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Knowledge of global patterns of genetic diversity is essential for biodiversity conservation as this parameter describes the ability of a species to respond to environmental changes. Ichneumonoids parasitoid wasps are among the few taxa showing an anomalous latitudinal diversity gradient. Using the largest georeferenced molecular dataset for this group, we used a macrogenetics approach to examine latitudinal patterns and predictors of intraspecific genetic diversity. We calculated the mean nucleotide diversity of mitochondrial DNA barcode sequences at three geographic levels: grid cells, latitudinal bands and climatic zones. Nucleotide diversity values were consistently higher at northern temperate latitudes, peaking at 50°. We found a positive but weak relationship between intraspecific diversity and the latitude, between intra- and interspecific diversity, and a positive effect of the temperature range. Examining the spatial relationship between different levels of biodiversity and its drivers is particularly relevant considering climate change and its impact on species distribution. Yet, in insects, it has been challenging to integrate ecological, evolutionary and geographical components when analysing the processes leading to species richness gradients., (© 2024 The Author(s). Journal of Animal Ecology published by John Wiley & Sons Ltd on behalf of British Ecological Society.)

Missing observations in trait datasets pose an obstacle for analyses in myriad biological disciplines. Considering the mixed results of imputation, the wide variety of available methods, and the varied structure of real trait datasets, a framework for selecting a suitable imputation method is advantageous. We invoked a real data-driven simulation strategy to select an imputation method for a given mixed-type (categorical, count, continuous) target dataset. Candidate methods included mean/mode imputation, k-nearest neighbour, random forests, and multivariate imputation by chained equations (MICE). Using a trait dataset of squamates (lizards and amphisbaenians; order: Squamata) as a target dataset, a complete-case dataset consisting of species with nearly complete information was formed for the imputation method selection. Missing data were induced by removing values from this dataset under different missingness mechanisms: missing completely at random (MCAR), missing at random (MAR), and missing not at random (MNAR). For each method, combinations with and without phylogenetic information from single gene (nuclear and mitochondrial) or multigene trees were used to impute the missing values for five numerical and two categorical traits. The performances of the methods were evaluated under each missing mechanism by determining the mean squared error and proportion falsely classified rates for numerical and categorical traits, respectively. A random forest method supplemented with a nuclear-derived phylogeny resulted in the lowest error rates for the majority of traits, and this method was used to impute missing values in the original dataset. Data with imputed values better reflected the characteristics and distributions of the original data compared to complete-case data. However, caution should be taken when imputing trait data as phylogeny did not always improve performance for every trait and in every scenario. Ultimately, these results support the use of a real data-driven simulation strategy for selecting a suitable imputation method for a given mixed-type trait dataset., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 May et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Metagenomics and total RNA sequencing (total RNA-Seq) have the potential to improve the taxonomic identification of diverse microbial communities, which could allow for the incorporation of microbes into routine ecological assessments. However, these target-PCR-free techniques require more testing and optimization. In this study, we processed metagenomics and total RNA-Seq data from a commercially available microbial mock community using 672 data-processing workflows, identified the most accurate data-processing tools, and compared their microbial identification accuracy at equal and increasing sequencing depths. The accuracy of data-processing tools substantially varied among replicates. Total RNA-Seq was more accurate than metagenomics at equal sequencing depths and even at sequencing depths almost one order of magnitude lower than those of metagenomics. We show that while data-processing tools require further exploration, total RNA-Seq might be a favorable alternative to metagenomics for target-PCR-free taxonomic identifications of microbial communities and might enable a substantial reduction in sequencing costs while maintaining accuracy. This could be particularly an advantage for routine ecological assessments, which require cost-effective yet accurate methods, and might allow for the incorporation of microbes into ecological assessments., (© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Postglacial dispersal and colonization processes have shaped community patterns in sub-Arctic regions such as Churchill, Manitoba, and Canada. This study investigates evolutionary community structure within the beetle (Coleoptera) families of Churchill and tests whether biological traits have played a role in governing colonization patterns from refugial and southerly geographic regions. This study quantifies sub-Arctic beetle phylogenetic community structure for each family using the net relatedness index (NRI) and nearest taxon index (NTI), calculated using publicly available data from the Barcode of Life Data Systems (BOLD); compares patterns across families with different traits (habitat, diet) using standard statistical analysis (ANOVA) as well as phylogenetic generalized least squares (PGLS) using a family-level beetle phylogeny obtained from the literature; and compares community structure in Churchill with a region in southern Canada (Guelph, Ontario). These analyses were also repeated at a genus level. The dominant pattern detected in our study was that aquatic families were much better represented in Churchill compared to terrestrial families, when compared against richness sampled from across Canada and Alaska. Individually, most families showed significant phylogenetic clustering in Churchill, likely due to the strong environmental filtering present in Arctic environments. There was no significant difference in phylogenetic structure between Churchill and Guelph but with a trend toward stronger clustering in the North. Fungivores were significantly more overdispersed than other feeding modes, predators were significantly more clustered, and aquatic families showed significantly stronger clustering compared to terrestrial. This study contributes to our understanding of the traits and processes structuring insect biodiversity and macroecological trends in the sub-Arctic., Competing Interests: The authors declare that there are no conflicts of interest., (© 2022 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

DNA barcoding and metabarcoding are now widely used to advance species discovery and biodiversity assessments. High-throughput sequencing (HTS) has expanded the volume and scope of these analyses, but elevated error rates introduce noise into sequence records that can inflate estimates of biodiversity. Denoising -the separation of biological signal from instrument (technical) noise-of barcode and metabarcode data currently employs abundance-based methods which do not capitalize on the highly conserved structure of the cytochrome c oxidase subunit I (COI) region employed as the animal barcode. This manuscript introduces debar, an R package that utilizes a profile hidden Markov model to denoise indel errors in COI sequences introduced by instrument error. In silico studies demonstrated that debar recognized 95% of artificially introduced indels in COI sequences. When applied to real-world data, debar reduced indel errors in circular consensus sequences obtained with the Sequel platform by 75%, and those generated on the Ion Torrent S5 by 94%. The false correction rate was less than 0.1%, indicating that debar is receptive to the majority of true COI variation in the animal kingdom. In conclusion, the debar package improves DNA barcode and metabarcode workflows by aiding the generation of more accurate sequences aiding the characterization of species diversity., (© 2021 John Wiley & Sons Ltd.)

