Molecular genetic markers of intra- and interspecific divergence within starfish and sea urchins (Echinodermata)

A fragment of the mitochondrial COI gene from isolates of several echinoderm species was sequenced. The isolates were from three species of starfish from the Asteriidae family (Asterias amurensis and Aphelasterias japonica collected in the Sea of Japan and Asterias rubens collected in the White Sea) and from the sea urchin Echinocardium cordatum (family Loveniidae) collected in the Sea of Japan. Additionally, regions including internal transcribed spacers and 5.8S rRNA (ITS1–5.8S rDNA–ITS2) were sequenced for the three studied starfish species. Phylogenetic analysis of the obtained COI sequences together with earlier determined homologous COI sequences from Ast. forbesii, Ast. rubens, and Echinocardium laevigaster from the North Atlantic and E. cordatum from the Yellow and North Seas (GenBank) placed them into strictly conspecific clusters with high bootstrap support (99% in all cases). Only two exceptions–Ast. rubens DQ077915 sequence placed with the Ast. forbesii cluster and Aph. japonica DQ992560 sequence placed with the Ast. amurensis cluster–were likely results of species misidentification. The intraspecific polymorphism for the COI gene within the Asteriidae family varied within a range of 0.2-0.9% as estimated from the genetic distances. The corresponding intrageneric and intergeneric values were 10.4-12.1 and 21.8-29.8%, respectively. The interspecific divergence for the COI gene in the sea urchin of Echinocardium genus (family Loveniidae) was significantly higher (17.1-17.7%) than in the starfish, while intergeneric divergence (14.6-25.7%) was similar to that in asteroids. The interspecific genetic distances for the nuclear transcribed sequences (ITS1–5.8S rDNA–ITS2) within the Asteriidae family were lower (3.1-4.5%), and the intergeneric distances were significantly higher (32.8-35.0%), compared to the corresponding distances for the COI gene. These results suggest that the investigated molecular-genetic markers could be used for segregation and identification of echinoderm species.     

基于线粒体细胞色素c氧化酶亚基I基因序列的帘蛤科贝类分子系统发育研究

对21种帘蛤科贝类线粒体细胞色素c氧化酶亚基Ⅰ(cytochrome c oxidase subunit I,COI)基因核苷酸序列进行了分析,以探讨这一序列在种质鉴定、分子系统发生研究中的应用价值。测序结果表明,所有物种扩增片段长度均为707 bp(含引物),序列A+T含量(62.4%—67.8%)明显高于G+C含量。物种间共有变异位点379个,其中简约信息位点334个;此区段共编码235个氨基酸,种间共有氨基酸变异位点100个。以COI基因片段序列为标记,用中国蛤蜊(Mactra chinensis)作外群,构建了35种帘蛤科贝类(其中14种贝类COI序列从GenBank下载)的系统发生树,结合拓扑结构分析和序列比对分析,结果表明:支持将短文蛤(Meretrix petechinalis)和丽文蛤(M.lusoria)订为文蛤(M.meretrix)的同物异名的观点,建议将丽文蛤和短文蛤订为文蛤的地理亚种;支持将薄片镜蛤(Dosinia corrugata)和D.angulosa订为2个独立种的观点;认为将波纹巴非蛤(Paphia undulata)和织锦巴非蛤(P.textile)订为2个独立种是合适的。COI基因序列含有丰富的遗传信息,适合作为帘蛤科贝类种群遗传结构和系统发生研究的分子标记。     

The Effectiveness of Three Regions in Mitochondrial Genome for Aphid DNA Barcoding: A Case in Lachininae

Background

The mitochondrial gene COI has been widely used by taxonomists as a standard DNA barcode sequence for the identification of many animal species. However, the COI region is of limited use for identifying certain species and is not efficiently amplified by PCR in all animal taxa. To evaluate the utility of COI as a DNA barcode and to identify other barcode genes, we chose the aphid subfamily Lachninae (Hemiptera: Aphididae) as the focus of our study. We compared the results obtained using COI with two other mitochondrial genes, COII and Cytb. In addition, we propose a new method to improve the efficiency of species identification using DNA barcoding.

