The aim of this study was to collect information about 2011 genetic activities in Italy, with the purpose of providing guidance to the national health systems in order to improve genetic services.A web-based survey was carried out to achieve the information.Data were collected from 268 macrostructures hosting 517 services and employing 3246 persons. About 295,000 cytogenetic, 35,000 immunogenetic and 263,000 molecular genetic analyses of 902 genes were recorded. Seventy-four percent of the services were accredited with institutional bodies and 57 % were also certified according to ISO 9001 standard. Twenty percent of cytogenetic laboratories had participated in an European External Quality Assessment (EQA) while 44 % participated in a national EQA. Only 28 % of the molecular laboratories had participated in a national Cystic Fibrosis EQA. The percentage of diagnoses confirmed by genetic tests varied among disorders, ranging from 52 % for coeliac disease to 4 % for fragile X syndrome.This study highlights the need for reorganizing the Italian genetic services network, improving EQA participation and developing national plans for implementing next generation technologies. Concerted effort has to be addressed in the education of the professionals prescribing tests to improve appropriateness and to inform patients, who now have exposure to direct-to-consumer multifactorial genetic testing where clinical utility is unproven. The Italian Society of Human Genetics (SIGU) launched in 2012 a survey designed to scrutinize the 2011 activities of the Italian Medical Genetic Services, to compare the results with previous data collections, and to update the national scenario. SIGU had previously carried out six surveys to gather information on cytogenetic and molecular testing and clinical genetics activities, with the aim of providing guidance to the national and regional health systems to improve the organization of genetic services [Here, we report the results of this last study, providing an overview of the changes occurred over a 4-year period, highlighting the need for reviewing and improving the Italian genetic structure network, in line with the current public health requirements.ad hoc questionnaire (http://docs.biomedia.net/SIGU/Schema-Censimento_SIGU-2011.pdf) sent to genetic centers hosted by universities, research hospitals (Istituti di Ricovero e Cura a Carattere Scientifico - IRCCS), general hospitals, local health centers  and commercial laboratories. The enrolled genetic centers were those registered and updated from the 2007 survey, integrated with others recruited through Orphanet database (www.orpha.net) or joining voluntarily to this initiative after learning of it during national scientific conferences or through the SIGU website (www.sigu.net). A verbal informed consent was obtained from participants.The survey refers to the medical genetic activities performed in Italy from January 1st to December 31st, 2011. This study was carried out by the Bambino Gesù Children Hospital in Rome, in collaboration with Orphanet-Italy, on behalf of SIGU. Ethical approval for this study was granted by SIGU. Data were gathered by an Questions from previous surveys were reiterated and new ones added, in order to achieve information on the activities of immunogenetic laboratories and diagnostic tests becoming available in more recent years (i.e. microarrays). Entered data were based on self-certification. Information was collected online from March to July 2012, using forms available in a dedicated website. Each participating center accessed the website through a private username and password assigned during the registration process. Requested data included total number and type of performed cytogenetic and molecular genetic tests, clinical genetic activities such as the number and type of genetic counseling sessions. Data concerning the personnel attending laboratories and clinical activities were also collected. The quality management system of the responding centers in terms of national accreditation standards and international certifications was investigated, together with national and international External Quality Assessment enrolment. By comparing the number of analyses with the number of diagnoses confirmed by the test results, appropriateness was evaluated for the following entities: achondroplasia, Angelman, DiGeorge, fragile X-mental retardation, Noonan, Prader-Willi and Williams syndromes, ankylosing spondylitis and coeliac diseases. Appropriateness was investigated also for CGH (Comparative Genomic Hybridization) and SNPs (Single Nucleotide Polymorphisms) array tests.Overall, 268 genetic centers, hosting 517 genetic services (genetic testing and counselling) were surveyed Table . CentersForty-six percent of cytogenetic laboratories were located in the Northern regions, compared with 21 % in Central regions, 20 % in Southern regions, and 11 % in the Islands Table . This diCancer cytogenetic tests increased from 2000 onwards with the greater contribution for this escalation being attributable to an increase of FISH (Fluorescent In Situ Hybridization) and ISH (In Situ Hybridization) analyses.One aspect of a Quality Management System is the participation in an accredited external quality assessment (EQA) scheme. Only 30 of 153 censused laboratories (20 %) had taken part in an international accredited Cytogenetic European Quality Assessment  Fig.  and 68  participated in either a national or international cystic fibrosis EQA schemes. It is likely that not all the 198 censused laboratories offered genetic testing for cystic fibrosis, but the majority did, although the participation in EQA was lower than desirable.A total of 35,446 molecular genetic analyses had been performed by 21 immunogenetic laboratories in 2011, indicating a sharp and constant increase during the last 12 years  disclosed pathogenic Copy Number Variations (CNVs), including 407 deletions resulting in known deletion syndromes.http://www.salute.gov.it/imgs/c_17_pubblicazioni_908_allegato.pdf). The total number of genetic services in Table The survey of the Italian medical genetic centers in year 2011 was carried out on the behalf of SIGU. This study aimed at providing indications to the national and regional public health systems, in agreement with the Regional Health System request to rationalize and integrate the public genetic laboratories under the pressure of the spending review and the need to improve efficiency. Indeed, the national health system is fully supporting the cost of genetic testing requested by the specialists, with some limitations for prenatal testing in women under the age of 35 years and/or without any specific indication. Accordingly to previous reviews, the number of genetic services was exceeding that required against the approximately 60,000,000 total inhabitants. Indeed the Minister of Health and the Italian Regions suggested that the maximums should be 1,000,000 inhabitants for every clinical genetics facility between 500,000 and 1,000,000 inhabitants for every cytogenetic laboratory and not less than 3,000,000 inhabitants for every molecular genetic laboratory . The Italian state of quality systems within genetic testing services is similar to the European overview provided by Berwouts et al. [In the last decade guidelines regulating the activities of clinical and laboratory genetic services were jointly developed by the Ministry of Health and the Italian Regions, establishing roles and functions of genetic services . Accordis et al. . Again, Collected data, showed that only 20 % of cytogenetic laboratories participated in the accredited CEQAS (formerly CEQA) and 44 % in the Istituto Superiore di Sanità  schemes. Only a minority of the molecular laboratories stated they participated in an EQA scheme for cystic fibrosis. The participation in EQA is an established tool for improving quality, educating laboratory staff and directing laboratories towards best practices. National rules establish that medical laboratories shall participate in EQA scheme in order to be institutionally accredited, but appropriate checks to verify the participation of the genetic laboratories in EQA are only occasionally carried out by responsible bodies.As shown in this report, the coverage of the Italian territory by genetic testing facilities is one of the highest in Europe. Table The need for a revision of the national molecular diagnostic network and for a diversification of the offer are supported by the results of the present survey, showing that testing of 15 disease-genes only accounted for 70 % of all molecular analyses in 2011. In addition, the demand of molecular tests has increased by a figure of 20 % from 2007 onwards, and cytogenetic analyses has reached a plateau of around 300,000 tests per year. Compared with the results registered by the 2007 census, the request of molecularly testing the susceptibility to common disorders and complex phenotypes was not increased, the analysis of thrombophilia cascade genes and HLA accounting for about 30 % of all molecular tests.The request of constitutional prenatal and postnatal chromosome analyses has decreased in recent years. In particular, the number of genetic tests in amniocytes, which had been stable between 2004–2007, decreased in the years preceding the present survey, while the number of test on chorionic villi, increased by about 2500 per year in 2007–2011, stabilizing at around 25,500. It is expected that the decreasing trend of invasive prenatal tests will be further corroborated in the coming years, due to introduction of Non-Invasive Prenatal Testing (NIPT) into the diagnostic set.The reduction of constitutional postnatal cytogenetic tests appears mainly related to the decrease of FISH analyses, which was expanded in the early 2000s, in parallel with the increased availability of commercial probes testing for cryptic deletions and supernumerary markers. A similar trend was also seen prenatally, with a consistent decrease of FISH tests. Between 2007–2011, FISH analyses had been progressively substituted by microarrays testing, as shown by the 8290 postnatal and 1862 prenatal tests  in 2011, compared to 1443 and 393 respectively in 2007. The reduction in FISH prenatal tests was related also to the widespread use of QF-PCR for detecting major aneuploidies.By contrast, oncological cytogenetic tests had increased by 58 %, mainly as a consequence of the wider introduction of FISH interphase analysis in genetic centers as documented by Table As to previous concerns on the appropriateness of genetic testing requested by referring clinical geneticists, the 2011 results show an increase in the confirmed diagnoses for DiGeorge/Velocardiofacial and Williams syndromes, and a significant decrease for Prader-Willi syndrome compared to 2007  have not yet entered in the diagnostics setting and remain restricted to a research application. Public healthcare in Italy is now administrated on a regional basis and a concerted effort must be made to review and rationalize the network of genetic services nationally, taking into account the results of this present survey. In fact, this study provides a real and detailed representation about the logistic of the genetic centers, the number of performed genetic tests, the quality of provided services, the appropriateness of genetic test requests and the number of employees. Within this setting, a number of recommendations can be given for improving genetic services in Italy, which is at present oversized and nonharmonious. Some priority issues are emerging, including management of quality systems in terms of accreditation, certification, and EQA participations; accessibility of pre-test and post-test genetic counseling; the need of increasing availability of genetic testing for rare diseases; the development of national plans and strategies for implementing next generation technologies and advising on the clinical utility of genetic testing for complex disorders. The quality standard for genetic structures elaborated by SIGU may be adopted by a national competent body responsible for the specific accreditation and quality assurance of the genetic services. Another challenge, focused by Battista et al. , refers This study provides a real and detailed representation about the logistics of the genetic centers operating in Italy, the quality of their provided services, the number of performed genetic tests, and the number of employees. There are too many genetic services given the population size of Italy and they are not equally distributed across the country. Quality assurance of the genetic services may be improved through a specific accreditation standard. Good clinical practice and evidence based medicine may be implemented through the education and training of the people involved in the prescriptions of genetic tests. The finding that 70 % of all molecular analyses performed in 2011 was related to only 15 disease-genes, strongly supports the need for a revision of the national molecular diagnostic network. All together, these results provide information useful for improving genetic services in Italy.


