Mitochondrial 16S Ribosomal DNA Research Articles

Prevalence of Rickettsia spp., Anaplasma spp. and Ehrlichia spp. in Ornithodoros lahorensis from southern Xinjiang, China

Ticks are important vectors for pathogens and transmit a variety of diseases in humans and animals. Numerous studies have reported the presence of Ixodidae ticks and tick-borne pathogens in southern Xinjiang. However, data pertaining to argasid ticks and argasid tick-associated pathogens are sparse. This study collected a total of 407 ticks from southern Xinjiang. The tick species were identified on the basis of their morphological and mitochondrial 16S ribosomal DNA (16S rDNA) characteristics. The citrate synthase gene (gltA), 17-kilodalton antigen gene (17-kDa), and outer membrane protein A and B genes (ompA, ompB) were amplified using four sets of primers. The species of rickettsiae were further identified on the basis of concatenated sequences. To detect Anaplasma and Ehrlichia, nested PCR was used to amplify the Ehrlichia and Anaplasma 16S rRNA genes, respectively. All 407 ticks were Ornithodoros lahorensis. A total of 109 Rickettsia-positive samples and 19 Anaplasma-positive samples were detected in DNA samples from the 407 ticks. R. sibirica, R. massiliae, Candidatus R. barbariae, A. ovis, A. marginale, and A. phagocytophilum were found. All DNA samples from Or. lahorensis were tested for the presence of the Ehrlichia spp. nucleotide, but none of the samples were positive for such pathogens. This is the first time that R. sibirica, R. massiliae, and A. marginale were detected in Or. lahorensis from southern Xinjiang.

Morphological and phylogenetic study of Acanthosquilla Manning, 1963 (Stomatopoda: Nannosquillidae) mantis shrimps, with description of two new species from the Ryukyu Islands, Japan.

Three species of nannosquillid mantis shrimps, including two new species, Acanthosquilla ryukyuensis n. sp. and Acanthosquilla shoheii n. sp., are described based on specimens collected from the Ryukyu Islands, Japan. The two new species resemble A. derijardi Manning, 1970, but can be distinguished from A. derijardi by the following features: 1) rostral plate anterolateral corner forms almost a right angle; 2) the distal tip of the antennular somite dorsal process reaches or slightly falls short of proximal half of rostral plate; and 3) eighth thoracic somite (= TS8) posterior margin is black medially. Furthermore, A. ryukyuensis n. sp. and A. shoheii n. sp. are easy to identify by the bifurcated lateral tooth of the telson, and by the posterodorsal pattern of the telson, respectively. In this study, molecular analyses based on partial sequences of mitochondrial 12S and 16S ribosomal DNA, cytochrome oxidase subunit I (COI), and the partial nuclear gene of 28S ribosomal DNA recovers these three species of Acanthosquilla and A. multifasciata (Wood-Mason, 1895) (the type species of the genus) in a single clade. The resulting trees also suggest the polyphyly of Nannosquillidae but with low nodal support. Detailed examinations of the morphological and color features and DNA barcoding results allowed us to delineate intraspecific variations and interspecific differences. The number and shape of setae under the dorsal spine of raptorial claw carpus was found to be useful in distinguishing A. shoheii n. sp. from A. derijardi and A. ryukyuensis n. sp., while combinations of the coloration of the rostral plate, posterior margin of TS8 and posterodorsal surface of telson are useful to distinguish the three species.

Effect of substrate on the proliferation of Myxobolus cerebralis in the mitochondrial lineages of the Tubifex tubifex host

The study goal was to examine the effects of sand and mud on the propagation of Myxobolus cerebralis, the whirling disease agent, in four mitochondrial 16S ribosomal DNA lineages (I, III, V, VI) of its oligochaete host, Tubifex tubifex (Tt). In all the lineage groups held continuously in either substrate (non-shifted) or transferred from sand to mud (shifted), substrate influenced parasite proliferation only in lineage III. Sporogenesis and release of triactinomyxon spores (TAMs) were more prevalent in lineage III Tt in mud compared to sand. Low-infection prevalence and lack of parasite development in lineage I is associated with the greater number of resistant worms and were not affected by substrate type. Substrate did not impact Tt from lineages V and VI that failed to develop any parasite stages in either substrate even after shifting from sand to mud. The relationship between the microbial community in the substrate and parasite proliferation in lineage III was described but not analyzed due to small sample size. Substrate-associated bacteria were hypothesized as essential dietary source for the oligochaete host feeding selectively on fine (mud)-microflora. Progeny was produced by all lineage groups shifted to mud with disparate survival profiles in lineage V and VI and high mortalities in lineage III. Our study demonstrates that substrate type can alter parasite proliferation in lineage III. Conversely, parasite development and infectivity were not altered in lineage V and VI that are refractory to the parasite nor among the more resistant phenotypes (I), regardless of substrate type.

