Inter-specific Sequence Differences Research Articles

Sequence analyses of mitochondrial gene may support the existence of cryptic species within Ascaridia galli.

Ascaridia galli (Nematoda: Ascaridiidae) is the most common intestinal roundworm of chickens and other birds with a worldwide distribution. Although A. galli has been extensively studied, knowledge of the genetic variation of this parasite in detail is still insufficient. The present study examined genetic variation in the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene among A. galli isolates (n = 26) from domestic chickens in Hunan Province, China. A portion of the cox1 (pcox1) gene was amplified by polymerase chain reaction separately from adult A. galli individuals and the amplicons were subjected to sequencing from both directions. The length of the sequences of pcox1 is 441 bp. Although the intra-specific sequence variation within A. galli is 0-7.7%, the inter-specific sequence differences among other members of the infraorder Ascaridomorpha were 11.4-18.9%. Phylogenetic analyses based on the maximum likelihood method using the sequences of pcox1 confirmed that all of the Ascaridia isolates were A. galli, and also resolved three distinct clades. Taken together, the findings suggest that A. galli may represent a complex of cryptic species. Our results provide an additional genetic marker for the management of A. galli in chickens and other birds.

Genetic Characterization of Argas persicus From Iran by Sequencing of Mitochondrial Cytochrome Oxidase I (COX1) and 16s rRNA Genes

Background: Argas persicus has a great importance for health and veterinary, it can transmit many infectious agents such as Borrelia anserina (avian spirochetosis) and Aegyptianella pullorum. Distinguishing Argasidae due to close morphological relationship is difficult. OBJECTIVES: In the present study, we performed molecular analyses based on PCR and sequencing of Amplicon derived from 16S rRNA and COX1 genes of A. persicus specimens in several provinces of Iran. Methods: Out of seventy Argas persicus collected and confirmed morphologically, eight ticks were chosen from five provinces of Iran for gene analysis. Their DNA were extracted and amplificated using primers derived from 16 S ribosomal RNA and COX1 genes using PCR. Then the amplicons were sequenced and analyzed by Chromas software and sequence alignment program (Clustal W). Phylogenetic analysis was also conducted using MEGA ver. 6.06 with a maximum-likelihood method. RESULTS: Sequencing results indicated that all eight samples belonged to A. persicus species. Their nucleotide sequencing revealed that the interspecific sequence differences of both genes (16S rRNA genes and COX1) between our isolates were very infrequent. All isolates from different provinces were conserved across regions except for one isolate that exhibited a difference of only 1 nucleotide. Within Phylogenetic tree, A. persicus formed a clade with A. persicus from other regions of the world (South Arica, Italy, China, and South Australia). CONCLUSIONS: Our findings suggested a very close phylogenetic relationship between A. persicus specimens obtained from different regions of Iran. Keywords:

Molecular characterization of hard tick Haemaphysalis longicornis from China by sequences of the internal transcribed spacers of ribosomal DNA

In the present study, the entire first and second internal transcribed spacer (ITS-1 and ITS-2) regions of nuclear ribosomal DNA (rDNA) of Haemaphysalis longicornis from China were amplified by polymerase chain reaction. The 45 representative amplicons were sequenced, and sequence variation in the ITS was examined. The ITS sequences of H. longicornis were 3644 bp in size, including the part of 18S rDNA, 28S rDNA sequences and the complete ITS-1, 5.8S rDNA and ITS-2 sequences. Sequence analysis revealed that the ITS-1, 5.8S rDNA and ITS-2 of this hard tick were 1582, 152, and 1610 bp in size, respectively. The intra-specific sequence variations of ITS-1 and ITS-2 within H. longicornis were 0–2 and 0–2.2%; however, the inter-specific sequence differences among members of the genus Haemaphysalis were significantly higher, being 35.1–55.2 and 37–52% for ITS-1 and ITS-2, respectively. The molecular approach employed in this study provides the foundation for further studies of the genetic variation of H. longicornis from different hosts and geographical origins in China.

