A study was conducted on the identifications of the degraded samples of sika deer （Cervus nippon） and red deer （Cervus elaphus） by phylogenetic and nucleotide distance analysis of partial Cytb and 12s rRNA genes s...A study was conducted on the identifications of the degraded samples of sika deer （Cervus nippon） and red deer （Cervus elaphus） by phylogenetic and nucleotide distance analysis of partial Cytb and 12s rRNA genes sequences. 402 bp Cytb genes were achieved by PCR-sequencing using DNA extracted from 8 case samples, and contrasted with 27 sequences of Cytb gene downloaded from GenBank database. The values of three nucleotide distance between three suspected samples and sika deer were identical （0.026±0.006）, which was smaller than the smallest nucleotide distance between eastern red deer and sika deer （0.036）. Furthermore, phylogenetic analysis of sika deer and red deer indicated that the evidences located within the same cluster as sika deer. The evidences were sika deer materials. As the same way, other three suspected samples were derived from red deer. The results were further confirmed by phylogenetic and nucleotide distance analysis of 387 bp 12s rRNA gene. The method was powerful and less time-consuming and helpful to reduce the related cases with wildlife.展开更多
A new COX1 primer for soil nematode metabarcoding was designed,and this primer outperforms other commonly used COX1 primer pairs in species recovery and quantity of PCR products.•The lack of reference database is the ...A new COX1 primer for soil nematode metabarcoding was designed,and this primer outperforms other commonly used COX1 primer pairs in species recovery and quantity of PCR products.•The lack of reference database is the main reason that led to the low species recovery in COX1 metabarcoding.•We expanded current NCBI database by adding 51 newly generated COX1 reference sequences.Microscopic nematodes play important roles in soil ecosystems and often serve as bioindicators of soil health.The identification of soil nematodes is often difficult due to their limited diagnostic characters and high phenotypic plasticity.DNA barcoding and metabarcoding techniques are promising but lack universal primers,especially for mitochondrial COX1 gene.In this study a degenerated COX1 forward primer COIFGED was developed.The primer pair(COIFGED/JB5GED)outperforms other four commonly used COX1 primer pairs in species recovery and quantity of polymerase chain reaction(PCR)products.In metabarcoding analysis,the reads obtained from the new primer pair had the highest sequencing saturation threshold and amplicon sequence variant(ASV)diversity in comparison to other COX1 as well as 18S rRNA primers.The annotation of ASVs suggested the new primer pair initially recovered 9 and 6 out of 25 genera from mock communities,respectively,outperformed other COX1 primers,but underperformed the widely used 18S NF1/18Sr2b primers(16 out of 25 genera).By supplementing the COX1 database with our reference sequences,we recovered an additional 6 mock community species bringing the tally closer to that obtained with 18S primers.In summary,our newly designed COX1 primers significantly improved species recovery and thus can be supplementary or alternative to the conventional 18S metabarcoding.展开更多
Ciliates in the subclass Hypotrichia have long been difficult to classify as they are one of the most polymorphic and highly differentiated groups,leading to their systematics remaining unresolved.Phylogenetic relatio...Ciliates in the subclass Hypotrichia have long been difficult to classify as they are one of the most polymorphic and highly differentiated groups,leading to their systematics remaining unresolved.Phylogenetic relationships within the hypotrich family Strongylidiidae have been ambiguous due to discordance between the morphological and genetic data.In this study,a new strongylidiid genus Heterouroleptus is established,mainly based on the novel mode of origin of the ventral cirral rows:left ventral cirral row(LVR)originates from frontal-ventral-transverse cirral anlagen(FVTA)Ⅲ(anterior portion),IV(middle portion),and V(rear portion);right ventral cirral row comes from the entire FVTA VI.A new species,Hetero-uroleptus weishanensis gen.nov.,sp.nov.,is investigated along with the morphometric and molecular data from a population of Strongylidium wuhanense.Eight new sequences and nuclear gene markers(single-gene and multi-gene)are provided to analyze the phylogenetic relationships of strongylidiids,with the COI gene utilized to uncover further genetic information at species level and below.The results reveal that:(1)Strongylidiidae is monophyletic and has a close relationship with Dorsomarginalia;(2)Heterouroleptus gen.nov.forms a clade that is sister to all the other strongylidiids;(3)Hemiamphisiella Foissner,1988 and Pseudouroleptus Hemberger,1985 should not be synonyms,and both genera should be subdivided due to their variable morphological characteristics;(4)LVR originating from three anlagen is a plesiomorphy of Strongylidiidae.The discovery of the origin of the LVR not only contributes to the establishment of the genus Heterouroleptus,but also helps to improve the diagnosis of the family Strongylidiidae.展开更多
