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The globally endangered saker falcon (Falcocherrug) is currently being re-introduced in Bulgaria, where the falcons are bred in captivity and released through the hacking method. We relied on the birds' pedigree when forming the breeding pairs from 2011. In 2021-2022, we had the opportunity to evaluate our captive population via DNA tests. We performed the first genetic assessment of the sakers in the WRBC through a genome evaluation of the most important founders (n = 12) and, in 2022, we executed a microsatellite analysis on 30 saker falcons from the programme. We compared the results with the known pedigree and history of the saker falcons. The genetic tests helped to assign relatedness to some birds with missing or incomplete pedigrees, indicating the test can complement that information and lead to better management of the captive group. One pair was separated as a precaution as it was indicated by one the tests that the two birds are more closely related than expected. The research could be beneficial to other raptor captive breeding programmes dealing with a similar group composition.

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The importance of Oreochromis in worldwide aquaculture and regional fisheries motivates the study of their genetic diversity in their native range. In this article, all mitochondrial cytochrome c oxidase subunit I gene (COI) sequences of Oreochromis species are retrieved from Barcode of Life Data system to quantify the available DNA barcoding information from wild individuals collected within the native ranges of the respective species. It is found that 70% of the known species in the genus still lack a COI barcode, and only 15% of the available sequences are from within the respective native ranges. Many of the available sequences have been produced from specimens acquired from aquaculture and introduced, naturalized populations, making the assessment of variation within the original native range challenging. Analyses of the wild-collected fraction of available sequences indicated the presence of cryptic lineages within Nile tilapia Oreochromis niloticus and O. schwebischi, the occurrence of potential introgressive hybridization between O. niloticus and blue tilapia O. aureus, and potential ancestral polymorphism between Karonga tilapia O. karongae and black tilapia O. placidus. This article also reports a case of misidentification of O. mweruensis as longfin tilapia O. macrochir. These results stress the importance of improving the knowledge of genetic variation within the native ranges of Oreochromis species for better-informed conservation of these natural resources.

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Rabbitfishes are reef-associated fishes that support local fisheries throughout the Indo-West Pacific region. Sound management of the resource requires the development of molecular tools for appropriate stock delimitation of the different species in the family. Microsatellite markers were developed for the cordonnier, Siganus sutor, and their potential for cross-amplification was investigated in 12 congeneric species. A library of 792 repeat-containing sequences was built. Nineteen sets of newly developed primers, and 14 universal finfish microsatellites were tested in S. sutor. Amplification success of the 19 Siganus-specific markers ranged from 32 to 79% in the 12 other Siganus species, slightly decreasing when the genetic distance of the target species to S. sutor increased. Seventeen of these markers were polymorphic in S. sutor and were further assayed in S. luridus, S. rivulatus, and S. spinus, of which respectively 9, 10 and 8 were polymorphic. Statistical power analysis and an analysis of molecular variance showed that subtle genetic differentiation can be detected using these markers, highlighting their utility for the study of genetic diversity and population genetic structure in rabbitfishes.

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In anticipation of the current biodiversity crisis, it has become critical to rapidly and accurately assess biodiversity. DNA barcoding has proved efficient in facilitating the discovery and description of thousands of species and also provides insight into the dynamics of biodiversity. Here, we sequenced a portion of the mitochondrial cytochrome c oxidase subunit I (COI) gene from all morphospecies of reef brittle stars collected during a large-scale biodiversity survey in the southwestern Indian Ocean (SWIO). Three methods of species delineation (Automatic Barcode Gap Discovery, Generalized Mixed Yule Coalescent model, and Bayesian Poisson Tree Processes) showed concordant results and revealed 51 shallow reef species in the region. Mean intraspecific genetic distances (0.005-0.064) and mean interspecific genetic distances within genera (0.056-0.316) were concordant with previous echinoderm studies. This study revealed that brittle-star biodiversity is underestimated by 20% within SWIO and by >40% when including specimens from the Pacific Ocean. Results are discussed in terms of endemism, diversification processes, and conservation implications for the Indo-West Pacific marine biodiversity. We emphasize the need to further our knowledge on biodiversity of invertebrate groups in peripheral areas.

