Sympotthastia annularis sp. nov. from Oriental China (Diptera: Chironomidae: Diamesinae) with a worldwide key

Dear Editor, Multiple infections with small liver flukes and minute intestinal flukes are the serious public health concern in the lower Mekong basin [1,2]. Although the epidemiological survey for those trematode infections are primarily carried out based on copro-parasitological examination, detection/identification of fecal eggs/worms is a tedious job and often problematic because of the morphological similarities of eggs/worms. Along with the popularization of PCR-sequencing methods, copro-DNA diagnosis and molecular phylogenetic identification/speciation have been introduced in epidemiological studies. Among various genes and non-coding lesions of nuclear and mitochondrial DNAs, mitochondrial cytochrome c oxidase subunit I (COXI) is one of the most widely used inter- and intra-species marker. Using COXI and some other markers, Lee and his colleagues performed molecular phylogenetic analyses on small liver flukes (Lee SU, Huh S. Variation of nuclear and mitochondrial DNAs in Korean and Chinese isolates of Clonorchis sinensis. Korean J Parasitol 2004; 42: 145-148) and on minute intestinal flukes (Lee SU, Huh S, Sohn WM, Chai JY. Sequence comparisons of 28S ribosomal DNA and mitochondrial cytochrome c oxidase subunit I of Metagonimus yokogawai, M. takahashii and M. miyatai. Korean J Parasitol 2004; 42: 129-135). The COX1 gene sequences appeared in those articles are; Clonorchis sinensis ({type:entrez-nucleotide,attrs:{text:AF184619,term_id:296940320,term_text:AF184619}}AF184619, {type:entrez-nucleotide,attrs:{text:AF181889,term_id:285026682,term_text:AF181889}}AF181889, {type:entrez-nucleotide,attrs:{text:AF188122,term_id:285809842,term_text:AF188122}}AF188122), Metagonimus yokogawai ({type:entrez-nucleotide,attrs:{text:AF096230,term_id:297039733,term_text:AF096230}}AF096230), Metagonimus takahashii ({type:entrez-nucleotide,attrs:{text:AF096231,term_id:297039734,term_text:AF096231}}AF096231), Metagonimus miyatai ({type:entrez-nucleotide,attrs:{text:AF096232,term_id:297039735,term_text:AF096232}}AF096232), Pygidiopsis summa ({type:entrez-nucleotide,attrs:{text:AF181884,term_id:288563287,term_text:AF181884}}AF181884), and Stellantchasmus falcatus ({type:entrez-nucleotide,attrs:{text:AF181887,term_id:285804435,term_text:AF181887}}AF181887). In addition, Park [3] compared his COXI sequence of Opisthorchis viverrini Laotian isolate (AY055 382) to those of Gymnophalloides seoi ({type:entrez-nucleotide,attrs:{text:AF096234,term_id:297039736,term_text:AF096234}}AF096234) and Neodiplostomum seoulense ({type:entrez-nucleotide,attrs:{text:AF096233,term_id:285026845,term_text:AF096233}}AF096233) registered in the DNA database (Lee et al. unpublished). For the phylogenetic analyses of COXI gene of minute intestinal flukes of our own data, we have downloaded all those above mentioned COXI of Lee et al. and aligned them including our own COXI sequence of Haplorchis taichui ({type:entrez-nucleotide,attrs:{text:EF055885,term_id:119855482,term_text:EF055885}}EF055885) [4] and Paragonimus bangkokensis ({type:entrez-nucleotide,attrs:{text:AB354227,term_id:155369203,term_text:AB354227}}AB354227) [5]. Surprisingly, those sequence data were divided into 2 distinct groups without any similarities (Fig. 1). Eventually, we realized that this astonishing result is due to the reverse complementary sequences of COXI data deposited by Lee et al. (in the bottom half of the figure). We also noticed similar mixed-up deposition of the forward and reverse sequences of COXI gene of Fasciola spp., which were also included in Fig. 1 ({type:entrez-nucleotide,attrs:{text:AJ628024,term_id:88319716,term_text:AJ628024}}AJ628024, {type:entrez-nucleotide,attrs:{text:AJ628039,term_id:88319746,term_text:AJ628039}}AJ628039, {type:entrez-nucleotide,attrs:{text:FJ469984,term_id:238631966,term_text:FJ469984}}FJ469984; Zhu XQ et al. unpublished). Fig. 1 The DNA sequence alignment of the partial COXI gene of some trematodes obtained from the GenBank. Seven sequences from the top are the forward strands with JB3 primer sequence, and 2 in the middle are the forward strands without primer. Eight sequences ... For the determination of partial COX1 sequences of Platyhelminthes, the primer set of JB3 (5'-TTT TTT GGG CAT CCT GAG GTT TAT-3') and JB4.5 (5'-TAA AGA AAG AAC ATA ATG AAA ATG-3') [6] was widely used for investigating the inter- and intra-species variations of trematodes and cestodes. We noticed the mixed-up of the forward and reverse COXI sequences by Lee et al. as well as Zhu et al. because of the presence of the characteristic feature of this primer set (boxed in Fig. 1) in the sequences. The primer sequence should be deleted from the sequence data because it is not always identical with the real DNA sequence of the gene and the inclusion of the primer sequences sometimes causes the misreading in phylogenetic analyses [7]. In 3 reverse sequences, {type:entrez-nucleotide,attrs:{text:AF181884,term_id:288563287,term_text:AF181884}}AF181884, {type:entrez-nucleotide,attrs:{text:AY055380,term_id:22203992,term_text:AY055380}}AY055380, and {type:entrez-nucleotide,attrs:{text:AF096233,term_id:285026845,term_text:AF096233}}AF096233 seems to contain also the partial sequence of the cloning vector, which should be trimmed off before deposition. In general, raw data of forward and reverse sequences obtained from the sequencer should be aligned manually by cross-checking of the wave patterns because some 10-20 bases downstream from the forward primer and upstream from the reverse primer often contain erroneous base pairs [8]. Deposition of the reverse sequence means that those sequences were not aligned against forward sequence and not quite reliable. Since each sequence data in GenBank are opened for the public use, an accuracy of the sequence data is critically important for the mutual reliability of the scientists. The scientists should aware how to deposit accurate sequence data to the DNA data base. The reappraisal and correction of those sequences mentioned above is urgently necessary.

