Paracheirodon axelrodi), the most important fish in the ornamental world market, is distributed in both basins. Here we investigated P. axelrodi phylogeography, population structure, and potential routes of migration and connectivity between the two basins. A total of 468 bp of the mitochondrial gene (COI), 555 bp of the nuclear gene fragment (MYH6), and eight microsatellite loci were analyzed. As a result, we found two major genetic clusters as the most likely scenario (K = 2), but they were not discreetly distributed between basins. A gradient of genetic admixture was observed in Cucui and São Gabriel da Cachoeira, between the upper Negro River and the upper Orinoco. Samples from the middle-lower Negro River were highly structured. Cucui (Negro basin) was more similar to the Orinoco than to the rest of the Negro basin populations. However, substructure was also observed by the discriminant analysis, fixation indices and other hierarchichal structure analyses (K = 3 − 6), showing three major geographic clusters: Orinoco, Cucui, and the remaining Negro basin. Unidirectional migration patterns were detected between basins: via Cucui toward Orinoco and via the remaining of the Negro basin toward Orinoco. Results from the Relaxed Random Walk analysis support a very recent origin of this species in the headwater Orinoco basin  with a later rapid colonization of the remaining Orinoco basin and almost simultaneously the Negro River via Cucui, between 0.115 until about 0.001 Ma. Historical biogeography and population genetic patterns observed here for Cardinal tetra, seem to be better explained by river capture, physical, or ecological barriers than due to the geographic distance.The Neotropics contain one of the most diverse assemblages of freshwater fishes worldwide. Part of this diversity is shared between the Orinoco and Amazon basins. These basins have been separated for a long time due to the Vaupes Arch, rising between 10–11 Ma. Today, there is only one permanent connection between the Orinoco and Negro (Amazon) basins, known as the Casiquiare Canal. However, alternative corridors allowing fish dispersion between both basins have been proposed. The cardinal tetra ( The Neotropics contain one of the most diverse assemblages of freshwater fishes in the world . Within The origins of this ichthyofauna date back to the middle Miocene where most of the area of what is now the western Amazon comprised the “Paleo Amazonas-Orinoco” drainage. It is believed that this system drained northward into the Magdalena River, and, after the rise of the Cordillera Oriental and Sierra de Perijá, into Lake Maracaibo . This anThe rise of the Vaupes Arch in the late Miocene (between 10–11 Ma) severed this connectivity, resulting in the formation of two basins: the Orinoco, running north and northeast, and the Amazon, flowing towards the east, until it reached its current delta in the Atlantic Ocean, once the Purús Arch ruptured . At presAlong its course, the Casiquiare Canal has a gradient of water types, ranging from clear water (waters low in organic matter and sediment) near its source in the upper Orinoco, to black waters (waters rich in organic matter but low in sediments) near its confluence with the Negro River . This envia the Essequibo River and the Rupununi savanna during the high-water season. This alternative corridor has been used to explain the dispersion of the ancestors of marine species that colonized the freshwater environments of the two basins , nuclear sequences (MYH6), and eight microsatellite loci. Additionally, we test the hypothesis of basins connectivity and provide support regarding potential routes of gene flow and colonization between the two basins.In this study we provide the first phylogeographic hypothesis and analysis of population structure for P. axelrodi. The samples were collected by ornamental fish fishermen from four localities on the Orinoco basin (Colombia) including San José del Guaviare (n = 20), Puerto Carreño (n = 20), Puerto Gaitan (n = 21), and Puerto Inirida (n = 21) (n = 21), Santa Isabel (n = 20), Barcelos (n = 20), and São Gabriel da Cachoeira (n = 20)  were collected along the Orinoco and Amazon basins on the distribution area for (n = 21) , and fou(n = 20) . Sampleshttps://www.avma.org/resources-tools/avma-policies/avma-guidelines-euthanasia-animals). This procedure is recommended for small-bodied  tropical and subtropical stenothermic fish like cardinal tetra. Then, all individuals and tissue samples were preserved in ethanol at 95% and stored at 4 °C. Genomic DNA was extracted using MoBio UltraClean® Tissue and Cells DNA Isolation Kit or following a phenol/chloroform protocol  on ice, following the American Veterinary Medical Association (AVMA) guidelines for the euthanasia of animals: 2020 Edition , 1.5 µL of 10x buffer ((NH4)2SO4), 1.2 µL de dNTPs (10 mM), 1.5 µL of both primers (2 µM), 0.3 µL of Taq DNA Polymerase (1 U/ µL) and 1 µL of DNA (∼50 ng/µL). PCR conditions were as follows: 72 °C for 1 min, 35 cycles of 94 °C for 10 s, 50 °C for 35 s and 72 °C for 90 s, with a final extension period of 72 °C for 5 min.In this study, 96 mitochondrial fragments of the cytochrome oxidase I (COI) gene corresponding to 468 base pairs (bp) length each one , were amplified using the primers designed by P. axelrodi representing unique haplotypes for COI gene within each locality, previously identified as described in the “Phylogeographic analyses” section. We used the primers designed by 2O, 1.5 µL of BSA (10 mg/mL), 1.2 µL of MgCl2 (25 mM), 1.5 µL of 10x buffer ((NH4)2SO4), 1.2 µL of dNTPs (10 mM), 1.5 µL of each primer working solution, 0.5 µL of Taq DNA Polymerase (1 U/ µL) and 1 µL of DNA (∼50 ng/ µL). The 2nd-PCR reaction was carried out in a final volume of 15 µL and contained 8.3 µL of ddH2O, 1.2 µL of MgCl2 (25 mM), 1.5 µL of 10x buffer ((NH4)2SO4), 1.2 µL de dNTPs (10 mM), 1.5 µL of both primers (2 µM), 0.3 µL of Taq DNA Polymerase (1 U/ µL) and 1 µL of 1st PCR product. PCR reagents were obtained from Fermentas .Nuclear genome sequences corresponding to 555 bp of exonic region myosin heavy polypeptide 6 cardiac muscle alpha (MYH6), were amplified only for samples of PCR conditions for nuclear genes were as follows: 1st PCR - 68 °C for 1 min, 30 cycles of 93 °C for 10 s, 48 °C for 35 s and 68 °C for 90 s, with a final extension of 68 °C for 7 min. 2nd–PCR: 68 °C for 1 min, 35 cycles of 93 °C for 10 s, 55 °C for 35 s and 68 °C for 90 s., with a final extension period of 68 °C for 7 min. PCR products were purified using EXO-SAP,  following the manufacturer’s suggested protocol . All PCRPa7, Pa13, Pa19, Pa26, Pa27, Pa32, Pa33, Pa37) developed for P. axelrodi by 2O, 1.0 µL of MgCl2 (25 mM), 1.0 µL of 10x buffer((NH4)2SO4) ,1.0 µL of dNTPs (10 mM), 1.0 µL of reverse primer (2 µM), 0.5 µL of forward primer (2 µM), 0.5 µL of FAM –labelled M13 primer (2 µM), 0.3 µL of Taq DNA Polymerase (1 U/ µL) and 1 µL of DNA (∼25 ng/µL). PCR reagents were obtained from Fermentas . Amplification conditions were as follows: 94 °C for 1 min, 25 cycles of 94 °C for 30 s, 60 °C for 30 s and 68 °C for 40 s, 30 cycles of 94 °C for 20 s, 52 °C for 30 s and 72 °C for 1 min, with a final extension of 72 °C for 30 min. Then, 1 µL of PCR product was combined with one mL of ROX size standard . Null allele frequencies were estimated in FreeNA Software .Fst among localities. Additionally, the number of alleles per locus (NA), the observed (Ho), and the expected heterozygosity (HE) for every locus were estimated. Deviations from Hardy-Weinberg equilibrium (HWE) were calculated for each locus for each sampling locality. Similar analyzes were done for COI, including pairwise Φst index, haplotype (h) and nucleotide (π) diversity. In all cases, significance  were calculated using 10,000 non-parametric permutations. P-values were adjusted using Bonferroni correction for multiple comparisons  and geographic (km) distances among the sampled localities, using the Arlequin v3.5 software (http://cmpg.unibe.ch/software/arlequin35/man/Arlequin35.pdf). The geographical distances were obtained following the course of the rivers via the coordinates of each location using GoogleEarth Pro (Google Inc). The analyses were performed to evaluate if the spatial processes are driving population structure, or, in other words, if the IBD is the best explanation to the observed genetic structure patterns for the sedentary P. axelrodi.Additionally, isolation by distance (IBD) was analyzed for COI and microsatellites, using the Mantel test from a matrix of genetic . Assignment analyses were executed with 1 million steps Markov-Chain-Monte-Carlo (MCMC) with a burn-in of 10%. The convergence of the MCMC was inferred from the plot of α value of each independent run. The runs were analyzed in the program Structure Harvester v0.6.92  in the complete genotype matrix of the 163 individuals; through a discriminant analysis of principal components (DAPC) using R package Adegenet , and folNm) between theme, using the program Migrate v3.6.11 ; and a conservative substitution rate for fish microsatellites of 1.0 ×10−5 mutations per locus per generation . The COI