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Current understanding of ecological and evolutionary processes underlying island biodiversity is heavily shaped by empirical data from plants and birds, although arthropods comprise the overwhelming majority of known animal species, and as such can provide key insights into processes governing biodiversity. Novel high throughput sequencing (HTS) approaches are now emerging as powerful tools to overcome limitations in the availability of arthropod biodiversity data, and hence provide insights into these processes. Here, we explored how these tools might be most effectively exploited for comprehensive and comparable inventory and monitoring of insular arthropod biodiversity. We first reviewed the strengths, limitations and potential synergies among existing approaches of high throughput barcode sequencing. We considered how this could be complemented with deep learning approaches applied to image analysis to study arthropod biodiversity. We then explored how these approaches could be implemented within the framework of an island Genomic Observatories Network (iGON) for the advancement of fundamental and applied understanding of island biodiversity. To this end, we identified seven island biology themes at the interface of ecology, evolution and conservation biology, within which collective and harmonized efforts in HTS arthropod inventory could yield significant advances in island biodiversity research., The iGON Working Group was organized by the iBioGen project, which has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement no. 810729.

Metabarcoding of vertebrate DNA obtained from invertebrates (iDNA) has been used to survey vertebrate communities, but we here show that it can also be used to study species interactions between invertebrates and vertebrates in a spatial context. We sampled the dung and carrion fly community of a swamp forest remnant along a disturbance gradient (10 sites: 80-310m from a road). Approximately, 60% of the baited 407 flies yield 294 vertebrate identifications based on two COI fragments and 16S sequenced with Illumina and/or MinION. A bipartite network analysis finds no specialization in the interaction between flies and vertebrate species, but a spatial analysis revealed that surprisingly 18 of the 20 vertebrate species can be detected within 150m of the road. We show that the fly community sourced for iDNA was unexpectedly rich (24 species, 3 families) and carried DNA for mammals, birds, and reptiles. They included common and rare ground-dwelling (e.g., wild boar, Sunda pangolin), and arboreal species (e.g., long-tailed macaque, Raffles’ banded langur) as well as small bodied vertebrates (skinks, rats). All of our results were obtained with a new, greatly simplified iDNA protocol that eliminates DNA extraction by obtaining template directly through dissolving feces and regurgitates from individual flies with water. Lastly, we show that MinION- and Illumina-based metabarcoding yield similar results. Overall, flies from several families (calliphorids, muscids and sarcophagids) should be used in iDNA surveys because we show that uncommon fly species carry the signal for several vertebrate species that are otherwise difficult to detect with iDNA.

Synchronous multispecific coral spawning generally occurs annually and forms an integral part of the coral life cycle. Apart from spawning times and species participation, however, much else remains unknown. Here, we applied environmental DNA (eDNA) metabarcoding to study two tropical reef sites of contrasting coral cover before, during and after coral spawning. Using coral-ITS2 and vertebrate-12S markers, we evaluated eDNA as an alternative monitoring tool by assessing its capabilities in detecting spawning species and tracking relative abundances of coral and fish eDNA. Over 3 years, elevated eDNA coral signals during the event (proportional read increase of up to five-fold) were observed, detecting a total of 38 coral and 133 fish species with all but one of the coral species visually observed to be spawning. This is also the first demonstration that eDNA metabarcoding can be used to infer the diurnal partitioning of night- and day-time spawning, spawning in coral species overlooked by visual surveys, and the associated changes in fish trophic structures as an indicator of spawning events. Our study paves the way for applied quantitative eDNA metabarcoding approaches to better study ephemeral and important biological events.

Environmental DNA (eDNA) with metabarcoding or metagenomics will likely become a major biomonitoring tool in the 21st century, perhaps even more so in the face of increased coastal urbanization and its associated effects such as pollution, land reclamation, and seabed dredging. Together, these impacts and the consequent high turbidity pose severe challenges to traditional survey techniques that rely heavily on visual observations. We here demonstrate that eDNA can be used for biomonitoring in turbid waters, using Singapore as a case study. The cytochrome c oxidase subunit I (COI) locus was used to detect 525 metazoan molecular operational taxonomic units (MOTUs) based on eDNA obtained from 52 L of filtered seawater. Of these, 130 MOTUs (24.7%) were identifiable to species, including ecologically significant species that in some cases were invasive or rarely observed. Metazoan signals also enabled discrimination of discrete, but connected, environments from intertidal and subtidal zones. Taxa with known habitat preferences were found to have left trace eDNA at sites that matched putatively suitable habitats. Moreover, no coast‐specific signals were detected in open water samples, which suggest that intermixing of water was limited. The study confirms that eDNA metabarcoding is a viable biomonitoring tool for coastal areas with highly sedimented waters.

New techniques for the species-level sorting of millions of specimens are needed in order to accelerate species discovery, determine how many species live on earth, and develop efficient biomonitoring techniques. These sorting methods should be reliable, scalable, and cost-effective, as well as being largely insensitive to low-quality genomic DNA, given that this is usually all that can be obtained from museum specimens. Mini-barcodes seem to satisfy these criteria, but it is unclear how well they perform for species-level sorting when compared with full-length barcodes. This is here tested based on 20 empirical data sets covering ca. 30,000 specimens (5500 species) and six clade-specific data sets from GenBank covering ca. 98,000 specimens ($>$20,000 species). All specimens in these data sets had full-length barcodes and had been sorted to species-level based on morphology. Mini-barcodes of different lengths and positions were obtained in silico from full-length barcodes using a sliding window approach (three windows: 100 bp, 200 bp, and 300 bp) and by excising nine mini-barcodes with established primers (length: 94–407 bp). We then tested whether barcode length and/or position reduces species-level congruence between morphospecies and molecular operational taxonomic units (mOTUs) that were obtained using three different species delimitation techniques (Poisson Tree Process, Automatic Barcode Gap Discovery, and Objective Clustering). Surprisingly, we find no significant differences in performance for both species- or specimen-level identification between full-length and mini-barcodes as long as they are of moderate length ($>$200 bp). Only very short mini-barcodes ($200 bp and the congruent mOTUs contain ca. 75% of all specimens. Most conflict is caused by ca. 10% of the specimens that can be identified and should be targeted for re-examination in order to efficiently resolve conflict. Our study suggests that large-scale species discovery, identification, and metabarcoding can utilize mini-barcodes without any demonstrable loss of information compared to full-length barcodes. [DNA barcoding; metabarcoding; mini-barcodes; species discovery.]

Invertebrate biodiversity remains poorly understood although it comprises much of the terrestrial animal biomass, most species and supplies many ecosystem services. The main obstacle is specimen-rich samples obtained with quantitative sampling techniques (e.g., Malaise trapping). Traditional sorting requires manual handling, while molecular techniques based on metabarcoding lose the association between individual specimens and sequences and thus struggle with obtaining precise abundance information. Here we present a sorting robot that prepares specimens from bulk samples for barcoding. It detects, images and measures individual specimens from a sample and then moves them into the wells of a 96-well microplate. We show that the images can be used to train convolutional neural networks (CNNs) that are capable of assigning the specimens to 14 insect taxa (usually families) that are particularly common in Malaise trap samples. The average assignment precision for all taxa is 91.4% (75%-100%). This ability of the robot to identify common taxa then allows for taxon-specific subsampling, because the robot can be instructed to only pick a prespecified number of specimens for abundant taxa. To obtain biomass information, the images are also used to measure specimen length and estimate body volume. We outline how the DiversityScanner can be a key component for tackling and monitoring invertebrate diversity by combining molecular and morphological tools: the images generated by the robot become training images for machine learning once they are labelled with taxonomic information from DNA barcodes. We suggest that a combination of automation, machine learning and DNA barcoding has the potential to tackle invertebrate diversity at an unprecedented scale.