Molecular Taxonomy of Serranidae, Subfamily Epinephelinae, Genus Plectropomus (Oken, 1817) of Andaman Waters by DNA Barcoding Using COI Gene Sequence

Co-overexpression of the epidermal growth factor (EGF) receptor (EGFR) and c-Src frequently occurs in human tumors and is linked to enhanced tumor growth. In experimental systems this synergistic growth requires EGF-dependent association of c-Src with the EGFR and phosphorylation of Tyr-845 of the receptor by c-Src. A search for signaling mediators of Tyr(P)-845 revealed that mitochondrial cytochrome c oxidase subunit II (CoxII) binds EGFR in a Tyr(P)-845- and EGF-dependent manner. In cells this association involves translocation of EGFR to the mitochondria, but regulation of this process is ill-defined. The current study demonstrates that c-Src translocates to the mitochondria with similar kinetics as EGFR and that the catalytic activity of EGFR and c-Src as well as endocytosis and a mitochondrial localization signal are required for these events. CoxII can be phosphorylated by EGFR and c-Src, and EGF stimulation reduces Cox activity and cellular ATP, an event that is dependent in large part on EGFR localized to the mitochondria. These findings suggest EGFR plays a novel role in modulating mitochondrial function via its association with, and modification of CoxII.

The Satyrinae is a subfamily of Nymphalid butterfly, which is morphologically and ecologically the most diverse group, occurring in all habitats. In the present study, Cytochrome c oxidase subunit I (COI) gene of seven species of Satyrinae was sequenced, aligned, and used to construct phylogenetic trees. The molecular identification of these Satyrinae species was confirmed by comparing the related sequences in the National Center for Biotechnology Information (NCBI) GenBank. The base compositions of the COI sequences were 39.07% T, 16.44% C, 29.83% A, and 14.64% G, revealing a strong AT bias (68.9%). The sequence distance among Satyrinae species ranged from 0.09% to 0.18%. Phylogenetic trees were constructed by the neighbor-joining (NJ) and maximum likelihood (ML) methods, using Orthetrum sabina as an outgroup. Both trees had almost identical topologies. The sampled species in Satyrinae exhibited the following relationships: Melanitis leda + [(Mycalesis mineus+(Mycalesis gotama+Mycalesis anaxias)) + (Ypthima baldus + (Lethe chandica+Elymnias hypermnestra))], suggesting that M. leda might be distantly related with the rest of the Satyrinae species. This clustering result is almost identical to current traditional classification. This study confirms that the COI based DNA barcoding is an efficient method for the identification of butterflies including Satyrinae species and, as such, may further contribute effectively to biodiversity and evolutionary research.
 Jahangirnagar University J. Biol. Sci. 8(1): 67-74, 2019 (June)

