Divergence Threshold Research Articles

Pitfalls of establishing DNA barcoding systems in protists: the cryptophyceae as a test case.

A DNA barcode is a preferrably short and highly variable region of DNA supposed to facilitate a rapid identification of species. In many protistan lineages, a lack of species-specific morphological characters hampers an identification of species by light or electron microscopy, and difficulties to perform mating experiments in laboratory cultures also do not allow for an identification of biological species. Thus, testing candidate barcode markers as well as establishment of accurately working species identification systems are more challenging than in multicellular organisms. In cryptic species complexes the performance of a potential barcode marker can not be monitored using morphological characters as a feedback, but an inappropriate choice of DNA region may result in artifactual species trees for several reasons. Therefore a priori knowledge of the systematics of a group is required. In addition to identification of known species, methods for an automatic delimitation of species with DNA barcodes have been proposed. The Cryptophyceae provide a mixture of systematically well characterized as well as badly characterized groups and are used in this study to test the suitability of some of the methods for protists. As species identification method the performance of blast in searches against badly to well-sampled reference databases has been tested with COI-5P and 5′-partial LSU rDNA (domains A to D of the nuclear LSU rRNA gene). In addition the performance of two different methods for automatic species delimitation, fixed thresholds of genetic divergence and the general mixed Yule-coalescent model (GMYC), have been examined. The study demonstrates some pitfalls of barcoding methods that have to be taken care of. Also a best-practice approach towards establishing a DNA barcode system in protists is proposed.

Time-Dependent Infectivity and Flexible Latent and Infectious Periods in Compartmental Models of Plant Disease

Compartmental models have become the dominant theoretical paradigm in mechanistic modeling of plant disease and offer well-known advantages in terms of analytic tractability, ease of simulation, and extensibility. However, underlying assumptions of constant rates of infection and of exponentially distributed latent and infectious periods are difficult to justify. Although alternative approaches, including van der Plank's seminal discrete time model and models based on the integro-differential formulation of Kermack and McKendrick's model, have been suggested for plant disease and relax these unrealistic assumptions, they are challenging to implement and to analyze. Here, we propose an extension to the susceptible, exposed, infected, and removed (SEIR) compartmental model, splitting the latent and infection compartments and thereby allowing time-varying infection rates and more realistic distributions of latent and infectious periods to be represented. Although the model is, in fact, more general, we specifically target plant disease by demonstrating how it can represent both the van der Plank model and the most commonly used variant of the Kermack and McKendrick (K & M) model (in which the infectivity response is delay Gamma distributed). We show how our reformulation retains the numeric and analytic tractability of SEIR models, and how it can be used to replicate earlier analyses of the van der Plank and K & M models. Our reformulation has the advantage of using elementary mathematical techniques, making implementation easier for the nonspecialist. We show a practical implication of these results for disease control. By taking advantage of the easy extensibility characteristic of compartmental models, we also investigate the effects of including additional biological realism. As an example, we show how the more realistic infection responses we consider interact with host demography and lead to divergent invasion thresholds when compared with the "standard" SEIR model. An ever-increasing number of analyses purportedly extract more biologically realistic invasion thresholds by adding additional biological detail to the SEIR model framework; we contend that our results demonstrate that extending a model that has such a simplistic representation of the infection dynamics may not, in fact, lead to more accurate results. Therefore, we suggest that modelers should carefully consider the underlying assumptions of the simplest compartmental models in their future work.

At least two indigenous species of the Bemisia tabaci complex are present in Brazil