The Sepsidae is, with approximately 300 described species, a relatively small family of cyclorrhaphan flies whose behaviour, morphology, and development have been extensively studied. However, currently the only available tree for Sepsidae is more than 10 years old and was based entirely on morphological characters. Here, we present the results of parsimony and Bayesian analyses based on 75 species, ten genes, and morphology. Parsimony and Bayesian analyses produce largely congruent and well-supported topologies regardless of whether indels are coded as 5th character states, as missing values, or all sites with indels are removed. The tree confirms the monophyly of Sepsidae and identifies the Ropalomeridae as its sister group. With regard to higher-level relationships, we identify widespread conflict between the morphological and the DNA sequence data. The proposed hypothesis based on both partitions largely reflects the signal in the molecular data. Particularly surprising is the rejection of two relationship hypotheses with strong morphological support, namely the sister group relationship between Orygma and the remaining Sepsidae and the monophyly of the Sepsis species group. Our partitioned Bremer support (PBS) analyses imply that indel coding has a stronger effect on the relative performance of individual gene partitions than the exclusion of alignment-ambiguous sequences or the location of a gene on the mitochondrial or nuclear genome. However, these analyses also reveal unexpectedly strong fluctuations in PBS values given that indel treatment has only a minor effect on tree topology and jacknife support. These unexpected fluctuations highlight the need for a comparative study across multiple data sets that investigates the influence of conflict and indel treatment on PBS values. © The Willi Hennig Society 2008.

A significant number of Southeast Asian mammal species described in the 19th and 20th century were subsequently synonymized and are now considered subspecies. Many are affected by rapid habitat loss which creates an urgent need to re-assess the conservation status based on species boundaries established with molecular data. However, such data are lacking and difficult to obtain for many populations and subspecies. We document via a literature survey and empirical study how shotgun sequencing of faecal DNA is a still underutilized but powerful tool for accelerating such evaluations. We obtain 11 mitochondrial genomes for three subspecies in the langur genus Presbytis through shotgun sequencing of faecal DNA (P. femoralis femoralis, P. f. percura, P. siamensis cf. cana). The genomes support the resurrection of all three subspecies to species based on multiple species delimitation algorithms (PTP, ABGD, Objective Clustering) applied to a dataset covering 40 species and 43 subspecies of Asian colobines. For two of the newly recognized species (P. femoralis, P. percura), the results lead to an immediate change in IUCN status to Critically Endangered due to small population sizes and fragmented habitats. We conclude that faecal DNA should be more widely used for clarifying species boundaries in endangered mammals.

Summary Distal-less has been repeatedly co-opted for the development of many novel traits. Here, we document its curious role in the development of a novel abdominal appendage (“sternite brushes”) in sepsid flies. CRISPR/Cas9 deletions in the homeodomain result in losses of sternite brushes, demonstrating that Distal-less is necessary for their development. However, deletions in the upstream coding exon (Exon 2) produce losses or gains of brushes. A dissection of Exon 2 reveals that the likely mechanism for gains involves a deletion in an exon-splicing enhancer site that leads to exon skipping. Such contradictory phenotypes are also observed in butterflies, suggesting that mutations in the conserved upstream regions have the potential to generate phenotypic variability in insects that diverged 300 million years ago. Our results demonstrate the importance of Distal-less for the development of a novel abdominal appendage in insects and highlight how site-specific mutations in the same exon can produce contradictory phenotypes., Graphical Abstract, Highlights • Distal-less is necessary for the development of a novel abdominal appendage • CRISPR/Cas9 editing produced both losses and gains of novel abdominal appendages • Gains of appendages result from mutations in exonic splicing enhancer (ESEs) sites • ESE mutations likely led to exon skipping and an altered Distal-less protein, Genetics; Molecular Biology; Evolutionary Biology; Developmental Biology

DNA barcodes are useful for species discovery and species identification, but obtaining barcodes currently requires a well-equipped molecular laboratory, is time-consuming, and/or expensive. We here address these issues by developing a barcoding pipeline for Oxford Nanopore MinION™ and demonstrate that one flowcell can generate barcodes for ∼500 specimens despite high base-call error rates of MinION™. The pipeline overcomes the errors by first summarizing all reads for the same tagged amplicon as a consensus barcode. These barcodes are overall mismatch-free but retain indel errors that are concentrated in homopolymeric regions. We thus complement the barcode caller with an optional error correction pipeline that uses conserved amino-acid motifs from publicly available barcodes to correct the indel errors. The effectiveness of this pipeline is documented by analysing reads from three MinION™ runs that represent three different stages of MinION™ development. They generated data for (1) 511 specimens of a mixed Diptera sample, (2) 575 specimens of ants, and (3) 50 specimens of Chironomidae. The run based on the latest chemistry yielded MinION barcodes for 490 specimens which were assessed against reference Sanger barcodes (N=471). Overall, the MinION barcodes have an accuracy of 99.3%-100% and the number of ambiguities ranges from 90%). We estimate that up to 1000 barcodes can be generated in one flowcell and that the cost of a MinION barcode can be

Biologists frequently sort specimen-rich samples to species. This process is daunting when based on morphology, and disadvantageous if performed using molecular methods that destroy vouchers (e.g., metabarcoding). An alternative is barcoding every specimen in a bulk sample and then presorting the specimens using DNA barcodes, thus mitigating downstream morphological work on presorted units. Such a "reverse workflow" is too expensive using Sanger sequencing, but we here demonstrate that is feasible with an next-generation sequencing (NGS) barcoding pipeline that allows for cost-effective high-throughput generation of short specimen-specific barcodes (313 bp of COI; laboratory cost

Biodiversity is in crisis due to habitat destruction and climate change. The conservation of many noncharismatic species is hampered by the lack of data. Yet, natural history research—a major source of information on noncharismatic species—is in decline. We here suggest a remedy for many mammal species, i.e., metagenomic clean-up of fecal samples that are “crowdsourced” during routine field surveys. Based on literature data, we estimate that this approach could yield natural history information for circa 1,000 species within a decade. Metagenomic analysis would simultaneously yield natural history data on diet and gut parasites while enhancing our understanding of host genetics, gut microbiome, and the functional interactions between traditional and new natural history data. We document the power of this approach by carrying out a “metagenomic clean-up” on fecal samples collected during a single night of small mammal trapping in one of Alfred Wallace’s favorite collecting sites., Natural history research is in crisis and non-charismatic species are increasingly ignored; this Community Page article argues and demonstrates that shotgun sequencing of serendipitously obtained faecal samples could reverse this trend for 1000 mammal species within 10 years.

