Evaluating the functional, sexual and seasonal variation in the chemical constituents from feces of adult Iberian wolves (Canis lupus signatus).

Chemical signals deposited in feces play an important role in intraspecific and interspecific communication of many mammals. We collected fresh feces of adult wolves from wild breeding groups. All samples visually identified as belonging to wolves were subsequently identified to species level by sequencing a small fragment of mtDNA and sexed typing DBX6 and DBY7 sex markers. Using gas chromatography-mass spectrometry (GC-MS), we identified 56 lipophilic compounds in the feces, mainly heterocyclic aromatic organic compounds, such as indole or phenol, but also steroids, such as cholesterol, carboxylic acids and their esters between n-C4 and n-C18, aldehydes, alcohols and significant quantities of squalene and α-tocopherol, which would increase the chemical stability of feces on humid substrates. There was variability in the number and proportions of compounds between sexes, which could be indicative of their function as chemical signals. We also found variability in different reproductive states, especially in odorous compounds, steroids and α-tocopherol. Feces with a presumed marking function had higher proportions of α-tocopherol and steroids than feces with non-marking function. These compounds could be involved in intragroup and intergroup communication of wolves and their levels in feces could be directly related with the wolf's sex and physiological and reproductive status.

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.

Molecular assays to reliably detect and quantify predation on a forest pest in bats faeces.

Targeted molecular methods such as conventional PCR (cPCR) and quantitative PCR (qPCR), combined with species-specific primers and probes, are widely applied for pest species detection. Besides, the potential of qPCR to quantify DNA in samples makes it an invaluable molecular tool to infer the predation levels on specific prey by analysing predators' stools. Nevertheless, studies on the diet of bats failed to find any empirical relationship, and it remains to be evaluated. Thus, we developed and evaluated two species-specific PCR assays to detect and quantify DNA of a major forest pest, the pine processionary, Thaumetopoea pityocampa, in bats' faeces. Further, we empirically compared a range of different known DNA concentrations (input) of the target species mixed with mocks and bat faecal samples against DNA abundances yielded by qPCR (output) for a quantitative assessment. Overall, cPCR showed a lower detection rate than qPCR, but augmenting the replicate effort from one to three replicates led to a greater increase in the detection rate of the cPCR (from 57 to 80%) than the qPCR (from 90 to 99%). The quantitative experiment results showed a highly significant correlation between the input and output DNA concentrations (t = 10.84, p < 0.001) with a mean slope value of 1.05, indicating the accuracy of our qPCR assay to estimate DNA abundance of T. pityocampa in bat faeces. The framework of this study can be taken as a model to design similar assays applicable to other species of interest, such as agricultural pests or insects of public health concern.

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.