Methodology/Principal Findings

Three mitochondrial genes (COI, COII and Cytb) were sequenced and were used in the identification of over 80 species of Lachninae. The COI and COII genes demonstrated a greater PCR amplification efficiency than Cytb. Species identification using COII sequences had a higher frequency of success (96.9% in “best match” and 90.8% in “best close match”) and yielded lower intra- and higher interspecific genetic divergence values than the other two markers. The use of “tag barcodes” is a new approach that involves attaching a species-specific tag to the standard DNA barcode. With this method, the “barcoding overlap” can be nearly eliminated. As a result, we were able to increase the identification success rate from 83.9% to 95.2% by using COI and the “best close match” technique.

Conclusions/Significance

A COII-based identification system should be more effective in identifying lachnine species than COI or Cytb. However, the Cytb gene is an effective marker for the study of aphid population genetics due to its high sequence diversity. Furthermore, the use of “tag barcodes” can improve the accuracy of DNA barcoding identification by reducing or removing the overlap between intra- and inter-specific genetic divergence values.     

Barcoding forest insect pests in South Korea: Constructing a basic endemic species dataset

A total of 103 barcode (mitochondrial COI) sequences were newly provided for 77 forest insect pests from 66 genera belonging to Coleoptera, Hemiptera, and Lepidoptera. All 77 species had distinct COI sequences, revealing low intraspecific genetic divergence (< 1.20%) and high interspecific genetic divergence (> 7.30%). Among the 66 genera, 32 COI sequences of 25 species belonging to 16 genera were compared with 280 COI sequences of 117 species belonging to the same 16 genera archived in GenBank, showing that most species were clearly distinguished by barcode sequences. Based on these results, we conclude that a DNA barcode is effective for identifying forest insect pest species.     

Molecular phylogeny of Trissolcus species (Hymenoptera: Scelionidae)

Trissolcus species (Hymenoptera: Scelionidae) are the most promising biological control agents against sunn pest. The accurate identification of natural enemies is crucial in order to develop successful biological control programs. This paper presents phylogenetic analyses of Trissolcus species based on data sets consisting of 18S, 28S, ITS1, ITS2 and cytochrome oxidase subunit I (COI) genes. The most commonly used genetic loci in Trissolcus species identification is the cytochrome oxidase I gene (COI). Also restriction fragment length polymorphism analyses of PCR amplified COI gene have been developed to discriminate closely related species. We suggest that Trissolcus grandis Thompson and Trissolcus semistriatus Nees split significantly into two different genetic groups while there is another species diverging from T. semistriatus which is decsribed as ‘Trissolcus flavotibialis Kocak & Guz, n. sp.’     

Slow molecular evolution in an ancient asexual ostracod

Genetic variability of the non-marine ostracod species Darwinula stevensoni was estimated by sequencing part of the nuclear and the mitochondrial genome. As Darwinulidae are believed to be ancient asexuals, accumulation of mutations should have occurred, both between alleles within lineages and between lineages, during the millions of years of parthenogenetic reproduction. However, our sequence data show the opposite: no variability in the nuclear ITS1 region was observed within or among individuals of D. stevensoni, despite sampling a geographical range from Finland to South Africa. Lack of allelic divergence might be explained by concerted evolution of rDNA repeats. Homogeneity among individuals may be caused either by slow molecular evolution in ITS1 or by a recent selective sweep. Variability of mitochondrial cytochrome oxidase (COI) was similar to intraspecific levels in other invertebrates, thus weakening the latter hypothesis. Calibrating interspecific, genetic divergences among D. stevensoni and other Darwinulidae using their fossil record enabled us to estimate rates of molecular evolution. Both COI and ITS1 evolve half as fast, at most, in darwinulids as in other invertebrates, and molecular evolution has significantly slowed down in ITS1 of D. stevensoni relative to other darwinulids. A reduced ITS1 mutation rate might explain this inconsistency between nuclear and mitochondrial evolution in D. stevensoni.     

Maintenance of genetic and morphological identity in two sibling Syrrhopodon species (Calymperaceae,Bryopsida) despite extensive introgression

