Deep sympatric mtDNA divergence in the autumnal moth (Epirrita autumnata)

Deep sympatric intraspecific divergence in mtDNA may reflect cryptic species or formerly distinct lineages in the process of remerging. Preliminary results from DNA barcoding of Scandinavian butterflies and moths showed high intraspecific sequence variation in the autumnal moth, Epirrita autumnata. In this study, specimens from different localities in Norway and some samples from Finland and Scotland, with two congeneric species as outgroups, were sequenced with mitochondrial and nuclear markers to resolve the discrepancy found between mtDNA divergence and present species‐level taxonomy. We found five COI sub‐clades within the E. autumnata complex, most of which were sympatric and with little geographic structure. Nuclear markers (ITS2 and Wingless) showed little variation and gave no indications that E. autumnata comprises more than one species. The samples were screened with primers for Wolbachia outer surface gene (wsp) and 12% of the samples tested positive. Two Wolbachia strains were associated with different mtDNA sub‐clades within E. autumnata, which may indicate indirect selection/selective sweeps on haplotypes. Our results demonstrate that deep mtDNA divergences are not synonymous with cryptic speciation and this has important implications for the use of mtDNA in species delimitation, like in DNA barcoding.     

DNA barcoding of endoparasitoid wasps in the genus Anicetus reveals high levels of host specificity (Hymenoptera: Encyrtidae)

The genus Anicetus includes economically important biocontrol agents that are introduced for control of soft and wax scale insect agricultural pests (Ceroplastes spp.). Understanding of host–parasitoid associations is critical to the successful outcome of their utilization in biological control projects. However, identification of these parasitoids is often difficult because of their small size and generally similar morphological features, and hence, studies on the host–parasitoid associations. Here, nucleotide sequence data were generated from the mitochondrial COI gene and the D2 region of 28S rRNA to assess genetic variation within and between species of Anicetus occurring in China. The results of this study support the use of the COI and the D2 region of 28S rRNA gene as useful markers in separating species of Anicetus, even in cases where morphological differences are subtle. On the other hand, the COI gene is also useful in recognizing species with much variation in morphology. DNA barcoding reveals high levels of host specificity of endoparasitoids wasps in the genus Anicetus. Our results indicate that each Anicetus species is adapted to a limited set of host species, or even are monospecific in their host choice.     

Molecular phylogeography of two sibling species of <Emphasis Type="Italic">Eurema</Emphasis> butterflies

The common yellow butterfly Eurema hecabe is widely distributed in East Asia, and is one of the most burdensome species for taxonomists due to the numerous geographic and seasonal wing colour patterns. Moreover, within this species, individuals with a yellow wing fringe that occur in temperate regions of Japan (Y type) proved to be biologically different from others that occur widely in subtropical regions of Japan and all over East Asia (B type). To unveil the genetic variation within and between the two types, a total of 50 butterflies collected at 18 geographic localities in East Asia were examined for nucleotide sequence variation of three mitochondrial regions: cytochrome c oxidase subunit I (COI), cytochrome c oxidase subunit III (COIII) and NADH dehydrogenase subunit 5 (ND5). In addition, they were also examined for infection status with the endosymbiotic bacteria Wolbachia. The three mitochondrial sequences consistently showed that (i) Y type and B type were highly divergent, (ii) nucleotide variation within B type was very small although sampled from a geographically wide range, and (iii) a weak association existed between mitochondrial DNA haplotypes and Wolbachia infection status.     

In Memoriam: Professor Walter James Harman, PhD (1928–2002)

The Mediterranean spiny lobsters, the common spiny lobster Palinurus elephas (Fabricius, 1758) and the pink spiny lobster P. mauritanicus Gruvel, 1911, are important target species for commercial fisheries. In this study, we focus our attention on the DNA sequence variation of the mitochondrial cytochrome oxydase I gene (COI) in the two species of Palinurus. Spiny lobster DNA samples from four Mediterranean localities were analysed to examine the genetic variability at both the intra- and interspecific level. Furthermore, the phylogenetic relationships within the family Palinuridae (among the two species of Palinurus, most of the species of Panulirus and all the species of Jasus) are examined.     

