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Population genomics analysis holds great potential for informing conservation of endangered populations. We focused on a controversial case of European whitefish (Coregonus spp.) populations. The endangered North Sea houting is the only coregonid fish that tolerates oceanic salinities and was previously considered a species (C. oxyrhinchus) distinct from European lake whitefish (C. lavaretus). However, no firm evidence for genetic-based salinity adaptation has been available. Also, studies based on microsatellite and mitogenome data suggested surprisingly recent divergence (c. 2500 years bp) between houting and lake whitefish. These data types furthermore have provided no evidence for possible inbreeding. Finally, a controversial taxonomic revision recently classified all whitefish in the region as C. maraena, calling conservation priorities of houting into question. We used whole-genome and ddRAD sequencing to analyse six lake whitefish populations and the only extant indigenous houting population. Demographic inference indicated post-glacial expansion and divergence between lake whitefish and houting occurring not long after the Last Glaciation, implying deeper population histories than previous analyses. Runs of homozygosity analysis suggested not only high inbreeding (FROH up to 30.6%) in some freshwater populations but also FROH up to 10.6% in the houting prompting conservation concerns. Finally, outlier scans provided evidence for adaptation to high salinities in the houting. Applying a framework for defining conservation units based on current and historical reproductive isolation and adaptive divergence led us to recommend that the houting be treated as a separate conservation unit regardless of species status. In total, the results underscore the potential of genomics to inform conservation practices, in this case clarifying conservation units and highlighting populations of concern.
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BACKGROUND: Buoyancy and balance are important parameters for slow-moving, low-metabolic, aquatic organisms. The extant coelacanths have among the lowest metabolic rates of any living vertebrate and can afford little energy to keep station. Previous observations on living coelacanths support the hypothesis that the coelacanth is neutrally buoyant and in close-to-perfect hydrostatic balance. However, precise measurements of buoyancy and balance at different depths have never been made. RESULTS: Here we show, using non-invasive imaging, that buoyancy of the coelacanth closely matches its depth distribution. We found that the lipid-filled fatty organ is well suited to support neutral buoyancy, and due to a close-to-perfect hydrostatic balance, simple maneuvers of fins can cause a considerable shift in torque around the pitch axis allowing the coelacanth to assume different body orientations with little physical effort. CONCLUSIONS: Our results demonstrate a close match between tissue composition, depth range and behavior, and our collection-based approach could be used to predict depth range of less well-studied coelacanth life stages as well as of deep sea fishes in general.
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Peixes , Animais , Oceano ÍndicoRESUMO
Evolution sometimes proceeds by loss, especially when structures and genes become dispensable after an environmental shift relaxes functional constraints. Subterranean vertebrates are outstanding models to analyze this process, and gene decay can serve as a readout. We sought to understand some general principles on the extent and tempo of the decay of genes involved in vision, circadian clock, and pigmentation in cavefishes. The analysis of the genomes of two Cuban species belonging to the genus Lucifuga provided evidence for the largest loss of eye-specific genes and nonvisual opsin genes reported so far in cavefishes. Comparisons with a recently evolved cave population of Astyanax mexicanus and three species belonging to the Chinese tetraploid genus Sinocyclocheilus revealed the combined effects of the level of eye regression, time, and genome ploidy on eye-specific gene pseudogenization. The limited extent of gene decay in all these cavefishes and the very small number of loss-of-function mutations per pseudogene suggest that their eye degeneration may not be very ancient, ranging from early to late Pleistocene. This is in sharp contrast with the identification of several vision genes carrying many loss-of-function mutations in ancient fossorial mammals, further suggesting that blind fishes cannot thrive more than a few million years in cave ecosystems.