Among the Ixodid ticks, Hyalomma anatolicum is a well-known vector that transmits various pathogens to terrestrial and semi-terrestrial vertebrates including humans, and its population differ in ecology and vector competence. Expansion of this tick to new areas changes the genetic structure, and lead to affect the vector-pathogen interaction and disease outcomes. The present study was designed to infer the haplotype diversity, demographic dynamics, gene flow and genetic differentiation, and phylogeny of H. anatolicum from different countries based on the cytochrome oxidase I (COI) and 16S rDNA sequences. A total of 320 ticks were collected from cattle, buffaloes, and sheep in five districts of Khyber Pakhtunkhwa, Pakistan, morphologically identified as H. anatolicum, and subjected to genetic analysis. A total 85 and 138 sequences for COI and 16S rDNA, including 11 and 2 sequences generated in this study, respectively, were analyzed to assess haplotype network, population structure and divergence, demographic changes, and phylogenetic analysis. Analysis based on COI sequences yielded 29 haplotypes in which haplotype 1 and 15 were the predominant consisting of 35 and 20 sequences, respectively, from Pakistan, India, China, Bangladesh, Iraq, Saudi Arabia, Kazakhstan and Egypt. The 16S rRNA yielded 30 haplotypes in which haplotype 1 was predominant consisting of total 86 sequences from Pakistan, India, China, United Arab Emirates, Tajikistan, Kazakhstan, Turkey, Egypt, and Iraq. Complete haplotype network based on COI and 16S rRNA confirmed stellate structure, together with high haplotype diversity (COI 0.77899, 16S rRNA 0.60774) and low nucleotide diversity (COI 0.00445, 16S rRNA 0.00431), which support recent population expansion. Similarly, neutrality indices for the whole dataset, Tajima’s D (COI − 2.36363**, 16S rRNA − 2.54127***), Fu and Li’s D (COI − 5.72992, 16S rRNA − 6.31313*), and Fu and Li’s F (COI − 5.04435*, 16S rRNA − 5.56085*) were negative, indicating deviation from neutrality and recent population dispersal. In the phylogenetic tree based on the COI and 16S rDNA sequences, with exception of one sequence for a single haplotypes, which appeared independently, there is a single main clade that includes the largest number of sequences for all other haplotype. Based on COI and 16S rDNA sequences, the present study provided first detail information about the population genetics and haplotype networks of H. anatolicum.

With their diverse species, mosquitoes are known to transmit the causal agents of diseases such as malaria, dengue, and yellow fever. Their high adaptability, attraction to humans, and variable adult behaviors make them a significant health concern. The focus on Aedes aegypti is significant for reducing vector-human contacts, monitoring insecticide resistance, and developing innovative vector management strategies. Given the scarcity of studies on Ae. aegypti in the western region of Saudi Arabia, this research is a significant step forward. The study aims to analyze the genetic variations and conduct a phylogenetic study of forty Ae. aegypti samples collected from Taif and Jeddah governorates of Saudi Arabia. The mitochondrial cytochrome c oxidase subunit I (COI) locus was targeted for genetic variance and phylogenetic analysis. Sequences of COI of Ae. aegypti isolates were submitted to the DNA Data Bank of Japan (DDBJ) and National Center for Biotechnology Information (NCBI) Genbank and compared with other global Aedes species isolates. The phylogenetic analysis shows that Ae. aegypti samples from Jeddah have identities ranging from 96.9% to 99.8%, closely related to the Peru (MN299016) and Cambodia (MN299014) isolates.Taif isolates have genetic similarities ranging from 97.5% to 99.8%, closely related to theGermany (KY022526) isolate. Sequence alignment and pairwise comparison show variation among the populations of Ae. aegypti from Taif and Jeddah regions (74.24-98.84%) with a genetic divergence distance of 0.008-0.12. In comparison, ranges slightly change with other Ae. aegypti (79.92-95.96%, 0.008-0.01) as well as Ae. albopictus populations (74.13-83.58%, 0.13-0.20) found in the Genbank database. According to our findings, the present study provides information for a local variation of Ae. aegypti in the western region of Saudi Arabia that could help in disease mapping and risk mitigation, thereby enhancing our ability to manage disease vectors effectively.