Cryptic species in the Neotropical fish genus Curimatopsis (Teleostei,Characiformes)

Detritivores of the fish family Curimatidae are assigned to eight genera, one of which, the Curimatopsis, with only five species, is the least speciose genus and sister to other seven genera in the family. Ongoing morphological investigations reveal, however, the likely existence of additional species. In this study, fifty‐one specimens of Curimatopsis from multiple rivers of the Amazon, Paraguay and Suriname drainages were identified morphologically according to the present species concepts and then barcoded using the universal cytochrome c oxidase subunit I (COI) mitochondrial marker. Species delimitation analyses were conducted using Bayesian methods through the general mixed Yule‐coalescent analysis combined with conventional likelihood, genetic distance and haplotypic diversity approaches. We found eleven well‐supported clusters that represent four of the named species and seven cryptic, undescribed species of Curimatopsis. Our results show a clear delimitation of species boundaries constrained by distinct Amazonian river ecotones that may have promoted intrageneric lineage diversification. This is the first of a series of genetic studies applicable to future taxonomic, phylogenetic and evolutionary studies across the Curimatidae.     

Molecular identification of Bactrocera passiflorae (Diptera: Tephritidae): Challenge and solution for DNA barcoding

Fruit flies cause significant damage to crop and fruit production worldwide. Therefore, it is essential to identify these organisms to species level; however, immature stages are often impossible to be identified morphologically; thus, the application of DNA barcoding has greatly assisted in species identification. Nuclear, mitochondrial pseudo-COI (NUMT) can be co-amplified with mitochondrial DNA when using generic primers and therefore impair the efficacy of DNA barcoding. This study detected two types of NUMTs from Bactrocera passiflorae, one of them is novel. Therefore, the new finding will assist future species identification by avoiding misidentification using ambiguous NUMT sequences. In addition, this study has developed primers to target the COI gene of B. passiflorae, not the NUMT copies. The newly designed primers have demonstrated its efficiency in amplifying the Mt-COI of B. passiflorae and can be used in routine diagnostics.     

Integrative taxonomy of the freshwater worm Rhyacodrilus falciformis s.l. (Clitellata: Naididae), with the description of a new species

The genetic and morphological variation within Rhyacodrilus falciformis Bretscher, 1901 (Clitellata: Naididae) in Europe was explored using an integrative approach, with three unlinked genetic markers [the mitochondrial cytochrome c oxidase subunit I (COI), the nuclear histone 3 (H3) and internal transcribed spacer region (ITS)] combined with morphology, to investigate whether this taxon constitutes a single or several species. Using Automatic Barcode Gap Discovery on the COI data set, the specimens were divided into seven clusters, used as hypothetical species that were further tested with the other data sources. Single‐gene trees were estimated for all three markers, using coalescence analysis and they were in many parts incongruent with each other. Only one of the clusters was supported by all trees; it was also morphologically differentiated from the other clusters by the shape of its modified penial chaetae. This group consists of two specimens from the Crotot Cave in south‐eastern France, and morphologically they fit a previously described but invalid variety, ‘pigueti’, which is here described as a new species, Rhyacodrilus pigueti Achurra & Martinsson sp. n. The study highlights the fact that a single data source (e.g. COI barcodes) seldom provides a sufficient basis for taxonomic decisions such as species delimitation.     

Sky island diversification meets the multispecies coalescent – divergence in the spruce‐fir moss spider (Microhexura montivaga,Araneae, Mygalomorphae) on the highest peaks of southern Appalachia

Microhexura montivaga is a miniature tarantula‐like spider endemic to the highest peaks of the southern Appalachian mountains and is known only from six allopatric, highly disjunct montane populations. Because of severe declines in spruce‐fir forest in the late 20th century, M. montivaga was formally listed as a US federally endangered species in 1995. Using DNA sequence data from one mitochondrial and seven nuclear genes, patterns of multigenic genetic divergence were assessed for six montane populations. Independent mitochondrial and nuclear discovery analyses reveal obvious genetic fragmentation both within and among montane populations, with five to seven primary genetic lineages recovered. Multispecies coalescent validation analyses [guide tree and unguided Bayesian Phylogenetics and Phylogeography (BPP), Bayes factor delimitation (BFD)] using nuclear‐only data congruently recover six or seven distinct lineages; BFD analyses using combined nuclear plus mitochondrial data favour seven or eight lineages. In stark contrast to this clear genetic fragmentation, a survey of secondary sexual features for available males indicates morphological conservatism across montane populations. While it is certainly possible that morphologically cryptic speciation has occurred in this taxon, this system may alternatively represent a case where extreme population genetic structuring (but not speciation) leads to an oversplitting of lineage diversity by multispecies coalescent methods. Our results have clear conservation implications for this federally endangered taxon and illustrate a methodological issue expected to become more common as genomic‐scale data sets are gathered for taxa found in naturally fragmented habitats.     

