Introduction


The purpose of this forum is to introduce notable papers and books published by you and other persons. The work can be new or old, but it should be of wide interest and high quality. A brief comment on the significance of the work should be attached. The current categories of the subjects are (1) adaptation, (2) behavioral evolution, (3) dosage compensation, (4) evo-devo, (5) gene evolution, (6) genomic evolution, (7) molecular phylogeny, (8) natural selection, (9) phenotypic evolution, (10) sensory receptors, (11) sex chromosomes, (12) sex determination, (13) speciation, (14) symbiosis and evolution, and (15) horizontal gene transfer. However, new categories can be added if necessary. Emphasis will be given on the biological work rather than on the mathematical. Any person may post a paper by sending it to one of the editors listed below. We also welcome your comments on posted work, but we moderate all the comments to control spam. This forum is primarily for scientific discussion and to construct a database for good molecular evolution papers.


Friday, March 14, 2014

Horizontal Gene Transfer Takes a Turn: Expansins from Plants to their Bacterial and Eukaryotic Parasites

Contributed by: Dimitra Chalkia


Genetic material is inherited from parents to offspring and this process is known as vertical transmission. However genetic material can be transferred form one organism to another in a non-genealogical fashion. Such type of transmission is defined as horizontal transmission or gene transfer (HGT) (1). Although mechanisms for the transfer of genetic material between organisms were known from the early years of molecular biology and genetics research, and the theoretical potential of cross-species gene transfer in evolution was proposed in the 1980s, the concept of HGT emerged in the 1990s (2). It was invoked as an alternative explanation for rarely observed incongruent phylogenetic relationships between species (2). However, the recent availability of genome sequence information and the thorough study of multiple pro- and eukaryotic genomes has revealed that HGT is pervasive and powerful among microbes (1,2,3). Additionally, more recent studies have shown that HGT is also evident between animals and bacteria, with the bacteria being the donor species (4,5). In plants, HGT has been relatively well documented, and in most cases involves the transfer of genetic material from a parasite to its host plant. Yet, HGTs with the plant species being the donor have rarely been documented.

Recently Nikolaidis et al (6) reported a rare case of HGT from plants to multiple plant parasites or free living microorganisms. Specifically, they found that members of the plant expansin gene family, which code for plant cell-wall loosening proteins and are comprised of two distinct protein domains D1 and D2, were transferred from plants to bacteria, fungi, and unicellular eukaryotes (amoebozoa).

Having previously established that the bacterial protein EXLX1 from Bacillus subtilis is structurally and functionally very similar to plant expansins (7,8,9), Nikolaidis et al investigated the evolution of the expansin family in-depth. To do so they used the bacterial EXLX1 sequence as their primary-sequence database interrogator.  Like expansins, EXLX1 protein contains two domains, D1 and D2, which are tightly packed structurally with a conserved open surface spanning both of them (Figure 1a,b). To ensure that the resulted raw sequence alignments of their exhaustive similarity searches are not random hits, the authors employed a set of established strict search criteria.  Remarkably, they identified numerous sequences from bacteria, fungi, and amoebozoa that align to both EXLX1 domains and therefore may share ancestry with it. If so, the identified sequences are EXLX1 homologs. By employing proven phylogenetic tools and methods, as well as protein domain and fold recognition programs, the authors confirmed that all identified proteins contain both expansin domains, and showed that the predicted protein structures are very similar to both B. subtilis EXLX1 and plant expansins, further supporting the homology inference (Figures 1 and 2).



Figure 1. The EXLX1 homologs are predicted to contain two domains, fold similarly to the Zea mays EXPB1 (a) and the B. subtilis EXLX1 (b), and contain a conserved long hydrophobic surface. (c, d) structural alignments of the three-dimensional models of the EXLX1 homologs from Ralstonia and Erwinia with the EXLX1structure. Surface (e) and ribbon (f) representations of the EXLX1 structure are colored according to conservation in 70 EXP domain sequences from bacteria and fungi (blue to red with increasing conservation). From Nikolaidis et al. (2014) (6).

