Restriction Site-associated DNA Research Articles

The Genetic Diversity of Mink (Neovison vison) Populations in China.

The American mink (Neovison vison) is a semiaquatic species of Mustelid native to North America that is now widespread in China. However, the knowledge of genetic diversity of mink in China is still limited. In this study, we investigated the genetic diversity and identified significant single nucleotide polymorphisms (SNPs) in mink populations of five different color types in three different mink farms in China. Using double-digest restriction site-associated DNA sequencing, we identified a total of 1.3 million SNPs. After filtering the SNPs, phylogenetic tree, Fst, principal component, and population structure analyses were performed. The results demonstrated that red mink and black mink grouped, with separate clustering of all other color types. The population divergence index (Fst) study confirmed that different mink populations were distinct (K = 4). Two populations with different coat colors were subjected to the selection signature analysis, and 2300 genes were found to have a clear selection signature. The genes with a selection signature were subjected to Gene Ontology (GO) categorization and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis, the results revealed that the genes with a selection signature were enriched in the melanogenesis pathway. These study's findings have set the stage for improved breeding and conservation of genetic resources in real-world practical mink farming.

Mapping and validation of a new quantitative trait locus (QTL) for fruit size in watermelon (Citrullus lanatus)

Watermelon (Citrullus lanatus) is one of the top ten fruit-bearing species in the world and is mainly grown for its edible fruit. However, the understanding of watermelon fruit size is still incomplete, with relatively few studies on the genetic and molecular mechanisms of this trait. In the present study, three segregation populations from one novel germplasm with small fruit and different inbred lines with medium fruit, big fruit and giant fruit, respectively, were constructed to locate and fine map one new quantitative trait locus (QTL) on chromosome 8 for fruit size in watermelon. Inheritance analysis of these three populations suggested that small fruit was controlled by a single recessive gene in watermelon. We located a new QTL for fruit size on chromosome 8, and developed markers to conduct fine mapping of the genomic region and to identify candidate genes. Specifically, we constructed a high-density genetic linkage map using restriction-site associated DNA (RAD)-based high-throughput genotyping data from a second-filial generation (F2) mapping family derived from novel germplasms with small fruit and big fruit. The total genetic map length was 1315.53 cM with an average inter-locus distance of 0.18 cM, which contained 7,272 high-quality single nucleotide polymorphism (SNP) markers distributed on 11 linkage groups corresponding to the number of chromosome pairs in watermelon. Genome wide QTL mapping revealed one significant QTL (fs-chr8) on chromosome 8 associated with fruit size. One major QTL (fs-B171) on chromosome 8 was also identified in another BC1 population derived from giant watermelon and small watermelon, which overlapped with fs-chr8. Moreover, based on the markers obtained from next-generation sequencing of the parental lines, we conducted fine mapping of the interval to a 304 kb region (from 26.83 to 27.14 Mb) on chromosome 8 using 7 polymerase chain reaction (PCR) based markers. The PCR based markers confirmed the accuracy of our mapping results for watermelon fruit size. Finally, candidate genes underlying fruit size were identified from the reference genome 97103v2, including genes related to cytochrome P450 family enzymes, gibberellin-regulated proteins, transcription factor and basic leucine zipper (bZIP) transcription factor family proteins. Our study provides fine mapping results for watermelon fruit size and will lay the foundation for the identification of candidate genes to elucidate the molecular mechanism underlying fruit size in this species.

Complex Patterns of Diversification in the Gray Zone of Speciation: Model-Based Approaches Applied to Patagonian Liolaemid Lizards (Squamata: Liolaemus kingii clade).

In this study we detangled the evolutionary history of the Patagonian lizard clade Liolaemus kingii, coupling dense geographic sampling and novel computational analytical approaches. We analyzed nuclear and mitochondrial data (restriction site-associated DNA sequencing and cytochrome b) to hypothesize and evaluate species limits, phylogenetic relationships, and demographic histories. We complemented these analyses with posterior predictive simulations to assess the fit of the genomic data to the multispecies coalescent model. We also employed a novel approach to time-calibrate a phylogenetic network. Our results show several instances of mito-nuclear discordance and consistent support for a reticulated history, supporting the view that the complex evolutionary history of the kingii clade is characterized by extensive gene flow and rapid diversification events. We discuss our findings in the contexts of the "gray zone" of speciation, phylogeographic patterns in the Patagonian region, and taxonomic outcomes. [Model adequacy; multispecies coalescent; multispecies network coalescent; phylogenomics; species delimitation.].