Molecular phylogeny of Phebalium (Rutaceae: Boronieae) and related genera based on the nrDNA regions ITS 1+2

Parsimony analyses of the internal transcribed spacer regions of nuclear ribosomal DNA (ITS 1 & ITS 2) for 38 taxa sampled from the Phebalium group (Rutaceae: Boronieae) and two outgroups confirm that, with the exception of Phebalium sensu stricto and Rhadinothamnus, six of the currently recognised genera within the group are monophyletic. The data indicate that Phebaliums. str. is paraphyletic with respect to Microcybe, and Rhadinothamnus is paraphyletic with respect to Chorilaena. Rhadinothamnus and Chorilaena together are the sister group to Nematolepis. Drummondita, included as an outgroup taxon, clustered within the ingroup as sister to Muiriantha and related to Asterolasia.The phylogeny suggests that the evolution of major clades within a number of these genera (e.g. Phebalium) relates to vicariance events between eastern and south-western Australia. Leionema is an eastern genus, with the most basal taxon being the morphologically distinct Leionema ellipticum from northern Queensland. Leionema also includes one species from New Zealand, but this species (as with some others) proved difficult to sequence and its phylogenetic position remains unknown. Taxonomic changes at the generic level are recommended.The authors wish to thank Paul G.Wilson, PERTH, for advice and discussion, and Paul Forster, BRI, for collecting and providing material of Leionema ellipticum. The project was supported by a Melbourne University Postgraduate Award (to BM), the Australian Biological Resources Study (ABRS), Australian Systematic Botany Society and Wolf Den (Australia) Investments.     

Cryptic diversity in flathead fishes (Scorpaeniformes: Platycephalidae) across the Indo‐West Pacific uncovered by DNA barcoding

Identification of taxonomical units underpins most biological endeavours ranging from accurate biodiversity estimates to the effective management of sustainably harvested, protected or endangered species. Successful species identification is now frequently based on a combination of approaches including morphometrics and DNA markers. Sequencing of the mitochondrial COI gene is an established methodology with an international campaign directed at barcoding all fishes. We employed COI sequencing alongside traditional taxonomic identification methods and uncovered instances of deep intraspecific genetic divergences among flathead species. Sixty‐five operational taxonomic units (OTUs) were observed across the Indo‐West Pacific from just 48 currently recognized species. The most comprehensively sampled taxon, Platycephalus indicus, exhibited the highest levels of genetic diversity with eight lineages separated by up to 16.37% genetic distance. Our results clearly indicate a thorough reappraisal of the current taxonomy of P. indicus (and its three junior synonyms) is warranted in conjunction with detailed taxonomic work on the other additional Platycephalidae OTUs detected by DNA barcoding.     

Development of internal COI primers to improve and extend barcoding of fruit flies (Diptera: Tephritidae: Dacini)

Accurate species-level identifications underpin many aspects of basic and applied biology;however,identifications can be hampered by a lack of discriminating morphological characters,taxonomic expertise or time.Molecular approaches,such as DNA"barcoding"of the cytochrome c oxidase(COI)gene,are argued to overcome these issues.However,nuclear encoding of mitochondrial genes(numts)and poor amplification success of suboptimally preserved specimens can lead to erroneous identifications.One insect group for which these molecular and morphological problems are significant are the dacine fruit flies(Diptera:Tephritidae:Dacini).We addressed these issues associated with COI barcoding in the dacines by first assessing several"universal"COI primers against public mitochondrial genome and numt sequences for dacine taxa.We then modified a set of four primers that more closely matched true dacine COI sequence and amplified two overlapping portions of the COI barcode region.Our new primers were tested alongside universal primers on a selection of dacine species,including both fresh preserved and decades-old dry specimens.Additionally,Bactrocera tiyoni mitochondrial and nuclear genomes were compared to identify putative numts.Four numt clades were identified,three of which were amplified using existing universal primers.In contrast,our new primers preferentially amplified the"true"mitochondrial COI barcode in all dacine species tested.The new primers also successfully amplified partial barcodes from dry specimens for which full length barcodes were unobtainable.Thus we recommend these new primers be incorporated into the suites of primers used by diagnosticians and quarantine labs for the accurate identification of dacine species.     

