Similis Populations Research Articles

Pectate lyase genes from Radopholus similis and their application in pathotype identification

Radopholus similis is a destructive, migratory, and endophytoparasitic nematode. It has two morphologically indistinguishable pathotypes (or physiological races): banana and citrus pathotypes. At present, the only reliable method to differentiate the two pathotypes is testing the infestation and parasitism of nematodes on Citrus spp. via inoculation. However, differences in inoculation methods and conditions adopted by different researchers complicate obtaining consistent results. In this study, the parasitism and pathogenicity of 10 R. similis populations on rough lemon (Citrus limon) seedlings and the tropism and invasion of rough lemon roots were tested. It revealed that populations SWK, GJ, FZ, GZ, DBSR, and YJ were citrus pathotypes, which showed parasitism and pathogenicity on rough lemon and could invade rough lemon roots, whereas populations XIN, ML, HN6, and HL were banana pathotypes, having no parasitism and pathogenicity on rough lemon and they did not invade the rough lemon roots. Four pectate lyase genes (Rs-pel-2, Rs-pel-3, Rs-pel-4, and Rs-pel-5) belonging to the Class III family from these populations were amplified and analysed. The gene Rs-pel-3 could be amplified from six citrus pathotype populations and was stably expressed in the four developmental stages of the nematode, whereas it could not be amplified from the four banana pathotypes. Rs-pel-3 expression may be related to the parasitism and pathogenicity of R. similis on rough lemon. Hence, it can be used as a molecular marker to distinguish between banana and citrus pathotypes and as a target gene for the molecular identification of these two pathotypes.Key points• Four pectate lyase genes (Rs-pels) from Radopholus similis were cloned and analysed.• The expression of Rs-pels is different in two pathotypes of Radopholus similis.• A molecular identification method for two pathotypes of Radopholus similis using pectate lyase gene Rs-pel-3 as the target gene was established.

Resistência horizontal e vertical da bananeira ao nematoide cavernícola depende do nível de agressividade ou da virulência da população do nematoide

Abstract The burrowing nematode Radopholus similis is among the most damaging pathogens. Resistant plants are one of the most promising approaches for nematode control, and knowledge of resistance and aggressiveness components is essential to understand resistance genetics and developing new cultivars. Therefore, this study aimed to assess the degree of resistance of eight banana accessions to populations of R. similis to verify differences in the reproduction capacity between nematode populations and to study the horizontal/vertical resistance and aggressiveness/ virulence components of Musa spp. and R. similis pathosystem. The accessions 4249-05, 4279-06, Yangambi Km5, 0323-03, 0337-02, 1304-06, Borneo and Grande Naine were inoculated with one of three R. similis populations from the Brazilian states of Pernambuco, Distrito Federal, and Santa Catarina and kept in a greenhouse. Accessions 4249-05, Yangambi Km5, 0323-03, and 4279-06 showed different resistance levels, and the Pernambuco population had the highest reproductive capacity. Using Griffing Model IV, evidence of vertical and horizontal resistance was observed, with accessions 4249-05 and Yangambi km5 showing the highest levels of horizontal resistance.

Study on burrowing nematode, Radopholus similis, pathogenicity test system in tobacco as host

Radopholus similis (Cobb 1893) Thorne (1949) is a destructive migratory endoparasitic plant nematode. In this study, the pathogenic process of R. similis infection in Nicotiana benthamiana (tobacco) was studied using quartz sand culture in laboratory. The results showed that R. similis mainly parasitised the root cortex, leading to cortical cell decomposition and tissue decay. We optimised the inoculation conditions to establish a method for determining the pathogenicity of R. similis as follows: (1) a glass culture tube was filled with quartz sand (about 1/3 of the height) and sterilised twice; (2) 20-day-old N. benthamiana seedlings were transplanted into test tubes and cultivated for 10 days at (25±1)°C; (3) R. similis female nematodes were inoculated in the root rhizosphere at a rate of 150 nematodes per plant; (4) the number of nematodes, disease severity, and growth of the plant at 30 days post-inoculation (dpi) were determined. The pathogenicity of eight R. similis populations from different hosts was determined, which proved the feasibility of this method.