Attack of the clones: Population genetics reveals clonality of Colletotrichum lupini, the causal agent of lupin anthracnose.

Colletotrichum lupini, the causative agent of lupin anthracnose, affects lupin cultivation worldwide. Understanding its population structure and evolutionary potential is crucial to design successful disease management strategies. The objective of this study was to employ population genetics to investigate the diversity, evolutionary dynamics, and molecular basis of the interaction of this notorious lupin pathogen with its host. A collection of globally representative C. lupini isolates was genotyped through triple digest restriction site-associated DNA sequencing, resulting in a data set of unparalleled resolution. Phylogenetic and structural analysis could distinguish four independent lineages (I-IV). The strong population structure and high overall standardized index of association (r̅d ) indicates that C. lupini reproduces clonally. Different morphologies and virulence patterns on white lupin (Lupinus albus) and Andean lupin (Lupinus mutabilis) were observed between and within clonal lineages. Isolates belonging to lineage II were shown to have a minichromosome that was also partly present in lineage III and IV, but not in lineage I isolates. Variation in the presence of this minichromosome could imply a role in host-pathogen interaction. All four lineages were present in the South American Andes region, which is suggested to be the centre of origin of this species. Only members of lineage II have been found outside South America since the 1990s, indicating it as the current pandemic population. As a seedborne pathogen, C. lupini has mainly spread through infected but symptomless seeds, stressing the importance of phytosanitary measures to prevent future outbreaks of strains that are yet confined to South America.

Population structure and adaptive differentiation in the sea cucumber Apostichopus californicus and implications for spatial resource management.

A growing body of evidence suggests that spatial population structure can develop in marine species despite large population sizes and high gene flow. Characterizing population structure is important for the effective management of exploited species, as it can be used to identify appropriate scales of management in fishery and aquaculture contexts. The California sea cucumber, Apostichopus californicus, is one such exploited species whose management could benefit from further characterization of population structure. Using restriction site-associated DNA (RAD) sequencing, we developed 2075 single nucleotide polymorphisms (SNPs) to quantify genetic structure over a broad section of the species' range along the North American west coast and within the Salish Sea, a region supporting the Washington State A. californicus fishery and developing aquaculture production of the species. We found evidence for population structure (global fixation index (FST) = 0.0068) with limited dispersal driving two patterns of differentiation: isolation-by-distance and a latitudinal gradient of differentiation. Notably, we found detectable population differences among collection sites within the Salish Sea (pairwise FST = 0.001-0.006). Using FST outlier detection and gene-environment association, we identified 10.2% of total SNPs as putatively adaptive. Environmental variables (e.g., temperature, salinity) from the sea surface were more correlated with genetic variation than those same variables measured near the benthos, suggesting that selection on pelagic larvae may drive adaptive differentiation to a greater degree than selection on adults. Our results were consistent with previous estimates of and patterns in population structure for this species in other extents of the range. Additionally, we found that patterns of neutral and adaptive differentiation co-varied, suggesting that adaptive barriers may limit dispersal. Our study provides guidance to decision-makers regarding the designation of management units for A. californicus and adds to the growing body of literature identifying genetic population differentiation in marine species despite large, nominally connected populations.

Introgressive hybridization in the west Pacific pen shells (genus Atrina): Restricted interspecies gene flow within the genome.

A compelling interest in marine biology is to elucidate how species boundaries between sympatric free-spawning marine invertebrates such as bivalve molluscs are maintained in the face of potential hybridization. Hybrid zones provide the natural resources for us to study the underlying genetic mechanisms of reproductive isolation between hybridizing species. Against this backdrop, we examined the occurrence of introgressive hybridization (introgression) between two bivalves distributed in the western Pacific margin, Atrina japonica and Atrina lischkeana, based on single-nucleotide polymorphisms (SNPs) derived from restriction site-associated DNA sequencing. Using 1066 ancestry-informative SNP sites, we also investigated the extent of introgression within the genome to search for SNP sites with reduced interspecies gene flow. A series of our individual-level clustering analyses including the principal component analysis, Bayesian model-based clustering, and triangle plotting based on ancestry-heterozygosity relationships for an admixed population sample from the Seto Inland Sea (Japan) consistently suggested the presence of specimens with varying degrees of genomic admixture, thereby implying that the two species are not completely isolated. The Bayesian genomic cline analysis identified 10 SNP sites with reduced introgression, each of which was located within a genic region or an intergenic region physically close to a functional gene. No, or very few, heterozygotes were observed at these sites in the hybrid zone, suggesting that selection acts against heterozygotes. Accordingly, we raised the possibility that the SNP sites are within genomic regions that are incompatible between the two species. Our finding of restricted interspecies gene flow at certain genomic regions gives new insight into the maintenance of species boundaries in hybridizing broadcast-spawning molluscs.

