Cytochrome C Oxidase Subunit I Region Research Articles

A comparative assessment of 16S ribosomal RNA and Cytochrome C Oxidase Subunit I (COI) Primers for Amphibian DNA Barcoding

Amphibians, a diverse and ecologically important group, are facing global declines due to various factors, including habitat loss and climate change. Accurate species identification is crucial for effective conservation efforts, and DNA barcoding has emerged as a powerful tool in this regard. This study compares the efficacy of two DNA barcoding primer sets, targeting the 16S ribosomal RNA gene and the Cytochrome Oxidase I (COI) gene, for identifying 20 amphibian species. While both primer sets successfully amplified sequences, the 16S rRNA gene region identified all 20 samples, whereas the COI region identified 14. The amplified sequences, approximately 550 base pairs for 16S rRNA and 658 base pairs for COI facilitated precise taxonomic placement within amphibian families using Neighbor-Joining phylogenetic trees. These findings enhance DNA barcoding methodology and aid in understanding amphibian diversity, crucial for effective conservation strategies amidst global declines drives by habitat loss, diseases, and climate change.

Evolutionary history of a cold-adapted limnephilid caddisfly: Effects of climate change and topography on genetic structure

The distribution of organisms is influenced by complex factors such as the phylogenetic evolutionary histories of species, the physiological and ecological characteristics of organisms, climate, and geographical and geohistorical features. In this study, we focused on a caddisfly, Asynarchus sachalinensis (Trichoptera: Limnephilidae), which has adapted to cold habitats. From phylogeographic analyses based on the mitochondrial DNA (mtDNA) cytochrome c oxidase subunit I (COI) and 16S rRNA regions and the nuclear DNA (nDNA) 18S rRNA, 28S rRNA, carbamoyl-phosphate synthetase (CAD), elongation factor-1 alpha (EF1-α), and RNA polymerase II (POLII) regions, two distinct genetic clades were detected. Clade I was shown to be widely distributed from Sakhalin to Honshu, whereas Clade II was only distributed within Honshu. The distributions of these clades overlapped in Honshu. The habitats were located at relatively lower altitudes for Clade I and higher altitudes for Clade II. The divergence time of these clades was estimated to be during the Pleistocene, indicating that repeated climatic changes facilitated distributional shifts. Haplotype network and demographic analyses based on the mtDNA COI region showed contrasting genetic structures in the two clades. It was indicated that the population sizes of Clade I had expanded rapidly in a recent period, whereas Clade II had maintained stable population sizes. The habitats of Clade II were typically isolated and scattered at high altitudes, resulting in restricted migration and dispersal because of their discontinuous “Sky Island” habitats. The habitats of Clade I were located at relatively low altitudes, and it was assumed that the populations were continuous, which resulted in a higher frequency of migration and dispersal between populations. Thus, differences in the spatial scale of the adapted habitats of each clade may have resulted in different patterns of population connectivity and fragmentation associated with repeated climatic changes during the Pleistocene. Our study provided new insight into the distributional patterns of cold-adapted aquatic insects in the Japanese Archipelago. Furthermore, the distributional shifts predicted by ecological niche modeling under future climatic change conditions were different for each clade. Therefore, different principles are required in the assessment of each clade to predict temporal changes in their distributions.

In Silico Evaluation of the Taxonomic Resolution and Coverage of the COI Region and Alternative Barcode Markers for Echinoderms

