Pairwise Genetic Divergence Research Articles

Complete mitochondrial genome of golden variant of freshwater fish Labeo rajasthanicus (Cypriniformes: Cyprinidae): endemic to India

The complete mitochondrial genome of the freshwater fish species Labeo rajasthanicus was obtained, using Illumina NovaSeq 6000 with 2 × 150 bp paired-end sequencing. The mitogenome of L. rajasthanicus is 16,738 bp in length (GenBank accession no.: OQ834146), comprised of 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and a control region, i.e. D-loop. The arrangement of genes was found to be identical to other Cypriniformes fish mitogenome, available in the NCBI database. The taxonomic status of L. rajasthanicus as a valid species was debated by some researchers and it was considered a synonym of L. boggut. However, phylogenetic analysis in the present study supports the species validity of L. rajasthanicus, as it showed a distinct node well separated from L. boggut and supported by a high bootstrap value. Furtherly, the pairwise genetic divergence among studied species showed the divergence between L. rajasthanicus and L. boggut as 1.6% whereas the minimum divergence was found to be 0.13% with L. dussumieri followed by L. fimbriatus (0.58%) and L. gonius (0.63%). The complete mitogenome of L. rajasthanicus will also be useful as a baseline reference genome for the reconstruction and annotation of the mitogenome of other Labeo species.

Reassessing the genetic variability of Tectona grandis through high-throughput genotyping: Insights on its narrow genetic base.

Teak (Tectona grandis Linn. f.) is considered one of the most expensive hardwoods in the world. The dispersion of the species over the years has taken the teak beyond its first sources of diversity and little is known about the genetic origin and genetic variability. Thus, this study aimed to investigate the genetic diversity and genetic population structure existing in a representative teak germplasm bank collection. DNA was extracted from young leaves and each sample were genotyped by whole genome sequencing at 3 giga bases per sample, the sequences are aligned using the genome, and SNPcalls and quality control were made. To study the population structure of the genotypes, Bayesian variational inference was used via fastStructure, the phylogenetic tree was based on the modified Euclidean distance and the clustering by the UPGMA hierarchical method. Genetic diversity was analyzed based on the pairwise genetic divergence (Fst) of Weir and Cockerham. Genotyping by sequencing resulted in a database of approximately 1.4 million of variations SNPs were used for analysis. It was possible to identify four populations with considerable genetic variability between and within them. While the genetic variability in teak is generally known to be narrow, this study confirmed the presence of genetic variability scale in teak, which is contrary to what was initially expected.

Two new karst-adapted species in the Cyrtodactyluspulchellus group (Reptilia, Gekkonidae) from southern Thailand.

The exploration of unsurveyed areas in southern Thailand discovered two new karst-adapted species, Cyrtodactylussungaiupesp. nov. and Cyrtodactyluswangkhramensissp. nov., from Thung Wa and La-ngu Districts, Satun Province, respectively. These new species are members of the C.pulchellus group that occur along the Thai-Malay Peninsula. The new species can be distinguished from all other congeners by their key morphological characters and genetic divergence. Morphologically, Cyrtodactylussungaiupesp. nov. and Cyrtodactyluswangkhramensissp. nov. can be diagnosed from other members by having a combination of differences in body size; degree of dorsal tuberculation; absence of tubercles on ventral surfaces; number of ventral scales, paravertebral tubercles and femoroprecloacal pores in males only; deep precloacal groove only in males; absence of a scattered pattern of white dorsal tubercles; number of dark body bands; and the extent of caudal tubercles on an original tail. Although the two species are sister taxa and have nearly identical morphologies, they are considered to be different species, based on a relatively high uncorrected pairwise genetic divergence of the mitochondrial ND2 gene (6.59-6.89%), statistically significant univariate and multivariate morphological differences (PERMANOVA and ANOVA) and diagnostic characteristics of caudal tuberculation on the original tail. Moreover, Cyrtodactylussungaiupesp. nov. and Cyrtodactyluswangkhramensissp. nov. are currently restricted to their karstic type localities which may serve as a geographic barrier to dispersal and gene flow.

