Taenia saginata is the most common human Taenia in Thailand. By cox1 sequences, 73 isolates from four localities in north and northeast were differentiated into 14 haplotypes, 11 variation sites and haplotype diversity of 0.683. Among 14 haplotypes, haplotype A was the major (52.1%), followed by haplotype B (21.9%). Clustering diagram of Thai and GenBank sequences indicated mixed phylogeny among localities. By MJ analysis, haplotype clustering relationships showed paired-stars-like network, having two main cores surrounded by minor haplotypes. Tajima's D values were significantly negative in T. saginata world population, suggesting population expansion. Significant Fu's Fs values in Thai, as well as world population, also indicate that population is expanding and may be hitchhiking as part of selective sweep. Haplotype B and its dispersion were only found in populations from Thailand. Haplotype B may evolve and ultimately become an ancestor of future populations in Thailand. Haplotype A seems to be dispersion haplotype, not just in Thailand, but worldwide. High genetic T. saginata intraspecies divergence was found, in contrast to its sister species, T. asiatica; among 30 samples from seven countries, its haplotype diversity was 0.067, while only 2 haplotypes were revealed. This extremely low intraspecific variation suggests that T. asiatica could be an endangered species. Taenia saginata and T. solium. A third species, T. asiatica, is an additional source of intestinal infection in a number of Asian countries. Cattle are the most common source of T. saginata infection, while the most common cause of both T. solium and T. asiatica infection is swine. T. solium metacestodes, called Cysticercus cellulosae, reside in the animal's muscle, whereas T. asiatica metacestodes, called C. viscerotropica,  parasitize the liver. In its evolution, speciation of Taenia appears to be linked primarily to host switching among carnivore definitive hosts [Taenia and humans is thought to have developed about 10,000 years ago, coincidental with the development of agriculture and the domestication of food animals, like cattle and pigs [Taenia tapeworms by humans happened independently twice, both times by host switching from carnivore definitive hosts to primate definitive hosts [T. saginata + T. asiatica, and the other is T. solium. Geographical distribution has been extensively modified by European exploration and colonization since the 1500s and by ongoing globalization of agriculture and the changing patterns of human migration [Human taeniasis occurs worldwide. It is caused by ve hosts . This asand pigs . Contracve hosts . One is igration .T. solium revealed two separate groups: Asian and African/Latin American genotypes [T. solium has been found to be minimal [T. solium. On the other hand, cattle, an intermediate host of T. saginata, are found in herds in a wide range of pastures. With different farming methods of this intermediate host, the population structure of T. saginata requires investigation.Understanding the genetic population structure of parasites helps to elucidate parasite transmission patterns and develop control measures . The popenotypes . Intrasp minimal , 8. Livec oxidase subunit I (cox1) genes of mitochondrial DNA have been commonly used for studying phylogenetic relationships among taeniid cestodes. Distinct intraspecific variations have been detected among various species, for example, Echinococcus granulosus [E. multilocularis [T. taeniaeformis [cox1 genetic variation within a human Taenia species. A minor variation was observed in one isolate of T. saginata from Kenya and Poland [ cox1 and nuclear rDNA 28S sequences. In this study, we focused on the genetic variation of T. saginata among samples collected from various geographical localities in north and northeast Thailand, where this parasite is highly prevalent among local inhabitants [T. saginata specimens in Thailand have never been conducted. Indeed, the genetic structure among populations of this species and its evolution in Thailand and throughout the world remains limited. Our aim, using partial sequencing of the mitochondrial cox1 gene, was to examine intraspecific variations and the population genetics of T. saginata in Thailand. The genetic divergence of the sister species, T. asiatica, from Thailand was also considered.Cytochrome anulosus , E. multocularis , and T. aeformis . Howeverd Poland , which cabitants , 14. In-Parasites were collected during the years 2009-2010 from four sites in north and northeast Thailand. The two sites in the north—one in the lowlands, the other in the upland hill tribe communities—are both in the Thung Chang district, Nan province, an area on the northern border with Lao PDR. The two sites in the northeastern region are in two different provinces: Ubon Ratchathani and Khon Kaen. Ubon Ratchathani shares borders with both southern Lao PDR and northern Cambodia. Khon Kaen lies closer to the center in the upper half of Thailand .T. saginata was collected from both Taenia egg-positive persons and from persons who spontaneously discharged gravid proglottids. The worms were identified morphologically as T. saginata by scolex and/or gravid proglottids. Male infection rates were almost double females, at a ratio of 43 : 23. The ages of the infected individuals ranged between 12 and 83 years. The worms were fixed in 70% ethanol for molecular analysis. This study was approved by the Ethics Committees of the Faculty of Tropical Medicine, Mahidol University, and the Ministry of Public Health, Thailand. Informed consent was obtained prior to subject participation. T. asiatica, previously collected from Kanchanaburi Province [T. saginata. The cox1 sequences of 33 T. saginata and 18 T. asiatica from various different countries in the world, deposited in the GenBank database . The PCR amplicons were amplified using two oligonucleotide primers: cox1 (forward), 5′-CATGGAATAATAATGATTTTC-3′, and cox1 (reverse), 5′-ACAGTACACACAATTTTAAC-3′. These primers were designed from the alignment of T. saginata and T. asiatica mitochondrial cox1 genes . PCR amplicons were produced in 50 μL of reaction mixture, containing: 10 ng genomic DNA; 0.5 µM of each primer; and 1x TopTaq Master Mix Kit  . Amplification conditions were as follows: initial heating at 94°C for 3 min, followed by 30 amplification cycles, consisting of denaturation at 95°C for 30 sec, annealing at 53°C for 30 sec, and elongation at 72°C for 50 sec. PCR products were run into 1.2% agarose gel and visualized with a UV illuminator. The PCR amplicons were purified and sequenced by dideoxytermination method, using an ABI3730XL sequencer and BigDye v 3.1  at Macrogen Inc. . DNA sequences were aligned using the BioEdit program, version 7.0 [Partial proglottid fragments of individual strobila were separated and washed with distilled water to remove any ethanol remaining from the fixation process. The genomic DNA of each worm was extracted using a Genomic DNA Mini Kit  per the manufacturer's instructions. DNA was resuspended in 50 sion 7.0 . There icox1 gene population genetics of T. saginata samples from four different localities was analyzed. Genetic diversity values, including polymorphic sites between populations (S), haplotype numbers (h), haplotype diversity (Hd), nucleotide diversity (π), theta-w  (θw), and theta-π (θπ) estimators to measure DNA polymorphism [D test and Fu's Fs test [The 924 bp morphism –19, weremorphism  and the morphism , 22. TheFs test .cox1 sequences were aligned by ClustalX version 2.0 [p-distance model by MEGA version 5.0 [cox1 haplotypes was illustrated by Network 4.5.1.6 Software (Fluxus Technology Ltd. (http://www.fluxus-engineering.com/)). The T. saginata world populations  and northeast  Thailand  sequences confirmed that they were all T. saginata (GenBank accession nos. JN986646 to JN986718). The 924 bp cox1 sequences of these samples were divided into 14 discrete groups, represented as haplotypes A–N, and revealed 11 segregation (polymorphic) sites (S). Percentage intraspecific variation was 1.2%, with 1–5 nucleotide substitutions , was 0.683 ± 0.05; the nucleotide diversity (π) was 0.00146 ± 0.00017.  θw was greater than θπ. Tajima's D test of neutrality showed no significant value  in these samples. A significant Fu's Fs value was, however, revealed   and 23 polymorphic sites (S) were revealed. Entire intraspecific variation was 2.5%. Mean haplotype diversity (Hd) was 0.686, and mean intrapopulation nucleotide diversity (π) was 0.00155. θw was greater than θπ (T. saginata in GenBank data for Thailand(7) and the other countries analyzed (China(2), Korea(1), Japan(1), Indonesia(2), Nepal(1), Ecuador(1), Brazil(2), Ethiopia(1), and Belgium(1)). The nucleotide sequence of Haplotype B was unique among the Thailand isolates, that is, 16 samples in this study and 3 from GenBank. The frequency of Haplotype A was 53.8% (57/106 samples); the frequency of Haplotype B was 17.9% (19 from 106 samples). Our sample sequences of Haplotype C–N and the GenBank sequences of Haplotype O–Q were unique among the Thailand isolates  were observed  . The coxdatabase  were inc than θπ . The nucisolates . Some saisolates . In the observed .T. asiatica, significant differences between T. asiatica and T. saginata were discovered. The 924 bp cox1 gene sequences of the 12 isolates from Kanchanaburi Province, Thailand, were all identical. Among 30 samples from seven different countries—China (4), Taiwan (1), Korea (2), Japan (3), the Philippines (1), Indonesia (3), and Thailand (16)—only two haplotypes of the cox1 gene were found, where the major haplotype comprised 29 samples. Only one sample from China had other haplotypes, and only one polymorphic site was found. Haplotype diversity Hd was 0.067. However, the value of nucleotide diversity π of each of the two species was very low , northeast (n = 25), and west (n = 8)  of Thailand were mixed together. Intraspecific variations in cox1 genes have been reported among T. saginata samples from a number of different localities, including China, Ethiopia, France, Indonesia, Korea, Lao PDR, the Philippines, Taiwan, Thailand, and Switzerland. Global genetic divergence was 1.2–1.8% in the nucleotide variant positions of the