Neophocaena asiaeorientalis asiaeorientalis) is becoming increasingly small and fragmented, and will be at high risk of extinction in the near future. Genetic conservation of this population will be an important component of the long-term conservation effort. We used a 597 base pair mitochondrial DNA (mtDNA) control region and 11 microsatellite loci to analyze the genetic diversity and population structure of the YFP. The analysis of both mtDNA and microsatellite loci suggested that the genetic diversity of the YFP will possibly decrease in the future if the population keeps declining at a rapid rate, even though these two types of markers revealed different levels of genetic diversity. In addition, mtDNA revealed strong genetic differentiation between one local population, Xingchang–Shishou (XCSS), and the other five downstream local populations; furthermore, microsatellite DNA unveiled fine but significant genetic differentiation between three of the local populations  and Tongling (TL)) and the other local populations. With an increasing number of distribution gaps appearing in the Yangtze main steam, the genetic differentiation of local populations will likely intensify in the future. The YFP is becoming a genetically fragmented population. Therefore, we recommend attention should be paid to the genetic conservation of the YFP.Ecological surveys have indicated that the population of the critically endangered Yangtze finless porpoise (YFP,  Neophocaena asiaeorientalis asiaeorientalis) which was reclassified as a subspecies of the narrow-ridged finless porpoise  in 2009  values were very large at K = 3, which indicated that K = 3 was not the optimal clustering  values were slightly smaller, they were very stable  and observed heterozygosity (Ho)) that would be retained after a 150-year period, with a stable population size characterized by an initial genetic diversity equal to the level we observed in the empirical data, showed that the allelic diversity declined considerably faster than Ho  compared with the finless porpoise population in the Yellow Sea  indicated that the current nuclear genetic diversity of the YFP was moderate compared with the marine finless porpoise populations  and som= 0.011, ; the sho= 0.018, ; and the 0.0106, ), which  0.0106, ,17. Furt0–0.795, ) and som7–0.705, ; the bot4–0.784, ). The la4–0.784, . Thus, tFst values  and nucleotide diversity (π) of the XCSS were zero. Compared to other local populations, the microsatellite diversity of the XCSS was also relatively low based on number of alleles detected per locus (N) and the allelic richness (AR)  . On the t values . No signt values .Fst values from the microsatellite DNA analysis  across the distribution range, including three from Shanghai (SH), 17 from Tongling (TL), five from Anqing (AQ), 91 from the Poyang Lake (PY), 15 from Ezhou–Huangshi (EZHS), three from Wuhan (WH), 14 from Honghu–Paizhou (HHPZ), 20 from the Dongting Lake (DT), and 18 from Xingchang–Shishou (XCSS) . Blood sTotal genomic DNA was isolated from blood samples using the Whole Genome DNA Extraction Kit  according to the manufacturer’s instructions. For tissue samples, total genomic DNA was extracted using standard proteinase K digestion and phenol/chloroform extraction protocols .A mtDNA control region segment of 597 base pair was amplified with the forward (5'-GAA TTC CCC GGT CTT GTA AAC C-3') and the reverse primers (5'-GGT TTG GGC CTC TTT GAG AT-3') designed in the present study by Primer Premier 5.0 . The PCR amplification was carried out in a 25 μL reaction containing 10–100 ng of genomic DNA, 2.5 μL of 10× buffer, 0.6 μM of each primer, 0.25 mM dNTPs and 1 U of Taq DNA polymerase . The amplifications were carried out as follows: 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 45 s, annealing at 60 °C for 45 s, and extension at 72 °C for 90 s, and a final extension step at 72 °C for 7 min. The PCR products were purified and then sequenced on an ABI3130 DNA sequencer .N. p. asiaeorientalis [N. phocaenoides [Phocoena phocoena [Eleven polymorphic and steadily amplified loci were used in this study: YFSSR1, YFSSR5, YFSSR40, YFSSR42, and YFSSR59 from ientalis ,31; NP39aenoides ,33; and phocoena . Each fophocoena  was usedh) and nucleotide diversity (π) [Ho and He) and the number of alleles. FSTAT version 2.9.3.2 [AR) and inbreeding coefficient (Fis). Hardy-Weinberg equilibrium was assessed across all microsatellite loci for each local population via an exact probability test implemented in GENEPOP version 4.0. The significance values were adjusted for multiple comparisons using the Bonferroni correction. To detect whether the population suffered a rapid reduction in allele numbers, BOTTLENECK version 1.2.02 [et al. [i.e., lack of low-frequency alleles) for each local population [For mitochondrial DNA, all mtDNA control region sequences were analyzed with ClustalX ,36. The sity (π)  of the esity (π) . For micsity (π)  was used 2.9.3.2  was usedn 1.2.02  was usedn 1.2.02 . Under t [et al. ). A one-pulation .Fst was calculated using ARLEQUIN version 3.0 based on both the mtDNA and microsatellite DNA, and tested for significance with 10,000 permutations [P(D), the probability of the data. The most likely number of subpopulations was taken when the Ln P(D) reached a plateaus. We chose an admixture model because it is more feasible to consider complex problems of real populations and hybrid zones in a natural manner [K from 1–10. Five independent runs were performed for each K. An ad hoc statistic ΔK was also used to help to find the best K [To estimate the degree of genetic differentiation between the local populations, the utations ,46. Nextutations . STRUCTUl manner . STRUCTUe best K .Fst/(1 − Fst) in order to get a linear relationship. Mantel test was performed using ARLEQUIN version 3.0 and 10,000 iterations were used to determine the statistical significance of the results [Mantel test  was also results .Ne of at least 500, which was recommended by Franklin and Frankham [Ne values made temporally stable over a period of 150 years, with other simulation parameters held constant . In addition, special conservation consideration should be given to the porpoise populations living in the Poyang and Dongting Lakes. Our analysis indicated that the porpoises in the two lakes contained 50% of the mtDNA diversity and 85% of the microsatellite allelic diversity. Carrying out effective protection of these two local populations will yield significant effects on the genetic diversity conservation of the entire population.Genetic diversity provides the raw material for the adaptation to environmental change ,54. LossSecond, effective conservation measures, such as habitat restoration and translocation, should be carried out to facilitate more widespread gene flow. This approach could not only reduce the loss of genetic diversity but also reduce the extinction risk of the small local populations because significant genetic differentiations have been detected between some local populations , and additional genetic differentiation may occur in the Yangtze main stream, where distribution gaps have appeared. Furthermore, the Poyang and Dongting Lakes are undoubtedly a crucial habitat that connects the different local populations in the Yangtze main stream, thus restricting human activities in the mouth area of these two lakes to restore the lake-river migration of the YFP will greatly benefit gene flow across the entire distribution range.Lipotes vexillifer) [Human activities have caused extinction to the Yangtze River dolphin (illifer) ,58. A si


Batillaria attramentaria, to these changes, using the snail as a model species in the northwest Pacific. We applied phylogeographic and divergence population genetic approaches to mitochondrial COI sequences from B. attramentaria. To cover much of its distributional range, 197 individuals collected throughout Korea and 507 publically available sequences (mostly from Japan) were used. Finally, a Bayesian skyline plot (BSP) method was applied to reconstruct the demographic history of this species. We found four differentiated geographic groups around Korea, confirming the presence of two distinct, geographically subdivided haplogroups on the Japanese coastlines along the bifurcated routes of the warm Tsushima and Kuroshio Currents. These two haplogroups were estimated to have begun to split approximately 400,000 years ago. Population divergence analysis supported the hypothesis that the Yellow Sea was populated by a northward range expansion of a small fraction of founders that split from a southern ancestral population since the last glacial maximum , when the southern area became re-submerged. BSP analyses on six geographically and genetically defined groups in Korea and Japan consistently demonstrated that each group has exponentially increased approximately since the LGM. This study resolved the phylogeography of B. attramentaria as a series of events connected over space and time; while paleoceanographic conditions determining the connectivity of neighboring seas in East Asia are responsible for the vicariance of this species, the postglacial sea-level rise and warming temperatures have played a crucial role in rapid range shifts and broad demographic expansions of its populations.To address the impacts of past climate changes, particularly since the last glacial period, on the history of the distribution and demography of marine species, we investigated the evolutionary and demographic responses of the intertidal batillariid gastropod,  From a paleoceanographic perspective, the seas surrounding the Korean peninsula, including the Yellow Sea, the East Sea/the Sea of Japan, the South Sea of Korea, and the East China Sea, have been profoundly affected by climate and sea-level changes during the Quaternary period. In particular, during the last glacial maximum (LGM) approximately 26,000–19,000 years ago (26–19 ka) , is widely distributed along the entire coastlines of Japan, Korea, and eastern China. As described above, this region encompasses highly complex oceanographic features such as numerous islands, widespread shallow seas, and narrow and shallow straits, which have significantly amplified the effects of climate changes during the Quaternary period, particularly on the position of coastlines. As the habitats of B. attramentaria are confined to narrow intertidal zones with bottoms of rocks, boulders, and sandy mud along coastlines  to obtain a more comprehensive phylogeography of B. attramentaria with a special focus on a detailed distributional and demographic history. To this end, we applied coalescent-based Isolation with Migration (IM) and Bayesian skyline plot (BSP) methods to obtain a more realistic and detailed history of B. attramentaria at the population level from COI sequence data.Previously, the phylogeography of a et al. ; howeverBatillaria attramentaria were collected from fifteen sites representing the widest distribution around the Korean peninsula in 2006–2007 , following the manufacturer's protocols.A total of 197 individual COI gene (619 bp) was amplified by polymerase chain reaction (PCR). The reaction