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This study revises the taxonomy and species limits of Chondrina taxa living in the Cantabrian mountain region of N Spain, based mainly on molecular data but supplemented with morphological data. On...

Knowledge of feeding ecology of declining species, such as farmland birds, is essential to address their conservation requirements, especially when their habitats are suffering important reductions of trophic resources. In this study, we apply a metabarcoding approach to describe the diet composition of six of the most significant farmland birds inhabiting European cereal pseudo-steppes: little bustard, great bustard, pin-tailed sandgrouse, black-bellied sandgrouse, red-legged partridge, and common quail. We further studied seasonal diet variations (autumn to spring) in all species but the common quail, whose diet was studied during spring and summer. We show that study species´ diets mostly consisted of plants, although in the case of little bustard and great bustard arthropods are also highly relevant. Among arthropods, we found high proportions of thrips, arachnids, and springtails, which were previously unreported in their diet, and some taxa that could be used as antiparasitic food. Moreover, we report that little bustard’s diet is the least rich of that of all studied species, and that diet of all these species is less diverse in winter than in autumn and spring. Diet composition of these declining species supports the importance of natural and semi-natural vegetation and landscape mosaics that can provide a wide variety of arthropods, plants, and seeds all year-round., This paper contributes to the REGRESSEDS project (CGL2016-75278-R funded by MINECO, Spain). This study is also a contribution to project Sistemática, Biogeografía, Ecología del comportamiento y Evolución (IT1163-19) funded by Basque Country Government. Additional funds for this study were provided by the projects 201630E096 funded by CSIC (recipient BA) and AGROPERDIZ (SBPLY/17/180501/ 000245) funded by the “Junta de Comunidades de Castilla-La Mancha” and “Fondo Europeo de Desarrollo Regional” (recipient FM & BA). Xabier Cabodevilla was supported by a Ph.D. grant, financed by the Basque Country Government (grant no. PRE_2018_2_0273).

Plants on oceanic islands can evolve insular syndromes such as secondary woodiness, a generalized trend found in island floras worldwide. This phenomenon occurs through evolution in situ. It is triggered by ecological and physiological stimuli that transform herbaceous annuals into woody perennials. However, well-dated and informative fossils that could help track and frame the evolution of this syndrome are lacking. Remarkably, in Madeira Island (Portugal), there are good examples of Apiaceae that evolved secondary woodiness, like the giant neoendemic Melanoselinum (≡ Daucus). Apiaceae has a very scarce fossil record, despite being a cosmopolitan family and an economically important crop. Here we describe the oldest Daucus s.l. fossil known to date and the first fossil evidence of a plant with insular woodiness. The fossils are preserved as mummified/compressed mericarps within 1.3-million-year-old fluvio-lacustrine sediments of the Funchal unit, Upper Volcanic complex, near Porto da Cruz. We assign them to the extant neoendemic species Melanoselinum (≡ Daucus) decipiens. The mericarp morphology shows remarkable stasis since the Calabrian stage of the Early Pleistocene. Our results demonstrate that in the Madeiran Daucinae clade, insular woodiness developed at least 1.3 million years ago, indicating a coeval or earlier immigration to Madeira Island of a Daucus sp. Our results reinforce the role of palaeobotanical research in oceanic islands, supported by stratigraphy and geochronology studies, as a key element for the understanding of plant palaeobiogeography, ecology and evolution worldwide. We expect this contribution to shed light on the evolutionary origins of carrots, and related plant groups, an important element of human food, and to better comprehend the evolution of plant insular woodiness.

To accurately delimit species the use of multiple character types is essential as all speciation processes are not equally reflected in different data (e.g. morphological, molecular or ecological characters). With the introduction of geometric morphometrics methods and advances in 3D technology, a comprehensive combination of molecular and morphological data has been enabled in groups where exhaustively quantifying and measuring morphological shape change was not possible before such as gastropod shells. In this study, we combined multilocus coalescent species delimitation methods with 3D geometric morphometrics of shell shape to delimit species within the land snail genus Pyrenaearia. A new taxonomic scheme was constructed for the genus identifying ten species. Two nominal species were synonymized and a hitherto unrecognized cryptic species was identified. Our findings support the importance of combining multiple lines of evidence as molecular and morphological data on their own do not yield the same information. Further, the integration of morphological and molecular data shows the importance of allometry in shell shape and suggests a combined effect of population history and selection in different environments on shells morphological variation. Our new taxonomy and phylogenetic reconstruction suggest that, besides the glacial cycles of the Pleistocene, passive dispersal and rock substrate complexity could also have been involved in the speciation of the genus.

Agriculture expansion and intensification are having a huge impact on plant and arthropod diversity and abundance, affecting food availability for farmland birds. Difficult food access, in turn, can lead to immunosuppression and a higher incidence of parasites. In studies designed to examine changes in the diet of birds and their parasites, metabarcoding is proving particularly useful. This technique requires mini-barcodes capable of amplifying the DNA of target organisms from faecal eDNA. To help understand the impact of agricultural expansion, this study sought to design and identify mini-barcodes that might simultaneously assess diet and intestinal parasites from the faeces of farmland birds. The capacity to identify diet and parasites of two existing and three developed mini-barcodes was tested “in silico” in relation to the behaviour of a reference eukaryotic primer set. Of the mini-barcodes tested, MiniB18S_81, one of our designs, showed the higher taxonomic coverage of eukaryotic taxa and a greater amplification and identification capacity for diet and parasite taxa sequences. This primer pair was tested on faeces samples from five different steppe bird species. According to our data, this barcode shows good taxonomic resolution of the most relevant diet and parasite phyla, Arthropoda, Nematoda, Platyhelminthes and Apicomplexa, at the order level. The mini-barcode developed emerges as an excellent tool to simultaneously provide detailed information regarding the diet and parasitology of birds, essential for conservation and management.