Genetic Variation versus Morphological Variability in European Peatland Violets (Viola epipsila-V. palustris Group).

In Europe, the V. epipsila-V. palustris group comprises V. epipsila Ledeb., V. palustris L., V. pubifolia (Kuta) G. H. Loos (=V. palustris subsp. pubifolia Kuta), interspecific hybrids, and putative introgressants. The genetic affinity of V. pubifolia to V. palustris, and their shared origin via hybridization followed by polyploidization, were confirmed using inter simple sequence repeat (ISSR) markers, restriction site-associated DNA sequencing (RAD-Seq), and a low-copy nuclear gene, GPI, which encodes glucose-6-phosphate isomerase. The other taxa of subsect. Stolonosae were not identified as putative parents of V. pubifolia by GPI. Our analyses indicated that V. pubifolia can be included in the morphological and genetic variation of V. palustris. The ISSR, RAD-Seq, and genome size value separated well V. palustris from V. epipsila and hybrids. The results also reopen the discussion on intraspecific variation in the context of taxa ranks and species concepts. The reduced tolerance of V. epipsila in Europe to changing environmental conditions might result from low genetic differentiation and heterozygosity, as well as the increased number of interspecific hybrids (V. epipsila × V. palustris), and eventually can possibly lead to its extinction. The disappearance of populations/individuals of this species may indicate anthropogenic changes occurring in peatlands.

Phenotypic characterization of the wheat temperature-sensitive leaf color mutant and physical mapping of mutant gene by reduced-representation sequencing

Few available leaf color mutants in crops have greatly limited the understanding of photosynthesis mechanisms, leading to few accomplishments in crop yield improvement via enhanced photosynthetic efficiency. Here, a noticeable albino mutant, CN19M06, was identified. A comparison between CN19M06 and the wild type CN19 at different temperatures showed that the albino mutant was temperature-sensitive and produced leaves with a decreased chlorophyll content at temperatures below 10 °C. Genetic analysis suggested that the albinism was controlled by one recessive nuclear gene named TSCA1, which was putatively assigned to the region of 718.1–729.8 Mb on chromosome 2AL using bulked-segregant analysis and double-digest restriction site-associated DNA. Finally, molecular linkage analysis physically anchored TSCA1 to a narrowed region of 718.8–725.3 Mb with a 6.5 Mb length on 2AL flanked by InDel 18 and InDel 25 with 0.7 cM genetic interval. Among the 111 annotated functional genes in the corresponding chromosomal region, only TraesCS2A01G487900 of the PAP fibrillin family was both related to chlorophyll metabolism and temperature sensitivity; therefore, it was considered the putative candidate gene of TSCA1. Overall, CN19M06 has great potential for exploring the molecular mechanism of photosynthesis and monitoring temperature changes in wheat production.

Niche evolution in a northern temperate tree lineage: biogeographical legacies in cork oaks (Quercus section Cerris).