The wasp family Vespidae comprises more than 5000 described species which represent life history strategies ranging from solitary and presocial to eusocial and socially parasitic. The phylogenetic relationships of the major vespid wasp lineages (i.e., subfamilies and tribes) have been investigated repeatedly by analyzing behavioral and morphological traits as well as nucleotide sequences of few selected genes with largely incongruent results. Here we reconstruct their phylogenetic relationships using a phylogenomic approach. We sequenced the transcriptomes of 24 vespid wasp and eight outgroup species and exploited the transcript sequences for design of probes for enriching 913 single-copy protein-coding genes to complement the transcriptome data with nucleotide sequence data from additional 25 ethanol-preserved vespid species. Results from phylogenetic analyses of the combined sequence data revealed the eusocial subfamily Stenogastrinae to be the sister group of all remaining Vespidae, while the subfamily Eumeninae turned out to be paraphyletic. Of the three currently recognized eumenine tribes, Odynerini is paraphyletic with respect to Eumenini, and Zethini is paraphyletic with respect to Polistinae and Vespinae. Our results are in conflict with the current tribal subdivision of Eumeninae and thus, we suggest granting subfamily rank to the two major clades of "Zethini": Raphiglossinae and Zethinae. Overall, our findings corroborate the hypothesis of two independent origins of eusociality in vespid wasps and suggest a single origin of using masticated and salivated plant material for building nests by Raphiglossinae, Zethinae, Polistinae, and Vespinae. The inferred phylogenetic relationships and the open access vespid wasp target DNA enrichment probes will provide a valuable tool for future comparative studies on species of the family Vespidae, including their genomes, life styles, evolution of sociality, and co-evolution with other organisms.

Macroinvertebrates that are collected in large numbers pose major problems in basic and applied biodiversity research: identification to species via morphology is often difficult, slow and/or expensive. DNA barcodes are an attractive alternative or complementary source of information. Unfortunately, obtaining DNA barcodes from specimens requires many steps and thus time and money. Here, we promote a short cut to DNA barcoding, that is, a nondestructive PCR method that skips DNA extraction (‘direct PCR’) and that can be used for a broad range of invertebrate taxa. We demonstrate how direct PCR can be optimized for the larvae and adults of nonbiting midges (Diptera: Chironomidae), a typical invertebrate group that is abundant, contains important bioindicator species, but is difficult to identify based on morphological features. After optimization, direct PCR yields high PCR success rates (>90%), preserves delicate morphological features (e.g. details of genitalia, and larval head capsules) while allowing for the recovery of genomic DNA. We also document that direct PCR can be successfully optimized for a wide range of other invertebrate taxa that need routine barcoding (flies: Culicidae, Drosophilidae, Dolichopodidae, Sepsidae; sea stars: Oreasteridae). Key for obtaining high PCR success rates is optimizing (i) tissue quantity, (ii) body part, (iii) primer pair and (iv) type of Taq polymerase. Unfortunately, not all invertebrates appear suitable because direct PCR has low success rates for other taxa that were tested (e.g. Coleoptera: Dytiscidae, Copepoda, Hymenoptera: Formicidae and Odonata). It appears that the technique is less successful for heavily sclerotized insects and/or those with many exocrine glands.

Avermectins are potent and popular veterinary pharmaceuticals used globally to fight parasites of livestock and humans. By disturbing ion channel transport through the membrane, avermectins are effective against endo- and ectoparasitic round and horsehair worms (Nematoida), insects, or ticks (Arthropoda), but not against Plathelminthes, including flatworms (Trematoda) and tapeworms (Cestoda), or segmented worms (Annelida). Unfortunately, excreted avermectins have strong nontarget effects on beneficial arthropods such as the insect community decomposing livestock dung, ultimately impeding this important ecosystem function to the extent that regulators mandate standardized eco-toxicological tests of dung organisms worldwide. We show that the ancient phylogenetic pattern and qualitative mechanism of avermectin sensitivity is conserved and compatible with most recent phylogenomic hypotheses grouping the Nematoida with the Arthropoda as Ecdysozoa (molting animals). At the species level, we demonstrate phylogenetic clustering in ivermectin sensitivities of 23 species of sepsid dung flies (Diptera: Sepsidae). This clustered 500-fold quantitative variation in sensitivity may indicate recent lineage-specific responses to selection, but more likely reflects pre-existing genetic variation with pleiotropic effects on eco-toxicological responses to pollutants. Regardless, our results question the common practice in eco-toxicology of choosing single test species to infer detrimental effects on entire species communities, which should ideally assess a representative taxonomic sample.

Male abdomen appendages are a novel trait found within Sepsidae (Diptera). Here we demonstrate that they are likely to have evolved once, were lost three times, and then secondarily gained in one lineage. The developmental basis of these appendages was investigated by counting the number of histoblast cells in each abdominal segment in four species: two that represented the initial instance of appendage evolution, one that has secondarily gained appendages, and one species that did not have appendages. Males of all species with appendages have elevated cell counts for the fourth segment, which gives rise to the appendages. In Perochaeta dikowi, which reacquired the trait, the females also have elevated cell count on the fourth segment despite the fact that females do not develop appendages. The species without appendages has similar cell counts in all segments regardless of sex. These results suggest that the basis for appendage development is shared in males across all species, but the sexual dimorphism is regulated differently in P. dikowi.

We here test the proposition that changes in the barcoding region of COI are commonly involved in speciation through intergenomic conflict. We demonstrate that this is unlikely given that even with incomplete taxon sampling, 78–90% of closely-related animal species have identical COI amino acid sequences. In addition, in those cases where amino acid substitutions between closely related species are observed, the inter- and intra-specific substitution patterns are very similar and/or lack consistent differences in the number, position and type of amino acid change. Overall, we conclude that there is little evidence for a widespread involvement of the barcoding gene in speciation.

We present SequenceMatrix, software that is designed to facilitate the assembly and analysis of multi-gene datasets. Genes are concatenated by dragging and dropping FASTA, NEXUS, or TNT files with aligned sequences into the program window. A multi-gene dataset is concatenated and displayed in a spreadsheet; each sequence is represented by a cell that provides information on sequence length, number of indels, the number of ambiguous bases ("Ns"), and the availability of codon information. Alternatively, GenBank numbers for the sequences can be displayed and exported. Matrices with hundreds of genes and taxa can be concatenated within minutes and exported in TNT, NEXUS, or PHYLIP formats, preserving both character set and codon information for TNT and NEXUS files. SequenceMatrix also creates taxon sets listing taxa with a minimum number of characters or gene fragments, which helps assess preliminary datasets. Entire taxa, whole gene fragments, or individual sequences for a particular gene and species can be excluded from export. Data matrices can be re-split into their component genes and the gene fragments can be exported as individual gene files. SequenceMatrix also includes two tools that help to identify sequences that may have been compromised through laboratory contamination or data management error. One tool lists identical or near-identical sequences within genes, while the other compares the pairwise distance pattern of one gene against the pattern for all remaining genes combined. SequenceMatrix is Java-based and compatible with the Microsoft Windows, Apple MacOS X and Linux operating systems. The software is freely available from http://code.google.com/p/sequencematrix/. © The Willi Hennig Society 2010.

