A glutaminase isoform switch drives therapeutic resistance and disease progression of prostate cancer.

Cellular metabolism in cancer is significantly altered to support the uncontrolled tumor growth. How metabolic alterations contribute to hormonal therapy resistance and disease progression in prostate cancer (PCa) remains poorly understood. Here we report a glutaminase isoform switch mechanism that mediates the initial therapeutic effect but eventual failure of hormonal therapy of PCa. Androgen deprivation therapy inhibits the expression of kidney-type glutaminase (KGA), a splicing isoform of glutaminase 1 (GLS1) up-regulated by androgen receptor (AR), to achieve therapeutic effect by suppressing glutaminolysis. Eventually the tumor cells switch to the expression of glutaminase C (GAC), an androgen-independent GLS1 isoform with more potent enzymatic activity, under the androgen-deprived condition. This switch leads to increased glutamine utilization, hyperproliferation, and aggressive behavior of tumor cells. Pharmacological inhibition or RNA interference of GAC shows better treatment effect for castration-resistant PCa than for hormone-sensitive PCa in vitro and in vivo. In summary, we have identified a metabolic function of AR action in PCa and discovered that the GLS1 isoform switch is one of the key mechanisms in therapeutic resistance and disease progression.

N-Myc promotes therapeutic resistance development of neuroendocrine prostate cancer by differentially regulating miR-421/ATM pathway.

BACKGROUND: MYCN amplification or N-Myc overexpression is found in approximately 40% NEPC and up to 20% CRPC patients. N-Myc has been demonstrated to drive disease progression and hormonal therapeutic resistance of NEPC/CRPC. Here, we aim to identify the molecular mechanisms underlying the N-Myc-driven therapeutic resistance and provide new therapeutic targets for those N-Myc overexpressed NEPC/CRPC. METHODS: N-Myc overexpressing stable cell lines for LNCaP and C4-2 were generated by lentivirus infection. ADT-induced senescence was measured by SA-ß-gal staining in LNCaP cells in vitro and in LNCaP xenograft tumors in vivo. Migration, cell proliferation and colony formation assays were used to measure the cellular response after overexpressing N-Myc or perturbing the miR-421/ATM pathway. CRISPR-Cas9 was used to knock out ATM in C4-2 cells and MTS cell viability assay was used to evaluate the drug sensitivity of N-Myc overexpressing C4-2 cells in response to Enzalutamide and ATM inhibitor Ku60019 respectively or in combination. RESULTS: N-Myc overexpression suppressed ATM expression through upregulating miR-421 in LNCaP cells. This suppression alleviated the ADT-induced senescence in vitro and in vivo. Surprisingly, N-Myc overexpression upregulated ATM expression in C4-2 cells and this upregulation promoted migration and invasion of prostate cancer cells. Further, the N-Myc-induced ATM upregulation in C4-2 cells rendered the cells resistance to Enzalutamide, and inhibition of ATM by CRISPR-Cas9 knockout or ATM inhibitor Ku60019 re-sensitized them to Enzalutamide. CONCLUSIONS: N-Myc differentially regulating miR-421/ATM pathway contributes to ADT resistance and Enzalutamide resistance development respectively. Combination treatment with ATM inhibitor re-sensitizes N-Myc overexpressed CRPC cells to Enzalutamide. Our findings would offer a potential combination therapeutic strategy using ATM kinase inhibitor and Enzalutamide for the treatment of a subset of mCRPC with N-Myc overexpression that accounts for up to 20% CRPC patients.

Correction to: N-Myc promotes therapeutic resistance development of neuroendocrine prostate cancer by differentially regulating miR-421/ ATM pathway.

Following publication of the original article [1], the authors reported that name that appeared in published online version is incorrect. Aifeng Wang should be Aifen Wang. Corrected name is provided in the author group section above.

Targeting glutamine metabolism network for the treatment of therapy-resistant prostate cancer.

