Morphometric Evidence for Three Juvenile Stages in Some Species of Xiphinema americanum sensu lato

One to two hundred nematodes from each of seven Xiphinema americanum-group populations were measured to determine the range of stylet and body lengths for juveniles and adults. First-stage juveniles were identified by the position of the replacement odontostyle (i.e., the tip of the replacement odontostyle overlapped the base of the odontophore). Nematodes were identified as second stage if the functional odontostyle was the same length as the replacement odontostyle of the first stage. Subsequent stages were similarly identified by establishing the range of corresponding replacement and functional odontostyle lengths. In all populations examined, this procedure created natural divisions that clearly grouped nematodes by stylet and body length. Presumably these groups identified all juvenile and adult stages. Populations of X. americanum, X. rivesi, and X. californicum from the United States had three juvenile stages, but a population of X. pachtaicum from Bulgaria had four juvenile stages.     

New insight into the identification and molecular phylogeny of dagger nematodes of the genus Xiphinema (Nematoda: Longidoridae) with description of two new species

The genus Xiphinema constitutes a large group of about 260 species of plant‐ectoparasitic nematodes. The group is polyphagous and distributed almost worldwide. Some of the species of this genus damage agricultural crops by direct feeding on root cells as well as by transmitting nepoviruses. Species discrimination in Xiphinema is complicated by phenotypic plasticity leading to potential misidentification. We conducted nematode surveys in cultivated and natural environments in Spain from 2009 to 2012, from which we identified 20 populations of Xiphinema species morphologically close to the virus‐vector nematode species Xiphinema diversicaudatum, three apomictic populations tentatively identified as species from the complex Xiphinema aceri‐pyrenaicum group, and one population morphologically different from all others that is characterized by a female tail elongate to conical and absence of uterine differentiation. We developed comparative multivariate analyses for these related species by using morphological and morphometrical features together with molecular data from nuclear ribosomal DNA genes [D2‐D3 expansion segments of large ribosomal subunit 28S, internal transcribed spacer 1 (ITS1), and partial small ribosomal subunit (18S)]. The results of multivariate, molecular, and phylogenetic analysis confirmed the morphological hypotheses and allowed the delimitation and discrimination of two new species in the genus described herein as Xiphinema baetica sp. nov. and Xiphinema turdetanensis sp. nov. , and ten known species: Xiphinema adenohystherum, Xiphinema belmontense, Xiphinema cohni, Xiphinema coxi europaeum, Xiphinema gersoni, Xiphinema hispidum, Xiphinema italiae, Xiphinema lupini, Xiphinema nuragicum, and Xiphinema turcicum. Multivariate analyses based on quantitative and qualitative characters and phylogenetic relationships of Xiphinema spp. based on the three molecular ribosomal markers resulted in a partial consensus of these species grouping as nematode populations were maintained for the majority of morphospecies groups (e.g. morphospecies groups 5 and 6), but not in some others (e.g. position of Xiphinema granatum), demonstrating the usefulness of these analyses for helping in the diagnosis and identification of Xiphinema spp. The clade topology of phylogenetic trees of D2‐D3 and partial 18S regions in this study were congruent in supporting the polyphyletic status of some characters, such as the female tail shape and the degree of development of the genital system in species with both genital branches equally developed. This is the most complete phylogenetic study for Xiphinema non‐americanum‐group species. Agreement between phylogenetic trees and some morphological characters (uterine spines, pseudo‐Z organ, and tail shape) was tested by reconstruction of their histories on rDNA‐based trees using parsimony and Bayesian approaches. Thus, integrative taxonomy, based on the combination of multivariate, molecular analyses with morphology, constitutes a new insight into the identification of Xiphinema species. © 2013 The Linnean Society of London     

Diversity of Xiphinema americanum-group Species and Hierarchical Cluster Analysis of Morphometrics

Of the 39 species composing the Xiphinema americanum group, 14 were described originally from North America and two others have been reported from this region. Many species are very similar morphologically and can be distinguished only by a difficult comparison of various combinations of some morphometric characters. Study of morphometrics of 49 populations, including the type populations of the 39 species attributed to this group, by principal component analysis and hierarchical cluster analysis placed the populations into five subgroups, proposed here as the X. brevicolle subgroup (seven species), the X. americanum subgroup (17 species), the X. taylori subgroup (two species), the X. pachtaicum subgroup (eight species), and the X. lambertii subgroup (five species).     

