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March 31, 2015

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Sialyltransferases are responsible for the synthesis of a diverse range of sialoglycoconjugates predicted to be pivotal to deuterostomes’ evolution. In this work, we reconstructed the evolutionary history of the metazoan α2,3-sialyltransferases family (ST3Gal), a subset of sialyltransferases encompassing six subfamilies (ST3Gal I–ST3Gal VI) functionally characterized in mammals. Exploration of genomic and expressed sequence tag databases and search of conserved sialylmotifs led to the identification of a large data set of st3gal-related gene sequences. Molecular phylogeny and large scale sequence similarity network analysis identified four new vertebrate subfamilies called ST3Gal III-r, ST3Gal VII, ST3Gal VIII, and ST3Gal IX. To address the issue of the origin and evolutionary relationships of the st3gal-related genes, we performed comparative syntenic mapping of st3gal gene loci combined to ancestral genome reconstruction. The ten vertebrate ST3Gal subfamilies originated from genome duplication events at the base of vertebrates and are organized in three distinct and ancient groups of genes predating the early deuterostomes. Inferring st3gal gene family history identified also several lineage-specific gene losses, the significance of which was explored in a functional context. Toward this aim, spatiotemporal distribution of st3gal genes was analyzed in zebrafish and bovine tissues. In addition, molecular evolutionary analyses using specificity determining position and coevolved amino acid predictions led to the identification of amino acid residues with potential implication in functional divergence of vertebrate ST3Gal. We propose a detailed scenario of the evolutionary relationships of st3gal genes coupled to a conceptual framework of the evolution of ST3Gal functions.

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Dimorphic mating-type chromosomes in fungi are excellent models for understanding the genomic consequences of recombination suppression. Their suppressed recombination and reduced effective population size are expected to limit the efficacy of natural selection, leading to genomic degeneration. Our aim was to identify the sequences of the mating-type chromosomes (a1 and a2) of the anther-smut fungi and to investigate degeneration in their nonrecombining regions. We used the haploid a1 Microbotryum lychnidis-dioicae reference genome sequence. The a1 and a2 mating-type chromosomes were both isolated electrophoretically and sequenced. Integration with restriction-digest optical maps identified regions of recombination and nonrecombination in the mating-type chromosomes. Genome sequence data were also obtained for 12 other Microbotryum species. We found strong evidence of degeneration across the genus in the nonrecombining regions of the mating-type chromosomes, with significantly higher rates of nonsynonymous substitution (dN/dS) than in nonmating-type chromosomes or in recombining regions of the mating-type chromosomes. The nonrecombining regions of the mating-type chromosomes also showed high transposable element content, weak gene expression, and gene losses. The levels of degeneration did not differ between the a1 and a2 mating-type chromosomes, consistent with the lack of homogametic/heterogametic asymmetry between them, and contrasting with X/Y or Z/W sex chromosomes.

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Meiotic chromosome segregation is critical for fertility across eukaryotes, and core meiotic processes are well conserved even between kingdoms. Nevertheless, recent work in animals has shown that at least some meiosis genes are highly diverse or strongly differentiated among populations. What drives this remains largely unknown. We previously showed that autotetraploid Arabidopsis arenosa evolved stable meiosis, likely through reduced crossover rates, and that associated with this there is strong evidence for selection in a subset of meiosis genes known to affect axis formation, synapsis, and crossover frequency. Here, we use genome-wide data to study the molecular evolution of 70 meiosis genes in a much wider sample of A. arenosa. We sample the polyploid lineage, a diploid lineage from the Carpathian Mountains, and a more distantly related diploid lineage from the adjacent, but biogeographically distinct Pannonian Basin. We find that not only did selection act on meiosis genes in the polyploid lineage but also independently on a smaller subset of meiosis genes in Pannonian diploids. Functionally related genes are targeted by selection in these distinct contexts, and in two cases, independent sweeps occurred in the same loci. The tetraploid lineage has sustained selection on more genes, has more amino acid changes in each, and these more often affect conserved or potentially functional sites. We hypothesize that Pannonian diploid and tetraploid A. arenosa experienced selection on structural proteins that mediate sister chromatid cohesion, the formation of meiotic chromosome axes, and synapsis, likely for different underlying reasons.

