Ilias Kappas

Evolution and Speciation

The brine shrimp Artemia comprises a group of bisexual and parthenogenetic, morphologically similar, species very likely to have diverged from an ancestral form living in the Mediterranean area some 5.5 million years ago (Abreu-Grobois and Beardmore, 1982; Abreu-Grobois, 1987; Badaracco et al. 1987), though this estimate, based on allozymes, could be somewhat conservative as compared to that based on mitochondrial DNA (Perez et al. 1994). At that time the area was, according to geological indications, the only place in the world exhibiting for significant periods of time the very high salinity required for Artemia to thrive (Abreu-Grobois, 1987; Badaracco et al. 1987). The hypothesis of the Mediterranean as the centre of radiation for Artemia is also supported by the diversity of Artemia types currently found in the area, i.e. bisexuality and parthenogenesis on the one hand, together with diploidy and polyploidy on the other (see below).

Is polyploidy a persevering accident or an adaptive evolutionary pattern? The case of the brine shrimp Artemia

Asexual organisms are confronted with substantial drawbacks, both immediate and delayed, threatening their evolutionary persistence. Yet, genetic associations with asexuality may refresh the gene pool promoting adaptation of clonal lineages; polyploidy is one of them. Parthenogenesis itself and/or polyploidy are responsible for the maintenance and spread of clones in Artemia, a sexual-asexual genus of halophilic anostracans. We applied flow cytometry, microsatellite genotyping, and mtDNA sequencing to 23 asexual populations. Artemia parthenogens have evolved multiple times either through hybridization or spontaneously. Nine out of 23 populations contained clones of mixed ploidy (2n, 3n, 4n). Most clones were diploid (20/31) while two and nine clones were triploid and tetraploid, respectively. Apomictic triploids and tetraploids formed two distinct groups of low genetic diversity compared with the more divergent automictic diploids. Polyploidy is also polyphyletic in Artemia, with triploids and tetraploids having independent origins from different sexual ancestors. We discern a pattern of geographical parthenogenesis with all clonal groups being more widespread than their closest sexuals. In favour of a specialist model, asexual diploids are restricted to single locations and are strikingly segregated from generalist triploids and tetraploids occupying a variety of sites. This is a rare pattern of mixed life-history strategies within an asexual complex.

Porous genomes and species integrity in the branchiopod Artemia

Over the years, studies on interspecific hybridization have highlighted cases where gene exchange between taxa continues for a significant amount of time after speciation. The reasons for this lag of reproductive isolation relative to genetic isolation are largely unclear, and the question still remains whether the resulting hybrids represent novel biological (and taxonomic) diversity or merely an evolutionary liability. We provide strong indications in the branchiopod Artemia that hybrids between distantly related species may not be evolutionary inconsequential. Based on a global sampling of published and newly derived nuclear (ITS1) and mitochondrial (16S rRNA) sequence data from all representatives of the genus, we have identified natural hybrids between Artemia species (A. persimilisxA. franciscana, A. salinaxA. franciscana) separated by evolutionary interludes of tens of millions of years. Our combined analytical framework of cladistic and network methods provides evidence that hybridizations are the result of recent secondary contact following pronounced allopatric differentiation. The detection of mitochondrial introgression from A. persimilis to A. franciscana attests F(1) hybrid fertility. The reasons for this apparent unidirectionality of introgression are currently unknown but a likely explanation is provided based on morphometric divergence. We discuss the evolutionary implications of our results within the broader context of continental zooplankters.

Sharp Phylogeographic Breaks and Patterns of Genealogical Concordance in the Brine Shrimp Artemia franciscana

Genealogical concordance is a critical overlay of all phylogenetic analyses, irrespective of taxonomic level. To assess such patterns of congruence we have compiled and derived sequence data for two mitochondrial (16S rRNA, COI) and two nuclear (ITS1, p26) markers in 14 American populations of the hypersaline branchiopod Artemia franciscana. Cladistic analysis revealed three reciprocally monophyletic mitochondrial clades. For nuclear DNA, incomplete lineage sorting was evident presumably as a result of slower coalescence or male-mediated dispersal. Our findings capture the genealogical interval between gene splitting and population divergence. In this sense, strong indications are provided in favour of a superspecies status and ongoing speciation in A. franciscana.

