Campylobacter jejuni is a leading foodborne pathogen worldwide. Despite the fastidious nature of C. jejuni growth, increasing numbers of human campylobacteriosis suggest that C. jejuni may possess unique mechanisms to survive under various stress conditions. C. jejuni possesses only three sigma factors  and lacks stress-defense sigma factors. Since FliA and RpoD are dedicated to flagella synthesis and housekeeping, respectively, in this study, we investigated the role of RpoN in C. jejuni's defense against various stresses.rpoN mutant was compared with the wild-type C. jejuni under various stress conditions. While the growth of the rpoN mutant was as comparably as that of the wild type in shaking cultures, the rpoN mutant exhibited significant survival defects when cultured statically. The rpoN mutant was more sensitive to osmotic stress (0.8% NaCl) with abnormally-elongated cell morphology. Compared to the wile type, the rpoN mutant was more susceptible to acid stress (pH 5) and more resistant to hydrogen peroxide. However, the rpoN mutation had little effect on the resistance of C. jejuni to alkaline pH, heat, cold and antimicrobials.Survivability of an C. jejuni's defense against various stresses which this bacterial pathogen may encounter during transmission to and infection of humans.The results demonstrate that RpoN plays an important role in  Campylobacter is a leading cause of human gastroenteritis and is annually responsible for estimated 400-500 million cases of human infection worldwide [Campylobacter species, C. jejuni is the major human-pathogenic species, accounting for more than 90% of human campylobacteriosis [C. jejuni infections are primarily caused by the consumption of contaminated poultry, because the spillage of intestinal content containing a large number of C. jejuni during slaughter can contaminate cooling water, knives and poultry meat in the processing plant [C. jejuni from animals, primarily poultry, to humans, this important zoonotic foodborne pathogen encounters various stresses, such as non-growth temperatures, starvation, hypo- and hyper-osmotic stress, and desiccation [Campylobacter is a fastidious bacterium, human campylobacteriosis cases have significantly increased presumably due to the ability of this pathogen to survive under harsh environmental conditions [Campylobacter spp. and the ability to transform into a viable-but-non-culturable state may enhance its adaptability to unfavorable growth conditions [Campylobacter under stress conditions [Campylobacter.orldwide . Among Cteriosis ,3. Humaniccation . Despitenditions -10 in adnditions . For exanditions ,8. AdditEscherichia coli, Salmonella, Pseudomonas and Vibrio significantly impair bacterial ability to resist various stresses, such as starvation, low pH, oxidative stress, hyperosmolarity, heat and cold [E. coli, RpoS is involved in resistance to high osmolarity in stationary-phase cells and survival in cold-shock by regulating one set of RpoS-dependent genes, including otsA and otsB, which are necessary for synthesis of internal trehalose as an osmoprotectant and important for survival at low temperature [E. coli by modulating gadC, a gene involved in the glutamate-dependent low pH-resistance, hdeAB, encoding pH-regulated periplasmic chaperons, and cfa, a gene for cycloporpane fatty acid synthesis [E. coli, Pseudomonas and Salmonella [Salmonella reduces bacterial survival and growth in macrophages by the loss of RpoE-dependent gene expression such as htrA, a gene required for oxidative stress resistance [Listeria and Bacillus [In many bacterial species, alternative sigma factors play an important role in regulation of stress-defense genes under hostile environmental conditions . Becauseand cold -17. In Eperature ,19. In aynthesis . As anotlmonella ,22. The sistance ,24. To dsistance . The altBacillus ,17. The Bacillus .C. jejuni genome sequence revealed that C. jejuni does not possess stress-related sigma factors and has only three sigma factors  [rpoN mutation affects the formation of flagellar secretory apparatus [C. jejuni [C. jejuni. In this work, we investigated the effect of rpoN mutation on the resistance of C. jejuni under various environmental stresses.Although alterative sigma factors involved in stress defense are available in many bacteria, the nd RpoN) . RpoD annd RpoN) ; thus, tpparatus , and thepparatus . In addi. jejuni . Since RrpoN mutant and a complementation strain, bacterial motility was determined to verify the success of the rpoN mutation, because an rpoN mutation is known to make Campylobacter aflagellate and non-motile [rpoN mutant showed significant defects in motility with complete restoration by complementation . These results show that RpoN affects the survival of C. jejuni under aeration-limited static culture conditions.After construction of an n-motile ,33. Consg Figure ; howevery Figure . To verie Figure , suggestCampylobacter to various osmolytes [rpoN mutant to osmotic stress in this study. When grown on Mueller-Hinton (MH) agar plates containing a high concentration (0.8%) of NaCl, the rpoN mutant exhibited significant growth defects , suggesting the rpoN mutant suffers more osmotic stress than the wild type under the same stress condition. We used transmission electron microscopy (TEM) to investigate bacterial morphology under the osmotic stress. Interestingly, 79.3 ± 9.0% of rpoN mutant cells were abnormally elongated after exposure to osmotic stress. The rpoN mutant was approximately several times longer (approximately > 5 μm) than the wild type in the presence of 0.8% NaCl, and the morphological change in the rpoN mutant was restored by complementation . This suggests that the survival defect of the rpoN mutant under the static culture condition was not caused by its loss of motility. Instead, the survival defects of the rpoN mutant may be related to the ability to respire under oxygen-limited conditions, because the levels of oxygen dissolved in broth media are lower in static culture than shaking culture. C. jejuni rarely encounters an active aeration system in its natural habitat , which may be more similar to static culture than shaking culture. The rpoN mutation significantly impairs C. jejuni's ability to colonize the intestines of chicken because of poor attachment of the aflagellated rpoN mutant to the epithelial cells in the intestines [rpoN mutation, the survival defects in the static culture condition may also be responsible for the colonization defect of the rpoN mutant. Molecular mechanisms of the survival defect in the rpoN mutant are currently being investigated in our group.Many bacterial pathogens have multiple sigma factors to regulate gene expression efficiently in response to environmental changes . In thisy Figure , whereastestines ,36. In aListeria monocytogenes [rpoN mutant and the wild type. NaCl is a common food additive used to inhibit microbial growth, and significantly impairs the culturability of Campylobacter at concentrations greater than 2.0% [C. jejuni was substantially inhibited even by 0.8% NaCl  NaCl concentration, whereas the rpoN mutant was significantly elongated compared to the wild type at the same NaCl concentration hydration under osmotic stress and may influence bacterial gene expression; however, understanding its molecular mechanisms still awaits further investigation.Because RpoN is known to be important for osmotolerance in some bacteria, such as ytogenes , resistahan 2.0% . In thisl Figure . TEM anan Figure . The morn Figure , suggesttrations . In Lacttibility . Althougmolality . The prerpoN mutant was highly susceptible to acidic stress (pH 5.5) compared to wild type , KatA , AhpC , Dps (DNA-binding protein from starved cells), the multidrug efflux pump CmeG, and PerR [rpoN mutant was more resistant to H2O2 than the wild type, and complementation restored the H2O2 susceptibility to the wild-type level  stress resulted in obvious morphological changes in the rpoN mutant  and acidic pH. Interestingly, the rpoN mutation rendered C. jejuni more resistant to H2O2 than the wild type. Notably, the rpoN mutant exhibited significant survival defects in the static culture conditions. Although understanding of molecular mechanisms for stress tolerance may exceed the scope of our present work, in this study, we provided new insights into the role of RpoN, one of the three sigma factors of C. jejuni, in the survivability of this bacterial pathogen under various stress conditions.As a zoonotic foodborne pathogen, C. jejuni 81-176 was used in this study. The strains, plasmids, and primers used in this study are listed in Table C. jejuni 81-176 and its derivatives were routinely grown at 42°C on MH agar plates or MH broth with shaking at 180 rpm under microaerobic condition  adjusted by the MART . To investigate the effect of rpoN disruption on C. jejuni growth, C. jejuni was cultured in 50 ml MH broth either in conical tubes without shaking or in Erlenmeyer flasks with shaking. Occasionally, culture media were supplemented with kanamycin (50 μg ml-1) or chloramphenicol (10 μg ml-1) where required.C. jejuni mutants were constructed with C. jejuni 81-176 as the parental strain by performing electroporation of suicide plasmids [cat) was amplified from pRY112 using primers of catF(SmaI) and catR(SmaI), and Vent Polymerase (New England Biolabs). To construct C. jejuni FMB1116, a DNA fragment containing rpoN and flanking region was amplified using primers rpoN_F and rpoN_R, and then ligated into SmaI-digested pUC19. The resultant plasmid was digested with SmiI, and then cat cassette was inserted into that digested site. The orientation of the cat cassette was confirmed by sequencing, and the plasmid in which the orientation of cat cassette was same to rpoN was designated as pUC-rpoN::cat. This plasmid was used as a suicide plasmid to construct C. jejuni FMB1116. For the rpoN complementation, an extra copy of rpoN was integrated into the chromosome by the methodology reported elsewhere [rpoN and its putative