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Funder: U.S. Department of Health & Human Services | NIH | National Institute of Biomedical Imaging and Bioengineering (NIBIB), DddA-derived cytosine base editors (DdCBEs) use programmable DNA-binding TALE repeat arrays, rather than CRISPR proteins, a split double-stranded DNA cytidine deaminase (DddA), and a uracil glycosylase inhibitor to mediate C•G-to-T•A editing in nuclear and organelle DNA. Here we report the development of zinc finger DdCBEs (ZF-DdCBEs) and the improvement of their editing performance through engineering their architectures, defining improved ZF scaffolds, and installing DddA activity-enhancing mutations. We engineer variants with improved DNA specificity by integrating four strategies to reduce off-target editing. We use optimized ZF-DdCBEs to install or correct disease-associated mutations in mitochondria and in the nucleus. Leveraging their small size, we use a single AAV9 to deliver into heart, liver, and skeletal muscle in post-natal mice ZF-DdCBEs that efficiently install disease-associated mutations. While off-target editing of ZF-DdCBEs is likely too high for therapeutic applications, these findings demonstrate a compact, all-protein base editing research tool for precise editing of organelle or nuclear DNA without double-strand DNA breaks.

BACKGROUND: Disorders of the sense of smell have received greater attention because of the frequency with which they occur as a symptom of SARS-CoV-2 infection. Olfactory dysfunction can lead to profound reduction in quality of life and may arise from many different causes. METHODS: A selective literature review was conducted with consideration of the current version of the guideline issued by the Association of the Scientific Medical Societies in Germany. RESULTS: The cornerstones of diagnosis are the relevant medical history and psychophysical testing of olfactory function using standardized validated tests. Modern treatment strategies are oriented on the cause of the dysfunction. While treatment of the underlying inflammation takes precedence in patients with sinunasal dysosmia, olfactory training is the primary treatment option for other forms of the disorder. The prognosis is determined not only by the cause of the olfactory dysfunction and the patient’s age, but also by the olfactory performance as measured at the time of diagnosis. CONCLUSION: Options for the treatment of olfactory dysfunction are available but limited, depending on the cause. It is therefore important to carry out a detailed diagnostic work-up and keep the patient informed of the expected course and prognosis.

Normalizing flows model probability distributions through an expressive tractable density. They transform a simple base distribution, such as a Gaussian, through a sequence of invertible functions, which are referred to as layers. These layers typically use neural networks to become very expressive. Flows are ubiquitous in machine learning and have been applied to image generation, text modeling, variational inference, approximating Boltzmann distributions, and many other problems. Here, we present normflows, a Python package for normalizing flows. It allows to build normalizing flow models from a suite of base distributions, flow layers, and neural networks. The package is implemented in the popular deep learning framework PyTorch, which simplifies the integration of flows in larger machine learning models or pipelines. It supports most of the common normalizing flow architectures, such as Real NVP, Glow, Masked Autoregressive Flows, Neural Spline Flows, Residual Flows, and many more. The package can be easily installed via pip and the code is publicly available on GitHub.

We present Multiscale Multiview Vision Transformers (MMViT), which introduces multiscale feature maps and multiview encodings to transformer models. Our model encodes different views of the input signal and builds several channel-resolution feature stages to process the multiple views of the input at different resolutions in parallel. At each scale stage, we use a cross-attention block to fuse information across different views. This enables the MMViT model to acquire complex high-dimensional representations of the input at different resolutions. The proposed model can serve as a backbone model in multiple domains. We demonstrate the effectiveness of MMViT on audio and image classification tasks, achieving state-of-the-art results.

Supplemental material, sj-docx-1-vet-10.1177_03009858231171906 for Hepatic proinflammatory myeloid phenotypes are a hallmark of Ebola virus Kikwit pathogenesis in rhesus monkeys by Anna E. Tseng, Mariano Carossino, Hans P. Gertje, Aoife K. O�Connell, Suryaram Gummuluru, Vijaya B Kolachalama, Udeni B. R. Balasuriya, John H. Connor, Richard S. Bennett, David X. Liu, Lisa E. Hensley and Nicholas A. Crossland in Veterinary Pathology

Group fairness is a popular approach to prevent unfavorable treatment of individuals based on sensitive attributes such as race, gender, and disability. However, the reliance of group fairness on access to discrete group information raises several limitations and concerns, especially with regard to privacy, intersectionality, and unforeseen biases. In this work, we propose a "group-free" measure of fairness that does not rely on sensitive attributes and, instead, is based on homophily in social networks, i.e., the common property that individuals sharing similar attributes are more likely to be connected. Our measure is group-free as it avoids recovering any form of group memberships and uses only pairwise similarities between individuals to define inequality in outcomes relative to the homophily structure in the network. We theoretically justify our measure by showing it is commensurate with the notion of additive decomposability in the economic inequality literature and also bound the impact of non-sensitive confounding attributes. Furthermore, we apply our measure to develop fair algorithms for classification, maximizing information access, and recommender systems. Our experimental results show that the proposed approach can reduce inequality among protected classes without knowledge of sensitive attribute labels. We conclude with a discussion of the limitations of our approach when applied in real-world settings.

Supplemental material, sj-docx-1-vet-10.1177_03009858231171906 for Hepatic proinflammatory myeloid phenotypes are a hallmark of Ebola virus Kikwit pathogenesis in rhesus monkeys by Anna E. Tseng, Mariano Carossino, Hans P. Gertje, Aoife K. O�Connell, Suryaram Gummuluru, Vijaya B Kolachalama, Udeni B. R. Balasuriya, John H. Connor, Richard S. Bennett, David X. Liu, Lisa E. Hensley and Nicholas A. Crossland in Veterinary Pathology

The role of visual representations in aiding children's understanding of biological variation in the scope of genetics inheritance

Many years have passed in oil field development but primary challenges faced by the X reservoir are the rapid decline of formation pressure and the significant solution gas released from the formation, which impairs production. Based on these challenges, a compositional simulation model of the X reservoir was constructed and run to establish the future development plans. The basic reservoir data collection and processing, quality assurance of the data, characteristic pressure-volume-temperature (PVT) matching by ECLIPSE PVTi, and simulation of various adjustment strategies to forecast development plans, as well as data sensitivity analysis and optimization has been included in this study. In addition, to establish a desirable development plan, the simulation model is set up in great consistency with the geological model resulted from the seismic and logging interpretations. Also, emphases are paid on establishing matches with the reported lab data from production wells by PVTi. Results revealed that the specific reservoir development plan intends to reinstate or maintain formation pressure of the X reservoir. All design and optimization studies are set to comprehend the reservoir with the numerical model.

Vision transformer (ViT) and its variants have achieved remarkable successes in various visual tasks. The key characteristic of these ViT models is to adopt different aggregation strategies of spatial patch information within the artificial neural networks (ANNs). However, there is still a key lack of unified representation of different ViT architectures for systematic understanding and assessment of model representation performance. Moreover, how those well-performing ViT ANNs are similar to real biological neural networks (BNNs) is largely unexplored. To answer these fundamental questions, we, for the first time, propose a unified and biologically-plausible relational graph representation of ViT models. Specifically, the proposed relational graph representation consists of two key sub-graphs: aggregation graph and affine graph. The former one considers ViT tokens as nodes and describes their spatial interaction, while the latter one regards network channels as nodes and reflects the information communication between channels. Using this unified relational graph representation, we found that: a) a sweet spot of the aggregation graph leads to ViTs with significantly improved predictive performance; b) the graph measures of clustering coefficient and average path length are two effective indicators of model prediction performance, especially when applying on the datasets with small samples; c) our findings are consistent across various ViT architectures and multiple datasets; d) the proposed relational graph representation of ViT has high similarity with real BNNs derived from brain science data. Overall, our work provides a novel unified and biologically-plausible paradigm for more interpretable and effective representation of ViT ANNs., Comment: 11 pages,7 figures, submitted to 36th Conference on Neural Information Processing Systems (NeurIPS 2022)

Supplemental material, sj-docx-1-cmg-10.1177_26317745221136775 for Serrated polyp detection rate in colonoscopies performed by gastrointestinal fellows by Matthew Heckroth, Michael Eiswerth, Mohamed Elmasry, Khushboo Gala, Wenjing Cai, Scott Diamond, Amal Shine, David Liu, Nanlong Liu, Sudaraka Tholkage, Maiying Kong and Dipendra Parajuli in Therapeutic Advances in Gastrointestinal Endoscopy

Artificial neural networks (ANNs), originally inspired by biological neural networks (BNNs), have achieved remarkable successes in many tasks such as visual representation learning. However, whether there exists semantic correlations/connections between the visual representations in ANNs and those in BNNs remains largely unexplored due to both the lack of an effective tool to link and couple two different domains, and the lack of a general and effective framework of representing the visual semantics in BNNs such as human functional brain networks (FBNs). To answer this question, we propose a novel computational framework, Synchronized Activations (Sync-ACT), to couple the visual representation spaces and semantics between ANNs and BNNs in human brain based on naturalistic functional magnetic resonance imaging (nfMRI) data. With this approach, we are able to semantically annotate the neurons in ANNs with biologically meaningful description derived from human brain imaging for the first time. We evaluated the Sync-ACT framework on two publicly available movie-watching nfMRI datasets. The experiments demonstrate a) the significant correlation and similarity of the semantics between the visual representations in FBNs and those in a variety of convolutional neural networks (CNNs) models; b) the close relationship between CNN's visual representation similarity to BNNs and its performance in image classification tasks. Overall, our study introduces a general and effective paradigm to couple the ANNs and BNNs and provides novel insights for future studies such as brain-inspired artificial intelligence.