Morphological and molecular differences in two closely related Calotes Cuvier, 1817 (Squamata: Agamidae: Draconinae) with the first record of Calotes medogensis Zhao & Li, 1984 from India

We studied the morphological and genetic differences within Calotes jerdoni,a widespread species across northeast India.Our results suggest the presence of two distinct species in this region,one being Calotes jerdoniand the other being Calotes medogensis, which we reportfor the first time from India. We designate a lectotype for Calotes jerdoni and provided extended description based on freshly collected materials. Previously undetermined diagnostic characters were identified and are discussed here in detail. The aforementioned species show an interspecific pairwise genetic divergence of 13-14% in the ND2 mitochondrial gene.

Genetic Diversity in Successive Age Cohorts of Ilex paraguariensis in Southern Brazil

Abstract Ilex paraguariensis A. St. Hil. (yerba mate) is a native species widely consumed in southern Brazil, a region that presents a current scenario of forest fragmentation. The objective was to evaluate the genetic diversity, genetic structure, and gene flow among cohorts of I. paraguariensis in two forest remnants. Genetic analyses were conducted using microsatellite markers from leaves from adult (Ad), regenerating (Reg), and seed-derived (Se) populations in Santa Catarina (SC) (Chapecó’s National Forest [CCO]) and Rio Grande do Sul (RS) (Teixeira Soares’ Municipal Natural Park [PTS]). We found unique alleles in both populations, eleven in the CCO and eight in the PTS. There was no significant difference in genetic diversity indexes, whereas high and significant fixation indexes were observed (FCCO = 0.394; FPTS = 0.319). The proportion of genetic variation obtained between populations was lower (8%) than the proportion within populations (92%). The presence of significant genetic structure was verified in the cohorts of I. paraguariensis for two study areas. Moreover, all pairwise genetic divergence between Ad and Reg, and Ad and Se cohorts was significant, except for Ad versus Reg in PTS. Aiming to maintain diversity over generations, we recommend the conservation of remnants and expansion of connectivity between fragments.

Redescription of Hemidactylus giganteus Stoliczka, 1871 with the description of three new allied species (Squamata: Gekkonidae: Hemidactylus Goldfuss, 1820) from peninsular India.

We revise the systematics of Hemidactylus giganteusan Indian endemic leaf-toed gecko based on recent collections from the type locality and other localities in peninsular India. We provide a comprehensive redescription of H. giganteus sensu stricto and describe three new allied species from the Karnataka and Telangana states in India. We analysed the phylogenetic relationships between the three new species, H. raya sp. nov., H. saxicolus sp. nov., and H. aemulus sp. nov., and found them to be nested within the H. giganteus sensu lato branch. The pairwise genetic divergence between these new cryptic taxa from H. giganteus sensu stricto ranged from 5.88% to 10.95% for the Cyt b gene. The new species also differ from H. giganteus sensu stricto based on number of femoral pores on each thigh and poreless scales separating them (1823 femoral pores and seven or eight poreless scales versus 17 femoral pores and seven poreless scales in H. raya sp. nov., 26 or 27 femoral pores and nine poreless scales in H. saxicolus sp. nov., and 2225 femoral pores and nine to ten poreless scales in H. aemulus sp. nov.), corroborated by linear discriminant analysis based on 29 morphometric and meristic characters. Our species delimitation analysis shows the presence of three additional putative new species awaiting formal description within the H. giganteus sensu lato branch.

Population genetic diversity and structure of the pecan scab pathogen, Venturia effusa, on cv. Desirable and native seedlings, and the impact of marker number

AbstractScab (Venturia effusa) is the major cause of economic loss in pecan in the south‐eastern USA. We explored population genetic diversity and structure among orchards of cv. Desirable and native seedlings, and within‐orchard variability among trees of all cultivars sampled. We compared the ability of 30, 15 and 7 previously developed microsatellites to characterize the population genetic diversity and structure of V. effusa. Analyses of molecular variance (AMOVA) provided little evidence of structure dependent on cultivar, but there was some evidence of structure between orchards of a cultivar based on distance. Individual orchard AMOVA showed that three of 11 orchards had between‐tree population structure. Among six populations from cv. Desirable, a Mantel test showed that geographic distance was related to the pairwise genetic divergence (R2 = 0.84). Among 11 orchards of various cultivars there was little difference in diversity using 30, 15 or 7 markers, or population structure based on AMOVA. Some minor differences in population structure were seen based on discriminant analysis of principal components, or dendrograms. Thus, depending on the objectives, future studies may use as few as 15 or 7 markers without losing ability to discern population genetic diversity or structure. More populations exhibited linkage disequilibrium when using 15 or 30 markers compared to when using seven markers. Knowledge of population genetics of V. effusa in relation to host genotype is needed to understand pathogen population interactions and gene flow, knowledge that will help underpin future breeding efforts to develop durable resistance in this long‐lived orchard tree.