Advanced and aggressive prostate cancer (PCa) depends on glutamine for survival and proliferation. We have previously shown that inhibition of glutaminase 1, which catalyzes the rate-limiting step of glutamine catabolism, achieves significant therapeutic effect; however, therapy resistance is inevitable. Here we report that while the glutamine carbon is critical to PCa survival, a parallel pathway of glutamine nitrogen catabolism that actively contributes to pyrimidine assembly is equally important for PCa cells. Importantly, we demonstrate a reciprocal feedback mechanism between glutamine carbon and nitrogen pathways which leads to therapy resistance when one of the two pathways is inhibited. Combination treatment to inhibit both pathways simultaneously yields better clinical outcome for advanced PCa patients.

Transcription recycling assays identify PAF1 as a driver for RNA Pol II recycling.

RNA Polymerase II (Pol II) transcriptional recycling is a mechanism for which the required factors and contributions to overall gene expression levels are poorly understood. We describe an in vitro methodology facilitating unbiased identification of putative RNA Pol II transcriptional recycling factors and quantitative measurement of transcriptional output from recycled transcriptional components. Proof-of-principle experiments identified PAF1 complex components among recycling factors and detected defective transcriptional output from Pol II recycling following PAF1 depletion. Dynamic ChIP-seq confirmed PAF1 silencing triggered defective Pol II recycling in human cells. Prostate tumors exhibited enhanced transcriptional recycling, which was attenuated by antibody-based PAF1 depletion. These findings identify Pol II recycling as a potential target in cancer and demonstrate the applicability of in vitro and cellular transcription assays to characterize Pol II recycling in other disease states.

Phylogeography and species limits in the red-shouldered hawk (Buteo lineatus): Characterization of the Northern Florida Suture Zone in birds.

The North American red-shouldered hawk, Buteo lineatus, is comprised of two widely allopatric eastern and western populations with an additional well-marked subspecies in the Florida peninsula. The two eastern populations meet in northern Florida, the location of a well-known suture zone in many nonavian organisms. We sequenced the complete mitochondrial ND2 gene and two nuclear introns to investigate its genetic population structure and species status. No mitochondrial haplotypes were shared between the eastern and western populations, and genetic variance among 14 populations was 0.42; almost all of this (0.40) was distributed among the three regions. A clade of haplotypes very common in the Florida peninsula decreased in frequency elsewhere and, when modeled as a hybrid zone, had an estimated width of 1,158 km with a center near Ocala, FL. Ecological niche modeling suggests the western, eastern, and Florida peninsula populations were geographically isolated during the last glacial maximum. We consider these to represent three phylogenetic species. A coalescent analysis incorporating incomplete lineage sorting and gene tree uncertainty also suggested the divergence between the western and eastern populations is consistent with species-level divergence. With the addition of this hawk, four avian species are now known to hybridize along the Gulf Coast of the United States in or near the Northern Florida Suture Zone. The widths of these avian zones vary substantially (176-1,158 km) and appear to reflect magnitude of gene flow, rather than extent of genetic differentiation. None of these birds was suggested as possible exemplars in the original description of the suture zone. Of the six species that were so identified, three have been surveyed to date, but none of those was found to be genetically differentiated.

ATM deficiency promotes progression of CRPC by enhancing Warburg effect.

ATM is a well-known master regulator of double strand break (DSB) DNA repair and the defective DNA repair has been therapeutically exploited to develop PARP inhibitors based on the synthetic lethality strategy. ATM mutation is found with increased prevalence in advanced metastatic castration-resistant prostate cancer (mCRPC). However, the molecular mechanisms underlying ATM mutation-driving disease progression are still largely unknown. Here, we report that ATM mutation contributes to the CRPC progression through a metabolic rather than DNA repair mechanism. We showed that ATM deficiency generated by CRISPR/Cas9 editing promoted CRPC cell proliferation and xenograft tumor growth. ATM deficiency altered cellular metabolism and enhanced Warburg effect in CRPC cells. We demonstrated that ATM deficiency shunted the glucose flux to aerobic glycolysis by upregulating LDHA expression, which generated more lactate and produced less mitochondrial ROS to promote CRPC cell growth. Inhibition of LDHA by siRNA or inhibitor FX11 generated less lactate and accumulated more ROS in ATM-deficient CRPC cells and therefore potentiated the cell death of ATM-deficient CRPC cells. These findings suggest a new therapeutic strategy for ATM-mutant CRPC patients by targeting LDHA-mediated glycolysis metabolism, which might be effective for the PARP inhibitor resistant mCRPC tumors.

