Unraveling hierarchical genetic structure in a marine metapopulation: A comparison of three high‐throughput genotyping approaches

Marine metapopulations often exhibit subtle population structure that can be difficult to detect. Given recent advances in high‐throughput sequencing, an emerging question is whether various genotyping approaches, in concert with improved sampling designs, will substantially improve our understanding of genetic structure in the sea. To address this question, we explored hierarchical patterns of structure in the coral reef fish Elacatinus lori using a high‐resolution approach with respect to both genetic and geographic sampling. Previously, we identified three putative E. lori populations within Belize using traditional genetic markers and sparse geographic sampling: barrier reef and Turneffe Atoll; Glover's Atoll; and Lighthouse Atoll. Here, we systematically sampled individuals at ~10 km intervals throughout these reefs (1,129 individuals from 35 sites) and sequenced all individuals at three sets of markers: 2,418 SNPs; 89 microsatellites; and 57 nonrepetitive nuclear loci. At broad spatial scales, the markers were consistent with each other and with previous findings. At finer spatial scales, there was new evidence of genetic substructure, but our three marker sets differed slightly in their ability to detect these patterns. Specifically, we found subtle structure between the barrier reef and Turneffe Atoll, with SNPs resolving this pattern most effectively. We also documented isolation by distance within the barrier reef. Sensitivity analyses revealed that the number of loci (and alleles) had a strong effect on the detection of structure for all three marker sets, particularly at small spatial scales. Taken together, these results illustrate empirically that high‐throughput genotyping data can elucidate subtle genetic structure at previously‐undetected scales in a dispersive marine fish.     

Modified low‐salt CTAB extraction of high‐quality DNA from contaminant‐rich tissues

The increasing use of high‐throughput sequencing platforms has made the isolation of pure, high molecular weight DNA a primary concern for studies of a diverse range of organisms. Purification of DNA remains a significant challenge in many tissue and sample types due to various organic and inorganic molecules that coprecipitate with nucleic acids. Molluscs, for example, contain high concentrations of polysaccharides which often coprecipitate with DNA and can inhibit downstream enzymatic reactions. We modified a low‐salt CTAB (MoLSC) extraction protocol to accommodate contaminant‐rich animal tissues and compared this method to a standard CTAB extraction protocol and two commercially available animal tissue DNA extraction kits using oyster adductor muscle. Comparisons of purity and molecular integrity showed that our in‐house protocol yielded genomic DNA generally free of contaminants and shearing, whereas the traditional CTAB method and some of the commercial kits yielded DNA unsuitable for some applications of massively parallel sequencing. Our open‐source MoLSC protocol provides a cost‐effective, scalable, alternative DNA extraction method that can be easily optimized and adapted for sequencing applications in other contaminant‐rich samples.     

A roadmap for high‐throughput sequencing studies of wild animal populations using noninvasive samples and hybridization capture

Large‐scale genomic studies of wild animal populations are often limited by access to high‐quality DNA. Although noninvasive samples, such as faeces, can be readily collected, DNA from the sample producers is usually present in low quantities, fragmented, and contaminated by microorganism and dietary DNAs. Hybridization capture can help to overcome these impediments by increasing the proportion of subject DNA prior to high‐throughput sequencing. Here we evaluate a key design variable for hybridization capture, the number of rounds of capture, by testing whether one or two rounds are most appropriate, given varying sample quality (as measured by the ratios of subject to total DNA). We used a set of 1,780 quality‐assessed wild chimpanzee (Pan troglodytes schweinfurthii) faecal samples and chose 110 samples of varying quality for exome capture and sequencing. We used multiple regression to assess the effects of the ratio of subject to total DNA (sample quality), rounds of capture and sequencing effort on the number of unique exome reads sequenced. We not only show that one round of capture is preferable when the proportion of subject DNA in a sample is above ~2%–3%, but also explore various types of bias introduced by capture, and develop a model that predicts the sequencing effort necessary for a desired data yield from samples of a given quality. Thus, our results provide a useful guide and pave a methodological way forward for researchers wishing to plan similar hybridization capture studies.     