Morphological identification and molecular characterization of economically important ticks (Acari: Ixodidae) from North and North–Western Egypt

Ticks represent a major source of growing economic and public health concern, especially in the tropics and sub-tropics. Towards evidencing ticks’ invasion in the North and North–Western parts of Egypt, the present study aimed to investigate the morpho-molecular aspects of those ectoparasites using stereomicroscopy, scanning electron microscopy (SEM) and sequencing of the mitochondrial 16S ribosomal DNA gene (rDNA) and nuclear second internal transcribed spacer (ITS2). Our analysis confirmed the presence and well–distribution of Rhipicephalus sanguineus s.l. infesting dogs and sheep (Alexandria governorate), Rhipicephalus annulatus infesting cattle (Beheira governorate), and Hyalomma dromedarii infesting camels (Marsa Matruh governorate) from North/North–Western Egypt. 16S rDNA and ITS2 sequences of the ticks were amplified using universal and gene–specific sets of primers, sequenced and analyzed. Lengths of amplified 16S rDNA sequences in all examined tick species were found to be similar in size (approximately 460 bp); however, they differed in base pair constitutions, whereas ITS2 lengths were 1,500 bp, 1,550 bp, and 1,800 bp for Rh. annulatus, Rh. sanguineus s.l., and Hy. dromedarii, respectively. Phylogenetically, based on the 16S rDNA results, Rh. sanguineus s.l. ticks clustered with the southeastern Europe lineage from Romania and Greece, Rh. annulatus ticks were similar to Turkish populations, and Hy. dromedarii were close to the isolates from Tunisia. Similarly, based on ITS2 sequences, Rh. sanguineus s.l. from dogs were showing 99% similarity to Nigerian populations; however, those collected from sheep were closer to Iranian populations with 4.1% nucleotide divergence between the two populations of different hosts. Rh. annulatus ticks were identical to a population from Romania, whereas Hy. dromedarii was close by 99.7% similarity to a population from Kenya. This is the first study reporting nucleotide sequences of 16S rDNA and ITS2 in integration with morphological identification of ticks from this part of Egypt.

Molecular phylogeny of bivalve families (Arcidae, Chamidae, Margaritidae, Ostreidae, Veneridae) in the Persian Gulf

Bivalvia is one of the least studied classes of the Persian Gulf. A survey and molecular analysis was conducted to determine bivalve species diversity in the Persian Gulf. To the best of our knowledge, this is the first report of barcoding data on bivalves of the Persian Gulf. We examined 40 individuals representing 8 species, 6 genera and 5 families. We collected samples from Hengam, Larak and Qeshm Islands and Bandar Lengeh in Persian Gulf, Iran. After DNA extraction, mitochondrial 16S ribosomal DNA (16SrDNA) and Cytochrome Oxidase subunit I were amplified by polymerase chain reaction (PCR). Based on obtained 16SrDNA and COI gene sequences and maximum parsimony, neighbor joining and maximum likelihood trees of these genes there was no overlap between maximum Kimura 2- parameter distance among conspecifics. Most species formed agglutinate sequence units with a small amount of changes. Eventually, comparison of the 8 selected studied species with metadata from India, Brazil, Japan, China, and America exposed that these species in Persian Gulf are classified in sister clades with high bootstraps except Pinctada. Since there is not much work on bivalves identification in the Persian Gulf, larger sampling and more research is needed to investigate mollusc diversity in this area.

