Phylogenomic analyses reveal incongruences between divergence times and fossil records of freshwater snails in East Asia.

Fossils provide important insight into our understanding of phylogenetic history by serving as calibration points for divergence time estimation. However, uncertainties in the fossil record due to parallel evolution and convergent evolution can critically affect estimates of node ages. Here, we compare and contrast estimates of phylogenetic divergence with geologic and fossil history for two freshwater snail genera of the family Viviparidae in East Asia (Cipangopaludina and Margarya). Cipangopaludina species are commonly widely distributed species in East Asia, but extant Margarya species are endemic to the ancient lakes in Yunnan, China. According to some previous studies, parallel evolution or convergent evolution of shell morphology has occurred in the family several times which may affect divergence time estimation using fossil records. In this study, we used SNP data derived from ddRAD-seq loci to investigate population demographic history of both genera. Our results show a common pattern of lake endemic lineages diversifying from widely distributed lineages in the Miocene, and multiple colonization to a single ancient lake occurred in the Pleistocene. Our results indicate substantial incongruence among estimated phylogenomic divergence times, some fossil records, and formation ages of ancient lakes. These findings suggest some fossil records may be misidentified in these groups and highlight the need to carefully evaluate geological evidence and fossil records when using these for divergence time estimation.

Population collapse in viviparid gastropods of the Lake Victoria ecoregion started before the Last Glacial Maximum.

Ecosystems of Lake Victoria and riparian communities have been strongly disrupted by the introduction of the invasive Nile perch and its fishing industry. Beyond this invasion and other recent anthropogenic stressors, the Lake Victoria ecoregion also underwent phases of pronounced aridity over the Late Pleistocene, lastly during the Last Glacial Maximum (LGM). The consequences of recent and historic environmental change have been canvassed for the adaptive radiation of haplochromine cichlids occupying the ecoregion, but their effect on freshwater invertebrate diversity remains largely unknown. Here, we use 15 microsatellite loci and approximate Bayesian computation to test whether viviparid gastropods experienced a population bottleneck during the LGM, as did cichlids. Clustering analyses support three viviparid gene pools in the Lake Victoria ecoregion, gathering specimens from 1) Lake Albert and the White Nile, 2) the Victoria Nile and Lake Kyoga and 3) Lake Victoria and tributaries. The last group contains the highest genetic diversity, but all groups have a considerable number of private alleles and are inferred to predate the LGM. Examinations of demographic history reveal a 190- to 500-fold population decline that started ~ 125-150 ka ago, thus substantially before the LGM bottleneck documented in haplochromine cichlids. Population collapses in viviparids are an order of magnitude more severe than declines in cichlids and have not been halted by the re-establishment of freshwater ecosystems since the LGM. Recent anthropogenic ecosystem deterioration is causing homogenization of previously diversified microhabitats, which may contribute to (local) extinction and enhanced gene flow among species within gene pools.

Role of ancient lakes in genetic and phenotypic diversification of freshwater snails.

Endemic organisms of ancient lakes have been studied as models to understand processes of speciation and adaptive radiation. However, it remains unclear how ancient lakes play roles in genetic and phenotypic diversity of freshwater mollusks. In the present study, we focus on viviparid freshwater snails in the ancient lakes of East and Southeast Asia (Japan and China) to address this question. Using molecular phylogenetic analyses based on mitochondrial (COI, 16S) and nuclear genes (18S, 28S, H3), we show that patterns of species diversification in viviparid lineages. Colonization to ancient lakes occurred independently in China and Japan at least four times, with subsequent diversification into more than two species within each lake group. Morphological analyses of fossil related viviparids suggest parallel phenotypic evolution occurred in the different lakes and ages. Each lake contained a single lineage which was phenotypically diversified relative to those from other sites. Using genome-wide SNPs obtained by MIG-seq, we also examined the genetic structure of three Japanese viviparids, including two endemic species of ancient Lake Biwa. The results suggest that these two species diversified from the population of the third species living in wetlands surrounding the lake. These findings suggest that rapid diversification of lineages and phenotypic divergence can occur in ancient lakes compared to other habitats. Formation of large lakes probably promotes speciation and phenotypic divergence as a result of adaptation into different microhabitats. High numbers of ancient lakes could be a driver of species diversity in Asian viviparid snails.

