The value of idiosyncratic markers and changes to conserved tRNA sequences from the mitochondrial genome of hard ticks (Acari: Ixodida: Ixodidae) for phylogenetic inference

Idiosyncratic markers are features of genes and genomes that are so unusual that it is unlikely that they evolved more than once in a lineage of organisms. Here we explore further the potential of idiosyncratic markers and changes to typically conserved tRNA sequences for phylogenetic inference. Hard ticks were chosen as the model group because their phylogeny has been studied extensively. Fifty-eight candidate markers from hard ticks (family Ixodidae) and 22 markers from the subfamily Rhipicephalinae sensu lato were mapped onto phylogenies of these groups. Two of the most interesting markers, features of the secondary structure of two different tRNAs, gave strong support to the hypothesis that species of the Prostriata (Ixodes spp.) are monophyletic. Previous analyses of genes and morphology did not strongly support this relationship, instead suggesting that the Prostriata is paraphyletic with respect to the Metastriata (the rest of the hard ticks). Parallel or convergent evolution was not found in the arrangements of mitochondrial genes in ticks nor were there any reversals to the ancestral arthropod character state. Many of the markers identified were phylogenetically informative, whereas others should be informative with study of additional taxa. Idiosyncratic markers and changes to typically conserved nucleotides in tRNAs that are phylogenetically informative were common in this data set, and thus these types of markers might be found in other organisms.     

A re-evaluation of relationships in the Metastriata (Acari: Parasitiformes: Ixodidae)

A systematic analysis of generic/subgeneric relationships in the ixodid lineage Metastriata was conducted based on 82 structural and developmental characters, 15 of which have multiple states. Results of the analysis show considerable agreement with current classifications of the Ixodidae, including good support for monophyly of the Metastriata, the genera Ixodes and Haemaphysalis, and the grouping of Hoogstraal's Rhipicephalinae and Hyalomminae, and fair support for the genus Hyalomma. Our results differ from these classifications most notably by rejecting monophyly of the genera Aponomma and Amblyomma. Instead, the analysis provides weak support for a hypothesis in which the basal two to three lineages in the Metastriata are all composed of Aponomma species, and in which a fourth lineage within that genus, Aponomma elaphense, clusters with Amblyomma quadricavum to form a more derived lineage. Resolution is insufficient to support or reject monophyly of the genera Dermacentor and Rhipicephalus, or to resolve the position of the genus Haemaphysalis relative to the other Metastriata. Overall resolution within the Rhipicephalinae/Hyalomminae is quite good, but it is quite poor among the Amblyomminae and Haemaphysalinae. These results are similar to the results of recent 16S and 18S rDNA based molecular analyses. There are few indications of host specificity among the lineages analysed. The basal metastriate lineages contain a large number of squamate associates, but the pattern is not sufficiently clear to reject the hypothesis that these ticks will feed on all available vertebrate hosts. On the other hand, nearly all of the basal taxa are Australian. This suggests a possible origin of the Metastriata in Australia, with subsequent dispersal to other parts of the world.     

Species identification of <Emphasis Type="Italic">Ixodes granulatus</Emphasis> (Acari: Ixodidae) based on internal transcribed spacer 2 (ITS2) sequences

To investigate the genetic specificity of Ixodes granulatus ticks collected from Taiwan, the genetic identities and phylogenetic relationships were analyzed by comparing the sequences of the internal transcribed spacer 2 (ITS2) region obtained from 27 strains of ticks representing twelve species of Ixodes. Five major clades can be easily distinguished by neighbour-joining analysis and were congruent by maximum-parsimony method. All these I. granulatus ticks collected from Taiwan and Japan were genetically affiliated to a monophyletic group with highly homogeneous sequences (95.8–99.5% similarity), and can be discriminated from other species and subgenera of Ixodes ticks with a sequence divergence ranging from 13.6% to 62.9%. Moreover, interspecific analysis revealed that four distinct lineages are evident between Ixodes ticks, and all these I. granulatus ticks collected from Taiwan and Japan belong to the same lineage. Our results provide the first investigation on the genetic specificity of I. granulatus ticks, and demonstrate that all these I. granulatus ticks represent a unique lineage distinct from other species and subgenera of Ixodes ticks. The feasibility of ITS2-based genetic analysis for species-specific identification of I. granulatus ticks around East Asia was highly anticipated.     