Comparing the performance of three ancient DNA extraction methods for high‐throughput sequencing

The DNA molecules that can be extracted from archaeological and palaeontological remains are often degraded and massively contaminated with environmental microbial material. This reduces the efficacy of shotgun approaches for sequencing ancient genomes, despite the decreasing sequencing costs of high‐throughput sequencing (HTS). Improving the recovery of endogenous molecules from the DNA extraction and purification steps could, thus, help advance the characterization of ancient genomes. Here, we apply the three most commonly used DNA extraction methods to five ancient bone samples spanning a ~30 thousand year temporal range and originating from a diversity of environments, from South America to Alaska. We show that methods based on the purification of DNA fragments using silica columns are more advantageous than in solution methods and increase not only the total amount of DNA molecules retrieved but also the relative importance of endogenous DNA fragments and their molecular diversity. Therefore, these methods provide a cost‐effective solution for downstream applications, including DNA sequencing on HTS platforms.     

Comparative phylogeography of two co‐distributed but ecologically distinct rainbowfishes of far‐northern Australia

Aim

To test the influence of historical and contemporary environment in shaping the genetic diversity of freshwater fauna we contrast genetic structure in two co‐distributed, but ecologically distinct, rainbowfish; a habitat generalist (Melanotaenia splendida) and a habitat specialist (M. trifasciata).

Location

Fishes were sampled from far northern Australia (Queensland and Northern Territory).

Methods

We used sequence data from one mitochondrial gene and one nuclear gene to investigate patterns of genetic diversity in M. splendida and M. trifasciata to determine how differences in habitat preference and historical changes in drainage boundaries affected patterns of connectivity.

Results

Melanotaenia splendida showed high levels of genetic diversity and little population structure across its range. In contrast, M. trifasciata showed high levels of population structure. Whereas phylogeographic patterns differed, both species showed a strong relationship between geographical distance and genetic differentiation between populations. Melanotaenia splendida showed a shallower relationship with geographical distance, and genetic differentiation was best explained by stream length and a lower scaled ocean distance (11.98 times coast length). For M. trifasciata, genetic differentiation was best explained by overwater distance between catchments and ocean distance scaled at 1.16 × 10⁶ times coast length.

Main conclusions

Connectivity of freshwater populations inhabiting regions periodically interconnected during glacial periods appears to have been affected by ecological differences between species. Species‐specific differences are epitomized here by the contrast between co‐distributed congeners with different habitat requirements: for the habitat generalist, M. splendida, there was evidence for greater historical genetic connectivity with oceans as a weaker barrier to gene exchange in contrast with the habitat specialist, M. trifasciata.     

Testing models of speciation from genome sequences: divergence and asymmetric admixture in Island South‐East Asian Sus species during the Plio‐Pleistocene climatic fluctuations

In many temperate regions, ice ages promoted range contractions into refugia resulting in divergence (and potentially speciation), while warmer periods led to range expansions and hybridization. However, the impact these climatic oscillations had in many parts of the tropics remains elusive. Here, we investigate this issue using genome sequences of three pig (Sus) species, two of which are found on islands of the Sunda‐shelf shallow seas in Island South‐East Asia (ISEA). A previous study revealed signatures of interspecific admixture between these Sus species (Genome biology, 14 , 2013, R107). However, the timing, directionality and extent of this admixture remain unknown. Here, we use a likelihood‐based model comparison to more finely resolve this admixture history and test whether it was mediated by humans or occurred naturally. Our analyses suggest that interspecific admixture between Sunda‐shelf species was most likely asymmetric and occurred long before the arrival of humans in the region. More precisely, we show that these species diverged during the late Pliocene but around 23% of their genomes have been affected by admixture during the later Pleistocene climatic transition. In addition, we show that our method provides a significant improvement over D‐statistics which are uninformative about the direction of admixture.     

jackalope: A swift,versatile phylogenomic and high‐throughput sequencing simulator

High‐throughput sequencing (HTS) is central to the study of population genomics and has an increasingly important role in constructing phylogenies. Choices in research design for sequencing projects can include a wide range of factors, such as sequencing platform, depth of coverage and bioinformatic tools. Simulating HTS data better informs these decisions, as users can validate software by comparing output to the known simulation parameters. However, current standalone HTS simulators cannot generate variant haplotypes under even somewhat complex evolutionary scenarios, such as recombination or demographic change. This greatly reduces their usefulness for fields such as population genomics and phylogenomics. Here I present the R package jackalope that simply and efficiently simulates (i) sets of variant haplotypes from a reference genome and (ii) reads from both Illumina and Pacific Biosciences platforms. Haplotypes can be simulated using phylogenies, gene trees, coalescent‐simulation output, population‐genomic summary statistics, and Variant Call Format (VCF) files. jackalope can simulate single, paired‐end or mate‐pair Illumina reads, as well as reads from Pacific Biosciences. These simulations include sequencing errors, mapping qualities, multiplexing and optical/PCR duplicates. It can read reference genomes from fasta files and can simulate new ones, and all outputs can be written to standard file formats. jackalope is available for Mac, Windows and Linux systems.     