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.     

Morphological and Morphometrical Characterization of Meloidogyne incognita from Different Host Plants in Four Districts of Punjab,India

The population of M. incognita, the root knot nematode (RKN) was found infesting five different host plants (okra, banana, sunflower, bottle gourd, and brinjal) out of 24 examined from four districts of Punjab, India (Gurdaspur, Ludhiana, Patiala, and Hoshiarpur). Morphological and morphometrical characterization indicated that in the case of mature female, the characters of body length and width, neck length, ratio ‘a’, anus to tail terminus (ATT), interphasmid distance (IPD), and perineal pattern were recorded as stable characters. These taxonomic characters can be reliable for identification. All characters of second-stage juvenile (J2) such as body length, stylet length, head to median bulb length (H-MB), distance from median bulb to excretory pore (MB-EP), tail length, anal body width (ABW), and ratios C and C’ were highly variable. Analysis of interpopulation morphometric characters of mature female of M. incognita, namely, body length, width, and ratio ‘a’ were moderately variable characters (CV 0.26% to 20%) and stylet length, neck length, length of median bulb (LMB), and width of median bulb (WMB) were highly variable (CV 1.0% to 36.1%). In the perineal pattern, the two characters ATT and IPD were moderately variable (CV 8.8% to 17.6%) and two characters, anus to vulval slit (AVS) and length of vulval slit (LVS), were highly variable (CV 2.1% to 40.5%). In J2, body length, stylet length, H-MB, MB-EP, ABW, tail length, ratios C, and C’ were highly variable characters (CV > 12%).     

Morphological and Molecular Characterization of Longidorus americanum n. sp. (Nematoda: Longidoridae), a Needle Nematode Parasitizing Pine in Georgia

We describe and illustrate a new needle nematode, Longidorus americanum n. sp., associated with patches of severely stunted and chlorotic loblolly pine, (Pinus taeda L.) seedlings in seedbeds at the Flint River Nursery (Byromville, GA). It is characterized by having females with a body length of 5.4-9.0 mm; lip region slightly swollen, anteriorly flattened, giving the anterior end a truncate appearance; long odontostyle (124-165 µm); vulva at 44%-52% of body length; and tail conoid, bluntly rounded to almost hemispherical. Males are rare but present, and in general shorter than females. The new species is morphologically similar to L. biformis, L. paravineacola, L. saginus, and L. tarjani but differs from these species either by the body, odontostyle and total stylet length, or by head and tail shape. Sequence data from the D2-D3 region of the 28S rDNA distinguishes this new species from other Longidorus species. Phylogenetic relationships of Longidorus americanum n. sp. with other longidorids based on analysis of this DNA fragment are presented. Additional information regarding the distribution of this species within the region is required.     

Molecular phylogeny,diagnostics, and diversity of plant‐parasitic nematodes of the genus Hemicycliophora (Nematoda: Hemicycliophoridae)