Genome‐wide single‐nucleotide polymorphism data reveal cryptic species within cryptic freshwater snail species—The case of the Ancylus fluviatilis species complex

DNA barcoding utilizes short standardized DNA sequences to identify species and is increasingly used in biodiversity assessments. The technique has unveiled an unforeseeably high number of morphologically cryptic species. However, if speciation has occurred relatively recently and rapidly, the use of single gene markers, and especially the exclusive use of mitochondrial markers, will presumably fail in delimitating species. Therefore, the true number of biological species might be even higher. One mechanism that can result in rapid speciation is hybridization of different species in combination with polyploidization, that is, allopolyploid speciation. In this study, we analyzed the population genetic structure of the polyploid freshwater snail Ancylus fluviatilis, for which allopolyploidization was postulated as a speciation mechanism. DNA barcoding has already revealed four cryptic species within A. fluviatilis (i.e., A. fluviatilis s. str., Ancylus sp. A–C), but early allozyme data even hint at the presence of additional cryptic lineages in Central Europe. We combined COI sequencing with high‐resolution genome‐wide SNP data (ddRAD data) to analyze the genetic structure of A. fluviatilis populations in a Central German low mountain range (Sauerland). The ddRAD data results indicate the presence of three cryptic species within A. fluviatilis s. str. occurring in sympatry and even syntopy, whereas mitochondrial sequence data only support the existence of one species, with shared haplotypes between species. Our study hence points to the limitations of DNA barcoding when dealing with organismal groups where speciation is assumed to have occurred rapidly, for example, through the process of allopolyploidization. We therefore emphasize that single marker DNA barcoding can underestimate the true species diversity and argue in strong favor of using genome‐wide data for species delimitation in such groups.     

Barcoding of biting midges in the genus Culicoides: a tool for species determination

Biting midges of the genus Culicoides (Diptera: Ceratopogonidae) are insect vectors of economically important veterinary diseases such as African horse sickness virus and bluetongue virus. However, the identification of Culicoides based on morphological features is difficult. The sequencing of mitochondrial cytochrome oxidase subunit I (COI), referred to as DNA barcoding, has been proposed as a tool for rapid identification to species. Hence, a study was undertaken to establish DNA barcodes for all morphologically determined Culicoides species in Swedish collections. In total, 237 specimens of Culicoides representing 37 morphologically distinct species were used. The barcoding generated 37 supported clusters, 31 of which were in agreement with the morphological determination. However, two pairs of closely related species could not be separated using the DNA barcode approach. Moreover, Culicoides obsoletus Meigen and Culicoides newsteadi Austen showed relatively deep intraspecific divergence (more than 10 times the average), which led to the creation of two cryptic species within each of C. obsoletus and C. newsteadi. The use of COI barcodes as a tool for the species identification of biting midges can differentiate 95% of species studied. Identification of some closely related species should employ a less conserved region, such as a ribosomal internal transcribed spacer.     