promoter region was amplified with rpoNC_F(XbaI) and rpoNC_R(XbaI) primers. The PCR product was digested with XbaI and cloned into pFMB, which carries rRNA gene cluster and a kanamycin resistance cassette. The constructed plasmid was delivered to the bacterial cell, FMB1116, by electroporation.plasmids . The antlsewhere . BrieflyC. jejuni cultured on MH agar plate with or without NaCl was absorbed onto a 400 mesh carbon-coated grid, negatively stained with 0.2% aqueous uranyl acetate (pH4.0), and observed in an EF-TEM  at an accelerating voltage of 80 kV.Bacterial cell suspension of each C. jejuni strains were inoculated into MH broth to an OD at 600 nm (OD600) of 0.1. After culturing to the early mid log phase (about 5 hr), OD600 was adjusted to 0.2. The aliquots of bacterial cells were exposed to several different stress conditions. The resistance to osmotic and pH shock was measured by culturing serially-diluted bacterial cells for 24 hr on MH agar plates containing 0.8% NaCl or at pH levels of 5.5 and 7.5. To test the susceptibility to oxidative stress, C. jejuni strains were exposed to the final concentration of 1 mM of H2O2 under microaerophilic condition for 1 hr. For heat and cold stresses, bacterial cells were incubated at 55°C and -20°C for 15 min or 1 hr, respectively. After exposure to each stress conditions, serially diluted culture aliquots were plated on MH agar and incubated at 42°C for 2 days to count viable cells. The viability was presented in percentage compared with the CFU of the sample without being exposed to stress.Minimal inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs) of erythromycin, cefotaxime, gentamicin, polymyxin B, rifampicin and ampicillin were determined by a microtitre broth dilution method as described previously ,50.The authors declare that they have no competing interests.SH, BJ, JY, and SR conceived and designed the study. SH carried out the experimental work and wrote the manuscript. JY designed the mutant construction. SH, BJ, and SR analyzed and interpreted the data. SR and BJ revised the manuscript critically for important intellectual content. All authors read and approved the final manuscript.rpoN mutantFigure S1. Loss of motility in the . The diameter of each motility zone was measured after 36 hr incubation of C. jejuni strains on 0.4% motility agar plates at 42°C.Click here for filerpoN mutation on C. jejuni's resistance to alkali, heat and cold stressesFigure S2. Effect of the . (A) Resistance to alkali stress. The growth under different pHs was examined by dotting 10 μl of serially-diluted bacterial cultures. pH 7 was used as a control. (B) Heat and cold resistance. Bacteria were exposed to 55°C and -20°C. After exposure, the viability changes were measured by dotting 10 μl of bacterial cultures on MH agar plates.Click here for filerpoN mutantTable S1. Antimicrobial susceptibility of the .Click here for file


Corynebacterium ulcerans may cause diphtheria in humans and caseous lymphadenitis in animals. We isolated nontoxigenic tox-bearing C. ulcerans from 13 game animals in Germany. Our results indicate a role for game animals as reservoirs for zoonotic C. ulcerans. Corynebacterium species C. diphtheriae, C. ulcerans, and C. pseudotuberculosis form the C. diphtheriae group, as shown by 16S rRNA gene sequence analysis (tox-encoded diphtheria toxin (DT)  25923 and the CAMP test by using Rhodococcus equi ATCC 33701 according to standard procedures on Columbia sheep blood agar   by using the amplification primers. Sequence analysis was performed by using the BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) sequence analysis tool. Additionally, coryneform isolates in which C. ulcerans was suspected were analyzed by using Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) and by using Biotyper version 3.3.1.0 . The database used (DB 4613) comprised spectra from 71 Corynebacterium species including C. diphtheriae, C. ulcerans, and C. pseudotuberculosis. For Fourier-transform infrared (FT-IR) spectroscopy, bacterial isolates were harvested and prepared as described . Both strands of the  spectra .C. ulcerans were isolated from 12 wild boars and 1 roe deer in 4 states of Germany  strains as shown by positive tox-PCR, and negative Elek test and Vero cell cytotoxicity results. Partial rpoB and partial tox sequences for all 13 isolates were identical to those submitted to GenBank for C. ulcerans strain CVUAS 4292 .Conventional biochemical tests showed congruent results for catalase activity, urea hydrolysis, and glucose acidification (positive) and for esculin hydrolysis and nitrate reduction (negative). Additional reactions and API and VITEK test results are shown in C. ulcerans because they had score levels of 2.0–2.2. The comparison of the IR spectra of the 13 strains from game animals with a collection of reference strains showed a clear separation in 2 main branches for the 2 species C. pseudotuberculosis and C. ulcerans (C. ulcerans branch, all isolates from game animals clustered compactly together and were closely adjacent to a group of spectra formed by reference strains from humans.By using MALDI-TOF MS, all isolates were identified to the species level as ulcerans . Inside C. ulcerans is one of the most notable members of the genus and was referred to as an emerging pathogen in 2011 (tox-positive C. ulcerans strains is documented for only 4 cases, involving 2 dogs, 1 cat, and 1 pig (With respect to its zoonotic potential, C. ulcerans infection in wild animals. To our knowledge, 3 reports regarding tox-positive C. ulcerans infection in wildlife have been published: 1 involved 2 European otters from 2 widely separated regions within the United Kingdom (C. ulcerans with unknown toxigenicity in wildlife pertains to an outbreak among 350 squirrels from Canada, 63 of which had clinical disease (Limited information is available concerning C. ulcerans strains from game animals in Germany. The finding of infected game in the center of Middle Europe suggests an even wider occurrence and distribution in other European countries. Misdiagnoses of C. ulcerans isolates as C. pseudotuberculosis in the past because of similar pathology and similar phenotype cannot be excluded. Our finding of C. ulcerans in a wild boar specimen from 1997 could indicate that this pathogen has not only recently infected wildlife.Here, we provide comprehensive data on 13 NTTB C. ulcerans and C. pseudotuberculosis might be problematic, and basic conventional tests may not properly discriminate between the 2 species (C. pseudotuberculosis. For correct understanding of epidemiology and host range and for unequivocal determination of the involved pathogen to species level, additional methods such as FT-IR and MALDI-TOF MS or DNA sequencing should be used. Because partial rpoB sequencing is more discriminatory than 16S rDNA sequencing, a cutoff value of ≤95% similarity proved suitable for species identification within Corynebacterium (tox- and rpoB-gene sequencing demonstrated a very close relationship between the 13 strains because no variations in these sequences were found (As also shown in this study, biochemical differentiation between C. ulcerans strains from wildlife, there is no information available. With respect to wild boars infected with C. ulcerans, however, it is noteworthy that 3 diphtheria cases occurred in humans who had occupational contact with pigs (Concerning the zoonotic potential for tox-positive strains has been described (C. ulcerans strains might occur in game animals, providing a reservoir for this microorganism. Because the C. diphtheriae and C. ulcerans DT sequences are quite similar, it might be reasonable to offer diphtheria toxoid vaccination to persons who have direct contact with game animals to prevent diphtheria-like illness caused by tox-positive C. ulcerans (Lack of DT expression in


Ixodes scapularis (L.) is the most abundant tick species encountered by humans and the primary vector of B. burgdorferi, whereas in the southeastern region Amblyomma americanum (Say) is the most abundant tick species encountered by humans but cannot transmit B. burgdorferi. Surveys of Borreliae in ticks have been conducted in the southeastern United States and often these surveys identify B. lonestari as the primary Borrelia species, surveys have not included Arkansas ticks, canines, or white-tailed deer and B. lonestari is not considered pathogenic. The objective of this study was to identify Borrelia species within Arkansas by screening ticks (n = 2123), canines (n = 173), and white-tailed deer (n = 228) to determine the identity and locations of Borreliae endemic to Arkansas using PCR amplification of the flagellin (flaB) gene.In the Eastern and Upper Midwestern regions of North America, Borrelia using PCR to amplify the flaB gene. A subset of the positive samples was confirmed with bidirectional sequencing.Field collected ticks from canines and from hunter-killed white-tailed were identified to species and life stage. After which, ticks and their hosts were screened for the presence of Ixodes scapularis, 161 A. americanum, 88 Rhipicephalus sanguineus, 50 Amblyomma maculatum, 19 Dermacentor variabilis, and 13 unidentified Amblyomma species) produced a Borrelia flaB amplicon. Of the positive ticks, 324 (22.7%) were collected from canines  and 259 (37.2%) were collected from white-tailed deer . None of the larvae were PCR positive. A majority of the flaB amplicons were homologous with B. lonestari sequences: 281 of the 296 sequenced ticks, 3 canines, and 27 deer. Only 22 deer, 7 canines, and 15 tick flaB amplicons  were homologous with B. burgdorferi sequences.In total 53 (21.2%) white-tailed deer, ten (6%) canines, and 583 (27.5%) Ixodid ticks (252 B. burgdorferi and B. lonestari; however, B. lonestari was significantly more prevalent in the tick population than B. burgdorferi. Results from this study suggest that the majority of tick-borne diseases in Arkansas are not B. burgdorferi.Data from this study identified