AbstractBemisia tabaci is one of the most important global agricultural insect pests, being a vector of emerging plant viruses such as begomoviruses and criniviruses that cause serious problems in many countries. Although knowledge of the genetic diversity of B. tabaci populations is important for controlling this pest and understanding viral epidemics, limited information is available on this pest in Brazil. A survey was conducted in different locations of São Paulo and Mato Grosso states, and the phylogenetic relationships of B. tabaci individuals from 43 populations sampled from different hosts were analysed based on partial mitochondrial cytochrome oxidase 1 gene (mtCOI) sequences. According to the recently proposed classification of the B. tabaci complex, which employs the 3.5% mtCOI sequence divergence threshold for species demarcation, most of the specimens collected were found to belong to the Middle East‐Asia Minor 1 species, which includes the invasive populations of the commonly known B biotype, within the Africa/Middle East/Asia Minor high‐level group. Three specimens collected from Solanun gilo and Ipomoea sp. were grouped together and could be classified in the New World species that includes the commonly known A biotype. However, six specimens collected from Euphorbia heterophylla, Xanthium cavanillesii and Glycine maxima could not be classified into any of the 28 previously proposed species, although according to the 11% mtCOI sequence divergence threshold, they belong to the New World high‐level group. These specimens were classified into a new recently proposed species named New World 2 that includes populations from Argentina. Middle East‐Asia Minor 1, New World and New World 2 were differentiated by RFLP analysis of the mtCOI gene using TaqI enzyme. Taq I analysis in silico also differentiates these from Mediterranean species, thus making this method a convenient tool to determine population dynamics, especially critical for monitoring the presence of this exotic pest in Brazil.

Spider: An R package for the analysis of species identity and evolution, with particular reference to DNA barcoding

Spider: SPecies IDentity and Evolution in R is a new R package implementing a number of useful analyses for DNA barcoding studies and associated research into species delimitation and speciation. Included are functions essential for generating important summary statistics from DNA barcode data, assessing specimen identification efficacy, and for testing and optimizing divergence threshold limits. In terms of investigating evolutionary and taxonomic questions, techniques for assessing diagnostic nucleotides and probability of reciprocal monophyly are also provided. Additionally, a sliding window function offers opportunities to analyse information across a gene, essential for marker design in degraded DNA studies. Spider capitalizes on R's extensible ethos and offers an integrated platform ideal for the analysis of both nucleotide and morphological data. The program can be obtained from the comprehensive R archive network (CRAN, http://cran.r-project.org) and from the R-Forge package development site (http://spider.r-forge.r-project.org/).

Beam Properties of Visible Proton-Implanted Photonic Crystal VCSELs

We investigate the optical properties of proton-implanted photonic crystal (PhC) vertical-cavity surface-emitting lasers (VCSELs) emitting in the visible spectrum. The fabricated lasers have a threshold current of 1.3 mA and single-mode output power greater than 1 mW at room temperature. The incorporation of a PhC into the top facet of a proton-implanted VCSEL results in a stable single-fundamental-mode operation with a side-mode suppression ratio larger than 30 dB and a constant beam divergence independent of injection current level or ambient temperature. Using an index-step optical fiber model, we compare the effects of different hole etching depths to variations in output beam divergence. By varying the design and etching depth of the hole pattern, the lasers can either be optimized for low beam divergence or low threshold currents. The controllable refractive index guidance effect from the PhC allows for precise engineering of the optical properties of these visible VCSELs for consumer and imaging applications.

Shape and sound reveal genetic cohesion not speciation in the New Zealand orthopteran, Hemiandrus pallitarsis, despite high mitochondrial DNA divergence

Levels of neutral mitochondrial (mt)DNA sequence diversity play a prominent role in alpha taxonomy, with divergence thresholds of approximately 3% widely being accepted as indicative of species differences. However, a number of studies of New Zealand invertebrates conclude that intraspecific mtDNA divergences are commonly much higher. We investigated the diversity and distribution of mitochondrial cytochrome oxidase I DNA sequences from populations of the New Zealand ground weta Hemiandrus pallitarsis with respect to variation among ten morphological characters and analysis of acoustic signalling associated with mate recognition. We looked for correlation between neutral mtDNA sequence variation and potential indicators of species differences. Despite high genetic distances (> 8.0% uncorrected) among mtDNA haplotypes in this species, morphological and male precopulation signals refute a hypothesis that multiple species exist. Other possible explanations for high genetic diversity, including accidental sampling of nuclear paralogues and elevated substitution rate, are not supported, whereas there is evidence for stable population size through time. We conclude that generalizations about genetic diversity and species status are overly simplistic. In some circumstances, notably southern hemisphere invertebrate taxa, species cohesion appears to have been retained throughout the Pleistocene, indicating the persistence of a relatively high population size. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105, 169‐186. ADDITIONAL KEYWORDS: DNA barcoding ‐ genetic diversity ‐ insect signalling ‐ phylogeography ‐ Pliocene ‐ population size ‐ speciation.

