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    Health assessment of important tributaries of Three Georges Reservoir based on the benthic index of biotic integrity

    Investigation method
    From March 2015 to December 2018, we surveyed 36 important tributaries of the TGR (Fig. 3) and conducted an investigation of macroinvertebrates. For the sake of convenience, we labeled tributaries from the reservoir dam to its tail area sequentially as R01–R36, i.e., R01 (Xiangxi River), R02 (Qinggan River), R03 (Shennong River), R04 (Baolong River), R05 (Guandu River), R06 (Daning River), R07 (Daxi-F River), R08 (Caotang River), R09 (Meixi River), R10 (Changtan River), R11 (Modao River), R12 (Tangxi River), R13 (Xiao River), R14 (Zhuxi River), R15 (Rangdu River), R16 (Ruxi River), R17 (Huangjin River), R18 (Dongxi River), R19 (Chixi River), R20 (Long River), R21 (Bixi River), R22 (Quxi River), R23 (Zhenxi River), R24 (Wu River), R25 (Lixiang River), R26 (Longxi River), R27 (Taohua River), R28 (Yulin River), R29 (Wubu River), R30 (Changtang River), R31 (Chaoyang River), R32 (Jialing River), R33 (Huaxi River), R34 (Yipin River), R35 (Daxi-J River), and R36 (Qi River).
    Figure 3

    Schematic diagram of important tributaries of TGR and sampling points (plotted by ArcGIS 10.5, https://www.32r.com/soft/16101.html).

    Full size image

    This study was approved by the Environmental Protection Bureau of the Three Georges Reservoir.
    A total of 175 sampling points were set up in all tributaries. Four parallel samples were taken from each sampling point. At least one sample was taken from each microhabitat (mainly including four microhabitats, i.e., shoal, deep pool, pebble and aquatic habitat). Parallel samples from the same sampling point were mixed together. The quantitative and qualitative sample collection methods were combined in this study. The quantitative collection was performed first, and then the qualitative collection for the same sampling point. The qualitative samples were collected by D-net. Quantitative samples of wadable sampling points were collected using a Surber net with an area of 0.3 m × 0.3 m. Quantitative samples of non-wadable sampling points were collected using a D-net with a bottom side length of 0.3 m. The collected samples were put into sample bottles (bags) and fixed with 5% formaldehyde solution. Then the samples were identified and classified under the laboratory conditions.
    Selection of reference sites and impaired sites
    A reference site refers to a sampling point with no or little anthropogenic disturbance, while a impaired site refers to a sampling point subject to obvious anthropogenic disturbance29. A total of 15 reference sites and 160 impaired sites were selected from 175 sampling points based on anthropogenic disturbance, vegetation coverage, population distribution, and the distribution of industry and agriculture in the vicinity of the sampling site6,8 (Table 7, Supplementary Fig. 1).
    Table 7 Assessment criteria for reference sites and impaired sites and the assessment outcomes.
    Full size table

    Creation and selection of the assessment metric index system
    With reference to the river health assessment indexes in China13,15,18,24, North America6,24 and Europe5, and based on the ecological characteristics such as species composition and abundance, sensitivity, tolerance and functional feeding groups, we constructed 26 candidate metrics (Table 8) for B-IBI. These candidate metrics have significant or noticeable response to human activities, and normally, can be applied to relatively large geographic areas; therefore, they can be used to indicate the ecological quality of rivers6,23,24. Among these metrics, 17 were associated with species composition and abundance, which included the total number of taxon, the number of EPT taxa, the number of crustacean and mollusca taxa, the number of ephemerida taxa, the number of pteroptera taxa, the number of trichoptera taxa, the number of diptera taxa, the number of chironomidea taxa, the percentage of EPT, the percentage of crustacean and mollusca, the percentage of ephemerida, the percentage of pteroptera, the percentage of trichoptera, the percentage of dipteral, the percentage of chironomidea, the percentage of oligochaeta and the Shannon-Weiner diversity index. Species composition and abundance-related indexes reflect the diversity of macrobenthic communities. An increase in species diversity is associated with the improvement of community health, which indicates that the niche space and food sources are sufficient to support the survival and reproduction of multiple species. The candidate metrics related to sensitivity and tolerance in this study were the number of sensitive taxa, the number of tolerant taxa, the percentage of dominant species and the percentage of the top three dominant species. Different zoobenthos show different degrees of sensitivity and tolerance to the influencing factors in the river habitat, for which these characteristics can be used to assess the health status of the river. In addition, the taxa and percentage of functional feeding groups are closely associated with their living environment, and the parameters that were used to represent functional feeding in this study were the percentages of shredders, herbivores, filterers, scrapers and predators. Some of the representative images of the identified taxa were shown in Supplementary Fig. 1E,F.
    Table 8 Candidate parameters for B-IBI and their response to anthropogenic disturbance.
    Full size table

