text:
Evaluating the quality of routinely reported data on malaria commodity stocks in Guinea, 2014–2016
Yu Sun1,
Timothée Guilavogui2,
Alioune Camara2,
Mohamed Dioubaté2,
Babacar Deen Toure2,
Claude Bahati3,
Marie Paule Fargier3,
Jessica Butts4, 5,
Patrick Condo6,
Abdoulaye Sarr4, 5 and
Mateusz M. Plucinski4, 5Email authorView ORCID ID profile
Malaria Journal201817:461
Received: 3 June 2018
Accepted: 29 November 2018
Ensuring malaria commodity availability at health facilities is a cornerstone of malaria control. Since 2013, the Guinea National Malaria Control Programme has been routinely collecting data on stock levels of key malaria commodities through a monthly routine malaria information system (RMIS). In parallel, biannual end-user verification (EUV) surveys have also assessed malaria commodity availability at a subset of health facilities, potentially representing a duplication of efforts.
Data on 12 malaria commodity stock levels verified during four EUV surveys conducted between 2014 and 2016 was compared to data for the corresponding months submitted by the same health facilities through the RMIS. The sensitivity and specificity of the RMIS in detecting stock-outs was calculated, as was the percent difference between average stock levels reported through the two systems.
Of the 171 health facilities visited during the four EUV surveys, 129 (75%) had data available in the RMIS. Of 351 commodity stock-outs observed during the EUV in the sampled reporting health facilities, 256 (73%) were also signaled through the corresponding RMIS reports. When the presence of malaria commodity stocks was confirmed during the EUV surveys, the RMIS also reported available stock 87% (677/775) of the time. For all commodities, the median percent difference in average stock levels between the EUV and RMIS was 4% (interquartile range − 7 to 27%).
The concordance between stock levels reported through the RMIS and those verified during the EUV visits provides certain evidence that RMIS data can inform quantification and procurement decisions. However, lower than acceptable rates of reporting and incomplete detection of stock-outs from facilities that do report suggest that further systems strengthening is needed to improve RMIS reporting completeness and data quality.
Stock-out
Artemisinin-based combination therapy
Rapid diagnostic test
Logistics management information system
Malaria transmission occurs year-round throughout Guinea and is the principal cause of healthcare seeking in the public sector, responsible for 31% of all outpatient consults [1]. The country has made substantial progress in malaria control in recent years. The national prevalence of malaria infection in children under 5 years of age, as measured by microscopy, fell from 44% in 2012 [2] to 15% in 2016 [3]. Malaria incidence in 2016 was estimated to be 87/1000 population by the Guinea National Malaria Control Programme (NMCP) [4].
The Guinea NMCP aims to reduce malaria morbidity and mortality through a combination of interventions aimed at prevention, consisting of the distribution of long-lasting insecticidal nets (LLINs), seasonal malaria chemoprevention, and intermittent preventive treatment of malaria during pregnancy, as well as interventions to ensure timely diagnosis and correct treatment of confirmed malaria cases. Ensuring effective treatment requires a steady and reliable supply of malaria tests and medicines at health facilities.
Key malaria commodities include malaria rapid diagnostic tests (RDTs), medicines used for treatment of uncomplicated malaria by artemisinin-based combination therapy (ACT), quinine tablets, injectable forms of quinine and artemisinin for treatment of severe malaria, sulfadoxine–pyrimethamine (SP) for intermittent preventive treatment in pregnancy, and LLINs for routine distribution to pregnant women and infants. These commodities are financed and centrally procured by the government of Guinea and donors, primarily the Global Fund to Fight AIDS, Tuberculosis and Malaria (GF) and the U.S. President's Malaria Initiative (PMI), and stored at the central medical stores. From there, they are distributed to regional depots, from which Guinea's roughly 400 public hospitals and health centres are supplied when they directly request commodities from the regional depot based on needs, using a "pull system". Health centres in turn supply the community health workers and health posts in their catchment area. Malaria commodities are provided free of charge to patients of all ages.
Monitoring stocks of malaria commodities throughout the system is a priority activity for the NMCP and its partners. Stock availability is an important indicator of health system readiness and sudden interruptions in the supply chain may affect the quality of service and health programmes [5]. Moreover, frequent stock-outs of essential anti-malarial drugs due to improperly managed supply chains can affect healthcare provider prescription behavior [6], increase patient healthcare costs [7], and impede universal access to quality malaria case management [5, 8, 9]. However, while there has been work on developing tools for malaria supply chain management in endemic countries [10], few formal studies of optimal supply chain policies have been performed [11–13].
During initial expansion of malaria control efforts in Guinea, the country's logistic management and information system (LMIS) at the time was originally targeted to manage and improve management of commodities at the level of the central medical stores and regional depots. Because the LMIS was not designed to collect data on commodity stocks at the health-facility level, the Guinea NMCP decided to include data on malaria commodity stocks as part of its monthly routine malaria information system (RMIS), parallel to and independent of the larger health management information system. A parallel malaria-only reporting system was deemed necessary in the absence of a functional health management information system at the time. The RMIS is supported by PMI and GF and was rolled out in 2013, reaching nationwide coverage in 2014. District-level reporting improved from 66% completeness in late 2014, shortly after expansion, to consistent 100% completeness since early 2016. Data on stock levels at the beginning of the month, stock levels at the end of the month, number of days of stock-out, and consumption rates, including use, expiration, and loss, are reported through the RMIS by hospitals and health centres for 12 malaria commodities. Monthly data are recorded in paper forms at health facilities, and then transferred to the district health authorities, where they are inputted into an electronic database and sent to the national level. The Guinea NMCP routinely analyses the reported data to identify areas with under- and over-stock of commodities and monitors stock levels and consumption rates to forecast commodity needs. The malaria monthly bulletin issued by NMCP has included district-level commodity indicators since its inception in late 2014 and is a key tool in allowing NMCP and its partners to track malaria commodity levels.
When PMI was launched in Guinea in 2012 and the RMIS was not yet operational, PMI implemented periodic end-use verification (EUV) surveys to monitor malaria commodity availability at regional warehouses and in peripheral health facilities [14]. Originally, the EUV surveys were only implemented in the 19 health districts supported by PMI, but starting July 2014, implementation expanded nationwide. During these surveys, teams from the national level visited a cross-sectional sample of hospitals and health centres to record stocks of malaria commodities, examine stock cards, document storage conditions, and review outpatient registries. The surveys were implemented by the PMI-funded Systems for Improved Access to Pharmaceuticals and Services project, but included participation from NMCP and district health authorities. They followed a standard methodology to track PMI-procured commodities from source to end-user.
Since 2016, the improved timeliness and completeness of the RMIS have raised the possibility that it might already be collecting sufficiently reliable commodity data to provide the same kind of accountability that the EUV surveys were originally designed to provide. If that were the case, then the resources currently devoted to the EUV surveys could be redirected to other monitoring and evaluation activities. To explore this possibility, NMCP and its partners undertook a systematic assessment comparing the RMIS commodity reporting to the results of the EUV.
A retrospective analysis was conducted comparing data on commodity stock-outs and stock levels reported through the RMIS to data collected in health facilities during four EUV surveys. Using the EUV data as the gold standard, the RMIS data were evaluated on reporting completeness, sensitivity and specificity of stock-out detection, and the relative difference in reported stock levels. The RMIS data were obtained from the NMCP national malaria database, and the EUV data were provided by the PMI-funded Systems for Improved Access to Pharmaceuticals and Services project in Guinea.
Period and area
Four EUV surveys that occurred since the scale-up of the RMIS were included in the analysis: July 2014, December 2014, October 2015, and August 2016. The number of health facilities visited during the surveys varied from 31 in the two 2014 surveys to 64 in the most recent survey in August 2016. In total, the four surveys comprised 171 hospital and health centre visits throughout Guinea; 71 (42%) of these were to visits to health facilities visited more than once during the four rounds. Hospitals represented 19% of all health facility visits across the four surveys. The number of regions visited during the surveys was six in July 2014, increasing to seven in December 2014, and finally including all eight regions in the October 2015 and August 2016 surveys.
For each health facility visited during the EUV surveys, the RMIS database was searched by matching on health facility name and district. Variations in spelling and health facility name required manual matching of health facilities in the two databases. Data on day-of-visit commodity stock levels verified during the surveys were abstracted from the EUV database. Data on beginning-of-month and end-of-month stock levels and the number of days with stock-out were abstracted from the RMIS for the month corresponding to the EUV visit for each health facility in the EUV. Data on twelve malaria commodities were analysed: RDTs, the four age/weight formulations of amodiaquine–artesunate (AS–AQ), oral quinine tablets, injectable quinine, injectable artesunate, intramuscular artemether, SP, and LLINs. Data on stocks of AL, which was not in wide use in Guinea in the period covered by the assessment, were considered separately. The July 2014 and December 2014 EUV surveys did not capture data on AL, so indicators for AL for these surveys were not calculated. Moreover, in the period covered by the four surveys, the RMIS data recording and reporting forms did not stratify AL stocks by its four age/weight formulations, so data on AL stocks collected during the EUV surveys, which did stratify by the different formulations, were considered together; AL was considered in stock if any formulation of AL was documented, and AL stock levels were considered to be the sum across all AL formulations.
Reporting completeness of the RMIS was assessed by calculating the proportion of the health facilities included in each EUV survey that had submitted their monthly malaria report through the RMIS for the month of the survey.
The sensitivity and specificity of the RMIS to detect stock-outs of malaria commodities were also assessed. Sensitivity was calculated as the proportion of health facilities with a stock-out during the EUV survey that also reported a stock-out in the RMIS report for the corresponding month. The specificity was defined as the proportion of health facilities without a stock-out during the EUV survey that did not report a stock-out in their corresponding RMIS report. Health facilities that had not submitted RMIS data during the month of the survey were excluded from the analysis of specificity and sensitivity. Stock-outs in the EUV were defined as zero stock level recorded during the day of the survey visit. In the RMIS data, a health facility was considered to have reported a stock-out if beginning-of-month stock levels were reported as zero, end-of-month stock levels were reported as zero, or the number of days of stock-out during the month were greater than zero. The sensitivity and specificity were calculated separately for each commodity and each survey.
The stock levels reported through the RMIS and counted during the EUV visits were compared separately for each survey and then jointly across all surveys. Because day-of-visit data were not available from the RMIS, the average monthly stock levels were analysed, defined as the mean of the beginning-of-month and end-of-month stock levels. For each commodity, the total mean monthly stock level per health facility was calculated for the RMIS dataset, and the average percent difference between the RMIS and EUV datasets was calculated. Additionally, for each individual health facility, the percent difference between the EUV and RMIS data was calculated for each commodity. The distribution of health-facility-level percent difference was visualized using boxplots, stratifying by commodity. The proportion of health facilities with percent difference < 25%, 25–50%, and > 50% was calculated. Health facilities that had not submitted RMIS data during the month of the survey were excluded from the analysis of stock levels.
Abstracted data were collected in an Excel (Microsoft, Redmond, USA) spreadsheet and data analysis was performed using R version 3.3.2 (R Foundation for Statistical Computing, Vienna, Austria).
Completeness of health facility reporting
Out of the 171 health facilities visited during the four EUVs, 75% (129/171) had submitted corresponding monthly reports through the RMIS (Table 1). Completeness ranged from 67% (30/45) in the October 2015 survey to 87% (27/31) in the July 2014 survey.
Proportion of health facilities visited during four end-user verification (EUV) surveys that also submitted reports through the routine malaria information system, Guinea
Reporting completeness
EUV Jul 2014
EUV Dec 2014
EUV Oct 2015
EUV Aug 2016
n/N
Stock-out detection
There were 351 instances of stock-outs for the 12 malaria commodities assessed recorded during the four EUV surveys in health facilities that had submitted RMIS reports. Of these commodity stock-outs, 256 (73%) were also signaled through the corresponding RMIS reports (Table 2). The sensitivity of the RMIS in detecting stock-outs of all commodities ranged from 68% (119/176) in the August 2016 survey to 84% (53/63) in the July 2014 survey. Across all surveys, sensitivity of the RMIS was highest in detecting injectable artemether stock-outs (sensitivity 98%, 51/52) and least sensitive for SP (sensitivity 31%, 5/16). The RMIS was also highly specific in capturing stock availability. For facilities visited during the EUV for which there was a corresponding RMIS report, there were 775 instances across all commodities and all four surveys where the EUV reported available stock. In the RMIS database, 677 (87%) of these instances were reported having stock available during the corresponding months. The specificity for all commodities ranged from 84% (173/205) in October 2015 to 91% (130/143) in December 2014. The RMIS was most specific for detecting stock availability of the ASAQ formulations and AL, with specificity exceeding 88% for each. Specificity was lowest for injectable artemether, 43% (6/14).
