Endre Willassen

What can biological barcoding do for marine biology

The idea of using nucleotide sequences as barcodes for species identification has stirred up debates in the community of taxonomists and systematists. We argue that barcodes are potentially extremely useful tools for taxonomy for several reasons. Barcodes may, for example, help to identify cryptic and polymorphic species and give means to associate life history stages of unknown identity. Barcode systems would thus be particularly helpful in cases when morphology is ambiguous or uninformative and would provide tools for higher taxonomic resolution of disparate life forms. Comparative analysis of short DNA sequences may also represent heuristic access cards to a deeper understanding of evolutionary relationships between organisms. However, barcodes are the “essence” of species identities no more than taxonomic holotypes are “the species”. It makes no sense to think that morphology and other biological information about organisms can be made obsolete by barcode systems. The biological significance of matching or diverging nucleotide sequences will still have to be the subject of taxonomic decisions that must be open for scrutiny. It is imperative, therefore, that barcodes are associated with specimen vouchers.

Midges as quantitative temperature indicator species: lessons for palaeoecology.

Calibration data sets give a unique opportunity to establish patterns of biological existence and their statistical associations with environmental variables. By use of calibration data sets, environmental variables can be inferred quantitatively. The resulting long time-series may assist in distinguishing natural environmental variability from human-induced variability, both in terms of climate change and biotic turnover. However, the validity of the palaeoenvironmental reconstructions depends on their accuracy, precision and sensibility. Before performing palaeoenvironmental inferences, key mechanisms controlling contemporary species’ distribution, abundances and dynamics should be identified and understood. An inference model is developed to produce reconstructions. A major challenge lies in validating and interpreting the reconstructions. Calibration data sets involving midges (Diptera: Chironomidae) suggest that climate has a broad-scale, regional control over midge existence and abundance, often over-riding the influence of local within-lake variables. In recent years, the use of midges as quantitative indicators of past temperatures has greatly expanded. As the number of reconstructions increase, especially in Fennoscandia and North America, it seems the among-site variability is so large that it is unlikely to be due only to local differences in climate. Hence, we question whether the long climate gradients in calibration data sets can accurately be used to calibrate local variables, when most local gradients in time and space are short. Ten Holocene chironomid-inferred temperature curves from Fennoscandia are compared. We illustrate some general principles in palaeoecology by identifying factors that may cause bias. Especially, we consider how calibration data sets simplify the complexity of the real world by maximizing single ecological gradients and by not taking into account co-varying variables. We give some recommendations and criteria that chironomid analysis should meet in order to improve the reliability of the temperature inferences. Finally, we discuss how the complex interactions between species and environment may have implications when we aim at predicting future biodiversity.

Local scale DNA barcoding of bivalves (Mollusca): a case study

Divergence in cytochrome c oxidase 1 (COI), the genetic marker proposed for DNA barcoding, was investigated in marine bivalves from the genera Ennucula, Nucula, Yoldiella and Thyasira. No overlap in levels of intra‐ and interspecific variation was found. The levels of divergence found suggest that barcodes from COI will be useful in distinguishing between the species investigated in this study. The insufficiency of blast searches in GenBank to assign many of the obtained sequences to correct phylum was noted and clearly demonstrates the need for better search strategies specifically targeted at identification using DNA barcodes.

Effects of within-lake variability of fossil assemblages on quantitative chironomid-inferred temperature reconstruction

Abstract We studied the effects of within-lake variation of subfossil chironomid assemblages on chironomid-inferred temperatures in five lakes in southern Norway. In each lake six replicate surface-sediment samples in the deepest part of the lake basin and two transects of seven samples each towards the littoral were analysed for subfossil chironomid assemblages. A quantitative chironomid–July air temperature inference model applied to the subfossil assemblages inferred temperatures with a standard deviation of 0.35–0.54°C for the six mid-lake samples and of 0.38–0.59°C for all 20 sediment samples. This variation seems high when compared with the cross-validated root mean square error of prediction (RMSEP) of the model of 1.04°C. However, we show that the variability of the replicate mid-lake samples can only account for a maximum of 15% of the model prediction error if the RMSEP is decomposed into a component due to uncertainties associated with sampling the chironomid assemblage of a lake and into a residual error component. Thus, according to our results analysing multiple samples in the model lakes to obtain better estimates of their subfossil assemblage composition may only slightly reduce the model prediction error. It was difficult to detect a clear pattern of chironomid-inferred temperatures with respect to water depth in any single lake due to the high variability of inferred values. However, a comparison of all five study lakes revealed that, relative to the mid-lake samples, cooler temperatures were inferred at intermediate depths and warmer temperatures in the shallowest parts of the lakes, although two shallow-water samples with very low inferred temperatures were exceptions in this respect. This depth-related bias of inferred temperatures in our relatively shallow study lakes indicates that chironomid–temperature models calibrated on mid-lake samples should only be applied with caution to near-shore sediments and that a systematic offset in the inferred temperatures may occur.

