Population structure and microscale morphological differentiation in a freshwater snail from the Chilean Altiplano.

BACKGROUND: The diversity and population genetic structure of many species have been shaped by historical and contemporary climatic changes. For the species of the South American Altiplano, the historical climatic changes are mainly related to the wet events of great magnitude and regional influence that occurred during the Pleistocene climatic oscillations (PCOs). In contrast, contemporary climate changes are associated with events of lesser magnitude and local influence related to intensifications of the South American Summer Monsoon (SASM). Although multiple studies have analyzed the effect of PCOs on the genetic patterns of highland aquatic species, little is known about the impact of contemporary climate changes in recent evolutionary history. Therefore, in this study, we investigated the change in population structure and connectivity using nuclear and mitochondrial markers throughout the distribution range of Heleobia ascotanensis, a freshwater Cochliopidae endemic to the Ascotán Saltpan. In addition, using geometric morphometric analyses, we evaluated the concomitance of genetic divergence and morphological differentiation. RESULTS: The mitochondrial sequence analysis results revealed the presence of highly divergent co-distributed and geographically nested haplotypes. This pattern reflects an extension in the distribution of groups that previously would have differentiated allopatrically. These changes in distribution would have covered the entire saltpan and would be associated with the large-scale wet events of the PCOs. On the other hand, the microsatellite results defined five spatially isolated populations, separated primarily by geographic barriers. Contemporary gene flow analyses suggest that post-PCO, climatic events that would have connected all populations did not occur. The morphometric analyses results indicate that there is significant morphological differentiation in the populations that are more isolated and that present the greatest genetic divergence. CONCLUSIONS: The contemporary population structure and morphological variation of H. ascotanensis mainly reflect the post-PCO climatic influence. Although both markers exhibit high genetic structuring, the microsatellite and morphology results show the preponderant influence of fragmentation in recent evolutionary history. The contemporary genetic pattern shows that in species that have limited dispersal capabilities, genetic discontinuities can appear rapidly, erasing signs of historical connectivity.

Shape, Microstructure, and Chemical Composition of Pearls from the Freshwater Clam Diplodon chilensis Native to South America.

The capability to produce pearls is widespread in the phylum Mollusca, including bivalves of the superfamily Unionoidea. Here, we identified and characterized natural pearls formed by Diplodon chilensis, a freshwater clam native to southern South America, using samples obtained from two lakes located in the Chilean Patagonia. Pearls were studied using light and scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), Fourier transform infrared spectroscopy (FTIR), and Raman spectroscopy. Naturally formed pearls were found in both male and female D. chilensis specimens. Pearls are produced in different shapes, including spherical, ellipsoidal, buttoned, and bumpy, ranging in size from 200 µm to 1.9 mm. The internal microstructure is composed of irregular polygonal tablets, about 0.40 to 0.55 µm in thickness. EDX analysis showed that pearls are composed of calcium carbonate. FTIR and Raman spectra recorded several peaks attributable to the aragonite in pearls of this species, as has been shown in other mollusks. In addition to these results, pearls of different colors are illustrated.

Genetic Diversity, Morphometric Characterization, and Conservation Reassessment of the Critically Endangered Freshwater Snail, Heleobia atacamensis, in the Atacama Saltpan, Northern Chile.

Evaporitic ecosystems of the Atacama Desert contain a rich endemic fauna, including mollusk species. A recent study performed in the freshwater snail Heleobia atacamensis, endemic to the Atacama Saltpan, revealed a strong interdependence of genetic patterns with climatic fluctuations and landscape physiography. The species is currently listed as Critically Endangered at regional scale and as Data Deficient on the International Union for Conservation of Nature (IUCN) Red List. Here, we studied genetic diversity and demographic history of several populations of the species occurring on a connectivity gradient, including snails from new peripherical localities (Peine and Tilomonte), which were compared with topotype specimens. In addition, we reassessed the conservation status using the IUCN Red List categories and criteria considering species-specific idiosyncrasy. Phylogenetic and phylogeographical analyses indicated that snails from Peine and Tilomonte belong to H. atacamensis. We discovered significant differentiation in shell morphology, which was generally greater in geographically isolated populations. We also inferred six genetic clusters and a demographic expansion congruent with the wet periods that occurred at the end of the Pleistocene. Considering the highest risk category obtained, H. atacamensis was reassessed as Endangered at regional scale. Future conservation plans should consider the genetic assemblages as conservation units.