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Relógios Circadianos/genética , Peixes/genética , Mutação com Perda de Função , Toupeiras/genética , Pigmentação/genética , Visão Ocular/genética , Animais , Cavernas , Pseudogenes , Seleção Genética , Peixe-ZebraRESUMO
Conservation and management of marine biodiversity depends on biomonitoring of marine habitats, but current approaches are resource-intensive and require different approaches for different organisms. Environmental DNA (eDNA) extracted from water samples is an efficient and versatile approach to detecting aquatic animals. In the ocean, eDNA composition reflects local fauna at fine spatial scales, but little is known about the effectiveness of eDNA-based monitoring of marine communities at larger scales. We investigated the potential of eDNA to characterize and distinguish marine communities at large spatial scales by comparing vertebrate species composition among marine habitats in Qatar, the Arabian Gulf (also known as the Persian Gulf), based on eDNA metabarcoding of seawater samples. We conducted species accumulation analyses to estimate how much of the vertebrate diversity we detected. We obtained eDNA sequences from a diverse assemblage of marine vertebrates, spanning 191 taxa in 73 families. These included rare and endangered species and covered 36% of the bony fish genera previously recorded in the Gulf. Sites of similar habitat type were also similar in eDNA composition. The species accumulation analyses showed that the number of sample replicates was insufficient for some sampling sites but suggested that a few hundred eDNA samples could potentially capture >90% of the marine vertebrate diversity in the study area. Our results confirm that seawater samples contain habitat-characteristic molecular signatures and that eDNA monitoring can efficiently cover vertebrate diversity at scales relevant to national and regional conservation and management.
ADN Ambiental de Vertebrados Tomado del Agua Marina para Realizar Biomonitoreos de los Hábitats Marinos Resumen La conservación y el manejo de la biodiversidad marina depende del biomonitoreo de los hábitats marinos, pero las estrategias actuales requieren de muchos recursos y de diferentes estrategias para diferentes organismos. El ADN ambiental (ADNa) extraído de muestras de agua es una estrategia eficiente y versátil para detectar animales acuáticos. En el océano, la composición del ADNa refleja la fauna local a escalas espaciales finas, pero se sabe poco sobre la efectividad del monitoreo basado en el ADNa de las comunidades marinas a grandes escalas. Investigamos el potencial del ADNa para caracterizar y distinguir las comunidades marinas a escalas espaciales grandes mediante una comparación de la composición de especies de vertebrados entre los hábitats marinos de Qatar, en el Golfo Arábigo (también conocido como el Golfo Persa), con base en el meta-código de barras del ADNa extraído de muestras de agua de mar. Realizamos análisis de acumulación de especies para estimar cuánta de la diversidad de vertebrados logramos detectar. Obtuvimos secuencias de ADNa de diversos ensamblajes de vertebrados marinos, los cuales abarcaron 191 taxones de 73 familias. Estos taxones incluyeron a especies raras y en peligro de extinción y cubrieron el 36% de los géneros de peces óseos previamente registrados en el golfo. Los sitios con tipos similares de hábitat también fueron similares en cuanto a la composición del ADNa. Los análisis de acumulación de especies mostraron que el número de réplicas de muestras fue insuficiente para algunos sitios de muestreo, pero sugieren que unos cientos de muestras de ADNa podrían capturar potencialmente >90% de la diversidad de vertebrados marinos en el área de estudio. Nuestros resultados confirman que las muestras de agua marina contienen firmas moleculares características del hábitat y que el monitoreo de ADNa puede cubrir eficientemente la diversidad de vertebrados a escalas relevantes para la conservación y el manejo nacional y regional.
