Diversity and origin of freshwater amphipods of Mediterranean islands

The Mediterranean islands are considered to be natural laboratories of evolution and places with extremely high level of endemism. Even though the fresh waters are among most diverse and also the most endangered ecosystems, still little is known about its biodiversity in the Mediterranean islands, as most of the studies on insular fauna focus mainly on terrestrial and marine biota. One of the most abundant organisms, being often keystone species in the freshwater macroinvertebrate communities are gammarid amphipods. In my PhD thesis, I investigated the diversity and origin of the freshwater gammarids of the Mediterranean islands. In the first part of my PhD, the available resources on the diversity of the freshwater malacostracan crustaceans from the Mediterranean islands were investigated and gathered together. Chapter I, is arguably the first such an extensive study on the freshwater fauna of the Mediterranean Islands, with valuable insight on its biogeographical affiliations. The findings indicate that amphipods are the most speciose group, being the most species-rich order on each of studied islands and archipelagos, with also one of the highest rate of endemism. In the main core of the PhD thesis, the diversity and origin of the freshwater gammarids from Aegean islands including Crete as well as Sicily were studied by gathering both morphological and molecular characteristics and by reconstructing the time-calibrated phylogenies using multimarket dataset. Chapter II provides the first evidence of the presence of freshwater populations of Gammarus on six Aegean islands with at least three endemic species, most probably new to science. The molecular methods were implemented to analyse their evolutionary history combining the information from the adjacent regions, including populations of recently described Gammarus plaitisi from Crete. Moreover, the divergence of these new species is strongly connected with the geological history of the Aegean region and its islands. In Chapter III, the substantial level of intraspecific diversity was detected within each of the Sicilian gammarid morphospecies. Moreover, these results support the different timescales and separate colonisation events of the gammarid fauna of Sicily. Altogether, these results indicate connectivity of the evolutionary history of the insular freshwater gammarids with the geological history of the islands as well as the entire Mediterranean region. Moreover, the level of overlooked diversity detected supports the importance of using molecular tools in the biodiversity assessments. The results of this thesis also highlight the need for future studies on the insular freshwater Mediterranean biota and deliver a valuable insight for better understanding of the mechanisms of the diversification of the freshwater organisms.Narodowe Centrum Nauki - projekt nr 2015/17/N/NZ8/01628 oraz 2018/28/T/NZ8/0002

Revisiting the past − novel insight into intraspecific molecular diversity and phylogeny of widespread

Echinogammarus berilloni (Catta, 187

Dragonflies (odonata) of high mountain habitats

Celem tego opracowania jest charakterystyka odonatofauny gór oraz analiza dotycząca zmian w różnorodności gatunkowej ważek na obszarach górskich, w różnych częściach świata. Ważki występują na wszystkich kontynentach oprócz Antarktydy, a największa ich różnorodność występuje w strefach tropikalnych oraz orientalnych. Jednakże skamieniałości odnalezione na Antarktydzie wskazują na to, że 150-200 mln lat temu ważki były tam obecne. Ze względu na trudne warunki środowiskowe panujące na dużych wysokościach, ważki siedlisk wysokogórskich wykształciły szereg przystosowań, dzięki którym mogą rozwijać się i rozmnażać na wysokości nawet 5000 m n.p.m. Nie byłoby to jednak możliwe, gdyby na takiej wysokości nie było żadnego zbiornika wodnego, a to ze względu na to, że ważki są owadami dwuśrodowiskowymi. Tak więc podstawowym czynnikiem ograniczającym ich występowanie jest brak dostępu do wody. Najwyższym jak dotąd rekordem wysokości, na jakiej znaleziono ważkę pozostaje wysokość 6300 m n.p.m., na której w Himalajach znaleziono pojedynczego osobnika z powszechnie występującego gatunku Pantala flavescens (Fabricius, 1798). W rozmieszczeniu ważek zasiedlających pasma górskie w różnych częściach świata widoczne są zarówno pewne różnice jak i podobieństwa. Na każdym kontynencie liczba gatunków maleje wraz ze wzrostem wysokości bezwzględnej. Jednakże skład gatunkowy ważek występujących na określonych wysokościach różni się w zależności od kontynentu oraz pasma górskiego. Tak więc np. na wysokości 3500 m n.p.m. inne gatunki są obecne w Andach, a inne w Himalajach czy Kordylierach. Różnice te wynikają przede wszystkim z położenia tych łańcuchów górskich w różnych strefach klimatycznych i na różnych kontynentach, co przekłada się na skład gatunkowy w danym rejonie.The aim of this article is to characterize the odonatofauna in the mountainous areas and to examine variations in the diversity of Odonata species in different regions of the world. Dragonflies are widespread on all continents except of Antarctica. However, the fossils found in Antarctica suggest that dragonflies were there present 150-200 million years ago. Their diversity reaches its peak in the tropics and in the Oriental regions. Due to the hard environmental conditions present at high altitudes, dragonflies of high mountain habitats have developed a number of adaptations that enable their development and reproduction even at the height of 5000 m. Dragonflies are merolimnic, so the main factor limiting their occurrence is the lack of water. The highest altitude at which any dragonfly was found was in Himalayas, where at 6300 m a single individual of the common species Pantala flavescens (Fabricius, 1798) was collected. In the distribution of the odonatofauna in high mountain habitats in different world regions there occur both some differences and similarities. On the every continent, with an increase in the altitude the number of species diminishes. However, the diversity of dragonflies occurring at certain heights varies depending on the continent and the mountain chain. So, for example, at the altitude of 3500 m different species are present in the Andes Himalayas, and Cordillera. These differences are mainly due to location of the mountain ranges in different climate zones and on different continents

