Cochliomyia hominivorax, is an ectoparasite and one of the most important myiasis-causing flies endemic to the Americas. The geographic distribution of NWS has been reduced after the implementation of the Sterile Insect Technique (SIT), being eradicated in North America and part of Central America. In South America, C. hominivorax is controlled by chemical insecticides, although indiscriminate use can cause selection of resistant individuals. Previous studies have associated the Gly137Asp and Trp251Leu mutations in the active site of carboxylesterase E3 to resistance of diethyl and dimethyl-organophosphates insecticides, respectively. Here, we have sequenced a fragment of the carboxylesterase E3 gene (ChαE7), comprising part of intron iII, exon eIII, intron iIII and part of exon eIV, and three mitochondrial gene sequences , of NWS flies from 21 locations in South America. These markers were used for population structure analyses and the ChαE7 gene was also investigated to gain insight into the selective pressures that have shaped its evolution. Analysis of molecular variance (AMOVA) and pairwise FST analysis indicated an increased genetic structure between locations in the ChαE7 compared to the concatenated mitochondrial genes. Discriminant analysis of principal components (DAPC) and spatial analysis of molecular variance (SAMOVA) indicated different degrees of genetic structure for all markers, in agreement with the AMOVA results, but with low correlation to geographic data. The NWS fly is considered a panmitic species based on mitochondrial data, while it is structured into three groups considering the ChαE7 gene. A negative association between the two mutations related to organophosphate resistance and Fay & Wu’s H significant negative values for the exons, suggest that these mutations evolved under positive selection.Livestock production is an important economic activity in Brazil, which has been suffering significant losses due to the impact of parasites. The New World screwworm (NWS) fly,  Cochliomyia hominivorax  38], werecytochrome c oxidase subunit I (COI) and subunit II (COII) and the B domain of the control region (CR), were amplified and sequenced using the same protocols and conditions for 54 flies that were not previously analyzed by [ChαE7 fragment previously characterized, including 157 bp from intron iII, the exon eIII (150 bp), the intron iIII (63 bp) and 282 bp from exon eIV, was amplified from all the individuals using the forward primer 7FIn2 (5´-ACCATCGGTGAGTTGAGAG-3´) and reverse primers RN2 (5´-AACAGTAATCCCTCGTACG-3´) or 7R3a (5´-ATCCTTATCATTATTTTCACCC -3´)  and ~15–20 ng of DNA. An initial denaturing step of 3 minutes was performed at 96°C, followed by 35 cycles of 1 minute at 95°C, 1 minute at 56°C and 2 minutes at 72°C, and a final elongation step of 10 minutes at 72°C. The PCR products were purified and sequenced as before. Sequences were trimmed, evaluated for quality and assembled in contigs using the Geneious 6.0.6 software . When heterozygotes with alleles containing insertions/deletions (indels) of different lengths within intron iII were detected, PCR amplifications were repeated and the products were purified and cloned into the pGEM-T vector (Promega Corp). At least 6 clones from each individual were sequenced, in order to obtain sequences from both alleles.Fragments of three mitochondrial genes, the lyzed by . Part ofCCC -3´) , leadingChαE7 exons (eIII and eIV) were checked for open reading frames using Mega 5.2 [ChαE7 intron iII sequences, each indel was considered as a single mutation and all indels were recoded as single positions in the final alignment. Individual sequences were collapsed into unambiguous haplotypes using FaBox [Sequences from all the 201 individuals were aligned with the algorithms of ClustalX2  and MuscMega 5.2 . For theng FaBox , and theng FaBox –40.ChαE7) independently using the DnaSP 5.0 [General diversity indices, such as number of haplotypes, haplotype diversity or expected heterozygosity (Ĥ) and nucleotide diversity (π) , were canaSP 5.0  and ArlenaSP 5.0  softwareST. This was performed using the Arlequin 3.5 software [a priori population model that identifies clusters (K) of individuals independent from geographic origin. The analysis was performed considering K = 2 to K = 10.The genetic structure of NWS fly populations was first investigated using the Analysis of Molecular Variance (AMOVA) and pairwise Fsoftware  separatesoftware  implemensoftware . This isSC, FST and FCT indices were plotted to investigate the number of groups (K) that best represent the spatial distribution of genetic variability.In order to describe genetic and geographically concordant groups of sampling locations we used spatial analysis of molecular variance (SAMOVA) . This waST's [Mantel tests  were perST's , and a gST's  through ChαE7 gene separately  using DnaSP 5.0 [Tajima’s D , Fu’s FsnaSP 5.0 . The sigRaggedness index (rg) were used to measure the adjustment to a population expansion model.Mismatch distribution analysis based on the distribution of pairwise differences between sequences was performed to better understand the demographic scenario of NWS fly samples. A unimodal distribution indicates recent expansion, while a multimodal distribution indicates population contraction, structure, influence of migration or subdivision –69. ThisChαE7 positive selection signals was done based on the population structure of the sampling locations.The investigation of The frequencies of polymorphic nucleotides in the two codons associated to insecticide resistance (Gly137Asp and Trp251Leu) and the Hardy-Weinberg equilibrium between these were obtained with the Arlequin 3.5 software .D. melanogaster (GenBank accession number: NM079537) was used as the outgroup and statistical significance was accessed by coalescence through 1000 replicates, considering a confidence interval of 95%.Fay and Wu’s H neutrality test  was perfChαE7 sequences from five individuals were obtained (GenBank accession numbers: KR067547- KR067551), although two of these were partial due to the absence of the initial sequence of intron iII. Sequences were approximately 3 Kb, comprising 111 bp of exon eII, around 2000 bp of intron iII (variable sizes due to presence of indels), 150 bp of exon eIII, 63 bp of intron iIII and 318 bp of exon eIV  is presented in M. domestica was excluded due to absence of sequence.Nucleotide composition of exons (eIII and eIV) and introns (iII and iIII) of the five NWS fly individuals and the other Muscomorpha species , while this was negative for most of the individuals in the case of the introns. However, both AT-skew and GC-skew were near to zero for intron iII (0.01–0.05), indicating that A and T nucleotides, as well as G and C, occurred in similar proportions.L. cuprina had a more similar pattern to the NWS fly, consistent with their closer phylogenetic relationship, with an A+T content of 64.67% and 56.29% for eIII and eIV exons, respectively. The AT-skew was negative and the GC-skew positive for both exons of L. cuprina. However, the values were near to zero for exon eIV (-0.04 and 0.01).D. melanogaster and M. domestica showed a more balanced distribution of nucleotide frequencies. However, D. melanogaster also had a high A+T content for intron iIII and iII sequences with values of 64.91 and 76.19%, respectively.The CR, COI and COII fragment lengths were 488, 731 and 511 bp after alignment, defining 59, 44 and 35 haplotypes, respectively. Among these haplotypes, 10 of CR, 5 of COI and 1 of COII sequences were novel. The GenBank accession numbers are KR067531—KR067540 for the CR, KR067541—KR067545 for COI and KR067546 for COII. The mean genetic distance among haplotypes (uncorrected p-distance) was 0.005 for CR (range 0–0.015), 0.003 for COI (range 0–0.010) and 0.004 for COII (range 0–0.012). Next the CR, COI and COII sequences were concatenated in a 1726 bp fragment, defining 96 haplotypes that had been found previously  and 19 tChαE7 gene were 157, 150, 63 and 282 bp length, respectively, resulting in a total sequence length of 652 bp. Based on this, we defined 49 haplotypes (GenBank accession numbers: KR067482—KR067530). Haplotypes 1 (133/402), 2 (132/402) and 3 (52/402) were the most frequent  and haplotype diversity/expected heterozygosity (Ĥ) estimated from concatenated mitochondrial and ChαE7 gene, with fixation indexes (ΦST) of 0.235 and 0.094 for the ChαE7 and mitochondrial datasets respectively  (Pairwise F137/210) , decreas137/210) . In the Examination of the mitochondrial data showed that the NWS fly populations were genetically and geographically homogeneous, according to DAPC and SAMOVA analyses. In addition, application of the Mantel test showed no correlation between geographic and genetic distances  gene according to DAPC and SAMOVA analyses, with K = 3 as the most probable geographic arrangement  and aspartate (coded by GAC or GAT) in position 137 are associated, respectively, with susceptibility and resistance to diethyl-organophosphates in dipterous species. The presence of tryptophan in position 251 (coded by TGG) defines the susceptible genotype, while serine and leucine  are associated with dimethyl-organophosphates resistance. The substitution of the amino acid tryptophan by a leucine in position 251 was associated previously with dimethyl-organophosphates resistance in  cuprina . Howeveryptophan , suggestChαE7 gene.Residue combinations in these two positions for 25 susceptible alleles and 24 resistant alleles did not reflect the real frequency of susceptibility and resistance in field populations. To explore this