Objective To make up the limitations of traditional morphological classification methods, we identified vector fleas by DNA barcoding in Qinghai Province. Methods The mt DNA cytochrome c oxidase subunit Ⅰ(COⅠ) gene was amplified by PCR from 36 muscle tissues of fleas in 3 states, 2 cities and 5 counties of Qinghai Province, and the obtained COⅠ gene fragments were sequenced and aligned. The intra- and inter-species genetic distances were calculated with Mega 6 software using K2-P model and a phylogenetic tree was constructed with neighbor-joining (NJ) method. Results Totally 36 COⅠ gene sequences of 2 superfamilies, 4 genera and 6 kinds of vector fleas were measured, the average genetic distance was 0.119, and the intraspecific distance was 0.002 - 0.027, the interspecific distance was 0.039 - 0.207, and the interspecific genetic distance was significantly greater than the intraspecific genetic distance. NJ tree showed the same species had formed a single line with high support rate and interspecific branch was clear. Conclusion DNA barcoding is suitable for identification of vector fleas in Qinghai Province, may make up the limitations of traditional morphological classification methods. Key words: Siphonaptera; Cytochrome c oxidase subunit Ⅰ; DNA barcoding

Muscid Flies (Diptera: Muscidae) are of great forensic importance due to their wide distribution, ubiquitous and synanthropic nature. They are frequently neglected as they tend to arrive at the corpses later than the flesh flies and blow flies. Moreover, the lack of species-level identification also hinders investigation of medicolegal purposes. To overcome the difficulty of morphological identification, molecular method has gained relevance. Cytochrome c oxidase subunit I (COI) gene has been widely utilized. Nonetheless, to achieve correct identification of an unknown sample, it is important to survey certain muscid taxa from its geographic distribution range. Accordingly, the aim of this study is to contribute more geographically specific. We sequenced the COI gene of 51 muscid specimens of 12 species, and added all correct sequences available in GenBank to yield a total data set of 125 COI sequences from 33 muscid species to evaluate the COI gene as a molecular diagnostic tool. The interspecific distances were extremely high (4.7-19.8%) in either the standard barcoding fragment (658 bp) or the long COI sequence (1,019-1,535 bp), demonstrating that these two genetic markers were nearly identical in the species identification. However, the intraspecific distances of the long COI sequences were significantly higher than the barcoding region for the conspecific species that geographical locations vary greatly. Therefore, genetic diversity presented in this study provides a reference for species identification of muscid flies. Nevertheless, further investigation and data from more muscid species are required to enhance the efficacy of species-level identification using COI gene as a genetic marker.

Dragonflies are insects in the order Odonata. They inhabit freshwater ecosystems and are found in the UAE. To date, few checklists have been published for the local dragonflies and the used identification keys are not comprehensive of Arabia. The aim of this study was to provide a molecular identification of a dragonfly based on the mitochondrial Cytochrome c Oxidase subunit I (COI) gene using the National Center for Biotechnology Information (NCBI) database and the Barcode of Life Data Systems (BOLD) in comparison with the morphology. The insect’s DNA was extracted and the PCR was performed on the target gene. The insect was identified initially as Anax imperator based on the NCBI database and as Anax parthenope based on the BOLD. However, the morphological identification was in agreement with the one produced by the BOLD. The results of this study is a demonstration of how, in some cases, the DNA-based identification does not provide a conclusive species designation and that a morphology-based identification is needed.