    The selection of core metrics for B-IBI mainly includes three steps: analysis of distribution range of candidate metrics, analysis of discriminant ability of candidate metrics and analysis of correlation between candidate metrics23.
    Analysis of distribution range of candidate metrics
    According to the numerical value of each biological metric in the reference site, an initial analysis was conducted to exclude the following two types of metrics: metrics with excessive nought values, which did not meet the requirement for a universal applicability; metrics with a scatter value distribution, and a standard deviation greater than or equal to the mean, indicating that the standard deviation of this value was relatively big and unstable, thereby unsuitable to be used as biological metrics6.
    Analysis of discriminant ability of candidate metrics
    After analyzing the distribution range of candidate metrics, those unsuitable for biological evaluation were eliminated. The distribution of the remaining eligible metrics for the reference site and the impaired site was analyzed using the box-plot, to mainly compare the distribution range of the 25th quantile to the 75th quantile of the reference site and the impaired site and the overlap of “box” InterQuartile Range (IQR), and judge which biological metrics could best distinguish between the reference site and impaired site. An IQ value ≥ 2 indicates a small overlapping part between the reference site and the impacted site, which means a significant difference in the related parameter between the reference site and the impacted site, suggesting a noticeable response to human activity6,24. The IQ scoring criteria were as follows6,24: 3 point, no overlapping between the two box bodies; 2 points, the box bodies have a small part of overlapping, but the median of neither body falls within the limits of its counterpart; 1 point, most parts of the box bodies overlap, and the median of at least one box body lies within the limits of its counterpart; 0 points, one box body falls within the limits of the other, or the medians of each body are within the other’s limits.
    Correlation analysis of candidate metrics
    Pearson correlation analysis was further performed of the metrics that met the preliminary conditions. If the correlation coefficient (left| {text{r}} right|) between two metrics is greater than 0.75, and they are intrinsically linked. Then most of the information reflected is overlapping. Therefore, it is OK to select one of them. If no intrinsic connection is found between two metrics, then both metrics can be selected even if the correlation coefficient is greater than 0.758.
    After screening through the above three steps, core metrics of the B-IBI are finally determined.
    Construction of B-IBI
    The core biological metrics screened out by the above method were used as the metrics for final biological assessment. The metrics used for biological assessment were standardized using the ratio scoring method, to unify the evaluation metric23.
    (1)
    For a metric that decreased with increasing interference, the metric was normalized by dividing the value of this metric at each sample point with the 95% quantile of all sample points:

    $${text{V}}_{{text{i}}}^{prime } = {text{V}}_{{text{i}}} /{text{V}}_{{{95}% }} ;$$

    (2)
    For a metric that increased with increasing interference, the metric was normalized by using the 5% quantile of this metric at all sample points as the reference object:

    $${text{V}}_{{text{i}}}^{prime } = left( {{text{V}}_{{{text{MAX}}}} – {text{V}}_{{text{i}}} } right)/left( {{text{V}}_{{{text{MAX}}}} – {text{V}}_{{{5}% }} } right),$$

    where Vi′ is the normalized value of the metric at the ith sampling point; Vi the actual value of the metric at the ith sampling point; V95% the 95% quantile of the metric; V5% is the 5% quantile of the metric; VMAX is the maximum value of this metric in all sampling points. The health thresholds of 5% quantile and 95% quantile can eliminate extreme abnormal values and retain most of biological information.

