Partial Mitochondrial Gene Research Articles

Phylogeny of Polycladida (Platyhelminthes) based on mtDNA data

A phylogenetic analysis of Polycladida based on two partial mitochondrial genes (cox1 and 16S) is provided. The analysis includes 30 polyclad terminals that represent species from the two taxa which traditionally divide the groups Cotylea and Acotylea. Our phylogenetic analyses produced a well-supported hypothesis that confirms the monophyly of Polycladida, as well as Acotylea and Cotylea. Within Acotylea, there are two lineages not highly supported: on one hand, Leptoplanoidea (excluding Hoploplana elisabelloi) and one Stylochoidea member (Pseudostylochus intermedius) (classification sensu Faubel, 1983, 1984), and on the other hand, Stylochoidea members together with Discocelis tigrina and H. elisabelloi. The genera Stylochus and Imogine are not monophyletic. Within Cotylea, Pseudocerotidae and Euryleptidae are monophyletic, though not highly supported, while Prosthiostomidae is not. Euryleptoidea is paraphyletic. The genera Pseudobiceros and Pseudoceros are monophyletic and highly supported. Our results suggest that, within Acotylea, the prostatoid organs of Discocelis may have been derived from a prostatic vesicle. The genus Hoploplana could be included in Stylochoidea. Within Cotylea, the common ancestor of Euryleptidae and Pseudocerotidae might have been an aposematic animal with tentacles.

Remarkable genetic homogeneity supports a single widespread species of Hoplosternum littorale (Siluriformes, Callichthyidae) in South America

Hoplosternum littorale is a widespread, non-migratory, air-breathing armored catfish (Callichthyidae, Siluriformes) present in most river drainages of tropical South America. This species has invaded aquatic habitats out of its native range. The distribution and unity of this species is supported by strong morphological evidences, but only few molecular data are available. As for February 2017, 45 partial mitochondrial gene sequences for H. littorale were deposited at the U.S. National Center for Biotechnology Information (NCBI) GenBank. The nearly complete mitochondrial genomes from two individuals of H. littorale sampled in geographically isolated populations were sequenced and compared to the intraspecific divergence encountered in a related species, Corydoras nattereri. Tamura-Nei corrected distances between the mitochondrial genomes from the two H. littorale was 0.004, equivalent to the distance found among individuals from the same population of C. nattereri (0.002) and considerably smaller than the distance between C. nattereri and C. schwartzi (0.123). An insertion of 30 nucleotides between the ATPase 6 and the COIII genes was identified in H. littorale. This insertion is homologous to the one previously noted in two species of Corydoras. This work supports H. littorale as a single and widespread species of Callichthyidae catfish. The new genetic resources for H. littorale are now available to support further studies with larger sampling sizes.

The complete mitochondrial DNA of three monozoic tapeworms in the Caryophyllidea: a mitogenomic perspective on the phylogeny of eucestodes

BackgroundExternal segmentation and internal proglottization are important evolutionary characters of the Eucestoda. The monozoic caryophyllideans are considered the earliest diverging eucestodes based on partial mitochondrial genes and nuclear rDNA sequences, yet, there are currently no complete mitogenomes available. We have therefore sequenced the complete mitogenomes of three caryophyllideans, as well as the polyzoic Schyzocotyle acheilognathi, explored the phylogenetic relationships of eucestodes and compared the gene arrangements between unsegmented and segmented cestodes.ResultsThe circular mitogenome of Atractolytocestus huronensis was 15,130 bp, the longest sequence of all the available cestodes, 14,620 bp for Khawia sinensis, 14,011 bp for Breviscolex orientalis and 14,046 bp for Schyzocotyle acheilognathi. The A-T content of the three caryophyllideans was found to be lower than any other published mitogenome. Highly repetitive regions were detected among the non-coding regions (NCRs) of the four cestode species. The evolutionary relationship determined between the five orders (Caryophyllidea, Diphyllobothriidea, Bothriocephalidea, Proteocephalidea and Cyclophyllidea) is consistent with that expected from morphology and the large fragments of mtDNA when reconstructed using all 36 genes. Examination of the 54 mitogenomes from these five orders, revealed a unique arrangement for each order except for the Cyclophyllidea which had two types that were identical to that of the Diphyllobothriidea and the Proteocephalidea. When comparing gene order between the unsegmented and segmented cestodes, the segmented cestodes were found to have the lower similarities due to a long distance transposition event. All rearrangement events between the four arrangement categories took place at the junction of rrnS-tRNAArg (P1) where NCRs are common.ConclusionsHighly repetitive regions are detected among NCRs of the four cestode species. A long distance transposition event is inferred between the unsegmented and segmented cestodes. Gene arrangements of Taeniidae and the rest of the families in the Cyclophyllidea are found be identical to those of the sister order Proteocephalidea and the relatively basal order Diphyllobothriidea, respectively.

