Characterizing restriction enzyme‐associated loci in historic ragweed (Ambrosia artemisiifolia) voucher specimens using custom‐designed RNA probes

Population genetic studies of nonmodel organisms frequently employ reduced representation library (RRL) methodologies, many of which rely on protocols in which genomic DNA is digested by one or more restriction enzymes. However, because high molecular weight DNA is recommended for these protocols, samples with degraded DNA are generally unsuitable for RRL methods. Given that ancient and historic specimens can provide key temporal perspectives to evolutionary questions, we explored how custom‐designed RNA probes could enrich for RRL loci (Restriction Enzyme‐Associated Loci baits, or REALbaits). Starting with genotyping‐by‐sequencing (GBS) data generated on modern common ragweed (Ambrosia artemisiifolia L.) specimens, we designed 20 000 RNA probes to target well‐characterized genomic loci in herbarium voucher specimens dating from 1835 to 1913. Compared to shotgun sequencing, we observed enrichment of the targeted loci at 19‐ to 151‐fold. Using our GBS capture pipeline on a data set of 38 herbarium samples, we discovered 22 813 SNPs, providing sufficient genomic resolution to distinguish geographic populations. For these samples, we found that dilution of REALbaits to 10% of their original concentration still yielded sufficient data for downstream analyses and that a sequencing depth of ~7m reads was sufficient to characterize most loci without wasting sequencing capacity. In addition, we observed that targeted loci had highly variable rates of success, which we primarily attribute to similarity between loci, a trait that ultimately interferes with unambiguous read mapping. Our findings can help researchers design capture experiments for RRL loci, thereby providing an efficient means to integrate samples with degraded DNA into existing RRL data sets.     

Host‐associated genomic differentiation in congeneric butterflies: now you see it,now you do not

Ecotypic variation among populations may become associated with widespread genomic differentiation, but theory predicts that this should happen only under particular conditions of gene flow, selection and population size. In closely related species, we might expect the strength of host‐associated genomic differentiation (HAD) to be correlated with the degree of phenotypic differentiation in host‐adaptive traits. Using microsatellite and Amplified Fragment Length Polymorphism (AFLP) markers, and controlling for isolation by distance between populations, we sought HAD in two congeneric species of butterflies with different degrees of host plant specialization. Prior work on Euphydryas editha had shown strong interpopulation differentiation in host‐adapted traits, resulting in incipient reproductive isolation among host‐associated ecotypes. We show here that Euphydryas aurinia had much weaker host‐associated phenotypic differentiation. Contrary to our expectations, we detected HAD in Euphydryas aurinia, but not in E. editha. Even within an E. aurinia population that fed on both hosts, we found weak but significant sympatric HAD that persisted in samples taken 9 years apart. The finding of significantly stronger HAD in the system with less phenotypic differentiation may seem paradoxical. Our findings can be explained by multiple factors, ranging from differences in dispersal or effective population size, to spatial variation in genomic or phenotypic traits and to structure induced by past histories of host‐adapted populations. Other infrequently measured factors, such as differences in recombination rates, may also play a role. Our result adds to recent work as a further caution against assumptions of simple relationships between genomic and adaptive phenotypic differentiation.     

Using noninvasive metagenomics to characterize viral communities from wildlife

Microbial communities play an important role in organismal and ecosystem health. While high‐throughput metabarcoding has revolutionized the study of bacterial communities, generating comparable viral communities has proven elusive, particularly in wildlife samples where the diversity of viruses and limited quantities of viral nucleic acid present distinctive challenges. Metagenomic sequencing is a promising solution for studying viral communities, but the lack of standardized methods currently precludes comparisons across host taxa or localities. Here, we developed an untargeted shotgun metagenomic sequencing protocol to generate comparable viral communities from noninvasively collected faecal and oropharyngeal swabs. Using samples from common vampire bats (Desmodus rotundus), a key species for virus transmission to humans and domestic animals, we tested how different storage media, nucleic acid extraction procedures and enrichment steps affect viral community detection. Based on finding viral contamination in foetal bovine serum, we recommend storing swabs in RNAlater or another nonbiological medium. We recommend extracting nucleic acid directly from swabs rather than from supernatant or pelleted material, which had undetectable levels of viral RNA. Results from a low‐input RNA library preparation protocol suggest that ribosomal RNA depletion and light DNase treatment reduce host and bacterial nucleic acid, and improve virus detection. Finally, applying our approach to twelve pooled samples from seven localities in Peru, we showed that detected viral communities saturated at the attained sequencing depth, allowing unbiased comparisons of viral community composition. Future studies using the methods outlined here will elucidate the determinants of viral communities across host species, environments and time.     

