Taxonomic and Molecular Identification of Mesocriconema and Criconemoides Species (Nematoda: Criconematidae)

Populations of Mesocriconema curvatum, M. kirjanovae, M. onoense, M. ornatum, M. sphaerocephala, M. surinamense, M. vadense, M. xenoplax, and Criconemoides informis from different geographical areas in the continental United States were characterized morphologically and molecularly. A new ring nematode from Washington County, Arkansas, is also described and named Mesocriconema ozarkiense n. sp., This new species is characterized by females with small flattened submedian lobes, lower than or at the same level as the labial disc, vagina straight, very well developed spermatheca without sperm, no more than one anastomoses, L=379-512 μm, V=89-93, stylet length = 49-61 μm, R=107-119, annuli with slightly crenate margins on tail portion and a simple anterior vulval lip. The molecular characterization of M. ozarkiense n. sp. using the ITS rRNA gene sequence and the phylogenesis relationship of this new species with the ring nematodes included in this study are provided.     

First report of Xiphinema brevicolle Lordello et Costa, 1961 (Nematoda, Longidoridae) in Japan

Mixed populations of Xiphinema americanum-group species were detected from a root zone soil sample of Japanese holly, Ilex crenata, during a survey for plant-parasitic nematodes of commercial ornamental plant nurseries in Chiba Prefecture, Japan. From the result of the morphological study, the species were identified as Xiphinema brevicolle and Xiphinema sp. This is the first record of Xiphinema brevicolle in Japan. Morphometrics of Xiphinema brevicolle generally agree with those of the type specimens and the topotype specimens. Xiphinema sp. morphometrically resembles Xiphinema paramonovi except for tail length. The mitochondrial COI region, the nuclear 18S rDNA and the nuclear large subunit rDNA D2/D3 region of the species were sequenced and compared in the molecular study. For the COI region, PCR primers were newly designed to obtain longer sequences, ca. 900 bp, than previously used. Sequence identities of COI, 18S and D2/D3 regions between these two populations were 84.0-84.1%, 99.9% and 98.1-98.2%, respectively. Phylogenetic analyses of maximum likelihood trees were carried out to compare genetic relationships among the group and some suggestions were made on the Xiphinema brevicolle-subgroup.     

Ecological and Morphological Differentiation Among COI Haplotype Groups in the Plant Parasitic Nematode Species Mesocriconema Xenoplax

DNA barcoding with the mitochondrial COI gene reveals distinct haplotype subgroups within the monophyletic and parthenogenetic nematode species, Mesocriconema xenoplax. Biological attributes of these haplotype groups (HG) have not been explored. An analysis of M. xenoplax from 40 North American sites representing both native plant communities and agroecosystems was conducted to identify possible subgroup associations with ecological, physiological, or geographic factors. A dataset of 132 M. xenoplax specimens was used to generate sequences of a 712 bp region of the cytochrome oxidase subunit I gene. Maximum-likelihood and Bayesian phylogenies recognized seven COI HG (≥99/0.99 posterior probability/bootstrap value). Species delimitation metrics largely supported the genetic integrity of the HG. Discriminant function analysis of HG morphological traits identified stylet length, total body length, and stylet knob width as the strongest distinguishing features among the seven groups, with stylet length as the strongest single distinguishing morphological feature. Multivariate analysis identified land cover, ecoregion, and maximum temperature as predictors of 53.6% of the total variation (P = 0.001). Within land cover, HG categorized under “herbaceous,” “woody wetlands,” and “deciduous forest” were distinct in DAPC and RDA analyses and were significantly different (analysis of molecular variance P = 0.001). These results provide empirical evidence for molecular, morphological, and ecological differentiation associated with HG within the monophyletic clade that represents the species Mesocriconema xenoplax.     

MOTUs, Morphology, and Biodiversity Estimation: A Case Study Using Nematodes of the Suborder Criconematina and a Conserved 18S DNA Barcode

DNA barcodes are increasingly used to provide an estimate of biodiversity for small, cryptic organisms like nematodes. Nucleotide sequences generated by the barcoding process are often grouped, based on similarity, into molecular operational taxonomic units (MOTUs). In order to get a better understanding of the taxonomic resolution of a 3' 592-bp 18S rDNA barcode, we have analyzed 100 MOTUs generated from 214 specimens in the nematode suborder Criconematina. Previous research has demonstrated that the primer set for this barcode reliably amplifies all nematodes in the Phylum Nematoda. Included among the Criconematina specimens were 25 morphologically described species representing 12 genera. Using the most stringent definition of MOTU membership, where a single nucleotide difference is sufficient for the creation of a new MOTU, it was found that an MOTU can represent a subgroup of a species (e.g. Discocriconemella limitanea), a single species (Bakernema inaequale), or a species complex (MOTU 76). A maximum likelihood phylogenetic analysis of the MOTU dataset generated four major clades that were further analyzed by character-based barcode analysis. Fourteen of the 25 morphologically identified species had at least one putative diagnostic nucleotide identified by this character-based approach. These diagnostic nucleotides could be useful in biodiversity assessments when ambiguous results are encountered in database searches that use a distance-based metric for nucleotide sequence comparisons. Information and images regarding specimens examined during this study are available online.     