The dipteran clade Calyptratae is comprised of approximately 18 000 described species (12% of the known dipteran diversity) and includes well-known taxa such as houseflies, tsetse flies, blowflies and botflies, which have a close association with humans. However, the phylogenetic relationships within this insect radiation are very poorly understood and controversial. Here we propose a higher-level phylogenetic hypothesis for the Calyptratae based on an extensive DNA sequence dataset for 11 noncalyptrate outgroups and 247 calyptrate species representing all commonly accepted families in the Oestroidea and Hippoboscoidea, as well as those of the muscoid grade. DNA sequences for genes in the mitochondrial (12S, 16S, cytochrome c oxidase subunit I and cytochrome b) and nuclear genome [18S, 28S, the carbamoyl phosphate synthetase region of CAD (rudimentary), Elongation factor one alpha] were used to reconstruct the relationships. We discuss problems relating to the alignment and analysis of large datasets and emphasize the advantages of utilizing a guide tree-based approach for the alignment of the DNA sequences and using the leaf stability index to identify ‘wildcard’ taxa whose excessive instability obscures the phylogenetic signal. Our analyses support the monophyly of the Calyptratae and demonstrate that the superfamily Oestroidea is nested within the muscoid grade. We confirm that the monotypic family Mystacinobiidae is an oestroid and further revise the composition of the Oestroidea by demonstrating that the previously unplaced and still undescribed ‘McAlpine’s fly’ is nested within this superfamily as a probable sister group to Mystacinobiidae. Within the Oestroidea we confirm with molecular data that the Calliphoridae are a paraphyletic grade of lineages. The families Sarcophagidae and Rhiniidae are monophyletic, but support for the monophyly of Tachinidae and Rhinophoridae depends on analytical technique (e.g. parsimony or maximum likelihood). The superfamilies Hippoboscoidea and Oestroidea are consistently found to be monophyletic, and the paraphyly of the muscoid grade is confirmed. In the overall relationships for the calyptrates, the Hippoboscoidea are sister group to the remaining Calyptratae, and the Fanniidae are sister group to the nonhippoboscoid calyptrates, whose relationships can be summarized as (Muscidae (Oestroidea (Scathophagidae, Anthomyiidae))).

Tan, D. S. H., Ang, Y., Lim, G. S., Ismail, M. R. B. & Meier, R. (2010). From ‘cryptic species’ to integrative taxonomy: an iterative process involving DNA sequences, morphology, and behaviour leads to the resurrection of Sepsis pyrrhosoma (Sepsidae: Diptera). —Zoologica Scripta, 39, 51–61. The increased availability of DNA sequences has led to a surge of ‘cryptic species’ in the literature. These units are usually proposed based on finding genetically distinct lineages within species that were initially defined based on morphological characters. However, few authors attempt to confirm whether these ‘cryptic’ units are species and even fewer authors are explicit about which species concept is applied. Here, we use an example from Sepsidae (Diptera) to demonstrate how cryptic species can be validated by an iterative process involving several data sources and an evaluation of the data under different species concepts. A phylogeographic analysis based on 50 specimens for five species of the flavimana group revealed deep mitochondrial splits within Sepsis flavimana which was suggestive of a cryptic species. We resolve the initial conflict between DNA sequences and morphology by adding new morphological data as well as behavioural evidence and tests for reproductive isolation. One cryptic species is confirmed and Sepsis pyrrhosoma, a former synonym of S. flavimana, is here shown to be a valid species under most species concepts. We can thus document that the same data can lead to similar conclusions under conflicting concepts once different kinds of data are integrated.

Freshwater prawns of the genus Macrobrachium are free-living decapod crustaceans that are commonly encountered in tropical streams and lakes. We present a molecular phylogenetic analysis of the diverse Southeast and East Asian fauna based on >3 kb sequence data from three nuclear and two mitochondrial markers for almost 50% of the described fauna. We reconstruct the phylogenetic relationships and track the evolution of key life history traits. Our tree suggests that the last common ancestor of the Asian Macrobrachium laid numerous small eggs and had prolonged larval development (“PLD”) in saline coastal waters after which the adults matured in freshwater habitats. We also argue for five independent losses of the marine larval phase to yield five clades of species that develop entirely in freshwater and have fewer and larger eggs than the species with PLD. These species have either semi-abbreviated (two origins) with at least one free-swimming stage or abbreviated larval development (“ALD”: three origins) which lack free-swimming larvae. A Shimodaira–Hasegawa test rejects all trees that would imply a single loss of the marine larval phase, but alternative and equally parsimonious optimizations exist that imply a smaller number of losses. However, these scenarios would require the re-acquisition of free-swimming larvae. A concentrated-change test supports Pereira and Garcia’s [Pereira, G.A., Garcia, J.V., 1995. Larval development of Macrobrachium reyesi Pereira (Decapoda, Palaemonidae), with a discussion on the origin of abbreviated development in palaemonids. J. Crust. Biol. 15, 117–133] hypothesis of a significant correlation between living in freshwater and the origin of semi-abbreviated and abbreviated larval development. Our phylogenetic tree also reveals that Asian Macrobrachium have independently become cavernicolous at least twice, and invaded the highly acidic waters of freshwater and peat swamps two or three times.

N-methyl-D-aspartate (NMDA) glutamate receptors play crucial roles in neuronal synaptic plasticity, learning and memory. However, as to whether different NMDA subunits are implicated in specific forms of memory is unclear. Moreover, nothing is known about the interspecific genetic variability of the GRIN2A subunit and how this variation can potentially explain evolutionary changes in behavioral phenotypes. Here, we used 28 primate GRIN2A sequences and various proxies of memory across primates to investigate the role of GRIN2A. Codon-specific sequence analysis on these sequences showed that GRIN2A in primates coevolved with a likely ecological proxy of spatial memory (relative home-range size) but not with other indices of non-spatial learning and memory such as social memory and social learning. Models based on gene averages failed to detect positive selection in primate branches with major changes in relative home-range size. This implies that accelerated evolution is concentrated in specific parts of the protein expressed by GRIN2A. Overall, our molecular evolution study, the first on GRIN2A, supports the notion that different NMDA subunits may play a role in specific forms of memory and that phenotypic diversity along with genetic evolution can be used to investigate the link between genes and behavior across evolutionary time.

Coral taxonomy and systematics continue to be plagued by a host of problems. Due to high phenotypic variability within species, morphological approaches have often failed to recognize natural taxa, and molecular techniques have yet to be applied to many groups. Here, we summarize the levels of paraphyly found for scleractinian corals and test, based on new data, whether paraphyly is also a significant problem in Faviidae, the second-most speciose hermatypic scleractinian family. Using both DNA sequence and morphological data we find that, regardless of analysis technique (maximum parsimony, maximum likelihood and Bayesian likelihood), many conventional taxonomic groups are not monophyletic. Based on two mitochondrial markers (COI and a noncoding region) that we amplified for 81 samples representing 41 faviid species and 13 genera, five genera that are represented by more than one species are paraphyletic, as is the family Faviidae. The morphological characters currently used to identify these corals similarly fail to recover many genera. Furthermore, trees based on both data types are incongruent, and total evidence analysis does little to salvage conventional taxonomic groupings. Morphological convergence, phenotypic variability in response to the environment, and recent speciation are likely causes for these conflicts, which suggest that the present classification of corals is in need of a major overhaul. We propose more detailed studies of problematic faviid taxa using standardized morphological, mitochondrial, and nuclear genetic markers to facilitate combining of data.

The All Birds Barcoding Initiative aims to assemble a DNA barcode database for all bird species, but the 648-bp 'barcoding' region of cytochrome c oxidase subunit I (COI) can be difficult to amplify in Southeast Asian perching birds (Aves: Passeriformes). Using COI sequences from complete mitochondrial genomes, we designed a primer pair that more reliably amplifies and sequences the COI barcoding region of Southeast Asian passerine birds. The 655-bp region amplified with these primers overlaps the COI region amplified with other barcoding primer pairs, enabling direct comparison of sequences with previously published DNA barcodes.