High‐throughput SNPs for all: genotyping‐in‐thousands

Understanding the genetic structure of species is essential for conservation. It is only with this information that managers, academics, user groups and land‐use planners can understand the spatial scale of migration and local adaptation, source‐sink dynamics and effective population size. Such information is essential for a multitude of applications including delineating management units, balancing management priorities, discovering cryptic species and implementing captive breeding programmes. Species can range from locally adapted by hundreds of metres (Pavey et al. 2010 ) to complete species panmixia (Côté et al. 2013 ). Even more remarkable is that this essential information can be obtained without fully sequenced or annotated genomes, but from mere (putatively) nonfunctional variants. First with allozymes, then microsatellites and now SNPs, this neutral genetic variation carries a wealth of information about migration and drift. For many of us, it may be somewhat difficult to remember our understanding of species conservation before the widespread usage of these useful tools. However most species on earth have yet to give us that ‘peek under the curtain’. With the current diversity on earth estimated to be nearly 9 million species (Mora et al. 2011 ), we have a long way to go for a comprehensive meta‐phylogeographic understanding. A method presented in this issue by Campbell and colleagues (Campbell et al. 2015 ) is a tool that will accelerate the pace in this area. Genotyping‐in‐thousands (GT‐seq) leverages recent advancements in sequencing technology to save many hours and dollars over previous methods to generate this important neutral genetic information.     

A likelihood-based comparison of population histories in a parasitoid guild

Little is known about the stability of trophic relationships in complex natural communities over evolutionary timescales. Here, we use sequence data from 18 nuclear loci to reconstruct and compare the intraspecific histories of major Pleistocene refugial populations in the Middle East, the Balkans and Iberia in a guild of four Chalcid parasitoids (Cecidostiba fungosa, Cecidostiba semifascia, Hobbya stenonota and Mesopolobus amaenus) all attacking Cynipid oak galls. We develop a likelihood method to numerically estimate models of divergence between three populations from multilocus data. We investigate the power of this framework on simulated data, and—using triplet alignments of intronic loci—quantify the support for all possible divergence relationships between refugial populations in the four parasitoids. Although an East to West order of population divergence has highest support in all but one species, we cannot rule out alternative population tree topologies. Comparing the estimated times of population splits between species, we find that one species, M. amaenus, has a significantly older history than the rest of the guild and must have arrived in central Europe at least one glacial cycle prior to other guild members. This suggests that although all four species may share a common origin in the East, they expanded westwards into Europe at different times.     

Sequence capture using AFLP‐generated baits: A cost‐effective method for high‐throughput phylogenetic and phylogeographic analysis

Target sequence capture is an efficient technique to enrich specific genomic regions for high‐throughput sequencing in ecological and evolutionary studies. In recent years, many sequence capture approaches have been proposed, but most of them rely on commercial synthetic baits which make the experiment expensive. Here, we present a novel sequence capture approach called AFLP‐based genome sequence capture (AFLP Capture). This method uses the AFLP (amplified fragment length polymorphism) technique to generate homemade capture baits without the need for prior genome information, thus is applicable to any organisms. In this approach, biotinylated AFLP fragments representing a random fraction of the genome are used as baits to capture the homologous fragments from genomic shotgun sequencing libraries. In a trial study, by using AFLP Capture, we successfully obtained 511 orthologous loci (>700,000 bp in total length) from 11 Odorrana species and more than 100,000 single nucleotide polymorphisms (SNPs) in four analyzed individuals of an Odorrana species. This result shows that our method can be used to address questions of various evolutionary depths (from interspecies level to intraspecies level). We also discuss the flexibility in bait preparation and how the sequencing data are analyzed. In summary, AFLP Capture is a rapid and flexible tool and can significantly reduce the experimental cost for phylogenetic studies that require analyzing genome‐scale data (hundreds or thousands of loci).     