First endemic freshwater Gammarus from Crete and its evolutionary history—an integrative taxonomy approach

The Mediterranean islands are known as natural laboratories of evolution with a high level of endemic biodiversity. However, most biodiversity assessments have focused mainly on terrestrial and marine fauna, leaving the freshwater animals aside. Crete is one of the largest islands in the Mediterranean Basin, with a long history of isolation from the continental mainland. Gammarid amphipods are often dominant in macrozoobenthic communities in European inland waters. They are widely used in biomonitoring and exotoxicological studies. Herein, we describe Gammarus plaitisi sp. nov., endemic to Cretan streams, based on morphological characters and a set of molecular species delimitation methods using mitochondrial cytochrome oxidase subunit I and 16S rRNA genes as well as nuclear 28S rDNA, ITS1 and EF1-alpha genes. The divergence of the new species is strongly connected with the geological history of the island supporting its continental origin

Morphology, nuclear SNPs and mate selection reveal that COI barcoding overestimates species diversity in a Mediterranean freshwater amphipod by an order of magnitude

DNA sequence information has revealed many morphologically cryptic species worldwide. For animals, DNA-based assessments of species diversity usually rely on the mitochondrial cytochrome c oxidase subunit I (COI) gene. However, a growing amount of evidence indicate that mitochondrial markers alone can lead to misleading species diversity estimates due to mito-nuclear discordance. Therefore, reports of putative species based solely on mitochondrial DNA should be verified by other methods, especially in cases where COI sequences are identical for different morphospecies or where divergence within the same morphospecies is high. Freshwater amphipods are particularly interesting in this context because numerous putative cryptic species have been reported. Here, we investigated the species status of the numerous mitochondrial molecular operational taxonomic units (MOTUs) found within Echinogammarus sicilianus. We used an integrative approach combining DNA barcoding with mate selection observations, detailed morphometrics and genome-wide double digest restriction site-associated DNA sequencing (ddRAD-seq). Within a relatively small sampling area, we detected twelve COI MOTUs (divergence = 1.8–20.3%), co-occurring in syntopy at two-thirds of the investigated sites. We found that pair formation was random and there was extensive nuclear gene flow among the ten MOTUs co-occurring within the same river stretch. The four most common MOTUs were also indistinguishable with respect to functional morphology. Therefore, the evidence best fits the hypothesis of a single, yet genetically diverse, species within the main river system. The only two MOTUs sampled outside the focal area were genetically distinct at the nuclear level and may represent distinct species. Our study reveals that COI-based species delimitation can significantly overestimate species diversity, highlighting the importance of integrative taxonomy for species validation, especially in hyperdiverse complexes with syntopically occurring mitochondrial MOTUs

Double origin of the racer goby (Babka gymnotrachelus) in Poland revealed with mitochondrial marker. Possible implications for the species alien/native status