Made available in DSpace on 2018-12-11T16:50:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-04-15 Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) The Cretaceous trachydacites and dacites of Chapecó type (ATC) and dacites and rhyolites of Palmas type (ATP) make up 2.5% of the ~ 800.000 km3 of volcanic pile in the Paraná Magmatic Province (PMP), emplaced at the onset of Gondwana breakup. Together they cover extensive areas in southern Brazil, overlapping volcanic sequences of tholeiitic basalts and andesites; occasional mafic units are also found within the silicic sequence. In the central region of the PMP silicic volcanism comprises porphyritic ATC-type, trachydacite high-grade ignimbrites (strongly welded) overlying aphyric ATP-type, rhyolite high- to extremely high-grade ignimbrites (strongly welded to lava-like). In the southwestern region strongly welded to lava-like high-grade ignimbrites overlie ATP lava domes, while in the southeast lava domes are found intercalated within the ignimbrite sequence. Characteristics of these ignimbrites are: widespread sheet-like deposits (tens to hundreds of km across); absence of basal breccias and basal fallout layers; ubiquitous horizontal to sub-horizontal sheet jointing; massive, structureless to horizontally banded-laminated rock bodies locally presenting flow folding; thoroughly homogeneous vitrophyres or with flow banding-lamination; phenocryst abundance presenting upward and lateral decrease; welded glass blobs in an ‘eutaxitic’-like texture; negligible phenocryst breakage; vitroclastic texture locally preserved; scarcity of lithic fragments. These features, combined with high eruption temperatures (≥ 1000 °C), low water content (≤ 2%) and low viscosities (104–7 Pa s) suggest that the eruptions were characterized by low fountaining, little heat loss during collapse, and high mass fluxes producing extensive deposits. Instituto de Geociências e Ciências Exatas Universidade Estadual Paulista (UNESP) Faculdade de Ciências and Instituto Dom Luiz Universidade de Lisboa Instituto de Geociências e Ciências Exatas Universidade Estadual Paulista (UNESP) CAPES: PDSE-2998/13-0 CNPq: 484657/2011-9

In oceanic islands, well age-constrained deposits containing arthropod somatofossils (body fossils) are rare. However, when available, these are important for providing empirical and independent minimum ages for molecular phylogenetic dating and complementary data on taxonomy, evolution and palaeobiogeography information of the biological groups found as fossils. This is especially important for taxa that speciated within oceanic islands, many becoming single island endemics (SIE). Recently, associated with a 1.3 Ma (Calabrian) fluvial and lacustrine sedimentary deposit from Porto da Cruz in Madeira Island (Fig. 1), a wing, putatively identified as Hymenoptera, was found. Here we describe this wing fossil as belonging to Ichneumonidae, a group with ca. 30% of SIE in Madeira Island. Moreover, this is the first somatofossil of ichneumonid parasitic wasps found in Madeira Island and in Macaronesian islands (i.e. Azores, Madeira, Canaries and Cabo Verde). Since the 19th century, oceanic island attracted several naturalists due to the high probability of finding taxonomical novelties (e.g. Vieira, 2005). Darwin (1859) amplified this interest, as oceanic islands biota presented a central role to explain evolution. Today oceanic islands became the ideal locations to study evolution, biogeography and ecology (e.g. Whittaker et al., 2017). Madeira Island (Central Atlantic Ocean; Fig. 1), geologically a shield volcano of 7 Ma (Ramalho et al., 2015 and references therein), is considered an insect diversity hotspot where 3019 species and subspecies are known, of which 665 are SIE (Borges et al., 2008). This diversity is most probably explained by stepping-stone through palaeo-Macaronesian islands and isolation (Triantis et al., 2010; Fernández-Palacios et al., 2011). Palaeoentomological records are rare in Madeira. The only known record is from the Mio-Pleistocene deposit of São Jorge (see Góis-Marques et al., 2018), where Heer (1857) described an extinct coleopteran, Laparocerus wollastoni, based on fossilized elytra. Machado (2006) in a taxonomic review of Laparocerus considers this taxon as nomen dubium, due to the missing holotype and the impossibility of reapraising its taxonomy. On other Macaronesian archipelagos, especially in the Canaries Islands, several deposits with insect ichnofossils have been described (e.g. Edwards & Meco, 2000; Meco et al., 2011; La Roche et al., 2014). In Azores only xylophagous ichnoentomological traces in charcoal wood are known (Góis-Marques et al., 2019b). The fossil wing was found within laminated lacustrine fine sandstone, associated with plant fossils. The sediments are constrained by two 40Ar-39Ar dates to 1.3 Ma, Calabrian stage (Góis-Marques et al., 2019a). Fossils are kept in the palaeobotanical collection at the Madeira University herbarium (UMad-P) with the numbers UMad-P500a (part) and UMad-P500b (counter-part). The wing fossil was studied under a stereo microscope, and its identification was performed through several sources (e.g. Goulet & Huber, 1993) and specific guidebooks (Prehn & Raper, 2016). Wing description follows the Comstock-Needham system as described by Quicke (2015)