Vibrio parahaemolyticus and Vibrio cholerae are ubiquitous to estuarine and marine environments. These two species found in Mediterranean coastal systems can induce infections in humans. Environmental isolates of V. cholerae (n = 109) and V. parahaemolyticus (n = 89) sampled at different dates, stations and water salinities were investigated for virulence genes and by a multilocus sequence-based analysis (MLSA). V. cholerae isolates were all ctxA negative and only one isolate of V. parahaemolyticus displayed trh2 gene. Most Sequence Types (ST) corresponded to unique ST isolated at one date or one station. Frequent recombination events were detected among different pathogenic species, V. parahaemolyticus, V. cholerae, Vibrio mimicus, and Vibrio metoecus. Recombination had a major impact on the diversification of lineages. The genetic diversity assessed by the number of ST/strain was higher in low salinity condition for V. parahaemolyticus and V. cholerae whereas the frequency of recombination events in V. cholerae was lower in low salinity condition. Mediterranean coastal lagoon systems housed V. cholerae and V. parahaemolyticus with genetic diversities equivalent to the worldwide diversity described so far. The presence of STs found in human infections as well as the frequency of recombination events in environmental vibrios populations could predict a potential epidemiological risk. Vibrio groups gram-negative halophilic bacteria that are found in marine and estuarine environments, among which some species are responsible for human infections   and/or tdh-related (trh1 and trh2) hemolysin genes. Some pathogenic clones encountered an epidemic success that led to the dissemination of the pandemic O3:K6 clone in many countries   and its tributary the Vistre River (598 km2) goes into the Ponant and Vidourle lagoons before flowing to the sea via the Ponant grau and the Grau du Roi channel, respectively. Numerous streams flow directly into the Mauguio or the Prévost lagoons, forming the watersheds of Mauguio and Palavas lagoons, respectively. To the west, the Lez watershed (535 km2) is formed by the Lez River and by its main tributary the Mosson River. Fresh water from Lez watershed doesn't flow directly into the Prévost lagoons but cross them via the Lez channel. In the latter system, salinity variations during heavy rainfalls are lower than those observed for the Ponant and Vidourle lagoons that directly receive large rivers. Sampling stations were situated in lagoons , in channels connecting lagoons  or rivers  to the sea, and on beaches . Surface water (5 L) was collected in July and September 2011 at 12 stations, and in November 2011 at six stations. Temperature and salinity were recorded in situ using a WTW LF 196 conductimeter at the time of sampling. Samples were transported in coolers (15–18°C) to the laboratory and processed within 4 h after collection.The studied lagoon system is presented in Figure V. parahaemolyticus, and V. cholerae in water samples according Cantet et al. (V. cholerae and V. parahaemolyticus were selected for further identification.A Most Probable Number (MPN) method was applied to detect t et al. . EnrichmV. cholerae and V. parahaemolyticus were performed as previously described by PCR using specific primers for 16S–23S rRNA Intergenic Spacer Regions (ISR)  and V. parahaemolyticus  were amplified as previously described by Octavia et al.  were separated in 1.5% (w/v) agarose gel in 0.5X Tris Borate EDTA (TBE) buffer. Amplification products were sequenced using forward primers on an ABI 3730xl automatic sequencer . Sequencing using reverse primer was performed when the sequences obtained with forward primers were not found in databases. The ssDNA sequences were deposited in the GenBank database with accession numbers from http://www.expasy.org). The length of sequences used for further analyses is indicated in Table Gene sequences were codon-aligned using ClustalX after translation with TRANSLATE (http://www.pubmlst.org). Allele profiles were also analyzed using goeBURST v3 software (http://goeburst.phyloviz.net/) to determine clonal complexes (CC) defined as sets of related strains that share at least five identical alleles on the 7 loci. The singleton (S) STs corresponded to STs differing from all the others by 3 or more of the 7 loci. The Phyloviz program employing eBURST algorithm was used to determine Minimum Spanning Tree that showed the relationships between STs (http://goeburst.phyloviz.net/). Dataset for each species are constituted of strains of this study and pubMLST isolates with complete MLST information: i.e., 228 isolates for V. cholerae, and 1513 isolates for V. parahaemolyticus.The alignment obtained for phylogenetic treeing was used for assignation of each isolate to a Sequence Type (ST) according to their allelic profiles and compared to accessible sequences on pubmlst databases (http://www.ub.edu/dnasp/). The number of synonymous (dS) and non-synonymous (dN) substitutions per site was determined on codon-aligned sequences using SNAP software (http://www.hiv.lanl.gov/content/sequence/SNAP/SNAP.html). Program LIAN 3.5 (http://pubmlst.org/) was used to determine the h score of genetic diversity at each locus. The mean genetic diversity (H) was then calculated as the arithmetic mean of the h values for all loci. Rarefaction analysis was carried out using the freeware program aRarefactWin (http://www.uga.edu/strata/software/Software.html). Program LIAN 3.5 was also used to evaluate the recombination by calculation of the standardized index of association IA(sIA) and to test the null hypothesis of linkage disequilibrium. The homoplasy index φw test was done on pub MLST web site (http://www.pubmlst.org) or split tree. Recombination events were detected from aligned concatenated sequences using seven different methods with default parameters of the RDP v3.44 .The number of polymorphic sites was calculated using DnaSP version 5.10   detected by MLST, the majority being represented by unique STs found only once and in one isolate  by 2 to 5 isolates. All STs but two, represented by multiple isolates (n = 13), grouped strains originating from different sites, the most represented being ST481, ST2000 and ST2005  by goeBURST confirmed the high level of genetic diversity within the two species. The population of s Figure . Seven Cs Figure .V. cholerae and V. parahaemolyticus, respectively. The genetic diversity at individual loci (h) and the number of single nucleotide polymorphism (SNP) are given in Table The genetic diversity (H) was 0.9082 ± 0.0369 and 0.9706 ± 0.0081 for V. cholerae and V. parahaemolyticus shared 3 identical loci, gyrB, pntA, and pyrC. The levels of polymorphism of gyrB and pntA were similar in the two species whereas pyrC displayed a much higher polymorphism in V. cholerae than in V. parahaemolyticus. In the two species, the numbers of SNPs at the 7 loci were close, 414 (12.9%) for V. cholerae and 498 (13.5%) for V. parahaemolyticus. None of these SNPs produced premature stop codon. The frequency of non-synonymous mutations was low for each locus in both species, the highest dN/dS ratio being 0.1386 for the gene adk in V. cholerae . H and ST/strain scores were similar in a collection sampled during 6 months in Mediterranean lagoons and in Vibrio collections from databases. The H score was 0.8836 in database vs. 0.9082 in this study for V. cholerae and H was 0.9536 in database vs. 0.9705 in this study for V. parahaemolyticus. Therefore, the very high genetic diversity observed locally in the coastal lagoon systems for the two Vibrio species was similar to that observed in strains collected from many countries (five continents) over the past 60 years (data not shown).We compared the multi-locus genetic data of the V. cholerae population, ST2 was the only ST previously deposited in the database. It corresponded to ctx-strains of serotype O49 isolated in Bangladesh in 2000 in a context of sporadic digestive infections  from 1997 to 2012. They presented the virulence genotype tdh-/trh-, except for one environmental strain isolated in USA (Pacific coast) in 2006 that was tdh+/trh+. The ST363 that contained 2 strains isolated in sites 5 and 10 in July 2011 was composed of clinical strains  isolated in a patient with gastro-enteritis in Thailand in 1991. The ST3, which represented 15% of the strains deposited in MLST database and corresponded to the pandemic virulent strain O3:K6, was not detected in this study.For V. cholerae were equally distributed whatever the month of sampling, the station or the salinity  when the salinity was low. For V. parahaemolyticus, the ST/strain score was higher in September 2011 (0.95) than in July 2011 (0.795) but the difference was not significant (p = 0.41)  when the salinity was low. Both species' genetic diversity was also significantly higher