Shortcut learning is common but harmful to deep learning models, leading to degenerated feature representations and consequently jeopardizing the model's generalizability and interpretability. However, shortcut learning in the widely used Vision Transformer framework is largely unknown. Meanwhile, introducing domain-specific knowledge is a major approach to rectifying the shortcuts, which are predominated by background related factors. For example, in the medical imaging field, eye-gaze data from radiologists is an effective human visual prior knowledge that has the great potential to guide the deep learning models to focus on meaningful foreground regions of interest. However, obtaining eye-gaze data is time-consuming, labor-intensive and sometimes even not practical. In this work, we propose a novel and effective saliency-guided vision transformer (SGT) model to rectify shortcut learning in ViT with the absence of eye-gaze data. Specifically, a computational visual saliency model is adopted to predict saliency maps for input image samples. Then, the saliency maps are used to distil the most informative image patches. In the proposed SGT, the self-attention among image patches focus only on the distilled informative ones. Considering this distill operation may lead to global information lost, we further introduce, in the last encoder layer, a residual connection that captures the self-attention across all the image patches. The experiment results on four independent public datasets show that our SGT framework can effectively learn and leverage human prior knowledge without eye gaze data and achieves much better performance than baselines. Meanwhile, it successfully rectifies the harmful shortcut learning and significantly improves the interpretability of the ViT model, demonstrating the promise of transferring human prior knowledge derived visual saliency in rectifying shortcut learning, Comment: NeurIPS2022 Under Review

PURPOSE Patients with indolent non-Hodgkin lymphoma typically respond well to first-line immunochemotherapy. At relapse, single-agent rituximab is commonly administered. Data suggest the immunomodulatory agent lenalidomide could increase the activity of rituximab. METHODS A phase III, multicenter, randomized trial of lenalidomide plus rituximab versus placebo plus rituximab was conducted in patients with relapsed and/or refractory follicular or marginal zone lymphoma. Patients received lenalidomide or placebo for 12 cycles plus rituximab once per week for 4 weeks in cycle 1 and day 1 of cycles 2 through 5. The primary end point was progression-free survival per independent radiology review. RESULTS A total of 358 patients were randomly assigned to lenalidomide plus rituximab (n = 178) or placebo plus rituximab (n = 180). Infections (63% v 49%), neutropenia (58% v 23%), and cutaneous reactions (32% v 12%) were more common with lenalidomide plus rituximab. Grade 3 or 4 neutropenia (50% v 13%) and leukopenia (7% v 2%) were higher with lenalidomide plus rituximab; no other grade 3 or 4 adverse event differed by 5% or more between groups. Progression-free survival was significantly improved for lenalidomide plus rituximab versus placebo plus rituximab, with a hazard ratio of 0.46 (95% CI, 0.34 to 0.62; P < .001) and median duration of 39.4 months (95% CI, 22.9 months to not reached) versus 14.1 months (95% CI, 11.4 to 16.7 months), respectively. CONCLUSION Lenalidomide improved efficacy of rituximab in patients with recurrent indolent lymphoma, with an acceptable safety profile.

The move from reading to writing the human genome offers new opportunities to improve human health. The United States National Institutes of Health (NIH) Somatic Cell Genome Editing (SCGE) Consortium aims to accelerate the development of safer and more-effective methods to edit the genomes of disease-relevant somatic cells in patients, even in tissues that are difficult to reach. Here we discuss the consortium's plans to develop and benchmark approaches to induce and measure genome modifications, and to define downstream functional consequences of genome editing within human cells. Central to this effort is a rigorous and innovative approach that requires validation of the technology through third-party testing in small and large animals. New genome editors, delivery technologies and methods for tracking edited cells in vivo, as well as newly developed animal models and human biological systems, will be assembled-along with validated datasets-into an SCGE Toolkit, which will be disseminated widely to the biomedical research community. We visualize this toolkit-and the knowledge generated by its applications-as a means to accelerate the clinical development of new therapies for a wide range of conditions.

Cytosine base editors and adenine base editors (ABEs) can correct point mutations predictably and independent of Cas9-induced double-stranded DNA breaks (which causes substantial indel formation) and homology-directed repair (which typically leads to low editing efficiency). Here, we show, in adult mice, that a subretinal injection of a lentivirus expressing an ABE and a single-guide RNA targeting a de novo nonsense mutation in the Rpe65 gene corrects the pathogenic mutation with up to 29% efficiency and with minimal formation of indel and off-target mutations, despite the absence of the canonical NGG sequence as a protospacer-adjacent motif. The ABE-treated mice displayed restored RPE65 expression and retinoid isomerase activity, and near-normal levels of retinal and visual functions. Our findings motivate the further testing of ABEs for the treatment of inherited retinal diseases and for the correction of pathological mutations with non-canonical protospacer-adjacent motifs.

Additional file 1: Appendix S1. Organization of the PROUD Study Investigative Teams.

Additional file 1. Table S1. Exclusion criteria. Table S2. Algorithm for identifying immune-related adverse events. Table S3. Summary of immune-related adverse events. Figure S1. Duration of response, assessed by the blinded independent review committee, for the population with other solid tumors. Figure S2. Kaplan-Meier curves showing progression-free survival, assessed by the blinded independent review committee, for the overall study population and the primary efficacy population (PEP).

Colorectal cancer is one of the most common cancers in the United States and treatment options are limited for patients who develop liver metastases. Several chemotherapeutic regimens have been used for transvascular liver-directed therapy in the treatment of colorectal liver metastases without clear evidence of superiority of one therapy over another. We describe the development of a novel nanoemulsion through combining irinotecan (IRI), a first line systemic agent used for the treatment of colon cancer, with lipiodol, an oily contrast medium derived from poppy seed oil, and evaluated its pharmacokinetic and biodistribution profile as a function of portal venous chemoembolization (PVCE) versus transarterial chemoembolization (TACE) delivery. The Tessari technique was used to create a stable emulsion (20 mg IRI mixed with 2 mL lipiodol) with resultant particle size ranging from 28.9 nm to 56.4 nm. Pharmacokinetic profile established through venous sampling in Buffalo rats demonstrate that the area under the curve (AUC0−∞) of IRI was significantly less after PVCE with IRI-lipiodol as compared to IRI alone (131 vs. 316 µg*min/mL, p-value = .023), suggesting significantly higher amounts of IRI retention in the liver with the IRI-lipiodol nanoemulsion via first-pass extraction. Subseqent biodistribution studies in tumor-bearing WAG/Rjj rats revealed more IRI present in the tumor following TACE versus PVCE (29.19 ± 12.33 µg/g versus 3.42 ± 1.62; p-value = .0033) or IV (29.19 ± 12.33 µg/g versus 1.05 ± 0.47; p-value = .0035). The IRI-lipiodol nanoemulsion demonstrated an acceptable hepatotoxicity profile in all routes of administration. In conclusion, the IRI-lipiodol nanoemulsion via TACE showed promise and warrants further investigation as an option for the treatment of metastatic colorectal cancer.

Additional file 1. Supplementary figures.

Additional file 4: Appendix S4. Quarterly PROUD debrief between Site PI/PM and PROUD Implementation Monitoring Team.

Additional file 2: Appendix S2. Baseline PROUD implementation monitoring information.

Additional file 9. Review history.

CRISPR-Cas-guided base editors convert A•T to G•C, or C•G to T•A, in cellular DNA for precision genome editing. To understand the molecular basis for DNA adenosine deamination by adenine base editors (ABEs), we determined a 3.2-angstrom resolution cryo-electron microscopy structure of ABE8e in a substrate-bound state in which the deaminase domain engages DNA exposed within the CRISPR-Cas9 R-loop complex. Kinetic and structural data suggest that ABE8e catalyzes DNA deamination up to ~1100-fold faster than earlier ABEs because of mutations that stabilize DNA substrates in a constrained, transfer RNA-like conformation. Furthermore, ABE8e's accelerated DNA deamination suggests a previously unobserved transient DNA melting that may occur during double-stranded DNA surveillance by CRISPR-Cas9. These results explain ABE8e-mediated base-editing outcomes and inform the future design of base editors.

Evolutionary algorithms borrow from biology the concepts of mutation and selection in order to evolve optimized solutions to known problems. The GENETIS collaboration is developing genetic algorithms for designing antennas that are more sensitive to ultra-high energy neutrino induced radio pulses than current designs. There are three aspects of this investigation. The first is to evolve simple wire antennas to test the concept and different algorithms. Second, optimized antenna response patterns are evolved for a given array geometry. Finally, antennas themselves are evolved using neutrino sensitivity as a measure of fitness. This is achieved by integrating the XFdtd finite-difference time-domain modeling program with simulations of neutrino experiments., Comment: 8 pages including references, 6 figures, presented at 36th International Cosmic Ray Conference (ICRC 2019)

Supplemental material, Liu_Successes_2020_Supplementary_Material_Revised for Successes and Struggles with Computational Reproducibility: Lessons from the Fragile Families Challenge by David M. Liu and Matthew J. Salganik in Socius

Rhesus macaques are physiologically similar to humans and, thus, have served as useful animal models of human diseases including cardiovascular disease. The purpose of this study was to characterize the distribution, composition, and phenotype of macrophages in heart tissues of very young (fetus: 0.5 years, n=6), young adult (2-12 years, n=12), and older adult (13-24 years, n=9) rhesus macaques using histopathology and immunofluorescence microscopy. Results demonstrated that macrophages were uniformly distributed throughout the heart in animals of all age groups and were more prevalent than CD3-positve T-cells and CD20-positive B-cells. Macrophages comprised approximately 2% of heart tissue cells in the younger animals and increased to a mean of nearly 4% in the older adults. CD163-positive macrophages predominated over HAM56-positive and CD206-positive macrophages, and were detected at significantly higher percentage in the animals between 13 and 24 years of age, as well as in heart tissues exhibiting severe histopathology or inflammation in animals of all age groups. In vivo dextran labeling and retention indicated that approximately half of the macrophages were longer lived in healthy adult heart tissues and may comprise the tissue-resident population of macrophages. These results provide a basis for continued studies to examine the specific functional roles of macrophage subpopulations in heart tissues during homeostasis and in cardiovascular disease for then developing intervention strategies.

The development of site-specific recombinases (SSRs) as genome editing agents is limited by the difficulty of altering their native DNA specificities. Here we describe Rec-seq, a method for revealing the DNA specificity determinants and potential off-target substrates of SSRs in a comprehensive and unbiased manner. We applied Rec-seq to characterize the DNA specificity determinants of several natural and evolved SSRs including Cre, evolved variants of Cre, and other SSR family members. Rec-seq profiling of these enzymes and mutants thereof revealed previously uncharacterized SSR interactions, including specificity determinants not evident from SSR:DNA structures. Finally, we used Rec-seq specificity profiles to predict off-target substrates of Tre and Brec1 recombinases, including endogenous human genomic sequences, and confirmed their ability to recombine these off-target sequences in human cells. These findings establish Rec-seq as a high-resolution method for rapidly characterizing the DNA specificity of recombinases with single-nucleotide resolution, and for informing their further development., The development of site-specific recombinases as genome editing tools is limited by the difficulty of altering their DNA sequence specificity. Here the authors present Rec-seq, a method for identifying specificity determinants and off-target substrates of recombinases in an unbiased manner.

Reproducibility is fundamental to science, and an important component of reproducibility is computational reproducibility: the ability of a researcher to recreate the results in a published paper using the original author's raw data and code. Although most people agree that computational reproducibility is important, it is still difficult to achieve in practice. In this paper, we describe our approach to enabling computational reproducibility for the 12 papers in this special issue of Socius about the Fragile Families Challenge. Our approach draws on two tools commonly used by professional software engineers but not widely used by academic researchers: software containers (e.g., Docker) and cloud computing (e.g., Amazon Web Services). These tools enabled us to standardize the computing environment around each submission, which will ease computational reproducibility both today and in the future. Drawing on our successes and struggles, we conclude with recommendations to authors and journals.