Hippoboscoidea is a superfamily of Diptera that contains the Glossinidae or tsetse flies, the Hippoboscidae or louse flies, and two families of bat flies, the Streblidae and the Nycteribiidae. We reconstruct the phylogenetic relationships within Hippoboscoidea using maximum parsimony and Bayesian methods based on nucleotide sequences from fragments of four genes: nuclear 28S ribosomal DNA and the CPSase domain of CAD, and mitochondrial 16S rDNA and cytochrome oxidase I. We recover monophyly for most of the presently recognized groups within Hippoboscoidea including the superfamily as a whole, the Hippoboscidae, the Nycteribiidae, the bat flies, and the Pupipara (=Hippoboscidae+Nycteribiidae+Streblidae), as well as several subfamilies within the constituent families. Streblidae appear to be paraphyletic. Our phylogenetic hypothesis is well supported and decisive in that most competing topological hypotheses for the Hippoboscoidea require significantly longer trees. We confirm a single shift from a free-living fly to a blood-feeding ectoparasite of vertebrates and demonstrate that at least two host shifts from mammals to birds have occurred. Wings have been repeatedly lost, but never regained. The hippoboscoid ancestor also evolved adenotrophic viviparity and our cladogram is consistent with a gradual reduction in the motility of the deposited final instar larvae from active burrowing in the soil to true pupiparity where adult females glue the puparium within the confines of bat roosts.

The phylogeny and biogeography of the Malayan freshwater crab genus Johora was studied using two mitochondrial genes, 16S rRNA (560 bp) and cytochrome oxidase subunit I (COI) (616 bp), and one nuclear gene, histone 3 (H3) (328 bp). Johora is shown to be monophyletic and composed of three clades that correspond with the topography of the Malay Peninsula. The three clades were estimated to be of similar age (c. 11 million years ago (mya)). The Malayan island of Pulau Tioman (with five species) was determined to have been colonised independently by two separate clades (at c. 11 and 5 mya, respectively), one of which evolved semiterrestrial habits, possibly in response to competition by the second. A partitioned Bremer support (PBS) analysis reveals that most of the support for the phylogenetic tree comes from the COI gene fragment and that the nuclear protein-encoding genes H3 is useful for reconstructing the relationships of Johora.

We here argue that data from comparative studies of genome size and karyotypes provide important information for planning comparative research on genome evolution. We document for 39 species of sepsids that there is a four-fold difference in genome size (151-618 Mbp). Mapping genome sizes onto a phylogenetic hypothesis identifies that this range is the result of five genome expansions and four genome contractions that we here define as changes in genome size of more than 50 Mbp. We then generate karyotype data for 10 species and find no changes in chromosome number. The study reveals that the "Oriental" clade of sepsids is a promising system for studying genome evolution because it has experienced three genome expansion events. These events can be compared with an expansion in the "Neotropical" clade in order to reveal the mechanisms that underlie genome expansion in Sepsidae. A review of the literature on genome sizes and karyotypes reveals that they have been poorly documented in Metazoa. This means that researchers interested in the evolution of genome expansions and contractions are currently not being able to identify appropriate target taxa for genome sequencing. We thus argue for more comparative research on genome sizes and karyotypes and point out that historically species were chosen for genome sequencing for reasons not related to genome evolution (e.g. small genome size, model species status, phylogenetic position, interesting phenotypes). We believe that it is now time to use a more genome-centric selection criterion, where species for whole genome sequencing are selected based on their importance for understanding genome evolution.

DNA barcoding and DNA taxonomy have recently been proposed as solutions to the crisis of taxonomy and received significant attention from scientific journals, grant agencies, natural history museums, and mainstream media. Here, we test two key claims of molecular taxonomy using 1333 mitochondrial COI sequences for 449 species of Diptera. We investigate whether sequences can be used for species identification ("DNA barcoding") and find a relatively low success rate (< 70%) based on tree-based and newly proposed species identification criteria. Misidentifications are due to wide overlap between intra- and interspecific genetic variability, which causes 6.5% of all query sequences to have allospecific or a mixture of allo- and conspecific (3.6%) best-matching barcodes. Even when two COI sequences are identical, there is a 6% chance that they belong to different species. We also find that 21% of all species lack unique barcodes when consensus sequences of all conspecific sequences are used. Lastly, we test whether DNA sequences yield an unambiguous species-level taxonomy when sequence profiles are assembled based on pairwise distance thresholds. We find many sequence triplets for which two of the three pairwise distances remain below the threshold, whereas the third exceeds it; i.e., it is impossible to consistently delimit species based on pairwise distances. Furthermore, for species profiles based on a 3% threshold, only 47% of all profiles are consistent with currently accepted species limits, 20% contain more than one species, and 33% only some sequences from one species; i.e., adopting such a DNA taxonomy would require the redescription of a large proportion of the known species, thus worsening the taxonomic impediment. We conclude with an outlook on the prospects of obtaining complete barcode databases and the future use of DNA sequences in a modern integrative taxonomy.

The beaches of the North Atlantic and North Pacific are home to kelp flies of the Coelopa frigida/nebularum complex, which consists of one to three different species depending on whether the two nominal species are accepted and a cryptic species proposed by Remmert is counted. The morphological differences between two populations of C. frigida (Fabricius, 1805) from the North Sea and the Baltic Region and two populations of C. nebularum Aldrich, 1929 from Alaska and Japan are described and discussed for small, medium, and large specimens. Crossing experiments are used to demonstrate that, under laboratory conditions, no isolation mechanisms between either population exist. Coelopa frigida and C. nebularum are therefore regarded as a single biological species, a conclusion that is congruent with the observation that the genetic distances based on Ef 1-α and 16S rDNA indicate lower levels of differentiation within C. frigida/nebularum than between undisputed Coelopa species. The substantial morphological, breeding and genetic information on the C. frigida/nebularum species complex is then applied to six different species concepts popular in the modern systematic literature. According to the Biological, Hennigian and Recognition Species Concepts, only a single species would be recognized. The Evolutionary Species Concept is too vague to be applicable and under two variants of the Phylogenetic Species Concept, C. frigida and C. nebularum would constitute separate species. This result confirms that Phylogenetic Species Concepts lead to a higher species number than concepts based on reproductive isolation. Practical and theoretical problems with the various species concepts are briefly discussed. Zusammenfassung Die Strande des nordlichen Atlantiks und Pazifiks sind der Lebensraum der Tangfliegen des Coelopa-frigida/nebularum -Komplexes, der je nachdem ob man Remmert's kryptische Ostseeart zahlt, 1-3 Arten umfasst. Die morphologischen Unterschiede zwischen zwei Populationen von C. frigida aus dem Nordseegebiet und dem Baltikum und zwei Populationen von C. nebularum aus Alaska und Japan werden fur kleine, mittelgrosse und grosse Exemplare dargestellt und diskutiert. Anhand von Kreuzungsversuchen wird demonstriert dass unter Laborbedingungen keine postgametischen Isolationsmechanismen zwischen diesen Poplationen bestehen. Coelopa frigida und C. nebularum mussen daher als eine einzige biologische Fortpflanzungsgemeinschaft betrachtet werden. Diese Folgerung steht mit der Beobachtung in Einklang, dass bei EF1α und 16SrDNA die genetischen Distanzen innerhalb des C. frigida/nebularum -Komplexes geringer sind als innerhalb ‘‘guter’’ Coelopa -Arten. Die breite Datenbasis zu Morphologie, Fortpflanzung/Zucht und Genetik im C. frigida/nebularum- Komplex wird ferner im Licht von sechs in der gegenwartigen Literatur gelaufigen Artkonzepten diskutiert. Gemas dreier dieser Artkonzepte ist nur eine einzige Art anzuerkennen. Ein weiteres Konzept ist zu unscharf formuliert fur eine Entscheidung, und unter zwei Varianten des Phylogenetischen Artkonzepts erscheinen C. frigida and C. nebularum als getrennte Arten. Dieses Ergebnis bestatigt, dass Phylogenetische Artkonzepte zur Anerkennung einer grosseren Anzahl von Arten fuhren als Konzepte, die sich auf reproduktive Isolation als Kriterium stutzen. Praktische und theoretische Probleme der verschiedenen Artkonzepte werden kurz diskutiert.