한국산 붉바리 집단에서 유전적 구조와 계통 유연관계를 mtDNA COI 유전자 서열의 다형성을 이용하여 조사하였다. COI 유전자 서열을 결정하였고 기존에 보고된 서열들과 비교하였다. 본 연구를 통해 결정된 COI 서열들은 기존에 보고된 EF607565에 대하여 99.1-99.8%의 동일성을 나타내었다. 전체 20가지의 haplotype들이 발견되었고, 한국산 붉바리 집단은 19가지의 haplotype을 나타내었다. 이들 중 Hap_03과 Hap_08은 각각 제주도와 중국-특이적인 COI 서열들을 보였다. 반면, Hap_07은 한국에서 채집된 시료들과 홍콩과 대만에서 보고된 기록 등 여러 COI 서열들을 포함하였다. COI haplotype들의 다형성에 근거한 계통 유전학적 분석을 통해 작성된 NJ tree는 Epinephelus 속 내에서 단계통적인 분지양상을 나타내었고, 이는 붉바리 집단들이 공통의 모계 선조에서 진화한 것임을 나타내었다. 또한 중국해에서 보고된 COI 서열만을 포함하였던 Hap_08은 NJ tree의 중앙부에서 위치하였고, Hap_07의 서열들과도 근연의 관계임을 보여주었다. 이 결과는 중국산 붉바리 역시 동아시아의 다른 집단들과 모계적으로 연관되어있음을 보여주었다. 결과적으로, 동아시아 붉바리 집단들은 모계적으로 연관되어있을 뿐만 아니라 공통의 진화 역사를 공유하고 있으며 여전히 동아시아 해류(Kuroshio 해류)에 의해 영향을 받는 집단이라고 할 수 있다. 본 연구는 붉바리의 유전적 구조와 계통 유연관계를 이해하는 데 도움을 줄 수 있으며, 인공증식과 산업화에 관련된 연구에 있어 중요한 역할을 담당할 것으로 기대된다. The genetic structure and phylogenetic relationship were investigated in Korean red spotted grouper populations using the nucleotide sequence polymorphisms of the mitochondrial DNA (mtDNA) cytochrome c oxidase subunit I (COI) gene. The COI gene was sequenced showed 99.1-99.8% identity with the EF607565 sequence previously reported. A total of twenty haplotypes were found, and the Korean population showed nineteen haplotypes. Among those, Hap_03 and Hap_08 showed Jeju-do and China-specific COI sequences, respectively. However, Hap_07 had twelve COI sequences from South Korea and records from Hong Kong and Taiwan. Neighbor-joining (NJ) trees constructed from the phylogenetic analyses based on the polymorphisms of the COI haplotypes showed a monophyletic branching pattern within the genus Epinephelus. This indicated that the red spotted grouper populations had evolved from common maternal ancestors. In addition, the Hap_08, which had the COI sequence recorded only from China Sea, was found in the middle of the NJ tree nearby Hap_07 and showed a close relationship with Hap_07. This indicates that Chinese red spotted grouper is also maternally related to other populations in East Asia. Consequently, East Asian red spotted grouper populations are maternally related, as well as sharing the same evolutionary history, and are still affected by the East Asian ocean current (Kuroshio). These findings help to explain the genetic structure and phylogenetic relationship of red spotted grouper and also contribute to research on artificial breeding and industrialization.

DNA barcoding identifies Eimeria species and contributes to the phylogenetics of coccidian parasites (Eimeriorina, Apicomplexa, Alveolata)

The genus Merodon Meigen, 1803 (Syrphidae, Diptera), with more than 50 European species, is primarily distributed in the Mediterranean region, there being 34 species that occur in the Iberian Peninsula. The morphological variation found within some species from the Iberian Peninsula prompted us to test their taxonomic status by integrating morphological and molecular data. We generated partial sequences of the mitochondrial, protein-coding gene cytochrome c oxidase subunit I (COI), the nuclear, internal transcribed spacer (ITS2) region, and the D2 region of the nuclear 28S rRNA gene. COI and ITS2 sequences were obtained for most included taxa. The variability of the COI sequences showed great differences between the studied species groups, exhibiting an interspecific range from 0.29% to 12.5% between ingroup taxa. Closely related taxa of the aureus complex (e.g. M. quercetorum and M. legionensis) presented identical COI sequences. The obtained ITS2 sequences showed low intraspecific variability, and only a few taxa presented more than one genotype. Species status and delimitation were discussed for all taxa in the light of available morphological and molecular character information. Using the obtained sequence data for COI and 28S we inferred the phylogenetic relationships of the included taxa, using parsimony analysis. Separate analysis of the COI sequences identified four, quite wellsupported clades within Merodon, the desuturinus, albifrons, nigritarsis and aureus groups. Combined analysis of the COI and 28S genes produced a topology similar to the COI topology.

