Juan Antonio Balbuena

PACo: A Novel Procrustes Application to Cophylogenetic Analysis

We present Procrustean Approach to Cophylogeny (PACo), a novel statistical tool to test for congruence between phylogenetic trees, or between phylogenetic distance matrices of associated taxa. Unlike previous tests, PACo evaluates the dependence of one phylogeny upon the other. This makes it especially appropriate to test the classical coevolutionary model that assumes that parasites that spend part of their life in or on their hosts track the phylogeny of their hosts. The new method does not require fully resolved phylogenies and allows for multiple host-parasite associations. PACo produces a Procrustes superimposition plot enabling a graphical assessment of the fit of the parasite phylogeny onto the host phylogeny and a goodness-of-fit statistic, whose significance is established by randomization of the host-parasite association data. The contribution of each individual host-parasite association to the global fit is measured by means of jackknife estimation of their respective squared residuals and confidence intervals associated to each host-parasite link. We carried out different simulations to evaluate the performance of PACo in terms of Type I and Type II errors with respect to two similar published tests. In most instances, PACo performed at least as well as the other tests and showed higher overall statistical power. In addition, the jackknife estimation of squared residuals enabled more elaborate validations about the nature of individual links than the ParaFitLink1 test of the program ParaFit. In order to demonstrate how it can be used in real biological situations, we applied PACo to two published studies using a script written in the public-domain statistical software R.

Interrelationships of the Haploporinae (Digenea: Haploporidae): A molecular test of the taxonomic framework based on morphology

The taxonomic framework of the Haploporidae is evaluated and the relationships within the Haploporinae are assessed for the first time at the generic level using molecular data. Partial 28S and complete ITS2 rDNA sequences from representatives of six of the nine recognised genera within the Haploporinae were analysed together with published sequences representing members of two haploporid subfamilies and of the closely related family Atractotrematidae. Molecular analyses revealed: (i) a close relationship between the Atractotrematidae and the Haploporidae; (ii) strong support for the monophyly of the Haploporinae, Dicrogaster and Saccocoelium, and the position of Ragaia within the Haploporinae; (iii) evidence for rejection of the synonymy of Saccocoelioides and Lecithobotrys and the validity of the Dicrogasterinae; and (iv) support for the distinct status of Saccocoelium in relation to Haploporus. The wider sampling within the genera Dicrogaster and Saccocoelium confirmed the distinct status of the included species, thus rejecting previously suggested synonymies. Saccocoelioides, recently transferred to the Chalcinotrematinae, was nested within the Haploporinae and this was largely associated with the position of Forticulcita, resolved as the most basal haploporine genus. Forticulcita also possesses a well-delimited eversible intromittent copulatory organ, a feature unique in the Haploporidae which has not been previously considered an important apomorphy. This, in association with the present hypothesis of the Haploporinae based on molecular data, led us to erect Forticulcitinae subf. n. for Forticulcita; this resolved Saccocoelioides and, by extension the Chalcinotrematinae, as sister groups to the Haploporinae.

DESCRIPTION AND MORPHOMETRICAL VARIABILITY OF A NEW SPECIES OF LIGOPHORUS AND OF LIGOPHORUS CHABAUDI (MONOGENEA: DACTYLOGYRIDAE) ON MUGIL CEPHALUS (TELEOSTEI) FROM THE MEDITERRANEAN BASIN

A comparative morphological study of specimens of Ligophorus spp. from Mugil cephalus in western Mediterranean and the Black Sea localities has been carried out, indicating the presence of 2 distinct forms, i.e., Ligophorus chabaudi and Ligophorus cephali n. sp. A detailed description of the latter and an up-to-date redescription of L. chabaudi are provided. The existence of these 2 morphological species was additionally supported by principal component analysis based on 19 metric characters of 87 specimens arranged in samples defined by parasite species and geographical locality. The analysis indicated consistent differences between species but not between localities; this pattern was well supported by jackknife procedures. Linear discriminant analyses showed that the main metric differences between the 2 species were attributable to the lengths of the dorsal and ventral bars and to the uncinulus length; use of the first 2 variables was enough to allocate all specimens studied to either form. The erection of L. cephali n. sp. raises the number of sympatric Mediterranean species of Ligophorus on M. cephalus to 3. The known geographical ranges of these species are compared.