Expression characteristics of prostate-derived Ets factor support a role in breast and prostate cancer progression.

The purpose of this study was to understand the characteristics of prostate-derived Ets factor (PDEF) protein expression in breast and prostate cancer progression. A polyclonal antibody specific to PDEF was raised and reacted with tissue microarrays consisting of benign breast, in situ ductal, invasive ductal, and invasive lobular breast carcinomas. The antibody was also reacted with tissue microarrays, including benign prostate, prostate intraepithelial neoplasias (PINs), and prostate carcinomas. Increased expression of PDEF was identified in 18%, 50%, 46%, and 51% of benign breast tissues, intraductal, invasive ductal, and invasive lobular carcinomas, respectively. Importantly, in matched samples of benign breast vs tumor, 90% showed higher expression of PDEF in the tumor tissue. Moreover, in invasive breast carcinomas, increased PDEF expression tended to correlate with Her2/neu overexpression. Increased expression of PDEF was also found in 27%, 33%, and 40% of benign prostate tissues, PIN samples, and prostate adenocarcinomas, respectively. Again, in matching samples of cancer vs benign and cancer vs PIN, 68% and 70%, respectively, showed increased expression in the malignant tissue. Moreover, PDEF was found to be more highly expressed in tumors with intermediate or high Gleason score compared with low-grade tumors (P < .01). In addition, R1881 treatment induced PDEF expression in the LNCaP prostate tumor cell line, suggesting regulation of PDEF by androgens in vivo. Together, these results for the first time show frequent increased expression of PDEF protein in breast and prostate tumors and support a role for PDEF in breast and prostate cancer progression.

Friend leukaemia integration-1 expression in malignant and benign tumours: a multiple tumour tissue microarray analysis using polyclonal antibody.

BACKGROUND: Friend leukaemia integration-1 (FLI-1) antibody is a useful marker for Ewing's sarcoma/primitive neuroectodermal tumour (EWS/PNET) and vascular tumours. However, it is also expressed in subsets of lymphoblastic lymphoma, Merkel cell carcinoma (MCC) and desmoplastic small round cell tumour (DSRCT). AIM: To determine expression of FLI-1 in various benign and malignant neoplasms, by immunohistochemical analysis on 4323 tumours using multiple tumour microarrays, as well as on whole sections. RESULTS: FLI-1 was expressed in 46/62 EWS/PNETs, 2/3 olfactory neuroblastomas, 7/102 small cell carcinomas of the lung, 10/34 MCCs, 1/14 rhabdomyosarcoma, 19/132 non-Hodgkin's lymphomas, 2/3 DSRCTs, and in 53/74 benign and malignant vascular tumours. In addition, 27/508 squamous cell carcinomas, 19/837 adenocarcinomas, 10/400 urothelial bladder cancers, 1/40 basal cell carcinomas, 3/29 liposarcomas, 1/40 glioblastoma multiforme and 9/29 medullar carcinomas of the breast expressed FLI-1. The sensitivity and specificity of FLI-1 to distinguish EWS/PNET from all types of malignancies were 74.2% and 96.0%, respectively. Finally, the sensitivity and specificity of FLI-1 to distinguish EWS/PNET from other small round cell tumours (SRCTs) were 74.2% and 91.6%, respectively. CONCLUSION: This study was the first to show that FLI-1 can be seen in a variety of solid tumours, some of which had never been explored before. This finding should be kept in mind, especially when using FLI-1 as a marker for finding the primary origin of poorly differentiated metastatic tumour. Finally, despite the expression of FLI-1 in numerous malignancies, it is still considered to be highly sensitive and specific in distinguishing EWS/PNET from other tumour types in general and from other SRCTs in particular.

