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

Morphological and genetic differentiation of old world Artemia strains

Recording biodiversity and studying its processes are based on a number of different scientific fields. This task can be greatly facilitated by following multidisciplinary approaches, especially for organisms like Artemia with sexual and parthenogenetic forms. The genus Artemia (addressed by many scientists as “aquatic Drosophila”) can be used as a model system for evaluating different methods assessing biodiversity. This is due to a number of unique characteristics piled within the genus: 1) two types of reproduction (i.e., sexual and parthenogenetic), 2) oviparity (production of cysts) and ovoviviparity (release of nauplii), 3) different ploidy levels in parthenogenetic strains, 4) increased chromosome number in the bisexual species A. persimilis (2n = 44) and 5) distinctive distributions of the different forms. The phylogenetic relationships in the genus Artemia are still not clear. Difficulties in acquiring acceptable karyotypes, different ploidy levels, possible hybridization between A. franciscana and A. persimilis, varying patterns of reproductive isolation in Eastern Old World, cases of sympatry and/or coexistence between sexual and parthenogenetic forms and a plethora of newly discovered populations have made the elucidation of kinship among species an arduous task. Despite the detailed recording of Artemia biodiversity the last few years materialised by the use of multidisciplinary approaches, a robust account of the morphological and genetic differentiation of Old World Artemia populations has been missing. Additionally, there is a need for evaluating available analytical tools as well as new techniques for the characterization of Artemia species and/or strains. The novelty of this work lies within the combined analysis of two molecular markers (16S rDNA and ITS1) and morphometry aiming to characterize Old World Artemia populations. Furthermore, it is the first time that sequencing of ITS1 is used as a potential phylogenetic marker in Artemia population genetics. More specifically, the targets of this study were: 1) The analysis of morphological differentiation and the identification of diagnostic variables majorly responsible for the discrimination of different species/strains. 2) The study of the genetic differentiation and the phylogenetic relationships between Old World Artemia populations using mitochondrial (16S rDNA) and nuclear (ITS1) DNA markers. 3) The evaluation of the utility of the previous markers as tools for characterizing unknown Artemiα populations. 4) The study of biogeographic history and ages of all recognized species in the genus, with emphasis in the origin of parthenogenetic forms.