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Monitoramento Biológico , DNA Ambiental , Animais , Biodiversidade , Conservação dos Recursos Naturais , Código de Barras de DNA Taxonômico , Ecossistema , Monitoramento Ambiental , Água do Mar , Vertebrados/genéticaRESUMO
The order Ophidiiformes is a large but not very well known group of fishes, unique among teleosts for showing high diversity in both deep sea and shallow reef habitats. The current classification includes more than 500 species, 115 genera and four families, based primarily on mode of reproduction: viviparous Aphyonidae and Bythitidae vs oviparous Carapidae and Ophidiidae. Since 2004 we revised the bythitid tribe Dinematichthyini, described more than 100 new species and noticed that this group has unique morphological characters, perhaps supporting a higher level of classification than the current status. Here we study the viviparous families phylogenetically with partial mitochondrial (nd4, 16s) and nuclear (Rag1) DNA sequences (2194bp). We use a fossil calibration of otolith-based taxa to calibrate the age of the clade comprising bythitid and dinematicththyid representatives, together with fossil calibrations adopted from previous phylogenetic studies. The separation of the order into two major lineages, the viviparous Bythitoidei and the oviparous Ophidioidei is confirmed. At the familial level, however, a new classification is presented for the viviparous clades, placing Aphyonidae as a derived, pedomorphic member of Bythitidae (new diagnosis provided, 33 genera and 118 species). The current subfamily Brosmophycinae is considered polyphyletic and we propose family status for Dinematichthyidae (25 genera, 114 species), supported by unique, morphological synapomorphic characters in the male copulatory apparatus. Previous use of the caudal fin separation or fusion with vertical fins is ambiguous. Age estimates based on calibrated molecular phylogeny agrees with fossil data, giving an origin within the Cretaceous (between 84 and 104mya) for a common ancestor to Ophidiiformes.
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Peixes/classificação , Animais , Sequência de Bases , Evolução Molecular , Feminino , Proteínas de Peixes/genética , Peixes/anatomia & histologia , Peixes/genética , Fósseis , Especiação Genética , Genitália/anatomia & histologia , Masculino , Tipagem de Sequências Multilocus , Oviparidade , Filogenia , RNA Ribossômico 16S/genética , Viviparidade não MamíferaRESUMO
Non-invasive computed tomography (CT) of an adult sand tiger shark Carcharias taurus Rafinesque, 1810 is used to provide an interactive three-dimensional 'general' shark (Selachimorpha) anatomy atlas. Given its post-cranial body morphology, the sand tiger shark appeared to be a well-chosen candidate and through comparison of the sand tiger shark with several other representatives of all eight established orders of sharks, we confirm that the relatively large degree of mineralization of the endoskeleton, along with the overall size, makes the sand tiger shark an ideal candidate for skeletal segmentation and construction of a skeletal atlas using conventional CT. This atlas both increases accessibility to the internal morphological features of the sand tiger shark and provides a more generalized overview of the skeletal anatomy of sharks and can aid as a supplement to destructive fresh dissection of specimens in the future and the construction of future skeletal atlases of other less mineralized sharks.
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Stoat (Mustela erminea) and weasel (M. nivalis) are hard to monitor as they are elusive of nature and leave few identifying marks in their surroundings. Stoat and weasel are both fully protected in Denmark and are thought to be widely distributed throughout the country. Despite this stoat and weasel were listed on the Danish Red List as Near Threatened in 2019, as their densities and population trends are unknown. Using a modified novel camera trapping device, the Double-Mostela, a wooden box comprising a tracking tunnel and two camera traps, we attempted to obtain density estimates based on identification of individual stoats and weasels. We deployed camera traps both inside Double-Mostela traps and externally in three different study areas in northern Zealand, Denmark, and tested commercial, American scent-based lures to attract stoat and weasel. We obtained very low seasonal trapping rates of weasel in two study areas, but in one study area, we obtained a seasonal trapping rate of stoat larger compared to another study using the Mostela. In one study area, both species were absent. We observed no effect of scent-based lures in attracting small mustelids compared to non-bait traps. Potential reasons behind low capture rates of weasel and stoat are suboptimal habitat placement and timing of deployment of the Double-Mostelas, land-use changes over the last 200 years, predation from larger predators, as well as unintended secondary poisoning with rodenticides. Due to the scarcity of weasel and stoat captures, we were unable to make density estimates based on identification of individuals; however, we identified potential features that could be used for identification and density estimates with more captures.