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Gene regulatory variation is an important driver of the evolution of physiological and developmental responses to the environment. The abscisic acid (ABA) signaling pathway has long been studied as a key component of the cellular response to abiotic stresses in plants. We identify two haplotypes in an Arabidopsis thaliana transcription factor, AREB1, which plays a central role in ABA-mediated response to osmotic stress. These two haplotypes show the sequence signature of long-term maintenance of genetic diversity, suggesting a role for a diversifying selection process such as balancing selection. We find that the two haplotypes, distinguished by a large number of single nucleotide polymorphisms and the presence or absence of four small insertion/deletions in AREB1 intron 1 and exon 2, are at roughly equal frequencies in Arabidopsis, and show high linkage disequilibrium and deep sequence divergence. We use a transgenic approach, along with mRNA Sequencing-based assay of genome-wide expression levels, and find considerable functional divergence between alleles representing the two haplotype groups. Specifically, we find that, under benign soil–water conditions, transgenic lines containing different AREB1 alleles differ in the expression of a large number of genes associated with pathogen response. There are relatively modest gene expression differences between the two transgenic lines under restricted soil water content. Our finding of pathogen-related activity expands the known roles of AREB1 in A. thaliana and reveals the molecular basis of gene-by-environment interaction in a putatively adaptive plant regulatory protein.

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Long noncoding RNAs (lncRNAs) do not code for proteins but function as RNAs. Because the functions of an RNA rely on either its sequence or secondary structure, lncRNAs should be folded at least as strongly as messenger RNAs (mRNAs), which serve as messengers for translation and are generally thought to lack secondary structure-dependent RNA-level functions. Contrary to this prediction, analysis of genome-wide experimental data of human RNA folding reveals that lncRNAs are substantially less folded than mRNAs even after the control of expression level and GC% (percentage of guanines and cytosines), although both lncRNAs and mRNAs are more strongly folded than expected by chance. In contrast to mRNAs, lncRNAs show neither the positive correlation between folding strength and expression level nor the negative correlation between folding strength and evolutionary rate. These and other results support that although RNA folding undoubtedly plays a role in RNA biology it is also important in translation and/or protein biology.

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Major challenges for illuminating the genetic basis of phenotypic evolution are to identify causative mutations, to quantify their functional effects, to trace their origins as new or preexisting variants, and to assess the manner in which segregating variation is transduced into species differences. Here, we report an experimental analysis of genetic variation in hemoglobin (Hb) function within and among species of Peromyscus mice that are native to different elevations. A multilocus survey of sequence variation in the duplicated HBA and HBB genes in Peromyscus maniculatus revealed that function-altering amino acid variants are widely shared among geographically disparate populations from different elevations, and numerous amino acid polymorphisms are also shared with closely related species. Variation in Hb-O2 affinity within and among populations of P. maniculatus is attributable to numerous amino acid mutations that have individually small effects. One especially surprising feature of the Hb polymorphism in P. maniculatus is that an appreciable fraction of functional standing variation in the two transcriptionally active HBA paralogs is attributable to recurrent gene conversion from a tandemly linked HBA pseudogene. Moreover, transpecific polymorphism in the duplicated HBA genes is not solely attributable to incomplete lineage sorting or introgressive hybridization; instead, it is mainly attributable to recurrent interparalog gene conversion that has occurred independently in different species. Partly as a result of concerted evolution between tandemly duplicated globin genes, the same amino acid changes that contribute to variation in Hb function within P. maniculatus also contribute to divergence in Hb function among different species of Peromyscus. In the case of function-altering Hb mutations in Peromyscus, there is no qualitative or quantitative distinction between segregating variants within species and fixed differences between species.