Species diversity and phylogeographical affinities of the Branchiopoda (Crustacea) of Churchill, Manitoba, Canada.

The region of Churchill, Manitoba, contains a wide variety of habitats representative of both the boreal forest and arctic tundra and has been used as a model site for biodiversity studies for nearly seven decades within Canada. Much previous work has been done in Churchill to study the Daphnia pulex species complex in particular, but no study has completed a wide-scale survey on the crustacean species that inhabit Churchill's aquatic ecosystems using molecular markers. We have employed DNA barcoding to study the diversity of the Branchiopoda (Crustacea) in a wide variety of freshwater habitats and to determine the likely origins of the Churchill fauna following the last glaciation. The standard animal barcode marker (COI) was sequenced for 327 specimens, and a 3% divergence threshold was used to delineate potential species. We found 42 provisional and valid branchiopod species from this survey alone, including several cryptic lineages, in comparison with the 25 previously recorded from previous ecological works. Using published sequence data, we explored the phylogeographic affinities of Churchill's branchiopods, finding that the Churchill fauna apparently originated from all directions from multiple glacial refugia (including southern, Beringian, and high arctic regions). Overall, these microcrustaceans are very diverse in Churchill and contain multiple species complexes. The present study introduces among the first sequences for some understudied genera, for which further work is required to delineate species boundaries and develop a more complete understanding of branchiopod diversity over a larger spatial scale.

Species delimitation in fungal endophyte diversity studies and its implications in ecological and biogeographic inferences

The estimation of species diversity in fungal endophyte communities is based either on species counts or on the assignment of operational taxonomic units (OTUs). Consequently, the application of different species recognition criteria affects not only diversity estimates but also the ecological hypotheses that arise from those observations. The main objective of the study was to examine how the choice and number of genetic markers and species delimitation criteria influence biodiversity estimates. Here, we compare approaches to defining species boundaries in three dominant species complexes of tropical endophytes, specially Colletotrichum gloeosporioides agg., Pestalotiopsis microspora agg. and Trichoderma harzianum agg., from two Amazonian trees: Hevea brasiliensis and H. guianensis. Molecular tools were used to describe and compare the diversity of the different assemblages. Multilocus phylogenetic analyses [gpd, internal transcribed spacer (ITS) and tef1] and modern techniques for phylogenetic species delimitation were overlaid with ecological data to recognize putative species or OTUs. The results demonstrate that ITS alone generally underestimates the number of species predicted by other nuclear loci. These results question the use of ITS and arbitrary divergence thresholds for species delimitation.

DNA barcodes discriminate freshwater fishes from the Paraíba do Sul River Basin, São Paulo, Brazil

Background and aims: Considering the promising use of DNA barcoding for species identification, the importance of the freshwater fish fauna of the Paraíba do Sul River Basin, and its advanced stage of degradation, the present study evaluated the effectiveness of DNA barcoding to identify the fish species in this basin.Materials and methods: A total of 295 specimens representing 58 species belonging to 40 genera, 17 families, and 5 orders were sequenced.Results: The DNA barcodes discriminated all species analyzed without ambiguity. The results showed a pronounced difference between conspecific and congeneric pair-wise sequence comparisons, demonstrating the existence of a “barcode gap” for the species analyzed. The nearest-neighbor distance analysis showed only three cases with Kimura two-parameter values lower than a 2% divergence threshold. However, the patterns of divergence observed in each case remained sufficient to discriminate each species, revealing the accuracy of DNA barcoding even cases with relatively low genetic divergence. At the other extreme, three species displayed high genetic sequence divergence among conspecifics. For two cases, Characidium alipioi and Geophagus proximus, barcoding proved effective at flagging possible new species. For another case, Astyanax bimaculatus, the use of DNA barcoding of the comparison of shared freshwater fish fauna between different basins revealed itself as highly useful in disclosing that the previously identified A. bimaculatus “cluster A” probably represents the species Astyanax altiparanae.Conclusion: The present study is among the first to assess the efficiency of barcoding for the Brazilian freshwater fishes. The results demonstrate the utility of barcoding to identify the fauna from this basin, contribute to an enhanced understanding of the differentiation among species, and to help flag the presence of overlooked species.