Using Proales similis (Rotifera) for toxicity assessment in marine waters.

There is a need to develop more animal species for assessing toxicity in marine environments. Cyst‐based toxicity tests using invertebrates are especially fast, technically simple, cost‐effective, and sensitive to a variety of toxicants. Over the past 30 years, a variety of toxicity endpoints have been measured using the marine rotifer Brachionus plicatilis hatched from cysts, including mortality, reproduction, ingestion, swimming, enzyme activity, and gene expression. A consensus has developed that the most ecologically relevant toxicity measurements should be made using more than one species. Furthermore, it has been noted that the rotifer species toxicant sensitivity distribution is much broader than which endpoint is measured. This implies that toxicity should be measured with the simplest, fastest, least expensive test available on as many species as feasible. If a battery of test species is to be used to estimate toxicity, diapause egg‐based toxicity tests that do not require culturing of test animals will be key. In this paper, we describe how diapause eggs of a new marine rotifer, Proales similis, can be produced, stored and hatched under controlled conditions to produce animals for toxicity tests. Methods are described for quantifying the toxicity of copper, mercury and cadmium based on mortality, ingestion, reproduction, and diapause egg hatching endpoints. We found that reproduction and ingestion endpoints were generally more sensitive to the metals than mortality or diapause egg hatching. When the copper sensitivity of P. similis was compared to Brachionus manjavacas and B. plicatilis using an ingestion test, similar EC50s were observed. In contrast, the B. rotundiformis ingestion EC50 for copper was about 4× more sensitive. Although diapause egg hatching was not the most sensitive endpoint, it is the most ecologically relevant for assessing sediment toxicity. Our discovery of diapausing eggs in the P. similis life cycle has created a conundrum. We have not observed males or sex in P. similis populations, which is a direct contradiction to the orthodox view of the monogonont rotifer life cycle. Work is needed to clarify how diapause egg production is accomplished by P. similis and whether sexual reproduction is involved.

Genome Announcement: The Draft Genomes of Two Radopholus similis populations from Costa Rica.

Radopholus similis is an economically important pest of both banana and citrus in tropical regions. Here we present draft genomes from two populations of R. similis from Costa Rica that were created and assembled using short read libraries from Illumina HiSeq technology.

Nematicidal protease genes screened from a soil metagenomic library to control Radopholus similis mediated by Pseudomonas fluorescens pf36.

Controlling Radopholus similis, an important phytopathogenic nematode, is a challenge worldwide. Herein, we constructed a metagenomic fosmid library from the rhizosphere soil of banana plants, and six clones with protease activity were obtained by functionally screening the library. Furthermore, subclones were constructed using the six clones, and three protease genes with nematicidal activity were identified: pase1, pase4, and pase6. The pase4 gene was successfully cloned and expressed, demonstrating that the protease PASE4 could effectively degrade R. similis tissues and result in nematode death. Additionally, we isolated a predominant R. similis-associated bacterium, Pseudomonas fluorescens (pf36), from 10 R. similis populations with different hosts. The pase4 gene was successfully introduced into the pf36 strain by vector transformation and conjugative transposition, and two genetically modified strains were obtained: p4MCS-pf36 and p4Tn5-pf36. p4MCS-pf36 had significantly higher protease expression and nematicidal activity (p < 0.05) than p4Tn5-pf36 in a microtiter plate assay, whereas p4Tn5-pf36 was superior to p4MCS-pf36 in terms of genetic stability and controlling R. similis in growth pot tests. This study confirmed that R. similis is inhibited by the associated bacterium pf36-mediated expression of nematicidal proteases. Herein, a novel approach is provided for the study and development of efficient, environmentally friendly, and sustainable biocontrol techniques against phytonematodes.

