Hosta sieboldiana and Hosta albomarginata across large portions of their geographic ranges in the Japanese archipelago. Our objective was to compare the phylogeographic histories and phylogeographic structures of the two congeneric species in the Japanese archipelago. The location of the study is Japanese archipelago. We sequenced 1380 bp of noncoding cpDNA from 45 populations of H. sieboldiana (n = 362) and 55 populations of H. albomarginata (n = 436) to assess genetic variations within and among populations across almost the entire distributions of the species in Japan. Extant patterns of geographic structure were analyzed using statistical parsimony networks and spatial analysis of molecular variance (SAMOVA). We also used Monmonier's algorithm to detect genetic barriers between regions. Relationships between the populations were examined using a neighbor-joining (NJ) method. Four haplotypes were found for H. sieboldiana, whereas eight haplotypes were identified for H. albomarginata. Total genetic haplotype diversity (hT) and within-population haplotype diversity (hS) for H. sieboldiana were 0.352 and 0.040, respectively, while the values for H. albomarginata were 0.529 and 0.085, respectively. The population differentiations (GST) for H. sieboldiana and H. albomarginata were 0.839 and 0.886, respectively. The SAMOVA analysis revealed two clusters in H. sieboldiana and four clusters in H. albomarginata. Differentiations between and among the clusters were supported by the BARRIER analysis and the NJ tree. We detected differences in the population genetic structure between the two species. We found that H. sieboldiana had lower haplotype diversity than H. albomarginata. These results may be partially explained by the difference in ecological habitats and geographic distributions between the species. Hosta albomarginata is more widely distributed than H. sieboldiana in East Asia including Russia, and this large distribution range would enable more chances to intraspecific gene flow.We analyzed variations in chloroplast DNA (cpDNA) in the widespread herbaceous species  Over the past decade, the geographic distributions of intraspecific cpDNA variations have been examined in a variety of plant species to elucidate their postglacial migration history : Sakhalin, the Kuriles, the Korean peninsula, and the Ryukyu Islands. These connections might have affect on the genetic structures and distributions of many plants Hotta . Phylogend Senni  conclude. Fujita  classifiH. sieboldiana along the Sea of Japan have been treated as H. sieboldiana, although other populations were regarded as another species, H. montana . We sampled individual plants occurring a few meters apart to prevent the collection of the same genets. Leaf materials were kept in an ultracold refrigerator (−70°C) in Ziplock plastic bags until DNA extractions were carried out.Leaf samples were collected from 1 to 10 individuals from each of 45 and 55 populations of 2, 10 mmol/L Tris–HCl (pH 8.3), and 0.375 units of Taq DNA polymerase . To examine the geographic distribution of cpDNA in H. sieboldiana and H. albomarginata, we amplified two noncoding regions (1380 bp) of cpDNA, the trnS(GCU)–trnG(UCC) spacer and the trnL–rpl32F spacer, using the primers reported by Hamilton (trnL–rpl32F intergenic region), 60°C for 60 s (the trnS(GCU)–trnG(UCC) intergenic region), and 72°C for 30 s, with a final extension at 72°C for 5 min. After amplification, the PCR products were purified using a Gene Clean Kit II . We sequenced the purified PCR products using a BigDye™ Terminator Cycle Sequencing Ready Reaction Kit  and analyzed them on Genetic Analyser Model 3100 and 3700 (Applied Biosystems) according to the manufacturer's protocol. Sequencing was conducted from both ends using the same primers in the trnL–rpl32F spacer as in PCR. In this study, because we could not sequence the total range of trnL–rpl32F which was too long, we tried to sequence the region from each side as long as possible. Thus, we separately showed the sequence as two regions from each end in trnL–rpl32F. We designed several internal sequencing primers in the trnS(GCU)–trnG(UCC) intergenic region to read from one direction: trnS2: 5′-AGTCCCCTCAGCCATCTCTC-3′; trnG2: 5′-GTGTTGACACTTTGTCTCAC-3′; trnG3 5′-GTCCCTTGAACAAGTAAATGAG-3′. For all minor variants, we performed the sequencing at least twice to verify the variations.Genomic DNA was extracted from the leaves according to the CTAB method of Doyle and Doyle . PCR ampHamilton  and TabeHamilton , which hhS), total species diversity (hT), and GST values were computed according to the method of Nei  were performed to identify groups of populations that are geographically homogeneous and maximally differentiated from each other using SAMOVA 1.0 . Assuming that the final configuration was influenced by the initial configuration, 100 initial conditions were used as recommended by Dupanloup et al.  could not derive the group structure  and the geographic structure were inferred from the configuration with the highest FCT that did not contain any single population group. We performed SAMOVAs for the data both with and without indels  were detected among sequences for H. sieboldiana , as was the case when indels were included  for H. sieboldiana  for H. albomarginata  included haplotype e when indels were included, and this haplotype corresponded to haplotype c, which occupied narrow regions when indels were excluded. When indels were excluded, haplotype c showed one mutation difference from haplotype b in the four groups identified by the SAMOVA, which is widely distributed in Honshu. One group in the north–central part of Honshu (populations 30 and 31) included haplotypes b, c, j, and q when indels were included, and when indels were excluded, these haplotypes all matched haplotype b, which dominates Honshu. These haplotypes exhibited different repeat numbers in a polyT region in the sequencing data. Thus, when indels were excluded, haplotypes b, c, j, and q corresponded to only haplotype b in the four groups identified by the SAMOVA. One group in the relatively remote areas (populations 45 and 55) contained haplotype q when indels were included, and this haplotype corresponded to haplotype b in the four groups identified by SAMOVA. Consequently, if we exclude indels from the sequence analysis, many haplotypes are assigned to specific haplotypes, such as haplotype b, which was common in the phylogeographic structures. As a result of the analysis of cpDNA noncoding regions in H. sieboldiana and H. albomarginata, we found only a few substitutions. We thought that it is difficult to elucidate the phylogeographic history of two species with only a few substitutions. Therefore, we tried to include all the information for the analysis of phylogeographic study as possible as we can. However, regardless of including indels or excluding indels in our analysis, we did not find large differences.Three additional groups were detected for H. sieboldiana, we detected the boundaries between Hokkaido populations and the other regions at the first boundary  based on cpDNA data across all populations was much higher than the average within-population haplotype diversity  in both species  has higher values than H. sieboldiana (hT = 0.136), and given the average within-population SAMOVA group diversity (hS) based on SAMOVA group data, the value was 0 in the two species  of H. sieboldiana and H. albomarginata showed almost the same values  Hokkaido and (2) Honshu, Shikoku and Kyushu. The BARRIER results also supported the SAMOVA results. According to previous phylogeographic studies, some plants in the Japanese archipelago show phylogeographic differentiation between Honshu and Hokkaido  in Group 2 included private haplotypes. Iwasaki et al. (Group (2) covers most of the distribution area of i et al.  hypothesi et al.  indicateH. albomarginata. Conversely, when indels were excluded from the sequence data, the SAMOVA revealed four phylogeographic groups. Regardless whether indels were included, the SAMOVA identified three common groups. The BARRIER analysis also supports the SAMOVA results. The range of the first group comprised most of the populations on Hokkaido except for population 6, the southeast population. Similar to H. sieboldiana, H. albomarginata may have been derived from a refugium in Honshu to Hokkaido through the Tsugaru Strait, although unlike H. sieboldiana, H. albomarginata is widely distributed on Hokkaido. Thus, H. albomarginata might exhibit more diverse genetic structuring than H. sieboldiana.When indels were included, the results of SAMOVA revealed seven phylogeographic groups in Picea alcoquiana and postulated that bottleneck events could have occurred around these areas and that the peripheral populations isolated by mountains experienced a recent bottleneck, thus reducing genetic diversity. The populations of this group might have also experienced the same range shifts as P. alcoquiana in response to the Quaternary climate changes.The second group occupied only the central part of Honshu and harbored private haplotypes. Although attributing this finding to a single factor is difficult, one explanation for the private haplotype observed in this group is that these areas experienced bottleneck events. Aizawa et al.  studied Aesculus turbinata in Japan might have consistently retained sufficiently large distribution ranges to harbor several haplotypes during the Quaternary climatic oscillations, causing the southwestern populations of A. turbinata to exhibit local phylogeographic structures or unique haplotypes. Hosta albomarginata might have experienced a similar range distribution history as A. turbinate, and some parts of these areas might have been a refugium of H. albomarginata during the Quaternary climate oscillations. Given the SAMOVA results and the BARRIER results, we hypothesized that a refugium existed within northern Honshu. The wide distribution of haplotype b might have been generated over a longer period prior to the LGM, which would explain the present haplotype distribution pattern. The population in the southeast district on Hokkaido (population 6) of the Honshu group differs from others on Hokkaido. Previous studies of Veratrum album ssp. oxysepalum showed the same phylogeographic pattern (Kikuchi et al. Veratrum album ssp. oxysepalum, H. albomarginata populations of the central refugium may have spread widely throughout northern Honshu and the southernmost part of Hokkaido.The third group consists of Kyushu and southwestern Honshu populations. Previous phylogeographic studies revealed the same patterns (Sugahara et al. H. albomarginata may have migrated to the Japanese archipelago from the Eurasian continent via a landbridge from the Sakhalin and expanded throughout the Japanese archipelago before the last glaciation. This would mean that more than two refugia existed during the last glaciation: One in the south part of the Japanese archipelago and the other on Hokkaido. To test this hypothesis, one must include Russian populations in the analyses; however, such samples are not currently available. Czerepanov (H. albomarginata and H. rectifolia are well-differentiated species based on morphological features and habitats in Russia. Hosta albomarginata occurs only south of the Amurskaya and Primorsky Territories of the Eurasian continent, while H. rectifolia is distributed in Sakhalin and the Kurils (Gage et al. H. albomarginata in the northern parts of Honshu and Hokkaido as H. rectifolia. The extent of the differences between the genetic structures of H. rectifolia and H. albomarginata in Russia is not currently known. An analysis of this genetic differentiation would provide a more detailed understanding of the phylogeographic patterns of H. albomarginata.If the three distinct distributions of cpDNA haplotypes that we identified originated in the glacial era, erepanov  reportedH. sieboldiana and H. albomarginata. The SAMOVA identified two groups in H. sieboldiana and four in H. albomarginata, and H. sieboldiana exhibited significantly lower haplotype diversity than H. albomarginata. The lower haplotype diversity in H. sieboldiana may have resulted from its relatively restricted habitat and geographic distribution. Hosta sieboldiana mostly occurs on cliffs around waterfalls and steep clines, thus restricting the interpopulation gene flow and leading to a smaller effective population size. Conversely, H. albomarginata widely inhabits wetlands, where regional exchanges among gene pools are more likely, leading to a relatively large effective population size. At a continental scale, H. sieboldiana is native to the Japanese archipelago, whereas H. albomarginata is distributed outside of the Japanese archipelago within the Eurasian continent, Sakhalin, and the Kurils of Russia. The relatively restricted geographic range of H. sieboldiana, combined with the relatively narrow distribution of appropriate habitat within its range, would have exacerbated the restrictions to gene exchanges after the LGM. In contrast, the larger distribution range of H. albomarginata might have enabled the maintenance of a larger gene pool.We analyzed the genetic structures of Even though these two species exhibit different phylogeographic structures, we determined that they show the same distinct genetic structure on Hokkaido. Therefore, these species might have experienced the same historical distribution range across the Tsugaru Strait during the LGM (Aizawa et al. H. sieboldiana and H. albomarginata, the possibility exists that these species have hybridized (Takahashi H. sieboldiana and H. albomarginata.Hybridization has been suggested to provide the adaptive genetic variation needed to invade postglacial habitats Stebbins . AlthougHosta sieboldiana and H. albomarginata may have experienced different distributional range shifts during the last glaciation. Similar conclusions were reported by previous studies (Maliouchenko et al. H. albomarginata samples from Russian populations are necessary to test the validity of the comparative phylogeographic histories of the two species.