Lineage divergence detected in the malaria vector Anopheles marajoara (Diptera: Culicidae) in Amazonian Brazil

BackgroundCryptic species complexes are common among anophelines. Previous phylogenetic analysis based on the complete mtDNA COI gene sequences detected paraphyly in the Neotropical malaria vector Anopheles marajoara. The "Folmer region" detects a single taxon using a 3% divergence threshold.MethodsTo test the paraphyletic hypothesis and examine the utility of the Folmer region, genealogical trees based on a concatenated (white + 3' COI sequences) dataset and pairwise differentiation of COI fragments were examined. The population structure and demographic history were based on partial COI sequences for 294 individuals from 14 localities in Amazonian Brazil. 109 individuals from 12 localities were sequenced for the nDNA white gene, and 57 individuals from 11 localities were sequenced for the ribosomal DNA (rDNA) internal transcribed spacer 2 (ITS2).ResultsDistinct A. marajoara lineages were detected by combined genealogical analysis and were also supported among COI haplotypes using a median joining network and AMOVA, with time since divergence during the Pleistocene (<100,000 ya). COI sequences at the 3' end were more variable, demonstrating significant pairwise differentiation (3.82%) compared to the more moderate 2.92% detected by the Folmer region. Lineage 1 was present in all localities, whereas lineage 2 was restricted mainly to the west. Mismatch distributions for both lineages were bimodal, likely due to multiple colonization events and spatial expansion (~798 - 81,045 ya). There appears to be gene flow within, not between lineages, and a partial barrier was detected near Rio Jari in Amapá state, separating western and eastern populations. In contrast, both nDNA data sets (white gene sequences with or without the retention of the 4th intron, and ITS2 sequences and length) detected a single A. marajoara lineage.ConclusionsStrong support for combined data with significant differentiation detected in the COI and absent in the nDNA suggest that the divergence is recent, and detectable only by the faster evolving mtDNA. A within subgenus threshold of >2% may be more appropriate among sister taxa in cryptic anopheline complexes than the standard 3%. Differences in demographic history and climatic changes may have contributed to mtDNA lineage divergence in A. marajoara.

Divergent photic thresholds in the non-image-forming visual system: entrainment, masking and pupillary light reflex

Light is the principal cue that entrains the circadian timing system, but the threshold of entrainment and the relative contributions of the retinal photoreceptors-rods, cones and intrinsically photosensitive retinal ganglion cells-are not known. We measured thresholds of entrainment of wheel-running rhythms at three wavelengths, and compared these to thresholds of two other non-image-forming visual system functions: masking and the pupillary light reflex (PLR). At the entrainment threshold, the relative spectral sensitivity and absolute photon flux suggest that this threshold is determined by rods. Dim light that entrained mice failed to elicit either masking or PLR; in general, circadian entrainment is more sensitive by 1-2 log units than other measures of the non-image-forming visual system. Importantly, the results indicate that dim light can entrain circadian rhythms even when it fails to produce more easily measurable acute responses to light such as phase shifting and melatonin suppression. Photosensitivity to one response, therefore, cannot be generalized to other non-image-forming functions. These results also impact practical problems in selecting appropriate lighting in laboratory animal husbandry.

Analysis of Genetic Diversity and Sites of Recombination in Human Rhinovirus Species C

Human rhinoviruses (HRVs) are a highly prevalent and diverse group of respiratory viruses. Although HRV-A and HRV-B are traditionally detected by virus isolation, a series of unculturable HRV variants have recently been described and assigned as a new species (HRV-C) within the picornavirus Enterovirus genus. To investigate their genetic diversity and occurrence of recombination, we have performed comprehensive phylogenetic analysis of sequences from the 5' untranslated region (5' UTR), VP4/VP2, VP1, and 3Dpol regions amplified from 89 HRV-C-positive respiratory samples and available published sequences. Branching orders of VP4/VP2, VP1, and 3Dpol trees were identical, consistent with the absence of intraspecies recombination in the coding regions. However, numerous tree topology changes were apparent in the 5' UTR, where >60% of analyzed HRV-C variants showed recombination with species A sequences. Two recombination hot spots in stem-loop 5 and the polypyrimidine tract in the 5' UTR were mapped using the program GroupingScan. Available HRV-C sequences showed evidence for additional interspecies recombination with HRV-A in the 2A gene, with breakpoints mapping precisely to the boundaries of the C-terminal domain of the encoded proteinase. Pairwise distances between HRV-C variants in VP1 and VP4/VP2 regions fell into two separate distributions, resembling inter- and intraserotype distances of species A and B. These observations suggest that, without serological cross-neutralization data, HRV-C genetic groups may be equivalently classified into types using divergence thresholds derived from distance distributions. The extensive sequence data from multiple genome regions of HRV-C and analyses of recombination in the current study will assist future formulation of consensus criteria for HRV-C type assignment and identification.