haplotype network did not show shared haplotypes between the Orinoco and Negro basins. However, no reciprocal monophyly was found between basins (MG384576 –MG384595), they were inferred 40 alleles, and the resultant network showed 25 alleles shared among populations between basins , between Inírida and Cucui, on the Vaupés Arch near the border of the Guiana Shield. The signal of both markers suggested that from there (approximately 0.115 Ma), that ancestral population of P. axelrodi dispersed faster along the Orinoco basin reaching all localities studied simultaneously. Later, around 0.019 Ma, it colonized the Negro River basin via Cucui, arriving in its middle basin through Santa Isabel in the Holocene, approximately between 0.003 Ma and 0.001 Ma. Finally, P. axelrodi reached the lower Negro River through Barcelos, the last location, to be colonized in the last 1,000 years.The analysis of phylogenetic reconstruction Relaxed Random Walk ; Fig. S3HE) and observed Heterozygosity (Ho), and number of alleles per population (n) were obtained from eight loci screened in all individual of P. axelrodi along with deviations from HW equilibrium. Microsatellite loci had between 2 and 22 alleles per locus. Heterozygosity values were very similar for Puerto Carreño and Puerto Gaitan while HE was the highest in Cucui and lowest in Barcelos  deviation from HWE was observed at the majority of loci in Orinoco basin populations except (Pa7). Among the Negro basin populations, deviation from HWE was observed most loci except   .FST pairwise population differentiation analysis showed that within Orinoco basin there was no differentiation between Puerto Gaitán and Puerto Inírida , showing panmixia between them. There was significant differentiation among the rest of populations with values ranging from ∼0.10 to 0.24; P < 0.05), showing structuring among them . According to the AMOVA, most of genetic variation in P. axelrodi was within populations . However, a small but significant portion  was occurring among populations.The ong them . Additiost index for COI showed a high degree of significant structuring, except among some Orinoco populations . For instance, in the Negro basin, each location was highly structured, showing an Φst index between ∼0.68 and 0.95 (P < 0.05). Similarly, pairwise differentiation values between basins, they ranged from 0.58 up to 0.98, indicating a highly significant structure (P < 0.05) (The pairwise Φ < 0.05) .Fst and geographic distances for the eight P. axelrodi populations was moderate but statistically significant . However, no robust evidence was found to infer Isolation by Distance, since only 35% of genetic differentiation observed among P. axelrodi populations can be explained by the geographic distance (R2 = 0.350211).According to the Mantel Test, the correlation between pairwise K = 2) as the highest posterior probability of population structure (Mean Ln(K) = −5063.080), according to the Evanno method (n = 103); and (2) the remaining of the Amazon River populations  (n = 60)  . Howeverto K = 6 , the Rioed above . Both, SNm =4.8 migrants per generation, Nm =6.58 migrants per generation, Nm =4.5 migrants per generation, https://doi.org/doi:10.5061/dryad.866t1g1vv.According to the genetic structure pattern observed, we tested in Migrate the gene flow among three biogeographical groups: Orinoco basin, Cucui and rest of Negro basin. As a result, we found that the unidirectional pattern was the best model explaining migration among these groups: Cucui toward Orinoco basin  is more associated with the Orinoco basin, while Santa Isabel and Barcelos are shown as another independent biological group, being São Gabriel da Cachoeira the transition area between them. This observed pattern (Orinoco+Cucui / rest of the Negro basin) is probably explained by an ancient geological event such as the formation of waterfalls and rapids located in São Gabriel da Cachoeira. Our hypothesis suggests that the waterfalls may be the first split event dividing the Orinoco basin + Cucui populations from the rest of the Negro basin populations . It is known that the waterfalls system and rapids near São Gabriel da Cachoeira act as a physical barrier structuring the populations of small species between the upper section and the remaining area of the Negro River .P. axelrodi between basins are also supported by the Mantel test, where the distance does not explain by itself the current patterns of genetic structuring.However, when the results are explored hierarchically in STRUCTURE (K3–K6) and DAPC, it is found a substructure that shows Cucui isolated from the Orinoco, currently showing itself as an independent population from the Negro River and Orinoco River. This suggests that after the São Gabriel waterfalls formation, a second more recent split event occurred between the headwaters of the Negro River (where Cucui is located) and the Orinoco, until then connected probably through the Inírida River. This separation event was most likely influenced by the final Vaupes Arch rising. It is known that this rising happened from west to east, towards the Guiana Shield, and studies evidenced the influence of this process on fish fauna divergence between the resulting basins . These sNannostomus eques (Carnegiella strigata (P. axelrodi is a blackwater fish (Another of the remarkable genetic structure patterns was the found for Santa Isabel and Barcelos. STRUCTURE (K2- K6), DAPC and fixation indices, coincide in showing Santa Isabel and Barcelos as a genetically different stocks from São Gabriel da Cachoeira and Cucui. Since the Isolation by Distance hypothesis was rejected, the explanation for this pattern of genetic structure may be associated with the influence of the whitewater river in the section that comprises the distribution of these two populations or other of ecological order. This pattern of high genetic structure can also be observed in ornamental fish such as us eques  and in Cstrigata . In thester fish , which ater fish .P. axelrodi. So, as there were no such strong barriers, this could have caused a more rapid, recent dispersal and establishment of populations, where there was not enough time to accumulate differences, as shown by Random Walk and pairwise FST indices. However, according to STRUCTURE (K4–K6) and DAPC, we found genetic structure among Guaviare and the rest of the Orinoco populations. This and other structuring process ongoing for the Cardinal tetra, may be explained by three factors. First, since P. axelrodi is a blackwater fish ) , which dter fish , it is lern area , at the ern area . This isistances .via the Casiquiare Canal and other blackwater tributaries  gene flow in a biological scale of ∼4 migrants per generation between them. These results suggest a connection via Cucui toward Inírida route , or through a Cucui independent route, via rest of the Negro basin toward Orinoco . Patterns of connection between the Orinoco and Negro basins, has also been suggested previously in Cardinal tetra by Cichla temensis, C. monoculus, and C. orinocensis  are connected with a 16 km marshy and swampy plain with the Pimichin creek, an affluent of the upper Guainia (Negro drainage) River. Another potential connection is via a Llanos floodplain, less than 10 km wide, that connects the left-bank affluents of the upper Guainia River and the Guacamayo creek that drain into the Inírida River (Orinoco basin). Yet another alternate connection is via the right bank affluent of the Conochirite River and tributaries of the Atacavi and Temi Rivers (Orinoco basin) separated by less than 10 km of flat, and at least seasonally marshy terrain. The Conochirite River is a left bank affluent of the Guainia River, and during the high-water season is also connected to the upper Casiquiare Canal. A fourth connection is between the floodplains of the Baria River  and the Maturaca River . The connection via the Pimichin isthmus was already mentioned by Alexander von Humboldt and confirmed by There are currently at least five alternative connections among the tributaries of the upper Orinoco and Negro to the west of the Casiquiare canal . One suci.e., uni- and biparental) can reveal the contrasting patterns of spatial distribution of the species’ genetic variation. As a result, stronger population structure can be detected with the mtDNA than with nuclear DNA ). In addition, localities such as São Gabriel da Cachoeira ) were not sampled for the COI marker due to low DNA quality, as explained above. Additionally, microsatellites are codominant markers that more robustly examine the demographic behavior of both genders in the population and not only the behavior of females (mtDNA) .P. axelrodi is in the Orinoco basin, as reported by In this study, according to mitochondrial and nuclear diversity indices, we found higher values in Orinoco River populations such as Puerto Inírida, Guaviare and Puerto Carreño. This may suggest that the ancestral origin of via Cucui), followed by the median and lower part of this river. These results are in accordance with the chronological Negro River formation proposed by P. axelrodi by Random Walk analysis confirmed