A new species of Cyrtodactylus (Squamata: Gekkonidae) from Ninh Thuan Province, southern Vietnam.

A new species of the Cyrtodactylus irregularis group is described from Ninh Thuan Province, southern Vietnam based on molecular divergence and morphological differences. Cyrtodactylus orlovi sp. nov. is distinguished from the remaining Indochinese bent-toed geckos by having the unique combination of the following characters: size medium (SVL 61.077.7 mm); dorsal tubercles in 1620 irregular rows; 3639 ventral scale rows; precloacal pores absent in females, 5 or 6 in males, in a continuous row; femoral pores absent; 38 enlarged femoral scales; postcloacal spurs 1 or 2; lamellae under toe IV 1619; a continuous neckband; a highly irregular transverse banded dorsal pattern; the absence of transversely enlarged median subcaudal scales. In phylogenetic analyses, the new species was revealed to be the sister taxon to a clade consisting of Cyrtodactylus cattienensis and the most recently described species from Vietnam, C. chungi, with 12.112.4% and 11.7 % pairwise genetic divergence from the two species, respectively, based on a fragment of the mitochondrial COI gene.

Morphometric and molecular differentiation between Egyptian Stellagama stellio vulgaris and S. stellio salehi (Reptilia: Agamidae)

From the seven recognised subspecies of Stellagama stellio, S. s. vulgaris is found in northwestern Egypt and S. stellio salehi in the south Sinai and are known to be very similar. A Principal Component Analysis was carried out for 14 morphometric fea­tures, PCA1 accounted for a little cumulative variation (87.03%) between the two subspecies with a strong canonical correlation (r = 0.999). PCA2 accounted for a high cumulative variation (98.04%) with a strong canonical correlation (r = 0.985) and head height (HH) was the only major parameter for the significant difference. Within 398 sites of 16S rRNA gene sequenced, 6 base substitutions were recorded between S. s. vulgaris and S. s. salehi and the pairwise genetic divergence was calculated as 1.5%, which is comparable to that found between some other conspecific agamids. Neither the morphometric nor the molecular data support the distinction of two different subspecies. No genetic difference was found between S. s. salehi and S. s. brachydactyla which occurs from northern Sinai over Jordan to Saudi Arabia.

DNA Barcoding and geographical scale effect: The problems of undersampling genetic diversity hotspots.

DNA barcoding identification needs a good characterization of intraspecific genetic divergence to establish the limits between species. Yet, the number of barcodes per species is many times low and geographically restricted. A poor coverage of the species distribution range may hamper identification, especially when undersampled areas host genetically distinct lineages. If so, the genetic distance between some query sequences and reference barcodes may exceed the maximum intraspecific threshold for unequivocal species assignation. Taking a group of Quercus herbivores (moths) in Europe as model system, we found that the number of DNA barcodes from southern Europe is proportionally very low in the Barcoding of Life Data Systems. This geographical bias complicates the identification of southern query sequences, due to their high intraspecific genetic distance with respect to barcodes from higher latitudes. Pairwise intraspecific genetic divergence increased along with spatial distance, but was higher when at least one of the sampling sites was in southern Europe. Accordingly, GMYC (General Mixed Yule Coalescent) single‐threshold model retrieved clusters constituted exclusively by Iberian haplotypes, some of which could correspond to cryptic species. The number of putative species retrieved was more reliable than that of multiple‐threshold GMYC but very similar to results from ABGD and jMOTU. Our results support GMYC as a key resource for species delimitation within poorly inventoried biogeographic regions in Europe, where historical factors (e.g., glaciations) have promoted genetic diversity and singularity. Future European DNA barcoding initiatives should be preferentially performed along latitudinal gradients, with special focus on southern peninsulas.

A new potentially endangered limestone-associated Bent-toed Gecko of the Cyrtodactylus pulchellus (Squamata: Gekkonidae) complex from northern Peninsular Malaysia.