DNA barcoding methods are potentially useful for the detection and identification of species difficult to identify using morphology-based taxonomy, such as the larvae of marine taxa. Combining barcoding methods with massively parallel sequencing and low-cost DNA library preparation improves the feasibility of processing several samples but limits the taxonomic resolution of resulting sequences. In light of these limitations, we looked for suitable barcode regions for echinoderm larvae, first by examining the cytochrome c oxidase subunit I (COI) region, the most commonly used barcode for echinoderms. Using a dataset containing over 4000 COI sequences representing nearly 400 echinoderm species across all five extant classes, the COI gene was found to lack a sufficiently conserved region for barcoding. Taxonomic resolution was also found lacking, with no clear barcode gap across classes and the results of Automatic Barcode Gap Discovery (ABGD) failing to reflect current taxonomic assignments. While sequences may be mislabeled, the aggregation of multiple species in one ABGD partition suggests that course-grained taxonomic resolution may still result from using COI alone. The search for short alternative mitochondrial regions across 110 genomes of distinct species using ecoPrimers software revealed that class-specific primers targeting 12S and 16S ribosomal RNA (rRNA) regions are prime candidates for barcoding echinoderm sequences. The results of in silico PCR across the 110 mitochondrial genomes indicate that compared to existing COI primers, the candidate 12S and 16S primers yield similar taxonomic coverage, with slightly lower resolution. Taking into consideration the previous analysis of COI, we suggest the use of multiple markers (COI, 12S, 16S) to adequately capture the understudied diversity of echinoderm larvae. In vitro PCR of the 12S and 16S primers is also needed to validate the results of the in silico analyses.

MetaBARFcoding: DNA‐barcoding of regurgitated prey yields insights into Christmas shearwater (Puffinus nativitatis) foraging ecology at Hōlanikū (Kure Atoll), Hawaiʻi

AbstractMorphological identification of digested prey remains from a generalist predator can be challenging, especially when attempting to match degraded remains to taxonomic keys. DNA techniques, whereby prey is sequenced and matched to large public nucleotide sequence databases, are increasingly being used to augment morphological identification. We used “metaBARFcoding” (DNA metabarcoding) to target a region of the cytochrome c oxidase subunit I (COI) mitochondrial gene to identify prey in highly digested regurgitations from Christmas shearwaters Puffinus nativitatis at Hōlanikū (Kure Atoll). Metabarcoding was used to bulk‐process 92 water samples from regurgitations collected from 2009 to 2017, providing an overview of the seabird's diet. We additionally Sanger‐sequenced 100 prey items from 50 randomly chosen regurgitations to verify that metabarcoding characterized key components of the diet. The metabarcoding technique identified 87 unique taxa from 29 families of fish and squid, spanning diverse taxa, including reef‐associated, pelagic–oceanic, and mesopelagic species. Rare prey (frequency of occurrence ≤5% of samples) constituted 66% of the species richness, demonstrating the highly diverse diet of this generalist predator. Overall, 81% of the families detected in the contemporary diet were previously documented in Christmas shearwater diets from the Northwestern Hawaiian Islands. Our results indicate that metabarcoding the COI region is useful in identifying a wide range of taxa from highly digested regurgitations, thus facilitating this approach to study seabird diets.

Phylogeography and shell morphology of the pelagic snail Limacina helicina in the Okhotsk Sea and western North Pacific

The pelagic snail Limacina helicina Phipps, 1774 is widely distributed in high-latitude seas and is a sensitive bioindicator of ocean acidification. It is known that the response patterns to ocean acidification differ among populations within or among species. Thus, it is important to understand their genetic population structure and identify the cryptic species. L. helicina displays different shell morphotypes, called “formae,” in different regions (North Atlantic, L. helicina forma helicina Phipps, 1774; Northwest Pacific, L. helicina f. acuta Spoel, 1967; Northeast Pacific, L. helicina f. pacifica Dall, 1871; Okhotsk Sea, L. helicina f. ochotensis Shkoldina, 1999: Spoel, 1967, pp. 257, 349). A recent study by Shimizu et al. (Journal of Molluscan Studies 84:30-37, 2018) showed clear genetic differentiation between two formae, L. helicina forma helicina (Svalbard in the Arctic Ocean) and L. helicina f. acuta (station K2 in the western North Pacific) (ΦCT = 0.59282), using partial mitochondrial cytochrome c oxidase subunit I (COI) gene sequences. However, whether other formae of this species are genetically distinct remain unknown. Here we focused on L. helicina f. ochotensis, which inhabits the Sea of Okhotsk, and compared its shell morphology and partial mitochondrial COI gene sequences with another forma, L. helicina f. acuta, which is found in the western North Pacific. We found differences in the shell morphology of L. helicina f. ochotensis collected from the Sea of Okhotsk (off the coast of Monbetsu, Hokkaido, Japan) and L. helicina f. acuta collected from the western North Pacific (station K2). However, molecular analysis of an mtDNA COI region (503 bp) revealed that most individuals collected from Monbetsu (Sea of Okhotsk) were haplotype 1 (75.5%), which is also the major haplotype found in L. helicina f. acuta (western North Pacific). Our results suggest that individuals of L. helicina are frequently dispersed between the Sea of Okhotsk and the western North Pacific by ocean currents. Thus, the two formae, L. helicina f. acuta and L. helicina f. ochotensis, are more likely to be attributable to phenotypic plasticity in response to different environmental parameters, such as temperature and salinity, than to differences in their genetic backgrounds.

