Molecular taxonomy of the suborder Bodonina (Order Kinetoplastida), including the important fish parasite,Ichthyobodo necator

Ichthyobodo necator is an important fish ectoparasite with a broad host and ecological range. A novel method, involving the use of an anesthetic, allowed the collection of large numbers of parasites from the skin and gills of hybrid striped bass (Morone saxatilis male x M. chrysops female). Genomic DNA from these samples was used to amplify and clone the 18S rRNA gene. The 18S rRNA gene was similarly cloned from Bodo caudatus, Bodo edax, Bodo saltans, an unidentified Bodo species, and Dimastigella trypaniformis. The resulting sequences were aligned with other representative kinetoplastid species using pileup and similarities in secondary structure. Phylogenetic relationships within the suborder Bodonina and representatives of the suborder Trypanosomatina were determined using maximum-likelihood statistics. The phylogenetic analyses strongly supported the order Kinetoplastida as a monophyletic assemblage consisting of at least two major lineages. One lineage consisted exclusively of L. necator, indicating that it may represent a new suborder. The second lineage consisted of all other kinetoplastid species. This second lineage appeared to contain at least 8 bodonine sublineages, none of which correlated with currently recognized families. For three sublineages, there was a close correspondence between the 18S phylogeny and the classical taxonomy of Dimastigella, Rhynchobodo, and Rhynchomonas. In contrast, Bodo and Cryptobia were polyphyletic, containing species in two or more sublineages that may represent separate genera.     

The survivability of the ectoparasitic flagellate Ichthyobodo necator on chum salmon fry (Oncorhynchus keta) in seawater and comparison to Ichthyobodo sp. on Japanese flounder (Paralichthys olivaceus)

Experimental studies revealed that a freshwater ectoparasitic flagellate Ichthyobodo necator (Henneguy, 1883) could survive and reproduce in seawater after infected chum salmon fry, Oncorhynchus keta (Walbaum), were transferred directly from fresh water to 33% seawater. Minor morphological changes (slight reduction in body width, loss of twistlike wrinkles on body surface, and reduction in contractile vacuoles) were observed in the attached form of I. necator following transfer to seawater. The field survey also confirmed that I. necator occurs on chum salmon fry in seawater estuaries (salinity 17-34%) and in freshwater habitats. It was assumed that I. necator acquired salinity tolerance as a result of adapting to the migratory behavior of its anadromous host. Two morphologically similar bodonids, I. necator from chum salmon and Ichthyobodo sp. from marine Japanese flounder, Paralichthys olivaceus (Temminck and Schlegel), were differentiated by cross-infection experiments. Thus, the parasite from marine flounder should be regarded as a separate species from I. necator.     

Molecular tools for the detection and identification of Ichthyobodo spp. (Kinetoplastida), important fish parasites

Ichthyobodo spp. are ectoparasitic flagellates of fish that may cause disease (ichthyobodosis), a common problem affecting the aquaculture industry worldwide. Ichthyobodosis in farmed fish is often associated with a range of other infectious agents and diagnosis in for example gill disease may be difficult. Sensitive and effective methods for detection and identification of Ichthyobodo spp. are needed to aid diagnosis of ichthyobodosis and epizootiological studies on Ichthyobodo spp. We have designed a specific quantitative real-time PCR assay targeting SSU rDNA for the detection of Ichthyobodo spp. infections. Also, several novel primer sets are presented for use in identification of Ichthyobodo spp. through PCR and sequencing. These PCR methods have been optimized and tested on samples from wild caught and farmed fish from different geographical areas in Norway. The real-time PCR assay has been tested for sensitivity and efficiency, and we present data demonstrating its use for absolute quantification of Ichthyobodo salmonis in tissue samples through RT-qPCR and qPCR. We demonstrate the use of the described set of molecular tools for the detection and sequencing of Ichthyobodo spp. from farmed and wild fish, and also show that they may aid the discovery of new Ichthyobodo species. The detection of light Ichthyobodo spp. infections through microscopy is time consuming and less sensitive compared to PCR methods. Initial real-time PCR testing and subsequent sequencing of positive samples is a powerful method that will increase diagnostic precision, aid carrier detection and promote species discoveries in the Ichthyobodonidae. Our preliminary observations indicate a high Ichthyobodo spp. diversity.     