Η περιγραφή της βιολογικής ποικιλότητας (ιδιαίτερα σε γένη που περιλαμβάνουν τόσο αμφιγονικά είδη όσο και παρθενογενετικά στελέχη, όπως η Artemia) δεν μπορεί να στηριχτεί μόνο σε δεδομένα που προέρχονται από ένα πεδίο. Τα τελευταία χρόνια η πολυπαραγοντική προσέγγιση για τη μελέτη των διαδικασιών της βιοποικιλότητας κερδίζει συνεχώς έδαφος. Η Artemia διαθέτει μια σειρά χαρακτηριστικών που δικαία της έχουν δώσει δικαίως το προσωνύμιο «Drosophila του νερού», όπως: 1) η ύπαρξη δυο τύπων αναπαραγωγής (αμφιγονία και παρθενογένεση), 2) εμφάνιση ωοτοκίας (παραγωγή κύστεων) ή ωοζωοτοκίας (παραγωγή ναυπλίων), 3) διαφορετικά επίπεδα πλοειδίας μεταξύ των παρθενογενετικών πληθυσμών, 4) αύξηση ενός αριθμού χρωμοσωμάτων σε ένα αμφιγονικό είδος, 5) εμφάνιση προσαρμογών σε ακραία και εφήμερα περιβάλλοντα, και 6) μικρή διάρκεια γενεάς και μεγάλος αριθμός απογόνων. Τα χαρακτηριστικά αυτά σε συνδυασμό με τη μεγάλη οικονομική σημασία των κύστεων της Artemia στις υδατοκαλλιέργειες και την αναζήτηση νέων «πηγών» την καθιστούν εξαιρετικό υλικό για τη μελέτη των φαινομένων της βιοποικιλότητας. Την τελευταία 20ετία, οι φυλογενετικές σχέσεις στο γένος έχουν περιπλακεί ακόμη περισσότερο. Η δυσκολία στην κατασκευή καρυοτύπων και ο διαφορετικός βαθμός πλοειδίας των παρθενογενετικών στελεχών, η πιθανότητα επιτυχημένου υβριδισμού μεταξύ των A. franciscana και A. persimilis σε περιοχές της Αργεντινής, η ανακάλυψη και προσδιορισμός τριών νέων αμφιγονικών ειδών του Παλαιού Κόσμου (A. sinica και Α. tibetiana και Artemia sp. από το Καζακστάν), η ύπαρξη συμπατρικών αμφιγονικών και παρθενογενετικών πληθυσμών και ο σχετικά μεγάλος αριθμός πληθυσμών που ανακαλύπτεται κάθε χρόνο είναι μερικοί από τους παράγοντες που υποστηρίζουν ότι ο χαρακτηρισμός των πληθυσμών Artemia (απαραίτητος για την εύρεση των φυλογενετικών σχέσεων) γίνεται ολοένα και πιο επίπονος. Παρά την ενδελεχή καταγραφή της βιοποικιλότητας του γένους Artemia και την υιοθέτηση του πολυπαραγοντικού χαρακτηρισμού των πληθυσμών Artemia, απουσιάζει η διερεύνηση των μορφολογικών και γενετικών διαφοροποιήσεων μεταξύ των πληθυσμών Artemia του Παλαιού Κόσμου. Επίσης, αναζητούνται εύκολοι, γρήγοροι, αξιόπιστοι και με μικρό κόστος μέθοδοι χαρακτηρισμού των διαφόρων στελεχών/ειδών. Η πρωτοτυπία της εργασίας έγκειται στην ταυτόχρονη χρήση δυο γενετικών δεικτών (ενός μιτοχονδριακού και ενός πυρηνικού) και της μορφομετρίας των πληθυσμών. Επίσης, είναι η πρώτη φορά που χρησιμοποιείται η πρωτοταγής δομή του ITS1 για την εύρεση των φυλογενετικών σχέσεων των ειδών Artemia και των παρθενογενετικών πληθυσμών. Ο σκοπός της παρούσας ερευνητικής μελέτης συνοψίζεται στα παρακάτω: 1) Μορφομετρική μελέτη των πληθυσμών και χρήση πολυμεταβλητών στατιστικών μεθόδων με σκοπό: α) τον εντοπισμό διαφορών μεταξύ τους, β) πιθανών ομαδοποιήσεων και γ) τον ταυτόχρονο προσδιορισμό εκείνων των μορφομετρικών μεταβλητών που παρέχουν ισχυρό διαχωρισμό μεταξύ των πληθυσμών. 2) Μελέτη της γενετικής διαφοροποίησης και των φυλογενετικών σχέσεων μεταξύ των πληθυσμών Artemia του Παλαιού Κόσμου με τη χρήση ενός μιτοχονδριακού γενετικού δείκτη (16S) και ενός πυρηνικού (ITS1) και τη σύγκριση των αποτελεσμάτων με παλαιότερες εργασίες. 3) Η εκτίμηση των δυο προαναφερθέντων γενετικών δεικτών ως εργαλεία για το χαρακτηρισμό αγνώστων πληθυσμών Artemia. 4) Μελέτη της βιογεωγραφικής ιστορίας και της ηλικίας των ειδών Artemia, με έμφαση στην προέλευση και την εκτίμηση του χρόνου εμφάνισης της παρθενογένεσης στο γένος

