Acropora palmata, to reveal fine-scale population structure and infer the major barrier to gene flow that separates the eastern and western Caribbean populations between the Bahamas and Puerto Rico. The exact location of this break had been subject to discussion because two previous studies based on microsatellite data had come to differing conclusions. We investigate this contradiction by analyzing an extended set of 11 microsatellite markers including the five previously employed and discovered that one of the original microsatellite loci is apparently under selection. Exclusion of this locus reconciles the results from the SNP and the microsatellite datasets. Scans for outlier loci in the SNP data detected 13 candidate loci under positive selection, however there was no correlation between available environmental parameters and genetic distance. Together, these results suggest that reef restoration efforts should use local sources and utilize existing functional variation among geographic regions in ex situ crossing experiments to improve stress resistance of this species.The advent of next-generation sequencing tools has made it possible to conduct fine-scale surveys of population differentiation and genome-wide scans for signatures of selection in non-model organisms. Such surveys are of particular importance in sharply declining coral species, since knowledge of population boundaries and signs of local adaptation can inform restoration and conservation efforts. Here, we use genome-wide surveys of single-nucleotide polymorphisms in the threatened Caribbean elkhorn coral,  There is an ongoing debate about the importance of local recruitment and barriers to gene flow in marine species. Many marine species reproduce via planktonic larvae and strong ocean currents have the potential to carry propagules over long distances. However, a high degree of self-recruitment has been found in a range of species with planktonic larval duration being a poor predictor of genetic structure . The devA cervicornis identified population structure within the Florida Reef tract . This is in contrast to microsatellite loci that consist of tandem repeats, in which allelic variation is determined by the number of tandem repeats and thus can be large. The limited number of alleles at each SNP locus requires a larger number of loci to be assayed to achieve the same power of detecting population genetic structure as a panel of microsatellite loci . The advef tract  where mief tract . Other fef tract  have yieef tract .Genome-scale genotyping can provide insights into genetic diversity within functional regions of the genome that may be under selection (those genomic regions that code for proteins or regulate transcription of genes). These regions are not commonly surveyed even though they are of interest to conservation managers who want to understand how much capacity there is in a species to adapt to changing conditions . StatistAcropora palmata is one of a few Caribbean coral species whose population genetic structure has been thoroughly investigated on local and range-wide scales  and two geographic regions in the eastern population (Puerto Rico and the US Virgin Islands (USVI)). We then compared the results to population structure derived from eleven microsatellite loci. We further aimed to produce a more comprehensive estimate of genetic diversity across the genome using SNPs and screened loci for signatures of selection.We sought to refine the location of the east–west population divide and test for the presence of finer scale population differentiation in A. palmata were collected between 2002 and 2010 and previously genotyped . Digested DNA was standardized to 100 ng for each sample before adaptor ligation. Samples were identified with eight 6-bp indices on the NlaIII (rare-cutter) P1 adapter  with the following modifications. Time of incubation in the extraction buffer was increased to 16–20 h and two 100 µl elutions were performed, the second of which was kept for library production as this fraction contained the high molecular weight DNA. Extracted DNA was then treated with 0.01 mg of RNase A . Extraction concentrations ranging from 500 ng to 6 µg were double-digested with 10 units of each of the restriction enzymes MluCI ( adapter . Samples adapter . The lib adapter . Each li adapter  and othe adapter .Stacks 1.21 (Raw sequence reads were filtered using the process_radtags in the pipeline cks 1.21 . Barcodecks 1.21 . Reads wAcropora digitifera genome (V1.0) (Stacks 1.30 with the following parameters. Each paired-end sequencing set was run separately through Stacks to compare results (designated Read1 and Read2) in a one-way ANOVA. The ANOVA used each paired-end read as a technical replicate of the same genomic region. We did this to assess whether we would retrieve similar estimates of FIS and heterozygosity from both reads, as expected.Processed sequences were then aligned to the e (V1.0)  with Bowe (V1.0)  within te (V1.0)  frameworm = 5. The number of mismatches allowed between loci when building the catalog was n = 4. SNPs with a log-likelihood of less than −10 were removed as reads with poor log-likelihoods tend to have sequencing error and/or low coverage. Two of the barcodes (TCGAT and CGATC) had few sequence reads across all four geographic regions with all Illumina lanes being affected, and samples with these barcodes were removed before assembly in Stacks.The number of raw reads required to report a stack was PRJNA407327.Sequencing reads are available under NCBI BioProject ID Bedtools  was usedp = 4). For each locus, we then set the minimum percentage of individuals in a region required to have data for that locus to 40% or 60% (option—r). Further, we set the minimum minor allele frequency (MAF) required to process a nucleotide site at a locus (option—min_maf) to 0.025, 0.05 and 0.075. A p-value correction was applied to FST scores, so that if a FST score was not significantly different from 0 (according to Fisher’s Exact Test) the value was set to 0. Additionally, only one random SNP from any RAD locus was written to the STRUCTURE export file in order to prevent linked loci from being processed. Read 1 and Read 2 STRUCTURE export files were combined and duplicate loci removed randomly between reads. FST  was calculated in Stacks. FIS and FST distributions are included in the We explored values for several parameters relevant to population genetic analyses. In the Populations module in Stacks 1.30 we required a locus to be present in all regions for all analyses  is the highest as described in STRUCTURE’s manual section 5.1. Results of the three structure runs were merged with Clumpak  at 10 and 11 microsatellite markers    were alscus 166)  using thFST pairwise matrix between geographic regions from Stacks, and the environmental dissimilarity matrix.Data on temperature, salinity, dissolved oxygen (ml/l), and phosphates was downloaded from the World Ocean Atlas 2013 , a candidate subset of selected loci  were identified and removed. Then the distribution of neutral FST was computed using 100,000 simulations and a bisection approximation algorithm  were blasted against the NCBI nr, UniProt, and Trembl databases with parameters of expected value = 0.00001, gap opening penalty = 11, gap extension penalty = 1, length of initial exact match (word size) = 6 and scoring matrix = BLOSUM62 using BLASTX 2.2.32 + . For each locus, the probability of it being under selection was inferred using the Bayes factor (BF). Based on The  2.0.5.2  to a GESF = 2.638, p > 0.1). The average % GC content for Read 1 and 2 was 41.7 and 39.6, respectively. The percentage of polymorphic sites per genomic region varied little among geographic regions, from 0.150 to 0.173% , indicating an excess of heterozygosity. Using the two paired-end read sets as replicates, a one-way ANOVA was performed for each variable  were retained on average . Pools ho 0.173% . The avebreeding . Howevervariable . Summarypth of 5 . All fouK = 3. Puerto Rico and the USVI were not distinguishable until K = 4,  values, regardless of whether the geographic region was used as a prior.A total of 390 SNPS were identified after filtering and including a minor allele frequency cutoff ≥0.05 . This inl K = 4, . Clumpakk Best K  indicateK = 2, a western  and an eastern cluster (including the USVI and Curacao) was evident  values, based on 11 microsatellite markers  values  values . Again, ) values . At the analysis .FST values and geographic distance  consistent with the microsatellite results (10 loci) from the Florida, Bahamas, Puerto Rico, and Curacao samples only   . AnnotatA. palmata population genetic structure using five, presumed neutrally evolving microsatellite markers showed that while most reefs are self-recruiting, A. palmata stands are not inbred and harbor high microsatellite genetic diversity  retained for population genetic analysis was lower than expected. This was due to more than a 10-fold increase in the number of fragments retrieved from the genome digest using the enzymes MluCI . The in-silico restriction predicted 19,067 to the actual 322,425 (read 1) and 276,753 (read 2) fragments retrieved. This under-prediction was most likely due to an early, incomplete genome draft and unknown genome size at the time of this study  failed to reveal population structure in A. digitifera collected from the Ryukyu Archipelago of Japan using Bayesian clustering based methods  consistent with the microsatellite results (10 loci) from the Florida, Bahamas, Puerto Rico, and Curacao samples  making it challenging to obtain the needed >25 genets per population recommended for FST analyses on a scale of a few km. Thus, genets were pooled over geographic regions to match the scale of the environmental data and yield sample sizes of at least 25 per location. Yet significant micro-environmental differences among colonies growing on the same reef have been documented  that is essential for coordinating the activity of Rho GTPases. Invertebrates are thought to contain a single myosin class IX gene (the exception is Drosphilia which has none) whereas most vertebrates have two with fishes having four  Bahamas vs Florida. (B) Bahamas vs Puerto Rico. (C) Bahamas vs USVI. (D) Florida vs Puerto Rico. (E) Florida vs USVI. (F) Puerto Rico vs USVI.Click here for additional data file.10.7717/peerj.4077/supp-3Figure S3y-axis) in a given cluster for each sample (x-axis) assuming values of K = 2 , K = 3 , and K = 4 . For each K, three replicate runs were performed. The most probable K was always 3 based on the mean estimated log probability of the data at a given K and the Evanno method. Setting the minor allele frequency to 0.025 (A–C) yields 632 SNPs  after combining Read 1 and Read 2. Setting the minor allele frequency to 0.075 (D–F) yields 213 SNPs  after combining Read 1 and Read 2. Reefs within geographic regions sorted by latitude.Shown is the probability of membership  in a given cluster for each sample (x-axis) assuming values of K = 2 (A), K = 3 (B), and K = 4 (C). Analysis of 299 SNPs  after combining Read 1 and Read 2. The most probable K was 3 (B). Minor allele frequency cut of value was 0.05 and percentage of missing data per geographic region was 40%. Reefs within geographic regions are sorted by latitude.Shown is the probability of membership  based on the mean estimated log probability of the data at a given K and the Evanno method . Reefs within geographic regions are sorted by latitude.Figure shows results of a Structure analysis of 4793 SNPs  after combining Read 1 and Read 2 and assuming a K of 2 (A), 3 (B) or 4 (C). The minor allele frequency was set to ≥0.05. The most probable Click here for additional data file.10.7717/peerj.4077/supp-6Figure S6Red, Florida; blue, Bahamas; yellow, Puerto Rico; green, USVI; purple, Belize and orange, Curacao.Click here for additional data file.10.7717/peerj.4077/supp-7Figure S7A. digitifera genome had sequence coverage at a stack depth of 5.On average 2.5% percent of the Click here for additional data file.10.7717/peerj.4077/supp-8Figure S8Acropora palmata samples from four geographic regions  were genotyped with 11 microsatellite markers, including the outlier locus, 166. y = 5∗10−6 ×  + 0.0137. R2 = 0.1147, p-value =0.1.Click here for additional data file.10.7717/peerj.4077/supp-9Table S1There were 12 pools with 8 unique barcodes in each. The Database ID is a unique identifier for each coral specimen. Given is also the total number of ramets for each genet in the Baumslab database. The indices are short DNA sequences that uniquely identify products in the final libraries.Click here for additional data file.10.7717/peerj.4077/supp-10Table S2Click here for additional data file.10.7717/peerj.4077/supp-11Table S3Click here for additional data file.10.7717/peerj.4077/supp-12Table S4A. dig scaffold identifies the A. digitifera scaffold where the A. palmata Stacks locus aligned to, followed by the basepair location in the next column. S start, sequence start; S end, sequence end; Lositan P, Lositan P-values (Simulated FST < sample FST). Bayescan q, Bayescan q-values; Ns, not significant.Stacks locus_bp is the Stacks program locus ID with the SNP location basepair after the underscore. Read category indicates whether the outlier SNP was found in read 1 or read 2 or the paired-end sequencing run. Click here for additional data file.10.7717/peerj.4077/supp-13Table S5The Database ID is a unique identifier for each coral specimen. Given is also is the geographic region and reef the samples were collected from, microsatellite allele call 1 and 2 for loci 166, 181, 182, 192, and 207 (in basebairs), and the coral genet ID. The last four columns show how the radseq samples were pooled and gives the inline barcode, the Illumina index, the Illumina sequence lane and chip.Click here for additional data file.10.7717/peerj.4077/supp-14Data S1Acropora palmata used in the population genetic analyses.DD-Rad Tag reads of Click here for additional data file.10.7717/peerj.4077/supp-15Data S2Acropora palmata. File is formatted for Structure.Microsatellite allele calls at eleven microsatellite loci of Click here for additional data file.10.7717/peerj.4077/supp-16Data S3Acropora palmata. File is formatted for Structure analysis.Allele calls at ten microsatellite loci of Click here for additional data file.