Parasitism and pathogenicity of Radopholus similis to Ipomoea aquatica, Basella rubra and Cucurbita moschata and genetic diversity of different populations

Ten populations of Radopholus similis from different ornamental hosts were tested for their parasitism and pathogenicity to water spinach (Ipomoea aquatic), malabar spinach (Basella rubra), and squash (Cucurbita moschata) in pots. The results showed all three plants were new hosts of R. similis. Growth parameters of plants inoculated with nematodes were significantly lower than those of healthy control plants. All R. similis populations were pathogenic to the three plants, but pathogenicity differed among populations from different hosts. The same R. similis populations also showed different pathogenic effects in the three different plants. RadN5 population from Anthurium andraeanum had the highest pathogenicity to the three studied plants. RadN1 from A. andraeanum had the lowest pathogenicity to squash and RadN7 from Chrysalidocarpus lutesens had the lowest pathogenicity to water spinach and malabar spinach. R. similis is usually associated with root tissues, but here we report that it could be found to move and feed in the stem bases of all three studied plants. Sequence and phylogenetic analyses of DNA markers of the 18S rRNA, 28S rRNA, ITS rRNA, and mitochondrial DNA gene sequences of ten R. similis populations revealed significant genetic diversity. RadN5 and RadN6 populations from anthurium showed a close genetic relationship and could be distinguished from other populations by PCR-RFLP. At the same time, RadN5 and RadN6 populations were the most pathogenic to three studied plants. These results confirm the existence of large biological variability and molecular diversity among R. similis populations from the same or different hosts, and these characteristics are related to pathogenic variability.

Molecular identification and functional characterization of the fatty acid- and retinoid-binding protein gene Rs-far-1 in the burrowing nematode Radopholus similis (Tylenchida: Pratylenchidae).

Fatty acid- and retinoid-binding protein (FAR) is a nematode-specific protein expressed in the nematode hypodermis. It is involved in nematode development, reproduction, and infection and can disrupt the plant defense reaction. In this study, we obtained the full-length sequence of the far gene from Radopholus similis (Rs-far-1), which is 828 bp long and includes a 558 bp ORF encoding 186 amino acids. A protein homology analysis revealed that Rs-FAR-1 is 75% similar to Mj-FAR-1 from Meloidogyne javanica. A neighbor-joining phylogenetic tree was inferred and showed that Rs-FAR-1 is most similar to Pv-FAR-1 from Pratylenchus vulnus. A fluorescence-based ligand-binding analysis confirmed that Rs-FAR-1 can combine with fatty acids and retinol. qPCR was used to assess Rs-far-1 expression levels at different developmental stages in different R. similis populations, and its expression was 2.5 times greater in the highly pathogenic Rs-C population than in the less pathogenic Rs-P population. The highest expression was found in females, followed by eggs, juveniles and males. When R. similis was treated with Rs-far-1 dsRNA for 36 h, the reproduction and pathogenicity decreased significantly. In situ hybridization revealed Rs-far-1 transcripts in the R. similis hypodermis. Additionally, R. similis treated with Rs-far-1 dsRNA or water were inoculated into Arabidopsis thaliana. Allene oxide synthase (AOS) expression in A. thaliana was upregulated during early infection in both treatments and then returned to the expression levels of the control plant. Compared with the control plant, AOS expression significantly decreased in A. thaliana inoculated with water-treated R. similis but significantly increased in A. thaliana inoculated with Rs-far-1 dsRNA-treated R. similis. This finding indicates that Rs-far-1 regulates AOS expression in A. thaliana. Rs-FAR-1 plays a critical role in R. similis development, reproduction, and infection and can disturb the plant defense reaction. Therefore, Rs-far-1 is an important target gene to control R. similis.