    B-IBI assessment criteria
    The 95% quantile of B-IBI distribution of all the sections/tributaries used for the health threshold can eliminate extreme abnormal values and retain most biological information. The distribution range lower than this value is divided into four portions, and the quartile close to the 95% quantile indicates a small disturbance. The biological integrity grade and the corresponding range of IBI6 are determined according to the 95% quantile and the quartile value, and the section/river health was classified into five grades, namely, excellent, good, fair, poor and very poor. More

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    Machine learning enables improved runtime and precision for bio-loggers on seabirds

    Video bio-logger hardware
    Supplementary Fig. 1a shows an example of the video bio-loggers used during this study. It measures 85 mm length × 35 mm width × 15 mm height. The bio-loggers were attached to either the bird’s back or abdomen by taping them to the bird’s feathers using waterproof tape. When attaching the bio-logger to a bird’s abdomen, a harness made of Teflon ribbon (Bally Ribbon Mills, USA) was also used. When working with streaked shearwaters, the bio-loggers used a 3.7 V 600 mAh battery and weighed approximately 26–27 g. When working with black-tailed gulls, the bio-loggers used a 3.7 V 720 mAh battery and weighed approximately 30 g.
    The bio-loggers are controlled by an ATmega328 MCU (32 KB programme memory, 2 KB RAM) and have an integrated video camera (640 × 480, 15 FPS) that can be controlled by the MCU, with the video data streamed directly to its own dedicated storage. Note that digital cameras such as the one used in this bio-logger have a delay of several seconds from powering on to when they can begin recording, which in the case of our bio-logger resulted in a 2- to 3-s delay between when the MCU signals the start of recording and the actual start of recording when attempting to save energy by powering off the camera when not in use (see also Yoshino et al.13 for another example of this camera delay). Our bio-loggers also include several low-cost sensors that are controlled by the MCU (see Supplementary Fig. 1b). Each of these sensors can be used by the MCU as input for AIoA applications (e.g., camera control) and can be archived to long-term storage for analysis upon device retrieval. The bio-loggers had an average battery life of approximately 2 h when continuously recording video and approximately 20 h when recording from all other (i.e., only low-cost) sensors.
    Activity recognition method
    We achieve AIoA by employing machine learning to conduct activity (behaviour) recognition on board the bio-logging devices. We do this by training an activity recognition model in advance using low-cost sensor data that has been labelled by an ecologist to identify the behaviours that he/she wants to capture. In the case of the black-tailed gulls, we use accelerometer-based features since they can be used to detect the body movements of the animals with only a small (e.g., 1-s) delay between when data collection begins and when behaviours can first be detected. Such features are often used when detecting body movements in human activity recognition studies in order to recognise activities such as running and eating37. For animal-based AI, such body movements can be useful to detect similar types of behaviours, such as flying and foraging38. See Fig. 3 for an example of how such accelerometer-based features can be extracted from raw data and used to train a decision tree classifier model. The features were extracted from 1-s windows of 25 Hz acceleration data, with the raw 3-axis acceleration data converted to net magnitude of acceleration data prior to feature extraction. The activity recognition processes were run once per second on the 1-s windows of data, allowing us to detect target behaviours within about 1 s of their start. See Supplementary Table 1 for descriptions and estimated sizes for all the features used in this study. In addition, Supplementary Fig. 5a shows the acceleration-based features ranked by their Normalised Gini Importance when used to classify behaviours for black-tailed gulls.
    Fig. 3: Generating decision trees from acceleration data.

    a We start by converting the raw three-axis data (row one) into magnitude of acceleration values (row two) and segmenting the data into 1-s windows. We then extract the ACC features listed in Supplementary Table 1 from each window. Rows three and four show examples of the features extracted, which can be used to differentiate between the behaviours. For example, Crest can be used to identify Flying behaviour, since its values are higher for Flying than for the other two behaviours. b An example decision tree generated from the feature values shown in the lower two rows of (a), with each leaf (grey) node representing a final predicted class for a 1-s segment of data. Supplementary Data 1 provides source data of this figure.

