Molecular evidence of cryptic species within the Liriomyza huidobrensis (Diptera: Agromyzidae)

Phylogenetic relationships among populations of the polyphagous pea leafminer, Liriomyza huidobrensis (Blanchard), were investigated using DNA sequence data. Maximum parsimony analysis of 941 bp of mitochondrial cytochrome oxidase I and II genes showed that L. huidobrensis contains two well-defined monophyletic groups, one composed of specimens from California and Hawaii and one composed of specimens from South and Central America together with populations that have been recently introduced into other parts of the world. The differentiation between the two clades within L. huidobrensis is equivalent to that seen between other agromyzid species, suggesting that L. huidobrensis as currently defined contains two cryptic species. This finding is consistent with field observations of differences in pest status and insecticide resistance between L. huidobrensis populations. Until additional studies are complete, no changes in L. huidobrensis taxonomy are proposed. However, researchers and quarantine officials may wish to consider the findings of the current study in designing research, pest management, and quarantine programs for L. huidobrensis.     

Molecular basis of hyperlipidemia in golden hamsters during experimental infection with Ancylostoma ceylanicum (Nematoda:Strongylidae)

An infection of golden hamsters with Ancylostoma ceylanicum, a hookworm parasite, induced profound hyperlipidemia, particularly hypertriglyceridemia, and the effect was directly related to the degree of infection. A significant increase was also noticed in serum cholesterol and phospholipid levels. The appearance of lipoprotein-X, an abnormal low density lipoprotein, was detected in the serum of hookworm-infected animals. The hyperlipidemia was further characterized by an increase in very low density lipoproteins (VLDL) and low density lipoproteins (LDL) with a concomitant decline in high density lipoproteins (HDL). Decreased lipolytic activities, especially triglyceride lipase, in hepatic tissue and induction of lipolytic activities in intestine and adipose tissues indicated mobilization of fats from adipose and jejunum with a defective removal of triglyceride-rich lipoproteins in hepatic tissues. Accumulation of lipids in liver and depletion in adipose tissue supported these results. The derangement may have a significant effect on host parasite interaction and is an important pathophysiological feature occurring during experimental ancylostomiasis.     

Molecular evidence for cryptic species in a narrowly endemic western North American springsnail (Pyrgulopsis gilae)

Pyrgulopsis gilae is a small springsnail that is narrowly distributed along the forks of the upper Gila River and currently being managed as a threatened and sensitive species by the State of New Mexico and United States Forest Service, respectively. A previous phylogeographic study of this species based on mitochondrial COI sequences delineated substantial divergence between several populations along the lower and upper reaches of the East Fork Gila River. The present study surveyed COI variation among a larger number of populations across the entire geographic range of P. gilae. Three haplotype groupings were delineated that were congruently resolved as clades by a Bayesian analysis. One of the clades was composed of populations along the lower East Fork and mainstem Gila River and corresponds to P. gilae as originally circumscribed. The other two clades were composed of populations along the Middle Fork and upper East Fork Gila River that were recently referred to P. gilae. These three geographically isolated clades do not share any haplotypes, have significant F_ST values, and are differentiated from each other by 3.9–6.3 % sequence divergence. Based on this evidence we suggest that the clades represent distinct species and should be managed as separate conservation units pending taxonomic revision of P. gilae. This study provides additional evidence that geographically disjunct subunits of Pyrgulopsis species often represent distinct monophyletic lineages that may warrant formal taxonomic recognition, and thus underscores the importance of fine-scale conservation genetics studies of these imperiled organisms.     

