Automated ISSR Fingerprinting Is a Cost-Effective Way to Assess Genetic Diversity and Taxonomic Differentiation—A Case Study on the Encephalartos eugene-maraisii Species Complex
<p>Map of the Limpopo province of South Africa showing the approximate location of the six members of the <span class="html-italic">Encephalartos eugene-maraisii</span> complex, as well as <span class="html-italic">E. hirsutus</span>, based on IUCN records (accessed December 2023).</p> "> Figure 2
<p>Boxplots showing the nanodrop readings for DNA concentration in ng/µL (<b>a</b>) and DNA fluorescence ratios indicating purity (<b>b</b>), in samples that were included in the study (blue plots) and those excluded (orange plots) due to unsuccessful PCR amplification. Means are denoted by X and medians by horizontal lines inside the boxes. Outliers are denoted by dots.</p> "> Figure 3
<p>STRUCTURE bar plots showing the proportion of membership of samples assigned to the optimum K within the <span class="html-italic">Encephalartos eugene-maraisii</span> complex. Results are based on ISSR fragments scored at a 50 relative fluorescence unit (rfu) cut-off value. The dataset was assessed using the standard STRUCTURE model (<b>a</b>) and the LOCPRIOR model (<b>b</b>), which account for known locality data prior to the run. Colours represent each of the predefined clusters to which each sample is assigned.</p> "> Figure 4
<p>Neighbor-Joining analysis of the <span class="html-italic">Encephalartos eugene-maraisii</span> complex based on ISSR markers with a minimum band intensity of 50 relative fluorescence units (rfu). Genetic distances were computed using the DICE coefficient. Bootstrap values exceeding 50% are indicated on the applicable nodes. The colour of each sample corresponds to its species, and sample names are represented by the first three letters of their species epithet. Sample duplicates, representing material obtained from the same plant, but extracted in a different DNA extraction batch, are indicated by the symbols.</p> "> Figure 5
<p>UPGMA dendrogram of the <span class="html-italic">Encephalartos eugene-maraisii</span> complex based on ISSR markers at a relative fluorescence unit (rfu) cut-off of 50 rfu. Bootstrap values exceeding 50% are indicated on the applicable nodes. Genetic distances were computed using the DICE coefficient. The colour of each sample corresponds to its species and sample names are represented by the first three letters of their species epithet. Sample duplicates, representing material obtained from the same plant, but extracted in a different DNA extraction batch, are indicated by the symbols.</p> "> Figure 6
<p>Median-Joining network of the <span class="html-italic">Encephalartos eugene-maraisii</span> complex based on ISSR markers with a minimum band intensity of 50 relative fluorescent units. Colours denote the species of each sample in this study.</p> ">
Abstract
:1. Introduction
The Conservation Status of Cycads in Africa–Encephalartos as a Case Study
The Encephalartos eugene-maraisii complex
2. Materials and Methods
2.1. Sampling
2.2. DNA Extraction
2.3. PCR Optimisation and Selection of ISSR-PCR Primers
2.4. PCR Reaction Conditions
2.5. Construction of Datasets for Analysis