The smallest and most strained member of a family of π‐conjugated cyclic porphyrin oligomers was synthesized by using pentapyridyl templates based on ferrocene and corannulene. Both templates are effective for directing the synthesis of the butadiyne‐linked cyclic pentamer, despite the fact that the radii of their N5 donor sets are too small by 0.5 Å and 0.9 Å, respectively . The five‐porphyrin nanoring exhibits a structured absorption spectrum and its fluorescence extends to 1200 nm, reflecting strong π conjugation and Herzberg–Teller vibronic coupling. In this work, we compared the ability of two pentadentate templates to direct the formation of this cyclic pentamer: FcT5 and T5 cor, which are based on ferrocene and corannulene cores, respectively  are 7.73 and 7.37 Å for FcT5 and T5 cor, respectively. The optimal N5 radius for binding c‐P5, computed by multiple methods, is 8.27±0.07 Å.T5 cor adopts the usual bowl conformation, but upon complexation in c‐P5⋅T5 cor, the bowl becomes flatter, thereby extending the radius of the N5 donor set by 0.17 Å to 7.54 Å. All five zinc centers are in the same plane in the ligand‐free c‐P5 nanoring, whereas they distort into an envelope conformation reminiscent of cyclopentane when c‐P5 binds FcT5 and T5 cor  geometry optimizations using Gaussian09/D.01 at the B3LYP/6‐31G* levelFcT5 was synthesized by phosphine‐activated palladium‐catalyzed aryl–aryl coupling,T5 cor was synthesized from corannulene by iridium‐catalyzed borylation,P1 to give the five‐porphyrin nanoring in yields of 4.0 % for cFc‐P5⋅T5 and 6.1 % for c‐P5⋅T5 cor . GPC analysis shows that the main byproducts in these reactions are larger linear and cyclic porphyrin oligomers (see the Supporting Information). The yield of c‐P5 is consistently higher when using T5 cor rather than FcT5 as the template, for both porphyrin monomers. Addition of excess pyridine quantitatively displaces both templates from their complexes, yielding the template‐free nanorings. The template complexes can be regenerated immediately by adding FcT5 or T5 cor to a solution of c‐P5.Both templates were prepared through transition‐metal‐catalyzed C−H activation Scheme . The ferr Scheme . We also1H NMR spectra of nanoring complexes cFc‐P5⋅T5 and c‐P5⋅T5 cor =6.45 ppm in both complexes .The r Figure  were ful1H NMR spectra, presumably because there is rapid interconversion between five degenerate envelope conformations for each complex. The symmetry of the ferrocene‐based template FcT5 is effectively C5v on the NMR timescale, owing to fast rotation of the isoquinoline substituents and of the phosphine oxide. This symmetry is retained in cFc‐P5⋅T5 and the rims of the nanoring become non‐equivalent, thereby resulting in four β‐pyrrole doublets  β‐pyrrole doublets  and three (rather than six) signals for the aryl protons. As mentioned above, DFT calculations  of the nanoring–template complexes reflect how well the templates fit the cavity of the five‐porphyrin nanoring. The nanoring–template complexes cFc‐P5⋅T5 and c‐P5⋅T5 cor are too stable for their formation constants to be determined by direct titration, so we measured Kf by displacing the templates with pyridine, giving log Kf values of 29.3±0.2 and 28.5±0.1 for cFc‐P5⋅T5 and c‐P5⋅T5 cor, respectively (see the Supporting Information). The chelate cooperativity of complex formation is quantified by the effective molarity (EM), calculated from the formation constant (Kf), the statistical factor (Kσ) of the complex, and the corresponding microscopic binding constant (K1) for the ligand site (isoquinoline for FcT5 and pyridine for T5 cor). The geometric average of the four effective molarities (EM‾) of the five‐coordinate complex can be calculated from Equation The formation constants EM‾=74±20 m),FcT5 and T5 cor for c‐P5.The effective molarities for c‐P5, which has a diameter of 2.1 nm. The corannulene‐based template gives a higher yield of c‐P5, despite being too small and having a lower affinity for c‐P5. The lack of correlation between the size of the template and its ability to direct the formation of c‐P5 may indicate that the transition state for template‐directed coupling is smaller than the final product. The five‐porphyrin nanoring exhibits highly structured absorption and fluorescence spectra and a low radiative rate, thus indicating that emission is strongly suppressed due to the high rotational symmetry of the lowest excited state, with the majority of the fluorescence arising from dynamic symmetry breaking through Herzberg–Teller coupling. This work demonstrates that perfect size‐complementarity is not essential in template‐directed synthesis, and it illustrates how templates can be used to synthesize strained π‐conjugated macrocycles.In conclusion, templates based on ferrocene and corannulene can be used to direct the synthesis of the five‐porphyrin nanoring As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer reviewed and may be re‐organized for online delivery, but are not copy‐edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors.SupplementaryClick here for additional data file.


Africanaspis edmountaini sp. nov., and redescribe Africanaspis doryssa on the basis of new material collected from the type locality of Africanaspis. The new material includes the first head shields of Africanaspis doryssa in addition to soft anatomy for both taxa. Hitherto Africanaspis was entirely described from trunk armour and no record of body and fin outlines had been recorded. In addition the first record of embryonic and juvenile specimens of Africanaspis doryssa is presented and provides a growth series from presumed hatchlings to presumed adults. The presence of a greater number of juveniles compared to adults indicates that the Waterloo Farm fossil site in South Africa represents the first nursery site of arthrodire placoderms known from a cold water environment. The preservation of an ontogenetic series demonstrates that variation within the earlier known sample, initially considered to have resulted from ontogenetic change, instead indicates the presence of a second, less common species Africanaspis edmountaini sp. nov. There is some faunal overlap between the Waterloo Farm fossil site and faunas described from Strud in Belgium and Red Hill, Pennsylvania, in north America, supporting the concept of a more cosmopolitan vertebrate fauna in the Famennian than earlier in the Devonian.Here we present a new species of placoderm fish,  Bothriolepis, groenlandaspidids and erroneously a phyllolepid and macropetalichyid ; ; The depositional environment has been interpreted as representing a brackish coastal back-barrier lagoonal system with significant fresh water input, sheltered by a sandy barrier bar system ; 19]; ; .Africanaspis fossils were initially collected during excavations by RG, between 1993 and 1995 from a thick layer (up to 6 meters) of laminated black shale lenses near Grahamstown, in the Eastern Cape Province of South Africa. This layer was exposed in 1985 during construction of the N2 road bypass, in a cutting at Waterloo Farm  , which has been the prime source of vertebrate material.12.70"E) . FollowiThe field work was done in South Africa with permission obtained from the National Roads Agency of South Africa (the land owners), under a South African Heritage Resources Permit (80/06/11/005/61 issued under section 35(4) of the National heritage Act, Act no.25 of 1999 to collect at this site.Fossils are preserved as near two-dimensional compressions, either as articulated, associated or isolated elements. The vertebrate bone was ultimately replaced by secondary metamorphic mica, which has variably been altered to kaolinite following uplift. Within the placoderms no remnants of the internal perichondral skeletal elements remain. Soft anatomy preservation occurs as white to grey/black impressions of variable clarity or as impressions.Impressions of soft tissues and body outlines occurred when pulses of mud buried their remains rapidly in fine-grained anoxic lagoonal bottom sediments. Regular, possibly riverine, supply of fine sediment is reflected in a thick stack of sediment . When deAfricaspis doryssa, and photographs thereof taken (by RG) prior to this latter preparation, were utilised in this study.Black shale layers were split apart and where necessary matrix covering the dermal plates, or soft tissue impressions, was manually removed. Specimens, prepared post 1997, were photographed under various light conditions and angles (with a Nikon D80 camera and a Micronikor 60 mm lens), in order to best show the morphological features. Previously collected specimens were prepared in 1994 and 1996 by washing the kaolinite away with 70% alcohol solution, manually removing clay particles, sealing the surface with a solution of 25% glyptal cement and acetone, and then making latex peels [Following preparation all specimens were registered in the collections of The Albany Museum as AM 5246; AM 5247; AM5905; AM 4907; AM 5924; AM 5923; AM 7502; AM7503; AM 5943; AM 5921; AM 5920; AM5242; AM5922. Specimen repository is located within the Albany Museum Somerset St, Grahamstown, 6139, South Africa.http://zoobank.org/". The LSID for this publication is: urn:lsid:zoobank.org:pub: 562DF258-104A-4B38-A731-BF151C1A0570. 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, 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 "ADL, anterior dorsolateral plate; ADL. un, anterior dorsolateral plate underlap; af, anal fin; AL, anterior lateral plate; AL ov., overlap area for the anterior lateral plate; APi, anterior pineal plate; art.c, articular condyle on the ADL; AVL, anterior ventrolateral plate; b.i., body impression; Ce, central plate; cf, caudal fin; csc, central sensory canal; df, dorsal fin; ?g, possible gut; ioc, infraorbital canal; kd, glenoid condyle; lc, main lateral line canal; M, marginal plate; M ov, overlap area for the marginal plate; MD, median dorsal plate; mt, mineralised tissue; Nu, nuchal plate; Or, orbit; orb m, orbital margin; ot c, otic capsule; pd, posterior denticles on the median dorsal plate; PDL, posterior dorsolateral plate; PDL. un, posterior dorsolateral plate underlap; pf, pectoral fin; PL, posterior lateral plate; PL ov, overlap for the posterior lateral plate; PM, post marginal plate; PM ov, overlap area for the post marginal plate; pmc, postmarginal sensory canal; PN, post nasal plate; PNu, paranuchal plate; pp, posterior pit line groove; PPi, posterior pineal plate; PrO, preorbital plate; PtO, postorbital plate; PtO ov, overlap area for the postorbital plate; PVL, posterior ventrolateral plate; R, rostral plate; rdg, ridge; SG, supragnathal SO, suborbital plate; soc, supraorbital sensory canal; Sp, spinal plate; vbw.AM, Albany Museum, Grahamstown.Placodermi Woodward, 1891 Arthrodira Woodward, 1891 Suborder Phlyctaenioidei Miles, 1973 Infraorder Phlyctaenii Miles, 1973 Family Groenlandaspididae Obruchev, 1964 Africanaspis Long, Anderson, Gess, Hiller, 1997 [er, 1997 Witpoort Formation  from Waterloo Farm, south east of Grahamstown, Eastern Cape, South Africa .Late Famennian, DevonianShort, high trunk covers the anterior body, posterior to thoracic armour the body tapers and there is a long whip-like tail. Anterior dorsolateral plate with relatively straight anterior margin. Step on anterior dorsolateral plates accommodates the anterior margin of the median dorsal plate. No overlap between left and right Anterior dorsolateral plates. Anterior and posterior dorsolateral plates with straight medial margins. Tall posterior dorsolateral plate, posterior margin concave in lateral view, distinct shoulder ventral to the posterior contact with the median dorsal plate. Median dorsal plate higher than long, pointed and slightly anteriorly posteriorly