Phylogeny of the hard ticks (Ixodidae) inferred from 18S rRNA indicates that the genus Aponomma is paraphyletic

We examined the phylogeny of ticks (Acari:Parasitiformes:Ixodida) and their closest known mite relatives (Acari:Parasitiformes:Mesostigmata and Holothyrida) using 18S rRNA sequences. In our analyses, we included sequences from 36 taxa. Sequences for 13 hard ticks (Family Ixodidae), 5 soft ticks (Family Argasidae), and 2 mesostigmatid mites were obtained from the GenBank database and we generated sequences for 15 hard ticks and 1 holothyrid mite. Ten of these tick species were endemic to Australia. Our analyses indicated that the suborder Holothyrida is more closely related to Ixodida than to Mesostigmata, the group used as outgroup in earlier molecular studies. This finding is consistent with Lehtinen's (1991) hypothesis that the Holothyrida rather than the Mesostigmata is the sister-group to the Ixodida. Within the hard ticks the genus Aponomma and thus the family Amblyomminae were paraphyletic. Taxonomic revision of these taxa is needed. The genus Amblyomma was paraphyletic without the inclusion of "typical" Aponomma species (Ap. latum and Ap. fimbriatum). There was a basal divergence between endemic Australian and other species in both the Metastriata and the Prostriata divisions of the hard ticks.     

Systematics and Biogeography of Hard Ticks, a Total Evidence Approach

Systematic relationships among the basal Ixodidae are examined using one morphological and three molecular data sets, 18S and 28S nuclear and 16S mitochondrial rDNA. Although different combinations of partitions are incompatible in a partition homogeneity test, combining them produces similar or better support for most major lineages through both additive and complementary effects. The different data sets are not complete for all taxa, but inclusion or exclusion of taxa with missing data for one or more data sets (8 of 29 ingroup taxa) does not influence overall tree topology and only weakly affects support levels. The only notable effect was based on gap treatment in the 28S data set. Gap treatment completely changes the arrangement and support levels for one basal node. The combined analyses show strong support for the Metastriata, a lineage including most endemic Australian Ixodes , and a lineage including the remaining Ixodes , but not for the Prostriata (= Ixodes s.l.). The distribution pattern of endemic Australian taxa (nearly all included in three exclusively Australian basal lineages) suggests that these lineages, and by extension the Ixodidae, originated after the isolation of Australia in the late Cretaceous, much more recently than previously indicated.     

Phylogenetic and phylogeographic relationships in Ixodes holocyclus and Ixodes cornuatus (Acari: Ixodidae) inferred from COX1 and ITS2 sequences

We inferred the phylogenetic and phylogeographic relationships in ticks, which were identified morphologically as Ixodes holocyclus and Ixodes cornuatus, from mitochondrial cytochrome oxidase subunit 1 (COX1) and nuclear internal transcribed spacer 2 (ITS2) sequences. We obtained COX1 (640bp) and ITS2 (527-568bp) sequences from 429 ticks from 49 localities in Tasmania, Victoria, New South Wales and Queensland, Australia. Our analyses show that there are two species of Ixodes in eastern Australia that cause paralysis in dogs and other mammals: I. holocyclus and I. cornuatus. Further, we conclude that the morphological criteria used to differentiate female I. holocyclus and I. cornuatus are equivocal but I. holocyclus can be distinguished from I. cornuatus using COX1 and/or ITS2 sequences. Intraspecific genetic variation in I. holocyclus and I. cornuatus was less than 0.86% and 0.19% for COX1 and ITS2, respectively. Ixodes holocyclus could be genetically distinguished between different geographic ranges. There were no significant genetic differences between I.cornuatus from Tasmania and mainland Australia, but there are some COX1 haplotypes of I. cornuatus from the mainland that were not detected in Tasmanian and vice versa.     

