| Eriophyoid mites are strictly phytophagous,distributed worldwide.Some of them are important economic pest mites in agriculture and forestry,not only causing direct damage,but vectoring many plant viruses.Eriophyoid mites belongs to the class Arachnida,subclass Acari,and superorder Acariformes.They are among the smallest of terrestrial arthropods,with more than 5,000 described species.However,morphology-based identification of eriophyoid mites has been heavily impeded by their small body size(～200μm body length)and simplified morphology,especially in taxa with species complexes;thus,these further hindered our understanding of their species diversity.In this study,we assembled the first extensive set of DNA barcode reference sequences for eriophyoid mites.We also assessed the efficacy of their molecular delimitation using fragment of the mitochondrial COI gene.Furthermore,we compared the efficiency of five broadly used delimitation methods and accounted for the extent to whicn intrinsic factors of the datasets affect delimitation results.Finally,we used DNA barcoding to explore species diversity in eriophyoid mites as well as their interactions with plant hosts.The results are summarized as follows:1.Assembled a comprehensive reference library of DNA barcode for eriophyoid mitesBy analyzing over 4,000 samples collected from 63 areas in Asia and North America,we assembled the first DNA barcode reference library of eriophyoid mites,including 1,850mitochondrial COI sequences and representing three families,99 genera,and 456morphological species.Our dataset provides a coverage for 45%of the 930 species of eriophyoid mites known from China,and for 45%of the 37 species from Canada.On average,four sequences were obtained per MTU.2.Confirmed the utility of the COI gene for species identification in eriophyoid mites.We tested and confirmed the utility of DNA barcoding for species identification in eriophyoid mites.Sequence analysis showed a clear barcode gap in nearly all species from the three families,reflecting the fact that intraspecific divergences averaged 0.97%versus a mean divergence of 18.51%between nearest neighbours.We constructed a neighbour-joining tree employing the K2P distance metric using the Taxon ID tree tool on BOLD.The NJ tree showed that COI sequences enabled discrimination of 99%of the eriophyoid species.BIN analysis showed that the 1,850 sequences were assigned to 531 barcode index numbers(BINs).3.Morphological methods often substantially underestimate their diversityAnalyses examining the correspondence between BINs and morphological species revealed that members of 45 morphospecies were assigned to two or more BINs,resulting in1.16 times more BINs than morphospecies.Comparing the species diversity in different ecological regions in China reaffirmed previous evidence that morphological studies often substantially underestimate mite diversity.The ICE projections based on the present data suggest that the Chinese fauna of eriophyoid mites is likely to include at least 2,345 species.The overall results indicate that the Chinese fauna of eriophyoids is substantially more diverse than expected,justifying the need for further investigation.The high incidence of BIN splits further suggests that cryptic species are prevalent,motivating further evaluation of their diversity.4.Eriophyoid mites are highly specialized,and switches in host usage were probably the main drivers behind their high levels of modularity and specializationHost plant analysis showed that 89%of these mites(BINs)attack only one or two congeneric host species,and only few have diverse hosts.Furthermore,host-mite network analyses demonstrate that eriophyoid mites are highly host-specific,and modularity is high in plant-mite networks.Taken together,these results confirm that most eriophyoid mites have high host specificity,and thus switches in host usage have likely been the main driver of the high levels of modularity and specialization in host-mite interactions.5.Five molecular species delimitation approaches have relatively high efficiency and indices of taxonomic congruenceWe compared the efficiency and consistency of five broadly used single-gene species delimitation methods for Eriophyoidea.The delimitation results for five approaches varied:in the dataset for the superfamily Eriophyoidea,438 OTUs were produced using ABGD,531from BIN analysis,442 using ASAP,487 from m PTP,and 447 from GMYC.All five methods resolved approximately 90%(ranged from 87.42%to 89.62%)of morphospecies,with the BIN method and m PTP having relatively high performance.The delimitation results of GMYC and ASAP showed high congruence to morphospecies(Ctax=0.93,i.e.,93%of all species agreed),whereas BINs had the lowest congruence(Ctax=0.85).We found that the BIN method had the most inflationist results:while it provided a superior resolution power,its tendency towards species inflation resulted in lower congruence with other methods analyzed.6.The extend to which intrinsic factors of datasets affect the efficiency of multiple molecular delimitation methods in EriophyoideaWe explored how intrinsic factors of the datasets,i.e.,(1)the taxonomic rank,(2)the mean number of haplotypes per species,(3 the number of host plants per species,and(4)sampling geographical distance,affect delimitation results.GLMEM results showed no significant differentiation among five delimitation methods(χ~2=7.30,p=0.12).In contrast,a complex scenario of intrinsic factors and their interactions with methods were revealed to affect the efficiency of the species delimitation methods.GLM results showed that the number of haplotypes significantly impacted the efficiency of species delimitation using the ASAP,BIN,and GMYC methods.The geographic distance positively affected the efficiency in ASAP,GMYC,and m PTP.Only BIN was not influenced by the number of host plants per species.Lastly,ABGD was not found to be significantly impacted by any other factors.7.Phylogenetic analysis revealed a new species of Calvittacus–Calvittacus spectabilus sp.nov.–from China,based on morphological characteristics and DNA barcodeThe genus Calvittacus includes only four known species:C.chenius,C.mollissimus,C.regiae,and C.swidanus.In this study,we combined morphological characterization and molecular approaches to delimitate Calvittacus species and provide the key to species of Calvittacus.We recovered one new species,Calvittacus spectabilus sp.nov.Fourteen COI sequences of C.spectabilus sp.nov.from three populations were nearly identical in composition with intraspecific distances(K2P)ranging from 0.000 to 0.001.Phylogenetic analysis of Calvittacus species rejected the monophyly of Calvittacus.The geographical distribution of all five known species of Calvittacus were from the Oriental Region,indicating the Calvittacus may be endemic in the Oriental Region.In summary,we assembled the first extensive set of DNA barcode reference sequences for eriophyoid mites.By coupling DNA barcoding with the BIN system,we examined both the extent of cryptic diversity in these mites and their host interactions.In addition,we described and illustrated Calvittacus species and recovered one new species,Calvittacus spectabilus sp.nov.We evaluated the performance of five delimitation methods and revealed factors affecting the efficiency of molecular delimitation.The present study underlines the mechanisms behind biodiversity formation in eriophyoid mites as well as challenges in their persistence,providing a theoretical framework for ecological and biodiversity research in mites. |
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