The alsodid ground frogs of the Eupsophus genus are divided into two groups, the roseus (2n = 30) and vertebralis (2n = 28), which are distributed throughout the temperate Nothofagus forests of South America. Currently, the roseus group is composed by four species, while the vertebralis group consists of two. Phylogenetic relationships and species delimitation within each group are controversial. In fact, previous analyses considered that the roseus group was composed of between four to nine species. In this work, we evaluated phylogenetic relationships, diversification times, and species delimitation within the roseus group using a multi-locus dataset. For this purpose, mitochondrial (D-loop, Cyt b, and COI) and nuclear (POMC and CRYBA1) partial sequences from 164 individuals were amplified, representing all species. Maximum Likelihood (ML) and Bayesian approaches were used to reconstruct phylogenetic relationships. Species tree was estimated using BEAST and singular value decomposition scores for species quartets (SVDquartets). Species limits were evaluated with six coalescent approaches. Diversification times were estimated using mitochondrial and nuclear rates with LogNormal relaxed clock in BEAST. Nine well-supported monophyletic lineages were recovered in Bayesian, ML, and SVDquartets, including eight named species and a lineage composed by specimens from the Villarrica population (Bootstrap:>70, PP:> 0.99). Single-locus species delimitation analyses overestimated the species number in E. migueli, E. calcaratus, and E. roseus lineages, while multi-locus analyses recovered as species the nine lineages observed in phylogenetic analyses (Ctax = 0.69). It is hypothesized that Eupsophus diversification occurred during Mid-Pleistocene (0.42–0.14 Mya), with most species having originated after the Last Southern Patagonian Glaciation (0.18 Mya). Our results revitalize the hypothesis that the E. roseus group is composed of eight species and support the Villarrica lineage as a new putative species.

Acid phosphatase (ACP) enzymes are involved in the mobilization of soil phosphorus (P) and polyphosphate accumulated in the fungal tissues of ectomycorrhizal roots, thereby influencing the amounts of P that are stored in the fungus and transferred to the host plant. This study evaluated the effects of ectomycorrhizal morphotype and soil fertility on ACP activity in the extraradical mycelium (ACPmyc), the mantle (ACPmantle) and the Hartig net region (ACPHartig) of ectomycorrhizal Nothofagus obliqua seedlings. ACP activity was quantified in vivo using enzymelabelled fluorescence-97 (ELF-97) substrate, confocal laser microscopy and digital image processing routines. There was a significant effect of ectomycorrhizal morphotype on ACPmyc, ACPmantle and ACPHartig, while soil fertility had a significant effect on ACPmyc and ACPHartig. The relative contribution of the mantle and the Hartig net region to the ACP activity on the ectomycorrhizal root was significantly affected by ectomycorrhizal morphotype and soil fertility. A positive correlation between ACPHartig and the shoot P concentration was found, providing evidence that ACP activity at the fungus:root interface is involved in P transfer from the fungus to the host. It is concluded that the spatial distribution of ACP in ectomycorrhizas varies as a function of soil fertility and colonizing fungus.

