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Sea ice is believed to be a major factor shaping gene flow for polar marine organisms, but it remains unclear to what extent it represents a true barrier to dispersal for arctic cetaceans. Bowhead whales are highly adapted to polar sea ice and were targeted by commercial whalers throughout Arctic and subarctic seas for at least four centuries, resulting in severe reductions in most areas. Both changing ice conditions and reductions due to whaling may have affected geographic distribution and genetic diversity throughout their range, but little is known about range-wide genetic structure or whether it differed in the past. This study represents the first examination of genetic diversity and differentiation across all five putative stocks, including Baffin Bay-Davis Strait, Hudson Bay-Foxe Basin, Bering-Beaufort-Chukchi, Okhotsk, and Spitsbergen. We also utilized ancient specimens from Prince Regent Inlet (PRI) in the Canadian Arctic and compared them with modern stocks. Results from analysis of molecular variance and demographic simulations are consistent with recent and high gene flow between Atlantic and Pacific stocks in the recent past. Significant genetic differences between ancient and modern populations suggest PRI harbored unique maternal lineages in the past that have been recently lost, possibly due to loss of habitat during the Little Ice Age and/or whaling. Unexpectedly, samples from this location show a closer genetic relationship with modern Pacific stocks than Atlantic, supporting high gene flow between the central Canadian Arctic and Beaufort Sea over the past millennium despite extremely heavy ice cover over much of this period.

Recent excitement over the development of an initiative to generate DNA sequences for all named species on the planet has in our opinion generated two major areas of contention as to how this ‘DNA barcoding’ initiative should proceed. It is critical that these two issues are clarified and resolved, before the use of DNA as a tool for taxonomy and species delimitation can be universalized. The first issue concerns how DNA data are to be used in the context of this initiative; this is the DNA barcode reader problem (or barcoder problem). Currently, many of the published studies under this initiative have used tree building methods and more precisely distance approaches to the construction of the trees that are used to place certain DNA sequences into a taxonomic context. The second problem involves the reaction of the taxonomic community to the directives of the ‘DNA barcoding’ initiative. This issue is extremely important in that the classical taxonomic approach and the DNA approach will need to be reconciled in order for the ‘DNA barcoding’ initiative to proceed with any kind of community acceptance. In fact, we feel that DNA barcoding is a misnomer. Our preference is for the title of the London meetings—Barcoding Life. In this paper we discuss these two concerns generated around the DNA barcoding initiative and attempt to present a phylogenetic systematic framework for an improved barcoder as well as a taxonomic framework for interweaving classical taxonomy with the goals of ‘DNA barcoding’.

We examined six historical specimens of the endangered North Atlantic right whale (Eubalaena glacialis) using DNA isolated from documented baleen plates from the late nineteenth and early twentieth centuries. Sequences from the mitochondrial DNA (mtDNA) control region from these samples were compared with those from a near-exhaustive survey (269 of approximately 320 individuals) of the remaining right whales in the western North Atlantic Ocean. Our results suggest that there has been only relatively modest change in maternal lineage diversity over the past century in the North Atlantic right whale population. Any significant reduction in genetic variation in the species most likely occurred prior to the late nineteenth century. One historical specimen was from the last documented female capable of propagating one of the maternal lineages in the population today; no females in the existing population have been found to carry this mtDNA haplotype. Analysis of the only specimens from the eastern North Atlantic right whale population ever to be examined revealed that eastern and western North Atlantic right whales may not have been genetically differentiated populations. Loss of gene diversity experienced by North Atlantic right whales over the last century has been modest, and the six decades of protection have been successful in maintaining much of the maternal lineage diversity that was present in the late nineteenth century. Resumen: Examinamos seis especimenes historicos de la ballena franca septentrional Eubalaena glacialis del Atlantico Norte, usando ADN aislado de placas de las barbas documentadas de finales de siglo diecinueve y principios del siglo veinte. Las secuencias del ADN mitocondrial (mtADN) de la region control de estas muestras comparadas con aquellas de una muestra casi exhaustiva (269 de aproximadamente 320 individuos) de las ballenas francas remanentes en el oceano Atlantico Norte Occidental. Nuestros resultados sugieren que solo ha habido ligeros cambios en la diversidad del linaje materno a lo largo del siglo pasado. Probablemente, cualquier reduccion en la variacion genetica en las especies ocurrio antes del final del siglo diecinueve. Uno de los especimenes historicos fue de la ultima hembra documentada capaz de propagar uno de los linajes maternos en la poblacion actual. Ninguna de las hembras en las poblaciones existentes ha sido identificada como portadora de este haplotipo de mtADN. El analisis de los especimenes de la poblacion de ballenas del Atlantico Norte Oriental examinados revelan que las ballenas francas del este y del oeste del Atlantico Norte podrian no haber sido poblaciones geneticamente diferentes. La perdida de diversidad genetica experimentada en las poblaciones de ballena franca del Atlantico Norte a lo largo del siglo pasado ha sido modesta y las seis decadas de proteccion han sido exitosas en el mantenimiento de la mayoria de la diversidad del linaje materno que estaba presente a finales del siglo diecinueve.