Bryophytes are a group of land plants in which the role of hybridization has long been challenged. Using genotyping by sequencing to circumvent the lack of molecular variation at selected loci previously used for phylogeny and morphology, we determine the level of genetic and morphological divergence and reproductive isolation between the sibling Syrrhopodon annotinus and S. simmondsii (Calymperaceae, Bryopsida) that occur in sympatry but in different habitats in lowland Amazonian rainforests. A clear morphological differentiation and a low (0.06), but significant Fst derived from the analysis of 183 single nucleotide polymorphisms were observed between the two species. Conspecific pairs of individuals consistently exhibited higher average kinship coefficients along a gradient of geographic isolation than interspecific pairs. The weak, but significant genetic divergence observed is consistent with growing evidence that ecological specialization can lead to genetic differentiation among bryophyte species. Nevertheless, the spatial genetic structures of the two species were significantly correlated, as evidenced by the significant slope of the Mantel test based on kinship coefficients between pairs of interspecific individuals and the geographic distance separating them. Interspecific pairs of individuals are thus more closely related when they are geographically closer, suggesting that isolation‐by‐distance is stronger than the interspecific reproductive barrier and pointing to interspecific gene flow. We conclude that interspecific introgression, whose role has long been questioned in bryophytes, may take place even in species wherein sporophyte production is scarce due to dioicy, raising the question as to what mechanisms maintain differentiation despite weak reproductive isolation.     

Analysis of the mitochondrial DNA variation in pond smelt Hypomesus olidus (Osmeridae)

Pond smelt Hypomesus olidus (Pallas, 1814), one of the five species of the genus Hypomesus, family Osmeridae, was examined for intraspecific variation of the mitochondrial DNA cytb (1062 bp) and COI (567 bp) genes. Among the ten single substitutions discovered, only one, leading to the substitution of isoleucine by valine, was nonsynonymous, while the remaining substitutions were synonymous. The degree of genetic divergence among pooled nucleotide sequences in H. olidus populations examined constituted 0.4% on average, ranging from 0.2 to 0.6%. These values were not higher than the levels of divergence between the individuals within the populations. Phylogenetic analysis of the populations examined did not reveal their subdivision depending of their geographic location, and pointed to the absence of intraspecific differentiation of the species.     

Molecular divergence between disjunct taxa in eastern Asia and eastern North America

Eastern Asian-eastern North American disjuncts in four genera were examined for allozyme divergence and sequence divergence of the internal transcribed spacers (ITS) of nuclear ribosomal DNA. The disjunct pairs of taxa include Caulophyllum robustum-C. thalictroid.es, Menispermum dauricum-M. canadense, Penthorum chinense-P. sedoides, and Phryma leptostachya var. asiatica-P. leptostachya var. leptostachya. Allozyme divergence was comparable in Caulophyllum and Penthorum (genetic identities of 0.534 and 0.546) and was considerably higher than between pairs of taxa in Menispermum (0.273) and Phryma (0.291). Caulophyllum and Penthorum, which have the highest genetic identities at allozyme loci, also have low ITS sequence divergences (1.30 and 1.65%, respectively). Phryma, which has low isozyme identity, also has the highest ITS sequence divergence (4.46%). The two taxa of Menispermum have low ITS sequence divergence (0.93%) despite having a low identity (0.273) at allozyme loci. The results suggest that divergence between the taxa in the four genera are not the result of a single historical event. Estimated divergence times are reasonably consistent with a late Miocene disjunction for Caulophyllum and Penthorum, whereas the age of the Phryma disjunction is calculated at over 20 million years. The nonconcordant divergences between allozymes and ITS sequences in Menispermum may be caused by concerted evolution in the latter or possibly longer generation time in the woody plants. Additional molecular data are needed to clarify the situation.     

DNA sequences of the mitochondrial COI gene have low levels of divergence among deep-sea octocorals (Cnidaria: Anthozoa)

We are analyzing genetic diversity in deep-seamount octocorals with the ultimate goal of studying the effect of retention and dispersal of larvae on genetic population structure. Here we report on the sequence diversity of the mitochondrial cytochrome oxidase I (COI) gene among 11 species. Uncorrected pairwise sequence divergences ranged from 0.4–10.3% for comparisons among species spanning the intrageneric to interordinal levels. Relative to other invertebrates, these divergences are very low, suggesting that COI may not be useful as a genetic marker for studying dispersal among deep-sea octocoral populations. Possible explanations for the reduced rates of divergence observed include a lower rate of evolution for octocoral mitochondrial genomes and the presence of a gene, mtMSH, which may code for a mitochondrial DNA mismatch-repair system. We report the finding of mtMSH in three deep-sea octocorals (Acanthogorgia sp., Corallium ducale, and Paramuricea sp.), which brings the total published observations of this gene to six species, all in the subclass Octocorallia.     