Supplemental material, DS1_SRD_10.1177_2378023119849803 for Successes and Struggles with Computational Reproducibility: Lessons from the Fragile Families Challenge by David M. Liu and Matthew J. Salganik in Socius

A. Phrynocephalus Taxonomic Recommendations Based on phylogenetic analyses, a few taxonomic changes are proposed. The following taxonomic changes are based on the principle of monophyly as well as genetic and geographic differences. In each case, the reason a change is suggested is provided, whether data are genetic, distributional or phylogenetic (ie. non-monophyly). 1). Phrynocephalus maculatus Complex as Previously Recognized Based on complete regional sampling, this study recognizes all previous subspecies of P. maculatus as distinct species on the minim-basis of non-monophyly. Phrynocephalus maculatus was described from the Iranian Plateau (Anderson 1872) with the type locality of “Awada, Shiraz, Persia ”; corrected to “Abadeh [31o 10' N, 52o 37' E; Fars Province, Iran], north of Shiraz” by Blanford (1876). Phrynocephalus maculatus longicaudatus was described by Haas (1957), with a type locality of “Doha Dhalum, Saudi Arabia ”. The sole known population of P. maculatus from Turkmenistan was reported by Bogdanov et al. (1974), and subsequently described as a distinct species, Phrynocephalus golubewii with the type locality of “vicinity of the Bami rail-road station” [= 7 km N railway station Bamy], (Shenbrot & Semenov 1990). It is recommended to recognize P. golubewii, P. longicaudatus, and P. maculatus as distinct species based on phylogenetic results: a). While Phrynocephalus golubewii (Shenbrot & Semenov 1990) may look like P. maculatus living in saltbed dry lakes, it is directly-related to the clade containing P. turcomanus and P. helioscopus (mt-DNA, bootstrap 91%, decay index 6; combined data, bootstrap 87%, decay index 7; allozyme presence/absence, bootstrap 78%, decay index 1; allozyme step matrix, bootstrap 75%, decay index 2; non-monophyly in RAG- 1); in fact P. turcomanus occurs within meters of P. golubewii and may overlap in distribution. b). Phrynocephalus maculatus (Anderson 1872) is restricted to salt-bed dry lakes on the Iranian Plateau where its type locality is located, and adjacent Baluchistan Plateau including the base of the Sulaiman Range of southern Pakistan. P. maculatus is related, yet outside and sister to a clade containing P. arabicus and P. longicaudatus (see below) based on both mitochondrial and nuclear RAG-1 DNA evidence (mt-DNA, bootstrap 100%, decay index 32; RAG-1, bootstrap 100%, decay index 13; combined data, bootstrap 100%, decay index 46; all of these taxa were not sampled for allozyme data). c). Phrynocephalus longicaudatus (Haas 1957) New Status is restricted to the Arabian Peninsula. It is the sister taxon to P. arabicus which is also endemic to the Arabian Plate and supported by mitochondrial and nuclear RAG- 1 DNA evidence (mt-DNA, bootstrap 100%, decay index 22; RAG- 1, bootstrap 100%, decay index 7; combined data, bootstrap 100%, decay index 30; these taxa were not sampled for allozyme data). These taxa in any combination do not form a monophyletic group. 2). Small Sand Dune Species of Southwest Asia and Caspian Basin Phrynocephalus interscapularis interscapularis (Lichtenstien 1856; type locality “Bucharei” = Uzbekistan) and Phrynocephalus interscapularis sogdianus (Chernov 1948; type locality “ Tadjikistan, vicinity of the Pjandzh village”, approximately 37o 14' N, 69o 05' E) are recognized here as full species P. interscapularis and P. sogdianus based on allozyme and DNA differences. The mt-DNA pair-wise uncorrected sequence distance between P. interscapularis and P. sogdianus is 3.2% and these taxa show one fixed allozyme difference at the carboxylic ester hydrolase locus (EST; Table 2). Phrynocephalus ornatus vindumi [Golubev, 1998; type locality “Iran, Khorasan Prov., 35 km. N of Gonabad on road to Torbat-E. Heydariyeh (ca. 34o 49' N, 58o 47' E), 850 m. elevation”; sampling here within ten kilometers of the type locality, see appendix 1] is elevated to species status as Phrynocephalus vindumi New Status. Phrynocephalus ornatus (Boulenger 1887; type locality “between Nushki and Helmand, Baluchistan”) and P. vindumi appear in a non-monophyletic arrangement as P. vindumi occurring on the northeastern portion of the Iranian Plateau is separated from P. ornatus that occurs south of the Hindu Kush in Afghanistan and southwestern Pakistan. In fact, P. vindumi is related to small sand dune species in the Caspian Basin to the north: P. interscapularis and P. sogdianus. In the mt-DNA parsimony analysis, P. vindumi appears in a sister position to the clade containing P. interscapularis and P. sogdianus, with those three taxa grouping with high support (bootstrap 98%, decay index 13). Alternatively, P. ornatus is sister to P. clarkourum (bootstrap 99%, decay index 21), and those two taxa are included in a clade with the two P. luteogutattus samples that also receive high support (bootstrap 100%, decay index 26). Allozyme and RAG-1 DNA data did not sample appropriately to test this question. 3). Gravel Dwelling Phrynocephalus Species in the Caspian Basin Previously, P. reticulatus, P. bannikovi, and P. strauchi were all considered subspecies of P. reticulatus (Eichwald 1831): The nominal subspecies Phrynocephalus reticulatus reticulatus [Eichwald 1831; type locality "Hab. ad Oxum, pristinum amnem, in caspii maris litore orietali", corrected to the Amu-Darja River by Golubev (1991)] and two additional distinct subspecies, Phrynocephalus reticulatus strauchi [Nikolsky 1899; restricted type locality “Khodzhent, 40o 17' N, 69o 37' E, northern Tajikistan ” by lectotype designated by Dunayev (1995)] and Phrynocephalus reticulatus bannikovi (Darevsky, Rustamov & Shammakov 1976; type locality “Tuarkyr Mountains, northwestern Turkmenistan ”), (see Bannikov et al. 1977; although P. reticulatus and P. strauchi were originally described as species). Here these gravel ecotypes are recognized as distinct species based on genetic difference and lack of monophyly. In our sampling, P. bannikovi and P. strauchi do not appear monophyletic with P. strauchi and P. rossikowi grouping with light support (mt-DNA, bootstrap 55%, decay Index 2). P. reticulatus from central Uzbekistan was not sampled in our study or the study of Solovyeva et al. (2014). If P. bannikovi and P. rossikowi were removed from the parsimony analysis performed on the mt-DNA data, the topology of the trees in this study and that of Solovyeva et al. (2014) would be congruent. Based on incomplete sampling, it is recommended that members of the P. reticulatus complex be recognized as separate species Phrynocephalus bannikovi (Darevsky, Rustamov & Shammakov 1976) New Status, P. reticulatus, and P. strauchi. Solovyeva et al. (2014) appear to have not sampled any species from Turkmenistan, which includes P. raddei raddei and P. rossikowi, and in our phylogenetic analyses are related to the P. reticulatus complex. The inclusion of P. raddei raddei and P. rossikowi in the sampling presented here indicates the traditional P. reticulatus complex is not monophyletic, deserving species recognition for the three former subspecies. In common with this study, Solovyeva et al. (2014) only sampled P. strauchi from the P. reticulatus complex. In the sampling of this study, P. bannikovi and P. raddei are geographically oriented disjunctive-adjacent to each other in eastern and west-central Turkmenistan, respectively, and are sister taxa receiving high support from mt-DNA (bootstrap 100%, decay index 23), nuclear RAG-1 DNA (bootstrap 91%, decay index 3), and combined data analyses (bootstrap 100%, decay index 27). Solovyeva et al. (2014) did not sample P. bannikovi, P. rossikowi, or P. raddei raddei from Turkmenistan. However, Solovyeva et al. (2014) and this study did sample P. strauchi from the Fergan Valley of eastern Uzbekistan or northern Tadjikistan. Sampling between this study and Solovyeva et al. (2014) differs in the subspecies of P. raddei: This study uses P. raddei raddei, (Boettger 1888: type locality “Perewalnaja an der transcaspischen Bahn", = Perevalnaja railroad station, southwestern Turkmenistan; near where this study's sample originated from), whereas Solovyeva et al. (2014) uses P. raddei boettgeri (Nikolsky 1905; type locality “Schirabad, Buchara orient” = Sherabad, approximately 37o 42' N, 67o 04' E, southern Uzbekistan) with a vague locality provided as “Turan Depression” [= Caspian Basin]; but occurs in extreme southern Uzbekistan and southern Tajikistan along the Afghanistan border (Bannikov et al. 1977). Until a single study samples both subspecies of P. raddei, it will be unclear how they relate to P. bannikovi, P. reticulatus, P. strauchi, and P. rossikowi. In Solovyeva et al. (2014) no detailed locality information is available, nor is an appendix with this information provided. All GenBank entries do not include locality data, so this information is not archival with NIH GenBank. The Zoological Museum of Moscow University (ZMMU) (http://zmmu.msu.ru/en/about-muzeum/ fonds/amphibians-&-reptiles-collections) in which specimens reported in Solovyeva et al. (2014) are housed, does not offer an obvious means of searching by voucher number on their website. Additionally, this institution does not currently participate in global databases which compile data from institutions across the globe and allow free public access to museum collections involving voucher number relating to locality information. VertNet, a global organization whose participants include 64 institutions spanning 13 countries including Russia, is one prominent database to organize museum collections around the world. The Global Information Biodiversity Facility (GBIF) also offers a forum for researchers to access information on specimens collected in select countries on every continent, which is an added resource. The Zoological Museum of Moscow University is not currently participating in any of these databases, and localities in the manuscript of Solovyeva et al. (2014) are very vague, crossing large geographic regions, and not informative for the difficulties of dealing with Phrynocephalus taxonomy and sampling. Solovyeva et al. (2014) present GenBank sequences as common courtesy in the scientific community, but in those sequences, no locality information is provided so comparing Solovyeva et al. (2014) results with this study is difficult across all issues, starting with taxonomy. Published localities in Solovyeva et al. (2014) such as Central and Middle Asia are not informative, as in the Russian style geography, Middle Asia refers to the Caspian Basin, and Central Asia refers to Tibet, Mongolia and western China. Localities of such gross generality do not provide any information for a study of this detail. 4). Phrynocephalus helioscopus Complex in the Caspian Basin Phrynocephalus helioscopus turcomanus [Dunayev, Solovyeva & Poyarkov 2012 ( in Solovyeva et al. 2012); type locality 40° 01′ 00′′ N 52° 58′ 00′′ E, current name Turkmenbashi, Turkmenistan] is recognized as a distinct species Phrynocephalus turcomanus [Dunayev, Solovyeva & Poyarkov 2012 ( in Solovyeva et al. 2012)] New Status from P. helioscopus based on a north-south distributional disjunction in the Caspian Basin with significant mt-DNA differences. P. helioscopus [Pallas (1771) listed the type locality as "in deserti australioris collibus ardentissimis"; restricted (with no lectotype) to the "Inderskija Gory, Gebiet des unteren Uralflusses" by Mertens & Müller (1928), now Inderskije (Inder) Mountains, Atyrau Region, northwestern Kazakhstan] here is sampled from the northern Aral Sea region of Kazakhstan, and P. turcomanus from southwestern Turkmenistan (Appendix 1). While no fixed allozyme differences are detected between populations sampled, there is a high mt-DNA difference. The mt-DNA pairwise uncorrected sequence distance between P. helioscopus and P. turcomanus is 4.5%; therefore significantly diverged and in the range of sister species divergence for this segment of the mitochondrial genome among amphibians and reptiles (for summary see table 6 in Weisrock et al. 2001). 5). Use of Names in the Low Elevation Chinese Desert—Northern Caspian Basin Clade a). Phrynocephalus salenskyi The name P. salenskyi Bedriaga 1907 is applied here for populations sampled from the Junggur Depression of northwestern China, and the Zaysan Depression of northeastern Kazakhstan. P. salenskyi is the oldest name available by date and page number for Phrynocephalus populations of the low elevation Chinese desert – northern Caspian Basin clade in this geographic region, see below: Phrynocephalus Salenskyi Bedriaga 1907: 141, 213.—Restricted type locality: “ Urungu Fluss ” [= Ulungur River, Xinjiang Uygur Autonomous Region, China]. Phrynocephalus Isseli Bedriaga 1907: 229.— Type locality: Desert in vicinity of Mt. Ssalburty (in Saur Mountains of the Tarbagatay Range, Xinjiang Uygur Autonomous Region, China). Phrynocephalus Haeckeli Bedriaga 1907: 237.— Type locality: Desert in vicinity of Mt. Ssalburty (in Saur Mountains of the Tarbagatay Range), in the Tarbagataï (= Tacheng County) district, northern Xinjiang Uygur Autonomous Region, China. Phrynocephalus Arcellazzii Bedriaga 1907: 307.— Type locality: “ Gaschun, in der Nähe von Gutschen in der Dschungarei ” [= Qitai (44° 00' N, 89° 50' E), Junggar Depression, Xinjiang Uygur Autonomous Region, China]. Phrynocephalus Grum-Grzimailoi Bedriaga 1909: 420.—Restricted type locality: “Gutschen” [= Qitai (44° 00' N, 89° 50' E), Junggar Depression, Xinjiang Uygur Autonomous Region, China]. Phrynocephalus bedriaga Tsarevsky 1926: 214.—Restricted type locality: Zaissan Lake region, eastern Kazakhstan. Phrynocephalus albolineatus K. Zhao 1979: 113.— Type locality: "45 kilometers south of Tacheng County, Xinjiang Uygur Autonomous Region, China; 584 m elevation". The Irtysh River flows out of Zaysan Lake linking the Junggar Depression with the Zaysan Depression, which makes a compelling case for sampling presented in this study representing P. salenskyi populations. b). Phrynocephalus przewalskii Complex in the Gobi Desert Phrynocephalus Przewalskii Strauch 1876: 10.— Type locality: “deserto Alaschanico ” [= Tengger Desert, Nei Mongol Autonomous Region, China]; corrected to eastern Tengger Desert west from 106° E, southwest Nei Mongol Autonomous Region, China in Barabanov & Ananjeva (2007). This taxon is generally a sand dwelling ecotype. P. frontalis Strauch 1876: 15.—Type locality: locality “Ordos dicta” [= Ordos Desert, Nei Mongol Autonomous Region, China]. This taxon is generally thought of as a gravel dwelling ecotype, and here considered a synonym of P. przewalskii (see below). A lack of genetic diversity between populations from these taxa (two populations each), confirm that only the name P. przewalskii is valid, whereas P. frontalis is a synonym of P. przewalskii (following Gozdzik & Fu 2009). This is supported by nuclear encoded allozyme data, and mt-DNA data. Alternative names exist as reviewed in Barabanov & Ananjeva (2007), but without detailed sampling of type localities, the validity of alternate names cannot be addressed. Across the 25 allozyme loci sampled here, we detected no fixed differences among the four populations of P. przewalskii, indicating a fair amount of gene flow between the populations. The average mt-DNA pair-wise uncorrected sequence distances between population 2 and the clade containing populations 1, 3, and 4 is 3.64% (range 3.41–3.79%). The average pair-wise comparison within the clade containing populations 1, 3 and 4 is 0.57% (range 0.51–0.63%). These results match the phylogenetic topology discovered in the mt-DNA parsimony analysis, with population 2 placed in a sister position to the clade containing populations 1, 3 and 4 (bootstrap 100%, decay index 19). No resolution was discovered among the four P. przewalskii populations in the RAG- 1 DNA parsimony analysis. Urquhart et al. (2009) suggests a vicariant break among P. przewalskii populations roughly corresponding to the Helan Shan (mountains). Data reported here do not corroborate that hypothesis. Sampled are a single population east of the Yellow River and three populations west of the Yellow River, which flows north to south in this region. Population 2 is sampled on the east side of the river, and population 3 is sampled on the west side of the river, on banks directly across from each other in Shapotou, Ningxia Autonomous Region, China. Population 2, the only population on the east side of the Yellow River is the most divergent, with populations 1, 3 and 4 on the west side of the river nearly identical in our sampling. Population 1 (Wujiachuan, Gansu Province, China) is west of the Yellow River and further south than sampling conducted in Urquhart et al. (2009) and Wang & Fu (2004), yet nearly identical to other populations sampled west of the Yellow River. In our sampling, only population 4 lies west of the Helan Shan (Wuwei, Gansu Province, China) in the Tengger Desert, the type locality of P. przewalskii. Sampling in this study suggests the Yellow River could be responsible for a genetic break in P. przewalskii populations, and not the Helan Shan, though more detailed, fine-scale sampling and analysis will need to be done to confirm this hypothesis. In the Zhongwei region of Ningxia Autonomous Region, there is an indication that east and west haplotypes as described above occur in the same locality, which could indicate a point of hybridization, but further detailed work is needed (Wang & Fu 2004). Sampling conducted in similar areas in Wang & Fu (2004) and Urquhart et al. (2009) did not completely overlap, making conclusions between these studies and this study very difficult for comparative interpretation, further indicating additional work is needed. In sampling of the P. przewalskii complex in Jin & Brown (2013), populations to the east are grouped, and are referred to as P. frontalis, with populations to the west grouping and referred to as P. przewalskii, not conflicting with results reported here. 6). Use of Names in the Northern Tibetan Plateau