Myriad environmental and biological traits have been investigated for their roles in influencing the rate of molecular evolution across various taxonomic groups. However, most studies have focused on a single trait, while controlling for additional factors in an informal way, generally by excluding taxa. This study utilized a dataset of cytochrome c oxidase subunit I (COI) barcode sequences from over 7000 ray-finned fish species to test the effects of 27 traits on molecular evolutionary rates. Environmental traits such as temperature were considered, as were traits associated with effective population size including body size and age at maturity. It was hypothesized that these traits would demonstrate significant correlations with substitution rate in a multivariable analysis due to their associations with mutation and fixation rates, respectively. A bioinformatics pipeline was developed to assemble and analyze sequence data retrieved from the Barcode of Life Data System (BOLD) and trait data obtained from FishBase. For use in phylogenetic regression analyses, a maximum likelihood tree was constructed from the COI sequence data using a multi-gene backbone constraint tree covering 71% of the species. A variable selection method that included both single- and multivariable analyses was used to identify traits that contribute to rate heterogeneity estimated from different codon positions. Our analyses revealed that molecular rates associated most significantly with latitude, body size, and habitat type. Overall, this study presents a novel and systematic approach for integrative data assembly and variable selection methodology in a phylogenetic framework.

Biological conclusions based on DNA barcoding and metabarcoding analyses can be strongly influenced by the methods utilized for data generation and curation, leading to varying levels of success in the separation of biological variation from experimental error. The 5' region of cytochrome c oxidase subunit I (COI-5P) is the most common barcode gene for animals, with conserved structure and function that allows for biologically informed error identification. Here, we present coil ( https://CRAN.R-project.org/package=coil ), an R package for the pre-processing and frameshift error assessment of COI-5P animal barcode and metabarcode sequence data. The package contains functions for placement of barcodes into a common reading frame, accurate translation of sequences to amino acids, and highlighting insertion and deletion errors. The analysis of 10 000 barcode sequences of varying quality demonstrated how coil can place barcode sequences in reading frame and distinguish sequences containing indel errors from error-free sequences with greater than 97.5% accuracy. Package limitations were tested through the analysis of COI-5P sequences from the plant and fungal kingdoms as well as the analysis of potential contaminants: nuclear mitochondrial pseudogenes and Wolbachia COI-5P sequences. Results demonstrated that coil is a strong technical error identification method but is not reliable for detecting all biological contaminants.

Aim: We construct a framework for mapping pattern and drivers of insect diversity at the continental scale and use it to test whether and which environmental gradients drive insect beta diversity., Location: Global; North and Central America; Western Europe., Time Period: 21st century., Major Taxa Studied: Insects., Methods: An informatics system was developed to integrate terrestrial data on insects with environmental parameters. We mined repositories of data for distribution, climatic data were retrieved (WorldClim), and vegetation parameters inferred from remote sensing analysis (MODIS Vegetation Continuous Fields). Beta diversity between sites was calculated and then modeled with two methods, Mantel test with multiple regression and generalized dissimilarity modeling., Results: Geographic distance was the main driver of insect beta diversity. Independent of geographic distance, bioclimate variables explained more variance in dissimilarity than vegetation variables, although the particular variables found to be significant were more consistent in the latter, particularly, tree cover. Tree cover gradients drove compositional dissimilarity at denser coverages, in both continental case studies. For climate, gradients in temperature parameters were significant in driving beta diversity more so than gradients in precipitation parameters., Main Conclusions: Although environmental gradients drive insect beta diversity independently of geography, the relative contribution of different climatic and vegetational parameters is not expected to be consistent in different study systems. With further incorporation of additional temporal information and variables, this approach will enable the development of a predictive framework for conserving insect biodiversity at the global scale., Competing Interests: None declared., (© 2019 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

A detailed morphological comparison was carried out among specimens of several samples of Archaebranchinecta Rogers Coronel, 2011 from the Altiplano of Peru, Bolivia, and Argentina. Surprisingly, striking differences were found between Peruvian samples collected near the western shore of Lake Titicaca, and those from Bolivia taken southwardly, near the east coasts of River Desaguadero and Lake Poopó. Accordingly, the new species Archaebranchinecta aimara sp. nov. is described, representing the second specific entity of a genus that so far included only A. pollicifera (Harding, 1940). The main differential features between both species include: (a) size and shape of the three processes of basal segment of the male second antenna; (b) shape and protrusion degree of the pair of medioventral bulges in the male genital segments; (c) presence or absence of strong ventrolateral spine on the second genital segment of female; and (d) presence or absence of a pair of ventrolateral outgrowths in the brood pouch. This research contributes to our understanding of the biodiversity and endemism of the unique Altiplano region of South America.

The evolutionary speed hypothesis (ESH) suggests that molecular evolutionary rates are higher among species inhabiting warmer environments. Previously, the ESH has been investigated using small numbers of latitudinally-separated sister lineages; in animals, these studies typically focused on subsets of Chordata and yielded mixed support for the ESH. This study analyzed public DNA barcode sequences from the cytochrome c oxidase subunit I (COI) gene for six of the largest animal phyla (Arthropoda, Chordata, Mollusca, Annelida, Echinodermata, and Cnidaria) and paired latitudinally-separated taxa together informatically. Of 8037 lineage pairs, just over half (51.6%) displayed a higher molecular rate in the lineage inhabiting latitudes closer to the equator, while the remainder (48.4%) displayed a higher rate in the higher-latitude lineage. To date, this study represents the most comprehensive analysis of latitude-related molecular rate differences across animals. While a statistically-significant pattern was detected from our large sample size, our findings suggest that the EHS may not serve as a strong universal mechanism underlying the latitudinal diversity gradient and that COI molecular clocks may generally be applied across latitudes. This study also highlights the merits of using automation to analyze large DNA barcode datasets.

Divergence times for species assemblages of Arctic marine invertebrates have often been estimated using a standard rate (1.4%/MY) of molecular evolution calibrated using a single sister pair of tropical crustaceans. Because rates of molecular evolution vary among taxa and environments, it is essential to obtain clock calibrations from northern lineages. The recurrent opening and closure of the Bering Strait provide an exceptional opportunity for clock calibration. Here, we apply the iterative calibration approach to investigate patterns of molecular divergence among lineages of northern marine molluscs and arthropods using publicly available sequences of the cytochrome c oxidase subunit I (COI) gene and compare these results with previous estimates of trans-Bering divergences for echinoderms and polychaetes. The wide range of Kimura two-parameter (K2P) divergences among 73 trans-Bering sister pairs (0.12%-16.89%) supports multiple pulses of migration through the Strait. Overall, the results indicate a rate of K2P divergence of 3.2%/MY in molluscs, 5%-5.2%/MY in arthropods, and 3.5%-4.7%/MY in polychaetes. While these rates are considerably higher than the often-adopted 1.4%/MY rate, they are similar to calibrations (3%-5%/MY) in several other studies of marine invertebrates. This upward revision in rates means there is a need both to reevaluate the evolutionary history of marine lineages and to reexamine the impact of prior climatic changes upon the diversification of marine life.