Mesocriconema kirjanovae (Nematoda: Criconematidae) from Southeastern Spain

An abundant female population of a criconematid species identified as Mesocriconema kirjanovae (Andr ssy, 1962) Loof &De Grisse, 1989 is reported from a natural habitat in southeastern Spain. Measurements and morpho-anatomy obtained with light microscopy and scanning electron microscopy are included and compared with previous data on this species.     

Discrimination of Six Pratylenchus Species Using PCR and Species-Specific Primers

A PCR-based assay for identification of six species of Pratylenchus common in California is described. In this assay, five forward species-specific primers were designed from the internal variable portion of the D3 expansion region of the 26S rDNA and were each used with a single, common reverse primer. The optimized species-specific primers produced unique amplicons from their respective target and did not amplify DNA from other Pratylenchus species. With this assay we were able to identify single females to species level. This method obviates the need for subsequent RFLP or sequence analysis of the PCR product and can be used as a rapid diagnostic tool in epidemiological and management studies.     

Phylogenetic Analysis of the Hoplolaiminae Inferred from Combined D2 and D3 Expansion Segments of 28S rDNA

DNA sequences of the D2-D3 expansion segments of the 28S gene of ribosomal DNA from 23 taxa of the subfamily Hoplolaiminae were obtained and aligned to infer phylogenetic relationships. The D2 and D3 expansion regions are G-C rich (59.2%), with up to 20.7% genetic divergence between Scutellonema brachyurum and Hoplolaimus concaudajuvencus. Molecular phylogenetic analysis using maximum likelihood and maximum parsimony was conducted using the D2-D3 sequence data. Of 558 characters, 254 characters (45.5%) were variable and 198 characters (35.4%) were parsimony informative. All phylogenetic methods produced a similar topology with two distinct clades: One clade consists of all Hoplolaimus species while the other clade consists of the rest of the studied Hoplolaiminae genera. This result suggests that Hoplolaimus is monophyletic. Another clade consisted of Aorolaimus, Helicotylenchus, Rotylenchus, and Scutellonema species. Phylogenetic analysis using the outgroup species Globodera rostocheinsis suggests that Hoplolaiminae is paraphyletic. In this study, the D2-D3 region had levels of DNA sequence divergence sufficient for phylogenetic analysis and delimiting species of Hoplolaiminae.     

中国拟家鼠分类与分布厘订

拟家鼠(Rattus pyctoris)由Hodgson在1845年命名,其模式产地为尼泊尔。该种的分类地位和是否在中国有分布都存在较大争议。近来有研究认为拟家鼠不分布于我国。2020年,在西藏吉隆县和札达县进行野外调查时采集到7号标本,皮毛、头骨和牙齿的形态特征与尼泊尔的拟家鼠相似,分子系统发育分析支持这些标本为单系(PP=1.0),与模式产地样品C_yt b基因的K2?P遗传距离为0~2.8%,确认这些标本为拟家鼠。这是第一次在我国境内采集到拟家鼠标本,丰富和完善了这个物种的生物地理资料。     

The complete mitochondrial genome sequence of the black-capped capuchin (Cebus apella)

The phylogenetic relationships of primates have been extensively investigated, but key issues remain unresolved. Complete mitochondrial genome (mitogenome) data have many advantages in phylogenetic analyses, but such data are available for only 46 primate species. In this work, we determined the complete mitogenome sequence of the black-capped capuchin (Cebus apella). The genome was 16,538 bp in size and consisted of 13 protein-coding genes, 22 tRNAs, two rRNAs and a control region. The genome organization, nucleotide composition and codon usage did not differ significantly from those of other primates. The control region contained several distinct repeat motifs, including a putative termination-associated sequence (TAS) and several conserved sequence blocks (CSB-F, E, D, C, B and 1). Among the protein-coding genes, the COII gene had lower nonsynonymous and synonymous substitutions rates while the ATP8 and ND4 genes had higher rates. A phylogenetic analysis using Maximum likelihood and Bayesian methods and the complete mitogenome data for platyrrhine species confirmed the basal position of the Callicebinae and the sister relationship between Atelinae and Cebidae, as well as the sister relationship between Aotinae (Aotus) and Cebinae (Cebus/Saimiri) in Cebidae. These conclusions agreed with the most recent molecular phylogenetic investigations on primates. This work provides a framework for the use of complete mitogenome information in phylogenetic analyses of the Platyrrhini and primates in general.     

