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.     

New evidence of cryptic speciation in the family Longidoridae (Nematoda: Dorylaimida)

Longidorid nematodes comprise more than 500 species, and Longidorus and Xiphinema are the most diversified, prevalent, and cosmopolitan genera within plant-parasitic nematodes. This increases the risk of species misidentification. We conducted an integrative morphometric and genetic study on two longidorid species to elucidate the existence of new cases of cryptic speciation within the genera Longidorus and Xiphinema. Detailed morphological, morphometrical, multivariate, and genetic studies were carried out, and mitochondrial and nuclear haploweb analyses were used to differentiate species within the L. iliturgiensis and X. hispanum complexes. Species delimitation using multivariate and haplonet tools of L. iliturgiensis species complex clearly separated L. tabernensis sp. nov. from L. iliturgiensis and L. indalus, and X. subbaetense sp. nov. from X. hispanum and X. adenohystherum. D2-D3, partial 18S, and partial coxI regions were used for inferring their phylogenetic relationships with other species in each genus. The present study provides new insights into the diversity of Longidorus and Xiphinema species detected in southern Spain, and new evidence of cryptic speciation in both genera. These results support our hypothesis that the biodiversity of Longidoridae in southern Europe is higher than previously supposed and is still not fully clarified.     

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 phylogenetic species delimitation in the aquatic genus Ottelia (Hydrocharitaceae) reveals cryptic diversity within a widespread species

Journal of Plant Research - Ottelia, a pantropical genus of aquatic plants belonging to the family Hydrocharitaceae, includes several narrowly distributed taxa in Asia. Although the Asian species...     

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.     

Hidden Mediterranean biodiversity: molecular evidence for a cryptic species complex within the reef building vermetid gastropod Dendropoma petraeum (Mollusca: Caenogastropoda)

The reef building vermetid gastropod Dendropoma petraeum inhabits the warmest waters of the Mediterranean Sea and is considered a threatened marine species. The aim of this study was to characterize its genetic structure throughout its whole distribution range using mitochondrial and nuclear sequence data. Because of its sessile adult lifestyle and lack of a pelagic larval stage, we expected a markedly subdivided population structure with limited levels of gene flow. Fragments of the mitochondrial genes cytochrome c oxidase subunit I (COI) and 16S rRNA (16S), were sequenced, along with the nuclear ribosomal cluster (internal transcribed spacer; ITS) in specimens from 18 localities. Our analyses identified four highly distinct phylogroups separated by a mean divergence of > 14% according to the COI sequence data or > 9% according to 16S, but differing only slightly in morphology. The nuclear data (ITS) indicated a lower substitution rate (divergence among groups of around 1%). These large genetic distances among the four lineages clearly point to the existence of a cryptic species complex within D. petraeum comprising at least four species. Differences in the characteristics of intracapsular larval development and protoconch were also detected among these lineages. The allopatric distribution of these cryptic species supports a predominantly vicariant-based cladogenetic pattern for the genus Dendropoma in the Mediterranean.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 96 , 898–912.     

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 evidence for co-occurring cryptic lineages within the Sepioteuthis cf. lessoniana species complex in the Indian and Indo-West Pacific Oceans

The big-fin reef squid, Sepioteuthis cf. lessoniana (Lesson 1930), is an important commodity species within artisanal and near-shore fisheries in the Indian and Indo-Pacific regions. While there has been some genetic and physical evidence that supports the existence of a species complex within S. cf. lessoniana, these studies have been extremely limited in scope geographically. To clarify the extent of cryptic diversity within S. cf. lessoniana, this study examines phylogenetic relationships using mitochondrial genes (cytochrome oxidase c, 16s ribosomal RNA) and nuclear genes (rhodopsin, octopine dehydrogenase) from nearly 400 individuals sampled from throughout the Indian, Indo-Pacific, and Pacific Ocean portions of the range of this species. Phylogenetic analyses using maximum likelihood methods and Bayesian inference identified three distinct lineages with no clear geographic delineations or morphological discriminations. Phylogeographic structure analysis showed high levels of genetic connectivity in the most widespread lineage, lineage C and low levels of connectivity in lineage B. This study provides significant phylogenetic evidence for cryptic lineages within this complex and confirms that cryptic lineages of S. cf. lessoniana occur in sympatry at both small and large spatial scales. Furthermore, it suggests that two closely related co-occurring cryptic lineages have pronounced differences in population structure, implying that underlying differences in ecology and/or life history may facilitate co-occurrence. Further studies are needed to assess the range and extent of cryptic speciation throughout the distribution of this complex. This information is extremely useful as a starting point for future studies exploring the evolution of diversity within Sepioteuthis and can be used to guide fisheries management efforts.     