Assessing the utility of whole genome amplified DNA for next‐generation molecular ecology

DNA quantity can be a hindrance in ecological and evolutionary research programmes due to a range of factors including endangered status of target organisms, available tissue type, and the impact of field conditions on preservation methods. A potential solution to low‐quantity DNA lies in whole genome amplification (WGA) techniques that can substantially increase DNA yield. To date, few studies have rigorously examined sequence bias that might result from WGA and next‐generation sequencing of nonmodel taxa. To address this knowledge deficit, we use multiple displacement amplification (MDA) and double‐digest RAD sequencing on the grey mouse lemur (Microcebus murinus) to quantify bias in genome coverage and SNP calls when compared to raw genomic DNA (gDNA). We focus our efforts in providing baseline estimates of potential bias by following manufacturer's recommendations for starting DNA quantities (>100 ng). Our results are strongly suggestive that MDA enrichment does not introduce systematic bias to genome characterization. SNP calling between samples when genotyping both de‐novo and with a reference genome are highly congruent (>98%) when specifying a minimum threshold of 20X stack depth to call genotypes. Relative genome coverage is also similar between MDA and gDNA, and allelic dropout is not observed. SNP concordance varies based on coverage threshold, with 95% concordance reached at ~12X coverage genotyping de‐novo and ~7X coverage genotyping with the reference genome. These results suggest that MDA may be a suitable solution for next‐generation molecular ecological studies when DNA quantity would otherwise be a limiting factor.     

multi‐dice: r package for comparative population genomic inference under hierarchical co‐demographic models of independent single‐population size changes

Population genetic data from multiple taxa can address comparative phylogeographic questions about community‐scale response to environmental shifts, and a useful strategy to this end is to employ hierarchical co‐demographic models that directly test multi‐taxa hypotheses within a single, unified analysis. This approach has been applied to classical phylogeographic data sets such as mitochondrial barcodes as well as reduced‐genome polymorphism data sets that can yield 10,000s of SNPs, produced by emergent technologies such as RAD‐seq and GBS. A strategy for the latter had been accomplished by adapting the site frequency spectrum to a novel summarization of population genomic data across multiple taxa called the aggregate site frequency spectrum (aSFS), which potentially can be deployed under various inferential frameworks including approximate Bayesian computation, random forest and composite likelihood optimization. Here, we introduce the r package multi‐dice , a wrapper program that exploits existing simulation software for flexible execution of hierarchical model‐based inference using the aSFS, which is derived from reduced genome data, as well as mitochondrial data. We validate several novel software features such as applying alternative inferential frameworks, enforcing a minimal threshold of time surrounding co‐demographic pulses and specifying flexible hyperprior distributions. In sum, multi‐dice provides comparative analysis within the familiar R environment while allowing a high degree of user customization, and will thus serve as a tool for comparative phylogeography and population genomics.     

Combined analyses of kinship and FST suggest potential drivers of chaotic genetic patchiness in high gene‐flow populations

We combine kinship estimates with traditional F‐statistics to explain contemporary drivers of population genetic differentiation despite high gene flow. We investigate range‐wide population genetic structure of the California spiny (or red rock) lobster (Panulirus interruptus) and find slight, but significant global population differentiation in mtDNA (Φ_ST = 0.006, P = 0.001; D_{est_Chao} = 0.025) and seven nuclear microsatellites (F_ST = 0.004, P < 0.001; D_{est_Chao} = 0.03), despite the species’ 240‐ to 330‐day pelagic larval duration. Significant population structure does not correlate with distance between sampling locations, and pairwise F_ST between adjacent sites often exceeds that among geographically distant locations. This result would typically be interpreted as unexplainable, chaotic genetic patchiness. However, kinship levels differ significantly among sites (pseudo‐F_16,988 = 1.39, P = 0.001), and ten of 17 sample sites have significantly greater numbers of kin than expected by chance (P < 0.05). Moreover, a higher proportion of kin within sites strongly correlates with greater genetic differentiation among sites (D_{est_Chao}, R² = 0.66, P < 0.005). Sites with elevated mean kinship were geographically proximate to regions of high upwelling intensity (R² = 0.41, P = 0.0009). These results indicate that P. interruptus does not maintain a single homogenous population, despite extreme dispersal potential. Instead, these lobsters appear to either have substantial localized recruitment or maintain planktonic larval cohesiveness whereby siblings more likely settle together than disperse across sites. More broadly, our results contribute to a growing number of studies showing that low F_ST and high family structure across populations can coexist, illuminating the foundations of cryptic genetic patterns and the nature of marine dispersal.     