Molecular Evolution and Phylogenetic Utility of the Internal Transcribed Spacer 2 (ITS2) in Calyptratae (Diptera: Brachycera)

The resolution potential of internal transcribed spacer 2 (ITS2) at deeper levels remains controversial. In this study, 105 ITS2 sequences of 55 species in Calyptratae were analyzed to examine the phylogenetic utility of the spacer above the subfamily level and to further understand its evolutionary characteristics. We predicted the secondary structure of each sequence using the minimum-energy algorithm and constructed two data matrixes for phylogenetic analysis. The ITS2 regions of Calyptratae display strong A-T bias and slight variation in length. The tandem and dispersed repeats embedded in the spacers possibly resulted from replication slippage or transposition. Most foldings conformed to the four-domain model. Sequence comparison in combination with the secondary structures revealed six conserved motifs. Covariation analysis from the conserved motifs indicated that the secondary structure restrains the sequence evolution of the spacer. The deep-level phylogeny derived from the ITS2 data largely agreed with the phylogenetic hypotheses from morphologic and other molecular evidence. Our analyses suggest that the accordant resolutions generated from different analyses can be used to infer deep-level phylogenetic relations.     

Trematode and Monogenean rRNA ITS2 Secondary Structures Support a Four-Domain Model

The secondary structure of rRNA internal transcribed spacer 2 is important in the process of ribosomal biogenesis. Trematode ITS sequences are poorly conserved and difficult to align for phylogenetic comparisons above a family level. If a conserved secondary structure can be identified, it can be used to guide primary sequence alignments. ITS2 sequences from 39 species were compared. These species span four orders of trematodes (Echinostomiformes, Plagiorchiformes, Strigeiformes, and Paramphistomiformes) and one monogenean (Gyrodactyliformes). The sequences vary in length from 251 to 431 bases, with an average GC content of 48%. The monogenean sequence could not be aligned with confidence to the trematodes. Above the family level trematode sequences were alignable from the 5′ end for 139 bases. Secondary structure foldings predicted a four-domain model. Three folding patterns were required for the apex of domain B. The folding pattern of domains C and D varies for each family. The structures display a high GC content within stems. Bases A and U are favored in unpaired regions and variable sites cluster. This produces a mosaic of conserved and variable regions with a structural conformation resistant to change. Two conserved strings were identified, one in domain B and the other in domain C. The first site can be aligned to a processing site identified in yeast and rat. The second site has been found in plants, and structural location appears to be important. A phylogenetic tree of the trematode sequences, aligned with the aid of secondary structures, distinguishes the four recognized orders. Received: 21 November 1997 / Accepted: 9 February 1998     

The Mitochondrial Genomes of Nuttalliella namaqua (Ixodoidea: Nuttalliellidae) and Argas africolumbae (Ixodoidae: Argasidae): Estimation of Divergence Dates for the Major Tick Lineages and Reconstruction of Ancestral Blood-Feeding Characters

Ixodida are composed of hard (Ixodidae), soft (Argasidae) and the monotypic Nuttalliellidae (Nuttalliella namaqua) tick families. Nuclear 18S rRNA analysis suggested that N. namaqua was the closest extant relative to the last common ancestral tick lineage. The mitochondrial genomes of N. namaqua and Argas africolumbae were determined using next generation sequencing and de novo assembly to investigate this further. The latter was included since previous estimates on the divergence times of argasids lacked data for this major genus. Mitochondrial gene order for both was identical to that of the Argasidae and Prostriata. Bayesian analysis of the COI, Cytb, ND1, ND2 and ND4 genes confirmed the monophyly of ticks, the basal position of N. namaqua to the other tick families and the accepted systematic relationships of the other tick genera. Molecular clock estimates were derived for the divergence of the major tick lineages and supported previous estimates on the origins of ticks in the Carboniferous. N. namaqua larvae fed successfully on lizards and mice in a prolonged manner similar to many argasids and all ixodids. Excess blood meal-derived water was secreted via the salivary glands, similar to ixodids. We propose that this prolonged larval feeding style eventually gave rise to the long feeding periods that typify the single larval, nymphal and adult stages of ixodid ticks and the associated secretion of water via the salivary glands. Ancestral reconstruction of characters involved in blood-feeding indicates that most of the characteristics unique to either hard or soft tick families were present in the ancestral tick lineage.     