Genetic distance between two sibling species of the Aedes mariae complex (Diptera, Culicidae)

Two sibling species of the mariae complex of the Aedes genus have been studied: Ae. mariae and Ae. zammitii. Thee mosquitoes are allopatric and show similar adaptations to rocky mediterranean coasts. The isolating mechanisms between the two species are well studied. A partial sterility of hybrid F1, limited to males, has been found with laboratory cross experiments. Strong pre-mating isolating mechanisms were shown in nature by means of release experiments, the frequency of hybrids never exceeding 2% without evidence of introgression. We analyzed genetic variation at 26 enzyme loci in a population of each species: 35% of loci were polymorphic, with an observed mean heterozygosity of 0.07 in Ae. mariae and 0.06 in Ae. zammitii; 6 loci allow discrimination between the two species at a probability of at least .99. Nei's measures of genetic identity and genetic distance are respectively I = 0.6096 and D = 0.2828. The distribution of genetic identities relative to loci is strongly bimodal, reflecting a different contribution of highly variable and fast evolving loci on one side and conservative and slowly evolving loci on the other. We consider, on the base of various experimental data (see Powell, 1975), in the first class ("fast evolving" loci) both variable substrate and regulatory enzymes, in the second ("slow evolving" loci) non regulatory enzymes. Values of genetic distance calculated separately for the "fast" (54% of the loci studied) and "slow" (46%) evolving loci are respectively Df = 0.41 and Ds = 0.03, showing that the main contribution to genetic distance in the first period of divergence is due to fast evolving loci, whose rate of evolution is about ten times more rapid with respect to slow evolving loci.     

萼花臂尾轮虫种复合体内三个姐妹种的形态差异

应用单因子方差分析和判别分析方法,分别比较了采自芜湖市3个自然水体的萼花臂尾轮虫种复合体内3个姐妹种(分别以克隆HE1和HE5、克隆HE3和SE1、克隆LE9为代表)的成体和幼体的形态差异。结果表明,克隆HE3成体的各形态参数均最小,而克隆HE1和LE9成体的各形态参数间无显著的差异;克隆HE1和HE3幼体的前中棘刺间距离和后侧棘刺长度、克隆LE9和HE1幼体的前侧棘刺间距离以及前中棘刺和侧棘刺间距离均无显著性差异,轮虫幼体的其它形态参数均以克隆LE9最长,克隆HE1次之,克隆HE3最短。对克隆HE1、HE3和LE9幼体有明显差异的形态参数所作的分布图显示,它们的分布区有很高程度的重叠。以判别分析建立的判别函数对3姐妹种成体的综合判别率中形态分析Ⅰ为47.8%,形态分析Ⅱ为55.6%;对3姐妹种幼体的综合判别率中形态分析Ⅰ和Ⅱ均为71.1%;两种形态分析对克隆HE1判别准确率均较低,分别为0-76.7%和0-74.2%;依据形态特征无法准确甄别萼花臂尾轮虫姐妹种。     

褶皱臂尾轮虫同胞物种复合类群分类的研究进展

褶皱臂尾轮虫包含复杂的同胞物种,对其分类研究经过了一个长期发展的过程。综述了轮虫分类手段的发展进程,介绍和分析了轮虫特别是褶皱臂尾轮虫同胞物种复合类群分类的研究进展。褶皱臂尾轮虫的L-型和S-型属于B.plicatilis和B.rotundiformis两个物种。这个复合类群中还包括更多的种类,如B.plicatilis sensu stricto、B.ibericus和B.rotundiformis,其中B.plicatilis sensu stricto属于L型,B.ibericus属于SM-型,B.rotundiformis属于SS-型。此类研究丰富了轮虫系统分类学,提高了水域生态调查的准确性。     

Molecular differentiation of sibling species in the Galactomyces geotrichum complex

PCR-analysis, multilocus enzyme electrophoresis and molecular karyotyping were used to characterize 52 strains belonging to the genus Galactomyces. The resultant data revealed that a PCR method employing the universal primer N21 and microsatellite primer (CAC)₅ is appropriate for the distinction of four Ga. geotrichum sibling species, Ga. citri-aurantii and Ga. reessii. Better separation was achieved with the UP primer N21; each species displayed a specific pattern with very low intraspecific variation. We propose to use the primer N21 for the differentiation of the six taxa composing the genus Galactomyces. Multilocus enzyme electrophoresis revealed genetic homogeneity of each sibling species within the Ga. geotrichum complex. On the other hand, the four sibling species, having from 41 to 59% of nDNA homology and similar phenotypic characteristics, are clearly distinguished based on their electrophoretic profiles using two enzymes: mannose-6-phosphate isomerase (MPI) and phosphoglucomutase (PGM). Despite the same number of chromosomal bands, different karyotype patterns were found in Ga. geotrichum sensu stricto and its two sibling species A and B. Within each sibling species, chromosome length polymorphism was observed, in particular for small bands, allowing discrimination to the strain level.     