LIGOPHORUS PILENGAS N. SP. (MONOGENEA: ANCYROCEPHALIDAE) FROM THE INTRODUCED SO-IUY MULLET, MUGIL SOIUY (TELEOSTEI: MUGILIDAE), IN THE SEA OF AZOV AND THE BLACK SEA

The monogenean Ligophorus chabaudi was originally described on the gills of the flathead mullet, Mugil cephalus, and was subsequently reported on the So-iuy mullet, Mugil soiuy. However, the morphology of sclerotized parts and multivariate statistical analyses suggest that the form from the So-iuy mullet represents a new species. This study provides a description of the new species Ligophorus pilengas n. sp. and provides additional morphological data concerning the morphology of the ventral bar that might be useful for the diagnosis of Ligophorus. Ligophorus pilengas n. sp. is the second species of Ligophorus reported on the So-iuy mullet. Zoogeographical records indicate that L. pilengas n. sp. was probably introduced to the Black Sea and the Sea of Azov from the western Pacific Ocean together with its host.

TAXONOMIC STATUS OF LIGOPHORUS MUGILINUS (HARGIS, 1955) (MONOGENEA: ANCYROCEPHALIDAE), WITH A DESCRIPTION OF A NEW SPECIES OF LIGOPHORUS FROM MUGIL CEPHALUS (TELEOSTEI: MUGILIDAE) IN THE MEDITERRANEAN BASIN

Ligophorus mugilinus was originally described on the flathead mullet Mugil cephalus from the Gulf of Mexico. The species was later recorded in the Mediterranean Sea on the flathead mullet and in the Caribbean Sea on the white mullet Mugil curema. After examination of the type material and additional specimens of Ligophorus from the northwest Atlantic coast, the Caribbean Sea, and the Mediterranean and Black Sea, we observed morphological differences concerning haptoral and male copulatory structures between L. mugilinus from the northwest Atlantic and the forms from the Mediterranean and Black Seas. Accordingly, the latter specimens are described as a new species, Ligophorus mediterraneus n. sp. A redescription of L. mugilinus, including new morphometric data, is also given. The morphometric analysis of the Caribbean specimens suggested that their ascription to L. mugilinus is uncertain, but additional material is needed to clarify their specific identity. The results of this study, together with previous zoogeographical evidence, indicate that flathead mullets from different seas harbor different species complexes of Ligophorus. Given the coastal habits of the host, it is suggested that open oceans could act as geographical barriers favoring speciation within disjointed flathead mullet populations.

Molecules and morphology reveal cryptic variation among digeneans infecting sympatric mullets in the Mediterranean

We applied a combined molecular and morphological approach to resolve the taxonomic status of Saccocoelium spp. parasitizing sympatric mullets (Mugilidae) in the Mediterranean. Eight morphotypes of Saccocoelium were distinguished by means of multivariate statistical analyses: 2 of Saccocoelium obesum ex Liza spp.; 4 of S. tensum ex Liza spp.; and 2 (S. cephali and Saccocoelium sp.) ex Mugil cephalus. Sequences of the 28S and ITS2 rRNA gene regions were obtained for a total of 21 isolates of these morphotypes. Combining sequence data analysis with a detailed morphological and multivariate morphometric study of the specimens allowed the demonstration of cryptic diversity thus rejecting the hypothesis of a single species of Saccocoelium infecting sympatric mullets in the Mediterranean. Comparative sequence analysis revealed 4 unique genotypes, thus corroborating the distinct species status of Saccocoelium obesum, S. tensum and S. cephali and a new cryptic species ex Liza aurata and L. saliens recognized by its consistent morphological differentiation and genetic divergence. However, in spite of their sharp morphological difference the 2 morphotypes from M. cephalus showed no molecular differentiation and 4 morphotypes of S. tensum were genetically identical. This wide intraspecific morphological variation within S. tensum and S. cephali suggests that delimiting species of Saccocoelium using solely morphological criteria will be misleading.

Real-time RT-PCR detection of betanodavirus in naturally and experimentally infected fish from Spain.

Infections with betanodavirus affect a wide range of wild and farmed fish species throughout the world, mostly from the marine environment. The aim of this work was to develop and validate real-time RT-PCR assays for sensitive and specific detection of nodavirus in diseased or carrier fish. The new detection assay was used to study the transmission and development of nodavirus infection in juvenile sea bass, Dicentrarchus labrax (L.), challenged by different routes, and also to screen for nodavirus in various farmed fish species. On average, the sensitivity was 10-100 times higher than a standard RT-PCR, and the assay was able to detect asymptomatic carrier fish that otherwise could have been classified as free of infection. Clinical signs of nodavirus infection were reproduced in fish infected following bath exposure or intramuscular injection, demonstrating horizontal transmission of the disease. Nodavirus was always detected in the brain of diseased fish but also in many recovered fish. The new assay enables us to confirm the presence of the virus at an early phase in the production cycle and may represent a useful tool to prevent or slow down the spread of nodavirus to new locations.