Genetic diversity of nuclear ITS1-5.8S-ITS2 rDNA sequence in Clonorchis sinensis Cobbold, 1875 (Trematoda: Opisthorchidae) from the Russian Far East

The present study examined the molecular organisation and sequence variation in the nuclear ribosomal DNA (rDNA) region, including the two internal transcribed spacers (ITS1 and ITS2) and the 5.8S gene of the Clonorchis sinensis from the Russian Far East. The relevant sequences from other parts of this species' area were downloaded from GenBank. The results showed 100% identity for all investigated 5.8S-ITS2 rDNA sequences. In contrast, two levels of intraspecific variations were revealed in the complete ITS1 sequences. The intra-genomic variation resulted from a C/T polymorphism in a single position. The inter-individual differences between the ITS1 sequences were both due to nucleotide and size polymorphisms resulting from a varying number of five-nucleotide repeats and followed by two ITS1 length variants. These variant frequencies correlate with the clonorchiasis level in some geographical localities. ITS1 differences, both in the mutation profile and mutation localisation, were revealed between northern and southern geographical samples. The presence of GC boxes that are identical to known regulatory motifs in eukaryotes was detected within the ITS1 sub-repeats. The predicted secondary structures for ITS1 consist of two large branches, one of which was invariable, while another depended on ITS1 length. The predicted secondary structure for ITS2 includes four helices around the core. The main differences between C. sinensis and other opisthorchids were localised on the tops of helices 2, 3, and 4. A phylogenetic MST reconstruction subdivided all ITS1 sequences into two well differentiated clusters, each with the major widespread ribotype, and showed that ribotype diversity in both Russia and Korea is much lower than in China. The results obtained demonstrate the feasibility of complete ITS1 sequences in C. sinensis population genetics and can be considered as a basis for further studies of the parasite infection because they may help to elucidate the molecular mechanisms of pathogen evolution and adaptation.     

Cryptic long internal repeat sequences in the ribosomal DNA ITS1 gene of the dinoflagellate Cochlodinium polykrikoides (dinophyceae): a 101 nucleotide six-repeat track with a palindrome-like structure

Extremely long PCR fragments were generated by PCR amplification of ITS and 5.8S rDNA from Cochlodinium polykrikoides against other dinoflagellates. These patterns were consistent among geographically different isolates of C. polykrikoies. DNA sequencing reactions revealed that the PCR products were 1,166 bp in length and consisted of 813 bp of ITS1, 160 bp of 5.8S rDNA and 193 bp of ITS2. Thus, the long length was caused mainly by the long ITS1 sequence. Cryptically, the ITS1 contained a tract of 101 bp that occurs six times in tandem. The six repeated elements had identical nucleotide sequences. ITS1, therefore, separated three distinct regions: the 5' end (122 bp), the six parallel repeats (606 bp), and the 3' region (85 bp). Interestingly, both the single and six-repeat sequences should be palindrome-like sequences. In inferred secondary structures, both repeat sequences formed a long helical structure. This is the first reported discovery of comparatively long internal repeats in the ITS1 of dinoflagellates.     

Molecular analysis of <Emphasis Type="Italic">Ixodes granulatus</Emphasis>, a possible vector tick for <Emphasis Type="Italic">Borrelia burgdorferi</Emphasis> sensu lato in Taiwan

The genetic identity of Ixodes granulatus ticks 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 19 strains of ticks representing seven species of Ixodes and two outgroup species (Rhipicephalus sanguineus and Haemaphysalis inermis). Four major clades could be easily distinguished by neighbour-joining analysis and were congruent by maximum-parsimony method. All these I. granulatus ticks of Taiwan were genetically affiliated to a monophyletic group with highly homogeneous sequences (92.2–99.3% similarity), and can be discriminated from other Ixodes species and other genera of ticks with a sequence divergence ranging from 11.7 to 30.8%. Moreover, intraspecific analysis revealed that two distinct lineages are evident between the same species of I. granulatus ticks collected from Taiwan and Malaysia. Our results demonstrate that all these I. granulatus ticks of Taiwan represent a unique lineage distinct from the common vector ticks (I. ricinus complex) for Borrelia burgdorferi spirochetes.     

