Federico Marrone

How do freshwater organisms cross the “dry ocean”? A review on passive dispersal and colonization processes with a special focus on temporary ponds

Lakes and ponds are scattered on Earth’s surface as islands in the ocean. The organisms inhabiting these ecosystems have thus developed strategies to pass the barrier represented by the surrounding land, to disperse and to colonize new environments. The evidences of a high potential for passive long-range dispersal of organisms producing resting stages inspired the idea that there were no real barriers to their actual dispersal, and that their distribution was only limited by the ecological characteristics of the available habitats. The development of genetic techniques allowed to criticize this view and revealed the existence of a more complex and diverse biological scenario governed by an assortment of historical and ecological factors. In this paper, we review the literature related to the passive dispersal of organisms producing resting stages among inland lentic ecosystems, with special emphasis to temporary ponds, which represent “isolated” ecosystems both in space and in time, and are characterized by high levels of biological diversity. The existence of a sharp decoupling between “dispersal potential” and “actual establishment rates” is stressed, thus urging a definitive overcome of the so-called “Everything is Everywhere” hypothesis in order to gain a proper understanding of the biogeography and ecology of inland water organisms.

Sister species within the Triops cancriformis lineage (Crustacea, Notostraca)

We investigated the phylogenetic relationships among the three presently recognized subspecies of the tadpole shrimp, Triops cancriformis, using mitochondrial 16S and 12S rDNA sequences. Our results indicate that the taxon is divided into two distinct lineages. One lineage is formed of T. c. cancriformis populations and samples from northern Spain that had been classified as T. c. simplex in the most recent literature. The second lineage comprises all populations of T. c. mauritanicus and northern African populations of T. c. simplex. These two main lineages separated 2.3 to 8.9 million years ago, based on the range of inferred molecular clocks recognized for crustacean mtDNA sequence divergence. Percentages of divergence are in the range reported for recognized species in other notostracan lineages and we therefore propose to recognize them as two species, Triops cancriformis and Triops mauritanicus. The latter would comprise two subspecies in northern Africa, one consisting of the Moroccan populations of the former T. c. mauritanicus, the other comprising the African populations of the former T. c. simplex. It also includes three as‐yet unnamed lineages. A comparison of morphological characters with the molecular data revealed that the former T. c. simplex cannot be reliably separated from T. c. cancriformis, using morphological characters that have hitherto been used to distinguish among subspecies of T. cancriformis. Our investigation is the first to demonstrate the presence of T. c. cancriformis in Africa (Tunisia). The genetic haplotypes of these populations are identical with haplotypes also occurring in Central and Western Europe, as well as in Sicily. Therefore, we hypothesize that the African populations of T. c. cancriformis represent a result of repeated long‐distance dispersal across the Mediterranean Sea.

Ecological characterization and cladocerans, calanoid copepods and large branchiopods of temporary ponds in a Mediterranean island (Sicily, southern Italy)

Temporary waters have been sporadically investigated in Sicily. These environments reflect the climatic features of the Mediterranean area with a winter ponding phase and a more or less prolonged dry period in summer. Their biota, especially those organisms strictly linked to aquatic environments and without any terrestrial life stage, have to exhibit special adaptations to survive the dry phases that are recurrent in such ecosystems. This study included more than 250 water bodies distributed on the whole Sicilian territory and on the small circum-Sicilian islands. This paper represents a first attempt to characterize Sicilian temporary waters from an ecological point of view and is mainly based on some of their limnological features and on the crustacean components of their communities. In particular, the groups which have been taken into consideration are those of cladocerans, calanoid copepods, and large branchiopods (notostracans, anostracans, and spinicaudatans).

Where are you from, stranger? The enigmatic biogeography of North African pond turtles (Emys orbicularis)

The European pond turtle (Emys orbicularis) is a Nearctic element in the African fauna and thought to have invaded North Africa from the Iberian Peninsula. All North African populations are currently identified with the subspecies E. o. occidentalis. However, a nearly range-wide sampling in North Africa used for analyses of mitochondrial and microsatellite DNA provides evidence that only Moroccan populations belong to this taxon, while eastern Algerian and Tunisian pond turtles represent an undescribed distinct subspecies. These two taxa are most closely related to E. o. galloitalica with a native distribution along the Mediterranean coast of northern Spain through southern France to western and southern Italy. This group is sister to a clade comprising several mitochondrial lineages and subspecies of E. orbicularis from Central and Eastern Europe plus Asia, and the successive sisters are E. o. hellenica and E. trinacris. Our results suggest that E. orbicularis has been present in North Africa longer than on the Iberian Peninsula and that after an initial invasion of North Africa by pond turtles from an unknown European source region, there was a phase of diversification in North Africa, followed by a later re-invasion of Europe by one of the African lineages. The differentiation of pond turtles in North Africa parallels a general phylogeographic paradigm in amphibians and reptiles, with deeply divergent lineages in the western and eastern Maghreb. Acknowledging their genetic similarity, we propose to synonymize the previously recognized Iberian subspecies E. o. fritzjuergenobsti with E. o. occidentalis sensu stricto. The seriously imperiled Moroccan populations of E. o. occidentalis represent two Management Units different in mitochondrial haplotypes and microsatellite markers. The conservation status of eastern Algerian pond turtles is unclear, while Tunisian populations are endangered. Considering that Algerian and Tunisian pond turtles represent an endemic taxon, their situation throughout the historical range should be surveyed to establish a basis for conservation measures.