Figure 2. The Bacillus subtilis EXLX1protein has many homologs in bacteria, fungi, and amoebozoa. (a) Phylogenetic relationships of representative B. subtilisEXLX1 homologs. Two different phylogenetic methods (NJ and ML) were used with gamma-distributed distances from the WAG substitution model with α = 1.72. Alignment gaps were excluded and the total number of sites used to construct the trees was 176. The numbers at the nodes are bootstrap values (NJ/ML). The biology of each species is shown with different symbols next to the species name. Species names abbreviations are given in supplementary table S1, Supplementary Material online. Only sequences producing BLAST hits with E-values lower than 10−4 and query coverage higher than 80% were used for the construction of these trees (b). Many EXLX1homologs contain additional domains. The domain organization of the EXLX1 homologs was identified using the Conserved Domains Database (CDD) database from NCBI coupled to fold recognition analysis. We define as expansin the domain that contains both D1 and D2 domains according to the EXLX1structure (Kerff et al. 2008; Georgelis et al. 2013). From Nikolaidis et al. (2014) (6).





However, sequence similarity is not sufficient for showing the genetic-material-transmission type—vertical or horizontal. Five significant observations led the authors to support the HGT type: a) the sporadic distribution of organisms harboring expansin homologs, b) the biological features of these organisms –plant pathogens, soil inhabitants, or cellulose producers, c) the incongruence between the phylogenetic tree derived from EXLX1 and its homologs and the established bacterial or fungal species tree, d) the fusion of additional and shared protein domains (cellulase GH5 or carbohydrate-binding modules) in several EXLX1 homologs, and e) the functional similarities between microbial and plant expansins, especially the lack of catalytic activity. The latter observation argues against convergence (independent fusion of D1 and D2 domains) because such a scenario would require the biochemically and evolutionarily improbable independent loss and gain of the same amino acid residues in multiple distant phyla.

Relaxing the criteria of their sequence similarity searches, the authors also examined whether sequences similar to each one of the two expansin domains exist. Their results were positive for both domains. Applying their phylogenetic/protein fold recognition methodology to the sequences similar only to the second expansin domain (D2), the authors showed that the fungal swollenin protein family is homologous to expansins. Swollenin proteins are composed of two domains, too. Interestingly, although their N-terminal D1 domain contains many conserved insertions and therefore bears very low similarity with the expansin D1 domain, the folding patterns are very similar.

Regarding the timing of the expansin HGT, the lack of any differences in parametric measures such as GC-content, amino acid or nucleotide usage, etc., in the EXLX1 homologs allowed the authors to conclude it was not recent. Two additional observations augmented this conclusion. First, the phylogenetic patterns revealed that the HGT of expansins was followed by vertical transfers during certain fungal or amoebozoan species evolution. Second, several bacterial and fungal distant species contain expansin genes fused with cellulose GH5 and carbohydrate-binding domains, respectively. According to the authors’ phylogenetic analysis these extra domains were most likely acquired independently by convergence. Therefore the HGT of expansins from plants to other organisms preceded the long-lasting and slow events of convergence and speciation. Hence it must be ancient.

Regarding the origin of the expansin gene family, the authors, following a reductio ad absurdum argumentation, favor the scenario of a single origin in the common ancestor of plants and subsequent horizontal transmission to non-plant species. The patchy distribution of EXLX1 homologs in a small percentage of the tested bacterial (128/4,281 or 3%) and fungal (28 /543 or 5.2%) genomes argues against a single origin in bacteria and subsequent vertical transmission, since such a scenario demands the absurd assumption of multiple independent gene losses.  Additionally, the authors’ previously reported functional similarities between plant and microbial expansins (8,9) argues against convergence, and therefore augments the single origin scenario.

Nikolaidis et al., offer a long list of logical and tightly woven arguments for the HGT scenario of non-plant expansins. However they do admit the difficulty of proving it beyond reasonable doubt. Another task—yet harder in the case of expansins—is to determine precisely the donor and recipient species, as well as its mechanism and timing. As more genomes are being sequenced we will probably be able to define at least a plant lineage that contributed its expansins to its intimately associated parasites. If so, then investigation for the potential mechanisms of HGT will be somehow eased.  A current plausible such mechanism includes a plasmid-mediated transfer (10).

Besides the mechanism and timing of expansins’ HGT, their adaptive significance for the recipient species is of essence. Experimental studies on the physiological role of non-plant expansins have started to shed light on this topic. The authors report several such studies and propose that the HGT of expansin proteins in plant-interacting microbes contributed new or alternative tools for colonization or infection. The latter hypothesis implies an adaptive advantage for the plant-infecting organisms and together with the results of other reports on the role of HGT to the emergence of new diseases suggests that the observed rarity of HGT is not indicative of its importance in organismal evolution.