multiple Borreliae genotypes in Arkansas ticks, canines and deer including  Ixodes scapularis (L.) is the most abundant tick species encountered by humans and the primary vector of Borrelia burgdorferi (causative agent of Lyme disease), whereas in the southeastern region Amblyomma americanum (Say) is the most abundant tick species encountered by humans but it cannot transmit B. burgdorferi[. Since its first description in the 1970s, Lyme disease is the most frequently reported vector-borne disease in the northern hemisphere [Borrelia genotypes other than B. burgdorferi and includes a range of symptoms and pathologies [B. lonestari is associated with Southern Tick Associated Rash Illness (STARI) or Masters disease [A. americanum; however, the etiology of B. lonestari remains undetermined [B. lonestari may not be pathogenic [In the Eastern and Upper Midwestern regions of North America, rgdorferi,2. Sincemisphere . Howeverhologies ,4. In th disease ,6. This termined  and recethogenic .I. scapularis), pathogens (B. burgdorferi) and hosts (Peromyscus species) are all present in the south [B. burgdorferi was isolated and characterized from field collected I. scapularis and cotton mice (Peromyscus gossypinus); and these field isolates were transmitted via I. scapularis to hamsters (Cricetidae) and mice (Muridae) [I. scapularis from Alabama could acquire, maintain, and transmit B. burgdorferi[B. burgdorferi[Borrelia in nine northeast Arkansas counties using indirect fluorescence antibody (IFA), both I. scapularis and A. americanum were identified as potential vectors, and deer mice (Peromyscus spp.) and marsh rice rats  as potential reservoir hosts [Borrelia transmission [Borrelia related diseases [Health professionals often question cases of Lyme disease from the southeastern United States because symptoms may be confused with other tick-borne illnesses and not all patients produce the erythema migrans or bull’s eye rash used for diagnosis ,17. Addirgdorferi and fielrgdorferi-22. Previr hosts . Althougdiseases ,15. The diseases .B. burgdorferi IFA positive canines geographically associated with I. scapularis infested deer, increase the likelihood of B. burgdorferi transmission to humans because the nymphs that feed on the deer may also feed on canines and their owners [A. americanum infesting canines and I. scapularis primarily infesting white-tailed deer [I. scapularis based on the 16 S mtDNA identified both the American and Southern lineages (formerly known as I. scapularis and I. dammini) on canines and white-tailed deer [I. scapularis lineage is more likely than the Southern I. scapularis lineage to transmit Lyme disease [Borrelia species within Arkansas ticks, canines, and white-tailed deer to determine the identity and locations of Borreliae endemic to Arkansas using PCR amplification of the flagellin (flaB) gene. The CDC defines an endemic county as “endemic for Lyme disease if there are at least two confirmed human cases that were acquired in that county or there are established populations of I. scapularis infected with B. burgdorferi”[Previous research indicates that r owners ,26. Receled deer . A populled deer , which i disease . The objgdorferi”.This project used the collections reported in Trout and Steelman . Brieflyet al.[flaB PCR to identify the presence of Borrelia DNA [To minimize DNA contamination, DNA extractions and PCR were conducted in different laboratories and reagents and equipment were dedicated to each procedure. Tick identification and extractions were conducted in the Veterinary Entomology Laboratory (VELUA) and PCR reactions were conducted in the Insect Genetics Laboratory (IGLUA) at the University of Arkansas. During each PCR, at least one blank reagent to detect contamination (negative control) and an appropriate positive control ensured PCR reagents and conditions were used. PCR and reaction product analyses were performed on the ticks according to the protocols of Trout et al.. After tet al.. If the elia DNA ,35.Borrelia DNA. The FTA card was cut into halves, where half of the card remained at VELUA for an initial screening and the other half was sent to the University of North Texas Health Science Center in Ft. Worth (UNTHSC) for verification screening. Each FTA card half was removed from its envelope and a 1.2 mm disc was punched from the card using a sterilized Harris Micro Punch and the paper disc was washed for DNA extraction according to the Whatman protocol . After the punch had dried at room temperature, it was subjected to PCR analyses. To ensure DNA detection from FTA punches were not inhibited in any manner, FTA punches were assessed for PCR amplification of host cytochrome b genes [Borrelia species by PCR in a genus specific manner [.Each canine and white-tailed deer FTA card was screened in duplicate to determine the presence of  b genes . As withc manner ,35.Borrelia[Summary statistics and relative abundance of each tick species were calculated to determine overall trends within the population using JMP (α = 0.05) . Fisher’Borrelia. Since sflaB positive samples, were sent to UNTHSC for DNA sequence analysis. The PCR reaction products were hydrolyzed with ExoSAP-IT  and sequence determination was performed using a BigDYE Terminator v.3.1 Cycle Sequencing kit  followed by capillary electrophoresis on a ABI PRISM 310 Genetic Analyzer  using the method of Williamson et al. [PCR products, from n et al. . SequencBorrelia DNA presence/absence data were georeferenced into ArcMap 9.0  and projected to the NAD 1983 UTM Zone 15 N of the GCS North American 1983 Geographic coordinate system. Boolean operations and symbology were used to identify locations with PCR positive ticks and hosts. The resulting Boolean map was overlaid on an existing county map of Arkansas [Canine and deer collection data along with their corresponding tick collections and Arkansas .I. scapularis (33%), A. americanum (31%), R. sanguineus (16 %), D. variabilis (9%), and A. maculatum (8%). An additional 3% were Amblyomma ticks that could not be identified to species because the tick specimens were damaged. The tick population was comprised of each life stage, but most were adults (83%) and more were collected from domesticated canines (67%) than from hunter-killed white-tailed deer (33%). Overall, 583 of the 2123 (27.5%) ticks tested by PCR produced an amplicon for the Borrelia flaB gene (Table flaB amplicons for each tick species were 36.4 % (252/692) I. scapularis, 27.9% (50/179) A. maculatum, 25.8% (88/341) R. sanguineus, 24.5% (161/657) A. americanum, and 10.3% (19/184) D. variabilis. Fifty percent of the 583 flaB amplicons from PCR positive ticks were sequenced, representing approximately one fourth of the specimens from each tick species , OK-strain HS-2 (FJ871032), Borrelia lonestari Clone Scc26 (DQ100451), isolate MO2002-V1 (AY850063), isolate TX076 (EF689742), strain NC/MD (AF273670), and clone AA115 (AY654945)  which was amplified from all five tick species. Locations of all Borrelia isolated from ixodid ticks collected from Arkansas canines and white-tailed deer during 2007 were mapped because B. burgdorferi ticks were few  and 22.7% (n = 324) were positive respectively. All ten positive canine flaB amplicons were sequenced and six samples were homologous to B. burgdorferi IP2 (AY345236), two were homolgous to B. lonestari NC/MD (AF273670), one was homologous to B. lonestari fragments MO2002-V1 (AY850063), and one was homologous to strain B. burgdorferi OK HS-2 (FJ871032)  such that I. scapularis (35.5%) had the highest flaB positive rate. The most frequently encountered tick on canines was A. americanum and 23.8% (151/634) were flaB PCR positive. Of the 173 canines, 87 canines did not have a positive tick, 27 canines had one positive tick, and 59 canines had more than one positive tick. Of the 59 canines with more than one positive tick, 18 canines had multiple specimens of the same species that were positive, 13 canines had specimens of two separate tick species positive, and two canines had specimens of three separate tick species PCR positive.Of the 173 canines and their 1427 ticks subjected to the B. lonestari strain NC/MD (AF273670) was also the most common amplicon and amplified in nymphs, males, and females. Positive ticks were identified primarily in northwest Arkansas where many of the canines were collected  and 37.2% (n = 259) were positive for flaB. Of the 49 white-tailed deer flaB amplicons, 24 amplicons were homologous to B. lonestari NC/MD (AF273670), 21 amplicons were homologous to B. burgdorferi OK HS-2 (FJ871032), 3 amplicons were homolgous to B. lonestari MO2002-V1 (AY850063), and one amplicon was homolgous to B. burgdorferi IP2 (AY345236) . flaB prevalence rates in ticks from white-tailed deer was 62.5% (5/8) in unknown Amblyomma, 41.7% (10/24) in R. sanguineus, 43.5% (10/23) in A. americanum, 37.5% (24/64) in A. maculatum, 36.6 % (209/571) in I. scapularis, and 16.7% (1/6) in D. variabilis. A majority of white-tailed deer did not have a positive tick (n = 91), but 70 white-tailed deer had more than one PCR positive tick of which 24 deer had more than one flaB positive tick species and one deer had three different tick species flaB positive. Fragments amplified from all five deer-collected tick species were most similar to the B. lonestari sequence AY850063. Positive ticks were identified throughout Arkansas  and it is homologous to the sequence previously isolated from a patient’s skin with Lyme-like symptoms [Borrelia in Arkansas. In 2007, four Arkansas counties reported human cases of Lyme disease . Ticks collected from these counties in this study primarily produced amplicons homologous to B. lonestari, and a few ticks produced amplicons homologous to B. burgdorferi. If these human cases of Lyme disease are ‘real’ then they are most likely either rare, acquired from a different location, or are false positives. Collections of field collected ticks near the site of transmission  would answer these questions. Data presented here