that ancient populations belong to the Orinoco basin, on the Western Guiana Shield, near Inírida. From here, it rapidly colonized the rest of Orinoco and almost simultaneously the upper Negro River . These river captures resulted in geodispersal of coastal-basin headwaters to the Amazon (and probably to Orinoco), thereby enriching the Amazonian biota, and vicariances isolating sister species on either side of the Western Guiana Shield-Amazon divide (reviewed in The colonization of Paracheirodon axelrodi) from the Orinoco and Amazon basins using coalescent- and frequency-based analyses of mitochondrial and nuclear DNA markers. Our results suggest that P. axelrodi is a young species that originated at Western Guiana Shield drainages (near Inírida) at late Pleistocene (∼0.255 Ma), with a very rapid and recent colonization of both basin from 0.115 Ma until about 1,000 years ago. Currently, the species is distributed between basins in a genetic structuring pattern of three geographical populations, even in the face of a restricted gene flow among them: (1) Orinoco , (2) Cucui (headwater Negro basin) and (3) rest of the Negro basin . Vaupés Arch is not a barrier to gene flow between basins for P. axelrodi. Instead, permanent connection between Orinoco and Negro basins, as the Casiquiare Canal or alternative black-water corridors drainages are allowing connection between both basins (currently or at least until a very recent past). This study provides key information to scientists, managers, and governmental agencies regarding the management of this fish as an important resource for ornamental industries in northern South American countries.This study presents the first comprehensive analyses of population structure, connectivity, and phylogeographic patterns for cardinal tetra  Puerto Carreño; (B) Puerto Gaitán; (C) San José del Guaviare; (D) Inírida) and Negro River basin ((E) Cucuí; (F) São Gabriel da Cachoeira; (G) Santa Isabel; (H) Barcelos), using COI and MYH6 exonic region, estimated under a HKY+GAMMA and GTR+GAMMA models of molecular evolution, respectively. The observed times are median heights into 95% credibility intervals, calibrated with the widely accepted mtDNA substitution rate for poikilotherm vertebrates in Click here for additional data file.10.7717/peerj.15117/supp-4Supplemental Information 4K = 2 as the most likely number of clusters. (C) Puechmille method estimators (MaxMed and MaxMean) showing K = 6 as the most likely number of clusters.(A) Evanno method (Δ K), identifying Click here for additional data file.10.7717/peerj.15117/supp-5Supplemental Information 5Click here for additional data file.10.7717/peerj.15117/supp-6Supplemental Information 6Click here for additional data file.10.7717/peerj.15117/supp-7Supplemental Information 7Click here for additional data file.10.7717/peerj.15117/supp-8Supplemental Information 8Click here for additional data file.10.7717/peerj.15117/supp-9Supplemental Information 9N = Cardinal sample size; for each locus: n = total number of alleles, Ho = observed heterozygosity, HE = expected heterozygosity. Loci out of equilibrium after Bonferroni correction (0.00078125) are shown in bold. CAR: Puerto Carreño; GV: San José del Guaviare; IN: Puerto Inírida; PG: Puerto Gaitán; CUC: Cucuí; SI: Santa Isabel; BAR: Barcelos.Click here for additional data file.10.7717/peerj.15117/supp-10Supplemental Information 10Paracheirodon axelrodi. Significantly different values (P < 0.05) are shown in bold. CAR: Puerto Carreño; GV: San José del Guaviare; IN: Puerto Inírida; PG: Puerto Gaitan; CUC: Cucui; SGC: São Gabriel da Cachoeira; SI: Santa Isabel; BAR: Barcelos.Pairwise FST values using eight microsatellites loci among Orinoco and Negro basin populations for Click here for additional data file.10.7717/peerj.15117/supp-11Supplemental Information 11P < 0.05) are shown in bold. Haplotype (h) and nucleotide (π) diversity ± standard deviation (SD) are shown on the diagonal for each geographic location. CAR, Puerto Carreño; GV, San José del Guaviare; IN, Puerto Inírida; PG, Puerto Gaitán; CUC, Cucuí; SI, Santa Isabel; BAR, Barcelos.Significantly different values (Click here for additional data file.10.7717/peerj.15117/supp-12Supplemental Information 12Click here for additional data file.


Aspergillus fumigatus is a ubiquitous mold and a common human fungal pathogen. Recent molecular population genetic and epidemiological analyses have revealed evidence for long-distance gene flow and high genetic diversity within most local populations of A. fumigatus. However, little is known about the impact of regional landscape factors in shaping the population diversity patterns of this species. Here we sampled extensively and investigated the population structure of A. fumigatus from soils in the Three Parallel Rivers (TPR) region in Eastern Himalaya. This region is remote, undeveloped and sparsely populated, bordered by glaciated peaks more than 6,000 m above sea level, and contained three rivers separated by tall mountains over very short horizontal distances. A total of 358 A. fumigatus strains from 19 sites along the three rivers were isolated and analyzed at nine loci containing short tandem repeats. Our analyses revealed that mountain barriers, elevation differences, and drainage systems all contributed low but statistically significant genetic variations to the total A. fumigatus population in this region. We found abundant novel alleles and genotypes in the TPR population of A. fumigatus and significant genetic differentiation between this population and those from other parts of Yunnan and the globe. Surprisingly, despite limited human presence in this region, about 7% of the A. fumigatus isolates were resistant to at least one of the two medical triazoles commonly used for treating aspergillosis. Our results call for greater surveillance of this and other human fungal pathogens in the environment.Aspergillus fumigatus is a ubiquitous pathogen capable of long-distance dispersal and growth in diverse environments. In this study, using A. fumigatus as a model, we investigated how localized landscape features contribute to genetic variations in fungal populations. Our results revealed that elevation and drainage isolation rather than direct physical distances significantly impacted genetic exchange and diversity among the local A. fumigatus populations. Interestingly, within each local population, we found high allelic and genotypic diversities, and with evidence ~7% of all isolates being resistant to two medical triazoles, itraconazole and voriconazole. Given the high frequency of ARAF found in mostly natural soils of sparsely populated sites in the TPR region, close monitoring of their dynamics in nature and their effects on human health is needed.The extreme habitat fragmentation and substantial environmental heterogeneity in the TPR region have long known to contribute to geographically shaped genetic structure and local adaptation in several plant and animal species. However, there have been limited studies of fungi in this region.  Aspergillus fumigatus is the most predominant Aspergillus human-pathogenic species and the primary cause of aspergillosis   -5. Whies of IA , 7. Withes of IA , 9. Theres of IA .Aspergillus  and the Quaternary climate oscillations. It’s also one of the global biodiversity hotspots . The patA. fumigatus around metropolitan Kunming, the capital of Yunnan, had a high prevalence of azole-resistant A. fumigatus (ARAF) while a lower frequency of ARAF was identified in outdoor environments associated with fewer human activities  markers, which have been widely used for genotyping strains of A. fumigatus  reveal the genetic structure of the A. fumigatus in the TPR region, (ii) explore the relative roles of geographical barriers and landscape features in shaping the population genetic diversities of this species, and (iii) investigate the prevalence of triazole-resistant strains and their potential mutations associated with drug resistance.The highly heterogenous topographic and climatic diversities within the TPR region provide an excellent opportunity to investigate the potential geographic effects on umigatus , were us2 of the TPR’s core area. The detailed information about the sampling sites is shown in A. fumigatus isolation followed the method described previously , Mekong (Lancang Jiang[LCJ]), and Salween (Nu Jiang[NJ]). Sampling sites belonging to the same river system were about 30 km apart from each other in numerical order. Along the three rivers, five sites along each river were located at similar altitudes as those from two other rivers (3 × 5 = 15 sites). The corresponding sites from different rivers were located very close to each other if measured based on map distance. At each site, 100 soil samples of about 10 g each were collected at a depth of 0–5 cm from the surface, then put into sterile zip-log plastic bags. Individual soil samples were about 2 m apart from each other. Together, the sampling sites covered about 21,323.25 kmeviously . The iniA. fumigatus isolates were genotyped by a panel of nine highly polymorphic STRs markers , to understand the