New 12S metabarcoding primers for enhanced Neotropical freshwater fish biodiversity assessment

The megadiverse Neotropical fish fauna lacks a comprehensive and reliable DNA reference database, which hampers precise species identification and DNA based biodiversity assessment in the region. Here, we developed a mitochondrial 12S ribosomal DNA reference database for 67 fish species, representing 54 genera, 25 families, and six major Neotropical orders. We aimed to develop mini-barcode markers (i.e. amplicons with less than 200 bp) suitable for DNA metabarcoding by evaluating the taxonomic resolution of full-length and mini-barcodes and to determine a threshold value for fish species delimitation using 12S. Evaluation of the target amplicons demonstrated that both full-length library (565 bp) and mini-barcodes (193 bp) contain enough taxonomic resolution to differentiate all 67 fish species. For species delimitation, interspecific genetic distance threshold values of 0.4% and 0.55% were defined using full-length and mini-barcodes, respectively. A custom reference database and specific mini-barcode markers are important assets for ecoregion scale DNA based biodiversity assessments (such as environmental DNA) that can help with the complex task of conserving the megadiverse Neotropical ichthyofauna.

Triplex real-time PCR assay for the authentication of camel-derived dairy and meat products

Authentication of dairy and meat products is important to ensure fair competition, consumer benefit, and food safety. The large difference in price between camel and cow milk may be an incentive to adulterate camel dairy products with cow-derived foodstuffs. However, no studies so far have used triplex real-time PCR with an endogenous control to identify camel and cow origins in dairy and meat products. In this study, we developed a triplex real-time PCR assay based on amplification of mitochondrial 12S ribosomal DNA for the authentication of camel-derived dairy and meat products. This method was applied to identify camel and cow DNA in milk, yogurt, cheese, milk powder, milk beverage, meat products, and mixtures with milk and meat. Concentrations as low as 1 to 5% and 0.1% camel milk and meat, respectively, were detected in the mixtures, and 1 to 5% and 0.1% cow milk and meat, respectively, were identified via this approach. The limits of detection were 0.005 to 0.0025 ng, 0.05 to 0.001 ng, 0.001 to 0.0005 ng, and 0.00025 to 0.0001 ng of DNA in camel milk, camel yogurt, commercial camel milk beverage, and camel meat, and from 0.0025 to 0.001 ng, 0.5 to 0.001 ng, 1 to 0.05 ng, 0.01 ng, 0.001 ng, 0.0005 to 0.00025 ng, 0.0005 to 0.00025 ng, and 0.005 ng of DNA from cow milk, yogurt, cheese, acidic whey, milk powder, beef, beef jerky, and beef sausage, respectively. Different dairy and meat samples of camel and cow origins had a range of authentication limits and limits of detection. The designed triplex real-time PCR assay was shown to be a specific, sensitive, and efficient technique for the identification of camel and cow DNA in foodstuffs.

Germ cells: a useful tool for the taxonomy of Rhipicephalus sanguineus s.l. and species of the Amblyomma cajennense complex (Acari: Ixodidae).

The systematics of several ticks species (Acari: Ixodidae) remains controversial. Many species, including those of the Amblyomma cajennense complex and Rhipicephalus sanguineus s.l., are given special attention since they are cryptic species complexes and are also important in human and veterinary medicine. The A. cajennense complex was recently reorganized into six valid species, among which Amblyomma patinoi and Amblyomma mixtum have been confirmed in Colombia. On the other hand, the taxonomic status of R. sanguineus s.l. is controversial since it is a cosmopolitan cryptic species complex with a high reproductive capacity and a broad range of hosts (including man). To address this challenge, the germ cells of male ticks display a diverse morphology that offers novel opportunities for taxonomy. This study describes the events of spermatogenesis in A. mixtum and R. sanguineus s.l. individuals collected during active feeding on domestic hosts in the department of Caldas, Colombia. The individuals were identified using dichotomous keys and through PCR amplification of a fragment of the mitochondrial 16S ribosomal DNA gene. The male reproductive systems of A. mixtum and R. sanguineus s.l. were fixed in 2.5% glutaraldehyde for 48h and dehydrated in increasing dilutions of ethanol. The samples were then embedded and mounted in historesin to obtain sections of 3μm that were stained with hematoxylin-eosin (HE), photographed, and visualized through optical microscopy. The results show that the morphology of mature germ cells displays excellent diagnostic traits that can be used for tick taxonomy.

