Species delimitation in the green algal genus Codium (Bryopsidales) from Korea using DNA barcoding

Codium, one of the largest marine green algal genera, is difficult to delimit species boundary accurately based on morphological identification only. DNA barcoding is a powerful tool for discriminating species of seaweeds. The plastid elongation factor TU （tufA） is considered as maker to perform DNA barcoding of green algal species than rbcL gene due to universality and rapid evolution rate. We conducted DNA barcoding application to Codium specimens from the Jeju Island, Korea to overcome the limit of morphological identification and to confirm the species diversity. As a result of applying tufA marker, we newly generated fifty-five tufA barcodes to resolve eight species. TufA marker exhibited 6.1%–21.8% interspecific divergences, wider than the gap of rbcL exon 1, 3.5%–11.5%. Molecular analysis of rbcL exon 1 sequences of Codium revealed eight distinct species like tufA analysis separated in five phylogenetic groups. DNA barcoding of the genus Codium using tufA marker is more helpful to overcome the limit of morphological identification, and this is more potential to reveal cryptic species and to resolve the relationships among subspecies than rbcL analysis alone. The complement of tufA barcoding and rbcL analyses including morphology for the genus Codium in the northwestern Pacific will give much more reliable achievement for discovering species diversity and resolving the phylogenetic relationships.

Delimitation of Countries,Regions and Cities Which the Tropic of Cancer Traverses and Its Development Strategies

The central value 23°26′17″ of the Tropic of Cancer is used for delimiting the standard position of the Tropic of Cancer as well as countries, regions and cities which the Tropic of Cancer traverses. It is confirmed that the Tropic of Cancer runs through 19 countries and regions in the world, and it traverses Yunnan, Guangxi, Guangdong, Fujian （sea area） and Taiwan provinces in China. At the same time, regions which the Tropic of Cancer traverses are divided into three regions, namely the extensive Tropic of Cancer region, the Tropic of Cancer region and the core area of the Tropic of Cancer. The series of delimitation and division aims to explore ways of sustainable development under the restriction of resources and environment and build collaborative development areas of the Tropic of Cancer at all leaves, especially setting up the collaborative de- velopment zone of the Tropic of Cancer in China and international collaborative development circle of the Tropic of Cancer.

Delimitation of Grain Production Functional Zones:An Empirical Analysis of Fangzi District in Weifang City

To solve some unreasonable problems in the acceptance process of the delimitation project of grain production functional zones,the acceptance results of grain production functional zones in Fangzi District,Weifang City were elaborated from the aspects of the third party organization,inspectors,expert group,acceptance criteria,acceptance method,acceptance contents,etc.,and the overall performance of the project was reviewed.The problems that need to be rectified during the implementation of the project were put forward,and the irrationality of the acceptance and the places that need to be improved were pointed out,which would provide a better guiding value for the delimitation of grain production functional zone and the acceptance and performance evaluation of similar projects.

Effect of natural prolongation with geological features on maritime delimitation

The United Nations Convention on the Law of the Sea entitled the coastal States to naturally extend the continental shelf, which has caused more drastic disputes of maritime delimitation. This paper devotes to clarifying the significant concept of natural prolongation through an effect method combing the legal principles and technical analysis. Firstly, the classic samples with respect of the development of geological features are traced. Based on these samples, the classification with a model is proposed in order to affirm the concept＇s significance under certain geomorphologic situations. Lastly, scientific analysis is used to present two potential prolongation situations and emphasize that all the technical analysis on maritime delimitation should be complied with international law and protect the common interest of all the mankind.

Influence of uncertainty in delimitation of seismic statistical zone on results of PSHA

The seismic hazard of research area is evaluated by probabilistic analysis method for three different seismic statis-tical zone scenarios. The influence of uncertainty in seismic statistical zone delimiting on the evaluation result is discussed too. It can be seen that for those local sites along zone's border or within areas with vast change of upper bound magnitude among different scenarios the influence on seismic hazard result should not be neglected.