Dietary specialization in mutualistic acacia‐ants affects relative abundance but not identity of host‐associated bacteria

Acacia‐ant mutualists in the genus Pseudomyrmex nest obligately in acacia plants and, as we show through stable isotope analysis, feed at a remarkably low trophic level. Insects with diets such as these sometimes depend on bacterial symbionts for nutritional enrichment. We, therefore, examine the bacterial communities associated with acacia‐ants in order to determine whether they host bacterial partners likely to contribute to their nutrition. Despite large differences in trophic position, acacia‐ants and related species with generalized diets do not host distinct bacterial taxa. However, we find that a small number of previously undescribed bacterial taxa do differ in relative abundance between acacia‐ants and generalists, including several Acetobacteraceae and Nocardiaceae lineages related to common insect associates. Comparisons with an herbivorous generalist, a parasite that feeds on acacias and a mutualistic species with a generalized diet show that trophic level is likely responsible for these small differences in bacterial community structure. While we did not experimentally test for a nutritional benefit to hosts of these bacterial lineages, metagenomic analysis reveals a Bartonella relative with an intact nitrogen‐recycling pathway widespread across Pseudomyrmex mutualists and generalists. This taxon may be contributing to nitrogen enrichment of its ant hosts through urease activity and, concordant with an obligately host‐associated lifestyle, appears to be experiencing genomewide relaxed selection. The lack of distinctiveness in bacterial communities across trophic level in this group of ants shows a remarkable ability to adjust to varied diets, possibly with assistance from these diverse ant‐specific bacterial lineages.     

Rapid genetic adaptation precedes the spread of an exotic plant species

Human activities have increasingly introduced plant species far outside their native ranges under environmental conditions that can strongly differ from those originally met. Therefore, before spreading, and potentially causing ecological and economical damage, non‐native species may rapidly evolve. Evidence of genetically based adaptation during the process of becoming invasive is very scant, however, which is due to the lack of knowledge regarding the historical genetic makeup of the introduced populations and the lack of genomic resources. Capitalizing on the availability of old non‐native herbarium specimens, we examined frequency shifts in genic SNPs of the Pyrenean Rocket (Sisymbrium austriacum subsp. chrysanthum), comparing the (i) native, (ii) currently spreading non‐native and (iii) historically introduced gene pool. Results show strong divergence in flowering time genes during the establishment phase, indicating that rapid genetic adaptation preceded the spread of this species and possibly assisted in overcoming environmental constraints.     

Plant host and soil origin influence fungal and bacterial assemblages in the roots of woody plants

Microbial communities in plant roots provide critical links between above‐ and belowground processes in terrestrial ecosystems. Variation in root communities has been attributed to plant host effects and microbial host preferences, as well as to factors pertaining to soil conditions, microbial biogeography and the presence of viable microbial propagules. To address hypotheses regarding the influence of plant host and soil biogeography on root fungal and bacterial communities, we designed a trap‐plant bioassay experiment. Replicate Populus, Quercus and Pinus plants were grown in three soils originating from alternate field sites. Fungal and bacterial community profiles in the root of each replicate were assessed through multiplex 454 amplicon sequencing of four loci (i.e., 16S, SSU, ITS, LSU rDNA). Soil origin had a larger effect on fungal community composition than did host species, but the opposite was true for bacterial communities. Populus hosted the highest diversity of rhizospheric fungi and bacteria. Root communities on Quercus and Pinus were more similar to each other than to Populus. Overall, fungal root symbionts appear to be more constrained by dispersal and biogeography than by host availability.     