Molecular phylogeny of the Notostraca

We used a combined analysis of one nuclear (28S rDNA) and three mitochondrial markers (COI, 12S rDNA, 16S rDNA) to infer the molecular phylogeny of the Notostraca, represented by samples from the six continents that are inhabited by this group of branchiopod crustaceans. Our results confirm the monophyly of both extant notostracan genera Triops and Lepidurus with good support in model based and maximum parsimony analyses. We used branchiopod fossils as a calibration to infer divergence times among notostracan lineages and accounted for rate heterogeneity among lineages by applying relaxed-clock models. Our divergence date estimates indicate an initial diversification into the genera Triops and Lepidurus in the Mesozoic, most likely at a minimum age of 152.3-233.5 Ma, i.e., in the Triassic or Jurassic. Implications for the interpretation of fossils and the evolution of notostracan morphology are discussed. We further use the divergence date estimates to formulate a biogeographic hypothesis that explains distributions of extant lineages predominantly by overland dispersal routes. We identified an additional hitherto unrecognised highly diverged lineage within Lepidurus apus lubbocki and three additional previously unknown major lineages within Triops. Within T. granarius we found deep differentiation, with representatives distributed among three major phylogenetic lineages. One of these major lineages comprises T. cancriformis, the T. mauritanicus species group and two hitherto unrecognised T. granarius lineages. Samples that were morphologically identified as T. granarius diverged from the most basal nodes within this major lineage, and divergence dates suggested an approximate age of 23.7-49.6 Ma for T. cancriformis, indicating the need for a taxonomic revision of Triassic and Permian fossils that are currently attributed to the extant T. cancriformis. We thus elevate T. cancriformis minor to full species status as Triops minorTrusheim, 1938 and include in this species the additional Upper Triassic samples that were attributed to T. cancriformis. We further elevate T. cancriformis permiensis to full species status as Triops permiensisGand et al., 1997.

Differences in gene flow in a twofold secondary contact zone of pond turtles in southern Italy (Testudines: Emydidae:Emys orbicularis galloitalica,E. o. hellenica,E. trinacris)

Using virtually range‐wide sampling for three pond turtle taxa (Emys orbicularis galloitalica, E. o. hellenica, E. trinacris), we analyse gene flow across their southern Italian contact zone. Based on population genetic analyses of 15 highly polymorphic microsatellite loci and a mitochondrial marker, we show that the general genetic pattern matches well with the current taxon delimitation. Yet, single individuals with conflicting genetic identity suggest translocation of turtles by humans. In addition, we identify in south‐western France and the vicinity of Rome populations being heavily impacted by introduced turtles. Cline analyses reveal that the major genetic break between E. o. galloitalica and E. o. hellenica corresponds well with the currently accepted intergradation zone in southern Italy. However, introgression is largely unidirectional from E. o. galloitalica into E. o. hellenica. In the distribution range of the latter subspecies, genetic footprints of E. o. galloitalica are evident along most of the Italian east coast. Our results corroborate that E. o. galloitalica was introduced long ago in Corsica and Sardinia and naturalized there. Gene flow between E. orbicularis and E. trinacris is negligible, with the Strait of Messina matching well with the narrow cline centre between the two species. This contrasts with other Mediterranean freshwater turtle species with extensive transoceanic gene flow. Compared to the two subspecies of E. orbicularis, the Sicilian E. trinacris shows an unexpectedly strong population structuring, a finding also of some relevance for conservation. The differences between the two taxon pairs E. orbicularis/E. trinacris and E. o. galloitalica/E. o. hellenica support their current taxonomic classification and make them attractive objects for follow‐up studies to elucidate the underlying mechanisms of speciation by comparing their properties.

Mitochondrial DNA Regionalism and Historical Demography in the Extant Populations of Chirocephalus kerkyrensis (Branchiopoda: Anostraca)