Historical mitochondrial genome introgression confounds species delimitation: evidence from phylogenetic inference in the Odorrana grahami species complex

Species delimitation is essential to informing conservation policy and understanding ecological and evolutionary processes.Most of our recent gains in knowledge on animal diversity rely on morphological characteristics and mitochondrial(mt)DNA variation.Concordant results based on both have led to an unprecedented acceleration in the identification of new species and enriched the field of taxonomy.However,discordances are also found commonly between morphological and mtDNA evidence.This confounds species delimitation,especially when gene flow or mt genome introgression has occurred.Here,we illustrate how mt genome introgression among species of the Odorrana grahami complex confounds species delimitation using the combined evidence of morphological characters,mt variation,and thousands of nuclear single-nucleotide polymorphisms(SNPs)from genotyping-by-sequencing(GBS).Fifty-eight samples across the distribution of the O.grahami complex were included.The mtDNA matrilineal genealogy indicated 2 clades,with O.grahami and Odorrana junlianensis clustered together.In contrast,all nuclear evidence including gene trees,species trees,and genetic structure analyses based on GBS data support 3 species with distinct genetic clusters.These 3 distinct genetic clusters also correspond to distinct morphological characters.They affirm the distinct taxonomic entities of both O.grahami and O.junlianensis,as well as a third clade distinct from either.Which species the third clade belongs to remains unclear and will require further testing.The nuclear genomic loci contradict the COI evidence,with indications of rampant historical mt genome introgression among the species of the O.grahami complex.These discordant signals previously confused species delimitation efforts in this group.Based on these findings,we recommend the integration of independent data,especially nuclear genomic evidence,in species delimitation so as to be robust against the pitfalls of mt introgression.

The BPP program for species tree estimation and species delimitation

This paper provides an overview and a tutorial of the BPP program, which is a Bayesian MCMC program for analyzing multi-locus genomic sequence data under the multispecies coalescent model. An example dataset of five nuclear loci from the East Asian brown frogs is used to illustrate four different analyses, including estimation of species divergence times and population size parameters under the multispecies coalescent model on a fixed species phylogeny （A00）, species tree estimation when the assignment and species delimitation are fixed （A01）, species delimitation using a fixed guide tree （A10）, and joint species delimitation and species-tree estimation or unguided species delimitation （A11）. For the joint analysis （A11）, two new priors are introduced, which assign uniform probabilities for the different numbers of delimited species, which may be useful when assignment, species delimitation, and species phylogeny are all inferred in one joint analysis. The paper ends with a discussion of the assumptions, the strengths and weaknesses of the BPP analysis [Current Zoology 61 （5）： 854-865, 2015].

Molecular phylogeographic analyses and species delimitations reveal that Leopoldamys edwardsi(Rodentia:Muridae)is a species complex

Leopoldamys edwardsi is a species with wide distribution ranges in southern China but is not discussed in stud­ies on geographic variation and species differentiation.We used 2 mitochondrial(Cytb,CO1)and 3 nuclear(GHR,IRBP and RAG1)genes to clarify species phylogeography and geographical differentiation.Maximum likelihood(ML)and Bayesian phylogenetic inference(BI)trees consistently indicated that L.edwardsi is a spe­cies complex containing 3 main lineages with high Kimura-2-parameter(K2P)divergences(i.e.lineages LN,LS and LHN)found in the northern and southern China and Hainan Island,respectively.The 3 species delimitation methods,automated barcoding gap discovery,Bayesian poisson tree process analysis and Bayesian phylogenet­ics and phylogeography,consistently supported the existence of cryptic species.Divergence times among the main lineages were inferred to be during the Pleistocene,with LHN/LS split at 1.33 Ma and LN/(LHN+LS)at 2.61 Ma;the diversifications of L.edwardsi complex might be caused by the rapid uplifts of Tibetan Plateau,paleo­climate change and complex topography.The divergence between LHN and LS was probably related to the sepa­ration of Hainan Island from the mainland via the formation of the Qiongzhou Strait.Lineages LN and(LS+LHN)likely diverged due to the Wuyi-Nanling mountain range forming a dispersal barrier.Our results suggested that L.edwardsi complex contains at least 3 distinct species:LHN represents L.hainanensis,endemic to Hainan Island and previously considered as a subspecies L.e.hainanensis;LS represents a cryptic species distributed through­out the southern Chinese continent;and LN represents the nominotypical species L.edwardsi.