Assessing Nuclear and Mitochondrial DNA Sequence Variation Within Steinernema (Rhabditida: Steinernematidae)

DNA sequence analysis was used to characterize the nuclear ribosomal DNA ITS1 region and a portion of the COII and 16S rDNA genes of the mitochondrial genome from Steinernema entomopathogenic nematodes. Nuclear ITS1 nucleotide divergence among seven Steinernema spp. ranged from 6 to 22%, and mtDNA divergence among five species ranged from 12 to 20%. No intraspecific variation was observed among three S. feltiae strains. Phylogenetic analysis of both nuclear and mitochondrial DNA sequences confirms the existing morphological relationships of several Steinernema species. Both the rDNA ITS1 and mtDNA sequences were useful for resolving relationships among Steinernema taxa.     

Morphologic and Genetic Identification of Diphyllobothrium nihonkaiense in Korea

Diphyllobothrium nihonkaiense was first described by Yamane in 1986 but the taxonomical features have been obscure due to lack of critical morphologic criteria in its larval and adult stages. In Korea, this tapeworm had long been known as Diphyllobothrium latum. In this study, we observed 62 specimens collected from Korean residents and analyzed them by morphological features and nucleotide sequences of mitochondrial cox1 gene as well as the ITS1 region. Adult tapeworms were examined after carmine or trichrome stain. Longitudinal sections of the gravid proglottids showed an obtuse angle of about 150 degree between the cirrus sac and seminal vesicle. This angle is known as a major differential point compared with that of D. latum. Nucleotide sequence differences between D. latum and the specimens from Koreans represented 17.3% in mitochondrial DNA cox1 gene. Sequence divergence of ITS1 among 4 Korean isolates was 0.3% and similarity was 99.7% with D. nihonkaiense and D. klebanovskii. All of the Korean specimens analyzed in this study were identified as being D. nihonkaiense (n = 62). We propose its Korean name as "Dong-hae-gin-chon-chung" which means ''long tapeworm of the East Sea'' for this newly analyzed diphyllobothriid tapeworm in Korea.     

Molecular Characterization and Phylogenetic Analysis of Hoplolaimus stephanus

Three Hoplolaimus stephanus populations were characterized morphologically, both by morphometrics and by SEM observations. These populations were used to develop a rapid and accurate molecular identification method for the species, which is useful because of the high level of morphological similarity between H. stephanus and H. galeatus. Species-specific primers for H. stephanus, amplifying a distinct fragment (260 bp) of the nuclear ribosomal internal transcribed spacer1 (ITS1), can be used in multiplex PCR along with previously developed primers for other common Hoplolaimus species. We also infer phylogenetic relationships among H. stephanus, the closely-related H. galeatus, and several other Hoplolaimus species, using sequences of the actin gene, ITS1 and LSUD.     

Steinernema diaprepesi n. sp. (Rhabditida: Steinernematidae), a Parasite of the Citrus Root Weevil Diaprepes abbreviatus (L) (Coleoptera: Curculionidae)

A nematode collected from Diaprepes abbreviatus is identified and described as a new species, Steinernema diaprepesi n. sp. The new species is closely related to S. feltiae, S. glaseri, and S. oregonense and can be distinguished from these species by the following characteristics: Males: Spicule averaging 79 (71-90) µm and spicule shape; D% (distance from anterior end to excretory pore/ esophagus length × 100) about 80; the ratio SW (spicule length/anal body width) about 1.8. Females: Vulva with short, double- flapped epiptygma; tail terminus usually with 5 papillae-like structures. Infective juveniles: Body averaging 1,002 (880-1,133) µm, EP (distance from anterior end to excretory pore) = 74 (66-83) µm; tail length = 83 (65-91) µm, and E% (EP/tail length × 100) = 89.6 (78-114). Lateral field pattern variable, the formula for the arrangement of ridges from head to tail is: 2, 6, 7, 8, 4, 2. The portion with eight ridges is the longest. This new species can be differentiated further from three closest species (S. feltiae, S. glaseri, and S. oregonense) by characteristic sequences of their ITS regions, including sequence lengths, ratios of similarity, composition, and differences in base characters in sequence alignment.     