Genotypic variation in Norway spruce correlates to fungal communities in vegetative buds

The taxonomically diverse phyllosphere fungi inhabit leaves of plants. Thus, apart from the fungi's dispersal capacities and environmental factors, the assembly of the phyllosphere community associated with a given host plant depends on factors encoded by the host's genome. The host genetic factors and their influence on the assembly of phyllosphere communities under natural conditions are poorly understood, especially in trees. Recent work indicates that Norway spruce (Picea abies) vegetative buds harbour active fungal communities, but these are hitherto largely uncharacterized. This study combines internal transcribed spacer sequencing of the fungal communities associated with dormant vegetative buds with a genome‐wide association study (GWAS) in 478 unrelated Norway spruce trees. The aim was to detect host loci associated with variation in the fungal communities across the population, and to identify loci correlating with the presence of specific, latent, pathogens. The fungal communities were dominated by known Norway spruce phyllosphere endophytes and pathogens. We identified six quantitative trait loci (QTLs) associated with the relative abundance of the dominating taxa (i.e., top 1% most abundant taxa). Three additional QTLs associated with colonization by the spruce needle cast pathogen Lirula macrospora or the cherry spruce rust (Thekopsora areolata) in asymptomatic tissues were detected. The identification of the nine QTLs shows that the genetic variation in Norway spruce influences the fungal community in dormant buds and that mechanisms underlying the assembly of the communities and the colonization of latent pathogens in trees may be uncovered by combining molecular identification of fungi with GWAS.     

Diet selection at three spatial scales: Implications for conservation of an endangered Hawaiian tree snail

Several recent studies suggest local adaptation in multiple taxa across Hawaii's steep environmental gradients. Restoration efforts in devastated tropical island ecosystems may be deficient if we lack an understanding of the interactions and dependencies in communities that occur along these gradients. Endangered Hawaiian tree snails are part of a snail–epiphyte–plant system where they graze fungi and other microbes on the leaf surface, a process difficult to observe using conventional techniques. Tree snails have undergone catastrophic decline due to introduced predators, removal by shell collectors, and human‐influenced habitat degradation. Prior to this study, little was known about the relationship among tree‐snails, their host plants, and the epiphytic microbes on which they feed. In this study, we identified scale‐dependent selection of substrates in Achatinella sowerbyana and Achatinella lila across the species’ ranges. We assessed: (1) within‐plant diet selection using high‐throughput DNA sequencing (micro‐scale); (2) among‐plant selection of tree host species (small‐scale); (3) and the influence of climate on this system (macro‐scale). Selection of substrates occurred at two scales: fungal communities in fecal samples differed in composition from those available on leaf surfaces; and at all sites, snail occurrence on Metrosideros polymorpha, a foundational forest plant, was significantly higher than expected based on availability. Habitat restoration efforts should focus on out‐planting of M. polymorpha, the preferred snail host tree, in degraded habitat. Fungal differences across sites suggest relocation efforts to predator‐free enclosures may be hindered by microbial shifts associated with geographic distance or differing environments.     

Distribution and population structure of the anther smut Microbotryum silenes‐acaulis parasitizing an arctic–alpine plant

Cold‐adapted organisms with current arctic–alpine distributions have persisted during the last glaciation in multiple ice‐free refugia, leaving footprints in their population structure that contrast with temperate plants and animals. However, pathogens that live within hosts having arctic–alpine distributions have been little studied. Here, we therefore investigated the geographical range and population structure of a fungus parasitizing an arctic–alpine plant. A total of 1437 herbarium specimens of the plant Silene acaulis were examined, and the anther smut pathogen Microbotryum silenes‐acaulis was present throughout the host's geographical range. There was significantly greater incidence of anther smut disease in more northern latitudes and where the host locations were less dense, indicating a major influence of environmental factors and/or host demographic structure on the pathogen distribution. Genetic analyses with seven microsatellite markers on recent collections of 195 M. silenes‐acaulis individuals revealed three main genetic clusters, in North America, northern Europe and southern Europe, likely corresponding to differentiation in distinct refugia during the last glaciation. The lower genetic diversity in northern Europe indicates postglacial recolonization northwards from southern refugia. This study combining herbarium surveys and population genetics thus uniquely reveals the effects of climate and environmental factors on a plant pathogen species with an arctic–alpine distribution.     