    Full size image

    The energy-saving microcontroller units (MCUs) in bio-loggers tend to have limited memory and low computing capability, which makes it difficult to run the computationally expensive processes needed for the pretrained machine learning models on board the bio-loggers. Therefore, this study proposes a method for generating space-efficient, i.e., programme memory efficient, decision tree classifier models that can be run on such MCUs. Decision trees are well suited for use on MCUs, since the tree itself can be implemented as a simple hierarchy of conditional logic statements, with most of the space needed for the tree being used by the algorithms needed to extract meaningful features from the sensor data, such as the variance or kurtosis of 1-s windows of data. In addition, since each data segment is classified by following a single path through the tree from the root node to the leaf node that represents that data segment’s estimated class, an added benefit of using a tree structure is that the MCU only needs to extract features as they are encountered in the path taken through the tree, allowing the MCU to run only a subset of the feature extraction processes for each data segment. However, the feature extraction algorithms needed by the tree can be prohibitively large, e.g., kurtosis requires 680 bytes (Supplementary Table 1), when implemented on MCUs that typically have memory capacities measured in kilobytes, e.g., 32 KB, which is already largely occupied by the functions needed to log sensor data to storage.
    Standard decision tree algorithms, e.g., scikit-learn’s default algorithm, build decision trees that maximise classification accuracy with no option to weight the features used in the tree based on a secondary factor such as memory usage39. The trees are built starting from the root node, with each node in the tree choosing from among all features the one feature that can best split the training data passed to it into subsets that allow it to differentiate well between the different target classes. A new child node is then created for each of the subsets of training data output from that node, with this process repeating recursively until certain stopping conditions are met, e.g., the subsets generated by a node reach a minimum size. Figure 4b shows an example of a decision tree built using scikit-learn’s default decision tree classifier algorithm using the black-tailed gull data, which results in an estimated memory footprint of 1958 bytes (Supplementary Table 1). Note that since the basic system functions needed to record sensor data to long-term storage already occupy as much as 95% of the bio-logger’s flash memory, incorporating a decision tree with this large of a memory footprint can cause the programme to exceed the bio-logger’s memory capacity (see the bio-logger source code distributed as Supplementary Software for more details).
    Fig. 4: Generating space-efficient trees.

    a Our process for the weighted random selection of features. We start with a list of features along with their required programme memory sizes in bytes (first panel). Each feature is assigned a weight proportional to the inverse of its size, illustrated using a pie chart where each feature has been assigned a slice proportional to its weight (second panel). We then perform weighted random selection to choose the subset of features that will be used when creating a new node in the tree. In this example, we have randomly placed four dots along the circumference of the circle to simulate the selection of four features (second panel). The resulting subset of features will then be compared when making the next node in the decision tree (third panel). b Example decision tree built using scikit-learn’s default decision tree classifier algorithm using the black-tailed gull data described in “Methods”. Each node is coloured based on its corresponding feature’s estimated size in bytes when implemented on board the bio-logger (scale shown in the colour bar). c Several space-efficient decision trees generated using the proposed method from the same data used to create the tree in (b). d Example space-efficient tree selected from the trees shown in (c) that costs much less than the default tree in (b) while maintaining almost the same accuracy.