re-curved. Median dorsal anterior margin smooth, posterior margin ornamented with a serration-like series of modified denticles. The anterior lateral plate is large, sutured to the entire width of the lateral trunk armour, and laterally angled.The diagnosis of this genus has been emended from Long et al. to complAfricanaspis doryssa Long, Anderson, Gess, Hiller, 1997 [er, 1997  .AM 5247, MD plate with displaced anterior dorsolateral plate; AM5905 complete partially dissociated juvenile; AM 4907 a & b complete skull roof with M, and PM plates, tooth plates, Sp and AL plates; AM 5924, MD plate; AM 5923, MD plate, PDL and ADL plates; AM 7502 largely articulated partially preserved, MD plate, ADL, PDL, AL, Sp, Nu plates and dorsal tail outline; AM7503 wholebodied hatchling. AM 5943; complete skull roof with cheek plates.A. doryssa. Thus the specimen AM4907 is here identified as A. doryssa, based on the revised diagnosis below whereas previously it had been assigned as a juvenile Groenlandaspis riniensis [The recent recovery of multiple arthrodire juveniles, including embryos, from Latvia, Canada and Australia –18, 26 hA. doryssa based on the similarity of the head shield plates with the above referred material, however no trunk shield plates were recovered with this specimen.AM 5943 is tentatively identified as Africanaspis with skull roof longer than wide . Head armour approximately twice the length of trunk armour in dorsal view contra [w contra . Rostralw contra ) with weThe diagnosis of this species has been emended from Long et al.  to complIn dorsal view the anterior margin of the skull roof is rounded . There iThe rostral plate is partially preserved in AM 4907b , its venThe postorbital plates predominately occupy a lateral position on the cheek Figs  and 3. TThe path of the supraorbital sensory line canals extends from the anterior of the preorbital plates, onto the central plates . There iGroenlandaspis, and in cross section it is bell-shaped. The single median dorsal plate is high with a narrow, bilobed base and in lateral view is triangular in shape    is consiTiaraspis and Turrisaspis. However, in the juvenile these are the largest plates of the skull roof, 1.5 times larger than the preorbital plates and approximately the same length as the paranuchal plates. Variation in the length of the posterior lobe on the central plate is seen between the adult specimens of A. doryssa. This variation is also present in Tiaraspis subtilis with well defined posterior lobes present in two specimens  but absent in Go 807-2  and in g 7 A-B; ). Variatg 7 A-B; , 39.Compagopiscis croucheri [A. doryssa. The presence of a well defined median ridge and the lack of a median boss on the nuchal plate is similar to T. elektor [Tiaraspis [Tiaraspis differs from the other genera in that the nuchal plate is club-shaped and the caudal region of the plate narrows.The juvenile (AM5905) nuchal plate is shorter and broader than in adults; a morphology also present in the eubrachythoracid arthrodire roucheri , 28. The elektor  and Tiariaraspis  althoughTiaraspis incorporate the postnasal plates into their anterior margin. The preorbital plates participate in the orbital margins. The orbits are intermediate in size, being smaller than in Tiaraspis but larger than Turrisaspis. In Tiaraspis sclerotic rings form the orbital margin, but these have not been reported in Turrisaspis and are not preserved in specimens of Africanaspis. The orbits of the juvenile of A. doryssa are larger than those of the adult as is common amongst vertebrates [The preorbital plates extend onto the face and as in tebrates , 12.A. doryssa is reconstructed as comprising the postorbital, marginal postmarginal and suborbital plates. The postorbital and marginal plates are larger than in Tiaraspis but as in Tiaraspis the marginal plate separates the postmarginal plate from the postorbital plate. In Turrisaspis the postorbital and postmarginal plates suture under the marginal plate, and form the ventral margin of the cheek and this feature is considered unique for the species [Tiaraspis subtilis with both suborbital and submarginal plates, although it is noted that these were not recovered and that the large orbits leave little space for them. Indeed the space filled by the submarginal in Schultze’s [Turrisaspis  althoug a bony iaraspis  and Turrrisaspis .A. doryssa is similar to Turrisaspis, which is the only other taxon reported where the path of the supraorbital sensory line canal disappears across the central plates and then resumes on the paranuchal plate, having presumably passed beneath the outer surface of the plate. In Turrisaspis this character was also noted to be variable [The path of the sensory line canals in variable . Signifivariable , 39.Trunk shield reconstruction Figs  and 4FA. doryssa [A. doryssa and A. edmountaini sp. nov. (see below).The original reconstruction of the dorsal and lateral trunk armour of  doryssa  is here A. doryssa is similar in proportions to those of Turrisaspis [Tiaraspis [Groenlandaspis thorezi [Groenlandaspis, the median dorsal plate of Groenlandaspis antarcticus forms a low cap uniting the left and right dorsolateral plates [Mithakaspis, another high median dorsal plate bearing arthrodire. In this taxon the ADL plates overlap anterior to the median dorsal plate. In addition Mithakaspis uniquely has ADL and PDL plates that overlap the median dorsal plate, contra the normal placoderm condition [The tall median dorsal plate of risaspis  Tiaraspiiaraspis  and Groe thorezi , all of l plates . Separatondition .A. doryssa comprises vertical striations whereas Turrisaspis [Tiaraspis [A. doryssa to have an ornament of both horizontal wavy rows and linear striations , whereaTurrisaspis, Tiaraspis and A. doryssa therefore share the character of high and narrow dorsolateral plates of the trunk shield. These are remarkably distinct from the low antero-posteriorly broad dorsolateral plates typical of Groenlandaspis , or themeraspis . Compare. The ane fig 2,). In A. iaraspis , 38 a ra,. In A. risaspis  and the andaspis  whereas risaspis . This hyn adults , 44.nd professor of geology at Rhodes University and childhood geological mentor of RGFor Edgar Donald Mountain 2“groenlandaspidid” Gess and Hiller [1995 d Hiller  p 285, fAfricanaspis doryssa Long et al. [1997 g et al.  p. 262, AM 5921 a & b Figs  completeAM 5920 a & b  completeWitpoort Formation , Waterloo Farm, Grahamstown, South Africa. Late Famennian .A. doryssa [AM5242, MD plate . MediumAfricanaspis median dorsal plate (AM 5242) had a distinctly different ornament and height to length ratio compared with other assigned median dorsal plates and suggested that more than one species could be present. However, it was rather concluded that this variation was commensurate with ontogenetic variation and due to small sample size it was decided not to place this one specimen within a new species [A. doryssa and A. edmountaini, we can now determine that AM 5242 does in fact represent a separate species, being conspecific with A. edmountaini.Long et al.  noted thlate AM 542 had a A. doryssa the dorsal and lateral trunk shield plates are best preserved, with the median dorsal plate being the most common. The median dorsal plate is high and broad (L/H 2:<3) with caudally directed short serrations  Figs . The venThe outline of the mid body region and tail are preserved as an impression in AM5921a and b Figs . A singlA. edmountaini sp. nov. and the outline of the head of A. doryssa has been used to guide the reconstruction. The median dorsal plate is shorter and laterally wider than in A. doryssa. It has a far more robust vertical linear ornament than that of A. doryssa and the serration-like row of denticles along the posterior margin are shorter and caudally directed, as opposed to those of A. doryssa which are more elongated and more vertically directed. The anterior process on the median dorsal plate is thinner and does not extend as far towards the glenoid process as in A. doryssa, though as in A. doryssa the anterior margins of the median dorsal plate and anterior dorsolateral plate form a straight profile. The anterior lateral plate is not as deep as that of A. doryssa. The post thoracic region of the body is long and the dorsal fin is positioned far back along the dorsal margin of the tail. The trunk region immediately posterior to the thoracic armour is reconstructed as it is ruptured, probably due to the expulsions of gases of decomposition, and the specimen has probably suffered some post-mortem shrinkage of the soft tissues c.f. [Currently there are no plates known from the head shield of ues c.f.  such thaA. doryssa specimen  commonly occurs posterior to the pelvic fins, above the claspers in arthrodires and more posteriorly in ptyctodonts [Parayunnanolepis xitunensis [Bothriolepis canadensis [In contrast to the poor preservation of the mineralised dermal skeleton of the Waterloo Farm fossils the body and tail are well preserved as 2D carbonaceous films, although as noted above there is no preservation of the axial skeleton Figs . Similarlaauweni , a jawleustralia , 47. In rocesses  and it arsal fin  and so tctodonts  and antitunensis  and Bothnadensis .Bothriolepis [The dorsal margin of the body appears to be intact, although the ventral body margin is not completely preserved and has ruptured to reveal a tubed shape containing a mass of dark carbonaceous matter, possibly the gut, in AM 135921 . In otheriolepis  and in ariolepis , which sAfricanaspis, conforms to this definition. Studies on extant show that few species are limited to a single nursery habitat, the majority utilizing a mosaic of connected habitats due to tidal conditions, feeding, protection and cover seeking behaviours [Contra [The sheltered estuarine depositional environment, in which the fossils have been recovered, was first suggested as a nursery site due to the abundance of antiarch and arthrodire juveniles . Furtherhaviours –58. The haviours . The ver [Contra  sites wi [Contra , 61. In  [Contra , 62, 63  [Contra , 64–66, Africanaspis remains unknown. The small size of AM7503, identified as a neonate, suggests hatching or birthing within the estuarine environment. However, although juveniles dominate this site, the numbers are not indicative of a spawning site such as Lode Quarry, Latvia or Tioga County, Pennsylvania. Internal fertilization with egg laying (oviparity) is a conceivable strategy. However, internal fertilization with live birth (viviparity) cannot be ruled out as a mode of reproduction because embryos recovered from within specimens of Incisoscutum [Africanaspis known. Although internal fertilization, with either oviparity or viviparity correspond with the number and size of embryos recovered for Africanaspis, to date no evidence of sexual dimorphism, an indicator of internal fertilization within vertebrates, is known for Africanaspis. It is however pertinent that the presence of intromittant organs is not a prerequisite for internal fertilisation. Modern coelacanths lack such organs but bear live young [Undina pencillata, [Despite the preservation of embryos the reproductive strategy of soscutum  fall witve young , 68, as cillata, Hyneria-like tristichopterid sarcopterygian, in addition to gyracanthid acanthodians, actinopterygians, a dipnoan, a species of Groenlandaspis, an Africanaspis-like arthrodire [Africanaspis-like arthrodire, actinopterygians, dipnoans and a tristichopterid sarcopterygian. In addition they both have acanthodii and a bothriolepid antiarch [68, [Olive et al.  have drathrodire , 72, andrch [68, . It is prch [68, , coupledrch [68,  permitterch [68, ,7,8 fromTurrisaspis, Tiaraspis and Africanaspis. Notably they share a distinctive high-spired median dorsal plate (H:L index >1) separating the ADL plates and high cranio-caudally shortened dorsolateral trunk armour. They share with some but not all species of Groenlandaspis, a raised crest running from the condyle of the ADL plate to centre of the caudal margin thereof and a nuchal plate lacking a median boss . Though unconfirmed in Tiaraspis they most likely also share the presence of two pineal plates. A close relationship between Africanaspis, Turrisaspis and Tiaraspis was previously questioned [Africanaspis, as described by Long et al. [Tiaraspis and Turrisaspis. In addition we note that this study further demonstrates that contra Long et al [et al[Africanaspis doryssa does not become broader in lateral view with age, as this interpretation resulted from confusion of two discrete species. Publication, for the first time of Africanaspis head material reveals further similarities between Africanaspis and Turrisaspis.We agree with Daeschler et al.  that it estioned  on the bg et al. , not being et al  and Olival [et al the MD pAfricanaspis, making it a rare component of the placoderm fauna. Newly prepared and collected material including one specimen with associated head and trunk shield plates (AM4907), an additional adult skull (AM 5943) and the complete armour of a juvenile (AM 5905) has resulted in a headshield originally attributed as a juvenile of Groenlandaspis riniensis [A. doryssa (AM 4907). In addition, impression of soft tissue morphology has allowed parts of the post thoracic body and fins to be described within the genus Africanaspis for the first time. Variation previously attributed to ontogeny within A. doryssa is diagnostic for a new species A. edmountaini. Full size ranges of specimens, from neonatal to presumed adults, suggest that Africanaspis was a resident reproducing within the estuarine setting of deposition. Intriguing similarities and differences are noted between the faunas of the Africanaspis-bearing Waterloo Farm, and Turrisaspis-bearing Red Hill, Pennsylvania and Strud Quarry, Belgium sites. The high paleolatitude and Gondwanan setting of Waterloo Farm contrasts with the equatorial paleolatitude and Laurasian setting of the latter sites. Marked similarities between them support possible reduction in faunal provincialism towards the end of the Devonian [Waterloo Farm has been interpreted as a high latitude estuarine environment and to date 370 identifiable fish fossils  have been recovered. The dominant preserved component of the fauna were placoderms (58%) and of these 76% were arthrodires, however, only 8% of arthrodire specimens have been identified as belonging to the genus iniensis  being reDevonian .


The rate of patients who underwent genetic testing increased from 60% in the pre-protocol time period to 77% in the post-protocol time period (p < 0.01). The rate of diagnosis of genetic conditions during the pre-protocol period was 26% versus 36% during the post-protocol period (p = 0.01). There was a reduction in cost to patients by $5105.59 per diagnosis during the post-protocol period. Patients with critical CHD in the post-protocol period were less likely to undergo multiple genetic tests and more likely to have a diagnosis of genetic disease. In addition there was a significant reduction in cost per diagnosis during the post-protocol time period. Genetic testing protocols for infants with critical CHD promoted more efficient use of genetic testing and increased the rate of diagnosis of genetic conditions in this population.Genetic testing is routinely performed on infants with critical congenital heart disease (CHD). This project reviewed the effect of implementing a genetic testing protocol in this population. Charts of infants with critical CHD were reviewed for genetic testing and results across two time periods: the time before implementation of a genetic testing protocol (pre-protocol) and the time after implementation (post-protocol). The use of karyotype, 22q11.2 Deletion testing, and chromosomal microarray were compared across these two time periods. Records of 891 infants were reviewed. 562 (63%) had at least one of the target genetic tests completed. During the pre-protocol time period, 66% of patients who had genetic testing underwent multiple tests versus 24% during the post-protocol time period ( Patients with critical congenital heart disease (CHD) have a high rate of copy number variants, particularly 22q11.2 Deletion Syndrome, yet clinical use of chromosomal microarray (CMA) in patients with CHD is unpredictable and not standardized . PreviouIn order to determine the rates of genetic testing in infants with critical CHD the genetic testing practices and yields for two time periods were examined. Baseline data were obtained through a project approved by the Institutional Review Board which resulted in the genetic testing protocol. Follow-up data were obtained as part of an Institutional Review Board exempt status quality improvement project determining the adherence to the protocol.The Society for Thoracic Surgeons Database was queried for infants who had CHD requiring surgical intervention at less than a year of age. The first time period, or “pre-protocol” time period, includes infants who underwent cardiac surgery at less than one year of age on or after January 1, 2010 who were born on or before December 31, 2014. The second time period, or “post-protocol” time period, includes infants born between January 1, 2015 and June 30, 2016 who had cardiac surgery at less than a year of age before June 30, 2016. Infants born at less than 30 weeks gestation or who underwent patent ductus arteriosus repair were excluded.All genetic testing recorded in the medical record was identified and was marked as normal or abnormal based on how it was clinically reported. Focus was on the three most commonly utilized genetic tests addressed by the protocol: G-Banded Karyotype, diagnostic testing for 22q11.2 deletion, and chromosomal microarray (CMA). The G-banded Karyotype has a resolution of identifying chromosomal abnormalities greater than five megabases. Karyotype testing can detect balanced translocations and structural changes not resulting in a change in copy number that could be missed by microarray and can detect low level chromosomal mosaicism. The method for diagnostic testing for 22q11.2 deletion included florescence in situ hybridization (FISH) for TUPLE1 and quantitative polymerase chain reaction (QPCR) for TBX1. FISH probe for TUPLE1 has a sensitivity of greater than 95% for 22q11.2 deletion syndrome and was available for the duration of the study. QPCR for TBX1 copy number became available to infants at Children’s Hospital of Wisconsin in August 2011 and has a sensitivity of greater than 95% for 22q11.2 deletion syndrome. In September 2012 this assay began to include copy number of CRKL to further enhance the sensitivity of detecting 22q11.2 deletions. The CMA utilized at Children’s Hospital of Wisconsin reports deletions larger than 200 kilobases and duplications larger than 500 kilobases. CMA is the most sensitive genetic test out of these options, and will pick up everything detectable by karyotype and 22q11.2 deletion testing with the exclusion of balanced translocations and low level chromosomal mosaicism which may only be detectable by karyotype.The genetic testing protocol was implemented on January 1, 2015. The protocol was introduced to the department of pediatrics and affected clinical divisions  through departmental lectures and division meetings starting in April 2014. Our basic testing protocol is demonstrated in Table All genetic diagnoses listed in the medical record were noted . Genetic diagnoses associated with congenital heart disease  were used to classify patients as being diagnosed with a genetic condition. For the specific protocol diagnosis rate the genetic diagnosis was only recorded if it was a direct result of the three target tests ordered. If a patient had a genetic diagnosis not made by the testing it was not recorded  and this protocol diagnosis rate was utilized to determine the calculation of cost per diagnosis. All charts were reviewed for genetic diagnosis by a single author for consistency (GCG).In order to determine the effect of the protocol on cost to patients we assessed cost and compared pre-protocol to post-protocol periods. To determine cost baselines we queried three large genetic testing providers on the same day and averaged the quoted prices of the three providers and averaged them to create a cost for each test. The average costs at a single discrete time point were utilized to standardize for changes in cost over time. These were $895.17 for karyotype, $669.92 for 22q11.2 Deletion Testing, and $2615.33 for CMA. To determine the effect on value we used the cost data per patient and per diagnosis.Descriptive statistics identified the proportion of specific CHD group for each classification system represented within the sample and the frequency of abnormal genetic test results in proportion to tests completed. Two-tiered Fisher’s exact test was utilized to determine statistical significance of changes between groups.Medical records of 891 infants were examined. There were 509 (57%) male infants and 382 (43%) female infants. Among these 891 infants 109 were diagnosed with Trisomy 21 and 36 were diagnosed with 22q11.2 Deletion Syndrome. Overall 562 (63%) had at least one of the three genetic tests completed. Population characteristics are summarized in Table p < 0.01) in utilization of multiple genetic tests. Testing patterns across the two time periods are demonstrated in Fig. During the pre-protocol time period 66% of patients who had genetic testing underwent multiple tests. During the post-protocol time period 24% of patients who had genetic testing underwent multiple tests. This was found to be a significant decrease p < 0.0 in utilip < 0.01) of patients undergoing genetic testing in the post-protocol period.During the pre-protocol time period 60% of patients had genetic testing. During the post-protocol time period 77% of patient had genetic testing. This was found to be a significant increase (p < 0.01) as was the increase in yield of 22q11.2 deletion testing (p = 0.03). There was no difference in yield of microarray between the pre-protocol and post-protocol time periods (p = 0.78). This is illustrated in Table In the pre-protocol time period the rate of abnormal karyotypes was 18%, the rate of abnormal 22q11.2 deletion testing was 9%, and the rate of abnormal microarray was 24%. In the post-protocol time period the rate of abnormal karyotype was 76%, the rate of abnormal 22q11.2 deletion testing was 26%, and the rate of abnormal microarray was 22%. The increase in yield of karyotype was found to be significant (g p = 0.0. There wp = 0.0005). We suspect this is a reflection of clinical ascertainment bias given that only 29% patients with septal lesions had microarray testing completed.The breakdown of testing patterns and yield of testing by lesion is demonstrated in Table p = 0.01). When patients with Trisomy 21 were excluded the rate of diagnosis of genetic conditions during the pre-protocol period was 16% and during the post-protocol period was 26%. This increase in diagnosis rate among patients without Trisomy 21 was also significant (p = 0.01).The rate of diagnosis of genetic conditions associated with congenital heart disease during the pre-protocol period was 26%. The rate of diagnosis of genetic conditions associated with congenital heart disease during the post-protocol period was 36%. This increase in diagnosis rate was significant (p < 0.01). As result, the average cost per patient for genetic testing was higher prior to the initiation of the protocol, $2720.77 verse $2403.27. When we analyzed the cost per