in seawater from beach stations than in samples from other sampling stations (p < 0.005) but this could be due to a sampling bias because of the low number of strains collected from seawater.Genetic data of strains of the two species were compared according to the month of sampling , the sampling stations  and water salinity  , containing five isolates, was found during the three campaigns in four different sites while two others (ST242 and ST255) were found in July and September 2011  grouped STs detected in different sampling campaigns but mostly at similar low salinities  are shown in Figures V. cholerae CC6 and V. parahaemolyticus CC4, which split into distinct parts in ML phylogeny. As observed above with goeBURST structuration, phylogeny showed only a few sporadic clusters of V. cholerae and V. parahaemolyticus composed by a few strains from a same date or a same station  or in V. metoecus and Vibrio parilis clades (gyrB and purM-based trees) remote from V. cholerae or on an independent lineage (metE-based tree). Comparison of metE and purM gene sequences of strains N1-12 and N5-13 with sequences of databases showed a best match of only 93% with other published sequences of Vibrio spp. The gyrB gene of N1-12 and N5-13 gave the best match with strains Vibrio sp. W0706-81 (HM009674), L8M (HM009590), and HB0308-2A5 (HM009568) non-affiliated to the species level and detected in coastal marine water in California and Hawaii. The third member of clade B, N6-9, was generally related to N1-12 and N5-13, except for purM phylogeny that placed it on a long independent branch remote from all other Vibrio spp. purM sequences, and for metE phylogeny that placed it in the V. cholerae clade.The e Figure  displayeV. cholerae in spite of the positive V. cholerae 16S–23S rRNA ISR PCR for isolates N1-12, N5-13, and N6-9 , and Vibrio sp. HB0308 and L8M (96.3%), which were previously found related to V. cholerae clade B strains  in gyrB phylogeny. This result indicated genetic exchanges between Vibrio sp. related to V. cholerae and V. parahaemolyticus. It is noteworthy that Vibrio sp. N5-13 and V. parahaemolyticus S5.61 were isolated in the same station in November and September, respectively. V. parahaemolyticus J8.24 and S11.38 were also placed on long branches due to very polymorphic dtdS and gyrB alleles, respectively. Comparison to databases showed that the best matches of isolate J8.24 for dtdS was 94% with Vibrio campbelli and Vibrio harveyi type strains. The recA gene of strain S11.38 displayed 98.8% identity with V. mimicus strain LMG 7896T and only 86% with the type strain of V. parahaemolyticus LMG2850T. Therefore, we described a genetic exchange occurring between two species of pathogenic vibrios, V. parahaemolyticus and V. mimicus but also between V. parahaemolyticus and particular genotypes related to V. cholerae.Two main clades were shown in the e Figure . The strV. cholerae , the homoplasy index φw test and by using RDP software. The sIA, expected to be null when a population is at linkage equilibrium, was significantly different from zero for both V. cholerae  and V. parahaemolyticus . No significant sIA difference was observed according to dates, stations, and salinities for both species (data not shown). The sIA values indicated significant linkage disequilibrium suggesting that V. cholerae and V. parahaemolyticus population conserved a clonal backbone despite frequent events of recombination. In contrast, the homoplasy index φw test, which discriminates between recurrent mutation and recombination, found statistically significant evidence for recombination (p = 0.0) in both V. cholerae and V. parahaemolyticus populations.Recombination was then evaluated in Vibrio populations were further analyzed by RDP software. In V. cholerae population detected by at least 4 out of the 7 methods performed by RDP, 34 strains (31.2% of isolates) underwent at least one recombination event  underwent at least one recombination event  and V. cholerae (0%) was lower, especially for V. parahaemolyticus, than that observed in waters collected from other French coastal areas (Hervio-Heath et al., ctx+ isolates in environmental strains (Zo et al., The isolates harvested in Mediterranean lagoons did not carry any major virulence factors, except for one isolate of V. cholerae and V. parahaemolyticus populations are extremely diverse and their genetic diversity under-reported. In this study, the distribution of STs, as well as the H and ST/strain scores, are in agreement with the overall diversity of V. cholerae and V. parahaemolyticus. Indeed, we observed locally a genetic diversity similar to or higher than that observed worldwide in the last fifty years. This has been previously shown for V. parahaemolyticus populations (Gonzalez-Escalona et al., V. cholerae populations. Zhang et al. (Databases comparison showed that most STs described in this study were detected for the first time, demonstrating that the g et al.  demonstrV. cholerae and V. parahaemolyticus populations collected in cholera-free or vibriosis outbreak-free regions. Moreover, some STs without virulence factors identified in this study had been previously detected as virulent strains, suggesting that the population described here could provide the genetic background for the emergence of pathogenic clones (Li et al., The vibrios diversity appeared independent from the type of sampling site and the type of lagoon system. Moreover, we did not detect any clones specialized locally according to a particular site in the lagoon system, but this could be due to the hydrodynamic conditions in these systems where water is extensively mixed. Some STs detected in Mediterranean lagoon systems were previously detected elsewhere in the world, in very different environmental conditions or in clinical samples. This suggests the absence of geographic or lifestyle specialization of V. parahaemolyticus according to local variation of environmental conditions. The level of genetic diversity of V. cholerae and V. parahaemolyticus was higher when the salinity was low. Some of V. parahaemolyticus CCs detected in both low and high salinity conditions are composed of closely related STs, each being detected in only one condition of salinity. Similarly, V. cholerae CCs detected in different sites are formed by closely related STs. These slight differences in nucleotide sequences suggest microevolution events spurred by adaptation to changing environmental conditions. In New Hampshire shellfish waters, the diversity in V. parahaemolyticus populations increased as water temperature increased (Ellis et al., To our knowledge, the study of Ellis et al.  and the V. cholerae (Hazen et al., V. cholerae, high chitin concentrations induce an increase of recombination rate (Meibom et al., V. cholerae. This could be related to the positive association between salinity and the rate of HGT detected by genomics (Jain et al., V. parahaemolyticus and V. cholerae populations. For some of the recombining STs, the donor STs were detected in the Mediterranean lagoon ecosystems. This demonstrates that, beside SNPs described above as related to changes in environmental conditions, recombination is a diversity-generating mechanism ongoing in Mediterranean lagoons. This has been previously described for environmental strains but not for clinical strains of V. parahaemolyticus (Chowdhury et al., V. cholerae to be a primarily clonal bacterial species, but the vast diversity in environmental strains showed a recombination rate of 6.5 times the mutation rate (Keymer and Boehm, Vibrio spp. populations form a reservoir where the diversity is efficiently enhanced by recombination in order to produce a generalist population from which specialized clones can emerge. Moreover, we showed that HGT of housekeeping genes occurred among species of potentially pathogenic vibrios in Mediterranean coastal systems. In multilocus phylogeny, such HGT leads to emerging branches similar to those supporting newly described vibrios species, V. mimicus (Davis et al., V. metoecus (Kirchberger et al., It is well-known that the emergence of pathogenic clones in vibrios is linked to HGT of virulence genes or genetic elements among strains as for phage CTX in V. cholerae and V. parahaemolyticus showing a genetic diversity equivalent to the worldwide diversity described so far. The current very low frequency of virulence genes in these populations indicates a low risk for human infection. But the presence of STs involved in human infections elsewhere, as well as the frequent recombinations observed in these transitional aquatic ecosystems, indicates that autochthonous V. cholerae and V. parahaemolyticus populations could be involved in epidemiological cycles and infectious hazard.Mediterranean coastal lagoons host 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.