issue further, we specifically analyzed the two residues associated with insecticide resistance at the nucleotide level and considering allele frequencies taking into account the three groups for the Considering codon 137, the nucleotide present in the second position defines the susceptibility  or resistance . Similarly, the nucleotide present in the second position of codon 251 defines susceptibility  and the two resistant types . The frequencies of each nucleotide in these two codon positions for each group of sampling locations are showed in Group I (consisting only of BTO) showed the same frequencies of Gly137-G and Ser251-C (56%), and Asp137-A and Trp251-G (44%). Group II showed the same pattern of complementary frequencies for the Gly137-G and Ser251-C (75%) and for Asp137-A and Trp251-G (25%). In group III, the frequency of Asp137-A was 64% and that of Trp251-G was 82%, while the frequencies of Gly137-G and Ser251-C were 36% and 16%, respectively. This showed that there was a higher frequency of individuals in groups I and II with a genotype conferring resistance to dimethyl-organophosphates, while there was a higher frequency of diethyl-organophosphates resistant flies in group III. Another difference between these groups is the low frequency (less than 3%) of Leu251-T that appeared only in group III.These two polymorphic codon positions (second codon positions from codons 137 and 251) were not in Hardy-Weinberg equilibrium in the three groups of sampled locations, as the observed heterozygosity was smaller than expected .Application of Fay & Wu’s H neutrality test showed statistically significant negative values for the exons eIII and eIV in groups II and III, indicating positive selection . Both exChαE7 gene allowed its use as a nuclear molecular marker for comparisons with previously studied mitochondrial markers [Partial characterization of the carboxylesterase E3  markers ,39 and fChαE7 sequence from L. cuprina [L. cuprina has a length of only approximately 280 bp [D. melanogaster is 85 bp [M. domestica is 63 bp [L. cuprina [D. melanogaster [M. domestica [D. melanogaster exon eIV presenting three more base pairs in the region analyzed, totalizing 321bp.Based on the homologous  cuprina , we expey 280 bp , which iis 85 bp  and thatis 63 bp . This in cuprina , 58 bp inogaster  and 62 bomestica . Unlike ChαE7 nucleotide composition was similar to that of L. cuprina, consistent with their closer phylogenetic relationship. Both species showed a bias toward A+T content, which was suggested previously to be a remarkable feature of the mitochondrial genome of Calliphoridae species [The NWS fly  species ,74. In g species .The low genetic distance among haplotypes in both datasets reflects the low nucleotide diversity (π), although haplotype diversity (Ĥ) was high in both cases. This pattern of diversity is typically observed in a demographic scenario of recent populations expansion .ChαE7 gene in relation to the mitochondrial gene sequences. This was indicated by the AMOVA ΦST value which was 2.5 times greater and a higher percentage of pairwise FST significant values for the ChαE7 gene. In a similar manner, the DAPC and SAMOVA data showed the ChαE7 genetic structure of NWS fly populations, revealing three geographically non-concordant groups (K = 3). There was also an apparent North-South pattern of variability distribution, which did not appear to result from isolation-by-distance (IBD), as indicated by the non-significant Mantel tests between genetic and geographic distances. A previous investigation suggested that IBD can lead to false positives in tests of population structure and detection of loci evolving under selection [ChαE7 groups could result from selection because of regional differences of insecticide usage.All population analyses pointed to an increased genetic structure of the carboxylesterase E3 election , althougST value was low and the BCR, BSA and APL locations showed the highest percentage of pairwise FST significant values. BCR is located in Mato Grosso do Sul (MS), the central region of Brazil, while BSA is located in Rio Grande do Sul (RS), the southern region of Brazil. Both states are noted for high Brazilian cattle production, being the second and tenth in amount of slaughter in 2013, respectively [ST values, indicating an unusual pattern of both locations which could be linked to the central points for cattle interchange between places. APL is the unique population sampled in Argentina (located near southern Brazil), which is also a region of high cattle production. The results of the DAPC and SAMOVA analyses supported a panmixia scenario for the mitochondrial gene data, consistent with a previous study [For mitochondrial data, the AMOVA Φectively . Not allus study , and theCydia pomonella exhibits a low level of genetic structure in four countries  based on seven microsatellite loci, probably due to its high capacity of migration, while a higher population differentiation was detected with insecticide resistance markers [Plutella xylostella), without evidence of spatial structure for resistance through IBD [These findings are similar to results from other insect pests. The codling moth  markers . In addiough IBD .ChαE7 gene indicates a different history which, in addition to the population analysis, supports the potential role of selection.Tajima’s D, Fu’s Fs and Fu & Li’s D* and F* significant negative values and unimodal distribution of pairwise differences indicate a history of population expansion according to mitochondrial gene data. However, the finding of no significant results for these neutrality tests and multimodal distribution of pairwise differences for the ChαE7 allele frequencies could reflect the usage of different insecticide compounds in each geographic location. Alternatively, they could reflect differences in alleles frequencies of a Modifier locus, which has been related to a fitness compensation of the negative effects of both resistant mutations Gly137Asp and Trp251Leu  in L. cuprina [The results of Fay & Wu’s H  neutrali cuprina –83.Aedes aegypti in three Brazilian states  [Musca domestica in three Argentinean populations [Haematobia irritans, showed high frequencies of the kdr mutation, associated with high levels of pyrethroid resistance, in southern Chile [High levels of insecticide resistance are also observed for d Ceará) ,85 and fulations . The horrn Chile . Our resChαE7 gene that confer resistance was detected, indicating that they are not present in the same allele. This was reported previously for L. cuprina [A negative association between the mutations in the carboxylesterase E3  cuprina  and NWS  cuprina .ChαE7 gene may be undergoing evolution under positive selection. The present findings indicate that this could be due to the use of organophosphate compounds on NWS fly populations in South America. This is consistent with previous studies on the demographic history of this species [Taken together, the observed low heterozygosity in the two polymorphic codon positions associated with susceptibility and resistance, the negative association between nucleotides in the same codons and the significant negative Fay & Wu’s H values for exons suggest that the  species ,39. Many species ,91).Livestock production began in the Americas around 500 years ago, resulting in the likely passive distribution and unclear geographic structure of this species and of some concordant insect pests. For example, approximately half of tick species have a genetic structure based mainly on the movement capacity of their respective hosts. Less mobile hosts typically support a low gene flow with highly structured tick populations. In contrast, highly mobile hosts allow a higher gene flow between ticks, leading to a low population structure . As in tChαE7 gene associated with organophosphate resistance in many species, Gly137Asp and Trp251Leu, are also related to this mechanism in NWS fly through Darwinian selection. Additionally, we showed that NWS fly populations from SAG have high frequencies of organophosphate resistant genotypes, which can represent a barrier for the success of a control program based on SIT.When selecting a target region for a SIT based program, it is important to analyze the insecticide susceptibility of populations. Here, we have confirmed that the two mutations in the carboxylesterase E3 The genetic data presented here showed that at least three groups of NWS fly populations can be found in SAG, but with no clear geographic correlation, and resistance to organophosphate insecticides is widespread and found in high frequency in most of the analyzed locations. These aspects may represent barriers to implement a successful control program based on SIT. New studies based on dense SNP genotyping will be necessary in order to refine previous results of population structure and to identify additional molecular markers that could have evolved under positive selection. This will allow the detection and monitoring of other genes involved in insecticide resistance and their frequencies in natural populations.S1 FigChαE7) gene.A) Mitochondrial data; B) carboxylesterase E3 ((TIF)Click here for additional data file.S1 TableStatistically significant values are in bold.(DOCX)Click here for additional data file.S2 TableStatistically significant values are in bold.(DOCX)Click here for additional data file.S3 TableChαE7) haplotype are shown. E3 hap: carboxylesterase E3 haplotypes; Mit hap: mitochondrial haplotypes.Additionally, mitochondrial haplotypes related to each carboxylesterase E3 ((DOCX)Click here for additional data file.