Aedes (Meigen) are frequently recorded out of their place of origin. Invasive Aedes species are proven or potential vectors of important Arboviruses and their establishment in new areas pose a threat for human and animal health. A new species of exotic mosquito was recorded in 2011 in north-eastern Italy: Aedes (Finlaya) koreicus [Hulecoeteomyia koreica]. The aim of this study was to characterize the biology, the environment and the current distribution of this mosquito in north-eastern Italy. Morphological details useful to discriminate this species from other invasive Aedes mosquitoes are also given .The container breeding species belonging to the genus All possible breeding sites for larval development were monitored. In addition, ovitraps and traps for adults were used to collect eggs and adults. The mosquitoes (larvae and adults) were identified morphologically and molecularly. Environmental data and climatic variables during the period of mosquito activity (from April to October) were considered.Aedes koreicus was found in 37 municipalities (39.4%) and was detected in 40.2% of places and in 37.3% of larval habitats monitored, in a range of altitude from 173 to 1250 m.a.s.l.. Garden centres were the most common locations (66.7%), followed by streets/squares (57.1%), private gardens (46.4%) and cemeteries (21.1%) (p < 0.01). The main larval habitats were catch basins (48.5%) and artificial water containers (41.8%). As for Aedes albopictus [Stegomyia albopicta], ovitraps were attractive for adult females resulting in the higher rate of positivity  among breeding sites. The period of Ae. koreicus activity ranged from March 29 to October 29.Ae. albopictus and colonizing areas over 800 m.a.s.l, not yet or sporadically reached by the tiger mosquito. The data collected are essential to assess the risk of colonization of other parts of Italy and Europe, as well as the risk of spreading of pathogens transmitted. These findings stress the importance of implementing entomological surveillance for early detection of invasive species, which is necessary for eradication or limitation of its further spread.The species is clearly established in the area and is now overlapping with other vectors such as  Aedes (Meigen) are frequently recorded out of their place of origin, mainly due to their diapausing eggs that survive to desiccation during the passive transport  represents the best example of colonization and spreading of an Aedes species in Europe   ,13, a spAe. albopictus. A quick guide for the identification of the species, reporting the main morphological features of the Italian population of Ae. koreicus, is included as Additional files, to help personnel involved in the entomological monitoring with a prompt recognition of this species.After this record, a more intensive surveillance was gradually extended from positive sites in surrounding areas to verify the distribution of this species, to attempt to trace back the possible route of entry and to collect biological data on its life cycle. In this work we report the results of two years of surveillance and the implication for the monitoring and control of a new species, which overlapped with another similar invasive mosquito, The monitoring started in 2011 from the valley  where the first mosquitoes were found and was extended in the whole Province of Belluno and in the neighbouring Province of Vicenza, Treviso and Trento in 2012  is an area of Veneto Region neighbouring Friuli Venezia Giulia and Trentino Alto Adige regions  and Austria. The population density in the province is one of the lowest of Italy (57.7 inhabitants/km2). The villages are quite small and only two have more than 10,000 inhabitants (Feltre and Belluno). The rest of the area monitored  is characterized by climatic and environmental conditions similar to Valbelluna valley.The valley is surrounded by Bellunesi Prealps and Dolomites, extending from north-east to south-west for 50 kilometres with an average elevation of 527.7 m.a.s.l.. The area has a sub-continental climate, characterized by temperate climate, with cold and often snowy winters and mild warm summers. The annual rainfall is above 1,000 mm. The Province of Belluno , Downy Oak (Quercus pubescens) and European Hophornbeam (Ostrya carpinifolia). Coniferous and pine forests are present on the mountains. Water bodies and lakes are abundant at the lowest elevation. The area is mainly covered by forest or natural vegetation (75.0%) and little used for agricultural activities (20.2%); the water bodies represent 0.5% of the area and only the 4.3% of the area is urbanized. The human settlings are small villages composed by country houses with private gardens and public parks, which offer many breeding possibilities to container breeding mosquitoes of the Aedes genus.Environmental data were obtained from Land Use Map (CORINE Land Cover 2006). Vegetation is dominated by Beech  were checked. The places visited in the villages included private and public places, i.e. gardens, garden centres and florists, tire markets, cemeteries, farms and houses.Aedes eggs were collected with standard ovitraps  with sticks of masonite™ as support for oviposition.2 and lure™, testing also the ability of the traps to catch this species. During larval collection, adults trying to land on the personnel were also collected. Adults and larvae obtained from the hatching of eggs were examined.Larvae collections were made using a standard larval dipper . The larvae collected were placed in 50 ml plastic tubes and transported alive to the laboratory in a cooler. The adults were collected using BG-Sentinel™ traps baited both by COAe. koreicus activity was defined when there were no more adults, larvae or positive ovitraps.The end of The larvae collected (either hatched from ovitraps or from direct collections) were stored in 70% ethanol for further identification. A small number of larvae were reared in the laboratory to obtain adults. The 4th-instar larvae were clarified in saturated solution of alcohol-phenol for 4-5 h and then transferred in phenol-balsam  used as mounting medium . The samAe. koreicus was present or obtained from eggs hatched in laboratory.Six females and six males were examined accurately; the legs were clarified, mounted on slides and length of white band measured. Twelve larvae were also examined. The specimens described were collected as larvae during the summer from two villages where only Ae. koreicus . Aedes koreicus was compared with the most similar species Aedes (Finlaya) japonicus  [Hulecoeteomyia j. japonica] [Ae. albopictus. The comparative study reports only the features that differ among the three species .Particular attention was paid to the morphological features useful to make a correct and rapid identification of larvae and adults of aponica] ,18,23 anIn case of doubtful morphological identification or findings in new areas, a molecular confirmation was carried out using a PCR targeting the nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 (ND4) gene and using the primers and protocol suggested by ,24.Ae. koreicus prevalence (only place/breeding sites monitored more than 10 times) according to the place of collection, larval breeding sites, altitude range. Prevalence distribution of Ae. koreicus was also compared with Ae. albopictus according to altitude.The chi-square test was used to test significance of the differences of The area was monitored from the discovery of the species, on May 24 through September 2011 and from March through October 2012. The altitude of the sampling places ranged from 14 to 1645 m.a.s.l..A total of 94 municipalities were monitored: 53 in the Province of Belluno (76.8%) and 41 in other villages in the Province of Vicenza, Treviso and Trento  in a range of altitude between 173  and 1,250 m.a.s.l. . Ae. koreicus was detected in 40.2% of the locations and in 38.0% of the larval habitats checked. Garden centres were the most common locations (66.7%), followed by streets/squares (57.1%), private gardens (46.4%) and cemeteries (21.1%) (Table Ae. koreicus larvae were catch basins (48.5%) and artificial water containers (41.8%). As for Ae. albopictus, ovitraps were attractive for adult females resulting in the higher rate of positivity  among breeding sites  (42%), Cx. hortensis  (2.3%), Aedes geniculatus (Olivier) [Dahliana geniculata] (0.8%), Culiseta longiareolata (Macquart) (0.8%) and Anopheles maculipennis s.l. (Meigen) (0.4%). Aedes albopictus was found in 57 (23.8%) out of 225 breeding sites monitored (including ovitraps), even in places not previously colonized.Six other mosquito species were identified in 152 collections (59.4%) (excluding ovitraps) during the survey: Ae. koreicus, larvae were identified as the only species in 80 cases (71%) and were associated with Cx. pipiens 17 times , Ae. albopictus 13 times , Cx. hortensis six times (5.3%) and Ae. geniculatus in one site. In 21 municipalities (22.3%) both Ae. koreicus and Ae. albopictus were present  whereas Ae. albopictus was more common in places under 200 m.a.s.l. (61.9%) (p < 0.01)  and was found in two sites above 1000 m.a.s.l., where Ae. albopictus was absent.Ae. koreicus activity remained unknown as the survey started after the first identification at the end of May; the last adults were collected on September 13, while ovitraps remained positive until October 13. In 2012 the first larvae were caught on March 29 and ovitraps were positive on May 5. The adults were active until the end of September and living larvae were observed until October 29. The period of activity of Ae. albopictus was July 7-September 8 in 2011 and May 23-September 28 in 2012.In 2011 the beginning of the Ae. koreicus activity (April-October) was 10.9°C and 10.8°C and the mean minimum temperature in the same period was 4.9°C and 6.2°C. The temperatures in South Korea showed the same trend  while the annual rainfall in Valbelluna is higher than in South Korea   and two representative sequences of our samples were submitted to GenBank database .All doubtful samples were identified by PCR and yielded the expected band of 283 bp. The sequences revealed 99-100% identity with the Belgium isolate (GenBank:JF430392) and with the South Korea isolates (GenBank:GU229925-27). The sequence of the first finding of Ae. koreicus is well established in an area of 2600 km2 of north-eastern Italy and confirm the invasive potential of this mosquito  in 1996 [Ae. koreicus. This is likely a consequence of the intensive trade of goods in one of the most developed industrial and commercial area of Italy. However, the intensive mosquito monitoring that followed recent vector-borne outbreaks in north Italy [Ae. albopictus[It’s interesting to note that this area of north-eastern Italy and in particular the Veneto Region has experienced several invasive mosquito introduction and spread in the past 20 years; in particular, ew years , Ae. atr in 1996 , detecteth Italy -53 couldAe. koreicus need urgently to be investigated in the next future, such as the feeding behaviour and the vector competence. The people resident in the areas where Ae. koreicus is the exclusive diurnal biting species complained due to mosquito bites (behaviour confirmed by the authors). Except humans, the host preference spectrum in this area remains unknown.Other aspects of the biology of Ae. koreicus vector competence is available; while experimental transmission of Japanese encephalitis virus (JEV) has been proven [Dirofilaria immitis[Culex and Aedes mosquitoes [Little information on n proven -56, the n proven . There ia immitis, which isquitoes ,60.Ae. koreicus will be demonstrated to be competent for some of these viruses, the risk of transmission will be extended in this new colonized area, especially at high altitudes, i.e. in places previously regarded at a negligible risk of animal and human outbreaks.Some arboviruses, such West Nile and Usutu viruses, are endemic in Veneto region ,52 and oAe. koreicus in northern Italy and reported some biological characteristics of the species, such as period and temperatures of activity and breeding sites preference. These data are important to model the risk of spreading in other part of Italy or Europe and to plan the future control of this invasive species, considering that the eradication of an establish species is unlikely.The findings of this survey defined horizontal and vertical geographical limits of the area colonized by Other research is urgently needed, especially to study the host preference spectrum and the vector competence of the species, to define the risk of infection transmission.This finding, once again, stresses the importance of implementing an integrated entomological surveillance system for early detection of invasive species, which is necessary for eradication or limitation of its further spread.The authors declare that they have no competing interests.FM, SM and GC conceived the study. FM, SM, MDP, ND, NFM, SCi carried out the samplings. SR and SCa carried out the molecular study. GC performed the statistical analysis. MM made the maps. FS, LC and FR supported this study. FM took the pictures and drew the drawings in Additional files Morphological features of Aedes koreicus. Description of the main characteristics of adults and larvae of Aedes koreicus, .Click here for fileAedes koreicus, compared to Aedes albopictus and Aedes japonicus. Table showing the main characteristics of Aedes koreicus , Aedes albopictus and Aedes japonicus .Click here for fileDrawings of the main characteristics considered for the identification of female culicinae mosquito. a - general aspect of a female mosquito; b – dorsal and lateral view of thorax. The arrows point only the characteristics useful for the identification of Aedes koreicus and Aedes albopictus.Click here for fileDrawings of main characteristics considered for the identification of larvae and male of Aedes species. c – hypopygium of Aedes; d – head of culicinae larvae; e – lateral view of the distal part of the larval abdomen of Aedes/Ochlerotatus species. The arrows point only the characteristics useful for the identification of Aedes koreicus and Aedes albopictus.Click here for fileComparison between larval features of Aedes albopictus and Aedes koreicus. Difference between Aedes albopictus (a) and Aedes koreicus (b); Figure S5, antennae and antennal setae 1A; Figure S6, comb scales; Figure S7, pecten.Click here for fileComparison between male features of Aedes albopictus and Aedes koreicus. Difference between Aedes albopictus (a) and Aedes koreicus (b); Figure S8, palps and proboscids; Figure S9, hypopygium.Click here for fileComparison between female features of Aedes albopictus and Aedes koreicus. Difference between Aedes albopictus (a) and Aedes koreicus (b); Figure S10, palps and interorbicular space; Figure S11, ornamentation on scutum; Figure S12, abdomen.Click here for fileComparison between legs of Aedes albopictus and Aedes koreicus. Difference between Aedes albopictus (a) and Aedes koreicus (b); Figure S13, 1-2-3 fore, mid and hind legs respectively.Click here for file


The unique breeding structure of the domestic dog makes canine genetics a useful tool to further the understanding of inherited diseases and gene function. Answers to the questions of when and where the dog was domesticated from the wolf are uncertain, but how the modern diversity of dog breeds was developed is documented. Breed development has resulted in many genetically isolated populations which are segregating for different alleles for disease and morphological and behavioral traits. Many genetic tools are available for dog research allowing investigation into the genetic basis of these phenotypes. Research into causes of diseases in dogs is relevant to humans and other species; comparative genomics is being used to transfer genetic information to them, including some studies on morphological and behavioral phenotypes. Because of the unique breed structure and well-maintained pedigrees, dogs represent a model organism containing a wealth of genetic information. Domestic dogs can be viewed as one of mankind's largest and longest running breeding experiments. The process has resulted in over 400 breeds with considerable morphologic and behavioral diversity compared to the gray wolf ancestor. The origin and time frame of domestication from the gray wolf are hotly debated. Early work using phylogenetic substitution rates in mitochondrial D-loop sequence suggests that dogs might have originated as early as 100,000 years ago ; howeverIdentifying the location of dog domestication has proven difficult, partly because it is confounded by the choice of samples from wild relatives for comparison to dog. Mitochondrial DNA (mtDNA) data has been used to support East Asia as the origin of all modern dog breeds , 4. VergThe domestication of the wolf established populations of native dogs in several places around the world, and these native populations existed for some time allowing some genetic diversity to rebuild after the original domestication bottleneck. The breed structure and relationship between dog breeds can be teased apart using SNP data such as that from vonHoldt et al. . The ancFor a dog to be classified as purebred, it has to be the offspring of purebred parents. Pedigree records are well documented, and dogs of mixed ancestry are excluded from any breed. This unique population structure results in a significant degree of inbreeding and strong population substructure. One factor that influences these processes is the popular sire effect. A popular sire is a male that is highly sought after for breeding purposes, usually from winning dog shows or herding competitions. A popular sire can produce hundreds of offspring, contributing significantly to the gene pool of the next generation . This reBreeding of dogs is easily manipulated and planned. Single animals with a rare disorder can be bred into disease colonies for research. Crosses between breeds can be used to place genes for genetic traits in different genetic backgrounds to allow the study of the influence of modifying genes on phenotype. This can be important when studying diseases with low penetrance or variable expression.Dogs have large haplotype blocks  than an outbred population which means that analysis of a canine phenotype will result in a stronger genetic signal. Mapping canine homologues of complex traits is therefore likely to identify single, high-effect loci as a result of the breeding structure . Dog genA typical mapping experiment in dogs would make use of an association study using SNP arrays on a trait or set of traits that exists in multiple breeds such as coat variation . In the http://www.affymetrix.com/estore/, http://www.illumina.com/). SNP arrays replace the need to laboriously type large numbers of microsatellites for whole-genome analysis. A comprehensive linkage map for all dog chromosomes is now also available that can be used in conjunction with whole-genome mapping [A 1.5x coverage sequence of a poodle  and a 7. mapping . The avaWith the development of next generation sequencing, which allows gigabases of DNA sequence to be generated from a single sample (see  for revihttp://server.vet.cam.ac.uk/index.html). Information on the genetic basis of common complex disorders such as cancers, heart diseases, and diabetes in the dog can be informative for disease gene identification in other species. A benefit of using the dog model for disease studies is the well-documented pedigrees providing information on relatedness, inbreeding coefficients, common ancestors, and thus high-risk family lines. This information can aid in the selection of samples for genetic studies on diseases and traits by allowing the researcher to identify potential carriers of a disorder.The gene complement of most eutherian mammals is very similar, and dogs have similar genetic diseases to those observed in large outbred populations, such as humans, from simple monogenic traits to complex disorders. The medical attention we provide our much loved canine companion has led to an extensive list of known disorders, second only to human and mouse (see : Inherithttp://www.eurolupa.org/). For example, there is a familial medullary thyroid cancer common in the Alaskan Malamute [Cancers of many different types exist in different breeds offering the potential for insight into disease mechanisms and treatment options for cancers in humans and other species and this is a major research focus of several groups, for example, LUPA  is a good example of a trait transferrable between species using comparative genomics. It causes increased muscle mass in several species, including dogs and horses (see [MSTN mutation has been found to increase racing ability in both whippets and racing horses [Loss-of-function mutations in myostatin (ses (see  for revig horses , 44. WhiWBSCR17, which in humans is involved in William-Beuren syndrome, a disease that includes mental retardation, ease with strangers, and a desire to be in groups. Such characteristics would make dogs easier to handle and could have been strongly selected early during the domestication process.One of the most remarkable characteristics of domestic dogs is their ability to pick up and understand human cues and emotions. Dogs show a strong attachment relationship with their caregiver and are more amenable to training than wolves raised in the same environment –47. ThisSLC1A2, which may be responsible for overly aggressive behaviour. These few examples show how canine genetics can be used to identify genes potentially affecting behaviours, which may assist in identifying similar genes affecting behaviour in other species.Individual dog breeds are enriched or fixed for innate behavioural characteristics including pointing, herding, and aggressive behaviour. While these breeds still require training for pointing and herding, they are far more responsive to the training than other breeds, which suggests that these traits have a degree of genetic predisposition. Mapping for these traits has identified genomic regions that appear strongly associated with these behaviours . DiffereDogs represent such a rich potential resource to further the understanding of diseases and genetic traits because of their history of domestication and breed development. Domestication of the dog has resulted in many isolated populations, much like a breeding experiment with gene mapping as the aim. Recent advances in understanding this genetic history are important for mapping genes for various phenotypes and traits. Breeds fixed or highly enriched for certain phenotypes already exist. Identifying the genetics responsible for breed-defining phenotypes is potentially as simple as collating the existing SNP array data and performing the analyses. A confounding issue that could pose a problem for mapping the phenotypes listed above is sample stratification and the large haplotype blocks that exists within breeds. However, canine genetics has significant potential to contribute to the understanding of genetic disorders and functional genomics in other species and will compete with other species as a genetic model organism.