Molecular evidence for further overlooked species within the Gammarus fossarum complex (Crustacea: Amphipoda)

The freshwater amphipod Gammarus fossarum Koch, in Panzer, 1836 is a locally abundant keystone species mainly occuring in European headwaters but also in larger rivers. Genetic studies in the past 25 years have revealed three cryptic species within nominal G. fossarum (types A, B and C). Assignments of specimens to these types were based on allozyme and 16S markers. Today, a fragment of the cytochrome c oxidase subunit 1 (CO1) is primarily used as a genetic marker for species assignments (‘DNA Barcoding’), yet not a single CO1 sequence of G. fossarum is available in the Barcode of Life Database. We analysed new CO1 and 16S data for German, Hungarian and Croatian G. fossarum specimens and compared them with 16S and CO1 sequences of G. fossarum from GenBank. Thereby, we close the gap between traditional allozyme- and 16S-based species assignments and modern CO1 barcoding. Studying genetic variation in 55 specimens from 29 populations, we identified between 11 and 23 novel and genetically distinct clades using distance- and tree-based methods. Our results suggest that G. fossarum comprises several additional, yet unrecognised, species in particular from the Balkan region. Therefore, a taxonomic revision and biogeographic reconsideration of the G. fossarum complex is urgently needed.     

Molecular evidence for a fourth species within the Isotoma viridis group (Insecta, Collembola)

For identification of single species within the Isotoma viridis group, we present polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) as a fast and efficient DNA-based molecular method. We used five PCR primers amplifying the cytochrome oxidase II (COII) region (760 bp) of the mitochondrial DNA. The sequences clearly separated four species ( I. viridis , I. riparia , I. anglicana and I. caerulea ) out of samples from Norway, Sweden, Germany and Switzerland. Examination of genetic variation and phylogenetic relationship did not support the separation of two colour pattern forms of I. viridis into distinct species. For RFLP, several restriction enzymes were tested for their ability to produce not only species-specific restriction fragment patterns but to discriminate more than one species per enzyme used with as few cleavage sites as possible. Such a design should render a clear fragment pattern when performing a double digest. These demands appear to be fulfilled best by the combination of the restriction enzymes Mfe I, Nci I and one of Aci I, Bst EII, Nde I, or Sfc I. From the enzymes tested in a previous study, Ase I proved to be reliable, whereas Mbo I can no longer be recommended.     

Molecular prospecting for cryptic species in Phyllodistomum lacustri (Platyhelminthes,Gorgoderidae)

Rosas‐Valdez, R., Choudhury, A. & Pérez‐Ponce de León, G. (2011). Molecular prospecting for cryptic species in Phyllodistomum lacustri (Platyhelminthes, Gorgoderidae). —Zoologica Scripta, 40, 296–305. Partial sequences of the 28S ribosomal RNA and cytochrome c oxidase I (COI) genes were compared among populations of a widely distributed and morphologically uniform digenean species in North America, Phyllodistomum lacustri, a parasite characteristically associated with ictalurid catfishes. Specimens were collected from the urinary bladder of ictalurid hosts in six localities of North America, spanning most of the latitudinal range of this freshwater fish group. Sequences of other congeneric species, including a potentially close relative, P. staffordi, were also obtained and used for comparison. Analyses of both molecular markers show very low or no intrapopulation variation within each sampling site. However, samples of P. lacustri from different hosts and regions exhibit varying levels of interpopulation genetic differences. Such differences are explained by the wide geographical distribution and host range of the ictalurids they parasitize, particularly in ictalurids distributed in Mexican freshwaters where they experienced a diversification process. Variation in both nuclear and mitochondrial genes and phylogenetic analyses, in conjunction with geographical and host information (drainage isolation and endemicity of the host species), indicate that at least three populations show potential as cryptic species.     