Sensitivity and specificity of the routine malaria information system (RMIS) in detecting stock-outs observed during four end-user verification (EUV) surveys, Guinea
Sensitivitya
Specificityb
RDT
4/4 (100%)
1/3 (33%)
17/17 (100%)
ASAQ infant
0/1 (0%)
ASAQ small child
ASAQ child
ASAQ adult
Injectable artesunate
Injectable artemether
Injectable quinine
Quinine tablet
LLIN
All commodities
152/172 (88%)
RDT Rapid diagnostic test, ASAQ artesunate–amodiaquine, AL artemether–lumefantrine, SP sulfadoxine–pyrimethamine, LLIN long-lasting insecticidal net
a Proportion of health facilities without stock during the EUV survey that had also reported a stock-out in their RMIS report
b Proportion of health facilities that were not stocked-out during the EUV survey that had not reported a stock-out in their RMIS report
Average commodity stock levels reported through the RMIS were close to the average stock levels verified during the EUV surveys (Figs. 1 and 2). The median percent difference in average stock levels between the EUV and RMIS across all commodities in the four surveys was 4%, with the interquartile range spanning − 7% to 27% (Fig. 3).
Average stock levels (individual commodity units) per health facility for 12 malaria commodities reported through the Guinea routine malaria information system (RMIS) and verified during four end-user verification (EUV) surveys conducted between 2014 and 2016, averaged across all four surveys, for the subset of health facilities with data available in both data sources. RDT Rapid diagnostic test, ASAQ artesunate–amodiaquine, AL artemether–lumefantrine, SP sulfadoxine–pyrimethamine, LLIN long-lasting insecticidal net
Average stock levels (individual commodity units) per health facility for 12 malaria commodities reported through the Guinea routine malaria information system (RMIS) and verified during four end-user verification (EUV) surveys for the subset of health facilities with data available in both data sources. RDT Rapid diagnostic test, ASAQ artesunate–amodiaquine, AL artemether–lumefantrine, SP sulfadoxine–pyrimethamine, LLIN long-lasting insecticidal net
Distribution of percent differences between stock levels reported through the routine malaria information system and those verified during four end-user verification surveys, averaged across all four surveys, Guinea. RDT Rapid diagnostic test, ASAQ artesunate–amodiaquine, AL artemether–lumefantrine, SP sulfadoxine–pyrimethamine, LLIN long-lasting insecticidal net. Boxes show median values and interquartile ranges. Whiskers show extreme values up to 1.5 times interquartile range and circles represent outliers outside this range
The concordance between average stock levels across the two data sources belied substantial variation at the individual health facility level (Fig. 3). However, the median percent difference was close to zero for all commodities, and, for all commodities with the exception of injectable artemether, more than half of all absolute percent differences were less than 50% (Table 3). The distribution of percent difference was stable across the four surveys (Fig. 4).
Distribution of percent differences between malaria commodity stock levels reported through the routine malaria information system compared to those verified during four end-user verification surveys, Guinea
Number of reports
< 25% difference
25–50% difference
> 50% difference
0/10 (0%)
Quinine tablets
Distribution of percent differences between stock levels reported through the routine malaria information system and those verified during four end-user verification surveys, Guinea. RDT Rapid diagnostic test, ASAQ artesunate–amodiaquine, AL artemether–lumefantrine, SP sulfadoxine–pyrimethamine, LLIN long-lasting insecticidal net. Boxes show median values and interquartile ranges. Whiskers show extreme values up to 1.5 times interquartile range and circles represent outliers outside this range
The RMIS data on overall malaria commodity stock levels generally agreed with the stock levels counted during the field visits. However, the detection of stock-outs through the RMIS is still incomplete. About a quarter of health facilities do not report through RMIS, and in the three quarters of health facilities that had submitted malaria reports for the months of the EUV visits, the RMIS was sufficiently sensitive to detect only three quarters of all stock-outs observed in the EUV. The RMIS reported stock-outs not captured by the EUV in 13% of cases.
The concordance between the two data sources should be interpreted in the context of the limitations of the methodology of the comparison. Because the EUV surveys only record the actual stock levels on the day of the health facility visit, the data from the monthly RMIS reports and the EUV data could not be compared directly. As a result, the definition of stock-outs differed between the two data sources, and the sensitivity and specificity reported here need to be interpreted in that context. In particular, the sensitivity could have been overestimated and the specificity underestimated since the RMIS can capture stock-outs that occur at any point during the month. In addition, the comparison of stock levels was not a one-to-one comparison, which could account for the observation that although mean stock levels were similar between the two sources, there was substantial variation at the individual health facility level. A further limitation is that there were differences in how AL stock levels were captured in the two systems. Finally, non-reporting health facilities might be more likely to have stock-outs or poor data recording practices, and thus the concordance between the EUV and RMIS stock levels reported here might be an over-estimate.
Ultimately, the complexities in validating the RMIS data against the EUV data reflect fundamental differences in the reporting systems' objectives and designs. The RMIS was designed to provide complete, timely data on key health facility malaria indicators for the entire country throughout the year. In contrast, EUV surveys were designed to provide periodic audits of health facilities for the purposes of accountability. The imperfect data completeness, sensitivity and specificity of the RMIS are balanced out by a much larger amount of data collected during the RMIS. For 2016, while the EUV survey visited a total of 64 health facilities, yielding 768 data-points on stock availability, the RMIS collected a total of 5280 monthly health facility reports across the entire year, for a total of 63,360 data points. The spatial and temporal scope of the RMIS allows continuous detection of stock-outs throughout the country. Similarly, the representativeness of the consumption and stock level data in the RMIS allows for accurate quantification and forecasting of malaria commodity needs. Finally, the fact that the RMIS is managed and implemented by the NMCP and district health authorities means that it is sustainable in the long term and permits the kind of routine analysis embodied by the production and dissemination of the monthly malaria bulletins.
Because it involves teams visiting health facilities, the EUV methodology allows for the incidental collection of data that are not normally available through the RMIS. Verification of commodity stock by survey teams allows for independent assessment of the availability of donor-sourced malaria commodities. In addition, survey teams assess other aspects of management of malaria commodities, such as storage conditions and the correct use of stock cards. EUV surveys in Guinea also include a retrospective review of outpatient registries, providing insight into malaria case management practices. Finally, EUVs provide a chance for capacity building for participating NMCP and district health authority staff, while also providing an incidental opportunity for supportive supervision of health facility staff.
The results presented here suggest that the data reported through the Guinea RMIS could be used to monitor commodity stocks in health facilities. In this context, the EUVs do not provide substantial additional actionable information on malaria commodity stock levels and the stock verification component of the EUVs might represent an unnecessary duplication of efforts, on the condition that the RMIS can routinely be queried to provide data on commodity stock levels and stock-outs. Instead, the added utility of the EUV surveys is concentrated in the secondary data incidentally collected during the surveys; for example, storage conditions and case management practices and their role in supervision and capacity building. In recent years, the Guinea NMCP has developed and rolled out a comprehensive supervision tool to be used during malaria-related supervisory visits to health facilities. The additional components of the EUV, such as those related to storage conditions and commodity management practices, are either already included in the supervision tool or could be easily integrated into the existing tool. Resources currently devoted to the implementation of the EUV survey could instead be repositioned to support routine supervisory visits, including increasing their frequency, expanding national- and district-level participation, and enhancing their focus on data quality. As malaria control efforts in Guinea mature, the focus of NMCP and its partners has been shifting from scale-up of interventions to monitoring and tracking their impact. In parallel, the periodic collection of data through household and health facility surveys has yielded to continuous, routine information systems.
Discontinuation of surveys, originally intended to be temporary measures in the absence of well-functioning routine systems, will need to be informed by systematic evaluations like the one reported here that can assess whether the sensitivity and specificity of routine information systems are adequate and can provide evidence that the routine information systems are sufficiently robust and accurate. Ultimately, the assessment of the commodity data in the RMIS reported here was made possible because of the unique opportunity to have an independent dataset on commodity stocks in the form of the EUV surveys. The ability to rely on the RMIS to provide high-quality commodity data will benefit from future assessments that cross-check independently collected data, such as from data quality audits, with the routinely collected RMIS data. These sorts of assessments are particularly timely as Guinea pursues a modernization of its ensemble HMIS, based on the increasingly popular open-source District Health Information System 2 (DHIS2) reporting platform. This new system has an integrated health-facility-level LMIS, whose framework is modeled on the commodity component of the RMIS. Eventually, the RMIS itself is planned to be fully integrated into the new HMIS, cementing it as a foundation of routine disease surveillance in Guinea.
MMP, YS, TG, AC, and JB planned and designed the study. YS, MMP, MD, BDT, CB, MPF, PC, and AS performed data analysis. MMP, YS, TG, and AC wrote the manuscript. All authors read and approved the final manuscript.
The authors would like to thank Rima Shretta for her comments and review of the report. Funding was provided by the U.S. President's Malaria Initiative. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. The authors declare they do not have any commercial or other associations that might pose a conflict of interest.
All data are available from the authors upon reasonable request.
The study was funded by the US President's Malaria Initiative.
Rollins School of Public Health, Emory University, Atlanta, GA, USA
National Malaria Control Program, Conakry, Guinea
Systems for Improved Access to Pharmaceuticals and Services, Conakry, Guinea
Malaria Branch, Division of Parasitic Diseases and Malaria, Centers for Disease Control and Prevention, Atlanta, GA, USA
U.S. President's Malaria Initiative, Centers for Disease Control and Prevention, Atlanta, GA, USA
U.S. President's Malaria Initiative, USAID, Conakry, Guinea
Guinea National Malaria Control Programme. National malaria strategic plan 2018–2022. Conakry. 2017.Google Scholar
Guinea National Statistics Institute. Demographic health survey. Conakry. 2012.Google Scholar
Guinea National Statistics Institute. Multiple indicator cluster survey. Conakry. 2016.Google Scholar
Guinea National Malaria Control Programme. Monthly malaria bulletin, December 2016. Conakry. 2016.Google Scholar
Wagenaar BH, Gimbel S, Hoek R, Pfeiffer J, Michel C, Manuel JL, et al. Stock-outs of essential health products in Mozambique—longitudinal analyses from 2011 to 2013. Trop Med Int Health. 2014;19:791–801.View ArticlePubMedPubMed CentralGoogle Scholar
Hensen B, Paintain LS, Shretta R, Bruce J, Jones C, Webster J. Taking stock: provider prescribing practices in the presence and absence of ACT stock. Malar J. 2011;10:218.View ArticlePubMedPubMed CentralGoogle Scholar
Mikkelsen-Lopez I, Tediosi F, Abdallah G, Njozi M, Amuri B, Khatib R, et al. Beyond antimalarial stock-outs: implications of health provider compliance on out-of-pocket expenditure during care-seeking for fever in South East Tanzania. BMC Health Serv Res. 2013;13:444.View ArticlePubMedPubMed CentralGoogle Scholar
Mikkelsen-Lopez I, Shango W, Barrington J, Ziegler R, Smith T. The challenge to avoid anti-malarial medicine stock-outs in an era of funding partners: the case of Tanzania. Malar J. 2014;13:181.View ArticlePubMedPubMed CentralGoogle Scholar
Sudoi RK, Githinji S, Nyandigisi A, Muturi A, Snow RW, Zurovac D. The magnitude and trend of artemether–lumefantrine stock-outs at public health facilities in Kenya. Malar J. 2012;11:37.View ArticlePubMedPubMed CentralGoogle Scholar
Strengthening Pharmaceutical Systems Program. Guide for malaria commodities logistic management system: applying the monitoring-training-planning approach for improving performance. Management Sciences for Health. Geneva: World Health Organization; 2013. p. 69.Google Scholar
Leung N-HZ, Chen A, Yadav P, Gallien J. The impact of inventory management on stock-outs of essential drugs in Sub-Saharan Africa: secondary analysis of a field experiment in Zambia. PLoS ONE. 2016;11:e0156026.View ArticlePubMedPubMed CentralGoogle Scholar
Githinji S, Kigen S, Memusi D, Nyandigisi A, Mbithi AM, Wamari A, et al. Reducing stock-outs of life saving malaria commodities using mobile phone text-messaging: SMS for life study in Kenya. PLoS ONE. 2013;8:e54066.View ArticlePubMedPubMed CentralGoogle Scholar
Bam L, McLaren Z, Coetzee E, von Leipzig K. Reducing stock-outs of essential tuberculosis medicines: a system dynamics modelling approach to supply chain management. Health Policy Plan. 2017;32:1127–34.View ArticlePubMedGoogle Scholar
President's Malaria Initiative. Guinea Malaria Operational Plan FY 2018. 2017.Google Scholar
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate. Please note that comments may be removed without notice if they are flagged by another user or do not comply with our community guidelines.