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Proteins that interact coevolve their structures. When mutation disrupts the interaction, compensation by the partner occurs to restore interaction otherwise counterselection occurs. We show in this study how a destabilizing mutation in one protein is compensated by a stabilizing mutation in its protein partner and their coevolving path. The pathway in this case and likely a general principle of coevolution is that the compensatory change must tolerate both the original and derived structures with equivalence in function and activity. Evolution of the structure of signaling elements in a network is constrained by specific protein pair interactions, by requisite conformational changes, and by catalytic activity. The heterotrimeric G protein-coupled signaling is a paragon of this protein interaction/function complexity and our deep understanding of this pathway in diverse organisms lends itself to evolutionary study. Regulators of G protein Signaling (RGS) proteins accelerate the intrinsic GTP hydrolysis rate of the Gα subunit of the heterotrimeric G protein complex. An important RGS-contact site is a hydroxyl-bearing residue on the switch I region of Gα subunits in animals and most plants, such as Arabidopsis. The exception is the grasses (e.g., rice, maize, sugarcane, millets); these plants have Gα subunits that replaced the critical hydroxyl-bearing threonine with a destabilizing asparagine shown to disrupt interaction between Arabidopsis RGS protein (AtRGS1) and the grass Gα subunit. With one known exception (Setaria italica), grasses do not encode RGS genes. One parsimonious deduction is that the RGS gene was lost in the ancestor to the grasses and then recently acquired horizontally in the lineage S. italica from a nongrass monocot. Like all investigated grasses, S. italica has the Gα subunit with the destabilizing asparagine residue in the protein interface but, unlike other known grass genomes, still encodes an expressed RGS gene, SiRGS1. SiRGS1 accelerates GTP hydrolysis at similar concentration of both Gα subunits containing either the stabilizing (AtGPA1) or destabilizing (RGA1) interface residue. SiRGS1 does not use the hydroxyl-bearing residue on Gα to promote GAP activity and has a larger Gα-interface pocket fitting to the destabilizing Gα. These findings indicate that SiRGS1 adapted to a deleterious mutation on Gα using existing polymorphism in the RGS protein population.

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Allele sharing between modern and archaic hominin genomes has been variously interpreted to have originated from ancestral genetic structure or through non-African introgression from archaic hominins. However, evolution of polymorphic human deletions that are shared with archaic hominin genomes has yet to be studied. We identified 427 polymorphic human deletions that are shared with archaic hominin genomes, approximately 87% of which originated before the Human–Neandertal divergence (ancient) and only approximately 9% of which have been introgressed from Neandertals (introgressed). Recurrence, incomplete lineage sorting between human and chimp lineages, and hominid-specific insertions constitute the remaining approximately 4% of allele sharing between humans and archaic hominins. We observed that ancient deletions correspond to more than 13% of all common (>5% allele frequency) deletion variation among modern humans. Our analyses indicate that the genomic landscapes of both ancient and introgressed deletion variants were primarily shaped by purifying selection, eliminating large and exonic variants. We found 17 exonic deletions that are shared with archaic hominin genomes, including those leading to three fusion transcripts. The affected genes are involved in metabolism of external and internal compounds, growth and sperm formation, as well as susceptibility to psoriasis and Crohn’s disease. Our analyses suggest that these "exonic" deletion variants have evolved through different adaptive forces, including balancing and population-specific positive selection. Our findings reveal that genomic structural variants that are shared between humans and archaic hominin genomes are common among modern humans and can influence biomedically and evolutionarily important phenotypes.

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Varicella-zoster virus (VZV) causes chickenpox and shingles, and is found in human populations worldwide. The lack of temporal signal in the diversity of VZV makes substitution rate estimates unreliable, which is a barrier to understanding the context of its global spread. Here, we estimate rates of evolution by studying live attenuated vaccines, which evolved in 22 vaccinated patients for known periods of time, sometimes, but not always undergoing latency. We show that the attenuated virus evolves rapidly (~10–6 substitutions/site/day), but that rates decrease dramatically when the virus undergoes latency. These data are best explained by a model in which viral populations evolve for around 13 days before becoming latent, but then undergo no replication during latency. This implies that rates of viral evolution will depend strongly on transmission patterns. Nevertheless, we show that implausibly long latency periods are required to date the most recent common ancestor of extant VZV to an "out-of-Africa" migration with humans, as has been previously suggested.