Phylogenetics of the common raven complex (Corvus: Corvidae) and the utility of ND4, COI and intron 7 of the β-fibrinogen gene in avian molecular systematics

The common raven (Corvus corax) is one of the most widely distributed and recognizable avian species in the world. Recent molecular work, however, described two mitochondrial lineages of the common raven, termed the Holarctic clade and the California clade, and questioned the monophyly of this taxon by placing the Chihuahuan raven (C. cryptoleucus) sister to the California clade. We evaluated this phylogenetic hypothesis with additional sequence data and increased taxon sampling. We used ～3.7 kb of DNA sequence data from sections of the mitochondrial coding genes COI, cyt b and ND4, a fragment of the non‐coding mitochondrial DNA control region, and the entire intron 7 of the nuclear β‐fibrinogen gene (β‐fibint 7). We combined these DNA sequence data to erect hypotheses of relationships for lineages of the common raven and related taxa. Maximum parsimony, maximum likelihood, and Bayesian methods yield a paraphyletic common raven. These analyses nest the Chihuahuan raven within the common raven, with strong support for a sister relationship between the Chihuahuan raven and the California clade. In addition, the pied crow (C. albus) is also nested within the common raven, and is sister to the Holarctic clade. Our analyses reveal the challenge of determining phylogenetic relationships and species boundaries in this morphologically conservative genus, and suggest that future molecular work with increased taxon sampling will uncover cryptic species and novel evolutionary relationships. Lastly, this survey is one of a growing number of avian phylogenetic studies to employ either β‐fibint 7 or COI, and the first to use ND4. We developed a simple procedure for comparing rates of evolution in molecular markers, and show that in Corvus the nuclear intron β‐fibint 7 is evolving at a considerably slower pace than the mitochondrial markers, while COI is evolving at a slower rate than cyt b, and ND4 approximately the same rate as cyt b. Hence, β‐fibint 7 and other individual nuclear introns may have limited utility in resolving relationships among recently evolved taxa, whereas both COI and ND4 should be useful in a wide range of avian molecular genetic investigations.     

DNA barcoding of bark and ambrosia beetles reveals excessive NUMTs and consistent east‐west divergence across Palearctic forests

A comprehensive DNA barcoding library is very useful for rapid identification and detection of invasive pest species. We tested the performance of species identification in the economically most damaging group of wood‐boring insects – the bark and ambrosia beetles – with particular focus on broad geographical sampling across the boreal Palearctic forests. Neighbour‐joining and Bayesian analyses of cytochrome oxidase I (COI) sequences from 151 species in 40 genera revealed high congruence between morphology‐based identification and sequence clusters. Inconsistencies with morphological identifications included the discovery of a likely cryptic Nearctic species of Dryocoetes autographus, the possible hybrid origin of shared mitochondrial haplotypes in Pityophthorus micrographus and P. pityographus, and a possible paraphyletic Xyleborinus saxeseni. The first record of Orthotomicus suturalis in North America was confirmed by DNA barcoding. The mitochondrial data also revealed consistent divergence across the Palearctic or Holarctic, confirmed in part by data from the large ribosomal subunit (28S). Some populations had considerable variation in the mitochondrial barcoding marker, but were invariant in the nuclear ribosomal marker. These findings must be viewed in light of the high number of nuclear insertions of mitochondrial DNA (NUMTs) detected in eight bark beetle species, suggesting the possible presence of additional cryptic NUMTs. The occurrence of paralogous COI copies, hybridization or cryptic speciation demands a stronger focus on data quality assessment in the construction of DNA barcoding databases.     

Exploring the use of cytochrome oxidase c subunit 1 (COI) for DNA barcoding of free-living marine nematodes

Background

The identification of free-living marine nematodes is difficult because of the paucity of easily scorable diagnostic morphological characters. Consequently, molecular identification tools could solve this problem. Unfortunately, hitherto most of these tools relied on 18S rDNA and 28S rDNA sequences, which often lack sufficient resolution at the species level. In contrast, only a few mitochondrial COI data are available for free-living marine nematodes. Therefore, we investigate the amplification and sequencing success of two partitions of the COI gene, the M1-M6 barcoding region and the I3-M11 partition.

Methodology

Both partitions were analysed in 41 nematode species from a wide phylogenetic range. The taxon specific primers for the I3-M11 partition outperformed the universal M1-M6 primers in terms of amplification success (87.8% vs. 65.8%, respectively) and produced a higher number of bidirectional COI sequences (65.8% vs 39.0%, respectively). A threshold value of 5% K2P genetic divergence marked a clear DNA barcoding gap separating intra- and interspecific distances: 99.3% of all interspecific comparisons were >0.05, while 99.5% of all intraspecific comparisons were <0.05 K2P distance.