Nassauvia subgen. Strongyloma is a characteristic element of the steppe, exhibiting a continuum of morphological variation. This taxon provides a relevant phylogeographical model not only to understand how past environmental changes shaped the genetic structure of its populations, but also to explore phylogeographical scenarios at the large geographical scale of the Patagonian steppe. Here, we (1) assess demographic processes and historical events that shaped current geographic patterns of haplotypic diversity; (2) analyze hypotheses of isolation in refugia, fragmentation of populations, and/or colonization of available areas during Pleistocene glaciations; and (3) model extant and palaeoclimatic distributions to support inferred phylogeographical patterns. Chloroplast intergenic spacers, rpl32–trnL and trnQ–5′rps16, were sequenced for 372 individuals from 63 populations. Nested clade analysis, analyses of molecular variance, and neutrality tests were performed to assess genetic structure and range expansion. The present potential distribution was modelled and projected onto a last glacial maximum (LGM) model. Of 41 haplotypes observed, ten were shared among populations associated with different morphological variants. Populations with highest haplotype diversity and private haplotypes were found in central-western and south-eastern Patagonia, consistent with long-term persistence in refugia during Pleistocene. Palaeomodelling suggested a shift toward the palaeoseashore during LGM; new available areas over the exposed Atlantic submarine platform were colonized during glaciations with postglacial retraction of populations. A scenario of fragmentation and posterior range expansion may explain the observed patterns in the center of the steppe, which is supported by palaeomodelling. Northern Patagonian populations were isolated from southern populations by the Chubut and the Deseado river basins during glaciations. Pleistocene glaciations indirectly impacted the distribution, demography, and diversification of subgen. Strongyloma through decreased winter temperatures and water availability in different areas of its range.The Patagonian steppe is an immense, cold, arid region, yet phylogeographically understudied.  Nassauvia Comm. ex Juss. subgen. Strongyloma (DC) Cabrera (Asteraceae) is one of these rare cases; it is a typical element throughout the immense territory of the Patagonian steppe.Phylogeographical reconstructions along large, extensive geographic areas are difficult to assess because they ideally need widely distributed, ubiquitous taxa, present at regional scale as a characteristic element of different local communities. Strongyloma by α taxonomy: Nassauvia axillaris (Lag. ex Lindl.) D. Don, Nassauvia fuegiana (Speg.) Cabrera, Nassauvia glomerulosa (Lag. ex Lindl.) D. Don, Nassauvia maeviae Cabrera, and Nassauvia ulicina (Hook. f.) Macloskie. The subgenus as a whole is easy to recognize in the field: it comprises small shrubs with a tendency to heteroblasty, with 1–5 white flowers per capitulum, and hairy cypselas  by considering Nassauvia subgen. Strongyloma as a monospecific taxon. The morphological combinations traditionally accepted as species , in Argentina (from Jujuy Province c. 22°S to northern Tierra del Fuego Province c. 54°S), and in Chile , including a variety of climatic zones but mainly adapted to extremely xeric environments  to northern Tierra del Fuego Province (c. 54°S) in Argentina, covering a total area of approximately 787,000 km2 (Correa c. 5–6 Ma), with ice reaching its maximum development in the Early Pleistocene  and the Last Glacial Maximum . The GPG resulted in the greatest expansion of ice sheets in the Patagonian steppe compared with LGM and other Pleistocene glaciations , covering most of the distribution of subgen. ts Table , Fig. 1.ts Table  and deporpl32–trnL  protocol , sequenced with BigDye 3 , and purified with Sephadex  before electrophoresis on an AB 3730xl automated sequencer housed in Brigham Young University's DNA Sequencing Centre. Electropherograms were edited and assembled using Sequencher 4.6 . Sequences were aligned with ClustalX 1.81 .DNA was amplified and sequenced with a profile consisting of 94°C for 3 min followed by 30 cycles of 94°C for 1 min, 50 or 52°C for 1 min for 0.9 Hall . Indels 0.9 Hall  as imple4 Müller . The twoeCA 1.2  was performed following Templeton et al.  and Temp Panchal . Althoug Panchal , it has  Panchal , 2009 anA haplotype network was constructed using TCS 1.21  between Agrio, Neuquén, and Negro rivers, and Chubut River, c. 38°–43°S; (3) between Chubut and Deseado rivers, c. 43°–47°S; (4) between Deseado and Chico rivers, c. 47°–50°S; and (5) south of Chico River, c. 50°S  were performed that consider genetic distances between haplotypes and their frequencies using A0°S Fig. , followiD . FS and R2 are the most powerful tests used to detect population growth  unit. Only 36 point localities of the total were geo-referenced using Google Earth (http://www.google.com/earth/index.html). Environmental data with a resolution of 2.5 arc minutes (5 km2) for current and past conditions were downloaded from the WorldClim database 1.4  and interpreting the resulting maps of each model in Diva-Gis.Current conditions are interpolations of observed data from climate stations around the world, representing a 50-year period from 1950 to 2000. Past conditions for the LGM are calibrated and statistically downscaled reconstructions based on the WorldClim data for current conditions. We chose the prediction of one of two global climate models tested for the LGM, the community climate system model (CCSM) and the Model for Interdisciplinary Research on Climate (MIROC), by evaluating the Meo Midpoint (http://www.geomidpoint.com/random/) and extracted environmental data in Diva-Gis. To avoid overestimation of climatic data that can lead to misleading results  and performed Principal Component Analyses in InfoStat 2.0 , isothermality (Bio 3), temperature annual range (Bio 7), mean temperature of the wettest quarter (Bio 8), mean temperature of the driest quarter (Bio 9), mean temperature of the coldest quarter (Bio 11), annual precipitation (Bio 12), precipitation seasonality (Bio 15), precipitation of the warmest quarter (Bio 18), and precipitation of the coldest quarter (Bio 19).We generated 2000 random points from across Argentina, Bolivia, and Chile using GStrongyloma, and then projected over a wider region from latitude 17°41′S to 55°49′S and from longitude 57°31′W to 75°43′O for the present and the LGM in Diva-Gis.The bioclimatic layers were trimmed to the surrounding areas of the known geographic distribution of subgen. axEnt was run using the following settings for current and past models: 10 replicates with auto features, response curves, jackknife tests, logistic output format, random seed, random test percentage = 25% , replicate run type = cross-validate, regularization multiplier = 1, maximum iterations = 500, convergence threshold = 0.00001, and maximum number of background points = 10,000. To determine the threshold value for each prediction, we used the value of the minimum training presence logistic threshold. Variable importance was determined comparing percent contribution values and jackknife plots.MStrongyloma ranged from 913 to 928 base pairs (bp) for rpl32–trnL (with two indels of seven and eight bp) and 916 to 925 bp for trnQ–5′rps16 (with one indel of nine bp). The three indels were coded as present or absent and appended to the end of the matrix. The combined matrix included 1856 characters, of which 37 were variable.DNA sequences of subgen. Statistical parsimony retrieved a well-resolved network in which three closed loops were resolved unambiguously Fig. . A totalAmong the total observed haplotypes Table , eight w–Andean region) were distributed through the Andes north of 38°S, clearly separated from the rest. Haplotypes in clade 2-3 (NP1: northern Patagonia 1 region) included the second most frequent, widespread haplotype (H1) and were distributed between 39° and 47°S, while haplotypes in clade 2-4 (NP2: northern Patagonia 2 region) included the fourth most frequent, widespread haplotype (H12) and were distributed between 40°25′ and 44°S. Haplotypes in clades 2-6  and 2-5  were scarce and distributed between 40° and 43°15′S, and 43° and 46°37′S, respectively. Haplotypes in clade 2-7 (SP1: southern Patagonia 1 region) included the most frequent, widespread haplotype (H25) and were distributed mainly south of 43°21′S, with one exception further north at 39°44′S. Finally, haplotypes in clade 2-2 (SP2: southern Patagonia 2 region) included the third most frequent, widespread haplotype (H18) and were distributed south of 45°28′S.The nested network resulted in 15 one-step clades, seven 2-step clades, two 3-step clades, and the total cladogram , all seven-two-step clades, the two-three-step clades, and the total cladogram showed significant levels of geographic association, while the geographic association hypothesis was rejected for the remaining nine-one-step clades . Processes of restricted gene flow and/or long-distance dispersal in intermediate areas not occupied by the species, or gene flow followed by extinction of intermediate populations were inferred for clade 1-5 (most haplotypes of NP1). Clades 1-7 (most haplotypes of SP2) and 3-1  have experienced long-distance colonization and/or fragmentation. Processes of restricted gene flow and/or long-distance dispersal were inferred for clade 1-9 (part of SP1). Isolation or long-distance dispersal was inferred for clade 1-10 (most haplotypes of SP1). Clades 2-2 (SP2) and 2-5 (CP2) have experienced restricted gene flow with isolation by distance. Finally, range expansion was inferred for clades 2-4 (NP2) and 3-2  ranged from 0 to 0.821 with an average of 0.532, nucleotide diversity (π) ranged from 0 to 0.00218 with an average of 0.00128, and mean number of pairwise differences among haplotypes within populations (p) ranged from 0 and 3.857 with an average of 1.175 , and in one population (24) located in south-eastern Santa Cruz. The highest values of p similarly occur in populations of central-western Patagonia, coinciding with most populations that showed higher haplotype and nucleotide diversity . Most populations with high genetic diversity also had exclusive haplotypes , followed by the CP2 region, which also showed the highest values of nucleotide diversity and pairwise differences , while the NP1 region showed the lowest values of h, π, and p .The same diversity indices were calculated for each geographic region Table , Fig. 3.P < 0.001), with the exception of regions located inside and outside the boundary of the GPG . By dividing the regions according to major rivers .Analyses of molecular variance  of suitable conditions was found in the Islas Malvinas/Falkland Islands, where these plants have never been observed.The predicted model for the present Fig. A was a fThe model of the geographic distribution for the LGM showed some important differences with the known current geographic distribution of the subgenus Fig. B. Low , indicating that this environmental variable contributed most to the construction of the model. In the jackknife test for the training data, the mean temperature of the coldest quarter (Bio11) presented the most useful information when used in isolation to build the model, causing the model to reach the highest gain and allowing a reasonable adjustment to the training data. The mean temperature of the wettest quarter (Bio8) contained a substantial amount of useful information that was not contained in the model when only the remaining variables were used, since excluding Bio8 in the model decreased training gain. Jackknife tests for the gain and for the AUC of the test data yielded similar results to the jackknife test for the gain of the training data, suggesting that mean winter temperature  helped MNassauvia subgen. Strongyloma were shaped mainly by three main different historical events: (1) isolation in refugia and restricted gene flow during Pleistocene glacial periods, followed by postglacial range expansions; (2) colonization of new available areas during Pleistocene glaciations and postglacial retraction of populations; and (3) fragmentation of populations in areas not reached by the ice sheet during Pleistocene glacial periods followed by postglacial range expansions.The phylogeographical and palaeomodelling analyses suggest that the genetic diversity and structure found in Nassauvia subgen. Strongyloma may have withstood the extreme cold and aridity during the Pleistocene glaciations in two kinds of refugia (Holderegger and Thiel-Egenter c. 39°30′ S 70°30′ W), north-western Chubut (c. 43°S 71°W), and north-western Santa Cruz (c. 47°S 71°W); and (2) lowland glacial refugia—at least four— located near the Chubut River in central-western Chubut (c. 44°S 69°30′ W), near the Deseado River in central-northern Santa Cruz (c. 46°30′ S 69°30′ W), and near the Chico River in central-western (c. 48°S 71°W), and south-eastern (c. 50°S 69°W) Santa Cruz.Our data suggest that populations of Strongyloma are consistent with long-term habitation such as would be expected from Pleistocene refugia , allowing for plant colonization  and the Deseado (c. 47°S) rivers. In fact, the geographic distribution of this subgenus, as in the steppe shrub M. spinosum  both fragmentation between northern and southern haploclades, and isolation with restricted gene flow of one southern haploclade during Pleistocene glaciations, produced the split of the original distribution into two groups of population, one north of the Chubut river basin, and one south of the Deseado river basin; and (2) successive range expansions after the Pleistocene glaciations, mainly toward warmer and wetter conditions in the center of the Patagonian steppe. The most likely posterior demographic expansions would be from both northern and southern populations toward central-eastern Chubut Province. The expansion of northern, central, and southern haploclades could have generated recent contact areas throughout the entire Patagonian steppe, but mainly between the Chubut and the Deseado rivers.Based on past river basin dynamics and climate conditions cited above, a significant difference could be expected between northern and southern haplotypes within subgen. c. 38°S was clearly separated from the rest, in accordance with a latitudinal break reported for a lizard species  coincides with the break at Limay River basin reported for another lizard species (Liolaemus bibronii, Morando et al. Within a general latitudinal geographic structure of haploclades, other phylogeographical breaks can be distinguished related to river basins. On one hand, the high-Andean haploclade located just north of the Agrio and Neuquén rivers at Finally, according to haplotype analyses, the four most widespread haplotypes and several other interior and tip haplotypes were shared among populations of different morphological variants, suggesting that there is no strict relation between morphology and genetic diversity. Furthermore, throughout the entire Patagonian steppe, some populations have haplotypes from different clades. This indication of distantly related haplotypes co-occurring suggests the existence of numerous contact zones Avise  throughoNassauvia subgen. Strongyloma appear to reflect climatic fluctuations and landscape changes that occurred during the Pleistocene having an impact on the distribution, demography, and diversification of different lineages within the group. Past events differentially affected areas within the immense geographic range of these shrubs. During glaciations, subgen. Strongyloma persisted in peripheral refugia along the eastern foothills of the Andes, in lowland refugia in central-western and south-eastern Patagonia, over the exposed Atlantic submarine platform toward the east, and in fragmented populations north and south the Chubut and the Deseado river basins. Related to those past scenarios, Pleistocene glaciations indirectly affected the demography of this group of plants through decreased winter temperatures and water availability in different areas of its immense geographic range. Future comparative phylogeographical studies should consider patterns found in subgen. Strongyloma to better comprehend the processes that have affected the Patagonian biota.Phylogeographical and palaeomodelling patterns found in


Epidendrum cinnabarinum and E. secundum adapted to Neotropical inselbergs of northeastern Brazil. Our goals were to identify major genetic divergences or disjunctions across the range of the species and to investigate potential demographic and evolutionary mechanisms leading to lineage divergence in Neotropical mountain ecosystems.Because of their fragmented nature, inselberg species are interesting biological models for studying the genetic consequences of disjoint populations. Inselbergs are commonly compared with oceanic islands, as most of them display a marked ecological isolation from the surrounding area. The isolation of these rock outcrops is reflected in the high number of recorded endemic species and the strong floristic differences between individual inselbergs and adjacent habitats. We examined the genetic connectivity of orchids E. cinnabarinum (FST = 0.644) and E. secundum (FST = 0.636). Haplotypes were not geographically structured in either taxon, suggesting that restricted gene flow and genetic drift may be significant factors influencing the diversification of these inselberg populations. Moreover, strong differentiation was found between populations over short spatial scales, indicating substantial periods of isolation among populations. For E. secundum, nuclear markers indicated higher gene flow by pollen than by seeds.Based on plastid markers, high genetic differentiation was found for The comparative approach adopted in this study contributed to the elucidation of patterns in both species. Our results confirm the ancient and highly isolated nature of inselberg populations. Both species showed similar patterns of genetic diversity and structure, highlighting the importance of seed-restricted gene flow and genetic drift as drivers of plant diversification in terrestrial islands such as inselbergs. Inselbergs are isolated rock outcrops typically harboring rupicolous vegetation and embedded within a landscape composed of contrasting plant communities. Because of their disconnected geographic nature, inselbergs are frequently compared with oceanic islands, since most of them display a marked ecological isolation from the surrounding area . The isoPhylogeographic studies of inselberg-adapted species have provided insights into evolutionary processes leading to diversification of lineages and species -11. SuchPorembsky et al.  have notPhylogeographic studies focused on organisms associated with mesic forest communities have indicated a strong association between the occurrence of glacial cycles and the fragmentation of forest-dwelling species, supporting the classical model of tropical refugia -22. On tThe Caatinga is the largest, most diverse dry seasonal tropical forest biome in the Neotropical region . DespiteEpidendrum cinnabarinum and E. secundum (Orchidaceae), two widespread, co-occurring species commonly found on inselbergs in northeastern Brazil. Epidendrum cinnabarinum, a polyploid species with 2n = 240 chromosomes [Epidendrum secundum exhibits a diploid chromosome count of 2n = 56 in most populations [E. cinnabarinum and E. secundum in light of results based on climate modeling, paleovegetation reconstructions and the island-like nature of populations distributed on inselbergs.In the present study, we used nuclear and plastid microsatellite markers to analyze phylogeographic structure and genetic diversity of omosomes , occurs ulations ; it has ulations . This spE. cinnabarinum and 12 haplotypes for E. secundum  and haplotype diversity  , and no apparent geographic structure was observed Figure C. The moFST = 0.644 and GST = 0.672. According to an analysis of molecular variance (AMOVA) (Table P = 0.704). Pairwise FST comparisons among populations . Haplotype differentiation between populations separated over short spatial scales was observed between populations QE and TO (12 km apart) and between BZ and FE (18 km apart) .High genetic differentiation was observed across populations, with A) Table , a high Analysis of plastid markers yielded a network lacking clear geographic structuring Figure D. HaplotE. secundum populations was also very similar to the patterns observed for E. cinnabarinum. AMOVA results indicated that a high proportion of uncovered genetic variability was found among populations , with only 36.36% attributed to within-population variability . Isolation by distance among populations was also not detected (P = 0.822).The only hypothesis of population differentiation significantly supported by AMOVA was that between populations from Chapada Diamantina and the remaining localities Table . The parST (0.114), GST (0.122) and G’ST (0.255). An AMOVA also generated different results than for plastid markers, with most of the genetic variation partitioned within  rather than among  populations (Table No sign of phylogeographic structure was detected among populations (P = 0.329). A Mantel test did not support isolation by distance among populations based on ΦST, GST or G′ST .Compared with plastid markers, nuclear markers exhibited lower levels of genetic differentiation as estimated by ΦST 0.11, GST  for population BZ showed strong assignment to cluster 2  obtained from simulations performed with different values of parameter θ, proportion of single-step mutations (pg) and average size of non-one-step mutations (Δg) was 0.589  and plastid (GST = 0.782) markers, the ratio of pollen flow to seed flow was estimated as 25.85. The value of this ratio suggests that gene flow via pollen in E. secundum is > 20 times higher than that occurring via seeds.Using values of genetic differentiation among populations BZ, DE, TO, FE and MC obtained for nuclear (E. cinnabarinum and E. secundum. As indicated by high area-under-the-curve (AUC) values (0.969 for E. secundum and 0.968 for E. cinnabarinum), the EEM analysis performed well. Climate variables contributed to both species models, with the E. cinnabarinum model additionally adjusted using a geological eras variable. Past distribution ranges differed between the two species, mainly at their peripheral limits. From the LIG to the present, E. cinnabarinum exhibited a pronounced decrease in its presence at Chapada Diamantina and other inland mountain ranges, with a more recent expansion towards northern Planalto da Borborema and coastal regions after the LGM  and in western portions of Planalto da Borborema. After the LGM, the species remained broadly distributed only within Chapada Diamantina and mountains to the east within the BAF biome. The model representing current climatic conditions indicated that E. secundum has a fragmented distribution within Planalto da