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A large proportion of duplicates, originating from ubiquitously expressed genes, acquire testis-biased expression. Identifying the underlying cause of this observation requires determining whether the duplicates have altered functions relative to the parental genes. Typically, statistical methods are used to test for positive selection, signature of which in protein sequence of duplicates implies functional divergence. When assumptions are violated, however, such tests can lead to false inference of positive selection. More convincing evidence for naturally selected functional changes would be the occurrence of structural changes with similar functional consequences in independent duplicates of the same gene. We investigated two testis-specific duplicates of the broadly expressed enzyme gene Aldehyde dehydrogenase (Aldh) that arose in different Drosophila lineages. The duplicates show a typical pattern of accelerated amino acid substitutions relative to their broadly expressed paralogs, with statistical evidence for positive selection in both cases. Importantly, in both duplicates, width of the entrance to the substrate binding site, known a priori to influence substrate specificity, and otherwise conserved throughout the genus Drosophila, has been reduced, resulting in narrowing of the entrance. Protein structure modeling suggests that the reduction of the size of the enzyme’s substrate entry channel, which is likely to shift substrate specificity toward smaller aldehydes, is accounted for by the positively selected parallel substitutions in one duplicate but not the other. Evolution of the testis-specific duplicates was accompanied by reduction in expression of the ancestral Aldh in males, supporting the hypothesis that the duplicates may have helped resolve intralocus sexual conflict over Aldh function.

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Lateral gene transfer (LGT) is an important mechanism of evolution for protists adapting to oxygen-poor environments. Specifically, modifications of energy metabolism in anaerobic forms of mitochondria (e.g., hydrogenosomes) are likely to have been associated with gene transfer from prokaryotes. An interesting question is whether the products of transferred genes were directly targeted into the ancestral organelle or initially operated in the cytosol and subsequently acquired organelle-targeting sequences. Here, we identified key enzymes of hydrogenosomal metabolism in the free-living anaerobic amoebozoan Mastigamoeba balamuthi and analyzed their cellular localizations, enzymatic activities, and evolutionary histories. Additionally, we characterized 1) several canonical mitochondrial components including respiratory complex II and the glycine cleavage system, 2) enzymes associated with anaerobic energy metabolism, including an unusual D-lactate dehydrogenase and acetyl CoA synthase, and 3) a sulfate activation pathway. Intriguingly, components of anaerobic energy metabolism are present in at least two gene copies. For each component, one copy possesses an mitochondrial targeting sequence (MTS), whereas the other lacks an MTS, yielding parallel cytosolic and hydrogenosomal extended glycolysis pathways. Experimentally, we confirmed that the organelle targeting of several proteins is fully dependent on the MTS. Phylogenetic analysis of all extended glycolysis components suggested that these components were acquired by LGT. We propose that the transformation from an ancestral organelle to a hydrogenosome in the M. balamuthi lineage involved the lateral acquisition of genes encoding extended glycolysis enzymes that initially operated in the cytosol and that established a parallel hydrogenosomal pathway after gene duplication and MTS acquisition.

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There are two distinct types of DNA sequences, namely coding sequences and regulatory sequences, in a genome. A recent study of the occupancy of transcription factors (TFs) in human cells suggested that protein-coding sequences also serve as the codes of TF occupancy, and proposed a "duon" hypothesis in which up to 15% of codons of human protein genes are constrained by the additional coding requirements that regulate gene expression. This hypothesis challenges our basic understanding on the human genome. We reanalyzed the data and found that the previous study was confounded by ascertainment bias related to base composition. Using an unbiased comparison in which G/C and A/T sites are considered separately, we reveal a similar level of conservation between TF-bound codons and TF-depleted codons, suggesting largely no extra purifying selection provided by the TF occupancy on the codons of human genes. Given the generally short binding motifs of TFs and the open chromatin structure during transcription, we argue that the occupancy of TFs on protein-coding sequences is mostly passive and evolutionarily neutral, with to-be-determined functions in the regulation of gene expression.