Morphological and molecular study of the genus Nosopsyllus (Siphonaptera: Ceratophyllidae). Nosopsyllus barbarus () as a junior synonym of Nosopsyllus fasciatus (Bosc d’Antic, 1800)

In the present work, a comparative morphological and molecular study of Nosopsyllus barbarus (Jordan & Rothschild 1912) and Nosopsyllus fasciatus (Bosc, d’Antic 1800) isolated from rodents from different geographical regions (Spain, Morocco, Belgium, France and Portugal) has been carried out. The Internal Transcribed Spacers 1 and 2 (ITS1, ITS2) and partial 18S rRNA, and partial cytochrome c oxidase 1 (cox1) and cytochrome b (cytb) mtDNA sequences were determined to clarify the taxonomic status of these two species and to assess intraspecific variation and inter-specific sequence differences. In addition, a phylogenetic analysis with other species of fleas using Bayesian inference was performed. We have found morphological differences between N. barbarus and N. fasciatus that did not correspond with molecular differences. Furthermore, any of the five molecular markers used in this study was able to discriminate between the two species. Thus, based on the phylogenetic and molecular study of three nuclear markers (18S, ITS1, ITS2) and two mitochondrial markers (cox1 and cytb), as well as the concatenated dataset of both species, we concluded that morphological characters traditionally used to discriminate between the two species do not provide solid arguments for considering these two “morphospecies” as two different species. Therefore, we propose N. barbarus should be considered as a junior synonym of N. fasciatus.

Genetic analysis of Toxocara cati (Nematoda: Ascarididae) from Guangdong province, subtropical China

Toxocara cati (cat roundworm) is a common parasitic nematode that infects humans and other hosts, causing toxocariasis. Although its significance as a pathogen, the epidemiology, genetics and biology of T. cati remain poorly understand in China. In the present study, genetic variation in mitochondrial (mt) cytochrome c oxidase subunit 1 (cox1) gene and internal transcribed spacer (ITS) of rDNA region among T. cati in Guangdong province, subtropical China was examined. A portion of the cox1 (pcox1) and the complete ITS (ITS1 + 5.8S rDNA + ITS2) were amplified separately from individual worms by polymerase chain reaction (PCR) and amplicons were then subjected to sequencing from both directions. The length of the sequences of pcox1, ITS-1, and ITS-2 were 308 bp, 462 bp, and 335 bp, respectively. The intra-specific sequence variations within T. cati were 0–3.6% for pcox1, 0–2.4% for ITS-1, and 0–2.7% for ITS-2. However, the inter-specific sequence differences were significantly higher, being 8.6%, 10.7%, and 11.3% for pcox1, ITS-1, and ITS-2, respectively. Phylogenetic analyses based on the pcox1 sequences indicated that all the isolates in Guangdong province were in genus Toxocara, which confirmed that these parasites represent T. cati. The molecular approach employed provides a powerful tool for elucidating the epidemiology, genetics, and biology of zoonotic T. cati in China and elsewhere.

Ctenocephalides felis and Ctenocephalides canis: introgressive hybridization?

AbstractIn the present work, a comparative molecular study of Ctenocephalides felis and Ctenocephalides canis isolated from dogs (Canis lupus familiaris) from different geographical regions (Spain, Iran and South Africa) was carried out. We found morphological variations in C. felis that do not correspond with molecular differences. The Internal Transcribed Spacers 1 and 2 (ITS1, ITS2) and 18S rRNA partial gene, and cytochrome c‐oxidase 1 (cox1) mtDNA partial gene sequences were determined to clarify the taxonomic status of these two species, and to assess interpopulation variation and interspecific sequence differences. In addition, a comparative phylogenetic study with other species of fleas using Bayesian, Maximum Parsimony and Maximum Likelihood analysis was performed. The 18S rRNA partial gene fragment was useful neither to discriminate C. canis and C. felis nor to infer phylogenetic relationships at this level, whereas ITS1 and ITS2 assessed for specific determination in the genus Ctenocephalides. The cox1 mtDNA sequences of C. felis revealed three main haplotypes and we suggest that there has been introgression of C. canis cox1 mtDNA into C. felis by Wolbachia pipientis. Based on cox1 sequences, restriction mapping identified many endonucleases that could be used to delineate different haplotypes of C. felis and to differentiate C. felis and C. canis.