Invasive black spiny‐tailed iguanas (Ctenosaura similis) on Gasparilla Island, Florida, USA

The native range of Ctenosaura similis extends from southern Mexico through Panama. From an initial introduction of 3 animals in 1979, the species now numbers in the thousands on Gasparilla Island in southwest Florida. In response to complaints of property damage from residents and threats to native species, local officials and the US Department of Agriculture Wildlife Services began a removal program in 2008. Through 2011, trappers removed 9467 ctenosaurs. The number removed declined from 32 iguanas/day in 2008 to 1.9 iguanas/day in 2011 despite no easing of the control effort. We necropsied 2757 ctenosaurs to document aspects of their natural history. Females outnumbered males overall, although the largest size class (>300 mm snout-vent length) included 32 males and just 2 females. Reproduction was seasonal. We found oviducal eggs in females from early Apr to early Jun, approximately 2 months later than C. similis in its native range. We trapped hatchlings from late Jul to early Oct coincident with the summer rainy season. Clutch size increased with female body size, with 62 being the largest clutch size recorded. In general, the biology of the invasive population on Gasparilla Island resembles native C. similis populations in Central America, except for the lack of large individuals. We suggest that shorter day length and colder temperatures create environmental conditions that are suboptimal for individual growth compared to those in the native range.

A comparative study of the reproductive fitness and pathogenicity of a population of Radopholus similis from Davao, Philippines, on banana (Musa spp.)

The in vitro reproductive fitness on carrot discs and the in vivo pathogenicity on selected FHIA (Fundación Hondureña de Investigaciónes Agrícola) banana hybrids of a population of Radopholus similis isolated from banana in Davao, Philippines, were investigated at the Bureau of Plant Industry, Davao City, Philippines. It was shown that on carrot discs following inoculation with 25 females, the Davao population had the highest population density and multiplication rate compared with two other R. similis populations from the Philippines (Los Baños and Quezon) and two exotic populations of R. similis (from Uganda and Indonesia). According to the Gompertz model, the Davao population also had a short lag phase and a high maximum specific growth. Following inoculation with one single female, the Davao population also had the highest multiplication rate and the highest proportion of juveniles and females compared with the other four populations included in the experiments. Nine weeks after inoculation with 1000 vermiforms, the R. similis population from Davao had reduced the plant height of the FHIA hybrids included in the experiment (FHIA-3, FHIA-4, FHIA-5, FHIA-18 and FHIA-23) on average by 37%, plant girth by 5.3–40.9%, shoot weight by 20.1–65.8% and root weight by 31.7–69.7%, indicating the high pathogenicity of this population on banana. FHIA-4 was tolerant to R. similis infection.

Variability in reproductive fitness and virulence of four Radopholus similis nematode populations associated with plantains and banana (Musa spp.) in Uganda

Burrowing nematodes (Radopholus similis) are among the most serious nematode pests affecting banana and plantain (Musa spp.). In Uganda, bananas, which are known locally as “matooke”, are the main staple. Radopholus similis populations collected in four banana-growing locations (Namulonge, Mbarara, Ikulwe and Mukono) were cultured monoxenically on carrot discs, and we compared the variability in reproductive fitness and virulence (as a function of time and inoculum level) of different populations of R. similis from Uganda. Their level of pathogenicity was determined by assessing the nematode reproductive ratio; that is, final population divided by the initial population. These in vitro experiments showed that the R. similis population from Mbarara had the highest reproduction ratio, while the population from Mukono had the lowest reproduction ratio. This assessment along with pathogenicity experiments on host Musa plants provides a means for defining pathogenicity groups among R. similis populations.

Analysis of the pathogenic variability and genetic diversity of the plant-parasitic nematode Radopholus similis on bananas

Radopholus similis is responsible for huge production losses of banana in Uganda, where a large proportion of the population are reliant on cooking banana as their main staple crop. Assessment of the pathogenic and genetic variability of seven populations of R. similis originating from banana plantations in Uganda demonstrated that their virulence on Musa cultivars differed, displaying pathotype-like variation. Using RAPD and AFLP techniques the populations could be grouped into two putative genomic groups, which corresponded with R. similis relative pathogenicity. Most, but not all, populations were able to reproduce and damage roots on the two widely confirmed sources of resistance, Yangambi km5 and Pisang Jari Buaya. Studies using RAPD techniques furthermore revealed putative markers for nematode virulence. Variation in virulence of R. similis populations was also demonstrated at the regional (Africa) level. Phylogenetic analysis most closely associated the Uganda populations with populations from Sri Lanka, indicating their likely source of origin. However, genetic variability between Uganda populations also indicate that R. similis was probably introduced into Uganda on separate occasions from different sources, which may help to explain the causes of variability in banana decline in Uganda. The results of this study have clear implications for the development and deployment of sustainable nematode management practices based on the use of resistance, which could help underpin ongoing management initiatives.