Oncorhynchus mykiss). This species was introduced to Chile during the early twentieth century for stocking and promoting recreational fishing; during the late twentieth century was reintroduced for farming purposes and is now naturalized. We used population- and individual-based inference from single nucleotide polymorphisms (SNPs) to illuminate three objectives related to the establishment and naturalization of Rainbow Trout in Lake Llanquihue. This lake has been intensively used for trout farming during the last three decades. Our results emanate from samples collected from five inlet streams over two seasons, winter and spring. First, we found that significant intra- population  genetic variance was greater than inter-population  genetic variance, downplaying the importance of spatial divergence during the process of naturalization. Allele frequency differences between cohorts, consistent with variation in fish length between spring and winter collections, might explain temporal genetic differences. Second, individual-based Bayesian clustering suggested that genetic structure within Lake Llanquihue was largely driven by putative farm propagules found at one single stream during spring, but not in winter. This suggests that farm broodstock might migrate upstream to breed during spring at that particular stream. It is unclear whether interbreeding has occurred between “pure” naturalized and farm trout in this and other streams. Third, estimates of the annual number of breeders (Nb) were below 73 in half of the collections, suggestive of genetically small and recently founded populations that might experience substantial genetic drift. Our results reinforce the notion that naturalized trout originated recently from a small yet genetically diverse source and that farm propagules might have played a significant role in the invasion of Rainbow Trout within a single lake with intensive trout farming. Our results also argue for proficient mitigation measures that include management of escapes and strategies to minimize unintentional releases from farm facilities.Knowledge about the genetic underpinnings of invasions—a theme addressed by invasion genetics as a discipline—is still scarce amid well documented ecological impacts of non-native species on ecosystems of Patagonia in South America. One of the most invasive species in Patagonia’s freshwater systems and elsewhere is rainbow trout ( Releases of non-native species can cause economic losses and ecological impacts to native ecosystems –3. HowevNb: [Knowledge about the genetic underpinnings of the invasion of salmonids in Patagonia has been recently accumulating –20 amid Nb: ), are alNb: , but estNb:  for an eOncorhynchus mykiss) is one of the most notorious invasive species around the world [Rainbow Trout  is an exemplary case to illustrate these stages . FollowiNb ,45.Nb) per year among introduced populations using a linkage disequilibrium method [Nb might be small as other invasive Rainbow Trout populations in Patagonia were likely founded two or three generations ago [In this study we used population- and individual-based inference from single nucleotide polymorphisms (SNPs) to address three objectives regarding the distribution of genetic variance within and among Rainbow Trout collections from Lake Llanquihue. First, we evaluated intra-population followed by inter-population genetic variance and their statistical significance from collections at five inlet streams off Lake Llanquihue . We hypom method . We hypoions ago . These hions ago . These mions ago ,51. Our Lake Llanquihue  before being photographed, measured, and weighed. We evaluated statistical differences in trout size  from different streams (and groups thereof) using non-parametric Mann-Whitney U tests in R [2 in size from the adipose fin, caudal fin, or both, were non-lethally obtained and preserved in ethanol 95%. Fish were put in a recovery tank containing well oxygenated water before release.Fish were anesthetized in a bucket containing a solution of 0.015% v/v of benzocaine  following the manufacturer’s instructions. We used a panel of 96 polymorphic SNPs ,55 whichrotocols ,56.Prior to estimate genetic diversity within and among naturalized samples, we implemented BAYESCAN  to identHO) and expected heterozygosities (HE) within naturalized collections. Allelic richness (AR) and inbreeding coefficients (f) were calculated in FSTAT [vegan in R [We used GENALEX to estimate observed  as well as inter-population θ (among streams) using GENEPOP [θ were significantly greater than zero, we estimated chi-square probabilities in CHIFISH [F statistics.We calculated intra-population  GENEPOP ,60. To t CHIFISH . We impl CHIFISH ) to evalK) in STRUCTURE [K, which ranged between one and five using an admixture model and a burn-in period of 50,000, followed by 250,000 Markov Chain Monte Carlo steps after burn-in. To evaluate the most probable value for K, we followed the Evanno criterion [Q-values) in R following cluster matching and permutation in CLUMPP [We used Bayesian inference to assign individual genotypes to a defined number of genetic pools  in naturalized collections using LDNE [proxy for genetic drift assuming selective neutrality, discrete generations, and closed populations. While the first might be met, the second might not as salmonid populations are age-structured and are likely to be connected by gene flow. When applied to age-structured populations, LD reflects a quantity closer to Nb per brood year rather than the effective population size per generation [Nb between groups of naturalized trout were performed through non-parametric Mann-Whitney tests in R.We estimated the contemporary annual number of effective breeders  rahttp://fisheries.org/guide-for-the-use-of-fishes-in-research). The protocol was approved by the Committee on the Ethics of Animal Experiments of the Universidad de Concepcion. Fishing permit #1715/14 was issued by the Chilean Undersecretary of Fisheries and Aquaculture.This study was carried out in accordance with the recommendations of the Guidelines for the Use of Fishes in Research . Also, trout from YER12S and YER13S were larger than trout caught at any of the remaining streams during spring (Mann-Whitney U test: U = 10098: p ˂ 0.001). Further, collections PES12S, YER12S and YER13S showed trout with skin ulcers, skin abrasions, eroded fins, and short opercula, suggestive of farmed fish  in more than 75% of collections (often in 100% of collections) for five groups of loci, some of which had known linkage relationships from previous studies [SNPs were removed if they were monomorphic in all populations, consistently deviated from HWE proportions, showed evidence of directional selection, or exhibited significant LD . Three m studies ,69 as pr studies . Signifi studies . We founHO between 0.312 (BLA13W) and 0.368 (YER13S), whereas values of HE ranged between 0.315 (BLA13W) and 0.369  and 1.984 (BAR13W). Trout from YER had higher HE than trout from any of the remaining collections (PERMANOVA: p < 0.001).We estimated values of θ among all collections with only three exceptions—BAR12 S vs. PES12S, BLA12S vs. BAR13W, and PES12S vs. BLA13W  and 0.004 (PES13W vs. BLA13W) and did not follow simple geographic patterns mediated by distance. For instance, θ values between inlet streams separated by less than 5 km  were larger than between inlet streams separated by more than 30 km . The highest θ values were found in inter-population pairwise comparisons involving all three YER collections. Hierarchical AMOVA suggested that both intra- and inter-population genetic variance were significant, and the latter was higher than the former if all collections were included in the analysis . Q-values among collections were relatively symmetric (0.45–0.55) when assigned to two clusters, with exception of YER13W that showed Q-values between 0.39 and 0.61.The number of gene pools inferred through Bayesian clustering varied depending on sampling season . We founNb were bound by finite 95% CIs, suggesting that the number of annual breeders contributing to all collections might be small .All estimates of be small . Point eθ did not follow simple geographic patterns mediated by distance. Second, individual-based Bayesian analyses further revealed that trout from one single stream, Yerbas Buenas (YER), drove genetic divergence among individuals during spring, but not in winter sampling seasons. Spring trout from YER were additionally more likely to show abnormalities and have smaller Nb than trout from any other collection, suggesting they might originate from farm broodstock. Third, our estimates of Nb showed that half of the collections had a breeding size of 73, consistent with previous findings of significant temporal genetic variance. We discuss these findings in detail in the following paragraphs.We used population- and individual-based inference of multilocus SNP genotypes to address three fundamental issues of the naturalization and invasion of Rainbow Trout in a lake historically impacted by trout stocking and farming practices. First, we found significant intra- and inter-population genetic variance, and the former  component seemed more important than the latter  component if highly distinct collections were excluded from the analyses. Spatial differentiation as revealed by estimates of inter-population Salmo salar: [S. trutta: [θ showed that intra- population  genetic variance was greater than inter-population  genetic variance, downplaying the importance of spatial divergence during the process of naturalization.With exception of Pink Salmon where temporal divergence is prominent between odd- and even-year populations, spatial divergence at various scales as a result of homing behavior is a trademark among salmonid systems in their native range ,73, usuao salar: ,76; brow trutta: ). For naNb).What is the explanation behind significant temporal genetic variance? We ruled out sources of confounding spatiotemporal effects such as family-biased sampling as no significant departures from HWE proportions or LD were evident within collections. One hypothesis is that winter and spring trout collections correspond to different cohorts and that genetic drift between cohorts was significantly higher than expected by chance. This is consistent with differences in size (and likely age) between spring and winter trout. Most spring trout likely comprised fish of age +1  and or even age +2 , whereas winter trout likely comprised young-of-the-year fish . Thus, dOur findings are limited to collections temporally spaced 8 to 13 months and they need not reflect patterns of genetic drift at longer temporal scales. Rainbow Trout of ages 4+ to 12+ (with a mode at age 6+) breeding at the same inlet streams we examined here  were described in an earlier study, suggesting an iteroparous life history . Multiplθ which was nearly universal among all collections and lack of geographic structure should be interpreted within the context of invasive populations that might not be at migration-drift equilibrium [θ will fail to translate into realistic measures of gene flow if populations are in disequilibrium [Lastly, significant inter-population ilibrium . This isilibrium  and Lakeilibrium . Estimatilibrium , which ailibrium ,36.-1), and one of the most productive inlet streams for Rainbow Trout (authors’ unpublished data). We speculate that the source of YER trout appears to be farm broodstock that migrated upstream to breed in spring, which explains why during winter they were apparently missing; it is beyond the scope of our study to determine whether YER12S and YER13S originated from one or two consecutive escaping events. Farm escapees are more likely to show abnormalities and disease that affect farmed trout [Nb, suggesting a highly diverse and yet genetically small source. A common disease affecting Rainbow Trout in freshwater is flavobacteriosis (Flavobacterium psychrophilum) with characteristic skin ulcers and erosions as reported here . These results are stimulating because farm propagules of Rainbow Trout can not only be identified and distinguished from naturalized individuals; they might establish in the wild and breed with other farmed or naturalized trout. Indeed, we observed several sexually mature trout from YER during spring (authors’ field observations), suggesting a time of breeding that begins in winter but extends into early spring [We identified two gene pools of Rainbow Trout coexisting in Lake Llanquihue during spring, but only one during winter sampling seasons. During spring, one of these pools comprised trout populating one single stream: Yerbas Buenas (YER), a narrow (1–2 m wetted channel width), fast-flowing  and Coho Salmon (Oncorhynchus kisutch), which are more conspicuous in lakes with low or no propagule pressure from aquaculture farms [While identifying farm escapees can be accomplished using other methods , SNP gena et al.  geneticare farms ,24. Our re farms .Nb suggest that half of naturalized trout collections have an annual breeding size of 73 individuals or less, which appears counterintuitive given their high levels of heterozygosity. This supports the notion that these populations might be experiencing substantial genetic drift and temporal genetic variance. Parallel estimates of LD Nb for Rainbow Trout populations in Patagonia were below 100 based on microsatellite DNA multilocus genotypes [HE among naturalized collections remained high despite small LD Nb. We performed a quick comparison with native populations from North America (n = 10) [p < 0.001; HE is also consistent with multiple “waves” of introduction in Lake Llanquihue likely originating from genetically distinct sources, a possible explanation of why invasive populations generally overcome founding effects and spread [Our estimates of enotypes . This is(n = 10)  genotyped spread .Nb below 100 in most cases, despite the fact of census population sizes (N) in the order of 1000s [N are unavailable for inlet streams of Lake Llanquihue, and these are necessary to comprehend ecological processes affecting these populations. In addition, the ratio Nb/N is crucial to assess population stochasticity in the face of recent global environmental change [The paradox of genetically diverse but small-size populations might not be exclusive of Rainbow Trout outside their native range and might suggest recently founded populations . Native of 1000s . To our l change . This thl change ,85,86. Ll change , which dNb) were below 73 in half of the five collections, suggesting genetically small and recently founded populations that might experience substantial genetic drift. Our findings reinforce the notion that naturalized trout originated recently from a small yet genetically diverse source and that farm propagules might have played a significant role in the invasion of Rainbow Trout within a single lake.Using population- and individual-based inference of SNP multilocus genotypes we provide insights on the genetic influence on the establishment and naturalization of Rainbow Trout in Lake Llanquihue, a Patagonian lake heavily affected by trout farming. First, we found that intra- population  genetic variance was greater than inter-population  genetic variance, downplaying the importance of spatial divergence during the process of naturalization. Temporal variance might be explained by allele frequency shifts between cohorts, consistent with variation in fish length between spring and winter collections. Second, individual-based Bayesian clustering suggested that genetic structure within Lake Llanquihue has been largely influenced by putative farm propagules from Yerbas Buenas (YER) stream. Yet, it is unclear whether interbreeding has occurred between “pure” naturalized and farm trout. Third, estimates of the annual number of breeders Click here for additional data file.S1 TableLinkage information between loci is emphasized using lower case letters.(XLSX)Click here for additional data file.