Molecular variability in the Celleporella hyalina (Bryozoa; Cheilostomata) species complex: evidence for cryptic speciation from complete mitochondrial genomes

The bryozoan Celleporella has been shown to be composed of multiple, often cryptic, lineages. We sequenced two complete mitochondrial (mt) genomes of the Celleporella hyalina species complex from Wales, UK and Norway (i) to determine genetic divergence at the complete mt genome level, and (ii) to design new molecular markers for examining the interrelationships amongst the major lineages. In addressing (i), we estimated genetic divergence at three levels: (a) nucleotide diversity (π), (b) genome size, and (c) gene order. Genes nad4L, nad6, and atp8 showed the highest levels of divergence, and rrnL, rrnS, and cox1 showed the lowest levels. Inter-genome nucleotide divergence of protein-coding and ribosomal RNA genes, measured as π, was 0.21. The two genomes differed substantially in size, with the Norwegian genome being 2,573 base pairs (bp) longer than the Welsh genome, 17,265 and 14,692 bp, respectively. This difference in size is attributable to long non-coding regions present in the Norwegian genome. Both genomes exhibit similar gene orders, except for the translocation of one transfer RNA (trnA). Considering the high nucleotide diversity, genome size difference and change in gene order, these mt genomes are considered sufficiently divergent to have originated from two distinct species. In addressing (ii) we designed PCR primers that flank the most conserved regions of the genome: 1,300 bp of cox1 and a contiguous 2,000 bp fragment of rrnL + rrnS. The primers have yielded products for tissue from Wales, Norway, New Zealand, Alaska and Chile and should provide useful tools in establishing species- and population-level diversity within the Celleporella complex.     

Inorganic elements in adult Ternidens deminutus (Nematoda: Strongylidae: Oesophagostominae) from humans and baboons

The present paper presents the results of an analysis of the inorganic elements detected in Ternidens deminutus from human and baboon hosts. It reports on the presence of Zn, Fe, P, Ca, Cu, Mg, Mn and K in the worms and notes especially that worms from humans contained a higher percentage of body weight of Zn than those from baboons, a finding in agreement with an earlier EDAX analysis of the pigment granules of this species.     

Molecular and cytogenetic evidence for cryptic speciation within a rare endemic Malagasy lemur, the Northern Sportive Lemur (Lepilemur septentrionalis)

Evolutionary relationships of different populations of the threatened malagasy lemur Lepilemur septentrionalis were assessed by sequence analysis of mitochondrial DNA (D-loop region and partial Cyt b gene). One hundred and fifty nine samples were collected from five main different localities in the northern part of Madagascar. We applied the phylogenetic species concept based on fixed diagnostic differences to determine the status of different geographical populations. No nucleotide site diagnoses Ankarana from Andrafiamena or Analamera. However, numerous fixed differences separate Sahafary from all other populations. These results were corroborated by phylogenetic trees. As previous cytogenetic studies, our molecular data suggest that two cryptic species of Lepilemur occur in the extreme north of Madagascar. This speciation is probably caused by chromosomal rearrangements in at least one of the evolutionary lineages. Our study comprises another striking example of how molecular genetic assay can detect phylogenetic discontinuities that are not reflected in traditional morphologically based taxonomies. Our study indicates that the Sahafary population is a hitherto undescribed endangered endemic species which urgently needs conservation efforts.     

New molecular evidence that Teladorsagia circumcincta (Nematoda: Trichostrongylidea) is a species complex

In the polymorphic Teladorsagia circumcincta (morphs circumcincta and trifurcata), a sheep and goat line (SGL) and a goat line (GL) have been previously described on the basis of the malate dehydrogenase allozyme polymorphism (MDH-2) and of the morphology of the dorsal ray. The GL were never found alone in 1 host, so the status of species was not given to these 2 lines. To investigate further whether there are other genetic markers that will delineate them, we collected T. circumcincta worms from goat and sheep at 8 farms in Touraine (west-central France). The worms were identified individually as being SGL or GL on the basis of MDH-2 polymorphism. This distinctiveness was corroborated by sequences of the beta-tubulin isotype I gene, the second internal transcribed spacer (ITS2) of their rDNA, and the nicotinamide dehydrogenase (ND4) gene of their mDNA. The extent of the divergence in the 3 additional genetic markers was such that SGL and GL may be considered as 2 species. A third putative species was found in the SGL line based exclusively on the ND4 gene. These findings suggest that T. circumcincta is a species complex and that further investigation is required on a wider geographic scale.     