Chironomid flies (non-biting midges) are among the most abundant and diverse animals in Arctic regions, but detailed analyses of species distributions and biogeographical patterns are hampered by challenging taxonomy and reliance on morphology for species-level identification. Here we take advantage of available DNA barcode data of Arctic Chironomidae in BOLD to analyse similarities in species distributions across a northern Nearctic - West Palearctic gradient. Using more than 260 000 barcodes representing 4666 BINs (Barcode Index Numbers) and 826 named species (some with interim names) from a combination of public and novel data, we show that the Greenland chironomid fauna shows affinities to both the Nearctic and the West Palearctic regions. While raw taxon counts indicate a strong Greenland - North American affinity, comparisons using Chao's dissimilarity metric support a slightly higher similarity between Greenland and West Palearctic chironomid communities. Results were relatively consistent across different definitions of species taxonomic units, including morphologically determined species, BINs, and superBINs based on a ∼4.5% threshold. While most taxa found in Greenland are shared with at least one other region, reflecting circum-Arctic dispersal, our results also reveal that Greenland harbours a small endemic biodiversity. Our exploratory study showcases how DNA barcoding efforts using standardized gene regions contribute to an understanding of broad-scale patterns in biogeography by enabling joint analysis of public DNA sequence data derived from diverse prior studies.

Ancient lakes are renowned for their exceptional diversity of endemic species. As model systems for the study of sympatric speciation, it is necessary to understand whether a given hypothesized species flock is of monophyletic or polyphyletic origin. Here, we present the first molecular characterization of the Hyalella (Crustacea: Amphipoda) species complex of Lake Titicaca, using COI and 28S DNA sequences, including samples from the connected Small and Large Lakes that comprise Lake Titicaca as well as from a broader survey of southern South American sites. At least five evolutionarily distant lineages are present within Lake Titicaca, which were estimated to have diverged from one another 12-20 MYA. These major lineages are dispersed throughout the broader South American Hyalella phylogeny, with each lineage representing at least one independent colonization of the lake. Moreover, complex genetic relationships are revealed between Lake Titicaca individuals and those from surrounding water bodies, which may be explained by repeated dispersal into and out of the lake, combined with parallel intralacustrine diversification within two separate clades. Although further work in deeper waters will be required to determine the number of species present and modes of diversification, our results strongly indicate that this amphipod species cloud is polyphyletic with a complex geographic history., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Plants and herbivorous (or parasitic) insects form the majority of macroscopic life. The specificity of interaction between host plant and parasitic insect depends on the adaptations of both the host and the parasite. Over time, these interactions evolve and change as a result of an 'arms race' between host and parasite, and the resulting species-specific adaptations may be maintained, perpetuating these interactions across speciation events. This can lead to specialisation between species or clades. With speciation and species sorting over time, complex interactions evolve. Here, we elucidate a three-tier method to test these interactions using the aphids (Hemiptera: Aphididae) and plants of Churchill (Manitoba, Canada) as a model system. We analyzed these interactions by testing for three patterns in host specificity: monophagy, phylogenetic clustering, and cophylogeny. We defined monophagy strictly as one species feeding exclusively upon a single host plant species (an association likely driven by arms races in morphology, chemical resistance/tolerance, and visual appearance) and observed this in 7 of 22 aphid species. In all the remaining 'polyphagous' cases, there was a strong trend toward monophagy (80% of individuals were found on a single host plant species). Second, we observed two separate examples of phylogenetic clustering where groups of closely related aphid species fed upon individual plant species. Finally, we found no support for cophylogenetic relationships where both aphids and plants cospeciate to form congruent phylogenetic trees (evidence of coadaptation through an ongoing arms race). One explanation for uncovering species-specific interactions in a recently deglaciated, subarctic locality is that the species involved in the associations moved north together. Testing different levels of specificity in the most predominant species-species interactions on the planet will allow us to elucidate these patterns accurately and gives us insight into where to direct future research., (© The Author(s) 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

During the past 50 years, the molecular clock has become one of the main tools for providing a time scale for the history of life. In the era of robust molecular evolutionary analysis, clock calibration is still one of the most basic steps needing attention. When fossil records are limited, well-dated geological events are the main resource for calibration. However, biogeographic calibrations have often been used in a simplistic manner, for example assuming simultaneous vicariant divergence of multiple sister lineages. Here, we propose a novel iterative calibration approach to define the most appropriate calibration date by seeking congruence between the dates assigned to multiple allopatric divergences and the geological history. Exploring patterns of molecular divergence in 16 trans-Bering sister clades of echinoderms, we demonstrate that the iterative calibration is predominantly advantageous when using complex geological or climatological events-such as the opening/reclosure of the Bering Strait-providing a powerful tool for clock dating that can be applied to other biogeographic calibration systems and further taxa. Using Bayesian analysis, we observed that evolutionary rate variability in the COI-5P gene is generally distributed in a clock-like fashion for Northern echinoderms. The results reveal a large range of genetic divergences, consistent with multiple pulses of trans-Bering migrations. A resulting rate of 2.8% pairwise Kimura-2-parameter sequence divergence per million years is suggested for the COI-5P gene in Northern echinoderms. Given that molecular rates may vary across latitudes and taxa, this study provides a new context for dating the evolutionary history of Arctic marine life.

Participants in the 7th International Barcode of Life Conference (Kruger National Park, South Africa, 20-24 November 2017) share the latest findings in DNA barcoding research and its increasingly diversified applications. Here, we review prevailing trends synthesized from among 429 invited and contributed abstracts, which are collated in this open-access special issue of Genome. Hosted for the first time on the African continent, the 7th Conference places special emphasis on the evolutionary origins, biogeography, and conservation of African flora and fauna. Within Africa and elsewhere, DNA barcoding and related techniques are being increasingly used for wildlife forensics and for the validation of commercial products, such as medicinal plants and seafood species. A striking trend of the conference is the dramatic rise of studies on environmental DNA (eDNA) and on diverse uses of high-throughput sequencing techniques. Emerging techniques in these areas are opening new avenues for environmental biomonitoring, managing species-at-risk and invasive species, and revealing species interaction networks in unprecedented detail. Contributors call for the development of validated community standards for high-throughput sequence data generation and analysis, to enable the full potential of these methods to be realized for understanding and managing biodiversity on a global scale.

