The intestinal digesta microbiota of tropical marine fish is largely uncultured and distinct from surrounding water microbiota.

Studying the gut microbes of marine fishes is an important part of conservation as many fish species are increasingly threatened by extinction. The gut microbiota of only a small fraction of the more than 32,000 known fish species has been investigated. In this study we analysed the intestinal digesta microbiota composition of more than 50 different wild fish species from tropical waters. Our results show that the fish harbour intestinal digesta microbiota that are distinct from that of the surrounding water and that location, domestication status, and host intrinsic factors are strongly associated with the microbiota composition. Furthermore, we show that the vast majority (~97%) of the fish-associated microorganisms do not have any cultured representative. Considering the impact of the microbiota on host health and physiology, these findings underpin the call to also preserve the microbiota of host species, especially those that may be exposed to habitat destruction.

The different fates of two Asian horseshoe crab species with different dispersal abilities.

Impending anthropogenic climate change will severely impact coastal organisms at unprecedented speed. Knowledge on organisms' evolutionary responses to past sea-level fluctuations and estimation of their evolutionary potential is therefore indispensable in efforts to mitigate the effects of future climate change. We sampled tens of thousands of genomic markers of ~300 individuals in two of the four extant horseshoe crab species across the complex archipelagic Singapore Straits. Carcinoscorpius rotundicauda Latreille, a less mobile mangrove species, has finer population structure and lower genetic diversity compared with the dispersive deep-sea Tachypleus gigas Müller. Even though the source populations of both species during the last glacial maximum exhibited comparable effective population sizes, the less dispersive C. rotundicauda seems to lose genetic diversity much more quickly because of population fragmentation. Contra previous studies' results, we predict that the more commonly sighted C. rotundicauda faces a more uncertain conservation plight, with a continuing loss in evolutionary potential and higher vulnerability to future climate change. Our study provides important genomic baseline data for the redirection of conservation measures in the face of climate change and can be used as a blueprint for assessment and mitigation of the adverse effects of impending sea-level rise in other systems.

From marine park to future genomic observatory? Enhancing marine biodiversity assessments using a biocode approach.

Few tropical marine sites have been thoroughly characterised for their animal species, even though they constitute the largest proportion of multicellular diversity. A number of focused biodiversity sampling programmes have amassed immense collections to address this shortfall, but obstacles remain due to the lack of identification tools and large proportion of undescribed species globally. These problems can be partially addressed with DNA barcodes ("biocodes"), which have the potential to facilitate the estimation of species diversity and identify animals to named species via barcode databases. Here, we present the first results of what is intended to be a sustained, systematic study of the marine fauna of Singapore's first marine park, reporting more than 365 animal species, determined based on DNA barcodes and/or morphology represented by 931 specimens (367 zooplankton, 564 macrofauna including 36 fish). Due to the lack of morphological and molecular identification tools, only a small proportion could be identified to species solely based on either morphology (24.5%) or barcodes (24.6%). Estimation of species numbers for some taxa was difficult because of the lack of sufficiently clear barcoding gaps. The specimens were imaged and added to "Biodiversity of Singapore" (http://singapore.biodiversity.online), which now contains images for > 13,000 species occurring in the country.

Roads to isolation: Similar genomic history patterns in two species of freshwater crabs with contrasting environmental tolerances and range sizes.

Freshwater species often show high levels of endemism and risk of extinction owing to their limited dispersal abilities. This is exemplified by the stenotopic freshwater crab, Johora singaporensis which is one of the world's 100 most threatened species, and currently inhabits less than 0.01 km2 of five low order hill streams within the highly urbanized island city-state of Singapore. We compared populations of J. singaporensis with that of the non-threatened, widespread, abundant, and eurytopic freshwater crab, Parathelphusa maculata, and found surprisingly high congruence between their population genomic histories. Based on 2,617 and 2,470 genome-wide SNPs mined via the double-digest restriction-associated DNA sequencing method for ~90 individuals of J. singaporensis and P. maculata, respectively, the populations are strongly isolated (FST = 0.146-0.371), have low genetic diversity for both species (also for COI), and show signatures of recent genetic bottlenecks. The most genetically isolated populations for both species are separated from other populations by one of the oldest roads in Singapore. These results suggest that anthropogenic developments may have impacted stream-dependent species in a uniform manner, regardless of ubiquity, habitat preference, or dispersal modes of the species. While signs of inbreeding were not detected for the critically endangered species, the genetic distinctiveness and low diversity of the populations call for genetic rescue and connecting corridors between the remaining fragments of the natural habitat.

The effects of Pleistocene climate change on biotic differentiation in a montane songbird clade from Wallacea.