mixture used in PCR was composed of 10X Taq polymerase buffer, 2.5 mmol/L dNTP mix, 10 μmol/L of each primer, the total extracted DNA, and 2.5 units per μL of Taq polymerase . PCRs were performed under the following conditions for 35 cycles: 94°C for 30 sec, 50°C for 60 sec, and 72°C for 60 sec, with a final extension step at 72°C for 7 min. Two primers were used: Bat-COI-4MF (5′-CAC GAC GTT GTA AAA CGA CTT AGC WGG TGT TTC CTC T-3′) and Bat-COI-2MR (5′-GGA TAA CAA TTT CAC ACA GGC CRA ATA ARG CRA AAA CAG C-3′). These two primers were designed with reference to the mitochondrial COI sequences of closely related species: B. attramentaria,B. zonalis,B. felctosiphonata, and B. multiformis (GenBank Accession: No. AB054364 to AB054367). PCR products were bidirectionally sequenced using an ABI 3730xl automatic sequencer .The mitochondrial COI sequences (507 specimens of 384 bp) from the Japanese population (GenBank Accession: No. AB164326 to AB164358) , the probability of two randomly chosen haplotypes being different (Nei π) (Tajima k), and their corresponding variances were calculated in Arlequin   and another pooled Korea–Japan dataset (337 bp). Network trees based on the most parsimonious connections of haplotypes were constructed in TCS v.1.21 , and ambiguous basecalls and indels were manually adjusted. Then, these sequences were compared to rent Nei , nucleot) Tajima , the meaD Tajima ,1989b anma FS Fu  were usema FS Fu . We recoN1, N2, NA), splitting time (t), and gene flow rates (m1 and m2). A 337-bp DNA segment that overlapped between the Korean and Japanese datasets was used for the IMa2 analysis in the HKY substitution model. As the mutation rate of the COI gene of B. attramentaria is not known, we used a range of nucleotide divergence rates of the COI genes from other marine gastropods: the genera of Concholepas (0.86% per nucleotide per million years)   implemented in IMa2 Hey  was applB. attramentaria were evaluated under the IM model for three populations ) might have also descended from the ancestral population in the following order: , JI). As Jeju Island first split off from the ancient coastline as it moved northward and as the Yellow Sea and the South Sea are connected along their coastlines both now and during the transgression, we set JI as a basal population. The order of divergence among more than three populations should be provided prior to running IMa2 (Hey n = 194) of the Tsushima haplogroup in Korea. The same mutation rates described above were used under the HKY substitution model to convert the model parameters into demographic terms. The IM run was performed as follows: MCMC with 10 million steps after 100,000 burn-in steps with 10 chains under a heating scheme according to the IMa2 manual. Finally, likelihood-ratio tests were performed on the nested model of gene flow according to Hey and Nielsen  analyses were conducted in BEAST v.1.7.5  was used as an a priori uniform distribution of the mutation rate of COI. Convergence to the stationary distribution and sufficient effective sampling sizes for each estimated parameter were checked using Tracer v.1.5 , and these haplotypes contained 24 polymorphic sites ≥619 bp in length. Haplotype compositions of the 15 sampling sites  was observed in the Jindo population  across the populations and groups showed that the populations of YS, SS, and NJI are less diverse than those of SJI (Tableh) of all Korean samples was 0.546, and the nucleotide diversity (π) was 0.0017. These two indices were lower than those of Japanese B. attramentaria  . On the other hand, the comparison between YS and NJI, which lie along a linear trajectory of the western coast of Korean peninsula, resulted in the lowest FST value (0.026), which is lower than that (0.155) of between the northern and southern areas of Jeju Island.Pairwise ups Table. The higB. attramentaria haplotypes found in Korea diverged into two lineages, one of which included only one rare haplotype (Bat17) . IMa2 results for bidirectional migration rates between the groups showed an absence of gene flow. The effective population sizes of the Kuroshio and Tsushima groups were estimated to be approximately 130,000  and 120,000 , respectively.The twenty unique 17) Figs., S1. Theshio Fig.. Bat17 cshio Fig.. Our IMashio Fig.. This tyCOI sequences collected from thee different groups of Korean B. attramentaria, the Yellow Sea (YS), the South Sea (SS), and Jeju Island (JI), were analyzed under the Isolation with Migration model in IMa2. The run resulted in convergent marginal posterior density curves for six model parameters of effective population sizes, bidirectional migration rates, and divergence times for all three groups and their ancestral populations . These summary statistics indicated that snail populations around the Korean peninsula might have experienced recent demographic expansions.The summary statistics Tajima's ely Table. Three oB. attramentaria  . The timing of the demographic expansions was estimated through the conversion of the resultant tau values  from the analysis of the mismatch distribution. The beginning time of the sudden population expansion of each Korean B. attramentaria group was calculated as follows: YS (49–18 ka), SS (78–28 ka), NJI (140–50 ka), and SJI (39–14 ka), and for the whole Korean population, (86–31 ka).Our parallel analysis of mismatch distribution based on a sudden expansion model also supported the results of the BSPs. Mismatch distribution curves of all four geographic groups  showed unimodal shapes for the expected distribution of pairwise sequence divergences under the assumption of a sudden demographic expansion model . In addition, the mismatch distribution of the four groups as a single pooled group indicated a demographic expansion  model for the divergence of multiple populations   in East Asia showed that there are four closely related species of snails around China, Japan, and Korea: Batillaria attramentaria , B. flectosiphonata Ozawa, 1996, B. multiformis , and B. zonalis   model Hey  suggestslow Table. The coelow Table. Alternalow Table. As mariB. attramentaria into the Tsushima and Kuroshio haplogroups. A similar pattern of disjunct distribution with a similar geographic boundary was observed in another gastropod species, the Japanese turban shell Turbo cornutus, which formed two divergent haplogroups that also exactly corresponded to the warm currents , the South Sea of Korea (SS), and the northern (N) and southern (S) areas of Jeju Island (JI)  Fig.. These g(JI) Fig.. As illu(JI) Fig.. As mentB. attramentaria as a series of population divergences from an ancestral population into the current geographical groups along the shifting coastlines over time. To this end, the Isolation with Migration (IM) model for more than three populations , JI)  Fig.. Becauseods Figs., 5, we aCOI sequences: effective population sizes, migration rates, and divergence times of the first and second divergences  were estimated as 24 ka (95% HPD: 85–10 ka) and 35 ka (95% HPD: 105–22 ka), respectively. In overall, the estimated duration of the divergences of the three geographic groups and the subsequent demographic increases of each group overlaps with the period of marine transgression in the Yellow Sea from the LGM onwards. The split time (t0) was not significantly difference from that of the prior split (t1), suggesting that the two divergences may reflect a process of very successive isolations among the three geographic groups  from the LGM onwards . Overall, our results demonstrate that the present genetic variation in Korean B. attramentaria could be traced to the initial splitting event of the most recent common ancestral population, approximately 35 ka and thus could correspond to the phylogeographic and demographic events occurred up to this time.The IMa2 analysis based on the reconstructed divergences enabled us to estimate the population demographic parameters of the consecutive divergences from the population ces Figs., S2. TheB. attramentaria should have occurred while the shifts of coastlines were resulting in expansion of habitats and the air temperature increased by approximately 8–13°C, reaching the present levels   is near the start of the increases in the demographic curves  analyses detected historical demographic expansions of  ka) Fig.. We appl ka) Fig.. In otherves Fig., suggestB. attramentaria occurred almost simultaneously throughout the entire distributional range of this species. Despite this wide and concurrent expansion, there was a slight difference in the time of initiation of the expansion among locations. For instance, the Kuroshio group began to increase approximately 35 ka, but the Tsushima group began at approximately 19 ka. The Kuroshio group of B. attramentaria was estimated to have increased about to fifty times the size it was before the LGM and, as a result, is five times larger than the Tsushima group  group with its relatively late initiation (approximately 15 ka). The estimates of degree and time are similar to those from IMa2 (about twelve times and 24 ka). The slight difference in time estimation between the methods may be due to differences in the exact meaning of the time. That is, the value of 24 ka is split between the YS and SS, rather than reflecting the true starting time of the demographic increase in the YS. As mentioned above, the demographic history of the YS group may illustrate the response of B. attramentaria to the most recent climate change since the LGM is exceptional. The BSPs reconstructed from the COI sequences clearly demonstrate that most of the variation was shaped during the demographic expansion since the LGM, but there are no further informative coalescent events in COI lineages beyond 35 ka, making it impossible to estimate effective population size. It is likely that older variation might have been erased due to severe bottlenecks during glacial periods before the LGM. This speculation is based in the immediate demographic response of B. attramentaria even to harsh environments. Finally, in relation to the time window of the BSP method, a single locus of mitochondrial DNA may be not sufficient to detect old coalescent events because the effective population size of mitochondrial loci is smaller than that of nuclear genetic loci. Thus, DNA sequence data of multiple nuclear loci will be invaluable to clarify the intriguing question on the role of the precedent glacial maxima before the LGM.Notably, the immediate and faithful demographic response of Turbo cornutus  the two deep lineages of the geographically separated groups, “Kuroshio” and “Tsushima,” of this species began to diverge approximately 400 ka and then have occupied geographically separated subregions in the north and south, (2) the geographic differentiation of Korean B. attramentaria could be explained by continuing divergences of an southern ancestral population into Jeju Island, the Yellow Sea, and the South Sea of Korea, (3) the estimates of population demographic parameters for these processes are remarkably consistent with the oceanographic history of marine transgression around the Korean peninsula after the LGM, and finally, (4) the results