The occurrence of plant fossils on Madeira Island has been known since the mid-nineteenth century. Charles Lyell and George Hartung discovered a leaf bed rich in Lauraceae and fern fossils at Sao Jorge in 1854. The determinations were controversial but a full review was never performed. Here we propose possible geological settings for the fossiliferous outcrop, and present an inventory and a systematic review of the surviving specimens of the Sao Jorge macroflora. The Sao Jorge leaf bed no longer outcrops due to a landslide in 1865. It was possible to establish the two alternative volcano-stratigraphical settings in the sedimentary intercalations from the Middle Volcanic Complex, ranging in age from 7 to 1.8 Ma. The descriptions of Heer (1857), Bunbury (1859) and Hartung & Mayer (1864) are reviewed based on 82 surviving specimens. From the initial 37 taxa, we recognize only 20: Osmunda sp., Pteridium aquilinum, Asplenium cf. onopteris, aff. Asplenium, cf. Polystichum, cf. Davallia, Woodwardia radicans, Fi...

The general dynamic model of oceanic island biogeography (GDM) predicts the immigration, speciation and extinction of terrestrial biota through geological time on oceanic islands. Additionally, the glacial-sensitive model of island biogeography (GSM) also predicts extinction due to eustatic and climate change within islands. However, well-documented and natural pre-Holocene plant extinctions are almost unknown for oceanic islands worldwide. To test these predictions, we have sampled the Early Pleistocene Porto da Cruz lacustrine and fluvial sediments for plant fossils that could confirm the GDM and GSM extinction predictions. Additionally, two new 40Ar/39Ar geochronological analyses were performed, constraining the age of the sediments to 1.3 Ma (Calabrian). Among the fossils, Eurya stigmosa (R.Ludw.) Mai (Theaceae) seeds were recognised and studied by scanning electron microscopy (SEM). E. stigmosa is the first report of a natural (non-anthropogenic) extinct plant in the fossil record for Madeira Island, and for an oceanic island, confirming the GDM and GSM predictions. Eurya spp. palaeobiogeography indicates wider distribution in Europe until the end of the Pliocene (2.58 Ma), becoming extirpated to small refugia and extinct thereafter. The Madeiran record expands the formerly unknown presence of E. stigmosa to the Macaronesian realm. The new dating of the deposit at 1.3 Ma (Calabrian) means that E. stigmosa in Madeira was already in a refugium. The extinction in Madeira is most probably a combination of island ontogeny and climate change due to Pleistocene glaciations. The palaeoecological role of this extinct shrub or tree is currently unknown, but it was a probably an element of the Madeiran laurel forest, as this community was already present in Madeira at least 1.8 My ago. This new information corroborates the predictive power of GDM and GSM and adds a new view on the importance of studying oceanic island palaeobotany, specially palaeocarpofloras.

This work was supported by the European Union’s Horizon 2020 research and innovation program under Grant 810856, National Funds Grant ERA-MBT/ 0003/2014, Ph.D. fellowship PD/BD/135187/2017 (to I. B. T.), and postdoctoral fellowships SFRH/BPD/94050/2013 and SFRH/BPD/93164/2013 (to E. M. and B. F. M.), through the FCT-Fundação para a Ciência e Tecnologia. The authors declare that they have no conflicts of interest with the contents of this article. Acknowledgments—We thank Isabel Pacheco for help in the purification of SfSIP and Dr. Américo Duarte for providing ferredoxin. We also thank all members of the Inorganic Biochemistry and NMR Laboratory for discussions and comments and feedback regarding the preparation of the manuscript. The NMR experiments were performed at CERMAX (Centro de Ressonância Magnetica António Xavier) and was financially supported by Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular) funded by FEDER funds through COMPETE2020-Programa Opera-cional Competitividade e Internacionalização (POCI), the European Community’s Seventh Framework Program (FP7/2007–2013)) under Grant Agreement 283570 (BioStruct-X). Beamtime at I04 at the Diamond Light Source and assistance from the beamline staff during the synchrotron data collections are gratefully acknowledged. Siderophores make iron accessible under iron-limited conditions and play a crucial role in the survival of microorganisms. Because of their remarkable metal-scavenging properties and ease in crossing cellular envelopes, siderophores hold great potential in biotechnological applications, raising the need for a deeper knowledge of the molecular mechanisms underpinning the siderophore pathway. Here, we report the structural and functional characterization of a siderophore-interacting protein from the marine bacterium Shewanella frigidimarina NCIBM400 (SfSIP). SfSIP is a flavin-containing ferric-siderophore reductase with FAD- and NAD(P)H-binding domains that have high homology with other characterized SIPs. However, we found here that it mechanistically departs from what has been described for this family of proteins. Unlike other FAD-containing SIPs, SfSIP did not discriminate between NADH and NADPH. Furthermore, SfSIP required the presence of the Fe 2+ -scavenger, ferrozine, to use NAD(P)H to drive the reduction of Shewanella-produced hydroxamate ferric-siderophores. Additionally, this is the first SIP reported that also uses a ferredoxin as electron donor, and in contrast to NAD(P)H, its utilization did not require the mediation of ferrozine, and electron transfer occurred at fast rates. Finally, FAD oxidation was thermodynamically coupled to deprotonation at physiological pH values, enhancing the solubility of ferrous iron. On the basis of these results and the location of the SfSIP gene downstream of a sequence for putative binding of aerobic respiration control protein A (ArcA), we propose that SfSIP contributes an additional layer of regulation that maintains cellular iron homeostasis according to environmental cues of oxygen availability and cellular iron demand. publishersversion published