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The importance of whole-genome multiplication (WGM) in plant evolution has long been recognized. In flowering plants, WGM is both ubiquitous and in many lineages cyclical, each round followed by substantial gene loss (fractionation). This process may be biased with respect to duplicated chromosomes, often with overexpression of genes in less fractionated relative to more fractionated regions. This bias is hypothesized to arise through downregulation of gene expression through silencing of local transposable elements (TEs). We assess differences in gene expression between duplicated regions of the paleopolyploid cotton genome and demonstrate that the rate of fractionation is negatively correlated with gene expression. We examine recent hypotheses regarding the source of fractionation bias and show that TE-mediated, positional downregulation is absent in the modern cotton genome, seemingly excluding this phenomenon as the primary driver of biased gene loss. Nevertheless, the paleo subgenomes of diploid cotton are still distinguishable with respect to TE content, targeting of 24-nt-small interfering RNAs and GC content, despite approximately 60 My of evolution. We propose that repeat content per se and differential recombination rates may drive biased fractionation following WGM. These data highlight the likely importance of ancient genomic fractionation biases in shaping modern crop genomes.

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Ultraconserved elements, unusually long regions of perfect sequence identity, are found in genes encoding numerous RNA-binding proteins including arginine-serine rich (SR) splicing factors. Expression of these genes is regulated via alternative splicing of the ultraconserved regions to yield mRNAs that are degraded by nonsense-mediated mRNA decay (NMD), a process termed unproductive splicing (Lareau et al. 2007; Ni et al. 2007). As all human SR genes are affected by alternative splicing and NMD, one might expect this regulation to have originated in an early SR gene and persisted as duplications expanded the SR family. But in fact, unproductive splicing of most human SR genes arose independently (Lareau et al. 2007). This paradox led us to investigate the origin and proliferation of unproductive splicing in SR genes. We demonstrate that unproductive splicing of the splicing factor SRSF5 (SRp40) is conserved among all animals and even observed in fungi; this is a rare example of alternative splicing conserved between kingdoms, yet its effect is to trigger mRNA degradation. As the gene duplicated, the ancient unproductive splicing was lost in paralogs, and distinct unproductive splicing evolved rapidly and repeatedly to take its place. SR genes have consistently employed unproductive splicing, and while it is exceptionally conserved in some of these genes, turnover in specific events among paralogs shows flexible means to the same regulatory end.

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We explored the potential of pooled sequencing to swiftly and economically identify selective sweeps due to emerging artemisinin (ART) resistance in a South-East Asian malaria parasite population. ART resistance is defined by slow parasite clearance from the blood of ART-treated patients and mutations in the kelch gene (chr. 13) have been strongly implicated to play a role. We constructed triplicate pools of 70 slow-clearing (resistant) and 70 fast-clearing (sensitive) infections collected from the Thai–Myanmar border and sequenced these to high (~150-fold) read depth. Allele frequency estimates from pools showed almost perfect correlation (Lin’s concordance = 0.98) with allele frequencies at 93 single nucleotide polymorphisms measured directly from individual infections, giving us confidence in the accuracy of this approach. By mapping genome-wide divergence (FST) between pools of drug-resistant and drug-sensitive parasites, we identified two large (>150 kb) regions (on chrs. 13 and 14) and 17 smaller candidate genome regions. To identify individual genes within these genome regions, we resequenced an additional 38 parasite genomes (16 slow and 22 fast-clearing) and performed rare variant association tests. These confirmed kelch as a major molecular marker for ART resistance (P = 6.03 x 10–6). This two-tier approach is powerful because pooled sequencing rapidly narrows down genome regions of interest, while targeted rare variant association testing within these regions can pinpoint the genetic basis of resistance. We show that our approach is robust to recurrent mutation and the generation of soft selective sweeps, which are predicted to be common in pathogen populations with large effective population sizes, and may confound more traditional gene mapping approaches.

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Over evolutionary time, both host- and virus-encoded genes have been continually selected to modify their interactions with one another. This has resulted in the rapid evolution of the specific codons that govern the physical interactions between host and virus proteins. Virologists have discovered that these evolutionary signatures, acquired in nature, can provide a shortcut in the functional dissection of host–virus interactions in the laboratory. However, the use of evolution studies in this way is complicated by the fact that many nonhuman primate species are endangered, and biomaterials are often difficult to acquire. Here, we assess how the species representation in primate gene data sets affects the detection of positive natural selection. Our results demonstrate how targeted primate sequencing projects could greatly enhance research in immunology, virology, and beyond.