Abstract of the original paper
Horizontal gene transfer (HGT) has been described as a common mechanism of transferring genetic material between prokaryotes, whereas genetic transfers from eukaryotes to prokaryotes have been rarely documented. Here we report a rare case of HGT in which plant expansin genes that code for plant cell-wall loosening proteins were transferred from plants to bacteria, fungi, and amoebozoa. In several cases, the species in which the expansin gene was found is either in intimate association with plants or is a known plant pathogen. Our analyses suggest that at least two independent genetic transfers occurred from plants to bacteria and fungi. These events were followed by multiple HGT events within bacteria and fungi. We have also observed that in bacteria expansin genes have been independently fused to DNA fragments that code for an endoglucanase domain or for a carbohydrate binding module, pointing to functional convergence at the molecular level. Furthermore, the functional similarities between microbial expansins and their plant xenologs suggest that these proteins mediate microbial–plant interactions by altering the plant cell wall and therefore may provide adaptive advantages to these species. The evolution of these nonplant expansins represents a unique case in which bacteria and fungi have found innovative and adaptive ways to interact with and infect plants by acquiring genes from their host. This evolutionary paradigm suggests that despite their low frequency such HGT events may have significantly contributed to the evolution of prokaryotic and eukaryotic species.

References
1. Goldenfeld N, Woese C (2007) Biology’s next revolution. Nature 445, 369
2. Boto L (2010) Horizontal gene transfer in evolution: facts and challenges. Proc R Soc B 277, 819–827
3. Syvanen M (2012) Evolutionary Implications of Horizontal Gene Transfer. Annu Rev Genet 46, 341–358
4. Dunning Hotopp JC (2011) Horizontal gene transfer between bacteria and animals. Trends Genet 27, 157–163
6. Nikolaidis N, Doran N, DJ Cosgrove (2014) Plant Expansins in Bacteria and Fungi: Evolution by Horizontal Gene Transfer and Independent Domain Fusion. Mol Biol Evol 31(2), 376–386
7. Kerff F, Amoroso A, Herman R, et al. (2008) Crystal structure and activity of Bacillus subtilis YoaJ (EXLX1), a bacterial expansin that promotes root colonization. Proc Natl Acad Sci U S A 105:16876–16881
8. Georgelis N, Tabuchi A, Nikolaidis N, Cosgrove DJ. 2011. Structure-function analysis of the bacterial expansin EXLX1. J Biol Chem 286: 16814–16823
9. Georgelis N, Yennawar NH, Cosgrove DJ. 2012. Structural basis for entropy-driven cellulose binding by a type-A cellulose-binding module (CBM) and bacterial expansin. Proc Natl Acad Sci U S A 109:14830–14835
10. Laine MJ, Haapalainen M, Wahlroos T, Kankare K, Nissinen R, et al. 2000. Thecellulase encoded by the native plasmid of Clavibacter michiganensis ssp. sepedonicus plays a role in virulence and contains an expansin-like domain. Physiol Mol Plant Pathol 57, 221–233




Friday, February 21, 2014

Evolution of a Behavioral Character in a Sparrow Species

            Although the mutational study of behavioral characters was initiated by Seymour Benzer in the 1960’s, his study was concerned primarily with laboratory mutations of fruitflies and did not give much insight into the evolutionary mechanisms of behavioral characters in the wild. For this reason, many authors are now investigating this problem at the molecular level. Horton et al. (2014) recently published an interesting result with respect to the social behavior in the white-throated sparrow Zonotrichia albicollis.

In the white-throated sparrow there are two polymorphic phenotypes with respect to the color pattern of the head crown: (1) tan-striped (TS) and (2) white-striped (WS) (Fig. 1). The male WS phenotype is known to be more aggressive than the male TS with respect to territoriality and mate-finding. In song-birds, these behaviors are typically dependent on sex steroid during the breeding season. WS birds have higher plasma testosterone than the same sex TS birds. However, morph differences in behavior cannot be entirely explained by these hormones, because the differences persist even when plasma levels are experimentally equalized. Therefore, individual variation in steroid-dependent behavior may be better explained by neural sensitivity to the hormones, for example by variation in the distribution and abundance of steroid receptors (Horton et al., 2014).
  From Horton et al. (2014).