Electrophoretic karyotype polymorphism of sibling species of the Paramecium aurelia complex

Variability of karyotypes is one of the main mechanisms of speciation in organisms. Electrophoretic karyotypes of the macronucleus (MAC) obtained by pulsed-field gel electrophoresis were compared for 86 strains of all 15 sibling species of the Paramecium aurelia complex in order to determine if karyotype differences corresponded to biological species boundaries. Because the electrophoretic karyotype of the MAC reflects indirectly the frequency and distribution of fragmentation sites in the micronuclear (MIC) chromosomes, any change in MAC electrophoretic karyotype may be a marker of certain chromosomal mutations in the MIC. Thirteen main variants of electrophoretic MAC karyotypes were observed in this species complex. Ten of them appeared to correspond to biological species, while the three other variants characterized several species each. Intraspecific polymorphism was observed for several species: in some cases a certain variant of MAC karyotype was specific for all strains from the same part of the world. Distribution of the MAC karyotype variants along molecular phylogenetic trees of the P. aurelia complex shows that isolation of each species or group of species of this complex was accompanied by divergence in the molecular organization of the genome.     

Characterization of the NADH:ubiquinone oxidoreductase (complex I) in the trypanosomatid Phytomonas serpens (Kinetoplastida)

NADH dehydrogenase activity was characterized in the mitochondrial lysates of Phytomonas serpens, a trypanosomatid flagellate parasitizing plants. Two different high molecular weight NADH dehydrogenases were characterized by native PAGE and detected by direct in-gel activity staining. The association of NADH dehydrogenase activities with two distinct multisubunit complexes was revealed in the second dimension performed under denaturing conditions. One subunit present in both complexes cross-reacted with the antibody against the 39 kDa subunit of bovine complex I. Out of several subunits analyzed by MS, one contained a domain characteristic for the LYR family subunit of the NADH:ubiquinone oxidoreductases. Spectrophotometric measurement of the NADH:ubiquinone 10 and NADH:ferricyanide dehydrogenase activities revealed their different sensitivities to rotenone, piericidin, and diphenyl iodonium.     

Distribution of sibling species of the Anopheles annulipes complex (Diptera: Culicidae) in the Townsville region of Australia

Abstract  As members of the Anopheles annulipes species complex have been implicated as possible malaria vectors in the past, their response to dams and urban development is of medical interest. We collected An. annulipes sensu lato (s.l.) Walker from the Ross River Dam and environs of tropical north Queensland, and scored them for 28 allozyme loci. Allozyme genotype data were analysed by the Unweighted Pair Group Method of Analysis applied to percentage fixed allelic differences (%FD) and by a Bayesian clustering approach. Both approaches revealed four groups, which differed by more than 25%FD, suggesting four species. Specimens sequenced for the Internal transcribed spacer subunit 2 of rDNA suggested that three of these groups were An. annulipes species D, species I and species O. Anopheles annulipes species D occurred in the Ross River and the Ross River Dam, An. annulipes species O inhabited rock pools, and An. annulipes species I inhabited both rock pools and artificial containers in domestic situations. Construction of the Ross River Dam appears to have benefited at least An. annulipes species D, and nearby housing development An. annulipes species I. Although An. annulipes s.l. does not appear to be a major medical risk, mosquito monitoring around Townsville may need to take into account the multispecies status of this taxon.     

Disparity in population differentiation of sex-linked and autosomal variation in sibling species of the Jaera albifrons (Isopoda) complex

The genetic variation at four enzyme loci is described for 22 populations of three Jaera species--J. albifrons, J. ischiosetosa, and J. praehirsuta--in the J. albifrons complex (Crustacea, Isopoda) in Denmark. The variation at three of the loci is similar, with the allele frequency spectra close to each other in all three species. An evolutionary tree based on the variation at these three loci revealed that the populations from the different species are completely intermixed in the tree. This was supported by hierarchical F-statistics where the between-species component was zero. At a fourth locus, Gpi (glucose phosphate isomerase), the species differ substantially. This locus is sex linked in J. ischiosetosa, but in the two other species, J. albifrons and J. praehirsuta, it is either found on autosomes or is sex linked with a high recombination rate between the locus and the centromere. An evolutionary tree for this locus partitions the populations into separate groups and a hierarchical F-statistic has a between-species component of about 50%. The results are attributed to introgression with a higher rate for autosomes than for sex chromosomes.     