A survey of a limestone forest at Gunung Baling, Kedah, West Malaysia lead to the discovery of an undescribed species of Bent-toed Gecko from the Cyrtodactylus pulchellus complex. Cyrtodactylus evanquahi sp. nov. can be distinguished from all other species in the C. pulchellus complex by a suite of morphological and color pattern characteristics: prominent tuberculation, higher number of dark body bands, and a smaller maximum SVL. It is further differentiated from all other species as follows; no tubercles on the ventral surface of the forelimbs, gular region, or in the ventrolateral folds; 31-34 paravetebral dorsal tubercles; 18-23 longitudinal rows of tubercles; 29-33 ventral scales; 22-23 subdigital lamellae on the fourth toe; 32-36 femoroprecloacal pores; a shallow precloacal groove in males; body bands and nuchal loop edged with a thin white line bearing tubercles; no scattered white spots on the dorsum; six or seven dark body bands much thinner than interspaces; 9-11 dark caudal bands on original tail; bands on the original tail separated by immaculate white caudal bands. It is further differentiated by an uncorrected pairwise genetic divergence of 6.50-15.67% from all other congeners in the C. pulchellus complex. It is most closely related to C. pulchellus from Penang Island ∼76 km to the southwest. In addition to the new samples from Gunung Baling, we added four samples of C. bintangrendah from the new locality of Belukar Semang, Perak. The discovery of yet another new species of the C. pulchellus complex from a limestone habitat continues to underscore the high degree of endemism and the importance of these unique habitats for biodiversity, and the continued need for their conservation.

The largest fish in the world's biggest river: Genetic connectivity and conservation of Arapaima gigas in the Amazon and Araguaia-Tocantins drainages.

Arapaima, pirarucu or paiche (Arapaima gigas) is one of the largest freshwater fish in the world, and has a long history of commercial exploitation in the Amazon region. To estimate levels of genetic variability and historical and recent connectivity in Arapaima, we examined variation in eleven microsatellite DNA markers in individuals from 22 localities in Brazil, Colombia, and Peru. The results of analysis of molecular variance, Bayesian clustering and discriminant analysis of principal components showed that Arapaima in our samples represents two major populations, one in the Amazonas and one in the Araguaia-Tocantins River basins. The Amazonas population is further structured by isolation-by-distance with the hydrologically largely unconnected Amapá locality representing the eastern-most extreme of this continuum; gene flow predominates at distances of less than 1500 km with localities separated by over 2000 km dominated by genetic drift and effectively forming different populations. We saw no evidence of multiple species of Arapaima in the Amazonas basin, and analysis of pairwise genetic divergence (FST) with Mantel tests and correlograms indicated that this largest population exhibits a large-scale pattern of isolation-by-distance, with which results from MIGRATE-N agreed. The degree and significance of genetic divergence indicates that most sampled localities represent demographically independent sub-populations, although we did identify several recent migration events between both proximal and more distant localities. The levels of genetic diversity were heterogeneous across sites, including low genetic diversity, effective population sizes, and evidence of genetic bottlenecks in several places. On average the levels of gene diversity and rarefied allelic richness were higher for localities along the Amazonas mainstem than in the tributaries, despite these being the areas of highest fishing pressure, while the lowest values were found in tributary headwaters, where landscape modification is a significant threat. We recommend that managers consider the regional and local threats to these populations and tailor strategies accordingly, strategies which should ensure the ability of young A. gigas to disperse through floodplain corridors to maintain genetic diversity among otherwise sedentary adult sub-populations.

PRDM9 Diversity at Fine Geographical Scale Reveals Contrasting Evolutionary Patterns and Functional Constraints in Natural Populations of House Mice.

One of the major challenges in evolutionary biology is the identification of the genetic basis of postzygotic reproductive isolation. Given its pivotal role in this process, here we explore the drivers that may account for the evolutionary dynamics of the PRDM9 gene between continental and island systems of chromosomal variation in house mice. Using a data set of nearly 400 wild-caught mice of Robertsonian systems, we identify the extent of PRDM9 diversity in natural house mouse populations, determine the phylogeography of PRDM9 at a local and global scale based on a new measure of pairwise genetic divergence, and analyze selective constraints. We find 57 newly described PRDM9 variants, this diversity being especially high on Madeira Island, a result that is contrary to the expectations of reduced variation for island populations. Our analysis suggest that the PRDM9 allelic variability observed in Madeira mice might be influenced by the presence of distinct chromosomal fusions resulting from a complex pattern of introgression or multiple colonization events onto the island. Importantly, we detect a significant reduction in the proportion of PRDM9 heterozygotes in Robertsonian mice, which showed a high degree of similarity in the amino acids responsible for protein–DNA binding. Our results suggest that despite the rapid evolution of PRDM9 and the variability detected in natural populations, functional constraints could facilitate the accumulation of allelic combinations that maintain recombination hotspot symmetry. We anticipate that our study will provide the basis for examining the role of different PRDM9 genetic backgrounds in reproductive isolation in natural populations.