Phylogenetic utility of five genes for dipteran phylogeny: a test case in the Chironomidae leads to generic synonymies.

In this study we examine the utility of three mitochondrial (COI, COII, 16S) and two nuclear (CAD and EF-1a) markers for estimating lower-level phylogenetic relationships within the dipteran family Chironomidae. As a test case we use species of the genus Micropsectra and the putatively closely related genera Krenopsectra, Parapsectra and Paratanytarsus. We also examine the phylogenetic evidence for the currently accepted species groups within the genus Micropsectra. In our results, highly variable EF-1a sequences within some species indicate the first find of paralogous gene copies in nematocerous Diptera. Among the other genes, COI is found to have the weakest while CAD contains the strongest phylogenetic signal. The resulting phylogeny displays a well-supported, but paraphyletic Micropsectra with regard to Krenopsectra acuta and five Parapsectra species, indicating taxonomic synonymy of these genera with 100% posterior probability. The genus Parapsectra is polyphyletic within Micropsectra while Paratanytarsus remains monophyletic although with low posterior probability. Micropsectra acuta, M. bumasta, M. fallax, M. nohedensis, M. mendli, M. uliginosa, M. chionophila, M. nana, M. styriaca and M. wagneri will all be new combinations as a consequence of the synonymy.

Mitogenomic sequences and evidence from unique gene rearrangements corroborate evolutionary relationships of myctophiformes (Neoteleostei)

BackgroundA skewed assemblage of two epi-, meso- and bathypelagic fish families makes up the order Myctophiformes – the blackchins Neoscopelidae and the lanternfishes Myctophidae. The six rare neoscopelids show few morphological specializations whereas the divergent myctophids have evolved into about 250 species, of which many show massive abundances and wide distributions. In fact, Myctophidae is by far the most abundant fish family in the world, with plausible estimates of more than half of the oceans combined fish biomass. Myctophids possess a unique communication system of species-specific photophore patterns and traditional intrafamilial classification has been established to reflect arrangements of photophores. Myctophids present the most diverse array of larval body forms found in fishes although this attribute has both corroborated and confounded phylogenetic hypotheses based on adult morphology. No molecular phylogeny is available for Myctophiformes, despite their importance within all ocean trophic cycles, open-ocean speciation and as an important part of neoteleost divergence. This study attempts to resolve major myctophiform phylogenies from both mitogenomic sequences and corroborating evidence in the form of unique mitochondrial gene order rearrangements.ResultsMitogenomic evidence from DNA sequences and unique gene orders are highly congruent concerning phylogenetic resolution on several myctophiform classification levels, corroborating evidence from osteology, larval ontogeny and photophore patterns, although the lack of larval morphological characters within the subfamily Lampanyctinae stands out. Neoscopelidae is resolved as the sister family to myctophids with Solivomer arenidens positioned as a sister taxon to the remaining neoscopelids. The enigmatic Notolychnus valdiviae is placed as a sister taxon to all other myctophids and exhibits an unusual second copy of the tRNA-Met gene – a gene order rearrangement reminiscent of that found in the tribe Diaphini although our analyses show it to be independently derived. Most tribes are resolved in accordance with adult morphology although Gonichthyini is found within a subclade of the tribe Myctophini consisting of ctenoid scaled species. Mitogenomic sequence data from this study recognize 10 reciprocally monophyletic lineages within Myctophidae, with five of these clades delimited from additional rearranged gene orders or intergenic non-coding sequences.ConclusionsMitogenomic results from DNA sequences and unique gene orders corroborate morphology in phylogeny reconstruction and provide a likely scenario for the phylogenetic history of Myctophiformes. The extent of gene order rearrangements found within the mitochondrial genomes of myctophids is unique for phylogenetic purposes.

Vibrio tapetis-like strain isolated from introduced Manila clams Ruditapes philippinarum showing symptoms of brown ring disease in Norway

The Manila clam Ruditapes philippinarum was introduced to Norway in 1987 and was produced in 2 hatcheries until 1991. Clam seed was planted at 6 sites. Two sites were on the Island of Tysnes, south of Bergen. Surviving adult Manila clams were recovered in 1995 and 1996. In the present study, Manila clams from the original seeding that displayed morphological signs of brown ring disease (BRD) were recovered in June 2003 (n=7) and in June 2004 (n=17). Samples from extrapallial fluid, tissues and haemolymph were inoculated on marine agar. Replicate subcultures on selective media were used to select potential Vibrio tapetis strains, and in total, 190 bacterial strains were isolated. One of these strains clustered within the V tapetis clade and was named NRP 45. DNA:DNA hybridisation with the type strain CECT4600 showed 52.7 and 57.3% DNA:DNA similarity. Hybridisation of NRP 45 and the V tapetis LP2 strain, isolated from corkwing wrasse Symphodus melops, produced 46.6 and 44.4% re-association. Partial gene segments encoding 16S rRNA, gyrase B protein (GyrB) and chaperonin 60 protein (Cpn60) were characterised and compared to CECT 4600. NRP 45 showed 5 differences in the 1416 nucleotides (nt) of the 16S rRNA encoding gene (99.6% similarity), while the GyrB encoding gene had 62 substitutions of 1181 nt compared (94.8% similarity) and the Cpn60 encoding gene had 22 substitutions out of 548 nt compared (96% similarity). This is the first finding of BRD and the first isolation of a V. tapetis-like bacterial strain from a bivalve in Norway.

