As revealed by AnnexinV-7/AAD double staining, when exposed to 45˚C for 10 minutes, more than 96% of HEPG2 and 91.6% HuH7 cells had survived after 24 hours without signs of apoptosis, whereas survival was decreased to 65.6%

and 87.6%, respectively, at 50˚C, and all cells died after exposure this website to 55˚C (Fig. 1A). Division indices (DI) of three HCC cell lines were determined after CFSE labeling, followed by FACS analysis. In all cell lines that were exposed to 50˚C, DI at day 5 (or 6) was significantly higher than in cells kept at 37˚C (Fig. 1B). This was paralleled by a significant increase of proliferation-related transcripts (Ki-67 and CyclinD1), with Ki-67 transcripts being already elevated in two cell lines after exposure to 45˚C (Fig. 1C). Distinct morphological changes, such as appearance of spindle-like cells (Fig. 2A), were only observed for HEPG2 cells exposed to 50˚C on day 5. Intracellular staining, followed by FCM, demonstrated that the cholangiocyte markers, CK7 and CK19, were

increased in HEPG2 cells at day 5 after exposure to 50˚C, whereas low baseline expression was observed in cells exposed to 37˚C or 45˚C (Fig. 2B). This was confirmed by western blotting for CK19 (Fig. 2C) and immunohistology (data not shown). Cell phenotype-related transcript levels were analyzed in the three HCC cell lines by using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR; Fig. 3). CK7 and 19 transcripts were dramatically elevated on day 5 day after exposure to 50˚C, compared to no or minor changes after treatment at lower temperatures, but this increase was transient Bortezomib and levels returned to or near baseline on day 12. Transcript levels of the putative stem cell and progenitor marker, CD133, showed similar kinetics, whereas the hepatocyte differentiation marker, albumin (ALB) was significantly reduced on day 5, to rise baseline on day 12. Expression of four central EMT markers (Snail, TWIST1, CHD1L, and COL1A1) was increased in all three HCC cell lines 5 days after treatment at 50˚C (Supporting see more Fig. 1). Most of these increases were significant or highly significant, especially for COL1A1. The transcript

level (cycle threshold [Ct] value) for COL1A1 almost resembled that found in the well-established, activated human hepatic stellate cell line, LX-2[33] (Supporting Fig. 2). Tissue inhibitor of matrix metalloproteinase, another EMT-related marker, showed a similar trend (Supporting Fig. 3). Of note, EMT-related transcript levels returned to baseline on day 12 post–heat treatment. EMT-like changes, enhanced invasiveness, and migration of HEPG2 cells were confirmed by a 5- to 8-fold increased protein expression of Snail at day 5, but also at day 12 after heat treatment, and a significantly enhanced level of in vitro HEPG2 and HuH7 invasions at day 5 (Fig. 4A,B). Preheating (50˚C) preheating increased Shc transcript levels 5.0-, 2.8-, and 5.1-fold at day 5 in HEPG2, HuH7, and HEP3B, respectively (Fig. 5A).

As revealed by AnnexinV-7/AAD double staining, when exposed to 45˚C for 10 minutes, more than 96% of HEPG2 and 91.6% HuH7 cells had survived after 24 hours without signs of apoptosis, whereas survival was decreased to 65.6%

and 87.6%, respectively, at 50˚C, and all cells died after exposure PF-6463922 cell line to 55˚C (Fig. 1A). Division indices (DI) of three HCC cell lines were determined after CFSE labeling, followed by FACS analysis. In all cell lines that were exposed to 50˚C, DI at day 5 (or 6) was significantly higher than in cells kept at 37˚C (Fig. 1B). This was paralleled by a significant increase of proliferation-related transcripts (Ki-67 and CyclinD1), with Ki-67 transcripts being already elevated in two cell lines after exposure to 45˚C (Fig. 1C). Distinct morphological changes, such as appearance of spindle-like cells (Fig. 2A), were only observed for HEPG2 cells exposed to 50˚C on day 5. Intracellular staining, followed by FCM, demonstrated that the cholangiocyte markers, CK7 and CK19, were