Genetic Diversity and Population Structure of the Eurasian Whiskered Tern (Chlidonias hybrida hybrida), a Species Exhibiting Range Expansion

Abstract. Contrasting the general trend of declining Afro-Palearctic migratory bird populations, the Whiskered Tern (Chlidonias hybrida hybrida) has recently exhibited spectacular demographic expansion and range shifts across its patchy European breeding distribution. To explore population connectivity of this species, individuals sampled from four sites in France, Poland, and Ukraine (n = 78) were genotyped at six microsatellite DNA loci and one partial mitochondrial cytochrome-b gene. Genetic variation was generally high (HE = 0.47-0.67; π = 1.29-2.26). Contrasting the historical absence of significant genetic structure (ΦST = 0.061-0.027), the microsatellite data indicated strong differentiation between France and the Eastern European sites (FST = 0.067-0.098). This, coupled with evidence of two distinct genetic clusters (K = 2; ΔK = 2) corresponding to little subdivision between Poland and Ukraine, has confirmed the presence of two European Whiskered Tern subpopulations (western and eastern) character...

A new dagger nematode, Xiphinema tica n. sp. (Nematoda: Longidoridae), from Costa Rica with updating of the polytomous key of Loof and Luc (1990)

A new dagger nematode, Xiphinema tica n. sp., is described and illustrated from several populations extracted from soil associated with several crops and wild plants in Costa Rica. The new dagger nematode is characterised by a moderate body size (3276–4240 μm), a rounded lip region, ca 13.5 μm wide, separated from body contour by a shallow depression, amphidial fovea large, stirrup-shaped, a moderately long odontostyle ca 135 μm long, stylet guiding ring located at ca 122 μm from anterior end, vulva almost equatorial (50–54%), well-developed Z-organ, with heavy muscularised wall containing in the most of specimens observed two moderately refractive inclusions variable in shape (from round to star-shaped), with uterine spines and crystalloid bodies; female tail short, dorsally convex-conoid, with rounded end and a small peg, with a c’ ratio ca 0.8, bearing two or three pairs of caudal pores and male absent. The unique and novel uterine differentiation based on the coexistence of a well-developed Z-organ mixed with uterine spines and crystalloid bodies in Xiphinema prompted us to update and include this combination of characters in the polytomous key of Loof and Luc (1990). Integrative diagnosis was completed with molecular data obtained, using D2-D3 expansion segments of 28S rDNA, ITS1-rDNA, partial 18S–rDNA and the partial mitochondrial gene cytochrome c oxidase subunit 1 (coxI). The phylogenetic relationships of this species with other Xiphinema spp. indicated that X. tica n. sp. was monophyletic to the other species from the morphospecies Group 4, Xiphinema oleae.

Detection and molecular characterization of the rice root-knot nematode Meloidogyne graminicola in Italy

The rice root-knot nematode Meloidogyne graminicola was detected in North Italy attacking roots of rice plants in lowland and upland fields. This nematode was previously reported only in South and Southeast Asia, China, South Africa, United States, Columbia and Brazil attacking rice and wheat. The present study provides an integrative diagnosis of the nematode on the basis of morphological and molecular data by sequencing the SCAR species specific marker, the ITS region and the partial mitochondrial coII-16srRNA genes. Morphological and morphometric traits of the new intercepted population of M. graminicola agree with the original description. The diagnostic SCAR marker was amplified showing the expected size fragment and unequivocally identified the Italian population as M. graminicola. Sequence analyses of ITS and coII-16SrRNA confirmed this identification. Sequence comparison of both molecular markers among Italian M. graminicola populations from lowland and upland rice fields revealed 100% identity. Phylogenetic analysis based on the ITS and coII-16SrRNA sequences revealed that Italian M. graminicola formed a well supported cluster with M. graminicola populations from GenBank confirming the high intraspecific variability among all populations. No correlation between genetic variability and geographic origins occurred. Furthermore, the phylogenetic trees suggest a common ancestor for most of the facultative meiotic parthenogenetic Meloidogyne species.