The conventional theory of concerted evolution has been used to explain the lack of sequence variation in ribosomal RNA(rRNA)genes across diverse eukaryotic species.However,recent investigations into rRNA genes in fla...The conventional theory of concerted evolution has been used to explain the lack of sequence variation in ribosomal RNA(rRNA)genes across diverse eukaryotic species.However,recent investigations into rRNA genes in flatfish genome have resulted in controversial findings.This study focuses on 18S rRNA genes of the widely distributed tongue sole,Cynoglossus abbreviatus(Pleuronectiformes:Cynoglossidae),aiming to explore sequence polymorphism.Five distinct 18S rDNA sequence types(Type A,B,R1,R2,and R3)were identified,suggesting a departure from concerted evolution.A combination of general criteria and variations in highly conserved regions were employed to detect pseudogenes.The results pinpointed Type A sequences as potential pseudogenes due to significant sequence variations and deviations in secondary structure within highly conserved regions.Three types(Type R1,R2,and R3)were identified as recombinants between Type A and B sequences,with simple crossing over and gene conversion as the most likely recombination mechanisms.These findings not only contribute to rRNA pseudogene identification but also shed light on the evolutionary dynamics of rRNA genes in teleost genomes.展开更多
Chaetoceros Ehrenberg is one of the most diverse genera of planktonic diatoms.The species in section Chaetoceros are characterized by cells and setae having numerous chloroplasts and being widely distributed.However,t...Chaetoceros Ehrenberg is one of the most diverse genera of planktonic diatoms.The species in section Chaetoceros are characterized by cells and setae having numerous chloroplasts and being widely distributed.However,the delimitations of some species are problematic because of limited morphological information in the classical descriptions.Monoclonal strains of the section Chaetoceros were established,morphological features were studied using light and electron microscopy,and the hypervariable D 1-D 3 region of the nuclear ribosomal large subunit gene was sequenced to address phylogenetic relationships.Fifteen species belonging to the section Chaetoceros were recorded,including two new species,C.hainanensis sp.nov.and C.tridiscus sp.nov.Chaetoceros hainanensis was characterized by straight chains,narrowly lanceolate to hexagonal apertures,sibling setae diverging in nearly right angles,stipule-shaped spines on terminal setae and arrowhead-shaped spines on intercalary setae.C.tridiscus had short straight chains,narrowly lanceolate apertures,arrowhead-shaped spines and circular poroids arranged in a grid pattern on terminal and intercalary setae.The phylogenetic analyses revealed six groups formed by 19 species within the section Chaetoceros,which was found to be monophyletic.The subdivision of the section is still not well understood.The morphological characters within each group varied considerably and molecular information on more species are needed to enrich the phylogenetic profiling.展开更多
The 18S ribosomal DNA gene （18S rDNA） sequences （approxtmately 1300 bp in length） were amplified from the DNA extracted from the free-living marine nematodes collected from the inter-tidal sediment of Qingdao coas...The 18S ribosomal DNA gene （18S rDNA） sequences （approxtmately 1300 bp in length） were amplified from the DNA extracted from the free-living marine nematodes collected from the inter-tidal sediment of Qingdao coast in bulk with nematode specific primers. The PCR products were cloned, re-amplified, digested with Rsa I and Hin61 restriction endonucleases and separated in agarose gel. Among 17 restriction fragment length types, types 1, 2 and 6 covered 61.2%, 14.4% and 9.3% of the clones analyzed, respectively, while the remaining 14 only covered 21 clones, which accounted for 15.1% of the total. Twenty-four representative clones were sequenced and phylogenetically analyzed by referring to those currently available in RDP and GenBank databases. Although it was hard to assign these sequences to known species or genera due to the lack of the 18S rDNA sequence data of known marine free-living nematodes, the obtained sequences were assigned to the nematodes of Adenophorea. Among them, twelve sequences were close to Pontonema vulgate and Adoncholaimus sp., four to Daptonema procerus and two （identical） to Enoplus brews. Our results showed that free-living marine nematode diversities could be determined by PCR retrieving and analysis of the 18S rDNA sequences and an 18S rDNA sequence could be assigned to a species or a genus only if the 18S rDNA sequences of the free-living marine nematodes were accumulated to some extent.展开更多
文摘A study was conducted on the identifications of the degraded samples of sika deer （Cervus nippon） and red deer （Cervus elaphus） by phylogenetic and nucleotide distance analysis of partial Cytb and 12s rRNA genes sequences. 402 bp Cytb genes were achieved by PCR-sequencing using DNA extracted from 8 case samples, and contrasted with 27 sequences of Cytb gene downloaded from GenBank database. The values of three nucleotide distance between three suspected samples and sika deer were identical （0.026±0.006）, which was smaller than the smallest nucleotide distance between eastern red deer and sika deer （0.036）. Furthermore, phylogenetic analysis of sika deer and red deer indicated that the evidences located within the same cluster as sika deer. The evidences were sika deer materials. As the same way, other three suspected samples were derived from red deer. The results were further confirmed by phylogenetic and nucleotide distance analysis of 387 bp 12s rRNA gene. The method was powerful and less time-consuming and helpful to reduce the related cases with wildlife.
