Isabel Blasco-Costa

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.

Fish pathogens near the Arctic Circle: molecular, morphological and ecological evidence for unexpected diversity of Diplostomum (Digenea: Diplostomidae) in Iceland.

Host-parasite systems at high latitudes are promising model systems for detecting and predicting the impact of accelerated environmental change. A major challenge is the lack of baselines for the diversity and distribution of parasites in Arctic wildlife, especially in the freshwater environment. Here we present the first known estimates of the species diversity and host associations of Diplostomum spp. in sub-Arctic freshwater ecosystems of the Palaearctic. Our analyses integrating different analytical approaches, phylogenies based on mitochondrial and nuclear DNA, estimates of genetic divergence, character-based barcoding, morphological examination, precise detection of microhabitat specialisation and host use, led to the discovery of one described and five putative new species that complete their life-cycles within a fairly narrow geographic area in Iceland. This increases the species richness of Diplostomum in Iceland by 200% and raises the number of molecularly characterised species from the Palaearctic to 17 species. Our results suggest that the diversity of Diplostomum spp. is underestimated globally in the high latitude ecosystems and call for a cautionary approach to pathogen identification in developing the much needed baselines of pathogen diversity that may help detect effects of climate change in the freshwater environment of the sub-Arctic.

Molecular approaches to trematode systematics: 'best practice' and implications for future study.

To date, morphological analysis has been the cornerstone to trematode systematics. However, since the late-1980s we have seen an increased integration of genetic data to overcome problems encountered when morphological data are considered in isolation. Here, we provide advice regarding the ‘best molecular practice’ for trematode taxonomy and systematic studies, in an attempt to help unify the field and provide a solid foundation to underpin future work. Emphasis is placed on defining the study goals and recommendations are made regarding sample preservation, extraction methods, and the submission of molecular vouchers. We advocate generating sequence data from all parasite species/host species/geographic location combinations and stress the importance of selecting two independently evolving loci (one ribosomal and one mitochondrial marker). We recommend that loci should be chosen to provide genetic variation suitable to address the question at hand and for which sufficient ‘useful’ comparative sequence data already exist. Quality control of the molecular data via using proof-reading Taq polymerase, sequencing PCR amplicons using both forward and reverse primers, ensuring that a minimum of 85% overlap exists when constructing consensus sequences, and checking electropherograms by eye is stressed. We advise that all genetic results are best interpreted using a holistic biological approach, which considers morphology, host identity, collection locality, and ecology. Finally, we consider what advances next-generation sequencing holds for trematode taxonomy and systematics.

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.

Host traits explain the genetic structure of parasites: a meta-analysis

Gene flow maintains the genetic integrity of species over large spatial scales, and dispersal maintains gene flow among separate populations. However, body size is a strong correlate of dispersal ability, with small-bodied organisms being poor dispersers. For parasites, small size may be compensated by using their hosts for indirect dispersal. In trematodes, some species use only aquatic hosts to complete their life cycle, whereas others use birds or mammals as final hosts, allowing dispersal among separate aquatic habitats. We performed the first test of the universality of the type of life cycle as a driver of parasite dispersal, using a meta-analysis of 16 studies of population genetic structure in 16 trematode species. After accounting for the geographic scale of a study, the number of populations sampled, and the genetic marker used, we found the type of life cycle to be the best predictor of genetic structure (Fst): trematode species bound to complete their life cycle within water showed significantly more pronounced genetic structuring than those leaving water through a bird or mammal host. This finding highlights the dependence of parasites on host traits for their dispersal, suggesting that genetic differentiation of parasites reflects the mobility of their hosts.

Swimming against the current: genetic structure, host mobility and the drift paradox in trematode parasites.

Life‐cycle characteristics and habitat processes can potentially interact to determine gene flow and genetic structuring of parasitic species. In this comparative study, we analysed the genetic structure of two freshwater trematode species with different life histories using cytochrome c oxidase I gene (COI) sequences and examined the effect of a unidirectional river current on their genetic diversity at 10 sites along the river. We found moderate genetic structure consistent with an isolation‐by‐distance pattern among subpopulations of Coitocaecum parvum but not in Stegodexamene anguillae. These contrasting parasite population structures were consistent with the relative dispersal abilities of their most mobile hosts (i.e. their definitive hosts). Genetic diversity decreased, as a likely consequence of unidirectional river flow, with increasing distance upstream in C. parvum, which utilizes a definitive host with only restricted mobility. The absence of such a pattern in S. anguillae suggests that unidirectional river flow affects parasite species differently depending on the dispersal abilities of their most mobile host. In conclusion, genetic structure, genetic diversity loss and drift are stronger in parasites whose most mobile hosts have low dispersal abilities and small home ranges. An additional prediction can be made for parasites under unidirectional drift: those parasites that stay longer in their benthic intermediate host or have more than one benthic intermediate hosts would have relatively high local recruitment and hence increased retention of upstream genetic diversity.

