Restriction Site-associated DNA Sequencing Data Research Articles

Differentiation of wild accessions within the Panax bipinnatifidus complex using newly developed polymorphic microsatellite markers

Abstract Panax L., renowned as ginseng genus, is a famous medicinal group of family Araliaceae. Within this genus, the taxa of Panax bipinnatifidus complex are mainly distributed in Himalayas and Hengduan Mountain areas. Due to the complex evolutionary history and short-term rapid radiation, the relationships among species within the complex have not been clearly resolved, and the taxa identification is difficult due to the intermediate morphological traits. This study aimed to use the available restriction-site associated DNA sequence data from 29 individuals of P. bipinnatifidus complex to mine high-polymorphic simple sequence repeat (SSR) markers, with the goal of evaluating their utility in taxa identification. Eleven polymorphic SSR loci were ultimately selected and validated through polymerase chain reactions amplifying across 63 individuals of P. bipinnatifidus complex and 13 individuals of three outgroup species. The subsequent genetic diversity analysis uncovered 76 alleles in total, ranging from 5 to 15 per locus. Observed heterozygosity spanned 0.241–0.512, while expected heterozygosity ranged between 0.345 and 0.644. The genetic kinship analysis revealed a sister relationship between Panax zingiberensis and Panax vietnamensis. The analysis result also supported the classification of samples from Hunan and Hubei provinces into a single genetic unit within the P. bipinnatifidus complex. These newly developed SSR markers will facilitate the identification of wild ginseng plants.

How do hydroelectric dams affect non-migratory fish?: genomic evidence for Cichla temensis (Perciformes: Cichlidae) in the Uatumã River, Amazonas, Brazil

Abstract The Balbina hydroelectric dam was built in the 1980s on the Uatumã River to supply electricity to Manaus, capital of the state of Amazonas, Brazil. Its construction caused a drastic change to the alluvial landscape, forming a large, lentic, artificial reservoir rather than a previously dynamic river system. The objective of our research was to evaluate the impact of the hydroelectric dam on the microevolutionary processes of fish, taking as a model Cichla temensis, an iconic non-migratory fish from the Amazon, abundant in this ecosystem. We collected double digest restriction site-associated DNA sequencing data and extracted single nucleotide polymorphisms, which we identified at neutral and adaptive loci, for subsequent analyses. The results showed that the connectivity of the population was affected historically by natural physical barriers, before the construction of the hydroelectric dam, recognizing two independent population units. We detected that the hydroelectric dam increased the differentiation between populations and affected the genetic diversity and effective population sizes, in addition to promoting a recent population expansion upstream of the dam, as a consequence of adaptation to the new environment. This study provides the first evidence that hydroelectric dams can promote local adaptation in some fish, such as non-migratory ones.

Low-coverage sequencing and Wahlund effect severely bias estimates of inbreeding, heterozygosity and effective population size in North American wolves.

vonHoldt etal. ((2024), Molecular Ecology, 33, e17231) (vH24) used low-coverage (average ~ 7X read depth) restriction site-associated DNA sequence data to estimate individual inbreeding and heterozygosity, and recent effective population size (Ne), in Great Lakes (GL) and Northern Rocky Mountain (RM) wolves. They concluded that RM heterozygosity rapidly declined between 1991 and 2020, and that Ne declined substantially in GL and RM over the last 50 generations. Here, we evaluate the effects of low sequence coverage and sampling strategy on vH24's findings and provide general recommendations for using sequence data to evaluate inbreeding, heterozygosity and Ne. Low-coverage sequencing resulted in downwardly biased estimates of individual inbreeding and heterozygosity, and an erroneous large temporal decline in RM heterozygosity due to declining read depth through time. Additionally, vH24's sampling strategy-which combined individuals from several genetically differentiated populations and across at least eight wolf generations-is expected to have resulted in severe downward bias in estimates of recent Ne for RM. We recommend using high-coverage sequence data ( 15-20X) to estimate inbreeding and heterozygosity. Carefully filtering individuals, loci and genotypes, and using genotype imputation or likelihoods can help to minimise bias when low-coverage sequence data must be used. For estimation of contemporary Ne, the marginal benefits of using more than 103-104 loci are small, so aggressive filtering of loci with low average read depth potentially can retain most individuals without sacrificing much precision. Individuals are relatively more valuable than loci because analyses of contemporary Ne should focus on roughly single-generation samples from local breeding populations.