Genetic variability among Hymenolepis nana isolates from different geographical regions in China revealed by sequence analysis of three mitochondrial genes

Hymenolepis nana is a common tapeworm that parasitizes in the small intestine of rodent animals and humans. The present study examined the sequence diversity of three mitochondrial (mt) genes namely NADH dehydrogenase subunits 5 (nad5), small subunit ribosomal RNA (rrnS), and ATPase subunit 6 (atp6) of H. nana from mice in different geographical regions of China. A part of the nad5 (pnad5), complete rrnS and atp6 genes were amplified separately from individual H. nana isolates using polymerase chain reaction (PCR) and then sequenced. The sequences of pnad5, rrnS, and atp6 were 710 bp, 704–711 bp, and 516 bp in length, respectively. The A + T contents of the sequences were 70.1–73.5% (pnad5), 70.1–71.7% (rrnS), and 76.6–77.9% (atp6). Sequence variation within H. nana was 0–1.4% for atp6, 0–1.7% for rrnS, and 0–0.7% for pnad5. The inter-specific sequence differences between H. nana and Hymenolepis diminuta were significantly higher, which was 31.6–31.7% (pnad5), 16.1–17.6% (rrnS), and 26.5–27.1% (atp6). Phylogenetic analysis based on the combined three sequences using the maximum parsimony (MP) method supported that H. nana is a species complex or “cryptic” species. These findings demonstrated clearly the usefulness of the three mtDNA sequences for population genetics and systematic studies of H. nana of human and animal health significance.

Obstacles to molecular species identification in sea anemones (Hexacorallia: Actiniaria) with COI, a COI intron, and ITS II

DNA barcoding has been successfully applied to a very large number of taxa, but remains problematic for basal diploblasts, and debates about suitable molecular markers are ongoing. Sea anemones (Anthozoa: Hexacorallia: Actiniaria) populate most any marine environment and often play an irreplaceable role as hosts to other animals. Three genetic markers were tested to assess their utility for molecular species identification in members of the Actiniaria, namely the cytochrome oxidase subunit I (COI), a COI Intron with a Homing Endonuclease Gene (HEG), and the Internal Transcribed Spacer II (ITS II). Both the power of COI and the COI Intron to distinguish species is limited by events of very low inter-specific sequence differences and not by high intra-specific diversity. This finding implies that more comprehensive taxon sampling will not resolve this problem and other markers need to be investigated in several families. Results should discourage the use of ITS II as alternative to COI for barcoding in Actiniarians, since it shows similar limitations to COI.

Sequence variation in two mitochondrial DNA regions and internal transcribed spacer among isolates of the nematode Oesophagostomum asperum originating from goats in Hunan Province, China

The present study examined sequence variability in two mitochondrial DNA (mtDNA) regions, namely cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1), and internal transcribed spacer (ITS) of nuclear ribosomal DNA (rDNA) among Oesophagostomum asperum isolates from goats in Hunan Province, China. A portion of the cox1 (pcox1), nad1 (pnad1) genes and the ITS (ITS1+5.8S rDNA+ITS2) rDNA were amplified by polymerase chain reaction (PCR) separately from adult O. asperum individuals and the representative amplicons were subjected to sequencing from both directions. The lengths of pcox1, pnad1 and ITS rDNA were 366bp, 681bp and 785bp, respectively. The A+T contents of gene sequences were 71.5-72% for pcox1, 73.7-74.2% for pnad1 and 58-58.8% for ITS rDNA. Intra-specific sequence variations within O. asperum were 0-1.6% for pcox1, 0-1.9% for pnad1 and 0-1.7% for ITS rDNA, while inter-specific sequence differences among members of the genus Oesophagostomum were significantly higher, being 11.1-12.5%, 13.3-17.7% and 8.5-18.6% for pcox1, pnad1 and ITS rDNA, respectively. Phylogenetic analyses using combined sequences of pcox1 and pnad1, with three different computational algorithms (Bayesian inference, maximum likelihood and maximum parsimony), revealed distinct groups with high statistical support. These findings demonstrated the existence of intra-specific variation in mtDNA and rDNA sequences among O. asperum isolates from goats in Hunan Province, China, and have implications for studying molecular epidemiology and population genetics of O. asperum.