The morphology and differential diagnosis of parasitic larvae of Triodontophorus (Nematoda, Strongylidae)

356 parasitic larvae of the genus Triodontophorus from Equidae (two Equus hemionus and one E. grevyi) have been investigated. They belong to three phenons, which differ from each other by the shape and dimensions of a stoma, the structure of teeth and other signs. That phenons belong to three different species: T. serratus, T. tenuicollis and T. brevicauda. The differential diagnosis of L4 of that species of Triodontophorus are given.     

Quantitative morphological evidence for incipient species within Lutzomyia quinquefer (Diptera: Psychodidae)

Morphological variation among geographic populations of the New World sand fly Lutzomyia quinquefer (Diptera, Phlebotominae) was analyzed and patterns detected that are probably associated with species emergence. This was achieved by examining the relationships of size and shape components of morphological attributes, and their correlation with geographic parameters. Quantitative and qualitative morphological characters are described, showing in both sexes differences among local populations from four Departments of Bolivia. Four arguments are then developed to reject the hypothesis of environment as the unique source of morphological variation: (1) the persistence of differences after removing the allometric consequences of size variation, (2) the association of local metric properties with meristic and qualitative attributes, rather than with altitude, (3) the positive and significant correlation between metric and geographic distances, and (4) the absence of a significant correlation between altitude and general-size of the insects.     

Detection by allozyme electrophoresis of cryptic species of Hypodontus macropi (Nematoda: Strongyloidea) from macropodid marsupials.

Allozyme electrophoresis of 98 Hypodontus macropi from eight different species of hosts using 24 enzymes revealed a complex of at least six sibling species, with 15-50% fixed genetic differences between taxa. Except for the taxon parasitizing Macropus rufus/M. robustus, pairs of parasite taxa were, in each case, sympatric at each locality examined, thus supporting the conclusion that they represent valid species. The existence of a series of host-specific nematode taxa explains many of the inconsistencies noted previously in the host distribution of H. macropi. Comparison of parasite allozyme phenograms with host phylogeny suggests that four of the speciation events could be attributable to cospeciation and two to host switching. A clear case of host switching between M. rufus/M. robustus and M. fuliginosus was found.     

Molecular approaches to identify cryptic species and polymorphic species within a complex community of fig wasps

Cryptic and polymorphic species can complicate traditional taxonomic research and both of these concerns are common in fig wasp communities. Species identification is very difficult, despite great effort and the ecological importance of fig wasps. Herein, we try to identify all chalcidoid wasp species hosted by one species of fig, using both morphological and molecular methods. We compare the efficiency of four different DNA regions and find that ITS2 is highly effective for species identification, while mitochondrial COI and Cytb regions appear less reliable, possibly due to the interference signals from either nuclear copies of mtDNA, i.e. NUMTs, or the effects of Wolbachia infections. The analyses suggest that combining multiple markers is the best choice for inferring species identifications as any one marker may be unsuitable in a given case.     

Genetic isolation and cryptic variation within the Lycaena xanthoides species group (Lepidoptera: Lycaenidae)

Species exist as biological entities with patterns of discontinuous phenotypic variation. However, the distinctness of taxa is called into question when morphological intermediates exist in areas of sympatry, reflecting either gene flow among variants of a species or hybridization between different species. Studying the partitioning of genetic variation provides a means to discern between the two possibilities. We used genetic and morphometric approaches to investigate the degree of isolation among the three members of the Lycaena xanthoides species group. Lycaena xanthoides, L. editha, and L. dione are predominantly allopatric and have been treated both as three separate species and as a single polytypic species. Using 618 bp of the mitochondrial gene COII, we found little phylogenetic resolution, but significant among-taxa genetic variance partitioning. Divergence among these taxa has been relatively recent, as evidenced by relatively low pairwise sequence divergence. Also, the existence of two well-supported clades within L. xanthoides sensu stricto, concordant with the Transverse Ranges of southern California, indicates divergence within this taxon, and a possible cryptic species. Significant morphological differentiation between L. editha and L. xanthoides supports the hypothesis that these taxa represent separate gene pools. Populations occurring in a narrow zone where the two species' ranges approach are characterized by intermediate morphology, suggesting incomplete morphological divergence or recent hybridization. These findings highlight the utility of genetic data in inferring species boundaries and the identification of cryptic lineages.     

