Daniele Salvi

Persistence across Pleistocene ice ages in Mediterranean and extra-Mediterranean refugia: phylogeographic insights from the common wall lizard

BackgroundPleistocene climatic oscillations have played a major role in structuring present-day biodiversity. The southern Mediterranean peninsulas have long been recognized as major glacial refugia, from where Northern Europe was post-glacially colonized. However, recent studies have unravelled numerous additional refugia also in northern regions. We investigated the phylogeographic pattern of the widespread Western Palaearctic lizard Podarcis muralis, using a range-wide multilocus approach, to evaluate whether it is concordant with a recent expansion from southern glacial refugia or alternatively from a combination of Mediterranean and northern refugia.ResultsWe analyzed DNA sequences of two mitochondrial (cytb and nd4) and three nuclear (acm4, mc1r, and pdc) gene fragments in individuals from 52 localities across the species range, using phylogenetic and phylogeographic methods. The complex phylogeographic pattern observed, with 23 reciprocally monophyletic allo- parapatric lineages having a Pleistocene divergence, suggests a scenario of long-term isolation in multiple ice-age refugia across the species distribution range. Multiple lineages were identified within the three Mediterranean peninsulas – Iberia, Italy and the Balkans - where the highest genetic diversity was observed. Such an unprecedented phylogeographic pattern - here called “refugia within all refugia” – compasses the classical scenario of multiple southern refugia. However, unlike the southern refugia model, various distinct lineages were also found in northern regions, suggesting that additional refugia in France, Northern Italy, Eastern Alps and Central Balkans allowed the long-term persistence of this species throughout Pleistocene glaciations.ConclusionsThe phylogeography of Podarcis muralis provides a paradigm of temperate species survival in Mediterranean and extra-Mediterranean glacial refugia. Such refugia acted as independent biogeographic compartments for the long-term persistence of this species, for the differentiation of its genetic lineages, and for the short-distance post-glacial re-colonization of neighbouring areas. This finding echoes previous findings from recent phylogeographic studies on species from temperate ecoregions, thus suggesting the need for a reappraisal of the role of northern refugia for glacial persistence and post-glacial assembly of Holarctic biota.

The role of post-natal ontogeny in the evolution of phenotypic diversity in Podarcis lizards.

Understanding the role of the developmental pathways in shaping phenotypic diversity allows appreciating in full the processes influencing and constraining morphological change. Podarcis lizards demonstrate extraordinary morphological variability that likely originated in short evolutionary time. Using geometric morphometrics and a broad suite of statistical tests, we explored the role of developmental mechanisms such as growth rate change, ontogenetic divergence/convergence/parallelism as well as morphological expression of heterochronic processes in mediating the formation of their phenotypic diversity during the post‐natal ontogeny. We identified hypermorphosis – the prolongation of growth along the same trajectory – as the process responsible for both intersexual and interspecific morphological differentiation. Albeit the common allometric pattern observed in both sexes of any species constrains and canalizes their cephalic scales variation in a fixed portion of the phenotypic space, the extended growth experienced by males and some species allows them to achieve peramorphic morphologies. Conversely, the intrasexual phenotypic diversity is accounted for by non‐allometric processes that drive the extensive morphological dispersion throughout their ontogenetic trajectories. This study suggests a model of how simple heterochronic perturbations can produce phenotypic variation, and thus potential for further evolutionary change, even within a strictly constrained developmental pathway.

One Species, Three Pleistocene Evolutionary Histories: Phylogeography of the Italian Crested Newt, Triturus carnifex

Phylogeographic patterns of temperate species from the Mediterranean peninsulas have been investigated intensively. Nevertheless, as more phylogeographies become available, either unique patterns or new lines of concordance continue to emerge, providing new insights on the evolution of regional biotas. Here, we investigated the phylogeography and evolutionary history of the Italian crested newt, Triturus carnifex, through phylogenetic, molecular dating and population structure analyses of two mitochondrial gene fragments (ND2 and ND4; overall 1273 bp). We found three main mtDNA lineages having parapatric distribution and estimated divergence times between Late Pliocene and Early Pleistocene. One lineage (S) was widespread south of the northern Apennine chain and was further geographically structured into five sublineages, likely of Middle Pleistocene origin. The second lineage (C) was widespread throughout the Padano–Venetian plain and did not show a clear phylogeographic structure. The third lineage (N) was observed in only two populations located on western Croatia/Slovenia. Results of analysis of molecular variance suggested that partitioning populations according to the geographic distribution of these lineages and sublineages explains 76% of the observed genetic variation. The phylogeographic structure observed within T. carnifex and divergence time estimates among its lineages, suggest that responses to Pleistocene environmental changes in this single species have been as diverse as those found previously among several codistributed temperate species combined. Consistent with the landscape heterogeneity, physiographic features, and palaeogeographical evolution of its distribution range, these responses encompass multiple refugia along the Apennine chain, lowland refugia in large peri-coastal plains, and a ‘cryptic’ northern refugium.