Phylogeography of Pterocarya hupehensis reveals the evolutionary patterns of a Cenozoic relict tree around the Sichuan Basin.

Environmental factors such as mountain tectonic movements and monsoons can enhance genetic differentiation by hindering inter- and intra-specific gene flow. However, the phylogeographic breaks detected within species may differ depending on the different molecular markers used, and biological traits may be a major confounding factor. Pterocarya hupehensis is a vulnerable relict species distributed throughout the Sichuan Basin. Here, we investigated the phylogeographic patterns and evolutionary history of P. hupehensis using chloroplast DNA and restriction site-associated DNA sequencing data from 18 populations around the Sichuan Basin. The 24 chloroplast haplotypes separated into western and eastern lineages at approximately 16.7 Mya, largely coincident with a strengthening of the East Asian monsoon system during the early to middle Miocene. Both cpDNA and nuclear DNA datasets consistently identified distinct western and eastern lineages whose phylogeographic break conformed to the boundary of the Sino-Himalayan and Sino-Japanese forest sub-kingdoms. However, in contrast to the nuclear gene data, the cpDNA data revealed further divergence of the eastern lineage into northern and southern groups along the Yangtze River, a result that likely reflects differences in the extent of pollen vs seed dispersal. During the temperature decline in the penultimate (Riss) glacial period of the Pleistocene epoch, P. hupehensis experienced a genetic bottleneck event, and ecological niche modeling suggests that a subsequent population expansion occurred during the last interglacial period. Our findings not only establish a basis for conservation of this species, but also serve as a case study for the effects of geography and climate change on the evolutionary history of wind-pollinated relict plants.

Origins of cryophilous lineages and the role of Beringia: evidence from the cosmopolitan angiosperm genus Stellaria L. (Caryophyllaceae)

Abstract The cosmopolitan angiosperm genus Stellaria L. (Caryophyllaceae) occurs across the Arctic and is most diverse in the high southern Asian mountains, including presence at the most extreme latitudinal and elevational limits of vascular plants. Using the primary fossil record and double digest restriction site-associated DNA sequencing data, we estimated divergence times of Stellaria and relatives across the Caryophyllaceae. We then used ancestral area and ancestral state reconstructions to interpret the biogeographical history of the group. We found that Stellaria originated in the Miocene and gave rise to repeated New World lineages, mostly from temperate Old World regions and probably via Bering Land Bridges. Circumboreal lineages were recovered of recent, Pleistocene origin and several might have originated in southerly mountains of both the Old World and the New World before subsequently colonizing the Arctic. Ancestral state reconstruction of inhabitance of cold vs. temperate climates and of wet vs. dry habitats revealed repeated evolutionary transitions across these extremes by members of the genus worldwide. Our study, which samples nearly all species within a diverse and cosmopolitan lineage of flowering plants, recovers a group characterized by niche lability and helps to support prior findings of temperate origins of many cryophilous plant lineages. Pre-adaptation to cold might have been a prerequisite for colonization of the Arctic.

Implications of headwater contact zones for the riverine barrier hypothesis: a case study of the Blue-capped Manakin (Lepidothrix coronata).

Rivers frequently delimit the geographic ranges of species in the Amazon Basin. These rivers also define the boundaries between genetic clusters within many species, yet river boundaries have been documented to break down in headwater regions where rivers are narrower. To explore the evolutionary implications of headwater contact zones in Amazonia, we examined genetic variation in the Blue-capped Manakin (Lepidothrix coronata), a species previously shown to contain several genetically and phenotypically distinct populations across the western Amazon Basin. We collected restriction site-associated DNA sequence data (RADcap) for 706 individuals and found that spatial patterns of genetic structure indicate several rivers, particularly the Amazon and Ucayali, are dispersal barriers for L. coronata. We also found evidence that genetic connectivity is elevated across several headwater regions, highlighting the importance of headwater gene flow for models of Amazonian diversification. The headwater region of the Ucayali River provided a notable exception to findings of headwater gene flow by harboring non-admixed populations of L. coronata on opposite sides of a < 1-km-wide river channel with a known dynamic history, suggesting that additional prezygotic barriers may be limiting gene flow in this region.