The art and science of species delimitation

DNA-based approaches to systematics have changed dramatically during the last two decades with the rise of DNA barcoding methods and newer multi-locus methods for species delimitation. During the last half-decade, partly driven by the new sequencing technologies, the focus has shifted to multi-locus sequence data and the identification of species within the frame-work of the multi-species coalescent （MSC）. In this paper, I discuss model-based Bayesian methods for species delimitation that have been developed in recent years using the MSC. Several approximate methods for species delimitation （and their limitations） are also discussed. Explicit species delimitation models have the advantage of clarifying more precisely what is being delimited and what assumptions we are making in doing so. Moreover, the methods can be very powerful when applied to large multi-locus datasets and thus take full advantage of data generated using today＇s technologies [Current Zoology 61 （5）： 846-853,2015].

Uprooting phylogenetic uncertainty in coalescent species delimitation： A meta-analysis of empirical studies

Phylogenetic and phylogeographic studies rely on the accurate quantification of biodiversity. In recent studies of taxonomically ambiguous groups, species boundaries are often determined based on multi-locus sequence data. Bayesian Phylogenetics and Phylogeography （BPP） is a coalescent-based method frequently used to delimit species; however, empirical studies suggest that the requirement of a user-specified guide tree biases the range of possible outcomes. We evaluate fifteen multi-locus datasets using the most recent iteration of BPP, which eliminates the need for a user-specified guide tree and reconstructs the species tree in synchrony with species delimitation （= unguided species delimitation）. We found that the number of species recovered with guided versus unguided species delimitation was the same except for two cases, and that posterior probabilities were generally lower for the unguided analyses as a result of searching across species trees in addition to species delimitation models. The guide trees used in previous studies were often discordant with the species tree topologies estimated by BPP. We also compared species trees estimated using BPP and ＊BEAST and found that when the topologies are the same, BPP tends to give higher posterior probabilities [Current Zoology 61 （5）： 866-873, 2015].

Practice and Exploration of the Delimitation and Implementation of Urban Development Boundary in Hangzhou

The urban development boundary is an institutional arrangement of overall planning and regulations of regional urban-rural space and ecological space, an important public policy which ensures the healthy and orderly development of urbanization, and one of the key contents that deepens the reform of the economic system. The delimitation of the urban development boundary is considered as a significant content for urban master plan revision and a fundamental basis for urban space layout and infrastructure layout in Hangzhou. As to the methods of delimitation, from the requirements of overall and long-term regional development, with the idea of "seeking the greatest common divisor," and in line with the layout requirements of population, facilities, and economic activities, the 18 space control elements sorted out according to the laws are integrated to form "one space map" that meets various requirements through interdepartmental negotiation and consultation. It should adhere to the principles including that it should follow related laws, should be implemented in space, should have multi-plan integration, and should be easy in operation. The Hangzhou Municipal Government formulates the Implementation Plan of the Hangzhou Urban Development Boundary, which, in line with the overall requirements of an intensive and highly efficient production space, a livable and moderate living space, and a pleasant and picturesque ecological space, classifies and disposes the present construction outside the urban development boundary, specifies the protection and utilization requirements for the space outside the urban development boundary, promotes the integration of functions inside and outside the urban development boundary, and establishes long-term management mechanisms including formulating regulations, dynamic monitoring, assessments of government performance, and social supervision.

DNA barcoding of fishes from Zhoushan coastal waters using mitochondrial COI and 12S rRNA genes