E. Phrynocephalus Size Diversity Phrynocephalus show a variety of snout vent lengths and tail lengths, (Table 6, Appendix 5). Body size per sample ranged from 106 mm for P. mystaceus to 35 mm for P. vindumi and P. interscapularis. Tail length per sample ranges from 133 mm for P. maculatus population 1 to 35 mm for P. luteoguttatus population 2. P. mystaceus and P. interscapularis are sympatric in the Caspian Basin, and habitat partition with P. mystaceus in large floating sand dunes, with P. interscapularis living on the edge of those large sand dunes, which may revolve around ecotype evolution in a predator-prey relationship. In northwestern Iran P. vindumi replaces the role of P. interscapularis, and is sympatric with P. mystaceus. Tail length appears to be the most sexually dimorphic characteristic. ......continued on the next page Snout vent lengths for clay ecotypes range from 62 mm for P. przewalskii population 1 to 49 mm for P. versicolor, with tail lengths ranging from 90 mm for P. przewalskii population 1 to 49 mm for P. persicus. The snout vent length for gravel ecotypes range from 64 mm for P. vlangalii population 1 to 40 mm for P. rossikowi, with tail lengths ranging from 90 mm for P. longicaudatus to 42 mm for P. theobaldi. Snout vent lengths for dry lakebed ecotypes range from 90 mm for P. maculatus population 1 to 60 mm for P. golubewii, with tail length ranging from 133 mm for P. maculatus population 1 to 78 mm for P. maculatus population 2. Snout vent lengths for small sand dune ecotypes range from 61 mm for P. vlangalii population 2 to 35 mm for both P. vindumi and P. interscapularis, with tail length ranging from 80 mm for P. przewalskii population 2 to 35 mm for P. luteoguttatus population 2. Snout vent lengths for large sand dune ecotypes range from 106 mm for P. mystaceus populations to 59 mm for P. salenskyi population 1, with tail lengths ranging from 110 mm for P. mystaceus population 1 to 70 mm for P. salenskyi population 1. The largest Phrynocephalus occupy large floating sand dunes, and the smallest Phrynocephalus species are found on the fringes of large floating sand dunes in the Caspian Basin and northern Iran. P. mystaceus occupying large sand dunes in these habitats coexists with the two smallest species that occupy the fringes of those sand dunes, P. interscapularis in the Caspian Basin, and P. vindumi in northwestern Iran. It has not escaped our attention that size and habitat partitioning may have occurred in these regions because it is likely that smaller species (P. interscapularis or P. vindumi) serve as a food source for larger species (P. mystaceus). Further work is needed for phylogenetic assessment of size evolution among Phrynocephalus species and populations considering the sexually dimorphic nature of this genus, both with body size and tail length.