increased in HEPG2 cells at day 5 after exposure to 50˚C, whereas low baseline expression was observed in cells exposed to 37˚C or 45˚C (Fig. 2B). This was confirmed by western blotting for CK19 (Fig. 2C) and immunohistology (data not shown). Cell phenotype-related transcript levels were analyzed in the three HCC cell lines by using quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR; Fig. 3). CK7 and 19 transcripts were dramatically elevated on day 5 day after exposure to 50˚C, compared to no or minor changes after treatment at lower temperatures, but this increase was transient Daporinad concentration and levels returned to or near baseline on day 12. Transcript levels of the putative stem cell and progenitor marker, CD133, showed similar kinetics, whereas the hepatocyte differentiation marker, albumin (ALB) was significantly reduced on day 5, to rise baseline on day 12. Expression of four central EMT markers (Snail, TWIST1, CHD1L, and COL1A1) was increased in all three HCC cell lines 5 days after treatment at 50˚C (Supporting selleck products Fig. 1). Most of these increases were significant or highly significant, especially for COL1A1. The transcript

level (cycle threshold [Ct] value) for COL1A1 almost resembled that found in the well-established, activated human hepatic stellate cell line, LX-2[33] (Supporting Fig. 2). Tissue inhibitor of matrix metalloproteinase, another EMT-related marker, showed a similar trend (Supporting Fig. 3). Of note, EMT-related transcript levels returned to baseline on day 12 post–heat treatment. EMT-like changes, enhanced invasiveness, and migration of HEPG2 cells were confirmed by a 5- to 8-fold increased protein expression of Snail at day 5, but also at day 12 after heat treatment, and a significantly enhanced level of in vitro HEPG2 and HuH7 invasions at day 5 (Fig. 4A,B). Preheating (50˚C) preheating increased Shc transcript levels 5.0-, 2.8-, and 5.1-fold at day 5 in HEPG2, HuH7, and HEP3B, respectively (Fig. 5A).

Darwin (1868) was also aware of the prolonged sperm storage in certain insects (below). There were other significant discoveries in reproduction that Darwin must (or ought to) have known of, including: (1) von Baër’s (1827) discovery of the mammalian ovum and his later description of sperm–egg interactions in sea urchins (von Baër, 1847); (2) Wagner’s (1837) documentation of the extraordinary diversity of sperm size and shape; (3) von Kölliker’s (1841) discovery that spermatozoa need to make contact with

the egg if fertilization is to occur; (4) rather later, Hertwig’s (1876) demonstration that fertilization in sea urchins involves the fusion of male and female nuclei. If he did not obtain it directly, Darwin’s most likely conduit for this type of anatomical and physiological NSC 683864 cell line information is Thomas Huxley. BVD-523 Not only did Huxley receive lectures from some of the key players, like Thomas Wharton, describing the new German cell theory, fertilization and embryo development (Desmond, 1994, p. 26), Huxley later translated into English several major German zoology text books, including Kølliker’s (1853)Manual of Human Histology, which contains

a very comprehensive description of the male and female reproductive system, including this: Nor, from the experiments of Prévost, Dumas, Schwann, and Leukart, and the later researches of Newport … can the least doubt be entertained that they [spermatozoa] are the true impregnating agent, and for the purpose of impregnation must necessarily come in contact with the ovum’. Because Darwin had access to up-to-date information on sexual reproduction, including the processes of insemination, sperm function and fertilization, it seems at first sight unlikely that ignorance could account for his reluctance to explore the evolutionary implications of female promiscuity. On the other hand, if one reads this website the section in Variation (1868, p. 352) on sexual reproduction in

relation to pangenesis, it is easy to see how Smith (1998) thought Darwin confused: The union of the two sexual elements seems at first sight to make a broad distinction between sexual and asexual generation …. [But] the now well-ascertained cases of Parthenogenesis prove that the distinction between sexual and asexual generation is not nearly so great as was formerly thought; for ova occasionally, and even in some cases frequently, become developed into perfect beings, without the concourse of the male’. Yes – parthenogenesis must have been confusing. Why the [female] germ … ceases to progress and perishes, unless it be acted on by the male element; and why conversely the male element, which in the case of some insects is enabled to keep alive for four or five years … .