Accurate species identification is a key component of biodiversity research.DNA barcoding is an effective molecular method used for fish species identification.We aimed to study the DNA barcoding of fish in Zhoushan coastal waters,explore the differences and applicability of two gene fragments(12S rRNA and COI)of DNA barcoding in fish species identification,and established a comprehensive fish barcoding reference database.Two hundred and eighty-seven captured fish samples from Zhoushan coastal waters were identified using morphological characteristics and DNA barcoding.A total of 26412S rRNA sequences(belonging to eight orders,31 families,55 genera,and 66 species)and 188 COI sequences(belonging to seven orders,30 families,48 genera,and 58 species)were obtained.The lengths of the 12S rRNA sequences ranged from 165 to 178 bp,and the guanine-cytosine(GC)content was 45.37%.The average 12S rRNA interspecific and intraspecific genetic distances(K2P)were 0.10%and 26.66%,respectively.The length of the COI sequence ranged 574–655 bp,and the content of GC was 45.97%.The average 12S rRNA interspecific and intraspecific genetic distances(K2P)were 0.16%and 27.45%,respectively.The minimum interspecific genetic distances of 12S rRNA and COI(1.23%and 1.86%)were both greater than their maximum intraspecific genetic distances(2.42%and 8.66%).Three molecular analyses(NJ tree,ABGD,and GMYC)were performed to accurately identify and delineate species.Clustering errors occurred when the 12S rRNA sequences were delimited using the NJ tree method,and the delimitation results of ABGD and GMYC are consistent with the final species identification results.Our results demonstrate that DNA barcoding based on 12S rRNA and COI can be used as an effective tool for fish species identification,and 12S rRNA has good application prospects in the environmental DNA(eDNA)metabarcoding of marine fish.

Continent-wide vocal leapfrog pattern in Collared Scops Owls obfuscates species boundaries

Much confusion has surrounded the taxonomy of the Collared Scops Owl(Otus bakkamoena)complex,distributed widely across tropical and subtropical Asia.Often divided into three species,modern taxonomies disagree on its treatment,as a lack of deep mitochondrial DNA divergence is seemingly in contrast with patterns of vocal differentiation.Analysing a dataset of territorial calls of 122 individuals across Asia,we identified a vocal leapfrog pattern in which owl populations at the eastern and western ends of the distribution sound more similar to each other but are separated by different-sounding populations in between.The boundaries between vocal types are not congruent with traditional species boundaries.Our data allow for a re-drawing of species boundaries and reveal that Collared Scops Owls follow a biogeographic pattern rare in Southeast Asian birds.However,this pattern is shared with several other owl complexes in that peninsular Malaysian populations form part of a continental Asian species centred further north in Southeast Asia rather than forming part of a Sundaic species centred in the adjacent Indonesian Archipelago.

Approach to Delimiting Metropolitan Regions' Boundary and Grading Urban Hierarchy Within a Metropolitan Region——A Case Study of Shanghai Metropolitan Region

In metropolitan regions, the change in the strength of "flows" between a core city and surrounding cities re-flects the range of the core city's influence, while the gravity between core city and other cities reflects the strength of potential relation between them. This article firstly attempts to delimit the boundary of metropolitan regions with the two dimensions measure combining "flows" and gravitation. The former is measured through the flow of people be-tween the core city and surrounding cities, and the latter is measured through both population and gross domestic products (GDP) of the core city and surrounding cities. The hierarchy of the cities within a metropolitan region is classified in order to emphasize the roles of the cities belonging to the metropolitan regions, different from the general way through population scale and administrative level, and is typical in China. This paper uses the Shanghai metropolitan region as a research case, determining boundary of this metropolitan region clearly and classifying hierarchy of the cities within the region. The final results are significantly different to previous work, even overthrowing the traditional system of urban hierarchy partly. It is helpful to highlight the function of cities in organizing the regional economy, the level structure of metropolitan regions, and each city's relative importance in a metropolitan region, which can be taken as scientific basis for planning integrated regions or urban systems.

Geological Anomaly of China

Geological anomaly is a geological body or geological body combination different from its surroundings in composition, structure, or genetic sequence. If there is a value (value interval ) that works as a valve representing the background field, exceeding or lower than the valve value,it is called a geological anomaly. Geological anomalies can be divided into high geological anomaly and low geological anomaly .The former shows the area that is more complex in composition, Structure or genetic sequence than its surroundings; while the latter just gives an opposite indication, i. e. much simpler. According to their distribution and delimitation markers, geological anomalies can be classified into (1)global geological anomaly; (2)regional geological anomaly; (3) local geological anomaly; (4) minor geological anomaly and (5)micro-geological anomaly .The methods for delimiting geological anomalies include (1)crustal elevation-subsidence coefficieat (G value ) determination; (2) geological complex coefficient (C value ) determination; (3) entropy (H value ) determination; (4) geological similarity coefficient (S value ) determination and (5 ) geological correlation coefficient (R value ) determination. As the result of the movements of Eurasian, Pacific and Indian Plates, the Chinese geological anomalies, including 11 regional ones and 32 main local ones, most Paleozoic in age, are distributed in strip on the margin or the outer and inner folded zones in the east and west arcs of the block symmetric arc tectonic system (BSATS), which controls the distribution of various minerals in China. Thus, geological anomaly theory is of great significance in searching for large, super-large and very large ore deposits.