Ceratitis capitata, is one of the major agricultural and economical pests globally. Understanding invasion risk and mitigation of medfly in agricultural landscapes requires knowledge of its population structure and dispersal patterns. Here, estimates of dispersal ability are provided in medfly from South Africa at three spatial scales using molecular approaches. Individuals were genotyped at 11 polymorphic microsatellite loci and a subset of individuals were also sequenced for the mitochondrial cytochrome oxidase subunit I gene. Our results show that South African medfly populations are generally characterized by high levels of genetic diversity and limited population differentiation at all spatial scales. This suggests high levels of gene flow among sampling locations. However, natural dispersal in C. capitata has been shown to rarely exceed 10 km. Therefore, documented levels of high gene flow in the present study, even between distant populations (>1600 km), are likely the result of human-mediated dispersal or at least some form of long-distance jump dispersal. These findings may have broad applicability to other global fruit production areas and have significant implications for ongoing pest management practices, such as the sterile insect technique.The invasive Mediterranean fruit fly (medfly),  Through globalization and increased economic trade, species are frequently transported outside of their natural ranges Ceratitis capitata (Weidemann) (Diptera: Tephritidae), one of the economically costly pest species worldwide C. capitata here and elsewhere. Amongst these are the species’ polyphagous life-history Ceratitis capitata may also have a broader climate niche compared to its congeners A case in hand concerns the Mediterranean fruit fly (medfly), Current management of medfly populations in South Africa predominantly relies on the use of insecticides, including bait application technique (food baits combined with pesticide), bait stations (food bait and pesticide placed in container or trap) and full-cover sprays Accurate information regarding population structure and movement of individuals is crucial in ensuring successful implementation of pest-control strategies. For example, incorrect estimates of the minimum size for area-wide pest management could result in a failed attempt and, given the cost involved, funding may not be available to complete or re-start eradication or control programmes Ceratitis capitata individuals were collected from eight locations in South Africa , 13 locations in the Western Cape  and 13 locations in the Ceres valley   , Table 1Bucket traps  were set up in fruit orchards and geo-referenced using a hand-held GPS. Traps were dry baited with a three-component attractant Biolure 3C (Chempac) consisting of putrecine, ammonium acetate and trimethylamine  gene was targeted for ten randomly selected individuals per sampling location where possible. This resulted in a sequence data set of 125 individuals from the Western Cape  and 74 iNh), haplotype diversity (h) and nucleotide diversity (π) DNA sequences were aligned and edited in GENEIOUS Pro™ 5.0 software . We calculated the number of haplotypes . We tested for isolation by distance (IBD) using a Mantel test Overall  and population pairwise Specimens were genotyped for 12 microsatellite markers obtained from previously published studies Microsatellite loci were tested for departures from Hardy-Weinberg equilibrium (HWE) and linkage disequilibrium using 10 000 permutations GENEPOP; . SignifiNA), expected heterozygosity  and observed heterozygosity  were calculated for each microsatellite locus and for each location , which is a measure of genetic diversity independent of sample size, was calculated in FSTAT v2.9.3.2 FIS) was calculated in GENETIX v4.05.2 with 10 000 permutations to assess deviations from the null hypothesis of no inbreeding (FIS = 0).Levels of genetic diversity were assessed by computing basic statistics for 11 microsatellite loci. The average number of alleles  and overall FST values were calculated in ARLEQUIN v3.5.1.2 using 10 000 permutations K), we ran 10 independent runs for each K value varying between 1 and 13. A burn-in period of 1 000 000 followed by 1 000 000 Markov Chain Monte Carlo (MCMC) permutations was run to allow statistical parameters to reach stability and gave consistent results over the 10 independent runs. To determine the true number of populations (K) in the dataset, the method described by Evanno et al. (2005) F-statistics. The standard error of the FST computation was estimated using 10 000 permutations. The results were visualized by plotting the FST/(1−FST) values against the geographic distance between sampled locations.To assess the degree of population differentiation , we used three complementary approaches: Ceratitis spp.; see e.g. We identified 67 distinct haplotypes for the 125 individuals included at the regional spatial scale. Haplotype diversity was notably high with an average value for the region of 0.980 . OverallΦST value was significant . Pairwise ΦST comparisons between sampling sites were mostly non-significant except for the Tulbagh sampling location which was significantly differentiated from four other sampling localities  . No correlation between genetic and geographic distances were found  indicating the absence of isolation by distance.The haplotype network showed no explicit spatial pattern of genetic variation across the Western Cape . Rather,son) see . A closeCeratitis spp.; see e.g. We identified 50 haplotypes for the 75 individuals included from across South Africa. Haplotype diversity was high with an average value for South Africa of 0.990  with nucΦST = 0.028; p = 0.04). Pairwise ΦST values between sampling sites were small and non-significant. The only significant comparison was between Stellenbosch and Makhado, situated more than 1 600 km apart  with relatively high levels of inbreeding indicative of non-random mating within sampling locations .NAP) except for Ceres 7 (HE), was 0.805 (AR) ranged from 3.991 (Ceres 5) to 4.147 (Ceres 6)  ranged between 0.185 (Ceres 1) and 0.256 (Ceres 12)  .FST value was 0.004 (p = 0.182), indicating no significant population differentiation. All of the pairwise FST values (quantification of genetic structure between populations) for sampling localities in the Ceres valley were not significant after corrections for multiple testing using False Discovery Rates. Both the Mantel test and the global F-statistics did not indicate any pattern of isolation by distance  cannot calculate a ΔK value at K = 1, as it uses the second order rate of change. Examination of the log of the posterior probability of the data [ln P(D)] for each K value revealed the highest ln P(D) value at K = 1, an indication of the lack of population differentiation.The investigation of population differentiation in the Ceres valley using a Bayesian clustering approach in STRUCTURE  showed aNAP) except for Lutzville and Citrusdal (HE) for C. capitata in the Western Cape was high (0.803) (AR) ranged from 3.950 (Lutzville) to 4.210   ranged between 0.138 (Lutzville) and 0.264   . Allelictrusdal) .FST comparisons were not significant after False Discovery Rate corrections (FST = 0.019). The overall FST value of 0.006 (p = 0.002) indicated weak but significant population differentiation possibly due to the few significant pairwise comparisons. Results from STRUCTURE for the Western Cape (F-statistics).Similar to the mtDNA findings, almost all of the pairwise rections . The higern Cape  indicateNAP) (HE) for C. capitata in South Africa was high (0.778) (AR) was 3.973  ranged between 0.138 (Lutzville) and 0.302 (Makhado)  . Genetic (0.778)  and the Makhado) .FST values were used to quantify the genetic structure between sampling locations and some of the comparisons were not significant after FDR corrections (FST = 0.083) was between Makhado and Port Elizabeth a distance of approximately 1300 km). The overall FST value was 0.021 (p = 0.0001), which indicated weak but significant population differentiation possibly due to the significant pairwise comparisons between a few localities. STRUCTURE results  to its entire range across South Africa. The values reported here are notably higher than those reported for other Three possible scenarios may account for these higher levels of genetic diversity in medfly. First, it may reflect the importance of propagule pressure and the effect of multiple introductions on their genetic diversity FIS (Ceres) ranged between 0.185–0.260; FIS (Western Cape) ranged between 0.138–0.260; FIS (South Africa) ranged between 0.138 and 0.302). Microsatellite estimates of FIS in medfly have only been reported once previously FIS in medfly. Although inbreeding seems unlikely given the large effective population sizes we report and that our results suggest that populations in South Africa may be expanding (Fu’s F-statistic) (possibly due to an increase in the area under fruit production), this possibility cannot be excluded as medlfy demonstrates lekking behaviour Despite high genetic diversity and proposed large population sizes, all locations sampled from South Africa showed slight levels of inbreeding  C. capitata populations in endemic Argan forests, predominantly driven by their occurrence at different altitudes. We suggest that the structure of medfly in South Africa may be the result of complex interactions among factors at play at local scales  and broad spatial scales, such as human-mediated or other forms of long distance dispersal. Indeed in South Africa large parts of the country are essentially climatically unsuitable for medfly and lacking in host plants; however, there are active commercial trade routes linking fruit distribution among these areas.In addition to high levels of genetic diversity, we find limited genetic differentiation among sampling locations across all spatial scales in South Africa. Given that the natural dispersal distances of C. capitata. Although limited genetic structure and higher levels of population connectivity may have been expected for the Ceres valley , this was an unexpected finding for our regional and broad scale investigation as sampling sites are geographically more distant, being up to 1600 km apart. Sharp and Chambers (1976) C. capitata can fly a maximum distance of 7–8 km, whereas most individuals only flew between 1–3 km within 2–3 hours. Similarly, Meats and Smallridge (2007) C. capitata of between 0.5–9.5 km through natural dispersal (mainly flight), but that only a very small percentage of individuals are likely to do so . Therefore, given their limited dispersal ability based on direct estimates, the gene flow estimates are perhaps higher than might be expected. However, direct estimates are typically measured under natural or semi-natural conditions at a specific time point and may exclude the movement of individuals under extreme conditions, over longer time scales or multiple generations Owing to its global pest status, many investigations have focused on quantifying the dispersal ability of C. capitata using their natural dispersal ability, although these are interspersed with large areas of unfavourable habitat. Therefore, these long distance dispersal events might occur in parallel or in series with human-mediated dispersal which can take place over even longer distances. The most likely vectors of human-mediated dispersal include fruit consignments, the movement of nursery material  and also the movement of fruit between different locations throughout the country The distribution of suitable host plants (both wild and cultivated) in South Africa is probably able to facilitate the movement of C. capitata around the country in order to eliminate this invasion pathway. Finally, it also suggests that import trade could be contributing to the high gene flow patterns detected (i.e. through propagule pressure). In this respect, it would be worthwhile to assess gene flow into South Africa from other adjacent countries or revisit global estimates of gene flow in medfly to determine if the global pattern previously documented C. capitata from taking refuge and recolonizing agricultural areas under control C. capitata, and in turn, could facilitate improved area-wide pest management programs for sustainable crop production in these and other geographic regions.If these results are assumed to be a reliable indication of movement patterns and population structure in South Africa, one potential implication of this work is that it suggests area-wide pest management should perhaps be undertaken at a broad scale, rather than on a fine scale , as is presently the case Figure S1Analysis of Ceratitis capitata individuals from (A) the Ceres valley, (B) the Western Cape and (C) South Africa using the Bayesian based method implemented in the program STRUCTURE. Each individual is indicated with a vertical line, the different shades of grey represent the individual’s estimated percentage membership to the K clusters. Genetic population structure is shown for K = 2.(PDF)Click here for additional data file.Figure S2Analysis of Ceratitis capitata individuals from (A) the Ceres valley, (B) the Western Cape and (C) South Africa using the Bayesian based method implemented in the program TESS. Each individual is indicated with a vertical line, the different shades of grey represent the individual’s estimated percentage membership to the K clusters. Genetic population structure is shown for K = 2.(PDF)Click here for additional data file.Table S1(PDF)Click here for additional data file.Table S2(PDF)Click here for additional data file.Table S3(PDF)Click here for additional data file.Table S4(PDF)Click here for additional data file.Table S5(PDF)Click here for additional data file.