corroborate with Texas collected ticks identified with B. lonestari DNA in A. americanumA. cajennense, A. maculatum, D. variabilis, I. scapularis, and R. sanguineus and relatively few samples of B. burgdorferi DNA (AE000783) [B. lonestari in Arkansas white-tailed deer serum [Borreliae were identified in field collected ticks, canines, and white-tailed deer throughout the state suggesting the bacteria (as a family) are endemic in Arkansas. Of interest was the diversity of s Figure , Table 2rgdorferi,2. Due tmic area . B. lonesymptoms  suggestiE000783)  and wither serum .Borrelia in five tick species infesting domesticated canines and white-tailed deer; consequently, Borreliae are likely endemic in Arkansas and in the southeastern U.S. by a more diverse tick repertoire than previously thought. Identifying Borrelia in ticks indicates that field collected ticks have fed on infected animals at some point in their life cycle and identifying Borrelia in canines and white-tailed deer indicates that infected ticks have transmitted Borrelia to these hosts. We observed a trend for ticks collected from canines to be positive with B. lonestari whereas ticks from deer could be positive with either B. lonestari or B. burgdorferi. This may be related to the species of tick collected on each host and the sampling periods, as canines were primarily infested with A. americanum and collected year round whereas white-tailed deer were primarily infested with I. scapularis and collected in the fall. Also, ticks feeding on white-tailed deer may actually be feeding on hosts that are incompetent reservoirs of Lyme disease [B. burgdorferi  [We documented  disease . Additio glands) ,20, but  glands) ,43 and E glands) ,39, perhB. burgdorferi and other tick-borne diseases is essential to minimize transmission and maximize management. Recent landscape changes in Arkansas are expanding urban environments into deer habitats [B. burgdorferi emerged independently in the Midwest and eastern United States [Monitoring tick populations on canines could provide reliable early detection of tick-borne disease outbreaks . Monitorhabitats ,46. Prevhabitats ; similard States -50.Borrelia spp. in the southeastern zoonotic cycle  should be conducted in order to determine the life cycle and etiology of the various Borreliae detected in this survey and what roles, if any, they may play relative to subsequent borreliosis observed in humans. Future work should also evaluate the interaction of A. americanum carrying B. lonestari, A. maculatum carrying B. burgdorferi, and I. scapularis carrying B. burgdorferi and what effect, if any, this might have on the pathogenicity of the Borreliae, as both tick species have been identified simultaneously infesting the same host [Additional research into the involvement of ame host ,51. It iBorrelia genotypes are endemic to Arkansas because flaB was amplified from ticks, canines, and white-tailed deer, but the exact role each genotype plays in transmission and epidemiology remains undetermined. There were significantly more amplicons of B. lonestari than B. burgdorferi present suggesting that the majority of tick-borne diseases in Arkansas are not B. burgdorferi.Data from this study indicate multiple flaB, flagellin gene; IFA, indirect fluorescence antibody; IGLUA, Insect Genetics Laboratory at the University of Arkansas; STARI, Southern tick associated rash illness; UNTHSC, University of North Texas Health Science Center in Ft. Worth; VELUA, Veterinary Entomology Laboratory at the University of Arkansas.The authors declare we have no competing interests with the work presented in this manuscript.RTF, CDS, and ALS conceived the study and designed the experiments. PMB and PCW conducted the work in Texas, and RTF conducted the work in Arkansas. RTF, KLK, PMB, and PCW analyzed the results. RTF and PCW wrote and edited the manuscript. All authors read and approved the final version of the manuscript.


Drosophila melanogaster subgroup shows that while ~70% of genes follow clock-like evolution, between 17–19.67% of loci show signatures of accelerated rates of evolution in recently formed species. These genes show 2-3-fold higher rates of substitution in recently diverged species compared to older species. This fraction of loci affects a diverse range of functions. Only a small proportion of reproductive genes experience speciation-related accelerated changes but many sex-and -reproduction related genes show an interesting pattern of persistent rapid evolution suggesting that sex-and-reproduction related genes are under constant selective pressures. The identification of loci associated with accelerated evolution allows us to address the mechanisms of rapid evolution and speciation, which in our study appears to be a combination of both selection and rapid demographical changes.The tempo and mode of evolutionary change during speciation have remained contentious until recently. While much of the evidence claiming speciation is an abrupt and rapid process comes from fossil data, recent molecular phylogenetics show that the background of gradual evolution is often broken by accelerated rates of molecular evolution during speciation. However, what kinds of genes affect or are affected by speciation remains unexplored. Our analysis of 4843 protein-coding genes in five species of the  The tempo and mode of evolutionary change during speciation have remained a contentious issue for more than five decades. Evidence for abrupt and rapid changes during speciation came from fossil evidence but lacked mechanistic explanations for such a process –7. SinceWhat remains unknown is the mode of change during speciation, that is, the kind of genetic changes associated with speciation. More specifically, the numbers and kinds of protein coding genes that change during speciation remain unexplored. Accelerated evolution is only observed in a fraction of genes analyzed by Pagel et al.  as well Taxa-wide evidence of sex- and reproduction-related genes evolving rapidly in sibling species , 20 has Divergence trends between recently formed species and old species provide a proxy to detecting signatures of speciation-related accelerated changes. Molecular divergences are expected to be proportional to the duration of a species' existence; newly formed species will have accumulated less molecular divergence compared to older species. Signatures of speciation-related accelerated evolution will be manifested as relatively higher rates of molecular evolution in newly formed species compared to species with longer postspeciation divergence times. Accordingly we asked the following questions: (1) are rates of molecular evolution in protein-coding genes affected by speciation events? That is, do genes show unexpectedly elevated rates of evolution in newly formed species relative to older species? (2) do sex-related genes preferentially exhibit accelerated speciation-related changes relative to non-sex related genes? And (3) do genes with accelerated rates of evolution show evidence of positive selection?since divergence of species pairs (recent versus older) to infer speciation-related accelerated changes.If evolutionary rates of protein coding genes are not affected by speciation and evolve at a constant rate, we should be able to find a correlation between the length of time that species have diverged and the proportion of molecular divergence between these species. Species diverged for longer lengths of time would have accumulated proportionally higher amounts of genetic changes relative to species diverged for shorter periods of time. Conversely, if rates of molecular evolution were indeed affected by speciation, then this correlation will be broken and newer species might show relatively higher evolutionary rates compared to older species. Our analyses therefore primarily exploit the nature of molecular changes  D. simulans and D. sechellia diverged about 0.3–0.6 Mya ) would fall within these divergence time-ranges. Genes that fall above the divergence time-ranges indicate accelerated evolution in the newer species and those falling below the lower limit are considered to evolve slowly in the newly formed species or to have much higher rates of evolution in the older species lineage.ere) see . We simia et al. . For insD. simulans-D. sechellia species pair grossly overestimated the number of genes in the accelerated rate category  have been contentious and unresolved [D. simulans-D. sechellia split using dS estimated from our dataset . We therefore employed a slightly more stringent approach by applying 95% confidence intervals to the existing divergence boundaries (using dS ratios), which consequently incorporated previous outliers  were obtained from the recently sequenced genomes available on FlyBase . Sequences were aligned according to the corresponding protein alignment using CLUSTALW ver. 1.8 [D. yakuba and D. erecta using PAML and restricted our analysis to genes for which the best tree involved D. yakuba and D. erecta as sister species. Pairwise estimates of dN and dS were determined using the program codeml in PAML [dN, dS, and ω along each lineage using branch site models and outputs of models M7  versus M8 (positive selection) were also determined using PAML [D. melanogaster-D. simulans versus and D. yakuba-D. erecta, and 4327 orthologs between D. simulans-D. sechellia versus D. yakuba - D. erecta comparisons as well as for 3988 genes from D. pseudoobscura-D. persimilis versus all melanogaster species pairs.All sequences . Genes that could not be classified into any tissue category were referred as unspecified. Based on EST data, we were able to classify tissue of expression for 3040 out of 4843 genes for comparisons involving D. melanogaster-D. simulans versus D. yakuba-D. erecta, 2686 genes for comparisons involving D. simulans-D. sechellia versus D. yakuba-D. erecta (Table S1).We classified genes according to their tissues of expression  by using the NCBI EST database  clock-like evolution: genes showing