Borborema, where most populations are found at the border between Caatinga and BAF biomes. Since the LIG, the distribution of this species has not been continuous between Chapada Diamantina and Planalto da Borborema.Distributions under LIB, LGM and current climatic conditions were successfully estimated for M Figure B and 3C.E. secundum, highlighting the important role of pollen dispersal, and consequently pollination services, in species cohesion. This result has also been observed in other plants [E. secundum suggest long-term stability of populations from Chapada Diamantina and the southern portion of Planalto da Borborema . The significant pairwise genetic differentiation among most populations, the absence of a pattern of isolation by distance, and the lack of significantly phylogeographically structured haplotypes suggest that genetic drift, as observed in other rock outcrop species [E. cinnabarinum and E. secundum. In finite populations, such as those inhabiting inselbergs and oceanic islands, genetic drift may be the predominant force governing allele frequencies of neutral loci [The most outstanding pattern recovered by the use of plastid markers was the deep among-population differentiation within  species , has beeral loci ,44. Becaral loci . In addiral loci ,46 couplE. secundum populations was higher for plastid than for nuclear markers  among most populations, indicating ancient dispersal events. Another explanation for the non-significance of phylogeographic structure is the fact that multiple, phylogenetically unrelated haplotypes are found in the same populations, especially in the Chapada Diamantina ecoregion. Chapada Diamantina is an old and very stable region; demographic changes in peripheral populations, such as those inhabiting the BAF and from mountains south of Chapada Diamantina, may have contributed to the exchange of unrelated haplotypes—a pattern observed in other studies [E. secundum, with a former range that includes mountains to the south and north of Chapada Diamantina.Despite the strong genetic differentiation observed between populations from Chapada Diamantina and the remaining localities Table , phyloge studies ,34,49. IE. cinnabarinum, a general pattern of expansion from the inland towards the seashore since the LIG is suggested  [The relictual nature of inselberg populations is also evident in the structure of the haplotype networks Figure C and 1D.istories , as demoistories ,29,31,32istories ,27,33,34etation) ,55.E. cinnabarinum and E. secundum reflects the isolated nature of their inselberg habitats. Similar to the results of studies of other inselberg species [E. secundum distribution has occurred in mountain ranges south of Chapada Diamantina. Future studies should consequently include a broad sampling of this species over adjacent mountain ranges to explore how such distribution oscillations have affected current levels of genetic diversity in these populations. In northeastern Brazil, inselbergs have different mineral origins and are distributed across different biomes and climatic clines. Thus, selection for divergent habitats may also be of great importance during lineage diversification within species under restricted gene flow. Given the contrasting patterns of genetic structure observed between nuclear and plastid markers, cytonuclear incompatibilities may contribute to the first stages of reproductive isolation among divergent lineages. Future efforts should take advantage of next-generation sequencing approaches [The population genetic structure recovered for both  species ,11,14,15 species , inselbeproaches  coupled proaches ,58 to clEpidendrum L. is the largest genus of Orchidaceae in the Neotropics, with about 1,500 species distributed from the southeastern United States to northern Argentina [Epidendrum cinnabarinum and E. secundum are perennial, terrestrial species commonly found on inselbergs whose locations include sea-level to mountain elevations. Both species belong to subgenus Amphyglottium[rgentina . The genrgentina . Epidendyglottium,61, a gryglottium). Althouyglottium).E. cinnabarinum and E. secundum were obtained from 11 and 9 locations, respectively . All polymerase chain reaction (PCR) amplifications were performed in an Applied Biosystems 2700 thermocycler  following the protocol described by Pinheiro et al. [Six plastid microsatellite loci were used to genotype samples from both species . For E.o et al. . Microsa[. Estimates of haplotype richness were corrected for differences in sample size using the rarefaction method.All sampled populations were characterized for levels of diversity based on plastid DNA markers. The number of haplotypes in each population, haplotype diversity and haplotype richness were estimated using RAREFAC v. 3.5 software . EstimaE. secundum populations was characterized according to number of alleles, number of private alleles, allelic richness, private allelic richness, expected heterozygosity and inbreeding coefficient [Nuclear microsatellite diversity of five fficient , which wfficient  and HP-Rfficient . Departufficient . The micfficient .http://www.fluxus-engineering.com). To assess whether observed genetic differentiation was due to drift, we tested the hypothesis that GST = RST[RST[RST significantly larger than GST), 10,000 permutations of RST values were performed. Partitioning of genetic diversity within and among populations and between populations from BAF  and Caatinga (inland) regions was assessed by AMOVA. For E. secundum, two additional models were considered, one corresponding to differentiation between populations from Chapada Diamantina and Planalto da Borborema, and the other considering populations from Chapada Diamantina separated from remaining localities. Genetic differentiation between populations was also measured by pairwise comparisons of FST using the program ARLEQUIN v. 3.5. Finally, the hypothesis that populations were differentiated because of isolation by distance was tested by assessing the correlation between pairwise geographic distances and pairwise FST values using a Mantel test in the program GENEPOP v. 4.0. Correlation significance was estimated after performing 10,000 permutations between pairwise geographic distance and pairwise genetic differentiation matrices.The geographical structure of genetic variation in plastid DNA was investigated for both species through several approaches. A median-joining network  based onGST = RST  and to estimate admixture proportions (Q) for each individual. A set of models was chosen in which individuals had admixed ancestries and correlated allele frequencies. Ten replicate runs were completed for each K value for K = 1–10. Markov chain Monte Carlo runs consisted of 600,000 generations after an initial burn-in of 250,000 generations. The most probable number of genetic clusters (K) present in the data were defined following Evanno et al. [Bayesian assignment analysis (in STRUCTURE v. 2.3.3 ) was useo et al.  using tho et al. .E. secundum with nuclear microsatellite-based M-ratios [M-ratios and critical values  obtained by simulating the distribution of M-ratios under specific demographic and mutational conditions using the software CRITICAL_M.EXE (http://swfsc.noaa.gov/textblock.aspx?Division=FED&id=3298). The critical value MC is set at the lower 5% tail of this distribution, and bottlenecks are detected when the M-ratio value is below the calculated MC threshold. Different values of MC were simulated by modifying θ , pg (0.1 and 0.3) and Δg (2.0 and 3.5). Low MC values are more conservative as a bottleneck must be of greater intensity to drop below this level [MC value was used to check bottleneck significance.Recent population size reductions (i.e. genetic bottlenecks) were tested in M-ratios  using ARis level ; for thiE. secundum populations, the parameter GST for nuclear and plastid loci was compared based on populations genotyped for both plastid and nuclear markers . Pollen/seed flow ratio was estimated following Ennos [To examine the relative contribution of pollen vs. seed flow to total gene flow among ng Ennos  and Peting Ennos  using EqTo test the hypothesis that inselbergs were refugia through late Quaternary climate cycles, we projected EEMs from the current situation onto late Quaternary scenarios. We used the maximum entropy algorithm of Maxent v. 3.3.3e ,86 to obE. cinnabarinum and 242 E. secundum occurrences from our field GPS records and georeferenced herbarium data extracted from the speciesLink project (http://splink.cria.org.br). All points were verified with Google Earth to ensure localities were not placed in heavily urbanized areas. The localities used for EEM analysis are available from the authors upon request to avoid illegal plant collecting.We obtained 226 EEMs: Environmental envelope models; LGM: Last Glacial Maximum; BAF: Brazilian Atlantic Forest; LIG: Last Interglacial; AUC: Area under the curve.The authors declare that they have no competing interests.FP participated in the study design, carried out the molecular genetic studies and drafted the manuscript. SC and CPS participated in the study design, and FB coordinated the study. LPF participated in the field work and sampling strategy design. MFF contributed to chemical supply procurement and analysis. DDM conducted the EEM analysis. All authors help to draft the manuscript, and read and approved the final version.Epidendrum cinnabarinum and 12 haplotypes of E. secundum, characterized at six cpSSR loci. For E. cinnabarinum, the presence (1) and absence (0) of a 16 bp fragment from intergenic region rps16–trnK is also reported. The frequency of occurrence (n) in total collection screened is indicated for both species.Description of 10 chloroplast microsatellite haplotypes of Click here for fileFST between populations of Epidendrum cinnabarinum based on plastid markers.Pairwise comparisons of Click here for fileFST between populations of Epidendrum secundum based on plastid markers.Pairwise comparisons of Click here for fileK from STRUCTURE analysis as a function of K  calculated according to the simulations described by Evanno et al. [K or the uppermost level of structure—in the present case, two genetic clusters.Magnitude of Δo et al. . The modClick here for file