Screening Musa germplasm for resistance to burrowing nematode populations from Uganda

Nematode pathogenicity experiments on Musa host plants were carried out in pot trials. Both the final nematode population densities and percentages root necrosis on different host plants were higher for the Radopholus similis population from Mbarara than for the populations from Namulonge, Ikulwe and Mukono (Uganda). The R. similis population from Mbarara managed to break the resistance of the diploid banana Pisang Jari Buaya, a known worldwide source of resistance to R. similis. The diploid banana hybrid TMB2× 9128-3 and the triploid dessert banana Yangambi km5 showed resistance against the four R. similis populations. These results indicate that differences in pathogenicity among different R. similis populations exist and should be taken into consideration for plantain and banana germplasm enhancement.

Monogenean parasites of introduced pumpkinseed Lepomis gibbosus (Centrarchidae) in the Danube River Basin

The pumpkinseed, Lepomis gibbosus, a centrarchid fish native to eastern North America, was introduced into Europe at the end of the 19th century. Lepomis gibbosus now represents one of the most successful of introduced fish species, having spread widely throughout many European countries. In this study, we collected monogenean parasites of L. gibbosus from four sites in the Danube River Basin. We recorded four monogenean species: Gyrodactylus sp., probably acquired in the area of introduction, and Onchocleidus similis, O. dispar and Actinocleidus recurvatus, species introduced together with their host. Onchocleidus similis dominated the parasite community at all sampling sites. Actinocleidus recurvatus has only been reported from southern Europe to date and, therefore, this is the first report of A. recurvatus from Central Europe and the Danube River Basin. We observed high morphological variability in both anchor pairs of A. recurvatus. Only in O. similis was there a positive correlation observed between haptor metric traits and fish host length. Differences in metric traits between native and introduced O. similis populations may reflect a difference in fish host size, the introduced fish host achieving a smaller size than fish from native populations. A detailed analysis of microhabitat distribution indicated a preference for the second gill arch for both dominant species O. similis and A. recurvatus, with dissimilar site segregation.

Burrowing nematodes from Colombia and their relationship with Radopholus similis populations, R. arabocoffeae and R. duriophilus

Abstract Two burrowing nematode populations from Colombia were characterised using morphological, morphometric and molecular criteria. The morphological and morphometric characters of the two populations did not differ from those of Radopholus similis. The phylogenetic analysis based on sequence comparison of 18S, D2D3 regions of these populations with those of Radopholus sp., R. similis and other Tylenchomorpha from GenBank confirmed the close molecular relationship between the genus Radopholus and the Hoplolaimidae. The consensus tree based on the comparison of ITS1-5.8-ITS2 rDNA sequences of the Colombian populations with R. similis (many sources), R. arabocoffeae and R. duriophilus from GenBank grouped the Colombian populations in a clade which also included R. similis from different localities. Other R. similis populations grouped in separated clades, which were closely related to the clade containing R. arabocoffeae and R. duriophilus. The high genetic variability among the R. similis populations was observed and confirmed by the high ITS1-5.8-ITS2 sequence divergence within the R. similis populations studied (5.4%) which was comparable to the divergence (4.2-6.9%) between the Radopholus species included in the analysis. These findings and the unresolved phylogenetic placement of R. arabocoffeae and R. duriophilus among R. similis suggests that the burrowing nematode could be a species complex. Limited information on the variability of the 18S and D2D3 region gives an indication of their possible value, besides ITS1-5.8-ITS, as molecular markers within Radopholus. No complete and comprehensible relation was found between the phylogenetic position of the Radopholus populations and their geographical origin or host.