Cytochrome C Oxidase Subunit I and II (COI and COII) Sequences Support Recognition of Papilio chikae Igarashi and Papilio hermeli Nuyda as Distinct Species

Papilio chikae and P. hermeli are two endemic swallowtail butterflies in the Philippines found primarily in the Cordillera region and Mindoro Island, respectively. Their species status is still subject to debate due to opposing interpretations of their genitalia and morphological traits. This study aims to delineate these taxa through the use of mitochondrial cytochrome C oxidase subunit I(COI) gene marker, with additional information on their cytochrome C oxidase subunit II(COII) gene. The COI nucleotide sequences from our Philippine P. chikae samples were surprisingly identical to P. hermeliEF514465.1 on the GenBank, whereas our P. hermelisamples showed a 100% similarity with P. hermeliEF514464.1.Based on our phylogenetic analysis, P. chikae and P. hermeli exhibit distinct COI barcodes supported by a high bootstrap value (85%). The P. hermeli voucher specimen with GenBank Acc. No. EF514465.1 clustered with our P. chikae samples, separate from the cluster containing our P. hermelispecimens. Nonetheless, the 3.1–3.4% genetic distance observed in the COI region supported by the formation of two separate clusters suggests species status for these taxa. Moreover, species-specific molecular markers obtained from the COII region displayed a 2.4% distance between these two taxa. These molecular markers could therefore be utilized to distinguish P. chikae from P. hermeli. Nevertheless, a thorough examination of their morphology, behavior, and ecology is still imperative to firmly establish their taxonomic status.

Saving DNA from museum specimens: The success of DNA mini-barcodes in haplotype reconstruction in the genus Anastrepha (Diptera: Tephritidae)

The fragmentation of DNA in historical specimens is very common, so obtaining sequences that allow molecular identification and the study of diversity is quite challenging. In this study, we used preserved and fresh specimens of the fruit fly genus Anastrepha, a genus of economic impact of fruit crops of the Neotropic. From these specimens, we evaluated: (1) the success PCR amplification rates of mini-barcodes fragments of the cytochrome c oxidase subunit I (COI) gene, and (2) the usefulness of mini-barcodes in the reconstruction of haplotypes for the identification of species and the diversity analysis. We used 93 specimens from 12 species, which had been preserved in 70% ethanol for more than 20 years. Internal primers were designed in the COI region and primers available in the literature were also evaluated. We obtained amplifications for 62.36% of the samples processed, and reconstructed haplotypes between 171 bp and 632 bp. Variable amplification rates between combinations of primers and between species were obtained, and molecular identification of some museum specimens was achieved. It was also possible to compare the haplotypes obtained in four species from which both fresh and museum samples were available. Our results also show the importance of the adjustment of the primers for the amplification, allowing to amplify fragments of up to 400 bp. The use available resources in biological collections is key to increasing knowledge of species of interest, and by means of the amplification of mini-barcodes, short sequences can be obtained that allow the molecular identification of specimens and the reconstruction of haplotypes with multiple purposes.