Integrating morphology and mitochondrial DNA for species delimitation within the spruce budworm (Choristoneura fumiferana) cryptic species complex (Lepidoptera: Tortricidae)

Species in cryptic complexes tend to be very difficult, if not impossible, to identify using morphological characters. One such complex is the spruce budworm (Choristoneura fumiferana Clemens, 1865) species group, an economically important group of Nearctic forest pests. Morphological, ecological, behavioural and genetic characters have been studied to try to understand the taxonomy of this group better, but diagnostic character states differ in frequency rather than being complete replacements between each species. We used mitochondrial DNA (mtDNA), together with a new morphology‐based character system that focuses on forewing colour components, to determine if one or a combination of character sources can be used for species diagnoses within the spruce budworm complex. We characterized 47 forewing morphometric measurements and sequenced a 470 bp region of cytochrome c oxidase I mtDNA for 111 ingroup individuals comprising five taxa within the complex. Larval host association and coloration or adult pheromone attraction were used as the prior method for grouping individuals. Our results showed that linear discriminant analysis of morphometric wing characters gave unique clusters for all species on the first and second canonical axes, except for a partial overlap between C. fumiferana and C. biennis, which are not sympatric in nature. In contrast, mtDNA distinguished C. fumiferana, C. pinus pinus Freeman, 1953 and a group of western species, but the three western species (C. occidentalis Freeman, 1967 , C. biennis Freeman, 1967 and C. lambertiana Busck, 1915) shared mtDNA haplotypes. On the basis of the linear discriminant analysis of the combined character set, this study supports the application of both morphology and mtDNA within a framework of integrative taxonomy as the most accurate method for species identification. Furthermore, it demonstrates the utility of quantitative colour analysis, which may be particularly helpful for groups in which colour characters are difficult to divide into discrete units due to intergrading hues.     

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.     

Wide distributions and cryptic diversity within a Microstomum (Platyhelminthes) species complex

Microstomum lineare is a common species of fresh and brackish waters found worldwide. Three genes (18S, CO1 and ITS) were sequenced from specimens of M. lineare collected from four countries, and the levels of cryptic diversity and genetic structuring were assessed. Results showed M. lineare has very wide haplotype distributions suggesting higher than expected dispersal capabilities. In addition, three new species were described on the basis of molecular taxonomy: Microstomum artoisi sp. nov., Microstomum tchaikovskyi sp. nov. and Microstomum zicklerorum sp. nov.     

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.     

Molecular and morphological evidence for multiple species within Paranoplocephala omphalodes (Cestoda, Anoplocephalidae) in Microtus voles (Arvicolinae)

Haukisalmi, V., Wickström, L. M., Henttonen, H., Hantula, J. & Gubányi, A. (2004). Molecular and morphological evidence for multiple species within Paranoplocephala omphalodes (Cestoda, Anoplocephalidae) in Microtus voles (Arvicolinae). —Zoologica Scripta, 33, 277–290. The present study was designed to test the hypothesis that the anoplocephalid cestode Paranoplocephala omphalodes (Hermann, 1783), a Holarctic parasite of Microtus voles, is a complex of host‐specific species, rather than a single host‐generalist species, using uni‐ and multivariate morphometrics and DNA sequence data from the mitochondrial cytochrome oxidase I gene. The phylogenetic methods applied to the mtDNA sequence data showed consistently that the cestodes morphologically recognizable as P. omphalodes include four well‐supported monophyletic groups, representing at least three distinct, largely host‐specific species. Multivariate morphometrics (discriminant analysis) successfully distinguished the four main mtDNA clades of P. omphalodes‐like cestodes. The true P. omphalodes is shown to be a parasite of Microtus arvalis, M. agrestis and Clethrionomys glareolus in Europe. Microtus oeconomus harbours two host‐specific, allopatric and possibly conspecific clades, one with a Holarctic and another with an (eastern) Beringian (Alaskan) distribution. The eastern Beringian endemic M. miurus is also parasitized with a host‐specific, morphologically divergent species of Paranoplocephala. The cestode clades recognized in M. oeconomus and M. miurus represent 2–3 undescribed species. Molecular phylogenetic analyses supported the monophyly of the ‘northern clade’ of Paranoplocephala spp., an assemblage including P. kalelai from Clethrionomys spp., P. macrocephala from Microtus spp. and all clades of P. omphalodes‐like cestodes except those representing the true P. omphalodes from Europe. The intra‐ and interspecific phylogeny within the northern clade is compared tentatively with the known evolutionary history of the hosts.     

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.