A NEW CYSTIDICOLID NEMATODE FROM MULLUS SURMULETUS (PERCIFORMES: MULLIDAE) FROM THE WESTERN MEDITERRANEAN

Ascarophis valentina n. sp. is described from Mullus surmuletus off the Valencian coast of Spain on the basis of both light and scanning electron microscopy. It can be distinguished from the other members of the genus by the length of the left (long) spicule of the males and by egg morphology. An updated grouping of the species of Ascarophis considered valid is provided with respect to these characters. The new species resembles Ascarophis capelanus, belonging to the group of species possessing eggs with a single polar knob with filaments, but is distinguished by the size of the body, the length of the esophagus (especially in relation to body length), the position of the vulva, and the size of the left spicule. The new species also shows substantial morphological differences compared with the 3 species, Ascarophis mullusi, Ascarophis upenei, and Ascarophis parupenei, previously described from mullid hosts.

Three new species of Ligophorus (Monogenea: Dactylogyridae) on the gills of Mugil cephalus (Teleostei: Mugilidae) from the Japan Sea

Comparative morphology and multivariate morphometric analysis of monogeneans collected on flathead mullets Mugil cephalus from 2 Russian localities of the Japan Sea revealed the presence of 3 new species of Ligophorus, namely, L. domnichi n. sp., L. pacificus n. sp., and L. cheleus n. sp., which are described herein. So far, only 1 species of dactylogyrid monogenean identified as Ligophorus chabaudi was known on flathead mullets in this sea, but after comparison with the present material, we propose that this form actually represents L. domnichi n. sp. Results support previous zoogeographical evidence, suggesting that flathead mullets from different seas harbor different species complexes of Ligophorus. One interesting finding is that the 3 new species have a U-shaped ovary, whereas ovate ovaries have been reported in previous descriptions of species of the genus, e.g., L. vanbenedenii, L. parvicirrus, L. imitans, and L. chongmingensis. The U-shaped ovary was revealed only when the worms were observed in lateral view. The additional examination of L. vanbenedenii, L. parvicirrus, L. imitans, and L. pilengas specimens from our collections also revealed a U-shaped ovary in these forms as well. Further studies should establish whether or not this character is shared by all members of the genus.

Corynosoma cetaceum in the Stomach of Franciscanas, Pontoporia blainvillei (Cetacea): an Exceptional Case of Habitat Selection by an Acanthocephalan

Adult acanthocephalans are typically found in the intestine of vertebrates, where they can readily absorb nutrients. However, Corynosoma cetaceum has been frequently reported in the stomach of cetaceans from the Southern Hemisphere. The ecological significance of this habitat was investigated by examining data on number, sex ratio, maturity status, biomass, and fecundity of C. cetaceum in different parts of the digestive tract of 44 franciscanas Pontoporia blainvillei. Individual C. cetaceum occurred in the pyloric stomach (PS) and, to lesser degrees, in the duodenal ampulla (DA) and the main stomach (MS). Females outnumbered males in all chambers, although the sex ratio was closer to 1:1 in the MS; there also was a higher proportion of nongravid females, with a smaller biomass in the MS than in the PS and the DA. This evidence suggests that cystacanths are released from prey tissues in the MS, where entire prey are reduced to semi-fluid chyme. The 3 chambers harbored gravid females that did not differ significantly in mean biomass or fecundity. The maturity status of females was nearly identical between the PS and the DA. In the MS, the higher proportion of non-gravid females is probably due to the occurrence of newly recruited females to this site. Mean biomass and fecundity of gravid females covaried strongly and positively among chambers within hosts. These results suggest that there are no major differences between the 3 chambers with respect to the suitability for reproduction by C. cetaceum. However, although the MS is the largest chamber, it harbored the smallest number of gravid females. Interestingly, worms were largely restricted to the aboral portion of the MS, a sheltered region where a concentration of chyme, and thus nutrient availability, likely occurs. Linear distribution differences of gravid female C. cetaceum at increasing intensities suggest that reproductive females occupy chambers according to available space. In summary, the stomach should be considered the main habitat for C. cetaceum. The choice of this habitat is puzzling because other Corynosoma species occur in the intestine, and because the stomach of cetaceans is not an absorptive site.