A Blind-Identification Test on Criconema annuliferum (de Man, 1921) Micoletzky, 1925 Species Complex Corroborate the Hyper-Cryptic Species Diversity Using Integrative Taxonomy.

Ring nematodes are obligate ectoparasites on crops and natural herbaceous and woody plants, and some species are of economic importance and cause damage to roots of several crops. Recent integrative taxonomical analyses recognized the existence of two cryptic species within the Criconema annuliferum morphotype in Spain. In this study, we corroborated that morphometric, morphological and a multi-locus analysis (including the ribosomal markers D2-D3 expansion segments of 28S rRNA, ITS rRNA, 18S RNA, and the mitochondrial DNA cytochrome oxidase I gene) identified a new lineage clearly separated from C. annuliferum, C. paraannuliferum and C. plesioannuliferum. The new lineage was described herein as Criconema pseudoannuliferum sp. nov., confirming that C. annuliferum species complex species complex comprises a hyper-cryptic species complex. This research analysed soil samples from the rhizosphere of maritime pine (Pinus pinaster Ait.) forests in Bermeja-Crestellina Mountain, located at the western part of Málaga province, southern Spain. The integrative taxonomical analyses revealed the occurrence of a new cryptic species identified using females, males and juveniles with detailed morphology, morphometry and molecular markers, described herein as Criconema pseudoannuliferum sp. nov. All molecular markers (D2-D3, ITS, 18S and COI) were obtained from the same individual that was also used for morphological and morphometric analyses. This research demonstrated the hidden diversity within the C. annuliferum species complex species complex can reach to four lineages under ribosomal and mitochondrial gene markers for one morphospecies group, which includes four species, viz. C. annuliferum, C. paraannuliferum, C. plesioannuliferum, and C. pseudoannuliferum sp. nov. Criconema pseudoannuliferum sp. nov. was detected in moderate soil density in two maritime pine forests (5 and 25 nematodes/500 cm3 of soil) suggesting that does not cause damage to maritime pine.

Detailed morphological structure and phylogenetic relationships of Degeeriella punctifer (Phthiraptera: Philopteridae), a parasite of the bearded vulture Gypaetus barbatus (Accipitriformes: Accipitridae).

Habitat loss is one of the main threats to species survival and, in the case of parasites, it is their hosts that provide their habitat. Therefore, extinction even at local scale of host taxa also implies the extinction of their parasites in a process known as co-extinction. This is the case of the bearded vulture (Gypaetus barbatus), which almost became extinct at the beginning of the twentieth century. After several attempts, this species was successfully reintroduced into the Alps at the end of the twentieth century. We collected 25 lice specimens from an electrocuted bearded vulture from Susa (Italian Alps) that were morphologically identified as Degeeriella punctifer. Six individuals were studied by scanning electron microscopy, with particular emphasis on their cephalic sensorial structures, while four further specimens were characterized at molecular level by amplifying partial regions of the 12SrRNA, COX1 and elongation factor 1 alpha (EF-1) genes. From a morphological perspective, the number, type and arrangement of the sensillae on the two distal antennal segments is quite similar to that of other species of the family Philopteridae (Phthiraptera: Ischnocera). The mandibles and tarsal claws allow lice to cling firmly to their host's feathers. Phylogenetic analyses help unravel the paraphyletic nature of the genus Degeeriella and demonstrate the clear differentiation between lice parasitizing Accipitriformes and Falconiformes, as well as the close relationship between D. punctifer, D. fulva, D. nisus and Capraiella sp. that, along with other genera, parasitize rollers (Aves: Coraciiformes).

DNA Barcoding and Morphometry Reveal Further Cryptic Bio-Diversity within the Pin Nematode Genus Paratylenchus (Nematoda: Tylenchulidae).