Mitochondrial phylogeography of the Bedriaga's rock lizard, Archaeolacerta bedriagae (Reptilia: Lacertidae) endemic to Corsica and Sardinia

Archaeolacerta bedriagae is a rock-dwelling lizard endemic to the Corso-Sardinian insular system. We investigated the phylogeography of the species by using the mitochondrial ND4 and flanking tRNAs genes from 94 specimens belonging to 19 populations. Phylogenetic, Barrier, and SAMOVA analyses revealed a highly structured pattern characterized by two levels of discontinuities in the geographical distribution of mtDNA diversity: (i) a deep phylogeographic break in Northern Corsica between Lineage A, restricted to northernmost Corsica, and Lineage B widespread all over the remaining range of the species, and (ii) some minor phylogeographic discontinuities within lineage B, which is sub-structured into six closely related haplotype clades with remarkable concordance with geography. The first evolutionary event concerning the split between the two main lineages from an ancestral population occurred in the Upper Pliocene (5.87-3.68 mya), while the divergence within lineage B would have started from the Upper Pleistocene (2.5-1.6 mya), between Corsican and Sardinian populations. Somewhat later (1.7-1.1 mya), the Sardinian ancestral population underwent fragmentation into population groups inhabiting North, Central, and South Sardinia. As inferred from previous allozyme surveys, the divergence among population groups would be driven by allopatric fragmentation, while the discrepancy concerning the major partition into two lineages inferred from mtDNA but not apparent in analysis of allozymes needs further investigation.

Molecular Phylogenetics and Systematics of the Bivalve Family Ostreidae Based on rRNA Sequence-Structure Models and Multilocus Species Tree

The bivalve family Ostreidae has a worldwide distribution and includes species of high economic importance. Phylogenetics and systematic of oysters based on morphology have proved difficult because of their high phenotypic plasticity. In this study we explore the phylogenetic information of the DNA sequence and secondary structure of the nuclear, fast-evolving, ITS2 rRNA and the mitochondrial 16S rRNA genes from the Ostreidae and we implemented a multi-locus framework based on four loci for oyster phylogenetics and systematics. Sequence-structure rRNA models aid sequence alignment and improved accuracy and nodal support of phylogenetic trees. In agreement with previous molecular studies, our phylogenetic results indicate that none of the currently recognized subfamilies, Crassostreinae, Ostreinae, and Lophinae, is monophyletic. Single gene trees based on Maximum likelihood (ML) and Bayesian (BA) methods and on sequence-structure ML were congruent with multilocus trees based on a concatenated (ML and BA) and coalescent based (BA) approaches and consistently supported three main clades: (i) Crassostrea, (ii) Saccostrea, and (iii) an Ostreinae-Lophinae lineage. Therefore, the subfamily Crassotreinae (including Crassostrea), Saccostreinae subfam. nov. (including Saccostrea and tentatively Striostrea) and Ostreinae (including Ostreinae and Lophinae taxa) are recognized. Based on phylogenetic and biogeographical evidence the Asian species of Crassostrea from the Pacific Ocean are assigned to Magallana gen. nov., whereas an integrative taxonomic revision is required for the genera Ostrea and Dendostrea. This study pointed out the suitability of the ITS2 marker for DNA barcoding of oyster and the relevance of using sequence-structure rRNA models and features of the ITS2 folding in molecular phylogenetics and taxonomy. The multilocus approach allowed inferring a robust phylogeny of Ostreidae providing a broad molecular perspective on their systematics.

Evolutionary history of the Maltese wall lizard Podarcis filfolensis: insights on the ‘Expansion–Contraction’ model of Pleistocene biogeography

The expansion–contraction (EC) model predicts demographic and range contraction of temperate species during Pleistocene glaciations as a consequence of climate‐related habitat changes, and provides a paradigm for explaining the high intraspecific diversity found in refugia in terms of long‐term demographic stability. However, recent evidence has revealed a weak predictive power of this model for terrestrial species in insular and coastal settings. We investigated the Pleistocene EC dynamics and their evolutionary consequences on temperate species using the Maltese archipelago and its endemic lizard Podarcis filfolensis as a model system. The evolutionary and demographic history of P. filfolensis as inferred from mitochondrial and nuclear sequences data does not conform to the EC model predictions, supporting (i) demographic and spatial stability or expansion, rather than contraction, of the northern and southern lineages during the last glacial period; and (ii) a major role for allopatric differentiation primed by sea‐level dynamics, rather than prolonged demographic stability, in the formation of the observed genetic diversity. When combined with evidence from other Mediterranean refugia, this study shows how the incorporation of Pleistocene sea‐level variations in the EC model accounts for a reverse demographic and range response of insular and coastal temperate biotas relative to continental ones. Furthermore, this cross‐archipelago pattern in which allopatric diversity is formed and shaped by EC cycles resembles that seen between isolated populations within mainland refugia and suggests that the EC model, originally developed to explain population fluctuations into and out‐of refugia, may be appropriate for describing the demographic and evolutionary dynamics driving the high genetic diversity observed in these areas.