CD24 expression is an independent prognostic marker in cholangiocarcinoma.

CD24 has been described as an adverse prognostic marker in several malignancies. This study evaluates CD24 expression in cholangiocarcinoma and correlates the findings with clinicopathologic data and patient survival. Between 1996 and 2002, 22 consecutive patients with cholangiocarcinoma were treated at our institution. Demographic data, SEER stage, pathologic data, treatment, expression of CD24, mitogen-activated protein kinase (MAPK), phosphorylated MAPK, and survival were analyzed. The majority of the tumors demonstrated CD24 (81.8%) and p-MAPK (87%) expression. A negative association was noted between the expression of CD24 and p-MAPK. Median survival for patients with low expression of CD24 was 36 months and high expression was 8 months. Median survival for patients who received chemotherapy with low CD24 expression was 163 months, and for seven patients with high CD24 expression, it was 17 months (p=0.04). With the addition of radiation therapy, median survival for patients with low expression of CD24 was 52 months and high expression was 17 months (p=0.08). On multivariate analysis, the use of chemotherapy (p=0.0014, hazard ratio 0.069) and the CD24 overexpression (p=0.02, hazard ratio 7.528) were predictive of survival. CD24 is commonly expressed in cholangiocarcinoma, and overexpression is predictive of poor survival and possibly of lack of response to chemotherapy and radiation therapy. These findings may improve selection of patients for the appropriate treatment modality and the development of CD24-targeted therapy.

Settling the name Diomedea exulans Linnaeus, 1758 for the Wandering Albatross by neotypification.

On-going conflict in use of the name Diomedea exulans Linnaeus, 1758 for different taxa of the great albatrosses (Wandering Albatross complex) is resolved by neotypification, fixing the name to the large subantarctic form formerly often known as D. chionoptera Salvin, 1896. Application of all scientific names in the complex is reviewed, an annotated synonymy for the large subantarctic form is provided, available names for smaller, temperate-zone forms are listed, and unavailable and otherwise invalid names referable to the complex are identified. Syntypes of D. chionoptera and D. spadicea J.F. Gmelin, 1789 are lectotypified as well, fixing their names as synonyms of D. exulans to prevent possible disturbance to in-use names for the smaller, temperate-zone forms.

Aberrant expression of novel and previously described cell membrane markers in human breast cancer cell lines and tumors.

PURPOSE: In a previous gene expression array study, we identified some 300 genes that were differentially expressed in human epidermal growth factor receptor tyrosine kinase 2 (HER2)-positive versus HER2-negative breast cancer cells. We have now done validation experiments on a group of three cell membrane components that had previously not been implicated in breast cancer. We also studied the expression of three other cell membrane proteins known to play a role in mammary neoplasia. EXPERIMENTAL DESIGN: By immunohistochemistry, we examined up to 130 archival breast carcinomas for Celsr2, E-cadherin, Kai1, and CD9 expression. The expression levels of NET-6 and TROP-2 were determined by quantitative reverse transcription-PCR in a subset of frozen tumors. We also studied fresh pellets and paraffin-embedded cell buttons of nine human breast cell lines. The relationship between the expression of all six membrane proteins and a variety of pathologic and biological variables, including estrogen receptor, HER2, and epidermal growth factor receptor status, was also examined. The NET-6 gene was transfected into a low-expressing cell line, and the effect on cellular morphology, growth, and invasion in vitro was recorded. RESULTS: Celsr2 was down-regulated in one cell line and in 7% of breast cancers. E-cadherin, Kai1, and CD9 were down-regulated in 35%, 76%, and 79% of tumors, respectively, confirming the important role of these markers in human mammary neoplasia. In breast cancer cell lines and tissues, TROP-2 was generally expressed at low levels, although a few specimens showed relative overexpression. NET-6 levels were lower in HER2-negative breast carcinoma cells. In addition, NET-6 was markedly down-regulated in estrogen receptor-negative breast cancers, and expression was lowest in "basal-like" tumors. Ectopic expression of NET-6 in low-expressing MDA-MB-231 cells altered cellular morphology, inhibited growth in vitro, and decreased invasion in a Boyden chamber assay. CONCLUSIONS: We have confirmed the expression of three new membrane markers that had previously not been implicated in human breast cancer, and one of them (NET-6) was correlated with HER2 and estrogen receptor status. NET-6 levels were decreased in estrogen receptor-negative and high-grade tumors, and ectopic expression of this gene had an inhibitory effect on proliferation and invasion. Thus, NET-6 may represent a novel breast cancer suppressor gene.