PCR identification and phylogenetic analysis of the medically important dust mite Suidasia medanensis (Acari: Suidasiidae) in Malaysia

The occurrence of Suidasia medanensis (= S. pontifica) mites in Malaysian house dust was first reported in 1984. The taxonomy of this storage mite is, however, quite confusing. Therefore, we need an accurate identification to resolve morphological problems due to its minute size and some overlapping characters between species. The purpose of this study was to demonstrate the application of partial mitochondrial cytochrome c oxidase subunit I (COI) sequences for the identification of S. medanensis by PCR. Identity of the mite was first determined by observing morphological characters under a light microscope. Genomic DNA of S. medanensis mites was successfully extracted prior to PCR and DNA sequencing using COI universal primers. The length of the COI sequences obtained was 378 bp. BLAST analysis of amplicon sequences showed that local S. medanensis COI region had 99% maximum identity with S. medanensis nucleotide sequence (AY525568) available in the GenBank. As the phylogenetic tree generated indicated, COI sequences from this study were clustered with S. medanensis from Korea and the UK in one major clade, supported with high bootstrap value (> 85%). Results of the phylogenetic analysis of this COI gene were congruent with the morphological identification and provided strong support for a single clade of local S. medanensis.

A reference cytochrome c oxidase subunit I database curated for hierarchical classification of arthropod metabarcoding data.

Metabarcoding is a popular application which warrants continued methods optimization. To maximize barcoding inferences, hierarchy-based sequence classification methods are increasingly common. We present methods for the construction and curation of a database designed for hierarchical classification of a 157 bp barcoding region of the arthropod cytochrome c oxidase subunit I (COI) locus. We produced a comprehensive arthropod COI amplicon dataset including annotated arthropod COI sequences and COI sequences extracted from arthropod whole mitochondrion genomes, the latter of which provided the only source of representation for Zoraptera, Callipodida and Holothyrida. The database contains extracted sequences of the target amplicon from all major arthropod clades, including all insect orders, all arthropod classes and Onychophora, Tardigrada and Mollusca outgroups. During curation, we extracted the COI region of interest from approximately 81 percent of the input sequences, corresponding to 73 percent of the genus-level diversity found in the input data. Further, our analysis revealed a high degree of sequence redundancy within the NCBI nucleotide database, with a mean of approximately 11 sequence entries per species in the input data. The curated, low-redundancy database is included in the Metaxa2 sequence classification software (http://microbiology.se/software/metaxa2/). Using this database with the Metaxa2 classifier, we performed a cross-validation analysis to characterize the relationship between the Metaxa2 reliability score, an estimate of classification confidence, and classification error probability. We used this analysis to select a reliability score threshold which minimized error. We then estimated classification sensitivity, false discovery rate and overclassification, the propensity to classify sequences from taxa not represented in the reference database. Our work will help researchers design and evaluate classification databases and conduct metabarcoding on arthropods and alternate taxa.

Molecular Identification of Four Members of the Anopheles dirus Complex Using the Mitochondrial Cytochrome C Oxidase Subunit I Gene.

Precise mosquito species identification is an essential step for proper management and control of malaria vectors. Misidentification of members in the Anopheles dirus complex, some which are primary malaria vectors in Thailand and mainland Southeast Asia, remains problematic because of indistinguishable or overlapping morphological characters between sibling species. Moreover, there is a need for alternative methods, since the existing molecular techniques in the literature are not entirely satisfactory in differentiating all members in the An. dirus complex. The nucleotide polymorphisms in the mitochondrial cytochrome c oxidase subunit I (COI) sequences were developed to identify the 4 species within the An. dirus complex using an allele-specific (AS) multiplex polymerase chain reaction (PCR). The identified primers amplified and clearly differentiated the 4 members of the complex found in Thailand, Anopheles dirus, An. cracens, An. scanloni, and An. baimaii with PCR products 428/104, 236, 625, and 428 bp, respectively. These results demonstrate that an AS-PCR based on the COI region can accurately identify 4 members of An. dirus complex and would be useful as an alternative PCR-based method for accurate species identification.