The evolution of powered flight is a major innovation that has facilitated the success of insects. Previously, studies of birds, bats, and insects have detected molecular signatures of differing selection regimes in energy-related genes associated with flight evolution and/or loss. Here, using DNA sequences from more than 1000 nuclear and mitochondrial protein-coding genes obtained from insect transcriptomes, we conduct a broader exploration of which gene categories display positive and relaxed selection at the origin of flight as well as with multiple independent losses of flight. We detected a number of categories of nuclear genes more often under positive selection in the lineage leading to the winged insects (Pterygota), related to catabolic processes such as proteases, as well as splicing-related genes. Flight loss was associated with relaxed selection signatures in splicing genes, mirroring the results for flight evolution. Similar to previous studies of flight loss in various animal taxa, we observed consistently higher nonsynonymous-to-synonymous substitution ratios in mitochondrial genes of flightless lineages, indicative of relaxed selection in energy-related genes. While oxidative phosphorylation genes were not detected as being under selection with the origin of flight specifically, they were most often detected as being under positive selection in holometabolous (complete metamorphosis) insects as compared with other insect lineages. This study supports some convergence in gene-specific selection pressures associated with flight ability, and the exploratory analysis provided some new insights into gene categories potentially associated with the gain and loss of flight in insects., (© The Authors 2017. Published by Oxford University Press.)

While pollinators are widely acknowledged as important contributors to seed production in plant communities, we do not yet have a good understanding of the importance of pollinator specialists for this ecosystem service. Determination of the prevalence of pollinator specialists is often hindered by the occurrence of cryptic species and the limitations of observational data on pollinator visitation rates, two areas where DNA barcoding of pollinators and pollen can be useful. Further, the demonstrated adequacy of pollen DNA barcoding from historical records offers opportunities to observe the effects of pollinator loss over longer timescales, and phylogenetic approaches can elucidate the historical rates of extinction of specialist lineages. In this Viewpoint article, we review how advances in DNA barcoding and metabarcoding of plants and pollinators have brought important developments to our understanding of specialization in plant-pollinator interactions. We then put forth several lines of inquiry that we feel are especially promising for providing insight on changes in plant-pollinator interactions over space and time. Obtaining estimates of the effects of reductions in specialists will contribute to forecasting the loss of ecosystem services that will accompany the erosion of plant and pollinator diversity., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

DNA barcoding has been used successfully for identifying specimens belonging to marine planktonic groups. However, the ability to delineate species within taxonomically diverse and widely distributed marine groups, such as the Copepoda and Thecostraca, remains largely untested. We investigate whether a cytochrome c oxidase subunit I (COI-5P) global pairwise sequence divergence threshold exists between intraspecific and interspecific divergences in the copepods plus the thecostracans (barnacles and allies). Using publicly accessible sequence data, we applied a graphical method to determine an optimal threshold value. With these thresholds, and using a newly generated planktonic marine data set, we quantify the degree of concordance using a bidirectional analysis and discuss different analytical methods for sequence-based species delimitation (e.g., BIN, ABGD, jMOTU, UPARSE, Mothur, PTP, and GMYC). Our results support a COI-5P threshold between 2.1% and 2.6% p-distance across methods for these crustacean taxa, yielding molecular groupings largely concordant with traditional, morphologically defined species. The adoption of internal methods for clustering verification enables rapid biodiversity studies and the exploration of unknown faunas using DNA barcoding. The approaches taken here for concordance assessment also provide a more quantitative comparison of clustering results (as contrasted with "success/failure" of barcoding), and we recommend their further consideration for barcoding studies.

Additive diversity partitioning (α, β, and γ) is commonly used to study the distribution of species-level diversity across spatial scales. Here, we first investigate whether published studies of additive diversity partitioning show signs of difficulty attaining species-level resolution due to inherent limitations with morphological identifications. Second, we present a DNA barcoding approach to delineate specimens of stream caddisfly larvae (order Trichoptera) and consider the importance of taxonomic resolution on classical (additive) measures of beta (β) diversity. Caddisfly larvae were sampled using a hierarchical spatial design in two regions (subarctic Churchill, Manitoba, Canada; temperate Pennsylvania, USA) and then additively partitioned according to Barcode Index Numbers (molecular clusters that serve as a proxy for species), genus, and family levels; diversity components were expressed as proportional species turnover. We screened 114 articles of additive diversity partitioning and found that a third reported difficulties with achieving species-level identifications, with a clear taxonomic tendency towards challenges identifying invertebrate taxa. Regarding our own study, caddisfly BINs appeared to show greater subregional turnover (e.g., proportional additive β) compared to genus or family levels. Diversity component studies failing to achieve species resolution due to morphological identifications may therefore be underestimating diversity turnover at larger spatial scales.

Insect lineages have crossed between terrestrial and aquatic habitats many times, for both immature and adult life stages. We explore patterns in molecular evolutionary rates between 42 sister pairs of related terrestrial and freshwater insect clades using publicly available protein-coding DNA sequence data from the orders Coleoptera, Diptera, Lepidoptera, Hemiptera, Mecoptera, Trichoptera, and Neuroptera. We furthermore test for habitat-associated convergent molecular evolution in the cytochrome c oxidase subunit I (COI) gene in general and at a particular amino acid site previously reported to exhibit habitat-linked convergence within an aquatic beetle group. While ratios of nonsynonymous-to-synonymous substitutions across available loci were higher in terrestrial than freshwater-associated taxa in 26 of 42 lineage pairs, a stronger trend was observed (20 of 31, p binomial = 0.15, p Wilcoxon = 0.017) when examining only terrestrial-aquatic pairs including fully aquatic taxa. We did not observe any widespread changes at particular amino acid sites in COI associated with habitat shifts, although there may be general differences in selection regime linked to habitat.