Current and historical events have both affected the current distribution patterns and intraspecific divergence of plants. While numerous studies have focused on the Qinghai-Tibetan Plateau (QTP), the impacts of such events on the flora of subtropical China remain poorly understood. Subtropical China is famous for its highly complex topography and the limited impact from glaciation during the Pleistocene; this may have resulted in a different genetic legacy for species in this region compared to fully glaciated areas.Primula obconica Hance, a widespread herbaceous montane species in subtropical China. The phylogenetic analysis revealed two major lineages (lineage A and lineage B), representing a west-east split into the Yunnan and Eastern groups, and the Sichuan and Central groups, respectively. The Eastern and Central groups comprised relatively new derived haplotypes. Nested Clade Analysis and Bayesian Skyline Plot analyses both indicated that P. obconica mainly experienced a gradual expansion of populations. In addition, the simulated distribution of P. obconica during the Last Glacial Maximum was slightly larger than its present-day distribution.We used plastid and nuclear DNA sequence data and distribution modeling to analyze the divergence patterns and demographic history of P. obconica. The gradual expansion pattern and a larger potential distribution range in cold periods detected for P. obconica indicate that the population expansion of this species is consistent with the phalanx model. In addition, the current patterns of genetic differentiation have persisted as a result of the extensive environmental heterogeneity that exists in subtropical China.Our results are the first to identify a west-east migration of  The distribution patterns and evolution of plants are profoundly impacted by their life history traits, environmental heterogeneity and historical events. However, the importance of these factors in the genetic divergence within and between species, along with the importance of changes in distribution patterns, remains a contentious issue Previous research on population expansions in China has mainly focused on the Qinhai-Tibetan Plateau (QTP), including the Himalaya-Hengduan Mountains (HHM). These studies indicated that the colonization patterns of most species in this region are consistent with the pioneer model, with populations on the QTP exhibiting relatively low genetic diversity and/or pure genetic haplotypes while those in the HHM exhibit high diversity Subtropical China is famous for its complex topography. There is a remarkable decrease in altitude from west to east, which divided China into three geographic zones: the QTP, with an average elevation of 4000–5000 m a.s.l.; the eastern plain, which lies below 1000 m a.s.l.; and a transitional belt between the QTP and the eastern Plain ranging from 1000–2000 m a.s.l Subtropical China itself is thought to have been one of the most important refugia during the middle Miocene extinction (ca. 15 Mya) As represented above, the population expansion of montane species in subtropical China might have occurred as described by the phalanx model, with species migrating down along mountain slopes and gradually expanding during cold periods but being forced to retreat upwards and form isolated island-like populations during the warm and moist interglacial periods. If this were the case, we would expect to detect a gradual expansion in effective population size, and a simulated species distribution in cold periods that is larger than at present.Primula exhibits the typical Chinese radiation pattern from the southwest to southeast Primula species in China are restricted to the southwest Primula obconica is a unique Primula species that has a relatively wide distribution range from southwest China (Yunnan and Sichuan provinces) to east China (Fujian province) stretching from 99° to 118°E and from 25° to 31°N, which means it is distributed in subtropical China obconica, ssp. begoniiformis (Petitm.) W. W. Smith & Forr.; ssp. parva  W. W. Smith & Forr.; ssp. werringtonensis (Forr.) W. W. Smith & Forr.; ssp. nigroglandulosa (W. W. Smith & Fletcher) C. M. Hu; and ssp. fujianensis C. M. Hu & G. S. He obconica is distributed throughout almost the entire range. The other subspecies are all endemic and sympatric with ssp. obconica with the exception of the most recently described subspecies ssp. fujianensis, which occurs solely in the easternmost Fujian province P. obconica was formally reported as diploid Primulafujianensis, which is isolated both in distribution and reproduction from other subspecies of P. obconicaThe genus P. obconica as a model to test competing hypotheses to explain the migration and evolution of species in subtropical China by examining two polymorphic plastid loci and the complete nuclear ribosomal Internal Transcribed Spacer (ITS) region. Specifically, we aimed to determine: (i) whether the species originated from southwest China (or HHM), then eastward migrated; (ii) whether the phalanx or the pioneer model better describe the species' population expansion and demographic history; and (iii) the impact of environmental heterogeneity on the species' present-day distribution.In the present study, we used Primula obconica is not endangered nor protected in the sampled area, and none of the sampled locations are privately owned or protected by any law.No specific permits were required for the described field studies, since all samples were collected by researchers following current Chinese regulations. P. obconica, representing four subspecies (Primula obconica ssp. parva and ssp. nigroglandulosa could not be sampled in this study because of their extremely limited distribution ranges trnL-trnF and rps16) and a nuclear marker (ITS) were amplified rps16 region (hc-p1: 5′-GGTATGTTGCTGCCATTTTG-3′) due to homopolymer runs. All bidirectional sequencing reactions were carried out by Invitrogen Trading Shanghai Co., Ltd.Twenty-three populations were sampled throughout the distribution range of bspecies , Table 1Sequence quality was checked against the original chromatogram and assembled using SeqMan™ (DNASTAR). For ITS sequencing, the presence of ‘double peaks’ at polymorphic sites for ITS in the chromatogram were check manually. Alleles (haplotypes) were first determined through haplotype subtraction Hd) and nucleotide diversity (π) were calculated using DnaSP 5.0 HS), total diversity (HT), and two other parameters of population differentiation (GST and NST) using PermutCpSSR 2.0 with 1000 permutations http://www.pierroton.inra.fr/genetics/labo/Software/PermutCpSSR/). GST is a parameter estimate based on haplotype frequencies; while NST takes into account the differences between haplotypes. The comparison of GST and NST was conducted by PermutCpSSR based on 1000 random permutations.Sequences for each fragment were aligned by Clustal X 1.18 Analysis of molecular variance (AMOVA) was performed to assess the genetic differentiation among groups and between populations within groups  using the program Arlequin 3.0 P. obconica. The MP analysis was conducted by PAUP* 4.0b10 with a heuristic search using the random addition of sequence method (1000 replicates) with the tree-bisection-reconnection (TBR) branch swapping, MULTREES, and BRANCHES COLLAPSED options selected Primula barbicalyx Wright, which, like P. obconica, belongs to the sect. Obconicolisteri Balf. f., and was used as an outgroup. In addition, a statistical parsimony network was constructed by TCS 1.13 Chlorotypes for the combined plastid fragments were generated by DnaSP 5.0 Dc) and the nested clade distance (Dn). These distances then were used to calculate an interior-tip statistic (I-Tc and I-Tn) within each nested category based on the coalescent theory described by Posada et al. http://darwin.uvigo.es/software/geodis.html).We defined hierarchic clades for Nested Clade Analysis (NCA) using the nested design rule P. obconica, two neutrality tests, Tajima's DFsD indicates an excess of low-frequency alleles that can arise from purifying selection, rapid population expansion, and selective sweeps Fs is expected to have significantly large and negative values under conditions of demographic expansion In order to test whether historical demographic expansion events have ever occurred in SSD) between the observed and the expected mismatch distributions Rag), were used to test the goodness-of-fit of the observation mismatch distribution to the expectation of a population expansion model tt), where μ is the substitution rate for DNA sequences.Mismatch distribution analyses were used to examine demographic changes. The distributions of the frequency of observed and simulated pairwise differences among sequences were plotted by Arlequin 3.0 Primula, a conventional molecular clock for the herb trnL-trnF region  under Juke-cantor model was calculated for trnL-trnF and the combined data, respectively.Despite the uncertainty of the plant molecular clock in general and the lack of reliable fossil record for the genus eN) of P. obconica since the time to the most recent common ancestor (TMRCA). We performed four MCMC runs for 10 million iterations, sampling genealogy and population size parameters every 1000 iterations and discarding the first 10% as burn-in. The linear growth model was selected for this analysis. We used an uncorrelated lognormal model θ  for the BSP.A Bayesian Skyline Plot (BSP), a method that does not rely on a prespecified parametric model of demographic history, was used to estimate past population dynamics over time from a sample of molecular sequences using a Markov chain Monte Carlo (MCMC) algorithm P. obconica, we inferred the distribution range using an Ecological Niche Model. Assuming the species has not changed its climatic preference, we reconstructed the range of P. obconica during the LGM according to its current distribution using a maximum entropy model , and sampling sites in this study were also added to cover the whole distribution range  on the distribution of on range . BioclimtrnL-trnF and rps16 were combined, including 77 site substitutions and 6 indels. The geographical distribution of the chlorotypes was highly structured (see Hd) varied among populations. High Hd values were found in central China (e.g. SZ-2) and southeastern China (e.g. RY-1 and RY-2), while populations from southwest China always exhibited relatively low haplotype diversity  was larger than the average within-population diversity (HS = 0.179±0.0435). High values of GST and NST  indicated that there is significant population differentiation in P. obconica. The species was confirmed to have significant phylogeographic structure as NST was significantly greater than GST . Results from analyses of the molecular variance (AMOVA) among groups and between and within populations are