Morphological study and molecular epidemiology of Anisakis larvae in mackerel fish

Objective:To examine and study the morphology,epidemiology,and molecular phylogeny of Anisakis larvae in blue mackerel[Scomber australasicus(Cuvier,1832)]and Indian mackerel[Rastrelliger kanagurta(Cuvier,1816)]using light microscope,scanning electron microscope,molecular phylogeny,and species delimitation methods for confirmation and investigation of Anisakis species and their evolutionary relationship.Methods:A total of 90 fish(45 per species)were purchased from a department store in Chiang Mai,Thailand.Anisakis samples were investigated for morphological characteristics using light and scanning electron microscopes.Molecular phylogeny and species delimitation methods based on the cox2 gene were performed.Results:The prevalence,mean intensity(Mean±SEM),and mean abundance of Anisakis larvae(Mean±SEM)in blue mackerel were 77.78%,6.74±1.320,and 5.24±1.107,respectively,and in Indian mackerel,these values were 13.33%,2.50±0.764,and 0.33±0.159,respectively.Scanning electron microscopy showed the detail of morphological characteristics and provided the different shapes of mucron and excretory pores in Anisakis larvae congruent with the phylogenetic tree.The species tree was congruent with the phylogenetic tree.Conclusions:The prevalence,mean intensity,and mean abundance of Anisakis larvae were higher in blue mackerel.To the best of our knowledge,this is the first time that Anisakis pegreffii was found in blue mackerel in Thailand.The phylogenetic tree also supported the morphological data of Anisakis larvae.However,species delimitation based on cox2 revealed 1-3 possible cryptic species in this genus.Anisakis spp.contamination of fish products is unpleasant and a health concern considering human infection with larvae(anisakiasis)arises.

Phylogeography and Cryptic Species Diversity of Paramesotriton caudopunctatus Species Group(Salamandridae: Paramesotriton) in Guizhou, China

The Paramesotriton caudopunctatus species group is mainly distributed in the karst mountain ecosystems of Guizhou, China. Although some species have been included in previous phylogenetic studies, the evolutionary relationships and divergence-time of members of this species group as a whole remain unexplored. In this study, we report the sequencing of one protein coding mitochondrial gene fragment(ND2) and one nuclear gene(POMC), and use a combination of phylogenetic analyses and coalescent simulations to explore the cryptic diversity and evolutionary history of the P. caudopunctatus species group. Phylogenetic relationships revealed that the P. caudopunctatus species group is composed of two major groups, i. e., East Clade and Western-South Clade. The divergence-time and ancestral area estimation suggested that the P. caudopunctatus species group likely originated in the Doupeng Mountains in Guizhou, China at 12.34 Ma(95% HPD: 8.30–14.73), and intraspecific divergence began at about 2.17 Ma(95% HPD: 1.39–2.97). This timing coincides with the orogenesis of the Miaoling Mountains during the Late Miocene to early Pleistocene. The delimitation of species in the P. caudopunctatus species group supports the existence of the currently identified species, and consensus was confirmed across methods for the existence of least to two cryptic species within what has been traditionally considered to be P. caudopunctatus species group. This study is of significance for understanding the species formation, dispersal, and diversity of the tailed amphibians in the karst mountains ecosystem of Guizhou and the role of the Miaoling Mountains as a geographical barrier to species dispersal.