Proposals for the classification of human rhinovirus species C into genotypically assigned types

Human rhinoviruses (HRVs) are common respiratory pathogens associated with mild upper respiratory tract infections, but also increasingly recognized in the aetiology of severe lower respiratory tract disease. Wider use of molecular diagnostics has led to a recent reappraisal of HRV genetic diversity, including the discovery of HRV species C (HRV-C), which is refractory to traditional virus isolation procedures. Although it is heterogeneous genetically, there has to date been no attempt to classify HRV-C into types analogous to the multiple serotypes identified for HRV-A and -B and among human enteroviruses. Direct investigation of cross-neutralization properties of HRV-C is precluded by the lack of methods for in vitro culture, but sequences from the capsid genes (VP1 and partial VP4/VP2) show evidence for marked phylogenetic clustering, suggesting the possibility of a genetically based system comparable to that used for the assignment of new enterovirus types. We propose a threshold of 13% divergence for VP1 nucleotide sequences for type assignment, a level that classifies the current dataset of 86 HRV-C VP1 sequences into a total of 33 types. We recognize, however, that most HRV-C sequence data have been collected in the VP4/VP2 region (currently 701 sequences between positions 615 and 1043). We propose a subsidiary classification of variants showing > 10% divergence in VP4/VP2, but lacking VP1 sequences, to 28 provisionally assigned types (subject to confirmation once VP1 sequences are determined). These proposals will assist in future epidemiological and clinical studies of HRV-C conducted by different groups worldwide, and provide the foundation for future exploration of type-associated differences in clinical presentations and biological properties.

A polymorphism in exon 2 of the δ-opioid receptor affects nociception in response to specific agonists and antagonists in mice selectively bred for high and low analgesia

This study searched for polymorphic sites in the murine μ-, δ- and κ-opioid receptors that presumably influence pain perception. We employed two mouse lines divergently bred for high (HA, high analgesia line) and low (LA, low analgesia line) swim stress-induced analgesia (SSIA). These mouse lines differ substantially in pain sensitivity, measured as hind paw withdrawal latency in the hot-plate test. We found a novel C320T transition in exon 2 of the δ-opioid receptor gene, resulting in an A107V substitution in the first extracellular loop (EL1) of the peptide chain. Using hot-plate and tail-flick tests, we found a significant association between the genotype of this locus and basal nociception and SSIA magnitude. Moreover, this transition affects the pharmacological effects of two specific δ-opioid receptor ligands, the agonist SNC80 and the antagonist naltrindole. The impact of the C320T polymorphism on the magnitude of SSIA was partially mediated by endogenous opioids. The effectiveness of exogenous δ-opioid receptor ligands was greater in the HA than in the LA line, and was greater in C320C homozygotes than in C320T heterozygotes within each line. Our results indicate that the C320T polymorphism in the δ-opioid receptor gene is at least partly responsible for the divergent nociceptive thresholds in HA and LA mice under both basal and post-stress conditions.

