genetic diversity was in congruence with the geographical diversity. The data strongly suggest that low genetic flow, geographic isolation and to some extent genetic drift are the major factors responsible for high genetic differentiation. Preservation of genetic diversity of S. trifoliatus is important, both to promote adaptability of the populations to changing environment as well as to preserve a large gene pool for future genetic improvement. The present study using RAPD, DAMD and ISSR profiles of S. trifoliatus provide the means of rapid characterization of accessions within the populations, and thus enable the selection of appropriate accessions for further utilization in conservation and prospection programs of this important plant genetic resource.