Simultaneous analysis of the intestinal parasites and diet through eDNA metabarcoding.

Agricultural 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 the 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 fecal environmental DNA. To help to understand the impact of agricultural expansion on biodiversity, this study sought to design and identify mini-barcodes that might simultaneously assess diet and intestinal parasites from the feces of farmland birds. The capacity to identify diet and parasites of 2 existing and 3 newly developed mini-barcodes was tested "in silico" in relation to the behavior of a reference eukaryotic barcode. Among the newly designed mini-barcodes, MiniB18S_81 showed the higher taxonomic coverage of eukaryotic taxa and a greater amplification and identification capacity for diet and parasite taxa. Moreover, when it was tested on fecal samples from 5 different steppe bird species, MiniB18S_81 showed high taxonomic resolution of the most relevant diet and parasite phyla, Arthropoda, Nematoda, Platyhelminthes, and Apicomplexa at the order level. Thus, the mini-barcode developed emerges as an excellent tool to simultaneously provide detailed information regarding the diet and parasites of birds, essential for conservation and management.

Environmental conditions experienced upon first breeding modulate costs of early breeding but not age-specific reproductive output in peregrine falcons.

Although once considered uncommon, there is growing evidence of widespread senescence in wildlife populations. However, few studies have examined the traits involved, inter-sexual differences, and environmental correlates of age-specific performance in raptors. We studied age-specific reproductive performance and actuarial senescence (decrease in survival probability with age) in a peregrine falcon population monitored for 21 years. We analysed changes with age in the number of offspring produced and incubation start date. We also inspected variation in lifespan and breeding lifespan (number of breeding occasions in a lifetime). In every case, we assessed associations between variations in traits and age, sex, recruitment age, and environmental conditions (cumulative rainfall during breeding season) experienced upon the first breeding attempt. We found scarce evidence for reproductive senescence. Only the incubation start date in females, which was delayed after approximately 8 cy (calendar years), suggested reproductive senescence in our study population. Regarding actuarial senescence, our data did not support it as we only found evidence of higher juvenile mortality. Furthermore, expected lifespan in peregrines recruited at 2 cy was associated with conditions experienced upon the first breeding attempt. The lifespan and breeding career of individuals recruited as yearlings and experiencing low rainfall upon first breeding did not significantly differ from those recruited as adults. However, those recruited as yearlings and experiencing poor environmental conditions upon the first breeding attempt showed reduced lifespan and breeding lifespan.

Molecular phylogeny of the genus Chondrina (Gastropoda, Panpulmonata, Chondrinidae) in the Iberian Peninsula.

Chondrina Reichenbach, 1828 is a highly diverse genus of terrestrial molluscs currently including 44 species with about 28 subspecific taxa. It is distributed through North Africa, central and southern Europe, from Portugal in the West to the Caucasus and Asia Minor in the East. Approximately 70% of the species are endemic to the Iberian Peninsula constituting its main center of speciation with 34 species. This genus includes many microendemic taxa, some of them not yet described, confined to limestone habitats (being strictly rock-dwelling species). They are distributed on rocky outcrops up to 2000 m.a.s.l. It is a genus of conical-fusiform snails that differ mainly in shell characters and in the number and position of teeth in their aperture. So far, molecular studies on Chondrina have been based exclusively on the mitochondrial Cytochrome Oxidase subunit I region (COI). These studies gave a first view of the phylogeny of the genus but many inner nodes were not statistically supported. The main objective of the study is to obtain a better understanding of the phylogeny and systematics of the genus Chondrina on the Iberian Peninsula, using multilocus molecular analysis. Partial sequences of the COI and 16S rRNA genes, as well as of the nuclear Internal Transcribed Spacer 1 (ITS1-5.8S) and Internal Transcribed Spacer 2 (5.8S-ITS2-28S) were obtained from individuals of all the extant Chondrina species known from the Iberian Peninsula. In addition to this, the newly obtained COI sequences were combined with those previously published in the GenBank. Phylogenetic relationships were inferred using maximum likelihood and Bayesian methods. The reconstructed phylogenies showed high values of support for more recent branches and basal nodes. Moreover, molecular species delimitation allowed to better definethe studied species and check the presence of new taxa.

Incorporating palaeogeography into ancestral area estimation can explain the disjunct distribution of land snails in Macaronesia and the Balearic Islands (Helicidae: Allognathini).