By the way, the WS and TS are associated with two inversion haplotypes of chromosome 2 (Thomas et al. 2008). That is, the genes controlling WS and TS are apparently located in the inverted segment of haplotypes ZAL2m and ZAL2. However, because WS is dominant over TS and the frequency of ZAL2m is relatively low, individuals can roughly be divided into two groups, WS (ZAL2m/ZAL2,) and TS (ZAL2/ZAL2), genotype ZAL2m/ZAL2m, being practically absent. It has also been inferred that ZAL2m was derived from ZAL2 about 2 million years ago by chromosomal inversion and therefore the polymorphism has existed for a long time. Note also that there is practically no recombination between two inverted chromosomes.
Horton et al. (2014) looked for steroid receptor genes in the inverted segment and found that the gene (ESR1) encoding estrogen receptor α (ERα) is located in the inverted segment and that the two proteins encoded by the ESR1 genes from the WS and TS phenotypes showed one amino acid difference but this difference did not affect the gene expression pattern appreciably. They then hypothesized that the phenotypic difference between WS and TS is caused by the difference in the gene regulatory region of the gene. In fact, when the binding sites of transcription factors in the cis-regulatory region upstream of the ESR1 gene were examined by a computer program, there was considerable difference between the WS and TS haplotypes (Fig. 2A).

 From Horton et al. (2014).

However, to prove that this difference is indeed responsible for the behavioral difference, it was necessary to show that the expression level of the ESR1 gene is higher in haplotype ZAL2m than in ZAL2. For this purpose, Horton et al. used several molecular techniques such as the luciferase reporter method with HeLa cells and radioimmunoassay. Their results showed that the expression level of the ESR1 gene is about 1.5 times higher in haplotype ZAL2m than in ZAL2 (Fig. 2B,C).
This finding indicates that the difference in expression level of a single major gene generates a clear phenotypic difference, which in turn affects an important behavioral character. At the present time, this type of data is rare, but it is possible that many behavioral characters are controlled by similar molecular mechanisms, and it is desirable that more studies will be conducted in the future. In practice, behavioral characters are generally controlled by many genes, and eventually we may be able to understand the molecular basis of the characters. Yet, the basic principle of gene expression could be simpler than our intuition suggests as in the case of the above example. In their “significance” statement, Horton et al. write:

In this series of studies, we provide a rare illustration of how a chromosomal polymorphism has affected overt social behavior in a vertebrate. White-throated sparrows occur in two alternative phenotypes, or morphs, distinguished by a chromosomal rearrangement. That the morphs differ in territorial and parental behavior has been known for decades, but how the rearrangement affects behavior is not understood. Here we show that genetic differentiation between the morphs affects the transcription of a gene well known to be involved in social behavior. We then show that in a free-living population, the neural expression of this gene predicts both territorial and parental behavior. We hypothesize that this mechanism has played a causal role in the evolution of alternative life-history strategies.

References
2. Horton, B. M., Hudson, W. H., Ortlund, E. A., Shirk, S., Thomas, J. W., Young, E. R., Zinzow-Kramer, W. M., & D. L. Maney. (2014). Estrogen receptor α polymorphism in a species with alternative behavioral phenotypes. Proc. Nat. Acad. Sci. USA. 111(4):1443-1448.