Phylogenomic data reveal cryptic diversity and deep phylogeographical structure within the common chuckwalla, Sauromalus ater (Squamata: Iguanidae)

Abstract Understanding how historical geological processes drive diversification and shape the contemporary distribution of species is fundamental to phylogeography. We take a genomic approach in order to elucidate the deep phylogeographical history and species limits of chuckwallas (Sauromalus), a conspicuous group of lizards of the arid lands of south-western North America. Phylogenetic and population genomic analyses of double digest restriction site-associated DNA sequencing data confirm the presence of at least two major lineages, peninsular and continental groups, within the widespread and morphologically variable common chuckwalla (Sauromalus ater). These lineages diversified in the vicinity of the head of the Gulf of California in north-eastern Baja California in the early Pliocene to late Miocene, during the formation of the northern gulf. The peninsular lineage of S. ater subsequently gave rise to the four insular endemic species of Sauromalus associated with the Baja California peninsula. Genomic analyses strongly support the continued recognition of the insular gigantics Sauromalus varius and Sauromalus hispidus as distinct species, although their relationship as sister species remains unresolved. Weaker phylogenetic signal for the insular species Sauromalus slevini and Sauromalus klauberi is provided by the genomic data; thus, it is advocated to continue recognizing these species until additional data can be analysed to evaluate their distinctiveness.

A phylogenomic analysis of Lonicera and its bearing on the evolution of organ fusion.

The ~140 species of Lonicera are characterized by variously fused leaves, bracteoles, and ovaries, making it a model system for studying the evolution and development of organ fusion. However, previous phylogenetic analyses, based mainly on chloroplast DNA markers, have yielded uncertain and conflicting results. A well-supported phylogeny of Lonicera will allow us to trace the evolutionary history of organ fusion. We inferred the phylogeny of Lonicera using restriction site-associated DNA sequencing (RADSeq), sampling all major clades and 18 of the 23 subsections. This provided the basis for inferring the evolution of five fusion-related traits. RADSeq data yielded a well-resolved and well-supported phylogeny. The two traditionally recognized subgenera (Periclymenum and Chamaecerasus), three of the four sections (Isoxylosteum, Coeloxylosteum, and Nintooa), and half of the subsections sampled were recovered as monophyletic. However, the large and heterogeneous section Isika was strongly supported as paraphyletic. Nintooa, a clade of ~22 mostly vine-forming species, including L. japonica, was recovered in a novel position, raising the possibility of cytonuclear discordance. We document the parallel evolution of fused leaves, bracteoles, and ovaries, with rare reversals. Most strikingly, complete cupules, in which four fused bracteoles completely enclose two unfused ovaries, arose at least three times. Surprisingly, these appear to have evolved directly from ancestors with free bracteoles instead of partial cupules. We provide the most comprehensive and well-supported phylogeny of Lonicera to date. Our inference of multiple evolutionary shifts in organ fusion provides a solid foundation for in depth developmental and functional analyses.

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.

Forest cover and geographical distance influence fine-scale genetic structure of leaf-toed geckos in the tropical dry forests of western Mexico

Abstract The biodiversity within tropical dry forests (TDFs) is astounding and yet poorly catalogued due to inadequate sampling and the presence of cryptic species. In the Mexican TDF, endemic species are common, and the landscape has been continually altered by geological and anthropogenic changes. To understand how landscape and environmental variables have shaped the population structure of endemic species, we studied the recently described species of leaf-toed gecko, Phyllodactylus benedettii, in coastal western Mexico. Using double-digest restriction site-associated DNA sequencing data, we first explore population structure and estimate the number of ancestral populations. The results indicate a high degree of genetic structure with little admixture, and patterns corresponding to both latitudinal and altitudinal gradients. We find that genetic structure cannot be explained purely by geographical distance, and that ecological corridors may facilitate dispersal and gene flow. We then model the spatial distribution of P. benedettii in the TDF through time and find that the coastline has been climatically suitable for the species since the Last Glacial Maximum. Landscape genetic analyses suggest that the combination of isolation by distance (IBD) and isolation by resistance (IBR; forest cover) has influenced the spatial genetic structure of the species. Overall, our genomic data demonstrate fine-scale population structure in TDF habitat, a complex colonization history, and spatial patterns consistent with both IBD and other ecological factors. These results further highlight the Mexican TDF as a diversity hotspot and suggest that continued anthropogenic changes are likely to affect native fauna.