Screening Respiratory Samples for Detection of Human Rhinoviruses (HRVs) and Enteroviruses: Comprehensive VP4-VP2 Typing Reveals High Incidence and Genetic Diversity of HRV Species C

Rhinovirus infections are the most common cause of viral illness in humans, and there is increasing evidence of their etiological role in severe acute respiratory tract infections (ARTIs). Human rhinoviruses (HRVs) are classified into two species, species A and B, which contain over 100 serotypes, and a recently discovered genetically heterogeneous third species (HRV species C). To investigate their diversity and population turnover, screening for the detection and the genetic characterization of HRV variants in diagnostic respiratory samples was performed by using nested primers for the efficient amplification of the VP4-VP2 region of HRV (and enterovirus) species and serotype identification. HRV species A, B, and C variants were detected in 14%, 1.8%, and 6.8%, respectively, of 456 diagnostic respiratory samples from 345 subjects (6 samples also contained enteroviruses), predominantly among children under age 10 years. HRV species A and B variants were remarkably heterogeneous, with 22 and 6 different serotypes, respectively, detected among 73 positive samples. Similarly, by using a pairwise distance threshold of 0.1, species C variants occurring worldwide were provisionally assigned to 47 different types, of which 15 were present among samples from Edinburgh, United Kingdom. There was a rapid turnover of variants, with only 5 of 43 serotypes detected during both sampling periods. By using divergence thresholds and phylogenetic analysis, several species A and C variants could provisionally be assigned to new types. An initial investigation of the clinical differences between rhinovirus species found HRV species C to be nearly twice as frequently associated with ARTIs than other rhinovirus species, which matches the frequencies of detection of respiratory syncytial virus. The study demonstrates the extraordinary genetic diversity of HRVs, their rapid population turnover, and their extensive involvement in childhood respiratory disease.

Diversity of Frankia populations in root nodules of geographically isolated Arizona alder trees in central Arizona (United States).

The diversity of uncultured Frankia populations in root nodules of Alnus oblongifolia trees geographically isolated on mountaintops of central Arizona was analyzed by comparative sequence analyses of nifH gene fragments. Sequences were retrieved from Frankia populations in nodules of four trees from each of three mountaintops (n = 162) and their levels of diversity compared using spatial genetic clustering methods and single-nucleotide or 1, 3, or 5% sequence divergence thresholds. With the single-nucleotide threshold level, 45 different sequences with significant differences between the mountaintops were retrieved, with the southern site partitioning in a separate population from the two other sites. Some of these sequences were identical in nodules from different mountaintops and to those of strains isolated from around the world. A high level of diversity that resulted in the assignment of 14 clusters of sequences was also found on the 1% divergence level. Single-nucleotide and 1% divergence levels thus demonstrate microdiversity of frankiae in root nodules of A. oblongifolia trees and suggest a partitioning of diversity by site. At the 3 and 5% divergence levels, however, diversity was reduced to three clusters or one cluster, respectively, with no differentiation by mountaintop. Only at the 5% threshold level do all Frankia strains previously assigned to one genomic group cluster together.

Identifying sharks with DNA barcodes: assessing the utility of a nucleotide diagnostic approach

Shark fisheries worldwide are mostly unmanaged, but the burgeoning shark fin industry in the last few decades has made monitoring catch and trade of these animals critical. As a tool for molecular species identification, DNA barcoding offers significant potential. However, the genetic distance-based approach towards species identification employed by the Barcode of Life Data Systems may oftentimes lack the specificity needed for regulatory or legal applications that require unambiguous identification results. This is because such specificity is not typically realized by anything less than a 100% match of the query sequence to an entry in the reference database using genetic distance. Although various divergence thresholds have been proposed to define acceptable levels of intraspecific variation, enough exceptions exist to cast reasonable doubt on many less than exact matches using a distance-based approach for the identification of unknowns. An alternative approach relies on the identification of discrete molecular characters that can be used to unambiguously diagnose species. The objective of this study was to assess the performance differences between these competing approaches by examining more than 1000 DNA barcodes representing nearly 20% of all known elasmobranch species. Our results demonstrate that a character-based, nucleotide diagnostic (ND) approach to barcode identification is feasible and also provides novel insights into the structure of haplotype diversity among closely related species of sharks. Considerations for the use of NDs in applied fields are also explored.

