Alfried P. Vogler

A plea for DNA taxonomy.

Abstract Taxonomy underpins all biological research, with implications for many basic scientific and applied fields. Insights into the stability or change of animal and plant guilds require species identification on a broad scale and biodiversity questions have become a major public issue. But this comes at a time when taxonomy is facing a crisis, because ever fewer specialists are available. Here, we explore the possibility of using DNA-based methodology to overcome these problems. The utility of DNA sequences for taxonomic purposes is well established. However, all current taxonomic approaches intend to use DNA, at best, as an auxiliary criterion for identifying a species or a taxon, but have not given it a central role. We propose a scheme in which DNA would be the scaffold of a taxonomic reference system, whilst maintaining the importance of the morphological information associated with whole specimens.

A Comprehensive Phylogeny of Beetles Reveals the Evolutionary Origins of a Superradiation

Beetles represent almost one-fourth of all described species, and knowledge about their relationships and evolution adds to our understanding of biodiversity. We performed a comprehensive phylogenetic analysis of Coleoptera inferred from three genes and nearly 1900 species, representing more than 80% of the worlds recognized beetle families. We defined basal relationships in the Polyphaga supergroup, which contains over 300,000 species, and established five families as the earliest branching lineages. By dating the phylogeny, we found that the success of beetles is explained neither by exceptional net diversification rates nor by a predominant role of herbivory and the Cretaceous rise of angiosperms. Instead, the pre-Cretaceous origin of more than 100 present-day lineages suggests that beetle species richness is due to high survival of lineages and sustained diversification in a variety of niches.

Accelerated Species Inventory on Madagascar Using Coalescent-Based Models of Species Delineation

High-throughput DNA sequencing has the potential to accelerate species discovery if it is able to recognize evolutionary entities from sequence data that are comparable to species. The general mixed Yule-coalescent (GMYC) model estimates the species boundary from DNA surveys by identifying independently evolving lineages as a transition from coalescent to speciation branching patterns on a phylogenetic tree. Applied here to 12 families from 4 orders of insects in Madagascar, we used the model to delineate 370 putative species from mitochondrial DNA sequence variation among 1614 individuals. These were compared with data from the nuclear genome and morphological identification and found to be highly congruent (98% and 94%). We developed a modified GMYC that allows for a variable transition from coalescent to speciation among lineages. This revised model increased the congruence with morphology (97%), suggesting that a variable threshold better reflects the clustering of sequence data into biological species. Local endemism was pronounced in all 5 insect groups. Most species (60-91%) and haplotypes (88-99%) were found at only 1 of the 5 study sites (40-1000 km apart). This pronounced endemism resulted in a 37% increase in species numbers using diagnostic nucleotides in a population aggregation analysis. Sample sizes between 7 and 10 individuals represented a threshold above which there was minimal increase in genetic diversity, broadly agreeing with coalescent theory and other empirical studies. Our results from > 1.4 Mb of empirical data suggest that the GMYC model captures species boundaries comparable to those from traditional methods without the need for prior hypotheses of population coherence. This provides a method of species discovery and biodiversity assessment using single-locus data from mixed or environmental samples while building a globally available taxonomic database for future identifications.

Towards writing the encyclopaedia of life: an introduction to DNA barcoding

An international consortium of major natural history museums, herbaria and other organizations has launched an ambitious project, the ‘Barcode of Life Initiative’, to promote a process enabling the rapid and inexpensive identification of the estimated 10 million species on Earth. DNA barcoding is a diagnostic technique in which short DNA sequence(s) can be used for species identification. The first international scientific conference on Barcoding of Life was held at the Natural History Museum in London in February 2005, and here we review the scientific challenges discussed during this conference and in previous publications. Although still controversial, the scientific benefits of DNA barcoding include: (i) enabling species identification, including any life stage or fragment, (ii) facilitating species discoveries based on cluster analyses of gene sequences (e.g. cox1=CO1, in animals), (iii) promoting development of handheld DNA sequencing technology that can be applied in the field for biodiversity inventories and (iv) providing insight into the diversity of life.