基金supported by the National Natural Science Foundation of China(Grant number 32001876).
文摘A new COX1 primer for soil nematode metabarcoding was designed,and this primer outperforms other commonly used COX1 primer pairs in species recovery and quantity of PCR products.•The lack of reference database is the main reason that led to the low species recovery in COX1 metabarcoding.•We expanded current NCBI database by adding 51 newly generated COX1 reference sequences.Microscopic nematodes play important roles in soil ecosystems and often serve as bioindicators of soil health.The identification of soil nematodes is often difficult due to their limited diagnostic characters and high phenotypic plasticity.DNA barcoding and metabarcoding techniques are promising but lack universal primers,especially for mitochondrial COX1 gene.In this study a degenerated COX1 forward primer COIFGED was developed.The primer pair(COIFGED/JB5GED)outperforms other four commonly used COX1 primer pairs in species recovery and quantity of polymerase chain reaction(PCR)products.In metabarcoding analysis,the reads obtained from the new primer pair had the highest sequencing saturation threshold and amplicon sequence variant(ASV)diversity in comparison to other COX1 as well as 18S rRNA primers.The annotation of ASVs suggested the new primer pair initially recovered 9 and 6 out of 25 genera from mock communities,respectively,outperformed other COX1 primers,but underperformed the widely used 18S NF1/18Sr2b primers(16 out of 25 genera).By supplementing the COX1 database with our reference sequences,we recovered an additional 6 mock community species bringing the tally closer to that obtained with 18S primers.In summary,our newly designed COX1 primers significantly improved species recovery and thus can be supplementary or alternative to the conventional 18S metabarcoding.
基金funded by the Science&Technol-ogy Innovation Project of Laoshan Laboratory(No.LSKJ202203201)National Natural Science Foundation of China(No.32030015,32370475)+1 种基金Researchers Supporting Project(No.RSP2024R10)King Saud University,Riyadh,Saudi ArabiaGuangdong Province Youth Innovative Talent Project 2022(2022KQNCX107).
文摘Ciliates in the subclass Hypotrichia have long been difficult to classify as they are one of the most polymorphic and highly differentiated groups,leading to their systematics remaining unresolved.Phylogenetic relationships within the hypotrich family Strongylidiidae have been ambiguous due to discordance between the morphological and genetic data.In this study,a new strongylidiid genus Heterouroleptus is established,mainly based on the novel mode of origin of the ventral cirral rows:left ventral cirral row(LVR)originates from frontal-ventral-transverse cirral anlagen(FVTA)Ⅲ(anterior portion),IV(middle portion),and V(rear portion);right ventral cirral row comes from the entire FVTA VI.A new species,Hetero-uroleptus weishanensis gen.nov.,sp.nov.,is investigated along with the morphometric and molecular data from a population of Strongylidium wuhanense.Eight new sequences and nuclear gene markers(single-gene and multi-gene)are provided to analyze the phylogenetic relationships of strongylidiids,with the COI gene utilized to uncover further genetic information at species level and below.The results reveal that:(1)Strongylidiidae is monophyletic and has a close relationship with Dorsomarginalia;(2)Heterouroleptus gen.nov.forms a clade that is sister to all the other strongylidiids;(3)Hemiamphisiella Foissner,1988 and Pseudouroleptus Hemberger,1985 should not be synonyms,and both genera should be subdivided due to their variable morphological characteristics;(4)LVR originating from three anlagen is a plesiomorphy of Strongylidiidae.The discovery of the origin of the LVR not only contributes to the establishment of the genus Heterouroleptus,but also helps to improve the diagnosis of the family Strongylidiidae.