Cell-lineage trees may contain information about spiralian phylogeny, as proposed by Guralnick and Lindberg (2001). Here we discuss this possibility further and conclude that the cell-division pattern must be known in greater detail and the coding methods refined before a possible phylogenetic signal can be identified.

With about 5000 species in ca. 180 genera, the Muscidae is the most species-rich family in the muscoid grade of Calyptratae (Diptera: Cyclorrhapha), the others being the Fanniidae, Scathophagidae and Anthomyiidae. Muscidae is remarkable for its young age, high species diversity in all biogeographic regions, and an unusually diverse range of feeding habits at the larval stage (e.g., saprophagy, phytophagy, carnivory, endoparasitism, haematophagy). We here review muscid classification and biology and present a molecular phylogeny based on four mitochondrial genes (12S, 16S, COI, CYTB) and three nuclear genes (28S, Ef1a, and CAD) for 84 species from 40 genera. Our analysis is the first to include species from all biogeographic regions and all currently recognised muscid subfamilies and tribes. We provide strong support for the monophyly of the Muscidae, and for the first time also for the first split within this family. The ancestral larval feeding habit is reconstructed to be saprophagy with more specialised coprophagous saprophagy, phytophagy, and carnivory evolving multiple times from saprophagous ancestors. The origins of carnivory in larvae are significantly correlated with a reduction of the number of larval instars from three (ancestral) to two and one. The genus Achanthiptera which was previously in its own subfamily is shown to be closely related to Azeliini. However, it appears that Azeliinae is paraphyletic because Muscinae is sister-group to the Azeliini while the azeliine Reinwardtiini are polyphyletic. Coenosiinae and Muscinae are monophyletic, but Muscini is paraphyletic with regard to Stomoxyini. Because many subfamilies are apparently para- or even polyphyletic, we review the history of muscid classification in order to reveal how the currently used classification originated.

Recent advances in scleractinian systematics and taxonomy have been achieved through the integration of molecular and morphological data, as well as rigorous analysis using phylogenetic methods. In this study, we continue in our pursuit of a phylogenetic classification by examining the evolutionary relationships between the closely related reef coral genera Merulina, Goniastrea, Paraclavarina and Scapophyllia (Merulinidae). In particular, we address the extreme polyphyly of Favites and Goniastrea that was discovered a decade ago. We sampled 145 specimens belonging to 16 species from a wide geographic range in the Indo-Pacific, focusing especially on type localities, including the Red Sea, western Indian Ocean and central Pacific. Tree reconstructions based on both nuclear and mitochondrial markers reveal a novel lineage composed of three species previously placed in Favites and Goniastrea. Morphological analyses indicate that this clade, Paragoniastrea Huang, Benzoni & Budd, gen. n., has a unique combination of corallite and subcorallite features observable with scanning electron microscopy and thin sections. Molecular and morphological evidence furthermore indicates that the monotypic genus Paraclavarina is nested within Merulina, and the former is therefore synonymised.

The egg morphology of 28 species of Diopsidae is described based on scanning electron microscopy and semi-thin sections for selected species from the following genera: Chaetodiopsis, Cyrtodiopsis, Diasemopsis, Diopsis, Eurydiopsis, Teleopsis, Teloglabrus, Trichodiopsis. Generic descriptions and a key to genera based on egg characters is presented. A data set for phylogenetic analysis is assembled and subjected to the first morphology-based numerical cladistic analysis for the family. The data set consists of 44 adult characters taken from the Diopsidae literature and 10 new egg characters. Cladistic analysis confirms the traditional concept of the family Diopsidae composed of the Centrioncinae (lacking eye stalks) and Diopsinae (with eye stalks) and the Syringogastridae are likely to be the sister group of Diopsidae. Within the Diopsinae, the genus Sphyracephala (including Pseudodiopsis) with its relatively short eye stalks is monophyletic and the sister group of the remaining Diopsinae. The following higher-level phylogenetic hypothesis is proposed (Centrioncinae (Sphyracephala ((Diasemopsis, Trichodiopsis, Chaetodiopsis) ((Teleopsis, Cyrtodiopsis) (Eurydiopsis, Diopsis))))). A high degree of congruence is observed for the morphological data and a recently assembled molecular data set. It is once more confirmed that egg characters provide valuable information for cladistic analysis and identification purposes.

Faecal samples are of great value as a non-invasive means to gather information on the genetics, distribution, demography, diet and parasite infestation of endangered species. Direct shotgun sequencing of faecal DNA could give information on these simultaneously, but this approach is largely untested. Here, we used two faecal samples to characterize the diet of two red-shanked doucs langurs (Pygathrix nemaeus) that were fed known foliage, fruits, vegetables and cereals. Illumina HiSeq produced ~74 and 67 million paired reads for these samples, of which ~ 10,000 (0.014%) and ~ 44,000 (0.066%), respectively, were of chloroplast origin. Sequences were matched against a database of available chloroplast 'barcodes' for angiosperms. The results were compared with 'metabarcoding' using PCR amplification of the P6 loop of trnL. Metagenomics identified seven and nine of the likely 16 diet plants while six and five were identified by metabarcoding. Metabarcoding produced thousands of reads consistent with the known diet, but the barcodes were too short to identify several plant species to genus. Metagenomics utilized multiple, longer barcodes that combined had greater power of identification. However, rare diet items were not recovered. Read numbers for diet species in metagenomic and metabarcoding data were correlated, indicating that both are useful for determining relative sequence abundance. Metagenomic reads were uniformly distributed across the chloroplast genomes; thus, if chloroplast genomes were used as reference, the precision of identifications and species recovery would improve further. Metagenomics also recovered the host mitochondrial genome and numerous intestinal parasite sequences in addition to generating data useful for characterizing the microbiome.

The Coral Triangle is widely considered the most important centre of marine biodiversity in Asia while areas on its periphery such as the South China Sea, have received much less interest. Here, we demonstrate that a small population of the knobbly sea starProtoreaster nodosusin Singapore has similarly high levels of genetic diversity as comparable Indonesian populations from the Coral Triangle. The high genetic diversity of this population is remarkable because it is maintained despite decades of continued anthropogenic disturbance. We postulate that it is probably due to broadcast spawning which is likely to maintain high levels of population connectivity. To test this, we analysed 6140 genome-wide single nucleotide polymorphism (SNP) loci for Singapore's populations and demonstrate a pattern of near panmixia. We here document a second case of high genetic diversity and low genetic structure for a broadcast spawner in Singapore, which suggests that such species have high resilience against anthropogenic disturbances. The study demonstrates the feasibility and power of using genome-wide SNPs for connectivity studies of marine invertebrates without a sequenced genome.