Data in this study confirm the validity of a species of muntjac, Muntiacus rooseveltorum , that has been controversial for 60 years. Diagnostic DNA characters are presented for each species examined including the M. rooseveltorum holotype. Three specimens of a recently collected small Laos barking deer have identical sequences to the type specimen of M. rooseveltorum . These DNA characters unambiguously diagnose the newly collected specimens as M. rooseveltorum . This study highlights the importance of continued field surveys in remote regions and the utility of diagnostic DNA characters in identifying species.

A new species of barking deer (Muntiacus spp.) is described from northern Myanmar. Diagnostic DNA character data are presented along with preliminary information on morphology, distribution, and phylogenetic relationships. This discovery contributes significantly to our knowledge of this poorly studied group and highlights the importance of continued field surveys in remote regions. Similar studies have resulted in a significant increase in conservation efforts in other parts of South-east Asia, and argue for additional conservation efforts for this region.

L1210 cell variants resistant to edatrexate (EDX) were isolated by selection in vivo during therapy with this folate analogue. Among the variants selected, seven (L1210/EDX-4 to −7 and L1210/EDX-12 to −14) were found to exhibit 2-23-fold lower levels of folylpolyglutamate synthetase (FPGS) activity compared with parental L1210 cells. Lower levels of FPGS activity in cell-free extract from these variants using EDX as substrate were characterized by the same relative decrease in value for Vmax with no change in apparent Km. The results of an analysis of FPGS activity in mixtures of variant and parental cell extract suggested that no endogenous inhibitors in the variant cells or stimulatory factors in parental cells accounted for the differences observed. Also, FPGS from variant and parental cells showed no difference in thermostability. Decreases in a 60-61-kDa protein as shown by immunoblotting with anti-FPGS peptide antibody were found to occur commensurately with the decrease in FPGS activity in cell extract from the variants compared with parental cells. However, no evidence was obtained for a difference in turnover of FPGS protein during measurement of the decay of FPGS activity in cycloheximide-treated variant and parental cells. In addition, Northern blotting of poly(A)+ RNA did not reveal any difference in the size or level of FPGS mRNA among these various cell types. Studies of in vitro translation of hybridization-selected FPGS mRNA from L1210 cells showed that both mitochondrial and cytosolic forms of FPGS were generated during the reaction. Moreover, FPGS mRNA from the variant cells was significantly less effective in mediating formation of the FPGS peptide product in a manner correlating with FPGS activity and protein found in the cytosol of the various cell types. These results suggest that FPGS gene expression in these variants is posttranscriptionally altered at the level of the cognate mRNA itself and that this alteration constitutively down-regulates the steady-state level of FPGS in these variants.

These studies in HL-60 cells examined the regulation of folylpolyglutamate synthetase (FPGS) activity at the level of gene expression during terminal maturation. Following addition of 210 mM Me2SO to cultures of HL-60 cells at a concentration that induces maturation of 85-90% of the cells, FPGS activity, but not folylpolyglutamate hydrolase (FPGH) activity, was reduced 2-7-fold within 1-5 days. The initial decline in FPGS activity preceded any effect of Me2SO on rate of growth and the increase in appearance of nitro blue tetrazoliumpositive cells, a marker of cellular maturation, and the decrease after 5 days of exposure to Me2SO was solely accounted for by a 7-fold decrease in value for Vmax. The same time and concentration dependence for Me2SO was shown for the decline in FPGS activity, increase in nitro blue tetrazolium-positive cells, and decline in the level of a 2.1-kilobase FPGS mRNA during exposure to this inducer. This decline in FPGS mRNA was reversible when Me2SO was removed from the culture medium but only until that time when an appreciable number of cells were committed to terminal maturation. Following growth of HL-60 cells with [3H]MTX, used as a model folate compound, a large reduction in its intracellular polyglutamate pools was shown during maturation which quantitatively reflected the decline in FPGS activity as well as folate transport inward (Sirotnak, F. M., Jacobson, D. M., and Yang, C-H.(1986) J. Biol. Chem. 261, 11150-11156). Other data showed that folate status or obviation of the folate requirement during growth of these cells strongly influenced the rapidity of the onset of maturation following exposure to inducer. Overall, these results show that FPGS activity in HL-60 cells is a marker for proliferative capacity and that the underlying basis for the decline in FPGS activity during maturation is altered cognate gene expression which is manifested as early reversible and late irreversible phases. They also suggest that the coordinate reduction observed in folate transport, FPGS activity, dihydrofolate reductase, and probably other folate related enzymes by limiting macromolecular biosynthesis may be early programmed events in the maturation process that influence the switch from proliferation to senescence in these cells.