Identification of Cultured Brachionus Rotifers Based on RFLP and SSCP Screening

The marine finfish industry worldwide depends greatly on the mass culture of Brachionus rotifers. Recently, molecular data have revealed a more complicated view about the species status of Brachionus rotifers than previous mainly morphological assessments. Under this view, Brachionus rotifers are comprised of many morphologically similar, albeit genetically differentiated, cryptic members of larger groups. A redefinition of the cultured rotifer species/biotypes is therefore needed if aquaculture is to reach higher levels of standardization and predictability. In this work, restriction fragment length polymorphism (RFLP) and single-strand conformational polymorphism (SSCP) methods are applied to the COI and 16S rRNA mitochondrial genes. A detailed COI restriction map was constructed, using sequence data from all known representatives of Brachionus phylogroups. Therefore, it is the first time that such an extended restriction database has been produced. Several restriction endonucleases are proposed for the discrimination of the different Brachionus species/biotypes. Furthermore, eight different SSCP gel alleles are described for the 16S region. Using these data, five Brachionus species/biotypes were identified in 78 samples collected from laboratories and hatcheries around the world.

Morphological and taxonomic demarcation of Brachionus asplanchnoidis Charin within the Brachionus plicatilis cryptic species complex (Rotifera, Monogononta)

Three well-defined groups, consisting of 15 species, have recently been ascribed to organisms historically identified as the Brachionus plicatilis species complex. One of these groups, the large clade, is composed of two named species (Brachionus plicatilis s.s. and Brachionus manjavacas) and two species identifiers (B. ‘Nevada’ and B. ‘Austria’). B. ‘Austria’ has been confirmed to be B. asplanchnoidis. As no type specimen exists for this species, and the original taxonomic description is lacking in detail, we give a detailed account of this species using material from Obere Halbjochlacke in Austria where B. ‘Austria’ was first identified genetically. Our analysis of B. asplanchnoidis populations was of global scope, an approach that revealed a great degree of morphological variability. However, combining aspects of both the dorsal and ventral surfaces clearly discriminated B. asplanchnoidis from the rest of the large-type members. This approach may prove useful in taxonomic studies of other cryptic species with relatively few morphological features. We also observed a geographic pattern of genetic divergence within B. asplanchnoidis. Average uncorrected COI divergences for a 554-bp fragment of the COI gene ranged from 3.9% within species to 17.5% between species of the large clade and indicate deep divisions within the cryptic species complex.

A Multiplex PCR Method for Rapid Identification of Brachionus Rotifers

Cryptic species are increasingly being recognized in many organisms. In Brachionus rotifers, many morphologically similar yet genetically distinct species/biotypes have been described. A number of Brachionus cryptic species have been recognized among hatchery strains. In this study, we present a simple, one-step genetic method to detect the presence of those Brachionus sp. rotifers that have been found in hatcheries. With the proposed technique, each of the B. plicatilis sensu stricto, B. ibericus, Brachionus sp. Nevada, Brachionus sp. Austria, Brachionus sp. Manjavacas, and Brachionus sp. Cayman species and/or biotypes can be identified with polymerase chain reaction (PCR) analysis. Based on 233 cytochrome c oxidase subunit I sequences, we reviewed all the available cryptic Brachionus sp. genetic polymorphisms, and we designed six nested primers. With these primers, a specific amplicon of distinct size is produced for every one of the involved species/biotypes. Two highly sensitive protocols were developed for using the primers. Many of the primers can be combined in the same PCR. The proposed method has been found to be an effective and practical tool to investigate the presence of the above six cryptic species/biotypes in both individual and communal (bulk) rotifer deoxyribonucleic acid extractions from hatcheries. With this technique, hatchery managers could easily determine their rotifer composition at the level of cryptic species and monitor their cultures more efficiently.

A Time-Calibrated Mitogenome Phylogeny of Catfish (Teleostei: Siluriformes)