Bacterial and protozoan pathogens/symbionts in ticks infecting wild grasscutters (Thryonomys swinderianus) in Ghana

Ticks and tick-borne pathogens constitute a great threat to livestock production and are a potential health hazard to humans. Grasscutters (Thryonomys swinderianus) are widely hunted for meat in Ghana and many other West and Central African countries. However, tick-borne zoonotic risks posed by wild grasscutters have not been assessed. The objective of this study was to investigate bacterial and protozoan pathogens in ticks infecting wild grasscutters. A total of 81 ticks were collected from three hunted grasscutters purchased from Kantamanto, the central bushmeat market in Accra. Ticks were identified as Ixodes aulacodi and Rhipicephalus sp. based on morphological keys, which were further confirmed by sequencing mitochondrial 16S ribosomal DNA (rDNA) and cytochrome oxidase I (COI) genes of specimens. Protozoan infections were tested by PCR amplifying 18S rDNA of Babesia/Theileria/Hepatozoon, while bacterial infections were evaluated by PCRs or real-time PCRs targeting Anaplasmataceae, Borrelia, spotted fever group rickettsiae, chlamydiae and Candidatus Midichloria mitochondrii. The results of PCR screening showed that 35.5% (27 out of 76) of I. aulacodi were positive for parasite infections. Sequencing analysis of the amplified products gave one identical sequence showing similarity with Babesia spp. reported from Africa. The Ca. M. mitochondrii endosymbiont was present in 85.5% (65 out of 76) of I. aulacodi but not in the five Rhipicephalus ticks. Two Anaplasmataceae bacteria genetically related to Ehrlichia muris and Anaplasma phagocytophilum were also detected in two I. aulacodi. None of the ticks were positive for Borrelia spp., spotted fever group rickettsiae and chlamydiae. Since I. aulacodi on wild grasscutters are potential carriers of tick-borne pathogens, some of which could be of zoonotic potential, rigorous tick control and pathogen analyses should be instituted especially when wild caught grasscutters are being used as foundation stock for breeding.

Molecular detection of virulent Mannheimia haemolytica and Pasteurella multocida in lung tissues of pneumonic sheep from semiarid tropics, Rajasthan, India

The present study was planned to detect the genetic elements of Mannheimia haemolytica and Pasteurella multocida in pneumonic sheep lungs. Pneumonia was diagnosed on the basis of gross pathological lesions. Lung tissues were collected at necropsy of sheep (n = 96) and subjected to isolation of total DNA. The M. haemolytica -specific PHSSA and Rpt2 genes, and the P. multocidaspecific KMT1 and the Omp87 genes were amplified using polymerase chain reaction (PCR). A housekeeping gene targeting the sheep cellular mitochondrial 12S ribosomal DNA was used as an internal control. PCR reactions were optimized using the positive and negative controls. Gene-specific PCR products were subjected to nucleotide sequencing for confirmation. The pneumonic lungs showed congestion and hemorrhagic changes with consolidation, which was most evident in the whole of the apical lobes and parts of the diaphragmatic lobes. PCR amplification showed detection of PHSSA (327 bp) and Rpt2 (~1022 bp) genes specific to M. haemolytica in 52 (54.1%), and the KMT1 (457 bp) and Omp87 (2627 bp) genes specific to P. multocida in 16 (16.6%) lung samples. Sequence analysis confirmed the PCR products for specific genes. This study highlighted the culture-independent, rapid, and confirmatory diagnosis of ovine pneumonic pasteurellosis caused by M. haemolytica and/or P. multocida .

Molecular identification and phylogenetic assessment of species under genus Parapenaeopsis Alcock, 1901, from Indian waters

Genus Parapenaeopsis constitute an important fishery resource in Indian waters. Parapenaeopsis stylifera, being morphologically very similar to Parapenaeopsis coromandelica, mainly differentiated using telsonic armature and it is subject of controversy regarding the number of armature for the species. The present study was aimed to resolve the taxonomic ambiguity between P. stylifera and P. coromandelica and to study phylogenetic relationship among five species under the genus Parapenaeopsis using partial sequences of mitochondrial 16S ribosomal DNA (rDNA) and cytochrome c oxidase subunit I (COI) genes. Sequence analysis revealed an average genetic divergence value ranging from 7.65 to 20.3% for COI gene and 2.4 to 12.0% for 16S rDNA gene between five species under study. The result shows that P. stylifera and P. coromandelica Alcock, 1906 are genetically a distinct species with average divergence value of 7.6% based on COI sequence and 2.4% based on the 16S rDNA. However, systematic relationship between the P. stylifera and P. coromandelica were closer than other species, thus formed separate clade in phylogenetic tree. The species specific molecular signatures developed in the present study will be helpful in effective fisheries management of the species in Indian waters.