Paratylenchus species are obligate ectoparasitic nematodes on cultivated and wild herbaceous and woody plants occupying numerous soil categories. Several species may cause damage to several crops (viz. P. dianthus, P. enigmaticus, P. microdorus, P. hamatus and P. epacris on carnation, lettuce, rose and walnut, respectively). This investigation proves and emphasizes the relevance of applying integrative taxonomy for the accurate detection of Paratylenchus species in mountainous wild environments in the Malaga province, Southern Spain. This research analyzed 45 soil samples of maritimus pine and one of green heather in southern Spain and identified fourteen Paratylenchus species, two of them are described herein as new species (P. paraaonli sp. nov., P. plesiostraeleni sp. nov.), six of them were first reports for Spain (P. canchicus, P. nainianus, P. neonanus, P. salubris, Paratylenchus sp. 2 SAS, and P. wuae), and six species (P. caravaquenus, P. microdorus, P. nanus, P. neoamblycephalus, P. sheri, and P. variabilis) have been already reported in Spain. Accordingly, these data increase the biodiversity of pin nematodes in Spain comprising a total of 47 species (33.1% out of 142 total species of this genus). Phylogenetic analyses based on ribosomal and mitochondrial markers (D2-D3, ITS, and partial COI) resulted in a consistent position for the newly described Paratylenchus species in this study (P. plesiostraeleni sp. nov., P. paraaonli sp. nov.). Paratylenchus plesiostraeleni sp. nov. grouped in a separated subclade as unequivocal species from the P. straeleni-complex species (including P. straeleni and P. parastraeleni), and P. paraaonli sp. nov. clustered with P. vitecus, but clearly separate from this species. This study indicates that Paratylenchus species diversity in natural environments may be higher than expected, and this study may help in accurate identifications.

Drivers of the Ectoparasite Community and Co-Infection Patterns in Rural and Urban Burrowing Owls.

Urbanization creates new ecological conditions that can affect biodiversity at all levels, including the diversity and prevalence of parasites of species that may occupy these environments. However, few studies have compared bird-ectoparasite interactions between urban and rural individuals. Here, we analyze the ectoparasite community and co-infection patterns of urban and rural burrowing owls, Athene cunicularia, to assess the influence of host traits (i.e., sex, age, and weight), and environmental factors (i.e., number of conspecifics per nest, habitat type and aridity) on its composition. Ectoparasites of burrowing owls included two lice, one flea, and one mite. The overall prevalence for mites, lice and fleas was 1.75%, 8.76% and 3.50%, respectively. A clear pattern of co-infection was detected between mites and fleas and, to less extent, between mites and lice. Adult owls harbored fewer ectoparasites than nestlings, and adult females harbored more lice than males. Our results also show that mite and flea numbers were higher when more conspecifics cohabited the same burrow, while lice showed the opposite pattern. Rural individuals showed higher flea parasitism and lower mite parasitism than urban birds. Moreover, mite numbers were negatively correlated with aridity and host weight. Although the ectoparasitic load of burrowing owls appears to be influenced by individual age, sex, number of conspecifics per nest, and habitat characteristics, the pattern of co-infection found among ectoparasites could also be mediated by unexplored factors such as host immune response, which deserves further research.

A Proposed New Species Complex within the Cosmopolitan Ring Nematode Criconema annuliferum (de Man, 1921) Micoletzky, 1925.

Ring nematodes are obligate ectoparasites on cultivated and wild herbaceous and woody plants, inhabiting many types of soil, but particularly sandy soils. This study explored the morphometrical and molecular diversity of ring nematodes resembling Criconema annuliferum in 222 soil samples from fruit crops in Spain, including almond, apricot, peach and plum, as well as populations from cultivated and wild olives, and common yew. Ring nematodes of the genus Criconema were detected in 12 samples from under Prunus spp. (5.5%), showing a low to moderate nematode soil densities in several localities from southeastern and northeastern Spain. The soil population densities of Criconema associated with Prunus spp. ranged from 1 nematode/500 cm3 of soil in apricot at Sástago (Zaragoza province) to 7950 and 42,491 nematodes/500 cm3 of soil in peach at Ricla and Calasparra (Murcia province), respectively. The integrative taxonomical analyses reveal the presence of two cryptic species identified using females, males (when available), and juveniles with detailed morphology, morphometry, and molecular markers (D2-D3, ITS, 18S, and COI), described herein as Criconema paraannuliferum sp. nov. and Criconema plesioannuliferum sp. nov. All molecular markers from each species were obtained from the same individuals, and these individuals were also used for morphological and morphometric analyses. Criconema paraannuliferum sp. nov. was found in a high soil density in two peach fields (7950 and 42,491 nematodes/500 cm3 of soil) showing the possibility of being pathogenic in some circumstances.