Cork oaks (Quercus section Cerris) comprise 15 extant species in Eurasia. Despite being a small clade, they display a range of leaf morphologies comparable to the largest sections (>100 spp.) in Quercus. Their fossil record extends back to the Eocene. Here, we explore how cork oaks achieved their modern ranges and how legacy effects might explain niche evolution in modern species of section Cerris and its sister section Ilex, the holly oaks. We inferred a dated phylogeny for cork and holly oaks using a reduced-representation next-generation sequencing method, restriction site-associated DNA sequencing (RAD-seq), and used D-statistics to investigate gene flow hypotheses. We estimated divergence times using a fossilized birth-death model calibrated with 47 fossils. We used Köppen profiles, selected bioclimatic parameters and forest biomes occupied by modern species to infer ancestral climatic and biotic niches. East Asian and Western Eurasian cork oaks diverged initially in the Eocene. Subsequently, four Western Eurasian lineages (subsections) differentiated during the Oligocene and Miocene. Evolution of leaf size, form and texture was correlated, in part, with multiple transitions from ancestral humid temperate climates to mediterranean, arid and continental climates. Distantly related but ecologically similar species converged on similar leaf traits in the process. Originating in temperate (frost-free) biomes, Eocene to Oligocene ranges of the primarily deciduous cork oaks were restricted to higher latitudes (Siberia to north of Paratethys). Members of the evergreen holly oaks (section Ilex) also originated in temperate biomes but migrated southwards and south-westwards into then-(sub)tropical southern China and south-eastern Tibet during the Eocene, then westwards along existing pre-Himalayan mountain ranges. Divergent biogeographical histories and deep-time phylogenetic legacies (in cold and drought tolerance, nutrient storage and fire resistance) thus account for the modern species mosaic of Western Eurasian oak communities, which are composed of oaks belonging to four sections.

Deep genome-wide divergences among species in White Cloud Mountain minnow Tanichthys albonubes (Cypriniformes: Tanichthyidae) complex: Conservation and species management implications

Identifying cryptic species is important for the assessments of biodiversity. Further, untangling mechanisms underlying the origins of cryptic species can facilitate our understanding of evolutionary processes. Advancements in genomic approaches for non-model systems have offered unprecedented opportunities to investigate these areas. The White Cloud Mountain minnow (Tanichthys albonubes) is a popular freshwater pet fish worldwide but its wild populations in China are critically endangered. Recent research based on a few molecular markers suggested that this species in fact comprised seven cryptic species, of which six were previously unknown. Here, we tested six of these cryptic species and quantified genomic interspecific divergences between species in the T. albonubes complex by analyzing genome-wide restriction site-associated DNA sequencing (RADseq) data generated from 189 individuals sampled from seven populations (including an outgroup congeneric species, T. micagemmae). We found that six cryptic species previously suggested were well supported by RADseq data. The genetic diversity of each species in the T. albonubes complex was low compared with T. micagemmae and the contemporary effective population sizes (Ne) of each cryptic species were small. Phylogenetic analysis showed seven clades with high support values confirmed with Neighbor-Net trees. The pairwise divergences between species in T. albonubes complex were deep and the highly differentiated loci were evenly distributed across the genome. We proposed that the divergence level of T. albonubes complex is at a late stage of cryptic speciation and lacking gene flow. Our findings provide new insights into cryptic speciation and have important implications for conservation and species management of T. albonubes complex.

A Phylogenetic and Morphological Evolution Study of Ribes L. in China Using RAD-Seq

Ribes L. belongs to the Grossulariaceae family and has important edible, medicinal, ornamental, and landscaping values. Taxonomic classification within this genus is difficult due to its large variety of species, wide distribution, large morphological variations, and presence of two complex taxonomic groups with bisexual or unisexual flowers. Our study aims to clarify the phylogenetic relationships of Ribes L. taxa in China, and further, to provide a reference for a revised global classification of it. The phylogenetic analysis of 52 Ribes L. samples from 30 species was constructed based on restriction site-associated DNA sequencing and single nucleotide polymorphisms. Afterward, two important taxonomic characters were selected for ancestral state reconstruction over the molecular phylogeny. The results showed that the 52 samples could be divided into six branches, i.e., six subgenera, which caused some controversy regarding the morphological classification of Ribes L. in China. The molecular phylogeny supported the separation of subg. Coreosma from subg. Ribesia and subg. Hemibotrya from subg. Berisia and validated the rationale for recognizing subg. Grossularia as an independent subgenus, the rationality of which was further verified by the reconstruction of ancestor traits. Gene flow among Ribes L. was identified and further confirmed our results.

Genome-wide analysis facilitates estimation of the amount of male contribution in meiotic gynogenetic three-spined stickleback (Gasterosteus aculeatus).