The role of urban greenspaces in fauna conservation: the case of the iguana Ctenosaura similis in the ‘patios’ of León city, Nicaragua

Private urban greenspaces, called ‘patios’ in Latin America, can act as important refuges for wildlife in scattered growing cities of the tropics. We studied the presence and abundance of the black spiny-tailed iguana (Ctenosaura similis) in patios of Leon city (Nicaragua). Forty patios were structurally characterized and abundances of iguanas were determined through surveys of inhabitants supported by observations of specimens and burrows. Patio area and maximum tree height were variables positively related with presence and abundance of iguanas and presence of burrows. The permeability of fences for iguana movements and the presence of preferred trees for food were also related to presence and abundance of iguanas. Stepwise selection model for abundance of iguanas included number of preferred trees, maximum tree height and permeability of fences. The presence of iguanas was only explained by maximum tree height. Our results show that the indigenous-root types of patios offer the most adequate conditions for the establishment and maintenance of iguana populations among the studied cases. In the light of these results, management practices to preserve the Ctenosaura similis populations in urban patios are suggested.

Radopholus duriophilus sp. n. (Nematoda: Pratylenchidae) from Western Highland of Vietnam

Abstract A new species of the genus Radopholus associated with durian (Durio zibetinus M.) in the Western Highland of Vietnam is described as Radopholus duriophilus sp. n. The new species is close to R. similis, but is distinguished from R. similis by the position of the excretory pore located posterior to pharyngo-intestine junction (vs at level of pharyngo-intestine junction), oval shape sperm (vs rod-like), four incisures terminating far behind position of phasmid (vs three incisures terminating at or just behind phasmid), and bursa in male never reaching tail terminus (vs bursa reaching tail terminus). Females of R. duriophilus sp. n. differ from R. nativus females by stylet length (16.5-19 vs 19-23 μm), oval or kidney-shaped sperm (vs rod-like), four incisures at level of phasmid (vs three) and their areolated lateral field (vs not areolated). The position of excretory pore of both female and male is located posterior to pharyngo-intestine junction (vs at level or anterior to pharyngo-intestine junction). Females of R. duriophilus sp. n. differ from R. clarus females by stylet length (16.5-19 vs 19-21 μm) and areolated lateral field (vs no areolation). Females of R. duriophilus sp. n. differ from R. musicola females by their lateral field with equidistant incisures at mid-body (vs two deep outer folds and two faint shallow inner incisures), oval or kidney-shaped sperm (vs rod-like), and rounded terminus tail (vs sharply pointed). The species also sp. n. differ in male stylet length (11.5-15 vs 8.8-12 μm). Females of R. duriophilus sp. n. differ from R. bridgei females by stylet length (16.5-19 vs 15-17.5 μm), median bulb length (11-16.5 vs 11-13 μm), length of hyaline tail (3-11 vs not more than 4 μm), and lateral field areolated for entire body (vs not areolated except irregularly on neck and tail). The male differs by stylet length (11.5-15 vs 10-12 μm) and length of the hyaline portion (4-9 vs 1-4 μm). In addition, the relatively high level of ITS sequence divergence of the new species from R. similis populations and the presence of nucleotide autapomorphies support a separate specific status for these durian populations. Results of surveys revealed that R. duriophilus sp. n. is rather widely distributed in durian orchards and associated with decline and death of trees in many durian nursery gardens. Densities of nematode population reached thousands of individuals per g of root samples.

