The proliferation of DNA data is revolutionizing all fields of systematic research. DNA barcode sequences, now available for millions of specimens and several hundred thousand species, are increasingly used in algorithmic species delimitations. This is complicated by occasional incongruences between species and gene genealogies, as indicated by situations where conspecific individuals do not form a monophyletic cluster in a gene tree. In two previous reviews, non-monophyly has been reported as being common in mitochondrial DNA gene trees. We developed a novel web service “Monophylizer” to detect non-monophyly in phylogenetic trees and used it to ascertain the incidence of species non-monophyly in COI (a.k.a. cox1) barcode sequence data from 4977 species and 41,583 specimens of European Lepidoptera, the largest data set of DNA barcodes analyzed from this regard. Particular attention was paid to accurate species identification to ensure data integrity. We investigated the effects of tree-building method, sampling effort, and other methodological issues, all of which can influence estimates of non-monophyly. We found a 12% incidence of non-monophyly, a value significantly lower than that observed in previous studies. Neighbor joining (NJ) and maximum likelihood (ML) methods yielded almost equal numbers of non-monophyletic species, but 24.1% of these cases of non-monophyly were only found by one of these methods. Non-monophyletic species tend to show either low genetic distances to their nearest neighbors or exceptionally high levels of intraspecific variability. Cases of polyphyly in COI trees arising as a result of deep intraspecific divergence are negligible, as the detected cases reflected misidentifications or methodological errors. Taking into consideration variation in sampling effort, we estimate that the true incidence of non-monophyly is ∼23%, but with operational factors still being included. Within the operational factors, we separately assessed the frequency of taxonomic limitations (presence of overlooked cryptic and oversplit species) and identification uncertainties. We observed that operational factors are potentially present in more than half (58.6%) of the detected cases of non-monophyly. Furthermore, we observed that in about 20% of non-monophyletic species and entangled species, the lineages involved are either allopatric or parapatric—conditions where species delimitation is inherently subjective and particularly dependent on the species concept that has been adopted. These observations suggest that species-level non-monophyly in COI gene trees is less common than previously supposed, with many cases reflecting misidentifications, the subjectivity of species delimitation or other operational factors.

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Environmental changes strongly impact the distribution of species and subsequently the composition of species assemblages. Although most community ecology studies represent temporal snap shots, long-term observations are rather rare. However, only such time series allow the identification of species composition shifts over several decades or even centuries. We analyzed changes in the species composition of a southeastern German butterfly and burnet moth community over nearly 2 centuries (1840-2013). We classified all species observed over this period according to their ecological tolerance, thereby assessing their degree of habitat specialisation. This classification was based on traits of the butterfly and burnet moth species and on their larval host plants. We collected data on temperature and precipitation for our study area over the same period. The number of species declined substantially from 1840 (117 species) to 2013 (71 species). The proportion of habitat specialists decreased, and most of these are currently endangered. In contrast, the proportion of habitat generalists increased. Species with restricted dispersal behavior and species in need of areas poor in soil nutrients had severe losses. Furthermore, our data indicated a decrease in species composition similarity between different decades over time. These data on species composition changes and the general trends of modifications may reflect effects from climate change and atmospheric nitrogen loads, as indicated by the ecological characteristics of host plant species and local changes in habitat configuration with increasing fragmentation. Our observation of major declines over time of currently threatened and protected species shows the importance of efficient conservation strategies.