Morphological and molecular characterization of the metacercaria of Paragonimus caliensis, as a separate species from P. mexicanus in Costa Rica

The trematode Paragonimus mexicanus is the etiological agent of paragonimiasis, a food-borne zoonotic disease in Latin America. This species, as well as Paragonimus caliensis, have been reported from Costa Rica, but it is not known if the two are synonymous. Two types of Paragonimus metacercariae from freshwater pseudothelphusid crabs from several localities in Costa Rica were recognized by light microscopy. Morphologically, these corresponded to descriptions of P. mexicanus and P. caliensis. Metacercariae of the former species lacked a membrane or cyst and their bodies were yellow in color. Those of P. caliensis were contained in a transparent thin cyst and were pink in color. Morphotypes of metacercariae were determined using scanning electron microscopy (SEM). Based on the number and distribution of papillae in the ventral sucker, three morphotypes were found for P. mexicanus and two for P. caliensis. Analysis of DNA sequences (nuclear ribosomal 28S and ITS2 genes, and partial mitochondrial cox1 gene) confirmed the presence of P. mexicanus and provided the first molecular data for P. caliensis. The two species are phylogenetically distinct from each other and distant from the Asian species. The confirmation of P. caliensis as a separate species from P. mexicanus raises several questions about the ecology, biological diversity, and epidemiology of the genus Paragonimus in Costa Rica.

Cerebral and non-cerebral coenurosis: on the genotypic and phenotypic diversity of Taenia multiceps.

We characterised the causative agents of cerebral and non-cerebral coenurosis in livestock by determining the mitochondrial genotypes and morphological phenotypes of 52 Taenia multiceps isolates from a wide geographical range in Europe, Africa, and western Asia. Three studies were conducted: (1) a morphological comparison of the rostellar hooks of cerebral and non-cerebral cysts of sheep and goats, (2) a morphological comparison of adult worms experimentally produced in dogs, and (3) a molecular analysis of three partial mitochondrial genes (nad1, cox1, and 12S rRNA) of the same isolates. No significant morphological or genetic differences were associated with the species of the intermediate host. Adult parasites originating from cerebral and non-cerebral cysts differed morphologically, e.g. the shape of the small hooks and the distribution of the testes in the mature proglottids. The phylogenetic analysis of the mitochondrial haplotypes produced three distinct clusters: one cluster including both cerebral isolates from Greece and non-cerebral isolates from tropical and subtropical countries, and two clusters including cerebral isolates from Greece. The majority of the non-cerebral specimens clustered together but did not form a monophyletic group. No monophyletic groups were observed based on geography, although specimens from the same region tended to cluster. The clustering indicates high intraspecific diversity. The phylogenetic analysis suggests that all variants of T. multiceps can cause cerebral coenurosis in sheep (which may be the ancestral phenotype), and some variants, predominantly from one genetic cluster, acquired the additional capacity to produce non-cerebral forms in goats and more rarely in sheep.

A new fish cell line derived from the caudal fin of freshwater angelfish Pterophyllum scalare: development and characterization