The morphology of Colpocephalum pectinatum (Phthiraptera: Amblycera: Menoponidae) under scanning electron microscopy.

Here, we describe under scanning electron microscopy (SEM) the morphology of Colpocephalum pectinatum (Phthiraptera, Menoponidae), an ectoparasite found in burrowing owls, Athene cunicularia. We devote particular attention to the morphology of the main structures of the head (antennae and mouth-parts) and legs (tarsi and femoral ctenidia). Moreover, we describe the main peripheral sensory organs, located in the labial palpi and the distal end of antennae. We also detected that the structure of antennae and antennal sensilla arrangement are very similar to that described for other Colpocephalum and Menoponid species, and we discuss the function of each type of sensilla. We suggest that SEM studies combined with other microscopy and physiological techniques could be useful for elucidate the function of each structure, lice behaviour, as well as their taxonomy.

Pratylenchus vovlasi sp. Nov. (Nematoda: Pratylenchidae) on Raspberries in North Italy with a Morphometrical and Molecular Characterization.

Root-lesion nematode species rank third only to root-knot and cyst nematodes as having the greatest economic impact on crops worldwide. A survey of plant-parasitic nematodes associated with decaying raspberries (Rubus sp.) in northern Italy revealed that root-lesion nematodes were the most frequently occurring species among other phytonematodes. Several Pratylenchus species have been associated with Rubus sp. in Canada (Quebec, British Columbia) and USA (North Carolina, Maryland, New Jersey) including P. penetrans and P. crenatus. In the roots and rhizosphere of symptomatic raspberries, nematodes of two Pratylenchus spp. were detected. Detailed morphometrics of the two root-lesion nematode isolates were consistent with Pratylenchus crenatus and with an undescribed Pratylenchus species. The extracted nematodes were observed and measured as live and fixed materials and subsequently identified by integrative taxonomy (morphometrically and molecularly). The latter species is described herein as Pratylenchus vovlasi sp. nov., resulting morphometrically closest to P. mediterraneus and phylogenetically to P. pratensis. The molecular identification of Pratylenchus vovlasi sp. nov. was carried out by sequencing the ITS region, D2-D3 expansion domains of the 28S rRNA gene and a partial region of the nuclear hsp90 gene. ITS-RFLP and sequence analyses revealed that Pratylenchus vovlasi sp. nov. had species-specific restriction profiles with no corresponding sequences present in the database. The phylogenetic relationships with ITS and D2-D3 sequences placed the Pratylenchus vovlasi sp. nov. in a clade with P. pratensis and P. pseudopratensis. This research confirms the occurrence of cryptic biodiversity within the genus Pratylenchus as well as the need for an integrative approach to the identification of Pratylenchus species.

Morphostatic Speciation within the Dagger Nematode Xiphinema hispanum-Complex Species (Nematoda: Longidoridae).

Dagger nematodes of the genus Xiphinema include a remarkable group of invertebrates of the phylum Nematoda comprising ectoparasitic animals of many wild and cultivated plants. Damage is caused by direct feeding on root cells and by vectoring nepoviruses that cause diseases on several crops. Precise identification of Xiphinema species is critical for launching appropriate control measures. We deciphered the cryptic diversity of the Xiphinema hispanum-species complex applying integrative taxonomical approaches that allowed us to verify a paradigmatic example of the morphostatic speciation and the description of a new species, Xiphinema malaka sp. nov. Detailed morphological, morphometrical, multivariate and genetic studies were carried out, and mitochondrial and nuclear haploweb analyses were used for species delimitation of this group. The new species belongs to morphospecies Group 5 from the Xiphinema nonamericanum-group species. D2-D3, ITS1, partial 18S, and partial coxI regions were used for inferring the phylogenetic relationships of X. malaka sp. nov. with other species within the genus Xiphinema. Molecular analyses showed a clear species differentiation not paralleled in morphology and morphometry, reflecting a clear morphostatic speciation. These results support the hypothesis that the biodiversity of dagger nematodes in southern Europe is greater than previously assumed.