shown in The total genetic diversity across all populations (HT = 0.994±0.0081) was larger than the average within-population diversity (HS = 0.571±0.0558).Individuals fixed for unique chlorotypes were sequenced with ITS primer pairs. ITS sequences from BS-2 and DJY-2 were not included in further analyses due to failure in amplification and/or sequencing. In cloning analyses, a total of 5–10 ITS clones was sequenced for each sample. A total of 220 sequences across the 110 individuals was obtained from the 21 populations we examined. The alignment length was 692 bp with 110 nucleotide substitution sites without indels. Sixty-six ribotypes were retrieved from the ITS matrix. The ribotypes in each population and the corresponding nucleotide parameters are shown in NST values (0.844±0.0380) compared to GST (0.425±0.0558), showing there is phylogeographic structure across the distribution range of P. obconica. AMOVA indicated that most of the variation (85.87%) related to differences between populations, while 15.13% of the variation was due to differences within populations  with strong supporting values  between Lineage A and Lineage B was estimated to be 0.008 when all plastid DNA fragments combined, approximately equal to that of trnL-trnF alone (0.0077). We then extended the assumed substitution rate of trnL-trnF to the combined plastid DNA data. We used the expression τ = 2μt to estimate the expansion ages of the Eastern group. Based on the aligned sequence length (1700 bp) and a estimated generation time of one year, the expansion of the Eastern group might occur at ca. 0.104 Myr (95% CI: 0.0495–0.169 Myr).Net average distance , except that Clade 1-1 contained chlorotypes  from the northern area of Guangdong (Nanling Mountain). According to the NCA analysis, Clade 1-1 evolved as a result of restricted gene flow due to isolation by distance, while most of the remaining clades probably resulted from previous gradual range expansions followed by fragmentation or contiguous range expansion (The most parsimonious network constructed by TCS contained two ambiguous connections (loops), which were resolved using the procedure described by Crandall et al. xpansion .Primula obconica is shown in P. obconica occur in southwest and central China. In addition, the inferred distribution during the LGM is slightly larger than the current distribution; for example, the areas of the Sichuan basin and the eastern plain of China that were suitable for the species were both slightly larger during the LGM (The inferred current and past (LGM) distribution of  the LGM .Primula obconica exhibited high genetic differentiation between populations, but extremely low genetic diversity within populations  . During P. obconica might result in the limited dispersal distance that allowed constant subsequent dispersal in a manner of the phalanx model Given the highly complex topography in Subtropical China P. obconica was further validated by modeling the distribution of the species, as it had a slightly larger distribution range in cold periods (i.e. during the LGM) than in warm periods (the present-day distribution)  . Range eP. obconica, different environments will have affected the historical demography of the species. For example, we found that the Eastern group has been expanding rapidly on the eastern plain of China approximately since the last 0.104 Myr  is consistent with the division between the HHM and the Yungui Plateau  occurred in the transitional belt between the Yungui Plateau and the eastern plain . In partP. obconica (the Yunnan and Sichuan lineages) are located in the HHM, which split geographically with the 29°N latitudinal line. Similar genetic patterns were also found in other alpine plants in this area, such as Primula secundifloraLigularia tongolensisAnother interesting division occurred in the HHM. Recent surveys of flora in this region have shown a major change in species composition across the 29°N latitudinal line in the HHM, dividing it into southern and northern sub-regions P. obconica, each comprising two subclades. We found that the species originated from Southwest China and then migrated to eastern and central China, since the relatively older lineages occurred in Southwest China. We also detected that P. obconica populations experienced a gradual expansion and had relatively larger distribution range during LGM than at present, which firstly provided the evidence to support the hypothesis of the phalanx model as a main expansion manner for montane species in subtropical China. Finally, we found that lineage divergence was highly correlated with many geographical divisions, highlighting the importance of environmental heterogeneity in maintaining the present-day lineage differentiation in subtropical China.In this study, we identified two main lineages in Figure S1Primula obconica.The nested cladogram of chlorotypes of (DOC)Click here for additional data file.Table S1GenBank accession numbers identified in this study.(DOC)Click here for additional data file.Table S2Primula obconica examined in this study.The distribution information of (DOC)Click here for additional data file.Table S3Nested contingency analysis of geographical structure based on 1000 permutation and chain of inference based on GeoDis inference key.(DOC)Click here for additional data file.


Gammarus fossarum is a key invertebrate in freshwater streams and contains several cryptic species. We examined the population genetic structure, genetic diversity and demographic history of two of them (type A and type B) using microsatellite markers and asked whether they show significant differences. We present results of population genetic analyses based on a total of 37 populations from the headwaters of two major European drainages, Rhine and Rhone. We found that, in both species, genetic diversity was geographically structured among and within drainages. For type A in the Rhine and type B in the Rhone, we detected significant patterns of isolation by distance. The increase of genetic differentiation with geographical distance, however, was much higher in type A than in type B. This result indicates substantial interspecific differences in population history and/or the extent of current gene flow between populations. In the Rhine, type B does not show evidence of isolation by distance, and population differentiation is relatively low across hundreds of kilometres. The majority of these populations also show signatures of recent bottlenecks. These patterns are consistent with a recent expansion of type B into the Rhine drainage. In summary, our results suggest considerable and previously unrecognized interspecific differences in the genetic structure of these cryptic keystone species.Cryptic species, i.e. species that are morphologically hard to distinguish, have been detected repeatedly in various taxa and ecosystems. In order to evaluate the importance of this finding, we have to know in how far cryptic species differ in various aspects of their biology. The amphipod  A comprehensive understanding of biodiversity requires knowledge about the species forming an ecosystem as well as the genetic diversity within these species. Identifying species may not be straightforward, e.g. when they are morphologically hardly distinguishable “cryptic species” Multiple cryptic species complexes have been identified in freshwater vertebrates e.g.  as well 2Amphipoda is an order of mostly aquatic crustaceans in which cryptic species have been found many times. Several amphipods play a central role in freshwater ecosystems as key shredders of decomposing material, important prey items for predators . However, G. fossarum may be threatened by extinction locally, for example due to pollution G. fossarum represents at least three cryptic species G. fossarum, but often focused on limited geographical regions containing only one of the cryptic species and/or were constrained by the limited availability of highly variable and selectively neutral markers Several population genetic studies have been conducted on G. fossarum from 36 sites (“populations”) within three major European drainages , it seems likely that these patterns reflect contamination during DNA extraction. Gammarids may be cannibalistic, they carry their eggs and young in a brood pouch, and we typically stored multiple individuals in the same tube, which could all have led to the occasional co-extraction of DNA from non-target individuals. We excluded these individuals, as well as those with unexpected peak size patterns  from the analysis .We amplified nine polymorphic, unlinked microsatellite loci  specifically designed for both cryptic species in 14 to 35 individuals per population following the protocol of Westram ST values calculated with and without these loci, respectively, were very similar (data not shown), indicating no major bias caused by the inclusion/exclusion of these loci. One locus (gf28) could not be scored in population G3A as peaks were irregular in size and shape. This population was excluded from analyses requiring data for all loci.In type B, locus gf27 was monomorphic, and locus gf21 showed evidence of null alleles (see below) and contained alleles which could not be reliably scored. We therefore excluded gf21 and gf27 from analyses of type B. These loci were also excluded from the type A data set when a direct comparison of results between species was desirable. However, FIS values (6660 randomizations) significantly deviating from Hardy-Weinberg equilibrium.Using the program Fstat (version 2.9.3.2), we tested all loci and sampling sites for FSTST is not independent of marker variability, we additionally calculated the newer measure DestGenetic distances between pairs of sampling sites were calculated as pairwise FAs a measure of genetic diversity, we calculated allelic richness per population based on 13 diploid individuals (the lowest sample size available) using rarefaction and averaged over loci in the program hp-rare We first asked whether geographically close sampling sites also cluster genetically. Because genetic divergence within species is large (see Results section), methods based on Hardy-Weinberg equilibrium, e.g. Structure To assess the relevance of the clusters identified in the trees, we used analysis of molecular variance AMOVA; , which pST/(1-FST) We tested for isolation by distance (IBD) within each species and drainage (Rhine/Rhone) using the program IBDWS The slope of the IBD relationship indicates how strongly genetic divergence increases with geographical distance. To test for interspecific differences in this respect, we analysed whether the 95% confidence intervals of the slopes overlapped between the two species.We identified highly divergent clusters in the phylogenetic trees for both species (see Results section). We hypothesized that gene