Analyses revealed one group of six populations to the west, above the Yarlung Tsangpo Grand Canyon (YTGC), and a second group to the east below the YTGC. Estimates of the timing of this east-west split indicate that these groups represent evolutionarily significant units that have evolved separately and rapidly in the middle Pleistocene, at the time of the Kunlun-Huanghe Movement A Phase and the Naynayxungla glaciation. Population dynamic analyses indicate that S. o’connori experienced a pronounced late Pleistocene expansion during the last interglacial period. The results of this study support the hypotheses that the QTP uplift and Quaternary climatic oscillations have played important roles in shaping the population genetics and dynamics of this endemic fish.The Qinghai-Tibet Plateau (QTP) is a biodiversity hotspot, resulting from its geological history, contemporary environment and isolation. Uplift of the QTP and Quaternary climatic oscillations are hypothesised to have influenced the genetic diversity, population structure and dynamics of all QTP endemic species. In this study, we tested this hypothesis by assaying variation at two mitochondrial DNA regions (cytochrome  Genetic diversity, the fundamental source of biodiversity, is a product of long-term evolution, with its distribution and extent in natural populations being mainly affected by gene flow, mutation, genetic drift and selection. The rapid development of molecular biology and the advent of new analytical approaches have dramatically improved our understanding of the evolutionary histories of many species, such that the contributions of landscape and ecological variation can now be linked to the extent and distribution of genetic diversity1Building on earlier work45457The Qinghai-Tibet Plateau (QTP) is the largest high-elevation ecosystem in the world. This region, along with southeast China and the Himalayan region, has been designated as one of the world’s 34 most important centres of biodiversity because of its high species richness and abundance of endemic species3112 and a length of 2,091 kmThe Yarlung Tsangpo River (YLTR) is the largest river system on the QTP, with a catchment area of 238,000 kmSchizothorax (Teleostei: Cyprinidae), also collectively known as snow trout, number more than 60 valid species from central and eastern Asia. In China, more than 30 Schizothorax species are widely distributed in different rivers and lakes on the QTP and its adjacent area14Schizothorax o’connori Lloyd 1908 is an endemic tetraploid fish that is distributed in the main streams and tributaries along the upper and middle reaches of the YLTR in TibetSchizothorax o’connori and Schizopygopsis younghusbandi, are thought to be distributed in both the western and eastern drainages of the YLTR13S. o’connori is an excellent model organism to examine the impact of uplift of the QTP and Quaternary climatic oscillations on the population structure and dynamics of species endemic to the QTP.Fishes of the genus Genetic diversity and population genetic structure across a species’ range reflect historical events such as range contraction and expansion (recolonisation) from glacial refugia, as well as current habitat fragmentation and dispersal131545S. o’connori from seven populations were investigated using mitochondrial cytochrome b (Cyt b) and control region (CR) sequences and twelve polymorphic nuclear microsatellite loci. The primary aim of our study was to test the hypothesis that the population structure and dynamics of S. o’connori are influenced by (coincident in timing with) the uplift of the QTP and Quaternary climatic oscillations. Secondarily, our aim was to compare the genetic uniqueness of S. o’connori in the western region with that in the eastern region to better understand the interplay between geological upheaval, geographic isolation and genetic diversity. Our results shed new light on the evolutionary history of the endemic QTP fish fauna and will also aid in the conservation of endemic fishes of this region.In this study, levels of mitochondrial and nuclear DNA genetic variability and population genetic structure of b and partial CR (control region) sequences were successfully amplified from 168 samples of S. o’connori. Cyt b (1141 bp) had 84 variable sites, 59 of which were parsimony informative: all variable sites were transitions, except one transversion. CR sequences were ~714 bp long and contained 67 variable sites, 47 of which were parsimony informative, with one indel: all variable sites were transitions, except two transversions. Because the Cyt b and CR regions have not reached saturation they are suitable for analyses employed here. The partition homogeneity test indicated no incongruence between the Cyt b and CR sequences; we therefore focussed on a concatenated mtDNA sequence of 1855 bp (5′-Cyt b-CR-3′) for analysis. The Cyt b and CR haplotypes were defined and their sequences submitted to GenBank (Accession numbers KT188614 – KT188672 for Cyt b and KT188673 – KT188752 for CR).Full Cyt b + CR sequences. The neighbour-joining (NJ) and maximum likelihood (ML) phylogenetic methods resulted in trees with similar topologies  plus the four CR haplotypes (H77–H80) from the eastern population (Bomi) formed another group. Interestingly, haplotypes from fish of the western region grouped firstly with outgroups  and then grouped with the haplotypes from the eastern region  distances and the pairwise  regions . Analysi regions .R = 0.222, P = 0.255), for the six western populations . IBD tests were no significant for all seven populations or the six western populations based on Cyt b only and CR only (results not shown here).Tests of isolation by distance (IBD) based on concatenated mtDNA sequence were not statistically significant for all seven populations  and raggedness index values , Nei’s gene diversity (H) and Shannon’s information index (I). The eastern population of Bomi had low observed values compared to the western populations.All 12 microsatellite loci displayed up to 4 bands per individual, as expected for a tetraploid genome, and were highly polymorphic . PopulatFST values were significant after Bonferroni correction for multiple testing, except that between Shigatse and Paizhen. FST values and Nei’s unbiased genetic distance values between pairs of western populations were generally lower than those between Bomi and any population in the western region  or for the six western populations .Tests of isolation by distance (IBD) were not statistically significant for all seven populations  which was similar to the previous results ~0.125 Ma (0.093–0.145 Ma)b data, for our work, and the use of only Cyt b in the earlier workS. o’connori populations on the QTP. The subsequent identification of the pronounced east-west divergence, even if based on only one eastern population, is novel and requires interpretation and explanation.The demographic history and divergence time of S. o’connori populations between the western (the YLTR) and eastern (PLTR) regions was estimated to be 1.19 Ma. This divergence date, which as noted above is based on only one eastern population, is associated with the Kunlun-Huanghe Movement A Phase (about 1.1 Ma) in the mid-Pleistocene1213S. o’connori. The YTGC formed a barrier between the western and eastern regions and prevented the dispersal of fish and resulted in isolation between populations on the two sides. The YTGC clearly forms a barrier to fish trying to migrate up the river (in a westerly direction), and whilst, in principle, fish may move down the river (in an easterly direction) although there is little or no evidence to indicate that this happens (see subsequent discussion of possible hybridisation). The Kunlun-Huanghe Movement has shaped the geomorphology of the area, making the topography of the YLTR very complex, with periodic uplift and repeated glacial-interglacial changes having a significant impact on the environment and climate of the QTPS. o’connori was shaped by the uplift events of the QTP and the associated climate change. The expansion and evolution of S. o’connori are therefore associated with the environmental changes and the violent upheaval of the QTPS. o’connori, in particular in the west.The divergence time of the S. o’connori in the east extends from the YTGC to Bomi (elevation ~2685 m) and covers the PLTR basin, which is the biggest tributary of the YLTR. However, some studies indicated that the PLTR was not the eastern tributary of the YLTR prior to 4 Ma, but the main stream of the YLTR in the eastSchizothoraxBuddleja crispaS. o’connori was formerly distributed in the PLTR (east) and in the YLTR (west). With river capture, reversal events and uplift of the QTP1524S. o’connori populations in the east and west were gradually isolated from each other.The distribution of S. o’connori (and other QTP river fishes) only lived in restricted areas in lower valleys of the western region, but once the glaciers retreated , S. o’connori expanded from restricted areas in the lower valleys of the YLTR to newly available habitat in the upper reaches of the river. The lower valleys and rivers in the western region of the YLTR are therefore likely to have been refugia for S. o’connori during the glacial period. The estimates of the expansion period in this study indicate that S. o’connori was present in the western region (e.g. Shigatse and Lhasa River) of the YLTR by the middle to late Pleistocene (~0.1 Ma). Thus, the demographic history of S. o’connori has been profoundly influenced by the Quaternary climatic oscillations. Consistent with the sequence of geological events in the region, the population expansion time estimate for S. o’connori is similar to that for other schizothoracine fishes living on the QTP (0.11 Ma for Schizopygopsis pylzoviGymnocypris fishesGymnocypris chilianensisSchizopygopsis younghusbandiThe estimate of the timing of population expansion (~0.1 Ma) reported in this study is consistent with the timing of the last interglacial that occurred 0.070–0.130 MaS. o’connori reveal pronounced population genetic structure at different hierarchical levels and a remarkable association with the two major geographic groups in the west and east. Whilst the mtDNA sequence and nDNA microsatellite results suggest that S. o’connori exhibits high levels of genetic variation in the west, in the east the Bomi population is characterised by low levels of genetic variation but a large number of private alleles. In the west, the Paizhen, Shigatse and part of the Zhaxue populations form one subgroup, whereas the Quxu, Shannan, Mainling and part of the Zhaxue populations form a second subgroup. There is no geographic underpinning for these two subgroups, with Paizhen being at the eastern end of the YLTR, Shigatse being at the western end of the YLTR, and with Zhaxue being on the Lhasa River, which is a tributary of the YLTR. The status of the Zhaxue population suggests that it is a mixture of the two subgroups within the YLTR. The Paizhen and Shigatse populations, which are geographically distant from each other, show a surprising degree of genetic similarity based on microsatellite variation. There is no obvious explanation for this, although we can rule out natural gene flow because this would be expected to affect populations in between these sites along the river. The most likely explanation for this anomalous result is human-mediated movement of fish from Shigatse to Paizhen, as part of a government-funded breeding programme set-up approximately 10 years ago (pers. obs. of the authors). Whilst it is impossible to confirm if human-mediated movement of fishes has occurred and that it has resulted in genetic mixing of different regional genotypes, we note that a similar pattern of population genetic similarity that is not related to geography  has been reported for another fish (Glyptosternum maculatum) from the same river system28Analyses of mtDNA sequence and microsatellite variation in S. o’connori, spanning ~700 km from Shigatse (elevation ~3842 m) to Paizhen (elevation ~2922 m), constitute a single group characterised by high genetic diversity and gene flow. In the western region, mtDNA analyses revealed high Hd and low π values for each population. Such results can be attributed to rapid population expansion after a period of small effective population size30Hd and low π values have originated in the Pliocene or early Pleistocene, but their mtDNA genealogies coalesce on a more recent time scale, perhaps the last few hundred thousand years45The western populations of S. o’connori populations have been gradually isolated from each other, such that populations in the east (PLTR) and west (YLTR) have evolved independently and formed the genetic structure observed today. Whilst additional studies of anatomy, physiology and molecular