Panthera leo) were reintroduced into 44 fenced reserves with little attention given to maintaining genetic diversity. To examine the situation, we investigated the current genetic provenance and diversity of these lions. We found that overall genetic diversity was similar to that in a large national park, and included a mixture of four different southern African evolutionarily significant units (ESUs). This mixing of ESUs, while not ideal, provides a unique opportunity to study the impact of mixing ESUs over the long term. We propose a strategic managed metapopulation plan to ensure the maintenance of genetic diversity and improve the long-term conservation value of these lions. This managed metapopulation approach could be applied to other species under similar ecological constraints around the globe.Population fragmentation is threatening biodiversity worldwide. Species that once roamed vast areas are increasingly being conserved in small, isolated areas. Modern management approaches must adapt to ensure the continued survival and conservation value of these populations. In South Africa, a managed metapopulation approach has been adopted for several large carnivore species, all protected in isolated, relatively small, reserves that are fenced. As far as possible these approaches are based on natural metapopulation structures. In this network, over the past 25 years, African lions ( Biodiversity is threatened worldwide. One of the major threats to biodiversity is habitat loss and resulting population fragmentation. However, population fragmentation is not always as problematic for a population as it may seem, since some species naturally occur as metapopulations (e.g. Melitaeini butterflies ). The teCervus elaphus nelsoni) [Puma concolor) [Panthera leo) [Metapopulation dynamics have been identified in species where human encroachment has caused landscape fragmentation, such as Rocky Mountain elk (nelsoni) , cougar oncolor)  and lionera leo) . Increasera leo) ). Howeveera leo) –11. IdeaLycaon pictus) management in southern Africa [Acinonyx jubatus) management plans [“Managed metapopulation” is a recent term in the scientific literature, first appearing with respect to African wild dog . They list two requirements for a metapopulation: 1) that the populations are geographically discrete and 2) that mixing between populations is less than within populations [Akçakaya, Mills & Doncaster  defined ulations .In this paper we explore the potential of a managed metapopulation approach to fenced, fragmented African lion populations in South Africa. This is important because some researchers have questioned the conservation value of reintroducing lions ; for altWe propose that a key requirement for conservation success would be for lions from these reserves to be managed in a way such that a) they are genetically similar to the populations that had been extirpated from the region as recommended by the IUCN/SSC Re-introduction Specialist Group  b) thereThis project was approved by Tshwane University of Technology Animal Research Ethics board, Pretoria, South Africa (AREC2010/11/004) and the National Zoological Gardens Ethics and Scientific Committee, Pretoria, South Africa (NZG/P12/04). Access to SANParks genetic samples was through a registered project (MILSM964).2) public or privately owned fenced reserves have been ongoing since the early 1990s [Historically lions roamed across most of Africa outside of the Sahara Desert, but their range has contracted markedly and is highly fragmented, and their overall numbers are declining ,23. The ly 1990s ,25. In 2ly 1990s . While tly 1990s , they wely 1990s ,24. ThisSeveral papers have summarized lion reintroductions into South African reserves ,16,25. H10.5061/dryad.f61vq). We used 301 of the previously genotyped samples in this study along with an additional 50 samples from Tembe Elephant Park, Hluhluwe-iMfolozi Park and Madikwe GR. These new samples were processed as detailed in Miller et al. [10.5061/dryad.fc2vv). The sampling included material from 17 small reserves, as well as from Kruger NP and Kgalagadi TP, which were included as reference gene pools unaffected by translocations.Samples from 351 free-roaming lions in southern Africa were genotyped at 22 microsatellite loci  as outlined in Miller et al. [As we did not have access to samples from Etosha NP directly, samples from Pilanesberg NP were used as Etosha NP surrogates as this population was established with lions translocated from Etosha NP in the early 1990s . In ordeSamples previously analysed in Miller et al.  from KruA subset of the data from unmixed populations  was analysed with STRUCTURE  to deterThe data set from reserve animals only (i.e. not including the reference populations) (n = 286) was run in STRUCTURE with K-values from one to eight with 100,000 of burn-in and data collection of 100,000 chains. The “Admixture Model” was applied as some reserves were founded from multiple sources and genealogical records showed subsequent mixing. This was replicated 100 times per value of K. STRUCTURE HARVESTER, CLUMPP and Distruct were used to process the data as above. However the “LargeKGreedy” algorithm was used with 100 iterations within CLUMPP. Samples with a Q value of less than 0.8 were designated as admixed.FIS) were determined using GenePop [Number of alleles, percentage of alleles present, allelic richness, observed and expected heterozygosities were determined for each reserve using the ‘divBasic’ command in the R package ‘diveRsity’ . Inbreed GenePop . SamplesThe program Coancestry was used to determine pairwise relatedness between all individuals . Both thSTRUCTURE analyses revealed four clusters that matched the four predicted populations of Kruger NP, Pilanesberg NP, Kgalagadi TP and Greater Mapungubwe TFCA . The EvaThe STRUCTURE analysis revealed many reserves to have a mix of sources . AdditioFIS = 0.213), had very low FIS values , the source of lions in reintroductions has been relatively historically accurate. Gene flow has been occurring (through translocations) as evidenced by mixed genetic provenances of many animals and other statistical analyses. All of these combined suggest that the genetic potential of the small reserve lions is enough to satisfy the requirement to be a metapopulation.Although there was a separate cluster in the HiP lions, this was presumably due to a founder effect as this lion population was started from very small numbers in the late 1950s, early 1960s with no additional founders until the late 1990s and early 2000s when some animals from Pilanesberg NP and Madikwe GR were added to address inbreeding concerns . As thisKruger NP, Kgalagadi TP, Greater Mabungubwe TFCA and Etosha NP are recognised as separate Lion Conservation Units (LCU) by the IUCN  and sepaA further complication affecting the genetics of these lions is the mixing of animals from separate ESUs. This is not ideal as it may erode genetic diversity and can lead to a loss of local adaptation . Long-teShort of removing all of the lions of mixed provenance and/or of Etosha NP provenance  there is no way to change the current genetic status of lions in South Africa’s small reserves. Thus we recommend that the current animals be administered as one managed metapopulation. This will result in a new genetic group that is a mix of southern African lions and will provide a useful system for future research into the effects of mixing ESUs. We also recommend that no new animals be sourced from Etosha NP. This will reduce the contribution of Etosha NP genetics, the least geographically accurate group in the metapopulation, over the long term.This population has not to date been formally managed as a metapopulation, however a Biodiversity Management Plan (BMP) for lions in South Africa is in the final stages of government approval . This BMWhen planning a managed metapopulation approach it is useful to have examples from nature on which to base models. The parallels between natural metapopulations and what we are trying to achieve with the managed lion metapopulation should not be ignored. What we are trying to achieve could be seen as a series of linked patchy populations, such populations being “distributed over a patchy… variable habitat, but in which high rates of dispersal effectively unite the patches into a single demographic entity” .With the implementation of the proposed managed metapopulation plan, there will be four nodes, or patches, around the country ,21. TherAycrigg & Garton  defined With strategic metapopulation management, lions on small reserves in South Africa could form another viable population of lions, containing genetic diversity from all of the remaining lion populations in South Africa and contributing to conservation of lions on the African continent. They do contain a mix of ESUs, which may not be ideal, but this gives conservationists an ideal opportunity to study the impact of mixing ESUs in the long term. It is hoped that the lessons we have learnt from studying these lions in South Africa could be applied to other lion populations across Africa and to other species that are subjected to similar constraints on natural ecological processes.


