Population Genomics of Wall Lizards Reflects the Dynamic History of the Mediterranean Basin.

The Mediterranean Basin has experienced extensive change in geology and climate over the past six million years. Yet, the relative importance of key geological events for the distribution and genetic structure of the Mediterranean fauna remains poorly understood. Here, we use population genomic and phylogenomic analyses to establish the evolutionary history and genetic structure of common wall lizards (Podarcis muralis). This species is particularly informative because, in contrast to other Mediterranean lizards, it is widespread across the Iberian, Italian, and Balkan Peninsulas, and in extra-Mediterranean regions. We found strong support for six major lineages within P. muralis, which were largely discordant with the phylogenetic relationship of mitochondrial DNA. The most recent common ancestor of extant P. muralis was likely distributed in the Italian Peninsula, and experienced an "Out-of-Italy" expansion following the Messinian salinity crisis (∼5 Mya), resulting in the differentiation into the extant lineages on the Iberian, Italian, and Balkan Peninsulas. Introgression analysis revealed that both inter- and intraspecific gene flows have been pervasive throughout the evolutionary history of P. muralis. For example, the Southern Italy lineage has a hybrid origin, formed through admixture between the Central Italy lineage and an ancient lineage that was the sister to all other P. muralis. More recent genetic differentiation is associated with the onset of the Quaternary glaciations, which influenced population dynamics and genetic diversity of contemporary lineages. These results demonstrate the pervasive role of Mediterranean geology and climate for the evolutionary history and population genetic structure of extant species.

Phylogenomics of Anguis and Pseudopus (Squamata, Anguidae) indicates Balkan-Apennine mitochondrial capture associated with the Messinian event.

A dated phylogenetic hypothesis on the evolutionary history of the extant taxa of the Western Palearctic lizards Anguis and Pseudopus is revised using genome-wide nuclear DNA and mitogenomes. We found overall concordance between nuclear and mitochondrial DNA phylogenies, with one significant exception - the Apennine A. veronensis. In mitochondrial DNA, this species forms a common clade with the earliest diverging lineage, the southern Balkan endemic A. cephallonica, while it clusters together with A. fragilis in nuclear DNA. The nuclear phylogeny conforms to the morphology, which is relatively similar between A. veronensis and A. fragilis. The most plausible explanation for the mitonuclear discordance is ancient mitochondrial capture from the Balkan ancestor of A. cephallonica to the Apennine population of the A. fragilis-veronensis ancestor. We hypothesize that this capture occurred only in a geographically restricted population. The dating of this presumed mitochondrial introgression and capture coincides with the Messinian event, when the Balkan and Apennine Peninsulas were presumably largely connected. The dated nuclear phylogenomic reconstruction estimated the divergence of A. cephallonica around 12 Mya, while the sister clade representing the A. fragilis species complex consisting of the sister species A. fragilis-A. veronensis and A. colchica-A. graeca further diversified around 7 Mya. The depth of nuclear divergence among the evolutionary lineages of Pseudopus (0.5-1.2 Mya) supports their subspecies status.

Fossil-calibrated time tree of Podarcis wall lizards provides limited support for biogeographic calibration models.

Podarcis wall lizards are endemic to the Mediterranean Basin where they represent the predominant reptile group. Despite being extensively used as model organisms in evolutionary and ecological studies their phylogeny and historical biogeography are still incompletely resolved. Moreover, molecular clock calibrations used in wall lizard phylogeography are based on the assumption of vicariant speciation triggered by the abrupt Mediterranean Sea level rise at the end of the Messinian salinity crisis (MSC). However, the validity of this biogeographic calibration remains untested. In this study we inferred a robust time tree based on multilocus data and fossil calibrations using both gene concatenation and species-tree approaches and including models with gene-flow. We found five deeply divergent, geographically coherent, and well-supported clades comprising species from i) Iberian Peninsula and North Africa; ii) Western Mediterranean islands, iii) Sicilian and Maltese islands; and iv-v) Balkan region and Aegean islands. The mitochondrial tree shows some inconsistencies with the species tree that warrant future investigation. Diversification of main clades is estimated in a short time frame during the Middle Miocene and might have been associated with a period of global climate cooling with the establishment of a marked climatic zonation in Europe. Cladogenetic events within the main clades are scattered throughout the time tree, from the Late Miocene to the Early Pleistocene, suggesting that speciation events in wall lizards reflect a complex interplay between regional topography, climate and geological history rather than a shared major climatic or paleogeographic event. Our absolute time estimates, as well as a relative dating approach, demonstrate that the assumption of a causal link between sea-level rise at the end of the MSC and the diversification of many island endemics is not justified. This study reinforces the notion that multiple dispersal and vicariant events, at different time frames, are required to explain current allopatric distributions and to account for the historical assembly of Mediterranean biota, and cautions against the use of biogeographic calibrations based on the assumption of vicariance.