molecular divergence that corresponds to species divergence times, and (3) slow evolution in younger species: genes showing lower rates of molecular divergence in newly formed species in comparison to species with longer divergence times  ce times . Table 1dN and dS estimates between D. melanogaster-D. simulans versus D. yakuba-D. erecta shows that the distribution of dN estimates is quite distinct between the accelerated, clock-like, and slow-rate categories while the distribution of dS estimates is not  fell into the clock-like rate category in all comparisons, indicating that evolutionary trajectories of most genes are unaffected by speciation events and their rates remained constant . A small Figures . This qu% fell inThese results quite unambiguously indicate two important trends: firstly, despite variances associated with divergence estimates, the 2-3-fold higher nonsynonymous divergence in newly formed species compared to species with much longer divergence times clearly represents increases in rates of protein evolution either during or immediately after speciation. Secondly, accelerated rates of molecular evolution are most apparent in nonsynonymous divergence and not in synonymous divergence see , a broadDrosophila genes, we used tissue of expression as a general and presumable indication of function . We tested the null hypothesis that genes expressed in each tissue-type are equally distributed within the accelerated, clock-like and slow rate categories. Because the expression data was determined using D. melanogaster data, only species from the D. melanogaster subgroup were analyzed. Given the long divergence time between the Obscura group and the melanogaster group (55 Mya), it is likely that patterns of gene expression may be radically different in D. pseudoobscura and D. persimilis. Identifying the range and, particularly, the kinds of loci in each rate category  provides a starting point to broadly address the effects of demographic factors and selection during speciation. Demographic factors  would affect a wide variety of loci whereas selection driven divergence would only affect specific functional classes of genes, such as sex-related genes which are expected to drive reproductive isolation in diverging populations , 46. DueD. melanogaster-D. simulans versus D. yakuba-D. erecta comparison, a significantly higher proportion of testis-specific genes occupy the accelerated and clock-like rate categories compared to the slow-rate category  among the different rate categories by implementing site models, branch-site and branch models in PAML [χ² = 0.88, df = 1, P > 0.05, accelerated, slow versus clock-like χ² = 0.93 and 2.46, df = 1, P > 0.05 and 0.35 after Bonferroni corrections, D. simulans in the accelerated rate category compared to the clock-like and slow rate categories . Branch model tests also show a greater proportion of genes with foreground ω > background ω in the accelerated rate category in the D. simulans branch and the branch leading to the D. melanogaster clade  while we found a significant paucity among the slow rate category . No significant effect is observed for the genes classified as rapidly evolving in the younger species .We compared the proportion of genes showing evidence of positive selection . There was no significant difference between the clock-like and slow rate categories . The D. simulans-D. sechellia/D. yakuba-D. erecta comparison showed no such effect of recombination in all rate categories .Gene evolution may be influenced by their chromosomal location , 51 as wd Kliman , we founDespite recent evidence linking accelerated rates of molecular evolution to speciation events , 13, theDrosophila is one of those groups that lack detailed fossil records and incomplete taxon sampling is an obvious but unavoidable problem in this, or any study of this nature. Apart from extinct taxa, undiscovered and undescribed taxa are an additional problem. We are also lacking the genome sequence of D. mauritiana, a member of the D. simulans-D. sechellia-D. mauritiana triad as well as the genome of D. santomea which recently split from D. yakuba [Drosophila transcriptome that show signatures of accelerated speciation-related changes that can be further investigated.This study analyzes the ratio of molecular evolutionary rates to the ratio of species divergence times, therefore clock-like evolution should not be taken as a sign of gradual evolution; it only implies a constant rate of evolution. This is exemplified by the large representation of testis-specific genes in the clock-like rate category, which are in fact evolving much faster than other genes in the same category. Furthermore, . yakuba . These fS in most genes fall under the clock-like category as opposed to dN is not entirely surprising, as we expect dN changes to be more sensitive to selection and may therefore show higher variance. Nevertheless, the 2-3 fold higher estimates of dN in the recently diverged species compared to species with much longer divergence times is a strict deviation from clock-like evolution despite the variance that may be involved. More importantly, our survey reveals that the more recently diverged species (D. simulans-D. sechellia) have a slightly greater proportion of genes that show accelerated rates of evolution  and a relatively small proportion of genes with lineage specific positive selection .D. sechellia has evolved an intricate ecological relationship with its plant host Morinda citrifolia, on which D. sechellia females oviposit their eggs [M. citrifolia fruit are toxic to other species of the melanogaster subgroup but not to D. sechellia larve that feed and grow on it [D. simulans-D. sechella pair, which might reflect rapid behavioral and sensory modifications that occurred during the host-plant specialization between D. sechellia and M. citrifolia. Genes involved in sensory perception , sensory organ development , detoxification (kraken), metabolism, and oogenesis  found to be involved in host specialization by Dworkin and Jones [According to the founder effect model of speciation , speciateir eggs . Compounow on it . A largend Jones  are amonD. sechellia data demonstrates the importance of founder-effect and subsequent ecological divergence in driving speciation and as a consequence, causing elevated rates of evolution of relevant protein coding genes associated with the speciation event. Such cases of speciation driven by founder-effect and subsequent resource specialization are not uncommon in Drosophila [D. sechellia, D. mauritiana and D. santomea (a recently described sibling species to D. yakuba) are all insular species within the melanogaster subgroup. Among these, D. sechellia and D. santomea show resource specialization having evolved special ecological relationships of utilizing specific host plants either for food or oviposition [D. simulans versus D. sechellia comparison; all of which fall in the clock-like rate category in the D. melanogaster versus D. simulans comparison . Many of these genes, as mentioned above, have been found to be involved in host-plant specialization [D. sechellia and D. simulans, as well as the inclusion of D. santomea genome (when completed) in a similar study should shed some light on our claim. However, it should be noted that while D. sechellia is isolated from D. simulans, D. santomea and its sibling species D. yakuba are sympatric on the Island of San Tome and there is substantial divergence of sexual traits between the two species [D. santomea and D. yakuba, much like what we observed between the sympatric D. melanogaster and D. simulans in our study.The osophila , 59, 65.position , 63–65, lization . Further species , 66–69. D. simulans-D. sechellia pair, our data reinforce previous evidence that founder effect and ecological specialization played important roles in their speciation process. Our results support the growing appreciation that speciation is often driven by a combination of demographic fluctuations, ecological adaptation, and sexual selection and can seldom be attributed to one single important factor [Signatures of speciation-related accelerated rates of evolution are detected in all newly evolved species analyzed in this study. This is not observed as a widespread genomic signature but is restricted to a fraction of the genome that affects widely different functions. The range and kinds of loci in this rapidly evolving fraction identified in this study complements the recently reported punctuational effect observed across a variety of taxa , 15 and t factor , 70.Additional file 1: Comparison between D. melanogaster – D. simulans / D. yakuba – D. erecta.Additional file 2: Distribution of protiendivergence log2 (P1/P2) to divergencetime ratio log2 (T1/T2), between D. melanogaster D. simulans vs. D. yakuba D. erecta (Supplementary Table 1). The upper limit of the clockwise category is given by the log2 ratio of of divergence times (6.5/8.1) and the lower limit by the log2 ratio 4.3/12.7. Color codes: Red circlesaccelerated evolution, Grey – clocklike, green slow evolution categories.ω estimates of genes falling under slow, clock-like and accelerated rate categories. Branch lengths represent the proportion of genes with ω values greater than background ω values. P-values for the different tests were corrected using a Bonferroni correction prior to the comparisons. mel: D. melanogaster, sim: D. simulans, sec: D. sechellia, simsecmel: branch leading to the D. melanogaster clade, yak: D. yakuba, ere: D. erecta, yakere, branch leading to the D. yakuba clade, ana: D. ananassae.Additional file 3: Dendrogram of average Additional file 4: Genes with evidence of positive selection using site models (M8 vs M7) from PAML.Supplementary data 5: Comparison of gene classification in between the D. simulans - D. Sechellia / D. yakuba – D. erecta and D. simulans – D. melanogaster / D. yakuba – D. erecta analyses.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.Click here for additional data file.