2.6. Methods to Assess Genetic Similarity and Diversity
2.6.1. Cluster Analysis
2.6.2. Statistical Analysis
2.6.3. STRUCTURE Analysis (Bayesian MCMC)
2.6.4. Network Analysis
3. Results
3.1. Sampling and ISSR Amplification Success
3.2. Statistical Analysis
3.3. STRUCTURE Analysis
3.4. Cluster Analysis
3.5. Network Analysis
4. Discussion
4.1. Statistical Analysis
4.2. Species Delimitation of the E. eugene-maraisii Complex
4.2.1. E. eugene-maraisii, E. dolomiticus, and E. dyerianus
4.2.2. E. nubimontanus and E. cupidus
4.2.3. E. middelburgensis
4.2.4. Anomalous Samples
4.3. Comparison of Analyses and Datasets
4.3.1. Datasets
4.3.2. Notable Differences among Analyses
4.4. Methodological Critique
4.4.1. Reproducibility of ISSR Amplifications
4.4.2. Sampling Effort and Cost-Reduction Strategies
4.5. Taxonomic Implications
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Rank | Country | Number of Publications | % of Total Publications |
---|---|---|---|
1 | India (8) | 1591 | 19.1 |
2 | China (4) | 1493 | 17.9 |
3 | United States of America (10) | 625 | 7.5 |
4 | Iran | 504 | 6.0 |
5 | Brazil (1) | 447 | 5.4 |
6 | Egypt | 384 | 4.6 |
7 | Türkiye | 275 | 3.3 |
8 | Italy | 246 | 2.9 |
9 | Saudi Arabia | 244 | 2.9 |
10 | Russian Federation | 225 | 2.7 |
11 | Poland | 186 | 2.2 |
12 | Spain | 181 | 2.2 |
13 | Germany | 179 | 2.1 |
14 | Japan | 164 | 2.0 |
15 | Mexico (5) | 153 | 1.8 |
16 | United Kingdom | 150 | 1.8 |
17 | France | 140 | 1.7 |
18 | Canada | 133 | 1.6 |
19 | Australia (6) | 129 | 1.5 |
20 | Portugal | 118 | 1.4 |
21 | Malaysia (15) | 109 | 1.3 |
22 | Thailand (20) | 102 | 1.2 |
23 | Indonesia (2) | 94 | 1.1 |
24 | South Korea | 86 | 1.0 |
25 | Argentina | 83 | 1.0 |
26 | Tunisia | 76 | 0.9 |
27 | Greece | 64 | 0.8 |
28 | Pakistan | 59 | 0.7 |
29 | South Africa (19) | 54 | 0.6 |
30 | Czech Republic | 52 | 0.6 |
ISSR Primer Name | 5′ Fluorescent Marker | Sequence |
---|---|---|
Manny | 6-FAM | CACCACCACCACRC |
812 | HEX | GAGAGAGAGAGAGAGAA |
Mao | TET | CTCCTCCTCCTCRC |
Omar | HEX | GAGGAGGAGGAGRC |
864 | 6-FAM | ATGATGATGATGATGATG |
856 | TET | ACACACACACACACACYA |
Primer | Minimum Fluorescence (rfu) | Total Number of Bands Obtained from All Samples | Mean Bands per Sample | Private Bands |
---|---|---|---|---|
ISSR Mao (TET) | 50 | 111 | 12 | 32 |
ISSR Mao (TET) | 100 | 83 | 7 | 22 |
ISSR Mao (TET) | 200 | 30 | 4 | 6 |
ISSR 864 (6-FAM) | 50 | 459 | 44 | 68 |
ISSR 864 (6-FAM) | 100 | 327 | 19 | 32 |
ISSR 864 (6-FAM) | 200 | 73 | 12 | 28 |
ISSR 856 (TET) | 50 | 93 | 11 | 21 |
ISSR 856 (TET) | 100 | 29 | 3 | 5 |
ISSR 856 (TET) | 200 | 10 | 1 | 1 |
Combined | 50 | 663 | 22 | 121 |
Combined | 100 | 439 | 10 | 59 |
Combined | 200 | 113 | 5 | 35 |
Species | Mean DNA Concentration (ng/μL) | Mean 260/280 | Mean 260/230 | Number of Samples | Percentage Successful Amplification |
---|---|---|---|---|---|
E. eugene-maraisii | 302.6 | 1.64 | 0.5 | 35 | 55.5% |
E. nubimontanus | 373.1 | 1.34 | 0.58 | 48 | 54.1% |
E. hirsutus | 328.2 | 1.65 | 0.53 | 2 | 100% |
E. dyerianus | 286.5 | 1.4 | 0.6 | 27 | 56% |
E. middelburgensis | 294.4 | 1.45 | 0.55 | 23 | 24.6% |
E. cupidus | 491.8 | 1.3 | 0.62 | 46 | 36.2% |
E. dolomiticus | 344.3 | 1.6 | 0.49 | 13 | 60% |
Omitted samples Batch 1 | 350.3 | 1.61 | 0.51 | 46 | 0% |