Shotgun metagenomics and microscopy indicate diverse cyanophytes,other bacteria,and microeukaryotes in the epimicrobiota of a northern Chilean wetland Nostoc (Cyanobacteria)

Prokaryotic Nostoc, one of the world's most conspicuous and widespread algal genera (similar to eukaryotic algae, plants, and animals) is known to support a microbiome that influences host ecological roles. Past taxonomic characterizations of surface microbiota (epimicrobiota) of free‐living Nostoc sampled from freshwater systems employed 16S rRNA genes, typically amplicons. We compared taxa identified from 16S, 18S, 23S, and 28S rRNA gene sequences filtered from shotgun metagenomic sequence and used microscopy to illuminate epimicrobiota diversity for Nostoc sampled from a wetland in the northern Chilean Altiplano. Phylogenetic analysis and rRNA gene sequence abundance estimates indicated that the host was related to Nostoc punctiforme PCC 73102. Epimicrobiota were inferred to include 18 epicyanobacterial genera or uncultured taxa, six epieukaryotic algal genera, and 66 anoxygenic bacterial genera, all having average genomic coverage ≥90X. The epicyanobacteria Geitlerinemia, Oscillatoria, Phormidium, and an uncultured taxon were detected only by 16S rRNA gene; Gloeobacter and Pseudanabaena were detected using 16S and 23S; and Phormididesmis, Neosynechococcus, Symphothece, Aphanizomenon, Nodularia, Spirulina, Nodosilinea, Synechococcus, Cyanobium, and Anabaena (the latter corroborated by microscopy), plus two uncultured cyanobacterial taxa (JSC12, O77) were detected only by 23S rRNA gene sequences. Three chlamydomonad and two heterotrophic stramenopiles genera were inferred from 18S; the streptophyte green alga Chaetosphaeridium globosum was detected by microscopy and 28S rRNA genes, but not 18S rRNA genes. Overall, >60% of epimicrobial taxa were detected by markers other than 16S rRNA genes. Some algal taxa observed microscopically were not detected from sequence data. Results indicate that multiple taxonomic markers derived from metagenomic sequence data and microscopy increase epimicrobiota detection.     

The population genomic basis of geographic differentiation in North American common ragweed (Ambrosia artemisiifolia L.)

Common ragweed (Ambrosia artemisiifolia L.) is an invasive, wind‐pollinated plant nearly ubiquitous in disturbed sites in its eastern North American native range and present across growing portions of Europe, Africa, Asia, and Australia. Phenotypic divergence between European and native‐range populations has been described as rapid evolution. However, a recent study demonstrated major human‐mediated shifts in ragweed genetic structure before introduction to Europe and suggested that native‐range genetic structure and local adaptation might fully explain accelerated growth and other invasive characteristics of introduced populations. Genomic differentiation that potentially influenced this structure has not yet been investigated, and it remains unclear whether substantial admixture during historical disturbance of the native range contributed to the development of invasiveness in introduced European ragweed populations. To investigate fine‐scale population genetic structure across the species' native range, we characterized diallelic SNP loci via a reduced‐representation genotyping‐by‐sequencing (GBS) approach. We corroborate phylogeographic domains previously discovered using traditional sequencing methods, while demonstrating increased power to resolve weak genetic structure in this highly admixed plant species. By identifying exome polymorphisms underlying genetic differentiation, we suggest that geographic differentiation of this important invasive species has occurred more often within pathways that regulate growth and response to defense and stress, which may be associated with survival in North America's diverse climatic regions.     

Phenological matching rather than genetic variation in host preference underlies geographical variation in host plants used by orange tip butterflies

An insect species that shows variation in host species association across its geographical range may do so either because of local adaptation in host plant preference of the insect or through environmentally or genetically induced differences in the plants, causing variation in host plant suitability between regions. In the present study, we experimentally investigate the host plant preference of Anthocharis cardamines (orange tip butterfly) in two populations from the UK and two from Sweden. Previous reports indicate that A. cardamines larvae are found on different host plant species in different regions of the UK, and some variation has been reported in Sweden. Host plant choice trials showed that females prefer to oviposit on plants in an earlier phenological stage, as well as on larger plants. When controlling for plant phenological stage and size, the host species had no statistically significant effect on the choice of the females. Moreover, there were no differences in host plant species preference among the four butterfly populations. Based on our experiment, the oviposition choice by A. cardamines mainly depends on the phenological stage and the size of the host plant. This finding supports the idea that the geographical patterns of host–plant association of A. cardamines in the UK and Sweden are consequences of the phenology and availability of the local hosts, rather than regional genetic differences in the host species preference of the butterfly.     