The genus Hemicycliophora (Nematoda: Hemicycliophoridae) contains 132 valid species of plant‐parasitic nematodes, collectively known as ‘sheath nematodes’. Hemicycliophora spp. are characterized morphologically by a long stylet with rounded basal knobs and a cuticular sheath, present in juvenile and adult stages. Populations of 20 valid and 14 putative species of Hemicycliophora and Loofia from several countries were characterized morphologically using light (LM) and scanning electron microscopy (SEM) and molecularly using the D2‐D3 segments of 28S rRNA and internal transcribed spacer (ITS) rRNA gene sequences. LM and SEM observations provided new details on the morphology of these species. PCR‐restriction fragment length polymorphisms (PCR‐RFLPs) of the D2‐D3 of 28S rDNA were proposed for identification of the species. Phylogenetic relationships within populations of 36 species of the genus Hemicycliophora using 102 D2‐D3 of 28S rDNA and 97 ITS rRNA gene sequences as inferred from Bayesian analysis are reconstructed and discussed. Ancestral state reconstructions of diagnostic characters (body and stylet length, number of body annuli, shape of vulval lip and tail), using maximum parsimony and Bayesian inference, revealed that none of the traits are individually reliable characters for classifying the studied sheath nematode. The Shimodaira–Hasegawa test rejected the validity of the genus Loofia. This is the most complete phylogenetic analysis of Hemicycliophora species conducted so far. © 2014 The Linnean Society of London     

Inter- and Intraspecific Variation in Wild-type and Single Female-derived Populations of Xiphinema americanum-group Nematodes

Ten populations of Xiphinema americanum-group nematodes were reared from individual females to evaluate inter- and intraspecific variation under identical host and environmental conditions. Data indicated that morphometric variability of X. americanum was the result of genetic variation rather than phenotypic plasticity and that genetic heterogeneity was greater than previously thought. Morphometrics of single female derived (SFD) populations identified different genotypes present in the field populations. Stylet length was the least variable morphometric character of SFD populations, but collectively stylet measurements of all individuals formed an uninterrupted continuum ranging from 107-148 μm. Range and frequency of stylet measurements of field populations could be accounted for by the relative proportion of different genotypes in the population. Nine SFD populations were identified as X. americanum sensu stricto, and one SFD population was similar to X. californicum.     

Species diversity of the genus Osmundea (Ceramiales,Rhodophyta) in the Macaronesian region

Species diversity within the genus Osmundea in the Macaronesian region was explored by conducting a comprehensive sampling in the Azores, the Canary, and the Madeira archipelagos. Toward identification, all specimens were first observed alive to verify the absence of corps en cerise, a diagnostic character for the genus and morphometric data were measured (thallus length and width, first‐order branches length and width, branchlets length and width, cortical cell length and width in surface view, cortical cell length and width in transverse section). Specimens were sequenced for COI‐5P (39 specimens) and three species delimitation methods (Generalized Mixed Yule Coalescent, Automatic Barcode Gap Discovery method, and Poisson Tree Processes) were used to assess the threshold between infra‐ and interspecific relationships. Subsequently, one or several sequences of plastid‐encoded large subunit of RuBisCO (21 specimens) per delimited species were generated to assess the phylogenetic relationships among Macaronesian Osmundea. Moreover, for each delineated species, vegetative and reproductive anatomy was thoroughly documented and, when possible, specimens were either assigned to existing taxa or described as novel species. This integrative approach has provided data for (i) the presence of O. oederi, O. pinnatifida, and O. truncata in Macaronesia; (ii) the proposal of two novel species, O. prudhommevanreinei sp. nov. and O. silvae sp. nov.; and (iii) evidence of an additional species referred as “Osmundea sp.1,” which is a sister taxon of O. hybrida.     

Morphological variability of potato cyst nematodes (Globodera spp.) in the Canary Islands

Thirty populations of potato cyst nematode (Globodera spp.) from the Island of Tenerife and two populations from the UK were assessed for several morphometric and non-morphometric characters thought to discriminate between the species G. rostochiensis and G. pallida. Also 200 cysts from each population were analysed by isoelectric focusing of soluble proteins. Correlation analysis, analysis of variance and principal component analysis were used to investigate relationships between the morphometric characters, how the relationships varied between species and between populations, and which characters were most useful for discriminating between species. The two species differed significantly for each of the four morphometric characters: stylet length, fenestra length, anus-fenestra distance and the number of ridges. The stylet length and fenestra length also showed differences between populations of G. rostochiensis while stylet length and number of ridges showed differences between populations of C. pallida. In general, populations of G. pallida showed greater variation than populations of C. rostochiensis. Principal component analysis of the population means indicated that over 73% of the variation in the characters could be explained by the contrast of stylet and fenestra lengths against the anus-fenestra distance and number of ridges. A plot of the first two principal components separated the two species. Stepwise discriminant analysis provided a linear combination of these four variables which discriminated between the species. Stylet length was found to be the most useful characteristic for distinguishing the species whilst anus-fenestra distance was the least useful.     