A meta-analysis of molecular marker genetic datasets for eastern Africa trees supports the utility of potential natural vegetation maps for planning climate-smart restoration initiatives

Forest and woodland landscape restoration is a key undertaking of renewed interest for forestry and conservation practitioners, but is hampered by the lack of information on the distributions of tree species and of patterns of intra-specific genetic variation. Through the first meta-analysis of its type, we here tested the utility of a high-resolution potential natural vegetation (PNV) map for eastern Africa (vegetationmap4africa) for supporting restoration activities by comparison with 20 molecular marker genetic datasets, identified through literature review and other sources, for ten indigenous tree species. Our analysis indicated that site suitability and stability values from PNV-based ecological niche modelling involving current and past climate scenarios were positively related to population genetic diversity values revealed by molecular markers, supporting the value of PNV maps for the practical planning of restoration activities accounting for anthropogenic climate change. Furthermore, population pairwise genetic divergence was strongly positively correlated with population pairwise geographic distances for most datasets, indicating generalizable sampling implications for tree genetic resource conservation in the region. Population pairwise genetic divergence was however not well explained by sampling across PNV and wider physiognomic types, possibly due to molecular markers’ adaptive neutrality and high rates of recombination in trees, among other factors. Patterns of neutral molecular marker variation are thus no substitute for trials of adaptive variation for confirming or refuting the utility of vegetation boundaries in defining tree planting zones. We discuss the importance of results for eastern Africa and more widely.

Bajos niveles de variación isoenzimática en las poblaciones sureñas del arbusto endémico de Corea <em>Sophora koreensis (Fabaceae)</em>: implicaciones para su conservación

Se investigó la variación isoenzimática del arbusto raro Sophora koreensis en el condado de Yanggu (Corea del Sur), en el extremo sur de su área de distribución. Para obtener información sobre los procesos ecológicos a nivel del paisaje, se estudiaron dos o tres poblaciones para cada una de las cuatro localidades (en total, 10 poblaciones) en un radio de unos 6 km. Se encontraron bajos niveles de variación genética intra-poblacional (%P = 13,6, A = 1,14 y He = 0,026) y un grado moderado de diferenciación genética entre poblaciones (FST = 0,203). El análisis de la varianza molecular (AMOVA) reveló un porcentaje sustancialmente mayor de variación entre poblaciones dentro cada una de las localidades (17%) que entre localidades (5%). No hubo una relación significativa entre la divergencia genética y el logaritmo de la distancia geográfica entre pares de poblaciones (r = 0,032, P = 0,842). Estos resultados sugieren un flujo genético limitado entre poblaciones dentro de las localidades, además de indicar que la mejor estrategia para la preservación de la diversidad genética de S. koreensis es la conservación del máximo número de poblaciones posible.

The Seven-arm Octopus, Haliphron atlanticus Streenstrup, 1861 (Cephalopoda, Alloposidae), in the Fernando de Noronha archipelago, Brazil

The rare deep-sea octopus, Haliphron atlanticus is the only known species recognized within the genus. A fragment of H. atlanticus was found in the Fernando de Noronha archipelago, Brazil (South Atlantic). Both phylogenetic reconstruction and pairwise genetic divergence show that the specimen recorded in South Atlantic is closely related to individuals from North Pacific. However, there is a greater divergence among these specimens and a giant octopus from North Atlantic. This evidence suggests that Haliphron is not monospecific, with at least two species, both represented in the Atlantic Ocean.