The subfamily Leptaxinae is included within the highly diverse land snail family Hygromiidae. In the absence of clear diagnostic morphological differences, the subfamily status is currently based solely on molecular information and includes three disjunctly distributed tribes, Leptaxini, Cryptosaccini and Metafruticicolini. However, the phylogenetic relationships among these tribes are not fully resolved and the clustering of some of the genera to the tribes is not statistically supported. To resolve the relationships within Leptaxinae and their position within Hygromiidae, we reconstructed their phylogeny using a multi-locus approach with two mitochondrial genes and eight nuclear markers. The phylogeny was further calibrated and an analysis of ancestral area estimation was carried out to infer the biogeographic history of the group. We elevated Metafruticicolini to subfamily level (Metafruticicolinae) and we restricted Leptaxinae to Cryptosaccini and Leptaxini. The Lusitanian genus Portugala was moved to Leptaxini, previously containing only the Macaronesian genus Leptaxis. Within Cryptosaccini, a new genus strictly confined to the Sierra de la Cabrera (Spain) is described, Fractanella gen. nov. According to our results, Leptaxinae originated in the Early Miocene in the Iberian Peninsula, from which the Macaronesian Islands were colonized. Due to the old split recovered for the divergence between Macaronesian and Iberian lineages, we hypothesize that this colonization may have occurred via the once emerged seamounts located between the archipelagos and the European and African continents, although this could also have occurred through the oldest now emerged islands of Macaronesia. In the Iberian Peninsula, the climatic shift that began during the Middle Miocene, changing progressively from subtropical climate towards the present-day Mediterranean climate, was identified as an important factor shaping the subfamily's diversification, along with Pleistocene climatic fluctuations.

Aim We infer the evolutionary history of the land snail genus Allognathus from a molecular phylogeny. An approximate temporal framework for its colonization of the Balearic Islands and diversification within the archipelago is provided according to palaeogeographical events in the western Mediterranean Basin. Location The Balearic Islands, Western Mediterranean. Methods A 2461-bp DNA sequence dataset was generated from one nuclear and two mitochondrial gene fragments in 87 specimens, covering all nominal taxa of the genus Allognathus. Through maximum-likelihood and Bayesian phylogenetic methods along with a Bayesian molecular clock, we examined the evolutionary history of the group. Ancestral distribution ranges were estimated for divergence events across the tree using a Bayesian approach. We also used genetic species-delimitation models to determine the taxonomy of Allognathus. Results We provided the first molecular phylogeny of Allognathus, a genus endemic to the Balearic Islands. The origin of the genus in the Balearic Islands was dated to the middle Miocene based on palaeogeographical events in the Western Mediterranean. During the late Miocene and Pliocene, several diversification events occurred within the archipelago. The ancestral range of Allognathus was reconstructed as the north-eastern Tramuntana Mountains of Mallorca. Main conclusions Three species were delimited within the genus, one of which has at least five subspecies. The phylogenetic reconstruction showed a high degree of parallelism between the divergence of the main Allognathus lineages and the palaeogeography of the Balearic Islands. The genus appears to have colonized Mallorca from the south-east of the Iberian Peninsula during the middle Miocene. Sea level fluctuations that took place in the Western Mediterranean from the Messinian to the present are consistent with the diversification and secondary contacts of the phylogroups of Allognathus, as well as their distribution ranges. The middle Miocene could have been a period for the colonization of the Balearic Islands by other terrestrial organisms.

The Helicoidea is one of the most diverse superfamilies of terrestrial land snails. In this study we present a molecular phylogeny of the western Palaearctic Helicoidea obtained by means of neighbor joining, maximum likelihood and Bayesian analysis of the mitochondrial 16S rRNA gene fragment and the nuclear rRNA gene cluster including the 30 end of the 5.8S gene, the complete ITS2 region and 50 end of the large subunit 28S. Most of the morphologically-defined families were confirmed. We propose a revised phylogenetic classification so that families, subfamilies and tribes are monophyletic. The family Hygromiidae sensu Hausdorf and Bouchet (2005) is divided into three clades which are here given familial rank: Canariellidae and Geomitridae, which are recognized for the first time at familial rank, and Hygromiidae s.str. (including Ciliella and Trochulus) that is here restricted. The subfamilies Ciliellinae, Geomitrinae, Hygromiinae, Monachainae and Trochulinae recognized in current classifications were not recovered as monophyletic groups. The family Cochlicellidae is here given tribe rank (Cochlicellini) belonging to the Geomitridae. We describe a new tribe, Plentuisini. Three subfamilies are recognized within Helicidae: Ariantinae, Helicinae (including Theba) and Murellinae. New classification indicates that free right ommatophore retractor muscle arose only once within Geomitridae. The anatomy of the auxiliary copulatory organs of the reproductive system of families, subfamilies and tribes is highlighted. We estimate the origin of the Helicoidea at the end of the Early Cretaceous and its families as Late-Cretaceous to Paleogene. Western Palaearctic Helicoidea belongs to two different lineages that diverged around 86 Ma ago, both starting their diversification at the end of the Cretaceous (around 73–76 Ma). Radiation of some western Helicoidean families started during the Eocene.