Out of Asia: mitochondrial evolutionary history of the globally introduced supralittoral isopod Ligia exotica.

The native ranges and invasion histories of many marine species remain elusive due to a dynamic dispersal process via marine vessels. Molecular markers can aid in identification of native ranges and elucidation of the introduction and establishment process. The supralittoral isopod Ligia exotica has a wide tropical and subtropical distribution, frequently found in harbors and ports around the globe. This isopod is hypothesized to have an Old World origin, from where it was unintentionally introduced to other regions via wooden ships and solid ballast. Its native range, however, remains uncertain. Recent molecular studies uncovered the presence of two highly divergent lineages of L. exotica in East Asia, and suggest this region is a source of nonindigenous populations. In this study, we conducted phylogenetic analyses (Maximum Likelihood and Bayesian) of a fragment of the mitochondrial 16S ribosomal (r)DNA gene using a dataset of this isopod that greatly expanded previous representation from Asia and putative nonindigenous populations around the world. For a subset of samples, sequences of 12S rDNA and NaK were also obtained and analyzed together with 16S rDNA. Our results show that L. exotica is comprised of several highly divergent genetic lineages, which probably represent different species. Most of the 16S rDNA genetic diversity (48 haplotypes) was detected in East and Southeast Asia. Only seven haplotypes were observed outside this region (in the Americas, Hawai’i, Africa and India), which were identical or closely related to haplotypes found in East and Southeast Asia. Phylogenetic patterns indicate the L. exotica clade originated and diversified in East and Southeast Asia, and only members of one of the divergent lineages have spread out of this region, recently, suggesting the potential to become invasive is phylogenetically constrained.

Molecular identification of zoantharians from around Hormuz, Qeshm and Kish islands, the Persian Gulf

Zoantharians have an important ecological role in marine ecosystems. However, only a few studies have been conducted on the zoantharians of the Persian Gulf (PG). In the current study, mitochondrial 16S ribosomal DNA (16S rDNA) and cytochrome oxidase subunit I (COI) regions of zoantharian colonies from Hormuz, Qeshm and Kish islands were amplified by polymerase chain reaction (PCR) and directly sequenced. Based on obtained 16S rDNA and COI gene sequences, three species-level clades were identified including Zoanthus sansibaricus, Palythoa tuberculosa and Palythoa aff. mutuki, all of which have been previously reported from the PG. In spite of morphological similarity of Palythoa aff. mutuki to Palythoa mutuki, mitochondrial DNA sequences obtained from these specimens resolved in a highly supported species-level clade. Since zoantharian identification studies are scant in other parts of the PG, further studies using both nuclear and mitochondrial markers are required to investigate the origins of this putative new species and to further complete the knowledge of zoantharian diversity in the region.

Distribution and phylogeny of Hyalomma ticks (Acari: Ixodidae) in Turkey.

The genus Hyalomma includes some of the most medically and veterinarily important tick species in the world. To clarify and identify the current distribution of the species of Hyalomma, field studies were conducted in 65 localities in Turkey and five localities in Cyprus. Additionally, using mitochondrial 12S and 16S ribosomal DNA, specimens of Hyalomma from Turkey, H. excavatum from Cyprus, H. marginatum from Spain and Italy were evaluated together with the available sequences obtained from Genbank. Morphological and molecular analyses demonstrated the presence of four species in Turkey: H. marginatum, H. excavatum, H. aegyptium and H. asiaticum. Hyalomma marginatum is the dominant species in the Central and Northern parts of Turkey, whereas H. excavatum distributes mostly in the Southern parts. Hyalomma asiaticum is restricted to the Southeastern Anatolia. However, some sympatric regions were observed for these species. Phylogenetic trees obtained with Maximum Likelihood method demonstrated five clades. Data supported previous conclusions, but placed H. asiaticum, H. scupense, H. dromedarii and H. aegyptium in different clades with high bootstrap values. Specimens of H. anatolicum group and H. marginatum complex are sister groups. Pairwise distance analyses of these groups showed 2.8 and 3% differences for 12S rDNA and 16S rDNA, respectively. Therefore, additional analyses with the samples from different locations using different markers need to evaluate the exact status of the species of these groups.