Morphological analysis of three equivocal sibling species of Deprea (Solanaceae)

Taxonomic confusion among closely related and morphologically similar Deprea species has persisted in the literature and in the identification of species. Morphological variation among three closely related, monophyletic Deprea species was studied to determine if and how they can be distinguished. Their sympatric occurrence in Venezuela afforded an opportunity to couple field study with analysis of herbarium specimens representing their entire geographic range. An analysis of 94 morphological characters resulted in five vegetative and 13 reproductive taxonomically informative traits. Canonical variates analysis clearly separated the three species using six quantitative traits. We conclude that these taxa, although quite variable and similar morphologically, are taxonomically distinct. Results of character analysis indicated that D. orinocensis is morphologically more similar to D. bitteriana than either are to D. paneroi. In D. paneroi, small, sterile anthers on fruit-bearing plants and the absence of fruits on plants possessing large, plllen-bearing anthers, suggest cryptic dioecy. Based on these data, D. granulosa is considered to be a synonym of D. orinocensis: Athenaea bitteriana, a misapplied synonym, is the correct basionym and is applicable to many specimens identified as D. granulosa. We submit a new combination, D. bitteriana (Werderm.) Sawyer & Benítez, and designate a lectotype to accommodate these findings.     

A demonstration of allopatric sibling species within the Gymnophallidae (Digenea)

Meiogymnophallus minutus and Meiogymnophallus fossarum n. comb. both use the oystercatcher Haematopus ostralegus as their final host and Scrobicularia plana as the primary host. M. minutus uses only the estuarine cockle Cerastoderma edule as its second host, where it occurs enclosed in mantle epithelium in a wedge-shaped cavity beneath the umbo and causes no apparent damage to the host. In contrast, the metacercariae of M. fossarum occur in a variety of microhabitats in a wide variety of lagoon lamellibranchs, including Cerastoderma glaucum. In this host it occurs free in the extrapallial fluid usually beneath the periostracum along the edges of the two valves, where it severely inhibits shell growth, and also beneath the umbo. Comparative morphology, experimental cross infections, second host and microhabitat selection, together with ecological and geographical distribution, suggest that Meiogymnophallus minutus and M. fossarum n. comb. are allopatric sibling species. The former occurs in estuaries in northern and western Europe and the latter from lagoons along the French Mediterranean coast.

---

Resumé Meiogymnophallus minutus et Meiogymnophallus fossarum n. comb. admettent comme hôte définitif l'Hûitriez-pie, Haematopus ostralegus, et comme premier hôte intermédiarie Scrobicularia plana. Les métacercaries de M. minutus parasitent C. edule; elles sont entourées par des proliférations palléales à l'intérieur d'une cavité en forme de coin fichée sous la charnière du Mollusqui; elles n'ingendrent aucun dommage chez leur hôte. Par contre, les métacercaires de M. fossarum si répartisseut dans plusiers types de microhabitats, chez différents espèces de Lamellibranches euryhalins, en particulier chez C. glaucum. Chez ce dernier, les métacercaires vivent librement dans le liquide extrapalléal, souvent logées à l'intérieur du sillon périostracol bordant les lobes palléaux ce qui entraine des perturbations dans la croissance de la cofuille. On trouve aussi des metacercaries sous la charnière mais elles ni y sont jamais enveloppées. La comparaison des caractères morpho-anatomiques de ces deux espécis, les infestations éxperimentales croisées, la sélection des deuxièmes hôtes ainsi que des microhabitats, la répartition géographique et la distribution écologique suggèrent qu'il n'agit de deux espèces jumelles allopatriques. La première se répartit duos les estuaries du nord et de l'ouest de l'Europe, la seconde le long des côtes méditerraneenes francaises.

     

Evolutionary relationships of sibling tapeworm species (Cestoda) parasitizing teleost fishes

DNA/DNA hybridization and sequencing of rDNA (partial 18S rDNA and ITS1) were used to investigate phylogenetic relationships among seven host-specific Bothriocephalus parasites (Cestoda, Pseudophyllidae). The small nucleotide divergence between six of the seven bothriocephalids suggests that isolation and differentiation of Bothriocephalus lineages in the different host species probably occurred recently and over a short time span. Comparison of the molecular phylogeny of the parasite species to the phylogeny of their hosts (teleostean fishes) revealed little congruence between the branching patterns of hosts and parasites, suggesting that bothriocephalids have not cospeciated with their hosts.      