The major branches of life diversified in the marine realm, and numerous taxa have since transitioned between marine and freshwaters. Previous studies have demonstrated higher rates of molecular evolution in crustaceans inhabiting continental saline habitats as compared with freshwaters, but it is unclear whether this trend is pervasive or whether it applies to the marine environment. We employ the phylogenetic comparative method to investigate relative molecular evolutionary rates between 148 pairs of marine or continental saline versus freshwater lineages representing disparate eukaryote groups, including bony fish, elasmobranchs, cetaceans, crustaceans, mollusks, annelids, algae, and other eukaryotes, using available protein-coding and noncoding genes. Overall, we observed no consistent pattern in nucleotide substitution rates linked to habitat across all genes and taxa. However, we observed some trends of higher evolutionary rates within protein-coding genes in freshwater taxa-the comparisons mainly involving bony fish-compared with their marine relatives. The results suggest no systematic differences in substitution rate between marine and freshwater organisms., (© 2016 The Author(s). Evolution © 2016 The Society for the Study of Evolution.)

DNA barcoding--the sequencing of short, standardized DNA regions for specimen identification and species discovery--has promised to facilitate rapid access to biodiversity knowledge by diverse users. Here, we advance our opinion that increased global participation in genetics research is beneficial, both to scientists and for science, and explore the premise that DNA barcoding can help to democratize participation in genetics research. We examine publication patterns (2003-2014) in the DNA barcoding literature and compare trends with those in the broader, related domain of genomics. While genomics is the older and much larger field, the number of nations contributing to the published literature is similar between disciplines. Meanwhile, DNA barcoding exhibits a higher pace of growth in the number of publications as well as greater evenness among nations in their proportional contribution to total authorships. This exploration revealed DNA barcoding to be a highly international discipline, with growing participation by researchers in especially biodiverse nations. We briefly consider several of the challenges that may hinder further participation in genetics research, including access to training and molecular facilities as well as policy relating to the movement of genetic resources.

Studies examining phylogenetic community structure have become increasingly prevalent, yet little attention has been given to the influence of the input phylogeny on metrics that describe phylogenetic patterns of co-occurrence. Here, we examine the influence of branch length, tree reconstruction method, and amount of sequence data on measures of phylogenetic community structure, as well as the phylogenetic signal (Pagel's λ) in morphological traits, using Trichoptera larval communities from Churchill, Manitoba, Canada. We find that model-based tree reconstruction methods and the use of a backbone family-level phylogeny improve estimations of phylogenetic community structure. In addition, trees built using the barcode region of cytochrome c oxidase subunit I (COI) alone accurately predict metrics of phylogenetic community structure obtained from a multi-gene phylogeny. Input tree did not alter overall conclusions drawn for phylogenetic signal, as significant phylogenetic structure was detected in two body size traits across input trees. As the discipline of community phylogenetics continues to expand, it is important to investigate the best approaches to accurately estimate patterns. Our results suggest that emerging large datasets of DNA barcode sequences provide a vast resource for studying the structure of biological communities.

The 6th International Barcode of Life Conference (Guelph, Canada, 18-21 August 2015), themed Barcodes to Biomes, showcases the latest developments in DNA barcoding research and its diverse applications. The meeting also provides a venue for a global research community to share ideas and to initiate collaborations. All plenary and contributed abstracts are being published as an open-access special issue of Genome. Here, I use a comparison with the 3rd Conference (Mexico City, 2009) to highlight 10 recent and emerging trends that are apparent among the contributed abstracts. One of the outstanding trends is the rising proportion of abstracts that focus upon multiple socio-economically important applications of DNA barcoding, including studies of agricultural pests, quarantine and invasive species, wildlife forensics, disease vectors, biomonitoring of ecosystem health, and marketplace surveys evaluating the authenticity of seafood products and medicinal plants. Other key movements include the use of barcoding and metabarcoding approaches for dietary analyses-and for studies of food webs spanning three or more trophic levels-as well as the spread of next-generation sequencing methods in multiple contexts. In combination with the rising taxonomic and geographic scope of many barcoding iniatives, these developments suggest that several important questions in biology are becoming tractable. "What is this specimen on an agricultural shipment?", "Who eats whom in this whole food web?", and even "How many species are there?" are questions that may be answered in time periods ranging from a few years to one or a few decades. The next phases of DNA barcoding may expand yet further into prediction of community shifts with climate change and improved management of biological resources.

Background: Arctic ecosystems, especially those near transition zones, are expected to be strongly impacted by climate change. Because it is positioned on the ecotone between tundra and boreal forest, the Churchill area is a strategic locality for the analysis of shifts in faunal composition. This fact has motivated the effort to develop a comprehensive biodiversity inventory for the Churchill region by coupling DNA barcoding with morphological studies. The present study represents one element of this effort; it focuses on analysis of the spider fauna at Churchill., Results: 198 species were detected among 2704 spiders analyzed, tripling the count for the Churchill region. Estimates of overall diversity suggest that another 10-20 species await detection. Most species displayed little intraspecific sequence variation (maximum <1%) in the barcode region of the cytochrome c oxidase subunit I (COI) gene, but four species showed considerably higher values (maximum = 4.1-6.2%), suggesting cryptic species. All recognized species possessed a distinct haplotype array at COI with nearest-neighbour interspecific distances averaging 8.57%. Three species new to Canada were detected: Robertus lyrifer (Theridiidae), Baryphyma trifrons (Linyphiidae), and Satilatlas monticola (Linyphiidae). The first two species may represent human-mediated introductions linked to the port in Churchill, but the other species represents a range extension from the USA. The first description of the female of S. monticola was also presented. As well, one probable new species of Alopecosa (Lycosidae) was recognized., Conclusions: This study provides the first comprehensive DNA barcode reference library for the spider fauna of any region. Few cryptic species of spiders were detected, a result contrasting with the prevalence of undescribed species in several other terrestrial arthropod groups at Churchill. Because most (97.5%) sequence clusters at COI corresponded with a named taxon, DNA barcoding reliably identifies spiders in the Churchill fauna. The capacity of DNA barcoding to enable the identification of otherwise taxonomically ambiguous specimens (juveniles, females) also represents a major advance for future monitoring efforts on this group.