In this study, a new cell line derived from the caudal fin of the freshwater angelfish Pterophyllum scalare was developed and characterized. The cell line was designated angelfish fin (AFF) and subcultured 44 times since its development. These cells grew well in Leibovitz's -15 medium supplemented with 10% foetal bovine saline (FBS) at 28° C and the modal chromosome number (2n) was 48. The AFF cell-line is mainly comprised of epithelial cells as confirmed by immunocytological technique using anti-cytokeratin antibodies, an epithelial cell marker. This cell line was tested for growth in a temperatures range from 20 to 37° C and at various FBS concentrations of 5-20% at 28° C. The cell line was cryopreserved at different passage levels and revived successfully with 80% survival rate. Polymerase chain reaction amplification and sequencing of partial mitochondrial 16s rRNA and coI genes confirmed that the AFF cell-line originated from angelfish. Mycoplasma sp. contamination was not detected in AFF cells and checked by Hoechst 33258 fluorescence staining. At the 42nd passage the cells were transfected with 2 μg of pAcGFP1-N1 expression vector. The AFF cells exhibited cytotoxic effects when exposed to the bacterial extra cellular products from Serratia marcescens and Proteus hauseri. The AFF cells and cells from kidney and brain did not show cytopathic effect when exposed to cyprinid herpes virus2 and viral nervous necrosis virus. The newly developed AFF cell line will be useful for the isolation of viruses affecting angelfishes, such as iridoviruses, in the future.

Evolution in karst massifs: Cryptic diversity among bent-toed geckos along the Truong Son Range with descriptions of three new species and one new country record from Laos.

Species designated as 'cryptic' share a similar morphotype, and are often only clearly separable by molecular data. Cyrtodactylus, the most diverse gecko genus of the family Gekkonidae, is a prime example, because many morphologically similar taxa have only recently been identified as new species as a result of available genetic evidence. However, while cryptic diversity of Cyrtodactylus is already well documented on the Vietnamese side of the Truong Son range, only scarce data is available from central Laos. In this study, we address this issue by means of an integrative approach, which employs morphological, molecular, and ecological data to distinguish cryptic species of the Cyrtodacylus phongnhakebangensis species group primarily distributed along the northern Truong Son Range. Our analyses based on 12 selected morphological characters, a partial mitochondrial gene (COI), and five ecological parameters revealed three undescribed cryptic Cyrtodactylus species from Hin Nam No National Protected Area, which are described as Cyrtodactylus calamei sp. nov., Cyrtodactylus hinnamnoensis sp. nov., and Cyrtodactylus sommerladi sp. nov. A fourth discovered Cyrtodactylus population in Hin Nam No proved to be the first country record of C. cryptus for Laos. Our results highlight the importance of applying an integrative approach to resolving the taxonomy of complex and cryptic species groups, and the role of the Truong Son Range in maintaining the high level of biodiversity over time.

Genus Profundiconus Kuroda, 1956 (Gastropoda, Conoidea): Morphological and molecular studies, with the description of five new species from the Solomon Islands and New Caledonia

The genus Profundiconus Kuroda, 1956 is reviewed. The morphological characters of the shell, radular tooth and internal anatomy of species in Profundiconus are discussed. In particular, we studied Profundiconus material collected by dredging in deep water during different scientific campaigns carried out in the Solomon Islands, Madagascar, Papua New Guinea and New Caledonia. We reconstructed a phylogeny of 55 individuals based on partial mitochondrial cox1 gene sequences. The phylogeny shows several clades containing individuals that do not match any of the known species of Profundiconus based on their shell and radular morphologies, and are introduced here as five new species: Profundiconus maribelae sp. nov. from the Solomon Islands; P. virginiae sp. nov. from Chesterfield Plateau (New Caledonia); P. barazeri sp. nov. from Chesterfield Plateau and the Grand Passage area (New Caledonia); P. puillandrei sp. nov. from Norfolk Ridge (New Caledonia), Kermadec Ridge (New Zealand) and possibly Balut Island (Philippines); and P. neocaledonicus sp. nov. from New Caledonia. Furthermore, Profundiconus teramachii forma neotorquatus (da Motta, 1984) is raised to specific status as P. neotorquatus (da Motta, 1984).

Changing Names with Changed Address: Integrated Taxonomy and Species Delimitation in the Holarctic Colymbetes paykulli Group (Coleoptera: Dytiscidae).