The systematics and biogeographical history of the Eastern Mediterranean and Macaronesian land snail tribe Allognathini (Helicidae: Helicinae) is investigated based on mitochondrial and nuclear DNA sequence data. Our molecular phylogenetic analyses indicate that the genus-group systematics of the tribe needs to be revised. We show for the first time that the narrow-range endemics Lampadia and Idiomela from the Madeira Archipelago belong to Allognathini and represent together the sister group of the diverse Canary Island Hemicycla radiation. We therefore suggest synonymising Lampadiini with Allognathini. Sister to these Macaronesian genera was the Balearic Island Allognathus radiation. Pseudotachea was not recovered as a monophyletic group and the two currently recognised species clustered in Iberus. Similarly, Adiverticula was not recovered as a monophyletic group and clustered in Hemicycla. We therefore suggest synonymising Pseudotachea with Iberus and Adiverticula with Hemicycla. The six genera in Allognathini, which we distinguish here (Cepaea, Iberus, Allognathus, Hemicycla, Idiomela and Lampadia), originated in Western to South-western Europe according to our ancestral area estimation and the fossil record. The disjunct distribution of the Balearic Islands and Macaronesian sister clades and the mainly Iberian Iberus clade that separated earlier can be explained by the separation of the Betic-Rif System from the Iberian Peninsula during the late Oligocene to early Miocene, along with independent Miocene dispersals to the Balearic Islands and Macaronesia from the Iberian Peninsula, where the ancestral lineage became extinct.

Molecular phylogeny and biogeography of the land snail subfamily Leptaxinae (Gastropoda: Hygromiidae).

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.

Integrating multilocus DNA data and 3D geometric morphometrics to elucidate species boundaries in the case of Pyrenaearia (Pulmonata: Hygromiidae).

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.

Molecular phylogeny of Candidula (Geomitridae) land snails inferred from mitochondrial and nuclear markers reveals the polyphyly of the genus.

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.

Species limits, interspecific hybridization and phylogeny in the cryptic land snail complex Pyramidula: The power of RADseq data.

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 1,500,000bp 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.

Isolation by distance, resistance and/or clusters? Lessons learned from a forest-dwelling carnivore inhabiting a heterogeneous landscape.

Landscape genetics provides a valuable framework to understand how landscape features influence gene flow and to disentangle the factors that lead to discrete and/or clinal population structure. Here, we attempt to differentiate between these processes in a forest-dwelling small carnivore [European pine marten (Martes martes)]. Specifically, we used complementary analytical approaches to quantify the spatially explicit genetic structure and diversity and analyse patterns of gene flow for 140 individuals genotyped at 15 microsatellite loci. We first used spatially explicit and nonspatial Bayesian clustering algorithms to partition the sample into discrete clusters and evaluate hypotheses of 'isolation by barriers' (IBB). We further characterized the relationships between genetic distance and geographical ('isolation by distance', IBD) and ecological distances ('isolation by resistance', IBR) obtained from optimized landscape models. Using a reciprocal causal modelling approach, we competed the IBD, IBR and IBB hypotheses with each other to unravel factors driving population genetic structure. Additionally, we further assessed spatially explicit indices of genetic diversity using sGD across potentially overlapping genetic neighbourhoods that matched the inferred population structure. Our results revealed a complex spatial genetic cline that appears to be driven jointly by IBD and partial barriers to gene flow (IBB) associated with poor habitat and interspecific competition. Habitat loss and fragmentation, in synergy with past overharvesting and possible interspecific competition with sympatric stone marten (Martes foina), are likely the main factors responsible for the spatial genetic structure we observed. These results emphasize the need for a more thorough evaluation of discrete and clinal hypotheses governing gene flow in landscape genetic studies, and the potential influence of different limiting factors affecting genetic structure at different spatial scales.

Patterns of genetic variation in the endangered European mink (Mustela lutreola L., 1761).