of range shifts and demographic growths and the timing of the each event were largely consistent with those of BSPs. Finally, the Bayesian skyline plot (BSP) analyses revealed that the historical effective population sizes of local groups of B. attramentaria have drastically increased since the last glacial maximum (LGM: 26–19 ka). The relative concurrence and similarity in the modes of the demographic expansions at six representative geographic groups around Korea and Japan significantly coincided with the postglacial rise in sea level and seawater temperature in the region. Therefore, such widespread demographic expansions must have started as interglacial marine transgressions and warming temperatures generated of new habitats and more favorable water environments for B. attramentaria since the LGM. The remarkable association between the genetic variation of B. attramentaria and past environmental changes, particularly from the LGM onwards, could be attributable to the key features of this species: Habitats confined to narrow intertidal zones and a reproductive stage of directly developing larvae that limits dispersal capability. To our knowledge, B. attramentaria is the first marine species in the northwest Pacific for which historical distribution and demography have been resolved in detail over space and time, and its recent demographic expansion has been found to be directly associated with the coupled changes of coastlines and warming seawater temperatures since the LGM. Therefore, the current results could be used to generate working hypotheses for future phylogeographic and demographic studies of other marine species in the region.The molecular population data from the


La Hoya  was one of the most important villages of the Late Bronze and Iron Ages of the north of the Iberian Peninsula, until it was violently devastated around the 4th century and abandoned in the 3rd century B.C. Archaeological evidences suggest that descendants from La Hoya placed their new settlement in a nearby hill, which gave rise to the current village of Laguardia. In this study, we have traced the genetic imprints of the extinct inhabitants of La Hoya through the analysis of maternal lineages. In particular, we have analyzed the mitochondrial DNA (mtDNA) control region of 41 human remains recovered from the archaeological site for comparison with a sample of 51 individuals from the geographically close present-day population of Laguardia, as well as 56 individuals of the general population of the province of Alava, where the archaeological site and Laguardia village are located. MtDNA haplotypes were successfully obtained in 25 out of 41 ancient samples, and 14 different haplotypes were identified. The major mtDNA subhaplogroups observed in La Hoya were H1, H3, J1 and U5, which show a distinctive frequency pattern in the autochthonous populations of the north of the Iberian Peninsula. Approximate Bayesian Computation analysis was performed to test the most likely model for the local demographic history. The results did not sustain a genealogical continuity between Laguardia and La Hoya at the haplotype level, although factors such as sampling effects, recent admixture events, and genetic bottlenecks need to be considered. Likewise, the highly similar subhaplogroup composition detected between La Hoya and Laguardia and Alava populations do not allow us to reject a maternal genetic continuity in the human groups of the area since at least the Iron Age to present times. Broader analyses, based on a larger collection of samples and genetic markers, would be required to study fine-scale population events in these human groups. La Hoya was a settlement located in the region currently known as the Basque Country, in Northern Spain, from Late Bronze to Iron Ages (15th to 3rd centuries B.C). This fortified town located near Laguardia (Alava)  had an eIn the present study we analyzed the mtDNA hypervariable regions HVS-I and HVS-II of 41 human remains recovered in La Hoya archaeological site, as well as the complete mtDNA control region of the present-day populations of the village of Laguardia and the province of Alava. Laguardia is the extant population settled nearby the ancient village of La Hoya and Alava is a representative sample set of the province. The comparison between the ancient population of La Hoya and these extant Basque populations might allow assessing the maternal genetic continuity of these populations settled in the same area but separated by thousands of years.The archaeological excavation carried out during 1973 and 1989 revealed more than 260 human remains from different cultural periods [BIBAT Museum, the Archaeological Museum of Vitoria-Gasteiz .A total of 41 human remains from the Celtiberian Iron Age (5th century B.C.), belonging to 33 newborns or infants  –S3 Figs BIBAT Museum and Instituto Alavés de Arqueología) and approved by the Director of Cultural Heritage of the Department of Culture of the Basque Government, as issued by the resolution of 2nd August 2011.All necessary permits were obtained for the described study, which complied with all relevant regulations. The genetic analysis of these ancient remains was granted by the institutions that are in charge of the collection : 51 from Laguardia (LG) and 56 from the whole Alava province (AL). Voluntary donors were healthy and maternally unrelated individuals, whose ancestors (until the third generation back) were born in Alava or, specifically, in Laguardia for the 51 LG samples. All participants gave their written informed consent to participate in this study following the ethical standards of the Helsinki declaration. This study was approved with the favorable ethical report from the Faculty of Pharmacy of the University of the Basque Country, signed at 26th September 2008.To prevent and detect contamination, we followed precautions at all stages of the analysis . Appropr® DNA Purification Spin Columns . A final concentration step to 30–40 μl was carried out using Amicon-30 columns . Extracted DNA was quantified by real-time quantitative PCR (qPCR) using QuantifilerTM Human DNA Quantification kit  in an ABI PRISM 7000 Sequence Detection System (AB/LT/TFS) following the manufacturer’s recommendations.To eliminate possible contaminants from prior handling, the outer layer of the bones was removed with a sanding machine and teeth were cleaned with 10% bleach. Subsequently, bone fragments and teeth were 30 min UV-irradiated each side. Samples were then grounded into a fine powder in a 6750 Freezer Mill  under liquid nitrogen to avoid DNA degradation. Afterwards, about 500 mg of powder was used for a silica-based DNA extraction method  (modified of ) adaptedDNA extraction from modern samples was performed in a separate laboratory room, using the standard phenol: chloroform procedure . DNA was2 (50 mM), 0.5 μl dNTPs mix (2.5 mM), 1 μl BSA (10 mg/ml), 0.15 μl Taq (5 U/ μL) , 0.25 μl of each primer (10 μM), 3 μl of DNA sample, and sterile water up to a total volume of 25 μl. Amplification of each hypervariable segment was carried out using three overlapping pairs of primers, except for HVS-III for which one primer pair was used (Hypervariable segments I (HVS-I) and II (HVS-II) of the mtDNA control region were analyzed in all 41 samples from La Hoya. Additionally, hypervariable segment III (HVS-III) was also analyzed in some samples in order to deepen into haplogroup J subclassification. PCRs contained 2.5 μl Buffer 10X, 0.75 μl MgClwas used . Cycling® TerminatorTM v3.1 Cycle Sequencing Kit (AB/LT/TFS). Sequencing products were purified with BigDye® XTerminatorTM Purification Kit (AB/LT/TFS).Amplification products were subjected to electrophoresis on 1.5% agarose gels and visualized with GelRed (3% μL/ml)  and UV light (UVItec Cambridge). DNA products were purified using 0.5 μl EXO (Exonuclease I) and 2.5 μl SAP   to 10 μl of PCR product and sequenced in both directions using the same PCR primers. Sequencing reactions were performed with BigDye® Software v2.5 (AB/LT/TFS). Edition ranges are shown in www.phylotree.org, v16) [Capillary electrophoresis was performed on a 3130 Genetic Analyzer (AB/LT/TFS). Sequencing reaction products were analyzed with the Sequencing Analysis software (AB/LT/TFS). Sequences were aligned to the revised Cambridge Reference Sequence (rCRS)  using Serg, v16) .Complete mtDNA control region (CR) from modern samples was amplified, sequenced, and interpreted as reported in . All samAncient and modern samples assigned to R0 macrohaplogroup were additionally analyzed by minisequencing for subtyping the following H lineages: H1 G3010A), H1bh (C11377T), H2a C4769T), H3 (A6776G), H4 (C3992T), H5’36 (G456A), H6a (C3915T), H7 (T4793C), H8 (A13101C), H9 (T13020C), H10 (T14470A), H11 (A8448G), H12 (C3936T), H13a1 (A4745G), and H15 (T6253C). Amplification and minisequencing primers used are described in [T, H3 .In order to statistically evaluate differences in DNA yield and inhibition among adult and infantile samples we performed two chi-square tests based on type of sample (adult/child) and absence/presence of DNA or inhibition, using the PAST v3.06 software . In the To carry out comparative analyses, population data available in the literature of ancient populations from the Iberian Peninsula were compiled . A princST values based on haplotypes. Additional Basque populations from Alava, Gipuzkoa, Bizkaia, and Navarre were included in these calculations for comparison purposes to a local level , implemented in DIYABC 2.1  was used−6 and 1 x 10−8 mutations. Only the simulations generating summary statistics close to the observed were considered to estimate the posterior probabilities of the models and parameters. We performed the summary statistics for populations pairs based on FST [6 simulated data sets were used. Pre-evaluation of each scenario was performed by a PCA, based on the summary statistics, and checking the congruence between the observed and simulated data set. Logistic regression was used to compare posterior probabilities. Performance of the scenario parameters was assessed by means of type I  and type II  errors, and by computing the relative bias. We estimated the type I error probability as the proportion of cases where the selected scenario did not show the highest posterior probability compared with the competing scenario for the simulated datasets generated under the best-supported model [Uniform distributions for the historical parameters were defined. The mutation model chosen was the Hasegawa–Kishino–Yano model , as recod on FST . A totaled model . LikewisDNA from eight adults and 33 newborn and infant remains was extracted and quantified. Real-time qPCR analysis provided valuable information about DNA preservation, as well as possible presence of inhibitors in the samples. Positive quantification results were obtained in 15 out of 41 samples, with nuclear DNA yields up to 0.2010 ng/μl . Two outA chi-square test was performed in order to evaluate the differences observed in adult and child samples for DNA recovery and inhibition. No significant differences between adult and child samples based on the recovery of DNA (p = 0.355) were observed, but the same test based on absence or presence of inhibition did present significant differences (p<0.002). This fact could be related to the type of bone available and consequently analyzed. Infantile samples comprised small bones that easily disintegrated, and whose thin and fragile cortical layer hindered the sample processing.Due to the limited nuclear DNA recovery efficiency (36.59%) a strategy based on the analysis of short amplicons (164–207 bp) of mtDNA was followed for the 41 samples included in the study.MtDNA haplotypes were obtained in 25 of the 41 studied samples , with different range of sequence coverage achieved . NeitherSamples LHY099 and LHY181 shared the same HVS-I and HVS-II haplotype , indicating a possible common maternal ancestry. In addition, the aforementioned HVS-II haplotype was also observed in samples LHY112, LHY158, and LHY170. Furthermore, other 13 samples shared common  haplotypes: LHY249/LHY223/LHY227, LHY235/LHY236, LHY104/LHY245, LHY163/LHY213, LHY169/LHY244, and LHY097/LHY258. In some of these cases, the limited sequence range accomplished for HVS-I and HVS-II does not allow concluding a maternal relationship among them. Interestingly, two of these individuals, LHY235 and LHY236, were found in the same burial group, further supporting a maternal kinship between these samples, which correspond to two infants. The rest of individuals sharing mtDNA haplotypes were found in different burial places, nevertheless this not necessarily excludes a common maternal ancestry.The major haplogroups found in La Hoya were H and J, which were present in nine individuals each, being the subhaplogroups H1  and J1c2  the most prevalent ones . HaplogrAs previously mentioned, samples LHY235 and LHY236 sharing the same haplotype and belonging to J1 haplogroup were found in the same burial group. Thus, they were considered only once  due to aThe high presence of haplogroup J in the ancient remains of La Hoya is striking even though previous studies have already pointed out the high frequency of J and, probably, J1 lineages in the Basque Country , since tIn order to evaluate the haplogroup variability of La Hoya mtDNA lineages in a geographically and temporarily context, haplogroup frequencies were compared with those of other ancient historic and prehistoric populations from the Iberian Peninsula . Fig 3 sThe position of La Hoya (LH) in the PCA was mostly determined by the comparatively high frequency of haplogroup J and haplogroup R0 within the ancient context. The cemetery of Aldaieta (AL) also located in the Alava region and chronologically more recent (VI-VII A.D.) with regard to La Hoya, showed similar frequencies of haplogroups J and H. The same occurred with the pre-Roman and Neolithic archaeological sites of Catalonia (C1 and C2), although C2 is also influenced by the frequencies of haplogroups T and W. Moreover, the Basque Neolithic Los Cascajos (LC) shows a similar pattern too. The Neolithic and Chalcolithic sites of the Basque Country and Navarre, Pico Ramos (PR), Paternanbidea (PA), Longar (LO), and San Juan Ante Portam Latinam (SJ), along the component 2, harbour similar frequencies of haplogroups R0 and K. Finally, the sample from the Mirador Cave (AT) in Atapuerca shows marked differences in haplogroup frequencies with respect to the rest of ancient sites, especially in terms of haplogroups T and X.Complete mtDNA CR was obtained from two present-day populations from the village of Laguardia and the whole province of Alava . A totalH = 0.9882 ± 0.0064 and H = 0.9851 ± 0.0077, respectively) is in accordance with the genetic variability described in other autochthonous populations from Basque Country  and non significant  genetic distance for Laguardia and the Alava population sample. On the other hand, these two population groups showed significant differences (p<0.005) with the rest of Basque populations. However, the FST values as well as the limited sample size are not high enough to evidence a strong population substructure.Population comparison based on Fur group  was perfur group  were groThe classification of lineages into haplogroups revealed that native populations of Laguardia and Alava are solely represented by European lineages , except Laguardia and Alava populations share lineages belonging to HV0, J1, K1, U5, and X2 haplogroups, as well as from macro-haplogroup R0. Additionally, it is noticeably the identification in Laguardia of two individuals belonging to L1b and U6a lineages, which are found in African populations , 23. TheThe comparative analysis between La Hoya and the current populations of Laguardia and Alava did not show complete haplotype coincidences, except for lineages belonging to haplogroup H. Nonetheless, phylogenetically close haplotypes were identified, for instance the lineage J1c2 of LHY099/LHY181 , LG003 , and VG110  individuals from La Hoya, Laguardia, and Alava, respectively.The haplogroup distribution of the extinct and extant populations under study presents certain similarities . Indeed,We used Approximate Bayesian computation method to investigate the genealogical relationship between La Hoya and the present-day Basque populations of Laguardia and Alava. Two competing scenarios were designed in order to test whether La Hoya and Laguardia exhibit genealogical continuity (Scenario 1) or they are genealogically independent (Scenario 2) . The latThe reconstruction of population history through the study of present-day population data may not be able to infer important information derived from population replacements, genetic drift or minor migration events occurred in certain past periods. For this reason, ancient DNA (aDNA) studies are of great interest as they provide a direct access to prehistoric and historic genetic information. We have carried out the analysis of the mtDNA control region of ancient remains of 41 individuals recovered from the village of La Hoya, one of the most important Iron Age trade centers in the north of the Iberian Peninsula to trace back its connection to current populations living in the area.The discovery of well-preserved adult human skeletons from the Celtiberian Iron Age is of great historical and biological importance, since few intact adult remains of the Celtiberian culture have been previously recovered. This is consequence of the funeral rites used in that period for adult individuals, where cremation was the common practice. On the other hand, numerous remains from newborns and infants of the Celtiberian period were also found in La Hoya due to the established ritual to bury those young individuals who prematurely died under the eave of their houses . ConsequThe major haplogroup found in the archaeological samples from La Hoya was haplogroup H (9 out of 24), particularly subhaplogroups H1  and H3 , whose highest frequencies are observed in the Iberian Peninsula and, in particular, in the north of the Iberian Peninsula , 28. In Haplogroup J was the second most relevant lineage in La Hoya, particularly subhaplogroup J1 . Previous studies have proposed haplogroup J as a marker for the spread of Neolithic farmers from Near East around 10,000 years ago . Indeed,In La Hoya, haplogroup U5b has been observed in three individuals. In the Iberian Peninsula we have to travel back until the Mesolithic to find the most ancient remains bearing U5b, two individuals U5b2c1 in La Braña-Arintero (León)  and one Finally, haplogroups K and W were found in one individual each in La Hoya sample set. Haplogroup K can also be found in other archaeological sites of the Basque Country  and otheThe results of the principal component analysis based on haplogroup composition of historic and prehistoric Iberian populations, placed La Hoya near to other geographically and/or temporary close archaeological sites. Nevertheless, the reduced number of individuals in all ancient sample sets has to be considered cautiously for further relevant conclusions regarding population interrelations.http://empop.online/). The 16366T variant, not found in La Hoya lineages, has been detected in two ancient individuals belonging to J1c2e lineage from the Unetice culture  and Corded Ware culture  [The study of mtDNA control region of the geographically close populations, Laguardia and Alava, has provided some clues about the possible maternal genetic imprint of La Hoya inhabitants in present-day populations. In terms of mtDNA haplotypes, no exact match was found between the ancient and modern populations. Other mitochondrial aDNA studies have also faced the difficulty of finding identical haplotypes between ancient and modern populations . Phyloge50 B.C.) ,38. Thes50 B.C.) . FurtherComparative analyses of La Hoya and the present-day populations revealed so far that mitochondrial DNA haplogroups of La Hoya inhabitants are contained in the contemporary European genetic pool, and especially showing a distinctive frequency pattern in the autochthonous populations of the north of the Iberian Peninsula. Despite the archaeological evidences, the apparent genetic similarity of La Hoya and Laguardia, in terms of phylogenetically close lineages and haplogroup composition, is not enough to imply that modern Laguardia population could be the descendant of the once-living in La Hoya. In fact, ABC analysis seems to support the lack of genealogical continuity between these populations at the haplotype level. Nevertheless, the highly similar subhaplogroup composition detected between La Hoya and Laguardia populations do not allow us to reject a maternal genetic continuity in the human groups of the area since at least the Iron Age to present times.A more comprehensive study based on a larger collection of samples and genetic markers would help to further deepen into more local scale population events, like the founding of Laguardia by the former inhabitants of La Hoya.S1 FigLocation of the infant human remains found is shown by red dots.(TIF)Click here for additional data file.S2 FigLocation of the infant human remains found is shown by red dots.(TIF)Click here for additional data file.S3 FigLocation of the infant human remains found is shown by red dots.(TIF)Click here for additional data file.S4 FigScenario 1 corresponds to a genealogical continuity hypothesis between the ancient population of La Hoya and the modern population of Laguardia, and Alava not derived from this ancient population. Scenario 2 assumed that the ancient and modern populations are genealogically independent.(TIF)Click here for additional data file.S1 TablePrimers information for coding region positions 7028 and 11467 studied in some modern samples is also shown.(XLSX)Click here for additional data file.S2 TableChronology, population acronym, number of individuals and source reference are shown.(XLSX)Click here for additional data file.S3 TableType of sample (adult or child), DNA concentration (ng/μl) and inhibition level (IPC) for each sample are shown.(XLSX)Click here for additional data file.S4 Table(XLSX)Click here for additional data file.S5 Table(XLSX)Click here for additional data file.S6 TableGenetic diversity (H) is shown.(XLSX)Click here for additional data file.