Late weichselian chironomidae (diptera) stratigraphy of Lake Nedre Æråsvatn, Andøya, Northern Norway

A 5.6 m long sediment column from Lake Nedre Æråsvatn, Andøya, Northern Norway, spanning ca 20 000 to 11000 14C yr B.P. (Before Present) has been analysed with respect to chironomid remains. Head capsules are absent or rare in the lower, marine part of the sequence (ca 20 000 to 15 500 14C yr B.P.). From ca 15 500 14C yr B.P. onwards, a continuous representation of hygropetric or rheophilous taxa indicate the existence of stream habitats. The lake itself seemingly had no significant chironomid fauna until ca 12 800 14C yr B.P.The main taxa recorded in pre-12000 14C yr B.P. samples are Micropsectra and Metriocnemus. Increases of Micropsectra are positively correlated with climatic improvements, especially the Bølling amelioration (ca 12800 to 12000 14C yr B.P.). This may be due to a denser vegetation cover, and an increased influx of organic detritus.Chironomid concentration and influx values increased markedly from ca 12 500 14C yr B.P. onwards, attaining a maximum of 450 head capsules cm-3 in late Bølling. At ca 12000 14C yr B.P., dominance shifted to Tanytarsus lugens Kieffer. Increases of T. lugens, Psectrocladius, Procladius sp. and Heterotrissocladius subpilosus Kieffer at ca 11400 14C yr B.P. may point to slightly more mature lacustrine conditions.

Inconsistent results should not be overlooked: A reply to Brooks et al. (2012)

Empirical data in the form of many chironomid-based temperature reconstructions give an excellent opportunity to assess the chironomid approach to temperature reconstruction by testing its reproducibility. Brooks et al. (The Holocene 22(12) 2012 (this issue)) offer a critique of points discussed in Velle et al. (The Holocene 20 (2010) 989–1002), but fail to explain the poor reproducibility found when Holocene chironomid-based temperature reconstructions are compared. We discuss the issues raised by Brooks et al. (2012) and cite studies that demonstrate the complexity involved. We are grateful to Brooks et al. (2012) for contributing to the discussion. However, they overly rely uncritically on transfer functions and the resulting reconstructions as representatives of true patterns in nature. A major source of bias involved when chironomids are used as a palaeoenvironmental proxy is the response to confounding gradients. Many of the challenges discussed in the Forum Article, in the comment, and in the reply are also valid for other research fields within palaeoecology. The challenges should still be properly addressed in chironomid research.

Exploring Tanytarsini relationships (Diptera: Chironomidae) using mitochondrial COII gene sequences

Mitochondrial DNA sequences from COII and flanking regions were retrieved from 42 species of Chironomidae belonging to the genera Cladotanytarsus, Corynocera, Diplocladius, Eukiefferiella, Lauterborniella, Micropsectra, Orthocladius, Pagastiella, Paratanytarsus, Psectroladius, Prodiamesa, Rheotanytarsus, Stempellinella, Sergentia, Stictochironomus and Tanytarsus. Some Tanytarsini have unusually long intergenic regions between COII and the flanking tRNAs. Length variation of the COII gene is associated with hypervariable regions including internal indels. We discuss the possibility that flanking tRNAs and intergenic repeat patterns may make COII susceptible to small scale rearrangements. These mutational patterns are not assumed to disturb the functionality because the polypeptides can be folded to the 3D structure described by the Swiss Model, and putative copper binding active sites in Anopheles COII are conserved in the chironomids. Phylogeny was inferred on COII data using maximum parsimony and Bayesian analyses. A general time reversible model accounting for the proportion of invariable sites and with gamma correction for nucleotide substitution rate heterogeneity (GTR+Γ+Ι) was found as the best model for evolutionary change. Implementation of the model in Bayesian analyses recovered a phylogenetic consensus tree that corresponds well with the hypothesis of Tanytarsini relationships derived from morphological studies. High probabilities were estimated for monophyly of the genera examined, except that Corynocera ambigua becomes the sister taxon of Tanytarsus gracilentus. The tree is also in agreement with the taxonomic division of subtribes Tanytarsina and Zavreliina. However, the data and model only weakly support many of the suggested relationships between the genera of Tanytarsini.