Molecular evolution of the polyamine oxidase gene family in Metazoa

BackgroundPolyamine oxidase enzymes catalyze the oxidation of polyamines and acetylpolyamines. Since polyamines are basic regulators of cell growth and proliferation, their homeostasis is crucial for cell life. Members of the polyamine oxidase gene family have been identified in a wide variety of animals, including vertebrates, arthropodes, nematodes, placozoa, as well as in plants and fungi. Polyamine oxidases (PAOs) from yeast can oxidize spermine, N1-acetylspermine, and N1-acetylspermidine, however, in vertebrates two different enzymes, namely spermine oxidase (SMO) and acetylpolyamine oxidase (APAO), specifically catalyze the oxidation of spermine, and N1-acetylspermine/N1-acetylspermidine, respectively. Little is known about the molecular evolutionary history of these enzymes. However, since the yeast PAO is able to catalyze the oxidation of both acetylated and non acetylated polyamines, and in vertebrates these functions are addressed by two specialized polyamine oxidase subfamilies (APAO and SMO), it can be hypothesized an ancestral reference for the former enzyme from which the latter would have been derived.ResultsWe analysed 36 SMO, 26 APAO, and 14 PAO homologue protein sequences from 54 taxa including various vertebrates and invertebrates. The analysis of the full-length sequences and the principal domains of vertebrate and invertebrate PAOs yielded consensus primary protein sequences for vertebrate SMOs and APAOs, and invertebrate PAOs. This analysis, coupled to molecular modeling techniques, also unveiled sequence regions that confer specific structural and functional properties, including substrate specificity, by the different PAO subfamilies. Molecular phylogenetic trees revealed a basal position of all the invertebrates PAO enzymes relative to vertebrate SMOs and APAOs. PAOs from insects constitute a monophyletic clade. Two PAO variants sampled in the amphioxus are basal to the dichotomy between two well supported monophyletic clades including, respectively, all the SMOs and APAOs from vertebrates. The two vertebrate monophyletic clades clustered strictly mirroring the organismal phylogeny of fishes, amphibians, reptiles, birds, and mammals. Evidences from comparative genomic analysis, structural evolution and functional divergence in a phylogenetic framework across Metazoa suggested an evolutionary scenario where the ancestor PAO coding sequence, present in invertebrates as an orthologous gene, has been duplicated in the vertebrate branch to originate the paralogous SMO and APAO genes. A further genome evolution event concerns the SMO gene of placental, but not marsupial and monotremate, mammals which increased its functional variation following an alternative splicing (AS) mechanism.ConclusionsIn this study the explicit integration in a phylogenomic framework of phylogenetic tree construction, structure prediction, and biochemical function data/prediction, allowed inferring the molecular evolutionary history of the PAO gene family and to disambiguate paralogous genes related by duplication event (SMO and APAO) and orthologous genes related by speciation events (PAOs, SMOs/APAOs). Further, while in vertebrates experimental data corroborate SMO and APAO molecular function predictions, in invertebrates the finding of a supported phylogenetic clusters of insect PAOs and the co-occurrence of two PAO variants in the amphioxus urgently claim the need for future structure-function studies.

Evaluating the phylogenetic signal limit from mitogenomes, slow evolving nuclear genes, and the concatenation approach. New insights into the Lacertini radiation using fast evolving nuclear genes and species trees