The genus Candidula (Geomitridae), consisting of 28 species in Western Europe as currently described, has a disjunct distribution in the Iberian Peninsula, Italy, the Balkans, the Aegean Islands, and one species on the Canary Islands. Although the genus is seemingly well defined by characters of the reproductive system, the relationships within the genus are still unclear and some authors have indicated a possible subgeneric division based on the internal morphology of the dart sac. Despite substantial phylogenetic incongruence, we present a well-resolved molecular phylogeny of Candidula based on two mitochondrial genes (COI and 16S rRNA), the nuclear rDNA region (5.8S rNRA + ITS2 + 28S rRNA) and seven additional nuclear DNA regions developed specifically for this genus (60SL13, 60SL17, 60SL7, RPL14, 40SS6, 60SL9, 60SL13a), in total 5595 bp. Six reciprocally monophyletic entities including Candidula species were recovered, grouping into two major clades. The incorporation of additional geomitrid genera allowed us to unequivocally demonstrate the polyphyly of the genus Candidula. One major clade grouped species from southern France and Italy with the widely distributed species C. unifasciata. The second major clade grouped all the species from the Iberian Peninsula, including C. intersecta and C. gigaxii. Candidula ultima from the Canary Islands was recovered as separated lineage within the latter clade and related to African taxa. The six monophyla were defined as six new genera belonging to different tribes within the Helicellinae. Thus, we could show that similar structures of the stimulatory apparatus of the genital system in different taxa do not necessarily indicate a close phylogenetic relationship in the Geomitridae. More genera of the family are needed to clarify their evolutionary relationships, and to fully understand the evolution of the stimulatory apparatus of the genital system within the Geomitridae.

In Faial Island (Azores Archipelago, North Atlantic Ocean), charcoalified and mummified wood fossils have been reported within late Holocene (Meghalayan) pyroclastic deposits from the Caldeira Formation. Due to their recent age, a detailed study conveys a snapshot into Azorean palaeophytodiversity and palaeovegetation, ca. 7–5 centuries before the arrival of Portuguese settlers to the Azores Islands. Here we provide the first detailed anatomical and taxonomical study of these wood fossils. In total, 41 samples were collected from seven localities, mainly from a ~ 1200 yr BP ignimbrite. Field work revealed autochthonous and paraautochthonous assemblages, with tree trunks in upright position. The anatomical study of the fossil woods resulted in the identification of Juniperus brevifolia, Laurus azorica, Myrsine retusa, Morella faya, Picconia azorica, Prunus lusitanica subsp. azorica, and Vaccinium cylindraceum. Two fossil assemblages are comparable to the proposed potential natural vegetation (PNV) for the Azores. Surprisingly, P. lusitanica subsp. azorica was the second most abundant fossil wood suggesting that this tree was more abundant in a recent past in Faial Island and probably in the archipelago. This is corroborated by historical accounts, and its modern scarcity was certainly anthropically driven. Identifying Holocene plant macrofossils is essential to properly reconstruct oceanic islands terrestrial palaeoecosystems, especially where forests with high percentage of entomophilous taxa are underrepresented in palaeopalynological limnic record. Further work is necessary to reconstruct Faial Island and Azores archipelago palaeovegetation which is essential to provide an ecosystem base-line for restoration and management.

The role of southern European peninsulas as glacial refugia for temperate species has been widely established, but the role of cryptic northern refugia has only recently been addressed. Here, we describe the phylogeographic pattern of the forest-dwelling European pine marten (Martes martes), using a 1600-bp mitochondrial DNA (mtDNA) fragment from 287 individuals sampled across the entire distribution range of the species. To clarify the relationships between M. martes and its sister species the sable (Martes zibellina) in Fennoscandia and Russia, ten M. zibellina samples were also included in the analyses. Our results reveal the presence of 69 different haplotypes for M. martes and ten haplotypes for M. zibellina, which are split into three major assemblages: Mediterranean, central–northern European, and Fennoscandian–Russian clades, showing a global pattern of spatial segregation, with some area of overlap and genetic admixture. It is apparent that the Mediterranean phylogroup did not significantly contribute to the postglacial recolonization of most of the Palaearctic range of the species. Instead, most of Europe was colonized by the central–northern European phylogroup, which probably survived the last glaciations in northern cryptic refugia, as has previously been suggested by palaeontological studies. A highly divergent phylogroup has been discovered in Fennoscandia–Russia, which includes specimens from both Martes species. Calculations of divergence times suggest that the phylogroups split during the Pleistocene. Overall, our study indicates a complex phylogeographic history for M. martes, indicating a mixed pattern of recolonization of northern Europe from both Mediterranean and non-Mediterranean refugia, providing new insights into the existence of cryptic northern glacial refugia for temperate species in Europe. © 2013 The Linnean Society of London, Biological Journal of the Linnean Society, 2013, 109, 1–18.