基金The Basic Scientific Research Operating Expenses of Zhejiang Provincial Universities under contract 2021JZ003the Zhoushan Science and Technology Bureau under contract No.2021C21007+1 种基金the Natural Science Foundation of Zhejiang Province under contract Y21C190023the National Natural Science Foundation of China under contract 31272273.
文摘The conventional theory of concerted evolution has been used to explain the lack of sequence variation in ribosomal RNA(rRNA)genes across diverse eukaryotic species.However,recent investigations into rRNA genes in flatfish genome have resulted in controversial findings.This study focuses on 18S rRNA genes of the widely distributed tongue sole,Cynoglossus abbreviatus(Pleuronectiformes:Cynoglossidae),aiming to explore sequence polymorphism.Five distinct 18S rDNA sequence types(Type A,B,R1,R2,and R3)were identified,suggesting a departure from concerted evolution.A combination of general criteria and variations in highly conserved regions were employed to detect pseudogenes.The results pinpointed Type A sequences as potential pseudogenes due to significant sequence variations and deviations in secondary structure within highly conserved regions.Three types(Type R1,R2,and R3)were identified as recombinants between Type A and B sequences,with simple crossing over and gene conversion as the most likely recombination mechanisms.These findings not only contribute to rRNA pseudogene identification but also shed light on the evolutionary dynamics of rRNA genes in teleost genomes.
基金Supported by the Joint Fund of National Natural Science Foundation of China and Chinese Shandong Province(No.U 2106205)the National Natural Science Foundation of China(No.32170206)the National Key Research and Development Program of China(No.2022YFC3105201)。
文摘Chaetoceros Ehrenberg is one of the most diverse genera of planktonic diatoms.The species in section Chaetoceros are characterized by cells and setae having numerous chloroplasts and being widely distributed.However,the delimitations of some species are problematic because of limited morphological information in the classical descriptions.Monoclonal strains of the section Chaetoceros were established,morphological features were studied using light and electron microscopy,and the hypervariable D 1-D 3 region of the nuclear ribosomal large subunit gene was sequenced to address phylogenetic relationships.Fifteen species belonging to the section Chaetoceros were recorded,including two new species,C.hainanensis sp.nov.and C.tridiscus sp.nov.Chaetoceros hainanensis was characterized by straight chains,narrowly lanceolate to hexagonal apertures,sibling setae diverging in nearly right angles,stipule-shaped spines on terminal setae and arrowhead-shaped spines on intercalary setae.C.tridiscus had short straight chains,narrowly lanceolate apertures,arrowhead-shaped spines and circular poroids arranged in a grid pattern on terminal and intercalary setae.The phylogenetic analyses revealed six groups formed by 19 species within the section Chaetoceros,which was found to be monophyletic.The subdivision of the section is still not well understood.The morphological characters within each group varied considerably and molecular information on more species are needed to enrich the phylogenetic profiling.
基金This study was supported financially by the National Natural Science Foundation of China （No. 40176028）.
文摘The 18S ribosomal DNA gene （18S rDNA） sequences （approxtmately 1300 bp in length） were amplified from the DNA extracted from the free-living marine nematodes collected from the inter-tidal sediment of Qingdao coast in bulk with nematode specific primers. The PCR products were cloned, re-amplified, digested with Rsa I and Hin61 restriction endonucleases and separated in agarose gel. Among 17 restriction fragment length types, types 1, 2 and 6 covered 61.2%, 14.4% and 9.3% of the clones analyzed, respectively, while the remaining 14 only covered 21 clones, which accounted for 15.1% of the total. Twenty-four representative clones were sequenced and phylogenetically analyzed by referring to those currently available in RDP and GenBank databases. Although it was hard to assign these sequences to known species or genera due to the lack of the 18S rDNA sequence data of known marine free-living nematodes, the obtained sequences were assigned to the nematodes of Adenophorea. Among them, twelve sequences were close to Pontonema vulgate and Adoncholaimus sp., four to Daptonema procerus and two （identical） to Enoplus brews. Our results showed that free-living marine nematode diversities could be determined by PCR retrieving and analysis of the 18S rDNA sequences and an 18S rDNA sequence could be assigned to a species or a genus only if the 18S rDNA sequences of the free-living marine nematodes were accumulated to some extent.