total 1620 bp length [S, 1.2% displayed the results of isolates from global infection. However, among the worldwide T. saginata populations (n = 106), the Thai isolates (n = 73) and GenBank isolates (n = 33) used in this analysis, a high level of genetic variation  was found. The genetic diversity values, haplotype diversity (Hd), and nucleotide diversity (π) found in populations in this study and in other combined populations were similar.In this study, we investigated the population variation of p length , 27. In T. saginata world population. In the T. saginata sequences, 19 of 33 (57.6%) from GenBank were identical to Haplotype A. Consequently, the star-like expansion in the MJ network of the major haplotype confirms Haplotype A as an ancestor among the T. saginata world population. It also suggests that the subpopulation of minor haplotypes recently experienced a significant increase from its ancestors. Haplotype B and its star-like expansion network were unique to the Thailand isolates. Indeed, it is possible for Haplotype B diverged genetically to be the recent common ancestor of T. saginata in Thailand. However, the major Haplotype A contained half (n = 57) of the total population we analyzed. This means that T. saginata Haplotype A may share its genetic ancestry with populations from a variety of different geographical areas, in Asia as well as those in other continents. θπ less than θw indicates purifying selection, which results in selective removal of the deleterious allele in the population [D value is significantly negative in the world population but not in the Thailand population. The significant Fu's Fs value revealed in isolates from both Thailand and the world population, however, suggests that the population is growing and is hitchhiking due to population expansion and selective sweep [T. saginata populations worldwide.By MJ network, Haplotype B showed connectivity to Haplotype A in the pulation . Tajima've sweep . The samT. asiatica is the third Taenia tapeworm of humans and is reported only in Asian countries. It is distributed in specific areas across several countries, including Taiwan [T. saginata and T. asiatica diverged from other human tapeworms about one million years ago (Mya), 0.414–1.616 Mya. T. saginata lineages emerged at an earlier period than T. asiatica. Lineages of T. saginata emerged at 238,000 years, while that of T. asiatica were at 41,000 years [T. asiatica has been observed. Identical partial cox1 gene nucleotide sequences have been found in 5 isolates (366 bp) from unspecified areas of Taiwan [T. asiatica suggests populations of T. asiatica tapeworm to be small. The prevalence of T. asiatica is low when compared to T. saginata infections in most countries [D value revealed no gene flow in T. asiatica, a result which indicates obvious differences in the population structures of T. saginata and T. asiatica. To date, since the emergence of T. saginata and T. asiatica, cattle have been known to be the intermediate hosts for T. saginata and swine for T. asiatica. The livestock management of these two intermediate host species has been different, and this may be an impact factor for these parasites. Pigs are raised feeding in restricted shed areas; gene flow among parasite populations in pigs, therefore, is diminished. In cattle farming, especially in Thailand, the animals are herded by grazing on naturally grown pastures, particularly pastures of postharvest rice which often cover wide distances. Furthermore, such cattle are frequently untransported, while daily herds are moved to a main city slaughterhouse over a relatively long period of time. The chances of cattle coming into contact with contaminated Taenia eggs, whether from human carriers during grazing or whilst drinking stream water from place to place, remain. This supports no specific locality in T. saginata populations for each genotype. It may even suggest that T. saginata tapeworm populations migrated during host cattle farming. Gene flow among T. saginata may have been influenced by host population migration. The difference in population genetics found in T. saginata suggests that intraspecies populations are growing. On the other hand, its sister species, T. asiatica, reveals very low genetic diversity. Such low divergence may indicate a loss of potential adaptive alleles for surviving in a changing environment, which could lead to the overall reduction of T. asiatica populations.g Taiwan , Korea [g Taiwan , China [g Taiwan , Vietnamg Taiwan , Indonesg Taiwan , and Thag Taiwan . It is e00 years . Very lof Taiwan ; 17 isolf Taiwan ; and 12 f Taiwan . The lowountries –37. AlsoFasciola spp. Ichikawa et al. [nad1 gene in 88 adult Fasciola flukes from three localities in Myanmar and found 27 substitution sites that yielded 20 haplotypes. A major haplotype revealed 54.6% (48/88 flukes) frequency and was seen in all three areas regardless of locality. The intraspecies genetic variation in Fasciola spp. is thought to have been introduced to Myanmar through ancient anthropogenetic movements of domestic ruminants. This seems to be the main factor determining mixing of the parasite population. Likewise, cattle host movements suggest intraspecific genetic variations of T. saginata populations in our study. Despite the fact that cattle serve as the definitive host for Fasciola spp. but the intermediate hosts for T. saginata, this status of intermediate host and definitive host does not influence different genetic variation of parasite species.Cattle also act as definitive hosts for the liver fluke a et al.  investigT. saginata adult worm isolates from humans, from two locations in the north and two provinces in northeast Thailand, exploited intraspecific genetic variability, without correlation with the geographical region of origin. The phylogenetic network of cox1 sequences revealed 14 haplotypes from 73 samples. Thirty-three sequences from GenBank were added, and 23 haplotypes were exploited among the 106 samples. The genetic divergence of world T. saginata populations was 2.5%. Two main haplotypes, A and B, showed connectivity between them. Haplotype A seems to be an ancestor of T. saginata in the world population. Haplotype B and its dispersion are unique to the Thailand population. Intensive studies and a greater number of samples from different geographical areas are required to clarify the population genetics of T. saginata both in Thailand and worldwide.Our work shows that


Hyla japonica to understand patterns of variation in these traits across Korea and provide clues about the underlying forces for variation.In a species with a large distribution relative to its dispersal capacity, geographic variation in traits may be explained by gene flow, selection, or the combined effects of both. Studies of genetic diversity using neutral molecular markers show that patterns of isolation by distance (IBD) or barrier effect may be evident for geographic variation at the molecular level in amphibian species. However, selective factors such as habitat, predator, or interspecific interactions may be critical for geographic variation in sexual traits. We studied geographic variation in advertisement calls in the tree frog H. japonica in three breeding seasons from 17 localities including localities in remote Jeju Island. Call characters analyzed were note repetition rate (NRR), note duration (ND), and dominant frequency (DF), along with snout-to-vent length.We recorded calls of The findings of a barrier effect on DF and a longitudinal variation in NRR seemed to suggest that an open sea between the mainland and Jeju Island and mountain ranges dominated by the north-south Taebaek Mountains were related to geographic variation in call characters. Furthermore, there was a pattern of IBD in mitochondrial DNA sequences. However, no comparable pattern of IBD was found between geographic distance and call characters. We also failed to detect any effects of habitat or interspecific interaction on call characters.H. japoinca. Although we did not detect effects of habitat or interspecific interaction, some other selective factors such as sexual selection might still be operating on call characters in conjunction with restricted gene flow.Geographic variations in call characters as well as mitochondrial DNA sequences were largely stratified by geographic factors such as distance and barriers in Korean populations of  When a species has a wide distributional range relative to its dispersal capacity, geographic variation in a trait may be explained by the balance of gene flow and selection Selection, which varies temporally and spatially, is also an important evolutionary force for broad-scale geographic variation in a trait. Demonstration of a selective factor for geographic variation in a trait requires identification of the factor and differential response of the trait to the factor. When a trait is under selection, a predicted pattern of the trait may be discontinuous between different states of a selective regime. In addition to gene flow and selection, genetic drift might play a role in shaping geographic distribution in traits. However, the effect of genetic drift on phenotypic traits is difficult to study, because its effect may be weak in large populations Many studies of genetic diversity have concluded that gene flow is critical for geographic variation in amphibians Physalaemus pustulosus in central Costa Rica, geographic variation in population structure and advertisement calls were attributed to habitat difference in conjunction with lack of movement between lineages In most amphibian taxa, males produce advertisement calls that are attractive to conspecific females Hyla japonica in the Republic of Korea to understand evolutionary forces shaping the variation. The genetic diversity using mitochondrial COI gene sequences was also estimated to understand the population structure of H. japonica. We specifically tested two hypotheses about geographic variation in call characters. In the gene flow hypothesis, we predicted a pattern of IBD or the barrier effect on geographic variation in a call character as well as genetic differentiation. In the selection hypothesis, we predicted a discontinuous pattern on geographic