Role of geography and climatic oscillations in governing into-India dispersal of freshwater snails of the family: Viviparidae.

The indian subcontinent has experienced numerous paleogeological and paleoclimatic events during the Cenozoic which shaped the biotic assembly over time in the subcontinent. The role of these events in governing the biotic exchange between Southeast Asia and Indian subregion is underexplored. We aimed to uncover the effects the collision of the Indian and Asian plate, marine transgression in the Bengal basin as well as the paleoclimatic changes in the subcontinent and adjoining regions, on the dispersal of freshwater snail family Viviparidae from Southeast Asia (SEA) to Indian subregion. Extensive sampling was carried out throughout the Indian subcontinent to capture the current diversity of the targeted lineages. Three mitochondrial and two nuclear markers were sequenced from these samples and combined with published sequences to reconstruct global phylogeny of Viviparidae. Molecular dating and ancestral range estimation were undertaken to obtain the time frame for the dispersal events. Results from these analyses were contrasted with paleoclimate and paleogeology to better understand the biogeography of Indian viviparids. Results support at least two dispersal events into India from Southeast Asia. The earlier event is likely to have occurred during a warm and humid Eocene period before a permanent land connection was established between the two landmasses. While the more recent dispersal occurred post-suturing and overlapped with a time in late Tertiary to Quaternary when arid climate prevailed. However, we could not firmly establish how the marine transgressions influenced the dispersal events. Even though most biotic exchange between India and SEA are noted to be post-suturing, our results add to a growing body of work that suggests faunal exchange pre-suturing probably mediated by intermittent land connections.

Morphological stasis in an ongoing gastropod radiation from Lake Malawi.

Evolutionary processes leading to adaptive radiation regularly occur too fast to be accurately recorded in the fossil record but too slowly to be readily observed in living biota. The study of evolutionary radiations is thereby confronted with an epistemological gap between the timescales and approaches used by neontologists and paleontologists. Here we report on an ongoing radiation of extant Bellamya species (n = 4) from the African Rift Lake Malawi that provides an unusual opportunity to bridge this gap. The substantial molecular differentiation in this monophyletic Bellamya clade has arisen since Late Pleistocene megadroughts in the Malawi Basin caused by climate change. Morphological time-series analysis of a high-resolution, radiocarbon-dated sequence of 22 faunas spanning the Holocene documents stasis up to the middle Holocene in all traits studied (shell height, number of whorls, and two variables obtained from geometric morphometrics). Between deposition of the last fossil fauna (~5 ka) and the present day, a drastic increase in morphological disparity was observed (3.7-5.8 times) associated with an increase in species diversity. Comparison of the rates of morphological evolution obtained from the paleontological time-series with phylogenetic rates indicates that the divergence in two traits could be reconstructed with the slow rates documented in the fossils, that one trait required a rate reduction (stabilizing selection), and the other faster rates (divergent selection). The combined paleontological and comparative approach taken here allows recognition that morphological stasis can be the dominant evolutionary pattern within species lineages, even in very young and radiating clades.

More deterministic assembly constrains the diversity of gut microbiota in freshwater snails.

Growing evidence has suggested a strong link between gut microbiota and host fitness, yet our understanding of the assembly mechanisms governing gut microbiota remains limited. Here, we collected invasive and native freshwater snails coexisting at four independent sites in Guangdong, China. We used high-throughput sequencing to study the assembly processes of their gut microbiota. Our results revealed significant differences in the diversity and composition of gut microbiota between invasive and native snails. Specifically, the gut microbiota of invasive snails exhibited lower alpha diversity and fewer enriched bacteria, with a significant phylogenetic signal identified in the microbes that were enriched or depleted. Both the phylogenetic normalized stochasticity ratio (pNST) and the phylogenetic-bin-based null model analysis (iCAMP) showed that the assembly process of gut microbiota in invasive snails was more deterministic compared with that in native snails, primarily driven by homogeneous selection. The linear mixed-effects model revealed a significant negative correlation between deterministic processes (homogeneous selection) and alpha diversity of snail gut microbiota, especially where phylogenetic diversity explained the most variance. This indicates that homogeneous selection acts as a filter by the host for specific microbial lineages, constraining the diversity of gut microbiota in invasive freshwater snails. Overall, our study suggests that deterministic assembly-mediated lineage filtering is a potential mechanism for maintaining the diversity of gut microbiota in freshwater snails.