Phylogeography of the mediterranean green seaweed Halimeda tuna (Ulvophyceae, Chlorophyta).

Populations of many Mediterranean marine species show a strong phylogeographic structure, but the knowledge available for native seaweeds is limited. We investigated the genetic diversity of the green alga Halimeda tuna based on two plastid markers (tufA gene and a newly developed amplicon spanning five ribosomal protein genes and intergenic spacers, the rpl2-rpl14 region). The tufA sequences showed that Mediterranean H. tuna represents a single, well-defined species. The rpl2-rpl14 results highlighted a genetic separation between western and eastern Mediterranean populations; specimens collected from widely scattered locations in the Adriatic/Ionian region shared a haplotype unique to this region, and formed a group separated from all western Mediterranean regions. Specimens from Sardinia also formed a unique haplotype. Within the western Mediterranean basin, a gradual shift in the frequency of haplotypes was apparent along a West-East gradient. Our results represent the first clear evidence of an East-West genetic cleavage in a native Mediterranean macroalga and offer an interesting perspective for further research into fine-scale seaweed population structure in the NW Mediterranean Sea.

Mitogenomic phylogeny of cone snails endemic to Senegal.

Cone snails attain in Senegal one of their highest peaks of species diversity throughout the continental coast of Western Africa. A total of 15 endemic species have been described, all placed in the genus Lautoconus. While there is ample data regarding the morphology of the shell and the radular tooth of these species, virtually nothing is known regarding the genetic diversity and phylogenetic relationships of one of the most endangered groups of cones. In this work, we determined the complete or near-complete (only lacking the control region) mitochondrial (mt) genomes of 17 specimens representing 11 endemic species (Lautoconus belairensis, Lautoconus bruguieresi, Lautoconus cacao, Lautoconus cloveri, Lautoconus cf. echinophilus, Lautoconus guinaicus, Lautoconus hybridus, Lautoconus senegalensis, Lautoconus mercator, Lautoconus taslei, and Lautoconus unifasciatus). We also sequenced the complete mt genome of Lautoconus guanche from the Canary Islands, which has been related to the cones endemic to Senegal. All mt genomes share the same gene arrangement, which conforms to the consensus reported for Conidae, Neogastropoda and Caenogastropoda. Phylogenetic analyses using probabilistic methods recovered three major lineages, whose divergence coincided in time with sea level and ocean current changes as well as temperature fluctuations during the Messinian salinity crisis and the Plio-Pleistocene transition. Furthermore, the three lineages corresponded to distinct types of radular tooth (robust, small, and elongated), suggesting that dietary specialization could be an additional evolutionary driver in the diversification of the cones endemic to Senegal. The reconstructed phylogeny showed several cases of phenotypic convergence (cryptic species) and questions the validity of some species (ecotypes or phenotypic plasticity), both results having important taxonomic and conservation consequences.

Environmental niche divergence among three dune shrub sister species with parapatric distributions.

Background and Aims: The geographical distributions of species are constrained by their ecological requirements. The aim of this work was to analyse the effects of environmental conditions, historical events and biogeographical constraints on the diversification of the three species of the western Mediterranean shrub genus Stauracanthus , which have a parapatric distribution in the Iberian Peninsula. Methods: Ecological niche factor analysis and generalized linear models were used to measure the response of all Stauracanthus species to the environmental gradients and map their potential distributions in the Iberian Peninsula. The bioclimatic niche overlap between the three species was determined by using Schoener's index. The genetic differentiation of the Iberian and northern African populations of Stauracanthus species was characterized with GenalEx. The effects on genetic distances of the most important environmental drivers were assessed through Mantel tests and non-metric multidimensional scaling. Key Results: The three Stauracanthus species show remarkably similar responses to climatic conditions. This supports the idea that all members of this recently diversified clade retain common adaptations to climate and consequently high levels of climatic niche overlap. This contrasts with the diverse edaphic requirements of Stauracanthus species. The populations of the S. genistoides-spectabilis clade grow on Miocene and Pliocene fine-textured sedimentary soils, whereas S. boivinii , the more genetically distant species, occurs on older and more coarse-textured sedimentary substrates. These patterns of diversification are largely consistent with a stochastic process of geographical range expansion and fragmentation coupled with niche evolution in the context of spatially complex environmental fluctuations. Conclusions: : The combined analysis of the distribution, realized environmental niche and phylogeographical relationships of parapatric species proposed in this work allows integration of the biogeographical, ecological and evolutionary processes driving the evolution of species adaptations and how they determine their current geographical ranges.