Genetic Diversity and Geographic Differentiation of the Giant Tiger Shrimp (Penaeus monodon) in Thailand Analyzed by Mitochondrial COI Sequences

Genetic diversity and geographic differentiation of the giant tiger shrimp, Penaeus monodon, in Thai waters (Satun, Trang, Phangnga, and Ranong in the Andaman Sea and Chumphon and Trat in the Gulf of Thailand) were examined by COI polymorphism (N = 128). We observed 28 COI mitotypes across all investigated individuals. The sequence divergence between pairs of mitotypes was 0.00–20.76%. A neighbor-joining tree clearly indicated lineage separation of Thai P. monodon and large nucleotide divergence between interlineage mitotypes but limited divergence between intralineage mitotypes. High genetic diversity was found (mean sequence divergence = 6.604%, haplotype diversity = 0.716–0.927, π = 2.936–8.532%). F-statistics (F _ST) and an analysis of molecular variance (AMOVA) indicated that the gene pool of Thai P. monodon was not homogeneous but genetically differentiated intraspecifically (P < 0.05). Six samples of P. monodon could be allocated into three different genetic populations: Trat (A), Chumphon (B), and the Andaman samples Satun, Trang, Phangnga, and Ranong (C). Contradictory results regarding patterns of geographic differentiation previously reported by various molecular approaches were clarified by this study.     

Genetic Differentiation of the Mitochondrial Cytochrome Oxidase c Subunit I Gene in Genus Paramecium (Protista,Ciliophora)

Background

The mitochondrial cytochrome c oxidase subunit I (COI) gene is being used increasingly for evaluating inter- and intra-specific genetic diversity of ciliated protists. However, very few studies focus on assessing genetic divergence of the COI gene within individuals and how its presence might affect species identification and population structure analyses.

Methodology/Principal findings

We evaluated the genetic variation of the COI gene in five Paramecium species for a total of 147 clones derived from 21 individuals and 7 populations. We identified a total of 90 haplotypes with several individuals carrying more than one haplotype. Parsimony network and phylogenetic tree analyses revealed that intra-individual diversity had no effect in species identification and only a minor effect on population structure.

Conclusions

Our results suggest that the COI gene is a suitable marker for resolving inter- and intra-specific relationships of Paramecium spp.     

Identification of Channa species using the partial cytochrome c oxidase subunit I (COI) gene as a DNA barcoding marker

The snakehead fish of the genus Channa are an important food fish in China. However, the molecular identification and phylogeny of this genus is poorly understood. Here, we present the utility of partial sequences of the COI gene for use in DNA barcoding for the identification of Channa individuals, which includes four species: Channa argus, Channa maculata, Channa asiatica, and Channa striata. A total of 19 haplotypes were identified in this study. The interspecific K2P distances were higher than intraspecific distances. The lowest interspecific distance (0.091) was between C. argus and C. maculata while the highest interspecific distance (0.219) was between C. argus and C. striata. No intraspecific–interspecific distance overlaps were observed, and a distinct barcoding gap was found between intraspecific and interspecific distances in each species. Our results showed that the partial COI gene is an effective DNA barcoding marker for identifying Channa species.     

The gnd gene of Buchnera as a new,effective DNA barcode for aphid identification

DNA barcoding uses a standard DNA sequence to facilitate species identification. Although the COI gene has been adopted as the standard, COI alone is imperfect due to several shortcomings. The primary endosymbiont of aphids, Buchnera, has higher evolutionary rates and interspecies divergence than its co‐diverging aphid hosts, making it a potential tool for resolving the ambiguities in aphid taxonomy. We compared the effectiveness of employing two different DNA regions, gnd and COI, for the discrimination of over 100 species of aphids. The mean interspecific divergence of the gnd region was significantly higher than the mean intraspecific variation; there were nearly nonoverlapping distributions between the intra‐ and interspecific samples. In contrast, COI showed a lower interspecific divergence, which led to difficulties in identifying closely related species. Our results show that gnd can identify species in the Aphididae, which suggests that the gnd region of Buchnera is a potentially effective barcode for aphid species identification. We also recommend the 2‐locus combination of gnd + COI as the aphid barcode. This will provide a universal framework for the routine use of DNA sequence data to identify specimens and contribute toward the discovery of overlooked species of aphids.     