piano caster cups uk wood floor protection world digital forums.
battat take apart 4x4 toys amazon wholesale other radio replacement parts.
kidco childrens mesh bed rail healthcare full length protectors cot side.


Discover and exceptionally welcoming address in the heart of the city minutes away from the Abu Dhabi National Exhibition center, major shopping malls, landmark attractions. Ramee Royal Hotel Apartments provides modern and luxuriously appointed Studio & One bedroom apartments. All rooms are centrally air-conditioned. It has electronic door locks, king size beds, safe lockers, Iron Board, LCD TVs, DVD players, Dining table, sofa sets, fully equipped kitchen with washing machine, refrigerator, toaster, microwave, cooking range, utensils etc. The apartments are equipped with fire alarm and sprinkler system for your safety.


text:
Bible Friendly Books
News, products and services of interest to students of the Bible.
Mississippi votes on when life begins
On Tuesday, voters in Mississippi will decide when human life begins.
Not in actuality, or course. When human life begins is a scientific fact. It might be debated by scientists, but it won't be changed by a vote. What they will be voting on is a legality.
The official ballot summary of the measure reads:
"Initiative #26 would amend the Mississippi Constitution to define the word 'person' or 'persons', as those terms are used in Article III of the state constitution, to include every human being from the moment of fertilization, cloning, or the functional equivalent thereof."
While the Mississippi Constitution uses the word "person" several times, such as in section 22, "No person shall be deprived of life, liberty, or property except by due process of law," until now, it had never defined just exactly who or what a "person" was. Until our confusing times, it had apparently never needed to be defined.
But now, when even words like "man," "woman," and "marriage" have become ambiguous, it seems "person" needs a definition.
Surprisingly, considering the current climate of focusing on 'a woman's right to choose,' at least 38 states already have "fetal homicide" laws; that is, laws which penalize an act of violence which causes physical harm to an unborn child. Mississippi, for example, already has laws on the books that define manslaughter as including "the willful killing of an unborn quick child by an injury to the mother of such child." (Miss.Code Ann.§ 97-3-37) They also define murder to include murder that is done "with deliberate design to effect the death of an unborn child." (§ 97-3-19)
Political groups are lining up on both sides of the Mississippi debate but, really, they are just using the publicity to further their own agendas. There are already laws on the books that specifically exclude "acts committed by the mother, a medical procedure performed by a medical professional or lawfully prescribed medication." (§ 11.7.13)
Thus even if the new wording is adopted women may continue to ask for abortions, and doctors may continue to perform abortions, without fear of criminal action.
Supporters of the amendment are largely of a religious nature, such as Catholic Social and Community Services director Jennifer Williams, who helped gather the signatures to get the initiative on the ballot.
Opponents, not surprisingly, include groups such as Planned Parenthood, but also some who are concerned that the wording could cause difficulties for practitioners of fertility techniques that currently involve the destruction or discarding of unneeded embryos.
None of us are on the sidelines in this issue. While various groups attempt to make political hay out of it, the fact remains that we all started life as embryos; as zygotes, even. None of us would be here if steps had been taken to deliberately interrupt gestation.
If abortion were entirely about "a woman's right to choose," then removing an embryo would be no different from a woman electing to have her gall bladder removed; yet, if a woman with no pain went to a doctor for such an operation, the doctor would probably try to talk her out of it.
Significantly, however, the Bible describes an embryo as a person. David wrote of God: "Your eyes saw my embryo; and in your book all my members were written, the days they were formed, and not one was among them." (Ps 139:16) Notice he did not say 'my mother's embryo.' That verse also makes it clear that David considered himself to have been a person even before all his 'members were formed.'
Similarly, the Bible character Job poetically describes a day nine months before his birth when his mother may have exclaimed, `Aman-child hath been conceived.' (Job 3:3) Job did not consider his beginning as simply a bit of tissue that his mother had a choice to retain or discard.
Professor Jerome Lejeune, the researcher who discovered the genetic cause of Down Syndrome, testified before the Fifth Judicial District of Tennessee on September 21, 1989, that "as soon as he has been conceived, a man is a man."
And he wasn't a lone voice in the wilderness. Here's what some other scientists have said:
"A zygote is the beginning of a new human being. Human development begins at fertilization." - The Developing Human: Clinically Oriented Embryology, 6th ed. Keith L. Moore, Ph.D. & T.V.N. Persaud, Md.
"The penetration of the ovum by a spermatozoan and resultant mingling of the nuclear material each brings to the union … marks the initiation of the life of a new individual." - Human Embryology, 3rd ed. Bradley M. Patten
"The zygote thus formed represents the beginning of a new life." - J.P. Greenhill and E.A. Friedman Biological Principles and Modern Practice of Obstetrics
"A new genetically distinct human organism is formed when the chromosomes of the male and female pronuclei blend in the oocyte." - Ronan O'Rahilly and Fabiola Miller, Human Embryology and Teratology
The pro-choice crowd always manages to bring up very sensitive questions such as:
What if the mother's life is in danger?
What if she was raped?
What if she is too young or mentally unstable to be a mom?
How do you weigh the potential benefits of embryonic stem cell research against the lost life of an embryo?
How do you weigh the emotional needs of the wannabe mom against the lives of the unneeded embryos that end up being discarded after in vitro fertilization?
What about custody of the frozen embryos in cases of divorce?
In every case, challenging as the question may be, difficult as the answer may be, at its core it is a simple truth: an embryo is a human life.
Regardless of how the good people of Mississippi vote.
Please leave a respectful comment. If you would like to be notified when I post a new column click on Subscribe at the top of the page.
Read my previous article
Email me at bill.underwood@mail.com
Posted by Bill K. Underwood at 9:01 PM
Worldwide Letter-writing Campaign Protests Proposed Religious Ban
Spread the Word: Transfusion is NOT a "Lifesaving Procedure."
Should Christians celebrate Halloween?
Is Medical Marijuana for Christians?
How Do We Know the Signs of the Times are Being Fulfilled
Who invented Santa?
Would Jesus like your church?
Did Jesus die on a cross?
Landmark ruling from South Korean appeals court
The Church's role in World War One
Prince, fame, and religious controversy
How did your Bible get to you?
Wild bees joining honey bees, monarch butterflies ...
Is Mankind "evolving" about homosexuality?
Davis, Day, others fight to defend constitutional ...
About Time, part 5: Carbon-14 dating, the ice age,...
How do you determine right and wrong?
About Time, part four: The evolution of Language –...
Why won't racism go away?
About Time, part three: The Bible and the Big Bang...
About Time, part two: Bible history versus secular...
About Time: Chronology and Bible skeptics
Measles stirs debate between vaccinators and antiv...
Should the Exodus be re-dated?
Evils of religion: The cult of Islam versus Charli...
Government, Parental Rights, and medical treatment...
Evils of religion: Brigham Young and the Mountain ...
Evils of religion: Catholic Church changing its st...
Evils of religion: Land speculation, Danites, and ...
Evils of religion: How a sexual predator became a ...
Evils of religion: The scam that became Mormonism
Evils of religion: Protestantism's present-day sca...
Evils of religion: The brutality of the early Prot...
Evils of religion part 5: Catholicism's worst atro...
Evils of religion, part 4: The lie about the unbro...
Evils of religion, part 3: Catholicism's false pre...
Evils of religion, part 2: Was Peter the first Cat...
Evils done in the name of Religion: Part 1, Cathol...
No religion has a monopoly on evil
Are things getting better or worse?
"Things have always been this way!"
The UN's coming attack on Religion, Part 3
Is the UN a paper tiger or a sleeping lion?
Is the UN preparing to attack Religion?
Do camel bones disprove the Bible?
Blood, medical ethics, and the Bible
Real advances in blood technology
Blood Medicine, part three
Blood Medicine, part two
Advances in Blood Medicine
GMOs, gluten, and the poisoning of a planet's food...
The great wheat crisis
Is gluten killing you?
Tom Hanks has diabetes
How Monsanto will be beaten
If a company does evil things, are the people who ...
More on Monsanto
Is Monsanto retreating?
Lessons from a frog
Archaeologists find, argue about, evidence support...
If you believe in giving til it hurts, this may hu...
Before you give, what do you know about your chari...
How much Babylonish activity is too much?
Babylonish behavior is NOT limited to the Catholic...
The last Passover
Does the resignation of the pope fulfill Bible Pro...
Do you know the difference between wisdom and unde...
More scandal surrounding Papal resignation
Pope resignation releases a flood of rumors
How to not get shot
Should you get a gun? Part two
Should you get a gun?
What's wrong with Christmas? Part 4
What's wrong with Christmas? Part one
Atheists convene to invite others to "Come out!"
Government, religion, and birth-control
Is birth control a sin? Part 2
Is birth control a sin?
Does the Bible still matter? Part 3
Replies to Bible survey begin to arrive
Does the Bible still matter?
10 things to be thankful for now that Thanksgiving...
Penn State pedophile scandal continues to grow
An athiest's take on the Ten Commandments
More fun with evolution
Arguments against evolution
Some random observations on the tenth anniversary ...
South Korea backs away from lifting ban on conscie...
Are the Jews still God's chosen people?
Making ends meet in the current economy, part 3
Making a living, part two, postponed due to end of...
Making ends meet in the current economy
Work without college
How the economy works
Why vote?
Halloween and Satanism
Why Christians hate
Vampires, part two
The attraction of vampires, part one
Comparing the Koran, the Book of Mormon, and the B...
More on the Book of Mormon
Is the Book of Mormon from God?
Bible verse about stoning children causes a firest...
Is the Bible still relevant?
Bill K. Underwood
Bill is the author of several books including best seller Resurrection Day. He is a photographer, telecommunications consultant and columnist. He has been a student of biblical Greek for 30 years, and a student of how religion shapes our worldview for most of his life. He lives in Mesa, Arizona.
Read a preview: "UNBROKEN"
Read a preview of "RESURRECTION DAY":
To read a sample of Resurrection Day in ANY electronic format, go here!
Read a preview of "THE MINOTAUR MEDALLION":