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Dholes (Cuon alpinus) are endangered large carnivores found in scattered populations in Asia. One of the main threats to dholes is the decreasing prey availability throughout their distribution range. In the present study, we used camera trap data collected over 6 years to investigate the temporal activity patterns of dholes and their putative prey species in Baluran National Park in Java, Indonesia. We also explored the overlap in activity between dholes and the park's other remaining large carnivore the Javan leopard (Panthera pardus melas), as well as humans. Furthermore, we investigated potential differences in activity patterns between dholes in packs and dholes roaming in pairs or alone. We found a high temporal overlap between dholes and their wild ungulate prey species (ranging from Δ = 0.66-0.90), with the lowest overlap observed between dholes and bantengs (Bos javanicus) (Δ = 0.66), and the highest between dholes and muntjacs (Muntiacus muntjak) (Δ = 0.90). A very low overlap was found between dholes and domestic cattle (Bos indicus) (Δ = 0.27) whereas a moderately high overlap was found between dholes and leopards (Δ = 0.70) and dholes and humans (Δ = 0.62). We found a significant difference in activity patterns between dholes in packs and dholes roaming alone or in pairs (Δ = 0.78, p = .01). Single/pairs of dholes were more active both during the day and at night, whereas packs were predominantly active around sunrise and sunset. The high overlap with humans potentially has a negative effect on dhole activity, particularly for dispersing individuals, and the low overlap with domestic species questions the extent to which dholes are considered to predate on them.
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A new species of the zoarcid genus Pyrolycus Machida & Hashimoto, 2002, Pyrolycus jaco sp. nov., is described from a hydrothermal seep environment named Jacó Scar in the eastern Pacific of Costa Rica. Four specimens were collected in 2018 between 1746-1795 m among tubeworm colonies around the seep. The new species is differentiated from its two western Pacific congeners by having a shorter head, snout, jaw, and pectoral fins. It is further diagnosed by having three postorbital pores and two occipital pores. Molecular sequences of the cytochrome c oxidase I gene are provided and are the first for the genus. The character states indicating miniaturization in this species are discussed. This is the first vertebrate species known from this composite reducing ecosystem and is the fourth hydrothermally-associated zoarcid from the eastern Pacific.
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Ecossistema , Perciformes , Animais , Costa Rica , PeixesRESUMO
Over the past decades the sampling of environmental DNA (eDNA) - encompassing the DNA of all organisms present in an environmental sample1 - has emerged as a technique for biodiversity monitoring and discovery in a diversity of environments. Avoiding the physical collection and identification of biota, this approach is praised for its independence of taxonomic expertise and has changed the way biologists study biodiversity. However, a common result in eDNA studies is the finding of unexpected taxa which are often removed by conservative bioinformatic filters or disregarded, since the authors are uncertain about the result and rarely have the interest, time, skills, and/or resources to return to the field and confirm with actual specimens2. Here, we report a case in which an eDNA discovery led to the physical localization of a member of the Micrognathozoa (Figure 1B) - a rare group of limnic micrometazoans, and the animal phylum to be discovered last3, which is the sister group to rotifers4,5. To this day, Micrognathozoa still comprises only a single named species from Greenland and a few additional disparate places.
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DNA Ambiental , Animais , Código de Barras de DNA Taxonômico , Biodiversidade , Biota , DNA/genética , Monitoramento AmbientalRESUMO
Temporal partitioning in large carnivores have previously been found to be one of the main factors enabling co-existence. While activity patterns have been investigated separately at artificial waterholes and e.g., game trails, simultaneous comparative analyses of activity patterns at artificial waterholes and game trails have not been attempted. In this study, camera trap data from Maremani Nature Reserve was used to investigate whether temporal partitioning existed in a carnivore guild of four species (spotted hyena, leopard, brown hyena and African wild dog). Specifically, we investigated temporal partitioning at artificial waterholes and on roads and trails an average of 1,412 m away from an artificial waterhole. Activity patterns for the same species at artificial waterholes and roads/game trails were also compared. We found no significant differences in temporal activity between species at artificial waterholes. Temporal partitioning on game trails and roads was only found between spotted hyena (nocturnal) and African wild dog (crepuscular). Between nocturnal species (spotted hyena and leopard) no temporal partitioning was exhibited. Only African wild dog exhibited significantly different activity patterns at waterholes and roads/game trails. This indicates artificial waterholes may be a location for conflict in a carnivore guild. Our study highlights the impact of anthropogenic landscape changes and management decisions on the temporal axis of carnivores. More data on activity patterns at natural water sources such as ephemeral pans are needed to properly assess the effect of artificial waterholes on temporal partitioning in a carnivore guild.