Environmental DNA for Detection of Endangered Grouper Species (Epinephelus spp.)

Marine ecosystems nestle species or populations known to be threatened due to human
 overexploitation. Reliable detection and monitoring of threatened organisms is crucial for
 data-driven conservation actions. Furthermore, misidentification of species represents a major
 problem. Here, we investigate the potential of using metabarcoding of environmental DNA
 (eDNA) obtained directly from seawater samples to detect endangered grouper species
 (Epinephelus spp.). Cytochrome c oxidase subunit I (COI) fragment of mtDNA was used to
 detect groupers species in the Mediterranean Coasts. We conducted eDNA sampling at sites
 by underwater diving across the range of the Grouper species habitats in Northeastern
 Mediterranean (Antalya-Kas Region and Iskenderun Bay). eDNA was isolated from 2 liter
 seawater samples which were vacuum-filtered onto 0.45-mm membrane filters. Filters were
 then folded inwards, placed in 2 ml tubes and stored at -20 oC until DNA extraction, which
 took place within 24 hours. DNA was extracted from the water sample filters using the
 DNeasy Blood and Tissue Kit (Qiagen, USA). Manufacturer’s protocols were used during all
 steps. PCR amplification of eDNA samples were done using selective primers of COI region
 of mitochondrial DNA, and next-generation DNA sequencing of PCR application was
 conducted. For the successfully obtained COI sequences, maximum matching rates were
 revealed as 80% for Epinephelus marginatus, 78,95% for Epinephelus aeneus, 73,48% for
 Epinephelus costae, 63,45% for Epinephelus caninus, 60,12% for Mycteroperca rubra and
 57,12% for Hyporthodus haifensis. Despite the methodological challenges inherent in eDNA
 analysis, the results demonstrated that eDNA method may be proved to step towards a new
 beginning to detect and monitor endangered grouper species.

Barcode analysis using mini-amplicons strategy for museum samples of neotropical primates Callithrix spp.

The identification of species and the assignment of unidentified samples to a voucher reference database are among the most requested forensic analyses involving crimes against wildlife. Genomic DNA from forensic or museum samples, however, are frequently degraded, hampering the analysis of DNA fragments longer than 500bp. Among studies targeting non-human species identification, one of the most frequently used fragments is the mitochondrial Cytochrome C Oxidase subunit I (COI), in particular a ‘barcode' region containing approximately 650 base pairs (bp). The main objective of this work was to develop a set of primers to amplify five shorter overlapping COI fragments for the neotropical marmosets (Callithrix spp.) to be used in samples from museum specimens. Taxidermized skins, bones, and hair tissues were sampled out of 9 museum specimens with a range archival from 81 to 2 years; PCR were performed with five mini-amplicons primers pairs and with the primer pair for the whole barcode COI fragment, followed by Sanger sequencing. As results, no amplification was observed using the primer pair for the longer COI sequence; however, using the new set of mini-amplicon primers, all types of samples were amplified. We established optimal PCR conditions for the employed primers and conclude that the mini-amplicon strategy for COI region typing is best suited for hardly degraded samples in forensic research, and in particular for museum samples.

Temperature-related activity of Gomphiocephalus hodgsoni (Collembola) mitochondrial DNA (COI) haplotypes in Taylor Valley, Antarctica