    Full size image

    In this study, we propose a method for generating decision tree classifiers that can fit in bio-loggers with limited programme memory (e.g., 32 KB) that is based on the random forests algorithm40, which is a decision tree algorithm that injects randomness into the trees it generates by restricting the features compared when creating the split at each node in a tree to a randomly selected subset of the features, as opposed to the standard decision tree algorithm that compares all possible features at each node, as was described above. Our method modifies the original random forests algorithm by using weighted random selection when choosing the subset of features to compare when creating each node. Figure 4a illustrates the weighted random selection process used by our method. We start by assigning each feature a weight that is proportional to the inverse of its size. We then use these weights to perform weighted random selection when selecting a group of features to consider each time we create a new node in the tree, with the feature used at that node being the best candidate from amongst these randomly selected features.
    Using our method for weighted random selection of nodes described above, we are then able to generate randomised trees that tend to use less costly features. When generating these trees, we can estimate the size of each tree based on the sizes of the features used in the tree and limit the overall size by setting a threshold and discarding trees above that threshold. Figure 4c shows an example batch of trees output by our method where we have set a threshold size of 1000 bytes. We can then select a single tree from amongst these trees that gives our desired balance of cost to accuracy. In this example, we have selected the tree shown in Fig. 4d based on its high estimated accuracy. Comparing this tree to Fig. 4b, we can see that our method generated a tree that is 42% the size of the default tree while maintaining close to the same estimated accuracy. We developed our method based on scikit-learn’s (v.0.20.0) RandomForestClassifier.
    In addition, in order to achieve robust activity recognition, our method also has the following features: (i) robustness to sensor positioning, (ii) robustness to noise, and (iii) reduction of sporadic false positives. Note that robustness to noise and positioning are extremely important when deploying machine learning models on bio-loggers, as the models will likely be generated using data collected in previous years, possibly using different hardware and methods of attachment. While there is a potential to improve prediction accuracy by removing some of these variables, e.g., by collecting from the same animal multiple times using the same hardware, moving to more animal-dependent models is generally not practical as care must be taken to minimise the handling of each animal along with the amount of time the animals spend with data loggers attached34. See “Robust activity recognition” for more details.
    GPS features
    Due to the low resolution of GPS data (e.g., metre-level accuracy), GPS-based features cannot detect body movements with the same precision as acceleration-based features, but are useful when analysing patterns in changes in an animal’s location as it traverses its environment. For animal-based AI, these features can be used to differentiate between different large-scale movement patterns, such as transit versus ARS. In this study, we used GPS-based features to detect ARS by streaked shearwaters. These features were extracted once per minute from 1/60th Hz GPS data using 10-min windows. Supplementary Fig. 5b shows these features ranked by their Normalised Gini Importance when used to classify behaviours for streaked shearwaters. Supplementary Fig. 2 shows an example of two such 10-min windows that correspond to target (ARS) and non-target (transit) behaviour, along with several examples of GPS-based features extracted from those windows. Supplementary Table 1 describes all the GPS features used in this study along with their estimated sizes when implemented on board our bio-logger. Note that the variance and mean cross features were extracted after first rotating the GPS positions around the mean latitude and longitude values at angles of 22.5°, 45.0°, 67.5°, and 90.0° in order to find the orientation that maximised the variance in the longitude values (see Supplementary Table 1, feature Y: rotation). This was done to provide some measure of rotational invariance to these values without the need for a costly procedure such as principal component analysis. The primary and secondary qualifiers for these features refer to whether the feature was computed on the axis with maximised variance vs. the perpendicular axis, respectively.
    Robust activity recognition
    In this study, we also incorporated two methods for improving the robustness of the recognition processes in the field. First, we addressed how loggers can be attached to animals at different positions and orientations, such as on the back to maximise GPS reception or on the abdomen to improve the camera’s field of view during foraging. For example, during our case study involving black-tailed gulls, the AI models were trained using data collected from loggers mounted on the birds’ backs, but in many cases were used to detect target behaviour on board loggers mounted on the birds’ abdomens. We achieved this robustness to positioning by converting the three-axis accelerometer data to net magnitude of acceleration values, thereby removing the orientation information from the data. To test the robustness of the magnitude data, we evaluated the difference in classification accuracy between raw three-axis acceleration data and magnitude of acceleration data when artificial rotations of the collection device were introduced into the test data. In addition, we also evaluated the effectiveness of augmenting the raw three-axis data with artificially rotated data as an alternative to using the magnitude of acceleration data. These results are shown in Supplementary Fig. 6a. Note that the results for the magnitude of acceleration data are constant across all levels of test data rotation, since the magnitudes are unaffected by the rotations. Based on these results, we can see that extracting features from magnitude of acceleration data allows us to create features that are robust to the rotations of the device that can result from differences in how the device is attached to an animal.