The Effect of Geographical Scale of Sampling on DNA Barcoding

Abstract Eight years after DNA barcoding was formally proposed on a large scale, CO1 sequences are rapidly accumulating from around the world. While studies to date have mostly targeted local or regional species assemblages, the recent launch of the global iBOL project (International Barcode of Life), highlights the need to understand the effects of geographical scale on Barcodings goals. Sampling has been central in the debate on DNA Barcoding, but the effect of the geographical scale of sampling has not yet been thoroughly and explicitly tested with empirical data. Here, we present a CO1 data set of aquatic predaceous diving beetles of the tribe Agabini, sampled throughout Europe, and use it to investigate how the geographic scale of sampling affects 1) the estimated intraspecific variation of species, 2) the genetic distance to the most closely related heterospecific, 3) the ratio of intraspecific and interspecific variation, 4) the frequency of taxonomically recognized species found to be monophyletic, and 5) query identification performance based on 6 different species assignment methods. Intraspecific variation was significantly correlated with the geographical scale of sampling (R-square = 0.7), and more than half of the species with 10 or more sampled individuals (N = 29) showed higher intraspecific variation than 1% sequence divergence. In contrast, the distance to the closest heterospecific showed a significant decrease with increasing geographical scale of sampling. The average genetic distance dropped from > 7% for samples within 1 km, to < 3.5% for samples up to > 6000 km apart. Over a third of the species were not monophyletic, and the proportion increased through locally, nationally, regionally, and continentally restricted subsets of the data. The success of identifying queries decreased with increasing spatial scale of sampling; liberal methods declined from 100% to around 90%, whereas strict methods dropped to below 50% at continental scales. The proportion of query identifications considered uncertain (more than one species < 1% distance from query) escalated from zero at local, to 50% at continental scale. Finally, by resampling the most widely sampled species we show that even if samples are collected to maximize the geographical coverage, up to 70 individuals are required to sample 95% of intraspecific variation. The results show that the geographical scale of sampling has a critical impact on the global application of DNA barcoding. Scale-effects result from the relative importance of different processes determining the composition of regional species assemblages (dispersal and ecological assembly) and global clades (demography, speciation, and extinction). The incorporation of geographical information, where available, will be required to obtain identification rates at global scales equivalent to those in regional barcoding studies. Our result hence provides an impetus for both smarter barcoding tools and sprouting national barcoding initiatives—smaller geographical scales deliver higher accuracy.

DNA-based species delineation in tropical beetles using mitochondrial and nuclear markers

DNA barcoding has been successfully implemented in the identification of previously described species, and in the process has revealed several cryptic species. It has been noted that such methods could also greatly assist in the discovery and delineation of undescribed species in poorly studied groups, although to date the feasibility of such an approach has not been examined explicitly. Here, we investigate the possibility of using short mitochondrial and nuclear DNA sequences to delimit putative species in groups lacking an existing taxonomic framework. We focussed on poorly known tropical water beetles (Coleoptera: Dytiscidae, Hydrophilidae) from Madagascar and dung beetles (Scarabaeidae) in the genus Canthon from the Neotropics. Mitochondrial DNA sequence variation proved to be highly structured, with >95% of the observed variation existing between discrete sets of very closely related genotypes. Sequence variation in nuclear 28S rRNA among the same individuals was lower by at least an order of magnitude, but 16 different genotypes were found in water beetles and 12 genotypes in Canthon, differing from each other by a minimum of two base pairs. The distribution of these 28S rRNA genotypes in individuals exactly matched the distribution of mtDNA clusters, suggesting that mtDNA patterns were not misleading because of introgression. Moreover, in a few cases where sequence information was available in GenBank for morphologically defined species of Canthon, these matched some of the DNA-based clusters. These findings demonstrate that clusters of close relatives can be identified readily in the sequence variation obtained in field collected samples, and that these clusters are likely to correspond to either previously described or unknown species. The results suggest that DNA-assisted taxonomy will not require more than a short fragment of mtDNA to provide a largely accurate picture of species boundaries in these groups. Applied on a large scale, this DNA-based approach could greatly improve the rate of species discovery in the large assemblages of insects that remain undescribed.

DNA barcoding insect–host plant associations

Short-sequence fragments (‘DNA barcodes’) used widely for plant identification and inventorying remain to be applied to complex biological problems. Host–herbivore interactions are fundamental to coevolutionary relationships of a large proportion of species on the Earth, but their study is frequently hampered by limited or unreliable host records. Here we demonstrate that DNA barcodes can greatly improve this situation as they (i) provide a secure identification of host plant species and (ii) establish the authenticity of the trophic association. Host plants of leaf beetles (subfamily Chrysomelinae) from Australia were identified using the chloroplast trnL(UAA) intron as barcode amplified from beetle DNA extracts. Sequence similarity and phylogenetic analyses provided precise identifications of each host species at tribal, generic and specific levels, depending on the available database coverage in various plant lineages. The 76 species of Chrysomelinae included—more than 10 per cent of the known Australian fauna—feed on 13 plant families, with preference for Australian radiations of Myrtaceae (eucalypts) and Fabaceae (acacias). Phylogenetic analysis of beetles shows general conservation of host association but with rare host shifts between distant plant lineages, including a few cases where barcodes supported two phylogenetically distant host plants. The study demonstrates that plant barcoding is already feasible with the current publicly available data. By sequencing plant barcodes directly from DNA extractions made from herbivorous beetles, strong physical evidence for the host association is provided. Thus, molecular identification using short DNA fragments brings together the detection of species and the analysis of their interactions.