Correlated patterns of genetic diversity and differentiation across an avian family

Comparative studies of closely related taxa can provide insights into the evolutionary forces that shape genome evolution and the prevalence of convergent molecular evolution. We investigated patterns of genetic diversity and differentiation in stonechats (genus Saxicola), a widely distributed avian species complex with phenotypic variation in plumage, morphology and migratory behaviour, to ask whether similar genomic regions have become differentiated in independent, but closely related, taxa. We used whole‐genome pooled sequencing of 262 individuals from five taxa and found that levels of genetic diversity and divergence are strongly correlated among different stonechat taxa. We then asked whether these patterns remain correlated at deeper evolutionary scales and found that homologous genomic regions have become differentiated in stonechats and the closely related Ficedula flycatchers. Such correlation across a range of evolutionary divergence and among phylogenetically independent comparisons suggests that similar processes may be driving the differentiation of these independently evolving lineages, which in turn may be the result of intrinsic properties of particular genomic regions (e.g. areas of low recombination). Consequently, studies employing genome scans to search for areas important for reproductive isolation or adaptation should account for corresponding regions of differentiation, as these regions may not necessarily represent speciation islands or evidence of local adaptation.     

Community assembly of a euryhaline fish microbiome during salinity acclimation

Microbiomes play a critical role in promoting a range of host functions. Microbiome function, in turn, is dependent on its community composition. Yet, how microbiome taxa are assembled from their regional species pool remains unclear. Many possible drivers have been hypothesized, including deterministic processes of competition, stochastic processes of colonization and migration, and physiological ‘host‐effect’ habitat filters. The contribution of each to assembly in nascent or perturbed microbiomes is important for understanding host–microbe interactions and host health. In this study, we characterized the bacterial communities in a euryhaline fish and the surrounding tank water during salinity acclimation. To assess the relative influence of stochastic versus deterministic processes in fish microbiome assembly, we manipulated the bacterial species pool around each fish by changing the salinity of aquarium water. Our results show a complete and repeatable turnover of dominant bacterial taxa in the microbiomes from individuals of the same species after acclimation to the same salinity. We show that changes in fish microbiomes are not correlated with corresponding changes to abundant taxa in tank water communities and that the dominant taxa in fish microbiomes are rare in the aquatic surroundings, and vice versa. Our results suggest that bacterial taxa best able to compete within the unique host environment at a given salinity appropriate the most niche space, independent of their relative abundance in tank water communities. In this experiment, deterministic processes appear to drive fish microbiome assembly, with little evidence for stochastic colonization.     

Site‐specific responses of foliar fungal microbiomes to nutrient addition and herbivory at different spatial scales

The plant microbiome can affect host function in many ways and characterizing the ecological factors that shape endophytic (microbes living inside host plant tissues) community diversity is a key step in understanding the impacts of environmental change on these communities. Phylogenetic relatedness among members of a community offers a way of quantifying phylogenetic diversity of a community and can provide insight into the ecological factors that shape endophyte microbiomes. We examined the effects of experimental nutrient addition and herbivory exclusion on the phylogenetic diversity of foliar fungal endophyte communities of the grass species Andropogon gerardii at four sites in the Great Plains of the central USA. Using amplicon sequencing, we characterized the effects of fertilization and herbivory on fungal community phylogenetic diversity at spatial scales that spanned within‐host to between sites across the Great Plains. Despite increasing fungal diversity and richness, at larger spatial scales, fungal microbiomes were composed of taxa showing random phylogenetic associations. Phylogenetic diversity did not differ systematically when summed across increasing spatial scales from a few meters within plots to hundreds of kilometers among sites. We observed substantial shifts in composition across sites, demonstrating distinct but similarly diverse fungal communities were maintained within sites across the region. In contrast, at the scale of within leaves, fungal communities tended to be comprised of closely related taxa regardless of the environment, but there were no shifts in phylogenetic composition among communities. We also found that nutrient addition (fertilization) and herbivory have varying effects at different sites. These results suggest that the direction and magnitude of the outcomes of environmental modifications likely depend on the spatial scale considered, and can also be constrained by regional site differences in microbial diversity and composition.     