First occurrence of Psammophis (Serpentes) from Europe witnesses another Messinian herpetofaunal dispersal from Africa - biogeographic implications and a discussion of the vertebral morphology of psammophiid snakes.

We here describe abundant new snake material from the late Miocene (MN 13) of Salobreña, Spain. Vertebral morphology suggests a referral of the specimens to the extant psammophiid Psammophis, documenting the first occurrence of this genus in Europe. The diversity and disparity across the vertebral morphology of different psammophiid genera are discussed. We identify vertebral features that could diagnose Psammophis and therefore enable the recognition of the genus in the fossil record. A comparison of the new Spanish form with other taxa is conducted. We provide a detailed review of the psammophiid fossil record. Material previously described from the middle Miocene of Beni Mellal, Morocco is here tentatively referred to as ?Psammophis sp., an action that renders that occurrence as the oldest (probable) record of the genus and Psammophiidae as a whole, providing thus a potential calibration point. On the other hand, Eastern European Pliocene material that had been previously supposedly referred to Psammophis is here discarded as being rather fragmentary, not affording any more precise determination. The two psammophiid genera Psammophis and Malpolon appear almost simultaneously in the European fossil record (MN 13), with the former achieving only a short-lived and apparently geographically limited distribution in the continent, while the latter still exists in its modern herpetofauna. We assess biogeographic implications of the new find, suggesting a direct dispersal event from northwestern Africa to the Iberian Peninsula during the late Miocene, facilitated by the Messinian Salinity Crisis.

Phylogenetic Placement and Phylogeography of Large-Flowered Lotus Species (Leguminosae) Formerly Classified in Dorycnium: Evidence of Pre-Pleistocene Differentiation of Western and Eastern Intraspecific Groups.

The Mediterranean region is a center of species and genetic diversity of many plant groups, which served as a source of recolonization of temperate regions of Eurasia in Holocene. We investigate the evolutionary history of species currently classified in Lotus sect. Bonjeanea in the context of the evolution of the genus Lotus as a whole, using phylogenetic, phylogeographic and dating analyses. Of three species of the section, L. rectus and L. hirsutus have wide Mediterranean distribution while L. strictus has a disjunctive range in Bulgaria, Turkey, Armenia, Eastern Kazakhstan, and adjacent parts of Russia and China. We used entire nuclear ribosomal ITS1-5.8S-ITS2 region (nrITS) and a plastid dataset (rps16 and trnL-F) to reconstruct phylogenetic relationships within Lotus with an extended representation of Bonjeanea group. We analyzed the phylogeographic patterns within each species based on the plastid dataset. For divergence time estimation, the nrITS dataset was analyzed. Our results confirmed the non-monophyletic nature of the section Bonjeanea. They indicate that Lotus is likely to have diverged about 15.87 (9.99-19.81) million years ago (Ma), which is much older than an earlier estimate of ca. 5.54 Ma. Estimated divergence ages within L. strictus, L. rectus, and L. hisrutus (6.1, 4.94, and 4.16 Ma, respectively) well predate the onset of the current type of Mediterranean climate. Our data suggest that relatively ancient geological events and/or climatic changes apparently played roles in early diversification of Lotus and its major clades, as well as in formation of phylogeographic patterns, in at least some species.

Geo-Climatic Changes and Apomixis as Major Drivers of Diversification in the Mediterranean Sea Lavenders (Limonium Mill.).

The Mediterranean realm, comprising the Mediterranean and Macaronesian regions, has long been recognized as one of the world's biodiversity hotspots, owing to its remarkable species richness and endemism. Several hypotheses on biotic and abiotic drivers of species diversification in the region have been often proposed but rarely tested in an explicit phylogenetic framework. Here, we investigate the impact of both species-intrinsic and -extrinsic factors on diversification in the species-rich, cosmopolitan Limonium, an angiosperm genus with center of diversity in the Mediterranean. First, we infer and time-calibrate the largest Limonium phylogeny to date. We then estimate ancestral ranges and diversification dynamics at both global and regional scales. At the global scale, we test whether the identified shifts in diversification rates are linked to specific geological and/or climatic events in the Mediterranean area and/or asexual reproduction (apomixis). Our results support a late Paleogene origin in the proto-Mediterranean area for Limonium, followed by extensive in situ diversification in the Mediterranean region during the late Miocene, Pliocene, and Pleistocene. We found significant increases of diversification rates in the "Mediterranean lineage" associated with the Messinian Salinity Crisis, onset of Mediterranean climate, Plio-Pleistocene sea-level fluctuations, and apomixis. Additionally, the Euro-Mediterranean area acted as the major source of species dispersals to the surrounding areas. At the regional scale, we infer the biogeographic origins of insular endemics in the oceanic archipelagos of Macaronesia, and test whether woodiness in the Canarian Nobiles clade is a derived trait linked to insular life and a biotic driver of diversification. We find that Limonium species diversity on the Canary Islands and Cape Verde archipelagos is the product of multiple colonization events followed by in situ diversification, and that woodiness of the Canarian endemics is indeed a derived trait but is not associated with a significant shift to higher diversification rates. Our study expands knowledge on how the interaction between abiotic and biotic drivers shape the uneven distribution of species diversity across taxonomic and geographical scales.