Transnational migrants who transport and introduce landraces to new locations may catalyze a third type of conservation that combines both approaches. Resulting populations may support reduced diversity as a result of evolutionary forces such as genetic drift, selection, and gene flow, yet they may also be more diverse as a result of multiple introductions, selective breeding and cross pollination among multiple introduced varietals. In this study, we measured the amount and structure of maize molecular genetic diversity in samples collected from home gardens and community gardens maintained by immigrant farmers in Southern California. We used the same markers to measure the genetic diversity and structure of commercially available maize varieties and compared our data to previously reported genetic diversity statistics of Mesoamerican landraces. Our results reveal that transnational dispersal creates an opportunity for the maintenance of maize genetic diversity beyond its recognized centers of diversity.Contemporary germplasm conservation studies largely focus on  Conserving landraces and crop wild relatives is crucial for maintaining genetic resources for future crop improvement  sampled from Southern Californian home gardens and community gardens to local commercial varieties for sale. Likewise, we used genetic diversity data for Mesoamerican maize reported in the literature to determine whether the gardens were more or less diverse relative to those in centers of diversity. We chose maize because the genetic erosion of maize diversity in Mexico, its center of domestication and diversity, has gained increased attention as farmers abandon the cultivation of traditional landrace varieties either in favor of migration to urban centers or in favor of relatively homogenous improved lines. Our results provide an examination of the population genetics of maize in Southern California gardens and reveal that maintaining maize germplasm diversity is not restricted to its traditionally recognized centers of diversity.In this study, we compared maize genetic diversity , respondents distinguished two distinct varieties of maize within their garden. In these instances, 10 plants from each of the two varieties were sampled and treated as separate accessions. Thus, we sampled 10 populations and 10 accessions from Riverside and 10 populations and 12 accessions from Los Angeles. Fresh leaf tissue was frozen at −80°C until DNA extraction could be performed.We included five commercially available cultivars for comparison. Two sweet corn populations (HV1 and HV2) that are representative of locally available horticultural varieties for home gardeners were purchased from a Riverside garden retail store. We also obtained imported corn seed for human consumption from Mexico from a bulk food bin at Big Saver Foods supermarket in Riverside. The latter collection was motivated by interviews with respondents that revealed local ethnic markets supplying bulk seed for consumption were sometimes a source of seeds for their gardens. Finally, we included maize cultivars IV1 and IV2 to represent industrial maize cultivars. We germinated 10 randomly selected seeds from each cultivar in a temperature–controlled greenhouse. Fresh leaf tissue was harvested from germinated plants and stored at −80°C prior to extraction.DNA was isolated from finely ground tissue by use of the DNeasy plant Mini Kit  with the following modifications: 3 μl Rnase A stock solution was added to the ground tissue; at the lysation phase, cells were incubated for 15 min; and at the final elution step, 50 μl Buffer AE was added to the DNeasy membrane. Extracted DNA was quantified with use of NanoDrop spectrophotometer  and stored at −20°C.http://www.maizegdb.org.Five microsatellite markers previously described by Senior et al.  were selDNA amplification involved the use of a PTC–100 Thermo Cycler  in a 10 μl volume containing approximately 5.0 to 10.0 ng/μl template DNA, 10 μm each forward and reverse primer, 1 mM dNTPs, 10x Promega PCR Buffer, 25 μm magnesium chloride, and 5 units/μl Taq DNA polymerase. PCR conditions were 94°C for 5 min, 34 cycles of 94°C for 1 min, 55°C for 4 min, 72°C for 2.5 min, and a final cycle of 72°C for 15 min.Polymorphisms in the amplified PCR product were detected by electrophoresis in a 2% Agarose Super Fine Resolution (SFR) gel . Gels were prepared with 1X TBE Buffer and were stained with ethidium bromide before electrophoresis in a submarine gel system . Each well contained 4 μl PCR product and 1 μl 6X blue loading dye. Bands were visualized by use of GelDOC . Fragments were estimated by comparison with a 100–bp DNA ladder . Each gel was run with a control sample of a known fragment size.DNA for several individuals failed to amplify at certain loci, despite repeated amplifications, particularly B73. These data are treated as missing in our analyses.a), proportion of polymorphic alleles, percentage of polymorphic loci, expected heterozygosity (He), and fixation index (Fis) were calculated for each population and population group by use of the computer program PopGene v1.32  for the group was 0.55±0.11, with mean Fis for the group of 0.05. A single population, LACG1 (one accession of two sampled from a Los Angeles community garden), had the lowest number of observed alleles, 6, and LAHG3 (one accession of two sampled from a Los Angeles home garden) had the highest, 17.All garden populations were polymorphic (100%). The total number of alleles observed was 25 (mean 5.00±1.87). The mean expected heterozygosity  had the highest number of total alleles, 11. Two–tailed t–test revealed significant difference in diversity (He) of individual garden populations and individual commercial populations (including SV1) (p≤0.016). When we removed the imported SV1 variety from the commercial group statistics, the total number of alleles for the commercial populations decreased to 9 (mean 1.80±0.84). The mean He was 0.19±0.22, with the mean Fis for the group of 0.06.In contrast, commercial populations showed less genetic diversity than garden populations, regardless of population genetic measurement. Only four loci were polymorphic (80%). The total number of observed alleles was 14 (mean 2.80±1.10). The mean He for home gardens was 0.55±0.10; for community gardens and 0.51±0.10 for community gardens. The mean Fis for home gardens was 0.05 and 0.09 for community gardens.The contrast between home and community gardens was not nearly as strong. Both garden types were polymorphic at all five loci (100%). The total number of alleles in home gardens was 25 (mean 5.00±1.87), with 19 (mean 3.80±1.80) in community gardens. The mean HThe program STRUCTURE assigned genetic affiliations to the sampled individuals within preselected populations. The most likely number of groups for all garden populations (unresolved amplifications treated as missing) was K = 5 Fig. . GeneralSeparate analysis of the five commercial varieties gave a group assignment of K = 3 Fig. . FurtherAs compared with commercially available maize from Southern California, maize collected from garden populations in Southern California showed a considerable amount of genetic variation in all five microsatellite loci investigated Table . The resThis analysis of the structure of diversity in the gardens gave several interesting results. First, PopGene revealed greater relative diversity in gardens than in commercial populations. In the STRUCTURE analysis, the imported SV1 corn seed showed greater allelic similarity to maize sampled from gardens than any of the commercial varieties, so the garden populations may have a closer genetic affiliation to this imported seed from Mexico. PopGene analysis showed less overall diversity in the commercial populations sampled.Results of STRUCTURE analysis showed that the gardens exhibited greater admixture within and between populations than the commercial populations. While these results may indicate potential gene flow, it may also indicate a high degree of relatedness due to similar ancestry. When analyzing the commercial populations alone, SV1, the imported seed from Mexico, did not pair with the rest of the commercial varieties. The store labeling of this seed as an imported corn seed from Mexico suggests a potential landrace origin; if so, our finding of little genetic affiliation with other locally available commercial varieties is not surprising. On analyzing SV1 with the garden populations, SV1 showed a common affiliation with the populations LAHG5, LAHG7, LACG4, LACG1, LACG2, LAHG2, and LAHG3. The resulting affiliation suggests that the farmers who managed the populations might have used the supermarket variety in the past as a seed source or that some of the garden accessions share ancestry with the imported seed.We considered that a community garden with multiple plots and accessions might have increased potential for gene flow and admixture, thus showing a more diverse germplasm profile. Results from grouped PopGene analysis of home garden and community garden populations do not support our original assumption of greater variation. These results could be attributed to the small number of community gardens we sampled as compared with the large number of home garden populations. Sampling from a larger set of community gardens in future studies would shed light on whether a small sample size affects the comparative outcome or whether other factors are involved.Several existing studies have reported on the genetic diversity of landraces of maize in Mexico. Their results Table  on fixatex situ methods to preserve diversity in controlled environments. It has ignored the importance of human migration and the movement of important cultural food species in establishing new regions of germplasm diversity. Biodiversity research of urban gardens worldwide has been limited in two ways. Geographically, studies have mostly concentrated on tropical home gardens in Asia and South America. Research has also been limited by its focus on species richness and evenness within gardens without addressing the level of genetic diversity within or between species  and a UCMEXUS–CONACYT Collaborative Research Grant awarded to NCE. Cristina Martinez–Canton provided invaluable field support. Laura Smales, Jeffrey Ross–Ibarra, Daniela Soleri, and two anonymous reviewers provided helpful comments to improve earlier drafts of this manuscript.