variation in call characters relative to different states of habitat and degree of population overlap .We studied geographic variation in advertisement call characters of the tree frog Hyla japonica, in strict accordance with the recommendations of the Animal Behaviour Society H. japonica is not listed as an endangered species and, in fact, is one of the most abundant frog species in Korea.The Institutional Animal Care and Use Committee, which oversees animal experimentation at Ewha University, was not established when this study was conducted. However, we treated our study subject, Hyla japonica, one of the most common frog species in Korea, is distributed widely in East Asia, ranging from Manchuria to southern China, and from Mongolia to Japan. From May to August in Korea, H. japonica aggregates to breed in places that hold water, such as rice paddies and wetlands. Males produce species-specific calls that attract conspecific receptive females. In addition to H. japonica, there is another tree frog species, H. suweonensis, which is endemic to Korea. H. suweonensis is known to be distributed in small areas in the Gyeonggi Province , the Korean golden frog (R. plandyi chosenica), the bullfrog (Lithobates catesbeianus), and the narrow-mouth frog  in Korea. The breeding seasons of these species largely overlap, but the advertisement calls are species-specific.Province  [33]. ThH. japonica from 17 localities in the Republic of Korea. Geographic information and abbreviations of these localities are found in H. japonica may be classified into two types in this study: rice paddies  and non-rice paddies . In Korea, rice paddies are temporary wetlands where rice seedlings grow from May to October every year. They contain water usually between May and August and provide habitats for frogs and aquatic insects. Rice paddies are basically open, flat fields without any obstacles to sound transmission. By contrast, the topography of non-rice paddy habitats is generally more complex. Two localities on Jeju Island (J2 and J3) are surrounded by trees and shrubs. J1 is in a pond of a Buddhist temple and is in the vicinity of low buildings and other obstacles. SE is in a bush near a stream. In addition to the quality of sound transmission, the two habitat types may differ in pesticide use. Pesticides are applied regularly during the rice-growing season in rice paddy localities, whereas use of pesticides is unlikely in non-rice paddy localities. However, J1 might be exposed to pesticide use from nearby lawn or crops. We classified SN as a non-rice paddy habitat, even though this locality contained rice paddies. This locality was surrounded by pine and oak trees that are typical of deciduous forests in central Korea. There were four rice paddies, arranged like steps vertically, ranging in size from about 10 to 35 m2. Rice paddies and the pond were maintained not for crop production but to provide habitat for fireflies. Thus, agricultural chemicals were strictly prohibited in this wetland. Although rice seedlings were planted at the beginning of early June, weeds become dominant in the rice paddies by late August.We recorded advertisement calls of H. japonica. Two localities, DY and SC, are located in the southwestern region, and five localities, GN, UJ, AD, CN, and US, are located in the eastern region, east of the Taebaek-Sobaek Mountain ranges. The remaining seven localities are located in the western region of the mainland Korea.The Korean Peninsula is mostly mountainous and not arable. Lowlands lie along the coasts, particularly along the West Sea and South Sea. Along the East/Japanese Sea, the Taebaek Mountain Range runs north and south as a continuous ridge with an average height of about 1,000 m . The easH. japonica at three localities on Jeju Island, which is the largest island in Korea. This island was created entirely from volcanic eruptions approximately 2 million years ago and is dominated by Halla Mountain with a peak of 1,950 m. The vegetation of this island ranges from subtropical forests at the base of the mountain to alpine plants near the peak of the mountain with increasing altitude. J1 was a lawn, and J2 was a pond in the Jejudo Seogipo Natural Recreation Forest that contained trees of humid subtropical and humid continental climates. J3 was a highland wetland. The minimum distance between localities there was 4 km, that is, between J2 and J3 in Jeju Island, whereas the minimum distance between localities on the mainland was 28 km.In addition to localities in the mainland, we recorded calls of H. japonica usually called on the ground or tree branches, not in the water. After recording, we captured the recorded males and put them individually in plastic containers (7×7×4.5 cm) for morphological measurements. We measured the snout-to-vent length (SVL) of each individual, which was defined as the length from the tip of snout to the cloacal opening, to the nearest 0.1 mm, using digital calipers . For a tissue sample for independent species identification and population genetic structure, we clipped one of the frog's toes using sharp scissors. After toe clipping, antibacterial ointment was applied to the cut toe to prevent infection. The tissue sample was preserved in 90% alcohol. Based on DNA analysis, we verified that all male frogs recorded for this study were H. japonica. After morphological measurements and tissue collections were made, we released the captured frogs at their original localities, usually the day after recording.We recorded frog calls between dusk and midnight. All calls were recorded with one of two shotgun microphones , which was placed as close to singing males as possible. Output from a microphone was fed into one of two flash-memory recorders  with a 44.1-kHz sampling rate and 16-bit resolution. Calls were recorded for at least one min. One individual frog contributed to only one recording. During each recording night, we recorded the ambient air temperature and relative humidity at the time of the first and last recording using a thermo-hygrometer , which was placed 10 cm above the ground near the recording sites. We measured air temperature instead of water temperature in this study, because H. japonica is a train of notes  with body size as a response variable and locality as a predictor variable. We did the same analysis twice: first, using all samples, and second, using the mainland samples only, excluding samples from the three Jeju Island localities. A multiple regression analysis was used to understand the effects of spatial variables such as latitude, longitude and elevation on body size.H. japonica populations into four groups: western, southwestern, eastern and Jeju Island. The western group included CC, SE, IC, SN, SW, JC and CJ, and the eastern group contained GN, UJ, AD, CN and US. The southwestern group included DY and SC, and the Jeju Island group had three localities, J1, J2, and J3. To examine the effects of geographic barriers on call characters, we used multi-way multivariate analysis of covariance (MANCOVA) with response variables NRR, ND and DF. Barrier and locality were fixed factors, with locality nested within barrier. Covariates were SVL, temperature and humidity. We first tested the assumption of slope parallelism In Korea, geographic barriers such as mountain ranges and open sea may separate H. suweonenisis occurs sympatrically with H. japonica in Gyeonggi province  and allopatric localities . We limited the analysis of patry to the western group only, because some call characters of the western group differed from those of other groups  was performed on residuals for three call characters. Habitat was a predictor variable. In addition, province . Likewis see the . All staWe used four groups of the tree frog populations  as a priori grouping for the population genetic analysis. The population genetic study was conducted on samples of all but one locality, CJ . BecauseCOI (cytochrome oxidase subunit I) gene was amplified by polymerase chain reaction (PCR) using the primer pair LepF1 (5′-ACC AAT CAT AAA GAT ATT GGT-3′ ) and LepR1 (5′-CCT CTG GGT GTC CGA AAA ATC A-3′) COI sequences were aligned with AlignIR software  and then were trimmed manually. A total of 339 COI sequences were aligned using ClustalW, implemented with MEGA ver. 4.0 software COI data were assessed through Tajima's D and Fu's Fs statistics using Arlequin ver. 3.5.Genomic DNA was isolated from the tree frog toe samples using LaboPass™ Tissue Mini kits  following the manufacturer's protocol. A partial fragment of 657 base pairs (bp) from the mitochondrial The relationship between geographic distance and genetic distance between localities was tested by the partial Mantel test of matrix association. The geographic distance was calculated as the shortest distance between two localities using geographic coordinates. In addition, we also tested the relationship between geographic distance and difference in call character using partial Mantel tests. To estimate differences in call characters, we used the predicted values of the MANCOVA with barrier, locality, temperature, humidity, and SVL as predictor variables and covariates. The difference in a call character was the absolute difference in values of a call character between two localities. Mantel tests were performed with 1,000,000 permutations for both the full dataset and the mainland-only dataset.H. japonica measured as SVL ranged from 25.2 to 46.3 mm with a mean (± SD) of 33.9±3.58 mm. The distribution of SVL deviated from the normal distribution  . That is, frogs from the three Jeju Island localities were larger than frogs from the 14 mainland localities, making the distribution of SVL skewed to the right (Body size of f = 324) . Body sihe right . BecauseF16, 307 = 28.287, P<0.001; F3, 320 = 76.427, P<0.001; r2 = 0.417) was significantly affected by longitude . For the mainland-only dataset, locality was still a significant factor for SVL . However, SVL with exclusion of the Jeju Island samples  was only affected by longitude . Latitude  and elevation  were not significant factors for SVL.Using the full dataset, body size significantly differed among localities , between barrier and temperature , and between barrier and humidity . Thus, the assumption of slope parallelism was met. For the full dataset, the result of the MANCOVA without interaction terms showed that locality was a significant factor for all three call characters, adjusting for SVL, temperature and humidity  was a significant factor for DF, but not for NRR or ND. When SVL was set to 33.93 mm, temperature to 21.58°C and humidity to 93.18%, the estimated marginal means of DF were 3047±15 Hz in the western group, 2903±17 Hz in the eastern group, 2965±22 Hz in the southwestern group, and 2862±30 Hz in the Jeju Island group. There were significant differences in DF for every pair of groups , except between the eastern and Jeju Island groups (P = 0.271). Therefore, mountain ranges and open sea may have acted as geographic barriers to the tree frog species. Likewise, for the mainland-only dataset, barrier was  a significant factor for DF, but not for NRR or ND , the average nucleotide difference among individuals was very low (π = 0.0088±0.0047), falling below the general intraspecific level of divergence. The high haplotype diversity resulted in a complex haplotype network, shaped by several major haplotypes connected by numerous singular hypotypes , western and southwestern (0.0134), western and Jeju Island (0.011), eastern and southwestern (0.0093), eastern and Jeju Island (0.0079), and southwestern and Jeju Island (0.0071). The average value among individuals within each population (0.0055) was lower than that between groups (0.0098), indicating low gene flow between populations.Overall, the geographic distribution of the haplogroups appeared to be congruent with the hypothetical grouping of populations into four regions bounded by mountain ranges and sea. Pairwise comparisons of population ΦCT = 0.35; P<0.001) and among populations . It was also significant among groups  and among populations  for the mainland-only dataset, indicating apparent population genetic structures of the tree frog, H. japonica, in Korea.When partitioned into three hierarchical levels according to the four hypothetical groups , the highest proportion of genetic variation resided in the comparison of individuals within populations (50.8%), followed by among groups (35.2%), and was very low between geographically clustered populations within groups (14%) . The genCOI sequences was evaluated with two statistical tests, Tajima's D and Fu's Fs. Both tests revealed statistically significant negative values , suggesting a possible demographic population expansion of H. japonica in Korea during the recent past.The relationship between genetic and geographic distance showed a statistically very significant correlation both for the full dataset and for the mainland-only dataset , indicatH. japonica. The most dominant feature in the geography of the Korean Peninsula is the Taebaek Mountain Range, which runs north and south along the East Sea. During the three-year recording period, we explored the Taebaek Mountains to elevations over 1,000 m to record H. japonica, but this effort was not successful. Areas with elevation over 1,000 m in the Taebaek Mountains might not be suitable for H. japonica, and it is probably difficult for this species to disperse cross the mountain range. However, we recorded the calls of H. japonica in a locality with the elevation over 1,000 m in Jeju Island. Because Jeju Island is located in lower latitudes, its climate is much milder than those of the high ridges in the Taebaek Mountains. Although there is no empirical data about the dispersal ability of H. japonica, the Taebaek Mountains may act as a geographic barrier to its dispersal, resulting in longitudinal variation in call characters.The strongest findings for the gene flow hypothesis about geographic variation in call characters were the barrier effect on DF and longitudinal effect on NRR. Major mountain ranges and open sea may explain the geographic variation in DF and NRR of H. japonica. Significant genetic differentiation among groups indicated that mountain ranges and open sea may act as barriers to gene flow in H. japonica. Furthermore, the result of significant genetic differentiation among populations was consistent with the pattern of an IBD effect. Because amphibians are generally philopatric H. japonica in Korea.The population genetic structure was largely consistent with the findings of geographic variation in call characters of The tree frog populations in Jeju Island seemed to be distinct from the mainland populations in terms of body size, call characters and population genetics. There was a longitudinal effect on DF for the full dataset, but this effect disappeared for the mainland-only dataset after Jeju Island samples were excluded. In addition, the altitudinal effect on DF and NRR differed between in the full dataset and the mainland-only dataset. The two highest elevations in our study were localities on Jeju Island, and the tree frogs on Jeju Island were generally larger and had lower dominant frequencies than those on the mainland. Thus, a strong altitudinal effect may be responsible for the distinct variation in body size and DF of the tree frog populations in Jeju Island. Being separated since the Pleistocene glacial period, the island and mainland populations may have diverged by vicariance COI sequences in an uncorrelated manner H. japonica in Korea.Although we detected a pattern of IBD in a mitochondrial gene, no such corresponding pattern was observed between geographic distance and call characters. We doubt that lack of correlation between geographic distance and call characters was a result of inadequate sampling, as there was a significant IBD effect on the mitochondrial gene. The effect of IBD might have differently influenced call characters and mitochondrial H. japonica in Korea. However, this study was not designed to systematically examine habitat effect on call characters, and formal studies should be conducted with detailed measurements of habitat characteristics. Properties of acoustic signals are differentially distorted depending on the environment through which the signals are transmitted Our analyses did not indicate any habitat effect on variation in call characters of Anthropogenic interference such as noise or pesticide use may influence signal production and transmission in animal communication systems. For example, frogs in a mixed-species assemblage modulated call rates or suppressed calling behaviors in response to airplane flyby or motorcycle noise in central Thailand H. suweonensis. There is growing evidence that reproductive interaction between closely related species is a selective factor for geographic variation in the communication system of frogs H. japonica and H. suweonensis are genetically segregated to the level of two separate species H. suweonensis were not characterized, it is still premature to conclude that there is no pattern of reproductive character displacement in call characters of H. japonica.We also found no difference in any call character between areas of sympatry and allopatry in relation to Calopteryx splendens, a similar pattern was found: significant IBD for the molecular data, but not for the phenotypic traits C. splendens was largely the consequence of interpopulational sexual selection. In many frog species, male advertisement calls are targeted by both intra- and inter-sexual selection Another possible explanation for the lack of relationship between geographic distance and call character is that male advertisement calls may be under intense sexual selection pressure. In 12 populations of a sexually dimorphic damselfly D and Fu's Fs tests on population COI data showed a significantly high negative value, indicating sudden population expansion during the recent past. The star-like mtDNA haplotype network (Pelophylax nigromaculatus, in China was reported P. nigromaculatus in this region could not be assessed. Based upon the assumption that the Pleistocene climatic fluctuations had a pervasive influence on historical species distribution in East Asia, the present molecular genetic signature of recent demographic expansion of H. japonica provides an additional clue that past glacial events had widespread influence on the evolution and distribution of terrestrial amphibians in East Asia.Tajima's  network  resembleH. japonica, which is widespread in East Asia. Analyses of field recordings suggested that call characters were largely stratified by geographic distance and by barriers such as mountain ranges and open sea. These findings were consistent with the population genetic structure based on mitochondrial DNA sequences. Although we did not detect effects of habitat or interspecific reproductive interaction, some other selective factors such as sexual selection might be still operating on call characters of H. japonica in Korea, in conjunction with restricted gene flow.In conclusion, studies of genetic diversity using neutral molecular markers show that patterns of isolation by distance or barrier effect may be evident for geographic variation at the molecular level in amphibian species. However, selective factors such as habitat, predator, or interspecific interactions may be critical for geographic variation in sexually selected traits. We studied the effects of gene flow and selection on advertisement calls of