Nucleotide Substitution Patterning within the Meloidogyne rDNA D3 Region and Its Evolutionary Implications

Evolutionary relationships based on nucleotide variation within the D3 26S rDNA region were examined among acollection of seven Meloidogyne hapla isolates and seven isolates of M. arenaria, M. incognita, and M. javanica. Using D3A and D3B primers, a 350-bp region was PCR amplified from genomic DNA and double-stranded nucleotide sequence obtained. Phylogenetic analyses using three independent clustering methods all provided support for a division between the automictic M. hapla and the apomictic M. arenaria, M. incognita, and M. javanica. A nucleotide sequence character distinguishing M. hapla from the three apomictic species was a 3-bp insertion within the interior of the D3 region. The three apomictic species shared a common D3 haplotype, suggesting a recent branching. Single M. hapla individuals contained two different haplotypes, differentiated by a Sau3AI restriction site polymorphism. Isolates of M. javanica appeared to have only one haplotype, while M. incognita and M. arenaria maintained more than one haplotype in an isolate.     

Description of Discocriconemella mineira n. sp. and D. degrissei Males from Brazil

A new species of Discocriconemella and the male of Discocriconemella degrissei are described from Minas Gerais state of Brazil. Discocriconemella mineira n. sp. differs from D. discolabia by having a longer stylet (61-71 vs. 35-47 μm) and fewer body annuli (78-88 vs. 155-174), and from D. inarata by the longer stylet (61-71 vs. 51-61 μm) and shorter body length (253-342 vs. 354-486 μm). It differs from D. persae by having a shorter stylet, fewer body annuli, and a shorter post vulval body portion (Rv = 6-7 vs. 14-20). Discocriconemella mineira can be separated from D. degrissei by the head disc which is round in D. mineira and quadrilobate in D. degrissei. Males of D. degrissei resemble those of D. retroversa.     

Taxonomy of Discocriconemella (Nematoda: Criconematoidea) with a Redescription of D. mauritiensis

The form of the cephalic disc and its taxonomic significance at the species level in the genus Discocriconemella De Grisse &Loof, 1965 is discussed. By light (LM) and scanning electron microscopy (SEM), four groups of disc configurations in females and juveniles are distinguishable. The disc is either round with an uninterrupted margin (group 1), has a deep dorsal and ventral indentation (group 2), is indented medially and laterally giving a four-lobed appearance (group 3), or is round with paired dorsal and ventral projections (group 4). There is no apparent correlation between groups of cephalic discs and other characters such as tail shape or number of body annules. Discocriconemella mauritiensis (Williams, 1960) De Grisse &Loof, 1965 is redescribed from a sugar cane population collected in Mauritius, and the diagnosis of the genus is emended.     

Non-destructive species identification of Drosophila obscura and D. subobscura (Diptera) using near-infrared spectroscopy

The vinegar flies Drosophila subobscura and D. obscura frequently serve as study organisms for evolutionary biology. Their high morphological similarity renders traditional species determination difficult, especially when living specimens for setting up laboratory populations need to be identified. Here we test the usefulness of cuticular chemical profiles collected via the non-invasive method near-infrared spectroscopy for discriminating live individuals of the two species. We find a classification success for wild-caught specimens of 85%. The species specificity of the chemical profiles persists in laboratory offspring (87–92% success). Thus, we conclude that the cuticular chemistry is genetically determined, despite changes in the cuticular fingerprints, which we interpret as due to laboratory adaptation, genetic drift and/or diet changes. However, because of these changes, laboratory-reared specimens should not be used to predict the species-membership of wild-caught individuals, and vice versa. Finally, we demonstrate that by applying an appropriate cut-off value for interpreting the prediction values, the classification success can be immensely improved (to up to 99%), albeit at the cost of excluding a considerable portion of specimens from identification.     

Molecular Variation and Distribution of Anopheles fluviatilis (Diptera: Culicidae) Complex in Iran

Anopheles fluviatilis James (Diptera: Culicidae) is one of the known malaria vectors in south and southeastern Iran. Earlier ITS2 sequences analysis of specimens from Iran demonstrated only a single genotype that was identical to species Y in India, which is also the same as species T. We identified 2 haplotypes in the An. fluviatilis populations of Iran based on differences in nucleotide sequences of D3 domain of the 28S locus of ribosomal DNA (rDNA). Comparison of sequence data from 44 Iranian specimens with those publicly available in the Genbank database showed that all of the 28S-D3 sequences from Kazeroun and Khesht regions in Fars Province were identical to the database entry representing species U in India. In other regions, all the individuals showed heterozygosity at the single nucleotide position, which identifies species U and T. It is argued that the 2 species may co-occur in some regions and hybridize; however, the heterozygosity in the 28S-D3 locus was not reflected in ITS2 sequences and this locus for all individuals was identical to species T. This study shows that in a newly diverged species, like members of An. fluviatilis complex, a single molecular marker may not be sufficiently discriminatory to identify all the taxa over a vast geographical area. In addition, other molecular markers may provide more reliable information for species discrimination.