genetic diagnosis from testing ordered we found similar results with the average cost per genetic diagnosis being significant less after the protocol was initiated, $11,427.24 verse $6321.65. The cost of testing per time period is further demonstrated in Table Prior to the protocol initiation, fewer patients underwent genetic testing (60 vs. 77%), but of the patients who did undergo genetic testing, they underwent multiple tests more frequently  and 22q11.2 deletion testing (9–26%) after initiation of the protocol.There are still clinical nuances to choosing genetic testing this protocol does not full address. Microarray testing has increased resolution to detect clinically significant chromosomal anomalies compared to karyotype, but important limitations of microarray include the inability to detect balanced translocations or determine the structural location of an anomaly as well as the inability to detect low level chromosomal mosaicism, which can be important when considering mosaic conditions like Turner Syndrome. Microarray is also not always the best test for the situation. For example, testing with karyotype Trisomy 21 is still appropriate as karyotype is the recommended standard of care to assess for translocations . All of Our study does have limitations. We are a tertiary referral center, and as a result it is possible that additional testing following discharge was performed at an outside institution that was not available at the time of chart review. As all charts were reviewed for testing by a single author (GCG) with a consistent, standard method it is likely limitation would be uniform throughout each time period. In addition, cost and value calculations are highly variable and continue to evolve over time, making retrospective reviews challenging.Despite these limitations, we feel that our data support a standardized genetic testing protocol for infants with critical cardiac disease can significantly impact the rate of identification of chromosomal anomalies and is cost effective in this complex, high risk population. We recommend institutions consider implementing genetic testing protocols for this population, ideally in conjunction with a medical geneticist or genetic counselor.


Gasterosteus) populations in Otsuchi Town, which was one of the most severely damaged areas after the tsunami. Our environmental surveys showed that spring water may have contributed to the habitat recovery. Morphological analysis of the stickleback before and after the tsunami showed morphological shifts in the gill raker number, which is a foraging trait. Genetic analyses revealed that the allelic richness of one population was maintained after the tsunami, whereas that of another decreased in 2012 and then started to recover in 2013. Additionally, we found that the large tsunami and ground subsidence created new spring water-fed pools with sticklebacks, suggesting that the tsunami brought sticklebacks into these pools. Genetic analysis of this population showed that this population might be derived from hybridization between freshwater Gasterosteus aculeatus and anadromous G. nipponicus. Overall, our data indicate that tsunamis can influence morphologies and genetic structures of freshwater fishes. Furthermore, spring water may play important roles in the maintenance and creation of fish habitats, faced with environmental disturbance.On March 11, 2011, a large earthquake occurred, causing a tsunami which struck the Pacific coast of northeast Japan. We investigated the ecological and genetic effects of the large tsunami on the threespine stickleback (genus  New habitats can also be created by catastrophic events4, where founder organisms can invade and may rapidly change their phenotypes to adapt to the new environments7. As catastrophic disasters are usually unpredictable, few opportunities are available to investigate their before and after effects of the catastrophic events on natural populations.Natural disasters, such as typhoons, volcanic eruptions, earthquakes, and tsunamis, can cause catastrophic damage, not only to human livelihood but also to natural animal and plant populations. Such catastrophic events may lead to drastic changes in the habitat qualities of various organisms, resulting in a decrease or possibly an increase in the numbers of particular species, and also affect dispersal of organisms19. In these cases, the tsunamis disturbed the habitats of wild aquatic animals by changing landforms, deteriorating water and soil qualities, and bringing in sediments and debris. Tsunami waves also flushed several marine and freshwater organisms and displaced them from their native habitats to non-native places13. In addition, tsunami waves transported marine materials, such as seawater and sea bottom slime, into freshwater habitats15, and thereby, freshwater-adapted and stenohaline organisms, including several freshwater fishes, may become extinct in these salinized environments20. These effects can lead to biodiversity loss in tsunami-inundated areas.Huge tsunamis, triggered by immense earthquakes, are one of the largest catastrophic events and can disturb aquatic and terrestrial ecosystems along coastal areas. In the 21st century, several huge tsunamis have occurred: the 2004 Indian Ocean tsunami, the 2010 Chilean tsunami, and the 2011 Tohoku-oki tsunami. Previous studies have focused on the effects of tsunamis on aquatic ecosystems22. A tsunami may also induce gene flow between populations. Gene flow can homogenize genetic differentiation between locally-adapted populations and may reduce the mean fitness of each population, although gene flow from different populations can occasionally increase genetic variation and increase the ability of adaptive evolution23. Therefore, it is crucial to investigate the changes in genetic structures for a better assessment of the extinction risk of natural populations affected by a tsunami.Although the effects of tsunamis on ecosystems have been investigated, we know little about how they affect the phenotypes and genotypes of organisms. Catastrophic events can reduce the genetic diversity and effective population sizes, thereby, increasing the risk of extinction, as a result of multiple factors, such as inbreeding depression, reduction of the standing genetic variation, and the accumulation of deleterious mutations24. Otsuchi Town, which is located at a Pacific coastline of the Iwate Prefecture, Japan, was one of the most severely damaged areas of the 2011 tsunami  in Otsuchi Town. The threespine stickleback is a small cold-water fish, which is widely distributed in the coastal regions of the Northern Hemisphere25. The threespine stickleback has both marine/anadromous and freshwater resident-life histories; the ancestral marine/anadromous sticklebacks have colonized freshwater repeatedly in widespread regions, resulting in phenotypic diversification and the evolution of diverse ecotypes27. In addition, this fish has many types of species pairs29. Thus, the threespine stickleback is considered a model for evolution, ecological, and genetic research. In Japan, there is a unique species pair composed of G. aculeatus and G. nipponicus 30. These two species are thought to have diverged about 1.0 million years ago32. These sticklebacks are sympatric in several habitats and reproductively isolated with low levels of on-going hybridization. They have different life history characteristics; some G. aculeatus are freshwater residents and others are anadromous, while G. nipponicus is exclusively marine or anadromous34.The present study aims to elucidate the ecological and genetic effects of a large tsunami on the threespine stickleback  and typically ranges from 9.0 to 11.0 mS/m in this river ; at the present time, large numbers of mature adult sticklebacks and their offspring are found every year.In the Gensui River  analysis of the morphological traits of the adult sticklebacks was conducted, following body size correction of the external morphological traits, using the standard length Table . The firP < 0.0001). Interestingly, all PC scores showed similar temporal changes in both the Gensui and Namaisawa populations  and PC3 (P = 0.1736), whereas the effect of the year was significant in both PCs , while PC2 did not differ between 2012 and 2013 (P = 0.265). A scatterplot of PC1 and PC3 . Since hybridization can increase the phenotypic variance and change the phenotypic variance-covariance matrix40, we calculated the ratio between the length of the major and minor axis of the ellipse (eccentricity) and the size of the ellipse ; both the Gensui and Namaisawa populations in 2012 and 2013 had smaller gill raker numbers, than those populations before 2011 and the 2012 and 2013 new-pool populations . However, there was no significant difference between 2012 and 2013 in the new-pool populations  showed a substantial reduction in 2012  and recovered slightly in 2013  indicated that the most likely cluster number was three (K = 3)  of G. nipponicus in the Namaisawa River (nearly 100% cluster 3 assignment), but no G. nipponicus was observed in 2012 and 2013  in July of 2011 . In both places, therefore, immediate removal of debris by humans and a continuous supply of spring water potentially played a role in the recovery of the stickleback habitats. This idea is supported by our data of the temporal change in EC  and to freshwater .Our genetic analysis revealed that the genetic diversity of the Gensui population did not reduce after the tsunami Fig. . There mG. nipponicus (cluster 3). In fact, our genetic results revealed that G. nipponicus migrated into the Namaisawa River before the 2011 tsunami, but not after the tsunami . In 2013, fish belonging to cluster 1 increased, although the reason for this increase is currently unclear. Further long-term monitoring of the number of anadromous migrants, the allelic richness, and the genetic structures will help to identify the factors that affect the genetic structure in this population.In contrast, the allelic richness of the Namaisawa population reduced in 2012 Fig. . This reami Fig. . A reducG. nipponicus (cluster 3), and their hybrids , when the water levels of the rivers and pools rise due to spray tides and rainfall, which enables anadromous sticklebacks to migrate into these pools. These new pools have been maintained through present day by discharging spring water that comes from flowing wells.We found that the tsunami created new stickleback habitats Fig. . New pooids Fig. . Our hypG. aculeatus and anadromous G. nipponicus seem to be hybridizing in the new tsunami-formed pools (hybrids between cluster 1 and cluster 3). Hybridization can often increase phenotypic diversity and change the phenotypic variance-covariance matrix, which may help the hybrid populations adapt to new environments40. Our analysis of the new pool population did not show any apparent changes in the variances and the phenotypic matrix. However, further analysis of the morphological changes would be necessary to make any conclusions about the roles of hybridization in adaptation to the new pool habitats. Recently, stickleback adaptations to newly formed environments, following large earthquakes and tsunamis in the wild, were investigated in a different region, but the formation processes of the new habitats and populations were different. A previous study showed that new habitats were formed because of an island uplift, which was caused by the 1964 Great Alaskan Earthquake, and then anadromous G. aculeatus stickleback invaded the new habitats7. In our case, new habitats were formed by ground subsidence and spring water, and both anadromous G. nipponicus and freshwater G. aculeatus sticklebacks invaded. Thus, the new pool population in Otsuchi provides