Nuclear ribosomal DNA internal transcribed spacer 1 (ITS1) in Picea (Pinaceae): sequence divergence and structure

The nrDNA ITS1 of Picea is 2747-3271 bp, the longest known of all plants. We obtained 24 cloned ITS1 sequences from six individuals of Picea glehnii, Picea mariana, Picea orientalis, and Picea rubens. Mean sequence divergence within these individuals (0.018+/-0.009) is more than half that between the species (0.031+/-0.011) and may be maintained against concerted evolution by separation of Picea 18S-26S rDNA repeats on multiple chromosomes. Picea ITS1 contains three subrepeats with a motif (5'-GGCCACCCTAGTC) that is conserved across Pinaceae. Two subrepeats are tandem, remote from the third, and more closely related and significantly more similar to one another than either is to the third subrepeat. This correlation between similarity and proximity may be the result of subrepeat duplication or concerted evolution within rDNA repeats. In inferred secondary structures, subrepeats generally form long hairpins, with a portion of the Pinaceae conserved motif in the terminal loop, and tandem subrepeats pair with one another over most of their length. Coalescence of ITS1 sequences occurs in P. orientalis but not in the other species.     

Evolution of duplicate control regions in the mitochondrial genomes of metazoa: a case study with Australasian Ixodes ticks

To investigate the evolution pattern and phylogenetic utility of duplicate control regions (CRs) in mitochondrial (mt) genomes, we sequenced the entire mt genomes of three Ixodes species and part of the mt genomes of another 11 species. All the species from the Australasian lineage have duplicate CRs, whereas the other species have one CR. Sequence analyses indicate that the two CRs of the Australasian Ixodes ticks have evolved in concert in each species. In addition to the Australasian Ixodes ticks, species from seven other lineages of metazoa also have mt genomes with duplicate CRs. Accumulated mtDNA sequence data from these metazoans and two recent experiments on replication of mt genomes in human cell lines with duplicate CRs allowed us to re-examine four intriguing questions about the presence of duplicate CRs in the mt genomes of metazoa: (1) Why do some mt genomes, but not others, have duplicate CRs? (2) How did mt genomes with duplicate CRs evolve? (3) How could the nucleotide sequences of duplicate CRs remain identical or very similar over evolutionary time? (4) Are duplicate CRs phylogenetic markers? It appears that mt genomes with duplicate CRs have a selective advantage in replication over mt genomes with one CR. Tandem duplication followed by deletion of genes is the most plausible mechanism for the generation of mt genomes with duplicate CRs. Once duplicate CRs occur in an mt genome, they tend to evolve in concert, probably by gene conversion. However, there are lineages where gene conversion may not always occur, and, thus, the two CRs may evolve independently in these lineages. Duplicate CRs have much potential as phylogenetic markers at low taxonomic levels, such as within genera, within families, or among families, but not at high taxonomic levels, such as among orders.     

Molecular markers for gyrodactylids (Gyrodactylidae: Monogenea) from five fish families (Teleostei)

Thirty-one gyrodactylid species from five families of freshwater fish were examined and variable region V4 of the 18S small subunit ribosomal RNA gene and ribosomal RNA internal transcribed spacers ITS1 and ITS2 were sequenced. Both the V4 region and spacers ITS1 and ITS2 proved useful for gyrodactylid diagnosis. Sequences of these fragments exhibited interspecific variations and allowed clear determination at the species level. In some cases, the length of the ITS1 PCR fragment provided useful genetic markers. Species that yielded a short ITS1 fragment also showed distinct groupings in ITS2 and V4 sequences that were markedly different to sequences from species that contain a long ITS1. Repetitive sequences located in the ITS1 of Gyrodactylus gobii and Gyrodactylus vimbi accounted for some of the variations in length of PCR products. There was no evidence for intraspecific variation within these regions and short tandem repeats were not found in the other species studied. The number of polymorphic and intraspecific variations in nucleic acid sequences was low, therefore these variations did not affect species determination of gyrodactylids. Minor differences in the sequences between Western and Eastern European populations were detected for Gyrodactylus salaris/Gyrodactylus thymalli, Gyrodactylus teuchis and Gyrodactylus truttae, but these do not affect species diagnosis based on ribosomal DNA sequence. These results confirm the utility of both variable region V4 and the ITS as molecular markers for Gyrodactylus species.     