relationships among strains and populations of A. fumigatus from TPR. The number of tandem repeats at each locus for each strain was determined following the method described previously (A. fumigatus isolates from our previous studies and from other countries reported previously and deposited in the STRAf database (http://afumid.shinyapps.io/afumID) were extracted and compared with A. fumigatus from TPR in this study , rBarD, and the proportion of pairwise loci that were phylogenetically compatible (PrC) were calculated following the method described previously (We constructed two minimum spanning trees (MSTs) based on STR markers . One investigated the genetic relationships among isolates from TPR and those from our two previous studies including 233 strains from nine greenhouses in Jinning and 452 outdoor isolates from 19 geographic locations across Yunnan . The secfrom TPR . IBM SPSeviously .A. fumigatus isolated in this study following the methods described in the CLSI M38-A2 (cyp51A gene (including full-length coding and promoter regions) of all isolates obtained in this study.Two clinical azole drugs (ITR and VOR) commonly used for the treatment of aspergillosis were used to test the susceptibility of I M38-A2 , 32. PriI M38-A2  were useA. fumigatus strains were isolated from 1,900 soil samples from the 19 sites in the TPR core area. Two hundred fifteen alleles and 332 multilocus genotypes were identified across the nine STR loci. The number of alleles ranged from 10 to 48 per locus among the nine STR loci, with an average of 24. The STRAf3A locus had the most alleles (n = 48), followed by STRAf3C locus (n = 41). Among the total 215 alleles, 174 were shared between at least two of the sites from TPR. The remaining 41 alleles were found only in one site each. The 19 sampling sites from TPR differed in their total numbers of alleles, with the number of alleles ranging from 46 (JSJ5) to 99 (NJ5). Except three populations , private alleles were found in all other populations: 18, 14, and 9 private alleles were found in the Nu River, Jinsha River, and Lancang River, respectively , followed by the Jinsha River population (uh = 0.754), and the Lancang River population (uh = 0.705). However, analysis of variance using the mean diversity estimates of each river showed no significant difference in the pairwise comparisons among the three rivers  from the 19 local populations from TPR ranged from 3.401 (JSJ5) to 8.637 (NJ5), with an average of 5.455. Shannon’s Information Index range from 1.288 (JSJ5 population) to 2.094 (NJ5 population), with an average of 1.643. Simpson’s Diversity ranged from 0.563 (LCJ4 population) to 0.821 (NJ5 population), with an average of 0.705. Unbiased Diversity range from 0.594 (LCJ4 population) to 0.863 (NJ5 population), with an average of 0.751 . When tae rivers .P = 0.01 for both, P = 0.01), Lancang River , and Nu River . We further investigated the extent of genetic differentiation between pairs of TPR populations. We found that the genetic differentiation level of the Nu River population was lower than that of the other two rivers (P < 0.05) (P < 0.05). The biggest differentiation was found between LCJ4 population and NJ3 population , followed by that between LCJ4 population and NJ5 population . Similarly, significant genetic differentiation was also found between rivers, i.e., Jinsha River populations and Lancang River populations , Jinsha River populations and Nu River populations , and Lancang River populations and Nu River populations   contributing significantly to the overall genetic variations  . Among t= 0.001) .A. fumigatus was higher in regions among altitudes 1360–1450 m (uh = 0.860), followed by 1160–1290 m (uh = 0.803) and the lowest in 2740 m and higher region (uh = 0.689). When combining local populations scattered in similar latitude ranges as one meta-population, we found that the diversity of A. fumigatus was highest in population group in middle latitudes (ranging from 27.73 to 27.78 N) (uh = 0.804), followed by the group in high latitude area (ranging from 27.95 to 28.07 N) (uh = 0.792), and the lowest genetic diversity was found in population group with lowest latitude (ranging from 27.46 to 27.49 N) (uh = 0.722)  . HoweverP = 0.01) (P = 0.099) (P = 0.18) (P = 0.24) (P = 0.041), Simpson’s Diversity , and Unbiased Diversity , respectively  . In cont= 0.099) , longitu = 0.18) , and lat = 0.24)  distanceectively .A. fumigatus , consistent with recombination throughout the sites we have tested in the genome. However, the null hypothesis of random mating and recombination in the total sample was rejected (P < 0.001).Despite very different compositions of genetic elements among the river-affiliated populations in the TPR region, evidence for recombination was found in all samples. Specifically, in the total sample that includes all 19 local populations, none of the STR loci comparisons showed phylogenetic compatibility  deposited in AfumID (https://afumid.shinyapps.io/afumID/). Here, a total of 1,108 A. fumigatus strains were used. All samples were divided into 11 populations according to their geographical origins  (P < 0.05), and the populations from TPR were significantly different from all other geographic populations (P = 0.001). Among them, the populations with the highest and lowest degrees of genetic differentiation from the TPR population were those from Africa  and East Asia , respectively  . Pairwisectively . The patectively . STRUCTUectively . Our sam A and B . However A and B . Further A and B , and MST A and B  analyses A and B .50 = 0.5 μg/mL, MIC90 = 2.0 μg/mL) and 0.25 to ≥ 8 µg/ml , respectively. Among the 331 isolates, 6.95% (23/331) were resistant to one or both clinical triazoles ITR and/or VOR. Specifically, 6.34% (21/331), 3.63% (12/331), and 3.02% (10/331) were resistant to ITR (MIC ≥ 4 mg/L), VOR (MIC ≥ 4 mg/L), and both ITR and VOR, respectively  and they are found in almost all the TPR isolates, regardless of their susceptibilities to triazoles. The second group included distinct but diverse mutations found only among the triazole-resistant isolates, including L98H and S297T. Strains with the TR34 insertion all had MIC ≥ 4 mg/mL and all are cross-resistant to both triazoles  are among the most frequently used genetic markers for population genetic and epidemiological studies of a variety of organisms, including animals, plants, and eukaryotic microorganisms such as fungi. In A. fumigatus, the nine STR markers have shown to be highly reproducible and highly polymorphic and have helped revealing a diversity of local to global population genetic patterns, including providing evidence for both local clonal expansion and long-distance dispersals  of the total number of alleles and 97.0% (193/199) of the total number of genotypes, respectively, while the occurrence of those private genetic elements from the outdoor populations in Yunnan were a little lower, at 16.2%  and 94.1% (unique genotypes), respectively  had private alleles, accounting for 19.07% (41/215) of the total number of alleles, and the unique genotypes accounting for 98.8% (328/332) of the total number of genotypes. The proportions of private alleles and genotypes are similar to those of A. fumigatus populations from Yunnan’s greenhouses  in combination explained 76.63% of the total variation  contributed about 49.7–55.6% to total variation  in the Eastern Himalaya (Tuber indicum species complex: more than 80% (63/77) of the haplotypes were restricted to certain geographic populations in the Hengduan Mountains region in Southwest China  among the 358 A. fumigatus strains from the TPR populations. In the Cameroon population, 26.4% (14/53) isolates were found to harbor the same allele  (P = 0.001), comparable to our previously analyzed 19 geographic locations across Yunnan   of our pairwise comparisons among local TPR populations showed significant genetic differentiation ( < 0.05) . In addi= 0.001) . However= 0.001) . Our stusibility , contribsibility , 53. How complex . Our stuA. fumigatus populations from Nu River and other two rivers. Furthermore, Nu River populations had the highest genetic diversity and the largest number of private alleles, but the lowest genetic differentiation level. Frequent gene flow may have contributed to maintaining low genetic differentiation and high genetic diversity in Nu River populations. Lastly, the frequency of triazole resistance of A. fumigatus in the Nu River population was the highest, followed by the Jinsha River and Lancang River populations. Nu River is located westernmost in TPR area with the lowest altitude among the three main rivers, it is sandwiched between the Biluo Snow Mountain and the Gaoligong Mountain, which are parts of the Hengduan Mountains with an altitude of 4,500 m and 4,000 m, respectively. In addition, the road along the Nu River is the only way for local residents in the river valley to connect with the outside world, thus these areas with high genetic diversity were likely more influenced by anthropogenic factors, with sampling sites close to villages and towns and relatively more frequent human activities, which can promote the