Conclusion

The I3-M11 partition reliably identifies a wide range of marine nematodes, and our data show the need for a strict scrutiny of the obtained sequences, since contamination, nuclear pseudogenes and endosymbionts may confuse nematode species identification by COI sequences.     

Bayesian species identification under the multispecies coalescent provides significant improvements to DNA barcoding analyses

DNA barcoding methods use a single locus (usually the mitochondrial COI gene) to assign unidentified specimens to known species in a library based on a genetic distance threshold that distinguishes between‐species divergence from within‐species diversity. Recently developed species delimitation methods based on the multispecies coalescent (MSC) model offer an alternative approach to individual assignment using either single‐locus or multiloci sequence data. Here, we use simulations to demonstrate three features of an MSC method implemented in the program bpp . First, we show that with one locus, MSC can accurately assign individuals to species without the need for arbitrarily determined distance thresholds (as required for barcoding methods). We provide an example in which no single threshold or barcoding gap exists that can be used to assign all specimens without incurring high error rates. Second, we show that bpp can identify cryptic species that may be misidentified as a single species within the library, potentially improving the accuracy of barcoding libraries. Third, we show that taxon rarity does not present any particular problems for species assignments using bpp and that accurate assignments can be achieved even when only one or a few loci are available. Thus, concerns that have been raised that MSC methods may have problems analysing rare taxa (singletons) are unfounded. Currently, barcoding methods enjoy a huge computational advantage over MSC methods and may be the only approach feasible for massively large data sets, but MSC methods may offer a more stringent test for species that are tentatively assigned by barcoding.     

Limitations of mitochondrial gene barcoding in Octocorallia

The widespread assumption that COI and other mitochondrial genes will be ineffective DNA barcodes for anthozoan cnidarians has not been well tested for most anthozoans other than scleractinian corals. Here we examine the limitations of mitochondrial gene barcoding in the sub-class Octocorallia, a large, diverse, and ecologically important group of anthozoans. Pairwise genetic distance values (uncorrected p) were compared for three candidate barcoding regions: the Folmer region of COI; a fragment of the octocoral-specific mitochondrial protein-coding gene, msh1; and an extended barcode of msh1 plus COI with a short, adjacent intergenic region (igr1). Intraspecific variation was <0.5%, with most species exhibiting no variation in any of the three gene regions. Interspecific divergence was also low: 18.5% of congeneric morphospecies shared identical COI barcodes, and there was no discernible barcoding gap between intra- and interspecific p values. In a case study to assess regional octocoral biodiversity, COI and msh1 barcodes each identified 70% of morphospecies. In a second case study, a nucleotide character-based analysis correctly identified 70% of species in the temperate genus Alcyonium. Although interspecific genetic distances were 2× greater for msh1 than COI, each marker identified similar numbers of species in the two case studies, and the extended COI + igr1 + msh1 barcode more effectively discriminated sister taxa in Alcyonium. Although far from perfect for species identification, a COI + igr1 + msh1 barcode nonetheless represents a valuable addition to the depauperate set of characters available for octocoral taxonomy.     

Evolution of the Neotropical ant genus Linepithema

Abstract A comprehensive species‐level phylogeny of the ant genus Linepithema Mayr, a Neotropical group best known for the invasive Argentine ant L. humile (Mayr), is inferred for the first time using fragments from three nuclear loci [wingless (WG), long‐wavelength rhodopsin (LWR) and internal transcribed spacer (ITS‐2)] and the mitochondrial cytochrome oxidase subunit I (COI) gene. Monophyly of the genus is strongly supported in parsimony, likelihood and Bayesian analyses of the concatenated data, as is the monophyly of four species groups defined previously on the basis of morphology. An Andean species, L. oblongum (Santschi), is the sister taxon of the Argentine ant. Eight of the 11 species whose monophyly was testable in the analysis were inferred to be monophyletic. Several instances of species paraphyly and one case of mitochondrial introgression suggest that complex population genetic processes underpin the patterns of diversity in Linepithema, and that simple genetic approaches to taxonomy such as DNA barcoding should be treated with caution. A maximum likelihood reconstruction of ancestral distributions suggests that Linepithema is of southern South American origin and that populations in the Greater Antilles are the result of four independent colonization events.