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Canidae , Hyaenidae , Panthera , Animais , Ligante de CD40 , ExistencialismoRESUMO
Trinajstic et al., (Science, 16 September 2022, p. 1311-1314) describe exceptionally well-preserved organs in fossilized Devonian placoderms to infer the early evolution of the vertebrate heart. We argue that the report has numerous shortcomings and examples of mixed specimen codes. Further, we question whether there indeed is any evidence for a mineralized chambered heart in these placoderms.
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Wildlife tracking devices are key in obtaining detailed insights on movement, animal migration, natal dispersal, home-ranges, resource use and group dynamics of free-roaming animals. Despite a wide use of such devices, tracking for entire lifetimes is still a considerable challenge for most animals, mainly due to technological limitations. Deploying battery powered wildlife tags on smaller animals is limited by the mass of the devices. Micro-sized devices with solar panels sometimes solve this challenge, however, nocturnal species or animals living under low light conditions render solar cells all but useless. For larger animals, where battery weight can be higher, battery longevity becomes the main challenge. Several studies have proposed solutions to these limitations, including harvesting thermal and kinetic energy on animals. However, these concepts are limited by size and weight. In this study, we used a small, lightweight kinetic energy harvesting unit as the power source for a custom wildlife tracking device to investigate its suitability for lifetime animal tracking. We integrated a Kinetron MSG32 microgenerator and a state-of-the-art lithium-ion capacitor (LIC) into a custom GPS-enabled tracking device that is capable of remotely transmitting data via the Sigfox 'Internet of Things' network. Prototypes were tested on domestic dog (n = 4), wild-roaming Exmoor pony (n = 1) and wisent (n = 1). One of the domestic dogs generated up to 10.04 joules of energy in a day, while the Exmoor pony and wisent generated on average 0.69 joules and 2.38 joules per day, respectively. Our results show a significant difference in energy generation between animal species and mounting method, but also highlight the potential for this technology to be a meaningful advancement in ecological research requiring lifetime tracking of animals. The design of the Kinefox is provided open source.
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Animais Selvagens , Fontes de Energia Elétrica , Animais , Cães , Cavalos , Movimento , Luz SolarRESUMO
Recently, a unique mechanism for protecting the airway during lunge feeding was discovered in rorqual whales (Balaenopteridae). This mechanism is based on an oral plug structure in the soft palate with similarities in musculo-fatty composition to the nasal plugs protecting the respiratory tract of rorquals from water entry and barotrauma during diving. As a follow-up, we present here a developmental series on fetal, prenatal, juvenile and adult specimens across five species of rorquals, showing differential maturation of the nasal and oral respiratory protection plugs. Nasal plugs are fully formed to serve an immediate crucial function at birth. By contrast, the soft palate remains muscular until the onset of solid food intake, where a musculo-fatty oral plug is developed.
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Monitoring the distribution of marine nonindigenous species is a challenging task. To support this monitoring, we developed and validated the specificity of 12 primer-probe assays for detection of environmental DNA (eDNA) from marine species, all nonindigenous to Europe. The species include sturgeons, a Pacific red algae, oyster thief, a freshwater hydroid from the Black Sea, Chinese mitten crab, Pacific oyster, warty comb jelly, sand gaper, round goby, pink salmon, rainbow trout and North American mud crab. We tested all assays in the laboratory, on DNA extracted from both the target and non-target species to ensure that they only amplified DNA from the intended species. Subsequently, all assays were used to analyse water samples collected at 16 different harbours across two different seasons during 2017. We also included six previously published assays targeting eDNA from goldfish, European carp, two species of dinoflagellates of the genera Karenia and Prorocentrum, two species of the heterokont flagellate genus Pseudochattonella. Conventional monitoring was carried out alongside eDNA sampling but with only one sampling event over the one year. Because eDNA was relatively fast and easy to collect compared to conventional sampling, we sampled eDNA twice during 2017, which showed seasonal changes in the distribution of nonindigenous species. Comparing eDNA levels with salinity gradients did not show any correlation. A significant correlation was observed between number of species detected with conventional monitoring methods and number of species found using eDNA at each location. This supports the use of eDNA for surveillance of the distribution of marine nonindigenous species, where the speed and relative easy sampling in the field combined with fast molecular analysis may provide advantages compared to conventional monitoring methods. Prior validation of assays increases taxonomic precision, and laboratorial setup facilitates analysis of multiple samples simultaneously. The specific eDNA assays presented here can be implemented directly in monitoring programmes across Europe and potentially worldwide to infer a more precise picture of the dynamics in the distribution of marine nonindigenous species.