flow might be reduced between these genetic clusters, on top of the effect of geographical distance alone. To test for that, we performed partial Mantel tests in Fstat, including an indicator matrix specifying whether two populations were in the same (0) or different genetic clusters (1). These tests were only performed for type A and B in the Rhine.Recolonization from glacial refugia is thought to have proceeded from east to west in type A and from west to east in type B a priori. We therefore ran analyses with all possible mutation models  with 1000 replications each. We applied the Wilcoxon test (one-sided) implemented in BOTTLENECK as this test is powerful if the number of loci is small, and appropriate for low sample sizes Recent changes in effective population size leave signatures in the allele frequency distribution in the population. Specifically, bottlenecks cause a loss of rare alleles, leading to a heterozygosity excess compared to the expectation at mutation-drift equilibrium, while population expansions cause heterozygosity deficiency No specific permits were required for the described field studies. In Switzerland, France and Germany, work with Gammarus does not require permission and waterbodies are not private property if nothing else is indicated. Samples were not taken from streams where private property was indicated or from nature reserves. The field studies did not involve endangered or protected species.IS values in eight out of 20 type B populations , indicating the presence of null alleles. Otherwise, no locus showed consistently elevated FIS values across populations. For the French type B population SOF, we detected significantly elevated FIS values for six loci, so results for this population should be interpreted with caution. Other populations did not show deviations from Hardy-Weinberg equilibrium across multiple loci, suggesting the absence of within-population genetic substructure. .Locus gf21 showed elevated FST was higher in type A  than type B . This difference remained when we used the alternative measure Dest , demonstrating that the FST differences are not caused by interspecific differences in marker variability . Most pairwise FST values were significantly different from 0 . In type A, all non-significant comparisons were between geographically close populations within the Thur subdrainage of the Rhine. In type B, only comparisons within the same genetic cluster  and included two major drainages, overall FThe consensus tree for type A contained two major clusters with bootstrap support of 75% (A1) and 67% (A2) . The cluIn type B, we also detected two main geographically sorted clusters . All RhiThe AMOVA results showed that in both type A and type B the identified clusters explained a substantial part of genetic variation . In type A the variation observed between populations within clusters (16.7%) was larger than in type B .For the type A populations from the Rhine, we found evidence of IBD using Mantel tests ; Table 1The type B populations from the Rhone drainage also showed significant evidence of IBD ; Table 1We compared the slopes of the significant IBD relationships between type A (Rhine) and type B (Rhone). Independent of the distance measure used, the non-overlapping 95% confidence intervals indicated that the slopes were significantly different between the two species . For watA partial Mantel test showed that genetic clusters in type A explain a significant proportion of the genetic differentiation between populations, on top of the effect of geographical distance alone . For typ1,34 = 18.1, P<0.001).While the mean allelic richness did not differ between species , the range was larger in type A than in type B  than in the central subdrainage (Limmat)  under the TPM (SMM). Other type B populations from cluster B1  and from the Rhone did not show a clear pattern of heterozygosity excess or deficiency.We show that two morphologically cryptic species show similar population genetic patterns reflecting stream topology and postglacial colonization history. However, the two species differ substantially with regard to the extent of genetic differentiation between populations: While type B is characterized by moderate genetic differentiation across hundreds of kilometres, even geographically close populations are markedly distinct in type A, suggesting differences in the time since colonization and/or the extent of intraspecific gene flow. Our results demonstrate that genetic data may reveal differences hidden by morphological stasis and, in line with other studies, support the importance of considering cryptic species in basic research and conservation.In both species, the population genetic structure is shaped by drainage boundaries. In type A, we find that populations from the same Rhine subdrainages generally cluster together in the tree , which is geographically close, but distant along waterways. Similarly, the type A population from the Danube drainage (SP) clusters with Rhine populations (cluster A1.1), and the three Lake Geneva region populations cluster with most of the Rhine populations  is often remarkably high, with some FST values reaching the level of interspecific comparisons in other taxa. Similarly, earlier allozyme studies found large genetic differences between geographically close G. fossarum type A populations et al.G. fossarum is typically restricted to the upper reaches of streams, leading to a very patchy distribution. This may also explain why the degree of genetic structure was found to be higher in G. fossarum type A than in other Gammarus species with a more continuous distribution in the lower reaches of streams Genetic differentiation between G. fossarum dispersal, and may include additional obstacles like waterfalls and anthropogenic barriers.The pronounced restrictions to dispersal might reduce gene flow between distant sites to a minimum. However, our partial Mantel tests indicate that the distinct clusters detected in type A are genetically even more differentiated than expected based on geographical distance alone. This may be explained by the fact that geographically distant streams are usually connected by large rivers, which present a barrier to et al., unpublished data). Secondary contact between two distinct type B lineages, one from the north (B2) and one from the southwest/Rhone drainage (B1) is therefore plausible. Based on the high degree of differentiation and the fact that there is no evidence for intermediate populations, reproductive isolation between these clusters is an interesting possibility that deserves further investigation.In type B, we also find evidence for two strongly isolated groups within a cryptic species (clusters B1 and B2). B2 might represent a lineage of type B with a different geographical origin (e.g. from more northern parts of the Rhine drainage). Interestingly, the 16S mtDNA haplotypes of individuals from population VI (cluster B2) belong to a clade widespread in the north (Germany), while all other mtDNA haplotypes we obtained from Switzerland (from B1 populations) had not been observed in Germany  and type B (Rhone) show isolation by distance, the slope of the relationship is markedly steeper in type A than type B ; Table 1Gammarus populations and, consequently, a higher degree of population differentiation. However, in the Rhine drainage, where both species coexist, type B also shows much lower levels of differentiation between populations. Lower FST values in type B than type A were also found in a small-scale study within the Sense subdrainage of the Rhine, where the two species occur in close spatial proximity . These findings indicate that the differences in differentiation levels are species differences instead of geographical effects.Systematic differences between the east and the west of our study area could theoretically contribute to the observed pattern, as species and geographical area are not completely independent in this comparison. For example, the east could be characterized by more dispersal barriers, leading to more isolated Such differences could reflect interspecific differences in the number of migrants per generation, i.e. in migration rate and/or effective population size STs and steeper IBD slope in type A would be consistent with lower Ne and/or m compared to type B. Accordingly, other studies have shown that closely related species may differ in the extent of gene flow between populations, e.g. due to differences in dispersal ability To explain the observed interspecific differences in population structure purely as differences in the time since colonisation, we would have to postulate that colonization by type A was more recent than by type B, and involved strong founder events see . ConsequType B in the Rhine does not show evidence of IBD, but signatures of recent bottlenecks are detectable in a large number of populations. One possible explanation for this is a relatively recent colonization of this area by type B. Bottlenecks in the early stages of colonization followed by a rapid spread across the drainage might explain why IBD cannot be observed.As earlier studies have shown, amphipods may be characterized by strongly isolated populations, but on the other hand some species may rapidly spread across large geographical regions. We show that markedly different patterns of population genetic structure can even be observed between relatively closely related cryptic species.The observed interspecific differences have important implications. First, the lower degree of population differentiation in type B might reflect a higher degree of connectivity between populations, which might reduce extinction risk in a metapopulation Cryptic species are most likely present within many endangered taxa and keystone taxa of ecosystems. Two findings of this study, the presence of isolated clusters within both cryptic species and the interspecific differences in the degree of population differentiation, call for detailed investigations of cryptic species. This study underlines the importance of using molecular markers not only to identify them, but also to characterize their population genetic structure to detect other “cryptic” patterns which may be of ecological and evolutionary importance.Figure S1Isolation-by-distance plot for Gammarus fossarum type A. Each dot (population pair) is coloured based on the genetic cluster the two compared populations belong to (see (TIFF)Click here for additional data file.Table S1Gammarus fossarum sampling sites, allelic richness and results of bottleneck tests.(DOC)Click here for additional data file.Table S2ST  and Dest  values for FGammarus fossarum type A population pairs.(DOC)Click here for additional data file.Table S3ST  and Dest  values for FGammarus fossarum type B population pairs.(DOC)Click here for additional data file.