Gomez-Moliner, B.J., Elejalde, A.M., Arrebola, J.R., Puente, A.I., Martinez-Orti, A., Ruiz, A. & Madeira, MJ. (2012). Molecular phylogeny of the Helicodontidae and Trissexodontidae (Gastropoda). —Zoologica Scripta, 00, 000–000. In this study, we present a molecular phylogeny of the Trissexodontidae and Helicodontidae obtained by means of Maximum Parsimony, Neighbor Joining, Maximum Likelihood and Bayesian analyses of DNA sequences. Nearly 3 KB of sequence data of two mitochondrial genes (COI, 16S rDNA) and the nuclear rRNA gene cluster including ITS-1, the 3′end of the 5.8S gene, the complete ITS-2 region and 5′ end of the large subunit 28S were used to reconstruct the phylogeny of these two families. Monophyly of Trissexodontidae and Helicodontidae at the family level is well supported. A new classification of the genera in the Trissexodontidae is proposed. It includes two subfamilies: Gittenbergeriinae (monotypic for Gittenbergeria turriplana) and Trissexodontinae. The latter includes three strongly supported tribes: (i) Trissexodontini, including Mastigophallus, Trissexodon, Oestophorella and Suboestophora; (ii) Oestophorini, with Oestophora; and (iii) Caracollinini, with Caracollina, Gasulliella, Gasullia and Hatumia. The polytypic Oestophora and Suboestophora are recovered as two monophyletic genera. The anatomy of the auxiliary copulatory organs of the reproductive system is coherent with the new taxonomic interpretation of the Trissexodontidae. Further work, including some more taxa is needed to delimitate subfamilies within Helicodontidae. Finally, the addition of some sequences of other Helicoidea shows that the genus Ciliella is not closely related to Trissexodontidae, being grouped within the Hygromiidae, instead.

The increasing fragmentation of natural habitats may strongly affect patterns of dispersal and gene flow among populations, and thus alter evolutionary dynamics. We examined genetic variation at twelve microsatellite loci in the Agile frog (Rana dalmatina) from 22 breeding ponds in the Iberian Peninsula, the southwest limit of its range, where populations of this species are severely fragmented and are of conservation concern. We investigated genetic diversity, structure and gene flow within and among populations. Diversity as observed heterozygosities ranged from 0.257 to 0.586. The mean number of alleles was 3.6. Just one population showed a significant FIS value. Four populations show evidence of recent bottlenecks. Strong pattern of structure was observed due to isolation by distance and to landscape structure. The average degree of genetic differentiation among populations was FST = 0.185. Three operational conservation units with metapopulation structure were identified. Additionally, there are some other isolated populations. The results reinforce the view that amphibian populations are highly structured even in small geographic areas. The knowledge of genetic structure pattern and gene flow is fundamental information for developing programmes for the preservation of R. dalmatina at the limits of its geographic distribution.

The first data about the genus Cryptazeca were provided by Folin & Berillon (1877a) when they described azeca monodonta based on the study of two empty shells collected near Bayonne in southwestern France. Later they collected some live specimens and established the genus Cryptazeca for it, after study of its anatomy (Folin & Berillon, 1877b, 1891). This genus was included together with Ferussacia, Cecilioides, azeca (with Hypnophila), Cochlicopa, and five additional genera in the family Ferussaciidae (Pilsbry, 1908). Watson (1928) noticed that Ferussacia and Cecilioides had a sigmurethrous excretory system and placed them and the family Ferussaciidae in the infraorder Sigmurethra, whereas Steenberg (1925) and Zilch (1959) placed azeca, Cochlicopa, and Hypnophila in the family Cochlicopidae (infraorder Orthurethra). New live specimens of the genus Cryptazeca were collected in 1987 and its excretory system described as being of the orthurethrous type by Gomez & Angulo (1987). Consequently, these authors proposed to change the taxonomic position of this genus to be placed with azeca, Cochlicopa, and Hypnophila in the family Cochlicopidae. Detailed anatomical and histological studies of the reproductive system also corroborated the close relationships among Cryptazeca, azeca, and Hypnophila (Gomez & Angulo, 1987, 1990; Gomez, 1990b, 1991). Nevertheless, Schileyko (1976) considered that orthurethria was probably a primary condition for Stylommatophora, which could be retained in some taxa outside orthurethral groups. This led again to the placement of Cryptazeca within the family Ferussaciidae. As a result, the classification followed by the CLECOM project (Bank et al., 2001; Falkner et al., 2001) placed Cryptazeca (Cryptazecinae) with Ferussacia, Cecilioides, and Hohenwartiana (Ferussaciinae) within the Ferussaciidae, in the superfamMALACOLOGIA, 2010, 52(1): 163−168

Iberus gualtieranus is a species complex of land snails that is endemic to the Iberian Peninsula. The species taxonomy of the group is based merely on the basis of shell morphology, but validity of the existing taxonomy is uncertain. Using mitochondrial DNA (mtDNA) data (cytochrome oxidase I and 16S rRNA sequences) we were able to validate the observed phylogenetic taxa within the I. gualtieranus s.l. complex by means of the analysis of specimens of the different morphospecies, together with the study of topotypes. Strong incongruences were obtained between morphology and molecular data. The Iberus alonensis morphospecies comprised several genetically divergent but morphologically cryptic lineages. Considering (1) the allopatric distribution of the operational taxonomic units (OTUs), (2) the morphological differentiation, (3) the possible occurrence of hybridization among the different lineages, and (4) the strong differentiation of the mtDNA phylogroups, we suggest the main lineages obtained, for the time being, may be treated as evolutionary species. The robust phylogenetic reconstruction obtained allows us to consider I. alonensis s.s., Iberus campesinus, Iberus carthaginiensis, and Iberus gualtieranus s.s. as valid species. Two additional unnominated taxa of the alonensis shell type have also been identified. Further subdivisions are also considered, including Iberus gualtieranus mariae and Iberus gualtieranus ornatissimus. © 2008 The Linnean Society of London, Zoological Journal of the Linnean Society, 2008, 154, 722-737. ADDITIONAL KEYWORDS: 16S - COI - gastropoda - helicidae - Iberian Peninsula - molecular phylogeny - morphospecies concept.