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Appropriate management of invasive fungi requires adequate understanding of their global diversities and movement histories. The fungus Leptographium procerum is associated with root-colonizing forest insects in pine forests throughout the world, and may have contributed to the aggressive behaviour of the red turpentine beetle (Dendroctonus valens) in the beetle's invasive range in China. We used microsatellites and mating type loci to investigate the global diversity of L. procerum and the source population of L. procerum associated with D. valens in China. Clustering analyses supported the separation of the fungal data set into three genetically and geographically-distinct clusters: Europe, North America, and China. The fungus had the highest genetic diversity in Europe, followed by North America and China. Analyses using Approximate Bayesian Computation supported Europe as the most likely source of the North American and Chinese populations. Overall, the results suggested that Europe is the global centre of diversity of L. procerum. Furthermore, they suggested that L. procerum most likely arrived in China independently of D. valens and adopted this beetle as a vector after its introduction.

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Previous investigations have uncovered divergent mitochondrial clades within the blue-spotted maskray, previously Neotrygon kuhlii (Müller and Henle). The hypothesis that the blue-spotted maskray may consist of a complex of multiple cryptic species has been proposed, and four species have been recently described or resurrected. To test the multiple cryptic species hypothesis, we investigated the phylogenetic relationships and coalescence patterns of mitochondrial sequences in a sample of 127 new individuals from the Indian Ocean and the Coral Triangle region, sequenced at both the CO1 and cytochrome b loci. The maximum-likelihood (ML) tree of concatenated CO1+cytochrome b gene sequences, rooted by the New Caledonian maskray N. trigonoides, yielded 9 strongly supported, main clades. Puillandre's ABGD algorithm detected gaps in nucleotide distance consistent with the ML phylogeny. The general mixed Yule-coalescent algorithm partitioned the dataset into putative species generally consistent with the ML phylogeny. Nuclear markers generally confirmed that distinct mitochondrial clades correspond to genetically isolated lineages. The nine main lineages identified by ML analysis were geographically distributed in a parapatric fashion, indicating reproductive isolation. The hypothesis of multiple cryptic species is thus validated.

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Despite their importance in marine biodiversity, invertebrates are far less studied than vertebrates. Given the current global biodiversity crisis and insufficient taxonomic resources, sustained efforts need to be undertaken to assess species diversity, especially in the highly threatened 'biodiversity hotspots'. Réunion is a young volcanic island lying in the Mascarene Islands (south-western Indian Ocean, SWIO), a marine biodiversity hotspot. A substantial sampling effort was conducted around Réunion Island to document shallow water reef-associated ophiuroid (brittle-stars) diversity, a class recognised as the most diverse among echinoderms. A total of 33 species were documented, increasing the known species richness of the island by 56%. Findings include 15 new records for Réunion, 11 for the Mascarene Islands and 8 for the Indian Ocean. The most diverse family was Ophiocomidae, a family of large, abundant and conspicuous tropical species. Even in this well studied family, a new species was revealed by this survey. Morphological variants together with DNA sequence variations within several species revealed cryptic species. We compared our results with the known fauna of other Mascarene Islands and discuss biogeographic implications for the region.

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Due to their geographic isolation, biotas of oceanic islands are likely influenced by episodic long distance dispersal events, but such observations are scarce. In June 2012, fishermen from Réunion Island caught an unknown specimen of grouper, identified as dusky grouper Epinephelus marginatus (Lowe, 1834). This was highly unexpected considering the large distance of its closest verified occurrence (South Africa, ∼2500km). To identify the origin of this specimen and the mechanisms driving this potential long distance colonization, we combined genetic analyses and hydrodynamic connectivity modeling approaches. Molecular markers and samples from various locations across the distribution range resulted in the identification of three putative source populations. The Réunion specimen clustered genetically with South Africa. The estimated spawning period in relation to the connectivity modeling of larvae showed no possible direct connection between South Africa and Réunion. However, connectivity was predicted through intermediate stepping stone populations likely located around the southern tip of Madagascar, where the occurrence of the species has yet to be verified. The results further highlight the potential role of the cyclone Bingiza (February 2011) in the connection between Madagascar and Réunion. This shows that cyclones may be an important driver in long distance colonization of oceanic islands.

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The molecular clock hypothesis is fundamental in evolutionary biology as by assuming constancy of the molecular rate it provides a timeframe for evolution. However, increasing evidence shows time dependence of inferred molecular rates with inflated values obtained using recent calibrations. As recent demographic calibrations are virtually non-existent in most species, older phylogenetic calibration points (>1 Ma) are commonly used, which overestimate demographic parameters. To obtain more reliable rates of molecular evolution for population studies, I propose the calibration of demographic transition (CDT) method, which uses the timing of climatic changes over the late glacial warming period to calibrate expansions in various species. Simulation approaches and empirical data sets from a diversity of species (from mollusk to humans) confirm that, when compared with other genealogy-based calibration methods, the CDT provides a robust and broadly applicable clock for population genetics. The resulting CDT rates of molecular evolution also confirm rate heterogeneity over time and among taxa. Comparisons of expansion dates with ecological evidence confirm the inaccuracy of phylogenetically derived divergence rates when dating population-level events. The CDT method opens opportunities for addressing issues such as demographic responses to past climate change and the origin of rate heterogeneity related to taxa, genes, time, and genetic information content.