In a worldwide collaborative effort, 19,630 Y-chromosomes were sampled from 129 different populations in 51 countries. These chromosomes were typed for 23 short-tandem repeat (STR) loci  and using the PowerPlex Y23 System . Locus-specific allelic spectra of these markers were determined and a consistently high level of allelic diversity was observed. A considerable number of null, duplicate and off-ladder alleles were revealed. Standard single-locus and haplotype-based parameters were calculated and compared between subsets of Y-STR markers established for forensic casework. The PPY23 marker set provides substantially stronger discriminatory power than other available kits but at the same time reveals the same general patterns of population structure as other marker sets. A strong correlation was observed between the number of Y-STRs included in a marker set and some of the forensic parameters under study. Interestingly a weak but consistent trend toward smaller genetic distances resulting from larger numbers of markers became apparent. It is intended to be used in forensic casework, kinship analysis and population genetic studies. Advantageous features such as short fragment length and an uninterrupted repeat structure were taken into account when constructing the kit. Six new markers , two of which (DYS570 and DYS576) categorized as “rapidly mutating” ®kit . The first studies employing the new PPY23 kit revealed a markedly increased haplotype diversity and discriminatory power in comparison to other marker sets, including the MHT, SWGDAM , PowerPlex®Y12 (PPY12) and Yfiler panels www.yhrd.org).The PowerPlex22.1Between 9/2012 and 6/2013, a total of 19,630 Y-STR haplotypes were compiled in 84 participating laboratories. In particular, unrelated males were typed from 129 populations in 51 countries worldwide .Each participating laboratory passed a quality assurance test that is compulsory for all Y-STR studies to be publicized by, and uploaded to, YHRD. In particular, each laboratory analyzed five anonymized samples of 10 ng DNA each, using the PowerPlex2.3n = 19,630) and for all 23 markers of the PPY23 kit. To this end, DYS389II alleles were encoded by the difference, henceforth labeled DYS389II.I, between the total repeat number at DYS389II and the repeat number at DYS389I. DYS385ab haplotypes were treated as single alleles thereby ignoring the internal order of its two component alleles. Forensic parameters were calculated for the study as a whole and for meta-populations defined according to the continental or ethnic origin of the samples (see above).Forensic parameters were calculated for all samples  was calculated analogous to GD. Match probability (MP) was calculated as the sum of squared haplotype frequencies. The discrimination capacity (DC) was defined as the ratio between the number of different haplotypes and the total number of haplotypes. To benchmark the practical utility of the PPY23 panel for forensic casework, all haplotype-based analyses were repeated for various subsets of Y-STRs, namely the MHT (9 loci), SWGDAM (11 loci), PPY12 (12 loci) and Yfiler marker panels (17 loci). The Yfiler and PPY23 panels also were compared to one another after confining both panels to Y-STRs with an amplicon length <220 bp.In particular, allele frequencies and haplotype frequencies were estimated using the counting method. Single-marker genetic diversity (GD) was calculated as 2.4RST, thereby taking the evolutionary distance between individual Y-STR haplotypes into account n = 705, 3.6%), leaving 18,925 haplotypes for analysis (Supplementary Table S2). RST values resulting from continental grouping were compared among the PPY23, Yfiler, PPY12, SWGDAM, and MHT panels. Multidimensional scaling (MDS) analysis served to visualize differences in Y-STR genetic variation between populations and was based upon pairwise linearized RST values for PPY23, that is RST/(1 − RST). MDS is commonly used to investigate genetic similarities between populations and has been described in detail elsewhere The extent of population genetic structure in our data was assessed by means of analysis of molecular variance (AMOVA). More specifically, genetic distances between groups of males were quantified by RST values and for randomization-based significance testing of genetic distances  vegan v.2.0-10 was used maps v.2.3-6 mapdata v.2.2-2 All analyses were performed using R statistical software v2.15.3 33.1A high level of genetic diversity was observed in our study at all 23 Y-STRs of the PPY23 panel. Some 521 different alleles were observed in the 19,630 Y-chromosomes analyzed, with a median number of 16 alleles per marker and a range of 10 (DYS391) to 31 . Marker DYS385ab showed 146 different allele combinations (i.e. unordered haplotypes). A total of 133 null alleles occurred at 17 of the 23 loci, 75 intermediate alleles (18 loci) and 69 copy-number variants . Of the six markers that distinguish PPY23 from Yfiler, the DYS481 and DYS570 markers showed the largest numbers of different alleles  and DYS391 and DYS549 showing the lowest number (n = 1). Four of the six PPY23-specific markers  had comparatively high numbers of variant alleles. Only two single non-fractional off-ladder alleles  were observed in this study. On the other hand, only five of the 19 intermediate alleles observed for the six PPY23-specific markers  were included in the bin set of the allelic ladder (Table S3).Variant alleles not representing simple repetitions of the respective STR motif occurred at all loci (Table S3). This included null alleles, likely due to a deletion or primer site mutation, intermediate alleles comprising fractional repeats, and copy-number variants such as duplications and triplications of the whole locus. All variant alleles were confirmed by retyping or sequencing at the laboratory that had performed the original STR typing. The proportion of variant alleles differed greatly among markers , with DYn = 13). The structure of 11.1 at the DYS643 marker (observed in 11 samples in our study) has been reported previously Some 75 different intermediate alleles occurred at one of 18 Y-STR loci and were seen in 550 samples (Table S3). DYS458 was the locus with the highest proportion of intermediate alleles (16 different in 374 samples), followed by DYS385ab (12 different in 57 samples) and DYS448 (8 different in 23 samples). Of the PPY23-specific markers, DYS481 had the highest number of different intermediate alleles (5 in 26 samples) of which 25.1 was the most frequent (n = 59), followed by PPY23-specific markers DYS576 (n = 14), DYS481 (n = 11) and DYS570 (n = 11). In nine samples, a large deletion was detected at Yp11.2 encompassing the AMELY region that removed four adjacent loci . All these samples were of Asian ancestry, namely Indians from Singapore, Tamils from Southern India and British Asians with reported origins from Pakistan or India, where this type of deletion is frequent AMELY gene locus. Another large deletion located at Yq11 and encompassing the AZFa region A total of 133 null alleles were observed at 17 loci (Table S3), which corresponds to an overall frequency of 0.03%. The DYS448 locus showed the highest number of null alleles (n = 30) and DYS448 (28), followed by DYS481 and DYS570 . Note that, at DYS385ab, only copy numbers larger than two are conventionally counted. One triplication each of the DYS19 and DYS448 markers was observed in African American samples and a duplication comprising two intermediate alleles (15.2 and 18.2) at the DYS576 marker occurred in a European American sample. Duplications of several consecutive loci in the AZFa region There were 69 copy number variants, mostly duplications, observed at 21 loci . Copy number variants were most abundant at the markers DYS19 (3.3n = 1), Finland , Netherlands , Xuanwei , Kinyawa , South Africa , Peru , Northern Alaska  and Western Alaska  (data not shown).The 23 markers of the PPY23 panel were evaluated with respect to their haplotype diversity (HD), discrimination capacity (DC) and other forensic parameters such as random match probability (MP). In total, 18,860 different haplotypes were observed . Of the Of the meta-populations formed according to continental residency, Asia showed the highest DC (>0.97), followed by Europe and Latin America (DC ∼ 0.96), and finally Africa . Grouping by continental ancestry yielded similar DC values of >0.96 for Asians, Europeans and Mixed Americans. However, a decrease in DC was observed for Native Americans (0.83) and an increase for samples of African ancestry . Notably, 42 out of the 129 population samples (32.6%) contained only unique PPY23 haplotypes (‘complete resolution’), namely seven Asian, 23 European, six Latin America and six North America (i.e. no African populations).3.4r = 0.97 and r = 0.98, respectively). On average, each additional marker generated 754 new different haplotypes (p = 0.005 from linear regression) and 888 new unique haplotypes (p = 0.003) in the overall sample. The proportion of unique haplotypes worldwide increased from 31.0% for MHT via 77.8% for Yfiler to 92.9% for PPY23 (r = 0.97). HD showed a similar trend (r = 0.81) whereas MP decreased rapidly with increasing marker number (r = −0.81). Similar trends were observed in the meta-populations defined according to both continental origin and ancestry (Table S5). In summary, an increasing number of markers was found to be associated with an almost linear increase of all forensic parameters used to discriminate among individuals.We compared the haplotype-based forensic parameters for five different sets of Y-STR markers commonly used in forensic practice, namely MHT, SWGDAM, PPY12, Yfiler and PPY23. Not surprisingly, a strictly monotonous relationship emerged among all forensic parameters and the number of markers included in a panel . Both thor PPY23 . Corresp3.5The forensic parameters were compared of Y-STRs that have amplicons shorter than 220 bp and that are included in Yfiler  or PPY23 . A substantially stronger discriminatory power of PPY23 compared to Yfiler was evident for these short haplotypes, mostly due to the higher diversity of PPY23-specific markers DYS576, DYS481, DYS570 and DYS635. In particular, DC and the number of different short haplotypes were nearly twice as high for PPY23 as for Yfiler whereas MP was more than 4-fold smaller .3.6RST > 0.2 for PPY23, p < 10−4). Genetic distances between non-African meta-populations were much smaller although still significant (p < 10−4). The smallest genetic distance was noted for North and Latin America  than pairs of non-African populations or African populations  for all marker sets (data not shown).With an increasing number of Y-STRs included in a marker set, the genetic distances between meta-populations decreased monotonical. However, the Yfiler panel was exceptional in this regard in that it yielded smaller distances than PPY23 for pairs of African and non-African meta-populations, but larger distances than PPY12 for pairs of non-African meta-populations . NeverthRST, separately for the five marker sets, considering either all 129 populations or the 68 populations of European residency and ancestry alone. When assessed for the PPY23 marker panel, Kruskal's stress value showed a clear ‘elbow’ with increasing dimensionality in both population sets, pinpointing an optimal trade-off between explained variation and dimensionality. For the worldwide analysis, two MDS components were optimal with PPY23 whereas four components were deemed optimal for the Europeans-only analysis. Both solutions explained the haplotypic variation well, with R2 = 95.1% in the worldwide analysis and R2 = 99.2% in the Europeans-only analysis. For comparability, MDS analyses for other marker panels were carried out with two or four dimensions, respectively. Haplotypic variation among