Associating larvae and adults of Chinese Hydropsychidae caddisflies (Insecta:Trichoptera) using DNA sequences

The utility of hydropsychid (Trichoptera:Hydropsychidae) caddisfly larvae for freshwater biomonitoring has been demonstrated, but the major impediment to its implementation has been the lack of species-level larval descriptions and illustrations. A rapid and reliable molecular protocol that also uses morphology is proposed because conventional approaches to associating undescribed larvae with adults have been slow and problematic. Male adults were identified before DNA sequence analyses were used. These identifications established morphospecies boundaries that were mapped on phylograms constructed from 2 independent gene fragments: mitochondrial cytochrome c oxidase subunit I (COI) and large subunit (28S) nuclear ribosomal DNA expansion fragment D2 (D2). Species boundaries were confirmed if they were monophyletic on both molecular phylograms. Larval associations were made with reference to the phylogenetic analyses under 2 criteria: sequence identity across both genes or nested placement within a reference species boundary. A total of 133 individuals belonging to Chinese Hydropsyche sensu lato group (including Hydropsyche [Hydropsyche], Hydropsyche [Occutanspsyche], Ceratopsyche, Mexipsyche, Hydatomanicus, and Herbertorossia) were included in our study to test the new protocol. D2 sequences (all individuals) and COI sequences (101 individuals) were obtained, and 2 independent phylograms were constructed using neighbor joining. Both fragments provided enough nucleotide changes to differentiate independently most Hydropsyche sensu lato species, with ambiguity in only a few species that eventually could be resolved with additional sequences and specimens. COI diverges significantly within some species, suggesting a need for caution when applying typical genetic divergence thresholds in species diagnoses. The study enabled us to establish a procedure for delimiting species boundaries and associating larvae and adults using DNA sequences and morphological evidence. Ideal sampling strategies for larval–adult association are suggested. Associating larvae and adults of hydropsychids using DNA sequences appears to be promising in terms of both reliability and speed.

Towards a conceptual and operational union of bacterial systematics, ecology, and evolution.

To completely understand the ecology of a bacterial community, we need to identify its ecologically distinct populations (ecotypes). The greatest promise for enumerating a community's constituent ecotypes is held by molecular approaches that identify bacterial ecotypes as DNA sequence clusters. These approaches succeed when ecotypes correspond with sequence clusters, but some models of bacterial speciation predict a one-to-many and others a many-to-one relationship between ecotypes and sequence clusters. A further challenge is that sequence-based phylogenies often contain a hierarchy of clusters and subclusters within clusters, and there is no widely accepted theory to guide systematists and ecologists to the size of cluster most likely to correspond to ecotypes. While present systematics attempts to use universal thresholds of sequence divergence to help demarcate species, the recently developed 'community phylogeny' approach assumes no universal thresholds, but demarcates ecotypes based on the analysis of a lineage's evolutionary dynamics. Theory-based approaches like this one can give a conceptual framework as well as operational criteria for hypothesizing the identity and membership of ecotypes from sequence data; ecology-based approaches can then confirm that the putative ecotypes are actually ecologically distinct. Bacterial ecotypes that are demonstrated to have a history of coexistence as ecologically distinct lineages (based on sequence analysis) and as a prognosis of future coexistence (based on ecological differences), are the fundamental units of bacterial ecology and evolution, and should be recognized by bacterial systematics.

DNA Barcoding Will Often Fail to Discover New Animal Species over Broad Parameter Space

With increasing force, genetic divergence of mitochondrial DNA (mtDNA) is being argued as the primary tool for discovery of animal species. Two thresholds of single-gene divergence have been proposed: reciprocal monophyly, and 10 times greater genetic divergence between than within species (the "10x rule"). To explore quantitatively the utility of each approach, we couple neutral coalescent theory and the classical Bateson-Dobzhansky-Muller (BDM) model of speciation. The joint stochastic dynamics of these two processes demonstrate that both thresholds fail to "discover" many reproductively isolated lineages under a single incompatibility BDM model, especially when BDM loci have been subject to divergent selection. Only when populations have been isolated for > 4 million generations did these thresholds achieve error rates of < 10% under our model that incorporates variable population sizes. The high error rate evident in simulations is corroborated with six empirical data sets. These properties suggest that single-gene, high-throughput approaches to discovering new animal species will bias large-scale biodiversity surveys, particularly toward missing reproductively isolated lineages that have emerged by divergent selection or other mechanisms that accelerate reproductive isolation. Because single-gene thresholds for species discovery can result in substantial error at recent divergence times, they will misrepresent the correspondence between recently isolated populations and reproductively isolated lineages (= species).