Species distinctions among closely related strains of Eustigmatophyceae (Stramenopiles) emphasizing ITS2 sequence-structure data: Eustigmatos and Vischeria

ABSTRACT

There is an increasing interest in the Eustigmatophyceae, a class of stramenopile microalgae, because they offer a variety of high-value health-beneficial compounds, e.g. polyunsaturated fatty acids (PUFAs), while concomitantly producing high biomass. Clarification of the taxonomy of these organisms at the species level is important in order to achieve reproducible results and constant yields of valuable compounds in their exploitation. Here the distinction of the, so far exclusively, morphologically defined species of the genera Eustigmatos and Vischeria was tested. Distinctions inferred from almost full 18S and ITS2 rRNA as well as plastid-encoded rbcL gene sequences were evaluated following a morphological investigation. The ITS2 secondary-structure-based phylogenies separated independent lineages (species) with long internal branches. This recommends ITS2 as a promising marker for a DNA metabarcoding approach (culture-independent biodiversity assessment). In contrast, the 18S V4 region which is commonly used in metabarcoding was almost invariant, whereas the almost full length sequences distinguished eight groups/types of strains. Monophyly of the species was supported by shared ITS2 secondary structure features, making them distinct from other eustigmatophyte lineages in concordance with phylogenetic analyses. No groups of strains were congruently supported by all three markers. Consequently, the previous distinction of two genera on the basis of morphology cannot be retained and the species should be accommodated in a single genus, Vischeria. Taxonomic changes among the species with the definition of epitypes, on the basis of cryopreserved strains, are recommended. Two findings point to a more complex evolutionary history of the species. The rbcL and nuclear markers resulted in disparate groupings of strains. In three species divergent intragenomic ITS2 paralogues were revealed. Therefore, a still broader taxon sampling, in conjunction with a deep sequencing approach, is needed for a more comprehensive understanding of the complex evolution of eustigmatophyte species.     

The internal transcribed spacer of nuclear ribosomal DNA in the gymnosperm Gnetum

We analyze the structure of the internal transcribed spacers ITS1 and ITS2 of the nuclear ribosomal DNA in the gymnosperm Gnetum, using a phylogenetic framework derived mainly from an intron in the nuclear low-copy LEAFY gene. Gnetum comprises 25-35 species in South America, Africa, and Asia, of which we sampled 16, each with two to six clones. Criteria used to assess ITS functionality were highly divergent nucleotide substitution, GC content, secondary structure, and incongruent phylogenetic placement of presumed paralogs. The length of ITS1 ranged from 225 to 986 bp and that of ITS2 from 259 to 305 bp, the largest ranges so far reported from seed plants. Gnetum ITS1 contains two informative sequence motifs, but different from other gymnosperms, there are only few and short (7-13 bp) tandem repeats. Gnetum ITS2 contains two structural motifs, modified in different clades by shortening of stems and loops. Conspecific sequences grouped together except for two recombinant pseudogenes that had ITS1 of one clade and ITS2 of another. Most of the pseudogenic ITS copies, paralogs, and putative chimeras occurred in a clade that according to a fossil-calibrated chloroplast-DNA clock has an age of a few million years. Based on morphology and chromosome numbers, the most plausible causes of the observed high levels of ITS polymorphism are hybridization, allopolyploidy, and introgression.     

Molecular phylogenetic analysis of ixodid ticks based on the ribosomal DNA spacer, internal transcribed spacer 2, sequences

An internal transcribed spacer (ITS2) sequence between the 5.8S and 28S rRNA genes was used to estimate the phyletic relationships among Ixodes spp. tick vectors of Lyme disease-causing Borrelia spirochetes. Analysis indicates that Borrelia burgdorferi sensu lato species associated with Lyme disease are found mainly in ticks of the Ixodes ricinus species complex. Other closely related tick species are not known to transmit the Borrelia-that cause Lyme disease in humans, but they appear to have a specific association with other closely related Borrelia species. There is a high degree of concordance in the phylogenetics of Borrelia taxa and the phylogenetic relationships among Ixodes ticks.