Gynogenetic embryos - those inheriting only maternal DNA - can be experimentally created by fertilizing eggs with radiation-treated sperm containing inactivated paternal chromosomes. Diploidy in the zygotes can be maintained through prevention of the second meiosis or restored by preventing the first mitosis after the maternal chromosome complement has been replicated. These gynogenetic organisms are useful in many fields including aquaculture, evolutionary biology and genomics. Although gynogenetic organisms have been created in numerous species, the completeness of uni-parental inheritance has often been assumed rather than thoroughly quantified across the genome. Instead, when tests of uni-parental inheritance occur, they typically rely on well-studied genetically determined phenotypes that represent a very small sub-set of the genome. Only assessing small genomic regions for paternal inheritance leaves the question of whether some paternal contributions to offspring might still have occurred. In this study, the authors quantify the efficacy of creating gynogenetic diploid three-spined stickleback fish (Gasterosteus aculeatus). To this end, the authors mirrored previous assessments of paternal contribution using well-studied genetically determined phenotypes including sex and genetically dominant morphological traits but expanded on previous studies using dense restriction site-associated DNA sequencing (RAD-seq) markers in parents and offspring to assess paternal inheritance genome-wide. In the gynogenetic diploids, the authors found no male genotypes underlying their phenotypes of interest - sex and dominant phenotypic traits. Using genome-wide assessments of paternal contribution, nevertheless, the authors found evidence of a small, yet potentially important, amount of paternally "leaked" genetic material. The application of this genome-wide approach identifies the need for more widespread assessment of paternal contributions to gynogenetic animals and promises benefits for many aspects of aquaculture, evolutionary biology and genomics.

High-throughput sequencing confirms the boundary between traditionally considered species pairs in a group of lichenized fungi (Peltigeraceae, Pseudocyphellaria)

Abstract Accurate species delimitations are fundamental to our understanding of the genetic diversity on Earth and a vital part in evolutionary and conservation biology research. In lichenized fungi, species pairs have the same morphology and chemistry. They only differ in how they reproduce with one species using sexual reproductive structures and the other using asexual propagules. To classify these as one species or two has been a point of contention, and conclusions based on Sanger sequencing, where sequence data are limited and species boundaries are usually not observed, have been refuted after analysis with genome-scale data such as restriction site-associated DNA sequencing that tends to find fixed genetic differences between the two morphs. Pseudocyphellaria glabra and P. homoeophylla have long been considered a species pair that differ in geographical ranges but co-occur in New Zealand. We used restriction site-associated DNA sequencing data and generated thousands of genetic loci across 53 individuals. The RADseq data provided high-resolution phylogenetic and population genomic information. A maximum-likelihood phylogenetic reconstruction recovered both species as separate lineages, whereas population genetics indicated some evidence for admixture among P. glabra and P. homoeophylla from New Zealand. It is not clear whether the latter is due to ancient polymorphism or recent gene flow. Our study represents another example of the usefulness of RADseq to test species boundaries that segregate closely related species in lichenized fungi.

Characterization of a sex-determining locus and development of early molecular assays in Telfairia occidentalisHook. F., a dioecious cucurbit

Although there exist over 7000 crop species, only a few are commercially valuable and grown on a large scale in monocultures worldwide. However, underutilised crops (also called orphan crops) have significant potential for food security and Telfairia occidentalis Hook. F. (Cucurbitaceae) is one such orphan crop grown in West Africa for its nutritious leaves, oil and protein-rich seeds. In this dioecious crop, farmers like to eliminate male plants and keep mostly females to increase their yield. However, they face the challenge of determining sex due to limited morphological differences between females and males before flowering. This study used double digested restriction site-associated DNA sequencing data (ddRADseq) to examine the genetic diversity within and among landraces of T. occidentalis, identify common sex-determining loci, and establish reliable assays to characterize the sex of immature plants in the vegetative state. To differentiate males from females of T. occidentalis, two molecular assays were thereupon developed based on polymerase chain reaction (PCR) to genotype sex-specific sequence variation either through restriction by Mfe1 or the direct use of sex-specific primers. Both assays require standard laboratory conditions to reach a certainty of 94.3% for females and 95.7% for males from the studied samples. With the inclusion of additional landraces, medium to largescale farms growing T. occidentalis as a crop can readily benefit from an early determination of the sex of plants.

Multispectral Phenotyping and Genetic Analyses of Spring Appearance in Greening Plant, Phedimus spp.