Phylogenetic relationships within the superfamily Desmodoroidea (Nematoda: Desmodorida), with descriptions of two new and one known species

Three nematode species of the superfamily Desmodoroidea Filipjev, 1922, were isolated from beach sediments in Wellington, New Zealand, for morphological and molecular analyses. Two of these species, D esmodorella verscheldei sp. nov. and D racograllus ngakei sp. nov. , were new to science and are described herein. Epsilonema rugatum Lorenzen, 1973, comb. nov. , which was originally described from New Zealand material as a subspecies of Epsilonema dentatum from Chile, is redescribed and elevated to the rank of species based on cuticular ornamentation. The phylogenetic relationships amongst the three Desmodoroidea families are investigated based on new and existing sequences of the D2 and D3 expansions segments of large subunit (LSU) 28S rRNA gene and small subunit (SSU) of 18S rDNA gene. Our analyses suggest that the Draconematidae is a sister taxon to the Desmodorinae and Spiriniinae, with the Draconematidae forming a monophyletic crown group and the Desmodorinae and Spiriniinae forming a paraphyletic stem group. Phylogenetic relationships between the Epsilonematidae and Stilbonematinae, however, could not be determined with certainty. The SSU and D2‐D3 of LSU consensus trees indicate that the morphological resemblance between the Draconematidae and Epsilonematidae, which are both characterized by swollen pharyngeal body regions and mid‐posterior body regions with specialized setae, reflects distinct and independently evolved adaptations to their unusual mode of locomotion, with differences in the structure and distribution of specialized setae between the two families also consistent with convergent evolution. We show that the family Desmodoridae and superfamily Desmodoroidea as currently defined are not monophyletic. It was not possible to determine whether the Prodesmodorinae are more closely related to the Desmodoroidea or Microlaimoidea, although it is clear that they do not belong to the Desmodoridae. The single Molgolaiminae sequence available formed a distinct clade together with the superfamily Microlaimoidea, and should therefore be placed with the latter. Clarifying the phylogenetic relationships within the Desmodoroidea will require greater focus on the Pseudonchinae, Molgolaiminae, and Epsilonematidae, for which no or very few sequences are available at present. © 2016 The Linnean Society of London     

Distribution of Xiphinema americanum and Related Species in North America

All species of the Xiphinema americanum-group and their synonyms are listed. The North American species reported are listed by state or province. Among these species, X. rivesi has the most widely reported distribution. Six species (X. diffusum, X. floridae, X. laevistriatum, X. luci, X. shell, and X. tarjanense) have been reported from only Florida. The reports of X. pachtaicum, X. sheri, and X. luci did not include morphometrics and need to be confirmed; X. brevicolle from California was identified before Lamberti and Bleve-Zacheo described 15 new species in 1979 and similarly needs to be confirmed. Because of the proliferation of species in this group, reports of X. americanum (sensu stricto) before 1979 are questionable. Extraction techniques for longidorids are discussed.     

Revision of the plant bug genus Xenocylapus Bergroth (Hemiptera: Heteroptera: Miridae: Cylapinae), with a description of Henryfulvius gracilis – a new cylapine genus and species from Ecuador

The genus Xenocylapus Bergroth, 1922 is revised. Nine new species, Xenocylapus aculeatus sp. nov., X. aquilonius sp. nov., X. bicolor sp. nov., X. globulus sp. nov., X. heissi sp. nov., X. panamaensis sp. nov., X. serrulatus sp. nov., X. tenuis sp. nov., and X. waorani sp. nov., are described from Brazil, Honduras, Mexico, and Panama. New genus and species Henryfulvius gracilis gen. nov., sp. nov. is described from Ecuador. Illustrations of the male genitalia, color photographs of dorsal and lateral views of the adult of most species, scanning electron micrographs of selected structures of X. tenuis, and keys to species of the genus Xenocylapus are provided.     