A high‐quality chromosome‐level genome assembly of a generalist herbivore,Trichoplusia ni

The cabbage looper, Trichoplusia ni, is a globally distributed highly polyphagous herbivore and an important agricultural pest. T. ni has evolved resistance to various chemical insecticides, and is one of the only two insect species that have evolved resistance to the biopesticide Bacillus thuringiensis (Bt) in agricultural systems and has been selected for resistance to baculovirus infections. We report a 333‐Mb high‐quality T. ni genome assembly, which has N50 lengths of scaffolds and contigs of 4.6 Mb and 140 Kb, respectively, and contains 14,384 protein‐coding genes. High‐density genetic maps were constructed to anchor 305 Mb (91.7%) of the assembly to 31 chromosomes. Comparative genomic analysis of T. ni with Bombyx mori showed enrichment of tandemly duplicated genes in T. ni in families involved in detoxification and digestion, consistent with the broad host range of T. ni. High levels of genome synteny were found between T. ni and other sequenced lepidopterans. However, genome synteny analysis of T. ni and the T. ni derived cell line High Five (Hi5) indicated extensive genome rearrangements in the cell line. These results provided the first genomic evidence revealing the high instability of chromosomes in lepidopteran cell lines known from karyotypic observations. The high‐quality T. ni genome sequence provides a valuable resource for research in a broad range of areas including fundamental insect biology, insect‐plant interactions and co‐evolution, mechanisms and evolution of insect resistance to chemical and biological pesticides, and technology development for insect pest management.     

A DNA mini‐barcode for marine macrophytes

Studies focusing on marine macrophyte metabarcoding from environmental samples are scarce, due to the lack of a universal barcode for these taxa, and to their poor representation in DNA databases. Here, we searched for a short barcode able to identify marine macrophytes from tissue samples; then, we created a DNA reference library which was used to identify macrophytes in eDNA from coastal sediments. Barcoding of seagrasses, mangroves and marine macroalgae (Chlorophyta, Rhodophyta and Phaeophyceae) was tested using 18 primer pairs from six barcoding genes: the plant barcodes rbcL, matK and trnL, plus the genes ITS2, COI and 18S. The 18S gene showed the highest universality among marine macrophytes, amplifying 95%–100% of samples; amplification performance of the other barcodes was limited. Taxonomy was assigned using a phylogeny‐based approach to create an 18S DNA reference library. Macrophyte tissue sequences were accurately identified within their phyla (88%), order (76%), genus (71%) and species (23%). Nevertheless, out of 86 macrophytes tested, only 48% and 15% had a reference sequence at genus and at species level, respectively. Identification at these levels can be improved by more inclusive reference libraries. Using the 18S mini‐barcode and the reference library, we recovered eDNA from 21 marine macrophytes in sediments, demonstrating the barcode's ability to trace primary producers that contribute to blue carbon. We expect this barcode to also be useful for other ecological questions, such as tracing macro primary producers in marine food webs.     

In defence of model‐based inference in phylogeography

Recent papers have promoted the view that model‐based methods in general, and those based on Approximate Bayesian Computation (ABC) in particular, are flawed in a number of ways, and are therefore inappropriate for the analysis of phylogeographic data. These papers further argue that Nested Clade Phylogeographic Analysis (NCPA) offers the best approach in statistical phylogeography. In order to remove the confusion and misconceptions introduced by these papers, we justify and explain the reasoning behind model‐based inference. We argue that ABC is a statistically valid approach, alongside other computational statistical techniques that have been successfully used to infer parameters and compare models in population genetics. We also examine the NCPA method and highlight numerous deficiencies, either when used with single or multiple loci. We further show that the ages of clades are carelessly used to infer ages of demographic events, that these ages are estimated under a simple model of panmixia and population stationarity but are then used under different and unspecified models to test hypotheses, a usage the invalidates these testing procedures. We conclude by encouraging researchers to study and use model‐based inference in population genetics.     