On Sea Turtle‐associated Craspedostauros (Bacillariophyta), with Description of Three Novel Species

The current study focuses on four species from the primarily marine diatom genus Craspedostauros that were observed growing attached to numerous sea turtles and sea turtle‐associated barnacles from Croatia and South Africa. Three of the examined taxa, C. danayanus sp. nov., C. legouvelloanus sp. nov., and C. macewanii sp. nov., are described based on morphological and, whenever possible, molecular characteristics. The new taxa exhibit characters not previously observed in other members of the genus, such as the presence of more than two rows of cribrate areolae on the girdle bands, shallow perforated septa, and a complete reduction of the stauros. The fourth species, C. alatus, itself recently described from museum sea turtle specimens, is reported for the first time from loggerhead sea turtles rescued in Europe. A 3‐gene phylogenetic analysis including DNA sequence data for three sea turtle‐associated Craspedostauros species and other marine and epizoic diatom taxa indicated that Craspedostauros is monophyletic and sister to Achnanthes. This study, being based on a large number of samples and animal specimens analyzed and using different preservation and processing methods, provides new insights into the ecology and biogeography of the genus and sheds light on the level of intimacy and permanency in the host–epibiont interaction within the epizoic Craspedostauros species.     

Concordance of bacterial communities of two tick species and blood of their shared rodent host

High‐throughput sequencing is revealing that most macro‐organisms house diverse microbial communities. Of particular interest are disease vectors whose microbiome could potentially affect pathogen transmission and vector competence. We investigated bacterial community composition and diversity of the ticks Dermacentor variabilis (n = 68) and Ixodes scapularis (n = 15) and blood of their shared rodent host, Peromyscus leucopus (n = 45) to quantify bacterial diversity and concordance. The 16S rRNA gene was amplified from genomic DNA from field‐collected tick and rodent blood samples, and 454 pyrosequencing was used to elucidate their bacterial communities. After quality control, over 300 000 sequences were obtained and classified into 118 operational taxonomic units (OTUs, clustered at 97% similarity). Analysis of rarefied communities revealed that the most abundant OTUs were tick species‐specific endosymbionts, Francisella and Rickettsia, and the commonly flea‐associated bacterium Bartonella in rodent blood. An Arsenophonus and additional Francisella endosymbiont were also present in D. variabilis samples. Rickettsia was found in both tick species but not in rodent blood, suggesting that it is not transmitted during feeding. Bartonella was present in larvae and nymphs of both tick species, even those scored as unengorged. Relatively, few OTUs (e.g. Bartonella, Lactobacillus) were found in all sample types. Overall, bacterial communities from each sample type were significantly different and highly structured, independent of their dominant OTUs. Our results point to complex microbial assemblages inhabiting ticks and host blood including infectious agents, tick‐specific endosymbionts and environmental bacteria that could potentially affect arthropod‐vectored disease dynamics.     

Next‐generation DNA barcoding: using next‐generation sequencing to enhance and accelerate DNA barcode capture from single specimens

DNA barcoding is an efficient method to identify specimens and to detect undescribed/cryptic species. Sanger sequencing of individual specimens is the standard approach in generating large‐scale DNA barcode libraries and identifying unknowns. However, the Sanger sequencing technology is, in some respects, inferior to next‐generation sequencers, which are capable of producing millions of sequence reads simultaneously. Additionally, direct Sanger sequencing of DNA barcode amplicons, as practiced in most DNA barcoding procedures, is hampered by the need for relatively high‐target amplicon yield, coamplification of nuclear mitochondrial pseudogenes, confusion with sequences from intracellular endosymbiotic bacteria (e.g. Wolbachia) and instances of intraindividual variability (i.e. heteroplasmy). Any of these situations can lead to failed Sanger sequencing attempts or ambiguity of the generated DNA barcodes. Here, we demonstrate the potential application of next‐generation sequencing platforms for parallel acquisition of DNA barcode sequences from hundreds of specimens simultaneously. To facilitate retrieval of sequences obtained from individual specimens, we tag individual specimens during PCR amplification using unique 10‐mer oligonucleotides attached to DNA barcoding PCR primers. We employ 454 pyrosequencing to recover full‐length DNA barcodes of 190 specimens using 12.5% capacity of a 454 sequencing run (i.e. two lanes of a 16 lane run). We obtained an average of 143 sequence reads for each individual specimen. The sequences produced are full‐length DNA barcodes for all but one of the included specimens. In a subset of samples, we also detected Wolbachia, nontarget species, and heteroplasmic sequences. Next‐generation sequencing is of great value because of its protocol simplicity, greatly reduced cost per barcode read, faster throughout and added information content.