gene flow in A. fumigatus  insertion within the upstream regulatory region of cyp51A, as well as a broad spectrum of triazole susceptibilities in A. fumigatus populations around the world  were resistant to ITR and/or VOR, with 3.92% (10/331) being cross-resistant to both ITR and VOR. The prevalence of ARAF in this study was significantly lower than in our previous two studies in Yunnan. The mountainous terrains of this region are extremely unfavorable to agricultural activities, and the local supplies of vegetables and grains mainly rely on imports from surrounding areas. Consequently, the use of fungicides is relatively uncommon, which is consistent with the lower level of ARAF compared with our previous two studies in Yunnan. However, the prevalence of ARAF in the TRP region was still higher than the strains from hospital gardens, city parks, and farmlands outside Yunnan (1.4%) and that from greenhouses in Zhejiang province (4.1%) , 61. It A. fumigatus in Yunnan.Among the 331 isolates from the TPR region, 21 and 12 were resistant to ITR and VOR (MIC ≥ 4.0 μg/mL), respectively. Among them, 12 of 21 ITR-resistant isolates had MIC ≥ 16.0 µg/mL, none of 12 VOR-resistant isolates had MIC ≥ 16.0 µg/mL, and the MIC value distributions between ITR and VOR were also different. Further analysis showed that 10 of the 23 triazole-resistant strains were cross-resistant to both ITR and VOR while the rest were resistant to either ITR or VOR, the proportion of cross-resistant isolates was similar to what was found in our previous two studies , 32. Togcyp51A locus, five were broadly distributed in our TPR samples with or without triazole resistance. Whether these mutations are ubiquitous in the remote regions from Eastern Himalaya needs further study. Furthermore, diverse patterns of other mutations occurred in resistant strains, indicating potentially unique selection for azole-resistant strains in this region. Interestingly, the TR34/L98H mutation was the most common mutation associated with triazole (cross-)resistance in environmental samples from Yunnan province, similar to those reported from other parts of China (Among the diverse mutations found among our isolates at the of China , 70, as of China , 71.A. fumigatus populations at these sites. Specifically, the warm and raining season between June and October may increase the reproduction of A. fumigatus in these regions, leading to higher isolation frequencies of A. fumigatus. A recent study showed differences in thermal profiles among isolates from different regional populations of A. fumigatus (A. fumigatus populations in this region and the potential effects of seasons on population genetic structure of A. fumigatus.We note that our analyzed samples across the 19 sites were collected at a single time point, during 4 days in early January 2020. While this short period of sampling helps reduce the influence of potential confounding temporal factor in our analyses of geographic factors on population genetic variations, it’s also possible that obtained samples may not fully represent the genetic variations of umigatus . In addi


Introduction: Since Aedes aegypti invaded Yunnan Province in 2002, its total population has continued to expand. Shi et al. used microsatellite and mitochondrial molecular markers to study the Ae. aegypti populations in Yunnan Province in 2015 and 2016, found that it showed high genetic diversity and genetic structure. However, there are few studies on the population genetic characteristics of Ae. aegypti in Yunnan Province under different levels of human intervention. This study mainly used two common types of molecular markers to analyze the genetic characteristics of Ae. aegypti, revealing the influence of different input, prevention and control pressures on the genetic diversity and structure of this species. Understanding the genetic characteristics of Ae. aegypti populations and clarifying the diversity, spread status, and source of invasion are essential for the prevention, control and elimination of this disease vector.Methods: We analyzed the genetic diversity and genetic structure of 22 populations sampled in Yunnan Province in 2019 and 17 populations sampled in 2020 through nine microsatellite loci and COI and ND4 fragments of mitochondrial DNA. In 2019, a total of 22 natural populations were obtained, each containing 30 samples, a total of 660 samples. In 2020, a total of 17 natural populations were obtained. Similarly, each population had 30 samples, and a total of 510 samples were obtained.Results: Analysis of Ae. aegypti populations in 2019 and 2020 based on microsatellite markers revealed 67 and 72 alleles, respectively. The average allelic richness of the populations in 2019 was 3.659, while that in 2020 was 3.965. The HWE analysis of the 22 populations sampled in 2019 revealed significant departure only in the QSH-2 population. The 17 populations sampled in 2020 were all in HWE. The average polymorphic information content (PIC) values were 0.546 and 0.545, respectively, showing high polymorphism. The average observed heterozygosity of the 2019 and 2020 populations was 0.538 and 0.514, respectively, and the expected average heterozygosity was 0.517 and 0.519, showing high genetic diversity in all mosquito populations. By analyzing the COI and ND4 fragments in the mitochondrial DNA of Ae. aegypti, the populations sampled in 2019 had a total of 10 COI haplotypes and 17 ND4 haplotypes. A total of 20 COI haplotypes were found in the populations sampled in 2020, and a total of 24 ND4 haplotypes were obtained. STRUCTURE, UPGMA and DAPC cluster analyses and a network diagram constructed based on COI and ND4 fragments showed that the populations of Ae. aegypti in Yunnan Province sampled in 2019 and 2020 could be divided into two clusters. At the beginning of 2020, due to the impact of COVID-19, the flow of goods between the port areas of Yunnan Province and neighboring countries was reduced, and the sterilization was more effective when goods enter the customs, leading to different immigration pressures on Ae. aegypti population in Yunnan Province between 2019 and 2020, the source populations of the 2019 and 2020 populations changed. Mantel test is generally used to detect the correlation between genetic distance and geographical distance, the analysis indicated that population geographic distance and genetic distance had a moderately significant correlation in 2019 and 2020 .Conclusion:Ae. aegypti in Yunnan Province maintains a high degree of genetic diversity. Human interference is one reason for the changes in the genetic characteristics of this disease vector. Aedes aegypti originated in Africa , Lincang City , and Dehong Prefecture (Ruili City). To avoid inbreeding effects, we selected at least six larvae from each breeding site in a designated area within 100 m and mixed the larval mosquitoes. All the collected mosquitoes were brought back to the laboratory and separated by sampling site for rearing until they grew into adults, and then they were identified through analysis of morphological characteristics under a microscope to confirm that they were all Ae. aegypti  following the standard DNA extraction protocol provided by the manufacturer. The eluted DNA was measured by a spectrophotometer and stored in a freezer at −40°C for later use.In this study, nine microsatellite sites developed by Shi were used for analysis . The detAe. aegypti populations in Yunnan Province. With the primer set 5′-GGAGGATTTGGAAATTGATTAGTTC-3′ (F-COI) and 5′- CCCGGTAAAATTAAAATATAAACTTC-3′(R-COI) plus 5′- ATTGCCTAAGGCTCATGTAG -3′ (F-ND4) and 5′- TCGGCTTCCTAGTCGTTCAT-3′ (R-ND4), DNA amplification of 550 bp fragments of COI and 361 bp fragments of ND4 was performed on a T100 Thermal Cycler . The reaction system was 25 μL, which included 5 µL PCR buffer , 4 µL dNTPs , 1 µL primers , 0.5 µL PrimeSTAR HS DNA polymerase  and 15.75 µL ddH20. The PCR amplification programs were set as follows: predenaturation at 94°C for 2 min, followed by 40 cycles of denaturation at 94°C for 60 s, annealing at 55°C for 30 s and elongation at 72°C for 1 min, with a final elongation at 72°C for 10 min (COI gene), and predenaturation at 94°C for 2 min, followed by 35 cycles of denaturation at 94°C for 60 s, annealing at 52°C for 30 s and elongation at 72°C for 1 min, with a final elongation at 72°C for 10 min (ND4 gene). All PCR products were detected by 1.2% agarose gel electrophoresis and sequenced on an ABI 3730XL automatic sequencer .We used COI and ND4 gene markers of mitochondrial DNA to analyze the genetic structure of Ae. aegypti, some software programs were used to calculate the standard genetic parameters. All microsatellite markers were examined for polymorphism by determining their polymorphic information content (PIC) values via PIC-Calc version 0.6 (via Arlequin (version 3.5.2.2) and the p-value was obtained through multiple comparison corrections. The null allele frequency of each microsatellite locus was calculated by Micro-Checker software (version 2.2.3) (via Bottleneck software (version 1.2.0.2) (via STRUCTURE HARVESTER (via NTSYS (version 2.2), and then graphs of