The role of Pleistocene Ice Age in tropical diversification is poorly understood, especially in archipelagos, in which glaciation-induced sea level fluctuations may lead to complicated changes in land distribution. To assess how Pleistocene land bridges may have facilitated gene flow in tropical archipelagos, we investigated patterns of diversification in the rarely-collected rusty-bellied fantail Rhipidura teysmanni (Passeriformes: Rhipiduridae) complex from Wallacea using a combination of bioacoustic traits and whole-genome sequencing methods (dd-RADSeq). We report a biogeographic leapfrog pattern in the vocalizations of these birds, and uncover deep genomic divergence among island populations despite the presence of intermittent land connections between some. We demonstrate how rare instances of genetic introgression have affected the evolution of this species complex, and document the presence of double introgressive mitochondrial sweeps, highlighting the dangers of using only mitochondrial DNA in evolutionary research. By applying different tree inference approaches, we demonstrate how concatenation methods can give inaccurate results when investigating divergence in closely-related taxa. Our study highlights high levels of cryptic avian diversity in poorly-explored Wallacea, elucidates complex patterns of Pleistocene climate-mediated diversification in an elusive montane songbird, and suggests that Pleistocene land bridges may have accounted for limited connectivity among montane Wallacean biota.

Next-generation freshwater bioassessment: eDNA metabarcoding with a conserved metazoan primer reveals species-rich and reservoir-specific communities.

Freshwater habitats are of high conservation value and provide a wide range of ecosystem services. Effective management requires regular monitoring. However, conventional methods based on direct observation or specimen collection are so invasive, expensive and labour-intensive that frequent monitoring is uncommon. Here, we test whether the evaluation of environmental DNA (eDNA) from water based on a simple protocol can be used for assessing biodiversity. We use universal metazoan primers for characterizing water eDNA across horizontal and vertical spatial dimensions in two reservoirs with known species diversity for two key taxa. eDNA obtained directly from 42 samples × 15 ml water (total = 630 ml) per reservoir yielded DNA signatures for more than 500 metazoan species, of which 105 could be identified to species/genus based on DNA barcodes. We show that eDNA can be used to assign each water sample to its reservoir of origin, and that eDNA outperforms conventional survey methods in single-sample richness comparisons, while revealing evidence for hundreds of unknown species that are undetected by conventional bioassessment methods. eDNA also confirms the presence of a recently discovered invasive snail species and provides evidence for the continued survival of a rare native species of goby not sighted in that habitat since 2007. eDNA thus promises to be a useful addition to the bioassessment toolbox for freshwater systems.

'Direct PCR' optimization yields a rapid, cost-effective, nondestructive and efficient method for obtaining DNA barcodes without DNA extraction.

Macroinvertebrates that are collected in large numbers pose major problems in basic and applied biodiversity research: identification to species via morphology is often difficult, slow and/or expensive. DNA barcodes are an attractive alternative or complementary source of information. Unfortunately, obtaining DNA barcodes from specimens requires many steps and thus time and money. Here, we promote a short cut to DNA barcoding, that is, a nondestructive PCR method that skips DNA extraction ('direct PCR') and that can be used for a broad range of invertebrate taxa. We demonstrate how direct PCR can be optimized for the larvae and adults of nonbiting midges (Diptera: Chironomidae), a typical invertebrate group that is abundant, contains important bioindicator species, but is difficult to identify based on morphological features. After optimization, direct PCR yields high PCR success rates (>90%), preserves delicate morphological features (e.g. details of genitalia, and larval head capsules) while allowing for the recovery of genomic DNA. We also document that direct PCR can be successfully optimized for a wide range of other invertebrate taxa that need routine barcoding (flies: Culicidae, Drosophilidae, Dolichopodidae, Sepsidae; sea stars: Oreasteridae). Key for obtaining high PCR success rates is optimizing (i) tissue quantity, (ii) body part, (iii) primer pair and (iv) type of Taq polymerase. Unfortunately, not all invertebrates appear suitable because direct PCR has low success rates for other taxa that were tested (e.g. Coleoptera: Dytiscidae, Copepoda, Hymenoptera: Formicidae and Odonata). It appears that the technique is less successful for heavily sclerotized insects and/or those with many exocrine glands.

Galactose induction of the GAL1 gene requires conditional degradation of the Mig2 repressor.

Skp1 an essential component of the SCF (Skp1/cullin/F-box) E3 ubiquitin ligases, which target proteins for degradation by the 26S proteasome. We generated a skp1dM mutant strain that is defective for galactose induction of the GAL1 gene and we have found that galactose-induced protein degradation of the repressor Mig2 is defective in this strain. Mig2 degradation was also abolished in cells lacking the protein kinase Snf1 and the F-box protein Das1, suggesting that Snf1 triggers galactose-induced protein degradation of Mig2 by SCFDas1. Chromatin immunoprecipitation showed that Mig2 associates with the GAL1 promoter upon the galactose-induced exit of Mig1 in skp1dM cells, but not in wild-type cells, suggesting that the conditional degradation of Mig2 is required to prevent it from binding to the GAL1 promoter under inducing conditions. A galactose-stable deletion derivative of Mig2 caused a strong Mig (multi-copy inhibition of GAL gene expression) phenotype, confirming that galactose induction of the GAL1 gene requires the degradation of the repressor Mig2. Our results shed new light on the conflicting reports about the functional role of the degradation of transcriptional activators and indicate that gene expression studies interfering with proteasome degradation should take the stabilization of potential repressors into account.