Gomphiocephalus hodgsoni (Collembola) is the most common and widely distributed arthropod in the Dry Valleys of southern Victoria Land, and is genetically diverse with over 70 mitochondrial cytochrome c oxidase subunit I (COI) haplotypes. There is also considerable physiological variation among G. hodgsoni individuals in their cold tolerance and metabolic activity. Here, we assessed genetic haplotypes of G. hodgsoni relative to the environmental conditions during which individuals were active. We sequenced the COI region of 151 individuals collected in pitfall traps from three sites within Taylor Valley and found 19 unique haplotypes that separated into two distinct lineages (1.6 % divergence), with one lineage comprising 80 % of the sequenced population. During two-hourly sampling, air temperature was the strongest predictor of activity between the two lineages (R 2 = 0.56), and when combined with subsurface soil temperature, relative humidity and photosynthetically active radiation, explanatory power increased to R 2 = 0.71. With steadily increasing air temperatures predicted for much of Antarctica, it is likely that some haplotypes will have a selective advantage and potentially result in decreased genetic variability within populations. We suggest that temporal monitoring of the relative proportions of COI haplotypes or other appropriate genetic markers may provide a subtle measure of biological responses to environmental changes within Antarctic terrestrial ecosystems.

DNA barcoding application of mitochondrial COI gene sequence in medicinal fish of Culter (Pisces: Cyprinidae)

The sequence variation of medicinal fish of Culter (Pisces: Cyprinidae) was analyzed by using cytochrome c oxidase subunit I (COI) sequencing collected from different regions of the Yangtze River basin, and we examine whether barcoding of COI can be used to discriminate medicinal fish of Culter. The AT content in the COI region of medicinal fish of Culter was higher than that of GC, which was similar with other species of Cypriniformes. Ninty-six percent of nucleotide changes were observed at the 3rd codon position of COI sequence, but the amino acid compositions translated by COI sequences of all Culter fish stayed the same. It is suggested that most synonymous mutations might occur at the 3rd position. The average Kimura-2-parameter (K2P) distance within-species was lower than 1%, and the K2P distance of pairwise-species was 10 times as much as that of within-species. The phylogenetic tree estimated by Neighbour-joining method indicated that species within genera invariably clustered, and generally so did individuals within species. Individuals from operational taxonomic units designated as different Culter species, supporting morphological evidence for each of these being separate species. It is suggested that the COI barcoding can be used to identify medicinal fish species of Culter.

Identification of forensically important Sarcophaga species (Diptera: Sarcophagidae) using the mitochondrial COI gene

The identification of species of the forensically important genus Sarcophaga is very difficult and requires strong taxonomic expertise. In this study, we sequenced the mitochondrial cytochrome c oxidase subunit I (COI) gene of 126 specimens of 56W European Sarcophaga species and added GenBank data to our database to yield a total dataset of 270 COI sequences from 99 Sarcophaga species to evaluate the COI gene as a molecular diagnostic tool for species identification in this genus. Using two simple criteria (Best Match, BM and Best Close Match, BCM), we showed that the identification success using a mini-barcode region of 127bp was very low (80.7-82.5%) and the use of this region is not recommended as a species identifier. In contrast, identification success was very high using the standard barcode region (658bp) or using the entire COI region (1,535bp) (98.2-99.3%). Yet, there was a low interspecific sequence divergence (<2%) in six species groups so that for 16 out of the 99 species (nine of which are of forensic importance), the use of COI barcodes as species identifier should be done with care. For these species, additional markers will be necessary to achieve a 100% identification success. We further illustrate how such reference databases can improve local reference databases for forensic entomologists.

Species identification of Tanzanian antelopes using DNA barcoding

Efficient tools for consistent species identification are important in wildlife conservation as it can provide information on the levels of species exploitation and assist in solving forensic-related problems. In this study, we evaluated the effectiveness of the mitochondrial cytochrome c oxidase subunit I (COI) barcode in species identification of Tanzanian antelope species. A 470 base-pair region of the COI gene was examined in 95 specimens representing 20 species of antelopes, buffalo and domestic Bovidae. All the Tanzanian species showed unique clades, and sequence divergence within species was <1%, whereas divergence between species ranged from 6.3% to 22%. Lowest interspecific divergence was noted within the Tragelaphus genus. Neighbour-joining phylogenetic analyses demonstrated that the examined COI region provided correct and highly supported species clustering using short fragments down to 100 base-pair lengths. This study demonstrates that even short COI fragments can efficiently identify antelope species, thus demonstrating its high potential for use in wildlife conservation activities.