A. Phrynocephalus Taxonomic Recommendations Based on phylogenetic analyses, a few taxonomic changes are proposed. The following taxonomic changes are based on the principle of monophyly as well as genetic and geographic differences. In each case, the reason a change is suggested is provided, whether data are genetic, distributional or phylogenetic (ie. non-monophyly). 1). Phrynocephalus maculatus Complex as Previously Recognized Based on complete regional sampling, this study recognizes all previous subspecies of P. maculatus as distinct species on the minim-basis of non-monophyly. Phrynocephalus maculatus was described from the Iranian Plateau (Anderson 1872) with the type locality of “Awada, Shiraz, Persia ”; corrected to “Abadeh [31o 10' N, 52o 37' E; Fars Province, Iran], north of Shiraz” by Blanford (1876). Phrynocephalus maculatus longicaudatus was described by Haas (1957), with a type locality of “Doha Dhalum, Saudi Arabia ”. The sole known population of P. maculatus from Turkmenistan was reported by Bogdanov et al. (1974), and subsequently described as a distinct species, Phrynocephalus golubewii with the type locality of “vicinity of the Bami rail-road station” [= 7 km N railway station Bamy], (Shenbrot & Semenov 1990). It is recommended to recognize P. golubewii, P. longicaudatus, and P. maculatus as distinct species based on phylogenetic results: a). While Phrynocephalus golubewii (Shenbrot & Semenov 1990) may look like P. maculatus living in saltbed dry lakes, it is directly-related to the clade containing P. turcomanus and P. helioscopus (mt-DNA, bootstrap 91%, decay index 6; combined data, bootstrap 87%, decay index 7; allozyme presence/absence, bootstrap 78%, decay index 1; allozyme step matrix, bootstrap 75%, decay index 2; non-monophyly in RAG- 1); in fact P. turcomanus occurs within meters of P. golubewii and may overlap in distribution. b). Phrynocephalus maculatus (Anderson 1872) is restricted to salt-bed dry lakes on the Iranian Plateau where its type locality is located, and adjacent Baluchistan Plateau including the base of the Sulaiman Range of southern Pakistan. P. maculatus is related, yet outside and sister to a clade containing P. arabicus and P. longicaudatus (see below) based on both mitochondrial and nuclear RAG-1 DNA evidence (mt-DNA, bootstrap 100%, decay index 32; RAG-1, bootstrap 100%, decay index 13; combined data, bootstrap 100%, decay index 46; all of these taxa were not sampled for allozyme data). c). Phrynocephalus longicaudatus (Haas 1957) New Status is restricted to the Arabian Peninsula. It is the sister taxon to P. arabicus which is also endemic to the Arabian Plate and supported by mitochondrial and nuclear RAG- 1 DNA evidence (mt-DNA, bootstrap 100%, decay index 22; RAG- 1, bootstrap 100%, decay index 7; combined data, bootstrap 100%, decay index 30; these taxa were not sampled for allozyme data). These taxa in any combination do not form a monophyletic group. 2). Small Sand Dune Species of Southwest Asia and Caspian Basin Phrynocephalus interscapularis interscapularis (Lichtenstien 1856; type locality “Bucharei” = Uzbekistan) and Phrynocephalus interscapularis sogdianus (Chernov 1948; type locality “ Tadjikistan, vicinity of the Pjandzh village”, approximately 37o 14' N, 69o 05' E) are recognized here as full species P. interscapularis and P. sogdianus based on allozyme and DNA differences. The mt-DNA pair-wise uncorrected sequence distance between P. interscapularis and P. sogdianus is 3.2% and these taxa show one fixed allozyme difference at the carboxylic ester hydrolase locus (EST; Table 2). Phrynocephalus ornatus vindumi [Golubev, 1998; type locality “Iran, Khorasan Prov., 35 km. N of Gonabad on road to Torbat-E. Heydariyeh (ca. 34o 49' N, 58o 47' E), 850 m. elevation”; sampling here within ten kilometers of the type locality, see appendix 1] is elevated to species status as Phrynocephalus vindumi New Status. Phrynocephalus ornatus (Boulenger 1887; type locality “between Nushki and Helmand, Baluchistan”) and P. vindumi appear in a non-monophyletic arrangement as P. vindumi occurring on the northeastern portion of the Iranian Plateau is separated from P. ornatus that occurs south of the Hindu Kush in Afghanistan and southwestern Pakistan. In fact, P. vindumi is related to small sand dune species in the Caspian Basin to the north: P. interscapularis and P. sogdianus. In the mt-DNA parsimony analysis, P. vindumi appears in a sister position to the clade containing P. interscapularis and P. sogdianus, with those three taxa grouping with high support (bootstrap 98%, decay index 13). Alternatively, P. ornatus is sister to P. clarkourum (bootstrap 99%, decay index 21), and those two taxa are included in a clade with the two P. luteogutattus samples that also receive high support (bootstrap 100%, decay index 26). Allozyme and RAG-1 DNA data did not sample appropriately to test this question. 3). Gravel Dwelling Phrynocephalus Species in the Caspian Basin Previously, P. reticulatus, P. bannikovi, and P. strauchi were all considered subspecies of P. reticulatus (Eichwald 1831): The nominal subspecies Phrynocephalus reticulatus reticulatus [Eichwald 1831; type locality "Hab. ad Oxum, pristinum amnem, in caspii maris litore orietali", corrected to the Amu-Darja River by Golubev (1991)] and two additional distinct subspecies, Phrynocephalus reticulatus strauchi [Nikolsky 1899; restricted type locality “Khodzhent, 40o 17' N, 69o 37' E, northern Tajikistan ” by lectotype designated by Dunayev (1995)] and Phrynocephalus reticulatus bannikovi (Darevsky, Rustamov & Shammakov 1976; type locality “Tuarkyr Mountains, northwestern Turkmenistan ”), (see Bannikov et al. 1977; although P. reticulatus and P. strauchi were originally described as species). Here these gravel ecotypes are recognized as distinct species based on genetic difference and lack of monophyly. In our sampling, P. bannikovi and P. strauchi do not appear monophyletic with P. strauchi and P. rossikowi grouping with light support (mt-DNA, bootstrap 55%, decay Index 2). P. reticulatus from central Uzbekistan was not sampled in our study or the study of Solovyeva et al. (2014). If P. bannikovi and P. rossikowi were removed from the parsimony analysis performed on the mt-DNA data, the topology of the trees in this study and that of Solovyeva et al. (2014) would be congruent. Based on incomplete sampling, it is recommended that members of the P. reticulatus complex be recognized as separate species Phrynocephalus bannikovi (Darevsky, Rustamov & Shammakov 1976) New Status, P. reticulatus, and P. strauchi. Solovyeva et al. (2014) appear to have not sampled any species from Turkmenistan, which includes P. raddei raddei and P. rossikowi, and in our phylogenetic analyses are related to the P. reticulatus complex. The inclusion of P. raddei raddei and P. rossikowi in the sampling presented here indicates the traditional P. reticulatus complex is not monophyletic, deserving species recognition for the three former subspecies. In common with this study, Solovyeva et al. (2014) only sampled P. strauchi from the P. reticulatus complex. In the sampling of this study, P. bannikovi and P. raddei are geographically oriented disjunctive-adjacent to each other in eastern and west-central Turkmenistan, respectively, and are sister taxa receiving high support from mt-DNA (bootstrap 100%, decay index 23), nuclear RAG-1 DNA (bootstrap 91%, decay index 3), and combined data analyses (bootstrap 100%, decay index 27). Solovyeva et al. (2014) did not sample P. bannikovi, P. rossikowi, or P. raddei raddei from Turkmenistan. However, Solovyeva et al. (2014) and this study did sample P. strauchi from the Fergan Valley of eastern Uzbekistan or northern Tadjikistan. Sampling between this study and Solovyeva et al. (2014) differs in the subspecies of P. raddei: This study uses P. raddei raddei, (Boettger 1888: type locality “Perewalnaja an der transcaspischen Bahn", = Perevalnaja railroad station, southwestern Turkmenistan; near where this study's sample originated from), whereas Solovyeva et al. (2014) uses P. raddei boettgeri (Nikolsky 1905; type locality “Schirabad, Buchara orient” = Sherabad, approximately 37o 42' N, 67o 04' E, southern Uzbekistan) with a vague locality provided as “Turan Depression” [= Caspian Basin]; but occurs in extreme southern Uzbekistan and southern Tajikistan along the Afghanistan border (Bannikov et al. 1977). Until a single study samples both subspecies of P. raddei, it will be unclear how they relate to P. bannikovi, P. reticulatus, P. strauchi, and P. rossikowi. In Solovyeva et al. (2014) no detailed locality information is available, nor is an appendix with this information provided. All GenBank entries do not include locality data, so this information is not archival with NIH GenBank. The Zoological Museum of Moscow University (ZMMU) (http://zmmu.msu.ru/en/about-muzeum/ fonds/amphibians-&-reptiles-collections) in which specimens reported in Solovyeva et al. (2014) are housed, does not offer an obvious means of searching by voucher number on their website. Additionally, this institution does not currently participate in global databases which compile data from institutions across the globe and allow free public access to museum collections involving voucher number relating to locality information. VertNet, a global organization whose participants include 64 institutions spanning 13 countries including Russia, is one prominent database to organize museum collections around the world. The Global Information Biodiversity Facility (GBIF) also offers a forum for researchers to access information on specimens collected in select countries on every continent, which is an added resource. The Zoological Museum of Moscow University is not currently participating in any of these databases, and localities in the manuscript of Solovyeva et al. (2014) are very vague, crossing large geographic regions, and not informative for the difficulties of dealing with Phrynocephalus taxonomy and sampling. Solovyeva et al. (2014) present GenBank sequences as common courtesy in the scientific community, but in those sequences, no locality information is provided so comparing Solovyeva et al. (2014) results with this study is difficult across all issues, starting with taxonomy. Published localities in Solovyeva et al. (2014) such as Central and Middle Asia are not informative, as in the Russian style geography, Middle Asia refers to the Caspian Basin, and Central Asia refers to Tibet, Mongolia and western China. Localities of such gross generality do not provide any information for a study of this detail. 4). Phrynocephalus helioscopus Complex in the Caspian Basin Phrynocephalus helioscopus turcomanus [Dunayev, Solovyeva & Poyarkov 2012 ( in Solovyeva et al. 2012); type locality 40° 01′ 00′′ N 52° 58′ 00′′ E, current name Turkmenbashi, Turkmenistan] is recognized as a distinct species Phrynocephalus turcomanus [Dunayev, Solovyeva & Poyarkov 2012 ( in Solovyeva et al. 2012)] New Status from P. helioscopus based on a north-south distributional disjunction in the Caspian Basin with significant mt-DNA differences. P. helioscopus [Pallas (1771) listed the type locality as "in deserti australioris collibus ardentissimis"; restricted (with no lectotype) to the "Inderskija Gory, Gebiet des unteren Uralflusses" by Mertens & Müller (1928), now Inderskije (Inder) Mountains, Atyrau Region, northwestern Kazakhstan] here is sampled from the northern Aral Sea region of Kazakhstan, and P. turcomanus from southwestern Turkmenistan (Appendix 1). While no fixed allozyme differences are detected between populations sampled, there is a high mt-DNA difference. The mt-DNA pairwise uncorrected sequence distance between P. helioscopus and P. turcomanus is 4.5%; therefore significantly diverged and in the range of sister species divergence for this segment of the mitochondrial genome among amphibians and reptiles (for summary see table 6 in Weisrock et al. 2001). 5). Use of Names in the Low Elevation Chinese Desert—Northern Caspian Basin Clade a). Phrynocephalus salenskyi The name P. salenskyi Bedriaga 1907 is applied here for populations sampled from the Junggur Depression of northwestern China, and the Zaysan Depression of northeastern Kazakhstan. P. salenskyi is the oldest name available by date and page number for Phrynocephalus populations of the low elevation Chinese desert – northern Caspian Basin clade in this geographic region, see below: Phrynocephalus Salenskyi Bedriaga 1907: 141, 213.—Restricted type locality: “ Urungu Fluss ” [= Ulungur River, Xinjiang Uygur Autonomous Region, China]. Phrynocephalus Isseli Bedriaga 1907: 229.— Type locality: Desert in vicinity of Mt. Ssalburty (in Saur Mountains of the Tarbagatay Range, Xinjiang Uygur Autonomous Region, China). Phrynocephalus Haeckeli Bedriaga 1907: 237.— Type locality: Desert in vicinity of Mt. Ssalburty (in Saur Mountains of the Tarbagatay Range), in the Tarbagataï (= Tacheng County) district, northern Xinjiang Uygur Autonomous Region, China. Phrynocephalus Arcellazzii Bedriaga 1907: 307.— Type locality: “ Gaschun, in der Nähe von Gutschen in der Dschungarei ” [= Qitai (44° 00' N, 89° 50' E), Junggar Depression, Xinjiang Uygur Autonomous Region, China]. Phrynocephalus Grum-Grzimailoi Bedriaga 1909: 420.—Restricted type locality: “Gutschen” [= Qitai (44° 00' N, 89° 50' E), Junggar Depression, Xinjiang Uygur Autonomous Region, China]. Phrynocephalus bedriaga Tsarevsky 1926: 214.—Restricted type locality: Zaissan Lake region, eastern Kazakhstan. Phrynocephalus albolineatus K. Zhao 1979: 113.— Type locality: "45 kilometers south of Tacheng County, Xinjiang Uygur Autonomous Region, China; 584 m elevation". The Irtysh River flows out of Zaysan Lake linking the Junggar Depression with the Zaysan Depression, which makes a compelling case for sampling presented in this study representing P. salenskyi populations. b). Phrynocephalus przewalskii Complex in the Gobi Desert Phrynocephalus Przewalskii Strauch 1876: 10.— Type locality: “deserto Alaschanico ” [= Tengger Desert, Nei Mongol Autonomous Region, China]; corrected to eastern Tengger Desert west from 106° E, southwest Nei Mongol Autonomous Region, China in Barabanov & Ananjeva (2007). This taxon is generally a sand dwelling ecotype. P. frontalis Strauch 1876: 15.—Type locality: locality “Ordos dicta” [= Ordos Desert, Nei Mongol Autonomous Region, China]. This taxon is generally thought of as a gravel dwelling ecotype, and here considered a synonym of P. przewalskii (see below). A lack of genetic diversity between populations from these taxa (two populations each), confirm that only the name P. przewalskii is valid, whereas P. frontalis is a synonym of P. przewalskii (following Gozdzik & Fu 2009). This is supported by nuclear encoded allozyme data, and mt-DNA data. Alternative names exist as reviewed in Barabanov & Ananjeva (2007), but without detailed sampling of type localities, the validity of alternate names cannot be addressed. Across the 25 allozyme loci sampled here, we detected no fixed differences among the four populations of P. przewalskii, indicating a fair amount of gene flow between the populations. The average mt-DNA pair-wise uncorrected sequence distances between population 2 and the clade containing populations 1, 3, and 4 is 3.64% (range 3.41–3.79%). The average pair-wise comparison within the clade containing populations 1, 3 and 4 is 0.57% (range 0.51–0.63%). These results match the phylogenetic topology discovered in the mt-DNA parsimony analysis, with population 2 placed in a sister position to the clade containing populations 1, 3 and 4 (bootstrap 100%, decay index 19). No resolution was discovered among the four P. przewalskii populations in the RAG- 1 DNA parsimony analysis. Urquhart et al. (2009) suggests a vicariant break among P. przewalskii populations roughly corresponding to the Helan Shan (mountains). Data reported here do not corroborate that hypothesis. Sampled are a single population east of the Yellow River and three populations west of the Yellow River, which flows north to south in this region. Population 2 is sampled on the east side of the river, and population 3 is sampled on the west side of the river, on banks directly across from each other in Shapotou, Ningxia Autonomous Region, China. Population 2, the only population on the east side of the Yellow River is the most divergent, with populations 1, 3 and 4 on the west side of the river nearly identical in our sampling. Population 1 (Wujiachuan, Gansu Province, China) is west of the Yellow River and further south than sampling conducted in Urquhart et al. (2009) and Wang & Fu (2004), yet nearly identical to other populations sampled west of the Yellow River. In our sampling, only population 4 lies west of the Helan Shan (Wuwei, Gansu Province, China) in the Tengger Desert, the type locality of P. przewalskii. Sampling in this study suggests the Yellow River could be responsible for a genetic break in P. przewalskii populations, and not the Helan Shan, though more detailed, fine-scale sampling and analysis will need to be done to confirm this hypothesis. In the Zhongwei region of Ningxia Autonomous Region, there is an indication that east and west haplotypes as described above occur in the same locality, which could indicate a point of hybridization, but further detailed work is needed (Wang & Fu 2004). Sampling conducted in similar areas in Wang & Fu (2004) and Urquhart et al. (2009) did not completely overlap, making conclusions between these studies and this study very difficult for comparative interpretation, further indicating additional work is needed. In sampling of the P. przewalskii complex in Jin & Brown (2013), populations to the east are grouped, and are referred to as P. frontalis, with populations to the west grouping and referred to as P. przewalskii, not conflicting with results reported here. 6). Use of Names in the Northern Tibetan Plateau, Published as part of Macey, Robert, Schulte, James A., Ananjeva, Natalia B., Van, Erik T., Wang, Yuezhao, Orlov, Nikolai, Shafiei, Soheila, Robinson, Michael D., Dujsebayeva, Tatjana, Freund, Gabriel S., Fischer, Clayton M., Liu, David & Papenfuss, Theodore J., 2018, A molecular phylogenetic hypothesis for the Asian agamid lizard genus Phrynocephalus reveals discrete biogeographic clades implicated by plate tectonics, pp. 1-81 in Zootaxa 4467 (1) on pages 34-38, DOI: 10.11646/zootaxa.4467.1.1, http://zenodo.org/record/1442134, {"references":["Anderson, J. (1872) On some Persian, Himalayan, and other Reptiles. Proceedings of the Zoological Society of London, 1872 (2), 371 - 404.","Blanford, W. T. (1876) List of Reptilia and Amphibia collected by the late Dr. Stoliczka in Kashmir, Ladak, Eastern Turkestan and Wakhan, with descriptions of new species. Journal of the Asiatic Society of Bengal, 44 (3), 191 - 196.","Haas, G. (1957) Some amphibians and reptiles from Arabia. Proceedings of the California Academy of Sciences, 29 (3), 47 - 86.","Bogdanov, O. P., Atayev, Ch. & Shammakov, S. (1974) The finding of Phrynocephalus maculatus in the USSR. Zoologicheskij Zhurnal, Moscow, 53 (2), 304 - 305. [in Russian]","Shenbrot, G. I. & Semenov (as Semyonov), D. V. (1990) New species of the genus Phrynocephalus (Reptilia, Agamidae) from Turkmenia. Zoologichesky Zhurnal, Moscow, 69 (9), 154 - 156. [in Russian with English summary]","Chernov, S. A. (1948) Reptiles - Reptilia. In: Pavlovsky, E. N. & Vinogradova, B. S. (Eds.), The Animals of the USSR. Fol. 2. The desert zone. USSR Academy of Sciences, Moscow-Leningrad, pp. 127 - 161. [in Russian]","Golubev, M. L. (1998) A new subspecies of Phrynocephalus ornatus Boulenger (Reptilia: Agamidae) from Eastern Iran, with a key to South-Western and Middle Asian microphrynocephalids. Hamadryad, 23 (2), 162 - 168.","Boulenger, G. A. (1887) Catalogue of the Lizards in the British Museum (Natural History), Second Edition. Fol. 3. Lacertidae, Gerrhosauridae, Scincidae, Anelytropidae, Dibamidae, Chamaeleontidae. Trustees of the British Museum, London, 575 pp.","Eichwald, E. (1831) Zoologia specialis quam expositis animalibus tum vivis, tum fossilibus potissimum Rossiae in universum, et Poloniae in specie, in usum lectionum publicarum in Universitate Caesarea Filnensi habendarum. Pars posterior. Josephi Zawadzki, Vilnae, 396 pp.","Golubev, M. L. (1991) About the name Agama ocellata Lichtenstein in Eversmann, 1823 (Reptilia, Agamidae) with redescription of the types. Herpetological Researches, Leningrad, 1, 12 - 17. [in Russian with English summary]","Nikolsky, A. M. (1899) Reptiles and amphibians of the Turkestan general-governorship (Herpetologia Turanica). In: Fedschenko, A. P. (Ed.), Travel to the Turkestan of the member-founder of the A. P. Fedchenko Society, accomplished on behalf of the Imperial Society of Amateurs of natural history, anthropology, and ethnography. Zoogeographical Investigations. University Publishing, Moscow, Issue 23, Vol. 2, part 7, pp. 21 - 84. [in Russian]","Dunayev, E. A. (1995) Reviewed description of the types of Phrynocephalus strauchi Nikolsky, 1899 (Squamata, Agamidae) and materials on the history of its study, distribution, and variability. Russian Journal of Herpetology, 2 (2), 87 - 94.","Darevsky, I. S., Rustamov, A. K. & Shammakov, S. (1976) Status and distribution of Phrynocephalus reticulatus Eichwald (Sauria, Agamidae) in Central Asia. Teoreticheskije i prikladnyije aspekty okhrany prirody i okhotovedenija, Moscow, 84, 113 - 119. [in Russian]","Bannikov, A. G., Darevsky, I. S., Ischchenko, V. G., Rustamov, A. K. & Szczerbak, N. N. (1977) Opredelitel' Zemnovodnikh i Presmikayushchikhsya Fauni SSSR [Guide to the Reptile and Amphibian Fauna of the USSR]. Prosveshchenie, Moscow, 415 pp., figs. 1 - 108, 135 maps, 32 col. pls. [in Russian]","Solovyeva, E. N., Poyarkov, N. A., Dunayev, E. A., Nazarov, R. A., Lebedev, V. S. & Bannikova, A. A. (2014) Phylogenetic relationships and subgeneric taxonomy of Toad Headed Agamas Phrynocephalus (Reptilia, Squamata, Agamidae) as determined by mitochondrial DNA sequencing. Doklady Biological Sciences, 455, 119 - 124. https: // doi. org / 10.7868 / S 0869565214100260","Boettger, O. (1888) Uber die Reptilien und Batrachier Transcaspiens. Zoologischer Anzeiger, Leipzig, 11 (279), 259 - 263.","Nikolsky, A. M. (1905) Reptiles and amphibians of the Russian Empire (Herpetologica Rossica). Memoires de l'Academie Imperiale des Sciences de St. - Petersbourg, 17 (1), 518 pp. [in Russian]","Solovyeva, E. N., Dunayev, E. A. & Poyarkov, N. A. (2012) Interspecific taxonomy of Sunwatcher Toadhead Agama species complex (Phrynocephalus helioscopus, Squamata). Zoologicheski Zhurnal, Moscow, 91 (11), 1377 - 1396. [in Russian with English summary]","Pallas, P. S. (1771) Reise durch verschiedene Provinzen des Russischen Reichs. Bd. I. St. Petersburg: Kayserliche Acade-mie der Wissenschaften, 540 pp.","Mertens, R. & Muller, L. (1928) Liste der Amphibien und Reptilien Europas. Abhandlungen der Senckenbergischen Naturforschenden Gesellschaft, 41 (1), 1 - 62.","Weisrock, D. W., Macey, J. R., Ugurtas, I. H., Larson, A. & Papenfuss, T. J. (2001) Molecular phylogenetics and historical biogeography among salamandrids of the \" true \" salamander clade: Rapid branching of numerous highly divergent lineages with the rise of Anatolia in Mertensiella luschani. Molecular Phylogenetics and Evolution, 18 (3), 434 - 448. https: // doi. org / 10.1006 / mpev. 2000.0905","Bedriaga, J. (1907) Amphibien und Reptilien (Reptilia Przewalskiana). Wissenschaftliche Resultate der von N. M. Przewalski nach Central-Asien unternommenen Reisen. Zoologischer Theil, Bd. 3, Abth. 1, Lfrg. 2, St. Petersburg, pp. 71 - 278.","Bedriaga, J. (1909) Amphibien und Reptilien (Reptilia Przewalskiana). In: Wissenschaftliche Resultate der von N. M. Przewalski nach Central-Asien unternommenen Reisen. Zoologischer Theil, Bd. 3, Abth. 1, Lfrg. 3, St. Petersburg, pp. 279 - 502.","Tsarevsky (as Carevskij), S. (1926) Contributions sur la systematique et la distribution des lezards du genre Phrynocephalus (Reptilia). Comptes Rendus de l'Academie des Sciences de l'URSS, 1926, 211 - 214. [in Russian with French title]","Zhao, K. (1979) A survey of the classification and distribution of the toad-headed agamids (Phrynocephalus) in China. Acta Scientiarum Naturalium Universitatis Intramongolicae, Hohhot, 1979 (2), 111 - 121. [in Chinese with English summary]","Strauch, A. A. (1876) Part III. Reptilia and Amphibia. In: Imperial Russian Geographic Society (Ed.), Przevalsky N. M., Mongolia and the Tangut Lands, a Three-Year Journey in Eastern High Asia. Fol. II. Reptiles and Amphibians. V. S. Balashov printing house, St. Petersburg, pp. 1 - 55. [in Russian]","Barabanov, A. V. & Ananjeva, N. B. (2007) Catalogue of the available scientific species-group names for lizards of the genus Phrynocephalus Kaup, 1825 (Reptilia, Sauria, Agamidae). Zootaxa, 1399, 1 - 56. https: // doi. org / 10.11646 / zootaxa. 1399.1.1","Urquhart, J., Wang, Y. & Fu, J. (2009) Historical vicariance and male-mediated gene flow in the toad-headed lizards Phrynocephalus przewalskii. Molecular Ecology, 18 (17), 3714 - 3729. https: // doi. org / 10.1111 / j. 1365 - 294 X. 2009.04310. x","Wang, Y. & Fu, J. (2004) Cladogenesis and vicariance patterns in the toad-headed lizard Phrynocephalus versicolor species complex. Copeia, 2004 (2), 199 - 206. https: // doi. org / 10.1643 / CG- 03 - 082 R 1","Jin, Y. - T. & Brown, R. P. (2013) Species history and divergence times of viviparous and oviparous Chinese toad-headed sand lizards (Phrynocephalus) on the Qinghai-Tibetan Plateau. Molecular Phylogenetics and Evolution, 68 (2), 259 - 268. https: // doi. org / 10.1016 / j. ympev. 2013.03.022","Bedriaga, J. (\" 1905 \" 1906) Verzeichnis der von der Central-Asiatischen Expedition unter Stabs-Kapitan W. Roborowski in den Jahren 1893 - 1895 gesammelten Reptilien. Annuaire du Musee Zoologique de l'Academie Imperiale des Sciences de St. - Petersbourg, 10 (3 - 4), 159 - 200.","Jiang, Y. - M., Huang, Q. - Y. & Zhao, E. - M. (1980) A new subspecies of Phrynocephalus vlangalii (Strauch) and preliminary observation on its ecology. Acta Zoologica Sinica, Beijing, 26 (2), 178 - 183. [in Chinese with English summary]","Jin, Y. - T., Brown, R. P. & Liu, N. - F. (2008) Cladogenesis and phylogeography of the lizard Phrynocephalus vlangalii (Agamidae) on the Tibetan plateau. Molecular Ecology, 17 (8), 1971 - 1982. https: // doi. org / 10.1111 / j. 1365 - 294 X. 2008.03721. x","Pang, J., Wang, Y., Zhong, Y., Hoelzel, A. R., Papenfuss, T. J., Zeng, X., Ananjeva, N. B. & Zhang, Y. - P. (2003) A phylogeny of Chinese species in the genus Phrynocephalus (Agamidae) inferred from mitochondrial DNA sequences. Molecular Phylogenetics and Evolution, 27 (3), 398 - 409. https: // doi. org / 10.1016 / S 1055 - 7903 (03) 00019 - 8","Qi, Y., Yang, W., Lu, B. & Fu, J. (2013) Genetic evidence for male-biased dispersal in the Qinghai toad-headed agamid Phrynocephalus vlangalii and its potential link to individual social interactions. Ecology and Evolution, 3 (5), 1219 - 1230. https: // doi. org / 10.1002 / ece 3.532","Wu, Y., Fu, J., Yue, B. & Yin, Q. (2015) An atypical reproductive cycle in a common viviparous Asia Agamid Phrynocephalus vlangalii. Ecology and Evolution, 5 (21), 5138 - 5147. https: // doi. org / 10.1002 / ece 3.1783","Staub, N. L., Brown, C. W. & Wake, D. B. (1995) Patterns of growth and movements in a population of Ensatina eschscholtzii platensis (Caudata: Plethodontidae) in the Sierra Nevada, California. Journal of Herpetology, 29 (4), 593 - 599. https: // doi. org / 10.2307 / 1564743","Jackman, T. R. & Wake, D. B. (1994) Evolutionary and historical analysis of protein variation in the blotched forms of salamanders of the Ensatina complex (Amphibia: Plethodontidae). Evolution, 48 (3), 876 - 897. https: // doi. org / 10.1111 / j. 1558 - 5646.1994. tb 01369. x","Wake, D. B. & Schneider, C. J. (1998) Taxonomy of the plethodontid salamander genus Ensatina. Herpetologica, 54 (2), 279 - 298.","Noble, D. W. A., Qi, Y. & Fu, J. (2010) Species delineation using Bayesian model-based assignment tests: A case study using Chinese toad-headed agamas (genus Phrynocephalus). BMC Evolutionary Biology, 2010 (10), 197. https: // doi. org / 10.1186 / 1471 - 2148 - 10 - 197"]}