Rana sphenocephala and R. pipiens. We primarily use bioacoustic and molecular data to characterize the new species, but also examine other lines of evidence. This discovery is unexpected in one of the largest and most densely populated urban parts of the world. It also demonstrates that new vertebrate species can still be found periodically even in well-studied locales rarely associated with undocumented biodiversity. The new species typically occurs in expansive open-canopied wetlands interspersed with upland patches, but centuries of loss and impact to these habitats give some cause for conservation concern. Other concerns include regional extirpations, fragmented extant populations, and a restricted overall geographic distribution. We assign a type locality within New York City and report a narrow and largely coastal lowland distribution from central Connecticut to northern New Jersey (based on genetic data) and south to North Carolina .We describe a new cryptic species of leopard frog from the New York City metropolitan area and surrounding coastal regions. This species is morphologically similar to two largely parapatric eastern congeners,  In order to develop clear understandings of species and their ecologies, distributions, and conservation needs, they must first be properly identified and accurately delimited Considerable effort has been given to identifying and cataloging new species, cryptic and otherwise, over the past few decades. In the case of amphibians, these efforts carry added urgency in the face of severe global declines and extinctions and also reveal strongholds of undocumented species, often in areas of tropical species richness or poorly known composition et al.Rana pipiens complex and occupies parts of the lower Northeast and mid-Atlantic US within the densely populated and heavily industrialized Interstate-95 (I-95) corridor. This is one of the largest human population centers on earth The species we describe here was first identified by Newman et al.R. pipiens complex, comparisons to similar species, and information on distribution, ecology, and conservation status.Here, we expand upon the initial genetic results presented by Newman R. pipiens complex has long been a source of taxonomic uncertainty and nomenclatural debate in eastern North America Although one of the most well-known and best-studied amphibian groups on earth, the R. pipiens complex spans several centuries and has been fueled largely by a lack of scientific consensus and changing species concepts across those years. This has led to numerous synonyms and conflicting species frameworks over time R. sphenocephala and R. pipiens, received lasting consideration and taxonomic recognition in the east Rana sphenocephala, the southern leopard frog, has a reported range from extreme southeastern NY to Florida (FL) and west from Texas to Iowa Rana pipiens, the northern leopard frog, ranges from eastern Canada, New England, and the northern mid-Atlantic, west to the Pacific Coast states and British Columbia et al.The unsettled taxonomic history of the R. pipiens complex can be traced to the Northeast and mid-Atlantic US R. pipiens itself Much of the historical discord and confusion surrounding the R. sphenocephala and R. pipiens at that time. Kauffeld R. pipiens to conclude that three distinct species did occur across the Northeast and mid-Atlantic US. He classified the northernmost species as R. brachycephala and reassigned R. pipiens – the binomial typically associated with the northernmost species – to his proposed central species ; R. sphenocephala was maintained as the southernmost species. Despite acknowledging the potential taxonomic confusion and backlash this could cause, Kauffeld R. pipiens fell within southeastern New York, where his reported central species occurred, not the northernmost species.In 1936, Kauffeld th Century single-species interpretation that classified all North American leopard frogs as R. pipiensR. pipiens complex that returned to a two-species arrangement in the eastern US, echoing arrangements prior to Kauffeld's work R. sphenocephala (referred to as R. utricularia by Pace) to the south, and R. pipiens to the north, with a species boundary centered in the NY/NJ-metro area. Pace's arrangement remained largely intact over subsequent decades, particularly across the eastern US.Kauffeld's three-species framework and taxonomic changes received some initial recognition et al.R. sp. nov., does indeed occur between populations of R. sphenocephala and R. pipiens in the NY/NJ-metro area. They also reported mitochondrial data showing this species to be most closely related to the pickerel frog, R. palustris, a morphologically distinct and readily identifiable species R. sphenocephala, the species to which it had been included based on morphological similarity; nuclear data regarding interspecific relationships were inconclusive.Occasional discussion of distinct populations, potential intergradation, and cryptic species in the NY/NJ-metro area continued after Kauffeld R. pipiens, R. palustris, R. sp. nov., and R. sphenocephala each occur in succession along a narrow 90-km west-to-east transect between Berks County, PA and Burlington County, NJ In retrospect, the long history of taxonomic and nomenclatural confusion in the NY/NJ-metro area was likely due to the unrecognized presence of a cryptic species occurring in close proximity to several similar congeners. For example, in the Philadelphia region – an area replete with historical confusion and variation reported among leopard frogs The species described here was discovered during research activities conducted under an Institutional Animal Care and Use Committee Protocol (IACUC) from Rutgers University (#07-024). Additional field work and collection of the holotype specimen occurred under New York State Collect or Possess permit #969 (to MDS) in compliance with Yale University IACUC protocol #2012-10681.Rana versus a recently proposed replacement name, Lithobates, which has been applied to a number of North American ranid frog species Rana for all North American ranid frogs, including the R. pipiens complex.We briefly point to an area of unresolved taxonomic debate within the herpetological community. This debate centers on use of the historical genus name R. sp. nov., R. sphenocephala, R. pipiens, and R. palustris), 30 US counties, seven eastern states, and Quebec, Canada . We collected morphometric measurement data from 283 specimens, including the holotype (YPM 13217) and 282 other museum specimens across four species , reducing the number of frogs with complete measurements to 264. Thus, we omitted these specimens from our DFA. All analyses were conducted in R, v. 2.15.2 and v. 3.0.2 We looked for univariate differences in species morphology using boxplots and one-way ANOVAs followed by Tukey HSD post-hoc pairwise comparisons. We used discriminant function analysis (DFA) to examine variation in multivariate space and determine which variables best discriminated among species. This was followed by a MANOVA to look for multivariate differences among species, and then Tukey HSD post-hoc pairwise comparisons. Because body size varied substantially among specimens, we removed this effect in our statistical analyses by using the residuals of a regression of snout-vent length on each morphometric variable. Foot length was not available for some specimens  into ArcMap 10.0 R. sp. nov. and R. sphenocephala, including 1) dorsal spot shape (round or elongate), 2) snout spot (present or absent), and 3) skin color (three color categories). We categorized a dorsal spot as ‘elongate’ if it was at least 2.5 times longer than wide at its widest point, but excluded eyelid spots from this analysis because the curvature of the eye made them difficult to assess. Lastly, we compared pigmentation on the posterior dorsal surface of the femur (thigh) among specimens of R. sp. nov., R. sphenocephala, and R. pipiens. This character was previously used to distinguish leopard frogs in regions where R. sp. nov. occurs We also examined color and patterning differences between leopard frog species. We compared dorsal spots  between the new species and its closest morphological congener, et al.et al.et al.Following the methods described in Newman R. sphenocephala, R. pipiens, R. palustris, and an acoustically similar species outside the leopard frog complex, R. sylvatica. We examined two populations of R. sphenocephala , one population of R. pipiens , one population of R. palustris , and three populations of R. sylvatica . We did not collect frogs used in our call analysis, but deposited call vouchers at YPM  and analyzed calls with RAVEN Pro v. 1.4 s at YPM .et al.et al.et al.R. sp. nov. in place of R. pipiens and R. palustris because both species were recorded at only one site each under a single temperature regime, and thus lacked sufficient variation for us to generate their own species-specific regression equations.We measured seven variables: call length , call rate , call rise time , call duty cycle , pulse number , pulse rate , and dominant frequency . We mostly follow parameters and terminology from Cocroft and Ryan http://zoobank.org/”. The LSID for this publication is: urn:lsid:zoobank.org:pub:2E7F07A6-19B1-4352-B5B7-A227A93A37CD. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central and LOCKSS.The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix “urn:lsid:zoobank.org:act:149ED690-FA7D-4216-A6A1-AA48CC39B292.YPM 13217, adult male , Table 1YPM 13559, subadult male  and YPM 13560, adult male ; both collected from Wangunk Meadows in Portland, CT by T. Mahard and M. Blumstein on 15 September 2010; genetically confirmed within the same clade as the holotype in situ within a field enclosure on Long Island, NY, for a separate research project); genetically confirmed within the same clade as the holotype YPM 13920, juvenile ; collected as an egg by J. A. Feinberg from the type locality on 27 March 2009  advertisement call (Distribution), 4) geographic distribution  ribution  and S3.R. sphenocephala and the snore-like calls of R. pipiens and R. palustris. The quivering ‘quack’ of R. sylvatica is superficially similar but consists of discrete bouts of 2–4 rapidly pulsed notes that are never accompanied by secondary ‘groans’ as occasionally emitted by R. kauffeldi. Although sympatric with R. kauffeldi, R. sylvatica is morphologically and genetically distinct and typically calls from smaller canopied wetlands and forested pools whereas R. kauffeldi usually calls from larger, open-canopied wetlands.The advertisement call is a single-noted unpulsed ‘chuck’  that is R. kauffeldi possess very large, laterally paired external vocal sacs that distinguish them from all similar congeners except R. sphenocephala. Additionally, R. kauffeldi has a dark femoral reticulum  and R. pipiens from the northeastern US and Canada (n = 46), and was 88.6% consistent in R. sphenocephala from NJ (n = 35). The diagnostic value of this character may be limited to northern regions, however, as Moore R. sphenocephala is broadly distributed.Adult male eticulum a whereaeticulum b. This Rana kauffeldi may be further distinguished from R. sphenocephala by a tympanic spot that is typically duller, less well-defined, and rarely pure white ; from R. pipiens by a light spot in the center of the tympanum that is often small and faint ; and from R. palustris by pale inner thighs without deep yellow coloration and round, unaligned dorsal spots.I1 – 2II1+ – 2⅓III1+ – 3+IV3 – 1V following Savage Body moderate and robust; head longer than wide. Dorsal outline of snout acuminate; lateral snout profile round. Nares dorsolaterally oriented, slightly protuberant, around two-thirds closer to tip of snout than anterior corner of eye. Canthus rostralis distinct and angular; loreal region steep and slightly concave. Eyes large and protuberant; diameter slightly less than combined eye-to-naris and naris-to-snout distances. Internarial distance nearly equal to eye-to-naris distance. Tympanum distinct and relatively large (>65% diameter of the eye); bordered dorsally and posteriorly by faint supratympanic fold. Distinct dorsolateral fold runs uninterrupted from posterior eye to pelvic insertion of femur. Forearms relatively short and robust; unwebbed fingers; relative length III>I>II>IV. Fingers lack fringes; tips rounded without expansion; subarticular tubercles small, round, and moderately prominent. No palmer tubercles appear present. First finger slightly swollen at base with faint nuptial pad; all other fingers slender. Hindlimbs relatively long, moderately robust; thigh and shank length nearly equal. Relative toe lengths IV>V>III>II>I; toes have rounded tips without expansion; subarticular tubercles small, round, and prominent. Inner tarsal fold connects tarsus to large, distinct, elliptical, elevated inner metatarsal tubercle. Indistinct, small outer metatarsal tubercle faintly evident. Toe IV very long and slender; toe V slightly fringed; webbing present between all toes; webbing formula In photographs taken before preservation, dorsal ground color of holotype varies from mint-gray in bright lighting a to ligGenerally similar to that in life with several notable distinctions. Ground color dark olive green in holotype c but caR. kauffeldi clade . The mitochondrial phylogeny suggests that R. kauffeldi is most closely related to R. palustris. Average pairwise mitochondrial sequence divergence (uncorrected p) is similar to genetic divergences between other closely related species in the R. pipiens complex  falls within the Rana kauffeldi is known from three states  based on genetic samples Rana kauffeldi appears to occur parapatrically in this core area. Beyond the core area, we depict an extended area of potential occurrence  and R. pipiens (in the north) based on genetic, bioacoustic, and specimen sampling  and Bronx County, NY (n = 7). Based on our examination, 29 of these 34 frogs were R. kauffeldi. Two other individuals, from xeric parts of Long Island, NY (Suffolk County), appeared to be R. sphenocephala . The remaining three frogs were R. pipiens, two of which  came from the Bronx County site previously noted by Pace et al.R. kauffeldi  were also collected historically. The third was a lone individual from western Long Island, in Queens County, NY (AMNH 36651). We also examined specimens (n = 9) from two presumably extirpated sites in southeastern CT (New Haven County)  . All werR. kauffeldi and R. sphenocephala, R. pipiens, and R. palustris (Rana kauffeldi had 1) the shortest eye-to-naris distance , 2) shortest thigh length , and 3) shortest shank length  of the four species examined. Rana kauffeldi had 4) narrower eyes , 5) a wider head , 6) and longer interorbital distance  than R. sphenocephala and R. pipiens. Rana kauffeldi also had 7) a shorter head than R. sphenocephala and a longer head than R. pipiens, , 8) a longer internarial distance than R. sphenocephala and a shorter internarial distance than R. pipiens , 9) a larger tympanum diameter than R. pipiens and R. palustris , 10) a shorter naris-to-snout distance  than R. pipiens, and 11) a wider snout angle than R. sphenocephala . The unadjusted summary data for all 13 morphometric characters are also presented . The DFA correctly classified 78.0% of specimens (p<0.0001) except for R. sphenocephala and R. palustris (p = 0.9966). The first discriminant function accounted for 58.4% of the variation in the data with tympanum diameter loading most heavily, while the second function accounted for 31.4% of the variation with eye-to-naris distance having the greatest load   and less dorsal surface covered by spots   (n = 42) of R. kauffeldi had one or more elongated spot compared to 61.16% (n = 67) of R. sphenocephala examined. Further, snout spots were present in 32.86% (n = 70) of R. kauffeldi versus 16.88% (n = 77) of R. sphenocephala. Lastly, we found considerable categorical color differences between R. kauffeldi (n = 75)  and R. sphenocephala (n = 94) . Multi-colored frogs were categorized by their lightest color.Previous studies report fewer and smaller dorsal spots among leopard frogs from areas where <0.0001) . Dorsal R. kauffeldi is typically emitted in evenly spaced, repeated series that can include up to 27 ‘chucks’ over 22 s. Calls were recorded at multiple locations within the type locality. Five males , R. sphenocephala (18.30°C±7.80), R. pipiens (18.00°C±0), R. palustris (15.00°C±0), and R. sylvatica (9.68°C±0.94).We compared summary data for all  species . Frogs w species , reflectRana kauffeldi had 1) a lower pulse rate  and 2) shorter call duration than all other species , and 3) a lower pulse number  and 4) a lower call rise time than all species except R. sylvatica . Rana kauffeldi also had 5) a lower call duty cycle than all species except R. pipiens , and 6) a call rate that was higher than R. pipiens and R. palustris and lower than R. sylvatica . Dominant frequency did not differ significantly among the five species .Our univariate analysis revealed significant differences among species in 6 of 7 call parameters . Rana kaF4,40 = 323.7, p<0.0001). The only classification errors were two R. sylvatica classified as R. kauffeldi (p<0.001) except for R. kauffeldi and R. sylvatica (p = 0.9991). Pulse rate was excluded from the DFA because R. kauffeldi has only one pulse per call. The first discriminant function accounted for 61.0% of the variation in the data with call rise time loading most heavily, while the second function accounted for 24.3% of the variation with call length contributing the greatest load , especially on warmer days. Thereafter, chorusing tapers to a more episodic nocturnal and precipitation-based regime from mid-April through early June (in NY). We have not observed opportunistic mid-summer chorusing as we and others R. sphenocephala, but we have observed occasional second breeding periods with the onset of cooler autumn temperatures and precipitation (late August through November).sensu lato) tend towards darker nocturnal shading and brighter, more vivid diurnal colors (as a putative mode of camouflage). Some degree of seasonal color change also appears to exist in R. kauffeldi; we often observed frogs with darker, drabber color and fainter tympanic spots in the early spring, and more vivid and varied overall color and brighter, more defined tympanic spots later in the season.Individuals may exhibit a limited degree of color change around a general base color that can vary widely between frogs, from light green to dark brown. Holmes Pseudacris crucifer). Thus dense aggregations may have compensatory value, especially when faced with noisy conditions R. pipiens) from Philadelphia and NJ, respectively, that we consider R. kauffeldi.During breeding, males congregate in concentrated groups, or possible leks R. kauffeldi, but leopard frogs have been described as being fairly terrestrial on Staten Island Little is known about non-breeding activity or dispersal in R. kauffeldi brings the current number of New World leopard frogs to 19  and the total number of native ranid frog species from the US mainland and Canada to 30 R. kauffeldi and the Cajun chorus frog, P. fouquettei,R. kauffeldi is the first anuran from the US Atlantic coast since the New Jersey chorus frog, P. kalmi, was originally recognized (as a subspecies) in 1955 The description of R. kauffeldi was first identified, the New York City metropolitan area  is also significant. It provides an example of new species discovery, not from a tropical biodiversity hotspot or poorly studied region, but rather the glacially impacted urban Northeast; one of the most developed, heavily settled, and well-inventoried places on earth. Novel and undescribed vertebrate species are unexpected here (particularly amphibians) and thus carry considerable interest and value. The last amphibian described from NY or New England was the Fowler's toad, Bufo fowleri, in 1882 R. kauffeldi follows the northern cricket frog, Acris crepitans, in 1854 R. kauffeldi and P. fouquetteiThe specific region where R. kauffeldi is a cryptic species, it is a relatively large, conspicuous, non-fossorial species nonetheless, and acoustically distinct. That it remained ill-defined and poorly documented within one of the largest population centers on earth Hyla versicolor and H. chrysoscelis. Despite being arboreal, smaller, and less conspicuous than leopard frogs, these two congeners were recognized as separate and distinct species nearly 50 years earlier (in 1966) by differences in their calls Although R. kauffeldi may have been due to its narrow and fragmented range, short and cold-season calling regime, and low frequency (less audible) call. Repeated acoustic misidentification may have also played a concealing role; many colleagues with whom we communicated recalled unusual calls from frog populations now known to be R. kauffeldi. Some attributed these calls to R. sylvatica in unusual habitats; others presumed call variation within R. sphenocephala. Given these examples and the generally stereotyped and species-specific nature of frog calls In part, the