populations within continental groups was lower than between continental groups . For all five marker sets, the first MDS component clearly separated the African populations from the non-African populations  analysis was performed based upon linearized RST values  (Table S6). In strong contrast, African Americans from Illinois, the Southwest and the whole of the US were quite distant to Africans from Ibadan (Nigeria) . Although likely not to represent the true source population, the distance between a group of Tamil from India and the Texan Gujarati population was as low as RST = 0.008, while the distance between the Tamils and a migrant Indian population in Singapore equalled 0.01. Finally, the distance between European Americans from Illinois, Utah and the whole USA on the one hand, and the Irish on the other was found to be consistently small . A similar trend applied to other European source populations and to European migrant populations in South America. Thus, Argentineans of European ancestry from Buenos Aires, Formosa, Mendoza and Neuquen showed virtually zero genetic distance to Spaniards from Galicia .Finally, the question was addressed of how closely related selected source and migrant populations might be in terms of their extant Y-STR haplotype spectra. A comparison between Han Chinese from Colorado (USA) and Han Chinese from Beijing, Chengdu (both China) and Singapore, respectively, yielded non-significant PPY23-based 4®Y23 kit  were compiled and analyzed. As expected, PPY23 provided higher discriminatory power for forensic purposes than other marker sets in our data. Remarkably, in almost one third of the populations studied, each sample could be identified unambiguously because all haplotypes in the population were unique. Most of the non-unique haplotypes were detected in populations that either passed through a recent bottleneck  were much more variable. This difference is clearly due to the high mutation rates of four of the six markers specific to PPY23 selected for a short amplicon length. Thus, it is likely that the PPY23 kit will greatly improve the analysis of aged or otherwise damaged DNA samples.The present study revealed a considerable number of null and duplicated alleles that were caused either by non-allelic homologous recombination between paralogous DNA sequences Previous population genetic analyses consistently revealed that Y-chromosomal haplotypes have a highly non-uniform geographical distribution characterized by less variation within, and more variation between, population groups than autosomal markers RST, between Africa and the four non-African meta-populations were of similar magnitude. These results confirm a previous study of 40,669 haplotypes from 339 populations typed only for the nine markers of the MHT panel For all five forensic marker sets studied here, samples of African ancestry were clearly separated genetically from all other continental meta-populations. Pairwise genetic distances, measured by Europe was the most intensively sampled continent in the present study and made up ∼60% of the overall sample size. A separate MDS analysis of samples of European residency and ancestry recapitulated the outcome of previous studies with smaller marker sets It must be emphasized that this population genetic analysis was based upon marker sets that were designed for forensic purposes, and that shared several markers. That all five sets yielded a similar picture of the geographic distribution of Y-STR haplotypes may therefore indicate that, in terms of population structure, the effects of markers included in the MHT  dominate those of more mutable markers, such as PPY23-specific STRs DYS576, DYS570 and DYS481. Indeed, it has been shown recently that haplotypes comprising only rapidly mutating markers lack strong signals of population history .®Y23 system provides unprecedented discriminatory power for forensic applications but at the same time shows similar patterns of population structure as established forensic marker sets. These patterns coincide well with groupings according to prior information on geographical and ethnic origin. In some cases, relatively large genetic distances were found for pairs of migrant and potential source populations, such as African Americans and autochthonous Africans. For forensic casework involving these populations, separate reference databases need to be established and used. On the other hand, populations showing small genetic distances, such as Western or Eastern Europeans, Arabs from Iraq and Lebanon or Mestizos from Peru and Bolivia may be merged into meta-populations for the purpose of reference databases. The annotated PPY23 data used in this study have been fully integrated into the YHRD database as of October 2013 .In summary, the recently introduced PowerPlex


Littorina saxatilis, occupying contrasting habitats exposed to either wave action or crab predation. This divergence is associated with barriers to gene exchange but, nevertheless, genetic variation is more strongly structured by geography than by ecotype. Using approximate Bayesian analysis of sequence data and amplified fragment length polymorphism markers, we show that the ecotypes are likely to have arisen in the face of continuous gene flow and that the demographic separation of ecotypes has occurred in parallel at both regional and local scales. Parameter estimates suggest a long delay between colonization of a locality and ecotype formation, perhaps because the postglacial spread of crab populations was slower than the spread of snails. Adaptive differentiation may not be fully genetically independent despite being demographically parallel. These results provide new insight into a major model of ecologically driven speciation.Parallel evolution of similar phenotypes provides strong evidence for the operation of natural selection. Where these phenotypes contribute to reproductive isolation, they further support a role for divergent, habitat-associated selection in speciation. However, the observation of pairs of divergent ecotypes currently occupying contrasting habitats in distinct geographical regions is not sufficient to infer parallel origins. Here we show striking parallel phenotypic divergence between populations of the rocky-shore gastropod,  Gyrinophilus), a model of continuous gene flow during divergence was supported over an alternative that allowed for a period of allopatry  or by “outlier” analysis  and Lysekil  in Sweden, Dunbar  and Thornwick  in Britain and Burela  and Silleiro  in Spain. At each locality, we collected in both the “wave” and the “crab” habitats, spacing samples along the shore to avoid collecting related individuals. We worked with females to avoid confusion with the related species axatilis . Thirty-n = 26–30 per ecotype per location) were analyzed using 11 landmarks positioned on the digitized shell image following Each snail was photographed with a Leica MZ12 stereoscopic microscope and Leica digital ICA video camera. The presence of shell scars was noted, indicating that the individual had survived a crab attack . We predFor each individual, we measured centroid size (CS) and shape, using relative warps (RW). The relative warps were computed using the software packages TpsDig and TpsRelw , 2006, eWe performed a three-way analysis of variance (ANOVA) on size and shape variables, with fixed factors region  and ecotype , and locality as a random factor nested within the interaction between fixed factors. We used a G-test to compare scar frequencies between ecotypes and regions.−1.Head–foot tissue was used for DNA extraction, using a CTAB protocol . DNA conL. saxatilis partial mtDNA sequence . Sixteen individuals were sequenced for each ecotype in each locality.Primers designed from the annotated Littorina Sequence Database , elongation factor 1 α (ElFac), and thioredoxin peroxidase 2 (ThioPer) genes (Table S1). Each gene was amplified in 16 individuals per ecotype from each locality, polymerase chain reaction (PCR) products were cloned and inserts sequenced (see Table S1 for details).The candidate nuclear genes were chosen from the Database . Three ehttp://darwin.uvigo.es). Polymorphism estimates, neutrality tests, and other population genetic analyses were conducted in DnaSP 4.0 , aligned with ClustalX  and inspnaSP 4.0  or ArleqnaSP 4.0 . To obtanaSP 4.0 .Sequence data for nuclear loci were processed using the software Geneious Pro version 5.1.7 . Primers and vector were trimmed and sequences were aligned with the clustalw2 algorithm . All seqCal, ElFac, and ThioPer.The distinct alleles present in each alignment were identified with Arlequin. For each gene, the occurrence of no more than two alleles per individual was checked to identify PCR artifacts. Doubtful individuals were first amplified, cloned and sequenced a second time, and ultimately discarded if ambiguities could not be resolved. Finally, only one allele was kept per individual and per gene. For all further analyses, the exonic regions were trimmed and all indels were deleted. The final alignments were composed of 192, 187, and 189 sequences of length 376, 314, and 341bp, respectively for D and Fu's Fs tests, implemented in Arlequin, and the significance was assessed with 10,000 simulations. A sequential Bonferroni correction for multiple tests was applied to the neutrality tests. In addition, a recombination test was performed for each gene with IMgc Online .The haplotype and nucleotide diversities were estimated in Arlequin. To obtain summary statistics for the ABC analysis, we used the Kimura 2-parameter model for consistency with our simulated data. The neutrality of the nuclear loci was verified with the Tajima's c Online . Recombiion 1.21  and editCal HG792757–HG792783, ElFac HG792716–HG792756, and ThioPer HG792699–HG792715. The mtDNA fragment corresponds to GenBank Accession AJ132137, starting at position 710. A haplotype file for individuals studied here is available at Dryad: doi:10.5061/dryad.m186r.Nuclear sequence data have been submitted to GenBank with accession numbers: EcoRI and PstI) to minimize homoplasy  was carried out in a final volume of 11.6 μL 1× Eco buffer (New England Biolabs) containing 100 ng of genomic DNA and 3 μg of bovine serum albumin. Samples were digested for 3 h at 37°C. Then, 5.5 μL of 1× ligation buffer  containing 0.5U of T4 DNA ligase (Invitrogen), 0.9 μM Eco-adaptor, and 0.9 μM Pst-adaptor were added to the digestion reaction. Samples were ligated for 16 h at 16°C. Ligation reactions were diluted 1:4 and used as template for preselective PCRs. Six different selective PCRs were performed and 12 primer combinations were obtained (Table S1). Preselective and selective PCR conditions, electrophoresis, and scoring are described in All 32 individuals per locality and ecotype were used in AFLP analysis. Profiles were generated with two rare-cutter enzymes (omoplasy . The methttp://www.maths.bris.ac.uk/∼mamab/stuff/). Dfdist input files were created using the AFLP convert program (http://webs.uvigo.es/acraaj/tools.htm) and analyses were carried out following FST values and individual pair-wise relatedness, which was used to create multidimensional scaling plots, following the methodology of Outlier analysis was performed using the program Dfdist , we also used equal prior distributions. Prior distributions were log uniform except for mutation rates and time proportions , which were uniform, and ranges are given in Table S3.