Molecular Identification of Sibling Species of Sclerodermus (Hymenoptera: Bethylidae) That Parasitize Buprestid and Cerambycid Beetles by Using Partial Sequences of Mitochondrial DNA Cytochrome Oxidase Subunit 1 and 28S Ribosomal RNA Gene

The species belonging to Sclerodermus (Hymenoptera: Bethylidae) are currently the most important insect natural enemies of wood borer pests, mainly buprestid and cerambycid beetles, in China. However, some sibling species of this genus are very difficult to distinguish because of their similar morphological features. To address this issue, we conducted phylogenetic and genetic analyses of cytochrome oxidase subunit I (COI) and 28S RNA gene sequences from eight species of Sclerodermus reared from different wood borer pests. The eight sibling species were as follows: S. guani Xiao et Wu, S. sichuanensis Xiao, S. pupariae Yang et Yao, and Sclerodermus spp. (Nos. 1–5). A 594-bp fragment of COI and 750-bp fragment of 28S were subsequently sequenced. For COI, the G-C content was found to be low in all the species, averaging to about 30.0%. Sequence divergences (Kimura-2-parameter distances) between congeneric species averaged to 4.5%, and intraspecific divergences averaged to about 0.09%. Further, the maximum sequence divergences between congeneric species and Sclerodermus sp. (No. 5) averaged to about 16.5%. All 136 samples analyzed were included in six reciprocally monophyletic clades in the COI neighbor-joining (NJ) tree. The NJ tree inferred from the 28S rRNA sequence yielded almost identical results, but the samples from S. guani, S. sichuanensis, S. pupariae, and Sclerodermus spp. (Nos. 1–4) clustered together and only Sclerodermus sp. (No. 5) clustered separately. Our findings indicate that the standard barcode region of COI can be efficiently used to distinguish morphologically similar Sclerodermus species. Further, we speculate that Sclerodermus sp. (No. 5) might be a new species of Sclerodermus.     

Genetic divergence of Octopus vulgaris species in the eastern Mediterranean

Nucleotide divergence in cytochrome c oxidase subunit I (COI) gene of mitochondrial DNA was analyzed to determine interpopulational variation of common octopus (Octopus vulgaris) species sampled along eastern Mediterranean. Nucleotide composition and nucleotide pair frequency analyses of 440 individuals representing 11 populations were investigated. Estimation of evolutionary divergence between sequences was conducted using Tamura-Nei model. Considering 440 individuals, 35 (5%) variable sites and an average variation of 1.9% were found between specimens. Highest divergence was found between Med11 and Med04-Med07 populations; with a genetic distance value of 0.039 (3.9%). Estimation of transition/transversion bias (R) was made under the Tamura-Nei model and resulted as 2.32. Phylogenetic tree was constructed using four methods: neighbor joining (NJ), minimum evolution (ME), maximum likelihood (ML) and unweighted pair-group method of arithmetic average (UPGMA). Results from clustering patterns of the consensus tree, with the sum of branch length = 0.05189, pointed out a correlation between genetic and geographic distance.     

Genetic structure among Charadrius plovers on the African mainland and islands of Madagascar and St Helena

Colonization of islands by long-distance dispersers has great impact on genetic diversification among populations and may spearhead speciation events. We investigated intra- and interspecific divergence in Charadrius plovers with populations on mainland Africa, Madagascar and St Helena. We analysed microsatellite loci and sequence data from four nuclear and two mitochondrial gene regions. Charadrius plovers are shorebirds with high dispersal and mobility. Our results confirmed genetic differentiation between Madagascar and mainland populations of three plover species (White-fronted Plover Charadrius marginatus, Kittlitz's Plover Charadrius pecuarius and, based on sequence data only, Three-banded Plover Charadrius tricollaris) but highlight substantial variation in levels of intraspecific divergence among the three species. Namely, the Kittlitz's Plover, a dispersive habitat generalist with a polygamous mating system, exhibited lower island–mainland differentiation (0.05% COI sequence divergence) compared with the two monogamous species, the White-fronted Plover (0.6% COI divergence) and Three-banded Plover (1.6% COI divergence). In addition, past colonization of the islands of St Helena and Madagascar by ancestors of today's Kittlitz's Plover has led to the evolution of two endemic island species, the Madagascar Plover Charadrius thoracicus and the more closely-related St Helena Plover Charadrius sanctaehelenae. We discuss the factors driving species differences in island–mainland divergence and highlight the importance of conserving genetically unique island populations and island habitats to safeguard future evolutionary potential.