Resumen En este estudio se muestran evidencias moleculares de éxito en la hibridación entre Serinus canaria domestica (linnaeus, 1758) y Spinus barbatus (molina, 1782). Como parte de una secuencia de retrocruzamiento, se logró reproducir híbridos F2 a partir de hembras híbridas fértiles viables F1 x S. c. domestica. La F1 fue la descendencia entre P0 = Serinus c. domestica x Spinus barbatus. Las secuencias de nucleótidos de dos segmentos de ADN, citocromo b mitocondrial (Cyt b) y el receptor de tirosina quinasa muscular (MuSK) del ADN nuclear se obtuvieron de tres especímenes híbridos F2. Tanto las secuencias de Cyt b como de MuSK señalaron fuerte soporte filogenético a la condición genética híbrida de los tres embriones F2. De esta manera la evidencia molecular refleja el éxito en el cruce interespecífico entre S. barbatus con S. c. domestica y que es posible obtener híbridos fértiles viables F1 (en este caso hembras) y F2 entre estos dos linajes en poblaciones naturales. Abstract In this study we show molecular evidences of success in hybridization between Serinus canaria domestica and Spinus barbatus. As part of a sequence of backcrossing we have achieved to reproduce F2 hybrids of fertile hybrid females F1 with parental S. c. domestica. F1 was the offspring between, P0 = S. c. domestica x S. barbatus. In this study the space where the observations and the breeding success are carried out between these species, they are described: is a room with the following measures; 4,45 m (lenghty) x 1,60 m (width) x 2,30 m (high), located in an urban building; specifically, glassed-balcony at a height of 10,60 m above ground level, facing some ornamental and exuberant canopy trees of Platanus orientalis (L., 1753) and with direct entrance of natural sunlight, in urban city of Santiago, Chile, where his perceptual world develops. We took care to keep the biotic and abiotic factors under control; specifically, incoming natural light, ambient vegetation and temperature. On the other hand, we fed the individuals appropriately and provided them with plenty of clean water to drink and get clean, where there were no predators. In sum, the individuals were kept in a healthy environment. The backcrossing between the male S. c. domestica (generation 0) bred with two viable hybrid F1 offspring females, it gave as resulted in a total of three independent generations of F2 hybrids (n = 12). All the individuals came out healthy, and none of them died, despite being inbred lineages. Three embryos representative of the F2 hybrids were sacrificed and deposited in 99% alcohol. Nucleotide sequences of two DNA segments, mitochondrial cytochrome b (Cyt b) and muscle-specific receptor tyrosine kinase (MuSK) gene of the nuclear DNA were obtained from three F2 hybrid specimens. The model of molecular evolution with the greatest adjustment to the data obtained by jModeltest was GTR + I + G (I = 0,1450, G = 0,0930), according to the phylogenetic analysis of mitochondrial DNA (Fig. 1a), the female progenitor P0 is S.c.domestica. This conclusion is based on the evi- dence that mitochondrial DNA in birds, as well as in most animal groups, is exclusively inherited through the maternal ways. The same analysis carried out with the nuclear MuSK gene shows that the parental male P0 of the embryos analyzed is very much related to S. barbatus (Fig. 1b). The Cyt b and MuSK region show strongly support to hybrids genetic condition of the three F2 embryos. Intergeneric hybrids are usually sterile, but it is worth noting that genera of Fringillidae are very closely related (the Family as a whole is only 12 million years old, and most genera in the terminal canary-siskin group are <5 million years old). This is simply a case of poor classification with oversplitting or maybe the nature of these lineages are fractals, since in most of the Passeriformes, family-level taxa are more than 20 million years old, and in other groups of Aves families and genera are even older. Then hybridization takes place more easily in captivity. Many authors considered that hybridization in birds is not important because hybrids are formed in proportion 1/50,000 specimens. Despite this, many bird hybrids have been created in captivity. In addition, hybrids produced ex situ under controlled conditions would play an important role for reproductive success and subsequent interspecific viability. The main conclusions derived from this study are as follows: 1) The present report strongly indicates that hybridizations have occurred among S. c. canary x S. barbatus. 2) In this way the molecular evidence reflects and justifies the success in interspecific reproduction between S. barbatus with S. c. domestica and that it is possible to obtain viable fertile hybrids F1 (in this case females) and F2 between these two lineages, 3) And consequently the close genetic affinity between these two genera and the formation of hybrids in natural populations should not discard.

Speciation in a biodiversity hotspot: phylogenetic relationships, species delimitation, 2and divergence times of the Patagonian ground frogs of Eupsophus roseus group 3 (Alsodidae) 4 5 Abstract 12 13The alsodid ground frogs genus Eupsophus is divided into the roseus (2n=30) and 14 vertebralis (2n=28) groups, distributed throughout the temperate Nothofagus forests of 15 South America. Currently, the roseus group is composed by four species, while the 16 vertebralis group consists of two. Phylogenetic relationships and species delimitation 17 within each group are controversial. In fact, previous analyses considered that roseus group 18 was composed between four to nine species. In this work, we evaluated phylogenetic 19 relationships, diversification times, and species delimitation within roseus group using a 20 multi-locus dataset. For this purpose, mitochondrial (D-loop, Cyt b, and COI) and nuclear 21 (POMC and CRYBA1) partial sequences, were amplified from 164 individuals, 22 representing all species. Maximum Likelihood (ML) and Bayesian approaches were used to 23 reconstruct phylogenetic relationships. Species tree was estimated using BEAST and 24 singular value decomposition scores for species quartets (SVDquartets). Species limits 25 were evaluated with six coalescent approaches. Diversification times were estimated using 26 mitochondrial and nuclear rates with LogNormal relaxed clock in BEAST. Nine well-27 supported monophyletic lineages were recovered in Bayesian, ML, and SVDquartets, 28 including eight named species and a lineage composed by specimens from Villarrica 29 population (Bootstrap: >90, PP:> 0.9). Single-locus species delimitation analyses 30 overestimated the species number in E. migueli, E. calcaratus and E. roseus lineages, while 31 multi-locus analyses recovered as species the nine lineages observed in phylogenetic 32 analyses (>0.95). It is hypothesized that Eupsophus diversification occurred during Mid-33 Pleistocene (0.42-0.14 Mya), with most species originated after of the Last Southern 34 Patagonian Glaciation (0.18 Mya). Our results revitalize the hypothesis that E. roseus group 35 is composed by eight species and support to Villarrica lineage as a new putative species. 36 37 41 42 Introduction 43 44From the operational point of view, the notion of biodiversity encompasses several 45 different levels of biological organization, from the species' make up genetic to ecosystems 46 and landscapes, in which the species is the most significant unit. Species are used for 47 comparisons in almost all biological fields including ecology, evolution, and conservation 48 [1-3]; no doubt the central unit for systematics is also the species [4]. Furthermore, 49 biodiversity hotspots are selected on the basis of the species they possess, conservation 50 schemes are assessed on how many species are preserved, and conservation legislation and 51 politics are focused on species preservation [5,6]. 52Although the importance of species concepts debate [7,8], and that the species as 53 taxonomic hierarchy...

The newly identified EST-SSRs in G. avellana and the multi-pooling strategy involving eight markers will facilitate future studies of clone identification, genetic structure, and diversity.