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DNA Ambiental , Dinoflagellida , Oncorhynchus mykiss , Animais , DNA/análise , Dinoflagellida/genética , Monitoramento Ambiental/métodos , Água DoceRESUMO
Environmental DNA (eDNA) provides a promising supplement to traditional sampling methods for population genetic inferences, but current studies have almost entirely focused on short mitochondrial markers. Here, we develop one mitochondrial and one nuclear set of target capture probes for the whale shark (Rhincodon typus) and test them on seawater samples collected in Qatar to investigate the potential of target capture for eDNA-based population studies. The mitochondrial target capture successfully retrieved ~235× (90× - 352× per base position) coverage of the whale shark mitogenome. Using a minor allele frequency of 5%, we find 29 variable sites throughout the mitogenome, indicative of at least five contributing individuals. We also retrieved numerous mitochondrial reads from an abundant nontarget species, mackerel tuna (Euthynnus affinis), showing a clear relationship between sequence similarity to the capture probes and the number of captured reads. The nuclear target capture probes retrieved only a few reads and polymorphic variants from the whale shark, but we successfully obtained millions of reads and thousands of polymorphic variants with different allele frequencies from E. affinis. We demonstrate that target capture of complete mitochondrial genomes and thousands of nuclear loci is possible from aquatic eDNA samples. Our results highlight that careful probe design, taking into account the range of divergence between target and nontarget sequences as well as presence of nontarget species at the sampling site, is crucial to consider. eDNA sampling coupled with target capture approaches provide an efficient means with which to retrieve population genomic data from aggregating and spawning aquatic species.
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DNA Ambiental , DNA Mitocondrial , Genoma Mitocondrial , Tubarões , Animais , Sondas de DNA , Frequência do Gene , Catar , Água do Mar , Tubarões/genéticaRESUMO
Recently, a barcoding study and a molecular phylogenetic analysis of the Cuban species of the cave-fish genus Lucifuga Poey, 1858 revealed the existence of different evolutionary lineages that were previously unknown or passed unnoticed by morphological scrutiny (i.e., cryptic candidate species). In the present study, Lucifuga gibarensis is described as a new species restricted to anchialine caves in the northeastern karst region of the main island. The species was earlier described as a variety of Lucifuga dentata, but since the name was introduced as a variety after 1960, it is deemed to be infrasubspecific and unavailable according to the International Code of Zoological Nomenclature Art. 15.2. The new species differs from L. dentata by pigmented eyes vs. eyes absent and lack of palatine teeth vs. present. Lucifuga gibarensis seems to be most similar to the Bahamian species L. lucayana by showing pigmented eyes, 13 or 14 precaudal vertebrae and ten caudal fin rays. However, differs from it by a larger size of the pigmented eye (1.1-1.9 vs. 0.9-1.0% SL) and number of posterior lateral line neuromasts (30-33 vs. 34-35).