Population dynamics and damage potential of the burrowing nematode, Radopholus similis, on Anthurium andreanum grown in soil-less medium

AbstractIn a 14 month pot experiment with Anthurium andreanum cv. Sonate, grown in soil-less medium in glasshouse conditions, burrowing nematode (Radopholus similis) population development and crop damage were investigated. At 9 days after transplanting, plants were inoculated with R. similis at four initial densities (Pi): 0, 100, 1000 and 10 000 per pot. Three to 5 months after inoculation R. similis populations reached maximum densities ranging from 59 000 to 112 000 per pot in roots and from 500 to 1150 per litre of drainage water. The greatest densities occurred at the smallest Pi. Subsequently, nematode populations decreased in the decreasing root mass. Fourteen months after inoculation, root systems of nematode-inoculated treatments were almost completely necrotic and root weights were reduced by 84-91% compared to those of non-inoculated plants. In response to the deteriorating root systems, plant vigour of all nematode-inoculated treatments declined tremendously and flower production was significantly less compared to non-inoculated plants at 6 months after inoculation. During the last 2 months of the experiment, reductions in flowers ranged from 46-70% (number), 30-53% (weight), 19-33% (stem length) and 15-31% (width). The total yield losses of these flower growth parameters, calculated over the 14-month experimental period, ranged from 18-33, 15-35, 9-19 and 6-15%, respectively. Greatest reductions occurred in plants in the treatment with the greatest Pi. In these conditions, the damage threshold was calculated to be equal to or fewer than 100 R. similis per pot.

Detection and analysis of inter- and intraspecific diversity of Pratylenchus spp. using isozyme markers

The root-lesion nematodes of the genus Pratylenchus are an economically important group of plant parasitic nematodes that show high similarity among sibling species. Isozyme patterns obtained by isoelectrofocusing (IEF) were used to differentiate and establish genetic relatedness among Pratylenchus species. A total of 40 populations comprising 9 Pratylenchus species and Radopholus similis from broad host and geographic origins was examined to compare isozyme patterns of esterase (EST), hexoquinase (HK), isocitrate dehydrogenase (IDH), malate dehydrogenase (MDH), phosphoglucose isomerase (PGI), phosphoglucomutase (PGM) and superoxide dismutase (SOD). Of these systems, only EST, MDH, PGI and PGM were useful for differentiation of P. vulnus, P. goodeyi, P. penetrans, P. scribneri, P. thornei and R. similis populations. The greatest intraspecific diversity was found within P. coffeae based on the isozyme patterns for MDH, PGI and PGM. Intraspecific variability was also detected among R. similis populations, which showed two isozyme patterns in EST and PGI systems. Less intraspecific variation was found within the P. penetrans group. The P. goodeyi population from Cameroon differed from the other populations in this specific group in its MDH, PGI and PGM phenotypes. Highly similar banding patterns of EST, MDH and PGI activity were found among the P. scribneri populations and the one population of P. agilis. A cluster analysis of the 40 populations, generated from the four enzyme banding patterns, produced groupings that broadly matched the previous classification into specific groups, reflecting intraspecific variability in some cases. The results confirm the potential use of isozyme patterns as markers for these nematode species and their value for diagnostic application.

A reappraisal of the 'fracticornis-opacicollis' taxonomic complex of the genus Onthophagus Latreille using allozymes (Coleoptera, Scarabaeoidea)

The present taxonomic status of the so called fracticornis–opacicollis complex of the genus Onthophagus, Onthophagus fracticornis (Preyssler, 1790), Onthophagus similis (Scriba, 1790) and Onthophagus opacicollis Reitter, 1892 is still controversial. Discrepancies mainly involve the status of O. similis and O. opacicollis. A genetic analysis of nine populations of this complex using eight loci is presented, indicating that: (i) O. fracticornis is a good species, and (ii) the genetic distances between O. opacicollis and O. similis populations as a function of the biogeographic scenery (allopatry versus sympatry), indicate that they are not completely reproductively isolated across their entire geographic range, thus suggesting a still unfinished speciation in different areas. The data demonstrate both the existence of some gene flow between O. opacicollis and O. similis in one place (‘El Barraco’; Central Spain) and isolation in others (‘Sa Nevada’; South-east Spain). Geographic areas hosting populations of both taxa with intermediate genetic distances (‘Doñana’; South-west Spain) also exist in the Iberian Peninsula.