Molecular Systematics, Species Concepts, and Myrmecophytism in Cecropia (Cecropieae: Urticaceae): Insights from Restriction-Site Associated DNA

Abstract— Cecropia is a group of fast-growing pioneer trees that are important in forest regeneration and a common ant-plant mutualism in the Neotropics. To investigate the evolution of mutualism between Cecropia and associated ants, a phylogenetic framework is necessary. Cecropia species are difficult to distinguish morphologically and conventional genetic markers are insufficiently variable to resolve the phylogenetic relationships among species. Our study aimed to compare the phylogenetic utility of restriction site associated DNA (RAD) sequencing to prior work based on commonly sequenced gene regions. RAD sequence data resolved and supported species-level relationships better than previous studies. We identified a deeply divergent non-myrmecophytic clade including C. sciadophylla and African Musanga. Results from geographically widespread and morphologically heterogenous C. obtusifolia and C. angustifolia suggest that current synonymy has lumped phylogenetically divergent lineages. Reconstruction of ant associations on the highly supported Cecropia phylogeny inferred equal probability of the ancestor of Cecropia being myrmecophytic or not. More intensive genetic study is needed to refine species concepts in Cecropia.

Systematics of Lepidothrix manakins (Aves: Passeriformes: Pipridae) using RADcap markers

Although recent molecular phylogenetic analyses of Lepidothrix manakins (family Pipridae) have helped clarify their evolutionary relationships, the placement of several lineages remains in question because of low or conflicting branch support. In particular, the relationship of L. coronata to other members of the genus and relationships within the L. nattereri + L. vilasboasi + L. iris clade have been difficult to resolve. We used RADcap to collect restriction site-associated DNA sequence data and estimate the first subspecies-level phylogeny of the genus Lepidothrix (17 of 18 currently recognized subspecies), and we included extensive geographic representation of the widespread and phenotypically variable L. coronata. We found strong support for the phylogenetic position and monophyly of L. coronata, and we resolved two clades separated by the Andes that, along with previous divergence time estimates and our assessment of morphological and vocal evidence, suggest the presence of two biological species: Velvety Manakin (L. velutina) west of the Andes and Blue-capped Manakin (L. coronata) east of the Andes. Species-level relationships within the L. nattereri + L. vilasboasi + L. iris clade remained poorly resolved in concatenated and coalescent-based analyses, with SNAPP analyses suggesting that the lack of reciprocal monophyly is due to extensive allele sharing among these taxa. Finally, we confirmed a previously documented hybrid between L. coronata and L. suavissima as an F1 individual, consistent with the view that hybridization between these two species is a rare event and that postmating reproductive barriers prevent successful backcrossing.

Population Genomics of New Zealand Pouched Lamprey (kanakana; piharau; Geotria australis).

Pouched lamprey (Geotria australis) or kanakana/piharau is a culturally and ecologically significant jawless fish that is distributed throughout Aotearoa New Zealand. Despite its importance, much remains unknown about historical relationships and gene flow between populations of this enigmatic species within New Zealand. To help inform management, we assembled a draft G. australis genome and completed the first comprehensive population genomics analysis of pouched lamprey within New Zealand using targeted gene sequencing (Cyt-b and COI) and restriction site-associated DNA sequencing (RADSeq) methods. Employing 16 000 genome-wide single nucleotide polymorphisms (SNPs) derived from RADSeq (n = 186) and sequence data from Cyt-b (766 bp, n = 94) and COI (589 bp, n = 20), we reveal low levels of structure across 10 sampling locations spanning the species range within New Zealand. F-statistics, outlier analyses, and STRUCTURE suggest a single panmictic population, and Mantel and EEMS tests reveal no significant isolation by distance. This implies either ongoing gene flow among populations or recent shared ancestry among New Zealand pouched lamprey. We can now use the information gained from these genetic tools to assist managers with monitoring effective population size, managing potential diseases, and conservation measures such as artificial propagation programs. We further demonstrate the general utility of these genetic tools for acquiring information about elusive species.