Using data on all scientific publications from the Scopus database, we find a superlinear scaling effect for U.S. metropolitan areas as indicated by the increase of per capita publication output with city size. We also find that the variance of residuals is much higher for mid-sized cities  compared to larger cities. The latter result is indicative of the critical mass required to establish a scientific center in a particular discipline. Finally, we observe that the largest cities publish much less than the scaling law would predict, indicating that the largest cities are relatively unattractive locations for scientific research. Many phenomena scale with city size and do this in a non-linear way. Bettencourt et al. (2010) find that as city size increases, per capita quantities such as wages and GDP increase by approximately 15% more than the expected linear growth. Elsewhere, it was established that gross domestic expenditure on R&D (GERD) & gross domestic product (GDP) and GDP & POP (population) all exhibit strong scaling effects For knowledge production activities, scaling effects have been found to be even bigger. Depending on the indicator, Bettencourt et al. We show below that such an approach does not apply well to the production of scientific knowledge. Indeed, data on scientific output of U.S. cities show that the per capita number of scientific papers increases with city size and the exponent is exceptionally high. However, the statistical regularity is weak as many mid-sized cities publish much less than would be predicted by the scaling law, while for some other  “science cities,” publishing performance is much higher than would be predicted from their modest size.We also observe that the very large cities publish much less than the scaling law would predict, indicative of agglomeration disadvantages for very large cities. We interpret this result as evidence that, relatively, very large cities are unattractive locations for scientific research. This effect may well be due to high rental prices that both universities themselves, as well as their staff and students have to pay for being located in such very large cities.A disaggregated analysis at the level of scientific discipline further shows large disciplinary differences in the scaling behavior as well as in the goodness of fit of the alleged “scaling law”. In particular, we find that scaling applies best to disciplines with a strong local interaction with citizens or firms . We understand this result as reflecting that agglomeration advantages are most apparent in scientific research which is closely linked to a local user base.Bettencourt et al. In spatial economics, scaling patterns are often explained as productivity gains that result from economies of scale, the mobility of labor, knowledge spillovers, and other effects of agglomeration economies Agglomeration advantages in research are efficiency gains for a researcher or research institute stemming from co-locating in a geographical cluster, that is, in the vicinity of many other researchers or research institutes, respectively. Advantages stem primarily from cost advantages in search costs for partners and staff, sharing of infrastructure, and the availability of supporting services. Furthermore, the cost of collaboration is lower as travel costs increase with physical distance Y) as a function of the size of another quantity (N), such that:i (iY) as a function of the population of city i.A scaling law describes one quantity  collectively, are defined by the US Office of Management and Budget (OMB). Each area is uniquely referred to by a code called as the CBSA code. As the name implies, a CBSA is a group of adjacent areas that are socioeconomically close to an urban center. Note that, in this paper we accordingly use the terms “CBSA” and “city” almost interchangeably for mere convention. Accordingly we built a dataset that has 923 observations, each for another CBSA.Our geographical unit of analysis is a metropolitan and micropolitan statistical area. These areas, also known as the 95.25% of the approximately 8.11 million addresses found in Scopus. On the basis of fractional accounting this is equivalent to 93.1% of all documents.Using an off-line version of Elsevier’s Scopus database covering the time period 1996–2008, we extracted all (about 4.4 million) documents that have at least one author whose (at least one) affiliation reports an address in the US. Together with the delineation tables (version 2003) made available by the US Census Bureau that maps the association between city/town names and the CBSA codes, we were able to standardize N. Total number of publications in a city is derived from Scopus for the period 1996–2008, which reports author affiliation in the respective area. We use fractional counting meaning that a city occurring on a paper with x affiliations is counted only as 1/x. The total number of publications is further broken down to scientific disciplines according to the classification by Scopus at the 2 digit level (thus 27 disciplines). Publications that report multiple scientific disciplines are accounted for fractionally as well.For each CBSA, we use the US 2000 Census for urban population Methodically, the primary research question posed by this paper would not require anything more than running the aggregate-level regression specified by α and β) of both the non-weighted (thus heteroskedastic) and the weighted regressions.To deal with the problem of heteroskedasticity, we apply a weighting scheme such that the observations of scientific output of medium-sized cities are weighted more according to a function that estimates the error term as an inversely U-shaped function of urban population see . Below, β = 1.78 of the non-weighted regression (as well as β = 1.676 of the weighted one) clearly lies above 1 and is indicative of a superlinear relationship meaning that the per capita scientific output increases quickly with urban population.β = 1.34), patenting (β = 1.27) or inventors (β = 1.25) in U.S. cities Interestingly, the value of the exponent is found to be substantially higher than that of R&D employment  in doing science in larger cities compared to smaller cities. per se a further concentration of resources in larger cities.Alternative candidate explanations include the preference of staff and students to live in larger cities Two other important caveats apply as well. First, it is important to note that even though the scaling exponent is very high, the largest three cities  are much less productive than the scaling law predict. For example, the city of New York  with about 18 million inhabitants is predicted to produce about 774 thousand papers, while it only produces about 253 thousand. Hence, looking at the total set of U.S. cities, scientific production seems over-represented in larger cities reading from the estimated exponent of 1.676. However, this strong logic of urban scaling clearly no longer applies when looking only at the largest three metropolitan areas. This finding resonates with the historical trend of geographical deconcentration in scientific knowledge production found by Grossetti et al. 2R value of 0.63. In this, the observed pattern is very different from the high 2R values found for urban scaling laws regarding wages (2 = 0.96R) and private R&D employment (2 = 0.92R), and more in line with the lower 2R values found for patents (2 = 0.72R) and R&D establishments (2 = 0.76R) 13.12 in logarithmic scale as in Second, the statistical fit of the scaling law is rather weak as indicated by a To conclude, scientific output scales with city size but this pattern is not very systematic. In particular, there are two systematic deviations. First, the very large cities greatly underperform. Second, small cities tend to produce little science with some notable exceptions.β = 1.72), Biochemistry, Genetics and Molecular Biology (β = 1.54) and Engineering (β = 1.41). These disciplines also show the best fits reading from the 2R values . What these disciplines have in common is that these are applied sciences characterized by intensive interaction with local stakeholders, be it patients or companies. The high beta coefficients, then, may well suggest that research in medical and engineering sciences preferentially locates in larger cities benefitting from the local presence of patients and companies engaged in related research. That is, these disciplines look for “institutional complementarities” Some disciplines show strong signs of scaling, in particular Medicine .Interestingly, the city size with maximum variance in scientific production differs greatly per discipline  to construct from publication data. It is most likely that the explanatory power of the scaling function would then be much higher. This issue can be taken up in future research.However, this study does raise the additional question whether the specific type of research activity undertaken matters? This question is important because there are clear policy implications of this issue in terms of policies directed towards the concentration of innovation and knowledge development. The results show a great deal of heterogeneity across disciplines. While some disciplines  show low scaling values, other disciplines show strong signs of scaling, in particular Medicine, Biochemistry, Genetics and Molecular Biology and Engineering.What these disciplines have in common is that they are characterized by intensive interaction with local stakeholders, be it patients or companies in a local context of application. The high beta coefficients suggest that research in these fields preferentially locates in larger cities benefitting from the local presence of patients and companies engaged in related research. These patterns can be expected to be particularly relevant for new and divergent areas of knowledge production As a consequence, especially large metropolitan areas can benefit from the diversity of human resources and institutional complementarities that provide comparative advantages to yield greater output in terms of knowledge production. The key policy concerns would then become how to identify the new fields of knowledge production and how to foster such diversity of human resources and institutional complementarities.We also observed that there is a high variance in scientific output for mid-sized cities. For this class of cities, a few cities excel while most underperform. The exact maximum variance in scientific output is reached at different city sizes in different disciplines. In some disciplines  very small cities with less than 150,000 can already excel while for other disciplines , such high exceptional performance is only observed for city exceeding half a million inhabitants.The city size with maximum variance is indicative of a critical mass that would support the development of disciplinary research strength. Hence, for small cities, a strategic investment in research that displays high variance at large city sizes  entails a high risk. Reversely, for large cities, the opportunities to excel may be precisely in these sciences as little competition is expected from smaller cities. In particular, one may expect that diversified knowledge bases and various institutional complementarities may be important at the frontier of science, where new interdisciplinary directions of research emerge by bringing together diverse skills and diverse contexts of application. In these respects, large cities can be expected to have a comparative advantage.Figure S1Scatter plots of the linear regression residuals  and the respective variance estimated as function of city size. (A) All disciplines. (B) Arts and humanities. (C) Chemistry. (D) Engineering.(TIF)Click here for additional data file.Text S1On the inverted U-shaped heteroskedasticity of the linear regression residuals.(DOCX)Click here for additional data file.