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Environmental DNA (eDNA) extracted from water samples has recently shown potential as a valuable source of population genetic information for aquatic macroorganisms. This approach offers several potential advantages compared with conventional tissue-based methods, including the fact that eDNA sampling is noninvasive and generally more cost-efficient. Currently, eDNA approaches have been limited to single-marker studies of mitochondrial DNA (mtDNA), and the relationship between eDNA haplotype composition and true haplotype composition still needs to be thoroughly verified. This will require testing of bioinformatic and statistical software to correct for erroneous sequences, as well as biases and random variation in relative sequence abundances. However, eDNA-based population genetic methods have far-reaching potential for both basic and applied research. In this paper, we present a brief overview of the achievements of eDNA-based population genetics to date, and outline the prospects for future developments in the field, including the estimation of nuclear DNA (nuDNA) variation and epigenetic information. We discuss the challenges associated with eDNA samples as opposed to those of individual tissue samples and assess whether eDNA might offer additional types of information unobtainable with tissue samples. Lastly, we provide recommendations for determining whether an eDNA approach would be a useful and suitable choice in different research settings. We limit our discussion largely to contemporary aquatic systems, but the advantages, challenges, and perspectives can to a large degree be generalized to eDNA studies with a different spatial and temporal focus.
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For several hundred years freshwater crayfish (Crustacea-Decapoda-Astacidea) have played an important ecological, cultural and culinary role in Scandinavia. However, many native populations of noble crayfish Astacus astacus have faced major declines during the last century, largely resulting from human assisted expansion of non-indigenous signal crayfish Pacifastacus leniusculus that carry and transmit the crayfish plague pathogen. In Denmark, also the non-indigenous narrow-clawed crayfish Astacus leptodactylus has expanded due to anthropogenic activities. Knowledge about crayfish distribution and early detection of non-indigenous and invasive species are crucial elements in successful conservation of indigenous crayfish. The use of environmental DNA (eDNA) extracted from water samples is a promising new tool for early and non-invasive detection of species in aquatic environments. In the present study, we have developed and tested quantitative PCR (qPCR) assays for species-specific detection and quantification of the three above mentioned crayfish species on the basis of mitochondrial cytochrome oxidase 1 (mtDNA-CO1), including separate assays for two clades of A. leptodactylus. The limit of detection (LOD) was experimentally established as 5 copies/PCR with two different approaches, and the limit of quantification (LOQ) were determined to 5 and 10 copies/PCR, respectively, depending on chosen approach. The assays detected crayfish in natural freshwater ecosystems with known populations of all three species, and show promising potentials for future monitoring of A. astacus, P. leniusculus and A. leptodactylus. However, the assays need further validation with data 1) comparing traditional and eDNA based estimates of abundance, and 2) representing a broader geographical range for the involved crayfish species.
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Astacoidea/genética , Conservação dos Recursos Naturais/métodos , DNA/análise , Monitoramento Ambiental/métodos , Água Doce/química , Espécies Introduzidas , Animais , Ecossistema , Países Escandinavos e NórdicosRESUMO
Pisione is a scaleless group of small scale worms inhabiting sandy bottoms in shallow marine waters. This group was once considered rare, but now 45 described species can be characterized, among others, by their paired, segmental copulatory organs (one to multiple external pairs), which display a complexity of various accessory structures. The evolutionary significance of these unique organs was suggested in the late 1960s, but has been heavily debated since the late 1990s and remains controversial. In the present paper, we study the internal relationships within Pisione, employing combined phylogenetic analyses of both molecular and morphological data from 16 terminals of Pisione, as well as two terminals of Pisionidens, and eight additional scale worms as outgroups. Our taxon sampling covers all geographical areas where the genus has been reported, as well as most of their morphological and copulatory variability, including representatives of the "africana," "remota," "crassa," and "papuensis" groups, established previously by Yamanishi. We hereby provide a first insight into the relationships of the genus, testing previously proposed hypotheses on the evolutionary significance of male copulatory structures within Pisione, while attempting to understand patterns of distribution. The phylogenetic analyses using maximum likelihood and Bayesian methods consistently recovered two large clades spanning the East Atlantic (including the Mediterranean) and the Indo-Pacific-West Atlantic, respectively. Character optimization on our trees revealed a high degree of homoplasy in both non-reproductive and sexual characters of Pisione, with buccal acicula found to be the sole apomorphy among the morphological features assessed herein, with none defining the biogeographical subclades within. Overall, our comparative analyses highlight the high degree of morphological variation in this widely distributed genus, rejecting previous assertions of an increasing number and complexity of copulatory structures across the genus.