Molecular and morphological identification of a human biting tick, Amblyomma testudinarium (Acari: Ixodidae), in Taiwan.

Genetic identity and morphological features of a human biting tick, Amblyomma testudinarium, were determined for the first time in Taiwan. Morphological features of adult male and female ticks of Am. testudinarium were observed and photographed by a stereo- microscope. The genetic identity was analyzed by comparing the sequences of mitochondrial 16S ribosomal DNA gene obtained from 18 strains of ticks representing 10 species of Amblyomma, and four outgroup species of Dermacentor and Rhipicephalus ticks. Nine major clades could be easily distinguished by neighbour-joining analysis and were congruent by maximum-parsimony method. All these Am. testudinarium ticks collected from Taiwan and Japan were genetically affiliated to a monophyletic group with highly homogeneous sequence (99.8-100% similarity), and can be discriminated from other species of Amblyomma and other genera of ticks (Dermacentor and Rhipicephalus) with a sequence divergence ranging from 6.9 to 23.9%. Moreover, intra- and inter-species analysis based on the genetic distance (GD) values indicated a lower level (GD<0.003) within the same lineage of Am. testudinarium ticks collected from Taiwan and Japan, as compared with other lineage groups (GD>0.108) of Amblyomma ticks, as well as outgroup (GD>0.172) species. Our results provide the first distinguished features of adult Am. testudinarium ticks and the first genetic identification of Am. testudinarium ticks collected from humans in Taiwan. Seasonal prevalence, host range, and vectorial capacity of this tick species in Taiwan need to be further clarified.

Phylogenetic Relationships of Japanese Unionoida (Mollusca: Bivalvia) Based on Mitochondrial 16S rDNA Sequences

Japanese unionoid mussels are classified into 2 families (Margaritiferidae and Unionidae), 12 genera, and 18 species based on the morphological characteristics of both adults and larvae; however, there are some debates regarding their systematics. In this study, we determined mitochondrial 16S ribosomal DNA sequences (347-bp) for 60 specimens belonging to 18 species and constructed trees to elucidate phylogenetic relationships and evaluate the current systematics of Japanese unionoid mussels. Almost all species formed clades, except for Inversiunio yanagawensis, Sinanodonta lauta, S. japonica, and Margaritifera laevis, even though two or more specimens were collected from distant localities. All genera formed highly supported clades with the exception of the genus Sinanodonta. Phylogenetic relationships obtained in this study supported systematics based on morphological and larval traits. Therefore, the current phylogenetic relationships and systematics of Japanese unionoid mussels are stronger than they were before; now that they are corroborated by genetic data.

Molecular analysis of Rhipicephalus sanguineus (Acari: Ixodidae), an incriminated vector tick for Babesia vogeli in Taiwan.

The genetic identity of Rhipicephalus sanguineus tick was determined for the first time in Taiwan. The phylogenetic relationships were analyzed by comparing the sequences of mitochondrial 16S ribosomal DNA gene obtained from 32 strains of ticks representing six species of Rhipicephalus, two species of Dermacentor and two outgroup species (Haemaphysalis inermis and Ixodes ricinus). Seven major clades can be easily distinguished by neighbour-joining analysis and were congruent by maximum-parsimony method. All R. sanguineus ticks of Taiwan were genetically affiliated to the tropical lineage group of R. sanguineus sensu lato with highly homogeneous sequence (99.7-100% similarity), and can be discriminated from the temperate lineage group of Rhipicephalus sp. II and R. turanicus with a sequence divergence ranging from 1.7 to 5.2%. In contrast, the nucleotide variations among other Rhipicephalus spp. and other species/genus of ticks compared with the R. sanguineus ticks of Taiwan were measured from 10.6 to 25.5%. Moreover, intra- and inter-species analysis based on the genetic distance (GD) values indicated a lower level (GD<0.003) within tropical lineage group compared with temperate lineage group(GD>0.055) of Rhipicephalus, as well as other (GD>0.129) and outgroup (GD>0.236) species. Our results provide the first genetic identification of R. sanguineus ticks collected from Taiwan and demonstrate that all these R. sanguineus of Taiwan affiliated to the tropical lineage group of R. sanguineus sensu lato.