Species delimitation of geographically isolated forms is a long-standing problem in less studied insect groups. Often taxonomic decisions are based directly on morphologic variation, and lack a discussion regarding sample size and the efficiency of migration barriers or dispersal/migration capacity of the studied species. These problems are here exemplified in a water beetle complex from the Bering Sea region that separates North America from Eurasia. Only a few sampled specimens occur from this particular area and they are mostly found in museum and private collections. Here we utilize the theory of integrated taxonomy to discuss the speciation of the Holarctic Colymbetes paykulli water beetle complex, which historically has included up to five species of which today only two are recognized. Three delimitation methods are used; landmark based morphometry of body shape, variation in reticulation patterns of the pronotum exo-skeleton and sequence variation of the partial mitochondrial gene Cyt b. Our conclusion is that the Palearctic and Nearctic populations of C. paykulli are given the status of separate species, based on the fact that all methods showed significant separation between populations. As a consequence the name of the Palearctic species is C. paykulli Erichson and the Nearctic species should be known as C. longulus LeConte. There is no clear support for delineation between Palearctic and Nearctic populations of C. dahuricus based on mtDNA. However, significant difference in size and reticulation patterns from the two regions is shown. The combined conclusion is that the C. dahuricus complex needs a more thorough investigation to fully disentangle its taxonomic status. Therefore it is here still regarded as a Holarctic species. This study highlights the importance to study several diagnosable characters that has the potential to discriminate evolutionary lineage during speciation.

Genetic differentiation of populations of the threatened saproxylic beetle Rosalia longicorn,Rosalia alpina(Coleoptera: Cerambycidae) in Central and South-east Europe

Knowledge of patterns of genetic diversity in populations of threatened species is vital for their effective conservation. Rosalia longicorn (Rosalia alpina) is an endangered and strictly protected beetle. Despite a marked decline in part of its range, the beetle has recently expanded to the lowlands of Central Europe. To facilitate a better understanding of the species' biology, recent expansion and more effective conservation measures, we investigated patterns of genetic structure among 32 populations across Central and South-east Europe. Eight microsatellite loci and a partial mitochondrial gene (cytochrome c oxidase subunit I) were used as markers. Both markers showed a significant decline in genetic diversity with latitude, suggesting a glacial refugium in north-western Greece. The cluster analysis of the nuclear marker indicated the existence of two genetically distinct lineages meeting near the border between the Western and Eastern Carpathians. By contrast, one widespread mtDNA haplotype was dominant in most populations, leading to the assumption that a rapid expansion of a single lineage occurred across the study area. The genetic differentiation among populations from the north-western part of the study area was, however, surprisingly low. They lacked any substructure and isolation-by-distance on a scale of up to 600 km. This result suggests a strong dispersal capacity of the species, as well as a lack of migration barriers throughout the study area. That the lowland populations are closely related to those from the nearby mountains indicates repeated colonization of the lowlands. Our results further suggest that R. alpina mostly lives in large, open populations. Large-scale conservation measures need to be applied to allow for its continued existence.

Discovering Hidden Diversity of Characins (Teleostei: Characiformes) in Ecuador's Yasuní National Park.

BackgroundManagement and conservation of biodiversity requires adequate species inventories. The Yasuní National Park is one of the most diverse regions on Earth and recent studies of terrestrial vertebrates, based on genetic evidence, have shown high levels of cryptic and undescribed diversity. Few genetic studies have been carried out in freshwater fishes from western Amazonia. Thus, in contrast with terrestrial vertebrates, their content of cryptic diversity remains unknown. In this study, we carried out genetic and morphological analyses on characin fishes at Yasuní National Park, in eastern Ecuador. Our goal was to identify cryptic diversity among one of the most speciose fish families in the Amazon region. This is the first time that genetic evidence has been used to assess the species content of the Napo Basin, one of the richest regions in vertebrate diversity.ResultsPhylogenetic analyses of partial mitochondrial 16S ribosomal RNA gene (∼600 pb) DNA sequences from 232 specimens of the family Characidae and its closest groups revealed eight candidate new species among 33 species sampled, representing a 24% increase in species number. Analyses of external morphology allowed us to confirm the species status of six of the candidate species.ConclusionsOur results show high levels of cryptic diversity in Amazonian characins. If this group is representative of other Amazonian fish, our results would imply that the species richness of the Amazonian ichthyofauna is highly underestimated. Molecular methods are a necessary tool to obtain more realistic inventories of Neotropical freshwater fishes.