A taxonomic review of the catfish identified as Glyptothorax zanaensis (Teleostei: Siluriformes: Sisoridae), with the descriptions of two new species

This study re‐examined the taxonomic status of the sisorid catfishes usually identified as Glyptothorax zanaensis using a combination of morphometric and molecular data. Our results resurrect Glyptothorax longinema from the synonymy of G. zanaensis, and we describe two previously unnamed species as Glyptothorax granosus sp. nov. and Glyptothorax fucatus sp. nov. All four species are diagnosed and described in detail. Truss‐based morphometrics combined with principal component analysis (PCA) detected three principal components (PCs) that can explain 86% of the total variation amongst species, which mainly reflect the characteristics of body depth, related depth, adhesive apparatus length, pectoral‐fin length, caudal peduncle length, and barbel lengths. We also generated a phylogenetic hypothesis of these species using concatenated mitochondrial cytochrome b and d ‐loop gene sequences. Molecular dating analysis revealed a rapid speciation of Glyptothorax in the south‐eastern corner of the Qinghai‐Xizang Plateau from the middle Pliocene to early Pleistocene. A key to identify the Glyptothorax species from the Salween River drainage is also provided. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 165 , 363–389.     

A new viviparous acoel Childia vivipara sp. nov. with observations on the developing embryos, sperm ultrastructure, body wall and stylet musculatures

A free‐living viviparous acoel, Childia vivipara sp. nov., from the Gullmar fjord of the Swedish coast is described. The new species is assigned to the taxon Childia based on histological, ultrastructural and molecular sequence similarities. All available molecular markers (18S rRNA, 28S rRNA and histone H3) and several morphological characters, obtained using transmission electron microscopy and confocal scanning laser microscopy of whole mount specimen stained with TRITC‐labelled phalloidin, support the placement of C. vivipara in the taxon Childia. Childia vivipara and other Childia species share the following morphological synapomorphies: well‐developed copulatory organs built of tightly packed stylet needles, proximal part of the stylet inserted into the seminal vesicle, reversed body‐wall musculature, absence of ventral diagonal muscles, presence of dorsal diagonal muscles, and presence of ventral straight longitudinal muscles between frontal pore and mouth, 9 + 1 sperm axoneme structure, six distal sperm cytoplasmic microtubules, and extensive overlap of axonemes and nucleus. The new species can be easily distinguished from other Childia species by its viviparous mode of reproduction and single curved stylet. Observations on late embryonic development based on the oldest developing embryos are discussed.     

Systematic revision of the formerly monotypic genus Tanganikallabes (Siluriformes: Clariidae)

The monotypic genus Tanganikallabes, endemic to Lake Tanganyika, is a poorly known member of the family Clariidae. Examination of 142 specimens housed in museum collections has revealed the presence of at least two additional species in this genus. Tanganikallabes alboperca sp. nov. is distinguished from all congeners by the length of its pelvic fins, the presence of a depigmented vertical bar on the opercular margin, and a combination of additional morphometric (pectoral spine length, preanal length, body depth at anus) and meristic (dorsal and anal fin ray counts) characters. Tanganikallabes stewarti sp. nov. is distinguished from other Tanganikallabes species by having a relatively shorter, incomplete lateral line, and shallow body depth at the anus, as well as shorter prepelvic and preanal lengths, and a longer anal fin with a higher number of fin rays. Several morphological characters, as well as genetic data from cytochrome b (mitochondrial DNA) and 18S–ITS1–5.8S–ITS2–28S (ribosomal DNA), indicate that Tanganikallabes constitutes a monophyletic group within the Clariidae and support the recognition of additional species diversity. The monophyly of Tanganikallabes, coupled with the geographical isolation of this group to a single lake satisfy the requirements for its classification as a true species flock, the latest to be described from Lake Tanganyika. © 2012 The Linnean Society of London, Zoological Journal of the Linnean Society, 2012, 165 , 121–142.     