Sand fly species are traditionally identified using morphological traits, though this method is hampered by the presence of cryptic species. DNA barcoding is a widely used tool in the case of insects of medical importance, where it is necessary to know quickly which species are present in a transmission area. Here, we assess the usefulness of mitochondrial cytochrome c oxidase subunit I (COI) DNA barcoding as a practical tool for species identification, correct assignment of isomorphic females, and to evaluate the detection of cryptic diversity that occurs in the same species. A fragment of the COI gene was used to generate 156 new barcode sequences for sand flies from different countries of the Neotropical region, mainly Colombia, which had been identified morphologically as 43 species. The sequencing of the COI gene allowed the detection of cryptic diversity within species and correctly associated isomorphic females with males identified by morphology. The maximum intraspecific genetic distances ranged from 0 to 8.32% and 0 to 8.92% using uncorrected p distances and the Kimura 2-parameter (K2P) model, respectively. The minimum interspecific distance (nearest neighbor) for each species ranged from 1.5 to 14.14% and 1.51 to 15.7% using p and K2P distances, respectively. Three species had more than 3% maximum intraspecific distance: Psychodopygus panamensis, Micropygomyia cayennensis cayennensis, and Pintomyia evansi. They also were split into at least two molecular operational taxonomic units (MOTUs) each, using different species delimitation algorithms. Regarding interspecific genetic distances, the species of the genera Nyssomyia and Trichophoromyia generated values lower than 3% (except Nyssomyia ylephiletor and Ny. trapidoi). However, the maximum intraspecific distances did not exceed these values, indicating the presence of a barcode gap despite their proximity. Also, nine sand fly species were DNA barcoded for the first time: Evandromyia georgii, Lutzomyia sherlocki, Ny. ylephiletor, Ny. yuilli pajoti, Psathyromyia punctigeniculata, Sciopemyia preclara, Trichopygomyia triramula, Trichophoromyia howardi, and Th. velezbernali. The COI DNA barcode analysis enabled the correct delimitation of several Neotropical sand fly species from South and Central America and raised questions about the presence of cryptic species for some taxa, which should be further assessed.Graphical

DNA barcoding of Bryozoa or “moss animals” has hardly advanced and lacks reference sequences for correct species identification. To date only a small number of cytochrome c oxidase subunit I (COI) sequences from 82 bryozoan species have been deposited in the National Center for Biotechnology Information (NCBI) GenBank and Barcode of Life Data Systems (BOLD). We here report COI data from 53 individual samples of 29 bryozoan species collected from Korean seawater. To our knowledge this is the single largest gathering of COI barcode data of bryozoans to date. The average genetic divergence was estimated as 23.3% among species of the same genus, 25% among genera of the same family, and 1.7% at intraspecific level with a few rare exceptions having a large difference, indicating a possibility of presence of cryptic species. Our data show that COI is a very appropriate marker for species identification of bryozoans, but does not provide enough phylogenetic information at higher taxonomic ranks. Greater effort involving larger taxon sampling for the barcode analyses is needed for bryozoan taxonomy.

The mitochondrial cytochrome c oxidase subunit 1 (COI) gene of ciliates was first successfully sequenced in species of the genera Tetrahymena and Paramecium (Class Oligohymenophorea). The sequence of the COI gene is extremely divergent from other eukaryotes and includes an insert, which is over 300 nucleotides long. In this study, we designed a primer pair that successfully amplified the COI gene of ciliates from five different classes: Heterotrichea, Spirotrichea, Oligohymenophorea, Nassophorea and Colpodea. These classes represent the diversity of the phylum Ciliophora very well, since they are widely distributed on the ciliate small subunit rRNA tree. The amplified region is approximately 850 nucleotides long and corresponds to the general barcoding region; it also includes the insert region. In this study, 58 new COI sequences from over 38 species in 13 orders are analysed and compared, and distance trees are constructed. While the COI gene shows high divergence within ciliates, the insert region, which is present in all classes, is even more divergent. Genetic distances calculated with and without the insert region remain in the same range at the intraspecific level, but they differ considerably at or above genus level. This suggests that the entire barcoding region is under similar selective constraints and that the evolutionary rate of the ciliate COI is extremely high and shows unequal rate variation. Although many problems still remain regarding standardization of barcoding methods in ciliates, the development of a universal or almost universal primer combination for the Phylum Ciliophora represents important progress. As shown in four examples, the resolution of COI at the intraspecific level is much greater than that of any nuclear genes and shows great potential to (1) identify species based on molecular data if a reliable database exists, and (2) resolve the relationships of closely related ciliate taxa and uncover cryptic species.