the correlations between geographic distance and genetic distance were generated in Excel 2019 .To study the population genetic characteristics of sion 0.6 . The numsion 0.6 . In eachsion 0.6 . The obssion 0.6 . Departun 2.2.3) . The ste1.2.0.2) . STRUCTU1.2.0.2) . The parARVESTER . After tARVESTER . DiscrimARVESTER . IsolatiDNASP 5.0 software was used to calculate the genetic diversity parameters of the haplotypes, including haplotype diversity (Hd), nucleotide diversity (Pi) and average number of nucleotide differences (π) . The genA total of 22 locations were sampled from September to October 2019, including nine locations at Jinghong City, one location in Mengla County, one location at Daluo Port, one location at Mohan Port, three locations at Ruili City, one location at Nansan Town, Zhenkang County, three locations at Mengding Town, two locations at Qingshuihe Port and one location at Myanmar Muse.In 2020, a total of 17 locations were sampled from September to October in Yunnan Province, including three locations at Jinghong City, two locations at Mengla County, two locations at Daluo Port, one location at Mohan Port, four locations in Ruili City, one location at Nansan Town, three locations at Mengding Town, and one location at Qingshuihe Port. Detailed acquisition information was listed in the In this study, the above nine pairs of primers were used to amplify 660 samples collected in 2019 and 510 samples collected in 2020.Ae. aegypti samples collected in 2019 and 2020, a total of 67 and 72 alleles were obtained, respectively. On average, the 2019 populations obtained 7.44 alleles per locus, and the 2020 populations obtained eight alleles per locus. In 2019, SQM6 obtained 14 alleles, which was the highest number among all loci. The SQM5 and SQM9 loci had four alleles, which was the lowest number among all loci, while in 2020, the SQM2 and SQM6 loci had the most alleles (14), and SQM5 and SQM9 had only four alleles. The PIC values of the populations sampled in 2019 and 2020 were generally high, with average values of 0.546 and 0.545, respectively. Microchecker software revealed null alleles at nine loci in the 2 years, with frequencies ranging from 0.001 to 0.159, however, frequency p values <0.2 are considered to have no significant effect on the accuracy of data analysis by many studies . The statistical test (one-way ANOWA) of the populations collected in 2019 and 2020 found that p > 0.05 (p = 0.086), indicating that the gene richness didn’t change significantly between 2 years.In this study, Fstat software was used to analyze the allelic richness of 22 populations sampled in 2019 and 17 populations sampled in 2020 . The 201The number of alleles and allelic richness are both important indicators of population diversity. Comparing the 2-year sampling data, the average number of alleles obtained for the 22 populations in 2019 was 3.798, while the average number of alleles in 2020 was 4.353. The average allelic richness of the populations was 3.707 in 2019 and 3.965 in 2020. Regardless of the average number of alleles or the average allelic richness, the populations sampled in 2020 showed higher diversity than the populations sampled in 2019 .In this study, we used TPM and SMM models to analyze the bottleneck effect of samples collected in 2019 and 2020 , and a tΔK methods . Meanwhile, of the genetic variation in the populations sampled in 2020, 85.99% could be attributed to differences within individuals and 7.88% to differences among populations within groups .The AMOVA results  showed tp < 0.05 R = 0.6933; p < 0.05 R = 0.6506); the geographical distance and genetic distance of the populations were positively correlated in both years.The genetic distributions of 22 populations sampled in 2019 and 17 populations sampled in 2020 as obtained by the IBD model are shown in Based on COI gene analysis, 10 and 20 haplotypes were found in the populations sampled in 2019 and 2020, respectively.Analyzing the ND4 fragments of the populations sampled in 2019 and 2020, we obtained a total of 17 haplotypes in the 2019 populations and 24 haplotypes in the 2020 populations.The 30 haplotypes obtained for the COI gene of the populations sampled in the 2 years were compared and analyzed by MEGA software, revealing that the haplotypes H1, H2, H3, H4, H5, H6, H7, and H10 in 2019 were the same as H1, H7, H12, H2, H5, H20, H18, and H19 obtained from the 17 populations in 2020. Forty-one haplotypes of the ND4 gene were analyzed by the same method. Haplotypes H1, H2, H4, H5, H7, H10, H13, H15, and H17 obtained from the 22 populations in 2019 were the same as H1, H6, H15, H2, H19, H17, H20, H23, and H16 in the 2020 populations. GenBank IDs of COI haplotype are OM865371-OM865392. GenBank IDs of ND4 haplotype are OM877316-OM877347. Haplotype details were recorded in the Ae. aegypti collected in 2019 were mainly divided into two clades, and the Myanmar population had more frequent exchanges with the populations of Lincang City and Ruili City (Ae. aegypti collected in 2020 were roughly divided into two clades, one in Xishuangbanna Prefecture and the other consisting of Lincang City and Ruili City populations sampled in 2020 and the Myanmar population sampled in 2019. This pattern was different from the clustering results based on microsatellite markers.The haplotype network diagrams constructed with 653 COI fragments and 656 ND4 fragments obtained in 2019 showed that H1 and H2 were the most frequent haplotypes, and nearly all the other haplotypes derived were from H1 and H2 with several mutations. H1 haplotypes were mainly distributed in Xishuangbanna Prefecture, the other dominant haplotype H2 was mainly distributed in the Lincang City, Ruili City and Burmese populations. The COI and ND4 network diagram showed that the ili City . The hapili City . Analysip < 0.05, indicating that these populations had experienced bottleneck effects and then expanded. Based on the COI and ND4 fragments of the populations sampled in 2020 (Ae. aegypti underwent obvious population expansion in these areas.The results of the neutrality tests based on the COI and ND4 fragments of the sampled populations in 2019  showed t in 2020 , the neu in 2020  showed tAe. aegypti populations in the border areas of Yunnan Province (Ae. aegypti populations under different selection pressures. Ae. aegypti in the border areas of Yunnan were collected in 2019 and 2020, respectively, and this study focused on analyzing the changes in the genetic characteristics of Ae. aegypti populations under different immigration pressures, especially the outbreak of dengue that occurred in 2019, where an integrated vector management approach was implemented. Because of the COVID-19 pandemic, the border was closed and crossings were limited in 2020, which led to declines in person and logistic flows. The status of the populations over 2 years provides an opportunity to study the population structure of Ae. aegypti with prevention and control pressures and human intervention related to COVID-19, thereby revealing the impact of human intervention, which will have important guiding significance for the prevention and control of this population in the future.Molecular markers were previously used to analyze the genetic characteristics of Province . HoweverAe. aegypti populations sampled in 2019 had immigration. Considering that the flight distance of Ae. aegypti is usually short, it is possible that the immigrants mainly came from the flow of people or goods. In 2020, the average observed heterozygosity of the sampled populations was 0.514, and the average expected heterozygosity was 0.519, which were basically the same, indicating that the populations experienced inbreeding. This may be due to the outbreak of COVID-19 in early 2020. China quickly took strong measures to control the epidemic. However, the epidemic prevention abroad was not optimistic. To reduce the risk of foreign imports, the management of the movement of people and goods was strengthened at Chinese ports. As a result, the opportunity for invasion and number of invading individuals of Ae. aegypti from neighboring countries through the movement of people and goods were greatly reduced.Null alleles are one of the biggest defects of microsatellite markers; they mainly reduce population genetic diversity and increase genetic differentiation between populations . Yan et Ae. aegypti populations in Yunnan Province was relatively high. Stable populations have been established and have the ability to resist adverse external environmental influences and expansion. The flow restriction of people and goods had a certain impact on the populations of Ae. aegypti. If the populations cannot be fully supplemented in the long run, the diversity of the Ae. aegypti in border areas of Yunnan Province may decline.The diversity index of the populations sampled in 2019 and 2020 was relatively high after microsatellite marker analysis. In summary, the overall diversity of Ae. aegypti, as an invasive mosquito species, experienced population expansion in Yunnan Province in 2019 and 2020.Generally, only a few individuals of invasive species can enter new areas, and