Genetic variability among Syphacia obvelata isolates from laboratory mice in four different geographical locations of China revealed by sequence analyses of five mitochondrial genes

Syphacia obvelata is a rodent nematode with high prevalence in laboratory mice. In the present study, we examined the genetic variability of S. obvelata from naturally infected laboratory mice in four different provinces, China. Five mitochondrial (mt) DNA regions, namely cytochrome c oxidase subunit 1 (pcox1), cytochrome b (pcytb), large subunit ribosomal RNA (prrnL) and NADH dehydrogenase subunits 1 and 5 (pnad1 and pnad5), were amplified separately from individual nematodes by PCR, and then sequenced directly. The size of the sequences of pcox1, pcytb, prrnL, pnad1 and pnad5 was 628 bp, 555 bp, 548 bp, 548 bp and 561 bp, respectively. While the intra-specific sequence variations within S. obvelata were 0–1.0% for pcox1, 0–1.6% for pcytb, 0–2.8% for prrnL, 0–2.0% for pnad1 and 0–1.8% for pnad5, the inter-specific sequence differences among members of the Oxyuridae were significantly higher, being 14.0–17.5% for pcox1, 27.5–32.9% for pcytb, 35.8–37.2% for prrnL, 22.2–26.8% for pnad1 and 22.3–25.2% for pnad5, respectively. Phylogenetic analyses based on combined sequences of four mt protein-coding genes, using Bayesian inference (BI), maximum likelihood (ML) and maximum parsimony (MP) methods, indicated that all of the S. obvelata samples grouped together with high statistical support, but samples from the same geographical origin did not always cluster together. These findings demonstrated the existence of low-level intra-specific variation in five mtDNA sequences among S. obvelata isolates from laboratory mice, but no obvious geographical distinction among S. obvelata isolates from laboratory mice in different geographic regions in China. These results provide basic information for further studies of systematics and population genetics of S. obvelata.

Sequence variability in four mitochondrial genes among rabbit pinworm (Passalurus ambiguus) isolates from different localities in China

Passalurus ambiguus is a common pinworm which parasitizes in the caecum and colon of rabbits. This study examined genetic variability among P. ambiguus isolated from naturally infected rabbits in four different provinces in China. The partial mitochondrial (mt) cytochrome c oxidase subunit 1 (pcox1), cytochrome b (pcytb) and NADH dehydrogenase subunits 1 and 5 (pnad1 and pnad5) were amplified separately from individual nematodes by PCR and sequenced. The results showed that pcox1, pcytb, pnad1 and pnad5 were 714, 663, 645 and 546 bp in length, respectively. The intra-specific sequence variations within P. ambiguus were 0–1.1% for pcox1, 0–1.2% for pcytb, 0–0.6% for pnad1 and 0–1.3% for pnad5, whereas inter-specific sequence differences with other members of the Oxyuridae were 16.2–17.3% for pcox1, 27.8–30.4% for pcytb, 20.2–24.0% for pnad1 and 27.1–30.3% for pnad5. Phylogenetic analyses using Bayesian inference (BI), maximum likelihood (ML) and maximum parsimony (MP) methods, based on the combined sequences of the four partial mtDNA sequences, revealed that all the P. ambiguus samples form monophyletic groups. This study demonstrated the existence of low-level intra-specific variation in cox1, cytb, nad1 and nad5 genes among P. ambiguus isolates from different geographic regions in China, and these four mtDNA sequences can be used as genetic markers for the population genetic studies of P. ambiguus, as well as the differentiation of P. ambiguus from other oxyurid nematodes.

Sequence variability in four mitochondrial genes among pinworm Aspicularis tetraptera isolates from laboratory mice in four provinces, China

This research aimed at exploring sequence variability in four mitochondrial (mt) genes, namely, cytochrome c oxidase subunit 1 (cox1), cytochrome b (cytb) and NADH dehydrogenase subunits 1 and 5 (nad1 and nad5), among pinworm Aspicularis tetraptera isolates from laboratory mice in four different provinces, China. A part of the cox1 (pcox1), cytb (pcytb), nad1 and nad5 genes (pnad1 and pnad5) were amplified separately from individual pinworms by polymerase chain reaction (PCR) and sequenced to determine sequence variations and examine their phylogenetic relationships. Herein, the intra-specific sequence variations within A. tetraptera were 0–0.5% for pcox1, 0–1.4% for pcytb, 0–1.8% for pnad1 and 0–1.7% for pnad5, respectively. In contrast, the inter-specific sequence differences among members of the Oxyuridae were significantly higher, being 13.7–17.0% for pcox1, 24.5–34.7% for pcytb, 26.6–29.6% for pnad1 and 24.4–25.5% for pnad5, respectively. Three methods, namely, Bayesian inference (BI), maximum likelihood (ML) and maximum parsimony (MP), were used for phylogenetic analyses based on the combined sequences of the four mt gene sequences, and the results indicated that all A. tetraptera samples form monophyletic groups, but samples from the same geographical origin did not always cluster together. This study demonstrated the existence of low-level intra-specific variation in four mtDNA sequences among A. tetraptera isolates from laboratory mice in different geographic regions in China, indicating no obvious geographical distinction among A. tetraptera isolates in China. These findings have important implications for studying systematics, molecular epidemiology and population genetics of A. tetraptera.