Microevolution of the noble crayfish (Astacus astacus) in the Southern Balkan Peninsula

Abstract Background The noble crayfish (Astacus astacus) displays a complex historical and contemporary genetic status in Europe. The species divergence has been shaped by geological events (i.e. Pleistocene glaciations) and humanly induced impacts (i.e. translocations, pollution, etc.) on its populations due to species commercial value and its niche degradation. Until now, limited genetic information has been procured for the Balkan area and especially for the southernmost distribution of this species (i.e. Greece). It is well known that the rich habitat diversity of the Balkan Peninsula offers suitable conditions for genetically diversified populations. Thus, the present manuscript revisits the phylogenetic relationships of the noble crayfish in Europe and identifies the genetic make-up and the biogeographical patterns of the species in its southern range limit. Results Mitochondrial markers (i.e. COI and 16S) were used in order to elucidate the genetic structure and diversity of the noble crayfish in Europe. Two of the six European haplotypic lineages, were found exclusively in Greece. These two lineages exhibited greater haplotypic richness when compared with the rest four (of “Central European” origin) while they showed high genetic diversity. Divergence time analysis identified that the majority of this divergence was captured through Pleistocene, suggesting a southern glacial refugium (Greece, southern Balkans). Furthermore, six microsatellite markers were used in order to define the factors affecting the genetic structure and demographic history of the species in Greece. The population structure analysis revealed six to nine genetic clusters and eight putative genetic barriers. Evidence of bottleneck effects in the last ~5000 years (due to climatic and geological events and human activities) is also afforded. Findings from several other research fields (e.g. life sciences, geology or even archaeology) have been utilized to perceive the genetic make-up of the noble crayfish. Conclusions The southernmost part of Balkans has played a major role as a glacial refugium for A. astacus. Such refugia have served as centres of expansion to northern regions. Recent history of the noble crayfish in southern Balkans reveals the influence of environmental (climate, geology and/or topology) and anthropogenic factors

Additional file 6: of Microevolution of the noble crayfish (Astacus astacus) in the Southern Balkan Peninsula

Fixation indices for all microsatellite loci. Values of the fixation indices for all microsatellite loci based on infinite allele model (FIT, FIS and FST) and stepwise mutation model (RST). (DOC 30 kb

Additional file 15: of Microevolution of the noble crayfish (Astacus astacus) in the Southern Balkan Peninsula

Posteriors and priors densities plots for the past (N1) and present (N0) population sizes of each genetic cluster (cluster 1 to 9) and the global data set (samples from different demes were merged into one dataset; black color). (DOC 859 kb

Additional file 2: of Microevolution of the noble crayfish (Astacus astacus) in the Southern Balkan Peninsula

The modified conditions used for the PCR and amplification of each microsatellite loci are given. (DOC 32 kb

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 36° 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 plasticityThe current work has been performed through the visits of Olga Ruiz and Dra. Alba Papeschi to the Department of Genetics, Development and Molecular Biology (Aristotle University of Thessaloniki) within the framework of the EU project ICA4-CT-2001-10020 (INCO) on Artemia biodiversity.Peer Reviewe

Genetic identification of South African Artemia

Detailed molecular data of Artemia (Crustacea, Anostraca) from South Africa are scarce. Here, we provide for the first time genetic data on the species status and phylogeography of Artemia from this region after an extensive sampling expedition. Our 16S rRNA RFLP analysis of 27 Artemia populations, including seven from South African sites, provides evidence for the presence of a mixed bisexual and parthenogenetic component in this area. Phylogenetic and network analyses reveal that three (out of seven) Artemia populations from South Africa belong to the invasive A. franciscana, two of them belong to A. salina while the last two contain both parthenogenetic and bisexual (A. salina) individuals. From a total of 18 haplotypes identified, seven were recorded from South Africa. To our knowledge, this is the first confirmed report of A. franciscana in this region. Its invasiveness is reaffirmed by evidence for the complete replacement of the native A. salina population from Velddrif Saltworks. However, in other cases, parthenogens seem to modify the capacity of A. franciscana to replace natives. Results on the genetic identification of South African Artemia provide insights into the dynamics of invasion and co-occurrence and highlight effects on species interactions and on biodiversity in inland aquatic invertebrates