Darwin (1868) was also aware of the prolonged sperm storage in certain insects (below). There were other significant discoveries in reproduction that Darwin must (or ought to) have known of, including: (1) von Baër’s (1827) discovery of the mammalian ovum and his later description of sperm–egg interactions in sea urchins (von Baër, 1847); (2) Wagner’s (1837) documentation of the extraordinary diversity of sperm size and shape; (3) von Kölliker’s (1841) discovery that spermatozoa need to make contact with

the egg if fertilization is to occur; (4) rather later, Hertwig’s (1876) demonstration that fertilization in sea urchins involves the fusion of male and female nuclei. If he did not obtain it directly, Darwin’s most likely conduit for this type of anatomical and physiological selleck information is Thomas Huxley. MAPK inhibitor Not only did Huxley receive lectures from some of the key players, like Thomas Wharton, describing the new German cell theory, fertilization and embryo development (Desmond, 1994, p. 26), Huxley later translated into English several major German zoology text books, including Kølliker’s (1853)Manual of Human Histology, which contains

a very comprehensive description of the male and female reproductive system, including this: Nor, from the experiments of Prévost, Dumas, Schwann, and Leukart, and the later researches of Newport … can the least doubt be entertained that they [spermatozoa] are the true impregnating agent, and for the purpose of impregnation must necessarily come in contact with the ovum’. Because Darwin had access to up-to-date information on sexual reproduction, including the processes of insemination, sperm function and fertilization, it seems at first sight unlikely that ignorance could account for his reluctance to explore the evolutionary implications of female promiscuity. On the other hand, if one reads see more the section in Variation (1868, p. 352) on sexual reproduction in

relation to pangenesis, it is easy to see how Smith (1998) thought Darwin confused: The union of the two sexual elements seems at first sight to make a broad distinction between sexual and asexual generation …. [But] the now well-ascertained cases of Parthenogenesis prove that the distinction between sexual and asexual generation is not nearly so great as was formerly thought; for ova occasionally, and even in some cases frequently, become developed into perfect beings, without the concourse of the male’. Yes – parthenogenesis must have been confusing. Why the [female] germ … ceases to progress and perishes, unless it be acted on by the male element; and why conversely the male element, which in the case of some insects is enabled to keep alive for four or five years … .

Darwin (1868) was also aware of the prolonged sperm storage in certain insects (below). There were other significant discoveries in reproduction that Darwin must (or ought to) have known of, including: (1) von Baër’s (1827) discovery of the mammalian ovum and his later description of sperm–egg interactions in sea urchins (von Baër, 1847); (2) Wagner’s (1837) documentation of the extraordinary diversity of sperm size and shape; (3) von Kölliker’s (1841) discovery that spermatozoa need to make contact with

the egg if fertilization is to occur; (4) rather later, Hertwig’s (1876) demonstration that fertilization in sea urchins involves the fusion of male and female nuclei. If he did not obtain it directly, Darwin’s most likely conduit for this type of anatomical and physiological check details information is Thomas Huxley. EGFR phosphorylation Not only did Huxley receive lectures from some of the key players, like Thomas Wharton, describing the new German cell theory, fertilization and embryo development (Desmond, 1994, p. 26), Huxley later translated into English several major German zoology text books, including Kølliker’s (1853)Manual of Human Histology, which contains

a very comprehensive description of the male and female reproductive system, including this: Nor, from the experiments of Prévost, Dumas, Schwann, and Leukart, and the later researches of Newport … can the least doubt be entertained that they [spermatozoa] are the true impregnating agent, and for the purpose of impregnation must necessarily come in contact with the ovum’. Because Darwin had access to up-to-date information on sexual reproduction, including the processes of insemination, sperm function and fertilization, it seems at first sight unlikely that ignorance could account for his reluctance to explore the evolutionary implications of female promiscuity. On the other hand, if one reads selleck inhibitor the section in Variation (1868, p. 352) on sexual reproduction in

relation to pangenesis, it is easy to see how Smith (1998) thought Darwin confused: The union of the two sexual elements seems at first sight to make a broad distinction between sexual and asexual generation …. [But] the now well-ascertained cases of Parthenogenesis prove that the distinction between sexual and asexual generation is not nearly so great as was formerly thought; for ova occasionally, and even in some cases frequently, become developed into perfect beings, without the concourse of the male’. Yes – parthenogenesis must have been confusing. Why the [female] germ … ceases to progress and perishes, unless it be acted on by the male element; and why conversely the male element, which in the case of some insects is enabled to keep alive for four or five years … .