A very significant part of the world’s freshwater ichthyofauna is represented by ancient, exceptionally diverse and cosmopolitan ray-finned teleosts of the order Siluriformes. Over the years, catfish have been established as an exemplary model for probing historical biogeography at various scales. Yet, several tantalizing gaps still exist in their phylogenetic history, timeline and mode of diversification. Here, we re-examine the phylogeny of catfish by assembling and analyzing almost all publicly available mitogenome data. We constructed an ingroup matrix of 62 full-length mitogenome sequences from 20 catfish families together with four cypriniform outgroups, spanning 15,557 positions in total. Partitioned maximum likelihood analyses and Bayesian relaxed clock dating using fossil age constraints provide some useful and novel insights into the evolutionary history of this group. Loricarioidei are recovered as the first siluriform group to diversify, rendering Neotropics the cradle of the order. The next deepest clade is the South American Diplomystoidei placed as a sister group to all the remaining Siluroidei. The two multifamilial clades of “Big Asia” and “Big Africa” are also recovered, albeit nodal support for the latter is poor. Within “Big Asia”, Bagridae are clearly polyphyletic. Other interfamilial relationships, including Clariidae + Heteropneustidae, Doradidae + Auchenipteridae and Ictaluridae + Cranoglanididae are robustly resolved. Our chronogram shows that siluriforms have a Pangaean origin, at least as far back as the Early Cretaceous. The inferred timeline of the basal splits corroborates the “Out-of-South America” hypothesis and accords well with the fossil record. The divergence of Siluroidei most likely postdated the final separation of Africa and South America. An appealing case of phylogenetic affinity elaborated by biogeographic dispersal is exemplified by the Early Paleogene split between the Southeast Asian Cranoglanididae and Ictaluridae, with the latter radiating into North America’s freshwater realm by Eocene. The end of Cretaceous probably concludes the major bout of diversification at the family level while with the dawn of the Cenozoic a prolific radiation is evident at the generic level.

Genetic characterization of Argentinean Artemia species with different fatty acid profiles

The Anostracan genus Artemia is composed by several sibling species reproductively isolated, but identical or very similar in outward appearance. The genus shows also an underlying striking variability from the biochemical point of view, regarding especially the fatty acid profile of the cysts and nauplii. In Argentina, Artemia is represented by two bisexual species: A. franciscana and A. persimilis. Former studies have shown that A. franciscana is present in northern of 36º and that A. persimilis is constrained southwards of 37° S. In general, there is good agreement between morphological and cytogenetic comparisons of Argentinean populations with respect to species discrimination. However, new Argentinean Artemia populations are being analyzed morphologically and it becomes necessary to further investigate if the genetic adscription of these populations is congruent with the results obtained from the current morphological analyses. Restriction fragment length polymorphism (RFLP) analysis of a fragment of the 16S rRNA mitochondrial gene was used to investigate the genetic diversity and population structure of 10 new Artemia populations from Argentina. The mitochondrial DNA (mtDNA) results showed a similar pattern to that of previous cytogenetic and morphological analyses with the two Argentinean species appearing as highly divergent. The presence of A. persimilis in southern Argentina and the southernmost Chilean population was confirmed unveiling a novel picture of species distribution in the country. A. franciscana showed a unique haplotype. Populations of A. persimilis appeared highly structured, although their clustering did not follow a clear geographic pattern. The different Argentinean Artemia populations analyzed were characterized by high variability in their fatty acids, showing both marine- and freshwater-type profiles. For the first time, the investigation of the relatedness between the fatty acid composition in Artemia and genetic markers was attempted. The study aimed at the putative association of molecular markers with marine versus freshwater-type populations. A lack of correlation between RFLP patterns at mtDNA and the fatty acid (FA) profiles was found in the A. persimilis populations which was discussed from the point of view of two main genetic hypotheses and/or phenotypic plasticity.

Molecular and morphological data suggest weak phylogeographic structure in the fairy shrimp Streptocephalus torvicornis (Branchiopoda, Anostraca)

Inland aquatic organisms almost ubiquitously display a pattern of marked provincialism characterized by substantial population differentiation and genealogical discontinuities. This is the result of strong priority effects and local adaptation following dispersal and colonization of new habitats. We present a case that defies this biogeographic paradigm. We have investigated the phylogeography of the fairy shrimp Streptocephalus torvicornis across its circum-Mediterranean and Eurasian distribution. Based on three independent datasets, namely sequence variation at 12S and 16S rRNA, cyst morphology and male second antenna characters, we discern a pattern of extensive genetic and morphological homogeneity pointing to unhindered gene flow and widespread connectivity among populations. These intriguing findings may provisionally be explained by (i) a high dispersal frequency overwhelming the ability of a population to maintain resource monopolization, (ii) an outbreeding vigour opportunity offered to secondary immigrants, (iii) an ecological equivalence of genotypes generating long-term immigration–extinction equilibria and buffering genetic diversity over spatial scales, (iv) enhanced bird-mediated dispersal in open habitats as opposed to ponds surrounded by forests or shrub, or (v) a shallow population history with little time for substantial genetic differentiation.