Genetic diversity, paraphyly and incomplete lineage sorting of mtDNA, ITS2 and microsatellite flanking region in closely related Heliopora species (Octocorallia)

Examining genetic diversity and lineage sorting of different genes in closely related species provide useful information for phylogenetic analyses and ultimately for understanding the origins of biodiversity. In this study, we examined inter- and intraspecific genetic variation in internal transcribed spacer 2 (ITS2), partial mitochondrial gene (mtMutS), and nuclear microsatellite flanking region in two closely related octocoral species (Heliopora coerulea, HC-A and HC-B). These species were recently identified in a population genetic study using microsatellite markers. The two species have different reproductive timing, which ecologically promotes lineage sorting. In this study, we examined whether species boundaries could be detected by the commonly used nuclear ITS2 and mtMutS, as well as by possibly neutral microsatellite flanking sequences. Haplotype network analysis of microsatellite flanking region revealed that a possible ancestral haplotype was still shared between the two species, indicating on-going lineage sorting. Haplotype network analysis of ITS2 and microsatellite flanking region revealed shared haplotypes between the two lineages. The two species shared fewer ITS2 sequences than microsatellite flanking region sequences. The almost fixed point mutation at the tip of helix 3 of ITS2 was not associated with the secondary structure or compensatory base changes (CBCs). The phylogenetic tree of ITS2 showed paraphyly and that of the microsatellite flanking region indicated that lineage sorting for the two species may be incomplete. Much higher intra- and inter-individual variation of ITS2 was observed in HC-B than that in HC-A, highlighting the importance of examining ITS2 from multiple individuals to estimate genetic diversity. The mitochondrial mtMutS gene sequences from 39 individuals, including both species collected from Japan and Taiwan, showed no variation because of slow rates of mitochondrial nucleotide substitution. This study suggests caution is warranted when reciprocal monophyly in a phylogenetic tree is used as the criterion for delimiting closely related octocoral species based on ITS2 or mtMtuS sequences. Detection of boundaries between closely related species requires multi-locus analysis, such as genetic admixture analysis using multiple individuals.

Avian Schistosomes from the South American Endemic Gastropod Genus Chilina (Pulmonata: Chilinidae), with a Brief Review of South American Schistosome Species.

Our current knowledge of avian schistosomes from South America is scarce in all respects, including species and generic diversity, their life cycles, patterns of host use, potential to cause dermatitis outbreaks, and evolutionary affinities. As a step towards addressing this shortcoming, the goal of this study was to provide discrete reference points relating to snail hosts, locality records, morphological attributes, sequence for nuclear 28S and ITS, and partial mitochondrial cox1 genes, and phylogenetic relationships for schistosome cercariae recovered from different species of Chilina, which are gastropods endemic to South America. In total, 1,308 snails belonging to 6 species of Chilina were collected from 12 localities across Argentina. Thirty-eight snails (2.9%) had schistosome infections. Our data indicate the presence of 3 lineages of Chilina-transmitted schistosomes, all of which group within the major avian schistosome clade. However, none of the lineages grouped within or as sister to other known avian schistosome genera in the tree, indicating they probably represent undescribed genera. The relationships of these schistosomes from Chilina spp. are discussed in relation to their position in the global schistosome phylogenetic tree.

A multilocus molecular phylogeny of the endemic North American camel spider family Eremobatidae (Arachnida: Solifugae)

Camel spiders (Solifugae) are a diverse but poorly studied order of arachnids. No robust phylogenetic analysis has ever been carried out for the order or for any family within the Solifugae. We present a molecular phylogenetic analysis of the endemic North American family Eremobatidae Kraepelin, 1899, the first such analysis of a family of Solifugae. We use a multi-locus exemplar approach using DNA sequences from partial nuclear (28S rDNA and Histone H3) and mitochondrial (16S rRNA and Cytochrome c Oxidase I) gene loci for 81 ingroup exemplars representing all genera of Eremobatidae and most species groups within the genera Eremobates Banks, 1900, Eremochelis Roewer, 1934, and Hemerotrecha Banks, 1903. Maximum Likelihood and two Bayesian analyses consistently recovered the monophyly of Eremobatidae, Eremorhax Roewer, 1934 and Eremothera Muma, 1951 along with a group comprising all subfamily Eremobatinae Kraepelin, 1901 exemplars except Horribates bantai Muma, 1989 and a group comprising all Eremocosta Roewer, 1934 exemplars except Eremocosta acuitalpanensis (Vasquez and Gavin, 2000). The subfamily Therobatinae Muma, 1951 and the genera Chanbria Muma, 1951, Hemerotrecha, Eremochelis, and Eremobates were polyphyletic or paraphyletic. Only the banksi group of Hemerotrecha was monophyletic; the other species groups recognized within Eremobates, Eremochelis, and Hemerotrecha were paraphyletic or polyphyletic. We found no support for the monophyly of the subfamily Therobatinae. A time-calibrated phylogeny dated the most recent common ancestor of extant eremobatids to the late Eocene to early Miocene, with a mean estimate in the late Oligocene (32.2Ma).