Divergence of cryptic species of Doryteuthis plei Blainville, 1823 (Loliginidae, Cephalopoda) in the Western Atlantic Ocean is associated with the formation of the Caribbean Sea

Although recent years have seen an increase in genetic analyses that identify new species of cephalopods and phylogeographic patterns, the loliginid squid of South America remain one of the least studied groups. The suggestion that Doryteuthis plei may represent distinct lineages within its extensive distribution along the western Atlantic coasts from Cape Hatteras, USA (36°N) to northern Argentina (35°S) is consistent with significant variation in a number of environmental variables along this range including in both temperature and salinity. In the present study D. plei samples were obtained from a large number of localities along the western Atlantic coasts to investigate the distribution of these possible species in a phylogeographic context. Phylogeographic analyses were performed using the mitochondrial Cytochrome Oxidase I gene and nuclear Rhodopsin gene. Divergence times were estimated using Bayesian strict clock dating with calibrations based on fossil records for divergence from the lineage containing Vampyroteuthis infernalis (162mya), the probable origins of the North American loliginids (45mya), and the European loliginids (20mya) and fossil statolith from Doryteuthis opalescens (3mya). Our results suggest a deep genetic divergence within Doryteuthis plei. The currently described specie consists of two genetically distinct clades (pair-wise genetic divergence of between 7.7 and 9.1%). One clade composed of individuals collected in northwestern Atlantic and Central Caribbean Atlantic waters and the other from southwestern Atlantic waters. The divergence time and sampling locations suggest the speciation process at approximately 16Mya, which is in full agreement with the middle Miocene orogeny of the Caribbean plate, ending up with the formation of the Lesser Antilles and the adjacent subduction zone, coinciding with a particularly low global sea level, resulting in the practical absence of continental shelves at the area, and therefore an effective geographic barrier for D. plei. Furthermore, this study also provides evidence of previously undocumented sub-population structuring in the Gulf of Mexico.

Species limits in polymorphic mimetic Eniclases net-winged beetles from New Guinean mountains (Coleoptera, Lycidae).

Species delimitation was compared in a group of closely related lineages of aposematically colored Eniclases (Coleoptera, Lycidae) using morphology, genetic distances, and Bayesian implementation of the Poisson Tree Processes model. A high diversity of net-winged beetles was found in previously unsampled regions of New Guinea and ten new species are described: Eniclases bicolor sp. n., Eniclases bokondinensis sp. n., Eniclases brancuccii sp. n., Eniclases elelimensis sp. n., Eniclases infuscatus sp. n., Eniclases niger sp. n., Eniclases pseudoapertus sp. n., Eniclases pseudoluteolus sp. n., Eniclases tikapurensis sp. n., and Eniclases variabilis sp. n. Different levels of genetic and morphological diversification were identified in various sister-species pairs. As a result, both morphological and molecular analyses are used to delimit species. Sister-species with uncorrected pairwise genetic divergence as low as 0.45% were morphologically distinct not only in color pattern, but also in the relative size of eyes. Conversely, differences in color pattern regardless of their magnitude did not necessarily indicate genetic distance and intraspecific mimicry polymorphism was common. Additionally, genetic divergence without morphological differentiation was detected in one sister-species pair. Low dispersal propensity, diverse mimicry patterns, and mimetic polymorphism resulted in complex diversification of Eniclases and uncertain species delimitation in recently diversified lineages.

Characterization and utilization of microsatellites in the Coffea canephora genome to assess genetic association between wild species in Kenya and cultivated coffee

Coffee is an important beverage crop in the world and has a significant contribution to Kenya’s economy. Here, we analyzed the genome-wide distribution of microsatellites in the Coffea canephora genome. A total of 159,041 SSRs were identified, with an overall density of 308 SSRs per Mb. Tetra-nucleotide repeats are the most abundant, accounting for 32 % of the total SSRs. AT-rich motifs are dominant across all SSR repeat units, while GC-rich motifs were generally rare. A set of 100 SSRs was selected to amplify 96 coffee accessions, including 10 wild accessions collected from Mt. Marsabit (Kenya). Of these SSRs, 33 % generated clear polymorphic bands among all tested accessions, with an average of 3.9 alleles per SSR locus. Wild coffee species from Mt. Marsabit showed a close genetic similarity with cultivated accessions in Kenya, suggesting that the wild species in Mt. Marsabit played an important role in the domestication of cultivated coffee in Kenya. Significantly low pairwise genetic divergence was observed between cultivated and wild accessions in Kenya, suggesting a relatively narrow level of genetic basis among coffee germplasm in Kenya. In addition, cultivated and wild coffee accessions in Kenya show a great divergence from those in other countries. Our results not only provide molecular tools for genetic studies in coffee but are also helpful for conservation and coffee breeding programs in Kenya.