Extremely low genetic variability within and among locations of the greenfish holothurianStichopus chloronotusBrandt, 1835 in Okinawa, Japan

The greenfish sea cucumber Stichopus chloronotus is an economically and ecologically important sea cucumber species throughout its range. This species is widely distributed, inhabiting coral reefs of the Indo-Pacific Ocean. Our study evaluated population genetic structure and levels of genetic diversity in southern Japan. A total of 180 individuals were collected from eight locations from Okinawa and Okinoerabu Islands and sequenced using mitochondrial 16S ribosomal DNA (16S) and nuclear histone H3 (H3) gene. Only three 16S haplotypes were detected (518 bp) with haplotype diversity ranging from 0 to 0.56 and nucleotide diversity from 0 to 0.1%. H3 showed no variation among the studied locations. It is plausible that such results could be due to a shift to asexual reproduction. Additionally, the presence of the species on the east coast of Okinawa could only be detected in one location and all individuals consisted of a single haplotype. Genetic differences between the east and west coasts of Okinawa have been noticed in other coral reef organisms, and attributed to either ecological or biogeographical historical differences between the coasts due to differing levels of isolation during Pleistocene ice ages. Results from the present study should inform management and conservation policies of S. chloronotus in southern Japan.

A revision of two distinct species of Rhipicephalus: R. microplus and R. australis

ABSTRACT: Rhipicephalus ( Boophilus) species are monoxenous ticks with seasonal distribution in tropical and subtropical regions. For many years, Rhipicephalus micropluswas considered as a single species; however, further analysis split these ticks into two distinct species. Because R. microplusand R. australisshare similar attributes, it is hard to discriminate these two species and explain the changes in the classification of these parasites over the past decades. The reappearance of R. australisis an outcome of new research, which has afforded to better characterize these probably cryptic species. Evidence based on morphological features, the lack of conspecificity, microsatellite markers, mitochondrial 12S and 16S ribosomal DNA, and mitochondrial genome supports the re-classification of R. microplusas different species. Therefore, populations of R. microplusfrom Australia, Cambodia, Philippines, Indonesia, New Caledonia, Borneo, New Guinea, Tahiti and parts of Southeast Asia were recently reinstated as R. australis. Moreover, a better knowledge on the speciation between these two species could pave the way to important advances in tick control strategies.

New molecular data shed light on the global phylogeny and species limits of the Rhipicephalus sanguineus complex

The Rhipicephalus sanguineus complex is a group of closely related tick species distributed all around the world. In this study, using mitochondrial 16S ribosomal DNA, new specimens of R sanguineus sensu lato from Turkey and Rhipicephalus camicasi from Kenya, were evaluated together with available sequences of this complex in GenBank. Our objectives were to delimit the complex, re-evaluate its global phylogeny and develop a reconstruction of its biogeographic history. Given Turkey's geographical location and its neighboring status within Africa, Asia and Europe, molecular information of R. sanguineus s.l. species from this region could have important implications both on a regional and global scale. Phylogenetic trees obtained with three methods (Bayesian, Maximum Likelihood and Maximum Parsimony) were highly similar and consensus trees gave the same branching patterns and similar node support values. A total of four different clades with up to 9 Operational Taxonomic Units formed strong monophyletic groups. Biogeographic reconstructions demonstrated the importance of populations in Middle East (Turkey) in the spread of the group from Europe to Africa and Asia. Data supported previous conclusions on the existence of two species of R. sanguineus s.l. in South America and the strong molecular similarity between R. camicasi and the so-called tropical lineage of R. sanguineus s.l. These results point to the need of a re-evaluation of most specimens designated as R. sanguineus s.l. in East Europe, Middle East, Africa and Asia after an adequate re-description of this taxon.