redpajama_set_name: RedPajamaCommonCrawl


Treat yourself to a pair of comfortable sandals with a lot of sparkles! The Clarra Sandals are adorned with multiple shapes and sizes of light-reflecting rhinestones, offering a beautiful style while providing an all-day comfort with the cow suede strap and a super soft Mellow Mat footbed. They are your perfect choice for standing or walking in comfort and style!


text:
This site uses cookies to make the site simpler. Find out more about cookies
Get business finance
Developing a tech venture
A property developer
Process and timescales
Types of finance
Funds we manage
Performance and impact
People and teams
For co-investors
Economic Intelligence Wales
Angels Invest Wales
Business Wales
Giles Thorley
Before joining the Development Bank of Wales, Giles was a partner at private equity firm TDR Capital LLP focusing on deal origination activities. Prior to this he spent nine years with Punch Taverns plc -- the first year as Chair, and then as chief executive following the IPO of the business.
Previously he served as the chief executive of Unique Pub Company. Giles was also a founding member of the Principal Finance Group at Nomura International plc.
He has held non-executive director roles with Esporta, Ducati SpA, Tragus Holdings, TUI Travel plc and Matthew Clark Wholesale Ltd; Giles is currently Chair of D&D London Ltd and ZipWorld plc. He is a non-executive director of Incopro Ltd. He also acts as consultant/angel investor on a number of business start-ups; and is a long-serving trustee with the Rona Sailing Project.
Giles holds a law degree from the University of London and qualified as a barrister in 1990. He is a member of the Bar Council of England & Wales.
Giles Thorley latest news and events
Read the latest news and blogs from Giles Thorley. Find out about events they'll be attending.
We'd love to hear your feedback
Please don't include personal or financial information like your National Insurance number or credit card details.
Is there anything you particularly liked or disliked?
Leave this feed blank - feedback form
Email:info@developmentbank.wales
Welsh Language policy
Development Bank of Wales Plc (Banc Datblygu Cymru ccc) is the holding company of a Group that trades as Development Bank of Wales. The Group is made up of a number of subsidiaries which are registered with names including the initials DBW. Development Bank of Wales Plc is a development finance company wholly owned by the Welsh Ministers and it is neither authorised nor regulated by the Prudential Regulation Authority (PRA) or the Financial Conduct Authority (FCA). The Development Bank of Wales (Banc Datblygu Cymru ccc) has three subsidiaries which are authorised and regulated by the FCA. Please note that neither the Development Bank of Wales Plc (Banc Datblygu Cymru ccc) nor any of its subsidiaries are banking institutions or operate as such. This means that none of the group entities are able to accept deposits from the public. A complete legal structure chart for Development Bank of Wales Plc can be found here.
Development Bank of Wales Plc - Registered in England and Wales under number 4055414 at Unit J, Yale Business Village, Ellice Way, Wrexham LL13 7YL


This lovely fish-out-of-water story is nearly flawless. And whatever minor blemishes it does possess are wiped away by the porcelain-skinned performance of Saoirse Ronan and Nick Hornby's beautiful script. In fact, it is one of the best film portrayals of three different phenomena I've seen: abject home-sickness, puppy-love, and This-Room-Seemed-So-Much-Larger-When-I-Left-It awareness.
John Crowley has directed with a very light touch, thankfully, which allows the performances, the photography and the art direction to flourish and convince. The three environments of the film (Enniscorthy, Wexford; Brooklyn, NY; and the TransAtlantic steamer), all seem as authentic as the novelist, Colm Tolbin, intended and, as a result, it is easy to get lost in the subtle and heart-felt expressions of the lead actress.
Hornby's script is, likewise, minimalist yet safe in the hands of such fine performers. I don't recall such a subtle script from him previously, but I applaud the nuance…and I had deep reactions without ever feeling I was being instructed to do so or remotely manipulated.
Michael Brook's score, interlaced with late-forties and early-fifties dance-hall tunes, is lovingly (and appropriately) reminiscent of Carter Burwell's "Miller's Crossing" work. And, like Hornby's script, successfully enhances emotion. But there is a moment of singing within the framework of the script that will haunt me for some time. You will know what I mean when you see it (so kudos to Iarla O. Lionaird).
This is a tissue-box drama. It won't change the world, but it will remind you of how far you've come from that place you never thought you'd survive the leaving of.


Android Pay has seen its fair share of attention over the last couple of days, having been updated to include ATM access for Bank of America customers, and making it possible for Android Pay to be used as a form of payment on mobile websites. Today it was also discovered that users who have updated their software to the third developer preview of Android N can now use Android Pay thanks to the approval of the Compatibility Test Suite. Now, Google has just highlighted a set of new APIs it's rolling out for Android Pay to make things easier for developers, specifically making it easier for developers to integrate Android Pay into their applications if they haven't already done so.
A big part of these improvements includes the compatibility of Android Pay with Instant Apps which opens up the ability for one-tap purchases using Android Pay as the form of payment. This means faster checkouts and less time fiddling with payment so users can get on to what they were doing before placing an order. With the release of the Android Pay API to developers, the app integration also allows them to sell physical goods and services inside of apps as long as they sign up at the Android Pay developer web portal. Google notes that they have been teaming up with payment processor globally so that app integration is possible within a few hours.
Although it was already briefly mentioned yesterday in the post about ATM access with Android Pay, the PaymentRquest API is part of the enhancements as a whole that are rolling out to Android Pay for developers, which aims to improve the overall experience. Also briefly touched on recently was the inclusion of loyalty cards and rewards in the Android Pay app as part of the latest update. This function is opened up by the Save to Android Pay API, and with this developers can add deep links to initiate a single tap process for adding in loyalty, rewards, and gift cards to the Android Pay app. Google also explains that the deep links can be added to various places like emails and notifications, which should make things extremely simple for users if there is a specific loyalty or gift card that they want to add into Android Pay.