Preoperative Single-Fraction Partial Breast Radiation Therapy: A Novel Phase 1, Dose-Escalation Protocol With Radiation Response Biomarkers.

PURPOSE: Women with biologically favorable early-stage breast cancer are increasingly treated with accelerated partial breast radiation (PBI). However, treatment-related morbidities have been linked to the large postoperative treatment volumes required for external beam PBI. Relative to external beam delivery, alternative PBI techniques require equipment that is not universally available. To address these issues, we designed a phase 1 trial utilizing widely available technology to 1) evaluate the safety of a single radiation treatment delivered preoperatively to the small-volume, intact breast tumor and 2) identify imaging and genomic markers of radiation response. METHODS AND MATERIALS: Women aged ≥55 years with clinically node-negative, estrogen receptor-positive, and/or progesterone receptor-positive HER2-, T1 invasive carcinomas, or low- to intermediate-grade in situ disease ≤2 cm were enrolled (n=32). Intensity modulated radiation therapy was used to deliver 15 Gy (n=8), 18 Gy (n=8), or 21 Gy (n=16) to the tumor with a 1.5-cm margin. Lumpectomy was performed within 10 days. Paired pre- and postradiation magnetic resonance images and patient tumor samples were analyzed. RESULTS: No dose-limiting toxicity was observed. At a median follow-up of 23 months, there have been no recurrences. Physician-rated cosmetic outcomes were good/excellent, and chronic toxicities were grade 1 to 2 (fibrosis, hyperpigmentation) in patients receiving preoperative radiation only. Evidence of dose-dependent changes in vascular permeability, cell density, and expression of genes regulating immunity and cell death were seen in response to radiation. CONCLUSIONS: Preoperative single-dose radiation therapy to intact breast tumors is well tolerated. Radiation response is marked by early indicators of cell death in this biologically favorable patient cohort. This study represents a first step toward a novel partial breast radiation approach. Preoperative radiation should be tested in future clinical trials because it has the potential to challenge the current treatment paradigm and provide a path forward to identify radiation response biomarkers.

FAS Death Receptor: A Breast Cancer Subtype-Specific Radiation Response Biomarker and Potential Therapeutic Target.