In mid-1990s racer goby, Babka gymnotrachelus, penetrated to the Vistula and Western Bug rivers in Poland through the canal connecting the Baltic and the Black Sea basins, namely the Vistula and Dnieper drainages. In early 2000s, the species was reported from Polish section of the Strwiąż River that is an affluent of the Dniester River that drains to the Black Sea basin. According to the Polish legislation, the racer goby has been enlisted in as an alien invasive species that may pose threat to local biota. Our analysis of the mtDNA cytochrome b diversity revealed that the Vistula/Western Bug and Strwiąż populations are different genetic units. First one originated from the Dnieper River, while the second derives from the Dniester River. According to the results of mismatch analysis, both are in the stage of demographic and spatial expansion. The haplotype frequencies in population from the Vistula/Western Bug differ significantly from those in the source population in Dnieper, suggesting founder effect, possibly due to human-mediated introduction of low number of individuals. On the other side, the population in Strwiąż does not differ in structure from the one in Dniester, providing a hint towards spontaneous range expansion. Interpretation of our results in light of historical data lead to the conclusion that presence of racer goby in Strwiąż was probably overlooked in previous, spatially limited, studies. Thus, in Strwiąż the species should not be treated as alien, contrary to its status in the Vistula drainage. This double origin of racer goby populations in Poland creates a peculiar situation for national legislation procedures as one population is alien and invasive, while the other one is not. In light of our findings, the population from Strwiąż should be recognised as a special case. Steps should be undertaken to raise public awareness to prevent translocation of the gobies between the rivers to prevent deterioration of the evolutionarily isolated genetic pools of the Dnieper and of the Dniester basins. Our results illustrate the need for case studies upon genetic population structure, following appearance of new species in previously unoccupied water bodies, even on local scale. </p

Rapid development of increased neonicotinoid tolerance in non-target freshwater amphipods

The comprehensive assessment of the long-term impacts of constant exposure to pollutants on wildlife populations remains a relatively unexplored area of ecological risk assessment. Empirical evidence to suggest that multigenerational exposure affects the susceptibility of organisms is scarce, and the underlying mechanisms in the natural environment have yet to be fully understood. In this study, we first examined the arthropod candidate species, Gammarus roeselii that – unlike closely related species – commonly occurs in many contaminated river systems of Central Europe. This makes it a suitable study organism to investigate the development of tolerances and phenotypic adaptations along pollution gradients. In a 96-h acute toxicity assay with the neonicotinoid thiacloprid, we indeed observed a successive increase in tolerance in populations coming from contaminated regions. This was accompanied by a certain phenotypic change, with increased investment into reproduction. To address the question of whether these changes are plastic or emerged from longer lasting evolutionary processes, we conducted a multigeneration experiment in the second part of our study. Here, we used closely-related Hyalella azteca and pre-exposed them for multiple generations to sublethal concentrations of thiacloprid in a semi-static design (one week renewal of media containing 0.1 or 1.0 µg/L thiacloprid). The pre-exposed individuals were then used in acute toxicity assays to see how quickly such adaptive responses can develop. Over only two generations, the tolerance to the neonicotinoid almost doubled, suggesting developmental plasticity as a plausible mechanism for the rapid adaptive response to strong selection factors such as neonicotinoid insecticides. It remains to be discovered whether the plasticity of rapidly developed tolerance is species-specific and explains why closely related species – which may not have comparable adaptive response capabilities – disappear in polluted habitats. Overall, our findings highlight the neglected role of developmental plasticity during short- and long-term exposure of natural populations to pollution. Moreover, our results show that even pollutant levels seven times lower than concentrations found in the study region have a clear impact on the developmental trajectories of non-target species

Conservation of freshwater biodiversity in Tunisia in a climate change context: combining amphipod distribution data and molecular analyses to improve priorities

International audienceThe exceptional diversity of freshwater fauna of the Mediterranean Basin currently faces a crisis in which climate change combined with overexploitation of freshwaters heavily threatens the local fauna. In this context, it is urgent to define conservation priorities on how to best protect freshwater biodiversity. One of the main limits to define such actions remains the lack of knowledge in many countries. In this study, we test the usefulness of molecular data (COI gene) combined with morphological identification to better predict the pattern of biological diversity and threats of climate change on freshwater biodiversity. We focused our study on the freshwater amphipods as model organisms in order to define conservation strategies in Tunisia, one of the most threatened countries. Our results confirmed that amphipods diversity is largely underestimated with nine species identified by their morphology and 33-39 species assigned depending on delimited with the most parsimonious molecular delimitation method. The distribution of amphipods is mainly restricted to the northern part of Tunisia and seems to be positively correlated with precipitation and negatively correlated with thermal amplitudes and precipitation fluctuations. These environmental factors are sensitive to climate change and confirm that conservation strategies need to be redefined and adjusted in the face of future climate predictions. Moreover, the total diversity and spatial distribution patterns provided by molecular methods seem to be more detailed and accurate than results based on morphology alone and nicely complement traditional species assignment