Phylogeography and molecular species delimitation reveal cryptic diversity in Potamolithus (Caenogastropoda: Tateidae) of the southwest basin of the Andes.

The species of the genus Potamolithus inhabiting the southwestern basin of the Andes are difficult to distinguish due to small size and similar shell morphology. Only Potamolithus australis and Potamolithus santiagensis have been traditionally recognized in this region, but the occurrence of several morphologically similar undescribed populations could increase the regional richness. Here we delimit described and potentially undescribed cryptic species of the genus using partial sequences of the mitochondrial cytochrome c oxidase subunit I (COI) gene. Network analysis and diversity indices inferred six highly differentiated haplogroups, many of them sympatric and widespread in the study area. Phylogeographic analyses suggest a scenario of recent diversification and the occurrence of multiple refuges during the successive Pleistocene glaciations. Phylogenetic analysis also recovered six major clades that showed no relationship with physiography. Species delimitation analyses consistently recognized three or four candidate species apart from P. australis and P. santiagensis. Divergence times indicate that speciation of Chilean Potamolithus began at the end of the Pliocene, probably driven by climatic rather than geographic events. Considering the high inter- and intra-basin genetic diversity, conservation efforts should be focused on protecting sympatric taxa in the basins with the highest species richness.

Reproductive aspects of the poorly known and critically endangered freshwater snail Heleobia atacamensis (Gastropoda: Truncatelloidea).

Knowing the reproductive biology of threatened species is essential for conservation and to establish proper management plans. Heleobia atacamensis, a freshwater snail only known from two locations in the Atacama Saltpan, northern Chile, is currently classified as Data Deficient on the IUCN Red List and Critically Endangered by the Ministerio del Medio Ambiente of Chile. Based on size-frequency distribution, multivariate analysis of shell measurements, and microdissections, we studied the reproductive strategy, recruitment period, sex ratio and sexual dimorphism in this species. Heleobia atacamensis is an oviparous species, with direct development (non-planktotrophic). Females lay capsules of a single egg from which a juvenile resembling a miniature adult hatches after intracapsular metamorphosis is completed. The development type was confirmed by the observation of a paucispiral protoconch (= protoconch I) using scanning electron microscopy. Recruitment was observed across the four seasons of the year, with an increment at the end of austral summer. Results also showed that sex ratio was 1:1, whereas sexual dimorphism was not detected using univariate and multivariate analysis of the shell. The reproductive data provided in this study are a starting point for future management plans.

Symbiotic Interaction Enhances the Recovery of Endangered Tree Species in the Fragmented Maulino Forest.

Beneficial plant-associated microorganisms, such as fungal endophytes, are key partners that normally improve plant survival under different environmental stresses. It has been shown that microorganisms from extreme environments, like those associated with the roots of Antarctica plants, can be good partners to increase the performance of crop plants and to restore endangered native plants. Nothofagus alessandrii and N. glauca, are among the most endangered species of Chile, restricted to a narrow and/or limited distributional range associated mainly to the Maulino forest in Chile. Here we evaluated the effect of the inoculation with a fungal consortium of root endophytes isolated from the Antarctic host plant Colobanthus quitensis on the ecophysiological performance [photosynthesis, water use efficiency (WUE), and growth] of both endangered tree species. We also, tested how Antarctic root-fungal endophytes could affect the potential distribution of N. alessandrii through niche modeling. Additionally, we conducted a transplant experiment recording plant survival on 2 years in order to validate the model. Lastly, to evaluate if inoculation with Antarctic endophytes has negative impacts on native soil microorganisms, we compared the biodiversity of fungi and bacterial in the rhizospheric soil of transplanted individuals of N. alessandrii inoculated and non-inoculated with fungal endophytes. We found that inoculation with root-endophytes produced significant increases in N. alessandrii and N. glauca photosynthetic rates, water use efficiencies and cumulative growth. In N. alessandrii, seedling survival was significantly greater on inoculated plants compared with non-inoculated individuals. For this species, a spatial distribution modeling revealed that, inoculation with root-fungal endophytes could potentially increase the current distributional range by almost threefold. Inoculation with root-fungal endophytes, did not reduce native rhizospheric microbiome diversity. Our results suggest that the studied consortium of Antarctic root-fungal endophytes improve the ecophysiological performance as well as the survival of inoculated trees and can be used as a biotechnological tool for the restoration of endangered tree species.

Isolation and characterization of microsatellites for the endangered endemic tree Nothofagus alessandrii (Nothofagaceae).