In this paper, we describe the development of Hennig's most important phyloge? netic concepts, which culminated in the publication of the now famous Phylogenetic Systematics in 1966, Hennig proposed strict definitions of monophyly and phylogenetic relationships as early as 1950 in Grundzuge einer Theorie der phylogenetischen Systematise. However, in this edition of the book, the terms "apomorphic" and ''plesiomorphic" were introduced for taxa and not for characters. It was not until 1952 that these terms were consistently applied to characters, hereby for the first time allowing the reconstruction of phylogenetic relationships. But this is not Hennig's sole contribution to systematics, as revealed by a comparison of his Phylogenetic Sys? tematics with publications of the "new systematics" school (e.g., Huxley, 1940, The new system? atics, Oxford Univ. Press, Oxford, England; Mayr et al., 1953, Methods and principles of systematic zoology, McGraw-Hill, New York). Hennig was the person who redirected the interest of sys? tematics to the study of supraspecific taxa after years of focusing on species and infraspecific taxa. (History; phylogenetic systematics; cladistics; monophyly; Hennig; apomorphy; plesio- morphy.) The oldest field of the biological sci? ences, systematics, underwent dramatic changes in the middle of this century. These changes were in part initiated by an entomologist, Willi Hennig, who clarified or, depending on your systematic persua? sion, redefined the goals of phylogenetic systematics in his Phylogenetic Systematics (1966). The history of phylogenetic sys? tematics sensu Hennig has not been exten? sively studied. Surprisingly, most contri? butions that deal with this subject focus on systematists that anticipated Hennig's ideas, were influential in their develop? ment, or dealt with their reception in var? ious countries (Hull, 1988; Craw, 1992

Recently Lorenzen and Sieg (1991) criticized cladistic computer programs as unreliable because they fail to find “all most parsimonious trees of relationships” and they find “wrong” ones. These authors suggest criteria which a “perfect parsimony program” should meet. Their analysis is flawed because of their: 1. peculiar definition of parsimony and relationship, 2. use of an outdated version of PAUP, and 3. misinterpretation of the programs' output. It is shown that PAUP and HENNIG86 by and large meet their criteria and find all most parsimonious cladograms for the hypothetical data set used by Lorenzen and Sieg. Zusammenfassung In einer kurzlich publizierten Arbeit charakterisieren Lorenzen und Sieg kladistische Computer-Programme als nicht zuverlassig, weil sie nicht nur nicht alle sparsamsten Stammbaume finden, sondern zudem auch falsche Stammbaume produzieren. Lorenzen und Sieg schlagen Kriterien vor, die von “perfekten kladistischen Programmen” erfullt werden sollen. Ihre Kritik ist verfehlt, weil sie letztlich das Resultat einer 1. ungebrauchlichen Definition vom Sparsamkeitsprinzip und Verwandtschaftsdiagramm ist, 2. in ihrer Studie eine veraltete Version von PAUP benutzt haben und 3. die Ergebnisdateien der Programme falsch ausgewertet haben. Es wird gezeigt, das PAUP und HENNIG 86 die von Lorenzen und Sieg aufgestellten Kriterien weitestgehend erfullen und alle sparsamsten Stammbaume fur den hypothetischen Datensatz finden, den Lorenzen und Sieg in ihrer Arbeit verwendeten.

The current usage of taxon names is ambiguous because the same name is sometimes used for taxa including the ‘stem lineage’ and sometimes for taxa that only comprise the monophylum that comprises all extant species. The confusion that results and the rather cumbersome terminology currently in use is reviewed and a new terminology is proposed. To the name of the more inclusive taxon the prefix ‘pan’ should be added. The unchanged taxon name or the taxon name with an added ‘crown’ refers to the less inclusive taxon. Furthermore, the term ‘ancestral lineage’ is normally introduced to replace Ax's stem lineage sensu stricto and the term ‘para-taxon’ to refer to the stem lineage sensu lato. Examples demonstrate the necessity for precise terminology and usefulness of the proposed definitions. Zusammenfassung Der bisherige Gebrauch von Taxonnamen ist zweideutig. Derselbe Begriff wird zum einen auf das Monophylum beschrankt, welches ausschlieslich die Nachkommen der gemeinsamen Stammart aller rezenten Arten eines Taxons beinhaltet, zum anderen jedoch zur Benennung eines Taxons verwendet, welches zusatzlich die Vorfahren einschlieslich aller Seitenaste der Stammart des ersten Taxons umfast. Die Problematik dieser Benennung und fruhere Losungsvorschlage werden diskutiert. Eine neue Terminologie wird vorgeschlagen. Fur das erstgenannte Taxon wird mit Jefferies das Praefix ‚Krone’ vorgeschlagen, fur das Taxon, welches die Stammlinie mit einschliest, in Anlehnung an Lauterbach das Praefix ‚Pan’. Die Arten, die direkte Vorfahren einer ‚Krone-Gruppe’ sind – die Stammlinie sensu stricto nach Ax – sollen unter dem Begriff ‚Ahnenlinie’ zusammengefast werden. Der Terminus ‚Stammlinie sensu lato‚ nach Ax soll durch den Begriff ‚Para-Taxon’ ersetzt werden. Beispiele demonstrieren die Notwendigkeit einer prazisen Terminologie und die Praktikabilitat der vorgeschlagenen Definitionen.

DNA barcoding is one of the best funded and most visible large-scale initiatives in systematic biology and has generated both much interest and controversy. DNA barcoding has also attracted significant support from foundations that had previously shown little interest in systematics. Yet, the project is controversial because many systematists feel that currently the conceptual foundation of DNA barcoding remains weak. This problem can only be alleviated through additional research that can lead to improved tools and concepts. Here, we scrutinize a key concept of DNA barcoding, the so-called barcoding gap (Meyer and Paulay, 2005), and use empirical data to document that it needs to be computed based on the smallest instead of the mean interspecific distances. In the literature on DNA barcoding, the “barcoding gap” (Meyer and Paulay, 2005) refers to the separation between mean intraand interspecific sequence variability for congeneric COI sequences. The barcoding gap is so essential to barcoding that a widely cited publication was dedicated to documenting these gaps across major metazoan taxa (Hebert et al., 2003b). It is also regularly mentioned in articles promoting barcoding to a broader audience (Check, 2005; Cognato and Caesar, 2006; Dasmahapatra and Mallet, 2006) and is one of the few metrics included in the Web-based identification system BOLD, “The Barcode of Life Data System,” which is a major identification tool for the DNA barcoding community (http://www.barcodinglife.org; Ratnasingham and Hebert, 2007). Large barcoding gaps are routinely used to predict DNA-barcoding success for the taxon under study (Hebert et al., 2003a, 2003b, 2004a, 2004b; Hogg and Hebert, 2004; Powers, 2004; Zehner et al., 2004; Armstrong and Ball, 2005; Ball et al., 2005; Barrett and Hebert, 2005; Lorenz et al., 2005; Saunders, 2005; Smith et al., 2005, 2006; Ward et al., 2005; Cywinska et al., 2006; Hajibabaei et al., 2006a, 2006b; Lefebure et al., 2006; Clare et al., 2007; Seifert et al., 2007). However, here we argue and document that barcoding gaps are currently incorrectly computed and that the values reported in the barcoding literature are misleading. The main problem is that the barcoding gap is generally quantified as the difference between intraspecific and mean interspecific, congeneric distances, whereas we will argue here that for species identification only the smallest interspecific distance should be used. Other authors have also pointed out that the use of smallest interspecific distances would be more appropriate (see Sperling, 2003; Moritz and Cicero, 2004; Vences et al., 2005a, 2005b; Cognato, 2006; Meier et al., 2006; Meyer and Paulay, 2005; Roe and Sperling, 2007), but currently we lack a comparative study that documents that the two measures yield different results. Here we provide evidence based on 43,137 COI sequences from 12,459 Metazoan species that barcoding gaps based on mean interspecific distances are artificially inflated and that only smallest interspecific distances correctly reflect that species identification gets more difficult as more species are sampled. Using DNA barcodes for species identification is analogous to identifying an unidentified specimen by comparing it to a reference collection of identified specimens. Initially one may compare an unidentified specimen to all identified material in the same genus, but ultimately the identification problem pares down to deciding whether a specimen belongs to one of a few, very similar, congeneric species. Determining an unidentified specimen to species is straightforward if the intraspecific variability is small—i.e., the unidentified specimen is a good match to a referenced species—and the differences between the best-matching species and the next best match is large—i.e., the specimen is a good match to only one of the referenced species. Analogously, the ease with which a query sequence can be identified to species is only dependent on how different it is from the most similar allospecific sequence, whereas its distinctness from a hypothetical “average” congeneric species does not matter (see Sperling, 2003; Moritz and Cicero, 2004; Vences et al., 2005a, 2005b; Cognato, 2006; Meier et al., 2006; Meyer and Paulay, 2005; Roe and Sperling, 2007). Yet, DNA barcoding publications and BOLD continue to report the mean instead of the smallest interspecific distances for congeneric species.