newly invasive individuals will experience genetic bottleneck effects after arriving in the new areas, which will eventually lead to a decrease in genetic diversity . The botAe. aegypti in the port areas. The bottleneck effect experienced by the populations in Jinghong City may be due to the outbreak of dengue fever year after year, and the Ae. aegypti populations face great environmental pressure, such as disinfection and sterilization, so it is difficult for them to maintain a stable colonization state. The populations in other regions have not experienced bottleneck effects and were in a stable state. The nucleotide mismatch distribution analysis of the populations sampled in 2019 and 2020 revealed that Ae. aegypti in Yunnan Province had experienced expansion. This may be because although the populations sampled in 2020 received less gene flow, the population diversity was similar to that of the 2019 populations, and it also had the ability to expand.In the port areas of Yunnan Province, the bottleneck effect impacting the populations occurred in the 2 years, indicating continuous invasion of Ae. aegypti in Xishuangbanna Prefecture, and the other clade included the populations of Lincang City, Ruili City, Dehong Prefecture and Myanmar. The Myanmar population had the highest allelic richness. Generally, multiple invasions can increase the genetic diversity of the invading population, but the overall level will not be higher than that of the source populations. With spread, genetic diversity will further decline (Ae. aegypti in Myanmar is likely a source population of Ae. aegypti in Yunnan Province. Comparing the allelic richness of various populations in Yunnan Province sampled in 2019, the Lincang City populations had higher values than the Ruili City populations. Because the Lincang City and Ruili City populations were located in the border areas, the Lincang populations likely didn’t directly spread from the Ruili populations but invaded from Myanmar, which indicates that invasion followed not a single route but multiple routes. Jinghong City is far from the border, the Lancang River flows through the city, tourism is developed, and freight transport is frequent. Therefore, the populations of Ae. aegypti in Jinghong City may come from invasion by foreign populations with travelers and then spread to other areas of Xishuangbanna Prefecture. They may also come from Laos bordering Mohan Port and eastern Myanmar bordering Daluo Port.The cluster analysis results of the populations sampled in 2019 based on microsatellite markers and mitochondrial DNA showed that the populations can be divided into two major clades; one clade included the populations of  decline ; therefoAe. aegypti populations in the border areas of Yunnan Province in 2020 may have come from a new invasion of Myanmar, or they may have resulted from breeding of the Ae. aegypti population in Yunnan Province after natural selection and vector control in 2019. There was inbreeding in 17 populations sampled in Yunnan Province in 2020. Considering the situation at the border ports, due to the impact of COVID-19, the flow of people and goods was reduced, and the disinfection and sterilization of goods entering Yunnan through ports were more effective than before. As a result, the probability of Burmese Ae. aegypti population invasion with the flow of people and logistics decreased.Microsatellite molecular marker analysis showed that the Burmese population sampled in 2019 and the Yunnan populations sampled in 2020 could not be grouped together. However, the haplotype network map constructed by mitochondrial DNA molecular markers revealed that the Myanmar population sampled in 2019 and the populations in Lincang and Ruili sampled in 2020 shared haplotypes. The dominant haplotypes didn’t change between the 2 years. This phenomenon may be caused by the fact that the mutation rate of mitochondrial DNA molecular markers is lower than that of microsatellite markers. In summary, the Ae. aegypti in Yunnan Province was relatively high, and the populations were mainly divided into two major clusters. The main reason for the formation of the two clusters may be different invasion events. The continued invasion of Ae. aegypti in border areas maintained population diversity. Under different immigration pressures, prevention and control pressures, the structure and diversity of the Ae. aegypti populations could change. Human activities may be one cause of the expansion of Ae. aegypti in the border areas of Yunnan Province. These research results can provide some guidance for the prevention and control of Ae. aegypti in China.The genetic diversity of


Zea commonly named teosintes, comprise nine different taxa, distributed from northern Mexico to Costa Rica. Although this genus of plants has been extensively studied from a morphological, ecogeographical and genetic point of view, most contributions have been limited to the study of a few populations and taxa. To understand the great variability that exists between and within teosinte species, it is necessary to include the vast majority of known populations. In this context, the objective of this work was to evaluate the diversity and genomic structure of 276 teosinte populations. Molecular analyzes were performed with 3,604 plants and with data from 33,929 SNPs. The levels of genetic diversity by taxonomic group show a marked difference between species, races and sections, where the highest values of genomic diversity was found in ssp. parviglumis and ssp. mexicana. The lower values were obtained for the Luxuriantes section as well as ssp. huehuetenagensis of the section Zea. The results of structure show that there is a great genetic differentiation in all the taxonomic groups considered. For ssp. parviglumis and mexicana, which are the taxa with the largest number of populations, a marked genomic differentiation was found that is consistent with their geographic distribution patterns. These results showed a loss of diversity in several teosinte populations, making a strong case for further collection, and ex situ and in situ conservation. Also, this study highlights the importance of integrating genomic diversity and structure for the applications of conservation and management.The wild species of the genus  Zea mays ssp. mays L.), the Mexican Agreement on the Determination of Maize’s Centers of Origin and Diversity states that research should be carried on to characterize and monitor maize wild relatives’ genetic diversity at the population level [Zea (Poaceae).Landscape transformation, mainly due to anthropogenic activities, has led to decreased diversity, size, and connectivity of many populations of plant species –5, incluon level . Here, wZea includes annual and perennial diploids (2n = 20) and a perennial tetraploid species (2n = 40) [Luxuriantes and section Zea. Section Luxuriantes includes: perennial diploid species Zea diploperennis, perennial tetraploid Z. perennis, and the annuals diploids Z. luxurians, Z. nicaraguensis and Z. vespertilio. Z. diploperennis is distributed in very small populations exclusively in: (i) a small valley in the mountains of the Sierra Madre Occidental, in the locality of San Andrés Milpillas at Huajicori, Nayarit at an average altitude of 1,400 meters above sea level (m asl), (ii) at the slopes of Cerro de San Miguel, inside of natural protected area of Las Joyas at Sierra de Manantlán, municipality of Cuautitlán, Jalisco, at altitudes from 1,400 to 2,400 m asl. Z. perennis populations are restricted to El Fresno locality at Uruapan, Michoacán, at an average altitude of 1,380 m asl; and on the slopes of Volcán de Colima in the state of Jalisco at altitudes of 1,600–2,200 m asl. Zea luxurians is native to southeastern Guatemala, Honduras, El Salvador and southern México at altitudes between sea level and 1,100 m asl. Subsequently, Z. nicaraguensis was described by Iltis and Benz [Z. vespertilio was recently described at Murciélago Islands, Santa Elena Península, Guanacaste province of Costa Rica [Zea includes only three subspecies of Zea mays: Zea mays ssp. huehuetenangensis (to which we refer to as ssp. huehuetenangensis from now on), which is only found in western Guatemala at elevations of 900–1,650 m asl at San Antonio Huista, Jacaltenango, Santa Ana Huista and Nenton localities; and Zea mays ssp. parviglumis and Zea mays ssp. mexicana  which occupy a diverse geographic range in Mexico, showing some level of geographic overlap within their natural distribution in central and northern Mexico. Subspecies mexicana, is distributed among highlands of the Mexican High Plateau and Sierra Madre Occidental biogeographical provinces, and ssp. parvilgumis, is found among the Mexican southwest lowlands of the Balsas basin and Sierra Madre del Sur province [mexicana into four races [The genus 2n = 40) –12. The 2n = 40) , who ide2n = 40) –12: Sectand Benz  as a geosta Rica . The Secprovince . Effortsur races , 18: racZ. luxurians and Z. nicaraguensis [Z. perennis and Z. diploperennis [Striga spp. from Z. diploperennis [Z. mays ssp. mexicana Durango race, which allows it to survive extreme