Description of Geocenamus vietnamensis sp. n. (Nematoda: Merliniidae) from Vietnam.

A new species of the genus Geocenamus was isolated from soil and root samples from the rhizosphere of Casuarina equisetifolia (Casuarinaceae) in Quang Nam province, Vietnam. This species is characterized by a round-to-hexagonal labial disc, the presence of a labial region, which is continuous or slightly offset from the body with six sectors, lateral sectors of first labial annulus being smaller than the submedian sectors, the presence of six to seven labial annules; the absence of deirids; stylet length 24 to 28 µâ€Šm long, body length 776 to 979 µâ€Šm long; lateral field with six to eight lateral lines, without areolation at mid-body and with areolation in outer bands at the tail region and a pointed tail terminus. Geocenamus vietnamensis n. sp. most closely resembles G. boghiae in having a non-sclerotized head framework and lacking a bursa in the males. It can be clearly distinguished from all other species of the genus Geocenamus by these characteristics. The combination of morphology, morphometric features, and phylogenetic trees, based on D2-D3 of 28S and ITS rDNA sequences, showed that this new species can be clearly separated from all other sequenced species. This record is the first for Geocenamus in Vietnam.A new species of the genus Geocenamus was isolated from soil and root samples from the rhizosphere of Casuarina equisetifolia (Casuarinaceae) in Quang Nam province, Vietnam. This species is characterized by a round-to-hexagonal labial disc, the presence of a labial region, which is continuous or slightly offset from the body with six sectors, lateral sectors of first labial annulus being smaller than the submedian sectors, the presence of six to seven labial annules; the absence of deirids; stylet length 24 to 28 µâ€Šm long, body length 776 to 979 µâ€Šm long; lateral field with six to eight lateral lines, without areolation at mid-body and with areolation in outer bands at the tail region and a pointed tail terminus. Geocenamus vietnamensis n. sp. most closely resembles G. boghiae in having a non-sclerotized head framework and lacking a bursa in the males. It can be clearly distinguished from all other species of the genus Geocenamus by these characteristics. The combination of morphology, morphometric features, and phylogenetic trees, based on D2­D3 of 28S and ITS rDNA sequences, showed that this new species can be clearly separated from all other sequenced species. This record is the first for Geocenamus in Vietnam.

Diversity of root-knot nematodes of the genus Meloidogyne Göeldi, 1892 (Nematoda: Meloidogynidae) associated with olive plants and environmental cues regarding their distribution in southern Spain.

Root-knot nematodes of the genus Meloidogyne are recognised worldwide as a major production constraint in crops of primary economic importance. Knowledge of their diversity and prevalence, as well as the major environmental and agronomical cues for understanding their distribution in specific areas is of vital importance for designing control measures to reduce significant damage. We provide the first detailed information on the diversity, distribution and levels of Meloidogyne species infecting wild and cultivated olive soils in a wide-region in southern Spain that included 499 sampling sites. Overall Meloidogyne spp. were found in 6.6% of sampled olive plants, with 6.6% and 6.5% for cultivated and wild olive, respectively. We identified five previously described Meloidogyne spp. (Meloidogyne arenaria, M. baetica, M. hapla, M. incognita, M. javanica) and one new species (Meloidogyne oleae sp. nov.) which, characterized using integrative taxonomy, increases the known biodiversity of Meloidogyne spp. affecting olive. Meloidogyne arenaria and M. incognita were only found infecting cultivated olive varieties, while, M. baetica was only found infecting wild olive. Three major parameters drive the distribution of Meloidogyne spp. in cultivated olives in southern Spain, cover vegetation on alley, irrigation and soil texture, but different species respond differently to them. In particular the presence of M. incognita is highly correlated with sandy loamy soils, the presence of M. javanica with irrigated soils and cover vegetation, while the presence of M. arenaria is correlated with the absence of cover vegetation on alley and absence of irrigation. These parameters likely influence the selection of each particular Meloidogyne species from a major dispersal source, such as the rooted plantlets used to establish the orchards.