text:
The Genomics of Speciation in Drosophila: Diversity, Divergence, and Introgression Estimated Using Low-Coverage Genome Sequencing
Rob J. Kulathinal,
Affiliation Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, United States of America
Laurie S. Stevison,
Affiliation Biology Department, Duke University, Durham, North Carolina, United States of America
Mohamed A. F. Noor
* E-mail: noor@duke.edu
Rob J. Kulathinal Laurie S. Stevison Mohamed A. F. Noor
In nature, closely related species may hybridize while still retaining their distinctive identities. Chromosomal regions that experience reduced recombination in hybrids, such as within inversions, have been hypothesized to contribute to the maintenance of species integrity. Here, we examine genomic sequences from closely related fruit fly taxa of the Drosophila pseudoobscura subgroup to reconstruct their evolutionary histories and past patterns of genic exchange. Partial genomic assemblies were generated from two subspecies of Drosophila pseudoobscura (D. ps.) and an outgroup species, D. miranda. These new assemblies were compared to available assemblies of D. ps. pseudoobscura and D. persimilis, two species with overlapping ranges in western North America. Within inverted regions, nucleotide divergence among each pair of the three species is comparable, whereas divergence between D. ps. pseudoobscura and D. persimilis in non-inverted regions is much lower and closer to levels of intraspecific variation. Using molecular markers flanking each of the major chromosomal inversions, we identify strong crossover suppression in F1 hybrids extending over 2 megabase pairs (Mbp) beyond the inversion breakpoints. These regions of crossover suppression also exhibit the high nucleotide divergence associated with inverted regions. Finally, by comparison to a geographically isolated subspecies, D. ps. bogotana, our results suggest that autosomal gene exchange between the North American species, D. ps. pseudoobscura and D. persimilis, occurred since the split of the subspecies, likely within the last 200,000 years. We conclude that chromosomal rearrangements have been vital to the ongoing persistence of these species despite recent hybridization. Our study serves as a proof-of-principle on how whole genome sequencing can be applied to formulate and test hypotheses about species formation in lesser-known non-model systems.
The transformation of populations into distinct species depends on whether hybridization, recombination, and subsequent gene introgression can be suppressed between diverging species. We use partial genome sequences to reconstruct this evolutionary process in the Drosophila pseudoobscura species subgroup, which includes the hybridizing species pair D. pseudoobscura pseudoobscura and D. persimilis. Recent models suggest that chromosomal inversions can facilitate the persistence of hybridizing species because of their effects on recombination, whereby inverted regions would exhibit higher nucleotide divergence than non-inverted regions. Indeed, D. pseudoobscura-D. persimilis nucleotide divergence outside these inverted regions is lower than within or near inversions, resembling D. ps. pseudoobscura levels of within-species nucleotide diversity. We also observe that recombination suppression in F1 hybrids extends greater than 2 Mbp outside the inversion breakpoints. Furthermore, when genomic sequence of D. persimilis is compared to two sister subspecies—the hybridizing subspecies, D. ps. pseudoobscura, and a non-hybridizing control subspecies, D. ps. bogotana—autosomal divergence is lower in the former, demonstrating recent gene exchange. These lines of evidence support a speciation model in which the two hybridizing species persist despite the presence of recent genic introgression in collinear regions of the genome because of the reduced recombinational effects of the inversions that distinguish them.
Citation: Kulathinal RJ, Stevison LS, Noor MAF (2009) The Genomics of Speciation in Drosophila: Diversity, Divergence, and Introgression Estimated Using Low-Coverage Genome Sequencing. PLoS Genet 5(7): e1000550. https://doi.org/10.1371/journal.pgen.1000550
Editor: Michael W. Nachman, University of Arizona, United States of America
Received: March 18, 2009; Accepted: June 5, 2009; Published: July 3, 2009
Copyright: © 2009 Kulathinal et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This research was supported by NSF grants 0509780 and 0715484 and NIH grant GM076051 to MAFN. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
One of the most significant empirical insights in evolutionary biology is that a large number of species naturally hybridize with close relatives [see reviews in 1],[2], and these species pairs often exchange genetic material ("introgression"). These insights contrast previous assertions that considered interspecies hybridization as rare or anomalous [e.g., 3]. However, recognizing that hybridization and introgression are frequent suggests that genetic features may allow such species to remain distinct.
Several recent studies suggested that genomic regions of low recombination may provide a means to create "islands of differentiation" between species [4]–[10]. While introgression may homogenize parts of genomes, regions of low recombination in hybrids, such as those within chromosomal inversions, maintain their distinction despite the influx of foreign alleles in collinear regions. This pattern of localized differentiation should be particularly strong if regions of low recombination also harbor loci with divergently selected alleles or alleles conferring reproductive isolation. However, this hypothesis has been difficult to test rigorously on a genome-wide scale.
Assembled whole genome sequences and inexpensive resequencing technologies can complement locus-specific sequencing studies and genetic mapping studies for clarifying the role of regions of low recombination in species persistence. Fruit flies from the genus Drosophila have been a major focus of many studies of nucleotide divergence between closely related species and mapping studies of traits that prevent introgression such as hybrid sterility. The recent sequencing of multiple closely related Drosophila species [11],[12] places us in a stronger position to test hypotheses concerning gene flow and speciation in this model system.
A Speciation Model System: The Drosophila pseudoobscura Species Subgroup
The Drosophila pseudoobscura species subgroup is comprised of two D. pseudoobscura subspecies (D. ps. pseudoobscura and D. ps. bogotana), and two closely related species, D. persimilis and D. miranda. The D. pseudoobscura subspecies are geographically isolated (D. ps. pseudoobscura ranges across the western half of North America and D. ps. bogotana is restricted to Colombia in South America), share chromosomal arrangements, and represent the earliest stages of species divergence [13]. D. persimilis and D. miranda are restricted to the west coast of North America, where they co-occur with D. ps. pseudoobscura. Both D. pseudoobscura subspecies differ from the close relative species D. persimilis by fixed (or nearly fixed) chromosomal inversion differences on three of their major chromosome arms, and F1 hybrid males from crosses between these species are sterile (though females are fertile). In contrast, D. miranda is an outgroup species which cannot produce any fertile hybrids with D. pseudoobscura or D. persimilis [14]. The relative relationships of these species as (((D. ps. pseudoobscura-D. ps. bogotana) D. persimilis) D. miranda) is well established by DNA sequences, chromosomal inversions, and reproductive isolation [15],[16]. Overall, this system provides us with a pair of taxa that hybridize and have experienced introgression (D. ps. pseudoobscura and D. persimilis [17]–[19]), and two taxa that have not experienced recent introgression from any close relatives (ingroup, D. ps. bogotana, and outgroup, D. miranda).
Nucleotide divergence between the hybridizing species D. ps. pseudoobscura and D. persimilis is high within and near the three chromosomal inversions [20],[21], which are linked to factors conferring hybrid sterility, mating discrimination, and other barriers to gene flow [22],[23]. Based on these observations, we hypothesized that inversions facilitate the distinction of these species despite ongoing natural hybridization. However, it has been difficult to fully disentangle complications that result from ancestral polymorphisms shared between these species [21],[24] and underlying assumptions found in many statistical tests for introgression [25]–[27]. Two recent studies also reached differing conclusions about whether gene exchange between these species occurred during the initial divergence process or later [20],[21].
To better understand the genealogical history of this subgroup, we use published genome sequence assemblies of D. ps. pseudoobscura and D. persimilis [12],[28], along with three novel partial genomic sequences that we generated using 454/Roche technology (Table S1): one from D. miranda, one from a second strain of the North American subspecies, D. ps. pseudoobscura, and one from the South American subspecies, D. ps. bogotana. By providing controls for divergence in the absence of gene flow, these newly obtained genomic sequences allow for more robust analysis than previous studies. Our new results suggest that the chromosomal regions inverted between D. persimilis and D. ps. pseudoobscura arose in allopatry, and that D. pseudoobscura, D. persimilis, and D. miranda all diverged within a relatively short time frame. We also find compelling evidence for autosomal gene exchange between D. persimilis and D. ps. pseudoobscura in collinear regions since the split of D. ps. pseudoobscura and D. ps. bogotana, likely within the past 200,000 years. Overall, our analyses utilize genome sequence data in an existing framework to demonstrate the importance of chromosomal inversions in maintaining the persistence of hybridizing species and to consolidate previous tentative conclusions about divergence in this group. Further, this research serves as a model for how whole genome shotgun sequence data can be used with a reference genome sequence to address fundamental questions regarding evolutionary changes leading to the formation of species.
Chromosomal Gradients of Polymorphism and Divergence
Figure 1 presents sliding window estimates of polymorphism within the North American subspecies, D. ps. pseudoobscura, and divergence between D. ps. pseudoobscura and each of D. persimilis and D. miranda across four of the five major chromosome arms. Each datapoint within the sliding window represents the fraction of bases differentiating two genome sequences along a 500 kilobase pairs (kbp) interval, iterated every 100 kbp. Very similar plots were generated for intergenic regions or introns alone (not shown). We only scored positions for which aligned sequences were available for all four taxa (Table S2), hence eliminating the possibility that a particular region of high or low divergence would be represented in some estimates but not others. Nucleotide polymorphism estimated within D. ps. pseudoobscura was confirmed to be in the same range as that observed in polymorphism studies of focal genomic regions of this species [18] (see Table S3).
Figure 1. Genome-wide distribution of variation.
Divergence (Dxy) between the D. pseudoobscura (Dpse) and each of D. persimilis (Dper, black line) and D. miranda (Dmir, blue line) is estimated in overlapping 500 kbp windows (100 kbp steps) across chromosome arms 2, 4, XL, and XR. All chromosome arms are presented from the telomere to centromere (left-to-right), though the sequence assemblies start well outside both the telomere and centromere in each case. Nucleotide diversity (Pxy) is estimated as the pairwise differences between genomic sequences of two strains of D. ps. pseudoobscura (red line). The breakpoints of the inversions distinguishing D. ps. pseudoobscura and D. persimilis are superimposed on each chromosome (rounded brown rectangle) and on the graph (vertical brown lines). The region where recombination is suppressed in hybrids is boxed in gray. Contigs from chromosomes are oriented and ordered according to previous studies. Each chromosomal increment is 5 Mbp. The positive control locus, Adh, is highlighted on chromosome 4 group 1.
Fixed inversions on chromosomes XL, XR, and 2 distinguish D. ps. pseudoobscura and D. persimilis, and their breakpoints are superimposed on Figure 1. Corroborating previous work, nucleotide diversity within D. ps. pseudoobscura and divergence between D. ps. pseudoobscura and D. persimilis are low in regions near the centromere [21],[29]. The latter observation was previously interpreted "as reflective of ancestral patterns of polymorphism rather than the process of divergence between these species" [21]. Consistent with this interpretation, we observe that diversity within D. ps. pseudoobscura and divergence between D. ps. pseudoobscura and D. persimilis were correlated on every chromosome arm (r = 0.418–0.535, P<0.01 for each) [see also 24].
The species pair, D. ps. pseudoobscura and D. miranda, exhibit a different pattern. There was no consistent decline in divergence between these two species in regions near the centromeres. Furthermore, diversity within D. ps. pseudoobscura was not significantly correlated with divergence to D. miranda along any chromosome arm except chromosome 4 (r = 0.330, P = 0.018), suggesting that D. miranda and D. ps. pseudoobscura are not sharing many polymorphisms. According to a model where D. miranda is the outgroup, we predict that the range (maximum minus minimum) of divergences across windows should be greater for the purportedly more divergent species pair, D. miranda - D. ps. pseudoobscura, than the pair of more recently diverged species, D. persimilis - D. ps. pseudoobscura. While this prediction was met for windows along the collinear chromosome 4, we observed instead a greater range of divergences in the D. persimilis - D. ps. pseudoobscura pairing on the chromosome arms (XL, XR, and 2) that harbor inversions distinguishing these species (see also Table S4, Figure S1). These observations are inconsistent with a more recent divergence of this latter species pair, and are more consistent with the presence of multiple genealogical histories along the genome.
Recombination and Divergence around Inversion Breakpoints