The rapid increase of brain size is a key event in human evolution. Abnormal spindle-like microcephaly associated (ASPM) is discussed as a major candidate gene for explaining the exceptionally large brain in humans but ASPM's role remains controversial. Here we use codon-specific models and a comparative approach to test this candidate gene that was initially identified in Homo-chimp comparisons. We demonstrate that accelerated evolution of ASPM (omega = 4.7) at 16 amino acid sites occurred in 9 primate lineages with major changes in relative cerebral cortex size. However, ASPM's evolution is not correlated with major changes in relative whole-brain or cerebellum sizes. Our results suggest that a single candidate gene such as ASPM can influence a specific component of the brain across large clades through changes in a few amino acid sites. We furthermore illustrate the power of using continuous phenotypic variability across primates to rigorously test candidate genes that have been implicated in the evolution of key human traits.

Approximately 5% of the known species-level diversity of Diptera belongs to the Muscoidea with its approximately 7000 described species. Despite including some of the most abundant and well known flies, the phylogenetic relationships within this superfamily are poorly understood. Previous attempts at reconstructing the relationships based on morphology and relatively small molecular data sets were only moderately successful. Here, we use molecular data for 127 exemplar species of the Muscoidea, two species from the Hippoboscoidea, ten species representing the Oestroidea and seven outgroup species from four acalyptrate superfamilies. Four mitochondrial genes 12S, 16S, COI, and Cytb, and four nuclear genes 18S, 28S, Ef1a, and CAD are used to reconstruct the relationships within the Muscoidea. The length-variable genes were aligned using a guide tree that was based on the protein-encoding genes and the indel-free sections of the ribosomal genes. We found that, based on topological considerations, this guide tree was a significant improvement over the default guide trees generated by ClustalX. The data matrix was analyzed using maximum parsimony (MP) and maximum likelihood (ML) and yielded very similar tree topologies. The Calyptratae are monophyletic and the Hippoboscoidea are the sister group to the remaining calyptrates (MP). The Muscoidea are paraphyletic with a monophyletic Oestroidea nested within the Muscoidea as sister group to Anthomyiidae + Scathophagidae. The monophyly of three of the four recognized families in the Muscoidea is confirmed: the Fanniidae, Muscidae, and Scathophagidae. However, the Anthomyiidae are possibly paraphyletic. Within the Oestroidea, the Sarcophagidae and Tachinidae are sister groups and the Calliphoridae are paraphyletic.

The evolution rates of mtDNA in early metazoans hold important implications for DNA barcoding. Here, we present a comprehensive analysis of intra- and interspecific COI variabilities in Porifera and Cnidaria (separately as Anthozoa, Hydrozoa, and Scyphozoa) using a data set of 619 sequences from 224 species. We found variation within and between species to be much lower in Porifera and Anthozoa compared to Medusozoa (Hydrozoa and Scyphozoa), which has divergences similar to typical metazoans. Given that recent evidence has shown that fungi also exhibit limited COI divergence, slow-evolving mtDNA is likely to be plesiomorphic for the Metazoa. Higher rates of evolution could have originated independently in Medusozoa and Bilateria or been acquired in the Cnidaria + Bilateria clade and lost in the Anthozoa. Low identification success and substantial overlap between intra- and interspecific COI distances render the Anthozoa unsuitable for DNA barcoding. Caution is also advised for Porifera and Hydrozoa because of relatively low identification success rates as even threshold divergence that maximizes the “barcoding gap” does not improve identification success.

We examined the phylogenetic relationships among taxa of attine or "fungus-growing" ants (Tribe Attini) using parsimony analyses of molecular and morphological data. We sequenced a region of mitochondrial DNA from 13 taxa of attines and from one closely related outgroup species, Wasmannia auropunctata. Our study sequence included the 3' end of the cytochrome c oxidase subunit I (CO I) locus (183 to 198 total sites; 91 informative sites), an intergenic spacer region of variable size (0 to 152 sites), the tRNA leucine locus (65 to 74 sites), and the 5' end of the cytochrome c oxidase subunit II (co II) locus (292 total sites; 140 informative sites). The inferred relationships among the attine taxa based on an unweighted analysis of the nucleotide sequence data closely matched the relationships inferred from an analysis of larval morphological characters from 11 of the taxa. In fact, the inferred relationships were completely congruent among the protein parsimony analysis of amino acid data, the morphology analysis, and "total evidence" analysis combining the amino acid and morphology data. The congruent conclusions we obtained from two independent data sets increases our confidence in the reliability of our analyses.

A method for quantifying levels of observed homoplasy in cladistic analyses is proposed. The homoplasy slope is a function that describes the relationship between number of taxa in a cladistic analysis and the observed number of extra steps per character. The homoplasy slope ratio (HSR) relates the homoplasy slope of a cladistic analysis (real data) to that of an analysis based on random binary data sets of the same size. Tests show that HSR is not correlated with numbers of characters and taxa, and this quality makes the index particularly useful for comparing the levels of observed homoplasy among cladistic analyses based on data sets of different sizes. All of the other homoplasy indices currently available-including the consistency index (CI), retention index (RI), and homoplasy excess ratio (HER)-are correlated with number of characters and/or number of taxa. (Homoplasy indices; homoplasy comparison; homoplasy slope ratio; consistency index; homoplasy excess ratio; retention index.) The discussion of homoplastic evolution commonly has focused on how it compli- cates the reconstruction of phylogenies. Systematists distinguish three kinds of nonhomologous similarity or homoplasy (Futuyma, 1986): parallelism, convergence, and reversal. These homoplasies are not a major problem for phylogenetic recon- struction if they are identified prior to the analysis and recoded or excluded from the data set. Additional homoplasies may be discovered during the cladistic analysis as incongruent character states that lengthen the cladogram beyond the sum of the range of the characters (Lmin) by any number of extra steps (EXS). The number of extra steps is thus a measure of character-state distri- butions that do not fit the hypothesized phylogeny. The literature about homoplastic evo