Morphological and Molecular Characteristics of Pratylenchus haiduongensis sp. n., a New Species of Root-Lesion Nematodes Associated with Carrot in Vietnam.

Pratylenchus haiduongensis sp. n. is described as associated with carrot (Daucus carota subsp. sativus (Hoffm.) Schübl. & G. Martens) in Hai Duong Province, Vietnam. P. haiduongensis sp. n. is characterized by the lip region with three annuli and slightly separated from the body. Stylet knobs are rounded (never indented anteriorly). The lateral field includes four incisures, bearing areolation at the pharynx region and tail region and occasionally appears in the vulval region. Sometimes the appearances of oblique broken striaes divide the lateral field into five or six incisures. The ovary is distinct with one row of oocytes. Spermatheca is oval in shape with round central cavity, without sperm or reduced in some specimens. The postvuval uterine sac is long surpassing the vulva body diameter by 2 to 2.5 times (PUS = 31 to 65 µm). High vulva position with V = 66 to 75%. The tail shape can be subhemispherical with a smooth, slightly indented, broadly smooth, or cleft terminus observed in some specimens. The matrix code of P. haiduongensis sp. n. is: A2, B1, C4, D(1,3), E1, F(5,6), G(1,2), H(1,4); I(1,2,3,4), J1, K(1,2) according to Castillo and Vovlas (2007). The LSU-D2D3 segment and the ITS-rDNA region of this species were amplified and sequenced. The morphological characters and molecular phylogenetic analyses confirmed that this is a new species of the genus Pratylenchus in Vietnam.

Discopersicus n. gen., a New Member of the Family Tylenchidae Örley, 1880 with Detailed SEM Study on Two Known Species of the Genus Discotylenchus Siddiqi, 1980 (Nematoda; Tylenchidae) from Iran.

Discopersicus iranicus n. gen., n. comb., previously described from Iran as a new species under the genus Discotylenchus, is illustrated using light microscope and scanning electron microscope (SEM) observations and further studied using molecular characters. SEM studies revealed the newly proposed genus has oblique amphidial apertures on the lateral sides of the lip region. SEM images are also provided for two species of Discotylenchus, namely D. discretus and D. brevicaudatus, as the first SEM study of the genus. These results confirmed longitudinal amphidial aperture type on lateral sides of the lip region in genus Discotylenchus, as noted by Siddiqi while erecting the genus with D. discretus as the type species. Molecular phylogenetic analyses using partial small subunit (SSU) and large subunit (LSU) rDNA sequences revealed the affinity of the genus Discopersicus n. gen. with members of the subfamily Boleodorinae, as supported by morphological characters (mainly, the oblique amphidial opening).

Rotylenchus castilloi n. sp. (Nematoda: Hoplolaimidae), a new species with long stylet from northern Iran.

Rotylenchus castilloi n. sp., a new bisexual species is described and illustrated based on morphological, morphometric and molecular data. The new species is characterised by having a hemispherical, continuous lip region with an irregular corncob-like appearance under SEM, very long stylet (62-68 µm), vulva located at 49.7-62.2% of body length from anterior end, with a protruding double epiptygma, a rounded to convex-conoid (rarely bi-lobed) tail with 8-12 annuli and specific sequences of D2-D3 segments of 28S and ITS1-rRNA genes. Differences between the new species and four other species of the genus (R. mesorobustus, R. cazorlaensis, R. magnus and R. jaeni) are discussed. Morphologically, the new species can be separated from these species mostly by its body length, lip region characters, stylet length and location of phasmid. Phylogenetic analyses using 721 bp partial sequences of D2-D3 expansion segments of the 28S and 590 bp ITS1-rRNA genes revealed the new species forming a clade with two isolates of R. eximius and two isolates of R. unisexus, two morphologically unrelated species.