Inversions prevent gene exchange because the products of recombination are not recovered. We confirmed that recombinant products are not recovered within 2.1 megabase pairs (Mbp) of fixed inversions along chromosome XL, XR, and 2 in heterozygotes (D. ps. pseudoobscura - D. persimilis interspecies hybrids). We recovered 0.25%–0.55% recombinants at markers 2.8 Mbp outside of each inversion, indicating that complete recombination suppression extends greater than 2.1 Mbp, but not more than 2.8 Mbp outside inversions. Strong crossover suppression, resulting in less than one percent recombinants, is observed relative to one marker 3.35 Mbp outside of the XR chromosomal inversion. Crossing over is largely restored at 4.55 Mbp outside inversions, with a crossover rate greater than 5% observed from one marker on chromosome 2 (see Table S5).
The lack of recombination and introgression should produce a distinct signature in nucleotide divergence within and near chromosomal inversions. We found that, along the three chromosome arms bearing inversions, nucleotide diversity within D. ps. pseudoobscura was comparable to D. persimilis nucleotide divergence when estimated on sequence greater than 2.5 Mbp outside the inverted regions. In contrast, divergence between D. persimilis and D. ps. pseudoobscura was comparable to divergence between D. miranda and D. pseudoobscura in regions inside and within 2.5 Mbp flanking the inversions. The consistency of this pattern across independent chromosomal arms suggests either that all three inversions arose at approximately the same time as the split from the ancestor of D. miranda, or that the ancestral populations of these species were already separated (i.e., allopatric) when the inversions arose (see Discussion).
Recent Autosomal Gene Exchange between D. ps. pseudoobscura and D. persimilis
Recent gene flow is not expected between the South American D. ps. bogotana and either of the North American taxa D. ps. pseudoobscura or D. persimilis. Analyses of nucleotide sequence data suggests that the D. pseudoobscura subspecies diverged from a common ancestor 200,000 years ago [19],[30]. As such, we can use the isolated subspecies as a "negative control" to test for recent introgression between North American D. ps. pseudoobscura and D. persimilis.
Because of hybridization between the North American taxa, a very simple expectation is that D. persimilis (Dper) should be more similar in sequence to North American (NA) than South American (SA) subspecies of D. pseudoobscura (Figure 2). We limited the dataset to sites where we have 454/Roche sequence reads for both D. pseudoobscura subspecies, and tested this hypothesis using regions far from the inversion on chromosome 2 and all along collinear chromosome 4. Aligned bases were categorized as [Dper = NA≠SA] or [Dper = SA≠NA]. No two bases were scored that were within 500 bp of each other, hence reducing artifacts from non-recombining haplotype blocks. We observed an excess of the first category (7073 vs. 6797, Binomial Sign Test P = 0.0096), indicating that divergence is lower between D. persimilis and North American D. ps. pseudoobscura than between D. persimilis and South American D. ps. bogotana.
Figure 2. Divergence and gene flow between D. persimilis and D. ps. pseudoobscura.
Genic introgression between the two North American sister species, D. persimilis (Dper) and D. ps. pseudoobscura (Dpse-NA), was assayed by comparison to the non-hybridizing isolated South American subspecies, D. ps. bogotana (Dpse-SA). Recent genic introgression between Dper and Dpse-NA is expected to generate different levels of divergence (Dper-Dpse-NA<Dper-Dpse-SA) while an absence of gene flow is expected to produce equivalent levels of divergence (Dper-Dpse-NA = Dper-Dpse-SA). Differences in lineage-specific evolutionary rates were assessed using a nonhybridizing species outgroup, D. miranda (Dmir).
The above test does not account for possible faster divergence within the South American subspecies lineage, either through increased mutation rate or more frequent fixation of slightly deleterious alleles. Testing for differences in lineage rates, we did not observe greater divergence between the South American subspecies and the non-hybridizing species, D. miranda (Dmir), than between the North American subspecies and D. miranda (Dxy = 0.019 for both, P = 0.221). Nonetheless, we can test for recent gene exchange more rigorously by specifically counting "shared-derived" base pair substitutions polarized with D. miranda. Counts of [Dmir = SA≠Dper = NA] were compared to counts of individual base pairs in which [Dmir = NA≠Dper = SA], where the latter half of the inequality denotes potential shared-derived bases. Again, we observe a slight, borderline significant excess of the first category (219 vs. 185, Binomial Sign test P = 0.05), suggesting that D. persimilis and North American D. ps. pseudoobscura share more derived bases.
Finally, introgression between species is not expected to be homogeneous outside inverted regions. The Alcohol dehydrogenase (Adh) region has been reported to have introgressed recently between these species using analyses independent of divergence from the South American species, D. ps. bogotana [19]. Further, it can be introgressed in the laboratory and made homozygous in a foreign genetic background with no deleterious effects [23]. We examined base pair counts of [Dmir = SA≠Dper = NA] vs. [Dmir = NA≠Dper = SA] for this region. In this region which bears Adh (chromosome 4 "group1", extending 4 Mbp starting at position 14.4 million in Figure 1), we again observed a significant and dramatic excess of the first category (27 vs. 10, P = 0.00382).
Lack of Recent X-Chromosomal Gene Exchange between D. ps. pseudoobscura and D. persimilis
We applied the same analyses to test for recent gene exchange along X-linked regions from both XL and XR distant from the inversion breakpoints. We observed a nonsignificant difference in number of bases categorized as [Dper = NA≠SA] vs. [Dper = SA≠NA] on this chromosome (1200 vs. 1131, Binomial Sign Test P = 0.079). When we polarized the bases and compared (Dmir = SA≠Dper = NA) vs. (Dmir = NA≠Dper = SA), we observed a nonsignificant difference opposite in direction to our expectation (46 vs. 62). However, there was only 27% as much sequence to analyze more than 2.5 Mbp from inversions on the X-chromosome than on the autosomes.
Genome sequencing has recently become affordable for individual investigators, but how the resultant data can be applied to address evolutionary questions about species formation or diversification has been less clear. Here, we use partial genome sequence data to: 1) evaluate the role of chromosomal inversions in maintaining the distinction between two hybridizing Drosophila species, 2) estimate when gene exchange occurred between these species, and 3) clarify contradictory interpretations from earlier studies that attempted to address related questions. Overall, this research demonstrates how present-day patterns within genomic data can help to infer past processes involved in speciation.
Patterns of Differentiation between Species and a Model for Their Divergence
If two species share extensive polymorphism through introgression or incomplete lineage sorting resulting from a recent split, we predict that nucleotide sequence diversity within species should be correlated with average pairwise nucleotide differences between species. Extensive polymorphism sharing was shown previously in the case of D. ps. pseudoobscura and D. persimilis [18],[20],[21],[31]. In contrast, we find that nucleotide sequence differences between D. ps. pseudoobscura and D. miranda were uncorrelated with nucleotide sequence differences between two strains of D. ps. pseudoobscura. This finding suggests that our comparisons to D. miranda are not hindered by introgression or extensive shared ancestral polymorphisms [but see 32].
Previous DNA sequence-based studies observed that D. ps. pseudoobscura and D. persimilis share variation far outside the fixed inversions that distinguish these species [20]. Here, we note that divergence between D. ps. pseudoobscura and D. persimilis is higher and more comparable to differences between two strains of D. ps. pseudoobscura in regions distant from the inversions. In contrast, divergence between D. ps. pseudoobscura and D. persimilis is comparable to that between D. ps. pseudoobscura and the non-hybridizing outgroup, D. miranda, for regions inside and just outside the chromosomal inversions that separate them.
These new results can be used to formulate a hypothesis for the evolutionary history of these species and reconcile previously contradictory inferences. Machado et al [20] suggested that D. ps. pseudoobscura and D. persimilis largely speciated in allopatry, close in time to the split of these species from D. miranda, and recent secondary contact between the first two resulted in the dissolution of differences outside the inverted regions. In contrast, Noor et al [21] noted that significant differences in divergence among the XL, XR, and 2-chromosome inverted regions suggest instead that D. ps. pseudoobscura and D. persimilis speciated under a sympatric "divergence-with-gene-flow" model. In other words, differences in divergence between inversions reveal when each inversion arose as both species evolved in sympatry.
Our study recapitulates both sets of results and allows us to suggest a resolution. As in Noor et al [21], we observe that the XL chromosome arm inversion was most different in sequence between D. ps. pseudoobscura and D. persimilis, followed by chromosome 2 and finally chromosome arm XR (see Figure 1). However, we also observe that divergence between D. pseudoobscura and D. persimilis within each inverted region was similar to the divergence between D. ps. pseudoobscura and D. miranda (shown at single loci by [20]). Because we observe the same XL>2>XR ranking in D. ps. pseudoobscura divergence from D. miranda that was shown previously for divergence from D. persimilis [21], we now interpret this variation among chromosomes as reflective of differences in mutational processes rather than differences in time since separation. Our new, combined observations suggest two possible interpretations. First, the three inversions independently may have arisen very close in time (near the time of the split from D. miranda) from the D. pseudoobscura-D. persimilis ancestor, and these three derived forms segregated exclusively in D. persimilis. Alternatively, and arguably more parsimoniously, the three species diverged close in time, D. persimilis acquired three new inversions sometime after the split from D. pseudoobscura, and secondary contact between D. persimilis and D. pseudoobscura homogenized the noninverted regions.
Timing of Introgression between Species
Many recent studies have analyzed DNA sequence polymorphism and divergence to identify the statistical signature of recent introgression. However, these tests did not typically identify a time frame within which introgression occurred except as variance in the time of divergence [25],[26]. Instead, most tests merely reject or fail to reject a model of divergence in total isolation.
Here, we use a comparison between subspecies to infer the timing of introgression between D. persimilis and D. ps. pseudoobscura. One D. pseudoobscura subspecies co-occurs and hybridizes with D. persimilis while the other subspecies lives isolated on a different continent. Hence, we can attribute differences in divergence between D. persimilis and these D. pseudoobscura subspecies to hybridization that has occurred more recently than the split of the subspecies, estimated to have been 200,000 years ago [19],[30]. We observe a slight but statistically significant difference in divergence across uninverted (collinear) autosomal regions between D. persimilis and the two D. pseudoobscura subspecies, suggesting recent introgression between the co-occurring taxa, but we fail to detect such evidence for introgression across comparable regions of the X-chromosome.
Although we detected a statistically significant signature of introgression along autosomal loci, the signature was faint, suggesting that recent gene exchange has not been extensive. DNA sequence-based studies previously identified the statistical signature of historical introgression [18],[20],[31], but these studies interpreted this gene exchange as ancient based on the lack of longer shared haplotypes [33]. Similarly, an allozyme-based meta-analysis failed to detect differences between D. ps. pseudoobscura populations co-occurring with D. persimilis compared to those elsewhere in North America [34], suggesting a lack of extensive recent introgression. Given the high levels of gene exchange among populations within D. ps. pseudoobscura, the approach used by Kulathinal and Singh [24] does not have enough resolution to detect the low levels of gene flux we infer here. Again, our sparse genomic sequence data helps to refine these earlier results.
A significant difference between sex-linked and autosomal loci in introgression has been a recurring theme in divergence population genetics [e.g.], [ 35], [36]–[38]. However, in most systems, we lack knowledge of the karyotype (e.g., inversion differences) or other factors which may make the sex chromosomes and particular autosomes inappropriate for comparison. In D. ps. pseudoobscura and D. persimilis, however, we observe evidence for introgression on the autosomes while not on the X-chromosome in regions outside the inversions, suggesting that these differences may be reflective of sex-linkage per se. This observation may be consistent with a higher density of factors conferring hybrid sterility or other barriers to gene flow on the X-chromosome than on the autosomes [e.g., 39].
Use of Whole Genome Shotgun Sequences to Infer Processes of Speciation
In this study, we used sparse whole-genome shotgun sequences from multiple taxa to infer the evolutionary history of a species group and to identify genomic features associated with their divergence. Our system was well-leveraged in that we initially began the investigation already having an assembled and annotated full-genome sequence for two of the focal species [12],[28] as well as genetic mapping data localizing factors that reduced potential gene exchange [22],[23].