Intervention at the stage of bacterial attachment to the gastric mucosa could be an approach to improve the control/eradication rate of this infection. Materials and Methods:  Fractions of purified milk

fat globule membrane glycoproteins were tested in vitro for their cytotoxic and direct antibacterial effect. The anti-adhesive effect on H. pylori was determined first in a cell model using the mucus-producing gastric epithelial cell line NCI-N87 and next in the C57BL/6 mouse model after dosing at 400 mg/kg protein once or twice daily from day −2 to day 4 post-infection. Bacterial loads were determined by using quantitative real-time PCR and the standard plate count method. Results:  The milk fat globule membrane fractions Opaganib datasheet did not show in vitro cytotoxicity, and a marginal antibacterial effect was demonstrated for defatted milk fat globule membrane at 256 μg/mL. In the anti-adhesion assay, the results varied from 56.0 ± 5.3% inhibition for 0.3% crude milk fat globule membrane to 79.3 ± 3.5% for defatted milk fat globule membrane. Quite surprisingly, in vivo administration of the same milk fat globule membrane fractions did not confirm the anti-adhesive effects and even caused an increase in bacterial load in the stomach. Conclusions:  The promising anti-adhesion in vitro results could not be confirmed in the mouse

model, even after the highest attainable exposure. It is concluded that raw or defatted milk fat globule membrane fractions do not have any prophylactic or therapeutic potential against Helicobacter infection. “
“Helicobacter pylori infections check details have become increasingly difficult to treat. To examine whether amoxicillin selleck screening library and high-dose dexlansoprazole would reliably achieve an H. pylori eradication rate of ≥90%. An open-label prospective

pilot study of H. pylori eradication in treatment-naïve subjects with active H. pylori infection (positive by two tests). Therapy: amoxicillin 1 g and dexlansoprazole 120 mg each twice a day at approximately 12-hour intervals for 14 days. Success was accessed by urea breath test. An effective therapy was defined as a per-protocol treatment success of 90% or greater; treatment success of 80% or less was prespecified as an unacceptable result. After 13 subjects were entered (12 men, one woman; average age of 54 years), the prespecified stopping rule of six treatment failures was achieved (i.e., the 95% confidence interval excluded achieving the required 90% success rate even if the proposed study of 50 completed patients were entered) and enrollment was stopped. Per-protocol and intention-to-treat treatment success were both 53.8%; (7/13); 95% CI = 25–80%. Compliance was 100%. Three patients (23%) reported side effects, all of which were mild and none interrupted therapy. Theoretically, dual PPI plus amoxicillin should reliably eradicate H. pylori provided nearly neutral intragastric pH can be maintained.

Upon photobleaching, we observed rapid recovery from the nonbleached pool, with the original prebleached fraction of fluorescence clearly restored in less than 100 seconds (Fig. 4A,E). In contrast, in YFP-HBcAg and CFP-MxA cotransfected cells, the fluorescence recovery of YFP-HBcAg aggregated with CFP-MxA in the perinuclear region was dramatically slowed, with a great proportion of the initial YFP-HBcAg fluorescence in the region check details failing to recover, even a much longer time after photobleaching (Fig. 4B,E), suggesting this proportion of HBcAg was irreversibly bound to the compartment. The irreversible binding of HBcAg to the perinuclear pool was further confirmed

by fluorescence loss in photobleaching (FLIP). YFP-HBcAg fluorescence in a small cytoplasmic region of the cell expressing only YFP-HBcAg was bleached repetitively. After about 200 seconds, the fluorescence signals were completely lost in the areas outside the region, indicating that YFP-HBcAg diffused between the bleached and unbleached areas (Fig. 4C,F). In cells coexpressing YFP-HBcAg and CFP-MxA, repetitive

photobleaching of a similar cytoplasmic Galunisertib cell line pool failed to cause the loss of YFP-HBcAg from the perinuclear area in which it colocalized with CFP-MxA, indicating fixation of YFP-HBcAg in that area (Fig. 4D,F). These results indicate that the formation of MxA-HBcAg complexes is able to immobilize HBcAg in the perinuclear area. Interaction of MxA and viral nucleocapsid protein to generate complexes in the perinuclear compartment has been reported in many types of viral infection.7, 19 However, so far, the compartment has not been well-characterized. In an attempt to determine the essence of the perinuclear this website compartment where the MxA-HBcAg complex forms, we tested whether the MxA-HBcAg aggregates overlapped with the Golgi apparatus, the endoplasmic reticulum (ER), or the ER-Golgi intermediates, using distinct organelle