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On the basis of three isolates (strains FC6T [T = type strain], FC4, and RG1) of extremely thermophilic chemolithoautotrophic archaebacteria obtained from solfataric fields on Sao Miguel Island, Azores, the new genus Stygiolobus is described. These isolates grow obligately chemolithotrophically by reduction of So with H, (H,-SO lithotrophy) and are the first strictly anaerobic members of the order Sulfolobales. With a DNA G+C content of 38 mol%, the Stygiolobus isolates resemble Sulfolobus spp., which, however, are faculatively organotrophic and aerobic So oxidizers. The new isolates are also distinct from Acidianus spp., which resemble Stygiolobus by growing by H,-SO lithotrophy. However, Acidianus spp. can also grow aerobically by So oxidation and have G+C contents of 31 mol%. At this time, only one species of the genus Stygiolobus is known, Stygiolobus uzoricus sp. nov.; the type strain of S. uzoricus is strain FC6 (= DSM 6296).The thermophilic sulfur-metabolizing archaea (archaebacteria) (66; for reviews, see references 56 and 62) that occur in acid solfataric fields comprise the following three groups: (i) the order Sulfolobales (29,59; for a review, see reference 51); (ii) the order Thermoproteales (24, 70; for a review, see reference 22); and (iii) the genus Thermoplasma (9, 46, 55; for reviews, see references 50 and 62), which because of its isolated phylogenetic position probably represents an order that is distantly related to the methanogens (65, 67). The following four genera have been validly assigned to the order Sulfolobales: (i) the genus Sulfolobus (7, 551, which is the type genus and currently includes the species Sulfolobus acidocaldarius (type species) (7,55), Sulfolobus solfataricus (23, 71), and Sulfolobus shibatae (16); (ii) the genus Metallosphaera, with one species, Metallosphaera sedula (18,29); (iii) the genus Acidianus (47), which includes the species Acidianus infernus (type species) and Acidianus brierleyi (5,47,71); and (iv) the genus Desulfurolobus, with the single species Desulfurolobus ambivalens (26,72). Suifolobus and Metallosphaera spp. are aerobic or facultatively anaerobic, facultatively organotrophic chemolithoautotrophs that are capable of oxidizing molecular sulfur, sulfide, or tetrathionate to H, SO, (6, 8,18,54,68) and have relatively high DNA G + C contents (about 35 to 45 mol%).In contrast, Acidianus and Desulfurolobus spp. are facultatively anaerobic organisms that grow by either oxidation or reduction of elemental sulfur, forming H,SO, and H,S, respectively (52). Relatively low G+C contents (about 31 mol%) are also characteristic of these organisms. The features that distinguish the genera Acidianus and Desulfurolobus are not well defined. In fact, D . ambivalens closely resembles A. infernus in both phenotype and genotype (20,47,72). A level of DNA homology of about 60% with A . infernus (20; this paper) indicates (45) that D. ambivalens is another species of the genus Acidianus.As inferred from immunochemical features of their DNAdependent RNA polymerases, all of...

Extraction of DNA from Lepidoptera is a destructive procedure and curators are often reluctant to provide museum specimens for molecular investigations. On the other hand, dissection of abdomens and genitalia is a standard procedure for description and identification of species and generally accepted even for type material. We present a method that combines the investigation of morphological traits in genitalia with the analysis of DNA sequence information by modifying the dissection protocol. Maceration of abdomens in potassium hydroxide is replaced by enzymatic digestion of soft tissue followed by DNA extraction. DNA extracted from abdomens is suitable for sequencing, as shown for the mitochondrial COI gene appropriate for species identification. Enzymatically treated abdomens proved to be sufficient for preservation of morphological traits. Recommendations are given for appropriate treatment of collected specimens and for routine use of enzymatic digestion.

The number of insect species and insect abundances decreased severely during the past decades over major parts of Central Europe. Previous studies documented declines of species richness, abundances, shifts in species composition, and decreasing biomass of flying insects. In this study, we present a standardized approach to quantitatively and qualitatively assess insect diversity, biomass, and the abundance of taxa, in parallel. We applied two methods: Malaise traps, and automated and active light trapping. Sampling was conducted from April to October 2018 in southern Germany, at four sites representing conventional and organic farming. Bulk samples obtained from Malaise traps were further analyzed using DNA metabarcoding. Larger moths (Macroheterocera) collected with light trapping were further classified according to their degree of endangerment. Our methods provide valuable quantitative and qualitative data. Our results indicate more biomass and higher species richness, as well as twice the number of Red List lepidopterans in organic farmland than in conventional farmland. This combination of sampling methods with subsequent DNA metabarcoding and assignments of individuals according depending on ecological characteristics and the degree of endangerment allows to evaluate the status of landscapes and represents a suitable setup for large-scale long-term insect monitoring across Central Europe, and elsewhere.