biology are required to evaluate the systematic status of the eastern group, there is now clear evidence of profound nuclear and mitochondrial DNA differentiation consistent with inter-specific differences between the eastern and western groups. Whilst data from only one eastern population limits inferences that can be drawn, the low mtDNA sequence diversity and the low nDNA allelic diversity estimatesFollowing river capture, reversal events and uplift of the QTP1524S. o’connori. Barcoding of Cyt b and COI are effective for species identification in freshwater fishesb sequences, a COI average intraspecific K2P distance of 0.35% and an average congeneric K2P distance of 8.11% have been employed as the standard to identify fish speciesSchizothorax species from the western Himalaya region (India) has been reportedS. o’connori might be classified as a subspecies or a new species in the genus Schizothorax. This interpretation is consistent with similar high mtDNA Cyt b + CR K2P distances reported between subspecies of Cyprinus carpioS. o’connori is further supported by results of the phylogenetic analyses (both the NJ and ML trees) of the mtDNA sequences. Fishes from the western region first grouped with the outgroups , and then grouped with fishes of the eastern region. This suggests that the fish in the eastern region are ancestral to, and different from, the fish in the western region.All analyses of mtDNA and nDNA point to substantial differences between the eastern (Bomi) population and the six western populations of S. o’connoriS. o’connori is narrower than that of the western fish, and barbel length of eastern individuals is longer than that of western fishesS. o’connori, but only four rows of pharyngeal teeth in western fishesS. o’connori was collected at Shigatse in the western region of the YLTRS. o’connori. Fish from the eastern region (PLTR) that are nominally S. o’connori require detailed study to validate their systematic status and their taxonomy.Consistent with the molecular interpretation presented above, pronounced morphological differences exist between western and eastern individuals of The possibility exists, as noted above, of unidirectional gene flow from the west to the east. Thus, fish at Bomi may, in principle, be able to interbreed with fish from the western populations if such fish are able to survive the journey down the many waterfalls from the YLTR to the PLTR. The mtDNA sequencing data is not well suited to test the suggestion of hybridisation because of the maternal mode of inheritance . The nDNA codominant microsatellites are more informative as markers of hybridization. The majority of the Bomi fish exhibit multilocus genotypes that are quite distinct from those of the western fishes, suggesting that hybridization is not occurring. However, two Bomi fish exhibit multilocus genotypes that are intermediate between eastern and western population-specific multilocus genotypes, and one fish exhibits a multilocus genotype that is similar to genotypes of western fishes e.g., . At thisS. o’connori in the western and eastern regions would change the present situation whereby S. o’connori is the only species of endemic Schizothorax fish that is distributed in both the western and eastern drainages of the YLTRS. o’connori in the western and eastern regions is likely to result in the recognition of a new subspecies or species in the east that requires specific management and conservation. Clarification of the taxonomic status of the east S. o’connori population requires that such work encompasses molecular approaches  as well as morphological analysis, physiological and environmental assessments, and the integration of all findings to help determine the likely status of eastern populations of S. o’connori. Ongoing threats to the river systems of the QTP such as pollution, over-fishing and the introduction of non-native species make this work a priority if we are to better understand the phylogeography of the fishes of this biodiversity hotspot.Revision of the specific status on the S. o’connori are profoundly influenced by the uplifts of QTP and Quaternary climatic oscillations. The relevance of these findings for S. o’connori to other fishes on the QTP, in particular schizothoracine fishes, remains to be determined, as does the taxonomic status of S. o’connori from the eastern (PLTR) system which is substantially different from putative conspecifics in the west. In terms of conservation and fisheries management, regardless of taxonomic status, the fishes in the east and west are very clearly different evolutionary units, and must be managed and protected as such.Combined analyses of mtDNA and nDNA variation provide evidence that population genetics and dynamics of No specific permits were required for the field studies described here. We confirm that the study locations were not privately owned or protected, and the field sampling activities did not involve endangered or protected species beyond the focal species. The animal research oversight committee of Huazhong Agricultural University (HZAU) had knowledge of the fish sampling plans prior to their approval of the present animal research protocol. All experimental protocols were approved by the Institutional Animal Care and Use Committee of HZAU, the methods were carried out in accordance with the approved guidelines.During the 2012 to 2014, fin clips were obtained from fish at six locations in the middle reaches of the YLTR in the western drainages (above the YTGC) and one location in the tributary of the PLTR in the east drainage (below the YTGC), Tibet . Fin clib (1141 bp) sequences and partial mtDNA CR (~714 bp) sequences following Guo et al.b, we used primers L14724 (5′-GACTTGAAAAACCACCGTTG-3′) and H15915 (5′-CTCCGATCTCCGGATTACAAGAC-3′), which were used in He & ChenS. o’connori (GenBank accession no. KC513575). PCR products were sequenced on an ABI 3730xl.DNA from 168 individuals (Table 5) was used to amplify complete mtDNA Cyt et al.et al.For microsatellite analysis, a total of 322 individuals  were gen363738b + CR to combine the phylogenetic signal to maximise the possibility of finding an optimal topology.Sequences of mtDNA were multiply aligned using Clustal X 2.0b, CR and Cyt b + CR sequences, respectively. Haplotype diversity (Hd), nucleotide diversity (π), number of segregating sites (S), number of haplotypes (h) and number of nucleotide differences (K) were estimated for the whole data set and for each population using Arlequin 3.11b + CR provided most information we focus primarily on the concatenated sequence.Population genetic analysis was carried out on Cyt Φ) was tested with 10,000 permutations. Genetic distances within/between populations based on the K2P model were calculated using MEGA 5.0ΦST/(1 − ΦST) statistics and log ) for all seven populations, and for the six western populations  was performed to test for genetic differentiation between regions (west/east) or among populations using Arlequin 3.11. Statistical significance of fixation indices  independently using neighbour-joining (NJ) and maximum likelihood (ML) in MEGA 5.0. The best-fitting nucleotide substitution model with the lowest Bayesian information criterion (BIC) score was determined using MEGA 5.0. ML analyses were performed under the TN93 + G (Cyt b), T92 + G + I (CR) and HKY + G + I (Cyt b + CR) model. In the NJ analysis, the K2P model was used. The reliability of nodes was assessed using 1000 bootstrap replicates. The congeneric species S. waltoni (GenBank accession no. JX202592), S. macropogon (GenBank accession no. KC020113) and Schizopygopsis younghusbandi (GenBank accession no. KC351895) were designated as outgroups.To determine relationships among populations and if there was any evidence of historical dispersal, phylogenetic analysis was carried out on Cyt b and the concatenated data set (Cyt b + CR) independently. The clock model and tree prior model were compared by calculating Bayes factor (BF). BF was estimated according to the marginal likelihood estimated by using the smoothed harmonic mean estimatorhttp://beast.bio.ed.ac.uk/Tracer). In this study, we tested two clock models: a strict clock and an uncorrelated lognormal relaxed clock. Two tree prior models, Yule process speciation and coalescent Bayesian Skyline, were also compared using BF calculation. As a result, all the data sets were analysed using a HKY model of nucleotide substitution under an uncorrelated exponential relaxed clock with a coalescent Bayesian Skyline tree prior model. The data of each model are shown in S. o’connori and hindered by the exhibition of different mutation rates by different lineages, therefore the molecular dating based on a fixed rate of mutation is not particularly suitable in this fish but at least gives us an idea about the potential age of diversification events. Nucleotide substitution rates for Cyt b and CR were assumed to be 1.0% and 3.6% per site per million years according to the previous study50b + CR) in schizothoracine fishes4http://tree.bio.ed.ac.uk/software/figtree/) with S. waltoni and S. macropogon used as outgroups.A continuous phylogenetic analysis was conducted using BEAST 2.3.2FsSSD) and the raggedness index (r)gen, where μ is the mutation rate per nucleotide per year or million years; k is the average sequence length of the mtDNA region under study; and Tgen is the generation time in yearsgen). Generation time for S. o’connori was estimated to be 10 years based on approximate time at which females become matureHistorical population dynamics were estimated using coalescent-based Bayesian skyline plots (BSPs)S. o’connori is an autotetraploid or an allotetraploid species the microsatellite data were treated as phenotypic data  and were analysed in two ways. First, all bands at each locus were combined to form multilocus allelic phenotypes for each individual. These phenotypes record the presence and identity of each allele, but are not genotypes because they contain no information about allele copy numbers unless individuals are homozygous  or have four different alleles. Second, each microsatellite locus was treated as dominant and then the multilocus allelic phenotypes were transformed into binary - presence (1) or absence (0) - for each individual. Although there is some information loss using binary data, such data are free from unrealistic assumptions about allelic identities and may be used in analysis of molecular variance (AMOVA). These approaches are routinely employed to evaluate genetic diversity and population structure of polyploidy speciesBecause it is unknown if H), Shannon’s information index (I), and the percentage of polymorphic loci (PPL) were calculated using POPGENEFST) and analysis of molecular variance (AMOVA) were performed using Arlequin 3.11.Binary allelic diversity was measured as total number of inferred bands  and number of bands unique to a single population (Private bands) and they were calculated using GenAlEx 6.5. Nei’s gene diversity index (K) from 1 to 7. Further, we used the ΔK metrichttp://taylor0.biology.ucla.edu/structureHarvester/).Estimation of genetic distance between populations of polyploid organisms is problematical because there is no accepted methodology for the calculation of genetic distance with binary dataHow to cite this article: Guo, X.-Z. et al. Phylogeography and population genetics of Schizothorax o’connori: strong subdivision in the Yarlung Tsangpo River inferred from mtDNA and microsatellite markers. Sci. Rep. 6, 29821; doi: 10.1038/srep29821 (2016).