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Recently, 454 sequencing has emerged as a popular method for isolating microsatellites owing to cost-effectiveness and time saving. In this study, repeat-enriched libraries from two southern African endemic sparids (Pachymetopon blochii and Lithognathus lithognathus) were 454 GS-FLX sequenced. From these, 7370 sequences containing repeats (SCRs) were identified. A brief survey of 23 studies showed a significant difference between the number of SCRs when enrichment was performed first before 454 sequencing. We designed primers for 302 unique fragments containing more than five repeat units and suitable flanking regions. A fraction (<11%) of these loci were characterized with 18 polymorphic microsatellite loci (nine in each of the focal species) being described. Sanger sequencing of alleles confirmed that size variation was because of differences in the number of tandem repeats. However, a case of homoplasy and sequencing errors in the 454 sequencing were identified. These newly developed and four previously isolated loci were successfully used to identify polymorphic markers in nine other economically important species, representative of sparid diversity. The combination of newly developed markers with data from previous sparid cross-species studies showed a significant negative correlation between genetic divergence to focal species and microsatellite transferability. The high level of transferability we described (48% amplification success and 32% polymorphism) suggests that the 302 microsatellite loci identified represent an excellent resource for future studies on sparids. Microsatellite marker development should commonly include tests of transferability to reduce costs and increase feasibility of population genetics studies in nonmodel organisms.

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Compared to endemics, widespread species are of particular interest to retrace recent evolutionary history. These species have a large population size which provides a clearer genetic signature of past events. Moreover, their wide geographic range increases the potential occurrence of evolutionary events (expansion, divergence, etc.). Here, we used several coalescent-based methods to disentangle the evolutionary history of a widespread amphidromous goby (Sicyopterus lagocephalus), in the light of sea-level variations during the Pleistocene. Using 75 samples recovered from three biogeographic regions (Western Indian Ocean, Melanesia and Polynesia), we analysed a portion of the cytochromeb gene and confirmed three major haplogroups, each specific to a region. Furthermore, we found that: (1) the Melanesian haplogroup was the oldest while the two peripheral regions hosted daughter haplogroups; (2) two centrifugal colonisation events occurred from Melanesia to the periphery, each synchronised with periods of strong paleo-ENSO episodes; (3) the demographic contraction-expansion events were linked to Pleistocene sea-level changes; (4) Melanesia and Polynesia acted as efficient refuges during the Last Glacial Maximum. These results highlight the importance of studying widespread species to better understand the role of climate changes and paleo-oceanography on the evolution of biodiversity.

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BACKGROUND: Despite the impressive growth of sequence databases, the limited availability of nuclear markers that are sufficiently polymorphic for population genetics and phylogeography and applicable across various phyla restricts many potential studies, particularly in non-model organisms. Numerous introns have invariant positions among kingdoms, providing a potential source for such markers. Unfortunately, most of the few known EPIC (Exon Primed Intron Crossing) loci are restricted to vertebrates or belong to multigenic families. RESULTS: In order to develop markers with broad applicability, we designed a bioinformatic approach aimed at avoiding multigenic families while identifying intron positions conserved across metazoan phyla. We developed a program facilitating the identification of EPIC loci which allowed slight variation in intron position. From the Homolens databases we selected 29 gene families which contained 52 promising introns for which we designed 93 primer pairs. PCR tests were performed on several ascidians, echinoderms, bivalves and cnidarians. On average, 24 different introns per genus were amplified in bilaterians. Remarkably, five of the introns successfully amplified in all of the metazoan genera tested (a dozen genera, including cnidarians). The influence of several factors on amplification success was investigated. Success rate was not related to the phylogenetic relatedness of a taxon to the groups that most influenced primer design, showing that these EPIC markers are extremely conserved in animals. CONCLUSIONS: Our new method now makes it possible to (i) rapidly isolate a set of EPIC markers for any phylum, even outside the animal kingdom, and thus, (ii) compare genetic diversity at potentially homologous polymorphic loci between divergent taxa.

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