Background: Coleoptera is the most diverse order of insects (>300,000 described species), but its richness diminishes at increasing latitudes (e.g., ca. 7400 species recorded in Canada), particularly of phytophagous and detritivorous species. However, incomplete sampling of northern habitats and a lack of taxonomic study of some families limits our understanding of biodiversity patterns in the Coleoptera. We conducted an intensive biodiversity survey from 2006-2010 at Churchill, Manitoba, Canada in order to quantify beetle species diversity in this model region, and to prepare a barcode library of beetles for sub-arctic biodiversity and ecological research. We employed DNA barcoding to provide estimates of provisional species diversity, including for families currently lacking taxonomic expertise, and to examine the guild structure, habitat distribution, and biogeography of beetles in the Churchill region., Results: We obtained DNA barcodes from 3203 specimens representing 302 species or provisional species (the latter quantitatively defined on the basis of Molecular Operational Taxonomic Units, MOTUs) in 31 families of Coleoptera. Of the 184 taxa identified to the level of a Linnaean species name, 170 (92.4%) corresponded to a single MOTU, four (2.2%) represented closely related sibling species pairs within a single MOTU, and ten (5.4%) were divided into two or more MOTUs suggestive of cryptic species. The most diverse families were the Dytiscidae (63 spp.), Staphylinidae (54 spp.), and Carabidae (52 spp.), although the accumulation curve for Staphylinidae suggests that considerable additional diversity remains to be sampled in this family. Most of the species present are predatory, with phytophagous, mycophagous, and saprophagous guilds being represented by fewer species. Most named species of Carabidae and Dytiscidae showed a significant bias toward open habitats (wet or dry). Forest habitats, particularly dry boreal forest, although limited in extent in the region, were undersampled., Conclusions: We present an updated species list for this region as well as a species-level DNA barcode reference library. This resource will facilitate future work, such as biomonitoring and the study of the ecology and distribution of larvae.

DNA barcodes were obtained for 81 butterfly species belonging to 52 genera from sites in north-central Pakistan to test the utility of barcoding for their identification and to gain a better understanding of regional barcode variation. These species represent 25% of the butterfly fauna of Pakistan and belong to five families, although the Nymphalidae were dominant, comprising 38% of the total specimens. Barcode analysis showed that maximum conspecific divergence was 1.6%, while there was 1.7-14.3% divergence from the nearest neighbour species. Barcode records for 55 species showed <2% sequence divergence to records in the Barcode of Life Data Systems (BOLD), but only 26 of these cases involved specimens from neighbouring India and Central Asia. Analysis revealed that most species showed little incremental sequence variation when specimens from other regions were considered, but a threefold increase was noted in a few cases. There was a clear gap between maximum intraspecific and minimum nearest neighbour distance for all 81 species. Neighbour-joining cluster analysis showed that members of each species formed a monophyletic cluster with strong bootstrap support. The barcode results revealed two provisional species that could not be clearly linked to known taxa, while 24 other species gained their first coverage. Future work should extend the barcode reference library to include all butterfly species from Pakistan as well as neighbouring countries to gain a better understanding of regional variation in barcode sequences in this topographically and climatically complex region., (© 2013 The Authors. Molecular Ecology Resources published by John Wiley & Sons Ltd.)

The loss of flight ability has occurred thousands of times independently during insect evolution. Flight loss may be linked to higher molecular evolutionary rates because of reductions in effective population sizes (Ne) and relaxed selective constraints. Reduced dispersal ability increases population subdivision, may decrease geographical range size and increases (sub)population extinction risk, thus leading to an expected reduction in Ne. Additionally, flight loss in birds has been linked to higher molecular rates of energy-related genes, probably owing to relaxed selective constraints on energy metabolism. We tested for an association between insect flight loss and molecular rates through comparative analysis in 49 phylogenetically independent transitions spanning multiple taxa, including moths, flies, beetles, mayflies, stick insects, stoneflies, scorpionflies and caddisflies, using available nuclear and mitochondrial protein-coding DNA sequences. We estimated the rate of molecular evolution of flightless (FL) and related flight-capable lineages by ratios of non-synonymous-to-synonymous substitutions (dN/dS) and overall substitution rates (OSRs). Across multiple instances of flight loss, we show a significant pattern of higher dN/dS ratios and OSRs in FL lineages in mitochondrial but not nuclear genes. These patterns may be explained by relaxed selective constraints in FL ectotherms relating to energy metabolism, possibly in combination with reduced Ne.

Background: Biodiversity surveys have long depended on traditional methods of taxonomy to inform sampling protocols and to determine when a representative sample of a given species pool of interest has been obtained. Questions remain as to how to design appropriate sampling efforts to accurately estimate total biodiversity. Here we consider the biodiversity of freshwater ostracods (crustacean class Ostracoda) from the region of Churchill, Manitoba, Canada. Through an analysis of observed species richness and complementarity, accumulation curves, and richness estimators, we conduct an a posteriori analysis of five bioblitz-style collection strategies that differed in terms of total duration, number of sites, protocol flexibility to heterogeneous habitats, sorting of specimens for analysis, and primary purpose of collection. We used DNA barcoding to group specimens into molecular operational taxonomic units for comparison., Results: Forty-eight provisional species were identified through genetic divergences, up from the 30 species previously known and documented in literature from the Churchill region. We found differential sampling efficiency among the five strategies, with liberal sorting of specimens for molecular analysis, protocol flexibility (and particularly a focus on covering diverse microhabitats), and a taxon-specific focus to collection having strong influences on garnering more accurate species richness estimates., Conclusions: Our findings have implications for the successful design of future biodiversity surveys and citizen-science collection projects, which are becoming increasingly popular and have been shown to produce reliable results for a variety of taxa despite relying on largely untrained collectors. We propose that efficiency of biodiversity surveys can be increased by non-experts deliberately selecting diverse microhabitats; by conducting two rounds of molecular analysis, with the numbers of samples processed during round two informed by the singleton prevalence during round one; and by having sub-teams (even if all non-experts) focus on select taxa. Our study also provides new insights into subarctic diversity of freshwater Ostracoda and contributes to the broader "Barcoding Biotas" campaign at Churchill. Finally, we comment on the associated implications and future research directions for community ecology analyses and biodiversity surveys through DNA barcoding, which we show here to be an efficient technique enabling rapid biodiversity quantification in understudied taxa.

Background: Insect diversity typically declines with increasing latitude, but previous studies have shown conflicting latitude-richness gradients for some hymenopteran parasitoids. However, historical estimates of insect diversity and species richness can be difficult to confirm or compare, because they may be based upon dissimilar methods. As a proxy for species identification, we used DNA barcoding to identify molecular operational taxonomic units (MOTUs) for 7870 Hymenoptera specimens collected near Churchill, Manitoba, from 2004 through 2010., Results: We resolved 1630 MOTUs for this collection, of which 75% (1228) were ichneumonoids (Ichneumonidae + Braconidae) and 91% (1484) were parasitoids. We estimate the total number of Hymenoptera MOTUs in this region at 2624-2840., Conclusions: The diversity of parasitoids in this sub-Arctic environment implies a high diversity of potential host species throughout the same range. We discuss these results in the contexts of resolving interspecific interactions that may include cryptic species, and developing reproducible methods to estimate and compare species richness across sites and between surveys, especially when morphological specialists are not available to identify every specimen.