Nothofagus alessandrii (Nothofagaceae) is one of the most endangered trees from Chile due to high rates of habitat disturbance caused by human activities. Despite its conservation status, few molecular markers are available to study its population genetic, connectivity and to assist reproduction programs. Thus, the species needs urgent actions to restore its original distribution. Novel polymorphic microsatellites from the genome of N. alessandrii were isolated and characterized using high-through sequencing. A total of 30 primer pairs were synthesized and 18 microsatellites were amplified correctly. Polymorphism and genetic diversity was evaluated in 58 individuals from three populations of N. alessandrii. Sixteen of them were polymorphic and the number of alleles in the pooled sample ranged from 2 to 14, the mean number of alleles was 4.81. The mean values of observed heterozigosity (HO) and excepted heterozygosity (HE) are similar in all studied populations. Linkage disequilibrium was found between a few pairs of loci (five out of 263 tests) suggesting that most of the markers can be considered as independent. Significant deviations from Hardy-Weinberg equilibrium (P < 0.05) were found in four loci probably due to low sampling size. Transferability to the congeneric N. pumilio was successful in only four out of the sixteen polymorphic markers. The microsatellite markers developed in this study will be useful to study the genetic diversity and structure and to develop integrated management plans for the conservation of this endangered species.

Morphological and molecular analysis of cryptic native and invasive freshwater snails in Chile.

Species delimitation in minute freshwater snails is often difficult to perform using solely shell morphology. The problem intensifies when invasive species spread within the distribution range of morphologically similar native species. In Chile, the Truncatelloidean snails are represented by the native genera Heleobia and Potamolithus plus the invasive mudsnail Potamopyrgus antipodarum, which can easily be confused. Using an integrative approach, we performed molecular phylogenetic analysis and studied reproductive and morphological features to identify superficially similar forms inhabiting the central area of the country. Truncatelloidean snails were identified in 40 of 51 localities sampled, 10 containing Potamopyrgus antipodarum, 23 Heleobia and 7 Potamolithus. Based on these results and previously published data, the known distribution of the mudsnail in Chile encompasses 6 hydrological basins, including 18 freshwater ecosystems. The finding of the mudsnails in several type localities of native species/subspecies of "Heleobia" that were not find in situ suggests species replacement or significant extinction of native fauna, a hypothesis supported by the restudy of type material that shows that endemic forms belong to the genus Potamolithus. This study shows the usefulness of integrative taxonomy not only resolving complex taxa with cryptic morphology but also measuring the extent of an ongoing invasion.

Influenced but not determined by historical events: genetic, demographic and morphological differentiation in Heleobia ascotanensis from the Chilean Altiplano.

In the present study, we focus on the phylogeographic pattern, demographic history and morphological differentiation of Heleobia ascotanensis, a freshwater gastropod restricted to the Ascotán saltpan in the Chilean Altiplano. The current distribution of the species is limited to twelve isolated or partially isolated springs that were affected by transitions between humid and arid periods during last glaciations. The genetic analysis of 322 specimens showed that H. ascotanensis is subdivided into three genetically divergent populations, with low and moderate degrees of historical gene flow among them and incipient morphological differentiation as a consequence of genetic and geographical isolation. Molecular analyses revealed different demographic histories among populations which seem to respond independently to climatic events, probably due to an environmental imposition and idiosyncratic strategies developed to cope with water availability. The results of this study and co-distributed taxa support the hypothesis that contemporary and historical events have influenced microevolutionary differentiation of these snails, although there is a need to complement further information to predict genetic or morphological divergence at microgeographic scale.

A new species of Heleobia (Caenogastropoda: Cochliopidae) from the Chilean Altiplano.

Cochliopidae Tryon, 1866 is a diverse family of caenogastropods that lives in a wide variety of aquatic habitats primarily in the New World (Hershler &Thompson 1992). In Chile, the species of the group have been traditionally assigned to the genus Littoridina Souleyet, 1852 using conchological characters (Biese 1944, 1947; Stuardo, 1961; Valdovinos 2006) but according to anatomical studies and phylogenetic analysis the majority of them have been reassigned to the genus Heleobia Stimpson, 1865 (Hershler & Thompson 1992; Collado et al. 2011a; Kroll et al. 2012; Collado et al. 2013; Collado et al. 2016). Here we formally describe a new species of the genus Heleobia from Spring 1 in the Carcote saltpan, Chilean Altiplano, based on molecular and morphological characters. Snails from this locality were previously shown to be distinct based on DNA sequences (Collado et al. 2013; Collado et al. 2016).