Safeguarding sustainability of forest ecosystems with their habitat variability and all their functions is of highest priority. Therefore, the long-term adaptability of forest ecosystems to a changing environment must be secured, e.g., through sustainable forest management. High adaptability is based on biological variation starting at the genetic level. Thus, the ultimate goal of the Convention on Biological Diversity (CBD) to halt the ongoing erosion of biological variation is of utmost importance for forest ecosystem functioning and sustainability. Monitoring of biological diversity over time is needed to detect changes that threaten these biological resources. Genetic variation, as an integral part of biological diversity, needs special attention, and its monitoring can ensure its effective conservation. We compare forest genetic monitoring to other biodiversity monitoring concepts. Forest genetic monitoring (FGM) enables early detection of potentially harmful changes of forest adaptability before these appear at higher biodiversity levels  and can improve the sustainability of applied forest management practices and direct further research. Theoretical genetic monitoring concepts developed up to now need to be evaluated before being implemented on a national and international scale. This article provides an overview of FGM concepts and definitions, discusses their advantages and disadvantages, and provides a flow chart of the steps needed for the optimization and implementation of FGM. FGM is an important module of biodiversity monitoring, and we define an effective FGM scheme as consisting of an assessment of a forest population’s capacity to survive, reproduce, and persist under rapid environmental changes on a long-term scale. The worldwide trend of biodiversity loss at the global, regional, national, and local levels , and in the framework of this cooperation, during the last decade, joint projects and developments have been initiated that contribute to monitoring of some aspects of forest biodiversity , there has been a lack, at least at the natural population level, of approaches regarding the application of genetic indicators in national and international conservation policies , has been neglected in all forest monitoring programs to date, mainly because genetic diversity is difficult to measure directly  may be fulfilled by developing a systematic way to answer questions such as the following: (i) Are the levels of genetic diversity maintained within critical limits? (ii) Will the population be able to survive and reproduce over future generations? (iii) Are there significant changes in the mating system over years? (iv) Does directional change of genotypic frequencies occur over generations? (v) Is the effective population size changing over time? (vi) Are there changes in fructification intensity over the years?To accomplish the above goals, FGM should include, as a minimum requirement, systematic field measurements of growth traits of old stands and natural regeneration, assessment of the number of reproductively mature trees and seed quality, and recording of phenotypic traits crucial for reproduction and growth, as, for example, flowering onset, termination and intensity, bud break, and bud set. According to Koskela et al. , monitorBiodiversity has been mostly discussed to date at the species and ecosystem level. Genetic diversity within species often is not incorporated in such attempts, although it is the basis of diversity at the species level. Intraspecific genetic variation provides the basis for long-term evolutionary adaptation of populations, mainly through selection mechanisms, and has been assessed by studying adaptive traits (including life history traits) as well as by genetic studies. For example, networks of field trials have been established worldwide for the assessment and improvement of certain economically important traits  FAO . ResultsSeveral concepts of genetic monitoring in forests have been proposed since 1996. Namkoong et al.  amended Fagus sylvatica) and wild cherry (Prunus avium), as reported by Konnert et al. (http://www.fp7-forger.eu/). The monitoring protocols are still in the testing phase and will be further evaluated and compared with alternative approaches.The work of Namkoong et al. , 2002 prt et al. . The Gert et al. . Within t et al. , one worDuring the same year that the first results of the pilot study following the German Concept of a Genetic Monitoring for Forest Tree Species were published, Aravanopoulos  presentePicea abies) to predict the most likely autochthonous plant material for a certain Austrian region, and Geburek et al.  is to condense these sets of indicators and verifiers to the most meaningful and cost-effective ones in a transnational case study approach.The abovementioned concepts of FGM differ in the number of indicators and verifiers. Namkoong et al.  suggesteThere is a clear need  is one of the most obvious silvicultural practices that can influence the genetic structure of future forests, while the gene flow that will follow from the planted genetic material will have an impact on the genetic structure of neighboring forests , mating system , shade tolerance, etc. Some difficulties may also occur during genetic monitoring of tropical forest tree species because of their high number and identification ambiguities (Ratnam et al. Ratnam et al.  providedEuropean forests that have been strongly affected by numerous anthropogenic influences and are expected to be strongly affected by the ensuing climatic change are in immediate need of a genetic monitoring scheme. Several approaches exist, which have to be harmonized at the European scale. Within the LIFEGENMON project, guidelines will be developed on the basis of different intensity and cost levels and will be prepared for implementation in practice by training courses. However, due to the development of new genetic/genomic tools, contributing to the more detailed information on the genetic structure of forest tree populations, and of new quantification methods and means, FGM should remain open for further improvement.


Background. Worldwide predictions suggest that up to 75% of the freshwater fish species occurring in rivers with reduced discharge could be extinct by 2070 due to the combined effect of climate change and water abstraction. The Mediterranean region is considered to be a hotspot of freshwater fish diversity but also one of the regions where the effects of climate change will be more severe. Iberian cyprinids are currently highly endangered, with over 68% of the species raising some level of conservation concern.Methods. During the FISHATLAS project, the Portuguese hydrographical network was extensively covered  in order to contribute with valuable data on the genetic diversity distribution patterns of native cyprinid species. A total of 188 populations belonging to 16 cyprinid species of Squalius, Luciobarbus, Achondrostoma, Iberochondrostoma, Anaecypris and Pseudochondrostoma were characterized, for a total of 3,678 cytochrome b gene sequences.Results. When the genetic diversity of these populations was mapped, it highlighted differences among populations from the same species and between species with identical distribution areas. Factors shaping the contemporary patterns of genetic diversity were explored and the results revealed the role of latitude, inter-basin connectivity, migratory behaviour, species maximum size, species range and other species intrinsic traits in determining the genetic diversity of sampled populations. Contrastingly, drainage area and hydrological regime (permanent vs. temporary) seem to have no significant effect on genetic diversity. Species intrinsic traits, maximum size attained, inter-basin connectivity and latitude explained over 30% of the haplotype diversity variance and, generally, the levels of diversity were significantly higher for smaller sized species, from connected and southerly river basins.Discussion. Targeting multiple co-distributed species of primary freshwater fish allowed us to assess the relative role of historical versus contemporary factors affecting genetic diversity. Since different patterns were detected for species with identical distribution areas we postulate that contemporary determinants of genetic diversity (species’ intrinsic traits and landscape features) must have played a more significant role than historical factors. Implications for conservation in a context of climate change and highly disturbed habitats are detailed, namely the need to focus management and conservation actions on intraspecific genetic data and to frequently conduct combined genetic and demographic surveys. Freshwater biodiversity has declined faster than terrestrial and marine biodiversity over the last decades . Up to 7Most endangered Iberian cyprinids have extremely restricted geographical ranges and occur in temporary Mediterranean-type Rivers with autumn-winter floods and extended summer droughts, resulting in a series of disconnected pools . AlthougAlthough crucial, the application of genetics in the management of wild threatened species is still far from being common . Thus, gAs the distribution of the genetic diversity may have been shaped by historical and contemporary events we aimed to disentangle the factors underlying the observed patterns. As such, in parallel with the spatial patterning of genetic diversity, the broad-scale sampling also allowed for testing the effects of species intrinsic traits and environmental characteristics on observed levels of genetic diversity. Data obtained allowed us to address the following questions: (1) Is the genetic diversity of endemic fish populations influenced by the specific status of each population and by other characteristics that are intrinsic to the species, such as the maximum size or migratory behaviour? (2) Is the genetic diversity of each population influenced by the area and by the hydrological regime of the river drainages they inhabit? (3) Does latitude influence genetic diversity, given that populations from northern rivers are subjected to lower temperatures and fewer temperature fluctuations, contrasting with those from southern rivers which are exposed to higher temperatures, lower oxygen concentrations and cyclical regimes of floods and droughts ? (4) Do Anaecypris hispanica , Achondrostoma oligolepis , Achondrostoma occidentale , Iberochondrostoma lemmingii , Iberochondrostoma lusitanicum , Iberochondrostoma almacai , Pseudochondrostoma polylepis , Pseudochondrostoma duriense , Pseudochondrostoma willkommii , Luciobarbus microcephalus , Luciobarbus bocagei , Luciobarbus sclateri , Luciobarbus comizo , Squalius carolitertii , Squalius pyrenaicus , Squalius torgalensis  and Squalius aradensis . Of all the cyprinids native to Portugal, only four species  were not included a priori due to their hybridogenetic origin .A total of 17 Iberian endemic cyprinids were selected as target species: c origin , uncertac origin  or extrec origin . L. micrThirty four river basins were sampled in the Portuguese mainland hydrographical network. The six largest river basins  were further sub-divided into 47 sub-basins resulting in a total number of 81 geographical units sampled . PopulatFish were collected with standard wadable electrofishing procedures  and retub (cytb) gene was amplified using the primers LCB1-new ACTTGAAGAACCACCGTTG (adapted from the LCB1 primer described by KU366823– KU370500).Total genomic DNA was extracted from fin clips using REDExtract-N-Amp Tissue PCR kits (Sigma-Aldrich) following the manufacturer’s instructions. The mitochondrial cytochrome http://www.birc.au.dk/software/fabox).All sequences were edited and aligned using CodonCode Aligner v4.0.4 . The search for shared haplotypes and the listing of representative sequences was conducted by DNAcollapser  and the average percentage of private haplotypes per population for each target species (%NPH). Mean, standard deviation, minimum and maximum values of %NPH were obtained with EXCEL 2013 (Microsoft®).ARLEQUIN software package V.3.5  was usedh index, defined as the probability that two randomly chosen haplotypes are different in a sample; used as a measure of haplotype diversity for haploid data), nucleotide diversity  and mean number of pairwise differences  for each population. Analyses of molecular variance (AMOVA) were also performed with ARLEQUIN.ARLEQUIN was also used to estimate gene diversity (h index were mapped in the sampling areas by Yris Graphics (www.yrisgraphics.com). This index, which varies between 0 and 1, reflects the probability of two randomly chosen haplotypes being different in a sample, and was selected to illustrate haplotype diversity of sampled populations. Using this index, diversity classes  were established, allowing for an automatic visual inspection of the diversity level of distinct populations. The maps and database are available for download at the project’s webpage (www.fishatlas.net).Values obtained for the L. bocagei, L. sclateri, L. comizo, P. duriense, P. polylepis, P. willkommii, I. almacai, I. lusitanicum, I. lemmingii, S. carolitertii, S. pyrenaicus, S. aradensis, S. torgalensis, A. hispanica, A. oligolepis and A. occidentale), and the dichotomous variables (2) “hydrological regime,” (3) “latitude,” (4) “migratory behaviour,” (5) “species range,” and (6) “inter-basin connectivity.” The quantitative variables “drainage area” and “species maximum size” were also included in the matrix.Populations, regardless of species, were considered the unit of comparison. In order to test whether the obtained genetic diversity pattern was influenced by species’ intrinsic traits and extrinsic factors, data concerning 8 variables were collected and organized in a matrix . Six of http://sniamb.apambiente.pt/Home/Default.htm) by using the polygon area as implemented in QGIS 2.10.1 software. Whenever a population inhabits a sub-basin of a larger basin, the area of the sub-drainage was considered, as for the studied small fish species the main course of a large river may represent a natural barrier to gene flow.