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Numerous computational methods exist to assess the mode and strength of natural selection in protein-coding sequences, yet how distinct methods relate to one another remains largely unknown. Here, we elucidate the relationship between two widely used phylogenetic modeling frameworks: dN/dS models and mutation-selection (MutSel) models. We derive a mathematical relationship between dN/dS and scaled selection coefficients, the focal parameters of MutSel models, and use this relationship to gain deeper insight into the behaviors, limitations, and applicabilities of these two modeling frameworks. We prove that, if all synonymous changes are neutral, standard MutSel models correspond to $$dN/dS\le 1$$. However, if synonymous codons differ in fitness, dN/dS can take on arbitrarily high values even if all selection is purifying. Thus, the MutSel modeling framework cannot necessarily accommodate positive, diversifying selection, while dN/dS cannot distinguish between purifying selection on synonymous codons and positive selection on amino acids. We further propose a new benchmarking strategy of dN/dS inferences against MutSel simulations and demonstrate that the widely used Goldman–Yang-style dN/dS models yield substantially biased dN/dS estimates on realistic sequence data. In contrast, the less frequently used Muse–Gaut-style models display much less bias. Strikingly, the least-biased and most precise dN/dS estimates are never found in the models with the best fit to the data, measured through both AIC and BIC scores. Thus, selecting models based on goodness-of-fit criteria can yield poor parameter estimates if the models considered do not precisely correspond to the underlying mechanism that generated the data. In conclusion, establishing mathematical links among modeling frameworks represents a novel, powerful strategy to pinpoint previously unrecognized model limitations and strengths.

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The estimation of substitution and recombination rates can provide important insights into the molecular evolution of protein-coding sequences. Here, we present a new computational framework, called "CodABC," to jointly estimate recombination, substitution and synonymous and nonsynonymous rates from coding data. CodABC uses approximate Bayesian computation with and without regression adjustment and implements a variety of codon models, intracodon recombination, and longitudinal sampling. CodABC can provide accurate joint parameter estimates from recombining coding sequences, often outperforming maximum-likelihood methods based on more approximate models. In addition, CodABC allows for the inclusion of several nuisance parameters such as those representing codon frequencies, transition matrices, heterogeneity across sites or invariable sites. CodABC is freely available from http://code.google.com/p/codabc/, includes a GUI, extensive documentation and ready-to-use examples, and can run in parallel on multicore machines.