Nonetheless, the cost of next-generation sequencing is dropping for both model and non-model systems, even between the execution of this study and its publication. Because of cost constraints, our study approached these questions using light resequencing (effectively utilizing the power of millions of markers) but producing extensive gaps and a majority of aligned positions being covered by single sequence traces. However, our approach serves as a proof-of-principle for future genomic studies on lesser developed systems. We attempted to reduce systematic biases by applying stringent filters, specific tests (including averaging across 500 kbp windows) and by employing the use of a well-assembled reference genome sequence. Future, more rigorous approaches enabled by less-expensive sequencing technologies will allow researchers greater power to infer historical evolutionary processes such as speciation and historical introgression in non-model systems.
Sampled Genomes
In this comparative study, a total of five genomes representing four species of the obscura subgroup were sampled. Adult females from inbred lines of D. miranda (from Mather, California; San Diego stock #14011-0101.08) and the subspecies, D. ps. bogotana (from El Recreo, Colombia; San Diego stock #14011-0121.152) were each extracted and purified using the Gentra PureGene DNA isolation kit. For D. miranda, genomic DNA was nebulized and single stranded libraries generated before being sequenced at light coverage on a single Roche/454 Life Sciences GS-FLX run at Duke University's IGSP core sequencing facility, yielding approximately 100 Mbp of sequence (see Table S1). D. ps. bogotana genomic DNA was similarly sequenced in one half of one run at Duke University's IGSP core sequencing facility and one half of one run at 454 Life Sciences. These genome sequence traces were submitted to the NCBI Short Read Archive (SRA) as accession SRA008268. Additionally, two previously sequenced and assembled genomes, D. ps. pseudoobscura (Release 2) and D. persimilis (Release 1), were used for comparative analysis [12],[28]. Finally, to estimate nucleotide diversity within D. ps. pseudoobscura, previously sequenced Roche/454 reads (NCBI SRA accession SRA000268) from a second line (from Flagstaff, Arizona; San Diego stock number 14011-0121.151; [24]) were reassembled syntenically to D. ps. pseudoobscura.
Genomic Assembly, Alignment, and Annotation
All Roche/454 reads were syntenically aligned against reference D. ps. pseudoobscura (Release 2) linkage groups. Individual base calls were filtered to exclude nucleotides that are: within 3 base pairs of an alignment gap, harbor low quality scores (below 10), contain greater than 30% mismatches within a 7 base pair window, are in regions of high divergence (divergence to D. persimilis is greater than 30% in a 7 base pair window). Alignments from the two previously sequenced reference genomes, Drosophila ps. pseudoobscura and D. persimilis were obtained via chain files from the UCSC Genome Browser (genome.ucsc.edu). Site-specific annotation information such as intron and codon position was extracted from D. ps. pseudoobscura Release 2.3 annotations from FlyBase (flybase.org). Chromosome arms (including ordered contigs) 2, 4, XL, and XR were used (see [40] for contig details), representing roughly 80% of the total genome. We did not survey chromosome 3 because of complications from its inversion polymorphism within each of these species [41].
Chromosomal Inversions and Recombinational Mapping in the Hybrids
Chromosome arms XL, XR, and 2 differ by single inversions between D. pseudoobscura and D. persimilis, and the breakpoints of these inversions have been mapped [21],[42]. Using microsatellite markers that flank the sides of each inversion, we surveyed the extent of recombination in F1 hybrids between D. ps. pseudoobscura and D. persimilis. The published genome lines of both species (San Diego stock numbers #14011-0121.94 and #14011-0111.49) were used in this cross and recombinants were screened among 384 progeny of F1 females backcrossed to D. pseudoobscura. The following markers were used to assay recombination rate at varying distances from the inversions – chromosome 2 inversion: DPS2019 (2.77 Mbp from inversion on telomeric side), DPS2026 (associated with inversion) and DPS2031 (2.8 Mbp from inversion on centromeric side), XL inversion: DPSX_7446z (2.84 Mbp from inversion on centromeric side), DPSX046 (associated with inversion), DPSX008 (0.4 Mbp from inversion on telomeric side), and DPSXL_3a_0.8 (2.8 Mbp from inversion on telomeric side), XR inversion: DPSXR_6_2.7 (3.35 Mbp from inversion on centromeric side), DPSX063 (associated with inversion), DPSX037nA3 (1.4 Mbp from inversion on telomeric side), DPSX037N (2.1 Mbp from inversion on telomeric side), and DPSX058 (2.8 Mbp from inversion on telomeric side). Primer sequences are available upon request.
Ratio of divergence to polymorphism (Dxy/Pxy), and differences in divergence over 500 kbp windows. Inversion breakpoints for chromosomes 2, XL, and XR are shown in green. Polymorphism was estimated by comparing genomic sequences between two strains of D. ps. pseudoobscura. Divergence estimates were then divided by diversity, the former of which was measured between D. ps. pseudoobscura and each of D. persimils (red) and D. miranda (black). The difference in overall divergence between D. miranda and D. persimilis against D. ps. pseudoobscura is found in grey.
(0.11 MB TIF)
Table S1.
"Short-read" sequences used in this study.
(0.02 MB DOC)
Aligned and filtered nucleotides common to four species. The total aligned sequence per chromosome with scored bases for all of the following four samples: D. pseudoobscura (published genome), D. pseudoobscura (Flagstaff, AZ), D. persimilis (published genome), and D. miranda (Mather, CA). This common set of base pairs were used in Figure 1 and other associated analyses.
Comparison of D. ps. pseudoobscura nucleotide diversity across studies. Estimates of nucleotide diversity from D. ps. pseudoobscura from our study (focusing on intergenic regions) were compared to a previous study of nucleotide diversity in focal intergenic regions (Machado et al. 2002). The results show that roughly the same range of values of diversity were recovered from the two works (0.0026–0.0210 vs 0.0024–0.0179).
Percent difference from D. pseudoobscura across 400 kbp windows. Numbers indicate lowest and highest percent differences across windows for each chromosome arm separately, as well as the observed range (highest-lowest).
Markers surveyed to examine recombination fractions in hybrids for various points at known physical distances from the inverted regions. For each chromosome, markers are all listed from the centromere to telomere, including the marker within the inversion. Physical distances from each marker to the nearest inversion breakpoint and the recombination fractions observed from the marker within the inversion are also indicated.
We thank A. Chang, M. Nachman, and three anonymous reviewers for valuable comments on the manuscript.
Conceived and designed the experiments: RJK MAFN. Performed the experiments: RJK LSS MAFN. Analyzed the data: RJK LSS MAFN. Contributed reagents/materials/analysis tools: MAFN. Wrote the paper: RJK LSS MAFN.
1. Mallet J (2005) Hybridization as an invasion of the genome. Trends Ecol Evol 20: 229–237.
2. Rieseberg LH (2009) Evolution: replacing genes and traits through hybridization. Curr Biol 19: R119–R122.
3. Mayr E (1963) Animal Species and Evolution. Cambridge, Mass.: Belknap Press.
4. Butlin RK (2005) Recombination and speciation. Mol Ecol 14: 2621–2635.
5. Ortiz-Barrientos D, Reiland J, Hey J, Noor MAF (2002) Recombination and the divergence of hybridizing species. Genetica 116: 167–178.
6. Slotman MA, Reimer LJ, Thiemann T, Dolo G, Fondjo E, et al. (2006) Reduced Recombination Rate and Genetic Differentiation Between the M and S Forms of Anopheles gambiae s.s. Genetics 174: 2081–2093.
7. Ayala FJ, Coluzzi M (2005) Chromosome speciation: Humans, Drosophila, and mosquitoes. Proc Natl Acad Sci USA 102: 6535–6542.
8. Feder JL, Roethele JB, Filchak K, Niedbalski J, Romero-Severson J (2003) Evidence of inversion polymorphism related to sympatric host race formation in the apple maggot fly, Rhagoletis pomonella. Genetics 163: 939–953.
9. Michel AP, Grushko O, Guelbeogo WM, Lobo NF, Sagnon N, et al. (2006) Divergence with gene flow in Anopheles funestus from the Sudan Savanna of Burkina Faso, West Africa. Genetics 173: 1389–1395.
10. Carneiro M, Ferrand N, Nachman MW (2009) Recombination and Speciation: Loci near Centromeres Are More Differentiated than Loci Near Telomeres Between Subspecies of the European Rabbit (Oryctolagus cuniculus). Genetics 181: 593–606.
11. Begun DJ, Holloway AK, Stevens K, Hillier LW, Poh YP, et al. (2007) Population Genomics: Whole-Genome Analysis of Polymorphism and Divergence in Drosophila simulans. PLoS Biol 5: e310.
12. Clark AG, Eisen MB, Smith DR, Bergman CM, Oliver B, et al. (2007) Evolution of genes and genomes on the Drosophila phylogeny. Nature 450: 203–218.
13. Ayala FJ, Dobzhansky T (1974) A new subspecies of Drosophila pseudoobscura. Pan-Pacif Entomol 50: 211–219.
14. Dobzhansky T (1937) Further data on Drosophila miranda and its hybrids with Drosophila pseudoobscura. J Genet 34: 135–151.
15. Dobzhansky T, Powell JR (1975) Drosophila pseudoobscura and Its American Relatives, Drosophila persimilis and Drosophila miranda. In: King RC, editor. Invertebrates of Genetic Interest. New York: Plenum Press. pp. 537–587.
16. Powell JR (1997) Progress and Prospects in Evolutionary Biology: The Drosophila Model. New York: Oxford University Press. pp xiv+562.
17. Dobzhansky T (1973) Is there gene exchange between Drosophila pseudoobscura and Drosophila persimilis in their natural habitats? Am Nat 107: 312–314.
18. Machado CA, Kliman RM, Markert JA, Hey J (2002) Inferring the history of speciation from multilocus sequence data: the case of Drosophila pseudoobscura and its close relatives. Mol Biol Evol 19: 472–488.
19. Wang RL, Wakeley J, Hey J (1997) Gene flow and natural selection in the origin of Drosophila pseudoobscura and close relatives. Genetics 147: 1091–1106.
20. Machado CA, Haselkorn TS, Noor MAF (2007) Evaluation of the genomic extent of effects of fixed inversion differences on intraspecific variation and interspecific gene flow in Drosophila pseudoobscura and D. persimilis. Genetics 175: 1289–1306.
21. Noor MA, Garfield DA, Schaeffer SW, Machado CA (2007) Divergence Between the Drosophila pseudoobscura and D. persimilis Genome Sequences in Relation to Chromosomal Inversions. Genetics 177: 1417–1428.
22. Brown KM, Burk LM, Henagan LM, Noor MAF (2004) A test of the chromosomal rearrangement model of speciation in Drosophila pseudoobscura. Evolution 58: 1856–1860.
23. Noor MAF, Grams KL, Bertucci LA, Reiland J (2001) Chromosomal inversions and the reproductive isolation of species. Proc Natl Acad Sci USA 98: 12084–12088.
24. Kulathinal RJ, Bennett SM, Fitzpatrick CL, Noor MAF (2008) Fine-scale mapping of recombination rate in Drosophila refines its correlation to diversity and divergence. Proc Natl Acad Sci U S A 105: 10051–10056.
25. Hey J, Nielsen R (2004) Multilocus methods for estimating population sizes, migration rates and divergence time, with applications to the divergence of Drosophila pseudoobscura and D. persimilis. Genetics 167: 747–760.
26. Hey J, Nielsen R (2007) Integration within the Felsenstein equation for improved Markov chain Monte Carlo methods in population genetics. Proc Natl Acad Sci U S A 104: 2785–2790.
27. Wakeley J, Hey J (1997) Estimating ancestral population parameters. Genetics 145: 847–855.
28. Richards S, Liu Y, Bettencourt BR, Hradecky P, Letovsky S, et al. (2005) Comparative genome sequencing of Drosophila pseudoobscura: Chromosomal, gene, and cis-element evolution. Genome Res 15: 1–18.
29. Ortiz-Barrientos D, Chang AS, Noor MAF (2006) A recombinational portrait of the Drosophila pseudoobscura genome. Genet Res, Camb 87: 23–31.
30. Schaeffer SW, Miller EL (1991) Nucleotide sequence analysis of Adh genes estimates the time of geographic isolation of the Bogota population of Drosophila pseudoobscura. Proc Natl Acad Sci USA 88: 6097–6101.
31. Machado CA, Hey J (2003) The causes of phylogenetic conflict in a classic Drosophila species group. Proc R Soc Lond B 270: 1193–1202.
32. Charlesworth B, Bartolome C, Noel V (2005) The detection of shared and ancestral polymorphisms. Genet Res 86: 149–157.
33. Wang R-L, Hey J (1996) The speciation history of Drosophila pseudoobscura and close relatives: Inferences from DNA sequence variation at the period locus. Genetics 144: 1113–1126.
34. Kulathinal RJ, Singh RS (2000) A biogeographic approach for testing the role of reinforcement: The case of Drosophila pseudoobscura and D. persimilis. Evolution 54: 210–217.
35. Carling MD, Brumfield RT (2008) Haldane's Rule in an Avian System: Using Cline Theory and Divergence Population Genetics to Test for Differential Introgression of Mitochondrial, Autosomal, and Sex-Linked Loci across the Passerina Bunting Hybrid Zone. Evolution 62: 2600–2615.
36. Geraldes A, Basset P, Gibson B, Smith KL, Harr B, et al. (2008) Inferring the history of speciation in house mice from autosomal, X-linked, Y-linked and mitochondrial genes. Mol Ecol 17: 5349–5363.
37. Macholán M, Munclinger P, Šugerková M, Dufková P, Bímová B, et al. (2007) Genetic analysis of autosomal and X-linked markers across a mouse hybrid zone. Evolution 61: 746–771.
38. Sætre G-P, Borge T, Lindroos K, Haavie J, Sheldon BC, et al. (2003) Sex chromosome evolution and speciation in Ficedula flycatchers. Proc R Soc Lond B 270: 53–59.
39. Masly JP, Presgraves DC (2007) High-resolution genome-wide dissection of the two rules of speciation in Drosophila. PLoS Biol 5: e243.
40. Schaeffer SW, Bhutkar A, McAllister BF, Matsuda M, Matzkin LM, et al. (2008) Polytene Chromosomal Maps of 11 Drosophila species: The order of genomic scaffolds inferred from genetic and physical maps. Genetics 179: 1601–1655.
41. Dobzhansky T, Epling C (1944) Contributions to the Genetics, Taxonomy, and Ecology of Drosophila pseudoobscura and Its Relatives. Washington, DC: Carnegie Institute of Washington.
42. Bhutkar A, Schaeffer SW, Russo SM, Xu M, Smith TF, et al. (2008) Chromosomal rearrangement inferred from comparisons of twelve Drosophila genomes. Genetics 179: 1657–1680.
Chromosomal inversions
Is the Subject Area "Chromosomal inversions" applicable to this article?
Introgression
Is the Subject Area "Introgression" applicable to this article?
Invertebrate genomics
Is the Subject Area "Invertebrate genomics" applicable to this article?
Is the Subject Area "Comparative genomics" applicable to this article?
Is the Subject Area "Sequence alignment" applicable to this article?
Is the Subject Area "Hybridization" applicable to this article?
Nucleotide sequencing
Is the Subject Area "Nucleotide sequencing" applicable to this article?
Is the Subject Area "Genome evolution" applicable to this article?