The Extent of Intra-Clonal Genetic Diversity within the Myeloma Clone Is a Predictive Biomarker of Progression and Outcome after Treatment

▪Multiple myeloma (MM) is a disease characterized by the abnormal proliferation of plasma cells in the bone marrow. We and others have recently demonstrated the existence of different myeloma subclones phylogenetically related to the founding clone. This intra-clonal heterogeneity is the basis for disease progression, treatment resistance, and relapse. However, the clinical and biological relevance of the presence and diversity of different myeloma subclones has not been fully established.In this study, we used whole exome sequencing (WES) plus a pull down of the MYC, IGH, IGL and IGK loci as a tool to analyze the largest series of presenting cases of myeloma (463 patients) to date, which were entered into the Myeloma XI trial (NCT01554852). DNA from both tumor and peripheral blood samples were used in the exome capture protocol following the SureSelect Target Enrichment System for Illumina Paired-End Sequencing Library v1.5. Exome reads were used to call single nucleotide variants (SNVs), indels, translocations, and copy number aberrations. The proportion of tumor cells containing an SNV was inferred. The presence and proportion of subclones were defined in a subset of 437 patients using a genetic algorithm based-tool (GAUCHO), which also calculated different indices of clonal diversity: number of clones, mean pairwise genetic divergence, Shannon and Inverse Simpson diversity index and Berger-Parker dominance index. Based on these results, we aimed to determine the clinical implications of the number of mutations and the subclonal diversity of MM at presentation in progression free (PFS) and overall survival (OS).We found that MM patients with t(14;16) and t(14;20) had more exonic mutations (not including Ig variants) than the rest of samples (median 87 versus 43, p<0.001). Additionally, we found that MM patients with an APOBEC signature or with mutations in ATM/ATR had significantly more mutations than patients without these genetic lesions with a median number of 137 mutations (range 20-569) and 84.5 (range 33-319) respectively (p<0.001). Subsequently, we identified patients with high number of mutations (>59 mutations) that had a worse outcome in terms of OS (2-year OS rate of 71% (95% CI, 63-80%) vs. 82% (95% CI, 78-87%), p=0.02), but not progression free survival (median 22.5 (95% CI 18.7-30.2) vs. 27.5 (95% CI, 25.8-30.5) months, p=0.1)We reported recurrent mutated genes in myeloma with mutations being present at both clonal and subclonal levels (IRF4, RB1, DIS3, BRAF, KRAS, and NRAS), whereas other genes were mutated only at clonal (HIST1H1E, LTB, TP53 or EGR1), or subclonal levels (CYLD, TRAF3, MAX). These results give insights about the differences in mutation acquisition times and potential subclonal fitness.We inferred that the median number of clones present in this myeloma series was 5, and determined the prognostic value of the number and diversity of subclones in MM patients. The prognostic impact of having high number of clones was unclear as no significant differences were found. On the contrary, there was a significant difference in terms of outcome when calculating distinct measurements of subclonal diversity. Briefly, MM patients with high values of inverse Simpson diversity index had a significantly poorer PFS (median 13.2 (95% CI, 9.4-∞) vs. 26.9 months (95% CI, 24-30.2) months, p=0.02) and OS (66% (95% CI, 52-82%) vs. 81% (95% CI, 77-85%) alive at 2-years, p=0.01); and, alternatively, MM patients who did not have a dominant subclone accounting for >25% of MM cells (low values of Berger-Parker Dominance index, n=56) had a significantly shorter PFS than those with a dominant clone accounting for more than 25% of cells with a median of 22 (95% CI, 12.3-26.3) vs. 27.5 months (95% CI, 23.9-30.9) respectively, p=0.02.Our results show that mutational load and subclonal diversity are poor prognostic factors in myeloma. Previous studies from massive-parallel sequencing and single cell analyses of myeloma plasma cells already revealed that myeloma had the features of an evolutionary ecosystem, where different tumour subclones coexist and have differential response to treatment. We have demonstrated in this study that measures of tumor diversity have important clinical consequences. To our knowledge, this is the first time that the use of clonal diversity indices as predictive biomarkers of progression is proposed in haematological malignancies, and more specifically, myeloma. DisclosuresWalker:Onyx Pharmaceuticals: Consultancy, Honoraria.