Partial DNA sequences of two mitochondrial genes [cytochrome oxidase subunit I (COI) and 16S rRNA] from 59 specimens of Iberus were used to test the validity of the described morphospecies of this genus, and examine genetic divergences within and between main phylogenetic groups. Both gene fragments showed phylogenetic concordance. The COI gene was found to be faster evolving than the 16S gene and was fully protein-coding with no insertions or deletions. 16S rRNA was more informative than COI for resolving basal nodes. Both individual and combined analyses of the two gene fragments revealed five main phylogroups. These five groups are genetically unique lineages that are allopatrically distributed and considered to have full species status. Further subdivisions were also considered. Shell morphology was suitable for delimiting species boundaries, but several incongruences between morphology and mtDNA phylogeny were observed. These incongruences were considered consequence of hybridization between Iberus cobosi and Iberus marmoratus, and the result of shell shape polymorphism in Iberus rositai. According to spatial patterns of sequence divergence, life habits and shell morphology may be concluded that the keeled-flat shelled snails independently originated several times within Iberus and they could represent cases of similar shell adaptation to a karstic arid environment. Resumen Se han analizado dos fragmentos del ADN mitocondrial (COI y 16S rRNA) de 59 ejemplares de Iberus con el fin de determinar la validez de las morfoespecies de tamano medio descritas para este genero, asi como para analizar las divergencias geneticas existentes entre los principales grupos filogeneticos identificados. Los resultados obtenidos para ambos genes fueron similares. La tasa evolutiva fue mas rapida en el COI, fragmento codificante, no presentando inserciones ni deleciones. Por otra parte, el fragmento del 16S fue mas informativo en la resolucion de los nodos basales. Tanto los analisis individuales de ambos fragmentos como el analisis combinado permitieron identificar cinco linajes geneticos unicos que presentan una distribucion alopatrica y son considerados como cinco especies diferentes. Ademas, se contemplan otras subdivisiones adicionales. Se ha observado que existe concordancia entre las morfoespecies y los filogrupos obtenidos, indicando que la concha es un criterio taxonomico valido para la delimitacion de especies en este grupo. No obstante, se han observado varias incongruencias entre la morfologia y la filogenia obtenida mediante secuenciacion del ADNmt. Estas incongruencias parecen ser consecuencia de procesos de hibridacion entre I. cobosi e I. marmoratus, pero parecen ser resultado del polimorfismo de la concha en I. rositai. Teniendo en consideracion los filogramas obtenidos, el modo de vida y la morfologia de la concha, se puede concluir que las conchas aquilladas y aplanadas se originaron varias veces independientemente en Iberus y que parecen constituir ejemplos de adaptacion de la concha a ambientes karsticos aridos.

Restriction site-associated DNA sequencing (RADseq) was used to jointly assess phylogenetic relationships, interspecific hybridization and species delimitation in the cryptic, non-model land snail complex Pyramidula. A robust phylogeny was inferred using a matrix of concatenated sequences of almost 1,500,000 by long, containing >97,000 polymorphic sites. Maximum likelihood analyses fully resolved the phylogenetic relationships among species and drastically improved phylogenetic trees obtained from mtDNA and nDNA gene trees (COI, 16S rRNA, 5.8S rRNA, ITS2 and 28S rRNA sequence data). The best species delimitation scenario was selected on the basis of 875 unlinked single nucleotide polymorphisms, showing that nine Pyramidula species should be distinguished in Europe. Applying D-statistics provided no or weak evidence of interspecific hybridization among Pyramidula, except for some evidence of gene flow between two species. (C) 2016 Elsevier Inc. All rights reserved.