“Hydrological regime” was classified as either permanent or temporary, depending on whether or not the population occurs in river basins that maintain flowing water throughout the year; “latitude” as northern or southern ; “migratory behaviour” as non-migratory or potamodromous  were selected as candidate variables for the modelling process. Then, a hierarchical linear regression was performed, first including all selected variables (excluding “species”) with a stepwise method and, finally including the categorical variable “species.” This procedure sought to first analyse the effect of each of the variables regardless of the effect of “species.” This variable was then included to test whether other aspects intrinsic to the species, not measured by the remaining independent variables, were significant. This method was adopted independently for each of the three dependent variables: h, π and MNPD. We search for the presence of outliers and excluded them to avoid spurious trends or masking of valid ones.Predictor variables with significant effects  concerning their genetic diversity . All statistical analyses were conducted using IBM SPSS Statistics, version 22  and h = 0.936 ± 0.010 for (A. hispanica). The average nucleotide diversity and the average mean number of pairwise differences showed the same pattern  and S. pyrenaicus the highest .The average values of haplotype diversity ranged between  pattern : L. comiL. bocagei to 91.91% for A. oligolepis) could be attributed to differences among populations, while for the remaining seven species 53.26% (for P. polylepis) to 98.95% (for L. comizo) of the variation was explained by genetic differentiation within populations , S. pyrenaicus from Tagus-Erges (for MNPD only), and S. pyrenaicus from Tagus-Grande da Pipa, S. pyrenaicus from Sado-São Martinho, I. lusitanicum from Sado-São Martinho and A. hispanica from Guadiana-Ardila (for both π and MNPD). These populations show a higher number of point mutations, probably due to an admixture of individuals from distinct haplogroups, and those differences between the sequences are reflected in the higher values of π and MNPD obtained.Preliminary linear regressions retrieved no outliers when using h values showed that “species maximum size,” “inter-basin connectivity,” “species range,” “latitude,” “migratory behaviour” and “species” had a significant effect on the diversity of populations  in the linear regression, using a stepwise method, indicated that the best fit model only includes “species maximum size,” “inter-basin connectivity” and “latitude.” Non-significant predictor variables “range” and “migratory behaviour” were excluded from the analysis  and the best fit model explained 33.7% and 26.7% of the variance, respectively, for and MNPD .h = 0.413 ± 0.306, N = 135; mean π = 0.00121 ± 0.00120, N = 131; mean MNPD = 0.831 ± 0.856, N = 131) than those occurring in unconnected river basins . Finally, regarding “latitude,” southern populations exhibited higher levels of haplotype diversity and mean number of pairwise differences  than northern populations , a tendency which was already evident from the diversity mapping depicted for the National Genetic Atlas .It is worth mentioning that the variable “species” was significantly correlated with the three genetic diversity indices  and was I. lusitanicum and with “latitude” for L. bocagei and P. polylepis . The analysis of the genetic diversity variation highlighted differences among populations within species and also differences between species with identical distribution areas and threat categories. In general, the percentage of private haplotypes and the average values of genetic diversity per population were higher for small sized non-migratory species of the genera Anaecypris, Achondrostoma, Iberochondrostoma and Squalius than for the larger sized potamodromous Luciobarbus and Pseudochondrostoma species, raising the hypothesis of species intrinsic determinants of genetic diversity. Also, the analyses of molecular variance revealed that for some species most of the variance could be attributed to differences among populations, while in others to differentiation within populations. The spatial distribution of the genetic diversity was undoubtedly distinct for co-occurring species, but what are the underlying causes of such distinct patterns?The sampling of a broad number of populations throughout the distribution range of cyprinid species in Portugal allowed for the publication of the first National Genetic Atlas of native cyprinid ichthyofauna  historical geological/climatic processes , (2) species intrinsic traits , and (3) landscape features, including human-mediated habitat changes  . Determiversus contemporary factors affecting genetic diversity. Indeed, the origin of Iberian lineages of Anaecypris, ex-Chondrostoma, Luciobarbus and Squalius dates back to the Miocene, around 19–7.7 Mya , indicates that, at the time of the LGM, all contemporary species were already differentiated  the ice sheet reached the central part of the Portuguese territory, presumably as far as the Tagus River . Previouentiated . Additioentiated . Thus, aes e.g., . Howeveres e.g., . Since dAs referred above, species intrinsic traits and landscape features may influence current levels of genetic diversity. Habitat loss and fragmentation, for instance, may result in declining population sizes and genetic diversity depletion, ultimately leading to local extinctions . HusemanEcological traits such as tolerance to stagnant disconnected summer pools , prefereper batch (r-strategists) than larger sized fish that typically mature later and produce a smaller number of eggs (k-strategists). As a consequence, in disturbed environments that impose shorter than normal life spans, it is expected that small sized species would be favoured as they will most likely leave more progeny than larger species and will presumably be less prone to genetic depletion due to inbreeding and genetic drift .The species maximum size was negatively correlated with genetic diversity, a feature that was already reported for several taxa e.g., . Body siDrainage area and hydrological regime (permanent vs. temporary) were not significantly correlated with genetic diversity, while connectivity between sub-basins and latitude had a significant effect.The results show that populations occurring in sub-basins connected with other water bodies had higher genetic diversity than those occurring in unconnected river basins. As the drainage had no significant effect on the genetic diversity of the populations inhabiting them, the observed effect of the inter-basin connectivity may be related to (1) the possibility of inter-population gene exchange or (2) historical features. This later explanation relates to the fact that the colonization of Iberia by the ancestral lineages of primary freshwater fish seems to have followed a westward path (from the Pyrenees to the margins), with the major basins as vehicles for colonizers and playing a crucial role in the radiation of these species throughout the Peninsula e.g., . As a coFuture studies should also address connectivity at an intrabasin scale, by quantifying the number of unsurmountable barriers preventing gene flow. Predictions point to lower global levels of genetic diversity due to fragmentation and population declines expected to occur in highly impounded river basins e.g., .in situ survival in the southernmost populations, far from the glaciers , in southern latitudes (L. bocagei and P. polylepis) and in connected sub-basins (S. pyrenaicus and I. lusitanicum). These results are in agreement with the view of Finally, when looking to each isolated species rather than using populations as the comparison units, the results showed that environmental characteristics did not explain the genetic diversity variance for most of the species, with the exception of the potamodromous large sized In conclusion, as multiple intrinsic and extrinsic drivers may be acting synergistically, future studies should ideally be conducted for each species separately and should adopt sampling procedures that would allow for an exhaustive collection of data concerning habitat-, landscape- and species-related variables.Different populations of the same species exhibit not only distinct gene pools but also distinct genetic diversity levels, reinforcing the need to preserve them as individual entities and to establish Operational Conservation Units (OTU’s), as defined by taxa are not driven to extinction before genetic factors affect them adversely  and least concerned (S. carolitertii). The same occurs among Iberochondrostoma and Achondrostoma species, with the critically endangered I. almacai and A. occidentale showing lower haplotype diversity than their less threatened congeneric species I. lemmingii (endangered) and A. oligolepis (vulnerable). A similar pattern was also detected among Luciobarbus, with the endangered L. comizo and L. sclateri showing lower haplotype diversities than the least concerned L. bocagei. Interestingly, however, the results obtained in this study indicate that a higher conservation status is not necessarily synonymous with genetic depletion, as demonstrated by the high haplotype diversity values shown by the critically endangered A. hispanica and I. lusitanicum. The reverse was also not always observed since non-endangered L. bocagei, P. polylepis or S. carolitertii showed relatively low levels of haplotype diversity (when compared to their congeneric species). These results highlight the need to focus management and conservation actions on intraspecific genetic data, instead of erroneously concluding that a species with low genetic diversity is more susceptible to extinction than a co-occurring more diversified one. Frequent genetic monitoring is also crucial since it is known that there is a time lag between the action of factors causing genetic change and the change itself, i.e., changes or disturbances that impact populations may not be immediately reflected in genetic metrics (A high risk of extinction is commonly associated with low levels of genetic diversity and small effective population size e.g., . As argudversely , thus, e metrics . The comex situ conservation, define OUT’s, and propose ex situ and in situ actions to allow for the long-term survival of endangered species and preservation of their genetic integrity.The dataset produced under the scope of the FISHATLAS project is available to be used by managers, decision-makers and authorities not only in the present context of hydrological resources management aiming to minimize the effects of climate changes, but also for the implementation of conservation and management plans aiming to preserve native Iberian cyprinids. More specifically, these data may allow for the definition of priorities in conservation policies, when choices have to be made concerning which populations of each species must be preserved first, a decision that must take into account the maximization of genetic diversity. Although it is widely recognized that genetic data should be taken into account to draw conservation guidelines and prioritize conservation actions , studies10.7717/peerj.1694/supp-1Table S1Number of specimens collected in each river basin/sub-basin. Legend: CR, Critically endangered; EN, Endangered; VU, Vulnerable; LC, Least concerned; NT, Not Threatened.Click here for additional data file.10.7717/peerj.1694/supp-2Table S2Matrix of independent and dependent variables used for statistical analyses.Click here for additional data file.