    Next, we addressed the varying amount of noise that can be introduced into the sensor data stemming from how loggers are often loosely attached to a bird’s feathers. We achieved this noise robustness by augmenting our training dataset with copies of the dataset that were altered with varying levels of random artificial noise, with this noise added by multiplying all magnitude of acceleration values in each window of data by a random factor. We tested the effect of this augmentation by varying the amount of artificial noise added to our training and testing data and observing how the noise levels affected performance (see Supplementary Fig. 6b). Based on these results, the training data used for fieldwork was augmented using the 0.2 level. Note that at higher levels of simulated noise (test noise greater than 0.15) the training noise settings of 0.25 and 0.3 both appear to outperform the 0.2 setting. However, since these results are based solely on laboratory simulations, we chose to use the more conservative setting of 0.2 in the field.
    Reduction of sporadic false positives
    When activating the camera to capture target behaviour, it is possible to reduce the number of false positives (i.e., increase our confidence in the classifier’s output) by considering multiple consecutive outputs from the classifier before camera activation. We accomplish this using two methods. In the first, we assume that the classifier can reliably detect the target behaviour throughout its duration, allowing us to increase our confidence in the classifier’s output by requiring multiple consecutive detections of the target behaviour before activating the camera. We employed this method when detecting ARS behaviour for streaked shearwaters using GPS data, since the characteristics of the GPS data that allow for detection of the target behaviour were expected to be consistent throughout its duration, with the number of consecutive detections required set to 2.
    In the second method, we assume that the classifier cannot reliably detect the target behaviour throughout its duration, since the actions corresponding to the target behaviour that the classifier can reliably detect occur only sporadically throughout its duration. In this case, we can instead consider which behaviours were detected immediately prior to the target behaviour. When controlling the camera for black-tailed gulls, we assume that detection of the target behaviour (foraging) is more likely to be a true positive after detecting flying behaviour, since the birds typically fly when searching for their prey. Therefore, we required that the logger first detect five consecutive windows of flying behaviour to enter a flying state in which it would activate the camera immediately upon detecting foraging. This flying state would time out after ten consecutive windows of stationary behaviour. Note that in this case, while the intervals of detectable target behaviour may be short and sporadic, the overall duration of the target behaviour is still long enough that we can capture video of the behaviour despite the delay between behaviour detection and camera activation (see “Video bio-logger hardware” for details).
    Procedures of experiment of black-tailed gulls
    We evaluated the effectiveness of our method by using AIoA-based camera control on board ten bio-loggers that were attached to black-tailed gulls (on either the bird’s abdomen or back) from a colony located on Kabushima Island near Hachinohe City, Japan18, with the AI trained to detect possible foraging behaviour based on acceleration data. The possible foraging events were identified based on dips in the acceleration data. The training data used for the AI was collected at the same colony in 2017 using Axy-Trek bio-loggers (TechnoSmArt, Roma, Italy). These Axy-Trek bio-loggers were mounted on the animals’ backs when collecting data. Along with the AIoA-based bio-loggers, three bio-loggers were deployed using a naive method (periodic sampling), with the cameras controlled by simply activating them once every 15 min. All 13 loggers recorded 1-min duration videos.
    Sample size was determined by the time available for deployment and the availability of sensor data loggers. The birds were captured alive at their nests by hand prior to logger deployment and subsequent release. Loggers were fitted externally within 10 min to minimise disturbance. Logger deployment was undertaken by the ecologists participating in this study. Loggers that suffered hardware failures (e.g., due to the failure of the waterproofing material used on some loggers) were excluded.
    Ethics statement
    All experimental procedures were approved by the Animal Experimental Committee of Nagoya University. Black-tailed gulls: the procedures were approved by the Hachinohe city mayor, and the Aomori Prefectural Government. Streaked shearwaters: the study was conducted with permits from the Ministry of the Environment, Japan.
    Statistics and reproducibility
    Fisher’s exact tests were done using the exact2x2 package (v. 1.6.3) of R (v. 3.4.3). The GLMM analysis was conducted using the lmerTest package (v. 2.0–36) of R (v. 3.4.3). In regards to reproducibility, no experiments as such were conducted, rather our data are based on tracked movements of individual birds.
    Reporting summary
    Further information on research design is available in the Nature Research Reporting Summary linked to this article. More