BACKGROUND: The European mink (Mustela lutreola, L. 1761) is a critically endangered mustelid, which inhabits several main river drainages in Europe. Here, we assess the genetic variation of existing populations of this species, including new sampling sites and additional molecular markers (newly developed microsatellite loci specific to European mink) as compared to previous studies. Probabilistic analyses were used to examine genetic structure within and between existing populations, and to infer phylogeographic processes and past demography. RESULTS: According to both mitochondrial and nuclear microsatellite markers, Northeastern (Russia, Estonia and Belarus) and Southeastern (Romania) European populations showed the highest intraspecific diversity. In contrast, Western European (France and Spain) populations were the least polymorphic, featuring a unique mitochondrial DNA haplotype. The high differentiation values detected between Eastern and Western European populations could be the result of genetic drift in the latter due to population isolation and reduction. Genetic differences among populations were further supported by Bayesian clustering and two main groups were confirmed (Eastern vs. Western Europe) along with two contained subgroups at a more local scale (Northeastern vs. Southeastern Europe; France vs. Spain). CONCLUSIONS: Genetic data and performed analyses support a historical scenario of stable European mink populations, not affected by Quaternary climate oscillations in the Late Pleistocene, and posterior expansion events following river connections in both North- and Southeastern European populations. This suggests an eastern refuge during glacial maxima (as already proposed for boreal and continental species). In contrast, Western Europe was colonised more recently following either natural expansions or putative human introductions. Low levels of genetic diversity observed within each studied population suggest recent bottleneck events and stress the urgent need for conservation measures to counteract the demographic decline experienced by the European mink.

Inferring Population Genetic Structure in Widely and Continuously Distributed Carnivores: The Stone Marten (Martes foina) as a Case Study.

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.

Molecular phylogeny of the western Palaearctic Helicoidea (Gastropoda, Stylommatophora).

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 3' end of the 5.8S gene, the complete ITS2 region and 5' 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 86Ma ago, both starting their diversification at the end of the Cretaceous (around 73-76Ma). Radiation of some western Helicoidean families started during the Eocene.

Landscape genetics for the empirical assessment of resistance surfaces: the European pine marten (Martes martes) as a target-species of a regional ecological network.

Coherent ecological networks (EN) composed of core areas linked by ecological corridors are being developed worldwide with the goal of promoting landscape connectivity and biodiversity conservation. However, empirical assessment of the performance of EN designs is critical to evaluate the utility of these networks to mitigate effects of habitat loss and fragmentation. Landscape genetics provides a particularly valuable framework to address the question of functional connectivity by providing a direct means to investigate the effects of landscape structure on gene flow. The goals of this study are (1) to evaluate the landscape features that drive gene flow of an EN target species (European pine marten), and (2) evaluate the optimality of a regional EN design in providing connectivity for this species within the Basque Country (North Spain). Using partial Mantel tests in a reciprocal causal modeling framework we competed 59 alternative models, including isolation by distance and the regional EN. Our analysis indicated that the regional EN was among the most supported resistance models for the pine marten, but was not the best supported model. Gene flow of pine marten in northern Spain is facilitated by natural vegetation, and is resisted by anthropogenic landcover types and roads. Our results suggest that the regional EN design being implemented in the Basque Country will effectively facilitate gene flow of forest dwelling species at regional scale.

On the phylogenetic position of a rare Iberian endemic mammal, the Pyrenean desman (Galemys pyrenaicus).

The nucleotide sequences of the complete mitochondrial genome and nine partial nuclear genes of the Pyrenean desman (Galemys pyrenaicus) were determined in order to establish the relative phylogenetic position of this species at different taxonomic levels within the placental tree. Phylogenetic relationships of desman within the family Talpidae were inferred based on complete mitochondrial cytochrome b gene nucleotide sequence data. The Pyrenean desman was unambiguously recovered as sister group of the Russian desman (Desmana moschata) confirming the monophyly of the subfamily Desmaninae. However, phylogenetic relationships among major lineages within the Talpidae could not be confidently resolved. Phylogenetic analyses based on mitochondrial (at the amino acid level) and nuclear (at the nucleotide level) sequence data sets confidently placed desman within the Eulipotyphla (that also included moles, shrews, and hedgehogs), and partially recovered placental interordinal relationships. The monophyly of Laurasiatheria (including Eulipotyphla, Chiroptera, Carnivora, Pholidota, Perissodactyla, and Cetartiodactyla) was strongly supported. Mitochondrial amino acid sequences of Pholidota (pangolins) were found to bias phylogenetic inferences due to long-branch attraction effects. A Bayesian inference based on a combined mitochondrial and nuclear data set without Pholidota arrived at an almost fully resolved tree that supported the basal position of Eulipotyphla within Laurasiatheria.