Phylogeography of Dictyota fasciola and Dictyota mediterranea (Dictyotales, Phaeophyceae): unexpected patterns on the Atlantic-Mediterranean marine transition and taxonomic implications.

The Atlantic-Mediterranean marine transition is a fascinating biogeographic region, but still very poorly studied from the point of view of seaweed phylogeography. Dictyota fasciola and D. mediterranea (Dictyotales, Phaeophyceae) are two currently recognized sister species that share a large part of their distribution along the Mediterranean Sea and the Atlantic Ocean, representing a unique study model to understand the diversification processes experienced by macroalgae during and after Messinian at this marine region. In this study, we sampled 102 individuals of D. fasciola and D. mediterranea from 32 localities along their distribution range and sequenced the mitochondrial cox1 and the chloroplast rbcL-rbcS DNA regions for all the samples. Our data do not support the occurrence of two sister species but a morphologically variable and highly genetic diverse species or a complex of species. Most of the observed genetic diversity corresponds to the Mediterranean populations, whereas the Atlantic ones are much more homogeneous. The early-diverged lineages inferred from both mtDNA and cpDNA phylogenetic reconstructions were constituted by samples from the Mediterranean Sea. Together, these results suggest that the Mediterranean Sea acted as a refugium for the D. fasciola-D. mediterranea lineage during the geologic and climatic changes occurred on the region since the Miocene, subsequently dispersing to the Atlantic Ocean.

Multiple transgressions and slow evolution shape the phylogeographic pattern of the blind cave-dwelling shrimp Typhlocaris.

BACKGROUND: Aquatic subterranean species often exhibit disjunct distributions, with high level of endemism and small range, shaped by vicariance, limited dispersal, and evolutionary rates. We studied the disjunct biogeographic patterns of an endangered blind cave shrimp, Typhlocaris, and identified the geological and evolutionary processes that have shaped its divergence pattern. METHODS: We collected Typlocaris specimens of three species (T. galilea, T. ayyaloni, and T. salentina), originating from subterranean groundwater caves by the Mediterranean Sea, and used three mitochondrial genes (12S, 16S, cytochrome oxygnese subunit 1 (COI)) and four nuclear genes (18S, 28S, internal transcribed spacer, Histon 3) to infer their phylogenetic relationships. Using the radiometric dating of a geological formation (Bira) as a calibration node, we estimated the divergence times of the Typhlocaris species and the molecular evolution rates. RESULTS: The multi-locus ML/Bayesian trees of the concatenated seven gene sequences showed that T. salentina (Italy) and T. ayyaloni (Israel) are sister species, both sister to T. galilea (Israel). The divergence time of T. ayyaloni and T. salentina from T. galilea was 7.0 Ma based on Bira calibration. The divergence time of T. ayyaloni from T. salentina was 5.7 (4.4-6.9) Ma according to COI, and 5.8 (3.5-7.2) Ma according to 16S. The computed interspecific evolutionary rates were 0.0077 substitutions/Myr for COI, and 0.0046 substitutions/Myr for 16S. DISCUSSION: Two consecutive vicariant events have shaped the phylogeographic patterns of Typhlocaris species. First, T. galilea was tectonically isolated from its siblings in the Mediterranean Sea by the arching uplift of the central mountain range of Israel ca. seven Ma. Secondly, T. ayyaloni and T. salentina were stranded and separated by a marine transgression ca. six Ma, occurring just before the Messinian Salinity Crisis. Our estimated molecular evolution rates were in one order of magnitude lower than the rates of closely related crustaceans, as well as of other stygobiont species. We suggest that this slow evolution reflects the ecological conditions prevailing in the highly isolated subterranean water bodies inhabited by Typhlocaris.