Speciation of Iberian diving beetles in Pleistocene refugia (Coleoptera, Dytiscidae)

The Mediterranean basin is an area of high diversity and endemicity, but the age and origin of its fauna are still largely unknown. Here we use species‐level phylogenies based on ≈ 1300 base pairs of the genes 16S rRNA and cytochrome oxidase I to establish the relationships of 27 of the 34 endemic Iberian species of diving beetles in the family Dytiscidae, and to investigate their level of divergence. Using a molecular clock approach, 18–19 of these species were estimated to be of Pleistocene origin, with four to six of them from the Late Pleistocene (≈ 100 000 years). A second, lower speciation frequency peak was assigned to Late Miocene or Early Pliocene. Analysis of the distributional ranges showed that endemic species placed in the tip nodes of the trees are significantly more likely to be allopatric with their sisters than endemic species at lower node levels. Allopatric sister species are also significantly younger than sympatric clades, in agreement with an allopatric mode of speciation and limited subsequent range movement. These results strongly suggest that for some taxa Iberian populations were isolated during the Pleistocene long enough to speciate, and apparently did not expand their ranges to recolonize areas north of the Pyrenees. This is in contradiction to observations from fossil beetles in areas further north, which document large range movements associated with the Pleistocene glacial cycles hypothesized to suppress population isolation and allopatric speciation.

Why barcode? High-throughput multiplex sequencing of mitochondrial genomes for molecular systematics

Mitochondrial genome sequences are important markers for phylogenetics but taxon sampling remains sporadic because of the great effort and cost required to acquire full-length sequences. Here, we demonstrate a simple, cost-effective way to sequence the full complement of protein coding mitochondrial genes from pooled samples using the 454/Roche platform. Multiplexing was achieved without the need for expensive indexing tags (‘barcodes’). The method was trialled with a set of long-range polymerase chain reaction (PCR) fragments from 30 species of Coleoptera (beetles) sequenced in a 1/16th sector of a sequencing plate. Long contigs were produced from the pooled sequences with sequencing depths ranging from ∼10 to 100× per contig. Species identity of individual contigs was established via three ‘bait’ sequences matching disparate parts of the mitochondrial genome obtained by conventional PCR and Sanger sequencing. This proved that assembly of contigs from the sequencing pool was correct. Our study produced sequences for 21 nearly complete and seven partial sets of protein coding mitochondrial genes. Combined with existing sequences for 25 taxa, an improved estimate of basal relationships in Coleoptera was obtained. The procedure could be employed routinely for mitochondrial genome sequencing at the species level, to provide improved species ‘barcodes’ that currently use the cox1 gene only.

Sequence alignment of 18S ribosomal RNA and the basal relationships of Adephagan beetles: evidence for monophyly of aquatic families and the placement of Trachypachidae.

Current hypotheses regarding family relationships in the suborder Adephaga (Coleoptera) are conflicting. Here we report full-length 18S ribosomal RNA sequences of 39 adephagans and 13 outgroup taxa. Data analysis focused on the impact of sequence alignment on tree topology, using two principally different approaches. Tree alignments, which seek to minimize indels and substitutions on the tree in a single step, as implemented in an approximate procedure by the computer program POY, were contrasted with a more traditional procedure based on alignments followed by phylogenetic inference based on parsimony, likelihood, and distance analyses. Despite substantial differences between the procedures, phylogenetic conclusions regarding basal relationships within Adephaga and relationships between the four suborders of Coleoptera were broadly similar. The analysis weakly supports monophyly of Adephaga, with Polyphaga usually as its sister, and the two small suborders Myxophaga and Archostemata basal to them. In some analyses, however, Polyphaga was reconstructed as having arisen from within Hydradephaga. Adephaga generally split into two monophyletic groups, corresponding to the terrestrial Geadephaga and the aquatic Hydradephaga, as initially proposed by Crowson in 1955, consistent with a single colonization of the aquatic environment by adephagan ancestors and contradicting the recent proposition of three independent invasions. A monophyletic Hydradephaga is consistently, though not strongly, supported under most analyses, and a parametric bootstrapping test significantly rejects an hypothesis of nonmonophyly. The enigmatic Trachypachidae, which exhibit many similarities to aquatic forms but whose species are entirely terrestrial, were usually recovered as a basal lineage within Geadephaga. Strong evidence opposes the view that terrestrial trachypachids are related to the dytiscoid water beetles.