The change in appearance during the seasonal transitions in ornamental greening plants is an important characteristic. In particular, the early onset of green leaf color is a desirable trait for a cultivar. In this study, we established a method for phenotyping leaf color change by multispectral imaging and performed genetic analysis based on the phenotypes to clarify the potential of the approach in breeding greening plants. We performed multispectral phenotyping and quantitative trait locus (QTL) analysis of an F1 population derived from 2 parental lines of Phedimus takesimensis, known to be a drought and heat-tolerant rooftop plant species. The imaging was conducted in April of 2019 and 2020 when dormancy breakage occurs and growth extension begins. Principal component analysis of 9 different wavelength values showed a high contribution from the first principal component (PC1), which captured variation in the visible light range. The high interannual correlation in PC1 and in the intensity of visible light indicated that the multispectral phenotyping captured genetic variation in the color of leaves. We also performed restriction site-associated DNA sequencing and obtained the first genetic linkage map of Phedimus spp. QTL analysis revealed 2 QTLs related to early dormancy breakage. Based on the genotypes of the markers underlying these 2 QTLs, the F1 phenotypes with early (late) dormancy break, green (red or brown) leaves, and a high (low) degree of vegetative growth were classified. The results suggest the potential of multispectral phenotyping in the genetic dissection of seasonal leaf color changes in greening plants.

Genetic diversity and population structure of the upriver orange mangrove Bruguiera sexangula along the coastlines of Thailand

Upriver orange mangrove Bruguiera sexangula is a member of the Rhizophoraceae family and is usually distributed in the intertidal zones of the tropical and subtropical coastal areas. The analyses of genetic diversity and population structure of B. sexangula are essential for their conservation and management. In the present study, the genetic diversity and structure of 101 B. sexangula individuals from mangrove forests in Thailand were evaluated using single nucleotide polymorphisms (SNPs) through restriction site-associated DNA sequencing (RAD-seq). A total of 3482 high-quality SNPs were obtained after filtration for genetic study. All 3 genetic structure analyses (Bayesian analysis, principal components analysis, and maximum likelihood tree) revealed that these individuals can be clustered into 2 groups corresponding to geographical regions, namely the Andaman Sea and Gulf of Thailand. The level of genetic differentiation between the clusters was moderate (FST = 0.122, p < 0.001), and the analysis of molecular variance (AMOVA) indicated that the individual differences within groups were greater than the differences between the 2 groups. A moderate level of genetic diversity (Shannon's information index, I = 0.458; observed heterozygosity, Ho = 0.416; expected heterozygosity, He = 0.295) of B. sexangula was observed. These results give us a better understanding of the genetic variations and will provide a framework for the conservation of B. sexangula.

SNPs Detection and Genetic Analysis of Chionanthus retusus via Genotyping-by-Sequencing

Abstract Chionanthus retusus is one of the well-known ornamental trees in East Asia and America. Not only its value in the market but also had the potential as a source for producing antioxidant. However, due to uncontrolled exploitation, the number of wild C. retusus in China is decreasing rapidly. The genetic study of C. retusus is limited. In order to investigate the genetic diversity and the distribution of C. retusus in China, 47 samples from 8 different provinces have been sequenced via restriction site-associated DNA sequencing (RAD-seq). Totally, 31, 402 high-quality single nucleotide polymorphisms (SNP) were obtained. According to the phylogenetic tree and the principal component analysis, the samples were divided into four populations, including 3 major populations and 1 hybrid population. Population1 were the samples from Jiangsu and Yunnan province and the Population2 were mainly from northern and northeast of China including Liaoning and Hubei province, while the Population4 were from Shandong and Henan province, which were in central China. As the admixture showed, the population3 were the offspring of the other 3 populations by hybridization. The mean heterozygosity of Chinese Fringe tree from different province is 0.42 %, with the highest heterozygosity, which is as high as 0.63 %, from Jiangsu province and the lowest heterozygosity, which is only 0.19, from Henan province. This is the first report about the genetic diversity and relationship of Chionanthus retusus, which will provide value information for further genetic study, genomic study, conservation and breeding.

Genomic differentiation with isolation by distance along a latitudinal gradient in the spotted-leg treefrog Polypedates megacephalus.