Estimating the phylogeny of lacertid lizards, and particularly the tribe Lacertini has been challenging, possibly due to the fast radiation of this group resulting in a hard polytomy. However this is still an open question, as concatenated data primarily from mitochondrial markers have been used so far whereas in a recent phylogeny based on a compilation of these data within a squamate supermatrix the basal polytomy seems to be resolved. In this study, we estimate phylogenetic relationships between all Lacertini genera using for the first time DNA sequences from five fast evolving nuclear genes (acm4, mc1r, pdc, βfib and reln) and two mitochondrial genes (nd4 and 12S). We generated a total of 529 sequences from 88 species and used Maximum Likelihood and Bayesian Inference methods based on concatenated multilocus dataset as well as a coalescent-based species tree approach with the aim of (i) shedding light on the basal relationships of Lacertini (ii) assessing the monophyly of genera which were previously questioned, and (iii) discussing differences between estimates from this and previous studies based on different markers, and phylogenetic methods. Results uncovered (i) a new phylogenetic clade formed by the monotypic genera Archaeolacerta, Zootoca, Teira and Scelarcis; and (ii) support for the monophyly of the Algyroides clade, with two sister species pairs represented by western (A. marchi and A. fitzingeri) and eastern (A. nigropunctatus and A. moreoticus) lineages. In both cases the members of these groups show peculiar morphology and very different geographical distributions, suggesting that they are relictual groups that were once diverse and widespread. They probably originated about 11-13 million years ago during early events of speciation in the tribe, and the split between their members is estimated to be only slightly older. This scenario may explain why mitochondrial markers (possibly saturated at higher divergence levels) or slower nuclear markers used in previous studies (likely lacking enough phylogenetic signal) failed to recover these relationships. Finally, the phylogenetic position of most remaining genera was unresolved, corroborating the hypothesis of a hard polytomy in the Lacertini phylogeny due to a fast radiation. This is in agreement with all previous studies but in sharp contrast with a recent squamate megaphylogeny. We show that the supermatrix approach may provide high support for incorrect nodes that are not supported either by original sequence data or by new data from this study. This finding suggests caution when using megaphylogenies to integrate inter-generic relationships in comparative ecological and evolutionary studies.

Molecular phylogenetics in 2D: ITS2 rRNA evolution and sequence-structure barcode from Veneridae to Bivalvia

In this study, we analyzed the nuclear ITS2 rRNA primary sequence and secondary structure in Veneridae and comparatively with 20 Bivalvia taxa to test the phylogenetic resolution of this marker and its suitability for molecular diagnosis at different taxonomic levels. Maximum likelihood and Bayesian trees based on primary sequences were congruent with (profile-) neighbor-joining trees based on a combined model of sequence-structure evolution. ITS2 showed higher resolution below the subfamily level, providing a phylogenetic signal comparable to (mitochondrial/nuclear) gene fragments 2-5 times longer. Structural elements of the ITS2 folding, such as specific mismatch pairing and compensatory base changes, provided further support for the monophyly of some groups and for their phylogenetic relationships. Veneridae ITS2 folding is structured in six domains (DI-VI) and shows five striking sequence-structure features. Two of them, the Basal and Apical STEMs, are common to Bivalvia, while the presence of both the Branched STEM and the Y/R stretches occurs in five superfamilies of the two Heterodonta orders Myoida and Veneroida, thus questioning their reciprocal monophyly. Our results validated the ITS2 as a suitable marker for venerids phylogenetics and taxonomy, and underlined the significance of including secondary structure information for both applications at several systematic levels within bivalves.

The analysis of rRNA sequence-structure in phylogenetics: An application to the family Pectinidae (Mollusca: Bivalvia)

Several studies pointed out the relevance of integrating secondary structure information in sequence analysis and phylogenetics, both in terms of phylogenetic resolution and of marker suitability for phylogenetic reconstruction at higher taxonomic-rank. In this study we explore in a phylogenetic framework the primary and secondary structure information from nuclear (ITS2) and mitochondrial (16S) ribosomal DNA sequences from the Pectinidae, commonly known as scallops. Primary sequences were analysed under neighbour-joining, maximum parsimony, maximum likelihood, and Bayesian approaches. The individual RNA secondary structures were analysed alone and with primary sequences employing a combined model of sequence-structure evolution. The information from primary sequences and secondary structure of the ITS2 are concordant and provide good phylogenetic resolution, while the mitochondrial marker 16S fails to resolve the relationships between the major clades and shows a lack of structural signals. Our phylogenetic reconstruction provided evidence for the monophyly of the subfamily Pectininae and the tribes Aequipectinini and Pectinini while the subfamily Chlamydinae, although recovered in some analyses, did not receive good support. The secondary structure analysis of the derived pectinid ITS2 rRNA sequence revealed three striking differences between Pectininae and Chlamydinae subfamilies: (a) Chlamydinae ITS2 rRNA folding shows the typical four domains architecture, while the one of Pectininae only three; (b) the Pectinidae basal DI pairing shows a different sequence-structure consensus between Pectininae and Chlamydinae; (c) the Pectininae DIII domain holds a specific short secondary stem (Pec STEM). Furthermore, the scallop ITS2 rRNA folding analysis has shown the presence of a conserved sequence motif (invariably located on apical portion of the DIII domain) which emerges as a common feature across Bivalvia. The combined sequence-structure approach employed in this study, corroborates the deep significance of including the secondary structure information in phylogenetic analysis both as combined sequence-structure alignment as well as pointing out conserved elements of the RNA folding.