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    Nearshore neonate dispersal of Atlantic leatherback turtles (Dermochelys coriacea) from a non-recovering subpopulation

    Ethics statement
    All procedures for fieldwork in Pacuare Nature Reserve followed approved protocol under Monash University’s School of Biological Sciences Animal Ethics Committee (Protocol No. BSCI/2016/13), the University of Maryland Center for Environmental Sciences’ Institutional Animal Care and Use Committee (IACUC) (Research Protocol No. S-CBL-16–11), and the Costa Rican Ministerio Del Ambiente y Energia, Sistema Nacional de Áreas de Conservación (SINAC), Área de Conservación La Amistad Caribe (ACLAC) (RESOLUCIÓN SINAC-ACLAC-PIME-VS-R-022-2016; RESOLUCIÓN SINAC-ACLAC-PIME-VS-R-025-2016). The study was performed in accordance with the approved guidelines.
    Hatchling tracking
    To examine in-situ factors of turtle dispersal into the offshore environment, leatherback hatchlings were tagged with coded acoustic transmitters between 20 August and 3 September 2016 in Pacuare Nature Reserve, Limón Province, Costa Rica (Fig. 1). At Pacuare, hatchlings were obtained from hatchery, incubator-reared, and relocated nests. Hatchery nests consisted of eggs collected as they were laid, transported in plastic bags for less than 5 km, and reburied in two separate protected areas. In these protected, monitored enclosures, the eggs were safeguarded (e.g. from predation) and otherwise developed naturally. Relocated eggs were collected from nests laid the night prior, transported in plastic bags a short distance above the high tide line, reburied on the nesting beach and unmonitored thereafter, until such time as hatchlings emerged. Hatchlings from hatchery and relocated nests were collected as they naturally emerged from the buried nests. Incubator-reared eggs were collected as they were laid, transported up to 1 km in vacuum-sealed bags, and raised under 3 treatments: control, low-oxygen, and high-oxygen in accordance with the Williamson38 protocol. Eggs were incubated for the first 5 days of development in: hypoxia (1% O2) for the low-oxygen treatment and hyperoxia (42% O2) for the high-oxygen treatment. As they did not have to expend time and energy exiting a nest, incubator-hatched turtles were left to absorb their yolk for 2 days38. Turtles held post-emergence from their nest (hatchery and relocated hatchlings) or eggs (incubator-reared hatchlings) were kept in moistened, sand-lined incubators at approximately 30 °C to reduce energy expenditure prior to trial release and prevent potential decreases in swimming performance39. To minimise the influence of genetic relatedness, hatchlings were taken from all available nests (n = 9 in total from hatchery, relocated, and incubator nests) at the time of the study, resulting in parentage by nine females. Turtles were weighed and measured prior to trials using a scale and calliper. To prevent overheating on the boat, turtles were transported in a bucket covered by a wet towel with a moistened cloth inside.
    Acoustic tracking was conducted using Vemco V5-180 kHz transmitter tags (0.38 g in-water weight; 0.65 g in-air weight) and tethered to the turtle via a line-float-transmitter assembly (6.85 g in-air weight) and Vetbond based on Gearheart et al.24 and Hoover et al.25 (Fig. S1). The monofilament line in the line-float-transmitter assembly was a total length of 2 m; the first float was suspended 1.5 m behind the hatchling, and the second float was an additional 0.5 m. The brightly coloured orange floats (4.4 cm by 1.9 cm) allowed for visual tracking in the water when acoustic signal was insufficient. Tracking began outside the surf zone, approximately 0.4 km from shore, where turtles were taken via a small 6 m, 150 hp motorboat. The release location was the approximate midpoint of the two hatcheries where hatchlings were collected. Between sunrise and sunset, each turtle was followed at a distance of 10–20 m in the boat using a Vemco VR100 acoustic receiver and VH180-D-10M directional hydrophone22. The V5 tag detection range was approximately 200 m. The VR100 receiver stored the detections, and the data were downloaded to reconstruct hatchling movement paths. The mobile acoustic receiver allowed tracking of the turtles’ movements for a longer period and over a broader area than visual tracking alone because turtles were found acoustically when visual contact was lost.