The patterns of isolation by distance (IBD) entailing increased genetic differentiation among populations have aroused extensive concerns for evolutionary biologists. Although the IBD may act on spatial processes contributing to the genetic differentiation among populations in anuran species, the factors shaping the IBD of frogs among populations in natural systems are largely unknown. Here, we studied the genetic differentiation among six populations with 24 individuals of the spotted-leg treefrog along a latitudinal gradient (1860.31 km) based on 1020 single nucleotide polymorphisms from restriction site-associated DNA sequencing. The results showed that the genetic diversity differed significantly among populations and that the insular populations had higher genetic diversity than the mainland populations. Furthermore, we also found a significant genetic differentiation among populations (FST = 0.277) and no sign of inbreeding (FUNI = -0.145). The IBD was detected for all populations, and a higher degree of the IBD was indicated when controlling for the effects of the isolation between Hainan and mainland populations caused by the Qiongzhou Strait. Our findings suggest that the form of the Qiongzhou Strait plays a key role in shaping the genetic diversity and population differentiation in treefrogs.

Morphology, nuclear SNPs and mate selection reveal that COI barcoding overestimates species diversity in a Mediterranean freshwater amphipod by an order of magnitude.

DNA sequence information has revealed many morphologically cryptic species worldwide. For animals, DNA-based assessments of species diversity usually rely on the mitochondrial cytochrome c oxidase subunit I (COI) gene. However, a growing amount of evidence indicate that mitochondrial markers alone can lead to misleading species diversity estimates due to mito-nuclear discordance. Therefore, reports of putative species based solely on mitochondrial DNA should be verified by other methods, especially in cases where COI sequences are identical for different morphospecies or where divergence within the same morphospecies is high. Freshwater amphipods are particularly interesting in this context because numerous putative cryptic species have been reported. Here, we investigated the species status of the numerous mitochondrial molecular operational taxonomic units (MOTUs) found within Echinogammarus sicilianus. We used an integrative approach combining DNA barcoding with mate selection observations, detailed morphometrics and genome-wide double digest restriction site-associated DNA sequencing (ddRAD-seq). Within a relatively small sampling area, we detected twelve COI MOTUs (divergence=1.8-20.3%), co-occurring in syntopy at two-thirds of the investigated sites. We found that pair formation was random and there was extensive nuclear gene flow among the ten MOTUs co-occurring within the same river stretch. The four most common MOTUs were also indistinguishable with respect to functional morphology. Therefore, the evidence best fits the hypothesis of a single, yet genetically diverse, species within the main river system. The only two MOTUs sampled outside the focal area were genetically distinct at the nuclear level and may represent distinct species. Our study reveals that COI-based species delimitation can significantly overestimate species diversity, highlighting the importance of integrative taxonomy for species validation, especially in hyperdiverse complexes with syntopically occurring mitochondrial MOTUs.

Cryptic persistence and loss of local endemism in Lake Constance charr subject to anthropogenic disturbance.

In the welcome circumstance that species believed extinct are rediscovered, it is often the case that biological knowledge acquired before the presumed extinction is limited. Efforts to address these knowledge gaps, in particular to assess the taxonomic integrity and conservation status of such species, can be hampered by a lack of genetic data and scarcity of samples in museum collections. Here, we present a proof-of-concept case study based on a multidisciplinary data evaluation approach to tackle such problems. The approach was developed after the rediscovery, 40 years after its presumed extinction, of the enigmatic Lake Constance deep-water charr Salvelinus profundus. Targeted surveys led to the capture of further species and additional sympatric normal charr, Salvelinus cf. umbla. Since the lake had been subject to massive stocking in the past, an evaluation of the genetic integrity of both extant forms was called for in order to assess possible introgression. A two-step genomic approach was developed based on restriction site associated DNA (RAD). Diagnostic population genomic (single nucleotide polymorphism [SNP]) data were harvested from contemporary samples and used for RNA bait design to perform target capture in DNA libraries of archival scale material, enabling a comparison between extant and historic samples. Furthermore, life history traits and morphological data for both extant forms were gathered and compared with historical data from the past 60-120 years. While extant deep-water charr matched historical deep-water specimens in body shape, gill raker count, and growth rates, significant differences were discovered between historical and extant normal charr. These resulted were supported by genomic analyses of contemporary samples, revealing the two extant forms to be highly divergent. The results of population assignment tests suggest that the endemic deep-water charr persisted in Lake Constance during the eutrophic phase, but not one of the historical genomic samples could be assigned to the extant normal charr taxon. Stocking with non-endemic charr seems to be the most likely reason for these changes. This proof-of-concept study presents a multidisciplinary data evaluation approach that simultaneously tests population genomic integrity and addresses some of the conservation issues arising from rediscovery of a species characterized by limited data availability.