markers. Vero cells expressing CFP-MxA and YFP-HBcAg were either transfected with RFP-tagged ssKDEL, or stained by GM130 or p58 antibody. We found that the perinuclear MxA-HBcAg complexes colocalized with neither the ssKDEL-RFP used as an ER marker, nor the GM130, a Golgi matrix protein, nor the p58 as a marker of ER-Golgi intermediates (Fig. 5A). We also treated the cells with brefeldin A (BFA), a drug that disassembles the Golgi structure by inhibition of early steps in ER-Golgi transport.20 In the presence of BFA, when RFP-tagged galactosyltransferase, a Golgi-resident enzyme, redistributed to the ER, and the endogenous GM130 disassociated from the Golgi membranes (Fig. 5B), the MxA-HBcAg aggregation remained in the perinuclear region without notable changes (Fig. 5B), suggesting that the Golgi and the ER-Golgi intermediates are not involved in the formation of the aggregation. To further characterize the perinuclear compartment, we disrupted microtubules by treating the cells with nocodazole21 or by putting the cells on ice.

Upon photobleaching, we observed rapid recovery from the nonbleached pool, with the original prebleached fraction of fluorescence clearly restored in less than 100 seconds (Fig. 4A,E). In contrast, in YFP-HBcAg and CFP-MxA cotransfected cells, the fluorescence recovery of YFP-HBcAg aggregated with CFP-MxA in the perinuclear region was dramatically slowed, with a great proportion of the initial YFP-HBcAg fluorescence in the region Decitabine molecular weight failing to recover, even a much longer time after photobleaching (Fig. 4B,E), suggesting this proportion of HBcAg was irreversibly bound to the compartment. The irreversible binding of HBcAg to the perinuclear pool was further confirmed

by fluorescence loss in photobleaching (FLIP). YFP-HBcAg fluorescence in a small cytoplasmic region of the cell expressing only YFP-HBcAg was bleached repetitively. After about 200 seconds, the fluorescence signals were completely lost in the areas outside the region, indicating that YFP-HBcAg diffused between the bleached and unbleached areas (Fig. 4C,F). In cells coexpressing YFP-HBcAg and CFP-MxA, repetitive

photobleaching of a similar cytoplasmic Saracatinib solubility dmso pool failed to cause the loss of YFP-HBcAg from the perinuclear area in which it colocalized with CFP-MxA, indicating fixation of YFP-HBcAg in that area (Fig. 4D,F). These results indicate that the formation of MxA-HBcAg complexes is able to immobilize HBcAg in the perinuclear area. Interaction of MxA and viral nucleocapsid protein to generate complexes in the perinuclear compartment has been reported in many types of viral infection.7, 19 However, so far, the compartment has not been well-characterized. In an attempt to determine the essence of the perinuclear find more compartment where the MxA-HBcAg complex forms, we tested whether the MxA-HBcAg aggregates overlapped with the Golgi apparatus, the endoplasmic reticulum (ER), or the ER-Golgi intermediates, using distinct organelle

markers. Vero cells expressing CFP-MxA and YFP-HBcAg were either transfected with RFP-tagged ssKDEL, or stained by GM130 or p58 antibody. We found that the perinuclear MxA-HBcAg complexes colocalized with neither the ssKDEL-RFP used as an ER marker, nor the GM130, a Golgi matrix protein, nor the p58 as a marker of ER-Golgi intermediates (Fig. 5A). We also treated the cells with brefeldin A (BFA), a drug that disassembles the Golgi structure by inhibition of early steps in ER-Golgi transport.20 In the presence of BFA, when RFP-tagged galactosyltransferase, a Golgi-resident enzyme, redistributed to the ER, and the endogenous GM130 disassociated from the Golgi membranes (Fig. 5B), the MxA-HBcAg aggregation remained in the perinuclear region without notable changes (Fig. 5B), suggesting that the Golgi and the ER-Golgi intermediates are not involved in the formation of the aggregation. To further characterize the perinuclear compartment, we disrupted microtubules by treating the cells with nocodazole21 or by putting the cells on ice.

Demographic histories can also be estimated from genealogies (phylogenies) in a Bayesian statistical framework using BEAST (versions 1.4.6 and 1.7.2, Drummond and Rambaut 2007; http://beast.bio.ed.ac.uk). MODELTEST (Posada and Crandall 1998), using the Akaike information criterion, indicated that the nucleotide substitution model of Hasegawa et al. (1985) was appropriate Fludarabine when additionally allowing for unequal substitution rates among sites and for a proportion of sites to be invariable. When using BEAST, runs were of sufficient length

(typically 30 million or more) that effective sample sizes (ESSs) were always over 100, and usually very far over this value. Several runs were done for each input file to check for convergence. The program TRACER v1.4 (http://beast.bio.ed.ac.uk/) was used to analyze the output from BEAST. The first 10% of iterations in each run were discarded as burn-in. A coalescent exponential expansion model was specified and a randomWalkOperator selected for the exponential.growthRate parameter (Supplementary data files 2 and 3). If the 95% highest posterior density (HPD) of the growth rate parameter Selleck SAHA HDAC includes zero, a hypothesis of constant population size cannot be rejected (Marino et al. 2011; https://groups.google.com/d/msg/beast-users/y-ppM_dB5UI/uPybHlRMYc4J).