Investigation of genome-wide InDel distribution and segregation in Populus with restriction-site associated DNA sequencing data

Insertion or deletion polymorphism (InDel) is one of the main genetic variations in plant genomes. However, there are few studies on InDels across the whole genome in Populus. In this study, we investigated genome-wide InDels in Populus deltoides and Populus simonii and InDel segregation in their F1 hybrid population with restriction-site associated DNA sequencing (RAD-seq) data. A total of 119,066 InDels were identified in P. deltoides and P. simonii according to the reference genome of Populus trichocarpa, including 58,532 unique InDels in P. deltoides, 54,469 unique InDels in P. simonii, and 6,065 common InDels in both. Meanwhile, the distribution of these InDels was analyzed along chromosomes, indicating that the distribution patterns for both species were largely similar, but the average InDel density was slightly higher in P. deltoides than in P. simonii. GO annotation and enrichment analysis of those genes harboring InDels showed the same patterns between the two poplar species. It is interesting to find that the ratio (~ 46%) of the common InDels within genes to all common InDels was higher than that of the InDels within genes to all InDels in P. deltoides (~ 35%) or in P. simonii (~ 34%), possibly indicating that those InDels are more conservative between poplar species. Moreover, investigation of the InDel segregation patterns demonstrated that a large number of Mendelian InDels could be selected for genetic mapping in the F1 hybrid population. RAD-seq provides genome-wide insights into the InDel distributions in P. deltoides and P. simonii and the segregation patterns in their progeny, providing valuable genomic variation information for genetic and evolutionary studies in Populus.

Long evolutionary history of an emerging fungal pathogen of diverse tree species in eastern Asia, Australia and the Pacific Islands.

Emerging plant pathogens have been increasing exponentially over the last century. To address this issue, it is critical to determine whether these pathogens are native to ecosystems or have been recently introduced. Understanding the ecological and evolutionary processes fostering emergence can help to manage their spread and predict epidemics/epiphytotics. Using restriction site-associated DNA sequencing data, we studied genetic relationships, pathways of spread and the evolutionary history of Phellinus noxius, an emerging root-rotting fungus of unknown origin, in eastern Asia, Australia and the Pacific Islands. We analysed patterns of genetic variation using Bayesian inference, maximum-likelihood phylogeny, population splits and mixtures measuring correlations in allele frequencies and genetic drift, and finally applied coalescent-based theory using Approximate Bayesian computation (ABC) with supervised machine learning. Population structure analyses revealed five genetic groups with signatures of complex recent and ancient migration histories. The most probable scenario of ancient pathogen spread is movement from an unsampled population to Malaysia and the Pacific Islands, with subsequent spread to Taiwan and Australia. Furthermore, ABC analyses indicate P. noxius spread occurred thousands of generations ago, contradicting previous assumptions that this pathogen was recently introduced to multiple geographical regions. Our results suggest that recent emergence of P. noxius in eastern Asia, Australia and the Pacific Islands has probably been driven by anthropogenic and natural disturbances, such as deforestation, land-use change, severe weather events and/or introduction of exotic plants. This study provides a novel example of applying genome-wide allele frequency data to unravel the dynamics of pathogen emergence under changing ecosystem conditions.