Improved COI barcoding primers for Southeast Asian perching birds (Aves: Passeriformes)

The All Birds Barcoding Initiative aims to assemble a DNA barcode database for all bird species, but the 648-bp 'barcoding' region of cytochrome c oxidase subunit I (COI) can be difficult to amplify in Southeast Asian perching birds (Aves: Passeriformes). Using COI sequences from complete mitochondrial genomes, we designed a primer pair that more reliably amplifies and sequences the COI barcoding region of Southeast Asian passerine birds. The 655-bp region amplified with these primers overlaps the COI region amplified with other barcoding primer pairs, enabling direct comparison of sequences with previously published DNA barcodes.

Molecular species identification of morphologically similar mussel larvae reveals unexpected discrepancy between relative abundance of adults and settlers

Understanding the relative importance of larval supply vs. post-settlement mortality underlies studies of marine invertebrate recruitment, yet is often hampered by researchers' inability to identify species among morphologically similar larvae or early juveniles. In New Zealand, two species of co-occurring intertidal mytilid mussels have morphologically indistinguishable settlers: the blue mussel Mytilus galloprovincialis, which is often numerically dominant in the mid-zone of the rocky intertidal, and the ribbed mussel Aulacomya atra maoriana which is often much less abundant. In this study, we obtained samples of newly settled mussels from 6 sample dates April-May 2005 from the rocky intertidal in Wellington Harbour, New Zealand. We used PCR-RFLP of the cytochrome c oxidase subunit I (COI) mitochondrial gene region to identify settlers to species. Of a total of 224 settlers that could be identified, 64% were identified as Mytilus galloprovincialis and 36% as Aulocomya atra maoriana. The percentage of A. atra maoriana in the samples was unexpectedly high and ranged from 22–50% among the sample dates. This study reinforces the need to quantify larval supply at the species level to understand the relative importance of pre- and post-settlement mortality, and also demonstrates the usefulness of the COI region as a species-specific marker for identifying mussel larvae and juveniles.

RFLP Analysis of the mtDNA COI Region in Four Abalone Species

The cytochrome c oxidase subunit I (COI) gene region of mitochondrial DNA (mtDNA) was examined in four abalone species to estimate its utility as a genetic marker using restriction fragment length polymorphism (RFLP) analysis. The utility was evaluated in terms of genetic divergence and relationships among Haliotis discus hannai, H. rufescens, H. rubra, and H. midae in both hemispheres of the world. There was clear genetic divergence in the mtDNA COI region between all pairs of the four species. Moreover, relationships among the abalone species were reflected in their geographical distributions and morphological characteristics. Therefore, RFLP analysis of the mtDNA COI region is a suitable genetic marker for the estimation of genetic divergence and relationships among abalone species. However, it is not effective for the evaluation of genetic differences within abalone species.

A test of monophyly of the gutless Phallodrilinae (Oligochaeta, Tubificidae) and the use of a 573‐bp region of the mitochondrial cytochrome oxidase I gene in analysis of annelid phylogeny

A 573‐bp region of the mitochondrial gene cytochrome c oxidase subunit I (COI) of two species of Inanidrilus Erséus and four species of Olavius Erséus (Phallodrilinae, Tubificidae) is used in a parsimony analysis together with a selection of 35 other annelids (including members of Polychaeta, Pogonophora, Aphanoneura, and the clitellate taxa Tubificidae, Enchytraeidae, Naididae, Lumbriculidae, Haplotaxidae, Lumbricidae, Criodrilidae, Branchiobdellida and Hirudinea), and with two molluscs as outgroups. The data support the monophyly of the Olavius and Inanidrilus group, with a monophyletic Inanidrilus. However, parsimony jackknife analyses show that most of the other groups are unsupported by the data set, thus revealing a large amount of homoplasy in the selected gene region. Practically no information is given of within/between family relationships except for a few, closely related species. This suggests that the analysed COI region is not useful, when used alone, for inferring higher level relationships among the annelids.