Wednesday, February 5, 2014

Symbiosis and Genome Degeneration: Micro-niche Evolution

            Symbiotic association of bacteria with animals and plants are ubiquitous, and about 15% of insect species have been estimated to harbor bacterial species as symbionts (Oakeson et al. 2014). These bacterial symbionts generally have reduced genome size compared with ancestral free-living bacteria (McCutcheon and Moran 2011). However, these symbionts produce nutrients that are essential for survival of the host, and therefore a mutualistic symbiosis is generated. In many of these bacteria, the initiation of symbiosis occurred a long time ago, so that it is difficult to know how the genome size reduction occurred. In these bacteria, the rate of amino acid substitution is generally higher than that of ancestral free-living bacteria (Moran 1996; Lynch 1996), and this high rate has been attributed to advantageous mutation (Fares et al. 2002) or to Muller’s ratchet effect (Lynch 1996). However, Itoh et al. (2002) and Dale et al. (2003) suggested that the loss of DNA repair enzymes in these bacteria is responsible for the high rate of amino acid substitution. To resolve this controversy, however, it is important to know how gene loss occurs in the early stage of evolution of endosymbiosis.
             In recent years a number of investigators (e.g., Dale et al 2002; Burke and Moran 2011) have identified bacterial species which started a symbiotic life very recently so that they could study their early stage of genome reduction. In particular, the group of Clayton et al. (2012) and Oakeson et al. (2014) discovered a novel human-infective bacterium designated “strain HS.” This strain was isolated from a patient who had a hand wound following impalement with a tree branch. Phylogenetic analysis showed that the strain HS is a member of the Sodalis-allied clade of insect endosymbionts and that close relatives of strain HS gave rise to symbiotic association in a range of insect species. Using 165 rRNA genes, Clayton et al. (2012) showed that strain HS is closely related (by 98% sequence identity for synonymous sites) to the bacteria Solidas glossinidius and Sitophilus oryzae primary endosymbiont (SOPE) that are endosymbionts of grain weevils. The genome size (5.16 Mb) of HS was only slightly greater than those of S. glossinidius and SOPE (see Table 1). This result suggests that the latter two species have become endosymbionts only recently and HS is a free-living bacterium. (The symbiont bacterium (Buchnera aphidicola) of aphids has only 20% of the genome of the ancestral free bacteria.)

Table 1. General features of the strain HS, SOPE, and S. glossinidius genome sequences

            Clayton et al. (2012) and Oakeson et al. (2014) sequenced the genomes of HS and SOPE and compared the genomic sequences with the sequence of S. glossinidius, which was available from the literature. The results indicated that the number of pseudogenes has increased substantially in the symbiont bacteria whereas the number of intact genes (supposedly functional genes) has been reduced (see Table 1 and Fig. 1). Furthermore, a large number of mobile insertion sequences (IS) and a substantial number of duplicate genes have accumulated in the symbiotic bacteria.

Figure 1. Alignments of three regions of the S. glossinidius, strain HS, and SOPE chromosomes. Alignments of three regions of the S. glossinidius, strain HS, and SOPE chromosomes, corresponding to SG0948–SG0977 (A), ps_SGL0466–SG0918 (B) and ps_SGL0318–ps_SGL0330 (C) in the most recent S. glossinidius annotation [25]. Putative ORFs and intergenic regions are drawn according to scale, oriented according to their inferred direction of transcription and color-coded according to COG functional categories. While all of the depicted strain HS genes have intact reading frames, the status of their orthologs in S. glossinidius and SOPE are shown in the outer bars (green = intact, purple = inactivated). Nonsense mutations (premature stop codons) are depicted by purple diamonds, and frameshifting indels are depicted by purple triangles. Light grey connecting bars are syntenic nucleotide alignments, while brown bars illustrate IS-element acquisitions that occur more frequently in SOPE.