an opportunity to investigate mechanisms by which freshwater and coastal fishes disperse, hybridize, and adapt to new environments.Our genetic data indicate that the freshwater population of 49. However, it is worth noting that recovery projects, including natural disaster contingency planning, are now being performed in Otsuchi Town50. These activities may ironically threaten various ecosystems. For example, construction of temporary houses, after landfills of spring water . All experiments were performed in accordance with relevant guidelines and regulations.35, only the freshwater-resident G. aculeatus inhabits the Gensui River, while both freshwater-resident G. aculeatus and anadromous G. nipponicus were found in the Namaisawa River.We conducted field surveys at the Gensui River , which is a tributary of the Otsuchi River, and the Namaisawa River , which is a tributary of the Kozuchi River; both rivers run through a lowland area of Otsuchi Town , Iwate Prefecture, Japan Fig. . Downstr51. During our study, on July 22, 2012, we found threespine sticklebacks in new spring water pools in the urban area for the first time, so we have no samples before that time. We have sampled sticklebacks in these two pools , electrical conductivity  and pH were recorded using a multi-parameter water quality probe . These measurements were conducted nearly every month from April 2011 to August 2014. Before the 2011 earthquake, we had measured some of these parameters in both rivers. Also, we started these environmental measurements in the tsunami-formed pools (P1 and P2) after July 2012.Samples were collected using minnow traps for adults and dip-nets for juveniles. Adult fish used for morphological analyses were sampled from the Gensui River at survey site G2 on December 22, 1998, December 24, 2011, July 1, 2012, and May 19, 2013; from the Namaisawa River at survey site N3 on June 26, 2010, December 24, 2011, July 29, 2012, and May 20, 2013; and from the new-pool at survey site P1 on July 29, 2012 and May 19, 2013  and Namaisawa Rivers (N3) on June 26, 2010, May 26, 2011, August 21, 2012, and June 29–30, 2013, , and counted the gill raker number using a dissecting microscope , followed by pairwise post-hoc Mann-Whitney U-tests with Bonferroni corrections (α = 0.0009). These analyses were performed in R version 3.0.252.We used 233 adult sticklebacks (range: 18–41 for each group) for the morphological analyses , and the 5′-end of the reverse primers were tailed with GTTTCTT to increase the accuracy of the fragment length analysis54. Microsatellites were amplified with three combinations of primer sets, with three different dye colors, using the KAPA2G Fast Multiplex PCR Kit . After 3 min at 95 °C, 30 cycles of 95 °C for 15 sec, 60 °C for 30 s, and 72 °C for 30 s were performed, followed by 10 min at 72 °C. Amplified fragments were analyzed by BEX Co. Ltd. . Allele lengths were then determined using Peak Scanner Software . Micro-Checker was used to confirm the accuracy of genotyping55.We used 360 sticklebacks (range: 16–102 for each habitat) for the genetic analyses Table 53: Stn9056. Five simulations were run for each cluster number (K) from K = 1 through K = 10. We estimated the probable number of clusters by finding the K value with the highest log likelihood Ln(K) and the K value with the highest ΔK, which is the rate of change of Ln(K) between successive K values57. This analysis was performed using Structure Harvester58. Parameters were estimated after 500,000 iterations, following a burn-in of 50,000 iterations. The allelic richness  was calculated using FSTAT 2.9.3 software59. In order to confirm yearly changes in the allelic richness within each population, we conducted the Friedman test (α = 0.05), followed by pairwise post hoc Wilcoxon signed ranks tests with Bonferroni corrections (α = 0.0083), and Wilcoxon signed ranks test (α = 0.05). These analyses were performed in R version 3.0.2.52.Data were first analyzed using STRUCTURE, which uses Markov chain Monte Carlo simulations to identify groupings that minimize Hardy-Weinberg and linkage disequilibrium within cluster groupsSupplementary Information


The results from urban scaling in recent years have held the promise of increased efficiency to the societies who could actively control the distribution of their cities’ size. However, little evidence exists as to the factors which influence the level of urban unevenness, as expressed by the slope of the rank-size distribution, partly because the diversity of results found in the literature follows the heterogeneity of analysis specifications. In this study, I set up a meta-analysis of Zipf’s law which accounts for technical as well as topical factors of variations of Zipf’s coefficient. I found 86 studies publishing at least one empirical estimation of this coefficient and recorded their metadata into an open database. I regressed the 1962 corresponding estimates with variables describing the study and the estimation process as well as socio-demographic variables describing the territory under enquiry. A dynamic meta-analysis was also performed to look for factors of evolution of city size unevenness. The results of the most interesting models are presented in the article, whereas all analyses can be reproduced on a dedicated online platform. The results show that on average, 40% of the variation of Zipf’s coefficients is due to the technical choices. The main other variables associated with distinct evolutions are linked to the urbanisation process rather than the process of economic development and population growth. Finally, no evidence was found to support the effectiveness of past planning actions in modifying this urban feature. The regularity in city size distribution has been known for more than a century . Auerbaci of population double to that of j is more than twice the output of j. In other words, larger cities tend to be, on average, richer per capita. On the contrary, the amount of infrastructure scales sublinearly and shows economies of agglomeration in larger cities. The resulting aggregate amount of wealth or infrastructure in a system thus depends eventually on the distribution of sizes of the different cities in the system . These urban features can be linked back to the size distribution of cities in a given system [More recently, research on urban scaling laws has shown that some indicators of urban efficiency could be expressed as a power law of urban population , 5. For n system . This liHowever, before trying to inform policies in such a way, three questions arise: 1/ is it possible to control the distribution of city sizes in a country at all? 2/ What drives the value of this slope? and 3/ have previous policies ever been successful in modifying the slope of the rank-size rule? I will try to answer these questions, relying on the past 100 years of literature, starting by unpicking undesirable fluctuations from genuine variations in the measured values of urban unevenness. The deviations observed thus can arise from four sources: estimation biases, measurement “errors”, authentic spatio-temporal variations and as a result of planning policies.Pi the population of city i, Ri its rank in the ordered distribution of populations, ei the randomly distributed error term and b = ln(β).There are two ways of estimating the rank-size rule. In the tradition of Lotka , the popα′ = 1/α and b′ = ln(β′).In the economic tradition, authors have favoured the Pareto form, which expresses the rank of cities as a power law of their population . The regα [α is the way the regression equation is fitted to the data. Until recently, all estimations were produced using the Ordinary Least Squares (OLS) method for the linear regression of logged populations and ranks. Gabaix and Ibragimov [The two forms should give equivalent results after transformation of the exponent α . This pabragimov  found thα estimates which are due to data collection choices, can be summarised into two categories. The first one refers to the definition of cities for which population is collected. Indeed, because the concept of city is broad, there are various delineations which can be used as units i in the rank-size regression. For example, cities can be considered as local administrative entities such as municipalities, as morphological units defined by the continuity of the built environment or the connectivity of street intersections [α for the same country at the same date. The same holds true when local government reforms imply substantial mergers of municipalities [Measurement “errors”, or more generally differences in sections , or as mpalities .The minimum population criterion for inclusion of settled units into the set is the second choice which affects the estimation of the rank-size rule, especially when the logged rank-size curve is not completely straight but somehow convex or concave, which is frequent in empirical systems . Consequα higher.Deviations to the “law” have been understood either as proofs against the validity of the law, either as indications of urban particularities. For example, Moriconi-Ebrard et Pumain , 14 haveAlternatively, Morrill  and RoseFinally, and most interesting for my purpose here, systems of cities have been said to tend towards more unevenness of time , 11, 14,α? Instead of estimating yet another set of α from empirical data, I choose to build on the accumulated literature and to answer this question with a meta-analysis of the rank-size estimates published in previous studies. A meta-analysis of Zipf’s law exists already [Given these insights, is it possible to control for measurement errors and estimation biases to reveal the “true” effect of time, development and demographic growth on the evolution of  already , but it I add more papers to the analysis (published since Nitsch’s article in 2005 for instance), especially from the non-economics literature;I add more variables in the meta-regression to find spatio-temporal drivers of city size unevenness;I control for the regression method ;https://github.com/ClementineCttn/MetaZipf and along with a dedicated website which allows to reproduce the analysis: https://clementinegeo.shinyapps.io/MetaZipf/.I distribute the resulting database online under an open-licence: These significant additions lead to new insights regarding the dynamics of Zipf’s law and the potentials for urban planning in this context.In this section, I explain the selection of studies to be included in the meta-analysis, as well as the way the data was collected and formatted to test my hypotheses. I then give an overview of a century of publication of Zipf’s estimates.An important aspect of meta-analyses has to do with the selection of the studies to be included in the analysis. The selection criteria should be broad enough to allow a large number of studies to be considered, yet systematic enough to be reproducible.In a previous meta-analysis of Zipf’s estimates, V. Nitsch  used theIn this study, I take advantage of new technologies of interactive online tools and crowdsourcing to establish a pool of studies as exhaustive as possible in relation to Zipf’s law. All studies are included in the meta-analysis as long as:They contain at least one estimate of the rank-size exponent based on populationThe regression is made on empirical urban dataThe regression model is bivariate .At the time of writing, I had collected 86 such studies, and for each of them, recorded the series of specifications which characterises the estimation of Zipf’s law and could influence its value. The analysis is reproducible with this selection, although the database will continue to grow.As shown in REFERENCE: the reference ID, which links an estimate to the study where it was published. Each study contains from 1 to 255 empirical estimates.