Molecular phylogeny of the nematode genus Longidorus (Nematoda: Longidoridae) with description of three new species

The phylum Nematoda includes the genus Longidorus, a remarkable group of invertebrates that are polyphagous root‐ectoparasites of many plants including various agricultural crops and trees. Damage is caused by direct feeding on root cells as well as by transmitting nepoviruses. Species discrimination in Longidorus is complicated by phenotypic plasticity (intraspecific variability and minor interspecific differences) leading to potential misidentification. We conducted nematode surveys in cultivated and natural environments in southern Spain that detected 11 species of Longidorus. We developed a comparative study amongst these related species by considering morphological and morphometric features together with molecular data from nuclear ribosomal RNA genes [D2‐D3 expansion segments of large ribosomal subunit (28S), internal transcribed spacer 1 (ITS1), and partial small ribosomal subunit (18S)]. The results of our molecular and phylogenetic analyses confirmed the morphological hypotheses and allowed the delimitation and discrimination of three new species of the genus, described herein as Longidorus baeticus sp. nov. , Longidorus oleae sp. nov. , and Longidorus andalusicus sp. nov. , and eight known species (Longidorus alvegus, Longidorus crataegi, Longidorus fasciatus, Longidorus intermedius, Longidorus iuglandis, Longidorus magnus, Longidorus rubi, and Longidorus vineacola). Phylogenetic analyses of Longidorus spp. based on the three molecular markers resulted in a general consensus of these species grouping, as lineages were maintained for the majority of species (i.e. species with a conoid‐rounded lip region, amphidial fovea asymmetrically bilobed, female tail bluntly rounded), but not in some others (i.e. positions of L. crataegi, L. intermedius, and L. rubi were quite variable). To date, this is the most complete phylogenetic analysis for Longidorus and Paralongidorus species, with the highest number of species included. No correspondence between phylogenetic trees and morphological characters was found for ribosomal markers, with the exception of amphidial shape. Thus, polyphasic identification, based on integration of molecular analysis with morphology, is a tool beyond doubt in Longidorus identification. © 2013 The Linnean Society of London     

Integrative taxonomy of the stunt nematodes of the genera Bitylenchus and Tylenchorhynchus (Nematoda,Telotylenchidae) with description of two new species and a molecular phylogeny

Stunt nematodes are characterized by phenotypic plasticity, with overlapping morphology and morphometry leading to potential misidentification. Consequently, the application of integrative taxonomic approaches is useful to species delimitation based on a combination of different perspectives, e.g. morphology and DNA sequences. We conducted nematode surveys in cultivated and natural environments in Spain and the USA, from which we identified 18 known species of the family Telotylenchidae and two new taxa within the studied samples. These species were morphologically, morphometrically, and molecularly characterized. The results of light and scanning electron microscopic observations, and molecular and phylogenetic analysis also allowed two new species to be distinguished, described herein as B itylenchus hispaniensis sp. nov. and T ylenchorhynchus mediterraneus sp. nov. The phylogenetic analysis was carried out using molecular data from nuclear ribosomal DNA genes [D2–D3 expansion segments of the large ribosomal subunit (28S), internal transcribed spacer (ITS), and partial small ribosomal subunit (18S)]. We also provide here a test of alternative hypotheses that confirms the monophyly of both Tylenchorhynchus and Bitylenchus sensu Siddiqi's classification but does not support Fortuner & Luc's conceptual view of Tylenchorhynchus as a large genus. Ancestral state reconstructions of several diagnostic morphological characters using a maximum parsimony approach showed congruence in morphological and molecular evolution for stylet knob inclination and tail tip annulation. Our analysis emphasizes some of the problems related to the taxonomy and phylogeny of nematodes of Telotylenchinae. © 2014 The Linnean Society of London