Genetic differences between Chabertia ovina and C. erschowi revealed by sequence analysis of four mitochondrial genes

This study examined sequence differences in mitochondrial cytochrome c oxidase subunit 1 (cox1), large subunit ribosomal RNA (rrnL) and NADH dehydrogenase subunits 1 and 4 (nad1 and nad4) between Chabertia ovina and C. erschowi from yaks in Qinghai and goats in Shaanxi provinces, China. A part of the cox1 (pcox1), rrnL (prrnL), nad1 and nad4 genes (pnad1 and pnad4) were amplified separately from individual nematodes by PCR and sequenced. The length of the sequences of pcox1, prrnL, pnad1 and pnad4 was 441 bp, 450 bp, 526 bp and 914 bp for C. ovina, and 441 bp, 451 bp, 517 bp and 810 bp for C. erschowi, respectively. The intra-specific sequence variations within C. ovina were 0.2–2.9% for pcox1, 0–0.9% for prrnL, 0.6–2.3% for pnad1, and 0.4–2.0% for pnad4, and were 0.5–1.6% for pcox1, 0–1.1% for prrnL, 0.2–1.7% for pnad1, and 0.4–1.1% for pnad4 within C. erschowi. Whereas, the inter-specific sequence differences between the two species were obviously higher, being 11.6–12.9% for pcox1, 9.8–11.1% for prrnL, 14.4–15.9% for pnad1, and 16.4–17.7% for pnad4. Phylogenetic analyses using Bayesian inference (BI), based on combined sequences of four genes, indicated that the C. ovina and C. erschowi represent distinct species. These results demonstrate that these mt gene sequences provide novel genetic markers for the identificaiton and differentiation C. ovina and C. erschowi, and have implications for studying the population genetics and molecular epidemiology of Chabertia spp.

Sequence variability in three mitochondrial genes among four roundworm species from wild animals in China

Sequence variability in three mitochondrial DNA (mtDNA) regions, namely portions of cytochrome c oxidase subunit 1 (pcox1), NADH dehydrogenase subunit 1 (pnad1) and NADH dehydrogenase subunit 4 (pnad4), for Toxocara canis. Baylisacaris transfuga. Ascaris suum and Parascaris equorum from Canis lupus. Ursus thibetanus. Sus scrofa and Equus burchelli in China were examined. The lengths of the sequences of pcox1, pnad1 and pnad4 were 711 bp, 648 bp and 666 bp, respectively. No intra-species differences were detected in pcox1 for the four examined ascarid species, in pnad1 for T. canis. A. suum and P. equorum, and in pnad4 for B. transfuga and P. equorum. Sequence differences in pnad4 for six roundworm samples of T. canis and P. equorum were 0–0.1% and 0–0.3%, respectively, and were 0–0.3% in pnad1 for six roundworm samples isolate of B. transfuga. The inter-specific sequence differences among four species were 8.7–12.4% for pcox1, 13.9–17.7% for pnad1, and 14.0–25.7% for pnad4. Phylogenetic analyses suggested that the three mtDNA fragments could be used to identify ascarid species in families Ascaridiae and Toxocaridae.