Background: Various methods have been proposed to assign unknown specimens to known species using their DNA barcodes, while others have focused on using genetic divergence thresholds to estimate "species" diversity for a taxon, without a well-developed taxonomy and/or an extensive reference library of DNA barcodes. The major goals of the present work were to: a) conduct the largest species-level barcoding study of the Muscidae to date and characterize the range of genetic divergence values in the northern Nearctic fauna; b) evaluate the correspondence between morphospecies and barcode groupings defined using both clustering-based and threshold-based approaches; and c) use the reference library produced to address taxonomic issues., Results: Our data set included 1114 individuals and their COI sequences (951 from Churchill, Manitoba), representing 160 morphologically-determined species from 25 genera, covering 89% of the known fauna of Churchill and 23% of the Nearctic fauna. Following an iterative process through which all specimens belonging to taxa with anomalous divergence values and/or monophyly issues were re-examined, identity was modified for 9 taxa, including the reinstatement of Phaonia luteva (Walker) stat. nov. as a species distinct from Phaonia errans (Meigen). In the post-reassessment data set, no distinct gap was found between maximum pairwise intraspecific distances (range 0.00-3.01%) and minimum interspecific distances (range: 0.77-11.33%). Nevertheless, using a clustering-based approach, all individuals within 98% of species grouped with their conspecifics with high (>95%) bootstrap support; in contrast, a maximum species discrimination rate of 90% was obtained at the optimal threshold of 1.2%. DNA barcoding enabled the determination of females from 5 ambiguous species pairs and confirmed that 16 morphospecies were genetically distinct from named taxa. There were morphological differences among all distinct genetic clusters; thus, no cases of cryptic species were detected., Conclusions: Our findings reveal the great utility of building a well-populated, species-level reference barcode database against which to compare unknowns. When such a library is unavailable, it is still possible to obtain a fairly accurate (within ~10%) rapid assessment of species richness based upon a barcode divergence threshold alone, but this approach is most accurate when the threshold is tuned to a particular taxon.

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.

Explosive speciation in ancient lakes has fascinated biologists for centuries and has inspired classical work on the tempo and modes of speciation. Considerable attention has been directed towards the extrinsic forces of speciation--the geological, geographical and ecological peculiarities of ancient lakes. Recently, there has been a resurgence of interest in the intrinsic nature of these radiations, the biological characteristics conducive to speciation. While new species are thought to arise mainly by the gradual enhancement of reproductive isolation among geographically isolated populations, ancient lakes provide little evidence for a predominant role of geography in speciation. Recent phylogenetic work provides strong evidence that multiple colonization waves were followed by parallel intralacustrine radiations that proceeded at relatively rapid rates despite long-term gene flow through hybridization and introgression. Several studies suggest that hybridization itself might act as a key evolutionary mechanism by triggering major genomic reorganization/revolution and enabling the colonization of new ecological niches in ancient lakes. These studies propose that hybridization is not only of little impediment to diversification but could act as an important force in facilitating habitat transitions, promoting postcolonization adaptations and accelerating diversification. Emerging ecological genomic approaches are beginning to shed light on the long-standing evolutionary dilemma of speciation in the face of gene flow. We propose an integrative programme for future studies on speciation in ancient lakes., (© 2010 Blackwell Publishing Ltd.)

The copepod family Centropagidae is widely distributed and occurs in marine, estuarine, freshwater, and inland saline settings. Molecular phylogenies based upon the 16S and 28S genes demonstrate a complex biogeographic history, involving at least five independent invasions of continental waters from the sea. The first colonization was ancient, likely into part of Gondwanaland, and resulted in an inland radiation in southern genera via both vicariance and subsequent habitat shifting among different types of continental waters. Species occupying saline lakes are nested within freshwater clades, indicating invasion of these habitats via fresh waters rather than directly from the ocean or from epicontinental seas. In contrast with the great southern clade, all of the remaining continental invasions are northern, species poor, and quite recent, perhaps even Pleistocene. Long-lived evolutionary euryhalinity, a high propensity for inland invasion, continental vicariance, and in situ radiation within single continents have all played major roles in the diversification of the centropagids., (Copyright 2010 Elsevier Inc. All rights reserved.)

Background: This study reports progress in assembling a DNA barcode reference library for Ephemeroptera, Plecoptera, and Trichoptera ("EPTs") from a Canadian subarctic site, which is the focus of a comprehensive biodiversity inventory using DNA barcoding. These three groups of aquatic insects exhibit a moderate level of species diversity, making them ideal for testing the feasibility of DNA barcoding for routine biotic surveys. We explore the correlation between the morphological species delineations, DNA barcode-based haplotype clusters delimited by a sequence threshold (2%), and a threshold-free approach to biodiversity quantification--phylogenetic diversity., Results: A DNA barcode reference library is built for 112 EPT species for the focal region, consisting of 2277 COI sequences. Close correspondence was found between EPT morphospecies and haplotype clusters as designated using a standard threshold value. Similarly, the shapes of taxon accumulation curves based upon haplotype clusters were very similar to those generated using phylogenetic diversity accumulation curves, but were much more computationally efficient., Conclusion: The results of this study will facilitate other lines of research on northern EPTs and also bode well for rapidly conducting initial biodiversity assessments in unknown EPT faunas.

Molecular studies have enlightened our understanding of freshwater zooplankton biogeography, yet questions remain regarding the scale and commonality of geographic speciation. Here, we present a mtDNA-based phylogenetic hypothesis for 92 Daphnia species from all seven continents, with a focus on North and South America, Europe, and Australia, and use it to explore the frequency, scale, and geographical orientation of allopatric divergence events. Allopatric speciation can conservatively account for at least 42% of cladogenetic events among the species included in our study; most of these involve intercontinental splits. Closely related species pairs are concentrated in the circumarctic region and between northern and southern continents, aligned with bird migration routes, suggesting recent dispersal. By contrast, deeper phylogenetic patterns are consistent with vicariance scenarios linked to continental fragmentation. The possible reasons for the puzzling persistence of these ancient patterns in light of the eroding force of dispersal are considered. Our results demonstrate the high frequency and complex pattern of allopatric speciation in this ancient, passively dispersed genus.