ST  = 0.011) was detected between ocean-type populations of Pacific herring in the North West Pacific, except for a population in the Yellow Sea (ΦST  = 0.065). A moderate reduction in genetic diversity for both mtDNA and microsatellites in the Yellow Sea likely reflects founder effects during the last colonization of this sea. Reciprocal monophyly between divergent mtDNA lineages (ΦST  = 0.391) across the Alaska Peninsula defines the discontinuity across the North Pacific. However, microsatellites did not show a strong break, as eastern Bering Sea (EBS) herring were more closely related to NE Pacific than to NW Pacific herring. This discordance between mtDNA and microsatellites may be due to microsatellite allelic convergence or to sex-biased dispersal across the secondary contact zone. The sharp discontinuity between Pacific herring populations may be maintained by high-density blocking, competitive exclusion or hybrid inferiority.Pacific herring show an abrupt genetic discontinuity in the central North Pacific that represents secondary contact between refuge populations previously isolated during Pleistocene glaciations. Paradoxically, high levels of gene flow produce genetic homogeneity among ocean-type populations within each group. Here, we surveyed variability in mtDNA control-region sequences (463 bp) and nine microsatellite loci in Pacific herring from sites across the North Pacific to further explore the nature of the genetic discontinuity around the Alaska Peninsula. Consistent with previous studies, little divergence (Φ Untangling the influences that historical events and present-day processes have on phylogeographic patterns in marine species is a major focus of phylogeographic research Here, we focus on a deep geographic partition between populations of Pacific herring in the central North Pacific that has previously been detected with allozyme Surveys of molecular variability among populations have found only small differences between ocean-type populations within the NE and NW Pacific The goal of the present study was to use mitochondrial (mt) DNA and microsatellite DNA markers to examine various aspects of population structure in ocean-type populations of Pacific herring across the North Pacific. These two marker types have different mutation rates and enable us to detect contemporary gene flow and deeper separations between populations. Relatively smaller mutation rates for mtDNA allow the reconstructions of population events reaching back several thousand years, and larger mutation rates for microsatellites give insights into contemporary levels of gene flow. One objective was to investigate the population structure of herring in the NW Pacific, and in particular to estimate the relationship of herring in the Yellow Sea to other NW Pacific herring. If present-day populations in the Yellow Sea were established by a small number of founders, these populations may show reduced levels of genetic diversity and departures from neutrality. A second objective was to estimate the population structure of ocean-type herring in the NW Pacific. Previous studies of mtDNA sequence variability All experimental procedures involving fish were approved by the Institutional Animal Care and Use Committee of Ocean University of China (Permit Number: OUC-20120525). This study did not involve endangered or protected species and the sampling locations are not privately-owned or protected in any way.Tissue samples from 341 Pacific herring adults were collected in 2003–2010 at six locations in the NW Pacific, one location in the southeastern Bering Sea, and three locations in the NE Pacific , Fig. 1.5′-ACTCCCAAAGCTAGGATTCTG-3′ (forward) and CP-R: 5′-CTCAATTTGGTTGGTCGGTTC-3′ (reverse), which amplified a 480 base pair (bp) fragment. Polymerase chain reaction (PCR), purification of PCR product, and sequencing were carried out according to protocols in J.-X. Liu et al. www.dnastar.com) and deposited in the GenBank (Accession numbers JN796250-JN796404) (Table S1).Genomic DNA was extracted following Sambrook et al. Ten microsatellite loci were amplified with primers developed by Olsen et al. I = 0.60 and gamma shape parameter α = 0.66. A 95% confident parsimony haplotype network was constructed with TCS 1.15 Molecular diversity indices were estimated with ARLEQUIN 3.1 FCT and ΦCT in ARLEQUIN. Several models of population structure, based on adjacent-sample pooling, were considered to assess population structure. Genetic breaks between populations were identified by finding the largest significant values of FCT or ΦCT between population groups and the smallest and non-significant values of FSC and ΦSC among populations within these groups.Genetic divergence between populations was estimated with an analysis of molecular variation (AMOVA) FS0) and after (θ1) population growth, and a compound variable representing the time since population expansion, τ = 2ut, where u is the mutation rate over the whole sequence and t is the time since population growth or geographical expansion Deviations from neutrality were tested with Fu's FCT and ΦCT) between samples and geographical distance with Mantel's test for correlation between difference matrices in the online program IBDWS (isolation by distance web service at http://ibdws.sdsu.edu/~ibdws/) r) between genetic and geographical distances was larger than 0.0 was estimated with 1000 randomizations. Genetic distances, or geographical distances, or both, were log transformed to attempt to improve correlations. A log transformation of geographical distances tested for IBD on large geographical scales, and a log transformation of genetic distances tested for IBD at large values of genetic distance.Isolation by distance was tested by comparing standardized genetic distance values  equal to 2000 fish. The results of the simulations indicated a rapid increase in power with larger values of FCT  We used several models of population structure and AMOVA in ARLEQUIN to identify genetic breaks among populations. Pairwise population Pro gene at 5′ end was removed, 463 bp CR sequences were obtained for 342 specimens. These sequences were polymorphic at 110 nucleotide sites (76 parsimony informative sites), with 93 transitions, 28 transversions and six indels, which defined 155 haplotypes (113 singletons). Haplotype diversity ranged from h = 0.894 (TO) to 0.980 (CW) (mean h = 0.979), and nucleotide diversity (Θπ) ranged from 0.0098 (YS) to 0.0238 (CW) (mean Θπ  = 0.0208) (After a conserved 17 bp portion of the tRNA 0.0208) . Nucleot 0.0208) .FS was highly significant (P<0.001) for each lineage, and was significant for all the samples, except for YS, IB, NH, TO, and CW , and most of the comparisons involving YS were significant (ΦCT  = 0.039–0.108), except between YS and AK. Two comparisons, YS-NH and YS-TO, were not significant after Bonferroni correction.Values of rrection . In the FCT  = 0.029 (P<0.001) overall, but divergence was much larger, ΦCT  = 0.297 (P<0.001), when divergence between haplotypes was considered . The AMOVA for the three NE Pacific samples, detected no significant divergence among three samples extending from Sitka, Alaska to Washington .An AMOVA of haplotype variability among samples yielded an nsidered . This diFCT  = 0.019, P = 0.0001; ΦCT  = 0.027, P = 0.002); however, none of the FCT and ΦCT values between adjacent groups was significant  (P = 0.125) or ΦCT/(1-ΦCT) (P = 0.352)  . Log traFS was significant in samples from SH, NH, AK and WK in the NW Pacific, and in samples SI and SG in the NE Pacific.Additional analyses of the mtDNA sequences provided insights in the historical demographies of Pacific herring populations. The shapes of the sequence mismatch distributions were unimodal in YS and SH, but tended to be multimodal in more northern samples  . HoweverC. harengus, were used to estimate divergences between populations. The average number of alleles per locus (A) over samples ranged from 2.2 alleles for Cpa 110 to 13.6 for Cha 17 (Table S2). Heterozygosities were lower on average among samples from the NW Pacific (HE  = 0.672–0.807) than among those from the NE Pacific (HE  = 0.792–0.811). No geographical trend appeared overall in the levels of microsatellite diversity. However, the lowest levels of diversity appeared in the sample from the Yellow Sea. Ten of 100 locus-by-population tests exhibited significant departures from HWE with heterozygote excess, but none remained significant after correction for multiple tests. Locus Cpa110 was excluded from further analysis because significant linkage disequilibrium appeared between Cpa110 and Cpa108, Cpa110 and Cha17 over all populations.Eight microsatellite loci developed for Pacific herring, and two loci developed for the sister species Atlantic herring, Cpa111 and Cpa102 that indicate possible directional selection  to nine (K = 9) population groups. The largest ΔK appeared under the assumption of two groups. This analysis showed a large genetic discontinuity between NW Pacific and NE Pacific populations, but unexpectedly placed the sample from TO (eastern Bering Sea) into the NE Pacific population group  (FCT) ranged from 0.018 to 0.084 between the six samples from the NW Pacific. Divergences between samples from the NW and NE Pacific were generally larger, ranging from 0.070 to 0.168. In the NE Pacific, a small FCT value (0.036) was observed between SI and CW (FCT  = 0.073 (P<0.001)  and the smallest FSC . The significant FSC value also indicated differences within the two groups. When TO was separated from NE Pacific group, FCT was also large and significant . This echoes the results of the STRUCTURE analysis for K = 3. Less divergence was found between TO and the two samples from NE Pacific group than between TO and the six samples from NW Pacific group (FCT/(1-FCT) and geographical distance appeared between samples in the NW Pacific  and across the whole geographical range  . DivergeI and CW . An AMOVP<0.001) . The paric group . A signiThe results of this study provide insights into two general features of Pacific herring population structure. First, our estimates of population structure in the western North Pacific with control region mtDNA sequences and microsatellites show minimal population structure, except for the divergence of Yellow Sea populations from other NW Pacific herring populations. Second, our results for mtDNA confirm the presence of a genetic discontinuity between the eastern Bering Sea and Gulf of Alaska populations of Pacific herring. However, microsatellite markers show a discordant biogeographic pattern from mtDNA markers, in that Eastern Bering Sea herring are more similar to populations in the Gulf of Alaska than to NW Pacific populations.FCT  = 0.004 about 90% of the time. This level of divergence is much small that the level of divergence between the NW Pacific and NE Pacific genetic groups, and hence our results can provide further insights into the nature of the genetic discontinuity across the North Pacific. A second caveat is that, although we sampled ocean-type herring at several locations in the NW Pacific, we had fewer samples from the NE Pacific. Hence, our focus is on estimating