Although a standardized approach to radiotherapy has been used to treat breast cancer, regardless of subtype (e.g., luminal, basal), recent clinical data suggest that radiation response may vary significantly among subtypes. We hypothesized that this clinical variability may be due, in part, to differences in cellular radiation response. In this study, we utilized RNA samples for microarray analysis from two sources: 1. Paired pre- and postirradiation breast tumor tissue from 32 early-stage breast cancer patients treated in our unique preoperative radiation Phase I trial; and 2. Sixteen biologically diverse breast tumor cell lines exposed to 0 and 5 Gy irradiation. The transcriptome response to radiation exposure was derived by comparing gene expression in samples before and after irradiation. Genes with the highest coefficient of variation were selected for further evaluation and validated at the RNA and protein level. Gene editing and agonistic antibody treatment were performed to assess the impact of gene modulation on radiation response. Gene expression in our cohort of luminal breast cancer patients was distinctly different before and after irradiation. Further, two distinct patterns of gene expression were observed in our biologically diverse group of breast cancer cell lines pre- versus postirradiation. Cell lines that showed significant change after irradiation were largely luminal subtype, while gene expression in the basal and HER2+ cell lines was minimally impacted. The 100 genes with the most significant response to radiation in patients were identified and analyzed for differential patterns of expression in the radiation-responsive versus nonresponsive cell lines. Fourteen genes were identified as significant, including FAS, a member of the tumor necrosis factor receptor family known to play a critical role in programed cell death. Modulation of FAS in breast cancer cell lines altered radiation response phenotype and enhanced radiation sensitivity in radioresistant basal cell lines. Our findings suggest that cell-type-specific, radiation-induced FAS contributes to subtype-specific breast cancer radiation response and that activation of FAS pathways may be exploited for biologically tailored radiotherapy.

A phylogenetic hypothesis for passerine birds: taxonomic and biogeographic implications of an analysis of nuclear DNA sequence data.

Passerine birds comprise over half of avian diversity, but have proved difficult to classify. Despite a long history of work on this group, no comprehensive hypothesis of passerine family-level relationships was available until recent analyses of DNA-DNA hybridization data. Unfortunately, given the value of such a hypothesis in comparative studies of passerine ecology and behaviour, the DNA-hybridization results have not been well tested using independent data and analytical approaches. Therefore, we analysed nucleotide sequence variation at the nuclear RAG-1 and c-mos genes from 69 passerine taxa, including representatives of most currently recognized families. In contradiction to previous DNA-hybridization studies, our analyses suggest paraphyly of suboscine passerines because the suboscine New Zealand wren Acanthisitta was found to be sister to all other passerines. Additionally, we reconstructed the parvorder Corvida as a basal paraphyletic grade within the oscine passerines. Finally, we found strong evidence that several family-level taxa are misplaced in the hybridization results, including the Alaudidae, Irenidae, and Melanocharitidae. The hypothesis of relationships we present here suggests that the oscine passerines arose on the Australian continental plate while it was isolated by oceanic barriers and that a major northern radiation of oscines (i.e. the parvorder Passerida) originated subsequent to dispersal from the south.

The RAG-1 exon in the avian order Caprimulgiformes: phylogeny, heterozygosity, and base composition.

We sequenced 2.8 kb of the RAG-1 exon for most of the extant genera in the avian order Caprimulgiformes to investigate monophyly of the order and phylogeny within the traditional families. The order is not monophyletic: the Aegothelidae (owlet-nightjars) were the sister group of the Apodiformes (swifts and hummingbirds). There was no support for the monophyly of a clade containing the remaining families of Caprimulgiformes. However, the RAG-1 data strongly supported a relationship between the Podargidae (frogmouths) and Caprimulgidae (nightjars). Within the Caprimulgidae, the Australasian genus Eurostopodus was sister to the rest of the family, which in turn was composed of four major clades, three of which were restricted to the New World and primarily to the Neotropics. The Old World caprimulgids form a monophyletic clade embedded within the New World taxa; consequently, most Old World nightjars are probably the result of a single expansion out of the Neotropics. The genus Caprimulgus was not found to be monophyletic. Several species in the Caprimulgidae have both elevated heterozygosity and high GC3 content; it is likely that these are causally related.

Phylogeny of the Falconidae (Aves): a comparison of the efficacy of morphological, mitochondrial, and nuclear data.