Amplicon sequencing of 42 nuclear loci supports directional gene flow between South Pacific populations of a hydrothermal vent limpet

In the past few decades, population genetics and phylogeographic studies have improved our knowledge of connectivity and population demography in marine environments. Studies of deep‐sea hydrothermal vent populations have identified barriers to gene flow, hybrid zones, and demographic events, such as historical population expansions and contractions. These deep‐sea studies, however, used few loci, which limit the amount of information they provided for coalescent analysis and thus our ability to confidently test complex population dynamics scenarios. In this study, we investigated population structure, demographic history, and gene flow directionality among four Western Pacific hydrothermal vent populations of the vent limpet Lepetodrilus aff. schrolli. These vent sites are located in the Manus and Lau back‐arc basins, currently of great interest for deep‐sea mineral extraction. A total of 42 loci were sequenced from each individual using high‐throughput amplicon sequencing. Amplicon sequences were analyzed using both genetic variant clustering methods and evolutionary coalescent approaches. Like most previously investigated vent species in the South Pacific, L. aff. schrolli showed no genetic structure within basins but significant differentiation between basins. We inferred significant directional gene flow from Manus Basin to Lau Basin, with low to no gene flow in the opposite direction. This study is one of the very few marine population studies using >10 loci for coalescent analysis and serves as a guide for future marine population studies.     

Spatial and temporal population genetic structure of four northeastern Pacific littorinid gastropods: the effect of mode of larval development on variation at one mitochondrial and two nuclear DNA markers

We investigated the effect of development mode on the spatial and temporal population genetic structure of four littorinid gastropod species. Snails were collected from the same three sites on the west coast of Vancouver Island, Canada in 1997 and again in 2007. DNA sequences were obtained for one mitochondrial gene, cytochrome b ( Cyt b ), and for up to two nuclear genes, heat shock cognate 70 ( HSC70 ) and aminopeptidase N intron ( APN54 ). We found that the mean level of genetic diversity and long-term effective population sizes ( N _e) were significantly greater for two species, Littorina scutulata and L. plena , that had a planktotrophic larval stage than for two species, Littorina sitkana and L. subrotundata , that laid benthic egg masses which hatched directly into crawl-away juveniles. Predictably, two poorly dispersing species, L. sitkana and L. subrotundata , showed significant spatial genetic structure at an 11- to 65-km geographical scale that was not observed in the two planktotrophic species. Conversely, the two planktotrophic species had more temporal genetic structure over a 10-year interval than did the two direct-developing species and showed highly significant temporal structure for spatially pooled samples. The greater temporal genetic variation of the two planktotrophic species may have been caused by their high fecundity, high larval dispersal, and low but spatially correlated early survivorship. The sweepstakes-like reproductive success of the planktotrophic species could allow a few related females to populate hundreds of kilometres of coastline and may explain their substantially larger temporal genetic variance but lower spatial genetic variance relative to the direct-developing species.     

An inexpensive and high‐throughput procedure to extract and purify total genomic DNA for population studies

We describe here a procedure for the purification of high molecular weight genomic DNA that combines the economies of ‘do‐it‐yourself’, single‐tube protocols with the sample throughput and DNA quality of microplate‐based DNA extraction and purification kits from commercial suppliers. The procedure allows the preparation of genomic DNA of a quality suitable for polymerase chain reaction‐based studies of large populations at around one‐tenth of the cost of commercially available kits. Furthermore, 96 samples can be purified from crude tissue digests in around 30 min and are produced in microtitre plate format to allow efficient downstream processing of samples.     

Performance comparison of genetic markers for high‐throughput sequencing‐based biodiversity assessment in complex communities

Metabarcode surveys of DNA extracted from environmental samples are increasingly popular for biodiversity assessment in natural communities. Such surveys rely heavily on robust genetic markers. Therefore, analysis of PCR efficiency and subsequent biodiversity estimation for different types of genetic markers and their corresponding primers is important. Here, we test the PCR efficiency and biodiversity recovery potential of three commonly used genetic markers – nuclear small subunit ribosomal DNA (18S), mitochondrial cytochrome c oxidase subunit I (COI) and 16S ribosomal RNA (mt16S) – using 454 pyrosequencing of a zooplankton community collected from Hamilton Harbour, Ontario. We found that biodiversity detection power and PCR efficiency varied widely among these markers. All tested primers for COI failed to provide high‐quality PCR products for pyrosequencing, but newly designed primers for 18S and 16S passed all tests. Furthermore, multiple analyses based on large‐scale pyrosequencing (i.e. 1/2 PicoTiter plate for each marker) showed that primers for 18S recover more (38 orders) groups than 16S (10 orders) across all taxa, and four vs. two orders and nine vs. six families for Crustacea. Our results showed that 18S, using newly designed primers, is an efficient and powerful tool for profiling biodiversity in largely unexplored communities, especially when amplification difficulties exist for mitochondrial markers such as COI. Universal primers for higher resolution markers such as COI are still needed to address the possible low resolution of 18S for species‐level identification.