Genetic evidence for three species within Pseudoterranova decipiens (Nematoda, Ascaridida, Ascaridoidea) in the North Atlantic and Norwegian and Barents Seas

Genetic variation of 1017 specimens of codworm, Pseudoterranova decipiens, collected from fish and seals at 23 sampling locations in the North Atlantic and Norwegian and Barents Seas, was analysed on the basis of 16 enzyme loci. Three reproductively isolated species, provisionally designated P. decipiens A, B and C, were detected, showing distinct alleles at the following loci: Mdh-1, 6Pgdh, Np, Pgm, Est-2 (between species A and B); Mdh-3, 6Pgdh, Np, Sod-1, Adk, Pgm, Est-2, Mpi (between A and C); Mdh-1, Mdh-3, Sod-1, Adk, Pgm, Est-2, Mpi (between B and C). One F1 hybrid was observed between P. decipiens A and B, but this apparently does not lead to any gene exchange between the two species, which do not show any evidence of introgression. No hybrids or introgressed individuals were observed between P. decipiens C and either A or B. Genetic distances among conspecific populations were low (average Nei's D 0.001-0.005), even though they were collected thousands of kilometres apart, indicating high levels of gene flow within each of the three species. The values of Nei's index D were 0.44 between P. decipiens A and B, 0.57 between B and C, and 0.79 between A and C. Estimated evolutionary divergence times, using Nei's formula, range from 2 to 4 million years. Differences between P. decipiens A, B and C were also found with respect to genetic variability, morphology, geographical distribution and hosts. Mean heterozygosity values of 0.08, 0.05 and 0.02 were obtained for P. decipiens A, B and C, respectively. Preliminary morphological examination of adult males, previously identified by multilocus electrophoresis, revealed differences in the relative size and pattern of caudal papillae. P. decipiens B is widespread in the study area, whereas P. decipiens A was found only in the North-East Atlantic and Norwegian Sea. In this area P. decipiens A is most common in the grey seal, Halichoerus grypus, while the common seal, Phoca vitulina, is the main host for P. decipiens B. In Canadian Atlantic waters, where P. decipiens A is apparently absent, P. decipiens B infects both grey and common seals; a few specimens were also found in the hooded seal, Cystophora cristata. The only definitive host so far identified for P. decipiens C is the bearded seal, Erignathus barbatus; P. decipiens C appears to be widespread, occurring in both the North-West Atlantic and Barents Sea.     

Molecular and morphological evidence for an origin of the aberrant genus Milula within himalayan species of Allium (Alliacae)

Phylogenetic relationships between Allium and the monotypic Himalayan genus Milula were analyzed using sequences of the nuclear ribosomal DNA internal transcribed spacer (ITS) region and of the intergenic spacers from the chloroplast trnD(GUC)-trnT(GGU) region. Both marker systems unambiguously placed Milula spicata within Allium subgenus Rhizirideum, close to A. cyathophorum. Morphologically, the main difference between Allium and Milula is the conspicuous spicate inflorescence of Milula vs the mostly capitate or umbellate inflorescences in Allium. Anatomical investigations of leaf characters support a close relationship of Milula with A. cyathophorum and A. mairei, whereas root characters are distinctive from other species of section Cyathophora. To maintain Allium as monophyletic, Milula has been included as A. spicatum in Allium subgenus Rhizirideum.