We sequenced 2800+ bp of the RAG-1 exon for representatives of all the currently recognized genera in the avian family Falconidae. A phylogenetic analysis of these data was compared to prior analyses of mitochondrial (cytochrome-b) and morphological (syringeal) data. The nuclear RAG-1 sequences produced results that were in agreement with the morphological results, but differed from the mitochondrial results with regard to monophyly of the genus Micrastur. A reanalysis of the cytochrome-b (cyt-b) data suggested that this result was due to heterogeneity in base composition. Comparisons of data quality and quantity across the three data sets indicate that the nuclear DNA sequences and the morphological data have similar consistency and retention indices as well as noise distributions that are superior to those of cyt-b. However, the RAG-1 data identify more nodes with high bootstrap support indices than do either morphology or mitochondrial sequences. In the final assessment, RAG-1 sequences were superior in phylogenetic utility both to syringeal morphology (because of sheer number of characters) and to cyt-b sequences (because of reduced noise and homogeneity of base composition, but in spite of having many fewer characters).

Phylogeographic structure, gene flow and species status in blue grouse (Dendragapus obscurus).

We investigated the genetic population structure and species status of a relatively sedentary bird that is a permanent resident of western North American forests, the blue grouse (Dendragapus obscurus). Phylogenetic analysis of complete mitochondrial control region DNA sequences resulted in the identification of three basal clades of haplotypes that were largely congruent with well-known biogeographical regions. These clades corresponded to the parapatric sooty (D. o. fuliginosus) and dusky (D. o. obscurus) subspecies groups of blue grouse plus a previously unrecognized division between northern and southern dusky grouse populations; the latter does not correspond closely to any currently recognized subspecies boundary. Approximately 66% of the total genetic variation was distributed among these three regions. Maximum likelihood estimates of gene flow between the regions were low or asymmetric; gene flow has been insufficient to prevent genetic divergence between dusky and sooty grouse. Estimates of gene flow among populations within sooty grouse were large except across the Columbia River valley. Among populations of dusky grouse, estimates of gene flow were heterogeneous and asymmetrical, reflecting large-scale fragmentation of the distribution due to landscape features and associated vegetation. Genetic, morphological and behavioural evidence suggest that sooty and dusky grouse are species-level taxa; the specific status of a third clade remains ambiguous.

RAG-1 sequences resolve phylogenetic relationships within Charadriiform birds.

The Charadriiformes is a large and diverse order of shorebirds currently classified into 19 families, including morphologically aberrant forms that are of uncertain phylogenetic placement within non-passerine birds in general. Recent attempts using morphological characters have failed to recover a well-supported phylogeny depicting higher level relationships within Charadriiformes and the limits to the order, primarily because of inconsistency and homoplasy in these data. Moreover, these trees are incongruent with the relationships presented in the DNA hybridization tapestry of, including the location of the root and the branching order of major clades within the shorebirds. To help clarify this systematic confusion we therefore sequenced the large RAG-1 nuclear exon (2850 bp) from 36 species representing 17 families of shorebirds for which DNA was available. Trees built with maximum parsimony, maximum likelihood or Bayesian methods are topologically identical and fully resolved, with high support at basal nodes. This further attests to the phylogenetic utility of the RAG-1 sequences at higher taxonomic levels within birds. The RAG-1 tree is topologically similar to the DNA hybridization tree in depicting three major subordinal clades of shorebirds, the Charadrii (thick-knees, sheathbills, plovers, oystercatchers, and allies), Scolopaci (sandpipers and jacanas) and the Lari (coursers, pratincoles, gulls, terns, skimmers, and skuas). However, the basal split in the RAG-1 tree is between Charadrii and (Scolopaci+Lari), whereas in the DNA hybridization tree Scolopaci is the sister group to the (Charadrii+Lari). Thus in both of these DNA-based trees the Alcidae (auks, murres, and allies) are not basal among shorebirds as hypothesized in morphological trees, but instead are placed as a tip clade within Lari. The enigmatic buttonquails (Turnicidae), variously hypothesized as being allied to either the Galliformes, Gruiformes, or Charadriiformes, are shown to be a basal lineage in the more conventional Lari clade. Divergence times estimated with rate-smoothing methods and minimum time constraints imposed at nodes with key fossils suggest that Charadriiformes originated in Gondwanaland.