This is a placeholder page for Mari Falquez, which means this person is not currently on this site. We do suggest using the tools below to find Mari Falquez.
You are visiting the placeholder page for Mari Falquez. This page is here because someone used our placeholder utility to look for Mari Falquez. We created this page automatically in hopes Mari Falquez would find it. If you are not Mari Falquez, but are an alumni of Hayden High School Winkelman, AZ, register on this site for free now.


text:
Handcuffed man shoots officer 3 times with hidden gun, flees
MCKEESPORT, Pa. (AP) — A handcuffed man fired a gun police didn't know he had out of a police vehicle hitting an officer three times before fleeing from a western Pennsylvania police station, police said.
Koby Lee Francis, 22, of McKeesport, had been arrested after allegedly violating a protection from abuse order before Sunday afternoon's gunfire outside the McKeesport police station, Allegheny County Police Superintendent Coleman McDonough said Monday. McKeesport is located near Pittsburgh.
Officer Gerasimos Athans, 32, had wounds that weren't life-threatening and was expected to be discharged from the hospital in the next few days, McDonough said. Police are searching for Francis and said he should be considered armed and dangerous.
Francis had been served Sunday with the protection from abuse order but is accused of having quickly violated it twice, police said. He was arrested and a pistol was found in his vehicle. Francis was described as combative and police initially said he kicked through a window of a police vehicle window. On Monday, police said that earlier report was mistaken and he did not kick out the window.
On a video released by police, the officer is seen opening the passenger side door of the police vehicle parked outside the station and then staggering back after police said he took fire from within the vehicle. The video shows Frances then emerges from the vehicle with hands handcuffed in front of him and fires at the officer before fleeing.
McDonough said Francis "was searched, obviously" when he was arrested but "was able to secret a weapon and that was the weapon he used … to shoot (the officer)." Video confirmed that he had been handcuffed with his hands in back of him, but he somehow managed to get his hands in front of him before his escape, McDonough said.
After he was shot, the officer "emptied his weapon" in returning fire, but there was no evidence Francis was hit, McDonough said.
Francis was being sought on attempted homicide, escape, flight to avoid apprehension, aggravated assault and firearms charges. McDonough called the search for the fugitive, both locally and anywhere he might have fled, an "all hands on deck" effort that needed help from the community.
"Obviously this was an egregious act on his part," McDonough said. "He went to this address in violation of a (protection from abuse order). He went there angry. He went there armed. I think that it's in the community's interest to help us to bring him in."
As for the officer, who has been on the force for four years, McDonough said, "We're very happy that he's going to be able to spend Christmas with his family rather than what could have been a very tragic event."
This story has been corrected to note that police now say that earlier reports that the suspect kicked out a window of a police vehicle were incorrect.
More AP US News Stories
Crews work to pull car from frozen pond in Lexington
Rounds of snow enter the picture


Quick Review: Let the sun shine in. Open the curtains, raise the blinds, and spend time in rooms that get natural light. Turn off lights you're not using. Leave a room, hit the switch! Don't forget outdoor lights.
7. Run full dishwashers and washing machines. For extra laundry savings, use the cold cycle.
I dunno about you, I'll bet this comes back to me, like even the next time I just wash my hands….


text:
Q: Participle phrase at the end of the sentence When participle phrase comes at the end of the sentence, it usually modifies the subject.

1 He smiled nervously with a chocolate in his hands, thinking that the end
  has come.

Here, participle phrase "thinking that  the end has come"modifies "he", not hands. What I want to ask is, is it OK to put a participle phrase that would modify "hands"?

2 He smiled nervously with a chocolate in his hands, darkened with
  sweetness.

Here, I meant to make past participle phrase "darkened with sweetness" modify his hands.
The reason I am asking is because I realized that participle phrases can be used in the position of appositives:

3 His hands, darkened with sweetness, were holding a chocolate.

So does my sentence 2 work in the same way as the sentence 3, or is it impossible to use sentence 2 at all? Is it OK to use sentence 2?

redpajama_set_name: RedPajamaStackExchange