drought [Teosintes are relevant for agriculture because their genetic variation may be useful in maize breeding programs, for example: aerenchyma in the roots for adaptations to rains and floods of aguensis , 20; virperennis ; resistaperennis ; and ver drought . Additio drought , and not drought . Today,  drought , 16, 25.parviglumis and ssp. mexicana) [parviglumis, confirming a high level of gene flow among populations and a low, but clear level of genetic differentiation, due to geographic features. Hufford et al. [mexicana and maize occurred during the expansion of the crop into the highlands of central Mexico early in its domestication process, leading to the incorporation of adaptive alleles of ssp. mexicana into the early maize. Focusing on both ssp. mexicana and ssp. parviglumis, Pyhäjärvi et al. [mexicana and ssp. parviglumis), few sampling localities within taxa, and few samples per sampling locality [Zea (except Zea vespertilio) using SNP markers from 276 sampling localities and 3,604 individuals.Evolutionary scientific interest on teosintes has mostly focused on systematics , showingexicana) , 30. Theexicana)  determind et al.  suggestei et al.  found thi et al.  found thlocality , 31–34. Zea species and subspecies  was performed at the Institute for Genomic Diversity  following a GBS protocol . DNA washttps://doi.org/10.5061/dryad.2547d7wxp).The sequence data and the genotypic database of SNPs were processed in the Tassel-5-GBS Production Pipeline software . Using aHo) and expected heterozygosity (He) with the 4P v. 1.0 software [Ho and He index, we partitioned the values into percentiles and quartiles and identified the species that fall in the lowest and highest percentile based on four thresholds: 1) <10th percentile; 2) 10th percentile to 2nd quartile; 3) from the 2nd quartile to the 90th percentile and 4) >90th percentile. This classification criterion was made based on the proposal of Canteri et al. [We estimated genomic diversity indices in two ways: the first included independent estimates for each of the 276 populations, while the second grouped the 276 populations according to the taxonomic classification criterion including the levels to subspecies and races (13 taxa). We calculated the observed heterozygosity  were obtained with DnaSP [ST) , Polymorphic site (PS), Theta of Watterson (θw) and Heterozygosity sites (th DnaSP . Each ofth DnaSP . IsolatiaSP [ST)  and the aSP [ST) . This anquin 3.5 . For the package  of R wasadegenet package from R. Based on the best K group from the DAPC analysis, an AMOVA was performed to determine the differences between and within the assigned groups with Arlequin. And (5) additionally, to examine the level of admixture among individuals and between populations with higher detail, for ssp. parviglumis and ssp. mexicana, we run the software Admixture [We examined population structure to identify clusters of genetically related individuals within all teosinte taxa. For this, we: (1) generated a pairwise genetic matrix according to the Tamura-Nei distance using Mega 7 software ; (2) perdmixture  with K =A total of 276 populations of nine taxa were sequenced and genotyped using GBS. A total of 4,010 teosinte plants were sequenced, of which 80% met the molecular quality and filtering parameters. The final matrix used for analyses included 33,929 SNPs and 3,604 individuals.Hs presented the highest values of genomic diversity in ssp. parviglumis, followed by ssp. mexicana but with variation between races. Lower values were obtained for the Luxuriantes section as well as ssp. huehuetenangensis of the section Zea. For the taxa of the Zea section, the values of Ho are variable, with ssp. parviglumis showing the highest values but with a wide range among populations. For races of ssp. mexicana, the highest values were found within races Chalco and Central Plateau  divides the genus into the Luxuriantes and Zea sections, with ssp. huehuetenangensis as intermediate. Plotting coordinates 2 and 3 shows similar patterns, but with higher separation of the races that make up ssp. mexicana  included the 276 populations divided into thirteen taxonomic groups . The results show that there is a great genetic differentiation in all the levels considered which were significant (P <0.001). Of the total genetic variation, the highest percentage (62.51%) corresponds to the variation within individuals and the lowest variation was found between individuals within the same sampling locations (9.87%) .parviglumis populations, followed by 9 clusters of ssp. mexicana, and an independent group for ssp. huehuetenangensis. The last two clusters group Z. perennis and Z. diploperenis in one, and Z. luxurians and Z. nicaraguensis in another . In the DAPC analysis, in K = 25 was found to show the lowest levels of BIC . The fir another .The second AMOVA based on DAPC was similar to the first model; the percentage of variation among the 25 groups was 15.97%; higher than for the first model, while variation among groups was higher in the first model .parviglumis follows a longitudinal gradient but with some clearly differentiated populations particularly at the West and East of its range  and Nobogame and Durango (ssp. mexicana) populations are considerably isolated from the rest of the distribution, likely by natural historical processes. A previous study [parviglumis and ssp. mexicana and determined that it was caused by genetic drift. Interestingly, other populations showing relatively low genetic diversity in that study are at the limits of the environmental niche of the taxon, and thus where natural selection is driving local adaptation [Within ssp. l Mexico . There, l Mexico . Most weus study  also fouaptation . TherefoZea genus is highly structured, with genetic clusters generally matching taxonomic groups, but also subdivided in further clusters driven by geography , which are distributed in western Mexico mountain ranges. The second group is formed by Z. luxurians from Guatemala and Oaxaca, and by Z. nicaraguensis. Within the genetic clusters of Z. luxurians and Z. diploperennis, there is further genetic differentiation driven by geographic distance , while widely distributed taxa, like ssp. parviglumis, are at the smallest risk , Nicaragua and Guatemala, due to their very low and low levels of genetic diversity, respectively  as in cely Figs  and 4 anely Figs .Zea section there are also populations of high concern, both considering their genetic differentiation and human induced threats. These are ssp. huehuetenangensis, and the most differentiated and geographically restricted genetic clusters of ssp. mexicana and ssp. parviglumis. These subspecies were not considered threatened by the NOM-059 in 2010, because the evaluation was made at the species level, at which population sizes are considerably high. However, the more recent assessment by the IUCN in 2017 evaluated subspecies and races independently , which showed the lowest levels of genetic diversity within ssp. mexicana  into ssp. parviglumis [Populations of the largest genetic clusters include races Chalco and Central Plateau from ssp. he genus . Howeverhe genus , which ahe genus . This syviglumis .in situ and ex situ actions are required. However, considering that most populations of teosinte taxa are distributed within regions of high social conflict and presence of organized crime [ex situ conservation actions are particularly urgent, while social conditions for in situ conservation became better in key areas where the smallest populations are distributed. Based on previous teosinte collection work in Mexico [ex situ regeneration in greenhouses for later recovery in situ.As for taking conservation of teosintes genetic diversity into practice, both ed crime , ex situn Mexico , 70 in mIn situ conservation actions are more challenging, because there are several uncontrollable environmental factors  as well as human factors such as farmer’s management decisions and land use changes. In situ conservation actions should be thus further discussed and proposed in the social and environmental conditions of each region. What remains clear from the current study, is that some of the reduced levels of genetic diversity found are caused by natural processes driven by historical isolation and adaptation, but that also recent anthropic activities play a role. Some of the naturally isolated and relatively small populations within all teosinte taxa have been further fragmented by the growth of intensive agriculture (mostly of maize and avocado), opening of non-native grasslands for livestock and urbanization [in situ conservation. The effectiveness of ongoing protection measurements on some taxa should also be evaluated. Importantly, even if the currently work represents the most updated and representative teosinte sampling, most of it is more than 10 years old Click here for additional data file.S2 Fig(PNG)Click here for additional data file.S3 Fig(PNG)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.S2 Data(XLSX)Click here for additional data file.S3 Data(XLSX)Click here for additional data file.S4 Data(XLSX)Click here for additional data file.