A first insight into the barcodes for African diplostomids (Digenea: Diplostomidae): Brain parasites in Clarias gariepinus (Siluriformes: Clariidae)

Diplostomid trematodes comprise a large and diverse group of widespread digeneans whose larval stages are important parasitic pathogens that may exert serious impacts in wild and cultured freshwater fish. However, our understanding of their diversity remains incomplete especially in the tropics. Our study is the first application of a DNA-based approach to diplostomid diversity in the African continent by generating a database linking sequences for the mitochondrial cytochrome c oxidase subunit 1 (cox1) barcode region and ITS1-5.8S-ITS2 rRNA gene cluster for brain-infecting diplostomid metacercariae from the catfish Clarias gariepinus. Analyses of newly-generated partial cox1 sequences for 34 larval isolates of Tylodelphys spp. from Tanzania and Diplostomum spp. from Tanzania and Nigeria revealed three strongly supported reciprocally monophyletic lineages of Tylodelphys spp. and one of an unknown species of Diplostomum. The average intraspecific divergence for the cox1 sequences for each recognised novel lineage was distinctly lower compared with interspecific divergence (0.46-0.75% vs 11.7-14.8%). The phylogenetic hypotheses estimated from Bayesian inference and maximum likelihood analyses of ITS1-5.8S-ITS2 data exhibited congruent strong support for the cox1-derived lineages. Our study thus provides molecular-based evidence for the existence of three distinct brain-infecting species co-occurring in natural populations of C. gariepinus. Based on phylogenetic analyses, we re-allocated Diplostomum mashonenseBeverley-Burton (1963) to the genus Tylodelphys as a new combination. We also generated cox1 and ITS1-5.8S-ITS2 sequences for an unknown species of Diplostomum from another African fish host, Synodontis nigrita.

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.

Molecular phylogeny of Neotropical monogeneans (Platyhelminthes: Monogenea) from catfishes (Siluriformes)

BackgroundThe phylogenetic relationships of dactylogyrids (Monogenea: Dactylogyridae) parasitising catfishes (Siluriformes) from the Neotropical region were investigated for the first time.MethodsPartial sequences of the 28S rRNA gene of 40 specimens representing 25 dactylogyrid species were analysed together with sequences from GenBank using Bayesian inference, Maximum likelihood and Parsimony methods. Monophyly of dactylogyrids infecting catfishes and the Ancyrocephalinae was evaluated using the Approximately Unbiased test.ResultsThe Ancyrocephalinae is a paraphyletic group of species clustering in three main clades as follows: (i) clade A comprising freshwater dactylogyrids from the Holarctic parasitising perciforms clustering together with species (Ameloblastella, Unibarra and Vancleaveus) parasitising Neotropical catfishes; (ii) clade B including species of Dactylogyrus (Dactylogyrinae) and Pseudodactylogyrus (Pseudodactylogyrinae) along with Ancyrocephalus mogurndae, and marine dactylogyrids with cosmopolitan distribution, parasites of scorpaeniforms and perciforms, along with the freshwater Cichlidogyrus and Scutogyrus (infecting African cichlids [Cichlidae]) and (iii) clade C containing exclusively dactylogyrids of siluriforms, freshwater and marine, with Palaearctic, Ethiopian, Oriental and Neotropical distributions; species of Aphanoblastella and Dactylogyridae gen. sp. 4 from the Neotropical region clustering together with species allocated in the Ancylodiscoidinae, along with species of Cosmetocleithrum, Demidospermus and Dactylogyridae gen. spp.ConclusionsThe position of the Ancylodiscoidinae within a larger clade of dactylogyrids (ancyrocephalines) indicates that this subfamily does not represent a natural group. Instead, species allocated to this clade (dactylogyrids of siluriforms along with species of the Ancylodiscoidinae) should be considered as a separate subfamily within the Dactylogyridae. The erection of this taxon requires the search for morphological diagnostic characters in addition to phylogenetic information. A similar strategy should be considered for a new classification of the paraphyletic Ancyrocephalinae. Members of the three clades do not seem to share obvious morphological synapomorphies nor clear patterns in host-parasite associations, zoogeographical distribution or ecology. Clade A should be considered as the Ancyrocephalinae sensu stricto since it includes the type species Ancyrocephalus paradoxus Creplin, 1839. A new subfamily should be proposed to accommodate species currently allocated to Ancyrocephalinae clustering within clade B. Future attempts to propose a new classification of the subfamilies in the Dactylogyridae should include the phylogenetically diverse Neotropical dactylogyrids.