            These results suggest that when free-living bacteria entered into a host insect many genes of the free-living bacteria was nonfunctionalized because they were not necessary in the insect host. At the same time normally harmful IS elements have accumulated because the destruction of many functional genes by IS elements appear to be harmless under the condition of symbiosis. It is interesting to note that these evolutionary changes have occurred very rapidly because in the initial stage of symbiosis many useless genes can be pseudogenized but the pseudogenes may be retained in the genome for some time. (In the present case symbiosis was estimated to have occurred only about 28,000 years ago, though this estimate seems to be too low; Concord et al. 2008.) This rapid regressive evolution occurred apparently because a small number of free-living bacteria colonized in the bacteriocytes of the host and the number of vertically inherited bacteria has remained to be small. I would like to call this type of evolution small-niche or micro-niche evolution. In micro-niche evolution, the bacterial population was effectively homozygous, and many mutational changes are expected to be fixed at the rate of mutation unless they are deleterious under the symbiotic condition.
             Of course, if the evolutionary time becomes long, many pseudogenes and other useless DNA elements would be lost by deletion and the genome size is expected to become small as is observed in ancient endosymbionts. Furthermore, in the long run some mutations that would enhance the mutual dependency of the symbiont and the host are expected to occur, and eventually the symbiont bacteria and the host organism become inseparable.
             Note also that because many mutations are not harmful under the symbiotic condition, even DNA repair genes such as recA and recF may be lost (Shigenobu et al. 2000; Itoh et al. 2002). In fact, the loss of recA has been confirmed even in the symbiont bacteria S. glossinidius and SOPE (Dale et al. 2003). This suggests that a relatively high rate of amino acid substitution in endosymbionts has evolved in the early stage of symbiotic life.
             Microniche evolution is different from the evolution by population bottlenecks proposed by Mayr (1963), because in the latter theory population size shrinks temporarily but increases later to the original level whereas in the former theory the population size remains small throughout the evolutionary process. Micro-niche evolution occurs in many different organisms, and it is often associated with regressive evolution of various characters. For example, many organisms living in ancient caves have lost pigmentation because in the dark cave condition pigments are not necessary. In the case of Mexican cavefish Astyanax mexicanus even the eyes are degenerated. In this case a number of authors (e.g., Jeffrey 2009; Yoshizawa et al. 2012) proposed that the degeneration of eyes in A. mexicanus has occurred by positive Darwinian selection. Their argument is that in the dark cave condition there is no need of having eyes and therefore natural selection operates to reduce the eye size so that the nutrition saved by elimination of eye formation can be used for other purposes.
             I have opposed this view by noting that the first step of degenerative evolution is likely to be the reduction of eye size by destructive mutations in the small cavefish population and therefore the degeneration of cavefish eyes can easily be explained by micro-niche evolution (Nei 2013). Of course, positive selection after degeneration of the eyes may have occurred in the cave condition. For example, cavefish are known to have thin skin to cover the degenerated eyes. It is quite possible that any mutations that cause the development of this skin have been subjected to positive selection. However, the most important event of eye degeneration must be caused by degenerative mutations. In fact, this view is supported by the micro-niche evolution of symbiotic bacteria mentioned above.

References
1. Oakeson et al. (2014). Genome degeneration and adaptation in a nascent stage of symbiosis. Genome Biol Evol 6(1):76-93.
2. McCutcheon, J. P. & Moran, N. A. (2011). Extreme genome reduction in symbiotic bacteria. Nat Rev Microbiol 10(1):13-26.
3. Moran NA. 1996. Accelerated evolution and Muller's ratchet in endosymbiotic bacteria. Proc. Natl. Acad. Sci. USA 93:2873-2878.
5. Fares, M. A., Barrio, E., Sabater-Munoz, B., & Moya, A. (2002). The evolution of the heat-shock protein GroEL from Buchnera, the primary endosymbiont of aphids, is governed by positive selection. Mol Biol Evol 19(7):1162-70.
6. Itoh, T., Martin, W., & Nei, M. (2002). Acceleration of genomic evolution caused by enhanced mutation rate in endocellular symbionts. Proc. Natl. Acad. Sci. USA 99:12944-12948.
7. Dale, C., Wang, B., Moran, N., & Ochman, H. (2003). Loss of DNA recombination repair enzymes in the initial stages of genome degeneration. Mol Biol Evol 20(8)1188-1194.
8. Burke GR, and Moran NA. 2011. Massive genomic decay in Serratia symbiotica, a recently evolved symbiont of aphids. Genome Biol Evol 3:195-208.
10. Concord, C., Despres, L., Vallier, A., Balmand, S, Miquel C., et al. (2008). Long-term evolutionary stability of bacterial endosymbiosis in curculionoidea: additional evidence of symbiont replacement in the dryophthoridae family. Mol Biol Evol 25:859-868.
11. Shigenobu, S., Watanabe, H., Hattori, M., Sakaki, Y., & Ishikawa, H. (2000). Genome sequence of the endocellular bacterial symbiont of aphids Buchnera sp. APS. Nature 407(6800):81-6.
12. Mayr E. 1963. Animal species and evolution. Harvard University Press, Cambridge.

13. Jeffery WR. 2009. Regressive evolution in Astyanax cavefish. Annu Rev Genet 43:25-47.

14. Yoshizawa, M., Yamamoto, Y., O’Quin, K. E., & Jeffery, W. R. (2012). Evolution of an adaptive behavior and its sensory receptors promotes eye regression in blind cavefish. BMC Biology 10:108.
15. Nei, M. (2013). Mutation-driven evolution. Oxford University Press, Oxford.