population structure in the NW Pacific. Nevertheless, our array of samples allows us to further examine the genetic discontinuity across the N Pacific.Before discussing these findings in detail, we mention two caveats. First, even though sample sizes for microsatellites were small, these samples sizes were still large enough and the microsatellite marker polymorphic enough to provide power to detect population structure as low as Several oceanic and biological traits potentially influence contemporary levels of population structure. Shoreline complexity on regional scales may isolate populations. Semi-enclosed Okhotsk and Japan seas, numerous island archipelagos and peninsulas, such as the Korean Peninsula, potentially act as barriers to dispersal between populations. The attachment of early embryonic stages to kelps and sessile invertebrates can also reduce dispersal FCT  = 0.019, P = 0.0001; ΦCT  = 0.027, P = 0.002) and microsatellites  showed significant differences among populations in the NW Pacific, but particular comparisons between population groups did not show a clear geographical patterns. Nevertheless, isolation by distance (IBD) was detected for microsatellites , but was absent for mtDNA FCT  and ΦCT . This contrast may be due to differences in the mutation rates between the two markers. The larger mutation rates in microsatellites may allow the detection of short-term isolations between populations. For example, the seasonal movement of herring from offshore overwintering areas to inshore spawning areas In our study, the overall AMOVAs for both mtDNA  and microsatellite (HE  = 0.672 vs HE  = 0.736–0.811). No significant mtDNA differentiations were detected among different samples in previous study The sample from the Yellow Sea was drawn from Pacific herring living at the edge of the species' distribution. These fish had marginally lower genetic diversities for both mtDNA  indicates that post-glacial population expansions occurred at about the same time. In the NE Pacific, however, mismatches for the lineages B and C were larger than those for NW Pacific populations (π = 9.90–11.02), suggesting either an earlier expansion, or differences in metapopulation structure between the two regions. The NW Pacific experiences greater annual and decadal swings in climate and temperature than does the NE Pacific Three deep mtDNA lineages occur in Pacific herring that show control-region sequence divergences of 1.5% to 1.9% from one another n = 21) from the southern shore of the Alaska Peninsula showed intermediate between the two regional groups Previous studies of allozyme markers showed a strong genetic discontinuity between NW and NE Pacific populations The geographic pattern across the North Pacific for microsatellites in the study of O'Connell et al. Molecular genetic differences between the two large groups coincide with morphological and ecological differences between the two groups. Hay et al. The reason for the differences in growth and asymptotic sizes between populations in the NW and NE Pacific are uncertain. Hay et al. The strong mtDNA divergence between NW and NE Pacific populations, together with biparentally inherited microsatellites provide clear dispersal markers. The appearance of fish bearing mtDNA lineage A in the NE Pacific and fish with lineages B or C in the NW Pacific can be taken as evidence for long-distance dispersal across the secondary contact zone. However, the temporal scale of this dispersal is difficult to gauge, but dispersal likely occurs in a stepping-stone fashion over generations. Both males and females carry maternal mtDNA, but only females pass their mtDNA on to offspring, so mtDNA lineages in dispersing males die out in one generation. Dispersing females, however, can pass on out-of-basin haplotypes to their offspring. Future analysis of northern populations by sex may give insights into the frequency of long-distance dispersals and may provide insights into the reproductive successes of migrants.Several species show abrupt genetic discontinuities between populations Several mechanisms have been postulated to account for abrupt genetic discontinuities along more or less continuous marine habitats. One is that established populations resist the immigration of individuals from other populations through competitive exclusion based on adaptive differences between populations Discordance between mtDNA and nuclear markers in a secondary contact zone is a common feature in many species, and may be due to combinations of sex-biased dispersal asymmetries, incomplete mtDNA lineage sorting, adaptive introgression, demographic disparities between population groups In conclusion, Pacific herring are characterized by two parapatric genetically distinct groups that are in secondary contact after divergence in isolation during glacial maxima. Allozyme and mtDNA markers generally show genetic homogeneity among ocean-type populations within regions that has been interpreted to indicate significant amounts of gene flow between populations Table S1Haplotypes distribution of the three lineages in the ten populations.(DOCX)Click here for additional data file.Table S2Levels of genetic variability at ten microsatellite loci in the nine populations of Pacific herring and for all populations pooled.(DOCX)Click here for additional data file.


Zostera marina) forms the foundation of an important shallow coastal community in protected estuaries and bays. Widespread population declines have stimulated restoration efforts, but these have often overlooked the importance of maintaining the evolutionary potential of restored populations by minimizing the reduction in genetic diversity that typically accompanies restoration. In an experiment simulating a small-scale restoration, we tested the effectiveness of a buoy-deployed seeding technique to maintain genetic diversity comparable to the seed source populations. Seeds from three extant source populations in San Francisco Bay were introduced into eighteen flow-through baywater mesocosms. Following seedling establishment, we used seven polymorphic microsatellite loci to compare genetic diversity indices from 128 shoots to those found in the source populations. Importantly, allelic richness and expected heterozygosity were not significantly reduced in the mesocosms, which also preserved the strong population differentiation present among source populations. However, the inbreeding coefficient FIS was elevated in two of the three sets of mesocosms when they were grouped according to their source population. This is probably a Wahlund effect from confining all half-siblings within each spathe to a single mesocosm, elevating FIS when the mesocosms were considered together. The conservation of most alleles and preservation of expected heterozygosity suggests that this seeding technique is an improvement over whole-shoot transplantation in the conservation of genetic diversity in eelgrass restoration efforts.Eelgrass ( Genetic diversity provides the basis for populations to respond and adapt to environmental changes and plays an important role in reducing the extinction risks associated with inbreeding Seagrasses, a group of subtidal and intertidal marine angiosperms, are the foundation species for many coastal communities and provide critical habitat for commercially important fish, migrating and resident waterfowl, manatees, dugongs, and the sea turtles, as well as important ecosystem services such as stabilization of sediments, coastline protection, water purification and clarification Methods that accomplish the twin goals of maximizing genetic diversity and the number of established plants are worth pursuing, especially if the program developed is ecologically viable and economically efficient. To date, restoration methods for eelgrass fall into two main categories: those that rely on clonal expansion through rhizomal growth by transplanting whole shoots, and those that involve collecting, processing and sowing seeds Z. marina bearing maturing seeds are floated above the restoration area in mesh bags until, over time, matured seeds drop to the sediment surface. Compared to seed broadcasting, the method has fewer infrastructure requirements, and since seeds are released over time in a manner mimicking natural processes, it is less susceptible to failure caused by disruptive events. Therefore, BuDS should be a useful tool for small restorations if their use effectively conserves diversity.Large seed-broadcasting efforts require a great deal of labor and infrastructure to collect flowering shoots, float them in tanks until seeds drop, separate seeds from substrate at the bottom of the tanks, store seeds until the time to broadcast them, and coordinate the broadcasting itself. Broadcasting seeds at once also increases the potential that restoration efforts will fail due to chance events like large waves or weather conditions. Reynolds et al. Z. marina for several decades, and encompassing a range of environmental conditions  and life histories  found among the extant populations in the San Francisco Bay  with flow-through seawater at the Romberg Tiburon Center for Environmental Studies, central San Francisco Bay, from late July 2005 through December 2006. Salinities of the bay water near the intake averaged 26 (range 2–32) during this period, which included a very high rainfall spring. During the season of ripe seed release in late July and August 2005, we collected flowering shoots from three genetically differentiated source populations within sites known to be inhabited by isco Bay  [28], [2isco Bay . The botP-values (α = 0.05) to reduce the likelihood of type 1 errors resulting from multiple comparisons.Collection of tissues occurred in June 2006, when individual clones could still be differentiated. Care was taken to ensure that sampling units were from genetically unique individuals (genets) and not clonal shoots (ramets) from the same genet. This was accomplished by sampling seedlings before rhizomes had time to expand and overlap with adjacent neighbors. Therefore, there is high confidence that the samples are unique genets within the mesocosms. We collected 100–500 mg of leaf tissue from shoots in each mesocosm and stored them in 1.5 