Omitted samples Batch 2 | 610.4 | 1.20 | 0.62 | 23 | 0% |
-rfu Cut-Off Dataset | |||
---|---|---|---|
50 | 100 | 200 | |
Nucleotide diversity (π) | 0.111362 | 0.06931 | 0.154567 |
Segregating sites | 474 | 207 | 105 |
Tajima’s D statistic | −0.70597 | −0.84998 | −0.50775 |
Significance (p) | 0.743371 | 0.79021 | 0.673339 |
-rfu Cut-Off Dataset | |||
---|---|---|---|
50 | 100 | 200 | |
Variation among groups (%) | −1.89289 | −7.46642 | −2.86349 |
Fixation index ΦCT | −0.01893 | −0.07466 | −0.02863 |
Significance (1000 permutations): | 0.378 | 0.42957 | 0.529 |
Variation among species within groups (%) | 37.31296 | 46.16488 | 34.46871 |
Fixation index ΦSC | 0.3662 | 0.42957 | 0.33509 |
Significance (1000 permutations): | <0.001 | <0.001 | <0.001 |
Variation among species among groups (%) | 64.57993 | 61.30154 | 68.39479 |
Fixation index ΦST | 0.3542 | 0.38698 | 0.31605 |
Significance (1000 permutations): | <0.001 | <0.001 | <0.001 |
STRUCTURE Model | K | Reps | Mean LnP(K) | Stdev LnP(K) | Ln′(K) | |Ln″(K)| | Delta K |
---|---|---|---|---|---|---|---|
50 rfu dataset | |||||||
Standard | 3 | 10 | −10,285.48 | 2.26019 | 673.2 | 300.53 | 132.96688 |
4 | 10 | −9912.81 | 68.14371 | 372.67 | 4205.43 | 61.71413 | |
2 | 10 | −10,958.68 | 24.08724 | 762.99 | 89.79 | 3.7277 | |
LOCPRIOR | 4 | 10 | −9890.92 | 11.29796 | 394.28 | 2663.47 | 235.74778 |
3 | 10 | −10,285.2 | 5.27952 | 673.84 | 279.56 | 52.95178 | |
2 | 10 | −10,959.04 | 55.6032 | 762.26 | 88.42 | 1.5902 | |
100 rfu dataset | |||||||
Standard | 2 | 10 | −4331.6 | 0.80139 | 481.21 | 176.68 | 220.46758 |
3 | 10 | −4027.07 | 2.03909 | 304.53 | 323.73 | 158.76198 | |
4 | 10 | −4046.27 | 408.31331 | −19.2 | 387.34 | 0.94863 | |
LOCPRIOR | 3 | 10 | −4051.01 | 6.14138 | 318.47 | 1845.36 | 300.47967 |
2 | 10 | −4369.48 | 17.8674 | 443.28 | 124.81 | 6.98535 | |
7 | 10 | −4022.16 | 418.53457 | 976.32 | 1844.54 | 4.40714 | |
200 rfu dataset | |||||||
Standard | 2 | 10 | −2499.8 | 2.28619 | 214.9 | 118.48 | 51.8242 |
5 | 10 | −2209.93 | 11.79426 | 144.62 | 115.61 | 9.80223 | |
6 | 10 | −2180.92 | 18.74145 | 29.01 | 96.91 | 5.17089 | |
LOCPRIOR | 2 | 10 | −2487.96 | 1.1462 | 226.58 | 125 | 109.05587 |
3 | 10 | −2386.38 | 2.85299 | 101.58 | 44.26 | 15.51355 | |
4 | 10 | −2240.54 | 23.62359 | 145.84 | 191.7 | 8.11477 |
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Murphy, D.; Barker, N.P.; Frisby, A. Automated ISSR Fingerprinting Is a Cost-Effective Way to Assess Genetic Diversity and Taxonomic Differentiation—A Case Study on the Encephalartos eugene-maraisii Species Complex. Diversity 2024, 16, 507. https://doi.org/10.3390/d16080507
Murphy D, Barker NP, Frisby A. Automated ISSR Fingerprinting Is a Cost-Effective Way to Assess Genetic Diversity and Taxonomic Differentiation—A Case Study on the Encephalartos eugene-maraisii Species Complex. Diversity. 2024; 16(8):507. https://doi.org/10.3390/d16080507
Chicago/Turabian StyleMurphy, Deanne, Nigel Paul Barker, and Arnold Frisby. 2024. "Automated ISSR Fingerprinting Is a Cost-Effective Way to Assess Genetic Diversity and Taxonomic Differentiation—A Case Study on the Encephalartos eugene-maraisii Species Complex" Diversity 16, no. 8: 507. https://doi.org/10.3390/d16080507