α′ = 1/α. In the present paper, I choose to express all results using the Lotka form, thus interpreting the coefficient α as an index of unevenness of size in a given urban system.ALPHA: the value of the estimate. Some were produced using the Lotka form  and otheR2: the r-squared value of the regression. The information was available only half of the time.ESTIMATION: the method used to fit the regression model. OLS refers to the Ordinary Least Squares method, GI to Gabaix and Ibragimov’s trick of using rank—1/2, MC stands for Markov Chains and ML for Maximum Likelihood estimations.DATE: the year to which the population of cities refer (which is different from the publication year). The distribution of these dates goes from 1600 to 2014.TERRITORY: the administrative boundaries within which cities are drawn.URBANDEF: the way cities are defined in the study, using the original and heterogenous phrasing of the authors.N: the number of cities used in the regression. One third of that information is not reported.TRUNCATION: the minimum population of cities used in the regression. A quarter of that information is not reported.In addition to the specifications of the estimations, which can vary within a single study, I recorded some particularities of the study itself, in the same way as in . I used Because of the heterogeneity of the data and the way it is reported, an additional variable was constructed. URBANSCALE is the simplification of the URBANDEF categories. It has only four values which correspond to a conceptual way of defining cities. It takes the value “LocalUnits” when the URBANDEF corresponds to municipalities, communes or places, i.e. administrative way of delineating cities. It takes the value “MorphoCities” for definitions using the continuity of the built environment as a criterion to define cities (urban areas or urban agglomerations for example). It takes the value “MetroArea” when a functional criterion is used to define cities . It takes the value “VariaMixed” in all other cases.Finally, and this is how this new meta-analysis innovates, four topical variables were added to the technical variables classically used. For estimates obtained within national boundaries, I matched the meta-database with historical datasets from the United Nations and the World Bank to add the total population , GDP perThe resulting database is composed of 1962 estimates covering all the continents and spanning over 400 years. They come from 86 studies published in a variety of journals . There seems to have been a fashion in publishing empirical estimates over the last two decades, with 54 of the 86 studies included published since 2000, although I detect a peak in publication about empirical estimations of Zipf’s law in the 1980s too . ConsequThe studies with the most estimates published are comparative studies with measures for many countries in the world . AnotheIn terms of geographical coverage, the picture is one of large urban unevenness in the world. Some visual trends arise from α estimates with the characteristics of the estimations, studies and territories at the time of observation. There are various non-equivalent ways to do this meta-analysis.To conduct the meta-analysis, I related the values of Xi,j,t a vector of variables describing the estimate α in the observation i in study j at time t, and ϵ a randomly distributed error.For the static analysis, I use ordinary bivariate then multivariate linear models .αi,j,t=α is estimated. In multivariate models, I draw variables from three sets representing different regimes of explanation , there is evidence that built-up areas (or MorphoCities) are more uneven on average than local units. This pattern has been observed empirically when comparing the two definitions within the same territory [α, the choice of city definition also determines if the model is robust at all or not.Although city definitions have various frequencies  but shoα for data sets of less than 30 observations, which can be regarded as too small and inconsistent to be interpreted [The number of cities used in the regression is somehow dependent on the cutoff, but also on the size of the country. It is also massively under-reported . It shows a suspiciously high average erpreted , 26. Theerpreted , 18.α when authors have used the method of Gabaix and Ibragimov [A factor that has not been tested systematically so far is the impact of the estimation method on the value of the estimate, although the vast majority of published results were obtained using OLS. I find a significantly lower average value of bragimov . AdditioUsing variables describing the study where estimates were published, I find that:α (1.04), whereas studies published before 2000 have average estimates of 0.98 , whereas studies with less than 10 estimates have an average value of 0.96 .α (1.03), whereas studies for one year only have an average value of 0.93 .α (0.97), whereas studies for one country only (1.06) or more than five of them (1.01) report higher unevenness .Finally, my interest lies in the last set of variables describing urban systems and their territorial envelope.In terms of evolution, I find that estimates for city systems of 1940 and before are reported with significantly lower values on average compared to estimates for cities in the 1940s to 1990s. This result could be interpreted in two ways, which this simple model cannot disentangle. Firstly, this could confirm theories about the tendency of city systems to grow more uneven with time , 14. SecAlthough some scholars have argued that cities of smaller countries should be more unevenly distributed because powers are relatively more concentrated in the primate city , 16, I fR2 is low. Nonetheless, the relationship is ordered systematically, with countries with less than 1,000 current US dollars per capita having an average value of α equal to 0.976, whereas the average is 1.049 for intermediate countries and 1.073 for countries with more than 10,000 dollars per capita.Richer countries tend to be associated with more unevenness in city sizes, although the information is missing in 875 cases, and the α = 0.993), compared to newly urbanised systems (α = 1.094). One explanation from Evolutionary Urban Geography [Cities in territories urbanised early on appear more evenly distributed in terms of size  compared to intermediate levels of urban population (from 20 to 60%). Although not significantly, this value tends to increase again above 60% suggesting a phenomenon opposite to Kuznets’ curve of income inequality. According to Kuznets , countriα values , and to a lesser extent in France (R2 = 45%) and China (53%). On average, the characteristics of the study alone explain twice less variation in α, expect in the US where the models are systematically worse. However in this country, where the estimation was published (i.e. the publication type and content) is relatively more important in predicting the value reported! Finally, except for China, territorial variables have the lowest explaining power of the three sets of variables. Only in China (and maybe Brazil) does the hierarchy of city sizes seem to be influenced by territorial characteristics and so, possibly, to policy interventions. The United States (and India) represent the opposite situation.This observation is confirmed and reinforced by meta-analyses disaggregated at the country level. I selected countries with more than 50 estimations drawn from more than 10 different studies . This corresponds to five national cases: Brazil, India, France, China and the United States . When I α seems to depend on the specification chosen  in α is statistically correlated to urbanisation attributes rather than economic and demographic variables. Indeed, with an R2 of 45.7%, this model indicates that everything else being equal, the inequality coefficient of urban systems in highly urbanised countries is 0.029 point higher than systems where between 20 and 60% of the population is urban. On the contrary, the inequality coefficient of urban systems in highly urbanised countries is 0.091 point higher than systems systems where between 20 and 60% of the population is urban. I thus confirm the U-shaped relationship between the urban transition and city size unevenness.The random-effect panel model using time-dependent territorial variables confirms my previous findings that the value of α ≤ 0.75 at time t1), which is rather trivial as it is where it has more potential for growth. The model is based on 313 longitudinal observations and “explains” 16.2% of variance. However, deceptively, no territorial variable, static or dynamic, is associated with this evolution of urban inequality.Finally, a fixed effects panel model of average annual growth rates of Zipf’s coefficients shows only one statistically significant relation: unevenness in city size increases more where it started low  would lie in the control of the rural-urban migration rather than natural growth and economic development. However, the models show a high regularity in the value of α) over the course and just after such policies in country applying them. The expected countries in this categories should therefore be:The last set of dynamic models shows that there is a very large part of evolution left unexplained by the characteristics measured and included in the model (83% to be precise). This could be the effect of rebalancing policies, which would appear in the form of negative residuals (overestimated growth of the Soviet Union in the 1920s when the Desurbanists promoted territorial inequality and the reduction of large cities’ growth.the United Kingdom after 1940, when the Barlow commission assigned the task for public policies to rebalance the spatial economy from London and the South East to the Northern cities.France after 1960, when it implemented a policy of ‘métropoles d’équilibre’ or balancing metropolises. The government then fostered the development of universities, tribunals and other metropolitan functions in the second-tier cities.China after 1978, when Deng Xioaping’s reforms fostered decollectivisation of the agriculture and decentralisation of towns and cities.None of these appear in the most negative residuals of the fixed-effects panel model of growth rates of Zipf’s coefficient. There is therefore plenty of room for further theory on the drivers of city size unevenness. In particular, generative models need to be implemented to account for different values of rank-size slopes, but also different evolutions of this value. The non-spatial stochastic model of Gibrat is not enough when trying to account for empirical urban systems, which have clear spatial patterns of migrations for example.To conclude, there is evidence from this novel meta-analysis that no iron Zipf’s law exists for cities. Furthermore, I found that technical specifications play an unnecessary large role in varying the results and have hampered so far the identification of the territorial drivers of city size inequality.Using a new and open material for almost 2000 estimations of the rank-size relation, I confirm some of the previous findings, refute the importance of the country size and find that the urban hierarchy is a process that is more linked to the urban process than to economic development. Using residual analysis, I do not find evidence of the effect of planning policy to rebalance city sizes.As a cumulative contribution to the field, this study comes with open data and a tool for its exploration. The next step in this direction is to look for further spatial mechanisms susceptible to explain the evolution of urbanisation and the urban hierarchy. Without this knowledge, it seems complicated to design effective policies.