−8 per base per generation based on a substitution rate of 3% per million years from the fossil record for Littorina species , and arlsumstat to calculate summary statistics. For the AFLP loci, we used fastsimcoal to simulate 462 separate loci, each one with a 20 base sequence, and an in-house program that converted the resulting sequence data into a binary matrix of AFLP alleles and calculated summary statistics from this binary matrix. For each simulated AFLP locus, one 20bp haplotype was chosen at random and designated the “1” allele. All other haplotypes were designated as “0” alleles. AFLP phenotypes were then called assuming that genotypes 11 and 10 correspond to “band present” and genotype 00 corresponds to “band absent.” This process allowed us to simulate the asymmetrical mutation expected for presence and absence alleles at AFLP loci and the fact that loci fixed for the absence allele are not observed. Calculating summary statistics from the simulated phenotype matrix made them directly comparable to the summary statistics obtained from the real data.We used tsimcoal  to simulD, π, and ΦST based on Kimura 2-parameter distances between sequences, implemented in Arlequin/arlsumstat 3.5. For the AFLPs, we used heterozygosity, mean FST, standard deviation of FST across loci, and Jaccard distance. Because our summary statistics were numerous , we used the partial least squares (PLS) method described in Summary statistics used for the sequence data (for each sample or sample pair) were Tajima's Separate sets of PLS components were defined for simulations under each different model because of the variation in parameters. These PLS components were used to transform the summary statistics of the entire dataset of simulations, as well as the observed summary statistics, prior to the estimation stage. After retaining the closest 0.5–2% of simulations to the observed data based on PLS components, we took two approaches for the “regression adjustment” step, depending on whether we were interested in model comparison or parameter estimation. For model comparison, we used all summary statistics to perform postsampling adjustment, using the GLM method of P-values .For combined datasets, we retained the closest 10,000 simulations (1%) to the observed data based on the Euclidean distance between PLS-transformed observed summary statistics and PLS-transformed simulated summary statistics, and used these retained summary statistics to estimate the parameter values that best reproduce the real-world data using the ABC-GLM procedure of P-value was used as a measure of goodness-of-fit.Models were compared using Bayes Factors, which are equal to the ratio of the marginal densities between models, and posterior probabilities, which are approximately equal to the marginal density of the model of interest divided by the sum of the marginal densities of all models. The In addition to these tests, we also compared the distribution of summary statistics of retained simulated datasets to the summary statistics of the observed dataset, to check that the observed dataset lay well within the distribution of simulations. We did this for distributions of both PLS  and raw summary statistics (one variable at a time). The condition was satisfied for all reported models.To validate our model choice, we simulated 1000 (new) datasets from the original priors for each competing model and used these pseudo-observed datasets to test the robustness of discrimination between models . To validate our parameter estimates, we used the 1000 pseudo-observed datasets that were generated under each model to check for uniformity of the posterior quantiles .L. saxatilis shell size and shape, from two sites in each of three regions, showed remarkable concordance in the direction of phenotypic differentiation between samples from crab- and wave-dominated habitats . This difference was also greatest in Sweden . The northern locality in Spain (Burela) showed much higher diversity than the southern locality (Silleiro) whereas all other localities showed diversity similar to Burela. In no case was there strong differentiation between ecotypes (Table S2) and ecotypes were not differentiated overall (AMOVA by ecotype: ΦCT = 0). These patterns are consistent with previous observations  revealed lower overall diversity than mtDNA . Neutrality tests did not reveal departures from expectations for any of the three introns in any locality. As for mtDNA, there was evidence for differentiation between regions but not between ecotypes .Sequence data from introns in three single-copy nuclear genes (FST = 0–0.027) relative to differentiation among localities (FST = 0.021–0.134), and the highest genetic distances were between Spanish and northern European localities (FST = 0.107–0.134). As for the other marker types, overall differentiation was strong among regions but not between ecotypes .After quality checking and removal of loci with poor repeatability, the AFLP dataset included 614 loci. We excluded 152 of these loci that showed evidence for an influence of divergent selection between ecotypes within any locality. Fewer of these outliers were observed in Britain, where morphological differentiation was also less marked than in the other regions. There was slightly greater sharing of outliers between localities and between regions than expected by chance Table and Spanin situ in each region or locality, or to a single, older common origin whose genetic signal has been obscured by subsequent gene exchange. Ideally, data from all markers should be combined to answer this question. Therefore, we formalized the two alternative models  and fell within the range of postrejection simulated values (both untransformed and PLS), and the distributions of posterior quantiles did not show strong departures from uniformity, indicating a lack of bias in parameter estimation , low migration rates between localities (m∼10−5), and long times since the first population separation ∼105 generations . The parallel divergence models estimated the time since ecotype formation to be ∼104 generations, that is with long times between colonization and ecotype formation, and low migration between ecotypes (∼10−6 per generation). These estimates are referenced to a fixed mtDNA mutation rate .Parameter estimates for the within-region models using data for all markers had wide 95% highest posterior density intervals (Table S3). However, the median estimates from the parallel divergence model were consistent across regions in suggesting large effective population sizes . The robustness of discrimination between models was slightly greater in this case . In the parallel divergence model, local population sizes were again estimated to be ∼104, except where the southern Spanish Silleiro site was involved where estimates were greater (∼105). These models allowed the possibility of population expansion associated with ecotype formation but the posterior distributions did not support this (Table S3). Migration estimates between ecotypes were similar to the within-region estimates (∼10−6 per generation) but migration between sites in different regions was, as expected, estimated to be lower (10−6.5–10−8), with the lowest values involving the Silleiro site which appears be more distinct from the northern regions than the other Spanish site (Table S3). Averaging parameter estimates across comparisons (excluding Lysekil–Thornwick), the between-region models suggest separation of British and Swedish populations ∼6 × 105 generations ago and separation of northern from Spanish populations ∼1.4 × 106 generations ago, whereas ecotype separation is estimated to be much more recent (∼5 × 103 generations from Britain-Sweden comparisons and ∼2.6 × 104 generations from northern Europe–Spain comparisons), as for the within-region models (∼104 generations).To investigate ecotype origin at the regional level, we applied the same two models to the combined marker data for each of the possible between-region pairs of localities in turn Fig.B. We didL. saxatilis in response to contrasting crab and wave environments, both between localities within regions and among European regions. Our analyses of genetic data support the hypothesis that the ecotypes arose in parallel, without allopatric separation and after colonization of the different regions and localities, rather than divergence being old and predating colonization. This support depends on the effect on neutral loci of the barriers to gene flow between populations in the contrasting environments that are associated with phenotypic differentiation. It remains to be seen whether the alleles underlying adaptive traits evolved in parallel.Our results show strikingly parallel phenotypic differentiation in Our data confirmed previous observations, based on mtDNA, of greater differentiation between Spain and northern Europe than between Britain and Sweden and of a large genetic distance between the two Spanish sites . Reid etL. saxatilis population sizes had expanded recently using their mtDNA markers (∼10×). They inferred large population sizes and, as in our analyses, their IMa model fits implied gene flow between geographical regions. As in L. saxatilis changed during the Pleistocene glacial cycles but that the effective population size remained consistently large, partly as a result of gene flow over large distances. Littorina saxatilis is cold-tolerant and now has a distribution extending into the Arctic. Therefore, its populations may not have been impacted as severely as some species by the glaciations, resulting in early colonization of northern European shores with limited population expansion and allowing long-term separation from southern European populations.Our models gave little support to past population expansion, even suggesting a reduction in population size in Spain. Our analyses support the parallel divergence of ecotypes over the alternative old divergence model Fig., that isLittorina (FST reaches about 0.05) but those barriers that do also appear rapidly.Under the parallel model, the separation of ecotypes was in every case estimated to be recent relative to the separation of populations in different localities (itself presumably reflecting patterns of colonization). In the within-regions models, the time to ecotype separation was about 10% of the age of the local populations whereas for between-region models it was an even smaller fraction (1–2%), as expected. If the actual time of colonization of British and Swedish coasts was after the most recent glacial retreat  has a current northern distribution limit well south of the northern limit of L. saxatilis, whereas Pachygrapsus marmoratus (predator in Spain) is a relatively warm-water species. Alternatively, following local extinctions . Thus, the available evidence strongly suggests that the crab and wave ecotypes of Littorina fabalis, also point in this direction  species. Following Nm, may reflect large population sizes rather than high gene exchange (m). This is important because divergence under selection requires s > m, whereas divergence under drift requires low Nm. FST for loci putatively under divergent selection between crab and wave exposed habitats in Britain. They found that the estimated strength of selection was too high to be compatible with the apparently very small genomic regions of elevated differentiation. Using estimates obtained here (m ∼ 10−6 between morphs) implies much weaker selection on the outlier loci (s ∼ 10−3), which is more consistent with the observed genomic pattern of differentiation.Speciation is typically a protracted process during which many changes in geographic distribution, population size, and opportunity for gene flow are likely to occur . Gene exL. saxatilis ecotypes most likely diverged in the presence of gene flow and are certainly now maintained despite gene flow. The ABC analyses, combining information from multiple markers of different types, suggest that the ecotypes have originated repeatedly in different localities. This provides a firm foundation for understanding the genetic basis of divergent adaptation and the nature of other barriers that impact on patterns of gene flow across the genome.We conclude that the