DNA barcoding of the family Sparidae along the coast of China and revelation of potential cryptic diversity in the IndoWest Pacific oceans based on COI and 16S rRNA genes

Sparids are of considerable economic importance in marine fishery and aquaculture in China, and the species diversity of this group is considered relatively high. However, the accurate species identification and delimitation of sparids in China remain unaddressed. In this study, we used mitochondrial cytochrome oxidase subunit ?(COI) and 16S ribosomal RNA(16S) genes to conduct DNA barcoding and species delimitation in eleven sparid species from the coastal waters of China. Based on Kimura-2 parameter genetic distances, the mean intraspecific/interspecific variation for COI and 16S were calculated as 0.004/0.152 and 0.002/0.072, respectively. All the conspecific individuals formed monophyletic clusters in neighbour-joining trees of both markers. An obvious barcoding gap was detected for each species, and a common genetic threshold of 1.3% sequence divergence was defined for species delimitation in both markers. Although the sequence variation of 16S was generally lower than that of COI, the results indicated that sparid species could be ef fectively and accurately identified and delimited by COI as well as 16S. Thus, we propose that the COI gene serve as the standard DNA barcode for sparids, and that the 16S gene could also be an ideal candidate barcode. Moreover, each of the six sparid species( Argyrops spinifer, Rhabdosargus sarba, Dentex hypselosomus, Acanthopagrus latus, Acanthopagrus australis and Acanthopagrus berda) showed high intraspecific divergence(>1.3% genetic threshold) with the remarkable geographic lineages in the Indo-West Pacific oceans, which supported that potential unrecognized cryptic species were in them. The potential cryptic diversity revealed here might be primarily attributed to the allopatric divergences caused by the long-term geographic isolation between the Indian and West Pacific oceans or between the opposite sides of the Indian Ocean. The results further suggest that a revision of taxonomic status of these species is required, followed by development of a biodiversity conservation strategy.

Multiple lines of evidence supports the two varieties of Halenia elliptica(Gentianaceae)as two species

Delimiting species requires multiple sources of evidence.Here,we delimited two varieties of Halenia elliptica(Gentianaceae)using several lines of evidence,including morphological traits and mating system in a sympatric population,phylogenetic relationships based on nrITS and cpDNA(rpl16)data,and complete chloroplast genome sequences.Comparative analysis of 21 morphological traits clearly separates the two varieties of H.elliptica.Examination of the flowering process and pollination treatments indicate that H.elliptica var.grandiflora produces seeds via outcrossing,whereas H.elliptica var.elliptica produces seeds via mixed mating.Furthermore,hand-pollinated hybridization of the two varieties produced no seeds.Observations of pollinators showed that when bees began a pollination bout on H.elliptica var.grandiflora they preferred to continue pollinating this variety;however,when they began a pollination bout on H.elliptica var.elliptica,they showed no preference for either variety.Phylogenetic analysis confirmed the monophyly of H.elliptica,which was further divided into two monophyletic clades corresponding to the two varieties.A large number of variants from the chloroplast genomes reflected remarkable genetic dissimilarities between the two varieties of H.elliptica.We recommend that the two varieties of H.elliptica should be revised as two species(H.elliptica and H.grandiflora).Our findings indicate that H.elliptica varieties may have split into two separate species due to a shift in mating system,changes in flowering phenology and/or post-pollination reproductive isolation.

Discussion about the relationship between seismic belt and seismic statistical zone

This paper makes a summary of status of delimitation of seismic zones and belts of China firstly in aspects of studying history, purpose, usage, delimiting principles, various presenting forms and main specialties. Then the viewpoints are emphasized, making geographical divisions by seismicity is just the most important purpose of delimiting seismic belts and the concept of seismic belt is also quite different from that of seismic statistical zone used in CPSHA method. The concept of seismic statistical zone and its history of evolvement are introduced too. Large differences between these rwo concepts exist separately in their statistical property, actual meaning, gradation, required scale, and property of refusing to overlap each other, aim and usage of delimitation. But in current engineering practice, these two concepts are confused. On the one hand, it causes no fit theory for delimiting seismic statistical zone in PSHA to be set up; on the other hand, researches about delimitation of seismic belts with purposes of seismicity zoning and studying on structural environment, mechanism of earthquake generating also pause to go ahead. Major conclusions are given in the end of this paper, that seismic statistical zone bases on the result of seismic belt delimiting, it only arises in and can be used in the especial PSHA method of China with considering spatially and temporally inhomogeneous seismic activities, and its concept should be clearly differentiated from the concept of seismic belt.