ml microcentrifuge tubes at −80°C until DNA extraction. DNA was extracted from ∼100 mg frozen tissue from 128 samples  and scored at seven microsatellite loci, as described in P-values were evaluated for significance after sequential Bonferroni adjustment Clonal richness and the number of multilocus genotypes were calculated in Genclone 2.0 FIT), inter-individual differences within a population (FIS), among populations within a group (FSC), and differences among groups of populations (FCT). For the purposes of these tests, each mesocosm was a “population,” and “groups” were defined as (1) all mesocosms that shared both seed source  and treatment (+/− sediment inoculation), creating six groups, or (2) all mesocosms with a common seed source regardless of sediment treatment, creating three groups. Source population data for CB and BFI were obtained from Ort et al. Analysis of molecular variance (AMOVA) K = 3, using prior knowledge of population of origin to assist clustering. Thus, the program used the three source populations to “learn” the population genotypes. We then asked the program to assign to each mesocosm sample a probability of belonging to each of the three clusters, ignoring information about the origin of the mesocosm sample. Other parameters were set at default values with GENSBACK = 1.We investigated whether individuals from the mesocosms could be genetically traced back to the correct source population using a Bayesian assignment test implemented in STRUCTURE Seedlings had established in all 18 mesocosms by March 2006 and shoot densities were monitored monthly through December 2006, as described in Of the 128 samples collected in June 2006, 124 samples were successfully genotyped at seven loci, and four samples yielded six loci. Null alleles and linkage disequilibrium were detected in some loci, but not in the same loci in larger samples from the source populations all genets across all six of the mesocosms for a source population captured the characteristics of the source, so the individual is the replicate unit in these tests. None of the metrics used to examine genetic diversity showed significant differences between inoculated mesocosms and controls . There was no genetic differentiation between individual mesocosms or between inoculated and control groups from the same source (NM = 6) had marginally higher observed heterozygosity (HO) compared to PM (NM = 6)  and BFI (NM = 6) . No significant difference was detected in HO between PM and BFI . Expected heterozygosity (HE) was higher in the CB mesocosms than BFI  and trended higher compared to PM , while HE did not differ between PM and BFI .A summary of genetic diversity-related statistics is given in e source , so theyt = 3.02, P = 0.05).Overall, allelic diversity was low among the mesocosms – only three loci, GA5, CT20 and CT3, had more than one allele present at an overall frequency greater than 0.1. At most loci, PM and BFI mesocosms had one allele at high frequency and one or more alleles at low frequency, although the low frequency alleles were not always the same for both populations. BFI mesocosms were generally the least diverse and were monomorphic at locus CT19. Allelic richness rarified to the smallest sample size revealed differences among the mesocosms only in the CB-BFI comparison  between the mesocosms and their source populations . All three sets of mesocosm samples did show significantly elevated inbreeding coefficients, FIS. Among the source populations, only CB showed a significant departure from Hardy-Weinberg equilibrium (HWE) P1-tailed = 0.047), but this is close to what is expected by chance. No other tests in BOTTLENECK were significant.Reductions in genetic diversity were small using the BuDS technique. There were no significant reductions in the number of alleles sampled, allelic richness after rarefaction, nor ulations . HE alsM = 3 mesocosms per group for 6 groups) was 6.2% (FCT P<0.001), and was 8.5% when grouped only by the three sources . In FST analyses, all three sets of mesocosms grouped by source (NM = 6) were differentiated from each other but not from their respective sources . The genetic composition of the plants in the mesocosms was similar to their respective source populations. Population differentiation, which is pronounced among the source populations  sources . Assignm sources , Table 4Z. marina in mesocosms and maintained genetic diversity at close to the level found at in situ source populations, in terms of expected heterozygosity and allelic richness. In work on the Atlantic Coast, USA, Reynolds et al. Z. marina aids in maintaining genetic diversity and may represent an improvement over exclusively transplanting whole shoots.Restoration of populations for conservation often consists of collecting propagules from an extant population and transplanting them to another location to repair damage from human activity, reestablish an extirpated population, or establish a new one in suitable habitat. The subsequent reduction in genetic diversity in the restored population can range from mild to severe, with potential effects on the long-term success of the project Shearer et al. FIS) was significantly elevated in the mesocosms, indicating a deficiency of heterozygotes. Heterozygosity could be reduced if a large proportion of seeds collected in the field were a result of self pollination Z. marina populations, with evidence for multiple seed paternities within a single spathe FST  are also explained in this manner. Nevertheless, pooling data across mesocosms for some analyses was required because we were interested in how an array of BuDS would capture the diversity of the source populations in a restoration setting and not how individual BuDS units would perform. Finally, the move from a natural environment to a laboratory setting almost certainly involved a change in selective conditions, which could contribute to a shift in allele frequencies. All of these effects could be successfully mitigated in a larger restoration attempt by using multiple BuDS from each donor population and averaging the variance associated among individual BuDS. Mitigating losses in heterozygosity is especially important in San Francisco Bay, where Z. marina heterozygosity levels are already relatively low compared to other populations nearby The inbreeding coefficient . Flowering shoots with approximately 30 seed-bearing spathes from one of the three source populations, PM, CB, or BFI, were placed in a pearl net and floated in a mesocosm of 120 cm diameter and 42 cm depth, containing 10 cm of sand at the bottom, and with flow-through bay water. Six mesocosms were assigned to each of the source populations. Three of the six were inoculated with sediment plugs from the source location (+), and three were non-inoculated controls (−).(DOC)Click here for additional data file.


East Asia harbors substantial genetic, physical, cultural and linguistic diversity, but the detailed structures and interrelationships of those aspects remain enigmatic. This question has begun to be addressed by a rapid accumulation of molecular anthropological studies of the populations in and around East Asia, especially by Y chromosome studies. The current Y chromosome evidence suggests multiple early migrations of modern humans from Africa via Southeast Asia to East Asia. After the initial settlements, the northward migrations during the Paleolithic Age shaped the genetic structure in East Asia. Subsequently, recent admixtures between Central Asian immigrants and northern East Asians enlarged the genetic divergence between southern and northern East Asia populations. Cultural practices, such as languages, agriculture, military affairs and social prestige, also have impacts on the genetic patterns in East Asia. Furthermore, application of Y chromosome analyses in the family genealogy studies offers successful showcases of the utility of genetics in studying the ancient history. East Asia is a sub-region of Asia, with a vast and diverse landscape. About 22% of the world’s population lives in East Asia, and more than 200 languages in seven linguistic families  are spoken in East Asia, which makes this region one of the world's most important places for studying human evolution, genetic diversity and interrelationships between genetics and cultures of human populations.et al. used 19 stable and highly informative Y chromosome biallelic markers to assess the genetic structure of the paternal lineages in East Asia, and suggested that modern humans of African descent replaced the previous hominids living in East Asia[et al. examined 12,127 male individuals from 163 populations using three Y chromosome biallelic markers . They found that all the individuals carried a mutation at one of the three sites-YAP, M89 and M130[In the past few years, molecular anthropologists have made efforts to characterize the genetic diversity of East Asian populations by analyzing three types of genetic materials, that is, autosomal and X chromosomal DNA, the paternal Y chromosomal and maternal mitochondrial DNA (mtDNA). Autosomes and X chromosome are inherited from both the parents and are always jumbled by recombination. The non-recombining portion of the Y chromosome (NRY) is strictly inherited paternally and, therefore, is the best material to trace the paternal lineage of the populations with the additional advantages of small effective population size, low mutation rate, sufficient markers and population-specific haplotype distribution,3. The dast Asia. In 2001and M130. These tand M130. AlthougClimate has played an important role in human migrations, especially in the Last Glacial Period. The Last Glacial Period refers to the most recent glacial period from approximately 110 to 10 thousand years ago, covering the Paleolithic and Mesolithic periods of human history. During Here, we have focused on the migration histories of East Asian populations achieved by studying Y chromosome, and have discussed the patterns and microevolution during the initial human settlement and later migrations and expansions in East Asia. It is noteworthy at the very beginning that most time estimations mentioned in this review were achieved using Y chromosome short tandem repeats (STRs). Although this approach is correct in principle, there are still many ongoing debates about the best way to use STRs in haplogroup dating. In particular, there are two popularly used Y chromosome STR mutation rates, that is, the evolutionary rate,13 and tOnce it became generally accepted that modern humans evolved recently in Africa, the times and routes of migration to East Asia remained controversial. Three different models were insisted upon by different researchers. The first model postulated that northern populations of East Asia migrated to the south, and mixed with the Australian ancestors who had settled in Southeast Asia. The second model suggested that the northern populations of East Asia evolved from the southern settlers. However, a third model assumed that northern and southern East Asian populations evolved independently since the late Pleistocene more than 10,000 years ago,16.There are four dominant Y chromosome macro-haplogroups in East Asia - O-M175, C-M130, D-M174 and N-M231 - accounting for about 93% of the East Asian Y chromosomes  and three Y chromosome STRs in a large collection of population samples from a wide area of Asia. Principal component analysis of their study showed that all northern populations clustered together and were well included in the southern population cluster, and the southern populations were far more diversified than the northern populations. They concluded that the northern populations derived from the southern populations after the initial Palaeolithic peopling of East Asia. They also estimated the age of O3-M122 to be 18 to 60 thousand years using three Y-STRs under the single-step mutation model with a mutation rate of 0.18% per locus every 20 years, which might reflect the age of the bottleneck event leading to the initial settlement of East Asia[et al.[et al. examined the haplogroups O3a2b-M7 and O3a2c1a-M117 in Southeast Asian Mon-Khmer and Hmong-Mien speaking populations, and indicated a unidirectional diffusion through bottlenecks from Southeast Asia into East Asia about 19,000 years ago  during the Last Glacial Maximum[Su ast Asia. In 2005a[et al.. Recentl Maximum. A gener Maximum. TherefoThe age of haplogroup O in East Asia is no more than 30 thousand years when estimated from sufficient numbers (>7) of STR markers. Therefore, haplogroup O was not the earliest Y chromosome carried by modern human into East Asia. Haplogroup C-M130 may represent one of the earliest settlements in East Asia. Haplogroup C has a high to moderate frequency in Far East and Oceania, and lower frequency in Europe and the Americas, but is absent in Africa  and is associated with a short black Asian physical style. Haplogroups E and D are brother haplogroups. While haplogroup E was carried westwards to Africa by the tall black people, haplogroup D might have been carried eastwards to East Asia by the short black people Figure .Haplogroup D-M174 has high frequencies in the Andaman Negritos, the northern Tibeto-Burman populations and the Ainu of Japan, and also appears at low frequencies in other East and Southeast Asian and Central Asian populations  from Central Asia to North Asia, and a recent population admixture along the Silk Road since about 3,000 years ago[The subsequent debate focuses on how to interpret the Central Asia- and West Eurasia-related genetic components in East Asia. Zhong ears ago.The genetic patterns in human societies are often influenced by their cultural practices, such as residence patterns and subsistence strategies. Y chromosomes of East Asian populations have played an important role in documenting such influences; for example, relationships among patrilocal populations should have stronger associations with Y chromosomes than with mtDNA. East Asian languages show strong association with paternal lineages of Y chromosomes,25,41,42et al. argued that Atkinson’s claim was only supported when the phonemic diversities were divided into three or five levels. Analyses using raw data without simplification suggest a decline from central Asia rather than from Africa[et al.[et al.[Another interesting topic is about language expansion and Y chromosome distribution patterns, That is, whether modern languages underwent a serial founder effect during their expansion. Atkinson reported a decreased trend of phonemic diversity  from Africa that indicated the African exodus of modern languages. Howeverm Africa, althougm Africa. The dism Africa. Therefom Africa and Hamma[et al.,50; howel.[et al. and Undel.[et al.. Neverthl.[et al..et al. suggested that it may often be the language of the father that is dominant within the family group if the parents have different linguistic backgrounds[The association between languages and Y chromosomes, but not mtDNA, might reflect sex-biased migrations due to patrilocality. Patrilocality refers to the social system in which a married couple resides with or near the husband's parents. Forster kgrounds. Howeveret al. analyzed the genetic pattern of Y-chromosomes and mtDNA variation in 28 Han Chinese populations. According to their data, northern and southern Hans share similar Y chromosome haplogroup frequencies but exhibit more differences in their mtDNA lineages. The substantial movements of the northern immigrants fleeing from warfare and famine have led to the transition in genetic structure in southern China. They concluded that the pattern of the demographic expansion of Han people and the southward diffusion of Han culture was consistent with the demic diffusion model, and males played a bigger role than females in this expansion[Other cultural practices, such as agriculture, military affairs or social prestige, can also have impacts on genetic patterns. The spread of culture is explained by two alternative demographic scenarios: the demic diffusion model, which involves mass movement of people; and the cultural diffusion model, which refers to the movement of ideas and practices rather than people and involves limited genetic exchange between people. For exaxpansion.Population expansion can also be associated with specific social prestige, for example, the increased reproductive fitness as in Genghis Khan’s case. Genghis Khan (1162 to 1227) established the largest contiguous empire in history. He and his patrilineal relatives had numerous offspring due to their high social status, which led to the increase in frequency of their Y lineage. As a result, his Y chromosome  is widespread from the Pacific to the Caspian Sea and makes up an estimated 0.5% of the world’s male population today. Intereset al. collected some present clans with full records of 70 to 100 generations claimed to be descendants of the famous Chinese Emperor CaoCao or Marquis Cao Can, and validated the records by comparing the Y chromosomes. The Y chromosome haplotype of the descendants of Emperor CaoCao (O2-M268) is different from that of the Marquis Cao Can (O3-002611). Therefore, Cao Cao’s claim of aristocratic ancestry from Cao Can, which has been doubted for almost 1,800 years is, therefore, not supported by genetic evidence[Similar to the study of Genghis Khan’s lineage, Y chromosome can be used to study the eminent persons in history. Most people now inherit surnames from their fathers; and similarly, most men also inherit the Y chromosomes from their fathers. Therefore, men sharing the same surname are expected to have similar Y chromosomes. Combined with the study of stemma records, Y chromosome study can reveal the relationships of individuals in ancient history. Wang etevidence. Ancientan O3-0021. Therefet al. sequenced the Y chromosomes of two individuals separated by 13 generations and estimated the human Y chromosome base-substitution mutation rate as 3.0×10-8 mutations/nucleotide/generation[The application of genetics to the studies of the ancient history will continue to grow. In genealogy, for example, it would be useful to investigate the genetic links among clans with the same surname to establish the missing records of family stemma. The deeneration. Deeper The Y chromosome plays an important role in unraveling the entangled history of modern human populations in East Asia. Although many questions remain unresolved, a clear framework of the prehistory has been obtained. Four Y chromosome haplogroups C, D, O and N, accounted for more than 90% of the East Asian Y chromosomes, are suggested to have Southeast Asian origins, carried by three waves of migrations. The distributions of western Eurasia specific Y chromosome haplogroups E, G, H, I, J, L, Q, R and T in northwest China reflect the recent gene flows from the west and the probable northern route migration. A west-to-east decline of these western haplogroups was clearly observed.However, current Y chromosome research in East Asia is limited in two important aspects. The first limit is the poor resolution for those East Asian specific Y chromosome branches, such as haplogroup O-M175. Despite the huge population of haplogroup O, there have been fewer markers defined in haplogroup O than in haplogroups R and E. For instance, three Y-SNP markers, 002611, M134 and M117, represent about 260 million people in East Asia, but downstream markers are far from enough to reveal informative genetic substructures of those populations. The second limit is inaccurate estimation of lineage and population divergence time as mentioned at the beginning.The advent of next-generation sequencing technology made it possible to sequence the entire Y chromosome in numerous human individuals and in deep-rooting pedigrees. For instance, the 1000 Genomes Project Consortium has already sequenced the Y chromosomes at an average depth of 1.83 in 77 males in the low-coverage project, and 15.23 in the two trio fathers. FurtherASD: Average squared difference; Bp: Base pairs; LGM: Last Glacial Maximum; mtDNA: Mitochondrial DNA; NRY: Non-recombining portion of the Y chromosome; SNP: Single nucleotide polymorphism; STR: Short tandem repeat.The authors declare that they have no competing interests.CCW and HL wrote the manuscript and performed graphical work. Both the authors read and approved the final manuscript.Phylogenetic trees of Y chromosomal haplogroup O-M175, C-M130, D-M174, and N-M231.Click here for file