The Messinian stromatolites of the Sierra del Colmenar (Western Mediterranean): facies characterization and sedimentological interpretation.

A representative outcrop of the Messinian stromatolites belonging to the Terminal Carbonate Complex unit, from the northern sector of the Bajo Segura basin (Caja de Ahorros del Mediterraneo section, Sierra del Colmenar, SE Spain) has been studied. Here, we present a detailed analysis of the architecture, external morphology, and internal morphology in order to reconstruct the environmental and palaeoecological conditions for their growth. The stromatolites macrostructure consists of a continuously doming type morphology (build up and sheets areas). These developed close to the coast and acted as a palaeogeographic barrier, reducing physical stress, channeling the erosive effect of water and favoring restricted conditions. This stromatolitic macrostructure exhibits variations in its internal morphology, giving rise to seven subfacies, which are a product of the environmental changes experienced during the growth of the microbial mats. Although broadly suggesting a coastal environment, restricted and shallow during formation, the variation in internal morphology (mesostructure and microstructure) is evidence of minor changes in the physical environment that indicate a progressive shallowing.

Does the genetic structure of spring snail Bythinella (Caenogastropoda, Truncatelloidea) in Bulgaria reflect geological history?

Bythinella is a minute dioecious caenogastropod that inhabits springs in central and southern Europe. In the Balkans, previous studies have addressed its morphological and genetic differentiation within Greece and Romania while the Bulgarian species have remained poorly known. The aim of the present paper has been to expand the knowledge on the subject in Bulgaria. Shell morphology and anatomy of the reproductive organs were examined, and a fragment of the mitochondrial cytochrome oxidase subunit I (COI) gene and the nuclear ribosomal Internal Transcribed Spacer 1 (ITS-1) were sequenced from 15 populations. Additional sequences from eight previously studied populations were included in our analyses. Phylogenetic analyses revealed five main mitochondrial DNA clades, which were partly confirmed by analyses of the ITS-1 sequences. The genetic differentiation between the clades was found to be in the range p=2.4-11.8%. Most of the populations belonged to clade I, representing Bythinella hansboetersi, and were distributed in SW Bulgaria. Clades II and III inhabit areas adjacent to clade I and were most closely related with the latter clade. Much more distinct were clade V, found at one locality in NW Bulgaria, and clade IV, found at one locality in SE Bulgaria, close to the sea. Four populations were found in caves, but only one of these represented a distinct clade. Considering the observed pattern of interpopulation differentiation of Bythinella in Bulgaria, we can suppose that isolation between clades I, II and III may have been caused by glaciations during the Pleistocene. The time of isolation between the above three clades and clade IV coincides with the Messinian Salinity Crisis, and the time of isolation between the clade V and the other four most probably reflects the isolation of the Rhodopes from western Balkan Mts by the seawater of the Dacic Basin.

Paleomagnetic and paleoenvironmental implications of magnetofossil occurrences in late Miocene marine sediments from the Guadalquivir Basin, SW Spain.

Although recent studies have revealed more widespread occurrences of magnetofossils in pre-Quaternary sediments than have been previously reported, their significance for paleomagnetic and paleoenvironmental studies is not fully understood. We present a paleo- and rock-magnetic study of late Miocene marine sediments recovered from the Guadalquivir Basin (SW Spain). Well-defined paleomagnetic directions provide a robust magnetostratigraphic chronology for the two studied sediment cores. Rock magnetic results indicate the dominance of intact magnetosome chains throughout the studied sediments. These results provide a link between the highest-quality paleomagnetic directions and higher magnetofossil abundances. We interpret that bacterial magnetite formed in the surface sediment mixed layer and that these magnetic particles gave rise to a paleomagnetic signal in the same way as detrital grains. They, therefore, carry a magnetization that is essentially identical to a post-depositional remanent magnetization, which we term a bio-depositional remanent magnetization. Some studied polarity reversals record paleomagnetic directions with an apparent 60-70 kyr recording delay. Magnetofossils in these cases are interpreted to carry a biogeochemical remanent magnetization that is locked in at greater depth in the sediment column. A sharp decrease in magnetofossil abundance toward the middle of the studied boreholes coincides broadly with a major rise in sediment accumulation rates near the onset of the Messinian salinity crisis (MSC), an event caused by interruption of the connection between the Mediterranean Sea and the Atlantic Ocean. This correlation appears to have resulted from dilution of magnetofossils by enhanced terrigenous inputs that were driven, in turn, by sedimentary changes triggered in the basin at the onset of the MSC. Our results highlight the importance of magnetofossils as carriers of high-quality paleomagnetic and paleoenvironmental signals even in dominantly terrigenous sediments.