F. Phrynocephalus Species Diversity as Related to Geology, Habitat and Size Two regions show high species numbers in close proximity. Six species are found on the northern side of the Kopet-Dagh (mountains) along the southern margin of the Caspian Basin in Turkmenistan. Species that inhabit hard substrates are P. rossikowi, P. raddei, P. bannikovi, P. golubewii, and P. turcomanus, with P. interscapularis and P. mystaceus occurring in soft substrates (Table 5, Fig. 16). Among these species there are major size differences (Table 6). Six species are also found at the juncture of the Helmand Basin of southern Afghanistan with the Baluchistan Plateau including the base of the Sulaiman Range of southern Pakistan. Species that inhabit hard substrates are P. maculatus and P. scutellatus (Table 5, Fig. 16). Smaller species that inhabit soft substrates are P. clarkorum, P. ornatus, and P. luteoguttatus (Clade C in Figs. 4 and 8). A larger species inhabiting soft substrates in large floating sand dunes is P. euptilopus, which was not sampled in this study even though repeated efforts were made in both Afghanistan and Pakistan by the last author. Among these species there are major size differences (Table 6). Both of these examples share a plate tectonic past around the northern migration of the Arabian Plate and its impact on ancient tectonic plates to the north in Eurasia. The Kopet-Dagh (mountains), on the northern margin of the Iranian Plateau. descends into the Caspian Basin. This region of the Caspian Basin experienced the most dramatic changes via tectonic uplift, erosion, and sea floor drying of the Paratethys Sea creating unique and diversified habitats (see Figs. 3, 9–14; Table 4). Additionally, the Helmand Block of southern Afghanistan is being pushed south by impinging Arabia and further northern invasion of the Indian Plate (Fig. 9). This has created uplift in the Gulf of Oman creating the push-back of the Makron in southern Pakistan (Figs. 9–12). It is dynamic tectonics of this nature that created a series of habitats leading to speciation among Phrynocephalus. As adaptive niches were created, size diversity, morphological specializations, background matching, and defensive mimicry developed (Plates I – VIII). Future work on Phrynocephalus will need to take into account these aspects with particular attention to plate tectonic induced events in order to investigate phyletic patterns, taxonomy, and evolutionary diversity among one of the world’s most interesting and confusing lizard genera., Published as part of Macey, Robert, Schulte, James A., Ananjeva, Natalia B., Van, Erik T., Wang, Yuezhao, Orlov, Nikolai, Shafiei, Soheila, Robinson, Michael D., Dujsebayeva, Tatjana, Freund, Gabriel S., Fischer, Clayton M., Liu, David & Papenfuss, Theodore J., 2018, A molecular phylogenetic hypothesis for the Asian agamid lizard genus Phrynocephalus reveals discrete biogeographic clades implicated by plate tectonics, pp. 1-81 in Zootaxa 4467 (1) on page 51, DOI: 10.11646/zootaxa.4467.1.1, http://zenodo.org/record/1442134