Incompatibility Phylogenetic Signals between Double-Digest Restriction Site-Associated DNA Sequencing and Plastid Genomes in Chinese Curcuma (Zingiberaceae)—A Recent Qinghai–Tibetan Plateau Diversification Genera

Curcuma is of high economic value, credited to its medicinal, edible, and ornamental properties, which possess all signatures of adaptability, and rapid radiation, especially species of Curcuma (Chinese Curcuma, a recent Qinghai–Tibetan Plateau diversification genera) scattered in China. However, little is known about the incongruent phylogenetic signals within this genera from different inheritance patterns that will militate against the further development of this genera. In this research, we applied complete chloroplast genome data together with double-digest restriction site-associated DNA sequencing data (ddRAD-seq) strategy to investigate phylogenetic signals of Chinese Curcuma species, clustering using two RAD analysis pipelines (STACKS and pyRAD). Phylogenetic trees were obtained from each locus based on the maximum likelihood (ML) and multispecies coalescent (BEAST) methods. For visual comparison, multi-method and different datasets were used to infer the phylogeny. We discovered inconsistent relationships for the Chinese Curcuma with varying degrees of support using different methods and datasets.

Phylogeography of the East Asian grassland plant, Viola orientalis (Violaceae), inferred from plastid and nuclear restriction site-associated DNA sequencing data.

To elucidate the origin and migration history of the "Mansen elements," a group of temperate grassland plants mainly distributed in northeastern Asia, phylogeographic analyses based on chloroplast DNA markers and double-digest restriction site-associated DNA sequencing (ddRAD-seq) data were performed on Viola orientalis, one of the representative species of the group. Phylogenetic analyses using ddRAD-seq data revealed that the populations of V. orientalis were clustered into five clades, among which the continental clades made of populations from Russia and Korea diverged more than 100,000years earlier than the Japanese clades. The Japanese clade likely diverged during the last glacial period, followed by a further post-glacial divergence into the Kyushu and the Honshu subclades. Our study demonstrated that V. orientalis originated in the continental area of northeastern Asia and, during the last glacial period, has spread southward through the Korean Peninsula across the Japanese Islands. This finding supports the previously proposed evolutionary hypothesis regarding the origin and migration routes of the Mansen elements.

The demographic history of Madagascan micro-endemics: have rare species always been rare?

Extinction has increased as human activities impact ecosystems, yet relatively few species have conservation assessments. Novel approaches are needed to highlight threatened species that are currently data-deficient. Many Madagascan plant species have extremely narrow ranges, but this may not have always been the case—it is unclear how the island's diverse flora evolved. To assess this, we generated restriction-site associated DNA sequence data for 10 Madagascan plant species, estimated effective population size (Ne) for each species and compared this to census (Nc) sizes. In each case, Ne was an order of magnitude larger than Nc—signifying rapid, recent population decline. We then estimated species' demographic history, tracking changes in Ne over time. We show that it is possible to predict extinction risk, particularly in the most threatened species. Furthermore, simulations showed that our approach has the power to detect population decline during the Anthropocene. Our analyses reveal that Madagascar's micro-endemics were not always rare, having experienced a rapid decline in their recent history. This casts further uncertainty over the processes that generated Madagascar's exceptional biodiversity. Our approach targets data-deficient species in need of conservation assessment, particularly in regions where human modification of the environment has been rapid.

RADSex: A computational workflow to study sex determination using restriction site-associated DNA sequencing data.

The study of sex determination and sex chromosome organization in nonmodel species has long been technically challenging, but new sequencing methodologies now enable precise and high-throughput identification of sex-specific genomic sequences. In particular, restriction site-associated DNA sequencing (RAD-Seq) is being extensively applied to explore sex determination systems in many plant and animal species. However, software specifically designed to search for and visualize sex-biased markers using RAD-Seq data is lacking. Here, we present RADSex, a computational analysis workflow designed to study the genetic basis of sex determination using RAD-Seq data. RADSex is simple to use, requires few computational resources, makes no prior assumptions about the type of sex-determination system or structure of the sex locus, and offers convenient visualization through a dedicated R package. To demonstrate the functionality of RADSex, we re-analysed a published data set of Japanese medaka, Oryzias latipes, where we uncovered a previously unknown Y chromosome polymorphism. We then used RADSex to analyse new RAD-Seq data sets from 15 fish species spanning multiple taxonomic orders. We identified the sex determination system and sex-specific markers in six of these species, five of which had no known sex-markers prior to this study. We show that RADSex greatly facilitates the study of sex determination systems in nonmodel species thanks to its speed of analyses, low resource usage, ease of application and visualization options. Furthermore, our analysis of new data sets from 15 species provides new insights on sex determination in fish.