Genetic variability in the three mitochondrial genes among Oesophagostomum asperum isolates from different regions in Shaanxi and Hunan Provinces, China

The present study examined sequence variations in three mitochondrial DNA (mtDNA) regions, namely, NADH dehydrogenase subunit 5 (nad5), adenosine triphosphate subunit 6 (atp6) and cytochrome c oxidase subunit 3 (cox3), among Oesophagostomum asperum isolates from different regions in Shaanxi and Hunan provinces, China. The lengths for partial sequences of nad5 (pnad5), atp6 (patp6) and cox3 (pcox3) were 427 bp, 381 bp and 337 bp, respectively. The intra-specific sequence variations among all O. asperum samples were 0–2.11%, 0–1.84% and 0–1.48% for pnad5, patp6 and pcox3, respectively, while the inter-specific sequence differences among Oesophagostomum species in pig and small ruminants were 18–21.3% for pnad5, 18.3–24.5% for patp6 and 10.6–13.7% for pcox3. A phylogenetic analysis based on combined sequences of three mtDNA fragments indicated that all O. asperum isolates were grouped in one solid clade, and the Oesophagostomum spp. from pig were located in another clade. However, these mtDNA fragments could not reveal genetic relationships of geographical isolates of O. asperum in China. These results provided valuable information for studying population genetics of Oesophagostomum spp., and for controlling Oesophagostomum infection in animals as well as humans.

Genetic variability of Baylisascaris schroederi from the Qinling subspecies of the giant panda in China revealed by sequences of three mitochondrial genes

The present study examined the variations in three mitochondrial (mt) DNA sequences, namely cytochrome b (cytb), cytochrome c oxidase subunit 3 (cox3) and NADH dehydrogenase subunit 5 (nad5), among Baylisascaris schroederi isolates from the Qinling subspecies of the giant panda in Shaanxi province, northwestern China. No differences in length were detected in the three mt fragments from different isolates. The intra-specific sequence variations within all B. schroederi samples were 0–2.6% for pcytb, 0–1.8% for pcox3 and 0–2.1% for pnad5, while the inter-specific sequence differences among members of the genus Baylisascaris were 8.2–15.2%, 6.2–15.9% and 8.4–16.0% for pcytb, pcox3, pnad5, respectively. A phylogenetic analysis of the combined sequences of pcytb, pcox3 and pnad 5 showed that all B. schroederi samples in the present study were located in two large clusters, with one cluster containing samples from giant pandas in Sichuan province. These findings provide basic information for further study of molecular epidemiology and control of B. schroederi infection in the Qinling subspecies of the giant panda and throughout China.

Morphological, biometrical, and molecular characterization of Ctenocephalides felis and Ctenocephalides canis isolated from dogs from different geographical regions

In the present work, a comparative morphological, biometrical and molecular study of Ctenocephalides spp. isolated from dogs (Canis lupus familiaris) from different geographical regions (Spain, Iran, and South Africa) has been carried out. The internal transcribed spacer 1 (ITS1) sequences of Ctenocephalides felis and Ctenocephalides canis collected from dogs from different geographical regions have been determined to clarify the taxonomic status of these species and to assess intraspecific variation and interspecific sequence differences. In addition, a phylogenetic analysis based on ITS1 sequences has been performed. Four different morphological populations were observed in the individuals of C. felis collected from dogs from different geographical locations. Nevertheless, the comparative study of the ITS1 sequences of the different morphological populations observed in C. felis did not show molecular differences. The results showed clear molecular differences between C. felis and C. canis and some specific recognition sites for endonucleases were detected between both species. Thus, BfrBI and DraI sites have diagnostic value for specific determination in C. felis. The phylogenetic tree based on the ITS1 sequences of C. felis and C. canis revealed that all the populations of C. felis from different geographical regions clustered together and separated, with high bootstrap values, from C. canis. We conclude that ITS1 region is a useful tool to approach different taxonomic and phylogenetic questions in Ctenocephalides species.

Taxonomic circumscription of heterogeneous species Neogoniolithon brassica‐florida (Corallinales, Rhodophyta) in Japan