Allozymes characterize sibling species of bipolar Priapulida (Priapulus,Priapulopsis)

Electrophoretic polymorphism of glucosephosphate isomerase (Gpi^*) and phosphoglucomutase (Pgm ^*) polymorphisms were assayed in the bipolardisjunct species pairsPriapulus caudatus/P. tuberculatospinosus andPriapulopsis bicaudatus/Priapulopsis australis (phylum Priapulida). Numbers of genotypes fromGpi ^* alleles and 14Pgm ^* alleles generally did not show deviations from Hardy-Weinberg expectations in composite populations sampled over geographic distances up to 500 km linearly measured. This genetic pattern suggests efficient population cohesion in a phylum where pelagic larvae have not been observed. Sibling species in generaPriapulus orPriapulopsis from northern and southern polar seas did not share identical allozyme alleles.     

萼花臂尾轮虫种复合体内三个姐妹种生活史特征的比较研究

应用生命表统计学方法,在13℃、18℃、23℃和28℃下对采自芜湖市莲塘湖和荷花塘水体中的萼花臂尾轮虫三个姐妹种HE1、HE3和LE9的生活史特征进行了比较研究。结果表明,三个姐妹种生命表参数间的差异因温度的不同而异。13℃下,姐妹种LE9的生命期望、平均寿命和世代时间都显著长于姐妹种HE3,LE9的净生殖率和种群内禀增长率均最大,三个姐妹种所产后代中的混交雌体百分率无显著的差异。18℃下,姐妹种LE9的生命期望和平均寿命显著长于姐妹种HE3,姐妹种LE9的世代时间显著长于姐妹种HE1和HE3,姐妹种 HE3的种群内禀增长率显著高于姐妹种LE9,姐妹种HE3所产后代中的混交雌体百分率最高,三个姐妹种的净生殖率无显著的差异。23℃下,姐妹种HE1的生命期望、平均寿命和世代时间均最长,姐妹种HE1所产后代中的混交雌体百分率显著低于HE3,三个姐妹种的净生殖率和种群内禀增长率均无显著的差异。28℃下,三个姐妹种的生命期望、平均寿命、世代时间和种群内禀增长率均无显著的差异;姐妹种HE1的净生殖率最低;姐妹种HE3所产后代中的混交雌体百分率最高。三个姐妹种的生命表参数对升高的温度的响应也不相同。温度、姐妹种及两者间的交互作用对轮虫的世代时间、平均寿命、出生时的生命期望和轮虫所产后代中的混交雌体百分率均有显著的影响;温度和姐妹种对轮虫的净生殖率和种群内禀增长率均有极显著影响,但两者的交互作用对其无显著性影响。     

The Golden-spectacled Warbler: a complex of sibling species, including a previously undescribed species

The Golden-spectacled Warbler, usually treated as a single species, Seicercus burkii , is widely distributed in mountains of southern Asia. We argue that it should be treated as five different species, one of which is described here for the first time. Two species occur in sympatry in the Himalayas. The other three species are found in eastern Asia, where all three are sympatric in China, and two of these breed sympatrically also in Vietnam. In Burma and adjacent parts of India, one of the Himalayan species is sympatric with one of the east Asian species. We describe differences in morphology, vocalizations, altitudinal distribution and habitat between these five species, as well as results from playback tests, which show that all sympatric taxa differ and appear to be reproductively isolated from each other.     

A sympatric sibling species complex of Petrotilapia Trewavas from Lake Malawi analysed by enzyme electrophoresis (Pisces: Cichlidae)

The protein electromorphs of three taxa of Petrotilapia , referred to in earlier works as sibling species, were examined by starch-gel electrophoresis. The results indicated that, for the three taxa, no alternative fixed alleles occurred for any of the 25 loci (enzymes) examined. However, heterogeneous gene frequencies were indicated at seven polymorphic loci. This suggests that either the taxa are isolated 'sibling' species which recently diverged, or that they are 'incipient' species with minimal gene flow between morphs. The genetic and field data lend support to the hypothesis that sympatric splitting of morphs could be important in the explosive radiation of the cichlidae.