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Dear all, I would like to thank all of you in the evoldir community for your very helpful replies to my question on sending blood samples in ethanol internationally. We are still deciding exactly what to do, but I had some very good suggestions to send the blood+ethanol by courier (or even normal post) without the dry ice / ice packs, which would mean the packaging would be much smaller and lighter and hence cheaper. Provided IATA guidelines are followed*, some people had good experience taking samples as checked luggage, however it was pointed out to me that in the end the pilot has a final decision of whether to accept the luggage, even if all of the paperwork is in order (which could end up with quite a stressful experience at the airport!). Others had suggestions for alternative storage solutions including lysis buffer and RNAlater. And I had many people emphasising the importance of checking the regulations and ensuring that all the paperwork at both ends of the journey is prepared thoroughly. I have compiled all the replies below, following a copy of my initial message. Thank you again for your excellent ideas! Very best wishes, Anna asanture@gmail.com *from my understanding, in my case where we have eppendorfs of ~1mL of blood, these should be put in a hard container (i.e. an eppendorf box), sealed in a plastic bag, an absorbant material wrapped around and then sealed inside another plastic bag, and then up to 10 of these boxes (i.e. From what I have read, the alternative is to send as a dangerous good with one of the international carriers e.g. Fedex, however this is likely to be prohibitively expensive. An online quote suggests the chilly bin we have in mind would cost around NZD $2,500 to transport, and this is before I’ve even mentioned the ‘hazardous’ contents. Any help would be very much appreciated! With many thanks, Anna Santure University of Auckland, New Zealand *also a dangerous good… **apparently litres of duty free gin, vodka, wine and rum are not dangerous goods though! *Responses - thank you all again!* # I can only tell you something you probably won’t like so much: my experience is to best leave it to a carrier, which is indeed expensive. I use World Courier a lot, much more reliable than FedEx or DHL etc. they are known to loose a lot of their packages, I would not risk that. Dry ice/ ice packs are usually not permitted on flights and I can only say the very best about World Courier, they are fast and professional, which you can see in their prices :( contact their office and ask about prices, but you can expect about double than FedEx. I guess it really depends on how valuable those samples are, but they know how to handle transport and customs. # I’ve flown with ethanol samples several times before. The easiest thing to do is to pour off as much of the ethanol as possible before transporting them and then top them up once you arrive at your destination. Of course, this is a lot easier if the sample is tissue - I’m not quite sure how it would work with blood. Perhaps you could spin them down and pour the top ethanol layer off and then just transport them as blood samples’? # About your question, depending on how serious the checks you think will be and how important/unique are the samples, you could decide to just put them in your checked luggage (maybe paying also for an extra luggage) and cross your fingers. In case the material you’re transporting needs some sort of permit to be exported from the UK or imported in NZ, make sure you have one so that in case of problems you can always show them the documents. Otherwise, you can always have some sort of official letter. I doubt they’ll be willing to stick their face in blood samples to check if they’re in ethanol. At that point they’ll be more concerned if you’re bringing some sort of bio-hazard rather than the ethanol. So the idea is: you put them in your luggage and then you have some official document stating that those samples are safe and you’re allowed to transport them. I know this is not exactly sticking to the rules but I know some people who have done this in inter-continental flights. # Here, for DNA, ethanol and freezing are alternatives: we certainly don’t do both - not even in the lab (though I know others do). We haven’t tested every tissue in every taxon, of course, but ethanol’s certainly good for nucleated blood cells and surely penetrates most tissues fairly rapidly. We’re shipping samples globally in ethanol all the time because it’s so cheap and convenient. One litre is a lot of ethanol and a lot of samples (typically about 1 ml each), so we never exceed it. But you could obviously use separate consignments. We’re more worried about leakage spoiling labels so always use polypropylene tubes with screw lids with seals - typically screw-top microfuge tubes. We pack with absorbent paper and in a plastic bag, just in case. We have less experience with RNA, but RNAlater offers the same convenience, though the samples should be frozen for storage. # Regarding blood samples preserved in ethanol, I’m guessing it’s whole blood (that may or may not be on filter paper, or similar)? In the past, I’ve brought in whole blood on filter paper that had been stored in ethanol from Canada, but had my colleagues pour off/evaporate the ethanol before shipping it. But if NZ isn’t happy with that option these days (at the time, I’m sure they decided that the RBCs have been well and truly desiccated/fixed in the ethanol that they didn’t pose a risk), is it an option to completely evaporate the ethanol and then add RNA later, or similar? I’ve never tried it, but might be worth an ask? # Why don’t you use RNA later or any other conservative for blood. Is there a specific need for Ethanol? # Why do you need to keep the samples cold if they are already stored in ethanol? It is the fact that the ethanol draws all the water out of the blood and dessicates it that preserves the blood. In the field and in transport we keep our samples at room temperature. As long as you have had at least 5 x ETOH to blood the samples will be well preserved. At that point you can pour off the ethanol and just keep the dried blood in the eppendorf. Thus negating the need to fly around with lots of ethanol. You can always add more ethanol when the sample arrive if you feel worried. We have done this numerous times and the DNA we extract is still excellent. # This may come too late, but for future reference, I can recommend that you preserve and ship the samples in RNAlater instead of ethanol. We have shown that it does a better job than ethanol at preserving DNA’s and RNA’s integrity and will probably not be considered a hazardous substance. See our paper discussing these benefits, which also includes the recipe for an effective homemade RNAlater: MIGUEL CAMACHO-SANCHEZ, PABLO BURRACO, IVAN GOMEZ-MESTRE and JENN I FER A. LEONARD (2103) Preservation of RNA and DNA from mammal samples under field conditions. Molecular Ecology Resources 13, 663”673 doi: 10.1111/1755-0998.12108 #In Canada ethanol is a dangerous good but falls under the limited quantities exemption, which means that if it is in individual quantities of 1L). However, there are still a number of regulations that must be followed for it to be appropriate (e.g. gross mass of container via Gmail

Source: EVOLDIR