Monophyly of Australian dugongs was forced, and a prior of 115,000 yr (normal distribution ± 5,000) (date of the closure of Torres Strait to transit by marine organisms at the start of the last glacial period inferred from sea-level estimates; Fig. 2)

placed on the most selleck recent common ancestor (MRCA) of all Australian dugongs (see Supplementary data file 6). Trees generated during this analysis were examined for the strength of support given to the individual lineages. Bayesian skyline plots (BSPs) (Drummond et al. 2005) show changes in effective population size (NE(FEMALE)) over time, along with credibility intervals. A major advantage of this approach is that it avoids problems associated with choosing a single demographic scenario such as “constant population size” or “exponential growth.” Sample input files are in Supplementary data files 4 and 5. The mutation rate prior was specified (following the analysis above) as normally distributed with a mean of 24.8% per million years and lower and upper bounds of 13.89% and 37.46% per million years, respectively. Given that most samples came from distinct localities where sampling was possible, we simply used those localities as the basis for assigning individuals to “populations.” With some clustering of localities by geographic region if samples were few in number, we could define 11 populations on this basis (each represented by a pie chart in Fig. 1). There are no clear criteria for a priori grouping of these populations for a hierarchical analysis such as AMOVA (Excoffier et al. 1992).

Demographic histories can also be estimated from genealogies (phylogenies) in a Bayesian statistical framework using BEAST (versions 1.4.6 and 1.7.2, Drummond and Rambaut 2007; http://beast.bio.ed.ac.uk). MODELTEST (Posada and Crandall 1998), using the Akaike information criterion, indicated that the nucleotide substitution model of Hasegawa et al. (1985) was appropriate PD0332991 order when additionally allowing for unequal substitution rates among sites and for a proportion of sites to be invariable. When using BEAST, runs were of sufficient length

(typically 30 million or more) that effective sample sizes (ESSs) were always over 100, and usually very far over this value. Several runs were done for each input file to check for convergence. The program TRACER v1.4 (http://beast.bio.ed.ac.uk/) was used to analyze the output from BEAST. The first 10% of iterations in each run were discarded as burn-in. A coalescent exponential expansion model was specified and a randomWalkOperator selected for the exponential.growthRate parameter (Supplementary data files 2 and 3). If the 95% highest posterior density (HPD) of the growth rate parameter selleckchem includes zero, a hypothesis of constant population size cannot be rejected (Marino et al. 2011; https://groups.google.com/d/msg/beast-users/y-ppM_dB5UI/uPybHlRMYc4J).

Monophyly of Australian dugongs was forced, and a prior of 115,000 yr (normal distribution ± 5,000) (date of the closure of Torres Strait to transit by marine organisms at the start of the last glacial period inferred from sea-level estimates; Fig. 2)

placed on the most click here recent common ancestor (MRCA) of all Australian dugongs (see Supplementary data file 6). Trees generated during this analysis were examined for the strength of support given to the individual lineages. Bayesian skyline plots (BSPs) (Drummond et al. 2005) show changes in effective population size (NE(FEMALE)) over time, along with credibility intervals. A major advantage of this approach is that it avoids problems associated with choosing a single demographic scenario such as “constant population size” or “exponential growth.” Sample input files are in Supplementary data files 4 and 5. The mutation rate prior was specified (following the analysis above) as normally distributed with a mean of 24.8% per million years and lower and upper bounds of 13.89% and 37.46% per million years, respectively. Given that most samples came from distinct localities where sampling was possible, we simply used those localities as the basis for assigning individuals to “populations.” With some clustering of localities by geographic region if samples were few in number, we could define 11 populations on this basis (each represented by a pie chart in Fig. 1). There are no clear criteria for a priori grouping of these populations for a hierarchical analysis such as AMOVA (Excoffier et al. 1992).