The stone marten is a widely distributed mustelid in the Palaearctic region that exhibits variable habitat preferences in different parts of its range. The species is a Holocene immigrant from southwest Asia which, according to fossil remains, followed the expansion of the Neolithic farming cultures into Europe and possibly colonized the Iberian Peninsula during the Early Neolithic (ca. 7,000 years BP). However, the population genetic structure and historical biogeography of this generalist carnivore remains essentially unknown. In this study we have combined mitochondrial DNA (mtDNA) sequencing (621 bp) and microsatellite genotyping (23 polymorphic markers) to infer the population genetic structure of the stone marten within the Iberian Peninsula. The mtDNA data revealed low haplotype and nucleotide diversities and a lack of phylogeographic structure, most likely due to a recent colonization of the Iberian Peninsula by a few mtDNA lineages during the Early Neolithic. The microsatellite data set was analysed with a) spatial and non-spatial Bayesian individual-based clustering (IBC) approaches (STRUCTURE, TESS, BAPS and GENELAND), and b) multivariate methods [discriminant analysis of principal components (DAPC) and spatial principal component analysis (sPCA)]. Additionally, because isolation by distance (IBD) is a common spatial genetic pattern in mobile and continuously distributed species and it may represent a challenge to the performance of the above methods, the microsatellite data set was tested for its presence. Overall, the genetic structure of the stone marten in the Iberian Peninsula was characterized by a NE-SW spatial pattern of IBD, and this may explain the observed disagreement between clustering solutions obtained by the different IBC methods. However, there was significant indication for contemporary genetic structuring, albeit weak, into at least three different subpopulations. The detected subdivision could be attributed to the influence of the rivers Ebro, Tagus and Guadiana, suggesting that main watercourses in the Iberian Peninsula may act as semi-permeable barriers to gene flow in stone martens. To our knowledge, this is the first phylogeographic and population genetic study of the species at a broad regional scale. We also wanted to make the case for the importance and benefits of using and comparing multiple different clustering and multivariate methods in spatial genetic analyses of mobile and continuously distributed species. This study has been partially funded by the Basque Government through the Research group on "Systematics, Biogeography and Population Dynamics'' (Ref. IT317-10; GIC10/76; IT575-13). MV (Ref: RBFI-2012-446) and ARG (Ref: DKR-2012-64) were supported by a PhD and post-doctoral fellowships awarded by the Dept. of Education, Universities and Research of the Basque Government. MPB and CF acknowledge financial support from Fundacao para a Ciencia e Tecnologia through the PhD fellowship SFRH/BD/38410/2007, the Ciencia 2007 contract C2007-UL-342-CBA1, and the research project PTDC/BIA-BEC/101511/2008. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Iberian and southern French populations of the genus Gobio , considered in the past to be populations of Gobio gobio , are assigned to a new species ( Gobio lozanoi n. sp. ) based on genetic and morphological characters. This new species of the genus Gobio is found in the basins of the Rivers Adour in France and Bidasoa, Duero, Ebro, Guadalete, Guadiana, Guadalquivir, Jucar, Llobregat, Mondego, Mijares, Nalon, Nansa, Mino, Segura, Tajo, and Turia in the Iberian Peninsula. The new species is distinguished from Gobio gobio by a combination of the following characters: 36-39 scales on the lateral line, 3 scales below the lateral line. The distance between the pectoral and ventral fins is greater than that between the ventral and anal fins (VAL/PVL≤0.8). The preorbital distance is short and the head is wide (ED/PrOL≥0.56; PrOL/HH≤0.69; PrOL/HW≤0.68). Divergence distances in cytochrome b between Gobio gobio and the new species are “ p ”=4.8-5.9%.

Recent advances in molecular scatology have allowed the development of reliable and non-invasive methods that can be applied in monitoring of small carnivores, without disturbance of the animals. Here a method is described that can be used to differentiate European mink Mustela lutreola, polecat M. putorius and American mink M. vison based on the analysis of DNA extracted from faeces. It consists of a nested PCR of a region of the mitochondrial D-loop followed by digestion of the resulting 240 bp amplicons with the restriction enzymes RsaI and MspI. The restriction patterns of both enzymes, when used together, are found to detect species-specific sequence variation. Two different haplotypes for European mink (AA, AB), another two for the polecat (AC, AD) and one for American mink (BC) can also be discriminated by this technique. Two new haplotypes for the mitocondrial D-loop of mustelids are described after DNA sequencing.

The mean age of a population of agile frogs (Rana dalmatina) from the Iberian Peninsula was estimated using mark and recapture and skeletochronology. Life-history parameters, including growth rate, body length, age and size at maturity, sexual dimorphism and longevity, were studied. The regression between age and snout-vent length (SVL) was highly significant in both sexes. Males reached sexual maturity at two years of age, although sometimes they can reach it at only one year of age. The average SVL at maturity was 51.75 mm (standard error (SE)=0.71; n=45). Females reached sexual maturity at two years of age with an average SVL of 62.14 mm (SE=2.20; n=14). A subset of the female population reached sexual maturity at three years of age. Growth was rapid until sexual maturity was reached. There was an overlap of SVL between different age classes. Growth was continuous, fulfilling the conditions of Von Bertalanffy's model. The growth coefficient (K) was 0.840 in males and 0.625 in females. The maximum SVL was greater in females (73.00 mm) than in males (59.50mm). Sexual dimorphism was significantly biased towards females in all age classes. The maximum longevity observed was 6 years in females and 8 years in males. Management strategies for agile frogs should take into account factors such as these life-history characteristics.

The brown trout (Salmo trutta L.) is one of the best studied native salmonids of Europe. Genetic studies on this species suggest that a large proportion of the evolutionary diversity corresponds to southern European countries, including the Iberian Peninsula, where this study is focused. Stocking activities employing non-indigenous hatchery specimens together with the destruction and fragmentation of natural habitats are major factors causing a decrease of native brown trout populations, mostly in the Mediterranean basins of the Iberian Peninsula. The main aim of the present work is to examine the genetic structure of the brown trout populations of the East Cantabrian region, studying the consequences of the restocking activities with foreign hatchery brown trout specimens into the wild trout populations. We have based our study on the Polymerase Chain Reaction and Restriction Fragment Length Polymorphism technique conducted on a mitochondrial fragment of 2700 base pairs and on the lactate dehydrogenase locus of the nuclear DNA. Our results show higher introgression rates in the Ebro (Mediterranean) basin than in the Cantabrian rivers.