sustained concealment of R. kauffeldi, its discovery from the Northeast and mid-Atlantic US has direct consequences for three species across eight states . We also identified areas of sympatry between R. kauffeldi and R. sphenocephala in southeastern VA from bioacoustic evidence (North American Amphibian Monitoring Program), and suspect additional overlap in southern locales. Lastly, based on museum specimens from areas where leopard frogs are now extirpated, we note several isolated examples of possible R. sphenocephala from xeric eastern Long Island, NY, and R. pipiens from Queens and Bronx Counties, NY (R. kauffeldi) or possible human introductions; isolated geographic records can suggest captive releases R. pipiens occurrences from Distributional relationships vary between ties, NY . HistoriDetermining the distribution of new species is essential to the process of identifying and interpreting their broader biogeographic implications. In the case of cryptic species, identifying regional compositions and reassigning museum specimens can be challenging but important, especially in heavily impacted landscapes with extirpations or species overlap. In our work, leopard frogs were simply unavailable across vast landscapes due to habitat loss and extirpations. Where individuals were available, differentiating similar-looking congeners was difficult. To overcome such challenges, several strategies can provide pathways forward, including 1) using genetic and bioacoustic methods at sites where new species and their cryptic congeners still occur to delineate species and study habitats, interactions, and hybridization; 2) using genetics and morphology to identify subtle physical differences, if any, between species; and 3) applying these insights to museum specimens and extirpated locales to help assess historical compositions and distribution where populations no longer exist. These pathways  can link genetic and bioacoustic tools with museum specimens and morphology and can also help inform future conservation strategies and range map development.R. kauffeldi to the North American faunal record and species lists of at least eight US states will have implications at various regulatory and management levels. This will include possible threatened or endangered species considerations in certain areas, and may require further assessment of the status of R. kauffeldi and its cryptic congeners in some of these impacted areas. It may also provide further opportunity to investigate and verify species composition and boundaries throughout different parts of the range. This may be challenging, however, especially in states where leopard frogs (sensu lato) already receive legal protections and in areas where multiple species are found to co-occur. Thus, reliable, field-ready characters that distinguish similar taxa, and research on potential hybridization, are key priorities. We also leave open the possibility that R. kauffeldi may extend farther south.The addition of R. kauffeldi has several broad conservation implications. For one, it reaffirms that refined taxonomic information is essential for implementing proper conservation measures R. kauffeldi adds another important observation to the growing consensus that we must protect sensitive species where they occur, not just in pristine environments. Findings such as this also provide invaluable opportunities to highlight and enhance access for increasingly urban societies to experience new species discoveries and taxa of high conservation concern firsthand.The discovery of R. kauffeldi awaits further definition of distribution and habitat use and should be considered data deficient in the IUCN classification system. On-the-ground assessments, coupled with genetic and bioacoustic data, will be critical to this and allow for more complete mapping of boundaries and overlap with related taxa. If the distribution is indeed narrow and fragmented (as reported here), it may pose some cause for concern as geographically restricted species are often at risk of extinction due to demographic stochasticity R. kauffeldi populations in the NY/NJ-metro area vary from tenuous to stable, with the most vulnerable populations being those that are small and isolated and threatened by succeeding canopy closure and development. Second, dense breeding groups and strong metapopulation structure may be essential features of R. kauffeldi demography, but may also represent key vulnerabilities in the face of habitat impacts. Rorabaugh R. pipiens. Lastly, on a broader scale, climatic events  have the ability to alter coastlines and threaten proximate low-lying freshwater wetlands and any amphibian populations therein with potentially harmful saline inundation.The overall conservation status of sensu lato) have already vanished from some parts of North America R. kauffeldR. kauffeldi persists in several locales within New York City (Staten Island) and the adjacent NJ Meadowlands. These sites are heavily industrialized and have endured severe long-term anthropogenic impacts and invasion by the common reed, Phragmites australis. Most offer large habitat areas, however, which may provide an important clue to survival. The surprising persistence of populations within these urban landscapes, while not completely understood, is encouraging and may have implications for management and restoration possibilities elsewhere, in the future.Leopard frogs (R. kauffeldi), the incorrect species  would have been moved from known populations farther to the south that harbor R. sphenocephala, not R. kauffeldi. Thus, careful consideration of systematics and population genetics at both donor and recipient site ends is critical to responsibly conducting any such endeavors.We finish with a cautionary note regarding reintroductions, repatriations, and translocations. Moving species to restore extirpated populations is a common conservation and management practice, but one that can have unintended risks and consequences. For example, had a leopard frog restoration been implemented on Long Island before the 2007 discovery of extant populations on nearby Staten Island  potentially vulnerable with highly specialized and restrictive habitat needs; 2) locally abundant where present, but often only occurring in isolated and scattered locales; 3) having a restricted distribution across heavily populated, urbanized regions; and 4) having suffered extirpations from certain areas. Concerns over habitat loss and degradation continue today, along with a suite of other threats  that may pose additional future challenges.In diagnosing, describing, and defining the Atlantic Coast leopard frog, Figure S1Box and whisker plots comparing the size-corrected residuals of 12 morphological characters among four Rana species. Species include R. kauffeldi (kauf), R. palustris , R. pipiens (pipi), and R. sphenocephala (sphe). For whisker plots, black bars  =  median, boxes  =  25th–75th quartiles, whiskers  =  minimum and maximum values but exclude outliers (represented by open circles). For each character, species whose measurements differed significantly (P<0.05) in a one-way ANOVA are denoted with different letters atop the plot. Side notches in boxes indicate significantly different medians.(TIF)Click here for additional data file.Figure S2Discriminant function analyses (DFA). Left: DFA using 12 size-corrected morphological characters measured from 264 frogs examined across four Rana species. Right: DFA using six bioacoustic characters measured from 45 frogs examined across five Rana species. Species include R. kauffeldi (circles), R. sphenocephala (triangles), R. pipiens (plus signs), R. palustris (x-crosses), and R. sylvatica (red squares). Morphological characters include all variables from (TIF)Click here for additional data file.Figure S3Box and whisker plots comparing spot features between Rana kauffeldi (kauf) and R. sphenocephala (sphe). Left: total number of dorsal spots. Right: proportion of dorsal surface covered by spots. For whisker plots, black bars  =  median, boxes  = 25th–75th quartiles, whiskers  =  minimum and maximum values but exclude outliers (represented by open circles). Side notches in boxes indicate significantly different medians.(TIF)Click here for additional data file.Figure S4Box and whisker plots comparing seven bioacoustic characters among five Rana species. Species include R. kauffeldi (kauf), R. palustris , R. pipiens (pipi), R. sphenocephala (sphe), and R. sylvatica (sylv). For whisker plots, black bars  =  median, boxes  = 25th–75th quartiles, whiskers  =  minimum and maximum values but exclude outliers (represented by open circles). For each character, species whose measurements differed significantly (P<0.05) in a one-way ANOVA are denoted with different letters atop the plot. Call length and call rate were temperature-corrected.(TIF)Click here for additional data file.Table S1List of Rana specimens examined.(DOC)Click here for additional data file.Table S2List of Rana primary (advertisement) calls measured for bioacoustic data.(DOC)Click here for additional data file.Table S3Classification matrix for four Rana species using discriminant function analysis on morphometric variables.(DOC)Click here for additional data file.Table S4Coefficients for three discriminant functions (from four species of Rana) for each of 12 morphological characters: head length (HL), head width (HW), eye diameter (ED), tympanum diameter (TD), foot length (FOL), eye-to-naris distance (END), naris-to-snout distance (NSD), thigh length (THL), internarial distance (IND), interorbital distance (IOD), shank length (SL), and dorsal snout angle (DSA).(DOC)Click here for additional data file.Table S5Classification matrix for five Rana species using discriminant function analysis on bioacoustic variables.(DOC)Click here for additional data file.Table S6Coefficients for four discriminant functions (from five species of Rana) for each of six bioacoustic characters: call length (CL), call rate (CR), call rise time (CRT), call duty cycle (CDC), pulse number (PN), and dominant frequency (DF).(DOC)Click here for additional data file.Table S7Underlying (raw) morphometric data.(XLSX)Click here for additional data file.Table S8Underlying (raw) bioacoustic data.(XLSX)Click here for additional data file.Table S9Underlying (raw) data for color and pattern analyses.(XLSX)Click here for additional data file.Video S1A male Rana kauffeldi emitting its primary (advertisement) call in foreground with several other males calling in background .(MOV)Click here for additional data file.