The prospect of finding macroevolutionary trends and rules in the history of life is tremendously appealing, but very few pervasive trends have been found. Here, we demonstrate a parallel increase in the morphological complexity of most of the deep lineages within a major clade. We focus on the Crustacea, measuring the morphological differentiation of limbs. First, we show a clear trend of increasing complexity among 66 free-living, ordinal-level taxa from the Phanerozoic fossil record. We next demonstrate that this trend is pervasive, occurring in 10 or 11 of 12 matched-pair comparisons (across five morphological diversity indices) between extinct Paleozoic and related Recent taxa. This clearly differentiates the pattern from the effects of lineage sorting. Furthermore, newly appearing taxa tend to have had more types of limbs and a higher degree of limb differentiation than the contemporaneous average, whereas those going extinct showed higher-than-average limb redundancy. Patterns of contemporary species diversity partially reflect the paleontological trend. These results provide a rare demonstration of a large-scale and probably driven trend occurring across multiple independent lineages and influencing both the form and number of species through deep time and in the present day.

The Pleistocene glaciations represent the most recent and dramatic series of habitat changes since the Cretaceous. The impact of these events was particularly acute for aquatic taxa with poor powers of dispersal, but few organisms have evolutionary histories more intimately entwined with the advance and retreat of ice than the 'glacial relicts'. In this study, we used a mitochondrial gene, cytochrome c oxidase subunit I (COI), to examine and compare the phylogeographical structure of two glacial relict crustaceans (Limnocalanus macrurus and members of the Mysis relicta species group) across North America. In both cases, we found a sharp phylogenetic division between populations from inland lakes formed during glacial retreat, and arctic lakes isolated from polar seas via isostatic rebound. However, the depth of this phylogenetic partition varied between taxa. In L. macrurus, nucleotide sequence divergence of 2.2% between these zones is consistent with its current status as a single morphologically variable species, but in Mysis the split occurred among recently described, morphologically conserved species, at a divergence of 8.2%. The disparity in the depth of divergence indicates a history of recurrent freshwater invasions from the arctic seas, in concordance with previous studies of Eurasian glacial relicts. However, we suggest further consideration of a largely overlooked explanation that could account for some of the discrepancies between molecular divergences and glaciation events. Many cladogenetic events could have occurred in arctic seas prior to the transition to inland waters, a possibility supported both by the complex physical and ionic history of arctic seas and by high marine and estuarine lineage diversity in the north.

Although trends are of central interest to evolutionary biology, it is only recently that methodological advances have allowed rigorous statistical tests of putative trends in the evolution of discrete traits. Oligomerization is one such proposed trend that may have profoundly influenced evolutionary pathways in many types of animals, especially arthropods. It is a general hypothesis that repeated structures (such as appendage segments and spines) tend to evolve primarily through loss. Although largely untested, this principle of loss is commonly invoked in morphological studies of crustaceans for drawing conclusions about the systematic placements of taxa and about their phylogeny. We present a statistical evaluation of this hypothesis using a molecular phylogeny and character matrix for a family of crustaceans, the Chydoridae, analysed using maximum likelihood methods. We find that a unidirectional (loss-only) model of character evolution is a very poor fit to the data, but that there is evidence of a trend towards loss, with loss rates of structures being perhaps twice the rates of gain. Thus, our results caution against assuming loss a priori, in the absence of appropriate tests for the characters under consideration. However, oligomerization, considered as a tendency but not a rule, may indeed have had ramifications for the types of functional and ecological shifts that have been more common during evolutionary diversification.

While evolutionary trends have long received much attention and have been widely disputed, new methods are now allowing the testing of directional hypotheses with increased rigor. Here, we test a general hypothesis about the way many kinds of discrete characters are thought to evolve, termed oligomerization. This is the tendency for serial structures (such as arthropod body and appendage segments) or armature (such as spines) to evolve primarily through loss and fusion. Focusing on the Crustacea, we use maximum likelihood methods to test for directional evolution in a large sample (> 500) of discrete traits, analyzed against molecular-based phylogenies. We find evidence for a significant trend toward trait loss, in accordance with the reduction principle. However, this trend is far from ubiquitous, with many characters exhibiting a reconstructed bias toward gains. These results suggest that caution must be used before drawing conclusions about which taxa are "primitive" or about the directionality of morphological shifts in the absence of phylogenetic analysis. Nevertheless, oligomerization-as a trend rather than a law-may be an important process that influences evolutionary trajectories from both morphological and functional perspectives.

This study provides C-value (haploid nuclear DNA content) estimates for 31 species of ladybird beetles (representing 6 subfamilies and 8 tribes), the first such data for the family Coccinellidae. Despite their unparalleled diversity, the Coleoptera have been very poorly studied in terms of genome size variation, such that even this relatively modest sample of species makes the Coccinellidae the third best studied family of beetles, behind the Tenebrionidae and Chrysomelidae. The present study provides a comparison of patterns of genome size variation with these two relatively well-studied families. No correlation was found between genome size and body size in the ladybirds, in contrast to some other invertebrate groups but in keeping with findings for other beetle families. However, there is some indication that developmental time and/or feeding ecology is related to genome size in this group. Some phylogenetic patterns and possible associations with subgenomic features are also discussed.

It has long been known that polyploid organisms are more prevalent in arctic than in temperate environments. Past explanations for this geographical trend have focused on the role of glacial cycles in generating polyploids and the influence of abiotic factors in favouring polyploidy in the north. In combination, these mechanisms probably suffice to explain the observed geographical cline in ploidy levels in members of the Daphnia pulex complex in the Holarctic. While only diploid members of the D. pulex complex are found in the temperate regions of North America and Europe, allozyme and DNA quantification analyses indicate that the southern Argentine pulex-complex fauna is dominated by polyploids. Indeed, the present study is the first to document the presence of polyploid members of the D. pulex complex in any temperate climate. The results of phylogeographic analyses suggest that this difference in polyploid distribution between the northern and southern hemispheres is based more on ecological and historical contingencies than direct selection for polyploidy. Specifically, competition with diploid relatives probably limits the lower latitudinal range of polyploids in the north, but appears not to have occurred in Argentina. Because of these differences, the present study provides important insights into the diverse factors that determine the distributions and evolutionary fates of polyploid organisms.