OrthologID (http://nypg.bio.nyu.edu/orthologid/) allows for the rapid and accurate identification of gene orthology within a character-based phylogenetic framework. The Web application has two functions - an orthologous group search and a query orthology classification. The former determines orthologous gene sets for complete genomes and identifies diagnostic characters that define each orthologous gene set; and the latter allows for the classification of unknown query sequences to orthology groups. The first module of the Web application, the gene family generator, uses an E-value based approach to sort genes into gene families. An alignment constructor then aligns members of gene families and the resulting gene family alignments are submitted to the tree builder to obtain gene family guide trees. Finally, the diagnostics generator extracts diagnostic characters from guide trees and these diagnostics are used to determine gene orthology for query sequences.

Background The availability of sequences from whole genomes to reconstruct the tree of life has the potential to enable the development of phylogenomic hypotheses in ways that have not been before possible. A significant bottleneck in the analysis of genomic-scale views of the tree of life is the time required for manual curation of genomic data into multi-gene phylogenetic matrices. Results To keep pace with the exponentially growing volume of molecular data in the genomic era, we have developed an automated technique, ASAP (Automated Simultaneous Analysis Phylogenetics), to assemble these multigene/multi species matrices and to evaluate the significance of individual genes within the context of a given phylogenetic hypothesis. Conclusion Applications of ASAP may enable scientists to re-evaluate species relationships and to develop new phylogenomic hypotheses based on genome-scale data.

Background While Expressed Sequence Tags (ESTs) have proven a viable and efficient way to sample genomes, particularly those for which whole-genome sequencing is impractical, phylogenetic analysis using ESTs remains difficult. Sequencing errors and orthology determination are the major problems when using ESTs as a source of characters for systematics. Here we develop methods to incorporate EST sequence information in a simultaneous analysis framework to address controversial phylogenetic questions regarding the relationships among the major groups of seed plants. We use an automated, phylogenetically derived approach to orthology determination called OrthologID generate a phylogeny based on 43 process partitions, many of which are derived from ESTs, and examine several measures of support to assess the utility of EST data for phylogenies. Results A maximum parsimony (MP) analysis resulted in a single tree with relatively high support at all nodes in the tree despite rampant conflict among trees generated from the separate analysis of individual partitions. In a comparison of broader-scale groupings based on cellular compartment (ie: chloroplast, mitochondrial or nuclear) or function, only the nuclear partition tree (based largely on EST data) was found to be topologically identical to the tree based on the simultaneous analysis of all data. Despite topological conflict among the broader-scale groupings examined, only the tree based on morphological data showed statistically significant differences. Conclusion Based on the amount of character support contributed by EST data which make up a majority of the nuclear data set, and the lack of conflict of the nuclear data set with the simultaneous analysis tree, we conclude that the inclusion of EST data does provide a viable and efficient approach to address phylogenetic questions within a parsimony framework on a genomic scale, if problems of orthology determination and potential sequencing errors can be overcome. In addition, approaches that examine conflict and support in a simultaneous analysis framework allow for a more precise understanding of the evolutionary history of individual process partitions and may be a novel way to understand functional aspects of different kinds of cellular classes of gene products.

Literally hundreds of protocols for DNA preparation from various sources of tissue have been published over the last few decades. To display all of these preparations would take volumes of manual space so instead we present in this chapter several of the preparations that have been used successfully in our laboratories. We also present a few “classical” procedures that are “tried and true” and nearly always work. In addition the www is an excellent source for protocols. Some forums exist for the dissemination of protocols for DNA and RNA isolation (DNA isolation protocols forums: http://www.nwfsc.noaa.gov/protocols.html, http://bric.postech.ac.kr/resources/rprotocol/; RNA isolation protocols forum: http://www.nwfsc.noaa.gov/protocols/methods/RNAMethodsMenu.html).

A new phylogenetic hypothesis for Heliconius and related genera is presented, based on DNA sequence data from mtDNA combined with a region of the wingless gene. This study also adds eight new taxa to a previous cladistic hypothesis based on the mtDNA alone. Simultaneous phylogenetic analysis of the two gene regions together supports a topology largely in agreement with traditional views of heliconiine relationships based on morphology and suggests that the mtDNA support for the sister relationship between Eueides and H. charithonia is due to convergent evolution of homoplasious mtDNA sites.

DNA was isolated from an early twentieth century museum specimen of northern right whale baleen. A system of stringent controls and a novel set of cetacean specific primers eliminated contamination from external sources and ensured the authenticity of the results. Sequence analysis revealed that there were informative nucleotide positions between the museum specimen and extant members of the population and closely related species. The results indicate that museum specimens of baleen can be used to assess historical genetic population structure of the great whales.