Phenotypic anomalies are common in wild populations and multiple genetic, biotic and abiotic factors might contribute to their formation. Turtles are excellent models for the study of developmental instability because anomalies are easily detected in the form of malformations, additions, or reductions in the number of scutes or scales.Emys orbicularis. The proportion of anomalous individuals varied from 3% to 69% in local populations, with increasing frequency of anomalies in northern regions. We found no significant effect of climatic and soil moisture, or climatic temperature on the occurrence of anomalies. However, lower genetic diversity and inbreeding were good predictors of the prevalence of scute anomalies among populations. Both decreasing genetic diversity and increasing proportion of anomalous individuals in northern parts of the Iberian distribution may be linked to recolonization events from the Southern Pleistocene refugium.In this study, we integrated field observations, manipulative experiments, and climatic and genetic approaches to investigate the origin of carapace scute anomalies across Iberian populations of the European pond turtle, Overall, our results suggest that developmental instability in turtle carapace formation might be caused, at least in part, by genetic factors, although the influence of environmental factors affecting the developmental stability of turtle carapace cannot be ruled out. Further studies of the effects of environmental factors, pollutants and heritability of anomalies would be useful to better understand the complex origin of anomalies in natural populations. The occurrence of anomalies, malformations, or asymmetries in wild animals may serve as an indicator of developmental instability, a variable negatively correlated with fitness Most reptiles, and particularly turtles, are excellent models for the study of developmental instability because anomalies are easily detected in the form of malformations, additions, or reductions in the number of scutes or scales . Three nChrysemys picta and Chelydra serpentina, Chelydra serpentina chemicals including organochlorine pesticides, polychlorinated biphenyls and mercury were identified as main causes of developmental abnormalities in both embryos and hatchlings Most studies reporting the occurrence of carapace anomalies have focused on incubation conditions (temperature and moisture). High number of scute anomalies has been detected in previous experimental studies when eggs were exposed to partial drying during critical developmental stages  in determining turtle shell anomalies is largely unexplored.Emys orbicularis, Linnaeus 1758) is a freshwater species from the family Emydidae. It has a widespread distribution ranging from East and Central Europe, to the Mediterranean countries and the North of Africa E. o. fritzjuergenobsti; The European pond turtle (E. o. fritzjuergenobsti. We focused on the effects of (i) water potential during egg incubation period; (ii) latitude and climatic variables (temperature and precipitation); and (iii) genetic diversity, inbreeding and bottleneck events. By integrating experimental, climatic, and genetic data across Iberian populations, we infer the likely causes of shell anomaly proportions among populations and discuss other potential factors that may affect the developmental stability of turtle carapace.In this study we investigated the relative roles of environmental stressors and genetic diversity in shaping this geographic trend of carapace scute anomalies in Iberian populations of E. orbicularisµl of blood was obtained from the occipital venous sinus of each animal. Ribadavia is a key population to understand the causes of the origin of anomalies because it is the northernmost sampled Iberian population and the second highest anomalous population . For both tests, significance was assessed after sequential Bonferroni correction for multiple tests E. o. fritzjuergenobsti was assessed by estimating observed heterozygosity (HO) over all loci using the software GENETIXhp-rarer) within-populations, GENALEX v.6 We assessed Hardy-Weinberg equilibrium (HWE) by applying a Markov chain exact test BOTTLENECKM  to detect reductions in effective population size for a sample of microsatellite loci. This method considers that quantitative diversity  is reduced more quickly than spatial diversity  in bottlenecked populations. Three parameters are needed for the M-ratio test: effective population size (θ), percentage of mutations greater than one step (Δg), and average size of a non one-step mutation (r). Given the absence of reliable estimates of these parameters, we performed a sensitivity analysis of θ and Δg using a range of more and less conservative values found in the literature , which belong to the same genetic deme To further investigate the heritability of anomalies we split the dataset into two subsets  and estimated, for each subset and population, mean pairwise relatedness, To survey for a possible genetic basis for anomaly occurrence, we tested for statistical associations between microsatellite allele frequencies and anomalous phenotypic individuals  using the program STRAT v.1.0 E. orbicularis. We obtained these data as environmental layers at 30 s (∼1 km) resolution from WorldClim 1.4 The individuals surveyed in this study were from juveniles up to old adults (from one up to 40–60 years probably) and thus we used available temperature and moisture information for the last 50 years in all studied populations. Current information on soil moisture and temperature would not reflect the environmental conditions experienced during embryos' development in the individuals surveyed. On the contrary, and based on current knowledge of potential environmental factors influencing the occurrence of scute anomalies in reptiles, we used climatic layers for maximum temperature, minimum temperature, and precipitation from June to August (when embryonic development occurs in Iberian populations) Previous structure analyses indicated that each population in our dataset represents an independent genetic deme r = −0.986, P<0.0001). When it happens, the variables are collinear and cannot be considered independent. Principal Component Analysis is a widely used method for reducing multicollinearity when running GLMs. In this method, the original variables are transformed into a new set of orthogonal or uncorrelated variables called PCs. This transformation ranks the new orthogonal variables in order of their importance and then involves eliminating PCs with low cumulative explanatory power to effect a reduction in variance. After eliminating the least important PCs, we performed GLMs of the response variable against the first and most important PC. In this study, the PC1 of the four mentioned environmental factors had a cumulative percentage of 77.98%.We ran Generalized Linear Models - GLMs  including one genetic index in turn and the four environmental variables to estimate the relative strength of each factor on mean anomalies. Subsequently, we avoided multicollinearity among cross-correlated environmental variables by running analyses of Principal Components (PC) including correlated environmental variables. When two or more variables in GLMs are highly correlated, they both convey essentially the same information. All environmental variables in our study are cross-correlated  and the PC1 of environmental factors as explanatory variables, allowing the interaction between them. We used sample size as weight estimation in all models to correct for slightly different sampling effort among populations. Analyses were run using JMP 8.0 (SAS 2009).We ran GLMs to estimate the relative importance of genetic indices and environmental factors explaining mean anomalies in icularis . In eachE. orbicularis from four northwestern Iberian populations  during 2006–2007  with binomial distribution and logit link. The effect of treatment on incubation duration and hatchling weight was further tested with an ANOVA, including year as a random factor and population as a fixed factor. Statistical analyses were performed with Genstat 12.Once hatched, we calculated the proportion of successfully hatched eggs for every clutch (number of hatched eggs/number eggs of the clutch), and every treatment (number of hatched eggs/number of eggs in the treatment). We checked each hatchling for the presence of carapace anomalies, including a few embryos that died but whose carapace scutes were already formed. To test the effect of r) increasing from Doñana (0.11) to Ribadavia (0.52). The Ribadavia population has the lowest genetic diversity and highest relatedness, with individuals in this population showing relatedness equivalent to that of full sibs.One out of 63 χ-square tests showed significant deviation from Hardy-Weinberg equilibrium at the 95% confidence interval after sequential Bonferroni correction (D16 in Ribadavia population). As in the previous genetic study of Iberian populations, we found linkage disequilibrium  between loci D88 and D114  and loci D88 and D51 . We found a latitudinal genetic trend across Iberian populations, with genetic diversity , central (Madrid), and southern population (Doñana),  . Interesp>0.1). However, the chance of identifying loci associated with phenotypic traits increases with the number of unlinked markers surveyed, and our attempt for association mapping included only a few available microsatellites.None of the seven microsatellites analyzed in this study showed significant association with individual phenotypes across the seven Iberian populations , minimum temperature , precipitation , and maximum temperature ; although the last two factors need further investigation given the low p-values. In our GLMs, each including one genetic index and the four environmental variables, genetic indices were the only significant factors. Trends found for genetic indices influencing mean anomalies hold after excluding cross correlation among environmental factors using Principal Components in GLMs [Appendix 1: Ho , A-R , r ; environmental factors showing a non-significant effect in all models.Genetic measures were strong predictors of the mean proportion of anomalies across the Iberian peninsula: observed heterozygosity, Ho  from the residual deviance. The model is significant , but the only significant factor was population, due to the difference between Ribadavia and Arnoia populations . Thus, different incubation conditions did not affect hatchling anomalies.Overall, 59.0±8.1% of hatchlings (N = 28 clutches) were symmetric under humid conditions and 65.1±6.9% under dry conditions , year  and population . The average hatching success was 58.7±6.4% under humid conditions and 72.7±5.4% under dry conditions .The duration of embryonic development, from egg-laying to hatching, ranged from 61 to 69 days. Eggs incubated in the humid treatment hatched three days earlier  and treatment , but no effect of year  on weight. Carapace length was affected by year  and population  but not by treatment .Our treatment had a significant effect on hatchling weight . HatchliE. o. fritzjuergenobsti . In contrast, our study shows a significant relationship between population genetic diversity and scute anomalies  and wetter and colder climates in the north  and loamy soils are common in the north (e.g. Porriño and Ribadavia). However, Altentejo population (SW Iberia) is the third highest anomalous population measured in this study  and uses sandy soils to incubate eggs, suggesting that the influence of soil type on anomaly development should be investigated further.In addition, the presence of populations with high proportion of anomalous individuals at higher latitudes, where temperature is lower and precipitation higher , suggestThamnophis elegans atratus. We did not test temperature in our manipulative experiment because of the limited number of clutches we had to work with, although both temperature (minimum and maximum) and precipitation for each locality were considered in the bioclimatic survey.Both temperature and moisture may be correlated, and increased anomalies at higher temperatures have been observed experimentally in other species Our experimental treatments affected the duration of incubation (three days longer on average in wet treatment) and hatchling weight (heavier in the dry treatment), but not hatchling success, indicating that incubation conditions do affect hatchling phenotype.Emys orbicularis, and other chelonian taxa. Additional studies on experimental incubation of clutches from southern and eastern Iberian populations, controlling for both temperature and moisture, would be useful to disentangle the potential effects of genetics and environmental factors on scute anomalies in natural populations.In summary, we do not discard the influence of moisture and temperature on embryos development, but this need to be further explored in future experiments under more extreme treatments in In this study, we independently examined the potential causative factors  of scute anomalies in the turtle carapace to avoid confounding the influence of these factors. Environmental conditions may greatly affect turtle hatchling phenotypes E. orbicularis we sampled. Other environment stressors have also been suggested to explain fluctuating asymmetry in vertebrates  loss of genetic diversity, ii) degree of protein heterozygosity, iii) co-adapted gene complexes disrupted by hybridization, iv) directional selection, and v) mutant genes Mauremys leprosa, which is sympatric with E. orbicularisMauremys compared to Emys individuals across five sympatric Iberian localities The genetic contribution to carapace anomaly formation is likely not equal across species. Comparative studies on turtle anomalies must be done on species sharing similar ecological niches and evolutionary histories to address the genetic influence among turtle species. The Iberian peninsula offers an ideal scenario, because it also harbors a second freshwater turtle, the Mediterranean pond turtle, Emys orbicularis should focus on the role of chemicals and pollution and studies of the heritability of scute anomalies to further elucidate the complexity of anomalies formation in natural populations.The challenge of identifying genes that underlie phenotypic variation in natural populations is still one of the major objectives in evolutionary biology, and very few studies have demonstrated the link between genetics and phenotypic traits Table S1Generalized linear Models evaluating, in turn, the relative strength of each genetic index and environmental factors on mean anomalies. Here, cross-correlated environmental factors  were reduced into one variable using Principal Components.(DOC)Click here for additional data file.