F. Phrynocephalus Species Diversity as Related to Geology, Habitat and Size Two regions show high species numbers in close proximity. Six species are found on the northern side of the Kopet-Dagh (mountains) along the southern margin of the Caspian Basin in Turkmenistan. Species that inhabit hard substrates are P. rossikowi, P. raddei, P. bannikovi, P. golubewii, and P. turcomanus, with P. interscapularis and P. mystaceus occurring in soft substrates (Table 5, Fig. 16). Among these species there are major size differences (Table 6). Six species are also found at the juncture of the Helmand Basin of southern Afghanistan with the Baluchistan Plateau including the base of the Sulaiman Range of southern Pakistan. Species that inhabit hard substrates are P. maculatus and P. scutellatus (Table 5, Fig. 16). Smaller species that inhabit soft substrates are P. clarkorum, P. ornatus, and P. luteoguttatus (Clade C in Figs. 4 and 8). A larger species inhabiting soft substrates in large floating sand dunes is P. euptilopus, which was not sampled in this study even though repeated efforts were made in both Afghanistan and Pakistan by the last author. Among these species there are major size differences (Table 6). Both of these examples share a plate tectonic past around the northern migration of the Arabian Plate and its impact on ancient tectonic plates to the north in Eurasia. The Kopet-Dagh (mountains), on the northern margin of the Iranian Plateau. descends into the Caspian Basin. This region of the Caspian Basin experienced the most dramatic changes via tectonic uplift, erosion, and sea floor drying of the Paratethys Sea creating unique and diversified habitats (see Figs. 3, 9–14; Table 4). Additionally, the Helmand Block of southern Afghanistan is being pushed south by impinging Arabia and further northern invasion of the Indian Plate (Fig. 9). This has created uplift in the Gulf of Oman creating the push-back of the Makron in southern Pakistan (Figs. 9–12). It is dynamic tectonics of this nature that created a series of habitats leading to speciation among Phrynocephalus. As adaptive niches were created, size diversity, morphological specializations, background matching, and defensive mimicry developed (Plates I – VIII). Future work on Phrynocephalus will need to take into account these aspects with particular attention to plate tectonic induced events in order to investigate phyletic patterns, taxonomy, and evolutionary diversity among one of the world’s most interesting and confusing lizard genera.