An electronic tongue constructed using a combination of the nanoparticle-based sensors is capable to monitor the phenolic maturity of red grapes from véraison to maturity. Principal Component Analysis (PCA) can be successfully used to discriminate samples according to the ripeness. Regression models performed using Partial Least Squares (PLS-1) have established good correlations between voltammetric data obtained with the electrochemical sensors and the Total Polyphenolic Index, the Brix degree and the Total Acidity, with correlation coefficients close to 1 and low number of latent variables. An advantage of this system is that the electronic tongue can be used for the simultaneous assessment of these three parameters which are the main factors used to monitor the maturity of grapes. Thus the electronic tongue based on metal oxide nanoparticles can be a valuable tool to monitor ripeness. These results demonstrate the exciting possible applications of metal oxide nanoparticles in the field of electronic tongues.The maturity of grapes is usually monitored by means of the sugar concentration. However, the assessment of other parameters such as the phenolic content is also important because the phenolic maturity has an important impact on the organoleptic characteristics of wines. In this work, voltammetric sensors able to detect phenols in red grapes have been developed. They are based on metal oxide nanoparticles (CeO Grapes must be harvested at the optimal maturity point. The sugar content has a direct influence on the alcoholic degree of wines and it is the analytical indicator commonly used to decide the harvest date OIV, . It is pThe phenolic content of grapes also changes during ripening and the phenolic maturity has a direct impact on the organoleptic characteristics of wines, and it would be desirable to assess it routinely. However, the main changes in the phenolic content take place in skins and seeds, and the assessment of phenolic maturity requires the previous separation of seeds and skins using long and complex methods  are a new class of instruments which are gaining interest in the food industry  and to evaluate their capabilities to detect phenols. Using chemometric techniques such as Principal Component Analysis (PCA), the ET was used to monitor the ripeness of 8 different varieties of grapes, from véraison (the onset of ripening) to post-maturity. Mathematical models were built to establish correlations between the phenolic index measured by traditional chemical techniques and the results of the ET. PLS-1 was used to predict the polyphenol index of the grapes along ripening. The possibility to obtain information about other classical indicators of maturity such as the °Brix and Total Acidity was also analyzed.The aim of this work was to develop an electronic tongue (ET) formed by sensors based on metal oxide nanoparticles  were purchased from Sigma-Aldrich. All reagents and solvents were of high purity (Sigma–Aldrich). Deionized water (18.2 MΩ·cm−1) was obtained from a Millipore MilliQ purifier.Metal oxide nanoparticles of Titanium (IV), Cerium (IV), and Nickel (II) (TiOElectron Microscope TEM images were recorded using a high-resolution electron microscope (HRTEM: JEOL JEM 2200)  operating at an accelerating voltage of 200 kV. Sample images were processed using Image J image processing software (public software).Tempranillo, Garnacha, Cabernet-Sauvignon, Prieto Picudo, Mencia Regadio, Mencia Secano, Rufete, and Juan Garcia. Grapes were grown and harvested by the Agrotechnological Institute of Agriculture of the Regional Government (ITACyL) and the cellar Bodega Cooperativa de Cigales. Berries were collected in a weekly basis from véraison until grapes were completely mature. Ripeness rate was different from one variety to another. For this reason, the official harvest date indicated by traditional chemical parameters, varied from one variety of grape to another. Grapes from Tempranillo, Garnacha, and Cabernet Sauvignon were considered matured 6 weeks after véraison. The rest of varieties attained the optimal ripeness 5 weeks after véraison. In all cases several grape bunches were left in the vine and two extra samplings were collected in successive weeks in order to analyze over-ripened grapes.Eight varieties of red grapes typical from the Castilla-Leon region (Spain) were included in the study: Musts were prepared from 100 berries representative of the ripening state of the vineyard. Once collected, grapes were crushed, and peels and seeds were separated by decantation. 50 mL aliquots of musts thus obtained, were frozen at −20°C until used. Total Polyphenol Index (TPI), Brix degree (°Brix) and Total Acidity (TA) were analyzed following international standard methods OIV, . TPI was2 (TiO2NP-CPE), and CeO2 (CeO2NP-CPE). One unmodified carbon paste electrode (C-CPE) was also included in the array. Electrodes were prepared following the classical method by mixing graphite with the corresponding metal oxide nanoparticle (15% w/w of the MONP) Nujol was used as the binder until a paste was obtained. The paste was packed in a 1 mL plastic syringe and compressed. A metallic copper wire was used as a contact  was formed. Three electrodes were modified with metal oxide nanoparticles including NiO (NiONP-CPE), TiO−1 electrode acting as a reference electrode and a platinum wire as the counter electrode. The MONPs sensors were able to provide responses toward aqueous solutions of phenolic acids (vanillic and caffeic acids) and polyphenols  present in wines. The electrocatalytic properties and the detection limits (LD) were evaluated. The calibration curves were constructed measuring standard solutions with concentrations ranging from 10−4 to 10−6 mol·L−1. LDs were calculated following the 3 SD/m criterion. The reproducibility of data provided by the MONPs sensors was evaluated by comparing data provided by three sensors measuring standard solutions in different days. Cyclic voltammograms were registered at a sweep rate of 0.1 V·s−1 from −0.8 to +1.0 V. Four replicas of each sample were measured. Principal Component Analysis (PCA) and Partial Least Squares (PLS) models were built using the software Matlab v5.3. .Electrochemical experiments were carried out using a potentiostat  combined with a multiplexor system. CPEs (surface of 2 mm in diameter) served as working electrodes, with an Ag|AgCl/KCl 3 mol·LPrior to perform the statistical analysis, the number of variables was reduced using a data reduction technique based on predefined response “bell-shaped-windowing” curves called “kernels” . These parameters were used by oenologists to establish the optimal date of harvest. The °Brix/TA ratio was also calculated. This parameter is also commonly used to decide the optimal maturity date even if there is no biochemical relationship between the acidity loss and the sugar increase. The complete list of parameters is collected in Ripening was monitored by measuring °Brix, TA, and TPI in a weekly basis .TPI values increased sharply just after the −4 M), was evaluated  or di-phenols (catechol and caffeic acid) than in the tri-phenol .The responses on MONP-CPEs were consistent with those already reported in bare CPE electrodes  lower than 7% were found in all cases. From the above results it can be concluded that modification with metal oxide nanoparticles drastically increases the sensibility of voltammetric electrodes toward phenols present in musts.Limits of detection (LD) were calculated from the slope of the curves of the redox peaks registered at concentrations ranging from 10As stated before, each sensor showed a characteristic response which is linked to the electrocatalytic properties of the type of nanoparticle introduced in the carbon paste. This cross-selectivity can be used to construct a multisensor system coupled to a pattern recognition software able to provide a pattern or “digital fingerprint” for each sample studied.−4 M solutions of phenols. The PCA showed four distinct clusters in the plot of the two first principal components (PC1 61.31% and PC2 20.61%). The position of the clusters was related to the number of hydroxyl groups in the molecule. The monophenol (vanillic acid) was located at positive PC1 values, diphenols (catechol and caffeic acid) in the central part of the diagram and the triphenol  on the left side corresponding to positive PC2 values.Figure véraison, in a weekly basis, during 7–8 weeks. Due to the complexity of the samples and in order to improve the repeatability, musts were diluted 1:2 with deionized water. Coefficients of variation lower than 7% were obtained in all cases.The capacity of the electronic tongue to monitor the phenolic maturity of grapes was evaluated by analyzing juices obtained from grapes collected from 2NP-CPE sensors immersed in musts obtained from grapes of the variety Garnacha collected during 7 weeks after véraison.Figure 2NP-CPEs were sensitive to the acidity.As illustrated in the figure, voltammograms registered in must showed peaks in the 0.6–0.8 V region, produced by components with redox activity (mainly phenols). Other components present in musts also influenced the overall response. For instance, the peaks observed at negative potentials were affected by the acidity of the samples. The array of sensors showed an interesting cross-selectivity that was due to the different catalytic properties of the MONP selected. For instance, NiONP-CPEs were highly sensitive to phenols, whereas TiOThe chemical changes occurring during the ripening process could be followed using the MONP-based sensors. First, the intensity of the peaks increased, then intensity decreased and finally increased according with the tendency shown in Figure véraison, were positioned in a counterclockwise organization “drawing” a circle  and in prediction (R2V) of ca. 0.8. Additionally, low Root Mean Square Error of calibration (RMSEC) and of prediction (RMSEp) were accomplished. These results confirm that the ET can be used to monitor the phenolic maturity of grapes. The °Brix and Total Acidity which are the parameters commonly used to evaluate the maturity of grapes, could also be predicted using the PLS-1 models but with lower correlation coefficients in prediction (~0.7) and the models need a higher number of components. In spite of the lower correlation, the models are good enough to confirm that the electrochemical responses also reflected the acidity and the sugar concentration. This indicates that it is possible to obtain simultaneous information of the three main parameters used to monitor the maturity of grapes.In all varieties of grapes, good correlations were found with the Total Polyphenol Index. Using a low number of components, both the calibration and the prediction showed Squared Correlation Coefficients in calibration (RThe advantages of metal oxide nanoparticles as sensing materials for the detection of phenols has been evidenced. They rely on the excellent electrocatalytic properties of the metal oxide nanoparticles, that improved the sensitivity and limits of detection toward phenols with respect to unmodified sensors. Using these exceptional sensing properties, and the cross-selectivity shown by different MONPs, an electronic tongue based on metal oxide nanoparticles has been developed. The chemical changes occurring during ripening could be followed using the MONP-based multisensor system. The sensors based on different MONP show an excellent degree of cross-selectivity. Using PLS-1, correlations have been established between the signal of the multisensor system and the Total Polyphenol Index TPI, the °Brix, and the Total Acidity.According to this, simultaneous analysis of three important markers of maturity can be achieved using a multisensory system based on MONPs. This means that the ET can be a useful tool to establish with a better accuracy the optimal harvesting date in a single experiment. This method can be used as a supplementary tool to classical analytical techniques.The results shown here demonstrate the excellent opportunities offered by metal oxide nanoparticles as sensing materials in electronic tongues.MR-M: conceived the experiments an wrote the paper; CG-H, CM-P, CG-C, and YB: carried out the experiments; JF-E and EB-T: provided de grape samples and analyzed the grapes by chemical methods; MR-P and FM-P: carried out the chemometric analysis.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.