Background Mediterranean temporary water bodies are important reservoirs of biodiversity and host a unique assemblage of diapausing aquatic invertebrates. These environments are currently vanishing because of increasing human pressure. Chirocephalus kerkyrensis is a fairy shrimp typical of temporary water bodies in Mediterranean plain forests and has undergone a substantial decline in number of populations in recent years due to habitat loss. We assessed patterns of genetic connectivity and phylogeographic history in the seven extant populations of the species from Albania, Corfu Is. (Greece), Southern and Central Italy. Methodology/Principal Findings We analyzed sequence variation at two mitochondrial DNA genes (Cytochrome Oxidase I and 16s rRNA) in all the known populations of C. kerkyrensis. We used multiple phylogenetic, phylogeographic and coalescence-based approaches to assess connectivity and historical demography across the whole distribution range of the species. C. kerkyrensis is genetically subdivided into three main mitochondrial lineages; two of them are geographically localized (Corfu Is. and Central Italy) and one encompasses a wide geographic area (Albania and Southern Italy). Most of the detected genetic variation (≈81%) is apportioned among the aforementioned lineages. Conclusions/Significance Multiple analyses of mismatch distributions consistently supported both past demographic and spatial expansions with the former predating the latter; demographic expansions were consistently placed during interglacial warm phases of the Pleistocene while spatial expansions were restricted to cold periods. Coalescence methods revealed a scenario of past isolation with low levels of gene flow in line with what is already known for other co-distributed fairy shrimps and suggest drift as the prevailing force in promoting local divergence. We recommend that these evolutionary trajectories should be taken in proper consideration in any effort aimed at protecting Mediterranean temporary water bodies.

Molecular evidence for the presence of cryptic evolutionary lineages in the freshwater copepod genus Hemidiaptomus G.O. Sars, 1903 (Calanoida, Diaptomidae)

The pattern of morphological and mtDNA cytochrome b diversity of three calanoid copepod species belonging to the diaptomid genus Hemidiaptomus has been investigated with the aim of checking the reliability of the morphological characters currently used for species identification, and the possible presence of cryptic taxa. A sharply different molecular structuring has been observed in the studied species: while Hemidiaptomus amblyodon exhibits a remarkable constancy throughout the European range of its distribution area (maximum inter-populations cytochrome b divergence of 3%), observed distances between presumed conspecific lineages of Hemidiaptomus gurneyi (maximum divergence of 21.5%) and Hemidiaptomus ingens (maximum 19.1%) suggest that under these binomens are in fact included complexes of cryptic, or currently just unrecognized, independent evolutionary lineages. The application of the “4x rule” shows that the two lineages singled out within H. ingens are in fact independent evolutionary units, while the complex molecular structure observed in H. gurneyi s.l. could not be resolved based on the currently available data. Applying standard crustacean mtDNA evolutionary rates to the observed divergence values, the separation of the main lineages within both H. ingens and H. gurneyi might dates back to the Miocene; however, it has also to be considered that the rate of mtDNA evolution might be accelerated in copepods, as already observed in other arthropod taxa. Present results gives further evidences of the high potential for copepod speciation with no or little morphological changes, and stress the need of a revision of the most controversial Palaearctic diaptomid genera.

Cryptic invasion in Southern Europe: The case of Ferrissia fragilis (Pulmonata: Ancylidae) Mediterranean populations

Four populations of the North American ancylid gastropod Ferrissia fragilis have been found in Southern Italy, thus expanding the known Eurasian distribution range of this invasive gastropod to the Mediterranean area. Both mitochondrial markers used for the molecular identification of the sampled specimens (16S and COI) showed a modest to absent haplotypic diversity in the studied Ferrissia populations from Europe and Asia, and their perfect identity with some of the haplotypes observed in North American F. fragilis populations. In the light of the scarce molecular diversity observed in the whole Palaearctic area and of the growing evidences for the ongoing spreading of the species in Europe, the occurrence of a single invasion event from North America possibly linked with aquarium plant trade followed by a rapid spreading of the species in Eurasia is suggested. The record of F. fragilis in Southern Italy caused some doubts on the presence of autochthonous Ferrissia populations in Europe and on the actual existence of Ferrissia wautieri. A molecular analysis of Ferrissia specimens from the loci typici of F. wautieri (Northern Italy) and F. clessiniana (Egypt) is definitely opportune.

Does lake age affect zooplankton diversity in Mediterranean lakes and reservoirs? A case study from southern Italy

Zooplankton assemblages of 51 lacustrine environments located in the middle of the Mediterranean Region were analysed to evaluate the existence of an ‘age effect’ in determining their structure. The analysed datasets refer to two different geographic areas, one comprising 30 natural and artificial lakes in Sicily and the other an arrangement of 21 analogous aquatic ecosystems located at the bottom of the Italian Peninsula, a more pristine area called Southern Apennine region. Most of the natural lakes are of post-glacial origin. The artificial lakes in both datasets were built in the last century and offer the opportunity to evaluate the possible short-term effects of ageing on the structure of their zooplankton. A comparison of assemblages in the two regions by PERMANOVA and nMDS revealed that they are quite different; therefore they were analysed separately. An explorative analysis on the possible relationship between biological data and environmental data (including lake age) was performed on both datasets using DISTLM. The presence of an ‘age effect’ emerged only in the subset of artificial lakes of the Southern Apennine region; accordingly it was tested more in detail with an a posteriori PERMANOVA analysis in the subset of reservoirs that resulted positive in the first test. SIMPER allowed us to single out the main species responsible of changes in the zooplankton along the selected age groups. No age effect was evident in the Sicilian water bodies, where other variables, such as conductivity, trophic state, urbanisation and water level fluctuations proved to have a major role in shaping zooplankton assemblages. The results showed that the age effect is: (i) detectable only at a time scale of decades; (ii) masked by the human impact in the watershed.