E. Phrynocephalus Size Diversity Phrynocephalus show a variety of snout vent lengths and tail lengths, (Table 6, Appendix 5). Body size per sample ranged from 106 mm for P. mystaceus to 35 mm for P. vindumi and P. interscapularis. Tail length per sample ranges from 133 mm for P. maculatus population 1 to 35 mm for P. luteoguttatus population 2. P. mystaceus and P. interscapularis are sympatric in the Caspian Basin, and habitat partition with P. mystaceus in large floating sand dunes, with P. interscapularis living on the edge of those large sand dunes, which may revolve around ecotype evolution in a predator-prey relationship. In northwestern Iran P. vindumi replaces the role of P. interscapularis, and is sympatric with P. mystaceus. Tail length appears to be the most sexually dimorphic characteristic. ......continued on the next page Snout vent lengths for clay ecotypes range from 62 mm for P. przewalskii population 1 to 49 mm for P. versicolor, with tail lengths ranging from 90 mm for P. przewalskii population 1 to 49 mm for P. persicus. The snout vent length for gravel ecotypes range from 64 mm for P. vlangalii population 1 to 40 mm for P. rossikowi, with tail lengths ranging from 90 mm for P. longicaudatus to 42 mm for P. theobaldi. Snout vent lengths for dry lakebed ecotypes range from 90 mm for P. maculatus population 1 to 60 mm for P. golubewii, with tail length ranging from 133 mm for P. maculatus population 1 to 78 mm for P. maculatus population 2. Snout vent lengths for small sand dune ecotypes range from 61 mm for P. vlangalii population 2 to 35 mm for both P. vindumi and P. interscapularis, with tail length ranging from 80 mm for P. przewalskii population 2 to 35 mm for P. luteoguttatus population 2. Snout vent lengths for large sand dune ecotypes range from 106 mm for P. mystaceus populations to 59 mm for P. salenskyi population 1, with tail lengths ranging from 110 mm for P. mystaceus population 1 to 70 mm for P. salenskyi population 1. The largest Phrynocephalus occupy large floating sand dunes, and the smallest Phrynocephalus species are found on the fringes of large floating sand dunes in the Caspian Basin and northern Iran. P. mystaceus occupying large sand dunes in these habitats coexists with the two smallest species that occupy the fringes of those sand dunes, P. interscapularis in the Caspian Basin, and P. vindumi in northwestern Iran. It has not escaped our attention that size and habitat partitioning may have occurred in these regions because it is likely that smaller species (P. interscapularis or P. vindumi) serve as a food source for larger species (P. mystaceus). Further work is needed for phylogenetic assessment of size evolution among Phrynocephalus species and populations considering the sexually dimorphic nature of this genus, both with body size and tail length., Published as part of Macey, Robert, Schulte, James A., Ananjeva, Natalia B., Van, Erik T., Wang, Yuezhao, Orlov, Nikolai, Shafiei, Soheila, Robinson, Michael D., Dujsebayeva, Tatjana, Freund, Gabriel S., Fischer, Clayton M., Liu, David & Papenfuss, Theodore J., 2018, A molecular phylogenetic hypothesis for the Asian agamid lizard genus Phrynocephalus reveals discrete biogeographic clades implicated by plate tectonics, pp. 1-81 in Zootaxa 4467 (1) on pages 48-51, DOI: 10.11646/zootaxa.4467.1.1, http://zenodo.org/record/1442134

The spontaneous deamination of cytosine is a major source of transitions from C•G to T•A base pairs, which account for half of known pathogenic point mutations in humans. The ability to efficiently convert targeted A•T base pairs to G•C could therefore advance the study and treatment of genetic diseases. The deamination of adenine yields inosine, which is treated as guanine by polymerases, but no enzymes are known to deaminate adenine in DNA. Here we describe adenine base editors (ABEs) that mediate the conversion of A•T to G•C in genomic DNA. We evolved a transfer RNA adenosine deaminase to operate on DNA when fused to a catalytically impaired CRISPR-Cas9 mutant. Extensive directed evolution and protein engineering resulted in seventh-generation ABEs that convert targeted A•T base pairs efficiently to G•C (approximately 50% efficiency in human cells) with high product purity (typically at least 99.9%) and low rates of indels (typically no more than 0.1%). ABEs introduce point mutations more efficiently and cleanly, and with less off-target genome modification, than a current Cas9 nuclease-based method, and can install disease-correcting or disease-suppressing mutations in human cells. Together with previous base editors, ABEs enable the direct, programmable introduction of all four transition mutations without double-stranded DNA cleavage.

We recently developed base editing, the programmable conversion of target C:G base pairs to T:A without inducing double-stranded DNA breaks (DSBs) or requiring homology-directed repair using engineered fusions of Cas9 variants and cytidine deaminases. Over the past year, the third-generation base editor (BE3) and related technologies have been successfully used by many researchers in a wide range of organisms. The product distribution of base editing-the frequency with which the target C:G is converted to mixtures of undesired by-products, along with the desired T:A product-varies in a target site-dependent manner. We characterize determinants of base editing outcomes in human cells and establish that the formation of undesired products is dependent on uracil N-glycosylase (UNG) and is more likely to occur at target sites containing only a single C within the base editing activity window. We engineered CDA1-BE3 and AID-BE3, which use cytidine deaminase homologs that increase base editing efficiency for some sequences. On the basis of these observations, we engineered fourth-generation base editors (BE4 and SaBE4) that increase the efficiency of C:G to T:A base editing by approximately 50%, while halving the frequency of undesired by-products compared to BE3. Fusing BE3, BE4, SaBE3, or SaBE4 to Gam, a bacteriophage Mu protein that binds DSBs greatly reduces indel formation during base editing, in most cases to below 1.5%, and further improves product purity. BE4, SaBE4, BE4-Gam, and SaBE4-Gam represent the state of the art in C:G-to-T:A base editing, and we recommend their use in future efforts.

Automatic localization and labeling of vertebra in 3D medical images plays an important role in many clinical tasks, including pathological diagnosis, surgical planning and postoperative assessment. However, the unusual conditions of pathological cases, such as the abnormal spine curvature, bright visual imaging artifacts caused by metal implants, and the limited field of view, increase the difficulties of accurate localization. In this paper, we propose an automatic and fast algorithm to localize and label the vertebra centroids in 3D CT volumes. First, we deploy a deep image-to-image network (DI2IN) to initialize vertebra locations, employing the convolutional encoder-decoder architecture together with multi-level feature concatenation and deep supervision. Next, the centroid probability maps from DI2IN are iteratively evolved with the message passing schemes based on the mutual relation of vertebra centroids. Finally, the localization results are refined with sparsity regularization. The proposed method is evaluated on a public dataset of 302 spine CT volumes with various pathologies. Our method outperforms other state-of-the-art methods in terms of localization accuracy. The run time is around 3 seconds on average per case. To further boost the performance, we retrain the DI2IN on additional 1000+ 3D CT volumes from different patients. To the best of our knowledge, this is the first time more than 1000 3D CT volumes with expert annotation are adopted in experiments for the anatomic landmark detection tasks. Our experimental results show that training with such a large dataset significantly improves the performance and the overall identification rate, for the first time by our knowledge, reaches 90 %.

AimsTo examine the safety and effectiveness of buprenorphine+naloxone sublingual tablets (BUP, as Suboxone(®) ) provided after administration of extended-release injectable naltrexone (XR-NTX, as Vivitrol(®) ) to reduce cocaine use in participants who met DSM-IV criteria for cocaine dependence and past or current opioid dependence or abuse.MethodsThis multi-centered, double-blind, placebo-controlled study, conducted under the auspices of the National Drug Abuse Treatment Clinical Trials Network, randomly assigned 302 participants at sites in California, Oregon, Washington, Colorado, Texas, Georgia, Ohio, New York and Washington DC, USA to one of three conditions provided with XR-NTX: 4mg/day BUP (BUP4, n=100), 16mg/day BUP (BUP16, n=100, or no buprenorphine (placebo; PLB, n=102). Participants received pharmacotherapy for 8weeks, with three clinic visits per week. Cognitive behavioral therapy was provided weekly. Follow-up assessments occurred at 1 and 3months post-intervention. The planned primary outcome was urine drug screen (UDS)-corrected, self-reported cocaine use during the last 4weeks of treatment. Planned secondary analyses assessed cocaine use by UDS, medication adherence, retention and adverse events.ResultsNo group differences were found between groups for the primary outcome (BUP4 versus PLB, P=0.262; BUP16 versus PLB, P=0.185). Longitudinal analysis of UDS data during the evaluation period using generalized linear mixed equations found a statistically significant difference between BUP16 and PLB [P=0.022, odds ratio (OR)=1.71] but not for BUP4 (P=0.105, OR=1.05). No secondary outcome differences across groups were found for adherence, retention or adverse events.ConclusionsBuprenorphine+naloxone, used in combination with naltrexone, may be associated with reductions in cocaine use among people who meet DSM-IV criteria for cocaine dependence and past or current opioid dependence or abuse.

AimsTo compare long-term outcomes among participants randomized to buprenorphine or methadone.Design, setting and participantsFollow-up was conducted in 2011-14 of 1080 opioid-dependent participants entering seven opioid treatment programs in the United States between 2006 and 2009 and randomized (within each program) to receive open-label buprenorphine/naloxone or methadone for up to 24 weeks; 795 participants completed in-person interviews (~74% follow-up interview rate) covering on average 4.5 years.MeasurementsOutcomes were indicated by mortality and opioid use. Covariates included demographics, site, cocaine use and treatment experiences.FindingsMortality was not different between the two randomized conditions, with 23 (3.6%) of 630 participants randomized to buprenorphine having died versus 26 (5.8%) of 450 participants randomized to methadone. Opioid use at follow-up was higher among participants randomized to buprenorphine relative to methadone [42.8 versus 31.7% positive opioid urine specimens, P < 0.01, effect size (h) = 0.23 (0.09, 0.38); 5.8 days versus 4.4 days of past 30-day heroin use, P < 0.05, effect size (d) = 0.14 (0.00, 0.28)]. Opioid use during the follow-up period by randomization condition was also significant (F(7,39,600) = 3.16; P < 0.001) due mainly to less treatment participation among participants randomized to buprenorphine than methadone. Less opioid use was associated with both buprenorphine and methadone treatment (relative to no treatment); no difference was found between the two treatments. Individuals who are white or used cocaine at baseline responded better to methadone than to buprenorphine.ConclusionsThere are few differences in long-term outcomes between buprenorphine and methadone treatment for opioid dependence, and treatment with each medication is associated with a strong reduction in opioid use.

Main figures are referred to as Fig. # in the article. Figure legends are supplied on the next page. Supplementary figures with figure legends are referred to as Supplementary Fig. # in the article.

Primary Liver Tumors Hepatocellular carcinoma (HCC) is the most common primary malignancy of the liver; its incidence is increasing worldwide. It ranks as the sixth most common tumor and third most common cause of cancer-related mortality (1,2). Primary liver tumors include HCC and intrahepatic cholangiocarcinoma. Surgical resection is preferred over transplantation and is considered potentially curative in patients with resectable HCC and normal liver function (3). Transplantation is considered the gold standard for patients with unresectable HCC and whose disease is within the Milan criteria (4). Resection and transplantation have limited roles, given advanced disease (chronic liver disease and/or tumor extent) at presentation and limited organ availability (5–7). Chemoembolization and radiofrequency ablation represent standard therapies in treating patients and serve as a bridge to transplantation in selected patients (8,9). Radioembolization has an emerging role in “bridging” patients within criteria by delaying tumor progression. It has also been shown to downstage disease beyond the Milan, to within, transplant criteria (10–12). A recent study has demonstrated that radioembolization leads to longer time-to-progression and better toxicity profile when compared with chemoembolization (13). Patients with macrovascular tumor involvement have also exhibited evidence of clinical benefit after radioembolization (14).