    Hatchlings were tracked only during daylight hours over a 3-week period given hatchling and boat availability. Although hatchlings generally emerge during cooler, evening hours of the day in Costa Rica, no effect on the overall innate behaviour of hatchlings was anticipated18,40. The tracking data should still be indicative of the orientation and speed at which hatchlings are likely to swim. Nighttime tracking was logistically infeasible because of the hazards associated with the oceanic entry point. For a track to provide enough data for inclusion in the analysis, a minimum tracking time of 30 min was established. Turtles were tracked individually for approximately 90 min. Track duration was a trade-off between obtaining a large sample of tracks to account for individual variability, while providing robust speed and orientation information. At the end of each track, the turtle was recovered with a small net, the line-float-transmitter attachment was completely removed, and the turtle was released at the recovery location. The Velcro piece easily removed from the carapace, and there were no evident damages, marks, or lesions from this attachment method on the leatherback hatchlings. Handling was kept to a minimum to reduce any unnecessary stress on the turtles.
    Surface current trajectories
    Two drifters were used to obtain data on local sea surface currents to evaluate the effect of currents on hatchling movements. A Pacific Gyre Microstar drifter was deployed at the beginning of each turtle track (Fig. S2A). The drifter’s surface float was equipped with a GPS unit that used the iridium short burst data service to broadcast location coordinates every 5 min. A flag was attached to the surface float for increased visibility. Sea surface temperature was recorded by the drifter with a Pacific Gyre probe with 0.1 °C accuracy. The position and temperature data of each drifter release were retrieved from the Pacific Gyre website (https://www.pacificgyre.com). One drifter track was removed from analysis because it entered the surf zone and did not represent nearshore surface currents.
    A secondary drifter was launched when equipment permitted at the approximate halfway point during tracking of a turtle. This better estimated the immediate currents the hatchling was experiencing and was used to estimate shifts in the nearshore currents as the turtles headed offshore. This second drifter was constructed using a Davis Instruments aluminium radar reflector with 80 cm of parachute cord attached to a 20.3 cm diameter Panther Plast trawl float (Fig. S2B). The centre of the drifter sat 1 m from the water’s surface, similar to the depth of the Microstar drifter. A piece of wood affixed to the top of the float had a Samsung Galaxy Core Prime mobile phone attached in a waterproof bag. A GPS application was started with each drifter release to provide locations at one minute intervals. Foam tubing was zip-tied around the middle of the trawl float to maintain the GPS unit in an upright position. The float had a flag attached for visibility on the water. Positions were stored on the mobile phone and downloaded upon retrieval of the drifter. Both drifters were recovered at the completion of each individual turtle track.
    Environmental data
    To understand the influence of tidal states and bathymetry on local currents experienced by hatchling leatherbacks, daily tidal currents were obtained at Limón, Costa Rica (10.00° N, 83.03° W; https://tides.mobilegeographics.com). Periods of peak tides (i.e. high and low) were defined as one hour before and after the measured minima or maxima, with ebb and flow tides between those periods of peak tides. Tidal states were categorised as: high, ebb, low, and flow (Fig. S3A). High-resolution, near-shore bathymetry data were obtained at a 0.0011° resolution from the Global Multi-Resolution Topography Synthesis dataset (https://www.gmrt.org). Missing values were filled with data from the General Bathymetric Chart of the Oceans dataset (GEBCO-2014; https://www.gebco.net; 0.0042° resolution). Bathymetric values for each hatchling track were extracted with a ‘bilinear’ interpolation in the R ‘raster’ package41 (Fig. S3B). All analyses were conducted in the R environment42.
    Hatchling movement analysis
    An acclimation period of five min was applied to each turtle track to provide time for the hatchling to orient and adjust to the water temperature. Intervals greater than five min between recorded hatchling positions were removed to prevent erroneous calculations (0.03% of recorded positions). These time lapses occurred when the boat was actively searching for a hatchling. Despite the combination of surface floats and the directional hydrophone, maintaining visual and acoustic contact with turtles was challenging, even in calm waters. Many hatchlings dove for short periods ( More

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    First molecular examination of Vietnamese mudflat snails in the genus Naranjia Golding, Ponder & Byrne, 2007 (Gastropoda: Amphibolidae)

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