Colletotrichum gloeosporioides, and were used to analyze genetic diversity and population structure of 163 isolates from four different geographical regions of Ethiopia. These loci produced a total of 118 alleles with an average of 5.13 alleles per microsatellite marker. The polymorphic information content values ranged from 0.104 to 0.597 with an average of 0.371. The average observed heterozygosity across all loci varied from 0.046 to 0.058. The gene diversity among the loci ranged from 0.106 to 0.664. Unweighted Neighbor-joining and population structure analysis grouped these 163 isolates into three major groups. The clusters were not according to the geographic origin of the isolates. Analysis of molecular variance showed 85% of the total variation within populations and only 5% among populations. There was low genetic differentiation in the total populations (FST = 0.049) as evidenced by high level of gene flow estimate (Nm = 4.8 per generation) among populations. The results show that Ethiopian C. gloeosporioides populations are generally characterized by a low level of genetic diversity. The newly developed microsatellite markers were useful in analyzing the genetic diversity and population structure of the C. gloeosporioides populations. Information obtained from this study could be useful as a base to design strategies for better management of leaf and fruit spot disease of citrus in Ethiopia.Twenty three polymorphic microsatellite markers were developed for citrus plant pathogenic fungus,  Citrus spp.) are economically important fruit crops in Ethiopia. The sustainable production of citrus in Ethiopia is severely constrained by various fungal diseases including C. gloeosporioides, Phythophtora spp., Alternaria spp., Penicillium spp. and Pseudocercospora angolensis .Frequency-based genetic distances were calculated using shared alleles distance matrix , and useirad.fr) . The resirad.fr)  and viewK), a burn-in period of 25,000 was used in each run, and data were collected over 100,000 Markov Chain Monte Carlo (MCMC) replications from K = 1 to K = 10. The probability values were averaged across runs for each cluster. This procedure clusters individuals into populations and estimates the proportion of membership in each population for each individual [K value was determined by the log probability of data (Ln P(D)) based on the rate of change in LnP(D) between successive K. The optimum K value was predicted following the simulation method of Evanno et al. [The pattern of population structure and detection of admixture were inferred using a Bayesian model-based clustering algorithm implemented in STRUCTURE version 2.3.4 ,94 usingdividual . The K vo et al.  using tho et al. .C. gloeosporioides isolates. Multiplex PCR amplifications were performed using four to five SSR primer pairs in each PCR based on annealing temperatures  were rare (with frequency ≤ 0.05). The number of alleles per locus ranged from three to eight, with an average of 5.13 alleles. The allele size ranged from 55 to 265 bp (G) were 135, with a range of 3 to 9 for each microsatellite marker. The number of effective alleles was in the range of 1.0 to 2.3. The value of allelic richness (Rs) ranged from 1.25 to 3.01, with a mean of 2.26. Allelic richness is the number of alleles per locus which is a measure of genetic diversity indicative of a population's long-term potential for adaptability and persistence [C. gloeosporioides isolates; but CG22 marker detected high heterozygosity of 0.865. Nine SSR loci had no heterozygosity while six displayed less than 0.01 heterozygosity. The expected heterozygosity ranged from 0.042 to 0.554 (P<0.001), having levels of heterozygosity less than expected. Two loci (CG22 and CG23) did not show significant departure from HWE, and CG22 had a level of heterozygosity higher than expected. The Fisher’s exact test showed that 60% of the pairwise combinations had significant genotypic linkage disequilibrium using Bonferroni correction. Given that most of the test isolates of C. gloeosporioides used in this study were assumed to be obtained from asexual populations, these results were not surprising.The polymorphism and diversity of the different SSR loci are presented in o 265 bp . The PICsistence ,98,99. Tsistence . Gene disistence , varied to 0.554 . The HarC. gloeosporioides populations are summarized in P) and effective alleles (NE) averaged across all loci ranged from 1.70 to 3.26, 0.043 to 1.261, and 1.38 to 1.85, respectively for the four populations . The South Ethiopian (SE) population had the highest while the Northwest Ethiopian (NWE) population had the lowest Na, NP and NE values. Similarly, the SE (RS = 3.16) and NWE (RS = 1.62) populations had the highest and the lowest allelic richness over all pairs of loci, respectively. Private allelic richness was also the least in NWE (RP = 0.04) and the highest in SE (RP = 1.24) C. gloeosporioides populations. Average observed heterozygosity (HO) was in the range of 0.046 to 0.058, with a mean of 0.052 across all loci. Gene diversity was the lowest in NWE population (HE = 0.209) and the highest in SE population (HE = 0.403), and its value averaged over all populations and loci was 0.283 (SE = 0.022). The percentage of polymorphic loci (PL) ranged from 60.87% (NWE) to 100% (SE), with an average of 82.61%. Based on the Shannon's Index (I), higher diversity was observed in SE (I = 0.725) population, but it was least in NWE (I = 0.311) population. The Shannon's Index is an information statistic index, which assumes all types are represented in a sample and that they are randomly sampled. It combines both evenness and richness in a single measure [The genetic diversity indices for the four  measure ,102.ST = 0.049 at P< 0.001) was significant as indicated by the randomization test. Pairwise FST values of the genetic distance among all populations were significant (P< 0.01) (m) was 4.8. Gene flow is the movement of genes into or out of a population. Such movement may be due to the movement of gametes, or migration of individuals and even entire population. In the absence of natural selection and genetic drift, gene flow leads to genetic homogeneity among populations as it can introduce new alleles into a population. However, restricted gene flow promotes population divergence via selection and drift, which can lead to speciation [Analysis of molecular variance (AMOVA) showed that 84% of the total variation was due to differences among isolates within populations, 11% was due to heterozygosity within isolates, and the variation among populations accounted only 5% of the total variation . The genP< 0.01) . The aveeciation .C. gloeosporioides complex associated with citrus leaf and fruit disease in Ethiopia. Several subgroups were observed for populations indicating genetic variability within and among isolates in each population. In terms of locations-specific alleles among the isolates, 17 SSR loci  showed unique alleles for the isolates from SE, 12 SSR marker  displayed unique alleles for the isolates from SWE, three SSR markers  detected unique alleles for the isolates from CE, and only one SSR marker (CG11) detected a unique allele for the isolates from NWE.The unweighted Neighbor-joining dendrogram grouped the 163 isolates of the four populations into three major clusters . Of the et al. [K = 3 to be the most likely number of clusters  based on the 23 microsatellite loci . Evanno et al.  method oclusters .Colletotrichum diseases worldwide, but SSR markers are available for a few Colletotrichum species such as C. capsici, associated with chili pepper anthracnose disease [C. acutatum, the causal agent of post-bloom fruit drop on citrus [C. gloeosporioides SSR markers development and their uses for genetic diversity and population structure study. Information on pathogen genetic diversity and population structure on temporal and spatial scales are important to understand the potential of pathogen populations to spread and overcome host resistance [A large number of hosts are affected by  disease , and C. n citrus . The presistance ,106. Aresistance .C. gloeosporioides isolates from citrus. The polymorphisms detected by these SSR markers were in the range of slightly informative to highly informative. They displayed allelic diversity among the isolates, with 3 to 8 alleles per locus, and the PIC values ranged from 0.104 to 0.597. The PIC provides an estimate of the discriminatory power of a locus by taking into account the number and the relative frequencies of the alleles [O at 0.865 and several loci with the lowest value at zero. Higher number of private alleles was observed in the isolates from the South region. Private alleles, or alleles that are unique to certain species or geographic area, are useful in comparing diversity between species or population [C. gloeosporioides isolates. However, the gene diversity observed among the Ethiopian C. gloeosporioides populations was low. This might be attributed to the recent introduction of the fungus probably of a few genotypes. Detection of high regional genetic diversity within the south Ethiopian population can be explained by the earlier introduction of the disease (in late 1980s) in the region [et al. [C. capsici isolates using 27 sequence-tagged microsatellite site markers and found the highest gene diversity of 0.857 at a locus with up to 18 alleles among all the isolates and the differentiation ranged from 0.05 to 0.45. Ciampi et al. [C. acutatum. The authors found from 3 to 6 alleles per locus, and heterozygosity ranging from 0.093 to 0.590 across loci using nine polymorphic SSR markers.In this study, 23 polymorphic SSR markers were developed and evaluated for assaying the genetic diversity of  alleles . All thepulation . The AMOe region . Similare region . These de region . Ranathui et al.  reportedet al. [C. gloeosporioides isolates is high where native or naturalized host species occur compared with locations where the host species has been recently introduced.Various factors can affect genetic diversity . Mutatioet al.  demonstrC. gloeosporioides populations from different locations is valuable in enhancing the understanding of the biology of the pathogen and potentially adaptive genotypic diversity in the species. The C. gloeosporioides isolates from the four geographic regions of Ethiopia are closely related to one another as reflected by the high genetic identity among populations. Population genetic analyses supported subdivision of the Ethiopian C. gloeosporioides isolates into three inferred sub-populations. STRUCTURE analysis, PCoA, and the unweighted Neighbor-joining algorithm indicated admixture among the three populations. Geographical separation of isolates into distinctly isolated subpopulations was not observed as evidenced by admixture among isolates from the different regions. The relatively low FST value (0.049) between the C. gloeosporioides populations evaluated in this study indicated low differentiation among the groups that might be attributed to gene flow among regions, which is reflected by the high migration rate (Nm = 4.8) estimates. A low degree of differentiation in populations of C. gloeosporioides shown in this study may be attributed to the dispersal of the clonal inoculum over long distances that may allow for pathogen spread in citrus-growing areas in Ethiopia. The citrus fruit produced in the south and southwest parts of Ethiopia are transported to the central areas for marketing while those produced in the northwest are consumed in the same region. The wind direction in Ethiopia is generally from northeast to southwest during the dry season and vice versa during the rainy season. These may cause migration and gene flow between populations that resulted in admixture among isolates from the different geographic origin. The pathogen is cosmopolitan in distribution, and conidia are known to be disseminated over long distances by wind [et al. [C. gloeosporioides infecting yam in Nigeria inferred by 51 microsatellite loci was described as a single population with low intra-population differentiation, high genetic diversity, and evidence of high gene flow. Conversely, there was significant and high genetic differentiation and gene diversity of subpopulations of C. gloeosporioides infecting strawberry in the United States based on 40 RAPD or microsatellite markers [An insight into the structure of  by wind  and via  by wind . Abang e [et al.  reported markers .m is greater than one, there will be little differentiation among populations and migration is more important than genetic drift. Similarly, Wright [C. gloeosporioides, identification of population subdivision within a particular geographic area can be associated with the epidemiology of the disease, such as sources of inoculum and host or tissue specificity [Pathogen population divergence may occur as a result of genetic drift and local adaptation to increase relative fitness in local environments . McDermo, Wright  stated t, Wright ,114,122.cificity ,120.C. gloeosporioides isolates from citrus growing regions of Ethiopia. Despite regional differences, the observed genetic diversity in all four populations was lower than expected suggesting inter-regional exchanges of planting materials and dispersal of inoculum among the regions. Information generated in this study could be useful in understanding the pathogen biology and provide basis for other studies on disease development, host-pathogen interaction, and developing disease management strategies including development and use of resistant citrus varieties for citrus leaf and fruit spot. The SSR markers could be useful to characterize C. gloeosporioides isolates that infect other fruit crops.In conclusion, the microsatellite markers developed in this study were useful to comprehend the genetic diversity and population structure of S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.