The complete mitochondrial genome of the killifish Orestias sp. (Cyprinodontiformes, Cyprinodontidae) from the high Andean range

The killifish genus Orestias is endemic to freshwater ecosystems in the High Andes of Peru, Bolivia and Chile. Phylogenetic and phylogeographic studies with partial mitochondrial genes have failed to resolve relationship among species, needing more comprehensive approaches. In this study, we described the complete mitochondrial genome of Orestias sp. from Lirima, northern Chile, with the aim to provide useful data for phylogenetic purposes and species delimitation. The mitochondrial genome was assembled with 2.6 million of reads obtained through an Ion Torrent (chip 318) sequencer. The circular sequence of 16,617 bp showed the following nucleotide composition: A, 26.7%, C, 27.1%, G, 17.0%, and T, 29.2%. Gene composition and structure were similar to other fish sequences available, and comprised 13 protein-coding genes, 12S and 16S rRNA, 22 tRNA genes, and a control region.

Asian fish tapeworm, Khawia japonensis (Yamaguti, 1934), has expanded its European invasive range.

The invasive fish tapeworm, Khawia japonensis (Yamaguti, 1934) originally described in Japan, is reported for the first time in Slovakia. The tapeworm was found in farmed common carp (Cyprinus carpio L.) from East-Slovakian breeding fish pond. This finding is registered 4 years after the first announcement of this parasite in Europe (Po River Basin in Italy) in 2010 and increases its distribution area within this continent. Morphological characterization of K. japonensis supplemented with DNA sequences of cox1 and ribosomal lsrDNA genes is provided. Specimens from Slovakia phenotypically corresponded with those from feral and farmed carps from China, Vietnam, and Italy. Moreover, 100 and 98.7% identity of partial ribosomal lsrDNA gene and mitochondrial cox1 genes, respectively, were detected with K. japonensis from Japan. The invasive and pathogenic potential of K. japonensis in commercial breeding fisheries and its possible further spread in natural habitats is difficult to estimate for now. As yet, K. japonensis appears to be without a major impact on commercial breeding fisheries, but calls for more attention to the problem of biological invasions.

Description of Ruehmaphelenchus juliae n. sp. (Tylenchina: Aphelenchoididae) isolated from an ambrosia beetle, Xylosandrus crassiusculus (Motschulsky), from South Florida

During a survey of nematode associates of ambrosia beetles from dead and dying red bay and avocado trees affected by the laurel wilt epidemic in southern Florida, a Ruehmaphelenchus species was isolated from the non-native ambrosia beetle, Xylosandrus crassiusculus. The new species is characterised by its possession of an oral disc at the stoma opening, three lines in the lateral field, male spicule with clear dorsal and ventral limbs connected by elongated triangular cuticle, thin membrane-like tissue and cuticular bridge-like structure, conical tail with pointed tip of males and conical tail with digitate mucro of females. The new species is very similar to four previously described species: R. asiaticus, R. digitulus, R. thailandae and R. sirisus, and can be distinguished only by some minor morphological differences in male tail characters, i.e., spicule morphology, position of genital papillae and tail tip shape, and morphometric values. However, the new species is phylogenetically unique, i.e., it is the basal taxon of the Ruehmaphelenchus clade and close to Bursaphelenchus spp. Ruehmaphelenchus juliae n. sp. is therefore proposed based on its morphological diagnostic characters and molecular sequences of near-full-length of SSU, internal transcribed spacer region, D1, D2 and D3 expansion segments of LSU ribosomal RNA and partial mitochondrial COI genes.