Assessment and analysis of metal bioaccumulation in freshwater gastropods of urban river habitats, Saint Petersburg (Russia).

The aim of this study was to analyze the content of heavy metals in the shells of freshwater gastropods Viviparus sp.; to evaluate the correlation of the trace element accumulation degree in biological objects, bottom sediments, and water, and to assess the degree of pollution of minor rivers of St. Petersburg, as well as the applicability of the studied biological objects for this. Water samples, sediment samples, and gastropods were collected in July 2019 and August 2020 at seven sampling points located at five rivers in St. Petersburg. Analysis of metals in water samples, water extract from bottom sediments, was carried out with atomic emission spectrometry. Analysis of metals in the shells of molluscs Viviparus sp. carried out with atomic absorption spectrophotometry and atomic emission spectrometry. The data obtained indicate the current contamination of the Neva River Delta with a number of metals, such as Al, Mn, Fe, Cu, and Pb, and indicate a significant accumulative capacity of bottom sediments with Zn, Pb, Ni, Cu, Mn, and Fe. Gastropods showed an ability for intensive bioaccumulation, which depended on the properties of metals, the hydrology of the river, and the characteristics of bottom sediments. It was found that for the studied environmental conditions, Sr, Fe, Cr, and Mn have the best ability to bioaccumulate in gastropod shells.

Can fractal dimensions objectivize gastropod shell morphometrics? A case study from Lake Lugu (SW China).

Morphometrics are fundamental for the analysis of size and shape in fossils, particularly because soft parts or DNA are rarely preserved and hard parts such as shells are commonly the only source of information. Geometric morphometrics, that is, landmark analysis, is well established for the description of shape but it exhibits a couple of shortcomings resulting from subjective choices during landmarking (number and position of landmarks) and from difficulties in resolving shape at the level of micro-sculpture.With the aid of high-resolution 3D scanning technology and analyses of fractal dimensions, we test whether such shortcomings of linear and landmark morphometrics can be overcome. As a model group, we selected a clade of modern viviparid gastropods from Lake Lugu, with shells that show a high degree of sculptural variation. Linear and landmark analyses were applied to the same shells in order to establish the fractal dimensions. The genetic diversity of the gastropod clade was assessed.The genetic results suggest that the gastropod clade represents a single species. The results of all morphometric methods applied are in line with the genetic results, which is that no specific morphotype could be delimited. Apart from this overall agreement, landmark and fractal dimension analyses do not correspond to each other but represent data sets with different information. Generally, the fractal dimension values quantify the roughness of the shell surface, the resolution of the 3D scans determining the level. In our approach, we captured the micro-sculpture but not the first-order sculptural elements, which explains that fractal dimension and landmark data are not in phase.We can show that analyzing fractal dimensions of gastropod shells opens a window to more detailed information that can be considered in evolutionary and ecological contexts. We propose that using low-resolution 3D scans may successfully substitute landmark analyses because it overcomes the subjective landmarking. Analyses of 3D scans with higher resolution than used in this study will provide surface roughness information at the mineralogical level. We suggest that fractal dimension analyses of a combination of differently resolved 3D models will significantly improve the quality of shell morphometrics.

The complete mitochondrial genome of the mudsnail Cipangopaludina cathayensis (Gastropoda: Viviparidae).

We present the complete mitochondrial genome of Cipangopaludina cathayensis in this study. The mitochondrial genome is 17,157 bp in length, containing 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes. All of them are encoded on the heavy strand except 7 tRNA genes on the light strand. Overall nucleotide compositions of the light strand are 44.51% of A, 26.74% of T, 20.48% of C and 8.28% of G. All the protein-coding genes start with ATG initiation codon except ATP6 with ATA and ND4 with TTG, and 2 types of termination codons are TAA (ATP6, ND2, COX1, COX2, ATP8, ND1, ND6, Cytb, COX3, ND4) and TAG (ND4L, ND5, ND3). There are 29 intergenic spacers and 5 gene overlaps. The tandem repeat sequences are observed in COX2, tRNA(Asp), ATP6, tRNA(Cys), S-rRNA, ND1, Cytb, ND4 and COX3 genes. Gene arrangement and distribution are different from the typical vertebrates. The absence of D-loop is consistent with the Gastropoda, but at least one lengthy non-coding region is essential regulatory element for the initiation of transcription and replication.