SummaryThe nongeniculate species Neogoniolithon brassica‐florida (Harvey) Setchell et Mason is circumscribed as a polymorphic species with various gross morphologies due to it being synonymized with several previous species. However, small subunit rDNA and cox1 analyses showed that N. brassica‐florida was polyphyletic, and strongly imply that crustose species lacking any protuberances such as Neogoniolithon fosliei (Heydrich) Setchell et Mason and species with protuberances or branches such as N. brassica‐florida and N. frutescens (Foslie) Setchell et Mason should be treated as genetically different groups (species). Therefore, we propose the resurrection of N. frutescens. We also confirmed that N. trichotomum was distinguished from N. frutescens by slender uniform diameter branches, a conceptacle with a prominent ostiole, and large cox1 interspecific sequence differences. Male and female reproductive structures of N. trichotomum were illustrated for the first time. Neogoniolithon fosliei, was divided into three clades, each of which was recognized as a species complex based on interspecific level sequence differences within clade and morphological differences. Therefore, we treated the clade most similar to N. fosliei as N. fosliei complex (Clade B), and the other clades as respective complexes of N. cf. fosliei with yellow conceptacles (Clade A) or N. cf. fosliei with large conceptacles (Clade C). Of two species complexes (Clade A and B) were morphologically consistent with two entities of N. fosliei previously reported in the Ryukyu Islands, Japan, which is supported by their niche partitioning. DNA barcoding research of nongeniculate corallines can promote the finding of more reliable taxonomic characters and the understanding of their biological aspects.

Characterization of Dicrocoelium chinensis from domestic yaks in Gansu and Sichuan provinces, China, using genetic markers in two mitochondrial genes

Dicrocoelium chinensis is commonly known as the “lancet fluke” or “small liver fluke” which lives in the bile ducts and gall bladder of domestic and wild ruminants. In this study, we examined sequence variability in two mitochondrial (mt) genes, namely cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) genes, among D. chinensis isolated from domestic yaks in Gansu and Sichuan provinces, China. A portion of the cox1 (pcox1) and nad1 (pnad1) genes was amplified by PCR separately from the individual adult D. chinensis, and the amplicons were subjected to sequencing from both directions. The lengths of the sequences of pcox1 and pnad1 were 358 and 458 bp, respectively. The A+T contents of the sequences were 58.94–59.22% for pcox1 and 60.04–60.48% for pnad1, respectively. The intra-specific sequence variations within D. chinensis were 0–0.84% for pcox1 and 0–0.87% for pnad1. The inter-specific sequence differences among members of the examined trematodes were significantly higher, being 22.9–29.3% and 33.6–41.5% for pcox1 and pnad1, respectively. Phylogenetic analysis utilizing combined sequences of pcox1 and pnad1 using Bayesian inference revealed that all of the D. chinensis isolates grouped together with high statistical support. These findings demonstrated the existence of low intra-specific variation in mitochondrial DNA (mtDNA) sequences among the D. chinensis isolates from domestic yaks in China and had implications for studying the molecular epidemiology and population genetics of D. chinensis.

Sequence variability in four mitochondrial genes amongBunostomum trigonocephalumisolates from four provinces in China

The present study examined sequence variability in four mitochondrial genes, namely cytochrome c oxidase subunit (cox1), cytochrome b (cytb) and NADH dehydrogenase subunits 1 and 5 (nad1 and nad5), among Bunostomum trigonocephalum isolates from four different geographic regions in China. Ten B. trigonocephalum samples were collected from each of the four provinces (Heilongjiang, Jilin, Shaanxi and Yunnan), China. A part of the cox1 (pcox1), cytb (pcytb), nad1 and nad5 genes (pnad1 and pnad5) were amplified separately from individual hookworms by polymerase chain reaction (PCR) and were subjected to direct sequencing in order to define sequence variations and their phylogenetic relationships. The intra-specific sequence variations within B. trigonocephalum were 0-1.9% for pcox1, 0-2.0% for pcytb, 0-1.6% for pnad1 and 0-1.7% for pnad5. The A+T contents of the sequences were 69.6-70.4% (pcox1), 71.9-72.7 (pcytb), 70.4-71.1% (pnad1) and 72.0-72.6% (pnad5). However, the inter-specific sequence differences among members of the family Ancylostomatidae were significantly higher, being 12.1-14.2% for pcox1, 13.7-16.0 for cytb, 17.6-19.4 for nad1 and 16.0-21.6 for nad5. Phylogenetic analyses based on the combined partial sequences of cox1, cytb, nad1 and nad5 using three inference methods, namely Bayesian inference (Bayes), maximum likelihood (ML) and maximum parsimony (MP), revealed that all the B. trigonocephalum samples form monophyletic groups, but samples from the same geographical origin did not always cluster together, suggesting that there was no obvious geographical distinction within B. trigonocephalum based on sequences of the four mtDNA genes. These results demonstrated the existence of low-level intra-specific variation in mitochondrial DNA (mtDNA) sequences among B. trigonocephalum isolates from different geographic regions.